Minerals in Animal and Human Nutrition, 2nd Edition

ELSEVIER SCIENCE B.V. Sara Burgerhartstraat 25 P.O. Box 211, 1000 AE Amsterdam, The Netherlands © 2003 Elsevier Science B.V. All rights reserved. This work is protected under copyright by Elsevier Science, and the following terms and conditions apply to its use: Photocopying Single photocopies of single chapters may be made for personal use as allowed by national copyright laws. Permission of the Publisher and payment of a fee is required for all other photocopying, including multiple or systematic copying, copying for advertising or promotional purposes, resale, and all forms of document delivery. Special rates are available for educational institutions that wish to make photocopies for non-profit educational classroom use. Permissions may be sought directly from Elsevier's Health Science Rights Department, Elsevier Inc., 625 Walnut Street, Philadelphia, PA 19106, USA; phone: (+ I) 215 238 7869, fax: (+ I) 215 238 2239, E-mail: healthpermissions@ elsevier.com. You may also complete your request on-line via the Elsevier Science homepage (http://www.elsevier.com). by selecting 'Customer Support' and then 'Obtaining Permissions'. In the USA, users may clear permissions and make payments through the Copyright Clearance Center, Inc., 222 Rosewood Drive, Danvers, MA 01923, USA; phone: (+ I) (978) 7508400, fax: (+ I) (978) 7504744, and in the UK through the Copyright Licensing Agency Rapid Clearance Service (CLARCS), 90 Tottenham Court Road, London WIP OLP, UK; phone: (+44) 207 631 5555; fax: (+44) 207 631 5500. Other countries may have a local reprographic rights agency for payments. Derivative Works Tables of contents may be reproduced for internal circulation, but permission of Elsevier Science is required for external resale or distribution of such material. Permission of the Publisher is required for all other derivative works, including compilations and translations. Electronic Storage or Usage Permission of the Publisher is required to store or use electronically any material contained in this work, including any chapter or part of a chapter. Except as outlined above, no part of this work may be reproduced, stored in a retrieval system or transmitted in any form or by any means, electronic, mechanical, photocopying, recording or otherwise, without prior written permission of the Publisher. Address permissions requests to: Elsevier's Health Science Rights Department, at the phone, fax and e-mail addresses noted above. Notice No responsibility is assumed by the Publisher for any injury and/or damage to persons or property as a matter of products liability, negligence or otherwise, or from any use or operation of any methods, products, instructions or ideas contained in the material herein. Because of rapid advances in the medical sciences, in particular, independent verification of diagnoses and drug dosages should be made. First edition 2003 Library of Congress Cataloging in Publication Data A catalog record from the Library of Congress has been applied for. British Library Cataloguing in Publication Data A catalogue record from the British Library has been applied for. ISBN: 0 444 51367 I

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This book is dedicated, with appreciation, to my parents; my wife, Lorraine; my daughters, Suzannah, Joanna, and Teresa and their husbands and children; and to three great animal nutritionists (Raymond B. Becker, Tony J. Cunha and Jack K. Loosli) at the University of Florida for their practical knowledge of the livestock industry and for encouragement to write books.

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Preface The second edition of Minerals in Animal and Human Nutrition contains under one cover 19 chapters of concise, up-to-date information on mineral nutrition for livestock and poultry, with comparative aspects to laboratory animals and human nutrition. The first chapter is an introduction dealing with historical considerations, distribution in the body, general functions, mineral requirements and tolerances, methods of analysis, mineral bioavailability, status detection, and incidence of mineral deficiencies and toxicities. Chapters 2 through 14 discuss the properties and distribution, metabolism, functions, requirements, natural sources, deficiency, supplementation, and toxicity of the established and most common minerals. Chapter 15 is concerned with five toxic elements and their significance to various species and methods of control. In Chapter 16, chromium and newly discovered essential and other trace elements are discussed. Chapter 17 covers mineral sources, while Chapter 18 deals with maximum tolerance levels. The final chapter discusses mineral supplementation concepts. The present second edition has been completely and vigorously revised. Since the first edition in 1992, a great deal of new information has been generated in the field of minerals; this is reflected by the fact that more than half of all the references in the majority of chapters have been published since the first edition. The new edition continues to provide a large number of classic photographs that illustrate mineral deficiencies and toxicities that have been provided by distinguished scientists in the mineral research field. The purpose of this book is to provide, as both a college textbook and a reference source, a comprehensive text that contains current information on mineral nutrition. Most emphasis is centered on minerals in which naturally occurring deficiencies or excesses are of economic importance. A unique feature of this book is the practical implications of mineral deficiencies and excesses and the conditions under which they might occur in various animal species and humans. It is hoped that this book will be of worldwide use and will continue, as the first edition, to be used as a textbook and as an authoritative reference book for use by research and extension specialists, in the animal, poultry, and veterinary sciences fields and for feed manufacturers, teachers, students, and others. A comparison between the balance of chemical, metabolic, and functional aspects of minerals and their practical and applied considerations has been made. Unlike other textbooks, this one places strong emphasis on practical aspects of mineral supplementation in each chapter and devotes the last chapter to this subject. When preparing the two editions of this book, I obtained numerous suggestions from eminent scientists in both the United States and other countries. I wish to express my sincere appreciation to them and to those who supplied photographs and other material used. I am especially grateful to C. B. Ammerman, J. D. xv

xvi

Preface

Arthington, R. B. Becker, D. K. Beede, G. D. Butcher, G. E. Combs, J. H. Conrad, G. K. Davis, G. L. Ellis, P. Henry, J. F. Hentges, W. E. Kunkle, J. K. Loosli, P. G. Mallonee, R. M. Mason, R. D. Miles, R. O. Myer, W. M. Neal, E. A. Ott, A. L. Shealy, R. L. Shirley, H. D. Wallace, A. C. Warnick, and S. N. Williams (Florida); O. Balbuena and B. J. Carrillo (Argentina); B. Hetzel (Australia); E. Espinosa (Bolivia); N. dos Santos Fernandez, Jiirgen Dobereiner, Francisco Megale, and C. H. Tokarnia (Brazil); T. Ma (China); W. J. Miller and N. W. Neathery (Georgia); M. Anke (Germany); U.S. Garrigus (Illinois); S. P. Arora (India); W. M. Beeson (Indiana); D. V. Catron and V. C. Speer (Iowa); C. W. Absher, J. A. Boling, G. L. Cromwell, V. W. Hays, and D. E. Miksch (Kentucky); J. O. Evans (Kenya); J. Mtimuni (Malawi); G. F. Combs and W. Mertz (Maryland); C. Garcia Bojalil (Mexico); A. T. Forrester, E. R. Miller, and D. E. Ullrey (Michigan); L. E. Carpenter and H. S. Teague (Minnesota); B. O'Dell and R. L. Preston (Missouri); J. Kubota, M. L. Scott, and S. E. Smith (New York); K. M. Hambidge and J. D. Latshaw (Ohio); J. Adair, O. H. Muth, J. E. Oldfield, and F. M. Stout (Oregon); J. Zorrilla-Rios (Panama); R. M. Leach (Pennsylvania); M. Echevarria (Peru); O. E. Olson (South Dakota); B. D. H. Van Niekerk (South Africa); O. M. Mahmoud (Sudan); H. S. Ergun and K. Goksoy (Turkey); A. E. Olson and J. L. Shupe (Utah); J. C. Montero, D. Morillo, and E. A. Velasco (Venezuela); I. A. Dyer, J. W. Kalkus, and R. C. Piper (Washington); G. Bohstedt and M. L. Sunde (Wisconsin); and O. A. Beath (Wyoming). I am particularly grateful to Nancy Wilkinson and Pamela Miles for working on various sections and tables of the book and along with my wife (Lorraine McDowell) for their thorough editing assistance and useful suggestions. I wish to thank Mary Schemear, Shirley Levi, Patricia French and Sabrina Robinson for skillful typing. Finally, I am indebted to the Animal Sciences Department of the University of Florida for providing the opportunity and support for this undertaking. Lee Russell McDowell

xvi

Chapter 1

General Introduction

I. INTRODUCTION

All forms of living matter require inorganic elements, or minerals, for their normal life processes. All animal tissues and all feeds contain inorganic or mineral elements in widely varying amounts and proportions. Some confusion exists in use of the terms "minerals" and "elements" in nutrition and feeding. In practical nutrition, the term "mineral" is generally used to denote all the mineral inorganic elements. However, not all the elements are minerals (i.e., carbon, hydrogen, oxygen, and nitrogen), and minerals frequently found as salts (e.g., carbonates, oxides and sulfates) can be a combination of different inorganic elements. For the purpose of this book, the terms "mineral," "element," and "mineral element" are used interchangeably. The mineral elements are solid, crystalline, chemical elements, which cannot be decomposed or synthesized by ordinary chemical reactions. These inorganic elements constitute the ash that remains after ignition of organic matter. The common method of determining the total mineral or inorganic content of feeds consists merely of measuring the total ash remaining after high-temperature burning of the organic matter. This analysis is of little value either for expressing mineral requirements or for indicating the useful mineral content of foods, for two basic reasons. In the first place, body requirements are specific for certain inorganic elements. Secondly, ash may not be a measure of total inorganic matter present, because some organic carbon may be bound as carbonate and some inorganic elements, such as sulfur (S), selenium (Se), iodine (I), fluorine (F), and even sodium (Na) and chlorine (Cl) may be lost during combustion. In practice, the most important reason for the determination of total ash in a food is to permit calculation of the nitrogen-free extract by difference, as required in the proximate analysis of foodstuffs. Also, the ash analysis can be used in forages to estimate the amount of dust and soil that has been harvested with the feed. II. CLASSIFICATION OF MINERALS

Minerals are classified in a number of ways, with some classification schemes having a place in understanding their requirements and/or nutritional roles. Minerals that are needed in relatively large amounts are referred to as major or 1

General Introduction

2

TABLE 1.1 Essential Mineral Elements Traditional Established Minerals Macrominerals Calcium (Ca) Potassium (K) Sodium (Na) Sulfur (S)

Phosphorus (P) Magnesium (Mg) Chlorine (CI)

Microelements (trace minerals) Copper (Cu) Cobalt (Co) Chromium (Cr) Iron (Fe) Manganese (Mn) Iodine (I) Molybdenum (Mo) Selenium (Se) Zinc (Zn)

Newer Microelements (Discovered since 1970) Aluminum (AI) Boron (B) Fluorine (F) Lead (Pb) Nickel (Ni) Silicon (Si) Vanadium (V)

Arsenic (As) Bromine (Br) Germanium (Ge) Lithium (Li) Rubidium (Rb) Tin (Sn)

macrominerals. Others that are needed in very small amounts are referred to as trace minerals or microminerals. These terms do not imply any lesser role for the trace minerals. Rather, they represent quantity designations based on the amounts required in the diet and their generally low or "trace" concentrations in tissues. The major minerals are required in concentrations of greater than 100 ppm (parts per million) and often as a percentage of the diet (or g per kg), while trace elements are required at less than 100 ppm and are expressed as ppm and sometimes as ppb (parts per billion). Twenty-nine elements are known to be required by at least some animal species (Table 1.1). Seven elements are macrominerals and 22 can be referred to as microminerals or trace elements. The listing of some of the trace elements as essential is difficult and sometimes tentative. An essential element is one that is required to support adequate growth, reproduction, and health throughout the life cycle, when all other nutrients are optimal. Essentiality is less certain when there is only a small change in the rate of growth, when the environment is suboptimal, or when there is a microbial infection (O'Dell and Sunde, 1997). Observed improvements in performance upon supplementation with a mineral may be due to changes in the intestinal microflora, to a pharmacologic effect, or to interactions with other elements. The proof that each element is essential rests upon experiments with one or more species. In these experiments, clinical signs produced by diets adequate in all nutrients, except the mineral in question, have been prevented or overcome by adding that mineral to the diets. All the elements mentioned have not been tested with all species, but it is highly probable that there are few exceptions to the need for all of them by all higher animals. There is no disagreement concerning the essentiali ty of the trace elements chromium (Cr), cobalt (Co), copper (Cu), I, iron (Fe), manganese (Mn), molybdenum (Mo), selenium (Se), and zinc (Zn) although not all would present practical nutritional supplemental problems for livestock or humans.

Classification of Minerals

3

Whether an element is considered essential would depend on the criteria used. A viewpoint in human nutrition is that nutritional requirements should include consideration of the total health effects of nutrients, not just their roles in preventing deficiency pathology (Nielsen, 1996). Therefore, the terms "beneficial element" and "apparent beneficial intake (ABI)" are in use. In humans, for example, the ABI for maximal benefit of F relates to its proven benefits for dental health and its suggested role in maintaining bone integrity. The ABI seems more appropriate for the elements with beneficial, if not essential, actions that can be extrapolated from animals to humans; these elements include, in addition to F, arsenic (As), lithium (Li), nickel (Ni), silicon (Si), and vanadium (V). More recently discovered trace elements since 1970 are referred to as "new trace elements." These newer trace minerals are elements with an established or highly suspected requirement for one or more species, and include aluminum (AI), As, boron (B), bromine (Br), F, germanium (Ge), Li, Ni, lead (Pb), rubidium (Rb), Si, tin (Sn), and V. The essentiality of these last 13 elements is based on growth and other effects with animals under highly specialized conditions, such as improved procedures for purification of diets and use of metal-free isolator systems for raising animals. Furthermore, more precise and accurate methods of determining minute quantities of trace elements have been developed. Although markedly different in their chemistry, mode of action, and effective levels, the newer essential trace elements have in common the facts that they were first known for their toxic effects and that induction of a dietary deficiency is often difficult. An additional 20 to 30 trace elements occur regularly in feeds and animal tissue, and it is unknown whether they serve some useful purpose or are merely incidental contaminants. It is likely using advanced methodology that some of these elements one day will be considered essential. It is also possible that some of the more tentatively established essential elements may be declared non-essential with further studies. Eight mineral elements can also be classified as cations, including calcium (Ca), magnesium (Mg), potassium (K), Na, Fe, Mn, Cu, and Zn. Six other elements are either anions or are usually found in anionic groupings. These are chloride (CI-), iodine (1-), phosphate (PO~), molybdate (MoO;), selenite (SeO) and sulfate S04'. Likewise, they can be classified on the basis of valence number and on their group position in the periodic chart of the elements. These classifications can be useful because they describe physical and chemical attributes of importance in nutrition (Miller, 1979). For example, the monovalent cations, K and Na have a very high absorption percentage and major interrelationships exist between them. In contrast, the absorption percentage of the divalent cations (Ca, Mg and Zn) is much lower. Numerous factors may alter the availability of the essential anions and cations. The most soluble and absorbable form of any of the elements should be the simple ionic state of the atom or ionic group of atoms (for example, as Ca++, Mg++, Mn++). However, many electronegative compounds in nature are looking for a cation with which it can share its electrons, thereby forming a stable compound (Leeson and Summers, 200 I). Often the resultant compound is highly insoluble in water but nevertheless dissociates to a sufficient extent in the intestinal tract to

4

General Introduction

allow absorption of the essential cations. This is influenced by the gastric acidity of hydrochloric acid in the stomach, which converts the cations temporarily into chloride salts, which allows good absorption from the intestinal tract. Therefore, even Mn oxide, Cu sulfide, or Zn oxide, which are highly insoluble chemical compounds, are converted to Mn chloride, Cu chloride, or Zn chloride which are forms more easily absorbed.

III. mSTORY The purpose of this section is to provide an overview of the historical development of knowledge concerning the essential nature of mineral elements as related to deficiency and excess. Table 1.2 summarized chronologically the history of important events relating to the nutrition of mineral elements. Most research accomplishments will not be cited in the Literature Review, and the reader should consult additional reports (i.e., Maynard, 1937; McCollum, 1956; Underwood, 1966, 1981; McCay, 1973; Loosli, 1978, 1991; Georgievskii et al., 1981; McDowell, 1985; Underwood and Mertz, 1987) for a more comprehensive treatment of the subject. Likewise, historical treatment of the various minerals is covered in chapters 2 through 16 of this book. Mineral nutrition of domestic animals was considered to be of limited importance as late as the early 1900s (Ammerman and Goodrich, 1983). Armsby (1880) had concluded in his book, Manual of Cattle Feeding, that "In practice, in the feeding of mature animals intended to be kept in a medium condition, or to be fattened, a lack of the necessary mineral matters is scarcely ever to be feared. They are, indeed, generally in excess. Only common salt is in certain respects, an exception ..." The history of deficiency diseases must date from antiquity. However, before the middle of the 19th century, only the most nebulous ideas existed as to the nature, origin, and functions of the mineral constituents of plant and animal tissues (Underwood, 1981). In 1874, Forster observed that the minerals in the ash of tissues are required to support animal life (McCollum, 1956). This observation helped establish the dietary essentiality of mineral elements. It should be recognized that only when methods were devised to identify and measure mineral elements in body tissues and feeds and to characterize responses to pure elements, was it possible to replace supposition with facts about the essential nature of any nutrient. Much of the earlier knowledge about nutrition resulted from systematic observations stimulated by a need to solve critical health problems with people and their domestic animals. Often, a new scientific discovery has proven to be a confirmation of common beliefs of native people and an explanation of why the beliefs are true (Loosli, 1974). Much information about mineral needs of animals gained by trial and error over centuries was never recorded, and there is no way of learning what was practiced (Loosli, 1978). There is a view that the "fall of Thebes was hastened by heavy livestock mortalities caused by unidentified agents when grazing apparently luxuriant pastures" (Underwood, 1966). There is also the suggestion that part of

History

5

TABLE 1.2 History of Nutritional Importance of Mineral Elements 29 BC 40-120 AD 23-79 AD 1295 1669 Before 1680

1747 1748 1770 1784 1791 1811-1825 1823 1842 1847 1847 1850-1854 1869 1873 1880 1893-1899 1905 1919 1920 1922 1922 1924 1926 1928-1933 1931 1931-1933

The "Fall of Thebes" was hastened by heavy livestock mortalities caused by unidentified agents while grazing luxuriant pastures. Salt fed to domestic animals during the time of Plutarch. Virgil and Pliny recommended salts for milk production. Clinical signs of Se toxicosis were apparently described by Marco Polo as affecting grazing livestock in China. Brand isolated phosphorus from urine. Sydenham treated anemia with iron filings. Menghini found iron in blood. Gahn reported phosphorus present in bones. Scheele reported that bones contain calcium phosphate. Scheele reported sulfur in proteins. Fordyce showed that canaries need "calcareous earth" supplemented to grain diets. Work by Courtois, Coindet, and Boussingault led to the discovery of iodine, the effectiveness of iodine in burnt sponges and specifically that iodine was the only cure for goiter. Proust reported chlorine in the hydrochloric acid in gastric juice. Chossat found pigeons required calcium for bone growth. Liebig reported potassium in animal tissues. Boussingault conducted the first experiment that cattle need common salt. Chatin published studies relating environmental iodine deficiency to incidence of endemic goiter in man and animals. Raulin discovered the essentiality of zinc for the microorganism Aspergillus niger. Von Bunge put forward the hypothesis of antagonism between sodium and potassium and between sodium and chlorine. Forster demonstrated that animals require minerals, and feeding dogs only meat resulted in deficiencies. Von Bunge and Abderhalden showed that young animals receiving milk require supplemental iron. Babcock studied salt requirements of cattle, noting particular importance for lactating cows. Kendall isolated and named thyroxin from thyroid gland; the hormone was found to contain 65% iodine. Bertrand in France and McHargue in the United States initiated the use of purified diets to study the need and function of various minerals Bertrand and Berzon showed zinc was necessary for rat growth and hair development. McCollum and co-workers found that in addition to calcium and phosphorus, rickets is caused by vitamin D deficiency. Theiler and co-workers illustrated phosphorus deficiency for grazing cattle and found that supplementation corrected bone chewing, prevented death loss from botulism. and increased growth and reproductive rates Leroy showed that magnesium increases the growth of mice. Warburg established that respiratory enzymes in animals contain an iron porphyrin group. Neal, Becker and Shealy established copper as an essential element for ruminants. Kemerer and McCollum showed manganese was essential for rats and mice, a deficiency causing tetany. Sjollema related a licking disease in cattle to copper deficiency. (Continued)

General Introduction

6

TABLE 1.2 (Continued)

1935 1935 1935 1936-1937 1937

1938 1938-1942 1940 1946 1948 1950-1954 1953 1954 1955 1957 1958-1959 1959 1970-1997

Franke and Potter identified selenium as the factor in forage responsible for alkali disease in farm animals. Duncan and Huffman observed tetany in calves due to low magnesium content of milk. Underwood and Filmer and, independently, Marston and Lines found that enzootic marasmus in sheep was a cobalt deficiency. Wilgus, Norris and Houser reported that manganese deficiency resulted in a perosis in chicks. Becker and co-workers established that the "salt sick" condition of cattle in Florida (USA) was caused by a combination of pasture deficiencies of cobalt, copper and iron. Bennets and Chapman demonstrated that enzootic ataxia of newborn lambs resulted from ewes receiving insufficient copper during pregnancy. Ferguson, Lewis and Watson showed that molybdenum toxicity resulted in a severe diarrhea for grazing cattle. Hevesy and others began to use radioisotopes to study mineral metabolism. Keilin and Mann reported zinc as a component of the enzyme carbonic anhydrase. Moulton established that small concentrations of fluorine in drinking water prevented dental caries. Rickes and co-workers and, independently, Smith showed that Co is an integral part of vitamin B12 • Dick noted metabolic interrelationships among copper, molybdenum and inorganic sulfates in ruminants. Richert and Westerfield isolated molybdenum from the metalloenzyme xanthine oxidase. Needy and Harbaugh found that high fluorine concentrations in drinking water resulted in mottling of tooth enamel. Tucker and Salmon discovered that parakeratosis, a severe skin disease, was a zinc deficiency for swine. Schwartz and Foltz identified selenium as a factor that prevents liver necrosis in rats. Scott prevented exudative diathesis in poultry with selenium, while Muth, Oldfield, Remmert, McLean, Thompson, Claxton and others prevented white-muscle disease in ruminants with this element. Schwarz and Mertz showed that chromium was essential for glucose metabolism. The most recently discovered elements ("new trace elements") were established using highly purified diets and metal-free isolator systems. These elements included aluminum, arsenic, boron, bromium, fluorine, germanium, lead, lithium, nickel, rubidium, silicon, tin, and vanadium.

'Compiled from a number of sources, including Maynard, 1937; McCollum, 1956; Underwood, 1966, 1981; McCay, 1973; Loosli, 1978; Georgievskii et al., 1981; McDowell, 1985; Underwood and Mertz, 1987.

the reason for the "fall of Rome" was related to infertility caused by Pb toxicosis of the upper class due to use of metal versus clay cooking utensils. Common salt was an item of trade before recorded history to satisfy the salt cravings of grazing animals and for use to flavor foods. Wars were even fought and children were sold into slavery to obtain the precious commodity, salt. Feeding "salts" to domestic animals can be traced to the time of Plutarch (40 to 120 A.D.). Virgil and Pliny (23 to 79 A.D.) recommended salts for milk production. There are many references to the feeding of salt in Britain after 1750 following land enclosure. Phosphorus was isolated from urine in 1669 by Brand, and both Ca and P were shown to be constituents of bone by Gahn in 1748. In 1842, Chossat demonstrated

History

7

that Ca was a necessary supplement to a grain diet for bone development in birds. Many experiments were carried out in Europe and North America during the following 50 years on Ca and P metabolism and requirements as reviewed by Forbes and Keith (1914). After the importance of the Ca: P ratio became recognized, as well as the actual levels of these elements in the diet and the discovery of vitamin D in 1922, it became possible to prevent or cure rickets. This had been a serious disease of children and young animals produced in the winter when they were kept indoors to protect them from the cold, thus also preventing exposure to sunlight (Loosli, 1978; McDowell, 1985). In the early 1800s, mineral constituents of plants were shown to vary with soil type and stage of maturity of forages, and these changes were considered to be important for animals, a view Boussingault later demonstrated to be true. Bone chewing by cattle was recorded in Africa in 1780 and in Paraguay in 1838, but the relation of osteophagia to P deficiency in cattle was not clarified until Theiler et al. (1924) of South Africa published their classical experiments showing that the dried grasses were critically low in P and that P supplements corrected bone chewing, overcame death losses from botulism, and markedly increased growth rates and reproductive levels. Iron was shown to be present in blood by Menghini in 1747. As long ago as 1680, Sydenham is credited with having treated anemia with Fe solutions prepared by steeping steel filings in wine, but he did not know why it was effective. In 1867, Boussingault published data showing the Fe content of a number of different animals and various foods and beverages, since Fe was generally accepted by that date to be an essential element. An excellent example of the usefulness of common practices to alleviate nutritional deficiencies relates to I in South America. In 1824, Alexander Humboldt described goiter in Colombia and stated that native Indians knew of a salt deposit that was an effective remedy, which was not true for other salt sources. A sample taken to France was shown by Boussingault to contain I, which was absent from the other salts. In 1831, Boussingault advised the Colombian government to provide general distribution of the naturally iodized salt to the population. Iodine had been discovered by Courtois in 1811, and nine years later, he began prescribing it as a cure for goiter. Over dosages of I caused injury to some patients, and physicians condemned its use. It was a 100 years later before the general use of iodized salt was started in the United States with medical authority support (McCollum, 1956). In 1926, Leroy showed that Mg increased the growth of mice. McCollum and associates reported in 1931 that deficiency caused tetany in rats, and Duncan and Huffman observed similar signs in 1935 in calves fed milk as the only food. As a result of this discovery, the etiology of grass tetany, which had been described earlier, could now be clarified. Potassium was discovered in 1847, but due to its general abundance in most natural feeds, it is only since the 1960s that K deficiencies have been reported for livestock under practical conditions. Except for Fe and I, the trace elements were not studied until about 1928. Copper was shown to be essential for hemoglobin formation and Fe utilization in rats by

8

General Introduction

Hart and Elvehjem in 1928. Following this discovery, there was more than a decade of intensive research interest, which resulted in the discovery of other essential elements. Sjollema reported Cu deficiency in cattle and sheep in the Netherlands in 1933. Bennets and Chapman reported that enzootic ataxia of newborn lambs was Cu deficiency. Molybdenum was found to cause a severe diarrhea in cattle in 1938 by Ferguson and co-workers, with Dick in the early 1950s establishing the metabolic interrelationships among Cu, Mo, and inorganic sulfate for ruminants. Cobalt deficiency was first identified as the cause of a "wasting disease" and anemia in cattle and sheep in Australia in 1935 by Filmer and Underwood, and Marston and Lines. From Florida (USA), the first report of a Co deficiency in cattle was in 1937, associated with a condition known as "salt sick" (Becker et al., 1965). The "salt sick" condition in Florida was prevented only if all three of the deficient elements, Co, Cu, and Fe, were supplied in adequate quantities. Previously, in Florida Cu was established as essential to ruminants (Neal et al., 1931). Manganese stimulated growth of mice and reproduction of rats in studies by Kemerer and McCollum in 1931. In 1937, Wilgus and co-workers showed that chick perosis was caused by Mn deficiency, and in the same year, Lyons and Insko found that chondrodystrophy in chicks was caused by lack of Mn in the diets of laying hens. However, the importance of Mn for cattle was shown much later, in the early 1950s. A similar situation existed with Zn, which was shown to be essential for growth and hair development of mice and rats in 1922, but its need by pigs was not shown until 1955 and for calves not until 1957. Zinc deficiency for grazing cattle was reported in 1960 by Legg and Sears. In 1954, Neely and Harbaugh found that mottling of tooth enamel, a disease affecting humans and livestock, was produced by high F concentrations in drinking water. Evidence that F is essential for livestock is limited to recent reports showing that a deficiency causes skeletal abnormalities in female goats and poor growth in their offspring after 10 generations of low F diets (Anke et al., 1997b). Earlier tests demonstrated that traces in the drinking water (0.7 to 1.0 ppm) help to prevent dental cavities in children, but 2.0 ppm or more causes mottling of the teeth enamel. Higher intakes (4 to 6 ppm in water) increase bone density and help to prevent osteoporosis and associated incidence of collapsed or distorted vertebrae in people over 55 years of age. There is also evidence that extra F decreases the aorta calcification often seen in low-F areas. However, it is the toxicity of F that is of greater importance in animal nutrition (Shupe et al., 1974). Marco Polo apparently described clinical signs due to Se toxicosis while traveling in China late in the 13th century. It was not until 1935 that Franke and Potter identified the toxic principle causing "alkali disease" and "blind staggers" in grazing animals was due to excess Se. In 1957, Se was shown to be essential by preventing liver necrosis in rats. The following year Se was shown to prevent exudative diathesis in poultry and white muscle disease in young ruminants. Shortly after this, Se in combination with vitamin E, was shown to prevent certain specific muscular dystrophy conditions and other forms of tissue degeneration in a large number of species. Up to the early 1950s 13 minerals had been identified as essential; these being the major elements Ca, P, K, Na, CI, Sand Mg, and the trace (micro) elements;

Mineral Distribution in Body

9

Fe, I, Cu, Mn, Zn and Co. By 1959, Mo, Se, and Cr had been added to this list, as well as beneficial aspects for supplemental F for humans. Starting in the 1970s, 13 additional elements were shown to be required for animals. Most of these elements were known only for their toxic properties. The "new trace elements" were discovered using highly purified diets and metal-free isolator systems. To date, with few exceptions, these newer elements have not been shown to be essential for livestock or humans that are consuming typical diets. One of the latest elements to be established as essential is rubidium (Rb). Female goats fed < 280 ppb (~g/kg) Rb had abortions, lower birth weight and increased mortality among kids (Anke et al., 1997a).

IV. MINERAL DISTRIBUTION IN BODY It should be noted that 96% of body weight consists of the four organically bound elements (carbon, hydrogen, oxygen, and nitrogen). The principal cations and anions together account for 3.5% of body weight, the remainder comprising additional elements (Table 1.3). The percentages of the macromineral constituents of the body are indicated by the following data showing the average analyses of 18 steers of varying ages exclusive of the contents of the digestive tract (Hogan and Nierman, 1927). Element Calcium Phosphorus Potassium Sodium Sulfur Chlorine Magnesium

Percent

1.33 0.74 0.19 0.16 0.15 O.ll 0.04

Typically Ca represents about 46% and P about 29% of total body minerals. Potassium, S, Na, Cl and Mg together account for about 25%, while essential trace elements constitute less than 0.3% of the total. Mineral distribution within the body's tissues is not uniform, since some tissues selectively concentrate specific elements. However, the proportions of each mineral, expressed as amount of fatfree dry body substance, are very similar among species in adult mammals and poultry (Scott et al., 1982). Each organ, in accordance with its function, has a characteristic mineral composition, which again is very similar in all mammals. However, after a period of undernutrition or water deprivation, there is quite a sharp rise in the mineral content [fat-free dry matter (OM)]. It should be noted that the Na, K, and Cl concentrations of the body are constant during all stages of development from embryo to full

General Introduction

10

TABLE 1.3

Elemental Composition of Human Body" Element Oxygen Carbon Hydrogen Nitrogen

Percent 65.00 18.0

10.0 3.0

Macrominerals Calcium Phosphorus Potassium Sulfur Sodium Chlorine Magnesium

1.5 1.0 0.35 0.25 0.15 0.15 0.05

Trace Minerals Iron Zinc Manganese Copper Iodine

0.004 0.003 0.0003 0.0002 0.00004

"The major elements oxygen, carbon, hydrogen and nitrogen comprise 96% of the body; macrominerals are 3.45% and trace and other minerals, 0.55%.

development, whereas the Mg, Ca and P contents in the embryo are only one half of the respective concentration in the adult animal. Bone is the primary storage site for many of the essential elements. Between 80% and 85% of the total body mineral matter, or ash, of the body is located in the skeletal tissues and consists mainly of salts of Ca, P and Mg. Thus, 99% of the total Ca, 80 to 85% of P and some 70% of Mg occur in bone (Underwood, 1981). The thyroid gland is the most specific storage site for I, and no less than 80% of the total body I is normally found there. In contrast to Ca, P, and Mg in bone, I in thyroid tissue, and Co as part of vitamin B12, most minerals are distributed more evenly throughout the body where they exist in a variety of functional combinations and in characteristic concentrations. These elements must be maintained within quite narrow limits if the functional and structural integrity of the tissues is to be safeguarded, and health and production optimized.

V. GENERAL FUNCfIONS OF MINERALS

Unlike other nutrients; mineral elements cannot be synthesized by living organisms. Minerals have four broad functions: structural, physiological, catalytic, and hormonal or regulatory. The most obvious function of mineral elements in the body is to provide structural support (skeleton). Bone is formed through the

General Functions of Minerals

11

deposition of Ca and P as hydroxyapatite into a protein matrix. Calcium, P, Mg, F, and Si in bones and teeth all contribute to the mechanical stability. Another example of structural function is the use of Ca by birds to produce eggshells. The presence of P and S in muscle proteins further illustrates the function of structural components of body tissue for these minerals. Minerals such as Zn and P can also contribute structural stability to the molecules and membranes of which they are part. Only small fractions of the Ca, Mg, and P, and most of the Na, K, and CI are present as electrolytes in the body fluids and soft tissues. Electrolytes present in body fluids, such as blood or cerebrospinal fluid, serve important functions in maintaining acid-base and water balance, and osmotic pressure; they regulate membrane permeability and exert characteristic effects on the exitability of muscles and nerves. For example, a certain balance between Ca, Na, and K in the fluid which bathes the heart muscle is essential for the normal relaxation and contraction that constitute its beating. Also, profound disturbances in neuromuscular function arise in the animal when the levels of Ca and Mg in the blood plasma fall below certain limits. Calcium is also directly involved in the coagulation of blood. Mineral salts are sometimes fed to dairy and feedlot cattle beyond the established requirement because of their role as buffers. Buffers have been reported to improve feed intake, milk production, milk composition, and animal health (Rogers et al., 1982). Mineral salts used as buffers function to control the excess hydrogen ion concentration in the rumen, intestines, tissues and body fluids, or increase the rate of passage of liquids from the rumen, or both (NRC, 2001). In addition to its bone function, P participates in a multiplicity of metabolic reactions involving energy transfer. Phosphorus also is an integral part of the nucleic acids. In addition to P, several trace metals, such as Fe, Cr, Ni, Mn, and Zn are components of ribonucleic acid (RNA), the compound vital to all protein synthesis and, therefore, to life itself (Underwood, 1981). Essential trace elements are integral components of certain enzymes and of other biologically important compounds, such as Se in glutathione peroxidase, Fe in hemoglobin, Co in vitamin B12 and I in the thyroid hormones thyroxine and triiodothyronine. Also, the insulin molecule contains both Zn and S. Certain minerals have regulatory functions in that they exert some control on cell replication and differentiation: Ca, for example, influences signal transduction and Zn influences transcription, adding to long-established regulatory roles, such as that of the element I as a constituent of the thyroid hormones (Underwood and Suttle, 1999). Functions of minerals are interrelated and balanced against each other and most often cannot be considered as single elements with independent and self-sufficient roles in the organized bodily processes. The definite relationship of Ca and P in the formation of bones and teeth and the interrelationships of Fe, Cu, and Co (in vitamin B12) in hemoglobin synthesis and red blood cell formation serve as examples. Sodium, K, Ca, P, and CI serve individually and collectively in the body fluids. A number of trace elements (e.g., Cu, Zn, Fe, and Se), in addition to certain

12

General Introduction

vitamins (e.g., vitamins A, D, E, B6, and folacin) and other nutrients, are strongly related to adequate immune response. These nutrients act together and/or separately for different components of an active immune response.

VI. MINERAL REQUIREMENTS AND TOLERANCES It is not the purpose of this chapter to provide detailed information on mineral requirements for each area of livestock production. The two main sources of information on mineral requirements for various species are the U.S. National Research Council's (NRC) "Nutrient Requirements" series and the British (ARC) "Nutrient Requirements of Farm Livestock" series. For human mineral requirements, the latest edition of "Recommended Dietary Allowances" (RDA) and "Dietary Reference Intakes" (2001) should be consulted. The NRC publication "Mineral Tolerance of Domestic Animals" provides suggested toxic levels of minerals for livestock (NRC, 1980). Mineral requirements and tolerances for livestock and humans for specific minerals are presented in the present book in chapters 2 through 16 and in the appendix tables. Likewise, Chapter 18 deals with mineral tolerances for livestock. Mineral requirements are generally expressed in several ways, in amounts per day or per unit of product, such as milk or eggs, or in proportions of the dry matter of the diet consumed. The former method is more precise, but expressing minerals as proportions of diet dry matter has obvious practical advantages. The requirements are meant to define the lower limits of adequacy in each case and are arrived at by relating the growth, health, production, or other relevant criteria in the animal with varying dietary mineral concentrations. Dietary recommendations can be stated as ranges, rather than as single figures of intake. The statement of a range can take into account the differences of intake that are required to meet the requirement when supplied by typical diets of different bioavailability. That presentation takes into account the homeostatic regulation of higher organisms that tends to buffer marginally deficient or marginally excessive intake by changing the efficiency of absorption and excretion (Underwood and Mertz, 1987). The actual amount of mineral in the diet may also influence utilization. For example, if the diet contains more Ca than required, homeostatic mechanisms are brought into play with the efficiency of absorption being decreased. The mineral status of the animal may also influence absorption. A Fe-deficient animal is more efficient in the absorption of Fe than an animal with adequate Fe stores. Minimum mineral intakes must be sufficient to ensure the long-term maintenance of the mineral reserves of the body tissues and the amounts of those minerals in the edible products of the animal. Through homeostatic mechanisms, the animal body has the capacity to make some adjustment to suboptimal intakes by reducing the amount of the mineral in its products. As an example, most trace minerals are substantially reduced in milk when dietary intakes are low. However, for

Mineral Requirements and Tolerances

13

macrominerals such as Ca, P, Na, and K the concentration in milk remains constant so that these minerals are spared only by reducing milk production. To conserve Ca for egg-laying poultry, the shell strength can be reduced in order to maintain production. Therefore, the assessment of mineral needs has come to include determination of the minerals in the tissues, fluids and products, as well as such gross criteria as weight gains, milk yields and so on. Many factors affect mineral requirements, including kind and level of production, age, level and chemical form of elements, interrelationships with other nutrients, mineral intake, breed and animal adaptation. Mineral requirements are highly dependent on the level of productivity. Highproducing dairy cows require much more dietary Ca and P than low-yielding cows because of the richness of milk in those elements. However, the necessary percentages in the diet do not rise to the expected extent because total dry matter intakes increase with rising productivity of the cow almost as rapidly as do mineral requirements. The P requirements of laying hens tend to follow a similar pattern with increasing egg production but those of Ca do not. For example, a non-laying hen can normally meet its Ca needs from a diet containing 0.2 to 0.3% Ca on a DM basis, whereas some 8 to 10 times this concentration is necessary for a hen approaching maximum egg production (Underwood, 1981). Improved practices that lead to improved milk, egg and wool production and growth rates for poultry and livestock will necessitate more attention to mineral nutrition. Mineral deficiencies, often marginal under low levels of production, are likely to become important, and previously unsuspected nutritional deficiency signs may occur as production level increases (Long et al., 1969; Thornton et aI., 1969; Underwood, 1981). Mineral requirements vary also with the criteria of adequacy employed. As the amount of a mineral available to the animal becomes deficient as a result of inadequate intake or depletion of body reserves, certain processes fail in the competition for the inadequate supply. The priority of demand exerted by these processes for the mineral vary among different animal species and within species, with the age of the animal and the rapidity with which the deficiency develops (Underwood, 1981). For instance, in sheep, the processes of pigmentation and keratinization of wool appear to be the first to be affected by a low Cu status. Thus, if wool quality is taken as the criterion of adequacy, the Cu requirement of the sheep is higher than if growth rate is used as criteria. The criterion of adequacy is also important, as illustrated by the fact that minimum Zn requirements for spermatogenesis and testicular development in male sheep are higher than for growth (Underwood and Somers, 1969), and Mn requirement is similarly lower for growth than for fertility (Underwood, 1981). Important differences in mineral metabolism can be attributed to breed and to individual animal variation. This can be illustrated by only a certain percentage of growing pigs suffering from tissue degeneration after being fed the same Se-vitamin E deficient diets (McDowell et al., 1974). Likewise, the l-deficient clinical sign of goiter in calves and skin lesions of Zn-deficient cattle are sometimes seen in a small percentage of animals, while most animals in the herd appear normal

14

General Introduction

Fig. 1.I Parakeratosis from Zn deficiency showing genetic variation. Above: Pigs showing varying degrees of parakeratosis from Zn deficiency. Below: Same pigs after 16 days of receiving a diet containing 40 ppm of Zn. (Courtesy of V.W. Hays and V.C. Speer, Iowa State University, Ames)

(McDowell, 1985). Fig. 1.1 illustrates three pigs that had received a Zn-deficient diet, with skin lesions varying in pigs from severe to completely unaffected. Adequate intake of feed by animals is essential in meeting mineral requirements. For example, factors which greatly reduce forage intake, such as low protein « 7.0%) content and increased degree of lignification, likewise reduce the total minerals consumed by grazing animals. Information concerning the toxicity or tolerance of minerals is incomplete (NRC, 1980). According to available information, the toxic level of most major minerals is about 4 to 10 times the recommended level for young, growing livestock. The toxic levels of trace minerals appear to be highly variable, ranging between 4 and 1500

Methods of Mineral Analyses

15

times the recommended level (See Chapter 18). As was true with mineral requirements, a series of "safe" dietary levels of potentially toxic elements has been established, depending on the extent to which other elements that affect their absorption and retention are present (NRC, 1980).

VII. METHODS OF MINERAL ANALYSES Research findings with minerals have been greatly facilitated by the development of several sophisticated, powerful, analytical tools. Atomic absorption spectrophotometry has been especially helpful, since its cost is within the budget of most laboratories (See Fig. 1.2). Other methods, such as emission spectrometry and neutron activation analysis, have many advantages, although equipment needed is expensive. These methods have resulted in a marked reduction in time and labor required for analyses, thus encouraging scientists to carry out more complete programs of experimentation. More importantly, these analytical techniques have increased both sensitivity and accuracy of mineral analysis.

A. Sampling for Mineral Analyses Due to very low levels of most trace elements in biological samples, there is a high potential for gross contamination during sampling, storage, handling, and analysis. Precautions should be taken to use non-metal sampling devices, tools, and containers. Plastics are recommended, with Teflon and polyethylene being the most popular. The use of certified standards will quickly identify most errors but not necessarily those from laboratory milling, because the standard comes ready-milled (Underwood and Suttle, 1999). Significant levels of trace elements can be found in reagents, air, hair, skin, clothing, etc. The analyst must evaluate and eliminate potential sources of contamination in every step of the analytical methodology.

B. Analytical Techniques In an extensive world survey, Iyengar and Woittiez (1988) indicated that both atomic absorption spectrophotometry (AAS) and neutron activation analysis (NAA) are the most frequently used analytical techniques. Other techniques such as inductively coupled plasma optical emission spectroscopy (ICP-oES), x-ray fluorescence (XRF), isotopic dilution mass spectrometry (lDMS), proton induced x-ray emission (PIXE), and near infrared reflectance spectroscopy (NIRS) have been used less extensively. 1. ATOMIC ABSORPTION SPECTROPHOTOMETRY (AAS)

This is a very specific technique with few interferences, has good sensitivity and precision and is relatively low cost. The main drawbacks are limited linear calibration range and the fact that it is a single-element technique.

16

General Introduction

Fig. 1.2 Sample preparation and analysis. The upper photograph illustrates a wet digestion technique. The most widely used method of mineral analysis utilizes flame atomic absorption spectrophotometry (lower photo) (University of Florida).

The element to be analyzed is introduced into a flame where it becomes dissociated from its chemical bonds into an unexcited, un-ionized ground state as individual atoms. The element in this state is capable of absorbing radiation at discrete lines of narrow wavelength. When a light beam at one of these wavelengths is directed through the flame, the amount of this light absorbed as it passes through the flame is proportional to the concentration of the element being analyzed.

Methods of Mineral Analyses

17

Two mam types of atomization sources are used: flame and graphite furnace (GF-AAS). The graphite furnace improves the sensitivity of atomic absorption. With this device many elements can be determined at concentrations 1000 times lower than what can be detected by flame atomic absorption. This technique can be used in situations where only a small amount of sample is available. 2. NEUTRON ACTIVATION ANALYSIS (NAA)

The NAA procedure is a multi-element technique, but is almost exclusively limited to centers of analytical expertise due to the radiochemical techniques involved. This technique bombards the sample with neutrons so that the elements present become radioactive and can then be quantitatively detected. 3. INDUCTIVELY COUPLED PLASMA OPTICAL EMISSION SPECTROSCOPY (ICP-DES)

This is a technique using inductively coupled plasma generators as an atomization source for optical emission spectrometry. It has a high analytical sensitivity for about 70 elements with detection limits frequently in the ppb (ngjml) range, and calibration graphs rectilinear over 5 orders of magnitude with respect to analyte concentration (Dean et al., 1989). Some advantages of ICP-OES technology are the high temperature (5500 to 8000 K) that allows for the complete ionization of elements; therefore, minimizing chemical interferences, a wider linear working range, and detection limits lower than flame AAS, but poorer than GF-AAS. One disadvantage of ICP-OES is that it is less precise with a coefficient of variation of 2 to 3% versus 0.3% for flame AAS. 0

4. INDUCTIVELY COUPLED PLASMA MASS SPECTROMETRY (ICP-MS)

The coupling of an ICP with a mass spectrometric detector was achieved by Houk et al. (1980). It is a particularly promising method because it allows not only for total metal concentrations to be determined, but also for isotopic information to be elucidated. This ability allows the technique of isotope dilution analysis (IDA) to be applied and tracer studies to be performed using stable nonradioactive isotopes (Dean et al., 1989). ICP-MS has a similar sensitivity to AAS and GF-AAS and requires less sample preparation time and in the case of GF-AAS, less analysis time. 5. ISOTOPE DILUTION MASS SPECTROMETRY (ID-MS)

This procedure is one of the most widely used definitive methods, and is the only mass spectrometric method which produces highly accurate analytical results for a great number of elements (Heumann, 1985). In 10 analysis, the sample is treated with an accurately known amount of a stable enriched isotope, for example, the 206Pb isotope. From a knowledge of the isotope ratios in the fortified and unfortified samples and the degree of fortification, the amount of analyte in the sample can be calculated (Dean et al., 1989).

General Introduction

18

6. X-RAY FLUORESCENCE SPECTROMETRY (XRF) A more recent development in mineral analyses has been x-ray fluorescence. Used commonly in the mining industry, this technique seems to have great potential for rapid assay of minerals in feed, following minimal sample preparation (Valdes and Leeson, 1990). This procedure provides the means for the identification of an element by measurement of its characteristic x-ray emission wavelength or energy. The method allows the quantification of a given element by first measuring the emitted characteristic line intensity and then relating this intensity to elemental concentration. Today nearly all x-ray spectrometers use the fluorescence excitation method and employ a sealed x-ray tube as the primary excitation source (Jenkins, 1988). 7. NEAR INFRARED REFLECTANCE SPECTROSCOPY (NIRS) The use of NIRS for determining simple components in grains and oilseeds has been used routinely over the last few years. This procedure quickly became an ideal laboratory technique because it is very fast, inexpensive, there is no sample preparation except grinding, and the technique does not require trained people after the calibrations are developed. Several nutrients in a sample can also be analyzed simultaneously (Leeson and Summers, 2001). Near infrared reflectance spectroscopy (NIRS) can provide quick, nondestructive and quantitative analyses of an enormous range of organic and inorganic constituents of homogeneous plant and animal tissues. Near infrared spectra depend on the number and type of C-H, N-H, and 0-H bonds in the material being analyzed. The spectral features are then combined with reliable compositional or functional analyses of the material in a predictive statistical model. This model is then used to predict the composition of new or unknown samples. The accuracy and precision of the reference values for the calibration data set in part determines the quality of the predictions made by NIRS. However, NIRS analyses are often more precise than standard laboratory assays. 8. CONVENTIONAL METHODS For analysis of feeds for Ca, P, Na, K, Cl, Mg, Zn, and Mn, conventional methods (i.e., NFIA, 1991) have been in routine use in feed laboratories for many years. These methods are fully described in the official methods of analysis of the Association of Official Analytical Chemists (AOAC, 1997). Whetter and Ullrey (1978) developed an improved method for determining Se by a fluorometric procedure that is sensitive and accurate. No single technique is applicable to all trace element analyses. There are several factors to consider when choosing methodology and instrumentation: 1. 2. 3. 4.

the requirement for single-element or multi-element analysis, type (matrix) of biological samples and potential interferences, number of samples and time required per sample (automated or manual), sample size required,

Methods for Estimating Mineral Bioavailability and Requirements

19

5. availability, size, cost, ease of operation, and service of instrumentation, 6. skill, training, and experience of available personnel, and 7. whether the instrumentation will be shared with other laboratories and/or used by different personnel (WHO, 1996; Miles et al., 2001). C. Sample Digestion Analytical techniques such as NIRS, NAA, and XRF can sometimes be used with minimal sample preparation; however, atomic spectrometric techniques require a pretreatment to remove organic matter. This is usually accomplished by either a dry ashing or wet oxidation-digestion. High-temperature dry ashing consists of placing the sample into a suitable dish, drying the sample, and placing it into a muffle furnace. Often ashing aids and/or posttreatment with small amounts of acid are required to destroy the organic material completely (Miles et al., 2001). Wet digestion is usually faster than dry ashing and can be set up to do a number of digestions simultaneously (Fig. 1.2). Acids and reagents used must be of the highest purity, since large amounts are sometimes required. For ultratrace analysis, the control of the blank is often the determinant factor in the level of analysis that can be performed. The analyst must ascertain the purity of each batch of acid or reagents to be used. This is especially true if the acids are shipped or stored for any length of time in glass bottles.

D. Calibration Standards Atomic spectroscopic analytical methods determine the concentration of samples by comparing their analytical signals to those of a series of calibration standards. The analytical determinations are only as accurate as the standards. Matrix matching is the most accurate but is time-consuming and requires detailed knowledge of the sample matrix. Dilution of the samples and standards into a common matrix can eliminate the most obvious interferences and is suitable for large numbers of samples. Every analytical method must be validated for accuracy. This is best done by verifying results using a standard reference material (SRM) of the same composition as the samples to be analyzed. The SRM are available from the National Institute of Standard and Technology (U.S.), the International Atomic Energy Agency (Vienna, Austria), Community Bureau of Reference (Brussels), National Institute for Environmental Studies (Tsukuba), World Health Organization (Solna). They come with a certificate, which gives reference values of components plus confidence levels. VIII. METHODS FOR ESTIMATING MINERAL BIOAVAILABILITY

AND REQUIREMENTS Not all sources of elements are created equally. Different sources of the same element can vary from zero availability to a highly available element. Ammerman

General Introduction

20

(1995) has reviewed the most suitable methods for determining mineral bioavailability.

A. Absorption and Chemical Balance bsornti intake - total fecal excretion 100 A pparent a sorption = . k x mta e Digestion and absorption of mineral elements by an animal provides an estimate of its bioavailability. It is assumed that once a mineral is absorbed from the gastrointestinal tract, it is available for physiological processes or for storage. Absorption, however, cannot always be equated to bioavailability. For example, I in the form of 3,5-diiodosalicyclic acid was shown to be well utilized by rats but was an ineffective source of I for cattle (Miller et al., 1965). The I-containing compound was readily absorbed by both species, but cattle had a very limited capacity to remove the mineral from the organic part of the molecule. Absorption studies of several days in length in which intake and fecal excretion is measured have often been carried out with the macrominerals Ca, P, and Mg. Very few absorption studies have been carried out with microelements due to the large errors resulting from even the slightest contamination and unknown endogenous sources. 1.

ApPARENT ABSORPTION

Apparent absorption is used in the evaluation of sources of certain mineral elements and is defined as total intake minus total fecal excretion of the element. Values are usually expressed as a percentage of intake. The difference between intake and excretion represents net disappearance of the element from the gastrointestinal tract and does not correct for the portion of the element present in feces that resulted either from abrasion of mucosal cells or from excretion of the element back into the gastrointestinal trace (Ammerman, 1995). Apparent absorption is of limited value for elements where feces is the major pathway of excretion (e.g., Ca, P, Zn, Mn, and Cu). 2.

TRUE ABSORPTION

. T rue a b sorption

= (intake -

tot.fecal exc. - tot.endogenous fecal exc.) 100 . k x mta e

True absorption corrects for the portion of the element which has been absorbed into the animal's body and subsequently is excreted back into the gastrointestinal tract. This portion of the total fecal excretion can be designated as "total endogenous fecal excretion." Minimum endogenous fecal loss represents the minimal or inevitable loss from the animal's body (ARC, 1980). True absorption represents total intake minus total fecal excretion (tot. fecal exc.) from which total endogenous fecal excretion has been subtracted.

Methods for Estimating Mineral Bioavailability and Requirements

21

The value for true absorption is greater than that for apparent absorption and is a more valid estimate of the amount of an element available for physiological purposes. Total endogenous fecal excretion can be estimated by use of appropriate radioisotopes (Underwood, 1981). 3.

URINARY EXCRETION

Urine is the major pathway for Mg, I, and K excretion, but minor for Mn, Fe, Zn, and Cu. Urinary excretion can be a useful indicator of absorption for Mg and K and other elements with similar excretion characteristics. 4.

NET RETENTION

Net retention is defined as total intake minus total excretion (total fecal plus total urinary) of the mineral. Collection of urine during absorption studies allows net retention to be calculated. Although this information may be useful in interpreting results, net retention probably has limited value in determining bioavailability of a mineral. In many situations, the mineral excreted in the urine represents a portion that was potentially nutritionally effective and that has been involved in, or was available for use in metabolism (Ammerman, 1995).

B. Growth and Specific Tissue Response 1. GROWTH

Growth response resulting from lack of or adequacy of a specific element is often used to establish the need and requirement for a particular mineral. A disadvantage of growth rate assays lies in the fact that, for many elements, the method requires use of semipurified diets, which increases cost and which also may yield results not entirely applicable when practical diets containing natural ingredients are fed (Ammerman, 1995). The young chick is an ideal assay animal because of (a) limited nutrient stores; (b) lack of or minimal coprophagy; (c) rapid rate of growth and; (d) high nutrient demand. 2.

BONE DEVELOPMENT

Bone development, as usually measured by bone ash response in the very young chicken, has been considered for years as one of the most critical tests for estimating bioavailability of Ca and P compounds, as well as vitamin D. In general, the bone of choice has been the tibia, and bone ash has been expressed as either total tibial ash or as tibial ash concentration of the dry, fat-free bone. Bone ash and bone breaking strength (force required to fracture the bone) have also been used widely in swine for both Ca and P. Bones used most commonly are the metacarpals and metatarsals. Stone and Mcintosh (1977) reported that femur breaking strength in pigs was a much more sensitive indicator of skeletal development and susceptibility to bone fracture than was bone ash or dimensional characteristics. Williams et al.

General Introduction

22

(1991) demonstrated that chemical, physical, and mechanical properties of bone could be used to evaluate the P status of cattle. Noninvasive bone techniques such as dual photon absorptiometry, radiographic photometry, and ultrasound can be used to estimate bone mineral content and bone strength and thus status of P and Ca (Williams et al., 1991). 3. ESSENTIAL COMPOUNDS OR ENZYMES

Functional assays for bioavailability in which the mineral element is necessary for an essential compound have been used. Cobalt is needed for vitamin B12 production as Fe is required for hemoglobin synthesis. The enzymes glutathione peroxidase and cytochrome C oxidase are influenced by Se and Cu, respectively. Iodine is an essential component of the thyroid hormones (i.e., thyroxin and triiodothyronine). 4.

TISSUE ACCUMULAnON

Accumulation of specific mineral elements in various target organs (e.g., liver, bone) has been used for many years as a response criterion. It was reported by Watson et al. (1970) that bone Mn concentrations in chicks fed with semipurified diets were more directly related to dietary concentrations of the element than were growth rate or leg development. For evaluation of P status in ruminants, a rib biopsy procedure is available (McDowell, 1997). Trace element concentrations of Cu, Co, and Se in liver tissue (e.g., biopsy) has been used to evaluate the status of these elements for ruminants in many tropical countries (McDowell, 1997, 1999). Biological availability of several microelements for ruminants and poultry have been estimated by tissue uptake following high dietary levels, and short-term supplementation (Henry et aI., 1986). The advantages of this method are fewer animals are required to test for significant differences, due to higher dietary levels and there is no need for purified diets. A disadvantage with this method is that homeostasis mechanisms are not considered. As an example, an animal often will utilize a nutrient much more efficiently at a deficiency or low status level than when the nutrient is in excess. 5.

USE OF ISOTOPES

Accumulation of radioactive or stable isotopes in target organs can be used to estimate absorption. Reviews of methods for assessment of mineral utilization in humans and laboratory animals, including the use of stable isotopes and intrinsic and extrinsic labeling with radioisotopes, are available (O'Dell, 1984).

IX. DETECTION OF MINERAL STATUS

The detection of mineral element deficiencies or excesses involves clinical, pathological, and analytical criteria as well as response from specific element

Incidence of Mineral Deficiencies and Toxicities

23

supplementation. Clinical signs of mineral deficiencies along with soil, water, plant, and animal tissue analyses have all been used with varying degrees of success to establish mineral deficiencies and toxicities (McDowell, 1985, 1997; Mills, 1987; Suttle, 1988; Underwood and Suttle, 1999). Since mineral analyses are complicated and expensive, it is important to select and analyze the minimum number of plant and animal tissues (or fluids) that are most indicative of mineral status in animals. Methods of diagnosis of mineral deficiencies or toxicities and appropriate critical levels have been reviewed (NCMN, 1973; Miller and Stake, 1974; Egan, 1975; Underwood, 1979, 1981; McDowell, 1985, 1997, 1999). The publication "Minerals for Grazing Ruminants in Tropical Regions" lists analyses of considerable value and critical levels for assessment of mineral status of ruminants (McDowell, 1997). The most reliable method to confirm mineral deficiencies is response derived from specific mineral supplementation. However, supplementation studies are costly in time and resources if conducted with adequate control and assessment. For several decades, a major goal in mineral research has been to discover and/or develop simple and accurate biochemical measurements of the status of animals for the minerals in which there are important practical problems (Miller and Stake, 1974). Like soils and plants, animal tissue mineral concentrations are influenced by many factors. Nevertheless, when appropriate interpretation is made, animal tissue concentrations are often better indicators of the mineral status of livestock than either plant or soil concentrations (McDowell, 1976, 1985, 1997, 1999). When the evidence obtained from clinical, pathological, and biochemical examinations of the animal and from chemical analysis of the diet and its components is combined and assessed, it is usually possible to detect and define any nutritional abnormality of mineral origin, even when it is mild (Underwood and Suttle, 1999).

X. INCIDENCE OF MINERAL DEFICIENCIES AND TOXICmES Mineral deficiencies and imbalances for livestock are reported from almost all world regions. Wasting diseases (Fig. 1.3), loss of hair, depigmented hair, skin disorders, non-infectious abortion, diarrhea, anemia, loss of appetite, bone abnormalities, tetany, low fertility, and pica (Fig. 1.4) are clinical signs often suggestive of mineral deficiencies throughout the world. There is good evidence that a wasting disease in Colombia (Fig. 1.5), a periodontal disease in Brazil (Fig. 1.6) and paralysis condition in Venezuela (Fig. 1.4) are the result of mineral deficiencies or imbalances. The extent to which a lack of sufficient energy and protein is responsible for these clinical signs and disease conditions is still largely unanswered. However, numerous investigators have observed that livestock sometimes deteriorate in spite of an apparent adequate feed supply (Sutmoller et al., 1966). Ruminants grazing forages in a severe Co- or Cu-deficient area are even more limited by lack of these elements than either that of energy or protein.

24

General Introduction

Fig. 1.3 Animal in poor condition even though pasture is of good quality (top). Same animal 2 \I, months later (bottom) after access to a high-quality free-choice mineral supplement. (Courtesy of Juan Carlos Montero. Programa NUTRILUZ, University of Zulia, Maracaibo, Venezuela)

Incidence of Mineral Deficiencies and Toxicities

25

Fig. 1.4 Signs of mineral deficiencies. Pica is illustrated as bone chewing (A and B) and eating of bark (C). Often it is a characteristic of phosphorus deficiency. Photo D is a devastating disease condition in Venezuela referred to as "sind rome paraplejico" (bovine paraplegic syndrome). (Courtesy of: A ~ David Morillo, FONAIP, Estacion Experimental Zulia, Venezuela; B - Jiirgen Dobereiner and Carlos H. Tokarnia, EMBRAPjUFRRJ, Rio de Janeiro, Brazil; C - Juan Carlos Contero, Programa NUTRILUZ, University of Zulia, Maracaibo, Venezuela; D - L.R. McDowell, University of Florida, Gainesville)

26

Fig. 1.4

General Introduction

Continued.

Mineral nutrition disorders range from acute mineral deficiency or toxicity diseases, characterized by well-marked clinical signs and pathological changes to mild and transient conditions difficult to diagnose and expressed as a vague unthriftiness or unsatisfactory growth and reproduction. The latter assume great importance because they occur over large areas and affect a large number of animals. Mineral deficiency signs can be confusing, as the observed conditions can

Incidence of Mineral Deficiencies and Toxicities

27

Fig. I.S A wasting disease ("secadera") of cattle in the Llanos of Colombia. Animals are characterized by an emaciated condition in spite of good-quality available forage. (L.R. McDowell, University of Florida, Gainesville)

Ten-month-old Zebu (Gir) (left) in the region of Jaciara, Mato Grosso, Brazil, affected by Fig. 1.6 "cara inchada". Bi- or unilateral swelling of the maxillary bones in the advanced stage of periodontal disease is the reason for its popular name that means "swollen face". Right: Deep, mostly symmetrical lesions at the site of the Papilla interdentalis lingualis between the maxillary Pd 3 and Pd 4 characterize the progressing periodontal disease. (Courtesy of Jiirgen Dobereiner, EMBRAPjUFRRJ, Rio de Janeiro, Brazil)

28

General Introduction TABLE 1.4 Mineral Deficiencies or Toxicities of Ruminants in Tropical Counttes"

Required elements Calcium Argentina, Bolivia, Brazil, Colombia, Costa Rica, EI Salvador, Guatemala, Guyana, India, Malawi, Mexico, Panama, Peru, Philippines, Senegal, Surinam, Uganda, Venezuela, Zaire Argentina, Brazil, Chile, Colombia, Costa Rica, Guatemala, Guyana, Haiti, Magnesium Honduras, Jamaica, Kenya, Malawi, Peru, Surinam, Trinidad, Uganda, South Africa, Uruguay, Venezuela Antigua, Argentina, Bolivia, Botswana, Brazil, Ceylon, Chile, Colombia, Costa Rica, Phosphorus Cuba, Dominican Republic, Ecuador, El Salvador, Egypt, Ghana, Guatemala, Guyana, Haiti, Honduras, India, Indonesia, Jamaica, Kenya, Malagasy Republic, Malawi, Malaysia, Mexico, Nicaragua, Nigeria, Panama, Paraguay, Peru, Philippines, Puerto Rico, Senegal, Somalia, South Africa, Surinam, Swaziland, Tanzania, Trinidad, Uganda, Uruguay, Venezuela, Zaire, Zimbabwe Brazil, Haiti, Nigeria, Panama, Swaziland, Uganda, Venezuela Potassium Bolivia, Brazil, Chad, Colombia, Dominican Republic, Guatemala, Kenya, Malawi, Sodium New Guinea, Nigeria, Panama, Philippines, Senegal, Somalia, South Africa, Surinam, Swaziland, Thailand, Uganda, Uruguay, Venezuela, Zimbabwe Brazil, Colombia, Ecuador, Uganda Sulfur Argentina, Brazil, Colombia, Costa Rica, Cuba, Egypt, EI Salvador, Guyana, Cobalt Haiti, India, Indonesia, Katanga, Kenya, Malaysia, Mexico, Nicaragua, Northern Africa, Peru, Philippines, South Africa, Surinam, Uganda, Uruguay, Zaire Argentina, Bolivia, Brazil, Colombia, Costa Rica, Cuba, Dominican Republic, Copper (or molybdenum Ecuador, EI Salvador, Ethiopia, Guatemala, Guyana, Haiti, Honduras, India, toxicity) Indonesia, Kenya, Malaysia, Malawi, Mexico, Panama, Peru, Philippines, Senegal, South Africa, Sudan, Surinam, Swaziland, Tanzania, Trinidad, Uruguay, Venezuela, Zaire, Zimbabwe Worldwide Iodine Brazil, Costa Rica, India, Panama Iron Manganese Argentina, Brazil, Burma, Costa Rica, Panama, South Africa, Uganda Bahamas, Bolivia, Brazil, Colombia, Costa Rica, Dominican Republic, Ecuador, Selenium Guyana, Honduras, Indonesia, Malawi, Mexico, Paraguay, Peru, South Africa, Swaziland, Thailand, Uganda, Uruguay, Venezuela Zinc Argentina, Bolivia, Brazil, Colombia, Costa Rica, Dominican Republic, Ecuador, EI Salvador, Guatemala, Guyana, India, Indonesia, Kenya. Malawi, Mexico, Panama, Peru, Philippines, Puerto Rico, South Africa, Sudan, Swaziland, Uganda, Uruguay, Venezuela Toxic Elements: Fluorine Algeria, Argentina, Ecuador, Guyana, India, Kenya, Mexico, Morocco, Saudi Arabia, South Africa, Tanzania, Tunesia Manganese Brazil, Costa Rica, Indonesia, Peru, Surinam Selenium Argentina, Brazil, Central African Republic, Chad, Chile, Colombia, Ecuador, Honduras, India, Iran, Kenya, Madagascar, Mexico, Nigeria, Northern Africa, Peru, Puerto Rico, South Africa, Sudan, Upper Volta, Venezuela "McDowell (1976,1985); Fick et 1/1. (1978); McDowell et al. (1984).

Incidence of Mineral Deficiencies and Toxicities

29

involve more than one mineral and be combined with the effects of energy-protein deficiencies, various types of parasitism, toxic plants, and infectious diseases. Mineral deficiencies result most often when animals (and also humans) are confined within a given area and are thus closely dependent upon the structure of the soil and the plant life in a very limited space. They no longer have recourse to migrations in order to compensate for the insufficiencies of the soil or the climate. Various studies have shown that certain trace mineral deficiencies can be prevented when ruminants are allowed greater grazing opportunities and when monogastric species are given access to feeds produced from more than one geological region (McDowell, 1985). In human nutrition, the Se-deficient conditions in China of Keshan and Kaschin-Beck diseases are attributed to the people in the affected region consuming a monotonous locally produced diet (i.e., rice) with no foods introduced from more Se-rich regions. For both humans and animals, deficiencies of trace minerals (also true for vitamins) are less likely when a variety of foods or feeds produced from different geological regions are available. Allman and Hamilton (1949) gathered information from various parts of the world on locations of livestock nutritional deficiencies. Russell and Duncan (1956) and Underwood (1981) have reported selected world locations of mineral deficiencies and toxicities. Information on mineral deficiencies and excesses specifically for grazing livestock in Latin America was updated (Phillips, 1956; De Alba, 1971; McDowell, 1985, 1997, 1999). Table 1.4 lists reports of mineral deficiencies or toxicities for grazing livestock in tropical African, Latin American and Asian countries. Most information in Table 1.4 is a combination of reviews on the reported incidence of mineral deficiencies or toxicities in the developing tropical countries of the world. The numerous references to support Table 1.4 for reported incidences of deficiencies and toxicities are listed elsewhere (McDowell, 1976, 1985, 1997; Fick et al., 1978). An additional tropical country not listed in Table 1.4 is Australia, where reported mineral deficiencies for grazing livestock are P, Ca, S, Co, Cu, I, and Se (Stobbs and Minson, 1980; Minson, 1990). A wide range of mineral deficiencies and excesses has been established in many countries of the world on the basis of forage analysis (See Chapter 17 of this volume.). A summary of mineral concentrations of 2615 forage samples included in the 1974 "Latin American Tables of Feed Composition" indicated that mineral deficiencies were severe and widespread (McDowell et al., 1977). Based on mineral requirements for grazing beef cattle, the percentage of forage samples deficient were as follows: Ca, 31%; P, 73%; Na, 60%; Mg, 35%; Co, 43%; Cu, 47%; Fe, 24%; Mn, 21%; and Zn, 75%. Molybdenum was over 3 ppm in 14% of the samples. Both I and Se were known to be widely deficient, based on other criteria, but few analyses were available. Analyses from the past 20 years illustrate the widespread areas of Se deficiencies throughout the world (McDowell, 1997, 1999; Oldfield, 1999).

30

General Introduction

XI. REFERENCES Allman, R. T., and Hamilton, T. S. (1949). "Nutritional Deficiencies in Livestock," FAO Agriculture Studies No.5, Washington, D.C. Ammerman, C. B. (1995). In "Bioavailability of Nutrients for Animals" (C. B. Ammerman, D. H. Baker, and A. J. Lewis, eds.) p. 83, Academic Press, San Diego. Ammerman, C. D. and Goodrich, R. D. (1983). J. Anim. Sci. 57(Suppl. 2), 519. Anke, M., Arnhold, W., Muller, M., IlIing, H., Schafer, U, and Jaritz, M. (I 997a). In "Handbook of Nutritionally Essential Mineral Elements" (B. L. O'Dell, and R. A. Sunde, eds.) p 465. Dekker, New York. Anke, M., Gurtler, H., Neubert, E., Glei, M., Anke, S., Jaritz, M., Freytag, H., and Schafter, U. (I 997b). In "Proc. Ninth Symp. on Trace Elements in Man and Animals" (P. W. F. Fischer, M. R. L'Abbe, K. A. Cockell, and R. S. Gibson, eds.) p. 192. NRC Research Press, Ottawa, Canada. AOAC (1997). "Methods of Analyses." Association of Official Analytical Chemists. 16th Ed. Publ. AOAC. Washington, D.C. Armsby, H. P. (1880). "Manual of Cattle-Feeding." John Wiley and Sons, New York. ARC (1980). Agriculture Research Council. "Nutrient Requirements of Farm Livestock," No. 2 Ruminants. Her Majesty's Stationary Office, London, England. Becker, R. B., Henderson, J. R., and Leighty, R. B. (1965). "Mineral Malnutrition in Cattle." Bull.699, Fla. Agri. Exp. Stn., Gainesville, FL. Dean, J. R. Crews, H. M., and Ebdon, L. (1989). In "Applications ofInductively Coupled Plasma Mass Spectrometry" (A. R. Date, and A. L. Gray, eds.), p. 141. Chapman and Hall, New York. De Alba, J. (1971). "Feeding of Livestock in Latin America," 2nd Ed. La Prensa Medica Mexicana, Mexico. DRI (Dietary Reference Intakes). (2001). Panel on Micronutrients of Food and Nutrition Board. National Academy Press, Washington, D.C. Egan, A. R. (\975). In "Trace Elements in Soil-Plant-Animal Systems" (D. J. Nicholas, and A. R. Egan, eds.), p. 371. Academic Press, New York. Fick, K. R., McDowell, L. R., and Houser, R. H. (1978). In "Proceedings Latin American Symposium on Mineral Nutrition Research with Grazing Ruminants" (J. H. Conrad and L. R. McDowell, eds.), p. 149. Univ. Florida Press, Gainesville, FL. Forbes, E. B., and Keith, M. H. (\914). In "Phosphorus Compounds in Animal Metabolism," Ohio Agr. Exp. Sta. Tech. Bul. 5, p. 746, Wooster, OH. Georgievskii, V. I., Annenkov, B. N, and Samokhin, V. T. (1981). "Mineral Nutrition of Animals." Butterworths, London, England. Henry, P. R., Ammerman, C. B., and Miles, R. D. (1986). Poult. Sci. 65, 983. Heumann, K. G. (1985). Biomed. Mass Spectrom. 12,477. Hogan, A. G., and Nierman, J. L. (1927). In "Studies of Animal Nutrition - VI the Distribution of the Mineral Elements in the Animal Body as Influenced by Age and Condition." Missouri Agr. Exp. Res. Bul. 107, Columbia, MO. Houk, R. S., Fassel, V. A., Flesch, G. D., Svec, H. J., Gray, A. L., and Taylor, C. E. (1980). Anal. Chem. 52,2283. Iyengar, V., and Woittiez, J. (\988). Clin. Chem. 34(3), 474. Jenkins, K. (1988). In "X-Ray Fluorescence Spectrometry" (J. D. Winefordner, ed.), p. 51, John Wiley, New York. Leeson, S., and Summers, J. D. (2001). In "Nutrition of the Chicken," 4th Ed., University Books, Guelph, Canada. Legg, S. P., and Sears, L. (1960). Nature (London) 186, 1061. Long, M. I. E., Ndyanabo, W. K., Marshall, B., and Thornton, D. D. (1969). Trinidad Trop. Agr. 46, 201. Loosli, J. K. (\974). Proc. Nigerian Soc. Anim. Prod. 1,74. Loosli, J. K. (\978). In "Proc. Latin American Symposium on Mineral Nutrition Research with Grazing Ruminants" (J. H. Conrad and L. R. McDowell, eds.), p. 5. Univ. Florida Press, Gainesville, FL. Loosli, J. K. (1991). In "Handbook of Animal Science" (P.A. Putnam, ed.), p. 25, Academic Press, San Diego. Maynard, L. (1937). "Animal Nutrition," McGraw-Hill Book Co., New York. McCay, C. M. (1973). "Notes on the History of Nutrition Research" (F. Vergar, ed.), Hans Huber Publisher, Berne, Stuttgart, Vienna. McCollum, E. V. (1956). "A History of Nutrition." Houghton Millin, Boston, MA.

References

31

McDowell, L. R. (1976). In "Beef Cattle Production in Developing Countries" (A. 1. Smith, ed.). p. 216, Univ. of Edinburgh Press, Edinburgh, Scotland. McDowell, L. R. (1985). "Nutrition of Grazing Ruminants in Warm Climates." Academic Press, New York. McDowell, L. R. (1997). "Minerals for Grazing Ruminants in Tropical Regions", (3rd Ed.) University of Florida, Gainesville, FL. McDowell, L. R. (1999). "Minerais para Ruminantes sob Pastejo em Regi6es Tropicais, Enfatizando 0 Brasil." University of Florida, Gainesville, FL. McDowell, L. R., Conrad, J. H., and Ellis, G. L. (1984). In "Symposium on Herbivore Nutrition in SubTropics and Tropics - Problems and Prospects" (F. M. C. Gilchrist, and R. I. Mackie, eds.) p. 67. Pretoria, South Africa. McDowell, L. R., Conrad, 1. H., Thomas, J. E., Harris, L. E., and Fick, K. R. (1977). Trap. Anim. Prod. 2,273. McDowell, L. R., Kroening, G. H., Froseth, 1. A., and Haller, W. A. (1974). NUlI'. Rep. Int. 9, 359. Miles, P. H., Wilkinson, N. S., and McDowell, L. R. (2001). "Analysis of Minerals for Animal Nutrition Research," Department of Animal Sciences, University of Florida, Gainesville, FL. Miller, 1. x., Swanson, E. W., and Hansen, S. M. (1965). J. Dairy Sci. 48, 888. Miller, W. J. (1979). "Dairy Cattle Feeding and Nutrition." Academic Press, New York. Miller, W. J. and Stake, P. E. (1974). In "Proceedings Georgia Nutrition Conference for Feed Industry," p. 25. Univ. of Georgia, Athens, GA. Mills, C. F. (1987). J. Anim. Sci. 65, 1702. Minson, D. J. (1990). "Forage in Ruminant Nutrition." Academic Press, New York. NFIA (1991). "NFIA Laboratory Methods Compendium. Vol. I. Vitamins and Minerals." National Feed Ingredients Association, West Des Moines, IA. NCMN - Netherlands Committee on Mineral Nutrition (1973). "Tracing Mineral Disorders in Dairy Cattle." Centre for Agricultural Publishing, Wageningen, The Netherlands. Neal, W. M .. Becker, R. B.. and Shealy, A. L. (1931). Science 74, 418. Nielsen, F. H. (1996). J. NlIIr. 126, 2377S. NRC. (1980). "Mineral Tolerance of Domestic Animals." National Academy of Sciences - National Research Council, Washington, D.C. NRC. (200 I). "Nutrient Requirements of Domestic Animals, Nutrient Requirements of Dairy Cattle." 7th Ed. National Academy of Sciences - National Research Council, Washington, D.C. O'Dell, B. L. (1984). Nutr. Rev. 42, 301. O'Dell, B. L.. and Sunde, R. A. (1997). In "Handbook of Nutritionally Essential Mineral Elements," Marcel Dekker, Inc .. New York. Oldfield, J. E. (1999). In "Selenium World Atlas," Selenium-Tellurium Development Association, Grimbergen, Belgium. Phillips, R. W. (1956). "Recent Developments Affecting Livestock Production in Americas," p. 83. FAO Agriculture Development Paper No. 55, Washington, D.C. Rogers, 1. A., Davis, C. L., and Clark, 1. H. (1982). J. Dairy Sci. 65, 577. Russell, F. C; and Duncan, D. L. (1956). "Minerals in Pasture: Deficiencies and Excesses in Relation to Animal Health." Technical Communication No. 15, Rowett Institute, Aberdeen, Scotland. Scott, M. L. Nesheim, M. C.. and Young, R. J. (1982). "Nutrition of the Chicken." M.L. Scott and Associates, Ithaca, NY. Shupe, 1. L.. Ammerman, C. Boo Peeler, H. T., Singer, L., and Suttie, J. W. (1974). "Effects of Fluorides in Animals." National Academy of Sciences - National Research Council, Washington, D.C. Stobbs, T. H. and Minson, D. J. (1980). In "Digestive Physiology and Nutrition of Ruminants" 3rd Ed., (D.C. Church, ed.), p. 257. 0 & B Books, Corvallis, OR. Stone, B. A., and Mclntosh, C. H. (1977). Aust, J. Agric. Res. 28, 543. Sutmoller, P., Vahia de Abreu, A. van der Grift,1., and Sombroek, W. G. (1966). "Mineral Imbalance in Callie in the Amazon Valley." The Netherlands Communication No. 53. Department of Agricultural Research, Royal Tropical Institute, Amsterdam, The Netherlands. SUllie, N. F. (1988). S. Afr. J. Anim. Sci. 18(1), 15. Theiler, A., Green, H. H., and Du Toit, P. J. (1924). Union S. Afr. J. Dep. Agric. 8, 460. Thornton, D. D., Long, M. I. E., and Marshall, B. (1969). Trinidad Trap. Agr. 46, 269. Underwood, E. J. (1966). "Mineral Nutrition of Livestock." FAO Commonwealth Agricultural Bureaus, London, England. Underwood, E. 1. (1979). In "Proc, of the Florida Nutrition Conference," p. 203. Univ. of Florida, Gainesville, FL.

32

General Introduction

Underwood, E. J. (1981). "The Mineral Nutrition of Livestock." Commonwealth Agricultural Bureau, London, England. Underwood, E. J., and Mertz, W. (1987). In "Trace Elements in Human and Animal Nutrition" 5th Rev. Ed., Vol. I (W. Mertz, ed.) p. 1. Academic Press, New York. Underwood, E. J., and Somers, M. (1969). Aust. J. Agric. Res. 20, 889. Underwood, E. J., and Suttle, N. F. (1999). In "The Mineral Nutrition of Livestock" 3rd Ed., Midlothian, UK. Valdes, E. V., and Leeson, S. (1990). Poult. Sci. 69, 1803. Watson, L. T., Ammerman, C. B., Miller, S. M., and Harms, R. H. (1970). Poult. Sci. 49, 1548. Whetter, P. A., and Ullrey, D. E. (1978). J. Assoc. Off Anal. Chern. 61(4),930. WHO (1996). In "Trace Elements in Human Nutrition." World Health Organization, Geneva. Williams, S. N., Lawrence, L. A., McDowell, L. R., Wilkinson, N. S., Ferguson, P. W., and Warnick, A. c., (1991). J. Anim. Sci. 69, 1232.

Chapter 2

Calcium and Phosphorus I. INTRODUCTION Calcium (Ca) and phosphorus (P) are considered together because they constitute the major part of the mineral content of bone. They are very closely related; a deficiency or an excess of one wi11 interfere with the proper utilization of the other. The Ca: P ratio in the bone is slightly greater than 2: 1 and is approximately constant. In young animals and humans, shortage of Ca, P, or vitamin D results in rickets (Fig. 2.1), and in the adult or more mature animal, osteomalacia. Calcium and P are the two most abundant mineral elements in the animal body. They are frequently found in insufficient quantities in common feedstuffs to meet requirements of livestock. Phosphorus deficiency is predominantly a condition of grazing ruminants, especially cattle, whereas Ca deficiency is more a problem of animals fed mostly on concentrates, especially pigs and poultry, and also feedlot cattle finished on high-grain diets. For humans, owing to the consumption oflow Cadiets by both infants and adults, Ca deficiency is second only to iron (Fe) deficiency.

n.

mSTORY

Rickets in man and animals has been known since antiquity; however, the relationships of Ca, P, and vitamin D to rickets were not known until more modern times. In the 1800s, the association ofCa and P to rickets was becoming clear, but the relationship of the disease to vitamin D was not established until the 1920s. Dr. Clive M. McCay of Cornell University has written a very comprehensive review of the history of nutrition research, including that of Ca and P. Unless stated otherwise, early citations for Ca and P noted herein are presented in his review (McCay, 1973). Sir Humphrey Davey is credited with the discovery of Ca in 1808, but during the 1700s, a number of experiments were carried out with animal bones. In 1736, an English surgeon, John Belchier, found that boiling dyes with bran and feeding this to pigs led to the dyes being deposited in the bone. This discovery led to the study of bone calcification. Brandt, a German alchemist, first isolated P from human urine in 1669. By 1769, a Swedish chemist, Gahn, recognized P as an essential part of bone, and large quantities of P were prepared from bone ash in 1771 by Scheele. Bone composition was studied with growing interest during the eighteenth century. It was known that burning bones left an ash. Scheele and Gahn around 33

34

Calcium and Phosphorus

Fig. 2.1 Calves and young bulls with rickets. (Courtesy of Francisco Megale, Universidade Federal de Minas Gerais, Escola de Veterinaria, Belo Horizonte, MG, Brazil)

1770 discovered that the earthy matter of bone was calcium phosphate. By 1803, lime phosphate was being fed to children with rickets, and it was claimed to improve teeth and heal fractures. It was known by 1817 that the inorganic part of bone was largely calcium phosphate with a small amount of carbonate and some magnesium (Mg). In 1841, Boussingalt first stated, "The bones as we have seen, contain a large quantity of lime: it is required, therefore, that the elements of this salt, phosphoric acid and lime, should form part of the diet." He noted that missionaries observed South American Indian children eating soil, which was evidence of depraved appetite, but their typical foods such as corn were very low in Ca. This represents the attempts (although of little or no value) of primitive people to provide Ca supplementation. In 1842, Choussat reported the first direct experiment in which Ca was shown to modify the composition of bone. Pigeons fed a diet of wheat and water died with very fragile bones, particularly the sternum. When CaC0 3 was added to the diet, bones were normal. In 1973, Nessler found that fragile cattle bones had thin shaft walls, were light in weight and contained less Ca and P than did bones of healthy animals. In the late 1800s, rickets was produced in swine fed low-Ca diets. Rickets in calves was prevented or cured by feeding alfalfa hay. The need for adequate supplies of Ca for the nursing mother was well established by 1900. Every dentist knew that gestation and lactation weakened the mother's teeth. In the early 1900s,

Chemical Properties and Distribution

35

it was also becoming apparent that Ca alone was not responsible for rickets. By 1909 researchers were producing rickets by feeding low-P diets. In actuality, at the turn of the twentieth century, the greatest cause of rickets was from lack of sunlight or vitamin D. Phosphorus deficiency was observed and described in cattle as early as 1785. A great deal of the early work on P deficiencies was carried out in South Africa, where it was intimately associated with the occurrence of lamsiekte (literally lame sickness) and botulism. Le Vaillant (1976; cited by Butterworth, 1985), in his book entitled Travels into the Interior Part ofAfrica, noted lamsiekte and mentioned bone eating or osteophagia. It was left to Theiler (1920, 1927) finally to elucidate the etiology of lamsiekte, botulism, and P deficiency. This South African researcher studied cattle exhibiting subnormal growth, low reproduction, and a depraved appetite or pica illustrated by bone chewing. Theiler concluded that vegetation containing low levels of P induced a deficiency in the animal, which in turn provoked a depraved appetite (pica), causing the animal to become infected with the toxicogenic organism Clostridium botulinum when bone was consumed. Chicco and French (1959) cite Azara (1838) as reporting osteophagia in cows in Paraguay and claim that this was the first mention of P deficiency in Latin America. In the Gulf Coast area of Texas, Schmidt (1926) reported that a fatal disease of cattle, creeps, could be prevented by bonemeal and salt supplementation. Later, the disease stiffs or sweeney was shown to be caused by a deficiency of P in Florida (Becker et al., 1933). In recent years, a great deal of the research with Ca and P is associated with more accurately determining requirements of these nutrients, interrelationships with other minerals (and other nutrients), and metabolism. A new phase of Ca and P research began in the late 1960s with discovery of how active forms of vitamin 0 influence Ca and P metabolism.

III. CHEMICAL PROPERTIES AND DISTRmUTION

A. Calcium Calcium is a soft, silvery white metallic element found most widely in rocks as chalk, limestone, and marble. It is much harder than sodium (Na) but softer than aluminum (AI) or Mg. Calcium is an alkaline earth metal with an atomic weight of 40.08, and an atomic number of20. Its occurrence in the earth's crust is 3.64% (fifth element in order of abundance). It is found naturally only in compounds, chiefly as limestone (calcium carbonate), calcium fluoride, and calcium sulfate. More than 80% of the Ca found in the crust of the earth is in the form of limestone. Calcium is the most abundant mineral element in the animal body (I to 2%), with 99% of it occurring in bone and teeth and the remainder, constituting the physiologically active pool of free Ca, is found in the extracellular fluid and within cells. Variable amounts of Ca are present in almost all feedstuffs (see Section VII).

36

Calcium and Phosphorus

Calcium is generally deficient in grains and abundant in most forages. Its content in natural feeds varies widely, depending on the species of plant and plant part analyzed. Grains such as barley, com, sorghum, oats, and wheat are very low in Ca (0.02 to 0.10%). The nonlegume roughages such as grass hay and mature range forages are intermediate in Ca content (0.31 to 0.36%), and legume forages such as alfalfa and clover hay contain 1.2 to 1.7% Ca (NRC, 1980).

B. Phosphorus Phosphate is anyone of a number of chemical compounds that contain P and oxygen in the phosphate radical P04-3. Phosphorus has an atomic weight of 30.97, and its atomic number is 15; it has one naturally occurring isotope, 31p. It forms about 0.12% of the earth's crust. Phosphorus does not occur free in nature, as it is much too reactive. Essentially, all of the naturally occurring P compounds are phosphates and always occur on the surface of the earth in the form of orthophosphates. The ultimate source of P is igneous rocks (formed by solidification of molten rock), in four principal types of large deposits: (1) igneous apatites, (2) marine phosphorites, (3) phosphatized rock, and (4) guano. Two major groups of sedimentary deposits important for the production of feed phosphates are pellet phosphorite and guano. The major phosphate deposits are found in a form called pellet phosphorite in Florida (USA), Morocco, Israel, and North and South Carolina (USA). Guano phosphate originates from the action of P in the excrement of birds and bats with limestone beds, principally in Curacao and Christmas Island. In Mexico, the same general type of phosphate was formed from bat guano in caves. Most phosphates in nature occur as apatite, CalO(P04MF,CI,OHh or 3Ca3(P04h·Ca(F,CI,OHh The high level of fluoride (F) in the natural rock phosphates limits their effectiveness as P sources for animal nutrition (see Chapter 14). This is relatively unimportant for fertilizer-grade phosphates, as there is little plant uptake of F from soil. Phosphorus is the second most abundant mineral element found in the animal body, and 80 to 85% is in bones and teeth. Phosphorus is present in all common feedstuffs (see Section VII). Seeds are uniformly higher in P than are roughages and seed by-products, such as wheat bran and oil meals, are especially rich in P. Feeds containing milk and bone are high in both P and Ca.

IV. METABOLISM

A. Absorption Many factors influence Ca and P absorption, utilization and metabolism, including adequate levels of one to the other. A Ca : P ratio of I : I to 2: I is usually recommended, with a close ratio most critical if P intake is marginal or inadequate.

Metabolism

37

The absorption of Ca and P is throughout most of the intestinal tract with the duodenum and jejunum being the most active absorptive sites. The large intestine contributes to Ca absorption, with an estimation of total absorption of 11% for the rat (Bronner and Pansu, 1999). In rats, Ca was shown to be mostly absorbed in the small intestine, but when an insoluble Ca salt is not sufficiently absorbed in the small intestine, the large intestine compensates by absorbing greater quantities of Ca (Shiga et al., 1998). In the rat, 7% of the total vitamin D-dependent calcium binding protein (calbindin) is found in the large intestine (Escoffier, 1996) indicating a controlled Ca absorption. Contrary to many species, in the horse, the colon is the major site of absorption and reabsorption of P (Frape, 1998). Small amounts of Ca may be absorbed from the rumen (Yano et al., 1991). Generally, only 30 to 50% of ingested Ca is normally absorbed, whereas 70 to 80% of dietary P is absorbed (Arnaud and Sanchez, 1996; Ternouth and Coates, 1997). Calcium is absorbed according to need up to the limits set by the absorbability of the mineral in the diet; this is close to 90% for milk and probably rarely <50% of the total Ca supply of most dry feed sources (Underwood and Suttle, 1999). The absorption of Ca and P appears to take place by both active and passive absorption (diffusion) (Wasserman, 1981; Braithwaite, 1984). When dietary Ca is relatively low, most of the Ca absorption is by active transport (Goodrich et al., 1985). The major function of vitamin D is related to the maintenance of plasma Ca and P concentrations required for normal mineralization of the skeleton and other physiological functions (DeLuca and Zierold, 1998). Animals absorb Ca from their gut according to need, and they can alter the efficiency of absorption to meet a change in requirement. For example, young sheep with a high Ca requirement absorb Ca at a higher rate and with greater efficiency than do mature animals with a low requirement. In contrast to that of Ca, the percentage of P absorbed is not so closely tied to the needs of the animal (ARC, 1980). However, results of sheep and cattle studies suggested that through active absorption, the percentage absorbed increased in response to an increased demand for P (Braithwaite, 1984; Knowlton and Herbein, 2002). Calcium absorption is directly related to milk production, though in early lactation when demand is greatest, the increase in absorption falls short of the requirement, with the deficit being met by increased bone resorption. A high producing dairy cow may be in negative Ca balance for the first eight months of lactation, while being in negative P balance for the first two months. Apparent P and Ca digestibilities for pigs were dependent on their physiological status (Kemme et al., 1997). Percentage digestibilities of both P and Ca increased as pregnancy progressed and during lactation. Calcium digestibility was 13.4% at 60 days of pregnancy but 30.6% during lactation. Irrespective of the forms in which Ca and P are ingested, their absorption is dependent on their solubility at the point of contact with the absorbing membranes. Absorption of both Ca and P is thus favored by factors that hold them in solution. The solubility of Ca compounds, and hence the absorption of Ca, is favored by acid and hindered by alkaline conditions in the small intestine. Vitamin C supplementation to chicks increased Ca and P absorption at 21 days and thereby decreased percentage of mortality and chicks with leg weakness (Doan and Giang, 1998).

38

Calcium and Phosphorus

Lactose may promote absorption of Ca by interacting with the absorptive cells of the intestine to increase their permeability to Ca ions (Chonan et al., 1998). However, the percentage of absorption of Ca decreases with age, high F intakes, and high Ca intakes, or low vitamin 0 intakes. For rats, indigestible carbohydrates are also reported to promote Ca absorption in both the small and large intestine (Mineo et al., 2001). The level of dietary Ca influences Ca absorption, as high dietary levels depress the efficiency of absorption. Insufficient estrogen production (O'Loughlin and Morris, 1998) and inadequate dietary protein (Kerstetter et al., 1998)have been shown to reduce Ca absorption in humans. Phosphorus absorption is influenced by source of P, intestinal pH, animal age, intestine parasitism and dietary intakes of several other minerals including Ca, iron (Fe), AI, manganese (Mn), potassium (K), and Mg (MacRae, 1993; McDowell, 1997). However, for the horse, excessive Ca has little effect on P absorption as Ca and P are absorbed from different regions of the intestine (Briggs, 1998). Nevertheless, excess P in any form binds Ca and prevents its absorption. Large intakes of Fe, AI, and Mg interfere with the absorption of P by forming insoluble phosphates. Phytates decrease absorption of both P and Ca. Calcium, when combined with dietary oxalic acid, forms insoluble Ca oxalate (Weaver et al., 1997). Fatty acids may form insoluble Ca soaps, which are assimilated with difficulty, yet a certain amount of fat seems to favor the absorption of this element.

B. Control of Calcium and Phosphorus Homeostasis Blood Ca concentration is maintained within very narrow limits by several hormones that control Ca absorption and excretion, as well as bone metabolism. Tetany in humans and animals results if plasma Ca levels are appreciably below normal. Two hormones, thyrocalcitonin (calcitonin) and parathyroid hormone (PTH), function in a delicate relationship with the active form of vitamin 0 (Fig. 2.2) 1,25 dihydroxycholecalciferol (1,25-(OHhD) (0 refers to O 2 or 0 3) to control blood Ca and P levels. Production rate of 1,25-(OHhO is under physiological as well as dietary control. Calcitonin, contrary to the other two, regulates high serum Ca levels by (1) depressing gut absorption, (2) halting bone demineralization, and (3) reducing reabsorption by the kidney. Calcium is adjusted in response to requirement when a fall in plasma Ca concentration resulting from an increase in demand leads, in turn, to an increase in PTH release. This then stimulates the increased production by the kidney of 1,25(OHhD, which acts on the gut to increase the production of calbindin (calciumbinding protein, CaBP), and so accelerates Ca absorption. In a reverse manner, an increase in plasma Ca concentration causes suppression of PTH release, a reduction in 1,25-(OHhO production, and reduced Ca absorption. It appears that the same mechanism operates in ruminants, in that an increase in circulatory 1,25-(OHhO level precedes the increase in Ca absorption that occurs in cattle soon after parturition (Horst et al., 1978). Vitamin 0 brings about an elevation of plasma Ca and P by stimulating specific pump mechanisms in the intestine, bone, and kidney. For ruminants, saliva is an

Metabolism

39

u-v

light Ii Skin

HO 7-dehydrocholesterol

Intestine

OH

OH

Bone~

Kidney~

Kidney Mitochondria

1,25-dihydroxyvitamin O,(I,25-(OH)zO,)

25-hydroxyvitamin 0, (25-0H-O s)

Fig. 2.2 The functional metabolism of vitamin D 3 necessary to activate the target organs of intestine, bone, and kidney.

additional source of P. These sources of Ca and P thus provide reservoirs that enable vitamin 0 to elevate Ca and P in blood levels necessary for normal bone mineralization and for other functions of these minerals. 1.

INTESTINAL EFFECTS

Vitamin 0 stimulates active transport of Ca and P across intestinal epithelium. This stimulation does not involve PTH directly but involves the active form of vitamin D. PTH indirectly stimulates intestinal Ca absorption by stimulating production of 1,25-(OHhD under conditions of hypocalcemia. In humans, as the body becomes vitamin 0 insufficient, the efficiency of intestinal Ca absorption decreases from 30 to 50% to no more than 15%. The mechanism whereby vitamin 0 stimulates Ca and P absorption is still not completely understood. Wasserman (1981) indicates that I,25-(OHhD is transferred to the nucleus of the intestinal cell, where it interacts with the chromatin material. In response to the 1,25-(OHhD, specific RNAs are elaborated by the nucleus and when these are translated into specific proteins by ribosomes, the events leading to enhancement of Ca and P absorption occur (Scott et al., 1982). In the intestine, 1,25-(OHhD promotes synthesis of calbindin and other proteins and stimulates Ca and P absorption. Vitamin 0 has also been reported to influence Mg absorption as well as Ca and P balance (Miller et al., 1965). Administration of

40

Calcium and Phosphorus

1,25-(OHhD3 to rachitic animals has been shown to stimulate the incorporation of eH]leucine into several proteins of the intestinal mucosa. This apparent increase in protein synthesis was at least in part accounted for by the discovery that 1,25(OHhD induces synthesis of a specific intestinal protein that has been identified as calbindin. Calbindin is not present in the intestine of rachitic chicks but appears following vitamin D treatment. Intestinal calcium transport relies on the integrated effects of both genomic and nongenomic mechanisms of hormone action. Two kinds of mucosal proteins are dependent on vitamin D: (I) calbindin and (2) intestinal membrane calcium-binding protein (1MCal). IMCal is a membrane component of the translocation mechanism rather than a cytosol constituent (Schachter and Kowarski, 1982). It is proposed that the primary nongenomic mechanism by which 1,25-(OHhD regulates Ca transport across the luminal membrane of the enterocyte involves inducing a specific alteration in membrane phosphatidylcholine content and structure, which leads to an increase in membrane fluidity and thereby to an increase in Ca transport rate. The size of the villi and the microvilli increases upon 1,25-(OHhD3 treatment. The brush border undergoes noticeable alterations of structure and composition of cell surface proteins and lipids occurring in a time frame corresponding to the increase in Ca 2+ transport mediated by 1,25-(OHhD 3 (Collins and Norman, 1991). In addition to inducing calbindin and IMCal, 1,25-(OHhD 3• has been shown to increase levels of several other proteins in the intestinal mucosa. These include alkaline phosphatase, Ca-stimulated ATPase, and phytase enzyme activities (Collins and Norman, 1991). Once Ca is transported to the basolateral membrane, it is extruded from the cell against a IOOO-fold concentration gradient by Mgdependent Ca-ATPase, which is also increased by 1,25-(OHhD 3 (Bronner, 1987). Originally, it was felt that vitamin D did not regulate P absorption and transport, but in 1963, it was demonstrated through the use of an in vitro inverted sac technique, that vitamin D does in fact play such a role (Harrison and Harrison, 1963). Little is known about the actual mechanism of P transport, but P is transported against an electrochemical potential gradient involving sodium (Na) in response to 1,25-(OHhD3 . 2. BONE EFFECTS

Vitamin D plays roles both in the mineralization of bone as well as demineralization or mobilization of bone mineral. I,25-(OHhD is one of the factors controlling the balance between bone formation and resorption. In young animals during bone formation, minerals are deposited on the matrix. This is accompanied by an invasion of blood vessels that gives rise to trabecular bone. This process causes bones to elongate. During a vitamin D deficiency, this organic matrix fails to mineralize, causing rickets in the young and osteomalacia in adults. 1,25-(OHhD 3 brings about mineralization of the bone matrix and Weber et al. (1971) provided evidence that 1,25-(OHhD3 was localized in the nuclei of bone cells. Vitamin D plays another role in bone, that is, in the mobilization of Ca from bone to the extracellular fluid compartment. This function is shared by PTH

Metabolism

41

(Garabedian et al., 1974). However, little is known about the mechanism of bone reabsorption in response to these factors, although it may be similar or identical to the intestinal transport system. It is an active process requiring metabolic energy, and presumably it transports Ca and P across the bone membrane by acting on osteocytes and osteoclasts. Rapid, acute plasma Ca regulation is due to the interaction of plasma Ca with Ca-binding sites in bone mineral as blood is in contact with the bone. Changes in plasma Ca are brought about by a change in the proportion of high- and lowaffinity Ca-binding sites, access to which is regulated by osteoclasts and osteoblasts, respectively (Bronner and Stein, 1995). These cells in turn respond to hormonal signals by shape changes. Contraction of osteoclasts and corresponding expansion of osteoblasts make more high-affinity sites available, whereas osteoblast contraction and osteoclast expansion make more low-affinity sites available leading to a lowering or rising of blood Ca level, respectively. 3. PLACENTAL AND EGG TRANSFER

Calcium and P are transferred to the egg and though the placenta, but studies are limited. Transplacental movement of Ca increases dramatically during the last trimester of gestation in all species observed. It is well established that in most mammalian species, fetal plasma Ca levels are higher than maternal levels at term. The transfer mechanism for either Ca or P to the developing fetus has not been determined, but because there are binding proteins in the intestinal tract and mammary tissue, they probably also exist in placental tissue (Mahan and Vallet, 1997). In sheep, passive diffusion accounted for a minor component of placental Ca movement with active transport mechanisms responsible for greater than 90% (Braithwaite et al., 1972). In the pregnant rat, vitamin D as 1,25-(OHhD was a critical factor in the maintenance of sufficient maternal Ca for transport to the fetus and may playa role in normal skeletal development of the neonate (Lester, 1986). Itoh et al. (1967) determined that fetal Ca deposition was not greatly influenced by maternal dietary levels of Ca. The bone ash content of neonatal pigs was not affected when sows were fed dietary levels below and above those normally provided to gestating sows (Mahan and Fetter, 1982). This suggested that mineral reserves are diverted from maternal bone tissue to meet fetal development needs when the sow diet provides inadequate levels of these minerals. 4.

KiDNEY EFFECTS

There is evidence that vitamin D functions in the distal renal tubules to improve Ca reabsorption and is mediated by the Ca-binding protein, calbindin (Bronner and Stein, 1995). It is known that 99% of the filtered load of Ca is reabsorbed in the absence of vitamin D and the parathyroid hormone. The remaining I% is under control of these two hormonal agents, although it is not known whether they work in concert. It has been shown that 1,25-(OH)zD 3 functions in improving renal reabsorption of Ca (Sutton and Dirks, 1978). With intact parathyroids and without vitamin D, renal tubular resorption of inorganic P decreases, thereby increasing

42

Calcium and Phosphorus

phosphate clearance and resulting in hypophosphatemia, although the parathyroids maintain a normal plasma Ca level. With adequate vitamin D, greater resorption of P by the renal tubules occurs. Without intact parathyroids, vitamin D actually increases renal loss of P. 5.

PHOSPHORUS HOMEOSTASIS INCLUDING SALIVA EFFECTS IN RUMINANTS

Sheep fed roughage diets usually excrete little P in their urine (Scott and McLean, 1981), so control of P balance must therefore be achieved within the gut through control of either absorption or secretion or both. Saliva is the main contributor of P to the gut. Little or no net absorption of P appears to occur from either the forestomach or large intestine; the upper small intestine is the major absorptive site. Using sheep fitted with reentrant cannulas, several workers have shown that the salivary secretion of P is closely matched by net absorption in the small intestine. Ability to balance absorption against secretion is unaffected by wide variations in dietary Ca: P values (Scott and McLean, 1981). In cattle, inadequate dietary P markedly reduced P concentration in saliva (Jain and Chkopra, 1998). At one time, secretion of P in saliva had usually been viewed as a buffer against the volatile fatty acids produced in the rumen. Studies by Australian workers (Tomas, 1974) have suggested that salivary glands may also play an important role in P homeostasis by controlling the amount of P secreted in the gut. Evidence for this function was provided by sheep studies in which both parotid salivary ducts were ligated, a procedure that led to a small increase in urinary P excretion and a proportional reduction in fecal P excretion (Tomas and Somers, 1974). Similar changes in the pathway of P excretion were also seen in sheep in which part of the parotid salivary flow was collected and returned directly to the circulation. Ruminants have a higher renal threshold for P excretion than do monogastric species, and it is important to consider what advantage this confers. Poor quality roughage diets, apart from their low digestibility, also tend to contain little P (McDowell, 1997). Therefore, the ratio between the amount of P required for saliva production and dietary intake is wide. If the renal threshold for P excretion in ruminants were as low as in monogastric species, then at times when the concentration of inorganic P in the plasma rises in response to reabsorption, P would be excreted in the urine. This P would not, however, in any real sense be surplus to requirement and its loss would have to be met from a diet low in available P. Under such conditions, there is clearly an advantage to the ruminant in maintaining the high renal threshold for P excretion. Concentrate diets, especially those that include fish meal, contain much larger quantities of P than do roughage diets, to the point where intake may equal or exceed the amount secreted in the saliva. Under these conditions, need to control P absorption is clearly less critical and, as a result, a different level of control may operate. Increasing dietary P intake leads to increased absorption and increased urinary P excretion. Administration of large amounts of parathyroid hormone over several days has been shown to reduce fecal P excretion in cattle (Mayer et al., 1968). Whether this

Metabolism

43

was due to reduced secretion or increased absorption is not clear. In sheep, parathyroidectomy has been shown to result in a negative balance for both Ca and P, and it has been suggested that the effect on P balance was the result of a reduction in amount of salivary P reabsorbed (McIntosh and Thomas, 1978). 1,25(OH)zD has also been suggested as a possible regulator of intestinal P absorption in ruminants (Scott and McLean, 1981), though whether this was a primary effect or secondary to its effects on Ca absorption and deposition in bone is not clear.

C. Storage and Blood Concentrations of Calcium and Phosphorus Bone contains approximately 98 to 99% of the total body Ca and 80 to 85% of its total P, affording an enormous depot of Ca and P that guards the circulating Ca and P pools (Centrella and Canalis, 1985). Unlike the relatively inert mineral of tooth enamel (Daniell, 1983), bone undergoes constant remodeling and turnover. To illustrate the large storage potential for Ca in bone, the dairy cow can be fed a Ca-deficient diet for months or even years without a major decrease in blood plasma Ca level (Miller, 1985). If blood Ca concentration starts to decrease, Ca is quickly mobilized from the bone to bring the blood level back to normal. When Ca intake is deficient, animals deplete their bone reserves. Plasma Ca is distributed in three major fractions: ionized, protein bound, and complexed. The biologically active ionized form (Ca 2+) constitutes 46 to 50% of total Ca. The biologically inert protein-bound fraction is roughly equivalent to the ionized fraction. However, the Ca bound to albumin (80%) and globulin (20%) is an important reservoir of Ca. The fraction of Ca that is complexed to organic (e.g., citrate) and inorganic (e.g., phosphate or sulfate) acids is small ("-'8%). Serum Ca concentration varies little in spite of large changes in dietary Ca because of endocrine regulation (see Section IV,B). The blood cells are almost or entirely devoid of Ca, but the plasma, in health, contains from 9 to 12 mg per 100 ml in most species. In the laying hen, levels three or four times higher may occur during egg production. Whole blood contains from 35 to 45 mg of P per 100 ml, most of which is in the cells. Plasma P level is more easily changed by diet than is the Ca level; in health it generally lies between 4 and 9 mg per 100 ml, depending on the age and species. Serum P concentrations in young children are almost double those in adults and increase slightly with age in women (Arnaud and Sanchez, 1996).

