Culture of Coldwater Marine Fish

CULTURE OF COLD-WATER MARINE FISH Edited by E. Moksness E. Kjørsvik and Y. Olsen Fishing News Books An imprint of Bla...

Author: Erlend Moksness | E. Kjorsvik | Y. Olsen

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CULTURE OF COLD-WATER MARINE FISH Edited by

E. Moksness E. Kjørsvik and

Y. Olsen

Fishing News Books An imprint of Blackwell Science

CULTURE OF COLD-WATER MARINE FISH Edited by

E. Moksness E. Kjørsvik and

Y. Olsen

Fishing News Books An imprint of Blackwell Science

© 2004 by Blackwell Publishing Ltd Editorial Ofﬁces: Blackwell Publishing Ltd, 9600 Garsington Road, Oxford OX4 2DQ, UK Tel: +44 (0)1865 776868 Iowa State Press, a Blackwell Publishing Company, 2121 State Avenue, Ames, Iowa 50014-8300, USA Tel: +1 515 292 0140 Blackwell Publishing Asia Ltd, 550 Swanston Street, Carlton, Victoria 3053, Australia Tel: +61 (0)3 8359 1011 The right of the Author to be identiﬁed as the Author of this Work has been asserted in accordance with the Copyright, Designs and Patents Act 1988. All rights reserved. No part of this publication may be reproduced, stored in a retrieval system, or transmitted, in any form or by any means, electronic, mechanical, photocopying, recording or otherwise, except as permitted by the UK Copyright, Designs and Patents Act 1988, without the prior permission of the publisher. First published 2004 by Blackwell Publishing Ltd Library of Congress Cataloging-in-Publication Data Culture of cold-water marine ﬁsh / editors, E. Moksness, E. Kjørsvik, and Y. Olsen. p. cm. Includes bibliographical references (p. ). ISBN 0-85238-276-6 (hardback : alk. paper) 1. Marine ﬁshes. 2. Fish-culture. I. Moksness, Erlend. II. Kjørsvik, E. III. Olsen, Yngvar. SH163.C85 2004 639.34¢2—dc21 2003002265 ISBN 0-85238-276-6 A catalogue record for this title is available from the British Library Set in 10 on 13 pt Times by SNP Best-set Typesetter Ltd., Hong Kong Printed and bound in Great Britain using acid-free paper by Bath Press, Bath For further information on Blackwell Publishing, visit our website: www.blackwellpublishing.com

Contents

Preface List of Contributors 1 Introduction The Editors 1.1

References

2 Abiotic Factors B.R. Howell and S.M. Baynes 2.1 2.2 2.3 2.4

Introduction Oxygen and oxygen consumption Ammonia Temperature 2.4.1 Seasonal temperature cycle and spawning 2.4.2 Egg and larval development 2.4.3 Sex ratio 2.4.4 Growth and metabolism 2.5 Salinity 2.6 Hydrogen sulphide 2.7 Light 2.7.1 Growth and development 2.7.2 Reproduction 2.8 Algae blooms 2.9 Site selection 2.10 References 3 Microbial Interactions, Prophylaxis and Diseases O. Vadstein, T.A. Mo and Ø. Bergh 3.1

Fish–microbe interactions and implications in aquaculture 3.1.1 Disease-causing organisms 3.1.2 Normal ﬁsh–microbe interactions, infection pathways and pathogenesis 3.1.3 The immune system of ﬁsh

xiv xv 1 5 7 7 7 10 12 13 13 14 14 15 17 18 18 20 21 23 26 28 28 28 29 33

iv

Contents

3.2

3.3

3.4

3.5

3.6

3.7

3.8 3.9

Viral diseases: diagnosis 3.2.1 Infectious pancreatic necrosis virus (IPNV) 3.2.2 Nodaviruses 3.2.3 Other viruses Bacterial diseases: diagnosis 3.3.1 Vibrio species 3.3.2 Aeromonas species Parasitic protists and metazoans: diagnosis, prophylaxis and treatment 3.4.1 Protists 3.4.1.1 Amoebae 3.4.1.2 Apicomplexans 3.4.1.3 Microsporidia 3.4.1.4 Ciliates 3.4.1.5 Flagellates 3.4.2 Metazoans 3.4.2.1 Myxosporidia (parasitic Cnidarians) 3.4.2.2 Monogeneans 3.4.2.3 Cestodes 3.4.2.4 Trematodes 3.4.2.5 Nematodes 3.4.2.6 Acanthocephalans 3.4.2.7 Leeches 3.4.2.8 Crustaceans A strategy for microbial control 3.5.1 General considerations 3.5.2 A strategy for microbial control and important elements in such a strategy Improving environmental conditions 3.6.1 Non-selective reduction of microbes 3.6.2 The use of probiotics 3.6.3 Selection for desirable bacteria Improving the resistance of the ﬁsh 3.7.1 Modulation of speciﬁc immunity—vaccination 3.7.2 Modulation of non-speciﬁc immunity 3.7.3 The effect of nutrition and genetics on resistance against microbes Closing remarks References

4 Live Food Technology of Cold-Water Marine Fish Larvae Y. Olsen 4.1 4.2

Introduction Cultivation systems

35 35 36 37 38 39 39 40 41 41 41 42 43 44 44 44 45 46 46 47 48 48 49 50 51 55 57 57 58 59 61 61 62 63 63 64 73

73 75

Contents

4.3

4.4

4.5 4.6 4.7

Production of rotifers 4.3.1 Biological characteristics 4.3.1.1 General biology and life history 4.3.1.2 Feeding kinetics of B. plicatilis 4.3.1.3 Growth, mortality and egg ratio 4.3.2 Cultivation feed and feed treatments 4.3.3 Cultivation of rotifers 4.3.3.1 Maintenance of stock cultures 4.3.3.2 Inoculation phase 4.3.3.3 Early growth phase 4.3.3.4 Late growth phase—harvesting strategies 4.3.3.5 Production in batch culture 4.3.3.6 Production in continuous culture 4.3.4 High-intensity rotifer cultivation 4.3.5 Problems in rotifer cultivation 4.3.5.1 Feeding-related problems 4.3.5.2 Environmentally related problems 4.3.5.3 Disease and contamination 4.3.5.4 Problem identiﬁcation—diagnostic criteria 4.3.5.5 Counter-measures against undesirable situations 4.3.6 Biochemical composition during steady-state feeding and growth 4.3.6.1 Proteins and essential amino acids 4.3.6.2 Lipids and essential fatty acids 4.3.6.3 Vitamins and minerals 4.3.7 Short-term enrichment techniques to improve nutritional value 4.3.7.1 Proteins 4.3.7.2 Lipids and fatty acids 4.3.8 Stability of nutritional value Production of Artemia 4.4.1 Feeding and growth 4.4.2 Biomass and biochemical composition 4.4.3 Pre-enrichment cultivation 4.4.3.1 Disinfection of cysts 4.4.3.2 Decapsulation of cysts 4.4.3.3 Hatching of cysts 4.4.4 Enrichment and stability of n-3 fatty acids 4.4.4.1 n-3 HUFA enrichment 4.4.4.2 Stability of n-3 fatty acids post-enrichment 4.4.5 n-3 HUFA of Artemia juveniles 4.4.6 Vitamins and minerals Marine copepods Concluding remarks References

v

76 76 76 77 79 80 81 81 82 83 86 88 92 96 96 97 97 98 98 99 100 100 102 105 106 106 106 108 111 111 113 114 114 114 115 116 116 119 121 122 122 124 125

vi

Contents

5 Brood Stock and Egg Production D. Pavlov, E. Kjørsvik, T. Refsti and Ø. Andersen 5.1 5.2

5.3

5.4

5.5

Reproductive strategies Gonad maturation 5.2.1 Females 5.2.2 Males 5.2.3 Spawning once or many times? 5.2.4 Endocrine regulation Brood-stock management and egg production 5.3.1 Brood-stock nutrition 5.3.1.1 Ration size 5.3.1.2 Feed composition 5.3.1.3 Fatty acids 5.3.1.4 Micronutrients 5.3.1.5 Pigments and minerals 5.3.2 Photoperiod 5.3.3 Temperature 5.3.4 Present husbandry practices and egg collection 5.3.4.1 Cod 5.3.4.2 Turbot 5.3.4.3 Atlantic halibut 5.3.4.4 Wolf-ﬁsh Egg quality 5.4.1 Assessment of egg quality 5.4.1.1 Egg morphology 5.4.1.2 Fertilisation success and cortical reaction 5.4.1.3 Blastomere morphology 5.4.1.4 Egg size 5.4.1.5 Chemical content 5.4.1.6 Cytology 5.4.1.7 Oxygen consumption 5.4.1.8 Evaluating mammalian embryo quality 5.4.2 Factors affecting egg quality 5.4.2.1 Over-ripening 5.4.2.2 Viability of ovulated eggs in vivo 5.4.2.3 Viability of ovulated eggs in vitro 5.4.2.4 Changes in the eggs 5.4.3 Change in egg quality over the spawning season 5.4.4 Maternal effects 5.4.5 Conclusions Sperm production and quality 5.5.1 Features of sperm production and quality

129

129 132 134 135 136 138 142 143 144 145 146 147 149 150 152 152 153 154 154 155 156 157 159 159 161 164 166 167 168 168 168 168 169 170 170 172 173 174 175 175

Contents

5.6

5.7

5.8

5.5.1.1 Morphology 5.5.1.2 Gonadosomatic index and ejaculate volume 5.5.1.3 Concentration 5.5.1.4 Motility 5.5.1.5 Fertilising capacity 5.5.1.6 Biochemistry and oxygen consumption 5.5.2 Inﬂuence of environmental factors on sperm quality 5.5.3 Sperm storage Selective breeding 5.6.1 Expected beneﬁts 5.6.2 Phenotypic value and variance 5.6.3 Genotype by environmental interaction 5.6.4 Breeding goal 5.6.5 Growth rate 5.6.6 Feed efﬁciency 5.6.7 Disease resistance 5.6.8 Quality 5.6.9 Age at sexual maturation 5.6.10 Base population and brood-stock development 5.6.11 Inbreeding 5.6.12 Selection methods 5.6.12.1 Individual selection (mass selection) 5.6.12.2 Family selection 5.6.12.3 Progeny testing 5.6.12.4 Combined selection 5.6.13 Response to selection 5.6.14 Multi-trait selection Modern biotechnology and aquaculture 5.7.1 Molecular pedigree analysis 5.7.2 Genetic mapping and QTL analysis 5.7.3 Transgenic ﬁsh 5.7.4 Future prospects References

6 From Fertilisation to the End of Metamorphosis—Functional Development E. Kjørsvik, K. Pittman and D. Pavlov 6.1 6.2 6.3 6.4

Intervals of ﬁsh ontogeny and deﬁnitions of the organism 6.1.1 Relative duration of the various stages of development Egg classiﬁcation 6.2.1 Egg structure and composition Insemination and fertilisation Embryonic development and hatching

vii

175 176 177 178 179 179 180 181 182 183 183 184 185 185 185 185 186 186 186 186 186 186 187 187 187 188 188 188 189 191 192 193 193 204

204 207 208 209 212 214

viii

Contents

6.5

6.6

6.7

6.8 6.9

6.4.1 Cod (Gadus morhua) 6.4.2 Wolf-ﬁsh (Anarhichas lupus) 6.4.3 Embryo growth and yolk absorption From hatching to metamorphosis 6.5.1 To be a larva . . . 6.5.2 . . . or not to be a larva 6.5.3 The yolk-sac period—preparation for real ‘real life’ 6.5.4 Metamorphosis Functional development of organ systems from hatching to metamorphosis 6.6.1 Sensory system 6.6.1.1 Vision and the oculovestibular system 6.6.1.2 Chemosensory system 6.6.1.3 Lateral line 6.6.2 Digestive system 6.6.2.1 Gut, pancreas and liver differentiation 6.6.2.2 Digestive enzymes 6.6.2.3 Digestive physiology—lipids, proteins and carbohydrates 6.6.2.4 Stomach development and metamorphosis 6.6.3 Muscle and body skeleton 6.6.3.1 Swimming capacity and muscle development 6.6.3.2 Musculature changes during metamorphosis 6.6.3.3 Skeletal changes 6.6.4 Swim-bladder 6.6.5 Osmoregulation 6.6.6 Respiration and excretion 6.6.7 Neuroendocrine systems 6.6.8 Growth hormone, prolactin and cortisol 6.6.9 The immune system 6.6.10 Skin and pigmentation 6.6.11 Larval feeding behaviour 6.6.12 Larval growth 6.6.12.1 How can we express larval growth? 6.6.13 Inﬂuence of diet 6.6.14 Juvenile quality Hatchery design ` 6.7.1 The demersal eggs of wolf-ﬁsh 6.7.2 Pelagic eggs (cod, turbot, halibut) 6.7.2.1 Cod 6.7.2.2 Turbot 6.7.2.3 Halibut Critical aspects of larval cultivation References

215 221 224 225 225 226 226 228 229 230 230 232 232 233 234 236 239 240 241 241 243 244 245 247 248 250 255 256 256 257 260 261 262 264 265 265 265 266 266 266 267 269

Contents

7 First Feeding Technology Y. Olsen, T. van der Meeren and K.I. Reitan 7.1 7.2

7.3

7.4

7.5

Introduction Nutritional requirements of marine ﬁsh larvae 7.2.1 Essential fatty acids 7.2.2 Main lipid classes 7.2.3 Physiological basis of n-3 HUFA requirements 7.2.4 Protein and essential amino acids 7.2.5 Protein versus lipid nutrition Deﬁnitions and system description 7.3.1 Extensive systems: large closed nature-like systems 7.3.2 Semi-intensive systems: large suspended mesocosms, enclosures or outdoor tanks 7.3.3 Larval rearing in relatively small tanks: classical intensive hatchery techniques 7.3.3.1 Water treatment and supply 7.3.3.2 Production lines for live feed 7.3.3.3 Larval rearing systems 7.3.3.4 Automation and process control Larval rearing in ‘nature-like systems’ 7.4.1 Pioneer work 7.4.2 The ‘lagoon method’ as a production system 7.4.3 Larval food and feeding in mesocosms 7.4.3.1 Initiation of exogenous feeding: the ‘green gut’ 7.4.3.2 Prey selection 7.4.3.3 Feeding, growth and survival Larval ﬁrst feeding in intensive systems 7.5.1 Physical chemical environment 7.5.2 Feeding characteristics of ﬁsh larvae 7.5.2.1 Food selection 7.5.2.2 Feeding and functional response 7.5.2.3 Larval feeding rate of live feed 7.5.2.4 Larval feeding on microalgae 7.5.3 Feeding regime components for cold-water larviculture 7.5.3.1 Microalgae 7.5.3.2 Rotifers 7.5.3.3 Artemia naupli 7.5.3.4 Juvenile Artemia 7.5.4 Tentative feeding regimes for common species 7.5.4.1 Stocking densities 7.5.4.2 Live food rations 7.5.4.3 Atlantic cod and haddock 7.5.4.4 Atlantic halibut

ix

279

279 280 280 280 282 284 284 285 287 289 290 291 292 293 294 295 295 296 297 298 299 300 301 301 302 303 303 304 305 306 306 307 307 308 308 308 309 310 310

x

Contents

7.6 7.7

7.5.4.5 Turbot 7.5.4.6 Sole, wolf-ﬁsh and hake 7.5.5 Growth-rate characteristics during ﬁrst feeding 7.5.6 Nutritional challenges and conﬂicts 7.5.6.1 Criteria of nutritional value for live feed 7.5.6.2 Lipids and n-3 HUFA 7.5.6.3 Essential amino acids and proteins 7.5.6.4 Synergetic importance of lipids and proteins 7.5.6.5 Vitamins and minerals 7.5.6.6 General recommendations on larval nutrition 7.5.7 Microbial conﬂicts and challenges 7.5.7.1 Methods of non-selective reduction of bacteria 7.5.7.2 Methods for selective enhancement of favourable bacteria 7.5.8 Use of ‘green water’ techniques 7.5.8.1 Effects of ‘green water’ 7.5.8.2 Nutritional effects 7.5.8.3 Microbial effects 7.5.8.4 Live feed retention time in larval tanks Concluding remarks References

8 Weaning and Nursery J. Stoss, K. Hamre and H. Otterå 8.1 8.2 8.3

8.4 8.5

Introduction Developmental aspects of digestion in marine ﬁsh larvae Nutrition 8.3.1 Macronutrient composition 8.3.2 Composition of dietary protein 8.3.3 Composition of the lipid fraction 8.3.4 Vitamin supplementation Microparticulate diets Weaning and nursery stage, practical aspects 8.5.1 General 8.5.1.1 The role of early start feeding 8.5.1.2 Early weaning and co-feeding 8.5.1.3 Uptake and ingestion of formulated diets 8.5.1.4 Availability of particulate food 8.5.1.5 Tank hygiene 8.5.1.6 Rearing temperature and light 8.5.1.7 Vaccination against bacterial diseases 8.5.1.8 Handling of ﬁsh 8.5.2 Cod

312 312 313 314 314 315 319 322 323 324 325 326 327 328 329 331 332 332 333 333 337

337 338 341 341 342 344 344 345 346 346 346 347 348 348 348 349 350 351 352

Contents

8.6

8.5.2.1 8.5.2.2 8.5.2.3 8.5.2.4 8.5.2.5 8.5.3 Turbot 8.5.3.1 8.5.3.2 8.5.3.3 8.5.3.4 8.5.3.5 8.5.4 Halibut 8.5.4.1 8.5.4.2 8.5.4.3 8.5.4.4 References

Early weaning Weaning Cannibalism Gas bubble formation Nursery Early weaning Weaning Nursery Rearing density Tanks Growth Early weaning Weaning Nursery

9 On-Growing to Market Size M. Jobling 9.1 9.2 9.3 9.4 9.5 9.6 9.7 9.8 9.9 9.10 9.11 9.12

Introduction Analysis of feeds and feedstuffs Protein requirements and sources Lipids and lipid requirements Carbohydrates Micronutrients: vitamins and minerals Feed types and formulations Feeding regimes and practices Growth and feed conversion Nutrient deposition and body composition Concluding comments References

10 The Status and Perspectives for the Species T. Svåsand, H.M. Otterå, G.L. Taranger, M. Litvak, A.B. Skiftesvik, R.M. Bjelland, D.A. Pavlov, J.Chr. Holm, T. Harboe, A. Mangor-Jensen, B. Norberg and B. Howell 10.1

Atlantic cod 10.1.1 Introduction 10.1.2 Brood stock, egg production and incubation 10.1.3 Extensive production 10.1.4 Intensive production 10.1.5 On-growing

xi

352 353 354 354 354 355 355 356 357 358 358 359 359 360 360 361 361 363 363 364 367 377 383 385 391 396 402 413 422 423 433

433 433 433 435 436 440

xii

Contents

10.2

10.3

10.4

10.5

10.6

10.7

10.1.6 Future prospects 10.1.7 References 10.1.8 Further reading Haddock 10.2.1 Introduction 10.2.2 Brood stock, egg production and incubation 10.2.3 Larval rearing 10.2.4 Weaning and on-growing 10.2.5 Health 10.2.6 Commercial development 10.2.7 Future prospects 10.2.8 Further reading Hake 10.3.1 Introduction 10.3.2 Egg production and incubation 10.3.3 Larval rearing 10.3.4 Weaning and on-growing 10.3.5 Future prospects 10.3.6 References Wolf-ﬁsh 10.4.1 Introduction 10.4.2 Brood stock, egg production and incubation 10.4.3 Larval rearing and on-growing 10.4.4 Future prospects 10.4.5 References Halibut 10.5.1 Introduction 10.5.2 Brood stock, egg production and incubation 10.5.3 Larval rearing 10.5.4 Weaning and on-growing 10.5.5 On-growing systems 10.5.6. References Turbot 10.6.1 Introduction 10.6.2 Brood stock, egg production and incubation 10.6.3 Larval rearing 10.6.4 Weaning and on-growing 10.6.5 Future prospects 10.6.6 References Sole 10.7.1 Introduction 10.7.2 Brood stock, egg production and incubation 10.7.3 Larval rearing 10.7.4 Weaning and on-growing

442 442 443 444 444 446 447 448 449 449 450 450 451 451 451 452 453 454 454 454 454 455 456 458 459 461 461 462 464 466 466 466 467 467 468 468 469 470 471 471 471 472 472 473

Contents

10.7.5 10.7.6

xiii

Future prospects References

473 474

11 Marine Stock Enhancement and Sea-Ranching T. Svåsand and E. Moksness

475

11.1 11.2

11.3 11.4

11.5 11.6

Introduction Stock enhancement and sea-ranching in Europe and North America 11.2.1 Atlantic cod 11.2.2 Other cold-water species Stock enhancement and sea-ranching in Asia Prospects and limitations of enhancement and sea-ranching 11.4.1 Biological constraints 11.4.2 Economic constraints Recommendations and guidelines References

12 New Species in Aquaculture: Some Basic Economic Aspects R. Engelsen, F. Asche, F. Skjennum and G. Adoff 12.1

Introduction 12.1.1 Markets, productivity and production growth 12.1.2 The economics of a market and productivity 12.1.3 The evolution of the salmon industry 12.1.4 The evolution of the sea bass and sea bream industry 12.1.5 The evolution of the American catﬁsh industry 12.2 Cod 12.3 Haddock 12.4 European hake 12.5 Wolf-ﬁsh 12.6 Halibut 12.7 Turbot 12.8 Sole 12.9 Conclusions 12.10 References Index

475 477 477 479 480 482 482 483 485 485 487 487 489 491 493 495 496 496 501 501 503 504 507 511 512 515 517

Preface

Mariculture has a long history worldwide, but intensive production, as seen with salmon today, is a relatively new approach. The global production and marketing of salmon is a success story, and many other species have, partly because of that success, caught interest as potential candidates for future mariculture. In Norway, the attention on species other than salmonides increased in the 1980s, and signiﬁcant research efforts were made to develop future production technology for cold-water marine ﬁsh species. The work focused very much on early life histories and the main goal was to obtain a high and stable number of juveniles for further on-growing. As a result of this research activity, efforts were increased to educate students from high school to university. Besides our own research on mariculture, we have also been involved in the teaching of students at university level. Over the years we have all produced compendiums as textbooks for the students and when Blackwell Publishing approached us some years ago to ask if we were willing to turn our compendiums into a book, we accepted their offer immediately. This was mainly because we saw a great need for such a textbook for our students. We also realised that each of the sections covered in the book had developed so quickly that we needed to invite expert authors for the chapters to improve the quality of the book. All the invited authors responded positively, and we are very grateful to them for their contribution to the book. We would also like to thank all the students, technicians and colleagues, from all research institutions, who have contributed to the comprehensive research and technological developments, that have made this book possible. The book is dedicated to the late Professor Arne Jensen who was an enthusiastic driving force for research in developing aquaculture in Norway until his death in August 2000. The Editors

List of Contributors

Adoff, Grethe Rønnevik Bergen Aqua AS, Bredalsmarken 15–17, Møhlenpris, Box 2604, 5836 Bergen, Norway Andersen, Øivind Institute of Aquaculture Research, PO Box 5010, N-1432 Aas, Norway Asche, Frank Stavanger University College, Box 2557 Ullandhaug, N-4091 Stavanger, Norway Baynes, Stephen M. CEFAS Weymouth Laboratory, Banach oad, The Nothe, Weymouth, Dorset DT4 8UB, UK Bergh, Øyvind Institute of Marine Research, Austevoll Aquaculture Station, 5392 Storebø, Norway Bjelland, Reidun M. Institute of Marine Research, Austevoll Aquaculture Station, 5392 Storebø, Norway Engelsen, Rolf Bergen Aqua AS, Bredalsmarken 15–17, Møhlenpris, Box 2604, 5836 Bergen, Norway Hamre, Kristin Fiskeridirektoratets ernæringsinstitutt, PO Box 185 Sentrum, N-5804 Bergen, Norway Harboe, Torstein Institute of Marine Research, Austevoll Aquaculture Station, 5392 Storebø, Norway Holm, Jens Chr. Directorate of Fisheries, Strandgt. 229, PO Box 185, N-5804 Bergen, Norway Howell, Bari R. CEFAS Weymouth Laboratory, Banach oad, The Nothe, Weymouth, Dorset DT4 8UB, UK. Present address: 73 Plasturton Avenue, Pontcanna, Cardiff CF11 9HN, UK Jobling, Malcolm NFH, University of Tromsø, Dramsveien 201, N-9001 Tromsø, Norway Kjørsvik, Elin Department of Biology, Norwegian University of Science and Technology, N-7491 Trondheim, Norway

xvi

List of contributors

Litvak, Matt Centre for Coastal Studies and Aquaculture, University of New Brunswick, PO Box 5050, Saint John, NB, E2L 4L5, Canada Mangor-Jensen, Anders Institute of Marine Research, Austevoll Aquaculture Station, 5392 Storebø, Norway Meeren, Terje van der Institute of Marine Research, Austevoll Aquaculture Station, 5392 Storebø, Norway Mo, Tor Atle National Veterinary Institute, Ullevålsvn. 68, N-0454 Oslo 4, Norway Moksness, Erlend Institute of Marine Research, Department of Coastal Zone, Flødevigen Marine Research Station, N-4817 His, Norway Norberg, Birgitta Institute of Marine Research, Austevoll Aquaculture Station, 5392 Storebø, Norway Olsen, Yngvar Trondhjem Biological Station, Department of Biology, Norwegian University of Science and Technology, N-7491 Trondhjem, Norway Otterå, Håkon M. Institute of Marine Research, Department of Aquaculture, PO Box 1870 Nordnes, N-5017 Bergen, Norway Pavlov, Dimitri A. Moscow State University, Faculty of Biology, Department of Ichthyology, Moscow 119899, Russia Pittman, Karin Department of Fisheries and Marine Biology, University of Bergen, Bergen High Technology, N-5020 Bergen, Norway Refstie, Terje Akvaforsk, PO Box 203, N-6600 Sunndalsøra, Norway Reitan, Kjell I. SINTEF Fisheries and Aquaculture, N-7465 Trondheim, Norway Skiftesvik, Anne B. Institute of Marine Research, Austevoll Aquaculture Station, 5392 Storebø, Norway Skjennum, Finn Chr. Bergen Aqua AS, Bredalsmarken 15–17, Møhlenpris, Box 2604, 5836 Bergen, Norway Stoss, Joachim Stolt Seafarm AS, Øyeslatta 63, N-4484 Øyestranda, Norway Svåsand, Terje Institute of Marine Research, Department of Aquaculture, PO Box 1870 Nordnes, N-5017 Bergen, Norway Taranger, Geir L. Institute of Marine Research, Department of Aquaculture, PO Box 1870 Nordnes, N-5017 Bergen, Norway Vadstein, Olav Trondhjem Biological Station, Department of Biology, Norwegian University of Science and Technology, N-7491 Trondhjem, Norway

Chapter 1

Introduction The Editors

The annual global production from aquaculture exceeded 30 million metric tons in 1998, representing more then 20% of the total annual yield from ﬁsheries and mariculture. Asia is by far the largest producer of marine products, with respect to both ﬁsheries and aquaculture, and China has the dominant position by contributing more than 32% of the total yield. Aquaculture production in Europe is approximately 5% of that in Asia, and is dominated by anadromous salmonids (Atlantic salmon, Salmo salar, and rainbow trout, Oncorhynchus mykiss) and the marine species sea bass (Dicentrarchus labrax), sea bream (Sparus aurata) and turbot (Scophthalmus maximus). While these three marine species are mainly farmed in southern Europe, the main production of salmonids is in northern European countries, particularly Norway and Scotland. At the turn of this century, the total world production of salmonids reached more than 1 million metric tons, of which Norway produced 50%. The doubling of production over the last 10 years is evidence of the success of this form of farming, and is indicative of the potential that may exist for farming other species in the cold-water environment (FAO-statistics, www.fao.org). Environmental conditions are the natural limiting factors for aquaculture activity. In northern Europe the temperature can vary between 0 and 20°C during the season, whereas the salinity can vary between 10 and 34‰ in coastal waters. In this environment there is potential for farming several marine ﬁsh species, such as cod (Gadus morhua), haddock (Melanogrammus aegleﬁnus), hake (Merluccius merluccius), wolf-ﬁsh (Anarhichas spp.), halibut (Hippoglossus hippoglossus), turbot and sole (Solea solea). The enhancement of natural stocks and sea ranching are important current and future issues in northern Europe as well as in other regions of the world. In fact, these activities were the driving force behind the ﬁrst initiatives in the cultivation of cold-water marine ﬁsh, which started in 1882. A former ship’s ofﬁcer, Captain G.M. Dannevig, took the initiative and established a cod hatchery in southern Norway in the 1880s, with the main goal of improving and stabilising the local cod ﬁshery (‘Flødevigen Utklekningsanstalt’, now the Institute of Marine Research, Department of Coastal Zones, Flødevigen Marine Research Station). At the same time, the American S.P. Baird convinced the American Congress to build a hatchery for cod in Woods Hole, and the two hatcheries were in operation at approximately the same time. Dannevig collected brood stock of cod in the winter, transferred the ﬁsh to a larger spawning basin, and collected the newly fertilised eggs for further incubation in the laboratory. After hatching, the

2

Culture of cold-water marine ﬁsh

yolk-sac larvae were transported from the hatching boxes in the laboratory to different locations along the coast of southern Norway and released into the sea. A similar activity took place off the north-eastern coast of the USA, and during the period between 1920 and 1950 a large number of newly hatched cod larvae were released in the coastal waters of the two countries. In Norway, the activity started in 1883 and did not end until 1971 (Solemdal et al., 1984). In 1886, as part of the veriﬁcation that the cod larvae hatched in the Flødevigen hatchery were viable and able to grow and survive in nature through to the juvenile stage, yolksac larvae of cod were stocked in a 2500-m3 outdoor concrete enclosure. The experiment was a success, and generated the ﬁrst artiﬁcially produced juvenile cod. It was another 50 years before the next major achievement. Gunnar Rollefsen, who was in charge of the hatchery at the Trondhjem Biological Station, succeeded in feeding plaice (Platessa platessa) larvae, a local candidate for stock enhancement, on newly hatched Artemia. This was a major breakthrough in the development of intensive production methods for juvenile marine ﬁsh using readily produced live food. Rollefsen then became the ﬁrst director of the Institute of Marine Research in Bergen and was not able to continue this work. No further major progress was made in the ﬁeld during the next couple of decades, but an important step forward was the discovery by Japanese scientists (Ito, 1960, see review by Nagata & Hirata, 1986) that a ubiquitous brackish water rotifer, Brachionus plicatilis, could also be an effective live food for marine ﬁsh larvae. In Europe, British scientists pioneered the development of mass-rearing techniques for marine ﬂatﬁsh initially using Artemia nauplii to feed plaice and sole (Shelbourne, 1964), and subsequently demonstrating the utility of the smaller rotifer, B. plicatilis, for the smaller larvae of lemon sole (Microstomus kitt) (Howell, 1973) and turbot (Jones et al., 1974). Success with turbot was not achieved before the early 1970s. The use of Brachionus sp. was a key factor contributing to this success, but the crucial discovery was that signiﬁcant survival of turbot larvae could only be obtained if certain species of microalgae were added to the tanks along with rotifers and Artemia (Howell, 1979). This was also found to be important for other species, and led to an appreciation of the importance of dietary sources of long-chain polyunsaturated fatty acids for these species (Scott & Middleton, 1979). This was paralled by work in Japan that demonstrated that n-3 fatty acids were essential for marine ﬁsh (Fukusho, 1977; Kitajima & Koda, 1976), and that marine larvae in particular had high dietary requirements (Watanabe et al., 1978). These ﬁndings resulted in cultivation techniques that ensured a high n-3 fatty acid level in the live feed. Considerable efforts have been made worldwide during the last decade to improve and adapt the Japanese methods to species with commercial potential. This has resulted in major activity in mariculture worldwide for marine cold-water ﬁsh species. The challenges of developing suitable technology to rear marine ﬁsh are multidisciplinary, and the crucial factors involved in the process, from brood stock maintenance to the market place, are illustrated in Fig. 1.1. The production chain involves several steps, and the general challenges during production involve knowledge about: (1) the general biology of the ﬁsh species; (2) the chemical and physical environmental requirements;

Introduction

3

Figure 1.1 Schematic diagram of the production process and the knowledge needed for economically feasible mariculture production.

(3) the nutritional requirements; (4) the effects of the microbial environment. Comprehensive knowledge of all these fundamental issues, and on their interactions in ﬁsh culture, is needed to develop a feasible mariculture industry of cold-water ﬁsh species. The production of viable juveniles is still the main constraint on the development of new cold-water species for aquaculture. Early experience suggests that the on-growing stages are more straightforward for most species considered, and that it will be possible to take advantage of the infrastructure and services that are already established for Atlantic salmon. This has been possible to a limited extent for juvenile production. One reason for the difﬁculty in rearing marine ﬁsh larvae is illustrated in Fig. 1.2, which shows that the larvae are very tiny and immature at the time of hatching compared with those of salmon. The larvae are 3–22 mm in length when they start feeding. Larvae of wolf-ﬁsh, which is the only species able to feed on dry pellets from the very beginning, are the largest. Other species have to be fed live prey during the larval stage. However, this is also the case for other marine species such as sea bass and sea bream that are successfully farmed in southern Europe. An additional challenge with the cold-water species has been their high requirements of n-3 fatty acids, and the complicated life cycle of some targeted species such as the Atlantic halibut. It is clear, however, that major improvements have been made over the last few decades, and that further progress is made each year. Another issue that has delayed the development of the culture of marine species is the success of the Atlantic salmon industry in the northern hemisphere. Salmon aquaculture has

4

Culture of cold-water marine ﬁsh

Hake, Turbot, Sole Cod, Haddock Halibut

Wolf-fish

Salmon

5

10

15 20 Length (MM)

25

30

Figure 1.2 Relative size of the larvae of cold-water ﬁsh species that are considered as candidates for mariculture. Table 1.1 Year 1886 1976 1980 1983 1985 1986 1987 1988 1989 1990 1991 1992 1993 1994 1995 1996 1997 1998 1999 2000 2001

Overview of number of juveniles produced until 2001 of ﬁve selected cold-water marine ﬁsh species. Cod

Haddock

Wolf-ﬁsh

Halibut

Turbot

2 500 4 400 2 60 000 130 000 50 000 266 000 422 000 513 000 330 000 390 000 86 000 175 000 320 000 230 000 53 000 156 000 113 000 100 000 500 000 1 000 000

400 300 5 000 5 000 5 000 2 000 18 000 79 000 95 000 165 000

25 000 31 000 50 000

2 100 1 000 2 000 4 000 8 000 35 000 130 000 316 000 794 000 175 000 255 000 520 000 1 520 000 1 620 000 1 740 000 1 300 000

40 000 150 000 352 000 430 000 615 000 350 000 510 000 380 000 460 000 300 000

1 705 000 ~6 000 000 ~7 000 000

been very successful and is economically feasible, and has attracted private investment to the degree that this has inhibited developments with marine species. The key to successful cultivation of any ﬁsh species is to produce high and stable numbers of high-quality juveniles annually. Table 1.1 shows the number of juveniles produced annu-

Introduction

5

ally for the seven marine ﬁsh species which are being considered for future aquaculture. Turbot have been cultured for some years, and juvenile production is high and stable from one year to another, while haddock and hake still are at an early stage. The production of juvenile sole is not a problem, but the rather slow growth rate so far achieved during the on-growing stages has inhibited commercial developments. Both turbot and sole have been included in this book because they are important as model ﬁsh for the other species. Cod has been cultivated for more then 100 years, but the juvenile production is still too low and too unreliable. The ﬁsh is well known in the market, and the yield from ﬁsheries has declined signiﬁcantly over the years. These facts, combined with increasing prices in the market, mean that its potential in aquaculture is currently considered to be high. The key for future production is intensive production. Similarly, juvenile production of halibut has been too unstable to support a signiﬁcant industry. However, with a better understanding and control of intensive juvenile production, farmed halibut will soon be on the market in increasing numbers. Experiments with wolf-ﬁsh have taken place during the past 15 years, and of the two species considered, the spotted wolf-ﬁsh (A. minor) has been favoured because of its much higher body growth rate compared with that of the common wolf-ﬁsh (A. lupus). The production of hake juveniles has been very limited, and a lot of work still remains to be done before any form of commercialisation can be realised. This textbook describes the current state of knowledge on the cultivation of cold-water marine ﬁshes, and considers problems and solutions in the rearing of all life stages.

1.1 References Fukusho, K. (1977) Nutritional effects of the rotifer, Brachionus plicatilis, raised by baking yeast on larval ﬁsh of Oplegnathus fasciatus, by enrichment with Chlorella sp. before feeding. Bull. Nagasaki Pref. Inst. Fish, 3, 152–4 (in Japanese). Howell, B.R. (1973) Marine ﬁsh culture in Britain. VIII. A marine rotifer, Brachionus plicatilis Muller, and the larvae of the mussel, Mytilus edulis L., as foods for larval ﬂatﬁsh. J. Cons. Int. Explor. Mer, 35, 1–6. Howell, B.R. (1979) Experiments on the rearing of larval turbot, Scophthalmus maximus L. Aquaculture, 18, 215–25. Ito, T. (1960) On the culture of mixohaline rotifer Brachionus plicatilis O.F. Muller. Rep. Fac. Fish. Mie Pref. Univ., 3, 708–40 (in Japanese). Jones, A., Alderson, R. & Howell, B.R. (1974) Progress towards the development of a successful rearing technique for larvae of the turbot, Scophthalmus maximus L. In: The Early Life History of Fish (ed J.H.S. Blaxter), pp. 731–7. Springer, Berlin, Heidelberg, New York. Kitajima, C. & Koda, T. (1976) Lethal effects of the rotifer cultured with baking yeast on the larval sea bream, Pagrus major, and the increase rate using the rotifer recultured with Chlorella sp. Bull. Nagasaki Pref. Inst. Fish, 2, 113–16 (in Japanese). Nagata, W.D. & Hirata, H. (1986) Mariculture in Japan: past, present, and future prospectives. Mini Rev. Data File Fish. Res., 4, 1–38. Scott, A.P. & Middleton, C. (1979) Unicellular algae as a food for turbot (Scophthalmus maximus) larvae—the importance of dietary long-chain polyunsaturated fatty acids. Aquaculture, 18, 227–40. Shelbourne, J.E. (1964) The artiﬁcial propagation of marine ﬁsh. Adv. Mar. Biol., 2, 1–83.

6

Culture of cold-water marine ﬁsh

Solemdal, P., Dahl, E., Danielssen, D.S. & Moksness, E. (1984) The cod hatchery in Flødevigen— background and realities. In: The Propagation of Cod Gadus morhua L. (eds E. Dahl, D.S. Danielssen, E. Moksness & P. Solemdal), pp. 17–45. Flødevigen Rapportserie, 1. Watanabe, T., Kitajima, C., Arakawa, T., Fukusho, K. & Fujita, S. (1978) Nutritional quality of rotifer, Brachionus plicatilis, as a living feed from the viewpoint of essential fatty acids for ﬁsh. Bull. Jpn. Soc. Sci. Fish, 44, 1109–14 (in Japanese).

Chapter 2

Abiotic Factors B.R. Howell and S.M. Baynes

2.1 Introduction The abiotic environment is of critical importance in determining the performance of cultured ﬁsh. Marine waters are mainly characterised by their temperature and salinity, and these factors will largely determine the range of species that can be grown. Light also has a considerable impact on performance, but is rather more readily manipulated than either temperature or salinity. Fish growth and survival is also affected by a range of other water-quality factors, many of which are inﬂuenced directly or indirectly by the metabolic activity of the ﬁsh themselves. In intensive culture systems, for example, growth may be particularly impaired by sub-optimal oxygen and ammonia levels. Less direct adverse effects may arise from exposure to hydrogen sulphide or algae blooms, both of which may be a consequence of high environmental organic loadings arising from the activities of the ﬁsh farm. This chapter reviews the impacts of the major environmental factors affecting the performance of ﬁsh, and concludes with a review of the factors to be considered in site selection.

2.2 Oxygen and Oxygen Consumption Oxygen content is probably the most important aspect of water quality because of its central role in driving metabolic processes and the profound effects of deprivation on both the performance and the welfare of the ﬁsh. Sub-optimal dissolved oxygen (DO) levels can increase stress and disease susceptibility, reduce food intake, growth and food conversion efﬁciencies, and, of course, under extreme conditions cause mortalities. Regular monitoring of oxygen levels in culture systems is a clear imperative, but changes in behaviour may also provide an indication of the development of sub-optimal conditions. These may include reduced feeding activity, swimming near the surface and increased ventilation rates. In extensive systems such as ponds, photosynthetic activity plays an important role in determining DO levels and consequently considerable diel variations can occur which can threaten ﬁsh survival. In productive ﬁsh ponds in which dense concentrations of algae have developed, diel variations in DO can be as great as 7–8 mg l-1, with super-saturation occurring during the day and sub-saturation during the night (Boyd, 1979). In northern latitudes, however, intensive culture methods predominate, and in these systems the supply of oxygen

8

Culture of cold-water marine ﬁsh

OXYGEN CONCENTRATION (mg/l)

12 o

10 C o

20 C

10

8

6 0

10

20

30

40

SALINITY (g/l) Figure 2.1 The relationship between oxygen solubility and salinity at 10 and 20°C.

is dependent on water ﬂow to a much greater extent than that arising from photosynthetic activity. In intensive systems, stocking levels will be determined by the ability to maintain adequate oxygen levels. Maximum stocking densities in cages will be limited by water exchange rates, and with careful management diel variations rarely exceed 2–3 mg l-1, although these may still prove stressful (Beveridge, 1987). However, extreme reductions in DO may occur as a result of dense algae blooms, and these can result in ﬁsh kills. Particularly high stocking densities can be achieved in tanks with high and more stable DO levels achieved through the use of a diverse range of aeration devices or direct injection of oxygen. However, damaging ﬂuctuations may occur unless adequate control and fail-safe systems are in place. The solubility of oxygen in water depends on a variety of factors, but is largely determined by temperature, salinity and altitude, since solubility decreases as each of these factors increases. In a marine environment, altitude has negligible impact. The effect of temperature and salinity (Fig. 2.1) shows that saturation oxygen concentrations are higher in freshwater than in seawater at 35 p.p.t., and that there is a reduction in saturation oxygen concentration in each medium with increasing temperature. Pressure also has a signiﬁcant effect on DO levels. The saturation concentration of freshwater at sea level is 11.0 mg l-1 compared with 8.9 mg l-1 at an altitude of 2000 m (Shepherd & Bromage, 1988). Water acquires oxygen from the air by a process of diffusion at a rate which is dependent on water temperature, salinity, the degree of saturation of the water and the level of turbulence at the air–water interface. The last two factors inﬂuence the concentration gradient, which is the main driving force for oxygen transfer (Wheaton, 1977). The transfer of oxygen within a water mass is almost entirely dependent on water movement, since the diffusion of oxygen within water is extremely slow. For example, it has been calculated that to raise the oxygen content at a depth of 10 m from zero to 0.4 mg l-1 by diffusion alone would take about 600 years (Wheaton, 1977)! This emphasises the need to ensure

Abiotic factors

9

good mixing within culture systems to avoid the development of areas of low DO as a result of both the respiratory activity of the ﬁsh and the microbial degradation of organic material such as food, faeces and dead ﬁsh. The biological oxygen demand of organic material can be highly signiﬁcant in intensive culture systems, and measures that ensure its regular removal are an important imperative. Tolerance to DO levels varies considerably both within and between species. In general, levels of 5 mg l-1 are considered to be acceptable to aquatic organisms, although many species, such as some tilapias, can survive and grow well at DO levels below 2 mg l-1. Most ﬁsh species can tolerate 1–2 mg l-1 for short periods, but mortalities would occur if this continued for more than a few hours. For salmonids, minimum DO levels are considered to be 5.0–5.5 mg l-1 for ﬁsh and 7 mg l-1 for eggs (Shepherd & Bromage, 1988). For those species that have a relatively high oxygen requirement, conditions under which oxygen solubility is reduced, particularly high temperature and salinity, can place the ﬁsh at risk. High temperatures not only reduce oxygen solubility, but also increase oxygen demand from enhanced levels of feeding and swimming activity. Taking such factors into account, it has been calculated that salmon, Salmo salar, should never be fed at temperatures above 20°C (Shepherd & Bromage, 1988). The proportionately higher oxygen consumption of small ﬁsh relative to large ﬁsh is also an important factor that should be taken into account in this respect. Models have been developed that take these factors into account in estimating oxygen consumption, and they provide a valuable management tool that not only minimises the risk of ﬁsh experiencing extreme situations, but allow near optimal conditions to be maintained in order to maximise efﬁciency and hence proﬁtability. Actual DO levels in culture systems depend on the balance of the rate of consumption and the rate of supply. Rates of oxygen consumption vary not only with ﬁsh size, but also between species, and are strongly inﬂuenced by environmental conditions, particularly temperature. Rates of oxygen consumption shortly after fertilisation may range from 3.7 ng h-1 (cod, Gadus morhua) to 70 ng h-1 (rainbow trout, Oncorhynchus mykiss), but on average will increase 20-fold during the period from fertilisation to hatch (Rombough, 1988). Thus, early embryos may be relatively unaffected by low oxygen concentration, whereas in older embryos the retarding effect increases progressively (Kamler, 1992). At optimum temperatures, oxygen consumption is relatively high because of increased growth rates and levels of activity. At temperatures above the optimum, ﬁsh are stressed and their warning and defence systems are mobilised, thereby further signiﬁcantly increasing oxygen consumption (Wheaton, 1977). Thus, at temperatures above the optimum, further temperature increases cause stress that increases oxygen demand more rapidly than the initial increases in temperature. Similarly, and regardless of temperature, external sources of stress signiﬁcantly increase oxygen demand. This is why, for example, ﬁsh are easily asphyxiated during harvesting operations. The relationship between oxygen consumption and temperature, ﬁsh size and feeding rate is illustrated by recent experiments on the common wolf-ﬁsh (Anarhichas lupus) carried out by Steinarsson & Moksness (1996). At 7°C, the oxygen consumption of juveniles (0.5 kg) ranged from 37 to 62 mg O2 kg-1 h-1, whereas that of adults (6.9 kg) ranged from 29 to 44 mg O2 kg-1 h-1. This illustrates the decrease in weight-speciﬁc oxygen consumption with increasing ﬁsh size. These authors also demonstrated that oxygen consumption was not uniform

Culture of cold-water marine ﬁsh

OXYGEN CONSUMPTION (mg/kg/h)

10

70

60

FEEDING FEEDING

50

40

30 9 12 15 18 21

0

3

6

9 12 15 18 21

0

3

6

TIME OF DAY

Figure 2.2 Diel rhythm of oxygen consumption of wolf-ﬁsh fed daily to satiation. Redrawn from Steinarsson & Moksness (1996).

throughout a 24-h period, showing a distinct diel rhythm (Fig. 2.2). Oxygen increased steadily after feeding at 0900 h, and decreased markedly during the night. This diel rhythm in oxygen consumption was also evident on non-feeding days, indicating an association with photoperiod as well as with feeding times.

2.3 Ammonia In ﬁsh, the majority of nitrogenous excretion occurs at the gill surface, with ammonia and ammonium ions being the main excretory products. Because of its high solubility and small molecular size, ammonia diffuses extremely rapidly. It can be lost through any surface which is in contact with water, and need not be excreted by the kidney. Ammonia is the most toxic form of inorganic nitrogen. Other products of nitrogen metabolism (e.g. urea and creatine) are produced in smaller quantities and may be excreted in the urine, through the skin or via the gills. Some of the ammonia is produced in the liver and is transported to the gills by the blood, but some may also be produced in the gills themselves by deamination of plasma amino acids. Spotte (1979) provides a comprehensive account of the mechanism of ammonia excretion and the toxic effects that may be induced. The toxicity of ammonia is largely controlled by pH through its effect on the hydrolysis of ammonium ions (NH4+), unionised ammonia (NH3) being the most toxic form. The proportion of unionised ammonia (PUIA) is described by Equation 2.1, which represents the concentrations of ammonia and ammonium ions.

(

[

PUIA = [NH 3 ] [NH 3 ] + NH 4

+

])

(2.1)

This is inﬂuenced most importantly by pH, temperature and salinity, which affect the equilibrium and the constant KaS that is determined from the concentrations.

Abiotic factors

[

S

K a = [NH 3 ][H + ] NH 4

+

]

11

(2.2)

The pH has the greatest effect on the PUIA, which can be calculated from the pH and this constant at a given temperature and salinity:

[

(

S

PUIA = 1 + antilog pK a - pH

)]

-1

(2.3)

where pKaS = -log KaS An increase of one pH unit (e.g. from pH 7 to pH 8) causes the proportion of unionised ammonia to increase about ten-fold. An increase in temperature from 10 to 20°C produces approximately a doubling in the proportion of the unionised form. However, as the salinity increases there is a slight fall in the proportion of the unionised form, the change being about 10% for a difference in salinity of 15 practical salinity units (p.s.u.). The buffering capacity of seawater provides a relatively stable pH, usually above pH 7, whereas freshwater tends to have a less stable pH, often below 7. Therefore saltwater systems will have a greater proportion of dissolved ammonia in the toxic unionised form than equivalent freshwater systems. As a result, if ﬁsh are held with a limited exchange of water, the risk of ammonia toxicity may be greater in saltwater than in freshwater. This would be the case, for example, when ﬁsh are reared in recirculation systems or when they are transported in closed systems. The lack of buffering of freshwater is to some extent an advantage in these circumstances. Any increase in dissolved CO2 from respiration will reduce the pH of freshwater and lead to an increase in the proportion of ionised ammonia. This would tend to counteract the toxic effects of a rise in total ammonia as waste products accumulate in the system. On the other hand, the natural buffering capacity of seawater minimises any pH change due to CO2 levels, and provides less compensation for any increase in toxic ammonia. In general, however, the instability of the pH of freshwater normally poses a greater risk since surface waters can rapidly become alkaline if high rates of photosynthesis reduce the carbonate levels. In addition, the toxic effects of ammonia may be increased under conditions of low oxygen levels. This may be caused by elevated levels of ammonia interfering with the ability of haemoglobin to retain oxygen. Tolerance to ammonia in water is 30% less at a dissolved oxygen level of 5 mg O2 l-1 than it is at 8.5 mg O2 l-1. There is some uncertainty as to the way in which environmental levels of ammonia exert their effects. It seems unlikely that ammonia enters the animals across the gills, and more probable that the effects are indirect. As the concentration gradient between environmental and tissue levels of ammonia decreases, the rate of ammonia loss is reduced, causing elevated levels in the tissues. Whether toxicity is due to diffusion into the gills or retention of metabolic ammonia, the effects are the same. Broadly, these effects are histopathological changes in the gills and other organs, decreased resistance to disease and impairment of growth. The effects on the gills may include necrosis, thickening of the epithelium, increased mucus production and epithelial rupturing and haemorrhage. Any fusing of lamellae reduces the surface area of the gills with a consequent impairment of gaseous (O2 and CO2) exchange. Ammonia has also been implicated in non-speciﬁc conditions such as ﬁn and tail rot, anaemia and bacterial gill disease.

12

Culture of cold-water marine ﬁsh

The estimation of the lethal limits of ammonia is hampered by a number of factors and is known to vary with species, other water-quality parameters, experimental methods, and the age, acclimation history and the condition of the test animals. For example, it has been shown that rainbow trout and coho salmon (Oncorhynchus kisutch) acclimated to sub-lethal levels of ammonia become resistant to otherwise lethal concentrations. Lethal limits for teleosts appear to range from about 0.07 mg NH3-N l-1 for rainbow trout fry to 1.4 mg NH3-N l-1 for juvenile striped bass (Morone saxatilis). Much of the early work on ammonia toxicity was concerned with rainbow trout and other freshwater species. The increased level of farming marine ﬁsh in the last decade or so has stimulated a number of studies of ammonia toxicity in commercially important species such as seabass (Dicentrarchus labrax), seabream (Sparus aurata) and turbot (Scophthalmus maximus). These illustrate inter-species differences in ammonia toxicity. Person-Le-Ruyet et al. (1995) used a continuous-ﬂow method to determine the LC50s of unionised ammonia under optimal conditions of temperature, salinity, pH and dissolved oxygen concentrations for juveniles (6–163 g) of all three species. Median LC50s ranged from 1.7 mg NH3-N l-1 for seabass to 2.5–2.6 mg NH3-N l-1 for seabream and turbot. These values did not change signiﬁcantly from 24 to 96 h and were not related to ﬁsh size. These authors also found that blood plasma levels of ammonia were positively correlated with external concentrations, and that a 50% mortality occurred after a 4-day exposure when the increase in total ammonia nitrogen was four times the initial level in seabass, but ten times the initial level in seabream and turbot. This showed that seabass have a lower threshold of physiological disturbance than both seabream and turbot, and explains their greater sensitivity to ammonia. Similar studies with the larval stages of seabass and Senegal sole (Solea senegalensis) demonstrated inter-species differences, 24-h LC50s being 0.28 mg NH3-N l-1 for seabream and 1.32 mg NH3-N l-1 for Senegal sole. These data also support the view that the larval stages are more sensitive to ammonia than the juvenile stages. Twenty-four-hour LC50 values are by no means safe limits. If ﬁsh are exposed to ﬂuctuating levels of ammonia that span the 24-h LC50 levels, the resulting toxicity may be higher than expected from continuous exposure to such concentrations and more difﬁcult to predict. While the concentration of total ammonia in marine ﬁsh hatcheries is generally below 0.7 mg TAN l-1, in some cases it may be as high as 2 mg TAN l-1.

2.4 Temperature A change in temperature affects the rate of biological processes and consequently will affect an animal’s metabolic rate and activity. Between lower and upper thermal limits, the rate generally increases to a maximum as the temperature increases to the optimum, but above the optimum deleterious effects become more signiﬁcant and the rate falls. Animals are adapted to the niche they occupy in their environment. A species’ distribution and the seasonal changes are reﬂected in the characteristics of the life cycle and in optima for various processes such as growth, activity and aspects of reproduction. For particular species, optimal temperatures very commonly differ with the stage of development (viz. egg, larva, juvenile and adult) and even in individuals, related processes such as

Abiotic factors

13

appetite, digestion and growth may have different optima. This section will highlight particular examples from this variety of effects, with emphasis on their importance for cultivation.

2.4.1 Seasonal Temperature Cycle and Spawning The annual cycle of temperature change in north temperate surface waters lags behind the annual cycle of daylength by a month or two, with the minimum in February and March and the maximum in August and September. The range between the winter minimum and the summer maximum varies with latitude and the depth of the mixed water column. This annual change in ambient temperature is important with regard to the selection of species for particular sites, although water temperature can be controlled at a cost in pump-ashore sites (see Section 2.8). However, for broodstock ﬁsh, some exposure to low winter temperatures and increasing temperatures in spring is considered to be important. A period of low temperature when vitellogenesis is taking place is thought to improve egg quality in several species. Egg diameter is greater in cod held at low temperatures, and fecundity is considered to be better in common sole (Solea solea) that have experienced a temperature of less than 10°C. Female wolf-ﬁsh must be kept at a temperature below 10°C for at least 4 months before ovulation for normal egg maturation. Rising temperatures may help to initiate spawning, although reaching a threshold temperature does not necessarily trigger spawning in wild stocks. The manipulation of temperature together with photoperiod enables spawning period to be extended and good egg quality to be maintained, for example in cod and haddock (Melanogrammus aegleﬁnus). However, temperature affects the timing of spawning by inﬂuencing gonad development and growth rather than by providing a speciﬁc cue to set the time.

2.4.2 Egg and Larval Development Water temperature affects the efﬁciency with which yolk is converted into embryo tissues. Within the range of thermal tolerance of a species, eggs tend to demonstrate greater efﬁciency at lower temperatures than they do at higher temperatures (Kamler, 1992). Optimal temperatures for embryonic development are not necessarily the same for larval growth. If eggs or larvae are reared at the extremes of their temperature range, they very often have developmental abnormalities such as the poor articulation of the jaw of Atlantic halibut (Hippoglossus hippoglossus) when reared at 9°C instead of 6°C. Even within the range at which growth of the yolk-sac larva appears normal, a change in incubation temperature of a few degrees Celsius may cause signiﬁcant changes in the relative timing of organogenesis. Such changes may have consequences for the ﬁtness of the larvae, and care must be taken in evaluating an optimum. A temperature difference of a few degrees at egg incubation and the early larval stage (e.g. 5–8°C in Atlantic halibut) has a profound inﬂuence on the number and size of white muscle ﬁbres, which ultimately determines the muscle cross-sectional area. This can have long-term effects on the muscle growth in juvenile stages, and makes avoiding unplanned temperature changes during early rearing very important.

14

Culture of cold-water marine ﬁsh

2.4.3 Sex Ratio For several species, there is evidence that the temperature during early rearing can inﬂuence the phenotypic or functional sex of ﬁsh. It is unclear how widespread this effect is in gonochoristic ‘coldwater’ marine ﬁsh, but one species of interest, the hirame or Japanese ﬂounder (Paralichthys olivaceus), does demonstrate what appears to be thermolabile sex determination. It has been shown that a normal 50 : 50 male : female sex ratio occurs if juveniles are reared through the ﬁrst 120 days after hatching at 18°C. Higher water temperatures (20, 23 or 25°C) during the same period lead to approximately 75% males in the brood, although it is also reported that slower-growing ﬁsh became predominantly male and so the effect may not be entirely due to temperature.

2.4.4 Growth and Metabolism The many studies on the growth of ﬁsh show a marked inﬂuence of temperature. The overall effect on growth rate depends on the interaction of the effect of temperature on the appetite and digestion, and the difference in the effects on standard and active metabolic rates. Brett (1979), and Brett & Groves (1979) provide extensive reviews of how temperature, amongst other factors, affects metabolic rate and growth, and should be consulted for detailed accounts. The optimum temperature for growth rate very often changes as ﬁsh grow, but this relationship varies with the stage of development. In cod, for example, the optimum has been shown to increase from 9.7 to 13.4°C as larvae grow from 73 to 251 mg. The optimum for small (50–1000 g), immature ﬁsh is higher (11–15°C), but for large (1.5–2.5 kg), sexually mature cod the optimal temperature range is 9–12°C, which is slightly less than for larvae. This pattern of change with ontogeny is also common in other species. The most signiﬁcant relationships involved in on-growing ﬁsh are those between temperature and the rate of food uptake and between temperature and growth rate. The change in the efﬁciency of feed conversion (i.e. growth per unit ration) with temperature depends on how these two relationships interact. Fish fed a maintenance ration do not increase in size: the energetic equivalence of the diet is fully utilised in the swimming involved in feeding, digesting the food, excretion, osmoregulation, renewing tissue and so on, thus maintaining the status quo. Fish fed to satiation (when appetite no longer drives a feeding response) have an excess in energetic terms over what is needed for maintenance, and that provides scope for growth. Temperature does not affect the level of the ration required for maintenance in the same way as it affects appetite and the ration required for satiation. As temperature changes, so does the scope for growth. Figure 2.3 shows generalised curves that indicate the energetic value of the maximum and maintenance rations and the difference is that available for growth: this increases with temperature up to a maximum before decreasing rapidly. The change in the scope for growth is comparable to the pattern of the change in growth rate with temperature, and the peak occurs close to the optimum temperature for growth. If a situation is considered where the higher ration is less than satiation but greater than maintenance, the scope for growth is reduced and the peak occurs at a lower temperature. The optimum temperature for growth decreases as the ration is reduced.

Abiotic factors

15

Satiation ration

5

Maintenance ration Scope for growth

ENERGY (k cal)

4

3

2

1

0 0

5

10

15

20

25

30

TEMPERATURE (°C) Figure 2.3 Generalised curves that indicate the relation between temperature and ration plotted as the energetic value of the quantity consumed at satiation and maintenance levels. The difference between them is the energetic equivalence that is available for growth and is plotted as ‘scope for growth’. The temperature at which the maximum value in scope for growth is reached approximates to the optimum temperature for growth.

The efﬁciency with which food is converted into growth very often increases as the temperature is reduced, and it is not uncommon to have an optimum temperature for feed conversion efﬁciency which is somewhat lower than the optimum for growth. Much of the published work in this area is for salmonids, but the relatively small amount of information for cold-water marine ﬁsh supports these generalisations.

2.5 Salinity The salinity of seawater varies little offshore, and in general marine ﬁsh are adapted to live in this stable environment. There are a limited number of species that naturally occupy the more variable conditions found in estuaries, and their physiology is better suited to tolerate the daily changes associated with each tidal cycle. Salinity is measured on the practical salinity scale, which relates the conductivity of a sample to that of a standard potassium chloride solution. If seawater at 15°C has a conductivity equal to that of the standard, it is said to have a salinity of 35 p.s.u. Values given in p.s.u. are approximately equivalent to those recorded in parts per thousand (S‰), the units more commonly used in earlier literature. A salinity of 35 p.s.u. is generally accepted as the norm for offshore seawater, while in coastal waters, although the salinity is still quite stable, run-off from the land may lead to the norm being 32 or 33 p.s.u., for example. In estuaries, however, depending on the tidal

16

Culture of cold-water marine ﬁsh

range, ﬂuctuations measured in tens of units may occur twice a day. The difference in density of freshwater and seawater means that unless there is adequate mixing, the freshwater tends to ﬂoat over the salt water forming an oblique halocline, i.e. a discontinuity between the different salinity waters, that moves up and down the estuary with the tidal ﬂow. The location of a water intake or the position of cages in an estuary in relation to these salinity changes are important considerations when selecting the site of a marine ﬁsh farm or the choice of species for estuarine waters. The salts in the body ﬂuids of ﬁsh are maintained by the osmoregulatory system at a concentration of about one-third of that of full-strength seawater. This differential means that ﬁsh in seawater passively lose water by osmosis through the gills and body surfaces, whilst salts enter the body. To maintain homeostasis, the ﬁsh has to drink seawater continually and excrete the excess ions. Specialised chloride cells in the gills remove ions such as sodium and chloride, while the kidneys produce small volumes of very concentrated urine. Fish that live in estuaries in brackish water at less than 10–11 p.s.u. are subject to the opposite ﬂuxes: salts leave the body and water enters by osmosis. Fish that tolerate both environments are able to do so because chloride cells can take up monovalent ions to replace those lost, and the kidneys produce copious amounts of dilute urine to counteract the osmotic ﬂux. Unless the surrounding water is isotonic with the body ﬂuids, energy is constantly used to maintain the body ﬂuids’ composition. Embryos and larval ﬁsh are less well adapted to control the ﬂux of water and ions in and out of the body, and therefore do not tolerate changes in salinity as well as adult ﬁsh. The conditions required for successful reproduction are thus more closely deﬁned. Gametes within the adult’s body are in osmotic balance with body ﬂuid blood plasma, but at the time of spawning those of oviparous species are exposed to the osmotic stresses of their surroundings. The ways that species may be affected by, or are able to tolerate, the osmotic changes that occur during embryogenesis and later larval development have been reviewed by Alderdice (1988). The energy required for osmoregulation affects yolk utilisation efﬁciency and the growth rate of larvae. In some euryhaline species this can lead to larger larvae hatching at intermediate salinities compared with those reared in either fully fresh or saline water (e.g. newly hatched striped bass are longer at 5 p.s.u. than at either 1 or 10 p.s.u.). Survival is affected by salinity, and the salinity to which larvae are expected to be adapted is not necessarily optimal. For example, newly hatched Atlantic halibut survive best at 29–34 p.s.u., which is slightly lower than full-strength seawater, although there is no beneﬁt of reduced salinity after 30 days. The salinity of the water also affects the buoyancy of the eggs and larvae, and this may have consequences for survival that are not directly associated with osmoregulation. In species that need to gulp air to ﬁll the swim bladder, low salinities may mean that the larvae are not sufﬁciently buoyant to reach the surface and survival is reduced. In a ﬁsh-farming context, salinity can be manipulated to some advantage. The large eggs of Atlantic halibut, for example, are fragile and can be protected during embryo development by incubating them at their salinity of neutral buoyancy. Salinity and temperature do not have independent effects on development and ideally should be considered together. The effect of temperature inﬂuences the rate of reactions, and

Abiotic factors

17

the salinity inﬂuences the energy required to regulate the body composition. Where the interactions of these inﬂuences have been analysed rigorously, the optima for growth do not necessarily coincide with those for the most efﬁcient food conversion, and compromises in setting conditions must be made.

2.6 Hydrogen Sulphide Hydrogen sulphide is a noxious gas and is a product of anaerobic decomposition of organic material. Anoxic conditions develop when the rate of deposition is high and the supply of oxygen is insufﬁcient to meet the metabolic demands of the plant and animal communities inhabiting the sediments. Under such anoxic conditions, benthic communities dominated by low-oxygen-tolerant and anaerobic species develop, and potentially toxic chemicals, such as hydrogen sulphide, are produced. Almost all the hydrogen sulphide in the environment is produced by a specialised group of micro-organisms. The most widely distributed sulphate reducer is Desulfoxibrio desulfurcans, which is found in freshwater environments. A closely related species, D. estuarli, is its marine equivalent. The main requirements for bacterial sulphate reduction to hydrogen sulphide are the absence of oxygen, the presence of sulphate, the presence of oxidizable organic substrates to supply hydrogen atoms, and the presence of organic nutrients to support the growth of the bacteria, including vitamins, amino acids and nucleotides. Only a narrow range of organic molecules can be oxidised by the sulphatereducing bacteria. These include acetic and lactic acid, although some strains can oxidise hydrogen. The range of nutrient molecules used by sulphate-reducing bacteria is also narrow, and includes lactate, pyruvate, fumarate and malate. The sulphate reduction reaction in anaerobic environments can be represented by the following equation: 2CH 2O + 2H + + SO 4

2-

ﬁ H 2S + 2CO2 + 2H 2O

(2.4)

In this equation, CH2O represents carbohydrates; the equation becomes much more complicated if it is written more accurately to show the consumption of glucose (C6H12O6) or similar molecules. Hydrogen sulphide is highly soluble in water and is readily precipitated as ferrous sulphate (FeS2), producing the black colour characteristic of anoxic sediments. Hydrogen sulphide is highly toxic to ﬁsh, but is readily oxidised to a harmless form by exposure to oxygen. The toxicity is increased at higher temperatures and at pH values less than 8, when the largest percentage of hydrogen sulphide is in the toxic unionised form. The toxicity is based on the capacity of the molecule to inhibit the reversible binding of oxygen to haemoglobin by binding to, and inactivating, cytochrome oxidase. In addition, the release of hydrogen sulphide has been implicated as a causative agent of gill damage in caged Norwegian salmon stocks. Damage to the gills of brown trout fry, Salmo trutta, following exposure to low, chronic concentrations (2–5 mg l-1) of hydrogen sulphide includes thickened gill lamellae and bulbous tips. The 96-h LC50 for this species has been estimated to be 7 mg l-1. In an aquaculture context, hydrogen sulphide may present a problem in a wide variety of situations. It may be present in water sourced from wells, but this rarely presents problems under conditions of adequate aeration. In general, the source of hydrogen sulphide is more

18

Culture of cold-water marine ﬁsh

likely to be organic materials generated by the farming activities themselves than the water sources unless the siting of the farm has been less than judicious. Thus, any situation where organic material is permitted to accumulate is likely to become a source of hydrogen sulphide. This may occur, for example, in ﬁsh ponds, beneath ﬁsh cages or in recirculation systems. The severity of any problem will depend both on the rate of accumulation of the organic materials and the rate at which oxygen is supplied. The greatest problems may occur when anaerobic sediments are disturbed, for example during husbandry or harvesting operations, when large amounts of hydrogen sulphide may be released. In northern Europe, the accumulation of organic material beneath salmon cages has perhaps given the greatest cause for concern. Under normal conditions, hydrogen sulphide generated in the sediments would become oxidised back to sulphate at the sea/sediment interface. Under more extreme conditions of organic input, however, the boundary between the reduced and oxidised zone may lie much higher in the water column, and hydrogen sulphide gas may escape to the atmosphere before being oxidised. Despite the solubility of hydrogen sulphide, it has been detected 9 m above the bottom in the vicinity of salmon cages. Water ﬂows and the management of the operation will principally determine the extent of the problem.

2.7 Light The quality, intensity and photoperiod are the characteristics of light that affect ﬁsh, and the conditions needed to obtain optimum performance in culture differ with species and stage of development. The effects can be considered under two headings: the inﬂuence on growth and development, which is particularly important during the larval stages, and the inﬂuence on reproduction.

2.7.1 Growth and Development Boeuf & Le Bail (1999) have recently reviewed this subject. This section will concentrate on the information available for marine species. Light can have a direct inﬂuence on the hatching process. The hatching of Atlantic halibut, for example, is inhibited by light. After the eggs have been held in constant light until after embryo development is complete, a transfer to darkness results in rapid and synchronous hatching. It appears that light inﬂuences the control of the secretion of the hatching enzyme. This is also the case for other species, although the response is not the same: in some, hatching can be more common during the light phase. In some species, it is possible that even the rate of embryonic development before hatching can be affected by light. Walleye pollock (Theragra chalcogramma), a deep-water species, has embryos that develop more rapidly under constant darkness than under diel light conditions. The larvae of the marine ﬁsh being considered for aquaculture are primarily visual feeders, and thus light plays a signiﬁcant role in determining the success of early feeding. Larval vision is characterised by limited spectral sensitivity, although the range of wavelengths to which the larval eye is sensitive normally increases with age. Hatchery rearing of

Abiotic factors

19

embryos and larvae is generally under artiﬁcial white light, and it is the intensity that is particularly important for the success of rearing. Different species have different requirements. Atlantic halibut yolk-sac larvae held at 10 lux show signiﬁcantly better growth and survival than those kept at 1000 lux, probably because their activity is greater at the higher light intensity, resulting in less of the yolk reserves being available for growth. Yolk-sac cod larvae develop faster in constant darkness than larvae kept under a diel light cycle, since swimming activity is 6–10 times less in darkness. Once exogenous feeding begins, a suitable light level is necessary for active feeding to be successful. For example, ﬁrst-feeding larvae of greenback ﬂounder (Rhombosolea tapirina) held in total darkness die within 20 days of hatching. The optimum intensity varies with species. Cod larvae show maximum feeding incidence at 1 lux and a clear inhibition of feeding at light levels above 12 lux, and Atlantic halibut are reported to have a higher feeding success at 0.5 lux than at 50 lux. Turbot, a species that has a surface-orientated feeding behaviour, feeds best at light levels of 860 lux and higher, but feeds poorly at 12 lux. The optimum light level does vary with the type of food offered. Whiteﬁsh (Coregonus sp.) larvae fed an inert diet at 20 lux do not grow as well as those held at 300 or 500 lux, whereas there is no signiﬁcant difference under these different light intensities when the larvae are fed live Artemia nauplii. The movement of the swimming nauplii may enhance their visibility and attractiveness to the larvae even at low light levels. There is evidence that the colour of the tank in combination with the light intensity is important. Low light intensity in black tanks may give poorer growth and survival than in lighter coloured tanks because of the lack of contrast between the prey and the background. The light intensity can also inﬂuence feeding by affecting the distribution of larvae within the rearing tank, and hence the spatial overlap with the distribution of food. It has been suggested that Atlantic halibut larvae aggregate around the walls, the bottom and the surface in rearing tanks owing to a phototactic response to reﬂected light. Supplying additional UV light can result in a more even vertical distribution and a greater ingestion of Artemia. Diffuse light reduces the tendency of Artemia to swarm, and so generates a more even distribution of food within a tank, but an absence of high-density patches may lead larvae to ingest less. Daylength has been shown to affect the growth of some species; longer days increase the period over which visually feeding larvae are able to take food. However, 24-h light does not necessarily improve survival as well as growth. Larvae of sea bass show better growth, but poorer survival, in continuous light compared with a 9-h light period. On the other hand, for haddock, there appears to be no effect of photoperiod, although a period of inactivity during a dark phase may beneﬁt digestion and assimilation. However, a dark period is not only associated with feeding and growth. A diel light cycle appears to facilitate swim-bladder inﬂation, and therefore can contribute to better survival. In some species the swim-bladder is ﬁlled by the early larvae gulping air at the surface during the dark period, and in others gas secretion is frequently stimulated nocturnally. Where light is natural rather than artiﬁcial, the high intensity of sunlight and its spectral composition may have adverse effects on young ﬁsh unless it is ﬁltered. Ultraviolet radiation can cause biological damage to the cells of an organism. Proteins and nucleic acids absorb short-wave ultraviolet light, and the photochemical damage that results can disrupt

20

Culture of cold-water marine ﬁsh

protein synthesis and cell replication. Eggs and early larvae generally lack protective pigments and are particularly sensitive. The damage caused can depend on the cumulative dose received, and this will vary with the time of year and the weather conditions, as well as water depth and clarity. The effects may cause particular problems in extensive shallow-water rearing systems or where ﬁsh are held in cages near the surface without shading. The effect of photoperiod on juvenile growth is generally less pronounced than for larvae. A few species show a positive relationship between longer photoperiods and growth (e.g. turbot), but others do not (e.g. yellowtail ﬂounder, Pleuronectes ferrugineus). It is likely that the effect on growth is more pronounced when feeding is restricted. Daylength also plays a role in determining the age of ﬁrst maturity in turbot (as well as other species; see below). Exposure to an extended photoperiod during the ﬁrst winter decreases the proportion of males maturing in their ﬁrst year of growth compared with those in a natural photoperiod. This illustrates that growth and reproduction do affect each other, and the separation of these subjects in this account is rather artiﬁcial.

2.7.2 Reproduction The reproduction of cold-water ﬁsh is generally an annual event synchronised with the time of year when the chances of survival of the progeny are maximised. In most species changing daylength, in addition to the annual cycle of temperature change, provides the main cue that regulates the hormonal control of gonadal recrudescence, maturation and spawning. There has been considerable study of the effect of altered seasonal cycles of daylength or ﬁxed photoperiods on salmonid reproduction (Bromage et al., 1993), and most of the work with marine species is based on experience with salmonids. The ability to shift or prolong the spawning season is a great advantage for commercial ﬁsh cultivation provided that water temperatures allow early developmental stages to be reared out of their normal season. This allows hatchery facilities to be used more efﬁciently and production to be increased. Various approaches have been used to alter spawning time, and the simplest to understand is the use of an annual cycle of changing daylength that follows the normal pattern, but is phase-shifted by several months. For example, the hatchery could have three different groups of broodstock ﬁsh, one under a normal light cycle, one that is delayed by 4 months and another delayed by 8 months, leading to peak spawning by the three stocks in March, July and November, respectively (Fig. 2.4). Constant photoperiods and sudden changes from short days to long days will also advance or delay spawning and can be simpler to implement practically, but the timing of the change and the photoperiod history of the ﬁsh are very important. Bromage et al. (1993) summarise this succinctly. They suggest that the change in photoperiod entrains an endogenous clock that controls reproduction, and it is the perception of changing daylength rather than absolute photoperiod that is signiﬁcant. An abrupt change from a photoperiod of 18 h to one of 14 h light advances spawning by a period similar to that following a change from 10 h light to a 6-h photoperiod. Although this work is based mainly on studies with rainbow trout (Oncorhynchus mykiss), similar mechanisms operate in other species that exhibit an inﬂuence of photoperiod. Examples include work with Atlantic halibut using different phase-shifted annual cycles to either delay or advance spawning; compressed annual cycles can result in

Abiotic factors

4 months delay

LIGHT PERIOD (h)

18:00

8 months delay

21

Normal light cycle

14:00

10:00

06:00 Mar

May

Jul

Sep

Nov

Jan

Mar

May

Figure 2.4 Generalised curves showing the normal change in daylength during the year (at a latitude of about 51°N) and two comparable cycles, one phase-shifted by 4 months, the other by 8 months. Maintaining different groups of a species that spawn shortly after a period of increasing daylength (such as turbot) under each light cycle would provide gametes from the different stocks in July, November and March, respectively.

cod spawning twice in one year; spawning in turbot can be induced by a sudden change from a short-day to a long-day photoperiod regime. Not only are annual reproductive cycles affected by photoperiod, the initial maturation of ﬁsh can also be inﬂuenced. It is often convenient in commercial ﬁsh farming to delay the onset of sexual maturity and obtain better growth performance in the early years. It has been shown, for example, that exposure of cod age 1+ years to continuous light from the midsummer solstice onwards will delay gonad development and inhibit spawning at 2 years (see Section 10.1). This treatment also reduces the proportion of females in the group that spawn at 3 years of age, and somatic growth in the second year is better than normal. Data in the published literature has inconsistencies in the effects of photoperiod manipulation, and species-speciﬁc differences do exist. However, some reported variation within species may be attributable to the differences in the time of year the treatments were begun, or the previous photoperiod regime to which the ﬁsh were subjected prior to being switched to the treatment regime of interest.

2.8 Algae Blooms An algae bloom is the name given to the development of an occasional high concentration of planktonic algal cells that forms rapidly when water conditions are suitable. The presence of high cell concentrations, usually dominated by one species, may cause the water to appear coloured, leading to so-called ‘red tides’, usually caused by species of dinoﬂagellate, or ‘brown tides’, which tend to be associated with diatom blooms. Such blooms are not always

22

Culture of cold-water marine ﬁsh

toxic, and the name red tide is often used for any algal bloom, but the same names are often used to describe blooms which result in a build-up of toxins in the water. These toxins can cause ﬁsh kills, or become concentrated in ﬁsh and shellﬁsh rendering them toxic to humans. Blooms that lead to the release of toxins are frequently referred to as harmful algal blooms, or HABs. However, apart from producing toxins, algal blooms can be detrimental to ﬁsh and shellﬁsh in several ways. Their physical presence can damage ﬁsh gills, dissolved oxygen in the water can be severely depleted, and the ﬂavour of the ﬁsh can be adversely affected if they are harvested within a few days of being exposed to a bloom. The environmental conditions that can lead to blooms include high light levels, relatively warm water and sufﬁcient nutrients to allow rapid growth. Therefore, they often occur in spring and summer in temperate regions, and usually in coastal waters during relatively calm periods when the population of phytoplankton cells can multiply without being dispersed. They are frequently derived from the development of resting cells as conditions become suitable, leading to a rapid increase in cell numbers. The main impact of algae blooms on cultivated ﬁsh is often at cage-farm sites where there is no simple way of isolating the ﬁsh in the cages from the developing algae population. Even in pump-ashore farms, toxins and algae can reach the ﬁsh tanks before the problem has been recognised. Many of the serious problems caused have been associated with salmonids in net-pens, or in the culture of warmer-water ﬁsh such as striped bass. Even so, there is little reason to believe that cold-water marine ﬁsh will not be affected at some time as the industry expands. The information that follows provides a broad overview of the types of bloom that can form. Several different groups of algae have species that may form blooms. In marine waters, the most important groups are the dinoﬂagellates (Dinophyta), diatoms (Bacillariophyceae) and members of the Haptophyta. Species of Cyanobacteria (blue–green algae) that form blooms are uncommon in marine waters and rarely lead to ﬁsh kills, although some freshwater species are a signiﬁcant source of the toxins that can kill ﬁsh. The most important group, in terms of effects from toxins, is the Dinophyta. Many different dinoﬂagellate species may produce blooms, but only about a dozen produce toxins. In some species, toxins may be produced only if there is some imbalance in the nutrients in the water, such as a phosphate depletion. Signiﬁcant ﬁsh kills or contamination of ﬁsh as a result of HABs have tended to be in warm temperate coastal waters, but some of the species involved occur in cold temperate waters, and if conditions prove suitable HABs could develop. Several different classes of dinoﬂagellate toxins exist (see Stickney, 2000, for more details of their effects). Most are known through their effects on people who consume shellﬁsh that have concentrated the toxin; the most familiar names describing these are paralytic shellﬁsh poisoning (PSP) and diarrhetic shellﬁsh poisoning (DSP). PSP is caused by a saxotoxin produced by species of the genera Alexandrium, Pyrodinium and Gymnodinium. DSP is caused by okadaic acid produced by species of Dinophysis and Prorocentrum. Despite the names, the toxins can also accumulate in ﬁsh. Neurotoxic shellﬁsh poisoning is caused by brevetoxins that are produced by Ptychodiscus brevis, and these toxins have also been associated with ﬁsh kills along the east coast of the USA. Several genera produce gambiertoxin and ciguatoxin, which can lead to ciguatera in humans. This is poisoning as a result of eating

Abiotic factors

23

contaminated ﬁsh, which are usually of tropical origin, although it has been reported in saupe from the Mediterranean. Although the location of most HABs is largely outside the areas of culture of cold-water marine ﬁsh, algal species are prime candidates for transfer from one area to another in ballast water of ships, and their distribution can change rapidly. In addition, new algal species and new toxins are still being isolated, and it is possible that more may be found in cold temperate waters. As recently as the 1990s, the dinoﬂagellate Pﬁesteria piscicida was ﬁrst recognised as the causative agent in major ﬁsh kills in estuaries of the south-eastern United States. It is most likely to cause problems at temperatures above 26°C, but the toxin is effective as low as 12°C. The release of the toxin is actually stimulated by the presence of live ﬁsh. The toxin leads to ulcerative diseases at cell densities of 100–250 cells ml-1 and is lethal if the concentration of the P. piscicida is 250–300 cells ml-1 . There are a few diatom species that can cause HABs, particularly those of the genus Pseudonitzschia. They produce the toxin domoic acid, which is the cause of amnesic shellﬁsh poisoning. As well as occurring in shellﬁsh, it can accumulate in planktivorous ﬁsh and could therefore be a problem for consumers of the ﬁsh, but there are no reports of a direct effect causing signiﬁcant ﬁsh kills. Some non-toxic diatoms can cause one of the more signiﬁcant problems affecting cagecultured ﬁsh in cold temperate coastal water. High concentrations cause physical damage that contributes to ﬁsh mortalities. The silicaceous theca of species such as Skeletonema costatum and Chaetoceros convolutus have setae with small spines that can seriously damage the lamellae of ﬁsh gills. At concentrations of more than 5 cells ml-1 this causes mucus buildup and impaired gas exchange, that has resulted in mortalities of Paciﬁc salmon in cagefarm sites. At concentrations of less than 5 cells ml-1, the damage is sufﬁcient to give rise to increased mortality from vibriosis. Of the Haptophytes, Phaeocystis, Prymnesium and Chrysochromulina are the familiar genera that are associated with algal blooms and ﬁsh kills. Prymnesium parvum is mainly a problem of fresh water, but it does occur in low-salinity brackish waters. It produces a potent ichthyotoxin called prymnesin. Mass mortalities of salmon and trout have occurred as a result of blooms in Norwegian fjords, where the salinity at the surface was about 5 p.s.u. and toxin production was enhanced because of a phosphorus limitation in the water. Phaeocystis pouchetii has long been considered to be a problem when blooms of the colonial form occur, mainly through the massive increase in biological oxygen demand that occurs when the bloom collapses and the decaying colonies settle out of the water column. However, recent work has shown that a chemical is released by the cells that is toxic to cod larvae and remains in ﬁltered seawater. Production of the toxin increases when the cells are exposed to increased light levels.

2.9 Site Selection Decisions regarding the choice of site for an aquaculture enterprise are among the ﬁrst and the most important in determining the success of the operation. The characteristics of the chosen site can have a signiﬁcant bearing on the capital outlay, the running costs of the

24

Culture of cold-water marine ﬁsh

operation and the rate of production and mortality of the target species. Although the selection of the site is of fundamental importance to all forms of culture, Beveridge (1987) argues that the decisions are perhaps more crucial for water-based culture operations than those based on land, reasoning that there is some potential for improving the latter. For example, more bore holes may be drilled to increase the water supply, or sediment traps or ﬁlters can be installed to reduce suspended solids. There are fewer options for such manipulations with water-based systems, and consequently it is important to get it right from the outset. Treece (2000) points out that before a detailed appraisal of potential sites is made, it is important to determine the overall objectives of the enterprise. First and foremost, the market for the product needs to be assessed. It is important to know what the market channels are, and how the product is to be delivered to the market in good condition and without excessive cost. Having considered the market and other factors, the required production level and the pattern of that production need to be determined. The area of land required can then be estimated; this is a function of the number of crops per year, the total production and the production strategy. It should also be remembered that a farmer can select a site to suit a particular species, or conversely select a species to suit a particular site. The ﬁnal step of these preliminary considerations should be to assess the ﬁnancial returns of the project. Treece (2000) expresses the view that a return of 20%, or preferably 30–50%, per annum over a 12-year horizon should be required before progressing further with the site-selection procedure. If the likely return is less than that level, the project should be abandoned before further resources are committed. However, such decisions will depend on the goals of the company. Having decided to proceed, the characteristics of available sites need to be evaluated. Beveridge (1987) provides a comprehensive review of the factors to be considered in selecting a site. Although he focuses particularly on those of importance to cage culture, the majority are common to both cage and land-based tank or pond culture. The following overview is based largely on this detailed review unless otherwise attributed. It is self-evident that by far the most important resource to be considered for an aquaculture project is water supply. The availability and physical, chemical and biological characteristics of the water will be the principal determinant of the production characteristics of the site. A record of seasonal changes over a signiﬁcant time-period should be known in order to assess the degree to which the site characteristics are likely to match the requirements of the target species. Of prime importance is temperature, since this is the physical attribute of the water that will have the greatest controlling inﬂuence on growth rate. Seasonal changes in temperature may be important in planning production strategies, since it would be desirable, for example, for peak temperatures to coincide with high stock holdings to obtain the greatest weight gain from the most favourable conditions. The avoidance of temperature extremes will also be important, during both the summer and the winter months, to avoid prolonged periods of low growth and low food conversion efﬁciencies, and even mortalities under the most severe conditions. In this respect, any changes in the temperature preferences of the species over its life-cycle will be an important consideration. Although most marine ﬁsh are euryhaline, salinity can vary considerably in coastal areas, and extreme conditions can arise from a combination of excessive run-off from the land and poor mixing and ﬂushing rates. Freshwater run-off can also have a signiﬁcant impact on temperature. In

Abiotic factors

25

temperate regions, freshwater will have a cooling effect in the winter and a warming effect in the summer. Oxygen levels are rarely limiting, although areas of high organic input should be avoided. The pH of the water is an important factor because extreme values can directly damage gill surfaces. It is also important because it affects the toxicity of several pollutants, including ammonia and cyanide, and heavy metals such as aluminium. The ideal pH for most ﬁsh species is 6–8.5. This is not a problem at most marine sites because seawater is naturally alkaline, with a pH typically within the range 7.5–8.5, and because it is well buffered and is less prone to ﬂuctuations than freshwater. The effects of turbidity can be signiﬁcant, but depend to a large extent on the nature of the suspended particles. Some may have toxic properties, such as the salts of various metals, whereas those of an organic nature can have a signiﬁcant oxygen demand and cause oxygen depletion. High levels of certain types of suspended solids can cause gill damage and have also been implicated in diseases such as ﬁn-rot. In general, it is considered that levels below 100 mg l-1 have little effect, while areas where levels are signiﬁcantly above that should be avoided. Water contaminated with pollutants should clearly be avoided, although this is difﬁcult to guarantee. At the very least, sites should not be chosen which are close to industrial complexes or in areas susceptible to such developments. Organically polluted areas may increase the risk of disease since they seem to harbour more disease agents than unpolluted areas. For example, the pathogenic bacterium Vibrio parahaemolyticus has been found in exceptionally large numbers in sewage-polluted waters. Similarly, areas prone to phytoplankton blooms, which can generate adverse effects by clogging gills, reducing DO levels, tainting the ﬂesh or producing toxins, should be avoided, but may be difﬁcult to identify. Susceptibility should be assessed from discussions with local people and the relevant authorities, and nutrient-rich sites and those where the exchange period is more than a few days should be avoided. Good water exchange at a site is important to avoid the accumulation of wastes in the vicinity of the farm. This is unlikely to be a problem in relatively open-coast situations, but cage farms are often situated well within sea lochs or fjords in areas where exchange rates may not be adequate. Estimating the exchange rate is a complex process, and in lochs or fjords is dependent on the size and topography of the basin, the number, location and depth of sills, the magnitude of freshwater inputs and the tidal range. Beveridge (1987) reviews the calculation of exchange rates and recommends that marine sites should have good bottom as well as surface currents, and that the exchange period should be in days rather than weeks. The exchange rate of the water mass in which the ﬁsh are held is a critical factor, since this will, to a large extent, determine the rate at which oxygen is supplied and solid and dissolved wastes are removed. In tanks, the exchange rate is largely under the control of the farmer, who can vary ﬂow rates in accordance with the biomass loading, or more precisely the metabolic activity, of the ﬁsh to maintain near-optimal conditions. In contrast, exchange rates in cages are much less controllable, since they are almost entirely dependent on tideand wind-generated currents, and to some extent the activity of the ﬁsh. Current velocities in coastal marine sites vary widely, ranging from 0 cm s-1 at slack water to more than 250 cm s-1 at full ﬂood and ebb tides. It is clearly desirable that the period of slack water is as short as possible. Current velocity during the ebb and ﬂood tides will determine the density

26

Culture of cold-water marine ﬁsh

of ﬁsh that can be stocked, and hence will have an important bearing on the economics of the operation. However, excessive currents can adversely affect the behaviour of the ﬁsh and deform the cage, thus reducing its volume. In practice, it is recommended that current velocities should be below 100 cm s-1, and preferably between 10 and 60 cm s-1. Other environmental factors are also important determinants of site suitability for cages. Weather can be of considerable importance in that excessive rainfall can have a marked effect on salinity, and excessive wind can cause considerable damage to structures and limit access to the stock. One of the most damaging effects of wind is through wave generation, and the probable conditions at potential sites need to be carefully considered. Beveridge (1987) describes how this can be estimated from information on the long-term frequency and direction of surface wind speeds, fetch lengths corresponding to the directions of the strongest prevailing winds, and the depth of water along the fetch. The depth of water directly beneath the cages is of less importance, although it is important that cages should be well clear of the bottom at all states of tide to allow effective dispersal of waste food and faeces, and to avoid local deoxygenation and toxic effects (e.g. H2S) from the accumulation of these waste organic materials. Other factors of no less importance which must be considered in evaluating sites relate to legal issues, including access and rights, the provision of essential utilities such as electricity, freshwater and telephones, and the availability in the region of labour, contractors and possibly even research support. The nature and area of the land is of particular importance. In the case of a cage farm, sufﬁcient land should be available for the construction of buildings to house, for example, laboratories, stores, ofﬁces and accommodation for staff, preferably within sight of the cages. Mooring or launching facilities for boats will also be important. With regard to pump-ashore tank systems, the land should be as close to sea level as possible (but without risks of ﬂooding) to minimise pumping costs.

2.10 References Alderdice, D.F. (1988) Osmotic and ionic regulation in teleost eggs and larvae. In: Fish Physiology. Vol 11. The Physiology of Developing Fish. Part A. Eggs and Larvae. (eds W.S. Hoar & D.J. Randall), pp. 163–251. Academic Press, San Diego. Beveridge, M.C.M. (1987) Cage Aquaculture. Fishing News Books, Farnham. Boeuf, G. & Le Bail, P.Y. (1999) Does light have an inﬂuence on ﬁsh growth? Aquaculture, 177, 129–52. Boyd, C.E. (1979) Water Quality in Warm Water Fish Ponds. Auburn University Agricultural Experiment Station, AL. Brett, J.R. (1979) Environmental factors and growth. In: Fish Physiology. Vol VIII. Bioenergetics and Growth. (eds W.S. Hoar, D.J. Randall & J.R. Brett), pp. 599–675. Academic Press, San Diego. Brett, J.R. & Groves, T.D.D. (1979) Physiological energetics. In: Fish Physiology. Vol VIII. Bioenergetics and Growth. (eds W.S. Hoar, D.J. Randall & J.R. Brett), pp. 279–352. Academic Press, San Diego. Bromage, N., Randall, C., Duston, J., Thrush, M. & Jones, J. (1993) Environmental control of reproduction in salmonids. In: Recent Advances in Aquaculture. Vol. 4. (eds J.F. Muir & R.J. Roberts), pp. 55–65. Blackwell Scientiﬁc, Oxford.

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Kamler, E. (1992) Early Life History of Fish: An Energetics Approach. Chapman & Hall, London. Person-Le-Ruyet, J., Chartois, H. & Quemener, L. (1995) Comparative acute ammonia toxicity in marine ﬁsh and plasma ammonia response. Aquaculture, 136, 181–94. Rombough, P.J. (1988) Respiratory gas exchange, aerobic metabolism, and effects of hypoxia during early life. In: Fish Physiology. Vol XI. The Physiology of Developing Fish. Part A. Eggs and Larvae (eds W.S. Hoar & D.J. Randall), pp. 59–161. Academic Press, San Diego. Shepherd, J. & Bromage, N. (1988) Intensive Fish Farming. BSP Professional Books, Oxford. Spotte, S. (1979) Seawater Aquariums. Wiley, New York, Chichester, Brisbane, Toronto. Steinarsson, A. & Moksness, E. (1996) Oxygen consumption and ammonia excretion of common wolf-ﬁsh, Anarhichas lupus Linnaeus 1758, in an experimental-scale, seawater, land-based culture system. Aquaculture Res., 27(12), 925–30. Stickney, R.R. (2000) Dissolved oxygen. In: Encyclopaedia of Aquaculture (eds R.R. Stickney), pp. 229–32. Wiley, New York. Treece, G.D. (2000) Site selection. In: Encyclopaedia of Aquaculture (ed R.R. Stickney), pp. 869–79. Wiley, New York. Wheaton, F.W. (1977) Aquaculture Engineering. Wiley, New York.

Chapter 3

Microbial Interactions, Prophylaxis and Diseases O. Vadstein, T.A. Mo and Ø. Bergh

This chapter deals with microbes and infectious agents and their interactions with the ﬁsh at all developmental stages. Whereas several texts on this topic look primarily at diseasecausing organisms, we also try to deal with the natural interactions between microbes and hosts. Thus, we try to have both a veterinary and a microbial ecology perspective on the issue. The chapter starts with a general presentation of ﬁsh–microbe interactions (including both mutualistic/commensalistic and parasitic relationships, and some general immunology), followed by known problem organisms (virus, bacteria and parasitic proto- and metazoa). The second half of the chapter presents a general strategy for the control of infectious agents, and discusses how to improve environmental conditions and the resistance of the ﬁsh. The ontogeny of the immune system of the ﬁsh, and the differences between open and closed systems, are used throughout as a back-drop to a discussion of countermeasures.

3.1 Fish–Microbe Interactions and Implications in Aquaculture Compared to life in air, life in water exists in a far more hostile environment in a microbial sense. A ﬁsh has to handle typical bacterial concentrations of approximately 1 000 000 bacteria ml-1, of which 0.1–1% can be cultured on a non-selective agar. Concentrations of virus are one to two orders of magnitude higher than bacterial densities. However, only a few of these micro-organisms are harmful to higher organisms. By far the majority of viruses in aquatic environments are infective to other micro-organisms. Of the large numbers of different types of marine bacteria, only relatively few are known to be able to cause infections leading to disease.

3.1.1 Disease-Causing Organisms If an organism spends part or the whole of its life in association with another species it is called a symbiont. The majority of symbiotic relationships are neutral or even beneﬁcial for the host (cf. the importance of the normal ﬂora below). A relationship in which one symbiont beneﬁts while the other (the host) is neither helped or harmed is called commensalism, whereas when both symbionts beneﬁt the relationship is termed mutualistic. However, if the symbiont lives at the expense of, or harms, the host, it is a parasitic organ-

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29

ism. The parasitic way of life is so successful that it has evolved independently in most groups of organisms. Although viruses and bacteria are parasitic organisms, the term parasite usually refers to parasitic protists and metazoans. The ﬁsh-pathogenic bacteria, i.e. the bacteria that are able to cause disease in ﬁsh, are commonly divided into two different ecological categories. Obligate pathogens are specialised organisms that cannot survive by any other means than parasitising another organism. Conversely, opportunistic pathogens, sometimes referred to as facultative pathogens, possess a variety of other survival strategies and are not dependent on parasitising other organisms in order to ensure their own survival. Opportunistic pathogens are often naturally present in the environment, such as the water-column or sediments, and are characteristically able to take advantage of opportunities such as weakened ﬁsh or environmental conditions that may be favourable to the opportunist but unfavourable to the ﬁsh. Most ﬁsh-pathogenic bacteria belong to this category. In addition to bacteria considered to be pathogenic in a classical sense, some bacteria may colonize a host owing to either a weakened host and/or their presence at high concentrations. Because of over-colonization, these bacteria may be harmful for the host. Such bacteria may be termed opportunistic bacteria. Fish viruses are dependent on ﬁsh cells in order to reproduce, and thus should be regarded as obligate pathogens. In many cases, carrier states may be found where viruses are reproducing in a host (ﬁsh) with no external signs of disease. This is also true for many bacteria, including obligate pathogens. Fungi have generally not been associated with major problems for cultured marine ﬁsh. However, reports on Ichthyophonus spp. in several wild species suggest that one may expect problems caused by this group of organisms. In fact, Ichthyophonus have recently caused problems in reared salmon fed untreated, fresh marine ﬁsh. Eucaryotic parasites possess a wide variety of different life cycles. Often very complicated life cycles may be found where the ﬁsh is only an intermediate or ﬁnal host. Parasites may live both on the surface (ectoparasite) or inside (endoparasite) of its host. Virulence is a quantitative term that refers to the relative ability of a pathogen to cause disease, i.e. its degree of pathogenicity. Virulence factors are thus factors that contribute to the virulence, such as speciﬁc abilities to adhere to host cells or enter the host, or abilities to survive within the host, produce certain toxins etc. For example, pathogenic strains of Vibrio anguillarum, the causative agent of vibriosis, produce a number of proteases, hemolysins, cytotoxins and dermatotoxins. These compounds contribute to the ability of the bacterium to exploit the resources provided by the host, and their effects can be observed as lesions. Different strains of pathogens may differ with respect to which virulence factors are present, and non-pathogenic strains (of e.g. V. anguillarum) are common.

3.1.2 Normal Fish–Microbe Interactions, Infection Pathways and Pathogenesis Because parasitic organisms, by deﬁnition, are dependent on their host, the relationship between parasite and host is dynamic. In most cases the dynamics are not determined by host and parasite only, but also by other symbiotic organisms of the host. Either a commensalistic (neutral) or mutualistic (beneﬁcial) relationship with the host will inﬂuence the dynamics (Fig. 3.1). Shifts between the various types of symbiotic organisms will inﬂuence

30

Culture of cold-water marine ﬁsh

the health status of the host. The competitive ability of the mutualistic and commensalistic organisms, the number of parasites, the virulence of the parasite and the susceptibility of the host are decisive for whether the parasite will grow and multiply on or within the host, which is termed an infection. In order to cause disease in a ﬁsh, an infective organism must be successful through several stages of the infection cascade (Fig. 3.2). First, the pathogen must establish physical contact with the host and colonise epithelial cells or mucosa. The ability to adhere to the host, colonize the host and eventually enter into the host’s various tissues are thus vital to ﬁsh pathogens. There is considerable evidence that bacteria or viruses which are able to initiate infection are able to adhere speciﬁcally to epithelial cells. Many bacterial pathogens have been shown to be capable of adhering to ﬁsh cells and to grow in mucus in vitro. Generally, the gills and intestinal surfaces are important sites of adhesion and colonisation of ﬁsh pathogens, but entry through the skin is also possible. The second stage of infection involves cell damage, and may also include cell penetration and intracellular proliferation. Not all pathogens invade the host cells to cause damage and therefore enter the third stages, that entail systemic spread. The gills are constantly ﬂushed with water, and only possess a thin structure separating the blood from the water surrounding the ﬁsh. This makes the gill an important site of entry for parasitic organisms such as bacteria or viruses. The surface of the gills is a natural habitat for bacteria. Phagocytosis by macrophages occurs in the gills, and thus bacteria may be phagocytosed in this organ. Furthermore, the gill is an important organ of antigen uptake, particularly of particulate antigens such as vaccines. The intestine of ﬁsh is a natural habitat for bacteria, and many are useful to the ﬁsh. The intestinal microﬂora may be beneﬁcial to ﬁsh in three different ways: (1) They may participate in digestion, (2) they may synthesise essential growth factors and nutrients, and (3) they

MUTUALISM

When relationships move in this direction, the infectious disease process begins

COMMENSALISM

When relationships move in this direction, reestablishment of healthy host occurs

PARASITISM Figure 3.1 Symbiotic relationships between microorganisms and their host. Such relationships are highly dynamic, and shifts among them may occur due to shifts at the community level. Mutualism is most beneﬁcial, and occurs when both symbionts beneﬁt from the relationship. The most destructive is parasitism, in which one symbiont lives at the expense of, or harms, the host. Commensalism is the relationship in which one symbiont beneﬁts while the other (the host) is neither helped or harmed. Redrawn from Prescott et al. (1999).

Microbial interactions, prophylaxis and diseases

31

Access to mucosa

Stage 1

Stage 2

Stage 3

Resistance to host defences

Competition with commensals Cell damage

Adherence

Colonisation

Lateral spread

Intracellular proliferation

Cell penetration

Entry into blood-stream and lymph system

Mucus

Epithelium

Laminaria propria

Resistance to host defences

Proliferation

Systemic spread Figure 3.2 The three stages of infection by pathogens (redrawn from Williams et al., 1988).

may play an important role in protection against pathogens. Bacteria and viruses are engulfed by endocytosis in the intestinal epithelium. Intestinal bacteria may take part in the degradation of various compounds such as complex carbohydrates to molecules that may be taken up and utilised by the ﬁsh, or in a synthesis of molecules which are of beneﬁt to the ﬁsh, such as essential fatty acids or vitamins (Hansen & Olafsen, 1999; Ringø & Birkbeck, 1999). Intestinal bacteria may also play a part in protection against disease (cf. Ringø & Gatesoupe, 1998). In adult ﬁsh, a bacterium entering the ﬁsh via the intestine must ﬁrst pass through the stomach. The secretion of lethal substances and the low pH in the stomach are important barriers against pathogens using this route. In larval ﬁsh, however, the stomach is not well developed and the intestine is more accessible. Live food organisms such as rotifers and Artemia nauplii spp. naturally ﬁlter bacteria, and may contain large amounts of bacteria (Skjermo & Vadstein, 1993; Makridis et al., 2000a). Pathogens present in the live feed cultures may thus be presented to larval ﬁsh intestines in concentrated live-feed packages. Bacteria colonise the ﬁsh intestine even before active feeding commences. In halibut, the mouth is not developed when the larva hatches, but the intestine is still available to aquatic bacteria. In this case, the pseudobranchs is the site of entry. Bacteria are taken up from the water at rates that exceed drinking rates by one order of magnitude (Reitan et al., 1998). This uptake is therefore active and regulated by some unknown mechanism. Prior to the onset of exogenous feeding, larvae generally possess a non-fermentative intestinal ﬂora, whereas the onset of feeding generates processes which probably include a reduction in oxygen levels in parts of the intestine, leading to a ﬂora dominated by fermentative bacteria (Bergh et al., 1994).

32

Culture of cold-water marine ﬁsh

One of the main functions of ﬁsh skin is the separation of the internal tissues from the environment. The mucus secreted by cells in the skin contains immunoactive compounds. Specialised eucaryotic parasites, such as lice, may penetrate the skin, thus making it possible for bacterial pathogens to enter through the lesions. Salmon lice (Lepeophtheirus salmonis) infecting Atlantic salmon (Salmo salar) may act as a vector for the infectious salmon anaemia (ISA) virus (Nylund et al., 1994). It is likely that many parasites are important vectors transmitting pathogens, and that by causing damage to the ﬁsh skin they open the way for other pathogens. In some cases a bacterium can remain localised at mucosal surfaces and initiate damage by liberating toxins. This mode of action may be important for opportunistic bacteria. However, in most cases the pathogenic bacteria penetrate the epithelium and spread to other parts of the body, where growth is initiated. Viruses must enter host cells in order to reproduce, and are normally dependent on speciﬁc receptor molecules that ﬁt surface structures of the virus particle in order to enter the host cell. From the site of entry, pathogens are spread mainly via the blood or lymphatic systems. The ability to survive in blood and the action of phagocytes is of vital importance to the pathogen, although the actual mechanisms by which pathogens survive or even multiply are poorly understood. Survival in serum has been documented for several pathogens. Although bacteria may be phagocytosed, several pathogens have been shown to survive and multiply within the phagocytes. The outer layers of the ﬁsh egg, chorion and zona radiata, seem to be highly protective against bacteria. To date only Tenacibaculum ovolyticum (previously named Flexibacter ovolyticus), a pathogen to halibut eggs, has been shown to be able to penetrate these structures and thus get access to the resources inside the egg (Hansen et al., 1992). However, many other bacteria colonise the surface of the egg, and pathogens may be able to infect the larvae post-hatch (Bergh et al., 1992). Special attention should be paid to some bacteria that are spread intra ovum, i.e. within eggs. A ﬁsh pathogen that has been shown to occupy this ecological niche is Renibacterium salmoninarum, the causative agent of bacterial kidney disease (BKD) in salmonids. Interestingly, this is an obligate pathogen that seems highly specialised for the niches it occupies, which include growth within host macrophages. It is a very slow-growing bacterium, both in laboratory cultures and within the ﬁsh. The progress of the disease is slow, making it possible for the host to reproduce, thus spreading the genes of its parasite. It is likely that intra-ovulary pathogens are also present in marine ﬁsh, but they remain to be found. Viruses can also be transmitted vertically. This is the case for the nodavirus causing viral encephalopathy and retinopathy in Atlantic halibut (Grotmol et al., 1997). It is not known whether the virus is transmitted intra ovum or on the surface of eggs, but successful application of disinfectants suggests that the virus may be present externally, i.e. in the ovary ﬂuid. The possibility of an outbreak of disease is dependent on a large number of factors which interact with each other. The ﬁsh, the pathogen, and the physical and chemical environment may all inﬂuence the status of any of the others (Fig. 3.3). When trying to improve survival in aquaculture, work should be aimed at all these factors. Improvements in physio/chemical environmental conditions and strengthening the resistance of the ﬁsh will contribute to

Microbial interactions, prophylaxis and diseases

A: Factors determining viability (P V )

33

B: Manipulation to increase

Genetics, Nutrition, Immune system Larvae

Larvae

Pv

Microbial environ. Numbers Composition

Pv

Physiochemical environ.

Microbial environ.

Physiochemical environ.

Chemicals, Physical stress

Figure 3.3 A. The three factors of signiﬁcance for the probability of viable larvae (PV) and the conditions which inﬂuence these factors. B. The probability of viable larvae (PV) may be increased by using methods that push the circles towards each other. Redrawn from Vadstein (1997). Reprinted with permission from Elsevier Science.

increases in survival and viability, as will the elimination of pathogens and strengthening of the commensal microﬂora. However, microbial control cannot be regarded as absolute, but is more a question of probabilities. This is evident if we consider the three interacting factors that are decisive for the development of conditions that ensure good viability of the larvae (Fig. 3.3). The ﬁsh, and the biological and physiochemical environments are in turn inﬂuenced by several factors, and manipulating these conditions may increase the probability of viable larvae. However, it is clear that manipulation of only one of these three factors may have a limited effect. Therefore, in any strategy to achieve microbial control and thus improve the viability of the ﬁsh, it is important to use a range of counter-measures directed towards the different aspects shown in Fig. 3.3.

3.1.3 The Immune System of Fish The main function of the immune system is to protect the animal against disease-causing organisms. The immune system of ﬁsh shows clear similarities to that of mammals, and several reviews have covered various aspects of this subject (cf. Vadstein, 1997). The immune system comprises both non-speciﬁc and speciﬁc components, and involves both cellular and humoral factors (Fig. 3.4). This division into four constituents is somewhat misleading because all components are interwoven and mutually dependent. The non-speciﬁc defence mechanisms are part of all normal ﬁsh, and do not require prior contact with an antigen/pathogen to elucidate a response. On the other hand, the speciﬁc immune system requires activation, and there is thus a time-lag between the ﬁrst introduction to the antigen and the activation. This process is also temperature-dependent (Bly & Clem, 1992). It is believed that non-speciﬁc immunity is phylogenetically older than speciﬁc immunity, and one might therefore speculate that ﬁsh are more reliant on non-speciﬁc defence than higher vertebrates.

Culture of cold-water marine ﬁsh

Cellular

Humoral

34

Non-specific (innate)

Specific (acquired)

Lytic enzymes, e.g. lysozyme Complement Agglutinins and precipitins Enzyme inhibitors Growth inhibitors

Antibodies

Macrophages/monocytes Granulocytes Non-specific cytotoxic cells

B-cells T-cells

Figure 3.4 Simpliﬁed ﬁgure showing the main components of the non-speciﬁc and speciﬁc immune system.

The immune defence systems of the ﬁsh mature during the development of eggs, larvae and fry. Unlike mammals, for instance, that are born at comparatively advanced developmental stages, most ﬁsh hatch at ontogenetically primitive stages which are analogous to the early developmental stages of embryos of higher vertebrates. This is also true with respect to the immune system. However, the ontogenetic stage at which hatching takes place is highly variable between species. Halibut larvae hatch at a very primitive stage and the wolf-ﬁshes are relatively advanced at hatching, whereas cod and turbot, for instance, may be viewed as intermediate cases with respect to developmental stage at hatching. It is believed that the larvae of most ﬁsh species do not have the ability to develop speciﬁc immunity during the early stages of development. In this respect, they are reliant on passive immunisation from maternal antibodies. Although it has been reported that maternal transfer of speciﬁc immunity does not occur in salmonids (Ellis, 1988a), this mechanism has been experimentally demonstrated in tilapias (Mor & Avtalion, 1990; Sin et al., 1994). In any given species, size rather than age seems to be most critical for when speciﬁc immunity may be developed. Larvae of many species go through a process of self-recognition after hatching, where the speciﬁc immune system ‘learns’ to recognise the tissue of the individual. This is of importance in aquaculture, as speciﬁc immunostimulation, i.e. vaccination, when performed at these immature developmental stages, could induce immunosuppression rather than immunoprotection, with reduced survival as a result (Joosten et al., 1995). Vaccination of early life stages must therefore be carefully evaluated with respect to the maturation and status of the immune system of the species at different developmental stages (Fig. 3.5). The non-speciﬁc immune system is probably the major defence against micro-organisms in larvae. Although our understanding of the components of the innate defence system of ﬁsh is growing, relatively little is known about the functioning and ontogeny of the general immune system in marine larvae. In the few ﬁsh species that have been studied, the major lymphoid organs are not fully developed at the time of hatching, and the phagocytic activ-

Microbial interactions, prophylaxis and diseases

35

Immunoprotection

No protection Immunosuppression

Larval ontogenesis (time) Vaccination possible Protection induced by vaccination

Figure 3.5 Theoretical protection by vaccination as a function of the larval ontogenetic stage at which vaccination is carried out. Vaccination at early ontogenetic stages generally induces immunosuppression rather than immunoprotection, with a reduced survival rate as a result. Thus, vaccination should only be done after the ﬁsh has become sufﬁciently immunologically mature to ensure positive immunoprotection. Following vaccination, there is a time delay before protection is achieved because of the time that is necessary before the immune reaction has induced protection.

ity is mainly associated with gills, skin and gut. It is therefore possible that during the stages when the lymphoid organs are developing, the main cellular defence is by the phagocyte populations within the integument. The non-speciﬁc or innate immune system is regarded as the ﬁrst line defence of animals. Furthermore, it seems that the bacterial problems in larviculture are more often due to opportunistic bacteria than speciﬁc pathogens (Vadstein et al., 1993; Munro et al., 1995). This emphasises how reliant the larvae are on their non-speciﬁc defence under intensive hatchery conditions.

3.2 Viral Diseases: Diagnosis 3.2.1 Infectious Pancreatic Necrosis Virus (IPNV) Juvenile stages of several marine species, particularly halibut and turbot, are susceptible to infections by an aquatic birnavirus, infectious pancreatic necrosis virus (IPNV) (Biering et al., 1994; Mortensen et al., 1993). Only minor sero- and genotypic differences have been found between isolates from halibut and turbot and the N1 or Sp strain from Atlantic salmon (Biering et al., 1997). Moreover, isolates from salmonids have been found to establish an infection in halibut, and thus transfer of virus between salmon and marine species cannot be excluded. From challenge experiments with halibut fry and yolk-sac larvae, it can be concluded that the virus is the causative agent of disease. Temperature inﬂuences IPN

36

Culture of cold-water marine ﬁsh

mortality, as well as the developmental stage of the ﬁsh. In general, smaller fry are more susceptible to infection. IPNV infections in wild cod have been observed in Denmark and the Faroe Islands. Fish of 2–10 g have developed the disease, which is associated with high mortalities. The most characteristic clinical signs of disease in challenged halibut are distended stomach, uncoordinated swimming and trailing, and white faecal casts (Biering et al., 1994). The symptoms are most prominent in small fry, but were also observed in larger individuals. Pathological ﬁndings included focal necrosis of the liver, kidney and intestine, but the pancreatic tissue was unaffected. The absence of pathological ﬁndings in pancreatic tissue of challenged ﬁsh reported by Biering et al. (1994) was in disagreement with the ﬁndings from naturally infected halibut fry (Mortensen et al., 1990), where severe necrosis of the pancreatic acinar cells was found, together with nuclear pycnosis. Reports from turbot vary on this point, although samples from natural outbreaks in Norway demonstrated pathological ﬁndings in pancreatic tissue (Mortensen et al., 1993). The intestine may be the primary organ for virus entry and replication, as indicated by immunohistochemical observations in a challenge experiment with yolk-sac larvae (Biering & Bergh, 1996). However, these authors also pointed out that differences in pathology and susceptibility to IPNV infection generally occur between different developmental stages. As the term infectious pancreatic necrosis virus, by deﬁnition, implies necrosis of the pancreas, a condition not always found in affected halibut, the term aquatic birnavirus is probably more correct (Biering et al., 1994).

3.2.2 Nodaviruses Nodaviruses, a family of neuropathogenic viruses ﬁrst described from insects, are known to cause infections in many marine ﬁsh species in many parts of the world (reviewed by Munday & Nakai, 1997), including turbot and halibut. The virus has been known to cause disease in turbot fry since an outbreak in an extensive production lagoon in 1989, and the virus was originally described as a picornavirus-like agent (Bloch et al., 1991). Mortality appeared during weaning onto moist pellets. Diseased ﬁsh became lethargic, often lying abdomen-up on the bottom. Atypical swimming, such as rotating, spinning and horizontal looping, was observed when the ﬁsh were disturbed. The clinical signs indicated a disturbance of the central nervous system, and were followed by 100% mortality. Under electron microscopy, vacuolated cells were found in the brain and medulla of the diseased ﬁsh, with large numbers of crystalline virus particles. In halibut aquaculture, problems with nodaviruses have caused a decrease in Norwegian juvenile halibut production since 1995 (Bergh et al., 2001). An outbreak of a nodavirusrelated disease, viral encephalopathy and retinopathy (VER), in halibut was ﬁrst recorded in the summer of 1995, when two major hatcheries in western Norway were severely affected (Grotmol et al., 1995, 1997). Most larvae died in the period of early metamorphosis, approximately 60 to 70 days post-hatch. The ﬁrst clinical signs of VER seen in the larvae were reduced skin pigmentation and an empty, transparent intestine due to reduced food intake. Darkening of the skin could be seen. Abnormal behaviour such as spiral swimming and looping was observed in the early stages of a disease outbreak. Severely diseased larvae and

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juveniles became lethargic, often lying upside down on the bottom. Lesions and vacuolisation in the retina, brain and spinal cord, and in ganglia of the peripheral nervous system, were typical ﬁndings, with no lesions detected in other organs. There is no doubt that the nodavirus is the causative agent of the disease, as in challenge experiments the strain from halibut fry was able to replicate and cause VER in halibut yolksac larvae (Grotmol et al., 1999). Monitoring the progression of the infection following challenge suggested that the portal of entry into the larvae may have been the intestinal epithelium, while the route of infection to the central nervous system (CNS) may have been axonal transport to the brain stem through cranial nerves such as the vagus nerves. Diagnosis of nodavirus infections has so far been dependent on histological and immunohistochemical investigations (Grotmol et al., 1997, 1999). A reverse transcriptase–polymerase chain reaction (RT–PCR) assay based on the capsid protein nucleotide sequence of the halibut nodavirus strain has been developed (Grotmol et al., 2000). Vertical transmission occurs, but may be successfully counteracted by ozone disinfection of eggs (Grotmol & Totland, 2000). There are differences between nodavirus isolates from different ﬁsh species. Comparing the pathogenicity of a Norwegian strain from halibut and a Japanese strain from striped jack Pseudocaranx dentex, Totland et al. (1999) found that the halibut strain, which caused high mortality to halibut larvae, was incapable of replicating or inducing mortality in striped jack larvae, and vice versa. However, this difference might be the result of either hostspeciﬁcity or the difference in rearing temperature. Nodavirus strains affecting cold-water species from Norway and Japan are closely related, belonging to a separate clade, whereas nodavirus strains from temperate species, such as striped jack, may be genetically more distant. VER is by far the most important disease problem in present-day halibut aquaculture (Bergh et al., 2001). The outbreak in Norway coincides with a decline in halibut fry production, and is probably the major reason why Norway’s production of halibut fry has levelled out since 1995. No vaccine is currently commercially available. However, a recombinant vaccine has been shown to give signiﬁcant protection in challenge experiments with turbot, Scophthalmus maximus (Húsgarð et al., 2001). At present, one is left with traditional prophylactic countermeasures such as egg disinfection and improved hygiene.

3.2.3 Other Viruses The rhabdovirus VHSV, the causative agent of the so-called Egtved disease or viral heamorrhagic septicaemia in rainbow trout, may also cause problems in marine ﬁsh aquaculture, as there are reports of the isolation of VHSV-like viruses from cod and turbot. Nucleotide sequencing of the glycoprotein gene of VHSV from different geographical areas has conﬁrmed a link between VHS in farmed salmonids and viruses isolated from cod (Stone et al., 1997). Two virus isolates recovered from wild-caught cod off Shetland and from farmed turbot in Scotland showed 99.4% nucleotide sequence similarity with a virus associated with VHS in rainbow trout. Challenge experiments with turbot has conﬁrmed the aetiology of the disease. Typical signs of the disease in challenged turbot included darkening of the skin and the presence of haemorrhaging around the head and ﬁn bases

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(Snow & Smail, 1999). VHSV has also been found in wild-caught Paciﬁc cod, Gadus macrocephalus (Meyers et al., 1992). Another related virus, the hirame rhabdovirus, is known to cause disease in the hirame, or Japanese ﬂounder, Paralichthys olivaceus (Oseko et al., 1988). The signs of infection were congestion of the gonad, focal haemorrhage of skeletal muscle and ﬁns, and accumulation of ascitic ﬂuid. Histopathologically, the kidney indicated necrotic changes by nuclear degeneration of haematopoietic cells and haemorrhage in the interstitial tissue. The spleen showed necrosis and haemorrhage in the pulp, and skeletal muscle revealed hyperaemia and haemorrhage of capillary vessels. Hyperaemia and haemorrhage were observed in the interstitial tissue of the seminiferous duct and ovarian lamella, and in the connective tissues around the seminal duct and oviduct of the testis and ovary. Mucosa of the alimentary tract showed hyperaemia and haemorrhage. An iridovirus-like agent has been described in association with systemic infection in cultured turbot fry in Denmark (Bloch & Larsen, 1993). The initial signs of disease were reduced feed intake, lethargy and darkening in pigmentation, especially of the tail and ﬁns. Later, one may observe atypical swimming and spasms in the terminal stages. Investigation by electron microscopy of samples of ﬁn, gill, liver, kidney, spleen, heart, pancreas and intestinal collagen, and in one of three brain samples investigated, conﬁrmed the presence of an iridovirus-like agent ca. 170 nm in diameter. Another virus, Herpesvirus scophthalmi, which is 200–230 nm in diameter, has only been described under electron microscopy from turbot in Scotland and Wales (Buchanan & Madeley, 1978). An affected ﬁsh is lethargic, often lying with head down and tail up on the bottom. A virus suggested to be a member of the aquareovirus group was demonstrated by Lupiani et al. (1989), but the disease condition was described as of mixed bacterial and viral aetiology.

3.3 Bacterial Diseases: Diagnosis Bacteria may be present in large numbers on the surface of ﬁsh eggs. This epiﬂora seems to be dominated by members of the Cytophaga/Flavobacterium/Flexibacter group, while Vibrio spp. are not frequent (Hansen & Olafsen, 1989; Keskin et al., 1994; Bergh, 1995). The composition of the intestinal bacterial ﬂora associated with yolk-sac larvae resembles the egg epiﬂora, whereas a shift in the intestinal microﬂora from a generally non-fermentative towards a fermentative ﬂora dominated by the Vibrio/Aeromonas group coincides with the onset of exogenous feeding (Bergh et al., 1994; Bergh, 1995). The psychrotrophic Tenacibaculum ovolyticum (formerly named Flexibacter ovolyticus) was isolated from halibut eggs with high mortality (Hansen et al., 1992). It resembles the ﬁsh pathogen T. maritimum, but differs in several biochemical and physiological characteristics. Challenge experiments conﬁrmed that the bacterium is able to cause mortality to halibut eggs and yolk-sac larvae by penetrating the eggshell (Bergh et al., 1992). This bacterium has not been found on hosts other than halibut, despite the fact that it is able to cause mortality to cod, Gadus morhua, eggs and larvae (Bergh, 2000). Pathogenic Flexibacter- or Cytophaga-like organisms have also been described from cultured turbot (Mudarris &

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Austin, 1989) and wild-caught cod (Hilger et al., 1991). Other bacterial pathogens described from cod are Streptococcus parauberis (Romalde, 1999) and a Mycobacterium sp. (B. Hjeltnes, National Veterinary Institute, personal communication, 2002).

3.3.1 Vibrio Species Several species of Vibrio are able to cause mortality to yolk-sac larvae. V. anguillarum, V. splendidus and a V. salmonicida-like strain were tested in a challenge experiment with halibut larvae by Bergh et al. (1992). Unlike Tenacibaculum ovolyticum, the Vibrio spp. did not cause mortality to eggs, but infected the larvae post-hatch, causing mortality during the yolk-sac stage. Of these species, V. anguillarum, serotype O1 and O2, are the most potent, whereas the others must be present in relatively large numbers in order to cause signiﬁcant mortality (Ø. Bergh, unpublished results, 2002). Opportunistic bacteria affecting yolk-sac larvae typically induce changes in larval behaviour, reducing the ability of the larva to initiate exogenous feeding and decreasing their buoyancy, as demonstrated on halibut and turbot larvae by Skiftesvik and Bergh (1993). Thus, even though the opportunists may not kill the larva directly, the probability of the larva surviving the ﬁrst feeding period may be reduced. Investigating the pathogenesis of vibriosis in turbot larvae, Grisez et al. (1996) utilised V. anguillarum-enriched Artemia franciscana as vector organisms. The authors concluded that the anterior part of the intestine was the major port of entry, and that the bacteria were transported through the intestinal epithelium by endocytosis, after which the bacterium was released in the lamina propria. From there, the bacterium was transported by the blood to different organs, eventually leading to septicaemia and mortality. Apart from V. anguillarum, two more species within this genus have been described as causative agents of vibriosis in turbot: V. damsela (Fouz et al., 1992), and V. splendidus (Farto et al., 1999; Gatesoupe et al., 1999). In cod, V. anguillarum dominates among pathogenic isolates (Knappskog et al., 1993; Wiik et al., 1995). Isolates of V. anguillarum from Norwegian marine ﬁsh with vibriosis were found to be free of plasmids, strongly indicating that their virulence properties were chromosome-mediated (Wiik et al., 1989).

3.3.2 Aeromonas Species The causative agent of furunculosis in Atlantic salmon, Aeromonas salmonicida subsp. Salmonicida, is for most practical purposes apathogenic to halibut. In a ﬁeld survey during a major outbreak of furunculosis in the Atlantic salmon stock, all dead halibut at the station were subjected to investigation (Hjeltnes et al., 1995), but no indications of a transfer were found. An experimental challenge with typical A. salmonicida subsp. salmonicida administered to yolk-sac larvae gave a more complex result (Bergh et al., 1997). Signiﬁcant mortality did take place, but this was probably a result of the production of toxic exudates by the bacterium, as histological and immunohistochemical examinations of the larvae revealed no evidence of bacteria in affected tissues. In contrast to the A. salmonicida subsp. salmonicida, which comprises a homogenous group of strains, atypical A. salmonicida strains are heterogenous with respect to serologi-

40

Culture of cold-water marine ﬁsh

cal and biochemical characteristics (Wiklund & Dalsgaard, 1998). However, atypical strains of A. salmonicida are occasionally isolated from diseased halibut and turbot suffering from septicaemia, although at present it cannot be ruled out that some of these infections may be secondary. Recently, Ingilæ et al. (2000) reported signiﬁcant mortality in a challenge experiment with an atypical strain isolated from halibut administered to juvenile halibut and spotted wolf-ﬁsh, conﬁrming the pathogenicity of this bacterium. Mortality of turbot following challenge with atypical furunculosis has also been reported (Perez et al., 1996). Comparing the pathogenicity of one atypical and one typical strain of A. salmonicida to subadult halibut (weight range 154–254 g), Bricknell et al. (1999) found no mortality of halibut as a result of a bath challenge. Minimum lethal doses per halibut after intraperitoneal injection of bacterium were 106 (typical A. salmonicida) and 107 (atypical A. salmonicida). Following the challenge, a stress test of the survivors gave the result that 9 of the 87 halibut died, but all were culture-negative for A. salmonicida, indicating that no carrier state was present.

3.4 Parasitic Protists and Metazoans: Diagnosis, Prophylaxis and Treatment A parasite lives at the expense of another organism, a host, and is usually dependent on this host to complete its life cycle. The term parasite usually refers to unicellular and multicellular eucaryotes, and thus excludes bacteria, fungi and viruses. The damage caused by a parasite can vary from minor to serious and even life-threatening. Usually, ﬁsh that carry relatively few parasite specimens cannot be considered to be diseased. Disease occurs when a ﬁsh is infected with so many parasites, of one or more species, that its normal life functions are disturbed. There is no exact number of parasites that will cause disease. This depends on several parameters such as parasite virulence, host susceptibility and resistance, host size and age, relative parasite size compared to host, and many more. Some parasites may occur in hundreds or even thousands without causing severe pathology, while only one or two Lernaeocera branchialis may cause the death of a small Atlantic cod. Many parasites are present in farmed ﬁsh without ever causing clinical disease, but they can signiﬁcantly reduce ﬁsh growth. Such parasites can sometimes cause greater economic loss for the farmer than more pathogenic parasites, as the former will easily be overlooked while the latter will be treated at an early stage in a disease outbreak. Parasite infections may also facilitate infections by other organisms, both by creating injuries, which serve as infection routes, and by reducing the general ﬁsh disease resistance. Thus, prophylactic measurements to keep the number of parasites low may also have beneﬁcial effects against viral, bacterial, fungal and other parasitic infections. The identiﬁcation of parasites, at least to family or genus level, is often necessary to ensure that correct prophylactic methods and chemotherapeutic compounds are chosen. Many multicellular parasites can be observed macroscopically. The majority of parasites, however, can only be demonstrated by the use of a dissection or light microscope, or even an electron microscope. Identiﬁcation of ﬁsh parasites is still mainly based on morphology

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(shape) and morphometry (size), but an increasing number of parasites are identiﬁed by the use of molecular techniques. A large number of parasites can be found in farmed and wild marine ﬁsh. In this text, the most common parasite groups in ﬁsh are mentioned. For each group, only one or a few species causing disease are mentioned. At present, examples of parasitic diseases in cultured cold-water marine ﬁsh are few. That is why examples of parasitic diseases in cultured warmwater marine ﬁsh are given. It is likely that the cold-water marine ﬁsh farms will experience the same kind of parasitic diseases as production increases.

3.4.1 Protists 3.4.1.1 Amoebae Diagnosis/Identiﬁcation Amoeba is a generic term for protists that move with the aid of pseudopodia (temporary projections of protoplasm and cell membrane for moving and feeding). The term amoeba no longer represents a single taxon, as many different protists have amoeboid developmental stages. Most protists with amoeboid development stages belong to the phylum Rhizopoda. Multiplication mainly occurs by division into two equal parts, a process known as binary ﬁssion. Occasionally, a sexual process associated with ﬂagellated or amoeboid gametes occurs. Identiﬁcation is based on locomotive form and behaviour, the presence of ﬂagellated stages, cyst structure, and nuclear structure and division. Symptoms/Pathology Amoebas may be found in many organs. Amoeboid gill disease seems to be the most common. The pathology includes elevated mucus production, epithelial hyperplasia and metaplasia, and fusion of primary and secondary lamellae. Agents causing amoebic gill disease in clinically diseased turbot, Scophthalmus maximus, were recently identiﬁed (Dyková et al., 1999), and a similar amoeboid gill disease has also been observed in farmed halibut, but the causative organism(s) has not yet been identiﬁed. Prophylaxis/Treatment Lower stocking densities and net cleaning may be of preventative value. Treatment with freshwater has been effective against Paramoeba pemaquidensis associated with proliferative gill disease of farmed salmonids in seawater. Chemical baths have been relatively ineffective. 3.4.1.2 Apicomplexans Diagnosis/Identiﬁcation Most apicomplexans live intracellularly. In at least one of the stages in the life cycle, the apicomplexan cell has a set of organelles at the apex, the apical complex, used for

42

Culture of cold-water marine ﬁsh

penetration into a host cell. The complex life cycle usually involves both asexual and sexual reproduction. Fish apicomplexans are divided into three groups. Coccidia mainly develop in cells of the intestinal wall and are spread by direct transmission or the use of paratenic hosts. Adeleids and piroplasmids are found in the blood and are spread by blood-sucking invertebrates, mainly leeches. Identiﬁcation of coccidia is mainly based on the shape and size of oocysts containing sporozoites usually enveloped in sporocysts, while adeleids and piroplasmids are identiﬁed in fresh and stained blood smears. Symptoms/Pathology A number of apicomplexans cause pathology and mortality in wild marine ﬁsh, but also in farmed marine ﬁsh. The blood-living apicomplexan Haemogregarina sachai has caused severe pathology in farmed turbot (Kirms, 1980). Prophylaxis/Treatment Little is known about prophylaxis, but it could involve elimination of the leeches or parasitic crustaceans needed for transmission. Some chemical compounds have been effective. 3.4.1.3 Microsporidia Diagnosis/Identiﬁcation Microsporidians live intracellularly. The transmission stage is a spore that contains a hollow, evertible polar tube. Under an appropriate stimulus from a host, the polar tube is everted and the sporoplasm is propelled through the tube. The parasite penetrates host tissue and follows circulatory vessels to the ﬁnal site. Both asexual and sexual reproduction is involved in the life cycle. During the asexual phase, parasites may spread to a large number of host cells and a large part of an organ may be affected. Some microsporidians stimulate the infected host cell to enormous hypertrophy, resulting in a xenoma or ‘cyst’, often macroscopically visible (Fig. 3.6, see colour plate section). Microsporidians are transmitted directly between ﬁsh. Spores can retain their infectivity in water at 40°C for at least 1 year. Identiﬁcation is usually based on the shape and size of the spores, which are usually less than 7 mm in length. Symptoms/Pathology Tetramicra breviﬁlum has caused disease in farmed turbot in Spain and Great Britain. The outbreaks were associated with a drop in the water temperature. Affected ﬁsh showed erratic swimming behaviour, swelling of different parts of the body, darkening of the dorsal surface and overproduction of mucus. Heavily infected ﬁsh had jelly-like muscles (Figueras et al., 1992). A species of Nucleospora (formerly Enterocytozoon) has been found in dead and moribund halibut, Hippoglossus hippoglossus, in Norway (Nilsen, 1999). The spleen and kidney were swollen. The excretory and haematopoietic tissues in the kidney were degener-

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ated. In the head kidney, more than 50% of the lymphoblasts were infected with microsporidians in different developmental stages (Nilsen, 1999). An unidentiﬁed microsporidian has caused mortality in farmed sea bream Sparus aurata (Abela et al., 1996).

Prophylaxis/Treatment Because of the longevity of spores and direct transmission, infections by microsporidians are usually difﬁcult to prevent. Few chemical compounds are effective (Schmahl & Mehlhorn, 1989).

3.4.1.4 Ciliates Diagnosis/Identiﬁcation Ciliates can be found in any ﬁsh organ and on external surfaces. They are partly or completely covered by a large number of cilia, which are used for locomotion and feeding. They have a vegetative macronucleus and a generative micronucleus. Most species multiply by binary ﬁssion. Identiﬁcation is mainly based on structure of the oral apparatus, combined with some ultrastructural features.

Symptoms/Pathology Ciliates are commonly found as ectoparasites or endoparasites in farmed and wild marine ﬁsh. Many species are not strictly parasitic, as they only use ﬁsh for attachment, and feed on bacteria and small protists in the water. However, these ciliates may become true parasites when they occur in large numbers (Fig. 3.7, see colour plate section), often as a consequence of increased organic burden, bacteria or protist numbers in the seawater, and/or reduced host resistance and immune responses. Some examples are species of the genus Trichodina (Figs. 3.8 and 3.9, see colour plate section), and ‘permanently’ attached ciliates such as members of the genera Apiosoma, Epistylis and Riboscyphidia. A Uronema-like ciliate has repeatedly caused severe systemic ciliatosis (Fig. 3.7) in farmed turbot in Norway (Sterud et al., 2000), and could be the same species that has been found in farmed turbot in Spain (Dyková & Figueras, 1994). Another scuticociliatid, Philasterides dicentrarchi, has caused a similar systemic infection in farmed sea bass, Dicentrarchus labrax (Dragesco et al., 1995). Reduced growth in farmed turbot due to Trichodina sp. has been documented (Sanmartin Durán et al., 1991), while heavy infections with T. hippoglossi have been observed in farmed halibut larvae (Nilsen, 1995). Prophylaxis/Treatment Prophylactic measures include reduced host stress and reduced stocking density. A formalin bath (usually 1 : 4000) for half-an-hour is commonly used to treat ectoparasitic ciliatosis. Treatment with freshwater, reducing the salinity to 10 p.p.t. or lower, for 30–60 min may be effective. Oral drug administration has also been shown to be effective (Rapp, 1995).

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Culture of cold-water marine ﬁsh

3.4.1.5 Flagellates Diagnosis/Identiﬁcation Parasitic ﬂagellated protists belong to several distinct phyla. They all have one or several ﬂagella used for locomotion and host attachment. Multiplication is usually by binary ﬁssion. Many parasitic species have morphologically different free-living stages for transmission between hosts. The cell, excluding ﬂagella, in most species is between 10 and 40 mm. Identiﬁcation is based on the number and location of ﬂagella, the shape of the cell, the shape and size of nuclei, and other ultrastructural features. Symptoms/Pathology The most serious ﬂagellated protists in farmed ﬁsh are ectoparasites, which often cause skin and gill epithelial hypertrophy/hyperplasia and necrosis. Species of the genus Ichthyobodo have a special disc for attaching to the host cell, while species of the genus Cryptobia use ﬂagella for attachment. I. necator is a well-known pathogen from freshwater and marine cultures of salmonids. Similar but morphologically different species have been found on 25 marine ﬁsh species (Urawa et al., 1998). The genus Cryptobia includes both ectoparasitic (skin and gills) and endoparasitic (blood and gut) species. It is suggested that the endoparasitic ones should be included in the genus Trypanoplasma. The ectoparasitic Cryptobia have a direct transmission, while the endoparasitic ones need a leech. The blood ﬂagellate C. bullocki has caused pathology and disease in wild ﬂatﬁshes in Chesapeake Bay, USA (see Woo & Poynton, 1995). Dinoﬂagellates such as Amyloodinium spp. and Piscioodinium spp. often cause suffocation due to severe damage of the gill epithelium. Prophylaxis/Treatment A formalin bath (1 : 4000) for about 30 min has been the most commonly used treatment against ectoparasitic ﬂagellates. Sometimes it has been necessary to use a higher concentration (1 : 3000), possibly because of a high organic content in the cages.

3.4.2 Metazoans 3.4.2.1 Myxosporidia (Parasitic Cnidarians) Diagnosis/Identiﬁcation Previously myxosporidians were classiﬁed as protists, but recent scientiﬁc studies have shown that they are parasitic cnidarians (Siddall et al., 1995) and are thus metazoans (Smothers et al., 1994). Myxosporidians can be found in any ﬁsh organ. They undergo several vegetative (pre-sporogonic) stages before reaching the transmission stage, which is a multicellular spore (Figs. 3.10 and 3.11, see colour plate section). Probably most, but not all, myxosporidians alternate between two hosts, a ﬁsh and an invertebrate, in their life cycle. Most myxosporidians have spores between 10 and 20 mm in length. Identiﬁcation is mainly based on spore morphology.

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Symptoms/Pathology Many species can cause severe pathology, either in their pre-sporogonic stages or due to the spores. Spores of coelozoic species are mainly found in the gallbladder and in the urinary bladder and associated tracts, while spores of histozoic species, living in host tissue, may be found in large ‘cysts’ or spread in the skeletal muscles, resulting in an unappetising ﬁsh. Species of the genus Kudoa may elicit post-mortem myoliquefaction, and thus reduce the market value of infected ﬁsh products (Moran et al., 1999). Myxidium leei has caused severe pathology in farmed sea bream Sparus aurata (Diamant et al., 1994). Prophylaxis/Treatment Myxosporean spores are very resistant and can tolerate extended freezing. Different disinfectants can be used to kill free spores, while treatment by medicated pellets may reduce the infection. 3.4.2.2 Monogeneans Diagnosis/Identiﬁcation Monogeneans are mainly ectoparasites on ﬁsh ﬁns, skin and gills (Figs. 3.12 and 3.13, see colour plate section). They are attached to the host by a specialised attachment organ called an opisthaptor, which includes hold-fast structures such as hooks and/or clamps. The life cycle is direct, involving only one host. With the exception of the viviparous Gyrodactylids, monogeneans lay eggs which hatch into a swimming, infective oncomiracidium. This larvae attaches directly to, or moves to, the ﬁnal site where it feeds. Most monogeneans are between 0.5 and 20 mm in length. Identiﬁcation is mainly based on the shape and size of sclerites in the opisthaptor and structures associated with the male sex organ, which is present in all adult specimens as monogeneans are hermaphrodites. Symptoms/Pathology Because of their direct life cycle, many monogeneans are troublesome parasites in ﬁsh mariculture. This is especially true of the viviparous species of the genus Gyrodactylus. Different species of this genus have caused severe pathology and mortality in many Norwegian marine ﬁsh farms, including Atlantic and spotted wolf-ﬁsh, cod, halibut and plaice (personal observations, 1990–2000). A Microcotyle sp. has caused pathology and mortality in farmed sea bream (Sanz, 1992), and Diplectanum aequans has caused gill pathology in farmed sea bass (Cognetti Varriale et al., 1992). The relatively large Entobdella hippoglossi is easily observed macroscopically, and may cause skin irritation and ulcers in halibut. Prophylaxis/Treatment Reduced host density and increased water ﬂow may reduce infection abundance, especially for egg-laying species. A large number of drugs have been shown to be effective, although

46

Culture of cold-water marine ﬁsh

rarely completely (Santamarina et al., 1991; Cognetti Varriale et al., 1992; Schmahl, 1993). Most commonly used has been a formaldehyde bath (in 1 : 4000) for about 30 min. Freshwater may also be effective against marine monogeneans. Large monogeneans, such as E. hippoglossi, can effectively removed from the skin of a resting host by a pair of forceps. 3.4.2.3 Cestodes Diagnosis/Identiﬁcation Cestodes are endoparasites. They are long, ﬂattened and usually whitish worms with complex life cycles, using ﬁsh as ﬁnal or intermediate hosts. In the former case, the segmented adult worms occur in the digestive tract and may reach several decimetres in length (Fig. 3.14, see colour plate section). Larval cestodes, usually non-segmented, may be free-living or encapsulated in skeletal muscles, internal organs or the abdominal cavity. Cestodes found in ﬁsh usually use crustaceans as their ﬁrst intermediate host. Cestodes lack an alimentary canal and food is absorbed through the outer surface. Identiﬁcation is mainly based on the shape of the attachment organ at the anterior end, the scolex, and the shape and arrangement of the sex organs in the segments, the proglottids. Symptoms/Pathology Most cestodes using ﬁsh as ﬁnal hosts are not regarded as pathogens. However, when they occur in large numbers in the digestive tract, they can signiﬁcantly reduce the growth of the ﬁsh. Larval cestodes in viscera may cause severe pathology. Organs may be destroyed and/or important metabolic processes may be altered or reduced. Fish mortality due to larval cestodes is usually associated with intensity of infection. The occurrence of cestode larvae may cause extensive visceral adhesions. Prophylaxis/Treatment Oral treatment by adding chemicals to dry pellets is commonly used against cestodes in the digestive tract. Treatment against larval cestodes in ﬁsh tissue is generally not effective. 3.4.2.4 Trematodes Diagnosis/Identiﬁcation Trematodes, also known as ﬂukes, are endoparasites using ﬁsh as their ﬁnal or intermediate host (Fig. 3.15, see colour plate section). Adult trematodes usually have two suckers, one anteriorly and one at the mid-body. The anterior one is associated with the mouth opening. Blood ﬂukes usually lack suckers. Larvae are typically encapsulated in subepidermal tissue in gills, skin or ﬁns. Most ﬂukes are hermaphrodites, having both male and female sex organs. Trematodes have complex life cycles including two, but usually more, hosts. Most commonly ﬂukes use molluscs, especially snails, as their ﬁrst intermediate host, but other

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invertebrates such as oligochaetes may also be involved. Most ﬁsh trematodes are between 2 and 20 mm in length. Identiﬁcation is based on several structures such as the shape and position of suckers, external appendages, and the shape and size of sex organs and eggs. Symptoms/Pathology Adult trematodes mainly occur in the digestive tract and are generally not very harmful to the host. The exception is blood ﬂukes. Serious disease and mass mortality due to blood ﬂukes of the genus Paradeontacylix has been observed in the 0+ age group in farmed species of amberjack (Seriola spp.) (Ogawa & Egusa, 1986; Crespo et al., 1992). A related species, Aporocotyle simplex, is commonly found in the vascular system of several pleuronectid ﬂatﬁsh in Europe. The occurrence of large numbers of encapsulated trematode larvae, such as Cryptocotyle lingua, in the skin of farmed ﬁsh (Figs. 3.16 and 3.17, see colour plate section) may reduce ﬁsh growth, while the associated host response, including melano-macrophages which result in black spots, may reduce the market value of the ﬁsh. Prophylaxis/Treatment For prophylaxis, farms could be moved to locations with fewer ﬁrst intermediate hosts in the environment. Treatment against adult ﬂukes in the digestive tract is not usually carried out. Effective treatment against blood ﬂukes or larvae is difﬁcult. 3.4.2.5 Nematodes Diagnosis/Identiﬁcation Nematodes are endoparasites found in any host organ. They are thread-like and tapered at both ends, and covered by a rigid cuticle. Parasitic nematodes have complex life cycles involving several hosts. Fish can act as ﬁnal or intermediate hosts. The ﬁrst intermediate host is usually an invertebrate, primarily a crustacean. Most adult nematodes live in the ﬁsh intestine, while larval stages are mainly found in the ﬂesh and viscera. Identiﬁcation is based on external structures, mouth-associated structures, and internal organs such as the digestive tract. Symptoms/Pathology Adult nematodes in the intestine are not considered to be important pathogens, but larvae in the ﬂesh and viscera may cause disease and economic problems. To date, nematodes have rarely been found in marine ﬁsh reared in ﬂoating cages. In northern European mariculture, Hysterothylacium aduncum is occasionally found in intestine or viscera; this parasite uses ﬁsh as both a ﬁnal and a second intermediate host. Recently, Hysterothylacium sp. larvae were commonly found in small halibut just after their ﬁrst feeding on natural zooplankton ﬁltered from seawater. The relatively large larvae were coiled 2–3 turns in the abdominal cavity. Very few ﬁsh had more than one larva, which could indicate that two or more specimens are lethal to small halibut.

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Culture of cold-water marine ﬁsh

Prophylaxis/Treatment Prophylaxis could include measures to prevent potential intermediate hosts entering the ﬁsh tanks or cages. Chemical treatment against larval nematodes is usually very difﬁcult. If necessary, some orally administrated anthelmintics are effective against adult nematodes in the intestine. 3.4.2.6 Acanthocephalans Diagnosis/Identiﬁcation Acanthocephalans are endoparasites using ﬁsh as their ﬁnal or intermediate host. Adult worms are found in the intestine, while juvenile worms occur in the viscera, especially the mesentery and the liver when the ﬁsh is used as an intermediate host. Fish acanthocephalans use crustaceans as an intermediate host. Acanthocephalans lack an alimentary canal, and food is absorbed through the outer surface. They have an invaginable proboscis with hooks used for attachment to the host intestine (Fig. 3.18, see colour plate section). The size of the adult worm varies from a few millimetres to several centimetres, but most species are about 10 mm. Identiﬁcation is based on body shape, and on the hooks and spines of the proboscis, among other things. Symptoms/Pathology Acanthocephalans are generally not considered to be important ﬁsh pathogens. Fish can be heavily infected without showing signs of clinical disease, and it is not unusual to ﬁnd acanthocephalans that have penetrated the intestinal wall and protrude into the abdominal cavity of healthy ﬁsh. However, such infections probably cause reduced growth. A negative correlation between the number of Echinorhynchus gadi present and energy stores in cod, Gadus morhua, has been demonstrated (Buchmann, 1986). Prophylaxis/Treatment Acanthocephalans are good colonisers, and the prevention of exposure is the most effective method of limiting infections. Orally administered drugs mixed in food pellets have been shown to be effective (Taraschewski et al., 1990). 3.4.2.7 Leeches Diagnosis/Identiﬁcation Leeches are ectoparasites mainly found on the body and gills, and in the oral cavity. They are thread-like and usually have two disk-shaped suckers, one in the anterior and one in the posterior end. Many leeches are obligate parasites, but after a blood meal they may leave the host for a long period to digest the meal and to deposit cocoons containing eggs. Identiﬁcation is based on many different structures such as pigmentation, spots, setaes in cephalic segments, protrusible proboscis, and a pharynx with or without jaws.

Microbial interactions, prophylaxis and diseases

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Symptoms/Pathology Leeches alone are generally not considered to be important ﬁsh pathogens. The effects are usually localised, and restricted to attachment and/or feeding sites. However, leeches are important vectors for many blood-living (haematozoic) protists such as ﬂagellates and apicomplexans, but also some bacteria and viruses. Johanssonia arctica is important as a vector for a variety of haematozoa in northern seas. It transmits Trypanosoma murmarensis to a number of commercially important hosts, including Atlantic cod and American plaice, Hippoglossoides platessoides, and it also transmits intra-erythrocyte parasites of the genus Haemohormidium (Burreson, 1995). Prophylaxis/Treatment To date, there are only a few reports of leeches found on ﬁsh in mariculture, and little has been written about prevention and treatment. However, several bath treatments have been used effectively against freshwater leeches, mainly Piscicola geometra, and similar treatments could possibly be effective against marine leeches. 3.4.2.8 Crustaceans Diagnosis/Identiﬁcation Parasitic crustaceans are ectoparasites mainly found on the body and gills, and in the oral cavity. They have an exoskeleton, and are characteristically segmented with several appendages used for attachment to the host. Most parasitic crustaceans are similar in appearance to their free-living relatives, but some are so modiﬁed that only experts are able to identify them as crustaceans. The life cycle is mostly direct, involving only one host. It usually includes several stages, both free-living and parasitic, with a moult between each stage. Parasitic stages are anchored or can move freely on the host surface. The size of adult parasitic crustaceans ranges mostly between 1 and 30 mm. Larval stages can be less than 1 mm and difﬁcult to observe macroscopically. Identiﬁcation is mainly based on the shape and size of segments and appendages. Symptoms/Pathology Because of their direct life cycle, many parasitic crustaceans are troublesome parasites in ﬁsh mariculture. This especially refers to parasitic copepods moving freely on the host surface, such as the caligid genera Lepeoptheirus and Caligus (Figs. 3.19 and 3.20, see colour plate section), but also to many other species in different crustacean groups. When they are relatively numerous, parasitic crustaceans may cause the death of the host if untreated, but even a relatively low number of adult parasites may signiﬁcantly reduce the growth of the host. Anchored copepods may be a potential problem in mariculture. Adult females of Lernaeocera branchialis, anchored to a gill arch of cod and other gadids, have experimentally caused host disease and mortality, primarily as the result of anorexia, stress and blood loss (Khan, 1988). Moreover, it has been reported that wild ﬁsh infected by L. branchialis are

50

Culture of cold-water marine ﬁsh

20–30% underweight (Mann, 1952). Isopods of the genus Ceratothoa have caused reduced growth and mortality in farmed sea bass and sea bream (Sarusic, 1999). Prophylaxis/Treatment Prophylactic measures could include the production of 1-year-old ﬁsh only, and allowing the site to lie fallow for a period after slaughtering. In salmonid mariculture, cleaner-ﬁsh (mostly wrasse species) have been used successfully as a prophylactic measure against L. salmonis. Several chemical compounds are effective against parasitic crustaceans. Treatments are given orally in feed or as bath treatments. New compounds have been introduced almost yearly in the last decade.

3.5 A Strategy for Microbial Control Microbial problems seem to be qualitatively different for larval and on-growing stages, and these differences are discussed below. For most species, the production of juveniles is a major bottleneck. The main symptoms are poor reproducibility in terms of survival, growth and quality, and the problem seems to be the same for temperate and warm-water species of both ﬁsh and crustaceans. The symptoms indicate a lack of control of at least one factor. Nutritional factors and egg quality may be ruled out as the principal cause, because the lack of reproducibility is also manifested in replicate tanks with full sibling groups that are given the same treatment. This does not mean that nutritional factors and egg quality are optimal (cf. Chapters 4, 5 and 7). Recent scientiﬁc data and accumulating experience in commercial hatcheries indicate that the bacteria normally selected for in hatcheries may be the principle cause of problems associated with the production of juveniles (Vadstein et al., 1993; Bergh et al., 1992; Skjermo & Vadstein, 1999; Bergh, 2000). This is most probably due to opportunistic bacteria, because the reported incidences of speciﬁc pathogens are low (Munro et al., 1995), even though some pathogens may be overlooked as they have not yet been described. Also for on-growing stages, microbial problems are occasionally very severe. However, these have different symptoms, and traditional disease outbreaks are the normal phenomenon. Whether or not opportunistic bacteria or speciﬁc pathogens cause problems will have strong implications for the choice of strategy for microbial control. This is because of the different ways of controlling the import of microbes to cultivation systems, and how the immune system of the ﬁsh can be used prophylactically. Another important difference between larval and on-growing ﬁsh is that for many species the on-growing stages takes place in open or semi-open systems, whereas larval stages are kept under controlled conditions in tanks indoors. Obviously, the openness of the system has strong implications for the possibilities for microbial control. We have previously (Vadstein et al., 1993) proposed a strategy for microbial control that consists of three different elements (Fig. 3.21). Two of the elements involve environmental factors, whereas the third considers the ﬁsh itself. Different methods have been proposed for

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51

Improvement of larval resistance

Figure 3.21 Outline of the three elements in the strategy to obtain microbial control in the rearing of marine ﬁsh (redrawn from Vadstein et al., 1993). © Swets & Zeitlinger. Used with permission.

Non-selective reduction of bacteria

Selective enhancement of bacteria

the different elements of the strategy (cf. below). Before presenting the strategy in detail (Section 3.5.2.), together with examples of the use of the various methods (Sections 3.6. and 3.7), we present some important general considerations. However, the ﬁrst line of defence with animal diseases should always be legislative control, thus ensuring that pathogens are not introduced into new areas and environments. It must be emphasised that many outbreaks of disease are the unfortunate result of such introductions. Among the most well-known examples is the introduction of furunculosis to Norway by the import of smolts infected with Aeromonas salmonicida subsp. salmonicida from Scotland, leading to an epizootic event with large economic consequences. Most probably, the disease was originally imported to Europe from North America. Another example is the probable introduction to European crayﬁsh of the so-called crayﬁsh plague. The causative agent is a fungus which is not pathogenic to American crayﬁsh, but is lethal to European crayﬁsh. Hygiene routines are often underestimated as a method for avoiding microbial problems. There are numerous examples of aquaculture facilities that have signiﬁcantly improved their stability of production by simply implementing good hygiene practice. The advantage is that this does not require R&D or large investments. Moreover, the implementation of good hygiene routines will increase the awareness of microbe–ﬁsh interactions, and may improve the state of awareness regarding an emerging microbial problem. General hygiene rules are presented in Table 3.1. There is no further discussion about either legislative control or hygiene routines in relation to animal diseases in this chapter.

3.5.1 General Considerations As mentioned above, whether or not opportunistic bacteria or speciﬁc pathogens cause microbial problems, and also the degree of openness of the culture system, have strong implications for the choice of strategy for microbial control. Generally, microbes can be transferred to, or interact with, ﬁsh in a number of ways, which are illustrated for larvae in Fig. 3.22. These sources can be divided into two main categories, external and internal, which strongly affect each other. To what extent external sources inﬂuence the microbial conditions in the rearing environment depends on the openness of the system. For a cage system, the

52

Culture of cold-water marine ﬁsh

Table 3.1

General hygienic rules for aquaculture facilities.

General hygiene and disinfection The facility should be well organised with good cleaning routines and ﬁxed storage places for all equipment Hygienic zones should be established in an intelligent manner. Transport of people and equipment should be avoided All equipment should be washed, disinfected and dried after use. Disinfection cannot compensate for normal cleaning Infection hygiene Establish the possible sources of pathogens (biological materials, water, equipment, feed, personnel, visitors, including dogs, birds, etc.) Establish where pathogens could proliferate inside the facility Evaluate and establish possible counter-measures Establish and implement regular control routines. This will promote early detection Establish routines for documentation of hygienic practice Minimise stress and handling

External Internal live feed

water

microalgae

Figure 3.22 Important bacterial sources interacting with mucosal surfaces of larval ﬁsh. Modiﬁed from Salvesen (1999).

external factors are important because of the open contact with the sea, and the large input of food which has its own microﬂora. For indoors, ﬁrst-feeding tank systems, microbes from the live feed cultures are the main external input. Traditionally, there has been considerable attention to microbes in the intake water, where water treatment to reduce the density of bacteria is a more or less standard procedure. On the other hand, control measures directed towards microbes associated with the live feed have mostly been neglected. This is in spite of the fact that detrimental effects due to bacteria associated with the live feed are well known (Benavente & Gatesoupe, 1988). There are also strong interactions among the internal sources. Whereas the outer surface of the ﬁsh is colonised by bacteria in the water, the intestine is generally affected by bacteria entering the intestine by active uptake from the water, and by bacteria associated with the feed. The number of bacteria associated with dry feed pellets is fairly low, but the number may exceed 1010 cells per gram live feed (Skjermo & Vadstein, 1993). Live feed represents the heaviest bacterial load to the larvae, except for the ﬁrst days of the larval stage when active uptake directly from the water is signiﬁcant (Reitan et al., 1998). For on-growing

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53

stages, the density of bacteria in the feed will determine what is the most signiﬁcant source. The processing and storage methods may cause considerable variation in bacterial content, and soft feed is likely to be more vulnerable to bacterial growth than dry pellets. Even though the main sources of microbes may be identiﬁed, the various internal sources in a rearing environment will still interact with each other (see Fig. 3.22). In particular, defecation processes may be a signiﬁcant source of bacteria in the water. This is from ﬁsh, and also from live feed in the case of larval rearing. It is also well established that the bacterial ﬂora of live feed changes after their transfer to the tanks. This includes both a reduction in numbers and a change in the composition of the bacterial ﬂora (Øie et al., 1994; Olsen et al., 2000). The direct input of bacteria into a system is not the only manner in which the density of bacteria in a rearing system is affected. The direct or indirect input of organic matter is also decisive, since it serves as a substrate for bacterial growth. Bacterial growth in ﬁrst-feeding tanks may be fairly high, with production in the range of 0.5–2 divisions per day. The above discussion has mainly considered quantitative aspects, but the species of microbes that are present and dominant are more important than the actual number. However, numbers and composition cannot be discussed as separate issues, as the total bacterial community (numbers) is the sum of the individual populations (composition). It is obvious that the composition of the externally introduced microﬂora will inﬂuence the composition of the microﬂora of the rearing system. This inﬂuence will be stronger the more open the rearing system is, and open systems therefore have less possibility of effective management. Generally, the microbial management of open systems is connected to all aspects related to localisation, and to disease management strategies for early detection. Early detection may prevent a problem reaching epizootic proportions, and the initiation of countermeasures at an early stage is of vital importance. In more closed rearing systems, the impact of external sources of bacteria is still signiﬁcant, but they are more manageable because water sources and live feed can be treated before they enter the ﬁsh tanks. In more closed rearing systems, the internal sources are increasingly important. The internal sources may affect the bacterial composition in both a positive and a negative way. The presence of haemolytic bacteria in larvae may seed the water with these bacteria (Skjermo & Vadstein, 1999), which affect the water ﬂora negatively. On the other hand, the ingestion of algae by live feed may change their ﬂora to a more diverse one, which may have a positive impact on larval viability in the next stage (Olsen et al., 2000). To understand such interactions may be decisive in the design of a microbial management strategy. Our comprehension of interactions between bacteria at the species level and ﬁsh is inadequate, except perhaps for a few ﬁsh pathogens. However, this research ﬁeld is developing rapidly, and several reviews have recently been published (Ringø & Birkbeck, 1999; Gatesoupe, 1999; Hansen & Olafsen, 1999). Because of our limited knowledge of normal ﬂora and normal ﬁsh–microbe interactions, it may be adequate to classify bacteria based on a general ecological scheme. A differentiation between opportunistic and non-opportunistic species may be appropriate, as may also be the division between parasitic and mutualistic species (cf. Fig. 3.1). The former division has been applied to aquaculture with some success (cf. Section 3.6.3), and is theoretically formalised as the r/K-concept (cf. Andrews & Harris,

54

Culture of cold-water marine ﬁsh

Table 3.2 Some key characteristics of organisms with r- and K-strategies, and pioneer and mature (climax) communities. Characteristics

Maximum growth rate Biomass at carrying capacity Effect of enrichment Competitive ability at a low supply of substrate per individual Mortality Afﬁnity to substrate

r-strategist

K-strategist

High Unstable Rapid growth

Low Stable Slow growth

Poor Density-dependent, often catastrophic Low

Good Density-independent High

Characteristics

Biological control Stability to perturbations Diversity (species, biochemical) Niche width Specialisation

Pioneer community

Mature community

Low Poor Low Wide Low

High Good High (?) Narrow High

1986). The r/K-concept does not, of course, entail an either/or. r and K represents the two extremes in a continuum: r-selection occurs in an uncrowded environment with a large supply of substrate per capita (Table 3.2), and r-strategists are considered to be opportunistic species; K-selection, on the other hand, occurs in crowded environments with a low supply of nutrients per cell. Several characteristic properties of r- and K-selected species are given in Table 3.2, and these properties can be used both as a selective force and for diagnostic purposes, e.g. percentage opportunistic species (Salvesen & Vadstein, 2000). Pathogens are often characterised as r-strategists (Andrews, 1984), but the group probably contains a large number of non-pathogenic, opportunistic species that may cause some of the problems experienced in the rearing of marine larvae. At the community level, r- and K-selected species dominate in pioneer (developmental) and mature (climax) communities, respectively (Odum, 1971). These two types of community also have distinctly different characteristics (Table 3.2). Pioneer communities will generally be systems with low stability against perturbations and with low biological control. On the other hand, matured water inhabited by K-strategists will be stable systems, with high biological control and high resistance to perturbations below a speciﬁc level. The current intensive production methods used for marine ﬁsh tend to increase the carrying capacity of the system and to select for opportunistic microbes. This tendency is stronger the more intensive and closed the system is. The reasons for such an unfavourable development are a high load of organic matter, large oscillations in this load, and direct perturbations of the bacterial community. Critical factors that sustain such a development are decimation of bacteria in in-ﬂowing water without controlled recolonisation of the microbial

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55

community, high loads of bacteria and organic matter together with feed, and an internal load of organic matter as faeces from live feed and larvae, or as dead larvae. Intensive larval rearing systems may serve as an example of a system with major negative perturbations, whereas open cages with good water circulation are an example of the opposite.

