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THE ROLES OF AMINO ACID CHELATESIN ANIMAL NUTRITION

THE ROLES OF

AMINO ACID CHELATES

IN ANIMAL NUTRITION

Edited by

H. DeWayne Ashmead

Albion Laboratories, Inc.Clearfield, Utah

Reprint Edition

NOYES PUBUCATIONSWestwood, New Jersey, U.S.A.

Copyright © 1993 by Noyes PublicationsNo part of this book may be reproduced or utilized inany form or by any means, electronic or mechanical,including photocopying, recording or by any information storage and retrieval system, without permissionin writing from the Publisher.

Library of Congress Catalog Card Number: 92-25242ISBN: 0-8155-1312-7Printed in the United States

Published in the United States of America byNoyes PublicationsFairview Avenue, Westwood, New Jersey 07675

1098 7 6 5 432

Library of Congress Cataloging-in-Publication Data

92-25242CIP

1992

The Roles of amino acid chelates in animal nutrition / edited by H.DeWayne Ashmead.

p. em.Includes bibliographical references (p. ) and indexes.ISBN 0-8155-1312-71. Amino acid chelates in animal nutrition. 1. Ashmead, H.

DeWayne.SF98.A38R64636.08'52--dc20

INTRODUCTION

Th i s book wi 11 be of great interest to anyoneconcerned with animal feeds and feeding programs whetherone is studying bovine, porcine, equine, avian or lowervertebrate (fish and eel) nutrition. This informationis critical to the success of an animal feeding program.Somet imes the di fference between a successful and afailing program can be traced to mineral deficiencieswhich cause either abnormal growth, reduced milkproduction, interrupted fertility and breeding,compromised immune system integrity and/or decrement innormal hemoglobin concentration. Increasedmorbidity/mortality rates can make a profitable animalfeeding program into a financial failure overnight whenthe replacement costs for a prize animal are considered.These abnormalities, and others, are addressed in thepages that follow.

From 25 controlled studies by 42 different authorsin five different countries a diverse array of data ispresented. These data val idate the effect i veness ofmineral nutrients presented as amino acid chelates whencompared with the ionic forms derived from the inorganicsal ts. These stud ies further support the resul ts ofnumerous laboratory experiments showing increasedabsorption, assimilation and reduced toxicity of theforms of minerals chelated to amino acids. With littlecost and effort animals can be supplemented with aminoacid chelates which will promote, with little risk ofoverdose, a fuller genetic potential achievement as faras mineral requirements are concerned. Results of thissupplementation are reflected in increased growth,immunological integrity, and more consistentreproduction (increased ovulation and conception afterfirst service) as a result of increased bioavailabilityof these chelated forms.

v

VI Introduction

Of novel interest are the reports showing aprotein sparing as a result of amino acid chelatesupp1ementat ion. In the face of dwi ndl i ng protei nsources for animal feeds, this effect of chelatedminerals needs further scrutiny in feeding programs inother species.

Darrell J. Graff, Ph.D.Weber State UniversityOgden, Utah, U.S.A.

A NOTE TO THE READER

In the late 1800's, many of the fundamentalconcepts of che1at ion chemi stry were evo1vi ng. Chemi stsbegan to recognize that certain atoms could exist inmore than one valence state, but could not comprehendhow atoms with more than one valence could form a highlystable compound.

Alfred Werner, a German chemist, was the first tobreak with traditional thinking and propose an entirelynew molecular structure to describe these highly stablemolecules. He noted that certain structural entities,which he called "complexes", remained intact through aseries of chemical transformations. In 1893, Wernerwrote, "If we think of the metal ion as the center ofthe whole system, then we can most simply place themo1ecul es bound to it at the corners of anoctahedron."(1) For the first time a chelate had beendescribed.

Werner further refined this revolutionary conceptin the succeeding years. He concluded that a metal ionwas characterized by two valences. The first, which hecalled the "principal valency", is now termed theoxidation state, or oxidation number, of the metal. Thesecond valency, which he called the "auxiliary valency",represents the number of ligand atoms associated withthe central metal atom. This is the same as thecoord inat i on number of the metal. (2-7) Werner's conceptswere fundamental to the comprehension of chelates.

The term, "chelate", was finally used by Morganand Drew, in 1920, to describe the molecular structurediscovered by Werner. As noted above, the fi rstchelating molecules that had been discovered were those

VII

VIII A Note to the Reader

with two points of attachment. It was this caliper-likemode of attaching the ligand (the chelating molecule) tothe metal atom that led Morgan and Drew to suggest theword "chelate" to describe the molecule.(8) The word isderived from the Greek word "chele", meaning lobster'sclaw. The word, IIchelate ll

, was originally used as anadjective. It later became a more versatile word andtoday i s used as an adj ect i ve, adverb, or noun. Theligands are chelating agents, and the complexes theyform are metal chelates.

Because the claw, or ligand, held the cation, themetal was no longer free to enter into other chemicalreactions. Thus it quickly became evident that when ametal was che1ated, the chemi cal and phys i calcharacteristics of the constituent metal ion and ligandswere changed. This had far reaching consequences in therealms of chemistry and general biology. In spite ofthe knowledge of what chelation could do to and for ametal ion, it was not until the early 1960's that anyonethought seriously about using this molecule fornutritional purposes.

At that period, a handful of investigators,independent of each other, each conceived the idea thatif a metal ion could be chelated before feeding it toanimals, the ligand would sequester the cation andprevent it from entering into various absorptioninhibiting chemical reactions in the gut. Thetheoretical consequence was greater nutritional uptakeof the ions.

Two schools of thought quickly developed. One,led by the pioneering research of Albion Laboratories,Inc., proposed that amino acid chelates were the properchelates to enhance mineral absorption. As attested bya large number of research reports, lectures, andpublications based on the research efforts both

A Note to the Reader IX

coordinated and conducted by this organization, the useof amino acid chelates in animal nutrition were bothpositive and highly encouraging. At that point in timethese amino acids were called "metal proteinates"instead of chelates.

Concurrently, with the development of the aminoacid chelates, a second school of thought approachedanimal nutrition with synthetic chelates based onethylenediaminetetraacetic acid (EOTA). The theory wasthe same as before. The EOTA ligand would chelate thecation and protect it from chemical reactions in thegut. While it successfully accomplished its mission interms of protect ion, it genera11 y fa i1ed to enhancemineral nutrition because it formed chelates that weretoo stable. The biological ligands in the animals'bodies were incapable of extracting the cations from theEOTA chelates, even after they were absorbed into theblood. Thus, the EOTA chelates were returned to thelower bowels or excreted into the urine still protectingthe cations that the animal s were supposed to haveutilized. As Bates, et li., concluded, even thoughchelation plays a dominant role in mineral absorption,"chelation does not, in itself, insure efficient uptakebecause the absorption of the ferric chelates of EOTA,NTA, and gluconate were not significantly different thanthat of ferrous sul fate. ,,(9)

These synthetic chelates were heavily promoted inthe decade of the 60's and the early part of the 70's.When they could not deliver the enhanced mineralnutrition promised by the chelation concept, allnutritional products using the word "chelation" lostfavor with most animal nutritionists. The "c" wordbecame a word to avoid if one wished to amicably discussanimal nutrition.

x A Note to the Reader

It was for this reason metal proteinates became afavored description for the amino acid chelates. As aterm, the words evolved out of the concept of complexingmetal s wi th protei n. Metal protei nates becameacceptable terminology because they successfully avoidedmention of the "ell word. There was a problem with thatapproach, however. There was no official definition todescribe a metal proteinate.

By 1970, Albion Laboratories, Inc. had suppliedthe necessary research to allow the American Associationof Feed Control Officials (AAFCO) to officially definemetal protei nates as the product resul t i ng from thechelation or complexing of a soluble salt with aminoacids and/or hydrolyzed protein.

As greater numbers of manufacturers begancapital izing on the metal proteinate definition, itbecame evident that this definition was too broad toaccurately define Qllly those minerals that research hadproven were efficacious. Many companies were not makingchelates, but could still have their products defined asmetal proteinates. Other companies, who may have beenmaking chelates, were not making products that could beabsorbed. Thei r compounds were ei ther too bi g (achelate over 1,500 daltons can not be absorbed), or themineral was bonded to whole or partially hydrolyzedprotein (which had to be digested with subsequentrelease of the metal to competing reactions in the chymesimilar to those facing cations derived from any otherfeedstuff).

Because of the confusion among feed companies intrying to decide which metal proteinates were valuablesources of the added mineral nutrition, which metalproteinates were supported by scientific studies, andwhich were "me too" products that had no support data oftheir own, Albion Laboratories, Inc. applied to AAFCO

A Note to the Reader XI

for a new definition which accurately and morecompletely described an amino acid chelate. Realizingthe "e" word was still out of vogue among manynutritionists due to their earlier experiences withsynthetic chelates, Albion still decided to call theproducts by their true name - amino acid chelates.

After several years of debate within the AAFCOorganization, a debate which was primarily fueled bycompanies using Albion's research to promote dissimilarproducts ascribed to the proteinate definition, a newdefinition was ultimately approved. The new definitionfor a metal amino acid chelate rectified the loosenessof the metal proteinate definition by including absoluterequirements for molecular weights, molar ratios ofami no ac ids to metal s, and the abso1ute presence ofchelation. The amino acid chelate definition alsodisbarred the complexing of metals with protein orpeptides, both of which require further digestion beforeabsorption. The formation of chelates too large to beabsorbed was thus disallowed.

As defined by the American Association of FeedControl Officials, a metal amino acid chelate is litheproduct resulting from the reaction of a metal ion froma soluble salt with amino acids with a mole ratio of onemole of metal to one to three (preferably two) moles ofamino acids to form coordinate covalent bonds. Theaverage weight of the hydrolyzed amino acids must beapproximately 150 and the resulting molecular weight ofthe chelate must not exceed 800." (0

)

This book is about amino acid chelates. With fewexceptions, the research contained within it wasconducted by investigators independent of AlbionLaboratories, Inc. The organization with which eachinvestigator is affiliated is noted on the list ofcontributors and at the beginning of each chapter.

XII A Note to the Reader

The book is divided into several sections so thata reader, who may not wish to read the entire book, canquickly turn to his or her own area of primary interest.Separate sections are devoted to cattle, pigs, poultry,horses and fish. The beginning section discusses thefundamentals of amino acid chelation as they relate tothe various aspects of animal nutrition discussed ineach of the subsequent sections. It is stronglyrecommended that the reader who has primary interest inonly one species of animal still read this first sectionprior to addressing the species of interest. The firstsection will provide numerous basic concepts that willenhance the reader's comprehension of the data in thesubsequent sections.

For the animal nutritionist, veterinarian, andothers whose interests range further than a s i ngl especies, reading the book in its entirety isrecommended. As noted above, it is divided into fiveadditional sections beyond the introductory section plusa summary. The second sect i on deal s wi th severalaspects of dairy and beef cattle mineral nutrition.Some topics discussed include immunity, fertility,increased mi 1k product ion, greater growth rates, andimproved feed conversions. The third section addressesseveral important concepts of swine nutrition includingbaby pig anemia, improved reproductive capacity in oldersows, and leaner pork. Poultry is handled in the fourthsection. Topics include improvements in breeder/broileroperations, egg production and enhanced turkeyproduction. The next section deals with equinenutrition as it relates to fertility and proper growthand development of the legs. The last section dealswith enhanced performance in fish and eels.

Although the data are conclusive in most cases,the research reported in these sections is by no meanscomplete. In many instances the editor was faced with

A Note to the Reader XIII

making painful decisions as to whose research toinclude, or not to include, in order to avoid excessiverepetition. In spite of these efforts, some repetitionwas unavoidable, but hopefully not redundant.

The purpose of reporting this research in the formof a book has been two-fold. The first is to stimulateothers to piek up the torches that have been lighted bythe researchers who have contributed to this book and tocont i nue onward from where they stopped. The secondpurpose is to make the "e" word once again an acceptableword in animal nutrition circles.

H. DeWayne Ashmead

XIV A Note to the Reader

References

1. Werner, A., "Beitrag zur KonstitutionAnaorgan i scher Verbi ndungen," Z., anorg. u. all gem.Chern., 3:267, 1893.

2. Werner, A. and Miolati, A., Z. physik. Chern.(Leipzig), 14:506, 1894.

3. Werner, A. and Vilmos, Z. "Beitrag zur KonstitutionAnaorganischer Verbindungen," l. anorg. u. allegem.Chern., 21:153, 1899.

4. Werner, A., "Ueber Acetyl acetonverbi ndungen desPlatins," Ber. deut. chern. Ges., 34:2584, 1901.

5. Werner, A. ,Kobaltatoms.1911.

"ler Kenntnis des AsymmetrischenV," Sere deut. chern Ges., 45:121,

..6. Werner, A., "Uber spiegelbild-isomerie bei

chromverbi ndungen. I I I ," Ber. deut. chern. Ges.,45:3065, 1912.

7. Werner, A., "lur Kenntris des AsymmetrischenKobaltatoms XII. Uber Optische Aktivitat beiKoh 1enstoffrei en Verbi ndugen, II Ber. deut. chern.Ges., 47:3087, 1914.

8. Morgan, G. and Drew, H., "Research on residualaffinity and coordination. II. Acetylacetones ofselenium and tellurium," J. Chern. Soc., 117:1456,1920.

9. Bates, G., et li., "Facil itation of iron absorptionby ferric fructose," Am. J. Cline. Nutr., 25:983,1972.

10. Haas, E., et li., eds., Official Publication 1989(Atlanta: American Association of Feed ControlOfficials, Inc.) 159, 1989.

CONTRIBUTORS

Ashmead, H. DeWayneAlbion Laboratories, Inc.Clearfield, Utah, U.S.A.

Ashmead, Harvey H.Albion Laboratories, Inc.Clearfield, Utah, U.S.A.

Atherton, DavidThomson & Joseph LimitedNorwich, England

Biti, F. RicciUniversity of BolognaBologna, Italy

Boling, James A.University of KentuckyLexington, Kentucky, U.S.A.

Bolsi, DanielleUniversity of ParmaParma, Italy

Bonomi, AlbertoUniversity of ParmaParma, Italy

xv

Cagliero, GermanoAgrolabo, S.P.A.Turin, Italy

Coffey, Robert T.Newton, Iowa, U.S.A.

Corradi, FulvioUniversity of BolognaBologna, Italy

Cuiton, LouisProductos QuimicoAgropecuarios, S.A.Mexico City, Mexico

Cuplin, PaulIdaho State Fish and GameDepartmentBoise, Idaho, U.S.A.

Darneley, A. H.Dorset, England

Ferrari, AngeloZoopropylactic Institute ofPiedmontLiguria and Valle d'AostaItaly

XVI

Forfa, Richard J.University of MarytandCollege Park, Maryland

Formigoni, AndreaUniversity of BolognaBologna, Italy

Guillen, EduardoProductos QuimicoAgropecuarios, S.A.Mexico City, Mexico

Hardy, Ronald W.University of WashingtonSeattle, Washington, U.S.A.

Herrick, John B.Iowa State UniversityAmes, Iowa, U.S.A.

Contributors

Jeppsen, Robert B.Albion Laboratories, Inc.Clearfield, Utah, U.S.A.

Kropp, Robert J.Oklahoma State UniversityStillwater, Oklahoma, U.S.A.

Lucchelli, LuiginaUniversity of ParmaParma, Italy

Maletto, SilvanoUniversity of TurinTurin, Italy

Manspeaker, Joseph E.University of MarylandCollege Park, Maryland, U.S.A.

Hildebran, SusanWapakoneta, Ohio, U.S.A.

Hunt, JohnSugar Creek Veterinary ServiceGreenfield, Indiana, U.S.A.

Iwahasi, YoshitoShizuoka Prefectural FisheriesExperimental StationLake Hamanako BranchShizuoka, Japan

Ming Lian, FengBeijing Agriculture ScienceInstituteBeijing, China

Parisini, PaoloUniversity of BolognaBologna, Italy

Quarantelli, AfroUniversity of ParmaParma, Italy

Contributors XVII

Robl, Martin G.University of MarylandCollege Park, Maryland, U.S.A.

Sabbiono, AlbertoUniversity of ParmaParma, Italy

Sacchi, C.University of BolognaBologna, Italy

Shearer, Karl D.University of WashingtonSeattle, Washington, U.S.A.

Superchi, PaolaUniversity of ParmaParma, Italy

Suzuki, KatsuhiroShizuoka Prefectural FisheriesExperimental StationLake Hamanako BranchShizuoka, Japan

Takatsuka, TakeharuShizuoka Prefectural FisheriesExperimental StationLake Hamanako BranchShizuoka, Japan

Volpelli, L. A.University of BolognaBologna, Italy

Wakabayashi, TakaakiEisai, Co., Ltd.Tokyo, Japan

Xian-Ming, CaoBeijing Agriculture ScienceInstituteBeijing, China

Van Ping, ZhouBeijing Agriculture ScienceInstituteBeijing, China

Zunino, HugoUniversity of ChileSantiago, Chile

Notice

To the best of the Publisher's knowledge the informationcontained in this publication is accurate; however, thePublisher assumes no responsibility nor liability for errors orany consequences arising from the use of the informationcontained herein. Final determination of the suitability of anyinfonnation, procedure, or product for use contemplated by anyuser, and the manner of that use, is the sole responsibility ofthe user.

The book is intended for informational purposes only. Thereader is warned that caution must always be exercised whendealing with chemicals, products, or procedures which mightbe considered hazardous. Expert advice should be obtained atall times when implementation is being considered.

Mention of trade names or commercial products does notconstitute endorsement or recommendation for use by thePublisher.

XVIII

Contents

INTRODUCTION . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. VDarrell J. Graff

A NOTE TO THE READER VII

CONTRIBUTORS XV

SECTION 1AMINO ACID CHELATION

1. MINERALS IN ANIMAL HEALTH 3John B. Herrick

2. FACTORS WHICH AFFECT THE INTESTINALABSORPTION OF MINERALS 21

H. DeWayne Ashmead and Hugo Zunino

3. COMPARATIVE INTESTINAL ABSORPTION ANDSUBSEQUENT METABOLISM OF METAL AMINO ACIDCHELATES AND INORGANIC METAL SALTS 47

H. DeWayne Ashmead

4. INCREASING INTESTINAL DISACCHARIDASEACTIVITY IN THE SMALL INTESTINE WITHAMINO ACID CHELATES 76

Silvano Maletto and Germano Cagliero

XIX

xx Contents

5. EVALUATION OF THE NUTRITIONALEFFICIENCY OF AMINO ACID CHELATES 86

Silvano Maletto and Germano Cagliero

6. AN ASSESSMENT OF LONG TERM FEEDING OFAMINO ACID CHELATES 106

Robert B. Jeppsen

SECTION 2CATTLE

7. THE USE OF AMINO ACID CHELATES TO ENHANCETHE IMMUNE SYSTEM 117

Robert T. Coffey

8. THE USE OF AMINO ACID CHELATES IN BOVINEFERTILITY AND EMBRYONIC VIABILITY 140

Joseph E. Manspeaker and Martin G. Robl

9. THE ROLE OF COPPER IN BEEF CATTLE FERTILITY .... 154J. Robert Kropp

10. THE USE OF AMINO ACID CHELATES IN HIGHPRODUCTION MILK COWS 170

Andrea Formigoni, Paoli Parisini, and Fulvio Corradi

11. THE FEEDING OF AMINO ACID CHELATESUPPLEMENTS TO BEEF CALVES 187

James A. Boling

SECTION 3SWINE

12. THE ROLE OF IRON AMINO ACID CHELATE INPIG PERFORMANCE . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 207

H. DeWayne Ashmead

13. THE EFFECT OF IRON AMINO ACID CHELATE ONTHE PREVENTION OF ANEMIA 231

Cao Xian-Ming, Feng Ming Uan, and Zhou Yan Ping

14. THE EFFECT OF AMINO ACID CHELATED IRON INPREGNANT AND LACTATING SOWS 243

P. Parisini, F. Ricci Biti, L.A. Volpelli and C. Sacchi

Contents XXI

15. IMPROVING REPRODUCTIVE PERFORMANCE WITHIRON AMINO ACID CHELATE 251

A.H. Darneley

16. A NUTRITIONAL APPROACH TO MAXIMIZINGCARCASS LEANNESS 269

David Altherton

SECTION 4POULTRY

17. THE EFFECT OF AMINO ACID CHELATES IN CHICKMORTALITY 291

David Atherton

18. THE DYNAMICS OF FEEDING AMINO ACID CHELATESTO BROI LERS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 302

Alberto Bonomi, Afro Quarantelli, Paola Superchi,Alberto Sabbiono, and Luigina Lucchelli

19. GROWTH RATES AND FEED CONVERSION IN BROilERCt-IICKS FED AMINO ACID CHELATES 318

Louis Cuitun and Eduardo Guillen

20. THE USE OF AMINO ACID CHELATES IN GROWINGTURKEYS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 330

Alberto Bonomi, Afro Quarantelli, Paola Superchi,Alberto Sabbiono, and Danielle Bolsi

21. THE ROLE OF AMINO ACID CHELATES INOVERCOMING THE MALABSORPTION SYNDROMEIN POULTRY 349

Angelo Ferrari and Germano Gagliero

22. THE VALUE OF AMINO ACID CHELATES IN EGGPRODUCTION . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 366

Alberto Bonomi, Afro Quarantelli, Paoli Superchi andAlberto Sabbiono

23. THE ROLE OF AMINO ACID CHELATED MAGNESIUMIN EGG PRODUCTION 380

David Atherton

XXII Contents

SECTION 5HORSES

24. THE EFFECTS OF AMINO ACID CHELATES ONTHOROUGHBRED MARES 393

Martin G. Robl and Richard J. Forta

25. COPPER-RESPONSIVE EPIPHYSITIS AND TENDONCONTRACTURE IN A FOAL 400

Susan Hildebran and John Hunt

SECTION 6FISH

26. THE USE OF AMINO ACID CHELATES IN RAINBOWTROUT OPEN-FORMULA DIETS 413

Harvey H. Ashmead and Paul Cuplin

27. THE USE OF ZINC AMINO ACID CHELATES IN HIGHCALCIUM AND PHOSPHORUS DIETS OF RAINBOWTROUT 424

Ronald W Hardy and Karl D. Shearer

28. THE EFFECTS OF IRON AMINO ACID CHELATE INCULTURE EELS 440

Katsuhiro Suzuki, Yoshito Iwahasi, Takeharu Takatsukaand Takaaki Wakabayashi

SECTION 7SUMMARY AND CONCLUSION

29. SUMMARY AND CONCLUSION 457H. DeWayne Ashmead

NAME INDEX 473

INDEX 475

Section 1. AMINO ACID CHELATION

Chapter 1

MINERALS IN ANIMAL HEALTH

John B. Herrick*Iowa State University

Sci ent i sts have long known that traces of mostelements exist in animal tissue, but in many cases theybelieved that those minerals were contaminants ratherthan functional entities. As analytical methods haveimproved, many elements that were once thought to becontaminants have been shown to be essential to the lifeand well being of the animals.(1) Thus, a mineral is nowbelieved to be essential if (a) it is present in allhealthy tissue, (b) its concentration from one animal tothe next is generally constant, (c) its withdrawal fromthe body results in reproducible physiological andstructural abnormalities, (d) its addition prevents orreverses those abnormal it i es, (e) the defi ci ency- inducedabnormalities are accompanied by specific biochemicalchanges, and (f) these biochemical changes can beprevented or cured with the addition of the mineral .(2)

The three major roles of essential minerals are todirectly or indirectly function in supplying energy, toindirectly and directly aid in growth and maintenance ofthe body tissues, and fin~lly to assist in theregulation of body processes.() The need for chromiumto potentiate insulin and the involvement of phosphorusin the ATP mol ecul e are two exampl es of the di verseroles of minerals in energy production. In their rolesof growth and maintenance of tissues, mineralscontribute to the rigidity of the bones and teeth and

* The original draft was written while associatedwith Iowa State U. Dr. Herrick is currentlyretired and working as a consultant.

3

4 The Roles of Amino Acid Chelates in Animal Nutrition

are an important part of protein and lipid fractions ofthe animal body. As regul ators of body processesminerals preserve cellular integrity by osmoticpressures and are a component of many enzyme systemswhich catalyze metabolic reactions in biologicalsystems. Most minerals function in more than one role.As an example, calcium is used in large amounts by thebody for synthesis of osseous tissue. Phosphorus alsocontributes structurally and yet is a key element in theuse of energy by the body. Furthermore, cobalt has aregulatory role (through vitamin 8-12) rather thancontributing quantitatively to tissue synthesis, but itseffect on growth i s as dramat i c as that of cal ci urn.Other elements play essential roles, as well.

Minerals which are involved in several metabolicprocesses are more likely to be interrelated with otherminerals than are those involved in a single or a fewfunct ions. (4.5) Knowl edge of these i nterre1at ionsh ipsbetween minerals is increasing rapidly. As thatknowledge expands in the field of mineral metabolism,many additional interrelationships will probably beelucidated.

Animals obtain essential mineral nutrition fromtwo pri mary sources: (1) through natural feeds and,possibly, water, and (2) through supplementation offeeds and water. Even though a 1arge port i on of therequired minerals may be provided by the vegetativeanimal feedstuffs, mineral supplementation is generallya necessary practice for properly nourishing the animalsdue to dep1et i on and imba1ances of mi nera1sin thesoils, and consequently in plants.(6) The mineralcontents of a plant depend primarily on the plantspecies, the season, the abundance of the element in thesoil, the type of soil, and the conditions (pH,moisture, etc.) which affect the plant's ability toabsorb the mi nera1s. (7) Because of the many factorsaffecting this absorption, the mineral contents ofplants vary both within and among the species.

Minerals in Animal Health 5

Furthermore, mineral requirements of plants are not thesame as for animals, so ratios of one mineral to anotherand the Quantities of minerals, being proportioned fordifferent biological functions, may not meet the needsof the animal when used in feeds. The availability ofminerals in plants is also affected by phytic acid andoxalic acid found in the cellulosic cell membranes asopposed to cytoplasm. It should be remembered that thepresence of a mineral in plant tissue used for feed doesnot guarantee its absorption into the animal's system.As stated by Underwood, "the evaluation of feeds andfeed supplements as sources of minerals depends not onlyon what the feed contains, i.e. the total content orconcentration as determined physio-chemically, but onhow much of the total mineral can be absorbed from thegut and used by the animal's cells and tissues."(8)

Before the development of isotopic techniques,absorption mechanisms of minerals were poorlyunderstood. Without isotopes the primary difficulty forconfusion of mineral metabolism was due to absorption,excretion back into the gut, and reabsorption, often asa standard cycle. Conventional balance studies wereunable to indicate the net utilization of minerals.Absorption of mineral ions is dependent upon numerousfactors, including the levels of the elements ingested,the age of the animal, Ph of the intestinal contents andenvironment, the state of the animal with respect todeficiency or adequacy of the element, and the presenceof other antagonistic minerals or nutrients as well asseveral other conditions. (9) Earl ier investigators wereunable to consider these variables due to lack ofadequate technology, and thus their reports often lackedagreement. With the use of isotopes it became easier tofollow the movement of trace elements through thebody. (8)

In a practical sense, all animals are subject tomineral deficiencies. These may be caused by: (a) asuboptimal amount of a specific mineral in the feed; (b)


an imbalance of another mineral or nutrient includingcertain vitamins, amino acids and fats, any of whichcould decrease absorption; (c) any condition whichincreases the rate of passage of the minerals throughthe intestine, such as diarrhea; and (d) the presence ofa metabolic antagonist which causes the animal torequ ire a greater quant i ty of the needed element. (4)

Imbalances and deficiencies are not synonymousconditions, but either condition may lead to the other.The net result is less than optimum mineral nutritionfor the animal.

The pathways for excretion and rates of excretionof mineral elements vary. Some are excreted almostentirely in the feces via the lower bowel; others areeliminated almost entirely in the urine, while stillothers are excreted through both pathways. Certa inminerals are excreted in minute quantities through thesweat, and others are lost during the menstrual cycle.Integument losses from the animal's body may alsooccur. (4) Absorpt i on from the 1umen does not guaranteemineral usage in metabolism.(lO) In some cases mineralsare absorbed but later excreted in urine and feces asnon-metabolized wastes without ever being utilized inbiological processes.

Chelation of minerals (a process by which a metalatom is sequestered) is employed in some feedingreg imens in order to enhance the absorpt i on wi thoutregard to mineral metabolism. Ametal chelate is formedas a ring structure. It is produced by attractionbetween the positive charges of certain polyvalentcations and any two or more sites of highelectronegative activity in a variety of chemicalcompounds known co11 ect i vel y as 1igands. Ache1ategenerally requires both an ionic and a covalent bond.The covalent bond in particular, known as a "coordinate"bond, occurs because of peculiarities in electron shellsof transition metals and the capacity of the donatingatom of the ligand to contribute two electrons at the


same time. The word "chelate" is taken from the Greekword chele meaning "claw," a fairly good descriptiveterm for the manner in which polyvalent cations are heldby the metal bi nd i ng agents or 1igands. (12)

If any element is chelated by a ligand which willcarry the bonded mineral into the mucosal cell as anintact chelate, this chelate may indeed greatly enhanceabsorpt i on of that mi nera1. However, if ache1atemerely releases an ion at the intestinal wall, this isfrequently no more efficient than use of metal saltsbecause once the ion is released, it is subject to themi nera1 absorpt ion 1imi t i ng factors ment i oned above.Intact absorption is generally more efficient. However,due to high stability constants, some chelates which areabsorbed are never metabolized. cll

) If a chelate isabsorbed, it occurs because the chelate prevents thechemical reaction of the element with other substancesin the stomach and intestines to form insoluble chemicalcompounds. Che1at ion a1so prevents the strongadsorption of the mineral onto insoluble colloids in theintestine. This prevents the ion from being releasedback into the lumen. If a chelate is metabolized afterabsorption, it occurs because the ligand is capable ofeither releasing the cation to other metallic requiringcellular systems with subsequent metabolism of the freeligand, or the chelate is able to enter into a systemwhich requires both the metal and the ligand together.

There are basically three types of chelates whichare recognized as being essential in biological systems:

The first group includes chelates which transportand store metal ions. In these chelates the metal hasno current function of its own. It does not modify theproperties of the ligand, but instead requires the useof ligands with the chemical and physical propertieswhich will allow the metal to be absorbed, transportedin the bloodstream, and pass across cell membranes todeposit the metal ion at the site where needed. One


such chelate is transferrin. It chelates absorbed ironions which enter the blood and transfers them throughoutthe body. The presence of transferri n in the blood,however, does not guarantee that th i s wi 11 be theultimate destination of absorbed or otherwiseadministered iron. In the case of iron dextraninjections made to supplement inadequate body iron1eve1s , some of the iron i s i ncorporated intotransferrin, while the remainder is either eliminated inthe wastes or fixed in the connective tissues at thesite of injection. (13)

All amino acids are particularly effective metalbinding agents, and may be of primary importance in thetransport of minerals from the lumen into the mucosalcells as well as for storage of mineral elementsthroughout the an; rna1's body. As part of ache1atemolecule these amino acid ligands do not functionbiologically as individual amino acids, but as uniquetransfer molecules.

Ethylenediaminetetraacetic acid (EDTA) and similarsynthetic ligands may improve the availability of zincand certain other minerals for plants by protecting thecations from precipitating chemical reactions in thesoil. EDTA and similar variants are effectively used inmedicine to hasten the excretion of lead and other heavymetals from animals poisoned by these metal ions.(l4)Generally speaking, however, EDTA chelates and similaranalogs, do not enhance mineral nutrition in animals.(ll)

The second group of chelates are those which areessential to physiology. Many chelates exist in theanimal body in forms which allow the metal ion toperform its metabolic function(s). The chelated iron inhemoglobin and the chelated cobalt in vitamin 8-12 aresuch examples of this group of chelates. Without itsiron moiety, the hemoglobin molecule could not transportoxygen. On the other hand, if the i ron were notchelated, the hemoglobin could not effectively bind and


release the oxygen for metabolic use.(15) Metals whichare chelated into enzyme systems and function as part ofa metalloenzyme are other examples of metabolicallyessential chelates.(16)

The third group of chelates consist of those whichinterfere with utilization of essential cations. Manymetal chelates are probably formed "accidentally" andconsequently have no useful biologic value. In Table 1numerous enzyme systems are listed. Many can bedeactivated or inhibited when the wrong metal forms achelate within the enzyme.(l7) One should note thevariety of cations required for the catalytic functionsof these enzymes. Without them the enzymes will ceaseto function. This is a necessity which is frequentlyoverlooked by many who relegate mineral nutrition to itsstructural role in bones and teeth and are unaware ofthe importance of minerals as enzyme cofactors in basicmetabolism.

Table 1

Enzymes Whi ch Are Infl uenced by Mi nera1s (Modi fi ed from Schut te (lJ»)

Column I. The trace element (or mineral element) constitutes theprosthetic group.

Column II. The trace element (or mineral element) is an active part of theprosthetic group, or is incorporated into the enzyme itself.

Column III. Elements with integrating function(s) that are not understood,as yet. El ements need not be speci fi c and may replace eachother.

Column IV. Facultative Activators.Column V. Inhibitors of enzyme activity.

ENZYME

Carbohydrases

II III IV v

Na,K,Li,Br, F,ISr,Mg,Ca,Ba

u-Amylase (animal) Cl

Lysozyme,B-D-GlucosidaseHyaluronoglucosaminidase

KS203, CN ,S I ,CuSK,Na,Ca,Ba, F,MgFe,Mn,Br,I,SO.,N03


(Table 1 continued)

ENZYME II III IV V

Esterases

Deoxyribonucleases Mg,Mn,(Ca) Ca,BaZoolipase Ca,Mn,Cl Pb,Mn,NaLipoprotein lipase W,Mo,SiCholinesterase Ca Co,Ba,Mg,Cu, S F

MnAcetylcholinesterase NaClPhosphatidylcholin- Mgesterase

Alkaline phosphatase Zn,Co,Mn,Mg Ni , Fe++,Ca MgSH,CNAcid phosphatase Co Ca,Mg,Co,Ni F,MgAcid phosphomono- Mg Co Niesterase III

Fructose- Mg,Mn Co,Ni ,m,OJbisphosphatase

Nucleotidase Mg Zn,FeH-,A],Cu++

Ribonuclease FProstataphosphatase Ce,LaTropinesterase KC1,NaBr, F,CN

KCNS, MgSO.,CaC1 2 ,Na,Cl,NaI

Phosphoprotein Mophosphatase

Phosphatidate Mg C3,B3.,M],~

phosphataseArginine deaminase FeArylsulphatase NO,ClDeoxyribonuclease Mg,Mn Ca FType I

Deoxyribonuclease Mg FType II

Micrococcal Ca Fendonuclease

Amidases

Defence proteinases S SH Sch-MeProteinases in general KCathepsins S,CN,SH, Fe Sch-Me

S203' S03 (Hg,Cu)Coagulation factor Xa Ca


(Table 1 continued)

ENZYME II III IV V

(Amidases continued)

Trypsin Ca, Mn, NH.,MgSO.

Chymosin Ca Rare EarthsEnteropeptidase CaPepsin A H,ClAminoacyl-histidine Zn,Mn PO., P20J , Fdipeptidase

Peptidases Mg,Mn,Zn, Zn,CoFe++,Co++

Dehydropeptidase I S,CNFolic acid conjugase SHg,CaTripeptide aminopeptidase MnAminopeptidase Mn,Mg Mn,Zn CN, S, P2OJ ,

Fe++, Pb++,1-9++, Cu++

Aminopolypeptidase Co,Zn Zn,CoCysteinyl-glycine Mn,Co,Fedipeptidase

Glycyl-l-leucyl- Mndipeptidase II

Glyclglycin- Co,Mn ZndipeptidaseGlycyl-l-leucine- Mn Zn, PO. Ca,(Zn-~.)

dipeptidase IProline dipeptidase Mn,Cd po., PzOJ , F,

S,CN,AgProlyl dipeptidase Mn Sn,~,F,

Fb++,Cd++,P2 0J

Tryptic carboxy- Zn Mgpolypeptidase

Catheptic carboxy- CNpolypeptidase

Histidine ammonia-lyase Cd++,KCNNAD(P)+ nucleosidase N0 3 , N0 2 , NH. ZnArginase Cd,Mn Fe++,Co,Ni Ca,Fe,Ni B,Zn

Cd,VAsparaginase Cu,Hg,AgGlycocarbaminase Mn


(Table 1 continued)

ENZYME II III IV V

Phosphohydrolases

Pyrophosphatases Mg Mn Al,Mg,Zn,Zr, Ca,FTh,Pb,Fe,Co,La,Ce,Y,CN

Polyphosphatases MgAdenosinetriphosphatase Mg,K Mg Ca,NaTriphosphatase Mg,Fe,CoApyrase CaATP-Pyro- Cl,Brphosphatase

Oligometa- Mg,Mn,Co,Zn Ca,Ba,Al ,Ti, CN,Fphosphatase Fe,Ni ,Cu,Zr,

Th,Pb,La,Ce,Nd,Sm,Y,Pr

Polymeta- Mn,Mg,Zn Ag,Hg++phosphatase Ca,Pb

Phosphohalogenase Mn,Co Hg++,ZnCu++, Pb++(Mn)

Hydrolases with varyingsubstrate specificity

C-C-Hydrolases B6 -PO"Alkylhalidase Cl,BrIodine-tyrosine- Ideiodase

Di-isopropyl- Ca,Mg,Co,Mn Hg Hg++,Cu++fluorophosphatase

Transglycosylases

Phosphorylase Mg

Transphosphatases(Kinases)

Glucose-l- AsO"phosphate --->

amylose-trans-glucosidase

Minerals in Animal Health

(Table 1 continued)

13

ENZYME II III IV V

(Transphosphatases Kinases continued)

Saccharase ---> As04

ortho-phosphate-transglucosidase

Adenylate kinase MgCreatin kinase MgArginine kinase Mn Mg1,3-Diphospho- Mg K, NH4

glycerate --->ADP-Trans-phosphatase

Pyruvate kinase K, Mg, NH 4 ,

RbHexokinase Mg6-Phosphofructo- Mg K, NH4

kinaseGalactokinase Mg,MnPhosphoglucokinase Mg,MnGluconokinase MgRibokinase MgATP---> Nucleic Mg,F P04

acid-trans-phosphatase

Riboflavin kinase Mg,MnAdenosine kinase Mg,MnPhosphoglucomutase Mg FFMN adenylyl- Mgtransferase

FMN adenylyl- Mgtransferase

ATP ---> NAD- Mg,MnTransphosphatase

Aminotransferases

Amino transferases Mg,NH4 ,C02

Glutaminyl-peptide Mn, P04

-glutamyltransferaseAspartate aminotransferase Mn, P04

Carbamoyltransferases Mg


(Table 1 continued)

ENZYME II III IV V

Transmethylases

Transmethylase Mg,Ca CN,F,Ca

Acyltransferases

Acetyl CoA Mg K, NH., Rb Na,Cssynthase

Choline PO. ,Ca , Mg , Kacetyl transferase

Special Transferases

Transketolase Mg PO.Thiosulfate S203' Cu As03sulfurtransferase

Anaerobic Transhydrogenases

Succinate Fe PO. Ca,Al,Co,dehydrogenase Rare Earths

Oxalate Mgdehydrogenase

Choline Codehydrogenase

Thiamine Mgdehydrogenase

u-fJ-Unsaturated CuAcyl-CoA-reductase

Saturase Mn,ClMonodehydro- PO., AsO.ascorbatereductase

Alcohol Zndehydrogenase

Glutathione disulphide Mg, Mn, PO. NaClreductase (NAD(P)H)

Minerals in Animal Health

(Table 1 continued)

15

ENZYME II III IV V

Aerobic transhydrogenases

Amine oxidase Cu,Fe PO., Mo, Fe(flavin containing)

Amine oxidase Cu Co PO. CN,Ca(copper containing)

Xanthine oxidase Mo,Fe PO. CN,CuAldehyde oxidase Me NH., WAldehydmutase Mo

Anaerobic transelectronases

NADPH dehydrogenase PO.NADH dehydrogenase Fe++, PO. CN Cu,Zn,Mn,

Ca,~,P04'

P2 0"

V

Aerobic transelectronases

Cytochrome c oxidase Fe C1Monopheno1 Cu PO.,Ni ,CO,V K,Mg,Ca,Zn,monooxygenase Mn,A1,Fe

Urate oxidase Cu Mn CN,FLuciferin Mg PO., Mn P2 O,4-menooxygenase

Hydroperoxidases

Peroxidase Fe+++ NO), IDioxymaleic acid Mn,Fe+++ Foxidase

Ferro-peroxidase Fe++Lacto-peroxidase Fe+++Myclo-peroxidase Fe+++Catalase Fe

Special redoxases

Homogentisate Fe++1,2-dioxygenase

Lactonizing enzyme Mn


(Table 1 continued)

ENZYME

Decarboxylases

II III IV V

Oxylacetate-,Bcarboxylase

Malatedehydrogenase

Malonatedecarboxylate system

Succinatedecarboxylate system

Isocitratedehydrogenase

Pyruvatedehydrogenase

Triosephosphate-lyases

PO., Mn, Cd, MgCoMn Mg

PO. Mg,Mn

PO., Mn

PO., Mn, Co Mg

Mg Mn

Fructose-bisphosphatealdolase

Asparate ammonia- Mglyase

Hydratases and dehydratases

MeCuCo,Cu

Zn,Fe++,Co++,B

Aconitate hydrataseCN,S,F,Cu,Hg

Enolase MgCarbo-anhydratase Zn

Isomerases

Fe++

Mn,Zn F,HgCN,S

UDP-glucose-4-epimerasePhosphoglucomutasePhosphoglyceromutase

C-N- and C-S-lyasesand synthases

Cystathionine-lyase

Mg,Mn,Co

Zn,Mn,Mg

Mg


In summary, there are many types of chelates, bothnatural and synthetic. In addition to medicalfunctions, such as removal of certain isotopes orpoisonous metals from the animal's body, chelation canalso be used in the deactivation of bacteria throughmetal deprivation.(18) Nutritionally, amino acidchelates are used to enhance trace metal delivery to thebody. (19) For that to occur, the stab; 1; ty constants ofthe chelating bonds must be compatible for intactabsorption while maintaining availability fordegradation at the sites of metal usage in the body. (20)

The molecular weight of the chelate must also be keptlow to promote intact absorpt ion. (21)

The science of chelation as it relates to thenutrition of domestic animals is coming of age. Theadvantage of using amino acids to chelate essentialminerals and render them more biologically available tothe animal offers greater possibilities to regulate theamount of a given metal ion at the cellular level thantechniques heretofore used.


References

1. Underwood, E., Trace Elements in Human and AnimalNutrition (New York: Academic Press) 1-10, 1977.

2. Mertz, W., IISome aspects of nutritional traceelement research," Federation Proceedings,

Federation of American Societies for ExperimentalBiology, Soc. Exp. Biol. 29:1482-1488, 1970.

3. Guthrie, H., Introductory Nutrition (St. Louis:C.V. Mosby Company) 11, 1975.

4. Dyer, I., "Mineral Requirements" in Hafez, E. andDyer, I., eds., Animal Growth and Nutrition(Philadelphia: Lea &Febiger) 313, 1969.

5. Suttle, N., "Trace Element Interactions inAnimals,1I in Nicholas, D. and Egan, A., eds.,Trace Elements in Soil-Plant-Animal Systems (NewYork: Academic Press, Inc.) 271, 1975.

6. Schutte, K., The Biology of the Trace Elements(Philadelphia: J.B. Lippincott Company) 132,1964.

7. Loneragan, J., "The Availability and Absorptionof Trace Elements in Soil-Plant Systems and their

Relation to Movement and Concentration of TraceElements in Plants," in Nicholas, D. and Egan,A., eds., Trace Elements in Soil-Plant-AnimalSystems (New York: Academic Press, Inc.) 109,A75.

8. Underwood, E., The Mineral Nutrition of Livestock(Slough: Commonwealth Agricultural Bureau) 15,1981.


9. Ashmead, D., and Christy H., "Factors interferingwith intestinal absorption of minerals," AnimalNutr. and Health, 40:10, August, 1985.

10. Ashmead, H., et tl., "Chel ation does notguarantee mineral metabolism," J. App. Nutr.,26:5, Summer, 1974.

11. Miller, R., "Chelating Agents in PoultryNutrition," A paper presented at DelmarvaNutrition Short Course, Delmarva, 1968.

12. Mellor, D., "Historical Background andFundamental Concepts [of Chelation]," in Dwyer,F. and Mellor, D., eds., Chelating Agents andMetal Chelates (New York: Academic Press) 1,1964.

13. Jones, L., "Antianemic Drugs," in Jones, L., etli., eds., Veterinary Pharmacology andTherapeutics (Ames: Iowa State University Press)283, 1965.

14. Goth, A., Medical Pharmacology (St. Louis: C.V.Mosby Company) 675, 1974.

15. Eichhorn, G., "The Role of Metal Ions in EnzymeSystems," in Seven, M. and Johnson, L., eds.,Metal-Binding in Medicine (Philadelphia: J.B.Lippincott Company) 19, 1960.

16. Hughes, M., The Inorganic Chemistry of BiologicalProcesses (London: John Wiley & Sons) 105, 1972.

17. Schutte, op. cit., 17-23.

18. Ashmead, D., "Chel ation in nutrition," WorldHealth &Ecology News, 7:10, 1976.


19. Ashmead, D. , "The needminerals," Vet. Med.jSm.69:467, 1974.

for chelated traceAnimal Clinician,

20. Kratzer, F. and Vohra, P., Chelates in Nutrition(Boca Raton: CRC Press, Inc.) 19-32, 1986.

21. Tiffin, L., "Translocation of Micronutrients," inDinauer, R., et li., eds., Micronutrients inAgriculture (Madison: Soil Science Society ofAmerica, Inc.) 207, 1972.