D. Excretion In all species, the feces are a primary path for Ca excretion. Fecal Ca is a combination of unabsorbed dietary Ca and unabsorbed endogenous Ca from intestinal mucosal secretions; therefore, any factors that affect Ca absorption will affect the amount found in the feces. Urinary loss is minimal, owing to efficient reabsorption by the kidneys. The horse and rabbit (Cheeke, 1987) may, however, excrete considerable amounts of Ca in the urine when high levels of Ca are fed. Some Ca is lost during sweating; humans may lose up to 20 mg Ca per hr during

44

Calcium and Phosphorus

profuse sweating (Hafez and Dyer, 1969). In the horse, extended work and overheating can result in a loss of 350 to 500 mg Ca/hr (Frape, 1998). Feces is the primary path for P excretion in herbivores, but the urine is the principal path for carnivores and in humans. However, substantially more P has been reported in the urine of cattle fed high concentrate diets (Preston, 1977). In states ofP depletion, the kidney responds by reducing excretion virtually to zero, thus conserving body phosphate (Berner, 1997). Variable endogenous fecal excretion is an important homeostatic control route for P. In contrast, endogenous losses of Ca are relatively fixed, with the percentage absorbed varying with intake (Miller, 1985).

V. PHYSIOLOGICAL FUNCTIONS

A. Structure of Bone Related to Calcium and Phosphorus Calcium and P make up over 70% of the total mineral elements in the body. The relative content of Ca and P in the bone tends to remain fairly constant even when there is a dietary deficiency of only one of these elements. The Ca: P ratio in bone is nearly constant and somewhat greater than 2: 1 (Berner, 1997). Bone contains considerable amounts of carbonate and citrate and small amounts of Mg, Na, K, Cl, F, and traces of other elements. Bone is somewhat variable according to age, state of nutrition, and species; normal adult bone has the following approximate composition: water, 45%; ash, 25%; protein, 20% and fat, 10%. The organic matrix of bone in which the mineral salts are deposited consists of a mixture of proteins, principally ossein. As the animal ages, the water content decreases, while fat is variable. Thus, ash content is expressed most frequently for moisture-free, fat-free bone. In mammals, the ash is made up of Ca, 36%; P, 17%; and Mg, 0.8%. Mammalian young are born with poorly mineralized bones and, while they suckle, they do not receive enough Ca and P to fully mineralize the bone growth that energy-rich milk can sustain (AFRC, 1991). After weaning, there is normally a progressive increase in bone mineralization stimulated by increased load-bearing, thus providing added strength and reserves of both Ca and P (Underwood and Suttle, 1999). Most of the Ca and P in bones is in the form of Ca phosphate [Ca3(P04hl and hydroxyapatite [CalO(P04MOHhl. The Ca phosphate of bone is deposited within a soft fibro-organic matrix composed of collagen fibers and, to a much less extent, of mucopolysaccharide. The protein matrix in bone is calcified when the proper levels of Ca, P, Mg, and other minerals are present. Bone mineral consists of two chemically and physically distinct Ca phosphate pools - an amorphous phase and a loosely crystallized phase (Posner, 1973). The amorphous phase contains hydrated tricalcium phosphate as well as secondary calcium phosphate. The crystalline form resembles hydroxyapatite but contains about 3% carbonate and 1% citrate. Other bone mineral ions are thought to bind primarily at the surface of the apatite crystals.

Physiological FuncUons

45

Bone-length growth takes place at the junction of the epiphysis and diaphysis (Maynard et al., 1979). The cartilage in between is a temporary formation that grows by the multiplication of its own cells and continues to be replaced by calcified bone. In ossification, cartilage is replaced by osteoid, which is then calcified. The zone where this takes place is the proliferation zone of cartilage, or the zone of provisional calcification. Bone is a unique living tissue; it is not only rigid and resists forces that would ordinarily break brittle materials, but is also light enough to be moved by coordinated muscle contractions (Krane and Schiller, 1989). Cortical bone is composed of densely packed layers of mineralized collagen, which provides rigidity and is the major component of tubular bones. Trabecular (cancellous) bone is spongy, provides strength and elasticity, and constitutes the major portion of the axial skeleton (Schuette and Linkswiler, 1984). Bone undergoes a continuous process of resorption and formation with mobilization and restorage of Ca and P occurring throughout life. There is a continuous interchange ofCa and P between the bone, the blood supply, and other parts of the body. If more leaves the bone than is being deposited, the bone eventually becomes weak, porous, and may be deformed or broken by the weight of the animal, the pull of the body muscles, and the stress placed on the bone by movement. Resorption and formation are dependent on different types of bone cells, each with distinct functions. Osteoblasts form new bone on surfaces of bone previously resorbed by osteoclasts. The osteoclast is a multinucleated giant cell that is responsible for bone resorption. Osteoblasts are actively involved in the synthesis of matrix components of bone (primarily collagen) and probably facilitate the movement of mineral ions between extracellular fluid and bone surfaces. Once a strong bone is formed, it does not remain that way forever. The animal continually needs Ca, P, and other nutrients to maintain bone in a strong condition. During pregnancy and heavy lactation, bone supplies minerals secreted in the milk. The body's regulatory mechanism, which allows minerals deposited in the bone to be drawn on during periods of emergency needs, is an efficient one (Cunha, 1990). As animals and humans age, they lose peak bone mass. This is particularly a problem for humans due to their longer life span. Healthy bones of young humans do not break due to ordinary falls. However, there are about 270,000 hip fractures annually in the United States that have occurred as a result of a fall or even small trauma (Graves, 1993). This is due to age-related weakening of bones. About onethird of the 70 to 79-year-old and about half of the 80-year-old or older white, nonHispanic U.S. women have lost at least 25% of their femoral bone mass (Looker et al., 1995) and reached the osteoporotic state (Kanis, 1994). This degree of bone loss increases the risk of hip fracture by a factor of about six (Cummings et aI., 1993). It can be thought that if the highest or peak bone mass attained as a result of normal growth is great, it would take longer, even more than the life span, to reach the osteoporotic state (Vuori, 1996). Physical activity can increase peak bone mass and bone mineral density. Studies of athletes show that the bone mineral density (BMD) of loaded bones can be more

Calcium and Phosphorus

46

than 30% higher in most studies and between 5 and 20% higher at most sites than that of unloaded contralateral bones or of the same bones in nonathletic control subjects (Bouxsein and Marcus, 1994; Chilibeck et al., 1995; Kirchner et al., 1995; Taaffe et al., 1995). Part of the high BMD in athletes may be due to genetic factors, but studies of squash and tennis players show large side-to-side differences in BMD or bone mineral content in the players' arms but no significant differences in BMD in the nondominant arm of the players and control subjects (Haapasalo et al., 1994, Kannus et al., 1994), which indicate that the high BMD in the players' dominant arm is mainly due to training. For poultry, activity increased bone growth in broilers, which also developed increasingly mineralized bones as they aged (Bond et al., 1991); providing a perch increases bone mineralization in hens.

B. Calcium and Phosphorus in Soft Tissue Nonskeletal Ca and P play important roles in a wide variety of essential functions in body metabolism (Goodrich et al., 1985; Miller, 1985; Rasmussen, 1986; Arnaud and Sanchez, 1996; Bronner, 1997; McDowell, 1997; Underwood and Suttle, 1999). 1.

CALCIUM

The 1% of the body's Ca located outside of the bone is found in extracellular fluid, soft tissue, and as a component of various membrane structures (Bronner, 1997). Nonskeletal Ca occurs as the free ion bound to serum proteins and complexed to organic and inorganic acids. Calcium is essential for normal blood clotting; the Ca ion must be present for prothrombin to form thrombin, which reacts with fibrinogen to form the blood clot, fibrin (McDowell, 2000). Dietary Ca decreases gastrointestinal lead (Pb) absorption and thereby reduces the risk for Pd poisoning (Ballew and Bowman, 2001). Calcium has a role as a cofactor in many enzymatic reactions, acting as an activator [e.g., adenosine triphosphatase (ATPase)] or stabilizer of enzymes (Peo, 1976) and it is necessary for secretion of a number of hormones and hormone-releasing factors (Arnaud and Sanchez, 1996). Calcium can activate or stabilize some enzymes and contributes to regulation of the cell cycle (Hurwitz, 1966). Bakalli et al. (1996) report a role of Ca in immune responses of chickens. Hemagglutination inhibition titers for New Castle disease was significantly increased when dietary Ca was elevated from 0.60 to 1.20%. Synaptic transmission is likewise affected; a low Ca2+ concentration produces hyperexcitability of preganglionic and postganglionic elements and a tendency for spontaneous fiber discharge. Acetylcholine may not be liberated in the total absence of Ca 2+ (Scott et al., 1982). Lowered levels of Ca result in increased excitability, whereas higher levels result in a pseudo tranquilizing effect. Extremely low levels of serum Ca can result in tetany. This is followed by death, unless the cause is determined and treatment given. Calcium is needed for efficient weight gain and feed utilization. It is particularly important for milk production, egg production, and shell quality, as both milk and

Requirements

47

egg shells contain large amounts of Ca. In poultry, Ca performs the unique function of protecting the egg through the deposition of an eggshell during passage through the oviduct. The shell matrix is heavily impregnated with CaC0 3 and the need to furnish about 2 g Ca for every egg produced dominates Ca metabolism in the laying hen (Underwood and Suttle, 1999). 2.

PHOSPHORUS

Nonskeletal P is especially concentrated in red blood cells, muscle, and nerve tissues. Large amounts of P, other than in bones, are present mostly in organic combinations such as phospholipids, phosphoproteins, and nucleic acids; the hormonal second messengers cyclic adenosine monophosphate, cyclic guanine monophosphate, and inosital polyphosphates; and 2,3-diphosphoglycerate, which is the regulator of oxygen release by hemoglobin (Arnaud and Sanchez, 1996; Berner, 1997; Underwood and Suttle, 1999). Phosphorus is involved in almost all, if not all, metabolic reactions, and therefore may be the most versatile of all mineral elements. It is involved in almost every aspect of feed metabolism and utilization of fat, carbohydrate, protein, and other nutrients in the body. Phosphorylation and dephosphorylation regulate many activities within cells, including the function of enzymes, hormones, and the transcription of genetic information. These reactions are catalyzed by phosphorylases (kinases) and phosphatases, respectively. High-energy phosphate bonds, such as in ATP, provide energy to drive most metabolic reactions. Phospholipid formation allows fatty acids to be transported throughout the body. Phosphorus also functions in protein metabolism in nucleoproteins and phosphoproteins. Because P is a component of nucleic acids (RNA and DNA), it is necessary for genetic transmission. Phosphorus is an essential component in buffer systems in the blood and other body fluids, including those of the rumen, and is essential for proper functioning of rumina I microorganisms, especially those that digest plant cellulose (McDowell, 1985). Phosphorus is further involved in the control of appetite, and in the efficiency of feed utilization (Ternouth and Sevilla, 1990; Underwood and Suttle, 1999). Phosphorus is needed for normal milk secretion, muscle tissue synthesis, egg formation, and efficient feed utilization. Any limitation to the P supply will be reflected in a generalized impairment of body function.

VI. REQUIREMENTS Table 2.1 lists Ca and P requirements for various animal species and humans, with more complete requirement listings in Appendix Table I. Monogastric animals in general have higher Ca and P requirements than do ruminants, with the highest Ca requirement for egg production, because of the high Ca content of the shell. The levels of Ca and P that result in maximum growth rate are not necessarily adequate for maximum bone mineralization. The requirements for maximizing bone strength

Calcium and Phosphorus

48

TABLE 2.1

Calcium and Phosphorus Requirements for Various Species" Species

Purpose

Chickens b

Leghorn-type 0-6 wks Leghorn-type 6-18 wks Leghorn-type laying" Leghorn-type breeding C Broilers 0-8 wks All classes All classes All classes Lactating All classes All classes All classes All classes Growing Growing Growing All classes All classes Growing Growing All classes Children Adults Lactating

Japanese quail" Turkeys" Beef cattle Dairy cattle Sheep Horses Swine Mink Foxes Rabbits Cats Dogs Rats Mice Guinea pigs Nonhuman primates Humans"

Calcium 0.90% 0.80% 3.25% 3.25% 0.8-1.0% 0.8~2.5%

0.50-2.25% 0.16-1.53% 0.43-0.60% 0.20--0.82% 0.24--Q.68% 0.45--0.90% 0.3-1.0% 0.5--0.6% 0.40% 0.80% 0.59% 0.50% 0.40% 0.80-1.0% 0.50% 0.50--0.80 g/d 0.70--0.90 g/d 1.00-1.30 g/d

Phosphorus 0.40% 0.35% 0.25% 0.25% 0.35--0.45% 0.30--0.35% 0.25--0.60% 0.17--0.59% 0.31--0.40% 0.16--Q.38% 0.17-0.38% 0.40--0.70% 0.30--0.80% 0.22% 0.60% 0.44% 0.40% 0.40% 0.40--0.70% 0.40% 0.46--Q.50 g/d 0.70-1.25 g/d 0.70-1.25 g/d

Reference NRC (1994) NRC (1994) NRC (1994) NRC (1994) NRC (1994 NRC (1994) NRC (1994) NRC (1996) NRC (2001) NRC (I985b) NRC (1989) NRC (1998) NRC (1982) NRC (1982) NRC (1977) NRC (1986) NRC (I985a) NRC (1995) NRC (1995) NRC (1995) NRC (1978) DRI (2001) DRI (2001) DRI (2001)

"Expressed as per unit animal feed either on as-fed (approximately 90% dry matter) or dry basis (see Appendix Table I). "Based on nonphytate phosphorus. 'Based on intakes of 100 g per day. dHuman requirements expressed as gfday.

and bone ash content for swine are at least 0.1 percentage unit higher than the requirements for maximum rate and efficiency of gain (Combs et al., 1991a,b; NRC, 1998). However, maximization of bone strength by feeding large amounts of Ca and P to growing pigs does not necessarily improve structural soundness (Eeckhout et al., 1995), nor has it been shown to be necessary for good health or longevity. However, higher dietary Ca and P levels may be desirable over prolonged reproduction periods, but not for swine destined for market (Cera and Mahan, 1988). Requirements for mineral elements have been established by the factorial method and the feeding experiment method (ARC, 1980; Miller, 1983; AFRC, 1991). There is lack of agreement in regard to the Ca and P requirements of animals and appropriate methods for estimating these requirements (Braithwaite, 1983; Ternouth et al., 1996; Underwood and Suttle, 1999). There are differing views on the percentage of Ca or P that is available from feedstuffs (Miller, 1983). As an example, the ARC (1980) concluded that 68% of the Ca in most cattle feeds was

Requirements

49

available for absorption, while the NRC (2001) used a figure of 38%. The absorption rate for P was found to be 77% for unsupplemented grazing cattle and 82% for penned supplemented cattle (Ternouth et al., 1996). An additional error arises from assuming that total endogenous loss is constant, irrespective of feed intake, age, or physiological state (Braithwaite, 1984; Gueguen et al., 1989; Ternouth and Coates, 1997). Different Ca and P requirements may include safety margins in some NRC publications, but ARC values are minimums with no added safety margins. Adequate Ca and P nutrition depends not only on sufficient total dietary supplies, but also on the chemical forms in which they occur in the diet (see Sections VII and IX) and on the vitamin D in the diet. A dietary Ca : P ratio between 1 : 1 and 2: 1 is assumed to be ideal for growth and bone formation since this is approximately the ratio of the two minerals in bone. A suggested ratio of total calcium-to-total phosphorus for grain-soybean meal diets is between 1: 1 and 1.25 : 1. When based on available phosphorus, the ratio is between 2: 1 and 3: 1 (Qian et al., 1996). Lowering the Ca to P ratio from 1.5: 1 to 1.3: 1 or 1.0: 1 increased P utilization in low-P corn-soybean meal diets supplemented with microbial phytase for growing-finishing pigs (Liu et al., 1998, 2000). Liberal supplies of vitamin D reduce the significance of adverse Ca : P ratios and enable the animal to make the best use of limited intakes. The dietary vitamin D content can be important to livestock housed for long periods, particularly to high-yielding dairy cows and laying hens with their high Ca and P requirements (McDowell, 2000). The NRC (1994) for poultry suggests a ratio of approximately 2 Ca to 1 nonphytate P (weight/weight) which is appropriate for most poultry diets, with the exception of diets for birds that are laying eggs. Chandramoni et al. (1998) suggested that the Ca: P ratio is not of great importance for laying hens. When poultry are laying eggs, a much higher level of Ca is needed for eggshell formation, and a ratio as high as 12 Ca to 1 nonphytate P (weight/weight) may be correct. If the diet is marginal in Ca after egg production begins, the bones will be a major source of Ca and bone density will decrease. It must be realized that the amount of Ca required for maximum egg production throughout the laying cycle is much less than the requirement necessary to maintain maximum quality of the egg shell. Also, if maximum bone ash in laying hens is a goal, then the Ca requirement for high bone ash and density greatly exceeds to the requirement for maximum egg shell quality. The balance of the minerals is often upset when legumes with a Ca : P ratio of 6 to 10 : 1 are fed, and a wide ratio exists when only overly mature forages, particularly those low in P, are available to grazing livestock during extended dry seasons. However, Ca in alfalfa is only 50 to 70% as available to dairy cattle as that from inorganic sources (Cunha, 1984). Actually ruminants can tolerate a wider range of Ca: P, particularly when their vitamin D status is high. Nine Ca: P ratios ranging from 0.41: 1 to 14.3: 1 were tested by Wise et al. (1963); dietary ratios below 1: 1 and over 7 : 1 adversely affected growth and feed efficiency. Cohen (1980) suggested that low Ca concentrations in forages will prevent responses from P supplementation because low dietary Ca will trigger 1,25-(OHhD production and mobilization of both Ca and P from bone. Greater responses to P supplementation are reported

50

Calcium and Phosphorus

in the South African grazing studies with cattle on calcareous soils than in beef cattle grazing on noncalcareous soils in Australia. Phosphorus requirements recommended by the NRC may be too high for grazing beef cattle. Ternouth et al. (1996) suggest that the requirements of cattle consuming forage diets were 40 to 50% lower than those published by the Agricultural and Food Research Council (AFRC, 1991). From Utah (USA), no difference in average weight gains (0.45 kg/day), feed efficiency or appetite was observed between Hereford heifers fed for 2 years a diet containing 0.14% P (66% of NRC recommendation) and comparable heifers receiving the same diet supplemented with monosodium phosphate to provide a total of 0.36% P (Call et al., 1978). After 8 months on a 0.09% P diet, however, some appetite reduction and decreased bone density were observed. On the contrary, studies from Florida (USA) demonstrated that 0.12 to 0.13% P was inadequate for growing Angus heifers in a 525 to 772-day experiment. Animals receiving the low-P diet had lower gains (205 versus 257 kg), exhibited pica, and had bone demineralization (Fig. 2.3) (Williams et al., 1990, 199Ia,b,c).

Fig. 2.3 Typical dual photon absorptiometry (DPA) scans of third metacarpals from cattle fed a (left) low phosphorus (0.12% P) or (right) adequate phosphorus (0.20% P) diet (dry basis). The metacarpal from the adequate P group shows little bone mineral loss in either its proximal or distal end indicated by the lack of dark areas, whereas both proximal and distal ends indicate either loss of labile trabecular bone in the low phosphorus metacarpal or that bone mineral was not deposited in a normal fashion. (Courtesy of S.N. Williams and L.R. McDowell, University of Florida, Gainesville)

Requirements

51

Both the Utah and Florida studies could be criticized because they were done in dry lot, feeding chopped or pelleted diets, and may not be indicative of requirements for cattle under grazing conditions. Animals required to walk to obtain forage would probably have greater requirements for structural minerals in bone. Also, the Utah and Florida studies used beet pulp and citrus pulp, respectively, as major diet ingredients. Phosphorus availability from these feeds would seem to be higher when compared to forages (particularly tropical) with high cell-wall contents (e.g. lignin). Other factors influence Ca and P requirements besides the dietary levels of these minerals, the ratio of the two elements, the presence and quality of other minerals (e.g., Fe, AI, and Mn), and vitamin D. High-fat diets increased fecal Ca losses through the formation of soaps (Oltjen, 1975; Juma and Arjmandi, 1999) and thus increase dietary requirements. Parasitic infestations of nematodes caused demineralization of the skeleton in sheep (Underwood and Suttle, 1999). Infections of the small intestine (e.g., by Trichostrongylus colubriformis) can reduce the absorption of dietary and endogenous phosphorus by about 40% and induce hypophosphatemia (Wilson and Field, 1983). The P requirements of monogastric species, but not of ruminants with functioning rumens, increase as the proportion of dietary P occurring as phytate increases (see Section VII, IX and Chapter 19). However, the NRC requirements (e.g., for swine and poultry) for total Ca and total P are based on a fortified, corn-soybean meal diet and take into account the fact that a significant quantity of the P in feedstuffs of plant origin is unavailable (e.g., phytates). A large number of recent studies have shown that microbial phytases can improve retention from 30 to over 45% in swine and poultry diets, and thus greatly reduce P requirements (BASF, 1997;Rodehutscord, 1998).Not only do dietary concentrations of energy and protein influence total intake of feed, but they also have a direct effect on mineral retention. High dietary energy and protein concentrations greatly increased Ca and Pretention in growing sheep, compared to diets low in energy and protein (49.9 versus 26.8% Ca and 72.5 versus 54.4% P, respectively) (Rosero et al., 1983). Environmental factors including stress from disease, overcrowding, poor ventilation and inadequate temperature control have a profound effect on Ca and P requirements. Owings et al. (1977) concluded that diets containing about 0.19% available P were adequate to maintain egg production, but that at least 0.28% available P was necessary to prevent cage layer fatigue syndrome. Calcium and P requirements are influenced by animal age and genetics. Young dogs (4 to 5 months), compared to older dogs, were unable to adjust the digestibility of Ca at either excessive or insufficient intakes (Dobenecker, 2002). Calves (Miller, 1979) absorb 90% of the Ca in milk. In older cattle, true absorption is quite variable, ranging from 22 to 55%, but averaging about 45%. Young animals apparently absorb Ca more efficiently (Hansard et al., 1957). However, it is hard to distinguish whether higher absorption for young animals is primarily an age consideration or a greater need for the nutrient, and therefore, caused by homeostatic mechanisms coming into play. As an example, an adult animal deficient in Ca (or most other nutrients) will absorb more of the nutrient than will a nondeficient animal. Different strains or breeds of poultry and livestock have different requirements for certain nutrients. The Leghorn chick was found by

52

Calcium and Phosphorus

Edwards and Lanza (1981) to have a higher requirement for P than does the broiler chick. They also reported that the Single Comb White Leghorn chick was able to utilize considerably more phytate P than did the broiler chick. Work with triplet sheep suggested the possibility of a sizable genetic difference in P absorption (Field et al., 1983). Chavez et al. (1989) compared Angus and Simmental cows and found apparent absorption (% of intake) of P to be lower for the Simmental breed. Calcium and P requirements are highly dependent on the level of productivity and the physiological state of the animal (NRC, 1996,2001). High-yielding milking cows obviously require much more dietary Ca and P than do low-yielding cows. Buchannan-Smith (1978) estimated the Ca and P requirements for a dairy cow weighing 450 kg and producing 4 kg milk daily to be 18.4 g Ca and 17.6 g P. However, a similar cow producing 10 or 30 kg of milk daily would require 34 and 86 g Ca and 29 and 67 g P, respectively. Increasing growth gains of finishing pigs by use of porcine somatotropin likewise increased P requirements to maximize bone traits and carcass lean deposition (Carter and Cromwell, 1998,a,b). Young pregnant animals that are lactating and still growing have high Ca and P needs. A young pregnant beef cow during her first lactation would have substantially higher mineral requirements than would a mature dry cow. The requirements recommended for growth in turkeys and broilers have shown an upward trend in recent years, mainly because the type of bird has changed and rate of growth has increased considerably (NRC, 1994). Human recommendations for Ca in different countries vary widely from a low of 400 mg/day for women in Thailand to a high of 1000 for both sexes over 75 years of age in the Netherlands. For humans, the DRI (2001) allowance for Ca is 700 to 900 rug/day, and for lactation 1000 to 1300 mg/day (Table 2.1). The subcommittee accepts that a 1 : 1 ratio of Ca to P will provide sufficient P, but if the Ca intake is adequate, the precise ratio of these minerals is less important. For most human diets, deficient Ca intakes are much more likely than are inadequate P intakes. The results of several studies suggest that women do not need extra Ca during breast feeding (Prentice, 1998). The Ca requirements for elderly people suggested by the RDA (1989) may be low (Wood et al., 1995). Calcium intakes by girls and adult females should approximate or exceed the Recommended Dietary Allowances so that peak bone mass can be achieved during early adulthood and bone mass can be maintained thereafter until menopause (Anderson, 1996). In a recent Ca supplementation trial, prepubertal girls with spontaneous calcium intake below 900 mg/day, when given additional calcium, had greater increases in bone mineral density than other girls. This lends support to the recently recommended adequate intake of 1300 mg/day Ca for American and Canadian children (Carter and Whiting, 1997).

Vll. NATURAL SOURCES Concentrates and roughages vary widely in their content of Ca and P. Calcium concentrations in forages are much greater than those in cereal grains. Legumes

Natural Sources

53

contain large amounts of Ca, while cereal grain straws contain relatively low amounts. Cereal grains are extremely low in Ca. Legume seeds, notably soybeans, are higher, and the same is true of the oilseed meals. All seeds and their products, however, are poor sources of Ca in terms of animal body requirements. Grass hays, such as timothy, are also poor in contrast to legume hays, which are rich. On a dry basis, milk exceeds grass but not legume hays in Ca content. Much richer in Ca than any other feeds are the animal by-products containing bone, such as animal carcass residue (tankage), meat scrap, and fishmeal. A 60% protein tankage will furnish four or five times as much Ca as will legume hay or skim milk and twenty times as much as will vegetable protein concentrates such as the oilseed meals (Maynard et al., 1979). These same feeds are very different in P content. Here the seeds are uniformly higher than the roughages; and seed by-products, such as rice bran and oilseed meals, are especially rich in P. Dried milk and animal products containing bone can be classed as rich in both Ca and P. Tankage supplies a high amount of Ca and P. The milled flours are lower in both Ca and P than whole seeds. Milk is a natural source of high-quality Ca and P for growing mammals. Calcium and P concentrations in milk are relatively constant and little affected by diet, but they differ between regular milk and colostrum. Salih et al. (1987) reported Ca and P concentrations to be significantly higher in colostrum than in 3 months postpartum milk for Brahman beef cattle, 0.143 versus 0.128% Ca and 0.127 versus 0.090% P, respectively. While composition data in feed tables are useful to show the differences among the various kinds of feeds, they are not exact values because the Ca and P contents of feeds, especially the roughages, are highly variable. Concentrations of Ca and P in crops and forages are dependent on soil factors, plant species, stage of maturity, yield, crop management, climate and soil pH (McDowell, 1985; see also Chapter 17). Variations in mineral content of grasses occur with increasing age of the plant (Gomide, 1978). In most circumstances, P concentrations decline dramatically as forages mature. Forage Ca concentration is less affected by advancing maturity (Gomide, 1978), thereby resulting in a detrimental widening of the ratio of Ca with other elements (i.e., a wide Ca: P ratio). It has been assumed that about one third of the P in feedstuffs of plant origin is biologically available to nonruminants. Research, however, has demonstrated major differences in the biological availability of P. The long accepted rule-ofthumb that one third of the P in plant feedstuffs is available, is not true for all feedstuffs. Table 2.2 presents an estimate of the bioavailability of P in feedstuffs for pigs (Cromwell, 1989). Availability of P in corn in seven experiments was found to be very low, ranging from 9 to 29% with an average of 14% (Cromwell, 1989). High-moisture grains had more available P. The P in high protein meals of animal origin is considerably more available than that in meals of plant origin. For example, in fish meal and blood meal, the availability estimates approach 100%. For ruminants, broiler litter and swine wastes were reported to be good sources of available P (Cook and Fontenot, 1990).

Calcium and Phosphorus

54

TABLE 2.2 Bioavailability of Phosphorus in Feedstuff for Swine" Ingredient

Cereal grains Corn Corn. high moisture Corn. pelleted Grain sorghum (milo) Grain sorghum, high moisture Barley Oats Wheat Grain by-products Hominy feed Corn gluten feed Distillers grains with solubles Wheat bran Rice middlings Rice bran

Average availability? (%)

14 49 II

19 43 31 30 50 14 59 71 35 45 25

Ingredient

Average availability (%)

High-protein meals (plant) Soybean meal, dehulled Cottonseed meal Peanut meal Canola meal Safflower meal Palm kernel cake High-protein meals (animal) Fish meal Meat and bone meal Blood meal Dried whey Miscellaneous Alfalfa meal Soybean hulls Inorganic phosphates Dicalcium phosphate Deftuorinated phosphate Steamed bone meal

25 15 12 21 3 II

102 76 92

76 <100 78 105 87 82

a Adapted

from Cromwell (1989). are relative to the availability of P in monosodium phosphate. which is given a value of 100. Values are based on slope-ratio assays of bone ash and/or bone-breaking strength in pigs. b Averages

Availability of Ca and P may vary considerably according to their chemical combination or physical association with other compounds in feeds. Phytic acid, which is formed from six phosphate molecules combined with myo-inositol, hinders intestinal absorption of P, Ca, and other minerals, including Fe, Mn, and Zn (see Chapter 17). One molecule of phytic acid can bind an average of 3 to 6 moles of Ca to form insoluble phytates at the pH of the intestine. In grains and plant protein supplements, about one half to two thirds of the P is in a less available phytate form. The proportion of total P contained as phytate P may range from 59 to 70% in cereal seeds, 20 to 46% in legume seeds, and from 34 to 66% in oilseed meals (Eeckhout and de Paepe, 1994). Utilization of phytate P is influenced by phytase present in plant materials or synthesized by ruminal microflora, and by intake of vitamin D, calcium, and Zn, as well as by such factors such as alimentary tract pH and dietary ratio of Ca and P (Biehl and Baker, 1997; Qian et al., 1996, 1997; Li et al., 1999). Calcium exaggerates the inhibition of Zn absorption by phytate while vitamin D needs are higher to counteract high phytate intakes.

Natural Sources

55

Studies with swine and poultry have indicated that increasing the dietary Ca : total P ratio has a negative effect on phytate utilization from added phytase. High levels of dietary Ca have also been shown to decrease phytate utilization in hens (Van der Klis and Versteegh, 1996). For broilers and turkeys, ratio of Ca: P above 1.4: I had an adverse effect on the release of P from phytate; whereas added Vitamin 0 3 had a small positive effect (Kornegay, 1996). Phytin is especially high in bran of cereal grains and oilseed meals. About 20 to 50% of phytin P is available to the pig. A good guide is to assume that no more than about 50% of the P in plant feeds is available to the pig (Cunha, 1977). There is disagreement concerning the ability of poultry to utilize phytin P (NRC, 1994). Most data, however, indicate that the utilization of phytin P, by young or adult poultry, is negligible if dietary Ca concentrations are sufficient to meet the birds' requirements. Some reports suggest, however, that the older bird has ability to use most of the phytin P. Many cereal grains contain the enzyme phytase, which is capable of splitting P from myo-inositol and leaving the P, Ca, and other minerals attached to it available for absorption. Rye, in particular, and also wheat contain enough phytase to lead to considerable destruction of phytic acid. Thus, although 50% or more of the P in the original whole grain may be in the form of phytic acid, the amount in the final product may be very much less. At the other extreme, oats contain little phytase. Phytin P may be almost totally unavailable to the pig unless the phytase of grains or other sources is present in the diet. Ruminants utilize phytin P quite satisfactorily because of consumption of dietary phytases and abundant phytase production by ruminal microorganisms (Morse et al., 1992). Before the rumen is developed, phytate is less utilized. In recent years, great strides have been made to develop microbial phytases to break down phytate in natural feed sources. The addition of microbial phytase, produced from a genetically modified Aspergillus niger strain, to high phytate corn-soybean meal diets fed to poultry (Denbow et al., 1995; Yi et al., 1996; Orban et al., 1999; Urn et al., 1999) and swine (Cromwell et al., 1993; Lei et al., 1993; Radcliffe and Kornegay, 1998; Rodehutscord et al., 1999) has been beneficial. Some studies have shown the bioavailability of phytate P to be improved by 20 to 30%. Lei et al. (1993) reported that supplemental microbial phytase improved phytate P utilization by pigs and reduced fecal phosphorus excretion. Cereal phytase from wheat bran fed from weaning through finishing pigs was also shown to be almost as effective as microbial phytase in improving phytate P utilization for body weight gain, but not for bone mineralization (Han et al.. 1997). Rodehutscord (1998) reported P digestibility increased in soybean meal and corn by 40% and canola meal by close to 50% for swine fed microbial phytase. Coon and Leskey (1998) determined the effect of phytase on phytate degradation in eight common feed ingredients with chicks. For all feeds studied, microbial phytase improved P retention by 41.2%, varying from 44.3 to 54.8% for barley, soybean meal, corn and wheat middlings, and 30.7 to 34.4% for wheat, canola meal, sorghum and rice bran. In addition to improving P retention, improved Ca retention of broilers (Schoner et al., 1993) and laying hens (Gordon and Roland,

56

Calcium and Phosphorus

1998) has been found with supplemental phytase. For Rainbow trout, microbial phytase increased P digestibility from 25 to 57% (BASF, 1999). Forages are often satisfactory sources of Ca for grazing livestock; particularly when they contain leguminous species. Reviewing the world literature on forage Ca (1263 samples), Minson (1990) reported that 31% of forages were low in Ca «0.3% OM). A similar proportion of 1123 samples of forage collected in Latin America (McDowell et al., 1977) and 103 Caribbean grass samples (Devendra, 1977) contained less than 0.3% OM Ca. Temperate forages generally contain more Ca than those grown in the tropics. Hay from Ireland had a mean Ca concentration of 0.8% OM (Wilson et al., 1968), which is similar to that reported by the Pennsylvania State Forage Test Service for over 9500 forage samples grown in 5 years (Adams, 1975). The mean level ofCa was 0.84% OM, with mean values of 0.4 and 1.15% OM for grass and legume hays, respectively. Legumes are significantly greater sources of Ca than grasses. The leaves often contain twice as much Ca as the stems. From temperate pastures (growing perennial ryegrass and white clover) fed fresh to sheep, apparent availability of Ca and P was found to be 21 and 17%, respectively (Grace and McCrea, 1972). Alfalfa meal is quite high in Ca (1.8%), but several studies indicate that the Ca is poorly available. The low availability of Ca in alfalfa may be owing to the presence of oxylates in alfalfa. Ward et al. (1979) followed the fate of Ca oxalate in alfalfa hay. They found that approximately 20 to 30% of the Ca in alfalfa was Ca oxalate. In the case of spinach for humans, there is usually enough oxalic acid present to render all of its Ca unavailable (Maynard et al., 1979; Weaver et al., 1997). On the other hand, the Ca of kale, which contains practically no oxalic acid, is nearly as well assimilated as that of milk. Certain tropical grasses contain high levels of oxalate (Kiatoko et al., 1978; Pimentel and Thiago, 1982). Swartzman et al. (1978) reported that 1% oxalic acid in equine diets reduced Ca absorption approximately 66%. Field cases of Ca deficiency have been reported in horses grazing grasses containing 2.6 to 4.3% oxalate (Groenendyk and Seawright, 1974). Most feedstuffs commonly used in swine and poultry diets contain very limited amounts of Ca. For example, corn contains only 0.01% Ca, and soybean meal, about 0.25% Ca; hence, the Ca contributed by these two ingredients in a typical corn-soybean meal diet is only about 0.05%. Whether the Ca in these feedstuffs is highly, moderately, or poorly available is probably of little consequence, because the Ca contributed by these natural ingredients represents such a small fraction of the total dietary Ca requirement (Cromwell, 1989). In many livestock-grazing regions, forages are deficient in P. In the 1974 edition of Latin American Tables of Feed Composition, the number of forages containing average P values was 1129. Of the forages from these feed tables, 72.8% of the P values «0.30%) were borderline to deficient in this element for most classes of grazing livestock (McDowell et al., 1977). Table 2.3 summarizes forage Ca and P concentrations from seven countries of Latin America, Africa, and Asia. Most samples were deficient in P «0.25%), with many forages also low in Ca, depending on the season of the year. From Costa Rica, 1468 forage samples contained 0.18%

Deficiency

57

TABLE 2.3 Forage Calcium and Phosphorus for Selected Warm-Climate Regions Calcium

Location

Season

Sample number

Malawi"

Dry Wet Dry Wet Dry Wet Dry Dry Wet Dry Wet Dry Wet

21 48 8 16 20 84 69 36 35 84 84 18 18

Bolivia" Bolivia" Dominican Republic"

Colombia" Guatemala! Indonesia"

Phosphorus

Mean

(Percentage of samples below 0.30%)

Mean

(Percentage of samples below 0.25%)

0.63 0.25 0.49 0.44 0.21 0.25 0.48 0.16 0.16 0.29 0.34 0.36 0.44

13 81 8 17 90 57 24 95 100 71 32 39 28

0.19 0.25 0.20 0.18 0.15 0.12 0.17 0.16 0.17 0.23 0.26 0.17 0.18

75 56 75 89 100 100 83 92 83 57 60 89 89

"Mtimuni et al. (1990). bMcDowell et al. (1982). 'Peducasse et al. (1983). dJerez et al. (1984). 'Vargas et al. (1984). 'Tejada et al. (1987). 'Prabowo et al. (1991).

P in the wet season, and 1335 samples averaged 0.11% P in the dry season (Vargas and Fonseca, 1989). Forage concentrations from II ranches in Venezuela averaged 0.08% P during the wet and 0.06% P for the dry season (Velasquez, 1978, Faria et al., 1981). Young growing grass pastures are often adequate in P (>0.3%) during early growth but then decline rapidly in P at increased stages of maturity (McDowell, 1997).

VIII. DEFICIENCY

A. Effects of Deficiency Failure of normal Ca and P nutrition may occur at any time of life when the supply of these elements and the factors concerned in their assimilation, notably vitamin D, are not adequate to meet functional needs. The outstanding disease of Ca and P deficiency is rickets, a decreased concentration of Ca and P in the organic matrices of cartilage and bone. A deficiency of Ca and P results in signs and symptoms similar to those of a lack of vitamin D. In the adult, osteomalacia is the

Calcium and Phosphorus

58

counterpart of rickets and, because cartilage growth has ceased, is characterized by a decreased concentration of Ca and P in the bone matrix. Clinical signs of Ca or P deficiency are seen mainly in the young. Deficiency results in an inhibition of growth, loss of weight, and reduced or lost appetite before characteristic signs in the bone system become apparent. There is decreased mineralization of the bones, resulting in lameness and fractures, which are alike at all ages, though during the formative stage, abnormalities of growth that result in misshapen bones are more common. During periods of inadequate intake, withdrawal of minerals does not take place equally from different parts of the skeleton (Underwood and Suttle, 1999). The spongy bones, ribs, vertebrae, and sternum, which are the lowest in ash, are the first to be affected, together with the cancellous ends of the long bones. The compact shafts of the long bones, such as humerus, femur, and tibia, and of the small bones of the extremities, are the last reserves to be used. In each case, the essential change is a reduction in the total mineral content of the bones, with little alteration in the proportions of the minerals in the remaining ash. Inadequate nutrition for bone growth results not only in an arrest of its normal development but also in various structural abnormalities. In severe and prolonged failure of adequate nutrition, the tension of the muscles pulls the weakened bones out of shape, and the weight of the body causes the leg bones to bend and even to fracture. Rapid growth accelerates the development of rickets, probably caused in part by the demands for P in muscle formation (Maynard et al., 1979). The same disruption of the orderly processes of bone formation with Ca, P, or vitamin D deficiency occurs in animals as it does in humans. Outward signs of rickets include the following skeletal changes, varying somewhat with species depending on anatomy and severity: I. 2. 3. 4.

Weak bones, causing curving and bending of bones, Enlarged hock and knee joints, Tendency to drag hind legs, and Beaded ribs and deformed thorax.

Although there are species differences in the susceptibility of different bones to such degenerative changes, differences that probably reflect bodily conformation (e.g., pig compared with sheep) and stance (e.g., humans compared with the common quadrupeds), there is nevertheless an apparent common pattern (Abrams, 1978). Spongy parts of individual bones, and bones relatively rich in such tissues are first and worst affected. As in simple Ca deficiency, the vertebrae and the bones of the head suffer the greatest degree of resorption. Next come the scapula, sternum, and ribs. The most resistant bones are metatarsals and shafts of long bones. Naturally occurring deficiencies of Ca and P in domestic animals usual1y develop in quite different circumstances. Phosphorus deficiency is predominantly a condition of grazing ruminants, especially cattle, whereas Ca deficiency is more a problem of hand-fed animals, especially pigs and poultry. Extensive grazing areas in the world contain forages deficient in P (McDowell, 1985, 1997). Calcium deficiency is inevitable for swine and poultry fed low-Ca, cereal grain-based diets, and for

Deficiency

59

high-yielding dairy cows given concentrate diets, unless they are appropriately supplemented. The disparity between the Ca and P intakes of animals consuming unsupplemented grain diets is apparent from the concentrations of these minerals in corn and wheat; corn typically contains 0.02% Ca and 0.3% P, and wheat 0.04% Ca and 0.4% P (McDowell et al., 1974). During less extreme deficiency, the first clinical sign is reduced appetite (anorexia). In P deficiency, the feed ingested and digested is used less efficiently than in the normal animal, presumably owing to a disturbance in energy metabolism. In fact, feed efficiency can be affected even more than feed intake (Long et al., 1957). No such disturbances in energy metabolism appear to arise in Ca deficiency. Reduced intake and/or feed efficiency will reduce weight gain or production of milk and eggs. Fertility is likely to be reduced in females, animals may appear listless and may have dull, dry hair coats, and pica (depraved appetite) may eventually occur. 1.

SWINE

Failure to provide adequate Ca, P, or vitamin 0 in the diet of the growing pig results in abnormalities in bone structure. A deficiency of anyone or all three of these nutrients produces rickets in young, growing animals, and osteomalacia in older swine. During growth, a deficiency of Ca, P, or vitamin 0 causes poor growth (Fig. 2.4), lameness, stilted gait, a general tendency to go down or lose the use of the limbs (posterior paralysis), frequent cases of fractures, softness of bones, bone deformation, beading of the ribs (Fig. 2.5), enlargement and erosion of joints, and unthriftiness (Cunha, 1977). Poor growth is primarily a sign of a P deficiency, while growth retardation results only when Ca is severely deficient over a long period while P is adequate. Bones may also be deformed by the weight of the animal and the pull of body muscles. Researchers have reported that rapid fetal bone development during the last month of gestation and long lactation periods with high milk production result in high physiological mineral requirements, which contribute to inward bone-tissue depletion of Ca and P reservoirs. Researchers have also found that bones of the axial skeleton were more responsive to demineralization process than were the long bones or the skeletal extremities. Therefore, posterior paralysis resulting from vertebra demineralization to support the sow's reproductive Ca and P demands is a logical response to the nutrient insufficiency of these minerals (Mahan and Vallet, 1997). The problem occurs most frequently in sows producing high levels of milk toward the end or just after the termination of lactation. Foot lesions are known to be closely associated with lameness and have increased in confinement-housed swine (Penny et al., 1963; Fritchen, 1979). Surveys by Jones (1967) have indicated that 20 to 30% of sows are culled from the herd because of lameness and posterior paralysis, with the prevalence higher in older sows. Although few long-term studies have been conducted, older sows have had more leg and vertebra fractures that appeared to be exacerbated when low dietary Ca and P levels were fed.

60

Calcium and Phosphorus

Fig. 2.4 Phosphorus deficiency. Litlermates received purified diets for five weeks. (Right) received diet with 0.2% phosphorus; (left) received diet with 0.6% phosphorus. (Courtesy of D.E. Ullrey, Michigan State University, East Lansing.)

Fig. 2.5 Beading of the ribs. Large knobs develop at the juncture of the bony with the cartilaginous parts of the ribs. This is caused by a calcium or phosphorus deficiency. (Courtesy of the late G. Bohstedt, University of Wisconsin)

The chemical changes in the bones that precede or accompany the structural changes are striking (Fig. 2.6). Growing pigs fed a corn-soybean diet containing 0.34% P for 9 weeks had bones that were less well mineralized. Pigs receiving 0.34 compared to 0.54% P had the following rib bone analyses; total ash, 53.2 versus 60.4; Ca, 20.1 versus 22.5; and P. 10.0 versus 11.5 (Harmon et al., 1970). Total bone

Deficiency

61

Fig. 2.6 X-ray reproductions of the left femur of pigs 35 days of age receiving various levels of phosphorus for a 28-day period. The number at the end of the row represents the level of phosphorus (0.10, 0.14. 0.20. 0.28. and 0.40%) in the diet. Note the increased density as the level of phosphorus in the diet increased. (Courtesy of G.E. Combs. University of Florida. Gainesville and Iowa State University, Ames)

ash contents, breaking load, trabecular bone volume, and mineral apposition rate all decreases as the Ca intake decreased in growing pigs (Eklou-Kalonji et al., 1999). A dietary Ca: P ratio of less than I : I may result in excessive bone resorption in relation to deposition, with replacement of bone by fibrous tissue (fibrous osteodystrophy) (Brown et al., 1966). This is a manifestation of nutritional secondary hyperparathyroidism induced by continued low serum Ca concentration is generalized throughout the skeleton and, in severe cases, results in spontaneous fracture of long bones and of ribs, as in rickets. The high P content of many plant materials probably contributes to the frequent occurrence of this nutritional imbalance. Prevention is achieved by dietary Ca adequacy and by maintenance of a Ca: P ratio greater than I : l. Low dietary levels of Ca and P fed to gestating swine can reduce litter size (Kornegay et al., 1973; Harmon et al., 1975) without affecting neonatal bone ash (Mahan and Fetter, 1982). The importance of P in reproduction was illustrated by

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McCrea et al. (1979). Feeding higher than NRC recommendations for Ca and P to first-litter gilts during 3 years of a project resulted in 1 to 1.5 more pigs weaned per litter compared with similar gilts fed those minerals close to the NRC suggested values. However, most studies have demonstrated that when dietary Ca and P levels meet or exceed NRC (1998) standards, pig birth weight and litter size (total born, live born, stillbirths, number weaned) were not affected (Mahan, 1990). 2.

POULTRY

Little difference exists among poultry species in relation to clinical signs of deficiency. Clinical signs in all poultry species would be rickets and lowered growth rate, egg production, and hatchability. Anorexia is one of the first clinical signs of a P deficiency, but it does not specifically indicate a P deficiency. Death, particularly in laying hens, will occur when P is deficient. Likewise, duck mortality was high (65%) along with severely stunted growth and lack of appetite (Cui et al., 1997). Loss of appetite, weakness, and death can occur in a severe P deficiency within 10 to 12 days (Scott et al., 1982). A less severe deficiency causes rickets and growth failure (Fig. 2.7). The principal clinical sign of both Ca and P deficiency in growing poultry

Fig. 2.7 Phosphorus deficiency. Poult on the right received inadequate phosphorus. (Courtesy of G.D. Butcher, University of Florida. Gainesville)

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is reduction of bone mineralization, with bones becoming fragile; ash and Ca contents can be reduced to about one-half normal. No minerals are more important to commercial egg producers in maintaining eggshell quality and embryonic development than Ca and P. The effects ofCa levels in breeder diets on embryonic development is primarily through eggshell quality. Poor shells due to Ca-deficient breeder diets result in excessive egg weight loss, increased contamination and embryos with stunted growth, poor bone development, and increased mortality at the end of incubation (Wilson, 1993). Phosphorus is necessary to support normal embryonic bone development and hatchability (Waldroup et al.. 1967). Breeders on litter floors appear to recycle considerable amounts of P by coprophagy (Wilson et al., 1980). Excess P intake through a combination of dietary and coprophagic sources can reduce shell quality and, indirectly, decrease hatchability. Roland (1985) summarized many references pertaining to the numerous clinical signs associated with the effects of Ca and P deficiency for the laying hen. Clinical signs included (I) cessation or reduction of egg production; (2) reduced feed consumption and efficiency; (3) reduced eggshell quality (i.e., decreased breaking strength, egg specific gravity, shell thickness, and shell weight); (4) inferior egg quality (i.e., blood spots, yolk mottling); (5) decreased egg size and weight; and (6) impaired reproduction (i.e., reduced hatchability, dead, weak, or deformed offspring, decreased mating activity, delayed sexual maturity). Skeletal abnormalities included increased bone resorption, cage fatigue, osteoporosis, rickets, osteomalacia, soft beak, osteodystrophy fibrosa, paralysis, muscular stiffness, lameness, beaded ribs, enlarged and painful joints, weak bones, easily broken or bent and abnormal posture, and mishappen bone (i.e., arching of back, rigidity, stiffness, shafts of bone bent outward, bowleggedness). Pullets, at the beginning of the laying period, undergo considerable metabolic stress with the need to supply approximately 2.4 g Ca daily to the oviduct for shell formation (NRC, 1994). Withdrawal of Ca from the skeletal reserves takes place normally in response to the intense demand of egg-laying. Some birds mobilize large amounts of Ca from their skeleton during this period; the bones may become so demineralized that the birds are unable to stand and appear paralyzed. The sternum and rib bones are frequently deformed, and all bones are easily broken. Dietary management to prevent this condition (generally termed cage-layer fatigue but more precisely described as osteoporosis) has not been devised (NRC, 1994). Birds affected with cage-layer fatigue appear paralyzed and cannot rise or stand on their legs. They eventually die of starvation. Even though the exact cause for cage layer fatigue is yet to be identified, a disturbance of the hen's mineral-electrolyte balance is thought to be involved. It is important for laying hens to consume adequate daily concentrations of Ca, P, and vitamin D so as to decrease the incidence of cage layer fatigue. Osteomalacia (rickets of adult birds) is commonly present with this condition and in layers under 30 weeks of age, the cause is usually a temporary Ca deficiency resulting from delayed feeding of high Ca feeds during egg production peaks above 80%. If the hen's daily feed intake does not provide adequate Ca for egg shell formation, more bone is utilized and osteoporosis develops. The bones are

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more subject to fracture and in many cases the thigh bone will fracture. If this occurs, the hen is crippled and unable to stand and in this instance cage layer fatigue is easily diagnosed (Schwartz, 1977). It is also common to see field cases of cage layer fatigue without fractures and it has been suggested that a lack of exercise in the cages can lead to fatigue in some hens. In these cases, if the hens with "fatigue" are placed in floor pens they usually recover. Manifestations of Ca deficiency in the laying hen can become evident within a few days or at most a few weeks on the deficient diet (Buckner et al., 1930). A gradual diminution in the ash content of the eggshells produced by hens abruptly deprived of dietary Ca is apparent in the experiments of Deobald et al. (1936). Egg production had virtually ceased by the twelfth day after removal of dietary Ca, and the ash content of the eggshells from some of the birds was less than 25% of normal. Phosphorus deficiency is similarly manifested by a decline in egg yield, hatchability, and shell thickness, but it is much less likely than Ca deficiency because of the smaller requirement and higher levels found in concentrate diets. In broilers, the bone condition known as tibial dyschondroplasia is related to Ca and P balance. Investigations into the role of Ca and P in tibial dyschondroplasia in young chicks by Edwards and Veltmann (1983) indicated high P levels seem to promote the development of the lesion. Increasing the Ca level in the diet prevents development of the lesion. 3.

RUMINANTS

Lack of Ca, P, or vitamin D results in improper bone mineralization; bones become weak, soft, and lack density. Signs include swollen, tender joints; enlargement of the ends of bones; an arched back; stiffness of the legs; and development of beads on the ribs. For caribou, P deficiency limited growth of antlers (Moen et al., 1998). Shupe et al. (1988) reported the effects of feeding low-P diets to cattle for a 10-year period; the animals developed an insidious and subtle complex syndrome characterized by weight loss, rough hair coat, abnormal stance, and lameness. Spontaneous fractures occurred in the vertebrae, pelvis, and ribs. In severely affected cows, fractures did not heal properly. Some bones were demineralized markedly, and the surfaces were porous, chalky white, soft, and fragile. Chemical, physical (Fig. 2.3), and mechanical properties of bone illustrate extreme bone demineralization when ruminants consume inadequate P (Williams et al., 1990, 1991a,b). If conditions leading to rickets are not corrected, calves will develop bowed and deformed legs, because of the effects of muscle tension and weight on the weak, soft leg bones. Dairy cows that have lactated heavily and that have been fed insufficient Ca are prone to develop osteomalacia, resulting in fragile, easily fractured bones (Fig. 2.8) and reduced milk yields (Arnold and Becker, 1936). A deficiency of Ca can also lead to muscle weakness and eventually tetany if the deficiency is prolonged and severe enough. Generally, both Ca and P are not limiting in the same animals. Grazing ruminants are most likely deficient in P owing to low concentrations of the element

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Fig. 2.8 Both hips of the cow shown above were broken (knocked down) as a result of a low-Ca ration. Her skeletal reserve of Ca was depleted to the point that her weakened bones were broken easily. The right photo shows the pelvic bones of a herd mate broken in three places. (Courtesy of the late R.B. Becker, University of Florida, Gainesville)

in forages, while ruminants consuming high-concentrate diets (i.e., animals on finishing diets and high-producing dairy cows) would be most likely to be deficient in Ca. Calcium deficiency may occur in sheep fed largely grain diets, with limited grazing available. Severe growth stunting, gross dental abnormalities, and some deaths were observed in lambs and young, weaned sheep fed a wheat-grain based diet without additional Ca (Franklin et al., 1948). Milk fever (parturient paresis) is a paralyzing metabolic disease caused by hypocalcemia near parturition and initiation of lactation in high milk-producing dairy cows. Milk fever is an impaired metabolic condition that is related to Ca status, previous Ca intake, and malfunction of the hormone form of vitamin D 1,25-(OHhD and PTH. Milk fever usually occurs within 72 hr after parturition and is manifested by circulatory collapse, generalized paresis, and depression of consciousness. The most obvious and consistent abnormality displayed is an acute hypocalcemia in which the serum Ca drops from a normal of 8 to 10 mg% to levels of 3 to 7 mg% (average, 5 mg%). Early in the onset, the cow may exhibit some unsteadiness as she walks. More frequently the cow is found lying on her sternum with her head displaced to one side, causing a kink in the neck, or turned into the flank. The eyes are dull and staring, and the pupils dilated. If treatment is delayed many hours, the dullness gives way to coma, which becomes progressively deeper, leading to death. Milk fever in dairy cows is caused by a temporary imbalance between Ca availability and high Ca demand following the onset of lactation (Oetzel, 1996).

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Calcium leaves the extracellular fluid to enter the mammary gland faster than it can be replaced by intestinal Ca absorption or bone Ca resorption (Goff and Horst, 1993). Despite much research, milk fever incidence has remained steady in the United States at 8 to 9% (Goff, 1989). Research has shown that milk fever is associated with dramatic increases in incidence of mastitis, ketosis, dystocia, displaced abomasum, and retained placenta (Curtis et al., 1983). Milk fever is an economically important disease and can reduce the productive life of a dairy cow by 3.4 years. Each case of milk fever leads to a loss of $334 to the producer by way of treatment charges and milk loss (Horst et al., 1997). If left untreated, about 60 to 70% of the cows die. Aged cows are at the greatest risk of developing milk fever. Heifers almost never develop milk fever. Older animals have a decreased response to dietary Ca stress due to both decreased production of 1,25-(OHhD and a decreased response to the 1,25-(OHhD. Target tissues of milk fever cows may have defective hormone receptors and the number of receptors declines with age. In the older animal, fewer osteoclasts exist to respond to hormone stimulation, which delays the ability of bone to contribute Ca to the plasma Ca pool (Goff, 1989). The aging process is also associated with reduced renal lc-hydroxylase response to Ca stress, therefore, reducing the amount of 1,25-(OHhD produced from 25-0HD (Goff et al., 1991 a). Attempts to prevent milk fever, which have been quite successful, include prepartum diets with a narrow Ca: P ratio and diets higher in anions than in cations (Horst et al., 1997; Goff and Horst, 1998; Pehrson et al., 1998; Vagnoni and Oetzel, 1998; Dhiman and Sasidharan, 1999). Parturient paresis, a disturbance of metabolism in pregnant and lactating ewes, is characterized by acute hypocalcemia and the rapid development of hyperexcitability, ataxia, paresis, coma, and death. The disease occurs any time from 5 weeks before to 10 weeks after lambing, principally in highly conditioned older ewes at pasture. The onset can be associated with an abrupt change of feed, a sudden change in weather, or short periods of fasting imposed by circumstances such as shearing or transportation. Prevention is largely a matter of avoiding the predisposing causes, with treatment consisting of injection of Ca (often with some Mg). As with dairy cattle, use of a more anionic diet may be useful for parturient paresis control in sheep (Wilson et al., 1998). Deficiencies of Ca and P are influenced by availability of different sources and interrelationships with additional mineral elements or nutrients. In India, Ca deficiency is reported for cattle fed straw, because of the large amounts of oxalates in this feed (Ray, 1963). Other countries reporting deleterious effects to cattle from oxalates are Costa Rica (Kiatoko et al., 1978) and Brazil (Pimental and Thiago, 1982). There is some suggestion that unfavorable cation-anion balance is a greater problem than oxalates (Sanchez et al., 1998). In many tropical countries, high amounts of soil Fe, Ca, and Al accentuate a P deficiency by forming insoluble phosphate complexes. Soils in humid tropics are characteristically acid with high percentages of Fe and exchangeable AI, which complexes with P, making it unavailable to plants. Also, direct soil consumption by grazing animals can result in adverse effects due to high intakes of Fe and AI (Rosa et al., 1982).

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Calcium deficiency is much less an area problem for grazing livestock than is P inadequacy. Todd (1967) states that, with the exception of cereals and their byproducts, most animal feeds contain between 0.2 and 1.0% Ca in the dry matter, and that uncomplicated Ca deficiency is rare in grazing livestock unless the pasture contains less than 0.2% Ca. Compared to P inadequacies, Ca deficiency is uncommon in grazing cattle, with the exception of high milk-producing cows or those grazing on acid, sandy, or organic soils in humid areas where the herbage consists mainly of quick-growing grasses with no legumes. Calcium deficiency is infrequently reported in grazing beef cattle even during lactation (Loosli, 1978). A lower incidence of Ca- than of P-related disorders is attributable to three major factors: (I) a higher concentration of Ca than of P in the leaves and stems of most plant species; (2) a wider distribution of P-deficient than of Ca-deficient soils, and (3) a lesser decline in the concentration of Ca and of P with advancing maturation of the plant (McDowell, 1997; Underwood and Suttle, 1999). For grazing livestock, the most prevalent mineral element deficiency throughout the world is lack ofP (McDowell, 1976, 1985, 1997; Underwood and Suttle, 1999). In a review (McDowell et al., 1984), P deficiency was reported in 46 tropical countries in Latin America, Southeast Asia, and Africa (see Chapter I of the volume). In most livestock-grazing areas of tropical countries, soils and plants are low in P. Many grass species with more than 0.3% P during early stages of growth are available to grazing livestock for only short periods. For the greater part of the year, mature forages contain less than 0.15% P. In the veld country of South Africa, where the classical studies of bovine aphosphorosis were made, herbage concentrations fall typically from between 0.13 and 0.18% P in the wet summer to as low as 0.05 to 0.07% P in the dry winter and can remain low for 6 to 8 months of the year (Underwood, 1981). Signs of P deficiencies are not easily recognized except in severe cases characterized by fragile bones (Fig. 2.9), general weakness, weight loss, emaciation,

Fig. 2.9

Lamb fed a P-deficient diet and showing a typical knock-kneed condition. (From NRC, 1975)

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stiffness, reduced milk production, and chewing of wood, rocks, bones (Fig. 2.10), and other objects. In one study, a complete blocking of the desire to eat bones was achieved within an hour by intravenous infusion of sodium phosphate sufficient to raise serum inorganic P to normal (Underwood, 1981).

Fig. 2.10 Bone chewing is often associated with a phosphorus deficiency. A cow (top) chewing bone in the Llanos regions of Santa Maria de Ipire, state of Guarico, Venezuela (L.R. McDowell, University of Florida, Gainesville). A bone (bottom) that was being consumed in Argentina. Saliva could still be seen on the bone when the photo was taken. (Courtesy of Bernardo Jorge Carrillo, C.I.C.V., INTA, Castelar, Argentina)

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Reduction in feed intake is a major consequence of P deficiency in ruminants (Field et al., 1975; Bortolussi et al., 1988; Ternouth and Sevilla, 1990). Dry matter intake in lambs was reduced 40 to 41% for animals receiving a low-P diet compared to controls (Ternouth and Sevilla, 1990). Also, lambs offered low-P diets had lower dry matter digestibility coefficients than those offered the high-P diets. Four possible feedback mechanisms to the satiety center that may result in reduced feed intake have been proposed (Ternouth, J .H., 1991, personal communication): 1. Low ruminal P results in reduction in fiber digestion by limiting microbial activity (i.e., the same phenomenon that occurs in a dietary nitrogen deficiency). 2. Low ruminal P reduces microbial protein synthesis, thus reducing the intestinal absorption of amino acids (AA) so that animal becomes AA deficient. 3. Low P in metabolically active tissues (for example, muscle and liver) results in reduced P for intermediary metabolism. 4. Low P in metabolically active tissues results in reduced RNA synthesis, affecting the metabolic activity of cells. Lower feed intake for grazing livestock with a P deficiency may relate to lameness (Jubb and Crough, 1988). Animals that must graze extensive areas to meet nutrient needs would be unable to walk the necessary distance if they were lame as a result of P deficiency. In northeastern Australia, the descriptive term peg-leg is used for the P-deficient grazing cattle, whose stiff-gaited movements hamper their ability to secure feed and water and can result in death from exhaustion (U nderwood, 1981). Many reports from diverse regions, dating back to the early part of the century, have revealed the beneficial effects of P supplementation on overall performance. Reports of improved weight gains by P-supplemented cattle have been summarized for various regions (Tokarnia and Dobereiner, 1973; Cohen, 1975; Fick et al., 1978; Karn, 1997; McDowell, 1985, 1997, 1999). In Peru, Echevarria et al. (1973) obtained weight gains of 0.59 kg in steers supplemented with dicalcium phosphate and 0.27 kg for controls. Bolivian cattle gained 96.4 kg when receiving a source of P versus 79.4 kg for controls (McDowell et al., 1982). Heifers receiving higher dietary P of 0.20 versus 0.12% had higher gains (257 versus 205 kg) and produced calves with greater birth weights (26.9 versus 22.7 kg), respectively (Williams et al., 1992). Estevez-Cancino (1960) reported increased milk production (up to 24%) with bonemeal supplementation on farms that had P-deficient forages. The most devastating economic result of P deficiency is reproductive failure. Failure to reproduce is associated with loss of body weight and body condition, which are the result of decreased intake of feed. The decreased intake of feed could be caused by decreased appetite, impaired locomotion, or both. It remains unclear whether reduced reproduction is caused by lack of P per se or is mediated through decreased body condition (Dunn and Moss, 1992). In two-year observations of 200 South African cattle, calf crops increased from 51 % in control cattle to approximately 80% for cattle supplemented with bone meal or other P sources (Theiler et al., 1928). A Colombian study included data

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TABLE 2.4 Effect of Phosphorus Supplementation on Production Performances Rate of phosphorus supplementation High Group" Low Group

Number of farms

Abortions (%j"

Calf mortality (%)d

Yearly gain (kg)

8 8

9.7 18.8

2.7 11.6

83.2

51.6

'CIAT (1979). data summarized by W. Miles (unpublished data). from 16 ranches in the llanos of Colombia. "Most ranchers used a prepared mix that also contained calcium, sodium. iodine. and cobalt in relatively adequate quantities. while copper and zinc were at such low levels as to have little favorable effect. C Abortions are a percentage of total pregnancies. dCalf deaths during first month as a percentage of total births.

on mineral supplementation and animal performance for selected ranches [Centro Internaccional de Agricultura Tropical (CIAT) 1979]. Miles (unpublished data, 1977) summarized this information to divide 16 ranches on the basis of high or low P supplementation (Table 2.4). The impact of mineral supplementation on reducing abortions and calf deaths is impressive, with a 61% higher weight gain for those with higher intakes. Increasing reproductive performance due to mineral supplementation was summarized for 17 different experiments in Latin America, Africa, and Asia (McDowell et al., 1984; McDowell, 1985). An average from 17 experiments of 52.6% calving for animals receiving salt only contrasted with 75.6% for those receiving additional supplemental minerals. The specific mineral or minerals responsible for increasing reproductive performance in the given experiments is unclear for some experiments; however, P most likely contributed most to this improvement. The effects of undernutrition on fertility were apparent from studies in Zimbabwe and northern Australia (Lamond, 1970). With few exceptions, lactating cows were not calving two years in succession. In P-deficient areas, if a calf was produced, cows might not come into a regular estrus again until body P levels were restored, either by feeding supplementary P or by cessation of lactation. In South Africa in the early 1900s, pioneer P-supplementation studies (Van Niekerk, 1978) revealed that bovine botulism and aphosphorosis were the result of a severe P deficiency, with cattle exhibiting subnormal growth and reproduction and a depraved appetite or pica, as illustrated by bone chewing. Besides South Africa, other countries reported death from botulism as a result of bone chewing include Argentina (Fig. 2.11), Brazil, and Senegal. In the area of Piaui, Brazil, an estimated 2 to 3% of approximately 10,000 cattle die annually of botulism (Tokarnia et al., 1970). At present, botulism, rabies, and plant poisoning are the three most important causes of adult cattle mortalities in Brazil (Dobereiner et al., 1992). 4.

HORSES

A deficiency of Ca, P, or vitamin D can cause bone deformities in the horse from the weight of the animal and the pull of the body muscles on weak, porous bones.

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Fig. 2.11 A cow in Argentina suffers from botulism as a result of eating bones. The animal is weak and has difficulty rising. (Courtesy of Bernardo Jorge Carrillo, CLC.V., INTA, Castelar, Argentina)

Rickets in horses is characterized by reduced bone calcification, stiff and swollen joints, stiffness of gait, bone deformities (Le., crooked long bones), and frequent fractures. El Shorafa et al. (1979) observed early stages of rickets in ponies to include decreased bone ash, decreased cortical area and bone density, and delayed epiphyseal closure. In the mature horse, inadequate dietary Ca resulted in weakening of the bones and an insidious shifting lameness (Briggs, 1998; Frape, 1998). Lameness is frequently a characteristic of resorption of cortical bone through loss of support for tendons and ligaments. Horses fed low levels of Ca but excessive amounts of P develop a condition called nutritional secondary hyperparathyroidism (also called big head and bran disease). This causes a great deal of Ca to be removed from the facial bones, followed by fibrous connective tissue invading the area, causing enlarged facial bones (see Section X). From a survey of the severity of metabolic bone disease in yearlings and diet analyses on 19 Ohio and Kentucky horse farms, Knight et al. (1985) reported a negative linear relationship between dietary Ca intake and perceived severity of metabolic bone disease in young horses. Yearlings having the lowest incidence of metabolic bone disease were fed diets containing 1.2% Ca, whereas yearlings with the most severe metabolic bone disease were fed diets with 0.2% Ca. Nielsen et at. (1998) reported that benefit was derived from feeding a greater amount of Ca in relation to NRC requirements to the young racehorse at the onset of training. Phosphorus deficiency results in loss of appetite, which results in a craving for and eating or chewing of bones, wood, hair, rocks, clothing, and other materials. Some horses may chew up an entire mineral box. The animals also become weak,

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emaciated, lose weight, and eventually die. In certain range-grazing areas, weakened horses die after eating decayed bones from diseased animals (Cunha, 1990). Other signs of P deficiency include retarded growth rate, reduced efficiency of feed utilization, lowered milk production, failure to exhibit estrus or heat, and low conception rate. 5. OTHER ANIMAL SPECIES

a. Dogs. Rickets with typical bone lesions is readily produced in dogs (NRC, 1985a). The dog was one of the first animals in which rickets was produced experimentally. In 1922, Mellanby of Great Britain produced rickets in dogs by feeding them oatmeal (NRC, 1985a). Rickets in dogs is similar radiographically, histopathologically, and biochemically as in other animals, or human beings with the disease. Rate of bone loss and osteoporosis depends on the skeletal region involved. Jawbones show earliest signs, followed by other skull bones, ribs, vertebrae, and finally the long bones. Loss of Ca from the jawbones can lead to recession of alveolar bone and gingiva. Detachment of the teeth and other signs of deficiency may appear before compression of vertebrae and fractures of long bone. Rather severe Ca deficiency is characterized by excessive bone resorption. Calcium deficiencies may result in tetany and convulsions, reproductive failure, and spontaneous fractures. An uncomplicated deficiency of P seldom occurs in dogs except under experimental conditions. In young dogs, low P intake will lead to rickets, poor growth and a depraved appetite.

b. Cats. Severe rickets in kittens results in enlarged costochondral junctions (rachitic rosary) with disorganization in new bone formation and excessive osteoid

(NRC, 1986). Calcium deficiency in all cats eventually results in disturbance of locomotion with limping and ultimate reluctance to move. Radiographic and dualenergy x-ray absorptiometry measurements provide evidence of generalized decrease in bone density and mass, loss of fine trabeculation, and thinning of cortices (Rowland et al., 1968; Morris and Earle, 1999). The most commonly encountered nutritional bone disease in cats results from nutritional secondary hyperparathyroidism (Bennett, 1976). It is seen most often in kittens and young adult cats and is commonly associated with the feeding of incorrectly supplemented, meat-rich diets. This can be a problem also with wild feline populations (e.g., lions and tigers) kept in zoos and fed only meat instead of a combination of meat plus bone. Meat by itself is extremely deficient in Ca (0.025% dry basis), with a Ca : P ratio of I : 20. The carnivorous nature of felines precludes development of P deficiencies. c. Laboratory Animals. Bones of rachitic rats showed decreased or absent calcification with wide areas of uncalcified cartilage at the junction of diaphysis and epiphysis. Bone ash may be less than half normal (NRC, 1995). With a low-Ca diet

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(0.01 % Ca), rats have shown retardation of growth, decrease in feed consumption, increase in basal metabolic rate, reduced activity and sensitivity, osteoporosis, rearleg paralysis, and internal hemorrhage. Males failed to mate, and females did not lactate properly. In a number of rat studies, reproduction was poor, with either abnormal Ca: P ratios or low dietary content of each (McCoy, 1949). Typical rickets lesions for guinea pigs occur in the zone of cartilage proliferation at the epiphyseal plate of long bones and ribs. Also, incisors of guinea pigs exhibited a high degree of enamel hypoplasia, while enamel and dentin were disorganized and irregular with poor calcification (NRC, 1995). When guinea pigs were fed low Ca (0.03%) and P (0.02%) diets, 9 of 21 did not survive for 60 days (Howe et al., 1940). The teeth of all animals developed extreme enamel hypoplasia. For hamsters, Stralfors (1961) obtained a 54% decrease in the incidence of dental caries when the Ca content of the diet was increased from 0.40 to 0.60%. Low-Ca diets in mice resulted in decreased weight gain and bone ash. However, these effects were much less marked in mice than in rats, because the mice increased the concentration of Ca-binding protein in the intestine to provide improved Ca utilization (NRC, 1995). They also compensated by reduced skeletal growth, so that growth reduction rather than osteoporosis was a more prominent sign of deficiency. d. Rabbits. In rabbits fed deficient diets, the bones became highly demineralized and very susceptible to fractures (Cheeke, 1987). The backbone may break spontaneously. In mature animals, osteoporosis results from excessive mobilization of bone salts to maintain normal blood Ca and P levels. Although not so common as in dairy cattle, milk fever (parturient paresis) sometimes occurs shortly after parturition. With this condition, does were found lying on their sides, with jerking of the hind limbs, muscle tremors, and ear flapping. Plasma Ca (5.8 mgjlOO ml) and P (2.9 mgjlOO ml) levels were very low, while Mg (1.96 mgjlOO ml) was normal. Injection of Ca gluconate induced a rapid recovery within 2 hr, with plasma Ca and P levels returning to normal (Barlet, 1980).

e. Foxes and Mink. Rickets can be produced in both foxes and mink by feeding diets with low vitamin D content and abnormal Ca: P ratios (NRC, 1982).The main sign for mink receiving Ca-deficient diets is abnormal bone growth. Mink receiving a higher Ca: P ratio of 1.9 had reduced weight gains and the skins were significantly shorter compared to lower Ca : P ratios of 0.9 and 1.3 (Hansen et al., 1992). Fox pups on a rachitogenic diet develop a stiffness of the rear legs and begin walking on their pasterns rather than on their toes. Their leg joints swell, and the leg bones become bent. Harris et al. (1945) have supplied evidence that Ca-deficient foxes progressively show lameness, recurrent spasms, crooked legs, and enlargement of the cranial bones, especially the maxillae and palatines. The muzzles enlarge, the gums swell, and the teeth loosen.

f Fish. Less dietary-induced Ca deficiency has been described in fish, as requirements can be met by absorption from water (NRC, 1993). The Japanese eel

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and Red Sea bream had depressed growth and poor appetites and feed conversions when fed Ca-deficient diets (NRC, 1993) In most fish, the main P-deficiency signs include poor growth, feed conversion, and bone mineralization (NRC, 1993). Further signs of deficiency in carp include an increase in some gluconeogenic enzymes, increases in carcass fat with decreased water content, reduced blood phosphate levels, deformed head (frontal bone), abnormal calcification of ribs and soft rays of the pectoral fins, and a curved or abnormal spine. A low-P intake by Red Sea bream also causes curved, enlarged vertebrae; increased serum alkaline phosphatase activity; higher lipid deposition in muscle, liver, and vertebrae; and reduction in liver glycogen content (Sakamoto and Yone, 1980). For Atlantic salmon receiving a P-deficient diet, whole-body P content was reduced to 65% of the initial value, and Ca to 40% within a few weeks (Baeverfjord et al., 1998). The reductions in mineral content were most severe in the mineral-rich tissues, the bones and scales. On gross examination, all bony structures were abnormally soft. Ribs were wrinkly, and the spine displayed scoliotic changes. These changes were correlated to the reduction in bone mineralization, and were manifest at the time when reductions in growth rate could be noted. Vielma and Lall (1998) showed bone deformities and low vertebral Ca and P content in Pdeficient Atlantic salmon. g. Nonhuman Primates. Rickets has been observed in many species of nonhuman primates and is characterized by softening, demineralization, and fibrous dysplasia of bone (NRC, 1978). Most research involving rickets involved lack of available vitamin D versus lack of Ca and P. 6.

HUMANS

Deficiency ofCa, P, or vitamin D leads to the pathological bone condition of rickets, which is characterized by disordered cartilage cell growth and enlargement of the epiphyseal growth plates in the long bones. There is also a prominent accumulation of unmineralized bone matrix (osteoid) on trabecular bone surfaces. Classic bone symptoms associated with rickets, such as bowlegs (Fig. 2.12), knock-knees, curvature of the spine, and pelvic and thoracic deformities, result from the application of normal mechanical stress to demineralized bone (Collins and Norman, 1991; McDowell, 2000). Beading of the ribs, referred to as the "ricketic rosary," is almost a constant sign after the age of 6 months, and is caused by the swollen cartilaginous ends of the ribs. The chest may be narrow and rather funnelshaped, and described as a "pigeon" chest. In severe cases, this may interfere with breathing. Enlargement of bones, especially in the knees, wrists, and ankles, and changes in the costochondral junctions also occur. With these defects, the bones become structurally weak, they bend under weight of the child, and are liable to fracture. Rickets also results in inadequate mineralization of tooth enamel and dentin. If the disease occurs during the first 6 months of life, convulsions and tetany can develop.

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Fig. 2.12 Child with rickets with marked bowlegs. Note the angle of the feet. (Courtesy of Alan T. Forrester, "Scope Manual on Nutrition." The Upjohn Company, Kalamazoo, Michigan)

Osteomalacia and osteoporosis are two chronic diseases of the skeleton. Osteomalacia is the adult counterpart of rickets. Osteomalacia (softening of the bone) is the result of deficiency of Ca or vitamin D; in practice, P is not deficient in typical human diets. Osteoporosis, an atrophy of bone, results from defective formation of the protein matrix on which the minerals in bone are laid down. Osteoporosis is the bone disease most commonly encountered in clinical practice and is associated with endocrine disorders. Both osteomalacia and osteoporosis refer to demineralization of bones, and the terms, correctly or incorrectly, are often used interchangeably. Osteomalacia occurs after skeletal development is complete. As in rickets, even though bone mineralization has ceased, collagen formation continues, this results in formation of uncalcified bone matrix. In adults, the bones no longer grow in length but are constantly remodeled. The main symptoms of osteomalacia are muscular weakness and bone pain. As the disease progresses, bone fractures occur. Osteoporosis is characterized by an absolute decrease in bone mass that results in an increased susceptibility to fracture, bone deformity and localized pain, especially at the wrist, spine and hip. Fractures are most commonly the result of trauma, which may be trivial, in the elderly with osteoporosis. It is common in the elderly, especially in postmenopausal women and in elderly persons of both sexes and

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constitutes an important pubic health problem (Arnaud and Sanchez, 1996; Bronner, 1997). Age-related bone loss is believed to be a general phenomenon in humans, beginning as early as the fourth decade in females and about the sixth decade in males. The more bone mass available before the period of age-related bone loss, the less likely it will decrease to a level at which fractures will occur (Parfitt, 1983; O'Brien, 1998). Bone mass increases from infancy until the late teens, thereafter remains at a plateau until the mid-thirties, and then begins to decline. In women, there occurs a rather drastic decline in the years following menopause, whereas in men, the decline is more gradual. Some 10 years after menopause, the rate of decline of bone mass in women parallels that in men. Total bone mass is lower in women than in men. Women, in general, are more prone to osteoporosis than are men because of the small skeletal mass at maturity and because of the period of rapid bone loss following menopause. Several generalizations characterize the segment of the population that develops osteoporosis (Schuette and Linkswiler, 1984; Wardlaw, 1993; Bronner, 1997; O'Brien, 1998): (1) the bone loss that leads to osteoporosis is owing, in part, to a relative estrogen deficiency resulting in an increase in bone resorption over formation; (2) osteoporotic patients generally show a rate of higher bone turnover, which exacerbates the disparity between resorption and formation; (3) patients with osteoporosis may have a greater impairment of Ca absorption due to a reduction in the conversion of vitamin D forms 25-0H-D to 1,25-(OHhD than do other elderly individuals; (4) patients with osteoporosis may be less able to conserve body Ca by reducing urinary losses when Ca intake is low; and (5) a low level of physical activity or immobilization will accelerate the rate of bone loss. There is a controversy as to the role of dietary Ca in the development of osteoporosis. Some epidemiological studies do not show a relationship between dietary Ca and incidence of osteoporosis, but inadequate Ca intake is still believed to be a contributing factor. In one study, cortical bone loss was independent of Ca intake, but estrogen accompanied by Ca arrested the loss of trabecular bone characteristics of menopause (Bronner, 1997). However, in study of men and women over 65 years of age, daily supplementation with 500 mg Ca and 700 IV vitamin D for 3 years moderately reduced bone loss at several sites and significantly decreased the rate of nonvertebral fractures, compared with a placebo group (O'Brien, 1998). A study of two similar populations in Yugoslavia showed increased bone density and reduced fracture rate in the population with greater Ca intake (Markovic et al., 1979). The hip-fracture incidence in the Yugoslav district with a high Ca intake was 50% lower than in the low-Ca district. It was possible to demonstrate an inverse rank-order relationship between Ca intakes and osteoporotic vertebral fracture frequency (Nordin, 1966). Japanese women whose Ca intake averaged 400 mg/day had the highest frequency of fracture, whereas Finnish women with the highest intake 1300 mg/day had the lowest fracture frequency. Kidney stones are associated with low dietary Ca intake (Martini and Heilberg, 2002). A five-year clinical study found recurrence of kidney stones was higher in stone-formers on a low dietary Ca treatment.

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Assessment of Calcium and Phosphorus Status Calcium and P concentrations of feeds, in comparison to requirements of various species, is useful in determining the status of these nutrients. Inadequate intake of Ca will cause weakened bones, slow growth, a decline in egg production, hatchability and shell thickness, low milk production, and tetany (convulsions) in severe deficiencies. Severe cases of P deficiency demonstrate fragile bones, general weakness, weight loss, emaciation, stiffness, reduced milk production, lowered reproduction, and chewing of wood, rocks, bones, and other objects. Abnormal chewing of objects (pica) may occur, however, with other dietary deficiencies as well. A number of response criteria have been used to evaluate Ca and P status of livestock and humans including growth rate, feed intake, and feed efficiency; serum Ca, P, and alkaline phosphatase levels; and numerous dimensional, compositional, and mechanical criteria for several bones. The first known response to a dietary deficiency of P is a fall in the inorganic P fraction of the blood plasma and a withdrawal of Ca and P from the reserves in the bones. Normal values for plasma P are 4.5 to 6 mg/lOO ml for adults and somewhat higher, often 6 to 8 mg/lOO ml for very young animals. After a few weeks or months of a P-deficient diet, or even days with laying hens, the concentrations fall to 2 to 3 mgjlOO ml, and 1 to 2 mg/lOO ml has been recorded in milking cows suffering from severe deficiency. Values consistently below 4.5 mgjIOO ml in cattle and sheep are indicative of P deficiency. However, the NCMN (1973) did not consider serum inorganic P to be sufficiently sensitive to recommend it for diagnosing problems with cattle, because forage analyses give earlier and more detailed information. Serum P is a good indicator of P status of ruminants only if stress factors, time of sampling and blood preparation (i.e., hemolysis, temperature, and serum separation time) can be strictly controlled (McDowell, 1985, 1997). The homeostatic or physiological mechanisms regulating serum Ca are more effective than those for P or for most other minerals. In most species, serum Ca is maintained closely about 10 mg/lOO ml by the regulatory actions of PTH, calcitonin, and the active metabolite of vitamin D (l,25-(OHhD). Laying hens have higher and more variable serum Ca values (20 to 30 mg/lOO ml) than nonlaying hens or other species (9 to 12 mg/lOO ml), except during the period of shell calcification, when values decline. Most research has suggested that bone criteria are more sensitive to P status than are other parameters. Williams et al. (1991c) analyzed P concentration in blood, milk, feces, bone, saliva, ruminal fluid, various tissues, and hair of growing cattle fed adequate (0.20%) or deficient (0.12%) dietary P. Of the parameters studied, rib bone P concentrations best reflected dietary P intake. The Ca-P requirement for maximizing most bone criteria is higher than that required for maximizing growth and feed efficiency. Thus, bone criteria are generally more responsive over a wider range of dietary Ca-P levels than are growth rate and feed efficiency or blood components. The incomplete calcification of the skeleton is easily detectable with x-rays, but like other production-related signs,

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would not be specific for Ca, P, or vitamin D deficiency versus other nutrient inadequacies. With the advent of noninvasive techniques to assess potential indicators of Ca adequacy, such as bone mineral content (BMC) or bone mineral density (BMD), it becomes easer to evaluate Ca and P status. For elderly humans, it is important to be able to predict risk of fracture with osteoporosis. Because risk of fracture is not easy to assess, BMC or BMD may be the best indicator of adequacy or to risk of fracture due to osteoporosis (Yates, 1998). The dried, fat-free ash content of several long bones from all classes of swine is generally increased when dietary Ca and P levels increased (Eklou-Kalonji et al., 1999). Mechanical measurements that characterize strength of bone are good indicators of dietary Ca and P status. Stone and McIntosh (1977) reported that femur breaking strength in pigs was a much more sensitive indicator of skeletal development and susceptibility to bone fracture than was bone ash or dimensional characteristics. For growing pigs, shear testing of bones was better than bend testing for estimating bone strength (Combs et al., 1991 b). Williams et al. (199Ia) demonstrated that chemical, physical, and mechanical properties of bone could be used to evaluate the P status of cattle. Breaking load and breaking strength were significantly higher for the adequate (0.20% P) versus low P (0.12% P) diets, 1348 versus 1179 kg and 202.5 versus 189.2 MPa (unit of measurement for stress), respectively. For the same diets, rib bone density (g/crrr') and mineral content expressed on a per unit volume basis (mg/cm ') were found to be sensitive parameters ofP status. Little and Shaw (1979) suggested that P levels of 120 mg/cnr' indicated deficiency, while levels of approximately 150 mgjcm ' indicated adequacy. Noninvasive bone techniques such as dual photon absorptiometry, radiographic photometry, and ultrasound can be used to estimate bone mineral content and bone strength and, thus, status ofP and Ca (Williams et al., 199Ib). Attempts to identify a Ca status indicator in human blood have not been very successful. As with other animals, long-term adequacy of Ca intake in humans influences bone-mass measurements. Fasting urinary Ca :creatinine ratios may be an easy, inexpensive method to indicate recent Ca status in human patents (Weaver, 1990).

IX. SUPPLEMENTATION For most classes of livestock, the two most important mineral supplements are Ca and P. All domestic livestock species generally require supplementation of both Ca and P. In monogastric animals such as swine and poultry, there is a much greater likelihood for a dietary deficiency of Ca than of P, because grain and protein supplements (i.e., corn-soybean meal) make up a very large portion of their feed. Corn and soybean meal account for over 80% of the feed grains (including protein supplements) fed to swine and poultry in the United States. By comparing the content of feeds and the requirements of swine and poultry for Ca and P, one can

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readily see why Ca is more apt to be lacking in these diets. This is even more true for laying hens, which have an extremely high Ca requirement for egg production. A laying hen requires 3.25% dietary Ca, but the two main feed ingredients for swine and poultry diets are corn and soybean meal, which contain 0.03 and 0.34% Ca, respectively. Ruminants that receive high-concentrate diets (e.g., finishing cattle and high producing dairy cows) will also have a greater need for supplementary Ca than for P. Generally, feed grains and especially oil meal protein supplements, are relatively rich in P; therefore, animals receiving high-concentrate diets will meet a high proportion of their requirements from these feeds. Unfortunately, the majority (60 to 80%) of the P in corn and soybean meal, as well as in other grains and oilseed meals, is organically bound as phytin P, a form that is not well utilized by nonruminants (see Sections VI and VII). Low availability of phytate P poses two problems: (I) the need to add inorganic P supplements to diets, and (2) the excretion of large amounts of P in manure. Land application of manure has been the primary method of disposal of livestock and poultry waste due to its fertilizing value. There is a potential environmental impact from excessive P buildup in soil, with surface run-off and erosion of soil Pinto rivers, lakes and streams. Excess P causes algae and other aquatic plants to have unchecked growth which reduces and/or changes the oxygen and carbon dioxide levels in water and causes other aquatic life to suffer. Because of this, many state and local governments have or are enacting legislation to reduce P pollution. Providing phytases to monogastric diets makes plant sources of P more available with less inorganic P required and less of the mineral in feces to be detrimental to the environment. For monogastrics, P excretion can be reduced by many nutritional strategies including feeding closer to P requirements, formulating diets based on available P, feeding by age group, phase and split-sex feeding, and supplementing with microbial phytase. The dietary P requirements of monogastric species consuming typical grainoilseed meals can be reduced considerably with the use of microbial phytase. Almost 2/3 of P in grains and 34 to 66% in oilseed meals is in the phytate form (Eeckhout and de Paepe, 1994). A number of studies with swine (Lei et al., 1993; Radcliffe and Kornegay, 1998; Rodehutscord et al., 1999) and poultry (Yi et al., 1996; Orban et al., 1999; Um et al., 1999) have shown bioavailability of P improved by 20 to 45%. Improved P bioavailability has also reduced P excretion (~30%). By making phytate P more available, less inorganic P is needed in supplementation programs. If there was a 45% higher P availability due to phytase coupled with a 20% reduction in inorganic P, this would result in a 30 to 50% reduction in P excretion (Coelho, 1994). In addition to use of phytase for reducing use of inorganic P, high available P corn varieties are available (Sands et al., 2001). Increasing environmental concerns and proposed regulations have also stimulated renewed interest in the role of P in dairy cattle and feedlot rations (Spears, 1996; Erickson et al., 1999; Satter and Wu, 1999; Valk and Sebek, 1999; Knowlton and Herbein, 2002). Environmental regulations, which limit the quantity of P applied to land, are either in place or are being considered. Because P is the most

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expensive mineral supplement for livestock and of environmental concern, it is not logical to exceed animal requirements for this nutrient. Proper supplementation strategies of P will assist feedlots and dairy operations in becoming more environmentally sustainable. Extensive field trials in the UK and The Netherlands have shown than P surplus can be reduced by up to 90% by dietary manipulations without apparent detrimental effect on milk production (Valk et al., 2000). Recently published data showed dietary P level of 80% of the current feeding practices had no effect on milk production or reproductive performance (Wu and Satter, 2000). For the grazing ruminant, P supplementation needs are greater than those for Ca. Many forages are relatively good sources of Ca (particularly legumes). However, forages, particularly mature ones, are generally very deficient in P in relation to grazing ruminant requirements (see Section VI-VIII; McDowell, 1985; 1997; McDowell and Valle, 2000). Factors that influence requirements and therefore supplementation needs of Ca and P are summarized as follows: I. 2. 3. 4. 5. 6.

7. 8. 9. 10. II.

12.

Variability of ingredient nutrients; Nutrient availability; Animal performance potential; Energy level of the feed; Ambient temperatures; Stress of disease, overcrowding, poor ventilation, and inadequate temperature control; Compensatory growth; Interaction of ingredients; Interaction of nutrients; Variability in animal response; Variability in management; and Adequacy of vitamin D intake and adequacy of liver and kidney integrity to convert it to the proper hormonal form of vitamin D.

Safety margins for Ca and P are needed because of some of the factors outlined above. The greater the variability of the nutrients (or antagonists, e.g., phytin and oxalates) in the ingredients used in a diet, the greater the margin of safety needed. If these growth effects are exerted through higher feed consumption, the requirements expressed as dietary Ca and P concentrations are little affected, but if feed efficiency also is improved, as is usual, requirements in terms of dietary concentrations rise correspondingly (Underwood and Suttle, 1999). The apparent higher incidence of leg problems in the breeding herd and the selection of faster growing, leaner and more efficient swine have led researchers to conclude that swine kept for the breeding herd (developing boars and gilts) require a higher Ca and P level than is necessary for maximizing growth rate and feed efficiency (Fammatre et al., 1977; Hollis, 1984). Although available data are limiting, there may be a slight benefit from elevated dietary Ca and P levels provided to growing and finishing swine on structural soundness scores, which appear to be more noticeable at older ages (Mahan, 1990).

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Several researchers have demonstrated that approximately a 0.1% higher dietary Ca and P level is needed to attain the maximum percentage bone ash than to attain maximal growth and optimal feed: gain ratios during the growth phase (Mahan, 1990; Combs et al., 1991a). However, the greatest weight gain and most efficient feed utilization are generally attained at a lower Ca and P level than those required for maximal bone development. The requirement, and thus supplementation, probably is best described as that level of Ca and P that maximizes performance and results in adequate bone mineralization for optimal performance of growing pigs and maximal longevity of breeding animals in the herd. Energy levels of concentrates, ambient temperatures, restricted feeding programs, and housing arrangements all will influence Ca and P requirements. Monogastric species consume diets to meet energy requirements, and if energy densities are high, less total diet, including minerals will be consumed. Cold environments increase feed consumption, whereas hot temperatures depress intake. For laying hens, the NRC (1994) requirement of 3.25% Ca is adequate under most conditions. However, hens subjected to high temperatures (33°C or more for several weeks) probably need 3.5 to 3.75% Ca along with a proportionately higher available P level because of reduced feed intake. When poultry and pregnant sows are administered restricted feeding programs, the levels of Ca and P (and certain other nutrients) should be increased. Cage-housed birds and swine on slats have higher P requirements than floor-raised animals because they have less chance to recycle P through coprophagy (Singsen et al., 1962). Several supplemental sources of Ca are used in animal diets, the most common being ground limestone or calcium carbonate. Other common sources include oyster shell, Ca sulfate, Ca chloride, Ca phosphates, and bone meal. These range in Ca content from 16 to 38%. Relative bioavailability of Ca supplements is summarized in Chapter 19. Many of the materials used as P supplements supply significant amounts of Ca, and most P supplements contain more Ca than P. However, when only liquid supplements are used to provide added minerals, adequate Ca may not be provided because of the low solubility of Ca. Pullets, at the beginning of the laying period, undergo considerable metabolic stress since their metabolism is aimed totally at egg production. The demand for Ca for egg shell formation increases rapidly as the first egg is formed. Prior to the onset of egg production, it is not uncommon to feed a prelay diet, which contains a higher Ca concentration than the grower diet, but less than the layer diet. A typical prelay diet fed starting at about 16 weeks of age will contain 2.25 to 2.5% Ca. In some instances, the first layer diet containing 3.5% Ca is also fed instead of a prelay diet. The increased Ca will aid in the synthesis of medullary bone, which acts as a Ca reserve during egg shell formation. It benefits the bird greatly to have a considerable amount of medullary bone so she can better survive the pressures on mineral metabolism as egg laying begins. Particle size of Ca supplements can be important for some species. Increasing the fineness of grind of limestone increases the amount of surface area exposed for digestion in the animal. However, it has been suggested that particle size, in some species, must be sufficient to allow for optimal retention and therefore maximal Ca

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bioavailability. Particle size can be important in the choice of Ca supplements, at least in relation to eggshell quality. The high Ca needs of laying hens are commonly met by supplementation of the diets with limestone, a relatively cheap and plentiful source of this mineral. Replacing one-half to two-thirds of the pulverized limestone in layers' diets with large particles of sources of Ca, such as oyster shell, has a Casparing effect, so that a diet supplying 3% Ca is equal, in respect to shell quality, to one containing 4% Ca supplied as finely ground limestone (Roland et al., 1974). Apparently large particles of Ca remain in the gizzard longer and provide a more constant supply than do finely ground sources (Scott et al., 1971). The reason is that Ca is continually released during dark hours when shell deposition is actively occurring. Cheng and Coon (1990) indicated that layers fed smaller particles of limestone needed an extra g/day to produce the same shell quality as hens fed larger particle limestone. A number of supplemental sources of P are available, including calcium phosphates (dicalcium phosphate, monocalcium phosphate, defluorinated rock phosphate, bone meal, guano-origin phosphate), ammonium phosphates (monoammonium phosphate, diammonium phosphate, ammonium polyphosphate), sodium phosphates (monosodium phosphate, disodium phosphate, sodium tripoIyphosphate), and phosphoric acid. These sources ofP are readily available, but there is considerable variation in the biological availability of P from sources within these types, especially within the Ca phosphates. A review on the biological availability of P to livestock is available (Soares, 1995). Also, the percentage of P and relative bioavailability ofP supplements are noted in Chapter 19. Colloidal phosphate or soft phosphate with colloidal clay is significantly less available than other sources. Ground rock phosphate, particularly when obtained from continental deposits, is not only less palatable and less physiologically available, but it also contains 3 to 4% F and can result in F toxicosis, as discussed in Chapter 14. Fertilizer phosphates are sometimes used to provide supplemental P to livestock (Coates, 1994; McDowell and Valle, 2000). Triple superphosphate (TSP) containing 21% P and 2% F is quite commonly used in world regions where feed-grade phosphates are difficult to obtain or prohibitively expensive. Jubb and Crough (1988) reported that TSP is quite commonly used as a P supplement in northern Australia because it is cheap and available. The economics of beef production in northern Australia, with the high costs of transport and the low value of the cattle, force the use of cheaper P supplements with marginal F concentrations. This often is true in developing countries, with use of fertilizer phosphates to provide part of the P requirement. Fertilizer phosphates can be used for short periods or mixed with safe P sources (with low F) to ensure no detrimental effects from F. Recommendations for using fertilizer phosphates to provide supplemental P for livestock are found in Chapter 19. Superjuices are supplemental P sources made from P fertilizers that are safe (low F) and economical to use (Jubb and Crough, 1988). The superjuices are made by mixing the fertilizers with water and allowing the slurry to settle for 12 to 24 hr and then using the supernatant only. This allows most of the toxic F to precipitate as CaF2 •

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Calcium and P deficiencies can be prevented or overcome by direct treatment of the animals through supplementation of the diet or the water supply or, indirectly, by appropriate fertilizer treatment of the soils on which the plants to be consumed are grown. The choice of supplementation procedure depends on the condition of husbandry. Methods of mineral supplementation are reviewed in Chapter 19. For most classes oflivestock including swine, poultry, feedlot cattle, and dairy cows, Ca and P mineral supplements are incorporated into concentrate diets, which generally ensures that animals are receiving required quantities. For grazing livestock, where concentrates are economically prohibitive, other methods of P and Ca supplementation are required. On sparse P-deficient grazing, the direct supplementation method is preferred because use of phosphate fertilizers involves high transport and application cost, and herbage productivity is usually limited by climate or soil problems. In more climatically favored and intensively farmed areas, phosphate fertilizer applications designed primarily to increase pasture yields also increase P concentrations. In Australian studies, superphosphate applications at the rate of 125 kgJha doubled pasture yields, and increased forage P by some 50% (Underwood, 1981). From pastures grown on soils from Florida, USA, Kirk et al. (1970) reported increases in the P content of pangola grass (D. decumbens) from 0.07 to 0.10% DM to 0.25 to 0.30% DM due to P fertilization. Phosphorus fertilization to raise forage P would not be effective for very acid soils, as the fertilizer P would be tied up in the soil, and forage levels of the element would be increased only slightly (McDowell, 1985; McDowell and Valle, 2000). Australian research (Underwood, 1981) has shown that not only does superphosphate fertilizer increase herbage P but it also results in improved palatability and digestibility of the forage. However, unless there are definite forage yield increases that can be utilized effectively by grazing herbivores, use of mineralcontaining fertilizers is economically prohibitive. Individual dosing or drenches with P supplements in a squeeze chute or corral ensures the right dosage for each type of animal and therefore maximal economy. However, it is tedious, costly in labor and requires frequent handling of the stock. This procedure has therefore limited applicability unless it can be linked to other practices such as dipping of cattle once weekly for control of ticks. With two individuals administering the drench, it is claimed that up to 200 animals/hr could be dosed (Theiler et aI., 1924). Dissolving soluble phosphates in the water supply is applicable only where the access of animals to water is controlled. It is impracticable where rivers, streams, lakes, or ponds are available. Water-soluble phosphates, such as disodium phosphate (Na zHP04 ) or ammonium polyphosphate, are more expensive than less-soluble but well-utilized Ca phosphates employed in free-choice mineral mixtures. Water solubility is important in fish nutrition. Although dicalcium or defluorinated phosphate may be used as a source of supplemental P in channel catfish diets, defluorinated phosphate may be desirable because of its low solubility in water (Robinson et al., 1996). Studies on supplying disodium phosphate through the drinking water were carried out by Black and his colleagues working at the King Ranch in Texas (USA)

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from 1941 to 1946 (Black et al., 1943; Reynolds et al., 1953). Disodium phosphate sufficient to supply P at approximately I gj5 I drinking water was given to heifers, and the performance of these animals was compared with that of cattle with access to bonemeal in self feeders. The authors concluded that both methods were practical and that the bonemeal-fed cows produced 133 kg weaned calfjha while those administered disodium phosphate in the drinking water produced 164 kgjha (this compared with 107 kg for cows that received no supplement and cows on pasture fertilized with triple superphosphate). The most widely used method of direct administration is providing mineral feeders and allowing free-choice consumption of mineral supplements. The greatest problem with free-choice supplements is individual variation in mineral consumption. A comprehensive discussion of the method of free-choice mineral supplementation is found in Chapter 19 and elsewhere (McDowell, 1985, 1997). Season of the year affects supplementing P for grazing livestock. Since forages contain less P during the dry season (winter), it is logical to assume that grazing ruminants would be most likely to suffer mineral inadequacies during this time. On the contrary, numerous reports, including those from Kenya (Hudson, 1944), Brazil (Correa, 1957), and South Africa (Van Niekerk, 1978), noted specific mineral deficiencies more prevalent during the wet season. Grazing cattle were more prone to develop P deficiencies, and the clinical signs were severest after the rains when pastures were green and plentiful. In South Africa, Van Niekerk (1978) noted that the beneficial effect of supplemental P was primarily during the wet season, although the P content in the grass was at its highest. Increased incidence of mineral deficiencies during the wet season is less related to forage mineral concentration than to the greatly increased requirements for these elements by the grazing animal. During the wet season, livestock gain weight rapidly because energy and protein supplies are adequate, and, thus, the mineral requirements are high, while during the dry season, inadequate protein and energy result in animals losing weight, which lowers mineral requirements. There are notable exceptions as to season of the year when mineral supplementation is most critical. As an example, in the wet llanos of Venezuela, northern Colombia and Bolivia, as water recedes in the dry season, cattle enter the lowlands to graze a great variety of plant species. For this reason, breeding and calving are more frequent during the dry season than in the rainy season (Stonaker et al., 1974), so mineral deficiencies would not be expected to be more prevalent during the wet season. Special Ca and P supplementation considerations need to be implemented for high-producing dairy cows to prevent parturient paresis (milk fever). Parturient paresis can be prevented by feeding a prepartum low-Ca and adequate-P diet. Prepartum low-Ca diets are associated with increased plasma PTH and 1,25(OHhD2 plus 1,25-(OHhD3 concentrations. Green et ai. (1981) suggested that these increased PTH and I,25-(OHhD concentrations resulted in prepared and effective gut and bone Ca homeostatic mechanisms at parturition that prevented parturient paresis.

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Unfortunately, low-Ca diets are difficult to formulate from available feedstuffs, particularly when it is desirable to have cows consume as much bulky feed (often high in Ca) as possible during late gestation. Also, iflow-Ca diets are continued for long periods, skeletal mineral reserves will be dangerously depleted. Supplemental vitamin D has been used to prevent milk fever in dairy cows for a number of years. Treatment with high levels of vitamin D has been successful, but toxicosis problems have sometimes resulted; for some animals, the disease has been induced by the treatment. Because of the extreme toxicity of vitamin D 3 in pregnant cows and the low margin of safety between doses of vitamin D 3 that prevent milk fever and doses that induce milk fever, Littledike and Horst (1982) concluded that vitamin D 3 cannot be used to prevent milk fever when injected several weeks prepartum. A report from the same laboratory suggested that injection of 24-F1,25-(OHhD3 (fluoridation at the 24R position) delivered at 7-day intervals before parturition can effectively reduce incidence of parturient paresis (Goff et al., 1988). This compound, however, never advanced beyond the experimental stage. An analogue that has been marketed in Israel and used with varying degrees of success was la-hydroxycholecalciferol (ltX(OH)D 3l (Sachs et al., 1977). This analogue was developed as a precursor in the chemical syntheses of 1,25-(OHhD3 and, fortunately, can be activated by the body following 25-hydroxylation in the liver to form 1,25-(OHhD3 . Use of this analogue, however, shared the disadvantages of earlier compounds: hypercalcemia was induced, and the endogenous synthesis of 1,25-(OHhD3 was inhibited. Hodnett et al. (1992) used a combination of 25-0HD 3 plus le-hydroxycholecalciferol to reduce parturient paresis in dairy cows fed high dietary Ca. The incidence of the disease was reduced from 33 to 8%. Supplementation of lc-hydroxycholecalciferol is less costly to produce than 1,25(OHhD 3 • Anion--eation balance of prepartum diets (sometimes referred to as acidity or alkalinity of a diet) also can influence the incidence of milk fever (Gaynor et al., 1989; Horst et al., 1997; Vagnoni and Oetzel, 1998; Pehrson et al., 1999). Diets high in cations, especially Na and K, tend to induce milk fever, but those high in anions, primarily C1 and S, can prevent milk fever. The incidence of milk fever depended on the abundance of the cations Na+ and K+ relative to the anions C1and so,>. This concept is now generally referred to as the cation-anion difference (CAD). Because most legumes and grasses are high in K, many of the commonly used prepartum diets are alkaline. There are large variations in the mineral content of roughages fed on different farms, and the mineral content of grass, and consequently the CAD of a diet can be significantly altered by different types of fertilization (Pehrson et al. 1999). Addition of anions to a prepartal diet is thought to induce a metabolic acidosis in the cow, which facilitates bone Ca resorption and intestinal Ca absorption (Horst et al., 1997). Diets higher in anions increase osteoclastic bone resorption and synthesis of 1,25-(OHhD3 in cows (Goff et al., 1991b). Both of these physiological processes are controlled by PTH. Workers at the Rowett Research Institute (Abu Damir et al., 1994) have also recently reported that 1,25-(OHhD3 production is enhanced in cows fed acidifying diets.