3.5.2 A Strategy for Microbial Control and Important Elements in such a Strategy The multiple aspects discussed above, which need to be considered when developing a strategy to obtain microbial control in the cultivation of marine ﬁsh, emphasise the necessity of not focusing on one counter-action only. Solving a multidimensional problem requires a multidimensional strategy. In the case of microbial control, this is even more important because control cannot be regarded as absolute, but is a matter of probability. Thus, by increasing the number of counter-actions, an increased probability of obtaining and maintaining microbial control is achieved. The proposed strategy for microbial control consists of three different elements (see Fig. 3.21). Two of the elements involve environmental factors, whereas the third relates to the ﬁsh itself. Both quantitative and qualitative aspects of the bacterial ﬂora are included. These two aspects are strongly dependent on each other, and it is not obvious which of these affect the ﬁsh larvae. Several different methods have been proposed for different elements of the strategy (Table 3.3), and there are examples in the literature on the use of some of them (Sections 3.6 and 3.7). Non-Selective Reduction of Bacteria The methods used for non-selective control can be put into two groups. Some methods are aimed at a non-permanent or non-stable reduction (disinfection and grazer control), whereas

Table 3.3 Examples of methods that can be used for the different elements in the microbial management strategy suggested. Modiﬁed from Vadstein et al. (1993). © Swets & Zeitlinger. Used with permission. Non-selective reduction of microbes Surface disinfection of eggs Reduction in input of organic matter Removal of organic matter Grazer control of bacterial biomass Selective enhancement of microbes Selection for desirable bacteria Addition of selected bacteria to tanks Incorporation of selected bacteria in feed Improvement of resistance against microbes Stimulation of general immune system Stimulation of speciﬁc immune system (vaccination) Modulation of general and speciﬁc maternal immunity Nutritional supplements to improve susceptibility to microbes and wound healing

56

Culture of cold-water marine ﬁsh

others are aimed at a reduction in the carrying capacity of the system (reduction in input and removal of organic matter). Selective Enhancement of Bacteria The application of probiotic bacteria in livestock production and for humans has aroused considerable interest (Vanbelle et al., 1990), and to some extent this is also true in aquaculture (Gatesoupe, 1999). Probiotics can be deﬁned as micro-organisms that are able to colonise the digestive tract, to maintain or increase the natural gut ﬂora to prevent colonisation by pathogenic organisms, and to secure optimal utility of the feed (Vanbelle et al., 1990). Their use can improve the health and productivity of farmed animals. Probiotics can be administrated both by direct addition of the selected organisms to the tanks, or by incorporation of the organisms into live or formulated feed. Regulation of the composition of the bacteria by selective measures differs from the probiotica concept by the fact that no organisms are added to the system. Instead, a physical, chemical or biological factor is used as a selective force. Thus, the selection is based on the statement of the pioneering microbiologist Martinus W. Beijerinck that ‘everything is everywhere, the environment selects’. It must be emphasised that this statement should not be interpreted in such a way that attempts to hinder the spread of pathogenic agents are neglected. Biogeographical differences with respect to the occurrence of pathogens are well documented. Improvement of Resistance Against Bacteria The natural ability of animals to resist potentially harmful bacteria, and the possibility of stimulating this ability, are used extensively in both human medicine and animal husbandry. Generally, such approaches are called immunotherapy, and include methods that utilise immunological principles to prevent or treat diseases. The best-known method is vaccination, which is the stimulation of the speciﬁc immune system, and has also been applied in aquaculture with some success (Ellis, 1988b; Anderson, 1992). In addition to speciﬁc stimulation targeted at a speciﬁc pathogen (i.e. vaccination), it is also possible to stimulate the non-speciﬁc immune system (Vadstein, 1997). An example of non-speciﬁc immunostimulation is macrophage activation. Fish larvae do not have the ability to develop speciﬁc immunity, as their immune system is not mature. For speciﬁc immunity they rely on immunoglobulins from the mother (maternal immunity). Therefore, one possibility is to manipulate speciﬁc antibody composition and levels in eggs and larvae through immunisation of the mother. The nutrition of ﬁsh has an indirect impact on their resistance against pathogens, as several nutritional factors inﬂuence the immune system. These include fatty acids, minerals and vitamins (Landolt, 1989; Blazer, 1992). Resistance to pathogens also has a genetic component, with signiﬁcant differences between families and strong heredity of such differences (Gjedrem et al., 1991; Fjalestad et al., 1995). Examples of the use of various methods included in Table 3.3 are presented in Sections 3.6 and 3.7.

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3.6 Improving Environmental Conditions The physical, chemical and biological environments signiﬁcantly affect the health status, and hence the viability, of reared ﬁsh. We treat only biological environmental conditions in this chapter, and give examples of methods used for microbial management (see Chapter 2).

3.6.1 Non-Selective Reduction of Microbes Bacteria colonise the surface of ﬁsh eggs, and these microbes may serve as a reservoir for the horizontal transfer of pathogens from brood stock facilities to hatcheries. A safe and effective method to disinfect the surface of ﬁsh eggs is therefore a prerequisite for the establishment of a hygienic barrier between brood stock and ﬁrst feeding. Salvesen & Vadstein (1995) tested the potential of four different chemicals for use as surface disinfectants of marine ﬁsh eggs. These were two chlorine-releasing compounds (sodium hypochlorite and chloramine-T), an iodophore (buffodine) and an aldehyde (glutaraldehyde). The experiments included eggs of place, cod and Atlantic halibut, and revealed that glutaraldehyde was the most promising of the four chemicals. When eggs were treated for 5–10 min at a concentration of 400–800 mg l-1, a high bactericidal effect was observed without adverse effects to eggs or yolk-sac larvae. Survival at the egg stage and the viability of yolk-sac larvae was improved. The other three chemicals caused adverse effects on eggs and larvae at the concentrations where a satisfactory bactericidal effect was obtained. A further evaluation of glutaraldehyde (Harboe et al., 1994; Salvesen et al., 1997) conﬁrmed these results and revealed several positive effects in addition to providing a hygienic barrier. These positive effects included increased hatchability (Fig. 3.23), more synchronised hatching, increased stress-tolerance and survival of larvae, and improved growth during ﬁrst feeding. Surface disinfection of eggs with glutaraldehyde has been implemented in many marine ﬁsh facilities. It should be noted that the activity of glutaraldehyde is very temperaturesensitive (see Salvesen & Vadstein, 1995), and the procedures applied for cold-water ﬁsh cannot be used directly for other temperature regimes. The mechanisms of the differences

100

Halibut Turbot Cod Plaice

% hatch

80

Figure 3.23 Hatchability after disinfection with glutaraldehyde in seven egg batches of different marine ﬁsh species (redrawn from Salvesen, 1999).

60 40 20 0

Not disinfected

Disinfected

58

Culture of cold-water marine ﬁsh

observed between disinfected and non-disinfected eggs are not clear. Both the easier exchange of gases and matter between the egg and the environment, and the reduced probabilities of over-colonisation by microbes or unfavourable colonisation of the larvae are possible explanations. Recent investigations concluded that aldehydes have little effect against ﬁsh pathogenic viruses (Arimoto et al., 1996). Therefore, Grotmol & Totland (2000) studied the effects of ozonation of halibut eggs, and concluded that this procedure efﬁciently hindered the transfer of nodavirus to the larvae both in cases of naturally infected broodstock and in experimentally infected eggs. Because nodaviruses are the main disease problem in Norwegian halibut aquaculture, ozonation has replaced glutaraldehyde disinfection in some facilities. There are not many examples of other non-selective reduction methods in the literature. Maeda & Nogami (1989) demonstrated the signiﬁcant role of grazers in controlling the density of bacteria during cultivation of crab larvae (Portunus tridentatus). No other studies treating this subject are known. Both Brachionus plicatilis (Rotifera) and Artemia franciscana (Branchiopoda) are able to graze bacteria to some extent (Vadstein et al., 1993; Makridis & Vadstein, 1999), and could therefore affect the biomass of bacteria in rearing tanks. With densities of ﬁve and three individuals per millilitre of B. plicatilis and A. franciscana, respectively, these organisms may clear between 5% and 140% of the tank volume of bacteria per day. Thus, we may conclude that whereas B. plicatilis does not seem to have a great impact on bacterial density, A. franciscana may do so.

3.6.2 The Use of Probiotics Gatesoupe (1999) has recently reviewed the use of probiotics in aquaculture. To date limited information exists on this topic, but existing data and experience are promising, particularly when one considers that the ﬁrst work was done only 15 years ago (Kozasa, 1986). Knowledge of terrestrial organisms indicates that the application of probiotics has to follow some basic principles (Fuller, 1989). These include the facts that the probiotic is part of the autochthonous ﬂora, has the ability to establish itself and proliferate in the intestine, and is able to resist the environmental conditions in the intestinal tract (e.g. lytic enzymes, low pH and bile salts). Frequently, this knowledge has not been fully implemented in the limited number of attempts to apply the probiotica concept in aquaculture. Generally, probiotic bacteria may be supplied either by direct addition to the water or encapsulated in feed. Encapsulation is possible in both formulated feed and live feed. Whereas direct addition to the water is possible for larval stages or other situations with tank rearing at low ﬂow-through rates (Ringø et al., 1996; Ringø & Vadstein, 1998; Makridis et al., 2000b), encapsulation in feed is the only possible method of administration in open or high-ﬂow-through systems. It has been reported that it is possible to incorporate selected bacteria in both B. plicatilis and A. franciscana, and that added bacteria persist for some time in association with the live feed after transfer to tank conditions (Makridis et al., 2000a). However, there are differences between various strains of bacteria, and therefore each different case requires validation.

Microbial interactions, prophylaxis and diseases

59

It has been shown that administered bacteria colonise the intestine for a signiﬁcant period of time (Strøm & Ringø, 1993; Olsson, 1995; Munro et al., 1995; Andlid et al., 1995; Austin et al., 1995; Ringø et al., 1996; Ringø & Vadstein, 1998; Makridis et al., 2000b). These studies include several hosts (ﬁsh and shellﬁsh) and several probiotic bacteria candidates. Moreover, these studies were conducted at both larval and juvenile stages, and by administration via water and encapsulated in feed. However, still there is limited knowledge on the stability and persistence of such manipulations of the mucosal ﬂora. A few studies have investigated the effect of probiotic bacteria in challenge tests with a pathogen. Some of these studies showed increased survival, but the effects are not always reproducible (cf. Gatesoupe, 1999). In some studies the mortality rate was not truly reduced as only a delay in mortality was observed. Also, there are few reports to date of an improvement in survival (Ringø & Vadstein, 1998) or growth (Byun et al., 1997; Gatesoupe, 1997). However, the application of probiotic bacteria in aquaculture seems promising, but considerable research is still needed for a full evaluation of the possibilities and constraints. The main obstacles are limited knowledge about their function and normal microbial interactions in the intestinal tract of marine ﬁsh, and a good strategy for the selection of candidate bacteria.

3.6.3 Selection for Desirable Bacteria The application of a rearing technology where a selective force is used to promote beneﬁcial bacteria has been reported in the literature (Vadstein et al., 1993; Skjermo et al., 1997; Salvesen et al., 1999). Such selection in relation to, for example, the r/K-concept, is possible by applying a selective force such as a low supply rate of organic matter per bacteria, and produces a community with slowly growing bacteria and a strong ability to resist high pulses of organic matter (Salvesen et al., 1999; Salvesen & Vadstein, 2000; O. Vadstein, unpublished results, 2002). These authors have termed this concept for microbial maturation of water. In an experiment with yolk-sac larvae of Atlantic halibut, the effect of applying microbially matured water or ﬁltered seawater for maintaining yolk-sac larvae at high densities was compared (20–30 larvae per litre; Vadstein et al., 1993). Both types of water were applied with or without the addition of an antibiotic (25 p.p.m. ﬁnal concentration of oxytetracycline). Whereas similar average survival rates were observed for the two treatments with matured water and the treatment with ﬁltered water with added antibiotic (range 87–90%), the average survival rate was only 45% for the treatment with ﬁltered water without antibiotic. The addition of antibiotics to ﬁltered water considerably reduced the variability between replicates, with a coefﬁcient of variation of 15 and 75% for the treatments with and without oxytetracycline, respectively. Much lower variability was observed for the two treatments with microbially matured water (CV 3 and 8%, respectively). In ﬁrst feeding experiments with turbot (Skjermo et al., 1997; Salvesen et al., 1999), microbially matured water produced a signiﬁcantly bigger larval size just 5 days after hatching (Fig. 3.24). At this stage the larvae had been eating for 2–3 days, and on average larvae in matured water were approximately 20% larger in size. The differences in size were main-

60

Culture of cold-water marine ﬁsh

µg C larva-1

35 Exp.I Exp.II Exp.III

30 25 20 15 10 F

F+A

M

M+A

Figure 3.24 Effect of microbially matured water on the size of turbot larvae on day 5 after hatching in three ﬁrst-feeding experiments. Larvae were reared in ﬁltered water (F), ﬁltered water with added microalgae (F + A), matured water (M) and matured water with added microalgae (MA). Data from Skjermo et al. (1997) and Salvesen et al. (1999).

tained until metamorphosis, and clearly demonstrate the effect of water quality on the viability of larvae during ﬁrst feeding. Improved survival in ﬁrst-feeding experiments was also observed for larvae reared in matured water, but the differences were not statistically signiﬁcant because of low statistical power. The mechanism for these effects is not understood, but several explanations are possible. The bacteria in the matured water may be beneﬁcial from a nutritional perspective by providing nutrients or digestive enzymes (Ringø & Birkbeck, 1999), or they may support the establishment of a beneﬁcial primary intestinal ﬂora that efﬁciently prevents colonisation by detrimental bacteria through competition (Tannock, 1984; Dopazo et al., 1988; Westerdahl et al., 1991; Olsson et al., 1992). Based on the theoretical background of the approach, the second hypothesis is the most likely. A large number of studies have concluded that the use of algae in larval rearing improves growth and survival, and several hypotheses have been proposed as the mechanism for these observations (cf. Reitan et al., 1997). One of the hypotheses is the selective effect that algae have on the bacterial community (Skjermo & Vadstein, 1993). It is well known from studies with algal cultures that each alga has its speciﬁc bacterial ﬂora (e.g. Bell, 1983; Salvesen et al., 2000). Moreover, ﬁsh larvae reared in tanks with algae present have a bacterial colonisation pattern that is different from the one that exists without the addition of algae (Skjermo & Vadstein, 1993; Skjermo et al., 1997; Salvesen et al., 1999). Although some of the positive effects due to the addition of algae are caused by other factors, the bacterial hypothesis is most likely a part of the ‘green water’ complex, and represents one way of selecting for beneﬁcial bacteria. The positive effect of algae on the magnitude and composition of the bacterial ﬂora has also been demonstrated by the ‘treatment’ of live feed in algal suspensions (Olsen et al., 2000). By the short-term incubation of Artemia franciscana in water to which the green algae Tetraselmis sp. had been added, bacterial numbers associated with A. franciscana were dramatically reduced and the composition of the bacterial community changed in a positive way. The change in composition was from a low-diversity community dominated by one Vibrio species and haemolytic bacteria to a higher diversity and a reduced dominance of haemolytic

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strains. These differences were also manifested in Atlantic halibut larvae during ﬁrst feeding by feeding the larvae with untreated or algal-treated A. franciscana.

3.7 Improving the Resistance of the Fish Imperfect rearing conditions easily induce stress (cf. Angelidis et al., 1987), stress inﬂuences the immune system of the animals, and stressed animals are more susceptible to intestinal infections than healthy animals (Tannock, 1984). This clearly emphasises the relevance of including resistance to infections in a microbial management strategy. This can be done by stimulation of the immune system, either directly by the use of immunostimulants, or indirectly by nutritional factors. Both speciﬁc and non-speciﬁc immunostimulation involves the use of immunostimulants. An immunostimulant may be deﬁned as an agent that stimulates the non-speciﬁc (innate) immune mechanisms when given alone, or the speciﬁc immune system when given together with an antigen (vaccination). Substances used as immunostimulants include bacteria and bacterial products, muramyl dipeptides, polysaccharides and synthetic chemicals (Vadstein, 1997).

3.7.1 Modulation of Speciﬁc Immunity—Vaccination Scientiﬁcally based vaccines have had tremendous signiﬁcance for humans since Louis Pasteur and Charles Chamberland developed the ﬁrst vaccine in 1884: an anthrax vaccine. In aquaculture, effective vaccines have been developed during the last few decades (Gudding et al., 1999), and vaccination is one of the most important prophylactic measures against disease. The salmon industry in Norway is a good example of the great impact vaccination may have. In 1987, almost 50 000 kg of antibiotics was used to produce 50 000 tons of salmon. In the same year, a vaccine against cold-water vibriosis was introduced, and later on programmes for vaccination against yersiniosis and furunculosis were implemented. The impact of these vaccination programmes was tremendous, and in 1997 only 746 kg of antibiotics was used to produce 316 000 tons of ﬁsh. Thus, whereas 1000 g antibiotics was used per ton produced in 1987, this was reduced to 2 g per ton produced in 1997. To date, relatively few pathogens that cause disease in marine ﬁsh have been discovered (cf. Section 3.5). However, this is most likely due to the fact that aquaculture of cold-water ﬁsh is still in its infancy. As the industry develops, new pathogens will be discovered. To date, it seems that the knowledge developed for salmonids may also be used to some extent for cold-water ﬁsh. However, there are clear indications that vaccines should be designed according to species, because different pathogens affect different species. The obvious positive effect of vaccination is reduced mortality, but for sustainable biological production the reduced need for medication is also signiﬁcant. One side-effect of vaccination by injection is local reactions in the peritoneal cavity (Midtlyng et al., 1996). The magnitude of such side-effects is dependent on the formulation of the vaccine. Studies of the vaccination of marine species are scarce, and little has been published. Ingilæ et al. (2000) demonstrated protection in challenge experiments with halibut and

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spotted wolf-ﬁsh vaccinated intraperitoneally with oil-emulsiﬁed vaccines against atypical Aeromonas salmonicida. Vaccination of cod against Vibrio anguillarum has been shown to increase survival in challenge experiments as well as in ﬁeld trials (B. Hjeltnes, National Veterinary Institute, personal communication, 2002), and the same has been shown for halibut (Bergh et al., 2001). Several vaccines designed speciﬁcally for marine ﬁsh species are now available. Vaccines against viral diseases are presently at the experimental stage. Húsgarð et al. (2001) showed protection in turbot in challenge experiments with nodavirus following vaccination with a recombinant vaccine. For salmonids the change from a freshwater stage to a seawater stage creates some beneﬁts for disease control. For marine ﬁsh that stay in seawater during their whole life cycle, it is therefore desirable to vaccinate at an early stage. However, the stress induced by vaccination at a young age may entail immunosuppression and increased susceptibility to pathogens, and it may also reduce performance in other areas such as growth (Lillehaug et al., 1999). The problems connected with vaccination at early stages, and the fact that the speciﬁc immune system is not fully developed at this stage, may be counteracted by the fact that egg/larvae ‘inherit’ speciﬁc immunity from the mother: maternal immunity. It has been demonstrated that it is possible to manipulate speciﬁc antibody composition and levels in eggs and larvae through immunisation of the mother. Immunisation of tilapia (Oreochromis aureus) broodstock with different proteins resulted in a considerable increase in antibody activity (Mor & Avtalion, 1990). The maximum increase was 10–13 log2 units in embryos that hatched 15–35 days after immunisation. In a second study with the same species, vaccination of broodstock with live tomites of a ciliated protozoa 1 month before hatching resulted in protection in challenge experiments of >75% (Sin et al., 1994). Atlantic salmon broodstock vaccinated against yersiniosis showed maternal transfer of speciﬁc antibodies to eggs and yolk-sac lavae, but at low levels which were insufﬁcient to protect the offspring against yersiniosis (Lillehaug et al., 1996). Too little is known to evaluate the full potential of the stimulation of maternal immunity as a method in microbial management. However, it is reasonable to believe that at least for some diseases, vaccination or the secondary stimulation of mothers with appropriate vaccines before the spawning season could protect the larvae against disease in the ﬁrst period after hatching.

3.7.2 Modulation of Non-Speciﬁc Immunity There are two situations where stimulation of the non-speciﬁc immune system is appropriate. First, for larval stages which have not developed speciﬁc immunity, but which have a functional general immune system. Second, in a situation with a general microbial problem (no speciﬁc pathogens identiﬁed) or with a high stress level. In fact, both these criteria seem to be fulﬁlled during ﬁrst feeding. A large number of substances are known to act as immunostimulants, and with variable speciﬁcity and activation mechanisms (Vadstein, 1997). Since the ﬁrst publication in 1985 (Oliver et al., 1985), considerable data have been accumulating on stimulation of the non-speciﬁc immune system of ﬁsh (Anderson 1992; Secombes, 1994; Vadstein, 1997). Moreover, non-speciﬁc stimulation has been detected over a wide range of complexity levels, including humoral, cellular and organism parameters

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Table 3.4 Type of effects reported after the stimulation of the nonspeciﬁc immune system of ﬁsh. Summarised from Vadstein (1997). Humoral effects Increased levels of lytic enzymes Increased levels of complement Production of interleukine-like molecules Cellular effects Increased phagocytosis and killing of bacteria Increased production of oxidative compounds Organism-level effects Increased survival in challenge test with bacteria Increased growth rates Counteraction of immune suppression

(Table 3.4). It is interesting to note that at the organism level, increased resistance to pathogens, improved growth and a reversal of immune suppression have been documented. Positive effects (Table 3.4) have been obtained by baths, injections and oral administration. In these studies, a large number of ﬁsh species have been investigated. The fact that nonspeciﬁc immune stimulation has been demonstrated with a variety of stimulants, administration methods, response parameters and species strongly demonstrates the robustness of the method. However, to date, most studies have not provided sufﬁcient information for industrial application. This includes most aspects of the administration regime, including concentrations needed, administration pathway, time and frequency of administration, and what type of conditions that are appropriate for treatment. However, one may conclude that stimulation of the non-speciﬁc immune system will be part of the microbial management regime aimed at reducing mortality in cold-water aquaculture.

3.7.3 The Effect of Nutrition and Genetics on Resistance Against Microbes As mentioned above, both nutritional and genetic factors have indirect inﬂuences on the immune system, wound healing and resistance to infections. In addition, some components in commercial diets have been shown to reduce the resistance to infections (Tacon, 1985). For more details on nutrition, see Chapters 7 and 9.

3.8 Closing Remarks This chapter has tried to emphasise both beneﬁcial/neutral and detrimental interactions between ﬁsh and microbes/infectious agents, and that the prevention of problems through good routines and appropriate and sustainable countermeasures is the best strategy for building an industry. Man has a tendency to search for ‘the solution’, but with such a complex and diverse problem, no ‘magic solution’ exists. The problem organisms (cf. Sections 3.2–3.4) have a high diversity of infection strategies, the vulnerability of ﬁsh species and

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developmental stage varies, and open and closed cultivation systems need different control strategies. In fact, this complex problem requires a diversity of countermeasures. Therefore, we believe that to minimise microbial problems, a general strategy for microbial management is required (see Section 3.5). Such a strategy should involve two steps. First, to analyse the expected or realised problem. Second, to decide on at least two or three countermeasures that could effectively reduce the probability of the problem developing, or prevent the expansion of the problem (see Table 3.3). Once again, the focus should always be on preventive efforts. Aquaculture of cold-water marine species is both a fairly new scientiﬁc discipline and an industry in its infancy. As a result, there are large gaps in the knowledge required for establishing an economically sound and sustainable industry. Disease problems have been identiﬁed as a major bottleneck, in particular in the production of juveniles. However, the various microbial problems of different species and developmental stages have not yet been fully identiﬁed, and only a limited number of organisms causing disease in cold-water species have been described. As a consequence, the topics dealt with in this chapter are part of an area that is developing very quickly, and which requires considerable future research efforts.

3.9 References Abela, M., Brinch-Iversen, J., Tanti, J. & Le Breton, A. (1996) Occurrence of a new histozoic microsporidian (Protozoa, Microspora) in cultured gilt head sea bream Sparus aurata L. Bull. Eur. Assoc. Fish Pathol., 16, 196–9. Anderson, D.P. (1992) Immunostimulants, adjuvants, and vaccine carriers in ﬁsh: applications to aquaculture. Annu. Rev. Fish Dis., 2, 281–307. Andlid, T., Juarez, R.V. & Gustafsson, L. (1995) Yeast colonizing the intestine of rainbow-trout (Salmo gairdneri) and turbot (Scophthalmus maximus). Microb. Ecol., 30, 321–34. Andrews, J.H. (1984) Relevance of r- and K-theory to the ecology of plant pathogens. In: Current Perspectives in Microbial Ecology (eds M.J. Klug & C.A. Reddy), pp. 1–7. American Society for Microbiology, Washington. Andrews, J.H. & Harris, R.F. (1986) r- and K-selection in microbial ecology. Adv. Microb. Ecol., 9, 99–147. Angelidis, P., Baudin-Laurencin, F. & Youinou, P. (1987) Stress in rainbow trout, Salmo gairdneri: effects upon phagocyte chemoluminescence, circulating leucocytes and susceptibility to Aeromonas salmonicida. J. Fish Biol., 31 (Supplement A), 113–22. Arimoto, M., Sato, J., Maruyama, K., Mimura, G. & Furusawa, I. (1996) Effect of chemical and physical treatments on the inactivation of striped jack nervous necrosis virus (SJNNV). Aquaculture, 143, 15–22. Austin, B., Stuckey, L.F., Robertson, P.A.W., Effendi, I. & Grifﬁth, D.R.W. (1995) A probiotic strain of Vibrio alginolyticus effective in reducing diseases caused by Aeromonas salmonicida, Vibrio anguillarum and Vibrio ordalii. J. Fish Dis., 18, 93–6. Bell, W.H. (1983) Bacterial utilization of algal extracellular products. 3. The speciﬁcity of algal– bacterial interactions. Limnol. Oceanogr., 28, 1131–43. Benavente, G.P. & Gatesoupe, F.J. (1988) Bacteria associated with cultured rotifers and Artemia are detrimental to larval turbot, Scophthalmus maximus L. Aquacult. Eng., 7, 289–93.

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Bergh, Ø. (1995) Bacteria associated with early life stages of halibut, Hippoglossus hippoglossus L., inhibit growth of a pathogenic Vibrio sp. J. Fish Dis., 18, 31–40. Bergh, Ø. (2000) Bacterial pathogens associated with early life stages of marine ﬁsh. In: Microbial Biosystems: New Frontiers (eds C.R. Bell, M. Brylinski & P. Johnson-Green), Proceedings of the 8th International Symposium on Microbial Ecology, 1999. Atlantic Canada Society for Microbial Ecology, Halifax. Bergh, Ø., Hansen, G.H. & Taxt, R.E. (1992) Experimental infection of eggs and yolk-sac larvae of halibut, Hippoglossus hippoglossus L. J. Fish Dis., 15, 379–91. Bergh, Ø., Naas, K.E. & Harboe, T. (1994) Shift in the intestinal microﬂora of Atlantic halibut (Hippoglossus hippoglossus) larvae during ﬁrst feeding. Can. J. Fish. Aquat. Sci., 51, 1899– 903. Bergh, Ø., Hjeltnes, B. & Skiftesvik, A.B. (1997) Experimental infection of turbot, Scophthalmus maximus, and halibut, Hippoglossus hippoglossus, yolk-sac larvae with Aeromonas salmonicida subsp. salmonicida. Dis. Aquat. Org., 29, 13–20. Bergh, Ø., Nilsen, F. & Samuelsen, O.B. (2001) Diseases, prophylaxis and treatment of the Atlantic halibut, Hippoglossus hippoglossus: a review. Dis. Aquat. Org., 48, 57–74. Biering, E. & Bergh, Ø. (1996) Experimental infection of Atlantic halibut, Hippoglossus hippoglossus L., yolk-sac larvae with infectious pancreatic necrosis virus: detection of virus by immunohistochemistry and in situ hybridization. J. Fish Dis., 19, 405–13. Biering, E., Nilsen, F., Rødseth, O.M. & Glette, J. (1994) Susceptibility of Atlantic halibut, Hippoglossus hippoglossus, to infectious pancreatic necrosis virus. Dis. Aquat. Org., 20, 183–90. Biering, E., Melby, H.P. & Mortensen, S. (1997) Sero- and genotyping of some marine aquatic birnavirus isolates from Norway. Dis. Aquat. Org., 28, 169–74. Blazer, V.S. (1992) Nutrition and disease resistance in ﬁsh. Annu. Rev. Fish Dis., 2, 309–29. Bloch, B.L. & Larsen, J.L. (1993) An iridovirus-like agent associated with systemic infection in cultured turbot, Scophthalmus maximus, fry in Denmark. Dis. Aquat. Org., 15, 235–40. Bloch, B., Gravningen, K. & Larsen, J.L. (1991) Encephalomyelitis among turbot associated with a picornavirus-like agent. Dis. Aquat. Org., 10, 65–70. Bly, J.E. & Clem, L.W. (1992) Temperature and teleost immune functions. Fish Shellﬁsh Immunol., 2, 159–71. Bricknell, I.R., Bowden, T.J., Bruno, D.W., MacLachlan, P., Johnstone, R. & Ellis, A.E. (1999) Susceptibility of Atlantic halibut, Hippoglossus hippoglossus (L.), to infection with typical and atypical Aeromonas salmonicida. Aquaculture, 175, 1–13. Buchanan, J.S. & Madeley, C.R. (1978) Studies on Herpesvirus scophthalmi infection of turbot Scophthalmus maximus (L.): ultrastructural observations. J. Fish Dis., 1, 283–95. Buchmann, K. (1986) On the infection of Baltic cod (Gadus morhua L.) by the acanthocephalan Echinorhynchus gadi (Zoega) Müller. Nord. Veterinaermed., 38, 308–14. Burreson, E.M. (1995) Phylum Annelida: Hirudinea as vector and disease agents. In: Fish Diseases and Disorders. Vol. 1. Protozoan and Metazoan Infections (ed P.T.K. Woo), pp. 599–629. CAB International, Wallingford. Byun, J.W., Park, S.C., Benno, Y. & Oh, T.K. (1997) Probiotic effect of Lactobacillus sp. DS-12 in ﬂounder (Paralichthys olivaceus). J. Gen. Appl. Microbiol., 43, 305–308. Cognetti Varriale, A.M., Cecchini, S. & Saroglia, M. (1992) Therapeutic trials against the Diplectanum aequans (Monogenea) parasite of seabass (Dicentrarchus labrax L.) in intensive farming. Bull. Eur. Assoc. Fish Pathol., 12, 204–206. Crespo, S., Grau, A. & Padros, F. (1992) Sanguinicoliasis in the cultured amberjack, Seriola dumerilli Risso, from the Spanish Mediterranean area. Bull. Eur. Assoc. Fish Pathol., 12, 157–9.

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Diamant, A., Lom, J. & Dyková, I. (1994) Myxidium leei n. sp., a pathogenic myxosporean of cultured sea bream, Sparus aurata. Dis. Aquat. Org., 20, 137–41. Dopazo, C.P., Lemos, M.L., Lodeiros, C., Bolinches, J., Barja, J.L. & Toranzo, A.E. (1988) Inhibitory activity of antibiotic-producing marine bacteria against ﬁsh pathogens. J. Appl. Bacteriol., 65, 97–101. Dragesco, A., Dragesco, J., Coste, F., Gasc, C., Romestand, B., Raymond, J.C. & Bouix, G. (1995) Philasterides dicentrarchi n. sp. (Ciliaphora, Scuticociliatida), a histophagous opportunistic parasite of Dicentrarchus labrax (Linnaeus, 1758), a reared marine ﬁsh. Eur. J. Protistol., 31, 327–40. Dyková, I. & Figueras, A. (1994) Histopathological changes in turbot, Scophthalmus maximus, due to a histophagous ciliate. Dis. Aquat. Org., 18, 5–9. Dyková, I., Figueras, A. & Novoa, B. (1999) Epizoic amoebae from the gill of turbot, Scophthalmus maximus. Dis. Aquat. Org., 38, 33–8. Ellis, A.E. (1988a) Ontogeny of the immune system in teleost ﬁsh. In: General Principles of Fish Vaccination (ed A.E. Ellis), pp. 20–31. Academic Press, London. Ellis, A.E. (ed) (1988b) Fish Vaccination. Academic Press, London. Farto, R., Montes, M., Perez, M.J., Nieto, T.P., Larsen, J.L. & Pedersen, K. (1999) Characterization by numerical taxonomy and ribotyping of Vibrio splendidus biovar I and Vibrio scophthalmi strains associated with turbot cultures. J. Appl. Microbiol., 86, 796–804. Figueras, A., Novoa, B., Santarem, M., Martinez, E., Alvarez, J.M., Toranzo, A.E. & Dyková, I. (1992) Tetramicra breviﬁlum, a potential threat to farmed turbot, Scophthalmus maximus. Dis. Aquat. Org., 14, 127–35. Fjalestad, K.T., Larsen, H.J.S. & Roed, K.H. (1995) Antibody response in Atlantic salmon (Salmo salar) against Vibrio anguillarum and Vibrio salmonicida O-antigens: heritabilities, genetic correlations and correlations with survival. Aquaculture, 145, 77–89. Fouz, B., Larsen, J.L., Nielsen, B., Barja, J.L. & Toranzo, A.E. (1992) Characterization of Vibrio damsela strains isolated from turbot Scophthalmus maximus in Spain. Dis. Aquat. Org., 12, 155–66. Fuller, R. (1989) Probiotics in man and animals. J. Appl. Bacteriol., 66, 365–78. Gatesoupe, F.J. (1997) Siderophore production and probiotic effect of Vibrio sp. associated with turbot larvae, Scophthalmus maximus. Aquat. Living Resourc., 10, 239–46. Gatesoupe, F.J. (1999) The use of probiotics in aquaculture. Aquaculture, 180, 147–65. Gatesoupe, F.J., Lambert, C. & Nicolas, J.L. (1999) Pathogenicity of Vibrio splendidus strains associated with turbot larvae, Scophthalmus maximus. J. Appl. Microbiol., 87, 757–63. Gjedrem, T., Salte, R. & Gjøen, H.M. (1991) Genetic variation in susceptibility of Atlantic salmon to furunculosis. Aquaculture, 97, 1–6. Grisez, L., Chair, M., Sorgeloos, P. & Ollevier, F. (1996) Mode of infection and spread of Vibrio anguillarum in turbot Scophthalmus maximus larvae after oral challenge through live feed. Dis. Aquat. Org., 26, 181–7. Grotmol, S. & Totland, G.K. (2000) Surface disinfection of Atlantic halibut (Hippoglossus hippoglossus) eggs with ozonated sea-water inactivates nodavirus and increases survival of the larvae. Dis. Aquat. Org., 39, 89–96. Grotmol, S., Totland, G.K., Kvellestad, A., Fjell, K. & Olsen, A.B. (1995) Mass mortality of larval and juvenile hatchery-reared halibut (Hippoglossus hippoglossus L.) associated with the presence of virus-like particles in vacuolated lesions in the central nervous system and retina. Bull. Eur. Assoc. Fish Pathol., 15, 176–80. Grotmol, S., Totland, G.K., Thorud, K. & Hjeltnes, B.K. (1997) Vacuolating encephalopathy and retinopathy associated with a nodavirus-like agent: a probable cause of mass mortality of cultured larval and juvenile Atlantic halibut, Hippoglossus hippoglossus. Dis. Aquat. Org., 29, 85–97.

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Grotmol, S., Bergh, Ø. & Totland, G.K. (1999) Transmission of viral encephalopathy and retinopathy (VER) to yolk-sac larvae of the Atlantic halibut, Hippoglossus hippoglossus: occurrence of nodavirus in various organs and a possible route of infection. Dis. Aquat. Org., 36, 95–106. Grotmol, S., Nerland, A.H., Biering, E., Totland, G.K. & Nishizawa, T. (2000) Characterisation of the coat protein gene of a nodavirus from Atlantic halibut, Hippoglossus hippoglossus: detection of the virus with RT–PCR. Dis. Aquat. Org., 39, 79–88. Gudding, R., Lillehaug, A. & Evensen, O. (1999) Recent developments in ﬁsh vaccinology. Vet. Immunol. Immunopathol., 72, 203–12. Hansen, G.H. & Olafsen, J.A. (1989) Bacterial colonisation of cod (Gadus morhua L.) and halibut (Hippoglossus hippoglossus L.) eggs in marine aquaculture. Appl. Environ. Microbiol., 55, 1435–46. Hansen, G.H. & Olafsen, J. (1999) Bacterial interactions in early life stages of marine cold water ﬁsh. Microb. Ecol., 38, 1–26. Hansen, G.H., Bergh, Ø., Michaelsen, J. & Knappskog, D. (1992) Flexibacter ovolyticus sp. nov., a pathogen of eggs and larvae of Atlantic halibut, Hippoglossus hippoglossus L. Int. J. Syst. Bacteriol., 42, 451–8. Harboe, T., Huse, I. & Øie, G. (1994) Effects of egg disinfection on yolk-sac and ﬁrst feeding stages of halibut (Hippoglossus hippoglossus L.) larvae. Aquaculture, 119, 157–65. Hart, S., Wrathmell, A.B., Harris, J.E. & Grayson, T.H. (1988) Gut immunology in ﬁsh: a review. Dev. Comp. Immunol., 12, 453–80. Hilger, I., Ullrich, S. & Anders, K. (1991) A new ulcerative ﬂexibacteriosis-like disease (‘yellow pest’) affecting young Atlantic cod, Gadus morhua, from the German Wadden Sea. Dis. Aquat. Org., 11, 19–29. Hjeltnes, B., Bergh, Ø., Wergeland, H. & Holm, J.C. (1995) Susceptibility of Atlantic cod, Gadus morhua, halibut, Hippoglossus hippoglossus, and wrasse (Labridae) to Aeromonas salmonicida subsp. salmonicida and the possibility of transmission of furunculosis from farmed salmon, Salmo salar, to marine ﬁsh. Dis. Aquat. Org., 23, 25–31. Húsgarð, S., Grotmol, S., Hjeltnes, B.K., Rødseth, O.M. & Biering, E. (2001) Immune response to a recombinant capsid protein of striped jack nervous necrosis virus (SJNNV) in turbot, Scophthalmus maximus, and Atlantic halibut, Hippoglossus hippoglossus, and evaluation of a vaccine against SJNNV. Dis. Aquat. Org., 45, 33–44. Ingilæ, M., Arnesen, J.A., Lund, V. & Eggset, G. (2000) Vaccination of Atlantic halibut, Hippoglossus hippoglossus L., and spotted wolf-ﬁsh, Anarhichas minor L., against atypical Aeromonas salmonicida. Aquaculture, 183, 31–44. Joosten, P.H.M., Aviles-Trigueros, M., Sorgeloos, P. & Rombout, J.H.W.M. (1995) Oral vaccination of juvenile carp (Cyprinus carpio) and gilthead seabream (Sparus aurata) with bioencapsulated Vibrio anguillarum bacterin. Fish Shellﬁsh Immunol., 5, 289–99. Keskin, M., Keskin, M. & Rosenthal, H. (1994) Pathways of bacterial contamination during egg incubation and larval rearing of turbot, Scophthalmus maximus. J. Appl. Ichthyol., 10, 1–9. Khan, R.A. (1988) Experimental transmission, development, and effects of a parasitic copepod, Lernaeocera branchialis, on Atlantic cod, Gadus morhua. J. Parasitol., 74, 586–99. Kirms, P. (1980) Observations on the pathogenicity of Haemogregarina sachai Kirms, (1978), in farmed turbot, Scophthalmus maximus (L.). J. Fish Dis., 3, 101–14. Knappskog, D.H., Rødseth, O.M., Slinde, E. & Endresen, C. (1993) Immunochemical analyses of Vibrio anguillarum strains isolated from cod, Gadus morhua L., suffering from vibriosis. J. Fish Dis., 16, 327–38. Kozasa, M. (1986) Tyocerin (Bacillus toyoi) as growth promotor for animal feeding. Microbiol. Aliment. Nutr., 4, 121–35.

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Landolt, M.L. (1989) The relationship between diet and the immune response of ﬁsh. Aquaculture, 79, 193–206. Lillehaug, A., Sevatdal, S. & Endal, T. (1996) Passive transfer of speciﬁc maternal immunity does not protect Atlantic salmon (Salmo salar L) fry against yersiniosis. Fish Shellﬁsh Immunol., 6, 521–35. Lillehaug, A., Nygaard, S., Eggset, G. & Poppe, T. (1999) Vaccination. In: Fish Health and Fish Diseases. (ed T. Poppe), pp. 288–301. Universitetsforlaget, Oslo (in Norwegian). Lupiani, B., Dopazo, C.P., Ledo, A., Fouz, B., Barja, J.L., Hetrick, F.M. & Toranzo, A.E. (1989) New syndrome of mixed bacterial and viral etiology in cultured turbot, Scophthalmus maximus. J. Aquat. Anim. Health, 1, 197–204. Maeda, M. & Nogami, K. (1989) Some aspects of the biocontrolling methods in aquaculture. In: Current Topics in Marine Biotechnology (eds S. Miyachi et al.), pp. 395–98. Japan Society for Marine Biotechnology, Tokyo. Makridis, P. & Vadstein, O. (1999) Food size selectivity of Artemia franciscana at three developmental stages. J. Plankton Res., 21, 2191–201. Makridis, P., Fjellheim, A., Skjermo, J. & Vadstein, O. (2000a) Control of the bacterial ﬂora of Brachionus plicatilis and Artemia franciscana by incubation in bacterial suspensions. Aquaculture, 185, 207–18. Makridis, P., Fjellheim, A., Skjermo, J. & Vadstein, O. (2000b) Colonization of the gut of ﬁrst feeding turbot larvae (Scophthalmus maximus L.) by bacterial strains added to the water or bioencapsulated in rotifers (Brachionus plicatilis). Aquacult. Int., 8, 367–80. Mann, H. (1952) Lernaeocera branchialis (Copepoda parasitica) und seine Schadewirkung bei einigen Gadiden. Arch. Fischereiwiss., 4, 133–44. Meyers, T.R., Sullivan, J., Emmenegger, E., Follett, J., Shorts, S., Batts, W.N. & Winton, J.R. (1992) Identiﬁcation of viral hemorrhagic septicemia virus isolated from Paciﬁc cod, Gadus macrocephalus, in Prince William Sound, Alaska, USA. Dis. Aquat. Org., 12, 167–75. Midtlyng, P.J., Reitan, L.J., Lillehaug, A. & Ramstad, A. (1996) Protection, immune responses and side effects in Atlantic salmon (Salmo salar L.) vaccinated against furunculosis by different procedures. Fish Shellﬁsh Immunol., 6, 599–613. Mor, A. & Avtalion, R.R. (1990) Transfer of antibody from immunized mother to embryo in tilapias. J. Fish Biol., 37, 249–55. Moran, J.D., Whitaker, D.J. & Kent, M.L. (1999) A review of the myxosporean genus Kudoa Meglitsch, 1947, and its impact on the international aquaculture industry and commercial ﬁsheries. Aquaculture, 172, 163–96. Mortensen, S.H., Hjeltnes, B., Rødseth, O., Krogsrud, J. & Christie, K.E. (1990) Infectious pancreatic necrosis virus, serotype N1, isolated from Norwegian halibut (Hippoglossus hippoglossus), turbot (Scophthalmus maximus) and scallops (Pecten maximus). Bull. Eur. Assoc. Fish Pathol., 10, 42. Mortensen, S.H., Evensen, Ø., Rødseth, O.M. & Hjeltnes, B. (1993) The relevance of infectious pancreatic necrosis virus (IPNV) in farmed Norwegian turbot (Scophthalmus maximus). Aquaculture, 115, 243–52. Mudarris, M. & Austin, B. (1989) Systemic disease in turbot, Scophthalmus maximus, caused by a previously unrecognized Cytophaga-like bacterium. Dis. Aquat. Org., 6, 161–6. Munday, B.L. & Nakai, T. (1997) Special topic review: nodaviruses as pathogens in larval and juvenile marine ﬁnﬁsh. World J. Microbiol. Biotechnol., 13, 375–81. Munro, P., Barbour, A. & Birkbeck, T.H. (1995) Comparison of the growth and survival of larval turbot in the absence of culturable bacteria with those in the presence of Vibrio anguillarum, Vibrio alginolyticus or a marine Aeromonas sp. Appl. Environ. Microbiol., 61, 4425–8.

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Nilsen, F. (1995) Description of Trichodina hippoglossi n.sp. from farmed Atlantic halibut larvae, Hippoglossus hippoglossus. Dis. Aquat. Org., 21, 209–14. Nilsen, F. (1999) Microspora (mikrosporidier). In: Fiskehelse og Fiskesykdommer (ed T.T. Poppe), pp. 186–9. Universitetsforlaget, Oslo. Nylund, A., Hovland, T., Hodneland, K., Nilsen, F. & Løvik, P. (1994) Mechanisms for transmission of infectious salmon anaemia (ISA). Dis. Aquat. Org., 19, 95–100. Odum, E.P. (1971) Fundamentals of Ecology. Saunders, Philadelphia. Ogawa, K. & Egusa, S. (1986) Two new species of Paradeontacylix McIntosh, 1934 (Trematoda: Sanguinicolidae) from the vascular system of cultured marine ﬁsh, Seriola purpurascens. Fish Pathol., 21, 15–19. Øie, G., Reitan, K.I. & Olsen, Y. (1994) Comparison of rotifer culture quality with yeast plus oil and algal-based cultivation diets. Aquacult. Int., 2, 225–38. Oliver, G., Evelyn, T.P.T. & Laillier, R. (1985) Immunity to Aeromonas salmonicida in coho salmon (Oncorhynchus kisutch) induced by modiﬁed Freund’s complete adjuvant: its non-speciﬁc nature and the probable role of macrophages in the phenomenon. Dev. Comp. Immunol., 9, 419– 32. Olsen, A.I., Olsen, Y., Attramadal, Y., Christie, K., Birkbeck, T.H., Skjermo, J. & Vadstein, O. (2000) Effects of short-term feeding of microalgae on the bacterial ﬂora associated with juvenile Artemia franciscana. Aquaculture, 190, 11–25. Olsson, J.C. (1995) Bacteria with inhibitory activity and Vibrio anguillarum in the ﬁsh intestinal tract, Fil. Dr. Thesis, Göteborg University, 141 pp. Olsson, J.C., Westerdal, A., Conway, P.L. & Kjelleberg, S. (1992) Intestinal colonization potential of turbot (Scophthalmus maximus)- and dab (Limanda limanda)-associated bacteria with inhibitory effects against Vibrio anguillarum. Appl. Environ. Microbiol., 58, 551–6. Oseko, N., Yoshimizu, M., Gorie, S. & Kimura, T. (1988) Histopathological study on diseased hirame Japanese ﬂounder, Paralichthys olivaceus, infected with Rhabdovirus olivaceus (hirame rhabdovirus, HRV). Fish Pathol., 23, 117–23. Perez, M.J., Fernandez, A.I.G., Rodriguez, L.A. & Nieto, T.P. (1996) Differential susceptibility of turbot and rainbow trout and release of the furunculosis agent from furunculosis-affected ﬁsh. Dis. Aquat. Org., 26, 133–7. Prescott, L.M., Harley, J.P. & Klein, D.A. (1999) Microbiology, 4th edn. McGraw-Hill, Boston. Rapp, J. (1995) Treatment of rainbow trout (Oncorhynchus mykiss Walb.) fry infected with Ichthyophthirius (Ichthyophthirius multiﬁliis) by oral administration of dimetridazole. Bull. Eur. Assoc. Fish Pathol., 15, 67–9. Reitan, K.I., Natvik, C. & Vadstein, O. (1998) Drinking rate, uptake of bacteria and micro-algae in turbot larvae. J. Fish Biol., 53, 1145–54. Reitan, K.I., Rainuzzo, J.R., Øie, G. & Olsen, Y. (1997) A review of the nutritional effects of algae in marine ﬁsh larvae. Aquaculture, 155, 207–21. Ringø, E. & Birkbeck, T.H. (1999) Intestinal microﬂora of ﬁsh larvae and fry. Aquacult. Res., 30, 73–93. Ringø, E. & Gatesoupe, F.J. (1998) Lactic acid bacteria in ﬁsh: a review. Aquaculture, 160, 177–203. Ringø, E. & Vadstein, O. (1998) Colonization of Vibrio pelagius and Aeromonas caviae in early developing turbot, Scophthalmus maximus (L.) larvae. J. Appl. Bacteriol., 84, 227–33. Ringø, E., Birkbeck, H., Munro, P.D., Vadstein, O. & Hjelmeland, K. (1996) The effect of early exposure to Vibrio pelagius on the aerobic bacterial ﬂora of turbot, Scophthalmus maximus (L.) larvae. J. Appl. Bacteriol., 81, 207–11.

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Romalde, J.L. (1999) Genetic analysis of turbot pathogenic Streptococcus paruberis strains by ribotyping and random ampliﬁed polymorphic DNA. FEBS Microbiol. Lett., 179, 297–304. Salvesen, I. (1999) Microbial ecology in early life stages of marine ﬁsh: Development and evaluation of methods for microbial management in intensive larviculture. Dr. Sci. Dissertation, Norwegian University of Science and Technology. Salvesen, I. & Vadstein, O. (1995) Surface disinfection of eggs from marine ﬁsh. Evaluation of four chemicals. Aquacult. Int., 3, 155–71. Salvesen, I. & Vadstein, O. (2000) Evaluation of plate count methods for determination of maximum speciﬁc growth rate in mixed microbial communities, and its possible application for diversity assessment. J. Appl. Bacteriol., 88, 442–8. Salvesen, I., Øie, G. & Vadstein, O. (1997) Surface disinfection of Atlantic halibut (Hippoglossus hippoglossus L.) and turbot (Scophthalmus maximus L.) eggs with glutaraldehyde: evaluation of concentrations and contact times. Aquacult. Int., 5, 249–58. Salvesen, I., Skjermo, J. & Vadstein, O. (1999) Growth of turbot (Scophthalmus maximus L.) during ﬁrst feeding in relation to the proportion of r/K-strategists in the bacterial community of the rearing water. Aquaculture, 175, 337–50. Salvesen, I., Reitan, K.I., Skjermo, J. & Øie, G. (2000) Microbial environments in marine larviculture: impacts of algal growth rates on the bacterial load in six microalgae. Aquacult. Int., 8, 275– 87. Sanmartin Durán, M.L., Fernandez Casal, J., Tojo, J.L., Santamarina, M.T., Estevez, J. & Ubeira, F. (1991) Trichodina sp.: effect on the growth of farmed turbot (Scophthalmus maximus). Bull. Eur. Assoc. Fish Pathol., 11, 89–91. Santamarina, M.T., Tojo, J., Ubeira, F.M., Quinteiro, P. & Sanmartin, M.L. (1991) Anthelmintic treatment against Gyrodactylus sp. infecting rainbow trout, Oncorhynchus mykiss. Dis. Aquat. Org., 10, 39–43. Sanz, F. (1992) Mortality of cultured seabream (Sparus aurata) caused by an infection with a trematode of the genus Microcotyle. Bull. Eur. Assoc. Fish Pathol., 12, 186–8. Sarusic, G. (1999) Preliminary report of infestation by isopod Ceratothoa oestroides (Risso, 1826), in marine cultured ﬁsh. Bull. Eur. Assoc. Fish Pathol., 19, 110–12. Schmahl, G. (1993) Up-to-date chemotherapy against Monogenea: a review. Bull. Fr. Peche Piscic., 328, 74–81. Schmahl, G. & Mehlhorn, H. (1989) Treatment of ﬁsh parasites. 6. Effects of Sym. Triazinone (Toltrazuril) on developmental stages of Glugea anomala Moniez, 1887 (Microsporidia): a light and electron microscopic study. Eur. J. Protistol., 24, 252–9. Secombes, C.J. (1994) Enhancement of ﬁsh phagocyte activity. Fish Shellﬁsh Immunol., 4, 421– 36. Siddall, M.E., Martin, D.S., Bridge, D., Cone, D.M. & Desser, S.S. (1995) The demise of a phylum of protists: Myxozoa and other parasitic Cnidaria. J. Parasitol., 81, 961–7. Sin, Y.M., Ling, K.H. & Lam, T.J. (1994) Passive transfer of protective immunity against ichthyophthiriasis from vaccinated mother to fry in tilapias, Oreochromis aureus. Aquaculture, 120, 229– 37. Skiftesvik, A.B. & Bergh, Ø. (1993) Changes in behaviour of halibut (Hippoglossus hippoglossus) and turbot (Scophthalmus maximus) yolk-sac larvae induced by bacterial infections. Can. J. Fish. Aquat. Sci., 50, 2552–7. Skjermo, J. & Vadstein, O. (1993) The effect of microalgae on skin and gut bacterial ﬂora of halibut larvae. In: Fish Farming Technology (ed H. Reinertsen, L.A. Dahle, L. Jørgensen & K. Tvinnereim), pp. 61–7. A.A. Balkema, Rotterdam.

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Chapter 4

Live Food Technology of Cold-Water Marine Fish Larvae Y. Olsen

4.1 Introduction Successful and economically feasible production of marine ﬁsh juveniles is highly multidisciplinary, requiring adequate competence in both larval and live-food technology. The live-feed developments include zootechnical, nutritional and microbial aspects, and these have been a major challenge (see also Chapter 7). Only two zooplankton families have so far shown that they can be produced regularly at an acceptable cost: the rotifer Brachionus sp. and the brine shrimp Artemia sp. (e.g. Lubzens, 1987; Sorgeloos et al., 1986). The most commonly used species within these groups are Brachionus plicatilis and Artemia franciscana, which are used alone or in appropriate combinations as live feed for most marine ﬁsh species in culture. There is an increasing use of the smaller rotifer species Brachionus rotundiformis in marine ﬁsh larviculture, but this species has only recently been used for cold-water species, and will be covered only brieﬂy in this chapter. There is a well-established international live-food production technology for Brachionus and Artemia. This technology has been modiﬁed for use for marine cold-water species of ﬁsh, and was taken up in the 1970s in northern countries (e.g. Howell, 1979). This chapter describes the basic cultivation techniques and methods for manipulation of nutritional value that are suitable for marine cold-water ﬁsh larvae. In the developmental work, the biochemical composition, or nutritional value, of the cultivated live feed was inspired by that of natural zooplankton, since marine copepods are believed to be important as a natural food for many larval ﬁsh species. In particular, the chapter will present the developments and ﬁndings of recent Norwegian research programmes. Methods for rotifer production and nutritional manipulation that are suitable at low temperatures will be the main focus. Both nutritional and microbial aspects are covered, and marine copepods have served as a nutritional reference. These cold-water methods deviate from the normal procedures used world-wide on some points: the production and n-3 HUFA (highly unsaturated n-3 fatty acids; see Section 7.2) enrichment of rotifers can readily been done simultaneously and not in successive steps. This has been found to be feasible for cold-water species. The international Artemia technology forms the basis of the cold-water adaptations, and these methods are already well described (see manuals by Sorgeloos et al., 1986; Lavens & Sorgeloos, 1996). In this chapter, the focus on Artemia will primarily involve speciﬁc problems of n-3 HUFA enrichment and stability. These problems

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Feeding system

Oxygen monitoring system, optional Seawater

Aeration tube

Heating Temperature control

Freshwater

Mixing Disinfection Temperature acclimatisation Aeration Electrical control, security

Water exchange tube

Drainage valve

Figure 4.1 Schematic view of the cultivation equipment and supporting functions needed for rotifer and Artemia production and enrichment.

have become evident during the rearing of Atlantic halibut (Hippoglossus hippoglossus) larvae, an important species for aquaculture in northern countries (Olsen et al., 1999; Shields et al., 1999b). Microalgae are important components of ﬁsh larval diets, either directly, or indirectly as food for Brachionus and Artemia. The methods of cultivation and further use during rearing of cold-water ﬁsh larvae are not different from those used for rearing other species. Other authors have provided detailed descriptions of the production technology of microalgae (see papers in Fulks and Main, 1991; Coutteau, 1996), and such methods will not be covered in this chapter. The speciﬁc use of microalgae during ﬁrst feeding is covered in Chapter 7.

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4.2 Cultivation Systems There is no uniﬁed technical design of cultivation systems for rotifers and Artemia, and all producers tend to use their own approach. However, there is a common core of system design and function that is generally useful during live feed production (see Lavens & Sorgeloos, 1996). The speciﬁc pre-treatments needed for the process water of live-feed cultures depend on the local water quality. Figure 4.1 illustrates one possible technical arrangement, including water supply and pre-treatment, tank design, feeding system and control functions. A vital requirement for the process water is the complete removal of zooplankton and larvae of marine organisms. Standard ﬁlter units (e.g. sand ﬁlters) will not retain zooplankton individuals as small as 20–30 mm (e.g. many ciliates). This group can create serious problems in rotifer cultures. Speciﬁc disinfection treatments may be needed as a protection against disease and contamination by small zooplankton species. Most strains of B. plicatilis have their optimum for growth in brackish water (e.g. 10–15 p.p.t.). If reduced salinity is used during cultivation, freshwater should be added and mixed with the seawater before disinfection and temperature acclimation. It is important that the process water is temperature-acclimated and thoroughly aerated before being added to the culture tanks in order to avoid super-saturation of nitrogen. B. plicatilis and Artemia can be cultivated in almost all types of tank. Cylindrical–conicalshaped tanks allow efﬁcient precipitation and easy removal of organic wastes from the bottom during intensive feeding and high animal densities, but other types of concrete or glass-ﬁbre tanks of various shapes and sizes have been used in commercial hatcheries. Some companies may use tanks larger than 100 m3. The volume of the cultivation units used is accordingly highly variable, but cultivation and lipid enrichment in very small units (e.g. <50 l) is not feasible. High zooplankton densities require continuous or pulsed water exchange during cultivation. This can be carried out using a central ﬁltering tube (pore size depends on animal sizes, 艌50 mm) which retains the animals, but allows the water and small residual particles to pass through. Temperature is an important variable that must be controlled. Suspended thermostat units which are suitable for smaller cultivation tanks are readily available, and 300–500 W m-3 is an appropriate energy supply in colder regions. Warmer regions may also require cooling, which is most easily done by circulating cold water in tubes suspended in the culture. In many cases, temperature control may most easily be achieved by controlling the room temperature or by water-bath-type solutions, which are suitable if a system for energy recycling or heat exchange is available. If high-voltage electricity is used, special attention must be paid to security to prevent accidents caused by a short circuit following accidental damage of the electrical system. All tanks must be carefully earthed, monitored and controlled. These precautionary measures are very important because sudden damage to the electrical system may cause immediate death. A low-voltage electricity supply is generally recommended. Efﬁcient aeration and culture mixing will normally ensure a satisfactory oxygen supply to cultures, but supplementary pure oxygen should be considered if the production is very intensive (e.g. >1000 rotifers ml-1). Air can be supplied by airstones or another diffuser

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system suspended close to the walls and bottom of the tanks. A 200 l culture will need one small air outlet, a 4-m3 culture will need more than four big airstones. Equipment for monitoring and controlling culture conditions is not strictly necessary for low densities of rotifers and Artemia, but very intensive cultures will require monitoring. Adequate aeration of cultures is probably among the main problems of producing rotifers in large and intensive units (>2 m3) and of n-3 HUFA enrichment of Artemia. The biotechnology industry has developed very intense aeration systems for very large production units of yeast and bacteria. The full adoption of this technology to rotifers and Artemia is still incomplete. Commercial large-scale production of live feed requires the installation of partly automated feeding systems, a cleaning and washing system for the live feed, enrichment systems, and systems for the cooling, storage and concentration of cultures. Labour costs may constitute over two-thirds of the live-feed costs. Appropriate equipment will therefore reduce the costs of production considerably, and allow the staff to concentrate on the more important tasks of rotifer and Artemia production. Transport can be mediated by gravity, vacuum, or thoroughly tested pumps.

4.3 Production of Rotifers In the early 1960s, the Japanese pioneered the use of Brachionus plicatilis as live feed for cultured marine larvae (Ito, 1960; review by Nagata & Hirata, 1986). The rotifer technology for cold-water species was mainly developed during the early 1990s. The descriptions involve general biology, cultivation techniques, and methods that were established to manipulate the biochemical composition of rotifers. These methods deviate to some extent from the methods normally used for temperate and warm-water ﬁsh larvae. The use of rotifers during ﬁrst feeding is treated in Chapter 7, and the importance of the microbial communities of livefeed cultures is treated in Chapter 3.

4.3.1 Biological Characteristics 4.3.1.1 General Biology and Life History Rotifers are small metazoa, and most species live in fresh water. A layer of keratin-like proteins forms their epidermis and is termed the lorica. The form and characteristics of the lorica are important taxonomic criteria. A rotary organ or corona, recognised by its annular ciliation, ensures locomotion in a whirling water movement that facilitates the rotifers feeding on small organisms in the water. The rotifer strains most commonly used as live feed for marine ﬁsh larvae are Brachionus plicatilis and Brachionus rotundiformis. The former morphotype was previously termed the L-type Brachionus, and is characterised by a lorica length of 130–340 mm. The latter morphotype has been termed the S-type, and is characterised by a typical lorica length of 90–210 mm. These strains are apparently not genetically isolated, but the S-type seems to have higher optimum temperatures for growth than the L-type (see Fu et al., 1991; Dhert, 1996). B. plicatilis is by far the most commonly used species in aquaculture. Most of the examples in this chapter are representative of the L-strain isolate of B. plicatilis (termed the

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SINTEF strain, lorica length 250–330 mm) that has been thoroughly studied and used in ﬁrstfeeding trials with turbot, Atlantic halibut and cod. The strain has both an asexual (amictic or parthenogenetic) and a sexual (mictic) life cycle, but males and females carrying resting eggs have only been observed on a very few occasions. The normal production temperature and salinity is 20–22°C and 20 p.p.t., respectively. Under these conditions, the average amictic females produce their ﬁrst eggs 1.4 days after hatching. Thereafter, they produce 21 eggs during the following 6.7 days. The developmental time of the eggs is 0.41 days. The post-reproductive period of the rotifer is 2.4 days, giving a total average life span of 10.5 days (Korstad et al., 1989a). 4.3.1.2 Feeding Kinetics of B. plicatilis B. plicatilis can feed on a wide range of food particles, including bacteria, microalgae, protozoa and dead organic material. The upper size level of food particles that can be ingested by the rotifer depends on the shape and nature of the food. B. plicatilis is able to ingest bacteria <1 mm, but its efﬁciency at harvesting the smallest bacterial cells is low compared with its ability to ingest suitably sized algae (Vadstein et al., 1993). It is very important to consider the size of the food particles, during both cultivation and nutrient enrichment. The alga Isochrysis galbana (T-iso strain) is a suitable food for B. plicatilis, and its feeding rate of this alga is well described as a Type-1 functional response (Holling, 1966). The rotifer ingestion rate increases in proportion to algal concentration (C) in the lower range of concentrations, and remains constant for food concentrations above a critical value, which is denoted as the ‘incipient limiting food concentration’ (ILC; Fig. 4.2A). The term clearance rate is deﬁned as the volume of water cleared of food particles per unit of time and rotifer biomass. At low food concentrations (C < ILC), the rotifers clear water at maximum efﬁciency, and the clearance rate is at its maximum level (CRmax, Fig. 4.2B). Above this concentration, the clearance rate decreases asymptotically towards zero as food concentration increases. Models of different complexities have been used to describe food intake in other Brachionus species (e.g. Rothhaupt, 1990a,b), but the simple rectilinear model used in Fig. 4.2A and B is unambiguous and convenient (Holling Type-I response). Using this model, the ingestion rate (I) is proportional to the food concentration (C) at food concentrations below the incipient limiting concentration (ILC). The slope is identical to the maximum clearance rate (CRmax). Above the ILC, the ingestion rate is maximal and constant independent of the food concentration (Imax), whereas the clearance rate (CR), expressed as the ratio between ingestion rate and food concentration, decreases asymptotically with increasing food concentration. The general equations of the model are accordingly I = CR maxC

C < ILC

(4.1)

I = I max

C ⭓ ILC

(4.2)

CR = I C

C ⭓ ILC

(4.3)

CR max = I max ILC

C < ILC

(4.4)

78

Culture of cold-water marine ﬁsh

Clearance rate, mL ind-1 day-1 200

Feeding rate, ngC ind-1 day-1, 500

B

400

150

300 100

200 100

50

A

ILC

0 5 10 15 Food concentration, mgC L-1

0

20

0 0

20

Growth and mortality rate, day-1 0. 6

ER, eggs rotifer-1 0.8

C

D

0.6

0. 4

0.4

0. 2

0.2

0

0

-0.2 0

5 10 15 Food concentration, mgC L-1

0.1 0.2 0.3 0.4 Specific growth rate, day-1

0

0.5

0.1 0. 2 0.3 0. 4 0.5 0. 6 0.7 ER, eggs rotifer-1

Net grow th Mortality Gross growth Figure 4.2 Feeding and growth characteristics of B. plicatilis. A. Model curve for the feeding rate of the algae I. galbana as a function of its concentration (based on Korstad et al., 1989b). B. Model curve for the food clearance rate of the algae I. galbana as a function of its concentration (based on Korstad et al., 1989b). C. Population steadystate egg ratio (eggs rotifer-1) as a function of population growth rate (day-1) (data from Øie and Olsen, 1997). D. Model curves for gross growth rate, net growth rate and mortality rate (day-1) as a function of the egg ratio (eggs rotifer-1).

Appropriate coefﬁcient values for the SINTEF strain of B. plicatilis are given below (modiﬁed from Korstad et al., 1989b). ILC = 2.6 mgC -1

(4.5)

I max = 430 ngC individual -1 day -1

C 艌 2.6 mgCl -1

(4.6)

CR max = 165 ml individual -1 day -1

C 艌 2.6 mgCl -1

(4.7)

Live food technology of cold-water marine ﬁsh larvae

79

The maximum ingestion rate expresses the maximum amount of food that can be consumed by the rotifers per individual per day. This variable is particularly important in the practical production of rotifers. 4.3.1.3 Growth, Mortality and Egg Ratio The net speciﬁc growth rate of rotifers maintained in batch and continuous cultures (see below) is a balance between gross speciﬁc growth rate and mortality rate, which is signiﬁcant compared with the gross growth rate because of the short life-span of the rotifer (10.5 days). The gross growth rate (mgross) can accordingly be expressed as the sum of the net growth rate (mnet, or simply m) and mortality rate (m): m gross = m net + m

(4.8)

This equation must be used with some care for partly synchronised, non-steady-state cultures. If the average rotifer lives for 10.5 days when the food supply is high, the corresponding mortality rate will be approximately 0.1 day-1. This rate is representative of well-fed rotifers. An independent estimate of mortality rate for severely starved steadystate cultures is 0.16 day-1 (see below). The initial age distribution of a rotifer population inoculated in a batch culture will affect the initial mortality rate and the net growth rate. If the inoculum contains a low fraction of post-reproductive individuals, or a high fraction of juvenile individuals, the initial mortality rate may be close to zero. On the other hand, the mortality rate may be >0.16 day-1 if the inoculum contains a high fraction of senile rotifers. This calls for careful use of Equation 4.8 under non-steady-state growth conditions. The egg ratio (ER, eggs per rotifer) of rotifer cultures has proved to be a very useful tool in mass cultivation. Eggs per rotifer can easily be counted using a microscope, and the value can be interpreted as a dynamic variable reﬂecting the growth and production rates of the cultures. The linear relationship between the average egg ratio and the net speciﬁc growth rate of steady-state cultures is illustrated in Fig. 4.2C. This ﬁgure indicates that an egg ratio of 0.18 eggs rotifer-1 is to be expected for zero net growth rate of the culture, and that cultures growing at the maximum net growth rate of 0.45 day-1 is expected to have an average egg ratio of 0.68 eggs rotifer-1. Figure 4.2D shows the terms of Equation 4.8 as a function of the culture egg ratio. The net growth rate relationship is simply the inverse of the curve in Fig. 4.2C extrapolated to zero egg ratios. The curve suggests a negative population growth rate of -0.16 day-1 for cultures of rotifers without eggs. Because the gross growth rate must be zero for animals without eggs, this value can be interpreted as the mortality rate for starved cultures. The mortality rate of well-fed cultures is 0.1 day-1 (see above), and the mortality curve in Fig. 4.2D assumes linearity between mortality rate and egg ratio. The gross growth rate is the sum of mortality and net growth (Equation 4.8), and the following equations describe growth and mortality rates as functions of the egg ratio of the present rotifer strain. m net = 0.90 ER - 0.16

(4.9)

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Culture of cold-water marine ﬁsh

m = 0.16 - 0.087 ER

(4.10)

m gross = 0.81 ER

(4.11)

Again, the equations are only strictly valid for steady-state conditions. The relationship between net growth, gross growth and mortality is important in practical production, for example during attempts to predict future production and to diagnose problems, because both reduced gross growth rate and enhanced mortality rate may affect the net rotifer production. Even though both events will result in low production, the counter-measures needed to overcome these problems may be quite different. The coefﬁcients in Equations 4.9–4.11 are believed to be independent of the food used, because the egg developmental time is independent of the food quality and quantity (King & Miracle, 1980). If for some reasons the rotifers exhibit a higher net growth rate with another feed, the egg ratio is expected to increase proportionally. The above equations are valid for the temperature range 20–22°C. However, changes in temperature will most likely affect the developmental time of the eggs, and in turn the relationship between egg ratio and speciﬁc growth rate. It will also affect the metabolic activity of the rotifer.

4.3.2 Cultivation Feed and Feed Treatments B. plicatilis will consume most food particles of an appropriate size for consumption. Efﬁcient cultivation feeds must also cover the nutritional demands of the rotifers and secure proper hygienic conditions in the cultivation tanks. Appropriate live feeds are microalgae, baker’s yeast, used alone or in combination with dispersed, emulsiﬁed (or even crude) marine oils, and formulated diets that are commercially available. Crude and cheap diets such as micronised ﬁshmeal and dried yeast powder are not efﬁcient, probably because of extensive leakage of organic compounds that create losses in nutritional value, poor hygiene conditions and enhanced rotifer mortality. Many species of microalgae are excellent food for rotifers, but their production costs are high. Some producers give microalgae as a component of the diet (1–5%), normally together with formulated diets or live baker’s yeast. Small supplements of microalgae can contribute to better rotifer health and viability, and thereby reduce risks. It is a common belief that even a moderate use of microalgae, in an appropriate combination with another principal feed, will tend to make the rotifer production more predictable and the rotifers more viable. Japanese pioneers learned that live baker’s yeast was an efﬁcient and cheap feed for rotifers (Nagata & Hirata, 1986). To overcome an essential n-3 fatty acids deﬁciency in ﬁsh larvae, they later suggested that yeast should be supplied together with emulsiﬁed marine oil (Fukusho, 1977; Kitajima & Koda, 1976). For many reasons, this principal technique has proved feasible for producing rotifers for cold-water species of ﬁsh. The mixture of yeast and oil can be added in controlled, high-quantity rations, which is not very easy with microalgae. This is important in order to run controlled and predictable large-scale production of rotifers. It is also noteworthy that baker’s yeast and crude marine oils are very cheap diets, and that the rotifer feed costs will also remain low if a commercial formulated emulsiﬁed diet is used instead of the crude marine oil (e.g. Selco-type, INVE Aquaculture, Belgium).

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81

Before addition to the rotifer tank, the yeast should be mixed with lukewarm fresh water (<200 g wet yeast per litre) for 10–15 s in a blender. If emulsiﬁed or crude marine oil is added along with the yeast, the oil should be mixed with the water for 20 s before the yeast is added. A suitable proportion of fresh baker’s yeast and oil is 10 : 1 (fresh weight). Lipids will then constitute approximately 25–30% of the total dry weight of the mixed diet, and 95% of the dietary lipids will originate from the oil. The fatty acid composition of the oil will then almost completely determine the fatty acid composition of the rotifers (see below). Formulated diets for rotifer cultivation are commercially available, and detailed descriptions of their composition and use in rotifer production are worked out by the manufacturers. The culture Selco, manufactured by the INVE Group, is probably the most commonly used product in Western countries and appears to be efﬁcient, even for cultures with a very high rotifer density (Suantika et al., 2000). The formulated diets are more expensive than baker’s yeast and crude marine oil, but the costs of the cultivation feed will affect the ﬁnal costs of juvenile ﬁsh in Western countries to a small extent only if the production efﬁciency is adequate. The extra cost that follows a crash in rotifer production and low and variable larval survival and quality are far more important.

4.3.3 Cultivation of Rotifers The process of rotifer production is a combined production and n-3 HUFA enrichment, and involves several phases (Fig. 4.3). (1) Maintenance of stock cultures. (2) Inoculation phase: start of new cultures based on inocula from stock cultures, or more commonly from production cultures. (3) Early growth phase: the critical phase when the food rations and rotifer density are increased gradually. (4) Late growth or production phase: the ﬁnal phase when cultures are harvested. Rotifer density may increase, decrease or remain constant, but the food ration is kept constant. 4.3.3.1 Maintenance of Stock Cultures Stock cultures should be kept physically isolated from the production facility of microalgae and rotifers in order to counteract contamination and the transfer of diseases. A sound precautionary approach that may reduce the risk of culture collapse and disease is to renew production cultures from pure stock cultures at least once a year. Such a renewal should go along with complete hatchery disinfection. Stock cultures of B. plicatilis must not be contaminated by other zooplankton species. Most aquatic bacteria are harmless to rotifers and are acceptable in stock cultures, but wellknown pathogenic bacteria should be excluded. Algal cultures used to feed the stock cultures of rotifers must also be free from harmful contaminants. Contaminated algal cultures are most easily rinsed by plating techniques using solid agar (Coutteau, 1996). Many species of microalgae used in aquaculture will grow on solid agar; other species are most easily purchased from culture collections that take professional care of the isolates (Smith et al., 1993). Stock cultures of B. plicatilis can be maintained in small units (0.1–1 l). The water used must be sterilised. A stock culture is initiated by transferring 5–10 ml of mature stock culture

82

Culture of cold-water marine ﬁsh

STOCK CULTURES (maintenance)

INOCULUM (grown from stock cultures)

INOCULATION PHASE

INOCULUM selected and rinsed

EARLY GROWTH PHASE Batch culture, stepwise increase of food ration

LATE GROWTH PHASE PRODUCTION Batch or continuous culture, constant food ration

HARVESTED LIVE FEED rinsing, cooling, enrichment, other treatments

Figure 4.3 Schematic view of the deﬁned phases of rotifer cultivation.

to a beaker containing 0.1–0.5 l of sterilised water. The cultures can be maintained at room temperature, but the necessary feeding and renewal frequencies are lower if the rotifers are kept in the light at 7–10°C (strain-dependent). The stock cultures will need to be renewed approximately once every month, or even less frequently at low temperatures. If all the stock cultures become contaminated by other zooplankton, single rotifers should be selected under the microscope, carefully and repeatedly washed in sterilised water, and then transferred to small units containing sterilised water and microalgae. A thorough inspection is then needed to conﬁrm success. 4.3.3.2 Inoculation Phase New cultures may be started using an inoculum taken from a production culture or from a stock culture (see Fig. 4.3). The quality of the production culture is important (e.g. egg

Live food technology of cold-water marine ﬁsh larvae

83

rotifer-1, abundance of harmful micro-zooplankton, concentration of organic particles). A critical evaluation of the inoculum quality is an efﬁcient precautionary measure against problems during later rotifer cultivation. One part of inoculum culture to 10 parts of water is suitable, and the culture (>30 rotifers ml-1) may be fed normal feed rations. The risk of severe rotifer mortality is highest during the ﬁrst phase of the cultivation process. The risk may be reduced if the rotifers are fed >1–2 mgC l-1 of microalgae (visible colour) during the ﬁrst 1–2 days after inoculation. Another way to reduce risks is to increase the initial inoculation density to >100 rotifers ml-1. A combination of high initial rotifer density, careful rinsing of the inoculum, and initial feeding by microalgae will normally secure success. The number of rotifers that is available is low if the inoculum is taken from stock cultures. A suitable method is then to grow the rotifers with microalgae only during the ﬁrst few days (densities >2 rotifers ml-1). This is easily done by inoculating them with microalgae in illuminated rotifer tanks (light tubes or other sources, >100 W m-3) at 10% strength of a normal algal medium (formulations in Smith et al., 1993; Coutteau, 1996). The main production feed should be added before the algae become grazed down by the rotifers, but the rotifers should be fed algae only for ca. 2 days. This method is also appropriate if the quality and viability of the production cultures are poor. The green algae Tetraselmis spp. have been shown to support culture self-cleaning quite efﬁciently. Many contaminating micro-zooplankton species cannot ingest the large Tetraselmis cells very efﬁciently, and will probably be out-competed by rotifers after the change in food source. 4.3.3.3 Early Growth Phase The ﬁrst 2–6 days of the cultivation process, when food rations and rotifer densities are raised (see Fig. 4.3), has been shown to be the most critical phase of rotifer production. As mentioned above, prophylactic measures to counteract potential problems are high initial rotifer densities and thorough quality evaluation of the inoculum. A further serious problem of the initial phase is related to a mismatch between the food ration offered and the rotifers’ food requirements for growth. Feeding a high speciﬁc food ration (SFR, food per rotifer per day) is important in order to obtain a rapid growth rate and viable rotifers. On the other hand, overfeeding may cause unfavourable environmental conditions (low oxygen, high reactive ammonia and extensive bacterial growth) and enhanced rotifer mortality. Feeding during the early phase of growth must be based on the rotifers’ actual food requirements for growth. The speciﬁc growth rate of rotifer cultures for a given feed is a function of the speciﬁc food ration (SFR, e.g. mg yeast rotifer-1 day-1) supplied. The relationships between growth rate and speciﬁc feeding rate obtained during batch and continuous cultivation are shown in Fig. 4.4A. Figure 4.4B shows the same relationship, but using feeding rate per day (day-1, food C per rotifer C and day), which is assumed to be less straindependent than the former expression. The ﬁgures reveal that the rotifer cultures must be fed approximately 0.5 mg baker’s yeast (plus 0.05 mg oil) per rotifer per day, or 0.5 day-1 (oil included) in order to maintain a positive net growth rate. The growth response is comparable for batch and continuous cultures when the feeding rate is below 1 mg yeast per rotifer per day, yielding growth rates below 0.2 day-1. For higher food rations, the growth response for a given food ration becomes dependent of the cultivation method used. The lower spe-

84

Culture of cold-water marine ﬁsh

Growth rate, day-1 0.6

Growth rate, day-1 0.6 0.5

A

0.4

0.4

0.3

0.3

0.2

0.2

0.1

B

0.5

Continuous Batch

0.1

Continuous Batch

0

0

0

1 3 5 0 2 4 Feeding rate, mg Yeast (rotifer day)-1

0.5

1 1.5 2 2.5 Feeding rate, day-1

3

3.5

Figure 4.4 Relationship between rotifer feeding and growth rates. Speciﬁc population growth rate (day-1) as a function of feeding rate expressed (A) in terms of mg wet yeast rotifer-1 day-1, and (B) in terms of day-1 (mgC day-1 mg rotifer-C-1).

ciﬁc growth rates obtained in continuous cultures are primarily the result of higher feed losses compared with those of closed batch cultures. Cultures harvested continuously at a high rate are characterised by high water turbidity (see below), which means high losses during water exchanges. The batch cultures are closed and the food is better utilised under high feeding conditions. The curves shown in Fig. 4.4A can be described by the following empirical equations: m net = 0.377 (SFR - 0.488) SFR

(continuous cultures)

(4.12)

m net = 0.305[ln(SFR ) + 0.715]

(batch cultures)

(4.13)

where mnet is the net speciﬁc growth rate of the rotifer cultures (day-1), and SFR is the speciﬁc food ration (mg yeast rotifer-1 day-1). The equations relate the growth rate and food rations given to the rotifers, and the net growth rate together with measured rotifer densities per unit volume (RD) are needed for predictions of production (P). P = m net RD

(4.14)

Equations 4.12 and 4.13 can be rearranged to express the speciﬁc food ration needed to sustain a given net growth rate. SFR = 0.184 (0.377 - m net ) SFR = 0.489(26.6)

m net

(continuous cultures)

(4.15)

(batch cultures)

(4.16)

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85

Table 4.1 Detailed procedures of rotifer feeding during the early phase of growth. Equations and deﬁnitions are given in the text. Internal control method 1 Make decision on culture growth rate (for example 0.3 day-1, the recommended range is 0.2–0.4 day-1) 2 Estimate the corresponding speciﬁc food ration (SFR) using Equation 4.16 (mnet = 0.3 day-1 requires 1.3 mg yeast individual-1 day-1, SFR range for the recommended mnet-range (0.2–0.4 day-1) is 0.94–1.8 mg yeast individual-1 day-1) 3 Count rotifer density (RD) per unit volume 4 Estimate the culture food ration (FR, food per unit volume and day) (FR = SFR · RD) 5 Feed the estimated ration to the culture (in one, two or several portions) 6 Repeat steps 3–6 with constant SFR for the next few days until the maximum sustainable feed ration (FRmax), which has already been determined, is reached. Feed the cultures their maximum sustainable feed ration from that day External control method 1 Make decision on culture growth rate (for example 0.3 day-1, the recommended range here is 0.2–0.4 day-1) 2 Estimate the corresponding SFR using Equation 4.16 (mnet = 0.3 day-1 requires 1.3 mg yeast individual-1 day-1, SFR range for the recommended mnet-range (0.2–0.4 day-1) is 0.94–1.8 mg yeast individual-1 day-1) 3 Count initial rotifer density per unit volume (RD0) 4 Estimate the initial culture food ration (FR0, food per unit volume and per day on day 0) (FR0 = SFR · RD0) 5 Feed the estimated day 0 culture ration (FR0) to the culture (in one, two or several portions) 6 For the next day (day 1) and later (days 2, 3, . . .), estimate the food ration for day n according to Equation 18. The daily percentage increase in FR depends on the predetermined growth rate. A growth rate of, for example, 0.3 day-1 requires a daily increase in the food ration (IR) of 35% per day (Equation 4.17) 7 If the quality of the cultures is acceptable (see below), feed the day n dose 8 Repeat steps 6–8 until the maximum sustainable feed ration, (FRmax) which has already been determined, is reached. Feed the cultures their maximum sustainable feed ration from that day

A thorough knowledge of rotifer food requirements is the basis for safe and reproducible production. Two basically different methods, which are operationally quite close, may be used to raise rotifer densities of cultures from low to high levels during the early phase of production in batch culture (Table 4.1). Internal Control Method The rotifers are fed a constant speciﬁc food ration (SFR, mg food individual-1 day-1) all through the early growth phase. The estimated culture food ration (FR, g food m-3 day-1) will then depend on the actual growth response of the rotifer population. This means that the actual growth response of the rotifers controls feeding. The cultures must be counted daily. External Control Method The initial feeding conditions are estimated in the same way as for the internal control method, but thereafter the rotifers are fed according to a ﬁxed feeding regime where the culture food ration (FR) is increased exponentially to sustain the expected exponential increase in rotifer density. Further control is therefore mainly in the hands of the producer. The cultures do not need to be counted daily, but the quality of the cultures should be evaluated visually before the food is added. The exponential rate of increase in food rations (IR, % day-1) must be kept within

86

Culture of cold-water marine ﬁsh

the growth capabilities of the rotifers. A net growth rate range of 0.20–0.40 day-1 corresponds to an IR range of 22–49% day-1, as estimated by Equation 4.17. IR = (e mnet - 1)100%

(4.17)

The food ration for day n (FRn) can be calculated as FR n = FR 0 ◊ (IR 100 +1)

n

(4.18)

where FR0 is the food ration at the initial day (n = 0) and RI is the daily increase rate of the food ration. For both methods, cultures should be inspected visually and only be fed if the food is consumed and the culture quality is satisfactory (e.g. low turbidity caused by small particles). Cultures of poor quality should be treated speciﬁcally, and optionally brought out of production (see below). It is also important to realise that there will always be a critical upper value for the culture food ration (FRmax, g m-3 day-1). This critical value for sustainable rotifer production will depend on cultivation method, system design, feeding mode, food quality and oxygen supply. The maximum sustainable ration for the simplest techniques and small production units (<1 m3) is typically about 250 g wet yeast m-3 day-1. These rotifer cultures will contain 200–800 rotifers ml-1 in normal operation, and aeration will not be required during internal transport. Both methods have yielded similar growth curves when tested in two successive experiments run during training courses (Fig. 4.5). The rotifers in the ﬁrst test were fed to grow at 0.4 day-1 (see Table 4.1 and legend), and the measured average growth rate was 0.39 day-1 for both cultivation methods (Fig. 4.5A). In the second trial, the cultures were not fed on day 0, and the growth rate was lower than expected, but still identical for both methods. For both methods and trials, the egg ratios showed a marked peak 2 days after inoculation, reaching values well above the steady-state value corresponding to maximum growth rate. The time courses obtained are typical, and the oscillations reﬂect the changing age structure and culture synchrony, with lower culture mortality rates in the initial phase than at the end. The general lesson is that both methods are suitable for rotifer production, and that the cultures should be fed from the ﬁrst day. 4.3.3.4 Late Growth Phase—Harvesting Strategies The methods used to produce rotifers after the early phase of increasing rotifer densities are highly diverse, and cannot easily be generalised. From a general point of view, however, we may deﬁne two distinctively different methods, and claim that most methods used are combinations of these principal cultivation methods. Batch Production Production in a closed culture system supplied only with the resources needed for growth. Cultures are completely harvested at a certain developmental stage or time in the late phase of growth.

Live food technology of cold-water marine ﬁsh larvae

Rotifer density, ind mL-1

Rotifer density, ind mL-1 100

87

Int1

A

Int1

B

200

Int2

Int2

200

Int3

Int3

100

Ext1

Ext1

100

Ext2 Ext3

50

Ext2

50

Ext3 30

20

30 0

2

4

6

0

8

2

4

6

8

Days

Days

Egg ratio, eggs ind-1

Egg ratio, eggs ind-1

1

1

C

Int1

0. 8

Int2

0. 6

Int3

0. 6

Ext1 0. 4

Int1

D 0. 8

Ext3

Int3 Ext1

0. 4 Ext2

Ext2 0. 2

Int2

0. 2

Ext3

0

0 0

2

4 6 Days

8

0

2

4 Days

6

8

Figure 4.5 Rotifer growth and egg ratio developments during the testing of a comparison of the internal control method (Int) and the external control method (Ext) for the early growth phase of B. plicatilis. A, B. Increase in rotifer numbers with time for Int (three replicates, solid lines) and Ext (three replicates, dotted lines) methods tested in two trials. C, D. Variation in egg ratio with time for Int (solid lines) and Ext (dotted lines) methods tested in two trials.

Continuous Production Production in an open culture system that is supplied with the necessary resources and harvested regularly by replacing a ﬁxed volume of the culture by seawater once daily, or continuously, during the late phase of growth. Both methods and appropriate combinations are feasible for production. The priorities of the producer regarding rotifer quality, costs, risks and hatchery/laboratory routines may affect the choice of method. Adequate knowledge of rotifer biology, their nutritional and environmental requirements, and the traits of the speciﬁc cultivation system are important for sustainable and safe mass cultivation of rotifers.

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Culture of cold-water marine ﬁsh

4.3.3.5 Production in Batch Culture Growth in batch culture during the early and late phases involves variable and transient conditions for the rotifers. The animals are initially exposed to sufﬁcient food and a promising prediction for life (high SFR). Later, when the cultures are fed the maximum sustainable feed ration, they experience severe food limitations, reduced growth rates and enhanced mortality. In the transition phase, they undergo major changes in their individual biomass, biochemical composition and nutritional value. This is how life is in batch cultures, and efforts to reduce transitions to a minimum will pay off during mass production. Models for Growth and Production The speciﬁc net growth rate (mnet) of rotifers grown in batch cultures can be expressed as a function of the rotifer density (RD). m net = m max (1 - RD CC)

(4.19)

RD CC = 1 - m net m max

(4.20)

which is equivalent to

where mmax is the maximum net speciﬁc growth rate of the culture, and CC is the carrying capacity for the actual resource supply rate. CC is the maximum steady-state biomass obtained when feeding the maximum sustainable food ration (FRmax). Both coefﬁcients can be determined experimentally. Equation 4.19 shows that the growth rate will be inversely related to the rotifer density, which can be expressed as a function of time by RDt = CC [1+ (CC RD0 - 1)e - mmaxt ]

(4.21)

where RDt is the rotifer density on day t, and RD0 is the initial rotifer density (day 0). The speciﬁc net growth rate multiplied by the rotifer density expresses the net rotifer production (Pt). Pt = m net RDt = m max (1 - RDt CC)RDt

(4.22)

This function exhibits an optimum when the rotifer density is 50% of the carrying capacity. The corresponding net growth rate is then 50% of the maximum growth rate (mnet = mmax/2). The optimum net production (Pmax) is then expressed as Pmax = 0.25CCm max

(4.23)

The optimum production (Pmax) is proportional to both the maximum growth rate and the carrying capacity, and the latter variable is also proportional to the food ration. The value of CC may be substituted by the maximum sustainable food ration (FRmax, see Equations 4.24 and 4.25, below) to express the optimum production as a function of this

Live food technology of cold-water marine ﬁsh larvae

89

maximum food ration. Moreover, Equation 4.8 may be extended to include mortality and gross growth rates (see example below), whereas Equations 4.9–4.11 can be used to substitute egg ratio for growth rate. The model can be used for an analysis of production efﬁciency and several other aspects of rotifer production. Rotifer Density and Feed Ration As discussed above, the rotifer density will stabilise at a constant level when a constant amount of feed is added daily to the rotifer culture for a period of time. This level denotes the carrying capacity (CC) at the given feeding conditions. Model curves expressing the rotifer biomass in batch cultures fed variable maximum food rations are shown in Fig. 4.6A, whereas the general relationship between maximum feed ration and rotifer yields are shown in Fig. 4.6B and C. The maximum feed ration used during the late phase of growth obviously affects the ﬁnal stationary biomass level but not the initial speciﬁc growth rate of the cultures, which is determined by the feeding conditions during the early phase (see above). The relationship between maximum food ration and carrying capacity clearly shows that CC is expected to increase linearly with increasing food ration. The curves shown are expressed by CC Ind = 2.9FR Yeast

(4.24)

CCC = 1.8FR C

(4.25)

where CCInd is the carrying capacity expressed in terms of 106 individuals m-3, FRYeast is the food ration in terms of g fresh yeast m-3 day-1 (+10% oil, by weight), CCC is the carrying capacity in terms of g rotifer-C m-3, and FRC is food ration in terms of g feed-C m-3 day-1 (oil included). The quantitative relationship between food ration and carrying capacity should be established for the cultivation system used, because feed losses, rotifer strain and feed quality are among the variables that may affect the relationship. However, the curves in Fig. 4.6 are believed to be general. This implies that the carrying capacity can be assumed to be proportional to the food ration for a wide range of ration values. Some care should be taken, but increased sedimentation of feed and an enhanced density of dead particles in the cultures will most probably accompany deviations from the general linear relationship. Development over Time, and Window for Optimal Harvesting Rotifer production in batch cultures calls for a harvesting strategy that secures both efﬁcient production and acceptable live-food quality. The simulations in Fig. 4.7 are an attempt to illustrate a feasible time-window for harvesting cultures (vertical lines). The increase in rotifer density is exponential in the initial phase, and levels off at a stationary value after 2 weeks cultivation (Fig. 4.7A). Meanwhile, there is a steady non-linear decrease in the rates of gross and net growth rates, and a minor increase in the mortality rate (Fig. 4.7B). The gross growth rate and the mortality rate are levelling off to an identical value after 2 weeks, while the resulting net growth rate levels off at zero.

90

Culture of cold-water marine ﬁsh

Rotifer density, ind mL-1

CC, mill ind m-3

A

B

1 500

100 0 1 000

500

300 0 0

100

200

300

400

500

600

FR, g Yst m-3 day-1

100

CC, gC m-3

C

200 150

30

100

75 150 300 600

50

10 0

5

10

Days

15

20

0 0

20

40

60

80

100

120

FR, gC m-3 day-1

Figure 4.6 B. plicatilis growth and biomass yields in batch cultures fed baker’s yeast and capelin oil (10% of weight) (20 p.p.t., 20°C). A. Model growth curves for batch cultures fed various ﬁnal rations of food (75–600 g yeast m-3day-1). B. Carrying capacity in terms of mill rotifers m-3 as a function of feed ration expressed as g yeast m-3 day-1. C. Carrying capacity in terms of gC m-3 as a function of feed ration expressed as gC m-3day-1. Simulations are based on Equations 21, 24 and 25.

The gross production of the cultures (Fig. 4.7C) shows a maximum after 10 days, and remains relatively stable and high in the time that follows. The mortality increases as an effect of the population increase and the increased speciﬁc mortality rate, reaching the same level as the gross production rate at the end. The net production shows a very distinct optimum after 8 days, and values close to zero after 2 weeks of cultivation, which may seem surprising. The patterns of variation in biomass and activity shown in Fig. 4.7A–C are important in order to deﬁne an optimal time-window for harvesting batch cultures. The nutritional value of the rotifers should also be considered. The individual biomass, energy contents and protein contents are dynamic variables exhibiting considerably lower values in starved cultures than in well-fed ones. This is a combined effect of variable egg ratio and body weight. Figure 4.7D illustrates dry matter and protein per rotifer during growth. Fast-growing rotifers are beneﬁcial, because their energy and protein contents are higher than for starved rotifers. The suggested harvesting window is therefore questionable from a nutritional point of view. The overall evaluation may be as follows. From a production efﬁciency perspective, harvesting should not be undertaken before the culture has reached its optimum for net pro-

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Figure 4.7 Culture growth characteristics of B. plicatilis as a function time during growth in batch culture. A. Rotifer numbers (individuals ml-1). B. Net speciﬁc growth rate, speciﬁc mortality rate and gross speciﬁc growth rate (gross growth rate) (all day-1). C. Net production rate, mortality rate and gross production rate (all individuals ml-1 day-1). D. ng dry matter and protein per individual rotifer. Vertical lines indicate the suggested time-window for harvesting.

duction. At that time, the rotifer density will be 50% of the carrying capacity, the net growth rate will be half the maximum speciﬁc growth rate, and the predicted egg ratio will be 0.43 egg rotifer-1. The net production rate is also acceptable during the ﬁrst days following the production optimum. The nutritional value is rapidly reduced after 5–7 days, but may still be acceptable at the production optimum on Day 8. In practical rotifer production, a timewindow for harvesting (TWH) is more feasible than one single day. An overall evaluation

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suggests that batch cultures should be harvested when the rotifer density is between 50 and 75% of the carrying capacity of the culture (see vertical lines). If no post-enrichment is undertaken, or if no algae are added along with the rotifers in the larval tanks (see Chapter 7), harvesting should be undertaken closer to the 50% CC level. The suggested window represents a trade-off between production efﬁciency and nutritional quality, and all producers will have to do their own evaluations. 4.3.3.6 Production in Continuous Culture The growth conditions in continuous cultures involve far less variability than those in batch cultures. The standing rotifer biomass, as well as the food supply, are relatively constant, and the animals are exposed to a steadier food limitation. Gross and net growth rates, mortality and environmental conditions are theoretically becoming constant and independent of time. Cultures may be maintained for a long time under such conditions provided that techniques and cultivation systems are adequate. Operational Conditions A constant fraction of the culture is harvested and replaced by water each day during production in continuous culture. Water may be continuously exchanged with culture by the use of a pump system which keeps the total volume of the culture constant. This is the strict principle of a chemostat culture, which is widely used as an ecological model system. Alternatively, the water may be exchanged in pulses, the most common approach in rotifer cultivation presumably being one daily dilution. For practical reasons, continuous cultivation is deﬁned as including one daily dilution in the present chapter. The culture may be added with the food ration and dispersed in the new water, or it may be added separately. The ﬁrst approach is appropriate if the rotifers are fed an algal culture. Conversely, with yeast or formulated feed it may be most convenient to add the food separately. It may then be added in a continuous mode, in one daily ration, or in any other intermediate mode. A general recommendation is one daily water exchange and a relatively continuous mode of feeding, which is particularly important if the rotifer densities are high (>1000 rotifers ml-1). It should be noted that anything other than one daily water exchange and one daily feeding require partial automation of the cultivation system. Under conditions of steady oxygen supply, there is a marked drop in the ambient oxygen of the cultures, which reaches a minimum 4–6 h after the addition of food. This drop is probably related to increased defecation and microbial activity in the period after food supply and increased feeding rate. This oxygen drop is highly reproducible and is related to the food dose, and the culture may collapse if the concentration becomes lower than the critical value for the strain (e.g. 2–3 mg O2 ml-1). Continuous feeding will reduce this potential problem. Models for Growth and Production in Continuous Cultures The variation in rotifer density with time (dRD/dt) in cultures that are harvested (diluted) and fed at constant rates can be expressed as

Live food technology of cold-water marine ﬁsh larvae

dRD dt = RD(m net - D)

93

(4.26)

where RD is the rotifer density, mnet is the net speciﬁc growth rate, and D is the speciﬁc dilution rate, deﬁned as dV/(dt V) if dV/dt is the daily volume exchanged and V is the culture volume. Cultures of rotifers, or any other microorganisms maintained in continuous culture, tend to reach a steady state of growth characterised by constant numbers of organisms independent of time. This implies that dRD/dt = 0, meaning that the speciﬁc growth rate of the rotifers is identical to the culture dilution rate. m net = D = dV (dt V )

(4.27)

The mathematical relationships become somewhat more complicated if the process of dilution is not strictly continuous in time. If the cultures are diluted at a frequency of t days, the growth rate is given by the following general equations: m net = ln [V (V - dV )] t

(4.28)

m net = - ln (1 - dV V ) t

(4.29)

which is equivalent to

Reorganisation of Equation 4.29 gives dV V = 1 - e - mnett

(4.30)

where t = 1 for one dilution per day. The above equations relate the operational variable (harvested volume, dilution rate) to the growth rate of the cultures. It must be emphasised that the equations are only strictly valid for steady-state conditions. The rotifer density just before harvesting should then be constant and independent of time. The harvesting rate in continuous cultures (F%) is a practical term that can be deﬁned as F% = dV V 100%

(4.31)

The steady-state relationship between harvesting rate and the speciﬁc net growth rate of the rotifers is F% = 100%(1 - e - mnett )

(4.32)

m net = - ln(1 - F% 100) t

(4.33)

which is equivalent to

The above equations are fundamental for predicting the growth and production of rotifers in continuous steady-state cultures, and are valid for all harvesting frequencies (t is days between dilutions).

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The daily net production in continuous cultures (Pd) can be predicted as rotifer density multiplied by the net growth rate: Pd = RDad (e mnett - 1)

(4.34)

Pd = RDbd (1 - e - mnett )

(4.35)

or

where RDad and RDbd are rotifer densities just after and before dilution, respectively. Alternatively, the daily rotifer production of continuous cultures can be estimated as rotifer density at the time of harvesting (RDbd) multiplied by the harvested volume (dV). Pd = dV ◊ RDbd

(4.36)

The theoretical optimal harvesting rate of continuous cultures is the rate that corresponds to half the maximum growth rate of the rotifer culture, as in batch culture. The maximum speciﬁc growth rate is variable (temperature, salinity, strain), but is easily measured at given conditions. This fact makes the optimisation of production in continuous rotifer cultures fairly easy. The optimal production can be estimated by substituting 0.5 mmax for mnet in Equations 4.34 and 4.35. The replaced volume (dV) that results in optimal production efﬁciency (dVopt) can be estimated as dVopt = V (1 - e -0.5mmaxt )

(4.37)

Finally, it should be noted that the production at a given harvesting rate will also depend on the food ration given, because this food ration will affect the rotifer density (RD). As a ﬁrstorder approximation, the rotifer density, and therefore also the production, can be assumed to be proportional to the food ration at any dilution rate (Equation 4.24). Dynamics of Growth in Continuous Rotifer Cultures As mentioned above, the rotifer density of the culture will become constant after some time (a few days or a week), if a constant fraction of the culture is harvested and replaced by seawater each day. This means that the net growth balances the losses from harvesting, and that the speciﬁc growth rate of the rotifers is constant (steady state of growth). Typical time-courses found in cultures harvested at rates of 5, 18 and 27% day-1 are illustrated in Fig. 4.8A. The average rotifer densities are inversely related to the harvesting rate or the culture growth rate. Figure 4.8C and D shows an inverse, linear relationship between the speciﬁc growth rate and the rotifer biomass expressed in terms of numbers and carbon biomass. Low growth rates result in relatively dense cultures. The carrying capacity can be found if the curves are extrapolated to a zero harvesting rate. High growth rates result in low rotifer density, and the maximum speciﬁc growth rate is indicated if the curves are extrapolated to zero density.

Live food technology of cold-water marine ﬁsh larvae

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Figure 4.8 Characteristics of B. plicatilis grown at various rates in continuous cultures. A. Development of rotifer densities with time in cultures diluted at various rates (mill individual m-3). B. ng dry matter and protein per individual rotifer. C. Steady-state rotifer densities (mill individual m-3). D. Steady-state rotifer biomass (gC m-3). E. Steady-state rotifer production rates (mill individual m-3 day-1). F. Steady-state carbon production rates (gC m-3 day-1).

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Figure 4.8E and F shows the measured and predicted production of the same cultures (see legend). The optimum production level for intermediate growth rates is clearly indicated. The measured and predicted values are very close for growth rates below the optimum level, but deviations become more pronounced for the higher range of values. The nutritional value of the rotifers increases with growth rate (Fig. 4.8B), with intermediate values at the optimum growth rate for production. It is important to recognise that the rotifer growth rate, their nutritional value, and their production efﬁciency can all be controlled during continuous production simply through selection of the dilution rate of the culture. Continuous production of rotifers therefore allows a more efﬁcient control of both production and food quality than production in batch culture. Finally, the equations presented for batch and continuous production allow a wide range of further simulations and calculations that cannot be further elaborated here.

4.3.4 High-Intensity Rotifer Cultivation The next generation of rotifer production technology with very high rotifer densities is now being developed in research laboratories. Japanese scientists were the pioneers of this developmental work, and they have been able to produce and maintain B. rotundiformis in densities higher than 100 000 individuals per ml in 1000-l batch cultures. This has been made possible by feeding the cultures concentrates of Chlorella, and the implementation of strict environmental control of the cultures (Yoshimura et al., 1997). A second attempt to develop highly intensive rotifer cultivation systems was made with B. plicatilis fed with the formulated feed Culture Selco. Belgian scientists were the ﬁrst to maintain the bigger B. plicatilis in densities of 7–10 thousand in stable cultures that were harvested throughout many weeks. Environmental control in the cultures is achieved by circulating the culture water in an external loop in which it is thoroughly puriﬁed. The rotifers remain in the culture tank, and can be harvested in batch or continuous modes independently of the water circulation rate (200–500% day-1). The circulating water is ﬁrst sterilised with ozone, then treated several times by protein skimmers, and ﬁnally treated and recolonised by bacteria in a bioﬁlter before being transferred back to the culture. Feasibility studies have shown that the technology reduces the costs of rotifer production compared with traditional techniques (Suantika et al., 2000). Industry will always be hesitant to change their live-food technology, and it is not surprising that high-density cultivation systems for rotifers are still not being used in commercial hatcheries. Both of the cultivation methods and systems described above have been thoroughly tested. The technologies will be further improved, and will probably be implemented in commercial hatcheries in the coming years. Skilled staff is a precondition, and lower production costs are a driving force.

4.3.5 Problems in Rotifer Cultivation A common problem in rotifer cultivation is overfeeding, which in turn may cause acute oxygen deﬁciency, high concentrations of reactive ammonia, or the invasion of microzooplankton (e.g. ciliates). Another problem is inadequate environmental control that results

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97

in bad water and culture quality. These problems may sometimes be difﬁcult to diagnose. A third main problem is speciﬁc infections or contamination that results in fatal diseases and enhanced mortality. 4.3.5.1 Feeding-Related Problems Two types of cultivation problems are related to the food and the feeding process. The food may be nutritionally inadequate, which means that limitations in the supply of essential compounds will occur during long-term cultivation. The second main problem is indirect, and is the result of a disparity between food supply and food consumption. Baker’s yeast and marine oil, microalgae and commercial formulated feeds have all been shown to support rotifer growth and reproduction. The use of dried yeast and other crude powder diets is not recommended because of their high losses of organic compounds in seawater. A high organic load may create microbial problems, and physical/chemical stability in seawater is an important requirement for a formulated rotifer diet. Emulsiﬁed marine oils create fewer microbial problems than powder diets. In general, the oil droplets are probably less available as a bacterial substrate than dissolved organic compounds and organic particles, and emulsiﬁed oils tend to be more inert than crude oils. A disparity between food ration and food consumption is probably the most common problem in rotifer cultivation, particularly during the critical early phase of growth. The rotifers may occasionally be either over-fed or starved. Severe mortality is likely to occur with sudden changes in the speciﬁc food ration (SFR). Sudden decreases in SFR will often result in reduced viability and enhanced population mortality. On the other hand, sudden increases to levels above the maximum consumption capacity of the rotifers may cause very unfavorable hygiene conditions and harmful invasions of micro-zooplankton in the cultures. The feeding frequency of the rotifer cultures may also affect the quality of the cultures. There is an obvious limit to the maximum single food ration that can be added (see above). The present strain has tolerated 250 g yeast plus 25 g oil m-3 day-1 without experiencing a fatal oxygen deﬁciency or abnormal feeding activity, but such a high dose is not generally recommended. The mode of food supply may also affect the competition between rotifers and micro-zooplankton species, but the general mechanisms are not well understood. 4.3.5.2 Environmentally Related Problems Pollution of the process water may cause severe toxicity problems, the nature of which is beyond the scope of this chapter. Many problems which are related to water quality are difﬁcult to diagnose precisely unless a speciﬁc hypothesis on the nature of the problem can be established. However, most of the problems that originate with bad water quality will become expressed in reduced rotifer egg ratio or birth rate. Acute oxygen deﬁciency may occur when the food ration becomes high, and maintenance of an adequate oxygen supply is more challenging in large cultures than in small. The wall and bottom zones of tanks may rapidly become anaerobic. This may, in turn, inhibit population growth in the entire culture. Continuous feeding is a way to avoid fatal oxygen deﬁ-

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ciency. Reactive ammonia may also accumulate in the late growth phase, but B. plicatilis apparently has a relatively high tolerance to reactive ammonium. However, some care should be taken to ensure that the pH does not become low while the cultivation temperature is high. 4.3.5.3 Disease and Contamination Parasites, bacteria or viruses that may cause rotifer death, as well as pathogens for the ﬁsh larvae, may infect the rotifer cultures. Specialists must carry out the diagnostic work in the case of serious infections (see Chapter 3), and detailed knowledge is important in choosing an efﬁcient counter-measure, which may involve complete disinfection of the entire production system for live feed and ﬁsh. Some micro-zooplankton species are harmful to rotifers, at least temporarily. Some ciliate species tend to stress the rotifers by becoming attached to their ﬁltering system. The rotifers react by rapid swimming movements (rotating and swimming in circles) and enhanced mortality. Some of the common ciliates in rotifer cultures are too big, or they swim too fast, to be eaten efﬁciently by the rotifers. Many the species normally cannot compete efﬁciently for suspended food particles because they are benthic species that are only able to survive in pelagic waters if the food concentration is high. Some ciliate species, which amongst others colonise and eat dead rotifers in the sediments, may cause serious problems during cultivation. The most harmful species are those which interact physically with the rotifers, either by disturbing their ﬁltering activity, or by colonising and killing them directly. There are some indications that these attacks primarily occur when the rotifers are in bad shape for some reason. 4.3.5.4 Problem Identiﬁcation—Diagnostic Criteria Some easily measurable characteristics of rotifer cultures can be used to diagnose problems. These are:

• egg ratio • swimming speed • turbidity of culture • abundance of suspended micro-zooplankton, mostly ciliates • abundance of attached micro-zooplankton If the net increase in rotifer numbers throughout is lower than the net growth rate predicted based on the egg ratio, it may indicate an enhanced mortality rate in the cultures. On the other hand, if an unexpected reduction in net growth rate is accompanied by a correspondingly low egg ratio (birth rate), the problem is normally related to sub-optimal food quality/quantity or unsatisfactory environmental conditions. Some rotifer producers use swimming speed as a criterion for rotifer viability (Snell et al., 1987). The biological basis for this method is the fact that old post-productive rotifers tend to swim more slowly than younger individuals. The average swimming speed can therefore give information on the age distribution and growth potential of the culture.

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The culture turbidity is a fast and useful predictor of culture quality and performance. The rotifers may perform very well with a relatively high abundance of larger suspended organic particles in the culture, but a high abundance of small particles can be an early sign of coming problems (unless there is obvious over-feeding). Both a high abundance of bacteria and micro-zooplankton, as well as leftovers of feed, may cause high turbidity, and a closer examination under a microscope may then be useful. If the turbidity is caused by yeast cells and oil droplets, and if the egg ratio of the rotifers is high, the situation may not be threatening. However, high turbidity along with an egg ratio that is lower than expected indicates reduced food consumption and birth rate (gross growth rate). The situation may be threatening, and the environmental conditions may be unsatisfactory (e.g. low oxygen, high reactive ammonia, and other pollution). Inadequate food quality is another explanation. However, this is unlikely if the food has already proved to be satisfactory in rotifer production. If high culture turbidity caused by food particles combined with low rotifer egg ratio is a common problem, nutritional factors should also be considered. A high abundance of micro-zooplankton (e.g. ciliates) in the rotifer cultures is an indication of over-feeding or inadequate cultivation routines. The situation is not necessarily threatening, but immediate measures should be taken to reduce the abundance of these organisms. A high abundance of ciliates that are attached to the rotifers is threatening. In any event, the situation is undesirable and will require some kind of action. 4.3.5.5 Counter-Measures Against Undesirable Situations Appropriate counter-measures against identiﬁed problems may involve changes in feeding procedures, direct rinsing or treatment of the cultures, the termination of cultures, or measures of a more prophylactic nature. An efﬁcient cultivation system, along with thorough monitoring, is probably the best way to avoid problems. A prophylactic measure already recommended during cultivation is to use microalgae as a supplementary food just after inoculation. Another prophylactic measure is to rinse the inoculum taken from production cultures very thoroughly. When cultures remain turbid, the normal measure will be to skip feeding. This treatment is also recommended if the abundance of suspended micro-zooplankton is high. Some other measures may also be efﬁcient against a high abundance of most micro-zooplankton species and bad hygiene conditions in general. These measures, which may be used alone or in combination, are:

• change of cultivation tank • change to algal-based feeding • starvation and cooling • freshwater treatment Infected cultures may become healthy by simply changing the cultivation tank. Starvation or an appropriate change in food source can be combined with this treatment. Cooling (10–15°C) in combination with starvation may work, but washing harvested rotifers for 5

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–10 min in fresh water is probably more efﬁcient. B. plicatilis will survive this treatment, whereas some of its competing ciliates will die. However, it is difﬁcult to get rid of harmful contaminants by washing, starvation and cooling alone. The problems may be temporarily reduced, but repeated treatments will normally be required. An application of microalgae as the only feed may be surprisingly efﬁcient in many cases.

4.3.6 Biochemical Composition During Steady-State Feeding and Growth The biochemical composition of rotifers used as live feed for marine larvae, and in particular their content of essential compounds, is paramount for their ability to sustain larval growth and survival. It is important to recognise that the biochemical composition is highly dynamic and variable throughout the phases of cultivation, nutrient enrichment and postenrichment. To control live feed quality throughout production, it is important to understand the fundamental biological mechanisms which mediate these changes. The overall integrated nutritional and environmental status of the rotifers is most efﬁciently expressed through their speciﬁc growth rate. Another important issue is the terms used to express nutritional value, e.g. essential amino acids and protein. The nutritional value may be expressed by:

• percentage of amino acid or total amino acids (weight or molar, the so-called proﬁle) • weight of amino acid per prey biomass (dry matter or other biomass expression) • weight of amino acid per individual prey The same applies to fatty acids and other compounds. The last expression relates nutrients to the individual prey, which is also the item that the larvae relate to during feeding. This term is the most variable and is sensitive to treatment, and may also be the most adequate term to express nutrient quality. Nevertheless, it may be a sound approach to include all terms in research work (Øie et al., 1997; Øie & Olsen, 1997; Makridis & Olsen, 1999). 4.3.6.1 Proteins and Essential Amino Acids Nitrogen-based estimates of protein derived using the common conversion factor of 6.25 mg protein/mg N yields overestimates, whereas amino acid-based estimates are likely to be slight underestimates. A conversion factor of 4.2 mg protein/mg N has been established through testing and analysis of amino acid proﬁles. This factor yields more equal results for estimates made for both zooplankton and ﬁsh larvae (Øie & Olsen, 1997). Protein is a major component of the biomass, and the content will to some extent reﬂect the energy level of the organism. The protein content per individual rotifer is a dynamic variable that is related to food availability and speciﬁc growth rate (Fig. 4.9A, modiﬁed from Øie & Olsen, 1997). The values may be relatively scattered, but several studies conﬁrm that fast-growing rotifers typically contain twice as much protein as slow-growing, starved rotifers, and a common range of variation for the present strain is 100–200 ng protein per individual. A positive relation to growth rate is also found for protein per dry matter, but this

Live food technology of cold-water marine ﬁsh larvae

Protein contents

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Figure 4.9 Protein contents and amino acid distribution of B. plicatilis grown at various rates. A. Protein contents expressed in terms of dry matter (open symbols) and individuals (solid symbols). B. Relationship between single amino acid (AA) contents in starved (m = 0.05 day-1) and well-fed (m = 0.22 day-1) rotifers. Solid symbols show amino acid values in percentage AA of total AA (proﬁles, slope 1.02, not signiﬁcantly different from 1, P < 0.05). Open symbols show amino acid values in terms of ng AA individual-1 (slope 1.53). The dotted line shows a 1 : 1 relationship.

Amino acids of well fed rotifers

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relationship is often not statistically signiﬁcant because protein is also a substantial part of the dry matter (Øie et al., 1997; Øie & Olsen, 1997). A very common misunderstanding is that the protein and amino acid contents of rotifers are constant and independent of growth conditions. This belief originates in their very stable amino acid proﬁles (Lubzens et al., 1989). Figure 4.9B illustrates that the percentage amino acid content of total amino acids is independent of feeding conditions (solid symbols, close to the 1 : 1 line), whereas protein per rotifer is signiﬁcantly higher in the well-fed than in the starved animals (open symbols, slope of curve 1.53) (data from Makridis & Olsen, 1999). The amino acid content is 53% higher in well-fed rotifers grown at 0.22 day-1 than in starved

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rotifers grown at 0.05 day-1. It is therefore an oversimpliﬁcation to conclude that protein and amino acid contents are constant. The contents per dry matter and per rotifer are indeed dynamic variables. Some authors have emphasised the importance of free amino acids during the very early larval stages (Fyhn, 1990, 1993). B. plicatilis exhibits relatively high levels of free amino acids (4–6% of the total amino acids, Øie et al., 1997; Øie & Olsen, 1997). In addition, its protein tends to disintegrate rapidly in the larval guts (Hjelmeland et al., 1993). 4.3.6.2 Lipids and Essential Fatty Acids The main fraction of the rotifer lipids is storage lipids with minor physiological functions (40–80%). This is different from the proteins, and it is therefore to be expected that lipids will relate differently to growth rate than protein. Figure 4.10A illustrates that the lipid contents per dry matter of rotifers shows a reduction with increasing growth rate, whereas the amount per individual remains constant. The range of lipid contents for fast-growing and starved rotifers, cultured and fed as described above, is typically 100 and 150 mg per g DW, respectively. The content per individual is typically 50 ng. This pattern is the opposite to that found for proteins, and the result is a more variable ratio of protein to lipid, which is positively related to the growth rate of the cultures (Fig. 4.10C). Although lipid per individual remains constant and is independent of the growth rate, the fatty acid percentage composition shows a substantial variation. The percentage n-3 HUFA becomes reduced when the growth rate increases, whereas the percentages of saturated and mono-unsaturated fatty acids become higher (Fig. 4.10B). Looking more closely into the group of n-3 HUFA, it is notable that DHA (22:6 n-3) is reduced twice as fast as EPA (20:5 n-3). The corresponding quantitative reduction of DHA is substantial. The characteristic patterns of lipids and fatty acids that are shown in Fig. 4.10 are the result of lipid metabolism, and not of variable dietary lipids. The higher lipid level per gram dry matter of severely starved animals is in apparent conﬂict with their severe food limitation. This must therefore imply that rotifers cannot utilise lipids efﬁciently during severe starvation, and that starved rotifers retain essential n-3 HUFA more efﬁciently than well-fed ones. This ﬁnding suggests that starved rotifers are nutritionally beneﬁcial from an n-3 HUFA perspective, which conﬂicts with their protein contents and the need for efﬁcient production. This is important knowledge that may have to be considered. The enrichment studies described below are all representative for rotifers grown at a rate of 0.1–0.2 day-1, which is a sub-optimal rate from a production efﬁciency perspective, but an optimal rate from an n-3 HUFA and lipid retention perspective. For low growth rates, the lipid content of the rotifers is positively related to the lipid content of their food, as shown in Fig. 4.10D. The response of enhanced food lipids on rotifer lipids is relatively moderate for naturally occurring lipid levels (<200 mg gDW-1), but stronger at higher levels. The present rotifer strain did not grow at food lipid levels above 30 mg gDW-1. It is important to note that the relationship between food and rotifer lipids may differ for fast-growing rotifers. The fatty acid composition of rotifer tissues is mainly a function of the composition of its dietary lipids and the inherent metabolic and genetic traits. B. plicatilis is presumably evolutionarilly adapted to warm water (Olsen, 1999a), and its requirements for n-3 fatty

Live food technology of cold-water marine ﬁsh larvae

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Feed lipids, mg g DW-1

DHA/EPA Protein/Lipid

Figure 4.10 Characteristics of lipids and fatty acid contents in B. plicatilis grown at various rates and feed lipid contents. A. Lipid contents expressed in terms of dry matter (open symbols) and individuals (solid symbols). B. Percentage n-3 HUFA and percentage saturated plus mono-unsaturated fatty acids. C. Ratios of DHA to EPA and of protein to lipids. D. Total rotifer lipids as a function of food lipid content.

acids are low. Dietary lipids will then affect the composition of both its triacylglycerides and its phospholipids. This means that diet composition is paramount, whereas the metabolic activity becomes less important, but still signiﬁcant (see Fig. 4.10A–C). This makes B. plicatilis very well suited to fatty acid manipulation, because its fatty acid composition becomes equal to that of its dietary lipids. Figure 4.11A and B illustrate the fatty acid composition of a dietary lipid emulsion and of rotifers that are fed that emulsion and baker’s yeast for >10 days (0.1–0.2 day-1). The fatty

104

Culture of cold-water marine ﬁsh

B

ASaturated

Saturated Monounsaturated

Monounsaturated

n-6

n-6

18:3 n-3

18:3 n-3

18:4 n-3

18:4 n-3

20:4 n-3

20:4 n-3

20:5 n-3

20:5 n-3

22:5 n-3

22:5 n-3

22:6 n-3

22:6 n-3

Sum n-3

Sum n-3 0 10 20 30 40 50 60

Dietary lipids

0 10 20 30 40 50 60 Rotifer lipids

Sum n-3 in rotifers, % of total FA 80

C 60

40

20

0 0

20 40 60 80 Sum n-3 in emulsion, % of total FA

100

Figure 4.11 Fatty acid distribution of steady-state rotifers grown at rates of 0.05–0.2 day-1. A & B Steady-state relationship between dietary (A) and rotifer (B) percentage fatty acid distribution. C. Steady-state percentage of n-3 fatty acids of rotifers as a function of the percentage n-3 fatty acid distribution of their dietary lipids (the dotted curve shows the 1 : 1 ratio).

acid proﬁles are almost identical; percentage DHA is slightly lower, while percentage DPA (22:5 n-3) and saturated fatty acids are slightly higher in the rotifer than in the emulsion. The total percentage of n-3 fatty acids remains almost equal. This close relationship between dietary and rotifer n-3 fatty acids is relatively independent of the n-3 content of the food, as illustrated in Fig. 4.11C. The response is close to unity and linear for n-3 contents <60% of total fatty acids. This implies that the rotifer contents of essential n-3 fatty acids can be completely controlled by careful selection of the dietary

Live food technology of cold-water marine ﬁsh larvae

105

oil used during cultivation. When the percentage n-3 fatty acid composition of the oil is known, the rotifer composition may be estimates as follows: Rotifer%n-3 = 0.91 Feed %n-3

(4.38)

Rotifer%EPA = 0.88 Feed %EPA

(4.39)

Rotifer%DHA = 0.72 Feed %DHA

(4.40)

These equations may be used to predict the n-3 HUFA contents of rotifers that are enriched during cultivation. The general concept of linearity is probably valid for all types of food and rotifer strains. The coefﬁcients are also likely to be valid for other types of easily digestible feed, but the coefﬁcients of the equations will depend on rotifer growth rate (here 0.05–0.2 day-1) and the cultivation time (>equilibrium time, 8–10 days). 4.3.6.3 Vitamins and Minerals Information about the vitamins and minerals in B. plicatilis is scarce, but accumulating (e.g. Sandnes et al., 1994; Merchie et al., 1995; Lie et al., 1997). A high number of components are involved, and fundamental knowledge about ﬁsh larval requirements is indeed very limited. This calls for a pragmatic approach, and the use of general knowledge and indexes established for cultured ﬁsh (e.g. Lall, 1989; NRC, 1993; see also Table 8.3 in Chapter 8). As for protein and lipids, there is a relationship between the vitamin and mineral contents of the feed and the resulting contents of the rotifer. This is exempliﬁed in Table 4.2 for the vitamin contents of rotifers grown on a number of different diets (data from Sandnes et al., 1994; Lie et al., 1997). None of the rotifers are obviously deﬁcient in vitamins, and the water-soluble ascorbic acid and thiamin become enhanced in rotifers that feed on the microalga I. galbana in the larval tanks (see ‘green water’ technique, Chapter 7). Minerals and trace elements are also found in reasonable amounts in the rotifers. A sub-optimal supply

Table 4.2 Vitamin contents (mg g DW-1) of B. plicatilis fed different diets (data from Sandnes et al., 1994; Lie et al., 1997).

Ascorbic acid Thiamin Riboﬂavin Pantothenic acid Niacin Pyridoxine Biotin Vitamin B12 Vitamin A (retinol) Vitamin E (alpha-tocopherol)

1 Baker’s yeast + capelin oil

2 Baker’s yeast + Super-Selco

3 Baker’s yeast + DHA Super-Selco

4 Diet 3 + Isochrysis galbana, 72 h

267 16 23 118 167 2 3 1 <1 122

167 16 26 107 214 7 3 2 4 640

372 5

1514 51

86 122

84 187

1

2

4 641

3 692

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Culture of cold-water marine ﬁsh

of vitamins and minerals is not believed to be a major problem during larval rearing, but speciﬁc sub-optimal components and limitations cannot be completely ruled out. Moreover, there is ample evidence that, for example, sub-optimal levels of ascorbic acid for growth may have positive effects on larval viability (Merchie et al., 1995).

4.3.7 Short-Term Enrichment Techniques to Improve Nutritional Value The earliest Japanese attempts to use B. plicatilis as live food for marine ﬁsh larvae failed because the organism was nutritionally inadequate with respect to n-3 HUFA. The introduction of methods of short-term n-3 fatty acid enrichment to overcome the nutritional deﬁciency of the live feed solved this problem (Watanabe et al., 1978, 1983). The current method involves exposure to n-3 HUFA-rich feed given in high concentrations for short time, normally less than 24 h. Normally, this treatment will not result in a signiﬁcant growth response, but only in a major change in biochemical composition. This technique was later applied for proteins as well as for vitamins. Methods to manipulate vitamins and trace metals in rotifers are not described in more detail here, but in principle they are comparable to the methods for lipids and fatty acids (Dhert, 1996; Lie et al., 1997). 4.3.7.1 Proteins The possibility of enhancing the protein level of rotifers through short-term enrichment is normally overlooked. The content may be enhanced in rotifers which have a low initial content, which is typical of slow-growing severely starved rotifers, but not in rotifers that have already obtained a high protein content during cultivation. Rotifers of a slow-growing culture (0.05–0.1 day-1) will typically contain 100 ng protein. If these rotifers are fed a high dose of microalgae, or a balanced formulated feed (e.g. Protein Selco, INVE Aquaculture, Belgium), they may increase their protein content by 30–50%, or to 130–150 ng protein per individual, over 24 h. This treatment will contribute to a higher energy content as well, which is important during ﬁrst feeding (see Chapter 7). Unlike protein per prey and dry matter, the amino acid proﬁle (percentage of total amino acids) is primarily genetically controlled, and cannot be manipulated through any type of enrichment. 4.3.7.2 Lipids and Fatty Acids Rotifers that are grown on dietary lipids that do not have an appropriate fatty acid composition must be short-term enriched before being used as live feed for cold-water ﬁsh larvae. The same may be true for rotifers maintained at a very high growth rate during cultivation (low DHA, see Fig. 4.10C). It should be noted that the lipid content of the diet is of moderate importance for the fatty acid distribution of the rotifer, since the most important feature is the fatty acid composition of the feed. Therefore, if care is taken while selecting dietary lipids, the rotifers may have reached satisfactory n-3 HUFA levels during cultivation. Their lipid and fatty acid levels, however, will become enhanced during short-term enrichment with fat diets. The quantitative responses in lipids, total fatty acids, EPA and DHA after adding variable rations of emulsiﬁed diets (Selco types, INVE Aquaculture, Belgium) to rotifer cultures are

Live food technology of cold-water marine ﬁsh larvae

R otif er lip id s, mg g D W -1

Fa tt y a cid s, % of t ot al FA 80

A

300

107

80

B

S at M ono

60

60

H UF A n–3

200

EP A

40

40 DHA

100

20

0

20

0

0

0

0 .2

0 .4

0 .6

0 .8

1

E nr ic h me n t r atio n, µ g in d -1 Lipids F atty a cids EPA D H A

0

0.2

0.4

0.6

0.8

1

E n r ich m en t r atio n, µg in d -1 S at M ono

H UFA n – 3

E PA DH A

Figure 4.12 Lipid, fatty acid and n-3 HUFA contents and percentage fatty acid distribution as a function of the enrichment of the food ration (pooled data from two trials). A. Total lipids, total fatty acids and quantitative contents of EPA and DHA. B. Percentage contents of important species and groups of fatty acids (right hand panel shows % values for feed).

illustrated in Fig. 4.12A. All components increase with increasing speciﬁc lipid ration (SLR), but the response levels off above a ration of 0.4 mg emulsion per rotifer per day. The pattern of increase is logarithmic and highly predictable for all the components shown. Equations describing the responses are given below. Lipid (mg gDW -1 ) = 39.7 ln(SLR ) + 309

(4.41)

Total FA (mg gDW -1 ) = 37.8 ln(SLR ) + 223

(4.42)

EPA (mg gDW -1 ) = 9.97 ln(SLR ) + 48.6

(4.43)

DHA (mg gDW -1 ) = 16.9 ln(SLR ) + 80.1

(4.44)

The patterns of increase found for lipids and total fatty acids are general and robust, but may depend on the initial contents. The equations for EPA and DHA will depend on the fatty acid contents of each diet, and therefore will not be general. The percentage contents of n-3 HUFA and other fatty acid groups as function of the food ration are shown in Fig. 4.12B. The general message is that the fatty acid percentage contents approach the composition of the diet as the ration increases (see right-hand panel). Percentage n-3 HUFA, DHA and EPA increases with food ration, whereas percentage saturated and monounsaturated fatty acids decreases. Figure 4.12 also shows that short-term enrich-

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Culture of cold-water marine ﬁsh

ment of rotifers is very predictable. In the present case, the percentage distributions were less affected than the quantitative contents of lipids and fatty acids. This was because the rotifers used were fed the same lipid emulsion during growth as in the later short-term enrichment. If rotifers with a poorer n-3 HUFA status had been used in the experiments, i.e. their initial percentage level would have been lower, their percentage content would have increased much more strongly. Combined cultivation and n-3 HUFA enrichment represent a simpliﬁcation of the production process, and there are other advantages as well. It is important to ask whether shortterm n-3 HUFA enrichment is needed when both lipid level and fatty acid composition can be controlled during cultivation. An important feature is that the lipid content of the rotifers becomes lower; 13–15% of dry matter, with extreme values of 10–25% (see Fig. 4.10). Short-term enrichment using emulsiﬁed oils will normally bring the lipid level to above 25% of dry matter. Such fat rotifers exhibit low tolerance when transferred to cold water (10–14°C), and a major fraction may die immediately. Some concern is also related to the fact that the natural food of many cold-water species is copepods with typical lipid contents of 10% of dry matter (see below). This may not be very important, but these copepods have served as a model for the biochemical composition of the cultivated live feed.

4.3.8 Stability of Nutritional Value Post-enrichment, the biochemical composition of the rotifers is a dynamic variable which speciﬁcally reﬂects temperature and food availability. The ﬁnal process of rotifer production may involve the rinsing and storage of cultures for some time before their ultimate use as live feed, and the treatment may involve starvation. The rotifers may also survive in larval tanks for some time before being eaten (see Chapter 7). This will happen because many ﬁsh larvae are not very efﬁcient at capturing live prey when they start to search for food. A practical measure against long residence times in the larval tanks is a high exchange rate of water, but this is impossible for the very early and fragile stages of many marine ﬁsh larvae. The delay between feeding and consumption will then involve starvation unless microalgae are added along with the rotifers in the larval tanks. Quantitative knowledge of the catabolic rates of essential compounds in the rotifer during starvation is useful to secure adequate post-harvest treatment and storage conditions for rotifer cultures. The loss of a biochemical component during starvation is an exponential process, which means that a ﬁxed fraction is lost per unit of time, not a ﬁxed amount. The nutrient content (Qt nutrient per rotifer) in a given period of starvation (t days) can generally be expressed as Qt = Q0 e Lt

(4.45)

ln(Qt ) = ln(Q0 ) + tL

(4.46)

which is equivalent to

where Q0 is the nutrient content at time zero (at harvest), and L is the speciﬁc loss rate of the nutrient, which may be lipids, proteins or other components. The time-courses illustrat-

Live food technology of cold-water marine ﬁsh larvae

ng DHA or EPA per rotifer

ng Protein or lipid per rotifer 100

ng Protein ind-1

109

4 ng DHA ind-1

ng Lip ind-1

ng EPA ind-1

3

80 60

2

40 1

20

B

A 0

1

2

3

4

5

0 0

1

2

Days

Loss rate for individuals, day-1 0.7

4

Fatty acids Sum n-3 0.8

EPA

0.5 0.4

0.6

0.3

0.4

DHA

C

0.2

5

Loss rate for individuals, day-1 1

Carbon Lipid Protein

0.6

3 Days

D

0.2

0.1

0

0 0

5

10 15 20 Temperature, ∞C

25

0

5

10 15 20 Temperature, ∞C

25

Figure 4.13 Quantitative losses of nutritional value in B. plicatilis during starvation. A. Losses of protein and lipid per individual as a function of starvation time at 18°C. B. Losses of DHA and EPA per individual as a function of starvation time at 18°C. C. Speciﬁc loss rates of body carbon, lipid and protein per individual as a function of starvation temperature. D. Speciﬁc loss rates of total fatty acids, sum of n-3 fatty acids, DHA and EPA per individual as a function of starvation temperature. The model curves are based on relations published by Olsen et al., 1993, and Makridis and Olsen, 1999.

ing the quantitative losses of some key components are illustrated in Fig. 4.13A and B, which show simulations for 18°C and no food addition. The model used is based on data published by Olsen et al. (1993) and Makridis & Olsen (1999). Both lipid per rotifer and protein per rotifer decrease rapidly with time of starvation (Fig. 4.13A). These are major nutrient components, and are decisive for the energy content of the rotifer and its general food value. The time-courses of EPA and DHA (Fig. 4.13B) show an even faster reduction of these essential fatty acids. A considerable amount is lost during the ﬁrst 24 h, which is well within the normal residence time of rotifers in larval tanks in the

110

Culture of cold-water marine ﬁsh

very early phase (Reitan et al., 1993; Øie et al., 1997; Chapter 7). The curves suggest that DHA is reduced slightly faster than EPA, and they clearly illustrate that the biochemical composition is a dynamic variable. All metabolic processes will be affected by temperature, including the rate of loss of nutrients during starvation. The loss rates (L) of different components are plotted as a function of the temperature during starvation in Fig. 4.13C and D. The relationship with temperature is almost linear for carbon and protein per rotifer, and closer to exponential for total lipids and the other lipid components (Fig. 4.13D). Lipids are lost faster than proteins above 10°C, but not at lower temperatures. Carbon per rotifer shows the slowest loss, which is not surprising because of the relatively constant amount of carbon retained in the exoskeleton. It is also clear that essential n-3 fatty acids are lost faster than the average fatty acid. DHA exhibits the highest rate of loss throughout the range of temperatures. The model curves ﬁt well with experimental data in the temperature range 5–20°C, but must be used with care for more extreme temperatures. Rotifer mortality during starvation may be very high below 3–4°C (Olsen et al., 1993), and extrapolation below this level is not recommended. The other constraints mentioned above for earlier models should also be considered. Within the recommended range of temperatures, the losses of nutrient components per individual rotifer during starvation may be predicted as follows. (1) Estimate the loss rate for the target temperature and component from Figure 4.13C and D. (2) Measure the initial nutrient content (Q0), i.e. the content before starvation, or make an educated guess. (3) Estimate the nutrient content after t days of starvation by inserting the initial nutrient level for Q0, number of starving days (t), and the estimated value for L in Equation 4.45 above. It is important to note that the curves in Fig. 4.13 are not directly valid if nutrient contents are expressed in terms of nutrient per dry matter. Another set of loss-rate values is then needed, some of which have been made available by Olsen et al. (1993). However, the procedures are the same. The above curves suggest that the biochemical composition of the rotifer is relatively constant with time if the temperature is low. The losses of all components remain less than 10% per day if the temperature is below 8°C. This is important information if rotifer cultures need to be stored after being harvested and rinsed. Storage for <24 h is relatively common in hatcheries, and the above results suggest that stored cultures should be cooled and maintained at 5–8°C. Components such as vitamins and minerals will show the same general pattern of decay during starvation as lipids and proteins. The conceptual model of variability is therefore the same, but the coefﬁcients, or the actual rates of decay, are different and must be measured if needed. As a general approach, however, it is reasonable to assume that energy content, protein and n-3 HUFA will become sub-optimal for larval growth before speciﬁc vitamins. If not, severe nutrient deﬁciency should also be expected when well-fed rotifers are used.

Live food technology of cold-water marine ﬁsh larvae

111

4.4 Production of Artemia Artemia production is different from that of rotifers because Artemia are hatched from resting cysts that are commercially available. There is no need for biomass production and the maintenance of cultures as for rotifers, and the biochemical composition and nutritional value are far more stable and reproducible in hatched nauplii than in rotifers. The challenges in making Artemia nutritionally adequate for cold-water ﬁsh larvae are primarily to manipulate n-3 HUFA content and to establish very strict routines for their production and use. Companies which supply cysts take the main responsibility for cyst quality, and comprehensive commercial programmes have been run to develop suitable feeds and production processes for the cysts. Last but not least, the scientiﬁc efforts which have been made to establish Artemia as live food in shrimp and ﬁsh mariculture have been considerable. These have been led by the pioneering Prof. Patrick Sorgeloos, together with colleagues and co-operators. In 1986, his group published a manual for the use of Artemia in aquaculture (Sorgeloos et al., 1986), and this manual has recently been revised and extended to cover other parts of the rearing process as well (Lavens & Sorgeloos, 1996). We now give a brief overview of some selected topics of Artemia technology, including issues that have become particularly important for the commercial rearing of marine coldwater ﬁsh species. These involve the main challenge of adapting established international n3 HUFA enrichment technology for Artemia to marine cold-water ﬁsh species, and also some contributions to fundamental studies of Artemia feeding and growth. The main problem has been related to n-3 HUFA enrichment and the poor stability of DHA in post-enriched Artemia, technologies which could not be adapted directly from temperate and warm-water species.

4.4.1 Feeding and Growth The Artemiidae are continuous, non-selective, obligate phagotrophic ﬁlter-feeders which start to ingest food at instar II stage (metanauplius I) using their larval antennae. The feeding function is gradually taken over by the multifunctional thoracopods, which are fully developed at the pre-adult stage. The morphological stages of A. franciscana differ in their efﬁciency at capturing, handling and ingesting food particles. Aggregations of food can be observed along the food groove of the animals shortly after the feed is offered (18–34 s). The youngest individuals will then need some 20 s before the food appears in the gut, whereas larger juveniles may need as much as 3.5 min (Evjemo & Olsen, 1999). The gut passage time is 24–29 min for well-fed animals. The food ingestion rate of A. franciscana increases with increasing food concentration to a maximum level, where it remains constant and independent of food concentration (Fig. 4.14A). The incipient limiting food concentration where the animals reach their maximum ingestion rate (Imax) is of the order of 5–7 mg Cl-1 (ILC, see models in the rotifer section). The clearance rate of the animals (ml individual-1 h-1) is also dependent on food concentration (Fig. 4.14B). Unlike rotifers, the clearance rates of Artemia showed maximum values in the intermediate range 0.5–5 mgC l-1, and lower values both below and above this range. The higher incipient limiting food concentration for A. franciscana than for B. plicatilis illus-

112

Culture of cold-water marine ﬁsh

W dw, µ g dry wt ind -1

C -In ges tion rate, n g C in d-1 h-1 3 0 00

A

2 5 00

200

3

C

5

2 0 00

100

1 5 00

7

1 0 00

50 5 00

10

0 1

2

3

5

10

20

30

F oo d, m gC L -1

20

20

Clearan ce ra te, µL in d-1 h -1 5 00

4 00

10

B 5

3 00

2 00

2 1 00

1

0 0. 1

0.3

1

3

F oo d, m gC L -1

10

30

0

2

4

6

8

10

12

T im e, days

Figure 4.14 Feeding and growth characteristics of Artemia franciscana fed various concentrations of Isochrysis galbana (T. iso) (26–28°C). A, B. Feeding rate (A) and food clearance rate (B) of post-metanauplius II and III (7 days old, 2.74 0 ± 0.11 mm length) as a function of food concentration. C. Increase of individual body dry matter with time for groups fed various concentrations of food algae. The legends express the food concentration in terms of mgC l-1. Data from Evjemo and Olsen, 1999, and Evjemo et al., 2000.

trates very clearly the higher food requirements of the former. The positive feeding response to increasing food concentration in the lower range of values indicates a sigmoidal functional response, or a type-3 functional response (Holling, 1966; Evjemo et al., 2000). The food (I. galbana) ingestion rate of A. franciscana (I, ngC individual-1 day-1) can be expressed as a function of quantity (C, mgC l -1) and animal length (L, mm). I = [998 (1 + 270e -C )]( L - 0.91)

(4.47)

(Evjemo & Olsen, 1999). This relationship was established for individuals of 0.3–5 mm length.

Live food technology of cold-water marine ﬁsh larvae

113

The growth rate of A. franciscana during its life cycle depends on the food concentration (Fig. 4.14C). Well-fed animals do not show a net increase in body weight earlier than 2 days after hatching, and their speciﬁc growth rate thereafter tends to increase gradually up to days 4–5. They then grow at an apparent maximum rate (mmax = 1.05 day-1, days 4–7) up to a biomass of >150 mg dry weight individual-1. A. franciscana fed at the lower food concentrations grow more slowly and do not reach pre-adult size, and animals fed 0.2 mgC l -1 (not shown) did not show any positive weight increase. Figure 4.14C shows that a food concentration of 10 mgC l -1 will support the maximum growth rate, whereas 7 mgC l -1 is sub-optimal. Not only feeding and growth rate, but also the assimilation efﬁciency of the animals, show pronounced variations with stage and food concentration. The assimilation efﬁciency changes as the animals grow to maturation, and a low food concentration and ingestion rate is partly compensated for by increased assimilation efﬁciency. Efﬁciencies as high as 60–80% are found for post-metanauplius, which grow particularly fast between day 4 and day 7. Both earlier stages and pre-adults (after day 8) show far lower assimilation efﬁciencies (<33%, Evjemo & Olsen, 1999).

4.4.2 Biomass and Biochemical Composition The biomass and biochemical composition of newly hatched Artemia cysts are highly reproducible compared with those of rotifer cultures. Artemia strain, harvesting batch and location will introduce some variations, but normally these can easily be controlled by suitable purchasing routines (Table 4.3). Both the biomass and composition of older stages will depend on the age and stage of the animal, and on the composition and digestibility of the food used. The fatty acid composition can be particularly variable. Empirical equations have been established to estimate biomass in terms of dry matter and carbon, based on length measurements of Artemia individuals. The relationship between length (L, mm), individual dry weight (Wdw, mg individual-1) and carbon biomass (Wc, mg individual-1) are given below (Evjemo & Olsen, 1999). Wdw = 0.75 e (1.22 L )

(4.48)

Wc = 0.34 e (1.18 L )

(4.49)

Table 4.3 Typical biometry and composition of a newly hatched Artemia franciscana nauplii (EG cysts, INVE Aquaculture) (J.O. Evjemo and A.I. Olsen, pooled unpublished values, 1999). Biomass/composition

Typical value

Length Dry weight Carbon Nitrogen Protein Lipids n-3 HUFA

0.63 mm 2.2 mg per individual 1.0 mg per individual 0.26 mg per individual 1.0 mg per individual 0.44 mg per individual <0.01 mg per individual

114

Culture of cold-water marine ﬁsh

The nitrogen content per dry matter weight of Artemia is relatively constant for groups that are fed adequately. This means that the protein content can be estimated based on the nitrogen content of dry matter biomass with a precision that is satisfactory for most purposes. With an average nitrogen to dry matter content of 9.7% and a conversion factor to protein of 4.2 mg protein mg N-1 (see rotifer section), the following equation can express the protein in Artemia individuals (Wprotein) as a function of their length (L). Wprotein = 0.31e (1.2 L )

(4.50)

Equations 48–50 should be used with some care, but will be accurate enough for most producers of live feed and marine ﬁsh juveniles. The main problem with protein measurements is the principle method used. The classical conversion based on nitrogen and estimates based on the sum of amino acids yields very different values. The value predicted by Equation 4.50 will be intermediate.

4.4.3 Pre-Enrichment Cultivation Cysts that are harvested from saline lakes are the current source of Artemia made available for the aquaculture industry, and a large proportion of commercial cysts are harvested from Great Salt Lake, Utah, USA. The biology of Artemia and procedures for pre-enrichment are described in detail by Van Stappen et al. (1996). Here, we will simply give a review of the practical methods of disinfection, decapsulation and hatching of Artemia cysts. 4.4.3.1 Disinfection of Cysts Disinfection of cysts before hatching will contribute to better microbial control of the Artemia culture in the later phases. Both the Artemia and the ﬁsh larvae are strongly affected by the microbial community (see Chapter 3). Disinfection of cysts is recommended. Decapsulation of the Artemia cysts is an even more efﬁcient way of disinfection, and this treatment is therefore a suitable alternative (see below). The principal methods recommended by Van Stappen et al. (1996) for disinfection of Artemia cysts are:

• Preparation of 200 p.p.m. hypochlorite solution (see cited manual for details). • Soak cysts for 30 min, 50 g cysts per litre hypochlorite solution. • Wash cysts thoroughly in fresh water on a 125 mm screen. The cysts are then ready for further hatching. Proper disinfection will contribute to a more reproducible hatching of the cysts. 4.4.3.2 Decapsulation of Cysts The decapsulation procedure involves the complete removal of the shell that encloses the dormant Artemia embryo. This treatment is beneﬁcial for many reasons. Indigestible cyst

Live food technology of cold-water marine ﬁsh larvae

115

shells are removed, so that they will not interfere with further processing or during use of the Artemia for ﬁsh larvae. The hatching process of the nauplii will require less energy, resulting in more energy-rich nauplii and an optional higher percentage hatching. Last but not least, decapsulation results in complete sterilisation of the cysts, and the use of decapsulated cysts is accordingly the best possible starting point for a controlled bacterial re-colonisation of the Artemia. The procedure of decapsulation involves hydration of the cysts, removal of the shell with a hypochlorite solution, and a ﬁnal rinsing and deactivation of the remaining hypochlorite. The principal methods recommended by Van Stappen et al. (1996) for the decapsulation of Artemia cysts are described below. Hydration

• Incubate 100 g cysts per litre in aerated water for 1 h at 25°C. • Collect the hydrated, rinsed cysts on a 125-mm screen. Removal of Capsules

• Prepare a hypochlorite/alkaline solution (0.5 g active hypochlorite g cysts, see manual cited above for details). Cool • the solution to 15–20°C using iced water. • Incubate the hydrated cysts for 5–15 min with aeration. The reaction is exothermic, and -1

•

the temperature must be kept below 40°C. Check the process of decapsulation using a microscope. Remove the cysts from the incubation solution when they turn grey/orange (depending on the alkaline product used) or when examination under a microscope shows almost complete dissolution of the cyst shells.

Washing and Deactivation

• Rinse with water until no chlorine smell is detected. • Deactivate any traces of hypochlorite by dipping for 1 min in 0.1 N HCl, or in 0.1% Na S O solution, then rinse again with water. • Repeat this procedure until all traces of hypochlorite have been removed. 2 2

3

The cysts may be hatched immediately or may be stored for a few days in a refrigerator (0–4°C). Long-term storage will require subsequent dehydration in saturated brine solution (300 g NaCl and 100 g cysts l-1 brine solution). Decapsulated cysts may also be used directly for marine larvae. 4.4.3.3 Hatching of Cysts The process of cyst hatching is now highly intensiﬁed, and several environmental and technical factors affect the ﬁnal result. The process is basically very simple, but processing large

116

Culture of cold-water marine ﬁsh

quantities with high reproducibility and survival yields requires strict and efﬁcient routines. A method for hatching of Artemia cysts based on the recommendations by Van Stappen et al. (1996) is described below.

• Use transparent, cylindro-conical tanks to facilitate aeration, illumination, harvesting and optional drainage of wastes. • Air should be supplied from the surface to the bottom of the cone. Strong aeration is needed. The oxygen concentration must be >2 mg l , and preferably 5 mg l . • Install a harvesting valve at the tip of the tank. • Use a hatching temperature of 25–28°C; 33°C will result in irreversible damage and high mortality. • The pH should be 8–8.5, and can be controlled by sodium bicarbonate or carbonate (<2 g l NaHCO ). • Use a minimum illumination of 2000 lux by ﬂuorescent light tubes close to the water surface. • Use seawater or brackish water at a salinity >5 p.p.t. Seawater is the most practical, but the energy requirements of the nauplii for hatching is lower in brackish water. • Disinfect or decapsulate the cysts prior to hatching. • Incubate the cysts at densities of 2 g l (maximum 5 g l ). • Incubation should be for a ﬁxed time period, e.g. 20 h, depending on the hatching characteristics of the cysts used. • Harvest and separate nauplii from unhatched cysts and hatching wastes. Rinse thoroughly. -1

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The results of the hatching process must be closely monitored and evaluated. Common evaluation criteria are hatching efﬁciency or percentage, hatching rate, hatching synchrony and hatching output. The methods are more thoroughly described and evaluated by Van Stappen et al. (1996).

4.4.4 Enrichment and Stability of n-3 Fatty Acids As with rotifers, there is a strong need to increase the content of essential n-3 HUFA in Artemia to obtain a live-feed quality that can meet the requirements of the ﬁsh larvae. It is a current belief that many cold-water carnivore ﬁsh species exhibit high DHA requirement in the larval stage. It is a general conclusion that Artemia may be very efﬁciently enriched with n-3 HUFA using appropriate emulsiﬁed oils, but a major problem has been that Artemia tend to catabolise DHA selectively at a far higher rate than other fatty acids. This problem has not been solved satisfactorily, but improvements in technology have been made during the last few years. 4.4.4.1 n-3 HUFA Enrichment Short-term enrichment of Artemia nauplii is by far the most important enrichment technique used, but few efforts have been made to develop techniques for combined growth and n-3 HUFA enrichment of juvenile Artemia (Olsen, 1999b). The situation for Artemia differs

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from that of rotifers because the production is more standardised, starting from the purchased cysts. This means that the starting point is a relatively fat, newly hatched nauplii with only a low initial content of n-3 HUFA, if any. The time available during short-term enrichment does not allow for the establishment of the same degree of equilibrium between the fatty acid proﬁle in feed and in Artemia as for rotifers. The general mechanisms are the same, but high predictability, such as in the enrichment of rotifers, cannot be achieved so easily. The enrichment techniques and detailed protocols become more important. Moreover, additional enrichment studies with Artemia clearly showed that efﬁcient n-3 HUFA enrichment can only be obtained with diets with a very high proportion of fat. Emulsiﬁed lipid diets (e.g. Selco-type or other emulsiﬁed oils) are by far the most common. Microalgae have many attractive traits for marine larviculture, but their lipid and n-3 HUFA contents are normally too low to obtain the n-3 HUFA levels needed for larval cultures of marine cold-water species. A typical procedure for n-3 HUFA short-term enrichment of Artemia nauplii is described below.

• Suitable cylindro-conical, strongly aerated tanks are ﬁlled with ﬁltered seawater and heated to 28°C. The installation of a water exchange system is beneﬁcial (see Fig. 4.1). • Hatched and carefully rinsed nauplii are transferred to the tanks in typical densities of 150–300 individuals ml within 4–6 h after hatching. • The emulsion is dispersed and treated as recommended by the manufacturer, and is added -1

• • •

in rations of 200 g m-3 culture 4–6 h after the harvest of hatched nauplii. Oxygen is monitored (>2–3 mg l-1). Another similar food ration is supplied after 6–12 h, or earlier if the ﬁrst ration has been consumed by the Artemia. Oxygen is monitored (>2–3 mg l -1). The Artemia nauplii are harvested using suitable harvesting gear before the enrichment diet has been completely removed (12–24 h, longer enrichment periods require further additions of food). Animals are carefully rinsed, and used as live feed immediately. Prolonged post-harvest storage will require cooling facilities.

Typical response patterns for lipids and fatty acids during the ﬁrst 2 days of enrichment are shown in Fig. 4.15. The lipid diets used in these two training trials, Super Selco (SS, Fig. 4.15A and B) and DHA Super Selco (DSS, Fig. 4.15C and D) differ in their contents of saturated and monounsaturated fatty acids (S + M), DHA and n-3 HUFA (the right-hand panels express percentage composition of emulsion). Lipids and fatty acids show a similar pattern of accumulation in Artemia independently of the diet used (Fig. 4.15A and C), which is to be expected because the diet rations are identical. The quantitative accumulation of DHA was highest with the use of DSS, the enrichment diet that was richest in DHA. The general pattern of variation in percentage fatty acid contents (Fig. 4.15B and D) is similar to that found for rotifers. The fatty acid composition approaches the composition of the dietary oil. Two distinct features illustrate the main differences between rotifers and Artemia. The DHA level of the Artemia tends to level-off at a value less than 50% of the level of the oil, and the response to an enhanced DHA level in the oil is therefore relatively moderate, although positive. The situation is different for EPA, which, unlike DHA, reaches

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Figure 4.15 Lipid, fatty acids and n-3 HUFA contents of Artemia franciscana as a function of enrichment time (two trials). A (Trial 1, enrichment diet Super-Selco). Total lipids, total fatty acids and quantitative contents of EPA and DHA. B. Percentage distribution of n-3 HUFA and other groups of fatty acids from trial 1. C. (Trial 2, enrichment diet DHA Super-Selco). Total lipids, total fatty acids and quantitative contents of EPA and DHA. D. Percentage distribution of n-3 HUFA and other groups of fatty acids from trial 2.

the same percentage composition in Artemia as in the feed. The differences in n-3 HUFA accumulation between the species are therefore to a great extent a result of low DHA accumulation. The fact that EPA reaches levels higher than those in the oil must be a result of animal metabolism, and experiments suggest that DHA is efﬁciently catabolised to EPA in n-3 HUFA-enriched Artemia during starvation. The percentage saturated plus monounsaturated fatty acids (Sat + Mono) in Artemia are reduced for both diets, but do not reach the lower level of the diets during the enrichment period, and equilibrium in fatty acid distribution is never reached. The general features and variability in Fig. 4.15 illustrate the extent of predictability and the enrichment levels that may be obtained during short-term n-3 HUFA enrichment of Artemia. It is important to note, in this regard, that short-term enrichment should be run for >12 h, but never for >48 h. There is reasonable room for manipulation of the Artemia lipid

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composition, but there are also clear constraints, in particular for DHA and total n-3 HUFA. Artemia differs from the natural food organisms of cold-water ﬁsh larvae such as marine copepods on this point (Olsen, 1999a) (see below). The combined effect of DHA catabolism and EPA accumulation in Artemia results in a markedly lower DHA : EPA ratio in Artemia than in rotifers and copepods. Further developments in enrichment techniques, the use of DHA-rich oils, and the application of extremely DHA-rich/EPA-poor oils containing phospholipids or salts of fatty acids (Tocher et al., 1997; Han et al., 2000; Harel et al., 1999) have resulted in higher percentage DHA levels and higher DHA:EPA ratios. These modiﬁcations may be feasible as long as the expenses are kept within an acceptable level for commercial larval rearing. 4.4.4.2 Stability of n-3 Fatty Acids Post-Enrichment As for rotifers, most nutritional components of Artemia decrease during starvation, and the quantitative loss rates of most components are comparable to those found for rotifers (Olsen et al., 1993; Evjemo et al., 2001). The fact that Artemia is hatched from cysts characterised by a reproducible initial biochemical composition and high energy content should imply that Artemia will not become nutritionally and energetically inadequate unless starved for a long time. However, the common commercially available strain of Artemia (A. franciscana), and most other identiﬁed strains as well, have one negative and critical property from a larval nutrition point of view. It is well documented that by some mechanism, these Artemia strains lose DHA very rapidly from their tissues, not only from their gut, after being enriched by n-3 HUFA (e.g. Dhert et al., 1993; Evjemo et al., 1997, 2001). This pattern seems to be independent of the enrichment diet and takes place for all developmental stages of the animal (Olsen, 1999b). These losses must originate in selective DHA catabolism. An enhanced lossrate of DHA compared with other fatty acids is more clearly expressed for Artemia than for B. plicatilis, which also exhibits a moderate catabolic preference for DHA over other fatty acids during starvation (see Fig. 4.13D). The rate of DHA catabolism is positively related to the temperature. Losses are very high at high temperatures, and it is notable that virtually no DHA is left after 1 day at a temperature of 25°C, which is a common temperature for warm-water species. This fact is not normally considered during larval rearing, but may be critical for some species, in particular for fast-growing carnivores in the very early stages when food is not consumed immediately after being added. Artemia cultures must be maintained at temperatures <10°C if storage of the enriched culture is needed. This will reduce losses of the accumulated DHA, although the losses are still signiﬁcant. Figure 4.16 compares the phases of DHA enrichment (0–24 h) and DHA stability postenrichment (24–96 h) for two Artemia species, one of which is a non-commercial Chinese strain which exhibits high DHA retention (Evjemo et al., 1997). The DHA stability of marine copepods during starvation is illustrated as a reference for comparison (Fig. 4.16C). The pattern of DHA accumulation during the initial 25 h enrichment period is comparable for both species, but is slightly less efﬁcient in A. sinica, which is the non-commercial strain (Fig. 4.16A and B).

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Figure 4.16 DHA enrichment and stability in Artemia strains and in marine copepods. A. Time-course of quantitative DHA accumulation during 24 h enrichment (28°C) and later starvation for 3 days (12°C) in Artemia franciscana and Artemia sinica. B. Percentage DHA accumulation during 24 h enrichment (28°C) and later starvation for 3 days (12°C) in A. franciscana and A. sinica. C. Percentage DHA in two species of marine copepods maintained in starvation conditions. Data from Evjemo et al., 1997.