Chapter 2

FACTORS WHICH AFFECT THE INTESTINALABSORPTION OF MINERALS

H. DeWayne AshmeadAlbion Laboratories, Inc.

Hugo ZuninoUniversity of Chile

The Periodic Table contains at lrast 104 elements,81 of which are considered minerals.() With certaintyseventeen of these minerals (and probably two others)are deemed essential for the vital functions of animals.The concept of determining if a mineral is essential ornot has evolved as a consequence to the development ofmore sophisticated analytical techniques andequipment.(2) It is probable that as scientificmethodologies are perfected and artificial diets becomemore sophisticated, additional mi~erals will be added tothis 1ist of essential elements. ( )

The primary source of essential mineral elementsfor all biological systems is the soil.(4) With theexception of animals retrieving minerals from thissource through natural salt licks, only some plants andcertain microorganisms are able to extract simpleorganic compounds and ions from the soil, withoutdepending on organic metabolites which have beenprefabricated by other organisms. Life forms having thecapability to extract soil ions for direct use (otherthan as salt licks) are termed autotrophic. For themost part, animals, which are heterotrophic, mustsecondarily find (or catch) their metal nutrients inprepackaged forms. Ultimately all animals are dependantupon plants as the primary source of theirunsupplemented mineral nutrition. Any deficienciesexperienced by plants have far-rea~hing consequences tothe animals which depend on them.()

21


Animal feedstuffs can be organized into six basicgroups: protei n, carbohydrates, 1i pi ds, vi tami ns,mi nera1s, and water. (6) The mi nera1 group exerc i sesthree bas i c funct ions. (7)

The first function relates to the role of mineralsin the growth and maintenance of both hard and soft bodytissues. Obviously, elements such as calcium andphosphorus as well as magnesium, contributesubstantially to the hardness of bones and teeth byformi ng mat ri xes or comp1exes that are fundamenta11 yinorganic in nature, while others such as phosphorus,sulfur, zinc, and magnesium make up important componentsof soft tissue; fluorine, zinc, and silicon play rolesin the formation of proteins and fats that compose thebody. (8)

Minerals also preserve cellular integrity throughtheir roles in maintaining the osmotic pressure betweenthe intra- and extracellular fluids, the acid-basebalance, membrane permeability and tissue irritability.Although not directly related to animal growth, one ofthe most dramatic examples of the types of rolesminerals can play in growth is seen in the developmentof the bacterium, Escherichia coli. A culture of thismicroorganism can double in size every 20 minutes in amedium containing only glucose and minerals. In thatperiod of time, the chemical components of the mediumbecome incorporated into the expanding protoplasmaticmass and are converted, through an intricate series ofbiochemical reactions, into approximately 2,500 proteinsof differing compositions including a wide range ofnucleic acids and over 1,000 organic non-proteincompounds. (9)

The second basic function played by minerals is inthe regulation of physiological and biological processesin the animal. For example, the same calcium which isessential for bone development, is equally necessary inthe proper functioning of the nervous system, for blood

Factors Which Affect the Intestinal Absorption of Minerals 23

coagulation, for regulating permeability in cellularmembranes, for the contraction of cardiac muscle, etc.Vanadium, as an essential trace element, regulatescholesterol and phospholipid synthesis. Copper isrelated to the synthesis of hemoglobin, regulatingthereby the oxidative processes in the animal.

In their role as regulators of body processes, theessential minerals function as catalysts in enzyme andhormone systems and as integral and specific componentsof metalloenzymes such as those seen in Table 1. Theymay also function as less specific activators withincertain metalloenzymes.<e.l0) In a metalloenzyme themetal is chelated to the protein moiety with a fixednumber of metal atoms per mole of protein. Upon removalof the metal, enzyme activity ceases.


I Table 1 ISome Essential Metalloenzymes in Animals

Metal Enzyme Function

Iron Ferredoxin PhotosynthesisSuccinate dehydrogenase Aerobic oxidation of carbohydratesCytochromes Electron transferCatalase Protection against HzOz

Copper Cytochrome oxidase Terminal oxidaseLysyl oxidase Lysine oxidationCeruloplasmin Iron utilizationSuperoxide dismutase Dismutation of the superoxide free

radical (Oz -:)

Zinc Carbonic anhydrase COz format ionAlcohol dehydrogenase Alcohol metabolismCarboxypeptidases Protein digestionAlkaline phosphatase Hydrolysis of phosphate estersThymidine kinase Thymidine triphosphate formationRNA and DNA polymerases Synthesis of RNA and DNA chains

Manganese Pyruvate carboxylase Pyruvate metabolismSuperoxide dismutase (as above)

Molybdenum Xanthine oxidase Purine metabolismSulphite oxidase Sulphite oxidation

Selenium Glutathione peroxidase Remova1 of HzOz

The third major function of minerals lies in thegeneration of energy. This does not mean that mineralsof themselves are sources of energy; nevertheless, theyparticipate as essential co factors in enzymaticreactions which chemically transform foods into othermetabolites, thus freeing energy to be used in otherfunctions. To illustrate this concept in part, itshould be noted that calcium, magnesium, phosphorus,manganese, and vanadium are all utilized in one form oranother in the synthesis and formation of high-energybonds in compounds such as ATP. (11) In the case of


phosphorus, every physiological event involving gain orloss of energy and almost every form of energy exchangewithin all animal cells includes the making or breakingof high energy phosphate bonds and requires thatphosphorus be present. (e)

Accord i ng to the tota1 amount requ i red by theanimal, essential minerals are normally classified intotwo groups: macronutrients and trace elements.Although some believe that because of the low quantityof micronutrients required as compared tomacronut ri ents, the former are not important in thedaily diet. This is not true. For example, calcium, amacroelement, is used in great quantities in bonesynthesis for growth, but without the trace element,cobalt, growth is retarded as if there were a manifestdeficiency of dietary calcium. A cobalt deficiency canstart a chain reaction in the animal that can lead toinadequate metabolism of both protein and lipids whichi s further man i fested as a lower growth rate. (12)

For the above reasons, as well as others that areoutside the scope of this discussion, it is extremelyimportant that there be little or no interference in theintestinal absorption of essential minerals. While lackof interference would be desirable, this is generallynot the case. There are numerous antagonistic factorswhich cause less than optimal absorption of manyminerals. Consequently, there are numerous variationsin the absorption rates and levels of the same mineralunder different gastrointestinal conditions.(13)

It is commonly known that certain minerals caninteract with each other and mutually affect eachother's absorption and metabolism.(3) The more metabolicprocesses in which a certain mineral is involved, thegreater will be the possibility of its interacting withother minerals. Some of these interactions are shown inFigure 1.(3) The arrows indicate antagonisms betweenmi nera1s as they compete for intest ina1 absorpt ion.


These interactions may be grouped into six basiccategori es. (14)

p Co

Na

Ag

CdBe

Cu

Figure 1. Mineral interrelationships in animalmetabolism. The arrows indicate antagonism betweenelements. For example, calcium is antagonistic tozinc. Magnesium and calcium are mutuallyantagonistic.

The first group consists of interactions whichproduce insoluble precipitates as a reaction product.


This may occur when two or more minerals in the lumencompete for the same anionic electron-bearing ligand.The ligand may be an organic compound, such as phyticacid, or an inorganic compound, such as phosphate. (15.16.17)

Mineral competition for a specific dietary liganddepends on various factors including the associationconstants of the mineral-ligand compound and thesol ubi 1i ty of the product formed. (14)

When a soluble mineral salt is ingested, it isnormally ionized in the stomach. The acid pH of thestomach tends to encourage solubility. However, as thepH elevates in the intestines, the solubilitycharacteristic is lost, and the metal tends to bind withan an i on or 1igand. (13) Th is genera11 y occurs in thejejunum and ileum where the metal ion is sequestered bysuch molecules as metal-acid radical complexes which arevery stable and highly insoluble, thus rendering themineral unavailable for absorption. In order to beabsorbed, the metal ion must be soluble in theintestinal medium. (17) For example, in a precipitatedphytic acid salt state, the mineral cannot betransported by carri er protei ns across the cell ul armembrane of the mucosal ce11 s. (18) As the 1eve1 ofphytic acid increases in the feedstuffs, the absorptionof certain essential elements, especially calcium andzinc, proportionately decreases.(lg)

The same general principle discussed in relationto phyt i c ac id app1i es to other substances that arefound in the normal diet which can also form complexesor insoluble salts with various cations. For example,calcium, magnesium, zinc, manganese and iron all reactwith organic phosphates to form low solubility products.Some phosphates, such as calcium phosphate, which aresomewhat soluble in the acid environment of the stomachmay interfere with the uptake of other minerals when thedissociated phosphate anion reacts with another cation,such as iron, to form an insoluble precipitant: ironphosphate. In the particular case of iron, the


precipitation with phosphate can result in a drasticdecrease in absorption, and if severe enough, the animalcoul d exh i bi t signs of iron defi ci ency anemi a. (20.21.22.23)

Table 2 demonstrates the influence of phosphates and toa lesser degree, calcium, on the absorption of iron. (24)

Table 2

Influence of the addition of inorganic calcium andinorganic phosphorus on the absorption of iron

Mineral content of meal (mg)

Ca P

59Fe absorpt i on*

(%)

2424

202202

40414

40414

2.21.51.50.6

*All mean iron absorption rates tested weresignificantly different at the P<O.005 level.

The second group of mineral interactions involvescompetition between ions for the active transportcarriers which convey the cations from the lumen to thecytop1asm of the intest ina1 ce11 s. (14.25) The carri ermolecules traverse the intestinal mucosal cellmembranes. These molecules are composed of tinyprotei ns. Through thei r funct i ona1 e1ectron-beari nggroups, they have ample capacity to complex and/orchelate the free cations from soluble salts which arefound in the intestinal tract. (26) Since the ions mustbond with the carrier proteins in order to be absorbed,a physio-chemical competition between the cations forthe active protein sites can result. This competition


can occur between trace elements, macroelements, orboth. (27)

Figure 1 demonstrates these types of interactions.(3) The affinity of each essential mineral ionfor the electron-bearing atoms of the carrier proteindepends on their electronic configuration and theirpositions in the Periodic Table of Elements.(28,31,32)Thus, iron and copper are mutually antagonistic, sinceboth elements share the same carrier molecule in thecell membrane. (29) Norma11 y, there is a suffi ci entamount of the transport molecule, transferrin, to carryboth elements across the mucosal cells. However, ifiron and copper salts in the diet are excessivelyincreased (i.e. through a heavy intake of unbalancedmineral supplements), iron absorption is inhibited bythe copper because of a greater affinity of copper fortransferrin.(29) In this case the animal can displaysymptoms of iron deficiency anemia due to excessivedietary copper intake. This has been shownexperimentally in Table 3,(~) where both hemoglobin andhematocrit levels drop, and iron is sequestered in theliver.(30) In this particular experiment, a basal dietof 8.5% mixed grain protein was supplemented with ironalone, copper alone, or iron and copper together.

I Table 3 IEffect of supplementing the Basal Diet (8.5%mixed-grain protein) with Iron or Copper, orboth Iron and Copper

Basal +Fe +Cu Fe+Cu

Hemoglobin (%) 10.4 9.2 11.3 9.8

Hematocrit (%) 31 28 38 30

Liver Fe (ppm) 183 198 187 211


The th i rd group of mi nera1 interact ions i nvo1vethe reduction in the capacity of body cells tosynthesize metal-binding proteins, due to interferencesproduced by spec i fi c react ions to some non-essent i a1heavy metal s. (33.34) The enzymat ic act i on necessary forthe building of a carrier protein can be blocked bydisplacement of a certain specific cation activated byanother exogenous metal. When this happens, the enzymemay function equally well, or may be completelyinhibited, depending on the particular enzymatic systeminvolved, and the mineral displaced.(lO) To illustrate,lead has an inhibiting effect on the anabolic routeregulating the synthesis of the porphyrin fraction ofthe hemoglobin molecule.(35) In the first stages inhemoglobin formation, the glycine is converted intoalpha amino levulinic acid, two molecules of whichcondense to form one molecule of pyrrol. Ultimately,two pa irs of pyrro1s bond together formi ng a 1argeporphyritic ring, which later undergoes a slight changein structure in one of the lateral groups of the ring.This change is a requirement for the reaction with iron.The most important enzyme catalyzing the condensation ofalpha-amino levulinic acid in pyrrol is activated byzinc, but inhibited by lead.(36) Excessive amounts oflead can result in a lower production of hemoglobin andpromote anemi a. (37)

A fourth group of mineral interactions is relatedto the previous one. In the previous group, the metalactivates the enzyme. In the fourth group, the metal isan integral part of the enzyme, thus forming ameta11 oenzyme. In these compounds, as the metal isreplaced or removed, the enzymatic action can either beaccelerated or blocked. For example, as shown in Table1, in the metalloenzyme, carboxypeptidase, zinc is anintegral part. The zinc may be inhibited from enteringinto the enzyme by cabal t . Wi thout the zinc, thepeptidase activity is reduced.(~) When cobalt replacesthe zinc in the enzyme, the peptidase activity of theenzyme will double. If either manganese or nickel


replace the zinc, peptidase activity is retarded.(39)Thus, the replacement of the zinc by other metals inthat particular metalloenzyme has the potential ofultimately affecting the protein nutrition of the animalbecause carboxypeptidase is proteolytic, catalyzing thehydrolyses of carboxy-terminal peptide bonds in peptidesand proteins.

The fifth group of mineral interactions is relatedto the transport and excretion of minerals which occurspecifically in the cells of the intestinal mucosa.Even though taken up by the intestinal cells, mineralsmay be returned to the lumen by those same cells withoutever being utilized by the body. This stagnation andexcretion can result in the replacement of a variety ofmetals at the same time.(40) The phenomenon usuallyoccurs only where specific interrelations exist betweenmetals promoting competition for specific transportmechan isms. (41)

The sixth group of mi nera1 interact ions i nvo1vechain reactions consequential to the previous groups.In the previous examples, each group of reactions wasconsidered as being separate. Generally, however, morethan one such interaction will be operating at the sametime. For example, if a certain cation is precipitatedby a given ligand making it insoluble in the intestine,then it cannot be absorbed, as has been indicated.Therefore, certain enzymatic reactions in which thatspecific metal is essential will be affected and may behalted or retarded. These enzymatic reactions may berelated to the production of other proteinaceoussubstances: hormones, carri ers, or enzymes that arerequired at the intestinal mucosa for the absorption ofsome other metals. At that point, the efficientabsorption of the other minerals is also affected. Inthis manner, the original precipitating ligand may blockthe absorpt i on of more than one essent iale1ement,although the 1igand only precipitated one mineral. (14)


Huisingh, et ~., demonstrated the complexity ofthese interactions while working with copper,molybdenum, and sulfur in ruminants and nonrumi nants. (.8) They cons idered three separateinteractions, the formations of unavailable CuMoO. andCuS and the i nh i bi t i on of MoO.2- Uptake by SO.2-- Theyshowed that these mechani sms are dependant upon eachother since the formation of either CuMoO. and Cu2

+ andMoO.2- or S2-from SO.2-1 n the gut woul d 1imi t the abi 1i ty ofMoO.2- and SO.2- to precipitate in the 504

2- x MoO.2

interact i on . (14)

Not only do interactions between mineralsinterfere with cation absorption, but vitamins may alsohave both negative and positive effects on mineraluptake. It is commonly known that vitamin D influencesthe absorption of calcium. (33) The effect of vitamin Con the absorpt i on of iron i sal so we11-known. (.3) Whenthese vitamins are absent, the absorption ofcalcium/iron, which are ingested as metal salts,significantly diminishes. On the other hand, the excessof another vitamin, such as niacin, can inactivatevitamin D which is necessary for the absorption ofcalcium.(··) Niacin can thus cause hypocalcemia, eventhough the levels of calcium from a salt and vitamin Din the diet appear to be adequate. Some of the clearlyelucidated synergistic relationships between vitaminsare shown in Fi gure 2. (.4)


A

PantothenIc

Niacin

K

B 12

D

BIotin

Figure 2. The synergistic relationships ofvitamins. Dietary increases in Vitamin E, Bl, andNiacin have no effects on Vitamin A, folic acid, andVitamin Bl, respectively. In all other cases adietary increase of a specific vitamin willinfluence body requirements for the other vitaminsconnected to that vi tami n by the 1i nes in th isfigure.

The quantity of fat in the feed can also affectthe absorption level of a given mineral. High fat dietspromote the formation of insoluble soaps of fatty acidsand calcium. This results in steatorrhea and areduction in calcium absorption.(46) Most of theseinterferences obviously depend on the quality of the fatin the diet and the chemical form in which the mineralelement i s found. (14,47)


Another important consideration in dietaryformulations which may affect mineral absorption is thequantity of non-digestible fiber in the feedstuffs. Ithas been demonstrated that fiber may reduce theabsorption of many minerals. In nutritional balancestudies, there were appreciable decreases in theintestinal absorption of calcium, magnesium, zinc andphosphorus in the presence of high fi ber diets. (49) Theloss of these elements through the feces correlated wellwith the increase in dry fecal material (which wasdirectly proportional to the increase of non-digestiblefi ber in the diet). These stud ies in non-rumi nantsshowed that the minerals were physically adsorbed andchemi call y bonded by the fi ber, and reta i ned in thefeces. Since some high-fiber feeds contain high levelsof phytic acid and, in some cases, oxalic acid, mineralabsorpt i on may be further reduced through theprecipitation of metal ions bonded to these organicacids as previously noted.

The chemical environments of the stomach andintestines are additional conditions affecting thepercent of mineral absorption. In unpublished research,it was shown that absorption of magnesium increased whenthe rumen was buffered wi th MgO. (49) Di fferent areas ofthe small intestine vary in their capacities to absorba given metal ion due to changes in pH. A solutioncontaining 45Ca was introduced into the intestinallumens of twelve individual rats. Thirty minutes laterthe intestinal segments were excised, ligated into nineequal segments (segment II includes the duodenum, fromthe pylobus to the ligament of Treitz), and assayed forcalcium uptake in a gas flow B-counter. The results areshown in Figure 3 and demonstrate the decreasedabsorption of calcium as it descends through the smalli ntest i ne. (51) The lower absorpt ion is a funct i on of pHand not morphological changes. (13)


80...---------------------..,

~ 70oC

641~C 5{J

ECDo 40Gi

D....

c: 3(J.2a..o 2Gfl)..act: 10

xDCVIIIrvII01....----"-----"----1....---......10....----'------&--.....0....------"

I v VI VI

Segment Number

Figure 3. The absorption of calcium as it descendsthe small intestine.

With the exception of the al kal ine-earth ions (Na,K, Ca, Mg), the metallic ions tend to form insolubleprecipitates as the pH increases, which processincreasingly occurs in the distal portion of the smallintestine. Generally speaking, the more alkaline thechemical environment of the lumen, the lower is mineralabsorpt ion. (50,51) Certa in dietary components may increasethe pH of the gut to a higher than normal physiologicallevel, which further inhibits the absorption ofessential mineral elements when ingested as salts.Figure 4 demonstrates this concept. (52)


-Strongly acid Neutral Strongly alkaline

I I -PHOSPHORUS

I I -POTASSIUM

I I I ISULFUR

I ICALCIUM

I I IMAGNESIUM

I IIRON

I IMANGANESE -.......- I II IBORON -....

I ICOPPER a ZINC

I I IMOLYBDENUM

I I -

Figure 4. The solubility of minerals at differentpH levels.

The absorption of a given mineral from the lumento the interior of the mucosal cells depends in part onthe capacity of the element to be bonded to thetransporting proteins embedded within the membrane ofthe intestinal cells. Any factor that inactivates thecarrier proteins or affects the chemical bondingcapacity of the cation, such as reducing its solubilityin the digestive tract, will cause a consequentreduction in the absorption of the cation. There are noexi st i ng general rates of absorpt ion that areuniversally accepted for essential elements in the formof salts.(lJ) Absorption varies in each particular case.According to Suttle,(14) these variations are primarilydue to the chemical form of the ingested mineral and thedegree of structural similarity it has to the effective


chemical form in which it is absorbed from theintestinal content. In other words, in the finalanalysis, the chemical interactions between the nutrientin the diet and the components of the intestinalenvi ronment are what determi ne the degree of absorpt ion.There is more than one mechanism available to transportan essential mineral through the intestinal wall towardsthe bloodstream. The system used wi 11 depend on thechemical form of the element when it reaches the mucosalmembrane of the intestinal cells.

This concept regarding the chemical form of themineral and its relative susceptibility to absorption isshown in Figures 5 through 8. In a series ofexperiments , metal sin di fferent chemi cal forms wereexposed to the intestinal mucosa for specific times.Absorptions of these different forms of the same metalwere compared as a function of time in vitro. (35,53) Inthis particular series of experiments, all potentiallyi nterferi ng factors normally found in the i ntest ina1environment were removed, thus allowing for optimumabsorption. The following figures clearly demonstratethe di fferent rates of absorpt i on of the di fferentchemical forms of the same metal.


350-----------------,

300Ea.E;250Z

Q 200I--t:l.a:o 150enCD~

Z 100oa:

50

AAe

15 30

SECONDS

Figure 5. The intestinal absorption of iron fromdifferent sources.

250

E200 Me0-SZo 150 -i=0-a:0CJ) 100 - co,CD« so.(.) O2ZN 50 -

015 30 45 60

SECONDS

Figure 6. The intestinal absorption of zinc fromdifferent sources.


35--------------------.MC

e30 -a.0.

~25 oi=a.. 20 -cr:o~ 15 «cr:W 10a..0-

S 5

15 30 45 60

SECONDS

Fi gure 7. The i ntest ina1 absorpt i on of copper f"omdifferent sources.

100,.....----------------.....,~

Ea.~ 80 -Z0~a.. 60a:0enco«

40::E::JCi5Wz 20C'«~

015 30 45 60

AAC

SECONDS

Figure 8. The intestinal absorption of magnesiumfrom different sources.


These figures show the increased absorptionresulting from an essential mineral in the form of aminoacid chelate. The absorption of iron from the aminoacid chelate increased 1.7 times compared to ferrouscarbonate, 3.8 times compared to ferrous sulfate, and4.9 times compared to ferric oxide. The amino acid zincchelate was absorbed in the intestine 2.2 times morethan zinc as a carbonate, 2.3 times more than zinc aszinc sulfate, and 2.9 times more than zinc as zincoxide. In the case of copper, 4.1 times more metal wasabsorbed from the chelate than from the oxide, 2.7 timesmore than from copper carbonate and 2.8 times more thanfrom the sul fate. The magnes i urn from the ami no ac idchelate was absorbed from the intestine 1.2 times betterthan magnesium as a carbonate, 2.6 times better than asa sulfate, and 4.1 times better than as an oxide.Clearly the absorption rate of a metal is dependant onits chemical form.

It is important to understand the complexity ofmineral absorption before examining forms of mineralsthat optimize absorption and circumvent the majority ofthe problems discussed above. These amino acid chelateswill be examined in detail in the following studiesillustrating their potential and effectiveness both inthe therapeutic field as well as in the field ofprophylactic nutrition.


References

1. Phipps, D., Metals and Metabolism (Oxford:Clarendon Press) 1, 1976.

2. Davies, I., The Clinical Significance of theEssential Biological Metals (Springfield:Charles C Thomas) 1-15, 1972.

3. Dyer, I., "Mineral Requirements," in Hafez, E.and Dyer, I., eds., Animals Growth and Nutrition(Philadelphia: Lea &Febiger) 312, 1969.

4. Zunino, H., et li., "Measurement of metalcomplexing ability of poly functionalmacromolecules: Adiscussion of the relationshipbetween the metal-complexing properties ofextracted soil organic matter and soil genesisand plant nutrition," Soil Science 119:210,1975.

5. Egan, A., liThe Diagnosis of Trace ElementDeficiencies in the Grazing Ruminant, II inNicholas, D. and Egan, A., eds., Trace Elementsin Soil-Plant-Animal Systems (New York: AcademicPress, Inc.) 371, 1975.

6. Wilson, E., et li., Principles of Nutrition (NewYork: John Wiley &Sons) 4, 1979.

7. Guthrie, H., Introductory Nutrition (St. Louis:c.v. Mosby Company) 11, 1975.

8. Underwood, E., The Mineral Nutrition of Livestock(Slough: Commonwealth Agricultural Bureaux) 3,1981.

9. White, A., et li., Principles of Biochemistry(New York: McGraw Hill Book Company) 279, 1973.


10. Schutte, K., The Biology of the Trace Elements(Philadelphia: J.B. Lippincott Company) 17-23,1964.

11. Gallagher, C., Nutritional Factors and Enzymological Disturbances in Animals (Philadelphia:J.B. Lippincott Company) 12-38, 1964.

12. Ibid, 76-77.

13. Ashmead, H. D., et ~., Intestinal Absorption ofMetal Ions and Chelates (Springfield: Charles CThomas) 13-26, 1985.

14. Suttle, N., "Trace Element Interactions inAnimals," in Nicholas, D. and Egan, A., eds.,Trace Elements in Soil-Plant-Animal Systems (NewYork: Academic Press, Inc.) 271, 1975.

15. Williams, S., Nutrition and Diet Therapy (St.Louis: C.V. Mosby Co.) 137, 1977.

16. Cabell, C. A. and Earle, I. P., "Additive effectof calcium and phosphorus on utilization ofdietary zinc," J. Animal Sci., 24:800, 1965.

17. Vohra, P., et ~., "Phytic acid-metal complexes,"Proc. Soc. Exp. Biol. Med., 120:447, 1965.

18. Witt, W., Biology of the Cell (Philadelphia:W.B. Saunders Co.) 433, 1977.

19. Oberleas, D., et ~., "The Avail abil ity of Zincfrom Foodstuffs," in Prasad, A., ed., ZincMetabolism (Springfield: Charles C Thomas) 225,1966.

20. Pond, W. G., et ~., "Effect of dietary Ca and Plevels from 40 to 100 Kg body weight on weight


gain and bone and soft tissue mineralconcentrations," J. Animal Sci., 46:686, 1978.

21. Pond, W. G., et li., "Weight gain, feedutilization and bond and liver mineralcomposition of pigs fed high or normal Ca-P dietsfrom weaning to slaughter weight," J. AnimalSci., 41:1053, 1975.

22. Sell, J. L., "Utilization of iron by the chick asinfluenced by dietary calcium and phosphorus,"Poultry Sci., 44:550, 1965.

23. Waddell, D. G. and Sell, J. D., "Effects ofdietary calcium and phosphorus on the utilizationof dietary iron by the chick," Poultry Sci.,43:1249, 1964.

24. Monsen, E. R. and Cook, J. D., "Food ironabsorption in human subjects IV. The effects ofcalcium and phosphate salts in the absorption ofnonheme iron," Am. J. Clin. Nutr., 29:1142,1976.

25. Hill, C. J. and Matrone, G., "Chemical parametersin the study of in vivo and in vitro interactionsof transition elements," Fed. Proc., 29:1474,1970.

26. Ashmead, H.D., Ope cit., 103.

27. Starcher, B. C., "Studies on the mechanism ofcopper absorption in the chick," J. Nutr.,97:321, 1969.

28. Ahrland, S., et li., "The relative affinities ofligand atoms for acceptor molecules and ions,"Chern. Soc. London Quart. Rev., 12:265, 1958.

29. El-Shobaki, F. and Rummer, W., "Binding of copperto mucosal transferrin and inhibition of


intestinal iron absorption in rats," Res. Exp.Med., 174:187, 1979.

30. Caster, W. and Resurreccion, A., IIInfluence ofCopper, Zinc, and Protein on Biological Responseto Dietary Iron," in Kies, C., ed., NutritionalBioavailability of Iron (Washington, D.C.:American Chemical Society) 103, 1982.

31. Ashmead, H., "Tissue transport of organic traceminerals," J. Appl. Nutr. 22:42, 1970.

32. Little, P., "A Role of Chelated Minerals in theBody," in Ashmead, D., ed., Chelated MineralNutrition in Plants, Animals. and Man(Springfield: Charles C Thomas) 264, 1982.

33. Templin, V. M. and Steenbock, H., "Vitamin D andthe Conservation of Calcium in the Adult. II.The effect of vitamin D on calcium conservationin adult rats maintained on low calcium diets,"J. Biol. Chern., 100:209, 1933.

34. Blunt, J. and Deluca, H., "Biologically ActiveMetabol ites of Vitamin D," in Deluca, H., ed.,The Fat Soluble Vitamins (New York: PlenumPress) 69, 1978.

35. Ashmead, H., et li., "Trace Mineral Interrelationships, New Techniques of Detecting leadand Other Heavy Metals in Animals, and The Roleof Organic Chelated Trace Elements Playas EnzymeCatalysts," Presented at Okla. Vet. Med.Convention, Tulsa, 1971.

36. Nieburg, P. I., et li., "Red blood cell-delta.aminolevulinic acid dehydrase activity; an indexof body lead burden," Am. J. Dis. Child.,127:348, 1974.


37. Hoffbrand, A. and Konopka, l., "Haem synthesis insideroblastic anemia," in Porter, R. andFitzsimons, D., eds., Iron Metabolism (Amsterdam:Elsever) 269, 1977.

38. Deluca H. F., "Vitamin D and Calcium Transport,"Ann. N.Y. Acad. Sci., 307:356, 1978.

39. Hsu, J., "Biochemistry and Metabolism of Zinc,"in Zarcioglu, Z. and Sarper, R., eds., Zinc andCopDer in Medicine (Springfield: Charles CThomas) 70, 1980.

40. Hendri x, T., "The Absorpt i ve Funct i on of theAlimentary Canal," in Mountcastle, V., ed.,Medical Physiology (St. louis: C.V. Mosby) V2,1117, 1974.

41. Deluca, H., "The Control of Calcium andPhosphorus Metabolism by the Vitamin D EndocrineSystem," in Levander, o. and Cheng, L., eds.,Micronutrient Interactions: Vitamins, Minerals,and Hazardous Elements (New York: N.Y. Academyof Science) 12, 1980.

42. Huisingh, J., et li., Abstract #3833 "Sulfatereduction in mixed and isolated rumen bacteria,"Fed. Proc., 32:900, 1973.

43. Conral, M. and Schade, S., "Ascorbic acidchelates in iron absorption: A role forhydrochloric acid and bile," Gastroenterology,55:35, 1968.

44. Patrick, H. and Schaible, P., Poultry Feeds andNutrition (Westport: AVI Publishing Co., Inc.)144, 1980.

45. Nicolaysen, R., et li., "Physiology of calciummetabolism," Physiol. Rev., 33:424, 1953.


46. Guthrie, Ope cit., 117.

47. Fleischman, A. I., et li., "Effects of dietarycalcium upon lipid metabolism in mature male ratsfed beef tallow," J. Nutr., 88:255, 1966.

48. Reinhold, J. G., et li., "Decreased absorption ofcalcium, magnesium, zinc and phosphorus by humansdue to increased fiber and phosphorus consumptionas wheat bread," J. Nutr., 106:493, 1976.

49. Beede, D., Personal communications, Sept., 1988.

50. Wa1dron-Edward, D., "Effects of pH and counteri onon absorption of metal ions," in Skoryna, S. andWaldron-Edward, D., eds., Intestinal Absorptionof Metal Ions, Trace Elements, and Radionuclides(Oxford: Pargamon Press) 381, 1971.

51. Wa1dron-Edward, D., et li., "Effects of thecounter i on and pH on intest ina1 absorpt i on ofcalcium and strontium," Proc. Soc. Exp. Biol.Med. 123:532, 1966.

52. Hsu, H., "Trace Mineral Availability to Plants,"in Ashmead, D., ed., Chelated Mineral Nutritionin Plants, Animals and Man (Springfield: CharlesC Thomas) 60, 1982.

53. Graff, D., et li., "Absorption of mineralscompared with chelates made from various proteinsources into rat jejunal sl ices in vitro, II

Presented at Utah Acad. Arts, Letters, Sci., SaltLake City, April, 1970.

Chapter 3

COMPARATIVE INTESTINAL ABSORPTION ANDSUBSEQUENT METABOLISM OF

METAL AMINO ACID CHELATES ANDINORGANIC METAL SALTS


As noted in the previous chapter, when consideredin its most basic terms, animal nutrition is the optimalintake of protein, carbohydrates, fats, vitamins,minerals, and water for growth and maintenance of bodytissues, for energy, and to regulate all of the bodyprocesses. Health and well being of the animal aredependant upon the opt i mum mi x and intake of thesenutrients. An absolute deficiency of anyone of themfor an extended period will result in death. Minerals,function in all of the above nutrient roles. Thisnecessitates having an intake amount of the essentialmineral nutrients to carry out their multitude ofassigned functions. Optimal level is the key. Too muchor too little has an equally deteriorative effect on theanimal as illustrated in Figure 1.

47


HEALTH FUNCTION

100%

MARGINAL

OPTIMAL

MARGINAL

-~~ NUTRIENT INTAKE ~

Figure 1. A dose response curve.

As seen in Figure 1, if there is an acutedeficiency or an extreme toxicity of an essentialmineral, death will occur. If the deficiency or thetoxicity is marginal, the health and well being as wellas the performance of the animal will be impaired. Thusit becomes absolutely necessary to provide minerals thatare "safe" and yet biologically available. The factthat a mineral is mixed in the feed and is chemicallypresent does not guarantee it wi 11 be absorbed andmetabol i zed. There are numerous factors that wi 11positively or negatively influence the bioavailabilityof essential minerals. Many of these intrinsic andextri ns i c factors have been revi ewed by Kratzer andVohra and are summarized in Table 1.(1)

Comparative Intestinal Absorption and Subsequent Metabolism 49of Metal Amino Acid Chelates and Inorganic Metal Salts

Table 1

Factors Affecting Mineral Bioavailability

Intrinsic factors:

1. Animal species and its genetic makeup2. Age and sex3. Monogastric or ruminant (intestinal microflora)4. Physiological function: growth, maintenance, reproduction5. Environmental stress and general health6. Food habits and nutrition status7. Endogenous ligands to complex metals (chelates)

Extrinsic factors:

1. Mineral status of the soil on which the plants are grown2. Transfer of minerals from soil to food supply3. Bioavailability of mineral elements from food to animal

a. Chemical form of the mineral (inorganic salt or chelate)b. Solubility of the mineral complexc. Adsorption on silicates, calcium phosphates, dietary fiberd. Electronic configuration of the element and competitive antagonisme. Coordination numberf. Route of administration. (oral or injection)g. Presence of complexing agents such as chelatesh. Theoretical (in vitro) and effective (in vivo) metal binding

capacity of the chelate for the element under considerationi. Relative amounts of other mineral elements

In the lumen:

1. Interactions with naturally occurring ligandsa. Proteins, peptides, amino acidsb. Carbohydratesc. Lipidsd. Anionic moleculese. Other metals

2. At and across the intestinal membranea. Competition with metal-transporting ligandsb. Endogenously mediating ligandsc. Release to the target cell


To review each of these factors is outside thescope of th i s chapter. The purpose of th i s presentdiscussion is to examine a single extrinsic factor inTable 1: the chemical form of the mineral as it affectsmineral bioavailability. It is well established that aninorganic metal salt has a different absorption levelthan a chel ate. (2) Even among chel ates, absorpt i on rateswill vary according to the ligand, stability constants,molecular weights, etc.()

Because there are numerous ligands including aminoacids, ascorbic acid, citric acid, gluconic acid,ethylenediaminetetraacetic acid, etc., for the purposesof this discussion the field of chelates will benarrowed to deal only with chelates resulting from thebinding of the polyvalent cation to the alpha-amino andcarboxyl moieties of an amino acid to form a fivemembered ring. The structure of the ring consists ofthe metal atom, the active carboxyl oxygen atom, thecarbonyl carbon atom, the alpha-carbon atom, and thealpha-nitrogen atom. The bonding is accomplished byboth coordinate covalent and ionic bonding. At leasttwo and sometimes three, amino acids can be bound to asingle metal ion, depending upon its oxidative state, toform bicyclic (Figure 2) and/or tricyclic ringedmolecules. Even though the oxidative states of certaincations would allow for a fourth amino acid to bechelated to the cation, the bonding angles and theatomic distances required for chelation would tend topreclude its occurrence.


Figure 2. A two dimensional drawing of a bicyclicchelate of iron with glycine and methionine asamino acid ligands.

As defined by the American Association of FeedControl Officials (AAFCO), such a metal amino acidche1ate as seen in Fi gure 2 is descri bed as, "theproduct resulting from the reaction of a metal ion froma soluble metal salt with amino acids with a mole ratioof one mole of metal to one to three (preferably two)moles of amino acids to form coordinate covalent bonds.The average weight of the hydrolyzed amino acids must beapproximately 150 and the resulting molecular weight ofthe chelate must not exceed 800."(4

) An amino acidchelate is not the same as a metal proteinate which islithe product resulting from the chelation of a solublesalt with amino acids and/or partially hydrolyzedprotei n. 11(15) The 1atter does not descri be ei ther thestability of the chelate, the molar ratio of aminoacids, or the molecular weight, all of which will affectmineral bioavailability.(J) The looseness of the metalproteinate definition allows for loosely definedproducts which may not provide reproducible results fromusage to usage. Conversely, since the metal amino acidchelates are more tightly defined, research results andexpectations in practical usage can be expected to bemore reliable.


In an experiment comparing absorption capacity ofthe amino acid chelates versus inorganic sources of thesame metals, jejunal segments from adult male SpragueDawley albino rats beginning ten (10) cm below thepylorus and continuing for another twenty (20) cm werede1i neated and removed. The segments were placed inpetri dishes containing a buffer solution and maintainedat 5° C. Each intestinal segment was cut into twocentimeter segments and then severed longitudinallyalong the mesenteric line. The segments wererandomized, washed, rinsed, and incubated in KRB (KrebeRingers Bicarbonate) solution at 37° C while 95.5%oxygen and 4.5% carbon dioxi de bubb1ed through thesolution via fritted glass tubes.

The randomized jejunal segments were then exposedto 50 ~g of copper, iron, magnesium, or zinc for twominutes. Each of the metals was either in the form ofa salt (carbonate, oxide, or sulfate) or an amino acidchelate all of which had been dissolved in simulatedgastric solutions before presentation to the intestinalsegments. At the end of the 120 second exposure period,the segments were washed, rinsed and then assayed fortheir metal contents by atomic absorptionspectrophotometry. The resul ts are shown in Tabl e 2. (4)

This table clearly shows that amino acid chelates arebetter absorbed into the intestinal mucosa than cationsfrom metal salts.


I Table 2 IJejunal Uptake of Different Mineral Forms·

Chelate SO. O2 Cal Control

Copper 35 8 11 6 TraceMagnesium 94 36 23 51 7Iron 298 78 61 82 53Zinc 191 84 66 87 14

~Data represent the means and are expressed as ppmof metal.

The above in vitro experiments were designed totest the absorption of minerals in an ideal intestinalenvironment in which the interfering factors wereeliminated in order to allow optimum absorption of themi nera1s. In vi vo work has demonstrated that, afteringestion, metal salts are generally ionized in thestomach, providing they are soluble. Assuming that nointerfering chemical reactions occur, the cations enterthe 1umen where they are bonded to carri er protei nsembedded in the luminal membranes of the mucosal cells.The minerals are then transported to the interior of themucosal cell by passive diffusion or by activetransport. This absorption can occur anywhere in thesmall intestine, but it generally takes place in theduodenum where most metal ions retain their solubilitydue to a lower pH. (16)

In the case of an amino acid chelate, the metalion in the molecule is chemically inert due to thecoordinate covalent and ionic bonding by the amino acidligands. It is not affected by different precipitatinganions as is the case with free metal ions from solublesalts. Fats and fibers do not interfere with the


absorption of the amino acid chelate due to the highformation constant (or the low dissociation constant).And finally, the absorption of the amino acid chelatedoes not require vitamin intervention for absorption asin the case of some metal ions.

As previously described in the AAFCO definition,amino acid chelates are formed with one or more aminoacids and must have a molecular weight of less than 800daltons. If the molecular weight were much larger, itwould be unable to traverse the intestinal mucosal cellmembrane wi thout requ i ri ng further hydro1ys isin theintestinal lumen.(l7) Since the amino acid chelates actlike low molecular weight peptides(2), it is useful tostudy them as such.

After digestion of the proteins found in the diet,they are rendered into amino acids, dipeptides, andtripeptides. They are absorbed from the lumen in thesethree states. When a small er mol ecul ar wei ght ami noacid chelate is formed, such as described in Figure 2,the resulting molecule assumes many of thecharacteristics of a dipeptide or tripeptide. Due totheir stability, most amino acid chelates are notaltered in chemical structure throughout the digestiveprocess. Therefore, the essential amino acid chelatedmetal travels through the intestine in the guise of adipeptide-like or tripeptide-like molecule. Its lowmo1ecul ar wei ght is the key to its absorpt i on as anintact molecule. In the case of a high molecular weightmetal proteinate or a metal complex, there is littlechance for survi val in the gastroi ntest ina1 envi ronment.When a metal is "sequestered" into these structureswhich have greater molecular weights than the amino acidchelate, the resulting molecule may not be able totransport the ion through the mucosal membrane. Ifabsorption were to occur, the proteinate must behydrolyzed in the intestinal lumen. When this happens,the bonds between the metal and the organic ligand arebroken, and the capacity to transport the sequestered


metal is lost. At the same time, the freed cationbecomes subject to all the i nterferi ng chemi calreactions found in the lumen that were discussed in theprevious chapter.

Besides being concerned with the molecular weightof an amino acid chelate, one must also address itsstability constant. The stability constant is alogrithemic number that describes how tightly the metalion is bonded to the ligand(s>. If an amino acid chelateis stabi 1i zed through a part i cul ar bufferi ng process(18),its stability constant is modified so that it is notaffected by the acid pH of the stomach and will beabsorbed as an intact mo1ecul e. When on1y one ami noacid is used to chelate the metal ion, it will not forma chelate bond that is as strong a chelate formed of twoor more ligands. Two chelating amino acids provide fourbonds (three amino acids provide 6 bonds) into a singlemetal atom. The combination of the bonds projecting outat tetrahedral angles and the steric hindrance of thechelating amino acid rings impede competingelectrophilic molecules or atoms from disrupting thechelate bond. Thus, two and three ringed amino acidchelates are the most stable. If the stability constantof the amino acid chelate is optimal, the amino acidche1ate wi 11 a1so be res i stant to the act i on of thepeptidases that break internal peptide bonds, due to thepresence of the metal atom in the mo1ecul e.Consequently, a properly prepared amino acid chelatemolecule is generally absorbed intact through theintestinal mucosa.

MacInni s et li. ,(19) have suggested that nearly allof the mucosal cells in the small intestine are capableof absorbing amino acids or dipeptides. The amino acidchelate is absorbed from the small intestine, primarilyfrom the jejunum, as if it were a dipeptide. It followsthe same absorption pathway through the mucosal cell asdoes a dipeptide. As illustrated in Figure 3, when the


intact amino acid chelate is absorbed into the mucosalcells, it is believed that through the amino acidchelate molecule forms a coordinating link with thegamma glutamyl moiety of glutathione, a tripeptide foundin the mucosal membrane cell s. (20,21,22) After theformation of a chelate-gamma-glutathione molecule, anenzymatic breakdown of this complex could then ensuewhich could result in the transport of the amino acidchel ate from the 1uminal side of the mucosa to thecytoplasm within the cell.

By the act i on of the gamma-gl utamyl transpept idaseenzyme, the che1ate-gl utath ione compound woul d thendegrade to gamma-glutamyl-amino acid chelate andcysteinyl-glycine. The next step would involve thecleavage of the chelate by the action of gamma-glutamyltransferase, degrading it to a 5-oxyoproline and to theoriginal amino acid chelate, with the same structure asthat present in the 1umen. (20,21,23.24) Us i ng th is pathway,the active transport of the amino acid chelate from thelumen to the cytoplasm could be accomplished through tworapid enzymatic steps. The process would require ATP torestore the glutathione transporting molecule, in orderto move the next molecule of the substrate amino acidchelate into the cell interior.


LLMEN!\MIt-«) ACID CtELATE

CYTCJlLASM

Figure 3. The membrane transport of an amino acidchelate demonstrating the biochemical transportpathway of an amino acid chelate through thece11 ul ar membrane. (24)

The final entry of the amino acid chelate into thecytoplasm could take place at the terminal web of themucosal cell and woul d be the resul t of pH changescoupled with enzymatic action which breaks the linkageof the transport molecule with the amino acid ligand ofthe chelate. The original molecule of the amino acidchelate would then be able to quickly traverse themucosal cell in the direction of the basement membraneand from there be moved directly into the plasma, as anintact molecule. There would be no requirement for anintracellular carrier required to transport the aminoacid chelates through the mucosal cell. Perhaps osmoticpressure and the kinetic energy of diffusion are factorsthat influence its movement.


In the cytoplasm of the mucosal cell a certainamount of peptide hydrolysis can take place.(23)However, the intracellular hydrolysis of the amino acidchelates occurs less frequently than the hydrolysis ofdipeptides without metal, probably due to the sterichindrance of the ring structure inherent in the chelatemolecule, as noted above in connection with restrainedluminal hydrolysis. The actual separation of the metalfrom its amino acid ligand molecules is believed togenerally take place at the sites of usage, ifhydrolysis is necessary. Most metals function in thebody as parts of amino acid chelates and complexes sohydrolysis may not be necessary. When hydrolysis doesoccur, the coordinating bonds break, possibly due toenzymatic action together with cellular pH changes thathave lowered the stability constants of the amino acidchelates, favoring the release of the cation.(27)

The above he1ps to exp1ain the differences inabsorption between the amino acid chelates and the metalsalts which were seen in the jejunal absorptionexperiment summarized in Table 2. The differential inabsorption rates can also be assessed visually. Todemonstrate the differences in the mucosal cell uptakeof an iron amino acid chelate and iron carbonate, fastedexperimental animals were fed iron as the chelate or asthe carbonate. Sect ions of thei r respect i ve mucosaltissues were excised and prepared for examination by xray dispersive microanalytical electron microscopy.Figure 4A shows the unabsorbed iron carbonate on themicrovilli after ingestion, whereas Figure 48 shows theiron amino acid chelate entering the mucosal cell. Thedifference in the i ron content of the two cell sisdramatic.(5) The data in Figures 4A and 48 alsodemonstrate that not only was the iron from the chelateabsorbed in greater quantities, but that its rate ofabsorption as a function of time was also greater. Whenconsidered in this light, the data in Table 2substantiate the same observation.


Figure 4A. The absorption of iron from ironcarbonate on the microvilli.

Figure 48. The absorption of iron from ironamino acid chelate on the microvilli.


As compared to the previously discussed jejunaluptake studies, in vitro perfusion studies usingisotopes represent a more sophisticated assessment ofthe absorption and transfer of metals from the mucosa tothe serosa. In these experiments, rat small intestineswere excised, washed, and everted in an oxygenated (95%O2 and 5% CO2 ) buffer solution. Fifteen-centimeter (15cm) segments were cut, tied at the ends and placed intandem in separate vessels at 37° C which contained 1 X10-4 zinc in the mucosal solution. One half of thesegments wereused to measure the transfer of ~Zn fromMZ nC1 2 from the mucosa to the serosa, and the other halfwere used to measure the mucosal to serosal transfer ofMZn after being chelated to the amino acid, histidine.Each hour, for a period of eight hours, a segment wasremoved, rinsed and digested prior to scintillationcount i ng to determi ne the total rad ioact i vi ty in thetissue. Each hour, for a period of five hours, a sampleof the serosal solution was removed and analyzed for theamount of uZn that had been transferred from the mucosato the serosa. Figure 5 shows the moles of zinc pergram of tissue in the duodenum (A) and in thejejunum(S), while Figure 6 shows the quantity of ~Zn

transferred to the serosal solution from eithersource. (6)


r;;. 7r--------------------...,I

o~ 6u

~ 5(f)

,~ 4~

~ 3........cN 2

If)(l) 1

~0

2 4 6 8 10 12

AAC

~urs

Figure SA. The transfer of ~Zn from the mucosato the duodenum.

AAC

CI:2'Ui' 4I

0,xu

a.>:J(f)(J)-~

cS"CN

(f)<1)-~

02 4 6 8 10 12 14 16

r-ours

Fi gure 58. The transfer of 65Zn from the mucosato the jejunum.


QI

5

3 ----

o L----__

o"X 4 ----u(1)::J(f)(f)

l

El'J"'cN

(f) 1 ---a>oL

Zn AmrnoAcid Chelate

ZnCI2

Figure 6. The transfer of MZ n to the serosalsolution from the mucosal solution.