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Collectively, these data suggest that a major underlying cause of milk fever is metabolic alkalosis, which causes an inability of cow tissues to respond adequately to PTH (Horst et al., 1997). This lack of response in turn reduces the ability of the cow to draw on bone Ca stores and production of the second Ca-regulating hormone 1,25-(OHhD, which is needed for active transport of Ca within the intestine. The presumption is that metabolic alkalosis somehow disrupts the integrity of PTH receptors on target tissues. Low CAD diets prevent metabolic alkalosis, increasing target tissue responsiveness to PTH, which controls renal Inhydroxylase and resorption of bone calcium. Several options exist regarding methods for the control of milk fever (Horst et al., 1997). The current understanding of the CAD concept suggests that milk fever could be managed more effectively if dietary K was reduced (Goff and Horst, 1997). Calcium chloride has been used to reduce blood pH (Dhiman and Sasidharan, 1999; Schonewille et al., 1999). This reduction is beneficial but excessive oral Ca chloride can induce metabolic acidosis (Goff and Horst, 1994), which can cause inappetence at a time when feed intake is already compromised. Dietary acidity can be monitored via the pH of urine, which should be below 7.5. Calcium propionate treatment has been beneficial in reducing subclinical hypocalcemia in all trials and reduced the incidence of milk fever in a herd having a problem with milk fever (Goff et al., 1996; Pehrson et al., 1998). Commercial preparations of Hel mixed into common feed ingredients as a premix could offer an inexpensive and palatable alternative to anionic salts as a means of controlling the incidence of milk fever in dairy cows (Goff and Horst, 1998). Treatment of milk fever returns serum Ca concentration to the normal range and must be carried out at the earliest possible opportunity to avoid muscular and nervous damage to downer cows. This is facilitated by maintaining close surveillance over cows that have calved in the preceding 72 hr. Calcium borogluconate is most commonly used, with Mg added to the injectable Ca preparation when hypomagnesemia is in evidence. The product is preferably administered intravenously for rapid response, but subcutaneous administration permits slow absorption of the Ca ion and may lessen the danger of cardiac arrest. Preventative measures for parturient paresis have not been studied in ewes, but those which reduce the incidence of milk fever in dairy cows should be just as effective in ewes, provided they begin well before lambing (Underwood and Suttle, 1999). Another Ca supplementation consideration involves the role of mineral salts as buffers. Several products, generally classified as buffers, have been added to highconcentrate dairy and beef cattle finishing diets to control low ruminal pH found when high-grain diets are fed. These products include sodium bicarbonate, dolomitic limestone, ground limestone, bentonite, and magnesium oxide. Some of these products are much more effective than others. Limestone exerts little or no buffering effect in the rumen of dairy cows regardless of its reactivity rate or its mean particle size (NRC, 1989a). This lack of effect is probably owing to its low solubility in ruminal fluid at pH values above 5.5. Calcium and P supplementation is unnecessary to prevent rickets if children are provided with milk and milk products high in Ca and P and have access to sunlight

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to provide vitamin D. Milk is also a source of vitamin D, particularly when it is fortified with the vitamin as it is in the United States. Rickets has virtually disappeared from the more developed countries, but can still be a problem in developing countries. Preterm infants are more prone to bone mineral deficiency if the birth weight is low, and are in special need of Ca and P supplementation (Pohlandt, 1994). For adult humans, Ca is one of the most limiting dietary nutrients, even though rickets was never expressed in youth. Unfortunately, the effects of inadequate Ca during growth and development of bone mass are not realized until later in life. Although Ca deficiency is common among humans, deficiencies of P are rare. Most human foods are relatively rich in P; diets providing sufficient protein to meet nutritional needs generally contain generous amounts of P. Supplemental P is needed when a deficiency results from defects of renal tubular reabsorption of P or for small prematurely born infants fed human milk (Harrison, 1984). Human milk is much lower in P than milk from other animals, containing 150 mg/l in comparison to 1000 mgjl in cow's milk. Because the caloric density of the two milks is the same, 0.7 kcal/rnl, an isocaloric feeding of human milk provides about one-seventh the P of cow's milk. The P content of human milk is clearly sufficient for the full-term infant, but is inadequate for the growth requirements of the premature infant. Many human diets need Ca supplementation (Carter and Whiting, 1997). Diets that are characteristically rich in animal proteins and P with low Ca : P ratios may prove deleterious to bone, because they may promote hypercalciuria and stimulate the release of PTH with a resultant progressive decrease in bone mass (Metz et al., 1993). However, dietary protein may be as important as ample Ca and vitamin D in maintaining strong bones in the elderly (Tucker and Mayer, 2001). Research with 70- to 90-year-old men and women showed that those with the highest protein intakes lost less bone over a four-year period than those consuming half as much. Exercise or moderate physical activity is important for maximizing bone mass and, at an older age, slowing down loss of bone mass. Immobilization, or the relative inactivity that often attends the infirmities of age, promotes skeletal demineralization. Peculiar dietary habits may also lead to increased urinary Ca loss and a negative Ca balance, despite an adequate intake. Once maximal bone mass is achieved (age 25 to 30 years), it is maintained without much change for 10 to 20 years. Both men and women lose bone at a constant rate of 0.3 to 0.5%/year, starting at age 40 to 45 years, and during a period (10 years) immediately before and after menopause, women lose bone more rapidly than do men (2 to SOlo/year). This rapid rate of bone loss in menopausal women returns to the slower rate shared by the sexes after this lO-year period. No treatment is available to reverse bone loss that has already taken place. Estrogen replacement therapy and Ca supplementation, however, reduce the rate of bone loss by decreasing the overall rate of bone turnover, both resorption and formation (Recker et al., 1977). Combined daily treatment with 0.3 mg of conjugated estrogens plus 1 g Ca supplement has, however, been reported to be as effective as 0.6 mg of conjugated estrogen (widely accepted to be the minimal effective dose for

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prevention of bone loss) without added Ca (Ettinger et al., 1987). Clinical trials using low doses of 1,25-(OHhD in the treatment of osteoporosis have also been encouraging (Gallagher et al., 1982; O'Brien, 1998). Combined Ca and vitamin D supplementation reduces bone loss and fracture incidence in older men and women (O'Brien, 1998). Older persons are subject to osteoporosis because of inadequate Ca and/or vitamin D and would benefit from supplementation. Calcium and vitamin Dare absolutely essential for growing children, and they are also essential for maintenance of a healthy mineralized skeleton in adults. Older individuals do not get enough exposure to sunlight (Miller, 1983), and they require more sunlight to get the same vitamin effect as do young individuals (McDowell, 2000). Holick (1987) observed that one of the primary causes of poor vitamin D nutrition in the elderly in the United States is a decrease in or complete abstinence from consumption of milk, the principal food that is fortified with vitamin D. Also, milk and milk products are the principal sources of Ca and P in many diets. Low Ca intake during childhood has long-term consequences. In particular, Ca is required for accretion of bone mass, and hence bone strength, which occurs almost exclusively during childhood and adolescence. Thus, inadequate Ca intake in early life is expected to reduce peak bone mass and increase the risk of osteoporosis later in life (Allen and Wood, 1994; Prentice, 1995). There is even data suggesting that intakes much higher than the RDA may further increase bone mass accretion and hence reduce the risk of future fractures (Johnson et al., 1992; Roberts and Heyman, 2000). Adequate Ca intake of 1300 mg/day for children 9 to 18 years old will maximize peak bone mass and Ca retention (Carter and Whiting, 1997; Rourke et al., 1998). With adequate Ca, vitamin D, and physical activity throughout life, osteoporosis can be delayed or completely eliminated. Supplemental Ca has benefits not related to bone health. Calcium supplementation has been shown to reduce the risk of hypertension by lowering high blood pressure (Osborne et al., 1996)and diminishing the risk ofcolon cancer (Mobarhan, 1999).

X. TOXICITY Neither dietary Ca nor P is considered toxic when single large doses are consumed by animals (NRC, 1980). Under normal conditions, both elements are absorbed according to need, and the excess is excreted. Excess of either of these minerals may cause bone disorders and reduce feed consumption and gain. Assuming adequate dietary P and depending on age and production status, maximum tolerance levels for Ca are 2% for cattle, sheep, horses, and rabbits; 1% for swine; 1.2% for most poultry, and 4% for the laying hen. For P, the maximum tolerance levels are about 0.6% for sheep; 1.0% for cattle, horses, rabbits and poultry (except laying hens, 0.5%) and 1.5% for swine. When expressed as a percentage of the requirement, P has one of the lowest tolerances of any mineral. A high level of either Ca or P reduces the efficiency of utilization of the other and can also reduce the utilization of other minerals. The addition of excess Ca to an

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otherwise adequate diet may result in a deficiency of other essential elements, e.g., P, Mg, Fe, I, Zn, and Mn (NRC, 1980). In every case, it appears that the injurious effect of the Ca is attributable to an interaction rather than to a harmful effect of the Ca itself. Experimental results show that increasing the Ca content of the diet well above requirements without concurrently increasing dietary Zn results in severe parakeratosis in swine (see Chapter 12 of this volume) and that the high levels of Ca and/or P in the diets of swine and poultry intensify the effect of Mn deficiency (see Chapter II). Excess Ca not only decreases the utilization of P but also increases the pig's requirements for Zn in the presence of phytate. When the molar ratio of cations (Zn and Ca) was 2: I or 3: I with phytate, the formation of an insoluble complex was much greater (Oberleas and Harland, 1996). Excessive dietary Ca fed to dairy calves affected concentrations of Zn, Fe, Cu, and Mn in some body tissues, but the magnitude was relatively small (Alfara et al., 1988). Additional quantities of the elements that are affected by excess Ca will overcome the adverse effect of the Ca, similar to that observed by reducing the level of dietary Ca. Excess Ca has also been found to be antagonistic to nonmineral nutrients such as vitamin K. Hall et al. (1991) reported a hemorrhagic syndrome in growing pigs induced by feeding a high dietary Ca level (1.8 or 2.7%) with 0.9% P. This condition was prevented by adding vitamin K, suggesting an interactive effect of Ca on the availability of vitamin K to the pig. Experiments with growing pullets show that feeding diets containing more than 2.5% Ca during the growing period from 8 to 18 weeks of age results in a high incidence of nephrosis, visceral gout, Ca urate deposits in the ureters, and 10 to 20% mortality (Scott et al., 1982). Within I to 2 weeks after feeding the high-Ca diets, pullets develop hypercalcemia and hypophosphatemia. Parathyroid size is reduced, and its activity is greatly decreased. Feeding high levels of Ca reduces feed consumption and weight gains and delays sexual maturity. There are conflicting reports on the effect of excess Ca in the diet of laying hens. Gutowska and Parkhurst (1942) reported that high dietary Ca (3.95%) decreased egg production and feed efficiency. However, Macintyre et al. (1963) reported that levels of dietary Ca up to 6% did not depress egg production, feed efficiency, or egg weight. Harms and Waldroup (1971) also found that dietary Ca levels up to 5% did not affect egg production, egg weight, eggshell thickness, or feed consumption during the four-month feeding period. The North Central Regional Feedlot Committee (NRC-88) summarized results from a series of feedlot trials and observed that feed intake was reduced by 3.2%, weight gains by 1.8%, and feed efficiency by 1.59% when feedlot cattle received diets containing more than 1% Ca (Goodrich et al., 1985). For swine, high levels of Ca depress appetite and growth rate, with the effect most severe when the Ca: P ratio is wide (NRC, 1998). Ammerman et al. (1963) reported that a level of 4.4% dietary Ca caused a significant depression in protein and energy digestion by beef steers. Lewis et al. (1951) found that excess Ca and a diet borderline in P content reduced feed consumption and rate of gain in steer calves. A high incidence of bone and joint abnormalities (osteoperosis, vertebral ankylosis, and degenerative osteoarthritis)

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was reported in bulls fed three to five times recommended Ca levels (Krook et al., 1971 ). Some research has shown that excess Ca can reduce digestibility of fat and other organic nutrients (Jacobson et al., 1975). Excessive Ca apparently can adversely affect digestive tract physiology, especially in ruminant animals fed high amounts of grain and poorly buffered sources of fiber such as corn silage (Miller, 1985). The main pathological effect of ingestion of massive doses of vitamin D is widespread calcification of soft tissues, with inflammation and cellular degeneration. Diffuse calcification affects joints, synovial membranes, kidneys, myocardium, pulmonary alveoli, parathyroids, pancreas, lymph glands, arteries, conjunctivae, and cornea. More advanced cases interfere with cartilage growth. Kidney insufficiency is the most critical development of these processes. Initial kidney damage is the result of Ca deposition in distal tubules, causing inflammation and later obstruction, which in turn causes hypertension and pathology related to it. As would be expected, the skeletal system undergoes a simultaneous demineralization that results in the thinning of bones (McDowell, 2000). Grazing animals in several parts of the world develop calcinosis, a disease characterized by the deposition of Ca salts in soft tissues (Carrillo, 1973; Morris, 1982). The ingestion of the leaves of the shrub Solanum malaeoxylon by grazing animals causes enzootic calcinosis in Argentina and Brazil, where the disease is referred to as enteque seco and espiehamento, respectively. As few as 50 fresh leaves per day (200 g fresh leaves per week) over a period of 8 to 20 weeks are enough to develop the disease in cows (Fig. 2.13) (Okada et al., 1977).

Fig.2.13 Vitamin D toxicity ("enteque seco") in Argentina. Animal had consumed the shrub Solanum malacoxylon. Photograph illustrates calcium deposits in soft tissue. (Courtesy of Bernardo Jorge Carrillo, C.I.C.V., INTA, Castelar, Argentina)

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Another solanaceous plant, Cestrum diurnum (a large ornamental plant), causes calcinosis in grazing animals in Florida, while the grass Trisetum flavescens is the causative agent in the Alpine regions in Europe. Solanum torvum is suspected of causing calcinosis in cattle in Papua, New Guinea. A condition known as humpyback, in which clinical signs reminiscent of calcinosis occur, may be caused by sheep grazing the fruits of Solanum esuriale in Australia. In Jamaica, Manchester wasting disease and in Hawaii, Naalehu disease are seen in cattle, and are virtually identical to enteque seco in clinical and pathological signs (Wasserman, 1975). The calcinogenic factor in S. malacoxylon and C. diurnum is a water-soluble glycoside of 1,25-(OHhD 3 (Wasserman, 1975). The digestive system of the animal releases the sterol, which promotes a massive increase in the absorption of dietary Ca and P, such that accommodation of these by the normal physiological processes is ineffective, and soft-tissue calcification results (McDowell, 2000). Phosphorus is not considered to be toxic when single large doses are administered or consumed by animals; however, mild diarrhea may occur. Prolonged consumption of high-P diets, however, may cause severe metabolic problems because of disorders associated with Ca absorption and metabolism (Cunha, 1977; Goodrich et al., 1985). The maximal amounts of P that can be safely tolerated appear to be quite dependent on several other factors, including amounts of other nutrients fed, especially Ca and Mg. In wether lambs, too much P can greatly increase the incidence of urinary calculi (urolithiasis). Apparently, a Ca : P ratio that is too narrow can increase the incidence of urinary calculi (Preston, 1977). This malady appears to be much less prevalent in cattle. Presumably inadequate Mg would increase the incidence of a high-P diet. A high P diet (0.60%) to heifers may increase the risk of Mg deficiency by depressing Mg absorption (Schonewille, et al., 1994). Pronounced bone loss in adult animals can occur by feeding excess dietary P or insufficient dietary Ca. Feeding horses diets high in P and low or marginal in Ca for a prolonged period results in a severely depleted skeleton and nutritional secondary hyperparathyroidism (Krook, 1968; Schryver et al., 1971), a condition called big head disease. In this condition, the facial bones enlarge because the fibrous connective tissue invades the area from which the Ca was resorbed. The facial bones become porous, and a hollow sound is heard when tapped (Cunha, 1990). Big head was quite common when horses were fed high levels of wheat bran, which is high in P (1.15%) and low in Ca (0.14%), and it is sometimes called bran disease. Several workers (Harms et al., 1965; Charles and Jenson, 1975) have observed that high dietary levels of P (0.8 to 1.2%) depress the performance of laying hens (egg production and eggshell quality). McGillivray and Smidt (1974) reported the effect of excess P on broiler performance. They observed decreased weight gains and efficiency of feed utilization as dietary P levels were increased above 0.8% (Ca level at I%). Significant mortality was noted when the dietary P concentration was raised to 2% (approximately four times requirement). A purified diet containing 1.20% P and 0.12% Ca fed to beagles produced rapid loss of bone and easily detached incisor teeth. This did not occur with dogs fed the same diet containing 0.42% P and 0.54% Ca (Hendrikson, 1968). Dogs fed a high

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Ca diet (3.1%) developed hypercalcemia, with lower feed intake and lower absorption of Ca and P (Schoenmakers et al., 1999). Dogs fed diets high in Ca or a high Ca to P ratio could develop skeletal abnormalities, particularly in large breeds (Slater et al., 1992). High P levels in laboratory animals result in calcification in soft tissues, especially in the kidney, stomach, and aorta. It is estimated that diets containing 1% P or more may be nephrocalcinogenic in rats. In rats, nephrocalcinosis and injury to the proximal tubules were rapidly induced in rats fed a high P (1.5%) diet (Matsuzaki et al., 1997a). These researchers later found that increasing Mg intake prevented high P diet-induced kidney damage in young rats (Matsuzaki et aI., 1997b). Diets containing 0.9% P and 0.8% Ca or higher levels of P produced calcification in the soft tissues of guinea pigs (House and Hogan, 1955). As with livestock species, excess Ca in human diets interferes with other essential elements including P, Mg, Fe, I, Zn and Mn. As an example, higher levels ofCa are known to further exaggerate the inhibition of Zn absorption by phytate, resulting in the formation of Zn-Ca phytate complexes (Forbes et al., 1983). Excess P in relation to Ca intakes interferes with Ca metabolism. For humans, the recommended Ca: P ratio of 1: 1 (RDA, 1989) is almost impossible to achieve, especially in diets as high in protein and P as the average American diet. Americans also consume large quantities of soft drinks high in phosphoric acid (e.g., cola drinks). Diets high in protein and P inhibit development of peak bone mass (Metz et al., 1993). The average Ca: P ratio for the United States is estimated to be approximately 1: 2 (Page and Friend, 1978). Some Americans, however, consume as little as 400 mg Ca per day, resulting in Ca: P ratios as high as 1: 4. Those who drink milk and/ or consume large amounts of green leafy vegetables have a higher intake of Ca and, therefore, more favorable Ca: P ratios.

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Satter, L. D., and Wu, Z. (1999). In "Proc, Southwest Nutr. Management Con.," p. 10. University of Arizona, Tucson, AZ. Schachter, D., and Kowarski, S. (1982). Fed Proc. Am. Soc. Exp. Bioi. 41, 84. Schmidt, H. (1926). "Feeding Bonemeal to Range Cattle on the Coastal Plains of Texas." Texas Agric. Exp. Stn. Bull. 344, College Station, TX. Schoemakers, I., Hazewinkel, H. A. W., and Vanden Brom, W. E. (1999). J. Nutr. 129, 1068. Schoner, F. J., Hoppe, P. P., Schwarz, G., and Wiesche, H. (1993). J. Anim. Phys. Anim. Nutr. 69, 235. Schonewille, J. T., Van't Klooster, A. T., and Beynen, A. C. (1994). J. Anim. Phys. Anim. Nutr. 71, 15. Schonewille, J. T., Van't Klooster, A. T., Wouterse, H., and Beynen, A. C. (1999). J. Dairy Sci. 82,1317. Schryver, H. F., Hintz, H. F., and Craig, P. H. (1971). J. Nutr. 101,259. Schuette, S. A., and Linkswiler, H. M. (1984). In "Present Knowledge in Nutrition" 5th Ed. (R. E. Olson, H. P. Broquist, C. O. Chichester, W. J. Darby, A. C. Kolbye, and R. M. Stalvey, eds.). The Nutrition Foundation, Inc., Washington, D.C. Schwartz, Dwight L., 1977. "Poultry Health Handbook" 2nd Ed. The Pennsylvania State University, University Park, PA. Scott D., and McLean, A. F. (1981). Proc. Nutr. Soc. 40, 257. Scott, M. L., Hull, S. J., and Mullenhoff, P. A. (1971). Poult. Sci. SO, 1055. Scott, M. L., Nesheim, M. C; and Young R. J. (1982). "Nutrition of the Chicken." M. L. Scott and Associates, Ithaca, New York. Shiga, K., Hara, H., and Kasai. T. (1998). J. Nutr. Sc Vitaminology 44,737. Shupe, J. L., Butcher, J. E., Call, J. W., Olson, A. E., and Blake, J. T. (1988). Am. J. Vet. Res. 49,1629. Singsen, E. P., Spandorf, A. H., Matterson, L. D., Serafin, J. A., and Tlustohowicz, J. J. (1962). Poult. Sci. 41, 1401. Slater, M. R., Scarlett, J. M., Donoghue, S., Kaderly, R. E., Bonnett, B. N., Cockshult, J., and Erb, H. M. (1992). Am. J. Vet. Res. 53, 2119. Soares, Jr., J. H. (1995). In "Bioavailability of Nutrients for Animals:Amino Acids, Minerals and Vitamins" (C. B. Ammerman, D. H. Baker, and A. J. Lewis, eds.), p. 257. Academic Press, San Diego, CA. Spears, J. W. (1996). In "E. T. Kornegay (Ed.) "Nutrient Management of Food Animals to Enhance and Protect the Environment," p. 259. CRC Press, Boca Raton, FL. Stralfors, A. (1961). Odent. Rev. 12,236. Stonaker, H. H., Salazar, J. J., Bushman, D. H., Villas, J., Gomez, J., and Osorio, G. (1974). In "Proc. Potential to Increase Beef Production," p. 63. CIAT, Cali, Colombia. Stone, B. A., and Mcintosh, C. H. (1977). Aust, J. Agric. Res. 28, 543. Sutton, R. A. L., and Dirks, J. H. (1978). Fed Proc. Am. Soc. Exp. Bioi. 37, 2112. Swartzman, J. A., Hintz, H. F., and Schryver, H. F. (1978). Am. J Vet Res. 39, 1621. Taaffe, D. R., Snow-Harter, C; and Connolly, D. A. (1995). J. Bone Miner. Res. 10, 586. Tejada, R., McDowell, L. R., Martin, F. G., and Conrad, J. H. (1987). Nutr. Rep. Int. 35, 989. Ternouth, J. H., and Coates, D. B. (1997). J. Agri. Sci. 128,331. Ternouth, J. H., and Sevilla, C. D. (1990). Aust, J. Agric. Res. 41,175. Ternouth, J. H., Bortolussi, G., Coates, D. B., Hendricksen, R. E., and Mcl.ean, R. W. (1996). J. Agr. Sci. 126, 503. Theiler, A. (1920). J. Dept. Agric. Union S. Afr. 7,221. Theiler, A. (1927). 11th and 12th Rep. Dir. Vet Servo Anim. Inc. 7, 821. Theiler, A., Green, H. H., and Du Toit, P. J. (1924). Union S. Afr. J Dep. Agric. 8,460. Theiler, A., Green, H. H., and Du Toit, P. J. (1928). J. Agric. Sci. 18,369. Todd, J. R. (1967). Vet Rec. 81, 6. Tokarnia, c., and Dobereiner, J. (1973). Pesqui. Agropecu. Bras. Ser. Vet. 8, 1. Tokarnia, C. H., Langenegger, J., Langenegger, C. H., and Carvalho, E. V. (1970). Pesqui. Agropecu. Bras. 5, 465. Tomas, F. M. (1974). Aust, J. Agric. 25, 495. Tomas, F. M., and Somers, M. (1974). Aust, J. Agric. Res. 25,475. Traylor, S. L., Cromwell, G. L., Lindemann, M. D., and Knable, D. A. (2001). J. Anim. Sci. 79,2634. Tucker, K. L., and Mayer, J. (2001). Agri. Res., September, 23. Urn, J. S., Paik, I. K., Change, M. B.. and Lee, L. H. (1999). Asian-Austr. J. Anim. Sci. 12,203. Underwood, E. J., (1981). "The Mineral Nutrition of Livestock." Commonwealth Agricultural Bureaux, London, England. Underwood, E. J., and Suttle, N. F. (1999). In "The Mineral Nutrition of Livestock" 3rd Ed. Midlothian. UK. Vagnoni, D. 8., and Detzel, G. R. (1998). J. Dairy Sci. 81, 1643.

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Valk, H., Metcalf, J. A., and Withers, P. J. A. (2000). J. Environ. Qual. 29, 28. Valk, H., and Sebek, L. B. J. (1999). J. Dairy Sci. 82, 2157. Van der Klis, J. D., and Versteegh, A. I. (1996). In "Proc, BASF Technical Symposium," p. 71. Atlanta, GA. Van Niekerk, B. D. H. (1978). In "Proc, Latin American Symp. on Mineral Nutrition Research with Grazing Ruminants" (J. H. Conrad, and L. R. McDowell, eds.), p. 194 University of Florida, Gainesville. FL. Vargas, E., and Fonseca, H. (\989). "Contenido Mineral y Proteico de Forrejes." Editorial de la Universidad de Costa Rica, San Jose, Costa Rica. Vargas, R., McDowell, L. R., Conrad, J. H., Martin, F. G., Buergelt, c., and Ellis, G. L. (\984). Trop. Anim. Prod. 9, 103. Velasquez, J. A. (\978). In "Situacion de la Nutricion Mineral del Ganado Bovino en el Estado Monagas." Univ. de Oriente, Juspein, Venezuela. Vielma, J., and Lall, S. P. (1998) Aquaculture 160, 117. Vuori, I. (1996). Nutr. Rev. 54, 511. Waldroup, P. W., Simpson, C. F., Damron, B. L., and Harms, R. H. (1967). Poult. Sci. 46, 649. Ward, G., Harbers, L. H., and Blaha, J. J. (1979). J. Dairy Sci. 62, 715. Wardlaw, G. (1993). J. Am. Diet. Ass. 93, 1000. Wasserman, R. H. (\975) Nutr. Rev. 33, I. Wasserman, R. H. (1981). Fed Proc. Fed. Am. Soc. Exp. Bioi. 40, 68. Weaver, C. M. (1990). J. Nutr. 120, 1470. Weaver, C. M., Heaney, R. P., Nichel, K. P., and Packard, P. I. (1997). J. Food Sci. 62, 524. Weber, J. C., Pons, V., and Kodicek, E. (1971). Biochem J. 125, 147. Williams, S. N., Lawrence, L. A., McDowell, L. R., Warnick, A. C., and Wilkinson, N. S. (1990). J. Dairy Sci. 73, 1100. Williams, S. N., Lawrence, L. A., McDowell, L. R., Wilkinson, N. A., Ferguson, P. W., and Warnick, A. C. (\99Ia) J. Anim. Sci. 69, 1232. Williams, S. N., McDowell, L. R., Lawrence, L. A., Wilkinson, N. S., Ferguson, P. W., and Warnick, A. C. (199Ib). J. Anim. Sci. 69, 1243. Williams, S. N., McDowell, L. R., Warnick, A. C., Lawrence, L. A., and Wilkinson, N. S. (1992). Int. J. Animal Sci. 7, 137. Williams, S. N., McDowell, L. R., Warnick, A. C., Wilkinson, N. S., and Lawrence, L. A. (199Ic). Lives. Res. Rural Devel. 3(2), 67. Wilson, H. R. (1993). "Hatchability Problem Analysis." Univ. Fla. Ext. Bull. 1112. Wilson, H. Roo Miller, E. R., Harms, R. H., and Damron, B. L. (1980). Poult. Sci. 59, 1284. Wilson, K. L., Min, S.H, Revell, D. K., Lee, J., Machenize, D. D. S., Cottam, Y. H., and Davis, S. R. (1998). Proc. New Zealand Soc. Anim. Prod. 58, 192. Wilson, R. x., Flynn, A. V., and ConnitTe, D. (1968). Ir. J. Agric, Res. 7, 31. Wilson, S. E., and Field, A. C. (\983). J. Compo Path. 93, 61. Wise, M. B., Ordovesa, A. L., and Barrick. E. R. (1963). J. Nutr. 79,79. Wood, R. I., Suter, P. M., and Russell, R. M. (1995). An. J. Clin. Nutr. 62,493. Wu, A., and Satter, L. D. (2000). J. Dairy Sci. 83, 1052. Yano, F., Yano, H., and Breves, G. (\991). Calcium and phosphorus metabolism in ruminants. In "Proceedings of the Seventh International Symposium on Ruminant Physiology," pp. 227-295. Academic Press, New York. Yates, A. A. (1998). Nutr. Rev. 56, 529. Yi, Z., Kornegay, E. T., Ravindran, V., and Denbow, D. M. (1996). Poult. Sci. 75, 240.

Chapter 3

Sodium and Chlorine (Common Salt)

I. INTRODUCTION

Common salt has been known since ancient times as a required part of the diets for both livestock and humans. It contains both sodium (Na) and chlorine (Cl), and is the main source of both elements. It is convenient to consider Na and Cl together because of their broad similarities in functions, requirements and interactions with each other. In this chapter, the term "salt" will only refer to common salt or sodium chloride (NaCl). Salt has been recognized much longer than any other mineral as having a crucial role.

II. mSTORY The value of common salt for humans and their animals was probably recognized before recorded history. A comprehensive review of the importance of salt to livestock including a historical review has been written by Cunha (1987). Early records list salt as an important item of trade along with spices and clothing. Salt is mentioned over 30 times in the Bible. Caesar's Roman soldiers received part of their pay in common salt. Due to unequal world salt distribution, wars were fought for control of the trade in salt (Kaunitz, 1956). At one time, children and even wives (sometimes with reluctance) were actually sold into slavery in order to obtain quantities of salt. The strong craving for salt exhibited by grazing animals (Fig. 3.1) under most natural conditions could not have gone unnoticed by the earliest herders. Animals were observed to travel long distances to salt deposits to satisfy ravenous appetites which had been developed for it. Animals deprived of salt would risk great danger or resort to unusual behavior in order to obtain salt. Early nomads and hunters were able to lure and capture animals by locating areas with salt and waiting for animals to come there periodically (Cunha, 1987). The value of supplementary salt for cattle was first demonstrated experimentally by Boussingault in 1847 (cited by McCollum, 1956). However, it was not until 1905 that Babcock reported his classical studies of salt deprivation on lactating dairy cows and suggested the requirement for this species. Babcock (1905) observed that after 2 to 3 weeks without salt, lactating cows exhibited an abnormal appetite for it, but up 101

102

Sodium and Chlorine (Common Salt)

Fig. 3.1 Illustration of strong craving for salt by Indonesian cattle. At this location, salt was used as a management tool to keep livestock tame, with salt offered only one time per week. (L.R. McDowell, University of Florida, Gainesville).

to a year elapsed before any health effect was noted. Smith and Aines (1959) repeated and expanded this study and identified Na rather than CI as the element primarily concerned. During this period, the need for supplemental salt by pigs and poultry fed cereal-based diets was also established. Earlier, Osborne and Mendel in 1918 showed that the rat required Na and Orent-Keiles and co-workers in 1937 differentiated between Na and CI deficiency in this species. More recent studies have established the essential role of CI for lactating dairy cows independent of Na (Fettman et al., 1984; Coppock, 1986).

III. CHEMICAL PROPERTIES AND DISTRIBUTION

The atomic number of Na is II and its atomic weight is 22.99. Occurrence in the earth's crust is 2.83% by weight, the principal cation in the hydrosphere. The chemistry of Na in the body is almost entirely that of a strong monovalent cation. Sodium does not occur free in nature and combines directly with the halogens (CI in greatest abundance) and phosphorus (P). When freshly cut, Na is a light, silverywhite metal, but tarnishes on exposure to air. Chlorine has an atomic number of 17, an atomic weight of 35.45, and is in the halogen family after fluorine. It is a greenish-yellow gas with a suffocating odor. Chloride is abundant in igneous rock and is 1.9% of seawater (primarily as NaCl).

Metabolism

103

Chlorine acts as an electron-acceptor, combining readily with all elements except the rare gases (xenon excluded) and nitrogen. However, CI exists in nature mainly as sodium chloride. The combination of Na and CI as salt is widely distributed in nature, where it occurs not only in the sea (2.68%) and other saline waters but also in dry deposits as rock salt. The Dead Sea (24% solids) is nine times as salty as the ocean. Whereas table salt is the most common use of salt, it finds application in literally thousands of commercial processes that yield products containing either Na or CI (Standen, 1970). The body contains approximately 0.2% of Na. Some of this amount is localized in the skeleton in an insoluble, rather inert form, but by far the larger proportion is found in the extracellular fluids where it undergoes a very active metabolism. Differing from Na, CI is found in large concentrations both within and without the cells of body tissues. Blood cells contain about one-half as much CI as the plasma.

IV. METABOLISM

A. Absorption and Excretion Sodium and CI ions are readily absorbed principally from the upper small intestine. There appears to be no control on the absorption of dietary Na and CI with virtually all being absorbed, provided glucose is available for transport purposes. The transport of Na across intestinal epithelium appears to be dependent upon a system of "pumps" and passive "leaks" located in cell membranes (Fregly, 1984). Absorption of Na and CI also occurs from the rumen, with Neathery (1981) reporting that CI is almost completely absorbed and that absorption occurs throughout the digestive tract. Approximately 80% of the Na and CI entering the gastrointestinal tract arises from internal secretions such as saliva, gastric fluids, bile, and pancreatic juice (NRC, 1980). Sodium and Cl are principally excreted in the urine as salt with smaller amounts lost in feces and perspiration. Practically all of the ingested Na (85 to 90%) is generally excreted via the urine as chlorides and phosphates (Cunha, 1987). The intestine may also be important in regulating Na balance during a deficiency (Michell, 1995). Hens deficient in Na have been shown to have more than double the microvilli surface area in the intestine. This increased surface area greatly increased net Na transport, thus improving Na balance (Underwood and Suttle, 1999). In non-ruminants, including the horse, Na is the major cation in sweat and salt concentrations in sweat can reach 4.5%. Horses, mules, and donkeys sweat profusely when exercised, but the high loss of sodium balances the loss of water and provides a defense against hypernatremia. For humans at hard work, particularly in warm weather, salt lost in perspiration may represent by far the major portion of the total excretion.

104

Sodium and Chlorine (Common Salt]

Loss of salt through perspiration can be a major excretion route for species that perspire in large quantities. Considerable quantities of Na may also be lost via secretion in milk. Excessive losses of Na may occur from vomiting, diarrhea, or profuse sweating. B. Regulation of Body Content

When Na intake is inadequate, the body has a remarkable capacity to conserve this element (Luft, 1996). The amount of Na excreted in the urine declines rapidly to extremely low levels. On the other hand, high Na intake triggers greater excretion of Na and Cl by the kidneys and water needs increase. The human kidney may excrete as little as 1 g or as much as 40 g ofNaCI per day, depending on the intake. Chloride metabolism is controlled in relation to Na so that excess kidney excretion of Na is accompanied by Cl. Approximately 65% of the filtered load of Na is reabsorbed in the proximal tubule. The process is mediated by active Na transport and also modulated by alterations in luminal surface permeability. Chloride, which is passively reabsorbed, accompanies the actively transported Na and is the major ion transported to balance the electrochemical gradient that is established by the activity of the Na-K pump in the proximal tubule (Harper et al., 1997). About 25% of the filtered load of NaCI is reabsorbed in the loop of Henle. The countercurrent system for the reabsorption of water is dependent in part on the reabsorption of NaCI in the loop of Henle. The main work of the kidney is thus; salt and water reabsorption at a rate finely tuned so that changes in glomerular filtration rate do not cause major changes in Na excretion, being offset by parallel changes in reabsorption (glomerulotubular balance) (Michell, 1995; Luft, 1996). When NaCI intake is suddenly reduced to very low levels, urinary Na and Cl excretion decreases exponentially over 4 or 5 days to virtually zero (to match the intake). Chloride excretion is also influenced by bicarbonate ion, with a rise in plasma bicarbonate resulting in the excretion of a comparable amount of Cl. Studies have shown a close correlation between potassium (K), Na and Cl in the urine of cows (Paquay et al., 1969; Bannink et al., 1999). Regulation of body Na concentrations is controlled by hormones, including aldosterone and an antidiuretic hormone of the posterior pituitary. Both these hormones act to maintain a constant ratio of Na to K in the extracellular fluid. The hormone aldosterone, secreted from the adrenal cortex, regulates the reabsorption of Na from the kidney tubules, thus conserving Na in the animal body. Other control is exercised by the antidiuretic hormone of the posterior pituitary, which is responsive to changes in osmotic pressure of the extracellular fluid (NRC, 1980). Both hormones act to maintain a constant ratio of Na to K in the extracellular fluid. Salt appetite is an overriding driving force for Na and Cl intake in omnivores and particularly in herbivores. Serotonergic neuron pathways within cell bodies of the hind brain are involved in generation and inhibition of Na appetite (Franchini et al., 2002). Much evidence indicates that the brain renin-angiotensin system is important in this regard (Luft, 1996).

Physiological Functions

105

The renin-angiotensin-aldosterone (RAA) system is known to adjust distal tubular Na reabsorption in the kidney, and hence excretion, to balance the Na needs of the body (Collings and Spagenberg, 1980; De Luca et al., 2002). Renin is a proteolytic enzyme that breaks the leucine-leucine bond of angiotensinogen. The rate of production of renin substrate appears to be substantially stimulated during Na deficiency. Angiotensin II appears to be the major factor for secretion and release of aldosterone by the cortex of the adrenal gland. Angiotensin II, a potent vasoconstrictor, also operates on the nephron directly to promote Na and Cl retention. Feedback control cuts off further stimulus to the RAA mechanism when the increased reabsorption of Na restores Na homeostasis. Angiotensin also affects thirst and vascular tone. Sodium balance in ruminants may be affected by a loss of saliva. In cattle, where 50 to 100 liters of alkaline parotid gland saliva may be produced per day, most Na is salvaged by reabsorption from the digestive tract (Collings and Spagenberg, 1980). However, animals slobber considerably during elevated environmental temperature, such that loss of NaCI in salivary secretions can be considerable (Denton, 1965).

V. PHYSIOLOGICAL FUNCfIONS

Sodium and Cl, in addition to K, all function in maintaining osmotic pressure and regulating acid-base equilibrium. These elements function as electrolytes in body fluids and are specifically involved at the cellular level in water metabolism, nutrient uptake, and transmission of nerve impulses. Interstitial fluid and plasma are the two main components of extracellular fluid (ECF). No single solute, certainly no other element, is so important for the integrity of ECF as Na. Its abundance dictates the volume of ECF and its concentration is the key determinant in osmotic equilibrium between ECF and intracellular fluid (lCF). Abnormalities of plasma Na concentration imply the existence of accompanying abnormalities of cell volume because rises or falls cause movement of water from ICF to plasma or from plasma into cells (Michell, 1995). Acid-base status is determined by the difference between total cation and anion intake vs excretion. The major electrolytes of importance are Na, K, and Cl. Sodium and Cl play important roles in the acid-base balance of the body, with Na making up over 90% of the total blood cations and Cl two-thirds of the acidic ions. Sodium is a major component of salts in saliva to buffer acid from ruminal fermentation (NRC, 200 I). Likewise, Na and Cl are crucial to the maintenance of normal fluid volume, and osmotic pressure relationships. Sodium has a major role in the transmission of nerve impulses, and in maintaining proper muscle and heart contractions. In respiration and regulation of blood pH, Cl is transferred between plasma and erythrocytes through a process known as the "chloride shift." Respiration is based on the "chloride shift," whereby the K salt and oxyhemoglobin exchange oxygen (0 2) for carbon dioxide (C0 2) via bicarbonate in the tissue and reverses that process in the lung, where reciprocal Cl exchanges maintain the anion balance (Block, 1994).

106

Sodium and Chlorine (Common Salt)

Sodium and CI help control the passage of nutrients into the cells and waste products out. Sodium ions must be present in the lumen of the small intestine for absorption of sugars and amino acids (Grim, 1980). Lack of Na lowers the utilization of digested protein and energy. Froseth et al. (1982a) reported that low dietary Na appears to alter protein and basic amino acid metabolism in the young pig. Dietary electrolytes affect calcium (Ca) absorption and possible mobilization as related to parturient paresis (West, 1987). Acidic diets (excess anions) resulted in greater Ca absorption whereas diets containing excess cations, including those containing dietary buffers, reduced Ca availability to the cow. Not only is Na required for absorption of sugars and amino acids from the intestine, but the Na ion also plays a role in the uptake of sugars by renal tubular epithelium and of amino acids by a great variety of tissues and cells, muscle, bone, adipose, erythrocytes, fibroblasts, etc. (Grim, 1980). The absorption of bile salts in the ileum as a part of the enterohepatic circulation is also Na-dependent. Absorption of several water soluble vitamins (riboflavin, thiamin, and ascorbic acid) may be Nacoupled (McDowell, 2000). Water absorption in the intestines is also closely linked to Na ion transport. Nucleosides from ruminal microbial breakdown of nucleic acids are efficiently absorbed by Na t-dependent transport across the intestinal brush border membrane (Theisinger et al., 2002). Chlorine is a major anion of extracellular fluid and is also found in gastric secretions where its role in hydrochloric acid is important in protein digestion; it is also found in fairly large concentrations in bile, pancreatic juice, and secretions from the intestines. Chlorine is essential for activation of intestinal amylase (Ammerman and Goodrich, 1983).

VI. REQUIREMENTS

The Na requirements for various animal species and humans are presented in Table 3.1. For most species, the CI requirement has not been sufficiently studied and estimated requirements are only available for a few species. The apparent requirement for poultry is between 0.11 to 0.20% (NRC, 1994; Murakami et al., 1997) and swine between 0.08 to 0.25% (NRC, 1998). Mahan et al. (1999) suggested a dietary minimum of 0.38% total Cllevel during the initial 2 week post-weaning period. The comparative abundance and cheapness of common salt in most areas as a source of supplementary Na and CI, and its low toxicity, have encouraged recommendations for its use in excess of minimum requirements as determined experimentally. Also, because common salt is used to provide the Na requirement, there is less stimulus to study CI because requirements of CI are less than the amount in salt necessary to meet Na requirements. Sodium requirements for growth, ranging between approximately 0.05 and 0.25% of the diet, have been reported for rats, chicks, pigs, and calves. Mahan et al. (l996a,b) noted that early-weaned pigs required more Na and CI than previously thought. Thus, the estimated dietary common salt requirements were increased to 0.25% from 3 to 5 kg, to 0.20% from 5 to 10 kg, and to 0.15% from 10 to 20 kg

Requirements

107

TABLE 3.1 Sodium, Chlorine, and Common Salt (NaCI) Requirements" Species

Purpose

Chickens

Leghorn-type 0-18 wk Laying, breeding Broilers 0-8 wk Japanese Quail All classes Turkeys All classes Beef Cattle All classes Dairy cattle Lactating Growing Sheep All classes Goats All classes Horses Growing Working Growing Swine Gestation-lactation Mink Gestation-lactation Fox All classes All classes Rabbits Cats Growing Dogs Rats Nonhuman primates Humans

Sodium

Chlorine

0.15%

0.12-{).IS%

0.13--{).19% 0.12--{).18% O.IS% 0.12-{).17% 0.06-{).08% 0.19--{).23% 0.07-{).08% 0.09-{).I8%

0.1 1--{).I6% 0.12--{).20% 0.14% 0.12-{).IS% O.24-{).29% O. 10-{}. 12%

0.10% 0.30% O. 10-{}.2S% 0.IS--{).20%

0.08--{).2S% 0.12--{).I6%

O.OS%

0.19%

All classes All classes All classes

0.06% O.OS% 0.22--{).64%

0.04% O.OS% 0.27--{).62%

Children Adults

120-S00 mg/day 180-7S0 rug/day 7S0 mg/day SOO rug/day

Salt

Reference NRC (1994)

NRC (1994) NRC (1994) NRC (1994) NRC (\994) NRC (1996) 0.20% NRC (2001) 0.46% NRC (2001) 0.2S% NRC (l98Sb) 0.50% NRC (1981) O.SO% 0.So-I.0% NRC (1989) NRC (\989) 0.15--{).2S% NRC (\998) 0.So-{}.50% NRC (\998) 1.3-1.5% NRC (1982) NRC (\982) O.S% NRC (\977) O.S% Yu and Morris 0.13% (1997, 1999a,b) NRC (198Sa) O.IS% NRC (\99S) 0.13% NRC (\978) RDA (1989) RDA (\989)

'Expressed as per unit animal feed. either as as-fed (approximately 90% dry matter) or dry basis (see Appendix Table I). Human requirements expressed as mg/day. For humans, there is lessinformation on whichto base allowances; values given represent estimated safe and adequate intakes.

body weight. The exact figure depends on the species and certain mineral interrelationships in the diet. In growing chickens, 0.12% Na is necessary during rapid growth, while 0.05 to 0.06% is adequate for mature, non-laying birds (Shaw and Phillips, 1953). Sodium requirements for ruminants are between 0.06 and 0.25% of the diet, with the highest requirement for lactating dairy cows. The CI requirement for lactating dairy cows is estimated to be between 0.10 and 0.20% and would not exceed 0.27% (Fettman et al., 1984). However, Underwood and Suttle (1999) suggested that the CI requirement for lactating dairy cows should be substantially higher than the 0.15% Na requirement since cow's milk contains almost twice as much CI as Na. Cow's milk contains 630 ppm Na and 1500 ppm CI (Cunha, 1987). Coppock (1986) estimated that a dry dairy cow requires 0.04% dietary CI while a lactating cow would require about 0.20%. Lowering dietary CI in young growing cattle

108

Sodium and Chlorine (Common Salt)

from 0.5 to 0.38% caused significant changes associated with acid-base balance, but did not adversely affect health and growth for 7 weeks (Burkhalter et al., 1980). Various researchers, including Dirven (1963), reported that under tropical or hot, semi-arid conditions where large losses of water and salt occur in sweat, the salt requirement is higher. Requirements would vary depending on the sweating capabilities of various species and the level of activity of a particular animal. The horse sweats profusely, and its sweat contains about 0.7% salt. The more a horse exercises, the more salt is lost via sweat and the more salt is required (Cunha, 1990). The ranking of domestic livestock for sweating in descending order is horses, mules, donkeys, cattle, buffaloes, goats, sheep, and swine (McDowell, 1972). Livestock that receive some type of relief from hot temperatures by shade or some system of air movement will have lower salt requirements. The concentration of NaCI in sweat will decline with excessive sweating (Harper et al., 1997), however, large quantities of these minerals are still lost. In addition to higher NaCI needs for animals growing, lactating, and located in warm climates, additional factors that influence salt requirements include (Cunha, 1987; Berger, 1993): 1. The level of K in the diet - An excess of K will aggravate a deficiency of Na just as too much Na will heighten effects of K deficiency. This may occur with high forage diets as pastures (e.g., particularly immature and fertilized) contain much more K than Na; for example, certain pastures have up to 18 times more K than Na. Froseth et al. (1982b) showed that supplemental K depressed performance of young pigs on diets low or barely adequate in Na. Mallonee et al. (1982) reported significant interactions of dietary Na and K on milk yield and feed intake of lactating cattle. 2. Dry vs green forage - Voluntary consumption of salt is higher for succulent compared to mature forage. Cattle fed silage consume more salt than those fed hay (also, corn silage is very low in Na). Grazing cattle consume more than twice the salt level of those fed dry feeds. 3. The level of Na, CI and other minerals in water will influence the consumption of a free-choice mineral mixture. Animals do not generally like the taste of minerals with the exception of common salt (McDowell, 1997). If water is naturally high in common salt, their desire for these minerals have been met thereby reducing the consumption of a salt-based free-choice mineral mixture. 4. Genetic differences in animals - The Na content of milk is genetically controlled vs affected by amount in the feed (Kemp, 1964). Animals producing higher amounts of Na or CI in milk, likewise, have higher requirements for these elements. 5. Other body losses of Na and CI- Body depletion of salt occurs from diarrhea and vomiting, with less of these nutrients absorbed. Animals with kidney or adrenal gland damage have increased depletion of these elements. Adrenalectomized people or those with Addison's disease are unable to tolerate a very low salt intake. Their basal body NaCI content may fall too low to sustain life.

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Likewise, children with cystic fibrosis cannot conserve salt and will not survive on a low salt intake (Harper et al., 1997). 6. Resistance to disease and parasitism - Studies with poultry indicate that higher levels of Na and CI may be required for normal immunity and maximizing resistance to diseases (Pimental and Cook, 1987). Parasitism in the form of nematodiasis caused an ion imbalance with diarrhea, indicating a need for NaCI (Suttle et al., 1996). The parasite Haemonchus contortus affects the Na status of sheep (Ortolani, 2000). Voluntary intake of salt is not a guide for establishing requirements since it is highly palatable. Elevated dietary salt may promote increased growth rates simply by increasing palatability and thereby increasing total feed intake. Milk yield and liveweight gains were increased by salt fertilization, but not by direct salt supplementation in the concentrate (Chiy and Phillips, 1991). Grazing time on forage was increased by salt-fertilization, but only in cows not receiving salt. Cows selectively grazed the salt fertilized pastures when given an option. Ruminating time was increased both by salt fertilization and supplementation.

VII. NATURAL SOURCES Most plant and plant products contain relatively small amounts of Na in comparison to animal products. Most grains and vegetable protein concentrates are fairly low in Na, containing 0.01 to 0.06%. Feeds of animal origin (e.g., meat meal, fish meal, dried skimmed milk) usually contain relatively high concentrations of Na (0.1 to 0.8%). Forages usually range from 0.007 to 0.12% Na. The Food and Agriculture Organization (FAO) has published an extensive summary of the composition of tropical feeds (Gohl, 1975). High Na sources were salt bush 6.3% Na, creeping salt bush 4.7%, seaweed meal 2.9%, and sunflower meal 2.1%. There is only a small volume of analytical data on Cl content of feedstuffs (Appendix Table II). From limited data, it is easy to see why omission of salt from most diets results in a Na deficiency before a CI deficiency. Cereal grains generally provide more CI than Na and cereal straws contain 3- to 6-fold more CI than the grains (Underwood and Suttle, 1999). However, without supplemental salt both Na and Cl would be limiting. Coppock (1986) concludes that a diet based on barley, cane molasses, soybean meal, alfalfa hay, and timothy hay would have ample Cl but insufficient Na, but a diet based on corn grain or oats, 25% or more corn gluten feed, soybean meal, and corn silage would have ample Na but inadequate Cl. A number of factors affect NaCI concentrations in plants (McDowell and Valle, 2000). Use of potassium chloride (KCl) as a K fertilizer increased plant Cl but depressed Na in herbage due to the antagonism between K and Na (Reid and Horvath, 1980). From Rhodesia, Jones (1964) reported one variety of Chloris gayana to contain 300 ppm Na, while a second variety grown on the same site had a vastly different Na level of 3100 ppm. In most circumstances, Na and Cl concentrations decline as grasses mature (but less in legumes) (Underwood and Suttle, 1999).

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From a favorable aspect for Na, K plant concentrations decline at both a more rapid and dramatic rate than does Na concentration (McDowell, 1985). Sodium deficiency is more likely to occur in livestock grazing tropical pasture species, as these plants generally accumulate less Na than temperate species (Morris, 1980). Natural forages low in Na have been reported in numerous tropical countries throughout the world (McDowell, 1985, 1997; Table 3.2). The University of Florida summarized the available mineral analyses of 1615 Latin American forages; only 146 had been analyzed for Na. Of these, 59% contained less than 0.10% Na (McDowell et al., 1977). French (1955) concluded that NaCI is the most needed nutrient for livestock throughout East Africa. According to Siitmoller et al. (1966), the insufficiency of Na is the most widespread mineral deficiency in the Amazon Valley of Brazil ("'3.5 million square kilometers). Forage Na analyzed from cattle ranches in northern Mato Grosso, Brazil, during both the wet and dry seasons, were extremely deficient, being able to meet only between 14 and 30% of the animal's requirements (Sousa et al., 1982). In Bolivia, steers receiving salt had significantly higher average weights

TABLE 3.2 Forage Sodium Concentrations for Selected Tropical Regions

Location Argentina" Malawi"

Bolivia" Bolivia" Dominican Republic" Colombia!

Guatemala/ Mexico" Venezuela;

"Balbuena et al. (1989). "Mtimuni et al. (1990). cMcDowell et 01. (1982). dpeducasse et 01. (1983). "Jerez et 01. (1984). (Vargas et 01. (1984). "Tejada et 01. (1987). "Gartenberg et 01. (1990). 'Morillo et al. (1989).

Season Wet Dry Wet Dry Wet Dry Dry Dry Dry Wet Dry Wet Wet Dry Wet

Number of samples 341

21 48 8 16 20 84 33 36 35 84 84 37 12 9

Mean %

Percentages of samples below 0.06% ofNa

0.12 0.05 0.05 0.03 0.03 0.01 0.01 0.07 0.02 0.Q3 0.03 0.09 0.03 0.04 0.09

43 97 96 92 83 100 100 78 100 100 55 88 92 79 82

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than unsupplemented controls, 385 vs 370 kg, respectively (McDowell et al., 1984). Australian workers (Murphy and Plasto, 1973) noted dramatic responses in beef cows supplemented with salt. Cattle in Thailand supplemented with Na had higher weight gains, reduced calf mortality, and better reproductive performance than unsupplemented controls (Falvey, 1980).

vrn.

DEFICIENCY

Deficiencies of CI in animals in general have been unequivocally observed only on specially purified or concentrated diets. By contrast, extensive areas of Na deficiency in livestock occur worldwide. A dietary deficiency of Na is most likely to occur (i) in rapidly growing young animals fed cereal-based diets or forages inherently low in Na, (ii) during lactation as a consequence of Na (also CI) losses in milk, (iii) in tropical or hot regions conducive to loss of NaCI in sweat of particularly hard working animals that sweat in abundance, and (iv) for pastures fertilized with K that are high in K and low in Na. For all species, the initial sign of Na and CI deficiency is a craving for salt, demonstrated by the avid licking of wood, soil, and sweat from other animals and by the drinking of water.

A. Effects of Deficiency 1.

SWINE

Cromwell et al. (1981) reported that addition of Na only to swine fed a cornsoybean diet improved rate of gain and feed efficiency, but performance was not optimum until both Na and CI were added to the diet. Improved growth rates and gain:feed ratios resulted when supplemental Cl was increased to 0.38% (Mahan et al., 1999). When a diet was deficient in salt, decreased performance was evident within a few weeks. Poor appetite, poor rate of growth (Fig. 3.2), unthriftiness and reduced efficiency of feed utilization occurs (Cunha, 1987; Falowski et al., 1998). In studies from Purdue, pigs receiving no salt required 174 kg more feed per 100 kg of gain; the rate of gain was only half as fast as pigs receiving adequate salt (Vestal, 1945 to 1947). In this study, I kg of salt saved 287 kg of feed. Salt-deficient pigs were observed licking their pens for salt. Also, the pigs deficient in salt ate 12.5 times as much of a mineral mixture as pigs fed salt, reflecting the craving for something lacking in their standard diet. In a Florida trial (Combs, 1974), 5.9-kg pigs fed no salt gained 0.13 kg/day, consumed 0.45 kg of feed daily and required 1.6 kg of feed/kg of gain. The pigs fed 0.5% salt in the diet, gained 0.65 kg/day, consumed 1.80 kg of feed daily and required 1.2 kg of feed/kg of gain. A study showed that dietary K and Cllevels have an interactive effect on pig growth (Golz and Crenshaw, 1990). Pig birth weights and weaning weights were less when NaCI was reduced from 0.50 to 0.25% during

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(C) Fig.3.2 Salt deficiency. Note difference in size of the pigs (A) not supplemented with salt as compared to the pigs fed 0.5% dietary salt (B). Chicks (C) on left received 0.04%; middle, 0.092% and on right received 0.14% sodium. Chicks are two weeks of age. (Courtesy of: Swine photos - G.E. Combs, University of Florida, Gainesville; Chick photos - G.F. Combs, University of Maryland, College Park).

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gestation and lactation for two or more parities (Cromwell et al., 1989). Less than 0.5% salt also appeared to reduce litter size at birth and weaning. 2.

POULTRY

A deficiency of Na causes growth failure (Fig. 3.2), softening of the bones, corneal keratinization, gonadal inactivity, adrenal hypertrophy, changes in cellular function, impairment of feed utilization and a decrease in plasma fluid volume. In growing poultry, a Na deficiency is manifested within a few weeks by inappetence, growth retardation, inefficiency of feed use and increased water consumption and an impairment of protein and energy metabolism. Digestibility does not seem to be affected. Laying hens on low-salt diets lose weight and are prone to cannibalism. Egg production and egg size are reduced (Harms et al., 1995). An extreme NaCI deficiency causes reduced hatchability (Leeson and Summers, 1980). Reducing Na intake to 110 to 114 mg/hen vs the NRC (1994) requirement of 150 mg/hen resulted in reduced egg production beginning the second week (Kuchinski et al., 1999). As the low Na experiments continued, egg weight, feed intake, hatchability, and body weight gain decreased. Salt deficiency can increase the susceptibility of poultry to disease by suppressing the immune system. Pimental and Cook (1987) showed that broiler chicks fed diets containing less than 0.14% Na or 0.17% Cl had depressed immunity to injected sheep red blood cells as compared to chicks fed higher levels of NaC!. Differences in susceptibility to Na deficiency exist between strains of birds. Thus, Sherwood and Marion (1975) observed a more than 80% drop in egg production in one strain of hens fed a cereal-based diet low in Na for 8 weeks compared to a 67.6% drop in another strain. Experimental Cl deficiency has been produced in chicks (Leach and Nesheim, 1963) and turkeys (Kubicek and Sullivan, 1973) by feeding low Cl diets. The birds fed Cl-deficient diets exhibited extremely poor growth rate, high mortality, dehydration, and reduced blood Cl. Moreover, they showed nervous signs, which appeared to be a form of tetany commonly associated with alkalosis. When the Cl-deficient chicks were stimulated by a sharp noise or by handling, they pitched forward and extended their legs to the rear as if they were in tetany. 3.

RUMINANTS

The first sign of Na deficiency in ruminants is a pica or craving for salt, manifested by avid licking of wood, soil, or sweat of other animals. Depraved appetite can include consuming quantities of soil soaked with urine or the run-off from the manure pile, licking the barn walls and drinking the urine from other cows during urinations. Even untamed cattle are known to approach horseback riders in order to lick the sweat of the horses. Cattle deprived of salt may be so voracious that they often

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Sodium and Chlorine (Common Salt)

injure each other in attempting to reach salt (McDowell, 1997). An extreme appetite for salt can occur within 2 to 3 weeks of deprivation. Other signs may not develop for many months. Since milk contains a relatively high Na content, high-producing cows become deficient much sooner. A prolonged deficiency causes loss of appetite, decreased growth, unthrifty appearance (e.g., rough skin and unkempt hair coat), reduced milk production, and loss of weight (Fig. 3.3). Dietary deficiency of Na has been associated with decreased production and lower fertility in large ruminants (Olson et al., 1989; Minson, 1990). For camels, the main clinical sign of Na deficiency was high incidence of skin necrosis (Hemingway, 1995). More pronounced signs of Na deficiencies include shivering (low body temperature), incoordination, weakness, and cardiac arrhythmia, which can lead to death. Cows fed a Na-deficient diet for 2 years produced about 50% less milk each year compared with pretrial production with a Na-sufficient diet (Smith and Aines, 1959). When these clinical signs occur shortly after calving with a high-producing cow, her breakdown and death can be

Fig. 3.3 Salt deficiency in a dairy cow. Illustrated before (top) and after (bottom) I year of salt deprivation. (Courtesy of the late S.E. Smith, Cornell University, Ithaca, NY).

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very sudden (Cunha, 1987). If salt is supplied before collapse, there is a rapid and almost dramatic recovery. Experimentally produced Cl deficiency, independent of Na deficiency, results in clinical signs in dairy cows that include decreased body weight and milk production, depraved appetite, lethargy, anorexia, emaciation, constipation, cardiovascular depression, and milk dehydration (Fettman et al., 1984; Coppock, 1986). Chlorinedeficient cows also lick urine of other cows, eat bedding material, chew on wooden stall dividers and lick metal pipes. Neathery (1981) reported clinical signs of CI deficiency for young dairy calves to include anorexia, weight loss, lethargy, mild polydipsia, and milk polyuria (Fig. 3.4). Severe eye defects (scleral infection, sunken eyes, scaliness around eyes) and reduced respiration rate were observed as the deficiency progressed. Several experiments with ruminants in tropical areas have demonstrated no benefits from salt supplementation. As the Na deficiency develops, Na content in urine, plasma, saliva, and other fluids decreases. However, for ruminants that are growing, lactating, or working, Na-conserving mechanisms will eventually be inadequate, particularly in hot environments where low-Na forages are consumed. Goats receiving adequate salt, compared to controls, digested and retained more energy and consequently had higher body weight gains (Ogebe and Ogunmodede, 1996). Goats developed deficiency signs in 4 to 6 weeks after receiving a diet low in Na (Schelliner, 1972). After 224 days, the deficient goats weighed 20% less than those supplemented with salt. They consumed 6% less feed and required 18.5% more feed per unit of gain. Hagsten and Perry (1976) depleted lambs on a low-Na diet (0.012% Na) for several months until daily gains declined from about 0.3 kg to less than O. I kg. Salt-deficient sheep in India showed such an intense appetite for salt that often they would chase

Fig.3.4 This calf had been on the low-Cl diet and abomasal contents removed for 22 days when photos were taken. It died 2 days later. Notice the emaciated appearance and the spraddle-leg stance. The animal had difficulty in walking and maintaining its balance. (Courtesy of M.W. Neathery, University of Georgia, Athens).

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attendants carrying salt (Singh et al., 2000). Animals not having an opportunity to ingest salt exhibited continuous bleating, with a tendency to refuse to retire for the day. 4. HORSES

Horses deficient in NaCI will lick mangers, fences, dirt, rocks, and other objects. A severe NaCI deficiency, brought about by considerable sweating, causes horses to become fatigued and exhausted (Cunha, 1990). Horses often develop signs of NaCl deficiency when worked hard in hot weather. A horse (500 kg) doing light exercise (work) would produce 2.5 to 5 liters of sweat compared to over 25 liters for very heavy exercise (Harris et aJ., 1995). Horse sweat can contain 2.9 and 5.2 g/I of Na and CI, respectively. Salt needs would be great under hot/humid conditions with heavy exercise (Harris, 1998). Horses deprived of salt tired easily, stopped sweating, decreased milk production, and exhibited muscle spasms (Templeton, 1949; Harris, 1998). 5. OTHER ANIMAL SPECIES

a. Cats. Sodium-deficient kittens exhibited anorexia, impaired growth, polydipsia and polyuria (Yu and Morris, 1997). Kittens fed the Na-deficient diet had an elevated water intake. Adult cats on low-Na diets also exhibited anorexia with body weight loss (Yu and Morris, 1999a). Chlorine-deficient kittens became alkalotic as evidenced by increased blood pH, in addition kittens were hypochloremic and hypokalemic (Yu and Morris, 1999b). b. Dogs. Signs of salt deficiency are fatigue, exhaustion, inability to maintain water balance, decreased water intake, retarded growth, dryness of skin, and loss of hair (NRC, 1985a).

c. Laboratory animals. Rats fed a Na-deficient diet exhibited retarded appetite and growth, corneal lesions, and soft bones. Males became infertile after 2 to 3 months, and sexual maturity was delayed in females. Death occurred in 4 to 6 months. Chloride-deficient rats had depressed appetite, reduced body weight gain and marked kidney pathology (NRC, 1995). The gerbil fed a purified diet without added NaCl developed alopecia without weight loss within 30 days, with dramatic recovery when NaCI was provided (Gullen and Harriman, 1973). d. Mink. For mink, salt has been suggested to prevent "nursing sickness," a condition that sometimes occurs during the latter stages of lactation (NRC, 1982).

e. Rabbits. Harris et aJ. (1984) reported slightly better gains for rabbits receiving 0.5% NaCI compared to 10w-NaCI controls. Colin (1977) found CI-supplemented rabbits had greater gains and feed intake.

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There is not much information on Na, CI, or salt requirements or deficiencies for most laboratory animals, fish, rabbits, non-human primates, and as expected carnivorous animals such as dogs, cats, and foxes. 6.

HUMANS

Sodium and CI intakes can vary tremendously within human populations. The range of habitual daily intakes of salt compatible with a normal life span is from less than 2 g (Yanamamo Indians of the Amazon Basin) to 35 g (Northern Island of Japan) (Harper et al., 1997). There are people for whom either extreme of intakes would be dangerous, however, the problems lie primarily with limitations of homeostatic mechanisms (conservation or elimination) rather than any intrinsic property of salt. Under most circumstances, approximately 0.5 g of NaCI daily would represent an adequate intake. However, the peoples of most world regions consume 10 to 35 times this amount. For most humans, salt is derived from three general sources; 1/3 from food items, 1/3 from cooking or processing, and 1/3 from salt shakers. A simple dietary deficiency of CI in adults has not been reported (Dahl, 1972). However, infants fed formulations with low NaCI developed severe metabolic alkalosis because of CI deficiency (Roy and Arant, 1979). Sodium deficiency would only rarely be expected and only in populations that consume no salt and rely entirely on plant food grown in inland areas low in soil Na. Early signs and symptoms of a Na deficiency would be a progressive fall in urinary Na, and the dramatic reduction in work capacity and psychologic function (Harper et al., 1997). Ultimately, urine can become almost Na-free at < I mmol/day. Salt deficiency could result in individuals who sweat a great deal or be due to sicknesses that result in vomiting and diarrhea. Humans who sweat profusely in hot climates or those involved in strenuous activity, may temporarily develop a salt deficiency characterized by headaches, dizziness, fatigue, nausea, vomiting, muscle cramps, exhaustion, collapse and even death. Football and basketball players may lose from 3 to 7% of their body weight during the course of a contest and perhaps more during vigorous practice sessions with more than 95% of it being water loss (Cunha, 1987). Miners have been noted to lose 1.5 kg of sweat per hour, containing 2 g of NaCl (Maynard et al., 1979). The problem with Na depletion is greatest for people who are not heat acclimatized (NAS, 1993), because their Na losses in sweat and urine are greater than those who are acclimatized (Hubbard et al., 1990; Armstrong and Maresh, 1991). B. Assessment of NaCI Status Craving for salt is the earliest and most obvious criterion for Na deficiency. However, Na concentration in the urine and saliva are more accurate indicators of Na status. To diagnose CI deficiency, there is a reduction of the element in the blood, and metabolic alkalosis. The most certain means of diagnosing NaCl deficiency is fast response to appetite, appearance, and productivity when supplementary salt is

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Sodium and Chlorine (Common Salt)

supplied. Because of a ruminant's rapid reaction to deficiency long before clinical signs appear, the best criterion ofNa status assessment is concentration ofNa and K in saliva (NCMN, 1973). A deficiency causes a fall in Na and a rise in K. Skydsgaard (1968) reported the normal Na:K ratio in saliva to be from 17:1 to 25:1 and suggested that if it is between 10:1 and 15:1, Na deficiency can be suspected. Singh and Rani (1999) reported that saliva, urine and muzzle secretion ofNa and K all reflected Na status in buffaloes or dairy cattle. Muzzle secretions of Na and K were as indicative of Na status as was saliva and a more convenient diagnostic tool. Fecal Na has also been demonstrated as a good indicator of Na status for cattle (Khalili et al., 1992) and for the African elephant (Holdo et al., 2002).

IX. SUPPLEMENTATION

Coppock (1986) reported that omission of supplemental salt from many formulations for lactating cows would result first in a Na deficiency, but practical diets are fed where Cl would become first limiting. It seems apparent with available information that salt supplementation to satisfy the Na requirement will also satisfy the Cl requirement. The amount of supplemental salt needed varies with the amounts in the feeds and water animals consume. The main reason Na is often deficient in unsupplemented livestock diets is the low content ofNa in most natural feeds. Forage in many areas, especially where rainfall is low, often is quite high in salt. In such areas, including parts of the Western United States and much of Australia, supplemental dietary salt is not needed. However, tropical forages often do not contain sufficient quantities of Na to meet the requirements of grazing livestock (Table 3.2). Likewise plantderived concentrate diets produced in most world regions are low in Na. Carnivorous animals usually secure adequate Na and Cl in their diets. Supplementary Na is invariably supplied as common salt because of its palatability, relatively low cost, and ready availability. Unfortunately neither the cost nor availability of salt is always satisfactory in tropical, developing countries where its need may be the greatest. Factors which determine supplementation needs for livestock have been summarized (see also Section VI) by Cunha (1987) as follows: I. Geographical location - In most world regions forages, seeds, and seed products are low in Na. Tropical forages are often lower in Na than plants found in temperate regions. Inland areas distant from the sea are generally lower in soil Na. 2. Temperature and/or humidity - In warm climates animals lose NaCI through sweating. 3. Animal class and physiological status - Rapidly growing and high-producing animals require more NaCl. Lactating animals are in particular need of supplementation as milk contains high levels of NaCl. Even after prolonged, severe deficiency, NaCI levels secreted in milk remain high. Thus, lactating animals suffer most from lack of salt in the diet (Loosli, 1978).

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4. Type of feeds - Kinds of concentrates, pasture, hay, or silage being fed affects NaCl intake, with great variations among classes of feed ingredients and individual lots of the same feed. 5. Potassium concentrations in the diet - Excess K aggravates a Na deficiency. Potassium concentrations particularly in immature pastures are very high in relation to Na. 6. Water content of NaCI - Water found in most world regions provides only small amounts of NaCl. However, waters from some locations contain sufficient NaCl to meet animal requirements, while other water supplies contain toxic levels of salt. 7. Dry vs green forage - Apparently NaCI is needed more for grazing animals as voluntary consumption of salt is higher when forage is succulent, as compared to when it is more mature. Cattle fed silage consume more salt than those fed hay. 8. Genetic differences - Differences in susceptibility in Na deficiency between strains of hens are reported (Sherwood and Marion, 1975). However, data are lacking for NaCI in most species, but there are considerable differences in requirements and metabolism of other minerals as influenced by genetics. 9. Availability of Na and CI in feeds - Sodium and CI from common salt are readily absorbed and utilized by animals. Studies with poultry showed Na in chemically pure compounds to be 90 to 100% as well utilized as NaCl, and Na in defluorinated phosphate to be 85% as valuable (Henry, 1995). Chlorine either in ammonium chloride for poultry or in potassium chloride for swine was 95% as well utilized as NaCI. During hot temperatures, sodium bicarbonate was more beneficial to broilers than NaCl, resulting in increased body weights, feed intake, water intake, and improved feed conversion (Nagwa and Maghraby, 1995). For more normal temperatures, Murakamietal. (1997) found sodium bicarbonate equal to NaCI as a source ofNa. Little information exists concerning availability of Na and CI from natural feedstuffs. 10. Illness or disease - Many diseases cause body depletion of Na and CI. Among these are gastrointestinal losses due to diarrhea, vomiting, and urinary losses in animals with renal or adrenal damage. Based on present information, it appears that NaCllevels of 0.25 to 1.0% of the total diet, or proportionately higher in supplements, will meet the Na and CI needs of most animals (see Section VI). It is recommended that feedlot diets contain 0.25% added salt, one-half of the 0.5% level previously recommended. An advantage of the lower salt level in modern feedlot diets is the prevention of salt buildup in feedlot waste and lessening problems in waste treatment and utilization as a fertilizer. For poultry, supplemental salt is minimized in order to reduce the moisture level in the excreta (Hooge et al., 1999). Lower levels of salt would be sufficient in animals' diets where such animal protein supplements as fish meal, meat meal, and dried skimmed milk are provided, as they are higher in salt than plant protein sources.

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Sodium and Chlorine (Common Salt)

Although the usual way of providing supplemental Na is as common salt, Na in other Na compounds will also supply the needs of livestock. Where nitrogenous fertilizers are normally applied to pastures to raise forage yields, a secure method of raising Na intakes by grazing livestock would be use of a fertilizer containing Na. Sodium nitrate fertilizer is an effective source of both Na and nitrogen (N). Where the pasture is low in Na due to high K fertilization, the use of a low-grade fertilizer containing subsidiary Na, such as kainite, can be recommended. Sodium fertilizers have been shown to increase milk yield and reduce milk somatic cell count (Phillips et al., 2000). Also, magnesium (Mg) absorption has been shown to increase when Na was added to a high-K diet and grass tetany halved when NaCI fertilizer was applied to pastures grazed by beef cows (Berger, 1995). Although the addition of Na to soils will increase Na in forages, it is much easier to supply Na by feeding salt. For many classes of livestock, mineral supplements (including salt) are incorporated into a concentrate diet. However, for grazing livestock to which concentrate feeds cannot be economically fed, it is necessary to rely on self-feeding of mineral supplements (see Chapter 19). The salt needs of grazing cattle, for example, can easily be met with mineral mixtures containing 20 to 35% salt and consumed at a rate of 45 g/head daily (McDowell, 1997). For sheep, self-feeding a salt-based mineral mixture is also effective in preventing mineral deficiencies and is the most cost effective method of delivery (McDowell, 1996). Cunha (1987) suggests that even livestock receiving salt in a concentrate diet should also be self-fed so that they can consume additional quantities if the level in the diet is not adequate. Animals will not consume enough salt to cause harmful effects if plenty of water is available to drink. Because salt serves as a condiment as well as a nutrient, the intake tends to be highly variable and frequently in excess of needs. Its use as a condiment has physiological support in evidence that it stimulates salivary secretion and promotes the action of selected enzymes. Salt has a palatability effect and when lacking reduces both the appetite and weight of animals. Feed efficiency is dramatically reduced as a result of a salt deficiency. Salt provided to ruminants above the requirement has been reported to either have no detrimental effect or in some cases has increased digestibility of certain nutrients (Cunha, 1987). Feeding excess salt has been used as a management technique in the prevention of urinary calculi "waterbelly" (Price, 2000). Urinary calculi can be a problem for certain types of diets (e.g., milo rations), excess salt will increase water consumption and thereby reduce the chance of stone formation. There are advantages for providing salt to grazing livestock other than as a nutrient requirement. Salt is used as a management tool to control animal location in different pastures of a ranch. The use of salt blocks or free-choice supplements in scattered localities is a method to distribute grazing animals throughout a range area (Cunha, 1987). Also, salt can be placed in less frequently utilized forage areas to increase forage use. In many mountainous or inaccessible areas, salt-containing mineral blocks are dropped by planes to get minerals to animals and to help distribute grazing (Cunha, 1987).

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121

Salt at high levels may safely be used to limit free-choice consumption of protein or other supplements. Such mixtures are usually 10 to 50% salt depending on the desired amount of diet consumption. Livestock with high-salt intakes need access to an adequate source of water and in cold climates care should be taken to prevent freezing of water supplies. Because of its palatability, salt is the main "carrier" for free-choice mineral mixtures that are provided to grazing livestock (see Chapter 19). The main disadvantage of free-choice mineral mixtures is lack of uniform consumption by animals. For mineral blocks, hardness and palatability are the factors most related to intake. Valk and Kogut (1998) reported consumption of mineral blocks for dairy cows to vary from nearly 0 to a maximum of70 g/head daily. If mixtures contain 30 to 40% salt or more, they are generally consumed on a free-choice basis in sufficient quantities to supply supplementary needs of other minerals. Vitamins are also sometimes included with salt-based mineral supplements but caution is advised as certain vitamins (even those stabilized and protected) are eventually destroyed by minerals depending on mixing conditions, mineral concentrations, and moisture levels (McDowell, 2000). Products that contain Na are sometimes added to livestock diets not for the nutrients they provide but as buffers (see Chapter 19). Buffers are of most benefit for dairy and feedlot cattle that are receiving high concentrate diets in order to reduce digestive upsets by counteracting low ruminal pH. These products include sodium bicarbonate, sodium sesquicarbonate, and sodium bentonite, as well as some nonNa sources (i.e., carbonate forms of calcium and magnesium). Sodium propionate may be considered an effective treatment of metabolic acidosis in diseases such as ketosis because the added propionate can serve as a source of glucose for the cow (Bigner et al., 1997). In dairy cattle, dietary buffers have been shown to increase dry matter intake, milk yield and milk fat yield (Staples et al., 1986; NRC, 2001). Improvements in animal performance may be attributed to increased ruminal pH and osmolality, which enhance rumina 1 fermentation and increase ruminal fluid outflow (Rogers et al., 1982). The addition of sodium bicarbonate to highconcentrate diets was found to also decrease the acetate:propionate ratio. Even though the role of salt in human survival has been known since antiquity, human nutritionists in most regions of the world are not concerned with suboptimal intakes of supplementary salt. Because salt makes a difference in palatability or taste of many foods, excess is used. The overriding concern is to reduce salt intake in human diets. The need to reduce the Na content of human food reflects the association of dietary Na with hypertension (Krauss et al., 2001). This approach may be appropriate for individuals prone to hypertension, however, other evidence has shown Na restrictions to increase incidence of cardiovascular disease and overall mortality rates (see Toxicity, Section X). Clearly both Na and Cl are essential nutrients, and effects of supplemental salt in human nutrition needs further study. Individuals who sweat profusely need supplemental water and salt. The absorption of fluid is maximized when both carbohydrate and Na+ are present in solution (Gisolfi et al., 1990). During recovery from exercise, a fluid that contains Na will result in a faster plasma volume expansion than pure water (Nose et al., 1994).

Sodium and Chlorine (Common Salt]

122

X. TOXICITY Salt may be toxic when excessive quantities are ingested and water intake is limited. "Salt poisoning" is a misnomer because the condition usually occurs in conjunction with water deprivation. Table 3.3 presents the maximum tolerable level for salt (and Na fed as salt) to animals. Tolerance levels for NaCl and other minerals will vary with species, adaptation, duration of receiving the toxicants, age, physical condition of the animal, and many other factors. Most animals can tolerate large quantities of dietary salt when an adequate supply of water is available. There is little likelihood that livestock will consume toxic amounts of salt unless a saltstarved animal is suddenly exposed to an unlimited amount of salt or high levels of salt are fed without adequate water. Sodium chloride toxicosis is characterized by increases in water consumption, anorexia, weight loss, edema, nervousness, paralysis, and a variety of signs that are dependent on the animal species involved. As noted, livestock tolerate a comparably high amount of dietary salt when water is available. However, much less salt in water will produce toxicosis than the same quantity consumed with access to non-saline water. When the high salt intakes come from the feed the animal can compensate to some degree by increasing its intake of water, thereby increasing the salt-excreting capacity of the kidneys. When the water is itself rich in salt the animal is unable to exploit this capacity to the same extent. In large areas of the arid or semi-arid regions of Africa, Asia, America, and Australia millions of livestock subsist for many months of the year upon water supplies of high saline content (Underwood and Suttle, 1999). Much of the western half of the United States, and many other semi-arid regions, have soils that are high in salinity and saline groundwater, which may lead to saline water intoxication. Saline waters may also occur, however, where sea water contaminates ground water sources or in other special circumstances. Animals restricted to such waters may

TABLE 3.3 Maximum Tolerable Levels of NaCI and Na in Animal Diets" Species Cattle Lactating Nonlactating Sheep Swine Poultry

Horses" Rabbits"

Sodium chloride in total diet (%)

Sodium in total

diet" (%)

4

1.57

9

3.54 3.54 3.14

9 8

2

0.79

(3) (3)

( 1.18) ( 1.18)

"Modified from NRC (1980). bLevelof sodium fed as salt (NeCl), determined by multiplying NaCllevel by 0.393. 'This level was derived by extrapolation from that of other animals.

Toxicity

123

TABLE 3.4

Use of Saline Waters for Livestock and Poultry" Total soluble-salts content of waters (ppm) Comment Less tha n 1000

These waters have a relatively low level of salinity and should present no serious burden to any class of livestock or poultry. 1000---2999 These waters should be satisfactory for all classes of livestock and poultry. 3000-4999 These waters should be satisfactory for livestock, although they might cause temporary diarrhea or be refused at first by animals not accustomed to them. They are poor waters for poultry, often causing watery feces and (at the higher level of salinity) increased mortality and decreased growth, especially in turkeys. 5000---6999 These waters can be used with reasonable safety for dairy and beef cattle, sheep, swine, and horses. It might be well for pregnant or lactating animals to avoid those waters approaching the higher levels. They are not acceptable waters for poultry. 7000---10,000 These waters are unfit for poultry and probably for swine. Considerable risk may exist in using them for pregnant or lactating cows, horses, sheep, the young of these species, or for any animals subjected to heavy heat stress or water loss. More than 10.000 The risks with these highly saline waters are so great that they cannot be recommended for use under any conditions. "From NRC (1974).

suffer physiological upset or death. The ions most commonly involved in saline waters are Ca, Mg, Na, bicarbonate, CI, and sulfate. The toxicity of particular saline waters varies with the chemical nature of the constituent salts. Sodium chloride appears to be the least harmful and Mg salts are much more toxic than Na salts, including sulfates and carbonates (Underwood and Suttle, 1999). Research on the effects of saline waters on animals is limited, but enough data have been obtained for an NRC committee (NRC, 1974) to develop preliminary guidelines for use in animal production (Table 3.4). In reviewing the use of saline waters for livestock (NRC, 1974; Shirley, 1985) it was concluded that water containing salt concentrations up to 5000 ppm is safe for lactating cattle and up to 7000 ppm for non-lactating cattle and sheep. Saline waters containing 3000 to 5000 ppm soluble salts would be unsatisfactory for poultry causing watery feces and lowered production. Obvious methods of alleviating or preventing salt toxicosis would be to limit salt intake and provide, as much as possible, water with lower levels of NaCI. It is important to prevent water from standing in troughs for long time periods as evaporation concentrates minerals in water. Likewise, preventing animals from becoming "salt starved" and always providing water is essential. Chronic intakes of high levels of salt have been reported to raise blood pressure in some people and low-Na, high-K diets are often recommended. For some time, the American Heart Association has had specific guidelines for maintaining a normal

124

Sodium and Chlorine (Common Salt)

blood pressure. The association believes that available evidence indicates that a high intake of salt adversely affects blood pressure (Fleet, 2001; Krauss et al., 2001). Specifically, it has been hypothesized that Na intake is positively related to blood pressure, which in turn is positively related to the incidence of fatal and nonfatal cardiovascular disease. The relevant hypothesis is that reduced Na increases health. On the belief that a low-Na diet will reduce blood pressure, it has been hypothesized that a lower-Na diet will translate into a human health benefit. The Food and Drug Administration since 1993 has permitted labeling of some food products to state that diets low in Na may reduce the risk of high blood pressure. In sharp contrast, studies have recently shown adverse effects of Na restriction. An inverse association exists between a single 24-hour urinary Na excretion measurement and incidence of myocardial infarction and total cardiovascular disease (CVD) (Grassi et al., 1997). All mortality rates (also CVD mortality rates) adjusted for age and sex were significantly higher for low Na intakes (Alderman et al., 1998). For persons prone to hypertension, a massive reeducation program would be necessary to implement a large-scale reduction in salt intake (Battarbee and Meneely, 1978). Enthusiasts never tire of describing how easy this intervention is to achieve (Kaplan, 1994). Unfortunately, man responds to restricted salt intake not unlike one addicted to drugs. There is no solid evidence for the benefits of reducing salt intake for normal individuals who are not prone to hypertension.

XI. REFERENCES Alderman, M. H., Cohen, H., and Madhavan, S. (1998). Lancet 351, 781. Ammerman, C B., and Goodrich, R. D. (1983). J. Anim. Sci. 57 (Suppl. 2), 519. Armstrong, L. E., and Maresh, C. M. (1991). Sports Med (New Zealand) 12,302-312. Babcock, S. M. (1905). "The Addition of Salt to the Ration of Dairy Cows." Wisconsin Agr. Expt. Sta. Ann. Ret. 22, 129 Balbuena, 0., McDowell, L. R., Luciani, C. A., Conrad, J. H., Wilkinson, N. S., and Martin, F. G. (1989). Veterinaria Argentina 6(56), 365. Bannink, A., Valk, H., and VanVuuren, A. M. (1999). J. Dairy Sci. 82, 1008. Battarbee, H. D., and Meneely, G. R. (1978). In "Handbook Series in Nutrition and Food, Section E. Nutrition Disorders" Vol I (M. Rechcigl, Jr., ed.), p. 219. CRC Press, West Palm Beach, Florida. Berger, L. L. (1993). In "Salt and Trace Minerals for Livestock, Poultry and Other Animals" 5th ed. Salt Institute, Alexandria, Virginia. Berger, L. L. (1995). "Sodium Nutrition of Livestock and Poultry." Salt and Trace Minerals. Salt Institute, Alexandria, Va. Bigner, D. R., GolT, J. P., Faust, M. A., Tyler, H. 0., and Horst, R. L. (1997) J. Dairy Sci. 80, 2162. Block, E. (1994). J. Dairy Sci. 77, 1437. Burkhalter, D. L., Neathery, M. W., Miller, W. J., Whitlock, R. H., Allen, J. C, and Gentry, R. P. (1980). J. Dairy Sci. 63, 269. Chiy, P. C, and Phillips, C J. C (1991). Grass and Forage Science 46, 325. Chiy, P. C, and Phillips, C. J. C. (1995). In "Sodium in Agriculture" (C J. C. Phillips and P. C. Chiy, eds.). p. 107, Chalcombe Publications, Canterbury, U.K. Colin, M. (1977). Ann. Zootech. 26, 99. Collings, W. E., and Spagenberg, R. B. (1980). "Sodium Excretion Monograph", Sodium in Medicine and Health (C Moses, ed.), Reese Press, Baltimore, Maryland. Combs, G. E. (1974). In "Proc. Florida Nutrition Conf.," p. 36, Gainesville, Florida. Coppock, C. E. (1986). J. Dairy Sci. 69, 595.

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Khalili, M., Capper, B., and Lindgren, E. (1992). Swedish J. Agr. Res. 22,85. Krauss, R. M., Eckel, R. H., Howard, B., Appel, L. J., Daniels, S. R., and Deckelbaum, R. J. (2001). J. Nutr. 131, 132. Kubicek, J. J., and Sullivan, T. W. (\973). Poult Sci. 52, 1903. Kuchinski, K. K., Harms, R. H., Wilson, H. R., Russell, G. B., and McDowell, L. R. (\999). J. Appl. Anim. Res. 15, 25. Leach, R. M., Jr. and Nesheim, M. C. (\963). J. Nutr. 81, 193. Leeson, S., and Summers. J. D. (\980). Poult. Sci. 59, 935. Loosli, J. K. (\978). In "Proc. Latin American Symposium on Mineral Nutrition Research with Grazing Ruminants" (J. H. Conrad and L. R. McDowell, eds.), p. 54. University of Florida, Gainesville, Florida. Luft, F. C. (\996). In "Present Knowledge in Nutrition" (E. E. Ziegler and L. J. Filer, Jr., eds.) p. 265. ILSI Press, Washington, D.C. Mahan, D. Coo Newton, E. A., and Cera, K. R. (1996a). J. Anim. Sci. 74,1217. Mahan, D. Coo Weaver, E. M., and Russell, L. E. (\996b). J. Anim. Sci. 74(Suppl. 1),58. Mahan, D. C, Wiseman, T. D., Weaver, E., and Russell, L. (\999). J. Anim. Sci. 77, 3016. Mallonee, P. G., Beede, D. K., Schneider, P. L., Caputo, S. J., and Wilcox, C. J. (\982). J. Dairy Sci. 65(Suppl. I), 112. Maynard, L. A., Loosli, J. K., Hintz, H. F., and Warner, R. G. (1979). "Animal Nutrition" 7th Ed. McGraw-Hill, New York. McCollum, E. V. (1956). "A History of Nutrition." Houghton Mifflin, Boston, Massachusetts. McDowell, L. R. (1985). "Nutrition of Grazing Ruminants in Warm Climates." Academic Press, New York. McDowell, L. R. (\996). In "Detection and Treatment of Mineral Nutrition Problems in Grazing Sheep" (D. G. Masters and C. L. White, eds.). Australian Center for International Agricultural Research, Canberra, Australia. McDowell, L. R. (1997). "Minerals for Grazing Ruminants in Tropical Regions" (3rd ed.). University of Florida, Gainesville, Florida. McDowell, L. R. (2000). "Vitamins in Animal and Human Nutrition," (2nd ed.), Iowa State Press, Ames, Iowa. McDowell, L. R., Bauer, 8., Galdo, E., Koger, M., Loosli, J. K., and Conrad, J. H. (\982). J. Anim. Sci. 55,964. McDowell, L. R., Conrad, J. H., and Ellis, G. L. (1984). In "Symposium on Herbivore Nutrition in SubTropics and Tropics-Problems and Prospects" (F. M. C. Gilchrist and R. 1. Mackie, eds.), p. 67. Pretoria, South Africa. McDowell, L. R., Conrad, J. H., Thomas, J. E., Harris, L. E., and Fick, K. R. (\977). Trap. Anim. Prod. 2,273. McDowell, L. R., and Valle, G. (2000). In "Forage Evaluation in Ruminant Nutrition." (D. 1. Givens E. Owen, R. F. E. Axford and H. M. Omed, eds.) p. 373, CAB International, Wallingford, U.K. McDowell, R. E. (1972). "Improvement of Livestock Production in Warm Climates." W. H. Freeman, San Francisco, California. Michell, A. R. (\995). In "Sodium in Agriculture." (C. J. C. Phillips and P. C. Chiy, eds.) p. 91, Chalcombc Publications, Canterbury, United Kingdom. Minson, D. J. (1990). In "Forage in Ruminant Nutrition." p. 291. Academic Press. Inc., San Diego, California. Morillo, D., McDowell, L. R., Chicco, C. F., Perdomo, J., Conrad, J. H., and Martin, F. G. (1989). Nutr Rep. Int. 39, 1249. Morris, J. G. (\980). J. Anim. Sci. 50, 145. Mtimuni, J. P., Mfitilodze, M. W., and McDowell, L. R. (\990). Commun. Soil Sci. Plant Anal. 21(5 and 6),415. Murakami, A. E., Saleh, E. A., England, J. A., Dickey, D. A., Watkins, S. E., and Waldroup, P. W. (\997). J. Applied Poultry Res. 6, 128. Murphy, G. M., and Plasto, A. W. (\973). Aust. J. Exp. Agric. Anim. Hush. 13,369. Nagwa, A. A., and Maghraby, N. A. (\995). Egyptian J. Anim. Prod. 32, 103. Neathery, M. W. (1981). In "Proceedings Georgia Nutrition Conference for the Feed Industry." p. 78. Univ. of Georgia Press, Athens, Georgia. NCMN (Netherlands Committee on Mineral Nutrition) (1973). "Tracing Mineral Disorders in Dairy Cattle." Centre for Agricultural Publishing, Wageningen, The Netherlands. NAS (National Academy of Sciences) (\993). In "Nutrient Needs in Hot Environments" (8. M. Marriott, ed.) p.ll. National Academy Press, Washington, D.C.

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Templeton, G. S. (1949). "Mule-Feeding Experiments." Mississippi Agric. Exp. Stn. Bull 270, Starkville, Mississippi. Theisinger, A., Grenacher, B., Rech, K. S., and Scharrer, E. (2002). J. Dairy Sci. 85, 2308. Underwood, E. J., and Suttle, N. F. (\999). In "The Mineral Nutrition of Livestock" (3rd Ed.), Midlothian, United Kingdom. Valk, H., and Kogut, J. (\998). Livestock Prod. Sci. 56, 35. Vargas, D. R., McDowell, L. R., Conrad, J. H., Martin, F. G., Buergelt, C., and Ellis, G. L. (1984). Trop. Anim. Prod. 9, 103. Vestal, C. M. (\945-1947). Purdue University Agr. Exp. Stn. Mimeo Circulars No. 18,20,23 and 28. West Lafayette, Indiana. West, J. W. (1987). "Proc. Georgia Nutrition Conference," p. 150, Atlanta, GA. Yu, S., and Morris, J. G. (1997). J. Nutr. 127,494. Yu, S., and Morris, J. G. (1999a). J. Nutr. 129,419. Yu, S., and Morris, J. G. (1999b). J. Nutr. 129, 1909.

Chapter 4

Potassium I. INTRODUCTION

Potassium (K) is the third most abundant mineral in the body. In the past, K has not been widely studied in animal or human diets, because common plant food or feeds generally contain plentiful supplies. More recently, recognition that levels of K in some feeds are lower than expected, and that dietary requirements may be higher, has stimulated considerable research. Potassium supplementation has advantages in some diets for ruminant animals, especially dairy cattle. However, elevated dietary K has a role in hypomagnesemia (grass tetany) and milk fever (parturient paresis). In human nutrition high dietary K and low sodium (Na) favor lower blood pressure and less cardiovascular disease.

II. HISTORY Potassium has been known to be of nutritional significance to man and animals since the science of nutrition was in its infancy. Sir Humphrey Davey first isolated K in 1807. Recognition of the importance of K in the plant kingdom dates from Justis von Liebig's report to the British Association for the Advancement of Science in 1840. Sidney Ringer in 1883 first recognized the importance of K in animal tissue in perfusion experiments with frog hearts (Vellaire, 1965). He demonstrated that the perfused mammalian heart required a balance of Na, K, and calcium (Ca) to function effectively. Since that time, the physiological importance of K has been studied in far greater detail.

m.

CHEMICAL PROPERTIES AND DISTRIBUTION

The chemical symbol K is used for potassium because the Latin word for this element is kalium. Potassium is frequently referred to as potash because of an early source of this mineral. Organic matter was burned, leached, and then evaporated in pots, yielding potassium carbonate or potash. Potassium is the seventh most abundant element and makes up 2.6% of the earth's crust by weight. The concentration of K in soil is greater than the concentration of Na and chloride (CI), which are generally leached out of soils. In spite of the wide distribution of K in 129

Potassium

130

soils, it still must be applied as a fertilizer to many soil types to enhance plant growth. Potassium is a Group IA member of the periodic table of elements, known as the alkali metals, which also includes lithium, Na, rubidium, cesium, and francium. Potassium has an atomic number of 19 and is a light metal with an atomic weight of 39.1. Because K is one of the strongest reducing metallic elements, it is not found free in nature, but rather in combined forms. When purified by electrolysis, metallic K is a silver white metal with a brilliant luster; it ignites when dropped in water, and burns with hydrogen to produce a violet flame. After Ca and phosphorus (P), K is the third most abundant mineral in the animal body. It represents approximately 0.3% of the body's dry matter of which twothirds is located in the skin and muscle. Because skeletal muscle has the highest K concentrations, the total amount of K is closely correlated with lean body mass. About 2% of body K is outside cells (extracellular) whereas 98% resides inside cells. In contrast with Na, the main electrolyte in the plasma and extracellular fluids, K is present primarily inside the cells (Thompson, 1978). The blood cells contain approximately 25 times as much K as is present in the plasma. Muscle and nerve cells also are very high in K, containing over 20 times as much as that present in the interstitial fluid.

IV. METABOLISM

A. Absorption and Excretion Potassium is absorbed mainly by simple diffusion from the upper small intestine, but some absorption also occurs in the lower small intestine and large intestine (Church and Pond, 1974). In ruminants, absorption takes place from the rumen and omasum as well as from the lower gastrointestinal tract (Kay and Pfeffer, 1970; Mroz et al., 2002). Because large volumes of saliva (high in K) are continuously secreted by ruminants, a significant amount of K in the rumen is derived from saliva. True digestibility of K is relatively high (95% or higher) for most feedstuffs, and no apparent problems interfere with absorption in a normal, healthy animal (Hemken, 1983). Relative K availability in corn was 90 to 95% and soybean meal 97%, compared to K acetate at 100% (Hooge and Cummings, 1996). Diarrhea and other disturbances to the tissues of the gastrointestinal tract can interfere with normal absorption and thus increase the amount of K required. Entry into the bloodstream occurs largely via conductance channels in the basolateral membrane of the gut mucosa (Peterson, 1997). Potassium must enter cells against a concentration gradient, and thus an active metabolic process is required. Potassium is excreted into urine by both filtration and secretion (Berliner et al., 1954). It is freely filtered at the glomerulus, reabsorbed in the proximal tubule, and secreted in the distal tubule. Fecal loss accounts for only about 13% of the total loss of K in cows, with the remainder being excreted in the urine. In lactating cows, milk can account for 12% of the K lost from the body (Preston, 1985). In sheep,

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131

considerable K is present on the skin and in the fleece (Telle et al., 1964); almost 30% of the K excreted by sheep in hot and humid conditions is through the skin (Rai et al., 1981). In ruminants, K is the major cation in sweat. For broilers, Smith and Teeter (1987) indicated a 600% increase in K excretion when birds were exposed to 35 vs 24°C temperatures.

B. Homeostasis and Storage Adrenal hormones, particularly aldosterone, favor the reabsorption ofNa and the excretion of K by the renal tubules. Michell (1995) has suggested that aldosterone may often be more important to the grazing ruminant by controlling K excess than to protect the animal from a Na deficiency. In humans, Peterson (1997) notes that in the early 1800s the Irish population consumed almost exclusively potatoes, a high-K food. This type of diet would provide a toxic level of K (20 to 40 g), however, adaptation to a high-K diet is due to the enhanced capacity of the kidney to excrete K. Short-term adjustments to fluctuating K supply can be made through changes in the net flux of K into cells. Insulin increases the rate of cellular K uptake, thereby blunting the increase in plasma K concentration (Lindeman and Pederson, 1983). In certain diseases, an insufficient production of aldosterone results in an excessive loss ofNa and retention ofK. Conversely, the hyperactivity of the adrenal cortex, or the administration of aldosterone, results in excessive reabsorption of Na and urinary loss ofK. Normally, hormonal output is controlled by various receptors that are affected by osmotic pressure and concentration levels of the various electrolytes. Stress tends to increase circulating levels of aldosterone, resulting in the kidney conserving Na but increasing K excretion, with an excessive loss of K (Thompson, 1978). The K concentration in ruminant saliva is also controlled by aldosterone, generally in response to dietary Na (Blair-West et al., 1970). With increasing Na depletion from the body, Na is replaced by K in the saliva. Body regulatory mechanisms do not appear to be as well designed to prevent K deficiency as they are to prevent K toxicosis. In lambs deficient in K, there is a delay in renal K conservation as well as an obligatory fecal K excretion (Cowan and Phillips, 1973). As a fixed or inorganic cation, the chemical form of K is not changed by metabolism. Potassium remains unaltered chemically when ingested, retained, or excreted by the animal, whereas other inorganic elements such as Ca and P change their chemical form. In the body, K salts dissociate to yield K as a free cation or positively charged electrolyte, which must be balanced by an anion (negatively charged electrolyte). In contrast to Ca and P, K is not readily stored and must by supplied daily in the diet.

V. PHYSIOLOGICAL FUNCTIONS Potassium appears to carry out many of the same functions inside the cell that Na (see Chapter 3) performs in the plasma and interstitial fluid, i.e., maintenance of

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132

acid-base relationships and proper osmotic balance. Sodium, K, and Cl are the three major electrolytes in the body and function to maintain cation-anion balance. Sodium is the major extracellular cation, providing greater than 90% of the total cations in the plasma and interstitial fluid, whereas K, the major intracellular cation, provides approximately 75% of the total cations within the cell. Table 4.1 shows the concentration of cations and anions in the physiological fluids (Guyton, 1976; Berliner and Giebisch, 1979). Within each compartment, the net charge equals zero, and electroneutrality is maintained. Active transport (energy required) mechanisms regulate the concentration of specific electrolytes in the extracellular and intracellular compartments. The intracellular-extracellular separation of Na and K is handled by a Na pump (Wilde, 1962). Maintenance of these concentration gradients is important for transport of substrates into and out of the cell as well as regulation of osmotic pressure. Potassium is the major determinant of resting membrane potential (Peterson, 1997). The electrochemical gradients for K and Na drive or participate in a number of processes in the body. These include nerve conduction, synaptic transmission, muscle contraction, fluid transport, hormone release, and embryonic development. Potassium contributes 50% of the osmolality of intracellular fluid, whereas Na and Cl contribute 80% of extracellular osmolality (Guyton, 1976). Diffusion of water maintains equilibrium on either side of the membrane. If the concentration of molecules outside the cell is greater than the intracellular concentration, the cell loses water and dehydrates, while the extracellular fluid volume increases and edema develops. TABLE 4.1 Average Cation and Anion Concentrations in Physiological FluidsBody fluid compartments"

Cations Na+ K+ Ca 2+ Mg2+ Total

Plasma

Interstitial

Intracellular

142 4 5 2 153

145 4 3 2 154

10 159

103 28 4

117 31 4

I

I

I

40 210

Anions

ClHC0 3HPO/-, H2P04S044Protein Others Total

17 I

153

154

'Adapted from Berliner and Biebisch (1979), Guyton (1976). and Crenshaw (1983). bConcentration expressed as mEq/liter.

3 10 75 2 45 75 210

Requirements

133

Potassium is the principal base in tissues and blood cells and plays an important part in the regulation of acid-base balance. Extracellular pH is rigorously maintained within a narrow range (7.40 ± 0.05). Maintenance of this range is a complex process involving respiration, blood buffering, and renal excretion and reabsorption (Masero and Siegel, 1971). Potassium is important in the transport of oxygen and carbon dioxide through the blood and is responsible for at least half the carbon dioxide carrying capacity of the blood. Potassium is important in the transmission of nerve impulses to muscle fibers and in the contractility of the muscle itself. An ionic balance exists between K+, Na+, Ca 2+, and Mg2+. These ions affect capillary and cell function and the excitability of nerve and muscle (Thompson, 1978). For instance, K acts as a brake in regulating heart beat and suppresses heart flutter. It also helps prevent tetany, convulsions, and an unsteady gait. Potassium activates or functions as a cofactor in several enzyme systems. These include energy transfer and utilization, protein synthesis, and carbohydrate metabolism. Some of the enzyme systems influenced or activated by K include adenosine triphosphatase, hexokinase, carbonic anhydrase, salivary amylase, pyruvic kinase, and fructokinase. Potassium affecting the uptake of amino acids into cells may form the basis for the influence of K on growth. Normal intracellular K concentration is required in protein synthesis and cellular growth. There is a linear relationship between intracellular K concentration and cell growth and incorporation of amino acids into protein (Peterson, 1997). Inadequate protein biosynthesis is manifested as growth retardation in young animals.

VI. REQUIREMENTS

The wide range in requirements for livestock as recommended by the National Research Council (Table 4.2) indicates a difference between ruminant and nonruminant animals. Ruminant animals require approximately twice as much K in their diet as monogastric animals. For example, the requirements are 0.60 to 0.80% for beef cattle (NRC, 1996) and 0.17 to 0.30% for swine (NRC, 1998). The higher requirement of the ruminant may be related to ruminal function or utilization of ruminal fermentation products. Potassium is the mineral element present in highest concentrations in milk; 0.15% K compared to 0.11% Ca, 0.08% P. Because milk is a rich source of K for all of the species, lactating animals should have a higher dietary requirement. However, it is interesting that the K requirement for dairy cows is higher for maximal feed intake than it is for milk production, although K loss by cows amounts to 1.5 gjkg milk (Sasser et al., 1966). Also, a particular K requirement for lactating dairy cows might appear to be low as compared to that for sheep and feeder cattle, but highproducing dairy cows will consume feed at a much higher percentage of body weight, and thus the total K intake will be higher. Potassium requirements are dependent on the levels of other dietary nutrients. For growing chicks the K requirement was increased from 0.20 to 0.24 to 0.30%

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TABLE 4.2

Potassium Requirement for Various Species" Species

Purpose

Requirements

Reference

Chickens

Leghorn-type 0-18 wk Leghorn-type laying Leghorn-type breeding Broilers 0-8 wk All classes All classes All classes Lactating Growing All classes All classes All classes All classes Rainbow Trout Chinook Salmon All classes Growing All classes All classes All classes Growing All classes Adults Children Infants

0.25% 0.15% 0.15% 0.30% 0.40% 0.40-0.70% 0.60-0.70% 1.0-1.04% 0.46-0.48% 0.50-0.80% 0.40-1.00% 0.17-0.30% 0.30% 0.7% 0.8% 0.60% 0.40% 0.44% 0.36% 0.20% 0.50% 0.80% 1.60-2.0 g/day 65 mg/day 15-20 rngjday

NRC NRC NRC NRC NRC NRC NRC NRC NRC NRC NRC NRC NRC NRC NRC NRC NRC NRC NRC NRC NRC NRC RDA RDA RDA

Japanese Quail Turkeys Beef Cattle Dairy Cattle Sheep Horses Swine Minks Fish Rabbits Cats Dogs Rats Mice Guinea pigs Nonhuman Primates Humans

(1994) (1994) (1994) (1994) (1994) (1994) (1996) (2001) (2001) (1985b) (l989b) (1998) (1982) (1993) (1993) (1977) (1986) (l985a) (1995) (1995) (1995) (1978) (1989) (1989) (1989)

"Expressed as per unit animal feed either on as fed (approximately 90% dry matter) or dry basis (see Appendix Table I). Human requirements expressed as g or mg/day,

when high-energy diets were used (Leach et al., 1959)and up to 0.60 to 0.70% when the diet contained excessive amounts of chlorides (Nesheim et al., 1964). Growth rate in young pigs consuming a low K diet was inhibited when dietary Cl was increased from 0.03 to 0.60% (NRC, 1998). Significant interactions among dietary contents of Na, K, and Cl have been demonstrated with laying hens. Sauveur and Mongin (1978) showed that a dietary deficiency of Na in laying hens (0.05% Na) was aggravated by restriction of Cl to 0.08% and was partly compensated for by increasing the dietary K from 0.7 to 1.2%. A relationship between K requirement and protein was established (Leach et al., 1959) for chicks, with the K requirement increased as dietary protein is increased. For rats the K requirement was determined to be 0.18% in the presence of 0.1% Na. High levels of Na reduced the K requirement to 0.15%, hence Na spared K. When K levels were below 0.09%, K deficiency developed and no sparing action of Na was observed. Mabuduike et al. (1980) reported that K and Na had a sparing effect on the lysine requirements of growing pigs. Improved growth rates were

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135

observed when diets that contained low levels of lysine (0.4%) were supplemented with high levels of K (1.5%) or Na (0.9%). The effect of K on lysine metabolism in chicks is opposite to that in swine (Crenshaw, 1983). Ruminant species require between 0.5 and 1.0% dietary K, and lactating dairy cows under heat stress, 1.2%. Excitement tends to increase urinary loss of K, and diseases with fever or diarrhea further increase K loss. A study from Texas revealed increased weight gains for steers stressed by shipping when fed feedlot diets containing 1.0 to 1.5% K (Hutcheson, 1979). Other results substantiate advantages of higher dietary K levels for beef cattle grazing winter range. Karn and Clanton (1977) concluded that weaning calves should receive supplements containing at least 2% K. In Wyoming (U.S.A.), Waggoner et al. (1979) reported cows receiving supplements containing 4.15% K performed superiorly compared to those receiving 2.25% K. Florida (U.S.A.) studies indicated that 0.8% K is not adequate under heat stress, particularly with high-producing dairy cows (Beede et al., 1983b; Sanchez et al., 1994). These researchers concluded that lactating dairy cows may have a higher dietary requirement for K compared with other domestic animals because of lactational stress associated with higher milk production and high K content of milk, and because of heat stress, due to an increased loss of endogenous K through sweating and decreased daily K intake. Increasing K from 0.66 to 1.08% increased feed intake and milk yield in heat-stressed lactating cows, however, no further benefit was noted for 1.64% K (Beede et al., 1983b). Increasing the K level in the diet also increased the requirement for Na. Another study (Schneider et al., 1982) from Florida demonstrated increased feed intake and milk yield when increasing the K content of the diet from 0.8 to 1.2% for cows during heat stress. Heat stress is reported to also increase K requirements for poultry (Smith and Teeter, 1987). Broilers subjected to elevated temperatures (35°C) required a K intake of 1.5 to 2.0%. During high temperatures, K requirements for lactating dairy cows are elevated to 1.90% of dry matter (Sanchez et al., 1994; Preston, 1995). For adult humans the minimum K requirement is approximately 1.6 to 2.0 gjday with infants and children ranging from 15 to 65 mgjday (RDA, 1989). Potassium requirements would be higher for the increment needed to build new tissue during pregnancy and during lactation since milk contains about 500 mgjl. In some societies soil ingestion increases K requirements; ingestion of clay soil impairs intestinal K absorption (Peterson, 1997).