When the enrichment diet is removed, DHA decreases exponentially in A. franciscana, whereas the present isolate of A. sinica retains DHA very efﬁciently. The speciﬁc loss-rate of DHA from A. franciscana at 12°C (see Equation 45) is 1.2 day-1, representing a half-time of disintegration of 14–15 h. The fact that percentage DHA decreases (Fig. 4.16B) implies that DHA is lost faster than the average fatty acid, and in fact faster than any other fatty acid (Evjemo et al., 2000). In A. sinica, however, there is an increase in percentage DHA during starvation, as in marine copepods (Fig. 4.16C). This means that DHA is being lost at a lower rate than the average fatty acid in this strain. These results suggests that DHA is not a major essential phospholipid or membrane component in A. franciscana as it is in copepods and the present isolate of A. sinica. This must originate in genetic and fundamental metabolic differences between Artemia strains. Accordingly, the common marine copepods that are potential food for cold-water species show the same mode of DHA retention as is found in A. sinica, which is therefore more comparable to the natural food prey (Fig. 4.16C). A. sinica would therefore probably be a better replacement for marine copepods than A. franciscana. Regrettably, other isolates of A. sinica have not shown the same type of DHA kinetics. The severe DHA catabolism in A. franciscana calls for careful routines during postenrichment and during the very early stages of ﬁrst feeding. It is important to consider that even brief periods of shortage in the enrichment diet between enrichment and the later use as live feed may be critical, in particular if the temperature is not reduced immediately. In most cases, the larvae will consume the Artemia immediately they are fed, and this will contribute to a short residence time between the termination of enrichment and larval consumption of the live food. DHA catabolism will then be of minor importance if the feed preparation and feeding routines are adequate.

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However, the problem may become more severe when Artemia is provided as the ﬁrst live feed organism in the very early phase of ﬁrst feeding, before the larvae have started to feed. In this case, the n-3 HUFA content of the Artemia cannot be stabilised and controlled by adding microalgae to the larval tanks, as is done for rotifers (Olsen, 1999b), or by introducing a high water exchange rate. The inadequate DHA supply through Artemia is a problem in the cultivation of Atlantic halibut larvae. These larvae have high DHA requirements (Shields et al., 1999a), they are very fragile and may suffer severely during fast water exchange, they need some days to learn to feed, and they grow relatively slowly (see Chapter 7).

4.4.5 n-3 HUFA of Artemia Juveniles The technology to produce Artemia juveniles and adults at a very high density using cheap feed and automated feeding systems was ﬁrst introduced many years ago (see Sorgeloos et al., 1986). It is notable, however, that juvenile Artemia have only sporadically been used as live food for marine ﬁsh larvae. This may originate from the fact that the production technology is not that well known in the hatcheries, and that there are extra costs and risks involved in culturing Artemia. The technology has recently been adapted and fully demonstrated for Atlantic halibut (Hippoglossus hippoglossus, see Olsen, 1999b, and articles therein). Newly hatched halibut larvae are relatively large (10–12 mm), and they select actively for larger prey than Artemia nauplii approximately 20 days after ﬁrst feeding (3.0–3.5 mg dry weight per larvae). Figure 4.14C illustrates the growth potential of A. franciscana through all its developmental stages from hatching to adult. In the perspective of variable larval prey preferences, there is the potential to produce any size group of Artemia juveniles that are needed. The production of juvenile Artemia requires a complete diet that can adequately sustain growth. In addition, the diet must be relatively fat- and DHA-rich to secure an accumulation of DHA in Artemia tissues. The production method described below for juveniles 3 days or older is based on a mixture of ﬁshmeal and marine oil (modiﬁed from Olsen, 1999b). For an initial stocking density of 20 nauplii ml-1 (values must be scaled up for higher densities):

• Use 90 g m ﬁsh meal and 54 g m ﬁsh oil high in DHA (e.g. Pronova TG 1040, DHA Selco), giving 47% lipid in the diet. • Feed three times a day (twice on day 0), maintain a high food level, feed more frequently for higher Artemia densities. • Use a regular water-exchange regime. • Use a temperature of 26–28°C. • Ensure that the oxygen concentration is >2.5 mg O l . -3

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It appears to be difﬁcult to produce >4-day-old juveniles which also exhibit a high DHA content, but the feeding procedure above works well for younger stages, which are the optimal size groups for halibut larvae. Three-day-old A. franciscana juveniles show a satis-

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factory lipid and fatty acid content (19% lipid, about 19 mg DHA g DW-1) and a protein to lipid ratio of 3.4. The survival is obviously lower and more variable than for short-term enrichment, but is still acceptable under skilled management. The biomass per prey is more than twice as high as that of short-term enriched nauplii. High DHA catabolism is also a characteristic feature for juvenile stages of A. franciscana, and the nutritional problems encountered using juvenile Artemia are therefore more or less identical to those described for nauplii. It remains to be seen if juvenile Artemia will become used more frequently for feeding marine juvenile ﬁsh larvae such as Atlantic halibut.

4.4.6 Vitamins and Minerals Information on vitamins and minerals in Artemia is available (Léger et al., 1986; Lavens & Sorgeloos, 1996; Olsen et al., 2000). Fundamental knowledge on ﬁsh larval requirements is very limited, and information and indexes for cultured ﬁsh are needed as a reference (e.g. Lall, 1989; NRC, 1993). The mineral content of Artemia is believed to be adequate, but a recent study revealed that the selenium in cysts may be insufﬁcient. This was reported by Van Stappen et al. (1996), who also provided data suggesting that the vitamin content of Artemia cysts is sufﬁcient. Vitamins such as ascorbic acid may be efﬁciently enriched in Artemia nauplii through techniques comparable to those for lipids and n-3 HUFA. Van Stappen et al. (1996) and Olsen et al. (2000) reported that ascorbic acid and vitamin B6 are lost when starving A. franciscana. The thiamine content, however, remained, constant throughout severe starvation. The general pattern of variation for vitamins is comparable to that of n-3 HUFA, with clear effects of selective retention for some single components, and selective and faster catabolism for others. As for rotifers, a sub-optimal supply of vitamins and minerals is unlikely, but it cannot be ruled out completely. It is also clear that enhanced levels of ascorbic acid for growth may have positive effects on larval viability (Merchie et al., 1995).

4.5 Marine Copepods It is generally believed that the natural food for larvae of ﬁsh species such as turbot, cod and halibut consists of small species and young stages of marine copepods. These groups of zooplankton are available in the coastal waters of many countries, and an obvious approach in early efforts to feed marine ﬁsh larvae was to supply harvested copepods of an appropriate prey size for the ﬁsh larvae as live feed. This approach was successful to some extent in the pioneering period when the rotifer and Artemia technologies were immature, and the copepod technology was further developed and scaled-up to be applicable to the commercial production of ﬁsh juveniles (see Chapter 7). Harvested copepods are still being used in the commercial production of ﬁsh fry in northern countries, in particular for Atlantic halibut (van der Meeren & Naas, 1997). The

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zooplankton is cultured and harvested from coastal locations using automated pumping and concentration systems. The harvested, mixed zooplankton stock is further treated to isolate appropriate size fractions that are transferred to ﬁsh larvae maintained in suspended ﬁrstfeeding enclosures. This technology of ﬁrst feeding has worked to some extent, but it must be regarded as premature with a restricted potential. There are several possible pitfalls using copepods, such as the transient availability of suitable prey for the larvae, and the probable transfer of parasites and diseases. However, experience to date has supported the assumption that copepods, which are believed to be the natural food of the larvae, can meet the nutritional requirements of ﬁsh larvae, and in particular the requirements for n-3 HUFA or DHA (Shields et al., 1999a). A common and sound approach has therefore been to use such copepods as a main reference during efforts to develop rotifer and Artemia technology for marine cold-water species of ﬁsh. There is a considerable database available on the biochemical composition of marine copepods (e.g. Båmstedt, 1986; Sargent & Henderson, 1986; Mauchline, 1998), and speciﬁc sampling programmes have been run to characterise the harvested and isolated copepods in hatcheries that use the copepod technology. Some examples of the fatty acid contents of common species which are actually being used are illustrated in Fig. 4.17. The values obtained for newly hatched and short-term-enriched A. franciscana and B. plicatilis enriched by different techniques are shown for comparison. The cases of rotifers illustrated are extreme: B1 is the lower extreme value for rotifers enriched during cultivation, whereas B2 shows the high extreme value for rotifers successively enriched during short-tem enrichment (see Fig. 4.12). The lipid level of the younger stages of copepods is low and relatively independent of species, although it is slightly higher for C. ﬁnmarchicus. Additional studies have clearly shown that this is a robust conclusion (Evjemo et al., 2003). The low lipid level implies a high fraction of phospholipids, which is in agreement with a ubiquitous high percentage of DHA and EPA (45–60% of total fatty acids). The high percentage n-3 HUFA in the copepods is very stable because these fatty acids, as the main functional components of membrane phospholipids, are selectively retained during starvation (see Fig. 4.16C). Artemia may incorporate larger quantities of DHA and n-3 HUFA than copepods through efﬁcient enrichment procedures, but its tissues are notoriously fatter than those of copepods. It is difﬁcult to obtain both a low lipid level and a high n-3 HUFA level in Artemia using the established techniques for enrichment, and the percentage contents of n-3 HUFA are lower than in copepods. The consequence is that with an adequate n-3 HUFA content, Artemia will exhibit twice the lipid content of copepods. It is not clear how this will affect larval ﬁsh growth, survival and performance. Rotifers that are enriched according to the methods presented in this chapter can, in principle, be almost identical to marine copepods. B1 and B2 (Fig. 4.17) represent normal rotifers enriched with different normal commercial emulsiﬁed oils. The use of an emulsiﬁed oil with an identical fatty acid composition as in Temora sp. and Eurytemora sp. would have resulted in rotifers with a similar fatty acid composition (see Fig. 4.11). Additional short-term enrichment could have been used to adjust the quantitative level of fatty acids and lipid if necessary (see Fig. 4.12).

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Figure 4.17 Fatty acid contents of cultivated live feed and some marine copepods commonly used as live feed for marine ﬁsh larvae (copepod data from Evjemo and Olsen, 1997). A1, newly hatched Artemia nauplii; A2, Artemia nauplii short-term enriched for 24 h by DHA Selco; B1, rotifers enriched by Super-Selco during production (a lower range of values is normally obtained); B2, rotifers enriched during growth and thereafter during shortterm enrichment with DHA Super-Selco; C. Calanus ﬁnmarchicus, copepodid stages I, II and III; T, Temora longicornis; E, Eurytemora sp.

4.6 Concluding Remarks This chapter has described experience in production techniques and methods to manipulate the nutritional value of rotifers and Artemia to be used as live feed for marine ﬁsh larvae. Some further aspects of these issues that are more closely related to ﬁrst feeding are treated in Chapter 7. In addition, the microbial aspects of live feed cultures are treated in Chapters 3 and 7. It is important to emphasise that the microbial characteristics of a live feed culture are just as important as the nutritional characteristics for the successful breeding of marine

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larvae. Larval rearing is highly multidisciplinary, and success can only be obtained if all the important aspects of the live feed are adequately covered. Rotifer technology for marine cold-water ﬁsh species is well established, but the general principles and techniques will always have to be adapted to meet the requirements of a given species of ﬁsh larvae. The nutritional value of the rotifers can be controlled during production, during successive short-term enrichment, and in the phase of ﬁrst feeding. The main tools have been developed, but ﬁne-tuning of the nutritional as well as the microbial treatments for each species will be needed. The main developmental challenge is to intensify and automate the cultivation techniques. This may contribute towards a reduction in costs and the risks of production. A preliminary Artemia technology for cold-water species has been established, but further improvements are needed for some species of ﬁsh larvae. Unstable DHA after enrichment and the low efﬁciency of DHA incorporation from Artemia in halibut larvae are problems in the establishment of a controlled and economically feasible rearing of Atlantic halibut larvae that must be fed Artemia from the very beginning of ﬁrst feeding. The commercialisation of an Artemia strain which can retain DHA in its tissues more efﬁciently is one countermeasure, but introducing co-feeding, or inhibiting DHA catabolism by some means, are other options. The established technology is probably optimal for cod and turbot, which are fed rotifers in the initial phase.

4.7 References Båmstedt, U. (1986) Chemical composition and energy content. In: The Biological Chemistry of Marine Copepods (eds E.D.S. Corner & S.C.M. O’Hara), pp. 1–58. Clarendon Press, Oxford. Coutteau, P. (1996) Micro-algae. In: Manual on the Production and Use of Live Food for Aquaculture (eds P. Lavens & P. Sorgeloos), pp. 7–48. FAO Fisheries Technical Paper No. 361. Dhert, P. (1996) Rotifers. In: Manual on the Production and Use of Live Food for Aquaculture (eds P. Lavens & P. Sorgeloos), pp. 49–78. FAO Fisheries Technical Paper No. 361. Dhert, P., Sorgeloos, P. & Devresse, B. (1993) Contributions towards a speciﬁc DHA enrichment in the live food Brachionus plicatilis and Artemia sp. In: Proceedings from the International Conference on Fish Farming Technology (eds H. Reinertsen, L.A. Dahle, L. Jørgensen & K. Tvinnereim), pp. 109–15. Trondheim, 9–12 August 1993. Balkema, Rotterdam. Evjemo, J.O. & Olsen, Y. (1997) Lipid and fatty acid content in cultivated live feed organisms compared to marine copepods. Hydrobiologia, 358, 159–62. Evjemo, J.O. & Olsen, Y. (1999) Effect of food concentration on the growth and production rate of Artemia franciscana feeding on algae (T. iso). J. Exp. Mar. Biol. Eco., 242, 273–96. Evjemo, J.O., Coutteau, P., Olsen, Y. & Sorgeloos, P. (1997) The stability of docosahexanoic acid in two Artemia species following enrichment and subsequent starvation. Aquaculture, 155, 135–48. Evjemo, J.O., Vadstein, O. & Olsen, Y. (2000) Feeding and assimilation kinetics of Artemia franciscana fed Isochrysis galbana (clone T. iso). Mar. Biol., 136, 1099–109. Evjemo, J.O., Danielsen, T.L. & Olsen, Y. (2001) Losses of lipid, protein and n-3 fatty acids in enriched Artemia franciscana starved at different temperatures. Aquaculture, 193, 65–80. Evjemo, J.O., Reitan, K.I. & Olsen, Y. (2003) Copepods as live food organisms for marine ﬁsh larvae with special emphasis on nutritional value. Aquaculture, in press.

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Fu, Y., Hirayama, K. & Natsukari, Y. (1991) Morphological differences between two types of the rotifer Brachionus plicatilis OF. Müller. J. Exp. Mar. Biol. Ecol., 151, 29–41. Fukusho, K. (1997) Nutritional effects of the rotifer Brachionus plicatilis raised by baking yeast on larval ﬁsh of Oplegnathus fasciatus, by enrichment with Chlorella sp. before feeding. Bull. Nagasaki Pref. Inst. Fish, 3, 152–4 (in Japanese). Fulks, W. & Main, K.L. (eds) (1991) Rotifer and Microalgae Culture Systems. Proceedings of a US–Asia Workshop. Honolulu, Hawaii, 28–31 January. Argent Laboratories, Washington. Fyhn, H.J. (1990) Energy production in marine ﬁsh larvae with emphasis on free amino acids as a potential fuel. In: Animal Nutrition and Transport Processes. 1. Nutrition in Wild and Domestic Animals (ed J. Mellinger), pp. 176–92. Karger, Basel. Fyhn, H.J. (1993) Multiple functions of free amino acids during embryogenesis in marine ﬁshes. In: Physiological and Biochemical Aspects of Fish Development (eds B.T. Walther & H.J. Fyhn), pp. 299–308. University of Bergen, Bergen. Han, K.M., Geurden, I. & Sorgeloos, P. (2000) Enrichment strategies for Artemia using emulsions providing different levels of n-3 highly unsaturated fatty acids. Aquaculture, 183(3–4), 335–47. Harel, M., Ozkizilcik, S., Lund, E., Behrens, P. & Place, A.R. (1999) Enhanced absorption of docosahexaenoic acid (DHA, 22:6 n-3) in Artemia nauplii using a dietary combination of DHA-rich phospholipids and DHA-sodium salts. Comp. Biochem. Physiol., 124B(2), 169–76. Hjelmeland, K., Uglestad, I. & Olsen, Y. (1993) Proteolytic activity and post-mortem autolysis in prey for marine ﬁsh larvae. In: Physiological and Biochemical Aspects of Fish Development (eds B.T. Walther & H.J. Fyhn), pp. 229–32. University of Bergen, Bergen. Holling, C.S. (1966) The functional response of invertebrate predators to prey density. Mem. Entomol. Soc. Can., 48, 1–85. Howell, B.R. (1979) Experiments on the rearing of larval turbot, Scophthalmus maximus L. Aquaculture, 18, 215–25. Ito, T. (1960) On the culture of mixohaline rotifer Brachionus plicatilis O. F. Muller. Rep. Fac. Fish. Mie Pref. Univ., 3, 708–40 (in Japanese). King, C.E. & Miracle, R.M. (1980) A perspective on aging in rotifers. Hydrobiologia, 73, 13–19. Kitajima, C. & Koda, T. (1976) Lethal effects of a rotifer cultured with baking yeast on the larval sea bream, Pagrus major, and the increase rate using the rotifer recultured with Chlorella sp. Bull. Nagasaki Pref. Inst. Fish., 2, 113–16 (in Japanese). Korstad, J.E., Olsen, Y. & Vadstein, O. (1989a) Life history of Brachionus plicatilis fed different algae. Hydrobiologia, 186/187, 43–50. Korstad, J.E., Vadstein, O. & Olsen, Y. (1989b) Feeding kinetics of the rotifer Brachionus plicatilis fed Isochrysis galbana. Hydrobiologia, 186/187, 51–7. Lall, S.P. (1989) The minerals. In: Fish nutrition, 2nd edn (ed J.E. Halver), pp. 219–57. Academic Press, New York. Lavens, P. & Sorgeloos, P. (eds) (1996) Manual on the Production and Use of Live Food for Aquaculture. FAO Fisheries Technical Paper No. 361, pp. 295. Léger, P., Bengtson, D.A., Simpson, K.L. & Sorgeloos, P. (1986) The use and nutritional value of Artemia as a food source. Oceanogr. Mar. Biopl. Annu. Rev., 24, 521–623. Lie, Ø., Haaland, H., Hemre, G.I., Maage, A., Lied, E., Rosenlund, G., Sandnes, K. & Olsen, Y. (1997) Nutritional composition of rotifers following a change in diet from yeast and emulsiﬁed oil to microalgae. Aquacult. Int., 5(5), 427–38. Lubzens, E. (1987) Raising rotifers for use in aquaculture. Hydrobiologia, 147, 245–255. Lubzens, E., Tandler, A. & Minkov, G. (1989) Rotifers as food in aquaculture. Hydrobiologia, 186/187, 387–400.

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Makridis, P. & Olsen, Y. (1999) Protein depletion of the rotifer Brachionus plicatilis during starvation. Aquaculture, 174, 343–53. Mauchline, J. (1998) The biology of calanoid copepods. In: Advances in Marine Biology, Vol. 33 (eds J.H.S. Blaxter, A.J. Southward & P.A. Tyler), 710 pp. Academic Press, New York. Merchie, G., Lavens, P., Dhert, P., Deshasque, M., Nelis, H., De-Leenheer, A. & Sorgeloos, P. (1995) Variation of ascorbic acid content in different live feed organisms. Aquaculture, 134(3–4), 325–37. Nagata, W.D. & Hirata, H. (1986) Mariculture in Japan: past, present, and future perspectives. Mini. Rev. Data File Fish. Res., 4, 1–38. NRC (1993) Nutrient Requirements for Coldwater Fishes. Sub-Committee on Coldwater Fish Nutrition, National Research Council, Washington, DC. Øie, G. & Olsen, Y. (1997) Protein and lipid content of the rotifer Brachionus plicatilis during variable growth and feeding conditions. Hydrobiologia, 358, 251–8. Øie, G., Makridis, P., Reitan, K.I. & Olsen, Y. (1997) Protein and carbon utilization of rotifers (Brachionus plicatilis) in ﬁrst feeding of turbot larvae (Scophthalmus maximus L.). Aquaculture, 153, 103–22. Olsen, Y. (1999a) Lipids and essential fatty acids in aquatic food webs. What can freshwater ecologists learn from mariculture? In: Lipids in Freshwater Ecosystems (eds M.T. Arts & B.C. Wainman), pp. 161–202. Springer, New York. Olsen, A.I. (1999b) Development of production technology of juvenile Artemia optimal for feeding and production of Atlantic halibut fry. D. Phil. Thesis, Norwegian University of Science and Technology, Department of Biotechnology, Trondheim. Olsen, A.I., Mæland, A., Waagbø, R. & Olsen, Y. (2000) Effect of algal addition on stability of fatty acids and some water-soluble vitamins in juvenile Artemia franciscana. Aquacult. Nutr., 6(4), 263–73. Olsen, Y., Reitan, K.I. & Vadstein, O. (1993) Dependence of temperature on loss rates of rotifers, lipids, and w3 fatty acids in starved Brachionus plicatilis cultures. Hydrobiologia, 255/256, 13–20. Olsen, Y., Evjemo, J.O. & Olsen, A.I. (1999) Status of the cultivation technology for production of Atlantic halibut (Hippoglossus hippoglossus) juveniles in Norway/Europe. Aquaculture, 176, 3–13. Reitan, K.I., Rainuzzo, J.R., Øie, G. & Olsen, Y. (1993) Nutritional effects of algal addition in ﬁrst feeding of turbot (Scophthalmus maximus L.) larvae. Aquaculture, 118, 257–75. Rothhaupt, K.O. (1990a) Differences in particle size-dependent feeding efﬁciencies of closely related rotifer species. Limnol. Oceanogr., 35(1), 16–23. Rothhaupt, K.O. (1990b) Changes of the functional responses of the rotifers Brachionus rubens and Brachionus calyciﬂorus with particle sizes. Limnol. Oceanogr., 35(1), 24–32. Sandnes, K., Lie, Ø., Haaland, H. & Olsen, Y. (1994) Vitamin contents of the rotifer Brachionus plicatilis. Fisk. Dir. Skr. Ernœring, 6(2), 117–19. Sargent, J.R. & Henderson, R.J. (1986) Lipids. In: The Biological Chemistry of Marine Copepods (eds E.D.S. Corner & S.C.M. O’Hara), pp. 59–108. Clarendon Press, Oxford. Shields, R.J., Bell, J.G., Luizi, F.S., Gara, B., Bromage, N.R. & Sargent, J.R. (1999a) Natural copepods are superior to enriched Artemia nauplii as feed for larvae (Hippoglossus hippoglossus) in terms of survival, pigmentation and retinal morphology: relation to dietary essential fatty acids. J. Nutr., 129(6), 1186–94. Shields, R.J., Gara, B. & Gillespie, M.J.S. (1999b) A UK perspective on intensive hatchery rearing methods for Atlantic halibut (Hippoglossus hippoglossus). Aquaculture, 176, 15–25. Smith, L.L., Fox, J.M. & Granvil, D.R. (1993) Intensive algal culture techniques. In: CRC Handbook of Mariculture. Vol. 1. Crustacean Aquaculture, 2nd edn. (ed J.P. McVey), pp. 3–13. CRC Press, Boca Raton, FL.

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Snell, T.W., Childress, M.J. & Boyer, E.M. (1987) Assessing the status of rotifer cultures. J. World Aquacult. Soc., 18, 270–77. Sorgeloos, P., Lavens, P., Léger, P., Tackaert, W. & Versichele, D. (1986) Manual for the Culture and Use of Brine Shrimp Artemia in Aquaculture. Manual prepared for the Belgian Administration for Development Cooperation and the Food and Agriculture Organization of the United Nations. Artemia Reference Center, Faculty of Agriculture, State University of Ghent, 319 pp. Suantika, G., Dhert, P., Nurhudah, M. & Sorgeloos, P. (2000) High-density production of the rotifer Brachionus plicatilis in a recirculation system: consideration of water quality, zootechnical and nutritional aspects. Aquacult. Eng., 21, 201–14. Tocher, D.R., Mourente, G. & Sargent, J.R. (1997) The use of silages prepared from ﬁsh neural tissues as enrichers for rotifers (Brachionus plicatilis) and Artemia in the nutrition of larval marine ﬁsh. Aquaculture, 148, 213–31. Vadstein, O., Øie, G. & Olsen, Y. (1993) Particle-size-dependent feeding by the rotifer Brachionus plicatilis. Hydrobiologia, 255/256, 261–7. van der Meeren, T. & Naas, K.E. (1997) Development of rearing techniques using large enclosed ecosystems in the mass production of marine ﬁsh fry. Rev. Fish. Sci., 5, 367–90. van Stappen, G., Merchie, G., Dhont, J., Lavens, P., Baert, P., Bosteels, T. & Sorgeloos, P. (1996) Artemia. In: Manual on the Production and Use of Live Food for Aquaculture (eds P. Lavens & P. Sorgeloos), pp. 79–136. FAO Fisheries Technical Paper No. 361. Watanabe, T., Kitajima, C., Arakawa, T., Fukusho, K. & Fujita, S. (1978) Nutritional quality of rotifer, Brachionus plicatilis, as a living feed from the viewpoint of essential fatty acids for ﬁsh. Bull. Jpn. Soc. Sci. Fish, 44, 1109–14 (in Japanese). Watanabe, T., Kitajima, C. & Fujita, S. (1983) Nutritional values of live organisms used in Japan for mass propagation of ﬁsh: a review. Aquaculture, 34, 115–43. Yoshimura, K., Usuki, K., Yoshimatsu, T. & Hagiwara, A. (1997) Recent development of a high-density mass culture system for the rotifer Brachionus rotundiformis Tschugunoff. Hydrobiologia, 358, 139–44.

Chapter 5

Brood Stock and Egg Production D. Pavlov, E. Kjørsvik, T. Refsti and Ø. Andersen

Good brood-stock management and good farming practices are necessary to obtain highquality offspring in ﬁsh aquaculture. The primary requirement for the successful mass cultivation of ﬁsh is the availability of eggs and sperm of good quality. A reliable, large quantity of healthy and normal juveniles can only be obtained from brood stock which is kept under adequate environmental and nutritional conditions. In order to obtain successful gonadal growth, gamete maturation and spawning of captive ﬁsh, it is important to understand the reproductive physiology and the spawning processes of the ﬁsh. These processes are sensitive to changes in environmental conditions and to physiological stress, and how external factors may modify reproduction is important knowledge for effective brood-stock management. However, we have little or no information on controlled reproduction and recruitment for many of the approximately 1000 ﬁsh species that are cultivated. The scope of this chapter is to describe the reproductive biology of ﬁsh, with special emphasis on factors of importance for brood-stock husbandry and offspring quality of cold-water marine species in aquaculture.

5.1 Reproductive Strategies Fish reproductive patterns may be very species-speciﬁc. The more than 20 000 different ﬁsh species in the world inhabit a larger variety of habitats than any other vertebrate group. Fish can be found in polar seas and lakes, in tropical swamps, in the greatest depths of the oceans or at high altitudes in freshwater streams. They have subsequently evolved into a wide variety of forms and life styles, and so have their reproductive styles, developmental patterns and environmental needs. These different developmental patterns and reproductive styles may be classiﬁed according to spawning tactics and ecological niches for development. The Russian scientist Sergei Kryzhanovskii (1949) was the ﬁrst to propose a classiﬁcation based on the spawning features of some freshwater ﬁsh. Five ecological groups were classiﬁed according to their spawning substrates: lithophils (rock and gravel spawners), phytophils (plant spawners), psammophils (sand spawners), ostracophils (egg deposition inside mussels) and pelagophils (pelagic spawners). According to the main idea of Kryzhanovskii and his followers, the concept of ecological groups should not be regarded simply as

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distinct adaptations of eggs and young to environmental conditions. The adaptation of reproductive styles is reﬂected in the whole ontogeny, and will determine the features of adult ecology, migration and distribution. Later several classiﬁcation schemes were constructed for freshwater and marine ﬁsh, and these are described in reviews by Pavlov (1989) and Balon (1990). The most comprehensive evolutionary classiﬁcation of reproductive styles (or reproductive guilds, according to the author) was created by the Slovenian–Canadian scientist Eugene Balon, based on the ideas of Kryzhanovskii. The majority of known reproductive styles of ﬁsh are now included in three ethological sections, with two ecological groups in each (Table 5.1). Several reproductive groups are characterised by spawning substrate, and associated characters of eggs, embryos and larvae are included in each ecological group. Each ecological group includes several guilds (or sub-groups). For example, the ecological group (A.1) is composed of seven guilds: pelagic spawners (pelagophils), rock and gravel spawners with pelagic larvae (lithopelagophils), rock and gravel spawners with benthic larvae (lithophils), non-obligatory plant spawners (phytolithophils), obligatory plant spawners (phytophils), sand spawners (psammophils), and terrestrial spawners with the eggs scattered out of water on damp sod (aerophils). The concept of reproductive guilds reﬂects evolutionary lines to a certain extent. Both the succession of groups within the ethological sections and the succession of guilds in each ecological group represent a trend from a life style characterised by small unprotected eggs and high fecundity to a life style with larger eggs, lower fecundity and more complex protection of eggs and offspring. The eggs are spawned and develop independently from the parents for most ﬁsh species. Freshwater ﬁsh eggs are mostly demersal (develop on the bottom or on a substrate), whereas almost all pelagic eggs (ﬂoating freely in the water) are marine. However, parental care has developed in many species (in 3000–5000 species of teleost ﬁshes), and care of the offspring may continue after hatching (e.g. mouth-brooding). Increased parental investment in the individual offspring will generally lead to decreased fecundity, less larval specialisations and more advanced development at hatching (see also Chapter 6). Many littoral species guard their eggs (cottids, blennies, gobies), and care is more often carried out by the male than by the female. Nests may also be built and/or one of the parents may ventilate the egg mass, e.g. the wolf-ﬁshes (family Anarhichadidae) and the lumpsucker Cyclopterus lumpus. Viviparity has evolved independently in several groups of ﬁsh. It is a dominant mode of reproduction in cartilaginous ﬁsh. In teleosts, viviparity is widespread, but not so prevalent; about 510 species are known to be viviparous. The gestation (or ‘pregnancy’) may occur within the follicle (intrafollicular gestation) or within the ovarian cavity (intraluminal gestation). In species such as the redﬁsh (Sebastes) and guppies (Poecilia), embryonic nutrition depends solely on yolk reserve in the eggs, and ﬁrst-feeding larvae are extruded from the mother’s body (ovoviviparity). However, the redﬁsh possess intraluminal gestation, and guppies intrafollicular gestation. Another form of parental care is viviparity, where nourishment is supplied by maternal structures, and the offspring are often born as juveniles. The majority of the species used for cold-water marine aquaculture (from the orders Pleuronectiformes and Gadiformes) produce pelagic eggs, which are scattered in the water during spawning and not protected, and they can therefore be placed in the guild A.1.1 of

Brood stock and egg production

Table 5.1

Classiﬁcation of reproductive styles (guilds) in ﬁsh (modiﬁed after Blaxter, 1988 and Balon, 1990).

Ethological section

Ecological group

Reproductive guilds

A Non-guarders 1

Open and substratum spawners

1 2 3 4 5 6 7 1 2 3 4 5

Pelagic spawners Rock and gravel spawners with pelagic larvae Rock and gravel spawners with benthic larvae Non-obligatory plant spawners Obligatory plant spawners Sand spawners Terrestrial spawners, damp conditions Beach spawners, above waterline at high tides Annual spawners, eggs estivate Rock and gravel spawners Cave spawners Spawners in live invertebrates

1 2 3 4 1 2 3 4 5 6 7 8

Pelagic spawners, at surface of hypoxic waters Above-water spawners; male splashes around Rock spawners Plant spawners Froth nesters Miscellaneous substratum and materials nesters Rock and gravel nesters Glue-making nesters Plant material nesters Sand nesters Hole nesters Anemone nesters; at base of host

2

B Guarders 1

2

C Bearers 1

2

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Brood hiders

Substratum spawners

Nest spawners

External bearers

Internal bearers

1 Transfer brooders; eggs carried before deposition 2 Auxiliary brooders; adhesive eggs carried on skin under ﬁns, etc. 3 Mouth brooders 4 Gill-chamber brooders 5 Pouch brooders 1 Facultative internal bearers; occasional internal fertilisation of normally oviparous ﬁsh; eggs rarely retained long 2 Obligate lecithotrophic live bearers; no maternal– embryonic nutrient transfer 3 Matrotrophous oophages and adelophages; one or a few eggs developing at the expense of other eggs or embryos 4 Viviparous trophoderms; nutrition partially or entirely from female via ‘placental’ structures

pelagic spawners (pelagophils). The perciform genus of wolf-ﬁsh Anarhichas, which is regarded as promising for cold-water marine farming in northern Europe and Atlantic Canada (Brown et al., 1995), possesses internal fertilisation, and the fertilised eggs are released several hours after ovulation and copulation between spawners. Owing to the internal insemination and protection of the egg mass by a parent during the development of embryos, the species of this genus should be placed in guild C.2.1, as they can be consid-

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Table 5.2

Features of egg production in different marine ﬁsh species.

Species

Fertilisation Duration of spawning season of a female (days)

Number of spawned eggs

Atlantic salmon1 Wolf-ﬁsh2 Cod3 Turbot4 Halibut5

External

10 000–15 000

Internal External External External

1 1 50–60 12–38 50–60

Number Number of Periodicity of of egg eggs in a releasing egg batches batch (¥103) batches (days) 1

5 000–50 000 1 2.5–14.5 million 10–20 10–15 million 5–16 0.5–1.5 million 4–16

1.3–6.8 10–530 – 10–350

Egg diameter (mm)

–

5–7

– 2.5–3.1 2.0–4.0 2.9–3.8

4.31–6.38 1.16–1.89 0.97–1.10 3.00–3.80

1

Gjedrem (1993). Falk-Petersen et al. (1999); Tveiten and Johnsen (1999); Moksness & Pavlov (1996). 3 Kjesbu (1989); Kjesbu et al. (1991); Iversen and Danielssen (1984); Kjørsvik (1994); Olsen (1997). 4 Jones (1974); Howell (1979); Bromley et al. (1986); Bromley et al. (2000); Fauvel et al. (1993). 5 Haug et al. (1984); Norberg & Kjesbu (1991); Kjørsvik & Holmefjord (1995); B. Norberg, personal communication, 2002. 2

ered ethologically as bearers, and ecologically as facultative internal (or lecithotrophic) live bearers. According to this classiﬁcation, a short retention of fertilised eggs within the oviduct should not be considered as viviparity. The features of egg production in the different species considered in this book are summarised in Table 5.2. The species which do not protect their eggs, e.g. cod (Gadus morhua L.), turbot (Psetta maxima L.) and Atlantic halibut (Hippoglossus hippoglossus L.), show very high total fecundity or egg yield (more than 14 million, 18 million and 1.5 million eggs, respectively) and comparatively small egg diameters (Fig. 5.1a). The egg diameter of spotted wolf-ﬁsh (Anarhichas minor Olafsen) is substantially higher (5.0–6.0 mm), and absolute fecundity is less than 35 000 eggs.

5.2 Gonad Maturation In many ﬁsh species, the gonads in females are represented by paired ovaries attached to the dorsal surface of the body cavity. However, in some species only one ovary is developed. A single ovary, or paired ovaries fused in their caudal parts (as in wolf-ﬁsh), are common for species with internal insemination. In ovaries of the open type, the oocytes are moved into the body cavity after ovulation, and then are released into the water through the genital pore. This arrangement can be found in less derived teleost ﬁsh, e.g. salmonids. Ovaries of the closed type possess a cavity for the storage of oocytes after ovulation. The caudal parts of the paired ovaries transform into the oviducts, which join before reaching the genital pore. This arrangement is usual for the majority of teleost ﬁsh. In males of teleost ﬁsh, the genital system includes paired testes, and the urinary canal which runs from the urinary bladder joins with the spermiduct to form a canal opening into the urogenital pore. The testes consist of the testicular canals surrounded by a dense membrane. Two types of testes, cyprinoid and percoid, are deﬁned, based on the position

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133

A

B

Figure 5.1 (a) Absolute fecundity and egg diameter (before swelling), and (b) average total length at hatching and at the beginning of the larval and juvenile states in ﬁve marine ﬁsh species with different types of ontogeny.

of the testicular canals and spermiduct. Testes of the ﬁrst type have an oval form in crosssection, and the testicular canals begin from the gonad periphery and reach the spermiduct at the dorsal or dorso-medial part of the gonad. In cross-section, the testicular canals appear as ampoules of different shapes. This type is common for cyprinids, herrings, salmonids and other ﬁsh. Testes of the second type have a form which is similar to a triangle in crosssection. Radial testicular canals reach the spermiduct, which is located in the deep part of the gonad close to its dorsal surface. Testes of the latter type are found in perciform species, in some ﬂatﬁsh (Soleidae) and in several other species. Testes of a transitory type (between the two basic types) are also known (Makeyeva, 1992). It is therefore clear that the capacity of a ﬁsh species to produce gametes, and their fecundity, will be inﬂuenced by their reproductive styles. However, the basic principles of gonadal development and gamete maturation can be somewhat generalised. In the developing gonads, the germinal tissue will differentiate at an early stage in the life of the ﬁsh into oogonia in females and spermatogonia in males. The species considered in this book are all gonochoristic, i.e. they have the same sex throughout their whole life cycle. However, sex change is not uncommon in the ﬁsh world, whereby they ﬁrst differentiate into one sex, and then later

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Culture of cold-water marine ﬁsh

into the other. Species maturing ﬁrst as males are called protandrous, and species maturing ﬁrst as females are called protogynous.

5.2.1 Females Primordial germ cells will migrate into the ovary and form oogonia during embryonic development. When the gonad maturation process (oogenesis) begins, the female oogonia will undergo numerous mitotic cleavages and develop into previtellogenic primary oocytes (Fig. 5.2). These primary oocytes will enter the ﬁrst meiotic cleavage, which is arrested at the prophase. The oocytes will then undergo a long growth period with endocytotic accumulation of yolk material. This is due to the hepatic (liver) synthesis of yolk vitellogenin (Vtg), which is a large lipoprotein molecule (MW up to 600 000). This process is called vitellogenesis. Vtg may also be modiﬁed and act as a carrier molecule for lipids, carbohydrates, phosphates and several other yolk nutrient components. The Vtg is transported by the blood to the ovary, where it is taken up by the developing oocytes through the ovarian follicle cells that surround each oocyte. Vtg is enzymatically transformed into lipovitellin and phosvitin in the oocytes. Most of the increase in gonad growth and oocyte size takes place during the vitellogenic phase (Le Menn et al., 2000). Vtg is the main component of the yolk material, being up to 80–90% of the egg yolk dry matter. Lipoproteins other than Vtg probably also contribute to the incorporation of material such

Figure 5.2 (A) Different stages of oocyte growth and maturation during the ovarian cycle of ﬁsh, and (B) follicular layers enclosing a growing oocyte. FOM, ﬁnal oocyte maturation; GV, germinal vesicle (nucleus); GVM, GV migration; GVBD, GV breakdown; OD, oil droplet (Khan & Thomas, 1999). Reproduced with permission from Elsevier Science.

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135

as lipids into the oocytes. Vitellogenesis stops when the maturing oocytes have reached a certain size and a certain stage in development. Oocyte growth and maturation is illustrated in Fig. 5.2, and the development is well described by Khan & Thomas (1999). The ﬁnal oocyte maturation (FOM) in ﬁsh is associated with the resumption of meiosis via a hormonal signal. A polar body containing a haploid set of chromosomes is shed from the primary oocyte, resulting in a haploid secondary oocyte after the completion of the ﬁrst meiotic division (Wallace & Selman, 1990). The nucleus (also called the germinal vesicle) of the secondary oocyte enlarges and migrates from the centre to the animal pole of the oocyte, adjacent to the micropyle opening in the oocyte membrane (see e.g. Goetz, 1983; Kjesbu et al., 1996). The micropyle is the only opening in the egg envelope, and is just large enough to let one sperm cell penetrate during insemination. A ‘fertilisation cone’ is formed around the ﬁrst spermatozoan and blocks the entrance to the micropylar canal, thus protecting the egg against polyspermi. The nucleus migration is followed by a continuation of meiosis, which proceeds to the metaphase of the second meiotic division, before it is again arrested. This stage is characterised by the breakdown of the nuclear membrane (the nucleus becomes invisible). The lipid and yolk droplets will generally coalesce and the oocyte will appear more homogenous. One or several lipid droplets may also be formed at this stage. The cytoplasm will be situated at the periphery of the oocyte, in a layer between the yolk and the eggshell (see also Chapter 6). Numerous cortical alveoli in the cytoplasm can be found towards its outer membrane (the vitelline membrane). The completion of the second meiotic division, where the secondary oocyte will transform into an egg cell, will not take place until egg activation, i.e. the discharge of cortical alveoli, causing the separation of the egg envelope (also called zona radiata, chorion or eggshell) from the vitelline membrane. The polar bodies contain a haploid nucleus but very little cytoplasm, and will degenerate rapidly. In many marine ﬁsh species, especially in forms with pelagic eggs, oocytes enlarge substantially due to hydration (a massive inﬂux of water) during the ﬁnal maturation phase (Craick & Harvey, 1987; Mangor-Jensen, 1987). This seems to be regulated by the proteolysis of the yolk proteins into free amino acids by the lysosomal enzyme cathepsin L, which results in an osmotic pressure for hydration and a dramatic increase in oocyte diameter, especially in pelagic eggs (Thorsen et al., 1993; Carnevali et al., 1999). The water content in pelagic eggs increases from 50–70% in vitellogenic oocytes to 85–95% in hydrated oocytes (Craick & Harvey, 1987; Thorsen et al., 1996; Finn et al., 2000). The ovarian follicle will rupture when the oocytes have completed the ﬁnal maturation, and the oocytes are then ovulated into the ovarian lumen, or, as in salmonids, into the body cavity, where they are surrounded by ovarian ﬂuid until spawning. The eggs are ready to be fertilised after completion of the ﬁnal hydration. Both maturation and ovulation are triggered by hormonal control, but represents two comparatively independent processes (see Section 5.2.4).

5.2.2 Males Spermatogenesis is divided into four periods: (I) division, (II) growth, (III) maturation and (IV) formation of spermatozoa. Two meiotic divisions occur in the maturation period,

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resulting in the appearance of haploid spermatocytes of the second order, and then spermatids. During the ﬁnal (IV) period of spermatogenesis, a spermatozoon is formed from each spermatid. When male sexual maturation approaches, primary spermatocytes are formed by several mitotic divisions of the spermatogonia. The basal membrane in the sperm-producing tubuli consists of glycogen-rich Sertoli cells, which surround and support the increasing numbers of developing spermatocytes. The primary spermatocytes will undergo the ﬁrst meiotic division to haploid secondary spermatocytes, which will rapidly divide into the second meiotic division and form spermatids. Each of the primary spermatocytes will thus divide into four haploid spermatids. During the ﬁnal spermiogenesis, the spermatids will develop into spermatozoa, and the mature spermatozoa are released to the sperm ducts (spermiation). This process is generally followed by milt hydration, in which there is an increase in the water content of the seminal ﬂuid–spermatozoa suspension. Cell size decreases to approximately half from the secondary spermatocyte to the spermatid, with a subsequent decrease to the spermatozoon. The sperm can be released in a short time in some species, or continuously as several spermatogenic waves in others. The latter type is more common for batch-spawning species (Makeyeva, 1992). In most ﬁsh with internal insemination, the spermatozoa are grouped into spermatozeugmas or spermatophores. These are transferred through the anal ﬁn that is transformed into a gonopod. In some species, including ﬁsh from the Anarhichadidae family, the spermatozoa remain free and are transferred through a primary (urogenital papilla) copulative organ (Billard, 1986; Makeyeva, 1992). Fish spermatozoon morphology is very species-speciﬁc, and depends on the taxonomy, as well as on the reproductive biology of the species (Ginsburg, 1968; Turdakov, 1972; Baccetti, 1984; Emelyanova & Makeyeva, 1985; Jamieson, 1991; Mattei, 1991; Makeyeva, 1992). The spermatozoon includes an egg-shaped head, which consists mainly of the haploid nucleus. It also has a middle section with numerous mitochondria to produce the necessary energy for swimming, and a ﬂagellum (tail), which for teleost ﬁsh has a typical ﬂagellatestructure (two central tubuli surrounded by nine double tubuli). The teleosts lack an acrosome for the penetration of the spermatozoon through the egg envelope because of the presence of a micropyle, but possess specialised structures on the plasma membrane at the top of the spermatozoon head (Billard et al., 1995). Once the sperm has been shed, or as soon as it comes in contact with the water surrounding the ﬁsh, it will become activated and is very short-lived. Therefore, great care has to be taken not to allow the sperm to come into contact with water during the stripping procedure or during sperm storage.

5.2.3 Spawning Once or Many Times? Different species have different spawning strategies, and this will be reﬂected in variations in the gonad maturation patterns. Some, such as the Paciﬁc salmon or the anguillid eels, may spawn once in a life-time (semelparous species). Others may spawn more than once (iteroparous species), and most of these iteroparous ﬁsh will typically have one spawning season per year.

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Figure 5.3 Patterns of gamete development in ﬁsh with synchronous, group synchronous or multiple group synchronous ovarian development (Pankhurst, 1998).

Depending on the different spawning modes of the species, three different types of gonad development can be recognised: synchronous, group synchronous and multiple group synchronous (the latter is also called asynchronous) (reviewed by Tyler & Sumpter, 1996; Pankhurst, 1998). The gonad development pattern of males will follow the female type of oocyte development. The semelparous species only need to develop gonads once in their lifetime. The females all have one homogenous group of developing oocytes in the ovary, and this is described as synchronous ovarian development (Fig. 5.3). The iteroparous species will develop gonads several times during their life-span, and consequently their ovaries will contain a population of previtellogenic oocytes as well as developing oocytes that will be ovulated during the forthcoming spawning season. Such gonad development is called group synchronous (Fig. 5.3), and is found in a number of high-latitude and temperate species as well as in many deep-water ﬁsh. The wolf-ﬁsh (Anarhichas), the species of the genus Macrozoarces, the Atlantic herring (Clupea harengus), and the iteroparous salmonids all have group synchronous oocyte development: all vitellogenic oocytes grow in unison, particularly in the few weeks before ovulation, causing ovulation of all vitellogenous oocytes in a short time period, and the subsequent release of a single egg clutch. However, there is a further difference between ﬁsh spawning once per year and those spawning several batches of eggs per spawning season. This last group demonstrates multiple group synchrony (they are also called asynchronous) (Fig. 5.3), where previtellogenic

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oocytes are present together with several different stages of maturing vitellogenic oocytes. This is the most common pattern of gonad development among teleost ﬁsh, and is typical for tropical and warm temperate marine and fresh-water ﬁsh, as well as for some cold temperate species. The majority of marine ﬁsh species with pelagic eggs (e.g. cod and ﬂatﬁsh) have multiple group synchronous oocyte development, with subsequent spawning of multiple egg batches over a period of time (see Table 5.2 and references therein). A high variation in the number of egg batches is observed in different species and in different females of one species. The ovulatory rhythms are species-speciﬁc, and individual variations are observed in some species. The varying numbers of egg batches and the variable fecundity within a species are mostly due to ﬁsh size and environmental conditions, such as temperature, feeding conditions, the presence of stress factors, etc. The fecundity of ﬁsh is generally inversely correlated to the egg size; the more eggs a species produces, the smaller the eggs will be. Demersal eggs are generally larger than pelagic eggs, and the mean size of a pelagic egg is around 1 mm in diameter (Blaxter, 1988; Kendall et al., 1984). The halibut therefore produces some of the larger pelagic eggs (3 mm). Within a species, a large female will generally produce larger eggs than a smaller female. One factor complicates this picture: species producing multiple spawnings per season generally produce progressively decreasing egg sizes towards the end of the spawning season. For cod, a large ﬁsh will also produce larger eggs than a small ﬁsh, but the seasonal difference in egg size within a single female is generally larger than differences in egg size between females of different size. If egg size is to be studied in relation to ﬁsh size, the same spawning stage of the ﬁsh must be compared.

5.2.4 Endocrine Regulation Reproduction is controlled by internal (endogenous) rhythms, which are stimulated by environmental inﬂuences such as light, temperature and nutritional factors. In temperate waters, the seasonal changes in daylength are important cues for the onset of gonad maturation, thus making it possible to produce offspring during the season offering the best chances of nutrition and survival for the larvae and juveniles. The reproductive cycle is regulated mainly by hormonal production, and is controlled by the so-called brain–pituitary–gonad axis. Gonad maturation is really an orchestrated cascade of events, and it is also a beautiful example of how ﬁnely tuned and vulnerable physiological processes are. Light affects the internal rhythms of ﬁsh through the eyes and through the pineal gland in the uppermost part of the forebrain. The pineal gland secretes the hormone melatonin, and the levels ﬂuctuate according to light levels, with the greatest production during darkness. Fish in temperate waters are generally sensitive to changes in daylength (and in tropical areas even to lunar cycles), and the resulting seasonal changes in melatonin will probably act together with the endogenous rhythm of the ﬁsh to stimulate the production of gonadotropin-releasing hormones (GnRH) in the hypothalamus. GnRH is a key regulator in reproduction for all vertebrates, although the possible role of melatonin in ﬁsh reproduction is still speculative (Mayer et al., 1997; Bromage et al., 2001). The GnRH is transported to the pituitary gland (which is attached to the hypothalamus), where it will induce sexual

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maturation by stimulating special neuro-secretory cells in the pituitary to produce and secrete two different gonadotropin hormones, namely follicle stimulating hormone (FSH, also called Gth-I) and luteinising hormone (LH, also called Gth-II). FSH and LH are structurally similar to mammalian gonadotropins (Swanson, 1991; Tyler et al., 2000). These hormones are transported by the blood to the gonads, where in females they are bound to speciﬁc surface receptors for the different hormones in the follicle cells surrounding the oocytes. FSH appears to be involved in vitellogenesis (Tyler et al., 2000), whereas LH seems to be responsible for oocyte maturation and ovulation (Swanson, 1991; Nagahama, 2000). Thus, FSH is present in the blood during the early stages of gonad development, whereas LH is present during the maturational phase. The secretion of these two gonadotropins is also regulated differently. The FSH hormone regulates the production of maturing oocytes, and is secreted during most of the maturation cycle, with increasing blood plasma levels up to ﬁnal maturation. FSH will stimulate the ovarian follicle cells to synthesise the androgens and oestradiol-17b. Rising levels of oestradiol-17b and testosterone thus signal active development of the ovaries. Oestradiol-17b and testosterone are synthesised in a twocell model (Fig. 5.4), where the theca cells synthesise testosterone in response to FSH, and oestradiol-17b is subsequently produced in the granulosa cells from testosterone, which is aromatised by cytochrome P450 aromatase (Kagawa et al., 1982; Nagahama, 2000; Patiño et al., 2001). Oestradiol-17b is transported by the blood to the liver and stimulates hepatic production and secretion of the yolk protein vitellogenin (Vtg) and ‘zona radiata’ proteins (material for making the egg envelope, or chorion). These proteins are transported from the liver by the blood to the ovaries. The ‘zona radiata’ proteins are deposited around the oocyte (OppenBerntsen et al., 1992; Tyler et al., 2000), and Vtg is actively sequestered by receptor-mediated endocytosis through the oocyte membrane (Wallace & Selman, 1990; Specker & Sullivan, 1994; Mommsen & Walsh, 1998). The secretion of FSH and LH is regulated by feedback mechanisms of oestradiol-17b and testosterone (Peter & Yu, 1997). In addition, oestradiol17b will stimulate the differentiation of female sex characters, whereas testosterone also functions in the ﬁnal maturation of the oocytes and as a sexual pheromone both for females and males. The largest increase in oocyte size occurs during vitellogenesis, and this process may last for several months. Blood plasma levels of Vtg, oestradiol-17b and testosterone will increase gradually up to ﬁnal maturation. The ﬁnal maturation, ovulation and spawning are regulated by the luteinizing hormone (LH), and plasma levels of LH increase rapidly up to spawning. Ovulation can be described as the release of a mature oocyte from the follicle wall in the ovary. LH stimulates the gonads to produce progesterone hormones called the maturationinducing steroids (MIS), which initiate the ﬁnal oocyte hydration (Nagahama, 2000). The stimulation of ﬁnal maturation by MIS seems to occur by the activation of a maturation-promoting factor that consists of cyclin B and a catalytic kinase. MIS stimulates the synthesis and action of cyclin B by binding to a membrane-bound receptor on the oocyte surface (see e.g. Nagahama, 1987). MIS also stimulates the follicle cells in the ovary to produce prostaglandins. Prostaglandin F2a will induce contraction of the ovaries, and thus be responsible for ovulation. Prostaglandin and MIS may also stimulate the spawning

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Figure 5.4 Oocyte growth and ﬁnal maturation (FOM). During oocyte growth, oestradiol-17b and testosterone are synthesised in a two-cell model, where the theca cells in the ovarian follicle synthesise testosterone in response to the follicle stimulating hormone (FSH, produced in the pituitary), and oestradiol-17b is subsequently produced in the granulosa cells (also in the follicle) from testosterone, which is aromatised by cytochrome P450 aromatase. Oestradiol-17b is transported by the blood to the liver, and stimulates hepatic production and secretion of the yolk protein vitellogenin (Vtg) and zona radiata proteins (ZRP, material for making the egg envelope or chorion). These proteins are transported from the liver by the blood to the ovaries. The ZRP is deposited around the oocyte and Vtg is actively incorporated into the oocyte by receptor-mediated endocytosis. The ﬁnal oocyte maturation (FOM), ovulation and spawning is regulated by the luteinizing hormone (LH), and plasma levels of LH increase rapidly up to spawning. LH stimulates the gonads to produce progesterone hormones, which initiate the ﬁnal oocyte hydration. See text for a further explanation. Figures by Geir Lasse Taranger, Institute of Marine Research, Bergen.

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Figure 5.5 Plasma levels of vitellogenin (VTG), oestradiol-17b (E2) and testosterone (T) in laboratory-held mature female halibut prior to and during the reproductive period. Values are means +1 SE. Missing standard error bars are too small for presentation (Methven et al., 1992).

behaviour of both females and males (by acting as a post-ovulatory pheromone), and thus synchronising the spawning act between the sexes. Female release of MIS and prostaglandins into the water is registered by the males, which are reacting with increasing levels of male LH and a subsequent increased production and release of sperm. Several other factors are also up- and down-regulated in the ovaries around ovulation, and these are described by Goetz and Garczynski (1997). In ﬁsh with synchronous or group synchronous oocyte development, such as salmonids and wolf-ﬁsh, there is only one peak in the levels of Vtg and of the different steroid hormones, and then the levels decline gradually during the month preceeding ovulation (Jobling, 1995; Tveiten & Johnsen, 1999). For species with multiple group synchronous gonad maturation, such as cod and Atlantic halibut, the levels will increase up to the commencement of spawning. The levels of Vtg and the different hormones will then oscillate during the spawning season, indicating that vitellogenic oocytes are developing in the ovary at the same time as mature oocytes are ready to undergo ﬁnal maturation (Fig. 5.5). During male maturation, the pituitary gonadotropins LH and FSH will regulate steroidogenesis and spermatogenesis by activating receptors in the Leydig cells (LH receptors) and in the Sertoli cells (FSH receptors) (Schulz et al., 2001). The Leydig cells are responsible for the production of the steroid testosterone and its derivative 11-ketotestosterone (Nagahama, 1994). In male teleosts, the growth and development of the testes is thus associated with rising plasma levels of testosterone and 11-ketotestosterone. 11-ketotestosterone seems to be responsible for regulation of the spermatogenesis (development of sperm cells), it stimulates the development of secondary male sexual characters, and may also have growth-promoting effects (Borg, 1994; Nagahama, 1994). Males are very sensitive to the female secretion of MIS, which will stimulate sperm maturation and release, and also stimulate male production of MIS rather than 11-ketotestosterone. Male MIS is probably synthesised in the sperm cells, although the surrounding testicular tissue is necessary.

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Induced ovulation and spawning include injections of pituitary extracts, chorionic gonadotropin (HCG) and synthetic hypothalamic-releasing hormones (see e.g. Zohar & Mylonas, 2001). The injections of gonadotropin-releasing hormone agonists (GnRHa) is an effective alternative to pituitary extracts and HCG in the induction of oocyte growth, maturation and ovulation in a range of ﬁsh species. The gonadotropins can be under the control of an inhibitory hormone (dopamine). Treatment with antagonists of dopamine together with injections of synthetic analogues of GnRH leads to an enhanced release of gonadotropins. It is important to note that hormonal stimulation often leads to maturation and ovulation, but does not induce natural spawning. For the latter process, special environmental conditions are required. The majority of cold-water marine ﬁsh species which are regarded as interesting for aquaculture will undergo ovulation without hormonal stimulation, under controlled artiﬁcial environments in captivity. Implants of GnRHa were applied to males of the Atlantic halibut to synchronise the time of spermiation and egg ovulation (Vermeirssen et al., 1998). Without such injections, spermiation often commences about 1–2 months prior to female ovulation. However, the most interesting application of the GnRH implants was that they improved milt ﬂuidity late in the spawning season, when milt is often difﬁcult to use because of its very high viscosity.

5.3 Brood-Stock Management and Egg Production The environmental conditions of ﬁsh (e.g. temperature and feeding) may be not optimal for their development and growth in the wild, at least not during part of the year, but they should be close to optimal in captivity. However, the artiﬁcial conditions (unusual environment, including crowding, light, temperature, feeding and the absence of caves) may represent a stressful situation, which is most severe at the beginning of the establishing phase of the brood stock from wild populations. Conditions suitable for spawning (e.g. special habitats) are almost never reached in marine farms, which causes problems for natural spawning in captivity (i.e. courtship behaviour and spawning rituals, followed by the release of fertilised eggs). The term ‘natural spawning’is used in this chapter to describe the ovulation and release of eggs and milt by the ﬁsh without hormone treatment or stripping of the ﬁsh to obtain gametes. Stress may affect the reproductive process in ﬁsh, and was recently reviewed by Schreck et al. (2001).The stress response depends on the species of ﬁsh, the stage of maturity and the duration of the stress factor(s). According to the deﬁnition of Schreck et al. (2001), stress is ‘the response of the body, i.e. a physiological cascade of events that occurs when the organism is attempting to resist death or re-establish homeostatic norms in the face of an insult’. The stressors induce the pituitary synthesis and secretion of corticotropic hormones, which in turn stimulate the synthesis and secretion of cortisol, a glucocorticoid hormone. There is a possible direct link between gonadotropins (and their inﬂuence on egg maturation and reproduction) and stress hormones. The inﬂuence of stress can be manifested directly in the reduced survival of adult ﬁsh. The secondary effect of stress is the immunosupression that renders the ﬁsh vulnerable to pathogens owing to the action of cortisol, which depresses the ability of ﬁsh leucocytes to form antibodies. Under adverse conditions, a female can select between energy allocated for

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somatic growth and energy for reproduction. In the ﬁrst case, the resorption of the ovary and egg atresia can occur. There are several indications of reduced egg quality associated with unfavourable maintenance of brood ﬁsh (see review by Brooks et al., 1997). In particular, for spawning cod separated into pairs in small tanks, up to one-third of the ﬁsh showed signs of stress resulting in irregular spawning intervals and poor egg quality caused by over-ripening (Kjesbu, 1989; Kjørsvik & Holmefjord, 1995). Nevertheless, the females apparently possess a buffering mechanism which protects their eggs from the negative effects of stress (Schreck et al., 2001). For instance, a number of eggs can be resorbed, and the energy allocated to the rest of the developing oocytes. Parental stress may also cause reduced viability and survival of the offspring (see e.g. Morgan et al., 1999). However, little is known about such problems, and much more work remains to be done in this area.

5.3.1 Brood-stock Nutrition Brood-stock nutrition will affect the viability and health of the offspring as well as that of the brood-stock ﬁsh, and feeding the brood-stock a diet to fulﬁl its optimal reproductive potential is therefore of vital importance. Most work on the nutrient requirements of broodstock ﬁsh is limited to a few species (mainly salmonids and sparids). However, it is clear that many problems in ﬁsh culture, including low fertilisation rate and poor egg and larval quality, are directly related to the diet composition of the brood stock. Sexually maturing ﬁsh (and other animals) generally have increased requirements for speciﬁc nutrients (i.e. somewhat different nutritional demands than during the grow-out phase), and on-growing feeds are often not adequately covering the dietary requirements of broodstock. Only a few commercial brood-stock diets have been developed for cold-water marine ﬁsh aquaculture owing to the short history of this industry, and to the present small production volume of these species. It is often reported that brood-stock ﬁsh fed on ‘natural diets’ produce eggs of better quality than those on formulated commercial diets (Brooks et al., 1997). The nutrition of the brood-stock can be improved by feeding marine ﬁsh solely on fresh marine organisms (squid, cuttleﬁsh, mussels, krill and small crustaceans), or marine organisms in combination with commercial diets. However, the use of unprocessed products does not always provide adequate levels of nutrients, and also increases the risk of disease transmission. At the same time, the nutritional quality of commercially formulated feed designed for each species could be substantially improved. At present, feed is the largest production cost for commercial aquaculture, and research to develop substitutes for ﬁsh oil and ﬁsh meal is now focused on oil seeds (especially soybeans), meat by-products and microbial proteins (see also Chapter 9). However, vegetable proteins have an inappropriate amino acid balance, and they contain phytoestrogens. A brood-stock diet based on soybean meal could thus reduce nutrients which are essential for reproduction (Izquierdo et al., 2001), and phytoestrogens may have an adverse effect on reproductive development in both males and females. For ﬁsh, the primary driving force for reproduction is generally reﬂected in the investments made during gonad growth, and this is especially so for females. Many species, such

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as salmon and cod, tend to reduce (or stop) their feed intake during gonad maturation and/or spawning, and the energy and nutrients necessary for gonadal growth must be taken from their body reserves. A wild female salmon uses almost 90% of her fat and 50% of her muscle protein to build up the gonads. Other species, such as the gilthead seabream (Sparus aurata L.), will continue to feed throughout gonad maturation and the spawning period, and will therefore rely on both body reserves and diet to produce eggs (the egg biomass is often greater than their own body weight). Brood-stock nutrition is important in terms of both the quality and the quantity of the feed, but we know relatively little of speciﬁc brood-stock requirements for each species. When considering the effects of brood-stock nutrition on egg and sperm quality and offspring viability, knowledge of the reproductive pattern of the species in question is very important. It is of particular importance to know the timing and duration of the gonad maturation period (especially vitellogenesis), as this is the time when most nutrients and energy are incorporated into the oocytes. Also important is the reproductive strategy (synchronous, group synchronous or multiple group synchronous), the duration of the spawning period, and the feeding pattern of the ﬁsh during gonad maturation and spawning. These parameters will regulate how and when dietary brood-stock nutrients must be available to build up the gonads, and whether we must rely on the body reserves of the ﬁsh, or if we may use direct feeding during maturation and spawning to ensure optimal egg quality. Fish will then not be limited to using dietary nutrients during gonad maturation and spawning, and body reserves can be mobilised from different tissues for gonadal growth. A good brood-stock diet and feeding regime must therefore result in a good status of the body stores before the beginning of vitellogenesis. The cod, sea bass (Dicentrarchus labrax L.), gilthead seabream and wolf-ﬁsh species are all different types of spawner, and have thus different requirements in terms of when the nutrients required for gonad development should be available. Wolf-ﬁsh vitellogenesis starts approximately 3 months prior to spawning, and all eggs are shed in one batch (Tveiten & Johnsen, 1999). In cod, which is a continuous spawner with no feeding activity during spawning, ovarian development starts 8–9 months prior to the onset of spawning, and nutrients are incorporated into the oocytes up to the ﬁnal maturation of the different egg batches (Kjesbu et al., 1991). A similar pattern is seen for halibut and turbot. Gilthead seabream is also a continuous spawner (although not in cold water), but will continue its feeding activity during the spawning period. In this species, ovarian development starts 2 months prior to spawning, and feeding high-quality diets for 60 days prior to and throughout spawning has resulted in a positive effect on spawning results. However, even this period seems too short to fully alter the egg fatty acids composition, and it is now recommended to feed bream a special brood-stock diet for at least 3 months prior to, and throughout, the spawning period (Almansa et al., 1999). 5.3.1.1 Ration Size Food restriction may lead to an inhibition of gonad maturation, and feeding rations and growth rate have a signiﬁcant effect on the size and potential fecundity of the ﬁsh. As shown for cod,

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feeding conditions have a major inﬂuence on the fecundity and proportion of pre-vitellogenic oocytes, whose number is regulated by means of atresia. In addition, egg size decreases more rapidly during spawning in starving ﬁsh, and the egg viability is lower (Kjesbu et al., 1991; Kjørsvik, 1994; Karlsen et al., 1995). Growth during the months prior to spawning has been found to affect the time of spawning in cod, with well-fed ﬁsh commencing spawning earlier than less well-fed ﬁsh (Kjesbu et al., 1996, 1998). Cod with high condition factors produce more pre-vitellogenic oocytes than ﬁsh of the same size deprived of food. In general, rations less than 100% will not affect egg viability and fry survival, but will decrease fecundity, possibly delay the time of spawning, and reduce the proportion of ﬁsh that mature (Springgate et al., 1985; Cerdá et al., 1994). Rainbow trout egg size and initial larval growth seem to be affected by brood-stock ration size, but not egg and fry survival. In cod, however, female fecundity was affected by ration size, but no effects were found in egg size. The degree of atresia is correlated to the condition factor of the ﬁsh (Kjesbu et al., 1991). When cod were exposed to periodic starvation during vitellogenesis, with weights being reduced to around 60% of those fed full rations, the observed differences in fecundity were only due to differences in ﬁsh size, and the relative fecundity was the same for all ﬁsh. In other experiments, in pre-spawning starvation of recruit spawners of cod for up to 9 weeks, almost all ﬁsh matured (Karlsen et al., 1995). Starvation for 9 weeks did not affect spawning time or the relative weight of the testes, but did affect ovary weight. In addition, the mean fecundity of the ﬁsh starved for 9 weeks was signiﬁcantly lower than that in the well-fed control group. The difference in the results of the effect of starvation on cod egg production between this study and the previous investigations can be explained by a different sensitivity to food variation during vitellogenesis of repeat and recruit spawners. In several marine ﬁsh species, reduced fecundity is related to the inﬂuence of a nutrient imbalance on the endocrine system, or to a restriction in the availability of biochemical components for egg formation. In sea bass, reduced rations seem to result in a reduced fecundity and delayed spawning period, but not in reduced egg quality and larval viability (Cerdá et al., 1994). Feeding of brood-stock should be to satiation, according to the daily appetite of the ﬁsh. Depending on the species, it is important to maintain an adequate food supply until vitellogenesis or spawning is completed. Reductions in feed rations during the later stages of maturation or spawning may result in atresia (resorption of oocytes), and thus reduced fecundity. 5.3.1.2 Feed Composition Fish seem to be very ﬂexible in their ability to adapt to different levels of macro-nutrients such as protein and fat without a reduction in reproductive performance. However, this ﬂexibility seems to have certain limits, which are different from species to species. The important components of the diet, which may determine egg viability, are the lipids, essential polyunsaturated fatty acids (PUFA), protein, vitamins, carotenoids and various trace elements (see reviews by Kjørsvik et al., 1990; Bromage, 1995; Brooks et al., 1997). For rainbow trout, the dietary protein levels should be at least 33%, whereas for red sea bream (Pagrus major (Temmink et Schlegel)), gilthead seabream and seabass it is recom-

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mended to keep the brood-stock dietary protein level at a minimum of 45% to prevent a reduced egg quality (Watanabe, 1985; Cerdá et al., 1994; Tandler et al., 1995; FernándezPalacios et al., 1997). Cod and halibut have a protein demand that is more similar to that seabream and seabass than to salmonids (Lie et al., 1988). Large variations in dietary lipid levels seem to have little effect on reproductive performance for salmonids or for bass and bream (10–30%). However, there are speciﬁc requirements for certain essential fatty acids during gonad maturation, and therefore very low lipid levels in the feed may not be adequate. An elevated carbohydrate level may have a positive effect on reproductive performance. Glucose is an important energy source for maturing gonad tissues, and an improved glucose tolerance has been observed in ﬁsh during vitellogenesis. Dietary carbohydrate levels between 5 and 28% did not affect cod brood-stock growth, feed conversion rate or gonadal development, possibly because the requirements for protein and lipids were met (Hemre et al., 1995). However, too high dietary carbohydrate levels seems to affect egg quality negatively, by increasing the ovarian lipid stores. 5.3.1.3 Fatty Acids The (n-3) and (n-6) essential fatty acids (EFA) are very important for normal reproduction and the egg development, and the egg viability of several marine ﬁsh species have been improved by altering the lipid composition of brood-stock diet (see Fig. 5.6, with data from Watanabe, 1985; and reviews by Sargent, 1995; Rainuzzo et al., 1997). Such a deﬁciency will affect fecundity, egg quality, hatching success and the number of normal larvae, and will lead to deformations in the juveniles that are produced. When brood-stock of gilthead seabream were fed an EFA-deﬁcient diet for 2 months prior to spawning, the spawning period

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was reduced by 50%, and egg quality was signiﬁcantly reduced towards the end of the spawning season (Fig. 5.6a). In general, a certain level and a correct balance between the three essential fatty acids docosahexaenoic acid (DHA, 22:6(n-3)), eicosapentaenoic acid (EPA, 20:5(n-3)) and arachidonic acid (ARA, 20:4(n-6)) seem to be important for successful reproduction and embryonic development (Watanabe, 1985; Luquet & Watanabe, 1986; Hardy et al., 1989; Thrush et al., 1993; Harel et al., 1994; Fernández-Palacios et al., 1995, 1997; Almansa et al., 1999; Navas et al., 1997; Bell et al., 1997). DHA plays a fundamental role in embryonic development, especially for the development of membranes and of brain and nerve tissues. A high DHA supply seems particularly important for the rapidly growing pelagic marine ﬁsh eggs and larvae, as these larvae have a high percentage of neural tissues in a relatively small body mass. Certain dietary nutrients, in particular EPA and ARA levels, show a correlation with fertilisation rates. Two possible explanations of this effect are (a) sperm fatty acid composition (and sperm motility) depends upon the essential fatty acid content of the brood-stock diet, and (b) both EPA and ARA modulate steroidogenesis in the testis, and the timing of spermiation may be delayed and the fertilisation rate reduced in conditions of a deﬁciency of these fatty acids (Izquierdo et al., 2001). ARA has recently been recognised as an essential fatty acid for egg quality in ﬁsh. ARA is a precursor for prostaglandins, which are important in the ﬁnal maturation of oocytes. Prostaglandins also act as a pheromone for the stimulation of male sexual behaviour, and for the synchronisation of male and female spawning. The dietary ARA level may thus have a direct impact on spawning behaviour, and thus fertilisation success, in ﬁsh. However, high EPA levels compared with ARA levels may inhibit the production of eiconoids derived from ARA, which exempliﬁes the importance of a good balance between EPA and ARA in marine brood-stock diets. 5.3.1.4 Micronutrients Several vitamins and minerals are important for ﬁsh reproduction, and the most important are discussed in this section. Vitamin E is important for the control of reproduction, testes function, macrophage function and intracellular oxidation in mammals and ﬁsh. A deﬁciency of dietary vitamin E will affect the number of spawning ﬁsh as well as hatching success and juvenile survival (Watanabe, 1985). This vitamin is the most important of the cellular fatsoluble antioxidants in the body, and has a stabilising effect on the embryonic membranes. Vitamin E is transported to the oocytes by lipoproteins, and the main carrier seems to be the low-density lipoproteins (LDL) and not vitellogenin. Varying vitamin E levels in brood-stock feed has received special attention, because this vitamin is closely linked to lipid metabolism. The level of vitamin E in the gonads increases during vitellogenesis, and this level reﬂects that in brood-ﬁsh muscle prior to the start of gonad growth in salmon and turbot (Lie et al., 1994; Hemre et al., 1994). The egg concentration of vitamin E (measured as a-tocopherol) closely resembles the dietary levels in the brood-stock feed, and a positive effect from vitamin E on egg hatching success and juvenile viability has been demonstrated for several species. This is shown in

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Percentage of groups (%)

100

80

Hatching >70% Juvenile survival >90%

60

40

20

0 50

250

Vitamin E supplement in broodstock feed (mg/kg) Figure 5.7 An appropriate content of vitamin E is necessary in the brood-stock feed to ensure normal embryo development and larval survival in Atlantic salmon. A low content of vitamin E in relation to the total content of n-3 HUFAs in the brood-stock feed resulted in decreased hatching success and larval survival from hatching to start-feeding (data from Rønnestad & Waagbø, 2001).

Fig. 5.7 for salmon (data from Rønnestad & Waagbø, 2001), where dietary vitamin E levels increased from 50 to 250 mg/kg resulted in signiﬁcantly improved egg and fry survival when the ﬁsh were fed high levels of polyunsaturated fatty acids. Marine brood-stock diets normally contain high levels of polyunsaturated fatty acids, and should thus contain vitamin E levels that secure effective prevention of in vivo lipid oxidation. Vitamin C (ascorbic acid) requirements tend to increase during sexual maturation, and the deposition of ascorbic acid in the growing oocytes is important for the hydroxylation of protein-bound proline and lysine to give optimum collagen strength throughout the embryonic stages (Waagbø et al., 1989; Blom & Dabrowski, 1995). This vitamin is an important anti-oxidant, and is vital for bone and cartilage tissue formation and for the non-speciﬁc immune system. A deﬁciency will result in reduced egg production and reduced egg and sperm quality. The hatching success of rainbow trout, seabass and seabream were found to be dependent on the brood-stock dietary level of ascorbic acid, and vitamin C in the eggs may be transferred to the larva and support normal development if the start-feed is deﬁcient in this vitamin (Sandnes et al., 1984; Soliman et al., 1986; Blom & Dabrowski, 1996). Despite observed differences in the free amino acid proﬁles of eggs, egg strength and neutral buoyancy, no effect on fertilisation or hatching rates were detected for cod eggs from brood stock fed a low vitamin C diet (Mangor-Jensen et al., 1994). Ascorbic acid also has an important role in sperm quality, as it prevents oxidative damage to sperm cell DNA and thereby maintains the genetic integrity of the sperm cells (Dabrowski & Ciereszko, 1996). Too low a level of ascorbic acid will reduce sperm concentration and motility, which will reduce ﬁsh fertility, and it has been associated with a high percentage

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of abnormal offspring in rainbow trout (Ciereszko & Dabrowski, 1995; Ciereszko et al., 1999). 5.3.1.5 Pigments and Minerals Fish are unable to synthesise carotenoids, and so obtain them from their diet. The yellow, orange or red colours of the eggs of teleost ﬁsh are caused by the presence of carotenoids in the yolk, and intuitively the most intensely coloured eggs are often regarded as eggs of high quality. Within a species, the eggs of ﬁsh from the wild often have more intensive coloration and larger carotenoid content than the eggs from cultured brood stocks. However, carotenoid content measured quantitatively by a spectrophotometric method does not necessarily relate to the visual intensity of the pigment. For example, the eggs of wild Atlantic salmon, which contain astaxantin as their only pigment detectable by thin-layer chromatography, seem more intensively coloured to the eye than do the eggs of farmed Atlantic salmon, which contain only canthaxantin. Pelagic ﬁsh eggs are usually almost colourless, and demersal eggs possess more or less intensive coloration of the yolk. The difference is often explained as possibly based on the respiration function of the carotenoids: the oxygen supply of pelagic eggs is much better, and the presence of a large amount of pigment is not required. However, there is an alternative explanation of this phenomenon. In pelagic eggs, the colour of carotenoids represents a disadvantage by attracting predators, and they become transparent by converting the carotenoids into a colourless form. For example, the unripe ovaries of cod are bright orange or yellow due to the presence of carotenoids, but mature eggs are colourless. Vitamin A and astaxanthin are important for cell proliferation (growth), for the function of epithelial cells in the ovaries and testes (and other organs), and for the vision. Deﬁciencies in the brood-stock may lead to reduced fertility and offspring deformities. Little is known about the effects of brood-stock nutrition with this vitamin, but it is suggested that requirements for vitamin A can be covered by carotenoids, and in particular astaxanthin. Astaxanthin is a pro-vitamin A, and is an important antioxidant. It is also suggested that astaxanthin plays a role in the respiration processes in eggs. The possible functions of carotenoids in ﬁsh eggs have been discussed over a prolonged period (Craik, 1985; Mikulin, 2000). There are some obvious functions of carotenoids in eggs. They are the source of external pigment for chromatophores of the skin in larvae and juveniles, and the precursors of the vitamin A involved in light reception in the eye. The carotenoids may play some part in oxidative metabolism under conditions of environmental shortage of oxygen. The connection between carotenoid content and egg quality has mainly been studied on salmonid ﬁsh. High fertilisation and hatching rates can be achieved over a wide range of carotenoid levels. However, the egg quality substantially decreases when carotenoid content reaches a ‘critical level’. Astaxanthin is effectively transferred from brood-stock feed to eggs in salmonids and in cod (Grung et al., 1993), and positive effects of dietary astaxanthin on egg quality were found for red seabream and yellowtail (Watanabe & Miki, 1993; Verakunpiriya et al., 1997a). In rainbow trout (Oncorhynchus mykiss Walbaum), the carotenoid content may exceed 13 mg g-1, but the critical level seems to be 1–3 mg g-1. The carotenoid composition obviously has a great inﬂuence on egg quality,

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but the nature of the carotenoids necessary for the maintenance of high egg quality has not yet been fully deﬁned. Minerals are required for normal reproduction (Watanabe, 1985), but requirements above those needed for normal growth have not been established for ﬁsh.

5.3.2 Photoperiod Fish are governed by endogenous rhythms that make them able to spawn at approximately annual intervals, even under constant conditions of light and other environmental factors. Changes in daylight length regulates the reproductive cycle of our temperate ﬁsh species to a large extent, and manipulation of the photoperiods may be used to change to spawning periods which are not normal in order to provide egg production over an extended period of the year. This is shown in Fig. 5.8 for halibut. However, the temperature will also vary during the normal changing daylight cycles of the year, and the temperature range is critical for a successful maturation and spawning of viable eggs. Temperature control is therefore necessary for successful manipulation of photoperiods. The photoperiod is the principal determinant of the timing of maturation, and other environmental factors (e.g. temperature and nutritional status) act in a permissive way to enable maturation to proceed.

Figure 5.8 Duration of spawning, from ﬁrst to last observed egg batch, of Atlantic halibut females on simulated natural (open boxes), four-month advanced (hatched boxes) and 4-month delayed (ﬁlled boxes) annual photoperiod cycle during four sequential spawning seasons. Vertical dashed lines are drawn at 4-month intervals as a visual aid (after Björnsson et al., 1998). Reproduced with permission from Elsevier Science.

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The mechanism for the determination of the timing of ﬁnal maturation under the inﬂuence of photoperiod is not well understood. It is known that melatonin levels (indoleamine hormone from the pineal gland) rise during the night and in the autumn, and that the seasonal changes in melatonin levels correlate with changes in the levels of GnRH and gonadal steroids. A possible link between the pineal function and the endocrine cascade was recently reviewed by Bromage et al. (2001), who concluded that ‘the clear effects of photoperiod on the timing of reproduction on the one hand, and on the diel and seasonal patterns of melatonin on the other, provide strong circumstantial evidence that melatonin is the intermediary in these processes. Direct experimental evidence for this involvement is, however, far less convincing’. An extended spawning period by the use of several brood-stock groups with different maturation cycles may be beneﬁcial for the hatchery economy, as a higher number of production cycles per year will result in higher production and a more effective use of the facilities. The ﬁsh farms should use out-of-phase seasonal light cycles at least until the response of the ﬁsh to the photoperiod can be ascertained. For example, the seasonal light cycle may be compressed into periods of time shorter than 1 year. Once a degree of advance or delay is achieved, the stock can be maintained at 12-month seasonal cycles. Then portions of light cycles can be substituted with constant photoperiods, which are easier to apply at commercial farms. Light can also be used to prevent early maturation in young on-growing ﬁsh. The exposure of Atlantic salmon to continuous light affects the number of ﬁsh matured as grilse, and lighting techniques are widely used to reduce maturity levels and increase the growth of ﬁsh in cages (Taranger et al., 1998). Recent experiments showed that photoperiod manipulation changed the incidence of sexual maturation, spawning time, fecundity and egg size in cod (Hansen et al., 2001). Cod reared under a natural photoperiod spawned between January and April. Cod that were transferred from a natural photoperiod to continuous light in December spawned earlier, and had a lower fecundity and smaller eggs than cod reared under a natural photoperiod. Oocytes of females reared under continuous light from June were arrested in the cortical alveoli stage, and even in the second year of continuous light, very few females matured. The pattern of sexual maturation inﬂuenced the somatic growth pattern. At the age of 26 months, the weight of cod reared under a natural photoperiod and continuous light was 1.5 and 2.5 kg, respectively. A reduction in daylength is thus a vital environmental signal regulating the maturation and spawning of cod, and sexual maturation may be arrested or considerably delayed in its absence. Turbot eggs are now routinely produced commercially throughout the year by means of photoperiod manipulations, and several larger cod and halibut hatcheries have established brood-stock groups for the year-round production of eggs (see also Chapter 2). The effects of changing light regimes on the spawning cycle of Atlantic halibut is shown in Fig. 5.8 (from Bjørnsson et al., 1998). Preliminary experiments with common wolf-ﬁsh show that photoperiod apparently has a major inﬂuence on the ovulation time: the majority of females exposed to a 6 hours light and 18 hours dark (6L:18D) photoperiod from May did not mature in the following spawning season (Moksness & Pavlov, 1996). A review of literature devoted to the inﬂuence of photoperiod on the egg quality of salmonids and marine ﬁsh species showed that results were controversial (Brooks et al.,

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1997). Present knowledge indicates that the extent to which egg quality is affected by manipulating the photoperiod may depend on how well other environmental parameters are controlled, and possibly the time of year at which the advance/delay in spawning occurs.

5.3.3 Temperature Temperature and feed availability are two of the most important factors affecting ﬁsh fecundity, and the optimal temperature for the feeding and growth of brood ﬁsh is not necessarily suitable for normal gonad development and spawning. In many species, the best egg quality is obtained if spawning occurs in a certain temperature range. The optimal temperature during gonad maturation is very important, and temperatures which are too high during vitellogenesis or ﬁnal maturation may reduce fecundity and result in a high degree of atresia and poor egg quality. The time of ﬁnal maturation can be changed under the inﬂuence of temperature manipulations, and a small reduction in temperature during vitellogenesis may delay spawning signiﬁcantly. As seen for cod, this may not be critical for fecundity and offspring viability. The ovulation of females of common wolf-ﬁsh kept at a constant photoperiod (18L:6D) was observed over the entire year, and showed a peak in January. Maintaining the ﬁsh at a lower temperature (8–9°C vs. 10–14°C) for 3 months prior to spawning did not change ovulation time, but led to a substantial increase in egg quality, with the average proportion of normally developed eggs being 6.4% and 69.2%, respectively (Pavlov & Moksness, 1996a). More synchronous ovulation (within 1.5 months) was registered with a natural photoperiod. Females exposed to 8 or 12°C from May to September, and kept at 4°C afterwards, delayed ovulation by 4 or 5 weeks, respectively, compared with ﬁsh held continuously at 4°C (Tveiten & Johnsen, 1999). Water temperature seems to be a comparatively less important factor for the ﬁnal maturation of males: good-quality sperm was obtained from males kept at several temperatures ranging from 2 to 12°C (Pavlov & Moksness, 1994a). Turbot and common sole are especially susceptible to a comparatively high temperature (above 14–15°C) during gametogenesis and the beginning of the spawning period, and a decrease of about 3°C leads to a substantial improvement in egg quality (Devauchelle et al., 1987, 1988). In Atlantic halibut, the viability of eggs from females kept at a constant temperature of 6°C over a spawning season was consistently higher than those from females maintained under ﬂuctuating ambient temperature (Brown et al., 1995, cited from Brooks et al., 1997). This observation is apparently connected to the natural spawning habitat of Atlantic halibut in deep waters with a stable temperature (Haug, 1990).

5.3.4 Present Husbandry Practices and Egg Collection All species considered in this book will undergo normal oocyte maturation, ﬁnal egg maturation and ovulation without the use of hormonal injections. However, conditions which are suitable for normal courtship and spawning behaviour (e.g. special habitats in the spawning grounds) are almost never achieved in ﬁsh farms, which causes problems. Of the species which could be used for cold-water aquaculture, only a few will spawn naturally in captivity. Fertilised eggs from two species, cod and common sole (Solea vulgaris Quensel), are

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normally collected from naturally spawning brood stock. Natural spawning of Atlantic halibut and turbot brood stocks has been observed occasionally, but it is not common and the fertilisation and hatching rates are highly variable, apparently due to inadequate conditions in captivity. For common wolf-ﬁsh, natural spawning with subsequent guarding of the eggs by the male was observed once at the Tromsø Sea Aquarium, after the spawners had adapted to the artiﬁcial conditions over several years (Ringø & Lorentzen, 1987). In captivity, males are often inactive and females may shed unfertilised eggs. Species such as the salmonids, Atlantic halibut, turbot and the wolf-ﬁsh must therefore be stripped for a regular supply of eggs and milt, with subsequent artiﬁcial insemination of the eggs. Care must then be taken to strip the ﬁsh at the correct time in relation to ovulation in order to avoid ageing (or over-ripening) of the eggs. As will be described in Section 5.4, over-ripening of eggs before fertilisation is an important reason for poor egg viability in marine ﬁsh. When ﬁsh are stripped for gametes, a clean, dry container should be used for each of the batches of eggs and milt, and contact with (sea)water must be avoided to prevent activation of the sperm. Often, several ﬁsh will be stripped at the same time, and the gametes can be stored for a short period (at the same temperature as for ﬁsh, or colder) before they are fertilised. For pelagic marine eggs, ‘wet’ fertilisation is normally used by mixing the gametes with seawater for fertilisation. The male wolf-ﬁsh has a very small volume of semen, and the mature ﬁsh exhibits internal fertilisation. To fertilise wolf-ﬁsh eggs, the gametes are therefore mixed together for at least 4 h before the addition of seawater. Insemination is a process during which gametes are brought together. Fertilisation, in its broadest sense, is a process started with insemination, continued with egg activation and cortical reaction, and terminated with the fusion of male and female pronuclei. Only the latter process can be considered as fertilisation in a strict sense, and a further description of the fertilisation process can be found in Chapter 6. 5.3.4.1 Cod Most cod brood-stocks are still caught from the wild. Adult cod is normally caught in the autumn/winter and the ﬁsh are maintained in cages or large tanks. The volume of the cages ranges from 125 to 1000 m3, and the depth is about 2 m or more. Cod brood-stocks are normally held at temperatures between 5 and 14°C, and the temperature optimal for spawning is 4–6°C (Rosenlund et al., 1993; Jobling & Pedersen, 1995). The density of ﬁsh in the cages is about 30–35 kg m-3. The brood-stock ﬁsh are fed standard dry pellets, moist pellets or fresh ﬁsh to satiation two or three times a week. If they are kept in cages, the ﬁsh are transferred to spawning pens (80–150 m3 in volume) or to large tanks (e.g. 7–10 m cylindrical tanks) a few weeks before spawning. The sex ratio is normally maintained at three females to one or two males, and the stocking density is about 5 kg m-3. The brood-stock can be used for spawning over several years, and the ﬁsh are usually tagged before their ﬁrst spawning. Males and females can be distinguished by the appearance of the genital pore 2–4 weeks before spawning (not very reliable), or by ultrasound inspection of the developing gonads (most reliable). Cod showing courtship behaviour spawn naturally in captivity. Courtship may be observed at almost any time of the day, but spawning occurs mostly at night. As the spawning season

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progresses, the number of eggs per batch tends to decrease. The fertilised pelagic eggs will ﬂoat to the surface, and are collected daily with an air-lift system or separated from the outﬂow water of the tank. The number of eggs may vary between 400 000 and 750 000/l, with an average of about 500 000/l. Typically, a female in good condition produces about 1–1.5 l of eggs/kg body weight. The collected eggs are usually disinfected with either glutaraldehyde (400 p.p.m. for 10 min) or Buffodine (100 ml in 10 l water for 10 min) and rinsed thoroughly before being transferred to the incubators. Usually over 95% of the eggs are fertilised, but as many as 10–35% of the embryos show malformations (Jobling & Pedersen, 1995; Kjørsvik, 1994). 5.3.4.2 Turbot Turbot brood-stock are kept in large tanks (20 m2, 1 m depth), at a temperature ranging from 9 to 14°C (Suquet et al., 1998a). The upper temperature limit for normal maturation of turbot is approximately 16°C. If the temperatures are higher from 1.5 months prior to spawning, the eggs will not be viable (Devauchelle et al., 1988). The brood-stock ﬁsh are generally fed a mixture of moist pellets and ﬁsh, or commercial dry pellets. Turbot ovulate egg batches every 2–4 days over a period ranging from 12 to 38 days in different females. The ovulatory period differs between females, and, in addition, may differ from year to year, and even within a single season. However, a lack of constancy during the spawning season may be explained by unstable water temperatures. Experience with egg production from turbot shows that at the time of stripping, only a variable fraction of the eggs obtained will be buoyant, and of these only a variable fraction will be fertilised. Low viability of eggs is connected with a rapid post-ovulatory overripening of the eggs. A low ratio of ﬂoating eggs may be due either to the presence of ‘old’ eggs from a previous ovulation, or to over-ripening of the last ovulated batch (or both). However, the low fertilisation rate in some egg batches is compensated by intensive egg production in this species (with annual relative fecundity reaching 285–463 g eggs kg-1 body weight). Fertilisation success and egg viability will thus increase with closer monitoring of the brood-stock, as was also shown by McEvoy (1984). 5.3.4.3 Atlantic Halibut Atlantic halibut brood-stocks are normally maintained in circular tanks (diameter 3.5–15 m, 1–2-m depth, light-protected, 34‰ salinity), and at temperatures below 8°C during gonad maturation and spawning (Olsen, 1997; Shields, 2001). An increase in temperature during the spring, and changes in water temperature in general will affect spawning rhythm and egg quality negatively. Female brood-stock ﬁsh normally range between 20 and 80 kg, whereas males are much smaller. Stocking density is up to approximately 11 kg m-3, with sex ratios typically ranging from 1–2 males per female. Brood-stock ﬁsh are normally fed to satiation at least three times each week, and the diets are usually based on fresh ﬁsh, either presented whole, or in a ‘sausage mix’ with ﬁsh meal, oils and micro-nutrients. However, there has recently been a widespread transfer to formulated diets owing to concerns about batch variations in diet

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quality, and because of the risk of disease transmission through the fresh-ﬁsh diet. The prevailing method of diet preparation involves mixing ﬁsh meal with ﬁsh oil and water, then extruding it into a sausage. The development of dry pelleted diets is taking place separately. No feed is offered to ﬁsh during the spawning season. In Atlantic halibut, the egg viability is relatively stable for approximately 6–8 h postovulation, but declines thereafter due to post-ovulatory deterioration. If not stripped in time, females may shed their eggs into the tank. Females are therefore monitored by recording the timing and quantity of egg releases (in tank overﬂow collectors), and by observing the progressive distension of the abdomen between ovulations. An ultrasonographic technique (7.5-MHz linear transducer) may be used to observe the appearance of hydrating oocytes in the ovary. In Atlantic halibut, at least 90% of the stripped eggs will normally be fertilised. Although hatching success varies widely between egg batches, up to 75–80% of the fertilised eggs may hatch. However, egg batches with a high fertilisation success may have a very poor hatching success (see Section 5.4). 5.3.4.4 Wolf-ﬁsh Common wolf-ﬁsh brood-stock is maintained in tanks of 2.4 m3, at stocking densities lower than 14 ﬁshes m-3. The oxygen content is above 6 mg O2 l-1 in the outlet water, and the salinity ﬂuctuates between 32.0 and 34.7‰ (Moksness, 1994). The brood-stocks of spotted wolfﬁsh are kept in outdoor, partly covered tanks at ambient sea temperature (3–10°C), and in indoor facilities using a supply of cooled water during the warm months (before and during ﬁnal sexual maturation). The optimal temperature in this period should be close to 4°C. In brood-stock maintained in a simulated natural photoperiod, egg ovulation is registered from July to January, with a peak in October (Falk-Petersen et al., 1999). Common wolf-ﬁsh are fed commercial dry salmon pellets. Before ﬁnal maturation, 1–4 weeks prior to egg ovulation, females are kept in tanks about 600 l each at densities of 1–6 ﬁsh per tank, at a low light intensity, and they are not fed (Pavlov & Moksness, 1996a; Moksness & Pavlov, 1996). The presence of males does not seem to inﬂuence the maturation of females or the egg quality (Pavlov & Moksness, 1994a). In wolf-ﬁsh species, which all have internal insemination, the size of the female’s abdomen increases rapidly 1–2 days before ovulation. Opening of the genital pore indicates the time of ovulation. In females with a swollen abdomen, the time of the opening of the genital pore should be checked several times a day. Ovulated eggs can be inseminated in vivo by injecting sperm into the opened genital pore of the female. Following insemination in vivo, the eggs are released into water within 24 h and stick together in a clutch representing an egg-ball. This is formed by the female, whose curving body surrounds the clutch for at least 1–2 h (Pavlov & Radzikhovskaya, 1991). Such a clutch might be eaten by the female, and incubation of the eggs is difﬁcult: unfertilised and dead eggs cannot be removed, and all the eggs may die due to bacterial infection. Therefore, a method of insemination in vitro has been developed for common wolf-ﬁsh (Pavlov, 1994b; Moksness & Pavlov, 1996). After stripping of the eggs, the excess slimy ovarian ﬂuid is removed. To increase the probability of contact between gametes without damaging them, a special procedure involving cylindrical vessels is used for the insemination (Fig. 5.9). After an inversion of the vessel,

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Figure 5.9 Schematic representation of several operations for artiﬁcial insemination and the prevention of adhesiveness of common wolf-ﬁsh eggs. (a) Pouring sperm into a cylindrical vessel. (b) Placing the eggs in the vessel (minimum egg/spermatozoa ratio 1 : 200 000). (c) Covering the upper part of the vessel. (d) Repeated mixing of eggs with sperm by inversions of the vessel kept at 2–7°C for 4–6 h (note that liquid ovarian ﬂuid with the sperm appears in the upper part of the vessel, while eggs sink to the bottom). (e) Distribution of the eggs on the bottom of large trays with stagnant seawater to prevent contact between eggs for at least 6 h. (f) Transfer of eggs into upwelling incubators (Moksness & Pavlov, 1996, modiﬁed).

the eggs settle to the bottom after passing through the layer of sperm, and thus good mixing of gametes is achieved. The average fertilisation rate of wolf-ﬁsh eggs is about 95%. The wolf-ﬁsh eggs will become adhesive and will stick to each other immediately after contact with seawater. To prevent this stickiness, which lasts until the hardening process is completed, the eggs may be distributed in trays with stagnant marine water during the hardening process (Moksness & Pavlov, 1996).

5.4 Egg Quality Fish juvenile production is generally characterised by variable egg mortality, high and variable larval mortalities, and variable juvenile quality. The poor or variable output in juvenile production can often be linked to problems with egg and larval quality. A strengthened focus on these aspects and how they are related is therefore necessary to obtain a better understanding of the biological mechanisms and implications involved in offspring quality. Variations in egg quality leading to variable hatching rates are often encountered, and are well demonstrated in salmonids such as Atlantic salmon and rainbow trout, as well as in several marine species, e.g. Atlantic cod, turbot, halibut, gilthead seabream and wolf-ﬁsh. A study of rainbow trout egg batches from commercial hatcheries (Bromage, 1995) showed that the best egg batch gave a fry survival higher than 80% after 120 days, whereas the

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FERT EYE HATCH SWIM UP

100

Survival (%)

best

75

50 mean

25 Figure 5.10 Rainbow trout: fertilisation rates and survival at eyeing, hatch and swim-up of egg batches (n = 15) (after Bromage, 1995).

worst

0

20 30 50

120

Time (days)

poorest egg batch did not survive to hatching (Fig. 5.10). The mean fry survival 120 days after fertilisation was less than 30% (data from 15 egg batches). This situation is even more variable for marine ﬁsh, and especially among the multiple spawners. The eggs from the spawning ﬁsh stock may be regarded as the seed for the ﬁsh harvest, and egg and larval quality (or viability) is important for ﬁsh recruitment in the sea as well as for optimal juvenile production in aquaculture. Owing to the very high variability in captive ﬁsh, there has been an increasing interest in egg quality problems in aquaculture (see reviews by Kjørsvik et al., 1990; Bromage, 1995; Brooks et al., 1997). Egg quality in its strict sense can be deﬁned as the egg’s potential to produce viable fry (Kjørsvik et al., 1990). A more practical version of this deﬁnition would be that egg quality is the potential of the ovulated egg batch to produce viable fry. According to this deﬁnition, egg properties depend on the genotype of the mother, as well as on the morphological, chemical and physiological processes occurring in the egg. Fish egg quality can be affected by many factors (Fig. 5.11), e.g. maternal age and condition factor, the timing of the spawning cycle, over-ripening processes, genetic factors and also the intrinsic properties of the egg itself (Bromage et al., 1994). These egg-quality characteristics and properties, and the inﬂuence of some environmental factors on the egg’s potential, are discussed in this section.

5.4.1 Assessment of Egg Quality An important aspect of hatchery management is to assess whether your egg batches seem viable or not. Such an evaluation should be at an early stage in development, in order to avoid wasting valuable resources on a poorly performing group. Important questions for such an assessment will thus be:

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Ovulation Parental effects (mostly maternal) - age, size, condition factor - spawning stage - parental stress - genetics

Fertilisation

Viable embryo

Egg/embryo

Oocyte

Nutrition Induced spawning Overripening Environmental conditions - husbandry practices - temperature - light regimes - water quality

Non-viable embryo

Figure 5.11 Known causes for varying egg quality in ﬁshes.

• Are there any possible predictive criteria for egg quality which can be applied at an early stage? • If gametes are stripped from the mature ﬁsh, how long after ovulation may the unfertilised eggs be viable? • How may poor egg quality affect the viability of surviving larvae and juveniles? Much effort has been put into evaluation criteria for marine egg and larval quality, and during the last two decades, some reasonably reliable criteria for egg quality and the effects of egg over-ripening have been established based on empirical research. Egg quality is usually assessed after fertilisation despite the contribution of the paternal genes. However, if a large enough quantity of good-quality sperm is used for the insemination, the paternal effect can be excluded. Moreover, as is known (Ginsburg, 1968), paternal genes can be expressed mainly from the step of gastrulation. In hatcheries the quality of pelagic eggs is usually assessed by their ability to ﬂoat or sink in seawater. However, buoyancy is not a good criterion of egg quality for a number of marine ﬁsh species, in particular for Atlantic halibut. A preliminary assessment of egg quality can be made based on visual characteristics such as easy or hard stripping of the eggs, the amount and consistency of the ovarian ﬂuid, the elasticity and colour of the eggs, and the presence of damaged eggs. For example, in common wolf-ﬁsh, good quality eggs are usually accompanied by a comparatively large amount of ovarian ﬂuid (up to 100 ml in large females), which is essential for good mixing of eggs and sperm during internal insemination in the wild (Moksness & Pavlov, 1996). The resistance of the eggs to bacterial contamination may to a certain degree indicate their quality: poorquality eggs are more prone to bacterial contamination (Pavlov & Moksness, 1993; Kjørsvik et al., 1990). However, to date, for many marine species in ﬁsh farming, egg morphology,

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fertilisation success and the ratio of normal blastomeres at early stages of cleavage seem to be the most useful general tool for the assessment of egg quality, and these are also promising tools for prediction of the potential viability of developing embryos, larvae and juveniles for several species. 5.4.1.1 Egg Morphology The preliminary assessment of egg quality just after stripping can be illustrated for common wolf-ﬁsh eggs stripped from the brood-stock and inseminated in vitro. In mature unfertilised eggs, oil droplets have a comparatively low density, and they move freely to the upper side of the yolk. The presence of several oil droplets attached to the yolk cytoplasmic membrane (Fig. 5.12a) may indicate that some of the egg granules are not fused, and the egg is thus immature (Pavlov et al., 1992). The presence of a perivitelline space and blastodisc in the ovulated egg before exposure to seawater (Fig. 5.12b) means that a cortical reaction has occurred inside the female’s body and that fertilisation of this egg is impossible (see Chapter 6 for a more detailed description of ﬁsh egg terminology and development). Damage to the yolk cytoplasmic membrane leads to the outﬂow of the yolk (Fig. 5.12c), and all the contents of the egg become homogenous and whitish (Fig. 5.12d). Large numbers of whitish eggs indicate bad egg quality, but, in addition, damage to the yolk membrane may be caused by the stripping procedure. Several eggs have a deep–yellow or brown colour due to the presence of large oil structures that appear after the fusion of oil droplets (Fig. 5.12e). The presence of eggs covered by a follicular layer at the beginning of resorption (Fig. 5.12f) indicates a deterioration in the process of egg ovulation. Similar observations may be carried out for pelagic ﬁsh eggs, since whitish, non-transparent eggs that rapidly sink to the bottom when mixed with seawater are non-viable. However, it is quite common to obtain a small percentage of non-viable eggs when multiple spawners such as halibut and turbot are stripped, and eggs that are transparent and ﬂoating may be viable and of good quality (but not always!). The multiple spawners regularly ovulate new batches of eggs, and when stripped, some eggs from a previous batch may be present, together with the new egg batch. Eggs from several species contain oil globules; some have one and others have several. The speciﬁc number of oil globules seems to be a good measure of egg quality in some species, such as the striped jack (Pseudocaranx dentex) (Vassallo-Agius et al., 2001). 5.4.1.2 Fertilisation Success and Cortical Reaction Fertilisation success is widely used in commercial hatcheries to predict egg survival. Many species in captivity show a very high variability in fertilisation success. For some species, fertilisation success seems to be related to embryo viability and hatching success, and this seems to be valid, for example, for demersal freshwater salmonid eggs as well as for pelagic marine eggs from yellowtail ﬂounder (Manning & Crim, 1998), gilthead seabream (Almansa et al., 1999) and the striped jack (Pseudocaranx dentex, Fig. 5.13). For yellowtail, a relation was found between fertilisation success and the percentage of normal larvae, but not between fertilisation and hatching success (Verakunpiriya et al., 1997a,b). Such a clear correlation is

160

Culture of cold-water marine ﬁsh

Figure 5.12 Morphology of eggs just after stripping from a female of common wolf-ﬁsh. (a) Egg which appears normal, but the presence of several oil droplets distributed outside the upper part of the yolk and attached to the yolk indicate that the egg is immature; (b) cortical reaction and the formation of the blastodisc took place inside the female’s body; (c) constriction of yolk due to damage of the yolk membrane; (d) egg with damaged yolk membrane; (e) egg with the oil structure and constricted yolk; (f ) resorbing egg covered by a follicular layer. bl, blastodisc; mc, micropyle; od, oil droplets; os, oil structure; ps, perivitelline space; yl, yolk. The natural orientation of the eggs with a view from the side is given (Pavlov & Moksness, 1994a, modiﬁed). Reproduced with permission from Elsevier Science.

not always found for other species such as cod (Kjørsvik & Lønning, 1983; Kjørsvik et al., 1984), halibut (Shields et al., 1997; E. Kjørsvik, unpublished data, 1996), turbot (McEvoy, 1984; Kjørsvik et al., 2003) or seabass (Saillant et al., 2001). Low fertilisation rates are usually related to very poor hatching success. However, a high fertilisation success does not necessarily lead to a high egg and larval viability for all species. Although the fertilisation rate seems to be a reliable indicator of egg quality in salmonids

Brood stock and egg production

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100

H a tc h in g (% )

80

60

40 2

R = 0,7662

20

0 0

20

40

60

80

100

F e rtilis a tio n (% )

Figure 5.13 Fertilisation success and hatching in striped jack, Pseudocaranx dentex (ﬁgure made from data in Vassallo-Agius et al., 2001).

and several other species, it is a necessary, but not a sufﬁcient, egg quality criterion for marine ﬁsh eggs of many species (see reviews by Kjørsvik et al., 1990; Bromage, 1995; Brooks et al., 1997). For example, in several females of common wolf-ﬁsh brood-stock, the fertilisation rate was close to 100%, but the proportion of normally cleaved eggs ranged from 0 to 20% (Pavlov & Moksness, 1994a). In halibut egg batches with fertilisation rates close to 100%, the percentage of normally cleaved eggs and the hatching success may vary between 10 and 90% (Bromage et al., 1994; E. Kjørsvik et al., unpublished results, 1994–2002). The features of the cortical reaction, which is closely linked to the fertilisation process, may also be used as part of an egg quality evaluation. In cod and wolf-ﬁsh, it has been observed that the duration of the cortical reaction may vary in eggs from different females. In eggs of poor quality, the duration of this process was prolonged, and the cortical reaction was often incomplete, resulting in less hardening of the egg envelope and a smaller perivitelline space (Kjørsvik & Lønning, 1983; Kjørsvik et al., 1984; Pavlov & Moksness, 1996a,b). Remnants of visible cortical alveoli in the cytoplasm and in the blastomeres after cleavage are observed for several marine species in connection with a high incidence of abnormal and incomplete cleavage (Kjørsvik et al., 1990; Pavlov et al., 1992; E. Kjørsvik, unpublished results, 1994–2002). These observations suggest a similarity in the morphological deterioration of the eggs of different marine ﬁsh species. 5.4.1.3 Blastomere Morphology Kjørsvik et al. (1990) suggested that an assessment of cell symmetry at the early stages of cleavage (normal blastomeres) might be a possible general indicator of egg quality for marine ﬁsh. This morphological criterion seems to be the most reliable so far, and signiﬁcant positive correlations have been observed between normal blastomeres in the earliest cleavage stages, hatching rates and the viability of yolk-sac larvae hatching in species such as Atlantic cod, turbot and halibut (Shields et al., 1997; E. Kjørsvik, unpublished data, 1994–2002).

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Culture of cold-water marine ﬁsh

Figure 5.14 Irregular blastomere cleavage of Atlantic halibut (an irregular cell number (7) and cell shape; no symmetry), cod (irregular cell size and cell shape), and turbot (4 cells, poor contact between cells). Photograph Elin Kjørsvik.

Figure 5.15 Morphology of cells at the stage of 16 blastomeres in common wolf-ﬁsh. (a) Normal and (b–j) abnormal cleavage.

As noted in the previous section, there should be no (or very few) inclusions of vacuoles (cortical granules) in the cytoplasm. Pelagic eggs are generally easiest to observe during the 4- to 16-cell stages, because after subsequent cleavages, the blastomeres are divided into several cell layers and become too small to observe individually. These early cleavages should be synchronous, and cell divisions should be complete and result in the correct number of blastomeres. The early blastomeres should be regular in shape, with clear cell margins and good contact between adjacent cell membranes. Examples of irregular blastomere cleavages are shown in Fig. 5.14. Similar observations on blastomere morphology have been made for the demersal common wolf-ﬁsh eggs, where the patterns of cleavage can easily be assessed at the stages from eight blastomeres to morula. In normal eggs at the stage of 16 blastomeres, all cells can be distinguished clearly (Fig. 5.15a). In poor-quality eggs (Fig. 5.15b–j) cleavage is incomplete, the number of blastomeres is different, the blastomeres have irregular shapes, or the borders of some cells are not visible. Then empty spaces form in the blastodisc at morula stage (Pavlov & Moksness, 1994a), and the cells totally destroy before blastulation (Pavlov et al., 1992).

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A word of caution is necessary regarding the assessment of blastomere symmetry. In many ﬁsh species, the size of blastomeres may be different from the ﬁrst cell division. In good-quality eggs of cod, the cells may be of unequal size at the stage of two blastomeres (Makhotin, 1982). In good-quality eggs of common wolf-ﬁsh obtained from wild-caught spawners, at the four-blastomere stage, some cells may be twice as large as others, and at morula stage, internal cells are substantially smaller than the cells on the periphery of the blastodisc (Pavlov et al., 1992). According to Doronin (1985), asymmetrical cleavage due to uneven cell size in teleost ﬁsh is the rule rather than the exception. Therefore, an asymmetry of cleavages due to normal differences in the relative size of cells in the blastodisc may not indicate egg quality in many ﬁsh species, and the most reliable criteria are the correct cell number, regularly shaped cells, and clear margins between them. In addition, for Atlantic halibut eggs, Shields et al. (1997) described such parameters of egg quality as bilateral symmetry about the axes of the eight blastomeres (which seems a reliable criterion for this species), proximity of adjacent cell membranes, and the absence of inclusions of vacuoles between adjacent blastomere membranes or on the periphery of the blastodisc. Previous studies have revealed that hatching success is normally more reliably correlated to the rate of abnormal blastomeres (early cell development) than to the fertilisation rate in marine ﬁsh such as Atlantic cod (Kjørsvik & Lønning, 1983; Kjørsvik et al., 1984, 2003; E. Kjørsvik, unpublished data, 1994–2002), halibut (Shields et al., 1997; E. Kjørsvik et al., unpublished data, 1994–2002), wolf-ﬁsh (Pavlov & Moksness, 1994a) and turbot (Kjørsvik et al., 2003), and in planktonic samples of wild ﬁsh eggs (Westernhagen et al., 1988; Cameron et al., 1989). For most species investigated so far, the correlation between the proportion of normally developing early embryos (or blastomeres) and hatching success or survival rates is usually high, regardless of whether the eggs are spawned and fertilised in the wild or in captivity. For cod, it has also been demonstrated that normal blastomeres are related to larval survival in the yolk-sac stage (Fig. 5.16), and to larval viability when exposed to a 100 Larval survival day 6 Hatching

Percentage (%)

80

2

R = 0,6295

60

40

2

R = 0,7799

20

0 0

20

40

60

80

100

Normal blastomeres (%) Figure 5.16 Normal blastomeres of cod in relation to hatching success and survival of yolk-sac larvae up to startfeeding (6 days after hatching) at 5°C. Each point represents one egg batch from different females, either stripped immediately after trawl capture or obtained from brood-stock spawning (E. Kjørsvik, A. Thorvik and R. van der Braak, unpublished data, 1994).

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Culture of cold-water marine ﬁsh

100 2

R = 0,5421

Percentage (%)

80

60 2

R = 0,3712

40

Hatching 20

Norm 230 dd Normal newly hatched

0 0

20

40

60

80

100

Normal blastomeres (%) Figure 5.17 Normal blastomeres of halibut in relation to hatching success and percentage of normal larvae at hatching and at 230 day-degrees at 5°C. Each point represents one egg batch from a brood-stock female (E. Kjørsvik, T.E. Leren, Ø. Aaleskjær and T.F. Galloway, unpublished data, 2000–2002).

high-salinity functionality test. However, the signiﬁcance of normal blastomeres as an egg quality criterion is not always apparent in relation to hatching success. In halibut, normal blastomeres are not usually very well correlated to hatching success, but rather to the ratio of normal yolk-sac larvae (Fig. 5.17). Hatching success is therefore not necessarily a good measure of egg quality for all species. A recent study of egg quality in turbot further demonstrates a long-term effect of poor egg quality (Fig. 5.18, from Kjørsvik et al., 2003). Normal blastomere morphology was related to hatching success and larval viability (tested by a high-salinity functionality test). When larvae were reared to metamorphosis, there was also a signiﬁcant correlation between normal blastomeres, survival to the juvenile stage, and juvenile quality (measured as the completion of metamorphosis and correct pigmentation). Survival to the end of metamorphosis for these groups varied between 2 and 30%, but if only normally metamorphosed and normally pigmented juveniles were included, the juvenile production yield for these groups varied between 1.5 and 27% of the original number of larvae stocked in the start-feeding tanks. For some of these groups, only about half of the surviving juveniles had a normal appearance. For a farmer, the resources needed to rear all these larval groups will be the same, but the difference in income will be very large. It may therefore be of great economic importance to assess the egg quality in the hatchery. 5.4.1.4 Egg Size Egg size is generally positively correlated with the size of the larva. It is important to note that in the egg after swelling, the yolk diameter and the relationship between the cytoplasm volume (measured between the beginning of cleavage and the beginning of epiboly) and the

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100

Hatching (%) Time to 50% mortality (minutes)

Hatching Time to 50% larval mortality in stress test

80

2

R = 0,8587

60

40 2

R = 0,743

20

0 0

20

40

60

80

100

Normal blastomeres (%)

a

Survival day 37

100

Normal metamorphosis

2

R = 0,5146

Normal pigmentation

Percentage (%)

80 2

R = 0,639

60

40

20 2

R = 0,3406

0

b

0

20

40

60

Normal blastomeres (%)

80

100

Figure 5.18 Turbot egg quality. (a) Normal blastomeres in relation to hatching success and larval survival in a high-salinity functionality test on day 3 after hatching. (b) Normal blastomeres in relation to survival by the end of metamorphosis and in relation to success of normal pigmentation and completion of metamorphosis (from Kjørsvik et al., 2003). Reproduced with permission from Elsevier Science.

volume of the yolk are more important in the determination of larval parameters than egg size itself (Pavlov, 1989; Balon, 1999). In the wild, larger larvae are less vulnerable to predators, they eat a wider variety of food items, have a survival advantage owing to larger yolk reserves, and may have a higher growth rate. However, larger offspring would be more noticeable as prey (see reviews by Kjørsvik et al., 1990; Blaxter, 1992; Kamler, 1992; Brooks et al., 1997). The size of the eggs seems to have different importance for different species. In salmonids, larger eggs produce signiﬁcantly larger larvae, but this size advantage is generally lost shortly after ﬁrst-feeding (Bromage & Cumaranatunga, 1988). Experiments with offspring obtained from cod collected in the wild revealed positive relationships between egg and larval size

166

Culture of cold-water marine ﬁsh

and some larval viability characteristics. Larvae hatched from the largest eggs (>1.5 mm diameter) initiated feeding earlier and expressed a higher incidence of feeding during the ﬁrst days of exogenous feeding, they showed higher frequencies of swim-bladder occurrence (percentage of larvae with a developing swim-bladder on day 10 after hatching) and had a higher growth rate, at least during the ﬁrst 2 weeks (Marteinsdottir & Steinarsson, 1998). In cod, as in many other species, the smallest eggs are produced towards the end of the spawning cycle, and a more variable egg quality has also been observed during the last part of spawning (Kjørsvik, 1994). Since egg size is linked to ﬁsh size as well as to the spawning cycle, it may be difﬁcult to use egg size per se as a criterion for egg quality. In the brood-stock of common wolf-ﬁsh maintained at a comparatively high temperature, poor egg quality was associated with smaller egg diameter. However, in ﬁsh kept at a more appropriate temperature, egg diameter was not correlated with the proportion of normally cleaved eggs, but the coefﬁcient of variation (CV) of egg diameter was signiﬁcantly negatively correlated with this proportion. In addition, negatively skewed frequency distributions of egg diameter were registered, with a higher skewness for the poor-quality egg group (Pavlov & Moksness, 1994a, 1996a). These results suggest that an analysis of the value of the CV and the parameters of egg size distribution may be useful for the assessment of egg quality for some species. According to data from many authors obtained mainly for freshwater species, a high CV reaching 10–15% is associated with poor egg quality (see review by Zhukinskii & Gosh, 1988). 5.4.1.5 Chemical Content A biochemical evaluation of egg quality parameters shows that certain components are ‘essential’ for an organism, while other components are species-speciﬁc and may indicate a positive egg quality criterion for one species and poor egg quality for another. The most commonly studied chemical parameters are pigments and vitamins, as well as some inorganic and organic components. The role of these parameters in the assessment of the quality of mainly fresh-water ﬁsh and salmonids is discussed by several authors (Kjørsvik et al., 1990; Kamler, 1992; Bromage & Cumaranatunga, 1988). In general, differences in levels of mineral ions, amino and fatty acids, vitellogenin and carotenoids did not show any substantial relationship with egg quality (Bromage & Cumaranatunga, 1988), and the egg composition will, to a large extent, reﬂect the composition of the ﬁsh diet. As many other articles also demonstrate, differences in egg viability may be found if essential components in the brood-stock diet are below certain levels, and the possible effects of nutrition on egg quality are discussed in Section 5.3.1. However, several investigations have shown that in ﬁsh fed a uniform diet, egg viability may be connected with their chemical content. Devauchelle et al. (1988) reported greater total lipid percentages of the dry weight in non-viable (unfertilised) versus viable (fertilised) turbot egg batches from a brood-stock. In Atlantic halibut, a comparison of lipid class and fatty-acid compositions for viable and non-viable egg batches (determined according to fertilisation and hatching rates, cell symmetry and percentage of larvae to ﬁrst feeding) revealed that they were similar between egg categories with the exception of cholesterol, whose level was signiﬁcantly greater in the non-viable eggs (Bruce et al., 1993). A relationship between

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167

the egg quality of two ﬁrst-time spawners and of a repeat-spawning female of Atlantic halibut and their biochemical composition has been reported (Evans et al., 1996). Eggs from ﬁrsttime spawners with lower fertilisation rates were signiﬁcantly lower in total lipid, triacylglycerol and sterol contents. These eggs also had signiﬁcantly lower percentages of two essential fatty acids, i.e. docosahexaenoic acid and arachidonic acid. A reduced egg quality was also observed in ﬁrst-time spawners of cod compared with older spawning ﬁsh (Solemdal et al., 1995). Several recent papers have been devoted to the investigation of the chemical characteristics of ﬁsh eggs, which can be useful in an evaluation of their quality. Such studies may deal with variations in egg composition during spawning periods, egg maturation processes and composition in relation to mechanisms that are possible determinants of egg quality, and all are contributing to our increasing knowledge of how biological mechanisms are linked to offspring viability. In carp (Cyprinus carpio L.), for example, fertilisation rates were correlated to ovarian ﬂuid pH and protein content, egg respiration rate, pyruvate kinase activity and malate dehydrogenase activity. These enzymes are linked to the cell energy metabolism, and their activity level may thus affect normal development (Lahnsteiner et al., 2001). In turbot, the pH values of the ovarian ﬂuid were positively correlated with egg viability and fertilisation success (Fauvel et al., 1993). Thus, ovarian ﬂuid pH may be used as a predictor of over-ripening and fertilisation success in turbot. Likewise, in a study of egg quality in Perch (Perca ﬂuviatilis (L.)) (Kestemont et al., 1999), the cathepsin- activity in 7-day-old eggs increased during the spawning season, and the mean hatching success and larval viability declined during the spawning season. 5.4.1.6 Cytology Cytological methods are used to reveal chromosomal anomalies during the ﬁnal maturation of oocytes and at earlier stages of developing embryos. Ovulated oocytes of starred sturgeon (Acipenser stellatus Pallas) obtained by hormonal stimulation were at different stages of meiosis, showing desynchronisation between maturation and ovulation (Faleeva, 1987), and a clear correlation was found between chromosomal anomalies and fertilisation rate. According to the author, different sizes and shapes of the chromosomal spindle at metaphase II may indicate errors in meiosis. Desynchronisation of maturation and ovulation, as well as other cytological anomalies leading to reduced egg quality, have been described for several freshwater species bred in captivity (Detlaf, 1977; Detlaf et al., 1981; Korovina, 1986; Makeyeva et al., 1987). In ﬁsh embryos at early stages of development (before gastrulation), three types of chromosomal aberrations have been described: (1) delayed anaphases caused by delayed division of some of the centromeres, but the chromosomes in most cases reach the poles at late telophase; (2) chromosomes or their fragments remain in or near the equatorial plane; (3) some of the chromosomes do not divide, or do not divide properly, and remain in the equatorial plane forming a bridge between the dividing chromosomes (Kjørsvik et al., 1984). Correlations between the survival of the eggs and cytogenetic status are found for several species, but the number of cytological studies for marine ﬁsh is small, and is restricted mainly to toxicological investigations (see review by Kjørsvik et al., 1990).

168

Culture of cold-water marine ﬁsh

5.4.1.7 Oxygen Consumption In carp, the oxygen consumption rate was measured just after ovulation for 3–8 min using a polarographic method (Zhukinskii & Gosh, 1988). The oxygen consumption rate was substantially lower in over-ripe eggs and in eggs with different types of deterioration. Signiﬁcant positive correlations between oxygen consumption rate and the subsequent survival of embryos and larvae were registered. According to the authors, this method of egg quality assessment could be used in ﬁsh culture. A substantially lower oxygen consumption rate (61–70% of the rate in the control group of high-quality eggs) was registered in over-ripe eggs of smelt, Osmerus eperlanus (L.), at the initial stages of egg development (from two blastomeres to blastula) (Korovina, 1986). 5.4.1.8 Evaluating Mammalian Embryo Quality Embryo quality is also a well-known problem in human medicine as well as in animal husbandry. In vitro fertilisation (IVF) and embryo transfer techniques are often characterised by variable embryo quality and low pregnancy rates (Giorgetti et al., 1995), and in mice and in humans, 15–50% of embryos die during the pre-implantation period from mechanisms that are largely unknown (see Warner et al., 1998). Therefore several eggs may be implanted in order to increase the chances of pregnancy, resulting in high multiple-pregnancy rates. In order to avoid these problems, much work on estimating embryo quality has been undertaken for humans and for other mammals. The most frequently used embryo quality codes are also based on blastomere symmetry and blastomere size, colour and density, and on embryo developmental rate (Stringfellow & Seidel, 1998). Several studies have examined the mechanisms regulating cell cycles and development, and results related to embryo mitotic activity (Wurth et al., 1994) and ATP content (Blerkom et al., 1995) have suggested that embryo quality is linked to physiological mechanisms. There also seems to be a genetic basis for pre-implantation egg and embryo survival. Mouse and human embryos of poor quality exhibit a very high degree of apoptosis compared with normal embryos, and two genes that regulate apoptosis (programmed cell death) were expressed differently in embryos of different quality (Warner et al., 1998). Factors that stimulate oocyte maturation and embryo development, such as insulin-like growth factors (IGFs) and insulin, may also be important. Expression of IGFs and their receptors is now a potential marker for human embryo quality (Liu et al., 1997), as the activity of several of these genes correlates well with a morphological assessment of embryo quality. There are indications that the same may be true for ﬁsh embryos.

5.4.2 Factors Affecting Egg Quality 5.4.2.1 Over-ripening The process of over-ripening (or egg ageing) can be deﬁned as the deterioration of eggs that are retained after ovulation in the ovarian ﬂuid within the female’s body (in vivo) or after stripping (in vitro). Even in the early stages of modern ﬁsh culture history, over-ripe eggs were observed to be a problem (Grimm, 1916), and since then over-ripening has been studied

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in a large number of ﬁsh species (see review by Korovina, 1986). Over-ripening of eggs may occur due to stress both in captivity and in the wild if the requirements for spawning are not met, or the usual spawning habitats are destroyed. The problem of over-ripening is especially important for many batch-spawning marine species, which do not spawn naturally in captivity, and where the eggs should be stripped at the correct time. For example, Howell and Scott (1989) observed that in turbot, on average, 60% of eggs were not viable due to postovulatory deterioration in the female’s body. The deterioration registered in the ovulated eggs may be connected with an oxygen deﬁcit occurring after the release of the eggs from follicles surrounded by blood vessels. This hypothesis can be supported by the viability of ovulated eggs in living and dead ﬁsh: over-ripening is much faster in dead individuals (Korovina, 1986). 5.4.2.2 Viability of Ovulated Eggs In Vivo The viability of eggs retained in the female’s body in different ﬁsh species ranges from several hours to several days. The longest period of viability of ovulated eggs is registered in autumn-spawning ﬁshes, in particular salmonids. For instance, a high proportion of normally developing eggs (>90%) was registered in coho salmon (Oncorhynchus kisutch (Walbaum)) 20 days after ovulation (Fitzpatrick et al., 1987). However, in carp (Cyprinus carpio (L.)), a substantial decrease in egg quality (with a fertilisation rate 10–15% lower than that just after ovulation) was observed 1.5–2 h after ovulation (Korovina, 1986). A very short viability period for ovulated eggs (about 1 h) is also reported for striped bass (Morone saxatilis (Walbaum); Stevens, 1966). The optimum time for fertilisation of the ovulated eggs retained in the female’s body seems to be both species-speciﬁc and temperature-dependent. For instance, eggs of rainbow trout (Oncorhynchus mykiss) stripped between 4 and 6 days after ovulation at 10°C consistently achieved the highest rates of fertilisation (Bromage, 1995). The lower fertility of eggs stripped immediately after ovulation is not well understood. In some species, the eggs may need a maturation period after ovulation, as the presence of slightly immature or ‘underripened’ oocytes has been observed (Bromage & Cumaranatunga, 1988). However, in many batch-spawning marine ﬁsh species with a short period of egg viability after ovulation, the highest egg quality seems to occur just after ovulation. The periods of viability of ovulated eggs in several cold-water marine ﬁsh species are given in Table 5.3. In cod, which will spawn naturally in captivity, over-ripening may occur if the ﬁsh are stressed. When the mature cod were separated in pairs in smaller tanks, some spawned at irregular intervals, and low fertilisation rates and the occurrence of abnormal embryos were registered (Kjesbu, 1989). In turbot, post-ovulatory over-ripening of the eggs was registered even when the ﬁsh were stripped daily. A clear indication was obtained of the effect of temperature on the rate at which eggs lost their ability to be fertilised (Howell & Scott, 1989). The fertilisation rate 24 h after ovulation was reduced to about 80% at 14°C and to 30% at 18°C. Thus, maintaining turbot brood stock at a comparatively low constant temperature during the spawning period would reduce the rate of over-ripening and allow eggs of better quality to be obtained. In Atlantic halibut, accurate monitoring of the ovulatory cycles may allow the eggs to be stripped at times close to ovulation. A wide variation

170

Table 5.3

Culture of cold-water marine ﬁsh

Period of viability of ovulated eggs (h) in some marine ﬁsh species.

Species

Cod Turbot Halibut Wolf-ﬁsh

In vivo

In vitro

Viability

°C

Source

Viability

°C

Source

–

– 12–14 4.0 4.0

– McEvoy (1984) Bromage et al. (1994) Moksness & Pavlov (1996)

9 – 6 13

5.0 – 4.0 4.0

Kjørsvik & Lønning (1983) – Bromage et al. (1994) Moksness & Pavlov (1996)

10 12 >24

in the periods of over-ripening of eggs from different females is often observed, and indications of ‘under-ripeness’ have been reported for the eggs of female Atlantic halibut stripped very close to ovulation (Bromage et al., 1994; Holmefjord, 1996; E. Kjørsvik, unpublished data, 1996). In wolf-ﬁsh, the over-ripening of eggs has not been registered owing to the comparatively rapid (within 24 h) deposition of ovulated eggs by females. At the same time, eggs stripped just after the opening of the genital pore, indicating the onset of ovulation, were often of poor quality, and showed some incidence of ‘under-ripening’ (D.A. Pavlov, unpublished data). 5.4.2.3 Viability of Ovulated Eggs In Vitro Eggs stripped and stored in the ovarian ﬂuid seem to undergo over-ripening in a similar way to that which occurs in the female’s body. However, for the ovulated eggs of many species, the rate of their deterioration is higher when they are stored in vitro, apparently owing to a lower oxygen supply than in the female’s body, or to the inﬂuence of the components of atmospheric air. For example, ovulated eggs of rainbow trout retain viability for at least 10 days in vivo, but they may be kept in an external medium for a few hours only before fertilisation, and the best results are achieved if eggs are fertilised within 1 h of being stripped from the female (Bromage & Cumaranatunga, 1988). In Atlantic halibut, the periods of viability of ovulated eggs in vivo and in vitro seem to be similar (see Table 5.3). The eggs from multiple batch spawners such as cod, halibut and turbot must all be fertilised within a few hours after ovulation (see Fig. 5.20). 5.4.2.4 Changes in the Eggs The structural changes which are responsible for the deterioration of eggs during overripening are not well researched. In their review, Kjørsvik et al. (1990) noted that changes due to over-ripening were often described as visible discoloration or non-transparency, the fusion of cortical alveoli and a ‘dimpled’ appearance of the cytoplasm. However, such changes are visible only after the viability of the eggs is already signiﬁcantly reduced. Repeated inseminations of eggs stored in the ovarian ﬂuid show a decrease in the proportion of normally cleaved eggs within a few hours, and a steady increase in the proportion of eggs with abnormal cleavage and uncleaved blastodiscs (Fig. 5.19). During the storage of eggs for a longer time, the proportion of unactivated eggs also increases. The eggs with

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Figure 5.19 Morphology of common wolf-ﬁsh eggs before and after insemination. (a, a1) Egg before insemination; (b, b1) fertilized egg at 3 h after insemination; (c, c1) unfertilised egg 24 h after release into water. bl, blastodisc; ca, cortical alveoli; fs, ﬁbrous structure; gr, granule; mc, micropyle canal; mf, micropyle funnel; mp, micropyle pit; od, oil droplet; ps, perivitelline space; yl, yolk (Pavlov & Moksness, 1996a). Reproduced with permission from Elsevier Science.

an uncleaved blastodisc are activated by spermatozoa, but fertilisation (i.e. a fusion of male and female pronuclei) does not take place. At the same time, apparently abnormal cleavage is possible only after fertilisation. Abnormal cleavage of over-ripened eggs, as well as the formation of abnormal embryos, have been described in many fresh-water ﬁsh (see review by Korovina, 1986), as well as in cod (Kjesbu, 1989), Atlantic halibut (Bromage et al., 1994; Shields et al., 1997) and wolf-ﬁsh (Moksness & Pavlov, 1996). For cod and halibut, the effects of over-ripening (in vitro) are shown in Fig. 5.20, which shows that 8 h storage of unfertilised eggs in ovarian ﬂuid resulted in a signiﬁcant decline in normal blastomeres and hatching success. It is also worth noting that fertilisation success for halibut eggs remained reasonably high throughout 24 h egg storage in ovarian ﬂuid, thus illustrating that fertilisation rate is not a good criterion for halibut egg quality. The changes which are responsible for the appearance of unactivated eggs have been described in common wolf-ﬁsh. Most of the eggs kept in ovarian ﬂuid were unchanged, but in approximately 10%, a small perivitelline space had formed after 24 h at 7.0°C. The cortical reaction in these eggs was not complete: only some of the cortical alveoli were broken at the animal pole of the egg (Pavlov & Moksness, 1996b). Apparently, these eggs could not be fertilised. After being placed into water, the perivitelline space increased slightly, but the cortical alveoli remained, and the blastodiscs did not appear (Fig. 5.19c, c1). Therefore, the breakdown of at least some of the cortical alveoli in the egg represented an event which was associated with over-ripening.

172

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Cod

Halibut

100

Percentage

80 60 40 Fertilisation Normal blastomeres Hatching

20 0 0

5

10

15

20

25 0

5

10

15

20

25

Storage of unfertilised eggs in ovarian fluid (h) Figure 5.20 Effects of egg ageing (over-ripening) on fertilisation success, percentage of normal blastomeres and hatching success in cod and halibut. Eggs and milt were stripped from newly caught mature cod and from broodstock halibut. Over a time-period of 24 h, unfertilised eggs were stored in ovarian ﬂuid, and milt were stored dry (5°C). Samples were fertilised in seawater at set time intervals. Arrows indicates when the ageing effects become signiﬁcant, (E. Kjørsvik, A. Thorvik and L. Schei, unpublished data, 1996/1997).

However, the events leading to the appearance of abnormal cleavage remain unknown. Mechanisms regulating cell cycles and embryonic development will clearly be involved, and it has been established from several studies that embryo quality is linked to physiological mechanisms. One important aspect of normal cell cycles is the available energy charge in a cell, as measured by the adenylated phosphates (ATP, ADP, AMP). Recent investigations of egg ageing in cod (in vitro) showed that the ATP content of eggs during the process of over-ripening declined faster than that of any other parameter observed (E. Kjørsvik et al., unpublished data, 1996). There was a signiﬁcant reduction in the ATP content of the egg after only 4 h storage in ovarian ﬂuid before fertilisation, and fertilisation success and percentage of normal blastomeres were signiﬁcantly reduced after 8 h storage. Other biochemical and physiological changes in the eggs which occur during overripening are described in Section 4.4.1, and in the reviews by Bromage & Cumaranatunga (1988) and Kjørsvik et al. (1990).

5.4.3 Change in Egg Quality Over the Spawning Season Egg quality is often lowest in females that spawn at the beginning of the spawning season. However, the general trend is the highest egg quality at the beginning (except the ﬁrst batches in batch-spawning ﬁsh) or in the middle of the spawning season, and a decline in quality towards the end. For example, the eggs from several cod earlier spawners were smaller than eggs from later batches, and these eggs were characterised by lower quality (Kjesbu et al., 1990; Marteinsdottir & Steinarsson, 1998). At the same time, the mean egg size shows a

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steady decline throughout the spawning season, from approximately 1.4–1.5 mm to 1.2– 1.3 mm. This has been reported for ﬁsh from a brood-stock (Kjørsvik, 1994; Mangor-Jensen et al., 1994) and for those collected in the wild (Marteinsdottir & Steinarsson, 1998). According to the observations of the latter authors, egg size seems to be positively correlated with egg quality. Poor egg quality in the last batches of cod eggs was also observed by Kjesbu (1989). A decrease in egg diameter and an increasing proportion of abnormal eggs were registered over the spawning season of cod from the White Sea, based on ichthyoplankton samples (D.A. Pavlov, unpublished data). Substantial decreases in the dry weight of egg batches have been registered over the spawning season of captive Atlantic halibut (Evans et al., 1996). In common sole, egg size tended to decline during the spawning season both in captivity and in the wild, and the shortest larvae that hatched from the smallest eggs might not be able to accept Artemia nauplii as a ﬁrst food (Baynes et al., 1993). The smallest mean egg diameters (5.0 and 5.2 mm) with the largest CVs (7.8 and 5.8%) were observed in two common wolf-ﬁsh females which spawned at the beginning of the breeding season. For these females, no eggs cleaved normally (Pavlov & Moksness, 1996a). In the brood stock of common wolf-ﬁsh, the diameter of the eggs of repeat-spawning females was lowest at the beginning of the breeding season (November), increased towards the middle of the season (January–February) and decreased towards its end (April), but egg quality was stable throughout the season. The lower quality of the eggs towards the end of the spawning season in batch-spawned ﬁshes can be explained by an exhaustion of the energetic reserves of the females.

5.4.4 Maternal Effects In general, an improvement in egg quality from the ﬁrst to the second and third spawning seasons has been reported for several ﬁsh species (see review by Brooks et al., 1997). A decreased egg mortality in second spawners was shown in the same individuals of coastal cod from a brood stock (Solemdal et al., 1995). In this species, egg size increased signiﬁcantly from the ﬁrst to the second spawning season, with a smaller increase in third-time spawners kept at the same level of condition (Kjesbu et al., 1996). In cod collected in the wild, eggs of the largest size (>1.5 mm diameter) and with the highest quality tended to be produced by the largest females and, in general, by the older females. The female’s characteristics (length, weight, age, condition factor) were signiﬁcantly related to egg size, which in turn, was highly correlated with the components of larval viability (Marteinsdottir & Steinarsson, 1998). In Atlantic halibut, the fertilisation rate of eggs from a repeat spawner and two ﬁrst-time spawners was 81% and 56%, respectively. In addition, these two groups of eggs had different biochemical compositions (Evans et al., 1996). However, egg quality was not different in ﬁrst and repeat spawners of common wolf-ﬁsh (Pavlov & Moksness, 1996a). Preliminary data indicate that in a single population of rainbow trout, females that produce better-quality eggs in their ﬁrst spawning season (year) also do so in the subsequent season, suggesting that there are genetic inﬂuences on egg quality (Brooks et al., 1997). In turbot, the largest ﬁsh tended to produce the largest eggs, and in both subsequent years the same females produced the largest or the smallest eggs (Howell & Scott, 1989).

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5.4.5 Conclusions In conclusion, fertilisation success and blastomere morphology generally show good correlation with embryo and larval viability, and blastomere morphology in particular seems to be a valuable tool for the assessment of egg quality in ﬁsh. The principles of such morphological criteria are very similar in ﬁsh and in mammals, and these criteria represent the only non-invasive scoring system for egg or embryo quality evaluation. Egg quality is clearly linked to physiological mechanisms, and there seems to be an important genetic basis for offspring viability. Only few studies have examined the possible long-term effects of poor egg quality on juvenile viability, and more studies should aim at understanding the mechanisms controlling egg viability and the long-term effects of varying egg quality on the development and functionality of the growing ﬁsh. Owing to the very high variability in egg quality and offspring viability from brood-stock ﬁsh, egg-quality parameters should be included in future quality certiﬁcation from producers of juveniles. For the species considered in this book, a careful assessment of egg quality should be carried out when embryos are at the 8–16-cell stage (preferably) of cleavage, and the eggs should be divided into several categories, as shown in Fig. 5.21. In this scheme, the eggs with cortical alveoli without blastodiscs are referred to as ‘unactivated’ despite the breakdown of some cortical alveoli. Eggs with a non-cleaved blastodisc (one cell) are referred to as activated and unfertilised. Eggs with abnormal cleavage are apparently fertilised, and the fertilisation rate may therefore be much higher than the proportion of eggs which are able to undergo normal development. A normal good-quality egg batch may thus be described as exhibiting a high fertilisation rate, pelagic eggs have good buoyancy, the eggs are transparent, with no or very few visible cortical alveoli in the cytoplasm, the early cleavages are synchronous and produce the correct number of cells, and the early blastomeres are regular in shape with clear margins between them and good contact between adjacent cell membranes. An assessment of fertilisation success and the proportion of normal blastodiscs currently seems to be the only ‘universal’ criterion for egg quality in cold-water marine ﬁsh, and the quality characteristics used for ﬁsh are very similar to those used in the present assessment of embryo quality for in-vitro fertilisation of mammals (including humans). This procedure

Eggs

Activated

Normal cleavage (Fig. 5.15a)

Abnormal cleavage (Fig. 5.15b–j)

F e r t i l i s ed

Unactivated

Incomplete cortical reaction (Fig. 5.19c, c1)

Uncleaved blastodisc (Fig. 5.12b)

U

n

f

e

r

Damaged yolk membrane, whitish (Fig. 5.12d)

t

i

l

i

Yellow or brown oil structure (Fig. 5.12e)

s

e

Resorbing, with follicular layer (Fig. 5.12f)

d

Figure 5.21 Categories of artiﬁcially inseminated eggs obtained in common wolf-ﬁsh from a brood-stock.

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175

may be done in a few minutes per egg batch, and is easy to perform with a simple stereomicroscope.

5.5 Sperm Production and Quality Sperm quality and its productive characteristics, including mainly ejaculate volume, concentration and motility, has a decisive inﬂuence on the success of artiﬁcial reproduction in ﬁsh. As in spermatozoon morphology, these characteristics show a great variation in different species, depending on the mode of reproduction. In general, sperm quality is less related to husbandry conditions and environmental factors than egg quality.

5.5.1 Features of Sperm Production and Quality 5.5.1.1 Morphology Sperm composition and malformations in the structure of spermatozoa have been studied in a restricted number of species. In the sperm of turbot stripped from 47 to 80 days before the beginning of the spawning period of the females, numerous spermatids were present. Some spermatozoa had slightly condensed chromatin and a middle piece containing numerous vesicles, while others had condensed chromatin and a middle piece containing only a few vesicles. Mature cells had dense chromatin. After the end of the spawning season of the females, no spermatids were present and the middle piece always contained very few vesicles. Different degrees of chromatin expression were connected with the decondensation caused by the ageing process. In addition, some degenerated spermatozoa appeared in which the plasma membrane was broken or had disappeared. Thus, the sperm collected after the spawning period of the females was not suitable for artiﬁcial fertilisation (Suquet et al., 1998a). In Atlantic halibut, observations of spermatozoa at the end of the motility phase showed drastic distortions of the ﬂagellum. A spermatozoon of common wolf-ﬁsh from the brood-stock normally has one ﬂagellum, but in some spermatozoa two basal plates with two axonemes are observed. This structure is regarded as abnormal (Pavlov et al., 1997). However, the spermatozoa of two closely related species, Zoarces elongatus and ocean pout (Macrozoarces americanus), normally possess biﬂagellar tails (Koya et al., 1993; Yao et al., 1995). As in turbot, spermatids at different stages of transition to spermatozoa are found in the ejaculate of common wolf-ﬁsh mainly at the beginning of the breeding season. Large numbers of spermatids together with motile mature spermatozoa are observed in the seminal lobule lumen of Zoarces elongatus, a viviparous ﬁsh with internal fertilisation, and in several other ﬁsh species with ‘semi-cystic’ asynchronous spermatogenesis occurring partly outside cysts (Mattei et al., 1993). The presence of spermatids in the ejaculate is apparently not normal, and can be caused by the stripping procedure. In addition to spermatids and normal spermatozoa, smaller cells without ﬂagella showing agitated movements are observed in common wolf-ﬁsh ejaculate (Pavlov et al., 1997). The proportion increases towards the end of the breeding season, and they are probably represented by degraded spermatozoa.

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Table 5.4

Main sperm parameters in some marine ﬁsh species.

Species

Maximum GSI (%)

Ejaculate volume (ml)

Cod Turbot

16 0.8

– 0.2–2.2

– 0.7–11.0

Halibut

–

1–92

11.9–37.2

0.2–10.6

0.012–1.198

Wolf-ﬁsh

0.1

Ocean pout 2.2

5–20

Sperm concentration (¥109 spz ml-1)

0.00756–0.215

Spermatocrit (%) 59 40

Sperm motility

1 s–120 min 1–17 min

40–100

60–70 s

–

>48 h

0.8–1.8

>24 h

References

Suquet et al. (1994) Suquet et al. (1994, 1995) Billard et al. (1993); Suquet et al. (1994) Johannessen et al. (1993); Moksness & Pavlov (1996) Yao & Crim (1995); Yao et al. (1995)

5.5.1.2 Gonadosomatic Index and Ejaculate Volume In species with seasonal reproductive cycles, the gonadosomatic index (GSI, i.e. (gonad weight/body weight) ¥ 100) indicates the efﬁciency of spermatogenesis. GSI is related to spawning behaviour, and in the case of external insemination it is lowest in species spawning in stagnant water and in couples when a close contact between the genital openings of the male and female can be achieved. Among cold-water marine ﬁsh species with external insemination, the lowest GSI is registered in common sole and in turbot (0.2% and 0.8%, respectively), and these low values are apparently connected with the spawning behaviour of the species. As a rule, species with internal insemination possess a comparatively low GSI. For example, it is lowest in common wolf-ﬁsh in comparison with other marine and fresh-water species (Table 5.4). In addition, the common wolf-ﬁsh differs from the majority of ﬁsh with seasonal spawning by having a relatively stable GSI and spermatozoa production over the entire year. In the males of ocean pout, another ﬁsh with internal insemination, the values of the GSI and ejaculate volume are much higher than in common wolf-ﬁsh, and this may be connected with the different amounts of energy put into sperm production owing to features of parental care (the eggs are protected by the male in wolf-ﬁsh and by the female in ocean pout). The volume of sperm released by stripping (or collected with a catheter, as in ocean pout) is comparatively low in species with a low GSI, and depends on the phase of the breeding season and the stripping frequency. In turbot, both fortnightly and weekly stripping result in the release of decreasing sperm volumes per stripping as a function of time. However, an increase in collection frequency has no effect on the total sperm volume collected during a 2-month period (Suquet et al., 1994). In males of common wolf-ﬁsh stripped at approximately monthly intervals, ejaculate volume was minimal at the beginning and at the end of the breeding season and reached maximum values in August, before egg ovulation in the majority of females (Fig. 5.22a).

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177

a Ejaculate volume (ml)

‡

Sperm concentration (ⴛ106 ml–1)

b

Ap r May Jun

Jul

Aug

Sep

Oct

Nov

Dec Ja n

Feb Mar

Figure 5.22 (a) Volume of ejaculate, and (b) concentration of sperm in 20 males of common wolf-ﬁsh repeatedly stripped during the breeding season. Error bars are the standard errors (Moksness & Pavlov, 1996).

5.5.1.3 Concentration The concentration of spermatozoa is lowest in species with internal insemination (Table 5.4). For example, in common wolf-ﬁsh it is about 66 times lower than in Atlantic salmon, Salmo salar L. (Pavlov & Moksness, 1994b). In marine ﬁsh species with external insemination, sperm concentration is low in turbot, and this concentration is similar to that in tilapia (Oreochromis spp.), which has one of the lowest concentration values. Sperm concentration depends strongly on the phase of the breeding season and the stripping frequency. In turbot, sperm concentration increases during the spermiation period. However, after a sampling period of 2 months, the sperm concentration observed at the last stripping was signiﬁcantly lower in ﬁsh stripped weekly or fortnightly than in individuals stripped monthly (Suquet et al., 1994). In common wolf-ﬁsh, the maximum sperm concentration is registered during the peak of egg ovulation in females (Fig 5.22b). Stripping the sperm at different frequencies showed that sperm concentration did not decrease if the sperm was sampled every 12–14 days (Pavlov & Radzikhovskaya, 1991). Spermatocrit can reﬂect sperm concentration in the majority of species. For instance, spermatocrit of ocean pout with a very low sperm concentration is also low (see Table 5.4). In Atlantic halibut, a positive relationship between sperm concentration and spermatocrit has been reported (Tvedt et al., 2000). However, in turbot, despite a low sperm concentration,

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comparatively high spermatocrit values are found (see Table 5.4), which may be caused by the presence of spermatids and degenerated spermatozoa in the ejaculate. Total sperm production (i.e. the total number of spermatozoa in the ejaculate) does not seem to depend much on the weight, age or number of spawnings in males of marine species, e.g. cod, turbot and common wolf-ﬁsh. However, a high variation in sperm production is observed in many species, and some males are characterised by very low production characteristics over the entire breeding season, possibly showing the genetic effects of sperm quality. 5.5.1.4 Motility The motility of activated spermatozoa is usually assessed based on its duration (when all or most of the spermatozoa stop exhibiting progressive forward movement), or using ranked scores for percentages of motile spermatozoa. The rank ranges used by the authors are different (see reviews by Trippel & Neilson, 1992; Billard et al., 1995). The swimming speed of sperm was measured in cod using a video-recorder of enlarged images of sperm against a haemacytometer grid pattern. The average swimming speed of motile spermatozoa was 75 mm 30 s-1, and the fastest recorded swimming speed was 1000 mm 30 s-1 (Trippel & Neilson, 1992). Stroboscopic illumination and dark-ﬁeld microscopy was used to measure ﬂagellar beat frequency, cell velocity and the distance covered by spermatozoa of salmonids (Cosson et al., 1985). The intensity and duration of spermatozoa motility indicates the quality of the males, and can be used to predict fertilisation rate. However, high sperm motility is not always essential for successful fertilisation. For example, immotile vibrating spermatozoa obtained from some male cod showed a similar fertilisation level to that found when exclusively motile sperm was used (Trippel & Neilson, 1992). In marine ﬁsh with external insemination, the dilution of sperm in a hypertonic medium initiates motility in the spermatozoa. In turbot, sperm motility is triggered by dilution in both hypertonic and isotonic media. The duration of movement is lower in isotonic diluents, but the reasons for sperm activation in these diluents are not understood. A decreased percentage of motile turbot spermatozoa was registered after increasing the dilution of the sperm, and to protect spermatozoa against dilution the addition of proteins is necessary (Suquet et al., 1994). In marine ﬁsh, the duration of sperm motility is generally higher than that in salmonids and fresh-water species. In Atlantic herring, Clupea harengus L., and Paciﬁc herring, C. pallasi Val., spermatozoa show vibrating movements, and intensive forward movements are observed only in the micropyle area. The spermatozoa of these species remain viable for 7 and 1 days after release into water, respectively (Makeyeva, 1992). In Atlantic halibut at 18–20°C, the percentage of motile spermatozoa declines slowly in the ﬁrst minute of motion and abruptly thereafter. The duration of spermatozoa motility is associated with the decrease in ﬂagellum beat frequency and spermatozoa velocity. The beat frequency remains fairly stable (40–50 Hz) over the ﬁrst 55 s after dilution, but then drops suddenly to values around 10–15 Hz. Over the same period, spermatozoa velocity declines gradually. The possible physiological and biochemical reasons for the sudden drop in beat frequency of Atlantic halibut spermatozoa have been discussed (Billard et al., 1993, 1995). Spermatozoa of common wolf-ﬁsh and ocean pout, which are both characterised by internal insemination, are motile in undiluted ejaculate. They are apparently immotile in the genital tract of the male, and the reasons for their activation in the

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external medium are not known. Their motility lasts more than 1 day (see Table 5.4). Longlived spermatozoa are connected with the need for the sperm and the eggs to be very well mixed in the female’s ovarium in order to increase the probability of contact between gametes. Spermatozoa motility may depend on the phase of the spawning season and the stripping frequency. The motility of turbot spermatozoa decreased signiﬁcantly at the end, and for 2–3 months after the end, of the spawning period of the females (Suquet et al., 1998a). In this species, an increase in stripping frequency from monthly to weekly did not inﬂuence the duration of spermatozoa motility (Suquet et al., 1994). However, high-frequency stripping (every 3–14 days) in males of common wolf-ﬁsh led to a substantial decrease in the duration of spermatozoa motility (assessed after their dilution in marine water) during the breeding season (Pavlov & Radzikhovskaya, 1991). 5.5.1.5 Fertilising Capacity Fertilisation rate is the most reliable indicator of sperm quality. However, for an appropriate use of this test, the minimal sperm-to-egg ratio and the minimal contact time between gametes required for successful insemination should be assessed. These parameters are known for a limited number of species. The results of experiments with the insemination of turbot and common wolf-ﬁsh eggs using various dilutions and contact times are shown in Fig. 5.23. In turbot, when high-quality egg batches are used, about 6000 spermatozoa per egg are required to obtain maximum fertilisation success. This value is low compared to that in other ﬁsh species, and it is connected to the high fertilisation capacity of turbot spermatozoa. Owing to this capacity, sperm production in turbot is comparatively low. In addition, the sperm requirements of turbot eggs seem to depend on egg quality: more sperm is needed for the insemination of egg batches with lower viability. In common wolf-ﬁsh, the minimal sperm-to-egg ratio to achieve high fertilization rates depends on the contact time between gametes, and is approximately 200 000. This value is similar to that reported for salmonids. The large number of spermatozoa required for the successful (internal) fertilisation of wolf-ﬁsh eggs may be connected to their low velocity in the viscous ovarian ﬂuid of the female, and to the comparatively large diameter of the eggs. In turbot, to reach a high fertilisation rate, the contact time between gametes should be at least 3 min at a spermatozoa-to-egg ratio of 6000, and this time should be increased to 4–5 min at a ratio of 1500 spermatozoa per egg. An extremely long contact time between gametes (>2 h) is required for common wolf-ﬁsh. This contact time depends on the spermto-egg ratio: if this ratio is low, the contact time should be increased to 7 h. Thus, the spermto-egg ratio and the contact time between gametes are inversely related, and apparently an increase in the contact time is accompanied by a higher probability that the spermatozoa will reach the micropyle. However, in the majority of ﬁsh with external insemination, the contact time is restricted by the short life of spermatozoa in water. 5.5.1.6 Biochemistry and Oxygen Consumption The biochemical composition of sperm and seminal ﬂuid in ﬁsh is reviewed by Piironen & Hyvärinen (1983) and Billard et al. (1995). The biochemical composition and oxygen con-

Culture of cold-water marine ﬁsh

s

s

180

b

s

s

a

c

d

Figure 5.23 Fertilisation rate in relation to sperm-to-egg ratio and contact time between gametes in (a,b) turbot and (c,d) common wolf-ﬁsh. (a) Vertical dashed line indicates a ratio of 6000 spermatozoa per egg. The eggs are from different breeders with high fertilisation rates. (b) The eggs from ﬁve batches; sperm-to-egg ratio 6000. (c) The eggs from one female; contact time between gametes 2, 7 or 12 h. (d) The eggs from one female; sperm-toegg ratio >200 000 (Pavlov, 1994a, b; Suquet et al., 1995, modiﬁed). Reproduced with permission from Elsevier Science.

sumption of spermatozoa in relation to their quality were described by Gosh (1989). In particular, the level of lactic acid may indicate sperm quality: it is higher in the sperm of older ﬁsh, and a negative correlation was observed between this level and sperm concentration on the one hand and fertilisation rate on the other in some fresh-water ﬁsh species. An intensive forward movement of spermatozoa is possible only at a certain level of ATP. A restriction of the oxygen supply leads to a sudden decrease in the motility of spermatozoa. The resumption of good conditions for gas exchange is associated with an increasing synthesis of ATP, and the motility increases again. However, in turbot, no signiﬁcant decrease in ATP content in the sperm was recorded during the spermiation period despite observed differences in spermatozoa concentration and motility (Suquet et al., 1998a).

5.5.2 Inﬂuence of Environmental Factors on Sperm Quality Sperm quality depends on the husbandry and feeding conditions, but this relationship has rarely been investigated. The duration of spermatozoa motility depends on the temperature:

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it decreases at higher temperatures. For example, in Atlantic salmon, the duration of forward movements is 53 s at 5°C and only 22 s at 30°C (Makeyeva, 1992). The photoperiod during cultivation of the brood-stock seems to have little inﬂuence on sperm quality. In particular, in turbot males receiving an annual light and temperature cycle compared with those receiving a 6-month contracting schedule, the sperm parameters (ejaculate volume, sperm concentration and motility) were similar (Suquet et al., 1994). The difference in sperm volume and sperm concentration between common wolf-ﬁsh males kept in different light cycles (18L:6D and 6L:18D) for a year was not signiﬁcant (Moksness & Pavlov, 1996). The inﬂuence of osmotic pressure and pH on the motility of spermatozoa was studied in Atlantic halibut. The spermatozoa were motile in a range of osmotic pressure from 300 to 1150 mOsmol kg-1, and the percentage of motile cells increased from pH 6.5 to 8.5 and declined above pH 8.5. The duration of forward movement reached a maximum at pH 7.5–8.0 (Billard et al., 1993).

5.5.3 Sperm Storage In aquaculture practice, sperm storage is necessary when the periods of egg and sperm maturation in ﬁsh brood-stock are different, and this can be used for selective breeding of ﬁsh from different stocks. In general, fresh sperm collected from males can be stored in vitro for a short period of time. One reason for the fast deterioration of spermatozoa is an insufﬁcient oxygen supply, resulting in a lower level of ATP. During sperm storage, the amount of carbohydrates decreases and the level of lactic acid increases, indicating intensive glycolysis. An increasing amount of lactic acid has a negative effect on the viability of spermatozoa (see reviews by Gosh, 1989; Billard et al., 1995). Sperm storage can be improved by stocking fewer sperm with a larger volume of available air, and sperm concentration can be reduced by diluting the sperm with an extender (rich in K+ or sucrose) that does not activate spermatozoon motility (Billard, 1988). In Atlantic halibut semen stored on ice for 24 h, the percentage of motile spermatozoa did not exceed 40%. In this species, the fertilisation rate declined by about 20% over 16 h after the storage of sperm at 1–3°C (Martin-Robichaud & Rommens, 2000). The experiments showed that Atlantic halibut sperm remained viable after short-term freezing and thawing of diluted sperm in the presence of 10% propanediol. The capacity of the sperm to be frozen declined rapidly after collection and storage in vitro for a period of 7 h (Billard et al., 1993). In common wolf-ﬁsh, sperm stored in undiluted ejaculate at 4°C had a high fertilising ability for at least 10 h after collection. This ability might be longer because of the long duration of spermatozoa motility (over 2 days). However, to date only the combined effect of egg and sperm storage has been reported (Moksness & Pavlov, 1996). Sperm storage capacity may depend on the phase of the spawning season. For example, in seabass stored at 4°C, this capacity decreases from 70 h at the beginning of the spawning season to 30 min in the middle and at the end of this season (Billard et al., 1977). In turbot, the short- and long-term storage capacities of sperm decreased as the spermiation period progressed (Suquet et al., 1998a). Cryopreservation is used for the long-term storage of sperm, and the sperm from more than 200 ﬁsh species have been cryopreserved. Although few practical applications have

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been achieved to date (Billard et al., 1995; Chao & Liao, 2001), such techniques are in use in ‘gene banks’ which have been established to preserve the genes of different strains of wild salmon. Such techniques may also secure some sperm availability if sperm quality declines towards the end of the spawning period. During sperm collection for cryopreservation, several factors are essential in order to maintain sperm quality: (1) collect the sperm without any contamination by faeces, blood or scales; (2) hold the sperm by providing air or oxygen for respiration; (3) maintain the temperature of collected milt at 4°C. Collected sperm is diluted in a freezing medium containing extenders and cryoprotectants. In addition to the universal cryoprotectant dimethyl sulfoxide (DMSO), other single cryoprotectants, combined cryoprotectants, and cryoprotectants with egg yolk or sucrose have been used. The milt mixture is placed in a tube, frozen and stored in liquid nitrogen. An alternative method of freezing sperm is the formation of pellets in hollows on the surface of dry ice. The sperm is thawed in a waterbath with the addition of diluting solutions with an appropriate ion content and pH. The freezing and thawing protocols vary with species, and higher quantities of thawed milt are needed to obtain fertilisation rates which are comparable to those obtained when fresh milt is used. The results obtained for some species allow the use of cryopreservation in routine aquaculture practices. For example, turbot spermatozoa can be cryopreserved in DMSO mixed with egg yolk, or in a sucrose solution with 10% DMSO and 10% egg yolk. Sperm of this species was stored during a 9-month period in liquid nitrogen. No signiﬁcant differences in spermatozoa motility, fertilisation rate, hatching rate, survival and wet weight of larvae were observed using fresh or frozen–thawed sperm (Suquet et al., 1998b).