While at first glance the zinc from the chloridesource would appear to be absorbed better than from theamino acid chelate source (Figures 5A and 58), thedynamics of the intestinal transfer are betterelucidated with reference to Figure 6. Less 65Zn fromthe amino acid chelate is found in the intestinal tissueof the duodenum and jejunum than from ZnC1 2 because moreof the zinc has been transferred to the serosa. Whenchelated to amino acids, approximately four times asmuch zinc was transported to the serosa. From thesefigures it is easy to see that the movement of zinc fromthe mucosa to the serosa in the form of an amino acidchelate is not only more rapid than zinc as zincchloride, but the quantity of zinc absorbed and


transferred is significantly higher. When the mucosalcell is dependant upon internally generated carriermolecules to transport the zinc to the serosa, as in thecase of ZnC1 2 , the movement appears hindered, or evenblocked. Research demonstrates the possibility ofhigher concentrations of zinc from the chloride in themucosal cells in vivo where the zinc could possiblydiffuse back into the lumen or be lost as the mucosalcells containing the untransferred zinc died and weresloughed off. Similar results as these data presentedon zinc exist for iron.(2)

The following comparative studies involve the useof radioactive isotopes in vivo, in which labeled metalschelated to amino acids or as salts were ingested bylive animals and their absorptions and metabolismscompared.

In the first example, Sprague-Dawley male adultrats were fasted for 24 hours before being divided intotwo groups of eight animals each. While under lightether anesthesia each animal was given an intraperi tonea1 dose of 5.0 J-Lg of 65Zn as an ami no ac idchelate or as zinc chloride. Commencing 1.5 hours afterdosing and continuing every half hour thereafter forfour hours, blood from each an i rna1 was removed bysuborbital bleeding and assayed for ~Zn with a gammaray spectrometer. After the 1ast blood sampl e wasremoved, all of the animals were sacrificed and theirtissues a1so assayed for 65Zn. Fi gure 7 presents themean zinc 1eve1sin the blood of the two groups asplotted against time. Table 3 presents the amount of ~

Zn found in the assayed tissues of the animals. (7)


40-----------------------------

30------------- ---------------

AAC

C1220 --

OJ----""'----""""""--------"--------:--~--...I..--------'

8:00 8:30 8:45 9:00 10:00 10:30 11:00 12:00

~+oJ::JC

ELQ)Q.

U)+oJC

6 10u

Ti rre

Figure 7. Mean levels of 65Zn in the blood of ratsrecelvlng an oral dose of zinc as either an aminoacid chelate or as zinc chloride as a function oftime.


I Table 3 IMean 6SZ inc Levels in the Tissues

Tissue Zn amino acid Zn ch1ori de" % Increasechel ate"

Muscle 186 153 22Heart 1,457 1,433 2Liver 10,250 7,529 36Kidney 8,629 7,797 11Brain 541 444 22

.. Data are expressed as corrected counts/minute/g oftissue.

As can be seen from these data, both theabsorption and metabolism of zinc amino acid chelate isgreater than that of zinc chloride. The lower initiallevels of the chelated zinc in the blood (Figure 7)suggest that the chelate is absorbed at a locus in thesmall i ntest i ne that is different than that of thechloride. Whereas the majority of the ionic salt isabsorbed in the acidic environment of the duodenum~where solubility of the salt is enhanced, the bicyclicamino acid chelate is absorbed in the jejunum, intact,and incorporated into a dipeptide-like molecule. Thus,one would expect that initially there would be as1ight1y lower amount of 65Zn in the blood of thechelate group until intestinal motility moved themajority of the amino acid chelate to its primaryabsorption site in the jejunum. Until that occurred onewould expect the ~Zn blood levels from the ZnC1 2 to behigher since the majority of that zinc would be absorbedin the duodenum. It is significant to note that thenormal sites for absorpt; on of am; no ac; ds and smallpeptides and the apparent site of absorption of amino


acid chelates are in the jejunum. In the latter case,as an amino acid chelate, the metal is a stable part ofan organic molecule and is not only smuggled into themucosal cell as part of that molecule but is alsotransported to the blood in the same form. (2)

In a similar study to the one above on zinc,54manganese amino acid chelate was compared to54manganese chloride. Two groups of adult male SpragueDawley albino rats were fasted for 24 hours and thenorally administered a single dose of 32 ~g ofradioactive manganese as either the chelate or thechloride. Fourteen days following dosing, the animalswere sacrificed and their tissues assayed for ~Mn. Thedelay in sacrificing the animals and assaying theirtissues was to provide time for equilibration. When thebody is loaded with a stable manganese there is a rapidexcretion of old manganese via the bile and aredistribution of the new manganese within thetissues. (25) The fourteen days between dos i ng and tissueassay allowed the distribution of ~manganese to occurso that true metabolic comparisons between the aminoacid chelate and MnC1 2 could be obtained. The resultsare shown in Table 4. (I)


I Table 4 IMean 54Manganese Levels in Tissue

Tissue Mn amino Mn chloride" % Increaseacid

chel ate"

Heart 107 36 197Liver 106 52 104Kidney 97 80 21Spleen 397 190 109Lung 56 54 4Sm Intestine 141 89 58Muscle 28 22 27Bone 266 112 138

.. Data are expressed as corrected counts/minute/g oftissue.

As can be seen from Table 4, when the manganese ischelated to the amino acids, its retention within bodytissues is much greater than when it is gi ven as aninorganic salt. This suggests that the absorption ofthe chelate was also higher, and is further justified bythe fact that 33% more 54Mn from the ch1ori de wasactually recovered in the feces after non-absorption orexcretion back into the bile than from the amino acidchelate.

A third comparative in vivo isotope study wasdesigned using calcium in a similar experimental designas described above. Adult male Sprague-Dawley albinorats were divided into two groups. After fasting for 24hours each animal received an oral dose of 1 mg ofradioactive calcium as either calcium amino acid chelateor as calcium chloride. One week following the dosing,


all of the animals were sacrificed and their tissuesassayed for 45Ca. The delay in sacrificing the animalsafter dosing was not for the same reasons as themanganese study. In this case, the movement of calciumions from CaC1 2 across the mucosal cell requires thehormonal production of a vitamin Ddependant carrier. (26)

Consequently, time was provided for the movement ofcalcium from CaC1 2 out of the mucosal cells and into thetissues. Table 5 presents the results. (9)

I Table 5 II Mean 45Ca1ci urn Levels in the Tissues I

Tissue Ca amino acid Ca % Increase b

chelate" ch1ori de·

Bone 5,772 3,682 57Muscle 1,206 614 96Heart 932 642 45Liver 742 664 12R. Cerebrum 804 698 15Kidney 730 686 6Serum 31 8 288Erythrocytes 13 19 (-32)Whole Blood 44 27 63

• Data are expressed as corrected counts/minute/gof tissue.

b With the exception of the erythrocytecompartment.

The chelate group excreted 76% less 45Ca in theirfeces than the chloride group, indicating greaterabsorption. The data in Table 5 indicate that thecalcium amino acid chelate was translocated to thetissues in greater quantities than the chloride.


In a fourth series of studies, 4.4 ~c of ~Fe wereutilized as either the amino acid chelate or as FeC1 2 •

The experimental design was similar to those describedabove except that pregnant Sprague-Dawley female albinorats were used. Each of the 15 an imal sin the twogroups was fasted for 24 hours prior to administering asi ngl e ora1 dose of one of the two forms of iron.Approximately 72 hours after dosing and one day beforeexpected parturi t i on the an imal s were sacri fi ced andthei r tissues and fetuses were assayed for 59Fe. Thedelay between dosing and assaying of tissues was done toallow placental transfer of absorbed iron andincorporation of that iron into the fetuses. Table 6presents the resul ts. (10)

Table 6

Mean ~Fe Levels in the Tissues of Mothers andFetuses

Tissue Fe amino acid Fe chloridea % Increasechelatea

UterusLiverKidneySpleenHeartLungFetus (mean/Fetus)

4,9259,675

950325

1,4252,925

46

3,3338,167

567134333

1,367

16

481868

143328114

188

a Data are expressed as corrected counts/minute/g oftissue.

The data in Table 6 demonstrate that when iron ischelated to amino acids, its uptake, and utilization,


are significantly greater than when equivalent amountsof iron in the chloride form are ingested. An addedfinding in this study is the placental transport of theiron as the amino acid chelate. Traditionally,transplacental iron transport has been a difficult featduri ng pregnancy. These data i nd icate that when theamino acid chelate is ingested, more iron can betranslocated to the fetus from maternal blood. Otherstudies have indicated that placental transfer of theiron amino acid chelate effect is primarily due to themolecular weight of the absorbed chelate (approximately300 daltons) when compared to iron transferrin in theblood (approximately 86,000) which is derived from theiron chloride.(ll) Because the amino acid chelate isabsorbed into the blood intact in the same molecularform as when it was ingested, the smaller molecule ismore easily moved through the so-called "placentalbarrier" than iron attached to the much largertransferrin molecule.

In another study involving ~Fe and itsincorporation into hemoglobin, 22-day-old male pigs thathad been fasted for 24 hours were given a single dose of~Fe as either ferrous sulfate or iron amino acidchelate. The amount of iron given in either oral dosewas the same. Beginning one hour after dosing andcontinuing for the next 20 hours blood samples weretaken hourly and assayed for 59Fe in the hemogl obi n.Fi gure 8 presents the resul ts. (24)


- 60 .-------------------..U...........

Q)

j 50c

AAC

-~ 40en+-Jc:J 30ou

~ 20t-JUQ) 10LL

8o

so ~

1

Figure 8. Thehemoglobin.

2 3

Hours

incorporation

4

of

5

into

The data in Figure 8 demonstrate a greaterproduction of hemoglobin in the baby pigs when the ironamino acid chelate is fed versus the inorganic salt. Italso shows that more iron from the chelate was absorbedand incorporated into the hemogl obi n mo1ecul es.Interestingly enough, the data also substantiate theobservations noted in Figure 7 in regards to absorptionof zinc from the intestine as an amino acid chelate oras a soluble salt. There is a dip in the ironabsorption from the chloride as intestinal motilitymoves it into the jejunum just as there was with thezinc. On the other hand, while initially lower, theabsorption from the amino acid chelate shortly overtakesthe chloride and then continues to distance itself fromthe salt as a function of time.


In conclusion, it was shown in Table 1 that thereare numerous factors which will affect mineralbioavailability. One of the extrinsic factors isinteractions with amino acids. The above datademonstrated that if the interaction is that of aminoacid chelation, the absorption and metabolism ofessential polyvalent minerals are greater than that ofthe same metals in the form of the soluble salts.

In Figure 1, a dose response curve was presented.The animal fails to thrive if it obtains too little ortoo much of a specific metal. The greater uptake of theamino acid chelates could open the door to speculationthat mineral toxicity may occur more readily when thesechelates are ingested. Such is not the case asdemonstrated by numerous toxicity and pathology studiesincluding LD-50 studies, acute and sub-acute toxicityand pathology studies and long-term and multi-generationtoxicity and pathology studies. Some of these studieswill be presented later in this book. The detailedfindings will be presented further on in this book. Forthe present, suffi ce it to say that the ami no ac idchelates are considerably less toxic than equivalentamounts of metal salts.(12·13) When essential minerals arechelated and presented as intact units to the internalphysiological systems of animals, they are in effectencased in their protective carriers. These amino acidunits protect the metals from unwanted precipitatingreactions in the gut and have the additional benefit ofprotecting the mucous membranes and other tissuessuscept i b1e to i rri tat i on from the effects of nonconfined metals.

Thus the use of mi nera1s that are che1ated toamino acids are not only absorbed from the smallintestine in greater quantities due to their molecularpresentation, but that they are also less toxic in thisprotected, sequestered form.


References

1. Kratzer, F. and Vohra, P., Chelates in Nutrition(Boca Raton: CRC Press, Inc.) 35, 1986.

2. Ashmead, H.D., et li., Intestinal Absorption ofMetal Ions and Chelates (Springfield: Charles CThomas) 1986.

3. Miller, R., "Chelating agents in poultrynutrition," Proc. Delmarva Nutr. Short Course,1968.

4. Ashmead, H.D., Ope cit., 120 - 121.

5. Ibid, 155 - 157.

6. Fang, S., et li., "Bioavailability of zinc:Effect of amino acid chelation," in Ashmead, D.,ed. Chelated Mineral Nutrition in Plants,Animals, and Man (Springfield: Charles C Thomas)137, 1982.

7. Peck, A. and Graff, D. "Absorption of 65Zn inchelated and salt form into the blood of rats andits incorporation into tissues (in vivo)",Unpublished, 1973.

8. Ashmead, H., et li., "Stabil ity constants of~manganese chelates and their effects onmanganese metabolism", Unpublished, 1974.

9. Ashmead, H., et li., "The influence of stabil ityconstants on calcium metabolism in vivo,"Unpublished, 1974.


10. Graff, D., et li., "Placental transport of59i ron," Unpub1i shed, 1976.

11. Ashmead, D. and Graff, D. "Placental transport ofchelated iron," Proc. IPVS, Mexico, 207,1982.

12. Larson, A.," L.D. 50 studies with chelatedminerals," in Ashmead, D., ed., Chelated Mineralsin Plants, Animals, and Man, Ope cit., 163.

13. Jeppsen, R. "Assessment of Long-Term Feedi ng ofMetalosates in Sows," Unpublished, 1987.

14. Haas, E., et li., eds., Official Publication 1989(Atlanta: American Feed Control Officials, Inc.)162, 1991.

15. Ibid, 163.

16. Ashmead, H.D., Ope cit., 71-102.

17. Tiffin, L., "Translocation of Micronutrients inpl ants," in Dinauer, R., ed., Micronutrients inAgriculture (Madison: Soil Science Society) 207,1972.

18. Jensen, N., IIBiological Assimilation of Metals,"U.S. Patent No. 4,167,546, Washington D.C., Sept.11, 1979.

19. MacInnis, A., et li., "Specificity of amino acidtransport in the tapeworm Hymenolepis diminutaand its rat host," Rice University Studies62:183, 1976.

20. Meister, A., "On the Enzymology of amino acidtransport," Science 180:33, 1973.


21. Meister, A., "Biochemistry of Glutathione," inGreenberg, D. , ed. , Metabo1i sm of Sul furCompounds, Metabol ic Pathways (New York:Academic Press) 101-188, 1975.

22. Meister, A. and Tate, S. S., "Glutathione andRelated .gamma.-glutamyl compounds: biosynthesisand Utilization" In E. E. Snell, et li., eds.,Annual Review of Biochemistry 45:559, 1976.

23. Matthews, D. and Burstin, D., "Intestinaltransport of peptides," in Kramer, M. andLouterbach, F., eds., Intestinal Permeation(Amsterdam: Excerpta Medica) 136, 1977.

24. Ashmead, H.D., Ope cit., 216-221.

25. Underwood, E., Trace Elements in Human and AnimalNutrition (New York: Academic Press) 174-176,1977.

26. Guthrie, H., Introductory Nutrition (St. Louis:Time Mirror/ Mosby College Publishing) 219, 1986.

27. Rosevear, A. et li., Immobil i zed Enzymes andCells (Bristol: Adam Hilger) 25, 1987.

Chapter 4

INCREASING INTESTINAL DISACCHARIDASEACTIVITY IN THE SMALL INTESTINE WITH

AMINO ACID CHELATES

Silvano Maletto, University of TurinGermano Cagl i ero, Agrolabo, S.P.A.

The primary function of carbohydrates in theanimal is as a source of fuel. Carbohydrates aredegraded to carbon dioxide and lower sugars, and in thatprocess they release energy. Carbohydrates alsofunction as the starting material for the biologicalsynthesis of fatty acids and certain amino acids. Theyhave roles in the structure of glycolipids,glycoproteins, heparin, nucleic acids, etc. For thesereasons the efficient digestion and metabolism ofcarbohydrates is of paramount importance to the overallfeeding efficiency of the animal. A less than optimaldegree of carbohydrate digest i on and metabo1i sm wi 11result in poorer feed utilization and increased feedingcosts.

Carbohydrates are classified into three groups:monosaccharides, disaccharides and polysaccharides. Thedisaccharides and polysaccharides are converted tomonosaccharides by enzymatic action in the saliva(amylase), from pancreatic secretions (pancreatin), andfrom enzymes produced in the small intestine(di saccharidases). (1)

The polysaccharides are composed of glucose andother monosaccharides which are held together by twotypes of linkage, joining the 1 -- 4 carbons and the 1 - 6 carbons between each monosaccharide unit. The 1 -4 1inkages form straight chains whereas the 1 -- 6linkages occur on the straight chains at R?ints whereone stra ight cha in branches from another. () Th is isseen in Figures 1 and 2. (1,6)

76

Increasing Intestinal Disaccharidase Activity in the Small 77Intestine with Amino Acid Chelates

o

H OH H OH H t':)M

Fi gure 1. Glycogen conta ins 1i near amylose cha ins,made by linking carbons 1 and 4 of glucose throughoxygen. Branches occur in the amylose chains wherecarbon 6 and a residue are linked through oxygen inthe C-1 terminal of another chain segment.


Figure 2. A cross-section of glycogen showing thetree-l ike structure created by branched amyl asact inchains. The short inner segments are actually partof branches extending above and below the crosssection. The circles represent glucosyl residues.

The amylases of the saliva and pancreaticsecret ions attack the 1 -- 4 carbon 1i nkages as thepolysaccharides. These enzymes convert the bulk of theingested carbohydrates into disaccharides. These


disaccharides are then presented to the intestinalmucosa where the disaccharidases break the 1 -- 6 carbonlinkages resulting in monosaccharides. There are twoclasses of disaccharidase enzymes: glucosidases andgl actos idases. (1)

The disaccharidases are located on the brushborder of the mucosal cells of the intestine. They notonly digest the disaccharides into monosaccharides, butthey also facilitate the transfer of these sugars acrossthe membrane. Thus, they are integral to the absorptionof the carbohydrates. (1.2)

Because these enzymes serve more than onefunction, different enzymes occupy different membraneenvi ronments. Based on the bi 1i pi d membrane mode1containing integral and peripheral proteins as developedby Si nger, (3) some enzymes may extend through the bi 1ayerwhile others are located in half of the bilayer. Theenzymes all appear to require lipid fluidity in theirimmediate environments. (4) Some of the enzymes may havea tightly bound shell of a specific lipid which slowlyexchanges with the bulk lipid of the membrane, whereasother enzymes may have no direct interaction with theenvironmental 1ipids but may be peripheral proteinswhich are bound to the cell membrane by an interactionwith the integral proteins found in the membrane. Stillother enzymes may be attached, or lIanchored ll to a nonpolar region of the membrane by a hydrophobic tail. Inthis case, the functionally active region of themembrane extends out into the lumen away from contactwi th the membrane 1i pi ds. (2) These a1ternat i ves areill ustrated in Figure 3. (2.5)


Fi gure 3. A schemat ic representat i on of thelocalization of some of the enzymes in plasmamembranes. The active site of enzymes orbinding site on receptors is indicated byAbbreviations: GR, glucagon receptor; H,glucagon, hormone; A, catalytic unit ofadenylate cyclase; SP, (Na+ + K+)-stimulatedATPase; M, Mg 2 +-dependent ATPase; POI,Phosphodiesterase I; CPD, cyclic AMPphosphodiesterase; N, 5'-nucleotidase.

The absorption of digested carbohydrates from theintestine is influenced by the general conditionsrelating to the animal. This not only includes thegeneral heal th of that an imal, but a1so its currentnutrition. (1) If its mineral nutrition is eitherinadequate or chemically present but nutritionallyunavailable to the animal, the disaccharidase activityi s 1imi ted. (2,7,4)

Recognizing the need for these enzymes to promotefeed efficiency and that many of these essential enzymesare metal activated, an experiment was designed toascertain if the feeding of amino acid chelates to the


animal would increase enzymatic activity at the brushborder of the intestinal cell.

The research was conducted using eight adult maleWi star-Hazemann wh i te rats. Each rat wei ghedapproximately 100 grams. They were individually housedin wi re mesh cages. The rats were fed water and acommercial laboratory rat chow ad libitum for ten days,which was considered a stabilization period.

At the end of the ten-day period the rats werearbitrarily divided into four groups of two rats each.The first group continued to receive the commercial1aboratory rat chow as above wi th no changes in itsdiet. The second, third, and fourth groups received thesame laboratory rat chow plus 500 ppm, 1000 ppm and 1500ppm, respectively, of an amino acid chelate formulashown in Table 1.

Table 1

Composition of Amino Acid Chelate Supplements

% in Mineral Mix % Supplemented at Different Levels

Control ~ 1000 ppm 1500 ppm

Iron 9.00 % -- 0.0045 0.009 0.0135Copper 2.15 % -- 0.00107 0.00215 0.00322Zinc 3.00 % -- 0.0015 0.0030 0.0045Manganese 1.20 % -- 0.0006 0.0012 0.0018Cobalt 0.08 % -- 0.00004 0.00008 0.00012

The four groups of rats received the dosages ofamino acid chelates shown in Table 1 for ten consecutivedays immediately following the stabilizing period. Atthe conclusion of this second feeding period, all of therats were sacrificed by cervical dislocation.


The small intestines from the rats in each groupwere removed. The duodenum and an equal length of theupper jejunum which was attached to the duodenum wereexcised from each small intestine. The excised sampleswere cut along the mesenteric line and then rinsed withdistilled and deionized water to remove luminalcontents.

The mucus membrane was scraped away from thesecleaned and flattened intestinal segments and put inlabeled containers, each containing 4 ml of deionizeddistilled water. The membrane-water mixture washomogenized for twenty minutes and then stored at 00 Cuntil centrifuging at 3000 RPM for twenty minutes. Thesupernatant was collected and maintained at -18 0 C untilmeasurement of disaccharidase activity. Usingelectrophoresis methods discussed by Andrews,(9) thedisaccharidase activity in the mucus membrane wasdetermined by measuring the quantities of five membranebound disaccharidase enzymes. All comparisons forenzyme levels were measured against the control group asa standard. The findings are summarized in Table 2, andthe data are expressed as percents of the control.

I Table 2 IThe Mean Disaccharidase Activity of the Intestinal Mucous MembraneWith and Without Supplementation With Amino Acid Chelates

No Supplementation Amino Acid Chelate Supplement

Control ~ 1000 ppm 1500 ppm

Maltase 100 129 134 177Lactase 100 136 147 157Saccharase 100 178 163 135Trehalase 100 113 212 142Cellobiose 100 186 139 121


As can be seen from the data in Tabl e 2, theinclusion of amino acid chelates increased carbohydrateenzyme activity at the intestinal membrane an average of48% when the chelates were included at 500 ppm, 59% whenthe chelates were included at 1000 ppm, and 46% when theche1ates were inc1uded at 1500 ppm. A1though theenzymatic activity was increased over the control ineach case that amino acid chelates were supplemented inthe diet, the increases were not consistent. Somedisaccharidase enzymes were more responsive to highermetal intake than others. The rna1tase and 1actaseactivity increased with the higher amount of mineralspresent. In the case of the saccharase, trehalase andcellobiose, after a certain level, any increased intakeof metals suppressed their activity. Whether this is aresult of toxicity or simply providing the wrong mineralcombination and suppressing activity is unknown.


References

1. Latner, A., Clinical Biochemistry (Philadelphia:W.B. Saunders Company) 1, 1975.

2. Freedman, R. , "Membrane-bound enzymes, " inFinean, J. and Michell, R., eds., MembraneStructure (Amsterdam: Elsevier) 161, 1981.

3. Singer, S. J., "The molecular organization ofmembranes," in E.E. Snell, et li., eds., AnnualReview of Biochemistry, 43:805, 1974.

4. Poste, G. and Micholson, G., eds., DynamicAspects of Cell Surface Organization (Amsterdam:North-Holland Biomedical Press) 205-293, 1977.

5. Houslay, M. D. and Palmer, R. W., "Changes in theform of Arrheniums plots of the activity ofglucagon-stimulated adenyl ate cyclase and otherhamstar liver plasma-membrane enzymes occurringon hibernation," Biochem J., 174:909, 1978.

6. McGilvery, R., Biochemistry: A FundamentalApproach (Philadelphia: W.B. Saunders Company)1970.

7. Roth, S., et li., "Evidence for cell-surfaceglycosyltransferases; their potential role incellular recognition," J. Cell Biol., 51:536,1971.

8. Shur, B. D. and Roth, S., "Cell surfaceglycosyl transferases," Bi ochem. Bi ophys. Acta.,415:473, 1975.


9. Andrews, A., Electrophoresis: Theory, Techniques,and Biochemical and Clinical Applications(Oxford: Clarendon Press) 1981.

Chapter 5

EVALUATION OF THE NUTRITIONALEFFICIENCY OF AMINO ACID CHELATES

Silvana Maletta, University of TurinGermano Cagl iera, Agro/abo, S.P.A.

In the past few years we have reviewed field testsand other research published by Albion Laboratories andits associates in which it has reported that whenanimals are fed a formula containing Albion'sbiologically available amino acid chelates inconjunction with certain vitamins and other nutrients,the producer can reduce the protei n content of theanimals' feed and still obtain increased growth andperformance. (1)

For example,(1) a group of Durlock and Hampshirepigs were divided into three groups: (1) a confinedtreated group, (2) a confined control group, and (3) apasture control. All pigs in the study were weaner pigsand were assessed until market time (220 pounds or 100kg). The pasture group grazed free choice and did notreceive any supplemental commercial feed. The twoconfined groups received the same commercial feed exceptthat the treated group received an additional amino acidchelated mineral supplement formulas in three sequentialrations shown in Table 1.

86

Evaluation of the Nutritional Efficiency of Amino Acid Chelates 87

I Table 1 I

Mineral Supplement Formulas

Minerals as amino Weaning to 60 Pounds to 125 Poundsacid chelates 60 Pounds 125 Pounds to Market

Potassiuma 1.206 % 0.804 % 0.804 %Iron 0.205 % 0.120 % 0.120 %Zinc 0.213 % 0.134 % 0.134 %Magnesium 0.027 % 0.018 % 0.018 %Manganese 0.028 % 0.015 % 0.015 %Copper 0.021 % 0.014 % 0.014 %Cobalt 0.0012% 0.0005% 0.0005%

~Potassium was administered as an amino acid complex

The feed compositions used in this study are shownin Table 2. It should be noted that in each of thetreated feeds there was less total protein than in thecontrol feed. The purpose of this study was to measurethe effects of feed i ng the ami no ac id che1ates inalower protein feed.


I Table 2 II Confinement Feeding Formulas I

Control Treated

Birth to 60 lbs 125 lbs60 1bs to to

125 1bs Market

Chelated MineralSupplement(Table 1.) -- 90 1bs 60 1bs 60 1bsSoybean Mea1 300 lbs 300 lbs 200 lbs 100 1bsGrain 1700 lbs 1610 1bs 1740 lbs 1840 1bsTotal Protein

(calculated) 347 lbs 335 1bs 312 1bs 284 1bs

The effect that the amino acid chelates producedon the pigs receiving less protein in their feed issummarized in Table 3. The average daily gain, which inpart is a function of protein metabolism from dietaryprotein, was greater.

I Table 3 IProtein Sparing Effect of Amino Acid Che1ates

FinishedDays to Average Length Loin Percent

Treatment 220 Lbs Daily Gain Inches Eye Backfat Lean

(1 bs) (i n.) (sq. in.) (i n.) (%)Control Pasture 175 1.29 32.68 4.92 0.77 56.22Control Confinement 160 1.42 34.03 5.40 1.10 54.04Treated 153 1.51 32.40 4.95 1.05 53.80


While not all of the data were positive, theindications suggested that the amino acid chelatesenhanced protein utilization from the feed. Thatconcl us; on was conf; rmed ; n another test on 183 SPFFeeder pigs divided into two groups. The feed regimeswere the same as listed in Tables 1 and 2. The resultsare summarized in Table 4.(1)

I Table 4 II SPF Feeder Pig Farm Test I

Control Treated

Number of pigs started 30 153Starting weight/pig 94.1 1bs 91.6 1bsTotal feed consumed 9,251 1bs 44,390 1bsAverage feed/pig marketed 319 1bs 301.97lbsTotal pigs marketed 29 147Percent mortality 3.33 % 3.92 %Average market weight/pig 218.29 lbs 212.32 lbsAverage gain/pig 124.19 lbs 120.72 lbsAverage feed/pound of gain 2.569 lbs 2.501lbsAverage daily gain 1.678 lbs 1.472 1bsAverage days to market 74 days 82 daysFeed cost/pound of gain 18.08 ¢ 17.09 ¢

The protein sparing effect resulting from feedingthe amino acid chelates is evident. It was believedthat the equivalent growth rates of the pigs fed lessprotein compared to those fed higher protein levels wasdue to the mi nera1sin the che1ates caus i ng greateractivity in the multitude of enzymes related to proteinutilization that require these minerals. The greaterbio-availability of the amino acid chelated form ofthese minerals possibly allowed more of the minerals to


activate the proteinase enzymes as well as thoseenzymes, such as magnesium, that are involved inanabo1ism. (7) Thus, these che1ates become the mi nera1ssource of choice for a series of tests. Using severaldifferent species of animals, it was our desire toascertain how efficient protein metabolism was throughsupplementing the amino acid chelates. The tests wereconducted on chickens (broilers and layers) pigs, andcattle.

In the case of the broilers, 180 male birds weredivided into six groups of 30 each. Three differentdiets were studied: (1) Corn, soybean, and barley.Each diet had a control group of 30 birds and anexperi mental group of 30 bi rds. Each experi mental groupwas further subdivided into three groups of ten birdseach. Each experimental subgroup had the formula ofamino acid chelates shown in Table 5 included with thefeed at the rate of 50 ppm, 100 ppm, or 200 ppm per Kgof feed. The experiment was designed as shown in Table6.

I Table 5 IComposition of Amino Acid Chelate Supplements

% inMineral Mix % in Feed at Different

Formula Levels

50 ppm 100 ppm 200 ppm

Iron 9.00 % 0.0045 % 0.009 % 0.0180 %Copper 2.15 % 0.00107 %0.00215 % 0.0043 %Zinc 3.00 % 0.0015 %0.0030 % 0.0060 %Manganese 1.20 % 0.0006 % 0.0012 % 0.0024 %Cobalt 0.08 % 0.00004 % 0.00008 % 0.0002 %


I Table 6 II Experimental Design for Broilers I

NumberGroup Feed of Supplement

Birds 50 ppm 100 ppm 200 ppm

Control corn 30 -- -- --Experimental corn -- 10 10 10Control soybean 30 -- -- --Experimental soybean -- 10 10 10Control barley 30 -- -- --Experimental barley -- 10 10 10

All of the birds were housed in wire cages andconsumed both water and feed ad 1i bi tum. Pri or toreceiving the supplements each experimental and controlgroup received the same feed for a period of ten days.

All of the feeds contained Cr20J which was used asboth an indicator of the digestibility of the feed andas a proportionality constant to measure the amino acidcontent of the feces. The 1atter was measured by aBeckman automatic analyzer using acid-insoluble ashesand Cr20J as markers. Energy determinations wereaccomplished with a calorimetric bomb.

All of the broilers were sacrificed on the 60thday of treatment, and the intest ina1 content of theileocecal tract removed. Feces were collected in thismanner to avoid the presence of the urine which wouldhave added uric acid and urates to the feces and changedthe results.

In addition to fecal analysis, feed samples werea1so assayed to ascerta in the total energy and ami noacids potentially available to the birds after theadditions of the amino acid chelates. The differences


between the potential available amino acids and energyin the feed analysis and the amounts of each that wereactually used from the fecal analysis were determinedand the mean results obtained from calculations oflinear regressions summarized in Table 7. The data areexpressed as percent vari at ions of the experi mentalgroups compared to the control groups. The percentageincreases or decreases of metabolizable energy and thedigestibility of the amino acids in the feeds arepreceded by plus (+) for an increase and by minus (-)for a decrease as compared to the controls. An asterisk(*) indicates that the value is statisticallysignificant at the P<O.05 level, thus being differentfrom its respective control but not necessarilydifferent from the same constituent in the otherrations.


I Table 7 IIncreased Metabolizable Energy and Available Amino Acids fromBroiler Feeds Containing Amino Acid Chelates

Corn Diet % Soybean Diet % Barley Diet %

Metabolizable energy + 10.9 * + 7.1 * + 12.3 *Aspartic acid + 2.8 + 0.7 + 3.1 *Threonine + 1.7 + 1.2 + 4.3 *Serine + 1.3 + 0.4 + 2.6Glutamic acid + 8.8 * + 4.3 * + 9.8 *Proline + 7.1 * + 6.4 * + 8.1 *Alanine + 3.4 * + 1.1 + 2.5Valine + 3.1 + 4.3 * + 8.2 *Methionine + 3.7 * + 3.2 * + 7.6 *Isoleucine + 1.6 + 1.6 + 2.1Leucine + 10.4 * + 6.3 * + 12.7 *Tyrosine + 7.3 * + 3.9 * + 11.1 *Phenylalanine + 5.9 * + 2.5 + 2.8Lysine + 10.8 * + 7.7 * + 12.3 *Histidine + 3.9 * + 2.4 + 5.7 *Arginine + 2.3 + 0.8 + 3.4

I*P<O.05 I

As the data in Table 7 demonstrates, in everyinstance the presence of the amino acid chelates in thefeeds increased the metabolizable energy and theavailable amino acids over the same feeds without thechelates. The increases in utilizable energy were allstatistically significant. There were significantincreases in 58% of the amino acids measured.

In a second experiment 180 laying hens were used.The experimental design was identical to the design ofthe broiler experiment except the amounts of amino acidchelates fed to the three experimental subgroups were 75ppm, 150 ppm, and 225 ppm. The pre-supplementationconditioning period was ten days. The treatment periodlasted 50 days. All hens were sacrificed on the 51stday, and their feces and feed were assayed as describedabove. The differences between available energy and


amino acids from the feed and the actual amounts of eachused (fecal analysis) were determined and summarized inTabl e 8 as the mean 1inear regress ions (expressed aspercent variations of the experimental groups comparedto the control groups). Data marked with an asterisk(*) are statistically significant at the P<O.015 level.

I Table 8 IIncreased Metabolizable Energy and Available Amino Acids fromLayer Feeds Containing Amino Acid Chelates

Corn Diet % Soybean Diet % Barley Diet %

Metabolizable energy + 7.1 * + 5.3 * + 8.9 *Aspartic acid + 6.2 * + 2.2 * + 5.3 *Threonine + 3.1 + 1.1 + 3.7 *Serine + 4.7 * + 5.6 * + 7.4 *Glutamic acid + 5.5 * + 2.7 + 8.3 *Proline + 8.3 * + 4.9 * + 9.0 *Alanine + 3.8 * + 2.1 + 4.7 *Valine + 1.1 + 0.4 + 2.6Methionine + 5.3 * + 2.2 + 7.1 *Isoleucine + 6.4 * + 2.6 + 5.3 *Leucine + 8.5 * + 6.1 * +11.7 *Tyrosine + 3.8 + 1.6 + 4.2 *Phenylalanine + 4.7 * + 2.5 + 7.6 *Lysine + 8.3 * + 5.1 * + 8.9 *Histidine + 5.7 * + 4.8 * + 7.0 *Arginine + 2.8 - 0.6 - 3.1

I*P<O.015 I

The data in Table 8 demonstrate that when aminoacid chelates were included as part of the laying hens'feeds, the birds were able to obtain additional energyfrom the feeds. The increases were significant. Thepresence of the chelates in the feeds also made all ofthe amino acids in the feed except arginine, moreavailable to the chickens. Sixty-seven percent of thetime, the increase in amino acid availability wassignificant.


Having demonstrated that the amino acid chelatesimproved the nutritional value of feeds given tochickens by enhancing the availability of the aminoacids and energy contained in the poultry feed, we nextapplied this same concept with pigs. Two test periodswere studied. The first period used male, non-castratedpigs that were four weeks old and had just been weaned,and fo 11 owed them for 30 days. The second peri odstarted with feeder seven week old male non-castratedpigs that each had a beginning weight of approximately70 Kg. These were also followed for 30 days.

Thirty-six pigs raised in confinement were used ineach study. They were divided into two groups:experimental and control. The experimental groups werefurther subdivided into three groups of six pigs each.These subgroups were fed 50 ppm, 100 ppm, or 200 ppm ofamino acid chelates (Table 5) in their feed. Other thanthe inclusion of the amino acid chelates in the feeds ofthe experimental groups, the pigs received the same feedad libitum. The feed formulation without the amino acidchelates is shown in Table 9.


I Table 9 II Pig Feed Formula I

Period 1 Period 2

Corn flour 13 Kg 21 KgBarley flour 18 Kg 19 KgSoya flour (44 % protein) 15 Kg 13 KgFish meal 5 Kg 4 KgPowdered skim milk 9 Kg 7 KgCorn germ meal 4 Kg 4 KgWheat millings - 3 KgOat flour - 17 KgRice polishings 9 Kg 5 KgTorula yeast 1 Kg 1 KgSugar 2 Kg 2 KgCalcium carbonate 1 Kg 1.5 KgOicalcium phosphate 1.5 Kg -Sodium chloride 0.5 Kg 0.5 KgVitamin/mineral pack 1 Kg 1 Kg

Vitamin A 4,000,000 I.U.Vi tami n OJ 200,000 I.U.Vi tami n B. 250 mgVi tami n B2 1,000 mgVi tami n 86 200 mgVitamin 812 7 mgPyridoxine 5,000 mgVitamin K 250 mgD-Pantothenic acid 3,000 mgCholine chloride 100,000 mgDL-methionine 20,000 mgLysine 10,000 mgB.H.T. 1,000 mgCalcium 100 mgIron 30,000 mg

On the last day of each test period, the feces ofeach pig was collected for a 24 hour period. Thesesamp1es were measured by cal ori met ri c bomb for theenergy remaining in them and by a Beckman automaticanalyzer for the undigested protein. These data werecompared to similar assays of the feed samples toascertain the degrees of digestibil ity of the feeds withand without the presence of the amino acid chelates.


Tab1e 10 presents the summari zed mean resul tswhich were obtained from linear regression calculations.The data are expressed as plus (+) or minus (-) percentvariations of the experimental groups compared to thecontrols. Percentages followed by an asterisk (*) arestatistically significant at the P<O.05 level.

Table 10

Increased Metabolizable Energy and Available AminoAcids From Swine Feeds

Metabolizable energyAspartic acidThreonineSerineGlutamic acidProlineAlanineValineMethionineIsoleucineLeucineTyrosinePhenylalanineLysineHistidineArginine

I*P<O.05

First Period%

+ 8.4 *+ 3.8 *+ 0.6+ 5.7 *+ 3.2 *+ 8.9 *+ 0.8+ 4.5 *+ 7.7 *+ 0.2+ 8.9 *+ 5.9 *+ 5.3 *+ 8.8 *+ 4.5 *+ 0.3

Second Period%

+ 7.1 *+ 1.8 *+ 0.2+ 4.4 *+ 2.3+ 5.3 *+ 0.2+ 4.0 *+ 6.1 *

o+ 4.7 *+ 5.4 *+ 4.2 *+ 6.2 *+ 3.3- 0.4

The data in Table 10 demonstrate that when theamino acid chelates are included as part of the feed,there was greater utilization of both the energy and thenaturally occurring amino acids in the feed. Seventy-


three percent of the amino acids measured increasedsignificantly in the younger pigs whereas 60% of themincreased significantly in the older pigs. Thisenhanced utilization appears to be a function of the ageof the pigs. As they become older, the availability ofthe energy and amino acids tends to diminish somewhat.

This same trend of reduced utilization ofnutrients as a function of age also appeared in growingcalves. In this last series of experiments two groupsof calves were used. The first group consisted of 36bull Piedmont calves that were 15 days old at thebeginning of the study. They were divided into twogroups of 18 calves each, experimental and control. Theexperimental group received a different levels of theamino acid chelates, which are shown in Table 5, at therates of 150 ppm, 200 ppm, or 250 ppm mixed inreconstituted milk formula. This is shown in Table 11.


I Table 11 IReconstituted Milk Formula for Calves

Control Experimental

Powdered skim milk 70.0 % 70.0 %Fat (tallow 28%, lard 25%,palm oi 1 25%, coconut oi 1 25%) 18.0 % 18.0 %

Dextrose 6.0 % 6.0 %Corn starch 3.0 % 3.0 %Lecithin 1.0 % 1.0 %Dicalcium phosphate .4 % .4 %Sodium chloride .3 % .3 %Esterase sugar .285 % .285 %Vitamin pack 1.0 % 1.0 %

Vitamin A 2,700,000 I.U.Vi tami n D3 700,000 I.U.Vitamin E 2,500 mgVi tam; n 8. 600 mgVi tami n 82 500 mgVi tami n 86 200 mgVi tam"j n 812 5 mgVitamin K 2,500 mgPyridoxine 5,000 mgdl-methionine 30,000 mgLysine 20,000 mg

Amino Acid Chelate Package (Table 5) 150 ppm200 ppm250 ppm

In addition to milk, the calves were given corn,wheat, and hay as they matured sufficiently to consumethem. The experiment lasted ninety days which includeda ten-day stabilization period at the beginning of theexperiment during which time no amino acid chelates werefed.

The second group of cal ves cons i sted of fortybulls of the same breed as above. They were ten weeksold at the commencement of the study which continued foreighty days which included a ten day stabilizationperiod during which time no amino acid chelates werefed.


The calves were divided into two groups:experimental (30 calves) and control (10 calves). Theexperimental group was subdivided into three groups often animals each and fed the amino acid chelatesupplement shown in Table 5 at the rates of 100 ppm, 150ppm or 200 ppm. The chelates were blended into the dryfeed, the formulation of which is shown in Table 12.The above feed formula was blended so that in every 102Kg of feed there were 40 Kg of corn silage, 40 Kg ofalfalfa hay, 20 Kg of barley, and 2 Kg of the vitaminpack.

I Table 12 IFeed Formula for Ten Week Old Calves (excluding amino acid chelates)

40 Kg 40 Kg 20 KgCorn Silage Alfalfa Hay Barley

Moisture 57.96 % 11.76 % 12.0 %Crude protein 3.41 % 8.78 % 33.0 %Crude fat 1.41 % 2.52 % 1.0 %Crude fiber 13.17 % 30.24 % 7.0 %Ash 2.06 % 8.19 % 33.0 %Non-nitrogen extract 22.26 % 38.51 % 26.0 %Vitamin Pack

Vitamin A 600,000 I.U.Vi tami n DJ 6,000 I.U.Vitamin E 20 mgCholine 1,000 mgCobalt 2.5 mgIron 100 mgIodine 7.5 mgManganese 150 mgCopper 25 mgZinc 275 mgBHT 100 mg

On the last day of the eighty days of the testperiod, 24 hour samples of feces were taken from everycal f and assayed for the energy and the am; no ac idsstill remaining. The results were compared with feed


analyses according to the analytical procedures whichhave been previously described.

A summary of the mean results after linearregress i on cal cul at ions for the two groups of cal ves arepresented in Tabl e 13. The di fferences between theexperimental groups and controls are indicated by plus(t) or minus (-). Data that are statisticallysignificant at the P<0.05 level are indicated by anasterisk (*).

I Table 13 IIncreased Metabolizable Energy and Available AminoAcids From Cattle Feeds

New-born Calves Older Calves% %

Metabolizable energy + 7.8 * + 5.1 *Aspartic acid + 1.4 + 0.5Threonine + 3.6 * + 2.1Serine + 7.5 * + 3.9 *Glutamic acid + 4.9 * .0Proline + 0.7 - 0.5Alanine + 4.7 * + 4.4 *Valine 0 - 0.2Methionine + 5.0 * + 3.9 *Isoleucine + 0.3 + 1.5Leucine + 2.0 - 0.2Tyrosine + 5.1 * + 3.7 *Phenylalanine + 1.8 - 0.2Lysine + 6.9 * + 3.4 *Histidine - 1.7 + 0.6Arginine + 0.5 0

I*P<O.05 I


As seen in Table 13, when amino acid chelates wereincluded in bovine feed, the utilization of availableenergy and amino acids from those feeds was improved.Like the pigs, however, the older animals were not asefficient as the younger ones in digestion. Thedifferences in the two groups of calves may be due totwo digestive models. The younger calves aremonogastric, whereas in the older calves, the rumen hasdeveloped, and they have become polygastric.

Rumen material was removed from all of the olderca1ves and compared. An increase in the amount ofpropionic acid with corresponding decreases in theamount of methane ammonia in the experimental groups wasfound. The increase of propionic acid and decrease ofmethane result in greater obtainable energy from theration. The decrease in ammonia has a protein sparingeffect which produces greater utilization of the aminoacids in the feed for the production of meat.

Having seen the effects of the amino acid chelateson improving digestibility of animal feeds, andtheori zi ng that thi s was probably due to increasedbioavailability of these crelates to the mineraldependant digestive enzymes(2, ,4), we also wanted to seeif there was an increased utilization of the amino acidsat the cellular level.

Eight male Westar-Hagemann white rats weighingapproximately 100 g each were used in this study. Allwere fed the same commercial rat chow for ten days tostabilize them. They were then divided into four groupsof two rats each. One group was the cont ro1. Theremaining groups, labelled experimental, received 50ppm, 100 ppm, or 150 ppm of the amino acid chelatesshown in Table 5, mixed with their feed. These chelateswere fed to the three experimental groups for ten days.The control group received the same feed as theexperimental groups minus the amino acid chelates for


the same ten day peri od.provided ad libitum.

All feed and water was

At the end of ten days all of the rats weresacrificed. Their duodenums and livers were removed.The duodenums were cut longitudinally and washed withdistilled water. The mucus membrane was then scrapedoff and put in containers which each contained 4 ml ofdeionized/distilled water. One microcurie (1 microcurieequal sa. 1 ml) of (14)C was added to the membrane-watermixture which was then homogenized for 20 minutes. Thecontents were refrigerated at O°C for the incubationperi od. (5,6) The 1i vers of the rats were treated inasimilar fashion. They were homogenized for 20 minutesafter (14)C was added. They were then incubated. (5.6)

Protei n synthes; s was measured as the rate ofincorporat i on of the (14)C into the ami no ac idin thetissues. The labelled amino acids were then determinedby scintillation counting. The data, which areexpressed as corrected counts per minute are shown inTable 14.

I Table 14 IProtein Synthesis in the Liver and Duodenum


Levels ofAmino Acid

Chelates

500 ppm 100 ppm 150 ppm

Liver 765±51 923±103 962±89 lO38±94

Duodenum 264±44 316±65 385±61 409±75


The data in Tabl e 14 demonstrate that protei nsynthesis in the liver increased over the control 20.7% (at 50 ppm), 25.8 % (at 100 ppm), or 35.7% (at 150ppm) depending on the quantity of amino acid chelatesincluded in the feed. Protein synthesis in the duodenumincreased over the control 19.7 % (at 50 ppm), 45.8 %(at 100 ppm) or 54.9 % (at 150 ppm) according to theamount of amino acid chelate supplemented. It appearsthat there was more protein synthesis at the duodenumwhere there probably was a greater concentration of theamino acid chelates than at the liver.

All of the above studies tended to confirm thevalue of including amino acid chelates in the feeds ofanimals and birds. The data demonstrated that when theamino acid chelates are included, the animal or bird isable to extract more energy and amino acids from thefeed. Furthermore, th i s increased ami no ac idavailability results in increased deposition of aminoacids in the soft tissues of the body.