VII. NATURAL SOURCES Potassium is found in highest concentrations in plant leaves rather than the seeds (Appendix Table II). Grains contain 0.3 to 0.8%, the vegetable proteins 1.0 to 2.5% and animal products 0.3 to 2.0% K. Exceptionally low K-containing feeds are the

136

Potassium

by-product feedstuffs such as brewers dried grains, distillers dried grains, corn gluten meal, corn cobs and cottonseed hulls. In alkali-treated straw used as the source of roughage, treatment with ammonia or sodium hydroxide appears to reduce K concentrations by about 25% (Underwood and Suttle, 1999). Forage crop K content is affected by: (I) plant maturity; (2) species as well as variety within a species; (3) management procedures such as grazing or crop removal systems; (4) fertilization, particularly with K and nitrogen (N); and (5) soil and environmental conditions (McDowell, 1985; McDowell and Valle, 2000). Stage of maturity is probably the most important factor influencing forage K. Forages harvested at an early stage of maturity are one of the richer sources of K. Actively growing grass and legumes are usually high in K, containing I to 5%. However, mature pastures, winter pastures which have weathered or hay which has been exposed to rain and sun can have deficient K levels for good animal nutrition. Pfander (1972) reports that tall fescue pastures in Missouri (U.S.A.) utilized for winter grazing can decline to 0.4 to 0.5% K by late winter. These same forages contained between I and 2% in the late fall months. In Florida (U.S.A.) forage analysis from four regions indicated adequate K in the fall, but five of seven ranches sampled in winter contained considerably less than 0.6% (Kiatoko et al., 1982). Kalmbacher and Martin (1981) reported creeping bluestem (Schizachyrium stoloniferum) affected by seasonal patterns and low K concentrations ranging from 0.33 to 0.54%, with an average of 0.42%. Decline in K concentration with plant age appeared to be less pronounced in tropical grasses than in temperate species, but severity of decline will be primarily influenced by level of fertilizer N and available soil K status (Cherney et al., 1998). The commonly used grains are generally below the NRC requirements for ruminants particularly lactating dairy cows which require about 1.0% K (NRC, 2001). However, nonruminant species consuming predominately cereal grains generally would receive sufficient K in relation to requirements. This is particularly true when grains are supplemented with K-rich protein feeds (e.g., soybean and cottonseed meals). For humans, fruits and vegetables are the best sources of K (Luft, 1996). Considerable variability, nevertheless, does exist in K content of feedstuffs. In one report the K content of corn varied from 0.11 to 0.54% from samples over a 3-year period, with individual averages for the 3-years at 0.25, 0.41 and 0.37% (Hemken, 1983).

vm.

DEFICIENCY

For all species studied reduced appetite is one of the first signs of K deficiency. With K depletion in the body, there is depressed growth, muscular weakness, stiffness and paralysis. Continued K deficiency results in intracellular acidosis, degeneration of vital organs and nervous disorders. Potassium loss accompanies persistent diarrhea. Young animals with diarrhea develop acidosis and a K deficit more rapidly than do mature animals. Body stores of K are small, therefore, a deficiency can occur rapidly.

Deficiency

137

A. Effects of Deficiency 1. SWINE

Typical swine diets contain adequate K to meet requirements, with deficiencies not observed under practical farm conditions (Cunha, 1977). Reduced appetite and growth rate are the first indications of a deficiency in pigs fed semipurified low-K diets (Hughes and Ittner, 1942). For growing pigs, signs of K deficiency include rough hair coat, emaciation, inactivity, and ataxia (Jensen et al., 1961). Other clinical signs include intracellular acidosis, skeletal muscle weakness, gastric atony, reduced systemic blood pressure, nervous disorders, renal vasoconstriction and reduced urine concentrating ability and degeneration of vital organs due to lack of osmotic pressure and hydration (Hooge and Cummings, 1996). Electrocardiograms of K-deficient pigs show reduced heart rate and increased electrocardial intervals (Cox et al., 1966) with necropsy revealing no unique gross pathology. Heart rates of K-deficient pigs decreased during the 28-day feeding period from 163 to 83 beats per minute. 2.

POULTRY

Potassium deficiencies would not ordinarily be seen in commercially raised poultry due to relatively high K concentrations of most feedstuffs. Using low-K experimental diets, the main K deficiency sign in poultry was an overall muscle weakness characterized by weak extremities, poor intestinal tone with intestinal distension, cardiac weakness, weakness of the respiratory muscles and their ultimate failure (Scott et al., 1982; Leeson and Summers, 2001). Retarded growth and high mortality are common characteristics of K deficiency in the chick (Rinehart et al., 1969), and are attributed to both decreased feed consumption and impaired protein metabolism. Decreased feed consumption can occur within 24 hours in day-old chicks. There is also reduced bone ash in young chicks. Death can occur by day 50 for chicks on low K diets (Gillis, 1948). Prior to death there were tetanic seizures in which the muscles were unable to relax. Other clinical signs included weakness, loss of use of legs, excretion oflarge amounts of water and lesions of the kidneys and ureters. In laying hens, K deficiency resulted in reduced egg production, egg weight, shell thickness, and albumen content, followed by weakness, inability to stand, and death (Leach, 1974; NRC, 1994). Potassium deficiency appears to be related to severe stress. During stress, under the influence of the adrenal cortical hormone (aldosterone), K is excreted into the urine. Heat-stressed broilers receiving supplemental K improved gains over those of controls (Smith and Teeter, 1987). 3.

RUMINANTS

Ruminants are much more likely to develop K deficiencies than monogastrics, as they have higher dietary requirements for K than do other species (Table 4.2). Ruminants producing high levels of milk have greatest need for K, the mineral

138

Potassium

Fig. 4.1 The lamb on the left received a K-deficient diet (0.1 % K). (Courtesy of R.L. Preston. University of Missouri, Columbia)

element present in highest concentrations in cow's milk. Potassium deficiencies are more common for ruminants because of the increased use of dietary urea, which contains no K, as a N source. Various types of stress for ruminants have resulted in higher K requirements (see Section VI). The stress of shipping cattle has lowered subsequent gains in feedlot cattle (Hutcheson, 1979). During heat stress, K requirements are higher; however, feed intake is less (including K) (Collier et al., 1982). Increased sweating during hyperthermia results in increased loss of K in skin secretions, thus enhancing a K deficiency (Sanchez et al., 1994). Potassium deficiency for ruminants results in nonspecific signs such as slow growth (Fig. 4.1), reduced feed and water intake, lowered feed efficiency, muscular weakness, nervous disorders, stiffness, decreased pliability of hide, emaciation, intracellular acidosis, and degeneration of vital organs (Thompson, 1978). Reduced feed consumption appears to be an early indicator of marginal K deficiency, but the depression in feed intake is usually of relatively small magnitude, making it difficult to detect under field conditions (NRC, 1996). Only under experimental conditions where K is considerably below requirement levels have deficiency signs been described. Deficiency signs likely to be observed on farms would be lowered feed intake and a corresponding decrease in milk production, or decreased weight gains or losses. An analysis of diet composition is more likely to expose a suboptimal level of K than are obvious deficiency signs. In Florida (U.S.A.) studies Mallonee et al. (1982), Beede et al. (I 983b), and Sanchez et al. (1994) reported that K deficiency for lactating dairy cows resulted in dramatic reductions in feed and water intake, milk yield, and blood plasma K concentrations within 3 to 5 days after administration of a K-deficient diet. Near complete inanition, pica, tetany and death of three cows occurred (Fig. 4.2). Potassium therapy of deficient cows reversed the condition within 12 to 24 hours. In one cow signs of tetany were arrested by intravenous and rectal K and Mg therapy. A basic question unresolved is whether many of the clinical signs are due directly to a K deficiency or to starvation, because K-deficient cows stop eating immediately. Pradham and Hemken (1968) reported deficiency signs in about 3 to 4 weeks in dairy cows on low K (0.15% K) diets. These included partial to almost complete

Deficiency

139

Fig. 4.2 Potassium deficiency in a dairy cow. During 4 weeks while fed a K-adequate diet (1.1 % K), this cow (top) consumed an average 23.6 kg of diet dry matter and yielded 26.4 kg of milk per day. Abruptly switched to a K-deficient diet (0.12% K; same basal diet as adequate diet, equal in CI but without KCI), the same cows's feed intake dropped 60%, milk yield declined 54%, and water intake dropped 43% within 4 days. By the eighth day of K restriction, the cow had lost 109 kg of body weight (presumably much was a loss of gut contents). Potassium restriction resulted in pica (by day 3) and severe inanition. On the eighth day of restriction (12 hr after the bottom picture was taken) the cow died exhibiting tetany-like signs. (Courtesy of D.K. Beede and P.G. Malonee, Department of Dairy Science, University of Florida, Gainesville)

inanition and pica characterized by hair licking of stall mates, floor licking, and chewing of wooden partitions. Hair coats showed a gradual loss of glossiness and finally turned very rough. For beef cattle it was noted that on low K diets animals chewed wood on the pens and barns to such an extent that it became a real facilities maintenance problem (Roberts and 81. Omer, 1965). For sheep fed low K diets « 0.3% K) deficiency signs involved a marked decrease in feed intake, loss of weight, listlessness, impaired response to sudden

140

Potassium

disturbances. progressive stiffness from the hind legs to the forelegs. neck and back. and eventual death (Telle et al.• 1964). Brink (1961) fed lambs diets containing 0.1% K and produced K deficiency signs characterized by loss of weight, emaciation, loss of wool. muscular stiffness, lack of appetite. lack of alertness. hypoglycemia, increased liver glycogen, urinary calculi, and histological changes in the heart, kidney, adrenal gland, liver, masseter and tricep muscles, and rumen. There are very few confirmed reports of K deficiency for ruminants grazing exclusively forages. One report from Nigeria indicated clinical manifestations of K deficiency in 27 cattle, raised in the traditional semi-nomadic herding system. that consumed forages analyzing 0.20% K (Smith et al., 1980). Clinical signs quickly disappeared after administration of K. Low K concentrations have been reported for forages in Florida, Brazil, Panama, Nigeria, Swaziland. Uganda. and Venezuela (McDowell, 1997). In Brazil, average K content of six grasses at 4 weeks was 1.42% versus 0.30% at 36 weeks of age (Gomide et al., 1969). Even though mature forages and tropical forages are often low in K (see Section VII), usually grazing livestock do not exhibit a deficiency because there generally are other nutrients even more limiting (e.g., energy, protein, P and trace elements). 4. HORSES

Typical horse feeds are relatively high in K. Potassium deficiency in young horses results in decreased growth rate, reduced appetite and hypokalemia (low serum K). Stowe (1971) fed different levels of K to orphaned foals and found greatest gains at 0.8% K but optimal hematological characteristics at 1.0% K. There may be a need for higher K level for performing horses because of the K lost by sweating. 5. OTHER ANIMAL SPECIES

a. Dogs. Signs of K deficiency in dogs are poor growth, restlessness, muscular paralysis, a tendency to dehydration, and lesions of the heart and kidney (NRC, 1985a). b. Cats. Uncomplicated deficiencies are rare in cats (NRC, 1986). When a deficiency does occur in cats, depressed reflexes or cardiac failure may be observed. Potassium is also instrumental in the maintenance of the mechanisms to fight infection. c. Laboratory Animals. Potassium deficiency for rats resulted in poor growth, reduced feed intake, a rough fur coat, and death (Kornberg and Endicott. 1946). Animals become lethargic and comatose and may die within 3 weeks. Lesions are found in the heart, liver, kidney, spleen, adrenal cortex, intestine, and bone marrow. Increased dietary K was found to lower blood pressure in rats (Murphy and Cohen, 1999). Elevated extracellular K inhibited proliferation and migration of vascular smooth muscle cells and formation of free radical compounds in the rat carotid artery (Ma et al., 2000). Edema results from an increase in the "leaking" of water into the intracellular space in the absence of K. Pathological lesions are widespread

Deficiency

141

with K depletion. The initial noninflammatory degeneration of myocardial fibers is followed by necrosis and cellular infiltration. Renal lesions include cast formation in proximal convoluted tubules, sloughing of tubular epithelium in the medulla, and accumulation of hyalin droplets in the epithelium of the collecting tubules (NRC, 1995). Mice fed highly purified diets, deficient only in K, died within 1 week, after exhibiting outward signs of inanition. Lusterless eyes and hair coat, dry scaly tail, and general emaciation were observed in severe deficiency. Partial deficiencies resulted in poor growth and lack of "bloom" (NRC, 1995). d. Rabbits. Hove and Herndon (1955) found that K deficiency in rabbits resulted in a severe and rapidly progressing muscular dystrophy. Licois et al. (1978) suggested that the pathogenesis of diarrhea (induced by coccidiosis) in the rabbit is a result of a nutritional deficiency of K induced by a lack of K intake and conservation of K by the kidney. Low dietary K in rabbits exacerbated the severity of subliminal lesion development in coronary arteries (Ma et al., 1999). e. Fish. Signs of K deficiency are difficult to produce because fish readily absorb these elements from the surrounding aquatic medium. The signs of K deficiency in chinook salmon include anorexia, convulsions, tetany, and death. 6.

HUMANS

For humans K depletion may commonly be manifested by impaired neuromuscular function, varying from minimum muscular weakness to frank paralysis. Physical examination reveals muscular weakness, reduced or absent reflexes, mental confusion and soft, flabby muscles. Many chronically starved individuals are hypokalemic, suggesting that K losses in these patients are out of proportion to N losses so that true K depletion as well as decreased cell mass may result (Luft, 1996). Because of the reciprocal effects of Na and K, authorities have argued that a diet high in K and low in Na (low urinary Na-K ratio) favors lower blood pressure. Increase in dietary K (as the chloride salt) has been shown to decrease blood pressure in some hypertensive individuals (Luft, 1996). It is also possible that a low Na and high K diet would decrease the development of cardiovascular disease (Rettig et al., 1988; Falkner et al., 2000; Hermanson, 2000). Nocturnal blood pressure patterns relate to circadian rhythms and quantity and quality of sleep. High K and low Na can be beneficial for influencing circadian variation in blood pressure and resulting normal sleep (Sica, 1999). Potassium deficiencycan be a risk factor for osteoporosis. Tucker et al. (1999) reported that K, Mg, and fruit and vegetable intakes are associated with greater bone mineral density in elderly men and women. Potassium deficiencies are more commonly seen not as a result of nutritional deficiency but as a result of pathological conditions such as severe dehydration, diarrhea, bums, or other fluid losses and as a result of surgery (Fenn, 1949). Several studies have indicated that a K deficiency exists in perhaps as many as 20% of all hospitalized patients, and there is the suggestion that in elderly populations about

142

Potassium

half had a deficient intake of K. Physical activity in the elderly can increase muscle K and can assist in preventing sarcopenia in older women (Hansen and Allen, 2002). B. Assessment of Status

Low serum K analyses have some diagnostic value for establishing a dietary deficiency, but low serum K may also be caused by malnutrition, negative N balance, gastrointestinal losses, and endocrine malfunction. Beede et al. (I983a) found serum K as a poor indicator of K depletion with deficient dairy cows having abnormally high levels of the element. Routine processing procedures have resulted in leakage of K from the erythrocyte during standard 4-hour clotting times at room temperature, therefore, resulting in false-high values. For humans, Jones et al. (2001) suggest that urinary K determination is superior to food frequency questionnaires for assessing K intake. Evaluation of K deficiency for livestock is difficult. Reduced feed consumption appears to be an early sign of inadequate dietary K. Changes in electrocardiogram and muscle K content have been used with limited success (NRC, 1996). However, because reliable evaluations of K deficiency based on tissue analyses are not available, dietary K concentration appears to be the best indicator of K status.

IX. SUPPLEMENTATION

Potassium is a daily dietary essential. There are no appreciable reserves other than those in the muscle and nerve cells, in contrast to bone storage of Ca, P or Mg. The greatest need for K supplementation will be for ruminants receiving high concentrate diets, particularly animals stressed by heat, high milk production, and transport. Grain-soybean meal diets normally contain enough K to meet the requirements for swine and poultry. For example, a corn-soybean meal diet formulated to provide 16% crude protein contains approximately 0.75% K. The K in corn and soybean meal has been shown to be 90 to 97% available (Combs et al., 1985). Although K in grain-soybean meal diets is considered adequate, K supplementation has improved performance of pigs fed diets formulated with alternative protein sources (Coffey, 1987). Also, some feeder pig finishers feel it is advantageous to provide newly purchased pigs with supplemental K during the 2-week receiving diet, apparently related to stress-disease relationships (Jesse et al., 1988). Smith and Teeter (1987) reported that heat-stressed broilers receiving supplemented potassium chloride continuously gained more weight than unsupplemented birds. Ruminant animals whose diet consists of forages generally high in K are normally not subject to a K deficiency. The bioavailability of K is very high, with release of up to 99% of grass herbage K after 48 hr of ruminal incubation (Emanuele et al., 1991). Bioavailability of K in most forage grasses generally

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exceeds 85% (Miller, 1995). Beef cattle and sheep fed diets consisting mostly of concentrates, however, could be subject to a K deficiency because grains often contain less than 0.50% K. The situation would be worse for lactating dairy cattle, with a requirement between 1.00 and 1.90% K (Sanchez et al., 1994; NRC, 2001). Also, the highest milk producers in a herd of dairy cattle are usually fed a higher concentrate diet. Replacing protein supplements, such as soybean meal, with urea in high concentrate diets also exaggerates the problem of low K intake as does the practice of replacing the forage of hay or silage by fibrous material such as corn cobs, rice hulls, and gin trash, feeds which are very low in K content (Hemken, 1983). In many areas of the U.S., corn silage usage has increased and corn silage is generally lower in K content compared with other forages. Also, some dairy cattle operations are using complete diets with low K feeds such as cottonseed hulls or corn cobs as a roughage source. The likelihood of K deficiency for ruminants will increase as high-forage diets are replaced with greater quantities of low K-containing grains, by-products and urea. Generally, forages contain considerably more K than required by ruminants. However, mature pastures that have weathered or hay that has been exposed to rain and sun or was overly mature when harvested can have K levels that are less than adequate for good nutrition (Karn and Clanton, 1977; McDowell, 1985; McDowell and Valle, 2000). Potassium is a very soluble element, and dead material that is allowed to leach will have a reduced K content. Even though mature forages are low in K often a deficiency for ruminants does not occur if animals are provided in the winter or dry season with a molasses-urea supplement. Molasses counteracts low forage K as it contains a high K level of about 4.0%. At times when the price of molasses is too expensive for ruminant livestock supplementation and is not utilized, the chances for K deficiency are greatly increased. During the dry season in tropical regions, K forage concentrations are almost always less than reported ruminant requirements (McDowell, 1985; see also sections VII and VIII of this chapter). Mature tropical forages are often deficient in energy, protein, P, Na, Ca, and a number of trace elements. It is likely that a K deficiency will not be expressed as long as there are other nutrients that are even more deficient. Heat stress and high milk production are factors combining to increase the need for K supplementation. McDowell and Weldy (1967) reported 176% increase of water lost from the body by sweating in non-lactating Holstein cows at 30DC compared with cows at 20DC. Cattle and sheep sweat large quantities of K and losses from skin are related positively to ambient environmental temperatures. Rates of K loss from skin were almost five times greater for the unshaded as for shaded cows during the hottest part of the day (Mallonee et al., 1985). A high producing dairy cow secretes 25 to 40% of daily K intake in milk, depending on level of feed intake and milk yield (Beede et al., 1983a). Lactational stress places demands on the physiology of the cow no less stressful than temperature stress.

144

Potassium

Cattle subjected to the stresses of shipping suffer weight loss, primarily due to losses of body and digestive tract water. Diarrhea due to stress or disease is a common cause of acute K deficiency. When animals are stressed, the output of adrenal hormones is increased resulting in conservation of Na and excretion of K by the kidney. Under these circumstances substantial losses of body K can occur (Thompson, 1978). Research by Hutcheson (1990) shows that receiving diets for shipped, stressed calves should contain about 1.3% K. Supplemental K in cattle diets the first two weeks they are in the feedlot will help prevent death. Today many of our feed ingredients are processed differently than in the past, changing the nutritive value of the ingredients (e.g., K). Also, the relatively high variability in the K content of grains, forages, and many by-products creates one of the problems in balancing diets for K (Hemken, 1983). Animal nutritionists should consider carefully if K supplementation is warranted for their particular operation and feeding practices. Typical human diets contain adequate K; meat, fruits, and leafy vegetables are good sources. In human medicine, studies have demonstrated that the mortality rate from all causes is much higher in K-depleted patients (Krehl, 1966). Potassium salts are supplemented for treatment in the following circumstances: 1. Loss of K ion in gastrointestinal fluids, as in vomiting and diarrhea. 2. Loss of K in urine caused by diuretics. 3. Reduced plasma and extracellular K caused by electrolyte shifts (e.g., treatment of diabetic acidosis); 4. Cardiac dysfunction. Large amounts of K can be lost due to diarrhea or inappropriate use of laxatives because of the ability of colonic epithelial cells to secrete K. Potassium deficiency would be expected for these patients. Hypokalemia may not be observed because the patient will have inorganic metabolic acidosis, which causes K to leave cells and increase urinary excretion, thus masking the deficit (Peterson and Levi, 1996). Epidemiologic surveys suggest that populations ingesting diets that are low in K are more susceptible to the development of hypertension. Prevention of hypertension, and control of blood pressure in patients with hypertension, are necessary for the reduction of cardiovascular morbidity and mortality. Dietary approaches to stop hypertension demonstrate that a diet rich in fruits, vegetables, low-fat dairy products, fiber, and minerals (Ca, K, and Mg) produces a potent antihypertensive effect (Sutter, 1999; Falkner et al., 2000; Hermansen, 2000). High blood pressure is a major risk factor for stroke, which is the third leading cause of death worldwide. All three of the minerals Ca, Mg, and K, potentially contribute to blood pressure and stoke reduction; of the three minerals K apparently has the greatest effect (Bazzano et al., 2001; Hajjar et al., 2001; Massey, 2001; McCarron and Reusser, 2002). The association of increasing dietary K intake with decreasing mortality is predominant among black men, all hypertensive men, and the elderly (Luft, 1996; Fang et al., 2000). The poor and the elderly tend to have low K intake due to less than ideal diets. A high intake of processed ("fast") foods and avoidance of fresh fruits and

Toxicity

145

vegetables may contribute to this low K intake (Luft, 1996). Whelton (1993) noted a protective effect of K and a 40% reduction in a 12-year mortality statistic that was inversely related to intake of fruits and vegetables. Recent clinical and biochemical evidence support the hypothesis that consumption of dairy products may be associated with reduced blood pressure and risk of stroke. When dairy products are consumed intake of Ca, Mg, and K are improved, but K likely has the greatest effect (Massey, 2001). Selection of supplemental forms of K is influenced by economics, bioavailability, and palatability. Unfortunately supplemental forms of K are unpalatable, and therefore must be combined with more palatable ingredients (e.g., grains). Neathery et at. (1980) fed different forms of K to dairy calves and found that potassium chloride was more palatable than the carbonate form but less palatable than bicarbonate or acetate forms. However, several chemical forms of K, including the chloride, carbonate, bicarbonate, and orthophosphate sources, are approximately equal in bioavailability, approaching 100% (Peeler, 1972). Forage K also appears to be efficiently utilized. In practice, the chloride is the most frequently added supplement with the combination K-Mg-sulfate supplement also being used to increase dietary K in formulated diets.

X. TOXICITY

Toxic concentrations of K for most classes of animals have not been established. Maximum dietary tolerable levels of K of 3% have been suggested for sheep and cattle (NRC, 1980). They have also suggested the same dietary level of 3% for swine, poultry, horses and rabbits, but these recommendations were derived by extrapolation. Clinical signs of K toxicosis include cardiac insufficiency, edema, muscle weakness and death (NRC, 1980). For sheep, Jackson et at. (1971) supplemented diets with five levels of KCl to raise the K content from 0.7 to 3.0%. The effect of K was to linearly decrease weight gain. Other researchers would feel that ruminants can tolerate high dietary K levels, and even higher than 3.0%. For example, during the early part of the growing season, grazing ruminants consume immature forages that often contain 4 to 5% K. Because ingested K requirement is quickly excreted, K toxicosis, was once thought not to be a practical problem. More recently it has become established that moderately excess K can bring about or aggravate deficiencies of Na and predispose ruminants to milk fever and grass tetany. Diets high in cations, especially Na and K, tend to induce milk fever, but those high in anions, primarily Cl and S, can prevent milk fever. The incidence of milk fever is favored by the excess of the cations Na+ and K+ relative to the anions Cl " and SO/- (see Chapter 2). This concept is now generally referred to as the cationanion difference (CAD). Because most legumes and grasses are high in K, many of the commonly used prepartum diets are alkaline. The current understanding of the CAD concept suggests that milk fever could be managed more effectively if dietary K was reduced (Goff and Horst, 1997).

146

Potassium

Hypomagnesemia tetany or grass tetany is associated with consumption of forages with high K concentrations (see Chapter 5). Often young forages contain high concentrations of K, with K level an important risk factor in the development of tetany. Potassium decreases Mg absorption (Schonewille et 01.,1999). Potassium forage levels in the range of 4.0 to 5.0% are associated with a doubling of Mg requirements (Underwood and Suttle, 1999). The mechanism of K affecting Mg may be that high dietary K results in increased aldosterone secretion which lowers Mg absorption (Charlton and Armstrong, 1989). Potassium toxicosis has been studied by either intravenous or intraruminal infusion with some form of K. Potassium administered orally as a drench can have the same effect as that administered intravenously because of the rapid absorption of K. Neathery et al. (1979) infused KCI into the reticulorumen of six-month-old calves and reported doses of 1.73 gjkg of body weight or higher resulted in death. Doses greater than 0.58 g K per kg showed clinical toxicosis signs including excess salivation, muscular tremors of legs, and excitability. Doses greater than 0.29 gjkg caused increased plasma K and higher packed cell volume. Pigs can tolerate up to 10 times the K requirement if plenty of drinking water is provided (Farries, 1958). Intravenous infusion of KCI in pigs resulted in abnormal electrocardiograms (Coulter and Swenson, 1970). In the horse a muscular disease, hyperkalemic periodic paralysis, is a hereditary genetic defect that affects a Na ion channel. This results in high blood K, causing muscles to contract more readily than normal. To prevent paralysis and death, horses are managed by reducing dietary K. Equal amounts of hay and grain should be fed, avoiding large amounts of alfalfa to keep K levels lower (Spier, 1991).

XI. REFERENCES Bazzano, L. A., He, J., Ogden, L. G., Lonia, C, Vupputuri, S., Myers, L., and Whelton, P. K. (2001). Stroke (Stroke Online) Jul 32(7), 1473. Beede, D. K., Mallonee, P. G., Schneider, P. L., and Caputo, S. J. (1983a). In "Proc. Florida Nutrition Conference" p. 15, St. Petersburg Beach, Florida. Beede, D. K., Sanchez, W. K., and Wang, C. (1990). In "Proc. First Annual Florida Ruminant Nutrition Symposium" p. 48, University of Florida, Gainesville, FL. Beede, D. K., Schneider, P. L., Mallonee, P. G., Collier, R. J., and Wilcox, C. J. (l983b).ln "6th Annual International Mineral Conference (lMG)," p. 5, St. Petersburg, Florida. Berliner, R. W., and Giebisch, G. (1979). In "Best and Taylor's Physiological Basis of Medical Practice" (10th Ed.) (J. R. Brodeck, ed.), Williams & Wilkins, Baltimore, Maryland. Berliner, R. W., Kennedy, T. J., Jr., and Orloff, J. (1954). Arch. Int. Pharmacodyn. 97, 299. Blair-West, J. R., Coghlan, J. P., Denton, D. A., and Wright, R. D. (1970). In "Physiology of Digestion and Metabolism in the Ruminant" (A. T. Phillipson, ed.), Oriel Press, Newcastle, England. Brink, M. F. (1961). "Potassium Requirement of the Immature Bovine." Ph.D. Thesis, University of Missouri, Columbia, Missouri. Charlton, J. A., and Armstrong, D. G. (1989). Quarterly J. Expt. Phys. 74, 329. Cherney. J. H., Cherney, D. J. R., and Bruulsema, T. W. (1998). In "Grass for Dairy Cattle" (J. H. Cherney and D. J. R. Cherney, eds.) p. 137. CABI Publishing, New York. Church, D. C, and Pond, W. G. (1974). "Basic Animal Nutrition and Feeding." Corvallis, Oregon. Coffey, T. (1987). Feedstuffs 59(41), 14. Collier, R. J., Beede, D. K., Thatcher, W. W., Israel, L. A., and Wilcox, C. J. (1982). J. Dairy Sci. 65, 2213.

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Combs, N. R., Miller, E. R., and Ku, P. K. (1985). J. Anim. Sci. 60, 709. Coulter, D. B., and Swenson, M. J. (1970). Am. J. Vet. Res. 31, 2001. Cowan, T. K. J., and Phillips, G. D. (1973). Can. J. Anim. Sci. 53, 653. Cox, J. L., Becker, D. E., and Jensen, A. H. (1966). J. Anim. Sci. 25, 203. Crenshaw, T. D. (1983). In "Sodium and Potassium in Animal Nutrition" (T. J. Cunha, R. W. Hemken, and T. D. Crenshaw, eds.), National Feed Ingredients Association, West Des Moines, Iowa. Cunha, T. J. (1977). "Swine Feeding and Nutrition." Academic Press, New York, Emanuele, S. M., Staples, C. R., and Wilcox, C. J. (1991). J. Anim. Sci. 69, 801. Falkner, B., Sherif, K., Michel, S., and Kushner, H. (2000). Arch. Pediatr. Adolesc. Med. 154,918. Fang, J., Madhavan, S., and Alderman, M. H. (2000). Stroke (Stroke online) 31(7), 1532. Farries, F. E. (1958). 'The Nutrient Requirements of Pigs," p. 240, Agricultural Research Council, Commonwealth Agriculture Bureaux, Slough, England. Fenn, W. O. (1949). Sci. Am. 181(2), 16. Gillis, M. B. (1948). J. Nutr. 36, 351. Goff, J. P., and Horst, R. L. (1997). J. Dairy Sci. 80, 176. Gomide, J. A., Noller, C. H., Mott, G. 0., Conrad, J. H., and Hill, D. L. (1969). Agron. J. 61, 120. Guyton, A. C. (1976). "Textbook of Medical Physiology" 5th Ed. W.B. Saunders, Philadelphia, Pennsylvania. Hajjar, I. M., Grim, C. E., George, V., and Kotchen, T. A. (2001). Arch. Intern. Med. 161, 589. Hansen, R. D., and Allen, B. J. (2002). Am. J. Clin. Nutr. 75, 314. Hemken, R. W. (1983). In "Sodium and Potassium in Animal Nutrition" (T. J. Cunha, R. W. Hemken, and T. D. Crenshaw, eds.), p. I. National Feed Ingredients Association, West Des Moines, Iowa. Hermansen, K. (2000). Br. J. Nutr. 83(Suppl. I), SIB. Hooge, D. M., and Cummings, K. R. (1996). Feedstuffs 68(13), 12. Hove, E. L., and Herndon, J. F. (1955) J. Nutr. 35, 363. Hughes, E. H., and Ittner, N. R. (1942). J. Agric. Res. 64, 189. Hutcheson, D. P. (1979). Anim. Nutr. Health 34, II. Hutcheson, D. P. (1990). In "Proc, First Annual Florida Ruminant Nutrition Symposium," p. 8. University of Florida, Gainesville, Florida. Jackson, H. M., Kromann, R. P., and Ray, E. E. (1971). J. Anim. Sci. 33, 872. Jesse, G. W., Walker, J. R., Weiss, C. N., and Mayes, H. F. (1988). J. Anim. Sci. 66, 1325. Jensen, A. H., Terrill, S. W., and Becker, D. E. (1961). J. Anim. Sci. 20,464. Jones, G., Riley, M. D., and Whiting, S. (2001). Am. J. Clin. Nutr. 73, 839. Kalmbacher, R. S., and Martin, F. G. (1981). J. Range Manag. 34,406. Karn, J. F., and Clanton, D. C. (1977). J. Anim. Sci. 45, 1426. Kay, R. N. B., and Pfeffer, E. (1970). In "Physiology of Digestion and Metabolism in the Ruminant" (A. T. Phillipson, ed.), p. 390. Oriel Press, Newcastle, England. Kiatoko, M., McDowell, L. R., Bertrand, J. E., Chapman, H. L., Pate, F. M., Martin, F. G., and Conrad, J. H. (1982). J. Anim. Sci. 55, 28. Kornberg, A., and Endicott, K. M. (1946). Am. J. Physiol. 145,291. Krehl, W. A. (1966). Nutr. Today I, 20. Leach, R. M., Jr. (1974). J. Nutr. 104,684. Leach, R. M., Jr., Dam, R., Zeigler, T. R., and Norris, I. C. (1959). J. Nutr. 68, 89. Leeson, S., and Summers, J. D. (2001). In "Nutrition of the Chicken, 4th Ed." University Books, Guelph, Canada. Licois, D., Coudert, P., and Mongin, P. (1978). Ann. Rech. Vet. 9,453. Lindeman, R. D., and Pederson, J. A. (1983). In "Potassium: Its biological significance" (R. Whang, ed.), p. 45, CRC Press, Boca Raton, Florida. Luft, F. C. (1996). In "Present Knowledge in Nutrition" 7th Ed. (E. E. Ziegler and L. S. Filer, Jr., eds.) p. 272, ILSI Press, Washington, D.C. Ma, G., Young, D. B., and Clower, B. R. (1999). Am. J. Hypertension 12, 821. Ma, G., Young, D. B., Clower, B. R., Anderson, P. G., Lin, H., and Abide, A. M. (2000). Am. J. Hypertension 13, 1014. Mabuduike, F. N., Calvert, C. C., and Austic, R. E. (1980). J. Anim. Sci. 51(Suppl. 1),210 (Abstr.). Mallonee, P. G., Beede, D. K., Collier,R. J., and Wilcox, C. J. (1985). J. Dairy Sci. 68, 1479. Mallonee, P. G., Beede, D. K., Schneider, P. L., Caputo, S. J., and Wilcox, C. J. (1982). J. Dairy Sci. 65(Suppl. I), 112. Masero, E. J., and Siegel, P. D. (1971). "Acid-base Regulation: Its Physiology and Pathophysiology." W. B. Saunders, Philadelphia, Pennsylvania.

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Massey, 1. K. (2001). J. Nutr. 131, 1875. McCarron, D. A., and Reusser, M. E. (2002). Am. J. Hypertens. 14, 2065. McDowell, 1. R. (1985). "Nutrition of Grazing Ruminants in Warm Climates." Academic Press, New York. McDowell, 1. R. (1997). "Minerals for Grazing Ruminants in Tropical Regions" 3rd Ed. University of Florida, Gainesville, Florida. McDowell, 1. R., and Valle, G. (2000). In "Forage Evaluation in Ruminant Nutrition" (D. I. Givens, E. Owen, R. F. E. Oxford, and H. M. Omed, eds.), p. 373, CAB International, Wallingford, UK. McDowell, R. E., and Weldy, J. R. (1967). In "Biorneterology," Vol. 2, Part I, p. 414. Pergamon Press, New York. Michell, A. R. (1995). In "Sodium in Agriculture" (c. J. C. Phillips and P. C. Chiy, eds.), p. 91, Chalcombe Publications, Canterbury, UK. Miller, E. R. (1995). In "Bioavailability of Nutrients for Animals" (C. B. Ammerman, D. H. Baker, and A. J. Lewis, eds.), p. 295, Academic Press, Inc., New York. Mroz, Z., Reese, D. E., Overland, M., VanDiepen, J. T. M., and Kogut, J. (2002). J. Anim. Sci. 80, 681. Murphy, M. E., and Cohen, D. B. (1999). J. Hypertension 17, 1481. Neathery, M. W., Pugh, D. G., Miller, W. J., Gentry, R. P., and Whitlock, R. H. (1980). J. Dairy Sci. 63,82. Neathery, M. W., Pugh, D. G., Miller, W. J., Whitlock, R. H., Gentry, R. P., and Allen, J. C. (1979). J. Dairy Sci. 62, 1758. Nesheim, M. C., Leach, R. M., Jr., Zeigler, T. R., and Serafin, J. A. (1964). J. Nutr. 84, 361. NRC. (1980). "Mineral Tolerance of Domestic Animals." National Academy of Sciences - National Research Council, Washington, D.C. NRC. Nutrient Requirements of Domestic Animals. National Academy of Sciences - National Research Council, Washington, D.C. (1977). Nutrient Requirements of Rabbits. (1978). Nutrient Requirements of Nonhuman Primates. (1982). Nutrient Requirements of Mink and Foxes. (1985a). Nutrient Requirements of Dogs, 2nd Ed. (1985b). Nutrient Requirements of Sheep, 5th Ed. (1986). Nutrient Requirements of Cats, 3rd Ed. (1989). Nutrient Requirements of Horses, 5th Ed. (1993). Nutrient Requirements of Fish. (1994). Nutrient Requirements of Poultry, 9th Ed. (1995). Nutrient Requirements of Laboratory Animals. (1996). Nutrient Requirements of Beef Cattle, 7th Ed. (1998). Nutrient Requirements of Swine, 10th Ed. (2001). Nutrient Requirements of Dairy Cattle, 7th Ed. Peeler, H. T. (1972). J. Anim. Sci. 3S, 695. Peterson, 1. N. (1997). In "Handbook of Nutritionally Essential Mineral Elements" (O'Dell and M. Sunde, eds.), p. 213, Marcel Dekker, New York. Peterson, 1. N., and Levi, M. (1996). In "Renal and Electrolyte Disorders, 5th Ed" (R. W. Schrier, ed.), Chapter 5, Little Brown, Boston. Pfander, W. H. (1972). Feedstuffs 44(15), 15. Pradham, K., and Hemken, R. W. (1968). J. Dairy Sci. SI, 1377. Preston, R. 1. (1985). In "Eighth Annual International Minerals Conference," p. I. Daytona Beach, Florida, International Minerals and Chemical Corporation, Mundelein, Illinois. Preston, R. L (1995). Feedstuffs 67(12), 13. RDA (2001). "Recommended Dietary Allowances." Food and Nutrition Board, National Academy of Sciences, Washington, D.C. Rai, A. K., More, T., and Singh, M. (1981). J. Agric. Sci. 96, 7. Rettig, R., Ganten, D., and Luft, F. C. (1988). "Salt and Hypertension." Springer Verlag, Berlin, Germany. Rinehart, K. E., Featherston, W. R., and Rogier, J. C. (1969). Poult. Sci. 48, 320. Roberts, W. K., and St. Orner, V. V. E. (1965). J. Anim. Sci. 24, 902 (Abstr.). Sanchez, W. K., McGuire, M. A., and Beede, D. K. (1994). J. Dairy Sci. 77, 2051. Sasser, L. 8., Ward, G. M., and Johnson, 1. E. (1966). J. Dairy Sci. 49, 893. Sauveur, B., and Mongin, P. (1978). Br. Poult. Sci. 19,475. Schneider, P. 1., Beede, D. K., Wilcox, C. J., and Collier, R. J. (1982). J. Dairy Sci. 6S(Suppl. I), 112 (Abstr.),

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Shonewillle, J. T., Beynen, A. c., Van't Klooster, A. T., Wouterse, H., nad Ram, L. (I 999a). J. Nutr. 129, 2043. Schonewille, J. T., Van't Klooster, A. T., Wouterse, H., and Beynen, A. C. (1999b). J. Dairy Sci. 82, 1824. Scott, M. L., Nesheim, M. C, and Young, R. J. (1982). "Nutrition of the Chicken." M. L. Scott and Associates, Ithaca, New York. Sica, D. A. (1999). Blood Press. Monit. 4(Suppl. 2), S9. Smith, M. 0., and Teeter, R. G. (1987). Poult. Sci. 66,487. Smith, O. B., Kasali, 0.8., Adeyanju, S. A., and Adegbola, A. A. (1980). J. Anim. Sci. 51(Suppl. 1),396 (Abstr.). Spier, S. J. (1991). J. Am. Vet. Assoc. 20, 584. Stowe, H. D. (1971). J. Nutr. 101,629. Suter, P. M. (1999). Nutr. Rev. 57(3), 84. Telle, P. P., Preston, R. L., Kintner, L. D., and Pfander, W. H. (1964). J. Anim. Sci. 23, 59. Thompson, D. J. (1978). In "Proc. Latin American Symposium on Mineral Nutrition Research with Grazing Ruminants" (J. H. Conrad and L. R. McDowell, eds.), p. 47. University of Florida, Gainesville, Florida. Tucker, K. L., Hannan, M. T., Chen, H., Cupples, L. A., Wilson, P. W., and Kiel, D. P. (1999). Am. J. Clin. Nutr. 64, 727. Underwood, E. J., and Suttle, N. F. (1999). In "The Mineral Nutrition of Livestock" 3rd Ed. Midlothian, UK. Vellaire, C. D. (1965). "Potassium - the Alkali of Life. Better Crops with Plant Food." America Potash Institute, Washington, D.C. Waggoner, J. W., Jr., Kaltenbach, C. c., Smith, W. W., Varnel, T. R., Clark, D. H., Applegate, S. 1., Nelms, G. E., and Radloff, H. R. (1979). Proc. West. Sec. Am. Soc. Anim. Sci. 30, 284. Whelton, P. K. (1993). In "Hypertension Primer" (J. L. Izzo, Jr., and H. R. Black, eds.), p. 170, American Heart Association Press, Dallas, Texas. Wilde, W. S. (1962). "Mineral Metabolism" Vol. IIB (C. L. Comar and F. Bronner, eds.), Academic Press, New York.

Chapter 5

Magnesium I. INTRODUCfION All living organisms require magnesium (Mg). Deficiencies are uncommon, owing to generally adequate concentrations of the element in foods of monogastric animals and humans. The practical importance of Mg in ruminant nutrition is its relationship to the serious metabolic disorder, grass tetany (hypomagnesemia). Excellent and extensive reviews concerning hypomagnesemic tetany are available: Rendig and Grunes (1979), Fontenot et al. (1983), Grunes and Mayland (1984), and Underwood and Suttle (1999).

II. mSTORY Sell (1980) described some early history of Mg by reviewing the importance of the water from the English village of Epsom, a popular health spa in the early 1600s. Epsom possessed unusual properties generally thought to be beneficial for people, as an "internal remedy and purifier of the blood." The principal mineral component of the water was magnesium sulfate (Epsom salts). Magnesium was first isolated in metallic form by Davy in 1808. In the early 1900s various experiments isolated the mineral and demonstrated its importance as an essential element for animals (Osborne and Mendel, 1918). Magnesium was first shown by LeRoy in 1924 to be essential for normal growth in mice (Leeson and Summers, 200I). In 1932, Kruse and co-workers produced Mg deficiency in rats and described the specific clinical signs of the deficiency: vasodilation, hyperirritability, convulsions, and death. In 1934 the first descriptions of various disease conditions related to Mg depletion in humans was published (Shils, 1997). For humans, this was followed in the early 1950s by documentation that Mg ion depletion occurred in alcoholics and in patients receiving Mg-free intravenous solutions. The serious metabolic disorder in cows, known as lactation tetany or grass tetany, was shown to be associated with subnormal serum Mg values (Sjollema, 1930). With this discovery, research on Mg and its relationship with other elements such as calcium (Ca), phosphorus (P), potassium (K), and sodium (Na) was stimulated. 151

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III. CHEMICAL PROPERTIES AND DISTRmUTION Magnesium is an alkaline earth metal belonging to Group IIA of the periodic table, with atomic number 12 and atomic weight 24.31. Magnesium is one of the most plentiful elements in the earth's crust (2.1 % by weight) and is present in considerable concentrations in nonliving matter as well as in living organisms. Magnesium is a silver white metal with a hexagonal close-packed structure. It is found naturally only in the forms of its compounds in magnesite, carnallite, dolomite, epsomite, kieserite, and many others. It is found in sea water and in all plants and animals. Magnesium combines directly with nitrogen (N), sulfur (S), the halogens, P, and arsenic (As). Among the important chemical characteristics of Mg are the tendencies to form aquo-complexes in solution and to form stable complexes with various organic moieties (Phipps, 1976). The latter property is exemplified by Mg's central role in chlorophyll, the photosynthetic pigment of green plants: photosynthesis does not proceed when Mg has been removed from the chlorophyll molecule (Aikawa, 1959). Magnesium is abundant in most common feedstuffs, and is present in the animal body at approximately 0.05% of total weight, with about 60 to 70% found in the skeleton, and the remainder in the soft tissue and extracellular fluids. In the pig body, Mg content at birth (0.01%) was lower relative to that in older and heavier pigs (Mahan and Shields, 1998). Serum, for example, normally contains Mg at 2 to 4 mgjdI. The concentration of Mg within the cells of the body is higher than that of any other mineral except K.

IV. METABOLISM Studies of Mg have been limited by lack of a suitable Mg isotope at an economical price. The isotope most used is 28Mg, which is expensive and has a halflife in the range of 21 hr, resulting in complete disappearance 9 days after use.

A. Absorption Magnesium is absorbed throughout the digestive tract, and endogenous secretions containing variable amounts of the element are also reabsorbed (Phillipson and Storry, 1965). In simple-stomached animals, the small intestine is the main site for Mg absorption. Guenter and Sell (1973) observed that more than 50% of the Mg absorbed by chickens was taken up from the duodenum and first portion of the jejunum. However, studies in rats that used segment perfusion of everted intestinal sacs have mostly shown that Mg is better absorbed in the ileum and colon than in the jejunum (Hardwich et al., 1991). In stripped rat mucosa, Mg was secreted across the duodenum but absorbed in the ileum and in the colon, where both passive and nonpassive cellular transport processes exist (Karbach, 1989). There is a passive diffusional process for intestinal Mg

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absorption, in rats fecal excretion was nearly directly proportional to dietary Mg intakes (Coudray et al., 2002). For ruminant animals the major Mg absorption site is the reticulorumen portion of the digestive tract (Grace and MacRae, 1972; Emanuele et al., 1991; Robson et al., 1997). In monogastrics the rate for mature animals is much higher, from 30 to 60% (Meyer, 1976). Whittemore and Manson (1995) suggest a lower absorption rate for the growing pig to be 26%. Guenter and Sell (1974) estimated that the true absorption of Mg (%) in chickens was 55.7 from corn, 56.8 from wheat, 82.7 from oats, 54.5 from barley, 60.3 from soybean meal, 62 from dried skimmed milk, and 42.5 from rice. Availability values for Mg as high as 70% have been observed in young, milk-fed calves, but these decline to 30 to 50% in older calves (Peeler, 1972). In a review, Henry and Benz (1995) reported that apparent availability of Mg in fresh grasses or grass hays varied greatly from -4% to +66%. Suttle (1987) suggested that the difference in absorbability between fresh grass and hay + concentrate diets might be two- to three-fold. Dalley et al. (1997) showed that Mg had greater solubility at the site of Mg absorption for hay and concentrate than from fresh pasture. The solubility difference was greatest at a higher ruminal pH. Undoubtedly, the proportion of Mg absorbed declines with increasing dietary levels of this element (Heaton, 1960), and the Mg status of the animal alters Mg absorption (McAleese et al., 1961). Grace and MacRae (1972) reported that the system of feeding affected Mg absorption. Under continuous feeding, 89 to 98% of the Mg was absorbed in the stomach of sheep, whereas only 42 to 52% was absorbed when they were fed once daily. Factors that affect the ionization of Mg from salts or organic complexes and/or the availability of absorption sites influence Mg absorption. Data from children suggested that two separate transport systems participate in the absorption of Mg from the proximal small intestine (Milia et al., 1979), a carrier-mediated system at low intraluminal concentrations, and simple diffusion at higher concentrations. A number of dietary and physiological factors also influence Mg absorption. High levels of dietary P or Ca adversely affect Mg absorption. Chicco et al. (1973) found that fecal Mg excretion is increased and serum Mg decreased by feeding high Ca and P levels to sheep. Magnesium and Ca may compete for the same absorption sites along the small intestine (Alcock and Macintyre, 1962). Phosphorus may form relatively insoluble salts with Mg, thereby reducing its absorption. Dietary protein source and supplemental fat also have an effect on Mg metabolism. Greater protein intake raises apparent Mg absorption (Brink and Beynen, 1992; Verbeek et al., 1993). For swine, dietary casein resulted in a higher Mg retention than was observed with soy protein. The difference between these protein sources may have been due to the relatively high phytate content of soy protein (Miller et al., 1965; Brink et al., 1992). Fat supplementation has been shown to decrease Mg utilization. Hakansson (1975) observed a reduction in Mg retention and an increased Mg requirement in growing chicks when tallow or lard was included in diets. Similarly, supplementation with higher fatty

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acids lowered Mg absorption in dairy cows (Kemp et al., 1966). In steers, supplemental fat decreased apparent Mg absorption 41.7% (Ramirez and Zinn, 2000). Young, lush pastures could be detrimental to Mg absorption if they have high concentrations of protein and K. They are low in fiber, increasing the passage rate and thereby reducing absorption. High dietary K, consumed by ruminants grazing immature forages, will decrease Mg absorption from the gut and thereby induce hypomagnesemia and tetany (Ram et al., 1998; Schonewille et al., 1999a) (see also Sections VII and IX). When lambs were fed diets containing 4% K as potassium chloride (KCI), apparent Mg absorption was reduced to 14.6% from 44.6% (Reffett and Boling, 1985). Different K salts have different effects on Mg absorption in sheep. Potassium bicarbonate and citrate forms were more inhibitory on Mg absorption than KCI (Schonewille et al., 1999b). Other workers have emphasized the importance to Mg absorption of the Na:K ratio, ammonia concentrations in ruminal contents, and the binding of Mg by fatty acids or bacterial proteins. Feeding 1000 ppm of iron (Fe) to steers resulted in depressions in apparent absorption of Mg (Standish et al., 1971). Feeding a high level of salt (3% Na) increased apparent absorption of Mg in sheep (Mosley and Jones, 1974). This could be related to the antagonism of Na to K, which would modify the antagonism between K and Mg. On the contrary, Wachirapakron et al. (1996) concluded that excess Na reduced Mg absorption or retention and may exacerbate existing hypomagnesemia in sheep. Dennis (1971) reported aluminum (AI) concentrations of up to 1000 ppm in a few forage samples of winter-grazed cereals grown on soil containing free lime. These values could be attributed to soil contamination. No change in serum Mg occurred in steers fed diets containing up to 1200 ppm Al (Valdivia et al., 1978). In general, high N fertilization results in a lower serum Mg concentration and a higher incidence of grass tetany (Kemp, 1983). Hypomagnesemia of cows, especially in early spring, may arise from inadequate absorption of Mg associated with high ruminal ammonia concentrations (Martens and Rayssiguier, 1980). Organic acid concentrations (i.e., citrate and trans-aconitate) in several species of forages have been suggested to interfere with Mg absorption and may be a contributing factor to grass tetany. Tetany that resembled field cases of grass tetany was produced experimentally by dietary administration of KCI and citric acid or trans-aconitic acid to cattle (Bohman et al., 1969). For ruminants, more readily available carbohydrates have a favorable effect on Mg absorption (Fontenot et al., 1989). Apparent Mg absorption, 15% in sheep fed unsupplemented hay, increased to 35 to 38% for sheep supplemented with readily degradable carbohydrates (Giduck and Fontenot, 1987). Ruminal pH is important as it influences solubility and absorbability in the rumen. Ruminal solubility of Mg decreased from 80 to 20%, approximately, when pH was increased in vitro from 5 to 7% (Dalley et al., 1997). Ionophores facilitate passage of ions across all membranes, thus Mg absorption is positively affected. Monensin and lasalocid increased apparent absorption of Mg by approximately 10% in steers (Starnes et al., 1984). The ionophore laidlomycin

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propionate enhanced Holstein steer performance, with the response likely enhanced by increasing dietary Mg level (Ramirez et al., 1998).

B. Excretion and Storage Magnesium is excreted in both urine and feces and secreted in milk. Urine is the major excretory pathway for Mg after absorption (Sell, 1980). Endogenous Mg also reaches the feces by way of bile, saliva, gastric juices, pancreatic juices, intestinal secretion, and intestinal defoliation (Storry, 1961). Fecal Mg excretion varies with Mg intake in sheep. A correlation of 0.67 was obtained between dietary and fecal Mg and 0.95 between Mg absorption and urinary excretion (Chicco et 01., 1972). Even though milk is low in Mg, it was the main source of Mg losses from the body in milking cows (Meyer, 1976). Salih et al. (1987) reported Mg in the colostrum of Brahman cows to be 220 ppm and to decline to 61 ppm after 3 months of lactation. About 60 to 70% of total Mg is in the bones, with the remainder equally distributed between muscle and other soft tissues. The Mg content in bone ash varies from 0.7% in young animals to 0.5% in older ones (Meyer, 1976). The amount that can be mobilized from bone decreases with the age of the cow. Blaxter and McGill (1956) calculated that older cows with more than six calves were 14 times more likely to develop grass tetany than were first-lactation heifers, which relates to available bone reserves of Mg. By far the major portion of Mg in eggs occurs in eggshells and during incubation from 1 to 1.8 mg of the shell Mg is transferred to the embryo. Thus the shell seems to be a reservoir for both Ca and Mg for use by the embryo (Leeson and Summers, 2001).

V. PHYSIOLOGICAL FUNCfIONS Magnesium has many diverse physiological functions (AI-Ghamdi et al., 1994). The Mg in the skeleton is important for the integrity of bones and teeth. It is present mainly as the Mg ion and as Mg(OHh held within the hydration shell of the apatite crystal surface (Rook and Storry, 1962). Magnesium is the second most plentiful cation (after K) in intracellular fluids. Although only about 1% of the total Mg is in the extracellular fluid (blood plasma and interstitial fluid), this Mg bathes the body cells and is of great importance. When the Mg in the extracellular fluid declines substantially below normal, the consequences are quite serious (e.g., tetany). Magnesium is involved in at least 300 enzymatic steps in intermediary metabolism (Shils, 1996). Magnesium is an active component of several enzyme systems in which thiamin pyrophosphate (TPP) is a cofactor. Oxidative phosphorylation is greatly reduced in the absence of Mg. It is also an essential activator for the phosphate-transferring enzymes myokinase, diphosphopyridinenucleotide kinase, and creatine kinase. It also activates pyruvic acid carboxylase, pyruvic acid oxidase, and the condensing enzyme for the reactions in the Krebs

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cycle. Magnesium also appears to facilitate the transketolase reaction in the pentose mono phosphate shunt. In the beta oxidation of fatty acids, the first step involving acyl CoA synthetase requires ATP and Mg 2+. In fatty acid synthesis the commitment step is the carboxylation of acetyl CoA to malonyl CoA by the carboxylase that requires ATP, Mg2 + , and bicarbonate (Shils, 1996). Intracellularly, Mg is predominantly associated with the mitochondria. Its main role in this respect is as an activator of enzymes (Wacker, 1969). Magnesium is vitally involved in the metabolism of carbohydrates and lipids as a catalyst of a wide array of enzymes. Magnesium is needed for normal insulin sensitivity and may be involved in early molecular steps of insulin action in the liver (Reis et al., 2000). It is essential for cellular respiration, and in certain tissues, it is complexed with adenosine triphosphate (ATP), adenosine diphosphate (ADP), and adenosine monophosphate (AMP). It is clear that Mg is important in almost all functions of the body, since ATP is required in numerous different functions such as muscle contractions; protein, nucleic acid, fat, and coenzyme synthesis; glucose utilization; methyl group transfer; sulfate, acetate, and formate activation; in oxidation phosphorylation; and many more functions. Magnesium is required at various steps in the synthesis of DNA, RNA, and protein (Brody, 1999). Magnesium is involved in protein synthesis through its action on ribosomal aggregation, its roles in binding messenger RNA to 70S ribosomes, and in the synthesis and degradation of DNA. It is also essential for the formation of cyclic AMP and other second messengers (Watson et al., 1980). Magnesium plays an important role in neuromuscular transmission, acting at some points synergistically with Ca, while at others, as an antagonist. The functions of Mg can be illustrated by the diverse physiological properties modified during a deficiency, which include (I) growth, (2) immunity and allergy, (3) muscle contraction, (4) red blood cell survival, (5) occurrence of neoplasms, (6) metabolism of collagen-rich tissues, and (7) Na and K metabolism (Larvor, 1983). Results from South Africa even suggest a reproductive role for Mg in dairy cows (Dugmore et al., 1987). Calving intervals were significantly reduced from 394 days for control animals to 373 for Mg-supplemented animals while services to conception were respectively reduced from 1.94 to 1.54.

VI. REQUIREMENTS Dietary requirement for Mg set by the National Research Council are presented in Table 5.1. Values for the requirement of Mg for most classes of poultry and swine range from 0.04 to 0.06%. Sell (1980) proposed that the Mg requirements of laying hens depend on the type of diet and criteria used. Only 155 ppm is required for normal serum Mg levels, 255 ppm is required for high rates of egg production, while 355 ppm is needed for optimal egg weight, normal egg Mg concentrations, and maximal hatchability and liveability of chicks (Hajj and Sell, 1969).

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TABLE 5.1 Magnesium Requirement for Various Speeies" Species Chickens

Japanese quail Turkeys Beef cattle Dairy cattle Sheep Horses Swine Minks Fish Rabbits Cats Dogs Rats Mice Guinea pigs Nonhuman primates Humans

Purpose Leghorn-type 0--6 wk Leghorn type, 12-18 wk Leghorn-type laying Broilers 0--8 wk All classes All classes All classes Lactating All classes All classes All classes All classes Channel Catfish All classes Growing All classes All classes All classes Growing All classes Children Women Men

Requirements 0.06% 0.04% 0.05% 0.06% 0.03-0.05% 0.05% 0.10-0.20% 0.18-0.21% 0.12-0.18% 0.08-0.13% 0.04% 0.44% 0.04% 0.03-0.04% 0.4% 0.04% 0.05% 0.05% 0.10% 0.15% 80--130 mg(day 240--360 mg/day 240--420 mg(day

References NRC NRC NRC NRC

(1994) (1994) (1994) (1994)

NRC (1994) NRC (1994) NRC (1996) NRC (2001) NRC (l985b) NRC (1989) NRC (1998) NRC (l982a) NRC (1993) NRC (1977) NRC (1986) NRC (l985a) NRC (1995) NRC (1995) NRC (1995) NRC (1978) DRI (2001) DRI (2001) DRI (2001)

"Expressed as per unit animal feed either on as fed (approximately 90% dry matter) or dry basis (see Appendix Table I).

Minimal requirements of Mg for growth of grazing livestock can generally be met by pastures or diets containing 0.10 to 0.15%. A greater concentration, 0.18 to 0.21 %, is considered necessary for lactating cows. Magnesium requirements for gestating beef cows have been estimated to be between 7 and 9 g/day, and between 18 and 22 g/day during lactation (Fontenot, 1980). In order to meet the lactation requirement, forages must contain between 0.16 and 0.19% Mg. It appears that Mg required by beef cows is much lower during gestation than during lactation. The minimal dietary requirements depend on the species, the criterion of adequacy employed, the chemical form in which the element is ingested, and the nature of the rest of the diet. Shockey et al. (1984) suggested that sheep were 1.75 times more efficient at absorbing dietary Mg than cattle and they explained their findings by the higher ratio of ruminal surface area:ruminal contents in sheep. Many dietary factors influence Mg absorption, and therefore Mg requirements, and include K, N, Ca, P, AI, Fe, Na, protein, fat, organic acids, carbohydrate type, ionophores, Mg status, and frequency offeeding (Fontenot et al., 1989, and see also Section IV). Early research from the Netherlands indicated that a Mg requirement of 0.20% was a safe level. However, for high-yielding dairy cows on pastures

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heavily fertilized with Nand K, which lower the availability of Mg, Dutch workers state the Mg requirements as 0.30 to 0.38% of dry matter (NCMN, 1973). For poultry, Nugara and Edwards (1963) found that the Mg requirement of growing chicks increased from 460 to 560 ppm, as dietary P increased from 0.3 to 0.9%, and the Ca level was kept at 0.6%. Raising the Ca level to 1.2% caused an additional increase in dietary Mg needs. Wu and Britton (1974) determined Mg requirements of chicks to be 200 ppm at 0.3% dietary P, but 400 ppm Mg was required with diets containing 0.8% P. Requirements of laying hens are dependent on the criteria of adequacy selected. For normal serum Mg 155 ppm is required, for optimum egg production 225 ppm is needed, and for satisfactory egg weight and hatchability 355 ppm Mg is required (Hajj and Sell, 1969). There are reported genetic differences in Mg requirements in some species. When two white Leghorn strains of chicks were fed 150 ppm Mg, the mortality rate was much lower in one strain (Christensen et al., 1964). This greater survival was correlated with a greater amount of Mg in the shell, coupled with greater ability to remove Mg from the shell during incubation. In a comparison of beef breeds on a common diet, Aberdeen Angus were found to have relatively low serum Mg concentrations (Littledike et al., 1995). Marked cattle breed differences in Mg metabolism have been linked to differences in susceptibility to grass tetany. Brahman cows are less susceptible to death from grass tetany and metabolic disorders than British breeds of cattle, whereas cows with 50% or greater dairy breeding (e.g., Holstein and Jersey) are more susceptible than British or Brahman breeds when mainstreamed in beefproduction herds (Laurenz et al., 1988; Greene et al., 1989). Magnesium absorption has been shown to be greater; hence, requirements are lower in Brahman than in Jersey, Holstein, and Hereford cows. Greene et al. (1986) performed an experiment to determine if breed or dry matter (OM) intake influenced Mg availability to dry, nonpregnant, mature cows. Hereford, Holstein, and Jersey breeds excreted more fecal Mg and absorbed less Mg than Angus and Brahman breeds. The human requirement for Mg is not well defined because of the limited data and unsatisfactory methods for estimating requirement. Human Mg requirements (rug/day) are estimated to be 30 to 130 for children, 240 to 420 for adults, and vary from 310 to 400 for pregnancy and lactation (DRI, 2001).

VII. NATURAL SOURCES Most cereal grains are fair sources ofMg, varying from 0.13 to 0.22% (dry basis). Plant protein supplements are excellent sources (0.28 to 0.62% Mg) (Appendix Table II), whereas protein supplements of animal origin are more variable (0.11 to 1.22% Mg). By-product feedstuffs derived from plants tend to be good sources of Mg, while forages are even more variable in ranges of Mg concentrations (i.e., 0.03 to 0.50%). The Mg content of forage plants is normally higher in legumes than in grasses (NRC, 1982b). Mayland et al. (1976) estimated the tetany hazard of several forages

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and found wheat> oats> barley> rye. Magnesium levels of wheat were the lowest, whereas those of rye were the highest. For tropical herbage, Mg concentrations were relatively low, with slightly more than one-third of 288 forages included in the 1974 Latin American Tables of Feed Composition containing 0.2% Mg or less (McDowell et al., 1977). Forage mineral analysis from a large number of farms in four Latin American tropical countries indicated a considerable number of forages contained less than 0.20% Mg (dry basis): Bolivia (64%), Colombia (56%), Dominican Republic (33%), and Guatemala (76%) (McDowell, 1985). Temperature and light may affect forage Mg concentrations and grass tetany etiology. Overall shading probably reduces forage Mg availability; the incidence of grass tetany is greater when daily radiation levels are low (Mayland et al., 1976). Magnesium concentrations generally decline as the plant matures (Aleroft, 1954), but this lowered concentration is often less dramatic than for many other minerals. Stem tissue contributes appreciably to total plant Mg; that of early maturity stage of growth forms the bulk of plant dry matter, Magnesium concentrations were greater in stems than in leaves of dwarf elephant grass (Pennisetum purpureum) (Montalvo et al., 1987). Magnesium fertilizers can significantly increase pasture concentrations, usually without influencing yields. Calcined magnesite (MgO) at the very high rate of 3180 kg/ha doubled the Mg content of an English pasture, from an average of 0.2 to 0.4%, while lower and more economic rates of application resulted in intermediate increases (Aleroft, 1961). High fertilization with K and/or N, especially in soils with low Mg content, reduced the Mg content in grasses. Mayland and Grunes (1979) reported that high K application rates reduced Mg concentration by an average of 15 to 20% compared to that of untreated forage. The detrimental effects of N fertilization may be because N increases K concentrations. Rosero et al. (1975) fed orchard grass and a fescue-rye hybrid to sheep and found that N fertilization lowered intake, percentage Mg absorption, and Mg balance in lambs. Biological availability of different sources of Mg for ruminants varies considerably. Peeler (1972) reported that the availability of Mg ranged from 10 to 25% in forages and from 30 to 40% in grains and concentrates. Kemp et al. (1961) reported that, contrary to most minerals, Mg availability improves with increasing maturity of grasses and may be decreased by heavy K and N fertilization. Perdomo et al. (1977) studied the apparent digestibility in sheep of five minerals at three stages of regrowth for three tropical forages. Digestibility and retention of Ca, P, and Na by sheep tended to decline with maturity, while availability of Mg tended to increase at 56 days of age for the three species. Usually Mg in preserved forages is more available than in pastures. Apparent availability of Mg was significantly higher in ensiled than in frozen or artificially dried grass (Powley and Johnson, 1977). The availability of Mg may range from 5 to 33% in succulent feeds and from 10 to 40% in hay and concentrates (Wilkinson and Stuedemann, 1979). For human diets, excellent sources of Mg include nuts, legumes, and unmilled grains. Removal of the germ and outer layers from cereal grains, however, results in

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a Mg loss of about 80%. Green vegetables are good sources of the element for humans, whereas milk, fish, and meat contain relatively low Mg concentrations.

VIII. DEFICIENCY

A. Effects of Deficiency

Magnesium deficiency is manifested clinically by retarded growth, hyperirritability and tetany, peripheral vasodilation, anorexia, muscular incoordination, and convulsions. Most practical diets contain adequate Mg to promote optimal performance. The exception is grazing ruminants and especially mature lactating cattle, which are most susceptible to Mg deficiency and/or abnormal Mg metabolism. Because of adequacy of Mg for most species consuming typical diets, special dietary ingredients (i.e., purified diets) are used to study both requirements and deficiency in nonruminants. 1.

SWINE

Metabolically, Mg is an extremely important nutrient, but its requirement is so low, or its recycling is so efficient relative to other macrominerals that deficiency must be produced by use of diets not containing cereal grains and oilseed meals. The signs of a Mg deficiency in order of appearance are hyperirritability, muscular twitching, reluctance to stand, weak pasterns, loss of equilibrium, tetany, and finally death. There was a high mortality rate in baby pigs fed low-Mg diets. The Mg-deficient pigs also exhibit stepping syndrome (Fig. 5.1), which causes them to keep stepping or lifting their hind legs almost continuously while standing (Mayo et al., 1959). Miller et al. (1965) reported that pigs fed 25 or 75 ppm Mg showed slow growth, poor feed conversion, weakened pasterns, stepping syndrome, tetany, and death. Pigs fed 125 ppm showed all these signs except tetany and death. High dietary manganese (Mn) in addition to low Mg intakes greatly increased convulsive seizures and death in growing pigs (Miller et al., 2000). The high dietary Mn was found to greatly depress heart concentrations of Mg. 2.

POULTRY

Magnesium deficiency seldom occurs in poultry fed practical diets. When highly purified feedstuffs are fed to young chicks, one observes poor growth, lethargy, convulsions, and death, with rather large differences in response within a chick group. Deficient chicks often pant and gasp, and there is poor feathering, decreased muscle tone, ataxia, progressive incoordination, and convulsions followed by death (Bird, 1949). Newly hatched chicks fed a diet devoid of Mg lived only a few days. Ducklings fed no supplemental Mg showed signs of deficiency and died within 10 to 16 days (Van Reen and Pearson, 1953). Plasma Mg and Ca are reduced by Mg deficiency. Marginal deficiency may allow near-normal growth, but plasma Mg will be reduced, and when disturbed chicks show signs of neuromuscular

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Fig. S.l Magnesium deficiency in swine and sheep. Five-week-old pig (A) showing stepping syndrome; the pig keeps stepping almost continuously while standing. Weakness of pasterns is apparent. Magnesium deficiency in a lamb (8) illustrating unthriftiness. Note the stiff legs. (A - courtesy of: E. R. Miller and D. E. Ullrey, Michigan State University, East Lansing; 8 - courtesy of U. S. Garrigus, University of Illinois, Urbana)

hyperirritability. Chicks may have brief episodes of convulsions followed by coma, from which they usually recover. Mahoney et al. (1992) noted an 80% reduction in growth of broilers fed 0.02 vs 0.06% Mg. A deficiency of Mg in the diet of laying hens results in rapid decline in egg production, blood hypomagnesemia, and a marked withdrawal of Mg from bones of Mg-deficient hens. Egg size, weight of shell, and Mg content of yolk and shell are decreased owing to Mg deficiency (Leeson and Summers, 2001). Feeding a semipurified diet containing 56 ppm Mg decreased egg production and reduced serum Mg within 10 to 14 days (Sell et al., 1967). Fertilization of eggs was not affected, but hatchability of fertile eggs was reduced markedly. A decline in hatchability precedes decreases in egg production and diet consumption.

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Magnesium

RUMINANTS

Grass tetany (or hypomagnesemia) is a complex ruminant metabolic disorder affected by mineral composition of forage species, soil properties, fertilizer practices, season of the year, temperature, animal species, breed, and age. Grass tetany is quite rare for livestock consuming predominantly legumes, as they are generally higher in Mg than grasses. Grass tetany is not an entirely appropriate name because the disorder is not limited to animals receiving grass and is characterized by convulsions rather than tetany. A number of clinical syndromes in cows, sheep. and goats are included in the grass tetany syndrome. Grass tetany is also referred to as lactation tetany, grass staggers, wheat pasture poisoning, winter tetany, and milk tetany in calves. Signs of hypomagnesemic tetany are encountered both in grazing ruminants and in calves reared too long on milk without access to other feeds. The spring-calving cow is usually more susceptible to development of tetany within a few weeks after calving. Susceptibility to grass tetany is increased in older ruminants because of the decreased ability to mobilize skeletal Mg with increasing age (see Section IV). Consequently, an abrupt change from a normal diet to one with inadequate available Mg can result in hypomagnesemia within 2 to 18days, even though the previous feed was high in Mg (Dishington and Tollersrud, 1967). It seems to be most predominant in cows subsequent to the second lactation (third to fifth lactation). In the United States grass tetany is a greater problem with beef than with dairy cattle, probably because of greater Mg availability in concentrates, along with the substantial amounts of grain usually fed to lactating dairy cows. In many developing tropical countries, dairy cows often do not receive concentrates in substantial quantities. Usually, only female animals are affected with grass tetany, although it has been reported in calves and steers (Crookshank and Sims, 1955). Cows are particularly susceptible to tetany when nursing a calf or producing milk. Sometimes pregnant animals die from the condition. Grass tetany generally occurs during early spring, or a particularly wet autumn, among older animals grazing grass or small grain forages in cool weather. Cows are most likely to get grass tetany soon after they are turned out on spring pasture. However, there is also a winter type of tetany that affects cattle fed winter diets (sometimes poor quality hay) in confinement. Grass tetany has also been reported in fall-calving beef cows. In addition to low blood Mg, the fall tetany or winter tetany syndrome is often associated with ketosis (Boling, 1982). In New Zealand, where cows are pastured throughout the year, the disorder occurs most frequently in late winter and early spring (Underwood and Suttle, 1999). Tetany is seen where ruminant production is highly developed, high quality pastures are available, and high-yielding, quick-maturing stock are raised. Voisin (1963) suggested that farming methods have caused an imbalance in the soil and herbage, upsetting the Mg metabolism of grazing ruminants. The incidence of a Mg deficiency is influenced as much or more by management considerations as by geographical location.

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Although it is not characterized by death, nonclinical hypomagnesemia is far more common than clinical tetany, and economic consequences of lowered production are substantial (NCMN, 1973). The subclinical stage of Mg deficiency may be followed by spontaneous recovery from hypomagnesemia and does not invariably progress to the acute disorder (Underwood and Suttle, 1999). Chronic marginal deficiency of Mg will result in reduced feed intake and performance. Suboptimal performance can be related to a decline in cellulose digestion. Studies with lambs and steers suggest that feed intake and cellulose digestion decline faster than does serum Mg when Mg-free diets are fed (Chicco et al., 1973; Emery, 1976). Boling (1982) has described the external or visible signs of grass tetany syndrome in cows. Initially, the cow may have a depressed appetite and exhibit a dull, lethargic appearance. As the condition progresses, signs of stiffness as she walks, and ultimately, a staggering gait may become apparent. As the condition progresses, the cow becomes highly excitable and nervous and has readily visible muscular tremors. The head is held high with staring eyes with movements becoming stiff and stilted. Chewing, hypersalivation, and blinking of the third eyelid are particularly characteristic of hypomagnesemic tetany. In the most severe stage, the animal collapses to the ground with continuation of the tetanic muscular spasms. The legs will usually thrash the ground around the cow, uprooting forage. Death occurs after collapse if the animal does not receive medical treatment (Fig. 5.2). Crookshank and Sims (1955) note that 6 to 10hr are usually required from the time of the first clinical signs until the animal passes into a comatose condition. If treatment is not initiated before coma, there is little chance of recovery. McCoy et al. (2000) reported that hypomagnesemic tetany was associated with alterations in regional brain monoamine concentrations in cattle. Monoamines are important brain neurotransmitters. The cerebral cortex and cerebellum regions in the brain play an important role in both voluntary and involuntary motor function, and therefore these alterations may playa role in the etiology of hypomagnesemic tetany. Preconvulsive clinical signs of hypomagnesemia in sheep are less clearly defined than in cattle and can be confused with those of hypocalcemia or pregnancy toxemia. Hypomagnesemia tetany in sheep is almost exclusively a disease of the first 8 weeks of lactation, with ewes nursing twins most susceptible. The incidence is highest I to 4 weeks after lambing (Herd, 1966). In Australia, however, a high incidence of hypomagnesemia tetany in breeding ewes has been correlated with periods of rapid winter growth of pastures (Underwood and Suttle, 1999). A lamb with hypomagnesemic tetany may fall on its side with its legs alternately rigidly extended and relaxed. Frothing at the mouth and profuse salivation are evident, and death may occur. The signs of Mg deficiency in adult ewes are similar to those in younger animals, but death may occur more rapidly after convulsions. Clinical tetanies in cattle, milk-fed calves, and sheep can combine hypomagnesemia and hypocalcemia (low blood Ca). A high incidence of hypocalcemia has been reported with wheat-pasture poisoning (Bohman et al., 1983). Both Ca and Mg metabolism are interrelated in this malady.

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Fig.5.2 Cow in collapse stage of tetany and death from tetany. Note area around forelegs where ground has been thrashed during convulsions. (Courtesy of I.A. Boling, C.W. Absher, and D.E. Miksch, University of Kentucky, Lexington)

The economic importance of grass tetany arises from its high death rate and its sudden occurrence. Grass tetany causes an estimated $50 to 150 million in livestock production losses each year in the United States (Wood, 1999). In the United States the mortality among untreated clinical cases is 30% or more (Grunes et al., 1970). Clinical tetany is endemic in some countries, affecting only a small proportion of

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cattle (l to 2%). However, individual herds may report incidence of tetany as high as 20%. Hypomagnesemic tetany occurs in most European countries, North America, Australia, South Africa, and New Zealand. McDowell (1985) listed 17 developing tropical countries where Mg tetany is encountered or highly suspected. Reports of Mg deficiency are much more prevalent in temperate than in tropical regions. Some of the reasons for fewer reports of grass tetany in tropical regions include the prevalence of the condition during cooler temperatures (8 to 14°C), and for many tropical countries, the generally low productivity of pastures and of herds, and lack of fertilization of pasture with Nand K. Nevertheless, grass tetany can be a problem for grazing ruminants in tropical countries, since forages are often low in Mg. 4. HORSES

Horses grazing pastures that produce tetany in cattle are seldom affected. Foals fed a purified diet containing 8 ppm Mg developed clinical signs including hypomagnesemia, nervousness, muscular tremors, and ataxia followed by collapse, with hyperapnea (hard breathing), sweating, convulsive paddling of legs, and, in some cases, death (Harrington, 1974). Mineralization of arteries, elastic tissue, spleen, lungs, and heart were observed at necropsy. Foals fed Mg-deficient diets showed degeneration in the lung, spleen, skeletal muscle, and heart (Harrington, 1975). Skeletal muscle degeneration was consistently found in all the foals fed the Mg-deficient diet for 71 days or longer, although it was not extensive. In this study, a sharp decrease in blood serum Mg levels was detected within 24 to 48 hr after the foals were given the Mg-deficient diet; at necropsy there was a reduction in bone Mg concentration. 5. OTHER ANIMAL SPECIES

a. Dogs. Anorexia, vomiting, decreased weight gain, and hyperextension of the front legs were observed in puppies (7 to 9 weeks of age initially) that were fed a purified diet containing less than 5 ppm Mg for 3 weeks (Kahil et al., 1966). By 4 to 6 weeks the puppies fed this diet showed irritability, ataxia of hind legs, and convulsive seizures. In another study with puppies, similar clinical signs were observed, and at necropsy, aortas of these animals contained extreme mineralized lesions, primarily Ca and P deposits (NRC, 1985a). b. Cats. Magnesium deficiency has been reported in cats (Chausow et al., 1985). At 50 ppm of dietary Mg, kittens grew poorly and exhibited muscular weakness, hyperirritability, convulsions, anorexia, reduced bone and serum Mg concentration, and calcification of the aorta. c. Laboratory Animals. Deficiency of Mg was first studied intensively in the rat. In rats, lowering dietary Mg to 1.8 ppm resulted in vasodilation, hyperirritability, convulsions, and death (NRC, 1995). Renal calcification is common and may be

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detected within two days after initiating a markedly deficient diet. Tufts and Greenberg (1938) reported that lactating female rats fed a deficient diet were bred successfully but did not suckle their young. Magnesium deficiency has been shown to induce bone loss in the rat (Rude et al., 1998). Laurant et al. (1999, 2000) have reported that dietary Mg intake can affect mechanical properties of rat carotid artery. These mechanical alterations could contribute to the development of atherosclerosis, hypertension, and cardiovascular diseases. Rock et al. (1995) suggested that dietary Mg deficiency in rats enhances free radical production in skeletal muscle. Magnesium deficiency has been established in mice and guinea pigs. Alcock and Shils (1974) reported that Mg-deficient mice, without showing previous hyperirritability, developed rapid and usually immediately fatal convulsions. Clinical signs of deficiency in young guinea pigs fed low-Mg purified diets include poor weight gains, hair loss, decreased activity, poor muscular coordination and stiffness of rear limbs, elevated serum P, and anemia (NRC, 1995).

d. Rabbits. Kunkel and Pearson (1948) found Mg deficiency in rabbits caused poor growth and hyperexcitability with convulsions. Inadequate Mg may result in fur chewing, alopecia, blanching of the ears, and alteration of fur texture and luster in rabbits fed a diet containing 5.6 ppm Mg (NRC, 1977). e. Fish. Magnesium deficiency causes anorexia, reduced growth, lethargy, and reduced tissue Mg content in fish. Deficiency signs in rainbow trout included loss of appetite, decreased growth, lethargy, reduced bone ash, spinal curvature, and histological changes in muscle, pyloric caeca, and gill filaments (Cowey et al., 1977). Trout fed Mg-deficient diets (40 ppm) developed renal calcification at dietary Ca levels of 2.7% and a dietary Ca:P ratio of 1:1. Dietary Mg deficiency in channel catfish and common carp causes poor growth, anorexia, sluggishness, muscle flaccidity, high mortality, and depressed tissue Mg levels (NRC, 1993). Magnesium deficiency has not been demonstrated in fish in a seawater environment, where they obtain Mg by drinking the water.

f Nonhuman Primates. Vitale et al. (1963) induced Mg deficiency (6 ppm dietary Mg) in growing cebus monkeys (Cebus apella), characterized by weight loss, hyperirritability, and convulsions. Serum Mg fell from about 1.3 to 0.7 mg/dl, and at necropsy, marked sudanophilia and connective tissue plaques were observed in the aortas of the deficient animals. Magnesium deficiency in rhesus monkeys resulted in hypomagnesemia, hypocalcemia, and hyperirritability (NRC, 1978). 6.

HUMANS

For patients fed a low-Mg formula, urinary Mg fell sharply to levels no longer detectable within a week, and plasma Mg fell continuously. The most prominent and consistent symptoms and signs were nausea, muscle weakness, irritability, mental derangement, and myographic changes.

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Magnesium deficiency with or without symptoms has been reported in numerous disease states (Shils, 1997), including severe malabsorption, diabetes, chronic alcoholism, and malnutrition. Deficiency of Mg is associated with prolonged infusions of Mg-free parenteral fluids - usually in association with prolonged losses of gastrointestinal secretions - renal tubular dysfunction occurring in acute or chronic kidney diseases or secondary to drugs, childhood malnutrition, familial disorders or renal or intestinal conservation, hyperaldosteronism and hyperparathyroidism, especially in the immediate post-pararthyroidectomy period. In recent years the literature has been concerned with the possible preventive and therapeutic rolls of Mg in relation to coronary artery disease (CAD) (Shils, 1997; Ford, 1999). The argument has been advanced that the American public has a significant amount of asymptomatic Mg deficiency and that this is a contributing factor in the prevalence of CAD. Serum Mg concentrations were inversely associated with mortality from CAD and all-cause mortality (Ford, 1999). Magnesium deficiency may affect heart function through changes of K, Na, and Ca concentrations in extracellular and intracellular fluids. The clinical situation becomes more complex with coexisting myocardial disease and when primary electrolyte abnormalities are exacerbated by anoxemia and/or diuretic therapy (Shils, 1997). High blood pressure is a major risk factor for stroke and a recent study has identified K, Mg, and fiber as significant modulators of stroke for men (Suter, 1999; see also Chapter 4). Dietary Mg depletion can be induced in otherwise healthy women; it results in increased energy needs and adversely affects cardiovascular function during submaximal work (Lukaski and Nielsen, 2002).

B. Assessment of Magnesium Status For all species with a Mg deficiency, concentrations in serum, erythrocytes, and urine are depressed. Increasing dietary Mg for lambs resulted in a linear increase in serum Mg (Chester-Jones et al., 1989). Peak serum Mg levels were 3.0, 3.2,4.2, and 5.5 mg/dl for lambs fed 0.2, 0.6, 1.2, and 2.4% Mg, respectively. Bone concentrations of Mg are significantly reduced with a deficiency. This is less pronounced for older ruminants, as they are less able to mobilize Mg than are younger animals. Serum Mg is a good indicator of Mg status of various species, but Mg urinary excretion and erythrocyte concentrations are better indicators. The Mg content of the erythrocytes decreases to about one-half the normal amount during the early phase of depletion, but Mg concentration in serum does not decrease until there is a severe deficiency. In contrast, an excess or a lack of Mg is immediately reflected in daily excretion of Mg in urine; hence, daily urinary excretion is a better criterion of Mg supply than is serum Mg concentration. If Mg deficiency is suspected and if the serum values are normal, erythrocyte Mg content and 24-hr urinary excretion should be measured. When these values are normal, Mg deficiency is very unlikely. For humans with suspected Mg deficiency, Mg concentrations are evaluated in serum, urine, and blood mononuclear cells (Shils, 1996). The concentration of Mg in human mononuclear cells has been claimed to be a better guide to Mg nutriture

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than is the serum concentration. A load test, which is the urinary Mg concentration after an infusion of a Mg salt, is an accurate measurement of Mg status. For grazing ruminants, confirmation of grass tetany is justified only when blood or urine samples are low in Mg. A reduction in Mg content of cerebrospinal fluid « 1.6 mg/dl) is an accurate indicator of deficiency (Meyer, 1976). The ranges in serum or plasma Mg level (mg/lOO ml) for cattle and sheep are as follows: normal values, 1.8 to 3.2; slight hypomagnesemia, 1.2 to 1.8; and severe hypomagnesemia, 1.2 or less (NCMN, 1973). In calves showing the typical signs of Mg deficiency, serum Mg is frequently as low as 0.1 mg/dl compared with a normal of around 2.5 mg. Tentative criteria for Mg in urine are as follows: more than 10.0 mg/dl, adequate to liberal; 2.0 to 10.0 mg/dl, inadequate; less than 2.0 mg/dl, severe deficiency and danger of tetany. A rough assessment of supply for grazing animals can be obtained from the content of Mg, N, and K in pasture. This approach is more accurate when the pasture is sampled close to the date of grazing. If the dates are more than a week apart, the assessment in unreliable. This method can be used only for grazing cattle, whereas the urine method is reliable on indoor diets as well as pasture (NCMN, 1973).

IX. SUPPLEMENTATION Magnesium supplementation is most important for ruminants for the prevention and cure of hypomagnesemia tetany. Berger (1992) calculated that, in a 100-cow herd, preventing the loss from grass tetany of a single cow every three years would more than pay the cost of Mg supplementation. For other species Mg deficiency is uncommon and is brought about by special diets and various disease conditions. Normally, adequate Mg is present in practical diets to meet the requirements of poultry. Since feedstuffs commonly used for poultry contribute Mg levels of 0.11 to 0.24% to the diet, and the estimated requirement varies between 0.03 to 0.06, a need for Mg supplementation seems unlikely, but little research has been done (Sell, 1980). Assuming that swine utilize Mg from feedstuffs with about the same efficiency as chickens, the Mg supplied by common feed ingredients should meet their requirements. Nevertheless, some feed industry nutritionists feel that supplemental Mg may help to prevent hyperirritability and tail biting in confined pigs. Female ruminant livestock that develop tetany should receive medical treatment immediately by intravenous injections or enemas. Treatment can include s.c. injection of a single dose of 200 to 300 ml of a 20% solution of magnesium sulfate or i.v. injection of a similar dose of magnesium lactate (Underwood and Suttle, 1999). These treatments will restore serum Mg of an affected cow to near normal within about 10 min and are almost always followed by disappearance of signs of tetany. Serum Mg concentrations will decrease again unless the cow is immediately removed from the tetany-producing pasture and fed Mg-adequate diets. Most treatments include Ca in addition to Mg salts because hypocalcemia usually accompanies Mg deficiency. Some veterinarians use i.v. injections of chloral

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hydrate or magnesium sulfate to calm excited animals, and then follow with a Ca-Mg gluconate solution. If the animal again goes into convulsions, a second dose of Ca-Mg gluconate solution may be required. Intravenous injections should be administered slowly because there is danger of heart failure if given too rapidly (Grunes and Mayland, 1984). In acute cases, Mg enemas can be used to treat animals with tetany. An enema of 100 ml of 20% MgCh is effective because Mg absorption takes place also in the rectum and colon. The enema may be given with the probe inserted 25 em into the anus. Magnesium chloride is used instead of MgS04 for enemas because colonic transport of Mg requires a simultaneous transport of an anion (CI in this case). The colonic epithelium is impermeable to the sulfate ion, as evidenced by the cathartic action of magnesium sulfate in the gut (Bell and Oluokum, 1977). Treatment effectiveness depends on the time between development of clinical signs and treatment, but those given within an hour have a much greater chance of success. Irreversible pathological changes (possibly of the central nervous system) may develop, and the animal may die if treatment is delayed. Dietary supplementation of Mg in place of intravenous injections or enemas has not been effective in treating tetany cases since too much time is required for the Mg to reach that part of the GI tract where it can be absorbed. For follow-up treatment, the animal should be removed from the tetanyproducing pasture and fed hay and concentrates. Also, 30 g Mg (e.g., MgO) should be given daily (Grunes and Mayland, 1984). Force-feeding ofMg may be necessary, but after a week the amount can be greatly reduced. Cows that get tetany are likely to get it again. Commonly, the recommendation is to take the entire herd off the tetany-prone pasture, if it can be done without unduly exciting the cows. It is often better to bring Mg supplements to the cattle under range conditions. Since the cause of grass tetany is a metabolic deficiency of Mg resulting from either a simple deficiency of Mg or factors that lower the efficiency of utilization of the element, increasing Mg intake should prevent the disturbance (Fontenot, 1980). Several safe and practical means of raising the Mg intakes of animals enough to prevent losses from tetany have been devised. There is some agreement that 50 to 60 g MgO/day is the minimum secure prophylactic dose for adult dairy cattle; with 7 to 15 g/day for calves, and 7 g/day for lactating ewes (Underwood, 1981). When a diagnosis of grass tetany is made, one or more of the following practices may be useful in preventing new cases from occurring. A. Magnesium Fertilization Fertilizing pastures with relatively high levels of Mg or liming with dolomitic limestone for several years increases Mg in the forage. Incorporating dolomitic limestone below the soil surface will increase its effectiveness. The amount of fertilizer Mg required may vary considerably, depending on the soil pH, texture, and K content. Local recommendations should be followed to avoid using too much Nand K fertilizer. Fertilizer Mg and limestone (dolomite) as methods of control have limitations on many soil types and usually have to be accompanied by

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other means of supplying additional Mg. Plant breeders have also improved forage Mg concentrations. A new grass referred to as "Hi-Mag" in the United States is a tall fescue that contains 20% more Mg than other plants in the tested regions (Wood, 1999).

B. Foliar Mg Application Foliar dusting of pastures with fine calcined magnesite (MgO) before or during tetany prone periods has proved effective, provided it is applied at not less than 17 kgjha at not more than IO-day intervals (Rogers, 1979). Foliar application is more rapid and more effective in increasing the level of plant Mg than fertilization and, therefore, prevents tetany in the majority of the herd if applied when early signs of tetany are observed. A disadvantage of foliar Mg application is that the element is easily washed off by rain. To help keep rain from washing the MgO off the forage, a water slurry of 10% MgO and 1.5% bentonite can be applied to the grass with a suspension fertilizer applicator. Neither fertilization nor foliar application of Mg are the methods of choice where forage yields are low under extensive grazing conditions. particularly in developing countries, because of unfavorable cost-benefit relationships.

C. Oral Magnesium Supplementation For calves that are being fed concentrates, provision of 50 g of MgOjday in 300 to 400 g of concentrate mixture is adequate. Other nutritionists have recommended from 10 to 20 g of Mg daily per head of mature cattle. In New Zealand, the method of choice is to give 10 g Mg as magnesium chloride daily at milking time (Underwood and Suttle, 1999). Unlike common salt (NaC\), most Mg salts are quite unpalatable. Supplemental Mg, as part of a concentrate mixture, is the best way of ensuring adequate intake by ruminants. When concentrates are not fed, freechoice Mg feeding is recommended. The provision of special high-Mg mineral blocks or mineral salt mixtures on pasture was more effective in raising blood Mg levels quickly after the initial drop than was the Mg fertilization treatment (Reid et al., 1976). Various combinations of MgO with salt, protein supplements, molasses, other concentrate ingredients, and other feeds have been used to obtain optimal Mg intakes (Miller, 1979). From West Virginia average consumption of Mg by beef cows given a free-choice mixture of 40% salt, 40% dicalcium phosphate, and 20% MgO ranged from 1.3 to 4.2 g/head/ day (Reid et al., 1976). This compared to an intake level of 5 to 109 Mg from a similar mixture containing 20% dried molasses, or 4.1 to 8.8 g Mg from commercial molasses -- MgO blocks (15% Mg). Several relatively successful free-choice consumption formulas of both liquid and dry supplements are as follows: (1) MgO plus molasses at a ratio of I: 1; (2) 97% molasses plus 3% MgCI2 (often with urea and a source of P); (3) equal parts of MgO, salt, bonemeal, and grain; and (4) a 1:1 ratio of salt and MgO. In the southeastern United States, a complete mineral mixture with 25% MgO (14% Mg)

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has been effective in preventing grass tetany in beef cattle (Cunha, 1973). Licking wheels or licking belts are sometimes used to slowly dispense MgO or MgS0 4 in molasses. An oral Mg supplement is of value only during seasonal occurrences of grass tetany (Aleroft, 1961). Unfortunately, many commercial Mg-containing, freechoice mineral supplements are often of little value because (1) they contain inadequate quantities of Mg to protect against tetany during susceptible periods, and (2) provision of such supplements to normal animals during nonsusceptible periods is useless as a prophylactic measure, since additional Mg will not provide a depot of readily available Mg for emergency use. Some producers feed Mg supplements about a month before the Mg tetany season, to decrease the amount of Mg needed daily during the susceptible period. The success of cobalt (Co) bullets in providing supplemental Co led to the development of Mg alloy bullets. Different size bullets are available for cattle and sheep. Bullets are presumably retained by the ruminoreticular fold and release a limited amount of Mg (see Chapter 19). For cattle, Ritchie and Hemingway (1968) used bullets 3 inches in length, one inch in diameter and weighing approximately 100 g. These were composed of a metal Mg alloy containing 86% Mg, 12% AI, and 2% Cu. These were designed to release about I g Mg per day for about 50 days. The three treatment levels used by these workers were zero, two, and four bullets per cow. There were 16 cases of tetany (six deaths) from 169 untreated cows, and no tetany from those treated with two or four bullets. Egan (1969) reported that Mg alloy bullets controlled an outbreak of hypomagnesemia in ewes. Some studies have shown Mg bullets ineffective in preventing grass tetany (Kemp and Todd, 1970; Stuedemann et al., 1984). The main disadvantage of Mg bullets is that often the daily Mg released is insufficient. Also, some bullets are regurgitated, and bullet decomposition is variable (Fontenot, 1980).

D. Magnesium Supplemented in Water Addition of a soluble Mg salt to water has been successful in both increasing blood Mg and preventing grass tetany (Rogers and Poole, 1976). Magnesium sulfate has been used. Some diarrhea may occur, but this has not been a problem. Magnesium acetate or magnesium chloride may be used instead of magnesium sulfate. For this treatment to be effective, the drinking trough must be the only source of water. In addition to supplementation, some management procedures lessen the risk of grass tetany. Animals should be adapted slowly to tetany causing pastures in the spring; as an example, feed animals hay before turning them out on new pastures. Pastures that are most likely to cause grass tetany should be available to steers and dry stock versus lactating animals. Feed more legume hay and high-legume pastures to milking cows and cows nursing calves, since legumes are higher in Mg than are grasses. Supplemental Mg salts are frequently used orally as saline purgatives (cathartics). The main forms of Mg used as laxatives are MgS04 (Epsom salts) and Mg(OHh

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(milk of magnesia). Magnesium sulfate has been used externally to give relief to patients suffering from joint problems. Magnesium is also given to dairy and beef cattle as a buffer. Feeding large amounts of concentrate with small amounts of forage, or with forages that have been finely chopped, will decrease a cow's saliva output and increase the acid load on lactating dairy cows and beef cattle on finishing diets. Feeding dairy cows limited roughage decreases the percentage of milk fat. Supplemental MgO or sodium bicarbonate increases milk fat. Supplementing with either 0.36 kg sodium bicarbonate or 0.18 kg MgO per day prevented depression in milk fat (Emery et al., 1965).

Supplementation of Mg to cattle has been shown to reduce calving difficulties (Villalba, 1999). Administration of two subcutaneous injections (10 ml magnesium gluconate) of Mg during the last 45 days of pregnancy significantly reduced difficult calvings. Recent information has shown that Mg supplementation may have beneficial effects on meat quality. Stress before slaughter can lead to pale, soft, and exudative (PSE) pork by stimulating the rate of immediate postmortem acidification. Magnesium supplementation prior to slaughter resulted in calmer pigs with significantly improved meat quality and reduced incidence of PSE meat (D'Souza et al., 1998; Apple et al., 2000). Increasing dietary Mg levels beyond current recommendations increased marbling scores in cattle fed fat-supplemented diets (Ramirez and Zinn, 2000). A number of sources of Mg salts are available for dietary supplementation. The effectiveness of different chemical forms of the element have mostly been compared in ruminants (Henry and Benz, 1995). Magnesium phosphate, a calciummagnesium-phosphate and magnesium-ammonium-phosphate are all satisfactory sources. For sheep the oxide and hydroxide forms of Mg had lower bioavailability than magnesium sulfate (Henry and Benz, 1995). Previously, Ammerman et al. (1972) found that the biological availabilities of Mg supplements in reagent-grade magnesium carbonate, magnesium oxide, and magnesium sulfate were 43.8, 50.9, and 57.6°;{., respectively (see Chapter 19 of this volume). Storry and Rook (1963) studied Mg availability from various salts by measuring increase in urinary Mg excretion when the salts were fed to nonlactating dairy cows. Availability was highest for the citrate form, intermediate for the oxide, lactate, acetate, and nitrate forms, and lowest for sulfate, silicate, and chloride forms. Availability (apparent absorption) of Mg for cattle was much higher for MgO than for dolomitic limestone, 51.1 versus 14.3%, respectively (Gerken and Fontenot, 1967). Considering cost per unit and biological availability, magnesium oxide generally is the best form of Mg for supplementation (Wilkinson and Stuedemann, 1979). However, if supplementation is in water, more soluble forms of Mg including sulfate, chloride, and acetate must be used. Physiological state of animals, source, particle size, and processing of the supplement all have been shown to influence Mg availability from MgO. Magnesium oxide appeared to have higher Mg bioavailability than magnesium sulfate for pre-calving cows, but Mg bioavailability was not different postcalving

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(Grings and Males, 1988). For steers fed MgO, a smaller particle size was more available (Noller et al., 1987). Availability of Mg varies for different sources of MgO (Van Ravenswaay et al., 1989), and processing temperature, both inadequate and excessive, affect Mg availability (Beede and O'Connor, 1986). Magnesium deficiency may be fairly common in acutely or chronically ill patients. Inadequate intake or provision of Mg is associated with alcoholism, protein-calorie malnutrition (usually with infection), and incorrectly formulated parenteral preparations (Shils, 1996). Magnesium loss with the urine is increased by alcohol and by various diuretic medicines. Disease conditions requiring Mg supplementation include malabsorption disorders, renal tubular dysfunction, endocrine disorders (e.g., hyperaldosteronism and hyperparathyroidism), and genetic disorders. Stress, whether physical (i.e., exertion, heat, cold, traumaaccidental, or surgical burns), or emotional (i.e., pain, anxiety, excitement, depression), and dyspnea as in asthma increases need for Mg (Seelig, 1994). Genetic differences in Mg utilization may account for differences in VUlnerability to Mg deficiency and differences in body responses to stress. High blood pressure is a major risk factor for stroke. Potassium, Mg and fiber are modulators of stroke (Suter, 1999). The best strategy to achieve a high intake of Mg, K, and fiber is a diet rich in fruits and vegetables.

X. TOXICITY Magnesium toxicosis due to ingestion of natural feedstuffs has not been reported and does not appear likely, but would be most likely to occur from using excess levels of supplementary Mg (NRC, 1980). Certain levels of Ca and P in the diet protect the animals from toxicosis (Nugara and Edwards, 1963), as does K in ruminant diets (Fontenot, 1980). Clinical signs of Mg toxicosis for various species are lethargy, disturbance in locomotion, diarrhea, lowered feed intake and performance, drowsiness, and death. The tolerance of poultry and swine for high concentrations of dietary Mg is not well defined. With growing chicks, 0.64% Mg depressed growth and increased mortality, while 0.32% had no effect (Nugara and Edwards, 1963). In feeding layers 0.8% Mg, Hess and Britton (1997) described loss in egg production and body weight as a consequence of reduced feed intake. Decline in shell quality was associated with a 30% decrease in plasma Ca and a 100% increase in plasma Mg. Plasma P was increased by up to 80% suggesting that the bird was mobilizing bone in an attempt to meet demands for Ca. The use of dolomitic limestone (i.e., 10% Mg), especially in the diets of laying hens, may decrease egg production, produce eggs with thinner shells, and cause wet droppings. Increasing dietary Mg from 0.16 to 0.22% lowered rate and efficiency of gain in growing or finishing swine when they weighed 20 to 45 kg, but had no effect thereafter (Krider et at., 1975). Cattle and sheep should be able to tolerate 0.5% Mg (NRC, 1980). Oral administration of 0.5% Mg to wethers did not produce toxicosis, but 0.8% or higher resulted in signs of toxicosis. Chester-Jones et at. (1989) reported levels as

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low as 0.6% Mg produced diarrhea in wether lambs and depressed nutrient utilization; at 2.4% Mg the element was elevated in selected tissues. Severe diarrhea was reported in wethers receiving 0.30% magnesium sulfate (P. R. Henry, personal communication). Increasing dietary Mg (2.4 to 4.7%) in steers caused a progressive degeneration of the stratified squamous epithelium of ruminal papillae (ChesterJones et al., 1990). Although 0.5% Mg is a suggested tolerance level for ruminants, about 0.6% supplemental Mg (as MgO) has been used in low-roughage diets to correct milk fat depression without apparent harm, except for occasional diarrhea (Miller et al., 1972). Intensity of diarrhea is closely related to dietary Mg, with reduced feed consumption and gains in evidence for the higher (2 and 4%) Mg concentrations (Gentry et al., 1978). Seventy percent of calves fed 0.6% Mg and 30% of calves at 0.6% Mg plus NaC1 had stones in their kidneys consisting primarily of Ca apatite (Peters son et al., 1988). Christiansen and Webb (1990) reported that the feeding of high levels of MgO (1.5%) reduced intestinal absorption of amino acids in lambs. The presence of high Mg levels in water (about 1%) was reported to cause a weakening effect on humans and livestock in some areas of the United States (Allison, 1930). Generally large oral intakes of Mg are not harmful to people with normal renal function, but impaired renal function resulting in Mg retention is often associated with hypermagnesemia (Shils, 1997). Early symptoms of hypermagnesemia for humans include nausea, vomiting, and hypotension. At the most severe level of hypermagnesemia, respiratory depression, coma, and asystolic cardiac arrest may occur (Mordes and Wacker, 1978). Calcium infusion can counteract Mg toxicity. Avoidance of Mg-containing medications in patients with significant renal disease is recommended unless there is good reason and close monitoring (Shils, 1997).

XI. REFERENCES Aikawa, J. K. (1959). Proc. Soc. Exp. Bioi. Med. 100, 293. Alcock, N., and MacIntyre, I. (1962). C/in. Sci. 22, 185. Alcock, N., and Shils, M. E. (1974). Proc. Soc. Exp. Bioi. Med. 146, 137. Al-Ghamdi, S. M., Cameron, E. c., and Sutton, R. A. (1994). Am. J. Kidney Dis. 24, 737. Alcroft, R. (1954). Vet. Rec. 66, 517. Alcroft, R. (1961). Vet. Rec. 73, 1255. Allison, I. S. (1930). Science 71, 559. Ammerman, C. B., Chicco, C. F., Loggins, P. E., and Arrington, L. R. (1972). J. Anim. Sci. 34, 122. Apple, J. K., Maxwell, C. V., deRodas, B., Watson, H. B., and Johnson, Z. B. (2000). J. Anim. Sci. 78, 2135. Beede, D. K., and O'Connor, M. A. (1986). In "Proc. Florida Nutriton Conference" p. 191. Daytona Beach, FL. Bell, M. c., and Oluokum, J. A. (1977). Tenn. Farm Home Sci. Prog. Rep. 104,22. Berger, L. L. (1992). Salt Trace Minerals 24, 12. (Salt Institute, Alexandria, VA). Bird, F. H. (1949). J. Nutr. 39, 13. Blaxter, K. L., and McGill, R. F. (1956). Vet. Rev. Annot. 2,35. Bohman, V. R., Horn, F. P., Littledike, E. I., Hurst, 1. G., and Griffin, D. (1983). J. Anim. Sci. 57,1364. Bohman, V. R., Lesperance, A. L., Harding, G. D., and Grunes, D. L. (1969). J. Anim. Sci. 29,99. Boling, J. A. (1982). Anim. Nutr. Health 37, 20.