5.6 Selective Breeding In farm animals and plants, selective breeding has played an important role in domestication, increasing yields, survival rates and improving product quality. Today, one cannot really imagine any commercial animal husbandry or plant production without genetically improved livestock or plants. Quantitative genetics is the theoretical basis on which animal and plant breeding programmes are founded. Although it has been applied in agriculture since around 1920, the theory has only recently been applied to aquaculture. Prospects for genetic improvements in aquaculture species are even more promising than for domestic animals (reviewed by Gjedrem, 1975, 1992; Kinghorn, 1983; Gjerde, 1986), mainly owing to higher reproduction capacities and the large additive genetic variation seen for traits of economic importance (e.g. growth rate) in many species. Because of the high reproduction capacities, a small number of new breeders are needed in each generation, and high selection intensity can be consistent. The expected selection response also depends on the choice of selection method, of which several are available for obtaining additive genetic improvement. The methods will differ according to which type of breeder will provide the information needed for selection decisions. The objective of all methods is to maximise the probability of correctly ranking the animals with respect to their breeding value, which is

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an estimate of each individual’s ability to produce high/low-performing offspring. Which method to choose depends on several factors. Among the most important is the heritability of the trait(s), the nature of the trait (e.g. normally distributed or binary; whether records can be obtained on live individuals, etc.) and the reproductive capacity of the species. Owing to the high fecundity of aquaculture species, the selection methods usually applied are individual selection (mass selection), family selection or a combination of the two (combined selection). The last method is the most efﬁcient, where the best families, and subsequently the best individuals within the best families, are selected according to the breeding goal deﬁned. Several large-scale breeding experiments and breeding programmes have demonstrated a substantial response to selection for growth rate. The following estimates of genetic gain per generation have been reported (reviewed by Gjerde, 1986). Coho salmon 10% Rainbow trout 13% Atlantic salmon 10–25% Channel catﬁsh 12–20% Nile tilapia 17% The average of these estimates is 15% genetic gain per generation, indicating that the growth rate can be doubled in less than seven generations of selection. Marine species are still new in intensive ﬁsh farming, and to date no selection experiment or results from a commercial selection scheme have been reported. To optimise a breeding programme, knowledge about genetic parameters such as phenotypic, genetic variation and heritability for the relevant trait(s) is needed, together with estimates about genetic correlations between traits and in-breeding depression. Until this information is available, the construction of a selection scheme for marine species has to be based on knowledge of other ﬁsh species. These will be mainly salmonids, where large-scale breeding programmes have been applied since the early 1970s.

5.6.1 Expected Beneﬁts The beneﬁts of a genetic improvement in growth rate are reductions in both ﬁxed and variable production costs. The latter is due to a reduced energy requirement for maintenance throughout the production process. It is generally assumed that an improved feed conversion rate will be obtained as a correlated response to increased growth rate.

5.6.2 Phenotypic Value and Variance The value observed when a character is measured or scored for an individual is called the phenotypic value of the individual. The phenotypic value (which is the only component which can be measured) can be partitioned into two components, one attributable to the inﬂuence of genotype, i.e. the particular assemblage of genes possessed by the individual, and

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one attributable to the inﬂuence of the environment, i.e. all non-genetic circumstances that inﬂuence the phenotypic value. Symbolically, this is P =G+E

(5.1)

where P is the phenotypic value, G is the genotypic value and E is the environmental deviation. Thus, we can think of the genotype conferring a certain value on the individual, and the environment causing a deviation from, and masking, the genetic value of that individual. The mean environmental deviation in the population as a whole is then taken to be zero, so that the mean phenotypic value is equal to the mean genotypic value. The genetics of a character centre around a study of its variation. The variance in phenotypic values is the phenotypic variance (VP), which is also termed the total variance. The variance in genotypic values is termed the genetic variance (VG), and the variance caused by environmental deviation is the environmental variance (VE). Symbolically, VP = VG + VE + VE = VA + VD + VL + VES + VER

(5.2)

In the equation 5.2, VG is further partitioned into three separate components: VA is the additive genetic variance, or the variance due to the average additive value of the gene. VD is the dominance genetic variance, or the variance due to the value of intra-locus interaction among genes. VL is the interaction genetic variance, or the variance due to the value of interlocus interaction among genes. The sum of VD and VL is termed the non-additive genetic variance, and cannot be used in a selection scheme based on pure breeding for additive genetic improvement. The environmental, or non-genetic, component is divided into two separate components: VES is the variance due to the value of systematic recognisable environmental causes. Examples of systematic causes that are at least partly under experimental control in ﬁsh farming are age, nutritional factors, water temperature, tank, cage or pond effects, and sex effects. VER is the variance due to the value of unknown or random environmental causes, which therefore cannot be eliminated by experimental or testing design. The value of VG, which is the breeding value, cannot be measured but only estimated, and all other factors included in the model more or less mask the breeding value. The ratio VG/VP express the extent to which an individual’s phenotypes are determined by their genotypes. This is heritability (h2) in the broad sense. The ratio VA/VP expresses the extent to which an individual’s phenotypes are determined by the additive value of their genes. This is heritability in the narrow sense. Heritability (h2) determines the degree of resemblance between relatives, and is of the greatest importance in the additive genetics of animals. If the heritability for a trait is low, the most effective course is selection based on family selection, while individual selection might be effective when the heritability is medium or high.

5.6.3 Genotype by Environmental Interaction Genotype by environmental interaction implies that strains, progeny groups or individuals rank differently when kept under different environmental conditions. If the interaction is substantial, a separate breeding population may be needed for each particular environment.

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For both Atlantic salmon and rainbow trout, a signiﬁcant genotype by farm interaction has been reported for growth rate (Gunnes & Gjedrem, 1978, 1981). However, the interaction accounted for a relatively small proportion of the total phenotypic variance. The authors therefore concluded that only one breeding population of Atlantic salmon and one of rainbow trout are needed in Norway. A more serious genotype by environment interaction was reported when sibling groups of rainbow trout were reared in quite different temperatures (McKay et al., 1984) and production systems (Sylven et al., 1991). It is important that the genotype by environment interaction is investigated when a breeding scheme is planned for marine species.

5.6.4 Breeding Goal The traits to be improved through a selection programme have to be speciﬁed for each species, production condition and market. For a trait to be included as a breeding goal, the following prerequisites must hold:

• the trait must be of economic importance • it must be possible to measure or judge (score) the trait • the trait must show genetic variation between individuals When planning a breeding programme, the following breeding goals should be evaluated.

5.6.5 Growth Rate Growth rate is recognised as a principal factor in most aquaculture production, and this trait is included in most genetic improvement programmes for aquaculture species. An increased growth rate means shorter production cycles, reduced risks and the possibility of rearing bigger ﬁsh before they reach sexual maturity. Growth rate also shows a positive genetic correlation with feed conversion efﬁciency, which has a major impact on overall production efﬁciency.

5.6.6 Feed Efﬁciency Feed efﬁciency is probably the factor of highest economic importance in intensive production. This trait cannot be measured directly, but it is genetically linked to growth rate. This leads to a positively correlated response in feed-conversion efﬁciency when selection on growth rate is applied.

5.6.7 Disease Resistance An improved natural resistance against diseases might be an important breeding goal. Improving natural resistance will give a higher survival rate until harvesting. If a speciﬁc disease is stated as a breeding goal, it has to be combined with an adequate protocol for the testing and ranking of families or individuals.

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5.6.8 Quality Quality traits which can be improved through selection will be different for different species and have to be stated in accordance with the market.

5.6.9 Age at Sexual Maturation For some species, sexual maturation will develop before harvest size. Maturation will decrease the growth rate, and for some species maturation is also followed by reduced meat quality and increased mortality. The general breeding objective for this trait should therefore be a ﬁsh that reaches marked size before ﬁrst sexual maturation.

5.6.10 Base Population and Brood-Stock Development For many species, the base population has to be founded from a wild population. If so, the base population should have as broad a genetic variation as possible. If domesticated breeders are available, own brood-stock should be compared with other available stocks before forming the base population. However, a selection within the present brood stock should be initiated as soon as possible.

5.6.11 In-breeding In-breeding generally results in in-breeding depression, which is noticeable as reduced performance, particularly for traits connected with reproductive capacity and viability. However, in-breeding might also depress the growth rate (Kincaid, 1976a,b; Gjerde et al., 1983). It is therefore important to keep the rate of in-breeding at a low level in a breeding programme. Within a population under selection, the rate of in-breeding per generation is a function of the number of sires and dams used as parents for each new generation. Assuming there is no genetic relationship among sires and dams, 50 sires and 50 dams will give an increase in in-breeding coefﬁcient of 0.5% per generation. This is probably an acceptable rate of inbreeding for most traits.

5.6.12 Selection Methods 5.6.12.1 Individual Selection (Mass Selection). Individual selection is easy and inexpensive to practise, and its demand for test capacity is low. It represents a relatively efﬁcient selection method for traits of high heritability which display continuous phenotypic variation, and which can be recorded in live animals. Growth rate is the only trait that fulﬁls these requirements and thus can be improved efﬁciently through individual selection. A breeding programme based on individual selection should be designed to restrict the accumulation of in-breeding in the population under selection in order to avoid the negative effects of in-breeding depression and loss of genetic variability. This can only be done by securing an effective population size which is large enough to prevent in-breeding. To secure

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an effective population size, a ﬁxed number of progeny per selected pair can be stocked together for grow-out testing. Assuming a heritability for growth rate in the range 0.20–0.30, a selection scheme based on individual selection can be designed to keep the expected accumulation of in-breeding below 1% per generation. This will be sufﬁcient to avoid inbreeding depression and maintain the genetic variation, and hence ensure a steady genetic selection response. Another strategy is to develop two separate genetic lines, where selection is applied within each line. The dissemination of genetic material should then be achieved by crosses between lines. 5.6.12.2 Family Selection Family selection will be more efﬁcient than individual selection except for traits of very high heritability. When applying family selection, it is also possible to improve binary (either–or) traits (e.g. age at maturity, mortality) and traits which can only be recorded on dead animals (e.g. quality traits). To apply family selection, the relationships between all individuals in the population have to be known. To achieve this, families have to be reared separately until they can be tagged, or they have to be identiﬁed by genetic markers before selection. 5.6.12.3 Progeny Testing This method of selection is widely applied for farm animals such as cattle, sheep and goats. The selection is made among the parents on the basis of information from their tested progeny. As for family selection, the relationships between all individuals in the population have to be known, and the families have to be produced and identiﬁed. Progeny testing cannot be applied for species where mortality after spawning is total or high. This is not the case for the majority of marine species, and for marine species progeny testing can be applied in combination with family selection in large-scale breeding programmes. 5.6.12.4 Combined Selection This method optimally combines all the information that can add to our knowledge of the breeding value of an individual; it would include recorded information about the individual itself, and information about full sibs, half sibs and progeny, as well as pedigree information. It represents the general solution for obtaining the maximum rate of genetic gain, and other simpler methods are special cases of this method. Therefore, it is always the most efﬁcient method. Selection indices are the most efﬁcient way to combine information about an individual and its relatives, as well as information about speciﬁc traits. For combined selection, all the information is combined into an index of merit, where the traits are weighted according to their relative economic value. The index gives the genetic value of all individuals in the population, and breeders are selected in accordance with the index.

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5.6.13 Response to Selection The expected genetic gain (DG) or response to selection per generation depends on four parameters. The formula for individual selection is DG = i ¥ h2 ¥ sP

(5.3)

where i is the standardised selection differential (also called the selection intensity), h2 is the heritability of the trait and sP is the phenotypic standard deviation, i.e. the square-root of the phenotypic variance (VP). A more general formula, which is applicable to all methods of selection, is DG = i ¥ rTI ¥ sG

(5.4)

where i is as described above, rTI is the accuracy of selection, i.e. the correlation between the true and estimated breeding value, and sG is the additive genetic standard deviation, i.e. the square-root of the additive genetic variance (VA). The expected response to selection is directly proportional to the accuracy of selection and the selection intensity. The majority of marine species have a very high reproduction potential in culture, and a small number of individuals are needed as spawners. It is therefore possible to achieve high selection intensity, and thereby a high level of genetic gain.

5.6.14 Multi-Trait Selection When several traits are included as breeding goals, multi-trait selection is the most effective way to achieve genetic improvement in all traits. Multi-trait selection is based on selection indices (breeding value) estimated for each trait. The selection indices combine information about an individual and its relative, as well as information about several traits. All information is combined into an index of merit, where the traits are weighted according to their relative economic value. For all species in aquaculture where reproduction is controlled, full- and half-sib groups with a large number of individuals can be produced. This makes it possible to estimate breeding values and indexes with great accuracy. This fact, together with the probable high selection intensity, gives great opportunities for genetic progress. These opportunities should be used in effective selection schemes for all farmed species.

5.7 Modern Biotechnology and Aquaculture The genetic improvement of ﬁsh stocks represents a major challenge for aquaculture in order to enhance the use of available feed and land resources. In most, if not all, ﬁsh species the biological potential is considerable owing to the high heritable variation for production traits such as growth rate and disease resistance. Large-scale breeding programmes have successfully been implemented for several aquaculture species. Modern biotechnology will become increasingly more valuable as a supplementary tool towards achieving the growing demands

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of world aquaculture. Some potential applications of these sophisticated molecular methods are pedigree analysis by DNA ﬁngerprinting, gene-mapping studies, and the identiﬁcation of genetic markers or speciﬁc genes associated with desirable traits. All these approaches involve the ampliﬁcation of nuclear or mitochondrial DNA, and make use of the polymerase chain reaction (PCR). With this technique, the gene or genetic marker of interest is easily ampliﬁed to visible amounts for a reasonable price within less than 2 h (Fig. 5.24). Since tiny amounts of tissue containing only a few copies of the desirable gene are sufﬁcient as a template in this DNA replication process, the PCR technique has revolutionised all aspects of molecular studies, including those within aquaculture.

5.7.1 Molecular Pedigree Analysis Selective breeding programmes in aquaculture make use of family information, which requires that families are kept separately until the ﬁsh are large enough to be physically tagged. This imposes major economic and practical problems, and can induce environmental effects common to full-sibs. The identiﬁcation of family groups by their speciﬁc DNA ﬁngerprint may dramatically improve this situation. Fish from different families can then be reared together in the same tank even from the egg stage. This also allows larger numbers of families to be tested, and thus facilitates the use of higher selection intensities without a rapid increase in in-breeding. Repetitive DNA sequences, or so-called microsatellites, have successfully been used empirically to reconstruct pedigrees in ﬁsh populations with families mixed from hatching. Microsatellites are useful markers for genetic tagging, owing to their high number and variability within the genome. Hundreds of polymorphic microsatellites have already been identiﬁed in many ﬁsh species of economic value, but the numbers of potential microsatellite loci present in the ﬁsh genome are likely to be of the order of 100 000. In particular, AC dinucleotide repetitions were estimated to be present in numbers of 2.34 ¥ 105 at intervals of about 7000 base pairs in the genome of Atlantic cod (Brooker et al., 1994). The four most informative microsatellites identiﬁed in the Atlantic salmon genome were predicted to be sufﬁcient to assign at least 99% of the offspring to the correct pair with 100 crosses involving 100 males and 100 females. An additional polymorphic microsatellite was required to correctly assign 99% of the offspring when the 100 crosses were produced with 10 males and 10 females (Villanueva et al., 2002). This indicates that parental assignment is feasible with the genetic markers currently available in several ﬁsh species. Both the efﬁciency and the costs of microsatellite-based pedigree analysis should be considered before this method is included in a breeding programme. In practice, the parents and the mixed offspring are genotyped by PCR ampliﬁcation of the appropriate microsatellite loci from crude DNA extracts from small non-destructively sampled quantities of tissue such as ﬁsh scales, mucus or a ﬁn clipping. Using this protocol, 2000 ﬁsh from a mixture of 500 families can be screened for 10 markers in less than a month, allowing 99% of the ﬁsh to be parentally assigned. For the time being, offspring have been assigned to parents of known genotype. However, with sufﬁcient levels of variability, the identiﬁcation of families may also be achievable in the absence of parental information. It should be noted that substantially more markers need to be screened for resolving individuals compared with family

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Figure 5.24 Detection of microsatellite markers by polymerase chain reaction (PCR). This microsatellite consists of tandem repeats of the dinucleotide CA. The number of repeats in such microsatellites varies with the individual (e.g. four repeats in ﬁsh I, and eight repeats in ﬁsh II). These variations can be detected by PCR amplifying the repeated DNA fragment with the ﬂanking primers 1 and 2. This process yields ampliﬁed DNA products of different lengths as visualised by gel electrophoresis.

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discrimination. The steadily decreasing costs of genetic tagging may still not compete with traditional physical tagging. Because the genotype information is detached from the individual, genetic tagging implies that the ﬁsh has to be retyped each time its performance is evaluated or individuals are selected. In a selective breeding programme, each ﬁsh needs to be tested many times during its life cycle, and selection must be carried out at several stages, so this approach is not economic with current technology. Hopefully, the use of microchipbased genotyping may solve this problem. DNA ﬁngerprinting may also have several other applications within ﬁsh management, including evaluating in-breeding levels, stock identiﬁcation, and the movements of released ﬁsh and their possible genetic interactions with wild stocks. The rapid evolutionary rate of mitochondrial DNA makes these markers ideal for ﬁsh population studies.

5.7.2 Genetic Mapping and QTL Analysis All selective breeding programmes depend on genetic variation in the breeding stock, but no information about the functional genes inﬂuencing the phenotype is needed. Even though a quantitative trait is inﬂuenced by multiple genes, a variation in a few genes may be responsible for a major part of the variation in the phenotype. By quantitative trait loci (QTL) analysis, it is possible to identify the region(s), or QTLs, of the genome in which these so-called major genes are localised, still without knowing the identity of these genes. A QTL analysis is carried out by searching for linkages between variations in performance of experimental crosses and the alleles of genetic markers, which have been mapped to their positions or loci (singular locus) on the genome. The numerous, hypervariable microsatellites are ideal markers for creating genetic maps, since they are evenly spaced throughout the genome. Genes are also useful markers, but they are widely spaced with large gaps between them, and only a fraction of the total number of genes seems to exist in allelic forms. The more markers utilised and the more polymorphic the markers, the greater the likelihood of detecting an association with variation in a quantitative trait. A genetic map is established by studying the inheritance or segregation of markers in cross-breeding experiments. Based on this linkage analysis, the markers are put together within linkage groups, each group corresponding to a single chromosome. The mapping of ﬁsh genomes, including hundreds of genetic markers, has been worked out for several commercial ﬁsh species, including salmonids, Atlantic cod, Paciﬁc herring, sea bass and tilapia. At present, zebra ﬁsh show the most complete genetic map, including more than 2000 markers (Woods et al., 2000). The methodology for the detection of QTLs in ﬁsh is currently still being developed, and very few QTL markers have been identiﬁed. In rainbow trout, microsatellite markers linked to two QTLs for upper temperature tolerance have been reported (Jackson et al., 1998). The key role of the major histocompatibility complex (MHC) in immune defence, and the highly polymorphic nature of MHC, make this complex a candidate QTL for disease resistance in ﬁsh. Indeed, a strong association between MHC alleles and resistance against furunculosis has been identiﬁed in Atlantic salmon (Langefors et al., 2001). In tilapia, the length of two microsatellites located within the promoter of the prolactin gene was shown to be associated with prolactin expression and growth of salt-challenged ﬁsh (Streelman & Kocher, 2002). This knowledge of linkages between desirable phenotypic traits and QTL markers can be

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used for marker-assisted selection (MAS), which will improve the accuracy of selection and thus lead to increased selection responses. Even without knowing the identity of the genes within a QTL, we can obtain information about their actions and interactions, and estimate how much of the total variation is accounted for by the QTL variation. In the future, QTL analyses and MAS will certainly not replace traditional selective breeding, but they should be implemented in the breeding programmes as supplementary tools to estimate breeding values and evaluate breeding candidates.

5.7.3 Transgenic Fish The high fecundity of most ﬁsh and external fertilisation and embryonic development make them especially suitable for transferring speciﬁc genes. The production of transgenic ﬁsh is aimed at dramatically improving traits such as growth, disease resistance and environmental tolerance. Because they are inﬂuenced by multiple genes, the nature of such quantitative traits makes them difﬁcult to manipulate by gene transfer techniques. However, signiﬁcant growth enhancement of several ﬁsh species has been demonstrated after the introduction of the growth hormone gene as the major gene under the control of a strong promoter element (Melamed et al., 2002). It should be noted that the increased growth rate is mainly due to the large amounts of growth hormone produced in large tissues such as the liver, and not by the transfer of millions of gene copies. Surprisingly, the growth of transgenic wild-strain rainbow trout was shown not to surpass that of a non-transgenic domesticated strain being selected for fast growth (Devlin et al., 2001). Furthermore, introducing the growth hormone construct into this fast-growing domestic strain did not cause further growth enhancement. These results indicate that similar alterations in growth rate can be achieved both by selective breeding and by transgenesis, but that the effects are not additive, at least not in rainbow trout. There are several problems to be overcome before transgenic animals can be produced on a large scale. Indeed, in over 90% of the microinjected eggs, the transgene is not efﬁciently integrated into the genome at the one-cell stage. The result is a highly mosaic transgenic ﬁsh and low frequencies of germ-line transmission, since only the tissues developing from the transformed cell will carry the transgene. Furthermore, the injected DNA integrates at single or multiple random sites in the genome of the recipient embryo, and each develops into a unique hemizygous ﬁsh. Hence, the establishment of a stable transgenic broodstock will be a costly endeavour, requiring several generations. On the other hand, attempts to combat viral and bacterial pathogens which threaten commercial stocks by utilising DNA vaccines have been promising. This technique is based on the injection of DNA encoding part of the antigen, usually a bacterial outer membrane or viral capsid protein, in the ﬁsh muscle, where the protein will be synthesised and the production of antibodies induced. A signiﬁcant degree of protection against infectious hematopoietic necrovirus (IHNV) was found in Atlantic salmon after vaccination with a gene construct containing an IHNV glycoprotein (Traxler et al., 1999). Similarly, protection against viral haemorrhagic septicaemia virus (VHS) was induced in vaccinated rainbow trout (Lorentzen et al., 1999). The main disadvantage of these approaches is that they require quite detailed information about the structure, conformation and encoding sequence of the pathogen’s protein.

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An alternative way to increase the resistance of ﬁsh to pathogens is to target the non-speciﬁc immune response through the use of antimicrobial peptides, which are found in both vertebrates and invertebrates. Short peptides consisting of 30–50 amino acids with strong antimicrobial activity have been isolated from the skin mucus of several ﬁsh species, including Atlantic halibut and winter ﬂounder. Furthermore, the encoding sequences of the histonederived ‘hippoglossin’ of halibut have been determined (Birkemo et al., 2003). To date known attempts to produce transgenic ﬁsh carrying genes encoding antimicrobial peptides all relate to the lysozyme gene, which has a non-speciﬁc antibacterial effect.

5.7.4 Future Prospects The applications of molecular techniques in aquaculture are promising, but still somewhat uncertain. While high costs seems to be the only hindrance to the widespread application of genetic markers for identiﬁcation purposes and MAS, the situation regarding the commercial use of genetically modiﬁed ﬁsh is more complex. Although the potential importance of gene transfer technology is large, a major concern relates to the possible impact which release or accidental escape of gene-modiﬁed individuals may have on natural ecosystems. Other controversial aspects are related to animal welfare, food safety and the public perception of gene manipulation in general. To what extent such issues will constrain the future use of transgenic animals in applied aquaculture production remains to be seen.

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Chapter 6

From Fertilisation to the End of Metamorphosis—Functional Development E. Kjørsvik, K. Pittman and D. Pavlov

Adult ﬁsh show a large variation in morphology and ecology, and so do their offspring. When studying the appearance of the early life stages of ﬁsh, one is immediately struck by their diversity and morphological dissimilarity to adults, and that they most often inhabit totally different habitats from their parents. A knowledge of reproductive and developmental biology and ecology is therefore crucial for producing offspring of optimal quality, and for the successful mass rearing of juvenile ﬁsh fry. The early life stages of ﬁsh also have species-speciﬁc and stage-speciﬁc environmental and nutritional requirements. Developments in rearing technology and start-feeding have beneﬁtted signiﬁcantly from studies of the developmental biology and ecology of ﬁsh eggs and larvae. The following chapter will focus on the functional egg and larval development of cold-water marine aquaculture species, and on aspects that are particularly important for their cultivation. Their nutritional and environmental needs are thoroughly covered elsewhere in this book.

6.1 Intervals of Fish Ontogeny and Deﬁnitions of the Organism The basic developmental mechanisms are similar in all teleost species, but differences exist with regard to the relative timing of growth and the development of specialised cells and organs. Genetic differences, yolk size and environmental conditions all inﬂuence the developmental stage and larval size at hatching and at the transition to exogenous feeding. Some common criteria are needed to deﬁne the stages of early ﬁsh development in general. The variety of developmental patterns should be considered, and the deﬁnitions should apply to as many patterns as possible. In this chapter, we use the operational deﬁnitions listed below. Egg: Yolk-sac larva:

Larva:

an encapsulated embryo (from spawning to hatching). a free-living embryo with a yolk-sac which may or may not be absorbed before exogenous feeding (i.e. from hatching to the start of exogenous feeding). from the start of exogenous feeding to the completion of metamorphosis.

From fertilisation to the end of metamorphosis

205

Metamorphosis: the transitional stage between larva and juvenile. Juvenile: having the ﬁnal phenotype, but not sexually mature. Each of the stages can be divided into several substages (Fig. 6.1). In developmental biology, the offspring is generally regarded as an embryo up to the point where exogenous feeding begins, i.e. the embryonic stage includes the egg stage and the yolk-sac larva stage (Balon, 1999).

Figure 6.1 Early life-history stages of ﬁsh larvae, as shown for the jack mackerel (Trachurus symmetricus). From the original drawings of Ahlstrom & Ball, 1954.

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Ontogeny is the entire life cycle of an organism from egg activation (and subsequent fertilisation) to death, and it seems natural to distinguish intervals of ﬁsh development. However, the principles for the deﬁnition of these intervals have been discussed for at least half a century (Vasnetsov, 1953). The main problem for the subdivision of the ontogeny lies with the divergent answers to this question: Do ﬁsh develop gradually, or is their development non-gradual (saltatory) with distinguishable natural intervals separated by thresholds? According to the ‘gradualists’, ontogeny is a gradual process with continuous changes in the form, structure, physiology and behaviour of an organism. According to the concept of non-gradual ontogeny of ﬁsh (Vasnetsov, 1953; Kryzhanovskii et al., 1953) and the theory of saltatory (step-wise) ontogeny (Balon 1985, 1990), the life-history of ﬁsh can be separated into periods which may be deﬁned as the longest intervals of ontogeny separated by the most decisive thresholds (Table 6.1). This means that the organism develops through a series of rapid, almost sudden, changes in form and/or function, which are separated from each other by longer intervals during which changes are smaller, more continuous and rather insigniﬁcant. Between these periods of rapid change, the organism will prepare for the next rapid change, and the organs and tissues necessary for the next big level of change will develop to converge functionally at a certain threshold. Whether the functional development of ﬁsh larvae is saltatory or not is still a question of much debate among scientists. The main factor to remember is that larval requirements and environmental responses change during their development.

Table 6.1 Terminology of life history stages. From Kendall et al. (1984), in which the references can be found. Reproduced with permission of the American Society of Ichthyologists and Herpetologists

END POINT EVENTS Spawning

Blastopore closure

Tailbud free

Hatching

Yolk-sac absorbed

Full ﬁnray complement Attains Attains juvenile present, adult squamation body body begun, loss proportions, proportions, Notochord Notochord pigment, pigment, flexion Metamorph- of larval starts to complete osis begun characters habits flex habits

TERMINOLOGY Primary developmental stages

Transformation larva

Yolk-sac larva

Transitional stages Subdivisions

Juvenile

Larva

Egg

Early

Middle

Preﬂexion larva

Late

Flexion larva

Postﬂexion larva

Pelagic or special juven

OTHER TERMINOLOGIES Hubbs, 1943, 1958

Embryo

Postlarva

Prolarva

Prelarva

Hattori, 1970

Snyder, 1976, 1981 (phases)

Postlarva

Embryo

Nikolsky, 1963

Balon, 1975 (phases)

Prejuvenile

Larva

Sette, 1943

Cleavage egg

Embryo

Eleutheroembryo

Protopterygiolarva

Protolarva

Pterygiolarva Mesolarva

Metalarva

From fertilisation to the end of metamorphosis

207

The terminology applied to ﬁsh ontogeny, which has been used in most practical research, is based mainly on the concept of ontogeny as a sequence of ‘normal stages’, and has evolved from ﬁsheries and ichthyoplankton ecology (Table 6.1). The egg phase includes several ‘stages’, and the hatched free organism with a yolk can be called a yolk-sac larva or a prelarva. The larval phase consists of the ‘stages’ where a ﬁnfold is present and where the ﬁns are forming. The embryonic and yolk-sac periods are characterised by endogenous feeding. The transition to exogenous feeding (and not hatching) is therefore regarded by many as one of the most critical periods for survival.

6.1.1 Relative Duration of the Various Stages of Development The type of early ontogeny is related to the relative duration of the main developmental intervals. The relative durations in ﬁve species are presented in Fig. 6.2. As a reference point for a comparison of different developmental styles, reaching the ‘juvenile state’ (i.e. the stage of the disappearance of larval characteristics and the formation of the most juvenile characteristics, including skeletal ossiﬁcation) is used. Cod, turbot and Atlantic halibut possess a comparatively short embryonic phase of development inside the egg-shell, and a prolonged larval period. Among them, the Atlantic halibut has a prolonged yolk-sac stage of about 44 days at 9°C. The common wolf-ﬁsh is characterised by a prolonged embryonic phase ranging from 104 days in captivity under the inﬂuence of a comparatively high temperature to 9.5 months in nature. In addition, larval exogenous feeding in this species begins just after normal hatching.

Egg state

Yolksac larva

Larval state

Wolffish

Salmon

Halibut

Turbot

Cod

0%

20 %

40 %

60 %

80 %

100 %

Duration from fertilization to juvenile state

Figure 6.2 Scheme representing comparative features of early ontogeny in cod, turbot, Atlantic halibut, Atlantic salmon and common wolf-ﬁsh. The duration of development from egg activation to onset of juvenile state = 100% (approximately 59, 90, 108, 112 and 177 days, respectively).

208

Culture of cold-water marine ﬁsh

6.2 Egg Classiﬁcation There is a great variability in the reproductive styles of ﬁsh (see also Chapter 5), and these styles are determinants for the species differences in fecundity, egg size and egg type. The Norwegian scientist G.O. Sars (1869) was the ﬁrst to discover that ﬁsh may have pelagic eggs (i.e. eggs ﬂoating freely in seawater). When he described the pelagic nature of Atlantic cod eggs and larvae for the ﬁrst time, he shed new light on the wide variety of reproductive styles and early life history patterns of ﬁsh. Until then, all known ﬁsh eggs were demersal, i.e. developing on the bottom or attached to a substrate. The eggs of all ﬁsh have a polar distribution of yolk and cytoplasm and belong to the telolecithal type. The concentration of yolk at the vegetative pole of the egg is different in species from the various taxonomic groups. The eggs also have different features of yolk distribution in the cytoplasm, and based on this characteristic, two egg subtypes are known (Makeyeva, 1992): (1) (2)

Eggs with no separation of yolk Eggs with separation of yolk

The egg cleavage pattern varies according to the different ﬁsh groups, and the two subtypes are characterised primarily by different developmental patterns. In eggs of the ﬁrst subtype, the yolk is distributed in the cytoplasm as granules or conglomerates. These eggs will undergo full (holoblastic) cleavage, i.e. the whole egg goes through cell cleavage after fertilisation (with uneven cell sizes). Eggs of this subtype are common for lower bony ﬁsh such as lungﬁsh (e.g. Neoceratodus), Chondrostei (sturgeons) and Holostei (Amia and Lepisosteus). Fish with this egg type all spawn in fresh water. All teleosts, most of the sharks (Chondrichthyes) and the living coelacanth, Latimeria chalumnae, produce eggs of the second subtype, where the yolk is separated from the cytoplasm. Only the cytoplasm (and not the yolk) is subject to cleavage. Therefore, the cleavage is called meroblastic or discoidal. Most marine ﬁsh spawn pelagic eggs that are fertilised externally and ﬂoat individually near the surface of the sea. Pelagic eggs are generally small; the egg size may vary from about 0.6 to 4.0 mm in diameter, with a mean diameter of 1 mm (Kendall et al., 1984). However, there are many exceptions to this pattern. For example, the halibut has exceptionally large pelagic eggs with a diameter of about 3 mm. Halibut eggs also differ from typical pelagic eggs in buoyancy. The halibut spawn at great depths, and the eggs develop in the mesopelagic layer, at about 150–250 m deep (Haug et al., 1984), while most pelagic eggs ﬂoat in the upper surface layers. Pelagic eggs are generally spherical and transparent, as is shown for cod and halibut eggs in Fig. 6.3. This is the normal pattern for pelagic eggs, regardless of systematic position, whether the adult ﬁsh has pelagic or demersal habits, or if it lives in coastal or oceanic waters, or in tropical or boreal areas. Many coastal, freshwater ﬁsh and some marine lay demersal eggs, which are generally larger than pelagic eggs. Demersal eggs are often adhesive, and may be laid in some sort of nest or cluster. Most demersal eggs are also less transparent, and have a much thicker egg-shell (or egg envelope or chorion) than pelagic ones (Fig. 6.3).

From fertilisation to the end of metamorphosis

209

Figure 6.3 Cod, halibut and wolf-ﬁsh eggs shown according to their relative size. Cod eggs (in the middle) are the typical size of a pelagic egg with a diameter of about 1.3 mm, whereas halibut (3 mm, on the left) has very large eggs for its pelagic nature. The large demersal egg of the common wolf-ﬁsh (5.5 mm) is comparable in size to salmonid eggs. Pelagic eggs are more transparent than demersal eggs because they have a much thinner shell. Photographs: Elin Kjørsvik and Inger-Britt Falk-Petersen.

Within a species, there is little variation in egg characteristics such as size, number and size of oil globules, pigmentation and the morphology of the developing embryo. The development time is highly temperature-dependent and species-speciﬁc. Between species, egg size and fecundity tend to be inversely related (Blaxter, 1988). Species with larger eggs also generally have a longer incubation time, their larvae are much more developed at hatching, and their development may often go direct from hatching to the juvenile stage. The time-scale from fertilisation to hatching may be days for species with small pelagic eggs, the large demersal eggs of salmonids and wolf-ﬁsh tend to take several weeks or months, whereas the very large eggs of elasmobranchs may take even longer. The large difference in size between salmonids and wolf-ﬁsh versus marine pelagic eggs and larvae (see Fig. 1.2, Chapter 1; Table 6.2) illustrates how much more advanced the functionality and viability of the larger eggs and larvae may be. These differences in size, embryo development time and developmental stage at hatching are the main reasons for the difﬁculties experienced in marine larval rearing, as pelagic larvae are much less developed at the time of start-feeding than the larger demersal ones.

6.2.1 Egg Structure and Composition In general, mature unfertilised teleost eggs are soft, and do not tolerate much mechanical pressure. A ﬁsh egg (Fig. 6.4) consists of a large mass of yolk material surrounded by a thin layer of cytoplasm and an outer egg envelope (egg-shell or chorion). Numerous small

210

Spawning and egg characteristics of some cold-water aquaculture species.

Species

Fertilisation

Egg diameter (mm)

Egg type

Number of spawned eggs

External

5–7

Demersal

10–15 thou.

Wolf-ﬁsh

Internal

5.2–6.0

Demersal

Cod3

External

1.1–1.7

Pelagic

External

0.90–1.2

Pelagic

10–15 mill.

External

3.0–3.8

Mesopelagic

0.5–1 mill.

Atlantic salmon1 2

Turbot4 Atlantic halibut 1

5

Preferred developmental temperature (°C)

Hatching time (d°)

Size of newly hatched larvae (mm)

Hatching to start-feeding (d°)

6–8

510

17–20

300

5–50 thou.

5–8

900

23

2.5–14.5 mill.

5–12

90

4

27

13–18

75–102

2.7–3.1

36

4–7

82–85

6

Gjedrem, 1993. Falk-Petersen et al., 1999; Tveiten & Johnsen, 1999. 3 Kjesbu, 1989; Kjesbu et al., 1991; Iversen & Danielssen, 1984; Galloway, et al., 1999a. 4 Jones, 1974; Howell, 1979; Bromley et al., 1986; Bromley et al., 2000; Fauvel et al., 1993. 5 Haug et al., 1984; Pittman et al., 1990; Norberg et al., 1991; B. Nordberg, personal communication, 2002; Galloway et al., 1998. 2

0–30

220–270

Culture of cold-water marine ﬁsh

Table 6.2

From fertilisation to the end of metamorphosis

211

Egg shell

Cytoplasm Oil droplets Cortical alveoli

Yolk

Micropyle Figure 6.4 Schematic drawing of an ovulated, unactivated ﬁsh egg. Most of the egg content is yolk, which contains all the necessary nutrients for the development of the embryo and yolk-sac larva. In some species, the yolk may contain one or several oil droplets. The micropyle is situated at the animal pole of the egg, and it is the only opening in the egg shell (egg envelope) where a spermatozoon may enter to fertilise the egg cell. The nucleus is not visible in the mature egg. The narrow opening is a protection against polyspermi, and it becomes clogged after the entrance of the ﬁrst sperm cell.

granules or cortical alveoli are distributed in the peripheral cytoplasm of the egg. These alveoli contain the enzymes and macromolecules necessary for the egg activation. The cytoplasm of a mature oocyte contains a nucleus (the germinal vesicle) located at the animal pole directly under the micropyle, which is a small opening in the egg-shell. The micropyle is obstructed after the entrance of the ﬁrst sperm cell. The eggs of many ﬁsh species contain oil (lipid) droplets. Turbot eggs have one large oil droplet, whereas cod and halibut have none. Salmonid and wolf-ﬁsh eggs contain several lipid droplets. These droplets are ﬁxed at the animal pole of the egg in salmonids, and move freely within the yolk in turbot and wolfﬁsh species. The egg envelope is a non-living part of the egg, and consists mainly of latticed proteinaceous (keratin-like scleroproteins) concentric layers. The egg envelope is also the hardest part of the egg, and it provides physical protection to the developing embryo. Its structure is therefore related to the ecological conditions during egg development. Thus, pelagic eggs generally have thin egg envelopes relative to their diameter, whereas the envelopes of demersal eggs are thicker, more robust and are often supplied with extra outer layers that may be adhesive.The primary egg envelope (originating from the superﬁcial protoplasm of the oocyte) contains many tiny radial canals and is called the zona radiata. The

212

Table 6.3

Culture of cold-water marine ﬁsh

Egg weight and composition of some cold-water aquaculture species.

Species

Egg wet weight (mg)

Atlantic salmon1 Common wolf-ﬁsh2 Cod3 Turbot4 Halibut5

53–142 115 1 0.6 17

Ash content (% of DW) 4

Water content (%)

Lipid content (% of DW)

Protein content (% of DW)

60–67

30

66–74

FAA content (nmoles/egg)

950 13 10 9

90–93 90 87–90

5–13 25–32 12–15

47 40–52 55

250 65 2300

1

Kamler, 1992; Sargent, 1995; Berg et al., 2001. H.J. Fyhn, unpublished data, 2002. 3 Lønning et al., 1988; Finn et al., 1995a, b; Fyhn & Serigstad, 1987. 4 Falk-Petersen et al., 1989; Finn et al., 1991; Finn, 1994; Rønnestad et al., 1995. 5 Finn et al., 1991; Rønnestad & Fyhn, 1993. 2

secondary egg envelope is formed by the follicle cells and is called the chorion. The egg envelope forms an outer adhesive coat in demersal eggs of many ﬁsh species, e.g. the lumpﬁsh (Cyclopterus lumpus) and the wolf-ﬁsh. The nature of such adhesion is still unknown. The yolk contains the material necessary for development and metabolism during the embryo and yolk-sac larva stages. A comparison of the egg characteristics of various species is given in Table 6.3. The general pattern is that species with larger eggs contain more energy, have longer incubation times and produce larger larvae at hatching than species with smaller eggs. If egg size is similar, larvae hatching from demersal eggs will generally be larger and more developed than larvae hatching from pelagic eggs. This is also reﬂected in the egg composition, as demersal eggs generally contain less water and higher absolute levels of lipids (especially neutral lipids) than pelagic eggs. About 90–95% of the yolk mass in pelagic eggs is water, which makes them buoyant. However, the yolk must also contain all the necessary nutrients and energy for normal embryonic development, such as proteins, free amino acids, lipids (many species have oil droplets in the yolk), vitamins and minerals. The different yolk components are used sequentially, according to the changing needs of the developing embryo.

6.3 Insemination and Fertilisation Insemination is the process of mixing eggs and sperm to obtain close contact between the gametes. According to the deﬁnition of Balon (1990), fertilisation in its broadest sense is a process which starts with insemination, continues with activation and cortical reaction, the formation of the perivitelline space and bipolar differentiation, and ends with the fusion of male and female pronuclei. Only the latter process can be considered to be fertilisation in a strict sense. The fertilisation process contributes to profound changes in the egg characteristics, and the eggs are very vulnerable during this interval. Egg activation includes a complex of changes. First, the developmental block of meiosis at the metaphase of the second meiotic division is released. In addition, the permeability of

From fertilisation to the end of metamorphosis

213

the egg membrane increases. A transitory rise in intracellular Ca2+ occurs, starting at the animal pole and spreading towards the vegetal pole (Polzonetti et al., 2002). This Ca2+ wave seems to be necessary for metabolic activation and cell-cycle control in the egg. The cortical alveoli are activated by the Ca2+ wave, and their membranes fuse with the plasma membrane, resulting in an exocytosis of the alveolar content (cortical reaction or cortical wave, see Fig. 6.5) beneath the egg envelope (chorion). This process seems to be dependent on the presence of Ca2+ in the water (Lønning et al., 1984). In ﬁsh, egg activation is induced by the

Figure 6.5 Cortical reaction in a cod egg. The egg is activated by the entrance of a sperm cell through the micropyle, which can be seen as a small indentation in the upper part of the egg (the animal pole). The content of the cortical alveoli are released in a wave-like action starting in the micropyle area and spreading over the egg surface. This can be observed as the disappearance of the cortical alveoli, as these time-lapse photographs during the ﬁrst 10 min after sperm addition demonstrate. From Davenport et al., 1981.

214

Culture of cold-water marine ﬁsh

penetration of a spermatozoon, by contact with water (as in salmonids), or even by mechanical stimulation (Ginzburg, 1972). In the majority of marine ﬁsh species, eggs are activated by spermatozoa: without insemination, the cortical reaction either does not take place or is incomplete. The release of the cortical colloidal macromolecules contribute to an inﬂux of (sea)water through the chorion, leading to formation of the perivitelline space (a ﬂuid-ﬁlled space between the egg envelope and the embryo). The perivitelline space is normally narrow in pelagic eggs, but it may be wide in some species such as the long rough dab Hippoglossoides platessoides. In addition, the formation of the perivitelline space may be caused by partial shrinkage of the yolk, as has been observed in cod and in common wolf-ﬁsh. After this initial activation, the cytoplasm starts to become concentrated towards the animal pole, in preparation for the ﬁrst cell cleavage. The cytoplasm (cell material) is heavier than the yolk, and in pelagic eggs the developing cells can be observed below the yolk. In the egg, the second meiotic division is then completed and the female pronucleus appears. The spermatozoon head transforms into the male pronucleus. The pronuclei fuse, causing a resumption in diploid chromosome numbers. The egg envelope (or chorion) is highly permeable to small molecules such as water, ions and amino acids. It is impermeable to larger molecules, and is therefore an effective barrier to bacteria and viruses. It also undergoes chemical and structural changes during the activation and the fertilisation process. Owing to the uptake of water, the egg swells, and at the same time the egg-shell hardens, a process that results in a thinner membrane with a much higher rigidity. Swelling of wolf-ﬁsh eggs in seawater is accompanied by hardening of the egg envelope and adhesion to other eggs, but not to other substrates. In marine ﬁsh eggs, there seems to be a good correlation between egg hardness and envelope thickness. The demersal lumpsucker eggs, which have very thick egg envelopes, may resist a mechanical force of about 2 kg before breaking, whereas other pelagic eggs may only tolerate forces ranging from 100 to 400 g before breaking (Lønning et al., 1988). The egg envelope structure is species-speciﬁc, and its characteristics may be used for species identiﬁcation.

6.4 Embryonic Development and Hatching All the ﬁsh species which are regarded as promising candidates for marine aquaculture are teleosts. For the teleosts, embryogenesis from fertilisation to hatching tends to follow the same basic pattern (e.g. Kendall et al., 1984; Blaxter, 1988). Therefore, in this section, a brief description of the general patterns of embryonic development is given exclusively for eggs with a separated yolk showing meroblastic cleavage, using cod as a model (see Figs. 6.6 and 6.7). Other descriptions of cod development can be found in Sars (1869) and Fridgeirsson (1978), for example. The intervals of embryonic development are given according to Makhotin et al. (1984) and Makeyeva (1992). Comparative data will be presented for common wolf-ﬁsh, a species with an alternative type of early ontogeny (see Figs. 6.8 and 6.9).

From fertilisation to the end of metamorphosis

215

6.4.1 Cod (Gadus morhua) Mature cod eggs are spherical and transparent, often with a light orange colour. Their diameter ranges from 1.4 to 1.8 mm. After spawning, the eggs collect together just below the water surface. A narrow (0.4 mm) primary egg envelope (zona radiata) encases the thin cytoplasmic layer surrrounding the yolk. The animal pole with the micropyle is located in the lower part of the egg.

Step I. Activation, Hardening of the Egg Envelope, Formation of Perivitelline Space and Blastodisc Egg activation can be observed as a consecutive breakdown of the cortical alveoli from the animal to the vegetal pole, and the formation of the perivitelline space between the thin cytoplasmic layer surrounding the yolk and the internal surface of the egg envelope. The perivitelline space is small: the egg volume remains almost the same, and the wet weight of the egg increases to 15–20%. Then the cytoplasm aggregates below the micropyle forming the blastodisc (Fig. 6.6a). The second meiotic division is then completed, and female and male pronuclei fuse. The hardening of the egg shell is observed during the ﬁrst few hours after fertilisation.

Step II. Cleavage The concentration of cytoplasm after fertilisation has the appearance of one cell before the ﬁrst cleavage. The dividing cells are called blastomeres, and the ﬁrst series of cell cleavages transforms the single egg cell into a multicellular body called the blastodisc on the surface of the yolk. Cleavage represents a series of mitotic cell divisions in the blastodisc, leading to the formation of a semispherical cap above the yolk, composed of many blastomeres. The size of the cells decreases with each subsequent division. In many ﬁsh species, including cod, one cell appears to be slightly larger than the other after the ﬁrst cleavage (Fig. 6.6b). The second cleavage is perpendicular to the ﬁrst. The two sections of the third cleavage are parallel to each other, forming eight blastomeres. During the ﬁrst four cleavages, the cell divisions are incomplete, leaving a thin layer of uncleaved cytoplasm at the bases of the cells, thus forming the syncytial layer. The cell divisions become asynchronous from the 5 to 7th cleavage cycles. The 5th cleavage is often latitudinal (parallel to the egg equator). As a result, the 32 blastomeres are separated into upper cells, which are not connected to the yolk, and lower blastomeres, which join to each other by means of the syncytial layer (Fig. 6.6c). Later the lower blastomeres form a speciﬁc layer, the periblast, with giant nuclei surrounding the base of the blastodisc. The periblast is central during the process of blastoderm epiboly and later for yolk utilisation. The stages between 64 and 256 blastomeres are called large-cell morula (Fig. 6.6d), and as the blastomere size decreases, the stages may be called middle-cell or small-cell morula (Fig. 6.6e,f ). The entire cell mass is the blastoderm. In some species, periodic motoric contraction waves in the cytoplasm surrounding the yolk can be observed. This

Figure 6.6 Stages of the embryonic development of cod at 1.8°C. Scale bar = 1 mm. (Makhotin et al., 1984; with additional drawings by V.V. Makhotin). Note that the natural position of the animal pole with the blastodisc is below the yolk. (a) Formation of blastodisc, age 8 h from egg activation; (b) two blastomeres, age 10 h; (c) 32 blastomeres, age 20 h; (d) large-cell morula, age 1 day 6 h; (e) small-cell morula, age 1 day 16 h; (f ) small-cell morula, the periblast nuclei at the periphery of the blastodisc, age 1 day 21 h; (g) beginning of blastulation, age 2 days 2 h; (h) the middle of blastulation, age 3 days 3 h; (i, j) beginning of gastrulation, formation of the dense sector and the germ ring in the blastodisc, age 3 days 23 h; (k, l) appearance of the embryonic shield, age 4 days 9 h; (m) beginning of blastoderm epiboly, age 4 days 19 h; (n) end of gastrulation, blastoderm epiboly 30% of the yolk surface, age 5 days 10 h; (o) organogenesis, blastoderm epiboly 60% of the yolk surface, age 6 days 1 h; (p) ﬁrst somites, blastoderm epiboly 70% of the yolk surface, age 6 days 6 h; (q) eight somite pairs, formation of the yolk plug, age 6 days 11 h; (r) 28 somite pairs, Kupffer’s vesicle, age 8 days 13 h; (s) end of segmentation of the caudal part of the embryo, 50 myomeres, age 14 days 14 h; (t) appearance of pigment spots on the body of the embryo, age 15 days 5 h.

From fertilisation to the end of metamorphosis

217

motoricity leads to mixing of the perivitelline ﬂuid and better respiration of the blastoderm inside the egg envelope. Step III. Blastulation As the blastomere divisions continue, the ﬁrst cell differentiation becomes visible, and the embryo is called a blastula. The surface cells ﬂatten and become polygonal, forming an epithelial layer, the periderm. The contact between the deeper cells becomes weaker and spaces can be formed between them. However, the typical blastocoel found in sturgeons or lungﬁsh does not appear. The deep cells form short protrusions, called lobopodia, and show restricted movements relative to each other. At the blastula stages, the blastoderm begins to protrude downward into the yolk (Fig. 6.6g), and later the blastodisc has a spindle-like form (Fig. 6.6h). At the late blastula, both the upper and the lower blastoderm surfaces ﬂatten. The blastodisc diameter decreases to 0.6 mm (compared with 0.8–0.9 mm at the previous stage). The periblast zone around the blastodisc becomes narrower. This zone transforms the yolk nutrients used by the blastoderm cells. With each successive reduction in cell size with cleavage, the content of nuclear material (DNA) remains unchanged, and thus the ratio of DNA to cytoplasm increases. Step IV. Gastrulation Gastrulation is the process by which the initially uniform cells of the blastoderm separate into primary germ layers: the ectoderm (the outer layer) and the endoderm (the inner layer). Later, a third middle layer, the mesoderm, appears between the ectoderm and the endoderm. The ectoderm will develop into the epidermis and the nervous system, the endoderm into the alimentary canal and other digestive organs, and the mesoderm leads to the formation of muscles, the circulatory system and the sex organs. Gastrulation in teleost ﬁsh differs from that in other vertebrates (Ballard, 1973, 1982). Generally, gastrulation is associated with two types of cell movement: epiboly (the distribution of cells towards the vegetal pole of the egg) and axial latitudinal convergence to the anlage of the embryo (the embryonic shield). During gastrulation, the properties of the deep cells of the blastoderm change. The lobopodia transform into long protrusions, called philopodia, and the cells adhere to each other. In early gastrulation, some of the deep cells migrate to the blastodisc periphery, forming the embryonic shield and germ ring (Fig. 6.6i,j). In the germ ring, the deep cells of the blastoderm are separated into two layers: the epiblast adjacent to the internal surface of the periderm and the hypoblast lying on the periblast. The epiboly of the blastoderm begins from the migration of the periblast towards the vegetal pole of the egg. The marginal cells of the periderm become elongated and adhere to the periblast as it begins to migrate. During epiboly, the caudal end of the embryonic shield remains at the edge of the germ ring (Fig. 6.6m,n). At the end of epiboly, the margin of the spreading periderm forms the yolk plug (not homologus to blastopore in amphibians and sturgeons). Incubation temperatures can determine the developmental stages reached by the

218

Culture of cold-water marine ﬁsh

embryo at yolk plug closure. In many demersal eggs, the blastoderm epiboly terminates at more advanced stages of embryo development than in pelagic eggs. During epiboly, the cells of the hypoblast migrate towards the embryonic shield (axial convergence), and the cells of the epiblast migrate longitudinally with the periderm. The hypoblast cells are often separated into two layers: the lower layer (presumptive endoderm) composed of cells with intracellular spaces, and the upper layer (presumptive mesoderm) represented by several layers of tightly packed cells. When they reach the embryonic shield, the cells have predetermined locations in the ectoderm, mesoderm or endoderm. The embryo is covered by the ﬂattened peridermal cells. The determination of the cells occurs during blastulation, and the fate maps of various organ anlages are known for several ﬁsh species. Step V. Organogenesis Organogenesis starts with the formation of the notochord anlage. This anlage is seen as an inﬂation in the yolk. Depending on the ﬁsh species, organogenesis can begin before or after yolk plug closure. In cod, organogenesis starts when blastoderm epiboly reaches approximately 50% of the yolk surface and the embryo is clearly seen at 60% of the epiboly (Fig. 6.6o). The formation of optic vesicles, the neural keel and three brain vesicles (forebrain, midbrain and hindbrain) can be observed. The ﬁrst somites appear at 70% of blastoderm epiboly (Fig. 6.6p). Invagination of the optic vesicles forms when eight somite pairs appear in the embryo body (Fig. 6.6q). The yolk plug closes when 20 somite pairs are visible. After blastopore closure, the lenses of the eyes, the optic vesicles, the heart, the olfactory capsules, Kuppfer’s vesicle (in the caudal, ventral part of the trunk) and the ﬁrst melanophores on the dorsal surface of the head can be seen (Fig. 6.6r). Step VI. First Muscle Contractions of the Embryo In cod, weak movements of the caudal part of the embryo begin before the onset of heart contractions. The body length increases, and its segmentation reach the maximum of approximately 50 myomeres (Fig. 6.6s). The anlages of pectoral ﬁns, branchial arches and hatching glands (on the surface of the eyes and the head) can be seen at this stage. Towards the end of this period, melanophores on the embryo body are concentrated into several groups and the eyes become pigmented (Fig. 6.6t). The embryo sometimes moves inside the egg. Step VII. Preparation for Hatching and Hatching This period is characterised by intensive secretory activity of the hatching glands, which can be seen as many slightly granular circles in the head region long before hatching. The localisation of hatching glands differs between species. In cod, they are found in the head (Adoff, 1987), whereas in halibut they form a circular ring around the yolk sac (Helvik et al., 1991). These glands secrete an enzyme (chorionase) which weakens the egg envelope. A special hydrostatic organ, hydrosinus, is formed in the area from the head to the beginning of the dorsal part of the ﬁnfold. Guanine glossy pigment appears in the eyes. The swim-

From fertilisation to the end of metamorphosis

219

bladder, urinary bladder, liver and gallbladder can be seen. The embryo often rotates inside the egg. A high oxygen concentration in the water can lead to inhibited release of the chorionase, and to delayed hatching and even death of the embryo. A decreasing oxygen concentration stimulates secretion of the enzyme by means of both signals from the central nervous system and increasing embryonic movements. These motoric movements are important for the mixing of the perivitelline ﬂuid, which stimulates chorionase secretion. The destruction is observed in several areas of the egg envelope, and its strength decreases. The embryo hatches by energetic movements of the body. Normal hatching is associated with the appearance of the caudal part of the embryo from the egg envelope. During the hatching of abnormal embryos with restricted body movements, the head appears ﬁrst due to the local action of chorionase on the egg envelope in the area of the head region. Exposure to different environmental conditions may cause a large variation in the degree of development at hatching (even within a single egg batch). However, despite a high variability, the stage of hatching is species-speciﬁc, and the hatching event leads to a substantial change in the relation of the organism to its environment. Step VIII. The Endogenous Yolk-sac Period The embryo at hatching has a large yolk sac and hydrosinus in the dorsal pre-anal part of the body (Fig. 6.7a). There are 11–12 myomeres before the anus and 38–39 after the anus. The melanophores are distributed in several transverse rows on the body (including three rows in the caudal part) and on the dorsal surfaces of the midbrain and hindbrain. Several melanophores are seen on the swim-bladder and liver. The jaws are well developed, but the mouth is closed. The newly hatched yolk-sac larvae gather together near the surface of the water with the yolk sac uppermost. Sometimes they can move in a horizontal direction by means of undulations of the caudal part of the body. Phototaxis is absent. During the rapid absorption of the yolk, the ventral part of the head separates from the yolk-sac and the mouth opens (Fig. 6.7b). The ﬁrst blood cells begin to circulate in the main blood vessels of the body. There are no blood vessels on the surface of the yolk-sac. The embryos can move in different directions with the yolk-sac underneath, but they ﬂoat to the surface and rotate when they are still. The jaws of the embryo become mobile when the yolk-sac volume decreases to 60% of its volume at hatching, the eyes have some restricted movement. The swim-bladder ﬁlls with gas during the yolk-sac period. The volume of the hydrosinus increases, and yolk-sac larvae have a positive phototaxis. Their ventral part is always directed downwards, and they possess neutral buoyancy when they are still. Step IX. Mixed (Endogenous and Exogenous) Feeding The yolk-sac volume decreases to 70% between hatching and the ﬁrst exogenous feed uptake, and becomes narrow in the anterior area (Fig. 6.7c). The intestinal cavity is markedly expanded and the rectum is formed. The gall bladder is ﬁlled with gall and becomes green. The stomach and pyloric caecae are absent. Peristaltic waves can be seen along the gut. The

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Figure 6.7 Stages of the subsequent development of cod at 7.2°C (Makhotin et al., 1984, modiﬁed; drawings f and g are by S.G. Soin). (a) Embryo at hatching, TL (total length) of the embryo = 4.3 mm, age 19 days from egg activation; (b) beginning of blood circulation, TL = 4.6 mm, age 1 day 8 h from hatching; (c) transition to mixed (exogenous) feeding, TL = 4.8 mm, age 4 days; (d) movements of the pectoral ﬁns, TL = 5.2 mm, age 7 days; (e) transition to exclusively exogenous feeding, TL = 5.5 mm, age 10 days; (f ) formation of rays in the pectoral, caudal, anal and two dorsal ﬁns, TL = 6.7 mm; (g) formation of rays in all ﬁns except pelvic ﬁns, TL = 9.5 mm.

larvae are actively swimming and feeding. Movements of the pectoral ﬁns are registered when the yolk is almost completely resorbed (Fig. 6.7d). Nine paired primary neuromasts can be seen along the presumptive lateral line, but secondary neuromasts appear at later developmental steps. The mouth is open, with well-developed jaws. Step X. Exclusively Exogenous Feeding, Finfold Differentiation The yolk is totally resorbed (Fig. 6.7e). The hydrostatic organs of the embryo are represented by the hydrosinus, wide ﬁnfold and swim-bladder. The lower jaw is longer than the upper one. Diffuse yellow pigment appears in the body. The digestive tract still has a low degree of differentiation. Its separation into oesophagus, stomach, middle intestine and rectum is registered only during the juvenile period.

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Step XI. Development of Rays in the Fins The rays develop in all ﬁns with the exception of the ﬁrst dorsal ﬁn (Fig. 6.7f). The anlages of the pelvic ﬁns (below the pectoral ﬁns) can be seen. The melanophores are scattered on the body without marked spots. At 9.5 mm total length, the rays are formed in all ﬁns with the exception of pelvic ﬁns (Fig. 6.7g). The intestine is covered by an intensive internal black pigment.

6.4.2 Wolf-ﬁsh (Anarhichas lupus) Mature oocytes of White Sea common wolf-ﬁsh from a wild population, 5.0–5.7 mm (average 5.3 mm) in diameter, have a light yellow colour. The lipid droplets, up to 0.4 mm in diameter, are concentrated mainly in the upper area of the yolk and possess more intensive coloration. These droplets can move freely in the yolk. The egg envelope includes the zona radiata covered by a thin layer (zona pellucida). The eggs are inseminated and fertilised internally after copulation between the spawners, and they are released into the water 8–15 h before the ﬁrst cleavage. The swelling of the egg envelopes in the water is accompanied by stickiness, and the eggs form a ball-like clutch after the female has coiled around them for several hours. The eggs adhere to each other, but do not stick to any substratum. They develop in holes, and are protected by the male during their whole embryonic development. In general, the ﬁrst steps of embryonic development in common wolf-ﬁsh are similar to those in cod (Fig. 6.8a–c). The perivitelline space is very narrow at the beginning of embryonic development, but it increases during the process of yolk absorption. Organogenesis begins when blastoderm epiboly reaches 50% of the yolk surface (Fig. 6.8d). Yolk plug closure is observed when three somite pairs appear in the embryo (2.3 mm in length). The lipid droplets begin to fuse into larger ones when the number of somite pairs has increased to 19–21 (Fig. 6.8e). The embryo is located mainly in the lower part of the egg. A large lipid droplet can be seen at step VI (the beginning of muscle contractions in the embryo, and the formation of blood cells) (Fig 6.8f). At this step, the circulation of blood plasma without blood cells can be observed in the vessels of the embryo and in a vessel (the hepatic vitelline vein) located to the left of the embryo. At step VII (the appearance of a large number of blood cells), the right part of the hepatic vitelline vein is formed, and the zone of yolk-sac vascularisation increases (Fig. 6.8g). The last step of embryonic development (VIII, preparing to hatch and hatching) is characterised by intensive movements of the pectoral ﬁns, jaws and body of the embryo. The vascularisation of the yolk sac is almost totally complete (Figs. 6.8h, 6.9a,b). At later stages, ﬁn rays appear in all ﬁns (with the exception of the pelvic ﬁns, which are absent and represented only by skeletal rudiments), and the intensity of body pigmentation increases (Fig. 6.9c–f). Well-developed segmental blood vessels continue into the dorsal and anal ﬁns forming a complex respiration net. In addition, a special respiration organ, the pseudobranch, is developed behind each eye. The eggs become opaque several days before hatching. The embryos hatch at a total length (TL) of between 19 and 24 mm, with the yolk sac almost totally resorbed and many juvenile characters (Fig. 6.9g). They are pelagic and show

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Figure 6.8 Stages of the initial embryonic development of common wolf-ﬁsh at 4.9°C. Scale bar = 1 mm (Pavlov, 1986). (a) Formation of the perivitelline space and blastodisc, age 8 h; (b) early gastrulation, formation of the dense sector in the blastodisc, blastoderm diameter 3.4–3.6 mm, age 10 days; (c) gastrulation, formation of the embryonic shield, blastoderm epiboly 30% of the yolk surface, blastoderm diameter 3.8–4.3 mm, age 12 days; (d) early organogenesis, appearance of the neural keel, blastoderm epiboly 50% of the yolk surface, age 14 days; (e) 20 somite pairs in the embryo body, formation of the gill cover, fusion of the lipid droplets, age 23 days; (f ) 26 myomeres before the anus, 32 myomeres after the anus, heart pulsation, beginning of blood circulation, formation of a large lipid droplet, TL = 4 mm, age 36 days; (g) 26 myomeres before the anus, 49 myomeres after the anus, beginning of blood ﬂow in the right part of the hepatic vitelline vein, TL = 9 mm, age 57 days; (h) beginning of the jaw and pectoral ﬁn movements, diameter of the zone without blood vessels on the surface of the yolk sac = 2 mm, TL = 11 mm, age 84 days. bl, blastodisc; dp, pit-like depression in the yolk; ds, dense sector of the blastodisc; es, embryo shield; fs, ﬁbrous structure; pz, periderm zone; sd, small lipid droplets.

a positive phototaxis. The transition to mixed (exogenous) feeding is observed just after hatching. The juvenile period begins at approximately 32 mm TL and coincides with the transition to a mainly demersal mode of life. Ossiﬁcation of the majority of the skeletal elements is complete in specimens of this size. Individuals of a larger size have a body shape and colouration which are similar to those in adult ﬁsh (Fig. 6.9h). Thus, cod are characterised by a typical form of indirect development, with a pronounced metamorphosis during the transition from larvae with a low degree of morphologic development to juveniles. In wolf-ﬁsh, which have a transitory type of early ontogeny, the larvae possess a much higher degree of morphologic development and metamorphosis is almost absent. Owing to a prolonged period of development inside the egg envelope, the sub-period

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Figure 6.9 Stages of the subsequent development of common wolf-ﬁsh at 4.9°C for 83 days and 7.8°C afterwards. The embryos (a–f ) have been removed from the egg envelopes (Pavlov, 1986; modiﬁed). (a, b) The stage shown in Fig. 6.8h; (c) formation of ﬁn rays in the pectoral ﬁns and caudal ﬁn, gill ﬁlaments on the branchial arches, beginning of continuous movements of the pectoral ﬁns, TL = 14 mm, age 93 days; (d) formation of teeth on the jaws and rays in the dorsal and anal ﬁns, TL = 17 mm, age 100 days; (e) appearance of pigment spots on the body of the embryo, continuous movements of the jaws, TL = 17.8 mm, age 107 days; (f ) formation of rays in the dorsal part of the caudal ﬁn, TL = 18.3 mm, age 113 days; (g) larva at hatching, transition to mixed feeding, TL = 21 mm, age 135 days; (h) juvenile, appearance of pigment spots at the bases of the dorsal and anal ﬁns, TL = 45 mm, age 196 days. ps, pseudobranch.

of development outside the egg envelope (free-embryo phase or yolk-sac period) in this species is absent. In general, wolf-ﬁsh are much more protected from the external environment than cod. However, this protection has required several adaptations directed mainly towards solving the respiration problem of the embryo developing inside the egg envelope, including a complex blood circulation system and functioning pectoral ﬁns and branchial apparatus.

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6.4.3 Embryo Growth and Yolk Absorption Yolk is the major source of energy and the material for body formation in developing embryos of oviparous species. At the initial developmental stages, the transport of nutritional substances from the yolk to the blastodisc occurs by means of cytoplasmic threads. The periblast has a major role in the trophic function of the embryo, and this function continues until the yolk has been totally absorbed. In many ﬁsh species, the blood system at the surface of the yolk sac is used for the transportation of nutrients from the yolk to the embryo. Yolk is absorbed by the phagocytic activity of the inner part of the periblast (vitellolysis zone), where it is degraded into substances with a low molecular weight and transported into the blood. The initial growth of the embryo mass (without the yolk) and yolk absorption can be described by an exponential curve and a linear equation, respectively (Novikov, 2000). Thus, the comparative independence of the regulation processes governing embryo growth and yolk absorption can be expected. In cod, at the steps of cleavage and blastulation, the protein content in the blastoderm remains at a low and constant level, and begins to increase from mid-gastrulation. In salmonids, the beginning of protein growth is registered from organogenesis (Novikov, 2000). The growth rate of the embryo decreases until the beginning of exogenous feeding, and a negative growth (due to tissue absorption) can be observed in unfed larvae. In some species (e.g. wolf-ﬁsh), the rate of yolk absorption is more or less constant over the entire interval of development using yolk reserves. In other species, such as Atlantic salmon, yolk is consumed mainly after hatching, which coincides with an abrupt increase in breathing intensity and the energetic demands of the embryo. Embryo growth and yolk absorption rates increase at higher temperatures within the range which is suitable for normal development. However, the effect of temperature on the embryo size and the amount of yolk at identical developmental stages can be especially important for aquaculturists. An increase followed by a slowing down of embryo growth rate in eggs incubated at higher temperatures is often registered at the steps of gastrulation and organogenesis (e.g. cod), or at later steps (e.g. salmonids). During the second half of embryonic development, embryos at the same developmental stage at lower temperatures are larger and have less yolk than those at higher temperatures. In embryos of rainbow trout, the difference in embryo size between ‘cold’ and ‘warm’ groups can reach 10% by length and 50–100% by protein content (Novikov, 2000). At the same time, a difference in embryo size at hatching could be the result of a different relationship between embryo growth and yolk absorption at different temperatures, or because of the transition of the hatching event to a later developmental stage at lower temperatures. As a result of the ﬁrst reason, larvae from ‘cold’ groups are often larger at the stage of total yolk absorption and at later stages than those from ‘warm’ groups, and this may be an advantage in their subsequent growth and survival. However, the incubation of eggs at temperatures below a certain threshold can lead to a slowing down of embryo growth rate. This change in the correlation between the rates of embryo growth and yolk absorption at different temperatures does not occur in common wolf-ﬁsh: embryo size and yolk content at identical developmental stages remain the same regardless of incubation temperature (Pavlov & Moksness, 1995).

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6.5 From Hatching to Metamorphosis Embryos can hatch at various sizes and degrees of development, depending mostly on the size of the yolk (see Fig. 1.2, Chapter 1). As stated previously, newly hatched yolk-sac larvae from demersal eggs are generally more advanced in development than larvae from pelagic eggs of a comparable size. In species with large eggs, hatching may be delayed until the yolk sac is absorbed and the larvae are ready to feed at hatching. Larvae from pelagic marine eggs with small yolks are thus less well developed at hatching than larvae hatching from eggs with larger yolks. Newly hatched pelagic larvae generally lack a functional mouth, eye pigments, digestive system and differentiated ﬁns. Yolk-sac larvae (free embryos) rely on their yolk supplies until their sensory, circulatory, muscular and digestive systems develop to such a degree that they are able to capture food. They are either transparent or lightly pigmented, and they possess species-speciﬁc characters (pigment patterns, body shape and size) which are useful for identiﬁcation in ﬁeld investigations and for systematic studies.

6.5.1 To Be a Larva . . . Direct or indirect development makes a difference to when and how larvae will change to the adult form (Balon, 1985, 1990). A larva is a transitory form of life, which often inhabits an entirely different niche than the adult form. Larvae may have a different body shape from the adults, and they are characterised by temporary larval organs and tissues such as an unpaired ﬁnfold with respiratory blood vessels and a dorsal sinus. Spines and ﬁlamentous appendages to give buoyancy may be present. The process of metamorphosis, or remodelling of the organism, is often connected with substantial changes in the larval morphology owing to the transition to a new environment. For example, the transition from pelagic to demersal life in ﬂatﬁsh is associated with a ﬂattening of the body and the migration of one eye to the opposite side of the head, which becomes the upper side of the body. The body shape of the European eel (Anguilla anguilla) changes from leaf-like to eel-like during a prolonged migration from the Sargasso sea to the coast of Europe. These are typical examples of indirect development from larva to juvenile. Thus, a ﬁsh larva may be deﬁned as a free-living, non-reproductive post-embryonic stage that is markedly different in form from the adult, and which undergoes metamorphosis into the adult form. Many of the species currently farmed have indirect developmental patterns (all ﬂatﬁsh and cod) which are associated with high fecundity, small eggs, larval stages, prolonged metamorphosis and high mortality. One probably important reason why many marine ﬁsh species have numerous small eggs, resulting in small, vulnerable larval stages, is their ability to make use of the rich planktonic habitat. The open sea is an excellent place for dispersal of the offspring, and the larvae are literally hatched into a very nutrient-rich ‘soup’ of small zooplankton prey organisms. The larval characteristics change during development, and the transformation to the juvenile stage may be gradual or abrupt. The latter is often the case when they move from pelagic to demersal habitats.

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6.5.2 . . . or Not to Be a Larva Not all ﬁsh pass through a real larval stage. Ovoviviparous and viviparous species, and many species with large eggs, a large yolk/blastodisc and/or parental care may omit the larval stage completely. In oviparous species, the embryo may hatch at an advanced stage of development, and transit to exogenous feeding as a juvenile after a short period of time. Thus, the direct ontogeny is characterised by the absence of metamorphosis, and allows development without using energy to produce transient larval tissues. In wolf-ﬁsh, the onset of larval exogenous feeding coincides with hatching, and their ontogeny is regarded as direct by some and intermediate by others. In these species, the period between the start of exogenous feeding and the loss of larval characters (e.g. the yolk sac) is a very short interval of development. The intermediate mode of early ontogeny is a transitory type. At the transition to exogenous feeding, the organism possesses many juvenile characters, and as a result, the metamorphosis is not pronounced and the period to the loss of larval characters is comparatively short. For example, in salmonids the organism has both larval and juvenile characters in the period from the beginning of exogenous feeding to when it reaches the juvenile state, and this period is called the ‘alevin’ period (Balon, 1980, 1985). Salmonids can be said to go through two metamorphoses, one at the end of the larval stage and one at smoltiﬁcation (note that smoltiﬁcation is reversible but metamorphosis is not). The alevin period is analogous to a short larval period, and the distinction between these two terms is very slight, if it exists at all. At the start of exogenous feeding, salmonids and wolf-ﬁsh have a clear skin pigmentation, possess functional gills and ﬁns, and can be fed commercial pelleted diets from startfeeding because their digestive system is as well developed as in juveniles. The major difference between early ontogeny in salmonids and wolf-ﬁsh is that salmonids have a prolonged yolk-sac larval phase, which is absent in wolf-ﬁsh because they develop for a longer period in the egg, hatch at an advanced developmental stage and begin to feed externally just after hatching (Fig. 6.10). In viviparous ﬁsh, the young organism may be born as a juvenile. For example, the eelpout (Zoarces viviparus L.) and the redﬁsh (Sebastes sp.) possess juvenile characters and transit to exogenous feeding just after parturition. Despite the presence of a small yolk sac, these organisms can be deﬁned as juveniles. Species with direct ontogeny generally have low fecundity, with a low mortality in their large offspring, and they hatch at advanced stages of development. Thus, the transition from indirect to direct ontogeny represents a gradient to a more specialised type of development with a shortening of the most vulnerable larval period.

6.5.3 The Yolk-Sac Period—Preparation for ‘Real Life’ During the yolk-sac stage, the organism must develop in order to be able to eat, grow and survive. When yolk-sac reserves are depleted, the larva must be able to catch prey and avoid predators, and to digest the ingested food. To become a functional ﬁrst-feeding larva, several larval qualities must develop and become functional before yolk nutrients are depleted (e.g. Blaxter, 1988). Their visual acuity, activity level, swimming pattern, muscular development,

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Figure 6.10 Comparative development of Atlantic salmon (1) and common wolf-ﬁsh (2) at 10°C. A, formation of blastodisc (1.5 days); B, start of ﬁnfold formation (40 days); C, formation of dorsal, anal and caudal ﬁn rays (60 days); D, ﬁrst feeding (salmon 90 days, wolf-ﬁsh at hatching 100 days); E, start of juvenile period (102 and 110 days, respectively). Drawing from Pavlov and Moksness, 1994.

jaw development and digestive capacity must all be correctly in place in order to catch food and survive. At the time for ﬁrst feeding, ﬁsh larvae therefore have well-developed heads, brains, eyes and jaws, which are all essential features in order to survive and grow. The body is slender, with somatic muscles around the notochord. Pelagic ﬁsh larvae have no differen-

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Table 6.4 Organs that are present/not present in pelagic marine ﬁsh larvae such as cod, turbot and Atlantic halibut at the start of exogenous feeding. Present

Not present

Gut (no stomach) Liver and pancreas Large eyes and brain Well-developed olfactory and taste senses Neuromasts Finbuds Median ﬁnfold and subcutaneous space

Stomach (i.e. less efﬁcient protein digestion) Calciﬁed skeleton

Lateral line Functional gills Real ﬁns and ﬁn rays Skin and scales

tiated skin, but a median ﬁnfold ﬁlled with ﬂuid surrounds the body and yolk sac. Its function is probably both for buoyancy and for regulatory purposes. The gut is more or less a straight tube, which is connected to the liver and a discrete pancreas. A summary of the organs that are present/not present in pelagic larvae is shown in Table 6.4. A characteristic feature of larval development is allometric growth, which means that the growth rate of one body part or tissue may differ from the growth rate of the whole organism (Osse et al., 1997). Developing larvae have to use their limited energy resources economically, and allometric growth ensures that growth is concentrated in those tissues and functions that are most important for the survival of the larva. Allometric growth is typical of larval stages, whereas older ﬁsh grow isometrically (i.e. all body parts have similar growth rates). For a ﬁsh larva, it is important to grow as quickly as possible, as a larger size generally increases its survival potential. A larger larva will be able to catch larger prey (more energy in one bite), and it will swim faster and thereby be more succesful in hunting prey and avoiding predators. Swimming capacity is thus crucial for larval survival, and the early development of swimming organs must therefore have a high priority. Systems for feeding and swimming should develop simultaneously and in balance with each other. Recent experiments clearly demonstrate that the allometric growth and function of organs and structures suggests a functionally optimised growth that matches the expected necessary priorities for larval survival and growth. Thus, ﬁsh larvae apparently spend their available energy on the most important functions for survival.

6.5.4 Metamorphosis Metamorphosis means transformation, and has been used to describe rapid morphological and physiological changes following a stable period of slow development. In ﬁsh, as in frogs, metamorphosis marks the transition from larval to adult form. This functional transition affects almost every system from internal physiology and neurology to external phenotype, but not the reproductive structures (even though these may be indirectly affected by external conditions during this phase). When does metamorphosis start? The literature shows various answers, not all of which are deﬁned or compatible:

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• when larval allometric growth changes to isometric growth, indicating that this is a species-speciﬁc application; • when the primordial ﬁnfold, or the larval median ﬁnfold, is absorbed and median ﬁns are visible (species-speciﬁc); • when axial skeletal vertebrae develop, which requires histological examination or clearing and staining for degree of ossiﬁcation (species-speciﬁc); • when endocrine levels are driven by the larva´s own neuroendocrine stimuli, rather than reﬂecting hormones deposited by the female ﬁsh (species-speciﬁc).

There is general agreement about what metamorphosis involves, despite the lack of concensus on speciﬁcs. In general, it involves changes in the non-reproductive structures between the period of embryonic growth and sexual maturation, excluding embryonic development, growth, sexual maturation and ageing. This may include such features as the resorption of the median ﬁnfold, the development of the axial skeleton, the formation of a proper stomach, changes in neurology, vision and behaviour, changes in dermal structure and pigmentation, changes in the basis of respiration and osmoregulation, and the development of specialised muscles. These changes are not necessarily rapid, and are usually triggered by an external or internal cue. They also imply that during metamorphosis the ﬁsh occupies a different ecological niche from the preceding or adult stages. By convention, metamorphosis is now divided into at least four ‘stages’. (1) Pre-metamorphosis: the larval stage before changes in muscle and head structure are visible, and close to ﬁrst feeding. (2) Prometamorphosis: when allometric growth of the body commences and some structural changes can be seen. (3) Climax metamorphosis: when there is strong allometric growth of the body, and structural changes are well underway. (4) Post-climax metamorphosis: when the early juvenile form has been attained. This terminology will be used throughout the rest of this chapter. The process of metamorphosis is best understood by examining the changes in speciﬁc systems, and then looking at triggers that may stimulate, control or modify the course of change. In the following sections we will look at the individual larval organ systems and how each one changes.

6.6 Functional Development of Organ Systems from Hatching to Metamorphosis At the time of mouth opening, the ﬁsh larvae will soon deplete the relatively small supply of energy from the yolk sac, and must learn to catch food from their surroundings. Larvae from larger eggs have more time and energy available before irreversible starvation occurs (the so-called point of no return, PNR). If larvae are starving, or if the nutritional quality of the food is inadequate, the larvae may quickly reach PNR, be unable to ingest and digest

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prey, and die. In cod, the PNR appears to be just before 9 days after hatching, and exogenous feeding usually starts 4–5 days after hatching (Kjørsvik et al., 1991).

6.6.1 Sensory System Functional sense organs such as optical receptors, mechanoreceptors and chemoreceptors are vital for larval detection of prey. The sensory organs of pelagic larvae are incomplete at hatching, and the timing of their development may differ from species to species. This section is divided into vision and the oculovestibular system, chemosensory systems such as the nares, and the lateral lines. The eyes of ﬁsh with direct, indirect or intermediate development differ with respect to the timing of the formation of the photoreceptors (rods and cones), although there are many species-speciﬁc differences. The formation of visual cells is also related to the optical environment which the larvae and juveniles inhabit. In general, the pigmentation of the previously colourless eyes occurs simultaneously with ﬁrst feeding. This is actually the development of the pigment epithelium, which is a layer of dark pigment that can expand over the visual cells (cones), reacts to light intensity, and signiﬁcantly increases the conemediated acuity (sharpness of vision) during bright light (Evans & Fernald, 1990). In general, marine ﬁsh larvae inhabiting the pelagic are visual predators. They have retinas comprising cones that require high light intensities for vision and see in colour. The rest of the retina is not fully differentiated prior to metamorphosis, but visual images are nonetheless translated into neural impulses. Most ﬁsh larvae seem to have a pure cone retina at ﬁrst feeding, which may explain why so many teleost larvae feed at relatively high light levels. It is not known to what extent meso- and bathypelagic larvae have a pure cone retina, but this is the case for halibut, whose larvae are assumed to ascend from the mesopelagic to the epipelagic zone prior to metamorphosis (Fig. 6.11). Before metamorphosis the lens may be directly on the retina, but as the ﬁsh grows the lens separates. The ﬁeld of focus may be short and fairly ﬁxed, which affects the distance at which prey can be perceived. 6.6.1.1 Vision and the Oculovestibular System Fish regulate the amount of light entering the retina (not the eye, as mammals do) by extending or contracting the pigment epithelium over the tips of the cones and rods. The rods and cones themselves can extend or contract according to the light conditions. This is called retinomotor action, since the cells in the retina move relative to each other. Fish have another distinction from other visual systems in that they focus by moving the lens with small muscles (retractor lentis), not by changing the shape of the eye. The ecology of marine ﬁsh dictates the form and function of the eye. All elements of the eye and retina, including physical placement, photoreceptor speciﬁcity, biochemistry and physiology, can be affected by a change in the environment and the amount of illumination. As the ﬁsh moves from the epipelagic to deeper in the water column (which is generally the case), the eyes become larger in the need to capture the ever-decreasing amounts of light, which itself becomes more monochromatic (goes towards one colour; in the ocean, this colour is usually blue).

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Figure 6.11 Development of the halibut (Hippoglossus hippoglossus) retina from hatching through to metamorphosis. Initially, the neuroblastic cells are in contact with the lens. The pigment epithelium and the retinal layers develop at around 150 day-degrees. Lens separation, the cone mosaic, rod recruitment and the retractor lentis muscle appear at around metamorphosis (Kvenseth et al., 1996).

In prometamorphosis, in addition to the cones, rods begin to be recruited to the retina (the ﬁsh can see at lower light levels). Coincident with rod neurogenesis, phototaxy changes. A larva which previously avoided light will be drawn to it, while a larva which was positively phototaxic may now prefer lower light levels. It is thus possible that rods signal a physical migration of the ﬁsh from one area to another. In freshwater ﬁsh, rods generally appear before hatching, whereas in salmon, rods occur at ‘swim-up’ just prior to exogenous feeding (a good example of such a shift in phototaxy). Other muscles in addition to the retractor lentis are added, meaning that the eye has a greater range of movement and a wider ﬁeld of view. The distance at which prey may be detected also extends. The section of the visual ﬁeld in which most prey are taken may change with both the physical movement of the eyes relative to the cranium (migrating more anteriorly, or slowly migrating from one side as in ﬂatﬁsh) and the number of photoreceptors. The photoreceptors each contain a pigment which is photoreactive. In some species, these visual pigments may change during metamorphosis, and the change can be induced by thyroxin. In salmonids, there is a transient UV-sensitive cone associated with smoltiﬁcation.

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At climax metamorphosis, the cones reorganise and a mosaic is formed in a regular pattern of single and double cones, and sometimes accessory cones. The retina is fully formed, and further growth involves stretching the retinal surface and some recruitment of new cells. As the ﬁsh continues to grow, the distance between the lens and the retina extends, and is compensated for by the growth of the lens. Flatﬁsh metamorphosis involves the spectacular migration of one eye to the other side of the head, leaving the ﬁsh with an ocular and an abocular side. This migration of the eye necessitates a major reorganisation of the vestibulo-ocular pathways (nerves going from the eyes to the otolith labyrinths to the brain, which are meant to indicate which way is up, among other things). The optic nerve of the migrating eye begins to coil and elongate prior to eye migration. The otolith labyrinth nerves are recoupled with both the muscles around the eye and the optic tectum. In ﬂatﬁsh, even though the eye migrates, the corresponding vestibular system (otolith and labyrinth) does not. During this metamorphic change there is a substantial increase in the number of neurons projecting from the vestibular nerve to the vestibular neural complex in the brain stem, and many more of the vestibulo-ocular neurons project bilaterally to the rostral eye motor nuclei after metamorphosis in turbot (Jansen & Enger, 1996). The otoliths remain on either side of the head, but the neural reorganisation means that the ﬁsh has changed its neural deﬁnition of the physical meaning of ‘up’. There may be a permanent change in the optic tectum. The period of eye migration may be shorter than the period of metamorphosis, and is variable. 6.6.1.2 Chemosensory System Many larvae from demersal eggs have olfactory receptors at hatching, whereas in many pelagic larvae the olfactory organ (nares, or olfactory plates) usually develops shortly after hatching. The nares are initially ﬂat structures on the surface of the larva, with exposed chemosensory cells. These perceive impulses (detect amino acids) from any direction. In addition, pear-shaped taste buds in the mouth and pharyngeal region, as well as elsewhere, appear early in larval development. During development, they increase in number and size. The larvae are probably adapted to a certain taste and palatability of food, and these requirements have to be met in order to improve the acceptance of formulated diets. As the larva becomes prometamorphic, the nares (olfactory plates) descend and skin ﬂaps rise laterally. These ﬂaps fuse at around climax metamorphosis and form a tunnel over the chemosensory cells, which thus detect amino acids and other molecules from the direction of the water ﬂow entering the channel. It is possible that a change in chemoreception occurs during metamorphosis as different cells recruit and specialise. 6.6.1.3 Lateral Line Larval marine ﬁsh generally have numerous, relatively large, free neuromasts. Initially these neuromasts are single, and are mostly concentrated around the eye and olfactory cup, with some along the body and the head. These gradually increase in number and aggregation, with more external free neuromasts developing along the body axis prior to the development of the familiar lateral line of juvenile ﬁsh. These external neuromasts comprise small sensory

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hair cells at the base covered by a large gelatinous cupula that extends outward. The neuromasts perceive currents or vibrations in almost any direction. The number and complexity of neuromast assemblages increases with larval age, going from single to doublets to triplets in juveniles. Neuromasts also become encased in the developing lateral line canals at around metamorphosis. The canals on the head close over the exposed neuromasts ﬁrst (at around climax metamorphosis), followed by the gradual closure of the trunk (body) canal. While the density of neuromasts decreases, the directional speciﬁcity of their sensory signal increases owing to their enclosure in a canal aligned along the body. In ﬂatﬁsh, the position and number of neuromasts changes according to whether they are on the ocular or the abocular side. The increase in the directional speciﬁcity of the response accompanies the development of a species-speciﬁc behaviour pattern (Poling & Fuiman, 1997).

6.6.2 Digestive System By the end of the yolk-sac period, larvae must obtain exogenous food in order to have energy for the synthesis of new tissue (growth) and for their maintenance metabolism. This includes capture, ingestion and digestion of the prey, and absorption and assimilation of the nutrients. In aquaculture, ﬁnding the larval nutritional requirements for optimal growth is also imperative. The dietary needs of developing larvae are different from those of the adult ﬁsh, and therefore larval nutrition should always be considered according to the ontogenetic development of their digestive systems, their nutritional requirements and their behaviour. It is possible to distinguish three types of larva based on the development of their digestive functions (Govoni et al., 1986). (1) (2) (3)

A functional stomach is developed before they start feeding. No stomach develops during the larval stage, but it is developed at a later stage. No stomach is ever developed.

Salmonids and the wolf-ﬁsh are typical examples of ﬁsh that possess a functional stomach at the time of ﬁrst feeding. Both salmonids and wolf-ﬁsh can be fed formulated diets from the ﬁrst feed uptake, owing to efﬁcient protein digestion in their stomach. The pelagic marine larvae of cod, turbot and halibut are typical of larvae that develop their stomach later, and most larvae in this group gain a functional stomach during metamorphosis. Fish that have no stomach throughout their whole life are generally herbivores, often with a very long gut compared with species with functional stomachs. Their diet consists mainly of carbohydrates, which are less digestible and consequently need a longer gut passage time. The lack of a functional stomach is one of the main reasons why start-feeding has been a bottleneck in marine ﬁsh larval rearing, and why we have had problems in developing formulated feeds that are suitable for these pelagic marine larvae. The digestive capacity of pelagic marine ﬁsh larvae has therefore been classiﬁed as ‘undeveloped’, ‘simple’ or ‘primitive’, and some theories have maintained that these ﬁsh larvae are dependent on exogenous digestive enzymes from their prey organisms.

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Culture of cold-water marine ﬁsh

However, the natural diet of marine pelagic larvae consists of zooplankton and phytoplankton, and the larvae are already raptorial carnivores from the end of the yolk-sac stage. More recent studies have shown that these larvae do indeed develop a digestive apparatus that is very well adapted and specialised to their life-style and feeding patterns. 6.6.2.1 Gut, Pancreas and Liver Differentiation For animals in general, the enzymatic digestion of food occurs in the lumen of the stomach and the intestine. This is a complex chemical process, where several digestive enzymes catalyse the breakdown of large food molecules into simpler compounds (nutrients) that can be absorbed through the gut epithelial cells. The stomach contents are acidic, and the intestinal lumen is slightly alkaline. The enzymes have different optimal pH values according to their site of action in the digestive system. Digestive enzymes are secreted from the gastric glands in the stomach, from the exocrine pancreas, where the enzymes are secreted to the gut lumen through the pancreatic duct, or from the intestinal epithelial cells. At hatching, the digestive tract of pelagic marine larvae is a closed straight tube (the mouth and anus are not yet formed), and it is histologically undifferentiated throughout its length. The mouth and anus will form during the yolk-sac phase, and the gut will lengthen and differentiate into a buccopharynx, fore-gut, mid-gut and hind-gut (Figs. 6.12 and 6.13).

M N Sw E Mg

L

Hg

Fg

Figure 6.12 Gut differentiation in cod larvae. The drawing shows a cod larva at the onset of ﬁrst feeding. The lower picture shows a longitudinal histological section through the digestive tract of a 17-day-old cod larva. Drawing from Galloway et al., 1999a; the histological section was made by E. Kjørsvik. L, liver; Fg, foregut; Mg, midgut; Hg, hindgut; E, oesophagus; Sw, swim-bladder; N, notochord; M, muscle.

From fertilisation to the end of metamorphosis

Yolk sac larva

Final yolk absorption

Larva

Transformation

235

Juveniles & adults Oesophagus

Foregut Incipient gut

Stomach

Midgut

Anterior intestine

Hindgut

Posterior intestine

Figure 6.13 Development of the alimentary canal in larval ﬁsh. At hatching, the gut is an undifferentiated straight tube which is closed at mouth and anus. The liver and pancreas are formed at hatching, and become functional at the time of exogenous feeding. The yolk is absorbed through the yolk syncytium, which is connected to the liver. The gut is differentiated into three distinct regions by the start of feeding. During larval development, the gut length and thickness increase, and the stomach is formed as an expansion from the fore-gut. From Govoni et al., 1986.

During the ﬁrst feeding period, the gut will increase in length and width, thus considerably increasing its absorptive surface. The gut wall (mucosae) is a thin layer of smooth muscle, connective tissue and squamous epithelium, lined with numerous absorptive microvilli towards the gut lumen. The gut epithelial cells are called enterocytes. A small expansion may appear posterior to the oesophagus which is called a ‘post-oesophageal swelling’, and this is where the stomach will later develop. There are no digestive gland structures in the larval digestive system, and thus no pepsinogen is present (i.e. a digestive enzyme secreted in the stomach for breaking down complex proteins). The gut mucosa is very dynamic tissue which is involved in the hormonal and nervous activation of enzyme and bile synthesis and their subsequent secretion from the pancreas and liver. It is the main site of the digestion and absorption of nutrients (Fig. 6.14), and the epithelial cells of the gut are renewed more often than cells in other tissues. Ingested food particles are transported through the digestive tract by peristaltic movements in the gut smooth muscle. Even though the larval digestive system is less elaborate than that in adult ﬁsh, the functions and tissues which are essential for nutrient digestion, absorption and metabolism are developed at the beginning of exogenous feeding. The gut will continue to increase in volume and length throughout the whole larval phase, the intestinal folds become more obvious, and gut passage rates will increase. The liver, with its bile system, and the pancreas develop during late embryogenesis and the yolk-sac stage, and these organs are functional before mouth opening and ﬁrst feeding. The liver rises from the periblast, with a clear connection to the gall bladder and to the yolk. Unlike in most adult ﬁsh, the larval pancreas is most commonly described as a distinct organ, which will develop to a diffuse tissue towards the end of the larval phase or during the juvenile phase. This is the case for cod (Kjørsvik et al., 1991; Morrison, 1993), halibut (Kjørsvik & Reiersen, 1992), turbot (Segner et al., 1994) and common wolf-ﬁsh (Falk-Petersen & Hansen, 2001). The general sequence of digestive system development is shown in Fig. 6.15 for the Japanese ﬂounder (Paralichthys olivaceus). The developmental sequence of cell differentiation, like the synthesis of enzymes, appears to be genetically programmed at ﬁrst, while a

236

Culture of cold-water marine ﬁsh

Hindgut

Midgut

Proteins

Foregut

Lipids

Absorption Ingestion PI Extinction

Pv Transport

Digestion

Resynthesis

Accumulation LI

Accumulation GA

Transport

Ly N

a

GA

N

b

Figure 6.14 (a) Histological section of a turbot larva, showing mid-gut epithelium with lipid vacuoles (E. Kjørsvik and T. Bardal, Department of Biology, NTNU, unpublished data, 2002). (b) Schematic drawing of enterocytes in the different parts of the larval gut. Lipids are mostly absorbed in the mid-gut epithelial cells (enterocytes), and macromolecules from proteins are taken up by pinocytosis in the hind-gut epithelial cells. From Govoni et al., 1986.

dietary inﬂuence has been detected on both the organ structure and the enzyme levels once feeding has started. 6.6.2.2 Digestive Enzymes The pancreatic cells start to synthesise enzymes well before the onset of exogenous feeding, and this serves as conditioning for the ﬁrst feed intake (Hjelmeland et al., 1984; HoehneReitan et al., 2001a). All the digestive enzymes (except the stomach enzymes) seem to be present in pelagic marine larvae at the onset of ﬁrst feeding (Hoehne-Reitan & Kjørsvik, in press). These include pancreatic enzymes such as lipases, trypsin, chymotrypsin and amylase, which are responsible for the luminal digestion of food macromolecules, and enterocytic brush-border bound enzymes such as aminopeptidase, maltase and alkaline phosphatase, which complete the breakdown into absorbable monomeres. The digestive enzymes and the activities detected during ﬁrst feeding in larval turbot are shown in Fig. 6.16. Pancreatic and intestinal enzyme activities are generally low at the onset of ﬁrst feeding, but will increase exponentially after exogenous food intake has been established if the quality and quantity of food is satisfactory (Hoehne-Reitan et al., 2001a; Hoehne-Reitan & Kjørsvik, in press). Some of the most important larval digestive enzymes are proteolytic trypsin-like enzymes and lipolytic enzymes such as lipases. These are produced in the exocrine pancreas and are secreted to the gut, where they become active in the alkaline environment produced by the

From fertilisation to the end of metamorphosis

237

Figure 6.15 Schematic development of the diffuse pancreas in Japanese ﬂounder, Paralichthys olivaceus. Shaded areas indicate the distribution of the pancreatic tissue from a distinct to a diffuse organ. (a) 3 dph (days post-hatch); (b) 10 dph; (c) 20 dph; (d) 30 dph; (e) 45 dph (= metamorphosed). bd, bile duct; es, oesophagus; gb, gall bladder; hd, hepatic duct; in, intestine; li, liver; pa, pancreas; ph, porta hepatis; py, pyloric appendages; re, rectum; st, stomach (from Kurokawa and Suzuki, 1996). Reprinted with permission from Elsevier Science.

Trypsin Aminopeptidase Alkaline phosphatase Maltase Esterase Carboxypeptidase A, B Acid phosphatase Amylase Phosphodiesterase Lipase

Pepsin

Phospholipase A2

1 Hatching

3

6

23

28

Life prey Weaning

Figure 6.16 Digestive enzymes and activities detected during ﬁrst feeding in the intestinal tract of larval turbot (from Hoehne-Reitan & Kjørsvik, in press). Dph denotes the time (days) after hatching when the enzyme or the enzyme activity was detected for the ﬁrst time. Data from Segner et al., 1994, Ueberschär, 1993, Munilla-Morán et al., 1990 and Cousin et al., 1987.

238

Culture of cold-water marine ﬁsh

e

-1 Lipase (mg larva )

10

Artemia d cd

Rotifers bc Hatching 1

bc abc abc ab

a ab

*

ab Algae

0.1

-5

0

5 10 15 Days after hatching

20

25

Figure 6.17 Content of bile salt-dependent lipase in developing turbot larvae determined by an ELISA assay. Different letters indicate signiﬁcant differences between means of the fed larvae; * indicates a signiﬁcant difference between fed (䊉) and starved (䉱) larvae. An immunoreaction was also found in the egg stage (䊊) (from HoehneReitan et al., 2001b).

bile. Some of the few quantitative studies of larval digestive enzymes have shown that the pancreatic cells start the synthesis of trypsinogen (the inactive form of trypsin) and ‘bilesalt-dependent lipase’ (the most important lipase in ﬁsh) well before the onset of exogenous feeding, as is shown for cod and turbot in Fig. 6.17. The presence of these enzymes follow a similar pattern of development. Larval synthesis, content and activity of pancreatic enzymes are generally low at hatching. The yolk-sac phase is characterised by a food-independent increase in enzymes, which may be interpreted as an imprinted physiological event during development (Hjelmeland et al., 1984; Zambonino Infante & Cahu, 1994; Hoehne-Reitan et al., 2001b; Hoehne-Reitan & Kjørsvik, in press). During the ﬁrst days of exogenous feeding, a plateau, or even a decline, in larval enzymatic content has been observed in both fed and starved larvae, and larval development and the initial responses to feeding conditions seem critical during this short period (Hoehne-Reitan & Kjørsvik, in press). This phase is also generally characterised by increased mortality and low growth rates (Blaxter, 1988). In older larvae, the synthesis of enzymes increases exponentially if the larvae are fed adequate food, and the amount of enzyme secretion seems to be dependent on the larval feed intake, and possibly on the diet composition (e.g. Pedersen et al., 1987; Zambonino Infante & Cahu, 2001; Hoehne-Reitan et al., 2001a, b; Hoehne-Reitan & Kjørsvik, in press). It has been shown for several species that ﬁsh larvae produce enough digestive enzymes to be able to digest the ingested food. The contribution of digestive enzymes from the prey organisms seems to be very low during the start-feeding period, and generally does not exceed 6% of the total larval enzyme activity (Hoehne-Reitan et al., in press). A signiﬁcant contribution of exogenous enzymes from the prey organisms to ﬁsh larval digestion thus seem very unlikely in older larvae, although they could play a role in the initial phase of ﬁrst feeding (Table 6.5).

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239

Table 6.5 Contribution of exogenous enzyme activity from prey to the total activity found in marine ﬁsh larvae or the larval gut (% of total activity in the larvae/intestine). Modiﬁed from Hoehne, 1999. Enzyme

% activity of total

Days

Lipase

0

Lipase PLA2 WEH

0.5–1.1 (of larvae) 1.7–5.8 (of larvae) 0.1 (of larvae)

Lipase Amylase Trypsin

0.6–4.5 (of intestine) 2.5–23.4 (of intestine) 0.9–5.9 (of intestine)

Protease

0.6 (of intestine)

Trypsin

3 (of intestine)

Esterase Exonuclease Amylase Protease

89–94 (of intestine) 79–88 (of intestine) 17–24 (of intestine) 43–60 (of intestine)

Neutral lipase

3.9–5.2 (of larva) 1.6–2.3 (of larva) 2.6–4.1 (of intestine)

6 12/13

Phospholipase

7.4–9.9 (of larva) 1.7–2.9 (of larva)

6 12/13

Prey

Fish species 1

–

Rotifers

–

7–20

Artemia

Striped bass2

<26 5–39 <31

Rotifers

Walleye pollock3

10

Rotifers

Japanese sardine Sardinops melanoticus4

40

Artemia

Sea bass5

3

Rotifers

Turbot6

Rotifers

Turbot7

1

Kühle & Kleinow, 1985; 2 Ozkizilcik et al., 1996; 3 Oozeki & Bailey, 1995; 4 Kurokawa et al., 1998; 5 Cahu & Zambonino Infante, 1997; 6 Munilla-Morán et al., 1990; 7 Hoehne, 1999. WEH, wax ester hydrolase.

6.6.2.3 Digestive Physiology—Lipids, Proteins and Carbohydrates In ﬁsh larvae, neutral lipids and phospolipids are emulsiﬁed and broken down (hydrolysed) to mainly polar monoglycerides and free fatty acids in the gut lumen by the action of bile and digestive lipases. They are then absorbed by the microvilli membranes in the mucosal epithelial cells of the mid-gut. In these mid-gut cells, the fatty acids and monoglycerides are re-esteriﬁed into triglycerides in the agranular (smooth) endoplasmic reticulum, and they become temporarily deposited as large lipid droplets in the cells. These large vacuoles are probably a temporary lipid store, and their appearance depends on the nature of the ingested lipids. As observed in cod, lipid absorption and accumulation in larval enterocytes is low during the ﬁrst days of exogenous feeding, but increases rapidly as feed uptake and enzyme secretion increases during larval development. The lipids are surrounded by a thin layer of protein, which forms particles called chylomicrons (lipoproteins). Chylomicrons are transferred to the Golgi complex, from where they migrate to the lateral cell membrane, cross it by membrane fusion (exocytosis), and ﬂow into the extracellular cell space towards the blood vessels. The larva’s capacity to synthesise intracellular lipoproteins seems rather low, and the larvae are probably dependent on a certain dietary level of phospholipids for efﬁcient lipid digestion. In larvae and newly metamorphosed juveniles fed low levels of phospholipids, the gut

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Culture of cold-water marine ﬁsh

showed accumulations of large lipid vacuoles, indicating a slower lipid transport out of the cells, a subsequent overload of the gut epithelium and a suboptimal utilisation of the lipid. The ability to synthesise lipoproteins seems to improve after metamorphosis, and the ﬁsh may become less dependent on high dietary levels of phospholipids. Proteins are digested relatively slowly in the gut if only alkaline proteases from the pancreas are active, whereas digestion is much more efﬁcient if proteins are ﬁrst exposed to peptic digestion in the stomach. In ﬁsh larvae with no stomach, proteins are probably partly broken down to peptides and amino acids in the gut lumen by digestive proteases. The protein and peptide macromolecules are then absorbed (‘engulfed’) directly into the epithelial cells of the hind-gut by pinocytosis, and thereafter digested intracellularly in several steps (see Fig. 6.14). The pinocytotic vacuoles may be observed in the hind-gut for 10–24 h before intracellular digestion is complete. Protein pinocytosis seems to vary between developmental stages and between species. This type of digestion may have evolved as part of an immature digestive system without a stomach, since the phenomenon is also observed in suckling mammals. Pinocytosis is believed to disappear after metamorphosis, although it has been observed in juvenile halibut and turbot. The quantitative role of this type of digestion is not fully understood, and it is not clear whether small peptides or free amino acids may be absorbed directly through the epithelium. The digestive mechanisms of carbohydrates are poorly understood in ﬁsh larvae. The larval diet contains little carbohydrate, although recent results suggest that microalgae may play a role in very early nutrition, and carbohydrate digestive enzymes are most pronounced in larvae during the ﬁrst days of start-feeding. 6.6.2.4 Stomach Development and Metamorphosis Metamorphosis changes the general larval ﬁsh digestive system from a gut system to a near adult system with pyloric caecae of varying numbers, a diffuse pancreas, a stomach, and anterior and posterior intestines. The larval gut pH is alkaline, due to the effect of the bile which is secreted from the liver and transported to the gut via the gall bladder. In prometamorphic larvae, sphincters develop between the oesophagus, fore-gut and intestine. During climax metamorphosis, the fore-gut differentiates into pyloric caeca and a functional stomach. The intestinal epithelium thickens and forms complex folds and crypts, in which cells proliferate and become secretory structures. These secretory, or acinar, structures are small sacs lined with secreting cells. During post-climax metamorphosis, these acinar structures become gastric glands, the pH decreases gradually (Fig. 6.18), and there is a strong immunoreaction for pepsinogen. Lipid absorption may increase, and there are changes in the enzymes of the glycolytic pathway (oxidative and citric acid cycle). This indicates a switch away from the use of fatty acids or amino acids to metabolising glycogen. Many ﬁsh have lower ingestion rates and food requirements after metamorphosis because of their higher food storage and conversion efﬁciency. There are species-speciﬁc differences in the rate and stage at which the ﬁnal differentiation of the stomach occurs. In general, the differentiation of the stomach will be complete

From fertilisation to the end of metamorphosis

Day 11

Day 16

Day 24

Day 28

241

Day 31

Foregut

Midgut

Hind gut

Rotifers Mikrodiell Artemia

7.5 8.0

8.0

Artemia

7.5 8.0

Formul. feed

6.0 7.0 8.0

Formul. feed

4.0 5.0 6.0 7.0 8.0 9.0

Figure 6.18 Development of changes in pH in the fore-gut (stomach), mid-gut and hind-gut of turbot larvae in relation to days after hatching. The larvae were fed a standard feeding regime of enriched rotifers and Artemia nauplii, and were weaned on to a commercial formulated feed from Day 24. Data from Hoehne-Reitan et al., 2001a. Figure drawn by Katja Hoehne-Reitan.

when the median ﬁns have been fully formed, but this is not always the case. In cod, Gadus morhua, the stomach becomes functional well after the completion of ﬁn development and squamation, when the ﬁsh is about 40 mm long (Pedersen & Falk-Petersen, 1992). The pattern of folds seems to be genetically determined, but the timing of further differentiation is affected by the thyroid hormones, T3 and T4. For example, in summer ﬂounder (Paralichthys dentatus), gastric glands normally appear in early pro-metamorphosis, and pepsinogen appears at climax metamorphosis. However, when exogenous thyroid hormone is added, the appearance of gastric glands is stimulated in larvae that are still pre-metamorphic. This is called ‘heterochrony’ of events, meaning that instead of being synchronous, one metamorphic event can be induced to come earlier or later (Huang et al., 1998). This is also the case for eye migration, which can be arrested despite the general progression of metamorphosis in farmed halibut (Pittman et al., 1998). Heterochrony is a type of variation that is different from the normal variation in the rate of development, and it shows the plasticity of the tissues. Heterochrony of events occurs in many organ systems, and is a clear indication of the complexity of the changes underlying metamorphosis. Therefore, deﬁning the normal course of change is important in determining which external cues may be stimulating change, and the cascade of events leading to the adult phenotype.

6.6.3 Muscle and Body Skeleton 6.6.3.1 Swimming Capacity and Muscle Development In addition to detecting and digesting prey organisms, ﬁsh larvae must also be able to catch them. Larval marine ﬁsh generally swim slowly, but almost constantly, in an environment with a density 800 times and a viscosity 30 times that of air. As in adult ﬁsh, the axial musculature is the largest and fastest growing tissue in ﬁsh larvae, giving the larvae increased abilities to cruise, hunt and avoid predators during their development. As the larva grows,

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Culture of cold-water marine ﬁsh

the hydrodynamic forces acting on it will change, and rapid growth will help it to overcome some of the most restricting hydrodynamic forces it is exposed to. Reynold’s number (Re) indicates the relationship between the viscous and inertial forces acting on a moving object in water. For ﬁsh, this is calculated as Re = U*L u where U is the swimming speed (ms-1), L is the body length (m) and u is the kinematic viscosity of the water (the ratio of viscosity to density) (Webb & Weihs, 1986). When ﬁsh larvae are 3–7 mm long, their cruising speeds vary from 1 to 3 body lengths per second (BL s-1), whereas a burst of speed may exceed 30 BL s-1 (Osse & van den Boogaart, 1995). The viscous forces are dominant when larvae are small (Re <10), so that the larvae may feel as if they are swimming in ‘heavy syrup’. Under these conditions, an eel-like (anguilliform or S-shaped) swimming mode is advantageous, as each part of the body can then effectively push the water backwards relative to the direction of motion. As larval length and swimming speed increase (Re >200), inertial hydrodynamic forces will become more and more dominant, resulting in less resistance from the water. This change in hydrodynamic regime is often accompanied by the development of caudal ﬁns and a transition from an anguilliform (S-shaped movement) to a sub-carangiform (kick and glide) swimming mode where movements are concentrated on the posterior part of the body (Webb & Weihs, 1986). Larval somatic growth and mobility depends on the number and distribution of muscle ﬁbre and the types of ﬁbre in the skeletal muscles. Newly hatched larvae generally have an inner muscle mass surrounded by a thin layer of superﬁcial ﬁbres. The inner ﬁbres develop into white ﬁbres, and are used for rapid swimming bursts. The superﬁcial ﬁbres will become the red muscle ﬁbres used for normal, slow swimming (El-Fiky et al., 1987; Akster et al., 1995). The adult red muscle is characterised by its concentration under the lateral line (Fig. 6.19). It is used for slow and sustained swimming, has a rich vascularisation (many blood vessels) and the ﬁbres contain many mitochondria. Red muscle has an aerobic metabolism which may use glycogen and fat as energy. The larval red muscle has many similar characteristics, although it is distributed in one continous layer around the whole larva, and the rich vascularisation of the adult is not seen in larvae. The adult white muscle is used for fast swimming bursts. It has very little vascularisation, and the ﬁbres contain relatively few mitochondria. The white muscle has an anaerobic metabolism which uses glycogen as energy fuel. Larval white muscle is also used for fast burst activity. However, all larval muscle ﬁbres have an aerobic metabolism in the early stages, and maturation into red aerobic and white anaerobic ﬁbres normally develops during metamorphosis, probably in relation to the development of functional gills (Batty, 1984; El-Fiky et al., 1987). Species-speciﬁc variations in muscle ﬁbre differentiation at hatching will depend on egg incubation period, body length and larval developmental stage, as well as on environmental factors, but in general, ﬁbres have a mature appearance by the onset of ﬁrst feeding (Johnston, 1999; Galloway, 1999). Larval muscles consist of thin ﬁbres with few myoﬁbrils,

From fertilisation to the end of metamorphosis

a

243

b

W P R

RL IW

Figure 6.19 Schematic drawings showing the position of different muscle ﬁbre types in the tail of (a) an adult cod (40–50 cm) and (b) a cod larva at the onset of ﬁrst feeding (approximately 4.5 mm standard length). W, white ﬁbres; P, pink ﬁbres; R, red ﬁbres; IW, inner white muscle; RL, superﬁcial red muscle layer. From Galloway, 1999.

and the muscle mass grows by both hypertrophy (increase in ﬁbre size) and hyperplasia (increase in number of ﬁbres). Growth during the larval start-feeding phase is dependent on factors such as nutritional and environmental conditions. The somatic growth rates are generally positively correlated with white muscle ﬁbre hyperplasia (Fig. 6.20), irrespective of the cause of different growth rates. The increased hyperplasia results in an increased number of new small muscle ﬁbres, which in turn may improve the potential for further growth by hypertrophy. Fish grow by hyperplasia during a large part of their adult life (Weatherley et al., 1988). This growth pattern makes ﬁsh different from birds and mammals, where the number of skeletal muscle ﬁbres is usually ﬁxed at birth. Recent results indicate that the potential adult size of a ﬁsh may be determined by the muscle growth and differentiation in the early larval stages. 6.6.3.2 Musculature Changes During Metamorphosis At prometamorphosis, the larval muscle ﬁbres become thicker, the number of myoﬁbrils increases and the protein content also changes. There may be further differentiation of the ﬁbres to thin ‘red’ or thicker ‘white’ ﬁbres. Larval muscle proteins such as troponinT (TNT) change from mainly the 41.5-kDa isoform to predominantly the 33.5-kDa isoform. This transition of TNTs can be induced by thyroid hormone (Yamano et al., 1991). During metamorphosis, a thin superﬁcial band of red muscle aggregates along the mid-line, accompanied by an increase in the complexity of myomere folding which affects forward acceleration. After metamorphosis, the adult type of myosin molecules appears and gradu-

244

Culture of cold-water marine ﬁsh

Figure 6.20 (a) The relationship between somatic growth rate (daily percentage increase in dry weight) and rate of hyperplasia/daily percentage increase in the number of white ﬁbres per cross-sectional area) in cod larvae. (b) The number of white ﬁbres per cross-sectional area plotted against cod larval length. From Galloway, 1999.

ally replaces the larval type. The adult forms of white and red muscle ﬁbres have then differentiated, and the ﬁsh grows isometrically. This growth is sensitive to both temperature and diet. 6.6.3.3 Skeletal Changes The head and the bones associated with feeding develop fastest in ﬁsh larvae. Many studies have examined the ossiﬁcation (transition from cartilage to bone) of the cranial and axial skeletal development of ﬁsh, and a very general pattern has emerged: feeding structures, swimming structures (initially the caudal ﬁn), the start of cranial ossiﬁcation, dorsal and anal ﬁns, paired ﬁns, axial skeleton, and ﬁnally the pterygiophores. However, there are many, many variations on this theme. In teleost ﬁsh, the cranium develops from a cartilagenous neurocranium with unfused plates, but is soon changed by endochondrial bones ossifying in the cartilagenous framework, and by the addition of many membranous bones of dermal origin. Separate ossiﬁcations produce the upper jaw (palatine, pterygoids and quadrate), whereas dermal contributions give rise to the premaxilla, maxilla, jugal and the lower jaw in general (Bone et al., 1995). Thus, the skull, jaw and feeding apparatus of teleost ﬁsh are of complex derivation and structure. The axial skeleton is different. The vertebral column of teleosts is like that of elasmobranchs, having biconcave centra (vertebrae), but arising after the embryonic somites. The development and ossiﬁcation of the vertebral column is subsequent to that of the skull and feeding apparatus. In avian and human embryos, the somites establish a segmental pattern that becomes transferred to adjacent structures such as the peripheral nervous system and

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the vascular system. In these groups, each vertebra arises from three sclerotomic areas. The paired lateral ones give rise to the neural arches, the ribs and the pedicles of the vertebrae, whereas the vertebral body and the intervening disc develop from the axially located mesenchyme. The neural arches originate from the caudal half of one somite, whereas the vertebral body is made up of the adjacent parts of two somites. Interactions between notochord and axial mesenchyme are a prerequisite for the normal development of vertebral bodies and intervening discs (Christ & Wilting, 1992). Koumoundouros et al. (1999) found that in the common dentex, the development of the axial skeleton begins with the formation of the anterior hypural, neural and haemal arches shortly after hatching. The formation of the vertebral centrae followed the formation of the caudal ﬁn rays, but all elements, except the ventral ribs, were fully ossiﬁed only after metamorphosis. However, in yellowtail, notochord ﬂexion occurred and formed an individual centrum by 6.6 mm SL, and all 24 centra of the axial skeleton had completely ossiﬁed by 12.7 mm (Liu, 2001). In Dipliodus sargus, ossiﬁcation of the vertebral centra was initiated mid-way and spread caudally and rostrally, following ossiﬁcation of the ﬁns and the neural and haemal arches (Koumoundouros et al., 2001). Thus it would seem that the pattern of axial skeletal development displays a species-speciﬁc spatio-temporal difference in ossiﬁcation. The development seems to be somewhat plastic, and responds to diet and physical requirements for feeding and further growth. Developmental abnormalities of the skeleton, such as incomplete eye migration, short opercula, deformed jaw and spinal deformities become evident at metamorphosis. Early rearing protocols may affect the development of deformities, and thus of ﬁsh growth and consumer acceptance of the product. For example, excessive Vitamin A has been implicated in some forms of bone deformity in Japanese ﬂounder (Takeuchi, 2001). The speciﬁc causes of bone deformation have not been identiﬁed, although Divanach et al., (1996) suggested the enrichment of live prey with fatty acids and vitamins, and the control of environmental factors such as water temperature and ﬂow. Spinal deformities are often associated with problems of swim-bladder inﬂation, but a complex causative origin is indicated in the literature.

6.6.4 Swim-bladder In larvae, the swim-bladder may be essential to the development of the spinal vertebrae, to buoyancy and to the efﬁcient use of energy during prey searches. However, not all species have swim-bladders (e.g. halibut). The swim-bladder usually occupies about 5% of the volume of an adult marine teleost, but about 7% of that of a typical freshwater ﬁsh. Swimbladder inﬂation usually coincides with the time of yolk-sac depletion and the initiation of feeding. Two basic types dominate, physostomous and physoclistous, although there are suggestions that the two types are not mutually exclusive. Physostomous swim-bladders have a pneumatic duct between the swim-bladder and the gut. Typical species with physostome swim-bladders are herring, salmonids, pike, cyprinids, catﬁsh and eels. These species inﬂate the swim-bladder by forced inﬂation through the duct (gulping) at the water surface. Deﬂation of the swim-bladder is achieved by a reﬂex reaction forcing gas back into the oesophagus or by diffusion. In some species, the swim-bladder

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is transient but is necessary to vertebral column formation (turbot, Scophthalmus maximus), while in others the pneumatic duct normally atrophies after inﬂation (walleye, Stizostedion vitreum). In walleye, the lumen of the undifferentiated gut is the site for the adjacent openings of both the common bile duct and the pneumatic duct of the swim-bladder (Marty et al., 1995). Physoclistous swim-bladders do not have a duct, but have specialised structures associated with the respiratory and circulatory system for inﬂation or deﬂation, such as the rete mirabile. The afferent and efferent capillaries of the rete mirabile form a tight bundle using a counter-current exchange of blood gases for an increase or decrease in pressure in the bladder. Thus, physical principles such as the Bohr effect (lower blood pH provokes a loss in haemoglobin oxygen afﬁnity) and the Root effect (changes in haemoglobin oxygen capacity with positon of attachment) reﬂect the physiological condition of the ﬁsh and underlie swim-bladder inﬂation. Deﬂation is also accomplished by diffusing the high pressure in the swim-bladder to the lower pressure in the blood. This may often be done in the dorsal or posterior wall of the swim-bladder where there may be an oval patch of dense capillaries. Two-thirds of teleost species are classed as physoclistous. The gas in the swim-bladder lumen consists of oxygen, carbon dioxide from bicarbonate in the blood, and nitrogen. The structures of the swim-bladder (gas gland, wall and rete mirabile) contain neutral lipids and phospholipids, as well as glycoproteins containing mannose and/or glucose sugar, as found in both seabream, Sparus aurata, and seabass, Dicentrarchus labrax. Glycogen was observed only in the gas gland of Sparus aurata (Dinis et al., 1997). Proteins rich in different amino acids, except those rich in tryptophan, as well as carboxyl but not sulphated glycoconjugates were observed in both species (op. cit). It is unclear to what degree gas composition is important in the early life stages. In cod, the swim-bladder is connected to the mid-gut at hatching, and the lumen is not inﬂated (Meek, 1924; Hardy, 1978; Morrison, 1993). The swim-bladder is ﬁlled between day 5 and day 9 after hatching. At day 9, it is no longer attached to the gut wall, and the vascular network of rete mirabile is more developed than in newly hatched yolk-sac larvae (Hardy, 1978; Morrison, 1993). When the pneumatic duct is closed, the swim-bladder is regulated by glandular activity (Morrison, 1993; Doroshev et al., 1981; Roberts, 2001). Non-inﬂation of the swim-bladder leads to the production of post-larvae which are smaller in size and weight than normal ones. Hyperinﬂation (often towards the end of the larval phase) of the larvae generally leads to mass mortality, and in cod larval rearing both of these phenomena are seen in commercial hatcheries. The growth delay in larvae without a fully functional swim-bladder can be observed in sea bream, sea bass, cod and walleye. The causes of this problem may vary. Organic debris and large numbers of bacterial rods ﬁlled the noninﬂated swim-bladder of a 13-day-old walleye larva examined by electron microscopy (Marty et al., 1995). These authors hypothesised that ‘surfactant-like secretions from the adjacent common bile duct affected fragmentation of large ingested air bubbles for transfer into the relatively small-diameter pneumatic duct’. After the development of pyloric sphincters, the bile duct is in the intestine and the pneumatic duct is in the stomach in walleye, thus limiting the possiblity of inﬂation. Swim-bladder inﬂation by physostomous ﬁsh seems to take place in a speciﬁc developmental window, probably before the physical separation of the fore-gut, and is subject to physical blockage. However, bacterial infection does not seem to

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be the only cause of this problem in cod larvae (E. Kjørsvik, unpublished data, 2002). Late swim-bladder inﬂation can occur but does not alleviate spinal curvatures in ﬁsh with chronic lordosis starting at the larval phase. In practice, commercial marine juvenile farms have increased the rate of successful swim-bladder inﬂation through the use of surface skimmers, aeration and good hygiene. Regulation of the swim-bladder is at present a problem in cod larval rearing, and more studies to solve these problems should be undertaken. Swimbladder problems generally seem to be connected with environmental or nutritional problems, and further work is still needed on the species-speciﬁc differences and underlying mechanisms.

6.6.5 Osmoregulation When marine teleost eggs are spawned, they are isotonic to the blood of the mother ﬁsh (about 350 mOsm) and will experience a brutal change to a very hypertonic (seawater) medium. Normal seawater has a salinity of approximately 34 p.p.t. (ca 1000 mOsm). Therefore, the osmotic difference between the eggs and the surrounding water is very high; eggs in seawater may rapidly lose water, and eggs in fresh water may gain. Unfertilised eggs also have a very permeable plasma membrane, and no organs or cells are developed for active ionic regulation during the early embryonic stages. However, the embryos are able to maintain an osmolarity which is similar to that of adult ﬁsh, the yolk osmolarity does not change much during embryonic development, and the eggs are able to maintain their diameter (Alderdice, 1988; Lønning et al., 1988). The chorion is no barrier to water loss or water uptake, since the perivitelline ﬂuid in marine eggs is approximately iso-osmotic with the surrounding seawater, and in fresh-water eggs it is slightly hyperosmotic to freshwater (10–15 mOsm) due to colloids from the cortical alveoli. The osmoregulation in ﬁsh embryos is possible because the properties of the plasma membrane change profoundly during the cortical reaction from being very permeable to becoming one of the least permeable biological membranes known. This is also well demonstrated in cod eggs, for example, in which yolk osmolality was affected by the seawater salinity during fertilisation, but not if the eggs were exposed to a different salinity 24 h after fertilisation (Kjørsvik et al., 1984). The egg’s free amino acids content is probably also involved in the process of maintaining the osmolality of the body ﬂuid during early embryonic development by a relatively high content in the yolk during embryogenesis (Fyhn et al., 1987; Finn et al., 1991). Most current knowledge of teleost larval osmoregulation is from studies of ﬂatﬁsh, and was recently reviewed by Schreiber (2001). Active osmoregulation for marine pelagic ﬁsh eggs is possible only very late in embryogenesis when embryos/larvae may take up water by drinking and some chloride cells are present (Tytler & Ireland, 1994; Schreiber, 2001). Drinking water may even start before the mouth is open, as water may pass through the gill cavities, which have an open passage to the gut. In cod and halibut, for example, drinking has been observed in newly hatched larvae (Mangor-Jensen & Adoff, 1987; Tytler & Blaxter, 1988), whereas their mouths open much later after hatching. Drinking is also associated with water and ion uptake by the gut, and even in early larval stages the osmoregulatory mechanisms of the gut appear to be similar to those in adult ﬁsh (Brown & Tytler, 1993).

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The chloride-secreting cells in the gill ﬁlaments are the primary extra-renal osmoregulatory sites in adult teleosts (Evans et al., 1999). Gills are either absent or undeveloped in early larvae, and appear functional at around metamorphosis, but the larval skin contains numerous similar chloride secretion cells and is considered to be the main site for ion exchange (Tytler & Ireland, 1994; Segner et al., 1994). The distribution of these cells in larval skin changes dramatically during larval development, and they seem to be absent by the completion of metamorphosis when the gills are fully functional. According to Schreiber (2001), a decrease in salinity tolerance is often observed during the transition period when the skin begins to lose its osmoregulatory capacity and the regulatory mechanisms of the gills are not yet developed.

6.6.6 Respiration and Excretion In ﬁsh eggs and larvae, respiration may initially be cutaneous, and oxygen diffuses across 1–2 cell layers to the target organs. Fully functional gills in marine ﬁsh larvae generally appear after hatching, after ﬁrst feeding or, as in halibut, even after metamorphosis. During the embryo or larval phase, there is a shift in respiratory gas exchange from the body surface epithelium to the gills. In salmonids, functional gill ﬁlaments and secondary lamellae are formed during late embryogenesis or the early yolk-sac phase. Small pelagic marine ﬁsh larvae have passive gas exchange through the surface epithelium, and larval blood may have no erythrocytes (red blood cells with haemoglobin). Larvae that do have erythrocytes have a speciﬁc embryonic type that is different from the adult type. Larval erythrocytes are large and round with relatively small nuclei, in contrast to the elongate erythrocytes of adult ﬁsh. Turbot exhibit red blood cells around Day 12 after hatching, but for cod and halibut they are not visible before the end of the larval stage. Functional, but not fully mature, gills seem present during metamorphosis in cod and halibut. Embryonic and larval metabolism is thus generally aerobic. Pelagic marine eggs have very low O2 uptake during the ﬁrst few days after fertilisation, followed by an exponential increase in the embryonic and yolk-sac stages. Embryonic and larval metabolism is dependent on temperature, larval size, activity and feeding rate. The energy requirement of ﬁsh embryos can be determined by measurements of O2 uptake and NH3 excretion, as is shown for Atlantic halibut eggs and larvae in Figs. 6.21 and 6.22 (see also Davenport et al., 1983). Such studies indicate that embryonic energy production is basically linked to the aerobic breakdown of nitrogen (especially free amino acids, in which the yolk is very rich). In species with oil droplets in the yolk, such as turbot, free amino acids are combusted in the embryonic stage, whereas the combustion of lipids is the main energy substrate in the yolk-sac stage. As ﬁsh larvae grow larger and older, the skin thickens, respiration by diffusion becomes impossible, and both the gills and other oxygen transport systems must come into play in order for respiration (and life) to continue. It is for this reason that the transition from cutaneous to branchial respiration has sometimes been considered to be a critical period in ﬁsh development. In pre- and prometamorphosis, the blood cells are dominated by large, round larval erythrocytes. Immature adult erythrocytes are small and round or slightly elliptical with large

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Hatu 400

O2 uptake (nl/egg/hour)

350 300 250 200 150 100 50 0 0

2

4

6 8 Days post fertilization

10

12

14

Figure 6.21 Oxygen uptake rates of developing eggs of Atlantic halibut. Each point represents the average uptake rate of pooled groups of 5–10 eggs/larvae. Incubation of the eggs occurred in darkness at 5.7°C and a salinity of 34.5 p.p.t. From Finn et al., 1991. Reproduced with permission of Elsevier Science.

Figure 6.22 Relationship between the routine rates of oxygen consumption and ammonia excretion and dry body mass measured under conditions of light and darkness for developing yolk-sac larvae of Atlantic halibut. The dry mass is measured for the embryo proper after removal of the yolk. From Finn et al., 1995c.

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nuclei. In some species, these occur ﬁrst at metamorphic climax. At this point, the blood cells will be divided between a group of large (larval) cells and a group of smaller (immature adult) cells, as can be demonstrated by a size–frequency distribution. In fully metamorphosed juveniles, the blood cells have developed into elliptical cells with elliptical nuclei (Inui et al., 1995). Larval tilapia have one type of haemoglobin (oxygen-carrying red pigment), whereas adult tilapia have two types. Thyroid hormones are involved in erythropoeisis, and there are nuclear L-T3 receptors in adult-type erythrocytes. It has been suggested that the number of receptors is larger in pro-erythrocytes and mature erythrocytes. In some ﬂounders, oxygen consumption increases until metamorphosis, at which point it declines, and increases again after metamorphosis is complete. In halibut, a change in photoperiod has been shown to induce the early appearance of haemoglobin, indicating that this is subject to heterochrony (Solbakken & Pittman, in press). It is assumed that the appearance of haemoglobin in ﬂatﬁsh is not only a consequence of increased oxygen requirements as development proceeds, but is also preparatory to occupying a new ecological niche on the sea bed, where oxygen levels will vary more than in the water column.

6.6.7 Neuroendocrine Systems In general, marine ﬁsh larvae hatch because of the hormones deposited in the yolk by the female brood stock. Hormones are internal secretions by endocrine or ductless glands that stimulate speciﬁc actions, such as cell change or the production of another hormone, in target cells or organs which are distant from the point of secretion. Hormones (proteins, peptides and steroids) are generally carried by the blood, and exert their action on target organs by interacting with speciﬁc receptors either in the nucleus or in the extracellular membrane. The binding of the hormone to the receptor initiates a change in gene transcription that eventually leads to a change in form and/or function. When one hormone stimulates the secretion of another hormone, we speak of a hormone cascade, as in ﬁsh reproduction. When the larva’s own endocrine organs develop to a functional stage (see Figs. 6.23–6.25), the larva is capable of the enzymatic transformation of amino acids and other basic nutrients to neuroendocrine or endocrine products. The main endocrine organs in ﬁsh are the pineal, hypothalamus and pituitary (brain), the thyroid (follicles near the ventral aorta), the ultimobranchial body, the chromafﬁn tissue, interrenals and juxtaglomerular cells of the kidney, the pancreas, gut and gonad, the corpuscles of Stannius, and the uropophysis (caudal tip of the spinal cord). Both the uropophysis and the pituitary consist partly of neuron cells in the central nervous system. The pituitary produces the greatest and most potent variety of hormones, including prolactin, adrenocorticotrophic hormone (which stimulates cortisol), somatostatin, growth hormone (GH), thyroid stimulating hormone (TSH), gonadotrophic hormone, melanophore stimulating hormone (MSH), arginin vasopressin, isotocin and mesotocin. The gland can appear a few days after hatching in some species, but the three main parts of the pituitary (pars distalis, pars intermedia and neurohypophysis) generally develop by the end of yolk absorption. This event generally coincides with eye pigmentation. The pituitary gland begins

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Figure 6.23 A gross overview of the neuroendocrine organs in ﬁsh. Redrawn from Bone et al., 1995.

Premetamorphosis

Prometamorphosis

Climax

Post climax

Hormone concentration

GH T4 PRL Cortisol

pituitary thyroid interrenal

T3 Fertilization

Hatch

First feeding

Juvenile

Developmental Event Figure 6.24 The general pattern of the thyroid hormones (T4 and T3), growth hormone, prolactin and cortisol expressed in the early life of ﬁsh. The levels prior to ﬁrst feeding may either be maternal deposition in the yolk and/or some embryonic expression. Redrawn from Tanaka et al., 1995.

to protrude from the hypothalamus prior to metamorphosis, and is attached to the ventral brain at metamorphosis. When the interrenals, thyroid follicles and pituitary are functional, usually near ﬁrst feeding, the larva begins to produce its own prolactin, thyroid hormones, growth hormone and cortisol. These play a major role in the further development and growth of the ﬁsh, and are directly affected by environment (temperature, oxygen, salinity, light) as well as diet. In reared yellowtail tuna, the pituitary ﬁrst appeared in the ventral edge of the brain on Day 2, and hung below the brain on Day 16, although the GH immunoreactive cells had already been detected on the day of ﬁrst feeding, Day 4 (Kaji et al., 1999). In Japanese ﬂounder, Paralicthys olivaceus, the ratio of the prolactin (PRL) immmunoreactive part to the whole pituitary increased gradually during the larval stages, and reached a constant level during metamorphosis (Hiroi et al., 1997). In gilthead seabream, both GH mRNA and protein are expressed shortly after hatching (Funkenstein & Cohen, 1996). This indicates not only that larval endocrine systems are functional early in development, but that further brain and endocrine organ development is essential to growth and differentiation to the adult form.

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Halibut Hippoglossus hippoglossus

Cod Gadus morhua

Figure 6.25 Longitudinal sections of prometamorphosing (a) halibut and (b) cod, showing the placement of the thyroid follicles (inset) in relation to the hyoid bone and the ventral aorta. In cod, note the brain development, including the hypothalamus and pituitary. Heart tissue can be seen lower left in each overview. Photographs: Sæle, Erstad and Grøtan.

Herein lies the importance of feeding, which will be the sole source of the building blocks for many hormones and their transforming enzymes. Most endocrine systems are believed to respond directly to organic nutrients via hormone synthesis, secretion, conversion or reception (MacKenzie et al., 1998).

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Many hormones interact to initiate and complete metamorphosis, but the process is not well understood. However, four hormones are central to metamorphosis: thyroid hormones (thyroxine T4 and triiodothyronine T3), growth hormone, prolactin and cortisol. Much of the research into metamorphosis in several ﬁsh species has concentrated on thyroid hormones. The pituitary–thyroid axis controls ﬂounder metamorphosis just as it does in amphibians. The pituitary produces TSH, which will stimulate the growth of thyroid follicles near the ventral aorta, under the glossohyal bones. Brood-stock thyroid hormone levels inﬂuence the levels found in their eggs and larvae. The initial levels found in the eggs decrease until the time of ﬁrst exogenous feeding, and remain low until metamorphosis. Generally, the thyroid follicles either differentiate at the end of yolk absorption (late group, species with pelagic eggs such as halibut), or they are already in place at hatching (early group, species with demersal eggs). In ﬂatﬁsh, T4 tissue levels increase during the initial stage of metamorphosis and peak at the end of climax metamorphosis, while T3 remains almost undetectable until climax metamorphosis. This seems almost counter-intuitive, given that adult tissues arise before the degeneration of larval tissues, and that early adult cell differentiation is assumed to respond to thyroid hormones, especially T3. However, the experiments on which these statements are based analysed whole organisms, and the results may not represent either the free hormone levels or the levels presented to the changing tissues. Advances in analytical techniques will shed further light on this complex issue. The thyroid gland comprises diffuse follicles, and is not one distinct gland as it is in mammals. A follicle is made up of an outer layer of epithelial cells that are usually cuboid. This outer layer encapsulates a colloid with thyroglobulin where the hormone thyroxine (T4) is synthesised. Hormonogenesis takes place mainly in the thyroid follicles. Thyroxin (T4) is synthesised from phenylalanine and tyrosine (both amino acids) by iodination (addition of iodide) of the molecule. Phenylalanine is converted via the enzyme phenylalanine 4-monooxygenase to tyrosine. Tyrosine is either degraded to thyroxin, or converted via 3-monooxygenase to melanin, as well as becoming a precursor for adrenaline and noradrenaline, which are important for pigmentation patterns. Iodide (I-) is taken up from the blood and extracellular space by sodium cotransporter proteins in the basal cell membrane of the follicle cell (thyrocyte). The iodide moves along an electrical gradient to the other side, where it is released through iodide channels to the follicle lumen. In the lumen, mono- and diiodotyrosine (MIT and DIT) have been formed in the thyroglobulin, and these are thus further iodinated. They are then condensed within the lumen to produce either triiodothyronine (three iodines, T3, considered to be more biologically active) or tetraiodothyronine (four iodines, thryoxin, T4). These are part of the thyroglobulin molecule in the colloid of the thyroid follicle. In order to be released from the lumen to the blood, where the hormones can be transported, there is a pinocytosis of the thyroglobulin by the apices of the follicle cells. These vesicles fuse with lysosomes and there is a proteolysis of the thyroglobulin that releases some T3, but mostly free T4. The MIT and DIT released during proteolysis are generally degraded and the iodide is recycled. The number, size and epithelial cell height of the follicles increases with larval development (Solbakken et al., 1999). T4 can be deiodinated to T3 at the target

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organ or in peripheral tissues. Tissue thyroxine levels are generally well synchronised with follicular development. In many ways T4 can be considered to be a prohormone for T3. T4 is monodeiodinated to T3 (one iodide is removed to make T3, which is considered to be more biologically active than T4). This monodeiodination is reduced when plasma levels of T3 are elevated, suggesting an autoregulation of T3 production. Since the deiodination takes place mainly in the liver and kidney (and some target organs), non-thyroid hormones acting on the hepatic and renal systems may modify the autoregulation. This means that the blood levels of prohormone T4 may be regulated independently of the production levels of T3. It also means that the production of T3 may be adjusted to the needs of the organism without using the negative feedback loop to control blood levels of T4. Much of the hormone in plasma is bound to carrier proteins, and only a small percentage is actually free hormone that can act on receptor molecules. Thyroid hormone receptors (TR) are nuclear receptors, sitting in the nucleus of the target cells and having speciﬁc T3 binding sites. The coupling of the hormone to the receptor activates gene expression in the nucleus. Receptors are saturable, and can also be activated by retinoids, for example. In ﬁsh, four types of thyroid hormone receptor have been identiﬁed: two each of the a (alpha) and b (beta) forms (Yamano & Miwa, 1998). There is a pattern in the timing and location of the appearance of speciﬁc thyroid receptors suggesting ‘windows of opportunity’ during which change can occur. Thyroid hormone receptor gene transcripts are found at very low levels in fertilised eggs, but TR mRNAs are found in substantial amounts in pre-metamorphic larvae. TRaA gene transcripts increase rapidly in metamorphic climax and decrease rapidly post-metamorphosis. TRb expression increased during climax metamorphosis, peaked during post-climax and remained high in post-metamorphic juvenile Japanese ﬂounder (Yamano & Miwa, 1998). There is a distinct tissue speciﬁcity of the a and b sub-types in the ﬁsh body, so that the thyroid hormone exerts its effects directly on each tissue. The expression of the TR sub-types changes with time and location. Hormone levels determine the development of each tissue by acting on receptors that are expressed at different times in varying numbers. The thyroid receptor a in Japanese ﬂounder is found in the skeletal muscle, intestinal epithelial cells and evenly in the muscle tissue. In the supporting ﬁn structures, TRa is in the striated muscle between cartilage. By comparison, TRb is found in some skeletal muscle, and more commonly in myosepta separating the myotomes, the cartilage cells and the osteoblasts surrounding cartilage and bone. TRb is also found in the cartilage cells of the pterygiophores, on the surface of pterygiophores, in haemal spines and in the layer just beneath the dermis of the skin (where adult chromatophores develop during metamorphosis) (Yamano & Miwa, 1998). It has been suggested that TRaA in ﬁsh may play a decisive developmental role in metamorphosis by acting on the muscle and digestive system, while TRb may be very important to the developing skeleton and in adult pigmentation patterns. Factors other than the transcription and synthesis of proteins are involved in the process of metamorphosis. In general, adult cell types begin to appear in changing tissues before the larval cells have become apoptotic or begun to change. Apoptosis, or programmed cell death, is an integral part of the dynamics of metamorphosis, and is clearly linked to the response

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genes. An early developmental response to thyroid hormones is the appearance of adult structures, while a late response to thyroid hormones is the degeneration (apoptosis) of larval structures, implying different gene transcript series. The larva will therefore have both larval and adult cells during metamorphosis.

6.6.8 Growth Hormone, Prolactin and Cortisol Somewhat less is known about the action of these hormones during metamorphosis, but the mode of action is similar. Growth hormone (GH) and prolactin are peptide hormones secreted from the pituitary, whereas cortisol is secreted from the interrenal cells. Growth hormone research in ﬁsh has been expanding rapidly, and has been best studied in salmonids. It is a broad-spectrum hormone with many direct and indirect effects, among them the stimulation of insulin-like growth hormone, lipid metabolism, osmoregulation, skeletal growth and a probable role in maturation (Bjørnsson, 1997). In the species studied, growth hormone secretion from the pituitary is under inhibitory regulation by somatostatin from the hypothalamus, as well as a possible stimulatory effect from analogues to growth hormone releasing factor and possibly gonadotropin releasing hormone. Growth hormone also stimulates the release of insulin-like growth factor (IGF) from the liver (indicating a dual activating process for growth), and together these initiate an array of direct and indirect effects on the developing ﬁsh. It appears that receptors on cell membranes mediate the responses to GH in a variety of tissues. Metabolically, GH stimulates lipid metabolism and protein accretion by acting alone or in concert with prolactin, TH and cortisol. Stimulation of the secretion of GH can occur through a variety of environmental cues, of which photoperiod is best studied, indicating that GH may play a central role in the mediation of seasonal cues to the physiological development of ﬁsh. The GH receptor has recently been characterised in turbot (Calduch et al., 2001), showing it to be in the class I cytokine receptor superfamily with a sequence similar to that found in GH receptors throughout vertebrate evolution. More than 40 ﬁsh growth hormone sequences have been characterised. In ﬁsh larvae, GH family protein genes are expressed in the developing embryos prior to the formation of the pituitary gland (Yang et al., 1999). These authors also found expression of GH and somatolactin genes in other tissues even after the organogenesis of the pituitary gland. In general, GH gradually increases from ﬁrst feeding until after metamorphosis. The thyroid hormone receptor is involved in pituitary gene expression since both growth hormone mRNA and thyroid hormone receptor mRNA are found in the pituitaries of carp as early as 4 days after fertilisation (Farchy et al., 1995). This strongly suggests that this array of hormones may play an important role, which is yet to be identiﬁed, during embryonic development in ﬁsh. Prolactin studies in ﬁsh larvae are increasing. In teleosts, prolactin plays a role in osmoregulation as well as in ovarian steroidogenesis, and is a possible extra control mechanism for oestrogen synthesis as well as having many other effects. The prolactin receptor has been characterised in several species, and is expressed mostly in the kidney, gills, intestine, brain and gonads. As for cortisol, the ﬁsh studied to date have exhibited a post-hatch rise in endogenous cortisol as the result of de novo synthesis of cortisol by the larvae. In 4-day-old Asian

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seabass larvae (Lates calcifer), the interrenals are immunoreactive for adrenodoxin and cytochrome P450-21, and the pituitary for adrenocorticotrophic hormone (Sampath et al., 1997). These ﬁndings suggest that the pituitary–interrenal axis is functional even at this early stage. Cortisol is generally measurable at pre-metamorphosis, and peaks prior to the appearance of the thyroid peak in marine ﬁsh larvae. Prolactin exhibits a similar pattern, but it is not clear whether there is a distinct peak prior to climax metamorphosis.

6.6.9 The Immune System The immune system of developing larvae is only partially understood, but despite our incomplete picture, some generalities can be stated. Non-speciﬁc defence systems in the skin and gut (among others) play an important role in the immunity of larval ﬁsh (e.g. phagocytes, granulocytes, preciptins, agglutins and lysozymes), but the speciﬁc defence system is not well developed. The lymphoid organs (thymus, kidney, spleen) have species-speciﬁc development times, and their appearance is earlier than their mature lymphoid function (Schrøder et al., 1998). In turbot, the pronephritic kidney has primordial haematopoietic stem cells shortly after hatching (Padros & Crespo, 1996), and the kidney is usually the ﬁrst lymphoid organ to develop. The spleen generally comes later, and has rich capillaries, red blood cells and thrombocytes, whereas the thymus has lymphoblasts at the beginning of metamorphosis. At climax metamorphosis in some ﬁsh, the thymus will be differentiated into two parts, the outer part with mature lymphocytes and reticular cells, and the inner part with lymphoblasts and macrophagic cells. Marine and fresh-water ﬁsh may differ in the sequence of appearance of these organs. The involvement of cortisol in metamorphosis, and its effects on the immune system, should be considered for marine as well as for fresh-water ﬁsh. A more complete description of the immune system is given in Chapter 3.

6.6.10 Skin and Pigmentation Larval pigment cells, i.e. chromatophores, develop in the layer below the dermis. They are generally erythrophores (red), xanthophores (yellow) and melanophores (black), which can be formed in stellate or dendritic cells under neural control. Some stellate melanophores can be seen over the head and along the body in newly hatched cod larvae, for example, whereas the halibut yolk-sac larvae have no pigment whatsoever. By ﬁrst feeding, dermal pigmentation has generally begun, with both erythrophores and melanophores developing as ﬁne spots to ﬁll the stellate or dendritic cells. Iridophores, containing guanine and giving the ﬁsh a shiny appearance, generally occur at around metamorphosis. The pattern of pigmentation is species-speciﬁc (Burton, 1998). The thickness of the skin increases during metamorphosis (most of the increase will occur on the abocular side of ﬂatﬁsh). The pigment pattern will ﬁll the body surface, and there is a cytological differentiation of chromoblasts (pigment cells) on the ocular side of ﬂatﬁsh, and a destruction of chromoblasts on the abocular side. The number of mucous cells is great-

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est on the abocular side, or the area having the greatest contact with the bottom, allowing settling ﬁsh to avoid damage from contact with the substrate. The pigmentation pattern is largely controlled by adrenergic factors. Melanosomes must ﬁrst be formed and dispersed throughout the growing skin, and then aggregate into the species-speciﬁc pattern. Catecholamines mediate the aggregation of melanosomes via aadrenoreceptors, as is the case with winter ﬂounder (Pleuronectes americanus). Low concentrations of noradrenaline enhance melanosome dispersal. The dispersal and aggregation of melanosomes are probably controlled by a single adrenergic innervation (Mayo & Burton, 1998). This means that aggregation would be controlled by a-adrenoreceptors responding to catecholamines immediately after release from nerve endings, and after nerve activity stops, the declining concentration of catcecholamines will cause dispersion through badrenoreceptor mediation. Pigmentation usually proceeds apace with metamorphosis. However, since it is under adrenergic control, metabolism and metabolic rates may affect the pattern via temperature and other factors. In ﬂatﬁsh, pigmentation pattern is often used as a quality criterion in the purchase of juveniles, and may be the most obvious sign of developmental effects of rearing techniques. It is clearly affected by diet, and also by the timing of the introduction of various nutrients, as the many experiments on ﬂatﬁsh pigmentation illustrate (see for example, Shields, 2001), although speciﬁc requirements have not yet been determined. Even in established cultivation, turbot still exhibit malpigmentation. Recent investigations suggest that the critical periods for pigmentation determination by nutritional inﬂuences may be even earlier in larval development than was previously reported (Takeuchi, 2001).

6.6.11 Larval Feeding Behaviour Fish larvae must learn to capture prey. As most of the ﬁsh in mariculture are visual predators, ﬁrst-feeding usually coincides with full eye pigmentation (Fig. 6.26). Feeding generally occurs before the end of the yolk sac (EYS), and thus the larva grows on both endogenous (yolk) and exogenous (prey) sources. This stage is called mixed feeding. In general, however, food assimilation is low at the beginning of exogenous feeding because digestive capacity is limited. The use of the green-water technique, where algae are added to the tanks during ﬁrst feeding, has been credited with increased feed uptake, or with stimulated assimilation efﬁciency by triggering peptic enzymes, among other factors (Reitan et al., 1993, 1997; Cahu et al., 1998). Therefore, food conversion may also be improved under green-water conditions. The addition of algae also has an obvious impact on the water quality, the light environment, and the contrast between background colour and prey colour. These aspects are further discussed in Chapter 7. In general, lighting conditions will inﬂuence both the duration of feeding and the level of activity maintained. Continuous light allows the larva to feed continuously, whereas feeding generally stops in darkness. However, overall larval growth may beneﬁt from a period of darkness during which maintenance energy costs decrease, as is the case with sea bream (Papandroulakis et al., 2002). Light quality may also play a signiﬁcant role in larval feeding: haddock larvae have signiﬁcantly higher feeding success under blue (470 nm) light than under either full-spectrum white or green (530 nm) light, although prey will be taken under

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Figure 6.26 A 31-day-old cod larva and its Artemia nauplii prey. Photograph Tora Bardal, Department of Biology, Norwegian University of Science and Technology.

many light qualities (Downing & Litvak, 2001). It would therefore seem that marine ﬁsh larvae may have a predisposition to optimal feeding in a visual environment which is comparable to open-ocean nursery grounds. Feeding behaviour is species–speciﬁc, but there are some general patterns. As they move towards exogenous feeding, the larvae change behaviour and swimming patterns (Fig. 6.27). In early feeding, larval ﬁsh swim in quick short bursts and rest in between, but this pattern changes to one in which activity increases and swimming speed decreases. Slow and easily detectable prey items are preferred initially, with the size range correlated to mouth size (Noakes & Godin, 1988; van der Meeren, 1991; Fuiman, 1994; Olsen et al., 1999). Prey size for ﬁrst-feeding marine ﬁsh larvae is generally between 80 and 200 mm. As the larvae grow, they will also catch increasingly larger prey (Fig. 6.28). The amount of time spent swimming increases, thereby increasing the volume of water searched for prey, but also the metabolic costs. In ﬁrst-feeding cod up to 6 mm TL, there are long pursuit and attack times and distances, and slow swimming speeds, whereas larger and more developed larvae have shorter pursuit and attack times and distances, and faster swimming speeds. Speciﬁc metabolic rates during pursuit increase with increasing larval size, while speciﬁc energy expenditure decreases due to shorter pursuit times (von Herbing et al., 2001). Thus, although the larvae use more energy, they are more efﬁcient at detection and capture. Fish learn to be efﬁcient predators through different behaviours, and through searching a three-dimensional volume usually within 1–2 body lengths distance. In 94% of all foraging events by ﬁrst-feeding cod, prey items were perceived during glides, and the percentage of successful attacks increased with ﬁsh size. In all larval cod, a successful attack on prey occurred over shorter distances and at a quicker speed than did unsuccessful attacks (von

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Cod Gadus morhua

Turbot Scophthalmus maximus

Figure 6.27 Activity levels and swimming speed of fed (a) and starved (b) cod and turbot larvae. There are species-speciﬁc ways to conserve energy when faced with starvation. The behaviour of ﬁsh larvae under cultivation may give good indications of both the condition of the ﬁsh and the success of the rearing protocol (Skiftesvik, 1992).

Herbing & Gallagher, 2000). Reactive distances to prey are generally small in ﬁrst-feeding ﬁsh, but increase with both body size and prey width, and the ability of the larva to use several sensory systems to detect prey (Pankhurst, 1994). Impending starvation forces a more energy-conserving strategy: in some species, such as cod, swimming speed decreases, and both cod and turbot will reduce their activity levels (Skiftesvik, 1992). The behaviour of ﬁsh is one attribute that determines their ability to survive once the juvenile form is attained. The behaviour of farmed ﬁsh differs from that of wild ﬁsh in many aspects, and may be exacerbated by the naïve environment of the hatchery. Japanese researchers used indices of behaviour to assess ﬁsh quality prior to release for stock enhancement (Fushimi, 2001), and applied different keys to different species. The duration of off-bottom behaviour in ﬂounder is associated with higher predation risks, and is greater for hatchery-reared ﬁsh than for wild ones. Predator-avoidance behaviour such as aggregation and tilting also affect the success of stocking programmes in red seabream (Pagrus major), striped jack (Pseudocaranx dentex) and ayu (Plecoglossus altivelis). Fish survival in tanks also depends on size grading, adequate feeding and the subsequent reduction in cannibalism. Certain nutrients, such as taurine and DHA (Takeuchi, 2001), have been implicated in the development of feeding and settlement behaviour in ﬁsh.

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Figure 6.28 The smallest and largest prey eaten by cod larvae in a basin (E. Moksness, unpublished data, 1980).

6.6.12 Larval Growth Fish larvae are the smallest free-living vertebrates. Larval growth is generally fairly slow during the ﬁrst few days of exogenous feeding, and feed uptake is relatively small. This is followed by a period of rapidly increasing feed uptake and increased growth rates, and several ontogenetic changes contribute to this accelerated growth. During their development, ﬁsh larvae exhibit the highest known growth rates of all vertebrates. Some species may even exceed a 50% increase in body weight per day, and the larval dry weight of, for example, turbot and cod can increase 100-fold in 3 weeks. The relative growth and development of the head (brain, jaws, eyes, sensory organs) has a high priority in order to develop improved feeding abilities and the sensing of predators. Simultaneously, the relative length of the gut tends to show a strong allometric increase, and the gut mucosal surface will increase dramatically as the gut becomes wider and more ‘bulky’.This enables the larva to ingest and digest more food, thereby supplying more energy for growth. Also, as the sensory system of the larva becomes more advanced, it may develop a more selective feeding pattern. Since larvae show allometric growth patterns and much higher growth rates than adults, their nutritional requirements are different from those of adults. The quality and quantity of prey are the most important nutritional aspects to obtaining fast growth rates, and not whether the larvae are fed natural zooplankton or cultivated prey organisms. This section now focuses on speciﬁc patterns of larval growth, and further discussions of larval growth and nutritional requirements can be found in Chapter 7.

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Growth may be deﬁned as an increase in volume and weight over time. In aquaculture, the aim is to achieve the maximum growth (or increase in body weight) of each individual ﬁsh within a certain time. Knowledge of their nutritional requirements and digestive mechanisms is an essential key to obtaining good growth, and the growth rate and physiological efﬁciency of growth is largely determined by temperature, food quality and quantity. There are many ways of studying larval nutritional requirements, utilisation and digestion, such as measurement of larval growth and survival during different feeding regimes, physiological studies of larval metabolism, measurements of larval enzymatic capacity, and morphological studies of nutrient breakdown and digestion in the larval gut. All these methods are applied to obtain a better understanding of nutrition and growth in marine ﬁsh larvae.

6.6.12.1 How Can We Express Larval Growth? Growth in ﬁsh follows an apparent sigmoid curve. At ﬁrst, when the organism is small, there is an almost exponential increase in size, which levels out at a certain point. Several equations have been developed to describe general ﬁsh growth. However, rates of growth are usually more informative than actual growth. The growth of ﬁsh larvae can be expressed in terms of absolute growth rate (i.e. dW/dt, g day-1), or as speciﬁc growth rate (m, day-1). Since growth rate depends on the size of the ﬁsh, speciﬁc growth rate (SGR) is generally used.

SGR =

(ln Wt - ln W0 ) t

(6.1)

where W0 is the initial individual larval dry weight, and Wt is the individual dry weight at time t. Fish larval growth can be adequately expressed by this exponential function, which implies a constant speciﬁc growth rate during a speciﬁc time interval (see Chapter 7). The daily percentage increase in larval weight (%SGR) is calculated by %SGR = (e SGR - 1) ◊100%

(6.2)

Both equations can be used to calculate the growth rate between two successive determinations of biomass. The speciﬁc growth rate will change as the larvae grow, and several measures of growth should be made during the start-feeding stage. In order to study the effects of different feeding regimes or of food quality on larval growth, differences in growth rates must be observed. The relation between, for example, feeding ratios or nutrient levels and growth may then be expressed in dose–response curves. Larval growth is also often expressed as an increase in length. This is an easy parameter to measure, and may work well for certain purposes. However, length is only an indirect measure of biomass, and for some species such as cod, length seems more age-speciﬁc than growth-related, as young larvae of the same age and length may exhibit very different growth rates in terms of weight (Galloway et al., 1999a). Also, length cannot be used for nutritional and growth-efﬁciency studies.

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6.6.13 Inﬂuence of Diet Major research efforts have been directed towards the nutritional requirements of marine ﬁsh larvae, but they have been hampered in part by the difﬁculty of controlling the levels of proteins and lipids in live feed (Olsen, 1997; Shields, 2001). As discussed in section 6.6.3, growth is mainly an increase in muscle mass, i.e. mostly an increase in body protein. In experiments with turbot larvae, it was shown that high protein levels in the prey (rotifers) have a positive effect on larval survival and growth (Øie et al., 1997). Between 60% and 80% of larval dry weight consists of protein, which makes protein the largest body component (except for water). Fish larvae require relatively more protein (amino acids) than older ﬁsh, because of their higher growth rate (body protein) and their use of protein as the preferred energy fuel. The protein is continually renewed, and it is also the most costly component for the organism to synthetise. However, the main reason for the fast growth of ﬁsh larvae is their high rate of protein synthesis at a minimal theoretical cost (Houlihan et al., 1995; Pedersen, 1997; Conceicão, 1997). Larval amino acid requirements may also change during development due to the allometric development of organs (built up by proteins of different amino acid compositions). It is therefore important to understand the correlation between protein uptake, synthesis, digestion, assimilation and growth. The energy required for protein synthesis is a major part (50–70%) of the total larval energy consumption. Protein synthesis and oxidative metabolism are closely correlated, and the rapidly growing ﬁsh larvae seem to have both a very high oxidative capacity and a high capacity for protein synthesis. Free amino acids may be important, especially in early larval feeding, but their long-term effects have not yet been evaluated. It is not only protein that is important for the growth and development of ﬁsh larvae. A balanced diet, including micronutrients, which is suited to the stage-speciﬁc requirements of each species is necessary to obtain a high survival rate, normal development and optimal growth. Lipids are among the most essential feed ingredients for growth and development. Fish generally depend on a dietary supply of essential fatty acids, and especially docosahexaenoic acid (DHA, 22 : 6 (n-3)), eicosapentaenoic acid (EPA, 20 : 5 (n-3)), 18 : 3 (n-3) and arachidonic acid (ARA, 20 : 4 (n-6)). In marine ﬁsh larvae preying on wild zooplankton, more than 50% of the larval content of fatty acids will be 22 : 6 (n-3) and 20 : 5 (n-3). In several species, sub-optimal levels of essential fatty acids will generally result in retarded larval growth, abnormal development such as a non-inﬂated swim-bladder or spine deformities, and increased mortality (Rainuzzo et al., 1997; Sargent et al., 1997; Kanazawa, 1997; Izquierdo et al., 2000).The dietary levels of these fatty acids and the ratio between them are especially critical during larval development, as they are a major constituent in the developing eyes, brain, membranes and neural tissues, and they play a crucial role in the formation of important organs and tissues. Information on larval essential fatty acid requirements has been obtained for several species, although the speciﬁc requirements for most species are largely unknown. Studies of larval requirements for DHA, EPA and ARA have shown that both the quantity and the ratios between these fatty acids are important. In ﬂatﬁsh such as the Atlantic halibut, an increasing content of DHA is positively correlated with correct pigmentation, implying that the commonly observed malpigmentation in ﬂatﬁsh has a dietary cause

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(Kanazawa, 1997; Evjemo et al., 2003). HUFA content, but not the other fatty acid families, has been found to be responsible for the variation in growth and survival in striped bass: the higher the HUFA content, the better the survival and growth (Tuncer & Harell, 1992). The use of diets with a high DHA/EPA ratio (~1.5–2.0) generally shows better larval survival and growth than a low DHA/EPA ratio (less than 1), and cod larvae seem to be more susceptible than turbot. For turbot, there was a signiﬁcant positive correlation between fully pigmented fry and a high DHA/EPA ratio in the larvae at the end of the rotifer period (Rainuzzo et al., 1994; Reitan et al., 1994). Cod larvae were much more susceptible to low DHA levels during the ﬁrst days of exogenous feeding (the rotifer phase) than at later stages, and the difference in growth between groups was even more pronounced at metamorphosis (Galloway et al., 1999a; Hoehne, 1999; E. Kjørsvik et al., unpublished results, 1996). Of the essential fatty acids, it is clear that high DHA is important to neural development, but the required absolute amounts and EPA : ARA ratio seems to be species-speciﬁc. Micronutrients such as vitamin C, iodine and selenium may improve survival, normal development and stress-resistance in cultured ﬁsh. Diets differing in vitamin A source and content may induce differences in skeletal and eye structure in halibut. Food intake and endocrine function are usually closely related (Fig. 6.29). The thyroid, pancreas and pituitary glands all contain tissue arising from the embryological endoderm, the same as the gut (MacKenzie et al., 1998). Food deprivation will usually result in an

Brain: neuropeptide and catecholamine hormones neurotransmitters liver: growth factors interrenal: steroids

pituitary: protein and peptide hormones

carbohydrates, amino and fatty acids, micronutrients

gastrointestinal tract: GI peptide hormones thyroid: thyroxine

gonad: steroids

pancreas: pancreatic peptide hormones

Figure 6.29 Nutrient pathways to endocrine regulation. Marine ﬁsh larvae will not have all the organs depicted (e.g. gonads and fully functional liver), so the impact of initial diet is ﬁrst on the brain and its neurotransmitters and pituitary, as well as on the thyroid and interrenals. Redrawn and modiﬁed from MacKenzie et al., 1998.