The earlier cited data indicated that when aminoacid chelates were included as part of a swine feedformulation the average daily gain was between 6.3 and17.1 % better. Our data indicate that this gain is dueto the increased availability of supplemental andnatural amino acids in the feed and their subsequentincorporation into the tissues of the animals.


References

1. Albion Laboratories, "Pigs for Profit," A salesbrochure, 1983.

2. Schutte, K., The Biology of the Trace Elements(Philadelphia: J.B. Lippincott Co.) 17-23, 1964.

3. Ashmead, H.D., et ~., Intestinal Absorption ofMetal Ions and Chelates (Springfield: Charles CThomas) 1985.

4. Trier, J. and Madara, J., "Functional Morphologyof the Mucosa of the Small Intestine" in Johnson,L., et ~., Physiology of the GastrointestinalTract (New York: Raven Press) V 2, 925, 1981.

5. Appel, G., Uber die Proteinsynthesis der Leberund S. Kelettmuskwlatur bei Ratte und Hahn, Ph.D.Thesis, Hannover University, 1973.

6. Dey-Hazra, R., et ~., "Protein synthesis changesin the liver of piglets infused withStrongyloides ramsomi," Vet. Parasitol., 5:339,1979.

7. Guthrie, H., IntroductorY Nutrition (St. Louis:Times Mirror/Mosby College Publishing) 188, 1986.

Chapter 6

AN ASSESSMENT OF LONG TERM FEEDINGOF AMINO ACID CHELATES

Robert B. JeppsenAlbion Laboratories, Inc.

The practice of using chelated minerals tofacilitate the intestinal absorption of metals requiredby animals has been relatively recent. Chelates werefirst used in 1920,(1) although it was 1952 before thisclass of metals was proposed as a technique to increasemetal uptake.(2) Although chelates were first proposedfor use in plant nutrition, it was soon determined thatanimals similarly benefitted from better mineralabsorpt ion i f the mi nera1sin the i r feed were a1sochelated.(3,4) Prior to the introduction of amino acidchelates by Albion Laboratories, the major ligand usedin animal nutrition was ethylenediamine tetracetic acid(EDTA). While EDTA was a good chelating agent, its highstabi 1i ty constant precl uded its useful ness inmostspecies of animals, because the intact chelated mineralswere absorbed ~nd then excreted back into the colon orurine unused.() The use of amino acids as chelating1igands overcame the problem of stabil ity constantsincreased in mineral utilization as well asabsorpt ion. (6)

This increased mineral uptake created concern forsafety when the amino acid chelates were ingested. Overthe course of the past twenty years these amino acidchelates have been tested for structural validity,effi cacy, and safety both to handl ers and the targetspecies receiving the nutritive benefits of theproducts. (7,8) The present long term study of theeffects of amino acid chelates on breeder sowsrepresents a further validation of their historicalperformances.

106

An Assessment of Long Term Feeding of Amino Acid Chelates 107

the same breed (York Landrace) and were raised inconfined housing on similar farms in the samegeographical location. The test sows were the productsof multiple generations having their diets continuouslysupp1emented wi th ami no ac id che1ates of iron,magnesium, zinc, manganese, copper and cobalt (Table 1).They also received potassium as an amino acid complex.All of these products were manufactured by AlbionLaboratories, Inc.

Both groups of pigs were routinely inoculatedagainst erysipelas, leptospirosis, Parvo virus, andEscherichia coli. Pregnant sows were vaccinated twice,once at six weeks and aga in at three weeks beforefarrowing. All pigs were dewormed and treated for lice.

Health and breeding statistics between the farmscontaining the control and test pigs were similar. Thevarious feeds provided the two groups of pigs throughoutthe course of their lives were also similar except thatthe test pigs were fed the amino acid chelates as notedabove.(9) The Quantities of amino acid chelates providedto each test sow are shown in Table 1. These amino acidchelates were provided continuously in weaner, grower,nursing sow, and dry sow pig feeds given the test pigsover four generations.

In order to better assess the possible internal orcellular effects of the amino acid chelates, six breedersows with various histories of parentage on the chelatesand complimentary individual doses were selected for indepth analysis. Two concerns in these test sows wereassessed: (1) the long term effects of continuouslyfeeding amino acid chelates on single individuals, and(2) cumulative effects of amino acid chelates fromgeneration to generation (teratogenic effects). Threeof the test sows represented the fourth filialgeneration to continuously receive the amino acidchelates. Of the remainder, one was of the third andtwo were of the second filial generations. Since the


test animals of the fourth generation were much youngerthan those of the second or third generations, the totalamount of amino acid chelates ingested by those of thefourth generation was much less than the others. Thiswas especially true for two of these animals, which werenot old enough to have had more than one and two littersrespectively. Since the nursing sow ration containedmore of the amino acid chelates than the other rations,the pigs which had fewer litters ingested considerablyless of the chelates.

I Table 1 ITotal Amounts of Minerals as Amino Acid Chelates Fed to TestSows(~)

Fi 1ialGeneration Iron Magnesium Zinc Manganese Copper Cobalt Potassiumb

3rd 734 599 367 147 18 6 5994th 556 451 278 111 14 4 11512nd 580 409 290 116 15 5 4092nd 687 537 344 138 17 6 5374th 193 154 96 39 5 2 1544th 131 88 65 26 3 1 88

~All numbers represent the number of grams of metal received from birthto death.

bPotassium was provided as an amino acid complex.

All of the animals in the control group and thetest group were sacri fi ced on the same day. Thei rtissues, shown in Table 2, were excised forhistopathological evaluation. A blood sample was alsotaken at the time of sacrifice. The blood from each sowwas placed in individual lined vacutainers containingEDTA for the purpose of blood cell analysis.


I Table 2 ITissues Excised for Histopathological Examination

Brain Muscle Lymph Node (mesenteric)Duodenum Heart KidneyJejunum Liver OvaryIleum SpleenLarge Bowel Bone Marrow

At the t; me the an; rna1s were de1i vered to theslaughter house for sacrifice, a premortum examinationwas conducted by a veterinarian. He did not know whichgroup was the test group and whi ch was the control.Following euthanization by cerebral electrocution, apostmortum examination by the same veterinarian was alsoconducted. (10)

Four samples of each tissue shown in Table 2 wereexcised from each animal and immediately submerged insample bottles containing 10% formalin (pH 7). Thesamples were subsequently delivered to a pathologicallaboratory. There they were dried, embedded inparaffin, sectioned, and mounted on slides. A total of308 specimens were mounted for examination. Thepathologist who read the slides was unaware of whichpigs were in the control group and which were in thetest group. (11)

As noted above, a premortum examination wascarried out to determine the condition of each animal.All but three of the animals were judged to be in "goodcondition." Two of the three which did not receive the"good condition," rating came from the control group andone from the test group. In spite of this, the


histopathological findings were essentially the same forall of the animals.(lO)

The postmortum investigation included anexamination of each animal's carcass. The viscera werealso individually examined. On the whole, there werefew abnormalities. One control sow had polycysticovari es and another suffered from purul ent metri tis.One test sow had a spl eni c scar as a resul t of aprevious splenic injury, and another had a hepaticcyst.(10) All of the abnormalities found by theveterinarian in this examination were later assessed bythe hi stopathol ogi st and judged to represent normallesions which would tend to occur in any group ofpigs . (11)

Blood sampl es were assayed by a Canadi angovernment 1aboratory. (12) Most of the val ues for redblood cell count, white blood cell count, hemoglobincontent, packed cell volume, mean red cell volume,protein, immature neutrophils, mature neutrophils,monocytes, lymphocytes, eosinophils and basophils werewi th in the normal range. Of the ones wh i ch wereaberrant, equal numbers came from both the controls andtest animals within the same parameter. Thus, noapparent differences existed between the two groups asmeasured on the basis of blood cell analysis.(lo.12)

The data generated from the detailedhistopathological examination are easily summarized:there were no histopathological tissue alterationsobserved that could be attributed to minerals or aminoacid chelates. Furthermore, there were no tissuefindings which could distinguish the test group from thecontrol group. Both groups were histologically thesarne. (11)

Care was taken to provide sows in the controlgroup that were similar to those in the test group.Identical and objective measures for both test animals

An Assessment of Long Term Feeding of Amino Acid Chelates III

and controls were conducted. Specialists commissionedto apply their knowledge and skills to the assessmentswere kept blind as to which pigs belonged to whichgroup. The amino acid chelates listed in Table 1 hadbeen administered as a dietary supplement to pigs on afarm over the course of severa1 years, and the on1yeffects attr i t'utabl e to thei r use were improvements(such as, no ~eed for iron dextran injections in newbornpiglets, increase piglet weight at weaning, lowermorta1i ty, and higher product i on outputs). Severa1generations had been sired by ancestry which hadreceived these amino acid chelates continuously, and noapparent teratogenic effects could be attributed to themby gross observation. The resulting impartialconclusions were definitive: there were nohistopathological tissue alterations which could beattributed to the amino acid chelates; nor were thereany significant gross or microscopic differences betweenthe test and control groups of sows.

Thi s long-term assessment has shown that ami noacid chelates are safe for continual feeding asnutritive dietary supplements when applied under theconstraints of recognized and recommended dosages fornutritive minerals. This study indicates that they arefree of teratogenic effects and chronic morbidity at thehistological level, as well as at the level of practicalobservation.


References

1. Oddo, E. and Pollacci, G., "Influenze del nucleopirrolico sulla formazione della chlorofilla,"Gaz. Chim. Italiana, 50:54, 1920.

2. Jacobson, L., "Maintenance of iron supply innutrient solutions by a single addition of ferricpotassium ethylenediamine tetracetate, " PlantPhys i 01 ., 24: 411, 1951.

3. Dyer, I., "Mineral requirements," in Hiefez, E.and Dyer, I., eds., Animal Growth and Nutrition(Philadelphia: Lea & Febiger) 312, 1969.

4. Guthrie, H., Introductory Nutrition (St. Louis:Times Mirror/Mosby College Publishing) 250, 1986.

5. Miller, R., "Chelating agents in poultrynutrition," Proc. Del Marva Nutr. Short Course,1967.

6. Ashmead, H., et ~., Chelated Minerals in Plants,Animals, and Man (Springfield: Charles C Thomas)1982.

7. Larson, A., "L.D. 50 Studies with Chel atedMinerals," in Ashmead, D., ed., Chelated Mineralsin Plants, Animals, and Man (Springfield:Charles C Thomas) 163, 1982.

8. Ashmead, H. D., et ~., Intestinal Absorption ofMetal Ions and Chelates (Springfield: Charles CThomas) 1985.

9. Fisher, B., "Analysis of feed samples by MillerLaboratories," Private communication, August,1987.


10. Hurnik, D., "Premortum and postmortum examinationand blood cell reports of sows receiving long-term supplements of dietary mineralMetalosates®," August, 1987.

11. Kitchen, D., "Histopathologic examination oftissues and organs from sows on a nutri t i analstudy utilizing mineral Metalosates®," August,1987.

12. Veterinary Laboratory Services, Ontario Ministryof Agriculture and Food, August, 1987.

Section 2. CATTLE

115

Chapter 7

THE USE OF AMINO ACID CHELATES TOENHANCE THE IMMUNE SYSTEM

Robert T. Coffey

Whenever an animal is exposed to an antigen, itsbody must recognize the antigen as a foreign substance.At the same time, its immune system must be able torecognize the animal's own cells as not being foreignand then mount a selective immune response against theantigen invading substance while still being tolerant tothe animal's own cells. If this does not occur becausethe immune system fails to develop or is destroyed, thenthe animal will rapidly succumb to the disease.

Figure 1 demonstrates the essential features ofthe optimal immune system. It has four components.First, there is a method of identifying and processingthe ant igen. Second, through the use of ant igensensitive cells, a reaction specific to the antigen isinduced. Third, cells produce antibodies or participatein a cell mediated immune response. Lastly, the cellsretain a memory of the event and therefore reactspecifically and more quickly to that type of antigen infuture encounters. (1)

117


FOREIGN MATERIAL..Recognition

and processing

Ceil-mediatedimmunity Antigen-sensitive

cell~ I Tolerance 1-48

Figure 1. A simple flow diagram showing theessential features of the immune responses.

In spite of the idealistic concept presented inFigure 1, the animal does not always respond in apositive manner. These limitations can be divided intophysiochemical limitations and the pro8lems associatedwith nature of the foreign material. 3 Sometimes aphysiochemical 1imitation can cause an inhibition ofantibody production. On other occasions, with certainantigens, the immune system is unable to differentiatebetween the antigens and normal cells. When this occursthe disease continues ifs course in the animal in spiteof vaccinations, etc. (3

Frequently it is not a simple process to identifyan antigen of the causitive agent in a disease eventhough the outbreak of the disease suggests a specificcause. Nevertheless, the conditions allowing the agent

The Use of Amino Acid Chelates to Enhance the 119Immune System

to become established may be varied and seeminglyunrelated to the disease. In fact, these conditions mayoverrun the natural immune system so the animal isincapable of a positive response to exposure to aspecific antigen.

The incidence and types of diseases in cattleoften have several predisposing factors involved. Forexample, the stress of the sale barn, transportation, anew environment, a change in feed, etc., all seem toencourage respiratory infections; while cold, dampweather, lack of colostrum, etc., predispose calves toscours and pneumonia. Because of the complexity of thedisease factors, many producers experience frustrationwhen all efforts to prevent a specific disease includingvaccinations, environmental changes, etc., seem to be invain.

To illustrate, a few years ago a herd of calveswas purchased in California. They were vaccinated forshipping fever and then transported by truck to Idaho.Several of the calves died, primarily from pneumonia,immediately following transportation to Idaho. A postmortem revealed not only the respiratory infection butalso an extremely high level of lead in their bodies.The lead was acquired via a lead polluted environment air as well as feed and water - in the specific area ofCal i forn i a where they were fi rst ra i sed. When 1eadlevels are relatively high, this pollutant willinterfere with body metabolism of both copper and zinc.The latter two are essential enzyme co-factors in theimmune system. (4,5) Thus, the cal ves immune systems wereunable to respond to the organisms responsible for therespiratory infections. The incidence of lead thuscompromised the calves' natural immune processsufficiently to allow the infection to becomeestablished in spite of vaccinations being administeredagainst them. (5)


The preceding example raises some question on theimportance of si ngl e disease ent i ty di agnos is. Thetypes of organisms isolated from a culture provides theveterinarian with some useful information whichfrequently allows for effective treatment or prevention,but on many occasions there are several pathogenspresent in a problem herd. The opposite extreme canalso occur. A pathogen may never be isolated, and yetthe clinical symptoms lead the veterinarian to believehe is dealing with shipping fever, viral scours,sal mone 11 as is, or some other disease. Based on thatbelief he commences treatment and vaccination programswhich ultimately do not prove effective. Sometimes thedeficiencies of treatment can be isolated to theproperties of antigens listed in Table 1. Other timesthe problems are of an entirely different cause.

I Table 1 I

I Essential Features of Antigenicity IFeature Property

Size Larger is better.Diversity Simple polymers may be poor antigens.Stability Structural stability is mandatory.Degradability Very sensitive molecules are poor antigens.

Totally inert molecules are poor antigens.Foreign The more foreign the better.

For these reasons, it is essential for theveterinarian not only to isolate the disease causingantigens, but also to evaluate the immune system of theanimals with which he is working since strength in thatsystem is paramount to disease resistance. Inevaluating the possibility of immune incompetence ormalfunctioning in cattle, there are severalcharacteristics one or more of which are consistentlyassociated with the herd. These include:


(1) In high producing cattle with rapidly growingcalves, there is a greater incidence of diseasemorbidity and/or mortality which isstatistically significant.

(2) The time of year when the disease outbreakoccurs is usually the same.

(3) The herd response to vaccination and/ortreatment programs is less than anticipated,and occasionally, even detrimental.

(4) Based on symptoms, a clinical diagnosis is madealthough no antigen can be isolated.

(5) The illness occurs after feeding the herdensiled roughage, especially corn silage.

(6) Associated with number 5, above, a respiratorydisease may occur 30 to 120 days after beingfed the corn silage.

(7) The cattle experience a "vaccination break"which occurs after the use of a modified livevirus vaccine.

(8) The animals seem to crave salt and/or minerals.They have rough hair coats and are unable tomaintain weight gains. Their average dailygains are not what they should be based on thefeed ration.

As stated above, not all of these broad factors areusua11 y present at one time. When anyone' of theseconditions exists, however, and the veterinarian notesa poor response to convent iona1 treatment and prevent ionprograms, it is imperative that he evaluate the immunesystem of the cattle he is dealing with. Immunity is


influenced by genetics, the environment, and nutrition.Alarge amount of the clinical data which our laboratoryhas accumulated and which is associated with immunesystem breakdown, demonstrates that there aresignificant relationships between trace element levelsin the body and immunoglobulin production.(8) Byreviewing the eight factors listed above, it is obviousthat each one can contribute to or result from a tracemineral deficiency. For example, high producing orrapidly growing cattle require large amounts of mineralsjust to maintain that production or growth. On thesecond item, at certain times during the year, forageshave a lower mineral content or high mineral antagonistssuch as nitrates, heavy metals, or sulfates, than atother times of the year. On the third characteristic,a mineral deficient herd is handicapped in developing animmune system regardless of the vaccination program. Inthe fourth case, the symptoms resulting from amicroelement deficiency may mimic an infectious disease.In the fifth and sixth cases, the corn silage may behigh in certain minerals, such as aluminum or iron. Inexcess, these minerals are nutritionally andphysiologically antagonistic to the minerals essentialfor the development of the natural immune system. Thisantagonism may create an artificial deficiency whensupplemental mineral nutrition is inadequate. Finally,the craving of salt/minerals, rough hair, and poorweight gains may all be indications of poor mineralnutrition which may be contributing to a faulty immunesystem. When trace element metabolism is out of balanceor deficient, there may be immune incompetence orrna1funct i on . (9.10.11)

Immunologic competence is the ability to developimmunity when appropriately stimulated by an antigen.As summarized in Figure 1, in a normal animal afterentry of antigens into the body there can be recognitionof that antigen by the immune system. This recognition(humoral) stimulates the synthesis of specificcirculatory antibodies to each antigen. The antibodies


are protein molecules produced by plasma cells as aresult of an interaction between antigen-sensitive Blymphocytes and the specific antigen. This interactionproduces what are known as immunogl obul ins. (12) Theimmunoglobulins are classified as IgG, IgM, IgD, IgA,and IgE.(13'14) Table 2 summarizes the basiccharacteristics of the immunoglobul ins. (lS) Cattle are1imited to IgG, IgM, IgE, and IgA. (17)

I Table 2 IBasic Characteristics of the Major Immunoglobulin Isotypes in theDomestic Animals

Property IgM Ig6 IgA IgE IgD

Most usual 19S 7S lIS 8S 7Ssedimentationcoefficient

Molecular weight 900,000 180,000 360,000 200,000 180,000(daltons)

Electrophoretic f3 r f3-r f3-r rmobility

Heavy chain jj r • e 3antigen

Prominant organ Spleen and Intestinal Intestinal Spleenof synthesis lymph Spleen and and and and

nodes lymph respiratory respiratory lymphnodes tracts tracts nodes

The IgG (which can be further divided into foursubclasses in some animal species) and IgM classestogether make up approximately 80% of all of theimmunoglobul ins which are produced by the body. (14) Theproduct i on of these immunogl obul ins is controll ed byenzyme systems which have specific trace minerals aspart of the act i ve enzyme. (7,13,16)

Upon initial antigen stimulation, normally IgM isproduced. As a membrane bound molecule on the


1ymphocytes, (14) it serves as the fi rst 1i ne of defenseagainst pathogens by mediating the response of the Blymphocytes to the antigen stimulation.(14.11) Because oftheir large size they are confined primarily to theblood. Their presence in the blood probably contributesto their service in the first line of defense.

The secondary immune response comes from the IgGclass of antibodies and constitutes about 70% of thetotal immunoglobulins produced. It, too, resides inhigh quantities in the blood, but because of its smallermolecular size, it more easily escapes the blood streamand can impart a defense to the spaces between thetissue cells as well as on body surfaces. The branchingof IgG from the blood stream occurs only when a specificamount has accumulated.(l8) As IgM levels decline, theIgG increases upon secondary stimulation. A secondexposure to the same antigen results in a secondary, oranamnestic response in which IgM levels, which were oncedeclining, increase, as does the IgG immunoglobulin, asseen in Figure 2. (19,20)


19M

IgG

0L-.---.L..L..- ............-_~--..I

o 7 14 21 28 35 42

DAYS

100 .

1000 .

100000'-- ----.1. --,

W PRIMARY SECONDARYcr: 10000' .~ RESPONSE RESPONSE}-

>oocoi=z4:(!Jo-J

PRIMARY ANTIGEN CHALLENGE

Figure 2. Primary and secondary antibody responses.In a typical immune response the antibody levelfollowing secondary antigenic challenge: 1. appearsmore quickly and persists longer, 2. attains ahigher titre, 3. consists predominantly of IgG, asopposed to the antibody response following a primaryantigenic challenge. In the primary response theappearance of IgG is preceded by IgM.

IgD, representing less than 1% of the serumimmunoglobulin, is located on the cell surfaces of Blymphocytes. It is believed that IgO acts s;inilarly toIgM. It, has not been isolated in cattle, as seen inTable 3. 14)


I Table 3 IImmunoglobulin Isotypes and Subisotypes of Domestic Animals and Man

Immunoglobulin Isotypes

Species IgG IgA IgM IgE IgD

Horse Ga,Gb,Gc,G(B),G(T)a,G(T)b A M E ?Cattle Gl,G2(G2a,G2b7) A M E 7Sheep Gl(Gla?),G2,(G3?) Al,A2 M E ?Pig Gl,G2,G3,G4 AI,A2 M E DDog Gl,G2a,G2b,G2c A M E ?Cat Gl,G2 A M E ?Chicken Gl,(G2,G37) A M 7 DHuman Gl,G2,G3,G4 Al,A2 M E 0

IgA is known as a secretory antibody (sIgA)because it is found in the seromucous secretions, suchas sal iva, tracheobronchial secretions, colostrum, mil k,and genitourinary secretions. It forms about 15% to 20of the total serum immunogl obul in. (14)

IgE is only found in traces, but it is boundthrough specific receptors on the cell surface of mastcells and basophils. It is involved in protectingagainst atopic allergies and helminthic parasites.(14)

As noted above, several trace minerals - notablycopper, zinc, and manganese - can affect immunoglobulinenzyme act i vi ty. (7,13,16,22,23) Inadequate intake or use ofthese minerals can affect both T-cell and B-cellresponse. Excessive metal intake of such elements aslead, iron or aluminum can interfere with copper, zinc,and manganese metabolism and change T-cells fromantibody-producing to antibody-suppressing cells. (9,12,23)When th i s occurs the immune response of the ant igenstimulated animal is not at all what would normally beanticipated. The following case history vividlydemonstrates this.


A Limousin cow-calf herd which our laboratory(27)had been studying had annual calf losses ranging between12% and 20% over a two year period. With the increasingannual losses, there were always higher losses with thespring calves than with calves born in the fall. Mostillness resulting in mortality occurred between threeand ten weeks of age and were both respi ratory andenteric in nature. This disease pattern was puzzling.

Since most calves are born with a gammaglobulinemia, the colostral transfer of maternal IgG across thecalves' intestinal epitheliums, which takes place duringthe first 24 hours after birth, generally providesadequate passive immunity until such time as they canbuild their own immune systems. As calves mature andcolostral immunity diminishes, their own immune systemsshould respond to antigen stimuli. Table 4 summarizeswhat should occur in normal calves and are based on theuse of radioimmunodiffusion plates from over 2,000 serumsamp1es. (24)

I Table 4 I

Bovine Immunoglobulin Levels Based on Age

mg/ml ± range

Age of calf IgG1 IgG2 IgM

Newborn 24 - 50 .2 - .8 1 - 51-2 weeks 15 - 35 .2 - .6 .6 - 1.01 month 15 - 29 .1 - 1.5 .4 - .82 months 8 - 14 .1 - 2.0 .7 - 1.73 months 10 - 16 .1 - 2.1 1.8 - 3.26 months to adult 6 - 14 .4 - 2.1 1.7 - 2.9


In the fall of the first year of the study withthe Limousin cow-calf herd, a preventative vaccinationprogram was estab1i shed in hopes of reduc i ng spri ngcalving mortality. The cows were vaccinated withcolostridrium bacterin/toxoids, rota-coronavirusvaccine, killed IBR-PI 3 -BVD vaccine, and .L.. colibacteri n. All of the cows were pregnancy tested,coordinated with their AI breeding dates or pasturebreeding dates, and schedules established to give thema second dose of the applicable vaccines and bacterinsprior to calving.

In the spring, when the calves were born, theywere given oral rota-coronavirus vaccine and anintranasal IBR-PI 3 vaccine. The effectiveness ofattempting to immunize neonatal calves has beenquestioned based on the ability of their immune systemsto respond adequately, as noted above. There is alsothe possibility of interference to antibody productiondue to the passive immunity acquired from the colostrum.Nevertheless, utilization of oral rota-coronavirus orintranasal IBR-PI 3 at birth, or shortly thereafter, hasbeen shown in some cases to stimulate localized immunityagainst these viruses. For this reason they were usedin the calves.

During this spring calving season, if a specificcalf was not nursing within one to two hours, the cowwas milked out by hand, and her calf given one to twoquarts (approximately 2 liters) of colostrum. Thisprocedure was repeated in six to eight hours, ifnecessary.

The spring in which the study commenced was coldand rainy. Consequently, the cows and their calves werehoused in barns, which were maintained at above normalcleanliness during the inclement weather. Managementpract ices were deemed to be exce11 ent. When weatherpermitted, both the cows and calves were allowed tograze in a rye grass pasture.


By the latter part of April, many of the oldercalves had developed pneumonia. Their response to theconventional treatments was fair. For the next two tothree weeks, recurrent and new cases of pneumonia andscours deve loped. Then, the cal ves started dyi ng.Post-mortem findings of the first three animals includedenteritis, FA positive for coronavirus, and pneumonia.

The ent ire herd of 60 cal ves was treated wi thinjectable antibiotics on three different occasions.The pneumonia and scours would subside and then flare upagain. Post-mortems of additional deceased animalsrevealed necrotic enteritis, possible but unisolatedsalmonellosis, pasteurella pneumonia, hemophilus somnusinfection, mucoid ~ coli isolate, coccidia, andcoronavirus. By the end of May, 14% of the calf herdhad died in spite of all efforts that had been taken toprevent mortality.

Because of the lack of consistent and predictableresponse, it was believed that an immunologic disordermay have been the cause of the problems. At the end ofMay the 58 surviving calves were bled and tested forspecific levels of IgM utilizing radio-immunodiffusionkits. (24) Cal ves under four weeks of age were tested foradequate passive transfer of colostral immunoglobulins(lgG) using a sodium sulfite precipitation test.(24)Table 5 summarizes the conditions found in the calves.


I Table 5 IImmunologic Disorders in Spring Calves

Total spring calves born 66Total mortality (22 July) 11 (16.7%)Total abnormally sick calves 22 (33.3%)Total calves treated individually 34 (51.5%)Total tested immunoglobulins 58Total IgM deficient calves 39 (67.2%)Number of IgM deficient calves treated 31 (79.5%)

From these findings, it became apparent that themajority of the animals tested were not developingadequate immune responses . Real i zing that age i s afactor in developing an immune system in a young animal,it was decided to retest at a later date before takingany positive action.

Thirty-six days later, the test described abovewas repeated in hopes that the greater maturity of thecalves would result in a normal IgM.(25) Unfortunately,such was not the case. The IgM of one of the calveswhich was previously judged to be normal had dropped toan abnormal level, and seven other calves dropped fromabnormally low to critically low levels. One animal,which was originally normal, remained so, and threeothers, which had low IgM previously, rose to a normallevel. It was noted that those calves which showed adecrease in IgM over the 36 day testing period were alsoin poorer health than the rest of the herd.

In measuring the IgG immunoglobul ins and comparingtheir levels to normal levels, it was found that they,as an aggregate, were abnormally high. Typically theantibodies produced as a secondary response to a T-cell


dependant antigen have a higher average affinity thanthose produced in the primary response as was noted inFigure 2. When this occurs, there is a normal switchfrom IgM to IgG production since there is no maturationin the affinity of the IgM response. This can also beseen in Figure 2. Our blood tests demonstrated that thedegree of affinity maturation is dependant on theantigen dose administered. Where we administered a highdose, it produced a poor maturation and a lower affin1t~

response than a low antigen dose as seen in Figure 3. 2Had the immune system of these calves been functioningproperly, their IgM and IgG curves would have been morein line with Figure 2 rather than Figure 3.

rgG-high (Ag)

234 5 6 789

~ 1~11~~~~~~~~~~~~~~lgG_row(Ag)2->-....zu:u.«>ooCDt= 10-5

-. -------------.-..01 19Mz«z~ 0 ""'--~_~_~__~~~~~.....J

~

DAYS

Figure 3. Affinity maturation. The averageaffinity of the IgM and IgG antibody responsesfollow primary and secondary challenge with a Tdependent antigen are shown. The affinity of theIgM response is constant throughout. The affinitymaturation of the IgG response depends on the doseof the antigen. Low antigen doses (low Ag) producehigher affinity immunoglobulin than do high antigendoses (high Ag).


By the time the analyses of these data wascomplete, fall calving had begun. It was decided totest these cal ves and compare them wi th the spri ngcalves before attempting any radical changes that mayaffect the immune system of the herd. Because nothingdifferent was done while awaiting these tests, weexperienced a severe outbreak of BRD and BRSV infection.When the 28 calves born in that season and still livinghad attained an average age of 107 days, they were bledand their IgM and IgG levels measured. Table 6summarizes the IgM findings.

I Table 6 I

I Immunologic Disorders of Fall Calves I

Number of fall calves born 28Mortality 5 (17.9%)Total abnormally sick calves 13 (56.5%)Total calves treated individually 13 (100 %)Total IgM deficient calves 19 (82.6%)Number of IgM deficient calves treated 19 (100 %)

An analysis of the calves' IgG2 levels revealedthat they had surged as before when the IgM levels werebelow normal. These data suggested that the calves hada deficient immune system. Since drugs had notgenera11 y improved the heal th of the an imal s, and infact, may have worsened the problem, it was decided torevise the nutritional program. The entire cow/calfherd commenced recei vi ng the same rat i on as beforeexcept now it was supplemented with amino acid chelatedminerals with major emphasis on copper, zinc, andmanganese intake. These minerals were selected becausethey are directly related to the proper functioning ofthe immune system. (7,13,16,22,23)


For example, in a case regarding zincsupplementation related by Tizard, certain Black PiedDanish and Fresian cattle which carry an autosomalrecessive trait for thymic and lymphocytic hypoplasiahad calves that were born healthy, but by four to eightweeks began to suffer from severe skin infections. Ifleft untreated they would die with none surviving to theage of four months. The report cont i nued, "Affectedcalves have exanthema, hair loss on the legs andparakeratos i s around the mouth and eyes. There i sdepletion of lymphocytes in the gut-associated lymphoidtissue and atrophy of the thymus, spleen, and lymphnodes. It can be shown that these animals are deficientin T-cells and have depressed cell-mediated immunity butnormal ant i body responses. Thus they have a normalresponse to tetanus toxoid but respond poorly todinitrochlorobenzene or tubercul in. If these calves aretreated by oral zinc oxide or zinc sulfate, they recoverfully and acquire the ability to mount normal cellmediated responses. If, however, zinc supplementationis stopped, then the animals will relapse within a fewweeks."(!7) Based on the above observations relating tothe bovine response to zinc supplementation, zinc aminoacid chelate was selected for this present study becauseof its greater bioavailability than equivalent amountsof zinc as the oxide or sulfate salts. The samerat i ona1 app1i ed to the use of copper and manganeseamino acid chelates. These three amino acid chelateswere fed daily to the animals as shown in Table 7.


I Table 7 IAmino Acid Chelate Supplement Program for Cu, Mn, and Zn

Cu Mn Zn

LactationFirst 90 days 0.18 9 0.36 9 0.72 9Second 90 days 0.13 9 0.26 9 0.52 9Last 125 days 0.10 9 0.19 9 0.32 9

Dry cow 0.09 9 0.17 9 0.34 9Dairy calf starter 0.04 9 0.08 9 0.16 9Growing heifers 0.07 g 0.14 9 0.28 gBulls (continuous) 0.07 9 0.14 9 0.28 9(50 days prior to andthrough breeding) 0.13 9 0.26 9 0.26 g

The following year when the calves born in thespri ng had reached an average age of 165 days, theanimals were bled and their IgM and IgG levels measured.They had a mean IgM level of 1.10 mgjml with a standarddeviation of ± 0.4 mg/ml. This was a considerableimprovement over the previous spring calf herd which hadmean IgM level of 0.62 mg/ml with a standard deviationof 0.53 mg/ml.

When IgG2 levels were measured after the aminoacid chelate supplementation, they averaged 2.43 mg/ml± 0.62 mgjml compared to 1.65 mgjml ± 2.13 mgjml beforesupplementation. The mean level of IgG1 aftersupplementation was 10.43 mg/ml ± 2.13 mg/ml compared to10.98 mgjml ± 6.05 mg/ml before supplementation.

In the fall, the calf herd was bled and assayedagain. Their mean IgM and IgG levels continued toimprove. Their mean IgM level was 1.76 mg/ml comparedto 0.91 mg/ml a year earlier.

The calves continued to improve. The bleeding ofthe fall calves demonstrated the further effects in the


immune system after feeding the amino acid chelates.These results are summarized in Table 8.

I Table 8 Ii Two Year Herd Summary I

Year 1 Year 2Before Supplementation After Supplementation

Spring Fall Spring Fall

Calf morbidity 67% 54% 11% 0%Calf mortality 22% 17% 2% 0%

The dramatic changes seen in this table did notoccur until the mineral nutritional changes were made.General improvement in the condition of the entire herd,including the cows and bulls has also been seen, but nodata were maintained to quantify those improvements.

Based on these data, it is obvious that the immuneresponse depends upon a specific series of events bycertain body cells. These cellular biochemicalreact ions are regul ated by enzymes whi ch incorporatespecific trace elements in the enzyme either as a cofactor or as an integra1 part of the meta11 oenzyme.Copper and zinc, and to a lesser extent, manganese, havebeen identified as being involved in those enzymes thatare associated with the immune system.

Wh i1e there are a tremendous vari ety of si tuat ionsinfluencing trace element uptake and metabolism in thebovine, and not all diseases are related to a mineraldeficiency, nevertheless, in cattle with clinicalhistories similar to those described, it should be aroutine practice to screen 10% to 25% of the herd for


specific immunoglobulin levels and conduct a completefeed intake analysis.

From the above, it can easily be inferred thatthere is a movement to consolidate many differentdisease entities into a new category that concentrateson the immune function and trace element absorption andmetabolism as it relates to that function. Clinicalmanifestation of different diseases may depend on thesegment of the immune system being affected by mineralexcesses or deficiencies. This is a new science whichwill require tremendous effort to elucidate.Nevertheless, even though at present we are unable toidentify all of the factors relating to this discipline,we can effectively measure the positive resultsemanating from the feeding of the amino acid chelates tothe bovine in a balanced nutritional program.


References

1. Tizard, I., Veterinary Immunology (Philadelphia:W.B. Saunders Company) 6-7, 1987.

2. Roitt, I., et li., Immunology (St. Louis: C.V.Mosby Co.) 1012, 1985.

3. Tizard, Ope cit., 25.

4. Prasad, A., "Metabol ism of zinc and its deficiencyin human subjects," in Prasad, A., ed., ZincMetabolism (Springfield: Charles C Thomas) 292,1966.

5. Underwood, E., Trace Elements in Human and AnimalNutrition (New York: Academic Press) 418, 1977.

6. Ashmead, et li., "Trace Mineral Inter-Relationship,new techniques of detecting lead and other heavymetals in animals, and the role of organic chelatedtrace minerals playas enzyme catalysts," Papergiven at Oklahoma Vet. Med. Convention, Tulsa,February, 1971.


8. Coffey, R., et li., "Cl inical data onimmunoglobulin serum levels, trace elements infeedstuffs and liver copper levels in clinicallyill cattle," Unpublished, 1982 - 1985.

9. Underwood, E., Trace Elements in Human and AnimalNutrition (New York: Academic Press) 1977.


10. National Research Council, Mineral Tolerance ofDomestic Animals (Washington, D.C.: NationalAcademy of Sciences) 1980.


12. Gordon, B., Essentials of Immunology (Philadelphia:F.A. Davis Company) 1974.

13. Lontie, R., ed., Copper Proteins and Copper Enzymes(Boca Raton: CRC Press) V3, 1984.

14. Burton, D., "Structure and function of antibodies,"in Calabi, F. and Neuberger, M., eds., MolecularGenetics of Immunoglobulin (Amsterdam: Elsevier)1987.


16. May, P. and Williams, D., "Role of low molecularweight copper complexes in the control ofrheumatoid arthritis," in Sigel, H., ed., MetalIons in Biological Systems (New York: MarcelDekker, Inc.) V12, 283, 1981.


18 . Ti zard, 0 p. cit., 41.

19. Roitt, Ope cit., 8.1.

20. Nisonoff, A., Introduction to Molecular Immunology(Sunderland: Sinauer Assoc. Inc.) 1984.

21. Brigelius, R., et li., "Superoxide dismutaseactivity of low molecular weight Cuz+-chelatesstudied by pulse radiolysis," FEBS Letters 47:72,October, 1974.


22. Joester, K. E., et li., "Superoxide dismutaseactivity of Cu 2+-amino acid chelates," FEBS Letters,25:25, September, 1972.

23. Golub, E., The Cellular Basis of the ImmuneResponse (Sunderland: Sinauer Assoc. Inc.) 1981.

24. This technique was developed by Veterinary MedicalResearch and Development, Inc., Pullman,Washington, U.S.A.

25. McGuire, T., et li., "Failure of colostralimmunoglobin transfer in calves dying frominfectious diseases," JAVMA 169:713, 1976.

26. Roitt, Ope cit., 8.4.

27. Coffey, R., "Predisposition to disease: The interrelationship of the bovine immune response andtrace element physiology," Paper given at NationalCattleman's Association, 1986.

Chapter 8

THE USE OF AMINO ACID CHELATES INBOVINE FERTILITY AND EMBRYONIC VIABILITY

Joseph E. Manspeaker* and Martin G. Robl**University of Maryland

Infertility in the dairy cow is a major problem ofthat industry. In the United States, the economic cost,in losses per cow per year, is $116.25 or approximately$1.266 billion total per year. Only mastitis creates1arger losses. (1)

The causes of i nfert i 1i ty are many. Hormonedeficiencies or imbalances, diseases, insufficient orimproper nutrition, genetics, etc., can all contributeto an inability of the cow to conceive, progress throughthe gestation period, and ultimately deliver a healthynormal calf. One problem which most practicingveteri nari ans encounter frequently in repeat breedercows is endometrial scarring of the uteruses whichinhibits conception. Such a diagnosis does notsufficiently explain why the cow cannot conceive.Furthermore, the literature does little to elucidate therelationship other than to acknowledg} that endometrialscarring contributes to infertility.()

In an attempt to clarify this factor, the 160Holstein cow herd at the University of Maryland wasstudied during a seven year period. At the conclusionof the research period, of the original herd, 45 cowsstill remained in that herd and had not beenrep1aced. () The his tory of each cow was rev iewed,clinical examinations performed, and rectal palpationsconducted. Biopsy samples from each uterus were removedat 30 to 37 dats postpartum, and studiedhistopathologically.C3..5) Finall¥, in many cases,fiberoptic endoscopy was utilized.( )

* Deceased **Currently with Veterinary Specialty Diagnostics, Inc.

140

The Use of Amino Acid Chelates in Bovine Fertility 141and Embryonic Viability

The uterine biopsies helped to establish a basisfor the subsequent studies done with mineral nutritionwhich are reported here. As stated above, as each cowfreshened, at about 30 to 37 days postpartum, sampleswere taken from the med i a1 surfaces of the 1eft andright horns and the body of the uterus as close to thecervix as possible. The samples were then evaluatedmicroscopy using routine histologic techniques.

Bovi ne uteri ne tissue undergoes dynami c changes inlate gestation, at parturition, as well as during thesubsequent involution process.(5) Light microscopicevaluation of endometrial biopsy tissues is an effectivemethod to assess these endometrial changes occurring inindividual animals.(7) Through this technique, changescan be determined in (1) the stage and intensity of theestrus cycle; (2) the type, amount, and severity of anypathological changes; and (3) frequently, the horn inthe uterus in which the previous pregnancy occurred. (9)

In repeat breeders, this evaluation, along with rectalpalpation and other physical observations, endoscopicfindings, and breeding records together become importantbecause proper involution of the cow's uterus must takeplace before pregnancy can occur and be rna i nta ined. (7.8)

During the seven year study previously mentioned,it was determined that periglandular fibrosis, orendometrial scarring, was the most common endometrial1es ion, and endometri tis was the second most commonlesion. In spite of those findings, it was difficult tocorrelate the data with breeding, conception, andpregnancy performance. Furthermore, with many cows itwas impossible to predict with any degree of accuracythe actual "fert i1i ty" of the cow. Someth i ng wasmissing from our research. Some cows, which had lesspathological changes than the average cow (as developedby the above data), would cycle normally, but notconceive. Other cows, which had conceived, were unableto maintain their embryos and subsequently abort their


pregnancies. And finally, other cows in the herd whichhad demonstrated greater uteri ne pathology than theaverage cows, consistently conceived and had twelve tothirteen month calving intervals.(IO)

Since no disease oriented justification could begiven for the infertile conditions of the animals withthe available diagnostic techniques, and acknowledgingthat optimal nutrition is important and necessary forfertilitY,(ll) it was believed that perhaps someheretofore unelucidated basic nutritional factors wereinvolved in conception and in maintenance of theembryo.(I) For example, in studies with other animals,it had been noted that increased dietary copper sulfate(CuSO.) had successfully shortened the postpartum period7.3 days and decreased follicle maturation time.(12.13) Inother stud i es, it has been reported that manganesedeficiencies have contributed to infertility, delayedestrus, poor conception rates and embryonic mortality incattle.(lO) Iodine supplementation along with vitaminsA, E, and C have increased the number of cows cominginto heat as well as reduced the time to achieve firstestrus. (1•. 15.16) From these data, and others, it waspostul ated that perhaps i ntracell ul ar metabol ism and thetransfer of nutrients, and particularly minerals, to thereproductive organs and the embryo, may play moreimportant roles in bovine fertility than heretoforesuspected. (1)

In order to exami ne thi s concept in somewhatgreater deta i1, and wi th the cooperat i on of severa1researchers at Brigham Young University, individualcells from biopsied tissue from cows which were able toconcei ve and cows that coul d not, regardl ess of thepathological condition of the reproductive organs asdetermined by previous diagnostic techniques wereassayed by X-ray dispersion microanalysis. Magnesium(Mg), sodium (Na), aluminum (AI), phosphorus (P), sulfur(5), chloride (el), potassium (K), calcium (Ca),chromium (Cr), manganese (Mn), iron (Fe), cobalt (Co),


nickel (Ni), and zinc (Zn) were assayed by thistechnique. (17)

The data generated a few differences between thecows whi ch we\~e concei vi ng and those that were not.Zinc was generally absent in the cells of the biopsieduterine tissue of infertile cattle and present in thefert i1e cows. Neverthel ess, even though there weretrends indicated, there was no statistical correlationbetween the presence of zinc and fertil ity. Iron,copper, magnesium, and potassium also appeared to beessential for cellular performance. Less of thesemetals was found in the tissues of the infertile cattle,but again, no definite correlations could beestablished.

Manganese, on the other hand, was found to beabsent or severely deficient in the cells of biopsieduterine tissue of every animal that had been determinedto be infertile. Furthermore, in the repeat breeders,when tissue from the left and right uterine horns wasassayed, it was found that, in each case, conception hadoccurred in the uterine horn which contained anabundance of cellular manganese.(I]) While these datadid not reach statistical significance, they werebiologically significant(18) and tended to substantiatea previous study involving 218 cows in which manganesewas also found to be deficient in cows which were notconce i vi ng . (19)

It is recogni zed that the bovi ne reproduct i veorgans are not static during the estrus cycle. Theepithelial cells and connective tissue change withhormonal influences. For example, both the size andmorphology of the epithelial cells in the endometrialtissues change during the estrogenic or follicularstages as compared to the progesteronal or luteal stagesof the estrus cycle. The fluid content of theconnect i ve tissue is increased duri ng the estrogeni c


influence, but is reabsorbed or lost during theprogesteronal phase. Furthermore, the amount of bloodflow also changes during the different stages of thenormal estrus cycle, being most congested during theestrus or follicular stage.(7) Besides these changes,which certainly effect mineral nutrition to thereproductive organs, inadequate blood flow may alsocompromise the capability of supplying an optimummineral nutrient level to the reproductive organs duringthe negative nutritional phase of its lactationcycl e. (20)

For these reasons, as well as justification fromthe above analytical data, it was decided to design andconduct a study involving the dairy herd at theUniversity of Maryland. Heifers that were pregnant withtheir first calves were divided into two equal groupsaccording to their predicted parturition dates. Twentyof the heifers, the control group, were fed the standardprepartum balanced diet generally given the universitycows. The other twenty heifers formed the treated group.In addition to the standard prepartum diet, they weregiven an amino acid chelated mineral supplement. Theformula for this chelate supplement is shown in Table 1.

I Table 1 IAmino Acid Chelate Supplement Given Dairy Cows

Metal %

Magnesium 24.0Iron 10.0Manganese 5.7Copper 1.0Zinc 14.0Potassium 45.3

"Potass i urn was given as an amino acid complex.


The amino acid chelates were selected for use inthis study for the following reasons: (I) based on pastobservations, the inorganic minerals supplied in thebalanced prepartum diet may have been inadequate foropt imum reproduct i ve performance; (2) there was thepossibility that these inorganic minerals were adequateas formul ated, but that excess i ve amounts wereprecipitated as inert compounds in the rumen andintestines making them less accessible at the cellularlevel and a deficiency resulted; (3) to have tried tocreate adequaticy by increasing dietary levels of theinorganic minerals may have led to toxicity, whereas theamino acid chelates were shown to be non-toxic at theproposed increased levels; (4) the amino acid chelateshave been shown to be an adj unct to the inorgan icminerals, making both more accessible; and (5) the aminoacid chelated minerals may help optimize reproductiveperformance because they are more readily accessible atintracellular levels in the endometrial tissue. Theamino acid chelates have been shown to retain theiri ntegri ty in the rumen(23) and a1so bypass the pept idaseactivity of the lumen,(24) ultimately being absorbed asorganic molecules into the body tissues. Thus, theessential metals appear to be released within theendometrial cells as needed to perform vital metabolicreactions. (20)

Each hei fer in the treated group was fed twoounces (56.7 grams) daily of the amino acid chelatedmineral supplement in Table I for approximately 80 daysbefore expected parturition. After calving, these cowswere fed four ounces (113.4 grams) daily of the samesupplement until they conceived and their pregnancieswere confirmed by rectal palpations. These rectalpalpations were performed routinely every ten tofourteen days after their first calving. Detailedrecords of vaginal discharges, cervical and uterinesizes and times, and ovarian activity were kept on eachanimal. Records of any postpartum complications


including dystocias, retained placentas, trauma, etc.,breedings per conception (artificial insemination) andendometrial histopathology were maintained.