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Kemp, A. (1983). In "Role of Magnesium in Animal Nutrition" (J. P. Fontenot, G. E. Bunce, K. E. Webb, Jr., and V. G. Allen, eds.), p. 143.Virginia Polytechnic Inst. and State Univ., Blacksburg, VA. Kemp, A., Deijs, W. B., Hemkes, O. J., and Van Es, A. J. H. (1961). Neth. J. Agric. Sci. 9, 134. Kemp, A., Deijs, W. B., and Kluvers, E. (1966). Neth. J. Agric. Sci. 14,290. Kemp, A., and Todd, J. R. (1970). Vet. Rec. 86,463. Krider, J. L., Albright, J. L., Plumlee, M. P., Conrad, J. H., Sinclair, C. L., Underwood, L., Jones, R. G., and Harrington, R. B. (1975). J. Anim. Sci. 40, 1027. Kruse, H. D., Orent, E. R., and McCollum, E. V. (1932). J. Bioi. Chem. %, 519. Kunkel, H. 0., and Pearson, P. B. (1948). J. Nutr. 36, 657. Larvor, P. (1983). In "Role of Magnesium in Animal Nutrition," (J. P. Fontenot, G. E. Bunce, K. E. Webb, Jr., and V. G. Allen, eds.), p. 81. Virginia Polytechnic Inst. and State Univ., Blacksburg, VA. Laurant, P., Daile, M., Berthelot, A., and Rayssiguier, Y. (1999). Br. J. Nutr. 82, 243. Laurant, P., Hayoz, D., Brunner, H., and Berthelot, A. (2000). Br. J. Nutr. 84, 757. Laurenz, J. C.; Green, L. W., Byers, F. M., and Schelling, G. T. (1988). J. Anim. Sci. 66,463 (Abstr.). Leeson, S., and Summers, J. D. (2001). In "Nutrition of the Chicken." University Books, Guelph, Ontario, Canada. Littledike, E. T., Wittam, J. E., and Jenkins, T. G. (1995). J. Anim. Sci. 73, 2113. Lukaski, H. C., and Nielsen, F. H. (2002). J. Nutr. 132, 930. Mahan, D. c., and Shields, R. G. (1998). J. Anim. Sci. 76, 506. Mahoney, C. P., Alster, F. A., and Carew, L. B. (1992). Poult. Sci. 71, 1669. Martens, H., and Rayssiguier, Y. (1980). In "Digestive Physiology and Metabolism in Ruminants" (Y. Ruckebusch and P. Thivend, eds.), p. 447. AVI Publishing, Westport, CT. Mayland, H. F., and Grunes, D. L. (1979). In "Proc, Grass Tetany" (M. Stelly, ed.), p. 123. American Society of Agronomy, Madison, WI. Mayland, H. F., Grunes, D. L., and Lazar, V. A. (1976). Agron. J. 68, 665. Mayo, R. H., Plumlee, M. P., and Beeson, W. M. (1959). J. Anim. Sci. 18.264. McAleese, D. M., Bell, M. C., and Forbes, R. M. (1961). J. Nutr. 74, 505. McCoy, M. A., Young, P. B., Hudson, A. J., Davison, G., and Kennedy, D. G. (2000). Res. Vel. Sci. 69, 301. McDowell, L. R. (1985). "Nutrition of Grazing Ruminants in Warm Climates." Academic Press, New York. McDowell, L. R., Conrad, J. H., Thomas, J. E., Harris, L. E., and Fick, K. R. (1977). Trop. Anim. Prod. 2,273. Meyer, H. (1976). In "Symposium: Magnesium in Ruminant Nutrition" p. 35. Israel Chemicals Ltd., Tel Aviv, Israel. Milia, P. J., Aggett, P. J., Wolff, O. H., and Harries, J. T. (1979). Gut 20, 1028. Miller, E. R., Ullrey, D. E., Zutaut, C. L., Baltzer, B. V., Schmidt, D. A., Hoefer, J. A., and Luecke, R. W. (1965). J. Nutr. 85. 13. Miller, K. B., Caton, J. S., Schafer, D. M., Smith, D. J., and Finley, J. W. (2000). J. NUlr. 130,2032. Miller, W. J. (1979). "Dairy Cattle Feeding and Nutrition." Academic Press, New York. Miller, W. L Britton, W. M., and Ansari, M. S. (1972). In "Magnesium in the Environment" (J. B. Jones, Jr., M. C. Blount, and S. R. Wilkinson, eds.). Taylor County Publishing Co., Reynolds, GA. Montalvo, M. I., Viega, J. V., McDowell, L. R., Ocumpaugh, W. R., and Mott, G. O. (1987). Nutr. Rev. Int. 35, 157. Mordes, J. P., and Wacker, W. E. (1978). Pharmacol. Rev. 29, 273. Mosley, G., and Jones, D. I. H. (1974). J. Agric. Sci. 83, 37. NCMN (Netherlands Committee on Mineral Nutrition). (1973). "Tracing Mineral Disorders in Dairy Cattle." Centre for Agricultural Publishing, Wageningen, The Netherlands. Noller, C. H., Wheeler, L. J., and Patterson, J. A. (1987). J. Dairy Sci. 70(Suppi. 1),200 (Abstr.). NRC. (1980). "Mineral Tolerance of Domestic Animals." National Academy of Sciences-National Research Council, Washington, D.C. NRC. (l982b). "United States-Canadian Tables of Feed Composition" 3rd Ed. National Academy of Sciences-National Research Council, Washington, D.C. NRC. "Nutrient Requirements of Domestic Animals." National Academy of Sciences-National Research Council, Washington, D.C. (1977). Nutrient Requirements of Rabbits, 2nd Ed. (1978). Nutrient Requirements of Nonhuman Primates. (I 982a). Nutrient Requirements of Mink and Foxes. (l985a). Nutrient Requirements of Dogs, 2nd Ed. (l985b). Nutrient Requirements of Sheep, 5th Ed.

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Chapter 6

Sulfur I. INTRODUCTION

Sulfur (S) is one of the more abundant elements in nature; however, a shortage of S-containing amino acids is a worldwide problem in human and animal nutrition. In most animals it represents about 0.15 to 0.25% of the body weight. It is difficult to divorce the dietary requirements of inorganic S from dietary levels of S-amino acids since the supply of tissue sulfate may be derived entirely from S-amino acid catabolism. The greater part of S in animals is present in the two S-containing amino acids, methionine and cysteine, or in the double form of the latter, cystine. Of the many S compounds, all can be synthesized in vivo from the essential amino acid methionine with the exception of S-containing vitamins thiamin and biotin. However, microorganisms in the ruminant digestive tract are capable of synthesizing S-containing amino acids and thiamin and biotin from inorganic S sources. Nonruminant animals have few assimilatory microorganisms so that the major proportion of their S requirement must be in the form of amino acids. Diets for ruminants that are low in protein or that contain a large proportion of the nitrogen (N) requirement as nonprotein N (NPN) (e.g., urea) may be deficient in S. Sulfur is a more critical nutrient for ruminants than nonruminants from the standpoint of potential deficiency. In recent years, S toxicosis has become more common, partly due to its high concentration in many byproduct feeds. The disease conditions in ruminants of polioencephalomalacia (PEM) and copper (Cu) deficiency are both favored by high dietary S intakes. Sulfur nutrition has been reviewed by Muth and Oldfield (1970), Goodrich et al. (1978), NRC (1980), Baker (1987), Griffith (1987), and Underwood and Suttle (1999).

II. mSTORY

Sulfur has been used in several forms since antiquity, but not until recently has there been a real understanding of its biological significance. In 1784, Scheele reported S in proteins. This information was not greatly expanded until the 1930s when the essential amino acid, methionine, was shown to contain one atom of S per 179

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molecule (McCollum, 1956). Most of the nutritional history of S relates more to the requirements and metabolism of the S-amino acids. For ruminant nutrition, Loosli et al. (1949) first demonstrated that ruminal microorganisms of sheep fed urea as the sole nitrogen source were able to synthesize methionine from inorganic S and that the sheep grew and remained in positive nitrogen balance. This was expanded when researchers showed that dietary radiolabeled S as sodium sulfate (Na2S04) was incorporated into cystine and methionine in the rumen and utilized to form milk protein (Block et al., 1951). During the period 1945 to 1954, Dick (1956) noted metabolic interrelationships among Cu, molybdenum (Mo), and inorganic sulfates in ruminants, which resulted in Cu deficiency.

III. CHEMICAL PROPERTIES, DISTRIBUTION, AND USES Sulfur is a solid, nonmetallic element. It has an atomic number of 16 and an atomic weight of 32.064. Sulfur is brittle and has almost no taste. When it is rubbed or melted, it gives off a "rotten egg" odor. It does not dissolve in water but dissolves readily in carbon disulfide. Sulfur ignites at a low temperature and burns very quickly. It burns in air with a pale-blue flame and gives off sulfur dioxide, a colorless gas. When sulfur dioxide is exposed to moist air, it mixes with the moisture in the air and forms sulfurous acid. Both sulfur dioxide and sulfurous acid are constantly being formed in the air in cities that burn large amounts of coal and gas. Large quantities of S are found in the earth's crust, both in a pure state and in combination with other substances. Sulfur occurs in a pure state in volcanic regions. It combines with metals to form valuable metal ores. Gypsum, also called calcium sulfate, is an important mineral that contains S. Sulfur has many commercial uses. Pure S is used to make up a group of valuable substances known as S compounds. These S compounds include sulfuric acid, the sulfite salts, and sulfur dioxide. Sulfur mixes with saltpeter and charcoal to form gunpowder, and is used to some extent in the manufacture of matches. About 80% of the annual production of S is used for production of sulfuric acid, which is used in the manufacture of phosphate fertilizer, purification of gasoline, production of synthetic fibers (e.g., rayon), pesticides, steel processing, bleaching agents for paper pulp, sugars and vegetable oils, preservation of beverages and food, and producing rubber products and synthetic rubber (NRC, 1980). Large amounts of sulfuric acid are also used in storage batteries. Sulfur is used in various medicines, most importantly as sulfa drugs or sulfonamides. These medicines fight bacteria and other organisms and are effective for many diseases including: pneumonia, dysentery, meningitis, blood poisoning, urinary tract infections, and some venereal diseases. Normally, sulfa drugs do not actually kill bacteria. Instead, they prevent the bacteria from multiplying. Many bacteria need a chemical called para-aminobenzoic acid (PABA) to multiply. This acts like a necessary vitamin for these bacteria. The sulfonamide drugs have a chemical structure similar to PABA, but they have S atoms where PABA has

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carbon atoms. Bacteria cannot tell the difference between the two and absorb the sulfa drug rather than the PABA. The S atoms then stop one or more of the growth processes of the bacter~a, and the bacteria cannot multiply.

IV. METABOLISM

The metabolism of S differs markedly between monogastrics and ruminants, and an understanding of this difference is basic to an appreciation of the S cycle and of the nutritional value of S compounds. The major terrestrial source of S is mineral sulfide, which is converted to inorganic sulfate by weathering and to organic S by microbial action in the soil (Young and Maw, 1958). Sulfur enters into metabolic pathways at oxidation levels as sulfate or sulfide; and forms of S must be either oxidized to sulfate or reduced to sulfide before they are utilizable by ruminants. Other naturally occurring forms of S such as thiosulfate, polythionates, polysulfides, and elemental S must be either oxidized to sulfate or reduced to sulfide before they are available for biosynthetic reactions. Biologically, the interconversion of sulfate and sulfide is a reversible process and can be considered as two broad phases: sulfate reduction, the reduction of sulfate to sulfide; and sulfide oxidation, the oxidation of sulfide to sulfate. Sulfate reductions and sulfide oxidation constitutes an overall process termed the "S cycle." It is largely a microbial process; but both animals and plants make some application of the cycle. Because several groups of bacteria utilize sulfate reduction or sulfide oxidation in their energy metabolism, the involvement of inorganic S compounds in the nutrition of microorganisms is more extensive than in the nutrition of either plants or animals. Plants can reduce sulfate to sulfide as indicated by their growth with sulfate as their only source of S. Plants generally reduce only enough sulfate to sulfide to meet nutritional requirements. A limited group of anaerobic sulfate-reducing microbes produces large amounts of sulfide during growth with adequate sulfate available. This has been considered a respiratory sulfate reduction in that the sulfate serves as a terminal acceptor similar to oxygen respiration (Peck, 1970). Mammals are unable to reduce sulfate to sulfide for the biosynthesis of S-containing amino acids and the cofactors such as biotin, thiamin, and coenzyme A. However, mammals are generally capable of incorporating sulfate as such into various lipids, carbohydrates (e.g., mucopolysaccharides), and phenols and metabolizing and incorporating to a limited extent reduced S compounds (Peck, 1970). Inorganic sulfate is taken up by higher plants and converted to organic S in the form of the S-containing amino acids, which in turn serve as an organic S source for both monogastric and ruminant animals. Many bacteria, including the microbial flora of the ruminant, are also able to convert inorganic S to organic S in the form of methionine, cysteine, and cystine and hence for the many functions of S in the body. Monogastric animals have few, if any, intestinal assimilatory bacteria to form organic S from inorganic sources and, therefore, must rely upon the S-amino acid sources for their requirement of organic S.

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The proportion of total S flow into the rumen which is 'captured' as ruminal microbial protein varies widely and is determined by factors such as the S source (methionine being less degraded than other S-amino acids) (Bird, 1972) and by the coavailability of other substances (chiefly degradable N) (Beever, 1996). Optimal microbial synthesis and S capture occur when fermentable energy, degradable S, N, and phosphorus (P) are supplied at rates which match the synthetic capacity of the ruminal microbial biomass (Underwood and Suttle, 1999). In animals, S absorption occurs in the rumen and small intestine, with substantial amounts absorbed through the ruminal wall (Bray, 1969); absorption by active transport of the inorganic sulfate takes place in the small intestine, especially the ileum (Dziewiatkowski, 1970). Organic forms of S are readily absorbed in the small intestine. The absorption mechanism is very efficient. Morrow et al. (1952) demonstrated that rats excreted, in the urine, 41 to 64% of an oral dose of inorganic sulfate 35S within 8 hours of administration. Humans also rapidly excreted sulfate in the urine. An additional source of S to the animal is S secreted in saliva. In saliva S is a mixture of both inorganic and organic forms, with salivary S higher in cattle than sheep (Underwood and Suttle, 1999). Sulfur is recycled to the rumen, with similarities to the recycling system for the urea-N system. The amount of S recycled in sheep is much less than for cattle (Kennedy et al., 1975). Under grazing conditions particularly with mature grasses rather than with legumes, and possibly under more intensive conditions, recycled S becomes nutritionally very significant (Moir, 1979).

V. RELATIONSHIPS OF SULFUR TO OTHER ELEMENTS Sulfur is closely associated with N and the dietary elements Cu, Mo, and selenium (Se) (see Chapters 8 and 13).

A. Sulfur, Copper, and Molybdenum An interrelationship between S, Cu, and Mo was first reported by Dick and Bull (1945). They reported that dietary Mo decreased liver Cu storage. Dick (1953a) recognized that there was a third factor in alfalfa hay, but not in oat hay, that potentiated the Cu-Mo antagonism, and concluded that the factor was inorganic sulfate. Dick (1953b) also showed that sulfate influenced Mo excretion in the urine and level in the blood. In the presence of S, high intakes of Mo can induce a Cu deficiency due to formation of insoluble Cu-Mo-S complexes (e.g., thiomolybdates) in the digestive tract that reduce the absorption of Cu (Mason, 1986, 1990). Several pathways exist by which Cu-Mo-S interactions mediate Cu deficiency (Dick, 1956; Ryan et al., 1987). Some thiomolybdates are absorbed and impair the metabolism ofCu in the body. Larson et al. (1995) cited research suggesting that Cu-thiomolybdate-albumin

Relationships of Sulfur to Other Elements

183

complexes cause Cu metabolism failure in the body. Price et al. (1987) showed that tri- and tetrathiomolybdates were the S-Mo complexes responsible for reducing Cu absorption, while the di- and trithiomolybdates had the greatest effect on Cu metabolism in the body. Sulfur also reduces Cu absorption by the formation of insoluble copper sulfide in the rumen, independent of formation of thiomolybdates. The effect of S alone may be greater than the S-dependent effects of Mo (Underwood and Suttle, 1999). Ruminant animals are much more susceptible to MojCu imbalance than are nonruminant animals. The primary effect probably occurs in the rumen through the involvement of sulfide-generating bacteria and the consequent formation of unavailable compounds such as cupric thiomolybdate. Provision of additional dietary Cu overcomes adverse clinical responses by inhibiting absorption of thiomolybdate or its derivatives. Formation of thiomolybdates also affects the kinetics of S metabolism by affecting sulfide formation and absorption. Thiomolybdates rapidly react with particulate matter and proteins to form complexes that bind Cu strongly, reducing its solubility, decreasing the hydrogen sulfide (H 2S) concentration and thereby the rate of sulfide absorption (Gawthorne et al., 1985). 1.

SULFUR-SELENIUM INTERRELATIONSHIPS

Selenium and S are members of Family VI of the periodic table and they share many physical and chemical properties. Competition between Se and S is due to similarity in chemical structure. Selenoamino acids are molecules with similar chemical structure to the S-containing amino acids (Se is substituted for S) and they compete for reactive sites on enzymes. Schwarz and Foltz (1957) were first to demonstrate an interrelationship between Se and S-containing amino acids in animals. Shrift (1958) reported that Se interfered with enzymes of S metabolism which in some instances was nullified when S metabolites were added to the diet. Methionine and cysteine reduced the toxic effect of their Se analogs. Sulfate was shown to reduce the effectiveness of Se in preventing white muscle disease in lambs by Hintz and Hogue (1964). Schubert et al. (1961) suggested an antagonistic relationship between dietary Se and S after field studies had shown considerable muscular dystrophy in lambs born of ewes fed alfalfa and grass hay high in both Se and S. In a more recent study with sheep, van Ryssen et al. (1998) reported that increasing dietary S from 0.2 to 0.4% reduced hepatic Se. For dairy cattle, dietary S from sulfate reduced Se balance especially when cows were fed diets with less than 0.3 ppm Se (Ivancic and Weiss, 2001). Lane et al. (1979) fed Torula yeast diets to rats with four levels of Se (0.01, 0.06, 0.11, and 0.61 ppm) and three levels ofS (267,3567, and 5267 ppm). When the two high levels of S were fed, 0.11 and 0.61 ppm levels of Se were necessary to obtain maximum glutathione peroxidase (a Se-containing enzyme) activity in small intestinal tissue of rats. Glutathione peroxidase activity was depressed in the tissue when rats were fed corn-soybean meal diets low in Se (0.03 ppm) and supplemented with S. Selenium at 0.11 ppm in the diet was adequate to prevent the

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high levels ofS from depressing the enzyme activity. Van Vleet (1982) reported that ducklings fed over four weeks with a commercial starter mash having "adequate" levels of Se and vitamin E developed a Se deficiency syndrome if the diet was supplemented with 0.5% S. Perdomo et al. (1966) reported that as S in fertilizer was increased, the Se content in clover, millet, and corn decreased. Holstein steers grazing S-fertilized herbage had lower blood glutathione peroxidase activity than those grazing unfertilized herbage (Murphy and Quirke, 1997). Dietary S from sulfate reduced Se balance especially when lactating cows were fed diets with less than 0.3 ppm of Se dry basis (Ivancic and Weiss, 2001). Both the feces and urine are paths of S excretion. However, S is excreted principally in urine. In the urine three forms occur: inorganic sulfate, the principal fraction, which represents the final stage of oxidation of organic S; ethereal S, which is present in complex detoxication products; and neutral S, which occurs as cystine, taurine, thiosulfates, and other compounds. Sulfur excreted via feces, is largely in inorganic forms. Since excreted S arises primarily from protein catabolism, there is a rather constant ratio between S and the N in the urine. There is evidence that the excretion of neutral S is proportional to the basal metabolism.

VI. FUNCfIONS Strictly speaking, S is only an essential nutrient for plants and microbes, because only they can synthesize S-amino acids and hence proteins from degradable inorganic S sources (Underwood and Suttle, 1999). Sulfur is required for the formation of the many S-containing compounds found in essentially all body cells and, therefore, is an essential nutrient. The important body S compounds include the S-containing amino acids, other S-containing molecules, hormones, and vitamins. A. Sulfur Amino Acids Sulfur is an essential element for all animals as S-containing compounds have vital metabolic functions in all living cells. Methionine, cystine, cysteine, homocysteine, cystathionine, and taurine are S-containing amino acids. Liver enzymes are able to produce cystine and cysteine from methionine; but all living animals require methionine. Methionine may be demethylated to form homocysteine; then combined with serine to form cystathionine, which upon cleavage produces cysteine and homoserine. Thus cysteine and cystine are nonessential amino acids, but a large part of the S-amino acid requirement of animals can be met by cystine and cysteine. The S-amino acids play an important function in protein structure due to their incorporation into polypeptide chains. Also, free sulfhydryl groups participate in hydrogen (H) binding, and covalent disulfide bonds between cysteine structures of the protein molecule. Disulfide bonds contribute to the biological activity of

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enzymes and many proteins (Boyer, 1959). Sulfhydryl groups provide sites for H bonding, as well as sites for the attachment of prosthetic groups of enzymes to substrate and for binding of substrates to active sites of enzymes.

B. Other Sulfur-Containing Molecules Hemoglobin, cytochromes, coenzyme A, coenzyme M, lipoic acid, Sadenosylmethionine, glutathione, heparin, penicillin G, metallothionein, and sulfate polysaccharides including chondroitin all contain S. Chondroitin is a key component of cartilage, bone, tendons, and blood vessel walls. Hemoglobin is an oxygen carrier and the cytochromes are used in electron transport. Coenzyme A serves as a carrier of acyl groups in enzymatic reactions of fatty acid oxidation and pyruvate oxidation, and is involved in the acylation of choline to form acetylcholine. Coenzyme M is essential for growth of a strain (Methanobacterium ruminatum) of methane-producing bacteria (McBride and Wolfe, 1971). Coenzyme M is a 2-mercaptoethane-sulfonic acid, i.e., HS-CH rCHrS0 3; and is required for the formation of methane from methylcobalamin (Taylor and Wolfe, 1974). Lipoic acid is a coenzyme involved in the decarboxylation of pyruvic acid and other keto acids. S-adenosylmethionine is a methylating agent in the synthesis of methyl-containing substances such as N-methyl nicotinamide, creatine, choline, epinephrine, anserine, and glycocyamine. Glutathione participates in the maintenance of proper redox potentials in cells. Heparin is a blood anticoagulant and penicillin is an antibiotic. Cysteine-rich molecules, such as metallothionein, playa vital role in protecting animals from excesses of Cu, cadmium (Cd), and zinc (Zn) while others influence Se transport and protect tissues from Se toxicity (Underwood and Suttle, 1999). Dietary S has also been shown to enhance the humoral immune response of goats (Rao et al., 1999).

C. Hormones and Vitamins Sulfur-amino acids are prominent structural features of some hormones. Insulin has disulfide bonds both within and between each of its two-polypeptide chains. Oxytocin has a disulfide linkage between cysteine residues, which helps form an internal ring structure. Sulfur is a part of the vitamins thiamin and biotin (McDowell, 2000). Thiamin pyrophosphate participates as a coenzyme in the decarboxylation of both alpha-ketoglutarate and pyruvate, and in transketo lase reactions. Biotin participates in the metabolism of aspartic acid, in the decarboxylation of oxalacetic, oxalosuccinic and succinic acids, and acts as a carrier for carbon dioxide in carboxylation reactions.

VII. REQUIREMENTS Considerable research has and will continue to be conducted on the S-amino acid requirements of monogastric species, rather than S requirements. Monogastric

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species do not require S, but rather organic S (sulfur-amino acids, thiamin, biotin, etc.) sources in their diets. With the exception of thiamin and biotin, all other organic sources can be derived from methionine. The total absence of inorganic S from the diet may, however, increase the S-amino acid requirement, which suggests that S from the amino acids is used to synthesize other organic compounds containing S. For ruminant species, S requirements need to be established for both the ruminal microbes and the ruminant animal. A. Sulfur Requirement of Ruminal Microbes The microbes of the rumen can incorporate inorganic S into organic compounds. Ruminal microbes utilize inorganic S to make S-containing amino acids, which are incorporated into microbial protein, and following digestion of the microbes in the lower alimentary tract may become a part of the body tissue. Block et al. (1951) infused radioactive 35S into the rumen and measured the activity of the isotope in the protein of milk from ewes and goats. The milk proteins contained 80% of the 35S. Lewis (1954) found that sulfate was reduced to sulfide in the rumen of sheep, and that other oxidized S compounds were also reduced to sulfide. Kulwich et al. (1957) administered radioactive 35S to sheep and found it was absorbed into the blood rapidly and peaked at six hours. Most of the dose was excreted within four days, 31% in the feces and 49% in urine. Labeled S was found in all tissues sampled and much of the isotope was present in cystine and cysteine. Anderson (1956) concluded that sulfate-S, protein-S, and free amino acid-S was reduced to sulfide by ruminal microbes and the sulfide was incorporated into microbial amino acids. Cattle ruminal fluid was incubated in vitro with labeled sulfate and substrates representative of concentrate and forage diets by Emery et al. (1957). They observed that cystine synthesis was twice as rapid as methionine formation and that S incorporation into amino acids was more rapid with the concentrate than the forage substrate. Without an adequate amount of S the ruminal microbes have a decreased ability to function normally, and reduced digestibilities (e.g., cellulose) of feedstuffs and N retention occur (Thomas et al., 1951). The artificial rumen was utilized by Hunt et al. (1954) to show that elemental S, sulfate, cysteine, and methionine stimulated the synthesis of riboflavin and vitamin B 12 • Martin et al. (1964) found that in vitro cellulose digestion was less (1.6%) when ruminal inoculum from cattle fed a low-S diet was used than that which occurred (33.5%) when the inoculum was from cattle fed a S-adequate diet. Whanger and Matrone (1967) observed large amounts of lactate produced in vitro when ruminaI fluid from sheep fed a S-free diet was utilized. They stated that as much as 30% of the lactate was converted to propionate via this pathway by microbes from S-deficient sheep. It appears that the microbes of sheep fed the S-deficient diet did not have the ability to hydrogenate acrylate. All ruminal bacteria require S, but not all bacteria can utilize inorganic S. Emery et al. (1957) reported that only 5 of 10 strains studied utilized significant amounts of inorganic sulfate to synthesize organic S compounds, and only three strains

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incorporated inorganic sulfate into microbial protein when cysteine was present. Hume and Bird (1970) observed that ruminal protein synthesis increased from 82.1 to 86.9 g/day as the S content of the diet was increased from 0.075 to 0.237%. Cystine and sulfate gave similar quantities of microbial protein synthesis. Slyter et al. (1986) reported that when purified diets deficient in S were supplied to cultures in vitro, less methane (3.2 vs 32.6 mmol/I daily) was produced and fewer cellulytic microbes were present than in sheep ruminal fluid supplemented with S. This was interpreted to be due to recycling of S to the rumen where it is efficiently scavenged by ruminal bacteria. Evans and Davis (1966) observed the optimum S level to be 0.29% for cellulose digestion in the rumen of fistulated steers. Unfortunately, beef cattle requirements have been determined with high-starch diets, resulting in unreasonably low recommended S requirements (Bull, 1979).

B. Dietary Sulfur Requirement of Ruminant Animals Determining S requirements of ruminants has involved supplementing diets with methionine, cystine, sulfate salts, or elemental S in balance trials, feedlot trials, or by radioisotope studies. Loosli and Harris (1945) increased the growth rate of lambs fed a diet containing 6.55% crude protein by raising the level to 10.28% with urea plus sulfate, or urea plus methionine. Lofgreen et al. (1947) increased N retention when 0.2% methionine was added to a 10% crude protein basal diet in which 40% of the N was supplied from urea. Nitrogen retention and wool production were improved in sheep by supplementing their diets with methionine or with methionine and cystine (McLaren et al., 1965). Digestion of cellulose decreased when steers were fed purified diets deficient in S (Martin et 01., 1964). Bull and Vandersall (1973) demonstrated that sodium (Na) sulfate, calcium (Ca) sulfate, DL-methionine and methionine hydroxy analog (MHA) were equivalent in promoting cellulose digestion in vitro, and concluded that the optimum level of S was from 0.16 to 0.24% for ruminants. Yearling wethers fed 0.155% total S in diets had good rates of growth (Rendig and Weir, 1957). A sheep diet that contained II % protein was calculated to require 0.176% S by Moir et. al. (1968) based on a need for a N:S ratio of 10:I. Chalupa et al. (1973) found better weight gains by Angus steers when sodium sulfate or elemental S were added to increase S from 0.05 to 0.13%; and feeding these forms of S in steer diets up to about 0.6% had no deleterious effects. However, Bouchard and Conrad (1973) found that when dietary S as calcium sulfate exceeded 0.3% dairy cows had reduced feed intake; and Johnson et 01. (1968) reported that the addition of 0.5% S as calcium sulfate decreased gains of lambs. Bouchard and Conrad (1973) found that dry matter intake and dry matter digestibilities were improved when basal diets with 0.10 or 0.06% S were supplemented to provide 0.15 or 0.18%, respectively. It was estimated by these workers that diets with 0.12% S would result in S balance in cows producing between 9 and 37 kg milk daily. Johnson et 01. (1971) calculated endogenous fecal loss of S to be 0.15 g/Iamb/day and an equal amount of endogenous S was lost in

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the urine daily. The maintenance requirement for S, determined from metabolic urinary and fecal losses and content of wool growth, was calculated to be 0.48 g of retainable S daily. This amount of S would be supplied by 1.79, 0.86, or 0.69 g of S as elemental S, sodium sulfate or methionine, respectively. Lambs gaining 100 g daily with 0.15% dietary S would require 0.63 g of retain able S. Sulfur requirements for grazing ruminants appear to be between 0.10 and 0.32% (McDowell, 1985). Sulfur requirements according to ruminant and horse NRC publications are presented in Table 6.1 and range from 0.14 to 0.32%.

c. Dietary Nitrogen:Sulfur (N:S) Ratios

in Relation to Sulfur Requirements

Ruminants through their ruminal microbes may satisfy their metabolic requirements for dietary Nand S compounds from cellulosic materials that are inadequate nutritionally for monogastrics. Products of ruminants such as beef, lamb, and milk have N:S ratios of about 15:I, while wool has a ratio of 5:I. Plants have a ratio of approximately 13:I. Loosli (1952) suggested that S requirements be expressed relative to those for N, and recommended a N:S ratio of 15:I based on the relatively constant ratio observed in animal tissues. For maximal growth, dry matter intake and feed efficiencyof Alpine and Angora goats, the S requirement was 0.21 to 0.24% and the N:S ratio was 9.5 to 11.1(Qi et al., 1992). The N:S ratio may have little use in some situations such as grasses with low S bioavailability or heat damaged forages (Goodrich and Garrett, 1986). Moir et al. (1968) working with sheep and utilizing S additions that narrowed the N:S ratio from 12:1 for the basal diet to 9.5:1 for supplemented diets observed an improvement in N retention from 28.8 to 36.0%. As the N:S ratio of recycled Nand S in the rumen of sheep is between 70 and 80:I, Moir et al. (1968) suggested that there are instances where animals will not benefit from N recycling due to the concurrent S deficiency; and supplementation with nonprotein N is of no value unless additional S is provided. The N:S ratio of a feedstuff protein is not important in itself if the protein is totally degraded in the rumen. If it is completely degraded, then the ratio of N to total S is the critical value. Bouchard and Conrad (1973) reported that lactating dairy cows did not

TABLE 6.1

Dietary Sulfur Requirements" Animal species

Requirements (%)

Reference

0.15 0.20 0.14-0.18 0.18--0.26 0.16--n.32 0.15

NRC (1996) NRC (2001) NRC (1985)

Beef cattle Dairy cattle Sheep, mature ewes young ewes Goats Horses "Expressed as per unit animal feed, OM.

NRC (1981) NRC (1989)

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respond when N:S ratios were changed from 14:1 to 8.2:1, 12.0:1 to 6.6:1, 11.0:1 to 6.9: 1, and 14:1 to 8.4: 1 in several trials.

Sulfur requirements are higher and S deficiencies are more prevalent in wooltype sheep and Angora goats (Qi, 1988, 1992). Cattle needed less S than sheep to digest a urea-supplemented, low quality diet (Kennedy and Siebert, 1972). Both protein and S requirements are high for mohair and wool growth, because efficiency of use of microbial (or undegraded dietary) protein for fleece growth is only 26% compared with around 80% for most other purposes (Underwood and Suttle, 1999). Also, Merino flocks selected for high fleece production show greater responses to supplements of S-amino acids than those selected for low fleece weight (Williams et al., 1972; Qi et al., 1994). Anaerobic fungi may be particularly sensitive to S concentration in the rumen; additional S increased the fungal ruminaI concentration and S-amino acids leaving the rumen (Weston et aI., 1998).

YIn. NATURAL SOURCES

There is a wide variation of S concentrations in feeds (Appendix Table II). Sulfur concentrations in pastures and conserved forages can vary widely from less than 0.05 to higher than 0.50% (McDowell, 1985; Underwood and Suttle, 1999). Various types of forages have different nutrient S requirements for optimum yields. The critical S content of sugar cane is 0.04% or less (Stanford and Jordan, 1966) compared to approximately 0.22% for alfalfa (Pumphrey and Moore, 1965). The wide variations in S content of plants are due largely to the amount of S in plant protein (Allaway and Thompson, 1966). Genetic factors determine the amino acid composition of plant proteins. It appears that sulfate S does not build up in the plant to a significant extent until the S requirement of the plant for protein formation has been met. The S status of the plant may be indicated by sulfate S content. McClung et al. (1959) found that when S was not applied to soils of the central plateau of Brazil, plant growth was only 4 to 30% of that obtained when a complete fertilizer was applied. Forages grown on soils so deficient in S that yield is depressed will have very low concentrations of S, far below those needed by animals (Rendig, 1986). Hay yield of bermudagrass was lower when it contained less than 0.14% S, and crimson clover responded to S fertilization when it also had less than 0.14% S (Kamprath and Jones, 1986). The type of forage in the diet may also influence S requirement. Sulfur requirements may be higher for cattle grazing sorghum Sudan grass because S is required in the detoxification of the cyanogenic glucosides found in most sorghum forages. Sulfur bioavailability varies with the type of forage; fescue has a lower S availability than other grasses. Cattle consuming fescuehay will often respond with improved intake and fiber digestion following S supplementation (Berger, 1999). Ruminants consuming large quantities of corn silage will likely be receiving inadequate S. The amount of S in corn silage samples analyzed in the Georgia Forage Testing Laboratory in 1973-1974 averaged 0.07% in dry matter, with a

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range of 0.005 to 0.19%. More than 80% of corn silages had less than one-half the estimated 0.20% S needed for lactating cows (Miller, 1979). With S fertilization, total S concentrations in corn silage were increased by approximately 32% (Buttrey et al., 1986). Cereal grains are quite low in S, with corn, rice, rye, sorghum, and wheat ranging from 0.05 to 0.18% (Appendix Table II). Oilseed S concentrations were moderately high with cottonseed meal, rapeseed meal, and soybean meal ranging from 0.41 to 0.58% S. Molasses is a good source, ranging from 0.47 to 0.60% S. High-sulfur feed ingredients such as feathers, viscera, and fecal waste used in livestock diets or as a fertilizer are organic sources of S. Forages grown on biosolids (municipal sewage sludge)-amended soil frequently have increased S contents (McBride et al., 2000; O'Connor et al., 2001). As a result of a three-year experiment using biosolid fertilization, forage S was elevated to 0.4% and Cu deficiency was in evidence in cattle (Tiffany et al., 2000, 2002). Sulfates in the water can be a major source of S intake. For example, in one of the cases cited by Kung et al. (l998b), sulfates in the drinking water ranged from 2200 to 2800 ppm. When the water S intake was expressed as a percentage of the dry matter consumed, it averaged 0.67%. Smart et al. (1986) demonstrated that cows receiving sulfated water (0.35% total dietary S) had plasma Cu concentrations lower than cattle drinking nonsulfated water (0.20% total dietary S), and a dietary Cu concentration of 10 ppm was not enough to correct the problem. Digesti and Weeth (1976) proposed the maximum safe concentration of sulfates in drinking water for cattle is 2500 ppm. Water sulfate concentrations as high as 5000 ppm have been reported (Veenhuizen and Shurson, 1992).

IX. DEFICIENCY A. Effects of Deficiency 1.

MONOGASTRIC SPECIES

Despite the fact that S is a key mineral in many compounds essential for life, dietary inorganic S is not necessary for the health of monogastric animals. Pigs and poultry can do quite well with only organic S (S-amino acids, thiamin, biotin, etc.) sources in their diets. However, the total absence of inorganic S from the diet may increase the S-amino acid requirement, because the breakdown of S-amino acids is used to synthesize other organic compounds containing S. The dietary requirements of S are not stated for monogastric species but rather the requirement for methionine. Sulfur deficiency for monogastrics is more correctly a protein deficiency (i.e., methionine). Present evidence indicates that in humans, the requirement for synthesis of S-containing compounds can be met by a single, S-containing amino acid, methionine. It is possible that in infants, as in felines, there is an additional requirement for taurine, an amino sulfonic acid.

Deficiency

191

Breast-fed infants have higher plasma levels of taurine than infants fed casein-based formulas (Sturman et al., 1976). Clinical signs of deficiency for monogastric species are typical for protein deficiency in the various monogastric species. The following discussion on the effects of supplemental inorganic S will be limited to swine and poultry. a. Swine. Two-week-old pigs fed purified diets containing 0.8% methionine tolerated half the methionine being replaced with inorganic sulfate without affecting weight gain, feed efficiency or the collagen content in tendons (Robel, 1976). Karunskii et al. (1982) reported that pigs fed trace element-deficient diets supplemented with normal levels of Fe, Cu, cobalt (Co), Zn, and manganese (Mn) had increased weight gains and feed efficiencies compared to controls. The improved gains of pigs due to the trace elements were significantly higher when the elements were given as sulfates rather than as chloride or carbonate salts. The NRC (1998) for swine concludes that the S provided by S-containing amino acids seems adequate to meet the pig's needs for synthesis of S-containing compounds with exception of thiamin and biotin.

b. Poultry. Gordon and Sizer (1955) suggested that inorganic sulfate was of importance in poultry nutrition. They fed a basal diet to chicks that was deficient in cystine (0.08% cystine, 0.51% methionine) and sulfate-free and found that by adding 0.5% sodium sulfate there was a 31.4% growth increase (372 vs 488 g) over the basal diet by the end of the fifth week. However, inorganic sulfate could not replace cystine or methionine for protein synthesis. Simultaneous supplementation with 0.5% sodium sulfate and 0.22% methionine gave a 66.1% growth response over the basal diet (375 vs 617 g) indicating that poultry could satisfy part of their total S requirement with inorganic sulfate. Hinton and Harms (1972) utilized a similar basal diet fed to chicks and found that supplementation with 0.2% sodium sulfate resulted in a 14.5% growth increase over the basal diet. Sasse and Baker (1974) concluded that there is no dietary requirement for sulfate in chicks per se since they found no response to sulfate in the presence of adequate S-containing amino acids. Miles et al. (1983a,b) demonstrated a three-way interrelationship between methionine, choline, and sulfate in poultry. They concluded that inadequate dietary choline in studies involving methionine and sulfate would result in a slightly higher methionine requirement since more of the methionine would have to be used to meet the methyl-group requirement. Also, inadequate sulfate in diets designed to study the interrelationship between methionine and choline would lead to higher methionine requirement because methionine would be used to provide S. When the interrelationship between S-containing amino acids and inorganic sulfate is studied the choline level of the diet should be given proper attention. 2.

RUMINANTS

Outward signs of S deficiency (Fig. 6.1) include loss of appetite, reduced weight gain, reduced wool growth in sheep, excessive lacrimation, cloudy eyes, dullness, weakness, emaciation, and death (Thomas et aI., 1951; Kincaid, 1988; Qi, 1988,

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Fig. 6.1 Lambs fed a low-S diet. The lamb on the left received 3 g of S per pound of diet, whereas the other lamb received none. The deficient lamb exhibited excessivesalivation, lacrimation. and shedding of wool. (Courtesy of U.S. Garrigus, University of IIlinois, Urbana)

1992). With a deficiency wool or hair can be shed; adding S to deficient animals increased grease and clean mohair production, grease and clean mohair staple strength, and staple length (Qi et al., 1992). Rakes and Clark (1984) suggested that lameness in dairy cattle may be associated with S deficiency, as characterized by slower-growing, less flexible hooves. With a S deficiency, a number of workers have observed a reduction in ruminal bacterial numbers as well as changes in the types of bacteria present (e.g., fewer cellulytic microbes). It has been suggested (Kennedy et al., 1968) that the S requirement for cellulose digestion is greater than that for starch. Therefore, grazing ruminants that are consuming high-cellulose diets will more quickly develop a S deficiency. Due to lack of cellulose digesting bacteria, S-deficient forages characteristically have longer retention times in the reticulo-rumen and lower intake than S-adequate forages (Rees and Minson, 1978; Hegarty et al., 1994; Mathews et al., 1994). On S-deficient diets, ruminaI sulfide concentrations are low ( < 1 ug sulfide Simi of ruminal fluid), and much of this may be held by the cellular fraction. The type of fermentation is different when S-deficient diets were fed, lactic acid concentration of the rumina I fluid increased, and the production of butyric and higher chain acids was depressed (Whanger, 1972; Slyter et al., 1988). In vitro studies indicated that added sodium sulfide (31 mg suifide-S/IOO ml fluid) decreased methane production but increased the molar ratio of carbon dioxide to methane from 1.98 to 4.49 (Whanger, 1972). Sulfur-deficient regions are worldwide, with low herbage S being reported in the six continents (Tabatabai, 1986). Improved ruminant animal production has resulted following S supplementation (McDowell, 1976; Stobbs and Minson, 1980; Qi et al., 1994). From China, vast regions of the northeast, southwest, and southern pastoral regions are considered S-deficient for grazing sheep (Qi et al., 1994).

Deficiency

193

Requirements by sheep and goats for S, expressed as a percentage of S in dietary DM, were not met by any of the 27 dominant forages in these regions (Hou, 1982). Sulfur deficiency was recognized in the U.S. in the 1900s. In the United States, S deficiency occurs most frequently in temperate regions where S contributions from precipitation and irrigation water are low and soils have originated from moderately weathered volcanic parent materials (Qi et aI., 1994). Both these conditions exist in major portions of the western U.S. Sulfur deficiency seldom occurs in arid regions because in arid regions, productivity of plants is low and soluble S04 accumulates in the soil from ground water evaporation. Deficiencies occurred most frequently in sub-humid regions and humid regions where rainfall leaches soluble S below the rooting zone of plants. Soils of the tropics generally have low levels of S compared to those of temperate regions. Responses to fertilizer S are widespread in the tropics and have been recorded in 40 tropical countries with 23 different crops (International Fertilizer Development Center (IFDC), 1979). A limited amount of analysis has indicated that many tropical forages contained considerably less than an optimum S concentration of 0.20%. Sulfur analyses of 10 forage samples from the llanos rangelands of both Colombia and Venezuela were low, ranging from 0.032 to 0.088% (Miles and McDowell, 1983). Some Brazilian studies found very low levels of S «0.1 % S, DM) in at least 30% of tropical grasses analyzed, principally during the dry season (Cavalheiro and Trindade, 1992). From these regions, severe leaching of soils and frequent burning of grasslands led to the assumption that many, if not most, llanos rangeland forages will befound deficient in S. McClung et al. (1959) also suggested that very low soil S levels in many tropical regions are due to repeated burning of dry grass which caused losses of 75% of the S by volatization.

B. Assessment of Sulfur Status in Ruminants Since S deficiency in ruminants relates to the well-being of the ruminal microflora, the best diagnosis may be afforded by obtaining samples of ruminaI fluid by stomach tube and determining whether or not they contain sufficient sulfide for unrestricted microbial protein synthesis (Underwood and Suttle, 1999). A suggested critical level would be between 1.0 to 3.8 pg S/I (Weston et al., 1988; Hegarty et al., 1991). Low serum sulfate « 10 mg/l) has been suggested as an indicator of S deficiency. Whiting et al. (1954) found a normal serum sulfate level in range ewes fed a basal diet that contained 0.09% S. Since serum sulfate may be of endogenous origin its use as an indicator of dietary S status is questionable. If another factor such as N, is limiting, serum S values for a given S intake are increased (Underwood and Suttle, 1999). A lack of S also results in a microbial population that does not utilize lactate; therefore, lactate accumulates in the rumen, blood, and urine. Plasma concentrations of amino acids are influenced by a S deficiency. Calves fed a S-deficient purified diet had elevated levels of serine, citrulline, alanine, cystine and total dispensable amino acids, but less glycine and tyrosine in blood plasma (Chalupa et al., 1971). Concentrations of methionine in plasma and liver fell when

194

Sulfur

calves were given a diet sufficiently low in S (0.04%, OM) to retard growth (Slyter et al., 1988). It appears that the best indicator of S status of ruminants is the dietary S content and animal performance (e.g., growth and wool growth) after supplementation of S.

X. SUPPLEMENTATION For monogastric species S needs are met by selecting feeds to meet methionine requirements. Next to lysine, methionine is often the second most limiting amino acid. Synthetic methionine is available and is frequently added to swine and poultry diets. With the exception of low S-containing forages, natural protein feedstuffs for ruminants normally contain sufficient S to meet the S requirement of ruminal microbes and additional S need not be added to protein-adequate diets. However, if NPN sources are utilized such as urea then S may be needed for microbial protein synthesis. Unlike natural protein sources, urea contains no S. Loosli and Harris (1945) increased the growth rate of lambs fed a diet containing 6.55% crude protein by raising the level to 10.28% with urea plus sulfate, or urea plus methionine. Lofgreen et al. (1947) increased N retention by adding 0.2% methionine to a crude protein basal diet in which 40% of the N was supplied from urea. Lambs fed urea-containing diets without added S lost weight and were in negative N balance, while those supplemented with S were in positive N balance (Thomas et al., 1951). Nitrogen retention and wool production in sheep were improved by supplementing their diets with methionine or with methionine and cystine (McLaren et al., 1965). Hill et al. (1985) working with beef calves fed diets of corn silage supplemented with urea, with and without added S, found that the urea-supplemented diets with added S resulted in greater gains and a trend toward improved feed efficiency compared to those not receiving S. A review (Miles and McDowell, 1983) summarized four cattle S-supplementation trials in which control diets contained between 0.04 and 0.10% S. From these studies, intake by supplemented cattle increased between 7 and 260%, and production of milk and meat increased anywhere from 6 to more than 400%. There is no need to supply excess S when N in the diet exceeds the animal's requirement, and most feeds have adequate S. However, when high levels of NPN are added to diets to make up a N deficiency in the diet or supplement, then additional S is needed. Diets low in S (less than 0.1% OM) should be supplemented with 3 g of inorganic Sf 100g urea, which is the equivalent of 1 part S to 15 parts of NPN (NRC, 1976). A popular way of providing S (e.g., calcium sulfate) to grazing cattle is inclusion of S as part of a molasses supplement, in a "lick wheel" or as a molasses-urea-bran block. These molasses mixtures have the advantage of providing energy, protein, S, and other needed nutrients such as phosphorus (P), Co, and Cu. For ruminants in feedlot systems or high concentrate feeding dairy operations, dietary S supplementation can be provided as ruminal escape protein

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or ruminally-protected S-containing amino acids. There are advantages in providing S in the form of methionine analogues (e.g., malyi and methylmalyl methionine), which are not degraded by ruminal microbes. Where ruminal microbial protein synthesis cannot provide sufficient methionine to meet the needs of the animal and methionine is the most limiting amino acid, it may be more economical to feed such "protected" S sources than to feed S in expensive escape protein (bypass protein), such as fish meal (Underwood and Suttle, 1999). A major use of methionine analogues is for types of animal and production with high methionine requirements (e.g., wool production) (Coetzee et al., 1995). Bassett et al. (1981) reported that rumen-protected methionine increased feed intake, grease, and clean fleece weight of Angora goats. Rumen-protected methionine at 0.54% of dietary DM produced maximum clean mohair production. A major method of providing supplemental S to grazing livestock is by S fertilization of forage crops. The incidence of S deficiency in many countries is increasing due to the increasing use ofhigh-N-P-K, low-S-containing fertilizers and the increased need for S brought about by increases in yields (Coleman, 1966). Shifting from the use of ordinary superphosphate (12% S) to triple superphosphate (1 % S) eliminates a source of available S (lFDC, 1979). Although economically prohibitive in many tropical regions, S fertilization is an effective way of increasing forage S, as well as crop yields. An additional value of S fertilization is that some reports from tropical regions have indicated that S fertilization may increase forage intake by improving the palatability of less palatable species (Rees et al., 1974; Centro International de Agricultura Tropical (CIAT), 1981). With S-deficient Pangola grass, applying fertilizer S increased intake by 44%, compared with only 28% when the sheep were drenched with sodium sulfate (Rees et al., 1974). Sulfur fertilization to Desmonium ovalifolium in the Colombian llanos increased foliar S, N, and biomass production, decreased tannin content in leaves and, most importantly, increased intake of the forage (CIAT, 1981). Sulfur supplementation of early vegetative Kenhy tall fescue hay diets improved its utilization (Muntifering et al., 1984). Chestnut et al. (1986) observed that in orchard grass, S fertilization increased S content, changed plant tissue composition, and increased digestibility of phenolic constituents. Morrison et af. (1990) reported that dietary S affected DM intake and digestibility by sheep. The source of supplemental S can influence its bioavailability. Goodrich et al. (1978) gave the following ran kings from the most available to the least available: L-methionine > calcium sulfate> ammonium sulfate> sodium sulfate> molasses S > sodium sulfide> lignin sulfonate> elemental S. Most bioavailability experiments have been conducted with either sheep or cattle in growth or absorption studies or with in vitro ruminal fermentation techniques. Assuming these criteria, including S absorption, are valid indicators of bioavailability, most sources of S have been well utilized when compared with sodium sulfate as the standard. The biologically available S in L-methionine found by Johnson et al. (1971) and in DLmethionine by Albert et af. (1956) was higher than in other forms of S studied. Methionine hydroxy analog (MHA) compares favorably with methionine as a source ofS for ruminal microbes in vitro (Gil et al., 1973a,b). In concentrate feedlot

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diets, where considerable feed bypasses the ruminal fermentation, the methionine in feed may be absorbed by the lower tract and utilized. Bouchard and Conrad (1974) reported that sodium sulfate, calcium sulfate and a mixture of potassium (K) sulfate and magnesium sulfate were equally usable as supplementary S for lactating dairy cows. They recommended a levelof 0.17 to 0.20% S for lactating dairy cow diets. Approximately three times more S was needed in the elemental form for lambs than as methionine (Albert et al., 1956; Johnson et al., 1971). However, Chalupa et al. (1973) reported that elemental S and sulfate S were equivalent sources for cattle. The S in corn and corn silage basal diets was found to be less available than S in sodium sulfate, methionine, and MHA (Bull and Vandersall, 1973). Bouchard and Conrad (1973) found S in molasses was 65 to 75% digestible with dairy cows; which was 15 to 20% less available than in Na and Ca sulfates. Inorganic compounds including ammonium bisulfate, ammonium sulfate, calcium sulfate, sodium bisulfate, and sulfuric acid were generally equal to sodium sulfate as a source of S (Henry and Ammerman, 1995). However, elemental S was not utilized as well as sodium sulfate when tested in both cattle and sheep. Sulfur, as the highly insoluble elemental S or lignin sulfonate, is much less available, and it is suggested that elemental S (flowers of S) is utilized about one-third as efficiently as the sulfate or methionine forms (McDowell, 1985). Sulfur in corn and corn silage has been found to be less available than that in sodium sulfate, methionine, and MHA. An additional supplementation consideration relates to reducing odor in manure. Formulating diets to contain reduced S concentrations will likewise reduce S excretion, with a reduction of hydrogen sulfide gas and odor (Shurson et al., 1999).

XI. TOXICITY The toxicity of S is dependent upon its form and route of administration. Whereas elemental S is considered one of the least toxic elements, hydrogen sulfide rivals cyanide in toxicity (NRC, 1980). The variable tolerances to different amounts and sources of S in the literature partly reflect differences in the rate of ingestion of degradable S, the rate of sulfide absorption across the ruminal wall (which is pH-dependent) and the rate of sulfide capture by ruminal microbes (Underwood and Suttle, 1999). Simultaneous addition of urea can lessen the depression of appetite and digestibility caused by Salone. The toxic effects of dietary inorganic sulfate are believed due to its conversion to hydrogen sulfide by the gastrointestinal flora in both ruminants and non-ruminants. In the rumen S from various forms and sources can be readily converted to sulfide. Sulfur is much less toxic to monogastrics due to the limited ability of their microflora to generate sulfide from S products. In monogastrics, S is relatively inert and can therefore be tolerated at relatively high levels. In ruminants, the ingestion of large amounts of S can lead to acute S toxicosis resulting in death. The immediate signs of distress include thrashing, kicking at stomach, staggering, and moaning followed by subsequent death within 48 hours, suggesting a fairly high capacity to produce sulfide. High concentrations of sulfide in ruminal gas have been

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reported (McAllister et al., 1992) and have resulted in respiratory distress, reduced feed intake, and reduced ruminal motility (Bird, 1972). For ruminants, in the normal process of eructation (belching of gases), 60% of eructated gases are inhaled and enter the respiratory tract (Bulgin et al., 1996). Therefore, inhalation of hydrogen sulfide from high sulfate has been implicated in enhancing sulfide toxicity. The margin between S requirement and toxic concentrations for ruminants is extremely small. The requirement is suggested to be between approximately 0.1 to 0.25%. However, maximum dietary tolerable levels of S for ruminants were suggested at 0.4% by the National Research Council in 1980 (NRC, 1980). However, more recent research indicates the toxicity to be less, ranging from 0.3 to 0.4%. Zinn et al. (1999) suggested an even lower tolerance; steers on a concentrate diet were adversely affected when ammonium sulfate was used to increase dietary S from 0.2 to 0.25%. Bouchard and Conrad (1973) suggested that this level should be less than 0.30% for lactating cows. If high levels of S inhibit intake, extreme caution should be taken during the close-up and early lactation stages where DM intake is lower than desired. Sulfide is readily absorbed through the ruminal wall into the bloodstream (Bray, 1969). Once absorbed, sulfide inhibits the functions of carbonic anhydrase, dopa oxidases, catalases, peroxidases, dehydrogenases, and dipeptidases, adversely affecting oxidative metabolism and the production of ATP (Short and Edwards, 1989). Specifically, sulfide is also thought to block the enzyme cytochrome C oxidase. Sulfide also binds to hemoglobin, creating sulfhemoglobin, reducing the oxygen carrying capacity to tissues. Sulfide also has a paralyzing effect on the carotid body and therefore may also inhibit normal respiration (Bulgin et al., 1996). Acute reactions in response to increased levels of ingested elemental S have been reported in sheep (Bulgin et al., 1996). These animals had grazed on an alfalfa field that had been sprayed with elemental S (60 kgjha). Within two hours after being released onto this field, some of the animals began to show signs of distress and quickly died. Upon necropsy, it was noted that the rumina I pH was 6 to 6.5, there was an odor of rotten eggs and pulmonary edema was observed. Immediate deaths were probably from acute sulfide toxicosis. Excess S can also impair animal performance by reducing the availability of other minerals. For example, hydrogen sulfide in the rumen binds with Mo to form thiomolybdates. Thiomolybdates bind with Cu in the rumen to form an insoluble complex. Sulfur also reduces Cu absorption by the formation of insoluble Cu sulfide in the rumen, independent of the formation of thiomolybdates (see Section V-I). In addition to detrimentally affecting Cu metabolism, excess S interferes with Se metabolism. Fertilizing pastures with ammonium sulfate resulted in increased forage S and lower liver Cu in cows grazing the forage (Arthington et al., 2002). Excess fertilizer S can promote a Se deficiency resulting in a higher probability of white muscle disease (see Section V-2). Another problem that can occur when high dietary S leads to the production of excess sulfides in the rumen is polioencephalomalacia, or PEM (Lowe et al., 1996; Gould, 1998; McDowell, 2000). The disease affects the central nervous system.

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Clinical signs in mild cases include dullness, blindness, muscle tremors (especially of the head), and opisthotonos. The condition is characterized by circling, head pressing, and convulsions, and in severe cases, the animal collapses within 12 to 72 hours after onset of the disease. Gould et al. (1991) reported that steers with the highest ruminal fluid sulfide concentrations coincided with the onset of clinical signs of PEM. Several cases of PEM occurred when gypsum had been used as a feed intake limiter. It would appear that the sulfate ion of gypsum, during its conversion to sulfide, must pass through sulfite, which may destroy thiamin. The sulfite ion apparently will cleave thiamin at the methylene bridge, mimicking thiaminase. In lambs, PEM was induced by administration of a sulfide solution; neurological clinical signs included stupor, visual impairment, and seizures (McAllister et al., 1992). After 12 weeks of feeding a high-S diet (0.8% S) to sheep, all animals had developed PEM and the availability of Cu and Zn was depressed (Krasicka et al., 1999). The exact interaction between dietary S, thiaminase production, and PEM is not well understood; Kung et al. (1998b) postulated sulfates in the feed or water are converted to hydrogen sulfide in the rumen. When the hydrogen sulfide is eructated with the other ruminal gases, it is inhaled and can damage lung and brain tissues. In some studies, thiamin status was within normal ranges and giving thiamin injections did not prevent the signs of PEM in all cases. There is the suggestion that PEM from excess S differs in fine pathology and responsiveness to thiamin from that attributed entirely to a thiamin deficiency (Underwood and Suttle, 1999). Maximum tolerable levels for monogastrics are not definitive. Sulfur is less toxic for monogastrics because intestinal absorption of inorganic S compounds is low and much less sulfide is formed in the intestinal tract than for ruminants. Smith (1973) concluded that 0.69% S (inorganic and organic) for rat diets is the optimal level. Reduced growth for poultry resulted from 1.4% S (NRC, 1994). Paterson et al. (1979) reported that weanling pigs consuming drinking water that contained 600 ppm S as sodium sulfate had loose feces and diarrhea, but no effect on weight gain or feed conversion. Sows provided water containing up to 664 ppm S as sodium sulfate from 30 days post-breeding to 28 days of lactation had no problems with reproduction. Corke (1981) described accidental S poisoning in horses when flowers of S were fed to 14 horses ranging in age from 5 to 12 years. Consumption was 0.2 to 0.4 kg per horse. Twelve horses appeared dull and lethargic followed by purgation within 12 hours; the other two had mild signs 48 hours after administration. Two horses died from respiratory failure associated with cyanosis and terminal convulsions 48 hours after consuming the S. Many common feeds and sources of water can contain high levels of Sand/or sulfate. Some common feeds have moderate to high levels of S. For example, corn gluten meal, molasses (cane and beet), and brassicas (e.g., turnips) are high in S (0.43 to 0.72% S) (Kung et al., 1998a). Other feeds that contain high concentrations of S include, fish, feather, meat, and blood meals that are common sources of rumen undegradable intake protein. Condensed molasses fermentation solubles is a high sulfate byproduct that contains 5.75% S (Hannon and Trenkle, 1990). Water can also be very high in sulfates with levels in excess of 5000 ppm (Veenhuizen and

References

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Shurson, 1992). Digesti and Weeth (1976) suggested that it was safe for cattle to consume water containing 2500 ppm of sulfate. Recently, Wagner et al. (1998) reported lower intake and gains in steers fed water with 2000 ppm sulfate. In recent years, there has been increased incidence of excess S in ruminant diets. Part of this is due to fertilization practices. High forage S is the result of aggressive fertilization with ammonium sulfate. Ammonium sulfate production has increased significantly and it is often priced competitively, both as a fertilizer and feed ingredient. Laboratory analysis of Michigan forages grown in 1996 and 1997 had 1.5 to 3.0 times greater S than forages grown in 1994 and 1995 (Beede, 1999). Cattle grazing ammonium sulfate-fertilized bahia grass containing 0.50% S were less able to respond to Cu supplementation (Arthington et al., 2002). Upon removal from high-S forages, Cu-deficient cattle were able to rapidly respond to Cu supplementation.

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McBride, B. C, and Wolfe, R. S. (1971). Biochem. 109,2317. McBride, M. B., Richards, B. K., Steenhuls, T., and Spiers, G. (2000). J. Environ. Qual. 29, 848. McCollum, E. V. (1956). A History of Nutrition. Houghton Mifflin, Boston, MA. McClung, A. C, DeFreitas, L. M. M., and Lott, W. L. (1959). Soil Sci. Soc. Am. Proc. 23,2221. McDowell, L. R. (1976). In "Beef Cattle Production in Developing Countries" (T. Smith, ed.), p. 216. Centre for Tropical Veterinary Medicine, Edinburgh, Scotland. McDowell, L. R. (1985). Nutrition ofGrazing Ruminants in Warm Climates. Academic Press, New York. McDowell, L. R. (2000). Vitamins in Animal and Human Nutrition. Iowa State Press, Ames, IA. McLaren, G. A., Anderson, G. C., and Barth, K. M. (1965). J. Anim. Sci. 24, 231. Miles, R. D., Ruiz, N., and Harms, R. H. (I 983a). Poult. Sci. 62,495. Miles, R. D., Ruiz, N., and Harms, R. H. (l983b). Proc. Soc. Exp. Bioi. Med. 173, 32. Miles, W. H., and McDowell, L. R. (1983). World Anim. Rev. 45, 2. Miller, W. J. (1979). "Dairy Cattle Feeding and Nutrition." Academic Press, New York. Moir, R. J. (1979). In "The Second Annual International Minerals Conference," p. 93. International Minerals and Chemicals Corp., Mundelein, IL. Moir, R. J., Somers, M., and Bray, A. C (1968). Sulfur Institute J. 3, 15. Morrison, M., Murray, R. M., and Boniface, A. N. (1990). J. Agric. Sci. 115,269. Morrow, P. E., Hodge, H. C, Neuman, W. F., Maynard, E. A., Blanchet, Jr., H., Fassett, D. W., Birk, R. E., and Manrodt, S. (1952). J. Pharmacol. Exp. Therap. lOS, 273. Muntifering, R. B., Smith, S. I., and Boling, J. A. (1984). J. Anim. Sci. 59, 1100. Murphy, M. D., and Quirke, W. Q. (1997). Ir. J. Agric. Food Res. 36, 31. Muth, O. H., and Oldfield, J. E. (eds.) (1970). "Symposium: Sulfur in Nutrition." AVI, Westport, CT. NRC (1976). "Urea and Other Nonprotein Nitrogen Compounds in Animal Nutrition." NAS -NRC, Washington, D.C NRC. (1980). Mineral Tolerance of Domestic Animals. National Academy of Sciences - National Research Council, Washington, D.C. NRC Nutrient Requirements of Domestic Animals. National Academy of Sciences - National Research Council, Washington, D.C (1981) Nutrient Requirements of Goats. (1985) Nutrient Requirements of Sheep, 5th Ed. (1989) Nutrient Requirements of Horses, 5th Ed. (1994) Nutrient Requirements of Poultry, 9th Ed. (1996) Nutrient Requirements of Beef Cattle, 7th Ed. (1998) Nutrient Requirements of Swine, 10th Ed. (2001) Nutrient Requirements of Dairy Cattle, 7th Ed. O'Connor, G. A., Brobst, R. B., Chaney, R. L., Kincaid, R. L., McDowell, L. R., Pierzynski, G. M., Rubin, A., and Van Riper, G. G. (2001). J. Environ. Qual. 30, 1490. Paterson, D. W., Wahlstrom, R. C, Libal, G. W., and Olson, O. E. (1979). J. Anim. Sci. 49, 664. Peck, H. D., Jr. (1970). In "Symposium: Sulfur in Nutrition." (0. H. Muth and J. E. Oldfield, eds.), Avi Pub!. Co., Corvallis, OR. Perdomo, J. T., Shirley, R. L., and Robertson, W. K. (1966). Soil Crop Sci. Soc. Fla. Proc. 26, 131. Price, J., Will, A. M., Paschaleris, G., and Chesters, J. K. (1987). Br. J. Nut. 58, 127. Pumphrey, F. V., and Moore, D. P. (1965). Agron. J. 57, 364. Qi, K. (1988). Chinese J. Sheep Goat Sci. 1, 15. Qi, K. (1992). J. Anim. Sci. 70, 2828. Qi, K., Lu, C D., and Owens, F. N. (1992). J. Anim. Sci. 70(Supp!. 1),302. Qi, K., Owens, F. N., and Lu, C. D. (1994). Small Rum. Res. 14, 115. Rakes, A. H., and Clark, A. K. (1984). In "Proceedings, Florida Nutrition Conference," p. 153. Univ. of Florida, Gainesville, FL. Rao, T. V. S., Anandan, S., Dey, A., Nandi, S., Harbola, P. C, and Asgola, D. (1999). Small Rum. Res. 31, 19. Rees, M. C., and Minson, D. J. (1978). Brit. J. Nutr. 39, 5. Rees, M. C, Minson, D. J., and Smith, F. W. (1974). J. Agric. Sci. 82, 419. Rendig, V. V., (1986). In "Sulfur in Agriculture," (M. A. Tabatabai, ed.), Amer. Soc. of Agron., p. 635. Madison, WI. Rendig, V. V. and Weir, W. C (1957). J. Anim. Sci. 16,451. Robel, E. J. (1976). Nutr. Rep. Int. 14, 147. Ryan, J., McKillen, M., and Mason, J. (1987). Ann. Rech. Vet. 18,47. Sasse, C E, and Baker, D. H. (1974). Poult. Sci. 53, 652. Schubert, J. R., Muth, O. H., Oldfield, J. E., and Remmert, L. F. (1961). Fed. Proc. 20,689.

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Schwarz, K., and Foltz, C. M. (1957). J. Amer. Chern. Soc. 78, 3292. Short, S. B., and Edwards, W. C. (1989). Vet. Human Toxicol. 31, 451. Shrift, A. (1958). Bot. Rev. 24, 550. Shurson, J., Whitney, M., and Nicolai, R. (1999). Feedstuffs 71(4),12. Slyter, L. L., Chalupa, W., and Oltjen, R. R. (1988). J. Anim. Sci. 66, 1016. Slyter, L. L., Chalupa, W., Oltjen, R. R., and Weaver, J. M. (1986). J. Anim. Sci. 63, 149. Smart, M. E., Cohen, R., Christensen, D. A., and Williams, C. M. (1986). Can. J. Anim. Sci. 66, 669. Smith, J. T. (1973). J. Nutr. 103, 1008. Stanford, G., and Jordan, H. V. (1966). Soil Sci. 101, 258. Stobbs, T. H., and Minson, D. J. (1980). In "Digestive Physiology and Nutrition of Ruminants" (D. C. Church, ed.), p. 357. 0 & B. Books, Corvallis, OR. Sturman, J. A., Rassin, D. K., and Gaul\, G. E. (1976). Pediatr. Res. 10,415. Tabatabai, M. A. (ed.) (1986). "Sulfur in Agriculture." Amer. Soc. Agron. Madison, WI. Taylor, C. D., and Wolfe, R. S. (1974). J. Bioi. Chern. 249, 4879. Thomas, W. E., Loosli, J. K., Williams, H. H., and Maynard, L. A. (1951). J. Nutr. 43, 515. Tiffany, M. E., McDowell, L. R., O'Connor, G. A., Martin, F. G., Wilkinson, N. S., Cardoso, E. C., Percival, S. S., and Rabiansky, P. A. (2000). J. Anim. Sci. 78, 1331. Tiffany, M. E., McDowell, L. R., O'Connor, G. A., Martin, F. G., Wilkinson, N. S., Percival, S. S., and Rabiansky, P. A. (2002). J. Anim. Sci. 80, 260. Underwood, E. J., and Suttle, N. F. (1999). In "The Mineral Nutrition of Livestock" (3rd Ed.), Midlothian, UK. van Ryssen, J. B. J., van Malsen, P. S. M., and Hartmann, F. (1998). J. Agric. Sci. (Camb.) 130, 107. Van Vleet, J. F. (1982). Am. J. Vet. Res., 43, 851. Veenhuizen, M. F., and Shurson, G. C. (1992). J. Am. Vet. Med. Assoc. 201,487. Wagner, J. J., Loneragan, G. H., Gould, D. H., and Thoren, M. (1998). J. Anim. Sci. 751(Suppl. 1),272. Weston, R. H., Lindsay, J. R., Purser, D. B., Gordon, G. L. R., and Davis, P. (1988). Austr. J. Agric. Res. 39, 1107. Whanger, P. D. (1972). World Rev. Nutr. Diet 15,225. Whanger, P. D., and Matrone, G. (1967). Biochim. Biophys. Acta. 136,27. Whiting, F., Sen, S. B., Bezeau, L. M., and Clark, R. D. (1954). Can. J. Agric. Sci. 34, 261. Williams, A. J., Robards, G. E., and Saville, D. G. (1972). Austr. J. Bioi. Sci. 25, 1269. Young, L., and Maw, G. A. (1958). "The Metabolism of Sulphur Compounds", John Wiley & Sons, New York. Zinn, R. A., Alvarez, E., Monano, M., and Ramirez, E. (1999). J. Anim. Sci. 77(Suppl. 1),115.

Chapter 7

Iron

I. INTRODUCTION

Iron (Fe) deficiency is one of the most common deficiency diseases of swine and humans. Baby pigs fed only milk and reared in concrete-floored pens are highly vulnerable to Fe deficiency. Iron deficiency is one of the most common human deficiency diseases in the world. More people suffer from Fe deficiency anemia than any other nutrient deficiency. In many developing countries, nearly 40% of the female population is anemic. Females are most likely deficient owing to menstrual Fe losses and added requirements for pregnancy. Recent studies indicate that excess Fe can be a problem for humans who are beyond the growth and reproductive years. For livestock other than baby pigs, Fe deficiency is rarely a practical concern, except in circumstances involving blood loss or disturbances resulting from parasitic infestation or disease.

n, mSTORY From antiquity, man has recognized the special role of the metal Fe in health and disease (Loosli, 1978; Yip and Dallman, 1996; Beard and Dawson, 1997). Iron compounds were used by the Egyptians as early as 1500 BC, and by the Hindus, Greeks, and Romans somewhat later. The early Greeks recognized anemia and treated it by drinking water in which a sword had been allowed to rust. Hippocrates used Fe therapeutically for such diverse maladies as dermatitis, hemorrhoids, wounds, gout, diarrhea, vomiting, weakness, edema, fever, and cystitis. Guggenheim (1995) has reviewed the historical significance of chlorosis as it related to Fe deficiency. Chlorosis (hypochromic anemia) was a name used in 1615, from the Greek word chloros (meaning green), as patients had a greenish tint to their skin. It was also called green sickness or weak blood. The first description of hypochromic anemia was in 1554, when Lang described a girl who was weak and her face, which in the last years was distinguished by rosiness of cheeks and redness of lips, is somehow as if exsanguinated, sadly paled, the heart trembles with every movement of her body, trouble dancing and stair climbing. Lang called the disease Morbus virgineus, a condition peculiar to virgins. Clark (1887) was often struck by frequent occurrences of anemia or chlorosis in girls... among the rich and poor, 203

Iron

204

among the cultured and the rude ... among the idle and occupied ... independent of country and town. Clark also believed wearing tight corsets may have displaced the abdominal organs and possibly, purely by mechanical pressure, decreased the volume of food eaten. Also, the bowels became either obstructed, confined, or inadequately relieved. In the sixteenth century, Monarde was the first to suggest a relationship between Fe and blood. Menghini drew attention to the Fe content of blood by lifting particles of dried, powdered blood with a magnet (Yip and Dallman, 1996). The role of Fe in blood formation became apparent in the seventeenth century when it was shown that Fe salts were of value in the treatment of Fe-deficiency anemia in young women. In 1664, Syndenham showed that administration of salts of Fe would restore the pink color in the cheeks of people suffering from anemia. The mechanism involved was not understood until Zinoffsky discovered in 1886 that hemoglobin (horse) crystals contained 0.335% Fe. Similar concentrations of Fe were demonstrated for a range of animal species. Convincing proof that inorganic Fe could be used for hemoglobin synthesis came in 1932 from Castle and co-workers, who found that the amount of Fe given parenterally to patients with hypochromic anemia corresponded closely to the amount of Fe in circulating hemoglobin (Yip and Dallman, 1996). Boussingault (1872) was probably the first to recognize Fe as a nutrient for animals, and he determined the Fe content of carcasses of a number of species and of food sources. In 1981, Braasch described anemia in suckling pigs; however, McGowan and Chrichton (1923) first published evidence associating Fe deficiency with baby pig anemia. They successfully treated the anemia with large doses of ferric oxide. For many years, nutritional interest in Fe was focused on its role in hemoglobin formation and oxygen transport. Keilin and others established the presence of Fe in the hemeprotein enzymes, the cytochromes, and the role of these enzymes in the oxidative mechanisms of all cells (Underwood and Suttle, 1999).

m.

CHEMICAL PROPERTIES AND DISTRIBUTION

Iron is the second most abundant metal in the earth's crust after aluminum, about 5%. Most plant materials used in the feeding of farm animals contain large and variable concentrations of Fe. Iron is silvery-white or gray and is somewhat magnetic. It holds magnetism only after hardening (as alloy steel). It is stable in dry air but readily oxidizes in moist air, forming rust (chiefly oxide, hydrated). Iron is alloyed with other elements in forming steel. Iron is a transition metal with an atomic weight of 56. It has two stable oxidation states (+ 2 and + 3) and several unstable oxidation states in aqueous solutions, and widely variable redox potentials depending on the ligands (Conrad et al., 1980). A special property of Fe is how easily it changes between the two oxidation states, Fe2 + (ferrous) and Fe3+ (ferric). This property makes Fe complexes useful in electron-transfer reactions.

Metabolism

205

Iron is a component of every living organism. The Fe content of animals varies from birth to maturity. Adult humans (70 kg) are estimated to contain 4 to 5 g Fe or 60 to 70 ppm of the whole body (Bothwell et al., 1979). Most body Fe exists in complex forms bound to protein, either as porphyrin or heme (Fig. 7.1) compounds, particularly hemoglobin and myoglobin, or as nonheme protein-bound complexes such as transferrin, ferritin, and hemosiderin (see Sections IV and V).

IV. METABOLISM

A. Absorption and Transport

Various aspects of Fe metabolism are reviewed by Yip and Dallman (1996), Beard and Dawson (1997), and Brody (1999). A schematic outline of Fe metabolism is shown in Fig. 7.2. Animals have a limited capacity to excrete Fe; Fe homeostasis in the body is largely controlled by absorption. The absorption of Fe is affected by (1) the age, Fe status, and state of health of the animal or individual; (2) conditions within the gastrointestinal tract; (3) the amount and chemical form of the Fe ingested; (4) the amounts and proportions of various other components of the diet, both organic and inorganic; and (5) genetic control, at least for excess absorption (Burk et al., 2001). Iron is poorly absorbed, but is better absorbed from animal (heme form) than from plant foods (non-heme forms) (South et al., 2000; Lynch, 2002). Generally, as the level of dietary Fe increases, the percentage absorbed decreases (Wood and Han, 1998). The absolute amount may increase as dietary levels increase, but the Fe status of the animal has a greater influence on the amount of Fe absorbed (Van Campen, 1974). In Fe-deficient rats, 80% of the dietary Fe was absorbed, while in Fe-adequate rats, only 7 to 10% was absorbed. For adult humans, only 5 to 15% of food Fe is absorbed from ordinary mixed diets, but may increase to twice this level or more in children and in cases of Fe deficiency. Josephs (1958) reported that

HOOC-CHiCHi

CH, Fig. 7.1 The structure of the iron protoporphyrin complex heme.

206

Iron

Urine, Bile, Feces

Fig.7.2 Iron metabolism. (Courtesy of M.L. Scott, Cornell University, Ithaca, NY)

2 to 20% of an oral dose of radio-Fe was absorbed in normal human subjects, compared with 20 to 60% in patients with Fe-deficiency anemia. Iron absorption occurs throughout the gastrointestinal tract. The major sites are the duodenum and jejunum. Although not absorbed in the stomach, the stomach contributes hydrochloric acid, which not only helps to remove protein-bound Fe by protein denaturation but also helps in Fe solubilization and reduction of ferric Fe to the ferrous state. Iron is absorbed in the ferrous state, in the ferric form in feed, and also in combination with organic compounds. Ascorbic acid and cysteine in food may aid in reduction of Fe from the ferric to the ferrous state and enhance Fe absorption (Cook and Reddy, 2001). The mucosal block theory (Hahn et aI., 1943) states that only enough Fe is absorbed in normal animals to meet needs, and is rejected when stores are adequate. Iron taken into mucosal cells is converted into ferritin, and when the cells become physiologically saturated with ferritin, further absorption is impeded until the Fe is

Metabolism

207

released from ferritin and transferred to plasma. This theory has been modified since the ultimate regulator of Fe absorption appears to be the Fe concentration in the mucosal epithelial cells of the duodenum. Decreased mucosal Fe induces an increase in the intestinal Fe absorption in the early stage of Fe-deficiency. Thus, Fedeficient animals absorb ingested Fe almost directly into the blood with very little remaining in the musosal cells; Fe-adequate animals transfer only a small portion of Fe absorbed by mucosal cells to the blood (Conrad and Crosby, 1963). Iron absorption is favored by more acid conditions. Normal gastric secretion is necessary for optimal absorption of Fe by rats (Murray and Stein, 1970). Inorganic Fe forms complexes with normal gastric juice at a low pH. These complexes remain soluble when the pH is raised to neutrality. Decreased stomach acidity, due to overconsumption of antacids, ingestion of alkaline clay, or pathologic conditions such as achlorhydria or partial gastrectomy, may lead to impaired Fe absorption (Beard and Dawson, 1997). Absorption of nonheme forms of Fe is greatly influenced by various dietary chelates. Ascorbic acid promotes Fe absorption, and ethylenediaminetetraacetic acid (EDTA) inhibits its absorption. Histidine, lysine, and cysteine enhance ferrous Fe uptake. High dietary levels of phosphorus (P) reduce Fe absorption, presumably by the formation of insoluble ferric phosphate and phytate. Dietary phytate reduces bioavailabilities of Fe and other minerals and thus absorption (Stahl et al., 1999; Kamao et al., 2000) while a phytase will release Fe and other minerals from this complex (Stahl et al., 1999). High dietary levels of copper (Cu), manganese (Mn), lead (Pb), and cadmium (Cd) increase Fe requirements by competing for absorption sites in the intestinal mucosa. The enterocyte is a highly specialized, polarized, absorptive cell found on the intestinal villus that controls the passage of dietary Fe into the body. The passage of Fe through the enterocyte entails transport of the metal across the following three formidable cellular barriers: the apical membrane, intracellular translocation across the cytosol, and release of Fe across the basolateral membrane and thence into the circulation (Wood and Han, 1998). Ferrous Fe entering the blood plasma is quickly oxidized to the ferric state. The ferric form immediately complexes with a specific B1-globulin (transferrin), and is transported throughout the body. Plasma transferrin links various cycles of Fe metabolism and thus regulates body Fe distribution. Transferrin accepts Fe that is absorbed from the intestinal tract and released from sites of storage and from hemoglobin destruction. The second phase of transferrin-Fe transport is delivery to the bone marrow for hemoglobin synthesis, to the placenta for fetal needs, and to cells for Fe-containing enzymes. More than 70% of plasma Fe turnover goes to erythroid cells in bone marrow for hemoglobin synthesis. ' The discovery of iron regulatory proteins (lRPs) has provided a molecular framework from which to more fully understand the coordinate regulation of Fe metabolism. Cellular Fe homeostasis is achieved through the controlled synthesis of several proteins involved in the movement, storage, and utilization of Fe (Piero, 2001). These IRPs bind to Fe-responsive elements in specific mRNAs and regulate their utilization. Because IRPs are key modulators of the uptake and metabolic fate

208

Iron

of Fe in cells, they are focal points for the modulation of cellular Fe homeostasis in response to a variety of agents and circumstances (Eisenstein and Blemings, 1998). Placental Fe transport is unidirectional and increases rapidly as pregnancy progresses. In animals with the hemochorial type of placenta, which includes the rat, rabbit, guinea pig, and human, the rate of Fe transfer across the placenta from maternal plasma transferrin is sufficient to account for all Fe accumulated by the fetus (Bothwell et al., 1979). A progesterone-inducible purple protein possessing phosphatase activity, uteroferrin, has been isolated from porcine placental tissue and is proposed to have a role in transfer of Fe to the fetal piglet (Ducsay et al., 1984). Hemoglobin Fe conservation is achieved by reticuloendothelial cells of the liver, spleen, and bone marrow, which recruit Fe from senescent or nonviable erythrocytes. They phagocytize the erythrocytes for the purpose of breaking down hemoglobin and releasing Fe. Released Fe is either rapidly returned to the circulation via plasma transferrin or held in a slowly exchanging pool of storage Fe in the reticuloendothelial cell (Fillet et al., 1974). This system of Fe reutilization is an efficient mechanism whereby a constant source of Fe is available daily for resynthesis of body hemoglobin.

B. Storage Ferritin concentration in tissues, together with that of hemosiderin, reflects the animal's Fe status. These Fe storage compounds are present primarily in the liver, reticuloendothelial cells, and bone marrow. The liver contains about 60% of the body ferritin (Beard and Dawson, 1997). Apoferritin, ferritin without Fe, is synthesized in the presence of a positive Fe balance and becomes loaded with Fe deposits of ferric hydroxide and ferric phosphate. Ferritin is a nonheme protein (globulin) compound (containing up to 20% Fe), which is present throughout the body and particularly in the liver. A high positive correlation exists between human serum ferritin concentrations and body Fe stores (Walters et al., 1973). Hemosiderin is relatively amorphous, containing as much as 35% Fe primarily as colloidal ferric hydroxide with very little protein (Shoden and Sturgeon, 1961). In most species, hemosiderin is the predominant form at high tissue levels, and ferritin predominates at lower levels. Exchange of Fe from ferritin to transferrin is reversible, giving the body access to its Fe stores and permitting redistribution of body Fe. This process involves the reduction of ferritin-Fe'' " and can be accomplished by riboflavin, ascorbic acid, glutathione, or cysteine, and to a lesser extent by zanthine oxidase (acting as a dehydrogenase) (Conrad et al., 1980). Mobilization of Fe from Fe stores also requires the Cu-containing enzyme of the plasma ceruloplasmin (ferroxidase I), as discussed in Chapter 8. Iron storage in the newborn is influenced by the maternal diet during gestation; most storage occurs late in gestation. If the number of young born is larger than usual, for example, twins in humans and extra-large litters in hogs, the individuals's supply tends to be smaller. Even if the store is normal, a long nursing period

Physiological Functions

209

without supplementary Fe-rich food will exhaust it (Maynard et al., 1979; Yip and Dallman, 1996). C. Excretion and Blood Loss Absorbed Fe is retained with great tenacity and therefore is not readily lost from the body except through hemorrhage. Iron is released from hemoglobin during erythrocyte breakdown, carried to the liver, and secreted in the bile. Most bile Fe is reabsorbed and used again to form hemoglobin. Although absorbed Fe is retained with great tenacity and, in the absence ofbleeding, excretion is very small, the amounts lost are of nutritional importance, particularly for growing or pregnant animals. Iron losses from bleeding from injury or menstrual flow can be substantial. The normal menstrual flow is about 35 ml per period. This is equivalent to about 18 mg Fe, as blood contains 0.5 mg of Fe/ml. Excessive menstrual blood loss is the most common cause of Fe deficiency in women. The upper limit of the normal period is about 80 ml; excessive losses may rise over 200 ml per period (Brody, 1999). Iron is excreted in the feces and urine, in addition to losses through sweat, hair, and nails. Most of the total Fe present in feces is nonabsorbed food Fe; probably less than 3% is endogenous Fe. Although Fe excreted in feces and urine is the major excretory loss, there is a continual dermal loss in the sweat, hair, and nails. Most of this occurs in desquamated cells, but cell-free sweat contains some Fe.

V. PHYSIOLOGICAL FUNCI'IONS

Iron plays a key role in many biochemical reactions. It is present in several enzymes responsible for electron transport (cytochromes), for activation of oxygen (oxidases and oxygenases), and for oxygen transport (hemoglobin and myoglobin). The cytochrome system is a series of reactions in which oxidation occurs with production of adenosine triphosphate (ATP) and formation of water. Iron has oxidation-reduction activity and transports electrons. Bound Fe changes oxidation state and functions at the active sites of numerous oxidation-reduction enzymes and oxygen-binding proteins. Iron exists in the animal body mainly in complex forms bound to protein (hemoproteins) as heme compounds (hemoglobin or myoglobin), as heme enzymes (mitochondrial and microsomal cytochromes, catalase, and peroxidase), or as nonheme compounds (flavin-Fe enzymes, transferrin, and ferritin). Lactoferrin is an Fe-containing glycoprotein secreted by mammary cells that has an important role as an antibiotic agent in the gland (Troost et al., 2002). Calves fed lactoferrin consumed more calf starter and improved performance (Joslin et al., 2002). The hemoproteins contain Fe in which four of the six coordination positions around the Fe atom are occupied by the nitrogen atoms of a porphyrin, most frequently protoporphyrin (Fig. 7.1). Hemoglobin and the catalases contain four heme groups per molecule, whereas myoglobin, the cytochromes, and peroxidases contain one heme group per molecule.

210

Iron

The biosynthesis of heme begins with synthesis of d-aminolevulenic acid, the precursor to porphyrins, and ends with incorporation of Fe 2 + into the porphyrin ring by the enzyme ferrochetalase. Heme synthesis is impaired in Fe deficiency because of substrate insufficiency for ferrochetalase, and also in Cu deficiency owing to decreased activity of the Cu-dependent enzyme cytochrome oxidase, which reduces Fe3+ to Fe2+ before incorporation into the porphyrin molecule (Williams et al., 1976). Hemoglobin (blood) Fe represents approximately 60% of total body Fe, whereas myoglobin represents about 4% of total Fe (Brody, 1999). The molecular weight of hemoglobin is 68,000, and each molecule contains four atoms of Fe. Hemoglobin is a tetramer composed of four globin moieties each containing a heme unit bound loosely by noncovalent bonding of Fe and the imidazole nitrogen of a histidine residue in each protein chain. Myoglobin is an Fe-porphyrin protein of one Fe atom per molecule, with a molecular weight of about 17,000. It is present in muscle cells and has a higher affinity for oxygen than does hemoglobin, facilitating the transfer of oxygen from oxyhemoglobin to sites of oxidation in muscle cells (Fruton and Simmonds, 1958). Hemoglobin is compartmentalized in red blood cells (erythrocytes), and accounts for over 90% of the total protein of those cells (Davies, 1961). Erythrocytes are formed in the bone marrow; this process is called hematopoiesis. Erythrocytes are continuously destroyed and replaced, with an average life span of 120 days. Iron released by the normal bloodcell destruction can be used again to form hemoglobin, practically without loss. Hemoglobin binds oxygen; after release of its oxygen to the tissues, hemoglobin binds carbon dioxide in the venous blood. Carboxyhemoglobin releases carbon dioxide in the lung in exchange for oxygen. Enzymes containing Fe include catalase, cytochrome A, B, and C, lactoperoxidase in milk, and verdoperoxidase in leucocytes, succinate dehydrogenase, nicotinamide-adenine dinucleotide, reduced (NADH)-coenzyme QIO reductase, a phosphatase in swine uterine fluid, and glutamate formimino-transferase (Fruton and Simmonds, 1958; Conrad et al., 1980). Enzymes activated by Fe ions include tryptophan peroxidase-oxidase, aconitase, homogentisic oxidase, hyroxyanthranilate cleavage enzyme, phenylalanine hydroxylase, and histidine decarboxylase (Fruton and Simmonds, 1958). Catalase and peroxidase enzymes break down peroxide molecules in the presence of reducing agents. In normal feather pigmentation for certain breeds of poultry and coloration in mink, Fe may be an essential component of an enzyme involved in melanin formation. The cytochromes contain heme as the active site, with the Fe-porphyrin ring functioning to reduce ferrous Fe and ferric Fe with the acceptance of electrons. The ability of Fe to change between the divalent and trivalent state allows the cytochromes A, B, and C to participate in the electron transfer chain. The cytochromes function as electron carriers, linking the oxidation of substrate with the reduction of molecular oxygen in aerobic metabolism. Iron plays a significant role in the tricarboxylic acid (Krebs) cycle, as all of the 24 enzymes in this cycle contain Fe either at their active centers or as essential cofactors.

Requirements

211

Iron functions have been shown to be related to normal thyroid hormone production (Beard et al., 1989) and disease resistance (Weinberg, 1984; Kuvibidila et al., 2001). Iron is needed for learning and cognitive function in laboratory animals (Kwik-Uribe et al., 2000; Piero et al., 2001), monkeys (Golub et al., 2000) and humans (Krebs, 2000; Youdim, 2001). Some behavioral consequences of Fe deficiency relate to dopamine transporter functioning (Hunt et al., 1994; Erikson et al., 2000).

VI. REQUIREMENTS The net requirements for Fe are the sum of the amounts laid down in the blood and tissues in the process of growth and the amounts lost in feces, urine, and sweat, in blood loss, in parturition, and in milk and eggs. Conversion of physiological requirements into dietary requirements is made difficult by variations among individuals in absorptive capacity and among foods and food combinations in the Fe bioavailability. The position is further complicated by the ability of the body to increase Fe absorption during Fe deficiency (Brody, 1999; Underwood and Suttle, 1999). Iron requirements are influenced by the chemical form or combination in which the mineral is ingested and by the amounts and proportions of other components of the whole diet. Feeds of animal origin such as meat meal and fish meal are better sources of Fe per unit than feeds of plant origin. The availability of Fe in plant foods such as beans, peas, corn, bread, and rice are poor ranging from less than 1 to 10% (Brody, 1999). However, the non heme in meat, fish, chicken, and liver may be about 20% available. The Fe of simple Fe salts is better absorbed than the Fe in ordinary feedstuffs (see Sections V and IX). High levels of dietary P and phytate reduce Fe absorption, presumably by the formation of insoluble ferric phosphate and phytate; and high dietary levels of several divalent metals, notably Cu, Mn, Pb, and Cd, increase Fe requirements by competing for absorption sites in the intestinal mucosa (Underwood and Suttle, 1999). Estimated Fe requirements for various animal species and humans are presented in Table 7.1. The suggested Fe dietary (dry basis) requirement for ruminants is between 15 and 50 ppm. Young animals have higher requirements than do adults. Young ruminants fed on exclusive whole-milk diets (milk is low in Fe) can develop Fe-deficiency anemia within 2 to 3 months. Although quite variable, reserves of the calf generally are sufficient to prevent serious anemia if dry feeds are fed beginning in the first few weeks. Swine have the greatest problem maintaining Fe status because of low stores at birth and rapid growth rate. Because of the definite need to supply Fe to baby pigs born in confinement (see Sections VIII and IX), more accurate requirements are available for this class of animal than for other livestock species. The Fe requirements of swine decrease with age because of a decrease in the blood volume per unit of body weight and increased feed consumption (NRC, 1998). There is a large Fe requirement in chickens during high egg production. A chicken egg

Iron

212

TABLE 7.1 Iron Requirement for Various Species" Species

Purpose

Requirement

Reference

Chickens

Leghorn-type (}-6 wk Leghorn-type 6-18 wk Leghorn-type laying Leghorn-type breeding Broilers-all classes All classes All classes All classes Growing All classes All classes Growing Breeding, lactating Channel Catfish Growing All classes All classes Growing Adults, male Adults, female Adults, pregnant

80 mg/kg 60 rng/kg 50 mg/kg 60 mg/kg 80 mg/kg 60-120 rug/kg 50-80 mg/kg 50 mg/kg 15--43 mg/kg 30-50 mg/kg 40-50 mg/kg 40-100 mg/kg 80 rug/kg 30 mg/kg 80 mg/kg 35 mg/kg 35 mg/kg 50 rug/kg 8-11 mg/day 8-18 mg/day 27 rug/day

NRC (1994) NRC (1994) NRC (1994) NRC (1994) NRC (1994) NRC (1994) NRC (1994) NRC (1996) NRC (2001) NRC (1985b) NRC (1989) NRC (1998) NRC (1998) NRC (1993) NRC (1986) NRC (1995) NRC (1995) NRC (1995) DRI (2001) DRI (2001) DRI (2001)

Japanese quail Turkeys Beef cattle Dairy cattle Sheep Horses Swine Fish Cats Rats Mice Guinea pigs Humans

"Expressed as per unit animal feed either on as-fed (approximately 90% DM) or dry basis (see Appendix Table I).

contains about 1.1 mg Fe. A minimal Fe dietary requirement of 35 to 45 ppm was needed for laying hens to maintain a normal hematocrit, but for optimum hatchability, was somewhat higher at 55 ppm (Leeson and Summers, 2001). Typical diets (except milk) are generally high enough in Fe to supply requirements for farm livestock. During the reproductive years, adult women lose an average of 20 mg Fe monthly as a result of menstruation and approximately 800 mg Fe for each pregnancy. These losses are in addition to an approximately I mg daily Fe loss from exfoliation of intestinal epithelial cells (Fairbanks, 1978). Daily Fe requirements are 8 to II mg for adult males, 8 to 18 mg for females, 27 mg for pregnant females, and 6 to 12 mg for children (DRI, 2001).

VII. NATURAL SOURCES The Fe content of most feed ingredients is highly variable, reflecting differences in soil and climatic conditions as well as differences in variety or processing procedures. The Fe level in herbage plants is basically determined by the species and type of soil on which the plants grow and can be greatly affected by soil

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TABLE 7.2 Mean Forage Iron Concentratlons" Country Argentina"

Colombia" Dominican Republic" Bolivia" Guatemala' Malawi"

Season

Mean (ppm)

Critical concentration (cc)(%)

Percentage below cc

Wet Wet Dry Dry Wet Dry Wet Dry Wet Dry

384 139 171 154 \78 \22 378 661 207 195

50 30 30 30 30 30 30 30 50 50

3 0 0 0 0 0 0 0 6 3

'Critical concentrations are based on ruminant needs (McDowell, 1985). "Based on 340 samples (Balbuena et al., 1989). 'Based on 35 and 36 samples for the wet and dry seasons, respectively (Vargas et al., 1984). dBased on 69 samples (Jerez et al., 1984). "Based on 16 samples for the wet season (McDowell et al., 1982) and 84 for the dry season (Peducasse et al., 1983). 'Based on 84 samples for both the wet and dry seasons (Tejada et al., 1987). sBased on 48 and 21 samples for the wet and dry seasons, respectively (Mtimuni et al., 1990).

contamination. Mitchell (1963) indicated that soil contains 20 to 100 times the Fe content found in pastures grown on that particular soil. Acid soil conditions favor availability and plant uptake of Fe. Even plants grown on neutral or slightly alkaline soils often contain quite high levels of Fe. Typical Fe contents of livestock feedstuffs are given in Appendix Table II. Most forages contain Fe concentrations considerably in excess of the requirements of herbivorous animals (Table 7.2). Beeson (1941) reported that cultivated grasses and legumes range from 100 to 700 ppm Fe, although values in excess of 1000 ppm have been noted. Of 256 forage averages in the 1974 Latin American Tables of Feed Composition, only 3.5% contained less than 30 ppm Fe (McDowell et al., 1974). Of 192 and 120 forage samples collected during the dry and wet seasons in northern Mato Grosso, Brazil, mean Fe concentrations were 212 and 263 ppm, respectively (Sousa et al., 1981). However, other reports from Panama note that 7 out of 28 locations averaged less than 30 ppm Fe for samples of Hyparrhenia rufa (Chicco, 1972). Most cereal grains contain 30 to 60 ppm Fe, and species differences appear to be small, although 10 and 20 ppm have been recorded for Egyptian-grown corn and barley, respectively (Abou-Hussein et al., 1970). Legumes and oilseeds are richer in Fe than the cereal grains and may contain 100 to 200 ppm Fe. Bioavailability of Fe in cereal grains and oil seeds is reduced because ofphytate in these feeds (Hurrell et al., 1992; Zhou and Erdman, 1995). Supplemental microbial phytase is highly effective in releasing phytate-bound Fe (Stahl et al., 1999). Feeds of animal origin, other than milk and milk products, are rich sources of Fe. Meat meals and fish meals commonly contain 400 to 600 ppm, and blood meals,

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Iron

more than 3000 ppm Fe (Morris, 1987). Dried skimmed milk, whey, and buttermilk powders used in normal feeding practices vary greatly in Fe content because of variable contamination during processing and storage. In one study (Blaxter et al., 1957), milk, as it comes from the udder, varied from 0.18 to 0.31 ppm Fe. In contrast, values after the contamination of usual handling may be substantially higher, at an average of about 0.5 ppm (Underwood, 1977). In a study of Brahman beef cows, Fe concentration in colostrum milk was relatively high at 1.04 ppm, but declined to 0.51 after 3 months (Salih et al., 1987). Many of the minerals used to supply the Ca and P needs of animals contain Fe. Ground limestone, oyster shell, and many forms of calcium phosphate used as mineral supplements frequently contain 200 to 500 ppm Fe. Drinking water can also be a nutritionally important source of Fe, with considerable variation in Fe content (see also Chapter 17 of this volume). The richest Fe sources for humans are the organ meats (liver and kidney), egg yolk, dried legumes, cocoa, cane molasses, and parsley. Poor sources include milk and milk products, white sugar, white flour and bread (unenriched), polished rice, potatoes, and most fresh fruit (Morris, 1987). Boiling in water can reduce the levels of Fe in vegetables by as much as 20% (Skeets et al., 1931), while milling lowers the Fe content in white flour. Only limited information is available on Fe bioavailability of natural feed sources. Feedstuffs in which Fe is complexed to heme (i.e., animal by-products) show better biological availability than to those containing nonheme Fe (e.g., chlorophyll-poor plant ingredients) (see Section 6). However, meat was also shown to enhance nonheme Fe absorption in swine diets (South et al., 2000). Swain et al. (2002) suggest that enhancement of nonheme Fe by beef may be due to peptides produced during gastrointestinal digestion and that histidine content may be important. Fritz et al. (1970) found that Fe in fish-protein concentrates had relative biological values ranging from 8 to 53%. Relative biological values for animal byproduct feedstuffs may be estimated to be 50 to 60%, and probably higher for blood meals (Conrad et al., 1980).

VIII. DEFICIENCY

A. Effects of Deficiency Iron deficiency affects many systems through the reduction in tissue oxygenation resulting from decreased hemoglobin concentration. Anemia may occur whenever the available supply of Fe becomes deficient relative to the needs for hemoglobin formation. Signs of a lack of Fe, in addition to anemia and related blood changes, include lower weight gains, listlessness, inability to withstand circulatory strain, labored breathing after mild exercise, reduced appetite, and decreased resistance to infection. For humans and other species, Fe deficiency in the young can result in learning and cognitive limitation as well as behavioral consequences. Iron

Deficiency

215

deficiency is to be expected primarily in young, rapidly growing animals that have limited access to Fe in their environment and in their feed, and particularly during the suckling period, since milk is very low in Fe. Iron deficiency is of limited practical significance in farm animals other than suckling pigs. For humans, it is one of the most common deficiencies in both industrialized and developing countries. 1.

SWINE

Piglet anemia is an uncomplicated Fe deficiency, completely preventable by farrowing piglets in conditions that permit access to soil or pasture, or by direct administration of supplemental Fe to the newborn pigs (Kleinbeck and McGlone, 1999). Piglets denied access to sources of Fe other than sows milk develop anemia within 2 to 4 weeks of birth. The anemia is typically described as a hypochromicmicrocytic type. Blood hemoglobin levels fall from a normal of about 10 g/dl to as low as 4 g/dl, Mortality at this time is high, but surviving piglets begin a slow spontaneous recovery at 6 to 7 weeks, when they begin to eat the sows food and undertake foraging. Iron deficiency in the nursing pig may vary from a borderline chronic anemia to acute anemia. One of the first signs of chronic anemia is a roughness of haircoat. The hair is dull, coarse, and stands erect. The skin becomes wrinkled (Fig. 7.3), and the normally pink mucous membranes became pale. Pigs are listless; the head and upper eyelids droop; the ears and tail hang limp. Subcutaneous edema may appear in neck, shoulder, and limb areas (Conrad et al., 1980), and anemic pigs do not have the characteristic pink ears and snout. This may be seen before any other signs are evident (Miller, 1981). A sign of a more acute anemia is labored breathing or a spasmodic movement of the diaphragm muscles following exercise, referred to as thumps. Fast-growing pigs

Fig.7.3 Iron deficiency. Left, anemic pig. Note listlessness and wrinkled skin. These signs, along with paleness about eyelids, ears, and nose, as well as low hemoglobin, are typical of baby pig anemia. Right, normal pig given iron. (Courtesy of H.D. Wallace, University of Florida, Gainesville)

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Iron

may die suddenly of anoxia. Iron deficiency may lower the resistance of the pig to disease, and respiratory problems and enteritis may appear more frequently in chronically anemic pigs. Many reports have also demonstrated a reduced rate of weight gain by anemic pigs (Hannon, 1971; Conrad et al., 1980). Necropsy findings further confirm Fe deficiency (Conrad et al., 1980). Clear fluid is found in body cavities; the heart is dilated, pale, and soft; the lungs are edematous and may be collapsed. The liver is enlarged, mottled, and infiltrated with fat. All organs have a lighter than normal color. The blood is thin and watery. The baby pig develops a physiological anemia caused by a rapid increase in blood volume within the first day and a half of life. This poor start is compounded by an Fe deficit brought about by a combination of the following factors: (1) unusually low Fe stores at birth, compared with the newborn of most other species; (2) absence of the polycythemia of birth common to other animal species; (3) low levels of Fe in sows milk; and (4) very rapid early growth rate compared to other mammalian species (Underwood and Suttle, 1999). Piglets normally reach four to five times their birth weight at the end of 3 weeks and eight times their birth weight by 8 weeks, imposing Fe demands much greater than can be supplied by the sow's milk. Such a rapid growth rate requires the retention of 7 to II mg Fe/day, whereas only about I mg/day is obtained from milk alone (Venn et al., 1947). The young pig is therefore highly dependent on endogenous sources of Fe when consuming only milk. Anemia can occur in older pigs fed diets very high in Cu to promote growth and increase the efficiency of feed use, unless these diets are supplemented with Fe well above levels that are otherwise adequate (Gipp et al., 1974). Gipp and co-workers (1974) showed that the hypochromic, microcytic anemia induced by high dietary Cu is the result of an impairment of Fe absorption; this impairment is ameliorated by ascorbic acid. Dietary Cu levelsup to 60 ppm had no effect on liver Fe concentration, but 120 ppm Cu resulted in 50% decrease in liver Fe (Bradley et al., 1983). Iron deficiency in growing pigs has been found when a high dietary concentration of cottonseed meal is fed (Kornegay et al., 1961). Gossypol, a toxic component of cottonseed meal, forms a chelate complex with Fe, thereby severely reducing Fe absorption. Iron supplementation (see Section IX) counteracts gossypol toxicity. 2.

POULTRY

Most practical feedstuffs contain sufficient Fe to meet the nutritional requirements for poultry. A number of studies, however, have clearly demonstrated the need for Fe supplementation of certain semipurified diets (Conrad et al., 1980; Leeson and Summers, 2001). Hill and Matrone (1961) found that chicks fed a skim milk-sucrose-based diet containing 7 ppm Fe experienced anemia associated with increased abnormally shaped erythrocytes. For both chicks and poults, Fe deficiency results in a hypochromic macrocytic anemia. Birds deficient in Fe have lowered growth rates and reduced hemoglobin and hematocrit concentrations (AI-Ubaidi and Sullivan, 1963). Hill and Matrone (1961) also observed achromatrichia among Fe-deficient Rhode Island Red chicks, which normally produce red-brown plumage. For

Deficiency

217

turkeys, AI-Ubaidi and Sullivan (1963) reported that the Fe-deficient Broad Breasted Bronze poults also showed poor feathering and impaired plumage pigmentation. The loss of the normal feather color in poultry was the result both of the loss of an Fe-containing red pigment and of impairment in melanin synthesis in the feathers. For mature poultry, low-Fe diets did not affect body weight; with deficient diets, there were significant reductions in blood hemoglobin and hematocrit concentrations (Morek, 1978). Also by 3 weeks, Fe-deficient hens were found to deposit less than 50% as much Fe in egg yolk as control hens. Although Fe deficiency was not associated with a marked or consistent effect on egg production, the 36 to 46 week-old Fe-deficient hens were reported to experience a decrease in egg production sooner than did hens fed the Fe-supplemented diet. Working with laying hens, heart hypertrophy was observed in birds fed less than 55 ppm (Aoyagi and Baker, 1995). Iron deficiency in breeding hens is characterized by embryonic mortality during the ninth through fifteenth days of incubation. The peak in mortality was associated with severe hypochromic anemia in embryos surviving to 10 days of incubation (Morek, 1978). The few chicks that survived to hatching were anemic and had only about one-half of the hematocrit and hemoglobin concentrations of day-old chicks from Fe-adequate hens. 3.

RUMINANTS

Iron deficiencies are most likely to occur in young ruminant animals because milk is low in Fe. Hibbs et al. (1961) reported that calves may be deficient in Fe at birth since 30% of those born in a 12-year U.S. study had low hemoglobin values « 9 gj100 ml). Calves fed milk diets may develop anemia within 8 to 10 weeks. Newborn calves receiving milk have increased hemoglobin formation as a result of supplemental Fe (Thomas et al., 1954). Experimentally, Fe-deficiency anemia has also been produced in milk-fed lambs and in lambs raised on slotted wooden floors and fed a semipurified diet (NRC, 1985b). Twin calves are more likely to develop anemia than single calves, because they compete for a limited maternal supply of Fe (Kume and Tanabe, 1994). Young calves fed an exclusive milk diet exhibit a microcytic, normochromic, or hypochromic anemia with marked decreases in liver nonheme Fe concentration and serum Fe (Furugouri, 1978). Clinical signs include anemia, lower weight gains, listlessness, inability to withstand circulatory strain, labored breathing after mild exercise, reduced appetite, atrophy of the papillae of the tongue, and blanching of visible mucous membranes. In addition to low performance, calves with Fe deficiency have high susceptibility to disease (Mollerberg et al., 1975). The incidence of scours is higher in anemic than in normal calves. With Fe treatment, anemic calves respond promptly in performance, and hematological and tissue parameters for Fe are restored. Light-colored veal is associated with low levels of muscle myoglobin and restricted Fe intakes. An objective in the rearing of veal calves is to produce light pink meat to satisfy a growing consumer demand in the United States. Meat from

218

Iron

anemic veal calves contains less hemoglobin Fe, myoglobin, and cytochrome C than that from normal calves. Hemoglobin levels can fall to 50% of normal even with normal growth (Underwood and Suttle, 1999). Thus, there is a conflict between healthy veal calves and veal that appeals to consumers (Furugouri, 1978). Heavy infestation with intestinal parasites results in Fe-deficiency anemia in lambs and calves (Campbell and Gardiner, 1960). Anemia can result from the direct loss of blood via blood-sucking parasites, increased rate of degradation of blood cells, and a depression of hematopoiesis from toxic substances produced by the parasites. Iron deficiency seldom occurs in older ruminants unless there is considerable blood loss from parasitic infestations or disease. Iron deficiency is considered rare for grazing livestock due to generally adequate pasture concentrations and contamination of plants by soil (see Chapter 17). Soil contamination of forages and direct soil consumption often provide excess quantities of dietary Fe. In New Zealand, annual ingestion of soil can reach 75 kg for sheep and 600 kg for dairy cows (Healy, 1974). Iron deficiency may be a problem when ruminants are fed low quality forages, such as straw, for extended periods (Sen and Ray, 1964). These investigators noted that Fe supplementation reduced weight losses in lactating cattle and produced more rapid gains in suckling calves. Iron deficiency has been reported in Florida (Fig. 7.4) when cattle grazed forages grown on white and gray, sandy loam and fine

Fig.7.4 This weak (Fe-deficient) 12-year-old cow, which was grazing on a Blanton fine sand (yellow) soil in Florida, had to be helped up. Her hemoglobin was only 4.8 g/ I00 ml of whole blood. After being given supplemental Fe (ferric ammonium citrate), the hemoglobin value increased to 12.6 g/IOO ml, and she regained body condition and strength. (Courtesy of the late R.B. Becker. University of Florida. Gainesville)

Deficiency

219

sand soils (Becker et al., 1965). When Florida cattle are pastured on light, sandy soils, and have heavy insect or parasite infestations, the additional Fe has been useful. The hemoglobin levels, as well as the condition of the animals, have improved under such treatment (Davis, 1951). Other investigators who fed supplemental Fe revealed no production benefit when older animals had been consuming typical diets. However, Fe deficiency exists and, in some cases, supplemental Fe has produced marked improvement (Thomas, 1970). 4.