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inhibition of thyroid function, possibly by inﬂuencing the hypothalamic–pituitary–thyroid axis at several levels. This may also reduce the sensitivity of the gland to stimulation, as well as reducing the ability of the liver and kidney to convert T4 to T3. This way of producing T3 may be more sensitive to total energy or protein intake than the mechanisms that produce circulating T4 (MacKenzie et al., 1998). Endocrine effects may also be secondary responses to the effects of the diet on growth or metabolism. It has been suggested that thyroid hormone production in ﬁsh is inﬂuenced by proteins, lipids and carbohydrates. Protein may affect active thyroid hormone production, whereas high carbohydrate levels can elevate plasma T4 under certain conditions. Eales et al. (1993) suggested that carbohydrates may be a more important regulator of T4 secretion in salmonids, and that protein may serve as a signal to activate its formation (cited in MacKenzie et al. 1998). However, the addition of exogenous thyroid hormone did not improve survival rates in summer ﬂounder (Bengtson et al., 2000), although its use is known to accelerate stomach development, and to synchronise settling behaviour (Gavlik et al., 2002). Thyroid hormone deﬁciency has been induced by glucosinolates in the diet (Higgs & Eales, 1978, cited in MacKenzie et al., 1998). Such deﬁciencies can also cause thyroid hyperplasia in brook trout. Fat-soluble vitamin A is necessary for the (re)generation of the lightsensitive rhodopsin in the retina and for other essential functions. Retinoic acid is also intimately involved in the ontogeny of any organism through its effects on differentiation and gene expression. Finally, some additional factors such as time of day for feeding and photoperiod also affect the expression of thyroid hormones and developmental events. Thyroid hormones can be synchronised by photoperiod, but nutrient intake appears to be necessary for thyroidal rhythmicity. Feeding time can phase-shift the peaks in the endocrine cycle, thus encouraging synergistic interactions between hormones.

6.6.14 Juvenile Quality The ontogeny of ‘ﬁsh quality’ is an integrated and complex process. Quality, or the viability of the ﬁsh, can be affected by nutrition (proteins, HUFAs, phospholipids, vitamins and trace elements), environment (light, density, temperature, water quality and tank colour), physiology (endocrinology, retinoids) and brood-stock quality. It is continually necessary to reﬁne larviculture techniques for growth and survival, as well as continuing to investigate the molecular basis for morphogenesis, and to reﬁne behavioural indices of quality which bring together many developmental parameters. The impact of diet on the developing ﬁsh depends on the developmental stage, since the digestive capacity, the endocrine and receptor systems, and the building blocks required are dynamic. For example, the early introduction of retinoic acids, which compete with thyroid hormones, can result in albinism and bone deformities, although later feeding with vitamin A, DHA and phospholipids may counter act this effect (Takeuchi, 2001). Growth and survival are not synonymous with normal development and juvenile quality. The morphological and physiological characteristics of farmed ﬁsh are a measure of their ﬁtness, which, combined with their behaviour, represent their juvenile quality (Fushimi, 2001). A complex causative origin is indicated, and much more work is needed.

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6.7 Hatchery Design For aquaculture purposes, it is standard practice to incubate all ﬁsh eggs in separate incubation facilities, and good environmental control and hygiene are important. The main differences in hatchery design are related to the demersal or pelagic nature of the eggs. It is especially important to operate within the environmental requirements of the different species during these early life stages, as all tissues and organs are being developed (either to a functional stage or to so-called proforms), and serious effects from suboptimal conditions may not become visible until long after the malformation has been induced. An increasing amount of evidence now demonstrates that the effects of brief suboptimal conditions during the early egg and larval stages often surface only much later in the juvenile or adult phase. A good example of how serious even small elevations in incubation temperature can be was recently demonstrated for salmon. The normal rearing temperature for Atlantic salmon is about 8°C. By raising the temperature to about 10°C, the incubation time was shorter, and no differences could be seen in hatching rate, alevin survival or growth, and many hatcheries gradually increased the incubation temperature. However, this relatively small change in temperature at the egg stage was shown to be responsible for the high incidence of malformations observed at the late juvenile stages (G. Bæverfjord, Akvaforsk, unpublished results, 2002). For cod eggs, which were believed to develop normally at temperatures between 1 and 10°C, no effects from temperature were found on egg survival, hatching success or observed normal development when eggs were incubated at very low temperatures (1°C), but the larvae hatching from these eggs were much less viable than those hatching from eggs incubated at higher temperatures between 5 and 8°C (Galloway et al., 1998).

6.7.1 The Demersal Eggs of Wolf-ﬁsh The demersal eggs of wolf-ﬁsh may be incubated in a similar way to salmonid eggs. However, in contrast to salmonids, eggs from wolf-ﬁsh are incubated in up-welling systems (Moksness & Pavlov, 1996). To prevent bacterial diseases and the growth of epibiotic organisms on the egg-shells, the periodic treatment of eggs with glutaraldehyde at 600 mg l-1 every third to ﬁfth day of incubation is carried out (Pavlov & Moksness, 1993). Normal hatching of embryos occurs at lengths greater than 20 mm, when the colour of the eggs changes from light orange to an opaque grey. The embryos are ready to hatch when the egg-shells become transparent, but a light mechanical pressure is required to induce hatching. Without this pressure, the embryos can remain inside the egg-shells and die. This method seems to reproduce the hatching repertoire in several species of Zoarcoidei when the guarding male or female coils around the egg-mass. This behaviour, leading to mass hatching of larvae, was also observed in wolf-eel (Anarrhichthys ocellatus Ayres) kept in an aquarium.

6.7.2 Pelagic Eggs (Cod, Turbot, Halibut) Egg incubation of the pelagic eggs of cod and ﬂatﬁsh is conducted in up-welling systems, and their hatchery systems were recently reviewed by Shields (2001). Pelagic eggs should

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also be disinfected before hatching. The eggs of cod, Atlantic halibut and turbot are immersed in 400 p.p.m. glutaraldehyde for 5–10 min at 6–12°C, depending on species and incubation temperature (Salvesen & Vadstein, 1995). The disinfection improves both hatching rate and larval survival, and the risk of transferring pathogenic bacteria within and between hatcheries is reduced. An increased larval feeding rate has also been observed in halibut larvae hatching from disinfected eggs compared with those hatching from non-disinfected ones.

6.7.2.1 Cod A system designed primarily for cod (70–200 l volume) includes a conical base for the collection of dead eggs and other debris, a water inlet, generally in the upper part of the incubator, a drainage port protected with plankton netting to prevent the loss of eggs and larvae, and an airlift system for proportional distribution of the eggs. Cod eggs (~1.3 mm) are usually incubated in the dark at 5–7°C, and the 5-mm-long larvae hatch after 15–20 days. The larvae start feeding 4–5 days after hatching. The temperature in the feeding stage is normally around 11–12°C, and larvae are stocked into rearing tanks at densities varying between 5 and 40 larvae per litre. Recently, larval densities up to 100–150 larvae per litre have been used successfully, although some workers have reported poorer growth with such high densities. It is important to remember that very high larval densities will probably require a more frequent addition of feed compared with lower densities, as a food shortage in the tanks may soon become a problem, with poor growth and cannibalism as a result. Much technological research and development is still needed for a more efﬁcient, large-scale rearing technique for pelagic marine larvae.

6.7.2.2 Turbot Incubators for turbot eggs are similar to those for cod, and a standard stocking density is about 3000 eggs per litre. The incubation of turbot eggs has been reported to be between 10 and 20°C, with optimum results at around 13°C. The 1–1.2-mm eggs will then hatch 6–7 days after fertilisation, and the larvae rely on yolk-sac reserves for 2–3 days before starting on exogenous food. The newly hatched larvae are normally stocked into rearing tanks at a density of 30–40 larvae per litre. The rearing tanks could be several cubic metres in volume, and are usually constantly illuminated. During the yolk-sac phase, the temperature is normally higher than the initial egg incubation level. The preferred larval rearing temperature is 18–19°C, although a broader range between 16 and 21°C may be used.

6.7.2.3 Halibut Egg Incubation The large pelagic halibut eggs (3 mm diameter) tend to be less buoyant than turbot and cod eggs, and are incubated at 5–6°C in up-welling cylindrico-conical incubators ranging in volume from 80 to 450 l, with a density up to about 600 eggs per litre. Halibut egg

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buoyancy changes with exposure to light, and a sinking reaction is especially apparent after blastopore closure (Valkner, 2000). Gentle aeration may be used, and egg incubation is carried out in darkness (light will also disturb the hatching process). An up-welling water ﬂow is used to keep them ﬂoating freely, with a daily routine of adjustments to the water ﬂow rate and the removal of dead eggs. Live eggs can be separated from dead eggs by stopping the water ﬂow and adding hypersaline water to the bottom cone of the incubators. The live eggs will then ﬂoat above the saline water, and dead eggs will sink to the bottom and can be removed with the hypersaline water before the water ﬂow is turned on again. After yolk plug closure (ca 65 day degrees (d°)), the halibut eggs are usually moved from the incubators, disinfected and stocked in larger conical tanks (silos) that are used for the yolk-sac phase. An up-welling water ﬂow from the bottom is also used in these silos, with the outﬂow near the surface. The stocking density is somewhat dependent on the size of the system, with about 10 eggs per litre in the largest silos (5–10 m3) and >20 eggs per litre in the smaller (0.5–1.5 m3) ones. Yolk-sac Larva Incubation Halibut eggs hatch at about 82 d°. A synchronised hatch can be obtained by exposing the nearly hatched eggs to light for a short period (this inhibits hatching), and then turning off the light to start the hatching process. The newly hatched halibut larvae are relatively large (~6 mm), but very poorly developed, and they have a very long yolk-sac stage (>40 days) compared with other pelagic marine larvae. They are also relatively fragile, and are sensitive to suboptimal environmental inﬂuences (temperature, salinity, mechanical disturbance, light and microbial activity). The larvae are usually kept in silos, as previously described, at 5–6°C in darkness until exogenous feeding starts. The general idea of silo management is to provide a stable environment with good water quality, but without mechanical disturbance of the larvae. Variable survival rates and high mortalities are still common problems, and the larvae often develop with serious malformations such as gaping jaws and yolk-sac oedema. The water ﬂow rate may be adjusted according to the vertical position of the larvae in the silo, usually 0.8–1.5 tank volumes per day for the larger silos, and more often for the smaller ones. The larvae are kept in the silos until they start exogenous feeding. They are transferred to start-feeding tanks at 220–270 d°, and the temperature is gradually changed to 9–12°C.

6.8 Critical Aspects of Larval Cultivation From the information given in this chapter, it is possible to deduce several consequences for aquaculture based on our general knowledge of egg and larval development. Sensitivity varies during embryonic development, and the embryo is especially sensitive to environmental effects during gastrulation (or epiboly). If halibut eggs are transported during this stage, for example, severe mortality generally occurs. It is therefore a good rule

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of thumb to transfer marine ﬁsh eggs only after the yolk plug has closed, although it should be done a least a few days before hatching, and before the hatching enzymes have weakened the egg-shell. Salmonids and wolf-ﬁsh should only be transported after the ‘eyeing stage’, i.e. after pigmented eyes have become visible in the embryo. The most critical aspect of larviculture is generally the very variable survival rate during the larval rearing stages. In turbot, for example, catastrophic mortalities may occur in the diet transition phase between the ﬁrst and second week after hatching. For ﬂatﬁsh, developmental abnormalities such as incomplete eye migration, malpigmentation, skeletal deformities or incomplete operculum also often appear at metamorphosis, and this may be due to inadequate nutrition, microbial conditions or other physical factors. It is still a challenge to fully understand the interaction between the larvae and the prey, and between the larvae and the environment. Environmental factors may also affect muscle ﬁbre recruitment in ﬁsh embryos and larvae, but little is known about how the environment might inﬂuence the muscle cell precursor population, or how maternal investment may affect egg and larval growth mechanisms. Identifying the developmental stages at which muscle precursor cells are most sensitive to external factors, and how egg quality, and maternal and environmental factors might affect muscle differentiation and growth in larval ﬁsh should therefore be further studied. The changes in the digestive system have a direct impact on rearing protocols because the ﬁsh can eat and utilise different feeds at different stages. In ﬂatﬁsh and some pelagic ﬁsh, metamorphosis is accompanied by settling to near the bottom of the tank, and the feeds no longer need to be live organisms. However, if the stomach is not fully differentiated, there is a risk of the feed being too complex. In such cases, despite an adult external appearance, the immature internal development will leave the ﬁsh unable to break down or absorb complex feeds, or there will be a risk of gut damage due to dry feeds of inadequate quality. The high ‘weaning mortalities’ often observed in cultured ﬁsh changing from live prey to formulated feeds may partially be the result of an inadequate understanding of speciesspeciﬁc digestive development at this stage. However, the digestive capacity of start-feeding marine larvae is high, and there is a growing understanding that formulated feeds may change (partly or completely) the present need for live prey organisms. Much research in this ﬁeld is still necessary to obtain such a goal. Prior to the development of functional gills and visible erythrocytes, larval marine ﬁsh require high and stable oxygen levels in the hatchery water. The development of gills and adult-type erythrocytes with haemoglobin for binding oxygen means that metamorphosed ﬁsh can physically occupy environments with lower or more variable oxygen conditions than was possible during the larval stage. The tight correlation between temperature, the oxygencarrying capacity of the water, and the narrow temperature optimum for normal growth of most ﬁsh larvae indicate that conditions for larval growth must be well controlled. The larva’s initial inability to focus means that high prey densities are necessary for them to have a chance of entering the ﬁeld of focus of a marine ﬁsh larva. It is also necessary to have reasonably high light and contrast levels between the prey and the background colours. However, once rods recruit, usually at around metamorphosis, the light levels may be lowered and the ﬁsh may actively avoid or seek out lighted areas, depending on the species. The

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attachment of muscles to the lens during metamorphosis means that the ﬁeld of focus extends and prey densities may be lowered without affecting the rate of capture. Farmed ﬁsh usually need higher light levels during larval feeding than at later stages. Prey densities need to be high to enter the ﬁeld of focus. During and after metamorphosis, more directionally speciﬁc responses to the environment are possible when the nares have formed, and the cephalic and trunk lateral lines have formed as almost closed canals. With increasing visual acuity, ﬁsh become more sensitive to their environment. Malformations and endocrine effects during metamorphosis may be a response to factors occurring much earlier in the life history, such as the period of ﬁrst feeding. Any factor (nutrition, environment, maternal, viral) impinging on the development of organs and tissues such as the brain, and hence the pituitary–thyroid axis, will affect the ability of the developing ﬁsh to complete metamorphosis. Much more research is necessary in this area to fully understand the importance of larval nutrition, neural development and functional differentiation. Larvae generally have only a non-speciﬁc defence system. The speciﬁc immune system generally starts developing before climax metamorphosis, and is often mature only long after the completion of metamorphosis. This means that hygiene is very important for the early life stages, especially around ﬁrst feeding, when the bacterial challenge can be high and the immune system is still very immature. Vaccination too early can cause tolerance rather than immunity. An intimate knowledge of these factors is necessary for the establishment of any species-speciﬁc immunisation programme. The transition from the larval stage to the juvenile stage is marked by metamorphosis. Metamorphosis involves every organ system, and is a complex process involving internal and external rhythms and stimuli. There may be several overlapping critical periods during which the appropriate nutrition and environment will contribute to the quality of the developing juvenile. When any critical stimulus is insufﬁcient, viable but unusual postmetamorphic forms may occur. Further studies of the molecular basis of morphogenesis will aid in determining the ways in which rearing protocols affect juvenile quality.

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Papandroulakis, N., Divanch, P. & Kentouri, M. (2002) Enhanced performance of intensive sea bream (Sparus aurata) larviculture in the presence of phytoplankton with long photophase. Aquaculture, 204, 45–63. Pavlov, D.A. (1986) Developing the biotechnology culturing White Sea wolfﬁsh, Anarhichas lupus marisalbi. II. Ecomorphological peculiarities of early ontogeny. J. Ichthyol., 26(6), 156–69. Pavlov, D.A. & Moksness, E. (1993) Bacterial destruction of the egg shell of common wolfﬁsh during incubation. Aquacult. Int., 1, 178–86. Pavlov, D.A. and Moksness, E. (1994) Reproductive biology, early ontogeny, and effect of temperature on development in wolfﬁsh: comparison with salmon. Aquacult. Int., 2, 133–53. Pavlov, D.A. & Moksness, E. (1995) Development of wolfﬁsh eggs at different temperature regimes. Aquacult. Int., 3, 315–35. Pedersen, B.H. (1997) The cost of growth in young ﬁsh larvae: a review of new hypotheses. Aquaculture, 155, 259–69. Pedersen, B.H., Nilssen, E.M. & Hjelmeland, K. (1987) Variations in the content of trypsin and trypsinogen in larval herring (Clupea harengus) digesting copepod nauplii. Mar. Biol., 94, 171–81. Pedersen, T. & Falk-Petersen, I.B. (1992) Morphological changes during metamorphosis in cod (Gadus morhua) with particular reference to the development of the stomach and pyloric caeca. J. Fish Biol., 41, 449–61. Pittman, K., Bergh, Ø., Opstad, I., Skiftesvik, A.B., Skjolddal, L. & Strand, H. (1990) Development of eggs and yolk-sac larvae of halibut (Hippoglossus hippoglossus L.). J. Appl. Ichthyol., 6, 142– 60. Pittman, K., Jelmert, A., Næss, A., Harboe, T. & Watanabe, K. (1998) Plasticity of viable postmetamorphic forms of farmed Atlantic halibut, Hippoglossus hippoglossus L. Aquacult. Res., 29, 949–54. Poling, K.R. & Fuiman, L. (1997) Sensory development and concurrent behavior changes in Atlantic croaker larvae. J. Fish Biol., 51, 402–21. Polzonetti, V., Cardinali, M., Moscori, G., Natalini, P., Meisi, I. & Carnevali, O. (2002) Cyclic ADPR calcium signaling in sea bream (Sparus aurata) egg fertilization. Mol. Reprod. Dev., 61, 213–27. Rainuzzo, J.R., Reitan, K.I., Jørgensen, L. & Olsen, Y. (1994) Lipid composition in turbot larvae fed live feed cultured by emulsion of different lipid classes. Comp. Biochem. Physiol. A, 107, 699– 710. Rainuzzo, J.R., Reitan, K.I. & Olsen, Y. (1997) The signiﬁcance of lipids at early stages of marine ﬁsh: a review. Aquaculture, 155, 103–15. Reitan, K.I., Rainuzzo, J.R., Øie, G. & Olsen, Y. (1993) Nutritional effects of algal addition in ﬁrstfeeding of turbot (Scophthalmus maximus L.) larvae. Aquaculture, 118, 257–75. Reitan, K.I., Rainuzzo, J.R. & Olsen, Y. (1994) Inﬂuence of lipid composition of live feed on growth, survival and pigmentation of turbot larvae. Aquacult. Int., 2, 33–48. Reitan, K.I., Rainuzzo, J.R., Øie, G. & Olsen, Y. (1997) A review of the nutritional effects of algae in marine ﬁsh larvae. Aquaculture, 155, 207–21. Roberts, R.J. (ed) (2001) Fish Pathology. Saunders, Harcourt, London. Rønnestad, I. & Fyhn, H.J. (1993) Metabolic aspects of free amino acids in developing marine ﬁsh eggs and larvae. Rev. Fish. Sci., 1, 239–59. Rønnestad, I., Finn, R.N., Lein, I. & Lie, Ø. (1995) Compartmental changes in lipid content, lipid class and fatty acid composition in developing egg and yolk-sac larvae of Atlantic halibut (Hippoglossus hippoglossus L.). Aquacult. Nutr., 1(2), 119–30.

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Chapter 7

First Feeding Technology Y. Olsen, T. van der Meeren and K.I. Reitan

7.1 Introduction It is quite a few years since early pioneers such as Dannevig ﬁrst succeeded in culturing fry of marine ﬁsh in enclosed pelagic ecosystems which simulated ‘nature-like’ conditions with natural zooplankton as live feed (Rognerud, 1887). However, half a century had passed before Rollefsen (1940) ﬁrst demonstrated that plaice larvae could be fed a zooplankton species that was not a natural component of the marine food web. This zooplankton species was Artemia, which is available from cysts all the year round (see Chapter 4). In this way, Rollefsen may have taken the ﬁrst step towards developing the hatchery methods for marine ﬁsh larvae that are now used world-wide. Further major improvements in feeding techniques did not take place until Japanese scientists cultured a smaller zooplankton species, the rotifer Brachionus plicatilis, and later learned that ﬁsh larvae had high requirements for highly unsaturated n-3 fatty acids (n-3 HUFA). The artiﬁcial food chain of rotifers and Artemia was not nutritionally adequate, but the n-3 HUFA content of the zooplankton could be enhanced by feeding procedures later known as n-3 HUFA enrichment techniques. The ﬁnal key to successful larval rearing was an understanding of the strong interactions between normal non-pathogenic bacteria and the ﬁsh larvae. These interactions were widely accepted during the 1990s (see Chapter 3). The production of ﬁsh larvae and viable fry has been a major obstacle in the development of a marine aquaculture industry. The challenges of larval ﬁrst feeding are highly multidisciplinary, and the physical–chemical, nutritional and microbial conditions of the larvae must all meet their requirements. The main interacting factors are:

• The conditions in the rearing environment, the physical–chemical cultivation regime and the technology used to maintain that regime • Larval nutritional requirements and the feeding regime • Larval interactions with normal and pathogenic bacteria, and microbial hatchery management.

As well as these interacting factors, the quality and viability of eggs and yolk-sac larvae are paramount for rearing larvae successfully. A number of other fundamental aspects become crucial during ﬁrst-feeding, where the process line from brood stock, to egg and yolk-sac larvae meets the process lines of the live food components in a completely new environment.

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This chapter describes the cultivation systems and methods that have been used to feed marine cold-water ﬁsh larvae from post-hatching to metamorphosis, or to weaning onto formulated feed (see Chapter 8). A wide variety of methods are described, but the main focus is on methods based on traditional rotifer and Artemia technology (see Chapter 4). The nutritional and microbial factors and their interactions during ﬁrst feeding are of particular importance, and the fundamental aspects of these issues are presented in Chapters 3 and 4, respectively. Physical factors that are important in mariculture are generally addressed in Chapter 2, and are only covered brieﬂy here. The most fundamental issues of larval morphological and physiological development that are important to an understanding of the complex nature and constraints of larval feeding are presented in Chapter 6. However, this chapter includes a short section on lipid and protein requirements, since these are essential to any discussion of the nutritional aspects of cultivation.

7.2 Nutritional Requirements of Marine Fish Larvae 7.2.1 Essential Fatty Acids Fatty acids are characterised by the number of their carbon atoms and double bonds. Fatty acids that can be synthesised de novo by animals (non-essential fatty acids) include saturated and monounsaturated fatty acids, with no and one double bond, respectively. Animals cannot synthesise fatty acids with two or more double bonds (polyunsaturated fatty acids, PUFA), although these are essential for growth and development. These fatty acids must be supplied in the food, and are known as essential fatty acids (EFA). EFA are grouped into two families: the linoleic acid (n-6) and the linolenic acid (n-3) families (Fig. 7.1). Highly unsaturated fatty acids (HUFA) are PUFA with 20 or more carbon atoms. Both animal and plant cells can catabolise, elongate and desaturate fatty acids through successive steps. However, animal cells cannot desaturate fatty acids closer than carbon 9–10 from the methyl end (n-9). This implies that all n-6 and n-3 fatty acid bonds of marine origin have been formed by organisms of other kingdoms, among which marine algae are the most important. Many carnivorous ﬁsh species, including species that are attractive for aquaculture, have little metabolic capacity to elongate and desaturate the shorter C18-precursors of the n-6 and n-3 families. However, they are able to modify HUFA through catabolic chain shortening reactions (Sargent et al., 1991). High contents of n-3 HUFA, e.g. eicosapentaenoic acid (20 : 5 n-3, EPA) and docosahexaenoic acid (22 : 6 n-3, DHA), are found only in aquatic plants and animals. These fatty acids are essential components for neural tissues, cell membrane functioning and many regulatory functions. Generally, n-3 fatty acids dominate n-6 fatty acids by a factor 5–20 in marine food webs, but the n-6 HUFA arachidonic acid (ARA, Fig. 7.1A) is also essential for marine ﬁsh.

7.2.2 Main Lipid Classes Lipids are grouped as neutral or polar lipids depending on their polarity (Fig. 7.1B). Triacylglycerides (TAGs) and wax esters (WEs) are neutral and abundant storage lipids which

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A

281

FAMILIES OF ESSENTIAL FATTY ACIDS Linolenic acid family, n-3

Linoleic acid family, n-6

linolenic acid, 18:3 n-3

linoleic acid, 18:2 n-6

H C 3

COOH

HC 3

COOH

eicosapentaenoic acid, 20:5 n-3 (EPA)

arachidonic acid, 20:4 n-6 (ARA) COOH

HC 3

H C 3

COOH

docosahexaenoic acid, 22:6 n-3 (DHA) COOH HC 3

B

MAIN CLASSES OF LIPIDS IN MARINE ANIMALS

NEUTRAL LIPIDS: Tri-acylglycerids (di-, mono-, glyco-)

: COOH

: OH

POLAR LIPIDS: Phospholipids

Wax esters

P

R

Figure 7.1 Characteristics of essential fatty acids and abundant lipid classes. (A) Schematic overview of the main fatty acids of the n-3 and n-6 families of essential fatty acids. (B) Main lipid classes of marine animals.

provide metabolic energy through oxidative catabolism. The TAG molecule is the dominant energy and carbon storage product in higher animals. Many zooplankton species populating cold waters, either in the deep parts of the oceans or at high latitudes, store their surplus energy as WE, which is formed by a long-chain monounsaturated fatty alcohol moiety bound to a fatty acid moiety. WEs have a lower melting point and a higher energy content per unit weight than TAGs. Zooplankton that store WE may exhibit very high contents towards the end of the growth season (>50% of dry weight, Sargent & Henderson, 1986). The basic unit of phospholipids (PL) is a diacylglyceride molecule with a phosphate group bound to an organic group in a terminal position of the glycerol (R, Fig. 7.1B). Common organic groups of PL are ethanolamine, choline and inositol, and the respective PLs are denoted phospatidylethanolamine, phospatidylcholine and phospatidylinositol. Aquatic coldblooded animals are characterised by having PL containing a high fraction of PUFA. PL, together with cholesterol and sphingolipids (not shown), are ubiquitous constituents of cell membranes, and are therefore both structurally and functionally important.

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Feed lipids

Ingestion Digestion

Intestine

Assimilation

Tissues Cell membrane Metabolism (chain elongation synthesis and desaturation) Growth Regulation Exchange of molecules/ions

20:5 n-3 (EPA)

Synthesis of tissue hormones Prostaglandin G3 Prostaglandin G2

20:4 n-6 (ARA)

Regulatory functions

Figure 7.2 Schematic view of the uptake, assimilation, metabolism and functional roles of essential n-3 and n-6 fatty acids in animals.

7.2.3 Physiological Basis of n-3 HUFA Requirements Marine coldwater ﬁsh larvae need high proportions of n-3 HUFA to meet their requirements for growth and development. The experimental determination of the n-3 HUFA requirements for the early stages of marine ﬁsh larva fed live zooplankton has proved to be quite complicated. Therefore, major questions regarding their n-3 HUFA requirements still exist for many species. EFAs supplied in the food are crucial for the following metabolic and physiological processes (Fig. 7.2):

• Growth or de novo formation of biomass: tissue formation and differentiation • Membrane activity and metabolism: membrane transports, respiration and activities • Regulation of metabolism: prostaglandins and their hormonal functions

enzyme

The physiological justiﬁcation for the relatively high n-3 HUFA requirements of marine cold-water ﬁsh is associated with PL synthesis and the formation of cell membranes. PL is

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synthesised by enzyme systems that exhibit a higher afﬁnity for polyunsaturated fatty acids (e.g. n-3 HUFA) than for other fatty acids. Under conditions of excess supply, these fatty acids are preferentially esteriﬁed to the sn2 position of the glycerol in both TAG and PL, whereas saturated and monounsaturated fatty acids are more frequently esteriﬁed to the terminal sn1 and sn3 positions. This discrimination explains why one out of two fatty acids of PL is expected to be polyunsaturated, whereas one out of three tends to be polyunsaturated in TAG (Sargent et al., 1991). This enzyme mechanism, along with other differences in enzyme speciﬁcity, secure some degree of genetic control in the composition of the membranes. This is critical from a functional point of view. However, the enzymes may incorporate enhanced levels of monounsaturated and saturated fatty acids in the membrane PL under conditions of n-3 HUFA deﬁciency. This is expected to result in reduced n-3 HUFA contents in the membranes (Sargent et al., 1991), and will, at some point which is species-speciﬁc, result in the gradually reduced activity of all membrane-bound enzymes. As a consequence, physiological capacity or general health is reduced. Inadequate dietary n-3 HUFA supplies may therefore have a general impact on animal function and health. Larval requirements of n-3 HUFA are strongly related to growth and tissue differentiation, which include eye, brain and neural development. Neural tissues contain very high amounts of DHA (Mourente et al., 1991), implying that this fatty acid is particularly important for very young and fast-growing stages of ﬁsh larvae. The speciﬁc growth rates of ﬁsh larvae are normally much higher than those for the adult ﬁsh. Larvae of cold-water species, e.g. cod (Gadus morhua) and Atlantic halibut (Hippoglossus hippoglossus) will double their biomass within a week or two, and some fast-growing species such as turbot (Scophthalmus maximus) will do so in 3 days. From an evolutionary point of view, we may assume that the essential components that are found in high quantities in the eggs are important in order to maintain normal growth and development during the early stages of life, and eggs of marine species do contain high levels of DHA. It is also easy to imagine that an inadequate brain function and vision, as demonstrated for DHA-deﬁcient herring larvae (Sargent et al., 1993), will be fatal for marine larvae and other animals. It is likely that pronounced species-dependent differences exist in the n-3 HUFA requirements of marine ﬁsh larvae. Our quantitative knowledge of the n-3 HUFA requirements of marine ﬁsh larvae is inadequate, and there is no general conceptual understanding of the factors which determine these requirements. It is assumed that ﬁsh living at lower temperatures at high latitudes or in deep water will exhibit higher n-3 HUFA requirements than ﬁsh living in warmer water. Independent of temperature, it is further assumed that fast-growing species will have higher n-3 HUFA demands in order to develop functional membranes. Another factor is the ability of the species to elongate and desaturate shorter n-3 fatty acids to form EPA and DHA. A high metabolic ﬂexibility in order to modify short-chain n-3 fatty acids may contribute to reduced requirements for n-3 HUFA. Such metabolic ﬂexibility is apparently characteristic of many salmonids, including rainbow trout. Conversely, it is commonly believed that carnivorous marine ﬁsh have a lower ﬂexibility than omnivorous and detritivorous species (Olsen, 1999a). Presumably it has not been important for carnivorous ﬁsh species to retain the facility for fatty acid elongation and desaturation during evolution because they naturally eat a well-balanced diet according to their speciﬁc requirements. Most

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of the marine species of interest for mariculture are indeed carnivorous with high n-3 HUFA requirements. The optimisation of diets with respect to n-3 HUFA contents is therefore an important issue in marine larval ﬁsh nutrition. The C20-fatty acids EPA and ARA of the membrane phospholipids are precursors in prostaglandin synthesis (see Fig. 7.2). The prostaglandins are precursors for a number of regulating compounds known as tissue hormones. Prostaglandin G3 is synthesised from EPA and acts as a regulatory antagonist to prostaglandin G2, which is synthesised from ARA. Both ARA and EPA are derived from the membrane PL through the actions of phospholipases. The G3/G2 ratio, which is believed to modify many cellular processes, will depend on the ratio of EPA to ARA in the membranes, and ultimately in the diet. The relatively poor enzymatic control (i.e. genetic control) of PL composition (i.e. the ratio EPA to ARA), and the successive prostaglandin synthesis (G3/G2 ratio), implies that the fatty acid composition of the food will directly affect the regulatory hormonal processes of ﬁsh larvae and other animals.

7.2.4 Protein and Essential Amino Acids Amino acids are the fundamental components of enzymes, other proteins and nucleotides, and they are important precursors or N-sources for a wide range of biomolecules. Protein is quantitatively more important than lipids for larval growth, and protein malnutrition was understood long before lipid malnutrition. The amino acids that are assumed to be essential, or conditionally essential, for ﬁsh are arginine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, threonine, tryptophane, valine, cystine and tyrosine. Dietary proteins are the main source of essential amino acids. This chapter only presents results that clearly demonstrate the importance of protein nutrition during ﬁrst feeding (Section 7.5.6). The protein requirements of ﬁsh are covered thoroughly in Chapter 9, and the requirements and digestion capabilities of speciﬁc ﬁsh larvae are discussed in Chapter 6.

7.2.5 Protein Versus Lipid Nutrition There is a fundamental biological difference between protein and lipid nutrition. The sequence of amino acids of enzymes, and the further synthesis of biomolecules mediated by these enzymes, are strictly genetically controlled. This means that the regulatory functions of proteins are under the strict control of genes. In addition, the percentage amino acid composition shows little variability between species, and is only moderately affected by nutrition. This means that the amino acid composition of the prey will be relatively close to that of the predator. This is contrary to the situation for lipids, which have regulatory cell functions, e.g. prostaglandin precursors. The fatty acid composition of these lipids is only moderately controlled by genes, and the fatty acid composition in the food is far more variable than the amino acid composition. This is why deﬁciencies in speciﬁc n-3 HUFA is more readily expressed than deﬁciencies in speciﬁc EAA. Because of these fundamental differences in (1) variability in distribution and (2) genetic control of lipid and protein metabolism, there is a greater probability that marine ﬁsh larvae

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will encounter a deﬁciency of speciﬁc EFA than of EAA. However, proteins may still be supplied in sub-saturating quantities compared with growth requirements. An inadequate supply of protein is likely to occur, and will be expressed as a reduction in the speciﬁc growth rate of the larvae. Other disorders may occur with severe deﬁciencies. Conversely, symptoms and disorders caused by n-3 HUFA deﬁciency develop more gradually (see above), and a severely reduced growth rate is more likely.

7.3 Deﬁnitions and System Description The principal differences in the methods used by the pioneers of marine larviculture can still be seen in more recent methods of raising cold-water ﬁsh species. Two extreme cultivation concepts, and one intermediate one, can be deﬁned (Fig. 7.3). Concept 1. Larval Feeding in Large Closed Nature-like Systems Larval densities are low (<20 m-3), production yields are within the carrying capacity of nature, and production is highly seasonal. The degree of intervention and the degree of production control are accordingly low (e.g. no feeding). Accumulated empirical knowledge may be as important as fundamental knowledge about components and systems. Concept 2. Larval Feeding in Relatively Large Suspended Mesocosms, Enclosures or Outdoor Tanks Larval densities are relatively low (0.1–5 l-1) and production yields are enhanced through the addition of feed. Both harvested and cultivated zooplankton are used. The degree of intervention is higher than for Concept 1 (e.g. food addition), and the production is seasonal. Some degree of production control is achieved through feed addition and the use of smaller volumes. Some fundamental knowledge about systems and components is needed, as well as empirical knowledge. Concept 3. Larval Rearing in Relatively Small Tanks: Classical Intensive Hatchery Techniques Larval densities are relatively high (10–150 l-1) and production yields are strongly enhanced through intensive feeding, normally with cultivated zooplankton and microalgae. The system is completely artiﬁcial, and the degree of intervention is accordingly high. The production is thus continuous and independent of season. The objective is production control, and fundamental knowledge about system components and their interactions is therefore crucial. The intensive rearing method (Concept 3) represents the current international standard for marine larval rearing, and is the main focus of this chapter. The remaining concepts, which are frequently denoted extensive (Concept 1) and semi-intensive (or semi-extensive) (Concept 2) larval rearing, were important in the early phase of developing a fry production technology for many marine cold-water ﬁsh species such as Atlantic cod and Atlantic halibut.

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Figure 7.3 Illustration of the generalised production concepts that have been used to produce juveniles of coldwater ﬁsh species, here exempliﬁed for cod. Upper panel, schemes for larval rearing methods (van der Meeren & Naas, 1997). Lower panel, semi-extensive rearing unit suspended in a coastal lagoon.

Harvested copepods are still used in the commercial production of ﬁsh fry in some countries, in particular for Atlantic halibut (see van der Meeren & Naas, 1997). The zooplankton is normally harvested from coastal lagoons. This technology has never been shown to be economically feasible, and it has restricted potential for industrial development. However, experience to date has shown that copepods, which are believed to be the natural food of the ﬁsh larvae, can meet the nutritional requirements of cold-water ﬁsh larvae, and in particular the requirements for n-3 HUFA, and especially DHA (Shields et al., 1999). A frequent

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approach has therefore been to use copepods as a reference during the development of an intensive technology based on rotifers and Artemia for cold-water marine ﬁsh.

7.3.1 Extensive Systems: Large Closed Nature-like Systems Coastal lagoons are natural enclosed systems that may range from a few thousand to several million cubic meters. They often have a narrow inlet to the fjord outside, but the water on each side of the inlet is often different in, for example, species composition, diversity and hydrography. This may be the result of fresh-water run-off from land, or the water exchange rate as determined by the entrance size and tide height. Not all lagoons are suitable for rearing marine ﬁsh larvae. The infrastructure, the size of the lagoon, the depth-to-surface ratio, and the control of water ﬂow in the system are key factors. Converting a lagoon into an extensive production unit (see Fig. 7.3) means closing the system off from the open sea in order to control the hydrography, stimulate copepod production, and reduce the impact of potential competitors on larval prey (Øiestad et al., 1985). Lagoons that have been used for extensive commercial rearing of marine cold-water species (to date only Atlantic cod) are typically 60 000 to 1.6 million m3. For experimental purposes, extensive production of cod has also been carried out in 2500 and 20 000 m3 in land ponds. The most successful lagoons seem to be the smaller ones, i.e. <250 000 m3 and 10 m deep. This probably reﬂects the fact that plankton production and the hydrographic conditions are easier to control in these lagoons. The typical output of a lagoon system has been 1–2 juvenile per cubic metre. The construction and management of lagoons include several steps which depend on the chosen production strategy. First, fresh-water run-off from the land should be minimal, and the inlet should be narrow and shallow. A sealed concrete dam is constructed in this inlet, often with devices that allow a controlled exchange of water through grates with, for example, 4-mm holes. High-capacity pump systems (e.g. 12 m3 min-1) are often used for water exchange by pumping water in at the bottom of the lagoon, and ﬂushing excess water over the dam. For sizes up to 60 000 m3, it is possible to drain the system completely with pumps, and this may be necessary in order to eliminate predators or competitors. For bigger lagoons, draining is too time-consuming and expensive, and predator control must be achieved by using rotenone to kill them (Næss et al., 1991). Rotenone will not seriously affect the resting copepod eggs, which will be in the sediment. These resting eggs may even survive complete drainage of the lagoon as long as they are kept moist and unfrozen. Resting eggs may also survive the oxygen-depleted water which is often found just above the sediment in a lagoon. The resting eggs provide predictability in the timing of copepod production (hatching of nauplii) and the release of ﬁsh larvae into the lagoon. The management of lagoons during extensive larval rearing has developed and changed slightly over the last decade. The following description is from the present management procedures of the only lagoon that has been successful in the production of juvenile cod throughout the 1990s. This lagoon has a volume of 250 000 m3 and a depth of 10 m. To prevent oxygen depletion in the bottom layers, continuous mixing of the water column is carried out by a propeller mounted on a raft in the lagoon. The size of the lagoon allows drainage, and the addition of rotenone and the complete sealing of the dam in November gives predator

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control. No exchange of water takes place during the winter, and the rotenone is broken down into harmless compounds within the system. The propeller also mixes the water during the rotenone treatment. In early February, water is pumped into the bottom of the lagoon through a 1-mm ﬁlter. Excess water goes out over the closed dam. A few weeks later, the gratings in the dam are opened, allowing a tidal exchange of water. For smaller lagoons or ponds that can be drained by pumps, a complete exchange of water is undertaken, also during February before the spring bloom starts. The lagoons are therefore ready for conditioning at this time. Conditioning involves fertilisation with a Cl-free NPK complex (Naas et al., 1991). This is done to enhance phytoplankton growth and the oxygen conditions in the lagoon, and the fertilisation is usually accompanied by the addition of sodium metasilicate to encourage diatoms which may prevent the growth of poisonous dinoﬂagellates in the system. Fertilisation takes place throughout the production season. The eggs are normally hatched indoors in 70–250 l tanks. With cod, the yolk-sac larvae are released on Days 1–3 post-hatch in late March, shortly after the phytoplankton spring bloom. The gratings in the dam are then closed, but pumping of water into the bottom of the lagoon is continued. During the early developmental stages, the larvae will normally stay at the bottom of the lagoon during the daytime. This may be because there is a higher density of micro-zooplankton in the layer of water just above the bottom. It also prevents larvae from escaping from the lagoon through the overﬂow. Later (3–4 weeks post-hatch), the larvae may be seen more frequently in the surface layers where the larger copepod stages stay during the daytime. Larval growth and plankton densities should be monitored together with the hydrography. Actions such as increased pumping of water or adjusting the propeller angle and speed may be required to prevent oxygen depletion in the bottom layers. The larval food produced in the lagoons is mainly small copepod species (e.g. Temora longicornis or Eurytemora afﬁnis) in the shallow systems (5–10 m deep), while the larger copepod Calanus ﬁnnmarchicus is more important in deeper (10–20 m) lagoons. Rotifers and trochophora larvae may also be important food for the young larvae, but copepod nauplii are required to promote larval growth. The timing of larval release and the peak of naupliar hatching is important, but copepods and nauplii may also originate from water entering the system. The nauplii will hatch over an extended period, providing larval food outside the hatching peak as well. At later larval or early juvenile stages, the copepods will be grazed down and the ﬁsh either have to be caught and transferred to tanks for weaning onto formulate diets, or given additional live feed spread throughout the lagoon. The gratings in the dam may also be opened to allow a tidal inﬂux of copepods. Extra ﬁltration units outside the dam may be used to concentrate copepods from the sea and ﬂush these into the lagoon. Large plankton ﬁlters capable of ﬁltering more than 100 m3 water per minute are now commercially available, and copepods may be supplied over an extended period, even before prey depletion. The challenge of successful extensive rearing will be to balance the number of released larvae with the expected copepod production so that depletion of prey takes place as late as possible. The initial larval densities should therefore be somewhere below 20 larvae m-3. Normally, the addition of formulated feed to the lagoon starts around Day 40 post-hatch. The food is distributed in the current of the propeller mixing the lagoon water. Harvesting typically starts around Day 60 post-hatch. The cod juveniles are usually harvested with huge

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lift nets (10 m diameter and 4 mm mesh size) mounted on, for example, a hydraulic crane. This requires that the ﬁsh are of a certain size (>35 mm) in order to withstand the handling in the catching process. From the lift net, the ﬁsh fry are either pumped or netted into tanks on land, or into net pens outside the lagoon, where further growing-on takes place.

7.3.2 Semi-intensive Systems: Large Suspended Mesocosms, Enclosures or Outdoor Tanks Many of the lagoons and ponds used for semi-intensive fry production have also been used as extensive rearing units. The semi-intensive rearing systems are typically plastic bag systems ﬂoating in lagoons, or huge concrete tanks on land built up in conjunction with lagoons. The lagoon is then mainly used for live food production, and it appears that the dominant organisms produced in such enclosures are copepods. The densities of copepods can be far higher than in the open sea, up to 1600 copepod nauplii per litre have been observed in a 20 000 m3 pond. More normal densities will be 50–500 nauplii and 20–200 juvenile and adult copepods per litre, and production can occur from March to November. In contrast to extensive rearing, the larval food has to be supplied to the larval rearing units in semi-intensive systems, and fry production may in theory take place several times during a year. As for the extensive approach, lagoon management in the semi-intensive method involves the optimisation of plankton production. Unlike the extensive approach, semi-intensive lagoons will have a ﬁlter system on a raft inside the lagoon itself. This is to concentrate different size fractions of copepods according to the needs of the larval rearing unit. Wheel ﬁlters have generally been used (van der Meeren & Naas, 1997), and such ﬁlters seem to treat the plankton gently enough to sustain good survival of the copepods. Along with the ﬁlters, there is sometimes a holding system for the collected plankton. This could be tanks on the rafts or ﬁne-meshed net pens ﬂoating in the lagoon. Because of the high density of copepods accumulated in the holding system, the use of oxygen or aeration may be required. The collected copepods could also be supplied directly to the larval rearing units through tubes. The larval rearing units used are large plastic enclosures tied to a raft system either inside or outside the lagoon (Fig. 7.3, lower panel) or, alternatively, large tanks on land. It may be risky to suspend the rearing units inside the lagoon because any disease outbreaks in the enclosures may be recirculated in the lagoon. A typical semi-intensive rearing unit may consist of 4–8 large enclosures. Each enclosure is normally made of a 3-layer woven polyethylene (PEL) plastic sheet because of the strength relative to the weight of this material (van der Meeren et al., 1994). The bags are cylindrical, with a conical bottom. Bag volumes are in the range 40–120 m3, with 4–6 m diameter and 4–8 m depth. Water may be supplied either at the surface or through a ﬂexible tube entering the bottom in the centre of the bag. The outlet is a large sieve of plankton net inside the bag and mounted on a ﬂexible tube that connects to the outside water through the plastic bag wall. The sieve may be positioned either at the bottom or the surface, according to where new water enters the system. In the case of halibut, a plastic collar is used to shadow the surface water. The raft system with bags must be placed in a sheltered area, because the bags cannot stand even moderate waves.

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The bag enclosures should be conditioned 2–3 weeks before larval stocking. The addition of fertiliser will ensure phytoplankton production in order to maintain oxygen levels and reduce the ammonia produced by ﬁsh larvae and copepods. The temperature and salinity of the water used for renewal should be the same as that inside the bags. This is not a problem if the rearing unit is inside a well-mixed lagoon. If not, there may be a choice between using deep water or surface water for renewal. Deep water is normally saltier than the surface water, and this may put extra pressure on the plastic sheet. Deep water is also normally colder, which may lead to conditions of gas supersaturation in the enclosures or tanks. Surface water is therefore preferable, and should be ﬁltered to at least 80 mm to prevent unwanted organisms entering the bags. The possibility of a high water exchange rate is very limited, and for this reason no more than 1–2 larva per litre should be stocked in the bag. During rearing, the hydrography and feeding conditions should be closely monitored. In cases of high survival rates (e.g. <50%), there may be problems with high organic loads in the rearing unit in the late larval stages. In particular, this may be the case if a shortage of copepods requires that Artemia also has to be used as food. Because of the low temperature, Artemia tend to sink and accumulate in the bottom of the system before being eaten, while copepods will be distributed in the upper layers of the bag. The water exchange rate of the enclosures is restricted owing to the limited strength of the plastic sheet, and a substantial proportion of the organic particles in the bag need to be decomposed in the system. Although the bottom water is frequently removed by the pump systems, cleaning the bags is very difﬁcult, especially for larger units. Some action can be taken to improve rearing procedures, and this may involve the shape and size of the bags as well as the feeding procedures. A longer cone in the lower part of the bag may allow better bottom cleaning. A continuous supply of live food at the surface over time may allow the prey to be eaten constantly, thus reducing the organic load. Unless the bags have a proper system for the removal of organic wastes from the bottom, formulated feeds should not be used in bag systems. Harvesting the ﬁsh fry can take place in several ways. The catching process induces stress in the fry, since they tend to avoid the different gears or devices lowered into the bag water. Catching fry in landing nets and dry transfer to tanks or buckets should be avoided, particularly for small fry. Different kinds of lift nets, in combination with feeding or the use of light to attract the fry, may work well. Also, a reduction in the volume of the bag by pumping out water may help harvesting, and the use of ﬁsh pumps has also proved useful. The use of tanks on land allows slow drainage of water from the tanks until the fry can be caught with nets or buckets. After harvesting, the fry are weaned onto formulated diets in intensive rearing systems.

7.3.3 Larval Rearing in Relatively Small Tanks: Classical Intensive Hatchery Techniques The intensive hatchery techniques that are used for marine cold-water ﬁsh are very much the same as those used for other species, e.g. sea bass and sea bream. However, there are systematic differences in their biological requirements. Relatively few commercial hatcheries so far established for cold-water ﬁsh species have been based on international intensive cultivation technology. Most experience originates from experimental hatcheries and

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Recolonisation by bacteria (biofilter unit, flow through) Disinfection/sterilisation (UV, ozone, membrane filters)

Water treatment Microalgae

Hatchery processes Harvesting

Rotifers

Optional feeding (enrichment)

Rinsing Concentration Storage

Rinsing ST-Enrichment Cultivation Concentration LT-Enrichment Artemia

Hatching

Rinsing

Shor t-term enrichment

"Green water" technique

Fish larvae

Rinsing Concentration Storage

Figure 7.4 Schematic illustration of water processing and production process lines during intensive rearing of marine ﬁsh larvae. (Redrawn from Hoehne, 1999.)

research laboratories. The processes of water treatment and hatchery operation are illustrated schematically in Figure 7.4. It is important to emphasise that this scheme is simpliﬁed, and that virtually all hatcheries will have their own procedures and infrastructure characteristics. 7.3.3.1 Water Treatment and Supply The quality of the water source is important, and groundwater or seawater taken from deeper layers is better than surface water. The water is ﬁrst treated to remove larger particles such as zooplankton, large algal cells and dead organic particles. This can be carried out using successive ﬁltration through sand ﬁlters with variable pore sizes. The water is then disinfected or sterilised to remove small organisms, including pathogenic bacteria. The hatcheries use different methods for this, but the traditional method was to use UV disinfection with systems where the water ﬂow passes UV lamps. This method will only reduce bacterial biomass and activity, and its efﬁciency depends on light intensity and how the system is designed and maintained. High-pressure membrane ﬁlters that remove particles smaller than

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1 mm may readily remove more than 95% of the bacterial cells. Their efﬁciency in removing bacteria is higher than that for UV treatment. Treatment by ozone is very efﬁcient at killing bacteria and other organisms, and it may ultimately sterilise the water. There are still some doubts about UV and ozone treatments because there is the danger of enhancing the activity of free radicals that are toxic to living organisms. Membrane ﬁltration is thus an attractive method because it is a purely physical process. Experience to date has revealed that the above methods may all be adequate, but the issue should be kept under continuous review. The disinfected or sterilised process water should be re-colonised by useful bacteria before being used for ﬁsh larvae. Such controlled re-colonisation, or microbial maturation, is a simple security measure against an outbreak of fast-growing opportunistic bacteria that may be harmful to ﬁsh larvae (see Chapter 3 and below). Re-colonisation may be carried out by allowing the water to ﬂow through an aerated bioﬁlter system before going into the larval unit. Such systems are both cheap and easy to install as a ﬁnal step in the water treatment. Regrettably, the bacterial community of the process water will also go through a maturation process while passing through tubes in the hatcheries and in the larval tanks. If the water exchange rate of larval tanks is low, this will contribute to maturation and adequate water quality. However, a bioﬁlter is always a means to increase control by re-colonisation, and this will be additional security against an outbreak of opportunistic bacteria. There is a general need to maintain temperature control in larval cultures, but many hatcheries and research laboratories have not installed the equipment needed for maintaining such control. Instead they depend on the ambient water temperature. Temperature control may be carried out using adapted heat exchange systems that can deliver process water at a constant temperature. Such systems are technically varied, but it is important to recognise that the amount of water used in hatcheries is relatively low. The last treatment that is essential for the predictable production of fry is an efﬁcient removal system for excesses nitrogen gas in the water. This must always be done after heating the water, which should be carried out before bacterial re-colonisation. The process of recolonisation involves intensive aeration that will take care of the oxygen supply, mix the water in the bioﬁlter, and remove excess nitrogen. A sound strategy which is commonly used is to aerate the water again locally just before it enters the larval tank. This is always necessary if water at different temperatures is mixed just before entering individual tanks (see below). 7.3.3.2 Production Lines for Live Feed The schematic processing lines from the production of live feed to larval feeding are almost identical for all cultured marine ﬁsh species world-wide (Fig. 7.4). The main modiﬁcations needed to adapt the technology to cold-water species are related to the methods of n-3 HUFA enrichment of rotifers and Artemia (see Chapter 4). Microalgae are mainly used as a supplement which is added to larval rearing tanks in order to enhance and stabilise production (the ‘green water technique’; see below). The biomass of the algal cultures is measured using a variety of methods, but cell counts and

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measurements of culture turbidity, which are both convertible to biomass (dry matter or carbon), are probably the most common. The algal culture is added directly to the larval tanks and maintained in concentrations of <2 mgC l-1. Algae may also be used to enrich rotifers and Artemia post-harvest, in order to modify their nutritional value and microbial composition (see below). The process of rotifer supplementation (Fig. 7.4) involves cultivation, optional short-term enrichment and several rinsing steps. As shown, and discussed in Chapter 4 and below, it is likely that short-term enrichment of lipids can be omitted, because rotifers can be adequately enriched by n-3 HUFA during cultivation. This simpliﬁes the process of rotifer production, but there may be a need for other short-term treatments in order to improve the rotifers’ nutritional or microbial quality (see below). In all events, there is the need for a system that allows thorough rinsing and adjustment of rotifer density just prior to their use as live feed. This is a laborious process that requires adequate ﬁltration equipment that is partly automated. Filtration is carried out using speciﬁc devices equipped with nylon plankton net, cylindrical ‘stainless steel’ screens, and recently also a stainless steel-based textile of appropriate screen size for rotifers (see Dhert, 1996). The tank sizes used during live feed cultivation vary widely. Many hatcheries originally used large concrete tanks for rotifer production (>50 m3), but there is a clear development towards the use of smaller units and higher rotifer densities. A typical tank for the production of rotifers is currently 0.5–2 m3, and 1 m3 is a typical volume for high-density cultures (see Chapter 4). Short-term enrichment is done in tanks of about the same volume. Cultures are normally concentrated during rinsing and optional storage, particularly if the rotifer density during cultivation is low (e.g. <500 individuals ml-1). Tanks used during Artemia hatching and n-3 HUFA enrichment are generally of same volume as those used for rotifers. There is a need for other mesh sizes in the ﬁltration units, and more concern for the rapid cooling of Artemia nauplii that are stored (<24 h) than for rotifers (see Chapter 4). There is a ubiquitous need to control temperatures during all steps of production, ranging from the high temperatures needed for the hatching of Artemia (28°C) to the low temperatures needed for storage of live feed (5–8°C). Two factors are important in this regard. The amounts of water that must be heated or chilled are very small, and the live feed organisms are relatively robust. These facts imply that thermostats combined with heaters/coolers suspended in the individual tanks or local water reservoirs can take care of heating and cooling. However, a more sophisticated solution is to install systems for heat exchange to produce the extreme temperatures and to mix these sources of water to obtain the intermediate temperatures. Finally, it is an advantage if the room temperature of the hatchery is in the same range as the cultivation temperature of the larvae. 7.3.3.3 Larval Rearing Systems Suitable tanks for the cultivation of marine larvae are normally cylindrical, with a ﬂat or slightly conical bottom in order to simplify the control of organic wastes, and a depth of 1.0–1.2 m and a volume of 10–20 m3 (4–6 m diameter). Water is often carefully added from the surface to encourage a slight circular water movement and mixing, and the tanks are

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drained from a point sink, for example through a nylon screen system suspended in the upper water layers. A simple skimmer, driven by air, is needed to remove any organic surface ﬁlm. The light conditions used are highly variable, but the larvae are often exposed to typical indoor light intensities and a natural diurnal light cycle. Common tank colours are white, light green, light blue or even black. All hatcheries have developed and continuously improved their detailed protocols for larval rearing over the last decade, and we can only present some options. The stocking densities of larvae are typically 10–150 l-1, and feeding is of course dependent on the actual density applied as well as the species (see below). Live feed is still added manually in most hatcheries and research laboratories, and the daily rations may be supplied in 3–4 portions. Larval maintenance also involves thorough inspections of larval behaviour and growth, visual evaluation or counting of rotifer and Artemia densities, sampling for speciﬁc analysis, and the removal of accumulating wastes. The results of these evaluations and measurements will affect feeding and be the basis of measures taken during the course of ﬁrst feeding. It is often emphasised that high survival and growth depend on thorough and gentle rinsing procedures to remove accumulating wastes. 7.3.3.4 Automation and Process Control The production processes of live feed and fry in hatcheries of marine ﬁsh are to a small extent controlled and automated as in most other process industries. The work operations are mainly done manually, which probably reﬂects the fact that the industry still is very young. Future hatcheries for marine ﬁsh juveniles will probably beneﬁt from the developments made in process control and automation in other process industries such as the metallurgical and petrochemical industries, power plants and paper mills (Balchen, 1987). Some of the beneﬁts of a control system for marine juvenile hatcheries are:

• enhanced stability of critical processes, including microbial and nutritional conditions; • active suppression of random disturbances; • simulations and analyses of different process scenarios can be made at low cost, including the prediction of hazardous conditions; • evalution of the efﬁciency and optimisation of management strategy. A modern control system is based on mathematical descriptions (models) of how the process states evolve with time. In the case of a hatchery, several levels of modelling are needed. The system will include the hatchery processes involving water treatment and supply, feeding, hygiene, and the transport of water, cultures or ﬁsh in the hatchery. Some examples are the treatment of process water, automatic feeding, the production of live feed cultures (algae, rotifers, Artemia), rinsing and concentration of live feed cultures, temperature acclimation and storage, the feeding of ﬁsh larvae during ﬁrst feeding and weaning, cleaning production tanks, and transport related to grading and the transfer of ﬁsh fry to the nursery. An important issue during process automation is to identify the tasks that should still be carried out by humans, and those which can be automated without losses in production yields or fry quality.

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7.4 Larval Rearing in ‘Nature-like’ Systems 7.4.1 Pioneer Work The Norwegian Captain G.M. Dannevig successfully hatched and reared cod larvae to juveniles in a large outdoor enclosure in 1886 (Rognerud, 1887). His objective was to examine the viability of newly hatched cod larvae that were used in a large-scale release programme, which was started in 1884 to enhance the cod stock along the southern Norwegian coast. He wrote, When the doubt regarding the possibility of hatching cod roe artiﬁcially was clearly removed, another question was brought forward, namely, had the young ﬁsh, hatched in this manner, energy and power enough for further growth and development in the natural element?

He continued, The public wanted proof; and as the same public kept the institution going by their subscriptions, there was no choice but to set to work and bring such proofs.

Dannevig became convinced that the environmental conditions of cultures, in particular temperature and salinity, had to resemble those of the sea. The speciﬁc gravity of the water had to be at least 1.022 g cm-3 to avoid the cod larvae sinking because of their poor swimming ability during the early stages. He constructed a 2500-m3 concrete basin on land. From a release of approximately 500 000 yolk-sac larvae in late April 1886, he reported thousands of survivors brought successfully through the larval stage. He reported an average juvenile size of 11.5 cm in mid-October, and he also reported, for the ﬁrst time, cannibalistic behaviour among young cod juveniles (15–16 mm length) due to food deprivation, although decapod larvae (crab zoea and megalops stage) were quite abundant. The decapod larvae were simply too quick for the cod larvae to capture. This ﬁrst reported trial on the rearing of marine ﬁsh larvae in large nature-like enclosure systems illustrates in an excellent way the potential, but also the problems, of using such systems, and the availability of suitable food for ﬁsh larvae or juveniles. In this regard, the enclosure must be stocked at a level corresponding to the carrying capacity of the system, which is determined by the expected numbers of survivors at the time of harvest. In the case of cod, this will largely depend on the availability and production of suitable plankton in the mesocosm (Table 7.1). From simple bioenergetic modelling (e.g. Blom et al., 1991), it can be calculated that the 500 000 cod larvae that Dannevig stocked in his relatively small basin would need to eat about 90 million adult copepods (e.g. Centropages hamatus Lilljeborg) per day at Day 46 post-hatch, an age which corresponds to Dannevig’s ﬁrst observation of cannibalism at 15–16 mm length. This would require a daily copepod production of 35 adult copepods per litre, and an exponential increase in these numbers of 15–25% per day. Dannevig’s basin was incapable of producing that amount of food, even if only a small fraction (e.g. 10–20%) of the cod larvae survived to the juvenile stage. Dannevig’s pioneering work and his conclusions were essential for later success in using large enclosures to rear marine larval ﬁsh. He succeeded in showing the potential of survival

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Table 7.1 Production of cod fry in two Norwegian lagoon systems. Releases are yolk-sac larvae. Early juvenile mortality is the logarithmic decline between metamorphosis and estimates of juveniles in the lagoon before harvest. Food density is averaged for the period after metamorphosis. Data are from G. Blom (in preparation), van der Meeren & Naas, 1997, and H. Otterå (personal communication, 2001). Number of individuals

Release (millions)

Metamorphosis (thousands)

Juveniles (thousands)

Harvested (thousands)

Production density (N per m3)

Early juv. mortality (% per day)

Hyltropollen (60,000 m3) 1983 2.21 1984 6.4451 1985 1.81 1986 1.2 1987 1.8

800 750 600 500 200

130 – 200 50 –

74 70 120 47.5 60

1.23 1.17 2.00 0.79 1.00

5.9 – 3.5 6.8 –

Parisvatnet (270,000 m3) 1987 7.955 1988 17.741 1989 17.5 1990 11.072 1991 7.07 1992 5.55 1993 8.0 1994 3.5

250 680 4955 2100 1700 90 2000 500

30 430 390 210 400 – 320 290

5 240 170 71 317 4.52 190 270

0.08 0.89 0.63 0.26 1.17 0.02 0.70 1.00

7.4 1.4 8.0 7.3 4.8 – 6.1 1.7

1 2

Food density (N per m3) 157 – 973 – –

13 4283 10 12 88 – 16 –

Multiple releases. Bloom of the poisonous algae Alexandrium excavatum.

and growth in large nature-like systems, free from predators, but with an internal production of zooplankton sufﬁcient to support the feeding and survival of a large number of larvae during their early developmental stages. The next attempt to use enclosures to prove larval viability and survival were carried out in the 1930s by Rollefsen, who reared plaice larvae (Pleuronectes platessa L.) and plaice ¥ ﬂounder (Platichthys ﬂesus L.) hybrid larvae in a 400-m3 basin. Again, larval stocking densities were high compared to the carrying capacity of the basin, resulting in survival of 0.045 and 0.53%, respectively. The lack of sufﬁcient quantities of suitable larval food led Rollefsen to be the ﬁrst to suggest using Artemia as live ﬁrst feed for marine larvae (Rollefsen, 1940).

7.4.2 The ‘Lagoon Method’ as a Production System Since 1975, larvae of many marine species have been reared in enclosures, which are often called ‘mesocosms’. The breakthrough for cod reared by the extensive method in the lagoon ‘Hyltropollen’ in 1983 made lagoon systems a commercially interesting method at that time (Kvenseth & Øiestad, 1984). Cod, turbot (Scophthalmus maximus L.) and halibut (Hippoglossus hippoglossus L.) larvae have all been reared in mesocosms (semi-intensive methods) on a commercial scale, but only halibut larvae have been reared in such systems

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for an extended period, with a maximum of 380 000 juveniles being produced in Norway in 1994. In recent years, disease problems and variable successes with larval rearing have reduced mesocosm production of halibut fry. It is therefore doubtful whether mesocosm rearing of ﬁsh fry can compete with intensive rearing when these methods have been fully developed. Since 1987, the 270 000 m3 lagoon ‘Parisvatnet’ in Western Norway has been used regularly for the production of cod fry (see Table 7.1). Approximately one cod fry per cubic metre can be harvested from the lagoon system, but accidental events may alter this completely if the food production is not carefully controlled. For example, in 1992, a poisonous algal bloom of the dinoﬂagellate Alexandrium excavatum (Braarud) reduced the production of fry from the lagoon to only a few thousand. This illustrates one of the problems of the ﬁsh farming industry, which cannot accept such unpredictable ﬂuctuations in fry availability. Further, with a production potential of 1 fry per cubic metre in the case of cod, a large number of lagoons are required to produce sufﬁcient fry to support the demands of the on-growers. Parallel to the development of the extensive lagoon rearing method, cod, capelin (Mallotus villosus Müller), herring (Clupea harengus L.), halibut, sole (Solea solea L.) and turbot larvae have been reared in different types and sizes of plastic enclosures (Øiestad, 1985). Production is more reliable in a bag system rather than that undertaken directly in a lagoon system. However, the semi-intensive bag method is still subject to natural variations in plankton production and environmental variables, and this is probably why the method lacks commercial success.

7.4.3 Larval Food and Feeding in Mesocosms Whereas intensive fry production aims at full control with the simplest ‘food chain’ (see below), mesocosm rearing is far more complex. In lagoons, marine ﬁsh larvae can eat any organisms which are suitable for their developmental stage. Thus, the transition from endogenous to exogenous feeding can be smooth, and the larvae can select an optimum prey type and size according to their ontogenetic needs. Growing larvae need copepods at various stages, and in lagoon systems they seem to compete with different kinds of medusa for the copepods. Copepod production in mesocosms is variable, which may explain much of the variation in fry production numbers (see Table 7.1). The production potential and the carrying capacity of a mesocosm are restricted, and cannibalism may play a major role in reducing survival when food depletion occurs in the late larval or early juvenile stages. The quality of ﬁsh juveniles produced with lagoon zooplankton as food generally seems to be very good, in contrast to the quality obtained for some species using intensive rearing methods. This is most pronounced for ﬂatﬁsh species such as Atlantic halibut. Deformities such as lack of eye migration and malpigmentation (see Chapter 6) are much less frequent, or absent, when copepods are used as food either in semi-intensive or intensive rearing units. This might be related to their high, stable content of n-3 HUFA, in particular DHA and EPA, which may constitute 50–60% of the fatty acids of total lipids (see Chapter 4). The high fraction of phospholipids (60% of lipids) in the copepods may also play a signiﬁcant role in this respect. Copepods are the natural food for marine ﬁsh larvae, and their ‘immature’ digestive system is probably specialised to utilise such prey very efﬁciently (see Chapter 6).

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7.4.3.1 Initiation of Exogenous Feeding: The ‘Green Gut’ Hjort (1914) proposed that ﬁsh larvae in the earliest developmental stages are particularly vulnerable to limitations in their feeding conditions. The availability of suitable food during the transition from the utilisation of yolk reserves (endogenous feeding) to ingestion of prey (exogenous feeding) is believed to be critical to the ability of the larval population to survive and grow, and hence is a major factor controlling stock recruitment. This ‘critical period concept’ has been the subject of many discussions and speculations, and the hypothesis has so far neither been veriﬁed nor rejected (see Chapter 6). Whether a critical period exists in a given instance will depend on a number of environmental and species-speciﬁc factors (May, 1974). Some of these species-speciﬁc factors have been examined for cod based on experiments in lagoon systems. In several species, the initiation of exogenous feeding occurs at the termination of the yolk reserves. In other species, including cod, food is found in the larval gut before yolk depletion. At this developmental stage, cod larvae are morphologically and functionally capable of feeding and digesting. Behavioural changes in larval activity also indicate the utilisation of exogenous food while yolk is still present. A gradual shift to an external diet during yolk resorption may ease the transition from passive nutrient diffusion to active feeding. Such an adaptation will increase the probability that the larvae will establish successful feeding on zooplankton and thus avoid the ‘point of no return’: the point of irreversible starvation after delayed feeding (see Chapter 6). From the initiation of feeding, cod larvae are opportunistic predators which are able to ingest a variety of plankton groups, including algae, protozoans such as tintinnids and oligotrich ciliates, rotifers, polychaete larvae, and copepod eggs and nauplii, the latter being the main food of cod larvae. Algae have been found in larvae of cod and several other ﬁsh species. This consists mostly of larger algal cells, probably ingested by visual feeding, but smaller algae and ‘green food remains’ have also been observed. The green gut content frequently reported in young cod larvae collected from mesocosms was found to originate from ingestion of:

• algae and ﬂocculates of algae • protozoans containing functional chloroplasts • zooplankton with algae in their gut • zooplankton faecal pellets containing the remains of algae In 1-day-old cod larvae, accidental ingestion of algae has been attributed to algal clogging of the visceral arches. However, the apparent ﬁlter-feeding of alga smaller than 10 mm seems to be an active mode of feeding rather than accidental (van der Meeren, 1991), and has also been demonstrated in intensive cultures and other ﬁsh larval species (see below). The ﬁlter feeding of algae during early developmental stages enables the cod larva to utilise a food resource which is not considered to be of particular importance to ﬁsh larvae. Laboratory-reared cod larvae do not contain the same amount of fragmented algal material in their guts as larvae reared in nature-like lagoon systems. This may be because the algae used in cultures are too small for active ingestion. The ingested phytoplankton may

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serve as a source of essential nutrients or as a trigger of the digestion system. Autoﬂuorescent material, resembling the red ﬂuorescent colour of algal pigments, appears inside vacuoles of the hind-gut in young cod larvae from lagoon systems. Lysis of algal cells and pinocytotic absorption of algal material takes place in young turbot larvae (see Chapter 6). In salmonids, selective absorption from the diet and deposition of algal pigments in ﬂesh and skin are common. It is still unclear if these differences between intensive and nature like rearing systems affect the growth and survival of the larvae. 7.4.3.2 Prey Selection Cod larvae are generally selective feeders. For example, the shape and size of different algal cells may cause considerable variation in retention probabilities on the larval visceral arches. Spines of chain-forming diatoms and dinoﬂagellates may explain why such algal cells are not observed in the gut of cod larvae, which may be unable to swallow such cells. Selective feeding can also be a result of behavioural patterns of both ﬁsh larvae and motile algal cells. Algal extracellular metabolites may act as chemical stimuli on the larval olfactory and neuromast organs and induce a special pattern of feeding behaviour, as shown for larvae of turbot and sole. Cod larvae make a clear distinction between copepod nauplii and other prey categories of smaller sizes during the early larval development stage. The nauplii are among the largest catchable food items, and may be preferred owing to the higher energy gain compared with smaller micro-zooplankton. Owing to the greater abundance of smaller zooplankton, the total biomass of this group may not differ much from that of copepod nauplii. However, energy spent on searching and handling smaller prey exceeds the energy used on a corresponding biomass of larger prey. Thus, size selection by optimal allocation of time spent searching for and handling prey induces dietary changes that maximise the energetic return to the predator (Werner & Hall, 1974). A scarcity of energetically favourable food, such as copepod nauplii, induces nonselective feeding on particles in a broad size range in cod larvae (van der Meeren & Næss, 1993). Protozoans, such as tintinnids and oligotrich ciliates, are particularly frequent in the diet of cod larvae. In coastal waters, such organisms may occur in densities of 103–105 cells per litre. The minimum size of protozoans that cod larvae can ingest seems to be 30–40 mm, in accordance with the minimum size of particles that ﬁsh larvae theoretically manage to catch by visual feeding. Size-selection of prey seems to be a common feature in larvae of many ﬁsh species. Their searching ability and handling efﬁciency increase with their increasing size. Thus, rapid incorporation of successively larger prey may be expected from the rapid growth often observed in ﬁsh larvae. Accordingly, within only 2–3 weeks post-hatch, at the time when cod larvae show the highest growth rates, larger copepodids become more important than copepod nauplii as a food source. Escape behaviour, swimming speed and transparency of the prey may also result in selective feeding (Drenner et al., 1978; Shuvayev, 1979). A predator may maximise the rate of successful capture by preferentially selecting non-evasive prey (Vinyard, 1980). In mixed prey populations, the optimally foraging predator may select nonevasive prey as long as the rate of energy return per encounter balances the difference between the non-evasive prey and larger evasive prey. Thus, different decisional constraints

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regarding prey selection, such as prey size or the probability of prey capture, may not conﬂict with optimisation of energy intake in a predator. Prey densities are probably the most important factor controlling food selection in ﬁsh larvae. Selection for copepod nauplii when cod larvae have an excess of food is in accordance with optimal foraging theory (Eggers, 1977; Krebs, 1978), and the larvae of several marine ﬁsh species feed selectively on copepod nauplii at high prey abundances. Prey density has been shown to induce different selective mechanisms in ﬁsh. Ohguchi (1981) showed that non-selective feeding, selection for the most common prey type (apostatic selection) and selection for clearly distinguishable prey types (oddity selection) occurred at low, medium and high prey density, respectively. Apostatic selection was attributed to the formation of a search image, and oddity selection to the confusion of the predator. Together with prey density, prey size and the probability of prey capture, search image formation and confusion are probably fundamental to foraging behaviour in predators, including ﬁsh larvae. 7.4.3.3 Feeding, Growth and Survival The variability in larval feeding ecology and growth emphasises the importance of bioenergetic models. Such models include the relations between larval ingestion, growth, metabolism, excretion and egestion. The precision of such models depends on how precisely the different parameters, and hence the ratios between them (e.g. growth and assimilation efﬁciencies), are determined in their relation to larval ontogenesis, temperature and feeding conditions. Several of the parameters cannot be measured directly for the young stages of small marine ﬁsh larvae. Despite such difﬁculties, bioenergetic models have provided important information on growth dynamics in ﬁsh larvae, and have also been useful tools in the rearing of cod larvae in both mesocosms and intensive systems. A number of 5–10 copepod nauplii per litre is assumed to be the critical level for successful larval feeding of cod (Ellertsen et al., 1989). Larval predation in nature-like rearing systems can easily exceed the sum of internal production and external supply of live food. Thus, in aquaculture, the number of available prey per larvae and per unit of time, rather than the number of food items per litre, will determine whether larval energy requirements are fulﬁlled or not. The use of bioenergetic models that predict larval food consumption, depending on larval growth rate and body size, may therefore be important for successful rearing through the larval stages. The daily food requirements of cod larvae at the moment of initial feeding have been estimated to be 36–52 nauplii (data from Solberg & Tilseth, 1984), and daily requirements for larval cod during the ﬁrst 3 weeks post-hatch have been estimated to be 50–120 copepod nauplii (Blom et al., 1991). Model predictions show that a rapid increase in growth rates occur when food availability exceeds 50 nauplii per larvae per day, whereas high growth rates can be expected with more than 100 available nauplii per larva (Fig. 7.5) (van der Meeren & Næss, 1993). Growth in cod larvae may vary considerably depending on food availability. The weightspeciﬁc growth potential for cod larvae seems to exceed 20% per day. Realisation of this growth potential may occur at optimal temperatures when energetically favourable prey organisms, such as copepod nauplii, are available in excess. Rapid compensatory growth, independent of any former growth lapse, has also been observed in herring and turbot larvae.

S G R (% p e r d a y )

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24 22 20 18 16 14 12 10 8 6 4 2 0 1

10

100

1 000

10 000

100 000

Num ber of prey per larva Figure 7.5 Daily weight-speciﬁc growth rates (SGR) in relation to prey requirements of young cod larvae. Circles are values from the bag enclosure (䊊) and the basin (䊉) in van der Meeren & Næss (1993). The solid line is daily prey consumption as a function of growth rate in a cod larva, calculated from a bioenergetic model (Blom et al., 1991). The size of the larva is 0.12 mg dry weight, the temperature is 6°C and the number of prey consumed is expressed as numbers of copepod nauplii of 200 mm total length. The star (¥ +) is daily larval food consumption of cod at the initiation of feeding (Solberg and Tilseth, 1984).

Food availability in the late larval and early juvenile stages correlates well with the numbers of cod fry that are harvested from lagoons (see Table 7.1). A survival of 50% beyond metamorphosis is not uncommon, and up to 80% survival has been reported. Cannibalism in periods of food deprivation during early juvenile stages is probably the most important factor inﬂuencing the output of cod fry from nature-like systems (Folkvord et al., 1994). The balance between larval stocking densities and copepod production (or plankton collection capacity) during the early juvenile stage is therefore essential.

7.5 Larval First Feeding in Intensive Systems The process of ﬁrst feeding is a particular challenge in intensive larval rearing, and is a bottleneck for most cultured marine species. The process lines of cultured feed components and ﬁsh larvae meet during ﬁrst feeding. At the same time, environmental factors and the microbial environment of the larvae may show abrupt and pronounced variability. Each factor, and the interactions between them, is a challenge in itself for the fast-growing and metabolically efﬁcient, but physically fragile, larvae (see Chapter 6).

7.5.1 Physical Chemical Environment The importance of the environmental physical and chemical conditions has been intensively discussed in Chapter 2. This section will only summarise issues that are found to be especially important during ﬁrst feeding of marine cold-water larvae.

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Like all other larvae of marine ﬁsh, cold-water species are very fragile, and therefore also very sensitive to physical disruption, especially during the very early stages. This is why most species are maintained in stagnant or semi-stagnant conditions in the early feeding stage. Even very moderate water currents may kill larvae, that typically end up attached to the screen of the outlet sieve. This speciﬁc sensitivity has major implications for the entire process of ﬁrst feeding. Water exchange is the main means of maintaining environmental control during intensive cultivation. Nutritional and microbial control of larval cultures must be maintained by other means. The positive relationship between water exchange rate, stocking densities and production yields is well known for the older stages. For larvae of Atlantic halibut, there have been major problems in establishing an artiﬁcial light regime that gives suitable larval behaviour during the ﬁrst days of feeding. With subdued sunlight, the larvae normally distribute well in the middle water layers. Conversely, artiﬁcial light conditions in indoor tanks commonly causes the majority of the larvae to form dense schools in the surface layers, just under the light source. This normally leads to the mortality of the majority of the population within a week. The most efﬁcient countermeasure has been to introduce a component of UV-radiation, which may not be surprising considering previous experiences with sunlight. This, together with the speciﬁc physical arrangements of the light sources and the shading patterns of the tank, have reduced the behavioural problem for halibut larvae. Together with their special needs during the yolk-sac stage (see Chapters 6 and 10), this illustrates the challenges of culturing halibut fry. The light conditions have not been a problem to the same extent for other species. A ﬁnal conclusion, which has not been veriﬁed, is that there is a general need to maintain the physical–chemical environment of the larvae at a constant level during ﬁrst feeding. Temperature and salinity must of course be well within the tolerance of the species, but in addition it may be important to maintain these factors at a constant level. This experience was also reported with older ﬁsh (see Chapter 8), and it is therefore a sound precautionary approach to take active control of the physical rearing regime. This will have consequences for the costs of establishing a hatchery, but it will probably be worthwhile.

7.5.2 Feeding Characteristics of Fish Larvae During the phase of ﬁrst feeding, the immature ﬁsh larvae will be exposed to, and interact with, a number of other organisms, including bacteria, microalgae and zooplankton. If the eggs and yolk-sac larvae are incubated in separate incubation systems, as is the case for Atlantic halibut, the initiation of ﬁrst feeding also will involve sudden changes in light, temperature and other physical factors. However, many species, such as cod and turbot, are often hatched in the feeding tanks. The food web structure in the larval tanks is among the simplest possible, but it is important to emphasise that there are still four functional and interacting food web components present: microalgae, bacteria, rotifers or Artemia, and the ﬁsh larvae. Successful control of these interacting food web components and the transport of energy to the top larval predator is the key to successful fry production.

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7.5.2.1 Food Selection Fish larvae maintained in intensive culture will not normally have any options when selecting their food items because there are no alternatives. The beneﬁts and importance of selecting optimal food items have already been thoroughly discussed. When only one prey is present, there is a need for particular care because that prey may be inadequate with respect to size or nutritional value. For example, rotifers and other species of zooplankton with an exoskeleton may have a variable meat content in individuals of a similar size (see Chapter 4). Rotifer individuals can show 100% variation in protein or energy contents as a direct result of the cultivation conditions. Larvae of turbot are apparently unable to compensate for poor nutritional value by simply eating more rotifers, and increased mortality has been found as a result of feeding nutritionally exhausted rotifers (see below). In nature-like systems, the ﬁsh larvae normally will have options to compensate for such conditions by simply selecting a different prey. Fish larvae maintained in intensive larviculture and fed a mixture of rotifers and various size groups of Artemia have been shown to select between these prey organisms. It has been assumed that the optimum Artemia size for larvae of Atlantic halibut, which are relatively large at the onset of exogenous feeding, will increase with larval developmental stages during ﬁrst feeding. Moreover, even the largest strains of B. plicatilis (length 250–300 mm) are expected to be too small, although their nutritional value is superior to Artemia owing to their more optimal fatty acid content. Careful studies of prey selection have shown that halibut larvae show no preference between rotifers and 1-day-old A. franciscana during the ﬁrst 2 days of feeding (e.g. Olsen, 1999b). However, from Day 3, the larvae already select quite strongly for the smallest A. franciscana. When offered a mixture of different sizes of A. franciscana, the larvae selected newly hatched A. franciscana (length <0.6 mm) up to a larval weight of 3 mg DW (20–25 days old). Larvae larger than 4 mg DW (30 days old) selected strongly for A. franciscana individuals >1.2 mm in length. These types of biological constraint are important when establishing adequate feeding regimes for cultured halibut larvae as well as for other species.

7.5.2.2 Feeding and Functional Response Larvae of marine ﬁsh are classiﬁed as carnivorous zooplankton. There is a species-speciﬁc relationship between food concentration and feeding rate which is comparable to that described for the omnivore B. plicatilis (see Chapter 4). However, unlike the rotifer, it is generally believed that ﬁsh larvae are so-called number maximisers that consume prey close to the rate at which they are encountered (Lubzens et al., 1989; Hoehne, 1999). The prey consumption will then generally be positively related to the prey density, and not become saturated at high food concentrations, as shown for turbot (Hoehne-Reitan et al., 2001). This relationship, normally termed the functional response, differs from the situation for rotifers, which showed a constant (saturated) feeding rate at high food concentrations. An important ecological trait in both cases is the ‘critical food concentration’ which allows the maximum growth rate. It is important to note that age as well as environmental conditions (e.g. turbu-

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lence) will modify the functional response of the larvae. A high prey density can have a positive effect during the initial feeding period, but this is necessarily not the case later on when the larvae have an increased ability to search for food. If the prey concentration is far higher than the critical concentration for maintaining maximum growth rate, the larval digestion efﬁciency may become reduced, with the implication that the defecation rate increases. The degree of digestion of food is inversely related to the rate of gut evacuation in ﬁsh larvae (Werner & Blaxter, 1980). The evacuation rate is directly dependent on the ingestion rate, which again is dependent on prey density. In fact, a medium prey density resulted in the fastest growth rate for turbot larvae. The prey eaten by larvae exposed to the highest prey densities were not sufﬁciently digested owing to the short retention time in the gut, corresponding to a high ingestion rate (Hoehne, 1999). Another negative effect of high prey density is that the rotifers may reside too long in the larval tanks between feeding and consumption. The biochemical composition of the postenriched rotifers may be stabilised by adding microalgae to the larval tanks, whereas major losses of lipids and proteins take place if microalgae are not added (see below). Artemia will always lose n-3 HUFA, and in particular DHA (see Chapter 4), and these losses are severe at high temperatures. Most studies on larval rearing use the density of prey rather than the dose added per day and per larva, which is a better variable to describe food supply (see above). Turbot and cod larvae are normally maintained at rotifer densities ranging from 3000 to 7500 l-1, which are far above the concentrations of large zooplankton in nature-like systems. These concentrations are probably much higher than the ‘critical prey concentration’ for sustaining the maximum growth rate, but some over-feeding may ensure that food is not completely depleted between additions. However, it is important to note that focusing only on prey concentration just after feeding may hide the fact that the daily dose of feed added may still be below the quantitative requirements for maintaining the maximum growth rate. This is particularly important if the prey concentration drops to low levels in between feedings, which normally happens during the later stages. Thus, it is better to control the daily food dose than the prey concentration. 7.5.2.3 Larval Feeding Rate of Live Feed The feeding activity of marine ﬁsh larvae can be measured by methods that are more or less quantitative. Direct counting of numbers of prey in the gut, the evacuation rate of gut contents, and the rate of removal of prey from the water are methods that can be used under most rearing conditions. More sophisticated methods based on the use of radioactive or other labelling of the prey are also available for fundamental research. A general and simple mass balance method is applicable. The daily mean ingestion rate of rotifers by the larval population (IPOP) can be calculated based on the dynamics of the rotifer population (Reitan et al., 1993): I POP = F + G + AR - DR

(7.1)

where F is the daily numbers of rotifers fed to the larval population, G is the daily net increase in the rotifer population in the larval tank, AR is the difference in the ambient rotifer popu-

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lation in the tank between two consecutive days, and DR is the quantity of rotifers lost from larval tanks through water exchange, and is equal to zero in stagnant conditions. Daily records of the rotifer density in a larval tank allow an estimation of the rotifer standing stock per larval tank (R) and their egg ratio (eggs/rotifer). The speciﬁc net growth of the rotifer population (m) can be estimated based on measurements of the egg ratio of the population, and conversion to the speciﬁc net growth rate for the population using empirical relations (see Chapter 4). The net increase in the rotifer population (G) is then given by G =m◊R

(7.2)

The value of G is negative if the rotifer mortality rate is higher than the birth rate. The number of rotifers added to the tank and the daily number of rotifers washed out of the tank through water exchange are both easily measurable. This allows an estimation of IPOP according to Equation 7.1. The rotifer ingestion rate per individual larvae (IIND) can be estimated if the number of larvae in the tanks is known: I IND = I POP (number of larvae)

-1

(7.3)

This method has primarily been applied to turbot cultures during the ﬁrst few days of feeding (Days 2–5) when the larvae were maintained under stagnant conditions, and later under conditions of a slow water exchange rate (after Day 5 or 6), but it is also applicable to later stages. If the water exchange rate is low and larval density is high, the value of F becomes much bigger than the other terms in Equation 1, meaning that IPOP @ F. This method has been used to assess feed intake under variable feeding conditions for growing larvae. The rotifer consumption rate of turbot larvae increases signiﬁcantly from Day 3 to Day 10: it is estimated to be 50–180 rotifers day-1 larva-1 on Days 2–4, 200–400 rotifers day-1 larva-1 on Days 6–8 and 300–700 rotifers day-1 larva-1 on Days 9–10 (Reitan et al., 1994; Hoehne-Reitan et al., 2001). It is a general experience that turbot larvae feed faster on rotifers with algae added (‘green water’) than without algae (‘blue water’) (Reitan et al., 1993; Øie et al., 1997). Counting the numbers of prey in the gut of ﬁsh larvae is a widely used qualitative method to measure feed intake. It is not directly comparable to the above method, and comparisons have shown that there is an inverse relationship between feeding rate and number of prey per larvae. Whereas larvae of turbot maintained in green water showed the fastest intake of rotifers up to Day 6, larvae maintained in blue water showed the highest number of rotifers in their gut (Øie et al., 1997). 7.5.2.4 Larval Feeding on Microalgae As discussed above, larvae of several marine ﬁsh species maintained in nature-like systems normally ingest microalgae before they start feeding on bigger prey. This has also been well documented for yolk sac larvae of turbot, halibut and cod maintained in intensive culture. The highest algal ingestion rate of halibut larvae was found in the period 150–250 day–degrees (days since hatching multiplied by incubation temperature), which is after the

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mouth is expected to be functional, and just before the larvae normally start feeding on zooplankton (see Chapter 6). It has been questioned whether the uptake of microalgae is active or simply a consequence of water-drinking activity. Marine larvae, among them halibut, turbot and cod, drink water just after hatching. The drinking rate, as well as the algal uptake rate, varies with developmental stage. The drinking rate for 2–7-day-old turbot is in the range of 1–2 nl water mg-1 body carbon h-1. The clearance rate of alga cells from the water, i.e. the volume of water cleared of microalgae per larva and per unit of time, is found to be 10–1000 times higher than the drinking rate, showing that only <1–10% of the algal intake can be the result of drinking activity. These ﬁndings support the idea that during the late yolk sac or early feeding period, larvae act as ﬁlter feeders. This is further supported by the fact that the algal clearance rate increases with the size of the algal cells for young larvae of cod, halibut and turbot (see data by Howell, 1979; Mangor-Jensen & Adoff, 1987; van der Meeren, 1991; Reitan et al., 1994, 1998). The speciﬁc ingestion rate of microalgae is <5% (normally <1%) of the larval biomass per day, and the yolk-sac larvae of both halibut and turbot show a relatively low assimilation efﬁciency of ingested algae. The uptake is therefore much too low to represent a major energy source for growth. The fact that this uptake does takes place suggests that it may have a function in the developing larvae. For example, it may contribute to critical micronutrients, or trigger substances that affect the development of the immature digestive tract, as already demonstrated for larvae of seabass. It may also contribute to the establishment of a functional intestinal bacterial ﬂora, which is crucial for food digestion, growth and survival (see Chapter 3).

7.5.3 Feeding Regime Components for Cold-water Larviculture The food components used for cold-water larvae of ﬁsh are illustrated in Fig. 7.6. Copepods are not included, but harvested copepods are still used as live feed both in experiments and in pioneering commercial production. Formulated feeds and the change from live to formulated feed (i.e. weaning) is covered in Chapter 8.

7.5.3.1 Microalgae Microalgae are not strictly necessary for all species during larval feeding, but there is overwhelming documentary evidence that algae enhance the production yields and quality of many species (see below). The two species of microalgae that have commonly been used in ﬁrst feeding of cold-water marine ﬁsh larvae are Isochrysis galbana (Prymnesiophyceae) and Tetraselmis sp. (Prasinophyceae). Both species are easy to culture in the laboratory and in hatcheries, and both grow relatively fast. The species exhibit almost complementary n-3 HUFA compositions. Isochrysis spp. have DHA as the predominant n-3 fatty acid and almost no EPA. Tetraselmis spp. show the opposite composition, with EPA as the dominant n-3 fatty acid and a low content of DHA. These algal species can be used alone or in combination at a total density of <2 mg carbon l-1. The addition of microalgae to the water during early ﬁrst feeding is normally termed the ‘green-water technique’.

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Figure 7.6 Common food components used during intensive rearing of marine ﬁsh larvae, illustrated in their relative size scales.

7.5.3.2 Rotifers Rotifers are a major food component for most cold-water species of ﬁsh larvae, as they are for most other larval ﬁsh. It is important to emphasise that the food component ‘rotifers’ is not well deﬁned without including a variety of quality aspects such as size, nutritional value, viability and bacterial content. The species B. plicatilis (the so-called L-strain) is available in many sizes (lorica length 130–340 mm, mean 240 mm), as is the smaller Brachionus rotundiformis (the so-called S-strain, lorica length 100–210 mm, mean 160 mm) (Dhert, 1996, and Chapter 4). B. plicatilis has been used most frequently for all cold-water species, but B. rotundiformis is introduced to cod these days. The nutritional value (e.g. the content of energy, protein, lipid essential fatty acids, and micro-nutrients such as vitamins and minerals) totally depends on the cultivation method, feed composition and the optional postharvest treatment of the culture between cultivation and feeding (e.g. short-term enrichment, washing procedure, live feed storage). The content and composition of bacteria and the loading of organic carbon depend on the rotifer feed used, feeding routines, rotifer densities, rinsing procedures and other optional post-harvest treatments employed. 7.5.3.3 Artemia Naupli Artemia nauplii have been used for all cultured species of cold-water ﬁsh larvae, and Artemia franciscana is the most commonly used Artemia species. Artemia are available as resting cysts from a number of sources, and the majority of cysts are harvested from Great Salt Lake, Utah, USA. The cyst quality is variable, but because of cyst formation, the biochemical composition of Artemia is much more stable than that of rotifers. The basic nutritional

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composition, e.g. protein and lipid contents, is relatively constant from the hatching of the cysts to larval feeding. However, n-3 HUFA is virtually absent in the Artemia cysts, and this critical component must be carefully bioencapsulated by established enrichment techniques before the Artemia become adequate as live food for marine ﬁsh larvae (see van Stappen et al., 1996, and Chapter 4). A speciﬁc problem in this regard is the rapid DHA catabolism in Artemia, because DHA is considered to be the most important fatty acid for cold-water ﬁsh larvae. As for rotifers, it is paramount that the bacterial ﬂora and its composition is also considered for the Artemia nauplii before they are used as live food. Thorough rinsing and postincubation of the short-term-enriched Artemia with microalgae are methods that may work satisfactorily in this respect (Olsen, 1999b). 7.5.3.4 Juvenile Artemia Larger on-grown stages of Artemia (2–7 days old) have been tested as live food for the relatively large larvae of Atlantic halibut, but their use may also be advantageous for other species. Juvenile Artemia franciscana have a higher, and therefore a more attractive, ratio of protein to lipids than Artemia nauplii, but they still suffer from a high DHA degradation rate. A larger prey will theoretically be more optimal for the larger larval stages, but nauplii, which are easier and less expensive to cultivate, may still be adequate. The production technology for juvenile Artemia that are suitable for marine ﬁsh larvae has been established and described, although there is still room for improvements (see Olsen, 1999b). The need to consider the composition of the bacterial ﬂora is the same as with Artemia nauplii.

7.5.4 Tentative Feeding Regimes for Common Species The feeding regime can be deﬁned as the detailed protocol describing stocking densities, feed components and the amounts that are provided day by day to individuals or the population throughout the entire feeding phase, and the overall physical conditions maintained during feeding. The feeding regime for marine ﬁsh larvae covers the period from the very ﬁrst addition of live food to the weaning process, when the live feed is gradually changed to a formulated feed. The feeding regimes used for intensive rearing of cold-water species are generally not different from those used for other species of ﬁsh larvae, e.g. that used for sea bream. However, it is important to emphasise that the feeding regimes for some species are still very tentative. 7.5.4.1 Stocking Densities The term ‘intensive’ is a relative term expressing the fact that cultures are run close to the carrying capacity rather than that the biomass is above a certain level. A typical initial stocking density during intensive larval rearing is in the range 10–150 larvae l-1. Judged from a biomass perspective, this is certainly a low density. On the other hand, if ten juveniles are produced per litre of water (10% survival of an initial 100 larvae per litre), corresponding to 1 million juveniles per 100 m3, the ﬁgures become signiﬁcant. For ﬁsh larvae maintained

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at concentrations that do not result in direct negative interactions between individuals, two important and partly dependent factors constrain the stocking density during ﬁrst feeding. (1) The food ration must support the maximum growth rate: the ration is proportional to the stocking density and increases exponentially with time. (2) The carbon loading rate of the tanks is related to the food ration and there is an upper sustainable level. The individual larvae increase their biomass in an exponential manner before metamorphosis, and it is hard to imagine how quickly food requirements really increase with time. The need for live feed may become dramatically high towards metamorphosis at high stocking densities, and our perception of that situation is probably inadequate. Under-feeding, resulting in enhanced mortality, is a very common consequence. For high larval densities, feeding frequencies must be increased steadily in order to maintain an adequate food supply. Care must also be taken to avoid too high a prey density after each feeding, as this may result in reduced food digestion, increased defecation rate, and increased carbon loading rate in the larval tanks (see above). There is obviously an upper limit for the carbon loading rate from the live feed itself, and also from larval defecation, which allows microbial control and sustainable larval growth. The loading from the live feed can be minimised through proper rinsing, whereas defecation from the larvae can be minimised through adding appropriate concentrations of live food. The supply of food must be high enough to secure the maximum growth rate, but it should not be very much higher than this in order to minimise carbon loading through defecation. High larval densities therefore require great attention to feeding rations. Turbot larvae can be stocked at initial densities of 100 l-1, and be produced in ﬁnal densities of at least 30 metamorphosed 25-day-old fry in experimental systems without any reduction in growth, survival or fry quality. This requires close attention to both food rations and frequency of feeding. A common method to counteract potential problems of high feed doses in commercial production has been to increase the available tank volume of the population in the course of ﬁrst feeding. 7.5.4.2 Live Food Rations The discussion above clearly shows that strict control of feed rations and frequency of feeding is required during intensive ﬁrst feeding, particularly at high stocking densities. The easiest way to control feeding is to use a simpliﬁed bioenergetic model which predicts feed ration based on the biomass and the expected growth pattern of the ﬁsh larvae. When the optimum growth curve during the larval stage is known (see Fig. 7.8), we can estimate the daily biomass increase of the larvae (DW) as DWn = Wn+1 - Wn

(7.4)

where Wn is the biomass prediction on one day, and Wn+1 is the prediction for the next day. The food ration needed to support that growth (FR) can then be estimated by

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FRn = D Wn Y

(7.5)

where Y is the yield coefﬁcient, or the feed factor. An appropriate value for Y is 0.2 mg DW biomass produced per mg DW feed added, but users should adapt this yield coefﬁcient to ﬁt their own experience. The feed ration of the larvae expressed in terms of prey numbers (DRn) is then given by DRn = FRn WLF

(7.6)

where WLF is the biomass per prey. The model is most easily constructed using a spreadsheet, and may be extended to cover the whole population if survival is monitored (or predicted based on experience). Bioenergetic models are usually more complex than this one (see above), but this is sufﬁcient to predict the daily food ration far more accurately than without the support of a quantitative method. There is no need for such models if the larval concentration is low, e.g. if the prey concentration is reduced by 30–50% between feed additions. Monitoring the prey concentration may then be satisfactory. It is also clear that the user’s experience is needed to tune model variables and coefﬁcients to each actual case. 7.5.4.3 Atlantic Cod and Haddock First feeding of cod is normally performed at 10–12°C, and the feeding components involved are microalgae, rotifers and short-term-enriched Artemia nauplii (Rosenlund et al., 1993). Some initial efforts have been made to culture larvae of haddock (Melanogrammus aegleﬁnus, Castell et al., 2002), and to date there is no reason to believe that haddock should be treated differently. Some tentative alterations to the food components for cod (and haddock) larvae, are illustrated in Fig. 7.7. The initial stocking density is typically 30–40 larvae l-1, but densities may be much higher. Microalgae are added from Day 4 to Day 20 post-hatch, rotifers from Day 5 to Day 20, and Artemia nauplii from Day 17 until the larvae can be weaned onto formulated diets. The weaning of cod can be started at 35–40 days after hatching (see Chapter 8). The Artemia ration can be decreased gradually during weaning, while the ration of formulated feed is increased correspondingly. 7.5.4.4 Atlantic Halibut Unlike other species, the fry production of halibut involves a prolonged yolk-sac period when the larvae are incubated in darkness in silo-shaped systems with an up-streaming water current for 42–46 days at 6°C, or 260 day-degrees (d°). At 250–270 d°, the larvae can be transferred to ﬁrst feeding tanks (see Chapters 6 and 10). The initial larval density during ﬁrst feeding has normally been 1–10 larvae l-1. At the onset of ﬁrst feeding, the temperature is increased gradually to 11–13°C with a low rate of water exchange. Microalgae are almost always added for 20–30 days (Fig. 7.7). Halibut larvae are larger than both turbot and cod larvae and need larger prey. The length of large rotifers (300 mm) is comparable to the smallest theoretical prey particles of halibut larvae, but there are nutritional advantages in using rotifers during the ﬁrst few days. However, the larvae are normally given Artemia nauplii

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Figure 7.7 Generalised feeding regimes illustrated for Atlantic cod, Atlantic halibut and European turbot. The ﬁgure shows optional alterations in the pattern of food components for each species, but there will always be other alternatives (Ffeed, formulated feed; Jartemia, juvenile Artemia).

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from the start, and only a few producers use rotifers in the ﬁrst few days. Later, the larvae start to select for larger prey sizes, and Artemia juveniles can be used before the larvae are weaned onto a formulated feed. Some commercial producers of halibut fry use harvested copepods, such as juvenile stages of Calanus ﬁnmarchicus and Temora sp., together with Artemia when copepods are available. This method can only be used in the spring or when the abundance of small copepods is adequate. 7.5.4.5 Turbot Turbot is a well-established species in European aquaculture, and the commercial producers have established their own favourite feeding protocols. The normal stocking density is 10–100 larvae l-1, but initial stocking densities of 20–40 larvae l-1 are common. The temperature is increased gradually from 12–14°C at hatching to 18°C on Day 5 post-hatch. During the ﬁrst 5–7 days, the larvae are kept in stagnant water or at a low water exchange rate, before the water exchange is gradually increased. Turbot is very susceptible to mechanical damage caused by contact with the tank surface or the outlet sieve. Figure 7.7 shows that microalgae should be used together with rotifers from Day 1 to Days 12–14, and shortterm-enriched Artemia nauplii from Day 12 to Days 22–25 after hatching. Some producers may introduce Artemia at an earlier stage. During the weaning phase, Artemia nauplii or juveniles can be used together with the formulated feed for 1–2 weeks. Turbot will readily accept larger stages of Artemia as it grows towards metamorphosis, but this is not commonly used. 7.5.4.6 Sole, Wolf-ﬁsh and Hake Larval feeding methods for sole (Solea solea) have been established for a relatively long time. Larvae can be reared at 17–19°C and be fed Artemia from the beginning. Their n-3 HUFA requirements seem to be low compared with most other species, but appropriate enrichment of the Artemia is nevertheless recommended. Formulated feed can be introduced from Day 30. The species is greatly appreciated, but questions regarding its growth potential have made it less attractive for mariculture (see Chapter 10). Wolf-ﬁsh (Anarhichas spp.) have a unique reproduction strategy with internal fertilisation, and males protecting the spawned eggs prior to hatching (see Chapters 6 and 10). Compared with the other species that are treated here, wolf-ﬁsh larvae are more developed at the time of hatching, the fecundity is far lower, and the survival is higher (see Chapter 6). Larvae of common wolf-ﬁsh are maintained at temperatures of 11–14°C (for 4 weeks) during ﬁrst feeding, whereas spotted wolf-ﬁsh are maintained at somewhat lower temperatures (6–8°C, for 3–4 weeks). They are able to take larger prey and, like salmon, formulated feeds from the beginning, but a tentative optimal feeding regime should still involve the use of Artemia during the ﬁrst 3 weeks (see Chapter 10). Interest in rearing hake (Merluccius merluccius) is more recent, and experiences of larval feeding are limited. Larvae maintained at 12°C have been fed a mixture of enriched rotifers and copepod nauplii under ‘green water’ conditions, and to date a small number of juveniles have been brought through weaning to formulated feed (see Chapter 10).

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LARVAL WEIGHT, mg fresh weight 1000

Turbot Wolffish

Halibut

100

Cod 10

Haddock 1

Figure 7.8 Typical curves describing the increase in weight with time during larval feeding of some cultured species. The increase is exponential with time until metamorphosis, and the speciﬁc growth rate is constant, and equal to the slope of the curve.

0 0

20 40 60 DAYS OF FEEDING

80

7.5.5 Growth-Rate Characteristics During First Feeding The larval biomass (length) at hatching, as well as the growth rate during ﬁrst feeding, are species-dependent, but all species show the same fundamental type of growth kinetics (Fig. 7.8). The growth curves intercept the weight-axis at different points, and the rate of weight increase with time (slope) is variable. However, healthy larvae of all species show an exponential increase in weight during the initial phase of growth, normally until metamorphosis. However, the rate of increase, termed the speciﬁc growth rate (m), is different. Larval weight increases during the initial phase of ﬁrst feeding can be expressed as Wt = W0 e mt

(7.7)

where m is the speciﬁc growth rate (day-1), expressed by the initial slope of the curves in Fig. 7.8, W0 is the apparent initial larval biomass (biomass extrapolated to Day 0, and not identical to the biomass at hatching) and Wt is the biomass after t days. An equation that, with some care, may be used to predict ﬁsh growth in later stages is Wt = Wmax (1 + W0 e - mt )

(7.8)

where Wmax is the biomass of the largest adult individuals, and W0 and m are as deﬁned in Equation 7.7. Growth represents the ﬁnal integrated result of feeding, digestion and the

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allocation of energy to the de novo biomass, and the speciﬁc growth rate during the larval feeding stage can be used as the ultimate criterion of rearing success. If the feeding conditions are adequate, the larvae are expected to show an exponential increase in weight during this stage. Any deviations from an exponential growth pattern means that the feeding conditions are sub-optimal. The rate of increase may actually vary, for example because of variations in temperature (Otterlei, 2000) or species, but the pattern of biomass increase in healthy larval populations should nevertheless be exponential over time. Starvation resulting from inadequate food supplies often causes growth kinetics that deviate from the exponential pattern, but other more speciﬁc deﬁciencies may also play a role. A typical deviation from the exponential growth pattern is a reduced rate of biomass increase from the start of feeding, followed by an apparent recovery towards metamorphosis. Although this recovery may be real, it is frequently a result of selective mortality of the smallest individuals in the population. Another typical deviation characterised by a reduced growth rate during the later stages is the effect of under-feeding. The speciﬁc growth rate during ﬁrst feeding is one of a set of criteria used for evaluating rearing success. Some others are viability, survival and frequency of malformation, for example pigmentation in ﬂatﬁsh (see Chapter 6). Larval and fry viability is tested in different ways, such as an ability to survive physical stress or enhanced salinity. Larval viability is sometimes the most sensitive criterion for assessing nutritional deﬁciency. It is also very frequently found that survival appears to be more sensitive to inadequate feeding conditions in intensive cultures than growth rate.

7.5.6 Nutritional Challenges and Conﬂicts The nutritional challenges in producing viable fry of marine cold-water ﬁsh were initially mainly focused on lipids and n-3 HUFA nutrition. This focus was gradually turned towards speciﬁc problems related to DHA, which still needs to be optimised for some species. It has generally been assumed that protein nutrition is easier to manage during the larval stage than lipid nutrition, but more recent results have shown that a focus on dietary protein is important to ensure that the quantitative larval requirements for essential amino acids are fully met.

7.5.6.1 Criteria of Nutritional Value for Live Feed The nutritional value of formulated feeds for older ﬁsh is normally expressed in terms of quantitative contents per dry matter, and as relative proportions of components. Expression of nutritional value becomes more complex for live feed, because the larvae primarily relate to and catch individual prey organisms. This fact introduces the mass of nutrient per prey organism as an additional expression of nutritional value. Some ways to express the nutritional value of live feed are given below. Quantitative expressions:

• mass of nutrient per live feed biomass (e.g. mg lipids, or DHA per g dry matter) • mass of nutrient per individual prey organism (e.g. ng lipids, or DHA per rotifer)

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Qualitative expressions:

• percentage nutrient content (e.g. % DHA of total fatty acids, and % lysine per total sum of amino acids) • speciﬁc ratios of components (e.g. ratios DHA/EPA, n-3/n-6, and protein/lipid) A vital question is whether one or some of the above expressions is generally more feasible than the others, or if this depends on the actual nutrients and other conditions. The ﬁsh larvae catch individual prey organisms during feeding, and the individual organisms show a pronounced variability in biochemical composition, which is the principal difference from a stable formulated feed. Single rotifers exhibit variable meat contents. This results in a variable mass of nutrients per rotifer, but also a variable digestible fraction, because the exoskeleton is there and cannot be digested. This reduction in meat content per rotifer will not necessarily always affect nutrient per dry matter to the same extent, because the actual nutrient may show a co-variation with the dry matter if it is a major component. For example, it is typical that protein per dry matter and the relative proportions of amino acids (proﬁle) show less variability than protein per rotifer (or EAA per rotifer) when the proﬁle is in fact constant (see Chapter 4). With these questions in mind, a sound approach and a general recommendation is also to consider the question of how to express nutritional value as a part of nutritional studies, rather than as an established tool. 7.5.6.2 Lipids and n-3 HUFA From the beginning, problems relating to n-3 HUFA nutrition were a major obstacle for the culture of larvae of marine ﬁsh, and of cold-water species in particular. For most species, these problems are no longer a barrier to the development of an economically feasible mariculture industry. As well as technological challenges in the administration of an adequate n-3 HUFA supply, there are still issues related to lipid nutrition and juvenile quality that are not adequately understood. General Status The question of n-3 HUFA nutrition has two main aspects: (1) the composition of the live feed that provides the nutrients, and (2) the requirements of the larvae that are nutrient sinks with species-speciﬁc requirements. There is limited knowledge about the n-3 HUFA requirements of individual species of cold-water larvae, and some further information is needed to establish knowledge-based protocols for ﬁrst feeding. However, reliable experiments on larval feeding can only be performed when all the up-stream production lines and the interacting factors during ﬁrst feeding are satisfactorily understood and controlled. Rearing protocols to allow nutritional studies on turbot have been known for a decade, and halibut, and certainly cod, should now be ready for more extensive studies, whereas the remaining species still need some development before their speciﬁc nutritional requirements can be studied. The current overall status of our knowledge of live feed components is described below.

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(1) Microalgae are an important source of n-3 HUFA because they are added along with rotifers during early ﬁrst feeding (‘green water’ techniques). The knowledge needed and the technology used for the production of microalgae are well developed, and algal costs per fry are very low. This does not imply that there is no need for improvements, because microalgae are also living organisms characterised by a variable biochemical composition. (2) The methods for n-3 HUFA enrichment of the rotifer Brachionus plicatilis are well established. Biological understanding of the basic mechanisms of fatty acid kinetics and the methods for controlling both quantitative and percentage n-3 HUFA enrichment are well developed. The n-3 HUFA enrichment is relatively stable post-enrichment, and efﬁcient methods to control nutritional value until the rotifers are consumed by the larvae are described. (3) The enrichment methods for n-3 HUFA in Artemia to cover the requirements of coldwater species are relatively well developed. Both n-3 HUFA and DHA can readily be enriched to high levels, but the relative contents (percentage of total fatty acids), especially of DHA and the DHA/EPA ratio, are still lower than in copepods. Moreover, a high lipid level and a low protein to lipid ratio is always a consequence of efﬁcient enrichment, particularly in Artemia nauplii. (4) The enriched DHA and n-3 HUFA are not stable in Artemia post-enrichment, and the DHA level cannot easily be maintained and controlled using microalgae in the larval tanks during ﬁrst feeding, as it can for rotifers. This is a difﬁcult problem, because DHA is the most important fatty acid for the larvae. However, it becomes a severe nutritional problem only when Artemia are used for too long as the sole live feed. This is the case for Atlantic halibut, which, apart from turbot, are the cold-water species that have received the greatest attention and investment to date. This problem will almost certainly be solved for most other species, as it has been for turbot, but it should be considered when working out enrichment protocols. The developments made for cold-water species have taken great advantage of the scientiﬁc and commercial developments made for other marine species. If fact, the principal questions are to a great extent the same, and the important biological mechanisms are almost always the same. A fundamental difference for cold-water species with regard to n-3 HUFA is that temperature affects the functional properties of the cell membranes, with DHA, and PUFA in general, as the main active components in the adaptation to low temperatures (Olsen, 1999a). This is probably why high DHA catabolism in Artemia is a more severe problem for cold-water ﬁsh than for ﬁsh that are evolutionarily adapted to higher temperatures. Phospholipids There has been major concern about phospholipids (PL) during the development of formulated larval feeds that can partly or completely replace live feed. It has been shown that PL facilitates n-3 HUFA uptake and digestion in many species of marine larvae. Amongst other things, the PL acts as an emulsiﬁer in the gut, and PLs are almost always richer in n-3 HUFA

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than triacylglycerides (TAG), making them nutritionally more favourable. Copepods that are the natural food of many species, as well as rotifers and Artemia, contain signiﬁcant amounts of PL, like all aquatic organisms. The quantitative contents of PL in all these prey organisms are comparable (5–8 mg PL per g DW), but the relative proportion of PL in total lipids is different. The small copepods that are eaten by ﬁsh larvae are lean, and may typically contain 60% PL in their total lipids. This proportion is typically in the range 20–30% in well-enriched rotifers and Artemia. The speciﬁc requirement for PL in early larvae originates in the fact that the larvae are not able to synthesise their own PL for membranes, and instead have to incorporate undigested PL directly from their food (Bell et al., 2002). n-6 HUFA The importance of arachidonic acid (20:4 n-6, ARA acid, see Fig. 7.1) for marine larvae has been addressed more recently. It is highly questionable whether carnivorous marine ﬁsh can synthesise ARA by the elongation and desaturation of shorter n-6 moieties, implying that it will be an essential component of their food. ARA, together with EPA, is a crucial precursor in the prostaglandin synthesis of eicosanoids (potent tissue hormones). It is therefore also an essential component in membrane synthesis, because eicosanoids are synthesised based on EPA and ARA incorporated in the membranes. Experiments have suggested that a high ARA content may affect the pigmentation of ﬂatﬁsh and their resistance to physical stress (Koven et al., 2001). ARA may not be essential for maintaining functional cell membranes, but it is certainly essential for the synthesis of prostaglandin, and therefore is also critical for larval health. n-3 HUFA and DHA Deﬁciency Considerable efforts have been made to assess the effects of n-3 HUFA deﬁciency in ﬁsh larvae. These involve developmental and behavioural disorders, including incomplete dorsal pigmentation in ﬂatﬁsh, incomplete metamorphosis and eye migration, neural disorders related to eye and brain development, reduced stress tolerance, reduced survival and ﬁnally reduced capability for growth. A number of these disorders have been documented for halibut larvae in a recent study by Shields et al. (1999), who relate the problem to the low DHA content of Artemia. The nutritional problems caused by inadequate DHA levels during larval rearing are illustrated in Fig. 7.9. The left-hand panel shows the percentage DHA content of halibut larvae after 30–35 days feeding as a function of the DHA content of the live feed (percentage of total fatty acids), be it Artemia or copepods. There is a 1 : 1 relationship between DHA in food and in larvae, and it is noteworthy that newly hatched larvae have exactly the same DHA content (percentage of total fatty acids) as the small and young stages of copepods (data from Evjemo et al., 2002). It should also be noted that no similar unique positive correlation exists between the quantitative DHA content of food and larvae (mg DHA per g dry matter, data not shown). This ﬁgure, along with further characteristics of DHA deﬁciency, shows that halibut larvae are barely able to elongate shorter n-3 moieties and thereby increase their DHA content.

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Turbot fry, 23 days old

40

B

100

% Normal pigmentet in fry

DHA in larvae, % of total FA

Halibut larvae, 30-35 days old A

30

20

10

Artemia

80

60

40

20

Copepods 0

0 0

10

20

30

DHA in live feed, % of total FA

40

0

0.5

1

1.5

2

2.5

3

3.5

4

DHA/EPA in larvae

Figure 7.9 Characteristics of DHA nutrition in relation to quality of larvae and fry. (A) Percentage DHA of total fatty acid in tissues of halibut larvae as a function of the respective DHA of the live feed. The larvae were fed for 30–35 days, and the arrows indicate the type of prey (data from Evjemo et al. 2002). (B) The fraction of normally pigmented turbot fry as a function of the ratio DHA: EPA in larval tissues on Day 12. Pigmentation was assessed in 23-day-old fry (data from Reitan et al., 1994; Øie et al., 1997, and unpublished data, 1999). Both curves are based on compilations of many independent experiments.

Larvae fed high levels of DHA (>30% of total fatty acids, copepods) did not show any signs of DHA deﬁciency, whereas larvae fed low levels of DHA (<11%, inadequately enriched Artemia) showed severe signs of DHA deﬁciency, including reduced growth rate. Larvae fed food with intermediate levels of DHA (16–22%, well-enriched Artemia) showed more variable symptoms of DHA deﬁciency. Although the quantitative DHA levels were higher in Artemia than in copepods, this did not prevent the symptoms of DHA-deﬁciency developing, e.g. malpigmentation and low stress tolerance. The lower percentage DHA levels of the larvae must reﬂect reduced DHA contents in larval PL also, which is the main lipid component. In one experiment, the PL content of 15–35-dayold halibut larvae was found to be 104 mgPL gDW-1 (range 85–122 mgPL gDW-1) or 67% PL of total lipids (range 62–74%) (J.R. Rainuzzo, unpublished results, 1998). If the DHAdeﬁcient larvae which are fed the most DHA-deﬁcient Artemia are characterised by deﬁcient membranes and a reduced general state of health, it is noteworthy that these larvae also developed clear morphological and developmental signs of DHA-deﬁciency. Unfortunately, it is not possible to identify a critical lower value for percentage DHA in food that would allow normal growth and development. The right-hand panel in Fig. 7.9 shows that the DHA content of larval tissues can act as a predictor of its later development. The graph shows the relationship between the DHA : EPA ratio in 12-day-old larvae and the later pigmentation of the turbot fry. Low DHA to EPA corresponds with a low degree of successful pigmentation, although DHA may not be the only nutritional factor involved. All the turbot larvae from the experiments used in the ﬁgure were fed rotifers that were well enriched with n-3 HUFA, but the n-3 HUFA composition differed. Some larval groups which contained high DHA levels, but even higher levels of EPA, developed poor pigmentation in later developmental stages.

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According to the examples described above, it is quite clear that both halibut and turbot larvae may show signs of deﬁciency even when they are fed high quantitative amounts of DHA in their cultured live feed. This is clearest for halibut larvae, which cannot utilise the DHA in Artemia as efﬁciently as the DHA in copepods. The relationship between the observed disorder and the nutritional value becomes clearest when DHA is expressed in terms of percentage of total fatty acids, but the quantitative content is still important. There are still challenges to improve our fundamental biological knowledge and cultural methods. Automation and further standardisation will certainly contribute to more reliable live feed production. Several important biological issues relating to lipid nutrition must be considered in future developments. (1) A general nutritional challenge in rotifer production is to optimise the biochemical composition to meet the speciﬁc requirements of different species of ﬁsh larvae. The manipulation tools are there for n-3 HUFA, but more speciﬁc information is required about the larvae. (2) Fatty acid kinetics and enrichment methods for other strains or species of rotifers that may be found to be favourable for any of the larval species must be characterised (e.g. Brachionus rotundiformis). (3) It is essential for the future development of Artemia technology to gain more experience and feed-back on enrichment methods from ﬁrst-feeding trials, and from fundamental research on speciﬁc ﬁsh species. It will also be important to establish methods to inhibit DHA catabolism in Artemia post-enrichment. Another approach is to commercialise Artemia strains that do not show this enhanced DHA catabolism. Such strains have been identiﬁed (Evjemo et al., 1997), but are not readily available. (4) The mechanism of digestion and the incorporation of PL into cell membranes needs to be better understood, because this will be instrumental in improving enrichment technologies. 7.5.6.3 Essential Amino Acids and Proteins There been a general misunderstanding that the supplementation of adequate amounts of proteins of the required quality has not been a major obstacle in the rearing of marine juveniles. However, it is well known that protein is a major constituent of developing larval tissues, and larvae have a very high speciﬁc requirement for protein to maintain the rapid growth that we observe. Amino acids, and thus protein, are therefore the keys to growth in fast-growing larvae, and are quantitatively more important than lipids. Some fundamental aspects of growth and protein synthesis are treated in Chapters 6 and 9, and this section mainly describes status and some relevant points discovered during larval cultivation. General Status As for n-3 HUFA, the question of protein and essential amino acid nutrition has two main perspectives that must be covered: (1) the composition of the live feed that provides the nutrients, and (2) the requirements of the larvae that are nutrient sinks characterised by

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species-speciﬁc requirements. The general status of protein in larval feed components is described below (see Chapter 4). (1) Microalgae is a source of protein during ﬁrst feeding because it is commonly added to the larval tanks during the early phases of ﬁrst feeding (‘green water’ technique). The algae then tend to stabilise the protein contents of the live food. The technology of producing microalgae is satisfactory. (2) The protein content of cultured B. plicatilis is variable, and depends on its rate of food supply and growth. A high feeding rate and growth rate of cultures will secure a high protein level per rotifer and a high protein level per unit of dry matter. The relative distribution of amino acids remains constant regardless of the protein content, or the nutritional state of the rotifers, because rotifer amino acid composition is genetically determined. Our biological understanding of the basic mechanisms of protein accumulation and methods of long- and short-term enrichment by proteins have been established. There are also efﬁcient methods to control and secure protein values until the rotifers are consumed by larvae. (3) Artemia hatched from resting cysts show highly reproducible protein levels compared with cultured rotifers. The protein content and quality are probably adequate to meet the requirements of cold-water larval species, as demonstrated in many experiments and production trials in hatcheries. The normal protocols for Artemia used as live feed ensure that proteins are stable post-enrichment, and are delivered efﬁciently to the ﬁsh larvae. The differences in protein requirements between cold-water species and species that are evolutionarily adapted to warmer water are probably smaller than for lipid nutrition. The scientiﬁc and commercial developments made for other marine species can therefore also be used during the cultivation of cold-water ﬁsh larvae. Protein Quality It has been suggested that marine ﬁsh larvae should be supplied with free amino acids (FAA), peptides or proteins in their diet. It is known that free amino acids are a major substrate for energy generation in endogenously feeding yolk-sac larvae (Fyhn, 1990). It is also known that the digestive capabilities of the larvae are not fully developed in the early stages of ﬁrst feeding (see Chapter 6). These ﬁndings have given rise to the idea that the larvae also have speciﬁc requirements for FAA in their early exogenous diet, but this is still an issue of controversy. Experience gained from larviculture cannot answer these controversies, but such results may form a framework for the more fundamental studies that are needed, and in this way contribute with premises for discussion. Rotifers are fed to many species of marine ﬁsh larvae as the ﬁrst food, and many experiments and production trials have revealed that they may sustain both a high growth rate and survival. This is the ﬁrst indication that food components are nutritionally adequate. Live Brachionus sp. and Artemia contain signiﬁcant amounts of free amino acids (see Chapter 4). Proteases from the dead rotifers continue to catabolise its proteins post-mortem, and short

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peptides and free amino acids gradually become more dominant during the hours of decomposition. These compounds are available for assimilation provided that the gut evacuation time of the larvae is not too short. An analysis based on the mass balance between protein consumption and protein retention in tissues clearly shows that larvae have the capacity to consume much more protein than is needed to sustain their actual growth during the early phase of ﬁrst feeding. Experiments with turbot larvae have shown that the larvae need to incorporate only 6–9% of the protein that they consume into their own tissues from Day 2 to Day 6 of feeding in ‘green water’. Larvae that were maintained in ‘blue water’ consumed a lower number of rotifers, which also contained less protein. These larvae needed to incorporate 18–28% of the ingested proteins (Øie et al., 1997). Both treatments resulted in a high speciﬁc growth rate (0.21–0.29 day-1 at 18°C), 88–100% fully pigmented fry, and a high fry ability to sustain physical stress. Their survival after metamorphosis depended on the treatment (29–54% in ‘green water’ and 7–24% in ‘blue water’). These results suggest that the larvae have more than enough protein available for digestion and assimilation, especially if they are cultured with microalgae. There is a suggestion that turbot larvae cultured with microalgae may get a major part of their amino acid requirements for growth from the FAA in rotifers (see Chapter 4). This suggests that the discussion regarding requirements for FAA versus peptides and proteins in larval feeds is primarily relevant for formulated larval feeds. An issue discussed above is that the digestion and assimilation of protein and other essential components, and also those that are liberated through endoenzymatic activity in the live feed post-mortem, may depend on the gut evacuation rate. This rate is primarily controlled by the food concentration, which therefore also affects the assimilation efﬁciency of the feed. This means that a high food concentration may inhibit protein assimilation, and in the worst case also the growth and survival of ﬁsh larvae. This emphasises the need to maintain an optimal prey concentration— not too high and not too low—during early ﬁrst feeding. Protein Deﬁciency Figure 7.10 shows growth rate and survival as a function of the protein content of individual rotifers, and the curves support the general idea that a protein supply is crucial to rearing success. For all treatments, improved survival and growth were obtained for rotifers characterised by a high protein content. The same pattern was also apparent for pigmentation (unpublished results, and Øie et al., 1997). Values are shown for both ‘green water’ and ‘blue water’ systems. Both survival and growth are systematically higher with microalgae added, and the effect is in particular pronounced for survival. The positive relationships shown in Fig. 7.10 are also apparent when the protein content is expressed in terms of protein per unit of dry matter. However, the scatter is more pronounced because the range of values is narrower. The same pattern is not at all apparent if the protein food value is expressed as percentage of essential amino acids in total amino acids (or protein). It is important to emphasise that the lorica length of rotifers used in all trials were identical. The factor which varied was the meat content of individual rotifers, and therefore also the ratio of digestible to indigestible matter because the mass of the

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indigestible exoskeleton is constant. Feed ecologists will probably accept the relationships in Fig. 7.10, but the use of another rotifer strain with a shorter lorica length and a high meat content (equal digestible fraction) may have yielded different results. The potential problem in giving live feed with an inadequate protein content is real because it is very easy to produce rotifers with a very low meat and protein content (see Chapter 4). On the other hand, only very extreme Artemia or larval rearing protocols can make that situation likely for Artemia. The empirical ﬁndings of many hatcheries that rotifers should be harvested while growing at a high rate in batch cultures is fully compatible with the results in Fig. 7.10. 7.5.6.4 Synergetic Importance of Lipids and Proteins The biochemical feature that makes rotifers, and in particular Artemia, different from marine copepods is their higher lipid content relative to proteins. One reason for this is the need to incorporate high levels of n-3 HUFA in the cultured live feed. Young stages and small species of marine copepods are relatively lean, and the protein to lipid ratio of copepods used to feed marine larvae is typically 4:6 (Evjemo et al., 2002). Such values can be approached in rotifers provided they are enriched with n-3 HUFA during cultivation only, and not in successive short-term procedures. This is not possible for Artemia nauplii, because there is no time to break down the lipid reserves of the nauplii during the short time that is available for enrichment (<1 day). On-grown juvenile stages of Artemia may have more copepod-like protein to lipid ratios because their lipid content is lower than that of nauplii (Olsen, 1999b). If the general strategy of using marine copepods as a reference for cultured live feed is the conceptual basis for live feed production, it should ﬁrst be examined more carefully. A compromise must be made regarding methods of n-3 HUFA and protein incorporation into live feed.

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Figure 7.11 clearly indicates how this compromise should be established for turbot larvae. The ﬁgure shows survival in relative terms as a function of protein per rotifer and lipid per rotifer. Normalised values of both ‘green water’ and ‘blue water’ treatments are included, and the content of n-3 HUFA as a percentage of total fatty acids is high for all treatments. Protein per rotifer affects survival much more than lipid per rotifer, which is not very important when the relative content of n-3 HUFA is adequate. Survival is gradually improved as protein per rotifer increases, and the rate of increase is particularly high for intermediate protein values. The speciﬁc growth rate of the larvae showed basically the same pattern of variation with protein and with lipid per rotifer. The general relationship of the ﬁgure was also valid when protein and lipid contents were expressed in terms of dry matter, although it was less clear. 7.5.6.5 Vitamins and Minerals Data on the contents of vitamins and minerals in live feeds are accumulating, but veriﬁcation of the quantitative needs for these components in larviculture is scarce. One strategy is to assume that larval requirements of vitamins and minerals are identical to those of larger ﬁsh (for values, see NRC, 1993, and Table 8.3 in Chapter 8). For most metabolic compounds of live zooplankton and larvae that are not strictly genetically controlled there tends to be a

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positive relationship between the concentrations of the compound in the prey and in the larvae. Normally this means that the content of the compound can be manipulated to meet the requirements of the larvae by just incorporating it in the feed of the live feed. This can be achieved in two ways in practical rearing. The uncontrolled way is to use marine microalgae that apparently have a relatively balanced composition of micronutrients. These nutrients are passed on to the live feed, and then to the larvae when microalgae are added to feed together with rotifers and Artemia. The controlled way is to use microalgae and thereafter leave the problem of adding the compounds that potentially are limiting to the manufacturer of the feeds for rotifers and Artemia. The vitamin contents of the live feed used for intensive larval rearing in general seem to be adequate. However, some vitamins should be added in enhanced amounts. For example, it is known that sea bream take advantage of a higher vitamin C content in live feed than the level present in early formulations of feed for rotifers and Artemia. Enhanced levels resulted in a higher larval ability to tolerate physical disturbance, and some experiments have even demonstrated positive effects on larval growth rate (Merchie et al., 1995). Greater attention to the optimisation of vitamins and minerals for cold-water species can be expected when the feeding regimes and the entire commercial production line is working as well as it now is for sea bass and sea bream. A further ﬁne-tuning of these compounds is hardly cost-effective at the present stage. 7.5.6.6 General Recommendations on Larval Nutrition The general nutritional recommendation for feeding cold-water species of ﬁsh larvae must include a compromise between quantitative protein content and percentage n-3 HUFA in rotifers. (1) Use rotifers that are well enriched with n-3 HUFA during cultivation, and avoid using rotifers that are harvested from cultures growing at a rate that is lower than half their maximum growth rate (e.g. >0.2 day-1, 20°C). (2) The n-3 HUFA or protein content of rotifers may be additionally enhanced through short-term n-3 HUFA or protein enrichment post-harvest. (3) If rotifers are harvested while growing close to their maximum rate (>0.4 day-1, 20°C, 20 p.p.t. salinity), the protein content will be optimal. However, extra efforts and checks should be made to ensure an adequate DHA content. The DHA content may then be selectively enhanced using short-term enrichment. (4) Short-term enrichment with emulsiﬁed oil reduces the mobility and viability of rotifers and Artemia in cold water. (5) Use DHA rich algae (e.g. Isochrysis galbana) in larval tanks along with rotifers. (6) Use emulsiﬁed oil diets rich in DHA and with a low ratio of EPA to DHA for shortterm enrichment of n-3 HUFA in Artemia, and reduce the starvation phase postenrichment to a minimum for the Artemia. (7) The lipid content of the short-term-enriched Artemia nauplii may be higher than optimal. This may be acceptable, but can be counteracted by using a leaner Artemia grown on for 2–4 days.

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Finally, it is again important to emphasise that different species will have their speciﬁc requirements, and that the above recommendations are therefore only general.

7.5.7 Microbial Conﬂicts and Challenges There is ample evidence that the stocking density of cultures of marine larvae is constrained by bacterial activity in the larval tanks through interactions between the larvae and the bacterial community of the water and the live feed. The general mechanisms and status of these issues are treated in Chapter 3. This section is restricted to treating aspects that are particularly relevant during the phase of ﬁrst feeding. The concept of ‘microbial hatchery management’ is now well established, and actions to control both normal and pathogenic bacteria are taken in hatcheries of both ﬁsh and shrimps. Two issues are of particular importance:

• More insight is needed into how the different steps in the cultivation procedure could inﬂuence the bacterial ﬂora in a negative way for the larvae • Based on this knowledge, we need to establish and implement a range of methodological tools to achieve satisfactory control of the bacterial community in larval tanks

It has become clear that the microbial control which is needed during ﬁrst feeding cannot be achieved by a single countermeasure. It is also clear that we are only able to improve and optimise conditions, but not eliminate the problem entirely. For example, we may remove the bacterial loading and dissolved organic substrates from live feed cultures and process water very efﬁciently, but this will not remove the problem as both larvae and live feed release undigested carbon compounds that will boost the microbial food web in the larval tanks. It is therefore important to use a set of countermeasures that will all contribute to the objective of maintaining microbial control. The general conceptual understanding of the microbial problem of larval rearing is that a sudden enhancement of available substrates or decimation of the bacterial biomass will allow opportunistic r-selected bacteria to bloom. Such bacteria are believed to include bacteria that are potentially more harmful for the larvae than non-opportunistic K-selected bacteria (see Chapter 3). Our protocols for larval rearing should therefore consider that sudden pulses of available carbon must be avoided as far as possible. The bacteria and the organic carbon loading coming from all the main process lines will affect the microbial conditions during ﬁrst feeding. This involves bacteria and substrates that are supplied with the process water, bacteria and loads that originate from the larva itself, and bacteria and carbon loading delivered with the live feed cultures. Chapter 3 recommends a general strategy for the maintenance of microbial control:

• Improvements in larval resistance • Non-selective reduction of harmful and potentially harmful bacteria • Selective enhancement of favourable bacteria Successful immune stimulation of larvae using potent biopolymers derived from macroalgae and vitamin C enrichment has been demonstrated experimentally, but these techniques

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are still not commonly used (e.g. Skjermo et al., 1995). The issue of improving larval resistance is treated further in Chapter 3. 7.5.7.1 Methods of Non-Selective Reduction of Bacteria Some common means of non-selective reduction of bacterial growth potential and bacterial biomass are:

• The removal of bacteria from the water source by UV-disinfection, ozone-sterilisation or membrane ﬁltration • Surface disinfection of eggs • A reduction in carbon loading following hatching • A reduction in carbon and bacterial loading from the live feed cultures, the bacteria associated with the surface, and in the intestines of the live feed • A reduction in the internal carbon loading in tanks, and the removal of dead organisms and particulate organic compounds

Suitable water treatment is paramount for all phases of larval rearing. Such treatment is a major barrier to pathogenic bacteria, and a means of reducing the bacterial loading of the water. Many hatcheries have traditionally used a simple sand ﬁlter treatment for their process water, but there seems to be a trend towards the use of more powerful methods for removing bacteria as well. Ozone treatment has been used increasingly in the last few years, but it has not been ﬁnally veriﬁed that this treatment is feasible for ﬁsh larvae. Proper ozone treatment may kill virtually all the bacteria, whereas membrane ﬁltration will physically remove >95%. The experience of using membrane-ﬁltered water for ﬁsh larvae is generally positive, but both treatments may turn out to be used in the future. Proper UV-treatment may work, but it will never be as efﬁcient as the other methods. An efﬁcient procedure for the surface disinfection of eggs is important in order to achieve early control of the microbial ﬂora of the larval process line. Glutaraldehyde, which regrettably is a toxic substance with restrictions on its use, is the most efﬁcient chemical yet tested (Salvesen & Vadstein, 1995). Treatment for 5–10 min at concentrations of 400–800 mg l-1 is efﬁcient at the normal temperatures needed for eggs of cold-water species. There is no need for large amounts of the chemical, and the hatchery staff can run the disinfection procedure without being exposed themselves. Disinfection is particularly important for eggs that need a long time to develop before hatching, or if the eggs have been exposed to stagnant conditions or high ambient carbon concentration, for example during shipments. The most obvious effects of disinfection are higher hatching synchronicity, better reproducibility for replicates, and a better general performance for larval groups in later stages of ﬁrst feeding. When considering methods and protocols, it is essential to bear in mind that the carbon and bacterial loading should be minimised for all components and treatments. The importance of rinsing the live feed and cleaning larval tanks are widely accepted as countermeasures against microbial problems. One issue normally overlooked is the increased loading of carbon that follows the hatching of the eggs. A signiﬁcant fraction of the egg biomass (20–25% for turbot) is released as dissolved carbon compounds that are readily

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available for bacteria. A high water exchange rate or large water volumes (large tanks or a high volume per egg) are optional countermeasures, but it is also important to realise that this carbon pulse will always create unfavourable local conditions. 7.5.7.2 Methods for Selective Enhancement of Favourable Bacteria The following methods may be used to select for favourable bacteria:

• Controlled re-colonisation of favourable bacteria in the process water after disinfection • Supplementation of favourable bacteria with the live food or directly into the tank water The treated process water (see above) will contain very low bacterial numbers and an enhanced concentration of organic carbon due to the extensive cell disruption that is a consequence of the treatment. This is a situation that always results in severe blooms of opportunistic bacteria, and such water should never be used immediately for larval rearing. This has been extensively documented for many species and stages of marine larvae (see Chapter 3). The effect of sub-optimal bacterial composition is manifested after only a few days of feeding. Both speciﬁc growth rate and survival may be reduced, even when microalgae are used along with the live feed (see below). Controlled re-colonisation of the water should start immediately after the initial treatment with UV, ozone or ﬁltration. Bacteria that have colonised the tubes and tanks used on the way to the larval tanks will immediately start to re-colonise the water, and this may be satisfactory in some cases. Transport in tube systems and a slow exchange rate in larval ﬁrstfeeding tanks will both contribute to a dampening of the oscillations of opportunists in the closed environment of the larvae. However, the microbial conditions of the larvae cannot be controlled in an optimal way, and a precautionary approach is to re-colonise the water with harmless K-selected bacteria in a more controlled manner. This process of water treatment has been termed microbial maturation (Skjermo et al., 1997; Salvesen et al., 1999). Microbial maturation is a simple biological process that can be undertaken with simple equipment and at a low cost. It will contribute to improving the stability and predictability of fry production, and to enhancing growth, survival and the general performance of the fry. Figure 7.12 illustrates a system that can be used to re-colonise the process water just after disinfection. The main component is a bioﬁlter unit that is kept in a tank, and the process water ﬂows uniformly through the ﬁlter. The sizes of the ﬁlter and the tank depend on the amount of water and the ﬂow rate, and these questions are still the subject of research. However, ample experience has shown that such systems are surprisingly efﬁcient at recolonising the water with a bacterial community dominated by K-selected bacteria. The system will secure a main water supply which is characterised by an ability to accommodate carbon pulses without producing blooms of opportunistic bacteria. Another means of controlling bacterial composition is to recycle the water from the larval tanks after the removal of larger particles, but without disinfection. This method is widely used for Mediterranean species during the nursery phase in order to save energy, but there is still limited experience of its use in the rearing of larval stages. One problem is the accumulation of an organic load originating from microalgae. Combined ozone treatment and

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Figure 7.12 System for re-colonising process water by harmless K-selected bacteria. Inlet water is treated by membrane ﬁltration (alternatives are ozone or UV). It is then re-colonised by slow-growing non-opportunistic bacteria in a ﬂow-through bioﬁlter unit before it is used for ﬁsh larvae (redrawn based on a presentation by I. Salvesen).

protein skimming can efﬁciently remove the organic components, but that treatment will also kill the bacteria (Suantika et al., 2000). Documentation about the bacterial conditions in these systems is still limited. There is a common understanding that the use of antibiotics in aquaculture should be kept to a minimum. Conversely, the use of probiotic methods in larviculture of ﬁsh has attracted considerable attention the last few years. Many potentially favourable bacteria have been isolated, characterised and tested, and the preliminary results are promising (see Chapter 3). However, these are methods that must be further developed and tested in the future. We could say that the use of microbial maturated water is a kind of probiotic treatment that is simpler because we use the ecological laws of natural selection. Conversely, an enriched bacterial strain, or a mixture of strains, may rapidly become out-competed by naturally occurring strains because of the same ecological laws. However, there are many tailor-made applications that will probably be developed in the years to come. One example is to ensure that the live feed always has a gut content of favourable or harmless bacteria when added to the larval tanks. This method has already been described, characterised and tested for bacterial cultures and for the use of microalgae and their associated bacteria as probiotic agents (see below). Another tailor-made application is to add a beneﬁcial bacterial culture daily during the period when the microﬂora of the water itself is most important for the larvae. This will probably be the period between spawning (i.e. surface disinfection of the eggs) and the ﬁrst few days of ﬁrst feeding.

7.5.8 Use of ‘Green Water’ Techniques Since the early 1960s, microalgae has been widely used in the culture of marine ﬁsh larvae. There are three common applications of microalgae.

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(1) Microalgae have been used as food for the production of rotifers, either as the only feed, or in combination with cheaper feeds such as baker’s yeast. (2) Microalgae are used for short-term enrichment of rotifers and Artemia with n-3 HUFA and other components before their use as live feed. (3) Microalgae have been added to the larval tanks together with the live feed, normally in concentrations of <2 mgC l-1, which is the so-called ‘green water’ technique. There is no doubt that microalgae are among the better feeds for growing rotifers, but their relatively high cost has made other feeds more attractive for this purpose. However, concentrates of microalgae, which have recently become commercially available, have been used as the main feed during the development of methods for highly intensive rotifer cultivation (Yoshimura et al., 1997). Short-term enrichment using microalgae has been widely used, originally to enhance the n-3 HUFA content of the rotifers, and more recently to introduce the more favourable bacteria which are associated with the rotifers and the Artemia. The most cost-efﬁcient application of microalgae by far is to add the algae to the larval tanks together with the live feed during the early phase of ﬁrst feeding. This treatment has a tremendously positive effect on production yields of fry and on fry quality, and the quantitative need for microalgae is low. This method is known as the ‘green water’ technique. 7.5.8.1 Effects of ‘Green Water’ The ‘green water’ technique was invented by accident during early efforts to cultivate turbot in the late 1960s (B. Howell, personal communication, 2001). Many studies have since questioned the basic mechanisms behind the positive effect of adding microalgae along with rotifers or Artemia to ﬁsh larvae, especially in the early phase of ﬁrst feeding (see reviews in Reitan, 1994; Reitan et al., 1997). A typical effect of adding microalgae on the survival and growth pattern of turbot is illustrated in Fig. 7.13 (see also Fig. 7.10). The most pronounced and reproducible positive effect of microalgae is enhanced larval survival, but the increased speciﬁc growth rate during feeding on rotifers, and the improved ability of the fry to survive physical stress in later stages are other positive effects. The numbers of viable turbot fry normally become more than four times higher simply by adding small quantities of microalgae to the larval tanks. This is probably the most cost-efﬁcient single treatment that can be identiﬁed during larval rearing. It is also clear that this positive effect seems to be common, although variable, for most species of marine ﬁsh larvae that have been cultured, including the cold-water species. Many factors are believed to be involved in the improved yields and quality of the fry, and extensive research has suggested the beneﬁcial effects of those listed below. (1) Larvae in the early stage of ﬁrst feeding exhibit higher feeding rates of live feed with algae added than without (termed ‘blue water’). (2) Microalgae contribute to the n-3 HUFA enrichment of rotifers if the algae are rich in such fatty acids, in particular in the early stages of ﬁrst feeding when the retention time of the rotifers in the larval tanks is longer. This effect is not shown for Artemia nauplii.

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Figure 7.13 Typical curves describing the growth and survival of turbot larvae maintained in ‘green water’ and ‘blue water’. (A) Percentage survival as a function of time during larval feeding until metamorphosis. (B) Increase in larval carbon biomass as a function of time during larval feeding until metamorphosis.

(3) In the same way, the addition of microalgae will help to stabilise or improve the general nutritional value of the rotifers, e.g. the lipid and protein contents. This effect is not achieved for Artemia nauplii. (4) Microalgae may transfer essential micronutrients to rotifers and Artemia that are otherwise supplied in only sub-optimal amounts, e.g. vitamins, minerals, carotenoids or other compounds. (5) Cultures of microalgae contain bacteria that are favourable for surface and intestine colonisation of the live feed and ﬁsh larvae. (6) The microalgae in larval tanks will contribute to increased light scattering and therefore a change in the light conditions that seems to be beneﬁcial to the swimming behaviour of some ﬁsh larvae. (7) Microalgae may remove endoparasites from rotifers, and it is suggested that they generally contribute to improved larval health in some way. This is still speculation, but some biopolymers from macroalgae are very potent immune stimulators.

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Earlier studies have suggested that microalgae may remove ammonium and produce oxygen, actions that are both beneﬁcial for the larvae. This may happen, but this mechanism is only likely to be signiﬁcant in heavily carbon-loaded larvicultures, and such conditions are never optimal for larval rearing. Some of the effects identiﬁed will require that the microalgae are exposed to the live feed for some time before they can become effective. This will be particularly important for the nutritional effects, to some extent also for the microbial effects, which work through both the water and the algal cells, and to a lesser extent for the changes in the physical conditions, such as the light regime. 7.5.8.2 Nutritional Effects When rotifers and microalgae are mixed in larval tanks, this should be done before stocking the larvae. The rotifers will then ﬁll their gut in about 15 min if the food concentration is at saturation point (1–2 mg C l-1, see Chapter 4). The microalgae will become assimilated and allocated to the anabolic processes of growth and reproduction. The fatty acid proﬁle of the rotifers will change rapidly, and the kinetics and predictability of these changes are well understood (see Chapter 4). The response time for changing the fatty acid proﬁle is typically a few hours, and the fatty acid composition of the rotifers will then gradually approach that of the algae. We can almost completely control the fatty acid composition of the rotifers in the larval tanks simply by selecting algal species or mixtures of species with the n-3 HUFA proﬁle that we want. For example, the n-3 HUFA proﬁle of the rotifers will be completely stable if the proﬁle of the algae, or mixture of algae, is identical to the initial proﬁle of the rotifers. The changes in lipid and protein contents will occur more slowly, but most of the changes will take place within 1 day. The protein content, as mentioned earlier, is directly coupled to the growth rate of the rotifers, and the response time is slightly shorter than that for growth (1 day). The lipid content will normally respond faster. The nutritional beneﬁts of maintaining Artemia with microalgae are more doubtful. Artemia needs very high food concentrations in order to feed at its maximum rate (5– 10 mgC l-1), and such high concentrations are sub-optimal in larval tanks for many reasons. Nauplii that are efﬁciently short-term-enriched by emulsiﬁed marine oil will actually lose their n-3 HUFA more rapidly with microalgae added than without (see Olsen, 1999b). Therefore there are no obvious nutritional beneﬁts of the treatment in Artemia, at least for n-3 HUFA, lipids and proteins. The nutritional conditions in ‘blue water’, which is the alternative to ‘green water’, are obviously less favourable. The conditions of ‘blue water’ imply starvation and steady losses in nutritional value for both rotifers and Artemia. One day of starvation at 18°C typically results in 40% reduction in the individual dry weight of the rotifers. All types of metabolic components will become catabolised, although at variable rates (see Chapter 4). It is clear that ‘blue water’ conditions will have dramatic results if the live feed resides for several days in the larval tanks, especially if the temperature is high. In this case, warm-water species would be even more sensitive than cold-water species.

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7.5.8.3 Microbial Effects Algal cultures contain a different bacterial ﬂora than the process water and the live feed cultures. The composition and community density of bacteria are obviously highly variable, but there is ample evidence to indicate that bacteria which are associated with algal cells throughout are more beneﬁcial for ﬁsh larvae than bacteria that are associated with rotifer and Artemia cultures. This means that a bacterial ﬂora originating from microalgae will be more optimal for colonising the skin and intestine of the ﬁsh larvae than the ﬂora from the live feed (Skjermo & Vadstein, 1993). During ﬁrst feeding, ﬁsh larvae are exposed to bacteria that are suspended in the water and associated with their food, and this exposure through their food is most important when the larvae start to feed actively. These bacteria are associated with the surface and the gut of the live prey organisms. The direct ingestion of bacteria by turbot larvae is low compared with the amount of bacteria that are ingested together with prey. When rotifers and Artemia are exposed to microalgae, they will gradually change the bacterial ﬂora which are associated with their surface and gut contents. This is a relatively fast process, because the average gut residence time is <30 min for Artemia and <15 min for the rotifer. This is the time needed to exchange the gut contents completely. The time needed to reach a new pseudoequilibrium for the surface-associated bacterial ﬂora of the live prey is longer, but major changes in composition will occur within a few hours or days. This means that the microbial effect of adding microalgae will be relatively fast. The suspended bacterial ﬂora will immediately change into a more larva-friendly one. The bacterial ﬂora that are associated with the live feed will undergo major changes within 1 h, or even faster if the gut content is the most important component. 7.5.8.4 Live Feed Retention Time in Larval Tanks The exposure time between microalgae and live feed is longest in the early phase of ﬁrst feeding when the larvae still are learning feeding behaviour. It is then quite common that the live feed remain in the larval tanks for some time between addition and actual consumption. The newly hatched larvae are very susceptible to physical damage, and many species are maintained under stagnant or close to stagnant conditions during this time. This means that the rotifers, or the Artemia for Atlantic halibut, may live in the tanks for long time before they are consumed, especially if the stocking density of larvae is low. With a high water exchange rate, the prey organisms would have been washed out of the tanks. This does not happen under stagnant conditions, because only the processes of larval feeding and mortality remove the prey organisms. The average retention time of rotifers in tanks with turbot larvae maintained at a density of 10 larvae l-1 with algae added was estimated to be 1–2 days, whereas it was 6 days in tanks that did not have added algae. The difference between ‘green water’ and ‘blue water’ is mainly due to a higher larval consumption rate and rotifer reproduction rate in ‘green water’. When the water exchange rate is increased to >4 day-1, normally after 3–5 days of feeding (Days 5–7), the retention time of the rotifers will be about 4 h. Based on the above discussions about the exposure time that is needed between live feed and algae addition before the mechanisms can become active, it is clear that microalgae will

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affect the ﬁsh larvae in a number of ways even under relatively high water exchange rates. The nutritional effects will be particularly pronounced under stagnant or semi-stagnant conditions (<0.5 day-1). The differences in feeding conditions between ‘green water’ and ‘blue water’ will be most pronounced at the end of the stagnant phase.

7.6 Concluding Remarks The only cold-water species that is in stable production is turbot, which is strictly neither a cold- nor a warm-water species. Turbot is now well domesticated, and most of the fry production is taking place in Atlantic regions of southern Europe. The development of culture methods and a commercial fry production industry for the remaining cold-water species of ﬁsh have been slower than for the Mediterranean species sea bass and sea bream, that are large industries today. One reason for the slow development is the successful salmon industry, that has attracted the majority of private funds. Another is the strong focus on the Atlantic halibut, which has turned out to be among the most complicated larvae to culture. This species has attracted considerable attention and research activity. Experience gained with juvenile production of Mediterranean species were not adequate for developing halibut. However, these experiences will be more important for most of the remaining species that have received more attention during the last few years. The greater attention to Atlantic cod in the northern hemisphere is a result of over-exploitation of the natural stocks of Atlantic cod in both the north-east and north-west Atlantic. In Norway, there is currently considerable interest in investing in cod aquaculture developments, and these are moving fast. Unlike Atlantic halibut, the establishment of a feasible juvenile technology for cod can take advantage of the Mediterranean experience as well as cold-water experience with other species. The main challenge now is to work systematically to establish an economically feasible industry for juvenile cod and other species that may follow later. Investments must be phased in line with biological progress. Any mismatch where the shareholders must wait longer than they expected to earn money is extremely negative for such development. The biological knowledge gained on the cold-water species, the adapted and available live feed technology, and full realisation of the complexity of the ﬁrst feeding process will be important for a rapid, but most importantly a predictable development of the industry.

7.7 References Balchen, J.G. (1987) Bridging the gap between aquaculture and the information sciences. Model. Identiﬁcation Control, 7(4), 162–3. Bell, J.G., McEvoy, L.A., Estevez, A., Shields, R.J. & Sargent, J.R. (2002) Optimising lipid nutrition in ﬁrst-feeding ﬂatﬁsh larvae. Aquaculture, in press. Blom, G., Otterå, H., Svåsand, T., Kristiansen, T.S. & Serigstad, B. (1991) The relationship between feeding conditions and production of cod fry (Gadus morhua L.) in a semi-enclosed marine ecosystem in western Norway, illustrated by use of a consumption model. ICES Mar. Sci. Symp., 192, 176–89.

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Castell, J., Blair, T., Nail, S., Howes, K., Mercer, S., Reid, J., Young-Lai, W., Guillison, B., Dhert, P. & Sorgeloos, P. (2002) The effect of different HUFA enrichment emulsions on the nutritional value of rotifers (Brachionus plicatilis) to larval haddock (Melanogrammus aegleﬁnus). Aquacult. Int., in press. Dhert, P. (1996) Rotifers. In: Manual on the Production and Use of Live Food for Aquaculture (eds P. Lavens & P. Sorgeloos), pp. 49–78. FAO Fisheries Technical Paper No. 361. Drenner, R.W., Strickler, J.R. & O’Brien, W.J. (1978) Capture probability: the role of zooplankter escape in the selective feeding of planktivorous ﬁsh. J. Fish Res. Board Can., 35, 1370–3. Eggers, D.M. (1977) The nature of prey selection by planktivorous ﬁsh. Ecology, 58, 46–59. Ellertsen, B., Fossum, P., Solemdal, P. & Sundby, S. (1989) Relation between temperature and survival of eggs and ﬁrst-feeding larvae of northeast Arctic cod (Gadus morhua L.). Rapp. P.-V. Reun. Cons. Int. Explor. Mer, 191, 209–19. Evjemo, J.O., Coutteau, P., Olsen, Y. & Sorgeloos, P. (1997) The stability of docosahexanoic acid in two Artemia species following enrichment and subsequent starvation. Aquaculture, 155, 135–48. Evjemo, J.O., Reitan, K.I. & Olsen, Y. (2002) Copepods as live food organisms for marine ﬁsh larvae with special emphasis on nutritional value. Aquaculture, in press. Folkvord, A., Øiestad, V. & Kvenseth, P.G. (1994) Growth patterns of three cohorts of Atlantic cod larvae (Gadus morhua L.) studied in a macrocosm. ICES J. Mar. Sci., 51, 325–36. Fyhn, H.J. (1990) Energy production in marine ﬁsh larvae with emphasis on free amino acids as a potential fuel. In: Animal Nutrition and Transport Processes. 1. Nutrition in Wild and Domestic Animals (eds J. Mellinger), pp. 176–92. Karger, Basel. Hjort, J. (1914) Fluctuations in the great ﬁsheries of northern Europe viewed in the light of biological research. Rapp. P.-V. Reun. Cons. Perm. Int. Explor. Mer, 20, 1–228. Hoehne, K. (1999) Lipid digestive enzymes in developing larvae of the Atlantic cod (Gadus morhua) and turbot (Scophthalmus maximus). D. Phil. Thesis, University of Karlsruhe. Hoehne-Reitan, K., Kjørsvik, E. & Reitan, K.I. (2001) Bile-salt-dependent lipase in larval turbot, as inﬂuenced by density and lipid content of fed prey. J. Fish Biol., 58, 746–54. Howell, B.R. (1979) Experiments on the rearing of larval turbot, Scophthalmus maximus L. Aquaculture, 18, 215–25. Koven, W., Barr, Y., Lutzky, S., Ben-Atia, I., Weiss, R., Harel, M., Behrens, P. & Tandler, A. (2001) The effect of dietary arachidonic acid (20:4n-6) on growth, survival and resistance to handling stress in gilthead seabream (Sparus aurata) larvae. Aquaculture, 193, 107–22. Krebs, J.R. (1978) Optimal foraging: decision rules for predators. In: Behavioural Ecology. An Evolutionary Approach (eds J.R. Krebs & N.B. Davies), pp. 23–63. Blackwell, Oxford. Kvenseth, P.G. & Øiestad, V. (1984) Large-scale rearing of cod fry on natural food production in an enclosed pond. In: The Propagation of Cod, Gadus morhua L. (eds E. Dahl, D.S. Danielssen, E. Moksness & P. Solemdal), pp. 645–55. Flødevigen Rapportser., 1. Lubzens, E., Tandler, A. & Minkoff, G. (1989) Rotifers as food in aquaculture. Hydrobiologia, 186/187, 387–400. Mangor-Jensen, A. & Adoff, G.R. (1987) Drinking activity of newly hatched larvae of cod (Gadus morhua L.). Fish Physiol. Biochem., 3, 99–103. May, R.C. (1974) Larval mortality in marine ﬁshes and the critical period concept. In: The Early Life History of Fish (eds J.H.S. Blaxter), pp. 3–19. Springer, Berlin. Merchie, G., Lavens, P., Dhert, P., Deshasque, M., Nelis, H., De-Leenheer, A. & Sorgeloos, P. (1995) Variation of ascorbic acid content in different live feed organisms. Aquaculture, 134, 325–37. Mourente, G., Tocher, D.R. & Sargent, J.R. (1991) Speciﬁc accumulation of docosahexaenoic acid [22:6n 3] in brain lipids during development of juvenile turbot Scophthalmus maximus (L). Lipids 26, 871–7.