Biopsy samples from the left and right uterinehorns and the body of the uterus were collected fromeach animal between 30 and 80 days postpartum,i rrespect i ve of the stage of estrus cycl e. Thesespecimens were studied histopathologically and analyzedby X-ray dispersive microanalysis. (24) Vaginal, cervicaland uteri ne swabs for mi crobi a1 cul turi ng were a1sotaken at the time biopsy samples were collected.

The study continued for fifteen months, fromFebruary to June of the following year. As the studyprogressed, the differences between the two groups ofan i rna1s became pronounced, and in many cases, werestatistically significant. All of the differences werebiologically and economically significant.

Concept i on occurred on an average of 45 daysearlier in the treated group compared to the controlgroup. The involution and tone of the pregnant uterinehorns were better in the treated group than in thecontrol group. It was noted that in several of thetreated animals the pregnant horn was histologicallydistinguishable from the non-pregnant horn at 30 to 35days, while in the control group, the two horns werestill indistinguishable at 50 to 55 days postpartum.

In the control group eleven of the twenty animals(55%) had extended estrus cycles, and five of theseeleven (45%) had two or more cycles that lasted five tonine days longer than normal. Only six of the treatedgroup had extended estrus cycles (30%). Four of theseanimals (20%) had two or more cycles that were longerthan normal.

An extended estrus cycle would suggest 30%abortion for the treated group and 55% embryonic


mortality for the control group. However, followingartificial insemination, the control group experiencedconfirmed embryonic mortality of 30.8%. This occurredat 35 to 55 days after conception, Contrary toprojections based on the length of the estrus cycle,there were no confirmed embryonic mortalities in thetreated group. Embryonic mortal ity was determined byrectal palpation for embryonic depression in the uterinehorn where the fetus had been aborted. The depressionsin the control group were approximately 5 to 6centimeters in length, 3 to 4 centimeters in width, andelliptical in shape. The walls of the depressions weremuch thinner than the surrounding tissue. The abortionsin the control group occurred between two and ten daysbefore rectal palpation. The more distinct thedemarcation between the surrounding uterine tissue andthe area of depression, the more recent had the abortionoccurred.

At the initial postpartum rectal palpation, thetreated group had more ovarian follicular activity thanthe control group. These data are summarized in Table2. At the time that the biopsy samples were taken, 35%of the cows in the treated group had at least one matureovarian follicle (F3) and vaginal secretions whichindicated estrus. Twenty percent of the control animalshad a mature follicle and vaginal discharges. Thisdifference is significant as far as ovarian activity isconcerned. Si xty-four percent more of the treated groupwere considered to be ready to rebreed 30 days aftercalving than the control group.


I Table 2 II Dairy Cow Ovarian Activity I

Ovarian Condition Control Treated(%) (%)

Follicle 1 (small) 40.5 45.1Follicle 2 (medium) 11.9 19.6Follicle 3 (large) 0.0 1.9Corpus luteum 16.7 9.8No activity 26.2 21.6Cysts 4.9 1.9

Cultures were taken from the vagina, the cervixand the uterus. Bacteria were isolated from one treatedanimal and five of the controls. The culture from theone treated animal revealed a gram negative betahemolytic infection. The control group had Escherichiacoli, Clostridium perfringens, Lactobacillus, andCorynebacteri urn spp. cul tured. Norma11 y, thesebacterial infections lead to later and weaker estrus,and increased breedings per conception. As notedearlier, bacterial infections also contribute to, orcause, endometritis.(21)

Endometrial biopsies taken from the right and lefthorns and the body of the uterus were evaluated by lightmicroscopy. Chronic endometritis, or inflammation, wasmore severe and persisted for a larger period of time inthe control group compared to the treated group. Thisobviously relates to a more difficult time achievingconception in the control cows.

The incidence of periglandular fibrosis wasmarkedly reduced in the treated group which received the


amino acid chelates. Only two of twenty animals, or10%, were afflicted in the treated group compared toeleven of nineteen, or 58%, of the cows in the controlgroup. Using chi-square analysis it was determined thatendometrial scarring, or periglandular fibrosis, wassignificantly less (p<0.005) in the treated group.Fibrosis, or scarring, in endometrial tissue isconsidered a pathologic response when the tissue doesnot regenerate properly following an injury. Theprocess of parturition is considered to be a cause ofthis type of injury to endometrial tissue. Thepathologic examination also revealed that the horn ofthe previous pregnancy was evident in more cows in thecontrol group and evident for a longer period of timeafter parturition than in the treated group.Consequently, when endometrial fibrosis occured, itcould be interpreted that the scarred tissue was notregenerating properly. Although it is obvious that theamino acid chelates had a positive effect onregeneration of endometrial tissue, the exactrelationship of each mineral to this effect has not beenelucidated.(9)

The final histopathologic evaluation related tothe ce11 ul ar mi nera1 compos it i on of the uterus. Thenormal levels of intracellular minerals in thepostpartum endometri a1 tissue of cows have not beenestablished, so comparisons made were not made against"normal levels." The electron microscopic microanalysisrevealed clearly that there was more manganese in theendometri a1 tissues of the treated group compared to thecontrol.

Although not completely elucidated, manganesedeficiency has been associated with silent estrus,anestrus, infertility, abortion, immature ovaries, anddystocias.~) Cows seldom display signs of a manganesedeficiency. Nevertheless, absorption is limited to 10%to 18% of the total inorganic manganese in their diets


and subclinical intracellular manganese deficienciescould exist and in many cases, probably do.

Other minerals, including zinc and copper are alsore1ated to breed i ng effi ciency. Although there appearedto be higher levels of these minerals in the endometrialtissues of the treated group, no clear patterns could beestabl i shed. It was previ ously noted that coppersupplements had reduced follicle maturation time inother species of animals.(12.13) This same phenomenon wasalso observed in this bovine study in spite of the lackof consistent histopathologic data.

These data, however, did establish that amino acidchelated minerals are accessible at the intracellularlevels in the endometrium where they can be used, asneeded, in vital metabolic reactions.(IO) The roles thatthese minerals play alone or in synergism with othernutrients and how they maintain a proper uterineenvironment for a viable embryo are not completelyunderstood at present.

What i s understood are the pas it i ve resul ts incattle receiving the amino acid chelated minerals intheir diets. There were increased ovarian activity aswell as more effective involution and regeneration ofendometrial tissue. Fewer persistent bacterialinfections, endometritis, and periglandular fibrosiswere also noted when the cows were fed the amino acidchelates. Finally, the amino acid chelates reducedembryonic mortality. This study suggests that thenutritional and mineral status at the cell level arevital to endometrial health, embryonic viability, andoverall fertility in the bovine.


References

1. Manspeaker, J., et li., liThe effects of chelatedminerals on bovine fertility," Proceedings ofAVMA Annual Meeting, Las Vegas, July, 1985.

2. Manspeaker, J., "Breeder Pac in BovineReproduction," Proceedings of Albion Seminar atBovine Practioners Annual Meeting, Des Moines,1984.

3. Manspeaker, J. and Edwards, G., "Clinicalapplication of fiberoptic endoscopy in bovineinfertility," Proc. X Pan Amer. Cong. Vet. Med.Zootech., Vol 1, Sect. 1, No. 23A, 1985.

4. Manspeaker, J. E. and Haaland, M.,"Implementation of uterine biopsy in bovinereproduction: a practioner's diagnostic tool,"VM/SAC, 78:760, 1983.

5. de Bois, C. and Manspeaker, J., "Endometrialbiopsy of the bovine," in Morrow, D., ed.,Current Therapy in Theriogenology (Philadelphia:W.B. Saunders) 424, 1986.

6. Gier, H. T. and Marion, G. B., "Uterus of the cowafter parturition: involutional changes,"A. J. V.R., 29: 83, 1968.

7. Robl, M., et li., "Histopathologic evaluation ofthe bovine involution process using endometrialbiopsy technique," Proc. of IV Int. Symposium ofVet. Lab. Diagnosticians, Amsterdam, 1986.


8. Thatcher, W., et li., "Normal uterine physiologyand involution," Proc. Am. Assoc. Bovine Pract.,75, 1984.

9. Robl, M., et li., "Relationship of pathologicchanges and intracellular mineral levels in thepostpartum bovine endometrium," Prac. IVInternational Symp. Vet. Lab. Diag., VI, 247,1986.

10. Manspeaker, J., et li., "Chelated minerals:Their role in bovine fertility," Vet. Med.,82:951, 1987.

11. Rattray, P., "Nutrition and reproductiveefficiency," in Cole, H. and Cupps, P., eds.,Reproduction in Domestic Animals (New York:Academic Press) 553, 1977.

12. Fomina, E. L., "Influence of copper on the sexualfunction of mares," Animal Breed. Abstr., 38:380,2172, 1970.

13. Naumenkov, A. I., et li., "The effect of PMS onreproduct i ve funct i on of mares, and increasedactivity of this preparation," Animal Breed.Abstr., 37:571, 3258, 1969.

14. Koval'skii, V. V., et li., liThe Amurbiogeochemical region of the iodine deficiency,"Nutr. Abstr. Rev., 43:517, 4118, 1973.

15. Valjuskin, V., Animal Breed. Abstr., 41:698,1971.

16. Valyushkin, K. D., "The effect of vitamin A onthe postpartum period and conception rate incows," Animal Breed. Abstr., 43:127,1086,1975.


17. Chastain, H., "Breeder Pac in Bovine Production,"Proc. Albion Seminar at Bovine Practioners AnnualMeeting, Des Moines, 1984.

18. Robl, M., "Breeder Pac in Bovine Reproduction,"Proc. Albion Seminar at Bovine Practioners AnnualMeeting, Des Moines, 1984.

19. Hinze, P. and Ashmead, H., "The use ofMetalosates in bovine nutrition," Unpubl ished,1969.

20. Manspeaker, J., et li., "Prevention of earlyembryonic mortality in the bovine fedMetalosates," Proceedings of IV InternationalSymp. Vet. Lab Diag., Vol 1, 253, 1986.

21. Manspeaker, J., et li., "Incidence and degree ofendometrial periglandular fibrosis in parity 1dairy cows," VM/SAC, 78;943, 1983.

22. Pugh, D., et li., "A review of the relationshipbetween mineral nutrition and reproduction incattle," Bov. Prac. 20:10, 1985.

23. Beede, D., University of Florida, Personalcommunication, Sept., 1988.


Chapter 9

THE ROLE OF COPPER INBEEF CATTLE FERTILITY

J. Robert KroppOklahoma State University

The Oklahoma State University Beef Cattle Centerherd currently consists of over 250 head of producingfemales representing six different breeds (Angus, PolledHereford, Horned Hereford, Brangus, Limousin, andSimmental). Prior to 1986, and despite feeding proteinand energy levels in excess of 1984 NRC requirements,fertility, as measured by percentage of females pregnant90 days after the breeding season, was relatively low(less than 75%).

Because of this low fertility an intensiveinvestigation was initiated in an attempt to identifypossible reasons for the problem. Clinicalinvestigations did not generally reveal any significantmanifestations that would interfere with conception. Itwas noted, however, that an analysis of the blood serumrevealed that several of the minerals analyzed were low.These data are summarized in Table 1.

154

The Role of Copper in Beef Cattle Fertility 155

I Table 1 I

I Initial Blood Serum Analysis IMi nera1 Level Normal Range

Calcium, mg % 6.6 9-12Phosphorus, mg % 3.4 4.5-7.0Magnesium, mg % 1.4 2-3Potassium, mg % 19.4 15-23Selenium, ppm 0.047 .07-.30Copper, ppm 0.217 .7-.10Zinc, ppm 0.913 1.5-1.8Iron, ppm 1.317 .9-2.5Manganese, ppm 0.039 .01-.03

Low levels of calcium, phosphorus, magnesium,selenium, copper and zinc were noted. The liver copperlevel of a Hereford cow which was slaughtered was 8.4ppm (normal> 40 ppm). An Angus calf, which diedshortly after birth, had a liver copper level of 62 ppm(normal> 300 ppm). One of the first clinical symptomsin a copper deficient cow herd is achromotrichis (lossof hair color). Angus cattle will typically exhibitbrown pigmentation rather than black color,particularlyaround the eyes, tips of the ears, and over the top,especially behind the shoulders. This condition wasprevalent in the Angus herd.

All of these observations suggested that perhapsthe fertility problem in the university herd wasnutritionally oriented. Morrison has emphasized thatnutrition is particularly important for optimumreproduct ion. (1) Copper, in part i cul ar, is essent i a1duri ng gestat i on as trends in Fi gure 1 suggest. (2)


60

50

.........................................

cu CON"TC~ OFTIlEREPRODUcnveTISSUES

CU IN UITRUS.REPRODUcnvePRODU~ AM>MAMMARYGLANDS

CU IN PREGNANTRATS WITHOUTREPROOUCl1VEORGANS ANDTISSUES

CU ANABOUSM

CUINo~~~~"""-'-"-"""""""''''''"''''''''''''''""''''''''~ NON-PREGNANT0.7 2.2 8.7 RATS (X 100)

DIETARY CU (PPM)

Fi gure 1. Copper retent ion in rats duri ngpregnancy compared to non-pregnant control s atdifferent copper levels in the diet.

Soil samples, forage samples and feed grains wereanalyzed for nutrient composition and especially minerallevels. Following these analyses, a mineral mix wasformulated which contained 66% dicalcium phosphate, 29%trace mineral ized salt and 5% cottonseed meal. Thetrace mineral salt contained sodium chloride, manganousox; de, iron oxi de, ferrous carbonate, copper oxi de,ethylenediamine dihydroiodide, zinc oxide, cobaltcarbonate and technical white mineral oil. Theguaranteed analysis of the trace mineral salt is shownin Table 2.


I Table 2 IInorganic Trace Mineral Salt Composition

Salt (NaCl) Not more than 97.0%Salt (NaCl) Not less than 92.0%Manganese (MnO) Not less than 0.25%Iron (FeO and FeCO) Not less than 0.20%Copper (CuO) Not less than 0.033%Iodine (EDDI) Not less than 0.007%Zinc (ZnO) Not less than 0.005%Coba1t (CoCO) Not less than 0.0025%

The animals showed some visual improvements in thepreviously indicated traits. The improvements were notsufficient, however, to overcome the reproductiveproblems. Nevertheless, these improvements suggestedthat the fertility problems may be a result ofinadequate mineral nutrition, and that this could becorrected.

The late Professor Eric Underwood has pointed outthat "the evaluation of feeds and feed supplements assources of minerals depends not only on what the feedcontains, i.e. the total content or concentration asdetermined physicochemically, but on how much of thetotal mineral can be absorbed from the gut and used bythe animal's cells and tissue." C

) Due to a concern withbioavailability, it was decided to provide amino acidchelated minerals to the cows to see if they wouldabsorb more of these minerals than from th8 previousmineral supplement, and if that greater bioavailabilitywould correct the reproductive problem of the herd.

The herd was placed on a free choice mineral andvitamin supplementation program. The supplementconsisted of 50% salt (NaCl) and 50% minerals from both


the amino acid chelates and from inorganic sources. Theformulation is seen in Table 3. A three ounce (85g)intake of this supplement supplied 170 mg of copper.Copper was the only trace element, other than iron,which was significantly modified in the formulation inTable 3 compared to Table 2. The iron was removed fromthe revised mineral supplement because it interfereswith copper metabolism, and feed analysis had determinedthat the feed contained adequate iron and required nofurther supplementation.

Table 3

Mineral and Vitamin Composition of Salt Supplement

Calcium (CaP04 ), not more thanCalcium (CaP04 ), not less thanPhosphorus (CaPO.), not less thanPotassium (KCl and amino acid complex), not less thanMagnesium (MgO and amino acid chelate), not less thanZinc (amino acid chelate), not less thanManganese (amino acid chelate), not less thanCopper (amino acid chelate), not less thanCobalt (amino acid chelate), not less thanIodine (EDDI), not less thanSelenium (SeSO))Vitamin A, minimum per lb.Vitamin 03 , minimum per lb.Vitamin E, minimum per lb.

9.0 %8.0 %8.0 %3.0 %4.75%0.7 %0.3 %0.2 %0.0025%0.001 %0.002 %

280,000 IU59,000 IU

400 IU

Subsequent blood serum analysis suggested that thetrace mineral supplementation program, as provided inTable 3, resulted in increased blood serum copperlevels. All tested animals had an average blood serumcopper level of 0.8 ppm copper, which was well withinthe normal range and a four-fold increase from the firstanalysis.

These fi ndi ngs become very encouragi ng becauseanimals which had been grazing in copper deficientpastures were experiencing low fertility. The females


di spl ay depressed or del ayed estrus. In some caseswhere pregnancy did occur, the gestating mother aborteda small dead fetus. (4) The sign i fi cance of the copperami no ac id che1ate had an even greater impact whencopper availability from herbage was considered.Regardless of whether a pasture is deficient or not,fresh herbage is less effective in promoting body copperstores in cattle than hay or dried herbage or equivalenttota1 copper content. (5) Grace, et li., reported thatthe rate of conception in ruminants is directly relatedto the rate of uptake of copper. (6) From theseobservations it was reasonable to hypothesize that byproviding additional biologically available copper viaan amino acid chelate as part of a special mineralsupplement, fertility would increase in those animalsgrazing in copper deficient pastures.

Forty 2- and 3-year old first calf heifers locatedat the Oklahoma State University Purebred Beef CenterRange were utilized. Five different breeds wererepresented: Angus (15), Horned Herefords (12), PolledHerefords (5), Brangus (5), and Simmental (3). All ofthe females calved between January 31 and March 5. Inaddition, all of the females utilized in the study hadfree access to 50% salt and 50% of the mineral formulaseen in Table 3 for one year prior to the start of thestudy. Providing the amino acid chelates to all of theanimals in the study for one year prior to testing thevariables equal ized the starting point so that theheifer randomization would be more fair.

Pri or to the tri a1, all of the fema1es wererna i nta ined on dormant 01 d Worl d 81 uestem pasture andsupplemented with 20 pounds of mature native range grass(6% crude protein, 45% TDN) and 5 pounds of 20% crudeprotein range protein supplement per head per day, plusfree access to the 50% salt: 50% special mineralsupplement (Table 3). The base diet, excluding themineral supplement, contained 31 ppm zinc, 81 ppmmanganese, 5.6 ppm copper and 2.3 ppm molybdenum. The


copper:molybdenum and zinc:copper ratios were 2.4 and5.6, respectively.

A schematic diagram of the trial is presented inFi gure 2. On Apri 1 4 (the start of the tri a1), theinitial blood samples were drawn, and the cow-calf pairswere randomly assigned to one of two mineralsupplementation programs by breed, calving date, andbody condition score to minimize the effects acrosstreatments. In addition, for the controls the freechoice ration of 50% salt: 50% special mineralsupplement consisting of amino acid chelates, inorganictrace minerals, major minerals plus vitamins A, D3 , andE was removed from the pasture and rep1aced wi th amineral supplement containing 66% dicalcium phosphate,29% salt and 5% cottonseed meal.

April 4BloodSample #1•

April 18 April 27 June 17Blood Sample #2

5MB Implant Removal 5MB Blood Sample #3

l!nOrganiC/Amino Acid Chelate SUPPlemen~J

April 16 April 30

I~ 1 lb. Supplement ~Jr 75 Days 1

Figure 2. Schematic design of trial.

A gO-acre 01 d Worl d Bl uestem pasture was sp1itinto two 45 acre pastures by an electric fence. All ofthe females remained on the base nutritional program


(Old World Bluestem pasture, 20 pounds of native grasshay, and 5 pounds of a 20% crude protein rangesupplement.) The mineral treatments consisted of aminoacid chelates (copper, zinc, manganese, and magnesium)and potassium amino acid complex versus inorganic traceminerals (copper sulfate, zinc sulfate, manganese oxide,magnesium oxide and potassium chloride) to supplyident i ca1 1eve1s of copper, zinc, manganese andpotassium. The test diets were incorporated into a 20%crude protein range supplement fortified with 14,687I.U. of vitamin A, 3125 I.U. of vitamin D3 , 150 I.U. ofvitamin E, and 2.5 mg of selenium per pound. Bothtreatment supplements were fed at the rate of 1 poundper head per day. Including the basic nutritionalprogram, the diets were formulated to supply a total of18 ppm copper, 71 ppm zinc, 89 ppm manganese, 0.15%magnesium and 0.90 %potassium. The supplemental nativegrass hay and 5 pounds of 20% crude protei n rangesupplement were discontinued on May 1. The one poundper head daily treatment supplements were fed until June15.

A special formulation of amino acid chelates wasutilized 14 days prior to breeding at the level of 2ounces per head per day in one pound of ground corn.The amino acid chelated group received this formulationwhich contained 5.5% Mg (MgO), 3.8% K (KC1), 400,000I.U. of vitamin D3 , 85,000 I.U. of vitamin A, and 570I.U. of vitamin E per pound, as well as copper,manganese, zinc, and magnesium in the amino acidchelated forms and potassium as an amino acid complex.The inorganic trace mineral group received a similarformula except that the copper, manganese, zinc, andmagnesium amino acid chelates and potassium amino acidcomplex were replaced by inorganic sources (coppersulfate, manganese oxide, zinc sulfate, magnesium oxideand potassium chloride). These formulas are summarizedin Table 4.


I Table 4 ITreatment Group Levels (mg) of Copper, Manganese, Zinc, Magnesium,and Potassium

1 1b. 20% 2 oz. TotalSupplement Amino Acid Chelate Supplemental

Chelate Inorganic Chelate Inorganic Chelate Inorganic

CuSo4 -- 178 mg -- 48 mg -- 226 mgCu Chelate 154 mg 24 mg 45 mg 3 mg 199 mg 27 mg

MnO -- 275 mg -- 80 mg -- 355 mgMn Chelate 231 mg 44 mg 65 mg 15 mg 296 mg 59 mg

ZnS04 -- 563 mg -- 258 mg -- 821 mgZn Chelate 466 mg 97 mg 233 mg 25 mg 699 mg 122 mg

MgO -- 1352 mg -- 3660 mg -- 5012 mgMg Chelate 142 mg 1210 mg 368 mg 3292 mg 510 mg 4502 mg

KCL -- 823 mg -- 2452 mg -- 3275 mgK Complex 142 mg 681 mg 368 mg 2084 mg 510 mg 2765 mg

The amino acid chelates were supplied to treatmentgroups from April 17 to April 30. On April 17, all ofthe cows were injected wi th 2 cc of Synchro-Mate Bi njectabl e (Norgestomet pl us estradi 01 val erate) andimpl anted with the Synchro-Mate Bimpl ant (Norgestomet).The implant was removed on April 27 (Day 10). The cowswere observed four times daily for signs of estrusstarting on April 28. Those cows exhibiting estrus wereartificially inseminated approximately 12 hours afterthe observed heat.

Blood samples were obtained from all animals inthe experiment on April 4, April 27, and June 17, usingsodium heparinized trace element collection tubes and avacutainer collection needle and holder. A 7 cc sampleof blood from the jugular vein was utilized forhemoglobin, catalase, Cu-Zn superoxide dismutase, andglutathionine peroxidase analyses.


Serum samples were collected using B-D vacutainertrace element serum tubes. The serum was analyzed byatomic absorption spectrophotometry with a Zeemanbackground correction.

Whole blood hemoglobin was determined by use ofthe Si gma Total Hemogl obi n Procedure no. 525, (7) and aquantitative colormetric determination was accomplishedusing Drabkin's reagent at 540 nm.

Heparinized blood samples were centrifuged at 4800RPM for 5 mi nutes. Pl asma was siphoned off intoadequate storage containers and frozen at 100 C. Thebuffy coat was discarded and approximately 3 volumes ofsterile physiological saline were added to re-suspendthe erythrocytes. The sample was re-centrifuged and theprocedure repeated twice more.

Approximately three to four times the volume ofdouble distilled water (Type III) was added to the RVCpellet to hemolyze the red blood cell. The lysatehemoglobin value(7) was determined as described in step#2, and a portion of the sample was diluted toapproximately 1 mgjml total protein for the Lowry'sprotein assay no. P5656 which was determined with anabsorbance measured at 500 nm. (8)

The lysate was diluted to 5 mg proteinjml in pH7.8 and 0.05 Mpotassium phosphate buffer for use in thecata1ase and Cu-Zn superoxi de di smutase assays. Onehundred microliters of lysate was incubated with 100microliters of 1 mM copper sulfate for one hour at roomtemperature, and then a 5 mg proteinjml dilution wasmade. The pH was maintained at 7.8 by the 0.05 Mpotassium phosphate buffer. This procedure was used torerun the Cu and Zn superoxi de di smutase assays todetermine if there was complete copper saturation of theassay. The Cu and Zn superoxide dismutase assay was anNBT inhibition assay done according to the method ofOberly and Spitz.(9) One unit of superoxide dismutase


activity equals that amount of protein that gives halfmaximal inhibition of NBT reduction.

Two of the heifers receiving the amino acidchelate supplement were removed from the study becauseone became lame and the Syncro-Mate B implant fell outfrom the other. One cow receiving the inorganic mineralsupplement was removed from the study after her calfdied.

Because erythrocyte superoxide dismutase may be abetter potential index of copper deficiency than plasmacopper or ceru1op1asmi n act i vi ty ,(11) the copper and zincsuperoxide dismutase activity in the red blood cell isshown in Table 5. The red blood cell activity of the Cuand Zn superoxide dismutase, an erythrocyte enzyme, isprincipally dictated by the copper status of the animalat the time of erythrocyte synthes is. (10) Compari son ofsuperoxi de di smutase act i vi ty between treatmentsindicates an interesting relationship. The amino acidchelated supplemented females exhibited increasedsuperoxi de di smutase act i vi ty from the start to thefinish of the trial, while the inorganic trace mineralsupplemented females exhibited decreased enzymaticactivity.

I Table 5 IBlood Analysis of Copper and Zinc SuperoxideDismutase Activity

Cu and Zn-Superoxide dismutase activity(units/mg protein-RBC Lysate)

Bleeding Chelate Inorganic

April 15 52.9 59.0

June 17 59.3 56.8


Compari sons of the Cu and Zn, superoxi de di smutaseactivity within treatments indicates an increase incopper status within the amino acid chelate supplementedcows and a trend toward decreasing copper status in theinorganic supplemented cows. This suggests the aminoacid chelated copper to be more biologically availablethan the copper sulfate used in this trial.

The results of estrus activity, the first serviceconception rates, and the 20S-day weaning weights forthe calves, are presented in Table 6. The amino acidchelate supplemented cows exhibited more standing heatsand had a greater percentage of females conceiving onthe first service than did the inorganic mineralsuppl emented cows. Fourteen of the 18 ami no acidchelate supplemented females exhibited a standing heatafter removal of the Syncro-Mate B implant. Of the 14heifers that cycled, 10 conceived on the first service.The reproduction performance of the females receivingthe inorganic trace mineral supplement was significantlypoorer (P<0.05). Only 8 of the 19 females receiving theinorganic trace minerals cycled after removal of theSyncro-Mate B implant. Furthermore, only 2 of those 8conce i ved. Therefore, 55.6% of the tota1 ami no ac idchelate supplemented cows actually conceived, while only10.5% of the inorganic trace mineral supplementedfemales conceived on the first service.


Table 6

Estrus Activity, First Service Conception, and 205Day Adjusted Weaning Weights of Calves

Mineral Supplements

Condition

Females, totalFemales, exhibiting estrusPercent of females exhibiting estrusFemales, first service conceptionPercent of females exhibiting estrus

that conceived on first servicesPercent of total females conceiving

on first serviceBody condition scoreWeaning weight (205-day) all calves

Chelates

181477. 8~10

71.4c

55.6c

5.0575~

Inorganic

198

42.1 b

2

a.b Means in same row with different superscripts differ (P <0.05)c.d Means in same row with different superscripts differ (P <0.01)

The weaning weights of the calves were adjustedfor age in days to a 205-day bas is. No age of daycorrections were made since all females were first calfheifers. The amino acid chelate supplemented femalesweaned 47 pounds more calf than the inorganicsupplemented females. Based upon a 100 head cow herdcomparison and using current feeder calf prices, the 47pound per calf advantage in weaning weight could beworth an additional $4,700. In addition, due todifferences in first service conception rates, if 45more calves could be produced 21 days earlier and anaverage of 2 pounds per day gain could be realized, thenat current prices, $1,890 additional income could beadded to the program. The total input cost oradditional feed cost for 100 cows was $477.50.Therefore, the net return to the producer woul d be$6,112.50, or for every dollar invested, $12.80 net wasreturned.


The results of this trial indicate an improvedestrus act i vi ty and concept i on rate wi th cowssupplemented with amino acid chelates and particularly,copper ami no ac id che1ates. Though the number offemales were limited, the differences were large.


References

1. Morrison,(Clinton:1961.

F., Feeds and Feeding, AbridgedThe Morrison Publishing Co.) 124,

2. Krichgessner, M., et li., "Studies on the superretention of trace elements (Cu, Zn, Mn, Ni, Fe)during gravidity," in Kirchgessner, M., ed.,Trace Element Metabolism in Man and Animals(Freising: University of Munich) 441, 1978.

3. Underwood, E., The Mineral Nutrition of Livestock(Slough: Commonwealth Agricultural bureau) 15,1981.

4. Howell, J. and Hall, G., "Infertility associatedwi th experi mental copper defi ciency in sheep,guinea-pigs and rats," in Mills, C., ed., TraceElement Metabolism in Animals (Edinburgh: E & SLivingston) 106, 1970.

5. Hartmans, V. and Bosman, M., "Difference in thecopper status of grazing and housed cattle andtheir biochemical backgrounds," in Mills, C.,ed., Trace Element Metabolism in Animals(Edinburgh: E & S Livingston) 362, 1970.

6. Grace, N., et li., "Accumulation of Cu and Mo bythe foetus(es) and conceptus of single twinbearing ewes," in Hurley, L., et li., eds., TraceElements in Man and Animals (New York: PlenumPress) 311, 1988.

7. Sigma Chemical Company, Biochemicals OrganicCompounds for Research and Di agnost i c Reagents(St. Louis: Sigma Chemical Co.) 1836, 1989.


8. Ibid, 1751.

9. Oberley, L. and Spitz, D., "Assay of SuperoxideDismutase Activity in Tumor Tissue," Methods inEnzymology (Orlando: Academic Press) VI05, 457,1987.

10. Coffey, R., Personal communication, October 18,1988.

11. Suttle, N. and McMurray, C., "The use oferythrocyte superoxide dismutase and hair orfleece Cu concentrations in the diagnosis of Curesponsive conditions in ruminants," Sr. Vet. J.,1980.

Chapter 10

THE USE OF AMINO ACID CHELATES IN HIGHPRODUCTION MILK COWS

Andrea Formigoni, Paoli Parisini, and Fulvio CorradiUniversity of Bologna

There has been a continuous effort on the partsof many researchers throughout the worl d to invest igatevarious nutritional alternatives in an attempt tomaximize milk production and reproduction efficiency indairy cattle with high genetic potential. Thenutritional needs of such animals are very high becausethe dairy cow is an exceedingly complex animal involvinginnumerable biochemical processes, each of whichrequires a continuous source of many differentnutrients. Because it is not possible in thispresentation to consider the nutritional needs for eachindividual biochemical process,these processes aregrouped together and examined according to functionalcategories: (1) maintenance, (2) growth and fattening,(3) reproduction, (4) production of milk, and (5) workor exercise.(1) Many investigators have studied each ofthese functional categories using different types andquantities of nutrients in hopes of reducing nutrientdeficiencies within a category which in turn wouldincrease the yi e1d of the da i ry cow and irnprove itshealth.

One of the basic needs of the ruminant along witha source of nitrogen and a source of energy and perhapssome vitamins is a source of minerals.(2) While the needfor minerals is well established, the effects of mineralbioavailability on the various functional categories isnot. It is well documented that when essential traceelements, such as iron, cobalt, zinc, copper andmanganese, are chelated with amino acids, theirabsorption into the animal's body is increased.(3) This

170

The Use of Amino Acid Chelates in High Production Milk Cows 171

The absorpt i on of the ami no ac id che1ates i sincreased because:

1) The competition between metal ions forabsorption sites in the intestine is avoided. Thiscompetition is particularly evident between cationswhich are chemically similar, but have different bondingaffinities, such as copper and iron in relation totransferri n. (5)

2) The amino acid chelate avoids the sequesteringand subsequent precipitation of metal ions that wereingested as salts and then ionized in the rumen andintestines. Such precipitants include oxylates,phosphates, oxides, phytic acid, etc. An insolublemi nera1 compound i s not absorbed. (5)

3) The amino acid chelates are absorbed intactwithout degradation in the rumen. Because thesechelates do not require carrier proteins as do metalions to be absorbed, the potential problem of depressionin enzymatic generation of carrier proteins isavoided.(6)

4) The amino acid chelate is not dependant uponthe quantity of vitamins in the diet for absorption(such as vitamin D and calcium).

5) The level of fiber in the ration, or thetransit speed of the chyme through the digestive tractdoes not influence intestinal uptake of the amino acidchelate to the same degree it does with metal salts.

While the above list may itemize some benefits forusing amino acid chelates of metals for supplementalmineral nutrition, it also points out the many obstaclesthat can interfere with the absorption of inorganicsupplemental minerals when trying to achieve significantincreases in mineral uptake. Given these problems, thequantities of trace elements that are effectively


absorbed by the ruminant at any given time are difficultto ascertain with any degree of predictability. Thisoften results in deficiencies of these minerals in thediet, which are either primary deficiencies, or arefrequently, deficiencies as a result of poor absorption(secondary deficiency).

The fact that many cations are chelated to aminoacids in the lumen before absorption automaticallygenerates interest in a prechelated mineral to improvetheir absorption. As a manufactured product, chelationinvolves the formation of a ring structure between theamino acid and the metal ion through coordinant covalentbonding. If this chelation process is compatible withthe natural process of the animal itself, then itsabsorption should accomplish the following:

1) There should be reduced interference with theabsorption of the mineral by various components in thediet, such a oxylates, phytates, fiber, etc.(3)

2) There should be improved absorption of theminerals themselves, thus making them more available toparticipate in the functional categories.

3) As a result of greater absorption, there shouldbe greater production due to the prevention of mineralrelated diseases and biochemically greater utilizationof all of the nutrients due to synergism betweennutri ents. (7)

With these benefits in mind, it was decided todesign an experiment in which the greater absorption ofthe amino acid chelates was examined as it related totwo interrelated functions in the dairy cow:reproduct i on and mi 1k product ion. Other data havesuggested that definite advantages can be obtained inthese categories with the chelates.


When a cow has the genetic potential of greatermilk production, but is producing small quantities ofmilk, any positive nutritional changes will result inmore mi 1k. The use of these types of cows does notfa i rly eva1uate the effect i veness of the tested nutri entin increasing milk production. For this reason 46 highproducing dairy cows were selected for this study. Thebreed of the cows was Fresian. All were maintained inopen stables.

The cows were divided into two groups of 23animals each. The division was based on the number ofparturitions and the quantity of milk produced duringthe previous lactation. Assignment into one of the twogroups occurred ten days after calving. All of the cowsca1ved wi th ina six-month peri od. Group A was theexperimental group, and Group 8 was the control. 80thgroups received exactly the same feed which is shown inTable 1.

Tab1e 2 presents the cherni cal anal ys i s of the feedration. Two samples were taken for the analysis, andthe data averaged. Tab1e 3 presents the chemi calanalysis of the feed concentrate. Two samples of theconcentrate were taken, and the analytical dataaveraged. Every time a new batch of feed was given tothe cattle, its individual ingredients as well as thetotal feed were analyzed by taking two separate samplesand averaging the analytical results, in order to ensurethat it conformed to the feed specifications for thisexperiment. The data in Tables 2 and 3 are the means ofall of these analyses with their standard deviations.


1~~~~~~~~~;;;;;;;;T;;;;;;;;a;;;;;;;;bl;;;;;;;;e~l~~~~~~~~~1I Feed Formulation Given Cows Daily I

Basic formulation for 23 Kg of milk (50.6 lbs)

Corn silage 18 KgHay 6 KgWheat 1 KgFeed concentrate 7 KgWater 7 Kg

Supplemental FormationFeed concentrate

In Milking ParlorFeed concentrate

1 Kg (2.2 lbs) extra feedconcentrate for each 2.2Kg (4.8 lbs) of milkproduced beyond the 23 Kg(50.6 lbs) per day.

0.4 Kg per day

I Table 2 IChemi ca1 Analyses of Dairy Cow Feed Ration

Dry matter 46.83% ± 0.85Crude protein 13.10% ± 0.20Crude fiber 20.69% ± 1.10Crude fats 2.71% ± 0.02Ash 6.97% ± 0.09

Iron 221 ppmZinc 112 ppmManganese 66 ppmCopper 16 ppmIodine 2 ppmCobalt 1 ppm


I Table 3 IChemical Analyses of Feed Concentrate

Moistu "'e 12.70% ± 0.60Crude protein 19.90% ± 0.38Crude fiber 7.20% ± 0.12Crude fats 5.60% ± 0.09Ash 8.10% ± 0.13

Zinc 275 ppmIron 195 ppmManganese 102 ppmCopper 33 ppmIodine 5 ppmCobalt 2 ppm

Each cow in both the experimental and the controlgroups recei ved enough feed ab 1i bi tum to meet therequirements for their maintenance and the production ofthe first 23 kg (50.6 lbs) of milk. Through the use ofauto feeders, a supplementary ration was provided toeach cow on the basis of 1 kg (2.2 lbs.) of additionalfeed, as the feed concentrate, for each 2.2 kg (4.8lbs.) of additional milk production per day. In themilk parlor, each cow received an additional 0.4 kg (0.91bs.) of feed concentrate per day regardl ess of thequantity of milk produced. Group A, the experimentalgroup, also received a supplement of amino acidchelates. It was mixed with the feed concentrate thatwas given in the milking parlor at the rate of 12.5grams of ami no ac id che1ate per 400 grams of feedconcentrate. Table 4 presents the analysis of thischelate supplement.


I Table 4 II Amino Acid Chelate Analysis I

Minerals (mg/Kg)

Iron 23,400.0Cobalt 100.0Zinc 4,800.0Manganese 4,400.0Copper 3,300.0

Amino Acids (g/100g of protein)

Aspartic acid 9.44Treosine 3.27Serine 3.60Glutamic acid 25.83Proline 7.61Alanine 4.95Valine 5.43Creatinine 0.79Isoleucine 4.08Tyrosine 2.20Phenalanine 4.22Lysine 5.36Histidine 3.89Arginine 6.50Leucine 7.12

The study lasted 221 days. In the course of thistime, seven cows were removed because they fa i1ed toconceive upon rebreeding. Three cows were taken fromGroup A and four from Group B. Th is was cons iderednormal.

1)

2)

3)

4)


The mi 1k product i on for each cow was measuredweekly. These data were analyzed using ANOVA implyinga factorial of 30 x 2 (thirty weekly tests per twotreatments). In order to reduce the variability betweengroups (i. e., mul t i parous and pri mi parous), the mi 1kproduction values were reported in percentages ofaverage production from each cow in the first five daysfollowing the colostrum period.

During the course of the study, milk samples weretaken on three separate occasions. These samples weretested for pH, butterfat, protein, SH/50 acidity, cellcount, and sugar content. The data obtained from thesetests were stat i st i cally assessed us i ng ANOVA wi th afactorial of 3 x 2 (three samples per two treatments).In addition to collecting data on milk production,reproduct i ve effi ci ency was a1so measured. The datacollected included the following:

The interval between parturition and firstheatThe interval between parturition and firstinseminationThe interval between parturition andconceptionThe number of breedings (artificialinsemination) required to attain confirmedpregnancy.

All of the above reproduct i on parameters weremeasured and analyzed using chi-square analysis. Astatistically significant difference between the twogroups of cows was ascertained in regards to the numberof cows that showed fi rst heat wi th in 30 days aftercalving, the number of cows that could be rebred andconceive on the first breeding within 55 days aftercalving, and the number of cows that became pregnantwithin 80 days after calving. The chi-square analysis,as it related to the number of artificial inseminations


per pregnancy, was chosen to demonstrate any differencesbetween the number of cows belonging to the two groups.

In addition to reproductive data, information wasalso gathered on the conditions of birth; that is, thedegree of ease or di ffi cul ty. The pathology of thereproductive tract was determined as well. Because oftheir subjective nature, neither of these data fieldswere statistically analyzed. The milk production dataare shown in Table 5.

I Table 5 II Milk Production I

Experimental ControlGroup A Group B

Number cows at beginning 23 23

Number cows at conclusion 20 19

Average days to lactation after calving 9.9 10.2

Average initial milk production 34.19 Kg 34.08 Kg(75.21 lbs) (74.98 lbs)

Average final milk production 25.48 Kg 24.36 Kg(56.84 lbs) (53.59 lbs)

Average total milk production 33.29 Kg 32.74 Kg(73.23lbs) (72.03 1bs)

As shown in Table 5, there is a slight differenceof approximately 2% in favor of the experimental groupas far as tota1 mi 1k product ion i s concerned. Th i slevel of difference is too small to be statisticallysignificant although there may be some economicsignificance. In effect, the data described in Table 5,when analyzed weekly, showed a gentle see-sawingvariation in production which follows a common trend


between both groups. This trend appears not to be tiedto the use of the amino acid chelates, but rather thelength of time the cow has been in its lactation cycle(P<O.05). The production tends to increase graduallyand constantly for approximately two months, thendecline. For those reasons, as far as the production ofmi 1k i s concerned, the differences between the twogroups is negligible.

These data are not consistent with the datareported by Ashmead(8). He presented data on a 95 cowhigh production Wisconsin dairy in which milk productionwas significantly increased when the cows startedreceiving a supplement of amino acid chelates. In thiscase, the normal lactation curve described above did notoccur. Instead, milk production continued to increaseduring the 21 month study. These data are summarized inFigure 1. The experimental designs of the two studiesare slightly different. Different breeds of cattle werealso used in the two experiments. Finally, differentfeedstuffs were also provided in the two studies. Each,or a combination of all these factors, as well as otherunidentified factors, may have contributed to thedifferent results in the two studies.


THE EFFECT OF AMINO ACID CHELATESON MILK PRODUCTION

Without AminoAcid Chelates

w<D<t:a:ill>~

oa:wI

<Dz-..J--l .

oc:

With AminoAcid Chelates

.......................... O' ••

o 2 4 6 8 10 12 14 16 18 20

MONTH

Figure 1. The effect of amino acid chel ates onmilk production.

Returning to the current experiment, Table 6summarizes the qualitative data on the samples of milkthat were taken. There is some statistical significanceon the percentage of butterfat (P<O.OS) and protein(P<O.OI) as a function of the time of sampling as itrelates to the lactation curve. With the advancement ofthe lactation phase, there is an increase in thepercentage of protein and butterfat in the milk. Thesedifferences may also be related to the amino acidchelates in the diet.


I Table 6 II Qualitative Differences in Mi 1k I


pH 6.59 6.58

Butterfat 3.16% 3.12%a

Protein 3.06% 2.98%b

Cell cultures (X 1000) 297.86 326.83

Sugar content 4.89% 4.77%

aSignificant for P < 0.05bS ign i fi cant for P < 0.01

These observat ions concerni ng butterfat product ionseem to be confirmed in another study involving a dairycow herd. (8) As the 1actat i on peri ad advanced J butterfatproduction increased. During that period of increasethe cows were receiving amino acid chelates as asuppl ement. When the suppl ement was di scant i nued thebutterfat production declined even though the lactationperiod was still increasing. When the animals resumedreceiving the amino acid chelates, their butterfatproduction increased. These data are seen in Figure 2.


THE EFFECT OF AMINO ACID CHELATES ON BUTTERFAT PRODUCTION

900J£.

+' +oJc: a

~ 850 ~.....~

l~ 800I]

'+-

t~ 750:1

CD

~

0700

1J-J

650

600

Yleld Incr8eQe on

~rno Acid Chelat..S~IQf'Mnt

Drecontrr-...ed

2 3 4 5 6 7 8 9 10 11 12

MONTH

Figure 2. The effect of amino acid chelates onbutterfat production.

The reproductive data were also statisticallysignificant. These data are shown in Table 7, anddemonstrate clear differences between the two groups.The period between parturition and conception was almost25 days less for the experimental group which receivedthe amino acid chelate supplement. This reduction intime was due, in part, to the animals coming into heatsooner so that they could be rebred sooner.Furthermore, the number of times the experimental grouphad to be rebred to ach ieve concept i on was reducedalmost 24% compared to the control group. It took 2.05artificial inseminations for the experimental group and2.68 for the controls to achieve impregnation. Thenumber of confirmed pregnancies that occurred in thefirst 80 days after calving were 45 for the experimentalgroup compared to 15 for the control group. The 30


extra pregnancies amounted to a 300% increase and wasstatistically significant at P<0.05. The total numberof days that it took the entire group to conceive andhave a confirmed pregnancy was 19% less for theexperimental group.

I

Table 7

IReproductive Activity


Number of cows 20 19

Period from parturition 54.70:!: 15.38 52.89:!: 17.79to first heat (days)

Period from parturition to 67.45:t 36.47 66.37 :t 16.92to insemination (days)

Number of pregnancies in 45" 15"80 days from parturition

Total days for group from 102.25 ± 48.81 127.00 ± 44.59parturition to conception

Number of inseminations 2.05 ± 1.00 2.68 ± 1.70per insured pregnancy

.. The difference is significant for P< 0.05.

Even though this particular study did notdemonstrate significant increases in milk production,the qua1i ty of the mi 1k produced, and the earl i erpregnancies more than economically justify the use ofthe amino acid chelates. The establishment of apregnancy caused a reduct ion in mi 1k product ion. Insome cases, this was almost a month earlier for the cowsthat received the amino acid chelates in their diets.Cows in the experimental group, having begun theirgestation period earlier, had higher peaks in their


lactation levels than the controls. The peaks were nothigh enough to be statistically significant, however.

The final area of observation was concerned withthe general health of the cows. Table 8 summarizesthese observations. As noted earlier, the differenceswere not analyzed statistically. Nevertheless theexperimental group of cows, which had received the aminoacid chelates, had fewer problems than the controlgroup.

Table 8

Health Problems Related to Calving and Reproduction


Retained placentas 0 2

Metritis 6 5

Ovarian cysts 0 0

Persistent corpus luteum 0 0

Difficult birth 2 5

Trace element supplementation is gaining inimportance in the feed of dairy cattle. As the animalsare subjected to different environmental conditions,coupled with the quality of the diet and the varyingconcentrations of minerals in feedstuffs, manyvariations occur in the absorption and metabolism of theminerals in the cow. A deficiency may manifest itselfas much by the state of the cow's health (including its


abi 1i ty to reproduce) as by the breed i ng performanceitself. The breeding ability of the high production cowis greatly influenced by its diet. This has beenrepeatedly demonstrated with metabolic pathologies anddisturbances in reproductive efficiency.