HORSES

The primary signs of Fe deficiency in horses are microcytic and hypochromic anemia. In severe cases of anemia, the horse will breathe hard in an effort to get enough oxygen to the various tissues of the body, and to make the available hemoglobin carry as much oxygen as possible. Inadequate oxygen is especially critical to working or racing horses. An anemic horse is more susceptible to stress factors and diseases (Cunha, 1990) as well as reduced growth, scouring, and pneumonia. Anemic horses lack a healthy pink color, and their blood looks watery at necropsy. The horse also becomes weak, inactive, develops a rough hair coat, and tires very quickly. Although young, milk-fed foals are most susceptible to this anemia, Fe deficiency is not a practical problem in foals or mature horses at any performance level (NRC, 1989). An exception may occur when horses are heavily parasitized (Cunha, 1990). 5.

OTHER ANIMAL SPECIES

a. Dogs and Cats. For both puppies and kittens, a hypochromic, microcytic type of anemia is produced on low-Fe diets (NRC 1985a, 1986). After weaning, animals that receive a certain quantity of Fe-rich feeds (e.g., meat) should not develop anemia, unless blood loss is a problem.

b. Laboratory Animals. Iron deficiency results in a microcytic, hypochromic anemia in both rats and mice (NRC, 1995). Mice show reduced birth weights and litter sizes, and rats have white incisor teeth, cardiomegaly, splenomegaly, and enlarged cecum (Cusack and Brown, 1965). Black-haired rats fed a Fe- (or Cu-) deficient diet developed achromotrichia. Rats deficient in Fe have a compromised immune system including impaired phagocytosis and natural killer cell activity, and reduced antibody production (Hallquist et al., 1992). Mice that received a low Fe diet (2 to 10 ppm) for 30 days were characterized by low body weights, anemia, and suppressed T-Iymphocyte-dependent functions associated with antibody production (NRC, 1995). Marginal Fe-deficient mice demonstrated significantly lower grip strength and cognitive function (Kwik-Uribe et al., 2000). c. Rabbits. Iron deficiency in rabbits produces microcytic, hypochromic anemia (NRC, 1977). At birth, rabbits have a very large Fe reserve (liver), so the newborn

220

Iron

are not dependent on a supply of Fe in the milk. Iron deficiency is unlikely in rabbits under practical conditions owing to the generous distribution of the mineral in feedstuffs.

d. Faxes and Mink. In mink, Fe-deficiency anemia occurs if diets contain high levels of certain kinds of fish, e.g., coalfish, whiting, blue whiting, and hake (Furugouri, 1978). These diets cause an impairment of Fe absorption following overt Fe-deficiency anemia. Clinical signs of Fe deficiency include microcytic, hypochromic anemia, severe emaciation, growth retardation, high mortality, roughened fur, and lack of underfur pigmentation (cotton-fur syndrome, Fig. 7.5). For foxes, Fe-deficiency signs also include anemia and depigmentation of underfur (Rimeslatten, 1959). Both trimethylamine oxide and formaldehyde, found in the fish digestive tract, have been identified as causative factors of cotton fur (NRC, 1982). e. Fish. Iron deficiency has been shown to cause hypochromic, microcytic anemia in common carp, red sea bream, yellowtail, eel, and brook trout (NRC, 1993). For catfish, the onset of mortality was earlier for fish fed an Fe-deficient diet (Lim et al., 2000).

f Nonhuman Primates. Fitch et af. (1964) reported that diets containing soybean protein resulted in a microcytic, hypochromic anemia characteristic of Fe deficiency in monkeys. Amine et al. (1972) reported Fe-deficiency anemia in squirrel monkeys fed modified cow milk diets. For adolescent Rhesus monkeys, a combined Fe and zinc (Zn) deficiency affected behavior that was characterized by reduced activity, reduced participation in behavioral testing and slower response (Golub et al., 2000).

Fig. 7.5 Pelts of cotton (left) and normal (right) mink, parted to show underfur. The condition can be overcome by supplying iron parenterally. (Courtesy of F.M. Stout, J.E. Oldfield, and J. Adair, Oregon State University, Corvallis)

Deficiency

6.

221

HUMANS

Iron deficiency is one of the most common nutritional disorders in the world, affecting nearly two billion people (WHO, 1994), and the deficiency affects especially infants, young children, and women of the reproductive years. When Fe deficiency is sufficiently severe, red blood cell synthesis becomes impaired, and anemia results. Prolonged Fe deficiency results in the development of hypochromic, microcytic anemia accompanied by a normoblastic, hyperblastic bone marrow containing little or no hemosiderin. Iron deficiency in human adults is manifested clinically by listlessness and fatigue, palpitations on exertion, and sometimes by a sore tongue, angular stomatitis, erythema at the corners of the mouth (cheilitis), dysphagia, and koilonychia (spoon nail). In children, anorexia, reduced growth, and decreased resistance to infection are commonly observed, but oral lesions and nail changes are rare (Morris, 1987). Low Fe status among adolescents may limit their growth spurt (Brabin and Brabin, 1992). Iron deficiency results in a significant reduction in physical activity and performance. A prolonged cardiorespiratory recovery period after exercise has been observed in anemic women (Anderson and Barkue, 1970). Low income postpartum women bear a substantially greater Fe deficiency risk than never pregnant women (Bodnar et al., 2002). The prevalence of Fe deficiency anemia is estimated to be 25% in infants and children worldwide and 50% of women and children in the less developing countries (DeMaeyer and Adiels-Tegman, 1985). According to data from the third National Health and Nutrition Examination Survey (Alaimo et al., 1994), the prevalence of Fe-deficiency anemia in children I to 2 years old in the United States was 3% and the prevalence of Fe deficiency without anemia was 9%. In many tropical regions, anemia, Fe deficiency, malaria, and multiple helminth infections coexist and are interrelated. Sub-Saharan Africa epitomizes this situation, although similar situations exist in equatorial South America, and south and southeast Asia. In these communities, anemia is typically prevalent and severe, especially in pregnant women and young children, and is often an important cause of mortality (Stoltzfus, 1997). For example, the case fatality rate for children admitted with severe anemia to one hospital in rural Tanzania was 6.1 % (Alonso Gonzalez et al., 2000). The consequences of maternal anemia may be serious, with reported associations ranging from preeclampsia to low birth weight and increased risk of maternal deaths (Spinillo et al., 1994; Sapre and Joshi, 1996). The relationship between anemia or Fe deficiency anemia and increased risk of preterm delivery «37 wk gestation) has been supported by several studies (Scholl and Hediger, 1994; Zhou et al., 1998; Scholl and Reilly, 2000). Behavioral disturbances such as pica, characterized by abnormal consumption of nonfood items such as soil (geophagia) and ice (pagophagia), are often present in Fe deficiency. Physiologic manifestations of Fe deficiency have also been noted in immune function, cognitive performance and behavior, thermoregulatory performance, energy metabolism, and exercise or work performance (Beard and Dawson, 1997; Haas and Brownlie, 2001). Iron-deficiency anemia alters the ability

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Iron

of humans to maintain body core temperature during acute cold exposure. Investigations have documented clear alterations in thermoregulation, the thyroid system, and the sympathetic nervous system (Beard and Dawson, 1997). Symptoms of Fe deficiency include lethargy, lack of concentration, lower intelligence scores, and decreased cognitive and attentional processes (ldjadinata and Pollitt, 1993; Pollit, 1993;Walter, 1993).There is evidence that Fe plays a role in neurobiological processes. Various studies have proposed changes in neurotransmitter metabolism (Beard et al., 1994; Chen et al., 1995), myelin formation (Larkin and Rao, 1990) and hippocampal functioning (Rao et al., 1999). Recent studies in humans clearly demonstrate that in early life, the brain is quite susceptible to Fe deficiency (Kwik-Uribe et al., 1999; Rao et al., 1999; Piero et al., 2000). Verbal learning, memory, lower intelligence, and physical performance may be impaired in Fe-deficient adolescent girls (Bruner et al., 1996). A 21-week experiment showed that volunteers with borderline anemia, as measured by blood hemoglobin, were less able to concentrate than those with higher hemoglobin (Kretsch and Green, 2001). Girls between the ages of 12 and 16 with Fe-deficiency anemia posted significantly lower math scores than did non-anemic females or males (Lord, 2001). Infants and children in developing countries are particularly vulnerable to Fe deficiency (Fig. 7.6). Often nursing infants receive only their mother's milk, which is extremely low in Fe, until the next child comes along. The Fe reserve of the human infant is usually exhausted before the end of the sixth month. To complicate this, the weaned child frequently receives very little protein and Fe-rich food (e.g., meat), but rather high-carbohydrate, low-Fe foods including cassava, potatoes, bread, and white rice. Not only infants but whole populations, particularly in developing countries, have blood-sucking parasites, especially hookworms (Stoltzfus et al., 2000). The amount of blood lost varies with the type of parasite and with the number present. One hookworm may consume 1 ml of blood daily, and an infestation of over 100 results in devastating blood loss. In developing countries not only are Fe intakes low but the bioavailability of Fe from the diets is often very low, owing mainly to the low availability of factors facilitating nonheme Fe absorption (fish, meat, and ascorbic acid). An Fe-poor diet and rapid growth are prime causes of Fe deficiency in infants and preschool children. However, in many such children, intestinal bleeding (from hookworm infestation or from bovine milk sensitivity) may also be a factor. Similarly, the suboptimal Fe content of the diet of young women contributes to the high prevalence of Fe deficiency in this group, although blood loss is the major factor (Fairbanks, 1978). Iron need is increased when there is either rapid growth or accelerated Fe loss e.g., from bleeding, hemoglobinuria, pregnancy, or lactation. In adults, Fe deficiency must be taken as evidence of Fe loss, which is usually through gastrointestinal bleeding.

Assessment of Iron Status The characteristic Fe-deficiency anemia is of a hypochromic, microcytic type. However, hypochromic anemia may also occur when the total Fe content of the

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223

Fig. 7.6 A child with iron deficiency anemia in Montero, Bolivia. The vast majority of children in this tropical region suffered from the deficiency as a result of carbohydrate-rich diets, low in iron, as well as high infestations of the blood sucking hookworm parasite. Hematocrits of the population were very low, being in the 20s, with several values as low as 6. (L.R. McDowell, University of Florida, Gainesville)

body is normal. In livestock or humans with a typical Fe-deficiency anemia, the hemoglobin level is reduced, the red cells are smaller in size and contain less hemoglobin than normal. The concentration of Fe in plasma-bound transferrin is reduced, but the level of transferrin itself is increased (Hallberg, 1984). The serum ferritin level, which is related to the size of the Fe stores, is usually very low. Departure from normal levels of serum Fe, total Fe-binding capacity, percentage transferrin saturation value, and hemoglobin and hematocrit values can all be used to diagnose Fe deficiency in livestock. The most commonly used screening methods for the presence of Fe deficiency in the population are the measurements of hemoglobin or hematocrit concentration for the presence of anemia (WHO, 1994). These measurements are relatively simple and cheap, can be carried out under field conditions, and values below a certain cut-off point indicate or define that anemia is likely to exist. However, low hemoglobin and hematocrit values are not sensitive indicators of early Fe deficiency stages because they only occur when storage Fe is severely depleted. Their use is often limited to diagnosis and confirmation of Fe deficiency (Miller and Stake, 1974). Evaluation of bone marrow hemosiderin Fe is usually regarded as the reference standard for assessment of Fe stores (Holyoake et al., 1993). It is considered as the

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Iron

most sensitrve and specific indicator of body Fe stores and unlike other conventional laboratory measures of Fe status, it is not confounded by infection or inflammation. This technique, however, is costly, invasive, painful. and impractical for routine examinations (Ahuwalia et al., 2000). Serum ferritin and percentage saturation of transferrin are early indicators of Fe deficiency. For Fe overload plasma Fe, transferrin saturation and plasma ferritin are good diagnostic criteria (Beard and Dawson, 1997). An assessment of Fe deficiency in ruminants can be made using reduced transferrin saturation « 13 to 15%), serum Fe (<1.1 mg/l), and hemoglobin levels (<10 g/dl) (McDowell, 1976). For swine, normal hemoglobin (g/dl blood) would be 10 or above; at 8, borderline anemia; at 6, severe anemia, and below 4, anemia associated with an increased mortality rate (Cunha, 1977). For humans, hemoglobin < 12.0 g/dl is used for detection of Fe deficiency (Khusun et al., 1999).

IX. SUPPLEMENTATION

Iron supplementation is most important for humans and suckling pigs. For most species, feeds and foods normally contain adequate quantities of Fe to meet nutritional requirements. Based on total Fe content, typical livestock concentrate diets should be close to providing adequate levels of Fe. Grazing animals generally do not need Fe supplementation, as forages are generally high in Fe (McDowell, 1985). Livestock heavily infested with blood sucking insects or parasites or fed byproducts particularly low in Fe (e.g., straw), are an exception. Deficiencies can also result from dietary antagonists to Fe. For example, certain fish diets provide substances (see Section VIII) that result in an Fe deficiency in mink (cotton-fur syndrome). Excessive levels of calcium (Ca), phosphorus (P), Mn, Zn, Cu, or the presence of gossypols, phytins, or tannins will increase the level of supplemental Fe required. When copper sulfate (250 ppm diet) is utilized as a growth promoter, dietary Fe should be increased to 150 ppm. Iron supplementation is most warranted for grazing livestock when forages contain less than 100 ppm of Fe and/or if insects or parasites are causing substantial blood loss. Supplemental Fe can be provided in swine diets when the protein supplement is largely limited to cottonseed meal because the toxicant gossypol found in this protein source is inactivated by Fe (see Section VIII). Research indicates that a 1:1 ratio of Fe to gossypol is needed to be effective. A level of 400 ppm of dietary gossypol will kill pigs after 37 to 57 days (Clawson and Smith, 1966), but 400 ppm Fe prevented deaths or any harmful effects from the gossypol toxicity. It has been postulated that gossypol reacts with liver Fe, and the Fe-gossypol complex is then excreted via the bile (Buitrago et al., 1970). For livestock, the principal need for supplemental Fe is for young animals restricted to milk diets. Adding Fe at rates as low as 30 ppm to milk has improved blood Fe levels in calves (Berger. 1998). The suckling pig is most likely of the livestock species to develop Fe deficiency. Feeding supplementary Fe to the sow before or after farrowing is ineffective because such treatment does not significantly

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increase the Fe stores of the piglet at birth (Venn et al., 1947) or the Fe content of the sow's milk (Pond et al., 1965). Supplemental Fe for piglets may not be necessary in an outdoor production system (Kleinbeck and McGlone, 1999). Successful treatment involves direct increase in the Fe intake of the piglets. Supplemental Fe may be administered orally as follows (Miller, 1980): (1) placing soil in the farrowing pen; (2) swabbing the sow's udder with an Fe solution; (3) dosing the pig with Fe pills, paste, or liquids; (4) placing Fe-rich liquid, meal, pellet, moss, or block preparations in the creep area; (5) feeding high levels of Fe in the sow's diet and allowing pigs access to sow's feed and feces. These methods require repeated administration and have been largely replaced by more efficient Fe injections. Oral Fe from iron chelates is effective, if administered within the first few hours of life. Early administration before gut closure to large molecules is crucial (Thoren-Tolling, 1975). The most popular and effective method of providing supplemental Fe is by injection. Although Fe injections may be given i.m., i.p., or s.c., the i.m. method is preferred. Injection of Fe dextran, or dextrin ferric oxide (Pond et al., 1960), may be made in the ham muscle or neck muscle at the site precleaned with 70% ethyl alcohol. Injectable Fe should be dispensed with a clean syringe using a 0.5 to 1 inch (1.3 to 2.5 em) 20-gauge disposable needle. To help prevent run back of Fe from the injection site, the skin may be forced slightly to one side with the thumb just before making the injection. Injection of Fe should be made within the first 3 days of life (Miller, 1980). Without Fe supplementation, the hemoglobin concentration in piglets decreases to only 70% of the original value after 3 days and continues decreasing. Intramuscular injection of I to 2 ml of Fe dextran (100 to 150 mg Fe) is given at 3 days of age which restores the hemoglobin to the value at birth and that level is maintained throughout a 3-week nursing period (Miller et al., 1961). When the nursing period is to be more than 3 weeks, the original injection should be 200 mg, or a second injection should be given at 2 weeks. However, if the piglets are receiving adequate Fe creep feed by 3 weeks, the first injection would be sufficient. An excessive level (more than 200 mg) of injectable oral iron should be avoided because unbound serum iron encourages bacterial growth and results in increased susceptibility to infection and diarrhea (NRC, 1998). Iron-deficiency anemia occurs in young calves reared for veal on milk-based diets (see Section VIII) and in lambs similarly raised. Intramuscular injections of 150 mg Fe dextran into newborn lambs, or Fe at 26.4 mg/kg body weight into newborn calves, can produce significant improvements in hemoglobin levels and some improvement in body weight over several weeks (Rice et al., 1967). For lambs, Holz et al. (1961) reported that two injections, 150 mg of Fe each, given 2 to 3 weeks apart are preferable to a single injection. A number of inorganic sources of Fe are available for supplementation (Henry and Miller, 1995). In a review, Miller (1978) concluded that Fe retention or incorporation of Fe into hemoglobin, revealed good bioavailability of Fe from monohydrated, dihydrated, and heptahydrated ferrous sulfate, ferric citrate, ferric ammonium citrate, and ferric choline citrate for the young growing pig. Iron as

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ferrous chloride was well utilized, whereas that as ferric chloride was less available (Henry and Miller, 1995). Bioavailability estimates using Fe concentrations in liver, kidney, and spleen of sheep fed four sources of Fe were determined (Van Ravenswaay et al., 2001). Relative values after setting ferrous sulfate at 1.00 were 0.55, 0.00, and 0.20 for carbonates A to C, respectively. The marked differences among sources of carbonate were dramatic, varying from relatively good to low. Bioavailability of Fe from ferrous carbonate and reduced Fe was quite variable, with Fe from ferric oxide essentially unavailable to the pig. Iron is present as a contaminant in Ca and P supplements, but with variable bioavailability. Relative to ferrous sulfate (100%) Fe bioavailability for chicks (Deming and CzarneckiMaulden, 1989) was as follows: dolomite (34.3%), limestone (51.8%), oystershell (10.2%), steamed bone meal (-6.7%), defluorinated rock phosphorus (43.5%), dicalcium phosphate (54.8%), monocalcium phosphate (61.0%), and soft rock phosphate (-9.8%). The utilization of different Fe compounds by ruminants has been studied to only a limited extent. Ammerman and Miller (1972) reported that orally administered ferrous sulfate and ferric citrate are equal for ruminants. Using these sources as a standard of 100, ferrous carbonate and ferric oxide have relative values of 60 and 10%, respectively. Only limited research has been conducted on bioavailability of Fe in feeds and this has been almost entirely with chickens and rats. Estimated relative bioavailabilities have ranged from about 30 to 70% for forages with values for Fe in soybeans and in certain grains or grain products being somewhat higher (Henry and Miller, 1995). Increased bioavailability of Fe from grains and oilseed meals will result ifphytase is included in the diet (Stahl et al., 1999). Iron deficiency anemia during pregnancy is associated with significant morbidity for mothers and infants. Over 40% of pregnant women in developing countries suffer from Fe-deficiency anemia. It is also prevalent among adolescent girls because the growth spurt and onset of menstruation increase Fe requirements (Lynch, 2000). The low Fe stores in these young women of reproductive age will make them susceptible to Fe deficiency anemia during pregnancy because dietary intakes alone are insufficient, in most cases, to meet the requirements of pregnancy (Beard, 2000). Adolescent Fe requirements are even higher in developing countries because of infectious diseases and parasitic infestations that cause Fe loss, and because of low bioavailability of Fe from diets limited in heme Fe (Hunt, 2002). As an example, a review of Indian studies on anemia in adolescent girls revealed that 80 to 90% of adolescent girls in low income communities were anemic with hemoglobin levels < II g per dl (Kanani and Ghaneker, 1997; Kanani and Poojara, 2000). Although Fe status in early pregnancy may be improved if the period of supplementation continues up to the time of conception, supplementation before pregnancy should be viewed as an additional strategy to supplementation during the second and third trimesters (Lynch, 2000, 2002). Viteri et al. (1999) reported that weekly Fe supplementation over 7 months (30 doses) improved and sustained Fe nutrition at least as effectively and was better tolerated than 90 daily Fe supplements consumed during 3 months. In an effort to build Fe stores before pregnancy and reduce the high prevalence of anemia in Indonesia, the Ministry of Health/Indonesia and the MotherCare project

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implemented an anemia control program for newlywed women (Jus' at et al., 2000). Prior to granting a marriage certificate, women were counseled to consume Fe tablets. There was a decrease in the prevalence of anemia from 23.8 to 14.0% over the course of the program. Diet, physiological status, metabolic disorders, disease, and parasites all affect Fe status in humans. In some segments of the human population, the nutrient concentration of Fe in the diet has decreased because of a higher proportion of calories coming from fat and refined sugar. In the individual with Fe-deficiency anemia, one must suspect pathological causes of increased blood loss such as tumors or ulcers in the gastrointestinal tract, or tumors of the genitourinary tract (Hallberg, 1984). In addition, Fe malabsorption due to glutin-induced enteropathy, gastric achlorhydria, or a postgastrectomy syndrome must be considered. Once a diagnosis of Fe deficiency has been established, it is important to determine the cause. In very young children, it may be evident that dietary deficiency (for example, diet restricted to milk) is the cause. In some countries, it is customary for infants to nurse for a year or until the next child arrives. For postmenopausal women or for men, gastrointestinal bleeding must be evaluated, a careful search made for an anatomic lesion that may be amendable to surgery. It is important to exclude gastrointestinal bleeding as the cause of anemia in young children. In tropical or semitropical areas, an examination must be made for hookworm infestation, as even a relatively light infestation can result in substantial blood loss. Iron balance in women can be markedly affected by the use of oral contraceptive pills because those hormones usually reduce menstrual Fe losses by about 50% (Nilsson and Solvell, 1967). The use of intrauterine devices, however, increases Fe losses by about 50% (Liedholm et aI., 1975). For prophylaxis of Fe deficiency in menstruating women, a ferrous sulfate tablet (65 mg elemental Fe) can be taken two or three times weekly; for deficiency in pregnancy, the same dose of Fe should be taken daily throughout pregnancy (Fairbanks, 1978). It is also a common, and reasonable, practice to administer both Fe and folacin throughout pregnancy, since deficiency of both substances commonly occurs (Kanani and Poojara, 2000; McDowell, 2000). Iron nutrition in infants can be improved by early weaning, thus providing foods (e.g., cereals) higher in Fe. A second approach is the direct addition of Fe to formula or fruit juice, or even by dropper into the infant's mouth (Fairbanks, 1978). Since the 1960s, fortification of foods has become common. A portion of the Fe requirement is being met from Fe-enriched flour and bread, with relatively high fortification levels in some countries such as Sweden (Hallberg, 1981). In more recent years, in the United States and in other industrialized countries, intakes of daily vitamin (sometimes vitamins plus Fe) and mineral pills have greatly increased, ensuring adequate intakes of Fe for many individuals. However, due to interactions (e.g., Ca and Mg) with Fe, less Fe is absorbed from certain multivitamin-mineral supplements than from an equivalent amount of Fe given alone. It seems advisable to select products that contain ~ 60 mg of Fe, rather than the recommended dose of 30 mg, to allow for anticipated lower absorption (Yip and Dallman, 1996).

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X. TOXICITY

Iron toxicosis is usually not a problem in domestic animals, but as with all other nutrients, a sufficiently high level in feed or water is detrimental. Most livestock species have a high tolerance for Fe. Swine are more tolerant of excess Fe than are cattle, sheep, or poultry. The maximum tolerable levels of dietary Fe are 3000 ppm for swine and 1000 ppm for cattle and poultry (NRC, 1980). The more limited data available for sheep suggest a maximum tolerable level of 500 ppm dietary Fe. All Fe compounds are probably equally toxic per unit of soluble Fe (NRC, 1980). Apparently, cattle can safely consume substantially more Fe from natural feed sources than from soluble sources such as ferrous sulfate (Standish et al., 1969). The amount of Fe needed to produce undesirable changes depends on whether the level of other nutrients that might be adversely affected are already near the deficiency borderline. As an example, injectable Fe at a typical administered dose of 100 mg (as Fe dextran) is toxic to pigs from vitamin E-selenium-deficient sows (Patterson et aI., 1967; Hill et al., 1999; McDowell, 2000). Standish et al. (1971) reported a decrease in plasma P by high levels of dietary Fe. Wilgus and Patton (1939) found that addition of 1700 ppm of ferric citrate (332 ppm Fe) to a low-Mn chick starter caused perosis, apparently due to antagonism of Mn absorption. Iron levels of 300 to 600 ppm reduced plasma and liver Cu as well as ceruloplasmin in lambs (Prabowo et al., 1988). Characteristic signs of chronic Fe toxicosis for most species are reduced feed intake, growth rate, and efficiency of feed conversion (NRC, 1980). Clinical signs of acute toxicosis of Fe include anorexia, oliguria, diarrhea, hypothermia, diphasic shock, metabolic acidosis, and death (NRC, 1980). For pigs, clinical signs occurred 1 to 3 hours after dosing (0.6 g ferrous sulfate/kg body weight). Signs of the toxicosis were shivering, incoordination, hyperpnea, and tetanic convulsions. Some pigs developed posterior paralysis and had profuse diarrhea (Campbell, 1961). Liveweight gains of sheep decreased when sheep consumed 500 ppm Fe and efficiency of liveweight gains decreased as dietary Fe increased from 150 up to 2000 ppm dry matter (OM) (Wang, 1994). High dietary Fe had depressed the digestibility of OM. Vascular congestion of the gastrointestinal tract, liver, kidneys, heart, lungs, brain, spleen, adrenals, and thymus are the dominant histopathologic findings in most species (NRC, 1980). Iron toxicosis is characterized by excessive deposition of storage forms of Fe in tissues (siderosis) accompanied by high plasma Fe (hypersideremia) and cellular damage to the intestinal mucosa (Campbell, 1961). The major effects of Fe toxicosis in fish include reduced growth, increased mortality, diarrhea, and histopathological damage to liver cells (NRC, 1993). Conditions of Fe overload have been observed in humans and include (Crosby, 1978; Halliday, 1998): (1) in idiopathic hemochromatosis (genetic disorder in which excessive Fe is absorbed); (2) in transfusion hemosiderosis; (3) after prolonged Fe therapy, particularly in patients whose erythropoietic rate is accelerated; and (4) in Bantu natives who consume diets containing as much as 200 mg Fe/day (much of the Fe being derived from kettles used in the preparation of food).

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There is some concern in the United States that intentional, indiscriminate fortification of foodstuffs and self-therapy with Fe-rich mineral tablets (vitamins plus Fe) will result in siderosis. It is important to stress that Fe preparations must be kept out of reach of little children, as severe and even fatal Fe poisoning may follow ingestion of a child's handful of Fe tablets. For both animals and humans with Fe overload, treatments should be (I) return to low-Fe intake, and (2) reduction of body Fe load by induction of sideruria using renally excreted chelating agents. For humans, phlebotomy (removal of patient's blood) is the easiest, cheapest, safest, and most effective way to reduce siderosis (Crosby, 1978; Yip and Dallman, 1996). There is increasing evidence that excess dietary Fe may be a risk factor for various diseases including heart disease (Gable, 1992; Salonen et al., 1992) and colorectal cancer (Lund et al., 2001). A report suggests that high Fe intakes might rank second to smoking as a cause of heart disease. In a five-year study involving 1931 healthy Finnish men (ages 42 to 60), Salonen et al. (1992) tested the hypothesis that high serum ferritin concentration and high dietary Fe intake are associated with an increased risk of acute myocardial infarction. Chronic feeding of Fe was associated with an increase in free radical generation capacity in the colon and increased lipid peroxidation and thus a risk factor for colorectal cancer (Lund et al., 2001). The potential risks of an excess intake of Fe is associated with an increase in free radical generation. Because of the risk of excess dietary Fe, many nutritionists suggest that adult men and women past menopause should not consume daily multivitamin-mineral supplements containing Fe.

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Miller, E. R. (1978). Feedstuffs 50(35),20. Miller, E. R. (1980). Anim. Nutr. Health Nov.-Dec., p. 10. Miller, E. R. (1981). Anim. Nutr. Health June-July, p. 14. Miller, E. R., Ullrey, D. E., Ackermann, I., Schnidt, D. A., Lucke, R. W., and Hoefer, J. A. (1961). J. Anim. Sci. 20, 890. Miller, W. J., and Stake, P. E. (1974). In "Proceedings Georgia Nutrition Conference for Feed Industry," p. 25. Univ. of Georgia, Athens, GA. Mitchell, R. L. (1963). J.R. Agric. Soc. Engl. 124,75. Mollerberg, L., Ehlers, T., Jacobson, S., Johnson, S., and Olsson, I. (1975). Acta Vet. Scand. 16, 197. Morek, T. A. (1978). "Studies on the Iron Requirement of Laying and Breeding Hens." Ph.D. thesis. Cornell Univ., Ithaca, NY. Morris, E. R. (1987). In "Trace Elements in Human and Animal Nutrition" (W. Mertz, ed.), p. 79, Academic Press, New York. Mtimuni, J. P., Mfitilodze, M. W., and McDowell, L. R. (1990). Commun. Soil Sci. Plant Anal. 21 (5 and 6),415. Murray, J., and Stein, N. (1970). In "Trace Element Metabolism in Animals" (c. F. Mills, ed.), Vol. I, p. 321. Livingstone, Edinburgh, Scotland. Nilsson, L., and Solvell, L. (1967). Acta Obstet, Gynecol. Scand. 46 (Suppl. 8),n I. NRC (1980). Mineral Tolerance of Domestic Animals. National Academy of Sciences-National Research Council, Washington, D.C. NRC Nutrient Requirements of Domestic Animals. National Academy of Sciences-National Research Council, Washington D.C. (1977). Nutrient Requirements of Rabbits. (1982). Nutrient Requirements of Mink and Foxes. (1985a). Nutrient Requirements of Dogs, 2nd Ed. (1985b). Nutrient Requirements of Sheep, 5th Ed. (1986). Nutrient Requirements of Cats, 3rd Ed. (1989). Nutrient Requirements of Horses, 5th Ed. (1993). Nutrient Requirements of Fish. (1994). Nutrient Requirements of Poultry, 9th Ed. (1995). Nutrient Requirements of Laboratory Animals. (1996). Nutrient Requirements of Beef Cattle, 7th Ed. (1998). Nutrient Requirements of Swine, 10th Ed. (2001). Nutrient Requirements of Dairy Cattle, 6th Ed. Patterson, D. S. P., Allen, W. M., Thurley, D. C., and Done, J. T. (1967). Biochem. J. 104,2. Peducasse, C. A., McDowell, L. R., Parra, L. A., Wilkins, J. V., Martin, F. G., Loosli, J. K., and Conrad, J. H. (1983). Trop. Anim. Proc. 8, 118. Piero, D. J., Li, N. Q., Connor, J. R., and Beard, J. L. (2000). J. Nutr. 130,254. Piero, D. J., Li, N., Hu, J., Beard, J. L., and Connor, J. R. (2001). J. Nutr. 131,2831. Pollit, E. (1993). Annu. Rev. Nutr. 13, 521. Pond, W. G., Lowrey, R. L., Maner, J. H., and Loosli, J. K. (1960). J. Anim. Sci. 19, 1286. Pond, W. G., Veum, T. L., and Lazar, V. A. (1965). J. Anim. Sci. 24, 668. Prabowo, A., Spears, J. W., and Goode, L. (1988). J. Anim. Sci. 66,2028. Rao, S. G., de Ungria, M., Sullivan, D., Wu, P., Wobken, J. D., Nelson, C. A., and GeorgiefT, M. K. (1999). J. Nutr. 129, 199. Rice, R. W., Nelms, G. E., and Schoonover, C. O. (1967). J. Anim. Sci. 26,613. Rimeslatten, H. (1959). Dansk Pelsdyravl 22, 273. Salih, Y., McDowell, L. R., Hentges, J. P., Mason, R. M., Jr., and Wilcox, C. J. (1987). J. Dairy Sci. 70,608. Salonen, J. T., Nyyssonen, K., Korpela, H., Tuomilehto, J., Seppanen, R., and Salonen, R. (1992). Circulation 86, 803. Sapre, S., and Joshi, V. (1996). J. Obstet. Gyn. Fam. Wei. 2, 7. Scholl, T. 0., and Hediger, M. L. (1994). Am. J. Clin. Nutr. 59, 492S. Scholl, T. 0., and Reilly, T. (2000). J. Nutr. 130, 443S. Sen, K. c., and Ray, S. M. (1964). Bull. Indian Council Agric. Rds. 25. Shoden, A., and Sturgeon, P. (1961). Nature (London) 189,846. Skeets, 0., Frazier, E., and Dickins, D. (1931). Miss. Agric. Exp. Sin. Bull. 291, Starkville, MS. Sousa, J. C. de, Conrad, J. H., Blue, W. G., Ammerman, C. B., and McDowell, L. R. (1981). Pes. Agropecu. Bras. 16, 773. South, P. K., Lei, X., and Miller, D. D. (2000). Nutr. Res. 20, 1749.

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Spinillo, A., Capuzzo, E., Piazzi, G., Nicola, S., Colonna, L., and Lasci, A. (1994). Early Hum. Dev. 38,35. Stahl, C. H., Han, Y. M., Roneker, K. R., House, W. A., and Lei, X. G. (1999). J. Anim. Sci. 77,2135. Standish, J. F., Ammerman, C. 8., Palmer, Z. A., and Simpson, C. F. (1971). J. Anim. Sci. 33, 171. Standish, J. F., Ammerman, C. B., Simpson, C. F., Neal, F. C., and Palmer, Z. A. (1969). J. Anim. Sci. 29,496. Stoltzfus, R. J. (1997). Lancet 349, 1764. Stoltzfus, R. J., Chwaya, H. M., Montresor, A., Albonico, M., Savioli, L., and Tielsch, J. M. (2000). J. NUlr. 130, 1724. Swain, J. H., Tabatabai, L. B., and Reddy, M. B. (2002). J. Nutr. 132,24S. Tejada, R., McDowell, L. R., Martin, F. G., and Conrad, J. H. (1987). Trap. Agric. 64, 55. Thomas, J. W. (1970). J. Dairy Sci. 33, 1107. Thomas, J. W., Okamoto, M., Jacobson, W. C., and Moore, L. A. (1954). J. Dairy Sci. 37,805. Thoren-Tolling, K. (1975). Nord. Vet. Med. 27, 544. Troost, F. J., Saris, W. H. M., and Brummer, R. J. M. (2002). J. Nutr. 132,2597. Underwood, E. J. (1977). "Trace Elements in Human and Animal Nutrition" Academic Press, New York. Underwood, E. J., and Suttle, N. F. (1999). In "The Mineral Nutrition of Livestock" (3rd Ed.), Midlothian, UK. Van Campen, D. (1974). Fed. Proc. Fed. Am. Soc. Exp. Bioi. 33, 100. Van Ravenswaay, Henry, P. R., and Ammerman, C. B. (2001). Anim. Feed Sci. Tech. 90, 185. Vargas, D. R., McDowell, L. R., Conrad, J. H., Martin, F. G., Buergelt, c, and Ellis, G. L. (1984). Trop. Anim. Prod. 9, 103. Venn, J. A. J., McCance, R. A., and Widdowson, E. M. (1947). J. Compo Pathol. Ther. 5, 314. Viteri, F. E., Ali, F., and Tujague, J. (1999). J. Nutr. 129,2013. Walter, T. (1993). Eur. J. Clin. Nutr. 47,307. Walters, G. 0., Miller, F. M., and Worwood, M. (1973). J. Clin. Pathol. 26, 770. Wang, Z. (1994). "Responses to High Intakes of Fe in Sheep," Faculty of Agr., The University of Western Australia, Perth, Australia. Weinberg, E. D. (1984). Phys. Rev. 64, 65. Wilgus, H. S., Jr., and Patton, A. R. (1939). J. Nutr. 18, 35. Williams, D. M., Loukopoulos, D., Lee, G. R., and Cartwright, G. E. (1976). Blood 48,77. Wood, R. J., and Han, O. (1998). J. NUlr. 128, 1841. World Health Organization (1994). Indicators and Strategies for Iron Deficiency and Anemia Programmes. Report of the WHO/UNICEF/UNU Consultation. Geneva, Switzerland. Yip, R., and Dallman, P. R. (1996). In "Present Knowledge in Nutrition," 7th Ed. (E. E. Ziegler, and L. J. Filer, eds.), p. 277, ILSI Press, Washington, D.C. Youdim, M. B. H. (2001). Nutr. Rev. 59(8), S83. Zhou, J. R., and Erdman, J. W. (1995). Cril. Rev. Food Sci. Nutr. 35,495. Zhou, L. M., Yang, W. W., Hua, J. Z., Deng, C. Q., Tao, X., and Stolzfus, R. J. (1998). Am. J. Epidemiol. 148,998.

Chapter 8

Copper and Molybdenum

I. INTRODUCTION

Copper (Cu) is an essential nutrient as well as a toxicant. Although molybdenum (Mo) is an essential element, its role in metabolism is only partially understood; however, deleterious effects of excess dietary intake of Mo on animal health are well known. Copper and Mo are discussed together in this chapter because of the important nutritional and biochemical interactions between them. Under practical feeding conditions, grazing ruminants are most likely to suffer from Cu deficiency and/or Mo excess, whereas monogastric diets based on grains generally are adequate in Cu and low in Mo.

II. mSTORY

Archeological records suggest that Cu has played a significant role in civilization since its early use in hammered artifacts of the stone age. Historical aspects of the discovery and importance of Cu are reviewed by Prasad (1978) and Davis and Mertz (1987). Not until the early 1800s was Cu found in plant and animal tissues. Early investigators believed that plant and animal Cu concentrations represented accidental contamination from soil. In 1928, Hart and associates discovered that Cu was essential for growth and hemoglobin formation in rats. This important discovery was soon followed by evidence that Cu is essential for growth and for the prevention of a wide range of clinical and pathological disorders in all types of farm animals. These investigations ultimately led to the recognition that enzootic ataxia (swayback) of lambs, bovine falling disease, aortic rupture of rabbits, swine, guinea pigs, and chickens, depigmentation of hair and wool, and bone disorders were signs of Cu deficiencies and were preventable with Cu supplementation. Soon after demonstration of the essential role of Cu in hematopoiesis, several enzymes (e.g., tyrosinase, amine oxidase, and ascorbic acid oxidase) with oxidase functions were shown to contain Cu. The essentiality of Cu for ruminants was first established in 1931 when a Cu deficiency in grazing cattle (Fig. 8.1) was demonstrated in Florida (Becker 235

236

Copper and Molybdenum

Fig. 8.1 Jersey heifer E-15 had a hemoglobin reading of 5.98 g per 100 ml of whole-blood on October 21, 1930 (left). Administration of ferric ammonium citrate and copper sulfate (50 Fe:1 Cu) was started on Dec. IS, 1930; it was 13.74 g per 100 ml. E-15 was fully recovered by March 27, 1931, and became a normal cow in the dairy herd (right). This was the first bovine to confirm that Cu was essential for ruminants. Meanwhile, two herd mates, receiving Fe as the sole supplement to the basal ration died of anemia. (Courtesy of R. B. Becker, W. M. Neal, and A. L. Shealy, University of Florida, Gainesville)

et al., 1965). Cattle had exhibited a wasting disease (salt sick), which eventually was found to be caused by deficiencies in cobalt (Co) and iron (Fe) in addition to Cu. Salt sick was reported in Florida as early as 1872 (Dodge, 1874). In 1933, investigators in Northern Europe discovered that a wasting disease (lechsucht) characterized by diarrhea, loss of appetite, and anemia was caused by a Cu deficiency. These researchers found marked differences in Cu concentrations between healthy and sick areas and cured the condition with Cu therapy. Molybdenum was discovered about 1782, but biological interest in Mo began in 1938 with the discovery that a severe and debilitating scouring disease of cattle occurring in parts of England, known locally as teart, was caused by ingestion of high forage levels of Mo (Underwood and Suttle, 1999). This interest was heightened when the preventive and curative effects of large doses of copper sulfate were demonstrated in a Mo-Cu interaction. Conversely, the effect of Mo in limiting Cu retention was demonstrated in sheep when Dick (1952) reported that this effect was only exerted in the presence of adequate amounts of inorganic sulfate. Findings indicating that a three-way interaction between Cu, Mo, and sulfur (S) gave a great stimulus to further studies of dietary mineral interactions in livestock. Indications of an essential role for Mo came in 1953 when two groups of workers independently discovered that the flavoprotein enzyme, xanthine oxidase, is a Mocontaining enzyme, dependent for its activity on the presence of this element (Underwood, 1977). Other Mo-containing enzymes were later discovered and direct evidence was then obtained that Mo is essential in the diets of chicks, poults, rats and lambs. Attempts to produce Mo deficiency in rats and chicks were successful only when the diet contained massive amounts of tungsten (W), an antagonist of Mo metabolism (Nielsen, 1996). An indirect Mo requirement was shown when chronic Cu poisoning in sheep and cattle in eastern Australia resulted from forage

Chemical Properties and Distribution

237

low in Mo (Underwood and Suttle, 1999). The condition was controlled with supplemental Mo.

III. CHEMICAL PROPERTIES AND DISTRIBUTION Copper has an atomic number of 29 and an atomic weight of 63.5. It tends to occur in sulfide deposits, particularly in igneous rocks, with concentration in the continental crust of 50 ppm. Sandstones contain 10 to 40 ppm; shales, 30 to 150 ppm; and marine black shales, 20 to 300 ppm Cu. Because of the variety of conditions that influence Cu availability, total soil Cu is not an accurate indication of deficient or excess concentrations in plants. Copper in soil is most available to plants grown under conditions of poor drainage, an acid pH, high in clay, and low in other metallic antagonists (e.g., Mo). Copper availability is reduced by high organic matter (muck soils). Species and variety of the plant as well as seasonal and weather variations also influence Cu uptake by plants. Molybdenum has an atomic weight of 95.94 and is a dark-gray or black powder with metallic luster, or a coherent mass of silver-white color. The average abundance of Mo in the earth's crust has been placed at I ppm, but individual rock types may range from near zero to as high as 3000 ppm. Molybdenum is found at concentrations ranging from <0.5 to > 100 ppm dry matter (DM) in plants grown on soils containing high levels of Mo, either naturally or as a result of contamination (Gardner and Hallpatch, 1962). As was true for Cu, the total concentration in soils bears little or no relationship to concentration of Mo in plants. The Mo content of plants can vary greatly in response to soil Mo content, soil pH, and the season of the year. Molybdenum-toxic areas characteristically occur on poorly drained, neutral, or alkaline soils that favor Mo uptake by plants and reduce the availability of Cu. In solution, as well as in living organisms, Cu is found almost exclusively in the + 2 and + I valence states, with + 2 predominating. At neutral pH in aqueous media (as in most cells and organisms) Cu ions form hydroxides that precipitate out of solution unless chelated by organic molecules (Linder, 1996). The redox chemistry of Cu makes it particularly suited for releasing and accepting electrons and especially for the direct transfer of electrons to molecular oxygen. The most common form of inorganic Mo is the oxyanion molybdate, MoO~-. However, the numerous other stable oxidation states of the metal underlie its versatility as a biological redox catalyst. In biological systems, the valence status of + 6, + 5, and + 4 states are most commonly encountered (Johnson, 1997). Adult humans contain approximately 100 to 150 mg of Cu, or 1.5 to 2 ppm. In newborns, Cu concentrations of liver are much higher than in adults (~40 ppm), reflecting prenatal storage of the element (Linder, 1996). Molybdenum is present in low concentrations in all tissues and fluids of all species studied. Organs that retain the highest amounts of Mo are liver and kidney (Nielsen, 1996).

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IV. METABOLISM

A. Copper Absorption, Transport, Storage, and Excretion

In most animal species, Cu is poorly absorbed; the extent of absorption is influenced by its chemical form (Baker and Ammerman, 1995). Absorption is greater in young than mature animals and in Cu-deficient than Cu-sufficient animals. Generally, not more than 5 to 10% of the Cu in the diet is absorbed by adult animals, while young animals may absorb 15 to 30%. Often only 1 to 3% of Cu is absorbed in ruminants. The preruminant animal absorbs Cu as efficiently as monogastric species and more efficiently than the mature ruminant. In a study with lambs, the apparent availability of dietary Cu was 47% 2 weeks before weaning compared with 8 to 10% after weaning (Suttle, 1975). Lee et al. (2000) reported adult sheep to have a 6.5% Cu absorption rate. Depending on the species studied, Cu can be absorbed in all segments of the gastrointestinal tract. Although sites in the upper section of the small intestine appear to play the major role in Cu absorption, substantial absorptive activity has been demonstrated for the human stomach and the sheep large intestines (Davis and Mertz, 1987). Starcher (1969) investigated the mechanism of Cu absorption in the chick and found appreciable absorption of 64Cu from the proventriculus and the duodenum; absorption from the latter organ was five times that of the former. Homeostasis is affected by controlling the rate of absorption, which in turn is regulated by the intestinal mucosa. There is good evidence that the intestinal absorption ofCu is regulated by the need of the organism, and that metallothionein in the epithelial cells of the intestine may play a key role in that regulation. Absorption of Cu is higher during a Cu deficiency than it is at adequate nutritional status (Linder, 1996; Brody, 1999). In the laboratory rat, efficiency with which both Cu and zinc (Zn) are absorbed doubles during the terminal stages of pregnancy to meet increased maternal demand (Mills, 1980). Animals of similar age, breed, and physiological state reared in a common environment can show marked differences in their efficiency of Cu utilization. In grazing sheep, Wiener and Field (1970) have reported heritable differences among three breeds and their crosses in susceptibility to swayback (Cu deficiency) and Cu poisoning, with the breed most susceptible to swayback being least susceptible to Cu poisoning (see Section VI). Studies in a number of species have shown that the intestinal absorption of Cu is influenced by its chemical form and by a substantial number of interactions with other dietary factors. Dietary phytates, high levels of calcium (Ca), S, Fe, Zn, cadmium (Cd), or Mo reduced absorption. Other dietary and environmental factors that influenced Cu absorption include chelating agents (such as amino acids and citrate), which enhance absorption, and intestinal binding agents (such as bile and fiber), which inhibit absorption (Linder, 1996). One of the biggest deterrents to absorption are Cu-binding proteins (metallothioneins) that are synthesized in mucosal cells. By binding Cu, metallothionein prevents its serosal transfer. Zinc can obstruct Cu

Metabolism

239

absorption indirectly by enriching the level of metallothionein through induced synthesis and antagonizing Cu exiting from mucosal cells (Harris, 1997;Brody, 1999). High concentrations of metallothioneins in the intestine offer protection against accidental Cu overload and thus protect the organism against Cu toxicity (Harris, 1997). Cousins (1985) has reviewed the potential role of metallothionein as a regulator of Cu absorption and as the site of intestinal interaction between Cu and Zn. Water temperature influenced Cu uptake by rainbow trout, increasing the temperature from 16 to 19°C almost doubled Cu uptake (Clearwater et al., 2000). Recent studies have indicated that intestinal parasitism lowers Cu status in sheep (Suttle, 1996) and the spring hatch of nematode eggs may lower the value of spring pasture as a Cu source (Underwood and Suttle, 1999). Copper appears to be absorbed by two mechanisms, one saturable and the other unsaturable, suggesting active transport for the former and simple diffusion for the latter (Bronner and Yost, 1985). After being absorbed by the intestine, Cu rapidly enters the blood circulation and is quickly deposited mainly in liver. The protein, referred to as transcuprein, is involved in the initial distribution of incoming dietary Cu to liver and kidney (Liu et al., 2000). This protein has higher affinity for Cu than albumin, which is also involved in Cu transport, under physiological conditions. Copper is released from the cellular and subcellular fractions of the liver primarily for hepatic synthesis of ceruloplasmin, for synthesis of erythrocuprein by normoblasts of the bone marrow, and for incorporation into many enzymes (Beam and Kunkel, 1957). Nearly 90% of the Cu in mammalian plasma is in the form of the Cu metalloprotein, ceruloplasmin. Ceruloplasmin is synthesized in the liver, where it receives six Cu atoms, and is then secreted into the plasma. The protein is also made in white blood cells (Juan et al., 1997). The exact physiological role of ceruloplasmin remains unclear, but it is related somehow to the transfer of Fe in and out of ferritin. Ceruloplasmin is the carrier for the tissue-specific export of Cu from the liver to the target organs. The liver is the central organ of Cu metabolism; its concentrations reflect intake interactions with other minerals, and the Cu status of the organism. The storage form of Cu seems to be a mitochondrial cuprein, which is especially elevated in Wilson's disease. In addition, Cu is present in the cytosol bound to a metallothionein-like protein. In all species studied, a high proportion of ingested Cu appears in feces. Most of this is unabsorbed Cu, but most active excretion also occurs via the bile (Underwood, 1977). Intermediate quantities are excreted through urine, milk, and intestine, and small amounts are excreted in perspiration.

B. Molybdenum Absorption, Storage, and Excretion Molybdenum is readily and rapidly absorbed from most diets and supplement forms of the element. Molybdenum absorption in rats occurs rapidly in the stomach and throughout the small intestine, the rate of absorption being higher in the proximal than in the distal parts of the small intestine (Nielsen, 1996). Hexavalent water-soluble forms, sodium and ammonium molybdate and the Mo of high-Mo herbage, most of which is water soluble, are particularly well absorbed by cattle

240

Copper and Molybdenum

(Ferguson et al., 1943). Absorption of Mo from the disulfide (MoS 2) is poor, owing to the antagonistic effect that sulfate has on Mo absorption. The extent of the Mo absorption depends on the species, the age of the animals, and level of Mo in the diet, but its average value is 20 to 30% based on experiments involving stable and radioactive isotopes of Mo (Georgievskii et al., 1981). In one study, humans fed ammonium molybdate contained in liquid-formula component of a diet absorbed 83 to 93% of the Mo (Turnlund et al., 1993). Rates of Mo absorption, retention, and excretion are inversely related to the level of dietary S. In sheep, for instance, increasing the dietary S from 0.1 to 0.2% in a diet supplemented with 10 mg Mo per day decreased the Mo retention from 37 to 4%. A working hypothesis for the effect of S on Mo retention is that sulfate inhibits membrane transport of molybdate, thus decreasing absorption ofMo in the intestine and decreasing reabsorption of Mo by the renal tubules (Dick, 1956; Ryan et al., 1987). For sheep with a Mo intake of 0.3 mg per day, total body Mo decreased from 92.9 to 16.8 mg when sulfate was increased from 0.9 to 6.3 g per day (Dick, 1956). There is little storage of Mo, with the element present in low concentrations in all tissues and fluids, and most of the storage in bones and liver. Dietary protein, Fe, Zn, lead (Pb), W, ascorbic acid, and a-tocopherol influence level of Mo in tissues (NRC, 1980). Molybdenum is not only rapidly absorbed, but also very rapidly excreted, mainly in the urine and in part through the bile. Absorbed Mo is also excreted via milk from cattle and sheep in proportion to levels of orally and parenterally administered Mo.

C. Copper-Molybdenum-Sulfur Interrelationships The three-way interaction between Cu, Mo, and S is complex and not fully understood (see Chapter 6 of this volume). Ignoring possible animal variations, interrelationships may be summarized as follows: I. Molybdenum, and especially Mo in the presence of S, reduces the deposition of Cu in organs and the synthesis of ceruloplasmin; as a result, Cu excretion with bile decreases, but its excretion in urine increases. 2. An increase in dietary Cu reduces Mo deposition in liver. 3. When S level is increased, urinary Mo increases substantially, while its tissue deposition decreases correspondingly. Some of the Cu-Mo-S interactions take place in the digestive tract, whereas others occur at the site of metabolism. The primary site of the three-way interaction controlling Cu storage is believed to be in the gut. In the presence of S, high intakes of Mo can induce a Cu deficiency due to formation of insoluble Cu-Mo-S complexes (e.g., thiomolybdates) in the digestive tract that reduce the absorption of Cu (Mason, 1986; 1990). Several pathways exist by which Cu-Mo-S interactions mediate Cu deficiency (Dick, 1956; Ryan et al., 1987). Sulfur also exerts an independent effect on the availability of Cu to ruminants, and the effect of S alone may be greater than the S-dependent effects of Mo (Underwood and Suttle, 1999). In a three-year experiment, cattle consuming

Physiological Functions

241

forage high in S (0.4%) and low in Mo « I ppm) and Cu «5 ppm) had low liver and plasma Cu concentrations and clinical signs of Cu deficiency (e.g., faded hair coat) (Tiffany et al., 2000a, 2001,2002). Sulfides react with molybdate in the reduction medium of the rumen to replace oxygen, producing thiomolybdates. Thiomolybdate (MoS~-) is more readily absorbed than is the oxygen analog, molybdate (MoO~-), which forms the highly insoluble and non-utilizable Cu thiomolybdate, CuMoS 4 . The concept of the formation of a thiomolybdate ion and the tight complexing of this ion with Cu has similarly been proposed by Suttle (1975). Plasma Cu increases because of the formation of the copper thiomolybdate complex, and clinical signs of Cu deficiency result. A study with rats confirmed this model by showing that 500 ppm dietary Mo increased plasma Cu levels without relieving Cu deficiency pathology (Nederbragt, 1980). Allen and Gawthorne (1987) suggested that the thiomolybdate hypothesis for explanation of the Cu-Mo-S interaction in ruminants should be modified by including the association of tetrathiomolybdate (TTM) and Cu with proteins, and de-emphasizing the formation of insoluble Cu thiomolybdate. Experiments using S35-labeled metallothionein (MT) revealed that changes in the Cu profile were the result of removal of Cu from MT by stronger chelators produced by the association of TTM with high-molecular-weight proteins. These concepts of Mo-Cu antagonism in ruminants envisage that Mo acts, not by direct interaction with Cu, but as a secondary consequence of Mo affinity for sulfide generated within the rumen. Excessive quantities of dietary S (0.3 to 0.4%) as sulfate or elemental sulfur may cause toxic effects and, in extreme cases, can be fatal (Kandylis, 1984). The effects of soil ingestion and Fe excess on Cu absorption (Suttle et al., 1975, 1984) are believed to result from Fe binding of sulfide in the rumen, with subsequent release of sulfide in the intestine that interferes with Cu absorption. Ruminant animals are much more susceptible to Mo-Cu imbalance than are nonruminant animals. The primary effect probably occurs in the rumen through the involvement of sulfide-generating bacteria and the consequent formation of unavailable compounds such as cupric thiomolybdate. Provision of additional dietary Cu overcomes adverse clinical responses by inhibiting absorption of thiomolybdate or its derivatives. Formation of thiomolybdates also affects the kinetics of S metabolism by affecting sulfide formation and absorption. Thiomolybdates rapidly react with particulate matter and proteins to form complexes that bind Cu strongly, reducing its solubility, decreasing the H 2S concentration and thereby the rate of sulfide absorption (Gawthorne et al., 1985). V. PHYSIOLOGICAL FUNCfIONS

A. Copper Copper is required for cellular respiration, bone formation, proper cardiac function, connective tissue development, myelination of the spinal cord, keratinization,

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Copper and Molybdenum

and tissue pigmentation. Copper serves as an essential catalytic co-factor of several physiologically important metalloenzymes, including cytochrome oxidase, Iysyl oxidase, Cu-Zn superoxide dismutase, dopamine-B-hydroxylase, and tyrosinase. Linder (1996) reports 14 Cu-containing enzymes. Copper is surpassed only by Zn in the number of enzymes which it activates. Copper is considered as an antioxidant in vivo but it also has pro-oxidant activity in vitro and accumulation of tissue Cu may lead to oxidative stress (O'Conner et al., 2000; Rock et al., 2000). At least three Cu enzymes appear to have a role in antioxidant defense. These are the widely distributed intracellular and extracellular superoxide dismutases (SODs), extracellular ceruloplasmin, and intracellular Cu thioneins. Studies with purified copper thioneins indicate that these proteins formerly thought to function merely in storage and detoxification of Cu also have free radical scavenging activity (Felix et al., 1993). Due to the redox activity of Cu, this metal ion readily participates in the generation of hydroxyl radical, which damages nucleic acids, proteins, and membranes. Consequently, all cells must establish fine tuned homeostatic mechanisms to allow cells to accumulate sufficient Cu for essential biochemical reactions, yet prevent the accumulation of Cu ions to toxic levels (Thiele, 2000). Marginal or sub-optimal Cu status may, in the long term, precipitate a number of degenerative and inflammatory conditions including arthritis, cancer, osteoporosis, and coronary heart disease. 1. IRON METABOLISM AND CELLULAR RESPIRATION

Along with Fe, Cu is necessary for hemoglobin synthesis. Copper is not contained in hemoglobin, but a trace of it is necessary to serve as a catalyst before the body can utilize Fe for hemoglobin formation. Anemia can develop with either a Fe or Cu deficiency. With Cu deficiency, there is an apparent delay in maturation and shortened life span of red blood cells (Baxter and Van Wyk, 1953). Copper plays a key role in Fe absorption and mobilization. An important enzyme for erythrocytes is Cu/Zn superoxide dismutase for protection against oxygen radicals. Also, ceruloplasmin and another Cu-dependent enzyme, ferroxidase II, may also playa role in the flow of Fe that supports hematopoiesis (Linder, 1996, Gabrielli et al., 2000). Serum Fe levels tend to be low in Cu deficiency, and hypochromic anemia develops while intestinal mucosa and liver Fe levels are higher than normal. Ceruloplasmin (ferroxidase I), which is synthesized in the liver and contains Cu, is necessary for the oxidation of Fe, permitting it to bind with Fe-transport protein, transferrin. Ceruloplasmin is a multi-functional enzyme involved in Fe metabolism, transport of Cu, and regulation of certain amines (Evans, 1978). Ceruloplasmin promotes the incorporation of Fe into the storage protein, ferritin (Saenko et al., 1994). Iron must be converted to the ferrous form to be mobilized from stored ferritin and/or to be incorporated into hemoglobin or myoglobin. For storage as ferritin or for transport as transferrin, Fe must be converted to the ferric form, a reaction performed by ceruloplasmin. The hypothesis is that Fe released from storage sites (such as liver ferritin) would be in the Fe 2 + valence state and that

Phy~ologicalFuncUons

243

ceruloplasmin and ferroxidase II oxidize this iron to Fe3+ so that it can bind to its transfer protein in the plasma. A low activity of the ferroxidases would thus result in a diminished flow of Fe to bone marrow and thus reduce the rate of erythrocyte formation (Linder, 1996). Serum ceruloplasmin mediates Fe release from a variety of cell types. During pregnancy, Fe is transferred from mother to fetus across the placenta. Uptake is through transferrin-receptor mediated endocytosis (Danzeisen et al., 2000). Copper is a constituent of the important metalloenzyme, cytochrome oxidase. This enzyme is the terminal oxidase in the respiratory chain; it catalyzes the reduction of O 2 to water, an essential step in cellular respiration. 2.

CROSS-LINKING OF CONNECTIVE TISSUE

With a Cu deficiency, there is failure of collagen to undergo cross-linking and maturation (Cashman and Flynn, 1998). The key to Cu-containing enzyme in the formation of the cross-links in collagen and elastin is Iysyl oxidase, which is necessary to add a hydroxyl group to lysine residues in collagen, allowing crosslinking between collagen fibers. Therefore, it is fundamental to the functioning and formation of connective tissue, including that needed for wound healing and maintaining the integrity of blood vessels (strengthened by elastic fibers) (Linder, 1996; Rucker et al., 2000). These cross-links give the proteins structural rigidity and elasticity. Aortic aneurysms and ruptures result from failure to convert lysine to desmosine, the cross-linking residue in elastin.

3.

PIGMENTATION AND KERATINIZATION OF HAIR AND WOOL

The melanin polymer that protects skin against excess ultraviolet light and determines the pigmentation of our eyes and hair is formed from tyrosine with the aid of the Cu-containing enzyme tyrosinase. Achromotrichia (lack of pigmentation) is a principal manifestation of Cu deficiency in many species. It is commonly observed in the hair and wool of mammals, and is usually attributed to lack of tyrosinase (polyphenyl oxidase) activity. A breakdown in the conversion of tyrosine and melanin is the probable explanation. Impaired keratinization of hair and wool are noted in Cu-deficient animals. The characteristic physical properties of wool, including crimp, are dependent on disulfide groups that provide cross-linkages or bonding of keratin and on alignment or orientation of long-chain keratin fibrillae in the fiber. Straight steely wool has more sulfhydryl groups and fewer disulfide groups than normal (Marston, 1946). Copper is required for formation or incorporation of disulfide groups in keratin synthesis. 4.

CENTRAL NERVOUS SYSTEM

The link between Cu deficiency and the integrity of the central nervous system, e.g., swayback (enzootic ataxia) of lambs, results from the reduction in cytochrome oxidase activity and thus incomplete myelin formation (Howell and Davidson, 1959). Myelin is composed largely of phospholipid. Loss of cytochrome

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244

oxidase in Cu deficiency leads to depressed phospholipid synthesis by liver mitochondria. The inhibition of myelin synthesis results in the ensuing neurological disturbances. Other central nervous system effects of Cu deficiency are reduction of at least two neurotransmitters, dopamine and norepinephrine (O'Dell, 1984). 5.

REPRODUCTION

Reproductive failure is commonly observed in mammals fed Cu-deficient diets (Underwood, 1977). For rats and guinea pigs, Cu deficiency has resulted in fetal death and resorption. Embryos from Cu-deficient hens exhibited anemia, retarded development, a high incidence of hemorrhage after 72 to 96 hours of incubation, and a reduction in monoamine oxidase activity. The anemia, hemorrhages, and mortality are probably caused by defects in red blood cell and connective tissue formation during early embryonic development. 6.

HORMONE RELATIONSHIPS

The formation and inactivation of hormones appears to be another Cu enzymecatalyzed function. Biosynthesis of the catecholamines epinephrine (released from the adrenal medulla in stress) and norepinephrine (the major neurotransmitter of the sympathetic nervous system) is dependent on hydroxylation of dopamine by dopamine-Bsmonooxygenase (Linder, 1996). Copper may also aid in the action of some small peptide hormones (the enkephalins) by regulating an optimal conformation for binding to their receptors. The Cu-dependent enzyme, peptidylglycine-e-amidating monooxygenase has been reported in the brain of newborn rats from Cu-depleted dams. This enzyme has a control on the appetiteregulating hormones, gastrin and cholecystokinin (Prohaska and Bailey, 1995). 7. LIPID

METABOLISM AND CARDIAC FUNCTION

A number of studies have demonstrated the effect of Cu deficiency on lipid metabolism. Copper deficiency is associated with a severe cardiomyopathy and EKG abnormalities. In rats and swine, prolonged Cu deficiency provoked hemopericardium, hemothorax, and heart rupture (Mielcarz, 2000). Petering et al. (1977) reported that Cu deficiency results in elevated levels of serum triglyceride, phospholipids, and cholesterol in the rat. Altered heart function of rats fed low Cu is associated with alterations in lipid and long-chain fatty acid metabolism (Cunnane et al., 1987), which can be attributed to the predominant role of Cu in the superoxide dismutase enzyme system. In work with rats, it was concluded that a combination of depleted antioxidant protection, due to Cu deficiency, with excess Fe is responsible for oxidative stress which, in turn, has the potential to induce hypercholesterolemia and coronary heart disease (Fields and Lewis, 2000). The hypercholesterolemic effect of Cu deficiency in various experimental animal models is well documented, and it has been suggested that it arises from an increased rate of hepatic cholesterol synthesis. Copper deficiency has been shown to reduce hypercholesterolemia in rats and is associated with changes in

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245

lipoprotein distribution and results mainly from an increase in the HDL and LDL fractions of cholesterol (Mazur et al., 2000). Copper supplementation for broilers reduced cholesterol content 25% in meat (Skrivan et al., 2000) and significantly reduced cholesterol in eggs (Pesti and Bakalli, 1998). For finishing steers, backfat depth was lower in animals receiving supplemental Cu, but marbling scores were similar across treatments (Engle et aI., 2000). Percentage of unsaturated fatty acid in longissimus muscle was increased and percentage saturated fatty acid tended to be reduced in steers supplemented with Cu. Polyunsaturated fatty acid (18:2 and 18:3) concentrations were higher in steers supplemented with Cu.

8.

IMMUNE SYSTEM

Copper metabolism affects T and B cells, neutrophils, and macrophages. Copperdeficient mice had depressed T- and B-lymphocyte numbers, and the cells produced were functionally impaired (Harris, 1997). An impaired humoral immune response (e.g., decreased numbers of antibody-producing cells) was observed in mice with hypocuprosis (Prohaska et al., 1983). The magnitude of this impairment was highly correlated with the degree of its functional deficiency. In a review, Miller et al. (1979) concluded that the relationship of Cu to the immune system is through superoxide dismutase, a Zn-, Cu-, and Mn-dependent enzyme, and its role in the microbial systems of phagocytes. The respiratory burst activity of macrophages, an index of phagocytotic killing activity, also is compromised in Cu deficiency (Harris, 1997). In cattle affected by Cu deficiency induced by Mo, neutrophils were impaired in their ability to kill ingested Candida albicans (Boyne and Arthur, 1986). Molybdenum-induced hypocupremia has lowered the antibody response to Brucella abortus antigen in cattle (Cerone et al., 1995). The ability of polymorphonuclear leukocytes to phagocytose C. albicans in sheep with low Cu status is comparatively lower than that of sheep on a normal Cu diet (Olkowski et al., 1990). A decreased resistance to infection has been observed in sheep affected by Cu deficiency (Wooliams et al., 1986). Bactericidal activity can be influenced and occurs early in the development of Cu deficiency in cattle and sheep. Copper-deficient sheep had increased mortality from bacterial infection (Chew, 2000). Antibody titers to Brucella abortus and proliferation responses to concanavalin A and soluble antigen-stimulated mononuclear cells were lower in Cu-deficient heifers (Cerone et al., 1995). Ceruloplasmin, an acute-phase protein, did not have the normal post-inoculant (bovine herpesvirus-I) increases in Cu-deficient calves (Arthington et al., 1996). Plasma Cu concentration and ceruloplasmin activity increase during stress (Cousins, 1985). Ceruloplasmin can function as a free-radical scavenger and can directly modulate inflammatory responses (Cousins, 1985). Increases in plasma Cu and ceruloplasmin during stress were not as pronounced in Cu-deficient cattle (Genge1bach et al., 1997). Furthermore, Cu deficiency induced by feeding high dietary Mo did not greatly affect immune response in cattle (Arthington et al., 1995, 1996). It is probable that Cu deficiency affects specific immunity differently in

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ruminants than in other species, or maybe Cu deficiency alters specific immune response of ruminants only after extended periods of time (Ward and Spears, 1999; Minatel and Carfagnini, 2000).

B. Molybdenum Molybdenum has been identified as a component of six enzymes including three in mammals: xanthine oxidase, aldehyde oxidase, and sulfite oxidase (Bray, 1974). All known Mo metalloenzymes, with the exception of nitrogenase (a plant enzyme), use Mo in the form of the Mo cofactor (Brody, 1999). In mammals, they are involved in the metabolism of purines, pyrimidines, pteridines, and aldehydes, and in the oxidation of sulfite. Xanthine oxidase and aldehyde oxidase are involved in the electron transport chain in the cells, involving cytochrome C (Rajagopalan, 1980). Aldehyde oxidase may be involved in niacin metabolism. Sulfite oxidase oxidizes sulfite to sulfate for final excretion in the urine. Molybdenum of the Mo metalloenzyme is directly involved in the chemistry of catalysis. It changes its oxidation state and accepts and donates electrons during the catalytic cycle. During the event of catalysis, Mo is thought to cycle between the different ionic forms M0 6 + , M0 4 + , and Mo5+ (Brody, 1999). Hence, Mo may be compared with Fe and Cu, which also change their oxidation state during catalysis. Molybdenum metalloenzymes are also iron metalloenzymes.

VI. REQUIREMENTS A. Copper Dietary factors including Fe, Mo, S, Zn, Pb, Cd, and the protein source may influence the Cu requirement. Estimates of Cu requirements, established with a minimum level of common dietary antagonists, have been established by the National Research Council (Table 8.1). General recommendations for ruminant livestock cannot reasonably be made without reference to pasture Cu, Mo, and S concentrations. Most clinical signs attributed to the three-way interaction are the same as those produced by simple Cu deficiency and probably arise from impaired Cu metabolism. The tolerable risk threshold of Cu to Mo ratio in feed is not fixed but declines from 5:I to 2:I as pasture Mo concentrations increase from 2 to 10 ppm (Suttle, 1991). Bingley and Carrillo (1966), from Argentina, reported that when the Cu:Mo ratio of forages in the presence of adequate S is less than 2.8: I, hypocuprosis in the animals is evident. Miltimore and Mason (1971), reported that the critical Cu:Mo ratio in feeds appears to be 2.0: I, and feeds or pastures with lower ratios result in conditioned Cu deficiency, A Cu:Mo ratio of no less than 4:1 has been proposed (Alloway, 1973) to ensure that the Cu requirement will be met. Where soils have been limed, the Cu requirement of Merino sheep is 10 ppm, whereas in western Australia where soil liming does not occur and the Mo content

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247

TABLE 8.1 Copper Requirement for Various Species" Species Chickens

Japanese quail Turkeys Beef Cattle Dairy ca ttle Sheep Horses Swine Mink Fish Rabbits Dogs Cats Rats Mice Humans

Purpose

Requirement

Reference

Leghorn-type 0--6 wk Leghord-type 6-18 wk Broilers all classes All classes All classes All classes All classes All classes All classes Growing Breeding. lactating All classes Catfish Trout All classes All classes Growing All classes All classes Adults Lactating

5 mg/kg 4 mg/kg 8 mg/kg 5 mg/kg 6-8 mg/kg 10 mg/kg 9-11 mg/kg 7-11 mg/kg 10 mg/kg 3-6 mg/kg 5 mg/kg 4.5-6 mg/kg 5 mg/kg 3 mg/kg 3 rng/kg 2 mg/kg 5 mg/kg 5 rug/kg 6 mg/kg 0.9-1.0 mg/day 1.3 mg/day

NRC (1994) NRC (1994) NRC (1994) NRC (1994) NRC (1994) NRC (1996) NRC (2001) NRC (1985b) NRC (1989) NRC (1998) NRC (1998) NRC (1982) NRC (1993) NRC (1993) NRC (1977) NRC (l985a) NRC (1986) NRC (1995) NRC (1995) DRI (2001) DRI (2001)

"Expressed as per unit animal feed either on as-fed (approximately 90% dry matter) or dry basis (see Appendix Table I).

of the pastures is generally below 1.5 ppm, 6 ppm of Cu is adequate (Underwood and Suttle, 1999). Increasing soil pH favors plant uptake of Mo, while reducing concentrations of Cu. With very low levels of Mo (below 1.5 ppm), as little as 4 ppm Cu has been adequate for cattle. High levels of Zn and Fe depress Cu absorption and tend to increase the requirement (Underwood, 1977). Addition of 250 ppm Zn to hens' diets depressed hatchability when Cu was limiting and appeared to increase Cu requirement approximately twofold (Bird, 1966). In sheep, a diet high in Zn reduced Cu toxicosis and liver stores (Bremner et al.. 1976). Steers grazing a pasture sprayed with Fe, Zn, and Pb developed a marked Cu deficiency, whereas controls remained normal (Binot et al., 1969). For goats, a Cd-induced secondary Cu deficiency has been reported (Miller, 1979). When dietary conditions are optimal for utilization of Cu, 4 to 5 ppm Cu in swine and poultry diets and 8 to 10 ppm Cu in ruminant diets appear adequate (Mcfrowell, 1997). Young and growing animals have higher Cu requirements and, therefore, a higher deficiency incidence than do older livestock (Mills, 1966). M unro (1957) reported that Cu therapy increased the conception rate in heifers following one or, at most, two inseminations from 52 to 95%, but no improvement was noted for cows. Gender in at least one species, the rat, determines death or survival on a marginally Cu-deficient diet; males are much more susceptible to death from heart

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disease than are females, a situation reminiscent of a similar difference in human cardiovascular disease (Fields et al., 1986). Although the Cu requirement of swine is 6 ppm or less, a growth response is sometimes obtained when the element is fed at 100 to 250 ppm. High dietary levels of Cu stimulate increased growth rate and feed efficiency in young growing swine (Barber et al., 1955), yet the exact mode of action remains unknown. Shurson et al. (1987) concluded that the main effect of high-Cu feeding is antimicrobial in the gut since the response of high-Cu feeding tends to be like that in a germ-free environment. Braude (1967) summarized the results of many British studies and concluded that 250 ppm Cu improved daily gain 8% and feed efficiency 5.5% in pigs when compared to controls. North American research with high-level feeding of Cu to pigs has not given consistent good results as those in Britain, but likewise generally showed benefits from the supplemental Cu (Wallace, 1967). Bioavailability of Cu is affected by the genetics of ruminants as well as antagonists such as Mo and S. Studies with sheep have clearly demonstrated that the requirement for, metabolism of, and tolerance of Cu by sheep is substantially affected by the genetic makeup of the sheep (Field, 1984). There are marked variations within breeds in the efficiency of absorption of minerals from the diet, varying from 2 to 10% for Cu in adult sheep (Field, 1984). When different breeds of sheep grazed certain pastures in Scotland, one breed exhibited signs of Cu poisoning, whereas another showed signs of Cu deficiency (Wiener et al., 1977). Goonerante et al. (1987) reported that Cu deficiency in Simmental cattle from Canada was more frequent than in other breeds. Feeding high levels of combinations of Cu, Mo, and/or S resulted in greatly enhanced biliary Cu excretion in Simmental versus Angus cattle. Goats retain less Cu in their livers than sheep when receiving high concentrations. This may be because they share with cattle a propensity for biliary secretion (Zervas et al., 1990). The Cu requirements for specific biological processes increase in order as follows: hemoglobin formation, growth, hair pigmentation, and lactation. According to Bird (1966), 2.7 ppm of Cu supported egg production but was inadequate to maintain hatchability. A level of 4.7 ppm was adequate for hatchability and gave normal egg and plasma Cu levels. Availability of Cu in feeds affects the requirement for the element. For instance, more supplemental Cu is required when the main dietary source is from pasture than when dry forage or concentrates are fed. Ruminants are more likely to develop a Cu deficiency when grazing lush pastures (NCMN, 1973). The Cu content of forage declines with increased maturity. However, Cu status of cattle grazing more mature forage is better than in those grazing immature forage (Hartmans, 1969). This suggests low availability of Cu in immature forage, or a change in the relationship of Cu to interfering factors. It also appears that the antagonism of Mo in natural feedstuffs has a greater effect than that chemically added. For humans, the newest dietary recommendations (DRI, 2001) for Cu are 0.9 to 1.0 mg/day for adults with this increased to 1.3 mg/day for lactation (Russell, 200 I). Pregnancy has been found to improve Cu retention by about 4% or 0.08 mg/day

Natural Sources

249

which would be sufficient to accumulate the 21 mg of Cu needed for the fetus (WHO, 1996). Human dietary requirements decline approximately 45 to 15 llg/kg body weight/day between infancy and adulthood (Mills, 1990). B. Molybdenum Molybdenum deficiency is most often related to excess Cu, and animals perform normally on extremely low dietary levels of Mo. The minimum Mo requirements compatible with satisfactory growth and metabolic well-being cannot be given with any precision for most animal species. Some of the more accurate dietary requirement information is for goats (Anke and Rich, 1989) at 0.05 to 0.10 ppm, and for rats (Wang et al., 1992) at 0.20 ppm. It is also clear that the chick has an extremely low Mo requirement, apparently below 0.2 ppm (Higgins et al., 1956). For ruminants, an exact estimate of the Mo requirement is impossible since Cu and Salter Mo metabolism. Work by Ellis et al. (1958) with growing lambs showed faster gains and improvement in cellulose digestibility when dietary Mo was increased from 0.36 to 2.37 ppm. Many pastures grazed by ruminants, however, contain less than 0.35 ppm of Mo, with no evidence of a deficiency. Such low-Mo pastures favor accumulation of Cu in tissues of sheep, and under some conditions, can lead to chronic Cu poisoning. Tungsten competitively inhibits Mo for formation of xanthine dehydrogenases, and can therefore be used to study Mo requirements and metabolism. Leach and Norris (1957) reported increased growth in chicks to Mo in a diet containing 0.5 to 0.8 ppm, when W was also added to the diet.

VII. NATURAL SOURCES Typical Cu and Mo concentrations in feeds are presented in Appendix Table II. Copper occurs in plant material at concentrations of I to 50 ppm, dry basis. Concentrations of Cu in crops and forages vary geographically, and are dependent on soil factors, plant species, stage of maturity, yield, crop management, climate, and soil pH. These factors also influence Cu antagonists Mo, Fe, and S, which are equally as important as forage Cu concentrations (MacPherson, 2000; McDowell and Valle, 2000). Beeson et al. (1947) found the Cu content of 17 grass species grown together on a sandy loam soil to range from 4.5 to 21.1 ppm. The application of Cu-containing fertilizers to soils low in plant-available Cu invariably increases the Cu concentration in the herbage and often increases yield (Underwood and Suttle, 1999). In most circumstances, Cu concentrations decline as plants mature, and are lower from alkaline soils (McDowell, 1985). Cereal grains contain 4 to 8 ppm Cu, and the leguminous seeds and oilseed meals provided as protein supplements generally contain 15 to 30 ppm (Davis and Mertz, 1987). Grasses, averaging 5 ppm, tend to be lower in Cu than are legumes, which average 15 ppm. Grains tend to be higher than leaves or stems. Kincaid and

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Copper and Molybdenum

Cronrath (1983) reported that significant portions of Cu are associated with the fibrous versus leaf portion of alfalfa hay. However, Minson (1990) noted that leaves contain 35% higher concentrations than stems. The Cu availability in cereal grains may be 10 times greater than in forages (Suttle, 1986). This partially explains why Cu deficiency can be a problem with grazing bovines, but usually not with dairy cattle or finishing cattle that receive high amounts of concentrates in their diets (McDowell et al., 2001). Feeds from animal origin that are rich in Cu include oysters, liver meal, and meals from crustacea and shellfish. Meat meal and fish meal typically contain 5 to 15 ppm Cu, compared with 80 to 100 ppm in liver meal. The liver, blood, spleen, lungs, brain, and bones are particularly responsive to variations in dietary Cu intakes (Davis and Mertz, 1987). Milk and milk products are inherently low in Cu but are subject to increases from contamination with metals during processing and storage. Copper content of milk decreases as lactation period progresses. Colostrum Cu concentrations of Brahman cows were 0.49 mg/l compared to 0.32 mg/l 3 months later (Salih et al., 1987). Markedly subnormal Cu values as low as 0.01 to 0.02 mg/l, have been reported for milk of severely deficient cows and ewes (Beck, 1941). Numerous Cucontaining compounds used in agriculture and veterinary medicine, such as plant and animal fungicides, and foot baths for the control of foot rot in cattle and sheep, are also potential sources of Cu for livestock. The Cu residues in manure from Cu-supplemented swine and poultry litter are significant when this manure is recycled in livestock diets (Fontenot, 1972; Davis, 1974). Often overlooked, but nevertheless of potential significance, is the contribution Cu pipes can make to Cu intake from drinking water. This can vary from less than 0.1 mg/day in hard water areas to 10 times that level with some extremely acid soft waters (WHO, 1996). Molybdenum is present in small quantities in a wide range of plant and animal tissues. Legumes and their seeds have the highest and most variable content of Mo among animal feeds. In general, neutral or alkaline soils in association with high moisture and organic matter favor Mo uptake from soil (Parham and DeRenzo, 1978). It would seem impossible or highly unlikely for forage Mo concentrations to be at toxic concentrations for grazing livestock on soils that are acid and welldrained unless there was direct surface contamination (McDowell et al., 2001). As an example, heavy application of high Mo containing biosolids (municipal sewage sludge) to well drained, acid, sandy soils over a three-year period did not greatly elevate forage Mo (Tiffany et al., 2000a,b). The biosolids varied in Mo concentration from 12 to 56 mg/kg DM and were applied from 16.8 to 44.8 t/ha: none of the forages exceeded 1.25 ppm Mo DM. Excessivelyhigh herbage Mo concentrations (100 to 200 ppm) occur naturally only on alkaline soils. Forage Mo content can be increased two- or three-fold by liming a soil to raise the pH from 6.0 to 6.5 (McDowell, 1985). Although there is high variability in feeds, Mo concentrations (ppm, dry basis) for plant sources are as follows: cereal grains and straws 0.2 to 0.5; grasses 0.2 to 0.8; clovers and other legumes 0.5 to 1.5; vegetable protein concentrates 0.5 to 2.0 (Mills and Davis, 1987).

Deficiency

251

Molybdenum is generally low in animal tissues except liver and bone, and these can be increased (see Section III) by increasing dietary concentrations. Molybdenum in the tibia of chicks was increased 100-fold when a diet low in Mo was supplemented with 2000 ppm Mo as molybdate (Davies et al., 1960). The Mo content of milk is influenced by dietary Mo. Archibald (1951) reported a range of Mo from 18 to 120 /lgjl in cow's milk, increasing to 371 /lg/I after daily feeding of 500 mg of Mo as molybdate daily.

VIII. DEFICIENCY A. Effects of Deficiency The main manifestation of Cu deficiency includes anemia, diarrhea, bone disorders, reproductive failure, nerve disorders, cardiovascular disorders, achromotrichia (loss of hair pigment), and keratinization failure in hair, fur, and wool. Poor growth and appetite occur in Cu deficiency in all species but are not conspicuous or dominant as in Co deficiency or in Zn deficiency in ruminants. When the diet is deficient in Cu, abundant studies in animals indicate that three Cu proteins are the most immediately affected, ceruloplasmin, cytosolic SOD and cytochrome C oxidase (Linder, 1996). One of the hallmarks of severe Cu deficiency in mammals is the loss of integrity of elastic and connective tissue, resulting in increased fragility of the blood vessel wall, abnormal elastin, vascular lesions (especially in the arteries), and a greater likelihood of aneurysms. No clinical syndromes in man or in domestic farm animals are directly attributable to an uncomplicated Mo deficiency under natural conditions. 1.

SWINE

An early sign of Cu deficiency in the pig is a microcytic and hypochromic anemia. Teague and Carpenter (1951) in their early studies ofCu-deficiency produced by milk diets, noted decreased mean corpuscular volume and hemoglobin concentration. Besides anemia, an unusual leg condition develops in Cu-deficient pigs (Teague and Carpenter, 1951). The animals lack rigidity in the leg joints, with excessively flexed hocks; this forces the animal to a sitting position. Forelegs show various types and degrees of crookedness (Fig. 8.2). Microscopically, there is a marked reduction in osteoblastic activity, with failure of bone deposition on the calcified cartilage matrix. Cardiovascular and central nervous system disorders are reported in Cu-deficient swine (Miller et al., 1979; NRC, 1998). The heart increases 200% in size, and ruptures of major blood vessels are observed, suggesting a defect in elastin tissue. Ataxia has been observed in young pigs and is associated with low levels of liver Cu and demyelination of the spinal cord. These observations are probably due to a deficiency of cytochrome C oxidase which depresses phospholipid synthesis.

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Copper and Molybdenum

Fig.8.2 Copper deficiency. Note the drawing under of the rear legs and crookedness of the forelegs, the swelling in the region of the hocks, a turning of the rear legs, and the extreme weakness of the carpal joints in the foreleg. (Courtesy of L. E. Carpenter, H. S. Teague, and Hormel Institute)

Teague and Carpenter (1951) found rate of growth for Cu-deficient animals to be similar to controls until severe deficiency signs were observed. Few naturally occurring Cu deficiencies have been observed in pigs on typical diets (Miller et al., 1979). Molybdenum deficiency uncomplicated by high dietary W or Cu was produced in pigs fed diets containing <24 ng Mo/g. Deficiency signs were depressed feed consumption, depressed growth, and impaired reproduction characterized by infertility and elevated mortality in both mothers and offspring. 2,

POULTRY

Anemia is a general sign of Cu deficiency in poultry. Chicks frequently die from internal hemorrhage as a result of defective vasculature before they become severely anemic (O'Dell, 1979). However, turkey poults fed a Cu-deficient diet develop a mild anemia within 4 weeks (Savage et al., 1966). Young chicks fed a Cu-deficient diet became lame in 2 to 4 weeks, and bones were easily broken (Leeson and Summers, 2001). Gallagher (1957) reported that the long bones, especially the metatarsals, were bent and excessively fragile. The epiphyseal cartilage became thickened, and vascular invasion of the thickened cartilage was therefore suppressed. The defect in Cu-deficient bone is associated with the organic matrix and more particularly with the failure of collagen cross-linking. Turkey poults developed enlarged hocks (Fig. 8.3) and perosis when Cu deficient (Savage etal., 1966). Hens fed a severely Cu-deficient diet (0.7 to 0.9 ppm) for 20 weeks had reduced egg production and subnormal levels of Cu in the plasma, liver, and eggs, while hatchability dropped rapidly and approached zero in 14 weeks (Savage, 1968). The embryos from these hens displayed anemia, retarded development, a high hemorrhage incidence after 3 to 4 days of incubation and a reduction in monoamine oxidase activity.

Deficiency

253

Fig. 8.3 The effect of copper deficiency on feather pigmentation, growth rate and hock development is shown in turkey poult on the left, which was fed a diet containing less than I ppm of copper for four weeks. The control on the right consumed the same diet supplemented with copper. (Courtesy of Boyd O'Dell, University of Missouri, Columbia)

Baumgartner et al. (1978) observed abnormal eggs while feeding hens a low Cu (0.72 ppm) diet. About 10% of eggs were shell-less, and 40% had an abnormal shape or texture. Savage et al. (1966) observed lack of pigmentation in addition to depressed growth rate in bronze turkey poults receiving low-Cu diets. In chickens, Mo deficiency aggravated by excessive dietary W resulted in depression of Mo enzymes, disturbances in uric acid metabolism, and increased susceptibility to sulfite toxicity (Mills and Bremner, 1980). Under field conditions, a Mo-responsive syndrome was found in hatching chicks. This syndrome was characterized by a high incidence oflate embryonic mortality, mandibular distortion, anophthalmia, and defects in leg bone development and feathering. The incidence of this syndrome was particularly high in commercial flocks reared on diets containing high concentrations of Cu (a Mo antagonist) as a growth stimulant. 3.

RUMINANTS

A wide variety of disorders in ruminants are associated with a simple or induced (high Mo and S) Cu deficiency, including anemia, severe diarrhea, depressed growth, change of hair color, neonatal ataxis, temporary infertility, heart failure, and weak, fragile long bones that break easily (Underwood and Suttle, 1999). Dietary Mo (5 ppm) in cattle significantly delayed the occurrence of first ovulation (26 to > 46 weeks) compared with controls (Phillippo et al., 1984). The first world report indicating Cu essentiality for ruminants was illustrated with a Jersey heifer in Florida (Fig. 8.1) in 1931. The anemic animal had a hemoglobin reading of 5.9 gjlOO ml vs 13.7 gjlOO ml after Cu administration.

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Copper and Molybdenum

Fig. 8.4 Copper deficiency in sheep. Copper deficient lamb in Argentina (A) exhibits a delayedswayback condition. From Saltillo. Mexico (B) there is a high incidence of paralysis in lambs. Animals with this condition respond to copper supplementation (A - courtesy of Osvaldo Balbuena, INT A. Colonia Benitex, Argentina; B - L. R. McDowell, University of Florida. Gainesville.)

Demyelination of the central nervous system, or neonatal ataxia (swayback) is a nervous disorder of lambs and goats. Two types of neonatal ataxia are recognized in lambs: (I) a common acute form occurs in newborn lambs, and (2) a delayed type (Fig. 8.4) often occurs several weeks or sometimes months after birth. In both diseases, the signs are spastic paralysis, incoordination of the hind legs, a stiff and staggering gait, and an exaggerated swaying of the hindquarters (NRC, 1985b). Some lambs are completely paralyzed or ataxic at birth and die immediately. There is a degeneration of the myelin sheath of the nerve fibers. Lambs that are born weak may die because of their inability to nurse, because their central nervous systems developed during maternal Cu deficiency. Figure 8.4 illustrates a type of mild paralysis in sheep in Saltillo, Mexico that responded to Cu therapy. An inability to suckle, incoordination, stiff gait, and opisthotonos have been reported in young calves whose dams were Cu-deficient (Larson et al., 1995). A very sensitive index of a Cu deficiency is achromotrichia, or loss of pigment in hair and wool (Fig. 8.5). The pigmentation of sheep is so sensitive to changes in Cu intake that alternating bands of unpigmented and pigmented wool fibers can be produced, as Cu is withheld from and added to the diet. The formation of the black hair or wool can be blocked within 2 days when the functional level of Cu is inadequate or dietary Mo and S have been increased (Underwood and Suttle, 1999). For cattle, depigmentation usually shows up around the eyes and ears first unlike normal sun bleaching, graying hair is typical, but Hereford's red turns yellowish and Angus black is reddish brown. Growth and physical appearance of wool and hair are altered with inadequate Cu (Suttle and Angus, 1976). Sheep deficient in Cu fail to impart a crimp in the wool

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255

Fig. 8.5 Hair color changes with copper deficiency. Both A and B illustrate hair color changes as a result of copper deficiency. The dark color is normal when cattle receive adequate copper. Photo C illustrates faded haircoat of animal in Saltillo, Mexico. Animals received diets high in molybdenum and low in copper. (A and B - courtesy of B. J. Carrillo, C.I.C.V., INTA, Castelar, Argentina and C - L. R. McDowell. University of Florida. Gainesville.)

fibers, which results in an almost straight hairlike fiber called "stringy" or "steely" wool. Another Cu deficiency sign is the development of fragile bones (Fig. 8.6), particularly the long bones, which break easily, sometimes without apparent cause.

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Fig. 8.5

Continued

Fig. 8.6 Broken bones on the leg of this calf are the result of Cu deficiency. Swelling can be seen on one leg. (Courtesy of B. J. Carrillo, C.I.C.V, INTA, Castelar, Argentina.)

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257

Lameness in animals may also be a result of the deficiency. In extremely Cudeficient cattle, there is a swelling or enlargement of the ends of the leg bones, especially above the pasterns (fetlocks) (Becker et al., 1953, 1965). Cattle that show these skeletal abnormalities may move like a pacing horse (pacing gait) rather than like normal cattle. It is characterized by an apparent stiffness in the hocks and springiness in the pasterns. Such animals had no difficulty running, but they advanced both left legs and then both right legs alternately, hence the term "paces." Animals with advanced paces breathed with difficulty after normal exertion, such as when being driven short distances on the range (Becker et al., 1965). Other terms to describe the condition are pigeon-toed, stiff-legged, or bunny-hopping. These conditions probably arise through impaired collagen and elastin development, and are simply not a bone disorder but a combination of bone and connective-tissue disorders (Underwood and Suttle, 1999). A condition known as falling disease sometimes occurs in Cu-deficient cattle (Becker et al., 1965; Underwood and Suttle, 1999). Deficient cattle may die suddenly with exertion, and postmortem examination may reveal small lesions of the heart. The essential lesion is a slow and progressive degeneration of the myocardium with replacement fibrosis (Underwood, 1977). Falling disease does not occur in sheep or horses grazing the same Cu-deficient pastures and occurs only rarely elsewhere. Low fertility in cattle grazing Cu-deficient pastures, associated with delayed or depressed estrus, occurs in several widely separated areas, and infertility (or aborted small dead fetuses), has been reported in experimental Cu deficiency in ewes (Underwood and Suttle, 1999). Estrus in cattle may be delayed or depressed and the conception rate reduced (Becker et al., 1953; Underwood, 1977). Calving difficulties, retained placenta, and calves born with congenital rickets have been described when cows were Cu-deficient. In bulls, fertility may be reduced by poor quality semen (Larson et al., 1995). A Cu-responsive diarrhea is reported in cattle (Fig. 8.7) in various world regions. Often the diarrhea is more prevalent when excess Mo is a major cause of the Cu deficiency (Ward, 1978, 1991; Miller, 1979), especially when the diarrhea develops very rapidly and is quite severe, as low Cu does not cause this type of diarrhea. Histochemical and ultrastructural changes have been observed in the small intestinal epithelium in animals with the diarrhea. There is much less diarrhea in sheep than in cattle. Scouring has been observed in goats maintained on the pastures that induce scouring in cattle (Underwood and Suttle, 1999). Subclinical Cu deficiencies are thought to be very widespread and are likely to be of more economic significance than are easily recognized cases. With inadequate Cu, the animals may be unthrifty and have lower milk production, growth and reproduction efficiency, without readily recognizable signs (Underwood, 1977; Miller, 1979). Thornton et al. (1972) reported Cu supplementation during a sixmonth period increased liveweight gains in cattle by 10 to 70% over controls even though, with few exceptions, control stock showed no clinical signs ofhypocuprosis. Copper deficiency is a severe limitation to grazing ruminants and has been observed in many parts ofthe world. In reviews, McDowell (1985,1997,1999) listed a total of34 tropical countries in Latin America, Africa, and Asia with reported deficiencies, more

258

Copper and Molybdenum

Fig. 8.7 The animals pictured exhibit severe diarrhea as a result of excess dietary molybdenum and tOQ little copper. (Courtesy of B. J. Carrillo, CLCV, INTA, Castelar, Argentina.)

than those of any other mineral except P. In cattle, a Cu deficiency is most likely to occur in grazing young stock, especially yearlings (NCMN, 1973; Miller, 1979). Widespread Cu deficiencies based on plant and animal tissue concentrations occur in grazing ruminants. A total of 47% of forages included in the 1974 Latin American Tables of Feed Composition contained low concentrations « I 0 ppm) of this element (McDowell et ai., 1977). Table 8.2 illustrates forage Cu analyses from seven tropical countries, the vast majority of which were deficient in Cu. These investigations also found corresponding low liver Cu concentrations. Hill et al., (1962) noted that 80% of the surveyed cattle and buffalo in Malaysia were Cu deficient. From Trinidad, 106 grasses ranged from 2.2 to 11.9 ppm, with 98.1% containing less than a critical level of 10 ppm (Youssef et al., 1999). From three dairy cattle regions of western Kenya, all forage samples and the majority of serum samples were deficient in Cu (Oduor et al., 2000). For buffalo from Marajo Island, Brazil, Cu deficiency was severe with 49% of animals exhibiting a hypochromic anemia (Cardoso et al., 2001). From the Buenos Aires Province of Argentina, 59% of 1580 cattle serum samples were deficient in Cu (Titarelli et al., 1996). Sheep with outbreaks of neonatal ataxia, raised under the seminomadic conditions of the Sudan, were evaluated for Cu status (Idris et al., 1976); deficient serum concentrations «0.6 IJ.g/ml) were found in 50% of 500 sheep evaluated. Of the numerous world reports of Cu deficiency in ruminants, only a few are concerned with a deficiency induced by unusually low concentrations ofCu «3 ppm) in the feed. The majority are concerned with a conditioned Cu deficiency, in which normal amounts of Cu (6 to 16 ppm) are inadequate owing to other forage constituents such as Mo, S, and Fe, and other factors that block utilization of Cu

Deficiency

259

TABLE 8.2 Mean Forage Copper Concentrations Wet season

Location Argentina" Bolivia"

Brazil" Colombia"

Number of samples

Mean (ppm)

340 16 120 35

3.0 5.8 1.5 3.6

10

84 48

21 9.5

10 8

Dry season

cc"

Percentage below cc

8 10

96.5 94.4 100

Dominican Republic!

Guatemala" Malawi"

58 52.6

Number of samples

Mean (ppm)

84 192 36 69 84 21

1.3 2.1 2.8 9.0 8 3.1

cc

Percentage below cc

10

100

10 10 8 8

100 64.0 92.0 89.5

"Critical concentration (cc) (McDowell et at. 1984). hBalbuena et al. (1989) 'Peducasse et al. (1983) (wet season); McDowell et at. (1982) "Sousa et al. (1980). 'Vargas et ul. (1984). fJerez et at. (1984) "Tejada et at. (1987) hMtimuni et al. (1990)

(Russell and Duncan, 1956). Copper deficiencies usually occur when forage Mo exceeds 3 ppm and the Cu level is below 5 ppm (Cunha, 1973). Some studies indicate that ingestion of soils containing substantial amounts of Mo can be an important contributing factor in Cu deficiency, especially of sheep in certain situations such as those grazing winter pasture (Suttle et al., 1975).Water can also provide a significant quantity of Fe and S, and less frequently Mo (Wright et al., 2000). Copper intake is the primary interacting factor in Mo toxicity because sufficient Cu supplementation can counteract most all disorders associated with high Mo intakes (Clawson et al., 1972). In addition to Mo intake and availability, Ward (1994) identified dietary factors clearly related to molybdenosis, or Cu deficiency, as (I) Cu intake, (2) Cu availability, (3) S intake, (4) Fe intake, and (5) the physical form of the feed. In the presence of S, high intakes of Mo can induce a Cu deficiency due to formation of insoluble Cu-Mo-S complexes (e.g., tetrathiomolybdate) in the digestive tract that reduce the absorption of Cu (McDowell, 1997). Several pathways exist by which Cu, Mo, and S interactions mediate Cu deficiency. High S intake can also decrease Cu status independent of Mo status (Smart et al., 1986). The effect of S alone may be greater than the S-dependent effects of Mo (Underwood and Suttle, 1999). Tiffany et al. (2000a, 2002) fertilized high Mo-containing biosolids (municipal sewage sludge) to evaluate the effect on Cu status in cattle (see Section VII). Data from the three-year experiment indicated that the high Mo biosolids applications had little effect on forage. Forage Mo concentrations were generally less than 1 ppm. Although biosolids Mo had little effect on cattle Cu status, Cu deficiency was

260

Copper and Molybdenum

evident each of the three years based on low liver and plasma concentrations and the clinical signs of faded hair coat in years 2 and 3. Copper deficiency resulted because forage Cu concentrations were inadequate « 10 ppm) and there was elevated forage S (> 0.4%) as a result of the biosolids treatments. Forages grown on biosolids-amended soil frequently have increased S contents (McBride et al., 2000; O'Connor et al., 2001). Studies with cattle (Campbell et al., 1974) and sheep (Suttle and Peter, 1984) have suggested that the intake of Fe may depress Cu status of ruminants. Studies from tropical regions have indicated that forages grown on acid soils are extremely high in Fe, which may aggravate the low Cu status of grazing livestock in many regions. Copper deficiency of small ruminants in the Sultanate of Oman was found to result from high dietary S and Fe versus elevated Mo (Ivan et al., 1990). Impaired immune response manifested as poor response to vaccination, severe parasitism, and failure to respond to treatment, has been reported in cattle with a Cu deficiency (Larson et al., 1995). It is widely recognized that feeder cattle from Cu-deficient areas are less responsive to vaccinations for diseases associated with shipping fever compared to cattle that have had adequate Cu (Berger, 1996). Calves with Mo-induced Cu deficiency have lower percentages oflymphocytes than control or Cu-supplemented calves and tend toward decreased cytokine response to disease challenge (Gengelbach et al., 1997). However, Ward and Spears (1999) report that Cu deficiency seemed to alter immune function only after a prolonged period and then only minimally. This is in contrast to other species in which Cu deficiency causes marked immunosuppression. Copper level in the diet has been shown to affect the resistance of sheep to bacterial infections (Wooliams et al., 1986). Resistance to internal parasites is also compromised with Cu deficiency. For example, Hucker and Yong (1986) found that Cu-deficient lambs inoculated with Trichostronglyus axei and T. colubriformis had maximum fecal egg counts 2 weeks sooner and became more hypoalbuminemic than lambs receiving supplemental Cu. An uncomplicated Mo deficiency for goats has been established with a low dietary Mo concentration of <24 ngjg (Anke and Rich, 1989). Deficiency signs were greatly reduced intake, depressed growth, impaired reproduction, and elevated mortality of mothers and offsprings. 4. HORSES

Little information is available on Cu deficiency in horses. An apparent relationship between low serum Cu levels and hemorrhaging in aged parturient mares, suggested either reduced absorption of Cu with age or reduced ability to mobilize stores (NRC, 1989). Two recent studies have shown that Mo is unlikely to increase dietary Cu requirements and induce secondary Cu deficiency (Pearce et al., 1999; Rieker et al., 2000). Low Cu and high Zn diets have been shown to increase the development of osteochondrosis and skeletal abnormalities which occur early in the life of young horses (Bridges and Harris, 1988).

Deficiency 5.

261

OTHER ANIMAL SPECIES

a. Laboratory Animals. Anemia is a common expression of severe, prolonged Cu deficiency in laboratory animals. Achromotrichia is seen in Cu-deficient rats and guinea pigs. Offspring of guinea pigs fed Cu-deficient diets grew slowly; some became moribund or died suddenly during the first month post-partum. Intrathoracic or intraabdominal hemorrhage and aortic aneurysms were found at necropsy (NRC, 1995). Copper deficiency during early development can result in significant abnormalities in the cardiovascular, nervous, skeletal, reproductive, immune, and hematopoietic systems (Davis and Mertz, 1987; NRC, 1995). Weanling and adult rats fed Cu-deficient diets can develop alterations in platelet function, impairments in both the acquired and innate arms of the immune system, altered exocrine pancreatic morphology and function, alterations in prostaglandin synthesis, and impaired cardiovascular function. When Cu was restricted in the diet to rats prior to conception, fetal development ceased (Prohaska, 2000). Also, there appeared to be long-term behavioral consequences manifest in the repleted rats evidenced by a blunted auditory startle response. In rats, Mo deficiency aggravated by excessive dietary W resulted in the depression of Mo enzymes, disturbances in uric acid metabolism, and increased susceptibility to sulfite toxicity (Mills and Bremner, 1980).

b. Dogs and Cats. Copper found in most meats is efficiently utilized; therefore, a deficiency would be rare for dogs and cats unless diets were composed entirely of milk or vegetables. Anemia results from Cu deficiency in both dogs and cats. Bone abnormalities may occur in Cu-deficient cats. Signs of connective tissue lesions were seen in kittens from queens fed diets containing Cu less than 1 ppm (NRC, 1986). Reproduction problems with cats have been reported as a result of Cu deficiency (Fascetti and Morris, 2002). Litters were frequently stillborn or born prematurely. c. Fish. Gatlin and Wilson (1986) observed reduced heart cytochrome C oxidase and liver Cu-Zn superoxide dismutase activities in Cu-deficient catfish. Carp fed diets containing high-ash fishmeal without Cu supplement showed reduced growth and cataract formation (NRC, 1993). A low concentration of Cu was found in Atlantic salmon suffering from Hitra disease (NRC, 1993), which is a cold-water bacterial disease caused by Vibrio salmonicida. d. Rabbits. A Cu deficiency of rabbits resulted in anemia and graying of dark hair (Smith and Ellis, 1947). Bone abnormalities, including defective cartilage formation, have been noted in Cu-deficient rabbits. Achromotrichia (Fig. 8.8), alopecia, and dermatitis are the most sensitive index of Cu deficiency in the rabbit, occurring even before a pronounced anemia. 6.