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Naas, K.E., van der Meeren, T. & Aksnes, D.L. (1991) Plankton succession and responses to manipulations in a marine basin for larval ﬁsh rearing. Mar. Ecol. Prog. Ser., 74, 161–73. Næss, T., Naas, K.E. & Samuelsen, O.B. (1991) Toxicity of rotenone to some potential predators on marine ﬁsh larvae—an experimental study. Aquacult. Eng., 10, 149–59. NRC (Nutrition Research Council) (1993) Nutrient Requirements of Fish, 114 pp. National Academy Press, Washington, DC. Ohguchi, O. (1981) Prey density and selection against oddity by three-spined sticklebacks. Z. Tierpsychol. Beih., 23, 1–79. Øie, G., Makridis, P., Reitan, K.I. & Olsen, Y. (1997) Protein and carbon utilization of rotifers (Brachionus plicatilis) in ﬁrst feeding of turbot larvae (Scophthalmus maximus L.). Aquaculture, 153, 103–22. Øiestad, V. (1985) Predation on ﬁsh larvae as a regulatory force, illustrated in mesocosm studies with large groups of larva. NAFO Sci. Counc. Stud., 8, 25–32. Øiestad, V., Kvenseth, P.G. & Folkvord, A. (1985) Mass production of Atlantic cod juveniles Gadus morhua in a Norwegian saltwater pond. Trans. Am. Fish. Soc., 114, 590–5. Olsen, Y. (1999a) Lipids and essential fatty acids in aquatic food webs: what can freshwater ecologists learn from mariculture? In: Lipids in Freshwater Ecosystems (eds M.T. Arts & B.C. Wainman), pp. 161–202. Springer, New York. Olsen, A.I. (1999b) Development of production technology of juvenile Artemia optimal for feeding and production of Atlantic halibut fry. D. Phil. Thesis, Norwegian University of Science and Technology, Trondheim. Otterlei, E. (2000) Temperature- and size-dependent growth of larval and early juvenile Atlantic cod (Gadus morhua L.). D. Phil. Thesis, University of Bergen. Reitan, K., Rainuzzo, J.R., Øie, G. & Olsen, Y. (1993) Nutritional effects of algal addition in ﬁrst feeding of turbot (Scophthalmus maximus L.) larvae. Aquaculture, 118, 257–75. Reitan, K.I., Bolla, S. & Olsen, Y. (1994) A study of the mechanism of algal uptake in yolk-sac larvae of Atlantic halibut (Hippoglossus hippoglossus L). J. Fish Physiol., 44, 303–10. Reitan, K.I. (1994) Nutritional effects of algae in ﬁrst-feeding of marine ﬁsh larvae. PhD Thesis, University of Trondheim. Reitan, K.I., Rainuzzo, J.R., Øie, G. & Olsen, Y. (1997) Nutritional effects of algae in marine ﬁsh larvae. Aquaculture, 155, 207–21. Reitan, K.I., Natvik, C. & Vadstein, O. (1998) Drinking rate, uptake of bacteria and micro-algae in turbot larvae. J. Fish Biol., 53, 1145–54. Rognerud, C. (1887) Hatching cod in Norway. Bull. U.S. Fish Comm., 7 (8), 113–19. Rollefsen, G. (1940) Utklekking og oppdretting av saltvannsﬁsk. Naturen, 6–7, 197–217 (in Norwegian). Rosenlund, G., Meslo, I., Rødsjø, R. & Torp, H. (1993) Large-scale production of cod. In: Proceedings from the International Conference on Fish Farming Technology (eds H. Reinertsen, L.A. Dahle, L. Jørgensen & K. Tvinnereim), pp. 141–6. Trondheim, Norway, 9–12 August 1993. A.A. Balkema, Rotterdam, Brookﬁeld. Salvesen, I. & Vadstein, O. (1995) Surface disinfection of eggs from marine ﬁshes: evaluation of four chemicals. Aquacult. Int., 3, 155–71. Salvesen, I., Skjermo, J. & Vadstein, O. (1999) Growth of turbot (Scophthalmus maximus L.) during ﬁrst feeding in relation to the proportion of r/K-strategists in the bacterial community of the rearing water. Aquaculture, 175, 337–50. Sargent, J.R. & Henderson, R.J. (1986) Lipids. In: The Biological Chemistry of Marine Copepods (eds E.D.S. Corner & S.C.M. O’Hara), pp. 59–108. Clarendon Press, Oxford. Sargent, J.R., Bell, J.G., Bell, M.V., Henderson, R.J. & Tocher, D.R. (1991) The metabolism of phospholipids and polyunsaturated fatty acids in ﬁsh. In: Experimental Aspects of Aquaculture.

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Proceedings of the European Society of Comparative Physiology and Biochemistry, pp. 103–24. Sargent, J.R., Bell, M.V. & Tocher, D.R. (1993) Docosahexaenoic acid and the development of brain and retina in marine ﬁsh. In: Omega-3 Fatty Acids: Metabolism and Biological Effect (eds C.A. Drevon, I. Baksaas & H.E. Krokan), pp. 139–49. Birkhäuser, Basel. Shields, R.J., Bell, J.G., Luizi, F.S., Gara, B., Bromage, N.R. & Sargent, J.R. (1999) Natural copepods are superior to enriched Artemia nauplii as feed for larvae (Hippoglossus hippoglossus) in terms of survival, pigmentation and retinal morphology: relation to dietary essential fatty acids. J. Nutr., 129, 1186–94. Shuvayev, Y.D. (1979) Movements of some planktonic copepods. Hydrobiol. J., 14, 32–6. Skjermo, J. & Vadstein, O. (1993) The effect of microalgae on skin and gut bacterial ﬂora of halibut larvae. In: Proceedings from the International Conference on Fish Farming Technology (eds H. Reinertsen, L.A. Dahle, L. Jørgensen & K. Tvinnereim), pp. 61–7. Trondheim, Norway, 9–12 August 1993. A.A. Balkema, Rotterdam, Brookﬁeld. Skjermo, J., Defoort, T., Dehasque, M., Espevik, T., Olsen, Y., Skjåk-Bræk, G., Sorgeloos, P. & Vadstein, O. (1995) Immunostimulation of turbot fry (Scophthalmus maximus L.) with FMI administered by Artemia nauplii. Eur. Aquacult. Soc., Spec. Publ. 24, 502–5. Skjermo, J., Salvesen, I., Øie G., Olsen, Y. & Vadstein, O. (1997) Microbially maturated water: a technique for selection of a non-opportunistic bacterial ﬂora in water that may improve performance of marine larvae. Aquacult. Int., 5, 13–28. Solberg, T. & Tilseth, S. (1984) Growth, energy consumption and prey density requirements in ﬁrst feeding larvae of cod (Gadus morhua L.). In: The Propagation of Cod, Gadus morhua L. (eds E. Dahl, D.S. Danielssen, E. Moksness & P. Solemdal), pp. 145–66. Flødevigen Rapportser., 1. Suantika, G., Dhert, P., Nurhudah, M. & Sorgeloos, P. (2000) High-density production of rotifer Brachionus plicatilis in a recirculation system: consideration of water quality, zootechnical and nutritional aspects. Aquacult. Eng., 21, 201–14. van der Meeren, T. (1991) Algae as the ﬁrst food for cod larvae, Gadus morhus L.: ﬁlter feeding or ingestion by accident? J. Fish Biol., 39, 225–37. van der Meeren, T. & Næss, T. (1993) How does cod (Gadus morhua) cope with variability in feeding conditions during early larval stages? Mar. Biol., 116, 637–47. van der Meeren, T. & Naas, K.E. (1997) Development of rearing techniques using large enclosed ecosystems in the mass production of marine ﬁsh fry. Rev. Fish. Sci., 5, 367–90. van der Meeren, T., Jørstad, K.E., Solemdal, P. & Kjesbu, O.S. (1994) Growth and survival of cod larvae (Gadus morhua L.): comparative enclosure studies of Northeast Arctic cod and coastal cod from western Norway. ICES Mar. Sci. Symp., 198, 633–45. van Stappen, G., Merchie, G., Dhont, J., Lavens, P., Baert, P., Bosteels, T. & Sorgeloos, P. (1996) Artemia. In: Manual on the Production and Use of Live Food for Aquaculture (eds P. Lavens & P. Sorgeloos), pp. 79–136. FAO Fisheries Technical Paper No. 361. Vinyard, G.L. (1980) Different prey vulnerability and predator selectivity: effects of evasive prey on bluegill (Lepomis macrochirus) and pumpkinseed (L. gibbosus) predation. Can. J. Fish Aquat. Sci., 37, 2294–9. Werner, E.E. & Hall, D.J. (1974) Optimal foraging and the size selection of prey by the bluegill sunﬁsh (Lepomis macrochirus). Ecology, 55, 1042–52. Werner, R.G. & Blaxter, J.H.S. (1980) Growth and survival of larval herring (Clupea harengus) in relation to prey density. Can. J. Fish. Aquat. Sci., 37, 1063–9. Yoshimura, K., Usuki, K., Yoshimatsu, T. & Hagiwara, A. (1997) Recent development of a high-density mass culture system for the rotifer Brachionus rotundiformis Tschugunoff. Hydrobiologia, 358, 139–44.

Chapter 8

Weaning and Nursery J. Stoss, K. Hamre and H. Otterå

8.1 Introduction The weaning of pelagic marine ﬁsh larvae is the transition from a live food diet, such as rotifers, Artemia, or any cultured or harvested marine zooplankton to a formulated diet. Traditionally, this transition is started once the ﬁsh larva has completed metamorphosis. The diets used are technically similar to diets for bigger ﬁsh. In ﬂatﬁsh, metamorphosis is easy to recognise by the completion of eye migration, ﬂattening of the body and increased bottom dwelling. In ﬁsh such as Atlantic cod, all ﬁns are developed and visible. Common to all is the onset of gastric digestion, since the stomach ﬁrst develops into a functional organ at the end of metamorphosis. At this stage, the ﬁsh do accept and utilise formulated diets without any problems, and feeding with living organisms can cease. In general, it holds true that the bigger the ﬁsh at weaning, the easier the weaning process and the lower the losses. Often, the ﬁsh have reached sizes of 50–250 mg of wet weight when weaning takes place using food particles of around 0.3 mm. The term ‘early weaning’ is used when offering ﬁsh larvae formulated diets prior to the onset of gastric digestion. There are a number of reasons why the early use of formulated diets in the culture of marine ﬁsh is of interest. The production or capture of living organisms is a demanding and costly process, which complicates the hatchery operation considerably. The availability of zooplankton by harvesting is season-dependent, and can still be highly unreliable in terms of quantities and stages. Hygiene problems can also be encountered when using harvested zooplankton. Further, nutritional control of cultured organisms such as rotifers and Artemia has its natural limitations (see Chapters 4 and 7), and may not completely match the speciﬁc requirements of the cultured ﬁsh species. A key objective governing the development of larval diets has been to identify the factors which account for the critical differences between live food organisms and formulated diets. Is it the larvae digestion which limits the use of formulated diets, or are the causes really in the diets, which are not suitable for the tiny ﬁsh larvae? For a long time it seemed impossible to substitute live food in the early larval stages, but there have been interesting developments during the last few years. It was recently demonstrated that European sea bass larvae, Dicentrarchus labrax, which were fed exclusively on a microparticulate diet, showed both growth and survival (Cahu et al., 1998). Larvae of European sea bream, Sparus aurata, which were fed a microencapsulated diet after 6 days of rotifer feeding, showed subsequent

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survival and growth which was similar to that of the live-food controls (Yúfera et al., 2000). These results represent a breakthrough, and make it clear that formulated diets can be used very early, or even as ﬁrst feed for pelagic marine ﬁsh larvae. These are only experimental data, but there is considerable commercial interest in developing formulated diets which can replace most, if not all, live food organisms in marine ﬁsh production. To date, a combination of formulated diets with live food (co-feeding) has shown good results, and is increasingly used for many species. This is an important step towards reduced dependence on live food. In this chapter, Atlantic halibut, Hippoglossus hippoglossus (L.), Atlantic cod, Gadus morhua (L.), and turbot, Scophthalmus maximus (L.), receive particular attention. These three species have clear larval stages prior to metamorphosis, in contrast to many other species which do not have a larval period (see Chapter 6). While turbot have been cultured successfully for almost two decades, the farming of halibut and cod is still limited by the supply of juveniles. Larval nutrition, weaning and the nursery stage are key stages for improving the success in juvenile production of these species. We now take a brief look at the fundamental aspects of ﬁsh larvae digestion and nutrition in order to understand the limitations and possibilities of employing formulated diets. Many aspects of larvae nutrition and weaning are of a fundamental nature, and parallels between species must be drawn with care. Then, some practical aspects of weaning and juvenile rearing are presented from the point of view of marine hatchery production. For the ﬁsh culturist, weaning has to be seen together with the preceding start-feeding period as well as with the subsequent nursery and ongrowing stages. Because early weaning still is under development in the species of interest, weaning under production conditions still takes place at relatively late developmental stages.

8.2 Developmental Aspects of Digestion in Marine Fish Larvae In the search for explanations of why formulated diets have had such little success in early feeding of marine ﬁsh larvae, it has been suggested that pre-metamorphic larvae are not sufﬁciently developed to ingest and digest nutrients from formulated diets. Such larvae do not develop a functional stomach until metamorphosis (see Chapter 6). The absence of a stomach has consequences for the digestion of proteins, which takes place in the enterocytes of the intestine by pinocytosis. Further, larval vision is different from that of juveniles, which may trigger the capture of particles which are actively swimming while ignoring ‘dead’ particles. Our current knowledge of marine ﬁsh larvae is showing a differentiated picture of specialised developmental stage rather than characterising them all as ‘undeveloped’. As shown in Chapter 6, hatched alevins of wolf-ﬁsh (Anarhichas spp.) and salmon and trout (Salmo and Oncorhynchus spp.) ingest and digest formulated diets without complications. These ﬁsh do not pass through a larval stage, and have a ready-developed stomach from start-feeding. It is interesting to note that these alevins (not larvae) have a wet weight of somewhere around 50–150 mg, which corresponds to the weight of the metamorphosed larvae of turbot, cod and halibut. The former species also show a high growth rate during the larval stage (10–30% daily growth), which exceeds the growth in the larger alevins. This

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high growth rate in marine ﬁsh larvae, which is based on exogenous food, requires a special digestive system which is designed for speed and turnover rather than efﬁciency. Also, the nature of the food has to be such that the nutrients are readily available to ensure the necessary turnover. Turbot larvae clear their gut within a matter of 1–2 h during the ﬁrst few days of start-feeding at a temperature of around 20°C. Any food which delayed the digestive process would reduce growth directly, and is therefore not suitable for larvae nutrition. An understanding of larval digestive development and metamorphosis is essential in order to assess the transition from living zooplankton to formulated diets. Developmental aspects are thoroughly reviewed in Chapter 6. Here, we summarise the development of the digestive system of turbot larvae from start-feeding to metamorphosis as an example (Table 8.1). At the point of start-feeding (Stage 1), the larvae are sufﬁciently developed for the digestion and absorption of nutrients. With the exception of the stomach, all other organs are differentiated and functional. Most enzymes needed for the digestion and uptake of nutrients are active, but not pepsin owing to the lack of gastric digestion. The intestine is sub-divided into three sections, the fore-gut with the oesophagus and later the stomach, the mid-gut (anterior intestine) and the hind-gut (posterior intestine). A few hours after ﬁrst feeding, the enterocytes in the anterior intestine show active lipid absorption. In the posterior intestine, pinocytotic invaginations indicate the uptake of macromolecules from the lumen. Subsequent development is generally of a quantitative rather than a qualitative nature. There is a continuous increase in gut length, and also in the mucosal surface area of the entire intestine. The liver shows growth and increased glycogen and lipid deposition The enlargement of the digestive system increases the passage time and resorption area for ingested food, thus improving its overall efﬁciency. This can clearly be observed under cultural conditions. Rotifers, which are ingested by young larvae (Stage 1), are digested poorly, and partly intact animals are defecated. After a few days of feeding, rotifers are completely digested by the larvae. The development of gastric glands and the pyloric caecae are the main events in Stage 5. This implies that protein digestion changes from intracellular digestion in the hind-gut to extracellular digestion, which starts in the stomach. The ontogeny of the digestive organs in both Atlantic halibut and cod conﬁrm this picture, but the time aspects vary according to species and temperature. Table 8.2 shows the age or size when gastric development is completed in various species. From this stage on, use of formulated diets should be safe. The development of metamorphosis, and in particular of the digestive functions, can be altered by nutritional factors. An example of special relevance for larviculture and weaning is the recent research on Atlantic halibut. Larvae being fed on either Artemia or harvested zooplankton (mostly copepods) were compared. The Artemia-fed larvae showed a reduced fat resorption in the mid- and hind-gut and a lower fat deposition in the liver than the copepod group. This was explained by the lower digestibility of Artemia based on observations of the gut contents. Copepod-fed larvae also switched more readily to an adult-type digestion (functional stomach and resorptive intestine), while the Artemia-fed ﬁsh maintained a larvae-type digestion until around 83 days post-ﬁrst feeding (Shields et al., 1999). This is a signiﬁcant ﬁnding, which may explain some of the differences which are frequently observed in marine juveniles raised on either Artemia or copeods.

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Table 8.1 Development of the digestive system in turbot larvae (approx 18°C). Stages according to AlMaghazachi and Gibson (1984) and Segner et al. (1994). Stage 1

Stage 2

Stage description

Just prior to start-feeding, still yolk, open mouth and anus

Length Approx. age in days posthatch (dph)

Stage 3

Stage 4

Stage 5

Developed head First ﬁn rays, spines and swim swim bladder bladder fully inﬂated, body symmetrical but deeper in shape

Asymmetrical, eye migration starts, notochord slants dorsally

Metamorphosis complete, spines and swim bladder resorbed, completed eye migration, bottom dwelling

3 mm 3 dph

5 mm 8 dph

6 mm 12 dph

8mm 16 dph

17 mm 30 dph

Relative gut length

45

84, intestinal loop has formed

92, further 106 folding and elongation of gut

115

Swim bladder

Forms, not inﬂated

Inﬂated around Day 7/8

Inﬂated

Inﬂated

Deﬂated

Mucosal surface area in anterior and posterior intestine

X

X, increasing by XX the end of the stage

XX

XXX

Stomach and anterior intestine

Enterocyte ready for nutrient absorption, few hours after feed uptake lipid absorption, stomach anlage

Enhanced lipid absorption

Enhanced lipid absorption

Two pyloric caecae and gastric glands developing

Final differentiation of stomach (fundus, cardia, pylorus) and 2 pyloric caecae

Posterior intestine

Enterocytes ready for nutrient absorption

Enhanced pinocytotic uptake of macromolecules

Enhanced pinocytotic uptake of macromolecules

Cytologically fully developed enterocytes

Liver (liver status highly nutritiondependent)

High glycogen content, little lipid deposition

Glycogen deposition in hepatocytes reduced and lipid increase

Gradual increase Further lipid and Increased lipid of both glycogen glycogen increase. deposition, and lipid Growth. growth. Liver Liver somatic somatic index = index = 1.6% 2.7%

A delay in development to an adult-type digestion may thus inﬂuence the success of weaning onto a formulated diet. Unfortunately, such a comparison was not made in the studies cited. However, production experience from hatcheries does indicate that larvae raised on marine zooplankton such as copepods are often easy to wean, while Artemia-raised ﬁsh usually require a longer overlap between live food and formulated diets. Another concern about delayed metamorphosis is the occurrence of irreversible defects such as uncompleted eye migration in ﬂatﬁsh, skeletal deformities in ﬂatﬁsh and cod, or

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341

Development of gastric digestion in ﬁsh larvae.

Species

Start of gastric development/pylorus; days post-hatch (dph) at temp; approx length (mm)

Fully developed gastric digestion; days post-hatch at temp; approx SLb

Atlantic halibut, Hippoglossus hippoglossus Atlantic cod, Gadus morhua Japanese ﬂounder, Paralichthys olivaceus Japanese ﬂounder, Paralichthys olivaceus Turbot, Scophthalmus maximus Asian sea bass, Lates calcarifer Summer ﬂounder, Paralichthys dentatus Senegal sole, Solea senegalensis Dover sole, Solea solea

30–50 dpffa, 11°C

80 dpffa, 11°C

16–20 mm sl 16–17°C, 21–28 dph

30–50 mm sl Approx. 40–55 dph, 16–17°C

Approx. 25 dph

31 dph (stable stomach at pH 4)

Approx. 15 dph, 20°C, 7 mm (stage 4) 13 dph, temp?, 11 mm TL 31 dph, 20°C

Approx. 20–30 dph, 20°C (stage 5) 15 dph, temp?, 11.5 mm TL

13 dph, 0.6 mg, acid protease activity Between 80 and 200 dph at 20°C

33 dph, 25.2 mg ww Between 80 and 200 dph at 20°C

a b

Days post-ﬁrst feeding. Standard length.

abnormal pigmentation patterns. Such defects can affect the production performance under farming conditions, and are also likely to be important for successful weaning. This point is considered in more detail below.

8.3 Nutrition The nutrient requirements of marine ﬁsh larvae and juveniles may be different from those of larger ﬁsh. For example, the rapid growth of the larvae is thought to increase their requirement for protein, and the gradually developing digestive tract may have a limited capacity for the digestion and absorption of nutrients. To date, nutritional studies have been hampered by the lack of functional formulated diets for larvae. However, diets for juveniles are available, and the nutrient requirements of young juveniles may, with caution, be extrapolated to the larval stage. Since copepods are a natural diet, giving good growth and survival, the nutrient composition of copepods can be used as a reference for the formulation of weaning diets.

8.3.1 Macronutrient Composition The natural diet of carnivorous ﬁsh is low in carbohydrate, and these ﬁsh have a limited capacity for handling dietary carbohydrate owing to low activities of both digestive and metabolic enzymes. Further, juvenile ﬁsh and larvae are thought to have a high requirement for protein, owing to their high growth rates. The protein content in copepods has been measured to 55–58% of dry matter, lipid constituted 9–11% and carbohydrate levels are negligible (Table 8.3). The optimal macronutrient composition in a weaning diet for Atlantic halibut juveniles (0.5–10 g) was recently studied by K. Hamre (unpublished results, 2003). A three-component

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Table 8.3 Nutrient composition of natural zooplankton (mostly copepods) sampled from a fertilised seawater pond, and nutrient requirements of cold-water ﬁsh as given by the National Research Council, USA (NRC, 1993). Nutrient

Copepods

Artemia

Protein (% of dry wt.)

55–58

33–41

Free amino acids (% of protein) Essential AA (% of FAA)

20 45

10 10–20

Lipid (% of dry wt.) Polar lipid (% of lipid) Neutral lipid (% of lipid) DHA (% of fatty acids)

9–10a 66a 35a 38–45a

24–32 36 63 5–20

13–20 3–5 20–40 100–150 2–6 1–2 0.6–0.9 14–27 600–1000 50–200

6–12 6–10 60–180 180–250 2–13 2–5 2–5 30–60 400–500 100–800

Vitamins (mg/g dry wt.) Thiamin (B1) Folic acid Pantotenic acid Niacin Pyridoxin (B6) Cobalamin (B12) Biotin Riboﬂavin (B2) Vitamin C Vitamin E a

NRC 32–38

0.5–2% n-3 0.5–1 1–1.5 10–30 10–28 3–6 0.01 0.15–1 4–9 25–50 50–100

Young individuals.

mixture was used where protein, lipid and carbohydrate were varied between 530–830, 50–300 and 0–150 g kg-1, respectively (Fig. 8.1). Lowered growth rates and an accumulation of glycogen in the liver was found at carbohydrate levels above 5%. When carbohydrate was low, Atlantic halibut juveniles appeared to have good growth rates with lipid levels between 5 and 25%, but higher lipid levels caused growth depression, which is well documented. The requirement for protein in these ﬁsh thus seems to be about 60%, which is in agreement with the protein requirements of juvenile Atlantic salmon. In cod, the requirements for macronutrients have been studied in larger ﬁsh (150–300 g), with similar results as for juvenile halibut, i.e. an optimal carbohydrate level of 7–11%, and similar growth rates with lipid levels between 5 and 25%.

8.3.2 Composition of Dietary Protein Copepods contain high levels of free amino acids that are used in osmoregulation (see Table 8.3). Further, live feed contains proteolytic enzymes that may participate in digestion in the larval gut. Although most data indicate that the quantitative contribution of these enzymes to digestion is quite small, the live feed may provide easily available amino acids so that the need for larval digestion is reduced. First-feeding ﬁsh larvae of many species lack a functional stomach, and acid secretion and peptic enzyme activity are often not fully developed until metamorphosis has been completed. Although chymotrypsin and trypsin, or proteolytic activity with alkaline pH optima, seem to be present from ﬁrst feeding in a number of species, it is not clear when these

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Figure 8.1 Optimal composition of macronutrients for Atlantic halibut juveniles. Experimental design (Hamre et al., submitted).

enzymes become fully active. Rust (1995) administered radioactively labelled nutrients into the intestine of striped bass larvae by tube feeding, and measured the uptake of these nutrients in the larval body. He found that the assimilation of free amino acids was two to three times higher than that of protein at the early larval stages, while assimilation of peptides was intermediate. This supports the hypothesis that larval ﬁsh have a requirement for easily available protein in the form of free amino acids or peptides. However, the results from feeding trials with hydrolysed protein are conﬂicting. While improved growth was obtained in sea bass when some of the protein was replaced with hydrolysed protein, higher inclusion levels reduced larval performance. In Dover sole juveniles, 100% hydrolysed protein gave a better performance than lower ratios. On the other hand, sea bream larvae fed a non-supplemented diet performed better than on diets with 50% and 100% hydrolysed protein. In our own work, 0, 10 or 30% of the dietary protein was hydrolysed with pepsin, and the diets were fed to Atlantic halibut larvae from Day 40 post-ﬁrst feeding. Although it was not signiﬁcant, there was a tendency for better growth with 10% hydrolysed protein in the diet than with the control treatment, while the addition of 30% hydrolysed protein gave increased mortality and a tendency towards reduced growth. Thus, except for the experiment

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with Dover sole, it seems that moderate levels of hydrolysed protein are beneﬁcial to ﬁsh larvae, whereas high levels have a negative effect. To our knowledge, no feeding trials with graded levels of free amino acids have been performed, perhaps owing to their high leakage rates from formulated diets. Apart from being a source of easily available amino acids, peptides may induce intestinal maturation. Zambonino Infante et al. (1997) found that the ontogeny of digestive brush border enzymes was faster in sea bass larvae fed moderate levels of hydrolysed protein than in those fed the control diet based on ﬁsh meal as the only protein source. In weaning diets for larvae, the need for pre-digested protein should be further investigated. Marine ﬁsh juveniles have a fully developed digestive tract and can be fed intact protein, even though protein hydrolysates may have beneﬁcial effects on growth, as shown for Atlantic salmon. An alternative to adding pre-digested protein to the feed is to add exogenous enzymes that may participate in digestion in the larval gut, but this approach has also given conﬂicting results.

8.3.3 Composition of the Lipid Fraction Marine ﬁsh larvae have a high requirement for long-chain n-3 polyunsaturated fatty acids, as explained in Chapter 5 and 7. This is easily met in formulated diets by the addition of ﬁsh oil. However, feeding larvae with fats that contain mainly triacyglycerols leads to problems in fat assimilation. The larvae accumulate large lipid droplets in the enterocytes and sometimes in the liver, indicating that the lipid is absorbed from the intestine, while further transport into the blood is hindered. The accumulation of lipid droplets can be alleviated by adding phospholipid to the diet (Fontagné et al., 1998). It has been hypothesised that larvae have a limited capacity for phospholipid synthesis, and need exogenous phospholipid to cover their requirements for lipoprotein synthesis. Since phospholipids from marine sources are not commercially available, the phospholipid is usually added as soy- or egg-lecithin, where the fatty acid composition is unfavourable for the larvae. The level of supplementation therefore becomes a compromise between the larval requirements for n-3 fatty acids and phospholipids. The need for phospholipids is reduced as the larvae grow. Juvenile Atlantic halibut still showed a higher growth rate on a diet supplemented with 4% lecithin than on the control diet, but larger ﬁsh do not seem to have a requirement for phospholipids beyond that provided by the feed ingredients. The phospholipid content of copepods (young stages) was measured by McEvoy et al. (1998) as 66% of the lipid (see Table 8.3).

8.3.4 Vitamin Supplementation The micronutrient requirements of larvae and juveniles of cold-water marine ﬁsh have not been studied in detail because of the lack of experimental diets, and because the cultivation of these species is still in its infancy. However, the micronutrient composition of copepods and the requirements of larger ﬁsh can be used as an indication of their requirements. As can be seen from Table 8.3, vitamin levels in copepods are, with few exceptions, higher than the requirements given by NRC (1993). One area of uncertainty is whether the vitamin

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requirements of the fast-growing larvae and juveniles are higher than those of larger ﬁsh, since they are accustomed to a copepod diet.

8.4 Microparticulate Diets Artiﬁcial diets can be divided into conventional weaning diets for juvenile ﬁsh and diets for early weaning of larvae. The conventional diets are manufactured in almost the same way as diets for larger ﬁsh, and functional diets are commercially available. The small particles required for larvae introduce problems related to leakage due to their large surface-to-volume ratio. The leakage of low molecular weight, water-soluble nutrients such as vitamins and free amino acids is most pronounced, while lipid and high molecular weight nutrients such as protein are reasonably well retained within the particles when immersed in water. Another problem is the buoyancy of the particles. Too high a settling rate will cause the particles to sink to the bottom of the tank, where they are unavailable to the larvae. Uneaten feed will then create problems of bacterial fouling in the tanks. Microbound and microencapsulated diets are being developed for marine ﬁsh larvae. In the microbound diets, the dietary ingredients are embedded in a gelled hydrocolloid matrix such as gelatine, carrageenan, zein or alginate. These diets are fairly simple to manufacture, but have high leakage rates (Table 8.4). Microencapsulated diets contain dietary ingredients enclosed within a capsule wall. The early microcapsules were made by cross-linking nylon and protein on the particle surface. Owing to the potential toxicity of diamines and the Tween detergent used in this process, protein-walled microcapsules were developed, with the use of 1,3,5-benzenetricarbonyl trichloride and sebacoylchloride as cross-linking agents. These particles lost less than 5% of the encapsulated 14C-protein after 24 h in seawater. However, free amino acids are lost from the capsules at an appreciable rate, even though leakage is lower than from the microbound diets (Table 8.4). Lipid-walled capsules are made by the emulsiﬁcation of lipid with an aqueous core solution containing the nutrients to be encapsulated. Depending on the lipid source and the method of manufacture, they can retain the low molecular weight, water-soluble nutrients quite well when incubated in water (Table 8.4). However, the encapsulation efﬁciency, i.e. the amount of core solution that can be incorporated compared with the total volume of the capsule, is low. A complete lipid-encapsulated diet would therefore become too high in fat. On the other hand, lipid-walled capsules may be well suited for the encapsulation of water-soluble micronutrients, which can subsequently be incorporated into a complete diet Table 8.4 1994).

Loss of free amino acids (FAA) after incubation for 2 min in borate buffer (Lopez-Alvarado et al.,

Loss of FAA (% of initial amount) Microbound diets (carrageenan, alginate, zein) Protein-walled capsules Protein-walled capsules coated with triolein Lipid-walled capsules with triolein/tripalmitin Lipid-walled capsules with tripalmitin

81–91 59 ± 1 39 ± 2 47 ± 9 4±2

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(Ozkizilkik and Chu, 1996; Baskerville-Bridges and Kling, 2000b). If the capsules constitute a major fraction of the complete diet, one should take into account the high requirements for long-chain polyunsaturated fatty acids in marine larvae when choosing lipid-wall material. Until recently, feeding larvae with microparticulate diets has resulted in poor growth and survival rates, and some authors use short-term feeding experiments and acceptance studies to evaluate the diets. However, a Spanish group has now developed a cross-linked proteinwalled diet for gilthead sea bream (Sparus aurata L.) which, when used from Day 8 after hatching, gave similar growth and survival rates as rotifers (Yúfera et al., 1999). The leakage was estimated to be 1% of dry weight after 4 h, and the settling rate was 25 cm h-1. In our work with Atlantic halibut, we have used a diet based on minced ﬁsh ﬁllet, which is heat-coagulated in a microwave oven. This creates a matrix of coagulated protein which binds the pellet. This diet has considerable leakage problems (approximately 50% of soluble protein lost after suspension for 2 min), and owing to a high settling rate, was unsuccessful in feeding free-swimming halibut larvae. However, the diet has given good growth and survival rates for halibut when fed from approximately 30 days post-ﬁrst feeding (0.07 g ww; Hamre et al., 2001). These larvae can be forced to settle on the bottom of the tank by the use of low water levels, and consequently the settling rate of the feed particles becomes less critical. The development of formulated diets for marine ﬁsh larvae will reduce the need for live feed, and thereby simplify production procedures and probably reduce the costs of marine juvenile production. Further, a functional formulated diet will facilitate studies of the nutrient requirements of larvae, which until now have been hampered by the limited possibilities for manipulation of the live feed nutrient composition.

8.5 Weaning and Nursery Stage, Practical Aspects 8.5.1 General Success during weaning and the subsequent nursery stage depends on a set of husbandry techniques which are adjusted to the speciﬁc species, but also to the speciﬁc conditions at the rearing site. Although good diets for early weaning may be on the market, a hatchery may ﬁnd out that it is less demanding to continue to grow larvae on live food. Some pragmatic recommendations are now given for various species and developmental stages. 8.5.1.1 The Role of Early Start Feeding In ﬁsh farming, correlations between subsequent production stages are well known, but the scientiﬁc documentation may often lack experimental evidence to verify them. A ﬁsh which has good conditions for development during its early life stages is usually strong during the later stages. Likewise, the switch from living prey to formulated diets is a sensitive phase, which has an impact on the further performance of the ﬁsh. Larvae which do show good growth and survival during early rearing are usually easy to wean when gastric digestion starts. Any disturbances during the early rearing stages reduces the weaning success. This is

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documented for nutritional factors in turbot larvae, which were reared on newly hatched and unfed Artemia. They coped poorly with formulated diets during subsequent weaning. This is not surprising, since such Artemia contains insufﬁcient levels of DHA and EPA, which play a particular role in the development of the larva’s neural system and stress resistance (see Chapter 7). A deﬁciency of these essential fatty acids during early rearing will probably also have an impact on subsequent weaning success. As with malnutrition, poor environmental conditions, infections or starvation during early rearing weaken the larvae and make the weaning phase more demanding. In order to assess weaning results, it is therefore necessary to have good records from the early rearing phase. This fact is often neglected. Early weaning trials focus on survival results in particular. The growth achieved is generally well behind that of live food controls, and further development is rarely documented. For the ﬁsh farmer, such information is important in order to assess any long-term effects of the weaning phase. Data from halibut showed that reduced growth from early weaning was not caught up later in the juvenile phase. In ﬂatﬁsh larvae, periods of retarded growth can result in incomplete metamorphosis and associated defects in the skeleton or eye migration. Such individuals grow poorly during grow-out compared with normally metamorphosed ﬁsh. This is demonstrated by farm data for Atlantic halibut. At the age of 20 months postﬁrst feeding, the average weight of ﬁsh with incomplete eye migration was 323 g (n = 180), while ﬁsh with normal eye migration averaged 520 g (n = 1404) (J. Stoss, L. Berg & M. Dorenfeldt Jensen, unpublished results, 1998). Since growth is a key variable which determines economic success in production, the consequences of mistakes during the larval stages, including weaning, can be vital. For these reasons, both growth and survival must be supported during weaning. Unless one has good documentation, we recommend that there should be no compromise over growth during weaning for the sake of achieving some short-term savings in the live-food cost. 8.5.1.2 Early Weaning and Co-Feeding There are an increasing number of reports on the use of formulated diets in combination with live food in early larval stages, also referred to as co-feeding. Such techniques can reduce the quantitative needs of live food organisms. Recent experience with cod larvae described a rearing system with rotifers and microdiets only, thus eliminating the need for Artemia. A low ingestion of formulated diets combined with restricted rations of live feed are the probable reasons why some authors report reduced growth and increased mortality during co-feeding. However, as shown for turbot, larvae also ingest some of the diets offered when live food is offered without restriction. This supplementary feeding is reﬂected in the larvae’s body composition, for example, by a rapid increase in DHA, which is limited when feeding with Artemia only (Rosenlund et al., 1997). Co-feeding can improve the overall ingestion and promote growth, which exceeds the live-food reference in European sea bream, turbot and halibut. In sole, Solea senegalensis, a period of co-feeding is essential for subsequent weaning success (Canavate and Fernandez-Diaz, 1999), indicating that Artemia alone does not satisfy the larvae’s nutritional requirements. Therefore, co-feeding can also be regarded as essential to the larvae in order to improve results in later stages.

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8.5.1.3 Uptake and Ingestion of Formulated Diets Larvae of cod, halibut and turbot are visual feeders. They show an interest in particles and feed occasionally even on completely indigestible particles such as unhatched Artemia cysts, with fatal consequences. Locomotion is the key stimulus for larvae to attack particles. This can easily be observed when feeding larvae on both living and dead prey. A motionless particle can attract a larva if it is the correct size, does not sink rapidly and releases the right smell. Size preference studies with both turbot and halibut conﬁrm a clear size preference for particles of a speciﬁc size (see Chapters 4 and 7). Fish larvae have a good sense of smell, and chemoreception is important for the recognition of food. When a particle is attacked, it has to be of the correct texture and taste to prevent the larva from rejecting it. Turbot and cod larvae frequently ‘try’ ﬂoating particles, but spit them out again after a brief ‘tasting’. From studies with sole larvae, Appelbaum et al. (1983) suggest that the taste will decide whether a particle will be swallowed or rejected. A variety of natural substances are known to function as attractants and feeding stimulants, and are probably considered in the commercial diets which are offered today. A good weaning diet added to the tank water induces searching behaviour in larvae. Observation of this behaviour can help in preliminary assessments of new diets.

8.5.1.4 Availability of Particulate Food Larvae have a limited capacity to search a given water volume for food within a given time, and therefore one has to ensure that the concentration of particles is sufﬁcient to provide enough encounters (see also Chapters 7 and 4 regarding prey densities). It is obvious that both the buoyancy of the particles as well as the species and age of the larvae are governing variables. Cod larvae, for example, swim and search actively, while halibut larvae stay more passively in the water column waiting for a particle to approach, and turbot larvae are between these two extremes. The buoyancy of the particles should ensure that they sink to the bottom before leaching reduces their nutritious value. Rearing conditions such as water depth, velocity and exchange rate also interact to affect larval feeding. In early weaning, the ingestion rate of inert food particles is often low, which makes it necessary to co-feed with living organisms. In large volume rearing systems, in particular, there is a limit to how much the concentration of a particulate diet can be increased before the tank is overloaded with food. On the other hand, large-volume tanks may be necessary to maintain good stability in the living feed organisms and algae. For the species of interest here, there are no standardised rearing procedures. Therefore, on-site protocols for weaning have to be established after considering the variables described above.

8.5.1.5 Tank Hygiene In order to achieve an acceptable ingestion of weaning diets, feed has to be offered in excess. This applies particularly to the early larval stages. In large systems with little turnover of water, any accumulation of sedimented or ﬂoating food particles induces the growth of

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bacteria or higher organisms such as ciliates, which in turn increase the risk of poor water quality and infections in the larvae. Hygiene in larvae tanks is critical to the survival of the larvae (see Chapter 7), and in practice, feeding with live organisms is often continued in order to maintain good tank hygiene. This emphasises the importance of self-cleaning tanks once weaning starts. For this reason, it is preferable to wean ﬂatﬁsh in tanks with fairly shallow water, which allows a high water exchange rate and thereby good self-cleaning of the tank. Once the ﬁsh are settled on the bottom, they constantly stir up particles and thereby enhance particle removal to the drain. A water depth of between 40 cm and 1–2 cm can be used. Shallow water also promotes early settling of halibut larvae. It is obvious that self-cleaning tanks, which are based on a good water exchange rate and a high water velocity at the bottom, are best suited to bigger and stronger ﬁsh, and not to early weaning of larvae. During the last few years, specialised equipment has become available which cleans the bottoms of tanks continuously. A Norwegian company offers a start-feeding tank with a cleaning arm, which sweeps at a controllable velocity over the bottom. Holes in the arm are connected to the tank drain. Such systems could be a technical solution to maintaining acceptable hygiene conditions, in particular during the early weaning of cod in large-volume tanks. 8.5.1.6 Rearing Temperature and Light Both the temperature regime and its stability are important considerations during the weaning and nursery stages. Halibut and turbot have a clear and changing temperature optimum which is related to ﬁsh size. The smaller the ﬁsh, the higher the optimum temperature for growth. Such ontogenetic changes are known for a number of ﬁsh species, but have not been found in Atlantic cod within the size and temperature ranges 250–2000 g and 4–16°C, respectively. Some data from the literature are given in Table 8.5, and show that changes in temperature preference occur during relatively narrow growth periods. One also has to consider the possibility of genetic (stock) differences and the effect of acclimation periods. Sensitivity to temperature variations is also important. Small variations of 1–2°C can result in immediate changes in appetite. Sudden drops, such as from 18 to 8°C can be lethal when combined with handling stress in small turbot. In addition, disease outbreaks can often be traced back Table 8.5 Temperature optimum for growth at various size ranges in halibut and turbot. Species

Size range (g wet weight)

Optimum temp. for growth (°C)

Halibut

0.1–2 5–10 20–25 40–50 0.1–1 25–30 45–50 70–75

15 14.9 13.9 13.0 22 19 18–19 16

Turbot

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to periods of unstable temperature. A stable temperature always provides the best rearing conditions. The effect of photoperiod on growth in the larval stages is not well documented, but is obvious. As visual feeders, all three species would be unable to feed during darkness. With the high gut evacuation rate in larvae, longer periods of darkness result in empty guts, starvation and reduced growth. Continuous light during the start feeding and weaning phases is therefore a common practice for many species. Studies carried out during the juvenile stage show that the best growth occurs during long light days. In halibut juveniles ranging from 30 to 150 g, continuous light resulted in a faster growth rate than light regimes which included dark periods. In turbot juveniles ranging from 5 to 125 g, continuous light improved growth until they reached 60 g compared with a 16 h light 8 h dark regime or a natural light regime. Above 60 g, the 16:8 cycle produced better growth at 16°C. In cod of around 0.9 g, a 24 h light regime resulted in better growth than a 16 h light 8 h dark regime. The turbot example shows that one should be aware of the necessity to turn gradually to days with dark periods as the ﬁsh grow. The growth-promoting effect of light is clearly associated with an increase in growth hormone in the blood and improved feed conversion. The food uptake is not inﬂuenced (Björnsson, 1997). This means that a correct use of light during the larval and juvenile stages is an effective means of improving the overall production result by optimising both growth and feed conversion rate. 8.5.1.7 Vaccination Against Bacterial Diseases With the ﬁrst experiences of farming turbot, halibut and cod, it soon became clear that disease prevention is very important in the management of the vulnerable early life history stages. Since there is a high species speciﬁcity for bacterial pathogens, autogene vaccines, which are based on isolated pathogens from a given site, have been used widely and with good effect. Because of the present limited farming activity, commercial vaccines for these species have rarely been available, but similar products designed for salmonids have been used. Several bacterial diseases may occur during the weaning and nursery stages. Infections often develop very fast over one or a few days, and mortality rates can be very high. Usually, ﬁsh reduce or stop their feed uptake when the ﬁrst mortality occurs, which also hampers the use of medicated feed. In turbot, cod and halibut, vibriosis, caused by Vibrio anguillarium, is rather common during the size range 0.2–20 g. Vaccination is a very effective means of preventing vibriosis. There is good practical and theoretical evidence that ﬁsh from around 0.5 g show an immune response after vaccination. The immunity does not usually last very long, and revaccination is necessary after a few weeks. It is general practice to dip-vaccinate the ﬁsh according to the suppliers’ instructions, but bath vaccination is also applicable, particularly for the younger stages, which may be more difﬁcult to remove from the tank to apply a dip. Atypical Æromonas spp. infections are well known in the species of interest, and vaccination provides effective protection. The best method is injection with a long-lasting (oil-based) vaccine. For practical reasons, the ﬁsh need to be around 20 g to have an injection.

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Flexibacter spp. infections are also a major cause of larvae and juvenile losses. This group of infectious bacteria has not received much attention to date. Work on vaccines is in an early stage, and management procedures therefore have to focus on high hygiene standards, good husbandry and medication. For other aspects of disease prevention, the reader is referred to Chapter 3 in this book. 8.5.1.8 Handling of Fish The handling of marine ﬁsh larvae is necessary in connection with weaning, grading, vaccination and transport. Prior to metamorphosis, ﬂatﬁsh and cod larvae are very sensitive to any handling such as dip-netting. Their fragile ﬁns are easily injured, and secondary bacterial infections develop rapidly. Any handling of such fragile larvae should therefore be done in water. Fish can tolerate careful siphoning and transport through hoses or pipes. They can be subjected to grading by means of grids or perforated baskets as long as they are submersed in water. There is no standard equipment for such needs, and hatcheries have their own in-house solutions. Grading larvae into the size range 50–250 mg wet weight is often the ﬁrst step in order to make homogenous groups for weaning, and to separate the sizes in order to avoid cannibalism. Any further grading would depend on the size range which develops. A practical rule is to grade when the difference between the smaller and larger ﬁsh exceeds a factor of three. When ﬁsh reach 1 g, careful dip-netting can be used. The use of grading machines is possible for ﬂatﬁsh from this size on as long as water is constantly sprayed onto the grading belts. Cod should still be handled underwater, since the ﬁsh are more active during handling and thereby more subject to injury. One can also consider weak sedation of juveniles prior to handling. Flatﬁsh from about 5 g can be handled ‘dry’ for short periods. This means that the ﬁsh can be carried in baskets without water for at least a few minutes. Manual grading on moist surfaces is also possible. Flatﬁsh usually lie calmly on moist surfaces, and they can easily be slid on slippery, wet smooth surfaces (epoxy, pvc, etc.) without losing slime. Turbot can survive for many hours in a humid atmosphere without the presence of water, and successful long-distance transport of turbot juveniles is done using no water and thereby limiting the cargo weight. It is important that the ﬁsh are not covering each other. Halibut do not survive such dry handling for more than a few minutes. This may be because halibut have scales, which may limit cutane respiration compared with turbot. As for cod, any transport needs to be done in proper transport tanks with sufﬁcient aeration. Commercial ﬁsh counters, which are used in the production of salmon and trout, also work for cod and ﬂatﬁsh. However, ﬂatﬁsh tend to ‘stick’ to each other, which limits the choice of suitable counters. Proper testing should be done prior to the use of any counter. Prior to any handling, ﬁsh should be starved for a few hours (if under 1 g), or about 1–3 days for juveniles. Starved ﬁsh have a reduced metabolism, and this takes some of the stress out of handling. However, this procedure has to be evaluated against any aggression which may develop in starving juveniles. Prior to any transport, it is important that the gut is emptied completely to avoid faeces reducing the quality of the transport water. Grading ﬁsh with a newly ﬁlled stomach would also reduce the quality of the grading.

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8.5.2 Cod Weaning cod is not very different from the procedures used for other marine species, and large-scale intensive rearing techniques are also being developed for cod. Any differences are likely to become even smaller within a few years. However, two functional characteristics of cod should be mentioned. Cod develop a functional stomach at a relatively large size (see previous section), and cod have a tendency to become cannibalistic. The ﬁrst point may restrict the timing of weaning and also the feed quality. Cannibalism remains low as long as feeding conditions, the size regime of the ﬁsh, and the rearing conditions in general are kept within some optimal limits. However, weaning may be a critical period, and severe mortality due to cannibalism can arise if things go wrong.

8.5.2.1 Early Weaning In cod, the term early weaning is used for larvae up to 12 mm in length. At this size, the larval ﬁn fold disappears, but this is well before functional gastric digestion at around 40 mm. Early weaning has great potentials when rearing cod in extensive systems. Initial larvae survival is usually very high, but this is often reduced because of the limited abundance of natural food during later development. An early uptake of ﬁsh from the start-feeding units for early weaning would therefore considerably increase the production potential. In intensive rearing, early weaning is clearly the method to choose because it reduces the need for cultured live food. Signiﬁcant progress in the early weaning of cod was reported by Baskerville-Bridges and Kling (2000a,b,c). They co-fed a microparticulate diet and rotifers during Days 14–21 posthatch (10–11°C). On Day 21, the larvae were completely weaned at a size of about 8 mm standard length. At 71 days post-hatch, the survival was 35%, the length 20 mm and the dry weight approximately 10 mg (SGR = 8.2). This survival compares well with the Artemia reference group, but growth was almost double in the Artemia group (20 mg dry weight, SGR = 9.0). The ingestion rate of the formulated diet was reported to be very low when it was introduced on both Day 8 and Day 15 as long as live feed was present in signiﬁcant amounts in the tanks. These growth rates are still much lower than those estimated for cod fed on zooplankton, where dry weights of 32–44 mg at 56 days post-hatch (10°C) can be reached. The results suggest that formulated feed at such early stages still is inferior to natural plankton. In other studies, early weaning from 12 mm standard length (wet weight 15–20 mg) resulted in survival rates around 40% for the best treatments. The main limitation to survival and growth was a low feed intake during the ﬁrst days. A preference for moist feed was also noticed, and this emphasises the importance feed palatability has at this stage. No co-feeding was attempted in these experiments. When the initial larvae size was 50 mg wet weight and 19 mm standard length, survival rates increased to around 60%. Again, the growth rates achieved with formulated diets were considerably lower than the reference data from feeding with natural zooplankton. We can conclude that acceptable survival rates during early weaning of cod is possible with the feeds available today. However, growth rates are far behind their potential. The consequences of low growth rates in the larval stage for later production performance are not

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known, but we suspect that the juveniles might be of doubtful quality. Elongated periods with co-feeding of formulated and live feed may solve the growth problem before improved diets are available which can be used exclusively. The question of a possible correlation between growth rates in the larval stage and later production performance would be an interesting area for research. 8.5.2.2 Weaning Once the larva gets above 100 mg wet weight weaning survival improves considerably, and at around 400–500 mg weaning is done with no problems related to feed acceptance or subsequent growth. A survival of 80% or more during the weaning of intensively reared cod larvae onto an agglomerated commercial diet has been achieved. In this case, weaning took place when the larvae were 15–50 mm in length. Pond-reared cod can relatively easily be collected for transfer to sea cages from a size of about 0.5 g/40 mm. At this size, ﬁsh are already weaned onto a dry diet. Weaning can be initiated at 30 mm, and in practice the presence of zooplankton will give a co-feeding situation. The choice of weaning size is fundamentally a calculation of the cost for alternative live feed (Artemia or natural plankton) against the possible reduced growth and increased mortality if weaned too early. Several authoritative measurements of growth in larva under various temperature conditions are available. At 14–16°C, cod larvae of 5 mg wet weight have a maximum growth rate of about 25% day-1. Growth decreases to 7% day-1 for 0.5-g cod. Table 8.6 gives some recommendations for early weaning of cod considering optimal growth conditions. An indication of dry feed consumption can be calculated by using the same amount of dry feed as the estimated increase in ﬁsh biomass in wet weight. However, during the weaning period, low feed intake may occur, and quite heavy overfeeding may be necessary to ensure sufﬁcient feed intake. Close observation of feeding behaviour and tank hygiene is necessary during this period. Production problems encountered during late weaning are not primarily related to the uptake or quality of the weaning diet, but rather to cannibalism, gas bubble formation and bacterial infections. Table 8.6 Early weaning protocol for cod reared at optimum temperature (14°C). Growth data are taken from Otterlei et al., 1999. Larva age (days posthatch)

Larvae weight; dry wt. (mg)

Larvae weight; wet wt. (mg)

Larvae standard length (mm)

Daily growth (%)

Daily biomass increase (g wet weight) per 10 000 ﬁsh

Live-food feeding frequency/day

Particle size dry feed (mm)

7 14 21 28 35 42 49 56

0.04 0.15 0.9 3.8 12 49 85 148

0.22 0.83 5.0 21.1 66.7 272 472 822

5 9 11 15 20 30 35 40

17 25 25 23 16 11 7 5

7 21 125 485 1066 2994 3305 4111

Continuous Continuous 6 2 0 0 0 0

– 0.1 0.1 0.2 0.3 0.5 0.6 0.7

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8.5.2.3 Cannibalism Inferior feeding and rearing conditions in general will increase the size variation in the population and eventually lead to heavy mortality due to cannibalism. Clearly, factors such as growth rate, mouth size and prey size are critical in this respect. Once a few ﬁsh get to the size where they can prey on smaller ones, their growth is accelerated to a maximum, and size differences increase so much that one may end up with a few big ﬁsh only. This phenomenon has been researched, and ﬁsh in the size range 10–40 mm are most susceptible to such conditions. The most effective way to avoid cannibalism is to maintain optimal growth, which results in homogeneous sizes. Since variable growth rates are particularly likely from early weaning, frequent and early grading is necessary to prevent cannibalism becoming established in tanks. Experiences from extensive rearing with a good supply of natural food show little or no problems related to cannibalism.

8.5.2.4 Gas Bubble Formation During intensive rearing, high larval mortality is frequently observed. They ﬂoat on the surface with extensive gas formation in their gut. Degassing the intake water reduces the problem. Apparently, larvae in the size interval 20–40 mm are extremely sensitive to supersaturation of dissolved gases in the water. Even differences in the temperature between the water and the room were found to be important. However, this problem is not typical of extensive rearing, and other factors which inﬂuence the larva’s condition may well be involved.

8.5.2.5 Nursery Once cod are weaned and have reached the size of a few grams, rearing is known to be proceeding smoothly. Correct feeding is also the key in cod juvenile production in order to utilise their growth potential. An exact estimate of growth rates and corresponding feeding regimes is a challenge for a skilled operator. Continuous feeding and a good distribution of particles is important, since cod are active feeders given a suitable temperature and continuous light. Juvenile cod seem to have a temperature optimum for growth which is similar to that of larvae. Growth rates decline rapidly with ﬁsh size, as shown in Fig. 8.2. This ﬁgure shows observed growth rates, as well as some operational conditions during a production cycle for pond-reared cod in western Norway. Here, cod juveniles are transferred from the pond and kept in sea cages from a size of 0.5–10 g onwards. It is expected that similar procedures regarding grading and vaccination will also function well in units employing intensive methods for start feeding and weaning. Whether the juvenile ﬁsh should be kept in sea cages or in tanks remains a matter of local conditions (sea temperature, wind exposure etc.) and costs. Cannibalism disappears gradually, and is virtually absent above a size of about 10 g. Practical experience has shown that cod juveniles tolerate high densities. Although systematic studies are not available, operational conditions which are comparable to those used for salmonid ﬁsh seem to be possible.

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Figure 8.2 Some weight data and operational incidents from the rearing of juvenile cod in sea cages in Parisvatnet (Institute of Marine Research, Norway). Data from several years and groups are pooled. Ambient temperature ranged from about 15°C in June to 6°C in December. Growth rates, calculated from the ﬁtted growth curve, are shown by the solid curve (refers to the right-hand y-axis).

Since cod rearing is still in its early developmental stages, we can expect to see major improvements related to the weaning and nursery stages. Clearly, cod has the potential for largescale production. Growth rates in the juvenile phase are good compared with those of many other species, and cod adapt well to husbandry conditions that allow efﬁcient production.

8.5.3 Turbot Turbot have been cultured for almost 20 years, and a good deal of experience in weaning is available. However, this is limited to the few production hatcheries for turbot, of which there are currently around 15 in Europe, although new production centres are being developed in China. Under production conditions, turbot are usually weaned as metamorphosed ﬁsh, and there are well-functioning diets on the market. Early weaning is poorly documented, and to date no early weaning diets especially for turbot are on the market. Since turbot juveniles have a relatively high market value, there may be a reluctance to reduce the live-food period and risk lower survival and increased growth variability. 8.5.3.1 Early Weaning At start-feeding, turbot larvae feed well on living organisms, but they will also ingest particulate diets. Some survival and limited growth has been achieved under experimental

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conditions, but to date there are no practical methods of early weaning. It appears that the uptake of particles by larvae in general is low since the larvae select for organisms which show active swimming movements. Dhert et al. (1999) carried out weaning trials which were started on Day 11 post-hatching. Various test diets and a commercial diet were co-fed together with Artemia (restricted to half the ration) until Day 20 or 29. There were no differences in survival until Day 29, but the live food option showed superior growth. If Artemia were stopped on Day 20, there was a substantial growth reduction. Components such as added phospholipid and free amino acids did not improve the results. Again, the reader is reminded that retarded growth can increase the frequency of poor quality ﬁsh with various defects. In preliminary tests with an experimental larval diet developed by K. Hamre (unpublished results, 1999), we found feeding and growing larvae at around 10 mg. In contrast to our earlier observations, the larvae already showed an interest in ﬂoating particles at this small size. The yellowish colour of the diet was easy to see in the translucent larvae, and indicated which were feeding. In conclusion, there is little information on early weaning in turbot. There seems to be a lower size limit at around 30–40 mg wet weight before the ﬁsh are ready for formulated diets. At this size, turbot start gastric digestion. 8.5.3.2 Weaning One objective in developing weaning routines for turbot has been to identify the earliest possible size when weaning can be performed safely. In trials by Rosenlund et al. (1997), larvae of the same age (21 days post-hatching) but with different weights were co-fed with a restricted ration of Artemia and a weaning diet for 7 days. Larvae with an initial weight of 34 mg wet weight showed reduced growth and survival compared with those having live food (Artemia). Insufﬁcient feed uptake seemed to be the reason why some individuals starved. When weaning was initiated at 61 mg, growth and survival were identical for the dry diet and the reference group (370 and 380 mg wet weight and 1% mortality after 15 days). However, a difference was found in the size range. When weaning started at 34 mg, the minimum and maximum weights in the live-food group were 130 mg and 610 mg, respectively, while in the dry-feed groups they ranged between 20 and 920 mg, respectively. This result was consistent in all replicates and in both trials, and was also conﬁrmed in halibut. This clearly demonstrates that formulated diets can develop a growth potential well above that found with Artemia. As pointed out above, the quality of the larvae is closely connected with the ease of weaning. Turbot larvae raised on high-quality food such as natural zooplankton or correctly enriched rotifers and Artemia usually develop early into well-metamorphosed ﬁsh. At around 30–50 mg, such ﬁsh accept a formulated diet without the need for co-feeding. However, if the start-feeding period has resulted in weak ﬁsh for any reason, co-feeding with live food over longer periods can be necessary in order to avoid mortality and slow growth. The abovecited ﬁnding about retarded development of the digestive system in poorly nourished larvae provides a possible explanation (Shields et al., 1999). As shown above, co-feeding with Artemia results in a more even size and does reduce cannibalism. As long as live food

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Table 8.7

357

Weaning protocol for turbot (20°C).

Larva age (days post-hatch)

Larva wet weight (mg)

Daily growth (%)

Grams dry feed per day per 10 000 ﬁsh

2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40 42

0.3 0.3 0.7 1.5 2.9 5.3 9.5 16 26 42 64 94 133 185 250 325 410 510 620 740 860

0 42 38 33 30 29 26 24 23 22 19 17 17 15 13 12 11 10 9 8

0 0 0 0 0 0 0 0 24 46 90 231 305 376 426 476 557 605 655 646

Particle size (mm)

0.3 0.3 0.3–0.5 0.3–0.5 0.3–0.5 0.5 0.5 0.5–0.8 0.5–0.8 0.8–1.0 0.8–1.0 1.0

Artemia feeding frequency/day

12 12 12 12 12 12 8 6 4 0

organisms are fed, the formulated diet can be restricted, thus avoiding hygiene problems in the tanks. A combination of Artemia with high-quality weaning diets does improve the nutritional status of the larvae, as demonstrated for the improved incorporation of DHA and EPA. An example of a weaning protocol is given in Table 8.7. This shows the growth of turbot larvae at approximately 20°C. It is proposed that inert diets are introduced at around 40 mg. It is advisable to feed small amounts frequently in the interests of tank hygiene and general rearing conditions. Usually, it would take 3–5 days before the larvae eat considerable amounts. Often, a switch to bigger particles triggers an increased food uptake. From around 130 mg body weight, the metamorphosing larvae are increasingly settling on the bottom and are feeding efﬁciently. The daily food ration can now be calculated from the expected growth assuming a feed conversion rate of 0.8. From this stage, feeding intervals for Artemia can be further reduced or cut. Weaning protocols require good daily follow-up of food rations and tank conditions to ensure that the larvae develop to their full capacity. Further, frequent weight checks are necessary. A protocol is therefore only a guideline which has to be adjusted to the actual situation by a skilled operator. 8.5.3.3 Nursery Once the ﬁsh are well adapted to formulated diets, the management of the nursery stage is a well-deﬁned production phase. The objective of the nursery stage is to deliver a healthy,

358

Table 8.8

Culture of cold-water marine ﬁsh

Nursery stage in turbot (18°C).

Age (days post-hatch)

Weight wet (g)

Diet particle size (mm)

Density (kg m-2)

Comments

40 45 50 55 60 65 70 75 80 85 90 95

0.7 1.1 1.6 2.2 3.0 4.0 4.8 5.8 7.0 8.0 9.3 10.4

0.8–1.0 1.0 1.0–1.5 1.5 1.5–2.5 2.0–3.0 2.0–3.0 2.0–3.0 2.0–3.0 2.0–3.0 2.0–3.0 3.0–4.0

1 1 2 2 3 3 4 4 5 5 5 5

Dip vaccination against vibriosis Grading

Booster vaccination against vibriosis Grading

growing juvenile for on-grown production. A common sales size for turbot juveniles is around 5–10 g. Table 8.8 summarises the main events during the nursery stage at 18°C. The juveniles reach sales size at 3 months from hatching under good growth conditions. The ﬁsh are kept in constant illumination, and feeding is done continuously. 8.5.3.4 Rearing Density Turbot are bottom dwellers from around 0.3–0.5 g size. Stocking densities are therefore expressed in kg m-2. The proposed densities given in Table 8.8 are moderate values. Higher densities are possible, but can easily reduce growth if ﬂow conditions and ﬁsh distributions are not well controlled. 8.5.3.5 Tanks Tanks of around 20–50 cm water depth are usually used for juveniles post-weaning. There has been a tendency over the years for increased tank sizes in order to obtain more effective production units. The possibilities of draining ﬁsh out through valves or ports in the tank wall or bottom do make handling more efﬁcient and gentle. As turbot tend to hold on to the bottom, a good slope with a slippery surface and sufﬁcient water ﬂow are necessary to make the ﬁsh move to the desired point. Juveniles also thrive in tanks with a water depth of only a few centimetres as long as their basic requirements for water exchange are maintained. Shallow raceways can be put above each other like shelves. The obvious advantage of such a system is the saving in area, while some drawbacks may be reduced safety or in stability of the water parameters. It is probable that new rearing systems for ﬂatﬁsh will be developed which will focus on creating the best environmental conditions for production, efﬁcient solutions for handling, and an overall competitive economy. The relative requirements for resources such as space, food and manpower are small in the early juvenile stages compared with later grow-out. A well-developing juvenile, largely protected from instability in the environmental conditions

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or disease outbreaks, will usually perform best in the on-grown stage. For this reason, the main focus for juvenile stages should be on optimising rearing conditions rather than minimising costs.

8.5.4 Halibut Juvenile production of halibut has for many years been the main bottleneck in the development of this species for aquaculture purposes. Production results have been highly variable, but generally low from year to year, for the last decade. However, one farm on Iceland has managed to produce consistent quantities of halibut for some time, and provides a model for other hatcheries. The low availability of juveniles has resulted in the acceptance of juveniles with incomplete eye migration or malpigmentation. Weaning around metamorphosis is similar to that for cod or turbot and not a particularly critical stage, although results for early weaning still are preliminary. As for cod, halibut develop gastric digestion late and at the rather large size of approximately 100–200 mg (see Table 8.2). In practice, weaning is initiated at around 250 mg, which is considered to be a safe size. Because of the high value of halibut, any additional costs of live food production are usually accepted as long as they can improve subsequent weaning survival. It is also possible that problems during larval rearing can result in variable groups with considerable size variations. A prolonged live-food stage can improve survival in such groups. However, as pointed out above, the prolonged use of Artemia does limit the growth capacity compared with formulated diets, and also retards the development of the digestive system. 8.5.4.1 Growth The growth data available for halibut larvae and juveniles are less comprehensive than those for cod or turbot. In Tables 8.9 and 8.10, we have compiled the best data from various sources. Growth close to 100 mg wet weight after 28 days has been reported. A 10-g ﬁsh can be produced within 140 days from start-feeding at best. In practice there is much variation, which often indicates inadequate rearing techniques. However, there is a steady improvement in the reported growth of halibut, and the species is not far behind the growth of cod. Table 8.9

Weaning protocol for halibut, Hippoglossus hippoglossus L. (13°C).

Larva age (days postﬁrst feeding)

Larva wet weight (mg)

Daily growth (%)

Grams dry feed per day per 10 000 ﬁsh

0 7 14 21 28 35 42 49 56–63

6 11 22 40 95 160 250 360 500

10 11 10 10 8 6 5 5

0 0 0 0 300 600 600 600

Particle size (mm)

Live food feeding frequency/day

0.3 0.3–0.5 0.3–0.5 0.5–0.7

Continuous Continuous Continuous Continuous Continuous Continuous 8–4 0

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Table 8.10

Nursery stage in halibut.

Age (days postﬁrst feeding)

Wet weight (g)

Diet particle size (mm)

Density max (kg m-2)

Comments

60 70 80 90 100 110 120 130 140 150

0.5 0.8 1.2 1.9 2.5 3.5 5.3 7.5 10.8 15.5

0.5–0.7 0.7–1.0 1.0 1.0–1.5 1.0–1.5 1.5 1.5–2.0 2.0 2.0–2.5 2.5–3.0

1 1 2 3 4 4 5 5 7 7

Dip vaccination against vibriosis Grading

Grading Booster vaccination against vibriosis Grading

8.5.4.2 Early Weaning Næss et al. (2001) attempted to wean halibut larvae from 70 mg wet weight, and compared the results with weaning at 100 and 160 mg, and with a live-food control (Artemia). An experimental larval diet was used, and co-feeding with Artemia was limited to 1 week. The results after 53 days showed that weaning at 70 mg did reduce survival (64%) compared with later weaning and with the control group (96%). Growth was also reduced if ﬁsh were transferred earlier to a formulated diet. Further, weaning was only successful if carried out at a water level of 1–2 cm, a high water exchange rate, and frequent feeding at 30-min intervals. Halibut larvae at this size are not very active swimmers, and the shallow water improves the uptake of feed particles. These results indicate a potential to develop early weaning techniques in halibut. However, the survival rate is too low to be acceptable for practical applications at this point. 8.5.4.3 Weaning In the work by Næss et al. (2001) cited above, weaning at 160 mg wet weight also resulted in both lower growth and survival than feeding with live food until 365 mg, and weaning onto a dry diet from this size. These results seem to conﬁrm the general experience of weaning halibut not earlier than from around 250 mg. Halibut larvae do not feed aggressively once food is dispersed into the water, and it may be difﬁcult to assess their immediate feeding response. A continuous, unhurried food uptake can be observed, and feed particles are often picked up from the bottom after some time. The gut content is the best indicator of feeding status. In order to ensure sufﬁcient food uptake, the diet has to be offered continuously. This requires good and frequent spreading, which means overfeeding in terms of the related hygiene problems. A suggested weaning protocol for halibut is given in Table 8.9. The proposed use of dry food indicates a need for great attention to feed and tank hygiene, and the costs involved set the upper limit for halibut production. Co-feeding is recommended up to 500 mg unless the ﬁsh are well graded and growing satisfactorily on the diet offered.

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8.5.4.4 Nursery Once the halibut have completed metamorphosis and are settled on the tank bottom, further rearing is similar to the strategy used for other ﬂatﬁsh. Table 8.10 gives an indication of the growth which can be obtained under optimal rearing conditions. In order to prevent repetition, only a few comments which are applicable to halibut in particular are made below. Halibut juveniles can show aggression, and biting the tails or eyes of other ﬁsh can cause injuries and losses. Good grading, a high feed availability and moderate densities seem to reduce aggression. Once disturbed, halibut are active swimmers and can be dip-netted, drawn into a ﬁsh pump, or guided through pipes and channels. This allows effective handling of large quantities of ﬁsh. Halibut juveniles seem to accept high stocking densities, but one should be cautious in extrapolating data from small units or limited time periods. In Table 8.10, we suggest densities similar to turbot. Increases in the swimming behaviour of the ﬁsh usually indicates if the density in the unit is too high. Light and depth conditions may also have an effect. Increased activity also leads to jumping, which is a common cause of both injuries and losses. In general, halibut show a strong reaction to disturbances, and increased activity at changing or low light levels is commonly observed.

8.6 References Al-Maghazachi, S.J. & Gibson, R. (1984) The developmental stages of larval turbot, Scophthalmus maximus (L.). J. Exp. Mar. Biol. Ecol., 82, 35–51. Appelbaum, S., Adron, J.W., George, S.G., Mackie, A.M. & Pirie, B.J.S. (1983) On the development of the olfactory and the gustatory organs of the Dover sole, Solea solea, during metamorphosis. J. Mar. Biol. Assoc. UK, 63, 97–108. Baskerville-Bridges, B. & Kling, L.J. (2000a) Early weaning of Atlantic cod (Gadus morhua) larvae onto a microparticulate diet. Aquaculture, 189, 109–17. Baskerville-Bridges, B. & Kling, L.J. (2000b) Development and evaluation of microparticulate diets for early weaning of Atlantic cod, Gadus morhua, larvae. Aquacult. Nutr., 6, 171–82. Baskerville-Bridges, B. & Kling, L.J. (2000c) Larval culture of Atlantic cod (Gadus morhua) at high stocking densities. Aquaculture, 181, 61–9. Björnsson, B.T. (1997) The biology of salmon growth hormone: from daylight to dominance. Fish Physiol. Biochem., 17, 9–24. Cahu, C.L., Zambonino Infante, J.L., Escaffre, A.M., Bergot, P. & Kaushik, S. (1998) Preliminary results on sea bass (Dicentrarchus labrax) larvae rearing with compound diet from ﬁrst feeding. Comparison with carp (Cyprinus carpio) larvae. Aquaculture, 169, 1–7. Canavate, J.P. & Fernandez-Diaz, C. (1999) Inﬂuence of co-feeding larvae with live and inert diets on weaning the sole Solea senegalensis onto commercial dry feeds. Aquaculture, 174, 255–63. Dhert, P., Gonzalez Felix, M., van Ryckeghem, K., Geurden, I. & Thysen, F. (1999) Co-feeding of phospholipids to turbot, Scophthalmus maximus L., larvae as a tool to reduce live food consumption. Aquacult. Nutr., 5, 237–45. Fontagné, S., Geurden, I., Escaffre, A.M. & Bergot, P. (1998) Histological changes induced by dietary phospholipids in intestine and liver of common carp larvae. Aquaculture, 161, 213–23.

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Hamre, K., Næss, T., Espe, M., Holm, J.C. & Lie, Ø. (2001) A formulated diet for Atlantic halibut (Hippoglossus hippoglossus, L.) larvae. Aquacult. Nutr., 7, 123–32. Kvåle, A., Harboe, T., Espe, M., Næss, T. & Hamre, K. (2002) Effect of pre-digested protein on growth and survival of Atlantic halibut larvae (Hippoglossus hippoglossus, L.). Aquacult. Res., 33, 311. Kvenseth, P.G. & Øiestad, V. (1984) Large-scale rearing of cod fry on the natural food production in an enclosed pond. In: The Propagation of Cod, Gadus morhua L. Arendal (eds E. Dahl, D.S. Danielssen, E. Moksness & P. Solemdal), pp. 645–55. Flødevigen Rapportserie. Lopez-Alvarado, J., Langdon, C.J, Teshima, S.-I. & Kanazawa, A. (1994) Effect of coating and encapsulation of crystalline amino acids on leaching in larval feeds. Aquaculture, 122, 335–46. McEvoy, L.A., Næss, T., Bell, J.G. & Lie, Ø. (1998) Lipid and fatty acid composition of normal and malpigmented Atlantic halibut (Hippoglossus hippoglossus) fed enriched Artemia: a comparison with fry fed wild copepods. Aquaculture, 163, 237–50. Næss, T., Hamre, K. & Holm, J.C. (2001) Successful early weaning of Atlantic halibut (Hippoglossus hippoglossus L.) in small, shallow raceway systems. Aquacult. Res., 32, 163–8. NRC (1993) Nutrient requirements of ﬁsh. Nutrient requirement of domestic animals. National Research Council, National Academy Press, Washington, DC. Otterlei, E., Nyhammer, G., Folkvord, A. & Stefansson, S.O. (1999) Temperature- and size-dependent growth of larval and early juvenile Atlantic cod (Gadus morhua): a comparative study of Norwegian coastal cod and northeast Arctic cod. Can. J. Fish. Aquat. Sci., 56, 2099–111. Ozkizilkik, S. & Chu, F.-L.E. (1996) Preparation and characterization of a complex microencapsulated diet for striped bass, Morone saxatilis, larvae. J. Microencapsulation, 13, 331–46. Rosenlund, G., Stoss, J. & Talbot, C. (1997) Co-feeding marine ﬁsh larvae with inert and live diets. Aquaculture, 155, 183–91. Rust, M. (1995) Quantitative aspects of nutrient assimilation in six species of ﬁsh larvae. PhD Thesis, School of Fisheries, University of Washington. Segner, H., Storch, V., Reinecke, M., Kloas, W. & Hanke, W. (1994) The development of functional digestive and metabolic organs in turbot, Scophthalmus maximus. Mar. Biol., 119, 471–86. Shields, R.J., Bell, G.J., Luizi, F.S., Gara, B., Bromage, N.R. & Sargent, J.R. (1999) Natural copepods are superior to enriched Artemia nauplii as feed for halibut larvae (Hippoglossus hippoglossus) in terms of survival, pigmentation and retinal morphology: relation to dietary essential fatty acids. J. Nutr., 129, 1186–94. Yúfera, M., Pascual, E. & Fernandez-Díaz, C. (1999) A highly efﬁcient microencapsulated food for rearing early larvae of marine ﬁsh. Aquaculture, 177, 249–56. Yúfera, M., Fernandez-Díaz, C., Pascual, E., Sarasquete, M.C., Moyano, F.J., Diaz, M., Alarcon, F.J., Garcia-Gallego, M. & Parra, G. (2000) Towards an inert diet for ﬁrst-feeding gilthead seabream, Sparus aurata L., larvae. Aquacult. Nutr., 6, 143–52. Zambonino Infante, J.L., Cahu, C.L. & Péres, A. (1997) Partial substitution of native ﬁsh meal protein by di- and tri-peptides in diet improves sea bass (Dicentrarchus labrax) larvae development. J. Nutr., 127, 608–14.

Chapter 9

On-Growing to Market Size M. Jobling

9.1 Introduction Seafood production from traditional capture ﬁsheries is not expected to increase much beyond current levels, so it is probable that an increased consumer demand for marine ﬁnﬁsh will be met by an expansion of aquaculture. Currently, less than 10% of marine food production is derived from aquaculture. Even though aquaculture in marine and fresh waters is the most rapidly expanding sector of animal production, the annual harvest from aquaculture is still modest in comparison with the production of livestock such as poultry, pigs and beef cattle. Furthermore, marine and brackish-water ﬁnﬁsh aquaculture (ca. 1.5 ¥ 106 mt) represents a low proportion of the total ﬁsh production from aquaculture (ca. 20 ¥ 106 mt). Most marine aquaculture is carried out in warm waters, the leading producers of cultured marine ﬁsh being Japan, China, other south-east Asian countries and the Mediterranean countries. In temperate regions of the world, marine farming has principally been directed towards the intensive cultivation of anadromous salmonids; salmonid farming has expanded considerably since the late 1970s. In recent years, there has also been an up-swing in interest in farming high-value, cold-water, marine species; on-growing trials have been initiated to examine the feasibility of farming species such as Atlantic cod, Gadus morhua, wolf-ﬁsh, Anarhichas spp. and a range of ﬂatﬁsh. In common with the salmonids, the cold-water marine ﬁsh species that are currently being farmed are carnivores, and their feeds typically contain ca. 500 g protein per kg dry feed. Feed ingredients that are good sources of protein are expensive. Consequently, much effort is directed towards the development of feed formulations that result in an efﬁcient use of protein for growth (Cho et al., 1982, 1994; Hardy, 1989, 1996; Wilson, 1991; Jobling, 1994, 1998; Goddard, 1996; Stickney, 2000; Houlihan et al., 2001). Further efforts to improve efﬁciency are made via the development of feed types, feed delivery systems, and feeding routines that reduce the risk of nutrient loss and feed wastage (Jobling et al., 1995; Goddard, 1996; Blyth et al., 1997; Jobling, 1998; Houlihan et al., 2001). The feed should be water-stable, so that it does not disintegrate before it has had a chance of being consumed, it should be made available in a form that it attractive to the ﬁsh, and it should be delivered when the ﬁsh are eager to feed. The feed types, delivery systems and feeding routines that fulﬁl these goals are expected to vary from species to species, so distinct feed formulations and feeding strategies may have to be developed as knowledge

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about the dietary requirements and feeding behaviours of the various cold-water marine species improves. It has long been known that marine ﬁsh are an excellent source of protein for human nutrition, and seafood is also an excellent source of several vitamins and minerals (Love, 1988; Macrae et al., 1993; Lie et al., 1994). In recent years, however, there has been an increased focus on the possible roles of marine lipids in human development and health. Fish lipids have received much attention because of their content of (n-3) fatty acids. These fatty acids have vital functions within the brain and nervous system, and they may protect against cardiovascular, and some other, diseases (Connor et al., 1992; Drevon, 1992; Macrae et al., 1993; Uauy-Dagach & Valenzuela, 1996; Lauritzen et al., 2001). Consequently, it is important that cold-water marine ﬁsh produced under farming conditions can be shown to have the same nutritional characteristics as wild ﬁsh, so that cultured ﬁsh retain a ‘health food’ image. In the sections that follow, attention will be drawn to some of the factors that must be considered in the formulation of feeds, and in the feeding of farmed ﬁsh. There is a focus on proteins and lipids, feed types and feeding routines, methods for the assessment of growth are considered, and this chapter closes with a discussion of dietary inﬂuences on the chemical composition of farmed marine ﬁsh.

9.2 Analysis of Feeds and Feedstuffs Knowledge about the nutrient content and energy value of feedstuffs is central for the correct formulation of feeds for farmed animals, and such information is also needed for the detailed study of energy metabolism and growth. The major energy sources in feedstuffs are the organic constituents lipids, proteins and carbohydrates, but feeds and feedstuffs will also contain a range of other organic constituents in addition to inorganic minerals (Fig. 9.1). The organic constituents can be oxidised in a bomb calorimeter to yield carbon dioxide, water,

Figure 9.1 Hierarchical division of an animal feed illustrating the major chemical components. From Jobling (2001a).

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MACRONUTRIENTS Carbohydrate Lipid Protein

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+ O2

CO2 + H2O (+ Oxides) + Heat (DH) Carbohydrate (e.g. Glucose): C6H12O6 + 6O2 Æ 6CO2 + 6H2O (DH: 2833 kJ) GMW Glucose = (6 ¥ 12) + (12 ¥ 1) + (6 ¥ 16) =180 ‘Gross energy’ =DH/GMW = 2833/180 = 15.7 kJ g-1 Lipid (e.g. Palmitic acid—fatty acid C16:0): CH3(CH2)14COOH + 23O2 Æ 16CO2 + 16H2O DH = 10014 kJ GMW C16:0 = 256 ‘Gross energy’ = 10014/256 = 39.1 kJ g-1 Figure 9.2 Heat-producing oxidation reactions, and the calculation of gross energy (heat of combustion), of macronutrients. GMW, gram molecular weight.

oxides of nitrogen and sulphur, and heat (Fig. 9.2). Bomb calorimetry involves the rapid combustion of a sample in oxygen at increased pressure, and heat production is measured. This provides a measure of the heat of combustion, or gross energy, of the sample. The chemical composition of a feed or feedstuff is measured using a series of standard laboratory methods, and there are also additional chemical tests that may be performed to test the ‘quality’ of the ingredients to be used in a feed formulation (Osborne & Voogt, 1978; Hardy, 1989; AOAC, 1990; Macrae et al., 1993; Stickney, 2000; Ibanez & Cifuentes, 2001). When an analysis of the major components is undertaken, samples are usually dried or freeze-dried and pulverised, and the ash, lipid and protein contents are determined directly. The moisture content is determined at the time of drying or freeze-drying. Ash weight is the weight of mineral residues left after the burning of organic material. The chemical analysis of the major components—moisture, protein, carbohydrates, lipids and ash—of feeds, feedstuffs, and plant and animal tissues is usually termed a proximate analysis. Proximate analyses and bomb calorimetry give information about the chemical compositions and gross energy contents of feeds and feedstuffs, but biological trials are required to reveal the full value of feed ingredients and formulations to the various species held in culture. For example, digestibility studies are required to reveal the bioavailabilities of the various nutrients to ﬁsh, and growth trials remain fundamental to feed assessment, and for the determination of nutrient requirements (for a discussion of methods, see Cho et al., 1982, 1994; Baker, 1986; Hardy, 1989; Macrae et al., 1993; Jobling, 1994, 1998; Shearer, 1995, 2000; Friedman, 1996; Stickney, 2000; Houlihan et al., 2001). Nutrient bioavailability can be assessed by measuring the digestibility of a feed or feedstuff. This provides an estimate of the proportion of the ingested nutrients which are

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absorbed. Methods for digestibility determination can be divided into those which are direct or indirect. The direct method requires both a knowledge of the total amount of feed consumed and the collection of all the faeces produced. Feed consumption is frequently unknown, and total collection of faeces is usually impracticable, so most estimations of digestibility in ﬁsh have been made using the indirect method. This involves the inclusion of an inert marker in the feed, with digestibility being calculated from the marker to nutrient ratios in the feed and faeces: 100 - 100([ X A X B ]¥[YB YA ])

(9.1)

where XA and XB are the concentrations of the inert marker in the feed and faeces, respectively, YA is the nutrient concentration in the feed and YB is the concentration of the nutrient in the faeces. A number of conditions must be fulﬁlled for the indirect method to yield accurate results. The marker must be inert, i.e. it must be non-toxic and should neither interfere with the normal processes of feeding, digestion and absorption nor be absorbed or metabolised. The marker should also pass through the gut at the same rate as the other digesta, i.e. there should not be any separation of marker and other feed components during passage through the gut. An accurate analysis of the marker in both the feed and the faeces must be possible, and it is advantageous if analyses can be performed cheaply and effectively. Finally, the feed containing the marker must be fed over a sufﬁciently long time-period to allow representative sampling of faeces to be made, i.e. the samples collected should be totally free of contamination by faeces produced during the consumption of previously unmarked feed. Chromic oxide has been the most commonly used marker in digestibility studies with ﬁsh, but it is suspected of violating some of the prerequisites of an inert marker: it may cause disturbance to ash digestive function, it has carcinogenic properties, and it may separate from the other digesta during passage through the gut. Acid-insoluble ash (AIA), barium carbonate, yttrium and ytterbium oxides, and ferro-nickel microtracers have all been suggested as alternatives to chromic oxide as markers (Atkinson et al., 1984; Riche et al., 1995; Refstie et al., 1997; Kabir et al., 1998). Feeds and feed ingredients are usually evaluated on the basis of their ability to support growth, and ‘feed quality’ is assessed using some form of expression that indicates how efﬁciently a feed or feed ingredient has been retained as growth (Hardy, 1989; Goddard, 1996; Stickney, 2000; Houlihan et al., 2001). The calculation of an efﬁciency involves the collection of both growth and consumption data. The importance of having accurate information about feed consumption cannot be stated too strongly; efﬁciencies are expressed as gain (growth) divided by feed consumption, so errors in measurements of feed consumption may have serious consequences for estimates of feed efﬁciency. The simplest way to express feed efﬁciency is to calculate the gain : feed ratio as the wet weight gain per weight of feed consumed. A similar expression, the protein efﬁciency ratio (PER), is often used to describe how well a protein source is used to support growth: PER is calculated as weight gain per unit weight protein consumed. Both of these gain : feed ratios give rather crude estimates of efﬁciency because they do not account for possible differences in the chemical composition of the gain deposited by animals given different feeds.

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Energy, and nutrient, retention can be measured if the chemical composition of the animal is estimated at the beginning and end of the growth trial. This is known as the comparative slaughter method. A sample of animals is taken at the start of the trial, and a proximate analysis is carried out to determine their chemical composition and energy content. These data are then used to predict the initial composition of the animals used in the growth trial. At the end of the trial, the test animals are slaughtered, their chemical composition and energy content are determined, and the amounts of each component deposited during the course of the trial are estimated by difference. Assuming that feed intake has also been measured satisfactorily, the data can then be used to provide more detailed assessments of nutrient retention efﬁciencies. If both energy consumption and energy retention are known, it is possible to calculate a feed efﬁciency in terms of feed energy, which is a more satisfactory assessment than that based upon weight alone. Comparative slaughter also allows a better measurement of the utilisation of dietary protein than PER. Net protein utilisation (NPU), also known as protein productive value (PPV), may be calculated as the weight of protein gained per unit weight of protein consumed.

9.3 Protein Requirements and Sources Fish require an adequate supply of dietary protein if they are to thrive and grow well, but the requirement is for certain amino acids that make up the proteins, rather than for protein per se (Table 9.1) (for a general discussion, see Wilson, 1989; Cowey, 1994; Jobling, 1994; Stickney, 2000). Thus, the quality of a protein may be considered to depend upon its amino acid composition and the availability of these amino acids to the animal that consumes it (Macrae et al., 1993; Friedman, 1996; Hertrampf & Piedad-Pascual, 2000; Stickney, 2000). Consequently, it is determinations of amino acid composition and availability that should underlie all assessments of protein quality. In nutritional terms, an amino acid is usually classiﬁed according to whether or not protein synthesis and growth can proceed in the absence of a dietary supply, so the amino acids that are constituents of proteins can be classiﬁed as: (1) essential or indispensable: arginine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, threonine, tryptophan, valine; (2) conditionally indispensable: cystine, tyrosine; (3) non-essential or dispensable: alanine, asparagine, aspartic acid, glutamine, glutamic acid, glycine, proline, serine. The essential amino acids are those that the ﬁsh cannot synthesise, or cannot synthesise in sufﬁcient quantity to allow the maintenance of good rates of growth, whereas the nonessential, or dispensable, amino acids can be synthesised de novo from other compounds. The same ten amino acids are considered to be essential for the majority of animal species, although there is some evidence that other amino acids (e.g. taurine, tyrosine, or the basic amino acids involved in the urea cycle) may be essential for some animal species at certain stages of their life-cycle. Cystine and tyrosine are usually classiﬁed as being conditionally

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Table 9.1 Characteristics of the different series of amino acids. Indispensable (essential) amino acids are indicated in bold type, and conditionally indispensable amino acids are shown in italics. From Jobling (2001a). Series

Characteristics

Amino acid

Aliphatic

Aliphatic amino acids containing one carboxyl group and one amino group Aliphatic, monoamino–monocarboxylic amino acids containing sulphur Aliphatic, dicarboxylic amino acids; aqueous solutions are acidic Aliphatic amino acids giving basic aqueous solutions Amino acids with the aromatic, or benzenoid, ring Heterocyclic structure incorporating nitrogen; proline and hydroxyproline have an imino (NH) group, but no amino group

Gly, Ala, Ser, Val, Thr, Leu, Ile Met, Cys

Sulphur amino acids Acidic Basic Aromatic Heterocyclic

Asp, Glu Arg, His, Lys Phe, Tyr Trp, Pro, Hyp

indispensable (although they may be essential for some species) because they are synthesised from methionine and phenylalanine, respectively. Both methionine and phenylalanine are essential amino acids, but a dietary supply of cystine and tyrosine is not required if sufﬁcient quantities of these essential amino acids are available. In addition to being component parts of proteins, several of the amino acids are also precursors for the synthesis of other biologically active compounds: for example, histidine is decarboxylated to form histamine, tyrosine is iodinated to form the thyroid hormones, and is also used in the synthesis of the catecholamines, and tryptophan is the precursor of serotonin (5-HT) and melatonin. Whilst it is widely recognised that ﬁsh, and most other animals, have a dietary requirement for the same ten essential amino acids, relatively little is known about the quantitative requirements for these amino acids amongst ﬁsh species. Quantitative data are available for some of the most widely cultivated species, but even for these the data are incomplete (Wilson, 1989, 1991; Cowey, 1994). The essential amino acid requirements of certain ﬁsh species have been shown to correlate reasonably well with the essential amino acid proﬁle of their whole body, or muscle, protein (Mambrini & Kaushik, 1995; Kaushik, 1998). Consequently, body protein composition data may provide useful background information that can be used for the formulation of feeds when the essential amino acid requirements of the ﬁsh have not been determined. The essential amino acid patterns of body proteins appear to be quite similar across a wide range of ﬁsh species (Table 9.2) (Haard, 1992; Cowey, 1993; Lie et al., 1994; Mambrini & Kaushik, 1995; Kaushik, 1998), so it is not surprising that ﬁsh meals (Table 9.3), silages and other ﬁsh-based products are generally considered to be highquality sources of protein for farmed ﬁsh (Hertrampf & Piedad-Pascual, 2000; Stickney, 2000). Fish meal is a major ingredient in formulated feeds for carnivorous ﬁsh, and the current success of intensive ﬁsh farming is dependent upon the use of ﬁsh meal as a major source of dietary protein. Fish meals contain a high percentage of protein (usually 60–75%), an appreciable amount of mineral ash (10–20%) and a proportion of lipid (5–10%) (Macrae et al., 1993; Hertrampf & Piedad-Pascual, 2000). Most ﬁsh meal is produced from small, pelagic ﬁsh species such as sardines, anchovies, capelin, herring and menhaden, but meals are also produced from the smaller members of the cod family (Table 9.4), and from the wastes arising from the processing of the larger gadoids, the so-called ‘white ﬁsh’. The

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Table 9.2 The essential (indispensable) amino acid (EAA or IAA) compositions (expressed as g AA kg-1 protein) of the ﬁllet (muscle) of some cold-water ﬁsh species. Adapted from Lie et al., (1994).

Arginine Histidine Isoleucine Leucine Lysine Methionine Phenylalanine Threonine Tryptophan Valine

Atlantic salmon Salmo salar

Atlantic cod Gadus morhua

Wolf-ﬁsh Anarhichas lupus

Atlantic halibut Hippoglossus hippoglossus

Turbot Scophthalmus maximus

71 43 60 92 92 33 54 49 11 60

55 22 39 72 88 28 39 39 11 39

54 22 38 70 86 27 43 43 10 38

62 25 43 80 105 31 37 43 19 49

57 19 38 69 82 25 44 44 13 38

Table 9.3 The essential (indispensable) amino acid (EAA or IAA) compositions (expressed as g AA kg-1 protein) of the ﬁllet (muscle) of Atlantic salmon, Salmo salar, and of several protein sources used in the manufacture of ﬁsh feeds. From data in Macrae et al. (1993), Lie et al. (1994), Hertrampf & Piedad-Pascual (2000) and Stickney (2000).

Arginine Histidine Isoleucine Leucine Lysine Methionine Phenylalanine Threonine Tryptophan Valine

Atlantic salmon Salmo salar

Fishmeal

Soybean

Rape/canola

Maize (corn) gluten

Wheat gluten

71 43 60 92 92 33 54 49 11 60

59 29 44 75 81 30 40 43 12 54

73 28 47 75 61 14 50 40 17 49

79 33 43 61 66 23 38 56 8 43

37 29 35 114 18 23 66 38 10 44

36 19 35 70 15 16 50 37 11 39

Table 9.4 Chemical compositions (proximate analyses) of a variety of ‘trash’, or industrial, ﬁsh species commonly used both as feed for farmed cold-water ﬁsh species, and for the production of ﬁsh meals and oils. From Jobling (1994). Fish species

Capelin (‘winter’), Mallotus villosus Capelin (‘summer’), Mallotus villosus Brisling, Sprattus sprattus Sandeel (sand-lance), Ammodytes marinus Mackerel, Scomber scombrus Blue whiting, Micromesisteus poutassou Norway pout, Trisopterus esmarkii Polar cod, Boreogadus saida

Chemical component (%) Moisture

Protein

Lipid

Ash

76.6 72.2 68.5 72.0 62.7 76.3 73.0 76.9

13.5 12.4 16.1 16.6 17.4 16.7 16.3 12.9

8.4 14.6 13.0 9.5 20.6 3.5 8.5 6.4

1.9 1.7 2.7 2.2 1.9 3.5 2.7 2.6

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majority of the small, pelagic ‘forage’ ﬁsh species used for the manufacture of ﬁsh meals and oils show marked seasonal variations in body composition (Hislop et al., 1991; Takama et al., 1994; Van Pelt et al., 1997; Payne et al., 1999; Anthony et al., 2000; Pedersen & Hislop, 2001), and this will have an inﬂuence on the relative proportions of protein and ash present in the meal. Thus, the nutritional value of a ﬁsh meal will depend both upon the time of year at which it is produced, and upon the species of ﬁsh from which it has been prepared. Fish meals have been classiﬁed into four broad groups: herring-type, anchovy, menhaden and white ﬁsh. In addition to being inﬂuenced by ﬁsh type and season, the nutritional quality of a ﬁsh meal will also depend upon the freshness of the raw material used, and the processing conditions employed during manufacture. The best-quality meals are produced by processing fresh whole ﬁsh under low-temperature conditions, and the quality of the meals produced decreases if stale ﬁsh and/or high cooking and drying temperatures are used (Pike et al., 1990; Macrae et al., 1993; Jobling, 1994; Hardy, 1996; Aksnes & Mundheim, 1997; Hertrampf & Piedad-Pascual, 2000). The meals produced from ﬁlleting wastes and other ﬁsh processing by-products are also considered to be of poor quality owing to their relatively high ash (ca. 20%), and low protein (ca. 60–65%), content: bone ash contains large proportions of tricalcium phosphate and hydroxyapatite, and because these phosphatecontaining compounds are not digested and absorbed by the ﬁsh, they lead to increased nutrient loading of the recipient water body. Feeding ﬁsh with feeds prepared from ﬁsh meal produced from stale raw material may result in reductions in both growth rates and feed efﬁciencies in comparison with those of ﬁsh fed feeds prepared with meals manufactured from fresh raw material. For example, both growth and feed efﬁciency of halibut, Hippoglossus hippoglossus, were found to decrease when the ﬁsh were fed on feeds containing ﬁsh meal prepared from stale raw material. The freshness of the raw material used in manufacturing the ﬁsh meals was assessed by monitoring levels of the biogenic amine cadavarine, derived from the decarboxylation of lysine due to the actions of spoilage bacteria. Levels of cadavarine tended to increase as the raw material became staler, and there were negative correlations between dietary cadaverine levels and the two performance characters, feed efﬁciency and growth rate. However, it is unlikely that the observed decreases in growth rate and feed efﬁciency were a direct result of the effects of the increased levels of biogenic amines per se, and presence of other toxic agents resulting from the degradation of the raw material was suspected (Aksnes & Mundheim, 1997). Processing conditions also inﬂuence the quality of ﬁsh meals. High processing temperatures (100–150°C) result in meals which have lower nutrient bioavailability than those produced under lower temperatures (60–70°C) (Aksnes & Mundheim, 1997; Hertrampf & Piedad-Pascual, 2000). The reduction in nutrient bioavailability that results from processing at a high temperature may arise because of the formation of enzyme-resistant cross-linkages between protein chains, the oxidation of amino acids, such as methionine and tryptophan, or the reaction of lipid oxidation products with amino acids (Camire et al., 1990; Macrae et al., 1993). Reduced nutrient availability may, in turn, have a number of negative effects: nutrient loading of the recipient water body will be increased, the utilisation of feed protein

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for growth will be reduced, and there may also be a reduction in the growth of ﬁsh given feeds that contain ﬁsh meals produced using harsh processing conditions (Pike et al., 1990; Aksnes & Mundheim, 1997). Thus, there may be differences in growth performance between ﬁsh given feeds manufactured from high-quality ﬁsh meals (e.g. Norse-LT 94) and lowerquality meals (e.g. NorSeaMink); high-quality meals have been reported to give increased growth and feed efﬁciency in a range of species, including salmonids, marine ﬂatﬁsh, and the wolf-ﬁsh, Anarhichas lupus (Pike et al., 1990; Moksness et al., 1995; Aksnes & Mundheim, 1997). Fish and ﬁsh-processing wastes are also used to produce ﬁsh silages and ﬁsh protein concentrates (Arason, 1994; Haard & Simpson, 2000; Hertrampf & Piedad-Pascual, 2000; Kristinsson & Rasco, 2000). Fish silage is a liquiﬁed product manufactured from chopped ﬁsh, or ﬁsh-processing waste, mixed with acid. The enzymes present in the raw material digest and liquify the ﬁsh tissues, and the low pH created by the presence of the acid hinders the growth of spoilage bacteria and other micro-organisms. Either mineral (e.g. sulphuric) or organic (e.g. propionic, formic) acids can be used directly as preservative agents, but the fermentative capabilities of lactic acid bacteria may also be exploited in the production of ﬁsh silage. Fish silage is a relatively stable product that can be stored at ambient temperatures for prolonged periods, but as time progresses the enzymes in the silage mass will continue to break down the proteins present in the ﬁsh tissue. Thus, over a period of weeks or months, the ﬁsh proteins will gradually be hydrolysed to small peptides and free amino acids, and this may reduce the nutritional value of the silage. Therefore, following the initial liquifaction, the silage may be heated to about 85°C to prevent further enzymatic breakdown, and thereby stabilise the mixture. Fish silage tends to separate into three fractions: an aqueous fraction containing the hydrolysed protein, a lipid fraction and a bottom sediment. The silage may be used as it is, the different fractions may be separated, or the silage may be concentrated by partial drying to remove some of the water content. The fact that ﬁsh silage is liquid means that it must be mixed with dry ingredients, such as ﬁsh meal, vitamins and minerals, and binders, before being fed to the ﬁsh in the form of a moist pellet. This, together with the fact that acid preservation may lead to the destruction of some essential nutrients, such as methionine and tryptophan, tends to limit the use of ﬁsh silage as a feed ingredient. Further, acid residues present in the silage can lead to reduced palatability of the feed. When there is a ready supply of ﬁsh silage produced from ﬁshprocessing by-products (e.g. viscera and ﬁllet off-cuts), the silage may be either concentrated or dried to a meal prior to inclusion in a feed. Under these circumstances, the silage may be mixed with ﬁsh meal, animal by-products meal, or plant protein sources before being dried. Fish protein concentrate (FPC) is produced from by-products of the ﬁshery industry, and is based upon the conversion of insoluble ﬁsh protein into polypeptides, small peptides and amino acids by hydrolysis (Haard & Simpson, 2000; Hertrampf & Piedad-Pascual, 2000; Kristinsson & Rasco, 2000). The raw materials, which are usually the wastes from ﬁsh ﬁlleting and processing plants, are minced to give a large surface for the agents used for hydrolysis. Chemical hydrolysis of the proteins may be carried out using either acids or alkalis, but enzymatic hydrolysis is the technique most usually employed. Enzymatic hydrolysis may

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rely on the endogenous proteolytic enzymes present in the ﬁsh muscle and viscera, but it is more usual to add enzymes from other sources because this allows for a greater degree of control over the process. Following hydrolysis of the protein to smaller polypeptides, the insoluble material, such as bones and other solids, is removed, and the liquid fraction containing the polypeptides is retained for further processing. The product is de-fatted, concentrated, pasteurised and spray-dried to give the ﬁnal FPC. Although the ﬁnal product contains very little lipid, most of the fatty acids present are long-chain (n-3) fatty acids that are susceptible to oxidation. Consequently, antioxidants are usually added to the FPC to prevent lipid oxidation during transport and storage. Most of the world’s ﬁsh resources appear to be either fully exploited or over-exploited, so there would seem to be limited potential for expansion of the aquaculture industry based upon the increased use of ﬁsh protein as a feed ingredient. For example, the annual production of ﬁsh meal has stabilised at about 6–7 million mt, production is not expected to increase in the future, and increases in ﬁsh meal prices are expected (Pike et al., 1990; Hardy, 1996). Consequently, considerable effort has been directed towards the evaluation of protein sources that could provide an alternative to ﬁsh meal in formulated feeds for farmed ﬁsh (Hardy, 1989, 1996; Hertrampf & Piedad-Pascual, 2000; Stickney, 2000). Meals produced from a range of animal by-products (e.g. meat and bone meal, blood meal, feather meal, poultry by-product meal) are readily available and have been included in formulated feeds for ﬁsh (Hardy, 1989, 1996; Hertrampf & Piedad-Pascual, 2000). However, it is unlikely that their use will increase in the near future. In fact, there may be a move away from using such meals because of consumer opposition, and the possible risks of disease transmission when using meals processed under some conditions (e.g. at low temperatures). In addition, animal by-products meals vary widely in nutritional value depending upon the way they are processed and handled, and some by-product meals may contain high levels of unavailable mineral ash in the form of calcium phosphate salts. Blood meals are produced from the blood of slaughtered livestock, but blood coagulates quickly and deteriorates rapidly, so it must be preserved shortly after the slaughter of the animal if a meal of good quality is to be produced. Drying is the most effective method of preserving blood, but the processes used during drying inﬂuence the quality of the meal; the exposure of blood to a high temperature for a long time damages the blood proteins and reduces their bioavailability. For example, spray-drying results in meal of a better quality than does ﬂame-drying. Spray-dried blood meal is produced by evaporating the blood to 40–50% solids in a vacuum at ca. 50°C, and then spraying it into a hot air stream at ca. 310–320°C. Blood meal is a rich source of leucine, but a poor source of methionine and isoleucine. Feather meal and poultry by-product meals are mostly produced from by-products of the broiler (poultry meat) industry. Feathers contain about 90% protein, but most of it is tough, ﬁbrous keratin, so the bioavailability of the feather protein is low. The feathers are usually treated by pressure cooking to denature the proteins and improve bioavailability, and after cooking the material is dried at 60°C and then ground to produce the meal. The quality of the ﬁnal product depends upon the methods used during processing (i.e. pressure, temperature and length of time). Poultry by-product meal is made from the rendered parts of slaugh-

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tered poultry (e.g. heads, feet, undeveloped eggs and viscera). Raw materials are coarsely ground and then ‘dry-cooked’ at 100–125°C to kill potential pathogens, liquify and remove the fat, and evaporate most of the moisture. Following cooking, the material is ground to a meal. The composition of poultry by-product meals tends to be variable, depending upon the proportions of different raw materials used, and most of the protein in the meals derives from connective tissues. Meat by-products are the offal from slaughterhouses, scraps from the meat industry, and livestock casualties processed by rendering. A wide range of processes are used by rendering plants for the manufacture of meat by-product meals, and both processing methods and the composition of the ﬁnished product will depend upon the raw materials used for producing the meal. For example, high proportions of bones or fat in the raw material results in a high ash or fat content in the ﬁnal product, which reduces the protein concentration of the meal. Most of the protein sources currently being tested as possible replacements for ﬁsh meal are of plant origin, and they are usually existing feed ingredients that seem to have the potential for quality improvement via selective breeding, genetic modiﬁcation techniques (transgenics), additional processing or nutrient supplementation. Most of the protein source replacement trials are being performed on established aquaculture species such as salmonids, tilapias, cyprinids and ictalurids, and it remains to be seen whether the results obtained can be extrapolated to cold-water, carnivorous marine species. The most important and widely used protein sources of plant origin are oilseed meals, derived from the dried residues after the oil has been extracted from soybeans, cottonseed, rape/canola, and other materials used in the production of vegetable oils (Hertrampf & Piedad-Pascual, 2000; Stickney, 2000). However, oilseed meals may be deﬁcient in some of the essential amino acids, particularly methionine and lysine. Cereals are considered to have limited potential as protein sources in ﬁsh feeds owing to their high carbohydrate contents and poor essential amino acid proﬁle: cereals tend to be deﬁcient in lysine, and maize is deﬁcient in both lysine and tryptophan (see Table 9.3). The annual production of maize exceeds 500 million mt, and maize is an important crop grown both for human consumption and livestock feed. Ground maize, or maize meal, is a major feed ingredient for livestock, but it has a low protein content and most varieties are deﬁcient in several essential amino acids. Zein, the major protein component of maize, is low in most of the essential amino acids, but especially so in lysine and tryptophan, so the total protein of most varieties of maize is deﬁcient in these two essential amino acids. However, there are several genetic varieties of maize, and one of these, opaque-2, has an increased protein content coupled with a reduced level of zein and an increase in the proportion of another protein, glutelin. This results in opaque-2 maize having increased levels of lysine, tryptophan and most other essential amino acids, and high-lysine hybrids can be produced. However, the yields of the high-lysine hybrids are lower than those of several other varieties, and this means that they are not particularly widely grown. Maize gluten meal has a higher protein concentration (ca. 60%) than maize meal because it is produced from the by-products resulting from the extraction of the starch from maize kernels. However, as with maize meal, maize gluten is deﬁcient in the amino acids lysine and tryptophan. In addition to potential problems related to imbalanced amino acid proﬁles, the use of unmodiﬁed plant materials as feed ingredients may also be limited owing to the presence of a range of anti-nutritional factors (ANFs)

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(Table 9.5) (Hendricks & Bailey, 1989; Macrae et al., 1993; Liener, 1994; Higgs et al., 1995; Friedman, 1996; Burel et al., 2000; Hertrampf & Piedad-Pascual, 2000; Stickney, 2000; Francis et al., 2001). Soybeans are cultivated in many countries, and with an annual harvest in excess of 100 million mt, the production of soybean meal far exceeds that of ﬁsh meal. At present, soybean meal is the most important protein source in livestock feeds, and it is usually considered to be the best widely available plant protein source used in the formulation of ﬁsh feeds. However, soybeans have the disadvantage that they contain ANFs such as protease inhibitors, phytate, phytoestrogens, lectins (haemagglutinins), goitrogens, antivitamins, saponins (haemolytic agents), various oligosaccharides and antigenic proteins (allergens) (Table 9.5). Some of the ANFs can be removed by de-hulling, and others, such as the trypsin and chymotrypsin inhibitors, are partially destroyed or inactivated by the heating and drying procedures used in the production of soybean meal. Nevertheless, other ANFs, such as phytate, are less affected by the normal processing procedures. The incomplete destruction of the ANFs in soybeans under normal processing may reduce the potential for using conventional soybean meal in diets for some carnivorous ﬁsh species, and soybean protein concentrate may be a better alternative. The use of protein concentrates surmounts many of the problems associated with the presence of the ANFs, and provides the ﬁsh with a source of protein which has an essential amino acid proﬁle that seems to fulﬁl most of the requirements for growth. Thus, soybean may be a valuable source of protein, but its full nutritional potential may only be attained following processing to remove, denature or inactivate the various ANFs. Processing may therefore involve a range of chemical, enzymatic and physical treatments aimed at improving the nutritional value of the ﬁnished product (Liener, 1994; Anderson & Wolf, 1995; Hertrampf & Piedad-Pascual, 2000). A variety of legumes, such as peas, beans, lupins and lentils, have been used as ingredients in feeds for domestic animals, and tests have also been carried out to examine the suitability of some of these as protein sources in ﬁsh feeds (Hertrampf & Piedad-Pascual, 2000). One disadvantage of plant protein sources, such as legumes, oilseed meals and cereals, is that they may contain up to 70% of their phosphorus bound in phytate (Selle et al., 2000; Francis et al., 2001). Phytate is a cyclic derivative of inositol that contains six phosphate groups. The phosphorus in phytate is unavailable, or has a very low bioavailability (10–50%), to non-ruminant animals, including ﬁsh, because they lack the digestive enzyme phytase (Liener, 1994; Selle et al., 2000). Pre-treatment or supplementation of plant protein sources with phytase may result in increased bioavailability (45–80%), and thereby reduce faecal losses of phosphorus (Campbell & Bedford, 1992; Chesson, 1993; Cain & Garling, 1995; Oliva-Teles et al., 1998; Bedford, 2000; Selle et al., 2000). Not only is much of the phosphorus in untreated plant protein sources unavailable because it is bound within the phytate molecule, but phytate also chelates with di- and trivalent metals such as calcium, magnesium, zinc and iron. This leads to the formation of poorly soluble compounds that are not readily absorbed from the gastrointestinal tract. Thus, phytate in the diet may inﬂuence the availability of a range of micronutrients, and it also interacts strongly with basic residues of protein (Selle et al., 2000). As a result of this binding to proteins, phytate may have an inhibitory effect on several digestive enzymes, including pepsin, trypsin and amylase. Thus,

Table 9.5 Some anti-nutritional factors (ANFs) present in various plant ingredients that may be used in the formulation of ﬁsh feeds. X indicates that the ANF is present. Heat-labile ANFs are indicated by bold typeface. From data in Hertrampf & Piedad-Pascual, (2000), Stickney (2000) and Francis et al. (2001). Anti-nutritional factor Enzyme inhibitors Protease ‘Oilseed’ meals Soybean Rape/canola Cottonseed Sunﬂower Sesame

Phytate

Phytooestrogens

Saponins

X X X

X X X

X

Amylase

X X X

X

‘Grains’ Corn (maize) Wheat Sorghum

X X X

‘Tubers’ Potato Cassava

X X

X X

X X

X X

X X X

X

Cyanogens

Glucosinolates

X X

X

X X

X X X

X

X

X X

X X

X

X

Alkaloids

X

X

X X

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Legumes Lupin Alfalfa Faba bean

Lectins

X X

X X

Tannins

375

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Culture of cold-water marine ﬁsh

there may be several nutritional beneﬁts to be gained by pre-treating plant protein sources with phytase prior to their inclusion in formulated feeds for ﬁsh and other non-ruminants (Campbell & Bedford, 1992; Chesson, 1993; Liener, 1994; Cain & Garling, 1995; OlivaTeles et al., 1998; Bedford, 2000; Selle et al., 2000). Lupin seeds have a high protein content (ca. 35%), although as a source of protein for animals they are deﬁcient in methionine. They do not contain a such wide array of ANFs as soybeans (see Table 9.5), but lupins contain quinolizidine alkaloids. These alkaloids make lupin seeds bitter and potentially toxic. Lupin cultivars with a low alkaloid content have been developed, and it is these which seem to hold most promise as animal feedstuffs (van Barneveld, 1999; Burel et al., 2000; Hertrampf & Piedad-Pascual, 2000). Rape and canola have attracted attention because these brassicas grow in cooler climates than most of the other oilseed plants, but the inclusion of rapeseed meals in ﬁsh and livestock feeds may be limited because of the presence of ANFs such as glucosinolates and erucic acid (see Table 9.5) (Burel et al., 2000; Hertrampf & Piedad-Pascual, 2000; Francis et al., 2001). Plant breeders have developed rape cultivars with low levels of these ANFs, and these new cultivars have been called canola to distinguish them from the older varieties. However, canola meals contain phytate, tannins and other phenolics, and they have a high ﬁbre content; this may restrict the use of these meals in ﬁsh and livestock feeds. As a result, canola protein concentrates have been produced to reduce the levels of ﬁbre and other undesirable compounds. The protein concentrates have a high protein content (60–65%), and have amino acid proﬁles that resemble those of ﬁsh meals. Thus, it may be possible to use canola protein concentrates to partially replace ﬁsh meal in formulated feeds (Hendricks & Bailey, 1989; Higgs et al., 1995). Single-cell proteins (SCPs) refer to a wide range of products of microbial origin, and the term is used to describe feed protein ingredients derived from bacteria, yeasts and microalgae. As such, SCP is an imprecise term, and a more precise terminology exists to describe feed ingredients that derive from speciﬁc organisms produced under deﬁned sets of conditions (Hertrampf & Piedad-Pascual, 2000). Efforts have been made to develop fermentation processes for the large-scale production of several species of yeasts and bacteria. Protein production via fermentation of yeast or bacterial cultures is considered desirable because the organisms have rapid rates of growth, they can be grown on relatively inexpensive media (e.g. soluble wastes from breweries and distilleries, sewage processing and wood pulping operations, and ‘cracking products’ from the oil industry), they make efﬁcient use of the energy source, and ﬁnished products with a high protein content can be prepared relatively easily (Tusé, 1984; Hertrampf & Piedad-Pascual, 2000). Most SCPs are too expensive to form the main protein source in ﬁsh and livestock feeds, and there may also be high concentrations of undesirable compounds, such as heavy metals, in some crude SCP products. Traditionally, the largest producers of SCP have been the brewing and distilling industries; the yeast by-products are sold as protein and vitamin supplements for inclusion in animal feeds. Certain yeasts and algal products may also provide a source of carotenoids in ﬁsh feeds. Although an initial interest in the production of SCP using oil and petroleum fractions as the growth medium waned during the late 1970s, there is currently renewed interest in the production of SCP via bacterial fermentation using natural gas as the energy source and ammonia as the source of nitrogen for protein synthesis.

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9.4 Lipids and Lipid Requirements The lipids are a heterogeneous class of water-insoluble compounds. They can be broadly classiﬁed into three groups: simple lipids, compound lipids and derived lipids. Simple lipids are esters of fatty acids which produce two classes of compounds upon hydrolysis, compound lipids are fatty acid esters that produce three or more classes of compounds on hydrolysis, whereas derived lipids cannot be hydrolysed to give fatty acids (for overviews of lipids and lipid nutrition, see Sargent et al., 1989; Macrae et al., 1993; Henderson, 1996; Bell, 1998; Cunnane, 2000; Stickney, 2000). The main simple lipids are triacylglycerols (TAGs), steryl esters and wax esters. TAGs are the major form of storage lipid in most plants and animals. They are esters of the trihydric alcohol glycerol with three fatty acids. Many of the properties of TAGs are dependent upon the component fatty acids. For example, the melting point of a TAG reﬂects the melting point of the component fatty acids; three high-melting-point fatty acids yield a high-meltingpoint TAG, whereas TAGs containing low-melting-point fatty acids are liquid at room temperature. Further, the presence of unsaturated fatty acids (i.e. those with double bonds between some of the carbon atoms making up the fatty acid chain) makes the TAG susceptible to oxidation, in the same manner as the unsaturated fatty acids themselves (St Angelo, 1996). Fatty acids differ in the numbers of carbon atoms in the molecule, and in the number and positioning of the double bonds between the carbon atoms. They have the general formula CH3(CXHY)COOH, and when Y = 2X the hydrocarbon chain is saturated, i.e. lacks double bonds (e.g. palmitic acid; see Fig. 9.2). The fatty acids form distinct series, and a shorthand system has been devised for classifying these fatty acid series (Table 9.6). For example, the shorthand formula 14:0 represents a fatty acid with 14 carbon atoms and no double bonds. A fatty acid lacking double bonds between the carbons is known as a saturated fatty acid (SFA). The shorthand formula 18:3(n-3) represents an 18-carbon fatty acid with three double bonds, the (n-3) denoting that the ﬁrst double bond is found in the link between the third and fourth carbon atoms from the methyl end. Fatty acids with one or more double bonds are unsaturated fatty acids. Those having a single double bond are monounsaturated fatty acids (MUFAs), those with two or three double bonds in the molecule are often termed polyunsaturated fatty acids (PUFAs), and those with four or more double bonds may be described as being highly unsaturated fatty acids (HUFAs). Fatty acids found in terrestrial plants and animals generally have relatively low degrees of unsaturation and have carbon chain lengths of 14–18, but longer-chain fatty acids, with up to 22 carbon atoms, are commonly encountered in aquatic organisms (Table 9.7). For example, although many different fatty acids may occur in the lipids extracted from the muscle of a single ﬁsh species, the fatty acid composition is usually found to be dominated by a small number of saturated (14:0, 16:0 and 18:0) and monounsaturated (16:1, 18:1, 20:1 and 22:1) fatty acids, along with PUFAs and HUFAs. SFAs generally comprise 20–30% of the fatty acids in ﬁsh muscle lipids, MUFAs usually make up 20–50%, and the remaining fatty acids are PUFAs and HUFAs (Table 9.8) (Cowey, 1993; Lie et al., 1994). However, the relative proportions of SFAs, MUFAs, PUFAs and HUFAs in the total lipids extracted from ﬁsh muscle will differ with species depending upon the extent to which muscle tissue is used

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Table 9.6 Classiﬁcation and naming of some representative fatty acids. Note that in the scientiﬁc designation, anoic refers to a fatty acid without double bonds in the carbon chain, enoic to a fatty acid with one double bond, dienoic to two double bonds, trienoic to three, tetraenoic to four etc. The shorthand notation gives the number of carbons, the number of double bonds, and the position of the ﬁrst double bond counting from the methyl end. From Jobling (2001a). Trivial name (scientiﬁc designation)

Number of carbon atoms

Number of double bonds

Fatty acid series

Shorthand notation

Saturated fatty acids (SFAs) Lauric (dodecanoic) Palmitic (hexadecanoic) Stearic (octadecanoic)

12 16 18

0 0 0

Monounsaturated fatty acids (MUFAs) Palmitoleic (hexadecenoic) Oleic (octadecenoic) Erucic (docosenoic)

16 18 22

1 1 1

(n-7) (n-9) (n-9)

16 : 1(n-7) 18 : 1(n-9) 22 : 1(n-9)

Polyunsaturated fatty acids (PUFAs) Linoleic (octadecadienoic) g-Linolenic (octadecatrienoic) a-Linolenic (octadecatrienoic)

18 18 18

2 3 3

(n-6) (n-6) (n-3)

18 : 2(n-6) 18 : 3(n-6) 18 : 3(n-3)

Highly unsaturated fatty acids (HUFAs) Arachidonic (eicosatetraenoic) EPA (eicosapentaenoic) DHA (docosahexaenoic)

20 20 22

4 5 6

(n-6) (n-3) (n-3)

20 : 4(n-6) 20 : 5(n-3) 22 : 6(n-3)

12 : 0 16 : 0 18 : 0

Table 9.7 Selected fatty acids (as % total fatty acids) in a terrestrial animal fat, and some ﬁsh and vegetable oils. From data given by Macrae et al. (1993), Hertrampf & Piedad-Pascual (2000) and Stickney (2000). Fatty acid

Saturates 16 : 0 18 : 0 MUFA 16 : 1 18 : 1 20 : 1 22 : 1 PUFA and HUFA 18 : 2(n-6) 18 : 3(n-3) 20 : 5(n-3) 22 : 6(n-3)

Fat oil source Beef tallow

Anchovy

Herring

Capelin

Rape/canola

Soya

Palm

25 20

19.5 3.5

14.5 1

11 1.5

5 2

10 4

43.5 4.5

5 40

9 15 2.5 1.5

6 10 15.5 22

9 14 13 10.5

0.5 54 1 1

0.5 23 0.5

0.5 36.5

2 2

1 0.5 18 11

1.5 1.5 5 6.5

1 1 10 10

23 11

51 7

9 0.5

Linseed 5.5 4

20

12.5 53.5

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Table 9.8 Chemical composition (as % wet weight) and fatty acid classes (as % total fatty acids) in the ﬁllet (muscle) of some cold-water ﬁsh species. SFAs, saturated fatty acids; MUFAs, monounsaturated fatty acids. Adapted from Lie et al. (1994).

Moisture (%) Dry matter (%) Protein Lipid

Atlantic salmon Atlantic cod Wolf-ﬁsh Salmo salar Gadus morhua Anarhichas lupus

Atlantic halibut Turbot Hippoglossus Scophthalmus maximus hippoglossus

69 31 18 10

78 22 19 2.5

72 28 16 10

79 21 16 2.5

22 39 27 7

16 72 8 2

23 32 36 6

80 20 18 0.5

Fatty acid classes (as % fatty acids) SFAs 23 22 MUFAs 50 18 (n-3) 20 54 (n-6) 5 3

as a depot for storage TAGs. Fish which store large amounts of TAG within the muscle will tend to have higher proportions of SFAs and MUFAs in their muscle lipids than those species in which the muscle lipids are primarily structural, i.e. the phospholipids incorporated into cell membranes (Fig. 9.3). Over 75% of the PUFAs and HUFAs present in the lipids of marine ﬁsh species will usually be of the (n-3) series, and (n-6) fatty acids usually constitute less than 20% (Tables 9.7 and 9.8) (Cowey, 1993; Takama et al., 1994; Bell et al., 1999). In contrast, the PUFAs and HUFAs present in most plant oils, and other lipid sources of terrestrial origin, are generally of the (n-6) series, with usually less than 20% of the PUFAs and HUFAs being of the (n-3) series (Table 9.7) (Macrae et al., 1993; Hertrampf & Piedad-Pascual, 2000). Thus, not only do terrestrial and marine lipids differ in the degrees of saturation of the fatty acids, there are also differences in the unsaturated fatty acids typical of food chains in these environments. The (n-6) series fatty acids are typically found in terrestrial and freshwater environments. On the other hand, fatty acids of the (n-3) series are characteristic of marine ecosystems, and the unsaturated fatty acid proﬁles of marine phytoplankton and zooplankton (i.e. with a dominance of 18:3(n-3), 20:5(n-3) and 22:6(n-3) amongst the PUFAs and HUFAs) tend to be reﬂected at higher levels in marine food chains (Sargent et al., 1989; Drevon, 1992; Lands, 1992; Macrae et al., 1993; Uauy-Dagach & Valenzuela, 1996). This conservative transfer of some fatty acids in the neutral storage lipids forms the basis of the use of lipids as biomarkers in studies aimed at elucidating the foraging ecology and diets of organisms at various trophic levels within aquatic food chains (Sargent et al., 1989; Kirsch et al., 1998). The phospholipids are the major group within the complex lipids. Phospholipids are esters that contain glycerol, fatty acids and phosphoric acid, along with certain other compounds. Phosphatidylcholine, phosphatidylethanolamine, phosphatidylinositol and phosphatidylserine have fatty acids esteriﬁed to carbon positions 1 and 2 of the glycerol backbone, and the phosphate group esteriﬁed in position 3. These phospholipids combine hydrophilic

380 Culture of cold-water marine ﬁsh

Figure 9.3 Schematic representation of fatty acid transformations and metabolism in ﬁsh. SFA, saturated fatty acid; MUFA, monounsaturated fatty acid; PUFA, polyunsaturated fatty acid; HUFA, highly unsaturated fatty acid.

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(water-loving) and hydrophobic properties, making them surface-active and allowing them to play an important role as emulsifying agents in biological systems. The surface-active nature of the phospholipids explains their function as components of biological membranes. The major membrane phospholipids are phosphatidylcholine and phosphatidylethanolamine, whereas phosphatidylinositol appears to have a number of important roles in the transduction of hormonal signals through biomembranes. In ﬁsh, the two main membrane phospholipids tend to be rich in the fatty acids 20:5(n-3) and 22:6(n-3), whereas the (n-6) series fatty acid 20:4(n-6) is present in large amounts in phosphatidylinositol (Sargent et al., 1989, 1995; Takama et al., 1994; Sérot et al., 1998). The fatty acid composition of the diet of farmed ﬁsh will have some inﬂuence upon the fatty acid composition of the phospholipids (Bell, 1998; Bell et al., 1999), and changes in environmental conditions may also lead to some modiﬁcation in the fatty acid composition. For example, exposure to low temperature often leads to an increase in the incorporation of both MUFAs and HUFAs into membrane phospholipids at the expense of SFAs. Animals, including ﬁsh, have the ability to modify the fatty acids they obtain from their diet via chain-elongation and desaturation pathways, and fatty acids may also be used to fuel energy metabolism (see Fig. 9.3) (Sargent et al., 1989; Henderson, 1996). There may be boxidation of fatty acids followed by recycling of the carbon skeleton into de novo lipid synthesis, and there appears to be a preference for SFAs and MUFAs (e.g. 16:0, 18:1 and 22:1) as fuels for energy metabolism via the b-oxidation pathway (Henderson, 1996; Cunnane, 2000). The latter seems to be most pronounced when there is a readily available dietary supply of SFAs and MUFAs, and this results in conservation of PUFAs and HUFAs (Henderson, 1996). In addition, the activities of fatty acid desaturation and elongation enzymes may be inﬂuenced by the fatty acid composition of the diet (Henderson, 1996; Tocher et al., 2000, 2001; Bell et al., 2001). However, ﬁsh and other animals seem to be incapable of synthesising the fatty acids of the (n-3) and (n-6) series de novo, so these fatty acids are essential nutrients that must be supplied in the diet (for a discussion, see Cunnane, 2000). Thus, terrestrial animals require a dietary supply of both (n-6) and (n-3) fatty acids, and these are usually supplied as 18:2(n-6) and 18:3(n-3). The majority of terrestrial animals are able to convert and chain-elongate these fatty acids into the longer-chain HUFAs that are incorporated into the phospholipids (Fig. 9.4) (exceptions seem to be some carnivores, such as cats and lions), so both 18:2(n-6) and 18:3(n-3) have traditionally been considered to have essential fatty acid (EFA) properties (Connor et al., 1992; Drevon, 1992; Cunnane, 2000). From the general discussion of lipids given above, it should be clear that ﬁsh lipids contain relatively high proportions of HUFAs of the (n-3) series, principally docosahexaenoic acid (DHA; 22:6(n-3)) and eicosapentaeonic acid (EPA; 20:5(n-3)) (Takama et al., 1994), and it has been established that ﬁsh generally have a high dietary requirement for fatty acids of the (n-3) series (Sargent et al., 1989, 1995; Bell, 1998). Some ﬁsh species are able to chainelongate and desaturate 18:3(n-3), so much of the EFA requirement for the (n-3) series fatty acids can be met by providing this fatty acid in the diet. Other ﬁsh species, including the marine ﬁsh species studied to date, seem to have low activities of the D5-desaturase enzyme which is necessary for the formation of EPA and DHA from their precursor, 18:3(n-3) (Fig. 9.4). Moreover, the ability of marine ﬁsh to convert EPA to DHA seems to be limited (Tocher et al., 1992; Bell, 1998). The formation of DHA (22:6(n-3)) from EPA (20:5(n-3)) is

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Enzyme

D9 desaturase D6 desaturase elongase D5 desaturase elongase elongase D6 desaturase (b oxidation)

Fatty acid series (n-7)

(n-9)

16 : 0 Ø 16 : 1(n-7) Ø 16 : 2(n-7) Ø 18 : 2(n-7) Ø 18 : 3(n-7) Ø 20 : 3(n-7)

18 : 0 Ø 18 : 1(n-9) Ø 18 :2(n-9) Ø 20 :2(n-9) Ø 20 :3(n-9) Ø 22 :3(n-9)

(n-6)

(n-3)

18 : 2(n-6) Ø 18 :3(n-6) Ø 20 :3(n-6) Ø 20 :4(n-6) Ø 22 :4(n-6) Ø 24 :4(n-6) Ø 24 :5(n-6) Ø 22 :5(n-6)

18 : 3(n-3) Ø 18 :4(n-3) Ø 20 :4(n-3) Ø 20 :5(n-3) Ø 22 :5(n-3) Ø 24 :5(n-3) Ø 24 :6(n-3) Ø 22 :6(n-3)

Figure 9.4 Pathways for chain elongation and desaturation of fatty acids. The D6 desaturase enzyme has a preference for unsaturated fatty acids as substrate. In the absence of 18:3(n-3) and 18:2(n-6), 18:1(n-9) is desaturated and elongated, leading to an accumulation of long-chain fatty acids of the (n-9) series in tissues (this can be used as an indication of essential fatty acid deﬁciency). Carnivorous marine ﬁsh species seem to have low D5 desaturase activity and have a reduced ability to use 18C fatty acids as precursors for synthesis of (n-3) and (n-6) HUFAs.

thought to operate via D6-desaturation of 24:5(n-3) to 24:6(n-3) followed by chainshortening to 22:6(n-3) (Buzzi et al., 1997). This sequence of reactions seems to be too slow to supply the requirement of DHA for incorporation into the membrane lipids of rapidly growing larval and juvenile marine ﬁsh. Consequently, both EPA and DHA may need to be supplied pre-formed via the diet to meet the requirement for (n-3) HUFAs. Marine ﬁsh are generally considered to require 0.5–1% of the dry weight of their diet as (n-3) HUFA. While the dietary requirement for (n-3) HUFAs has been widely recognised, less attention has been paid to the possibility that marine species may also need to be provided with (n-6) HUFA via the diet (Castell et al., 1994; Bell et al., 1995; Bell, 1998; Furuita et al., 2000). In particular, there may be a dietary requirement for arachidonic acid (AA; 20:4(n-6)), because of the central role this fatty acid plays as a precursor for the formation of some of the biologically active eicosanoids (Drevon, 1992; Lands, 1992; Sardesai, 1992; Stickney, 2000). Animal fats and plant oils have been included in formulated feeds for several ﬁsh species (Hertrampf & Piedad-Pascual, 2000), but these would not seem to be particularly suitable lipid sources for cold-water marine ﬁsh. Fats from terrestrial animals, such as beef tallow, pork lard, mutton fat and poultry fat, contain high proportions of SFAs, and are poor sources of EFAs of the (n-3) and (n-6) series (see Table 9.7). Oils derived from the seeds of a number of plant species, such as soya and rape, contain quite high levels of unsaturated fatty acids of the (n-6) series, although linseed oil contains appreciable quantities of both 18:3(n-3) and 18:2(n-6) (see Table 9.7). Fish of many species can chain-elongate 18C fatty acids of the

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(n-3) and (n-6) series to HUFAs. These ﬁsh can utilise plant oils as a source of EFAs provided that the oils contain sufﬁcient 18:3(n-3), but cold-water marine ﬁsh seem to have a limited ability to chain-elongate 18 C fatty acids to the long-chain HUFAs, and these must be supplied pre-formed in the diet. The best sources of the long-chain HUFAs of the (n-3) series are marine ﬁsh oils (see Table 9.7); marine ﬁsh oils have traditionally been used as a lipid source in feeds for intensively farmed ﬁsh, but their inclusion in feeds for cold-water marine ﬁsh species would seem to be mandatory. Nevertheless, it may prove possible to meet the EFA requirements of cold-water marine ﬁsh by providing them with mixtures of oils derived from marine ﬁsh and oilseeds. In addition, it should be possible to modify the fatty acid compositions of plant oils by selective breeding or by the use of transgenic technologies. By using oils from several sources, it may be possible to formulate feeds which have similar fatty acid proﬁles to those of the natural prey of the ﬁsh. This may be considered to be important given that the fatty acid composition of ﬁsh lipids, particularly that of the storage TAGs, tends to reﬂect the composition of the diet (Lie et al., 1986; Haard, 1992; Kirsch et al., 1998; Sérot et al., 1998, 2001; Bell et al., 1999, 2001; Stickney, 2000; Jobling, 2001b; Morais et al., 2001), even though there will inevitably be some metabolism and modiﬁcation of the fatty acids obtained from the diet (see Fig. 9.3) (Sargent et al., 1989; Henderson, 1996).

9.5 Carbohydrates Carbohydrates are generally deﬁned as compounds that contain carbon, oxygen and hydrogen, and have the general formula CX (H2O)Y (e.g. glucose; see Fig. 9.2), although some compounds classiﬁed as carbohydrates do not comply with this general description. For example, some compounds not showing the 2:1 ratio of hydrogen to oxygen have many of the chemical properties of carbohydrates, e.g. deoxyribose (C5H10O4), and a number of compounds that contain small proportions of nitrogen and sulphur in addition to carbon, hydrogen and oxygen have characteristics that are considered typical for this class of nutrients (Macrae et al., 1993). Carbohydrates may be classiﬁed as simple sugars (monosaccharides) and their derivatives, oligosaccharides and polysaccharides. Monosaccharides occur naturally in only very small amounts, the simple sugars usually being polymerised into larger oligosaccharide (2–10 monosaccharide units) or polysaccharide molecules. Most polysaccharide molecules contain several hundred to several thousand monosaccharide residues. In nature, the carbohydrates are usually present as long-chain polysaccharides, the polysaccharides having either a structural or energy-storage function (for a general discussion, see Macrae et al., 1993). The main component of the dry matter of terrestrial plants is carbohydrate, but in contrast to plants, the carbohydrate content of the animal body is low. One of the main reasons for the difference in carbohydrate content between plants and animals is that the cell walls of terrestrial plants are made up of carbohydrates, largely cellulose, whereas the walls of animal cells are composed of protein and lipid. Furthermore, plants store most of their energy in the form of carbohydrates, such as starch, whereas an animal’s main energy store is in the form of lipid. Thus, the natural prey of carnivorous ﬁsh contain limited amounts of carbo-

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hydrate, so carbohydrate will form only a very small proportion of the diet of carnivorous species. However, carnivorous ﬁsh that consume invertebrate prey, such as crustaceans and polychaetes, will ingest a certain amount of chitin, and all carnivores will ingest small amounts of glycogen. The major carbohydrates of terrestrial plants, cellulose and starch, are both homoglycans that are broken down to their constituent glucose monomer units on complete hydrolysis. Glucose is a hexose sugar, i.e. has six carbon atoms (see Fig. 9.2). The main difference between cellulose and starch is the nature of the chemical bonds between the adjacent glucose monomer units. Cellulose, which is the most abundant organic compound in the biosphere, comprises a linear chain of glucose residues joined by b-1,4 linkages (i.e. a b link between C1 of a glucose unit and C4 of its neighbour). Starch, on the other hand, is made up of a long chain of glucose units joined by a-1,4 linkages, that may be either unbranched (amylose) or branched at an a-1,6 linkage (amylopectin). In other words, starch is a mixture of two glucose polymers, and both the molecular weights and the proportions of the two molecules making up the mixture differ between plant species. This imparts different properties to starches derived from different plant sources (Macrae et al., 1993; Hertrampf & Piedad-Pascual, 2000). Similarly, the different forms of chemical bonding between the glucose units making up starch and cellulose give rise to differences in physical and chemical properties between the two, including the ease with which they can be digested by animals. Animals produce a digestive enzyme, amylase, which attacks the a linkages in starch, but the b linkages in cellulose are more resistant to digestion. Cellulose is usually considered to be highly indigestible for monogastric animals, including carnivorous ﬁsh, but there may be some breakdown of cellulose in the gut due to bacterial activity. Glycogen is the main storage carbohydrate of animals, and it is stored mostly in the liver and muscles. It is similar in structure to amylopectin, the major difference being that glycogen has relatively large numbers of a-1,6 linkages in the molecule. Chitin is found in the exoskeletons of invertebrates such as insects, crustaceans and polychaetes, where it acts as a strengthening agent. The chitin molecule is composed of long chains of an amino-sugar, N-acetylglucosamine, which means that the chitin molecule contains atoms of nitrogen in addition to carbon, hydrogen and oxygen. Structurally, chitin is a close relative of cellulose in that the monomer units are joined to each other by b-1,4 linkages. In essence, the hydroxyl (–OH) group attached to the carbon atom at position 2 in the glucose residues of cellulose is replaced by an N-acetylamino (–NHCOCH3) group in the N-acetylglucosamine units that make up chitin. Despite the fact that carbohydrate makes up only a very limited part of the natural diet of carnivorous ﬁsh, some plant feedstuffs that contain high proportions of carbohydrate will be included in formulated feeds for these species (Hertrampf & Piedad-Pascual, 2000; Stickney, 2000; Jobling et al., 2001b). Carbohydrate may be considered a cheap energy source, but the prime reason for the inclusion of carbohydrates in formulated feeds is for their binding properties. The most usual carbohydrate sources included in formulated feeds for cold-water carnivorous ﬁsh are cereals (e.g. wheat), maize, potato and their by-products, all of which contain starches (Hertrampf & Piedad-Pascual, 2000; Stickney, 2000). During feed production, the mixture of ingredients will usually be subjected to a combination of moisture, heat, pressure and mechanical shear, and this treatment causes the starch granules

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to swell and rupture, a process known as gelatinisation (Hardy, 1989; Stickney, 2000; Jobling et al., 2001b). Gelatinisation, in addition to imparting thickening and gel-forming properties, also improves the ease with which the starch can be digested. Several other carbohydrates, in addition to starch, may be used as thickening or gelling agents. Those most commonly used in ﬁsh feeds are alginate, cellulose ethers and derivatives of lignin and hemicelluloses (Stickney, 2000). Alginic acid is a component of algal cell walls, and it represents up to 40% of the dry weight of brown seaweeds (Phaeophyceae). It is extracted commercially from kelps such as Macrocystis pyrifera, and is sold as the sodium alginate salt. The alginate forms a gel in the presence of calcium ions, and this makes it an effective binding agent in feeds. The cellulose ethers include carboxymethyl cellulose (CMC), methyl cellulose and ethyl cellulose, all of which are derivatives formed when cellulose is treated chemically under strongly alkaline conditions. CMC is formed when cellulose reacts with chloracetic acid, methyl cellulose is formed by reacting alkali cellulose with methyl chloride, and ethyl cellulose is produced by reacting alkali cellulose with ethyl chloride. CMC, which is indigestible, is the cellulose ether most commonly used as a binding agent in ﬁsh feeds. Carnivorous ﬁsh species have a low capacity to digest complex carbohydrates, even when they are present in feeds at low concentrations. Furthermore, their capacity to digest complex carbohydrates decreases with increasing concentration in the feed (Hemre et al., 1989). In addition, the inclusion of some complex carbohydrates, such as CMC, in feeds may reduce the capacity of the ﬁsh to digest and absorb protein and lipid (Yamamoto & Akiyama, 1995). However, several carnivorous ﬁsh species can utilise gelatinised starch when it is present in feeds at low concentrations, although there are large interspecies differences (Hemre et al., 1989, 1995; Wilson, 1994). As a consequence of this, it is generally believed that feeds for marine and cold-water carnivorous ﬁsh species should be formulated to contain relatively low levels (<20%) of digestible carbohydrate (Wilson, 1994).

9.6 Micronutrients: Vitamins and Minerals As the term implies, micronutrients are those dietary components that are required by animals in relatively small amounts for the maintenance of optimal health, growth and reproTable 9.9 Overview of the major essential minerals (macro-minerals), trace and ‘ultra-trace’ elements required by animals. Major essential minerals (required in relatively large amounts) Electrolytes Sodium, potassium, chlorine Skeletal (bone) minerals Calcium, phosphorus, magnesium Sulphur (constituent of sulphur-containing amino acids methionine and cysteine) Trace elements (required in small quantities) Zinc, iron, silicon, manganese, copper, ﬂuorine, iodine, chromium, cobalt ‘Ultra-trace’ elements (only required in very small amounts) Boron, molybdenum, selenium, nickel, vanadium, arsenic, tin, aluminium

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Table 9.10

Classiﬁcation of vitamins, and related substances, and examples of their biological functions.

Fat (lipid)-soluble vitamins Retinol (vitamin A) Cholecalciferol (vitamin D) Tocopherols (vitamin E) Menadione (vitamin K)

Normal growth, vision, reproduction Calcium and phosphate metabolism/regulation Antioxidant, muscle and RBC function Blood clotting

Water-soluble vitamins Thiamin (vitamin B1) Riboﬂavin (vitamin B2) Niacin (nicotinic acid) Pantothenic acid (vitamin B5) Pyridoxine (vitamin B6) Cyanocobalamin (vitamin B12) Biotin (vitamin H) Folacin (folate) Ascorbic acid (vitamin C)

Energy metabolism, nerve function Cellular energy metabolism Energy metabolism, nerve function Energy metabolism, nerve function Protein metabolism and utilisation Nerve function, RBC formation and function Fatty acid synthesis, glucose metabolism Embryonic development, gut function Antioxidant, collagen synthesis, immune responses

‘Vitamin-like’ substances Carotenoids Inositol (myo-inositol) Choline Carnitine

Table 9.11 elements.

Antioxidants, provitamin A Cell membrane phospholipids, chemical signal transmission Fatty acid metabolism, cell membrane phospholipids, neurotransmission functions Lipid/fatty acid metabolism

Biological functions of the essential minerals, with some examples of the roles of the different mineral

Biological or physiological role

Examples

Ionic regulation (electrolytes) Acid–base balance Structural functions Bone/skeletal tissue Cell membranes Nerve impulse transmission and muscle contraction Respiratory pigment (haemoglobin) Component of hormones Enzyme structure and function Component of enzyme Co-factor or component of co-factor Activator or regulator

Sodium, potassium, chlorine Calcium, sodium, chlorine Calcium, phosphorus, magnesium, sulphur

Calcium, sodium, potassium Iron Iodine, sulphur Zinc, selenium, cobalt, manganese, chromium, vanadium

duction. The micronutrients encompass essential minerals (Table 9.9) and the vitamins and related compounds (Table 9.10) (for a general discussion, see Halver, 1989; Lall, 1989; Macrae et al., 1993; Stickney, 2000). The essential minerals fulﬁl a variety of functions: some, such as calcium, magnesium and phosphorus, are structural components of the skeleton, others, such as sodium, potassium and chlorine, function in acid–base balance and in the maintenance of cell homeostasis, and several, including zinc and copper, act as co-factors in biochemical reactions involving a range of enzyme systems (Table 9.11). Vitamins are complex organic compounds that are required in trace amounts, and deﬁciency symptoms

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will occur if a single vitamin is omitted from the diet of a species that requires it. Several of the vitamins act as co-enzymes, but others do not have a single role and may play a part in several vital functions (Table 9.10). Determination of the vitamin and mineral requirements of ﬁsh is difﬁcult and time-consuming, and quantitative requirements are known for very few species. It may be difﬁcult to decide upon the correct criterion to be used when assessing these requirements. For example, if insufﬁcient quantities of a vitamin are included in the diet, deﬁciency symptoms or poor growth may be observed. These are the criteria generally used in requirement studies. However, given the involvement of the micronutrients in various aspects of general health and reproduction, it is open to question whether the development of deﬁciency symptoms or reduced growth are adequate criteria to use for the determination of micronutrient requirements. Given that the requirements are poorly known, it is usual for feeds to be fortiﬁed with vitamin and mineral mixtures under the assumption that the supplementation will be adequate to meet the requirements for the different micronutrients (Hardy, 1989; Stickney, 2000). The minerals required by animals for normal metabolism and growth can be broadly divided into the major, trace and ‘ultra-trace’ minerals, depending upon the quantities required (see Table 9.9). As the term implies, the major minerals are required in relatively large quantities. The major minerals include calcium, sodium, potassium, magnesium, phosphorus, chlorine and sulphur. Trace and ‘ultra-trace’ minerals are those required in lesser amounts, and include iron, iodine, manganese, copper, cobalt, zinc, selenium, molybdenum, chromium and ﬂuorine (for general reviews, see Lall, 1989; Schwarz, 1995). Not all the elements required by ﬁsh need to be supplied via the diet, because many of the elements that the ﬁsh need for survival and growth will be contained in solution in their ambient medium. This will be particularly true for marine ﬁsh, in which there will be an inﬂux of some minerals, such as sodium, chlorine and magnesium, from the environment, and excesses must be actively excreted. Marine ﬁsh may also be at risk of becoming hypercalcaemic due to the inﬂux of calcium from the surrounding medium; vitamin D appears to be involved in the regulation of the calcium and phosphate balance in ﬁsh, and may serve to reduce the absorption of calcium from the environment in marine species (Graff et al., 1999). The amount of a mineral present in a feed does not necessarily reﬂect the amount that is available for absorption into the body. For example, plant feedstuff components such as ﬁbre, phytates and polyphenolic compounds (tannins) may substantially reduce the bioavailability of minerals (Selle et al., 2000; Francis et al., 2001). The bioavailability of a mineral may also be inﬂuenced by the concentrations of other minerals present in the feed. For example, the bioavailability of manganese and zinc may be reduced if the dietary concentration of calcium is high. High phosphate concentrations may reduce the absorption of iron, and high dietary iron concentrations may reduce the bioavailability of zinc. Further, the chemical form in which a mineral occurs in the diet may inﬂuence bioavailability. For example, the bioavailability of the phosphorus present in ﬁsh meal, legumes and oilseed meals may be low owing to the chemical form in which the phosphorus is bound. Most of the phosphorus present in ﬁsh meals is derived from bone ash, and is bound as tricalcium phosphate and hydroxyapatite. Both of these chemical forms of phosphorus have relatively low bioavailability, with only 20–60% being absorbed from the gastrointestinal tract (Riche & Brown, 1996). On the other hand, the legumes and oilseed meals may contain most of their phosphorus bound in

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phytate (Selle et al., 2000), and phytate phosphorus has low bioavailability, e.g. 10–50%, for ﬁsh because they lack the digestive enzyme phytase (Riche & Brown, 1996). The vitamins have been divided into two groups based on their solubility characteristics, i.e. water-soluble and fat-soluble vitamins (see Table 9.10). These characteristics affect the distributions of the different vitamins in feedstuffs, and also inﬂuence the way in which the vitamins are absorbed from the gastrointestinal tract, transported and stored in the body tissues. The fat-soluble vitamins—A, D, E and K—are absorbed from the gut along with the lipid components of the diet, and may be stored in body tissues. This means that it may take some time before deﬁciency symptoms appear when animals are fed diets which are deﬁcient in fat-soluble vitamins. The storage of the fat-soluble vitamins within the body also leads to the possibility of toxic effects resulting from hypervitaminosis if diets containing excessive amounts of fat-soluble vitamins are fed (Halver, 1989; Stickney, 2000). Unlike the fat-soluble vitamins, the water-soluble vitamins are not stored in appreciable quantities in body tissues, so they must be supplied in the diet on a regular basis. Further, because excesses of the water-soluble vitamins are readily excreted, it is unlikely that toxicity will arise. Amongst the water-soluble vitamins, eight (the B-complex vitamins) are required in minute amounts. The B-complex vitamins function as co-enzyme metabolic catalysts, and there are profound aberrations in metabolism if insufﬁcient amounts of these water-soluble vitamins are present in the tissues. Ascorbic acid (vitamin C) is required in larger amounts, and is sometimes referred to as a macrovitamin. A number of other dietary compounds have important biological functions that are akin to those of the vitamins, and these compounds may be considered ‘vitamin-like’ (see Table 9.10) (for a general review of vitamin nutrition, see Halver, 1989). The fact that several of the vitamins are found in high concentrations in gonadal tissue points to them having important roles in reproduction (Sandnes, 1991; Albrektsen et al., 1994; Hemre et al., 1994; Blom & Dabrowski, 1996; Sandnes et al., 1998), but the exact roles of these vitamins in oocyte development and maturation, and their importance for embryonic and larval development, remain to be elucidated. High concentrations of vitamin C (ascorbic acid), vitamin E and some B-complex vitamins have been recorded in ovarian tissue. Vitamin C is involved in the synthesis and metabolism of gonadal sex steroids, but there is also deposition in the eggs (Sandnes, 1991; Blom & Dabrowski, 1996). The vitamin C deposited in the eggs may play an important role during early development since it functions as a co-factor in the synthesis of collagen, and also has antioxidant properties. Vitamin E also has antioxidant properties, and the effects of the two vitamins appear to be synergistic (Chan, 1993; Jacob, 1995; Benzie, 1996). The eggs of teleost ﬁshes contain lipids in the yolk, and the eggs of some species also contain oil globules. The lipoprotein yolk lipids are primarily phospholipids rich in the (n-3) HUFAs (Sargent et al., 1989; Wiegand, 1996; Bell, 1998; Furuita et al., 2000), and the (n-3) HUFAs are particularly susceptible to peroxidative damage (Benzie, 1996). Thus, the accumulation of high concentrations of vitamins C and E in the ovary during the development of the oocytes and eggs (Sandnes, 1991; Hemre et al., 1994; Blom & Dabrowski, 1996) may serve to mitigate the risk of peroxidation of the lipoprotein (n-3) HUFAs. During the course of the past couple of decades, there has been a focus on the roles of various micronutrients, both vitamins and minerals, in the protection of tissues from oxida-

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tive damage. It is customary to think of oxygen as a life-giving molecule upon which animals rely for the metabolism of macronutrients, to harness nutrient energy to perform work. However, this view ignores the fact that reactions involving oxygen can also lead to damage of vital biological macromolecules, such as the fatty acids of cellular membranes, various proteins and nucleic acids (Benzie, 1996). The protection of the cellular biological macromolecules against damage caused by activated oxygen is complex, involving an array of agents. Certain micronutrients, which may be termed ‘antioxidant nutrients’, occupy central positions in the protective mechanism (Hsieh & Kinsella, 1989; Chan, 1993; Macrae et al., 1993; Jacob, 1995). These ‘antioxidant nutrients’ include the trace minerals selenium, zinc, copper and manganese, the vitamins E (tocopherols) and C (ascorbic acid), and carotenoids. The antioxidants may be broadly classiﬁed into two groups: primary, or chain-breaking, antioxidants, which react with free radicals to convert them into more stable products, and secondary, or preventative, antioxidants, which retard autoxidation by a variety of mechanisms. The chain-breaking antioxidants are usually compounds that donate a hydrogen atom to the free radicals to produce more stable products, thereby hindering the propagation of the autoxidative chain reaction. As they exert their antioxidant activity by acting as donors, the chain-breaking antioxidants are consumed in performing their protective function. Antioxidants of this type usually have a phenolic ring in their molecular structure, and they include the tocopherols and ﬂavonoids. The secondary antioxidants usually do not have antioxidant activity per se, but exert their effects either via improving the effectiveness of the primary antioxidants, or by inhibiting the effects of potential pro-oxidants. The ﬁrst step in the oxidative reaction chain, i.e. initiation, involves the formation of a free radical from a biological macromolecule via removal of a hydrogen atom (Fig. 9.5) (Benzie, 1996; St Angelo, 1996). For example, the hydrogen atom may be removed from the macromolecule by reaction with a hydroxyl radical (• OH), the superoxide anion (O2•) may be the source of the oxidising power, and other oxidants, such as hydrogen peroxide (H2O2), can also serve to initiate the reactions. As such, the formation of free radicals may be eliminated or controlled by the removal of superoxide anions and other oxidants. This removal is carried out by enzyme systems; O2• is removed by superoxide dismutases (SODs), and H2O2 is removed by the actions of glutathione peroxidase and catalase. The mitochondrial SOD is a manganese-containing enzyme, whereas the cytoplasmic SOD depends upon copper and zinc for its activity, and glutathione peroxidase is a selenium-dependent enzyme. Thus, these enzymes depend upon trace minerals for their activity, and a deﬁciency of these ‘antioxidant nutrients’ may lead to a failure in the ﬁrst line of defence against the oxidative damage of biological macromolecules. Propagation of free radical oxidation occurs via reactions that consume oxygen and yield new free radicals that can initiate further reactions, thereby setting up a chain. The chain reaction may, however, be quenched via the donation of a hydrogen atom from a chainbreaking antioxidant, such as vitamin E (Fig. 9.5). Thus, the chain-breaking antioxidants act as a second line of defence against oxidative damage to cellular components. The tissue antioxidant system comprises several other components, including glutathione and melatonin, that are efﬁcient scavengers of free radicals, and metal-binding proteins, metallothioneins, that sequester iron and copper ions that may induce peroxidation reactions (Hsieh & Kinsella, 1989; Chan, 1993; Jacob, 1995; Benzie, 1996).

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Figure 9.5 Schematic diagram indicating the reactions involved in the oxidation of food components (e.g. unsaturated fatty acids), and the points of intervention of the various antioxidants. O2•, superoxide anion; R•, a free radical; ROO•, a peroxy radical; ROOH, a peroxide.

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The tocopherols (vitamin E and related compounds) provide antioxidant protection in the lipid phase, e.g. for the HUFAs of cell membranes, and ascorbic acid (vitamin C) is an important water-soluble antioxidant. The antioxidant properties of ascorbic acid and tocopherols appear to be synergistic, with the ascorbic acid seeming to play a role in the regeneration of tocopherols rather than acting directly to trap free radicals. Carotenoids are thought to provide antioxidant protection to lipid-rich tissues, but this protection may also result from interaction with the tocopherols rather than directly. Thus, there is evidence of a series of interactions between carotenoids, tocopherols and ascorbic acid, and the ﬁsh must be provided with a sufﬁcient dietary supply of these antioxidants to mitigate the risk of peroxidation of the tissue HUFAs.

9.7 Feed Types and Formulations Feed formulations used in intensive and semi-intensive culture of marine ﬁsh differ markedly in the proportions of the ingredients used in their manufacture, and in the manufacturing processes themselves. However, in broad terms three general categories of feeds are recognised: feeds are categorised according to their moisture content as wet (ca. 50–70%), moist (ca. 30–40%) or dry (less than 10%) (Hardy, 1989; Jobling, 1994: Goddard, 1996; Tucker, 1998; Stickney, 2000; Jobling et al., 2001b). Commercially produced dry pellet feeds are the ones most widely used in intensive ﬁsh farming. Nevertheless, whole or chopped, industrial (‘trash’ or ‘forage’) ﬁsh species (e.g. herring, capelin, mackerel, blue whiting, argentine and sandeel) (see Table 9.4), or wet and moist feeds manufactured on the farm site, are frequently used to feed certain marine species. Whilst some farmers feed their ﬁsh on whole or chopped ‘trash’ ﬁsh, the widespread use of this type of feed is limited by a number of factors. Although the purchase price of ‘trash’ ﬁsh may be low, the ﬁsh must be stored frozen to prevent rapid deterioration, and maintaining a freeze-storage facility on the farm site will substantially increase the real ﬁnal costs of the feed. Even when ‘trash’ ﬁsh are stored frozen, they will continue to undergo some deterioration, and the length of time the ﬁsh can be held under such conditions is limited to a few weeks. In addition, problems of nutritional imbalance may arise if there is reliance on a single, or a limited number of, ‘trash’ ﬁsh species as a feed source. The ‘trash’ ﬁsh species used as feed may differ widely in nutritional content, and some are pelagic ﬁsh with a high, and seasonally variable, lipid content (see Table 9.4) (Hislop et al., 1991; Takama et al., 1994; Van Pelt et al., 1997; Payne et al., 1999; Anthony et al., 2000; Pedersen & Hislop, 2001). Thus, the nutritional quality of the pelagic ﬁsh species may vary considerably depending upon when they are caught, and how they are handled and stored after capture. In addition to potential problems linked to seasonal variations in lipid content and rancidity, the ﬂesh of some of the species, such as herring, Clupea harengus, and mackerel, Scomber scombrus, contains high levels of the enzyme thiaminase. If not destroyed by heat treatment, this enzyme can lead to depletion of the vitamin thiamin. One consequence of this may be the development of thiamin deﬁciency in farmed ﬁsh fed for prolonged periods on unsupplemented feeds comprising a large proportion of herring or mackerel. It is also possible that diseases or parasites may be transmitted to the farmed ﬁsh when unpasteurised

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Table 9.12 Examples of wet and moist feed mixes that may be suitable for cold-water marine ﬁsh. Binder mix may contain cereal and potato products and starches, alginates, carboxymethylcellulose, and various plant/algal gums. Based upon information given in Hardy (1989), Jobling et al. (1991, 1994), Goddard (1996), Tucker (1998) and Hertrampf & Piedad-Pascual (2000). Ingredients (g kg-1) 1 Wet ingredients Trash ﬁsh Herring Capelin Fish off-cuts Fish silage Squid Dry ingredients Fish meal Dried ﬁsh solubles Shrimp/prawn meal Wheat middlings Vitamins/minerals Binder mix

600 240

4

300

290

350

400

5

6

150

220

100

100

530

430

5 60

80 20 40

42 20 40

50

80

148

47 34 9 4

31 42.5 14 5.5

28 35.5 21 5.5

ca. 17

ca. 18.5

45 20

50

150

270 50

50 5 100 35

Composition (%) Moisture Protein Lipid Ash

62 17 9.5 4

Protein as % dry matter mg protein: kJ energy (P : E)

3

565

Fish oil

Energy (kJ g-1)

2

ca. 9 45 19

5 250

53 25 7 5 ca. 10.5 53 24

10 100

48 29 5 7.5 ca. 10.5 58 27

ca. 12.5 64 27

62 25

49 19

‘trash’ ﬁsh are used as feed, although frozen storage may be sufﬁcient to destroy many parasites. Some of the problems encountered in using raw ﬁsh as feed can be eliminated, or at least substantially reduced, by using formulated wet or moist feeds. Wet (ca. 50–70% moisture) and moist (ca. 30–40% moisture) feeds are made by combining wet ingredients and dry meal mixes in different proportions, ranging from ca. 90 : 10 (wet : dry) for a wet feed to 50 : 50 for a moist feed. The wet ingredients comprise fresh, pasteurised or frozen ‘trash’ ﬁsh, together with squid, wastes from the processing of ﬁsh and crustaceans, and acid-preserved hydrolysed ﬁsh waste (silage). The dry meal mixes usually contain ﬁsh meal, vitamin and mineral premixes, and binding materials such as starches or alginates (Table 9.12). The onsite manufacture of wet or moist feeds allows advantage to be taken of cheap, locally available raw materials that can be mixed with other feed ingredients to produce a feed that is nutritionally balanced. One problem that may be encountered with wet and moist feeds is water-stability: such feeds may disintegrate rapidly, leading to loss of nutrients, feed waste and water fouling. The stability of the feed may be increased by heating pellets using electromagnetic radiation, or by using alginate (50 g kg-1) as the binder and treating the moist

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feed pellets with calcium chloride (passing them through a bath containing a 50 g l-1 calcium chloride solution). Moist pellets will be subject to bacterial and fungal attack, so antibacterial and antifungal agents will need to be added if the pellets are not used immediately, but are to be stored for a few days prior to use (Hardy, 1989; Stickney, 2000). In some countries, restrictions apply with regard to the inclusion of untreated trash ﬁsh, ﬁlleting wastes and offal in feeds prepared for farmed ﬁsh, and regulations also vary from country to country with regard to requirements for heat-treatment, pasteurisation or sterilisation of feed ingredients. Dry pellet feeds are by far the most commonly used feeds in intensive aquaculture, and the use of dry feeds is increasing in the farming of marine ﬁsh species. The widespread use of dry feeds results from the ease with which they can be distributed by manufacturers, their ease of storage and handling, and their relatively consistent quality (Hardy, 1989; Stickney, 2000). Dry feeds contain less than 10% moisture, and this means that they are easier to transport and store than are wet and moist feeds. Further, the nutritional balance of dry feeds is consistent, and these feeds also tend to be more water-stable than either wet or moist feeds. However, some species of ﬁsh appear to prefer wet or moist feeds to dry feeds, and there is circumstantial evidence that wet and moist feeds are more readily accepted by ﬁsh at low temperatures than are dry feeds. There are two main methods used in the manufacture of commercial dry pellet feeds: compressed steam pelleting and extrusion (or expansion) pelleting (Fig. 9.6) (Hardy, 1989; Stickney, 2000; Jobling et al., 2001b). Extrusion pelleting allows the manufacture of pellets with a greater range of physical and nutritional properties than does steam pelleting. Steam pelleting produces dense pellets that sink quite rapidly. The ingredients are ﬁnely ground, mixed, cooked brieﬂy (often ca. 30 s) with steam under pressure to raise the temperature to 70–95°C and the moisture to 15–18%, compressed and forced through a die. The pellets are cut, cooled, dried and may be top-dressed with oil. The mix does not expand much as it exits the die, and sinking pellets with 9–10% moisture are produced. Ingredients, compression, temperature and moisture levels can be varied to modify pellet hardness. Feed ingredients that contain starch must be included in the formulation because steam pelleting relies upon the gelatinisation of starch to bind the other ingredients together. Fibre and lipid reduce binding, and lipid levels during pelleting should be less than 10%. In extrusion processing the ingredients are ﬁnely ground, mixed and cooked (2–5 min) with steam under pressure to raise the moisture content to 20–30%, and the mixture is then forced through a die at high pressure. The steam expands as the mixture leaves the die, and the expanded pellets are cut, cooled and dried, and top-dressed with oil. There is almost complete gelatinisation of starch, so well-bound pellets can be produced even though inclusion levels of ingredients that contain starch (e.g. cereals, maize and potato meal) may be low. The expansion that occurs as the pellets leave the extruder die means that the pellets can readily absorb additional lipid applied as oil. Thus, it is possible to produce extruded pellets that contain over 25% lipid (up to ca. 40%), and very little carbohydrate. Further, by making alterations to the extrusion conditions, it is possible to inﬂuence the density of the pellet, and thereby produce pellets that either ﬂoat, or sink through the water column at different rates. Extruded pellets tend to be harder and more stable than compressed dry pellets, but require more time and expense to produce.