Because of the benefits of trace elements on thereproductive abilities of cows, it seems logical toinclude amino acid chelates in their diets. The resultsof this research conducted on 46 milk cows with highmilk production potential demonstrated that bysupplementing the diets of the cows with amino acidchelates, the period from calving to a new pregnancy wasreduced by about 25 days. The number of art i fi cia1inseminations per cow required, to achieve a confirmedpregnancy was reduced by 0.6 units. In this study, theamino acid chelates did not significantly modify milkproduction, although they stimulated a slight increasein the early phase of lactation and promoted a greaterdepress ion ina success i ve phase (due to an earl i erpregnancy) with statistically significant improvementsin milk quality.

The improvement in reproductive efficiency, whichincludes less breedings per pregnancy and a gestationperiod which begins earlier, remain the most interestingand economically viable aspects of these amino acidchelates in the feeding of dairy cattle.


References

1. Miller, W., Dairy Cattle Feeding and Nutrition(New York: Academic Press) 18-21, 1979.

2. McCullough, M., Optimum Feeding of Dairy Animalsfor Meat and Milk (Athens: University of GeorgiaPress) 64, 1973.


4. Suttle, N., "Trace Element Interactions inAnimals," in Nicholas, D. and Egan A., eds.,Trace Elements in Soil, Plant and Animal Systems(New York: Academic Press) 271, 1975.

5. Dyer, I., "Mineral Requirements," in Hafez, E.and Dyer, I., Animal Growth and Nutrition(Philadelphia: Lea &Febiger) 312, 1969.

6. Underwood, E., Trace Elements in Human and AnimalNutrition (New York: Academic Press) 7, 1977.

7. Baker, L., Bovine Health Programming (Cleveland:United Publishing Corp.) 1968.

8. Ashmead, D., "Nutritional Technology,"Proceedings of an Albion Laboratories Seminar,Salt Lake City, 1980.

Chapter 11

THE FEEDING OF AMINO ACID CHELATESUPPLEMENTS TO BEEF CALVES

James A. BolingUniversity of Kentucky

The body growth of an animal involves hyperplasia,which is the multiplication of the cells that make upthe tissues and organs of the animals, and hypertrophy,which is an increase in the size of each body cell fromreplication to maturity. During growth where body cellsare increasing in numbers and sizes, extracellularsubstances also accumulate.(1)

Feedstuffs necessary to sustain this growth can begrouped into six basic nutrients. These includeprotein, carbohydrates, lipids, vitamins, minerals, andwater. Through their involvement with protein asenzymatic catalysts, the vitamins, and frequentlyminerals, are indirectly involved in the growth andmaintenance of the animal's tissues. P~otein and oftenminerals are also directly involved.() Lipids (forenergy and structure) and carbohydrates (for energy) andwater are also required.

The growth of the animal is intimately related tothe anabolic synthesis of a wide variety of cellstructures from the above nutrients including thenuclei, nucleol i, chromosomes, centrioles, mitochondria,cytoplasm, organelles, enzymes, and cell membranes. Thebiochemical synthesis of the necessary macromoleculesfor hyperplasia and hypertrophy is closely associatedwith energy yielding reactions.(1)

The post-weani ng growth rate of cattl e is affectedby hereditary factors, the ambient temperature, theability of each animal to adapt to its environment, thesocial stress within the herd, and the availability ofessential nutrients from the feed.(1) A retardation of

187


the growth rate in a bovine is associated with thefailure of DNA to replicate, which causes a reduction incellular protein synthesis. Hormonal imbalances,chromosome anomalies, radiation, drugs, toxins, hypoxia,sympathetic over-activity, infection,{I) or a deficiencyof essential nutrients, such as zinc(3) may interferewith normal DNA replication. The reduction of proteinsynthesis may be caused by low protein intake, a proteinloss, impaired amino acid transport into the body cells,hormonal deficiencies, abnormal ratios of amino acids, (1)

or a lack of specific vitamins and minerals to functionwithin the enzymes which are necessary for proteinsynthes is. (4) When there are severe defi ci enc i es orexcesses of minerals, striking changes in the metabolismand deve1opment of the an i rna1 can occur. (I)

The absorption of nutrients from the intestine aswell as their uptake by body cells depend on minerals.Some vitamins, such as thiamine and vitamin B12 , containminerals as essential components. In addition to thesevitamins, hormones, enzymes, and other vital bodycompounds contain minerals as essential parts of theirstructure. Some minerals, such as calcium andphosphorus, are building elements for bovine tissue.Other minerals are indirectly involved in the growthprocess as catalysts to enzymatic reactions that lead tosynthesis of body compounds, or to the release of energywhich is necessary for growth. Adeficiency of a singlerequired mineral will result in retarded growth, even ifall of the other necessary nutrients are available,because there is a synergistic relationship between themi nera1s and the other nutri ents. (4,6)

Dyer has summarized the primary functions ofseveral essential minerals. These are seen in Table1. (5) As far as growth i s concerned, a mi nera1 isconsidered essential if it aids in that process. Theremust be improvement in growth when the mineral is addedto the diet in physiological amounts. There must also

The Feeding of Amino Acid Chelate Supplements to Beef Calves 189

be evidence of a deficiency when that mineral is removedfrom the diet. (4)

1::1=============T=a=b=le=l=============1

1 Summary of Primary Mineral Functions 1

Element

A. Macroelements

Some Functions

Calcium Osteogenesis; decreases nerve irritability; necessary forblood clotting; decreases cell membrane permeability;ATPase activator; needed for osmoregulation.

Chloride Maintenance of proper osmotic concentrations; carbondioxide transport; solubility of proteins; activatessalivary amylase.

Magnesium Osteogenesis; enzyme activator (kinases, mutases, enolase,ATPases, cholinesterase, alkaline phosphatases,isocitricdehydrogenase and arginase); helps decrease tissueirritability.

Phosphorus Osteogenesis; necessary for fat and carbohydrate metabolism(components of ATP, DNP +, glucose-l-phosphate,phosphoproteins, phospholipids, and nucleic acids).

Potassium Electrolyte and water balance; intracellular osmoticconcentrations; activates enzyme systems such as pyruvickinase and those having to do with phosphorylation ofcreatine; increases heart beat; increases tissueirritability.

Sodium Electrolyte and water balance (neutrality regulator);exchange with potassium ions during nerve and muscleaction; increases tissue irritability; osmotic pressureregulation.

Sulfur Component of cysteine, methionine, biotin, sulfolipids,cystine, sulfonated polysaccharides and many ot~er

metabolites.


Table 1 (continued)

Summary of Primary Mineral Functions

Element Some Functions

B. Microelements

Aluminum;arsenic;barium; boron;bromine No known function in animals.

Chromium Has an insulin-like effect on glucose metabolism.

Cobalt Activator of certain peptidases; required in thesynthesis of vitamin B12 •

Copper Prosthetic group of hemocyanins; increases ironabsorption; necessary for formation of erythrocytes;component of flavoproteins: cofactor for tyrosinase,ascorbic acid oxidase cytochrome oxidase, uricase,plasma monoamine oxidase, and ceruloplasmin.

Fluorine Prevention of dental caries; no specific growth functionknown.

Iodine Needed for thyroxin synthesis (homeostatic regulator).

Iron Component of hemoglobin and myoglobin; component ofcytochromes, cytochrome oxidase and xanthine oxidase.

Manganese Needed for synthesis of cholesterol (mevalonic kinase);osteogenesi s; norma,l funct; oni ng of the reproduct i vesystem; needed for bone phosphatase and may be acomponent of arginase; muscle ATPase and cholineesterase may depend on Mn++.

Molybdenum

Nickel

Needed for xanthine oxidase, aldehyde oxidase, and ironflavoproteins. Molybdenum deficiency has not beenproduced in mammals, but has been observed in birds.

No definitely known function in animals; may beassociated with pigmentation; bound to RNA.


1===========T=a=bl=e=l=(=c=on=t=i=nu=e=d=)=========~III Summary of Primary Mineral Functions I

Element

Selenium

Strontium

Tin; titanium

Vanadium

Zinc

Some Functions

B. Microelements (continued)

Needed in prevention of muscular dystrophy inlaboratory animals, exudative diathesis in chicks, andhepatic necrosis in rats.

No definitely known function in animals, although somesuggest its possible need for bone calcification.

No known function in animals.

Depresses cholesterol and phospholipid biosynthesis;reduces ATP, COA; stimulates MAO; may be necessary fornormal bone and teeth formation.

Cofactor of carbonic anhydrase, and certaindehydrogenases, and phosphatases; required for growth;chicks require zinc for bone and feather development;may be required for RNA synthesis.

Recognizing the need for minerals to effect thedigestion of other nutrients, Verini-Suppl izi, et li., (7)

at the University of Perugia fed 440 Kg yearling calves8 9 per head per day of a ration of amino acid chelateswhich is shown in Table 2. This mineral supplement wasin addition to the standard university diet which alsocontained minerals, but not in an amino acid chelateform. The controls received the same feed without theamino acid chelates.


Table 2

Amino Acid Chelate Formula Fed by the University ofPerugia

Mi nera1

IronZincManganeseCopperCobalt

mgjkg chelate

23,4004,8004,4003,300

100

Rumen mater; a1 was taken from both groups ofanimals and analyzed. It was found that those calvesthat had received the amino acid chelates had greatercellulases and amylase activity in the rumen with acorresponding decrease in methane production.

Those researchers then proceeded to demonstratethat the enhanced digestibility of the ruminantfeedstuffs, with the amino acid chelates present,resulted in greater growth. In this study, they dividedeleven- to twelve-month old bull Chianina beef calvesinto two groups of seven each. One group was thecontrol and the other was the experimental group. Theanimals had an average beginning weight of 440 Kg, each,and were cons idered homogeneous. Each an imal was fedthe same commercial feed ration plus alfalfa hay. Totalconsumption per animal was approximately 8 Kg per headper day. To the feed of the experimental group wasadded 8 gm per head, per day, of the amino acid chelateformula shown in Table 2.

Weight gains and feed consumption were measuredthroughout the 300 day experiment. Weighing of each


animal was done monthly after fasting from food andwater for twelve hours. At twenty months of age, whenthe animals reached a live weight of approximately 700Kg, they were slaughtered and their carcass qual itydetermined.

Table 3 shows the mean daily weight gains for eachgroup. Table 4 shows the mean monthly weight gains.The cattle which received the amino acid chelates grewat a faster rate and produced a total of 309 Kg moremeat on the same amount of feed.

I Table 3 II Mean Daily Weight Gains (Kg) I

Group Beg. DaysWgt.

32 62 96 128 152 188 216 246 276

Control 441 1.125 1.100 0.911 0.937 0.934 0.867 0.821 0.767 0.700

Experi- 439 1.156 1.167 1.059 1.031 1.000 0.933 0.921 0.808 0.734mental

I Table 4 II Mean Monthly Weight Gains (Kg) I

Group 11 12 13 14 15 16 17 18 19 20

Control 441 477 510 543 573 601 627 650 673 694

Experimental 439 476 511 547 580 610 638 664 688 719

Table 5 presents the monthly feed conversion data.The improvements were most significant during the lastmonths of fattening, when feed consumption increased but


growth rates decreased. There was 12. 2% 1ess feedconsumed by the group receiving the amino acid chelates.

I Table 5 IMonthly Feed Conversion (Kg feed/Kg weight)

12 13 14 15 16 17 18 19 20

Control 6.01 6.14 7.18 8.39 8.41 11.49 12.13 13.12 14.37

Exper; menta1 5.85 5.79 6.38 7.09 7.86 10.67 10.81 12.45 13.57

Finally, carcass quality was measured. Table 6presents those data. There was significant improvementin the carcass quality of those cattle which were fedthe amino acid chelate supplement.


I Table 6 II Carcass Quality Data I

Control Group

Hot ColdCar- Car- Appo-

I Live cass cass % si- CarcassSlaugh S. % S. % Loss tion Struc-

No. Wgt. Wgt. Yield Wgt. Yield Water Meat Color Fat ture

662 670 404 60.30 396 50.10 1.98 Strong Red-Pink Right Medium625 713 432 60.60 424 59.47 1.85 Light Red-Pink Right Medium640 723 441 61.00 433 59.89 1.81 Light Red-Pink Right Optimum636 692 422 60.98 410 59.25 2.84 Strong Red-Pink Right Medium624 709 431 60.79 427 60.26 0.93 Strong Red-Pink Right Optimum637 681 408 59.91 401 58.88 1.72 Strong Red-Pink Right Medium620 670 416 62.00 409 61.04 1.68 Strong Red-Pink Right Optimum--- --- --- ----- --- ----- ---- --------------- ----- -------

-X 694 60.80 58.41 1.83

Amino Acid Chelate Group

614 781 478 61.20 469 60.05 1.88 Light Red-Pink Right Optimum642 724 443 61.19 435 60.08 1.81 Light Red-Pink Right Optimum641 630 412 59.71 403 58.41 2.18 Light Red-Pink Right Optimum652 714 445 62.32 437 61.20 1.80 Light Red-Pink Right Medium627 687 425 61.86 418 60.41 1.65 Light Red-Pink Right Medium634 800 489 61.12 480 60.00 1.84 Light Red-Pink Right Optimum651 637 398 62.48 392 61.54 1.51 Light Red-Pink Right Medium--- --- --- ----- --- ----- ---- -------------- ----- -------

-X 710 61.41 60.24 1.81

In the above research, it was noted that theinclusion of amino acid chelated minerals in the dietsof those growing beef cattle increased the digestiveactivity in the rumen, particularly as it related tostarches and cellulose. Knowing that several digestiveenzymes are activated by minerals, and that magnesium,in particular, is essential for amino acid metabolism,(4)it was proposed that an experi ment (8) be des igned thatmeasured the value of this amino acid chelate, as well


chelate, as well as several other amino acid chelates,when fed to growing steers receiving different dietarylevels of protein.

Manganese is essent i a1 for both 1i pi d andcarbohydrate metabolism(9J, which in turn play roles inprotein metabolism as seen in Figure 1.(10) Manganese isalso essential in ruminant growth.(9) Consequently,manganese amino acid chelate was included in thismineral ration.

ABOMASUMAND SMALLINTESTINE

zUJ~:JII:

Figure 1. Some of the known enzyme systems thatare of importance in feed utilization byruminants.


Zinc, as an amino acid chelate, was also includedin the formulation because it plays key roles incarbohydrate metabolism, protein synthesis, and nucleicacid metabolism. Zinc deficient cattle grow more slowlythan those with optimum zinc consumption and have lesseffi ci ent feed ut i1i zat ion. (11)

Iron, copper and cobalt, as amino acid chelates,were also included in the feed supplement to approximatethe work of Verini-Suppl izi, et lie (7) These researchersshowed that when these amino acid chelates are fed tofattening calves, they grew faster with better feedconversion than calves receiving the same minerals asinorganic salts.

Forty Simmental X Angus or Hereford crossbredsteers were selected for our research at the Universityof Kentucky. Their average beginning weight was 731.5pounds (331.8 Kg). They were put into twenty pens oftwo steers each, according to breed and weight. All ofthe breeds were equally involved in the four replicas ofeach proposed treatment.

Fi ve di fferent feed formul as were fed to thereplicates as summarized in Table 7. There was onepositive control (Diet A) which met National ResearchCouncil recommendations for potassium, sulfur, calcium,phosphorous and trace mi nera1 sal t. The other fourdiets contained varying amounts of protein (all lessthan Diet A) plus magnesium, zinc, and manganese, iron,copper and cobalt as amino acid chelates.


Table 7

Composition of Experimental Diets

Diet

Ingredient, % A B C D E

Cottonseed hulls 30.00Cracked yellow corn 55.51Soybean meal (44% CP) 12.40Trace mineral salt 0.50Ground limestone 0.85Dicalcium phosphate 0.65Potassium chloride 0.07Manganese~ 0.59Magnesium~ 0.50Zinca 0.74Irona 0.50Coppera 0.12Cobalt~ 0.02Vitamin ADE premix 0.02

I· Supplied as an amino acid chelate.

30.0060.526.900.450.890.100.070.590.500.740.500.120.020.02

30.0060.766.900.480.860.430.070.590.500.740.500.120.020.02

30.0063.284.100.450.890.140.070.590.500.740.500.120.020.02

30.0063.524.100.480.470.470.070.590.500.740.500.120.020.02

Diets A through E had crude protein analysis of11.7%, 10.0%, 9.7%, 8.8%, and 8.6%, respectively. Allof the calves receiving Band D diets were first fedDiet B for the first 84 days of the trial and then halfof them had their protein intakes reduced to Diet D forthe remainder of the study. The calves in the C and Egroups were first fed Diet C for 84 days and then halfof the calves were given Diet E for the remainder of thetrial.

Each steer was weighed on two consecutive days, atthe beg inn i ng and end of the experi ment, and eachsteer's weight averaged for the two days. Each animalwas also weighed one time every 28 days throughout theexperiment. Hip heights were measured on days 0, 56,84, and the day before termination of the study.Termination occurred on the 84th day when the weight ofthe steers was projected to reach 1150 pounds (523 Kg).


The weight gains and performance of the steers areshown in Table 8. Daily feed intake was almost the samefor each group except C - E which was slightly higher.This is reflected in larger animals and a greateraverage daily gain. The actual feed consumed per poundof gain was lower than any of the other groups except c.The significance of these observations is that thecalves in the C - E group received less crude protein intheir diet than any of the other groups, and yet theyseemed to out perform the other animals. This suggeststhat when the amino acid chelates are supplemented, alower amount of di etary protei n ; s ut; 1; zed by thecattle. When additional protein is provided itinterferes with the growth of the animals because theirnutrition is out of balance.

I Table 8 II Weight Gains and Performance of Steers I

Control Dietary Treatment

A 8 C B-D C-E

No. steers 8 8 8 8 8

lQ§ JSg Ibs ~ Ibs JSg lQ§ lS9 Ibs .!S9

Initial wgt. 739.2 335.3 728.2 330.3 732.8 332.4 728.2 330.3 744.4 337.7

Final wgt. 1134.4 514.6 1128.0 511.6 1155.5 524. 1 1146.1 519.9 1162.0 527.1

Feed intake 26.59 12.06 26.38 11.97 26.39 11.97 25.98 11.78 28.22 12.80per day

Average 2.67 1.21 2.70 1.22 2.86 1.30 2.40 1.09 3.00 1.36daily gain

Feed/Gain 9.96 9.n 9.23 10.83 9.41

Days to 148 148 148 174 139final wgt.


Hip measurements are shown in Table 9. There wereno real differences between the animals, and it wasconcluded that skeletal development was similar. Thisis a reasonable conclusion since all of the animalsreceived similar amounts of calcium and phosphorous forosteogenesis.

I Table 9 IHip Measurements of Steers

Control Dietary Treatments

A B C B-D C-E

In Cm In Cm In Cm In Cm In Cm

Initial 46.9 119.1 45.9 116.6 46.7 118.6 46.8 118.9 47.0 119.4Day 56 49.2 125.0 48.0 121.9 48.9 124.2 48.6 123.4 49.1 124.7Day 84 50.5 128.3 49.3 125.2 49.8 126.5 49.5 125.7 49.8 126.5Final 51.5 130.8 50.2 127.5 51.3 130.3 51.1 129.8 50.9 129.3

The carcass data are shown in Table 10. Althoughthe differences in the groups were not great, Group C E, which had the lowest protein intake dressed out withthe most meat, almost twenty pounds (9.1 kg) more perhead than the control group with an identical carcassgrading.


I Table 10 II Carcass Characteristics of Steers I

Control Dietary Treatments

A B C B-E C-E

Carcass weight,(lbXKg) 1447.8 656.7 1423.76.5.8 1.58.1 661 .• 140•. 3 637.0 1502.7681.6Dressing % 58.0 57.2 57.2 55.6 58.6

Carcass grade 12.6 12.8 11.2 11.4 12.6

lYield grade 2.4 2.6 2.5 2.8 2.3Longissimus area (sqin),(cm 2

) 12.0 77.• 11.6 74.8 12.0 77.4 10.8 69.7 13.1 84.5Kidney fat, % 2.75 2.69 2.81 2.25 2.94Fat over ri b (in),(cm) 0.29 0.74 0.33 0.84 0.29 0.74 0.38 0.97 0.34 0.86Marbling 5.4 5.1 4.6 4.9 5.6Maturity 14.0 14.0 14.0 14.0 14.0

From these data it is obvious that the inclusionof amino acid chelates in the feeds of growing beef aidsin their growth. Not only do they grow faster withsuperior feed conversions, but they seem to be able togenerate superi or musc1e growth on lower 1eve1s ofdietary protein in their feeds than NRC recommendations.Whether the inclusion of the amino acid chelates causesimproved digestion of the cattle's feedstuffs, increasedabsorption of digested nutrients, enhanced metabol ism ofthe nutrients after absorption, or any combination ofall three possibilities is unknown at present.


References

1. Hafez, E., "Introduction to animal growth," inHafez, E. and Dyer, I., eds., Animal Growth andNutrition (Philadelphia: Lea & Febiger) 1-15,1969.


3. O'Dell, B., "Introduction," in Pories, et li.,eds., Clinical Application of Zinc Metabolism(Springfield: Charles C Thomas) 5, 1974.

4. Guthrie, Ope cit., 184-191.

5. Dyer, I., "Mineral Requirements," in Hafez, E.and Dyer, I., eds., Animal Growth and Nutrition(Philadelphia: Lea & Febiger) 317, 1969.

6. Baker, L., Bovine Health Programming (Cleveland:United Publishing Corp.) 53, 1968.

7. Verini-Supplizi, A., et li., "Metalosates andFlavophospholypol in the fattening of beefcalves,1I Unpublished, 1986.

8. Boling, J., et li., "Performance of finishingsteers fed diets containing amino acid chelatesand varying levels of protein," in 1988 BeefCattle Research Report #306 (Lexington:University of Kentucky) 68, 1988.

9. Hurley, L., and Keen, C., "Manganese", in Mertz,W., ed., Trace Elements in Human and AnimalNutrition (Sun Diego: Academic Press) 203-209,1987.


10. McCullough, M., Optimum Feeding of Dairy Animalsfor Meat and Milk (Athens: University ofGeorgia) 68, 1973.

11. Miller, W., Dairy Cattle Feeding and Nutrition(New York: Academic Press) 134-135, 1979.

Section 3. SWINE

205

Chapter 12

THE ROLE OF IRON AMINO ACID CHELATE INPIG PERFORMANCE


For years adequate body store of iron have beenrecognized as being essential for optimum pigletperformance. The main reasons for the extremesensitivity of the baby pig to iron deficiencies are itshigh growth rate and its relative poor endowment of ironat birth.(1) Normally a piglet reaches four to fivetimes its birth weight by the end of three weeks, andabout eight times its birth weight at the end of eightweeks. In order to achieve this, it must have adequateiron. The effects of iron supplementation on pigletdevelopment can be seen in Table 1.(2) One half thepi gs were fed 5 mg of iron, as ferrous sul fate, perpound of body weight per day (2.3 mgjkg). The otherhalf received no supplemental iron in its feed. As thedata in this table indicate, there was a 17% increase inweight gain when the piglets received supplemental iron.

I Table 1 IEffect of Feeding Iron on Whole Body Compositionof Three-week Suckling Pigs

Pigs fed Litter matesiron not fed iron

Number of pigs analyzed 3 3Weight gain from birth tothe end of third weeka 492.00 420.00

Iron i n bodyb 3.66 1.56Iron i n b1oodb 31.50 15.00Iron i n 1i verb 9.50 1.72Iron in remainder of bodyc 51.00 29.90

I- gmb mg/g C mg I

207


Iron's influence on growth rates is due to itbeing an integral component of numerous essentialmetabolic enzymes that make up the billions of cellswhich together form the pig's body. Its most widelyrecognized role, of course, is in respiration. Here, itis responsible for allowing the 02/C02 exchange from thealveolar sacs of the lungs and the reciprocal exchangeat all individual cells of the body. Iron is alsoinvolved in the cytochrome systems at the terminus ofoxidative phosphorylation in basic cell metabolism.This is part of the major energy gathering and storingenity of the cell. The oxidative and reductivecapacities of iron are absolutely required thisimportant in energy gathering.

Respiration does not explain iron's role in pigletgrowth, however. Growth is a non-respiration, aspect ofthe iron function. Iron's role in growth results fromits involvement is involved in the synthesis ofpuri nes, (3) the bas ic component of DNA and RNA in thebody ce11 s. (~) DNA and RNA are the keys to the synthes isof new cellular protein, or in other words, animalgrowth. (5) If an iron defi ciency occurs in the pig, theheme funct ions of i ron wi 11 be the 1ast to show thedefect. The first will be the growth functions. Purineproduct i on and the subsequent manufacture of new protei nwill be retarded, thus retarding the rate of growth.

Postpartum piglet growth is partially dependentupon fetal nutri t ion. Early research suggested thatplacental transfer of iron from the pregnant sow to thefetus was inadequate. Consequent1y, in modern swi neproduction, the standard therapeutic approach has beento administer 100 mg I.M. of iron dextran to the newbaby pigs. However, subsequent research hasdemonstrated i ron dextran inject ions may not be theoptimum approach because the fetus has the capacity tostore adequate iron for early post-natal requirements ifthe gestating mother is provided a form of iron that cancross the placenta.

The Role of Iron Amino Acid Chelate in Pig Performance 209

An examination of a gestating 1itter of pigsduring the last few weeks of pregnancy demonstrates thatprotein and iron demands of the fetuses are high.Indeed the developing fetuses achieve over half of theirgrowth duri ng the 1ast one-th i rd of the gestat i on peri odas shown in Tabl e 2. ca) These data were obta i ned fromPoland China gilts bred to the boar of the same breed.All were fed the same diet and had an average daily gainof 1 to 1.25 pounds (0.45 to 0.57 kg).

I Table 2 IComulative Batch Weights and Composition of a Litter of Eight PigsDuring Gestation

Week Total Fresh Crudeof Weight Protein Ash Calcium Phosphorus Iron

Gestation (gm) (gm) (gm) (gm) (gm) (gm)

1 99 1.5 0.06 0.0002 0.002 0.822 366 8.5 0.6 0.005 0.028 4.23 787 23 2 0.036 0.12 114 1,354 47 5 0.14 0.36 225 2,062 83 10 0.40 0.83 376 2,909 130 18 0.96 1.61 577 3,891 191 30 2.0 2.8 828 5,005 285 45 3.8 4.7 1139 6,251 356 66 6.6 7.2 149

10 7,625 462 92 10.9 10.6 19111 9,127 585 125 17.1 15.1 23912 10,755 726 165 26 21 29413 12,507 886 213 38 28 35514 14,385 1,065 269 54 37 42315 16,384 1,263 335 74 47 49916 18,504 1,483 411 101 60 581

When iron is supplied as a true chelate of aminoacids, intestinal absorption of this mineral issignificantly enhanced, as demonstrated in Table 3. Inthis in vitro experiment, intestinal exposure to 50 mcgof iron in various forms for 2 minutes demonstrated 4.9times greater absorption of the chelate than the oxide,


3.8 times more than the sulfate, and 3.0 times more thanthe carbonate. (6)

I Table 3 IMean Jejunal Absorption of Iron as an Amino Acid

Chelate a Carbonate, a Sulfate, and Oxide in vitro

Iron Source Jejunal Absorption

Iron amino acid chelate 298 ppmIron carbonate 82 ppmIron sulfate 78 ppmIron oxide 61 ppm

Intestinal absorption does not necessarilyguarantee physiological usability. Iron that has beenabsorbed may be excreted back into the lower bowel oreliminated in the urine without ever having beenutilized by the animal. Research utilizing radioactiveisotopes has suggested that not only is intestinalabsorption of amino acid chelated iron increased oursalts, but body assimilation and tissue distribution ofthis form of iron are also enhanced, as shown in Table4.(7) In this particular study a single dose of 4.4microcuries of iron as ferrous sulfate or as the aminoacid chelate was orally administered to two groups oflaboratory animals. The animals were sacrificed 72hours after dosing, and their tissues assayed for ~Fe.

All of the tissues of the animals receiving the ironamino acid chelate contained more iron than the tissuesof the animals administered FeS04 • There was, however,more excreted iron in the feces and urine of animalsthat received the iron sulfate.


I Table 4 IMean Comparisons of ~Fe Retention and Distributionfrom Ferrous Sulfate and Iron Amino Acid Chelate

Chelate/SulfateFerrous Iron Amino (Decrease)

Body Tissue Sulfate Acid Chelate % Increase

Heart 63 151 140 %Liver 136 243 79 %Leg muscle 2 54 2,600 %Jaw muscle 14 138 712 %Brain 31 130 319 %Kidney 2 327 16,250 %Testes 20 109 445 %Blood serum 700 1,797 157 %Red blood cells 724 2,076 187 %Whole blood 1,355 4,215 211 %Feces 302,400 214,000 (29 %)Urine 490 370 (24 %)

This increased iron metabolism from the chelatesuggests that there may a1so be increased placenta1transport of the chelated iron.

Radioactive isotope experiments on laboratoryanimals confirm this increased placental transport ofamino acid chelated iron, as shown in Table 5.(9)

Gestating dams were given a single dose of 5 microcuriesof ~Fe, as either the amino acid chelate or as ferroussulfate, 72 hours before expected parturition. Twentyfour hours before parturition the mothers weresacrificed, their fetuses removed, and both the motherand fetuses assayed for 59Fe. These data demonstratethat, at this late stage of pregnancy, essentially noiron from the sulfate was able to cross the placenta,


whereas iron from the amino acid chelate was capable ofdoing so.

I Table 5 IMean Comparison of Absorption and Retention of Ironin Gestating Mothers and their Fetuses

Iron Source Increase(Decrease) ofIron Amino Acid

591ron Ami no Versus FerrousAcid Chelate 59 FeSO.. Sulfate

Amount of iron retained bymother 70.4 % 42.7 % 65 %

Total amount of iron excretedin feces by mother 24.4 % 29.6 % (18 %)

Total amount of iron excretedin urine by mother 5.0 % 27.7 % (81 %)

Amount of iron passed on toyoung 0.2 % • 0 4130 %

(7.3 meg) (lee/min)(42.3 cc/min)

As indicated previously, the intestinal absorptionof i ron ami no acid chel ate is through a di fferentpathway than ferrous sulfate. The chelate arrives inthe blood as the same small molecule (approximately 300daltons) that was originally ingested, whereas the ironfrom the sulfate which arrives in the blood is bonded totransferrin, a relatively large molecule (approximately86,000 daltons). It is the very small molecular weightof the iron amino acid chelate that allows it to freelytraverse the mature placenta, while this tissueeffectively "screens out" the iron bound to the largertransferrin molecule.(24)

Swine field trials with iron amino acid chelatessupport the above isotope studies. In work reported byBrady, et ~., at Michigan State University, twelve sowswere fed 8.5 grams of iron amino acid chelate


(containing 0.85 grams of iron) daily, starting fourweeks prior to expected parturition. One week beforefarrowing the level was increased to seventeen grams ofchelate (1.7 grams of iron) daily. At birth, one pigfrom each litter was sacrificed, and the liver, spleen,and skeletal muscles were analyzed for their ironcontents by atomi c absorpt i on spectrophotometry. Thepiglets from sows receiving the iron amino acid chelateduring gestation had higher levels of iron in theirbodi es than the control s. Tabl e 6 shows the resul ts. (10)

Table 6

Mean Iron Concentration at Birth Among Pigs of SowsFed Diets Supplemented or Unsupplemented with IronAmino Acid Chelate

Piglet Tissue Iron Sow Diet

Hemoglobin, g/dlPlasma Fe, g/dlLiver Fe, ppmSpleen Fe, ppmMuscle Fe, ppm

- Chelate

8.2102.0155.0105.0

9.4

+ Chelate

9.2112.0208.0114.0

9.7

In another study similar findings were found. Inthis trial, however, the investigators fed one group often gestating sows 5 grams of iron amino acid chelate(500 mg of iron) per day, which was less iron than thepigs received in the study conducted at Michigan StateUn i vers i ty. The experi ment commenced 21 days beforeexpected farrowi ng. The other group, the cantro1s,received the same gestation diet as the treated groupexcept that the supp1emented i ron was from ferroussulfate. All piglets from both the experimental and


control groups were sacrificed - half from each group atbirth, and the other half at seven days of age. Table7 reports these findings.(l1) The differences in liveriron contents in Tables 6 and 7 are due to Table 7 databeing measured on a dry-weight basis, while Table 6values were measured on a wet-weight basis. Thecomparative differences of liver-iron contents of thetreated and control groups in both tests are thussimilar in direction although not identical.

Table 7

Mean Iron Concentration in Piglet Livers on Day ofBirth and at 7 Days of Life

Sows Fed IronControl Amino AcidSows Chelate

Number of sowsNumber of living piglets bornMean weight at birth (Kg)Mean content of iron in liverat birth (ppm) on dry weightbasis

Mean content of iron in liverat 7 days of life (ppm) ondry weight basis

10102

1.256

641

252

10991.387

1,225

458

The mean birth weight of the piglets in the twogroups should be noted. The piglets from the treatedgroup were 10% heavier than the controls. Based on therole of iron in purine production, which was discussedearl; er, a heavi er bi rth wei ght was to be expected.When fetal iron nutrition is increased, as suggested byTables 5, 6, and 7, then greater protein production andincreased fetal growth should occur because of iron's


metabolic role in the production of tissue protein,assuming the other essential nutrients, such as zinc,amino acid, etc., are equally available to the fetuses.

This increased fetal growth can be furtherobserved in another study involving 460 piglets. Inth is experi ment, the sows in the experi menta1 groupreceived 350 mg of iron daily, as an amino acid chelate,start i n9 28 days pri or to expected farrowi n9. Thecontrol group of sows received no supplemental gestationi ron a1though the iron in the feed met NRCrecommendations. At farrowing, piglets from the controlgroup were injected with iron dextran. No other ironwas suppl emented to ei ther group after parturi t ion.Piglets from the experimental group did not receive anyiron dextran injections. The mean piglet weights in thetwo groups from birth to eight weeks of age are shown inTab1e 8. (l2)

;

Table 8 IMean Weight Increase (Kg) of 460 Pigs from Birth toEight Weeks

Control Experimental % Increase

Birth 1.30 1.40 7.692 Weeks 4.33 4.70 8.555 Weeks 9.44 9.79 3.718 Weeks 11.77 12.13 3.06

A heavier birth weight has two major advantages:The first, as suggested from Table 8, is a larger pigthroughout 1i fe. As shown, the experi mental group,which received superior fetal iron nutrition, maintaineda heavier weight than the controls. Widdowson(lJ) foundthat insufficient intrauterine nutrition resulted in


curtailing the rate of body cell division in runtsduring gestation, which subsequently curtailed generalsomatic growth in runt pigs after farrowing. Anundernourished fetus had fewer cells per tissue or organfollowing farrowing, which became a permanent reductionand account for its smaller size. It is, therefore, notuncommon to farrow runt pigs that never attain the samefinal growth size as their litter mates. Because of thecurtailed cell growth in the fetuses receiving less thanadequate intrauterine nutrition, there is also apermanent reduction in the mature cell size of the cellscomposing the organs of the pig. Pigs which develop inthis kind of intrauterine environment carry these twocellular defects during their entire post-natallives.(lJ) Widdowson's research provides an explanationto an earl i er invest igat i on conducted at Iowa StateUniversity, which, on the average, showed that adifference of one pound (0.45 Kg) at birth wasaccompanied by a 7.78 pound (3.54 Kg) difference atwean i ng . (14)

The effects of chelated amino acid iron in fetalnutrition and its market consequences are borne out inanother study. One group of sows was fed 250 ppm ofchelated iron amino acid mixed in the of feed and feddaily during the last three weeks of gestation andthroughout 1actat ion. Tabl e 9 presents the wei ghtdifferences of those piglets compared to the controls,which received exactly the same treatment and feed,excluding the supplemental iron.(lS) The iron amino acidchelate was not included in any of the pig starter,grower, or fi nish i ng feeds of either group, so thesedata demonstrate only the positive consequences ofadequate placental transfer of iron during gestation.

The Role of Iron Amino Acid Chelate in Pig Performance 21 7

I Table 9 IMean Weight Difference from Birth to Market of Pigs from Sows FedIron Amino Acid Chelates or No Chelates

Chelated Iron Sows Not Fed Weight %Fed Sows Chelated Iron Difference Difference

Birth 3. 15 1bs . 2.95 lbs. 0.2 lbs. 6.7 %weight (1.43 Kg) (1.34 Kg) (0.09 Kg)

4 week 15.00 lbs. 14.40 lbs. 0.6 lbs. 4.2 %weight (6.82 Kg) (6.55 Kg) (0.27 Kg)

9 week 48.70 lbs. 46.20 lbs. 2.5 lbs. 5.5 %weight (22.14 Kg) (21.00 Kg) (1.14 Kg)

5'12 month 219.70 lbs. 208.50 lbs. 11.2 1bs. 5.7 %weight (99.96 Kg) (94.77 Kg) (5.09 Kg)

The group recelvlng amino acid chelated ironduri ng the pre-nata1 gestat i on peri od was not on1yheavier at birth, but remained approximately 5% heavierto market.

The second advantage of heavier birth weights inpigs is a lower piglet mortality. In an experimentinvolves 1,948 litters of pigs conducted at Iowa StateUniversity, it was shown that, when all other factorswere equal, piglet survival was directly related tobirth weight, as shown in Table 10.(16)


I Table 10 I

I The Effect of Birth Weight on Piglet Mortality IBirth Weight No. of % of % of

Pigs Population Survival

Under 2 lbs. (0.9 Kg) 1,035 6 42

2.°1bs . (0.9 Kg) to2.4 1bs . (1.1 Kg) 2,367 13 68

2.5 1bs . (1.1 Kg) to2.9 1bs . (1.3 Kg) 4, 197 24 75

3.°1bs. (1.4 Kg) to3.4 1bs. (1.5 Kg) 5,012 29 82

3.5 1bs . (1.6 Kg) to3.9 1bs. (1.8 Kg) 3,268 19 86

4.0 lbs. (I .8 kg)& over 1,734 10 88

Total 17,613 100 77 %

This research substantiates the fact that sows orgilts fed iron amino acid chelate during gestationcontribute to heavier pigs at birth which enjoyincreased liveability. Table 11 summarizes the effectsof heavier birth weights, due to fetal iron nutrition,and the resulting heavier weights and lower mortalityrates. (17) All these groups of sows rece; ved the samegestation diet except group 3 which received 250 ppm ofiron amino acid chelate in its feed for 30 days beforeexpected parturition. At farrowing, group 2 pigletsreceived an intramuscular injection of 100 mg of irondextran. Neither group 1 nor group 3 piglets received


iron dextran injections. Except for the iron dextran,and the i ron ami no ac id che1ate , all three groups ofpiglets were treated and fed identically. The resultsshowed 36% and 21% greater weights at weaning for thechelate group over the controls and the iron dextrangroup, respectively.

I Table 11 IThe Effect of Iron Amino Acid Chelate on Birth and Weaning

Weights and Piglet Mortality

Control Iron Dextran Iron ChelateI

Group 1 Group 2 Group 3

Number of pigs farrowed 144 285 168

Average birth weight 2.86lbs. 2.82Ibs. 2.99 Ibs.(1.3O Kg) (1.28 Kg) (1.36 Kg)

Number of pigs weaned 115 233 157

% pigs weaned 79.9 81.7 93.5

Avg. weaning weight 14.76Ibs. 16.53 Ibs. 17.43Ibs.(6.70 Kg) (7.50 Kg) (7.91 Kg)

Mortality 20.1 % 18.3 % 6.5%

Total average weaning 11.95Ibs. 13.51 Ibs. 16.28Ibs.weightjpigsfarrowed (5.42 Kg) (6.13 Kg) (7.38 Kg)

Other field studies, shown in Tables 12 and 13,also illustrate the reduced mortality of baby pigsfarrowed from mothers fed ami no ac id che1ated iron. 01.19)

These resul ts are attri buted to increased placenta1transfer of that particular iron during gestation.


I Table 12 IThe Effect of Iron Amino Acid Chelate onPiglet Mortality

% Livability

Number of Days Chelate Control Difference

III sows 108 sows

Farm 1 1 - 3 days 97.35 % 93.91 % + 3.44 %

3 days - 7 weeks 95.45 % 88.89 % + 6.56 %

birth - 7 weeks 92.92 % 83.48 % + 9.44 %

87 sows 73 sows

Farm 2 1 - 3 days 97.75 % 93.59 % + 4.16 %

3 days - 7 weeks 96.55 % 91.18 % + 5.37 %

birth - 7 weeks 94.38 % 85.90 % + 8.48 %


Table 13

The Effect of Iron Amino Acid Chelate onPiglet Mortality

35 35Trial ControlSows Sows

~verage total born per litter~verage born alive per litterAverage born dead per litterAverage reared to 3 wks. per litterMortality % of total bornBorn dead % of total born

(stillbirths)

11.9210.901.02

10.2913.678.56

11.209.891.319.81

16.8811.70

While the above program appears to offersignificant improvements to swine production, they arenot a panacea. I f the producer ignores good tota1nutrition, or good swine handling practices, or diseaseprevention and control, he cannot expect the equivalentperformance from his pigs. The presence of infectiousdiseases will also seriously hamper a pig's ability toutilize nutrients as seen in Figure 1.(20)


PHAGOCYTIC ACTIVITY

DEPRESSION OF PLASMA AMINO ACIDS AND ZN

SALUAESIS. RETENTION OF URINARY PO. AND ZN

INCREASED SECRETION OF GLUCOCORTJOCIDS & GROWTH HORMONEINCREASED DEIODINATION OF THYROXINEINCREASED SYNTHESIS OF HEPATIC ENZYMES

SECRETION OF -ACUTE PHASE- SERUM PROTEINS

CARBOHYDRATE INTOLERANCEINCREASED DEPENDENCE ON LIPIDS FOR FUEL

[

INCREASED SECRETION OF AlDOSTERONE AND ADH

fNEGATIVE BALANCES BEGIN - N, K, Mg,PO., Zn AND SO.

RETENTION OF BODY SALT AND WATER

JINCREASED SECRETION OF THYROXINE

.fDIURESIS .r RETURN TO

POSITIVEFEVER BALANCES

~ INCUBATION ILLNESS

l PERIOD

MOMENT OF EXPOSURE

CONVALESCENTPERIOD

Figure 1. The nutritional response of the host toan infection.

During the incubation period of the infectiousdisease, there i s a depres si on of the plasma ami noacids, iron and zinc. Following that, the animalcommences retaining urinary phosphates and zinc.Deiodination of thyroxine in conjunction with increasedsecretion of growth hormones retards or reverses weightgains with an increased synthesis of hepatic enzymes.

At the onset of illness (the acute phase, whenfever occurs) the animal will secrete serum proteins,there will be a carbohydrate intolerance and anincreased dependence on 1ipids from its own body forfuel. As the pig's illness and temperature begin topeak there wi 11 be an increase in the secret i on ofaldosterone and ADH. Negative balances of nitrogen,potassium, magnesium, phosphates, zinc, and sulfate willoccur, with retention of body salt and water.


As the body temperature and ill ness begi n tosubside the pig will begin to excrete thyroxine, anddiuresis will occur. During the convalescent period itwill return to positive balance.

The vari ety of nutri tiona1 responses duri ng ageneralized infection is broad enough to include mostmajor metabolic pathways of body cells. During theinfection, iron is removed from the plasma andaccumulated as hemosiderin within the liver cells, andas long as the infection persists, any iron giventherapeutically is also stored and not incorporated inmetabolic functions. The result is retarded proteinsynthesis, which manifests itself as slow or inadequategrowth. Anemia will also be manifested.

To illustrate Figure 1, infected sows were given12.5 grams of amino acid chelated iron per sow per day,three weeks before farrowing. The dosage was continuedfor one week after farrowing. At farrowing the babypigs from the treated sows were heavier than the pigletsfrom the control sows, as expected. However, as theexperiment progressed the mortality rates and weaningrates of the two groups of piglets were almostidentical, as seen in Table 14.(22) At first glance itmi ght appear that the che1ate had no effect on thepiglets. Such a conclusion would prove to besuperficial, however. The heavier birth weights of thepiglets from the treated sows showed that some initialbenefits from placental transfer of iron (from thechelate) accrued. The subsequent similarities observedin both groups appeared to result from an underlyinginfection as explained below.


Table 14

The Effect of Disease on Iron Metabolism in pigs

Control Sows Treated Sows

Number of sows 51 54

Total piglets born 492 534(9.647 per sow) (9.889 per sow)

Total dead piglets at birth 38 (0.745 per sow) 36 (0.667 per sow)

Piglet birth weight 1.385 Kg 1.402 Kg

Number of piglets weaned 420 456

Average weaned piglets perlitter 8.235 8.444

Percentage mortality 14.64 14.61

Piglet weaning weight 7.06 Kg 7.112 Kg(21 - 25 days)

The key to understanding these data rested in theobservation that both groups of piglets were found to beseverely anemic, and it was decided to inject them withiron dextran. After iron dextran injections were givento the piglets in both groups, hemoglobin levels wereraised only to 6.8 gldl, (22) whereas they should havebeen in the range of 10 g/dl. During on-going chronic,but active, infections, the administration of iron byei ther oral or parenteral routes is not on1yunnecessary, it is ineffective in reversing the anemiaof infection.(21) The injected iron was apparentlysequestered in the liver and not converted tohemogl obi n. Further exami nat i on of the pi gl ets andtheir dams revealed that they were all infected with asubclinical erysipelas and did not efficiently use theiron obtained via the placenta during gestation, or fromthe iron dextran after farrowing.


While the purpose of this discussion has been toexamine the non-heme role of iron rather than report onits role in preventing anemia, the above study has beenincluded because anemia touches directly on the non-hemerole of iron, as well. As noted earlier, iron isessential to the synthesis of purines, which form thebasis of animal production of protein for growth. Whenthe animal's iron level is insufficient, growth and feedefficiency are retarded. When it is sufficient, feedefficiency and more rapid weight gains are obtained, asshown at Table 15, which summarizes a study done at theUn i vers i ty of III ina is. (23.24) When i ron ami no ac idchelate was supplemented in the diets at the rate of 250ppm in the feed and fed daily to the growing pigs, theaverage gain was 15% more than the controls. It alsotook 7.7% less feed per pound (or Kg) of gain.


I Table 15 I42-Day Performance of Starting Pigs Supplementedwith Iron Amino Acid Chelate

TreatmentIron Amino Acid

Response Chelate Control Difference

First 21Days

Gain/Pig 16.40 lbs. 13.90 lbs. 2.50 1bs.(7.44 Kg) (6.30 Kg) (1.13 Kg)

Feed/Gain 1.66 1bs . 1.74 lbs. 0.08 lbs.(0.75 Kg) (0.79 Kg) (0.04 Kg)

Second 21Days

Gain/Pig 26.20 lbs. 23 . 19 1bs . 3.10 lbs.(11.88 Kg) (10.52 Kg) (1.41 Kg)

Feed/Gain 2. 15 1bs . 2.28 lbs. 0.13 lbs.(0.98 Kg) (1.03 Kg) (0.06 Kg)

Total 42Days

Gain/Pig 42.60 lbs. 37.00 lbs. 5.60 lbs.(19.32 Kg) (16.78 Kg) (2.54 Kg)

Feed/Gain 1.91 lbs. 2.07 1bs. 0.16 lbs.(0.87 Kg) (0.94 Kg) (0.07 Kg)

The supplementation began when the pigs weighed anaverage of 19.8 pounds. It therefore did not deal withfetal nutrition but still demonstrates the role of amino


acid chelated iron in pig growth. The radioactiveisotope and pathological data, which were examinedearlier, established the fact that when iron amino acidchelate is fed, there is an increase in the absorptionand metabolism of that iron, and that the absorbed ironcrosses over the placental barrier to become part of thefetal tissue iron. Additionally, while some of the ironis used for fetal growth and development duringpregnancy, much of it is stored for post-natal use, asshown in Table 7. Furthermore, the positive effects ofamino acid chelated iron supplements to growing pigs areseen in Table 15.