HUMANS

It is assumed that the incidence of Cu deficiency is low in the general population, but these results may not be reliable. Due to the lack of sensitivity of chemical tests presently used, actual Cu deficienciesare difficult to detect (Kehoe et al., 2000). Most

262

Copper and Molybdenum

Fig. 8.8 Gray hair is one sign of a copper deficiency. Other signs are an anemia, loss of hair, and a dermatosis. (Courtesy of S. E. Smith, Cornell University, Ithaca, NY)

of the obvious clinical signs ofCu deficiency already described for animals also have been observed in humans, beginning with the anemia, leukopenia, and especially neutropenia; decreases in ceruloplasmin and erythrocyte Cu-Zn SOD; hypercholesterolemia; increased turnover of erythrocytes, and development of abnormal electrocardiographic patterns (Linder, 1996). Recent studies have indicated that many diets provide low dietary Cu which suggests the possibility that marginal deficiency may occur more widely than hitherto appreciated (WHO, 1996). Severe Cu deficiency caused by inadequate dietary Cu intake occurs in infants. Signs of Cu deficiency were first observed in severely malnourished Peruvian infants rehabilitated with cow's milk (Cordano et al., 1964). The signs included anemia, neutropenia, osteoporosis, depigmentation, and neurological disturbances. Similar pathology has been observed in premature infants and patients maintained on total parenteral nutrition (Mason, 1979). In humans, a dementia, dysarthria with soft staccato speech, accompanied by involuntary movements and gait disturbances, has been observed in adult twin males that display abnormally low levelsof serum Cu and ceruloplasmin. Linke et al. (2000) reported that decreased level of Cu in the polyps tissue indicated that it is also possible that Cu deficiency will also influence the development of cancer. Copper status may affect aging, with a deficiency spurring sugar molecules to attach to proteins (non-enzymatic glycosylation) (McBride, 1999). This protein glycosylation is thought to cause much of the tissue damage in people with diabetes, and this increases in all humans as they age. Several experiments have attempted to induce Cu deficiency in adult men (Davis and Mertz, 1987). Two separate studies had to be prematurely terminated as some of the volunteers developed cardiovascular disturbances before developing classical

Deficiency

263

Cu deficiency signs of anemia, leukopenia, and bone changes. Klevay (1990) suggested that Cu deficiency is important in the etiology and pathophysiology of ischemic heart disease. Likewise, Kinsman et al. (1990) found that leucocyte Cu status had a significant link with atherosclerosis, which implies that Cu may be involved in the mechanisms associated with ischemic heart disease. Other forms of mild Cu deficiency are unrelated to the dietary Cu intake. One syndrome can result from excessive protein loss due to nephrosis or tropical sprue or from inadequate protein intake, as in kwashiorkor. Another, much more severe nonnutritional Cu deficiency is the Menkes' steely-hair syndrome, a hereditary disease in which intestinal Cu absorption and subsequent utilization is inadequate (Menkes et al., 1962). One clear case of Mo deficiency in humans has been documented. The mineral had been inadvertently omitted from a solution used for total parenteral feeding (TPN) (Abumrad et al., 1981). During the last 6 months of TPN, the patient developed a syndrome characterized by tachycardia, tachypnea, severe headache, night blindness, nausea, vomiting, and central scotomas leading to coma. Conclusive evidence for a dietary requirement for Mo was provided by certain genetic diseases involving a failure to synthesize sulfite oxidase or a failure to synthesize the Mo cofactor (Johnson, 1997). A failure to synthesize this cofactor leads to a lack of activity of the three Mo metalloenzymes. The genetic diseases lead to neurological damage, mental retardation, dislocation of the lens of the eye, and death.

B, Assessment of Copper Status In sharp contrast to Zn, Mn, and some other elements, good reliable procedures are available for diagnosing Cu status of livestock. Evaluation of Cu status in livestock by determination of dietary Cu has limited diagnostic value unless other elements with which Cu interacts, particularly Mo, S, Fe, and Zn, are determined also. Achromotrichia is one of the clinical signs of Cu deficiency in rats, rabbits, guinea pigs, cats, dogs, cattle, and sheep, but has not been observed in the pig. Lack of pigmentation in the fur of the rabbit and in the wool of sheep is a more sensitive index of Cu deficiency than is anemia, which is found in all species studied. Whole blood or plasma concentrations reflect the dietary Cu status, although the normal range is wide. For sheep, cattle, and goats, the normal Cu range is 0.6 to 1.5 llg/ml. It is widely accepted that whole blood or plasma Cu values consistently below 0.6 mg/ml are indicative of deficiency in sheep and cattle (NCMN, 1973). Copper deficiency is associated with a decline of the element in liver, kidney, rib, brain, and other tissues. Likewise, Cu toxicosis causes elevated tissue levels, particularly in the liver, so liver Cu concentrations could provide a useful index of the Cu status of the animal. Among ruminant livestock, liver Cu values in healthy sheep and cattle have a normal range of 100 to 400 ppm on a OM basis. In horses, pigs, domestic fowl and turkeys (as in humans), the normal range is lower, 10 to 50 ppm, with a high proportion of values lying between IS and 30 ppm. Liver Cu concentrations (Mills et al., 1976) are influenced by dietary proportions of

264

Copper and Molybdenum

Mo and S and by high intakes of Zn, Ca carbonate, and other dietary compounds. Copper values below 25 to 75 ppm of liver DM in ruminants should be used to differentiate deficient from normal animals. Activity changes of a number of Cu metalloenzymes in blood and tissues occur during Cu deficiency and offer diagnostic possibilities. Sample contamination is much less a problem with an enzyme than with plasma Cu. Todd (1970) showed that ceruloplasmin estimations on blood serum provide advantages over whole blood or plasma Cu determinations. The enzyme erythrocyte superoxide dismutase (ESOD) may be an even superior reflection of Cu status. Theoretically, SOD activities have a distinctive diagnostic contribution to make, since they decline at a slow linear rate during deficiency, compared with the rapid exponential decline in plasma or serum Cu (Underwood and Suttle, 1999). Low values confirm a prolonged deficiency, and high values a Cu overload. In human studies, ceruloplasmin and cytochrome C oxidase in platelets and mononucleated white cells were more sensitive indicators of Cu status than were plasma Cu or SOD (Milne et al., 1987). Kehoe et al. (2000) suggested that serum diamine oxidase activity is sensitive to changes in dietary Cu intakes and may also have the potential to evaluate changes in Cu status in healthy adult human subjects. Status evaluation of Mo is not well developed, but changes in Mo-dependent enzymes would seem useful. Patients with Mo cofactor deficiency lack the activities of sulfite oxidase, xanthine dehydrogenase, and aldehyde oxidase. Of these, the absence of sulfite oxidase is clearly the most devastating (WHO, 1996).

IX. SUPPLEMENTATION

Under most conditions, practical poultry and swine diets do not require Cu supplementation. Other monogastric species only rarely require Cu supplementation. Supplemental Cu for humans was once thought to be restricted to individuals with severe gastrointestinal malabsorption, genetic disorders, parenteral nutrition, and premature infants and infants fed exclusively milk diets. However, recent studies have indicated that marginal deficiency may occur (WHO, 1996). Typical swine diets contain adequate Cu to meet requirements unless young swine are restricted to milk diets, which are deficient in both Fe and Cu. Methods of anemia prevention in baby pigs that supply supplemental Fe also provide Cu, even as an impurity, to provide for the needs of the pig (see Chapter 7). Some farmers add Cu to swine diets far in excess of the requirement for this mineral (see Section VI). Growth responses with high levels (100 to 250 ppm) are the result of a pharmacological effect rather than that of the nutrient. High dietary levels of Cu have frequently improved growth parameters under the same conditions that antibiotics are also most effective. An additional benefit of higher Cu diets (e.g., 225 ppm) to swine is the improved odor characteristics of swine feces (Goihl, 2000). The microbial population of the intestine is affected and subsequent odor is reduced. As antibiotic use becomes more and more restricted by many

Supplementation

265

governments, many farmers may opt to increase the practice of elevated Cu in monogastric diets. Copper deficiency is widespread for grazing ruminants throughout the world (McDowell, 1985; see Section VIII). Both Mo toxicosis and Cu deficiency are generally corrected by providing additional Cu to the animal diets. If Mo content of the diet is less than 1 ppm (DM basis), the optimal level of Cu is between 6 and 8 ppm. Copper at 8 to 10 ppm is inadequate when Mo is between 1 and 3 ppm. With sheep, great care has to be taken to avoid over supply ofCu, because of the extreme sensitivity of this species to Cu toxicosis. When ruminants are pen fed or in dry-lot feeding, such as finishing beef cattle and lambs or milking cows, supplementary Cu is best supplied by incorporation of the element into a concentrate mixture. However, this method is usually economically prohibitive under grazing conditions (McDowell and Conrad, 1991). Copper can be added to the drinking water by a proportioner at the rate of 2 to 3 mg/l to prevent deficiencies (Farmer et al., 1982). Supplying Cu in the water probably has more problems and may be less satisfactory than other methods of supplementation (Smith and Moon, 1976). Under range conditions, Cu deficiency can be prevented by the provision of Cu-containing supplements, by dosing or drenching animals at intervals with Cu compounds, or by injection of organic complexes of Cu. Free-choice mineral supplements containing 0.1 to 0.2% Cu sulfate are generally consumed voluntarily by grazing animals in amounts sufficient to maintain adequate and safe total Cu intakes. Ruminant animals store Cu in their livers during periods of excess intake and draw on those stores when intakes are inadequate. Continuous ingestion of licks and frequent oral dosing is, therefore, less important than it is with Co. Drenching at monthly or longer intervals has been found satisfactory in Cu-deficient areas, except where Mo contents in the forages are sufficiently high to induce scouring (5 ppm or higher). In these circumstances, the Cu supplementation must be regular and more frequent (Underwood and Suttle, 1999). For grazing ruminants, the problem of Cu deficiency due to low forage Cu or a conditioned Cu deficiency (e.g., high forage Mo and/or S) is restricted to the usual six-month season for grazing of green forages. The condition is rarely seen during the feeding of stored forages in either beef or dairy cattle. Copper deficiency can be highly detrimental for ruminants grazing fresh forage in some regions, but when this same forage is dried as hay, there is no Cu deficiency (Huber et al., 1971; Allaway, 1977). These authors suggested that drying forage makes Cu more available for absorption and reduces the availability of Mo. Suttle (1980) evaluated Cu bioavailability of grazed pastures, dried grass, hay, and silage by responses in plasma Cu during repletion of hypocupremic ewes. Copper in cut hay and grass was more bioavailable than Cu in fresh grass and silage from the same field. Copper absorption in fresh grass ranged from 0.5 to 2.8% in three of the four grasses. Copper absorption was 0.9 to 1.9% for grass silage, 3.1 to 4.9% for dried grass, and 5.2 to 7.2% for hay. The ruminant groups perhaps at greatest risk of Mo-induced Cu deficiency are those that consume predominately fresh forages versus those being finished on

266

Copper and Molybdenum

concentrate diets or lactating cows receiving grains (McDowell, 1985; O'Connor et al., 2001). In more developed countries, regardless of the pasture forage species, the entire ration of the dairy cow rarely consists of more than 60% fresh forages because of the need to incorporate other feed ingredients into their diets to maximize milk production. Most large herds of dairy cattle do not graze pastures, and their diets remain fairly constant during all seasons of the year. Subcutaneous or intramuscular injections of some safe and slowly absorbed forms of Cu (e.g., glycinate) constitute satisfactory means of treating animals in Cu-deficient areas where the pasture Mo contents are moderate, even at intervals as long as 4 to 7 months (Bohman et al., 1984). There is considerable research on parenteral administration of Cu complexes with glycine, ethylenediaminetetraacetic acid (EDTA) and methionine (Hidiroglou et al., 1990). From severe Mo-toxic areas, injections of Cu compounds are often the preferred method of administration, because the primary site for Cu and Mo interaction is the gut (Suttle and Field, 1974). Copper oxide needles and Cu-containing controlled-release glass boluses have been used successfully. The application of Cu-containing fertilizers can raise forage Cu concentrations. Australian experience indicates that a single dressing of 5 to 7 kg/ha of CUS04 or its Cu equivalent in the form of cheaper Cu ores, is usually sufficient for 3 or 4 years, except on calcareous soils. Fertilization is not a dependable supplement method because sometimes problem pastures are flooded part of the year, and because Cu deficiency in grazing livestock is the result of poor uptake of Cu by plants (McDowell, 1985). Supplemental chemical forms of Cu that are rated as highly available are copper sulfate, copper lysine, copper proteinate, cupric carbonate, cupric chloride and cupric chloride-tribasic (McDowell et al., 2002). Cupric nitrate is intermediate in bioavailability, while cupric oxide and cupric sulfide are of low availability. Cupric oxide needles administered orally to ruminants, however, are effective sources of Cu for an extended period of time. Cupric carbonate is intermediate in absorption, but basic cupric carbonate [CuC0 3 Cu(OHhl is well absorbed (Baker and Ammerman, 1995). Bioavailability of dietary Cu from Cu proteinate was greater than from copper sulfate for calves fed diets containing Mo (Kincaid et al., 1986). Ward et al. (1993) reported that copper lysine and copper sulfate were of similar bioavailability when fed to cattle; however, Nockels et al. (1993) found that Cu lysine was more available than copper sulfate. A number of reports indicate that organic complexes of Cu appear to have equal bioavailability to copper sulfate. However, in two trials reported by Coffey et al. (1994) and Zhou et al. (1994), growth performance was greater in pigs fed growth-promoting levels of Cu from a Cu lysine complex than those fed copper sulfate. In cases where there is high dietary Mo, Cu in chelated form would have an advantage over an inorganic form as it may escape the complexing that occurs in the digestive system among Mo, Cu, and S (Nelson, 1988). For poultry, bioavailability of Cu lysine and Cu amino acid chelate were greater than for copper sulfate (Guo et al., 2001). Copper lysine at 16 ppm Cu vs copper sulfate was more beneficial for cattle that were borderline to deficient in Cu status

Toxicity

267

(Rabiansky et al., 1999). Pott et al. (1999) determined bioavailability of supplemental inorganic Mo sources and found sodium molybdate, ammonium molybdate, and molybdenum trioxide to be similar in availability but Mo metal was very poorly available.

X. TOXICITY A. Copper Considerable variation has been reported in the tolerance by various species of livestock to chronic Cu toxicosis (NRC, 1980), and some variation exists among breeds of animals; for instance, Merino sheep are more tolerant of dietary Cu than other breeds of sheep (Buck et al., 1973). Those most sensitive to Cu toxicity are the ruminants, while most nonruminants have relatively high tolerance for Cu. For cattle, the Cu tolerance level is 100 ppm and for sheep, 25 ppm (NRC, 1980). Horses appear to be more resistant to Cu toxicosis than either cattle, swine, sheep, or poultry. The apparent differences between ruminants and non ruminants seem in large part determined by their differences in S metabolism. Swine and poultry have routinely been given 100 to 250 ppm Cu (CUS04) as an antimicrobial agent and growth promoter, whereas these same levels would be toxic for calves and lambs. As many governments are now banning the use of antibiotics, more swine and poultry prod ucers will increase use of high levels of dietary Cu. Copper poisoning, acute or chronic, is encountered in most parts of the world. Animals may experience nausea, vomiting, salivation, abdominal pain, convulsions, paralysis, collapse, and death (NRC, 1980; Rojas et al., 1995). Hypercupremic conditions may also predispose the animal to anemia, muscular dystrophy, decreased growth, and impaired reproduction (NRC, 1980). Acute poisoning is usually observed after accidental administration of excessive amounts of soluble Cu salts, which may be present in antihelminthic drenches, mineral mixes, or improperly formulated diets. There are relatively few veterinary examples of acute Cu toxicoses (NRC, 1980). With high Cu intakes, the digestive tract is injured and there is damage to additional internal organs, including the kidney (renal tubules) and brain. Hemolysis of erythrocytes can occur and is a major response to acute toxic doses (Linder, 1996). Chronic Cu toxicosis is found in ruminants but not in monogastric species and only rarely in humans. However, with swine fed high-Cu (250 ppm) diets, other nutrients could be adversely affected, such as a destruction of natural feed tocopherols (Dove and Ewan, 1990). Chronic Cu toxicosis occurs under grazing conditions as a result of high Cu intake or very low Mo and S intakes. As an example, Cu toxicity in sheep under grazing conditions can occur where soil and pasture Cu concentrations are normal but Mo concentrations are very low (0.1 to 0.2 ppm). A Cu content in feed over 20 ppm can cause chronic poisoning in sheep (NCMN, 1973). Todd (1970) concluded that chronic Cu toxicosis in ruminants is almost entirely confined to sheep and calves. However, cattle can experience Cu

268

Copper and Molybdenum

toxicosis. When cattle consume excessive Cu, they may accumulate extremely large amounts in the liver before toxicosis becomes evident. Stress or other factors may result in the sudden liberation of large amounts of Cu from the liver to the blood, causing a hemolytic crisis. Such crises are characterized by considerable hemolysis, jaundice, hemoglobinuria (blood tinged urine), generalized icterus, widespread necrosis, and often death (NRC 1980,2001). Feeding of manure from swine or poultry fed high-Cu diets can cause Cu toxicity in ruminants, especially in sheep (Fontenot and Webb, 1974). Copper toxicosis in calves with undeveloped rumens have clinical signs and lesions sufficiently different from those produced in adult cattle primarily due to differences in ruminal function and development (Pusillo, 1997). Hemolytic crisis in calves with underdeveloped rumens is recognized by markedly decreasing numbers of circulating erythrocytes detected in blood analysis. Subclinical liver damage can occur several weeks prior to the hemolytic crisis (Sargison and Scott, 1996). If the excess dietary Cu is not corrected, the calves will not grow well and may develop a chronic toxic hepatosis. At slaughter, the calves may be icteric and the livers may be small and fibrotic, resulting in condemnation and economic loss to the producer. Performance of veal calves experiencing Cu toxicosis is impaired due to clinical diarrhea, pneumonia, and septicemia (Sullivan et al., 1991). Calves with chronic Cu poisoning exhibit the following clinical signs: depression, thirst, hemoglobinuria, pallor, jaundice or icterus, labored breathing, diarrhea, trembling, high morbidity, and high mortality. Copper finds its way into the environment in metal utensils, Cu coins, plumbing fixtures, bacteriocides, and numerous industrial applications. This has increased the probability of humans and animals becoming exposed to Cu at toxic levels (Harris, 1997). However, in humans excess intakes of Cu causing acute or even chronic toxic effects are rare. Nevertheless, there have been instances when children accidentally ingested CUS04 used as a pesticide on certain crops (e.g., fungicidal spray on grape vines). Also, there is a chronic Cu intake in the case of Indian childhood cirrhosis (Linder, 1996). It was determined that some of the women in India heated milk formula in brass pots that leached a great deal of Cu into the liquid. This resulted in the development of liver cirrhosis, one of the most common clinical signs of Cu toxicosis. Wilson Disease in humans, an inherited defect in Cu excretion, results in accumulation of Cu in the liver, brain, and other organs. Biliary Cu excretion is practically nonexistent in this genetic disorder. To treat Cu toxicosis, both Mo and S should be administered (Ivan et al., 1999). An effective treatment for lambs is to drench each lamb daily with 100 mg ammonium molybdate and 1 g sodium sulfate in 20 ml water (NRC, 1985b). Adding equivalent amounts of Mo and S to feed is equally effective. Either treatment usually requires a minimum of 5 to 6 weeks. Tetrathiomolybdate, the compound resulting from Cu-Mo-S interaction in the rumen, is also used to treat Cu toxicosis (Underwood and Suttle, 1999). High dietary concentrations of Zn protect against Cu intoxication. A diet of 100 ppm of Zn (OM basis) reduced liver Cu storage (Pope, 1971). Zinc is more protective against Cu toxicity for monogastric species than is a combination of Mo

Toxicity

269

and S. The occasional toxic effects from 250 ppm of Cu in swine diets are probably the result of a lack of Zn and Fe. The addition of 130 ppm of Zn and 150 ppm of Fe has prevented harmful effects from use of 250 ppm of Cu in the diet (Cunha, 1977).

B. Molybdenum The tolerance of animals and humans to high dietary Mo intakes varies with the species, the amount, and chemical form of the ingested Mo, the Cu status of the animal, and the diet, forms, and concentration of S in the diet. Cattle are the least tolerant species, followed closely be sheep, while pigs are the most tolerant of domestic livestock. However, humans and other monogastric species are much less affected by Sand Cu due to lack of the formation of insoluble Cu-Mo-S complexes (e.g., thiomolybdates) (See Section IV-C). For ruminants, the effects of excess Mo are largely those of Cu deficiency. However, excessive Mo (13.3 ppm) fed to steers reduced crude protein entering the small intestine, resulting in less protein absorbed postruminally (Boila and Golfman, 1991). Also, research suggests that relatively low levels of Mo can reduce growth and cause infertility in heifers independent of alterations in Cu metabolism (Phillippo et 01., 1987; Spears, 1991). A wide variation in susceptibility to Mo toxicity is caused by variations in concurrent dietary levels of Cu, Zn, S, Ag, Cd, and S-containing amino acids. Substantially higher levels of Mo would be tolerated in the presence of adequate Cu and S. Nonruminants are much more resistant to Mo toxicity, and under practical feeding conditions, only ruminants are affected by excess Mo. Ruminants also vary greatly in Mo tolerance from as low as 6.2 ppm in one study for growing cattle to approximately 1000 ppm in adult mule deer (NRC, 1980). Horses seem resistant to molybdenosis, for they can graze, without apparent problems, the same pastures that are known to cause diarrhea in cattle. There is a report of acute human Mo toxicosis of an individual who self-medicated with a Mo-chelated product (Momcilovic, 2000). After seven days, there were signs of anxiety and agitation followed by severe psychosis with strong audio and visual hallucinations, insomnia, intense craving for salt, diarrhea, and painful and cold extremities. The soils and resulting herbage in some areas have relatively high Mo levels that account for a regional incidence of molybdenosis in ruminants. An excessive intake of Mo will seriously deplete Cu reserves in cattle, quickly leading to scouring (Fig. 8.7), anorexia, anemia, loss of condition, and other signs associated with Cu deficiency. Depigmentation of skin and hair, with loss of crimp in wool, are the clinical signs of Cu toxicosis exhibited by sheep. Molybdenum levels of 5 to 6 ppm inhibit Cu storage and produce signs of molybdenosis (NRC, 1980). Even 2 ppm or less Mo can be toxic if forage Cu levels are sufficiently low. The chemical form of Mo may have an important effect on its toxicity. For example, Mo in pasture is much more toxic than a similar amount experimentally fed (Cunningham, 1950; McDowell et 01., 2002).

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(1985b). Nutrient Requirements of Sheep, 5th Ed. (1986). Nutrient Requirements of Cats, 3rd Ed. (1989). Nutrient Requirements of Horses, 5th Ed. (1993). Nutrient Requirements of Fish. (1994). Nutrient Requirements of Poultry, 9th Ed. (1995). Nutrient Requirements of Laboratory Animals. (1996). Nutrient Requirements of Beef Cattle, 7th Ed. (1998). Nutrient Requirements of Swine, 10th Ed. (2001). Nutrient Requirements of Dairy Cattle, 7th Ed. O'Conner, J. M., Bonham, M. P., Turley, E., Kchoe, C., Coulter, J. S., Faughnan, M. S., McKeown, A., McKelvey-Martin, V. J., Rock, E., Rayssiguier, Y., Mazur, A., Flynn, A., Cashman, K., Baker, A., and Strain, J. J. (2000). In Trace Elements in Man and Animals-IO (A. M. Roussel, R. A. Anderson, and A. E. Favier, eds.), p. 493, Kluwer Academic/Plenum Publishers, New York. O'Connor, G. A., Brobst, R. B., Chaney, R. L., Kincaid, R. L., McDowell, L. R., Pierzynski, G. M., Rubin, Alan, and Van Riper, G. G. (2001). J. Environ. Qual. 30, 1490. O'Dell, B. L. (1979). In Copper and Zinc in Animal Nutrition. Literature Review Committee, National Feed Ingredients Association, West Des Moines, IA. O'Dell, B. L. (1984). Nutrition Reviews: Present Knowledge in Nutrition 5th Ed. (R. E. Olson, H. P. Broquist, C. O. Chichester, W. J. Darby, A. C. Kolbye, and R. M. Stalvey, eds.), p. 506. The Nutrition Foundation, Inc., Washington, D.C. Oduor, F. D.O., Jumba, 1. 0., and Wandiga, S. O. (2000). In "Trace Elements in Man and Animals-IO" (A. M. Roussel, R. A. Anderson, and A. E. Favier, eds.), p. 786, Kluwer Academic/Plenum Publishers, New York. Olkowski, A. A., Goonerante, S. R., and Christensen, D. A. (1990). Res. Vet. Sci. 48, 82. Parham, M. R., and DeRenzo, E. C. (1978). In Handbook Series in Nutrition and Food, Section E: Nutrition Disorders Vol. II (M. Rechcigl, Jr., ed.), p. 319. CRC Press, West Palm Beach, FL. Pearce, S. G., Firth, E. c, Grace, N. D., and Fennessy, P. F. (1999). New Zealand J. Agr. Res. 42, 93. Peducasse, C. A., McDowell, L. R., Parra, L. A., Wilkins, J. V., Martin, F. G., Loosli, J. K., and Conrad, J. H. (1983). Trap. Anim. Proc. 8, 118. Pesti, G. M., and Bakalli, R. 1. (1998). Poultry Sci. 77, 1540. Petering, H. G., Murthy, L., and O'Flaherty, E. (1977). J. Agric. Food Chern. 25, 1105. Phillippo, M., Humphries, W. R., Atkinson, T., Henderson, G. D., and Garthwaite. (1987). J. Agric. Sci. 109,321. Phillippo, M., Humphries, W. R., Brenner, L., Atkinson, T. G., and Henderson, G. (1984). Trace Elem. Metab. Anim. Proc. Int. Symp. Sth, p. 17 (Abstr.). Pope, A. L. (1971). J. Anim. Sci. 33,1332. Pott, E. B., Henry, P. R., Rao, P. V., Hinderberger, E. J., and Ammerman, C. B. (1999). Anim. Feed Sci. Technol. 79, 107. Prasad, A. S. (1978). Trace Elements and Iron in Human Metabolism. Plenum, New York. Prohaska, J. R. (2000). In Trace Elements in Man and Animals-I 0 (A. M. Roussel, R. A. Anderson, and R. A. Favier, eds.), p. 909, Kluwer Academic/Plenum Publishers, NY. Prohaska, J. R., and Bailey, W. R. (1995). Proc. Society Exper. Biology and Med. 210, 107. Prohaska, J. R., Downing, S. W., and Lukasewycz, O. A. (1983). J. Nutr. It3, 1536. Pusillo, G. (1997). Feed Management 48(6),21. Rabiansky, P. A., McDowell, L. R., Velasquez-Pereira, J., Wilkinson, N. S., Percival, S. S., Martin, F. G., Bates, D. B., Johnson, A. B., Batra, T. R., and Salgado-Madriz, E. (1999). J. Dairy Sci. 82,2642. Rajagopalan, K. V. (1980). In "Molybdenum and Molybdenum-Containing Enzymes" (M. P. Coughlan, ed.), p. 241, Academic Press, New York. Rieker, J. M., Cooper, S. R., Topliff, D. R., Freeman, D. W., and Teeter, R. G. (2000). J. Equine Vet. Sci. 20, 522. Rock, E., Mazur, A., Rayssiguier, Y., Kehoe, C; O'Connor, J. M., Bonham, M. P., and Strain, J. J. (2000). In "Trace Elements in Man and Animals-IO" (A. M. Roussel, R. A. Anderson, and A. E. Favier, eds.), p. 475, Kluwer Academic/plenum Publishers, New York. Rojas, L. X., McDowell, L. R., Wilkinson, N. S., and Velasquez, J. B. (1995). Int. J. Anim. Sci. 10,41. Rucker, R. B., Cui, C. T., Eskouhie, E. H., Mitchell, A. E., Clegg, M., Uriu-Hare, J. Y., and Keen, C. L. (2000). In Trace Elements in Man and Animals-IO (A. M. Roussel, R. A. Anderson, and A. E. Favier, eds.), p. 186, Kluwer Academic/Plenum Publishers, New York. Russell, F. C., and Duncan, D. L. (1956). Minerals in Pasture: Deficiencies and Excesses in Relation to Animal Health. Technical Communication No. 15, Rowett Institute, Aberdeen, Scotland.

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Chapter 9

Cobalt

I. INTRODUCTION

Cobalt must be supplied in the diet of monogastric animal species and humans in its active form, vitamin B12. When these species receive adequate dietary vitamin B12, there is no convincing evidence of a requirement for or benefit from dietary Co. Cobalt is, however, a dietary essential for ruminants; ruminal microorganisms incorporate Co into vitamin B12 . A direct requirement for Co is found only in certain bacteria and algae; vitamin B12 is produced only by these microorganisms and not by higher plants or by animals. Prior to discovery of the need for Co in the 1930s, grazing ruminants could not be produced in many world regions due to deficient concentrations of this element in forages.

II. mSTORY Cobalt had been known to be present in plant tissue since the mid 1800s and was also found in animal tissues in the early 1900s. It was not until 1935, however, that Australian research established Co as a dietary essential for ruminants. The effects of Co deficiency had long been experienced in many parts of the world, resulting in "wasting diseases" and in general unsatisfactory conditions for raising sheep and cattle. The more severe forms of Co deficiency in sheep and cattle were given a variety of local names such as bush-sickness in New Zealand, coast disease and wasting disease in Australia, salt sick, neck ail, and Grand traverse disease in the United States, Nakuruitis in Kenya, pinning disease in Great Britain and toque, mal de colete, pest de secar, and sablose in Brazil. An appropriate designation for all these conditions was suggested as enzootic marasmus (muscular wasting) (Underwood and Suttle, 1999). In these regions sheep and cattle were found unable to thrive unless they were periodically moved to "healthy" areas. Ruminants remaining in the deficient areas stopped eating, became weak and emaciated and usually died. Because diseased animals could be cured by a change to "healthy" ground, it was rightly assumed that these were nutritional deficiency diseases. Iron (Fe) was first suggested as the nutrient effective in preventing the debilitating disease of "bush sickness" in cattle of New Zealand (Aston, 1924). The Fe 277

278

Cobalt

Fig. 9.1 A Co-deficient heifer (left) in Florida that had access to an Fe-Cu salt supplement. Note the severe emaciation. Her blood contained 6.6 g of hemoglobin per 100 ml on February 25. 1937. The same heifer (right) fully recovered with an Fe-Cu-Co salt supplement while on the same pastures. (Courtesy of the late R.B. Becker. University of Florida, Gainesville)

deficiency theory received support from other parts of the world where large doses of crude Fe salts or ores were found effective in preventing or curing similar maladies. Iron deficiency then became accepted as the cause of this and similar diseases until it became clear from the Australian studies that the efficacy of the Fe compounds resided in the Co with which they were contaminated (Filmer and Underwood, 1934). Research with an Fe-free extract prepared from one of the curative compounds (limonite) demonstrated that the Co found in the extract was just as potent as whole limonite. The first observation that Co was an essential trace element for livestock was made by Lines in 1934 who showed that it corrected unthriftiness in sheep grazing on Kangaroo Island, Australia (Lines, 1935). By 1935, Australian research had proven that Co was effective in preventing coast disease and wasting disease (Underwood and Suttle, 1999). In Florida (USA) in 1937, Co deficiency in cattle (Fig. 9.1) was established; deficiency of this element was responsible in part for the condition known as salt sick (Becker et al., 1965). Within a few years of these discoveries, Co supplements were found to be equally effective in the cure and prevention of all the diseases previously shown to respond to massive doses of Fe compounds, and the soils and herbage of the affected areas were shown to contain subnormal levels of Co (Russell and Duncan, 1956). However, from the mineral-deficient, sandy soils of Florida, the salt sick condition could be prevented only if all three of the deficient elements, Co, copper (Cu), and Fe, were supplied in adequate quantities. The action of Co in the body and reason for its necessity were not discovered until the simultaneous discovery of vitamin B12 by Smith (1948) in England and Rickes et al. (1948) in the United States. Both studies reported the presence of Co in their compounds, which were effective against pernicious anemia in humans. As a result of this finding it was shown that consequences of Co deficiency in ruminants are due to a deficiency of vitamin B l 2 in the tissues and can be averted by injection of vitamin B l 2 (Smith et aI., 1951).

Chemical Properties and Distribution

279

III. CHEMICAL PROPERTIES AND DISTRIBUTION Cobalt makes up only 0.001 to 0.002% of the earth's crust, and has a single isotope with an atomic weight of 58.9. Cobalt is a gray, hard, magnetic metal that exists in two allotropic forms. The hydrated salts of Co are red. Cobalt concentrations in soils and plants are highly variable, with plant Co concentrations affected by many factors including soil Co concentrations, fertilization, plant species, plant growth, and soil factors, including pH and drainage. Cobalt is widely distributed in the animal body, with high concentrations in liver, bone, and kidney (Underwood and Suttle, 1999). The average whole body Co content of a normal adult human was reported to be 1.1 mg (Yamagata et al., 1962). This value may be compared with the commonly accepted values of 12 to 20 mg for manganese (Mn), 80 mg for Cu, and 2000 mg for zinc (Zn). Typical Co concentrations for human and animal tissues have been reported by Smith (1987). However, tissue and fluid concentrations of the active form of Co, vitamin B\2, are of more significance. Foods of animal origin including liver, kidney, meat, fish, milk, and eggs are generally good sources of vitamin B\2' with these concentrations influenced by dietary intakes of vitamin B\2 and Co or injectable vitamin B 12 . Vitamin B\2 contains about 4.5% Co, and is also referred to as cobalamin. Vitamin B I2 has the general formula C63HggN14014PCO, with a molecular weight of 1355. The structure of vitamin BI2 or one cobalamin form, cyanocobalamin is shown in Fig. 9.2. Vitamin B12 resembles a porphyrin structure consisting of four pyrrole nuclei coupled directly to each other, with the inner nitrogen atom of each

CH 3 CHrCONH2 CHryH2

co I

NH2

Fig.9.2 Structure of vitamin Bl 2 (cyanocobalamin).

280

Cobalt

pyrrole coordinated with a single atom of Co. Cyanide, which lies above the planar ring, is attached to the Co atom, and thus, the name cyanocobalamin. Cyanocobalamin is not the naturally occurring form of the vitamin, however, it is the most widely used form of cobalamin in clinical practice because of its relative availability and stability. Deoxyadenosylcobalamin, hydroxycobalamin, and methylcobalamin are predominant forms of cobalamins in animal tissue (Farquharson and Adams, 1976). Vitamin B 12 is a dark-red crystalline hygroscopic substance, freely soluble in water and alcohol but insoluble in acetone, chloroform, or ether. Cyanocobalamin is the most complex structure and heaviest compound of all the vitamins. Oxidizing and reducing agents and exposure to sunlight tend to destroy its activity. Losses of vitamin B)2 during cooking are usually not excessive because it is stable at temperatures lower than 250°C.

IV. METABOLISM

A. Digestion, Absorption, and Transport Cobalt is well absorbed by small laboratory animals and humans. Mice were found to absorb 26.2% of an oral dose of labeled Co (Toskes et al., 1973), while human balance studies indicated a variable intestinal absorption ranging from 20 to 97% (Smith, 1987). Cobalt absorption by the ruminant is much less efficient than that in simple-stomached animals with about 3% converted to vitamin B l2 in the rumen (Smith and Marston, 1970). Following oral or intraruminal administration of labeled Co to sheep or cattle, 84 to 98% appeared in the feces within 5 to 14 days (Smith, 1987). Efficiency with which dietary Co is converted into vitamin B I 2 is inversely proportional to Co intake. Thus Smith and Marston (1970) reported 13 ± 5% conversion in sheep on a Co-deficient diet but only about 3% when intake was adequate. In nonruminants (and perhaps in ruminants), absorption of Co appears to share a common intestinal mucosal transport system with Fe; in which Co and Fe mutually inhibit one another's absorption, and Co absorption is enhanced in Fe deficiency (Underwood and Suttle, 1999). Of the total vitamin B 12 produced, only I to 3% is absorbed. For most species, the absorptive site of vitamin B12 is the lower portion of the small intestine. Features of vitamin B12 absorption found in nonruminant species such as an increase in absorptive capacity in pregnancy and in the colostrum-fed neonate, may well also occur in ruminants (Smith, 1997). Substantial amounts of B)2 are secreted into the duodenum and then reabsorbed in the ileum. The poor absorption in the rumen may be related to the rapid binding of Co by ruminal microorganisms. Also, absorption is reduced because Co is converted into nonvitamin B12 analogs that cannot be absorbed or used. It is probable that the balance of production between vitamin B)2 and its inactive analogues is influenced by other dietary variables.

Metabolism

281

Passage of vitamin B12 through the intestinal wall requires the intervention of certain carrier compounds able to bind the vitamin molecule. Vitamin B]2 in the diet is bound to food proteins. In the stomach, the combined effect of gastric acid and peptic digestion releases the vitamin, which is then bound to a nonintrinsic factor-eobalamin complex (cobalophilin). The B12 remains bound to cobalophilin in the slightly alkaline environment of intestine until pancreatic proteases (e.g., trypsin) partially degrade the cobalophilin protein and thereby enable B12 to become bound exclusively to intrinsic factor. Therefore, human patients with pancreatic insufficiency absorb B12 poorly (Jorgensen et al., 1991), and this malabsorption is completely corrected by administration of pancreatic enzymes or purified trypsin. Intrinsic factor is a glycoprotein (mucoprotein) synthesized and secreted by parietal cells of the gastric mucosa. Atrophy of the fundus, where intrinsic factor is produced, and lack of free HCI (achlorhydria) are usually associated with pernicious anemia (Behrns et al., 1994). Gastric juice defects are responsible for most cases offood-vitamin B12 malabsorption (Carmel, 1994). The intrinsic factorB I2 complex is transiently attached to an ileal receptor. There is transport of vitamin B I 2 from the receptor-intrinsic factor-Bj , complex through the epithelial cell to portal blood. The absorption of vitamin B12 is limited by the number of intrinsic factor-vitamin B12 binding sites in the ileal mucosa, so that not more than about I to 1.5 ug of a single oral dose of the vitamin in humans can be absorbed (Bender, 1992). When B I 2 enters the portal blood it is no longer bound to intrinsic factor but to specific transport proteins called transcobalamins (Seetharam et al., 1999). Three binding proteins have been identified in normal human serum and are designated as transcobalamin I, II, and III. Transcobalamin II appears to be primarily concerned with transport of vitamin B12, whereas transcobalamin I is involved in storage of the vitamin. The function of transcobalamin III is to provide a mechanism for returning vitamin B12 from peripheral tissues to the liver, as well as for clearance of other corrinoids without vitamin activity (e.g., undesired analogs of B I 2 ) , which may arise either from foods or the products of intestinal bacterial action, and be absorbed passively across the lower gut (Bender, 1992).These corrinoids are then secreted into the bile, bound to cobalophilins. Like dietary vitamin B12 bound to salivary cobalophilin, the biliary cobalophilins are hydrolyzed in the duodenum, and the released vitamin B 12 binds to intrinsic factor, permitting reabsorption into the ileum. To summarize the B12 absorption for most species studied, the following are required (McDowell, 2000): (1) adequate quantities of dietary vitamin B12 , (2) normal stomach for breakdown of food proteins for release of vitamin B12, (3) normal production of cobalophilin (nonintrinsic factor) secreted in saliva, (4) normal stomach for production of intrinsic factor for absorption of B12 through the ileum, (5) normal pancreas (trypsin) required for release of bound B12 prior to combining the vitamin with the intrinsic factor, and (6) normal ileum with receptor and absorption sites. Additional factors that diminish vitamin B I2 absorption include deficiencies of protein, Fe, and vitamin B6 , thyroid removal, and dietary tannic acid.

Cobalt

282

B. Tissue Distribution and Storage About 43% of body Co is stored in muscles and approximately 14% is in bone (Underwood, 1977),with the remainder in other tissues; the kidney and liver contain the most Co (Henry et al., 1997). Cobalt levels (dry basis) in kidney, liver, pancreas, spleen, and heart average 0.25, 0.15, 0.11,0.09, and 0.06 ppm, respectively. Andrews et al. (1960) reported that the proportion ofliver Co that occurs as vitamin B12 varies with the Co animal status. Under grazing conditions where there is adequate Co in the pasture, most liver Co can be accounted for as vitamin B12 , but in Co deficiency only about one-third of the liver Co exists in this form. This indicates that in Co deficiency, liver vitamin B12 is depleted faster than other forms of Co. Even though vitamin B12 is water-soluble, Kominato (1971) reported a tissue half life of 32 days, indicating a considerable degree of tissue storage. Cattle and sheep with normal stores of the vitamin in their liver can go for months on a Co-deficient diet without showing signs of a vitamin B12 shortage. Placental transfer is not great and liver vitamin BI2 concentrations in the lambs were less than half of those found in the mother (Grace et al., 1986). In normal human subjects, vitamin B I2 is found principally in the liver, the average amount is 1.5 mg. Kidneys, heart, spleen, and brain each contain about 20 to 30 ug (Ellenbogen and Cooper, 1991). Vitamin B12 is stored in the liver in the largest quantities for most animals that have been studied, but in the bat it is stored in the kidney. Vitamin B12 storage in humans can exceed the daily requirement by about 1000-fold.

C. Excretion In animals, both Co and vitamin B12 are mainly excreted in the feces, although variable amounts are secreted in urine (Smith and Marston, 1970). Lactating cows on a normal diet excrete 86 to 87.5% of all absorbed Co in the feces (mainly with the bile), 0.9 to 1.0% with urine and ll.5 to 12.5% with milk. For humans, the major route of excretion of both vitamin B12 and Co is via feces. Small amounts of Co are also lost in the urine, sweat, and hair.

V. PHYSIOLOGICAL FUNCTIONS The only known function of Co is its participation in metabolism as a component of vitamin B 12 ; thus the signs of Co deficiency are in reality signs of a shortage of vitamin B12 • Vitamin B 12 is an essential part of several enzyme systems which carry out a number of very basic metabolic functions. Most of the cobalamins occur as two coenzymatically active forms, adenosylcobalamin and methylcobalamin. Cyanocobalamin is converted within cells to either methylcobalamin, a coenzyme for methyltransferase, or adenosylcobalamin, the coenzyme for mutase. A number of vitamin B1rdependent metabolic reactions have been identified in microorganisms, however, only three vitamin B1rdependent enzymes have been

Physiological Functions

283

discovered in animals: methylmalonyl CoA mutase and leucine mutase which each required adenosylcobalamin, and methionine synthetase which requires methylcobalamin. Most reactions requiring adenosylcobalamin can be classified as rearrangement reactions of the carbon skeleton of several metabolic intermediates, a hydrogen atom moves from one carbon atom to an adjacent one in an exchange for an alkyl, acyl, or electronegative group, which migrates in the opposite direction (Ellenbogen and Cooper, 1991). In all these rearrangement reactions, adenosylcobalamin is an intermediate hydrogen carrier. The reactions requiring methylcobalamin involve transfer or synthesis of one-carbon units, for example, methyl groups. Most reactions of vitamin BJ2 enzymes involve transfer or synthesis of onecarbon units, for instance methyl groups. Though the most important tasks of vitamin B I2 concern metabolism of nucleic acids and proteins, it also functions in: (I) purine and pyrimidine synthesis, (2) transfer of methyl groups, (3) formation of proteins from amino acids, and (4) carbohydrate and fat metabolism (McDowell, 2000). Vitamin Bl 2 promotes red blood cell synthesis and maintains nervous system integrity, which are functions noticeably affected in a deficiency. Vitamin BI2 is metabolically related to other essential nutrients such as choline, methionine, and folacin, and functions in transmethylation and biosynthesis of labile methyl groups (Savage and Lindenbaum, 1995; McDowell, 2000). The purine bases (adenine and guanine) as well as thymine are constituents of nucleic acids and with a folacin deficiency there is a reduction in biosynthesis of nucleic acids essential for cell formation and function. Deficiency of BJ2will induce a folacin deficiency by blocking utilization of folacin derivatives. A vitamin BJ2-containing enzyme removes the methyl group from methylfolate, thereby regenerating tetrahydrofolate (THF), from which is made the 5,20-methylene THF required for thymidylate synthesis. Metabolism of labile methyl groups plays a significant part in biosynthesis of methionine from homocysteine. A vitamin B1rrequiring enzyme, 5-methyltetrahydrofolate-homocysteine methyltransferase, catalyses reformation of methionine from homocysteine. Activity of this enzyme is depressed in liver of vitamin B1rdeficient sheep (MacPherson, 1982). This defect could lead to a deficiency of available methionine which may account for impairment of nitrogen metabolism in vitamin B 12-deficient sheep. Overall synthesis of protein is impaired in vitamin BJ2-deficient animals. Wagle et al. (1958) demonstrated that rats and baby pigs deprived of vitamin B12 were less able to incorporate serine, methionine, and phenylalanine into liver proteins. Impairment of protein synthesis may be the principal reason for the growth depression frequently observed in these animals (Friesecke, 1980). In animal metabolism, propionate of dietary or metabolic origin is converted into succinate which then enters the tricarboxylic acid (Krebs) cycle. Propionate is a three-carbon, and succinate a four-carbon compound, therefore, this process requires the introduction of a one-carbon unit. Methylmalonyl CoA isomerase (mutase) is a vitamin BJ2-requiring enzyme (5' deoxyadenosylcobalamin) which catalyzes the conversion of methylmalonyl-CoA to succinyl-CoA.

284

Cobalt

Metabolism of propionic acid is of special interest in ruminant nutrition because of the large quantities produced during carbohydrate fermentation in the rumen. The main source of energy to ruminants is not glucose but primarily acetic and propionic acids. In Co or vitamin B12-deficiency, the rate of propionate clearance from blood is depressed, and methylmalonyl-CoA accumulates. This results in an increased urinary excretion of methylmalonic acid and also loss of appetite because impaired propionate metabolism leads to higher blood propionate levels inversely correlated to voluntary feed intake (MacPherson, 1982). Injection of Co-deficient animals with vitamin B 12 produces an overnight improvement in appetite, whereas oral dosing with Co takes from 7 to 10 days to produce the same effect. A further important function of vitamin B 12 in intermediary metabolism consists of maintaining glutathione and sulfhydryl groups of enzymes in the reduced state. The reduced activity of glyceraldehyde-3-phospate dehydrogenase, which needs glutathione as a coenzyme, is possibly responsible for carbohydrate metabolism being impaired in a vitamin B12 deficiency. Vitamin B I2 also influences lipid metabolism via its effect on the thiols. Cobalt-vitamin B 12 deficiency has been shown to influence the immune response in sheep (Vellema et al., 1996). Non Co-supplemented lambs had lower serum vitamin B 12 and lower lymphoblastic responses after paratuberculosis vaccination.

VI. REQUIREMENTS

A direct requirement for Co is found only in certain bacteria and algae. The Co requirement of ruminants is actually a Co requirement of ruminal microorganisms. The microbes incorporate Co into vitamin B 12, which is utilized by both microorganisms and animal tissues. If Co intake is sufficiently low to cause the Co concentration in the ruminal fluid to fall below about 0.5 ng/ml, vitamin B 12 synthesis by the microorganisms is inhibited (Smith and Marston, 1970). There is no convincing evidence that Co is needed when adequate vitamin B 12 is present in the diet for monogastric species, and therefore generally no requirements are recommended by National Research Council publications. A. Ruminant Requirements Under typical conditions a rumen synthesizes all B-vitamins at 6 to 8 weeks of age. Therefore, only young ruminants that do not have a fully developed rumen would be expected to require a dietary source of vitamin B 12. Estimated vitamin B I2 requirements of the dairy calf are between 0.34 and 0.68 J.tg/kg body weight (NRC, 2001). Dietary Co requirements have been established at 0.10 to 0.20 ppm (Table 9.1). The Agricultural Research Council (ARC, 1980) estimated the Co requirement for cattle and sheep to be 0.11 ppm. Precise estimates of minimum Co requirements

Requirements

285

TABLE 9.1 Dietary Cobalt Reqatremeats" Animal species Beef cattle" Dairy cattle Sheep Horses

Requirement (ppm) 0.10 0.10 0.10-0.2 0.10

Reference NRC NRC NRC NRC

(1996) (2001) (1985) (1989)

"Expressed as per unit animal feed, dry basis. "Stangl et al. (2000) suggest a requirement from 0.2 to 0.3 ppm.

applicable under all grazing conditions are difficult because of the influence of many variables such as seasonal changes in herbage Co concentrations, selective grazing habits, and soil contamination. Under grazing conditions, lambs are the most sensitive to Co deficiency, followed by mature sheep, calves, and mature cattle (Andrews, 1956). Ruminants have higher vitamin B)2 requirements than nonruminants, presumably because of its involvement in the metabolism of propionic acid. Sheep appear to be more susceptible than cattle to Co deficiency and wethers appear to be more susceptible than ewes (Shallow et al., 1989; Kennedy et al., 1995). The presence of gastrointestinal parasites may also increase the susceptibility of sheep to Co deficiency (Judson et al., 1985; Masters et al., 1992; Peverille and Judson, 1999). Experiments with sheep suggested an oral requirement for growing lambs of some 200 Ilgjday, which is about 10 times the reported oral requirement for other species per unit of food intake (Marston, 1970). The surprisingly high requirement of ruminants for Co arises partly from the low efficiency of production of vitamin B12 from Co by the ruminal microorganisms and partly from the low efficiency of absorption of vitamin B I2 (see Section IV). It has been reported that the dry matter of grass in healthy areas contains around 0.10 ppm of Co or more, on the average, as compared with 0.004 to 0.07 ppm for deficient areas. As little as 0.10 ppm has restored sick animals to health. Further evidence of 0.10 ppm as the dietary requirement for sheep was provided by Mohammed (1983), who fed various levels of Co and found that vitamin B)2 and propionic acid concentrations in rumen were maximal at 0.10 ppm. The dietary Co levels of about 0.10 ppm diet dry matter have widely been accepted as the minimum requirement for cattle. However, recent work (Kirchgessner et al., 1998; Stangl et aI., 1999b; Schwarz et al., 2000) indicates a higher Co requirement for growing beef cattle than is currently estimated by the NRC (1996). Furthermore, newer findings from a few authors also indicate the necessity to increase the amount of Co for ruminants to a level of 0.3 to 0.5 ppm dry matter for optimum ruminal microbial activity, fermentation and vitamin B12 synthesis (Paragon, 1993; Singh and Chhabra, 1995). Stangl et al. (2000) estimated the Co requirement of growing cattle on the basis of vitamin B I2 and folacin, plasma levels of homocysteine and methylmalonic acid (MMA) and hematological

Cobalt

286

parameters. On the basis of various criteria these researchers concluded the following: Basis

Requirement

Plasma vitamin B 12 To maximize liver vitamin B 12 To maximize liver folacin To minimize homocysteine MMA as response criterion Hemoglobin and hematocrit

0.26 0.20 to 0.23 0.17 to 0.19 0.16 0.13 to 0.16 0.11

A staggers syndrome in sheep and cattle that have grazed pastures in which the forage species, Pha/aris tuberosa, predominated have developed a disease known as "Phalaris staggers" (Underwood, 1977). A number of world regions, including Australia, New Zealand, and the United States (Florida and California), have reported this condition, with affected animals suffering irreversible and commonly fatal nerve degeneration (Ruelke and McCall, 1961; Smith, 1987). Cobalt supplementation has been shown to alleviate the "Phalaris staggers" condition and would, therefore, influence the Co requirement (Lee et al., 1957).

B. Monogastric Requirements Cobalt deficiency per se has never been clearly demonstrated in monogastric species and is essential only as a component of vitamin B 12 . Most monogastric species have only a limited ability to synthesize vitamin B12 in the lower portion of the intestinal tract. However, although not well quantified, synthesis of vitamin B I2 in the alimentary tract of the horse and its subsequent absorption from the large intestine have been shown (NRC, 1989). This finding was expected because horses have often been observed to remain in good health while grazing pastures so low in Co that ruminants confined to them have died. Although little is known about Co metabolism in the horse, it is indicated that 0.10 ppm dietary Co should be adequate (NRC, 1989). Rabbits also make good use of microorganisms in the digestive tract to synthesize vitamin B 12 from Co. Utilization of Co by the bacterial flora is much more efficient in rabbits than in ruminants. Vitamin B l2 requirements for monogastric species have been established by the various National Research Council publications and generally range from 3 to 50 Jlg/kg. For nonruminant species, dietary vitamin B 12 needs depend on intestinal synthesis (and absorption) and tissue reserves at birth. Intestinal synthesis probably explains frequent failures to produce a vitamin B I2 deficiency in pigs and rats on diets designed to be vitamin B12-free. The deficiency can be readily produced in rats, however, when coprophagy is prevented completely (Barnes and Fiala, 1958). Coprophagous animals and poultry on deep litter receive excellent sources of vitamin BI2 by direct absorption of the vitamin produced by bacterial synthesis in the intestine (NRC, 1994). However, the amount from this source is not reliable.

Natural Sources

287

Human vitamin B 12 requirements range from 2.6 to 2.8 Ilgjday (DRI, 2001). These requirements have been estimated from three different types of studies (Ellenbogen and Cooper, 1991): (I) the amount necessary to treat megaloblastic anemia from vitamin B I2 deficiency, (2) comparison of blood and liver concentration in normal and cobalamin-deficient subjects, and (3) body stores and turnover rates of the vitamin. Obviously, the requirement for B 12 will be substantially higher for humans lacking intrinsic factor or other conditions which affect absorption and metabolism of the vitamin (McDowell, 2000).

VII. NATURAL SOURCES Although most feeds are adequate in Co, the element is deficient in forages for grazing ruminants in many parts of the world (McDowell, 1985). Concentration of Co in crops and forages is dependent on soil factors, plant species, stage of maturity, yield, pasture management, climate, and soil pH. Cobalt deficiency is found in soils of diverse origin, including coarse, volcanic, sandy loams, and leached sands. Soil containing less than 2 ppm of Co is generally considered deficient for ruminants (Correa, 1957). Raising the pH by liming reduces the Co uptake by the plant and may increase the severity of the deficiency. Plants grown on a IS ppm soil that is neutral or slightly acidic may contain more Co than those grown on a 40 ppm alkaline soil (Latteur, 1962). Not only does liming reduce availability by causing greater adsorption on to the various exchange sites, resulting in the precipitation of various Co salts, but higher pH also encourages the establishment of better-quality grasses of lower Co content (MacPherson, 2000). High rainfall tends to leach Co from the topsoil. This problem is often aggravated further by rapid growth of forage during the rainy season, which dilutes the Co content. Waterlogging of soils allows the release of Co from the soil minerals into soil solution, so that forage on poorly drained soils can have up to seven times more Co than that from well-drained soils (MacPherson, 2000). Plants have varying degrees of affinity for Co, some being able to concentrate the element much more than others. Legumes, for example, generally have greater ability to concentrate Co than do grasses (Underwood, 1977; Singh and Aruna, 1994; MacPherson, 2000). Cobalt is needed by the N-fixing bacteria in the nodules of roots of legumes. The Co content of representative feedstuffs is shown in Appendix Table II. Among various feeds, green leafy vegetables are the richest and most variable in Co content, while refined cereals and sugar are the poorest. Typical values for the former group are 0.2 to 0.6 ppm (dry basis), and for the latter 0.01 to 0.03 ppm Co (dry basis). The organ meats, liver and kidney, commonly contain 0.15 to 0.25 ppm (dry basis), and the muscle meats about half those levels (Smith, 1987). The average Co content of normal cow's milk has been reported to be approximately 0.004 to 0.009 ppm (dry basis) (Underwood and Suttle, 1999). It is possible to increase these values several fold by heavy supplementation of the cow's diet with Co salts. Because the origin of vitamin B I2 in nature appears to be microbial synthesis, plant products are practically devoid of vitamin B 12 • The vitamin B 12 reported in

288

Cobalt

higher plants in small amounts may result from synthesis by soil microorganisms, excretion of the vitamin into soil, with subsequent absorption by the plant. There is no convincing evidence that the vitamin is produced in tissues of animals, and their intestinal microflora have an extremely limited capacity to synthesize vitamin B l2 at a point in the digestive tract where the vitamin can be absorbed. Any feed source that has been contaminated with feces is a source of the vitamin. Foods of animal origin, including meat, liver, kidney, eggs, and fish are reasonably good sources of vitamin Bl2 (McDowell, 2000). Kidney and liver are excellent sources, these organs are richer in vitamin B l2 for ruminants than for nonruminants. Vitamin B I2 presence in tissues of animals is due to the ingestion of vitamin B l2 in animal foods or from intestinal or ruminal synthesis. Among the richest sources are fermentation residues. High vitamin B l2 sources are activated sewage sludge and manure.

VIII. DEFICIENCY

A. Effects of Deficiency 1.

~ONOGASTmC SPEClliS

Vitamin B l2 is a dietary essential in monogastric species. The results of vitamin BJ2 deficiency in humans are megaloblastic anemia (pernicious anemia) and neurological lesions. A deficiency of vitamin B12 in man usually is conditioned by a deficiency of an intrinsic factor necessary for its absorption or consumption of strict vegetarian diets. Vitamin BI 2 deficiency is estimated to affect 10to 15% of people over the age of 60 (Baik and Russell, 1999). Problems, including sensory losses and dementia, have been documented with "low normal" levels of vitamin B l2 in the absence of anemia or very low serum levels, suggesting that deficiency may actually be more prevalent than previously thought (Tucker, 1995). For other animals, anemia is not characteristic of a vitamin B12 shortage. In rats, guinea pigs, swine, and poultry, vitamin Bl2 functions as a growth factor, although a mild anemia does occur in a small percentage of deficient swine. McDowell (2000) has reviewed vitamin B l2 deficiency signs and symptoms for various animal species and humans (Fig. 9.3). For all species there is, in general, reduction in body weight gain (Fig. 9.4), feed intake and feed conversion. Reproduction is affected in pigs with litter size and pig survival reduced. In poultry, hatchability is affected with embryos dying about the 17th day of incubation. 2. RUMINANTS

Before the recognition of Co deficiency in ruminants in many parts of the world, cattle could be maintained on deficient pastures only if they periodically moved to "healthy" ground. Cobalt deficiency could be prevented by taking animals every year for a few months to a "healthy" region, preferably during the rainy season. An

Deficiency

289

Fig. 9.3 Pernicious anemia. In addition to megaloblastic anemia, an additional sign with vitamin B12 deficiency is a pale, smooth tongue with inflammation (A). The normal tongue in (B) has papilla. The smooth tongue is found in one-third to one-half of pernicious anemia patients. (Courtesy of R.R. Streiff, Veterans Administration. University of Florida, Gainesville)

example of the necessity of periodically moving animals was illustrated in a disease condition known as "togue" in Espirito Santo, Brazil (Tokarnia et al., 1971,2000). The disease was observed when animals stayed for a period longer than 60 to 180 days on certain pastures. Sick animals isolated themselves from the rest of the herd, were

290

Cobalt

Fig.9.4 Vitamin BI2 deficiency. Top: pig deficient in vitamin B12 . Note rough hair coat and dermatitis. Bottom: control pig. (Courtesy of the late D.V. Catron and Iowa State University, Ames)

apathetic, showed loss ofappetite, rough haircoat, dry feces, and lost body condition. If the animals were not moved from the pasture, they died, but if they were taken to a pasture where the disease did not occur, the animals recovered quickly. Cobalt deficiency occurs in large areas of many countries and is often, but not exclusively, restricted to grazing ruminants that have little or no access to concentrates. With the exception of phosphorus (P) and Cu, Co deficiency is the most severe mineral limitation to grazing ruminants in tropical countries (McDowell, 1997).A total of 24 developing tropical countries or regions in Latin America, Africa, and Asia have noted Co deficiencies or low forage concentrations of this element (McDowell, 1985). Deficiency signs for Co are not specific, and it is often difficult to distinguish between an animal having a deficiency and malnutrition due to low intake of energy and protein, and an animal that is diseased or parasitized. Acute clinical signs of Co

Deficiency

291

Fig.9.5 A Co-deficient lamb (top) fed a diet containing 0.05 ppm of Co and weighing 22 kg at the end of the experimental period. A positive control lamb (bottom) that daily received, in addition, 0.1 mg of Co as the sulfate and weighs 42 kg. (Courtesy of S.E. Smith, Cornell Agricultural Experiment Station, Ithaca. NY)

deficiency include lack of appetite, rough hair coat, thickening of the skin, anemia (normocytic and normochromic), wasting away (Fig. 9.5), and eventually death. At necropsy the body of severely affected animals presents a picture of extreme emaciation, often with a total absence of body fat. The liver is fatty, the spleen

292

Cobalt

hemosiderized, and in some animals there is hypoplasia of the erythrogenic tissue in the bone marrow (Filmer, 1933). The anemia in lambs is normocytic and normochromic but the mild anemia is not responsible for the main signs of Co deficiency. Inappetence and marasmus invariably precede any considerable degree of anemia. The first discernible response to Co feeding, or vitamin BJ2 injections, is a rapid improvement in appetite and body weight. Subclinical deficiencies or borderline states are extremely common and are characterized by low production rates unaccompanied by clinical manifestations or visible signs. Subclinical deficiencies of Co often go unnoticed, thereby resulting in great economic losses to the livestock industry (Latteur, 1962). No estimate can be made of the effect of Co subdeficiencies on animal performance in general, but, in many areas of the world, it is one of the major causes of poor production. The long term effects of moderate Co deficiency in cattle were reported by Stangl et al. (1999a,b). In addition to reduced feed intake and daily gain, a significant number of changes in lipid metabolism occurred, affecting liver, brain, and erythrocyte lipids and lipoproteins. The diminished liver vitamin B I2 level resulted in significantly reduced folacin level, and dramatic accumulation of Fe and nickel in liver. Cobalt deficiency has been reported to reduce lamb survival and increase susceptibility to parasitic infestations in cattle and sheep (Ferguson et al.. 1988; Suttle and Jones, 1989). Cobalt deficiency has been associated with photosensitization of lambs characterized by a swollen head (Hesselink and Vellema, 1990). The condition responded to two injections of vitamin B J2 three weeks apart. Cobaltdeficient ewes produced fewer lambs and had more stillbirths and neonatal mortalities than Co sufficient controls (Fisher and MacPherson, 1991). Lambs from deficient ewes were also slower to start suckling. Auricular myocardial necrosis has been reported in Co-deficient sheep (Mohammed and Lamand, 1986). Two other conditions attributed to Co deficiency are ovine white liver disease and Phalaris staggers (Graham, 1991; Kennedy et al., 1994b). Ovine white liver disease is characterized by hepatic lipidosis and emaciation. At necropsy, affected lambs had pale, swollen, friable fatty livers, and showed accumulation of lipofuscin (Kennedy et al., 1994b). Alteration in choline synthesis presumably leads to impaired lipid mobilization, but white liver disease may be complicated by other factors. Kennedy et al. (1997) suggest that reduced activities of the vitamin BJ2dependent enzymes, methylmalonyl CoA mutase and methionine synthase, and lipid peroxidation are of likely pathogenetic importance in the development of the lesions. Similarly, the persistent neural effects of Phalaris spp., inducing Phalaris staggers, have been shown to be preventable with oral Co supplementation, but not with B 12 • Peducasse et al. (1983) evaluated the mineral status of two cattle-producing regions of Bolivia. From one region, all forages were above the Co requirement, while, for the second region, 47.6% of forages contained less than 0.1 ppm of Co. Vargas et al. (1984) analyzed forages for mineral content from three regions in the llanos of Colombia and found 72 and 31% of all forages below 0.1 ppm for the wet and dry seasons, respectively. On the contrary, of forage samples collected from

Deficiency

293

Fig.9.6 Cobalt deficiency. A - cobalt-deficient cattle in northern Mato Grosso, Brazil; B - an extreme condition of Co-deficiency of an Indu-Brazil steer in Sao Paulo, Brazil. (A - courtesy of Jiirgen Dobereiner and Carlos H. Tokarnia, EMBRAPA-UFRRJ, Rio de Janeiro. Brazil; B - Nelson dos Santos Fernandez. Instituto Biologico de Sao Paulo, Sao Paulo, Brazil)

northern Mato Grosso, Brazil, mean Co concentrations were deficient (0.08 ppm) in the dry season but adequate during the wet season (0.17 ppm) (Sousa et al., 1981 ). In Malaysia, Co deficiency was reported in cattle that were consuming a grasslegume pasture containing 0.01 ppm of Co ('t mannetje et al., 1976). There was a dramatic increase in liveweight gain as a result of either vitamin B12 or Co administration. Tokarnia and Dobereiner in Brazil (1978) reported Co deficiencies (Fig. 9.6) were widespread in the states of Sao Paulo, Ceara, Amapa, Espirito Santo, Maranhao, and Rio Grande do SuI. Lassiter et al. (1953) demonstrated vitamin B 12 deficiency in calves less than six weeks old that received no dietary animal protein. Clinical signs characterizing the deficiency included poor appetite and growth, muscular weakness, demyelination of peripheral nerves, and poor general condition. Young lambs (up to two months of age) if weaned early, likewise have a need for dietary vitamin B12 (NRC, 1985). In vitamin B12-deficient lambs, there is a sharp decrease of vitamin B12 concentrations in blood and liver before signs like anorexia, loss of body weight, and a decrease in hemoglobin concentration are observed. Sheep are more sensitive to Co deficiency than cattle and Mburu et al. (1993) suggested that sheep are likewise less resistant to low dietary Co than goats, based on blood parameters.

B. Assessment of Status Cobalt deficiency in ruminants, in its milder forms, is impossible to diagnose with certainty on the basis of clinical and pathological observations alone. The only evidence of the deficiency is a state of unthriftiness, lack of appetite, with usually no sign of anemia. A definite diagnosis of Co-vitamin BI2 deficiency can be achieved in these circumstances by measuring the response in temperament, appetite, and liveweight that follows Co feeding or vitamin B12 injections. Forage, soil, and animal tissue concentrations have all been used to determine the status of Co (Table 9.2). If forage concentrations consistently contain <0.08 ppm Co, Co-vitamin B I2 deficiency can be predicted with confidence. For all species, as

Cobalt

294

TABLE 9.2 Cobalt Sample Concentrations (Dry Basis)" Cobalt status - sheep

Liver Kidney Heart Soil Forage

Cobalt status - cattle

Healthy

Deficient

Healthy

Deficient

0.15 0.25 0.06

0.02 0.05 0.01

0.20

0.06

20.37 0.03

0.08 0.05

"Modified from Underwood (1977) and Correa (1957) for sheep and cattle, respectively.

a vitamin B 12 deficiency progresses, there is a concurrent depletion of vitamin B12 (or Co) in serum and tissue reserves. The Co level in the ruminal fluid also falls, as would be expected from the subnormal dietary intake of the element. When this has fallen below a critical level, tentatively set at <0.5 ng/ml (Smith and Marston, 1970), vitamin BI2 synthesis by the ruminal microorganisms is inhibited and the levels of the vitamin decline in the rumen, blood, liver, and other tissues. For ruminants, the best indicators of a Co deficiency are low levels of Co and vitamin B12 in tissues, loss of appetite, elevated blood pyruvate, and elevated urinary MMA. A vitamin B12 enzyme (methylmalonyl-CoA) is required for conversion of propionate to succinate and with the vitamin deficiency, methylmalonic acid is excreted. Methylmalonic acid concentrations would be higher in all species with a vitamin B12 deficiency, but particularly high in ruminants because of the large quantities of propionic acid metabolized. However, MMA may not be a good indicator of B 12 status in lactating ruminants and their offspring (Underwood and Suttle, 1999). A deficiency of B12 also impairs conversion of formiminoglutamic acid (FIGLU) to glutamic acid, hence FIGLU accumulates (Smith, 1997). In fact, urinary FIGLU concentration may be a better indicator of a B12 deficiency in calves than urinary MMA (Quirk and Norton, 1988). Milk B12 is another potential indicator of nutritional B12 status, and although concentrations of B12 in milk are low, they do vary with relative B12 status and may be a better indicator of status than serum B12 because of fewer analytical problems (Judson et al., 1997b; Underwood and Suttle, 1999). Other indicators of low vitamin B l 2 status have been increased plasma homocysteine (Kennedy et al. 1994b; Hirsch et al., 2002) and low liver methionine synthase activity in sheep (Kennedy et al., 1992). Mild hyperhomocysteinemia may result from a deficiency of folacin, vitamin B6 , or vitamin B\2. This is associated with vegetarianism, where high levels of serum homocysteine would be expected (Hung et al., 2002). A potential method for assessment of Co and B12 status is the increase in branch-chained fatty acids in tissues of Co deficient animals (Kennedy et al., 1994a). Serum vitamin B12 concentrations are lower in deficient than in Co-sufficient animals and have the advantage over liver analysis because of the ease and frequency with which blood samples can be obtained and the avoidance of any

Deficiency

295

necessity to kill the animal or to take biopsies. Field evidence suggests that for ewes and lambs, well established Co deficiency is associated with plasma vitamin B)2 values of 200 pg/rnl or less, and that 300 pg/rnl is, on the average, the threshold at which the first clinical signs of deficiency are likely to appear (Andrews and Stephenson, 1966). For sheep consuming low dietary vitamin B\2, plasma vitamin B)2 concentration decreased below the lower limit of normality after 6 weeks, and plasma MMA concentration increased above the upper limit after 10 weeks (Kennedy et al., 1994a). However, Fisher and MacPherson (1990) suggest that serum MMA concentrations are less variable and provide a more accurate diagnosis of Co deficiency than serum vitamin B)2. Normal concentrations of serum MMA are tentatively suggested as being <2 umol/l, subclinically Co deficient 2 to 4 umol/l and Co deficient >4 umol/l (Paterson and MacPherson, 1990). Graham (1991) suggested that for ruminants plasma B\2 is marginally deficient at 380 to 760 pmol/l and deficient when under 380 pmol/l. For goats serum vitamin B)2 concentrations below 200 pg/rnl is indicative of Co deficiency (Mburu et al., 1994). The levels of Co in the livers of sheep and cattle are sufficiently responsive to changes in Co intake to have value in the detection of Co deficiency (Table 9.2), with liver vitamin BIZ an even more reliable criterion. Values of 0.10 ug vitamin Blz/g wet weight or less are "clearly diagnostic of Co deficiency disease" (Underwood, 1977). Liver Co concentrations in the range of 0.05 to 0.07 ppm (dry basis) or below are critical levels indicating deficiency (McDowell, 1997). Vast regions of Brazil are Co deficient, as confirmed from cattle liver Co concentrations, with below 0.05 ppm indicating deficiency, from 0.05 to 0.12 ppm indicating marginal deficiency, and above 0.12 ppm as adequate (Tokarnia and Dobereiner, 1978). The diagnostic value of liver Co or vitamin B)2 is reduced if the Co deficiency is coexistent with other diseases or conditions resulting in loss of appetite (Andrews and Hart, 1962). Marston (1970) also found that loss of appetite occurred when the concentration of vitamin B)2 in the liver was reduced to about 0.1 ~g/g wet weight. While herbage and tissue analyses are helpful in diagnosing the deficiency, the definite proof is the prompt improvement in feed intake following injection of vitamin BIZ' Reduction of neutrophil activity, an indication of immune cell function, may be used to evaluate the status of Co (MacPherson et al., 1987). In sheep, changes in neutrophil activity became evident before plasma vitamin B)2 concentrations had fallen below "normal" (mean was around 400 pg/ml) and before there was any increase in serum MMA, i.e., before the first recognized signs of functional deficiency in propionate metabolism. For laying poultry, vitamin B)2concentrations in egg yolk can be used as a status indicator. Squires and Naber (1992) reported that egg yolk vitamin B)2 concentrations responded rapidly to dietary changes in the level of this vitamin and were indicative of the vitamin BIZ status of the hen. At four times the dietary requirement, efficiency of transfer to eggs remained nearly constant for vitamin BIZ (Naber and Squires, 1993). Hence, vitamin B 12 fortification of eggs is easily accomplished.

296

CobaJt

For humans, a serum concentration of vitamin B)2 below 110 pmoljL is associated with megaloblastic bone marrow, incipient anemia and myelin damage, and below 150 pmoljL there are early bone marrow changes (Bender, 1992). Frequently, serum folacin and vitamin B)2concentrations are both found to be low or low normal in a patient with megaloblastic anemia, making distinction between the two syndromes difficult (Lindenbaum et a1., 1994; Savage et aJ., 1994). Contrary to serum vitamin B)2, elevations of MMA and total homocysteine are very sensitive and specific in diagnosing vitamin B)2 deficiency and can be used to help differentiate vitamin B I2 deficiency from folacin deficiency (Stabler et aJ., 1996). Urinary MMA is a sensitive, convenient and noninvasive indicator for vitamin B)2 versus folacin deficiency and has been used as a screening procedure for elderly populations (Norman and Morrison, 1993; Morris et al., 2002).

IX. SUPPLEMENTATION Because many forages and some concentrated feeds do not supply a minimum of 0.10 ppm, which may be considered as minimum adequate dietary allowance, there is need for supplemental Co compounds. Generally, ruminants that are consuming concentrates are less likely to receive inadequate dietary Co than grazing livestock, however, growth responses to supplementary Co have been demonstrated in steers fed finishing diets based on barley grain (Raun et al., 1968) and sorghum grain and silage (Kirchgessner et al., 1998). Confinement feeding production of beef cattle is widely based on corn silage which has been shown to support inadequate amounts of Co for growing cattle (Kirchgessner et al., 1998). Available Co must be present in the animal diet for ruminal and intestinal synthesis of vitamin B l2 to occur. Cobalt deficiency in ruminants can be cured or prevented through treatment of soils or pastures with Co-containing fertilizers or by direct oral administration of Co to the animals through free-choice mineral supplements (McDowell, 1985, 1997). In deficient areas where the pastures require regular fertilizer applications, adequate Co intakes can usually be ensured by including Co salts or oxide ores. In some areas, the added Co will also increase yields in pasture legumes by stimulating the nitrogen-fixing rhizobia. Underwood (1977) stated that on most deficient soils, as little as 260 to 390 g cobalt sulfate per ha applied annually or biennially provided adequate levels of Co. However, on highly alkaline soils and heavily limed soils, and on soils high in manganese oxides that fix Co in an unavailable form, "cobaltized" fertilizers do not reliably raise herbage Co. Often, use of fertilizer Co is impractical and uneconomical for extensive range conditions in developing countries (McDowell et al., 1984). Cobalt deficiency in grazing ruminants can best be prevented by direct oral administration of Co through free-choice mineral supplements. Large and frequent injections of vitamin B I2 can effectively prevent or cure Co deficiency, but are much more expensive. Oral dosing or drenching with dilute Co solutions are likewise satisfactory if the doses are regular and frequent. Dosing sheep twice each week with 2 mg Co or once each week with 7 mg Co, or dosing cattle with 5 to 10 times

Supplementation

297

those amounts, depending on their size and age, is fully adequate for regions severely deficient in Co (Underwood, 1981). The need for continual Co supplementation is difficult to assess, as the incidence of Co deficiency can vary greatly from year to year, from an undetectable mild deficiency to an acute stage. Lee (1963) illustrated this variation in a 14-year experiment with sheep in southern Australia. Half the ewes, replacements, and progeny were dosed with Co and remained healthy. The undosed half had the following performance for the 14 years: in two years, lambs were unthrifty, but there were no deaths; in three years, growth rate of the lambs was slightly retarded; in four years, 30 to 100% of the lamb crop was lost; in five years, the performance of the remaining stock was as good as that of dosed animals. Intramuscular injections of vitamin B 12 at the rate of 100 ug each week or of 150 Ilg every second week produced a rapid remission of all signs of deficiency in lambs and were just as effective as Co administered orally at the rate of 7 mg/week (Andrews and Anderson, 1954). More recently it was concluded that S.C. injection of 2 mg of soluble vitamin B12 is effective for preventing Co deficiency in lambs for approximately four weeks (Grace et al., 1998). A long term injectable product has been developed where injections of microencapsulated vitamin B 12 in lactideglycolide copolymers are able to increase and maintain the vitamin B I 2 status of lambs for at least 210 days (Grace and Lewis, 1999). For rapid correction of Co deficiency in cattle, I.M. administration of vitamin B 12 at 500 to 3000 J.lg/head is recommended, which may be repeated weekly (Graham, 1991). Injection of Co-deficient animals with vitamin B 12 produces an overnight improvement in appetite whereas oral dosing with Co takes from 7 to 10 days to produce the same effect (MacPherson, 1982). Although vitamin B 12 injections will prevent Co deficiency in ruminants, it is often more convenient and cheaper to supplement the diet with Co, allowing the microorganisms to synthesize the vitamin for subsequent absorption by the host. An additional method of providing Co, developed in Australia, is the use of an orally administered, heavy pellet (bullet) made of Co oxide plus finely divided Fe that remains in the reticulorumen for an extended period. Pellets may be lost through regurgitation or become ineffective because of formation of a surface coating of calcium phosphate. The addition of a steel grinder, which provides an abrasive action, reduces the surface coating and extends the usefulness of the pellet. Judson et al. (1997a) reported that one cobalt pellet (30% Co oxide) will prevent vitamin B12 inadequacy in beef cows for between 28 and 57 weeks; two or four pellets will prevent inadequacy for 57 to 75 weeks. A multiple slow release trace element (including Co) ruminal bolus is available (Judson et al., 1988; Ritchie et al., 1991). The carbonate, chloride, sulfate, nitrate, and glucoheptonate forms of Co have been indicated to be effective supplemental sources of Co for ruminants (Ammerman et al., 1995). Using production of vitamin B 12 in semicontinuous ruminal in vitro cultures, different Co sources were evaluated (Kawashima et al., 1997b). Estimates of relative bioavailability of the Co sources based on multiple regression slope ratios of vitamin B 12 concentration on added Co were 100, 91,

298

Cobalt

84, and 0 for Co sulfate, carbonate, glucoheptonate, and oxide, respectively. Kawashima et al. (l997a) fed sheep reagent and feed grade Co sources to evaluate Co bioavailability. Based on liver and kidney Co concentrations, the Co in Co sulfate, two carbonates and the glucoheptonate was more available than that in the three oxide forms studied. Although Co oxide is of low availability in diets, orally administered heavy pellets made of Co oxide and clay, which remain in the reticulorumen for several months have been effective in supplying Co to sheep and cattle. Grazing ruminants deficient in Co receive some of their requirement through soil consumption. Sheep ingesting 100 g/day of New Zealand soil significantly increased liver vitamin B12 concentration (Grace et al., 1996). Rigg and Askew (1934) reported that as little as 10 g of soil twice weekly prevented Co deficiency in sheep. Pigs, poultry, humans, and other monogastric species that receive diets derived entirely from plant sources, and therefore containing little or no vitamin B 12, can utilize some dietary Co from which their intestinal flora can synthesize vitamin B12• However, the gastrointestinal synthesis, unlike that of ruminants, is inadequate to meet the vitamin B 12 needs of monogastric species, even in the presence of ample Co, so that signs of vitamin B12 deficiency may arise. Some monogastric animals will benefit from Co supplementation, particularly the horse. Fish most likely benefit from Co supplementation due to intestinal synthesis and subsequent coprophagy. Vitamin B 12 is normally added to diets of all classes of swine and poultry. Swine and poultry raised in confinement, in management systems where there is less access to feces for coprophagy, will have a greater dietary requirement for the vitamin. Although dietary supplements would be recommended, injections of vitamin B12 are often given to animals with a poor health appearance. Ruminants coming into a feedlot are sometimes given vitamin B12 injections, along with other vitamins, as insurance against animals not quickly adapting to new feeding regimens. The use of vitamin B12 may be warranted under certain conditions where stress, disease, or parasites lower feed intake, impair ruminal function, and/or reduce intestinal absorption. For human therapy, during the stage of relapse of pernicious anemia, intramuscular doses of vitamin B 12 at the rate of 15 to 20 ~g/day should be given. A single injection of 100 ug or more will produce complete remission in any patient whose vitamin B I2 deficiency is not complicated by unrelated systemic disease or other factors. When pernicious anemia is due to inadequate absorption, 1 ug of the vitamin by injection daily is adequate therapy. Remission is sustained for life by monthly injections of 100 ug of vitamin B 12 (Herbert, 1990). In patients treated successfully with intramuscular vitamin B12 , the first response occurs within about two days of the start of treatment and consists of an intense feeling of wellbeing and increase in appetite. Single, large oral doses (1000 ug) of vitamin B 12 without intrinsic factor have proven effective in treatment of pernicious anemia. Absorption of a small amount of B12 from massive doses is independent of the action of intrinsic factor and is believed to occur by a "mass-action" effect, resulting in diffusion of some of the

Toxicity

299

vitamin. An oral dose of at least 150 ug a day is deemed necessary to maintain the pernicious anemia patient. Single weekly oral doses of 1000 ug satisfactorily maintain some pernicious anemia patients (Ellenbogen and Cooper, 1991). For vegetarian patients who have a normal secretion of intrinsic factor, 1.0 to 1.5 Ilg daily of vitamin B12 orally is sufficient to prevent pernicious anemia. Vitamin B12 supplementation is particularly important for individuals consuming all vegetable diets. Supplementation of vitamin B 12 should be considered by vegetarians, particularly true for lactating mothers to provide vitamin B 12 to their offspring. If malabsorption and chronic diarrhea are combined with low dietary intake of vitamin B12, as is the case for many children in the Third World, depletion of vitamin B 12 stores may result (Paerregaard et al., 1990). Premature infants require additional folacin and vitamin B I 2 to reduce the severity of the anemia of prematurity (Worthington-White et al., 1994). When high amounts of supplemental folacin are administered (e.g., to prevent neural tube defects), the risk of an undiagnosed vitamin B 12 deficiency could be reduced by simultaneous supplementation with generous doses (1 mg/day) of vitamin B12 .

X. TOXICITY Cobalt has a low order of toxicity in all species studied. The maximum dietary tolerable level of Co for common livestock species suggested by the National Academy of Sciences (NRC, 1980) is 10 ppm. Becker and Smith (1951) concluded that 150 ppm Co (dry diet basis), or some 1000 times normal levels, can be tolerated by sheep for many weeks without visible toxic effects. Pigs have tolerated up to 200 ppm Co when added to a corn-soybean diet (Huck and Clawson, 1976). Characteristic signs of chronic Co toxicosis for most species are reduced feed intake and body weight, emaciation, anemia, hyperchromemia, debility, and increased liver Co (NRC, 1980). Some evidence suggests that cattle are less tolerant to high levels of Co than sheep (Howell, 1996). Cobalt toxicosis for cattle is characterized by a mild polycythemia; excessive urination, defecation, and salivation; shortness of breath; and increased hemoglobin, red cell count, and packed cell volume (NRC, 1996). Toxicological effects of Co include vasodilation and flushing in humans, a centrally mediated decrease in heat production in the mouse, a striking reduction in myocardial hypoxic contracture in the rat and cardiomyopathy in dogs (Smith, 1987). Human cardiomyopathy following industrial exposure has been recorded. Workers exposed to Co-containing metal dust have decreased pulmonary function (Smith, 1997). The toxicity of excessive Co, in part, appears to be a mineral antagonism with anemia resulting from depressed Fe absorption. In the rat, the intestinal absorption of Fe is reduced by almost two-thirds in the presence of a tenfold higher Co concentration. A IOO-fold excess of Co will almost completely suppress Fe absorption (Underwood, 1977). Reports of Co toxicity almost invariably involve management mistakes in formulating mineral mixtures.

300

Cobalt

XI. REFERENCES Agricultural Research Council (ARC). (1980). "The Nutrient Requirements of Ruminant Livestock." Commonwealth Bureaus, Slough, England. Ammerman, C. B., Baker, D. H., and Lewis, A. J. (1995). In "Bioavailability of Nutrients for Animals" p. 119, Academic Press, San Diego. Andrews, E. D. (1956). N. Z. J. Agric. 92, 239. Andrews, E. D., and Anderson, J. P. (1954). N. Z. J. Sci. and Tech. A35, 483. Andrews, E. D., and Hart, L. I. (1962). N. Z. J. Agr. Res. 5,403. Andrews, E. D., Hart, L. I., and Stephenson, B. J. (1960). N. Z. J. Agr. Res. 3, 364. Andrews, E. D., and Stephenson, B. J. (1966). N. Z. J. Agr. Res. 9, 491. Aston, B. C. (1924). N. Z. J. Agric. 28,215. Baik, H. W., and Russell, R. M. (1999). Ann. Rev. Nutr. 194,357. Barnes, R. H., and Fiala, G. (1958). J. Nutr. 65, 103. Becker, D. E., and Smith, S. E. (1951). J. Anim. Sci. 10,226. Becker, R. B., Henderson, J. R., and Leighty, R. B. (1965) Fl. Agric. Exp. Stn. Bull. 699. Behrns, K. E., Smith, C. 0., and Sarr, M. G. (1994). Digestive Dis. Sci. 39(2), 315. Bender, D. A. (1992). "Nutritional Biochemistry of the Vitamins" p. 294, Cambridge University Press, Cambridge, UK. Carmel, R. (1994). Digestive Dis. Sci. 12,2516. Correa, R. (1957). Arquivos de Instituto Biologicos 24, 199. DRI (Dietary Reference Intakes). (2001). Panel on Micronutrients of Food and Nutrition Board. National Academy Press, Washington, D.C. Ellenbogen, L., and Cooper, B. A. (1991). In "Handbook of Vitamins" (L. J. Machlin, ed.), p. 491. Dekker, New York. Farquharson, J., and Adams, J. F. (1976). Brit. J. Nutr. 36, 127. Ferguson, E. G. W., Mitchell, G. B., and MacPherson, A. (1988). Vet. Rec. 124,20. Filmer, J. F. (1933). Aust. Vet. J. 9, 163. Filmer, J. F., and Underwood, E. J. (1934). Aust. Vet. J. 10,83. Fisher, G. E. J., and MacPherson, A. (1990). Brit. Vet. J. 146, 120. Fisher, G. E. J., and MacPherson, A. (1991). Res. Vet. Sci. 50, 319. Friesecke, H. (1980). In "Vitamin B12" F. Hoffmann-La Roche & Co., Basel, Switzerland. Grace, N. D., Clark, R. G., and Mortleman, L. (1986). N. Z. J. Agric. Res. 29, 231. Grace, N. D., and Lewis, D. H. (1999). N. Z. Vet. J. 47, 3. Grace, N. D., Rounce, J. R., and Lee, J. (1996). N. Z. J. Agric. Res. 39, 325. Grace, N. D., West, D. M., and Sargison, N. D. (1998). N. Z. Vet. J. 46, 194. Graham, T. W. (1991). Vet. Clin. N. Amer.: Food Anim. Proc. 7, 153. Henry, P. R., Littell, R. C., and Ammerman, C. B. (1997). Nutr. Res. 17,947. Herbert, V. (1990). "Nutrition Reviews: Present Knowledge in Nutrition" (5th Ed.) (R. E. Olson, H. P. Broquist, C. O. Chichester, W. J. Darby, A. C. Kolbye, and R. M. Stalvey, eds.), p. 170. The Nutrition Foundation Inc., Washington D.C. Hesselink, J. W., and Vellema, P. (1990). Tijdschrift-Diergeeneskunde 115, J. Hirsch, S., de la Maza, P., Barrera, G., Gattas, V., Petermann, M., and Bunout, D. (2002). J. Nutr. 132,289. Howell, J. McC. (1996). In "Detection and Treatment of Mineral Nutrition Problems in Grazing Sheep" (D. G. Masters and C. L. White, eds.) p. 106, Australian Center for International Agricultural Research, Canberra, Australia. Huck, D. W., and Clawson, A. J. (1976). J. Anim. Sci. 43, 1231. Hung, C. J., Huang, P. C., Lu, S. C., u, Y. H., Huang, H. B., Lin, B. F., Chang, S. J., and Chou, H. F. (2002). J. Nutr. 132, 152. Judson, G. J., Brown, T. H., Beveridge, I., and Ford, G. E. (1985). In "Trace Elements in Man and Animals-TEMA 5" (c. F. Mills, I. Bremner, and J. K. Chesters, eds.), p. 549, Commonwealth Agricultural Bureaux, Slough, UK. Judson, G. J., Brown, T. H., Kempe, B. R., and Turnbull, R. K. (1988). Aust . J. Exp. Agric. 28, 199. Judson, G. J., McFarlane, J. D., Baumgurtel, K. L., Mitsioulis, A., Nicolson, R. E., and Zviedrans, P. (1997a). In "Trace Elements in Man and Animals-TEMA 9" (P. W. F. Fischer, M. R. L'abbe, K. A. Cockell, and R. S. Gibson, eds.) p. 310. NRC Research Press, Ottawa, Canada. Judson, G. J., McFarlane, J. D., Mitsioulis, A., and Zviedrans, P. (I 997b). Aust. Vet. J. 75, 660.

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Jorgensen, B. B., Pederson, N. T., and Worning, H. (1991). Aliment. Parmacol. Ther. 5, 207. Kawashima, T., Henry, P. R., Ammerman, C. B., Littell, R. c., and Price, J. (1997a). Nutr. Res. 17, 957. Kawashima, T., Henry, P. R., Bates, D. G., Ammerman, C. B., Littell, R. C., and Price, J. (1997b). Nutr. Res. 17, 975. Kennedy, D. G., Blanchflower, W. J., Scott, J. M., Weir, D. G., Molloy, A. M., Kennedy, S., and Young, P. G. (1992). J. Nutr. 122, 1394. Kennedy, D. G., Kennedy, S., Bianchflower, W. J., Scott, J. M., Weir, D. G., Molloy, A. M., and Young, P. B. (1994a). Brit. J. Nutr. 71,67. Kennedy, D. G., Young, P. B., Blanchflower, W. J., Scott, J. M., Weir, D. G., Molloy, A. M., and Kennedy, S. (1994b). Int. J. Vito Nutr. Res. 64, 270. Kennedy, D. G., Young, P. B., Kennedy, S., Scott, J. M., Molloy, A. M., Weir, D. G., and Price, J. (1995). Int. J. Vito Nutr. Res. 65, 241. Kennedy, S., McConnell, S., Anderson, H., Kennedy, D. G., Young, P. B., and Blanchflower, W. S. (1997). Vet. Path. 34, 575. Kirchgessner, M., Schwarz, F. J., and Stangl, G. 1. (1998). J. Anim. Physiol. Anim. Nutr. 78, 141. Kominato, T. (1971). Vitamins (Japan) 44,76. Lassiter, C. A., Ward, G. M., HulTman, C. F., Duncan, C. W., and Webster, H. D. (1953). J. Dairy Sci. 36,997. Latteur, J. P. (1962). "Cobalt Deficiencies and Sub-deficiencies in Ruminants" Centre D'Information du Cobalt, Brussels, Belgium. Lee, H. J. (1963). In "Animal Health. Production and Pasture" (A. H. Worden, K. O. Sellers, and D. E. Tribe, eds.), p. 662. Longmans, Green, New York. Lee, H. J., Kuchel, R. E., Good, B. F., and Trowbridge, R. F. (1957). Aust. J. Agric. Res. 8, 502. Lindenbaum, J., Rosenberg, I., Wilson, P., Stabler, S. P., and Allen, R. H. (1994). Am. J. Clin. Nutr. 60,2. Lines, E. W. (1935). J. Council Sci. Indus. Res. 8,117. MacPherson, A. (1982). "Roche Vitamin Symposium: Recent Research on the Vitamin Requirements of Ruminants" p. 1. Hoffmann-La Roche & Co., Basel, Switzerland. MacPherson, A. (2000). In "Forage Evaluation in Ruminant Nutrition" (D. I. Givens, E. Owen, R. F. E. Axford, and H. M. Omed, eds.). CAB International, UK. MacPherson, A., Gray, D., Mitchell, G. B. B., and Taylor, C. N. (1987) Br. Vet. J. 143,348. Marston, H. R. (1970). Br. J. Nutr. 24, 615. Masters, D. G., Street, K. A., and Dunsmore, J. D. (1992). Proc. Nutr. Soc. Aust. 17, 138. Mburu, J. N., Kamau, J. M. Z., and Badamana, M. S. (1993). Int. J. Vito Nutr. Res. 63, 135. Mburu, J. N., Kamau, J. M. Z., Badamana, M. S., and Mbugua, P. N. (1994). Bull. Anim. Health Prod. Africa 42, 141. McDowell, L. R. (1985). In "Nutrition of Grazing Ruminants in Warm Climates" (L. R. McDowell, ed.), p. 339. Academic Press, New York. McDowell, L. R. (1997). "Minerals for Grazing Ruminants in Tropical Regions" 3rd Ed. University of Florida, Gainesville, Florida. McDowell, L. R. (2000). In "Vitamins in Animal and Human Nutrition" 2nd Ed. Iowa State University Press, Ames, Iowa. McDowell, L. R., Conrad, J. H., and Ellis, G. L. (1984). In "Symposium on Herbivore Nutrition in Sub-Tropics and Tropics-Problems and Prospects" (F. M. Gilchrist and R. 1. Mackie, eds.), p. 67. Pretoria, South Africa. Mohammed, R. (1983). Ph.D. Dissertation, Univ. of Clermont, Clermont, France. Mohammed, R., and Lamand, M. (1986). Ann. Res. Vet. 17,447. Naber, E. and Squires, M. W. (1993). Poult. Sci. 72, 1046. Norman, E. J., and Morrison, J. A. (1993). Am. J. Med. 94, 589. NRC. (1980). "Mineral Tolerance of Domestic Animals" National Academy of Sciences-National Research Council, Washington, D.C. NRC. Nutrient Requirements of Domestic Animals. National Academy of Sciences-National Research Council, Washington, D.C. (1985). Nutrient Requirements of Sheep, 5th Ed. (1989). Nutrient Requirements of Horses, 5th Ed. (1994). Nutrient Requirements of Poultry, 9th Ed. (1996). Nutrient Requirements of Beef Cattle, 7th Ed. (2001). Nutrient Requirements of Dairy Cattle, 7th Ed.

c.

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Paerregaard, A., Hjelt, K., and Krasilnikoff, P. A. (1990). J. Pediatr, Gastroenterol. Nutr. 11, 35 I. Paragon, B. M. (1993). Recueil de Medecine Veterinaire de l'Ecole d'Alfort 169, 759. Paterson, J. E., and MacPherson, A. (1990). Vet. Rec. 126,329. Peducasse, C. A., McDowell, L. R., Parra, L. A., Wilkins, J. V., Martin, F. G., Loosli, J. K., and Conrad, J. H. (1983). Trop. Anim. Prod. 8, 118. Peverille, K. I., and Judson, G. J. (1999). In "Soil Analysis an Interpretation Manual" (K. I. Peverille, L. A. Sparrow, and D. J. Reuter, eds.), p. 319. CSIRO Publishing, Collingwood, Australia. Quirk, M. R., and Norton, B. W. (1988). J. Agric. Sci. Camb. 110,465. Raun, N. S., Stables, G. L., Pope, L. S., Harper, O. F., Waller, G. R., Renbarger, R., and Tillman, A. D. (1968). J. Anim. Sci. 27, 1695. Rickes, E. L., Brink, N. G., Koniuszy, F. R., Wood, T. R., and Folkers, K. (1948). Science 107, 396. Rigg, T., and Askew, H. O. (1934). Emp. J. Exp. Agric. 2, I. Ritchie, N. S., Lawson, D. c., and Parkins, J. J. (1991). In "Trace Elem. Man Anim. Proc. Int. Symp. TEMA 7" p. 15. Dubrovnik, Yugoslavia. Ruelke, O. c.. and McCall, J. T. (1961). Agron. J. 53, 406. Russell, F. c., and Duncan, D. L. (1956). "Minerals in Pasture: Deliciencies and Excesses in Relation to Animal Health" Commonwealth Bureau of Animal Nutrition Technical Communication No. 15, Rowett Institute, Aberdeen, Scotland. Savage, D. G .. and Lindenbaum, J. (1995). In "Folate in Health and Disease" (L. B. Bailey, ed.) p. 237, Marcel Dekker, Inc., New York. Savage, D. G., Lindenbaum, J., Stabler, S. P., and Allen, R. H. (1994). Am. J. Med. 96, 239. Schwarz, F. J., Kirchgessner, M., and Stangl, G. I. (2000). J. Anim. Physiol. Anim. NII/r. 83, 121. Seetharam, B.. Bose, S., and Li, N. (1999). J. Nutr. 129, 1761. Shallow, M., Ellis, N. J. S., and Judson, G. J. (1989). Aust. Vet. J. 66, 250. Singh, K. K., and Aruna, C. (1994). Indian J. Anim. Nutr. 11, 127. Singh, K. K., and Chhabra, A. (1995). J. Nuclear Agric, Bioi. 24, 112. Smith, E. L. (1948). Nature (London) 162, 144. Smith, R. M. (1987). In "Trace Elements in Human and Animal Nutrition" (W. Mertz, ed.), p. 143. Academic Press, New York. Smith, R. M. (1997). In "Handbook of Nutritionally Essential Mineral Elements" (B. L. O'Dell and R. A. Sunde, eds.), p. 357, Marcel Dekker, Inc., New York. Smith, R. M., and Marston, H. R. (1970). Br. J. Nutr. 24,879. Smith, S. E., Koch, B. A., and Turk, K. L. (1951). J. Nutr. 44, 455. Sousa, J. C. de, Conrad, J. H., Blue, W. G., Ammerman, C. B., and McDowell, L. R. (1981). Pesqui. Agropecu. Bras. 16,759. Stabler, S. P., Lindenbaum, J., and Allen, R. H. (1996). J. Nutr. 126, 1266s. Stangl, G. I., Schwarz, F. J., and Kirchgessner, M. (l999a). Int. J. Vito Nutr. Res. 69, 120. Stangl, G. I., Schwarz, F. J., and Kirchgessner, M. (1999b). Nutr. Res. 19,415. Stangl, G. I., Schwarz, F. J., and Kirchgessner, M. (2000). Brit. J. Nutr. 84, 645. Suttle, N. F., and Jones, D. G. (1989). J. Nutr. 119, 1055. Squires, M. W., and Naber, E. C. (1992). Poult. Sci. 71, 2075. 't mannetje, L., Singh Sidhu, A., and Murugaiah, M. (1976) MARDI Res. Bull. 4(1), 90. Tokarnia, C. H., and Dobereiner, J. (1978) In "Proceedings Latin American Symposium on Mineral Nutrition Research with Grazing Ruminants" (J. H. Conrad and L. R. McDowell, eds.), p. 163. Univ. of Florida, Gainesville. Tokarnia, C. H., Dobereiner, J., and Peixoto, P.V. (2000). Pesq. Vet. Bras. 20(3), 127. Tokarnia, C. H., Guimaraes, J. A., Canella, C. F. C; and Dobereiner, J. (1971). Pesqui Agropecu. Bras. 6, 61. Toskes, P. P., Smith, G. W., and Conrad, M. E. (1973). Am. J. Clin. Nutr. 26,435. Tucker, K. (1995). Nutr. Rev. 53, S9. Underwood, E. J. (1977). "Trace Elements in Human and Animal Nutrition." Academic Press, New York. Underwood, E. J. (1981). "The Mineral Nutrition of Livestock." Commonwealth Agricultural Bureaux, London, England. Underwood, E. J., and Suttle, N. F. (1999). In "The Mineral Nutrition of Livestock" 3rd Ed. CAB( Publishing, New York. Vargas, D., McDowell, L. R., Conrad, J. H., Buergelt, c., and Ellis, G. L. (1984). Trop. Anim. Prod. 9, 103.

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Chapter 10

Iodine I. INTRODUCTION No microingredient is more critical for metabolism and overall general health than iodine (I). Iodine is unique among the required trace elements in that it is a constituent of the thyroid hormones thyroxine and triiodothyronine. Iodine deficiency disorders (100) is the term now used, versus goiter (enlarged thyroid), to denote all the effects of I deficiency on growth and development that can be prevented by correction of I deficiency (Hetzel, 1989). Iodine deficiency is accepted as the most common cause of preventable mental defects in the world today (Hetzel and Wellby, 1997). In humans and farm animals, I deficiency is one of the most prevalent deficiency diseases, and it occurs in almost every country in the world (Fig. 10.1).

II. mSTORY Iodine is one of the oldest elements in terms of the recognition of its importance in animal and human functions. A comprehensive review of the historical aspects of goiter has been written by Langer (1960). One of the earliest references to goiter is attributed to a legendary Chinese emperor Shen-Nung (third millennium B.C.) who, in his treatise on herbs and roots, mentions seaweed as an efficacious remedy against goiter. Ancient Hindu accounts of medical literature likewise contain references to goiter from the period around 2000 B.c. Tumors of the neck were known and treated surgically in ancient Egypt, according to the Eber papyrus (about 1500 B.C.). Burnt sponges and seaweed were added to the diet to relieve goiter during the time of Hippocrates (460 to 370 B.C.). Many views on the causes and preventions of goiter abound in the literature. Properties and characteristics of water were often erroneously associated with goiter. Other causes were related to properties of the atmosphere (humidity, temperature, chemical composition, lack of sunshine, etc.), faulty nutrition, poverty and unsanitary living conditions, alcoholism, and consanguinity in marriage. In Bohemia and Germany it was held that goiter was the result of strenuous work or of frequent fits of coughing. A fairly widespread belief in Europe was that goiter was brought on by effects of the moon. In Ecuador it used to be customary to rub goiters with saliva at the time of the new moon. 305

306

Iodine

IV

)

Fig. 10.1 World map showing occurrence of iodine deficiency disorders (IDD), characterized by endemic goiter. Black indicates areas where endemic goiter has been found.

Courtois isolated this element in 1811 from the ashes of seaweed. By 1816, I had already been used in the treatment of goiter by Proust, and in 1820 Coindet independently recommended I preparations for this purpose. Soon, however, use of I in goiter treatment met with marked opposition because of its toxic side-effects. Coindet laid emphasis on correct dosage and Prevost (1790 to 1850) reduced the dosage further, observing that amounts as low as 0.9 to 2.0 mg daily produced a noticeable effect on goiter. It was not until shortly before 1900 that the view that I is an essential component of a protein molecule synthesized by the thyroid began to take form. By 1914, Kendall had isolated crystalline thyroxine from thyroid tissue. lodization of salt as a method of preventing goiter was first suggested by the French scientist Boussingault in 1831; however, mass prophylaxis was first attempted in Switzerland and Michigan (1922 to 1924). In Switzerland there was a widespread occurrence of a severe form of mental deficiency and deaf mutism (endemic cretinism). However, following the introduction of iodized salt in 1922, goiter incidence fell rapidly and new cretins were no longer born. For Michigan after five years of iodized salt, goiter rate fell from 38.6 to 9%. This practice of using iodized salt was soon transferred to the livestock industry. A further major development was the administration of injections of iodized oil in Papua, New Guinea to people living in inaccessible mountain villages. The successful prevention of goiter and subsequently cretinism was shown in controlled trials over the period 1959 to 1972 (Hetzel and Wellby, 1997).

III. CHEMICAL PROPERTIES AND DISTRIBUTION Iodine is a member of the halogen family after fluorine (F), chlorine (CI), and bromine (Br). Its atomic number is 53 and its atomic weight is 126.91. Iodine is lost due to volatilization by the action of sunlight and heat. Volatilization losses are

Metabolism

307

minimized by maintaining I in an alkaline mixture and by using potassium iodate vs less stable potassium iodide. Iodine is a relatively rare element in the earth's crust and it does not seem to be required by mono- and dicotyledonous plants, in which its concentration is low. It occurs in the dispersed state in air, soil, water, and living organisms: in air (0.7 ug/rrr'), soil (300 Ilgjkg), fresh water (5 Ilgjl), sea water (50 Ilgjl), and animal body (0.4 mgjkg) (Maynard et al., 1979; Matovinovic, 1984). The I in air is increased by the pollution from combustion of gasoline and oil (Vought et al., 1970). Sea water contains the largest total amount of I, mostly in the form of iodates. Solar light can oxidize iodide to I, and about 400,000 tons of I escape each year from the ocean into the air. Atmospheric I is then deposited on land and vegetation by rain and snow. Coastal air may contain up to 400 Ilgjm 3 . Vought et al. (1970) reported ambient air contained 0.74 ug Ijm 3 . Many soils of the world are low in I; goiter regions are located on every populated continent (Fig. 10.I). Low soil I levels are generally associated with three factors, anyone of which can be dominant: recent glaciation, distance from the sea, and low annual rainfall. During the last glaciation period, I-rich soil was swept away by glaciers and replaced with new crystalline soil, which, lacking humus, could not retain I. Iodine-deficient areas also arise from the effects of heavy rain on steep mountain slopes and from floods. The highest concentration of I (0.5 to 2.0 g per kg) is found in the deposits of Chilean nitrate, where it was brought by the Antarctic anticyclonic air flow from the Pacific Ocean. In general, the older an exposed soil surface is, the more likely it is to be leached of I, and the most likely to be leached are the mountainous areas (Hetzel, 1989).

IV. METABOLISM A. Absorption In feeds and water, I occurs largely as inorganic iodide; in this form it is almost completely absorbed throughout the gastrointestinal tract and transported by loose attachment to plasma proteins. Iodide and I are also readily absorbed in the lungs, and significant absorption of I through skin also occurs following application of tincture of I, iodophors, or organic I-containing compounds (Vidor, 1978). In addition to dietary I, endogenous sources such as I secreted in saliva, other gastrointestinal fluids, and breakdown products of I hormones are reabsorbed in the digestive tract. For ruminants, between 70 and 80% of the daily iodide intake is absorbed directly from the rumen and an additional 10% from the abomasum (Barua et al., 1964). Iodine in compounds that are relatively insoluble in ruminal fluid may be absorbed in the abomasum (Miller et al., 1975). Iodothyronine and other iodinated amino acids are also efficiently absorbed intact; however, more is lost in the feces than with the ionic form. In children, intestinal parasitic infections have been shown to interfere with I absorption (Furnee et al., 1997).