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Figure 9.6 Schematic representation of the steps involved in the manufacture of dry, pelleted ﬁsh feeds. From Jobling et al. (2001b).

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Table 9.13 Factors that have an inﬂuence on the stability of vitamins and vitamin-like substances. S, relatively stable under the conditions indicated; L, stability affected, with some loss or destruction. From data given in Macrae et al. (1993) and Stickney (2000). Heat

Light

Oxygen

Humidity

Acid

Alkali

Fat (lipid)-soluble Vitamin A Vitamin D Vitamin E Vitamin K

L L L S

L L L L

L L L S

L L S L

L S S L

S L L L

Water-soluble Thiamin Riboﬂavin Pyridoxine Pantothenic acid Niacin Folacin Cyanocobalamin Biotin Ascorbic acid

L L L L S L S L L

S L L S S L L S L

L S S S S L L S L

L S S L L S S S S

S S S L S L S L S

L L S L S S S L L

‘Vitamin-like’ Carotenoids Choline

L L

L S

L S

L S

S

L

Reducing agents

Trace minerals L L L L

L L L S L

S

L S L S S L L S L

S

One disadvantage with extrusion-pelleting is that processing occurs at greater temperatures, moisture levels and pressure than in conventional steam pelleting. This can lead to some nutrients becoming less available due to non-enzymatic browning (Maillard reaction). Extrusion processing may also increase the risk of destruction of some nutrients, such as heatsensitive vitamins (Table 9.13) (Camire et al., 1990; Macrae et al., 1993). In addition, the fact that extruded pellets may be top-dressed with oil to create a feed with over 25% lipid means that quite high levels of antioxidants must be added to prevent the feed becoming rancid during storage (Hardy, 1989; Stickney, 2000). The synthetic antioxidants that are added to feeds are mostly hydrogen-donating, phenolic, chain-breaking antioxidants, examples of which include butylated hydroanisole (BHA), butylated hydroxytoluene (BHT), gallic acid (pyrogallol) and the amine ethoxyquin (Hardy, 1989; Macrae et al., 1993; Stickney, 2000). The development of stable forms of some of the vitamins, such as the phosphate derivatives of ascorbic acid (Dabrowski et al., 1994; Hertrampf & Piedad-Pascual, 2000; Stickney, 2000), together with post-processing supplementation of feeds with both vitamins and antioxidants, has eliminated some of the problems associated with extrusion-pelleting techniques. The introduction of extrusion-pelleting techniques means that high-protein, high-energy feeds with reduced mineral ash, phytate and complex carbohydrate can be produced, and pellets containing up to 40% lipid can be manufactured. How far the lipid content should be increased in practice will depend upon the dietary balance between protein and energy that promotes the best growth and efﬁcient feed utilisation of the particular species, or life-history stage, being reared. For example, based on assessments of weight gain, lipid

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Culture of cold-water marine ﬁsh

deposition and feed utilisation in relation to feed costs, Morais et al. (2001) suggested that an extruded feed formulated to contain 48% protein and 16% lipid might be suitable for use in the on-growing of juvenile Atlantic cod, Gadus morhua. Protein is an expensive feed component, and the aim is to achieve a balance between dietary protein and energy that ensures that the protein is deposited as growth, rather than being used as metabolic fuel. If insufﬁcient non-protein energy is available in the feed formulation, dietary amino acids will be deaminated and used as metabolic fuel, rather than for protein synthesis and growth. On the other hand, if the dietary energy to protein ratio is excessively high, the ﬁsh may not consume sufﬁcient protein and essential nutrients to maintain maximal growth, and they may also accumulate large amounts of body lipid. Feeds prepared for marine ﬁsh species typically have ca. 500 g protein per kg dry feed, and have protein-to-energy (P : E) ratios of 20–30 mg crude protein per kJ gross energy (see Table 9.12) (Tucker, 1998; Morais et al., 2001). The protein requirement is expected to decrease with increasing ﬁsh size, so there will be a decrease in the optimal P : E as the ﬁsh increase in weight. One disadvantage with expressing feed protein and energy as P : E is that this ratio does not take account of the facts that protein bioavailability (digestibility) depends on the protein sources used in formulating the feed, and that the bioavailability of the energyproviding macronutrients (lipids, carbohydrates and proteins) is inﬂuenced by factors such as type, source and feed-processing conditions. Consequently, there are compelling arguments for taking nutrient bioavailability into account when making assessments of feeds with respect to optimising protein-to-energy ratios. Thus, the digestible protein-to-digestible energy ratio (DP : DE) is to be preferred over P : E. This may be especially critical for the accurate assessment of optimal protein-to-energy ratios when ﬁsh are fed high-protein, highenergy feeds containing highly digestible feed ingredients with high protein and lipid contents (Cho et al., 1994; Stickney, 2000). The formulation of feeds on the basis of nutrient bioavailabilities and digestible energy necessitates information about the digestibilities of different feed ingredients being available. Information about the digestibility of a feedstuff is usually obtained by substituting part of a ‘reference’ feed (for which nutrient digestibilities are known) with the feedstuff in question, estimating nutrient digestibilities of the ‘new’ feed, and then calculating the digestibilities of the nutrients in the feedstuff ‘by difference’ (Cho et al., 1982; Allan et al., 1999). An assumption behind this calculation is that the digestibilities of feed ingredients are independent and additive, i.e. the digestibilities of the macronutrients and energy estimated for a given feedstuff are not inﬂuenced by interactions with components derived from other feed ingredients (Cho et al., 1982, 1994; Allan et al., 1999). Nutrient interactions may occur, so the assumption of independent, additive digestibilities may not be valid, and checks should be carried out to estimate the degree of error introduced by adopting this method of calculation (Cho et al., 1982; Allan et al., 1999).

9.8 Feeding Regimes and Practices The provision of feed to aquatic animals poses some problems not encountered in the farming of terrestrial animals. Feeds need to be water-stable so that they remain intact until ingested,

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and there should also be minimal losses of water-soluble nutrients due to leaching. Although most feeds employed in intensive aquaculture are quite water-stable, some disintegration will occur within a relatively short period of time. This disintegration makes the feed unavailable to the ﬁsh, and can lead to water fouling problems. Further, a large proportion of the cost of aquaculture production is related to feeds and feeding, so there is an additional incentive for the farmer to attempt to ensure that feed is consumed rather than going to waste. Thus, feed provisioning should be timed to match the times at which the ﬁsh are eager to feed, and the quantities of feed supplied should meet the demand, without being excessive. The amount of feed consumed by ﬁsh is markedly affected by ﬁsh size and water temperature. Although ﬁsh size and water temperature have a major inﬂuence on the changes in feed intake observed during the course of the growth cycle, taking account of these two factors is inadequate to enable rational feed delivery on a day-to-day basis because a myriad other factors inﬂuence the feed intake of ﬁsh in the shorter term (Jobling, 1994; Jobling et al., 1995, 2001a; Alanärä et al., 2001; Kestemont & Baras, 2001). In other words, large variations in the amounts of feed consumed by groups of ﬁsh may be observed independently of changes in size and water temperature: a day of heavy feeding may be followed by a day in which the ﬁsh eat little, and any activities that stress the ﬁsh usually result in transient reductions in feed intake. Feed intake may also be inﬂuenced by season, photoperiod, stocking density, water quality and abrupt changes in rearing conditions. For example, feed intake may be depressed for a few days following handling or sorting procedures, and changes in dietary composition or feed type may also result in a short-term reduction in the amounts of feed consumed. The reproductive status of the ﬁsh will also inﬂuence feed intake, with ﬁsh of several species decreasing their rates of feed consumption in the weeks preceding spawning. Given the wide array of factors that inﬂuence feed consumption by ﬁsh, it is not surprising that widely different feeding methods and routines are adopted by farmers cultivating the same species in the same type of rearing system. Feeding methods range from hand-feeding to the use of different types of automatic, computer-controlled feeding systems. However, even when employing the same feeding method, farmers may opt to feed their ﬁsh at different times of the day, adopt different feeding frequencies, and apportion the feed in different meal sizes (Jobling et al., 1995; Goddard, 1996; Tucker, 1998; Houlihan et al., 2001). The feeding methods and routines adopted may have a signiﬁcant bearing upon the growth of the ﬁsh, and the production costs of the farm. The way in which the ﬁsh are fed will inﬂuence both the amount of feed that goes to waste, and whether or not all individuals within the rearing unit are given adequate opportunities to feed. The development of optimal feeding regimes for farmed ﬁsh might be aided by the incorporation of ideas relating to resource defence theory. According to the theory, an animal will defend a resource (such as food) when the beneﬁts of defence exceed the costs. Resources that are defended tend to be restricted in quantity, spatially clumped and predictable (i.e. the animal knows where and when the resource can be found) (Grant, 1993, 1997). Thus, within the context of ﬁsh farming, attempts should be made to avoid feeding regimes that incorporate unnecessary restrictions, and feed should be widely dispersed rather than being delivered at a point source (Jobling et al., 1995; Carter et al., 1996; Shelverton & Carter, 1998). Furthermore, costs associated with the defence of a resource tend to increase with increasing numbers

398

Culture of cold-water marine ﬁsh

of competitors, so attempts should be made to ensure that stocking densities are such that resource defence is uneconomic for individual ﬁsh. The distribution of feed by hand allows observations of feeding responses by ﬁsh which feed actively at the water surface, or are grown in shallow tanks containing clear water. This enables direct assessments of feed demand to be made, and feed can be distributed accordingly. Thus, over-feeding, and concomitant feed wastage, can be avoided if feed delivery is gradually reduced as the ﬁsh approach satiation. The main advantage of hand-feeding is that, when carried out carefully, feed supply is tightly linked to changes in the feed demands of the ﬁsh. Further, because this feeding method involves regular observation of the behaviour of the ﬁsh, it enables abnormalities to be detected, and remedial action taken. Thus, handfeeding may be effective, and relatively easy to use, when feeding those species of ﬁsh that show clear feeding responses, and feed avidly close to the water surface. However, the behavioural responses of the ﬁsh may vary depending upon the way in which the feed is presented (Ang & Petrell, 1998). Some species, such as cod, Gadus morhua, and marine ﬂatﬁsh, may feed rather slowly, making it difﬁcult to assess when they approach satiation. Thus, considerable experience may be required in order to feed these species successfully by hand. It may also be difﬁcult to successfully hand-feed ﬁsh in deeper water, or when visibility is poor. The problem of observing ﬁsh in deep water may be overcome by using an underwater video camera to monitor feeding response, and detect uneaten pellets (Ang & Petrell, 1997, 1998). The underwater camera is placed in the water column 1–2 m below the ‘feeding zone’ of the ﬁsh, and is connected to a surface viewing monitor. The lens of the camera is directed towards the water surface, so feed pellets and ﬁsh appear as distinct dark objects against a light grey background. As an alternative to video monitoring, a sediment trap may be used to assess whether or not the feed provided is being consumed. One sediment trap system (the LIFT-UP system made for ﬁtting to sea cages) has a sedimentation cone inserted into the base of a sea cage, and uneaten pellets and other wastes are transported to the surface using air-lift pumps. By monitoring the efﬂuent, it can readily be seen whether or not the feed is being eaten, and the supply of feed can be terminated once the return of pellets to the surface becomes excessive. A sediment trap system can be used under routine feeding procedures both to control feeding and to reduce the feed losses that lead to environmental pollution. The efﬁcacy of the LIFT-UP system has, for example, been tested when feeding medicated feeds to salmonids in sea cages. The system proved effective in collecting uneaten feed, thereby preventing excessive losses of medicaments to the environment (Ervik et al., 1994). The main disadvantages of hand-feeding are that it is labour-intensive and timeconsuming. However, large cod are able to maintain good rates of growth when fed to satiation three or four times a week, so it may be possible to employ hand-feeding in the rearing of this species. Nevertheless, labour and time constraints may mean that the method has limited application on large farms. A further disadvantage with hand-feeding is that it involves the provision of feed at times of the day that are predetermined by the farmer. This may have some undesirable consequences, because it is known that the timing of feed provision can inﬂuence a range of physiological processes, and have consequences for growth and nutrient partitioning (Boujard & Leatherland, 1992; Bolliet et al., 2001). One way that the farmer can overcome these potential ‘feeding-time’ effects is by dis-

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tributing feed at regular intervals throughout the day using automatic feeders (Goddard, 1996; Alanärä et al., 2001). Most automatic feeders are designed to distribute dry feeds, although some can handle moist diets. Traditionally, automatic feeding systems have been designed to dispense predetermined amounts of feed at pre-set time intervals (i.e. they are time-release systems). Such feeders vary in design, but most are electrically powered, and are operated from a central control unit. The feed is usually spread as it falls from the feed hopper, either mechanically or by being blown by compressed air, and this ensures that the feed is widely distributed over the surface of the rearing unit (Goddard, 1996). This widespread dispersal of feed is considered to be advantageous because it is thought that it gives all ﬁsh within the rearing unit access to some feed. If feeders are timed to release pellets at short time intervals, feed will be available almost continuously, but it is open to question whether a ‘little-and-often’ feeding regime will always be more effective than the provision of feed in the form of a few large meals. A further problem arises in that the farmer must decide the size of the portions of feed to be released to meet the possible variations in feed demand of the ﬁsh at different times of the day. This has risks, because an erroneous decision means that the ﬁsh may be either under-fed, leading to competition for feed, or over-fed, with increased feed wastage as a consequence. Thus, time-release automatic feeding systems may ease the workload associated with feed delivery, but they do not solve the problems associated with the development of optimal feeding regimes. The problems associated with variable feed demand during the course of the day can be reduced, if not completely solved, by using on-demand feeders. Such feeders enable the ﬁsh to adjust feeding regimes either directly (self-feeders) or indirectly (interactive feedback systems) (Alanärä et al., 2001). In a self-feeding system, the ﬁsh usually control the delivery of feed by actuating a trigger placed below the water surface (Alanärä, 1996), whereas interactive feedback systems may rely either upon the detection of waste feed, or on changes in the behaviour and distribution of the ﬁsh, to control the delivery of feed from automatic feeders (Fig. 9.7) (Blyth et al., 1993, 1997; Juell et al., 1993; Summerfelt et al., 1995; Goddard, 1996; Ang & Petrell, 1997, 1998). The simplest types of self-feeder consist of a feed hopper with an aperture, the opening of which is controlled by a movable gate. The trigger is attached to the gate, and lateral movements of the trigger lead to the release of feed from the hopper. One disadvantage with this type of system is that feed may be released by trigger actuations resulting from wave action or other mechanical disturbances, and this will lead to feed wastage. Further, selffeeders of this type deliver feed at a point source rather than providing a widespread distribution of feed over the water surface. This has the disadvantage that one, or a few, ﬁsh may establish feeding territories close to the feeder, and prevent the other ﬁsh from gaining access to the feed. Electronically controlled self-feeders are more sophisticated in design, and usually overcome the problems listed above. These self-feeders are usually designed in such a way that each time the ﬁsh activates the triggering system an electric pulse is generated and, through a relay, this leads to the delivery of feed from a hopper. Thus, the entire system consists of a trigger, a feed hopper (automatic feeder) and an interface between the trigger and the automatic feeder. This design reduces the risk of false trigger actuations due to mechanical disturbance, and also allows feed to be widely dispersed over the water surface in response to activation of the triggering mechanism. Self-feeders have been employed as

400

Culture of cold-water marine ﬁsh

a tool for the study of feeding rhythms in ﬁsh (Madrid et al., 2001), and have also been used in pilot-scale and commercial production units for species such as channel catﬁsh, Ictalurus punctatus, rainbow trout, Oncorhynchus mykiss, and European sea bass, Dicentrarchus labrax. Owing to their design and mode of operation, it might be envisaged that self-feeders would be best suited for feeding diurnal ﬁsh species that feed at the surface or in mid-water, but in practice, it has been shown that ﬁsh of a wide range of species, including some that are benthic and some that are nocturnal, can learn to operate self-feeders (e.g. Gwyther & Grove, 1981; Alanärä, 1996; Boujard et al., 1996; Boujard & Luquet, 1997; Burel et al., 1998; Jobling et al., 2001a). The interactive automatic feeding systems employ an alternative approach to feed delivery: the ﬁsh are provided with feed at regular intervals, and registration is made as to whether or not the feed is consumed. This may be done by direct observation of the feeding response of the ﬁsh, by following the fate of feed pellets using video recording, or by using special optical or hydroacoustic sensors to detect uneaten pellets. Feed delivery is stopped or modiﬁed according to feedback signals from the sensory system. In this way an attempt is made to control the delivery of feed in relation to the propensity of the ﬁsh to feed (Bjordal et al., 1993; Blyth et al., 1993, 1997; Juell et al., 1993; Summerfelt et al., 1995; Ang & Petrell, 1997). The most commonly used interactive automatic feeding systems consist of a control unit (a computer with the capacity to run several feeding programmes), a feed trap with a sensor capable of detecting uneaten feed, and a feed hopper. The operation of this type of

Figure 9.7 Schematic diagrams showing the basic principles of on-demand feeders. (A) Self-feeders. (B) Interactive feeding systems.

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system relies upon the detection of a certain quantity of feed waste, and feed release from the hopper can be regulated in accord with the amount of waste detected by the sensor. This can ensure that the delivery of the main bulk of the feed occurs at times at which the ﬁsh are feeding most avidly. Interactive automatic feeding systems can be extremely ﬂexible, and the computer may be programmed to operate a wide range of feeding regimes. For example, the system may be programmed to deliver food only during certain periods of the day, e.g. only during daylight hours. There is also the possibility of controlling ‘meal timing’, in that the system may be programmed to restart feed delivery at speciﬁed times after the termination of the previous feeding bout. Further, it is possible to incorporate differentiated step-up and step-down functions so that the rate of feed delivery is increased gradually when all feed is being consumed, and is terminated promptly once large numbers of uneaten pellets are detected by the sensor. This ensures that feed waste is reduced to a minimum as the ﬁsh approach satiation.

Figure 9.7 Continued.

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Culture of cold-water marine ﬁsh

The interactive automatic feeding systems would seem to be ideally suited for use in landbased rearing systems. This is because the ﬁtting of the sensor to the tank out-ﬂow should allow the detection of most, if not all, of the uneaten pellets as they are carried out of the tank in the efﬂuent (Summerfelt et al., 1995; Chen et al., 1999). Thus, feeders could be adjusted to dispense the feed over the entire water surface, and water ﬂow directed so that the current ensures an even distribution of feed throughout the water column. This should create conditions that give the majority of ﬁsh within the rearing unit very good opportunities to feed. However, the fact that uneaten pellets are carried out of the tank in a single outﬂow means that little waste will go undetected despite a widespread distribution of the feed within the rearing unit. In practice, limitations related to waste pellet detection in the water outﬂow have hindered the development of commercial interactive feeding systems for use in tanks, and these feeding systems are most widely used in sea-cage farming. Interactive systems are now being widely used in the sea-cage farming of salmonids, and tests have been carried out on warm-water marine species, such as barramundi, Lates calcarifer, and those reared in the Mediterranean countries. Interactive systems would seem to have a potential for use in the farming of a range of ﬁsh species, and are expected to meet with most success when ﬁsh either display clear circadian feeding rhythms, or consume large meals at irregular intervals. This is because of the ease with which the feeding and nonfeeding periods of such ﬁsh can be distinguished.

9.9 Growth and Feed Conversion Growth is deﬁned as a change in size, or stored body material. Numerical expressions of growth may be based on absolute changes in length or weight (absolute growth), or changes in length or weight relative to the size of the ﬁsh (relative growth) (for a general discussion, see Jobling, 1994; Goddard, 1996; Iwama, 1996; Stickney, 2000). Length almost always increases with time, whereas weight can either increase or decrease over a given time interval depending upon the inﬂuences of the various factors that affect the deposition and mobilisation of body materials. Measurements of growth in relation to time provide an expression of growth rate. When considered over the life-time of the ﬁsh, curves describing growth in terms of changes in either length or weight with time both approach an asymptotic value. Length growth can usually be modelled using an asymptotic curve which tapers off with increasing age. Weight growth is usually sigmoidal, i.e. the weight increments increase gradually up to an inﬂection point, from where they gradually decrease again. Thus, growth rates are constantly changing, and the absolute growth increments will be different for different sizes of ﬁsh. At any point on the growth curve, the instantaneous rate of growth in terms of weight, g, will be given by the differential equation g = d (ln W ) dt

(9.2)

where W is weight and t is time. For short intervals, this equation is approximated by the difference equation

On-growing to market size

g = (ln W2 - ln W1 ) (t2 - t1 )

403

(9.3)

When the instantaneous growth rate, g, is used as an expression of growth rate, the numerical value will decrease with increasing size and age of the ﬁsh. Growth is often expressed in terms of instantaneous rates, with the commonest form of expression being the speciﬁc growth rate (SGR = 100 g). SGR, which is equivalent to the percentage increase in weight per unit time, declines as body size increases, and the relationship between SGR and body size may be described by an allometric function SGR = aW b

(9.4)

where a and b are constants. The weight exponent, b, is negative, and in many empirical studies the value of the exponent has been found to be within the range -0.32 to -0.42 (Jobling, 1994; Imsland, 1997; Björnsson et al., 2001). For example, Björnsson et al. (2001) reported that the relationship between maximum SGR (i.e. the SGR at the optimum temperature for growth) and body weight of cod (weight range ca. 2–2200 g) could be described as SGR = 7.74W -0.404

(9.5)

The fact that SGR decreases with increasing size can create problems when attempts are made to use this expression of growth rate for comparative purposes. Consequently, attempts have been made to ﬁnd alternatives to SGR, and it has been suggested that a growth index 3 based on the analysis of changes in the cube root of weight ( W ) with time may be suitable for a comparison of growth rates of ﬁsh reared under different conditions (Iwama, 1996). Calculations based on the cube root of weight are thought to be particularly appropriate for 3 comparative purposes because W has a close relationship with length (assuming W = cL3), and this gives a means of calculating a relatively robust growth index. If length increase is linear over a given time interval, the modelling of growth in terms of the cube root of body weight gives a relationship of the form 3

3

W2 = W1 + Gs (t2 - t1 )

(9.6)

3

where Gs is the slope of the regression of W against time. This represents the growth index. This model may be modiﬁed to take account of the inﬂuence of temperature on growth, the thermal growth coefﬁcient (TGC) being calculated as

[(

3

3

W2 - W1 )(ST )

-1

] ¥ 1000

(9.7)

where ST is the sum day-degrees Celsius, and the multiplication factor (x 1000) is used to obtain numbers that can be managed easily. The modiﬁed growth model incorporating TGC is given by 3

W2 = [3 W1 + [(TGC 1000) ¥ ST ]]

(9.8)

404

Culture of cold-water marine ﬁsh

In calculating the TGC, it is assumed that there is a steady increase in growth rate with increasing temperature. This assumption is invalid, but may provide a reasonable approximation for much of the ascending limb of the temperature range over which a species is reared. Caution should therefore be exercised when applying the TGC for the prediction of ﬁsh growth. Nevertheless, a growth index based upon a plot of cube root of weight against time has certain advantages over SGR, and the model is increasingly being used for the analysis of growth data. For example, Iwama and co-workers have used this growth model to make predictions about the growth of salmonids under hatchery conditions, and the model may prove useful in production planning (Iwama, 1996; Alanärä et al., 2001). In simple terms, growth reﬂects the change in body weight that results from the difference between the feed nutrients that enter the body and the waste materials that leave it (Jobling, 1994, 1997; Shearer, 1995). This can be represented as an algebraic equation dW dt = pR - M

(9.9)

where dW/dt is the change in weight per unit time, R is the amount of food consumed (ration), p is a coefﬁcient indicating the bioavailability of nutrients (absorption efﬁciency or digestibility) and M represents metabolic losses. Both feed intake (ingestion rate) and metabolic rate are inﬂuenced by temperature, and the difference between the rate–temperature curves for ingestion rate and metabolic rate will give an indication of the resources available for growth under different temperature conditions (Fig. 9.8). An assumption being made here is that the bioavailability of nutrients changes little over the range of rearing temperatures considered. The plotting of growth rate–temperature relationships for ﬁsh that are feeding and growing maximally indicates that growth rate reaches a peak at a temperature that is slightly lower than that at which ingestion rate reaches its maximum (Fig. 9.8) (Jobling, 1997; Kestemont & Baras, 2001). The temperature at which growth rate peaks is known as the optimum temperature for growth, but growth optima do not remain ﬁxed throughout the entire life-cycle of the ﬁsh. In general, the optimum temperature for growth tends to become lower as the ﬁsh increase in age and size (Fig. 9.9) (Pedersen & Jobling, 1989; Fonds et al., 1992; Björnsson & Tryggvadóttir, 1996; Imsland et al., 1996; McCarthy et al., 1998, 1999; Jonassen et al., 1999; Björnsson et al., 2001); a decrease of 4–5°C does not seem to be uncommon over the life-cycle of coldwater marine species. For example, the optimum temperature for growth of Atlantic halibut, Hippoglossus hippoglossus, has been reported to decrease from 13–14°C in 10–60 g ﬁsh to 11.5°C for 100–500 g ﬁsh, and to 9.5–10°C for halibut weighing 3–5 kg. Furthermore, the domed form of the rate-temperature curve appears to become ﬂatter as ﬁsh became larger, suggesting that thermal sensitivity at temperatures close to the optimum may be reduced in large ﬁsh (Björnsson & Tryggvadóttir, 1996). In a similar way to the situation in halibut, Björnsson et al. (2001) reported that the optimum temperature for growth (TG) of cod decreased from 17°C in ﬁsh weighing ca. 2 g to ca. 8.5°C in ﬁsh of ca. 2200 g, giving a relationship described by the equation TG = 18.28 - 1.43 ln W

(9.10)

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405

Figure 9.8 Rate–temperature curves illustrating the effects of temperature on rates of ingestion, metabolism and growth. Note that the temperature at which ingestion rate reaches its maximum (A) is a few degrees higher than the optimum temperature for growth (B). From Jobling (1997).

In the preceding paragraph, discussion was directed towards the inﬂuence of temperature on the growth of ﬁsh provided with sufﬁcient food to enable them to feed maximally. However, it is important to distinguish between the effects of temperature per se, and the effects on growth induced by the interactions between temperature and limited food supply. It is also important to bear in mind that other environmental factors, such as those related to water quality, can inﬂuence feeding. Thus, feed intake may be depressed in ﬁsh exposed to a poor rearing environment even though it may appear that feed is being supplied in excess of requirements. Any such reduction in feed consumption will obviously be reﬂected in a reduction in growth. When the supply of food becomes more and more restricted, ﬁsh will grow best at lower and lower temperatures. The most obvious reason for this is the effect that temperature has on metabolic rate (see Fig. 9.8). With increasing temperature there is an increase in metabolic rate, and consequently the amount of food required to hold body weight constant (maintenance ration) increases. Thus, when ﬁsh are supplied with a ﬁxed, restricted quantity of food, the amount available to be directed towards growth (i.e. nutrients supplied in the

406

Culture of cold-water marine ﬁsh

Figure 9.9 Inﬂuence of temperature (T°C) on speciﬁc growth rate (SGR) of different weight classes (W) of halibut, Hippoglossus hippoglossus. (A) 5–10 g; (B) 20–25 g; (C) 40–50 g; (D) 60–70 g. Least-squares polynomial ﬁts to the growth data are shown for each size class: SGR = aT 2 + bT + c. The estimated optimum temperature for growth is indicated by the broken line. From Jonassen et al. (1999).

food minus those needed for maintenance) decreases as the temperature is increased. Consequently, as the feed supply becomes increasingly restricted, the amounts of nutrients available for growth are greatest at progressively lower temperatures. Further, the increase in metabolic rate that accompanies rising temperature accounts for the fact that ﬁsh lose weight more rapidly when held without food at high, rather than low, temperatures. The metabolic rates of well-fed ﬁsh are higher than those of ﬁsh that are deprived of food, and rates of oxygen consumption increase as the feeding conditions of the ﬁsh are improved. When ﬁsh are exposed to hypoxia, i.e. the level of dissolved oxygen in the water is below full saturation, oxygen availability may not be sufﬁcient to allow very high metabolic rates to be maintained (Jobling, 1994). One way in which a ﬁsh can reduce metabolic expenditure is to reduce its food intake. Thus, when ﬁsh are exposed to hypoxic conditions they tend

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to reduce their food intake, and this results in reduced rates of growth (e.g. Chabot & Dutil, 1999; Pichavant et al., 2000, 2001). Fish reduce their feed intake at levels of dissolved oxygen that are considerably higher than those that compromise survival, feed intake being gradually reduced as water becomes progressively more hypoxic. For example, when Atlantic cod were held at 10°C and fed to satiation three times each week, growth was compromised in ﬁsh exposed to water that was 65–70% saturated with oxygen, and growth became progressively poorer as the level of hypoxia was increased (Chabot & Dutil, 1999). Similarly, the growth of juvenile turbot was compromised when they were held in hypoxic water, growth being signiﬁcantly lower in ﬁsh exposed to ca. 5 mg O2l-1 than in those held in water with oxygen concentrations of 7–7.5 mg O2l-1 (Pichavant et al., 2000, 2001). Relative to feeding and growth under normoxia, there may also be reductions under conditions that oscillate between normoxia and hypoxia (Thetmeyer et al., 1999). Oscillating oxygen conditions are likely to be encountered in the cage farming of many species, and may therefore be expected to have an inﬂuence on production at many sites. Consequently, attempts should be made to ensure that levels of dissolved oxygen in rearing units remain close to saturation if the rates of feeding and growth of the ﬁsh are to be maintained close to maximal. The reduced solubility of oxygen in water at a high temperature, together with the increase in metabolic rate that accompanies an increase in temperature, may also explain why ﬁsh must reduce their food intake at high temperature even when exposed to water that is fully saturated with oxygen (see Fig. 9.8) (Jobling, 1997). Numerous studies have addressed the inﬂuence of salinity on the growth of marine and freshwater ﬁsh (Boeuf & Payan, 2001). Foss et al. (2001) reported that juvenile spotted wolfﬁsh, Anarhichas minor, were euryhaline, and grew equally well at all salinities within the range 12–34‰. There is also evidence that some species of cold-water marine ﬁsh grow best in water with a salinity lower than that of full-strength sea water (35‰) (Lambert et al., 1994; Gaumet et al., 1995; Imsland et al., 2001). For example, cod acclimated to 7–15‰ exhibited enhanced growth, and although the response was seasonally dependent, these ﬁsh never grew less well than ﬁsh held in full-strength sea water (Lambert et al., 1994). Similarly, Gaumet et al. (1995) reported that the growth of juvenile turbot was enhanced when they were held at 10‰, but Imsland et al. (2001) found an interaction between salinity and temperature in the governing of growth. The latter authors reported that the best temperature–salinity combination for the growth of juvenile turbot was ca. 22°C and 18.5‰. The basis of growth enhancement at reduced salinity remains open to speculation. The osmotic and ionic concentrations of ﬁsh body ﬂuids differ from those of the surrounding medium, so the ﬁsh is required to expend a certain amount of energy in order to meet the metabolic costs of ionic and osmotic regulation. However, there is, a lack of agreement concerning the magnitude of these costs (Boeuf & Payan, 2001), and it is unclear whether or not the rearing of ﬁsh in an isionic, isosmotic environment would lead to sufﬁcient reductions in metabolic costs to enable these to provide improvements in growth and feed utilisation. The enhanced growth of ﬁsh held in an isosmotic environment need not merely be a reﬂection of a reduction in the metabolic costs of iono- and osmo-regulation. Since growth is a net result reﬂecting the difference between energy provided by the ingestion of food and metabolic expenditure, any inﬂuences of changes in salinity on feed intake, digestive and absorptive processes, and swimming activity would also lead to effects on growth (Jobling, 1994; Dutil et al., 1997; Swanson, 1998; Boeuf & Payan, 2001).

408

Culture of cold-water marine ﬁsh

The growth of ﬁsh often varies with season and daylength, with growth being more rapid when ﬁsh are exposed to long or increasing photoperiods than when they are subjected to short or decreasing photoperiods, but because natural seasonal variations in daylength follow similar cycles to temperature, the effects of the two variables may be confused. However, under controlled conditions both constant long, and increasing, photoperiods have been found to have a growth-stimulatory effect on a range of temperate-zone anadromous and freshwater ﬁsh species, including salmonids, ictalurids, centrarchids and percids (see Jobling, 1994; Imsland, 1997; Boeuf & Le Bail, 1999). There is also evidence of a growth-promoting effect of extended photoperiod on the juveniles of some cold-water marine species (Imsland et al., 1997; Jonassen et al., 2000; Simensen et al., 2000), but the effects appear far less dramatic than those seen in the anadromous and freshwater species (Imsland, 1997; Boeuf & Le Bail, 1999). In some cases where the exposure of marine species to extended photoperiods has resulted in increased weight gain, they seem, at least in part, to be related to inﬂuences on the timing of the onset of maturation and spawning (Imsland et al., 1997; Hansen et al., 2001). Such inﬂuences have also been observed in freshwater and anadromous species (Boeuf & Le Bail, 1999). Thus, the inﬂuence of photoperiod on growth may be via several, not mutually exclusive, pathways. Firstly, an extended photoperiod may promote growth via stimulation of the hypothalamic-pituitary axis, as has been seen in juvenile salmonids (Boeuf & Le Bail, 1999). Secondly, ﬁsh that are in the early stages of maturation may grow faster than immature conspeciﬁcs, owing to the anabolic effects of low concentrations of sex steroid hormones, but as mature ﬁsh approach the time for spawning they may become anorexic, and growth rate falls to below that of immature individuals. The reasons for the differences between freshwater, anadromous and marine species are unknown, but it is possible that the responses of ﬁsh to photoperiod relate primarily to the synchronisation of an endogenous growth rhythm with prevailing environmental conditions, i.e. photoperiod is acting as a zeitgeber. If this is the case, then the photoperiodic response would be expected to be more marked in the temperate-zone freshwater species that occur in habitats that are highly variable on a seasonal basis than in those species that inhabit more stable environments. Under conditions prevalent in the wild there may be seasonal changes in prey availability, the growth of the ﬁsh will often be limited by food supply, and the ﬁsh may undergo pronounced seasonal cycles of depletion and repletion of energy reserves. Fish appear to be able to adapt to these feast-and-famine conditions by showing marked growth spurts, known as catch-up or compensatory growth, when food supplies are increased following a period of undernutrition. The response shown following a period of nutritional restriction seems to be common across vertebrate species: increased food intake (i.e. hyperphagia), rapid growth and the repletion of energy reserves (Broekhuizen et al., 1994; Jobling, 1994; Jobling & Johansen, 1999). It is usually the animals that are in the poorest condition that show the greatest response, and display the most rapid rates of weight gain, when adequate feeding conditions are restored (Fig. 9.10) (Pedersen & Jobling, 1989; Jobling et al., 1991, 1994), and amongst ﬁsh that have been held on restricted rations, the catch-up growth response appears to be inversely related to the severity of the feed restriction (Jobling, 1994; Jobling et al., 1999; Sæther & Jobling, 1999). The physiological mechanisms underlying hyperphagia and the onset of the catch-up growth response are not completely understood, but it is hypothesised that increased feed

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Figure 9.10 The effects of initial length (L cm) and condition (CF = [W/L3] ¥ 100) on weight (W) change in groups of Atlantic cod, Gadus morhua, undergoing catch-up growth. Note that the cod that were in the poorest condition at the start displayed the highest rates of weight gain, and at 18 weeks the body weights attained by cod within each initial length group were similar. From Jobling et al. (1994).

intake will occur when the animals sense that they are insufﬁciently fat or that muscle development does not conform to some pre-set genotypic norm (Broekhuizen et al., 1994; Jobling, 1994; Jobling & Johansen, 1999). Body tissues can be broadly divided into reserves (lipid depots and the mobilisable parts of the musculature) and structural components (skeleton, circulatory and nervous tissue). It is suggested that the balance between these two components is regulated, and that a shift in the balance induced by a period of undernutrition would

410

Culture of cold-water marine ﬁsh

invoke the changes in feed intake (i.e. hyperphagia) and growth that constitute the catch-up response. For example, when cod were deprived of food for a prolonged period they depleted their reserves of liver lipids, and liver and muscle glycogen, and then there was increased mobilisation of white-muscle proteins. The integrity of the red muscle, used for continuous swimming rather than burst performance, seemed to be maintained at the expense of the white muscle (Black & Love, 1986). When the cod were re-fed, the muscle was the ﬁrst to be repleted. This involved an increase in weight, a relative increase in lipid and glycogen, and a decrease in the percentage of muscle water. There was little repletion of the liver lipid store before muscle water fell below 82% (Black & Love, 1986). Thus, during the initial stages of recovery from a period of food deprivation, cod seem to give priority to the repletion of muscle tissue, and this results in a marked increase in the ﬁllet yield, and percentage ‘dress-out’, of cod that are undergoing catch-up growth (Jobling et al., 1991, 1994). There has been some speculation as to whether the ability of ﬁsh to display catch-up growth could be exploited in commercial production to control rates of weight gain, or to manipulate the composition of body tissues (Jobling, 1994). Cyclic feeding regimes, incorporating a variety of combinations of feed restriction or fasting, have been used in livestock production to inﬂuence commercially important parameters such as meat yield, lean-to-fat ratios, production times and feed efﬁciencies. It is possible that cyclic feeding regimes could also ﬁnd an application in the production of farmed ﬁsh for the manipulation of growth, ‘dress-out’ and ﬁllet yield, and the chemical composition of the tissues. Growth data are important because a farmer will wish to harvest the ﬁsh once they reach a given size, and information about size–frequency distributions is important because it gives an indication about the degree of size heterogeneity of the ﬁsh within a rearing unit. Thus, it is considered desirable to have information about both the average weight and the size distribution of ﬁsh within rearing units in order to allow rational planning of production and harvesting (for a general discussion, see Goddard, 1996; Iwama, 1996; Stickney, 2000; Alanärä et al., 2001). Reliable growth assessment depends upon accurate measurements of weight and length, and such measurements should be made at regular intervals. Attempts are being made to develop methods for growth and biomass assessment that do not require the removal of ﬁsh from the rearing unit (e.g. Petrell et al., 1997). Such methods generally rely upon morphometric measurements being made from video-recordings of ﬁsh swimming in situ, and an assessment of ﬁsh size is made using data from a control series of measurements made on ﬁsh of known size and body weight. Most usually, however, weight and length measurements are undertaken directly following removal of the ﬁsh from the rearing unit. When dealing with large numbers of ﬁsh in commercial rearing units, it will usually be impracticable to measure and weigh all individuals, and some form of sampling will be required. Estimates of average weights can be obtained by bulk weighing samples taken from the ﬁsh populations. Fish may be netted from the rearing unit, and after the weight of the sample has been recorded the ﬁsh are counted back into the unit. The total weight of the sample is divided by the number of ﬁsh to give an estimate of average weight. A sampling protocol of this type can provide useful information, but it does have the disadvantage that it gives no indication of the size–frequency distribution of the ﬁsh within the population. Information about size–frequency distributions can only be obtained by weighing and measuring ﬁsh individually. For this, a measuring

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board with a ruler incorporated into it can be tared on a balance, and measurements of length and weight made simultaneously. The ﬁsh within a rearing unit will not all grow at the same rates, and the disparity in growth among individuals can be assessed by examining the coefﬁcient of variation (CV) of the size–frequency distribution CV = (standard deviation mean) ¥ 100

(9.11)

CV is an expression of relative variability that relates sample variability to the mean of the sample, and may therefore be used to compare the variations of populations independently of the magnitude of their means. For any given population, a pronounced increase in CV with the passage of time is taken as indicating that the ﬁsh are growing at markedly different rates, something that would be deemed undesirable under farming conditions. There are numerous factors that may inﬂuence the development of size disparity within groups of ﬁsh, but as a generalisation, it may be said that when CV increases over time it indicates that some ﬁsh within the group are not growing to their full potential. Such a growth suppression (or growth depensation) can often arise within groups of ﬁsh held under conditions that promote competition (Purdom, 1974; Grant, 1993, 1997; Jobling et al., 1995, 2001a). For example, CV may increase for a period of time following the establishment of rearing groups, and then stabilise (Purdom, 1974), and restricted feeding may also result in increased size disparity among ﬁsh within a rearing unit. However, there are considerable species differences (Jobling, 1982; Jobling et al., 1995, 1999, 2001a; Boujard et al., 1996; Carter et al., 1996; Imsland, 1997; Shelverton & Carter, 1998; Jonassen et al., 1999). For those species which show growth depensation, information about growth rates and changes in the size– frequency distribution enable assessments to be made about the rearing environment to which the ﬁsh are being subjected. (1) High growth rates accompanied by stable CVs for body weight indicate a good rearing environment in which there is little competition for feed, and where there is no suppression of growth due to poor water quality. (2) Poor and disparate growth, i.e. reduced average growth rates accompanied by an increase in CV with the passage of time, may reﬂect competition for feed due to underfeeding. (3) Poor growth with little inter-individual variability, i.e. stable CV, is a reﬂection of a general growth depression, and will often be indicative of a sub-optimal rearing environment resulting from poor water quality. In addition to needing information about growth rates and size–frequency distributions, a farmer will also wish to know how efﬁciently the ﬁsh are utilising feed for growth. Estimations of feed efﬁciency require information about both feed consumption and the growth of the ﬁsh. The expression of efﬁciency is best given as weight gain per unit feed consumed (feed conversion efﬁciency), but in practical ﬁsh farming it is often given as the amount of feed required to produce a given weight gain (feed : gain ratio). Strictly speaking, feed conversion efﬁciency describes the relationship between feed consumption and growth, but an

412

Culture of cold-water marine ﬁsh

Figure 9.11 The inﬂuence of temperature on growth rate (SGR, % day-1) and feed conversion (CE, wet weight gain per weight dry feed) of juvenile Atlantic cod, Gadus morhua, (ﬁsh weight ca. 12 g). Calculated from data given in Björnsson et al. (2001).

accurate assessment of the amount of feed consumed by the ﬁsh will rarely be available. Consequently, feed conversion efﬁciencies are most usually an expression of the relationship between the observed weight gain and the amount of feed provided, with feed that goes to waste being included in the calculation of feed conversion efﬁciency. As a consequence, low feed conversion efﬁciencies (high feed : gain ratios) will be recorded under conditions where the ﬁsh are being over-fed and there is considerable feed waste. Low feed conversion efﬁciencies are also recorded when growth is poor, i.e. when ﬁsh are being under-fed or when food consumption is suppressed owing to poor water quality. When ﬁsh are being under-fed, the vast majority of the feed provided will be consumed but growth will be poor, and may be disparate due to high levels of competition for the limited food resource. Under these conditions, there will be only limited weight gain per unit of feed provided. On the other hand, when water quality is poor the situation will be exacerbated because both feed intake and growth will be reduced, and feed will be lost as waste. Low rates of growth accompanied by considerable feed waste lead to poor conversion (high feed : gain ratios). The efﬁciency with which ﬁsh utilise feed for growth is inﬂuenced by both ﬁsh size and water temperature. When examined over a wide temperature range, feed conversion efﬁciencies shown by ﬁsh fed on maximum rations are usually found to increase and then decrease with increasing temperature; this gradual change is often adequately described by a parabolic relationship (Fig. 9.11) (Jobling, 1994, 1997; Björnsson & Tryggvadóttir, 1996; Björnsson et al., 2001). The temperature at which feed conversion efﬁciency is maximized is, however, slightly lower that the optimum temperature for growth (Fig. 9.11) (Jobling, 1997). Further, the temperature at which the best feed conversion efﬁciency is observed tends

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to decrease with increasing ﬁsh size (Jobling, 1994, 1997; Björnsson & Tryggvadóttir, 1996; Björnsson et al., 2001). For example, Björnsson and Tryggvadóttir (1996) observed that the best feed conversion efﬁciency was attained at 14°C in 10–60 g Atlantic halibut, whereas 100–500 g ﬁsh had the best feed conversion efﬁciency at 10.5°C, and 3–5 kg halibut converted feed most efﬁciently at 5.5°C. Similarly, Björnsson et al. (2001) reported that the optimum temperature for feed conversion (TCE) by cod decreased from 16°C in ﬁsh weighing ca. 2 g to ca. 8°C in ﬁsh of ca. 2200 g body weight, giving a relationship described by the equation TCE = 15.92 - 1.22 ln W

(9.12)

Feed conversion efﬁciencies are usually calculated as the wet weight gain achieved per unit weight of feed provided. While such estimates may be suitable for most purposes, they do have limitations. It is, for example, inappropriate to make direct comparisons of feed conversion efﬁciencies obtained when using feed types that differ substantially in either moisture content (i.e. wet, moist and dry feeds) or nutrient density (i.e. low-energy compressed steam pellets and high-energy extruded feeds). Further, the calculation of feed conversion efﬁciency on the basis of growth expressed in terms of wet weight gain implies that the composition of ﬁsh tissues is constant, i.e. the assumption is made that a change in wet weight will accurately reﬂect changes in the contents of the major biochemical components. However, the composition of the ﬁsh body is not constant, and the relative proportions of protein, lipid, carbohydrate and moisture making up a given weight gain may vary with feeding conditions and dietary composition (Cowey, 1993; Shearer, 1994; Jobling 2001b). For example, lipid deposition tends to increase with increasing feed supply, so that the bodies of ﬁsh fed maximally may contain proportionately more lipid than those held on restricted rations. Similarly, ﬁsh tend to deposit increasing amounts of storage lipids as they grow older, so percentage body lipid is usually found to be higher in large, old ﬁsh than in small, young ﬁsh. Thus, in order to undertake a complete assessment of feed conversion efﬁciency, the researcher will need information about feed composition, feed consumption and nutrient bioavailability, and also about the chemical composition of the ﬁsh at the start and end of the growth trial.

9.10 Nutrient Deposition and Body Composition The proportion of protein in ﬁsh muscle, and in the body as a whole, is usually within the range 15–19% (see Tables 9.4 and 9.8) (Lie et al., 1988, 1994; Love, 1988; Hislop et al., 1991; Jobling et al., 1991; Haard, 1992; Cowey, 1993; Macrae et al., 1993; Payne et al., 1999; Schreckenbach et al., 2001), and the deposition of increased quantities of lipid leads to a more marked reduction in the proportion of body moisture than of protein (Jobling, 2001b; Schreckenbach et al., 2001). The sum of lipid and moisture often amounts to ca. 80%, both on a tissue-by-tissue and whole-body basis (see Tables 9.4 and 9.8; Figs. 9.12 and 9.13) (Jobling, 1982, 2001b; Haug et al., 1988; Lie et al., 1988; Hislop et al., 1991; Cowey, 1993; dos Santos et al., 1993; Payne et al., 1999; Anthony et al., 2000; Morais et al., 2001; Schreckenbach et al., 2001).

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Figure 9.12 The percentage of body fat (lipid) and water (moisture) in various tissues of Atlantic halibut, Hippoglossus hippoglossus. Circles indicate white muscle, squares are red muscle, diamonds are liver and the triangle is the ‘anal’ notch. Filled symbols are data for males, and open symbols are for females. The ‘fat–water’ line is described by the following regression: %Water = 76.24 - 0.826 %Fat (N = 7; R2 = 0.958). Data from Haug et al. (1988).

Figure 9.13 The percentage of body fat (lipid) and water (moisture) in samples of turbot, Scophthalmus maximus, taken at the start of a growth trial (open squares, start weight ca. 200 g), following feeding for 12 weeks (weight ca. 450 g) on either a high-fat (open circles, HF = 25% lipid) or low-fat (open triangles, LF = 17% lipid) feed, and then after 8 weeks (weight 559 g) of dietary reversal (i.e. ﬁlled circles, HF Æ LF; ﬁlled triangles, LF Æ HF). The ‘fat-water’ line is described by the following regression: %Water = 80.11 - 1.08 %Fat (N = 44; R2 = 0.866). Data from Sæther & Jobling (2001).

The skeletal muscle and liver are usually major sites of lipid storage in ﬁsh, but there are considerable species differences: in so-called ‘lean’ ﬁsh, such as the cod, Gadus morhua, large amounts of lipid may be stored in the liver, whereas in ‘fatty’ species, such as herring and anchovies, the skeletal muscle represents a major lipid depot (Love, 1988; Macrae et al., 1993; Jobling, 2001b). Where in the body the lipid is stored will inﬂuence several attributes of farmed ﬁsh: ‘dress-out’ losses during processing, ﬁllet texture and storage properties, and the nutritional value of the ﬁllet. For example, the cod is a ‘lean’ ﬁsh in which

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Table 9.14 Lipid and fatty acid compositions of the liver and muscle of Atlantic cod, Gadus morhua, fed on herring, Clupea harengus. SFAs, saturated fatty acids; MUFAs, monounsaturated fatty acids; PUFAs, polyunsaturated fatty acids; HUFAs, highly unsaturated fatty acids. Nd, not detectable. Adapted from data given by dos Santos et al. (1993). Herring

Composition (%) Lipid Moisture Lipid classes (%) Triacylglycerol Phospholipid Free fatty acids Fatty acids (%) SFAs 14 : 0 16 : 0 MUFAs 16 : 1(n-7) 18 : 1(n-9) 20 : 1(n-9) 22 : 1(n-11) PUFAs and HUFAs 18 : 2(n-6) 20 : 4(n-6) 18 : 3(n-3) 20 : 5(n-3) 22 : 6(n-3)

Atlantic cod Liver

Muscle

12.0 63.0

66.8 24.5

1.0 77.4

88 1 8

94 1 2

5 78 6

22.9 7.3 14.6 52.4 5.6 9.9 13.2 20.0 18.5 1.8 Nd 1.3 5.8 5.3

18.3 5.3 10.5 45.6 6.3 12.1 12.4 8.7 28.9 1.3 Nd 0.8 9.4 11.9

21.4 2.1 15.7 19.0 2.0 7.1 4.4 2.5 54.7 1.0 2.5 0.5 17.1 29.8

the ﬁllet lipids seldom exceed 1% of the ﬁllet mass (Tables 9.8 and 9.14) (Morais et al., 2001). The structural phospholipdis represent 65% or more of the muscle lipids, and there are only very small proportions of storage lipids, which are predominantly triacylglycerols (TAGs) (Lie et al., 1986; Love, 1988; dos Santos et al., 1993). The storage lipids of the cod are found in the liver, and farmed ﬁsh fed on lipid-rich moist feeds may develop large fatty livers. The liver may account for as much as 15–20% of the body weight, and have a lipid content of 70–75% by weight (Lie et al., 1986, 1988; Jobling et al., 1991, 1994; Grant et al., 1998). This is undesirable, because a large fatty liver will lead to increased ‘dress-out’ losses, and a concomitant relative reduction in ﬁllet yield. However, the lipids may be extracted from the liver to produce cod liver oil. Thus, the liver may yield a valuable commodity that is relatively rich in (n-3) HUFAs (Table 9.14) (Macrae et al., 1993; Morais et al., 2001), but the supplies of cod liver need to be regular and high to make oil extraction a commercially viable proposition. The yield of muscle tissue (ﬁllet) from ﬁsh is generally relatively larger than from terrestrial vertebrates. The locomotor muscle makes up ca. 30–70% of the body mass of ﬁsh, but the composition and nutritional value of the muscle will vary depending upon the species and the time of harvest (see Table 9.8) (Love, 1988; Jobling et al., 1991, 1994; Haard, 1992;

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Macrae et al., 1993; Lie et al., 1994; Takama et al., 1994; Jobling, 2001b). The locomotor muscle making up the ﬁllet comprises a lateral red component, used during low-speed cruising, and a much larger white muscle mass which is active during bursts of high-speed swimming. These two types of muscle differ in their metabolic enzyme spectra, degree of vascularisation and relative lipid content. The red muscle contains a higher percentage of lipid than does white muscle (see Fig. 9.12) (Haug et al., 1988; Love, 1988; Macrae et al., 1993; Jobling, 2001b), so ﬁllets taken from ﬁsh that have a relatively high proportion of locomotor ‘red’ muscle (e.g. mackerel and herring) tend to be both lipid-rich and susceptible to lipid peroxidation (see Fig. 9.5). Thus, these ﬁllets may easily become rancid during storage (Hsieh & Kinsella, 1989; Haard, 1992; Freeman & Hearnsberger, 1994; St Angelo, 1996). Depending upon species and season, the lipid content of the ﬁsh and its muscle can range from as little as 0.5% to over 20% (see Tables 9.4 and 9.8). From a human nutritional standpoint, ﬁsh can be classiﬁed into groups according to their muscle lipid content (Haard, 1992; Cowey, 1993; Macrae et al., 1993): lean (<2% lipid), low fat (2–4% lipid), medium fat (4–8% lipid) and high fat (<8% lipid). The muscle lipids of the lean species are mostly structural phospholipids that contain high levels of HUFAs (see Tables 9.8 & 9.14), whereas the muscle lipids of the ‘fattier’ species are dominated by storage TAGs. Farmed ﬁsh will usually contain relatively more lipid than their wild counterparts (Jobling, 1982; Haard,1992), and ﬁsh fed on lipid-rich feeds will usually deposit more storage TAGs than those fed on leaner diets (Cowey, 1993; dos Santos et al., 1993; Shearer, 1994; Higgs et al., 1995; Bell, 1998; Stickney, 2000; Jobling, 2001b; Regost et al., 2001). For example, farmed turbot, Scophthalmus maximus, were found to have a higher percentage of intramuscular lipid than wild turbot (1.06 vs. 0.64%), the difference being largely the result of an increased deposition of neutral lipids in the muscle of the farmed ﬁsh (0.52 vs. 0.24%) (Sérot et al., 1998). There were also differences between the farmed and wild ﬁsh in the fatty acid compositions of both the phospholipids and the neutral lipids, although the differences were most pronounced for the neutral lipids. The lipids of the farmed turbot had higher proportions of 18 : 2(n-6), 20 : 1 and 22 : 1, and lower proportions of 20 : 5(n-3) and 22 : 6(n3) than those of the wild turbot. This was clearly a reﬂection of the composition of the feed provided to the farmed ﬁsh: the feed contained 18 : 2(n-6) derived from cereals and plant oils, and 20 : 1 and 22 : 1 from the ﬁsh meal and capelin, Mallotus villosus, oil components (Sérot et al., 1998). Similarly, the fatty acid compositions of both the total lipid fraction and the phospholipids of juvenile turbot were found to differ among ﬁsh fed diets containing ﬁsh oil, linseed oil or olive oil as the main lipid sources (Bell et al., 1999). An inﬂuence of dietary fatty acid composition on ﬁllet fatty acids was seen in larger turbot by Sérot et al. (2001). Fillets of turbot given feed containing ﬁsh oil had relatively high concentrations of 22 : 6(n3), 20 : 1 and 22 : 1, ﬁllets from ﬁsh given soybean oil were high in 18 : 2(n-6), and ﬁllets of ﬁsh given feeds containing linseed oil had high concentrations of 18 : 3(n-3) and 18 : 2(n-6). Thus, the ﬁllet fatty acid proﬁles of these turbot closely resembled those of the feed oils (see Table 9.7 for the composition of feed oils). Turbot fed high-lipid feeds deposit more body lipid than those fed low-lipid feeds (Regost et al., 2001; Sæther & Jobling, 2001). For example, Regost et al. (2001) recorded that body

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lipid increased from 3% in turbot fed a 10% lipid feed to 4.8% in ﬁsh provided with a feed containing 25% lipid, but the authors were unable to discern any consistent trends in lipid deposition when individual tissues (liver, viscera, dorsal and ventral muscle, and skin) were examined. The trend towards increasing adiposity in turbot fed lipid-rich feeds can be reversed by changing the feed to one containing less lipid (see Fig. 9.13), i.e. percentage body lipid can decrease over time when ﬁsh are switched from a high-lipid to a low-lipid feed (Sæther & Jobling, 2001). When cod are fed on lipid-rich diets they tend to develop enlarged livers in which lipids, particularly TAGs, may represent over 65% of the liver weight (Lie et al., 1986, 1988; Jobling et al., 1991, 1994; dos Santos et al., 1993; Morais et al., 2001). The relative size of the liver has a direct relationship to the dietary lipid concentrations, or the quantity of lipid consumed. For example, when juvenile cod (ca. 250 g body weight) were fed on extruded feeds that differed in protein-to-lipid ratio (48 : 12, 48 : 16, 58 : 12 and 58 : 16) for 16 weeks there were minimal differences in weight gain, but ﬁsh given feeds containing 16% lipid tended to develop larger more lipid-rich livers (Morais et al., 2001). The fatty acid composition of the feed is most strongly reﬂected in that of the liver TAGs, but also inﬂuences the fatty acid composition of the phospholipids of the liver and muscle (see Table 9.14). For example, when cod were fed on either prawn, Pandalus borealis, or herring, Clupea harengus, the fatty acid compositions of the diets were reﬂected in the fatty acid proﬁles of the muscle lipids, ca. 75% of which were phospholipids (dos Santos et al., 1993). Thus, the muscle lipids of the cod fed on prawn had high levels of 18 : 1 fatty acids, whereas the muscle lipids of the ﬁsh fed on herring contained 18 : 1, 20 : 1 and 22 : 1. Whilst dietary type had an inﬂuence on the fatty acid proﬁles of the muscle lipids, the effects on liver lipids, most of which were TAGs, were much more pronounced. Cod fed on prawn, which contained relatively high levels of 16 : 0, 18 : 1 and 20 : 5, had liver lipids dominated by 16 : 0, 18 : 1 and 20 : 5, whereas the liver lipids of cod fed on herring had higher concentrations of 20 : 1 and 22 : 1, both of which were present in large quantities in the food (see Table 9.14). By the same token, cod fed a diet containing peanut oil with high concentrations of 18 : 1(n-9) and 18 : 2(n-6) deposited these fatty acids in the liver TAGs, whereas cod fed diets containing marine oils from either cod liver or Greenland halibut, Reinhardtius hippoglossoides, had much reduced concentrations of these fatty acids in their liver TAGs. The same trends were seen in the fatty acid compositions of the phospholipids of both the liver and muscle, in which 18 : 2(n-6) made up ca. 13% of the phospholipid fatty acids in the cod fed peanut oil, but only ca. 2% in the cod fed the marine oils (Lie et al., 1986). The composition of the feed has also been shown to inﬂuence the chemical composition of halibut, Hippoglossus hippoglossus. For example, the proportions of dry matter and lipid in the carcass tend to increase as the concentration of lipid in the feed is increased (Aksnes et al., 1996; Helland & Grisdale-Helland, 1998; Nortvedt & Tuene, 1998). In addition, lipid deposition tends to be higher in rapidly growing ﬁsh, and also increases with increasing ﬁsh size (Nortvedt & Tuene, 1998). Helland and Grisdale-Helland (1998) reported that when the lipid content of the feed was increased from 20 to 27% there was an increase in carcass lipid from ca. 8.5% to ca. 10.5%. In halibut, lipid concentrations tend to be highest in the liver (ca. 5–25%), in the ‘notch’ at the base of the anal ﬁn (sometimes in excess of 40%), and in

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the red muscle (ca. 10–20%) (see Fig. 9.12) (Haug et al., 1988; Berge & Storebakken, 1991; Cowey, 1993; Nortvedt & Tuene, 1998). There are also differences in lipid concentrations in different regions of the white muscle mass: the greatest relative amount of lipid is found in the abdominal area, with relative lipid content decreasing from the anterior towards the posterior regions (Haug et al., 1988; Hemre et al., 1992; Nortvedt & Tuene, 1998). Lipid accumulation in the area around the base of the anal ﬁn appears to be common amongst ﬂatﬁsh, and is attributed to fatty tissue, that is rich in TAGs ﬁlling the spaces between the muscles and the ﬁn rays. The lipid fractions of the liver, and the red and white muscle of the halibut are also dominated by TAGs, which make up ca. 60–70% of the total lipids in each of these tissues (Haug et al., 1988). The fatty acid composition of the storage TAGs most usually reﬂects the fatty acid composition of the feed (Lie et al., 1986; Sargent et al., 1989, 1995; Haard, 1992; Cowey, 1993; dos Santos et al., 1993; Sérot et al., 1998; Stickney, 2000; Jobling, 2001b), so it is possible to manipulate the fatty acid composition of the muscle (ﬁllet) storage TAGs via dietary means. The beneﬁcial effects of the (n-3) HUFAs EPA and DHA in human health have been extensively reported (Connor et al., 1992; Drevon, 1992; Lands, 1992; UauyDagach & Valenzuela, 1996; Lauritzen et al., 2001), and the natural abundance of these HUFAs in the lipids of ﬁsh has led to an increased consumer awareness of ﬁsh as a ‘health food’. However, TAGs usually contain lower proportions of HUFAs than do phospholipids, and the incorporation of HUFAs into the TAGs will usually be dependent upon them being provided (pre-formed) in the feed. Further, the HUFAs, which contain numerous double bonds within the molecule, are susceptible to peroxidative damage, and so must be protected by antioxidants to prevent the ﬁllet becoming rancid during storage (see Fig. 9.5) (Hsieh & Kinsella, 1989; Haard, 1992; Freeman & Hearnsberger, 1994; St Angelo, 1996). The requirement for antioxidants will obviously depend upon tissue levels of HUFAs at risk from peroxidation; the greater the concentration of HUFAs, the greater the need for antioxidant protection (Benzie, 1996; St Angelo, 1996). The extent to which the ﬂesh of farmed ﬁsh contains high levels of HUFAs will be governed by the relative proportions of red and white locomotor muscle in the ﬁllet, by muscle lipid content (i.e. whether the ﬁsh is one of the ‘lean’ or ‘fatty’ species), and by the fatty acid composition of the feed consumed. The majority of cold-water marine ﬁsh species under consideration for commercial farming (e.g. some gadoids, marine ﬂatﬁsh and wolf-ﬁsh) have a low proportion of red muscle in the ﬁllet. However, muscle lipid concentrations of farmed ﬁsh tend to be elevated in comparison with those of wild ﬁsh, and the ﬁllet of farmed ﬁsh is envisaged to have considerable potential as a vehicle for delivering HUFAs to human consumers. Consequently, attention needs to be paid to the potential risk of peroxidation of the HUFAs in the ﬁllets of farmed cold-water marine ﬁsh, and precautions must be taken to prevent the development of ﬂesh rancidity during transport and storage. Fatty acid oxidation products and their derivatives, e.g. alcohols and aldehydes, impart characteristic ﬂavours and odours to ﬁsh and ﬁsh products (Lindsay, 1990; Macrae et al., 1993; St Angelo, 1996; Haard & Simpson, 2000). The typical aroma of ﬁsh is related to volatile compounds arising from the oxidation of long-chain (n-3) fatty acids (PUFAs and HUFAs) through a combination of the actions of lipoxygenase enzymes and autoxidation reactions. Both aldehyde and alcohol derivatives of fatty acid oxidation contribute to the

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aroma of a ﬁsh ﬁllet, but which ﬂavour and odorant volatiles are produced will depend on the composition of the ﬁllet with respect to the relative proportions of (n-3) and (n-6) PUFAs and HUFAs. For example, Sérot et al. (2001) reported that the proﬁles of odour-active compounds in the ﬁllet of turbot differed depending upon the type of oil (ﬁsh, soybean or linseed) used to formulate the feed. Volatiles generated during the oxidation of (n-3) PUFAs and HUFAs contributed strongly to the odour of the ﬁllets of turbot given feeds containing ﬁsh oil and linseed oil, whereas hexanal, which is derived from the oxidation of (n-6) fatty acids, and decanal, which results mainly from 18 : 1(n-9) oxidation, were perceived as odorants in the turbot given feeds formulated to contain soybean and linseed oils. The ﬂavour of ﬁsh is also inﬂuenced by the presence of compounds arising from the hydrolysis of muscle proteins: free amino acids, di-, tri- and oligopeptides (Love, 1988; Macrae et al., 1993; Haard & Simpson, 2000). For example, enzymatic hydrolysis of proteins may lead to the production of ‘bitter’ hydrophobic peptides, but hydrolysis of these peptides to free amino acids decreases bitterness because the hydrophobic peptides impart a far more bitter taste than do mixtures of their constituent amino acids (Kristinsson & Rasco, 2000). The hydrolysis of the muscle proteins occurs following the slaughter of the ﬁsh, and the ﬁllet ﬂavour will develop and change as the small peptides and free amino acids are liberated over time (Fig. 9.14). If ﬁsh are held overlong under unfavourable conditions, there is a risk of the growth of bacteria. This eventually leads to spoilage due to the accumulation of ammonia and the acidic products of bacterial degradation, including those derived from the sulphur-containing amino acids (Macrae et al., 1993). Bacterial enzymes may also result in the decarboxylation of certain amino acids, e.g. tyrosine, arginine, lysine and histidine, resulting in increased concentrations of the biogenic amines tyramine, putrescine, cadaverine and histamine. As time progresses, the taste characteristics of the ﬁllet change from a sweet to a neutral ﬂavour, and then to the sour and bitter tastes of spoilage products. Concomitant with the chemical changes resulting from autolytic processes and the actions of the spoilage bacteria, the ﬁllet also undergoes a series of textural changes (Fig. 9.14) (Love, 1988; Haard, 1992; Macrae et al., 1993). Many of these changes in texture are related to alterations in the structure of the muscle proteins, particularly collagen and the actomyosin complex of the sacromere. The muscle is made up of muscle ﬁbres bound together by connective tissue, and ﬁsh muscle usually has 15–19% protein by weight (see Tables 9.4 and 9.8). The muscle comprises a limited number of abundant proteins, actin, myosin and collagen, together with several minor components such as elastin, troponin and actinin. Collagen is the main connective tissue protein, and the quantities present have a major inﬂuence on the texture of the ﬂesh. The proportion of collagen in ﬁsh ﬂesh is much lower than that in meat from terrestrial animals, making ﬁsh ﬂesh more tender than that of terrestrial animals. The other proteins than contribute to texture are the myoﬁbrillar proteins which make up the contractile unit, the sarcomere, of the muscle. The sarcomeres consist of thick ﬁlaments, mainly of myosin, that are interdigitated with thin ﬁlaments, which are predominantly actin. Thus, actin and myosin together form the actomyosin complex, which is responsible for muscle contraction and relaxation. The muscle proteins are often categorised based on their solubility characteristics. The water-soluble, or sarcoplasmic, proteins occur dispersed within the liquid medium of the

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Figure 9.14 Post-mortem changes in the ﬁllet (skeletal muscle) of a ﬁsh resulting in textural changes and the development of ﬂavour, and ﬁnally to spoilage.

muscle cells, so any loss of liquid that occurs during processing and storage of the ﬁllet will result in a loss of sarcoplasmic proteins. The myoﬁbrillar, contractile proteins are saltsoluble, whereas the stroma proteins, primarily collagen, are insoluble (Love, 1988; Haard, 1992; Macrae et al., 1993). Fish muscle tends to contain a relatively high proportion of

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myoﬁbril protein (65–75%), and lower proportions of sarcoplasmic (20–30%) and stroma (2–5%) proteins. Myosin comprises 50–60% of the myoﬁbrillar contractile proteins, and actin 15–30%. Thus, myosin is the most abundant of the muscle proteins, making up about 35–40% of the total. The proteins of the actomyosin complex play a major role in determining the textural properties of the ﬁsh ﬁllets because they are labile, and are inﬂuenced by processing and storage conditions. For example, actomyosin becomes progressively less soluble during frozen storage, and this results in the ﬁllet becoming increasingly tough. Depending on the workload of the muscle, its metabolism may be either aerobic or anaerobic, and in the struggle of capture anaerobic metabolism will predominate. At capture, the ﬂesh of the ﬁsh has a ﬁrm elastic feel, but textural changes begin to occur within a few hours of slaughter. After death, the blood no longer transports oxygen and nutrients to the muscle, nor is there any removal of metabolites and waste products of catabolism. Nevertheless, catabolic reactions continue in the muscle tissue, and there is anaerobic hydrolysis of muscle glycogen which leads to accumulation of lactic acid. Concomitant with the production of lactic acid, adenosine triphosphate (ATP) continues to be produced. As the reserves of muscle glycogen are depleted and lactate accumulates, there is a fall in muscle pH. At the same time, the concentration of ATP starts to fall as it is degraded via dephosphorylation and deamination reactions to ADP, AMP and inosine monophosphate (IMP) (see Fig. 9.14). These reactions are faster than the subsequent degradation reaction, and there is an accumulation of IMP. Within a few hours of death, the concentration of ATP falls to a level at which the enzymes that maintain the muscle in a state of readiness for contraction can no longer function effectively. The muscles start to contract, and the ﬁsh becomes stiff as it enters rigor mortis. Eventually, after a period of time that can range from ca. 2 h to over 100 h depending upon factors such as species, muscle condition and temperature, the muscles go limp again, and rigor is said to be resolved. This softening is not a reversal of rigor, but arises as a result of autolysis of the muscle protein, including the collagen of the connective tissue. As proteolysis results in a breakdown of the cross-links between the collagen ﬁbres within the connective tissue, the ﬂesh becomes increasingly soft in texture. The fall of muscle pH following death has a major inﬂuence upon the development of texture. Fish that develop a low post-mortem pH usually have a ﬁrm-textured ﬂesh, but a rapid fall in pH after death results in a rapid onset of rigor and this can have several undesirable consequences. Early onset of rigor may result in soft-textured ﬂesh with poor waterholding properties, and a ﬁllet that is prone to gaping (Love, 1988). Gaping refers to the separation of the muscle blocks (myotomes of the ﬁllet) that occurs when the connective tissue myosepta between the myotomes weaken and break. Whether or not this occurs depends upon the strength of the myosepta and the intensity of the muscular contraction that occurs with the onset of rigor; the risk of gaping may be high when the myosepta have been weakened due to partial hydrolysis and denaturation of their proteins, and this is combined with intense muscular contraction under rigor. Thus, the onset of gaping appears to be related to the cleavage of acid-labile cross-links between the collagen ﬁbres of the myosepta, leading to a weakening and rupturing of the connective tissue. The effects of the rupture of the

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myosepta will be observed as gaping once rigor has resolved and the ﬁllet starts to soften. At the time that rigor resolves, the texture of the ﬁllet should be ﬁrm, but the cooked ﬁllet can be chewed easily, and the ﬂavour should be sweet. Flavour nuances are mainly due to the presence of free amino acids, and the ﬂavour is enhanced by IMP, which is at its highest concentration at about this time (see Fig. 9.14). As time progresses there is dephosphorylation of the IMP, leading to a transient accumulation of inosine. Cleavage and degradation of the inosine leads to the production of ribose, ribose-1-phosphate and hypoxanthine. Measurements of the concentrations of hypoxanthine in the ﬁllet are sometimes used as a spoilage indicator. The accumulation of trimethylamine (TMA), produced via the bacterial reduction of trimethylamine oxide (TMAO) coupled with the oxidation of lactic acid, is also used to assess the degree of spoilage of ﬁsh ﬁllets. TMAO is used by some bacteria as the terminal electron receptor of the respiratory chain, enabling them to grow in the absence of oxygen, and the presence of TMAO in the muscle of marine ﬁsh may explain why their ﬁllets are particularly prone to spoilage. In addition to TMA, other products arising from bacterial activity include amines and ammonia from the degradation of amino acids, sulphides and methyl mercaptan from the sulphurcontaining amino acids, acidic products arising from the degradation of carbohydrates, and various products of fatty acid oxidation, such as hexanal, pentane and nonanal, arising from the hydrolysis and metabolism of lipids (Macrae et al., 1993; St Angelo, 1996; Haard & Simpson, 2000). Eventually, the accumulation of the acidic products of bacterial degradation causes the ﬁllet to become ‘sour’ and inedible.

9.11 Concluding Comments Although farming of several cold-water marine ﬁsh can be carried out proﬁtably, it is probable that both the dietary formulations and feeding regimes used are sub-optimal. Consequently, there may be considerable scope for increasing growth rates and improving feed efﬁciencies as more knowledge about the nutritional requirements and feeding behaviours of the different species becomes available. Hand-feeding has traditionally been the most widely used method for feeding farmed cold-water marine ﬁsh, this method being largely dictated by the use of trash ﬁsh, or wet and moist feeds. However, there is a trend towards a reduced reliance on these feeds, and an increased use of dry feeds is likely. The main reasons relate to the ease of storage and handling of dry feeds, the fact that they are easy to distribute using automatic feeding systems, their greater water stability than wet or moist feeds, and the fact that it is easier to maintain consistency of nutrient composition in dry feeds than in the other feed types. Most automatic feeding systems are designed to distribute dry feeds, and the increased reliance upon such feeds in the farming of cold-water marine ﬁsh will increase the focus on the way in which automatic feeding systems can be adapted to improve feeding routines. Thus, feed management will be given increased attention, leading to both improved feed efﬁciencies and reduced environmental pollution from feed waste. To this end, it is likely that interactive feeding systems that control feed delivery based upon the feeding response of the ﬁsh will be developed especially for use in marine ﬁsh farming.

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Dry feeds manufactured for intensively farmed carnivorous ﬁsh, including the cold-water marine species, have a high protein content; most of the protein is supplied via ﬁsh meal and most of the lipid via marine ﬁsh oils. Marine ﬁsh oils are the best sources of (n-3) HUFAs, and a source of these fatty acids must be included in diets formulated for cold-water marine ﬁsh because they are unable to chain-elongate and desaturate the 18C precursors of these HUFAs. The inclusion of marine ﬁsh oils in feeds for cold-water marine species should give marketing beneﬁts due to the deposition of the (n-3) HUFAs in the ﬂesh of the ﬁsh. The positive effects of the (n-3) HUFAs for human health have been extensively reported, so ﬁsh ﬁllets containing an abundance of these fatty acids could be marketed with a ‘health food’ image. The production of ﬁsh meals and oils is not expected to increase in the future, so there would seem to be limited potential for the expansion of the aquaculture industry based exclusively upon the increased use of ﬁsh products as feed ingredients. Numerous alternative protein sources, mostly of plant origin, are available, but few of these have been rigorously tested as feed ingredients for cold-water marine species. Thus, the search for, and testing of, protein sources that can act as alternatives to ﬁsh meal remain priorities for aquafeed development. Similarly, it is a major priority to ﬁnd alternative lipid sources that can be used in partial replacement of marine ﬁsh oils, without leading to any marked compromise in the composition and ‘quality’ of the ﬁnal product.

9.12 References Aksnes, A. & Mundheim, H. (1997) The impact of raw material freshness and processing temperature for ﬁsh meal on growth, feed efﬁciency and chemical composition of Atlantic halibut (Hippoglossus hippoglossus). Aquaculture, 149, 87–106. Aksnes, A., Hjertnes, T. & Opstvedt, J. (1996) Effect of dietary protein level on growth and carcass composition in Atlantic halibut (Hippoglossus hippoglossus L.). Aquaculture, 145, 225– 33. Alanärä, A. (1996) The use of self-feeders in rainbow trout (Oncorhynchus mykiss) production. Aquaculture, 145, 1–20. Alanärä, A., Kadri, S. & Paspatis, M. (2001) Feeding management. In: Food Intake in Fish (eds D. Houlihan, T. Boujard & M. Jobling), pp. 332–53. Blackwell Science, Oxford. Albrektsen, S., Waagbø, R., Lie, Ø. & Sandnes, K. (1994) Contents and organ distribution of vitamin B6 in Atlantic salmon (Salmo salar) and turbot (Psetta maxima) during the reproductive cycle. Comp. Biochem. Physiol., 109A, 705–12. Allan, G.L., Rowland, S.J., Parkinson, S., Stone, D.A.J. & Jantrarotai, W. (1999) Nutrient digestibility for juvenile silver perch Bidyanus bidyanus: development of methods. Aquaculture, 170, 131–45. Anderson, R.L. & Wolf, W.J. (1995) Compositional changes in trypsin inhibitors, phytic acid, saponins and isoﬂavones related to soybean processing. J. Nutr., 125, 581S–8S. Ang, K.P. & Petrell, R.J. (1997) Control of feed dispensation in seacages using underwater video monitoring: effects on growth and food conversion. Aquacult. Eng., 16, 45–62. Ang, K.P. & Petrell, R.J. (1998) Pellet wastage, and subsurface and surface feeding behaviours associated with different feeding systems in sea cage farming of salmonids. Aquacult. Eng., 18, 95–115. Anthony, J.A., Roby, D.D. & Turco, K.R. (2000) Lipid content and energy density of forage ﬁshes from the northern Gulf of Alaska. J. Exp. Mar. Biol. Ecol., 248, 53–78.

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Atlantic halibut (Hippoglossus hippoglossus) fed three pellet sizes and three dietary fat levels. Aquaculture, 161, 295–313. Oliva-Teles, A., Pereira, J.P., Gouveia, A. & Gomes, E. (1998) Utilisation of diets supplemented with microbial phytase by seabass (Dicentrarchus labrax) juveniles. Aquat. Living Resour., 11, 255–9. Osborne, D.R. & Voogt, P. (1978) The Analysis of Nutrients in Foods. Academic Press, London. Payne, S.A., Johnson, B.A. & Otto, R.S. (1999) Proximate composition of some north-eastern Paciﬁc forage ﬁsh species. Fish. Oceanogr., 8, 159–77. Pedersen, J. & Hislop, J.R.G. (2001) Seasonal variations in the energy density of ﬁsh in the North Sea. J. Fish Biol., 59, 380–9. Pedersen, T. & Jobling, M. (1989) Growth rates of large, sexually mature cod, Gadus morhua, in relation to condition and temperature during an annual cycle. Aquaculture, 81, 161–8. Petrell, R.J., Shi, X., Ward, R.K., Naiburg, N. & Savage, C.R. (1997) Determining ﬁsh size and swimming speed in cages and tanks using simple video techniques. Aquacult. Eng., 16, 63–84. Pichavant, K., Person-Le Ruyet, J., Le Bayon, N., Sévère A., Le Roux A., Quéméner L., Maxime V., Nonnotte G. & Boeuf G. (2000) Effects of hypoxia on growth and metabolism of juvenile turbot. Aquaculture, 188, 103–14. Pichavant, K., Person-Le Ruyet, J., Le Bayou, N., Sévère, A., Le Roux, A. & Boeuf, G. (2001) Comparative effects of long-term hypoxia on growth, feeding and oxygen consumption in juvenile turbot and European sea bass. J. Fish Biol., 59, 875–83. Pike, I.H., Andorsdottir, G. & Mundheim, H. (1990) The role of ﬁsh meal in diets for salmonids. IAFMM Tech. Bull., 24, 35 pp. Purdom, C.E. (1974) Variation in ﬁsh. In: Sea Fisheries Research (ed F.R. Harden Jones), pp. 347–55. Elek Science, London. Refstie, S., Helland, S.J. & Storebakken, T. (1997) Adaptation to soybean meal in diets for rainbow trout, Oncorhynchus mykiss. Aquaculture, 153, 263–72. Regost, C., Arzel J., Cardinal, M., Robin, J., Laroche, M. & Kaushik S.J. (2001) Dietary lipid level, hepatic lipogenesis and ﬂesh quality in turbot (Psetta maxima). Aquaculture, 193, 291–309. Riche, M. & Brown, P.B. (1996) Availability of phosphorus from feedstuffs fed to rainbow trout, Oncorhynchus mykiss. Aquaculture, 142, 269–82. Riche, M., White, M.R. & Brown, P.B. (1995) Barium carbonate as an alternative indicator to chromic oxide for use in digestibility experiments with rainbow trout. Nutr. Res., 15, 1323–31. Sæther, B.-S. & Jobling, M. (1999) The effects of ration level on feed intake and growth, and compensatory growth after restricted feeding, in turbot, Scophthalmus maximus L. Aquacult. Res., 30, 647–53. Sæther, B.-S. & Jobling, M. (2001) Fat content in turbot feed: inﬂuence on feed intake, growth and body composition. Aquacult. Res., 32, 451–8. Sandnes, K. (1991) Vitamin C in ﬁsh nutrition—A review. FiskeriDir. Skr., Ser. Ernæring, 4, 3–32. Sandnes, K., Rosenlund, G., Mangor-Jensen, A. & Lie, Ø. (1998) Contents and organ distribution of pantothenic acid in maturing turbot (Psetta maxima). Aquacult. Nutr., 4, 285–6. Sardesai, V.M. (1992) Biochemical and nutritional aspects of eicosanoids. J. Nutr. Biochem., 3, 562–79. Sargent, J., Bell, J.G., Bell, M.V., Henderson, R.J. & Tocher, D.R. (1995) Requirement criteria for essential fatty acids. J. Appl. Ichthyol., 11, 183–98. Sargent, J., Henderson, R.J. & Tocher, D.R. (1989) The lipids. In: Fish Nutrition (ed J.E. Halver), pp. 153–218. Academic Press, London. Schreckenbach, K., Knösche, R. & Ebert, K. (2001) Nutrient and energy content of freshwater ﬁshes. J. Appl. Ichthyol. 17, 142–4.

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Schwarz, F.J. (1995) Determination of mineral requirements of ﬁsh. J. Appl. Ichthyol., 11, 164–74. Selle, P.H., Ravindran, V., Caldwell, R.A. & Bryden, W.L. (2000) Phytate and phytase: consequences for protein utilisation. Nutr. Res. Rev., 13, 255–78. Sérot, T., Gandemer, G. & Demaimay, M. (1998) Lipid and fatty acid compositions of muscle from farmed and wild turbot. Aquacult. Int., 6, 331–43. Sérot, T., Regost, C., Prost, C., Robin, J. & Arzel, J. (2001) Effect of dietary lipid sources on odouractive compounds in muscle of turbot (Psetta maxima). J. Sci. Food Agric., 81, 1339–46. Shearer, K.D. (1994) Factors affecting the proximate composition of cultured ﬁshes with emphasis on salmonids. Aquaculture, 119, 63–88. Shearer, K.D. (1995) The use of factorial modelling to determine the dietary requirements for essential elements in ﬁshes. Aquaculture, 133, 57–72. Shearer, K.D. (2000) Experimental design, statistical analysis and modelling of dietary nutrient requirement studies for ﬁsh: a critical review. Aquacult. Nutr., 6, 91–102. Shelverton, P.A. & Carter, C.G. (1998) The effect of ration on behaviour, food consumption and growth in juvenile greenback ﬂounder (Rhombosolea tapirina: Teleostei). J. Mar. Biol. Assoc. UK, 78, 1307–20. Simensen, L.M., Jonassen, T.M., Imsland, A.K. & Stefansson, S.O. (2000) Photoperiod regulation of growth in juvenile Atlantic halibut (Hippoglossus hippoglossus L.). Aquaculture, 190, 119– 28. St Angelo, A.J. (1996) Lipid oxidation in foods. Crit. Rev. Food Sci. Nutr., 36, 175–224. Stickney, R.R. (ed) (2000) Encyclopedia of Aquaculture. John Wiley, New York. Summerfelt, S.T., Holland, K.H., Hankins, J.A. & Durant, M.D. (1995) A hydroacoustic waste-feed controller for tank systems. Water Sci. Technol., 31, 123–9. Swanson, C. (1998) Interactive effects of salinity on metabolic rate, activity, growth and osmoregulation in the euryhaline milkﬁsh (Chanos chanos). J. Exp. Biol., 201, 3355–66. Takama, K., Suzuki, T., Yoshida, K., Arai, H. & Anma, H. (1994) Lipid content and fatty acid composition of phospholipids in white-ﬂesh ﬁsh species. Fish. Sci., 60, 177–84. Thetmeyer, H., Waller, U., Black, K.D., Inselmann, S. & Rosenthal, H. (1999) Growth of European sea bass (Dicentrarchus labrax L.) under hypoxic and oscillating oxygen conditions. Aquaculture, 17, 355–67. Tocher, D.R., Bell, J.G., Dick, J.R., Henderson, R.J., McGhee, F., Michell, D. & Morris, P.C. (2000) Polyunsaturated fatty acid metabolism in Atlantic salmon (Salmo salar) undergoing parr–smolt transformation and the effects of dietary linseed and rapeseed oils. Fish Physiol. Biochem., 23, 59–73. Tocher, D.R., Bell, J.G., MacGlaughlin, P., McGhee, F. & Dick, J.R. (2001) Hepatocyte fatty acid desaturation and polyunsaturated fatty acid composition of liver in salmonids: effects of dietary vegetable oil. Comp. Biochem. Physiol., 130B, 257–70. Tocher, D.R., Mourante, G. & Sargent, J.R. (1992) Metabolism of [1-14C] docosahexaenoate (22:6n3), [1-14C] eicosapentaenoate (20:5n-3) and [1-14C] linolenate (18:3n-3) in brain cells from juvenile turbot, Scophthalmus maximus. Lipids, 27, 494–9. Tucker, J.W. Jr. (1998) Marine Fish Culture. Kluwer Academic, Boston. Tusé, D. (1984) Single-cell protein: Current status and future prospects. CRC Crit. Rev. Food Sci. Nutr., 19, 273–325. Uauy-Dagach, R. & Valenzuela, A. (1996) Marine oils: the health beneﬁts of n-3 fatty acids. Nutr. Rev., 54, S102–S108. Van Barneveld, R.J. (1999) Understanding the nutritional chemistry of lupin (Lupinus spp.) seed to improve livestock production efﬁciency. Nutr. Res. Rev. 12, 203–30. Van Pelt, T.I., Piatt, J.F., Lance, B.K. & Roby, D.D. (1997) Proximate composition and energy density of some North Paciﬁc forage ﬁshes. Comp. Biochem. Physiol., 118A, 1393–8.

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Wiegand, M.D. (1996) Composition, accumulation and utilization of yolk lipids in teleost ﬁsh. Rev. Fish Biol. Fish., 6, 259–86. Wilson, R.P. (1989) Amino acids and proteins. In: Fish Nutrition (ed J.E. Halver), pp. 111–51. Academic Press, London. Wilson, R.P. (ed) (1991) Handbook of Nutrient Requirements of Finﬁsh. CRC Press, Boca Raton. Wilson, R.P. (1994) Utilization of dietary carbohydrate by ﬁsh. Aquaculture, 124, 67–80. Yamamoto, T. & Akiyama, T. (1995) Effect of carboxymethylcellulose, a-starch and wheat gluten incorporated in diets as binders on growth, feed efﬁciency, and digestive enzyme activity of ﬁngerling Japanese ﬂounder. Fish. Sci., 61, 309–13.

Chapter 10

The Status and Perspectives for the Species T. Svåsand, H.M. Otterå and G.L. Taranger

10.1 Atlantic Cod 10.1.1 Introduction Atlantic cod is one of the most famous cold-water ﬁsh species in the world, and has recently been the subject of a biography, Cod. A Biography of the Fish that Changed the World, by Mark Kurlansky, who has written an exciting story about the ﬁsh that has been traded for a millennium on four continents. Atlantic cod is distributed on both sides of the Atlantic. In the west Atlantic, cod is found from Cap Hatteras north to the ice edge. In the east Atlantic, cod is found from the Bay of Biscay in the south to the northern part of the Barents Sea. Atlantic cod is distributed in several stocks, and each stock has its own distinct life history characteristics and migration patterns. The annual landings from most of the wild stocks have been declining, and several of the stocks are now very small compared with historical levels. In Canada, the ﬁshery for Atlantic cod was stopped in 1992 in some areas, and in the east Atlantic the annual landings in 2000 (894 000 metric tonnes) were only half of the average catches over the last 30–50 years. North-east Arctic cod is the largest of these stocks. Fish in this stock make long spawning migrations from feeding areas in the Barents Sea to the spawning grounds at Lofoten and along the Norwegian coast. As well as the large oceanic stocks, more stationary local cod stocks are found. In Norway, stock enhancement programmes and farming experiments have been carried out with local cod (coastal cod) as the brood stock. The ﬁrst cultivation experiments with Atlantic cod go back to the 1880s, when attempts were initiated in Norway, the USA and Canada to stock the sea annually with hundreds of millions of newly hatched yolk-sac larvae. This activity was continued for nearly 90 years, although the beneﬁts of the releases were not documented.

10.1.2 Brood Stock, Egg Production and Incubation Natural spawning is the easiest and most usual way to obtain cod eggs for aquaculture (Fig. 10.1). Cod easily spawn in captivity, and due to the high fecundity, large amounts of high quality eggs and larvae can be produced with little effort. Cod are a batch spawner that spawn

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Figure 10.1 System for collecting eggs from naturally spawning cod (Holm et al., 1991).

1,5

1800 1600

Amount of eggs (ml)

1200 1,4 1000 800 1,35 600 400

Diameter (mm)

1,45

1400

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200 0 00 20 .2. 22

1,25 00 20 .2. 27

00 .20 3.3

00 20 .3. 10

00 20 .3. 17

00 20 .3. 22

00 .20 4.4

Figure 10.2 Egg production (bars, left-hand scale) and egg diameter (points, right-hand scale) of a female cod kept in a spawning tank during the spawning season in 2000. The size of the female prior to the spawning season was 73 cm and 5.4 kg. Data from an EU project (Svåsand et al., 2000a).

multiple batches of small pelagic eggs approximately every 3 days during the spawning season from February to April (Fig. 10.2). Juvenile cod can be produced by extensive and semi-extensive methods where the larvae are start-fed on natural zooplankton, or in intensive systems where all parts of the production are controlled, and are not dependent on natural variations in controlling parameters such as temperature and zooplankton densities (Fig. 10.3, Table 10.1).

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Sea water enclosures -Norway Intensive production - Canad a

Semi-intensive - Norway Intensive production - UK

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Intensive production- Norway

600

Production in thousands

500 400 300 200 100

19 86 19 87 19 88 19 89 19 90 19 91 19 92 19 93 19 94 19 95 19 96 19 97 19 98 19 99 20 00 20 01

0

Figure 10.3 Production of juvenile cod (>10 g) in Norway between 1986 and 2001 (Anonymous, 1995; Kvenseth et al., 2000; Glette et al., 2002), and in the UK and Canada from 1999 (ICES, no data available for 2001).

Table 10.1 Overview of the main production methods used for production of cod in Norway. The table is based on results from the period 1990–1994, and production is calculated as the number of collected cod juveniles larger than 1 g (wet weight). The table is slightly modiﬁed from Svåsand et al. (2000b). Production method

Volume (m3)

Feed

Sea-water enclosure Tanks on land Plastic bags in sea-water enclosures Intensive production

37 000–510 000 450–800 64–200

Copepods etc.a Naturalb Collectedc

0.15–4

Cultivatedd

Prey density (plankton l-1)

Stocking density (larvae l-1)

2–500 10–300 1–100

0.01–0.32 1.4–2.8 1.2–2.2

1000–6000

10–30

Production (juveniles m-3) 0.02–1.6 3.8–30 33–546 0–416

a

Prey organisms were produced naturally in the system from resting eggs of copepods. Algae production was manipulated by the supply of nutrients and the degree of water turbulence. In some cases, zooplankton were supplied from the surrounding seawater. b Some prey organisms were produced naturally in the system. The main supply were ﬁltered from the surrounding sea. c A supply of zooplankton collected from the enclosure or from the surrounding sea. d Supply of cultivated algae, rotifers and Artemia.

10.1.3 Extensive Production The ﬁrst artiﬁcial production of juvenile cod happened in Norway in 1886, when several thousand cod juveniles were produced in a 2500-m3 basin at the Flødevigen Research Station (now part of the Institute of Marine Research). The purpose of that experiment was to test the viability of millions of hatched larvae released on the Norwegian Skagerrak coast between 1884 and 1971 (Solemdal et al., 1984). Similar cultivation experiments with Atlantic cod were done in the USA and Canada.

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Ninety years later, new efforts were put into the development of methods for the production of juveniles of marine ﬁsh in the basins at Flødevigen, and experiments in 1976 and 1977 showed that it was fully possible to produce cod juveniles fed natural plankton in mesocosms. The motive for using natural production systems was so that cod larvae would start to feed on the same natural zooplankton as wild cod, while the removal of predators would secure a high survival rate. In the 1980s, this research was moved to the Austevoll Marine Research Station, Institute of Marine Research, and in 1983 the researchers managed to produce more than 70 000 small cod in a 60 000-m3 sea-water enclosure. This was the start of ‘another cod period’ in Norway, and several different production systems were built at the lagoon Parisvatnet (Fig. 10.4). The main production results are summarised in Fig. 10.3 and Table 10.1. As part of the sea-ranching activity, cod juveniles were also produced in Sweden, Denmark and the Faroe Islands. The major obstacle in using extensive and semi-intensive production has been the large variations in the production of juvenile ﬁsh, which is often correlated to variations in the zooplankton community (Table 10.1). Therefore considerable effect has been put into studies of their ecosystems as a basis for a more efﬁcient production. However, since the supply of zooplankton seems to be the key factor, the development of more efﬁcient zooplankton ﬁltration systems will probably be the best way to increase production, together with a supply of Artemia and early weaning. The quality of the juvenile cod produced by extensive and semi-intensive systems has been good, and comparable with that of wild cod. However, although major efforts have been put into optimising these systems, the production results have been too small and unpredictable to be commercially viable.

10.1.4 Intensive Production In the 1980s, small-scale intensive production of Atlantic cod had already started in both the UK and Norway. Intensive juvenile production is based on the control of all aspects of production, including water exchange, light, density of both cod and feed organisms, the quantity and quality of feed organisms, and hygiene and health conditions. The newly hatched larvae are transferred to start-feeding units (tanks 50–1000 l) where they are startfed on intensively produced food organisms (microalgae, rotifers), and later Artemia and subsequently formulated feed. Systems for the intensive production of marine ﬁsh are explained in detail in Chapter 7. In Norway, large-scale intensive production was tried in 1990–1993 by BP Nutrition ARC/Salar A/S Bessaker in cooperation with several Norwegian research institutions. The large-scale production of rotifers and Artemia was developed, and a high number of metamorphosed cod were produced. However, mortality was high after metamorphosis, mainly as the result of cannibalism. Sub-optimal feeding conditions, but also the physical rearing conditions, were recognised as the main reason for the cannibalistic behaviour. Even though the results from these large-scale trials were promising, the trials were ended in 1993, mainly as a result of the general disbelief in cod as an economically feasible aquaculture species at that time.

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437

A

B Figure 10.4 Juvenile cod have been produced successfully in seawater lagoons such as the one at Parisvatnet (A) in western Norway (area, 50 000 m2; volume, 270 000 m3). Local cod are captured as brood stock (B). Fertilised eggs are incubated in the hatchery (early March), and 3–5 million yolk-sac larvae are released in a predator-free mesocosm in late March (treated with rotenone 4 months earlier). The cod larvae start to feed on naturally occurring zooplankton. After metamorphosis, the supply of zooplankton is through large ﬁlters (C, capacity >50 m3). Later formulated feed is also supplied (>0.5 g), and from a size of about 1 g (June), cod are collected from the mesocosm using a dip net (D), sorted, vaccinated and moved to net pens for on-growing to a size of 50–100 g (September– October), when they are sold and transported to ﬁsh farmers for on-growing to market-size ﬁsh (Photo: Leiv Aardal, Institute of Marine Research (SMR), Norway).

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C

D Figure 10.4 Continued.

In recent years, low catches from the wild ﬁsheries and the high market price for cod has renewed interest in cod farming. Intensive pilot-scale hatcheries have been built in both the UK and Canada. In 1999, the production of 30 000 juvenile cod was achieved in Scotland, and 50 000 in Newfoundland. Juvenile cod production has also been reconsidered in Norway, and the focus is now on intensive-rearing methods. In 2001, about 400 000 (40% of the total production) were produced by this method. Several large commercial cod hatcheries are now being planned, so it is expected that production will increase signiﬁcantly in the coming years. The renewed belief in intensive rearing methods for cod juveniles is due to the success in rearing temperate-water species such as sea bass and sea bream in such systems. The major challenge is to adapt and further develop these methods for cod at a scale that is

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439

economically proﬁtable. Important factors for the successful production of cod juveniles include:

• a year-round supply of eggs of high quality • feeding regimes and physical rearing conditions that prevent aggression and cannibalism • control of environmental and microbial conditions in the tanks • live feed enrichment, weaning diets and feeding regimes which are suitable for cod The results from several recent experiments suggest that cod can actually be reared successfully in intensive systems; cod larvae can have growth rates of about 25% day-1 if the temperature is optimal (close to 14°C). Experiments in small tanks have also shown that cod larvae can be reared successfully at high densities (300 l-1), and can be weaned onto formulated feeds at a stage which is comparable to that of other aquaculture species. It is probable that the wide-ranging effort that is now being put into the development of intensive production of Atlantic cod will result in large-scale commercial production (Fig. 10.5). However, it is important to keep in mind that it is more than 110 years since the ﬁrst juvenile cod were produced in a basin in Flødevigen (several thousand cod juveniles were produced), and 20 years since the ﬁrst breakthrough in juvenile cod production in a seawater enclosure at Austevoll (with nearly 70 000 cod juveniles produced). A ﬁnal breakthrough will depend on accumulated knowledge of Atlantic cod and of other marine species, and also on adequate resources being put into both scientiﬁc studies and full-scale production experiments.

1 - g ju ve n a

il e

s

la

rv ae

lar

v

g

l Yo

k-

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c

ac

Ju

-s Yolk

Rotifers

p

il e

ju v

e n il e s

1

-g

Se

n

esn uivle jn j1u-vge

Formulated feed Artemia

Yo lks ac

la r v a

Figure 10.5 The main components of a future full-scale production unit for juveniles of Atlantic cod.

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Culture of cold-water marine ﬁsh

10.1.5 On-Growing From the mid-1980s, sea-cage farming of cod has been attempted in Norway using similar technology to salmon farms (Fig. 10.6). However, problems with low prices, variable quality and slow growth have impaired the development of the industry. On the other hand, the recent shortage of wild cod in Europe has increased the interest in cod farming in Norway, as prices have increased dramatically in the last few years. Economic modelling has indicated production costs of cod in the range NKr 15–18 kg-1, and ﬁsh farmers have received approximately NKr 18–22 kg-1 cod recently, suggesting that cod farming can be a viable industry in the future (for further discussion, see Chapter 12). One of the main problems in on-growing farms has been precocious sexual maturation. Nearly 100% of the farmed cod mature at the age of 2 years after hatching, and a substantial proportion of the males can mature 1 year after hatching. In contrast, wild Norwegian coastal cod typically matures at a median age of 4–5 years in western Norway, and northeast Arctic cod mature at a median age of around 7 years in the Barents Sea. The onset of maturity leads to a signiﬁcant loss of body weight during the spawning season. This also affects the quality of the ﬁsh, as it reduces the proportion of edible ﬂesh of whole body weight, and alters the proximate composition of the ﬁllet, including a higher water content. The loss of growth during the spawning season means a longer time to reach the desired marked size, and the energy expenditure due to gonadal development, spawning and recovery after spawning obviously increases the amount of feed needed to produce ﬁsh of a certain size. The early maturity of farmed cod is probably due to the favourable growth conditions in aquaculture, as rapid growth is assumed to reduce age at ﬁrst maturity. The

Figure 10.6 Farming of Atlantic cod in sea cages is still carried out on a small scale. The picture shows Henrik Johan Tveit, one of the pioneers of cod farming in Norway (Photo: Leiv Aardal, IMR, Norway).

The status and perspectives for the species

441

rapid growth in farmed cod is also associated with a much larger energy deposition in the liver than that observed in wild populations, and also compared with that of sea-ranched cod, where the ratio of liver weight to whole body weight decreases from around 11% when the ﬁsh are put into the sea to around 3% after 1 year in the wild. The liver is the main lipid store in cod, as there are only small amounts of lipid in the ﬂesh. It has been suggested that lipid stores, or rate of lipid deposition, is an important factor in determining age at ﬁrst maturity in salmonids, and similar suggestions have also been made for cod. Attempts have therefore been made to reduce the energy deposition by restricted feeding, or the use of lowenergy diets, to delay maturity or reduce gonadal investment in cod. However, even prolonged periods of starvation or restricted feeding did not reduce liver size to the levels typically found in wild situations, and restricted feeding did not affect the relative fecundity. Although such regimes can reduce the proportion of mature 2+ cod to some extent, they also substantially reduce somatic growth. In contrast, it has been shown that the exposure of 1+ cod in indoor seawater tanks to continuous light (LL) from the summer solstice onwards delayed or arrested gonadal development and inhibited spawning at the age of 2 years. In addition, the cod spawned far fewer eggs than normal at the age of 3 years, indicating that the proportion of spawning females was very low. Exposure to LL also enhanced growth during winter and spring; no reduction in growth due to maturity was indicated at the 2+ stage (Hansen et al., 2001). Several experiments have recently been conducted to delay the puberty of cod in sea cages by exposing 1-year-old cod in sea cages to LL from July or September until the next summer. This appeared to delay spawning by 4–6 months and reduce the proportion of spawning ﬁsh. The treatment also enhanced growth: cod exposed to LL reached mean body weights of 2.90–3.13 kg within 28 months of hatching, whereas controls reached 2.20–2.42 kg in the same time. However, these trials, and also later trials where cod were exposed to LL at different times of the year, failed to arrest maturation completely at the 2+ stage in contrast to a study carried out in seawater tanks. Some possible reasons for these differences could be (1) differences in the swimming activity of cod in sea cages or in tanks, leading to different energy deposition, thereby affecting maturation, and (2) differences in the light intensity ratio between day and night between the two systems; the intensity of the LL used on the sea cages was well below that of natural daylight, whereas the tanks indoors received strong illumination and only small amounts of natural light through windows in the roof. The ﬁrst of these hypotheses was recently tested in a trial at the Institute of Marine Reaserch, Austevoll Aquaculture Research Station, where 1-year-old cod were exposed to either natural light (NL) or LL in 3-m circular tanks, in combination with either high (1 BL s-1) (BL, body length), medium (0.5 BL s-1) or low water current speed. The LL was supplied by ﬂuorescent tubes in light-proof tanks, so there were no diurnal changes in intensity. This revealed that almost all cod matured at the age of 2 years in the NL groups irrespective of water current, whereas no maturation appeared in the LL groups at this age irrespective of water current (Karlsen et al., 2000). These results suggest that the intensity and/or spectral composition of light may be the important difference between the situation in tanks and cages exposed to LL, and possibly that the difference in light intensity between day and night is critical for the ﬁsh to perceive the photoperiod as truly continuous (24L : 0D) or changing. Thus, it is hypothesised that the use of sufﬁcient light of an appropriate spectral composition

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Culture of cold-water marine ﬁsh

may delay maturation in cod to the 3+ stage in sea cages, also allowing the production of cod in the range of 4–5 kg body weight prior to sexual maturation.

10.1.6 Future Prospects To date, cod farming has been a sideline business for people in rural areas searching for alternative ways of making their living. Units have been small, and further development has been hampered by the lack of cod juveniles and low, or even negative, proﬁtability. Belief in the potential of cod farming as a viable industry has changed dramatically in the last few years in Norway, the UK and North America. The general shortage of white ﬁsh on the world market has led to increased prices, and large national and international ﬁsh companies are now considering investments in cod farming. Large investments are also planned in juvenile production, and if this bottleneck is removed, market situation and proﬁtability will be the major limiting factors for growth. In Norway, a national network ‘go for cod’ has been established, aiming to gather together cod farmers, the ﬁshing industry and scientists for a common goal: to develop cod farming into a viable industry. A short-term, and realistic production goal of 10 000 tons slaughtered per year in 2005 has been set. Currently, 100– 200 tons are slaughtered per year, and some of this comes from on-growing of wild-caught undersized cod. The potential for cod farming in the future depends on several factors. In the short term, the major limitation is juvenile production, but factors such as ﬂesh quality, early sexual maturation, growth enhancement and health also need to be addressed. It is also clear that breeding must be an integrated part of the production escalation. The overall success of cod farming will depend on how well these factors are dealt with—in other words, whether the production costs can be lower than the price the market is willing to pay.

10.1.7 References Anonymous (1995) Yngelproduksjon av torsk—Hva har resultatene vist? Sluttrapport Havbeiteprogrammet PUSH, Bergen, 37 sider. Glette, J., van der Meeren, T., Olsen, R.E. & Skilbrei, O. (eds) (2002) Havbruksrapporten 2002. Fisken og Havet, 3, 103 pp. Hansen, T., Karlsen, Ø., Taranger, G.L., Hemre, G.I., Holm, J.C. & Kjesbu, O.S. (2001) Growth and sexual maturation of Atlantic cod (Gadus morhua) reared under different photoperiods. Aquaculture, 203, 51–67. Holm, J.C., Svåsand, T. & Wennevik, V. (eds) (1991) Håndbok i torskeoppdrett. Stamﬁskhold og yngelproduksjon. Instititute of Marine Resaerch, Department of Aquaculture, 156 pp. Karlsen, Ø., Taranger, G.L., Dahle, R. & Norberg, B. (2000) Effect of exercise and continuous light on early sexual maturation in cod. In Proceedings of the 6th International Symposium on the Reproductive Physiology of Fish, Norberg, B., Kjesbu, O.S., Taranger, G.L., Andersson, E. and Stefansson, S.O. (eds.), Bergen, July 4–9, 1999 pp. 328–30. Kurlansky, M. (1997) Cod. A Biography of the Fish that Changed the World. Jonathan Cape, London. Kvenseth, P.G., Winther, U., Hempel, E. & Fagerholt, A.F. (2000) Torskeutredning for Statens næringsog Distriktsutviklingsfond SND. Report from KPMG Consulting, 7406 Trondheim, 110 pp. (in Norwegian). Solemdal, P., Dahl, E., Danielssen, D.S. & Moksness, E. (1984) The cod hatchery in Flødevigen—

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background and realities. In: The Propagation of Cod, Gadus morhua L. (eds E. Dahl, D.S. Danielssen, E. Moksness & P. Solemdal), pp. 17–45. Flødevigen Rapportserie, 1. Svåsand, T., Ajiad, A.M., Carvalho, G.R., Clemmesen, C., Dahle, G., Hauser, L., Hutchinson, W.F., Jakobsen, T., Kjesbu, O.S., Moksness, E., Otterå, O., Paulsen, H., Schnack, D., Solemdal, P. & Thorsen, A. (2000a) Demonstration of maternal effects of Atlantic cod: combining the use of unique mesocosm and novel molecular techniques. Results from ﬁrst-year experiment. ICES CM 2000/R, 8, 19 pp. Svåsand, T., Kristiansen, T.S., Pedersen, T., Salvanes A.G.V., Engelsen, R., Nævdal, G. & Nødtvedt, M. (2000b) The enhancement of cod stocks. Fish Fish., 2, 173–205.

10.1.8 Further Reading Baskerville-Bridges, B. & Kling, L.J. (2000a) Early weaning of Atlantic cod (Gadus morhua) larvae onto a microparticulate diet. Aquaculture, 189, 109–17. Baskerville-Bridges, B. & Kling, L.J. (2000b) Larval culture of Atlantic cod (Gadus morhua) at high stocking densities. Aquaculture, 181, 61–9. Blom, G., Otterå, H., Svåsand, T., Kristiansen, T.S. & Serigstad, B. (1991) The relationship between feeding conditions and production of cod fry (Gadus morhua L.) in a semi-enclosed marine ecosystem in western Norway, illustrated by use of a consumption model. ICES Mar. Sci. Symp., 192, 176–89. Braaten, B. (1984) Growth of cod in relation to ﬁsh size and ration level. In: The Propagation of Cod, Gadus morhua L. (eds E. Dahl, D.S. Danielssen, E. Moksness & P. Solemdal), pp. 677–710. Flødevigen Rapportserie, 1. Dahl, E., Danielssen, D.S., Moksness, E. & Solemdal, P. (eds) (1984) The Propagation of Cod Gadus morhua L. Flødevigen Rapportserie, 1 (1&2). Ekstrøm, P. & Meissl, H. (1997) The pineal organ of teleost ﬁshes. Rev. Fish Biol. Fish., 7, 199–284. Eliassen, J.E. & Vahl, O. (1982) Seasonal variations in biochemical composition and energy content of liver, gonad and muscle of mature and immature cod, Gadus morhua L., from Balsfjorden, northern Norway. J. Fish Biol., 20, 707–16. Holdway, D.A. & Beamish, F.W.H. (1985) The effect of growth rate and season on oocyte development and maturity of Atlantic cod (Gadus morhua L.). J. Exp. Mar. Biol. Ecol., 85, 3–19. Holm, J.C. & Andersen, E. (1989) Improved spawning pen for Atlantic cod. World Aquacult., 20, 107. Howell, B.R. (1984) The intensive rearing of juvenile cod, Gadus morhua L. In: The Propagation of Cod, Gadus morhua L. (eds E. Dahl, D.S. Danielssen, E. Moksness & P. Solemdal), pp. 657–75. Flødevigen Rapportserie, 1. Jakobsson, J., Astthorsson, O.S., Beverton, R.J.H., Björnsson, B., Daan, N., Frank, K.T., Meincke, J., Rothschild, B., Sundby, S. & Tilseth, S. (eds) (1994) Cod and Climate Change. Proceedings of a symposium held in Reykjavik, 23–27 August 1993. ICES Mar. Sci. Symp., 198, 699 pp. Jobling, M. (1988) A review of the physiological and nutritional energetics of cod, Gadus morhua L., with particular reference to growth under farmed conditions. Aquaculture, 70, 1–19. Karlsen, Ø., Holm, J.C. & Kjesbu, O.S. (1995) Effects of periodic starvation on the reproductive investment in ﬁrst-time-spawning Atlantic cod (Gadus morhua L.). Aquaculture, 133, 159–70. Kjesbu, O.S. (1989) The spawning activity of cod (Gadus morhua L.). J. Fish Biol., 34, 195–206. Kjesbu, O.S. (1994) Time of start of spawning in Atlantic cod (Gadus morhua L.) females in relation to vitellogenic oocyte diameter, temperature, ﬁsh length and condition. J. Fish Biol., 45, 719–35. Kjesbu, O.S. & Holm, J.C. (1994) Oocyte recruitment in ﬁrst-time-spawning Atlantic cod (Gadus morhua) in relation to feeding regime. Can. J. Fish Aquat. Sci., 51, 1893–8. Kjesbu, O.S., Klungsøyr, J., Kryvi, H., Witthames, P.R. & Greer Walker, M. (1991) Fecundity, atresia, and egg size of captive Atlantic cod (Gadus morhua) in relation to proximate body composition. Can. J. Fish Aquat. Sci., 48, 2333–43.

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Kjesbu, O.S., Kryvi, H. & Norberg, B. (1996) Oocyte size and structure in relation to blood plasma steroid hormones in individually monitored, spawning Atlantic cod. J. Fish Biol., 49, 1197–215. Love, R.M. (1980) The Chemical Biology of Fishes. Vol. 2. Advances 1968–1977. Academic Press, London. Øiestad, V., Kvenseth, P.G. & Folkvord, A. (1985) Mass production of Atlantic cod juveniles (Gadus morhua) in a Norwegian salt-water pond. Trans. Am. Fish. Soc., 114, 590–5. Otterlei, E., Nyhammer, G., Folkvord, A. & Stefansson, S.O. (1999) Temperature- and size-dependent growth of larval and early juvenile Atlantic cod (Gadus morhua): a comparative study of Norwegian coastal cod and northeast Arctic cod. Can. J. Fish. Aquat. Sci., 56, 2099–111. Rognerud, C. (1887) Hatching cod in Norway. Bull. US Fish Comm., VII, 113–19. Rosenlund, G., Meslo, I., Rødsjø, R. & Torp, H. (1993) Large-scale production of cod. In: Fish Farming Technology (eds H. Reinertsen, L.A. Dahle, L. Jørgensen & K. Tvinnereim), pp. 141–6. Balkema, Rotterdam. Shelbourne, J.E. (1964) The artiﬁcial propagation of marine ﬁsh. Adv. Mar. Biol., 2, 1–83. Stearns, S.C. (1992) The Evolution of Life Histories. Oxford University Press, Oxford. Svåsand, T., Jørstad, K.E., Otterå, H. & Kjesbu, O.S. (1996) Differences in growth performance between Arcto-Norwegian and Norwegian coastal cod reared under identical conditions. J. Fish Biol., 49, 108–19. Svåsand, T., Skilbrei, O.T., van der Meeren, G.I. & Holm, M. (1998) Review of morphological and behavioural differences between reared and wild individuals: implications for sea-ranching of Atlantic salmon, Salmo salar L., Atlantic cod, Gadus morhua L., and European lobster, Homarus gammarus L. Fish. Manage. Ecol., 5, 1–18. Thorpe, J.E. (1994) Reproductive strategies in Atlantic salmon, Salmo salar L. Aquacult. Fish. Manage., 25, 77–87.

10.2 Haddock M. Litvak

10.2.1 Introduction Haddock (Fig. 10.7), Melanogrammus aegleﬁnus (L.), is one of the most highly prized ﬁsh caught for the north-eastern North American market. Interest in haddock culture had its origins in New Brunswick, Canada, in the late 1980s. Salmon growers were looking for alternatives to Atlantic salmon that could be grown with existing cage systems. The proximity of the New Brunswick aquaculture industry, and their existing relationships with northeast US markets, created the initial interest in this species for culture. Current landings of haddock are still low (Fig. 10.8), and their value has increased in North America during the past 50 years. The existing market for haddock is for fresh dressed, fresh and frozen ﬁllets. Fillets are sold as combinations of skin-on, skinless, pinbone in and boneless. Prices vary with season and year, but generally products are sold in the following ranges: fresh dressed $1.20–1.70 per lb; fresh ﬁllets $4.00–7.00 per lb; frozen ﬁllets $3.50–4.25 per lb. The goal in the development of haddock in Atlantic Canada is to help both stabilise and grow the existing ﬁnﬁsh aquaculture industry. The ﬁrst aquaculturist to try haddock in a salmon cage was John Malloch of Harbour DeLoutre Products of Campobello, New Brunswick. He is a pioneer of salmon and alternate species culture in the region. He ﬁrst acquired wild juvenile haddock and tested them

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Figure 10.7 Adult haddock at the St Andrews Biological Station, Department of Fisheries and Oceans, in St Andrews, New Brunswick. Photograph by Steve Neil, St Andrews Biological Station, Department of Fisheries and Oceans. 600,000

Europe North America

500,000

Metric Tons

400,000

300,000

200,000

100,000

0

1970

1975

1980

1985

1990

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Year Figure 10.8 Haddock landings in Europe and North America. Data from FAO Fisheries Department, Fishing Information, Data and Statistics Unit, collected with FISHSTAT Plus.

in what is referred to as a Malloch cage, i.e. a hexagonal wooden foam-ﬁlled ﬂoatation collar with a suspended net. After this experience, he enlisted the help of the Biological Station of the Department of Fisheries and Oceans (DFO) in St Andrews, New Brunswick, to develop rearing protocols and produce juvenile ﬁsh for cage trials. Fish reared at the DFO showed good growth in cages at Malloch’s site, suggesting that haddock can adapt to culture conditions. Since then, there has been a tremendous amount of research and development on haddock aquaculture by industry (led by Dr Chris Frantsi of Heritage Salmon Ltd, Black’s

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Harbour, New Brunswick), government laboratories (provincial and federal) and university scientists. There follows a brief description of haddock aquaculture research to date, causes for concern, potential modes of culture and future prospects.

10.2.2 Brood Stock, Egg Production and Incubation Wild ﬁsh are caught with hook and line. There continues to be a problem with exophthalmia and other symptoms of gas saturation related to bringing adult haddock up from depth. To minimise the effect of gas bubble disease, ﬁsh are brought up slowly to allow more nitrogen to leave the blood. Haddock are not easily sexed. Fish are followed through a breeding cycle to allow for identiﬁcation of gender and then tagged. However, ultrasonography may be used to help in identiﬁcation of gender prior to the spawning season. Haddock, like many marine ﬁnﬁsh, is a highly fecund (r-selected) batch spawner, producing many (can be millions per female) buoyant small eggs (1.3–1.6 mm diameter) with limited yolk reserves. Haddock eggs are not stripped, as they are much more prone to handling damage than other gadids such as cod and pollock. Haddock is a serial volitional spawner like cod (see Section 10.1). Groups of males and females are held together in land-based tanks during the spawning season. Embryos are collected from tanks with a collection system similar to that shown in Fig. 10.1. Natural spawning of Atlantic Canadian populations occurs from April to June. Brood stock at the St Andrews Biological Station, Department of Fisheries and Oceans’ brood-stock facility, have produced millions of eggs over the past few years. Workers have been able to advance the breeding season by photoperiod manipulation. Eggs are now available from December to June. In all likelihood, it will be possible to extend egg and sperm production over an even greater time period through continual photoperiod advancement and thermal manipulation. A major problem with haddock brood stock has been the loss of ﬁsh at or around spawning (January–June). Most current brood stock are wild, and may already have been damaged during capture. The proximate cause of death is unknown, although in almost all cases hyperinﬂation of the swim bladder was observed. This mortality may be due to tank depth and stress, because hyperinﬂation was not observed in brood stock held in cages (John Malloch’s site on Campobello Island, New Brunswick). Wild haddock brood stock, which do not normally occur in shallow water, may have difﬁculty in regulating their buoyancy in shallow tanks, particularly during stressful periods such as spawning. Haddock also exhibit courtship swimming behaviour and vocalisation during mating. Haddock may require quiet, and larger tanks for spawning than are currently being provided. It is hoped that this stress response may disappear through the domestication process, which would minimise the cost of tanks and brood stock holding facilities. Alternatively, we could collect spawn directly from cages, as in the procedure with cod (Fig. 10.1). The collection of spawn from cages, although not as well controlled as in a brood stock facility, would also be considerably less expensive for the farmer. However, a successful extension of the breeding season would be extremely difﬁcult under these conditions. Embryos are disinfected with glutaraldehyde (400 p.p.m. for 5–10 min) following the cod protocol. Embryos have been incubated in a variety of set-ups, e.g. static, recirculation and ﬂow-through (up-welling). For production purposes, an up-welling cylindro-conical tank

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A

B

447

C

Figure 10.9 Haddock egg development gone wrong. An evaluation of blastomere form early in development determines the probability of batch success (Rideout et al., 2003). (A) Complete separation of blastomeres. (B) Poor adhesion between blastomeres. (C) Proper development at the 16-cell stage. Both (A) and (B) will lead to batch failure (Rideout et al., 2003). Photograph by Rick Rideout, Department of Biology and Centre for Coastal Studies and Aquaculture, University of New Brunswick.

equipped with a banjo ﬁlter is preferred. Embryos become negatively buoyant when they die, making removal from an up-welling tank simple; the up-welling ﬂow is turned off, and embryos that sink to the bottom of the incubator are removed. Depending on temperature, the 3–4-mm larvae will hatch in 2–3 weeks (5–8°C). As with incubation tank design, different light regimes have been used, all resulting in larvae at hatch. However, recent work suggests that although successful development does occur over a wide range of light environments, there is an effect of photoperiod, light intensity and spectral composition on the embryos. In terms of photoperiod, day-length was directly related to incubation time; embryos exposed to a longer day-length hatched signiﬁcantly earlier than those reared under a shorter day-length or continuous darkness. Embryos reared under a 18L : 6D photoperiod were larger at hatch than those reared under either continuous light or a 12L : 12D photoperiod. Embryos also hatched at a larger size when grown under a higher intensity. There is no effect of light spectra on development. If size does confer an advantage to growth and survival in a culture setting, then haddock larvae should be grown at high intensity white light with an 18L : 6D photoperiod. A major problem during the egg incubation stage is complete batch failure. This has been related to handling damage and/or egg, and possibly sperm, quality. Labour and incubator space is wasted while caring for a batch that later fails. However, recent work suggests that we can use an early diagnostic of blastomere cleavage pattern (Fig. 10.9) to determine the hatching potential of a batch at a very early stage in development. By removing batches with poor prospects of hatching, we will be able to maximise production per incubation unit. There has been little work on the prediction of egg quality from brood-stock quality, although we do know that egg size declines with batch number. However, no research has yet shown a correlation between egg size and larval survival (R. Rideout, personal communication, 2002).

10.2.3 Larval Rearing Currently, larvae are reared in intensive land-based systems utilising either ﬂow-through or recirculation. Some laboratories stock larvae after hatch, and others try to minimise handling

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damage and stock eggs just prior to hatch in small screen hatching boxes. After larvae hatch in the boxes they allowed to swim out, and the shells (chorion) are removed with the box from the culture tank. Initially, stocking density was set at the standard cod model of 20 larvae l-1. However, recent work at the University of Maine, Orono, ME, demonstrated that gadids experience higher survival and growth at very high densities of 150–300 larvae l-1. This higher survival was attributed to high clearance rates of the live food placed in the tanks. Since rotifer quality decreases with time, high clearance rates ensure that the food ingested is of a high quality, and that there is little or no detrital build-up in the tank. Light spectra, intensity and photoperiod all affect the growth and survival of haddock larvae. Light spectra inﬂuences their ﬁrst feeding behaviour, and a blue wavelength at high intensity promotes the greatest feeding activity. A continuous photoperiod is not required for haddock larvae. There is no difference in growth and survival to 40 days post-hatch (d.p.h.) for larvae reared under continuous light or an ambient photoperiod (14L : 10D). There is anecdotal evidence to suggest that a dark period is helpful for swim-bladder inﬂation. Currently, there is no artiﬁcial food that can be used to feed haddock from hatch. The earliest time to switch larvae to artiﬁcial diet and/or initiate co-feeding is still under investigation at Canada’s National Research Council Institute of Marine Biosciences. Thus, haddock, like many marine species being developed for aquaculture, depend on the production of live rotifers, Brachionus plicatilis. Larvae are fed twice per day on rotifers enriched with algae and/or a variety of enrichment media (see Chapter 4) until 20–25 d.p.h. They are then switched to Artemia, and at approximately 40 d.p.h. weaned on to a micro-diet.

10.2.4 Weaning and On-Growing The weaning process has not yet been reﬁned, and many larvae die during this stage. These larvae are often found with hyper-inﬂated swim-bladders struggling on the surface prior to death. Surviving larvae ﬁnish metamorphosis at approximately 25 mm standard length, and resemble the adult at this stage. Although there has recently been a quantum leap in the production of juvenile haddock (particularly at the New Brunswick Department of Agriculture, Fisheries and Aquaculture’s Centre Marine Shippegan and at the Institute of Marine Biosciences (IMB) of the National Research Council of Canada in Halifax, Nova Scotia), the optimal tank size, ﬂow regime and water quality (physical and biological parameters) have not yet been clearly identiﬁed, and mortality still remains high at the weaning stage. Currently, juveniles are transferred to cages at 3–5 g in either May or June. Heritage Salmon uses a cage-within-a-cage model. Ten thousand juveniles are stocked in a small (6 m ¥ 6 m ¥ 3 m) deep cage of 1.27 cm stretch treated mesh placed inside a larger salmontype cage. The salmon cage acts as a predator net and also as a breakwater to minimise the smaller cage’s movements due to wave action (C. Frantsi, personal communication, 2002). Fish are graded as they grow, and then are placed into larger ‘salmon’ cages. As with larvae, trials in the laboratory have shown that light plays an important role in the growth of juveniles. Fish grown under lower intensity continuous light showed the best growth rates. Trials at Heritage Salmon are being conducted to examine the suppression of gonadal development through the manipulation of light periodicity in order to maximise commercial yield.

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Diets for haddock are being developed at IMB. Haddock grow best with a moist feed, and this is used as the benchmark to test feeds under development. It is already clear that haddock, like cod, are prone to fatty liver disease. Haddock store their fats in the liver, providing a muscle lipid level of 1%. While this is an advantage for the market, it does mean that feeds must contain less than 14% lipids otherwise the ﬁsh will develop fatty liver disease.

10.2.5 Health Early trials with haddock in cages suggested that they were prone to ‘sun-burn’. This problem was quickly solved by placing a shade cloth over the cages. As previously mentioned, fatty liver disease is a problem for haddock juveniles. It is also a problem for brood stock. However, fatty liver disease can be defeated by the development of low-lipid diets. Vibrio has infected haddock. Haddock do not have the same immune response as salmon. Immunological work at IMB suggests that they do not show a strong immune response after vaccination, and to date it is not known whether immunisation confers any disease resistance. Haddock are also susceptible to Nodavirus, with a high probability of mortality during an outbreak. They are also prone to corneal damage from tank abrasions and hyperinﬂation of swim-bladders, which is probably a result of stress.

10.2.6 Commercial Development In addition to the value of haddock, it was hoped that they would have a similar anti-freeze system to cod. It appears that haddock does not have this level of protection, which is conferred by the anti-freeze or glycerol that is found in the blood plasma of cod. Therefore, locations for haddock culture will be restricted to areas that do not experience temperatures below -0.8°C. Heritage Salmon has developed a long-term plan (1995–2012) to increase the commercialisation of haddock. They currently have 500 000 haddock, representing 3 year-classes (2000, 2001, 2002), being grown in cages (C. Frantsi, personal communication). Heritage Salmon is running harvest and processing trials with these ﬁsh. To date, harvest has been in the autumn/winter in order to avoid spring, when ﬁsh give a poorer yield. The yield (currently 34–38%), is maximised not only through culture techniques and technology, but also by minimising loss during processing. They are currently attempting to develop better systems to maximise yield during the ﬁlleting process. In the development of any ﬁsh for culture, there is great concern about growth rates. In other words, will these ﬁsh grow fast enough to make a proﬁt? Haddock grown with our current capability attain a size of 2–2.5 kg in 3 years after hatch, which is almost twice as heavy at that age than a wild ﬁsh. However, it is hoped that growth rates will become much higher with further domestication through traditional breeding selection practices and improved husbandry and diets. Another approach to reduce the time to the table is through a hybrid land-based cage aquaculture model. It is obvious that heated water and land-based facilities are expensive. However, we can minimise these costs by growing haddock when they are small and do not require as much space in this type of system. It is clear that we can advance the egg pro-

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duction to the autumn. If we then grow larvae and juveniles at high temperatures on land in the winter, we can provide a much larger ﬁsh for delivery to cages in the spring and thus reducing the time to market.

10.2.7 Future Prospects The potential beneﬁts of the development of haddock for the existing aquaculture industry are great. Its value, market acceptance and ability to be used in existing grow-out systems are excellent. Additionally, the potential beneﬁts in the use of haddock for culture extend beyond the direct cash crop. Crop rotation is often used in agriculture to defeat epizootic outbreaks. A similar strategy may be necessary with the cage systems used for grow-out by Canadian aquaculturists. To accomplish this type of crop rotation, more ﬁsh species like haddock must be made available to the farmer. The future development of haddock depends on a clear and focused research and development strategy. Although we are learning a lot, in reality little is known about haddock, and thus a better understanding of the basic biology and culture of this ﬁsh is indicated. The success of the salmon industry today can in large measure be attributed to the tremendous amount of information collected over the past 100 years about this ﬁsh. The production of haddock will depend on an integrated research and development programme. Key to this development is the production of highly qualiﬁed personnel to continue the push towards development, and to run the high-tech marine ﬁnﬁsh industrial hatcheries of the future.

10.2.8 Further Reading Buckley, L.J., Bradley, T.M. & Allen-Guilmette, J. (2000) Production, quality, and low-temperature incubation of eggs of Atlantic cod, Gadus morhua, and haddock, Melanogrammus aegleﬁnus, in captivity. J. World Aquacult. Soc., 31, 22–9. Downing, G. (2002) Impact of spectral composition on larval haddock, Melanogrammus aegleﬁnus L., growth and survival. Aquacult. Res., 33, 251–9. Downing, G. & Litvak, M.K. (1999a) The effect of photoperiod, tank colour and light intensity on growth of larval haddock (Melanogrammus aegleﬁnus). Aquacult. Int., 7, 135–40. Downing, G. & Litvak, M.K. (1999b) The inﬂuence of light intensity on growth of larval haddock (Melanogrammus aegleﬁnus). N. Am. J. Aquacult., 61, 135–40. Downing, G. & Litvak, M.K. (2000) Inﬂuence of spectral composition on ﬁrst feeding, growth, and survival of larval haddock (Melanogrammus aegleﬁnus). Aquacult. Assoc. Can. Spec. Bull., 4, 37– 40. Downing, G. & Litvak, M.K. (2001) The effect of light intensity and spectrum on the incidence of ﬁrst feeding by larval haddock. J. Fish Biol., 59, 1566–78. Downing, G. & Litvak, M.K. (2002) Effects of light intensity, spectral composition and photoperiod on development and hatching of haddock (Melanogrammus aegleﬁnus) embryos. Aquaculture, 213, 265–78. Ewart, K.V., Blanchard, B., Johnson, S.C., Bailey, W.L., Martin-Robichaud, D.J. & Buzeta, M.I. (2000) Freeze susceptibility in haddock (Melanogrammus aegleﬁnus). Aquaculture, 188, 91–101. Frantsi, C., Lanteigne, C., Blanchard, B., Alderson, R., Lall, S., Johnson, S., Leadbeater, S., MartinRobichaud, D. & Rose, P. (2002) Haddock culture in Atlantic Canada. Bull. Aquacult. Assoc. Can., 102-1, 31–4.

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Hamlin, H.J. & Kling, L.J. (2001) The culture and early weaning of larval haddock (Melanogrammus aegleﬁnus) using a microparticulate diet. Aquaculture, 201, 61–72. Kim, J.-D. & Lall, S.P. (2001) Effects of dietary protein level on growth and utilization of protein and energy by juvenile haddock (Melanogrammus aegleﬁnus). Aquaculture, 195, 311–19. Laurence, G.C. & Rogers, C.A. (1976) Effects of temperature and salinity on comparative embryo development and mortality of Atlantic cod (Gadus morhua) and haddock (Melanogrammus aegleﬁnus (L.)). J. Cons., Cons. Int. Explor. Mer, 36, 220–8. Litvak, M.K. (1998) The development of haddock culture in Atlantic Canada. Bull. Aquacult. Assoc. Can., 98-1, 30–3. Nanton, D.A., Lall, S.P. & McNiven, M.A. (2001) Effects of dietary lipid level on liver and muscle deposition in juvenile haddock, Melanogrammus aegleﬁnus. Aquacult. Res., 32, 225–34. Reith, M., Munholland, J., Kelly, J., Finn, R.N. & Fyhn, H.J. (2001) Lipovitellins derived from two forms of vitellogenin are differentially processed during oocyte maturation in haddock (Melanogrammus aegleﬁnus). J. Exp. Zool., 291, 58–67. Rideout, R.M., Trippel, E.A. & Litvak, M.K. (2003) Predicting haddock embryo viability based on early cleavage patterns. Aquaculture, in press. Scott, W.B. & Scott, M.G. (1988) Atlantic ﬁshes of Canada. Can. Bull. Fish. Aquat. Sci., 219, 731 pp. Trippel, E.A. & Neil, S.R.E. (2003) Effects of photoperiod and light intensity on growth and activity of juvenile haddock (Melanogrammus aegleﬁnus). Aquaculture, 217, 633–45. Trippel, E.A., Doherty, C.M., Wade, J. & Harmon, P.R. (1998) Controlled breeding technology for haddock (Melanogrammus aegleﬁnus) in mated pairs. Bull. Aquacult. Assoc. Can., 98-3, 30–5.

10.3 Hake A.B. Skiftesvik and R.M. Bjelland

10.3.1 Introduction European hake (Merluccius merluccius L.) is a popular species in European ﬁsh markets, and large ﬁsh of high quality make very high prices. The total landings of hake in the northeast Atlantic is about 80 000 tonnes per year, but has been as high as 175 000 tonnes (Casey & Pereiro, 1995). In Norwegian waters, the total catch is about 500–1000 tonnes per year (Aglen & Monstad, 2000). There is a growing interest in farming hake, both in Europe (Merluccius merluccius L.) and in Chile (Merluccius australis, Hutton), but until recently there have been no successful attempts to rear European hake. The ﬁrst experiments on rearing hake were conducted at Austevoll Aquaculture Station in 1996, and have continued with improving results in 1997, 1998 and 1999. These efforts are summarised here.

10.3.2 Egg Production and Incubation Gametes for experiments were stripped from freshly captured wild ﬁsh on spawning grounds off the west coast of Norway during July and August. Hake become extremely agitated when caught in gillnets (the typical mode of ﬁshing for this species), and severe injuries result. For this reason, it has been impossible to obtain adults in a condition that would permit their use as captive brood stock. Pelagic hake eggs (Fig. 10.10) are approximately 1.1 mm in diameter and contain a single oil globule measuring 0.27 mm. Fertilised eggs are hydrofugal, and ﬂoat at the surface when in contact with the water–air interface. To prevent eggs at the surface

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Figure 10.10 0.27 mm.

Eggs of hake, diameter 1.1 mm, oil globule

Figure 10.11 Newly hatched hake larva (the standard length is 3 mm).

from drying out, a ﬁne water mist was provided above the rearing tanks. The eggs were incubated in 250-l up-stream conical rearing systems. The water ﬂow was maintained at 1 l min-1 and the temperature was 12°C. Under these conditions, hatching occurred after 5 days (~60 day-degrees). Hatching was highly synchronous.

10.3.3 Larval Rearing Larvae (Fig. 10.11) measured approximately 3 mm at hatching and were poorly developed. The eyes were unpigmented, the mouth and gut were closed, and there were no functional ﬁns. The larvae were pigmented at hatching, with three bands of black and yellow pigments in the caudal region, and a more diffuse pattern of pigment cells around the cranium and dorsal area of the yolk. A small number of pigment cells are also associated with the yolk and oil globule. On the day of hatching (0 days post hatch = 0 d.p.h.), the larvae were transferred to a tank of larger volume. This served to avoid the pathogen proliferation that is typically associated with an abundance of empty eggshells in the egg incubation tank. The larval rearing system

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consisted of 2.8-m3 conical up-stream silos in which water ﬂow was maintained at approximately 1 l min-1 and the temperature was 12°C. Larvae remained in the upper part of the water column for the ﬁrst 2–3 days. This was followed by a period during which their speciﬁc gravity appeared to increase and they sank to near the bottom of the silo. The situation reversed itself at around 5 d.p.h. when the larvae were again found near the surface. At this age the jaws were functional and the larvae were equipped to begin exogenous feeding. Hake larvae were offered a mixed diet of Tetraselmis algae (the ‘green water’ technique), enriched rotifers and natural zooplankton nauplii, and later copepods. Approximately 20% of 6-d.p.h. larvae had food in the gut (feeding incidence), and this rose to 100% by 11 d.p.h. The yolk was totally depleted by 8 d.p.h., while the oil globule was present until 11 d.p.h. The growth of hake larvae was unusual; for the ﬁrst 3–4 weeks post-hatching there was little growth in length, but the head and trunk exhibited pronounced changes that produced a larva with a big mouth and a large stomach. When growth in length did begin, the increase was rapid: hake larvae (Fig. 10.12) grew from 5 to 10 mm in length over the course of 1 week. The transition to the juvenile form began around 30 d.p.h., when the unpaired ﬁns became differentiated and the body became more ‘stocky’.

10.3.4 Weaning and On-Growing At 50 d.p.h., the juveniles (Fig. 10.13) measured approximately 20 mm and were transferred to small (50 l) tanks for weaning from live to formulated feed. Pellet size started at 0.6–

Figure 10.12 Hake larvae 33 days post-hatch (the standard length is 10 mm).

Figure 10.13 Hake juvenile 59 days post-hatch (the standard length is 24 mm).

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1.0 mm and was increased gradually to 2.5 mm in the following 2 months, at which point the juveniles reached a length of approximately 60 mm. Juvenile hake are demersal and behave in a very ‘domesticated’ manner. However, they ate only pellets that were sinking, not pellets that remained on the surface or the bottom.

10.3.5 Future Prospects Despite the preliminary success summarised above, much more work is required before it becomes feasible to rear European hake intensively in large quantities. The key challenges are obtaining and maintaining healthy brood stock, increasing survival during the larvae to juvenile transition, reducing or eliminating cannibalism, and starting the weaning to formulated feed earlier.

10.3.6 References Aglen, A. & Monstad, T. (2000) Andre marine ressurser. Havets ressurser 2000. Fisken og Havet (Særnummer 1), p. 105. Casey, J. & Pereiro, J. (1995) European hake (M. merluccius) in the north-east Atlantic. In: Hake. Biology, Fisheries and Markets (eds J. Alheit & T.J. Pitcher), pp. 125–47. Chapman & Hall, London.

10. 4 Wolf-ﬁsh (Anarhichas spp.) D.A. Pavlov

10.4.1 Introduction Wolf-ﬁsh are in the perciform suborder Zoarcoidei and family Anarhichadidae. The family includes two species in the north Paciﬁc (wolf-eel, Anarrhichthys ocellatus Ayres, and Bering wolf-ﬁsh, Anarhichas orientalis Pallas) and three species in the north Atlantic (common wolf-ﬁsh, Anarhichas lupus L., spotted wolf-ﬁsh, A. minor Olafsen, and blue wolf-ﬁsh, A. denticulatus Kröyer). In the northern Atlantic, the ranges of the three wolf-ﬁsh species overlap, reaching the western coast of Spitsbergen to the north. However, the common wolfﬁsh is distributed farther to the south, reaching north-western France in Europe and Long Island in the USA. The Atlantic species are most common at a depth ranging from 150 to 250 m, but they migrate to the coast in spring and summer to forage before spawning. The maximum body length registered in common, spotted and blue wolf-ﬁsh in the northern Atlantic is 120, 144 and up to 200 cm, respectively. Wolf-ﬁsh species possess a unique reproductive strategy: ovulated eggs are inseminated internally, and they are released into the water several hours after insemination (Johannessen et al., 1993; Pavlov, 1994). The egg mass is protected by the male until hatching. Wolf-ﬁsh are taken as a by-catch of the trawl ﬁshery and from long-line catches. From 1976 onward, wolf-ﬁsh species have shown a declining distribution, with lower catch rates and narrower ranges reducing to the north. The stocks of the most valuable species, spotted wolf-ﬁsh, are over-exploited. The ﬁrst suggestion to use wolf-ﬁsh in marine aquaculture was based mainly on the features of its early ontogeny: just after hatching from large eggs (3.7–6.4 mm diameter), the

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larvae of common wolf-ﬁsh (about 21 mm length) are at an advanced stage of ontogeny, they are able to feed on comparatively large objects, and their survival rate can be very high (Pavlov & Novikov, 1986). At present, only spotted wolf-ﬁsh is regarded as a promising candidate for cold-water marine farming in northern Europe and Atlantic Canada. The suggestion is based on the high quality white ﬂesh and the comparatively high growth rate in captivity. Common wolf-ﬁsh possess a lower body growth rate. At the same time, some aspects of the life cycle of the latter species have been well studied, and they can be used in wolf-ﬁsh culture because of the many similarities in captive biology in the two species.

10.4.2 Brood Stock, Egg Production and Incubation Several wolf-ﬁsh brood stocks were established in Norway in the 1990s, and initially the life cycle of common wolf-ﬁsh was studied in more detail than that of spotted wolf-ﬁsh. The individuals of common wolf-ﬁsh raised in captivity from larvae reach sexual maturation at 4–5 years. In spotted wolf-ﬁsh, a few ﬁsh (less than 10%) mature at the age of 3+, whereas most ﬁsh mature at age 4+ or later at a weight of 3–4 kg. A small percentage of ﬁsh may not mature until the age of 6+, reaching a weight of more than 10 kg. Absolute fecundity ranges from 1314 to 6765 eggs in common wolf-ﬁsh (female weight 0.6–2.9 kg), and from 8000 to 30 000 eggs in spotted wolf-ﬁsh (female weight 3.6–13.0 kg). Among males of both species, the ejaculate volume of sperm is low (from less than 1 ml to 8–11 ml), and the spermatozoa are motile in undiluted ejaculate. The problem of poor egg quality in common wolf-ﬁsh has been overcome by a reduction in water temperature during vitellogenesis and ﬁnal egg maturation (Pavlov & Moksness, 1996a). The time of egg ovulation can be controlled by photoperiod (see Chapter 5), and the results of preliminary studies showed that mature eggs can be obtained over the entire year by the manipulation of photoperiod (Moksness & Pavlov, 1996). In addition, the temperature during vitellogenesis and, to a lesser degree, prior to egg ovulation inﬂuences the ovulation time, and this event is delayed at higher temperatures (8 and 12°C vs. 4°C). The egg quality was best in common wolf-ﬁsh females kept at 4 and 8°C during vitellogenesis and at 4°C before ovulation (Tveiten & Johnsen, 1999; Tveiten et al., 2001). In females, steroid synthesis during the periovulatory period was also inﬂuenced by temperature, but in males the concentration of steroid hormones was low at all times (Tveiten et al., 2000; Tveiten & Johnsen, 2001). The artiﬁcial insemination of wolf-ﬁsh eggs represented the main cultural problem for a long time. Recent investigations have shown that ovulated eggs of common wolf-ﬁsh can be inseminated both in vivo (by injecting sperm into the genital pore of the female) and in vitro. In both cases, the contact time between eggs and spermatozoa should exceed 2 h (see Chapter 5). Insemination in vitro is the most reliable for aquaculture. Following the prevention of egg adhesiveness, which is conducted in large trays with stagnant water for 6–10 h, the eggs can be incubated in up-welling systems using periodical treatment with glutaraldehyde to restrict development of pathogenous bacteria and benthic organisms. To shorten the incubation period of common wolf-ﬁsh eggs (up to 9 months in nature), comparatively high temperatures (ranging from 5 to 13°C) have been tested. At a constant temperature of 11°C, embryos hatch at a less advanced stage of development, and skeletal

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Figure 10.14 Morphology of common wolf-ﬁsh larvae at hatching after different temperatures of egg incubation. (A) 11.0°C, TL = 20.0 mm; (B) 7.0°C, TL = 22.0 mm. TL, total length; fs, free space in the dorsal or anal ﬁn; mb, muscular buds; od, oil droplet; tr, terminal rays in the dorsal ﬁn; ur, undeveloped rays; ys, yolk sac. Bar = 1 mm (Pavlov & Moksness, 1995).

abnormalities are observed (Fig. 10.14). A sensitive period for the formation of these abnormalities has been found, and a temperature regime to minimise the egg incubation time (up to 104 days) and obtain normal offspring has been suggested (Pavlov & Moksness, 1996b). The eggs of spotted wolf-ﬁsh can be incubated at 4–8°C. The incubation period decreased from 220 days at 4°C to 177 days at 6°C and 150 days at 8°C. The proportion of normal embryos and the survival of eggs until hatching were highest when the eggs were incubated at 6°C. The transformation of yolk to body mass during incubation appeared to be most efﬁcient at 4°C, and the embryos hatched with a larger yolk sac at 6 and 8°C. The largest larvae hatched from the largest eggs and from the egg groups incubated at the lowest temperature (4°C) (Hansen & Falk-Petersen, 2001). Normal hatching of wolf-ﬁsh embryos occurs at lengths greater than 20 mm, when the colour of the eggs changes from light orange to opaque and grey, and the egg envelopes become transparent. However, a light mechanical pressure is required to induce hatching. In both wolf-ﬁsh species, premature hatching of the embryos in some egg batches represents a problem which is not fully understood.

10.4.3 Larval Rearing and On-Growing The larvae of common and spotted wolf-ﬁsh need exogenous feeding just after hatching at an average wet weight of 70 and 90 mg, respectively. They have many juvenile characteristics, possess a positive reaction to light, and move towards the water surface by means of continuous undulations of the trunk. Embryonic and osteological development from embryo to juvenile (Pavlov, 1986, 1997; Pavlov & Moksness, 1997), as well as organ differentiation in larvae, based on a histological study (Falk-Petersen & Hansen, 2001), are described in detail for common wolf-ﬁsh. Common wolf-ﬁsh larvae can be start-fed on natural zoo-

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plankton and Artemia salina nauplii, with survival being close to 100%. The larvae of spotted wolf-ﬁsh were start-fed in low-water-level raceways, and at the beginning of the culture practice, Artemia salina was offered for about 3 weeks followed by dry feed (0.3–0.5-mm, and later 0.5–0.8-mm, granules). The possibility of start-feeding common wolf-ﬁsh larvae on dry pellets only has been demonstrated in several experiments. During a 60-day experiment with start-feeding on dry pellets, the highest survival rate (82%) was obtained in a raceway system with a low water level (1.5 cm), at a very high initial stocking density (167 larvae l-1), using a dry feed with high levels of protein and lipid and low levels of ash and carbohydrate (Strand et al., 1995). The feed ﬂoated for several minutes, which was important for larval feeding from the water surface. Larvae fed with the formulated diet sometimes showed increased mortality and impaired growth during the ﬁrst 10 days, followed by a period of rapid growth, and may obtain a signiﬁcantly higher weight than groups fed only with Artemia. For the experimental aims, after hatching, the larvae of spotted wolf-ﬁsh were transferred to shallow raceways and weaned on a formulated dry feed (Nutra Pluss, Skretting) containing 53% protein, 20% fat and 10% carbohydrate. Subsequently, the ﬁsh were reared in raceways (0.4 m ¥ 2.2 m) with a water level of 8–10 cm, providing a total volume of 80 l. They fed on a commercial ﬂoating feed (Dan-ex 1547, Dana Feed, Horsens, Danmark) containing 47% protein, 15% fat and 21% carbohydrate (Foss et al., 2003). At present, start-feeding of spotted wolf-ﬁsh for commercial purposes entirely on dry pellets is the common practice at Troms Steinbit AS, the only commercial juvenile producer (I. Andreassen, personal communication, 2002). At 30–40 mm in length, the juveniles of both species lose their positive photoreaction and spend all the time at the bottom, with the exception of feeding, when the young wait for food near the water surface. Among 1-month-old larvae of common wolf-ﬁsh, the maximum growth rate is observed at comparatively high temperatures (11–14°C). Juveniles of 9–12 months have optimum water temperatures for growth and growth efﬁciency at 10–11 and 9–10°C, respectively (McCarthy et al., 1998). Spotted wolf-ﬁsh are more stenothermal. During the start-feeding period of this species (3–4 weeks), the best growth was registered at 8°C, but the survival was the highest at 6°C (Falk-Peterson et al., 1999). The larvae raised at 8°C from eggs incubated at the lowest temperature (4°C) showed the highest growth rates. The best survival of larvae was noted among batches incubated at 6°C (Hansen & FalkPetersen, 2001). Recently, the inﬂuence of low salinity on the growth performance of both wolf-ﬁsh species has been studied. The salinity range tested for common wolf-ﬁsh (initial wet weight 0.11 and 0.41 g) was from 0 to 35‰. No ﬁsh survived in fresh water, but the mortality was similar at salinities exceeding 7‰, and protein content and total length were signiﬁcantly higher at lower salinities (Francois et al., 2001). For spotted wolf-ﬁsh (initial wet weight 76 g), salinity ranging from 12 to 34‰ did not have a signiﬁcant effect on daily feeding rates, total food consumption, food conversion efﬁciency or protein efﬁciency ratio (Foss et al., 2001). The results indicate that the wolf-ﬁsh species are strong osmo-regulators that can be reared successfully within a wide range of salinity levels. Juveniles and adult ﬁsh can be kept in tanks and big race-way systems at extremely high stocking densities. For example, ﬁsh density in a shallow race-way may be as high as

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800 kg m3 (Øiestad, 1999). In such systems, a reduction in oxygen content and the accumulation of natural catabolites may affect growth performance, food conversion efﬁciency and the health status of the ﬁsh. An evaluation of the effect of water oxygen content on growth and food conversion efﬁciency in juvenile spotted wolf-ﬁsh (initial weight 68.5 g) for 11 weeks at 8°C showed that at the end of the experiment, the mean weight was signiﬁcantly higher at oxygen levels of 9.6 and 14.5 mg l-1 compared with the level (4.0 and 6.0 mg l-1) in the hypoxic groups. In the hyperoxic group (14.5 mg l-1), growth rate was only limited during the ﬁrst half of the experiment, and it increased throughout the experiment in the hypoxic groups, which suggests the possibility that the ﬁsh can adapt to both high and low ambient water oxygen content (Foss et al., 2002). In another experiment, juvenile spotted wolf-ﬁsh (initial weight 95.4 g) were reared at oxygen levels of 9.6 and 14.5 mg l-1 with the addition of a sub-lethal concentration of unionised ammonia (0.17 mg NH3 l-1). The growth rate was signiﬁcantly higher in the group with the higher oxygen level, showing that hyperoxic conditions may increase tolerance to ammonia (Foss et al., 2003). As well as environmental factors, body growth depends on both feed composition and protein quality. The protein content should be greater than 50%, and the carbohydrate content lower than 20%. An increased fat content in the feed results in increased hepatosomatic indices and increased fat deposition in the ﬁllet (Moksness et al., 1995). Mean survival rates obtained for common wolf-ﬁsh in the laboratory were as follows: 95% at fertilisation, 80% during egg incubation, 82% at ﬁrst feeding, and 100% for juveniles (Moksness & Pavlov, 1996). The same rates reported for spotted wolf-ﬁsh were more variable (e.g. 0–78% for egg incubation) (Falk-Peterson et al., 1999). However, the poor egg quality was probably connected with too high a temperature (9–10°C) during late vitellogenesis and egg ovulation.

10.4.4 Future Prospects The breeding of wolf-ﬁsh species can be simpler and cheaper than for other marine ﬁsh species, and even salmonids, because of the possibility of raising large larvae entirely on dry pellets immediately after hatching. In the farming strategy for wolf-ﬁsh, many similarities with salmonids can be found. The protein synthesis retention efﬁciency for juvenile common wolf-ﬁsh is higher than that in salmonid ﬁsh at similar water temperatures, and the growth rate of juveniles is lower than in salmonid ﬁsh of a comparable size (McCarthy et al., 1998, 1999). In identical conditions, the growth rate of spotted wolf-ﬁsh is substantially higher than that of common wolf-ﬁsh (Fig. 10.15). According to Falk-Petersen et al. (1999), spotted wolf-ﬁsh reach 2.7 kg in 3 years. However, an even higher growth rate was registered at Troms Steinbit A/S (Norway): 4.2 kg in 3 years at 4–8°C. Thus, at present the spotted wolf-ﬁsh is regarded as the only possible candidate for aquaculture among wolf-ﬁsh species. Based on observations in nature, a very high growth rate can be predicted in captive blue wolf-ﬁsh (Fig. 10.15). This ﬁsh has poor ﬂesh quality but, as is shown for common wolfﬁsh, the meat content depends very much on the food composition (Moksness et al., 1995), and this problem may be overcome in captivity. Unfortunately, experiments with blue wolfﬁsh have not been conducted.

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Figure 10.15 Size at age of common (Anarhichas lupus) and spotted (A. minor) wolf-ﬁsh in the laboratory and blue wolf-ﬁsh (A. denticulatus) in the wild, based on data from Barsukov (1959) and Moksness (1994).

The optimum temperature for growth of the spotted wolf-ﬁsh is between 4 and 10°C (Moksness, 1994; Lundamo, 1999). Therefore, cultivation of this species will be especially suitable in regions with a low mean temperature (approximately 5°C) throughout the year, which were previously regarded as being unsuitable for marine aquaculture. On-growing of the ﬁsh in cages is apparently not feasible owing to comparatively high temperatures in summer. Therefore, the use of land-based systems and a continuous supply of water, with a stable temperature from lower water levels, seems the most promising. Recirculation and cooling or warming at least part of the water ﬂow can be used to obtain the optimum temperature for speciﬁc intervals in the wolf-ﬁsh life cycle. Facilities for the commercial production of spotted wolf-ﬁsh were recently established in Norway. The production of spotted wolf-ﬁsh juveniles at Troms Steinbit AS reached 100 000 in 2002. Another plant is situated on the island of Tomma (Nesna Kommune, Helgeland). More than 20 000 individual spotted wolf-ﬁsh (from 5 to 3000 g) are kept in tanks and in a large race-way. Poor egg quality and a variable hatching rate are the main reasons for the inadequate supply of juveniles for commercial production. Thus, the captive biology of spotted wolf-ﬁsh should be intensively studied in the near future.

10.4.5 References Barsukov, V.V. (1959) The Wolf-ﬁsh (Anarhichadidae). Fauna SSSR, Moscow (Translation by the Smithsonian Institute, 1972). NTIS, Springﬁeld, VA. Falk-Petersen, I.-B. & Hansen, T.K. (2001) Organ differentiation in newly hatched common wolf-ﬁsh. J. Fish. Biol., 59, 1465–82. Falk-Petersen, I.-B., Hansen, T.K., Fieler, R. & Sunde, L.M. (1999) Cultivation of the spotted wolf-ﬁsh, Anarhichas minor (Olafsen)—a new candidate for cold-water ﬁsh farming. Aquaculture, 30, 711–18.

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Foss, A., Evensen, T.H., Imsland, A.K. & Øiestad, V. (2001) Effects of reduced salinities on growth, food conversion efﬁciency and osmoregulatory status in the spotted wolf-ﬁsh. J. Fish Biol., 59, 416–26. Foss, A., Evensen, T.H. & Øiestad, V. (2002) Effects of hypoxia and hyperoxia on growth and food conversion efﬁciency in the spotted wolf-ﬁsh, Anarhichas minor (Olafsen). Aquacult. Res., 33, 437–44. Foss, A., Vollen, T. & Øiestad, V. (2003) Growth and oxygen consumption at normal and O2 supersaturated water, and interactive effects of O2 saturation and ammonia on growth in spotted wolf-ﬁsh, Anarhichas minor (Olafsen). Aquaculture, 224, 105–16. Francois, N.R.L., Dutil, J.D., Blier, P., Lord, K. & Chabot, D. (2001) Tolerance and growth of juvenile common wolf-ﬁsh (Anarhichas lupus) under low salinity and hypoxic conditions: preliminary results. Bull. Aquacult. Assoc. Can., 4, 57–9. Hansen, T.K. & Falk-Petersen, I.B. (2001) The inﬂuence of rearing temperature on early development and growth of spotted wolf-ﬁsh, Anarhichas minor (Olafsen). Aquacult. Res., 32, 369–78. Johannessen, T., Gjøsæter, J. & Moksness, E. (1993) Reproduction, spawning behaviour and captive breeding of the common wolf-ﬁsh, Anarhichas lupus L. Aquaculture, 115, 41–51. Lundamo, I. (1999) Vekst og overlevelse hos ﬂekksteinbit (Anarhichas minor). Effect av temperatur og fotoperiode. Cand. Scient. Thesis, Norwegian College of Fishery Science, University of Tromsø (in Norwegian). McCarthy, I., Moksness, E. & Pavlov, D.A. (1998) The effects of temperature on the growth rate and growth efﬁciency of juvenile common wolf-ﬁsh. Aquacult. Int., 6, 207–18. McCarthy, I.D., Moksness, E., Pavlov, D.A. & Houlihan, D.F. (1999) Effects of water temperature on protein synthesis and protein growth in juvenile common wolf-ﬁsh (Anarhichas lupus L.). Can. J. Fish. Aquat. Sci., 56, 231–41. Moksness, E. (1994) Growth rates of the common wolf-ﬁsh, Anarhichas lupus L., and spotted wolfﬁsh, A. minor Olafsen, in captivity. Aquacult. Fish. Manage., 25, 363–71. Moksness, E. & Pavlov, D.A. (1996) Management by life cycle of wolf-ﬁsh, Anarhichas lupus L., a new species for cold-water aquaculture: a technical paper. Aquacult. Res., 27, 865–83. Moksness, E., Rosenlund, G. & Lie, Ø. (1995) The effect of ﬁsh meal quality on growth of juvenile wolf-ﬁsh (Anarhichas lupus). Aquacult. Res., 26, 109–15. Øiestad, V. (1999) Shallow raceways as a compact resource-maximising farming procedure for marine ﬁsh species. Aquacult. Res., 30, 1–10. Pavlov, D.A. (1986) Developing the biotechnology culturing White Sea wolf-ﬁsh, Anarhichas lupus marisalbi. II. Ecomorphological peculiarities of early ontogeny. J. Ichthyol., 26(6), 156–69. Pavlov, D.A. (1994) Fertilization in the wolf-ﬁsh, Anarhichas lupus: external or internal? J. Ichthyol., 34(1), 140–51. Pavlov, D.A. (1997) Development of head skeleton and paired ﬁn girdles in wolf-ﬁsh, Anarhichas lupus, at different temperature regimes. J. Ichthyol., 37(4), 294–303. Pavlov, D.A. & Moksness, E. (1995) Development of wolf-ﬁsh eggs at different temperature regimes. Aquacult. Int., 3, 315–35. Pavlov, D.A. & Moksness, E. (1996a) Repeat sexual maturation of wolf-ﬁsh (Anarhichas lupus L.) broodstock. Aquaculture, 139, 249–63. Pavlov, D.A. & Moksness, E. (1996b) Sensitive stages during embryonic development of wolf-ﬁsh, Anarhichas lupus L., determining the ﬁnal numbers of rays in unpaired ﬁns and skeletal abnormalities. ICES J. Mar. Sci., 53, 731–40. Pavlov, D.A. & Moksness, E. (1997) Development of axial skeleton in wolf-ﬁsh, Anarhichas lupus (Pisces, Anarhichadidae), at different temperatures. Environ. Biol. Fish., 49, 401–16.

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Pavlov, D.A. & Novikov, G.G. (1986) On the development of biotechnology for rearing of White Sea wolf-ﬁsh, Anarhichas lupus marisalbi. I. Experiments on obtaining mature sex products, incubation of eggs and rearing of the young ﬁsh. J. Ichthyol., 26(4), 95–106. Strand, H.K., Hansen, T.K., Pedersen, A., Falk-Petersen, I.B. & Øiestad, V. (1995) First feeding of common wolf-ﬁsh on formulated dry diets in a low-water-level raceway system. Aquacult. Int., 3, 1–10. Tveiten, H. & Johnsen, H.K. (1999) Temperature experienced during vitellogenesis inﬂuences ovarian maturation and the timing of ﬁnal maturation in common wolf-ﬁsh (Anarhichas lupus L.), J. Fish Biol., 55, 809–19. Tveiten, H. & Johnsen, H.K. (2001) Thermal inﬂuences on temporal changes in plasma testosterone and oestradiol-17 beta concentrations during gonadal recrudescence in female common wolf-ﬁsh. J. Fish. Biol., 59, 175–8. Tveiten, H., Scott, A.P. & Johnsen, H.K. (2000) Plasma-sulfated C21-steroids increase during the periovulatory period in female common wolf-ﬁsh and are inﬂuenced by temperature during vitellogenesis. Gen. Comp. Endocrinol., 117, 464–73. Tveiten, H., Solevåg, S.E. & Johnsen, H.K. (2001) Holding temperature during the breeding season inﬂuences ﬁnal maturation and egg quality in common wolf-ﬁsh. J. Fish Biol., 58, 374–85.