In both cases, the iron, if biologicallyavailable, affects the weight of the pig. The greaterthe weight on the part of the fetuses (within reason)the greater the growth, and the lower the mortal i tyrate. If the post natal diet is supplemented with ironamino acid chelate, which has been demonstrated to bebiologically available to the pig, and if sufficientattention is given to disease control, nutrition, andmanagement practices, the pig producer may well expectincreased growth rates and lower mortality rates in hisherd as well.


References

1. Underwood, E.J., Trace Elements in Human andAnimal Nutrition, (New York: Academic Press) 40,1971.

2. Jones, L.M., Veterinary Pharmacology andTherapeutics, (Ames: Iowa State UniversityPress) 387, 1965.

3. Briggs, G. and Calloway D., Nutrition andPhysical Fitness, (Philadelphia: W.B. SaundersCo.) 278, 1979.

4. Guthrie, H., Introductory Nutrition, (St. Louis:c.v. Mosby Co.) 147, 1975.

5. Roberts, E., et ~., Cell Biology (Philadelphia:W.B. Saunders Co.) 49, 1975.

6. Graff, D., et ~., "Absorption of mineralscompared with chelates made from various proteinsources into rat jejunal slices in vitro," paperpresented before Utah Academy of Arts, Lettersand Sciences, Salt Lake City, Utah, 1970.

7. Ashmead, H., et ~., "Chelation does notguarantee mineral metabolism," J. App. Nutr.26:25, Summer, 1974.

8. Cunha, T., Swine Feeding and Nutrition, (NewYork: Academic Press) 295, 1977.

9. Ashmead, H., et li., "Iron Metabolism as Relatedto Hemoglobin in Pregnancy," unpublished, 1975.


10. Brady, P. S., et li., "Evaluation of an aminoacid-iron chelate hematinic for the baby pig," J.Animal Science, 47:1135, 1978.

11. Cagl iero, G. and Garigl io, G., "Iron ProtalosatesResearch Results," (unpublished), 1975.

12. Kamm, P., "An Evaluation of Fetal Iron as an Aidto Increased Productivity," unpublished, 1979.

13. Widdowson, S., "Malnutrition During Pregnancy andEarly Neonatal Life," presented at Symposium onFetal Malnutrition, N.Y., 1970.

14. Forshaw, R.P, et li., J. Animal Science, 12:263,1953.

15. Svajgr, A., Personal Communication, July 20,1976.

16. Stevermer, E.J., Personal Communication, March 5,1979.

17. Ashmead, H.D., et li., "A new prophylacticapproach to reduction of piglet mortality,"Modern Veterinary Practice, 58:509, 1977.

18. Pederson, G., Personal Communication, December15, 1978.

19. Darnley, A.H., Chelated Iron Feeding Trials forSows, (unpublished study), 1979.

20. Beisel, W.R., "Magnitude of the host nutritionalresponses to infection," Am. J. Cl inicalNutrition, 30:1236, 1977.

21. Schneider, H., et li., Nutritional Support ofMedical Practice, (New York: Harper &Row) 361,1977.


22. Vollers, M., Personal Communication, May 21,1979.

23. Hodson, H., Personal Communication, August 30,1978.

24. Ashmead, H.D. and Graff, D., "Placental Transportof Chelated Iron," Proc. International Pig Vet.Society Congress, Mexico, 207, 1982.

Chapter 13

THE EFFECT OF IRON AMINO ACID CHELATEON THE PREVENTION OF ANEMIA

Cao Xian-Ming, Feng Ming Lian, Zhou Van PingBeijing Agriculture Science Institute

In its natural environment, the baby pig does notbecome iron deficient because it is able to root in thesoil with its snout and in that process, consumes largeamounts of iron, some of wh i ch i s absorbed. Irondeficiency anemia is thus associated with thedomestication and commercial production of pigs in anenvironment foreign to this natural habitat, such as onconcrete, where there is no contact with the soil.

In confinement, baby pigs are particularly proneto iron deficiency for three reasons. First, the sow'smilk contains low concentrations of iron which areinsuffici~nt to meet the needs of the nursingpiglet.(1,) Second, because of the so called "placentalbarrier," which can limit the amount of iron which themother can transfer to the fetus, baby pigs aretraditionally born with iron stores that are inadequatefor their needs.(1,2) Third, relative to its birthweight, the young pig grows at a phenomenal rate whichrequires a continual supply of iron to support thatgrowth. (3)

At birth, 47% of the iron in the baby pig's bodyis associated with blood, 15% with the liver, 1.6% withthe spleen, and the remainin~ 46.4% with the rest of thetissues in the pig's body. (4 After the early neonatalperiod, approximately 80% of the iron fo~nd in the pig'sbody is associated with hemoglobin.() Thus, theadequacy of hemoglobin levels becomes critical in modernswine husbandry. Many researchers have suggested thatthree week old piglets should have hemoglobin concentrations of at least 9 to 10 g/dl,(6) and that thisshould increase to at least 10 to 11 g/dl by the time

231


the pigs are eight weeks of age.(1) While someresearchers believe that hemoglobin levels of young pigsof 10 g/dl, or above, are normal, 9 gldl are requiredfor average performance, 7 gldl will retard growth, 6g/dl is considered to be severe anemia, and 4 gldl willresult in severe anemia with increasing mortalityrates, (5) other researchers report that pi gl ets wi thhemoglobin levels of less than 11 gldl are irondefi ci ent . (I)

If iron deficiency anemia affects suckling pigs,it can lead to poor growth rates, lack of vigor, lack ofluster in the hair and skin, including paleness of theskin and the mucous membranes, accelerated beating ofthe heart, difficulty in breathing and, ultimately, agreater susceptibility to respiratory and digestivetract diseases. All of this results in a significantfinancial loss to the swine industry.

Because of the consequences of baby pig anemia,many measures have been taken to correct the disease.These include adding iron preparations to the feed ofsows to effect some placental transfer, feedingsupplementary iron to the suckling pigs, or injectingiron intramuscularly into infant pigs. While each hassome advantage, each also has several disadvantages. Itwas for this reason that we elected to try an iron aminoacid chelate with both an enhanced rate ofassimilation(2) and the ability to achieve placentaltransfer(9) due to the un; queness of the chel atemolecule, itself. Measurements of hemoglobin levels andgrowth rates were selected to determine theeffectiveness of the iron amino acid chelate since bothare object i ve measurements of baby pi g anemi a. (10)

Fifteen pregnant sows, which were all expected tofarrow at about the same time, were selected for theexperiment. Since the sows were of two differentbreeds, Changbei and Beihe, each breed was divided inhalf so that one half of each breed was in the control

The Effect of Iron Amino Acid Chelate on the 233Prevention of Anemia

group while the other half was in the experimentalgroup. Each breed was maintained separately so that, ineffect, there were two control groups and twoexperimental groups. All of the sows had previouslyfarrowed once before. All were raised in confinement oncement floors.

All of the sows received the pre-parturition dietshown in Table 1. The iron amino acid chelate was addedto the experimental diet, and the ferrous sulfate wasadded to the control diet 28 days before expectedparturition. The diet was regulated so that each sowreceived 1 gram of iron per day from either the sulfateor the amino acid chelate in the feed.

I Table 1 IFeed Analysis of Sow Feed Fed During ExperimentalPeriod


Corn 66 % 66 %Soybean meal 10 % 10 %Wheat flour 7 % 7 %Fish meal 10 % 10 %Hay (grass) 5 % 5 %Bone meal 1.2 % 1.2 %Salt (NaCl) 0.5 % 0.5 %Zinc 0.02% 0.02%Copper 0.01% 0.01%Vitamins 0.25% 0.25%Oxytetracycline 0.02% 0.02%Iron (FeSO.) 0.03% ----Iron (amino acid chelate) ---- 0.03%


Blood samp1es were taken from each sow in theexperiment 28 days before parturition (at the beginningof the experiment), 14 days before parturition, and 14days after parturi t ion. The hemog1 obi n measurementsfrom these blood samples are shown in Table 2. Bothgroups' hemoglobin levels were considered normal at thecommencement of the experiment. The hemoglobin of thecontrol group 2 (CG2) dropped to what may be consideredsl ightly anemic at two weeks before parturition andremained so throughout the remainder of the experiment.The hemoglobin levels of the experimental groups werehigher than in the control groups, but the differenceswere not statistically significant (P>O.OS).

I Table 2 IThe Effect of Dietary Iron Amino Acid Chelate in Hemoglobin Levelsof Sows

Hemoglobin (g/dl)

Group # of 28 Days before 14 Days before 14 Days a.fterpigs parturition parturition parturition

I EG1 3 12.63 1: 0.98 12.47 1: 0.97 11.231: 1.42(Changbei)

CG1 3 12.20 + 1.40 12.23 + 0.98 10.53 + 0.06

I I EG2 5 13.861: 2. 71 12.20 1: 0.66 11.981: 0.84

(Beihe) CG2 4 12.50 + 0.20 10.10 + 1.64 10.68 + 1.53

Total EGs 8 13.40 1: 2.21 12.30 ± 0.54 11.70 ± 1.06

CGs 7 12.37 1: 0.84 11.01 1: 1.72 10.61 1: 1.09

When the sows farrowed all of the piglets weretreated identically. No supplemental iron was givenorally or intramuscularly to the piglets from eithergroup. All were farrowed in crates and raised on cementafter weani ng. Blood sampl es were taken from eachpiglet at 7 days of age, 21 days of age, and 45 days of


age, and the hemoglobin determined in each. No bloodsample was taken at birth because other research hadshown that normally hemoglobin concentrates at birth arebetween 11 and 14 g/dl and then drop to 8 to 10 g/dlduring the first 24 hours of life due to expansion ofplasma space. (11,12) Furthermore, hemogl obi n va1ues areexpected to generally drop to 2 to 4 g/dl by 3 to 4weeks of age when the piglets receive no supplementali ron and are ra i sed on cement. (13)

While no supplemental iron was given to eithergroup after parturition, the piglets received some ironfrom the sows milk and also additional iron from thestarter feed shown in Table 3. This starter feed wasgiven to all piglets on an ab libitum basis. This feedwas provided as a powder and continued throughout the 45day post-parturition period of the experiment.

I Table 3 II Starter Feed Formula I

Corn 66.0 %Flour 8.0 %Bean meal 12.0 %Peanut mea1 2.0 %Fish meal 8.0 %Hay (grass) 2.0 %Bone meal 1.0 %Salt (NaCl) 0.5 %Iron (FeSO.) 0.03 %Zinc 0.02 %Vitamins 0.25 %Oxytetracycline 0.20 %

The effect of feeding the iron amino acid chelateto the sows on the offspring can be seen in Table 4.


The mean hemoglobin levels are shown for each group.All of the increases were statistically significant(P<O.Ol.).

I Table 4 IChanges in Hemoglobin Levels of Baby Pigs

Hemoglobin (g/dl)# of

Group pigs 7-Days Old 21-Days Old 45-Days Old

I EG1 26 * * * * * *(Changbei) 9.62 ± 1.13 10.65 :t 0.89 11.30 :t 0.83

CG1 266.42 + 0.88 6.60 + 1.17 9.21 + 1.12

I I EG2 47 * * * * * *8.41 ! 1.91 9.59! 1.40 10.61:t 1.12

(Beihe) CG2 337.40 + 1.12 8.27 + 1.46 9.71 + 1.33

Total EGs 73 * * * * * *8.84! 1.76 10.01 ! 1.33 10.87! 1.07

CGs 596.89 ± 1.17 7.58 + 1.56 9.51 + 1.26

1* * P< 0.01 I

These data indicate that anemia existed in thesuckling pigs from the control group within two weeksafter birth. They also indicate that although the bloodof the sows in the control group exhibited a normalhemoglobin level, these sows were unable to transfersufficient iron to these piglets via their milk toprevent anemia.

The data in the previous tables indicate that wheniron is chelated to amino acids it is able to eithercross the placenta and be stored in higher quantities bythe fetus, and/or this iron is excreted into the sow'smilk in much higher quantities. Work done at MichiganState University(2) indicates that while higher amounts


of this form of iron is transferred through the sow'smilk, it is insufficient to sustain the blood picturepresented in Table 2 and 4. Statistical analysis showedthat there was a significant difference in thehemoglobin levels (P<O.OI) between piglets whose mothersconsumed iron amino acid chelates and those who consumedFeSO•. It is clear that the iron amino acid chelate cansupply significantly more iron through the placenta tothe fetus and into the milk. This insures sufficientiron for suckling pigs to produce adequate red bloodcells and prevent anemia.

The second measurement of the placental and milktransfer of the iron amino acid chelate is found in thegrowth rates of the baby pigs. A primary symptom of ananemi c pi gl et is a reduced rate of ga in. (14.15.16.17.11.19.20)

Based on the data presented above c.), the experimental

group, which had an average hemoglobin level ofapproximately 10 g/dl, should theoretically grow at amuch faster rate than the control group with an averagehemoglobin level of less than 8 g/dl (at which level,growth is reported to be retarded).

The growth rates of the baby pigs are presented inTable 5. All of the comparative data which have asingle asterisk (*) are significantly different atP<O.05. All data which have two asterisks (* *) aresignificant at P<O.OI.


I Table 5 II Growth Rates of Piglets I

21-DaysBirth Old 45-Days Old

Average Average Average Avg wgt. Avg wgt.II of weight weight weight gain per gain per

Group pigs (Kg) (Kg) (Kg) day (g) pig (Kg)

* * * * * * * *I EG1 26 1.49 1: .13 6.91 1: .41 12.601: 1. 16 245.4 11.04

(Changbei)CG1 26 1.37 ± .18 6.41 ± .38 11.19 ± .64 220.0 9.90

* * * * *I I EG2 47 1.42:t .11 5.95 :t .99 12.25:! 1.79 241.4 10.86

(Beihe) CG2 33 1.38 ± .10 5.73 ± .94 10.38 ± 1.12 199.3 8.97

* * * * * * *Total EGs 73 1.44 ± .12 6.33 ± .89 12.38 ± 1.58 242.7 10.92

CGs 59 1.37 1: .13 6.01 1: .83 10.70 ± 1.03 207.8 9.35

* * P< 0.01* P< 0.05

Regardless of the age (i.e., birth, 21 days or 45days), the weights of the piglets from the experimentalgroups are higher than those of the control groups. Thestatistical difference (P<O.Ol) is seen frequently andis highly significant. For example, in the EG2 groupthe average weight gain per day is 42.1 grams more thanthat of the pigs in CG2. These data fully demonstratethe importance of iron in the growth of suckling pigs.

In spite of the above comments, before the pigletswere 20 days old, the differences in weight were notvery obvious due to the size of the piglets. By thetime the pigs were 45 days old, the weight differences


were very obvious, even though the control pigletsrecei ved the same amount and type of iron in the; rstarter feed as the experimental piglets. This pointsout the long tfrm effect of prenatal iron insufficiencyon post-natal growth rates.

The effectiveness of iron amino acid chelate inpreventing baby pig anemia by feeding the iron to thegestating sow has significant positive benefits.Pi gl ets are farrowed heavi er and rna i nta in the extraweight advantage. They also have higher iron storesresulting in more hemoglobin production and preventionof baby pig anemia.

It is also obvious from these data that when ironamino acid chelate is fed to the lactating sow,additional iron is secreted into the sow's milk, thusproviding the suckling pig with supplemental iron. Theincreased growth rate of approximately 3 Kg of extraweight demonstrated the beneficial effects of thispractice.


References

1. Venn, J., et li., "Iron Metabolism in pigletanemia," J. Camp. Pathol. Therap. 57:314, 1947.

2. Brady, P., et li., "Evaluation of an amino acidiron chelate hematinic," in Report of SwineResearch 1976 (East Lansing: Michigan StateUniversity) 4, 1976.

3. Underwood, E., Trace Elements in Human and AnimalNutrition (London: Academic Press) 38, 1977.

4. Thoren Toll ing, K., "Studies on the absorption ofiron after oral administration in piglets," ActaVet. Scand. Suppl., 54, 1975.

5. National Research Council, Nutrient Requirementsof Swine (Washington, D.C.: National Academy ofScience) 1979.

6. Kernkamp, H., itA parenteral hematinic for thecontrol of iron deficiency anemia in baby pigs,"N. Amer. Vet., 38:6, 1957.

7. Zimmerman, D., et li., "Injectable iron dextranand several oral i ron treatments for theprevention of iron-deficiency anemia in babypigs," J. Anim. Sci., 22: 1409, 1959.

8. Rudolphi, K. and Pfau, A., "The determination ofsub-clinical iron deficiency in piglets usingwhole body 59Fe retention measurements," Proc.Int. Pig Vet. Congress, 9, 1976.

9. Ashmead, D. and Graff, D., "Placental transfer ofchelated iron," Proc. Int. Pig Vet. Congress,207, 1982.


10. Zimmerman, D., "Iron in Swine Nutrition," in Ironin Animal and Poultry Nutrition (West Des Moines:National Feed Ingredients Assoc.) 10, 1980.

11. Richardson, D., Effect of Nutrition Upon CertainComponents of Blood and Milk of Sows and Blood oftheir Litters, M.S. Thesis, Iowa StateUniversity, 1950.

12. Furugouri, K. and Tohara, S., "Studies on ironmetabolism and anemia in piglets. II. Bloodvol ume and mean corpuscul ar constants," Bull.Nat. Ins. Anim. Ind., Japan, 24:75, 1971.

13. Kernkamp, H., "Blood picture of pigs of so-called'anemia of suckling pigs,'" Minn. Agr. Expt. Sta.Tech. Bull. 86, 1932.

14. Moe, L., et li., "Supplementing soil with ironand copper for the prevention of anemia in youngpigs," J. Am. Vet. Med. Assoc. 87:302, 1935.

15. Harris, R., "Further investigations into thetreatment of piglet anemia with iron salts," PigBreeders Annual 19:30, 1939.

16. Vestal, C. and Doyle, L., The effect ofconfinement on suckling pigs and its influence onthe hemoglobin of their blood," Ind. Exp. Stat.Bull., 426, 1938.

17. Swales, E., et li., "The relationship of bloodhemoglobin concentrations to the rate of gain insuckling pigs," Canada J. Res., 20:380, 1942.

18. Barber, R., et li., "Comparisons of hemoglobinlevels in blood of pigs reared indoors andoutdoors on pastures," Vet. Rec. 67:543, 1955.


19. Swenson, M., et li., "A prel iminary report on theeffects of iron-dextran, injected intramuscularlyon the growth rate of newborn pigs," J. Am. Vet.Med. Assoc. 131:146, 1957.

20. Hannan, J., "Recent advances in our knowledge ofiron deficiency anemia in piglets," Vet. Rec.88 : 181, 1971 .

Chapter 14

THE EFFECT OF AMINO ACID CHELATED IRONIN PREGNANT AND LACTATING SOWS

P. Parisini, F. Ricci Biti, L.A. Volpell i, C. SacchiUniversity of Bologna

Pigs exhibit varying degrees of fertility. Thisis seen in Table 1. From the data in this table, it isobvious that a sow or gilt can produce two, six, twelveor more live pigs per litter, and that the number ofpregnancies per year can vary depending on how quicklythe female returns to estrus after weaning, and if sheconceives on the first time she is bred. Because theproduction of a small or large litter and more or lesslitters requires about the same amount of feed, space,labor, and equipment, it is evident that larger littersof live pigs with shorter return times to estrus andhigher conception rates will result in greater profit tothe pi g producer. (1)

I Table 1 Ii Reproduction of Female Swine iAge at puberty 5-8 monthsWeight at puberty 150-250 pounds (68.2-113.6 Kg)Estrus cycle 21 days (16-25 range)Length of estrus 2-3 days (1-5 range)Weaning to estrus 5 days (3-8 range)Ovulation time 40 hours (18-60 after onset of

estrus)Ovulation rate 12-30 ovaLength of gestation 114 days (111-117 range)Pigs per pregnancy 6-16Pregnancies per year 2-2.3Pigs per sow per year 14-20

243


There are several factors that wi 11 affect thereproductive capacity of the pig. These includefert i 1i ty, genet; cs, temperature and other env; ronmenta1considerations, management, disease, breeding practices,age, and nutri t ion. In the 1atter case, researchconducted at several un i vers it i es has shown that thediet fed during growth influenced the ability of thepi gs to concei ve, reproduce and 1actate many monthslater.(2)

The same general nutritional considerations mustbe accorded the mature pig as well. If its diet isseverely deficient in an essential nutrient such asriboflavin(J,4), niacin(4), pantothenic acid(5), or proteinquant i ty or qua1i ty(6) , effi ci ent reproduct ion i shindered. In other studies involving mineral nutritionsimilar results have been observed.(7) To illustrate, 50pregnant sows were divided into two groups. Both groupswere housed in identical conditions. All received thesame feed except that in the treated group received avitamin and an amino acid chelated mineral supplement,shown in Table 2, in its feed at the rate of 10,000 ppmfeed beginning three weeks before expected farrowing andcontinued through lactation and rebreeding.

The Effect of Amino Acid Chelated Iron in Pregnant 245and Lactating Sows

Table 2

Analysis of Vitamin and Mineral Supplement

Vi tami n A.......................................... 770, 000 IU/kgVitamin D3 •••••••••••••••••••••••••••••••••••••••••• 35,715 IU/kgVi tami n E............................................ 1,100 IU/kgMenadi one (Vi tami n K).................................. 550 mg/kgMagnesi urn (Mg)a 5.00 %Potassium (K)b 3.80 %Iron (Fe)a 12.5 mg/kgManganese (Mn)a 2.5 mg/kgZi nc (Zn)· 6.0 mg/kgCopper (Cu)a .03 mg/kgCabal t (Co)· 1.5 mg/kg

a Element given as an amino acid chelate.b Element given as an amino acid complex.

As the sows farrowed, the number of stillborn pigsfrom each farrowing were counted. Apiglet was includedin the stillborn count if it was born dead or if it diedwi thi n the fi rst 24 hours after farrowi ng. As thepiglets were weaned, the number of days each sowrequired to recycle and show signs of estrus wererecorded. Finally, records were maintained to documentthe number of artificial insemination services requiredbefore conception occurred. These data are summarizedin Table 3.


I Table 3 II Reproductive Study with 50 Sows I

Control Treated % Improvement

Number of stillbirths per litter 1.3 0.7 46.2 %

Days to recycle to estrus 7.5 5.0 33.3 %

% conception on first service 73.0 94.0 28.8 %

There are several nutrients in Table 2 that couldhave influenced results that were obtained in the study.One on which very little has been written, as it relatesto reproduction, is iron. Nevertheless, iron haspart i cul ar importance in the diets of pregnant sowsbecause its deficiency will be manifest as declines insow performance and piglet production. It may playakey role in swine reproduction by catalyzing theconvers i on of beta-carotene, the precursor of vi tami n A,to vitamin A.(·) Vitamin A is known to be essential forreproduction. (9) There are probably other biochemicalroles of iron in reproduction that have not as yet beenelucidated that are equally important.

The scope of this present research was todetermine if the iron amino acid chelate in the aboveformulation, when isolated by itself, had a positiveeffect on the reproduct i ve capac i ty of sows. Onehundred and twenty Large White second litter sows wereused in this study and were individually penned in asingle house in which the environment was controlled.All had free access to water and received the same feedration. During gestation each sow was limited to 2.2 Kgper day of th i s feed. Duri ng 1actat i on each sow hadfree access to as much feed as it desired. Thecalculated feed analyses for the gestation and lactation


diets were 2,888 and 2,935 Kcal/Kg of digestible energy,13.87 % and 16.52 % of crude protein, and 0.62 % and0.72 % of lysine, respectively.

The sows were arbitrarily divided into four groupsof thirty sows each. Any sow that miscarried or had tobe rebred was removed from the experiment. Differentlevels of iron as either ferrous sulfate (FeS04 or ironamino acid chelate were fed to the sows remaining in thestudy as part of thei r rat ions . All of the groupsreceived 100 ppm of iron in the feed as ferrous sulfatein order to meet NRC requ i rements. (4) Supp1ementa1 ironwas gi ven to the three treatment groups. The fi rstgroup, the control, consumed 100 ppm of iron as FeS04 ,

as noted above. The second group received the base 100ppm of iron plus an additional 200 ppm of iron for atotal of 300 ppm of iron, all of which was supplied byFeS0 4 • The th i rd group rece i ved 100 ppm of i ron asFeS04 and 150 ppm of iron amino acid chelate. The lastgroup recei ved 100 ppm of i ron from FeS04 pl us anadditional 200 ppm of iron as an amino acid chelate fora tota1 of 300 ppm of iron. The feed i ng of thesedifferent iron levels commenced on the day of conceptionand continued through gestation, lactation andreconception.

All sows were farrowed in farrowing crates. Eachsow was weighed when entering and exiting the farrowingcrate to ascertain the loss of weight during delivery.

The intervals between weaning and estrus andbetween weaning and the next conception for each sowwere recorded. The number of artificial inseminationsper subsequent pregnancy for each sow was also recorded.

As each sow farrowed, the number of stillborn pigs(24 hours) and the number of live pigs were recorded.The number of live born pigs that were weaned in eachlitter was also noted, thus allowing a total mortality


percentage to be calculated. The weight of the litterat birth and at weaning (which occurred on the averageat 26 days of life) were recorded.

All of the data thus gathered were statisticallyevaluated using the chi-square method.

The results of this study are seen in Table 4.

I Table 4 IReproductive Results in Sows and Piglets Fed DifferentQuantities and Forms of Iron as the Single Variable

GROUPS A B C D

Available iron:-from sulfate ppm 100 300 100 100-from amino acid chelate ppm - - 150 200

Sows number 19 23 24 29Length of lactation days 26.2 25.8 26.1 26.1Feed consumption duringlactation Kg/day 4.07 4.02 3.96 3.85

Loss of weight indelivery room Kg 34.74 29.14 26.85 33.28

Piglets born alive number 9.51 9.22 9.13 9.63Average birth weight Kg 1.42 1.43 1.42 1.37Piglets weaned number 7.73 8.00 7.88 8.26Average weight after

26 days of life Kg 5.22 5.20 4.84 4.97Mortality % 18.72 13.21 12.70 14.21Interval betweenweaning/estrus days 7.95 8.32 9.45 8.33

Interval betweenweaning/conception days 30.05 37.27 23.91 26.00

Inseminations/conception number 1.63 1.82 1.45 1.50Interpartal period days 171.47 178.68 164.82 167.17Births/year (projection) number 2.13 2.04 2.21 2.18Piglets/sow/year

(projection) number 20.26 18.81 20.18 20.99Piglets weaned/year

(projection) number 16.47 16.32 18.17 18.01

In analyzing the data in Table 4, it was foundthat none of the differences were statistically


significant. Nevertheless, there was economicsignificance as noted by the decline in mortality ratesof the piglets farrowed to sows receiving the iron aminoacid chelate. This observation has been confirmed byothers. Duri ng 1actat ion, the two groups of sowsreceiving the chelates consumed an average of 3.25 Kgless feed than those sows receiving the sulfate.

In examining the reproductive data, it was notedthat there was a greater time period between the time ofweaning and first estrus in the group that received theamino acid chelates. This is contrary to the data inTable 3. However, this trend was not sustained by thelength of time between weaning and conception or by thenumber of inseminations per pregnancy. To the contrary,the sows which received the iron amino acid chelates(300 ppm) became pregnant approximately ten days soonerand required 1/3 less inseminations per sow to achievepregnancy.

This study demonstrated that there is a shorterperiod between births when the sows are fed the ironamino acid chelate. Thus, the fertility of the sow isimproved as a result of the chelate. This improvementequates to approximately 1.5 more piglets per sow peryear when the iron amino acid chelate is fed as part ofthe feeding program. While not statisticallysign i fi cant, these improvements are economi call ysignificant.

It was not possible to obtain similar results whenferrous sulfate was fed at equivalent levels. When thisform of iron is fed beyond the normal requirements itdoes not modify productivity. Increased fertility wasachieved only with iron amino acid chelate.


References

1. Krider, J., et gl., Swine Production (New York:McGraw-Hill Book Co.) 171-173, 1982.

2. Cunha, T., Swine Feeding and Nutrition (New York:Academic Press) 29, 1977.

3. Miller, C., et gl., "The riboflavin requirementof swine for reproduction," J. Nutr., 51:163,1953.

4. National Research Council, Nutrient Requirementsof Swine (Washington D.C.: National ResearchCouncil) 1973.

5. Ullrey, D. E., et gl., "Dietary levels ofpantothenic acid and reproductive performance offemale swine," J. Nutr., 57:401, 1955.

6. Cunha, Ope cit., 155.

7. Albion Laboratories, Inc., "Breeder Pac & PigBooster," A sales booklet, 1984.


9. Cunha, Ope cit., 120.

Chapter 15

IMPROVING REPRODUCTIVE PERFORMANCEWITH IRON AMINO ACID CHELATE

A. H. Darneley, D.V.M.

A few years ago, a new generation of traceminerals, known as amino acid chelates, was introducedto the market in the United Kingdom. They differed fromother chelates then available in that the metals werechelated with amino acids from hydrolyzed protein andsubsequently stabilized. Thus, a low molecular weightamino acid chelate was formed, somewhat similar to thechelation process which occurs naturally in digestion inanimals.

It was claimed that by feeding this product to thegestating sow, better live piglet birth rates and lowerpiglet mortality could be achieved.(1) Thus, more pigscould be reared per litter, with higher average weaningweights. I decided to test the iron amino acid chelatein a herd.

I had previously noted in the proposed test herdthat the percentage of stillborn piglets was greater inthe litters of older sows. In examining forty of thoselitters more closely, I determined that the older sowshad longer farrowing times and longer intervals betweenbirthing individual piglets than younger sows withsimilar litter size. When farrowing was completedwithin two and a half hours, stillborn piglets wererare, but when farrowing took up to six hours, many ofthe piglets born towards the end were stillborn. Inaddition, thin sows had more prolonged farrowings withmore stillborn piglets than sows in good condition.Finally, I noted -that in earlier experiments whichmeasured parameters different than those for latefarrowi ng probl ems, sows that had been fed the iron

251


amino acid chelates had fewer stillborn piglets,regardless of age.

With these initial observations in mind I decidedto determine in the study if reproductive performance inlater farrowings could be improved by feeding gestatingpigs iron amino acid chelates. The research began withthe above observations in mind and with a background ofdetailed farm records extending back for several years.A summary of piglet mortality rates in terms offarrowi ng numbers for the two years previ ous to theinception of the study is shown in Table 1. Note therapi d increase of pi gl et mortal i ty experi enced after thesixth farrowing.

I Table 1 IIncreased Piglet Mortality as a Function of Farrowing Number

No. of Farrowing 1 2 3 4 5 6 7 8

% in Herd 21.9 17.21 15.78 13.16 13.66 10.01 5.76 2.52Avg. total born 10.7 11.0 12.1 12.7 13.0 13.1 13.6 13.5Avg. reared to 9.1 9.3 9.6 9.8 10.3 9.9 9.5 9.0

21 daysMortality % of 14.95 15.45 20.66 22.83 20.77 24.43 30.15 33.33total born

Average pigs reared per litter for all sows was 9.56.

Over the course of the two and a half year trialperi ad (as we11 as for the two years pri or to theinception of the trial) the same rations had been fed,with the same specifications and formulas and at thesame basic feed levels in pregnancy and lactation. Inaddition, the sows were housed under the same conditionsand had the same herd manager for the entire four and ahalf year period.

The 200 sow herd used for the trial comprised sowsof mixed Large White and Landrace blood which were back-

Improving Reproductive Performance with Iron Amino Acid Chelate 253

crossed to pure Large White or Landrace boars orreciprocally crossed crossbred boars. Many gilts wereintroduced to the herd annually to gain maximum geneticimprovement. Culling was rigorous at all sow ages,usually due to poor breeding and conformation,particularly poor legs.

Sows instead of gilts were initially selected forthe study for three reasons: 1) the percentage ofmortality was highest in their litters, as noted inTable 1, 2) the sows were tethered so individualsupplement feeding was easily accomplished, and 3) giltsdo not produce as many stillbirths as do sows, and thenumber of stillbirths was one of the parametersmeasured. Sows were farrowed in Solari type pens,weaned into yards in groups of four or five and tetheredon the day of the second service. They remained in thetether houses until four to ten days before farrowing.Straw was given three times weekly. The sows were fedhigh quality rations which retained the same compositionduring the research period, as previously noted. Afterservice, dry sow ration was fed twice daily at fivepounds (2.27 Kg) per sow per day. From November toMarch, the weekly ration was increased by 2.5 pounds(1.13 Kg), the extra food being given in two feeds of1.25 pounds (0.57 Kg) each during the week. Inlactation, a higher protein/energy ration was fed at alevel of up to twelve pounds (5.44 Kg) per day. Weaningtook place between nineteen and twenty-eight days,depending on piglet weight. Between weaning andservice, six to eight pounds (2.72 to 3.63 Kg) of feedwas fed each day. After service, any very thin sowsreceived 6.0 to 7.5 pounds (2.72 to 3.40 Kg) per day forthe fi rst four to six weeks of pregnancy unt i1 bodycondition was normal. This was rarely necessary afterthe trials had been under way for a year.

Selection of sows for the trial and controlgrouping was done by taking all sows due to farrow inone week for the trial group. Those due to farrow


during the following week were selected as controls.This prevented complications with cross-suckled piglets.

For the first nine months of the study, the aminoacid chelated iron was fed diluted one part in 33 withmeal. Each trial sow in the pilot study received twoounces (56.7 g) of this supplement once daily, as a topdressing, during the last 21 to 24 days of pregnancy(the average being 22 days). The sows in the controlgroup received 2.75 pounds (1.25 Kg) of unsupplementedmeal in one feed, ten days before farrowing. Thisequated to receiving an equivalent amount of meal as inthe trial group less the iron amino acid chelate.

For the second nine months of the experiment, thefeed was modified to supplement one part of the ironamino acid chelate in 25 parts of meal. This was stillgiven at the rate of two ounces (56.7 g) per sow perday, but for a longer period of time (between 27 and 30days, the average being 28 days).

Records were kept of the total number of pigletsborn, the piglets born alive and dead, and the totalnumber of piglets reared to three weeks. For thistrial, piglets that were stillborn or that died withinthe first twelve hours after farrowing were counted inthe 'Born Dead' category.

Table 2 shows the results of the initial pilotstudy of 35 control and 35 trial sows. There was a 27%reduction in piglet mortality from the sows in the trialgroup compared with the controls. This was surprisingbecause the total pigs born were greater in the trialgroup and a higher morta1i ty percentage woul d, thus,have been expected in that group. This reducedmortality was mainly due to reductions within the "BornDead" category.


I Table 2 II Improved Liveability on Pilot Trial I

35 Trial Sows 35 Control Sows

Average total born per litter 11.92 11.20Average born alive per litter 10.9 9.89Average born dead per litter 1.02 1.31Average redred to 3 weeks per litter 10.29 9.31Percent mortality of total born 13.67 % 16.88 %Percent born dead of total born 8.56 % 11.70 %

Because the results in Table 2 indicated that theiron amino acid chelate could reduce the percentage ofstillborn piglets, further studies were conducted to seeif this could be substantiated on a larger number ofpigs over a longer period of time. The amino acidchelate supplementation period was increased to 28 daysprior to farrowing.

It soon became obvi ous that meani ngful compari sonsbetween numbers reared, following trial or controltreatments, could only be made by comparing sows thathad farrowed the same number of litters. The researchcant i nued unt i1 a 1arge number of sows had farrowedfollowing trial or control treatments. Table 3 showsresults for this evaluation.


I Table 3 IDifferences in Farrowing Based on Number of Litters

Tri al Sows (148)

No. of Farrowings 2 3 4 5 6 7 8

No. of sows in group 22 30 20 19 31 11 15Avg. total born/litter 10.91 11.73 12.65 12.84 13.19 13.73 13.07Avg. born alive/litter 10.18 10.80 11.25 11.73 11.35 12.55 10.60Avg. born dead/litter 0.73 0.93 1.40 1.11 1.84 1.18 2.47Mortality as % oftotal born 12.47 12.79 16.60 13.94 23.96 21.85 22.95

Avg. reared/litter 9.55 10.23 10.55 11.05 10.03 10.73 10.07

Control Sows (152)

No. of Farrowings 2 3 4 5 6 7 8

No. of sows in group 39 27 25 18 16 11 16Avg. total born/litter 11.03 12.15 12.92 13.56 12.81 12.91 13.3Avg. born alive/litter 10.21 10.30 11.64 11.95 11.94 10.82 11.17Avg. born dead/litter 0.82 1.85 1.28 1.61 0.87 2.09 2.13Mortality as % oftotal born 14.69 19.26 17.34 19.32 14.60 28.20 22.93

Avg. reared/litter 9.41 9.81 10.68 10.94 10.94 9.27 10.25

% Less Mortality inTrail Group vs. Control 2.22 6.47 0.74 5.38 9.36 6.35 0.02

B = Better B B B B W B BW= Worse

There were reductions in piglet mortality rates ofbetween 15% and 30% in the second, third, fifth, andseventh parities. Fourth litter farrowings had a slightimprovement. Si xth 1i tter farrowi ngs showed reducedmortality in the control group. This was a smallergroup of pigs than the trial group. Among the sixthlitter farrowing trial group, there were 7 sows who werethin before they farrowed and who had exceptionally highlitter mortality. Furthermore, it was observed in thissixth litter farrowing that seven of the control pigshad not become pregnant on their first service and had


to be rebred six weeks later. This rest from pregnancyenabled them to farrow in better condition than usuallyexpected with the sixth litter farrowing and may alsohelp explain the lower mortality. No trial sows in thesixth farrowing had to be rebred, and therefore, theylacked the rest period afforded to some of the controlsows. It would thus appear that the hoped forphysiological similarities among pigs in the trial andcontrol groups were not present and the results, whichdid not follow the trend of the trial as a whole, couldbe due to an inner (though unintentional) bias.

The study was continued for an additional sixmonths. As a result of choosing each group on a weekto-farrow basis, some sows previously in a trial groupwere subsequently placed in a control group on the nextpregnancy cycle and vice-versa. This had the beneficialeffect of helping to cancel out the effects of sow tosow variabilities due to individual physiology andvigor. When this occurred, the results weredifferential between extremes of the two former groups.The mortality for the litters of former control sowsthat had become trial sows was less than for previouscontrol sow litters, although not as low as the currenttrial litters, as a whole. This suggested that thebeneficial effects of the iron amino acid chelate were,to some extent, carri ed over from one 1i tter to thenext. Table 4 shows the numbers born and reared and thepercentage mortal i ty of pi gl ets from sows and gi 1tsfarrowing in the first six months of the study. Duringthis period, 76% of sows were treated, and thisrepresents 60% of the total farrowing. Piglets born togilts and their subsequent survival were lower in numberas compared to the sows.


I Table 4 I

I Herd Results for Six Months IFarrowing 1 2 3 4 5 6 7 8

% in Herd 23.15 18.17 15.11 12.86 10.29 8.84 6.75 4.82Avg. total born 10.50 11.03 12.03 12.81 13.02 13.33 14.00 13.52Avg. reared to

21 days 8.66 9.43 11 .1 10.44 10.70 10.36 10.50 10.77Mortality %ofof total born 17.52 14.51 7.73 18.50 17.82 22.28 25.00 20.34

Average pigs reared per litter for all sows was 10.24.

Condition scoring records had establ ished the 1inkbetween poor sow cond it i on and lowered reproduct i veperformance, including numbers reared. Culling recordsfor two years showed that over 2/3 of the sows culledafter five litters were judged "too thin" as acontri butory reason for cull i ng. There had been anobserved connect i on between becomi ng "too thi nil anddemonstrating poor reproductive performance at the nextbreeding. Prior to the commencement of this experiment,the normal scenario for aging sows was to becomeprogressively thinner with most reaching the stage whereperformance was affected at about the sixth pari ty.After the sixth litter there would be a marked drop inthe number of piglets reared (as seen in Table 1).Thus, in previ ous years, most sows were cull ed afterthis litter. Other criteria for culling a sow from theherd were poor numbers of pi gl ets reared per 1i tter(under 9), and/or poor weights of growing piglets (whichdenoted poor milking or poor health of the mother).

Comparing Tables 1, 3 and 4, it can be seen thatmortality in piglets from sows not fed the iron aminoacid chelate increased dramatically after the sixthlitter. With the addition of the iron amino acidchelate supplementation, this no longer occurred (Tables3 and 4). The overall effect of the supplementation on


the ent ire herd was to ra i se the number of pi gl etsreared per litter by 0.41. This was done in spite ofthe fact that only 60% of farrowing females in the wholeherd had received the iron amino acid chelatesupplement, and in spite of a slight rise in thepercentage of gilts and their concomitantly lowernumbers reared in first farrowings.

The culling records for the first six month periodshowed that only 15% of the experimental sows had "toothin" as a contributory reason for culling (comparemortalities shown in Table 4). Over the period of theexperimentation, a large number of sows had their backfat measured ultrasonically at the level of the last ribat the P2 position. Table 5 shows the average P2 atselection (upon reaching 200 lbs.), at the point offirst service, and at each weaning, thereafter. Thismeasurement was in agreement with the visual assessmentof body fat percent, and showed that no furtherreduction in body fat occurred after the third litterwas weaned. The older sows remained in good conditionalthough no extra food was fed (see Figure 1). Only 2sows had P2 measurements as low as 11 mm out of the 80weaned toward the end of the research period.


Table 5

Backfat of Sows

Average P2 mm.

Gilts at 200 lbs.1st service1st weaning2nd weaning3rd weaning4th weaning5th weaning6th weaning7th weaning8th weaning

13.517.519.119.217.617.215.918.016.317.5

The retention of body fat may contribute to theobserved improvements in reproductive performance.There is some evidence that sows with P2 measurements of10 mm, and below, are at some risk of reproductivefailure in the near future. Ultrasonic measurementtogether with visual score have confirmed that the sowsdescribed as "too thin" in the culling records had a P2of 9-10 mm, or below.

After one year into the study, scoring showed animproved condition in the trial sows compared with thecontrol sows, especially where they had been on thetrial regime for two farrowings. Breeding performancein respect to returns to servi ce, farrowi ng rate tofi rst servi ce and reduct ion in empty days appearedimproved, as well as numbers reared in the trials sows.This is also seen in Figure 1.


TRIAL GROUPBACK FAT (P2) MEASUREMENT (mm)25~-------_

20V~'·/'·/'-.;/'·/'/"

15 .

10 .

5 .

F S F S F S F S F S F S F SF - FARM S - SERVICE

12 AVERAGE NUMBER OF PIGS REARED PER urrER

11.5 " . .

11 .

10.5 . -...........~.'~~';J,

10· .

9.5 .

2 3 4 5 6 7 8+FARROWING

"""*- TRIAL GROUP

CONTROL GROUPBACK FAT (P2) MEASUREMENT (mm)

25r------------...20. . .

15 . .

10 .

5 .

F S F S F S F S F S F S F S

F - FARM S - SERVICE

11 AVERAGE NUMBER OF PIGS REARED PER LITTER

10.5 .

10 ...•.. . ....•....

9.5 .

2 3 • 5 6 7 8+FARROWlNG

-8- CONTROL GROUP

Figure 1. The effect of amino acid chelated ironon the sow's body condition and productivity.

It was noted during the course of the study thatthe sows fed iron amino acid chelate appeared to loseless body condition during lactation than did thecontrols. For this reason, it was decided to includegilts in the study. Gilts generally lose more bodycondition than sows during lactation and, consequently,have a longer weaning to service interval, as well asmore returns to service and more empty days. Gilts werekept in groups of five in yards with individual feeders.This necessitated group feeding of the iron amino acidchelate supplement. By convention, it was fed for 28days before the first gilt was due to farrow.Consequently, a few gilts were thus supplemented forseven weeks, although this occurrence was rare.


Tabl e 6 shows the reproduct i ve performance ofgilts between the weaning of their gilt litter and beingserv; ced for the; r second 1; tter. It compares thevar; ous parameters w; th 1; ke f; gures from a surveyconducted in 60 herds of breeding pigs which was carriedout in 1979. The survey contained sows of all ages, butshows a similar picture to the controls in this currentstudy. The average total empty days category equals thetotal of all days wasted including weaning to servicedays, returns, and days from weaning to culling for anysow who does not farrow; divided by the number of sowswho do farrow.

I Table 6 II Reproductive Performance of Gilts I

Own Herd 2nd FarrowingReproductive Performance M&LC Survey

Tri al Control

Number of sows 84 76 1995Farrowing rate - 1st service 93 % 84 % 83.6Cull rate after 1st service 2.4% 6.6% 7Weaning - service interval (days) 7.11 8.79 7.8

Average total empty days includes 10.6 17.27 23.0weaning to service interval, dayswasted by returns and beforeculling.

The fo 11 owi ng general it i es for normal sowperformances can be extrapolated:

1) 9.5 pigs reared for the second 1itter

2) 114.9 days service to farrowing (M &LC Survey)

3) 28.3 days lactation (M &LC Survey)


By using these assumptions and the figuressummarized in Table 6, one can calculate the pigletsreared per sow per year. The variation is thendependant on empty days only. These calculations yieldthe following data:

M & LC Herds 20.86 pigs per sow peryear

Own Herd Control Gilts 21.61 pigs per sow peryear

Own Herd Trial Gilts 22.57 pigs per sow peryear

This gilt trial contains a low number of pigs.However, if one compares the breeding results for thewhole herd for the two years before the trials beganwith the experimental breeding results obtained aftermost sows were receiving the supplement (with theexcept i on of 2/3 of the tota1 gil ts wh i ch were notincluded in the experiment at that time), one can seeimprovement both in the number of pigs reared per litterand pigs per sow per year (see Tables 1,3,4,6). Weaningage had not altered, but the pi gs per sow per yearimprovement proved to be greater than can be explainedby litter size alone. This shows that empty days arereduced in the sows. The improvement in reproductiveperformance in the second year of the study equated toproducing the same number of weaners as in the firstyear with 8.7% fewer sows. Table 7 also shows that amuch lower percentage of sows in the second year wereculled for being "too thin" than occurred in the firstyear.

The number of sows requiring extra food to correcttheir body condition was also much lower in the secondyear than in the first year. This is reflected in theannual sow feed consumption figures shown in Table 9.