308

Iodine

B. Tissue Uptake and Distribution Absorbed I is quickly distributed to plasma where it then enters an iodide pool. From the plasma, I is transported to the thyroid, with over 90% administered I accounted for by thyroid uptake and urinary excretions. If I supply has been abundant, only about 10% or less of the I absorbed by the gut may appear in the thyroid, but with long-standing I deficiency the fraction cleared into the thyroid may approach 80% or more (Stanbury, 1996). The gland produces and releases thyroxine when stimulated by the pituitary thyroid stimulating hormone (TSH). All phases of biosynthesis and secretion of thyroid hormones are stimulated by TSH. The TSH acts on tyrosine-rich thyroglobulin, serving to unravel this protein to make tyrosine available for iodination via the enzyme, iodide peroxidase. The TSH stimulates the thyroid to release thyroxine which is transported to its target tissues (all the cells of the body) by way of a transport protein called thyroid binding protein. Upon delivery to the target cell the thyroxine is carried into the cell and deiodinated to triiodothyronine. The enzyme catalyzing this reaction is 5'-deiodinase, a selenium (Se)-containing enzyme. Triiodothyronine is the active form of the hormone, having at least ten times the activity of thyroxine (Berdanier, 1998). Additionally, increased TSH secretion, which accompanies falling thyroid function from various causes - for example, I deficiency - leads to hyperplasia of the follicular cells and enlargement of the thyroid gland, i.e., goiter. In the thyroid, I is trapped, concentrated, rapidly oxidized, and converted to organic I by combining with tyrosine. Iodine is present in the thyroid as inorganic I, monoiodotyrosine, diiodotyrosine (T2), triiodothyronine (T 3), tetraiodothyronine (thyroxine, T4), and other iodinated organic compounds (Fig. 10.2). Selenium deficiency will have a role in the control of thyroid hormone metabolism. The deiodinating enzyme, which produces most of the circulating T 3, type I iodothyronine 5'-deiodinase, is a selenoenzyme with most of the activity occurring in the liver, kidney, and thyroid. Deiodination of T 4 is also catalyzed by type II 5'-deiodinase, which produces T 3 primarily for local use, and occurs in the central nervous system, pituitary gland, and brown adipose tissue. The deiodinase enzymes and their activities have been reviewed (Kohrle, 1994). Type III deiodinase catalyzes the conversion of T4 to reverse T 3, and the conversion of T 3 to T 2. The physiological role of the type III enzyme is thought to be to protect the brain from possible toxic effects of active thyroid hormone (T 3) (Brody, 1999). Selenium plays an indirect role in the control of thyroid hormone synthesis because it is required by another selenoenzyme, Se-dependent glutathione peroxidase (GSH-Px). In the thyroid, GSH-Px is thought to be the main antioxidant system for neutralizing cytotoxic hydrogen peroxide (H 202) and its oxidative by-products. Hydrogen peroxide is produced by the thyroid as a cofactor in thyroid hormone synthesis. High I intake when Se is deficient may initiate thyroid tissue damage as a result of low thyroidal GSH-Px activity during thyroid stimulation (Hotz et al., 1997). Studies in animals and humans have shown that iron (Fe) deficiency anemia impairs thyroid metabolism, reducing both total T 4 and T 3 (Hess et al., 2002).

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309

Monoiodotyrosine

Diiodotyrosine

HoOOCH2-~:2_COOH I 3,5, 3' -Triiodothyronine (T3)

I

I

NH

H~CH2-dH=-COOH I

I 3,5,3',5'- Tetroiodothyronine(Thyroxine,T4 )

Fig. 10.2 The structures of monoiodotyrosine, diiodotyrosine, triiodothyronine (T 3), and thyroxine (T4).

The Fe deficiency lowers thyroid peroxidase activity which is a heme-containing enzyme that catalyzes the two initial steps in thyroid hormone synthesis. The metabolism of I in the mammary gland, the ovary, and the placenta seems to be adapted to favor the I economy of the fetus or newborn (Gross, 1962). Iodine is concentrated in the mammalian ovary, particularly during follicular development (Brown-Grant, 1961). During intensive follicle growth of laying hens, the ovaries absorb the same amount of 1311 introduced intravenously as the thyroid gland. In pregnant animals 1311 freely passes across the placenta and accumulates in the tissues and blood of the fetus. Transfer of thyroxine to the fetus occurs most readily in the latter stages of pregnancy in the rabbit and the guinea pig (Gross, 1962). C. Storage Iodide ions resemble chloride ions in that they permeate all tissues, but are concentrated mostly in the thyroid (70 to 80%). Thyroglobulin, an iodinated glycoprotein in the thyroid constitutes the storage form of the hormones and represents 90% of the total thyroid I. The ovaries, pituitary gland, and salivary glands contain high levels of I. The thyroid gland has a considerable capacity to

Iodine

310

store the element. Goiter has been prevented in children in goitrous areas for several years by feeding a single concentrated dose of iodized oil. D. Excretion Iodide not used or sequestered is either excreted as organic I in the feces or as free I in the urine. Excess I is excreted primarily in the urine, with smaller amounts in feces and sweat. There appears to be no renal threshold for iodide, since loss continues into the urine even in serious I deficiency. The urinary loss is 40 times greater than the fecal loss. In tropical areas of low dietary I status, losses in the sweat are significant. Lactating animals secrete large amounts of I in milk, with quantities varying with the amount ingested or the concentration in the body fluids. Temperature can affect transfer of I to milk in the goat; milk contains six times more I at 33°C than at 5°C (Lengemann, 1979). Milk I concentration varies with stage of lactation and generally increases with advancing stages oflactation (Hemken, 1978). Colostrum is four to five times higher in I than later milk (Lewis and Ralston, 1953).

V. GOITROGENS AND OTHER IODINE ANTAGONISTS Perhaps the presence of goitrogenic substances is of equal or greater importance than is low dietary I as a contributing factor toward I deficiencies. Goitrogenic substances in the feed may increase I requirements substantially (two to four fold) depending on the amount and type of goitrogens present. Goitrogens cause enlargement of the thyroid by interfering with thyroid hormone synthesis. The basic cause of the increased size of the thyroid is increased stimulation by TSH from the pituitary in an effort to increase thyroid hormone production. Important natural goitrogen sources are cassava, cabbage, disulfides of saturated and unsaturated hydrocarbons from geological organic sediments in drinking water, bacterial products of Escherichia coli in drinking water, soybean, cottonseed, flaxseed, peas, peanuts, and I excess in seaweed and brown and green kelp. Babassu (Orbignya phalerata), a palm-tree coconut fruit, mixed with mandioca (Manihot utilissima) are goitrogenic and are responsible for a high goiter prevalence in parts of Brazil (Gaitan et al., 1994). Two varieties of millet were shown to cause thyroid enlargement (Elnour et al., 1997). Even smoking of tobacco was associated with a two-fold increase in goiter frequency (Foo et al., 1994). Prolonged continuous ingestion of 10 to 100 times (or more) of the daily requirement of I in organic and inorganic forms appears necessary for the development of goiter and/or myxedema (Wolff, 1969). Excess consumption of seafood high in I for fishermen along the coast of Northern Japan resulted in goiter incidence of 6 to 12%. The goiters regressed when I consumption stopped. Large doses of iodide are goitrogenic in the laying hen and also to the developing chick embryo in the eggs thus produced (Wheeler and Hoffmann, 1949).

Goitrogens and Other Iodine Antagonists

311

Goitrogens inhibit the conversion of iodide to I, which is necessary for iodination of tyrosine; inhibit the iodination of monoiodotyrosine; or inhibit the coupling of diiodotyrosine molecules to form thyroxine. Cruciferous plants contain goitrogens which interfere with the process of hormonogenesis in the thyroid gland - a process which is either not reversible by additional I or only partly so (Underwood and Suttle, 1999). Many pasture plants contain cyanogenetic glycosides that are goitrogenic because of the conversion of the HCN into thiocyanate in animals. Thiocyanates act by inhibiting selective concentration of I by the thyroid, an action that can be offset or minimized as long as animal I intakes are sufficiently high. However, if the animal's diet is composed largely of highly goitrogenic feeds, such as kale or cabbage, the goiter incidence can be high, even where I intakes are otherwise adequate. Goitrogenic substances are much more prevalent in feeds than is generally recognized. From New Zealand, Sargison and West (1998) reported that modern varieties of white clover contain extremely high concentrations of thiocyanate goitrogen precursors which have affected sheep production including high lamb mortalities. Soybean meal and cottonseed meal both increase serum thyroxine losses to the intestinal tract. Linseed meal in swine diets has dramatically increased serum thiocyanate (Schone et al., 1996). High intake of corn silage resulted in calves with goiter (Hemken et al., 1971). Goitrogens in the milk of cows consuming cruciferous plants increase the likelihood of I deficiency in both calves and humans consuming the milk (Clements, 1957). Cassava is cultivated extensively in developing countries and is an essential source of energy. With high cassava consumption, development of goiter is critically related to the balance between supplies of I and cyanogenic glucosides (Tewe, 1994; Abuye et al., 1998). Brassica species (i.e., kale, cabbage, broccoli, rutabagas, mustard, cauliflower, turnip, brussel sprouts, and rape) produce active goitrogens. They are glucosinolates (also called thioglucosides); 100 different kinds are known to exist in the plant kingdom (Stoewsand, 1995). Many Brassica species are minor components of pastures (McDonald, 1968). Small aliphatic disulfides, the major volatile components of onions and garlic, also have marked antithyroid effects. The alkaloid mimosine, found in the tropical legume Leucaena leucocephala, is also a goitrogenic factor (Senani et al., 1994; Ranjhan, 1998). Other substances also are antagonistic to I. Underwood and Suttle (1999) reported interrelationships that interfere with I metabolism: (I) high dietary arsenic, fluorine (F), or calcium (Ca) levels; (2) deficient or high cobalt levels; and (3) low manganese. Jooste et al. (1999) reported a high incidence of goiter in towns in South Africa where drinking water was high in F. High-Ca diets or hard water high in Ca may increase the need for additional I. Fertilization with nitrogen has resulted in reduced forage I concentrations, from 0.41 to 0.27 ppm (Alderman and Jones, 1967). Increased dietary potassium increases urinary losses of I (Hemken, 1978). Synthetic flavonoids have also been shown to adversely affect thyroid hormones, with flavonoids crossing the placenta in the rat and decreasing the availability of T, to the fetus (van der Elst Schroder et al., 1998).

312

Iodine

VI. PHYSIOLOGICAL FUNCfIONS The only known role of I is in the synthesis of the thyroid hormones, thyroxine, and triiodothyronine. Thyroxine contains about 65% I. Thyroid hormones have multiple functions as regulators of cell activity and growth. They cross the placental barrier very early in human embryonic life to have presently undefined effects before the embryonic thyroid begins to function (Stanbury, 1996). Thyroid hormones have an active role in thermoregulation, intermediary metabolism, reproduction, growth and development, circulation, and muscle function; they control the oxidation rate of all cells. An increase in thyroid hormone levels results in an increase in the basal metabolic rate (BMR). The increase in BMR has been associated with increases in various reactions that use ATP. The increased use of ATP is matched by an increase in activity of the respiratory chain and in O 2 reduction. The two reactions most closely associated with the increase in BMR with higher levels of plasma thyroid hormones are that of Na, K-ATPase (the sodium pump) and those of fatty acid synthesis. Hyperthyroidism results in weight loss, despite a normal or increased energy intake. It may result in an exaggerated release of fatty acids from the adipose tissue with fasting. In hypothyroidism, the opposite trends in metabolism can occur. There may be decreases in BMR and body temperature, as well as a mild gain in weight (Brody, 1999). In animals with a hypo functioning thyroid, as in simple goiter, the rate of energy exchange and quantity of heat liberated by tissues are reduced and BMR declines. Thyroid hormones also: (1) influence physical and mental growth and differentiation or maturation of tissues; (2) affect other endocrine glands, especially the hypophysis and the gonads; (3) influence neuromuscular function; (4) have an effect on the integument and its outgrowths, hair, fur, and feathers; and (5) influence the metabolism of the food nutrients, including various minerals and water (Stanbury, 1996; Brody, 1999). Biological effects of thyroid hormones vary with different species, as well as at different stages of development within a species, and among different tissues. In the fetus, neonate, and young, thyroid hormones exert a major influence on cellular differentiation, growth, and development (Shambaugh, 1978; Stanbury, 1996), probably mediated by affecting gene expression and manifested via increased synthesis of new proteins and enzymes or activation of existing enzymes (Oppenheimer et al., 1976; Brody, 1999). Certain proteins appear to be more specifically regulated by thyroid hormones, including those associated with skin epidermis and hair production and also cartilage metabolism. The conversion of carotene to vitamin A appears to be regulated by thyroid hormones, and they also have an effect on stimulation of erythropoiesis (Chopra, 1981). The cellular function of thyroid hormone, mediated through triiodothyronine, is directed to the genome. The hormone/receptor complex then binds to special promoter regions of the chromosome and provokes changes in the rate of transcription of nearby genes. The function can be to turn on or off the activity of specific genes involved in synthesis of specific messenger ribonucleic acids, which

Requirements

313

control synthesis or inhibition of particular proteins involved in cell function (Refetoff et al., 1993; Stein, 1994). Nuclear receptors of vitamin A (retinoic acid) interact with steroid and thyroid hormone receptors (Ross, 1993; Leng et al., 1994; McDowell, 2000). Retinoic acid receptors in cell nuclei are structurally homologous and functionally analogous to the known receptors for steroid hormones, thyroid hormone (triiodothyronine) and vitamin D(I,25-(OHhD). The super family of nuclear proteins interacts with specific genes and regulates their transcription. Retinoic acid has been found to stimulate, synergistically with triiodothyronine, the production of growth hormone in cultured pituitary cells. The nuclear receptors bind the gene element responsive to retinoic acid, in addition to the one responsive to triiodothyronine, suggesting that retinoic acid and the thyroid hormone control overlapping networks of genes.

VII. REQUIREMENTS The I requirements for various livestock species as published by the NRC are presented in Table 10.1, but cannot be given with any accuracy. Iodine requirements for growth are not necessarily identical with those for reproduction and lactation, or for maintenance of the integrity of thyroid structure and function (Underwood and Suttle, 1999). Normal growth in chickens has been reported on diets as low as 0.07 ppm I, although 0.3 ppm was required for completely normal thyroid structure (Creek et al., 1954). Different breeds of turkeys have different I requirements (McDowell and Parkey, 1995) and the amount of I deposited in eggs depended on the chicken hens (Rys et al., 1997). Lactating animals require more dietary I because about 10% of the I intake is normally excreted in milk; this percentage may increase with the level of milk production (Miller et al., 1975). Iodine requirements are influenced by climate and environment. The rate of secretion of thyroid hormones in cattle has been shown to be inversely related to environmental temperature and there is a substantial decrease in thyroid hormones in cattle, sheep, and goats during the summer (ARC, 1980). The thyroid gland has a great capacity to adapt to low dietary I. The I-deficient gland clears I from blood more rapidly than does the I-sufficient gland and uses the I more efficiently. If a deficiency is not too severe, the increased efficiency of the enlarged thyroid gland in "trapping" I from the bloodstream may compensate for the low dietary concentration, so the production of thyroid hormones is normal although the thyroid glands are enlarged. Iodine requirements are greatly affected by the nature of the diet, particularly goitrogenic substances (see Section V). If the diet contains as much as 25% strongly goitrogenic feeds, especially Brassica forages such as kale, rape, and turnips, supplemental I should be at least doubled (Miller, 1979). Thiocyanate-type goitrogens are easily counteracted by additional I, but thiouracil types can only partially be suppressed by additional I.

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314

Table 10.1 Iodine Requirement for Various Species" Species

Purpose

Chickens

Leghorn-type 0-20 wk Leghorn-type laying" Broilers 0-8 wk

0.35 rug/kg 0.35 mg/kg 0.35 mg/kg

NRC (1994) NRC (1994) NCC (1994)

All classes All classes All classes Lactating Growing All classes All classes All classes All classes All classes Growing All classes All classes Growing

0.30 mg/kg 0.40 mg/kg 0.50 mg/kg 0.50 mg/kg 0.25 rng/kg 0.1Q-{).80 mg/kg 0.10 mg/kg 0.14 mg/kg 0.20 mg/kg 0.2 mg/kg 0.35 mg/kg 0.15 mg/kg 0.15 rng/kg 0.15 mg/kg

NRC (1994) NRC (1994) NRC (1996) NRC (2001) NRC (2001) NRC (l985b) NRC (1989) NRC (1998) NRC (1982) NRC (1977) NRC (1986) NRC (1995) NRC (1995) NRC (1995)

Japanese quail Turkeys Beef cattle Dairy ca ttle Sheep Horses Swine Mink Rabbits Cats Rats Mice Guinea pigs Nonhuman primates Humans

Req uirements

All classes Children Adults Lactating

2.00 mg/kg 90-120 !!g/day 150 !!g/day 290 !!g/day

Reference

NRC (1978) DRI (2001) DRI (2001) DRI (2001)

"Expressed us per unit animal feed either on as fed (approximately 90% dry matter) or dry basis (see Appendix Table I). bBased on intake of 100 gjday.

Recommended daily I intake for both sexes in humans is 110 ug for children aged up to six months, 130 ug from 6 to 12 months, 90 ug from I to 8 years, and 120 to ISO ug from 9 years onward (DRI, 2001). The requirements during pregnancy and lactation are 220 and 290 ug, respectively.

VIII. NATURAL SOURCES Iodine is distributed widely in nature and is present in both organic and inorganic substances in very small amounts. The level of I in the drinking water reflects the I content of the rocks and soils of a region and hence of the locally grown foods or feeds. Crops closer to the ocean have higher I concentrations. Karmarkar et al. (1974) found water I from goitrous areas in India, Nepal, and Ceylon to range from 0.1 to 1.2 Ilg/I, compared with a nongoitrous area in Delhi, India of 9.0 Ilg/l. The water itself does not normally contribute a significant proportion of total daily I intake, over 90% of which comes from the food in goiter and non-goiter areas alike (Underwood and Suttle, 1999).

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315

The concentration of I in common feedstuffs is highly variable. The oilseed meal protein supplements (i.e., soybean, cottonseed, linseed, and peanut) contain 0.11 to 0.20 ppm I while common cereal grains range from 0.04 to 0.10 ppm. Milk, milk products, eggs, meat, and fish (marine) contain larger quantities of I than do plant foods. Fish meal (marine) is especially rich in I. The edible flesh of sea fish and shellfish may contain 0.30 to 3.0 ppm of I (fresh basis) compared with 0.02 to 0.04 ppm for freshwater fish (Hetzel and Wellby, 1997). Plant concentrations of I depend on species, soil type (including its I content), fertilizer applied to the soil, and climatic conditions. Soils high in I generally produce plants richer in the element than I-low soils but attempts to correlate total soil with plant I have been only partially successful, presumably due to differences in the chemical forms in which I occurs in soils and their availability to plants (Underwood and Suttle, 1999). Application of Chilean nitrate of soda, the only mineral fertilizer naturally rich in I, can double or triple the I content of crops and pastures when applied in amounts required to meet their nitrogen needs. Heavy applications of seaweed, whether in the natural form, as compost or as the ash, can increase the I content of animal fodders (and vegetables for human consumption) 10 to 100 times (Gurevich, 1964). Soil contamination of forages influences I consumption for grazing livestock as most topsoils are higher in I than plants which grow on them (Underwood and Suttle, 1999). The I levels in pasture plant species have been reported to range between 300 and 1500 ppb (dry basis) (Hetzel and Wellby, 1997). Hartmans (1974), in a study of the factors affecting herbage I content in the Netherlands, found that dicotyledonous species had up to 14 times higher I contents than grasses, while the I content of grass species varied over a two-fold range. Butler and Johnson (1957) concluded that species was more important in determining forage I content than either soil or season. Thirteen perennial ryegrass plants taken at random from different strains in New Zealand gave I contents ranging from 185 to 2470 ppb dry basis. Plant maturity influences pasture I, there is a four- to five-fold reduction in I concentration which can accompany the advancing maturity of a sward (Alderman and Jones, 1967). The processes of ploughing, drainage, and liming involved in the improvement of upland pasture have been implicated in a reduction in their capacity to retain I in a bioavailable form (Lidiard, 1995). Miller and Ammerman (1995) conclude a review on I availability by saying that little is known about its bioavailability from plant sources. Animal products, particularly milk and eggs, readily reflect dietary sources of I. The average hen's egg contains some 4 to 10 ug of I, most of which is in the yolk. These amounts are reduced in goiter areas or in conditions of prolonged I deficiency but can be increased as much as 100-fold by feeding the hen with large amounts of I (Underwood and Suttle, 1999). When high concentrations, 100 to 500 mg/day, of I are fed to laying hens, the I content of eggs may reach 50 to 120 mg/kg (Marcilese et al., 1968). Moderate changes in dietary I are quickly reflected in milk I, but I in meat is relatively stable (Swanson et al., 1990). Iodine concentrations in milk of cows fed 0, 40, 81, 162, 405, or 810 mg supplemental I daily as ethylenediaminedihydriodide (EDDI) averaged 8, 361, 895, 1559,2036, and 2393 llg/I (Miller et aI.,

316

Iodine

1975). Prior to about 1960, milk was considered a rather poor source of I. Surveys in the United States of market milk indicated that milk is now a good source of the mineral. Surveys of I intake revealed that dairy products contribute 38 to 50% of the I for adults and 56 to 85% for young children (Hemken, 1981). The major cause of very high values was found to be the feeding of relatively high levels of the organic I form EDDI. Some farms used higher than the daily 50 mg that was recommended as a preventive and 200 mg as a therapeutic measure for foot rot and soft tissue lumpy jaw. Other sources for increased milk I include iodophor-antiseptics used as teat dips. lodophors are also used as antiseptic agents for udder washes, milking machines and tanks for handling, storing, and transporting of milk, and milk products (Sanchez, 1995). In addition to I from fish, milk, and eggs, human diets receive I from less traditional sources (Hampel et al., 1997). Erythrosine (2,4,5,7-tetraiodofluorescein), a food coloring (red dye) agent, contains 58% I and is widely used in foods. Potassium iodate is used as a dough conditioner or improver in bread making. Many products are seasoned with iodized salt.

IX. DEFICIENCY Iodine deficiency disorders including goiter in both animals and man occur in almost every country (Fig. 10.1), quite independently of climate, season, or weather (Kelly and Snedden, 1960). Goiter or other I deficiency signs are reported in animals in most areas where human goiter is endemic. Since animals usually receive feeds produced locally, they are often more susceptible to goiter than are human beings. Although not true today, Allman and Hamilton (1949) suggested that I deficiency was the most widespread of all mineral deficiencies in grazing stock. Some of the most notorious goiter and IDD regions are the high mountain regions - in Alpine valleys, in the Pyrenees, on the slopes of the Himalayas, and along the Cordillera of the Andes. However, goiter is also known to occur in comparatively low-lying areas and even at sea level. Iodine-deficient areas are found in the interior of countries, especially in areas where wind or rainfall are unable to carry traces of 1 from the sea to the soil and where the soil has been depleted by leaching by heavy I-poor rains or where there is little rainfall.

A. Effects of Deficiency 1. SWINE

Reproductive failure is the outstanding manifestation of I deficiency in swine. The birth of dead, or hairless pigs in breeding stock has long been recognized in goitrous areas (Fig. 10.3). Most of the pigs are born alive, but usually die within a few hours. At necropsy, the thyroid is enlarged (Fig. 10.4) and hemorrhagic.

Deficiency

317

Fig.10.3 Iodine deficiency. The top photo shows a litter of hairless pigs which were stillborn. The other shows a live hairless pig. (Courtesy of the late l.W. Kalkus, Western Washington Experiment Station, Puyallup)

Weak pigs may also be produced. These weak pigs may appear unusually large and fat when born because of a bloated condition and a pulpy, thickened skin, especially about the head and neck. Less severe deficiency may be reflected in less drastic disorders of the integument, such as dry skin and a rough, harsh hair coat. Older animals rarely show any signs of a lack of I. Growing pigs fed goitrogens showed visual evidence of hypothyroidism, including shortening of the leg bones, a dwarfed appearance, enlarged thyroid glands (Fig. 10.4) and extreme sluggishness in their activity (Sihombing et al., 1974). A diagnosis of congenital goiter was confirmed histologically in piglets which were born hairless and swollen, and with enlarged thyroid glands (de Welchman et al., 1994). The suspicion of an inherited disorder (autosomal recessive) was confirmed when a test mating of a suspect carrier boar and sow resulted in the birth of two affected piglets.

318

Iodine

Fig. 10.4 Iodine deficiency. The pig in front (top photo) was fed the basal diet plus 0.5% potassium thiocyanate for 51 days. The littermate pig in the back was fed the basal diet plus 0.2 ppm iodine for the same period. Signs of hypothyroidism were observed in the pig fed the goitrogen. The bottom photo is thyroids of pigs fed a corn-soybean meal basal diet with and without 0.2 ppm iodine for 51 days. The goitrous thyroids of pigs fed the basal diet averaged almost six times greater in weight than those of pigs given supplemental iodine. (Courtesy of G.L. Cromwell. University of Kentucky. Lexington)

2.

POULTRY

Very few cases of goiter, or enlarged thyroids, have been observed in poultry, probably because it is difficult to observe the glands under field conditions. Goiter has been produced experimentally in chickens by feeding a diet exceedingly low in I (about 0.025 ppm) to laying hens. Inadequate thyroid hormones resulted in poor growth, egg production, and egg size. Iodine deficiency in breeders result in low I egg content and, consequently, decreased hatchability, retardation in absorption of the yolk sac and thyroid enlargement in embryos. The glands may increase to many times their usual size. If the deficiency is not too severe, the increased efficiency of

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319

the enlarged gland in "trapping" I from the bloodstream may compensate for the low dietary concentration (NRC, 1994). Lack of thyroid activity or inhibition of thyroid action by thiouracil or thiourea caused hens to cease laying and to become very fat, and also caused the growth of abnormally long, lacy feathers (Scott et al., 1982). Thyroid insufficiency can be very conspicuous in some types of birds due to plumage changes. In the Brown Leghorn male the testes remain small and free of spermatozoa, the comb decreases in size, moulting is inhibited, and the characteristic male plumage is lost (Underwood and Suttle, 1999). Iodine deficiency can also be a problem in pet birds such as in Budgerigars (Schoemaker et al., 1999). Goiter was found as a result of I-deficient drinking water and provision of a seed mixture based on millet. Poultry appear to be able to withstand a considerable degree of I deficiency without any marked loss of production or hatchability of eggs. Thus Rogier (1958) maintained hens on an I-deficient diet for 35 weeks without affecting hatchability or embryo weight. However, when these hens received these low-I diets for two years, there was decreased hatchability, prolongation of hatching time, and retarded embryo development. Iodine deficiency appears rapidly when poultry diets contain goitrogenic substances. 3.

RUMINANTS

Goiter is illustrated in young ruminants in the tropical regions of Brazil and Indonesia and the USA (Fig. 10.5). Iodine deficiency in young ruminants is also manifested by general weakness, and animals may be born blind, hairless, or dead. Goiter may be a clinical sign of a less severe deficiency than the lack of hair or wool (Underwood and Suttle, 1999). Retardation of fetal brain development in lambs has been confirmed by histological studies (Potter et al., 1981). Reduced quantity and quality of wool growth have been widely associated with goiter in sheep. Iodine deficiency in the young lamb permanently impaired quality of the adult fleece, since normal development of wool-producing secondary follicles requires thyroid activity in excess of that needed for growth (Ferguson et al., 1956). Iodine deficiency in breeding animals results in irregularity or suppression ofestrus periods. Fetal development may be arrested at any stage resulting in early death and resorption, abortion and stillbirth, or the livebirth of weak young, often associated with prolonged gestation and parturition and fetal membrane retention (Allcroft et al., 1954). Low dietary I affected reproduction of cows, causing irregular estrus cycles, low conception rates as well as resorbed fetuses, abortions, stillbirths or calves may be born blind, weak, or with goiter (Sanchez, 1995). In a 5-cow mixed-breed, beef herd, four calves were stillborn or died four hours after birth (Wither, 1997). The gross changes in calves included enlarged thyroid glands, histological examination revealed a marked follicular hyperplasia of the thyroid gland. For sheep, supplementation improved reproductive performance, with 21 and 14% more lambs born to supplemented than to control mixed-age ewes in 1996 and 1997, respectively (Sargison et al., 1998). In goats, infertility and abortion characterized I deficiency (Zhang et al., 1994; Bires et al., 1996). Bires et al. (1996)

320

Iodine

Fig. 10.5 Iodine deficiency. Calf with severe goiter in Rondonapolis, Mato Grosso. Brazil (top left). Goats with goiters from Yogyakarta, Indonesia (top right) and the United States (bottom). (L.R. McOowell. University of Florida. Gainesville)

diagnosed goiter in 39 of 46 imported goats. Abortions occurred in the last months of pregnancy in 14 goats, 14 goats delivered stillborn kids, and 18 goats gave birth to 26 live kids, but 18 of these died within 12 to 24 hours of birth. Dead kids were hairless and had skin edema, very shortened limbs, and enlarged thyroid glands. A significant improvement from I therapy on first-service conception rate and in reducing irregular estrus was reported from 190 herds in goitrous areas in Finland (Hetzel and Wellby, 1997). In an I-deficient region of Canada, there was a marked

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improvement in first-service conception rate by feeding an organic I preparation (McDonald et al., 1962). Male fertility has also been related to I deficiency (Church, 1971). A decline in libido and a deterioration in semen quality have been associated with I deficiency in bulls and stallions, and a seasonal decline in semen quality in rams has been related to a mild I deficiency. For adult cattle more than a year may be required on low-I diets before deficiency signs are noticed (Swanson, 1972). Long-term deficiencies may result in decreased feed intake, milk fat test, milk yields, and some signs of hypothyroidism (Hemken et al., 1971). Hemken (1970) reported that I-deficient dairy cattle were less able to resist stress and may even have a higher incidence of ketosis. 4.

HORSES

Goiter usually appears in the young at birth. In addition to goiter, deficiency signs include weakness, persistent hypothermia, respiratory distress, and high neonatal mortality (Frap, 1998). There is an increased susceptibility to infectious disease and respiratory infections are frequent. Foals may be stillborn or exhibit extreme weakness at birth, resulting in an inability to stand and suckle the mare (Fig. 10.6). Foals born alive with an advanced goiter condition will usually die or will remain weaklings if they live (Cunha, 1990). Iodine-deficient mares may exhibit abnormal estrus cycles (Kruzkova, 1968) and males may have decreased libido and reduced semen quality (Hemken, 1981). 5. OTHER ANIMAL SPECIES

Fig. 10.6 Iodine-deficient foal. Foals deficient in iodine are born weak and usually die because of their inability to stand and suckle the mare. (Courtesy of the late l.W. Kalkus, Washington State Agricultural Experimental Station. Puyallup)

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a. Laboratory Animals. Female rats receiving low dietary I give birth to young with goiter, the deficiency generally inhibits reproduction (Feldman, 1960). lodinedeficient rats also had more coarse and less dense hair than controls (NRC, 1995). For mice, Abbassi and McKenzie (1970) reported that an I-deficient diet fed for six months resulted in thyroid glands three times the normal size, pituitaries twice the normal size, and serum thyrotropin concentrations 200 times more than normal. Hamsters, like other rodents, develop goiter when receiving I-deficient diets (NRC, 1995). Livingston et al. (1997) suggested that there was a genetic influence on the increased prevalence of goiter in Syrian hamsters.

b. Cats. For cats in addition to goiter, clinical signs of I deficiency include alopecia, abnormal Ca metabolism, fetal resorption, and death (NRC, 1986; Ranz et al., 2002). Estrus and libido were unaffected. c. Dogs. Goiter is the main sign of I deficiency, and cretinism in dogs has been reported in regions where goiter is endemic (NRC, 1985a). Myxedema appears in the skin, and there are skeletal deformities, hairlessness, dullness, apathy, drowsiness, and timidity. d. Fish. A deficiency of I causes goiter in fish. Histological indications of thyroid hyperplasia and increased mortality, particularly during extended feeding, are definite signs of I deficiency (NRC, 1993). Since 1972, polychlorinated biphenyls (PCBs) produced by the chemical industry but now banned, caused severe endemic goiter in predator fish of the Great Lakes (USA) waters (Matovinovic and Trowbridge, 1980).

e. Rabbits. Iodine-treated water had a favorable effect on growth, carcass weight, and social behavior of rabbits (EI-Mahdy and Karousa, 1995). Rabbits not receiving I had higher levels of aggressive behaviors including scratching and biting. 6.

HUMANS

Approximately 1.6 billion people worldwide may consume inadequate daily amounts of I (UNICEF, 1995) and are at risk for I deficiency disorders (IDD) (Hetzel and Pandav, 1994), which include enlargement of the thyroid (goiter) and a wide spectrum of mental, psychomotor, and growth abnormalities (Delange, 1994). Currently, there are an estimated 655 million cases of endemic goiter and 26 million cases of preventable mental deficiency, including 5.7 million cases of cretinism worldwide (WHO, 1991; Hetzel and Pandav, 1994). Seventy-five percent of people with goiter live in developing countries (Gaitan et al., 1991). Stanbury (1985) and Ramakrishnan (2002) suggest that one-quarter to one-third of the world's population subsists on I-deficient diets. Hetzel (1989) suggests up to one-third of the people in China are at risk for IDD. Follis (1966) reported that endemic goiter exists in all of the continental Latin American countries. In Asuncion, the capital city of Paraguay, at one time there was goiter in almost every

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home (Langer, 1960). In Ceylon and Nigeria, the incidence of goiter among females in selected villages ranged up to 56 and 72%, respectively (Wilson, 1953). Endemic cretinism is often associated with an I intake of <20 Ilgjday and is still widely prevalent, affecting up to 10% of the populations living in severely I-deficient areas in India, Indonesia, and China (Tai et al., 1982). In India it is estimated that 270 million people are at risk of IDD, 79 million have goiter, and 2.2 million suffer from cretinism (Ramachandran, 2002). Hetzel (1989) categorized IDD at three levels of severity: (1) mild IDD with goiter prevalence in the range of 5 to 20% (school children), with median urine I levels of 3.5 to 5.0 Ilgjdl; (2) moderate IDD with goiter prevalence up to 30% and some hypothyroidism with median urine I levels of 2.0 to 3.5 Ilgjdl; and (3) severe IDD indicated by a high prevalence of goiter (30% or more), endemic cretinism (prevalence I to 10%) with urine 2.0 Ilgjdl or less. Goiter (Fig. 10.7) is 20 or 30 times more common in women than in men, and it is most commonly seen in young girls at the age of puberty. Research has defined the considerable impact of I deficiency on brain development and function, particularly in the fetus and in early childhood during the period of rapid brain growth. The extreme consequence is endemic cretinism, but much more prevalent are lesser degrees of intellectual and neurological deficits, with a potential reduction of intelligence scores in affected communities of more than 10% (Kalk, 1998). Raja tanavin et al. (1997) evaluated 112 endemic cretins in Thailand and reported a mean intellectual quotient (I.Q.) of 30.8 ± 8.8. Mild-to-moderate intellectual impairment is an important consequence of I deficiency (Huda et al., 1999). An analysis of the effects of IDD on mental development found an average I.Q. deficit of 13.5 points among I-deficient populations (Bleichrodt and Born, 1994). From Russia, performance in mathematical tests in school children improved by 46.7% after I supplementation (Sukhinina et al., 1997). From South Africa, goitrous subjects scored consistently worse in their Zulu examination papers than those without goiter (Benade et al., 1997). In areas with a high prevalence of IDD, maternal and fetal I deficiency has been associated with increased rates of stillbirth, abortion, congenital anomaly, and infant mortality. Women with goiter in India had a higher incidence of reproductive problems (e.g., two or more unexplained abortions, stillbirths, or neonatal loss). Goiter was present in 87.5% of patients with severe I deficiency. Women with reproductive insufficiency were more likely to have goiter (37.9 vs 16.13% in the control group) (Chhabra and Hora, 1996). Both human and animal data indicate that I-deficiency presents a threat to fetal survival - stillbirths are increased and can be reduced by correcting I deficiency (Hetzel and Wellby, 1997). In districts where I-deficiency has been severely and continuously endemic for long periods of time and over many generations, the children of goiter-bearing parents may be born with an almost complete lack of thyroid secretion. The condition in its severest form is known as endemic cretinism (Fig. 10.7). Endemic cretins frequently survive to adult life, and in some countries the condition is so widespread as to constitute a distressing social and economic problem.

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Fig. 10.7 Iodine deficiency disorders. A and B - Goiter in an adult and child in Montero, Bolivia. The incidence of goiter in this small city of 10,000 inhabitants in 1965 was 40 percent. C - severe iodine deficiency. Left: A myxedematous cretin from Sinjiang, China who is also a deafmute. This condition is completely preventable. Right: The barefoot doctor of her village. Both are about 35 years of age. (A and B - L.R. McDowell, University of Florida, Gainesville; C - Photograph made available by Professor T. Ma of Tianjin, People's Republic of China and provided by Basil S. Hetzel, International Council for Control of Iodine Deficiency Disorders, Adelaide, Australia.)

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Endemic goiter results in impaired fetal, mental, and socioeconomical development, with stillbirths, abortions, congenital defects, and endemic cretinism, characterized by mental deficiencies, deaf mutism, spastic diplegia, and lesser neural defects (Hetzel, 1983). The most devastating effects of I deficiency are the congenital anomalies, increased prenatal and infant mortality, and cretinism. From a Himalayan goiter endemic region, Kochupillai et al. (1984) observed that a significant proportion of newborns suffered from brain damage due to thyroxine deficiency. From the Guizhou province of China, an endemic goiter region, YanYou and Shu-Hua (1986) reported that hearing of children is inferior to those in a non-endemic control area. Azizi et al. (1995) evaluated presumably normal school children in an I-deficient region of Iran. The study demonstrated that mild to moderate growth retardation and neurological, auditory, and psychomotor impairments occur in apparently normal subjects residing in areas of I deficiency. A common misconception is that IDD primarily affects only remote rural populations (Sullivan et al., 1997). This belief may have developed because goiter, the most common visible evidence of I deficiency, is usually most prevalent in rural populations. While goiter may be the most common visible evidence of IDD, this clinical sign is just the "tip of the iceberg" of the consequences of IDD, which include lower intelligence quotient, increased fetal, infant, and child mortality, poorer growth, and birth defects (Hetzel and Pandav, 1994). There is a misconception by some in developed countries that I deficiency has been virtually eliminated because of salt iodination. Unfortunately, I deficiency is still devastating to the health of many of the world's people. There are still regions of many countries with I deficiencies. Incidences of I deficiencies reported recently (since 1992) in various regions of selected countries for certain groups are as follows: Malaysia, 42.8% (Zaleha et al., 1998); South Africa, 17.5% (Jooste et al., 1997); Tunisia, 49.5% (EI-May et al., 1997); India, 20.5% (Pandav et al., 1997); Saudi Arabia, 22.0% (Al-Nuaim et al., 1997); Chad, 63.0% (Wyss et al., 1996); and Namibia, 34.5% (Jooste et al., 1992). From Reunion Island (Cirque de Salazie), the prevalence of goiter increased with age (0% in infants, 12.1 % in children, 23.3% in teenagers, and 38.4% in women (Jaffiol et al., 1997). The greatest factor affecting the prevalence of human IDD is the isolation of various populations and their sole dependence on locally grown foods low in I and/or high in goitrogens (see sections V and VIII). Goiter and other I deficiency symptoms are usually prevalent in locations remote from the sea, but the main consideration is I concentrations of soils and foods produced in specific regions.

B. Assessment of Iodine Status Severe I deficiency and thus status can be diagnosed on the clinical evidence of goiter alone. This would include the rate of palpable or visible goiter classified according to accepted criteria. Less severe forms of goiter or I deficiency are more difficult to diagnose, and thus weight and histological structure of the thyroid gland as well as serum I (largely thyroxine) are used in diagnosis. In the human neonate

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Iodine

<4 ug thyroxine/dl is regarded as prejudicial to brain development (Hetzel and Maberly, 1986). Caple et al. (1985) indicated that the time required for lambs to rewarm from a cold climate was related to plasma thyroxine and the relationship indicated that lambs with plasma thyroxine less than 40 nmol/l had markedly impaired abilities to rewarm. Serum protein-bound I values in adult cattle less than 3 to 4 Ilg/dl or total serum I values less than 5 to 10 Ilg/dl may indicate an inadequate I intake (Puis, 1981). When the I concentration in the thyroid of lambs and pigs falls below 0.12%, dry basis, hyperplastic changes characteristic of goiter can be expected (Andrews et al., 1948). A similar limit may be appropriate for other animal species. Milk I determinations have been used to establish the I and goiter status of an area (see Section VIII). Cattle milk I levels less than 10 to 20 Ilg/1 are associated with inadequate intake (Hemken et al., 1972). For sheep, milk I concentrations below 80 Ilg/1 pointed to a likely deficiency state (Mason, 1976), and for swine less than 50 Ilg/1 (Schone et al., 1997). Since I is excreted primarily in the urine, urinary levels of I should be useful, but little information from livestock is available. For humans, however, urinary I of >50 Ilg/g creatinine indicate an adequate status, whereas concentrations of <25 Ilg/g creatinine indicate a serious risk for endemic cretinism (Hetzel and Wellby, 1997). Many surveys to evaluate nutritional status have used urinary I as a criterion. The determination of the level of blood thyroid stimulating hormone (TSH) is proving to be an excellent indicator of I status (Hetzel and Wellby, 1997). Sullivan et al. (1997) suggested the use ofTSH testing in newborns to identify I deficiency.

X. SUPPLEMENTATION Iodine deficiency has declined in more recent years for both humans and animals due to use of iodized salt and other preventive measures. However, I deficiency is still today a serious health problem, particularly in developing countries. There are no reliable estimates on incidence of goiter for livestock in developing countries or indications of how subclinical I deficiency may be affecting livestock production. Iodine supplementation for livestock and humans is extremely important in goiterogenic areas or in a low-I region. Before I feeding was practiced in Montana (USA), goiter caused an annual loss of thousands of pigs (Maynard et al., 1979). Records from other areas show that serious losses in the sheep and cattle industries were largely prevented following the discovery of the lack of! as the causative factor. For livestock, salt is the predominant vehicle for providing supplemental I. The value of sea salt in comparison with certain inland deposits was recognized early. Recommended levels of salt iodization vary in different countries. For livestock the addition of 0.0076% I to the salt represents the customary and effective level. Supplemental I may be incorporated into the salt, mineral mixture, or concentrate feeds. It is more difficult to assure an adequate intake by grazing animals unless salt with I can be self-fed. In some inaccessible areas, salt blocks with I are dropped by low-flying planes.

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Several supplemental sources of I are relatively equal in availability (Miller and Ammerman, 1995). Potassium iodide, sodium iodide, and calcium iodate are readily available to ruminants but will leach or evaporate from salt blocks under hot, humid, tropical conditions. Sodium iodide, potassium iodide, and ethylenediamine dihydriodide (EDDI) are well utilized by animals as sources of I. Calcium iodate and pentacalcium orthoperiodate are also of high bioavailability and have greater physical stability and are not so rapidly lost from free-choice mineral mixtures. Diiodosalicylic acid (DIS) is a relatively stable compound which is bioavailable to rats but not well utilized by ruminants. For cattle, DIS had only 15% of the value of potassium iodide. The EDDI source of I is an organic iodide compound that is used at relatively high levels for treatment of footrot, lumpy jaw, and other conditions. This source is often given in daily doses of 50 and 200 mg per cow for prevention and for therapy of these conditions. The I is available but is not as stable as in other I compounds, and should be kept dry and cool. Iodized salt loses potency during storage, but is less subject to deterioration in mixed feeds because of the presence of proteins and unsaturated fats. One source of I loss is the migration of iodide to the cardboard or fabric of the container, which can be overcome by lining the bags with some material impervious to moisture (Iodine Educational Bureau, 1946). In the absence of sunlight, iodized salts undergo no serious loss of I when stored in atmospheres up to at least 50% relative humidity, but exposure of the salts to sunlight results in a considerable loss (Johnson and Herrington, 1927). Volatilization of I by action of sunlight may be reduced by rendering the mixture alkaline (e.g., addition of sodium bicarbonate) and is almost entirely prevented by iodizing the salt with potassium iodate. Exposure to heat, however, causes losses of I from acid, neutral, and alkaline iodized salts. Use of potassium iodate or a stabilized iodide form instead of potassium iodide prevents volatilization of the mineral. Loss of I from potassium iodide is reduced by the addition of stabilizers such as 0.1 % sodium thiosulfate and 0.1 % calcium hydroxide combined. Iodate has a remarkable stability even in impure salts and under adverse climatic conditions. Johnson and Frederick (1940) reported that a salt containing one part of iodide in 5000 parts of salt lost 40% or more of the I in 18 months, from oxidation of the iodide to free I, with subsequent volatilization through the agency of oxidizing impurities in the mineral mixture, chiefly chlorate, nitrate, and traces of iron. It is important to periodically provide fresh supplies of minerals to animals receiving free-choice mixtures outdoors. Other methods of providing I to livestock include oral dosing, intra-muscular injections of iodized oil, and the use of intraruminal devices. Injectable I compounds have proven effective for preventing goiter in both human and livestock studies. S. Lebdosoekojo (personal communication) studied an injectable iodized poppyseed oil administered to breeding goats in Yogyakarta, Indonesia. One injection was found to last for approximately 2 years or until after the third kidding. These goats had received coconut oil meal and rice bran that was thought to contain goitrogenic substances. Slow-release intraruminal devices have been developed; the I capsule employs a diffusion process to supply I to sheep (Judson, 1996). Solid I,

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which is contained in a polyethylene envelope, is in equilibrium with I vapor which diffuses through the plastic at a constant rate. The plastic offers enough resistance to the diffusion of the vapor to limit its escape to an amount sufficient to meet the I needs of sheep for up to 6 years; the maximum duration of effectiveness of a single injection of iodized poppy seed oil is about 2 years (Judson, 1996). Unless I nutrition is improved in communities of many developing countries, learning disabilities and other abnormalities may prevent millions of children from achieving their full potential even if learning opportunities are made available to them. For humans, iodized salt is the most widely used and simplest source of supplemental I. Contrary to popular belief, use of iodized salt has not eliminated 100. In 1964, goiter incidence in Bihar, India was 40.3%, after many years of utilizing iodized salt, however, in 1997, the incidence of goiter was still 11.6% (Umesh et al., 1997). In Haiti, iodized salt containing diethylcarbamazine is used to both control 100 in addition to controlling the parasite Wuchereria bancrofti (Beach et al., 2001). Difficulties are encountered in the production of iodized salt and in maintaining its quality so that it can be supplied to the millions ofI-deficient people. In Asia, the cost of iodized salt production and distribution at present is of the order of three to five cents per person per year (Hetzel and Wellby, 1997). There is the problem of the salt actually reaching the l-deficient subject. There may be a problem with distribution or preservation of the I content - it may be left uncovered or exposed to heat. Finally, there are difficulties in relation to the consumption of the salt. While the addition of I makes no difference to the taste, the introduction of a new variety of salt in an area where salt is already available and familiar, is likely to be resisted. In the Chinese provinces of Xinjiang and Inner Mongolia, the strong preference of the people for desert salt of very low I content led to a mass program of iodized oil injection in order to prevent cretinism (WHO, 1996). Also, some individuals have reduced salt intake to assist the control of hypertension. Iodized oil injections for humans in developing countries has been highly effective in the prevention of goiter and cretinism. A simple dose of iodized oil has been shown to correct I deficiency for three to five years (Hetzel, 1989). Iodized oil is singularly appropriate for isolated village communities so characteristic of mountainous endemic goiter areas. The striking regression of goiter following iodized oil injection ensures general acceptance of the measure (Hetzel and Wellby, 1997). In a suitable area, the oil (l ml usually contains 480 mg of I) should be administered to all females up to the age of 40 years and all males up to the age of 20 years. A repeat injection may be required in three to five years, depending on the dose given and the age of the subject. The need of children for I is greater than that of adults, and the recommended dose should be repeated in three years if severe I deficiency persists (WHO, 1996). Iodized oil can also be given by mouth. Recent studies in India and China reveal that oral iodized oil lasts only half as long as a similar dose given by injection (Hetzel and Wellby, 1997). Apart from iodized salt and iodized oil (via injection or oral) programs, I deficiency disorders may also be prevented by increasing the I content of animal feeds, thereby supplying humans with I via milk, eggs, and meat products. Feeding

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329

swine a high-I feed caused a much higher I content (three to seven times) in the muscle, heart, kidney, liver, serum, adipose tissue, and thyroid gland compared with the control group (Rambeck et al., 1997; Feng and Rambeck, 1998). Endemic goiter was widespread in the UK but has declined since the 1960s. Disappearance of endemic goiter was probably due to changes in farming practice, especially I supplementation in dairy herds which has resulted in I contamination of milk and milk products (Phillips, 1997). Other methods of I supplementation include the addition of I as iodide or iodate to various foods. The addition of iodide to sweets has been used in Mexico. The use of sugar as a vehicle for I fortification has been used successfully in Sudan (Eltom et al., 1995). Iodination of municipal water supplies is a relatively effective method of prophylaxis (Foo et al., 1996; Solomons, 1998). From Malaysia, after one year of iodization, goiter prevalances were reduced by 22.6 to 35.8% in three villages (Foo et al., 1996). In industrialized countries excessive I intake by humans, rather than I deficiency has become a health concern. Daily I intakes by adult Americans has been estimated at 150, 454, 548, and 696 ug for the years 1960, 1970, 1975, and 1976, respectively (Hemken, 1981). This increase may stem from the presence of iodates in baked goods, milk, and dairy products, and from increased consumption of I supplements and seafoods (see Section VII). However, recent and disturbing new data from the United States suggest that there has been a sharp decline in I intake during the last 20 years, especially in women of reproductive age (Lee et al., 1999).

XI. TOXICITY

Species differ widely in their susceptibility to I toxicosis, but all animals can tolerate I levels far beyond their requirements. Maximum suggested dietary tolerable levels of I for common livestock species are sheep and cattle (50 ppm), swine (400 ppm), poultry (300 ppm), and horses (5 ppm) (NRC, 1980). Horses are considerably less tolerant of excess I than cattle, sheep, swine, and poultry (Wehr et al., 2002). Iodine toxicosis has been noted in pregnant mares consuming as little as 40 mg of I per day. Goiter due to excess I was noted in both mares and their foals, and several cases have been associated with dietary inclusion of large amounts of dried I-rich seaweed (kelp). If the daily intake of the mare exceeds about 100 mg I for any length of time, especially during the last three months of pregnancy, her newborn foal is likely to show additional signs of hypothyroidism, expressed as weakness, lethargy, high neonatal mortality, poor muscular development and, most patently, osseous dysplasia of long bones (Frape, 1998). This includes angular deformity, tendon contraction, and hyperextension of the lower limbs with poor ossification of the carpal and tarsal bones. This extension causes the foal to walk on its heels with its toes raised from the ground. Abnormal growth of the bones occurs both at the epiphysis and in the shank of long bones. These bones are thin and small with cortical thickening. Observations of protruding jaws (mandibular prognathism) and parrot mouth (brachygnathia) have also been recorded.

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Iodine

For cattle, I toxicosis signs may appear when the diet consistently contains 50 to 100 ppm. Young stock are more sensitive to I toxicosis than lactating cows. The preruminant calf was found to tolerate up to 50 ppm I in milk replacer for five weeks postpartum (Jenkins and Hidiroglou, 1990). However, as I concentrations in plasma and nonthyroid tissues started to increase at 50 ppm I, an upper limit of 10 ppm would be preferable. Toxicosis signs include depressed appetite, dull, listless appearance, excessive tears, scaliness and sloughing of the skin, excessive lacrimation, difficulty in swallowing, excessive nasal discharge, and hacking cough. Feeding excessive I for an extended period to cattle resulted in impaired function of the humoral and cell-mediated immune systems, with reduced ability to form antibodies in response to disease organisms (Haggard et al., 1980). Toxicoses have been caused by misuse of EDDI in the oral treatment of foot-rot and softtissue lumpy jaw. Recovery from I toxicosis is rapid after the excessive I is eliminated from the diet. For sheep, signs of I toxicosis are depression, anorexia, hypothermia, and poor body weight gain (McCauley et al., 1973). Contrary to the NRC (1980) maximum dietary tolerable level for sheep of 50 ppm, levels of 267 mg I (as EDDI) and 133 mg I (as potassium iodide) per kg had no effect on live weight gain and feed intake of lambs during a 22-day treatment period (McCauley et al., 1973). Signs of toxicosis in the laying hen are reduced egg production, egg size, and hatchability (Arrington et al., 1967; Jiang et al., 1996). For swine, 400 ppm of dietary I does not depress gain or efficiency of feed utilization, but 800 ppm or more depresses growth rate, feed intake, hemoglobin level, and liver Fe concentration (NRC, 1998). For humans, I intakes of 2 mg/day should be regarded as harmful (Wolff, 1969). Inhabitants of coastal regions of both Japan and China consume large amounts of seaweed, which results in large intakes of I (i.e., 50 to 80 mg/day), In these regions, goiter induced by high I intake has been documented (Hetzel and Wellby, 1997). Consumption of excess I will inhibit organic I formation and results in hypothyroidism, because of feedback inhibition of T 3 synthesis.

XII. REFERENCES Abbassi, v., and MacKenzie, J. M. (1970). Metabolism 19,43. Abuye, C., Kelbessa, U., and Wolde, G. S. (1998). E. African Med. J. 75(3), 166. Agricultural Research Council (ARC). (1980). "The Nutrient Requirements of Ruminant Livesock.' Commonwealth Bureaux, Slough, England. Allcroft, R., Scarnell, J., and Hignett, S. L. (1954). Vet. Rec. 66, 367. Alderman, G., and Jones, D. I. H. (1967). J, Sci. Food Agric. 18, 197. Allman, R. T., and Hamilton, T. S. (1949). "Nutritional Deficiencies in Livestock," FAO Agric. Studies No.5, Washington, D.C. Al-Nuaim, A. R., AI Mazrou, Y., Kamel, M., Al Attas, 0., AI Daghari, N., and Sulimani, R. (1997). Annals Saudi Med. 17, 293. Andrews, F. N., Shrewsbury, C. L., Harper, C., Vestal, C. M., and Doyle, L. P. (1948). J. Anim. Sci. 7, 298. Arrington, L. R., Santa Cruz, R. A., Harms, R. H., and Wilson, H. R. (1967). J. Nutr. 92, 325.

References

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Azizi, F., Kalani, H., Kirniagar, M., Ghazi, A., Sarshar, A., Nafarabadi, M., Rahbar, N., Noohi, S., Mohajer, M., and Yassari, M. (1995). Int. J. Vito Nutr. Res. 65, 199. Barua, J., Cragle, R. G., and Miller, J. K. (1964). J. Dairy Sci. 47, 539. Beach, M., Streit, T. G., Houston, R., May, W. A., Addiss, D. G., and Lammie, P. J. (2001). Am. J. Trop. Med. Hyg. 64, 56. Benade, J. G., Oelofse, A., van Stuijvenberg, M. E., Jooste, P. L., and Weight, M. J. (1997). S. African Med. J. 87, 310. Berdanier, C. D. (1998). In "Advanced Nutrition Micronutrients," CRC Press, Boca Raton, FL. Bires, J., Bartko, P., Weissova, T., Michna, A., and Matisak, T. (1996). Vet. Med. 41, 133. Bleichrodt, N., and Born, M. P. (1994). In "The Damaged Brain of Iodine Deficiency-Cognitive,

Behavioral, Neuromotor, Educative Aspects" (J. B. Stanbury, ed.), Ch. 19, p. 279. Cognizant Communications Corp., New York. Brody, T. (1999). "Nutritional Biochemistry," 2nd Ed. Academic Press, San Diego. Brown-Grant, K. (1961). Physiol. Revs. 41,189. Butler, G. W., and Johnson, J. M. (1957). Nature (London) 179,216. Caple, I. V., Azuolas, J. K., and Nugent, G. F. (1985). In "Trace Elements in Man and Animals- TEMA 5" (c. F. Mills, I. Bremner, and J. K. Chesters, eds.), p. 609. Rowett Research Institute, Aberdeen, Scotland. Chhabra, S., and Hora, A. (1996). J. Obstet. Gynec. 16,242. Chopra, I. J. (1981). In "Triiodothyronines in Health and Disease: Monographs on Endocrinology," Vol. 18, p. 118. Springer-Verlag, Berlin and New York. Church, D. C. (1971). "Digestive Physiology and Nutrition of Ruminants," Vol. 2, Nutrition. D.C. Church and Oregon State Univ. Book Stores, Corvallis, Oregon. Clements, F. W. (1957). Med. J. Aust. 2, 645. Creek, R. D., Parker, H. E., Hauge, S. M., Andrews, E. N., and Carrick, C. W. (1954). Poult. Sci. 33, 1052. Cunha, T. 1. (1990). "Horse Feeding and Nutrition." Academic Press, New York. Delange, F. (1994). Thyroid 4, 107. de Welchman, D. B., Cranwell, M. P., Wrathall, A. E., and Christensen, P. (1994). Vet. Rec. 135, 589. DRI (Dietary Reference Intakes). (2001). Panel on Micronutrients of Food and Nutrition Board. National Academy Press, Washington, D.C. EI-Mahdy, M. R., and Karousa, M. M. (1995). Egypt J. Rabbit Sci. 5, 65. EI-May, M. V., Bourdoux, P., Slimane, F. B., Abdallah, M. 8., and Mtimet, S. (1997). Eur.J. Epid.13, 363. Elnour, A., Lieden, S. A., Bourdoux, P., Eltom, M., Khalid, S. A., and Hambraeus, L. (1997). Nutr. Res. 17, 533. Eltom, M., Elnagar, B., Sulieman, E. A., Karlsson, F. A., Thi, H. V., Bourdoux, P., and Gebre, M. M. (1995). Int. J. Food Nutr. 46, 281. Feldman, J. D. (1960). Am. J. Physiol. 199, 1081. Feng, J., and Rambeck, W. (1998). Acta Vet. Zoo. Sinica 29,17. Ferguson, K. A., Schinckel, P. G., Carter, H. 8., and Clarke, W. H. (1956). Aust. J. Bioi. Sci. 9, 575. Follis, R. J., Jr. (1966). Bioi. Of. Sanit. Panam. 60, 28. Foo, L. c., Zainab, T., Goh, S. Y, Letchuman, G. R., Nafikudin, M., Doraisingam, P., and Khalid, B. (1996). Biomed. Envir. Sci. 9, 126. Foo, L. c., Zainab, T., Letchuman, G. R., Nafihudin, M., Azriman, R., Doraisingah, P., and Khalid, A. K. (1994). Southeast Asian J. Trop. Med. Public Health 25(3), 575. Frap, D. (1998). "Equine Nutrition and Feeding" 2nd Ed., Blackwell Science Inc., Malden, MA. Furnee, C. A., West, C. E., Vander Haar, F., and Hautvast, J. (1997). Am. J. Clin. Nutr. 66, 1422. Gaitan, E., Cooksey, R. C, Legan, J., Lindsay, R. H., Ingbar, S. H., and Medeiros, N. G. (1994). Europ. J. Endocrin. 131, 138. Gaitan, E., Nelson, N. C., and Poole, G. V. (1991). World J. Surg. 15,205. Gross, F. (1962). In "Mineral Metabolism," Vol. II, Part B (C. L. Comar and F. Bronner, eds.), p. 221. Academic Press, New York. Gurevich, G. P. (1964). Fed. Proc. 23, T 511. Haggard, D., Stowe, H. D., Conner, G. H., and Johnson, D. W. (1980). Am. J. Vet. Res. 4, 539. Hampel, R., Gordella, A., Demuth, M., Zoolner, H., and Klinke, D. (1997). Zeitschrift-Ernahrungswissenschaft 36, 151. Hartmans, J. (1974). Neth. J. Agr. Sci. 22, 195. Hemken, R. W. (1970). J. Dairy Sci. 53, 1138. Hemken, R. W. (1978). J. Anim. Sci. 48, 981. Hemken, R. W. (1981). The Professional Nutritionist 13(4), II.

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Hemken, R. W., Vandersall, J. H., Oskarsson, M. A., and Fryman, L. R. (1972). J. Dairy Sci. 55, 931. Hemken, R. W., Vandersall, J. H., Sass, B. A., and Hibbs, J. W. (\971). J. Dairy Sci. 54, 85. Hess, S. Y., Zimmermann, M. B., Arnold, M., Langhans, W., and Hurrell, R. F. (2002). J. Nutr. 132, 1951. Hetzel, B. S. (1983). Lancet 2(8359), 1126. Hetzel, B. S. (1989). "The Story oflodine Deficiency: An International Challenge in Nutrition," Oxford University Press, Delhi. Hetzel, B. S., and Maberly, G. F. (1986). In "Trace Elements in Human and Animal Nutrition," 5th Rev. Ed., Vol. No.2 (W. Mertz, ed.), p. 139. Academic Press, New York. Hetzel, B. S., and Pandav, C. S. (\994). In "S.O.S. for a Billion: The Conquest of Iodine Deficiency Disorders" (B. S. Hetzel and C. S. Pandav, eds.), Oxford University Press, New York. Hetzel, B. S., and Wellby, M. L. (1997). In "Handbook of Nutritionally Essential Mineral Elements" (B. L. O'Dell and R. A. Sunde, cds.), p. 557, Marcel Dekker, Inc., New York. Hotz, C. S., Fitzpatrick, D. W., Trick, K. D., and L'abbe, M. R. (1997). J. Nutr. 127, 1214. Huda, S. N., Grantham-McGregor, S. M., Rahman, K. H., and Tomkins, A. (1999). J. Nutr. 129,980. Iodine Educational Bureau. (1946). "Iodine Facts," Fact No. 332. Iodine Educational Bureau, p. 215. Stone House, Bishopsgate, London, England. Jaffiol, c., Manderscheid, J. C., Rouard, L., Dhondt, J. L., Arguillere, S., and Bourdoux, P. (1997). Bul/. Acad. Nat. Med. 181, 1795. Jenkins, K. J., and Hidiroglou, M. (1990). J. Dairy Sci. 73,804. Jiang, Q. Y., Fu, W. L., and Chen, L. J. (1996). J. S. China Agr. Univ. 17, 33. Johnson, A. H., and Herrington, B. L. (1927). J. Agric. Res. 35, 167. Johnson, F. F., and Frederick, E. R. (\ 940). Science 92, 315. Jooste, P. L., Badenhorst, C. J., Schutte, C. H., Faber, M., van Staden, E., Oelofse, A., and Aalbers, C. (1992). South Afr. Med. J. 81, 571. Jooste, P. L., Langenhoven, M. L., Kriek, J. A., Kunneke, E., Nyaphisi, M., and Sharp, B. (\ 997). E. African Med. J. 74, 680. Jooste, P. L., Weight, M. J., Kriek, J. A., and Louw, A. J. (1999). Europ. J. Clin. Nutr. 53, 8. Judson, G. J. (1996). In "Detection and Treatment of Mineral Nutrition Problems in Grazing Sheep" (D. G. Masters and C. L. White, eds.), p. 57, Australian Center for International Agricultural Research, Canberra. Kalk, W. J. (1998). S. African Med. J. 88, 352. Karmarkar, M. G., Deo, M. G., Kochupillai, N., and Ramalingaswami, V. (1974). Am. J. Clin. Nutr. 27, 96. Kelly, F. c., and Snedden, W. W. (1960). "Endemic Goiter." World Health Organization Monograph Series, No. 44. Geneva, Switzerland. Kochupillai, N., Panda, C. S., and Karmarkar, M. G. (1984). Indian J. Med. Res. 80, 293. Kiihrle, J. (1994). Exp. Clin. Endocrinol. 102, 63. Kruzkova, E. (1968). Nutr. Abstr. Rev. 39, 807. Langer, P. (1960). In "Endemic Goiter," World Health Organizational Monographs Series 44,9. Lee, K., Bradley, R., Dwyer, J., and Lee, S. L. (1999). Nutr. Rev. 57,177. Leng, X., Blanco, J., Tsai, S. Y., Ozato, K., O'Malley, D. W., and Tsai, M. J. (\994). J. Bioi. Chern. 269, 31436. Lengemann, F. W. (1979). J. Dairy Sci. 62,412. Lewis, R. C., and Ralston, N. P. (\953). J. Dairy Sci. 36, 363. Lidiard, H. M. (1995). App. Geochem. 10,85. Livingston, R. S., Franklin, C. L., Lattimer, J. c., Dixon, R. S., Riley, L. K., Hook, R. R., and BeschWilliford, C. L. (1997). Lab. Anim. Sci. 47, 346. Marcilese, N. A., Harms, R. H., Valsecchi, R. M., and Arrington, L. R. (1968). J. Nutr. 94, 117. Mason, R. W. (1976). Br. Vet. J. 132,374. Matovinovic, J. (1984). In "Nutrition Reviews Present Knowledge in Nutrition" (R. E. Olson, ed.), p. 587. The Nutrition Foundation, Washington, D.C. Matovinovic, J., and Trowbridge, F. L. (1980). In "Endemic Goiter and Endemic Cretinism" (J. B. Stanbury and B. S. Hetzel, eds.), p. 37. J. Wiley, New York. Maynard, L. A., Loosli, J. K., Hintz, H. F., and Warner, R. G. (\979). "Animal Nutrition" 7th Ed. McGraw-Hill, New York. McCauley, E. H., Linn, J. G., and Goodrich, R. D. (1973). Am. J. Vet. Res. 37, 65. McDonald, I. W. (1968). Nutr. Abstr. Rev. 38, 381. McDonald, R. J., McKay, G. W., and Thomson, J. D. (1962). Prac. Int. Congr. Anim. Reprod. Artif. Insemin, 4th, Vol. 3, p. 679. McDowell, L. R. (2000). In "Vitamins in Animal and Human Nutrition" 2nd Ed. Iowa State University Press, Ames, Iowa.

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Chapter 11

Manganese

I. INTRODUCTION Manganese (Mn) is essential for plants and animals. Manganese deficiency can occur naturally on diets composed of normal feed ingredients in swine, poultry, and ruminants, and has been reported in humans associated with vitamin K deficiency. The Mn requirements of poultry are appreciably higher than those of other domestic livestock, and Mn deficiency is largely confined to these species.

II. HISTORY Manganese has been known since Roman times. The name derived from a Greek word for magic. Bertrand and associates were first to show (1913 to 1928) that Mn occurs in relatively constant amounts in tissues and organs of both plants and animals, and is especially concentrated in the reproductive organs (Scott et al., 1982). Manganese was first recognized as an essential mineral element for growth and reproduction in mice and rats in 1931. Interest in Mn nutrition was greatly stimulated a few years later by the discovery (Wilgus et al., 1936) that a deficiency of this element was largely responsible for a crippling disease of chickens known as perosis (slipped tendon). These findings gave a great stimulus to studies of basic functions and metabolism of Mn, but a number of years elapsed before a deficiency in ruminants was demonstrated (Bentley and Phillips, 1951). Since that time, Mn has been shown to be essential for many species of animals.

III. CHEMICAL PROPERTIES AND DISTRIBUTION The atomic number of Mn is 25 and its atomic weight is 54.938. It is the twelfth most abundant element, constituting 0.10% of the earth's crust. Soil Mn concentrations range from less than one to as much as 7000 ppm with an average of 500 to 600 ppm. Manganese is a steel gray lustrous metal that is hard and brittle. In chemical properties, it is similar to iron (Fe); its two most important valence states in biological systems are +2 and +3. However, Mn can exist in II oxidation states from -3 to +7. Similarity to Fe allows Mn to assume some properties of this 335

Manganese

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element, therefore Fe-Mn interactions in biological systems are well known (Leach and Harris, 1997). Liver, bones, pancreas, and kidney have relatively high levels of Mn and muscles have very little. The skeleton accounts for about 25% of total body Mn. Excess Mn intake is generally reflected in excess concentrations in hair, wool, and feathers, but deficient intakes are not consistently reflected in lower levels. The Mn concentration of most tissues is quite characteristic and not very responsive to changes in intake (Miller, 1979).

IV. METABOLISM

A. Absorption and Transport Manganese absorption apparently occurs equally well throughout the length of the small intestine (Keen and Zidenberg-Cherr, 1990) in two steps: uptake from the gut lumen, then transfer across mucosal cells. Manganese absorption, in all species studied, is relatively poor. Greenberg and Campbell (1940) reported that only 3 to 4% of orally administered radioactive Mn was absorbed by rats. Using 54Mn_ labeled test meals, Mn absorption in adult humans has been reported to range from 1 to 15% (Keen and Zidenberg-Cherr, 1996). Less than 0.1 % of an oral dose was apparently absorbed by avian species (Turk et al., 1982). Cattle absorb about 1% of ingested Mn (Abrams et al., 1977). However, Howes and Dyer (1971) showed that absorption in young calves is considerably higher than generally reported in other species. Low absorbability of Mn in grains has been associated with formations of complexes with phytate and fiber (Underwood and Suttle, 1999). Determination of Mn absorption is difficult as the element [also copper (Cu) and zinc (Zn)] are absorbed and quickly resecreted into the gut through bile (Finley et al., 1997). When this occurs, the unabsorbed nutrient and the absorbed and resecreted nutrient may mix in the gut, preventing quantitative calculation of either. Other minerals influence absorption, including phosphorus (P), magnesium (Mg), calcium (Ca) and Fe. Either Fe (Rodriguez-Matas et al., 1998), or Mg (SanchezMorito et al., 1999) deficiency will increase absorption of Mn. In birds, high dietary levels of calcium phosphate aggravate Mn deficiency owing to a reduction in soluble Mn (Davies and Nightingale, 1975). It is believed that P is the antagonist to Mn rather than Ca (Wedekind et al., 1991). The major impairment ofMn utilization in practice is likely to come from phytate (Underwood and Suttle, 1999). Vitamin D and/or dietary phytase will improve Mn absorption by negating the inhibition of phytates (Biehl et al., 1995). In chickens, high levels of dietary Fe can accentuate the severity of perosis, probably by decreasing Mn absorption (Wilgus and Patton, 1939). Manganese competes directly with cobalt (Co) and Fe for binding sites; thus excess Fe or Co could induce Mn deficiency, and excess Mn or Co could induce Fe deficiency. Estrogenic hormones increase Mn absorption. In sows, Mn absorption was increased during pregnancy (Kirchgessner et al., 1981). Coccidiosis infection in

Metabolism

337

chickens has also been reported to increase Mn absorption (Southern and Baker, 1983); the mechanisms of this effect are not understood. Absorbed Mn may either remain free or rapidly become bound to Olr macroglobulin before traversing the liver, where it is removed (Hurley and Keen, 1987). However, some of the Mn bound to cx2-macroglobulin may enter the systemic circulation, become oxidized to the manganic state, and become bound to transferrin. Davidsson et al. (1989), using 54Mn, concluded that transferrin is the major plasma carrier protein for Mn regardless of route of administration. Manganese is readily transferred, via the placenta from the mother to the fetus in swine (NRC, 1998). For humans, the fetus contained about half the Mn as did the mother (Tolonen, 1990). B. Storage In marked contrast to most other essential mineral elements, there does not appear to be any appreciable stores of Mn in the body (Keen and Zidenberg-Cherr, 1996). Manganese is widely distributed throughout the body but in low concentrations. It is found in higher concentrations in bone, liver, kidney, and pancreas than in skeletal muscle. Cytosol contains less than mitochondria. The storage capacity of the liver for Mn is limited compared with its capacity to retain Cu, Fe, and selenium (Se), and although the skeleton normally accounts for about one quarter of the total body content of Mn, this reserve is not readily used when dietary intake is low. Only a small change in tissue Mn level (twofold) has been brought about with a 200-fold increase in dietary level (Thomas, 1970). A number of researchers have observed an increase in liver Mn when dietary Mn is elevated (Watson et al., 1973). It appears that liver Mn changes readily up to a certain level (about double normal) and then resists further change (Carter et al., 1974). Watson et at. (1973) did, however, report that liver Mn increased 4.5 times over controls when lamb diets contained toxic levels (4030 ppm) ofMn. Likewise, Howes and Dyer (1971) found a marked change in liver Mn in newborn calves given supplemental Mn for 7 days; liver Mn increased from 4 to 943 ppm. This indicates that Mn metabolism in the newborn calf is different from that of older animals. C. Excretion Variable excretion and absorption are major homeostasis mechanisms by which animals maintain normal health and performance over the wide range of Mn intake. Britton and Cotzias (1966) suggested that variable excretion rather than variable absorption regulates tissue Mn. The Mn body pool is small, so that total body excretion is continuously very nearly equal to intake, with 25 to 50% of the body pool ingested daily (Lassiter et al., 1970). Thus, excess Mn intakes result in both decreased absorption efficiency and increased excretion rates. Oral Mn is excreted mainly in feces (95-98%), with only 0.1 to 0.3% usually excreted in urine (Thomas, 1970). Bile is the main route of Mn excretion

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(Papavasilion et al., 1979). Biliary excretion may be a major homeostatic mechanism for preventing both deficiency and toxicosis of Mn (Malecki et al., 1996). Carter et al. (1974) report that increasing dietary Mn in calves from 0.5 to 15 ppm (a 30-fold increase) for 5 days caused a 30-fold increase in bile Mn. Pancreatic juice, however, also contributes to the gut excretion of Mn as does secretion from the intestinal wall. Probably Mn is reabsorbed as bile-bound Mn. Each atom may recirculate several times before final excretion.

V. PHYSIOLOGICAL FUNCTIONS

A. Enzyme Activity Like other essential trace elements, Mn can function both as an enzyme activator and as a constituent of metalloenzymes. Manganese-containing enzymes include arginase, pyruvate carboxylase, and manganese-superoxide dismutase. While the number of Mn metalloenzymes is limited, the enzymes that can be activated by Mn are numerous. They include hydrolases, kinases, decarboxylases, and transferases (Groppel and Anke, 1971). Whether an activator or a component of the enzyme proper, Mn is often the priority cation, but another cation, especially Mg, can partially substitute for Mn with little or no loss in enzymatic activity. Thus, biotindependent enzymes such as pyruvate carboxylase continue to fix CO 2 during Mn deficiency because Mg substitutes for Mn in the enzyme.

B. Bone Growth In most species studied, Mn-deficient bones are considerably shortened and thickened. Manganese is essential for development of the organic matrix of the bone, which is composed largely of mucopolysaccharide. Impairment in mucopolysaccharide synthesis associated with Mn deficiency has been related to the activation of glycosyltransferases (Leach and Harris, 1997). These enzymes are important to polysaccharide and glycoprotein synthesis, and Mn is usually the most effective of the metal ions required. Manganese is essential for maintenance of bone mineralization. In long-term studies, Mn deficiency in rats resulted in lowered bone Ca concentrations and radiographic differences in bone mineralization (Strause et al., 1986).

C. Reproduction Effects on reproduction were among the first signs of Mn deficiency to be observed. The deficiency can cause an irreversible congenital defect in young chicks, rats, and guinea pigs characterized by ataxia and loss of equilibrium. Shils and McCollum (1943) found several stages of Mn deficiency in female rodents: (I) birth of viable young with ataxia; (2) nonviable young that die shortly after birth; and

Physiological Functions

339

(3) disturbance of estrus, with no reproduction. Impaired or irregular estrus has also been observed in cattle and swine. Hidiroglou (1975), on the basis of Mn tissuedistribution studies of the reproductive tract of normal and anestrus ewes, has suggested that Mn has a role in corpus luteum functioning. In laying hens, Mn deficiency has resulted in a decreased rate of egg production, poor shell quality, reduced hatchability, and an embryonic deficiency called chondrodystrophy. Testicular degeneration has been reported in Mn-deficient rats, mice, and rabbits (Leach, 1978).

D. Lipid Metabolism Underwood and Suttle (1999) suggest that pyruvate carboxylase sustains lipid as well as glucose metabolism, because the fat accumulation seen in Mn deficiency is also a feature of biotin deficiency and biotin activates the same enzyme. Defects in both lipid and carbohydrate metabolism have been reported in Mn-deficient rats and guinea-pigs. In a Mn deficiency in rats, manganese-superoxide dismutase activity and conjugated diene formation in the heart were reduced (Malecki and Greger, 1996). Manganese superoxide dismutase is an important mitochondrial antioxidant (Kuratko, 1997). Manganese was found to protect against heart mitochondrial lipid peroxidation in rats fed high levels of polyunsaturated fatty acids (Malecki and Greger, 1996). A metabolic association between Mn and choline has been known for some time. Fatty liver in rats induced by Mn deficiency is alleviated by either Mn or choline. Also, Mn deficiency increases fat deposition and backfat thickness in pigs. Both Mn and choline are needed for prevention of perosis in poultry. Manganese is involved in the biosynthesis of choline. Furthermore, the changes in liver ultrastructure that arise in choline deficiency are very similar to those in Mn deficiency (Bruni and Hegsted, 1970). Deficiencies of Mn and choline both appear to affect membrane integrity. E. Carbohydrate Metabolism

Glucose utilization is impaired by Mn deficiency. Necropsy has revealed gross abnormalities in the pancreas such as aplasia or marked hypoplasia of all cellular components, so Mn may in some way be involved in insulin formation or activity. Rats deficient in Mn had fewer insulin receptors per cell compared to controls (Baly et al., 1990). Biosynthesis of glycoproteins may be impaired in Mn-deficient animals. Prothrombin is a glycoprotein whose synthesis has long been known to be controlled by vitamin K. Manganese is also required, and a deficiency reduces the vitamin K-induced clotting response (Doisey, 1974).

F. Cell Function and Structure Abnormalities in cell function and ultrastructure, particularly involving the mitochondria, occur in Mn deficiency (Hurley and Keen, 1987). Manganese

340

Manganese

deficiency caused alterations in cell membrane integrity in the liver, pancreas, kidney, and heart in aged mice (Bell and Hurley, 1973). G. Immune Function Manganese plays a role in immunological function (Hurley and Keen, 1987). Interaction of Mn with neutrophils and macrophages has been demonstrated, possibly through interactions with the plasma membrane of cells employed in the immune response (Rabinovitch and Destefano, 1973).

H. Brain Function Manganese deficiency or toxicosis can affect brain function (Hurley, 1984). Manganese-deficient rats, whether they are ataxic or not, are more susceptible to convulsions (Hurley et al., 1963). Papavasiliou et al. (1979) reported that humans with convulsive disorders, including epilepsy, showed whole blood Mn concentrations significantly below normal.

VI. REQUIREMENTS

The minimum dietary requirements (Table ll.l) depend on species, criteria of adequacy employed, chemical forms in which the element is ingested, and the nature of the rest of the diet (Underwood, 1977). The higher Mn requirements of poultry compared with other species arise mainly from lower intestinal absorption. On the contrary, the Mn requirement in swine is considerably lower than the requirements for ruminants or poultry. Growth requirement for Mn in cattle is less than that for reproduction Dyer et al. (1964) and Rojas et al. (1965) concluded that the Mn requirements of cows for maximal fertility is in excess of 16 ppm. Bentley and Phillips (1951) concluded that 10 ppm Mn was adequate for growth but marginal for optimal reproduction. Cows on the unsupplemented diets were slower to exhibit estrus and to conceive with some calves born with weak legs and pasterns. Anke et al. (1973) further confirmed the lower requirements for growth than for fertility in female goats. Goats fed diets containing 20 ppm Mn in the first year and 6 ppm in the second year grew as well as those receiving 100 ppm of additional Mn, but the former exhibited greatly impaired reproductive performance. The requirements of pigs for satisfactory reproduction are substantially higher than those needed for body growth (NRC, 1998). A level of 1 ppm Mn appears to be adequate for growth but is inadequate for normal fetal development in mice or rats (Bell and Hurley, 1973). The levels of Ca and P in the diet affect Mn requirements. Excessively high intakes of Ca and P over a long period reduced weight gains of growing calves reared mainly on a milk diet, possibly due to interference with Mn absorption, but additional Mn (50 ppm) tended to counteract the excess (Hawkins et al., 1955). A total of 64% of chicks fed a diet containing 3.2% Ca, 1.6% P, and 37 ppm Mn

Requirements

341

TABLE 11.1 Manganese Requirement for Various Species" Species

Purpose

Chickens

Leghorn-type ~ wk Leghorn-type 6-20 wk Leghorn-type laying Leghorn-type breeding Broilers 0-8 wk All classes All classes All classes All classes All classes All classes Gestation-lactation Growing All classes All classes Growing All classes All classes Growing All classes All classes All classes Children Adults

Ducks Turkeys Beef cattle Dairy cattle Sheep Horses Swine Mink Rabbits Cats Rats Mice Guinea pigs Channel catfish Common carp Nonhuman primates Humans

Requirement

60 rng/kg 30 mg/kg 20 mg/kg 60 mg/kg 60 mg/kg 25-40 rng/kg 60 mg/kg 20-40 mg/kg 14--22 mg/kg 20-40 mg/kg

40 rng/kg 20 mg/kg 2-4 mg/kg 40-44 mg/kg 2.5-8.5 mg/kg 5 mg/kg 10 mg/kg 10 mg/kg

40 mg/kg 2.4 mg/kg 13 mg/kg 40 mg/kg 1.2~ 1.5 mg/day 1.9-2.6 mg/day

Reference

NRC (1994) NRC (1994) NRC (1994) NRC (1994) NRC (1994) NRC (1994) NRC (1994) NRC (1996) NRC (2001) NRC (1985) NRC (1989) NRC (1998) NRC (1998) NRC (1982) NRC (1977) NRC (1986) NRC (1995) NRC (1995) NRC (1995) NRC (1993) NRC (1993) NRC (1978) DRI (2001) DRI (2001)

"Expressed as per unit animal feed either on as-fed (approximately 90% dry matter) or dry basis (see Appendix Table I). Human requirements are unknown, with estimates expressed as mgjday.

developed perosis, whereas no perosis was observed when the diet supplied the same amount of Mn but only 1.2% Ca and 0.9% P (Schaible et ai., 1938). Freedom from perosis was achieved either by omitting the bone meal, without additional Mn, or by retaining the bone meal and increasing dietary Mn levels. Manganese requirements in poultry are dependent on physiological function. The level of Mn for prevention of perosis can be 50 ppm, but only 20 ppm for maximal growth. A total of 25 ppm is often considered normal to support maximal egg production, feed efficiency, egg weight, hatchability, and livability, but for maximal eggshell strength, the minimum requirement for laying hens is between 50 and 100 ppm (Cook and Crenshaw, 1983). On the basis of field evidence with cattle, unidentified factors other than Ca and P exist that are capable of producing conditioned Mn deficiency and thereby increasing Mn requirements (Dyer et al., 1964). Shupe et al. (1967) reported that cows ingesting Lupinus caudatus, Lupinus sericeus, lupine extracts, and sparteine sulfate gave birth to deformed calves. Alkaloids in the lupin us plants were toxic to animals, with teratogenic effects on calves similar to anomalies due to

342

Manganese

Mn deficiency (Rojas et al., 1965). Howes et al. (1973) showed that lupines (sparteine sulfate) had a significant effect on mineral metabolism. The reduction and/or increase in 54Mn and 65Zn activity caused by addition of sparteine sulfate suggests that this alkaloid causes a change in tissue turnover rate of Mn and Zn. The human requirement for Mn is not well defined, but the estimated safe and adequate intake is 1.9 to 2.6 mg per day for adults and 1.2 to 1.5 mg daily for infants and children (ORI, 2001).

VII. NATURAL SOURCES Concentration of Mn in crops and forages is dependent on soil factors, plant species, stage of maturity, yield, crop management, climate, and soil pH. Gomide et al. (1969), studying mineral composition of tropical grasses in Minas Gerais, Brazil, found significant year, species, and age interaction in Mn content. Poor drainage conditions increase forage Mn (Mitchell, 1963), but increasing soil pH decreases plant availability and uptake of Mn (Cox, 1973). Mitchell (1957) shifted soil pH from 5.6 to 6.4 by liming in an experiment with red clover and ryegrass. This shift in pH decreased the Mn in clover from 58 to 40 ppm and in the grass from 140 to 130 ppm. Where Mn concentrations are not above 60 ppm (OM), grass and legumes grown on the same site have similar levels. Above 60 ppm (OM), however, grasses tend to have considerably higher values than legumes (MacPherson, 2000). The Mn content of feedstuffs is shown in Appendix Table II. Rice bran, wheat middlings, alfalfa meal, and corn distillers dried solubles are the richest plant sources. Typical values for various grasses, clover, etc., range from 60 to more than 800 ppm on a dry basis (Underwood, 1977). Corn grain, and to a lesser extent sorghum and barley, are generally substantially lower in Mn than wheat or oats. Corn grain is typically very low at 5 ppm and barley moderately low at perhaps three times that of corn. Manganese is highly concentrated in the outer layers of grains so that the inclusion of mill products, bran and middlings, markedly increases Mn intakes. Protein supplements obtained from animal sources (i.e., meat meal, blood meal, and fish meal) are generally low in Mn, approximately 5 to 15 ppm. Milk and milk products are even lower in this mineral owing to the generally very low content of Mn in cow's milk (20 to 40 I!g/l). Colostrum is considerably richer in Mn than milk produced later in lactation. Archibald and Lindquist (1943) increased the milk Mn concentration two- to four-fold by feeding Mn sulfate to cows in amounts equivalent to 10 or 13 g/day, Manganese concentrations in sheep and goat milk are similar (20 to 50 I!g/l) to that of the cow, while human milk is low (4 to 15 I!g/l). Relatively high concentrations of Mn are found in tea and coffee (300 to 600 I!g/ml), and these sources can account for as much as 10% of daily Mn intake for some individuals (Keen and Zidenberg-Cherr, 1996). Whole egg contains roughly 0.7 ppm, with yolk being five times richer than the white. The Mn content of eggs varies widely with the Mn level of the diet, with supplemental Mn readily transmitted to the egg for use by the chick embryo.

Deficiency

343

Gallup and Norris (1939) increased dietary Mn in the hen's diet from 13 to 1000 ppm, which resulted in increased yolk Mn from 4 to 33 ug,

VIII. DEFICIENCY

A. Effects of Deficiency The main manifestations of Mn deficiency, namely impaired growth, skeletal abnormalities, disrupted or depressed reproductive function, ataxia of the newborn, and defects in lipid and carbohydrate metabolism, are displayed in all species studied, but their actual expression varies with the degree and duration of the deficiency and with the developmental stage during which the deficiency occurs. 1. SWINE

A deficiency of Mn in swine diets causes decreased growth, feed efficiency, and impaired reproduction. Growth was normal when pigs were fed a purified diet containing as little as 1.5 ppm Mn, and reproduction was satisfactory when the element was increased to 6 ppm. Plumlee et al. (1956) observed reduced skeletal growth, muscular weakness, increased fatness, irregular estral cycles, resorption of fetuses, birth of small and weak pigs, poor udder development, and poor milk secretion in female pigs fed 0.5 ppm Mn in a semipurified diet from 3 weeks of age throughout growing, gestation and lactation periods (Figure 11.1). The surviving newborn were ataxic, consequently their balance and locomotion were impaired. In Mn-deficient pigs the skeletal abnormalities were characterized by lameness and enlarged hock joints with crooked and shortened legs. Total litter weight at birth was less for sows fed a low-Mn, basal corn-soybean meal diet (10 ppm Mn) than for sows fed the basal diet plus 84 ppm Mn (Rheaume and Chavaz, 1989). Christianson et al. (1990) reported that birth weight of pigs was greater when sows were fed 10 to 20 ppm Mn than when they were fed 5 ppm. Also, return to estrus was improved by feeding 20 ppm Mn. 2.

POULTRY

Manganese deficiency in birds was studied, seeking a treatment for perosis and chondrodystrophy. Birds are considered to be more susceptible to the deficiency than mammals because they require more Mn to overcome or prevent the deficiency. Perosis (Fig. 11.2) in chicks is the most commonly observed Mn deficiency. The disease is a malformation of bones characterized by enlarged and malformed tibiometatarsal joints, twisting and bending of the tibia and the tarso metatarsus, thickening and shortening of the long bones, and slippage of the gastrocnemius or Achilles tendon from its condyles. One or both legs may be affected. Deficiencies of other nutrients including choline, biotin, and other B vitamins are involved in inducing perosis (McDowell, 2000). The disease is also markedly aggravated by high intakes of Ca and P (see Section VI).

344

Manganese

Fig. 11.1 Manganese deficiency. Top photo show 132-day-old gilt fed 40 ppm manganese since she weighed 8.5Ib. Middle photo shows a littermate gilt started at same weight and 132days old, fed 0.5 ppm of manganese. Note increased fat deposition due to low manganese. Pigs deficient in Mn showed weakness and poor sense of balance at birth. (Courtesy of the late W.M. Beeson, Purdue University, West Lafayette, IN)

Deficiency

345

Fig. 11.2 Manganese deficiency in a 4-week-old chicken. Photo on the left shows control chicken with normal tibiotarsus. Photo on the right shows chicken with perosis. Note swollen joints and twisted legs. The tibiotarsus from the manganese-deficient chicken is shortened, thickened and twisted. (Courtesy R.M. Leach, Jr., Pennsylvania State University, University Park)

Manganese deficiency in the diet of the breeding hen causes a condition in embryonic chicks known as nutritional chondrodystrophy. Chondrodystrophy is characterized by defective growth, edema, generalized bone disease, and high mortality (Leeson and Summers, 2001). The abdomen protrudes, the head is round, and the underdeveloped lower mandible makes these chickens look like parrots (Parrot beak).

346

Manganese

In young chicks, a deficiency of Mn produces nervous signs (ataxia) characterized by a star-gazing posture similar to that observed with thiamin deficiency (McDowell, 2000; Leeson and Summers, 2001). According to Erway et al. (1970), the otoliths of the inner ear are defective or absent. Chondrodystrophy in chick embryos and the disproportionate growth at the site of the otolithic matrix, resulting in abnormal development of the inner ear, both appear to be caused by faulty synthesis of mucopolysaccharides, Manganese deficiency in laying and breeding hens reduces egg production, markedly decreases hatchability and increases incidence of thin-shelled and shellless eggs (Leeson and Summers, 2001). Reduced eggshell thickness and strength, using the scanning electron microscope, revealed alterations in shell structure, Chemical analysis of the shells revealed changes similar to those found in the cartilage of embryos and young chicks. This deleterious effect on egg production and shell quality is accentuated by manipulations of dietary Ca and P levels. 3.

RUMINANTS

Although Mn deficiency has been produced experimentally in ruminants, with effects on skeletal development and reproductive performance, doubt had been expressed whether this deficiency arises under field conditions. However, Mn deficiency for ruminants under grazing conditions has been reported in the United States, Brazil, Costa Rica, The Netherlands, the United Kingdom, Union of South Africa, and Germany (McDowell, 1985). Clinical signs and conditions observed when ruminant diets contain insufficient Mn include suboptimal soft tissue and skeletal growth; decreased breaking strength of bones; abnormal bone shape; ataxia; muscular weakness; excess accumulation of body fat; reduced tissue storage of Mn in bone, liver, hair, and ovary; reduced milk production; reduced level of Mn in milk; delayed, irregular, or absent estrus; resorption of fetus; fetal deformities; and small birth weights (Thomas, 1970; Hidiroglou, 1980). The skeletal abnormalities result in crippling and incoordination in the offspring. The reproductive processes are particularly susceptible to lack of Mn. A report from Wisconsin notes that sterility exists in about 10% of the cattle of certain herds where low-Mn diets «20 ppm) are fed (Bentley and Phillips, 1951). The effect of Mn supplementation on cattle infertility in Great Britain was reported by Wilson (1965) in an experiment with 350 cows. Of those receiving Mn, 63% conceived after the first service, compared with 51 % of those not given Mn. In South Australia, supplementation increased the lambing percentage in herds in which reproductive performance had declined (Egan, 1972). Anke et al. (1973) reported that cows on diets low in Mn expressed estrus weakly, and 31% were pregnant from the first insemination, compared with 64% in the control herd. The nature and severity of the skeletal abnormalities that arise in Mn-deficient animals vary from a mild and generalized rarefaction of bone to gross and crippling deformities, particularly of the long bones (Underwood, 1981), Reports from

Deficiency

347

Fig. 11.3 Manganese deficiency in a newborn calf. Legs are weak and deformed. (Courtesy of the late I.A. Dyer and Washington State University, Pullman)

the western United States show a positive relationship between a low-Mn intake of gestating cows and the incidence of neonatal deformities in their calves (Dyer et al., 1964). Shupe et al. (1967) reports a crooked caifdisease in nine western states, similar to the clinical signs of Mn deficiency. A survey conducted over a 4year period with a total of 4000 head of cattle indicated that 2.7% of the calves born over this period were deformed. From South Africa, come reports of low forage Mn in addition to the clinical signs of deficiency in young cattle, including a stilted gait and straight hocks (Bisschop and Groenwald, 1963). Rojas et al. (1965) fed pregnant cows diets containing 15.8 or 25 ppm Mn. Calves born to cows fed the low-Mn diets were born with general weakness and deformities characterized by enlarged joints, stiffness, and twisted legs (Fig. 11.3). Deformed calves had shorter humeri, with greatly reduced breaking strength (Rojas et al., 1965). The likelihood of ruminant diets being deficient in Mn is less likely than for the trace elements Co, Cu, iodine (I), Se, and Zn. Generally most feeds consisting of grains, grasses, legumes, and silages are adequate in Mn. Grazing livestock have been reported to be deficient in Mn, as previously noted, but generally forages (particularly tropical forages) contain adequate to excess concentrations of the element. Table 11.2 illustrates high forage Mn from seven tropical countries. Forage Mn in these countries far exceeded the requirement of this element by grazing livestock. Of 293 average forage values from the 1974 Latin American Tables of Feed Composition, 21% contained less than 40 ppm, while only 5.2% contained less than 20 ppm (McDowell et al., 1974). 4.

HORSES

The Mn requirement of the horse in unknown; deficiencies have not been reported experimentally or under field conditions (NRC, 1989).

Manganese

348

TABLE 11.2 Mean Forage Manganese Concentrations" Manganese

Country

Season

Mean (ppm)

Malawi"

Wet Dry Wet Wet Dry Wet Dry Dry Wet Dry Wet Dry

98 245 183 365 339 209 264 151 92 93 112 103

Argentina"

Bolivia" Colombia" Dominican Republic"

Guatemala" Indonesia"

cc

20 20 40

20 20 40 40 30 40 40 40 40

Percentage below cc

6 3 5.5 0 0 0 0 10 32 24 13 20

'Critical concentrations (cc) are based on ruminant needs (McDowell and Conrad. 1977). bMtimuni et al. (1990). 'Balbuena (1988). dMcDowell et al. (1982).

"Vargas

et al. (1984).

[Jerez et al. (1984). "Tejada et al. (1987). hprabowo et al. (1991).

5.

OTHER ANIMAL SPECIES

a. Fish. Manganese deficiency was studied in common carp and rainbow trout by Ogino and Yang (1978). Deficient diets (4 ppm) caused depressed growth in both species, and abnormal tail growth and shortening of the body in rainbow trout. In broodstock rainbow trout, a fishmeal based diet without Mn supplement caused poor hatchability and low Mn concentration in the eggs (NRC, 1993).

b. Laboratory Animals. Rats, mice, and other laboratory animals are unable to grow normally when fed purified diets low in Mn. Mice fed a deficient diet later in life showed obesity and fatty livers, as well as altered integrity of cell membranes, swollen and irregular endoplasmic reticulum, and abnormal mitochondria (Bell and Hurley, 1973). A deficiency of Mn in mice during prenatal development can result in congenital irreversible ataxia, which is characterized by lack of equilibrium and retraction of the head (NRC, 1995). In rats, Mn deficiency results in defective bone mineralization, reduced feed consumption, and early death. Reproduction is characterized by testicular degeneration and defective ovulation (NRC, 1995). If reproduction does occur, many young of rats, mice, and guinea pigs are ataxic (Erway et al., 1970).

Deficiency

349

Fig. 11.4 The crooked front legs induced by a manganese deficiency. (Courtesy of S.E. Smith. Cornell University, Ithaca, NY)

c. Mink. Manganese deficiency in pastel mink results in screw necks or head tilting (NRC, 1982). Otoliths are reduced in size or absent. Animals displaying this defect have extreme difficulty in swimming and, depending on extent of the defect, may be completely unable to maintain equilibrium, and consequently drown.

d. Rabbits. Bone malformations, most evident as crooked front legs, occur in rabbits fed low dietary Mn (Fig. 11.4). The humeri are brittle, shorter than normal, and lower in ash, density, and breaking strength (Smith and Ellis, 1947). There are also marked histological changes. 6.

HUMANS

There is only one reported occurrence of Mn deficiency in humans (Doisey, 1974). In this study when a patient accidentally received a diet low in Mn, response to vitamin K supplementation was reduced. Clinical signs included inability to elevate depressed clotting proteins in response to vitamin K, hypocholesterolemia, slowed growth of hair and nails, weight loss, and reddening of hair and beard. Clinical signs were alleviated when the diet contained supplemental Mn. In some studies, deficiencies of Mn in Scandinavia have been connected with the incidence of cancer (Tolonen, 1990).

350

Manganese

B. Assessment of Manganese Status No single, simple diagnostic test permits the early detection of a deficiency in animals. The most sensitive noninvasive methods for evaluation of status include serum, urinary, and lymphocyte Mn, and superoxide dismutase activity (Leach and Harris, 1997). Although it is relatively expensive, magnetic resonance imaging (MRI) has been used to determine Mn toxicosis as the images associated with Mn toxicity are toxicosis specific (Hauser et al., 1994). The Mn values in the blood, bones, and liver decline in animals deprived of Mn, but they do not provide diagnostic criteria in the way that plasma and liver do for such elements as Cu and Se. Hidiroglou (1979) reported that whole blood concentrations substantially below 20 J.1gjml suggested the possibility of a dietary Mn deficiency in sheep and cattle. In the rat, low blood Mn was reflective of low tissue levels (Keen et al., 1983). Several investigators have reported that the Mndependent enzyme superoxide dismutase activity in several tissues (e.g., heart and liver) reflected dietary intake of Mn (Davis et al., 1990). Reduced activities of alkaline phosphatase are sometimes found when Mn is inadequate, but this is not definitive (Underwood and Suttle, 1999). Underwood (1981) believes that the Mn level in the liver is a useful but not an entirely reliable indicator of deficiency, unless the deficiency is severe. Van Koetsveld (1958) and Egan (1975) consider that ruminant liver Mn values below 10 ppm and 6 ppm, respectively, indicate a deficiency. Wool, feathers, and hair apparently reflect the dietary status of animals, but their diagnostic value is doubtful, at least at marginal intakes (Underwood, 1981). Lassiter and Morton (1968) reported a mean of 6.1 ppm Mn in the wool of lambs fed a low-Mn diet for 22 weeks, compared with 18.7 ppm in the wool of control lambs. Similarly, the skin and feathers of pullets fed a low-Mn diet for several months averaged 1.2 ppm Mn, compared with 11.4 ppm in comparable birds on a high-Mn diet (Mathers and Hill, 1968). For cattle, Tesink (1960) suggested hair Mn concentration of less than 5 ppm as a slight deficiency. Anke and Groppel (1970) found the hair of mature goats to reflect the Mn dietary supply better than any other parts of the body studied. Hair Mn levels continue to rise as dietary Mn intake increases. Costa Rican cattle consuming high Mn forages (» 100 ppm) had very elevated hair Mn levels compared with controls, 83.3 versus 18.6 ppm, respectively (Lang, 1971). McDowell (1976) concluded from the literature that ruminant Mn deficiency can best be detected by the combination of liver «6 ppm) and diet «20-40 ppm) analyses, while toxicosis is suspected when hair samples contain over 70 ppm.

IX. SUPPLEMENTATION Manganese deficiency is apt to occur in poultry and cattle consuming diets composed of natural feed ingredients. A deficiency is most easily produced in the chick with natural diets. For many mammals, prolonged feeding of depletion diets

Supplementation

351

containing less than 1 ppm is necessary to produce signs of Mn deficiency. Human Mn supplementation is limited as likelihood of a deficiency is remote, and much remains unknown concerning the requirement. Most feeds are adequate in Mn. However, when corn is the basic energy ingredient in poultry diets, and to a lesser extent sorghum and barley, Mn will be deficient unless supplementation with Mn or with Mn-rich feeds such as wheat bran or middlings is provided. The high-Ca and P diets normally fed to poultry are a contributing cause of perosis. Intake of Ca and P may be excessive if Ca-P supplements are provided free-choice or when meat-and-bone scrap are used as principal sources of protein. All ordinary diets (e.g., corn-soybean meal) that are otherwise adequate for the growth, health, and reproduction of pigs supply adequate Mn. Manganese supplementation is not normally necessary for swine, but virtually all commercially produced swine diets include supplemental Mn as an insurance against a deficiency of this element. For dairy cattle, if corn grain is a major portion of the diet for extended periods or if other low-Mn diets are used, sufficient Mn should be added to have at least 40 ppm in the total diet dry matter. Beef cattle receiving high concentrate diets based on corn may need supplemental Mn. For grazing ruminants, with the exception of Fe, Mn requires less attention than the commonly supplemented trace minerals, Co, Cu, I, Se, and Zn. Manganese supplementation for grazing ruminants will be dependent on forage Mn concentrations which are influenced by soil, plant, and management factors (see Section VII). In Germany, Mn deficiency in cattle developed only after heavy liming (Anke et al., 1973). Cox (1973) noted that in order to develop a Mn-deficient soil, soils must be leached of reduced forms of Mn, and soils frequently are limed, often in excess of crop requirements. Soils of tlie semiarid region of the state of Pernambuco, Brazil, are characterized by adequate Mn, with the chances of a deficiency remote (Horowitz and Dantas, 1966). However, in soils near the coastal Zona de Mata, where most of the cattle are located, available Mn is much less than 20 ppm, which is considered deficient (Dantas, 1971). In areas where Mn deficiency or conditioned Mn deficiency occurs in ruminants, remedial measures normally include careful control of liming, the use of crop sprays or fertilizers containing Mn, and the inclusion of Mn-containing supplements in the diet. Supplementation of the feed with Mn sulfate at the rate of 4 g for cows, 2 g for heifers, or 1 g per day for calves is sufficient for either the prevention or the cure of the deficiency (Underwood, 1981). Underwood (1981) also reported that treatment of pastures with Mn sulfate at the rate of 15 kgjha is completely effective in preventing Mn deficiency in the Netherlands. It appears that 40 ppm of forage Mn is considered adequate, 20 to 40 ppm borderline, and less than 20 ppm deficient for most ruminant species. Nevertheless, soil and water likewise provide Mn. Dyer (1961) reported that water from a ranch with normal calves contained 1.7 ppm, while that from a nearby ranch with deformed calves contained 0.02 ppm.

352

Manganese

Manganous oxide and manganese sulfate are the two most commonly used forms of supplemental Mn in animal feeds. In fertilizer applications, manganese sulfate is the preferred source; however, for feed applications, manganese oxide is preferred. Although manganese sulfate has better solubility, it contributes to additional acidity in the digestive system. For the feed industry a high-quality Mn supplement is required compared to that used for fertilizer application. Manganous oxide used in feeds must have no more than a maximum of 100 ppm of lead (Pb) or arsenic (As). Information on bioavailability of Mn from various sources has been reviewed (Ammerman and Miller, 1972; Henry, 1995; Costa et al., 1998). Black (1983) used a bioassay (bone and liver Mn accumulations) with chicks and determined that the sulfate form was most available followed by the oxide and carbonate forms. Setting the sulfate form as 100%, relative biological availability was 60-77% for the oxide and 32-36% for carbonate. In further bioavailability studies (using sheep), relative bioavailability of Mn from MnO, Mn02' and MnC0 2 averaged 57.7, 32.9, and 27.8%, respectively, compared with 100% for MnS04 (Wong-Valle et al., 1990). Korol et al. (1996) compared Mn sulfate to three different feed grades of Mn oxides fed to broilers, using Mn sulfate as 100% availability. The oxides ranged from 61 to 74% in availability. For broilers, Mn proteinate was found to have a value of 120% and Mn methionine 125% compared to the sulfate standard at 100%, Smith et al. (1995) and Henry (1995), respectively.

X. TOXICITY Manganese is often considered to be among the least toxic of trace elements for poultry and mammals. Maximum Mn dietary tolerable levels (NRC, 1980) for common livestock species are sheep and cattle (1000 ppm), poultry (2000 ppm), and for swine, horses and rabbits (400 ppm). Adverse health effects have not occurred in most species of animals fed dietary Mn concentrations of 1000 ppm or less. At 2000 ppm and above, growth retardation, anemia, gastrointestinal lesions, and sometimes neurological signs have been observed. Swine appear to be more sensitive to high levels of Mn, as 500 ppm to growing pigs retards growth and depresses appetite (Grummer et al., 1950). The pigs also showed stiffness of limbs and stilted gait toward the end of the experiment. High dietary Mn may exacerbate Mg deficiency in heart muscle and thus may be a complicating factor in the deaths observed in Mg-deficient pigs (Miller et al., 2000). The toxicity of excessive Mn appears to be a mineral antagonism manifestation, with Fe perhaps heading the list of antagonistic minerals. Low hemoglobin levels are reported as a result of excessive dietary Mn, with accompanying low tissue levels of Fe and elevated levels of liver Cu. Matrone et al. (1959) reported that Mn supplementation (50 to 250 ppm Mn) to a basal diet low in Fe (25 ppm) resulted in an inability of anemic pigs to restore normal hemoglobin levels. Further evidence of an antagonistic interaction between Mn and Fe has been obtained in studies of the Mn tolerance of lambs, with 1000 ppm dietary Mn greatly reducing serum Fe and preventing hemoglobin regeneration in anemic lambs (Hartman et aI., 1955).

References

353

Rats and poultry tolerated high dietary concentrations of Mn in a 240-day study with rats; growth and reproduction were normal with 4990 ppm Mn, and only growth was adversely affected at 9980 ppm (NRC, 1980). Hens tolerated 1000 ppm in the diet without ill effects, but 4800 ppm to young chicks was highly toxic (Heller and Penquite, 1937). The first observed effects of excessive dietary Mn in cattle are reduced feed consumption and slower growth, presumably caused by less feed intake (Underwood, 1981). Black et al. (1985) studied route of administration of Mn (diet versus capsule-dosed) to sheep and concluded that decreased feed intake associated with high Mn intake is apparently not related to palatability and is more a physiological response. It appears that 1000 ppm of dietary Mn is the maximum tolerable level for cattle and sheep (NRC, 1980). However, continuous grazing of forage containing 200 ppm or higher, produced on volcanic soils of Costa Rica, resulted in reproductive abnormalities in dairy cattle (Fonseca and Davis, 1968; Lang, 1971). These cattle that consumed high Mn forages had a lower calving percentages and required more services per pregnancy than did controls (Lang, 1971). Some evidence suggests that lowered reproduction efficiency could have resulted from excess Mn interfering with I metabolism. Manganese toxicosis occurs in workers in Mn mines who inhaled dust and fumes from the ores (Underwood, 1977; Keen et al., 1999). Manganese toxicosis in humans is characterized by a severe psychiatric disorder (locura manganica) resembling schizophrenia, followed by a permanently crippling neurological disorder clinically similar to Parkinson's disease (Hurley and Keen, 1987).

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