10.5 Halibut J. Chr Holm, T. Harboe, A. Mangor-Jensen and B. Norberg

10.5.1 Introduction The ﬁrst systematic, scientiﬁc attempts to establish basic protocols for the farming of Atlantic halibut in Norway were initiated in 1974 by Per Solemdal and co-workers (Solemdal et al., 1974). They managed to keep larvae alive until Day 60 after hatching. After a period of inactivity in the late 1970s, new trials in the 1980s gave promising results. In 1980, two metamorphosing halibut were harvested from a mesocosm at IMR Flødevigen Research Station, and a description of their early development was subsequently published (Blaxter et al., 1983). In 1985, two halibut fry survived at IMR Austevoll Aquaculture Research Station. From 1987, the scientiﬁc effort increased in order to solve the more speciﬁc problems of halibut production. A comprehensive bibliography of halibut larvae cultivation has been published by Mangor-Jensen et al. (1998). More than 25 years of scientiﬁc work, the last 13 years of which were both costly and intensive, have provided a foundation for commercial culture operations. In 2001, a total of 14 Norwegian farms produced nearly 450 000 1–5-g juveniles. In 1999, a similar quantity was produced by one Icelandic farmer, while 200 000 were produced in Scotland, and Canadian operations achieved 48 000 juveniles. The same year, the total estimated production was 950 000 juveniles, with three-quarters of these being produced intensively. In Norway, where extensive and semi-intensive production methods have been historically important, intensively produced juveniles have increased in numbers. This trend seems to be persisting in 2002, indicating that this new method of fry production has gained a foot-hold, although semi-intensive pond production still shows good results when climatic conditions are favourable. To put this in perspective, compared with the development of intensive

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warm-water marine ﬁsh aquaculture in southern Europe, the production of halibut fry for aquaculture will most certainly increase in volume to meet all the requirements of commercialisation. The establishment of a year-round production of gametes still represents a bottleneck in reaching this goal. In addition, fry quality in terms of pigmentation and eye-migration need to be improved for future production. The market situation for halibut is good, since both price and demand indicate that it will become an important species for aquaculture. The wild stocks of Atlantic halibut are decreasing. In the early 1990s, the total landing of Atlantic halibut was approximately 7500 tons per year, but since then landings have been decreasing. From 1995 to 2000 the total landing has been close to 4000 tons annually.

10.5.2 Brood Stock, Egg Production and Incubation Adult and sexually mature ﬁsh are normally caught on their spawning grounds, preferably by long-line ﬁshery. Candidate brood stock are brought to the farms before the hook is removed from either the oesophagus or the cardiac region of the stomach. Stainless steel hooks must be removed (they do not disintegrate as wrought iron does), and ﬁsh have to be properly anaesthetised during removal. Fish should be tagged and their sex determined, the latter either by stripping gametes or by ultrasonography. It is important to get the newly caught ﬁsh to feed properly. As the result of an increasing number of farming operations, ﬁsh produced in captivity (F1 generation) are now available. For these domestic brood stocks, steps should be taken to avoid in-breeding as well as disease transfer. When establishing groups of breeders, intensive health control should be practised. The removal of ectoparasites and the treatment of wounds and injuries should be done as soon as possible. The ﬁsh can be manipulated to spawn in or off season by exposure to natural, advanced or delayed annual photoperiod cycles. This ensures the availability of viable gametes and larvae throughout the year (Næss et al., 1996). Manipulated ﬁsh are usually exposed to a compressed or an extended annual photocycle, and then to a phase-shifted 12month annual cycle, resulting in an advanced or delayed spawning period, respectively. The water temperature must be held at around 6°C, both prior to and during the spawning season. Egg production is based on the stripping of individual females at ovulation (mean time span between ovulations 72–80 h, Norberg et al., 1991). Males are kept in the same tank as females. Females can be checked for maturity stage visually or by ultrasonography. Another method is to press the abdomen gently (Fig. 10.16) in order to detect ovulated eggs (when ovulated eggs are present, the gonads will be more ﬂexible than prior to ovulation). After ovulation, ﬁsh must be taken gently out of the water and stripped manually. In males, spermiation is continuous, and they can be stripped when eggs are obtained. Shortly before fertilisation, a minimum of 1 ml milt per 1 egg is suspended in 3 l full seawater, the eggs are added, and fertilisation is allowed to proceed for a period of 10 min. The fertilised eggs are kept in an up-welling incubation system. The development of the halibut embryos should be checked throughout the incubation period, so that egg groups showing low fertilisation or poor development may be discarded. Eggs are reared in darkness, at 6°C and at salinities similar to brood-ﬁsh conditions.

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Figure 10.16 Female halibut with a large abdomen during the checking procedure on a neoprene-covered stripping table (photograph Jens Chr. Holm).

Figuer 10.17 Halibut yolk-sac incubator. The seawater inlet is from the lowest valve under the cone. Low-salinity water is added through the oriﬁces in the vertically mounted pipeline to create a continuous salinity gradient from the lowest part of the inner pipeline to the surface. An outlet sieve is located close to the surface, and the second valve at the bottom is for taking out dead larvae (drawing by T. van der Meeren).

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In order to prevent viral infections of the egg surface, ozone treatment of the seawater has proved to be effective. Ozone treatment as surface disinfection inactivates any nodavirus and increases the survival of the larvae. The poorly developed halibut larva hatches after ca. 82 day-degrees. Hatching is an enzymatic process, and the zone of breakdown is very distinct (Helvik et al., 1991). Hence, eggs should not be exposed to turbulence or handling prior to hatching. Eggs should therefore be transferred to larvae silos 1 or 2 days prior to the expected hatching time (see next section). The hatching process is inhibited by light exposure (Helvik & Walther, 1992). To synchronise hatching, eggs are exposed to light from 75 day-degrees until 87 day-degrees. Hatching will then occur within 2 h.

10.5.3 Larval Rearing Halibut larvae have a long yolk-sac stage compared with other marine ﬁsh species. The period from hatching to ﬁrst feeding lasts approximately 44 days at 6°C. Newly hatched larvae are poorly developed and are very sensitive to handling, and the eggs are normally transferred to yolk-sac incubators before hatching (Fig. 10.17). Larval vertical distribution should be observed during and immediately after hatching in the silo. For eggs and larvae with low buoyancy, the ﬂow should be reduced prior to hatching, and stopped completely during hatching and for 2–3 days thereafter. Oxygenation of the water layer with a high larval density must be done gently if needed (Harboe et al., 1998). For heavier larvae, the water ﬂow and a dynamic gradient (30 p.s.u. in the outlet) should be kept intact during the period immediately after hatching since the larvae will not reach the upper level in the silo. A standard outlet sieve can be mounted near the surface prior to hatching, but operational procedures should always keep the larvae away from this sieve. After Day 7, larvae will start drinking and become more active, and will distribute themselves more evenly in the water volume. The density gradient is not necessary at this time, but oxygenation must occasionally be used if heavy larvae tend to aggregate near the bottom cone. Halibut larvae should be transferred to the ﬁrst-feeding units at 260–265 dC (days degrees) after hatching (Harboe & Mangor-Jensen, 1998). The size of the larvae should be at least 1 mg DW. In intensive systems, a combination of UV-A radiation for the ﬁrst 24 h and green-water techniques seem to initiate a high feeding incidence as early as possible. However, the single most important factor is to maintain a water velocity pattern that allows the larvae to position themselves facing the water current for easy capture of their prey. A ﬁrst-feeding temperature of around 12°C is recommended. Halibut larvae consume high numbers of Artemia (up to 2500 instar II per day). Quick growth and high consumption rates, and the fact that the enriched Artemia should be consumed within a few hours of introduction, underline the importance of using feeding regimes based on information on larval number, consumption rate and Artemia wash-out rate. Both the high feeding rate as well as the development from a pelagic to a more demersal phase increase the importance of cleaning and tending procedures that create a minimum of stress. This factor has resulted in modiﬁcations to traditional tanks, e.g. the development of automatic cleaning systems (Fig. 10.18) (Meeren, van der, et al., 1998).

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Figure 10.18 Examples of different cleaning devices in halibut larvae (empty dark tank) and juvenile rearing (photographs Jens Chr. Holm).

Intensive rearing methods, based exclusively on the use of enriched Artemia diets, make the halibut susceptible to pigmentation and eye migration abnormalities. The optimal ﬁrst-feeding diet for halibut is still to be developed. Both formulated diets and live-feed diets are composed based on the chemical composition of halibut larvae, the macro-nutrient composition of feed for halibut juveniles and the nutrient composition of copepods. The metamorphosis of halibut consists of several separate biochemical processes (growth, neural and skeletal transformation, pigmentation) and is mediated by thyroxine (Solbakken et al., 1999). The same authors suggest a ‘window of opportunity’ for inducing or inﬂuencing metamorphosis between 16 and 22 mm standard length. There is great plasticity in the viable post-metamorphic forms in halibut: at least four sub-lethal variations in development have been identiﬁed in pigmentation, eye migration, anterior dorsal ﬁn development and body shape (Pittman et al., 1998).

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10.5.4 Weaning and On-Growing Weaning to a formulated feed can be carried out in a modiﬁed ﬁrst-feeding tank (Meeren, van der, et al., 1998) or in shallow raceways. Weaning can be done at a small size (0.07 g wet weight; survival ca. 80%), which saves large amounts of live feed. Diets provided for these ﬁsh need to take into account the limited ability of the halibut larvae to assimilate protein and lipid. It is expected that the time period for the provision of live feed can be shortened even more (0.07-g size is achieved 3 weeks after the onset of ﬁrst feeding). Metamorphosed ﬁsh should be reared at temperatures near 12°C, and in continuous light to provide accelerated growth (Jonassen et al., 2000). These authors proposed that reduced activity (reﬂected by higher oxygen consumption during darkness) and the anabolic effects of photoperiod contribute to the increased growth and growth efﬁciency in ﬁsh subjected to continuous light. During this period, ﬁsh are very susceptible to eye loss (mainly the right eye), probably due to aggressive contacts with other ﬁsh. Because of the advantages of relatively high water temperatures, it is probable that the halibut will be kept in tank facilities with heated water until they reach sizes of 100–300 g. The optimum temperature for halibut decreases with size, and is 11.4°C for 280-g ﬁsh.

10.5.5 On-Growing Systems Both cages (net pens) and tanks (land-based) are used for the on-growth of halibut. Tanks are expensive, but ﬁsh are easier to control compared with net pens. In both operations, heating the water seems to be economically unfavourable. The larger (3–6 kg) ﬁsh have an optimal rearing temperature of 9.7°C. Feed formulations are implemented commercially, but it seems that during the on-growth period, ﬁsh can achieve their maximum growth rate with a protein content as low as 48% (1-kg ﬁsh and larger; Helland & Grisdale-Helland, 1998). Variations in the fat content of the feed between 10 and 39% of dry matter does not inﬂuence growth or gross feed conversion efﬁciency between 0.8 and 1.8 kg. The fat content in the feed should be around 40% before slaughter, and ﬁsh should not be slaughtered at sizes under 2 kg owing to sensory characteristics (Nortvedt & Tuene, 1998). The stocking rate should be moderate. In both tanks and cages, ﬁsh covering more than 200% of the bottom tend to show a reduced growth rate, probably due to physical contact and disturbance, and increased activity levels (Holm et al., 1998). It is expected that growth rate can be accelerated compared with current levels (from 0.3 to 5 kg in 30 months). Sexual maturation will reduce growth rates. This problem is more pronounced in males, as they will mature before reaching a commercially acceptable size. Continuous light stimulates growth, but it can also increase the incidence of sexual maturation in male halibut (Norberg et al., 2001).

10.5.6 References Blaxter, J.H.S., Danielssen, D.S., Moksness, E. & Øiestad, V. (1983) Description of the early development of the halibut (Hippoglossus hippoglossus) and attempts to rear the larvae past ﬁrst feeding. Mar. Biol., 73, 99–107.

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Harboe, T. & Mangor-Jensen, A. (1998) Time of ﬁrst feeding of Atlantic halibut larvae. Aquacult. Res., 29, 913–19. Harboe, T., Skår, S.Å., Naas, K.E. & Holm, J.C. (1998) Incubation of yolk-sac larvae improved by addition of freshwater and oxygen. Coun. Meet Int. Coun. Explor. Sea, L:13. Helland, S.J. & Grisdale-Helland, B. (1998) Growth, feed utilization and body composition of juvenile Atlantic halibut (Hippoglossus hippoglossus) fed diets differing in the ratio between the macronutrients. Aquaculture, 166, 49–56. Helvik, J.V. & Walther, B.T. (1992) Photo-regulation of the hatching process of halibut (Hippoglossus hippoglossus) eggs. J. Exp. Zool., 263, 204–209. Helvik, J.V., Oppen-Berntsen, D.O. & Walther, B.T. (1991) The hatching mechanism in Atlantic halibut (Hippoglossus hippoglossus). Int. J. Dev. Biol., 35, 9–16. Holm, J.C., Tuene, S.A. & Fosseidengen, J.E. (1998) Halibut behaviour as a means of assessing suitability of ongrowth systems. ICES C.M., L4, 11 pp. Jonassen, T.M., Imsland, A.K., Kadowaki, S. & Stefansson, S.O. (2000) Interaction of temperature and photoperiod on growth of Atlantic halibut, Hippoglossus hippoglossus L. Aquacult. Res., 31, 219–27. Mangor-Jensen, A., Harboe, T., Shields, R., Gara, B. & Naas, K.E. (1998) Review of halibut cultivation literature, 1997. Aquacult. Res., 29, 857–87. Meeren, T. van der, Harboe, T., Holm, J.C. & Solbakken, R. (1998) A new cleaning system for rearing tanks in larval ﬁsh culture. Coun. Meet Int. Coun. Explor. Sea, L:13. Næss, T., Harboe, T., Mangor-Jensen, A., Naas, K.E. & Norberg, B. (1996) Successful ﬁrst feeding of Atlantic halibut larvae from photoperiod-manipulated broodstock. Prog. Fish Cult., 58, 212–14. Norberg, B., Valkner, V., Huse, J., Karlsen, I. & Grung, G.L. (1991) Ovulatory rhythms and egg viability in the Atlantic halibut (Hippoglossus hippoglossus). Aquaculture, 97, 365–71. Norberg, B., Weltzien, F.-A., Karlsen, Ø. & Holm, J.C. (2001) Effects of photoperiod on sexual maturation and somatic growth in male Atlantic halibut (Hippoglossus hippoglossus L.). Comp. Biochem. Physiol. B, 129, 357–65. Nortvedt, R. & Tuene, S. (1998) Body composition and sensory assessment of three weight groups of Atlantic halibut (Hippoglossus hippoglossus) fed three pellet sizes and three dietary fat levels. Aquaculture, 161, 295–313. Pittman, K., Jelmert, A., Næss, T., Harboe, T. & Watanabe, K. (1998) Plasticity of viable forms of farmed Atlantic halibut (Hippoglossus hippoglossus). Aquacult. Res., 29, 949–52. Solbakken, S.S., Norberg, B., Watanabe, K. & Pittman, K. (1999) Thyroxine as a mediator of metamorphosis of Atlantic halibut, Hippoglossus hippoglossus. Environ. Biol. Fish., 56, 53–65. Solemdal, P., Tilseth, S. & Øiestad, V. (1974) Rearing of halibut. I. Incubation and the early larval stages. Int. Counc. Explor. Sea C.M. 1974/F:41, 5 pp. (mimeo).

10.6 Turbot B.R. Howell

10.6.1 Introduction The turbot, Scophthalmus maximus, is among the most valuable ﬁsh of the north-eastern Atlantic. It occurs in coastal waters to a depth of about 80 m from western Norway to southern Spain and throughout the Mediterranean. In the Black Sea, it is replaced by the equally valuable and closely related species S. maeoticus. Turbot does not occur in dense concen-

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10,000 9,000 8,000 7,000 6,000 5,000 4,000 3,000 2,000 1,000 0 1970

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Figure 10.19 European landings of turbot from 1970 to 2000 (source FAO).

trations, so the relatively modest annual European landings, that have ﬂuctuated between 3.4 and 9 thousand tonnes over the last 30 years (Fig. 10.19), are almost entirely generated as a by-catch of ﬁsheries targeting other species. The turbot is a top predator which feeds almost exclusively on other ﬁsh during its adult life. The potential of the turbot for farming was recognised during the early 1970s. The pioneering work of Shelbourne (1964) had demonstrated the feasibility of mass-producing juveniles of other ﬂatﬁsh species in hatcheries, but the turbot appeared to offer the best prospect for commercial exploitation because of its relatively rapid growth rate and high value. The following account is largely based on a recent review by Howell (1998).

10.6.2 Brood Stock, Egg Production and Incubation Early studies were supported by eggs fertilised at sea, but eventually captive stocks of mature ﬁsh were established. The relatively large size of the turbot seems to inhibit natural spawning in the type of facility (e.g. 10 m3 tanks) in which plaice and sole readily spawn, but the gametes can readily be hand-stripped and the eggs artiﬁcially fertilised. The viability of eggs and larvae obtained in this way varies considerably, which to a large extent is due to the rapid deterioration in the quality of the eggs after ovulation. Studies of ovulatory patterns and egg viability have provided a means of designing stripping schedules that minimise these effects. These studies, combined with those that led to the development of methods for securing year-round production of eggs by photoperiod control, were of central importance in supporting the development of commercial operations. The translucent eggs are about 1 mm in diameter, and are generally incubated in up-welling systems stocked at a rate of about 2–3000 l-1.

10.6.3 Larval Rearing The main obstacle to realising the potential for farming turbot was that the species could not be reared by the methods that had been developed for plaice, Pleuronectes platessa, and sole, Solea solea. Turbot larvae were found to be too small to ingest newly hatched nauplii of

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Artemia, and even though the smaller rotifer Brachionus plicatilis were voraciously ingested, the survival rates were very low. Success was only accomplished once it was realised that the nutritional value of cultured rotifers was inadequate, but was improved when the rotifers were fed particular species of unicellular algae. The algae were thought to provide certain polyunsaturated fatty acids that were known to be an essential dietary requirement for turbot and other marine species. More recent work has further explored the role of these fatty acids and demonstrated that they are not only important determinants of survival, but are also implicated in controlling the level of abnormal pigmentation, which is a common feature in cultured ﬂatﬁsh. Although various proprietary products, such as lipid emulsions, have been developed as a means of enhancing the lipid quality of live foods, the use of unicellular algae continues to be favoured by many commercial operators. In addition to the undoubted nutritional beneﬁts of adding algae to rearing tanks, other beneﬁts may be realised through, for example, modiﬁcation of the microbial environmental and light conditions.

10.6.4 Weaning and On-Growing The partial resolution of these nutritional problems also contributed to the development of consistent methods for weaning the ﬁsh from live to formulated feeds, which is an important step in the commercialisation of rearing procedures. The ﬁrst attempts to wean turbot from live to formulated feeds met with highly variable levels of success, but it was found that this variability was almost entirely attributable to the quality (in terms of lipid) of the live food on which the ﬁsh had previously been fed. The ﬁrst pilot-scale on-growing trials in the UK during the 1970s exploited the warmwater efﬂuent from nuclear power stations. Growth of turbot is optimal at 15–16°C, and in the UK this approach offered the best opportunity for providing optimal growing conditions. By the end of the 1970s, annual production had reached about 50 tonnes. Although small in scale, the success of these operations, combined with the gradually improving supply of juveniles from hatcheries, encouraged an expansion of the industry. The preferred option for this expansion was to exploit areas where ambient water temperatures were relatively favourable. This avoided some of the practical difﬁculties that had been experienced in the cooperative ventures with nuclear power stations as well as the adverse publicity that such methods might attract. Thus, during the 1980s, commercial activities became concentrated on the Atlantic coast of France and Spain, and particularly Galicia, where temperatures in the range 12–18°C could be found. In these areas, the preferred system for turbot production is in shore-based tanks and raceways supplied with pumped seawater. The tanks, which are typically about 1 m deep, are mainly constructed of concrete and usually covered to reduce light levels and limit the growth of algae that could cause fouling problems. The juveniles are stocked at a size of about 5 g and take a little over 30 months to grow to an average weight of 2 kg. The tank area required per tonne of output is approximately 25–30 m2, although this may be higher when supplementary oxygenation of the water is employed. Water requirements vary considerably from about 10 to 30 m3 per hour per tonne of stock held depending on factors such as temperature and whether or not the farms use pure oxygen.

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PRODUCTION (tonnes) PRODUCTION (tonnes)

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Other Other France France Spain Spain

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Figure 10.20 European production of farmed turbot (redrawn from Howell, 1998).

The majority of farms in Galicia were constructed between 1982 and 1986, and this resulted in a rapid increase in annual production that reached about 3000 tonnes by 1995. Over 90% of this originated from France and Spain (Fig. 10.20). This increase in production coincided with a marked reduction in market value, which was attributable not only to the limited markets for this species, but to the relatively poor quality, in terms of pigmentation, ﬂesh texture and fat content, of the ﬁsh being produced. A gradual improvement in juvenile quality combined with a gradual change from a wet diet of trash ﬁsh of variable quality to dry diets of more consistent quality seemed to rectify these difﬁculties and lead to a stabilisation of prices. During this period of commercialisation, the reducing price of turbot and the high initial investment costs of pump-ashore installations caused severe economic problems for the industry, and many farms were forced into liquidation. A signiﬁcant problem in this context was that hatchery production failed to meet expectations, and many farms were forced to operate well below their production capacity. In 1991, for example, farms could only access sufﬁcient juveniles to operate at about 30% of their production capacity. Continuous improvements in juvenile production allowed this to increase to 68% over the subsequent 4 years.

10.6.5 Future Prospects The industry is now well established and has attained relative stability. The current level of production of about 4000 tonnes per annum is likely to increase with the development of new markets as well as the adoption of novel production techniques. Recent developments in recycling technology are being exploited for turbot and other marine species in more

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northern European countries, such as The Netherlands and the UK. The degree of environmental control offered by such systems, and the relative independence of prime coastal locations they allow, are strong incentives for an expansion of this approach, but it remains to be seen whether technical and economic success will be realised.

10.6.6 References FAO (Food and Agriculture Organization of the United Nations Statistical Data) (FAOSTAT). http://apps.fao.org/ Howell, B.R. (1998) Development of turbot farming in Europe. Bull. Aquacult. Assoc. Can., 98–1, 4–10. Shelbourne, J.E. (1964) The artiﬁcial propagation of marine ﬁsh. Adv. Mar. Biol., 2, 1–83.

10.7 Sole B.R. Howell

10.7.1 Introduction Sole of the family Soleidae are among the most highly priced marine ﬁsh. In western Europe, the most important species, Solea solea and S. senegalensis, have long been considered prime candidates for farming. S. solea, variously known as the common, black or Dover sole, is widely distributed along the eastern Atlantic seaboard from southern Norway to Senegal and throughout the Mediterranean. Over the period 1970–2000, annual landings of this species have ﬂuctuated between 25 and 63 thousand tonnes, and are currently close to the average of 41 thousand tonnes (Fig. 10.21). The senegal sole, S. senegalensis, has a more restricted distribution, and is not found north of the Bay of Biscay or in the eastern Mediterranean.

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Figure 10.21 European landings of the common sole from 1970 to 2000 (source FAO).

2000

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Attempts to rear sole date back to the turn of the last century, but farming only became a realistic possibility in the 1960s when it was demonstrated that juveniles could be readily mass produced under hatchery conditions. Despite the comparative ease with which the species could be reared through its larval stages, commercial development was prevented because of the poor performance of the juvenile stages on ﬁsh-based formulated feeds. This problem was primarily related to the natural mode of feeding of the species. Unlike other important candidate species for farming, such as turbot and halibut, sole are not piscivorous, but browse on invertebrate benthic organisms. The important consequences of this are that the ﬁsh have a small stomach adapted to a ‘little and often’ mode of feeding and, perhaps more importantly, are not attracted to ﬁsh-based diets. In the 1970s, it was shown that these problems could be partly overcome by the inclusion in the feeds of invertebrate tissue or chemical taste attractants, but reproducible commercially acceptable on-growing techniques did not emerge. Consequently, in the early 1980s, efforts were diverted to less problematic species such as the turbot, Scophthalmus maximus. Recent advances in feed technology, however, have indicated that the problems of ongrowing sole may be less intractable than they once appeared. These developments have rekindled considerable interest in the possibility of farming sole, particularly among the ﬁsh farmers of southern Europe. The temperature conditions in this region would favour both the Dover sole and the Senegal sole, making these species attractive options for diversiﬁcation of the bass (Dicentrarchus labrax) and bream (Sparus aurata) industries. The following section brieﬂy reviews the current status of rearing methods for sole. This is based on the Dover sole, but is largely applicable to the Senegal sole, which has extremely similar characteristics. This account is largely based on the recent review of sole culture by Howell (1997).

10.7.2 Brood Stock, Egg Production and Incubation Sole are generally maintained in tanks of 10–15 m3 capacity, and fed on live or natural food such as mussels, Mytilus edulis, ragworm, Nereis sp., and lugworm, Arenicola marina (see review by Baynes et al., 1993). The ﬁsh spawn spontaneously in captivity, so that a regular supply of good-quality eggs is readily obtained. Annual egg production ranges widely from 10 to 140 eggs g-1 of female, with a tendency for the lower fertility rates to occur following relatively warm winters. Displacing the annual 6–8-week spawning period by manipulating photoperiod and temperature is readily accomplished, so that eggs can be made available throughout the year. The buoyant eggs are about 1 mm in diameter and are characterised by the presence of multiple clusters of oil globules. Hatching occurs after about 5 days at 12°C. At hatching, the larvae have a large yolk sac, no eye pigmentation, and the mouth is poorly developed and non-functional. During the subsequent 3–4 days, however, the larvae develop the capacity to feed.

10.7.3 Larval Rearing Rearing the larvae through to metamorphosis presents few problems, with survival rates of over 70% being regularly attainable in small-scale laboratory systems. A particular advan-

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tage of this species is that the larvae are large enough to be reared on a diet of Artemia nauplii from the outset, making the provision of the rotifer, Brachionus plicatilis, as a ﬁrst food unnecessary. The ease with which the larvae can be reared relative to some other species, for example the turbot, may in part be due to the consistently high quality of naturally fertilised eggs. However, it also appears that the sole has a less stringent dietary requirement for (n-3) HUFAs than many other species. Although sole have an essential dietary requirement for EPA, 20:5(n-3), high survival rates may be achieved on diets which are almost deﬁcient in DHA, 22:6(n-3). Thus, enhancing the lipid content is not a prerequisite of high survival in this species. However, survival, although clearly essential, may not be an adequate criterion by which to judge rearing methodology on its own. Recent studies described a link between the lipid quality of the larval diet and the low-temperature tolerance of the juveniles several months after having been weaned onto a formulated feed. The persistence of such an effect indicates that larval diet may be an important determinant of the phenotype of later developmental stages, and may therefore have an important impact on characteristics of economic importance such as growth rate, stress tolerance and disease resistance. Optimising the quality of larval diets against such criteria is an important priority, even when high survival rates are readily achieved.

10.7.4 Weaning and On-Growing It has long been known that the attractiveness of the diet is critical to the success of weaning in sole, but more recent work has indicated that the ability of the ﬁsh to utilise the diets may also be an important consideration. Consistently high survival and growth rates have recently been achieved using a readily digestible agglomerated feed. In one weaning trial, growth in length showed a progressive increase against a live-food standard (Fig. 10.22). This may reﬂect an increasing acceptance of the novel diet and/or an increase in the digestive competence of the ﬁsh. There is little documented information on the potential growth rate of Dover sole to market size under optimal conditions, or on the extent to which growth to market size may be achieved on commercially acceptable formulated feeds. Extrapolations from experimentally derived data indicate that under optimum temperatures of 18–20°C, 5-cm-long sole fed a live or natural food would reach a minimum market size of 24 cm (125 g) in less than 300 days. Even this relatively slow growth rate has not yet been matched by ﬁsh fed a formulated feed. In addition, Dover sole may be less suited to crowded conditions than some other species, since they respond to increasing density by slower growth and increased size variation. In contrast, the Senegal sole appears to be much faster growing and less affected by crowded conditions than the Dover sole. Recent trials indicate that a mean weight of about 450 g may be achieved within 14 months when sole are fed a formulated feed at temperatures around the optimum of 25°C. Methods of ameliorating these effects need to be developed.

10.7.5 Future Prospects The sole is probably the easiest marine ﬁsh to rear through its larval stages, and so juvenile supply would not be a constraint on commercial developments. Similarly, weaning onto for-

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65

MEAN STANDARD LENGTH (mm)

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20 25 DAYS

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Figure 10.22 The change in mean standard length of juvenile sole following an abrupt transfer (Day 0) from Artemia nauplii to a commercially prepared formulated feed. The vertical bars indicate standard deviations, and the broken line the ‘potential’ growth for each growth period as predicted from Irvin’s (1973) data (cited in Howell, 1997). Reproduced with permission from Elsevier Science.

mulated feeds can be achieved with minimal losses on a repeatable basis. The high growth rates of the Senegal sole on commercially acceptable formulated feeds indicate that this species will almost certainly be commercially farmed in the near future, particularly in southern European countries where temperature conditions are most favourable. Prospects for farming the slower-growing Dover sole will depend on the extent to which markets will demand that particular species in preference to the Senegal sole. However, farming of that species could only become a reality if faster growth rates could be achieved, and methods developed to ameliorate the effects of high stocking density. These may include regular grading to reduce size variation, and the adoption of feeding strategies that reduce opportunities for individuals to dominate the food supply.

10.7.6 References Baynes, S.M., Howell, B.R. & Beard, T.W. (1993) A review of egg production by captive sole, Solea solea (L.). Aquacult. Fish. Manage., 24, 171–80. FAO (Food and Agriculture Organization of the United Nations Statistical Data) (FAOSTAT). http://apps.fao.org/ Howell, B.R. (1997) A re-appraisal of the potential of the sole, Solea solea (L.), for commercial cultivation. Aquaculture, 155, 355–65.

Chapter 11

Marine Stock Enhancement and Sea-Ranching T. Svåsand and E. Moksness

11.1 Introduction Stock enhancement and sea-ranching of marine ﬁsh in modern times goes back more than 100 years. People living in coastal areas depended on the yield from the sea, and frequently experienced great ﬂuctuations in their annual landings, which affected their quality of living signiﬁcantly. They believed that by releasing artiﬁcially hatched larvae of marine species such as Atlantic cod, these ﬂuctuations could be reduced, which would result in repeated high annual yields in the ﬁshery. In 1878, the head of the US Fish Commission, Spencer F. Baird (1823–1887), succeeded in hatching cod eggs in Gloucester, Massachusetts, and in 1885 the US Fish Commission built its ﬁrst commercial marine hatchery at Woods Hole, Massachusetts (Shelbourne, 1964). In Norway, a captain of sailing vessels, Gunder Mathisen Dannevig (1841–1911), raised funds from local citizens and from the Norwegian legislature to build a cod hatchery in southern Norway (Solemdal et al., 1984). The hatchery was established in 1882 in Flødevigen, near the town of Arendal. In Flødevigen, mature female and male cod were held in an enclosed outdoor basin, and natural spawning resulted in viable fertilised eggs. The eggs were collected by a ﬁlter device and transferred to the laboratory for incubation (Fig. 11.1). Both the Woods Hole and the Flødevigen hatcheries were in full production in the 1890s, annually releasing millions of cod larvae (Fig. 11.2). As no tagging techniques for marking eggs were then available, it was not possible to prove that these early release programmes had signiﬁcant effects, and interest in these releases gradually faded. While the US release programme was terminated before World War II, the Norwegian programme continued until 1971. More recently, experimental releases of genetically marked yolk-sac larvae have shown that the beneﬁts of releasing yolk-sac larvae are very small (Kristiansen et al., 1997). An interest in cold-water marine stock enhancement and sea-ranching started again in the 1980s, along with the development of suitable tagging methods, and programmes were initiated in the Scandinavian countries, the UK and the USA (Blaxter, 2000). A major difference from the previous activity was that instead of releasing larvae, juveniles were used. The main marine cold-water species were Atlantic cod, European lobster and turbot (Psetta maxima). In the last 20 years, stock enhancement and sea-ranching have experienced increased interest world-wide, and now 27 countries are involved with the stocking of over 65 marine or brackish-water species (Fig. 11.3, see colour plate section) (Bartley, 1999).

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Figure 11.1 Photograph of the Flødevigen hatchery at Arendal, in the southern part of Norway.

3000

Number (In millions)

2500 USA

2000

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1000

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1951-1960

1941-1950

1931-1940

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1911-1920

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Figure 11.2 Number of released yolk-sac larvae of cod (in millions per 10 years) in the USA and Norway (Solemdal et al., 1984).

Stock enhancement and sea-ranching normally describe the release of hatchery-raised animals, and in general, the programmes focus on local coastal stocks rather than abundant open-sea stocks. In marine ranching, Japan is the leading country, with more than 90 marine species in their ranching programme. The individuals that are reared for stock enhancement and sea-ranching purposes have to be of high quality to compete and survive in the wild. In addition, the release strategy and

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an ability to adapt to the wild are of signiﬁcant importance for success (Blaxter, 1975; Howell, 1994; Svåsand et al., 1998). Release strategies affect survival after release and depend on factors such as:

• time and size at release • habitat selection • carrying capacity in the release area • prey availability • predators present Regarding an ability to adapt to the wild, the behaviour of individuals is a key factor with respect to both the capture of wild prey and the avoidance of predators. Reared animals are often found to be less well adapted to life in the wild compared with their wild-born counterparts.

11.2 Stock Enhancement and Sea-ranching in Europe and North America In the course of this century, more and more evidence has emerged indicating that the mortality rates of ﬁsh larvae and juveniles in their natural environment decline rapidly with increasing size. This was demonstrated using released genetically marked yolk-sac larvae (Kristiansen et al., 1997). Less than 1% of the larvae were alive 1 month after release, and less than 1 in 150 000 survived 1 year later. The effect of releasing yolk-sac larvae therefore seemed to be very small. By the 1970s in Norway, large numbers of juveniles could be reared, and therefore juveniles were released. The results of the ﬁrst cod juvenile release study were presented at the Flødevigen symposium on ‘The Propagation of Cod, Gadus morhua L.’ in 1983. Many scientists expressed interest in performing release experiments with the purpose of testing the hypothesis that releasing large numbers of reared juveniles could enhance local cod stocks, and release experiments were initiated in Norway and in other countries (Dahl et al., 1984).

11.2.1 Atlantic Cod Tagged, reared juvenile Atlantic cod were ﬁrst released on the Norwegian Skagerak coast in 1976 and 1977. These releases were scaled up in 1983. In 1985, the Norwegian Fisheries Research Board initiated an interdisciplinary research programme with the aim of identifying the potential for proﬁtable sea-ranching, and also to examine whether releases of juvenile cod could even out the natural ﬂuctuations in recruitment to cod stocks and thereby stabilise the ﬁsheries. This programme was further scaled up in 1990, when the Norwegian government decided to establish a programme for the development and promotion of searanching (Norwegian acronym, PUSH). This programme also included European lobster

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(Homarus gammarus), Atlantic salmon (Salmo salar) and Arctic char (Salvelinus alpinus) (Svåsand et al., submitted for publication). Between 1976 and 1995 more than 1 million tagged juvenile cod were released in different coastal and fjord ecosystems in Norway. The size of the ﬁsh varied between 8 and 37 cm. All ﬁsh were marked prior to release. In the 1990s, smaller groups of Atlantic cod were also released in Denmark, the Faeroe Islands, Sweden and the USA (Fig. 11.4). The major ﬁndings of the experimental stocking of Atlantic cod (Svåsand et al., 2000) are listed below.

M aine

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Figure 11.4 Release of reared juvenile cod in Europe and North America (Svåsand et al., 2000).

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(2)

(3)

(4) (5) (6) (7) (8) (9)

479

Cultured cod adapted well to the natural environment, and after only a few weeks their choice of prey was similar to that of wild cod. It was found that the released cod suffered from higher mortality rates than wild cod, and that time spent in captivity inﬂuenced the patterns of migration, and anti-predator and feeding behaviour after release. Norwegian studies indicated that genetic selection and genetic drift were a minor problem, and very few differences in genotype distribution and gene frequencies were observed between wild and reared cod. Efﬁcient methods for tagging and marking cod juveniles were developed, and included external anchor tags, chemical tags (alizarin complexon and oxytetracycline) and genetic markers. Most of the released cod remained in the release areas, although some variations were observed. In most of the release areas, a positive correlation between size at release and survival was observed. Recapture rates ranged from 0% to 30% depending on area, time and size at release. In Norway, it was found that growth rates were highest in the outer coastal areas, which was in accordance with ecosystem models. Ecological studies in Norway indicated that on average enough cod were recruited to utilise the food available. Despite relatively large variations in environmental conditions, cod production and ﬁshing mortality along the Norwegian coast, the results indicated that under the conditions that existed during the 1980s and 1990s, releases of juveniles did not signiﬁcantly increase cod production and catches.

In the future, however, increased ﬁshing pressure on the local ﬁshery resources in Norway due to recreational ﬁshing and tourism could make releases of cod a good way of increasing the attraction of ﬁshing for tourism.

11.2.2 Other Cold-water Species Early stages of plaice and sole were released in the UK from the 1890s, but, as with releases of Atlantic cod, any beneﬁcial effects were difﬁcult to prove (Blaxter, 2000). In Norway, Rollefsen (1940) released yolk-sac larvae hybrids from crosses of female plaice (Pleuronectes platessa) and male ﬂounder (Platichthys ﬂesus) into the Borgenfjord near Trondheim, and afterwards found that a considerable proportion of the metamorphosed ﬂatﬁsh in part of the fjord consisted of hybrids of plaice and ﬂounder. Since hybrids of plaice and ﬂounder had not been recorded in Norway, hybridity could be used as a natural tagging method (Rollefsen, 1940). However, experiments showed that the hybrids had far higher survival rates than plaice, but lower than ﬂounder. Therefore, the results obtained by releasing hybrids would not yield adequate information on the survival potential of released yolk-sac larvae of either of the parent species. The lesson to be learnt from these experiments is that efﬁcient marking techniques are a prerequisite for efﬁcient testing of the performance and yield of released cod juveniles (Pedersen et al., 1998)

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More than 400 000 turbot were released in Danish waters between 1991 and 1998 (Støttrup et al., 2002). Most were released as small juveniles (4–6 cm) tagged with alizarin, while smaller groups were tagged with T-bar tags and released at larger sizes (11–16 cm). The major ﬁndings of the stocking experiment on turbot in Danish waters (Støttrup et al., 2002) are given below.

• The released ﬁsh adapted well to the natural environment • Growth of the small ﬁsh tagged with alizarin was similar to, or higher than, that of wild • • • •

turbot; the larger ﬁsh tagged with T-bar tags had smaller growth rates than wild turbot, probably as a result of the external tag Mortality of the released turbot was variable and relative high, but was comparable to that of wild ﬁsh Most of the released turbot were recaptured close to the release area No evidence of displacement of wild stock was found The null hypothesis that stocking enhances the local population of turbot was not rejected

11.3 Stock Enhancement and Sea-ranching in Asia In Asia, Japan is the leading country, with large stocking programmes of warm- and coldwater marine species in coastal areas. The main reason for this activity is the signiﬁcant decline in coastal ﬁsheries due to over-ﬁshing and habitat degradation. Over the years, action such as the regulation of commercial ﬁsheries and habitat improvement has been taken, but with no measurable effect. Another way to restore reduced coastal stocks is by stockenhancement. The species selected were those that show economic potential and technological feasibility with regards to seed production and stocking. At present, Japan releases more than 90 million juveniles of 38 different marine ﬁsh species, including more than 24 million red sea bream (Pagrus major), 30 million Japanese ﬂounder (Paralichtyhs olivaceus), 6 million black sea bream (Acanthopagrus schlegeli) and 2.2 million tiger puffer (Takifugu rubripes). In addition, more than 2.9 billion shellﬁsh and 274 million crustaceans are released, as well as 71 million individuals of other species, such as sea-urchin (Echinoidea). To organise this activity, 16 national centres and 54 prefecture centres have been established. The explanation given for these large-scale releases is that Japan has been excluded from several distant ﬁshing ﬁelds, the country has the highest per capita consumption of ﬁsh in the world, and landings of important species in coastal ﬁsheries have decreased. Only a few cold-water marine ﬁshes are included in the stock-enhancement and searanching programmes (Table 11.1), and the areas of their release are shown in Fig. 11.5 (see colour plate section). In Japan, natural environmental conditions vary greatly from Hokkaido in the north, where ice ﬂoes can be observed, to Okinawa and the Ryukyu Islands located near the tropic of Cancer in the south. The cold-water ﬁsh have mainly been released around Hokkaido and in the north part of Honshu, were the water temperature drops to 7°C in the winter. The species with the highest number of juveniles released are herring and Japanese sandﬁsh, with releases of more than one million annually in Hokkaido and northern Honshu. Large releases of Jacopever also take place. However, even though the recovery rate of

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Table 11.1 The number of juvenile cold-water marine ﬁsh released in 1999 for sea-ranching purposes, grouped by species and area. Common and Latin names are given. (Dr Y. Yamashita, Tohoku National Fisheries Research Institute, Japan, personal communication, 2002). Species

Area

No. of release locations

No. released in 1999 (thousands)

Barﬁn ﬂounder (Verasper moseri)

Hokkaido Northern Honshu Northern Honshu Southern Japan Hokkaido Northern Honshu Hokkaido Northern Honshu Middle Honshu Northern Honshu Hokkaido Northern Honshu Hokkaido Northern Honshu

19 4 20 8 7 1 52 32 1 3 13 3 13 25

110 38 52 115 134 16 722 1117 10 248 3569 980 4373 4400

Spotted halibut (Verasper variegatus) Brown sole (Pseudopleuronectes herzensteini) Jacopever (Sebastes schlegeli)

Paciﬁc cod (Gadus macrocephalus) Paciﬁc herring (Clupea pallasi) Japanese sandﬁsh (Arctoscopus japonicus)

Jacopever is reported to be high, and it is a technically interesting ﬁsh, there is a problem about its economic effectiveness as a target species for stock enhancement because the market price is low. In general, there are three main aims when identifying which species to use in stock enhancement programmes: (1) (2) (3)

to increase the economic yield from the coastal ﬁshery; to increase and stabilise the annual landing of ﬁsh; to restore depleted stocks.

Among the cold-water marine ﬁsh species released around Hokkaido and northern part of Honshu (Fig. 11.5, see colour plate section), none can be considered to belong to category 1. A good example of category-1 species are chum salmon and scallop, both of which have also been released from Hokkaido. In both cases, a signiﬁcant relationship was found between the number of juveniles released and the numbers returned to the ﬁsh market. In addition, it has been documented that there is a signiﬁcant economic proﬁt from this activity (Kitada, 1999). The largest group belong to category 2, and include brown sole, Jacopever, Paciﬁc cod, Paciﬁc herring and Japanese sandﬁsh. However, both brown sole and Paciﬁc cod releases are still at an early experimental stage. Within this category are species with the highest number of juveniles released annually, but the impact of these releases is still unclear. The stocking of Paciﬁc herring is based on local stocks, of which there are several, and not on the historically much larger oceanic stock (the Hakkaido–Sakhalin stock). Herring is an important species in any ecosystem, as it transfers energy from lower to higher tropic levels. The main objective of the releases in Hokkaido and northern Honshu is to maintain or increase local populations. In addition, because the price is good, i.e. between 1500 and 2500 ¥ (13–21 €) per kilo in the local ﬁsh markets, there is the potential for economic success. However, the activity has not shown stability as it has for the Furen Lake stock,

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where releases started in 1982 and in 1 year reached an annual high release of 440 000 herring juveniles. The commercial landings from this local stock increased from less than 50 mt in the early 1980s to 530 mt in 1996. However, landings in 1998 were 100 mt, and have been low since then. The main cause of the increase was successful recruitment in the wild stock. In category 3, Barﬁn ﬂounder and spotted halibut are both high-price species, but are almost depleted due to over-ﬁshing.

11.4 Prospects and Limitations of Enhancement and Sea-ranching 11.4.1 Biological Constraints The survival and recapture percentages will often depend on the release habitat, as shown for releases of Atlantic cod in western Norway, and are correlated with the production in the area and number and sizes of predators. There is increased production when going from closed to open coastal areas, which is shown in increased growth (Fig. 11.6). However, increased production also implies more and larger predators, and the total recapture rate was lower in open, more productive, areas than in closed, less productive, areas (Fig. 11.7). The carrying capacity for a given stock can be deﬁned as the biomass level that can be supported by available food resources in the absence of harvesting (Frèchette, 1991). There are several reasons why stocks may under-exploit the carrying capacity. Stocks could suffer from a ‘recruitment bottleneck’, and consequently seldom reach the carrying capacity of the environment (Svåsand et al., 2000). This may occur when unfavourable environmental conditions generate high mortality in the early life stages, and favourable conditions in later stages. In such cases, releases of ‘older juveniles’ could be a way to enhance local stocks. Carrying capacity changes over time depending on the abundance of predators and the supply of food. Food supply is a function of the productivity of prey populations and com80 70 Length (cm)

60 50

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Øygarden

20 10 0 1

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Age (Years) Figure 11.6 Growth of recaptured cod from groups of cod released in western Norway. Heimarkspollen is a small nearly landlocked fjord, Masfjorden is a deep fjord and Øygarden is an open coastal area (modiﬁed from Svåsand et al., 2000).

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Length-at-release (cm) Figure 11.7 Reported recapture percentages from groups of cod released in western Norway (modiﬁed from Svåsand et al., 2000).

petition for that food from other predators. Changes in the biotic environment affect the distributions and productivity of all populations involved. It can happen that a system can only support a limited number and biomass of organisms. It has been suggested that the high number of Paciﬁc salmon released in Japanese waters has resulted in a size reduction, mainly due to density-dependent growth rates (depression). Similar effects might be expected in cases of large-scale releases of marine organisms. One of the most important lessons learnt from the Norwegian stock enhancement and sea-ranching programme was that it is difﬁcult to manipulate marine ecosystems because the many feedback mechanisms, such as densitydependent growth, mortality, etc. Svåsand et al. (2000) suggested that in order to evaluate whether a stock is suitable for enhancement, the following questions need to be considered:

• Is there a recruitment bottleneck? • How close is the wild stock to its carrying capacity? • How large are annual ﬂuctuations in carrying capacity? • What is the maximum production capacity of the stock? • What proportion of the biomass production of the stock is not harvested, but is eaten by predators? • What are the actual and optimal exploitation levels of the wild and enhanced stock? • What is the yield per released individual? 11.4.2 Economic Constraints Ranching projects might be successful from a biological point of view. In other words, one may be able to produce juveniles, release them, and have a signiﬁcant proportion of those juveniles

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grow, survive and be re-captured without displacing wild individuals. However, from an economic point of view, the same project might be an redundant disaster. A classical example of a ‘successful project’ is the introduction of the king crab (Paralithodes camtschatica) from the Paciﬁc to the north-east Atlantic during the period 1961–69 and in 1978. In 1995, the northeast Atlantic stock was estimated to consist of more than 600 000 animals, and investigations indicated a near doubling of the abundance every year. The stock continues to spread along the Norwegian coast and into the Barents Sea, thereby forming the basis for an important commercial ﬁshery in the future. However, the strategy should not be to introduce new species into an ecosystem, but to increase the abundance of high-value organisms native to the ecosystem. Examining the economics of ranching programmes is complicated, and requires exact numbers for production costs, ﬁshery costs, catches and market prices. One approach is to estimate the net present value (NPV) of a project. The NPV will depend on the return rate of the released animals, the duration of the project and the interest rate, and is a useful tool to check how the above-mentioned parameters affect the project’s economic results. Another approach is to do cost–beneﬁt analyses which include the political, social, recreational and tourism beneﬁts of the projects, and how the beneﬁts are allocated among different user-groups. However limited information is available regarding the economic feasibility of most stock enhancement and sea-ranching activities. In Japan, it has been concluded that releases of red sea bream in the Kagoshima Prefecture was an economic success, mainly due to the high return rate (15%) and the high market price of 30–50 € Kg-1. However, this is not necessarily the case for other red sea bream releases, because return rates are normally much lower. Japanese ﬂounder (Hirame) is another high-priced and high-quality product in Japan, with a value between 30 and 50 € Kg-1 on the ﬁsh market, and there have been estimated return rates of up to 30% for some releases. These two examples have indicated that ranching of Japanese ﬂounder is economically proﬁtable. However, the red sea bream reports lack high-quality data on re-capture rates, while high-quality data on re-capture rates of Japanese ﬂounder have been reported by Kitada et al. (1992) and Okouchi et al. (1999). The Norwegian ranching program for cod has included re-capture data for the last 20 years. However, there is no indication that the operation is economically feasible. There are two main reasons for such a conclusion: ﬁrst, the high production cost (~ 1 € per juvenile), and second, the low market value (~ 2.5 € Kg-1). The possibilities for ranching of lobster in Europe and North America have not been fully explored. However, there is an indication that such an activity might be economically feasible if juvenile production costs are signiﬁcantly reduced. In general, it can be concluded that sea ranching of cold-water marine ﬁsh can only be economically proﬁtable if juvenile cost and post-release mortality are signiﬁcantly reduced. Mortality can be reduced by an optimisation of the release strategy and increased ﬁtness of the juvenile ﬁsh. Recreational ﬁsheries have been known for a long time, and in some countries such as the USA and Denmark, a ﬁshing fee has been introduced and partly used to produce juveniles for release. In some countries, a new industry has increased signiﬁcantly over the past 20 years. This is called tourist ﬁshing, and is deﬁned as non-inhabitants ﬁshing in coastal waters all the year round. In Norway, almost 250 000 persons (in the season 2000/2001) participated in this activity, ﬁshing almost 15 000 tons of ﬁsh, of which approximately 60% was Atlantic cod. The annual increase has been estimated at more then 35%, indicating that by

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2005 almost 1 million persons might be participating. The economic value of this industry has been questioned, and the effect of tourist ﬁshing on coastal resources needs to be considered. The ﬁshery might be regulated by quotas or stabilised by stock enhancement. A third alternative might be stock enhancement and a regulated minimum size at landing. The value of tourist ﬁshing needs to be considered seriously, since its value will probably increase over the years.

11.5 Recommendations and Guidelines Guidelines for a ‘Responsible approach’ are given below (Blankenship & Leber, 1995). These guidelines prescribe several key components as integral parts in the development, evaluation and management of marine stock enhancement programmes. Each component is considered to be essential in order to control and optimise the effectiveness of hatchery releases in helping to conserve and expand natural resources.

• Establish methods for prioritising and selecting species to be enhanced • Create a management plan with long- and short-term goals, harvest regimes and genetic conservation objectives • Incorporate life histories and ecological attributes into enhancement strategies and tactics • Create a genetic resource management plan to minimise in-breeding and out-breeding depression, and to conserve genetic resources • Create a disease and health management plan • Deﬁne and use an empirical process for determining optimal release strategies • Deﬁne and implement means to identify hatchery-produced ﬁsh • Deﬁne quantitative measures of success, and assess the enhancement project in terms of stated objectives in the management plan • Deﬁne and evaluate socio-economic objectives • Use adaptive management principles to evaluate and improve management strategies and tactics

11.6 References Bartley, D.M. (1999) Marine ranching: a global perspective. In: Stock Enhancement and Sea Ranching (eds B.R. Howell, E. Moksness & T. Svåsand.), pp. 79–90. Fishing News Books, Blackwell Science, Oxford. Blankenship, H.L. & Leber, K.M. (1995) A responsible approach to marine stock enhancement. In: Uses and Effects of Cultured Fishes in Aquatic Ecosystems (eds H.L. Schramm Jr. & R.G. Piper), pp. 167–75. American Fisheries Society Symposium No. 15. Blaxter, J.H.S. (1975) Reared and wild ﬁsh—how do they compare? In: 10th European Symposium on Marine Biology, Ostend, Belgium, September 1975, Vol. 1. Mariculture (eds G. Persoone & E. Jaspers), pp. 11–26. Universal Press, Belgium. Blaxter, J.H.S. (2000) The enhancement of marine ﬁsh stocks. Adv. Mar. Biol., 38, 1–54.

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Dahl, E., Danielssen, D.S., Moksness, E. & Solemdal, P. (eds) (1984) The Propagation of Cod, Gadus morhua L. Flødevigen Rapportserie, 1(1–2), 895 pp. Frèchette, M.M. (1991) Carrying capacity and density dependence. ICES J. Mar. Sci., 192, 78. Howell, B.R. (1994) Fitness of hatchery-reared ﬁsh for survival in the sea. Aquacult. Fish. Manag., 25 (Suppl. 1), 3–17. Kitada, S. (1999) Effectiveness of Japan’s stock enhancement programmes: current perspectives. In: Stock Enhancement and Sea Ranching (eds B.R. Howell, E. Moksness & T. Svåsand), pp. 103–31. Fishing News Books, Blackwell Science, Oxford. Kitada, S., Taga, Y. & Kishino, H. (1992) Effectiveness of a stock enhancement program evaluated by a two-stage sampling survey of commercial landings. Can. J. Fish. Aquat. Sci., 49, 1573–82. Kristiansen, T.S., Jørstad, K.E. Otterå, H., Paulsen, O.I. & Svåsand, T. (1997) Estimates of larval survival of cod (Gadus morhua L.) by releases of genetically marked yolk-sac larvae. J. Fish Biol., 51 (Suppl. A), 264–83. Okouchi, H., Kitada, S., Tsuzaki, T., Fukunaga, T. & Iwamoto, A. (1999) Number of return rates of hatchery-released ﬂounder, Paralichthys olivaceus, in Miyako Bay—evaluation of a ﬁsh market census. In: Stock Enhancement and Sea Ranching (eds B.R. Howell, E. Moksness & T. Svåsand), pp. 573–82. Fishing News Books, Blackwell Science, Oxford. Pedersen, T., Kristiansen, T.S. & Svåsand, T. (1998) Evaluation of current results with Atlantic cod and further prospects. ICES CM 1998/L, 11, 19 pp. Rollefsen, G. (1940) Utklekking og oppdretting av saltvannsﬁsk. Naturen, 6–7, 197–217. Shelbourne, J.E. (1964) The artiﬁcial propagation of marine ﬁsh. Adv. Mar. Biol., 2, 1–83. Solemdal, P., Dahl, E., Danielssen, D.S. & Moksness, E. (1984) The cod hatchery in Flødevigen— background and realities. In: The Propagation of Cod, Gadus morhua L (eds E. Dahl, D.S. Danielssen, E. Moksness & P. Solemdal), pp. 17–45. Flødevigen Rapportserie, 1. Støttrup, J.G., Sparrevohn, C.R., Modin, J. & Lehmann, K. (2002) The use of releases of reared ﬁsh to enhance natural populations. A case study on turbot Psetta maxima (Linné, 1758). Fish. Res., 59, 161–80. Svåsand, T., Skilbrei, O.T. van der Meeren, G.I. & Holm, M. (1998) Review of morphological and behavioural differences between reared and wild individuals: implications for sea ranching of Atlantic salmon (Salmo salar L.), Atlantic cod (Gadus morhua L.), and European lobster (Homarus gammarus L.). Fish. Manage. Ecol., 5, 1–18. Svåsand, T., Kristiansen, T.S., Pedersen, T., Salvanes, A.G.V., Engelsen, R., Nævdal, G. & Nødtvedt, M. (2000) The enhancement of cod stocks. Fish Fishe., 2, 173–205.

Chapter 12

New Species in Aquaculture: Some Basic Economic Aspects R. Engelsen, F. Asche, F. Skjennum and G. Adoff

12.1 Introduction During the past 20 years we have seen substantial changes in the world ﬁsh market, with a large increase in the production of farmed ﬁsh. This is due to new technology, the decline in wild ﬁsh catches, over-exploitation and increasing demand for ﬁsh and seafood. Atlantic salmon, salmon trout (a large rainbow trout that is distinguished from the small, portionsized rainbow trout that is produced in small land-based farms in several European countries), American catﬁsh, sea bass and sea bream are ﬁve important species in the seafood markets in Europe and the USA. As the aquaculture industry grows, new species are likely to be farmed. However, one should bear in mind that most meat production in agriculture is based on fewer than ten species, and hence, one is not likely to see large quantities of more than a few species. The objective of this chapter is to look at the potential for the successful development of a selected group of new cold- and temperate-water species for ﬁsh farming, and the criteria to evaluate this potential. The species considered are Atlantic cod (Gadus morhua), haddock (Melanogrammus aegleﬁnus), hake (Merluccius merluccius), spotted wolf-ﬁsh (Anarhichas minor), Atlantic halibut (Hippoglossus hippoglossus), turbot (Scopthalamus maximus) and sole (Solea solea). One of these species, turbot is already farmed successfully. Cod is on the verge of semi-commercial fry production, and there is already some experience when it comes to on-growth. For halibut, fry production is on a semi-commercial scale, but there is limited experience when it comes to on-growth. Sole, wolf-ﬁsh, hake and haddock still are in a pre-commercial research and development stage. The following discussion will include all species, but with a closer look at cod, halibut and turbot. An analysis of the prospects of commercial ﬁsh farming of new species involves two questions as seen from an economic viewpoint: (1) Is there a market for the farmed ﬁsh? (2) Is it possible for the farmer to produce and sell the ﬁsh at a lower cost than the market price so that the operation is proﬁtable over time? Answering these questions will lead to new ones, such as, what will happen to prices and production costs as time goes by and production volume increases? The main goal for all participants in the aquaculture industry is proﬁtability (or alternatively, satisfying returns on the investments made). However, there is a debate about the best means to achieve this objective. One widely held opinion is to target the high-priced species

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as priority candidates for commercial ﬁsh farming, as the high prices would cover the high production costs. In Europe, salmon and sea bream/sea bass are major success stories in modern ﬁsh farming. These species initially belonged to the group of high-value ﬁsh when farming commenced, and may also have beneﬁted from a good reputation as high-quality species. The alternative view is to direct one’s efforts towards building up farming of low-cost species, as the price of these species is less sensitive to increased supplies. American catﬁsh, a low-value species, has been successfully developed through low-cost farming. In the discussion we look at seven species that can broadly be divided into two groups, high-valued species, i.e. turbot, halibut and sole, and lower-value ﬁsh, i.e. hake, cod, haddock and wolfﬁsh. However, hake, and to a certain degree wolf-ﬁsh, are obtaining high prices in certain segments of the market. One should note that this categorisation is not ﬁxed over time. In particular, cod has recently sold at all-time high prices. Knowledge about the market is important before starting production of farmed ﬁsh, but it is also important to consider costs and compare them with possible prices. The possible combinations of price levels and production costs can be conceptualised as in Fig. 12.1. The ideal initial situation is the possibility of picking candidates which satisfy the conditions in Box 2. None of our seven species are easily put into that group. In general, attempts to commercialise new species involve high production costs and high market prices, as presented in Box 1. Some projects have low production costs with a low market price (Box 4), whereas others struggle with high production costs and low prices (Box 3). Given a substantial increase in volume, the tendency over time is to move from the situation described in Box 1 to that in Box 4. A number of different factors affect the costs of production and development of ﬁsh farming. When establishing basic production and completing the rearing cycle, cost levels tend to be determined by the technology applied, the environmental conditions, biological complexity and scale. Hence, some argue for species farmed in sea-cages rather than costly land-based operations when deciding on new candidates for commercial aquaculture. Finally, the production of juveniles differs in complexity and cost. Such factors are barriers to new enterprises. For instance, the production of halibut juveniles has been regarded as costly, whereas ﬁngerlings of sea bream and sea bass are relatively cheap to produce. The early phase of the commercial development of ﬁsh farming is risky, and depends on whether the introductory economic ventures are proﬁtable or not. Proﬁtability is determined

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Figure 12.1 Initial combinations of price and production costs.

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by the relationship between market price and production cost. A high market price gives room for an inefﬁcient preparatory farming business with high production costs, as in the development of turbot farming in Spain. A low market price, on the other hand, requires the start-up of commercial farming to be efﬁcient in the sense of low production costs, as demonstrated by the troublesome development of commercial cod farming in Norway in the late 1980s. The choice of the form in which the species is marketed in may also inﬂuence the likelihood of success. For most species, whole fresh ﬁsh is the product form that gives the highest return to the farmer, and further processing is of interest when one needs to expand the market.

12.1.1 Markets, Productivity and Production Growth The successful development of commercial farming of new ﬁsh species has several stages. Research and development, aimed at closing the rearing cycle, constitute the foundation of the later commercial stages. However, technical competence is worthless without a market for the ﬁsh. Moreover, this market has to be willing to pay a price which is sufﬁciently high to cover the production and investment costs for the new species to become commercially viable. This, of course, is the reason why most ﬁrms will aim at Box 1 in Fig. 12.1. Subsequent growth in production is the next stage, and will depend on market and productivity growth. The relation between market and productivity growth in an economic context is explained brieﬂy in Section 12.1.2 and, although it is not necessary for the further reading of the chapter, we recommend that the reader becomes familiar with the concepts discussed there. For most new farmed species, one starts in a setting where there is a demand for the species but no supply because of the lack of production technology. Anderson (1985), Bjørndal (1990) and Asche et al. (2001) give more comprehensive descriptions of most elements in the development of a farming industry. Research can then result in production, which will create the potential for supply of the farmed product. Initially, knowledge of the species and the production technology is limited, and this tends to mean high production costs. This is one reason why highly priced luxury species often are targeted (Box 1 in Fig. 12.1), as these species are believed to be the easiest for commercially viable production. However, in general, there are very few consumers who are willing to pay high prices for any product, even if it is of superior quality. Thus, the quantities of highly priced products sold in a market tend to be low. In any market one can always increase the quantity sold by reducing the price. How much the price will be reduced depends on market size. In markets where a low quantity is sold, a small increase in the quantity supplied will still be a relatively large percentage of the total, and therefore lead to a substantial price reduction. In a large market, the size of the increase may be so small that in relative terms it makes no difference. For instance, a production increase of 500 tons more than doubles the supply of farmed halibut (2001), while an increase of 500 tons in the production of salmon barely inﬂuences the supply, and hence has virtually no impact on the market. Unfortunately, this implies that it may be difﬁcult to create an industry of a substantial size for species in Box 1 in Fig. 12.1, as prices will tend to decrease rapidly as supply increases. This effect can be avoided if one can make the market grow as much as production growth, and there is evidence that this has

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been possible for many new farmed species, but only up to a certain point. After this, market growth can only delay the drop in prices. Products with the right characteristics for Box 2 in Fig. 12.1, with a high price and low production costs, are generally virtually impossible to ﬁnd, and not only for seafood. The reasons for this are the prospects of immense proﬁtability, which make ﬁrms willing to invest in this industry, causing production increases while prices sink towards production costs. The only exceptions are when there are barriers to new ﬁrms entering such an industry. Such barriers can be legal, as with a patent, or due to the limited availability of natural resources. Of course, goods with the characteristics for Box 3 in Fig. 12.1 are not very attractive, since it is hard to make them proﬁtable. Fish products characterised as described in Box 4 are thus the only realistic alternative to species in Box 1. Because of the lower price, it will be much harder to make these species proﬁtable. Markets with low prices are usually large in volume, and growth will therefore be easier. In particular, the demand is likely to be less price-sensitive, and increased supplies of the aquaculture product will not have much inﬂuence on the price. In most cases, the size of the market will be determined by the quantity of wild ﬁsh supplied of the same or very similar species. With small quantities there will often be a distinction between wild and farmed ﬁsh that may or may not be to the beneﬁt of farmed ﬁsh. The same may be true in a large market if the quantity of farmed ﬁsh is small. If the supply of both farmed and wild ﬁsh is substantial, it is very hard to differentiate between the two. Low prices are mostly associated with large-quantity markets. If one can be proﬁtable in a market with low costs and relatively low prices, this is probably the type of species which is most likely to succeed in creating a big industry. Another possibility is to grow into this segment, as has been the case for salmon. The development of wild catches of the species in question can have a great inﬂuence on the price as well as other market conditions. In the market model, this can be interpreted as shifts in the demand schedule, or changes in the size of the market. Cod is a good illustration of this point. In 1969, the total landings were about 4 million tons, while annual landings now are approximately 1 million tons. Reduced quotas and a limited standing stock in the Barents Sea will affect the supply of cod for years to come. Hence, the market price could rise to a level where farming this species could become proﬁtable even if it is not at the current time. However, if landings increase substantially, the price can also decrease quite rapidly. As the volume of farmed products starts to outgrow the volume of wild landings, farmed products tend to set the overall market price. At this level of commercialisation, the structure of the value chain for the farmed product will become important in regulating the overall market price. The market is also sensitive to changes in consumer preferences. A change from meat to ﬁsh could lead to higher prices and a more favourable situation for introducing new species from ﬁsh farming. Yet competition in the market will increase by introducing additional species of farmed ﬁsh. Many of these species will be substitutes. An increased demand for one species could lead to a reduced demand for another. Consumer behaviour can be affected by marketing. To date, little work has been undertaken at the early stages of introducing new farmed species to the market. This is mainly because of the large amount of resources needed for this kind of work compared with the relatively small volumes being offered to the market.

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It is an important, although very difﬁcult, task to evaluate the market potential of a new species. In spite of this, it is equally important to analyse the potential for productivity gains, as the ﬁsh farming industry is still young. The experience of the industry so far shows a substantial and steady decrease in production cost for several species, and this is what seems to be fuelling the growth. Salmon, sea bream and sea bass are all examples of growth in productivity resulting in lower production costs.

12.1.2 The Economics of a Market and Productivity The simplest version of an economic market model consists of two curves, a demand function and a supply function. Further information on these topics can be found in any good intermediate economics textbook, and Varian (1999) is a good example. The demand function is downward sloping, as consumers are willing to buy a higher quantity of a good only if the price is reduced. The steeper the slope, the more the price has to be reduced to sell a higher quantity. The supply function is upward sloping, because producers are willing to produce a higher quantity only if they can cover all additional cost, and these are generally an increasing function of the quantity produced. Again we ﬁnd that with a steeper slope, a larger price change is necessary to change the quantity supplied. Market equilibrium is when the two curves intersect. This is shown in Fig. 12.2, where the demand curve is denoted as D and the supply curve as S. The intersection shows that the quantity sold in this market is Q at a price P. If the demand and supply curves do not cross, there will not be a market for this good. This can happen, for instance, if there is demand for a product but the production cost is too high for supply to be proﬁtable (i.e. the supply curve is always higher then the demand curve). This is shown in Fig. 12.3 with the supply schedule S1. However, the supply and demand curves are not static. They will shift due to changes in production costs, productivity growth, changes in prices for competing goods (substitutes) and/or market growth. In relation to aquaculture, productivity growth is probably the most interesting of these factors. Productivity growth is basically new production methods that allow the same quan-

Price S

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Figure 12.2 Supply and demand.

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tity to be produced at a lower cost. In the ﬁgures, this will show up as a downward shift in the supply curve so that at any price, a higher quantity is supplied. For instance, research on a new species can give better survival rates, which leads to a reduction in production costs as fewer resources are spent on ﬁsh that cannot be marketed. In Fig. 12.3, this is represented as a shift from S1 to S2. This productivity shift is what makes this species commercially viable, as a quantity Q2 now is sold at the price P2. Further productivity increases will shift the supply curve further down, e.g. to S3, which again gives a greater quantity produced (Q3) at a lower price. Hence, productivity growth induces production growth because it makes a good less costly to produce, and it will therefore be proﬁtable for producers to supply more even if the price is reduced. Moreover, an industry that experiences decreasing prices over a longer period can only be proﬁtable if there is productivity growth. Since many successful aquaculture industries have experienced substantial declines in prices, we can already deduce that there has been substantial productivity growth. Market growth may be as important as productivity growth, and even more beneﬁcial to producers. In Fig. 12.4, we start at the same point as in Fig. 12.3, with demand and supply curves, D1

P r ic e S1 S2 P2 S3 P3 D

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Figure 12.3 Productivity growth.

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Figure 12.4 Market growth.

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and S1, that do not intersect. The demand schedule will shift upwards if the price of competing goods increases, and also for most goods if people get richer. Moreover, one will actively try to shift the demand curve up when one is marketing a product. Whatever the reason for a demand schedule to shift upwards, it will constitute market growth. In Fig. 12.4, when we shift the demand curve from D1 to D2, this market growth is what makes the product in question commercially viable, as with the demand curve D2, a quantity Q2 will be sold at a price P2. Further market growth (e.g. to the demand curve D3) will further increase the price (P3) and the quantity (Q3) sold. Unfortunately, market growth can also be negative, as the price of competing goods can decrease, or competitors can be more successful in marketing their products. If so, both price and quantity will be reduced in the market in question as the demand curve shifts downwards.

12.1.3 The Evolution of the Salmon Industry The successful development and commercialisation of farmed ﬁsh requires research, technology and knowledge of the market. The production of salmon has been the most successful of the farmed species measured in quantity produced, with a substantial growth from the mid- and late 1970s, leading to its status as a commercially interesting species from the 1980s (Asche, 1997; Asche et al., 2001). The developments in the global production of salmon, together with the real price, are shown in Fig. 12.5. The initial situation was one of high market price, high production costs, and a limited supply of wild Atlantic salmon. In other words, the point of departure for producers of farmed salmon was the one described in Box 1, Fig. 12.1. In addition, there was an imbalance between demand and supply in favour of the supply side, as illustrated by the increase in production and relatively stable prices until 1985 (see Fig. 12.5). By then, the production had grown from zero to nearly 30 000 tons in Norway alone. The reasons were the luxury image of salmon at that time, the market channels available, the existing market segments with up-market restaurants, and the

Figure 12.5 Total production of farmed salmon world-wide and Norwegian export price (the real price is computed using the consumer price index from International Financial Statistics, International Monetary Fund).

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fact that wild salmon was only available seasonally, while farmed salmon was accessible all year round. Hence, one could sell farmed salmon to a market with little competition and exploit a situation of limited supply for several years. This situation changed in the mid-1980s as the original restricted supply was expanded by increased output from the salmon-farming industry. New technology allowed further productivity developments, so ﬁsh farmers could increase the level of production without augmenting costs. However, increased production has an important drawback, namely the eventual decrease in price unless there is an equal growth in the market. Despite intense marketing programmes that increased demand and increased the production of value-added products such as smoked salmon, price reductions became necessary to sell all the salmon produced. The output from the salmon farming industry increased from about 66 000 tons in 1986 to 1.1 million tons in 2000, while the real price fell by about 70% over the same period (Fiskeridirektoratet, 2001). However, growth was proﬁtable, despite the price decline, because of the substantial growth in productivity. From the estimated cost in 2001 in Fig. 12.6, one can see that the Norwegian production costs follow the price downwards relatively closely, and the production cost today is about one-third of what it was in the early 1980s. This suggests that the price of salmon to the farmer follows production costs to a large extent, as one would expect in a competitive industry. One can also see from Fig. 12.6 that the cost and price do not move in full synchrony. In particular, the differences between price and cost, i.e. the margin, is fairly small in 1986, 1991, 1997 and 2001, as well as being especially wide for one year between these years. Hence, some years are much more proﬁtable than others. This is a structure that one observes in many industries which are based on biological production processes, as well as other industries where there is a substantial time-lag between the decision to increase production and when that increased production hits the market. A high margin gives a signal from the market to increase the supply, but because of the time-lag in increasing the production, this signal can be quite persistent. This often leads to over-investment that causes the production

Figure 12.6 Real production costs and Norwegian export price.

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to increase too much and prices may fall to, or even below, production costs for a period. The low margins will then be a signal to reduce production. Again this takes time, and production will often be reduced too much, giving birth to a new period with very good margins. In a stable world, one would expect producers to ﬁgure out what production level gives normal margins, but the world is anything but stable, and the production volume that gives a normal margin is a moving target. This is due to productivity growth and other supply shocks, as well as to exchange-rate movements, demand shocks and market growth. With the delay in the response from the producers due to the production time, one will therefore expect to see boom and bust cycles at irregular intervals and with different strengths in industries like aquaculture.

12.1.4 The Evolution of the Sea Bass and Sea Bream Industry Sea bass and sea bream are two different species, but because the production and marketing methods are so similar, they are often treated as one species in analysis (see Asche et al., 2001, for further discussion and references). The situation at the onset of the sea bass and sea bream industry in the late 1980s was in some respects similar to that in the salmon industry in the late 1970s. The rearing cycle was closed, and market price and production costs were both high. The production of each of the two species grew to around 4500 tons in 1991, with stable or increasing market prices up to that year. The total production of the two species 9 years later was more than 50 000 tons. Prices fell considerably during this growth phase. This development is shown in Fig. 12.7. Again, we have an example of growth in production and decline in price, which can be explained by the growth in productivity due to technical improvements. There is evidence that in 2001 the sea bass and sea bream industries had reached the bottom of a bust cycle caused by over-estimating the productivity growth and therefore increasing production too much. However, if one looks at salmon (and also many agricultural products), one would not expect the period of poor proﬁtability to last long. Production growth will decelerate and possibly be negative for some time, and some companies may be bankrupted. When production has been brought back to a proﬁtable level, one will

Figure 12.7 Production of sea bream/sea bass and real Italian import price.

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again expect to see new expansion if the productivity growth continues. If productivity stops improving for any reason, one would expect that production and prices would stabilise.

12.1.5 The Evolution of the American Catﬁsh Industry In contrast to most other farmed species, catﬁsh had a low value and was not very well regarded when farming started in the 1970s. A good introduction to the economics of the catﬁsh industry was given by Kinnucan (1995). Moreover, there were no large supplies of wild ﬁsh, and catﬁsh therefore had to build new markets at the expense of other ﬁsh species or meat. The production of American catﬁsh (or channel catﬁsh) has increased from about 40 000 tons in 1982 to almost 300 000 tons in 2000. In the same period, the real price has been reduced to about €1.8 kg-1, but the magnitude of the price reduction is smaller than that for salmon, sea bass and sea bream. This development is shown in Fig. 12.8. Hence, even though catﬁsh did not have a clearly deﬁned market to grow in, the fact that it had a low price level enabled it to win market shares from many other products in large-volume markets without substantial reductions in price.

12.2 Cod Cod (Gadus morhua L.) has traditionally been the most important species in the north Atlantic ﬁsheries. In 1997, the global catch was about 1.3 million tons. However, catch rates have been declining over the past years, and over-ﬁshing is considered to be a threat to the wild stocks. On a general basis, cod has not been considered a high-value product, even though there are market segments which pay very good prices for cod. The species was early looked upon as a candidate for aquaculture, and research has produced a large amount of biological knowledge during the last few decades. In the 1980s, initial production of juveniles was carried out, and the combination of this together with a decrease in wild landings and a higher market price for cod led to great interest in cod farming in the late 1980s.

Figure 12.8 Price and volume of American catﬁsh, 1982–2000.

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However, farming was unpredictable and labour-intensive. When wild stocks rebounded in the early 1990s, with associated low prices, several bankruptcies occurred. The current situation (2002) is similar to the one in the late 1980s in that weak stocks are giving historically high prices. Moreover, recent improvements in cod farming have strengthened hopes of a substantial reduction in production costs. Hence, the likelihood for success is assumed to be larger this time due to higher prices and possible rapid productivity gains (R. Engelsen, unpublished data, 1996). The conditions for proﬁtable farming of cod depend on mastering the mass rearing of juveniles at low costs to permit the stocking of grow-out facilities several times a year. In 1983, the Aquaculture Research Station in Austevoll produced 75 000 juveniles using extensive production methods in lagoons. In the next few years, several private companies, as well as research institutions, attempted to commercialise the semi-intensive methods by using submerged bags in lagoons (Holm et al., 1991). One could argue that the rearing cycle was more or less closed by 1990. However, cod farming based upon semi-intensive techniques for juvenile production did not become a commercial success despite the production of fairly high numbers of juveniles. The main reason for this was ﬁnancial losses due to high production cost combined with a fall in market prices. This led to the termination of the majority of cod activities in the early 1990s. Figure 12.9 shows the development of juvenile production from 1986 to 2001. The history of cod farming shows that it is not enough to close the rearing cycle to commercialise a new species in aquaculture. The next step is to establish farming at a suitable level of productivity to make the activities proﬁtable. Rising cod prices due to diminishing stocks led to a revival of interest in cod farming around the year 2000. At the same time, the scope for cost reductions in cod farming was thought to be high by making use of production techniques and competence from the intensive juvenile production of sea bream and sea bass, as well as on-growing of salmon. The rise in productivity has to be achieved in ﬁngerling production as well as on-growing.

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The ﬁrst large-scale intensive hatchery for cod in the world, Cod Culture Norway (CCN), located outside Bergen, was completed in 2002. The technology is being adapted to cod from the sea bass/sea bream industry. Results so far indicate that it will be successful. The production at CCN is fully intensive, with complete control of production parameters such as temperature, oxygen and live feed production, thus allowing all year round production. The plant has a production capacity of >10 million fry/ﬁngerlings annually. The successful intensive production of cod ﬁngerlings implies a substantial productivity improvement, i.e. reduced cost per ﬁngerling. On-growing of cod will beneﬁt heavily from the traditional technology developed for large-scale farming of salmon, i.e. big cages, deep nets, a feed barge, automatic feeding, the use of light, monitoring systems, the use of well boats and others. This technology can be used for cod after some modiﬁcations in operational routines. The expected growth from 100 g, which might be an average stocking size, to a weight at slaughtering of 3–5 kg should be achieved in considerably less than 24 months after transfer (R. Engelsen, 1996). This is a promising up-front growth potential. Since the best price is currently paid for sizes between 3 and 5 kg gutted weight, this size is the main target for farmed cod. Sexual maturation usually occurs during the second winter of production. Then growth stagnates, and the ﬁsh might even lose weight before it starts eating again a few months later. However, there could be a market for maturing cod as well, and this has to be explored when it comes to the development of different market niches at an early stage of cod farming. The aim is still to reach harvesting size before sexual maturation occurs, as well as to postpone maturation. If not, production costs are likely to be unacceptably high, and will most likely result in ﬁnancial losses. Juveniles of variable size and quality have also been typical problems in the early stages of cod farming. However, recent research has shown promising results as the use of additional light may postpone sexual maturation until the cod reaches an acceptable harvest weight (G. Taranger, unpublished results, 2002). An alternative way to avoid early maturation is to stock the pens with larger juveniles. If the farmer could buy large juveniles (100–250 g) in the early spring, harvesting could start during the autumn the year after, and ideally most of the ﬁsh will be sold out before stocking again in the following spring (Bergen Aqua, Norway, unpublished data, 2001). Before cod can become a substantial commercial success, there are other barriers affecting productivity to break down. For instance, feed has to be especially designed for cod and have a sufﬁciently high level of digestible protein to exploit the growth potential and to avoid large livers. Another problem in cod farming is cannibalism, especially at the early stages, but high-quality ﬁsh feeds and frequent grading of ﬁsh will reduce these problems. However, if the industry is commercially viable for some years at current high prices, experience from other species suggests that there is a substantial ‘learning by doing’ effect that will result in productivity gains which will make the industry more competitive. Moreover, if it is large enough, the industry is more likely to attract interest from suppliers such as feed and pharmaceutical companies. This will enhance the productivity gains, as these companies research and produce products targeted at cod production. Based on initial experiences, economic simulations and comparisons with other marine ﬁngerling production, a cost level of €1.0 for a 100-g ﬁngerling of cod can be expected. In the long run, the cost is expected to become lower and become close to the level for salmon

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smolts. As salmon prices are currently (winter 2002) lower than cod prices in the freshﬁsh segment, the potential for proﬁtable farming is present if the on-growing phase is competitive. Some initial analyses show that the price to the farmer must be €2.50 kg-1, live weight ex farm, at the lowest to make cod farming proﬁtable (Kvenseth et al., 2000). Cod is often sold gutted without a head. A necessary price is thought to be at least €4 kg-1 to make farming proﬁtable. However, great uncertainty attaches to such analyses because of the lack of robust empirical data. If one can increase the scale of operation and productivity sufﬁciently before the wild stocks rebound, one is likely to see a substantial cod-farming industry in the future. Wild cod is currently mainly sold as a bulk commodity in a global market of about a million tons at fairly low prices, although the prices vary substantially with the quantity landed (Fig. 12.10). Traditionally, cod has been priced at about €1.0 kg-1 to ﬁshermen, but recently prices have increased to above €2.0 kg-1. With this market structure, even large quantities of farmed cod will have little or no effect on market prices. As noted in Section 12.1, an existing market can be an important advantage for new species. There are already indications in the market that a premium might be paid for farmed cod. This is particularly related to the freshness and/or the size of the ﬁsh. One may ask whether it is in the bulk market where farmed cod will compete, or if farming opens up the possibility of delivering fresh product forms in a separate market segment where most of the wild cod cannot compete because it has to be processed to reach the market at an acceptable quality (Asche & Salvanes, 2001). When cod can be supplied on a regular basis, all the year round, it can also be a very interesting product for processing plants and activities globally. This implies that there may exist at least two market segments for cod, one large segment for processed products with low prices, and one smaller segment for fresh cod that commands higher prices because of the preferred quality. Selected characteristics can lead to a potential price premium. For instance, skin pigmentation seems to be related to the speciﬁc strain of cod. Light-skin ﬁsh from cod caught

Figure 12.10 Volume and prices of cod (source: United States Department of Agriculture).

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in the North Sea is considered to be more delicate and of a higher quality, and therefore can be priced up to double that of darker-pigmented cod from the Baltic Sea. Farmed cod will be able to compete with the light-skin ﬁsh. Farmed cod also has the important advantage of being without parasites in the ﬁllets. Proper bleeding makes the ﬁllet appear whiter than in wild cod, which is regarded as an important aspect by consumers. The fact that farmed cod can compete in the prime quality market segments is important, since this gives a higher price, which enables farmers to cover higher production costs. Although the quantities have been very low, experience so far suggests that the price premium obtainable for farmed cod is substantial. Moreover, wild cod of very good quality also realises substantial price premiums. Hence, there is little doubt that there is a high-quality, high-price market segment for cod. However, it should also be noted that the price for farmed cod varies together with the price of wild cod, and therefore with the bulk market price. Accordingly, the high-quality market segment is not isolated from the bulk segment. If the price premium obtained in the upper-quality markets is high enough, one can initially put farmed cod into Box 1 in Fig. 12.1. Nevertheless, as the premium-quality segments are small in terms of quantity, there is no doubt that the price will decline towards the bulkmarket prices as production grows. If productivity does not improve, the product will then be in Box 3 in Fig. 12.1, and the industry is likely to die. If productivity improves sufﬁciently, it is likely to reach Box 4, and cod aquaculture will be a large, commercially viable industry. As already mentioned, the rebound in the wild stocks, with the associated reduced prices for farmed cod, basically killed the cod farming industry in Norway around 1990. This is also a valid concern today. The current high prices provide a window of opportunity that makes it easier to succeed with cod farming, since it is easier to make the industry proﬁtable. The recent growth of wild cod stocks does not seem very positive, and landings are therefore likely to remain low for some time (Fig. 12.11). Prices are therefore expected to

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stay high for a while, so that the window of opportunity for cod farming is likely to be open for a longer period today than it was around 1990. Even so, to ensure long sustainability of the industry at some scale, it is necessary that productivity increases so that it can survive when prices in the bulk market eventually drop again.

12.3 Haddock Haddock (Melanogrammus aegleﬁnus) is a well-known species in northern Europe and in North America. Scientiﬁcally, it belongs to the cod family, and is sold in many of the same markets as cod. Haddock is considered by many to be an interesting species for aquaculture. The wild catch has been restricted for the last 10 years, and the quota was set to 45 000 tons in 2000. Recruitment to the next year-classes of haddock is low, and spawning stocks are already over-ﬁshed (Litvak, 1998). The quality of haddock is considered to be high, and in countries such as Iceland and Canada, the species is preferred to cod as a fresh ﬁsh product. However, it is often used in value-added products such as ﬁllets, dried and salted ﬁsh, and ﬁsh cakes. In England, cod is used for ﬁsh and chips, while haddock is used in Scotland. The market for haddock is not in the high-price segments, but there is a large market for a constant supply of haddock. The wild catch is available all the year round, but prices have ﬂuctuated between €2.0 and 2.6 depending on supply and demand. Haddock is primarily sold gutted with the head off, indicating that reported prices only represent 65% of the round weight. Although no commercial aquaculture of haddock has been conducted so far, production technology is thought to be comparable to that used for cod, and it is likely to beneﬁt from the on-going developments in aquaculture when it comes to both juvenile production and on-growing technology. To date, trials have showed promising results for juvenile production. Unlike cod, haddock larvae showed very little cannibalism, and after 3 months the average weight was 4.8 g, which is twice as much as the weight of cod at the same level in the production process (van der Meeren & Ivannikov, 2001). Experiments on the growing stages, on the other hand, have uncovered problems with slow growth and high mortalities. However, the experiments are continuing, since more research seems to be necessary before the rearing cycle for haddock is closed. Since farmed haddock has not yet reached the level of commercialisation, there are no data on an economic evaluation of future prospects for the farming of this species. If we assume that the production technology will be similar to that for cod, the cost of production could be comparable for haddock, but unfortunately market prices do not favour haddock as it is generally priced lower than cod. Hence, the proﬁtable production of haddock represents a severe challenge because of the current market prices.

12.4 European Hake Hake (Merluccius merluccius L.) has been noted as an interesting new species in aquaculture due to the high market price in some niches. Hence, some researchers have stated that

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hake is one of the most promising candidates for farming among all the species yet to be farmed. There are several species of hake: North Paciﬁc hake, South African Cape hakes, Namibian hakes, European hake, Argentine hakes, Chilean hakes, Peruvian hake and New Zealand hake, among others. The total catch of hake in 1987 was 1.6 million tons. The hake have different product characteristics, are sold in different markets, and command different prices. Hake are also sold in a variety of product forms, both fresh and frozen, and today only wild-caught hake is available on the market. The one species looked at here is the European hake, which is the highest priced of the different types. The 1987 catch of European hake was 116 000 tons, while the total potential yield is estimated to be 175 000 tons (Alheit & Pitcher, 1995). To achieve a label of ‘excellent quality’ and the highest prices, the ﬁsh must possess the following characteristics: larger than 2 kg, white ﬂesh, ﬁrm ﬂaky texture, relatively small ‘fat layer’ and few parasites. European hake and some of the other hake species often meet these quality standards, and are thus often used as a fresh whole or ﬁllet product at expensive restaurants or retail markets. The most important market region is southern Europe. In 1990, over 30% of the world’s supply of hake was consumed in Europe, where the hake is established as a primary ground ﬁsh. Wholesale prices for hake (all species) show wide variations, ranging from less than 1 to over €15 kg-1 depending on species, product quality and product form. Spain is the largest market for hake, with an annual per capita consumption of 6.7 kg (1989). The Spanish harvest alone was 200 000 tons in 1989. In Spain, hake is not a substitute for cod as it is in many other Western countries, as it is considered to be a unique ground ﬁsh product. France is the second largest market for hake (Alheit & Pitcher, 1995). Prices for European hake on the Mercamadrid market were settled at around €14 kg-1 in the late 1990s, with a maximum of around €28 kg-1. These high prices are only connected to premium-quality fresh ﬁsh, caught on long lines. Hake has a relatively short shelf life, as the ﬂesh tends to become soft, even when stored on ice for short periods of time. Farmed hake is considered to have the potential to achieve the same quality as the very best of the wild-caught hake. No analyses are yet available regarding the size of the up-market high-price segment. Hence, there is no information regarding price sensitivity given an eventual increased output from the ﬁsh farming industry. Nevertheless, it is a widely held opinion in the aquaculture industry that excellent market opportunities may exist for farmed European hake. The commercialisation of farmed hake is now a question of closing the rearing cycle by mastering the different stages of production. The situation today (2002) is clearly that this species has a substantial way to go before it is ready for commercial farming. There are still major problems connected with the collection and holding of brood ﬁsh, as well as the different stages in ﬁngerling production. One of these is the problem of cannibalism at the weaning stage. As a result, few ﬁngerlings are produced. Little or nothing is yet known concerning the on-growing of hake, although it is expected that traditional sea-cage culture may be suitable. A number of questions are still to be answered, such as: Where should farming of hake in net pens take place? What are the required optimum temperature intervals? What about growth, mortality, feed conversion rates and so on?

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Hake is therefore still ﬁrmly in the research and development stage. The priority of this species among the actual candidates for introduction into commercial ﬁsh farming has to be founded on (1) thorough market analyses, and (2) more knowledge concerning the feasibility of farming hake, including both ﬁngerling and on-growing trials. There is a window of opportunity for farming hake because of the high market price. The initial cost of production could be high, and a major challenge will therefore be to succeed in effective farming to ensure productivity growth.

12.5 Wolf-ﬁsh Research and experiments on farming of the spotted wolf-ﬁsh (Anarhichas minor) started in Norway in the late 1980s, and is still in an early developmental phase. With the exception of small test volumes, farmed wolf-ﬁsh is not available on the market, and consequently very little effort has been directed towards evaluating the market potential for the farmed product. However, a few general market studies covering wolf-ﬁsh have been performed during recent years. The main markets for wolf-ﬁsh are in northern Europe, where the majority of the volume is sold as frozen ﬁllets. Norway and Iceland are the dominant participants in the wild catch of wolf-ﬁsh (a mix of grey and spotted), which amounts to 15 000 tons annually. The domestic consumption in Norway (3000–4500 tons per year) makes the country one of the larger consumers of wolf-ﬁsh in Europe, while major export markets for the ﬁsh are France and Britain. The market price of wolf-ﬁsh is generally considered to be in the ‘low to medium range’, as the species is normally more higher valued than white ﬁsh species such as pollack and plaice, but less appreciated than typical high-value species such as halibut, sole and turbot. The price to the ﬁshermen in Norway has always been low (approximately €1.0–1.50 kg-1 gutted, head off ). However, the export price for wolf-ﬁsh has shown an upward trend in recent years, and the demand for high-quality, fresh wolf-ﬁsh delivered to ‘up-market’ restaurants is also increasing. At European ﬁsh auctions in Denmark and The Netherlands, the market price for wolf-ﬁsh has been higher than in Norway, and has obtained prices in the range €3.2–4.0 kg-1 gutted. The present market trends, combined with recent test results with farmed wolf-ﬁsh, indicate the potential to obtain market prices of €4.5–6.0 kg-1 (fresh, gutted) in certain segments, given the development of a sustainable farming sector that can ensure a supply of high-quality products. Additional value can be gained from utilising byproducts, e.g. the skin. A Norwegian company is the only one currently developing the commercial production of wolf-ﬁsh. The key activities have been to concentrate on brood-stock performance and the rearing technology for juveniles. The production of juveniles, based on intensive hatchery techniques, increased from 3500 in 1998 to 26 000 in 1999 and 45 000 in 2002 (Sparboe et al., 1999). In the short term, the main problem for commercial development is the supply of eggs and the survival of larval stages. Owing to the size of wolf-ﬁsh larvae at ﬁrst feeding, more rapid progress than that experienced with other marine species is likely when commercial hatchery techniques develop. Financial analyses of an intensive

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wolf-ﬁsh hatchery have shown the potential of growing juveniles of 10 g at a cost of €1.3 each. On-growing trials with wolf-ﬁsh are still at a pre-commercial stage. To date, these activities have been performed in small-scale land-based pump-ashore facilities. It is too early to speculate on the basic on-growing technology in the future. Economic analyses of a mediumsize wolf-ﬁsh farm (200–300 tons), based on experience of cost ﬁgures from species such as halibut and turbot, combined with recent trial results on wolf-ﬁsh, indicate a potential for growing the species from 10 g to 4 kg in less than 35 months at an average production cost below €3.2 kg-1 round weight (excluding ﬁnancial costs) (Bergen Aqua, Norway, unpublished data, 2000). Recent progress in the development of wolf-ﬁsh technology in Norway, and a growing niche market demand for steady supplies of high-quality, fresh farmed products, indicate a possible closing of the rearing cycle of wolf-ﬁsh farming within a period of 5–10 years. In the long run, the decisive factors in wolf-ﬁsh farming are believed to be connected to marketing conditions, where product development and market prices will be the most important challenges.

12.6. Halibut Atlantic halibut (Hippoglossus hippoglossus L.) has been considered to be a high-quality ﬁsh in northern Europe for centuries. Atlantic halibut, and its relative Paciﬁc halibut (Hippoglossus stenoleptis L.), are also both well-recognised products in North America. Quotas, which normally lie between 25 000 and 45 000 tons annually, regulate the landings of Paciﬁc halibut, which mainly is sold frozen in domestic markets. The total wild landings of Atlantic halibut are on a downward trend in both Europe (Iceland, Greenland, Norway and the Faeroe Islands) and Canada, where the total annual catch is currently below 5000 tons, as shown in Fig. 12.12. Sales volumes of farmed halibut have so far been insigniﬁcant compared with those of wild landings, but are expected to increase as a consequence of more than 10 years’ effort in research and development directed towards the biological challenges of juvenile production aimed at closing the rearing cycle. The ﬁrst attempt to cultivate halibut took place in Norwegian and British research institutions in the 1980s. Stolt Sea Farm established the ﬁrst commercial halibut hatchery in 1989, and produced 10 000 juveniles in 1990. During the late 1980s and until the mid-1990s, several halibut hatcheries based on semi-extensive production in lagoons were established in Norway. Until 1994, these hatcheries produced increasing numbers of juveniles, but after this year the total Norwegian production stagnated because of lack of control of environmental factors, especially temperature. The ﬁrst intensive halibut hatchery was established in Iceland in the late 1980s, and this hatchery has produced increasing numbers of juveniles in the last 3 years (Fig. 12.13). The biological and technological processes of producing halibut juveniles are still in a pre-commercial phase, but large-scale development is predicted within a few years as a consequence of increased experience among the existing producers, particularly in Norway, Iceland and Scotland.

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The volumes of farmed halibut are not expected to increase rapidly during the next decade owing to the fact that juvenile production technology has been progressing slowly for several years, and that the species’ grow-out cycle typically takes 3–4 years to reach the preferred market size of 5+ kg. The total European production of approximately 800 000 halibut juveniles reported in 1999 could lead to harvest volumes of between 2000 and 2500 tons by 2003. A prognosis for the following years will mainly depend on the results from juvenile production. The development of halibut on-growing in both land-based and net-pen farms has been progressing in Norway since 1989, while farms in Iceland, the UK and Canada have been established in recent years. Currently, a large part of halibut on-growing occurs in land-based pump-ashore farms, but more farms are now being developed as sea-cages. The varying quality of juveniles and high prices have made it difﬁcult for on-growers to ensure proﬁtable production. None of the 20–30 smaller Norwegian halibut producers, of which many are net-pen farmers, are reported to be proﬁtable today. However, some of the larger integrated

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companies expect to generate a future proﬁt from selling between 150 and 300 tons annually during the next few years. A predictable supply of competitively priced juveniles, reduced sexual maturation of males and improved growth performance are the main obstacles for the future proﬁtability of halibut farming. Through the implementation of breeding and optimalisation programmes, these obstacles should be eliminated within 5–10 years. In the short term, it may be possible for smaller-scale net-pen farms, based on modiﬁed salmon-cage technology, to become proﬁtable as a result of the high market prices and the low capital required to enter the business. In the longer run, however, it is more likely that new land-based re-circulation farms creating optimal growth conditions will secure proﬁt margins to stimulate further growth of the halibut business. Since farming of halibut is in the early phase of commercial production, a standard for production costs has not yet been set. This is mainly because of the low and variable scale of the current operations as a result of the lack of a constant supply of juveniles. However, existing farms producing 200–300 tons per year are at a ‘break-even point’ today, with average market prices in the range of about €10 kg-1 gutted ﬁsh. Generally speaking, this situation will improve with the increased scale of operations and experience. It is expected that commercial-size operations of 300 tons or more will be able to achieve production costs of between €5 and €6 kg-1 round ﬁsh within a few years. This depends on sufﬁcient available juveniles to keep a steady biomass for efﬁcient production all the year around. The largest markets for halibut in Europe are the United Kingdom, Germany and Scandinavia. The UK market is currently consuming approximately 1500 tons of halibut per year. The majority are imported as frozen products and sold through auctions in larger cities such as London and Birmingham. Some wild Atlantic halibut are also sold through auctions in The Netherlands and Denmark. This ﬁsh is typically of a larger average size (>10 kg) than the farmed products. Farmed halibut, which is mainly produced in Norway in the size categories 3–10 kg, have been sold as fresh product to selected wholesalers in European cities. The wholesalers distribute the product to local restaurants and hotels. Smaller volumes have been directed towards retail chains in the UK, Germany, France and Spain, while near 50% of the farmed Norwegian halibut has been sold domestically. Farmed halibut has been well received in most markets, signifying a possible growth in the market. Landings of fresh Paciﬁc halibut have started to make an impact on the total sales volumes and market price of halibut in Europe during recent years (from 1996) because of the prolonged ﬁshing seasons for Paciﬁc halibut. Paciﬁc halibut may be a competitor to farmed halibut in the short term, but these circumstances will probably not continue in the longer term as the volume of halibut becomes larger. A market study from 1994 describes the halibut market in Europe as promising with regard to sales volumes, which are predicted to develop to 20 000 tons in a few decades. Fresh whole halibut has been among the higher-priced ﬁsh products in the north European seafood market. However, the market prices of wild-caught halibut depend on season, quality, including size, and local market conditions. Typical market prices can ﬂuctuate between €5 and €14 depending on the conditions mentioned above (Fig. 12.14). The wild landings of halibut occur mainly between April and November, which currently leads to lower sales of the higher-priced farmed halibut. The latter has achieved market prices between

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€7.50 and €11.25, with the larger halibut (7+ kg) being the most expensive product. The smaller farmed halibut are still considered to be new products that will probably require several years of marketing effort to become commercially viable. Wholesalers tend to substitute fresh halibut with farmed halibut as these products become available and consistent with respect to supply and quality criteria. Owing to the fact that wild landings, including Paciﬁc halibut, will continue to dominate the market for many years, it is not likely that future average market prices of farmed halibut will stay at the present high level if farmed volumes grow signiﬁcantly. According to economic theory, the intersection of demand and supply results in a certain price and quantity produced. In the case of halibut, there are several possible short-term equilibria. The prices of farmed halibut are barely affected by the increase in wild landings, so there appear to be different market segments, one for the farmed product and one for the wild one. As mentioned above, continued growth in the production of farmed halibut will eventually affect the high prices, but it will have the advantage of being deliverable all the year round as opposed to its wild counterpart. The large entrance barriers will tend to hold back the production of farmed halibut, so the increase in productivity will be slower than for species with smaller entrance barriers. The price is also relatively high, so the movement from Box 1 to Box 4, as described in Fig. 12.1, will eventually happen, but it will probably be at a slower pace than for species such as cod or salmon.

12.7 Turbot Turbot (Scophthalmus maximus L.) is a well-recognised gourmet ﬁsh, particularly in southern Europe, but the species is also appreciated among consumers in northern European countries such as the UK, Germany, the Benelux countries and parts of Scandinavia. Production of turbot started in the 1980s, followed by more intensive farming in the 1990s, leading to

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a proﬁtable product (Fig. 12.15). The barriers to starting production of farmed turbot are substantial. This has led to a more stable relationship between demand and supply than for other farmed species. We look at some of the aspects of juvenile production and on-growing of turbot in this section, but most of the discussion will be on the market for turbot. The main biological barriers related to brood-stock holding and larval production of turbot were solved during the 1980s, when both governmental institutions and private industry funded research and development to build up turbot juvenile production techniques (Howell, 1998). The ﬁrst farms that actually produced a large number (several hundred thousand) of turbot juveniles were established in Norway and the UK in the late 1980s. During this period, a fairly high number of juveniles were produced in the Norwegian lagoon systems, and were mainly fed on natural plankton produced within the lagoons. A similar system was developed in Denmark, and turbot juveniles were trucked from these hatcheries to on-growing farms in Spain. In the early 1990s, intensive juvenile production techniques were developed within landbased facilities where environmental conditions were under control, and live feed was secure all the year round. Intensive production techniques were developed in Norway, Spain and France, leading to the present situation where more than seven million juveniles for stocking are produced annually by three hatcheries. The juvenile technology for turbot became commercially viable in the 1990s, and the largest current producer is the French company France Turbot, followed by Stolt Sea Farm in Spain. The current market price of juveniles for stocking at the size of 10 g is approximately €1.25. The development of volumes and prices of farmed turbot is shown in Fig. 12.16. The optimal water temperatures for growth of turbot in southern Europe (11–20°C) have resulted in an on-growing technology in speciﬁc regions of Spain (Galicia), France (Atlantic Ocean) and in recent years Portugal (Atlantic Ocean). As turbot is a bottom-dwelling ﬁsh, and is susceptible to waves and ﬂuctuations in water temperature, nearly all commercial turbot in growing farms are in land-based pump-ashore facilities where holding tanks are made of concrete or ﬁbreglass. The difﬁculty of ﬁnding suitable sites for net-pen culture of

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ﬂatﬁsh, and the need for large investment in the construction of land-based ﬁsh farms, has resulted in a more balanced development between demand and supply of turbot than of other farmed species. As we have seen in the other cases, increased productivity has resulted in low production costs and a discrepancy between demand and supply in favour of the demand side, leading to reduced prices. This will probably also happen in the case of turbot, but as long as the barriers to entering production are as strong as they are today, this development will be belated. During the last few years, governmental institutions and private ﬁsh-farming groups world-wide have increased their effort towards the development of re-circulation and heatrecovery technology. It is believed that this kind of technology will become a more frequent solution for new establishments, allowing optimisation of farming conditions in less suitable sites. It is too early to say whether this technology will be competitive with regard to turbot production costs throughout the next decade, but this type of technology will probably help to reduce future logistic costs relating to the distribution of products by allowing farms to be constructed closer to the main markets, i.e. larger cities. The reported production cost for land-based turbot on-growing farms in Spain in 1998 was about €4.0, giving room for considerable proﬁt considering the current market prices as shown in Fig. 12.16. The total sales volume of turbot has been relatively stable in the last decade at 10 000– 12 000 tons per year (Fig. 12.17). During this period, the wild landings have decreased, but the reduction has been compensated for by an increase in the farmed volume. The two largest markets for turbot are Spain and France, where the total consumption has increased rapidly during the 1990s. In 1998, the total Spanish consumption was estimated at 2500 tons, which was dominated by the supply of fresh products from domestic farms. In comparison, French consumption was approximately 1500 tons, with about 1000 tons from domestic ﬁshermen. Most of the Spanish volume is distributed as fresh round ﬁsh through wholesale markets in Madrid (Mercamadrid) and Barcelona (Mercabarna). These municipal markets are intended to centralise supplies through a large number of wholesalers in the biggest Spanish cities. An estimated 65% of market volume is sold through these markets, of which 55% goes to

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1995

1997

1999

2001

Figure 12.17 Volumes and prices for farmed turbot (source: France Turbot).

restaurants and hotels, and 10% to small specialised retailers. Approximately 20% is sold directly to retailing chains, and the remaining 15% directly to restaurants, hotels and specialised outlets. Future prospects seem to indicate a canalisation of fresh farmed seafood through the retailing chains that are located in the big cities. This will again ensure the exposure of fresh products directly to households, and probably lead to an increased consumption of seafood. Turbot is among the highest priced ﬁsh species in the European seafood market. During the early 1990s, a rapid expansion of small turbot farms in Spain caused a temporary oversupply with falling market prices. In this period, the prices dropped to €5, with a modest supply of farmed turbot. The new competition led to a restructuring of ownership from many small companies to a few major producers. This reorganisation and expansion gave better control of the supply of farmed products, and the market price has gradually increased due to the marketing mix and the branding strategy. Today market prices vary between €8 and €15, depending on season, size and geographical markets. Large turbot, i.e. 2+ kg, command a substantially higher price than smaller ﬁsh with an average sales weight of around 1 kg. Since it is the largest consumer and the dominant producer of farmed turbot, the Spanish market has a strong inﬂuence on the price of turbot sold in most European markets. The increased availability of farmed versus wild products means that the current market price is less inﬂuenced by wild landings than it used to be. The supply of wild turbot is greatest during the period from October to January, which inﬂuences the price range and leads to some reduction in output of farmed products. The consistent supply, good quality and low prices of farmed turbot have led many wholesalers to replace the wild product with the farmed one. The European turbot market is expected to continue its growth as a result of farming technology and the fact that the present margins of commercial turbot farming are among the best within this business. The current leaders within the turbot farming business are a few

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fully integrated companies which control the whole process from breeding programmes and egg production to sales and marketing under speciﬁc brand names. Since the mid-1990s, the turbot farming business has been generating high proﬁt margins, and since turbot has always been among the most preferred gourmet ﬁsh species in Europe, it is reasonable to believe it will continue to be amid the higher priced ﬁsh species in the market. The leading companies control the more advanced technical aspects of the biological processes needed to expand future production of farmed turbot in the market. The structure of the current turbot farming business, the high investments needed for establishment and the continued reduction in landings of wild turbot and its substitutes lead us to believe that the future competition and growth of farmed turbot production in Europe will be moderate compared with other species where the entrance barriers are lower. However, a general price reduction will probably occur in the future as competition from new farming ventures is expected to increase over the years to come. In other words, the production of turbot is moving from Box 1 in Fig. 12.1 towards Box 2, and may ﬁnally end up in Box 4 with the other species. However, it has a larger proﬁt margin due to its status as a gourmet ﬁsh, and because of the large barriers to new producers going into production.

12.8 Sole Several species of sole are well known in the European market, and it is a highly appraised ﬁsh. The name refers to the family Soleidae, but more particularly to the common sole or Dover sole (Solea solea). The name sole is also used for other ﬂatﬁsh families, especially Pleuronectidae and Bothidae. In the United States, many ﬂounders are referred to as sole. Yellowﬁn sole, rock sole and lemon sole are other important species, but they do not belong to the family Soleidae (Howell, 2000). Sole generally obtains high prices in European markets. The wild catch has been relatively consistent over the past 10 years at about 300 000 metric tonnes, although some species have varied to some extent. In ofﬁcial listings of wild catch, sole is often listed in the category ‘other ﬂatﬁsh’, so the statistics do not give accurate ﬁgures of the catch. Sole is a well-known ﬁsh in many parts of the world. The market potential is therefore large if it can be produced at prices which can compete with those of the wild catch. The common sole (Solea solea) and S. senegalensis are considered to be the most interesting species for farming. The two species seem to be comparable, and cause similar problems in the cultivation process (Howell et al., 1995). It therefore seems likely that both species will beneﬁt from the current research on sole farming. Sole is marketed whole, gutted or ﬁlleted, and is usually a portion-size ﬁsh. Market prices are consistently high, but there may be some ﬂuctuation because of the variation in the wild landings from month to month. When farmed volumes are small, prices are affected by the inconsistency of the wild landings. Harvesting is therefore targeted at times when demand is high and the wild catch is expected to be low. In Spain in the 1990s, sole was priced at €10–13 kg-1, showing promising potential for future aquaculture. The restaurant segment is an important market for sole, but it is also commonly used in private households, and is found in most exclusive ﬁsh markets and on the ﬁsh counter of

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Culture of cold-water marine ﬁsh

supermarkets. Different ﬂatﬁsh species very often replace each other, although some ﬂounders are lower priced. Sole is a natural candidate for aquaculture, but it is still at a semi-experimental stage. Fertilised eggs are obtained from natural spawning. Juveniles are produced intensively using technology for live-feed production which is similar to that used for sea bass and sea bream. The main problems are currently connected with on-growing and difﬁculties related to feeding. Sole is not predatory, but is more of a passive feeder. It is also a nocturnal feeder, and difﬁcult to raise in the traditional on-growing facilities used for other ﬂatﬁsh (Howell, 2000). Several companies in Spain are interested in producing sole, but so far no one has succeeded in reaching a volume that is commercially viable. Sole is usually found in relatively warm waters, normally no further north than the British Isles. According to industrial experts, the optimal temperature for on-growing is 20°C. Farming of sole is thus likely to be situated in southern climates with high ambient temperatures, or in locations with access to warm industrial wastewater. Unlike many other species, spawning and juvenile production of sole is relatively uncomplicated. Eggs can be obtained by natural spawning, while the use of light-manipulation has been successful in out-of-season spawning. First feeding of the larvae is done with Artemia, and unlike most other marine ﬁsh larvae that have speciﬁc nutritional requirements, sole larvae can be reared on undeveloped Artemia. In small-scale experiments the survival rate is high, and juvenile production is not the restraining factor in commercial production. Juveniles can be produced in large quantities, and the problems of weaning to artiﬁcial diets have been solved (Ellis et al., 1997). The problem with on-growing of sole to formulated food has prevented the full closure of the rearing cycle. This will probably be the primary obstacle to an economically sustainable sole culture. Sole has been grown from juveniles of 5 g to 125 g in less than 300 days at an optimum temperature of 20°C, which is rather slow compared with other marine species such as turbot and halibut. Sole also seems to be less well suited to high stocking densities, with the result of low production per unit area in the rearing tanks. In addition, farmed sole has not yet reached the quality level of its wild counterpart. Juveniles are often malpigmented, and the texture as well as the taste of the meat is different. In the short term there might be a limited production of cultured sole. The relative ease in which juveniles can be produced might result in the availability of juveniles at a low cost. In addition, all year round production is an advantage to an on-growing producer who can stock the farm two to three times a year to exploit the production potential. A high market price for sole will justify high production costs in the initial stage of farming. If aquaculture of sole becomes a success, prices are likely to be affected, but by then sole will be able to beneﬁt from improved land-based production technology, and thus the reduction of production costs to a level similar to those of turbot or halibut.

12.9 Conclusions In this chapter, we have looked at the possibilities for farming seven new species of ﬁsh, some already in production, some in an initial commercial phase, and others still at the

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research and development level. Before one starts developing a new species, it is important to explore the biological and technical possibilities for farming and the market potential. The rearing cycle must be closed before commercialisation is possible. Moreover, production must be efﬁcient, i.e. technical competence and a likelihood of growth in productivity is a necessity in order to make an economically viable product. There must also be a market for the new goods with prices which are compatible with production costs. From an economic perspective, it is productivity, market status and prospects that are stressed. We can divide the market for ﬁsh and ﬁsh products roughly into two parts, one large bulk market with relatively low prices, and one possible niche market with higher prices. The seven species discussed in this chapter can broadly be divided into high value, such as turbot, halibut and sole, and lower value, such as hake, cod, haddock and wolf-ﬁsh. Some are also situated in several segments of the market, such as cod, hake and haddock, which can be found in the low-price bulk market, but also in smaller quantities of high-quality ﬁsh in the higher-priced niche markets. In the initial phase of ﬁsh farming, most producers ﬁnd themselves in a situation of high production costs and, in some cases, high market price, but as soon as productivity expands and production levels increase, prices drop. This was the case with salmon, for example, as there was a great unsaturated demand for the product in the ﬁrst 10 years, and although production increased with higher productivity, prices were relatively stable. As soon as the market was satisﬁed, however, prices dropped, and after a while prices and quantities stabilised at levels determined by production costs and demand. There are different stages in the development of farmed ﬁsh, starting with a research and development phase to see if the technological barriers are manageable, i.e. that the rearing cycle is, or can be, closed. Halibut, sole, wolf-ﬁsh, hake and haddock are still at this level, although halibut has moved towards the initial commercial phase, and in some cases also the take-off phase. In the next stage, the initial commercial phase, both research institutions and commercial interests are involved in judging the future prospects for farming of the particular species. Cod, halibut and to a certain extent wolf-ﬁsh can be found in this category. In the two ﬁnal stages, take-off and big growth, commercial interests operate alone, and only three of our seven species have reached the next to last phase, i.e. turbot, cod and halibut, while the last stage remains unexplored. In addition to these stages of production, there are also different challenges along the way. For the majority of the species, productivity is the most critical factor for success. As soon as technology allows for large-scale production at low cost, the chances of accomplishment rise considerably, as market prices in most cases are low or will decline. Cod is one species that has had ups and downs in the last 20 years because of variations in productivity, market prices and also wild landings. Today, there is a renewed and substantial interest in cod farming because of prospects of increases in productivity, market segments with relatively high prices, and a decline in wild landings. There is already a market for cod, which the farmed cod can take advantage of, and at the same time exploit the temporary niche for high-priced fresh cod. However, the challenge in the long run is to establish low production costs for farmed cod to be able to sell at competitive prices. Another interesting species for aquaculture may be haddock, which is both of high quality and a possible substitute for cod in many markets. Production techniques are developing,

514

Culture of cold-water marine ﬁsh

but production costs are expected to be too high in a low-price market. The future development of haddock is thus dependent on better productivity. Market prices for European hake are relatively high for high-quality ﬁsh, so the future market opportunities for the farmed species might be good. The problem is that the farming of hake is still in the research and development phase, with a long way to go within brood ﬁsh, ﬁngerling and juvenile production. Market analysts believe that there is a potential market for the species, but we do not know if productivity will be high enough for the species to be economically viable. Low prices are the overall tendency within the market for wolf-ﬁsh, with some segments of higher prices. The main challenges for the development of wolf-ﬁsh, which is still in the process of research, are ﬁngerling production and technology concepts for on-growing. The species has attracted some commercial interest, so there is a potential niche market if productivity reaches a satisfactory level. Halibut is also at the level of research and development when it comes to production techniques, but large-scale production is expected within a few years. Wild halibut obtains relatively high prices in the market, indicating bright prospects for the production of halibut, which is a ﬁsh regarded as being of high quality. Since production has only reached the ﬁrst stage in our scheme, it is hard to estimate production costs and market possibilities for farmed halibut, but there seems to be a substantial potential in the market as long as production reaches a level where cost and price are in balance. Turbot is the ﬁrst success story of our seven species. It was developed through the research phase in the 1980s and the commercial phase in the 1990s. Since the entrance barriers for farming turbot are relatively high due to the high investment costs, there will be a slower increase in production than for the other species, and, as we have seen, thereby better proﬁt in the ﬁrst years. Productivity will probably increase within a few years, causing lower prices and less proﬁt per unit produced, but as long as only a few producers control the more advanced technical aspects of the biological processes, and the species is perceived as a gourmet ﬁsh, this development will take time. Finally, we looked at sole, which is a relatively high-priced ﬁsh that is highly appraised. There is a large market potential for farmed sole, but nonetheless it is in a semi-experimental stage where production costs are high because of major problems with feeding at the ongrowing stage. Since juvenile production seems to be manageable, the critical factor for success will be feeding and on-growing. Given a solution to this problem, farmed sole should have the potential to become a viable commercial product within a few years. When choosing new species for farming, there are several aspects to consider. The biological and technical competence necessary to master the rearing cycle are the ﬁrst barriers along the way, followed by production costs and market price. These elements give the farming of the different species various pathways. As we have seen in the discussion, turbot, and possibly halibut and sole in cases of land-based production, demand high production costs and thus large investments. This gives large entrance barriers for new producers, and thereby relatively low quantities are produced. Since the market prices are high, production can be cost-effective and consequently we have a proﬁtable product. Cod, on the other hand, might be a low-cost product, produced in large quantities and selling at relatively low prices, which is a different direction. Thus, before farming can start we must consider the market

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price and the possibilities for production within these margins in order to create a commercially viable product.

12.10 References Alheit, J. & Pitcher, T.J. (eds) (1995) Hake—Biology, Fisheries and Markets. Chapman & Hall, London. Anderson, J.L. (1985) Market interactions between aquaculture and the common-property commercial ﬁshery. Mar. Resourc. Econ., 2, 1–24. Asche, F. (1997) Trade disputes and productivity gains: the curse of farmed salmon production? Mar. Resourc. Econ., 12, 67–73. Asche, F. & Salvanes, K.G. (2001) Noen vurderinger av torskeoppdrett. Nor. Fiskerin., 4, 82–7 (in Norwegian). Asche, F., Bjørndal, T. & Young, J.A. (2001) Market interactions for aquaculture products. Aquacult. Econ. Manage., 5, 303–18. Bjørndal, T. (1990) The Economics of Salmon Aquaculture. Blackwell, Oxford. Ellis, T., Howell, B.R. & Hughes, R.N. (1997). The cryptic responses of hatchery-reared sole to a natural sand substratum. J. Fish Biol., 51, 389–401. Engelson, R. (1996) Økonomisk analyse. Moderne lysstyrt produksjon, Torsk Matﬁsk. PUSH (Program for Utvikling og Stimulering av Havbeite). ISBN 82-91625-05-0. Fiskeridirektoratet (2001) Lønnsomhetsundersøkelse for matﬁskproduksjon laks og ørret 2000. økonomiske Analyser Fiskeoppdrett, 1, 1–71 (in Norwegian). Holm, J.C., Svåsand, T. & Wennevik, V. (eds) (1991) Håndbok i torskeoppdrett. Stamﬁskhold og yngelproduksjon. Instititute of Marine Research, Department of Aquaculture, 156 pp. (in Norwegian). Howell, B.R. (1998) Development of turbot farming in Europe. Bull. Aquacult. Assoc. Can., 98, 4–10. Howell, B.R. (2000) Sole culture. In: Encyclopaedia of Aquaculture (ed R.R. Stickney), pp. 889–92. Wiley. Howell, B.R., Beard, T.W. & Hallam, J.D. 1995. The effect of diet quality on the low-temperature tolerance of juvenile sole, Solea solea (L.). ICES CM 1995/F, 13, 9. Kinnucan, H. (1995) Catﬁsh aquaculture in the United States: ﬁve propositions about industry growth and policy. World Aquacult., 26, 13–20. Kvenseth, P.G., Winther, U., Hempel, E. & Fagerholdt, A.F. (2000) Torskeutedning for SND, Oslo. KPMG Oppdrag nr. 1286 (in Norwegian). Litvak, M.K. (1998) The development of haddock culture in Atlantic Canada. Bull. Aquacult. Assoc. Can., 98, 30–3. Sparboe, L.O., Fieler, R., Falk-Petersen, I.B., Lund, V., Arnesen, J.A. & Eggset, G. (1999) Utvikling av ﬂekksteinbit som kommersiell oppdrettsart—år 2 In: Akvaplan-niva report nr APN 630.98.1455, Tromsø (in Norwegian). Van der Meeren, T. & Ivannikov, V. (2001) Seasonal shift in spawning of cod broodstocks by light manipulation: egg quality and larval rearing. In: Larvi’01—Fish and Crustacean Larviculture Symposium. (eds C.I. Hendry, G. van Stappen, M. Wille & P. Sorgeloos), Gent, Belgium, 3–6 September, pp. 616–17. European Aquaculture Society, Special Publication No. 30. Varian, H. (1999) Intermediate Microeconomics. Norton, New York.

Index

a-adrenoreceptor 257 1,3,5-benzenetricarbonyl trichloride 345 11-ketotestosterone 141 abdomen 155, 462, 463 abnormal behaviour 36 abnormal cleavage 162, 170, 171, 174 abnormal embryo 169, 171, 219 abocular side 232, 233, 256 absolute growth 261, 402 accuracy of selection 188 acid insoluble ash (AIA) 366 Acipenser stellatus 167 acrosome 136 actin 419, 420 actinin 419 actomyosin 419, 421 additive genetic variance 184, 188 adenylated phosphates 172 ADP 172, 421 adrenaline 253 adrenergic 257 adrenocorticotrophic hormone 250, 256 adrenodoxin 256 aerobic metabolism 242 Aeromonas salmonicida 39, 51, 62 aerophil 130 age at sexual maturation 186 agglutin 256 ageing (egg) 153, 168, 172 algae 7, 21, 52, 53, 60, 77, 78, 83, 92, 112, 257, 280, 293, 298, 305, 307, 324, 329, 330, 435, 448, 453, 469 alimentary canal 235 alizarin complexon 479 alkali cellulose 385

alkaline phosphatase 236 allometric growth 228, 229, 260 Amberjack 47 American catﬁsh 487, 488, 496 American crayﬁsh 51 amine ethoxyquin 395 amino acid 10, 17, 100, 101, 106, 114, 135, 143, 148, 193, 212, 214, 232, 240, 246, 247, 248, 250, 262, 284, 315, 319, 342, 367, 413 aminopeptidase 236 ammonia 10, 25, 83, 96, 98, 249, 290, 376, 419, 422, 458 ammonia excretion 10, 249 amoeba 41 amoeboid gill disease 41 AMP 172, 421 amylase 236, 239, 374, 375, 384 Amyloodinium spp. 44 amylopectin 384 amylose 384 anaerobic metabolism 242, 421 anaerobic sediments 18 anal ﬁn 136, 221, 244, 417, 418 Anarhichas lupus 9, 221, 369, 371, 379, 454 Anarhichas minor 132, 407, 487, 503 Anarrhichthys ocellatus 265, 454 androgens 139 anaemia 32 Anguilla anguilla 225 animal pole 135, 171, 211, 213, 214, 215, 216 anorexia 49 anoxic conditions 17 anterior intestine 339, 340 antibiotic 59, 61 antibody 56, 62

518

Index

antifreeze 449 antifungal 393 antigen 30, 33, 374 antimicrobial peptide 193 antinutritional factors (ANF) 374, 375 antioxidant 147, 149, 372, 386, 388, 389, 391, 395, 418 anus 234, 340 apoptosis 168, 254 Aporocotyle simplex 47 arachidonic acid (ARA) 147, 167, 262, 280, 317, 382 Arctic char 478 Arenicola marina 472 arginine 284, 368, 369, 419 Artemia fransiscana 39 Artemia juveniles 121, 308 Artemia nauplii 2, 19, 116, 117, 241, 258, 293, 307 Artemia salina 457 artiﬁcial diets 345 artiﬁcial insemination 153, 156 ascorbic acid 105, 122, 148, 386, 388, 391, 395 ash content 212 asparagines 368 aspartic acid 368 assessment of egg quality 157 assimilation 113, 257, 262, 282, 300, 306, 321, 343 astaxantin 149 asymmetrical cleavage 163 Atlantic halibut 4, 13, 16, 18, 57, 59, 77, 121, 142, 150, 151, 152, 154, 162, 169, 173, 177, 207, 210, 228, 249, 310, 341, 349, 359, 369, 379, 461, 504, 505, 507 Atlantic herring 137, 178 Atlantic salmon 1, 32, 35, 39, 132, 148, 183, 210, 227, 369, 379 ATP 168, 172, 180, 181, 421 atresia 145, 152 autogene 350 axial convergence 218 axial mesenchyme 245 axial musculature 241 axial skeleton 229, 244, 245 Bacillariophyceae 22 Bacterial Kidney Disease (BKD) 32

bacterial sulphate 17 barium carbonate 366 Barramundi 402 base population 186 batch culture 79, 83, 84, 88, 90, 91, 96 batch production 86, 88 batch-spawning 136, 169 bathypelagic 230 bile duct 237, 246 bile synthesis 235 bile-salt dependent lipase 238 binding agent in ﬁsh feed 385 biotin 105, 386 blastocoel 217 blastoderm 216, 217, 218, 222 blastodisc 159, 160, 162, 163, 171, 174, 215, 216, 217, 224, 227 blastomere morphology 161, 174 blastopore 217 blastopore closure 217, 218, 221 blastula 168, 217 blastulation 217 blood cell 219, 221, 248 blood vessel 169, 219, 222, 225, 242 blue wolf-ﬁsh 454 blue-green algae 22 Bohr effect 246 bone deformity 245 borate buffer 345 Brachionus plicatilis 2, 58, 73, 316 Brachionus rotundiformis 73, 76, 307, 319 brain development 252, 317 brain vesicle 218 brain–pituitary–gonad axis 138 branchial arch 218 brood hiders 131 broodstock diet 143 broodstock management 142 brown tides 21 buccopharynx 234 buffering capacity of seawater 11 Buffodine 57, 154 butylated hydroanisole (BHA) 395 butylated hydroxytoluene (BHT) 395 C6H12O6 17 Cannibalism 259, 266, 295, 297, 301, 351, 352, 354

Index

Capelin 90, 105, 297, 368, 369, 378, 391, 392, 416 carbohydrate 146, 181, 239, 240, 264, 341, 342, 383, 457 carboxymethyl cellulose (CMC) 385 carotenoid 145, 149, 166, 330, 376, 386, 389, 391, 395 catecholamine 257 cathepsin L 135, 167 cestodes 46 CH3(CxHy)COOH 377 Chaetoceros convolutes 23 channel catﬁsh 183, 400, 496 chemosensory 230, 232 chloracetic acid 385 chloride cell 16, 247 cholesterol 166, 281 choline 281, 386, 395 chorion 32, 135, 139, 140, 209, 211 chorionase 219 chromafﬁn tissue 250 chromatophore 149, 254, 256 chromic oxide 366 chromoblast 256 chromosomal aberration 167 chromosome 135, 167 Chrysochromulina 23 chylomicron 239 chymotrypsin 236, 342, 374 ciguatoxin 22 ciliate 43, 98 climax metamorphosis 229, 232, 240 Clupea harengus 139, 178, 415, 417 cnidarians 44 coelocanth 208 coefﬁcient of variation (CV) 141 condition factor 145 continuous light 19, 21, 257, 350 conversion efﬁciency 15, 185, 240, 411, 412 cortical alveoli 135, 151, 161, 170, 171, 174, 213, 215 cortical reaction 153, 159, 160, 171, 212, 213, 247 corticotropic hormone 142 crossbreeding 191 cryopreservation 181 Cryptocotyle lingua 47 cyanobacteria 22

519

cyclin B 139 Cyclopterus lumpus 130, 212 cytochrome oxidase 17 cytochrome P450 139, 140, 256 deoxyribose (C5H10O4) 383 Desulfoxibrio desulfurcans 17 diatom 21, 299 Dicentrarchus labrax 1, 43, 144, 246 dietary carbohydrate 146, 341 dietary lipid 81, 102, 104, 106, 146, 417 digestive enzyme 234, 236 dinoﬂagellate 21, 44, 288, 297, 299 Dinophysis 22 dip-vaccinate 350 disinfection of eggs 37, 57, 326 dissolved oxygen (DO) 7, 406 DNA ﬁngerprint 189 DNA vaccines 192 docosahexaenoic acid (DHA) 147, 167, 262, 280, 381 early maturation 151, 498 ectoparasite 29 egg activation 135 Egtved disease 37 eicosapentaenoic acid (EPA) 147, 262, 280 endoparasite 29 enterocyte 235 erythrocyte 248 erythrophore 256 essential fatty acid (EFA) 147, 262, 381, 382 essential polyunsaturated fatty acids (PUFA) 145 esterase 239 ethyl cellulose 385 ethyl chloride 385 eucaryotic parasites 29, 30 European lobster 475 excretory products 10 exocrine pancreas 234, 236 exocytosis 239 eye migration 232, 241, 245, 268, 297, 317, 337, 340, 347, 359, 466 fat-soluble vitamins 386 feed conversion efﬁciencies 7, 15, 24, 185, 240, 411, 412, 413, 457, 458, 466

520

Index

ferro-nickel microtracers 366 ferrous sulphate (FeS2) 17 ﬁnal oocyte maturation (FOM) 135, 140 ﬁnfold 207 ﬁrst time spawner 167 ﬁsh meal 121, 368, 369, 392 ﬁsh oil 121, 378, 392, 423 ﬁsh protein concentrate (FPC) 371 ﬁsh-pathogenic bacteria 29 Flexibacter ovolyticus 32, 38 follicle stimulating hormone (FSH) 139, 140 formaldehyde 46 free amino acids (FAA) 342, 345 furunculosis 39, 51, 61 gallic acid (pyrogallol) 395 gamete development 137 gas gland 246 gastric gland 240 gastrulation 217 gene mapping 189 gene transcription 250 gene transfer techniques 192 genetic gain 183, 187, 188 genetic improvement 182, 183, 184, 185, 188 genetic interaction 191 genetic line 187 genetic map 191 genetic marker 187, 189, 191, 193, 479 genetic parameter 183 genetic tagging 189, 191 genetic value 184, 187 genetic variance 184, 187 genetic variation 182, 183, 185, 186, 187, 191 genital pore 132, 153, 155, 170, 455 genital system 132 genome 189, 191, 192, 193 genotype 157, 183, 184, 185, 189 germ layer 217 germ line transmission 192 germ ring 216, 217 germinal tissue 133 germinal vesicle 134, 135, 211 gill 10, 11, 16, 17, 22, 23, 25, 30, 35, 38, 41, 44, 45, 46, 48, 49, 131, 222, 223, 226, 228, 242, 247, 248, 255, 268 gill epithelium 44 gill ﬁlament 223, 248

gill lamellae 17 gilthead seabream 144, 145, 146, 156, 251, 346 glossohyal 253 glucose 17, 146, 246, 383, 384, 386 glucose tolerance 146 glucosinolate 264, 375, 376 glutamine 368 glutanic acid 368 glutaraldehyde 57, 58, 153, 265, 266, 326, 446, 455 glutathine 389 glutathione 389 glycogen 136, 240, 242, 246, 339, 340, 342, 384, 408, 410, 421 glycolysis 181 glycolytic pathway 240 glycoprotein 37, 192, 246 gobies 130 Golgi complex 239 gonad 20, 38, 132, 133, 137, 138, 139, 140, 144, 146, 147, 151, 153, 176, 250, 255, 263, 388, 441, 462 gonad development 13, 21, 137, 138, 139, 144, 152, 440, 441, 448 gonad growth 134, 143, 147 gonad maturation 132, 134, 136, 138, 141, 143, 144, 146, 152, 154 gonadal growth 129, 144 gonadal tissue 388 gonadosomatic index (GSI) 176 gonadotrophic hormone 250 gonadotropin-releasing hormone agonist (GnRHa) 142 gonadotropin-releasing hormone (GnRH) 138, 142, 255 gonadotropin 138, 139, 141, 142 gonochoristic 14, 133 gonopod 136 granulocyte 256 granulosa cells 139, 140 green water 60, 305, 312, 321, 323, 329, 330, 331, 332 green water technique 105, 257, 292, 306, 316, 320, 328, 329, 453, 464 greenback ﬂounder 19 Greenland halibut 417 group synchronous ovarian development 137, 138, 141, 144

Index

growth hormone 192, 250, 251, 253, 255, 350 growth hormone releasing factor 255 growth rate 5, 9, 14, 16, 24, 78, 79, 80, 83, 84, 85, 86, 88, 89, 91, 92, 93, 94, 95, 96, 98, 99, 100, 101, 102, 103, 105, 106, 113, 144, 165, 166, 182, 183, 185, 186, 187, 188, 192, 224, 228, 238, 243, 244, 260, 261, 262, 283, 285, 299, 300, 301, 303, 304, 305, 309, 313, 314, 318, 320, 321, 322, 323, 324, 327, 329, 331, 338, 341, 342, 352, 353, 354, 355, 370, 402, 403, 404, 406, 408, 411, 412, 422, 439, 448, 449, 451, 457, 458, 466, 468, 473, 474, 479, 480, 483 guanine 218, 256 guarder 131 guild 130, 131 guppies 130 gut 35, 44, 56, 102, 111, 119, 219, 228, 233, 234, 235, 236, 239, 240, 241, 245, 246, 247, 250, 256, 260, 261, 263, 268, 298, 299, 304, 305, 316, 321, 328, 331, 332, 339, 340, 342, 344, 350, 351, 354, 360, 366, 384, 386, 388, 452, 453, 498, 499, 501, 503, 506, 511 gutted 498, 499, 501, 503, 506, 511 gut epithelial cell 234, 235, 236 gut evacuation 304, 321, 350 gut content 298, 304, 328, 332, 339, 360 gut lumen 234, 235, 239, 240 gut mucosa 235, 260 gut pH 240 haddock 13, 19, 257, 310, 444–50, 487, 488, 501, 513, 514 haematopoetic 192 haemal spine 254 haemoglobin 11, 17, 246, 248, 250, 268, 386 Haemogregarina sachai 42 haemolytic bacteria 53, 60 Hake 310, 312, 451–4, 487, 488, 501–503, 513, 514 half sibs 187 halibut 13, 16, 18, 19, 20, 31, 32, 35, 36, 37, 38, 39, 40, 41, 42, 43, 45, 47, 57, 58, 59, 61, 62, 74, 77, 121, 122, 125, 132, 138, 141, 142, 144, 146, 150, 151, 152, 153, 154–5, 156, 158, 159, 160, 161, 162, 163, 164, 166, 167, 170, 171, 172, 173, 175, 176, 177, 178, 181, 193, 207, 208, 209, 210, 211, 212, 218,

521

228, 230, 231, 233, 235, 240, 241, 245, 247, 248, 249, 250, 252, 253, 256, 262, 263, 265, 266–7, 283, 285, 289, 296, 297, 302, 303, 305, 306, 308, 310–12, 315, 316, 317, 318, 319, 332, 333, 338, 339, 341, 342, 343, 344, 346, 347, 348, 349, 350, 351, 356, 359–61, 369, 370, 379, 404, 413, 417, 461–7, 472, 481, 482, 487, 488, 489, 503, 504–507, 513, 514 haploid 135, 136 hardening process 156 harvesting 9, 18, 86, 89–92, 93, 94, 113, 116, 117, 185, 288, 290, 337, 410, 482, 498, 511 harvesting strategies 86 hatching (evertebrates) 77, 113, 114, 115, 117, 121, 287, 288, 293, 307 hatching (ﬁsh) 14, 16, 18, 19, 34, 57, 59, 60, 62, 130, 133, 146, 147, 148, 149, 153, 155, 156, 159, 160, 161, 163, 164, 165, 166, 167, 171, 172, 182, 189, 204, 207, 209, 210, 212, 214, 218–19, 220, 221, 222, 223, 224, 225, 226, 227, 229, 230, 231, 232, 234, 235, 237, 238, 241, 242, 245, 246, 247, 248, 250, 251, 253, 256, 265–7, 268, 280, 295, 306, 310, 312, 313, 326, 356, 358, 440, 441, 447, 448, 452, 453, 454, 455, 456, 457, 458, 459, 461, 464, 472, 475 hatching gland 218 hatching of cysts 115 hatching rate 116, 148, 149, 153, 156, 161, 166, 182, 265, 266, 459 hatching success 146, 147, 148, 155, 159, 160, 161, 163, 164, 165, 167, 171, 172, 264 hatching time 210, 464 hatching enzyme 18, 268 heart 38, 218, 222, 252 hepatic vitelline vein 221, 222 herbivore 233 heritability 183, 184, 186, 187, 188 hermaphrodites 45, 46 Herpesvirus scophthalmi 38 herring 133, 137, 178, 191, 245, 283, 297, 300, 368, 370, 378, 391, 392, 414, 415, 416, 417, 480, 481 highly unsaturated fatty acids (HUFA) 280, 377, 415 hindbrain 218, 219 hind-gut 234, 236, 240, 241, 299, 339

522

Index

Hippoglossoides platessoides 49, 214 Hippoglossus hippoglossus 13, 42, 74, 121, 132, 213, 283, 296, 341, 359, 370, 404, 406, 414, 417, 487, 504 histamine 368, 419 holoblastic 208 Homarus gammarus 478 hormonal production 138 hormone cascade 250 husbandry practice 152 hydrating oocytes 155 hydrodynamic force 242 hydrogen peroxide (H2O2) 389 hydrogen sulphide 7, 17, 18 hydrolysed protein 343, 344 hyoid bone 252 hyperplasia 41, 44, 243, 244, 264 hypersaline 267 hypertonic 178, 247 hypertrophy 42, 44, 243 hypoblast 217, 218 hypothalamic releasing hormone 142 hypothalamic-pituitary-thyroid 264 hypothalamus 138, 139, 250, 251, 252, 255 hypural 245 Hysterothylacium aduncum 47 Ichthyophonus spp. 29 Ictalurus punctatus 400 immune system 28, 33–5, 50, 56, 61, 62, 63, 148, 256, 269 immunoreactive 251, 256 immunostimulation 34, 56, 61 inbreeding depression 183, 186, 187, 189, 191, 462, 485 incomplete cleavage 161 incubation time 209, 212, 265, 447, 455 incubator 154, 156, 266, 267, 447, 463, 464 indirect development 222, 225 individual selection (mass selection) 183, 184, 186, 187, 188 induced ovulation 142 infectious hematopoetic necrovirus (IHNV) 192 Infectious Pancreatic Necrosis Virus (IPNV) 35, 36 inheritance 191 inner ﬁbre 242 inner white muscle 243

inoculation phase 81, 82 inorganic nitrogen 10 inosine monophosphate (IMP) 421 insemination 131, 132, 135, 136, 153, 155, 156, 158, 170, 171, 176, 177, 178, 179, 212, 213, 454, 455 insulin-like growth factors (IGFs) 168, 255 insulin-like growth hormone 255 intensive hatchery techniques 285, 290–94, 503 intensive systems 8, 301–33, 434, 439, 464 internal bearers 131 internal control method 85, 87 internal fertilisation 131, 153, 175, 312 internal insemination 131, 132, 136, 155, 158, 176, 177, 178 interrenal 250, 251, 255, 256, 263 intestinal epithelium 31, 37, 39, 240 intestinal fold 235 intestinal tract 58, 59, 237, 374, 387, 388 intestine 30, 31, 36, 39, 47, 48, 52, 58, 59, 219, 220 intrafollicular gestation 130, 221, 234, 237, 239, 240, 246, 255, 326, 330, 332, 338, 339, 340, 343, 344 investment costs 470, 489, 514 in vitro fertilisation (IVF) 168, 174 ion exchange 248 ionic regulation 247, 386 iridophore 256 irregular blastomere 162 irregular spawning 143 isometric growth 228, 229, 244 iso-osmotic 247 isotonic 16, 178, 247 iteroparous 136, 137 jack mackerel 205 Japanese ﬂounder 14, 38, 235, 237, 245, 251, 254, 341, 480, 484 jaw 13, 48 jaw development 13, 219, 220, 221, 222, 223, 227, 244, 245, 260, 267, 453 Johanssonia arctica 48 juvenile quality 156, 164, 264, 269, 315, 470 juvenile state 133, 207 juvenile survival 147 juvenile viability 147, 174 juxtaglomerular 250

Index

kidney 10, 16, 32, 36, 38, 42, 43, 250, 254, 255, 256, 264 King crab 484 krill 143 Kuppfers vesicle 218 lactic acid 17, 180, 181, 371, 421, 422 larval nutrition 119, 233, 261, 269, 279, 324, 338, 339 larval activity 298 larval characters 207, 225, 226 larval feeding 257–9, 261, 266, 269, 280, 285, 292, 297–313, 314, 315, 330, 332, 457 larval feed 238, 313, 316, 320, 321, 330, 332 larval food 288, 289, 296, 297, 300, 301 larval growth 13, 100, 110, 145, 257, 260, 261, 262, 268, 284, 287, 288, 300, 309, 324 larval incubation 267 larval mortality 156 larval rearing systems 55, 293–4, 452 larval requirement 105, 122, 206, 279, 283, 323, 344 larval size 59, 165, 204, 248, 258 larval survival 81, 148, 163, 165, 228, 262, 263, 266, 329, 447 larval viability 53, 106, 122, 145, 160, 163, 164, 166, 167, 173, 174, 296, 314 Larval vision 18, 230–32 larval weight 261, 303, 313 lateral line 220, 228, 230, 232–3, 242, 269 Lates calcarifer 341, 402 Latimeria chalumnae 208 lecithotrophic 131, 132 lens 218, 230, 231, 232, 269, 398 Lepeophtheirus salmonis 32 Lepisosteus 208 Lernaeocera branchialis 40, 49 leucocyte 142 Leydig cell 141 light quality 257 light reception 149 light-manipulation 512 lipase 236, 237, 238, 239, 284 lipid-encapsulated diet 345 lipids 75, 81, 102–105, 106–110, 117–22, 123, 134, 135, 145, 146–8, 166, 167, 211, 212, 221, 222, 236, 239, 240, 246, 248, 255, 262, 264, 280–81, 284, 293, 297, 304, 307, 308,

523

314, 315, 316, 317, 318, 319, 320, 322, 323, 324, 330, 331, 339, 340, 341, 342, 344, 345, 364, 365, 368, 369, 370, 371, 372, 377–83, 385, 386, 388, 391, 392, 393, 395, 396, 409, 410, 413, 414, 415, 416, 417, 418, 422, 423, 441, 449, 457, 466, 469, 473 lipid absorption 239, 240, 339, 340 lipid class 166, 280–81 lipid droplet 135, 211, 221, 222, 236, 239, 344 lipid metabolism 102, 147, 255 lipid oxidation 148, 370, 372 lipid requirement 280–85, 315–20, 322–3, 324, 330, 331, 341, 342, 344, 377–83, 386, 392, 409, 410, 413, 416, 417, 418, 423, 473 lipolytic enzymes 236 lipoprotein 134, 135, 147, 239, 388 lipoprotein synthesis 239, 240, 344 lipovitellin 134 lithophil 129, 130 littoral 130 live food technology 73, 96 liver 10, 36, 38, 48, 134, 139, 140, 192, 219, 228, 234, 235, 237, 240, 254, 255, 263, 264, 339, 340, 342, 344, 384, 410, 414, 415, 417, 418, 422, 441, 449, 498 lobopodia 217 lobster 475, 477, 484 long rough dab 214 lordosis 247 low density lipoprotein (LDL) 147 low-energy feed 413, 441 low-lipid feed 416, 417 low-oxygen 17 low-temperature 370, 473 L-strain (rotifers) 76, 307 lumen 135, 175, 234, 235, 239, 240, 246, 253, 339 lumpﬁsh 212 lumpsucker 130, 214 lungﬁsh 208, 217 lupin seeds 374, 375, 376 luteinising hormone (LH) 139, 140, 141 lux 19, 116 lymphoblast 43, 256 lymphocyte 256 lymphoid organs 35, 256 lysine 148, 284, 315, 367, 368, 369, 370, 373, 419

524

Index

lysozyme 256 lysozyme gene 193 mackerel 205, 369, 391, 416 Macrocystis pyrifera 385 macronutrient 145, 341–2, 343, 365, 389, 396, 465 macrophagic cell 256 macrovitamin 388 Macrozoarces americanus 137, 175 major histocompatibility complex (MHC) 191 malate dehydrogenase 167 male maturation 135–6, 141 malformation 154, 175, 265, 267, 269, 314 malpigmentation 257, 262, 268, 297, 359 maltase 236 mammal 33, 34, 139, 147, 168, 174, 230, 240, 243, 253 mammalian embryo quality 168 marine copepods 73, 119, 120, 122–4, 322, 323 marker-assisted selection (MAS) 192 market price 436, 481, 484, 487, 488, 489, 490, 493, 495, 496, 497, 499, 500, 501, 503, 504, 506, 507, 508, 509, 510, 511, 512, 513, 514, 515 maternal age 157 maternal effect 173 maturation (ﬁsh) 13, 20, 21, 34, 129, 132–42, 144, 145, 146, 147, 148, 150, 151, 152, 154, 155, 167, 168, 169, 181, 185, 186, 229, 242, 255, 344, 388, 408, 440, 441, 442, 454, 455, 466, 498, 506 maturation (live feed cultures) 111 maturation cycle 139, 151 maturation-inducing steroids (MIS) 139, 140, 141 maturation-promoting factor 139 maximum stocking densities 8 mechanical pressure 209, 265, 456 mechanoreceptor 230 medulla spinalis 243 meiosis 135, 167, 213 meiotic cleavage 134 meiotic division 135–6, 213, 214, 215 melanin 253 Melanogrammus aegleﬁnus 13, 310, 444, 487, 501 melano-macrophages 47

melanophore 218, 219, 221, 256 melanophore stimulating hormone 250 melanosome 257 melatonin 138, 151, 368, 389 menhaden 368, 370 Merluccius australis, Hutton 451 Merluccius merluccius L 312, 451, 487, 501 meroblastic 208, 214 mesocosm 285, 289–90, 295, 296–301, 436, 437, 461 mesoderm 217, 218 mesotocin 250 metabolic enzymes 341, 416 metabolic rate 12, 14, 257, 258, 404, 405, 406, 407 metamorphic (change) 232, 241, 250, 254 metamorphic climax 250, 254 metamorphosis 36, 60, 164, 165, 204, 205, 222, 225, 226, 228–9, 230, 231, 232, 233, 240, 241, 242, 243, 245, 248, 250, 251, 253, 254, 255, 256, 257, 263, 268, 269, 280, 296, 301, 309, 312, 313, 314, 317, 330, 337, 338, 339, 340, 342, 347, 351, 359, 361, 436, 437, 448, 465, 472 metanauplius 111, 112, 113 metaphase 135, 167, 213 metazoa 28, 29, 40, 44–50, 76 methionine 284, 368, 369, 370, 371, 372, 373, 376, 385 methyl cellulose 385 microalgae 60, 74, 77, 80, 81, 82, 83, 97, 99, 100, 105, 106, 108, 117, 121, 240, 285, 292, 302, 304, 305–306, 308, 310, 312, 316, 320, 321, 324, 327, 328–33, 376, 436 microbial activity 92, 193, 267 microbial condition 51, 268, 279, 325, 327, 439 microbial control 50–56, 114, 302, 309, 325 microbial hatchery management 279, 325 microbial interactions 28, 59, 279, 325 microbial maturation of water 59, 291, 327 microbound diets 345 Microcotyle sp. 45 microencapsulated diets 337, 345 micronutrient 147, 262, 263, 306, 323, 330, 344, 345, 374, 385–91 microparticulate diets 337, 345–6, 352 micropyle 135, 136, 160, 171, 178, 179, 211, 213, 215

Index

microsatellite 189, 190, 191 microsatellite marker 190, 191 microsporidian 42–3 microvilli 235, 239 midbrain 218, 219 mid-gastrulation 224 mid-gut 234, 236, 239, 241, 246, 339 milt 136, 142, 153, 172, 182, 462 milt ﬂuidity 142 mineral 56, 105, 106, 110, 122, 147, 149, 150, 166, 307, 323, 324, 330, 364, 365, 368, 371, 372, 385–91, 392, 395 mitochondial DNA 189 mitotic cleavage 134 molecular pedigree analysis 189, 191 molecular techniques 41, 193 monoglyceride 239 monosaccharide 383 monounsaturated fatty acids (MUFA) 107, 117, 118, 377, 378, 379, 380, 405, 415 Morone saxitalis 12, 169 morphogenesis 262, 269 morphology 40, 44, 136, 158, 159, 160, 161–4, 171, 174, 175, 204, 209, 225, 456 morula 162, 163, 215, 216 mouth 31, 46, 47, 219, 220, 225, 229, 232, 234, 235, 247, 258, 306, 340, 354, 452, 453, 472 mouth brooding 130, 131 mouth size 258, 354 mucosa 30, 32, 38, 52, 59, 235, 239, 260, 339, 340 multi-trait selection 188 multiple group synchronous ovarian development 137, 138, 141, 144 multiple group synchrony 137 muramyl dipeptides 61 muscle 13, 38, 42, 45, 46, 144, 147, 192, 231, 234, 235, 241, 242, 243, 254, 262, 268, 269, 368, 369, 372, 377, 379, 384, 386, 405, 409, 410, 413–22, 449, 456 muscle ﬁbre 13, 242–4, 268, 419 muscle development 217, 218, 221, 225, 226, 229, 231, 232, 241–4, 268, 269 mussels 129, 143, 472 Mycobacterium sp. 39 myoﬁbril 242, 243, 419, 421 myomere 216, 218, 219, 222, 243

525

myosepta 254, 421, 422 myosin 243, 419, 421 myotome 254, 421 Mytilus edulis 472 myxidium 45 myxosporean spores 45 myxosporidia 44–5 n-3 fatty acids 73, 103, 104, 105, 106–108, 109, 116, 118, 119, 124, 146, 147, 148, 262, 279, 280, 281, 282, 283, 306, 344, 364, 372, 377–83, 418 n-3 HUFA enrichment 73, 76, 81, 106–110, 111, 116–22, 279, 292–3, 329, 331, 473 n-3 HUFA requirements 80, 106, 262, 280–84, 286, 312, 315–19, 322–3, 324, 342, 377–83 n-3 polyunsaturated fatty acids (HUFA) 73, 102–105, 106–108, 110, 111, 113, 116, 117, 118, 119, 121, 122, 123, 147, 148, 262, 279, 280–84, 285, 297, 304, 306, 308, 314, 315, 344, 377–83, 388, 415, 416, 418, 419, 423, 473 n-6 fatty acids 146, 147, 262, 280, 281, 282, 315, 317, 378, 379, 381–3, 416, 417, 419 N-acetylamino (-NHCOCH3) 384 N-acetylglucosamine 384 natural photoperiod 20, 151, 152, 155 natural spawning 142, 152, 153, 433, 446, 468, 475, 512 nature-like systems 285, 287–9, 296, 303, 304, 305 nauplii 19, 31, 111, 112, 113, 115, 116, 117, 121, 122, 124, 173, 241, 258, 287, 288, 289, 293, 298, 299–301, 307–308, 310, 312, 316, 322, 324, 329, 330, 331, 453, 457, 468, 473, 474 Neoceratodus 208 Nereis sp. 472 nerve tissue 147 nest spawners 131 net present value (NPV) 484 net protein utilization (NPU) 367 neural 147, 218, 222, 230, 232, 245, 256, 262, 263, 269, 280, 283, 317, 347, 465 neural arch 245 neural keel 218, 222 neural tissue 147, 262, 280, 283 neuroblast 231

526

Index

neurocranium 244 neuroendocrine 229, 250–56 neurogenesis 231 neurohypophysis 250 neuromast 220, 228, 232–3, 299 neuropathogenic viruses 36 neutral lipase 239 neutral lipids 212, 239, 246, 280, 342, 416 nitrogen metabolism 10 nitrogenous excretion 10 nonguarder 131 nonobligatory plant spawner 130, 131 non-pathogenic 29, 54, 279 non-speciﬁc defense system 33, 34, 256, 269 non-speciﬁc immune stimulation 56, 61, 62, 63, 193 non-speciﬁc immune system 33, 34, 56, 61, 62, 63, 148 non-viable (eggs) 159, 166 noradrenaline 253 normal blastomere 159, 161–5, 171, 172, 174 normal development 148, 167, 174, 224, 245, 262, 263, 264, 265 normal egg 13, 146, 162 normal larva 146, 159, 164 notochord 218, 227, 234, 243, 245, 340 number of eggs in a batch 132, 143, 154 nutrient imbalance 145 nutritional quality 92, 143, 229, 370, 391 nutritional requirement (larvae) 123, 204, 233, 260, 261, 262, 279, 280–85, 286, 315, 347, 512 nutritional requirement (ﬁsh) 422 nutritional value 73, 80, 88, 90, 96, 100, 106, 108, 109, 111, 124, 125, 293, 303, 307, 314–15, 316, 319, 330, 331, 370, 371, 372, 374, 414, 415, 469 obligate pathogens 29, 32, 48 ocean pout 176, 177, 178 ocular side 232, 233, 256 oculovestibular 230 oesophagus 220, 339, 462 oestradiol-17b 139, 140, 141 offspring quality 129, 156 offspring viability 144, 152, 167, 174 oil droplet 134, 146, 159, 160, 171, 211, 212, 248, 456

oil globule 159, 209, 388, 451, 452, 453, 472 olfactory 218, 228, 232, 299 olfactory plate 232 oligochaetes 47 oligopeptides 419 oligosaccharide 374, 383 Oncorhynchus kisutch 12, 169, 338 Oncorhynchus mykiss 9, 149, 169, 338, 400 ontogeny 14, 28, 34, 130, 133, 204–207, 214, 222, 226, 264, 339, 344, 455 oocyte 132, 134, 135, 137, 138, 139, 141, 144, 145, 147, 151, 155, 167, 169, 211, 221, 388 oocyte development 134–5, 137, 138, 139, 141, 143, 148, 388 oocyte growth 134–5, 137, 138, 139, 140, 141, 142, 144, 148 oocyte maturation 134–5, 137, 138, 139, 140, 141, 144, 147, 152, 167, 168, 388 oocyte size 134, 139 oogenesis 134 opportunistic microbes 54 opportunistic pathogens 29 optic nerve 232 optic tectum 232 optic vesicle 218 optical receptor 230 Oreochromis aureus 62 organogenesis 13, 216, 218, 221, 222, 224, 255 Osmerus eperlanus 168 Osmoregulation 14, 16, 229, 247, 255, 342, 407 osmotic balance 16 osmotic regulation 407 ossiﬁcation 207, 222, 229, 244, 245 ostracophil 129 otic vesicle 218 otolith 232 ovarian cycle 134 ovarian development 137, 144 ovarian ﬂuid 135, 155, 156, 158, 167, 168, 170, 171, 172, 179 ovarian ﬂuid pH 167 ovarian follicle cells 134, 139 overripening 143, 153, 154, 157, 158, 167, 168, 169, 170, 171, 172 oviduct 38, 132 oviparous 16, 131, 224, 226 ovoviviparity 130

Index

ovoviviparous 226 ovulated egg 155, 157, 159, 169, 170, 454, 455, 462 ovulation 13, 131, 132, 135, 137, 139, 140, 141, 142, 151, 152, 154, 155, 158, 159, 167, 168, 169, 170, 176, 455, 458, 462, 468 ovulatory period 154 ovulatory rhythm 138 oxidants 389 oxidation 147, 148, 365, 370, 372, 377, 389, 390, 418, 419, 422 oxidative capacity 262 oxygen consumption 7, 9, 10, 168, 179, 249, 250, 406, 466 oxygen requirement 250 oxytetracycline 59, 479 ozonation 58 ozone disinfection 37 Paciﬁc cod 38, 481 Paciﬁc herring 178, 191, 481 Paciﬁc salmon 23, 136, 483 Pagrus major 145, 259, 480 palatine 244 pancreas 36, 38, 228, 234, 235, 236, 237, 240, 250, 263 pancreatic duct 234 pancreatic enzyme 236, 238 paradeontacylix 47 Paralichthys dentatus 241, 341 Paralichthys olivaceus 14, 38, 235, 237, 341 Paralithodes camtschatica 484 Paramoeba pemaquidensis 41 parasitic crustaceans 42, 49, 50 parental care 130, 176, 226 pars distalis 250 pars intermedia 250 pectoral ﬁn 218, 220, 221, 222, 223 pedigree analysis 189 pelagic egg 130, 135, 138, 149, 154, 158, 162, 174, 208, 209, 211, 212, 214, 218, 225, 253, 265, 434 pelagic spawner 129, 130, 131 pelagophil 129, 130, 131 pelvic ﬁn 220, 221 pepsinogen 235, 240, 241 Perca ﬂuviatilis 167 perciform 131, 133, 454

527

periblast 215, 216, 217, 224, 235 periderm 217, 218, 222 peristaltic movement 235 perivitelline space 159, 160, 161, 171, 212, 214, 215, 221, 222 Pﬁesteria piscicida 23 phagocytic activity 34, 224 phenotypic standard deviation 188 phenotypic value 183, 184 phenotypic variance 184, 185, 188 phenylalanine 253, 284, 368, 369 phenylalanine 4-monooxygenase 253 Philasterides dicentrarchi 43 Philopodia 217 phospatidylcholine 281, 379 phosphatase 236 phosphatidylethanolamine 281, 379 phosphatidylinositol 281, 379 phosphatidylserine 379 phospholipase 239, 284 phospholipid 103, 119, 120, 123, 239, 240, 246, 264, 281, 284, 297, 316, 344, 356, 379, 381, 386, 388, 416, 417, 418 photoperiod manipulation 21, 151, 446 photoreceptor 230, 231 physoclistous 245, 246 physostomous 245, 246 phytate phosphorus 388 phytoestrogens 143, 374 phytolithophil 129 phytophil 129 phytoplankton 21, 287, 298, 379 pigment cell 256, 452 pineal function 151 pineal gland 138, 151 pinocytosis 236, 240, 253, 338 Piscicola geometra 49 Piscioodinium spp 44 pituitary gland 138, 250, 255, 263 point-of-no-return 229 polychaete 298, 384 polymerase chain reaction (PCR) 189 polysaccharide 383 polyunsaturated fatty acids (PUFA) 145 porta hepatis 237 postclimax metamorphosis 229 posterior intestine 339, 340 premetamorphosis 229, 248

528

Index

previtellogenic oocytes 137 previtellogenic primary oocyte 134 primary spermatocytes 136 probiotic bacteria 56, 58 prometamorphosis 229, 231, 243, 248 protein efﬁciency ratio (PER) 366 protein productive value (PPV) 367 protein-to-digestible energy ratio (DP : DE) 396 Prymnesium parvum 23 Ptychodiscus brevis 22 r/K-concept 53, 54 relative growth 402 Renibacterium salmoninarum 32 rete mirabile 246 Reynold’s number 242 rhabdovirus VHSV 37 riboﬂavin 105, 342, 386, 395 semelparous 136 shellﬁsh poisoning 22 skeletal deformities 268, 340 Skeletonema costatum 23 soybean protein 374 speciﬁc growth rate (SRG) 403, 406 spinal deformities 245 Streptococcus parauberis 39

thyroid stimulating hormone 250 triacylglycerols (TAG) 377, 415 tocopherol 147, 386, 389, 391 Trichodina sp. 43 triiodothyronine 253 trimethylamine (TMA) 422 trimethylamine oxide (TMAO) 422 Trypanosoma murmarensis 49 trypsinogen 238 vascularisation 242 Vibrio anguillarum 62 Vibrio parahaemolyticus 25 viral haemorrhagic septicaemia 37 vitamin A 105, 149, 245, 264, 386, 387 vitamin B 105, 386 vitamin C 105, 122, 148, 386, 388, 391, 395 vitamin E 105, 147, 148, 342, 386, 388, 389, 395 vitellogenic oocytes 135, 137 viviparity 130 water-soluble vitamins 386, 388 weaning protocol 353, 359 ‘wet’ fertilisation 153 white muscle 242, 410 xanthophore 256

Tenacibaculum ovolyticum 32, 39 testosterone 139, 140, 141 tetraiodothyronine 253 Tetramicra breviﬁlum 42 Tetraselmis algae 453 thiamin deﬁciency 391 thymus 256 thyroid gland 253

yolk plug 217 yolk protein 139 yolk syncytium 235 ytterbium oxides 366 zona pellucida 221 zona radiata 32, 135, 139, 211