Table 7

Comparison of Sow Performance Between the First and Second Test Years

First Year

Farrowing 1 2 3 4 5 6 7 8+

% in Herd 21.9 17.2 15.6 13.2 13.7 10.0 5.8 2.5Avg. no. reared 9.1 9.3 9.6 9.8 10.3 9.9 9.5 9.0Avg. pigs/litter 9.55

60% of cull sows "too thin"

Second Year

Farrowing 1 2 3 4 5 6 7 8+

% in Herd 21.2 17.8 15.3 12.7 13.5 5.9 4.3 9.3Avg. no. reared 9.0 9.4 11.1 10.4 10.7 10.4 10.5 10.4Avg. pigs/litter 10.24

10% of cull sows "too thin"

Improved Performances

Pigs/litter 7.11% increasePigs/sow/year 1.9 increaseRequired sows 8.7% less sows required to produce same number of

weaners

Many factors adversely affected by poor conditionappear to be improved by supplementation with the ironamino acid chelate. Table 8 lists some of the effectsof poor sow condition on reproductive performance andcorresponding beneficial effects of iron amino acidchelate supplementation.


I Table 8 IEffects of Iron Amino Acid Chelate on Sow Performance

Effects of Poor Condition on Effects of Amino Acid ChelatedReproductive Performance Iron Supplementation

* Reduced lactation yields causing * Some evidence of raised 3 weeklower 3 week weights. weights.

* Longer weaning to service * Reduced weaning to serviceintervals. intervals.

* More returns to service (at 21 * Fewer returns to service.days and longer intervals).

* Longer farrowing interval - * Shorter farrowing interval -fewer litters/sow/year. more litters/sow/year.

* Abortion in extreme emaciation. * No sows extremely emaciated.

* Increased culling. * Reduced culling.

* Reduced sow life. * Increased sow life.

This two and a half year study provided evidencethat sows fed amino acid chelated iron in the last monthof pregnancy reared more piglets, especially in laterfarrowings. The economic effect of raising more pigsper litter is seen in Table 9, which summarizes theamount of sow and boar food required per piglet reared.During the 2.5 years of the study the overall amount offood consumed per piglet reared was reduced by 17.7%.


I Table 9 IEconomic Effect of Iron Amino Acid Chelate Supplementation

Year Litters Per Pigs Reared Pigs Reared Sow Feed Sow FeedEnding Sows Per Per Litter Per Sow Per Per Year Per PigletAutumn Year Year in Tons in Kilograms

Year 1· 2.200 9.57 21.05 1.20 57.01Year 2 2.278 10.08 22.96 1.10 47.91Year 3 2.340 10.05 23.52 1.07 46.45

·Year 1 represents initiation values, prior to the beginning of theIron Amino Acid Chelate supplementation.

Notes: 10% fewer sows needed in year 3 to produce same number ofweaners as in year-1 17.7% less sow feed per piglet rearedin year-3 compared to year-I.

In conclusion, after two and a half years of studyit became obvious that the amino acid chelated iron hada positive effect on herd performance. The number ofpiglets reared per litter increased by more than 5%.The very high percentages of mortality in litters afterthe sixth farrowing, which were seen at the beginning ofthis study, did not occur in ensuing years. Many of theolder sows in the first year of the study became thinand necessitated culling. This also no longer occurred1ater in the study. Ul trasoni c measurement of sowbackfat in animals which received the amino acidchelated iron indicated that body fat remainedconsistent after the third litter. This may havecontributed to the reduced mortality in litters of oldersows. Reproduction also improved, with 39% less opendays between weaning and service. Furthermore, theincidence of sows conceiving on the first serviceincreased by almost 11%.

It appeared that supplementing the gestating pigsdiet with iron amino acid chelate for 28 days prior toexpected farrowing was adequate to improve reproductive


performance. The economic advantages of using the ironamino acid chelate in this test were obvious. Not onlywere sow effi ci enc i es improved, but the effects onoffspring viability were also marked. Thissupplementation was obviously not a substitute for goodmanagement, but in a well managed herd it was able toimprove performance and profitability.


References

1. Albion Laboratories, Inc., "Mr. Swine Producer",1982.

Chapter 16

A NUTRITIONAL APPROACH TO MAXIMIZINGCARCASS LEANNESS

David AthertonThomson and Joseph Limited

Originally, it was believed that the primary roleof the pig in livestock production was to economicallyconvert cheap feeds into fat. As the chemistry of fatsevolved and the cheaper vegetable oils became acceptablesubstitutes for pork lard, the swine industry changedfrom producing a lard-type carcass to an either moremeaty, heavier-muscled carcass or a leaner, bacon-typecarcass. (1)

With this evolution, the producer turned togenet ics to ass i st in those changes. Th i s work wasdirected primarily towards improving the efficiency ofpig production through repartitioning of growth towardslean meat deposition and away from fat. Genetistsdiscovered, for example, that by increasing the carcasslength, the b~ckfat thickness was also reduced as seeni n Figure 1. (1 )

269


Thlckness

2.1101' backt'at

Thlcknees

1 .45 01' 6 'defat

~m

96.7

_sooth of ce. 'caSl

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em

96

94

92

90

88

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3.8

3.6

3.4

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2.8

2.6

2.4

2.2

2.0

1.8

1.6

1.4~/

1929/30 341 39/ 441 49/ 54/ 59/ 64/ 69/ 71/ 72/ 73/

3S 40 45 50 55 60 65 70 72 73 74

Figure 1. Development of length of carcass,thickness of backfat, and thickness of sidefatfor Dani sh Landrace pigs sl aughtered at about200 pounds, live weight, from 1926 through 1974.As carcass length increases, backfat and sidefatare reduced.

Different breeds of pigs produce different levelsof fat. Even the source of genetic material within abreed has a bearing on actual incorporation ofcharacteri st i cs into progeny. It shoul d be noted,however, that the sows' genetics, while important, donot have the same genetic influences on a herd that aboar does. Thus, producers tended to concentrate on the

A Nutritional Approach to Maximizing Carcass Leanness 271

boar, believing that the characteristics of the boarwould permeate throughout the herd. With this in mind,the various breeds were analyzed for fat production inan attempt to establ ish a general profile that wouldmeasure pig performances and the data are summarized inTable 1.(3) Al~ of these animals were raised on slattedfloors and fe~ the same 16% protein and 3% fat diet.

I Table 1 IBody Profiles of Different Breeds of Pigs

Daily Lean LipidLean feed Feed Avg 10th gain %01

Total effi- eonsump- effi- daily rib Loineye Dressing day on loinno. ciency tion ciency gain backfat area percen- test muscle

Breed pigs (Ibs.) (Ibs.) (Ibs.) (Ibs.) (in.) (sq.in.) tage (Ibs.)

Berkshire 58 8.51 5.43 3.22 1.69 0.98 5.52 73.87 0.688 2.80

Dekalb 77 132 8.02 4.96 3.24 1.53 0.96 6.02 73.25 0.634 2.50

Duroe 213 8.28 5.37 3.14 1.71 0.94 5.64 73.01 0.678 3.25

(F1) Duroe- 141 8.46 5.28 3.27 1.62 0.98 5.60 72.87 0.634 2.84

Hampshire

Farmers 180 8.43 5.21 3.27 1.60 1.03 5.85 73.75 0.636 2.73Hybrid

Hampshire 199 8.00 5.14 3.17 1.62 0.91 5.83 72.71 0.655 2.48

PIC HY 89 7.89 4.97 3.11 1.60 0.89 5.81 72.82 0.654 2.14

PIC L-26 153 7.78 5.13 3.12 1.65 0.82 5.85 72.45 0.668 2.53

Poland China 92 8.70 5.29 3.38 1.57 1.01 5.51 73.11 0.614 2.72

Yorkshire 133 8.76 5.34 3.30 1.62 1.03 5.32 72.90 0.637 2.38

Mean 8.28 5.21 3.22 1.62 0.96 5.70 73.07 0.650 2.64

Standard 0.34 0.16 0.09 0.08 0.07 0.21 0.45 0.023 0.30Deviation

The data in Table 1 emphasize lean efficiency ascompared to feed efficiency. According to David Meekerof the National Pork Producer's Council, "Feedefficiency gives you too much credit for fat. Lean


efficiency is a better statistic to use because it talksabout feed requ i red per pound of ed i b1e product. II (3)

A survey among pig producers reported that theissue which has top priority in their minds is theproduction "of lean, high qual ity pork muscle. II

(4) Thegenetics are currently available to efficiently andeconomi call y produce a 1eaner pig. (3,4) However, evengenetics will not produce a leaner pig if the producerprovides the wrong feedstuffs to the animal. Feed isthe largest single factor in producing lean meat. Itrepresents 60% to 70% of the total farrow-to-finishcosts, 50% to 60% of the cost of raising feeder pigs,and 65% to 71% of the cost of grower-finisheroperat ions. (5)

Probably the most obvious change in feedingpractices for leaner meat has been the continuous risein the amount of crude protein being included in thefeeds. This trend has been designed to match the higherprotein deposition rates in lean meat. In spite ofthis, it has largely been a comprehension of the rolesof amino acids in the diet and particularly the optimumratios and levels of the amino acids (the so-calledideal protein), which have had the greatest impact onmaximizing the pigs' genetic potentials for proteindeposition.

Even with these feed modifications, there is ahigher cost of feeding pigs for lean meat at theirlatter stages of development. This is due to ametabolic tendency to increase body fat production atthe expense of protein development. These data are seenin Figure 2.(6)


70

60+.JCQ)

UL 50(l)Q.

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o

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0 0 0 00 --.."

r"\. "Ash

~ ~

-=

50 100 150 200 250 300 350

Empty body weight J pounds

Figure 2. Percentages of water, protein, fat, andash in the empty body of pi gs from 50 to 325pounds.

From the data in Figure 2 it can be seen that anynutrient that would flatten the slopes of the protein,water, and fat curves would be of interest to the swineproducer who is trying to increase the leanness of hispigs. In economic terms such a nutrient would result inreduced energy requirements since lean meat is composedlargely of water (80%) whereas fat contains less than10% water and has twice as much energy concentrated init as does lean meat. Thus, as the producer attempts to


raise leaner pigs, their feed efficiency should alsoimprove.

Manganese is a nutrient which is essential for1eaner carcasses and improved feed effi ci enci esmentioned above to occur. Previous research hasdemonstrated that both liver and bone fat are reducedwith manganese supplements.(7) Furthermore, a manganesedeficient diet has produced excessive fat deposition inpigs.(8,9) This may have been due in part to theessential role that manganese plays in the metabolism offatty acids. In its absence, fatty acids are notsynthesized from body tissue fat.(lO) When fatty acidsynthesis is impaired, dietary fat remains stored asbody fat rather than bei ng used for energy. (11) In fact,a clinical sign of a manganese deficiency in pigs isincreased fat deposition.(12)

In other research it has been demonstrated thatcont i nuous feedi ng of 2 ppm, 5 ppm, or 10 ppm ofmanganese as either manganese sulfate or as an aminoacid chelate resulted in 13.9% (2 ppm), 39.4% (5 ppm),and 155.7% (10 ppm) more manganese in the liver from thechelate source compared to the sulfate.(1J) Inradioactive isotope research involving laboratoryanimals, the group receiving a single dose of 32 mcg ofmanganese as an amino acid chelate had 239% more ~Mn

distributed throughout their bodies than a similar groupwhich had received an equivalent amount of ~Mn asmanganese sul fate. (14)

In a foundat i on work conducted by th i s authorunder the direction of J. Bibby Agricultural Ltd., U.K.,various contributions of Albion amino acid chelates wereassessed in pigs over the course of five years. In thearea of backfat reduction, zinc and manganese amino acidchelates were found to have greater effects withmanganese outstripping zinc. The following tablesdemonstrate the results relating to manganese amino acidche1ate. The tests were all conducted on grower and


finishing pigs because the preliminary studies alludedto above had demonstrated that this was the period inwhich the most fat is incorporated into the tissues.

The first trial to evaluate amino acid chelatedmanganese in finishing pigs involved PIC CamboroughBreeding stock. Twenty-four (24) pigs which were alleleven weeks old and had an average weight of 34 kg,were arbitrarily divided into two groups. Both groupsreceived the same diet, except that 20 mg/kg ofmanganese as the amino acid chelate was added to thefood of the treated group. The test continued for sixtydays, at which time all of the pigs were butchered.Table 2 summarizes the results.


I Table 2 IThe Effects of Adding Manganese Amino Acid Chelate to the Dietsof Finishing Pigs

Control Treated % Difference(Treated

Compared toControl)

No. pigs 12 12 ---Average start weight (kg) 34.21 34.08 -0.4 %Average final weight (kg) 85.50 87.04 101.8 %Average weight gain (kg) 51.29 52.96 103.3 %Days on test 60.8 59.1 ---Average daily gain (kg) 0.844 0.900 106.6 %Feed consumed/pig (kg) 143.58 138.82 -3.3 %Feed conversion rate 2.80 2.62 -6.4 %

Carcass Data

Average killing out (Kg) 74.83 73.17 -2.2 %Fat depth at Pz (T11T1) 10.67 10.08 -5.5 %Number grading AA1 5 10 200.0 %Number grading 61 7 2 -71.4 %

Economic Data

Average price/kg deadweight (p) 111.44 115.92 104.0 %Average return per pig (£) 71.30 73.60 103.2 %Feed cost/tonne (£) 140.57 141.24 100.5 %Average feed cost/pig (£) 20.18 19.61 -2.8 %Average gross margin/pig (£) 51.12 53.99 105.6 %Extra gross margin (£) -- +2.87 --

Another series of trials was made to ascertain ifsimi 1ar resul ts coul d be obta i ned wi th manganese sul fateas those observed with the manganese amino acid chelate.For this study, 48 pigs were used with an averagestarting weight of approximately 35 kg. They weredivided into four groups. Each group received the samepell eted feed except for the amounts and types ofmanganese included. Group 1 received 20 ppm of


manganese as manganese sulfate. Group 2 received 20 ppmof manganese as manganese sulfate and 20 ppm ofmanganese as manganese ami no ac i d che1ate. Group 3recei ved 40 ppm of manganese as manganese sul fate.Group 4 recei ved 100 ppm of manganese as manganesesul fate. Fi fty-fi ve days after commencement of thetest, all of the pigs were butchered. Table 3 presentsthe data.

I Table 3 IA Comparison Between Manganese Sulfate and Manganese Amino AcidChelate in Finishing Pigs

Results Treatment

Group 1 Group 2 Group 3 Group 4

No. of pigs 12 12 12 12Average start weight (kg) 36.00 35.00 34.80 35.92Average final weight (kg) 80.33 83.93 82.50 83.17Average weight gain (kg) 44.33 48.93 47.70 47.25Days on test 55 55 55 55Daily weight gain (kg) 0.806 0.890 0.867 0.859Average feed/pig (kg) 126.40 127.95 126.80 128.76Feed conversion 2.85 2.62 2.66 2.73

Carcass Data

Killing out % 73.00 73.00 72.60 74.33Fat depth Pz (rTlTl) 13.83 12.33 14.40 14.33

Economic Data

Price/kg dead weight (p) 99.50 101.17 97.50 97.83Returns/pig (£) 58.35 61.99 58.40 60.48Feed cost/tonne (£) 209.20 209.95 209.23 209.30Feed cost/pig (£) 26.44 26.86 26.53 26.95Margin over feed/pig (£) 31.91 35.13 31.87 33.53Extra gross margin/pig - +3.22 -0.04 +1.62(£r

• Groups 2, 3, and 4 compared to group 1.


These data demonstrate that the manganese aminoacid chelate is more bioavailable than the manganesesulfate. Similar results produced by the chelate couldnot be obtained when equivalent, or greater amounts, ofmanganese from the sul fate were used. The manganeseamino acid chelate improved carcass quality by reducingfat deposition. Elevated levels of manganese sulfatedid not. The manganese amino acid chelate also improvedgrowth and feed efficiency.

In a third trial, it was decided to compare theeffects of the manganese amino acid chelate withche1ated manganese gl uconate. It was bel i eved thatsince the latter was considerably less expensive, andwas also a chelate, there may be an economic advantage.Thirty-five eleven week old pigs averaging approximately31 kg each, were divided into three groups: negativecontrol, amino acid chelate, and gluconate. Allreceived the same pelleted finishing feed except for theinclusion of 20 ppm of manganese as either the aminoacid chelate or the gluconate in the pellets of the twotest groups' feed. At the concl us i on of the testperiod, all were butchered. Table 4 summarizes theresults.


I Table 4 IAn Evaluation of Manganese Amino Acid Chelate and Manganese GluconateAgainst a Negative Control in a Pelleted Finishing Feed

TreatmentControl Mn Amino Acid Mn Gluconate

Chelate

Number of pigs 11 12 12Average start weight (kg) 31.59 31.63 31.38Average final weight (kg) 89.86 89.88 92.08Average weight gain (kg) 58.27 58.25 60.70Days on test 65.82 65.50 70.17Daily weight gain (kg) 0.885 0.889 0.865Average feed/pig (kg) 153.92 152.80 165.30Feed conversion 2.64 2.62 2.72

Carcass Data

Killing out % 75.67 75.83 75.83Fat depth @ P2 (nm) 16.25 14.92 15.50Grade (%) 1 --- 8 ---Grade (%) 2 33 50 67Grade (%) 3 50 42 25Grade (%) 4 17 --- 8

Economic Data

Price/kg deadweight (p) 93.81 98.25 97.83Returns/pig (£) 63.79 66.96 68.31Feed cost/tonne (£) 193.30 194.10 194.10Feed cost/pig (£) 29.75 29.66 32.08Margin over feed/pig (£) 34.04 37.30 36.23Extra gross margin/pig (£)- --- 3.26 2.19

- Treatments compared to control.

As can be seen from these data, the best growthand feed convers i on were ach ieved when the manganeseami no ac id che1ate was fed. The P2 measurement wasreduced by 1.3 mm in the pigs given this manganesesupplement which resulted in better grading and improvedmargin over feed per pig. While the manganese gluconatemay act in a similar way to the amino acid chelate by


reducing backfat thickness, it is not as effective, andthus, the economic benefits are less.

Although they have not all been presented here,seven separate tri a1s were conducted us i ng manganeseamino acid chelate. The manganese amino acid chelateconsistently showed a reduced backfat effect at the P2probe position which, in some trials, was alsoaccompanied by an improved growth rates and feedutilization. The manganese amino acid chelate alsotended to demonstrate a greater response at a lowerlevel of supplementation (20 ppm of manganese from thechelate versus 100 ppm from the sulfate). That dosagerate has since represented the basis for commercialevaluation and usage.

In a final study, 36 Camborough XLarge White pigs(18 boars and 18 gilts) were divided into two groups:the control and experimental. Six pigs ( 3 boars and 3gilts) were housed in each pen. All were eleven weeksof age and weighed approximately 31 Kg at the beginningof the experiment. The length of the trial was 74 days.All pigs were weighed and measured every two weeksthroughout the trial period. Water was provided ad1ibitum via low pressure water nipple drinkers. Thefeed formul at ion gi ven to both groups was ident i ca1except for the source and quantity of manganese. Thecontrol and experimental groups both received 0.45 Kg ofmanganese ch1ori de per tonne of feed, but theexperimental group also received 0.20 Kg of manganeseamino acid chelate in each tonne of feed. The dietswere assayed at the beginning, during, and end of thetrial to verify the theoretical calculations.

At the end of the experiment, all pigs werebutchered. Assessment of each carcass was undertaken byA.F.R.C., Institute of Food Research. Water holdingcapacity was determined by taking a core sample from theLongissimus Dorsi Muscle and using the filter pressmethod of Grau and Hamm. (17,18) The resul ts were expressed


expressed as the ratio: Expressed Fluid Area/MuscleArea. The refl ectance measurements from the Longi ss i musDorsi Muscle and the Adductor Muscle of the hind legwere done using a fiber optic probe. The pH ultimatemeasurements were done from a core sample. Table 5presents the results.

i Table 5 IStatistical Comparison of Sample Means Between Control and ManganeseAmino Acid Chelate Treated Pigs

Control Mn Chelate SEM+ SIG

Start weight (kg) 31.97 31.61 0.40 NSFinish weight (kg) 95.00 94.50 1.10 NSDaily weight gain (kg/day) 0.852 0.850 0.013 NSFeed consumed (kg/pig) 173.595 170.909 1.13 NSFeed conversion 2.754 2.718 0.040 NSReturn/pig 59.88 61.54 1.54 NS

Carcass length (cm) 836 846 6.45 NSBackfat loin min (mm) 16.06 12.94 1.056 *Backfat 1oi n max (mm) (P2 ) 17.50 15.12 0.075 **Backfat mi d-back (mll) 14.61 15.65 1.029 NSBackfat shoulder max (mm) 33.06 32.53 0.92 NS

Fiber optic probe LD 31.92 32.19 1.70 NSFiber optic probe AD 26.23 29.60 1.75 NSWater holding capacity 1.59 1.62 0.102 NSPH ultimate 5.44 5.45 0.24 NS

+ SEMS standard error of the mean.** Significant at P<O.OI.* Significant at P<0.05.

These studi es demonstrated no sex re1ated responsedi fferences to the treatments. There were nostatistically significant differences between the groupsin terms of daily weight gains, feed consumption, orfeed conversion. There were statistically significantdifferences, however, in fat deposition in those pigsreceiving the manganese amino acid chelate. Thesedifferences were significant at the 1% level of P2 and


the 5% level for the loin maximum and the loin minimum,respectively. The fiber optic probe measurements showedthat the control pigs had lighter colored meat than themanganese amino acid chelate fed pigs, but thedifference was not significant.

The pigs that were fed the manganese amino acidchelate as part of their diets had, as in previoustrials, reduced levels of backfat (a 1.6 mm reductionover the control at P2). This improved carcass qualitycoupled with an improved feed conversion rate resultedin an approximate 15:1 cost effective ratio from the useof the manganese amino acid chelate. About two-thirdsof th i s was due to improved carcass qua1i ty and theremaining one-third due to better feed conversion.

On first examination, the physiological orbiochemical mode of action of the manganese amino acidchel ate is not readi ly apparent. Neverthel ess, bytaking the reduced backfat deposition as the primaryresponse indicator one can develop three possiblehypotheses:

(A), The manganese ami no ac id che1ate causes amodified nutrient reapportioning effect in favor of leandeposition at the expense of lipid deposition. In thiscase the limiting factor is the supply of availablemanganese to dependent enzyme systems such as thoseinvolved in fatty acid synthesis from tissue lipids.Inorganic manganese has an inherently lowavailability,(lS) being subject to a range ofantagonistic chemical reactions in the gut. Manganesewhich has been correctly chelated with amino acids hasa considerably higher availability as seen in Table6.(14) In this study a single dose of 82 Jjg of 54Mn wasadministered as a chloride or as an amino acid chelate.Fourteen days later, following a metabolic equilibrationtime, tissue analysis for 54Mn occurred.


Table 6

Mean Tissue Distribution of ~Mn from ManganeseAmino Acid Chelate and Manganese Chloride*

MnC1 2 Mn ChelateHeart 36 107Liver 52 106Kidney 80 97Spleen 190 397Lung 54 56Small Intestine 89 141Muscle 22 28

* Data are expressed as corrected counts perminute per gram of dry tissue.

(8), Initially the benefits associated with theamino acid chelation of manganese were thought to be dueentirely to an increase in the uptake of dietarymanganese from the digestive tract as discussed inhypothes i s (A). More recent1y, however, workers inFrance, using ~ manganese amino acid chelates fed torats have demonstrated that at least a portion of thismanganese is taken into the hypothalamus and pituitarygland. It is believed that this in turn may stimulatethe rel ease of thyrotrophi n whi ch then control s therelease of thyroxin by the thyroid gland. A flow sheetof these relations is shown in Figure 3.(19)


BRAIN

PEPTIDE STIMULATINGFACTOR

HYPOTHALAMUS

Thyrotrophin ReleasingHormone (TRH)

T

PITUITARY

Thyrotrophin (TSH)T

THYROID

Thyroxine (TH)T

MUSCULATURE - Enhances BasalMetabolic Rate

- Stimulates BReceptor Activity

NET EFFECT

Increased maintenance requirement- Fat used preferentially as a source of energy

Increased feed intakeIncreased growth rate

- Reduced carcass fatness

Fi gure 3. Proposed effect i ve path of 54Mn intovarious targeted tissues.


(C), As an additional factor to the manganese,there may also be an enhanced metabolic basal rateresul t i ng from a pept ide induced response. Certa inpeptides are known to exert defined metabolic effectswhich are generally under the control of the endocrinesystem. Evidence exists to demonstrate that the aminoac id che1ates are absorbed intact as dipept ide-l i kemolecules. (16) The observation that growth rates arereduced in a fixed feed intake system, with increasingmanganese intake well within the normal physiologicalrange, would tend to support this explanation. If thiswere the case, it may be that the manganese within thechelate is indirectly associated with the response,acting only as a stabilizing factor within the peptidemolecule. This last hypothesis has still to beval idated and may eventually be inval idated when onerecalls that the pigs receiving manganese gluconate didexhibit a response, albeit, not as great as thatobtained from the amino acid chelate. That observationsuggests that the bioavailability of the manganese is asignificant factor.

Even in the absence of a clear explanation for itseffect, manganese amino acid chelate has demonstratedsuperior performance in pigs fed 20 ppm of thismanganese as part of their grower or finisher feeds.The economic benefits include: (1) better feedconversion, (2) improved lean meat deposition, (3) lessdays to slaughter, (4) lower feed costs per pig, and (5)improved carcass quality.


References

1. Krider, J., et ~., Swine Production (New York:McGraw-Hill Book Company) 4, 1982.

2. Ibid, 7.

3. Miller, M., "Lean, Meaty, And more efficient.But ... ", Pork '89, 9:14, July 1989.

4. Pri ce, S., "Payment for 1ean: Where do we gofrom here?", Pork '89, 94:12, July 1989.

5. Krider, Ope cit., 369.

6. Hankins, o. and Titus, H., "Growth Fattening andMeat Production," u.s. Dept. Agr. Yearbook(Washington, D.C.: U.S.A.A.) 458, 1939.

7. Amdur, M. 0., et ~., "The Lipotropic Action OfManganese," J. Biol. Chern. 164:783, 1946.

8. Pl uml ee, M., et ~., "The effects of a manganesedeficiency on the growth and development ofswine," J. Ani. Sci., 14:996, 1954.

9. Plumlee, M., et ~., "The effects of a manganesedeficiency upon the growth, development andreproduction of swine," J. Ani. Sci., 15:352,1956.

10. Cotzias, G., "Manganese," in Comar, C. andBronner, F., Mineral Metabolism An AdvancedTreatise (New York: Academic Press), V2, 411,1962.



12. Teague, H., et li., Nutrient Requirements ofSwine (Washington, D.C.: National Academy ofSciences) 1979.

13. Vinson, J., "Bioavailability of Manganese,"Unpublished, 1982.

14. Ashmead, H., et li., Stability constants of54manganese and thei r effects on manganesemetabolism," Unpublished, 1974.

15. Ashmead, H.D., et li., Intestinal Absorotion ofMetal Ions and Chelates (Springfield: Charles CThomas) 25, 1985.

16. Ibid, 117.

17. Grau, K. and Hamm, R., "Eine einfache Methode zurBest i mmung der Wasserbi ngdung in Muske1, "Naturwissenschaften, 40:29, 1953.

18. Briskey, E., et li., "The chemical and physicalcharacteristics of various pork ham muscleclasses," J. Ani. Sci. 18:146, 1959.

19. Robbins, S., "Feeding for carcass qual ity," PigTopics, 7, Feb. 1989.

Section 4. POULTRY

289

Chapter 17

THE EFFECT OF AMINO ACID CHELATES INCHICK MORTALITY

David AthertonThomson and Joseph Limited

Most nutritionists accept the fact that thecomposition of an egg is, to a certain degree, dependantupon the diet of the hen that laid it. The egg willcontain all of the vitamins necessary for a chickembryo, but the levels of these vitamins in the egg aredirectly related to their levels in the layer feed. Thepercentages of fat and protein in the egg are affectedby their percentages and availability in the hen's diet.The dietary mineral levels can also be related to theirlevels in the egg.(1)

Ashmead, et ~., have demonstrated that when themineral composition of the egg is modified, it willaffect chick viabil ity and growth rates. (2) In theirstudy, one hundred and si xty-fi ve fert i 1e eggs weredivided into five groups. A small hole was drilled intothe large end of each egg shell exposing the air sac.Each hole was immediately sealed with sterile paraffin.Various mineral compositions were injected through thesterile paraffin seal into the eggs being careful not topenetrate the egg membrane. The injections did notoccur for two weeks following the drilling of the holesin order to confirm the viability of the embryos. Thesterile mineral formulation injected into the eggs isshown in Table 1.

291


Table 1

Minerals* Injected Into Eggs (mg)

Calcium .0079Zinc .014Manganese .004Iron .002Copper .001

*Dissolved in two microliters of sterile deionizedwater.

Group 1 received these mineral s as CaC1 2t FeSO. t

CuSO., and MnSO.. Group 2 received these same mineralsas amino acid chelates. Group 3 had the same sources ofminerals as Group 1 except that 59Fe, .5Ca and 54Mn wereused as radiotracers to ascertain the transfer of theminerals to the chicks after hatching. Group 4 had theabove radioisotopes included as part of the amino acidchelates. Group 5, the control group, was injected withsterile deionized water.

Following hatching all chicks in Groups 3 and 4were sacri fi ced and thei r tissues assayed forradioisotopes. Table 2 presents the results. The meandata are presented as corrected counts of radioactiveemissions per minute per mg of tissue. In almost everyinstance, more of the mineral was incorporated into thetissues of the chicks from the amino acid chelate sourcethan from an inorganic salt.

The Effect of Amino Acid Chelates in Chick Mortality 293

Table 2

Radioactive Minerals Transferred to the Tissues of Chicks

Skin & SkeletalGroup Mineral Bone Brain Feathers Liver Heart Muscle

3 45Ca1ci urn 18.9 0 .07 .04 .07 .17(Inorganic)

4 45Ca1ci urn 140.1 .11 .16 .09 .06 .17(Chelate)

3 59 1ron 9.2 .74 .38 3.86 .60 .38(inorganic)

4 59 1ron 18.2 1.34 .34 10.03 1.15 .57(Chelate)

3 54Manganese 19.8 1.83 .42 6.43 .96 .62(Inorganic)

4 54Manganese 37.0 3.65 .65 15.12 2.23 .78(Chelate)

Groups 1, 2 and 5, which did not receiveradioisotope injections in the air sacs, were hatchedand ra i sed to 175 days. The mortal i ty for Group 1(inorganic) was 12% compared to 8% for the chelate group(Group 2) and 19% for the control group (Group 5).

The chicks in each group were weighed weekly,beginning at hatching and continuing for 25 weeks (175days). At hatching, Group 1 had a mean weight of 45.8gm per bird. Group 2 (chelate) had a mean weight perchick of 46.5 gm, a 1.6% increase. The control group,Group 5, had a mean weight of 43.7 gm per bird.Clearly, the increased uptake of the amino acid chelatesthrough the egg shell membrane had a positive influenceon hatch wei ght. Furthermore, the more pronouncedabsorption continued to have a positive effect. At thetermination of the experiment, 175 days later, Group 1


had a mean weight of 1310 gm per bird. Group 2 had amean weight of 1394 gm, and the controls' average weightwas 1271 gm each.

The foregoing study was summarized in detailbecause it demonstrated the effects of the amino acidchelates on mortality rates and weight differences whenthose chelates are deposited in the eggs. This studydemonstrated that if more of an essential mineral can betransferred to the embryo before the shell is formed andthe egg is laid, then mortality rates of the hatchedchicks is reduced. Titus has indicated that mineraltransfer to the embryo is an essential requirement fora reduction in chick mortality.(l) This current studywas designed to address more completely the question ofthe transfer of amino acid chelates from the feed of thebreeder hen to the embryo before the egg is laid.

Two Cobb broiler breeder flocks on the same farm,containing 3,000 hens each, were selected for theexperiment. Both groups had identical housing andreceived the same feed, water and management practices.

At 20 weeks of age, the experimental groupreceived the amino acid chelated mineral formula shownin Table 3 as a feed supplement for 15 weeks, until theywere 35 weeks in age. The control group did not receiveany additional supplemental minerals. Both groups werefed a commercial ration which was deemed to containadequate mineral nutrition.

I Table 3 IAmino Acid Chelated Mineral Supplement Fed toExperimental Group of Breeder Hens

Copper 25 ppmZinc 25 ppmManganese 12 ppm


The chicks hatched from each broiler breeder groupwere reared separately on the same growing unit in orderto provide a direct comparison of their mortality rates.The handling and treatment of both groups of chicks wereidentical. Both groups received the same starter andgrower feeds. There were no disease problems in eithergroup.

Table 4 presents a summary of the mortality ratesof the chicks from the two groups. There wassubstant i a1 and cons i stent reduct ion in the morta1i tyrates of the growing chicks hatched from the breedersthat received the amino acid chelate as compared to thecontrol flock during the period of 27 to 35 weeks ofage. The overall chick mortality from the experimentalgroup was 7.41% lower than those from the control groupas seen in Figure 1.

I Table 4 IChick Mortality Rates

Flock Age Control Experimental % Mortality Diff(weeks) No. % No. % %

26 10,900 4.57 9,000 5.69 + 1.1227 10,300 5.90 10,300 5.74 - 0.1628 33,600 6.20 37,800 4.71 - 1.4930 38,700 5.52 38,600 4.75 - 0.7731 24,700 5.54 22,100 4.11 - 1.4332 10,300 6.53 12,200 4.94 - 1.5933 15,000 3.52 8,000 3.43 - 0.0934 10,200 4.74 10,500 5.32 + 0.5835 28,600 3.64 28,700 3.04 - 0.60

Total/mean % 182,300 / 5.13 177,200 / 4.64 - 0.49


10....-------------------,

20

10

I 0

:. ·10

020

IS II 27 21 21 10 11 U 13 M II

Breeder Flock Age (Weeks)

Figure 1. Growing flock mortality differentialwith breeder flock age.

A good correlation was also observed between thedegree of response and the absolute percent ofmortality. These data are summarized in Table 5 andFigure 2.


I Table 5 IPercent of Mortality and Absolute Differences

% Mortality % Difference

Flock Code Control Treated Absolute Comparative

530 9.14 5.62 - 3.52 - 38.5530 8.35 6.17 - 2.18 - 26.1531 6.53 4.94 - 1.59 - 24.3530 6.35 4.27 - 2.08 - 32.8530 5.90 5.74 - 0.16 - 2.7531 5.86 4.86 - 1.00 - 17.1531 5.67 5.09 - 0.58 - 10.2530 4.79 3.67 - 1.12 - 23.4531 4.74 5.32 + 0.58 + 12.2530 4.63 4.27 - 0.36 - 7.8531 4.57 5.69 + 1.12 + 19.7531 3.84 3.85 + 0.01 + 0.3530 3.79 3.41 - 0.38 - 10.0530 3.52 3.43 - 0.09 - 2.6530 3.50 2.67 - 0.83 - 23.7530 3.26 3.67 + 0.41 + 11.2

Total 5.28 4.54 - 0.74 - 11.1II

298 The Roles of Amino Acid Che/ates in Animal Nutrition

4Or-----------------,

J20 .

J 0 ..----....-, .............--~WIOIro&....•1-20 -.-.- -.. _ .

f-40 .• -80 .'#.

I 7 • •

Figure 2. Growing flock mortality differentialcompared to the percent of mortality in the controlflock.

Table 6 provides data demonstrating that whenmortal ity rates in chicks are in excess of 5%, thecomparative reductions gained through the inclusion ofthe amino acid chelates shown in Table 3 in the feed ofthe breeders were quite marked.


I Table 6 IFinal Mortality Bracket Comparison

%Mortality % DifferenceMortality No. ofBracket Flock Control Treatment Absolute Comparative

< 6.0 % 4 7.40 5.18 - 2.22 - 30.05.0 - 5.9 3 5.81 5.21 - 0.60 - 10.34.0 - 4.9 4 4.67 4.77 + 0.10 + 2.13.0 - 3.9 5 3.55 3.36 - 0.19 - 5.4

Mean Total 16 5.36 4.63 - 0.73 - 10.9

The results reported in this study tend to supportthe thesis that feeding certain amino acid chelates tobroiler breeder hens will result in a reduction in chickmortality. This presumably occurs because of thegreater absorption and translocation of the amino acidchelates from the physiological stores of the breederhen, but also implies a greater bioavailability of theami no ac id che1ates to the hen from her feed in thefirst place. This is particularly true during thestressful period of peak production. These conclusions,as refl ected in reduced ch i ck morta1i ty, wereparticularly noticeable during the lay period of 28 to32 weeks of age, when the control group mortality wasvery high.

Any stressful period results in significantalternations to body metabolism. Body reserves ofessential nutrients are used up rapidly. Whenreplacement is inadequate to meet the needs, thendefi ci enc ies occur. (3) In the case of the high produc i ngbreeder hen, not only is her own body harmed as hermineral stores are depleted, but there are also lessminerals provided to the embryo in the egg. This lowermineral level then results in higher mortality in thechicks as demonstrated by Ashmead, et li. (2)


When the amino acid chelates are provided as asupplement during this stressful period, their greaterbioavailability allows a greater transfer to the egg.Thus, the chick enters life with higher hatch weightsand a greater mineral reserve in its own body towithstand the initial stresses of its existence. Theresult is lower mortality rates and healthier birds.

The Effect of Amino Acid Chelates in Chick Mortality 30 1

References

1. Titus, H., The Scientific Feeding of Chickens,(Danville: The Interstate) 75, 1955.

2. Ashmead, H., et li., "The effect of amino acidchelated minerals on chicks," Unpublished, 1988.

3. Blackburn, G. and Bristrian, B., "Curativenutrition: Protein-caloric management," inSchneider, H., et li., eds., Nutritional Supportof Medical Practice (Haggerstown: Harper & RowPublishers) 80, 1977.

Chapter 18

THE DYNAMICS OF FEEDING AMINO ACIDCHELATES TO BROILERS

Alberto Bonomi, Afro Quarantelli, Paola Superchi,Alberto Sabbiono, and Luigina Lucchelli

University of Parma

In the past three decades there have beentremendous advances in the knowledge of the biochemicalroles of minerals, as testified by the numerousscientific publications on this subject. While theirnumerous roles are outside the scope of this discussion,it is still important to realize that, together with theother basic nutrients, minerals control the growth anddevelopment( of )poultry and assist them in utilizingthei r feed. 11-14

In spite of this influx of knowledge, there isvery little published on how various external factorsi nfl uence the metabol ism of mi cronutri ents. Many ofthese factors operate independantly or in synergisticmanners and, consequently, produce variable levels ofutilization of the same elements. This makes itdifficult to determine, with any degree of exactness thelevels of inorganic mineral salts required to insure thechicken's health and optimum production. Thus, therehas been a search for a form of mineral that approachesthe exact needs of poultry and is less vulnerable to theexternal influences that cause bioavailabilityvariations.

Among those minerals that have come to theforefront in meeting these needs, are the amino acidchelates of copper, cobalt, iron, manganese, and zinc.When compared to equivalent amounts of the same metalsfrom salts, the utilization of the amino acid chelatesis greater. This is due to the chelation process which

~~~~e~::i ~~~n~a;~ ~~ss:;soomc~::~~~~(115~nfl ~~~~~~eb~fm~~~~~302

The Dynamics of Feeding Amino Acid Chelates to Broilers 303

greater stability their absorption from the digestivetract is enhanced.(16-20

In the past, other types of chelates have beenused in poultry nutrition with varying degrees ofsuccess. Thesf ~nclude ethylenediamine tetraacetic acid(EOTA), diethy1enetriaminepentraacetic acid (OTPA), andmonosodium and disodium derivatives. The EOTA and OTPAhave a similar behavior to the amino acids in that theywill react with various cations to form soluble metalchel ates. C14J However, they do not guarantee an el evatedrate of mineral absorption because the bonds between themetal and ligands are much stronger than those of aminoacids in enzymes, tissues, etc., and metals. Thus, thecells within the bird are frequently unable to removethe metal from the EOTA or OTPA, and the chelate, al-

;~~~~~s~~~~r~~~a~~r~~i~i~~ ~~~~~~i ~~di ~~~ mttnea~~~~(qlJ5~

In studies with species of animals, other thanpoul try, superi or resul ts have been obta i ned throughamino acid chelate supplementation. (25-37} This includesswi ne, (38) cal ves, (39) hei fers, (40) rabbi ts, (41) turkeys, [42)and laying hens.(43) In the case of the turkeys, whenthe amino acid chelates were included in the feed at1000 ppm (100 ppm of metal) (the amino acid chelatescontain 10% metal) growth rates were improved 6.93%,feed conversion improved 5.94%, and the killingpercentage )increased 2.61% with an improved meat yieldof 5.69%.(42 When provided in the same dosage levels asthe turkeys, the ami no ac id che1ates increased thelaying of eggs in chickens by 5.8% with a 4.9% improvement in feed convers ion. (431 In that same study wi thlayers, it was noted that the birds grew to maturity 7%faster than the controls that received metal salts.

As a result of these studies it was decided toinvestigate the value of the amino acid chelates inbroilers. A chelate formula similar to that given tothe turkeys and layers was selected. The metal portion


contained 9% Fe, 2.15% Cu, 3% Zn, 1.2% Mn, and 0.08% Co,with the remainder being the ligand. The compositionof the amino acid chelate is shown in Table 1.

I Table 1 IChemical Analysis of the Amino Acid Chelate Formula

Moisture 6.96 %Ash 31.18 %Crude protein 25.26 %Crude fat 0.91 %Crude fiber 2.32 %Undigestible matter 33.37 %

The ami no ac id compos it i on of the ami no ac idchelate formulation is shown in Table 2. T44 ,45,51)


Table 2

Amino Acid Composition of Amino Acid Chelate Formula

Tryptophan 0.485 %Lysine 5.584 %Histidine 1.885 %Arginine 3.804 %Aspartic acid 8.227 %Threonine 3.318 %Serine 3.248 %Glutamic acid 26.839 %Proline 4.254 %Glycine 6.921 %Alanine 9.105 %Cystine 0.311 %Valine 5.911 %Methionine 1.077 %Isoleucine 4.813 %Leucine 7.850 %Tyrosine 2.609 %Phenalaline 3.678 %

A1though the guaranteed anal ys i s of the metalcontent of the amino acid chelates was given above, itshould be remembered that the source of the amino acidswas from a vegetable protein which itself has a certainmetal composition. Thus a mineral analysis of theche1ate was conducted by atomi c absorpt ionspectrophotometry, wi th the except i on of pho~Rhorus,

which was determined by colormetric technique. 6) Theresults are shown in Table 3.


I Table 3 ITotal Metal Contents of Amino Acid Chelate Formula

as Determined by Chemical Analysis

(per 100 g of amino acid chelate)

Calcium 0.47 9Phosphorus 0.36 9Magnesium 0.17 9Potassium 0.97 9Sodium 0.95 9Copper 1,581.76 mgCobalt 100.00 mgIron 7,067.25 mgZinc 2,195.39 mgManganese 1,103.46 mgIodine 150.00 mg

The experimental design involved taking 4,000 oneday old male chicks and dividing them into four groupsof 1, 000 each. Group 1 was the control group andreceived no amino acid chelates. Group 2 received thechelate formula at the inclusion rate of 500 ppm (50 ppmmetal) of the total feed. Group 3 received these sameamino acid chelates at 1000 ppm (100 ppm metal) of thetotal feed, while group 4 received them at 1500 ppm (150ppm metal) of the total feed.

The study lasted for 60 days and was broken intotwo periods: day 1 to day 30 for the first period andday 31 to day 60 for the second. Duri ng the fi rstthirty days, all of the chicks were fed the same feedwith the exception of the amounts of amino acidchelates. During the next thirty days, they were fedthe same grower feed, except, again for various


inclusions of the amino acid chelates. These two feedformulas can be seen in Table 4. The basic vitamin andmineral package, which was added to all of the feeds, isshown in Table 5.

Table 4

Chick Starter and Grower Feed Formulas (Kg/IOO Kg Feed)

Soybean mealPeanut mealSunflower flour (45% protein)Meat mealPeach flour*FatDyshidrosis cureCorn mealCorn glutenVitamin/mineral pack (Table 5)Calcium carbonateDicalcium phosphateSodium chloride

StarterFeed

13654422

6020.50.60.50.5

GrowerFeed

9435I22

7020.50.60.50.4

*The fat was composed of equal parts of lard, bovine suet, palm oil, andcoconut oil.


Table 5

Vitamin and Mineral Package (per Kg)

Vitamin AVi tami n D3

Vi tami n 81

Vi tami n 82

Vi tami n B6

Vi tami n 812

PeroxidoxinePantothenic acidCholine chlorideDL methionineManganeseIronZincCopperIodineCobaltQS with soybean meal to

2,500,000 IU400,000 IU

200 mg1,000 mg

200 mg3 mg

4.500 mg1,000 mg

100,000 mg50,000 mg20,000 mg5,000 mg5,000 mg

200 mg100 mg

50 mg1 kg

The compositions of the two feeds are shown inTable 6.

Table 6

Chemical Analysis of the Chick Starter and GrowerFeeds

Moisture. %Ash. %Crude protein, %Crude fat. %Crude fiber. %Indigestible material. %

Starter Feed

11.565.15

23.134.973.24

51.95

Grower Feed

12.114.61

20.095.383.07

55.04

All of the chicks were weighed at 30 days beforebeing fed the grower feed and again at 60 days at the


conclusion of the study. Data on feed consumption werealso recorded. At sacrifice, representative carcasseswere evaluated and the killing percentage recorded. Allof the data obtained ~ere ~tatistically evaluated by ananalysis of variance. 47-50

Table 7 shows the growth of the four groups ofbirds, their feed conversions for the sixty days, andtheir mortal ity rates for both the thirty-day periodand for the total of sixty days.

I Table 7 IGrowth Rates, Mortality Rates and Feed Conversions In Supplemented Chicks

30 Days 60 Days 60 DayFeed

Group Mean Live Weight Mortality Mean Live Weight Mortality Conversion(grams)

1 1,192.621: 69.34 18/982 (1.8%) 2,771.131: 102.36 27/973 (2.3%) 2.48

2 1,205.711: 52.63 23/977 (2.3%) 2,793.191:121.19 30/970 (3.0%) 2.45

3 1,263.271: 73.29 10/990 (1.0%) 2,960.371: 113.46 20/980 (2.0%) 2.33

4 1,271.441: 59.30 15/985 (1.5%) 2,972.12 1: 129.67 22/978 (2.2%) 2.32

LS.D. 31.50 46.24(P<0.05

At thirty days, there were statisticallysignificant difference (P<0.05) in the growth ratesbetween group 4 and group 1. Each bird in group 4 hada mean weight of 6.61% more than group 1. Group 2 andgroup 3 birds had mean weight increases of 1.10% and5.92%, respectively over group 1, but neither of theseincreases was statistically significant.

At sixty days, the differences between the fourgroups were even greater. Group 4 bi rds were 7.25%heavier than group 1. This was also significant at theP<0.05 level. Groups 2 and 3 had total mean weight


increases of .8% and 6.83%, greater, respectively, thangroup 1 birds. Group 3 weight gains were statisticallysignificant (P<O.05) compared to the gains of group 1.

As Table 7 summarizes, there were also differencesinch ick mortal i ty . Even though groups 3 and 4 had18.52% and 25.93% lower mortality rates than group 1birds, the decreases were not statistically significant.The differences were economically significant, however.

Finally, from the data in Table 7, it is obviousthat the inclusion of the amino acid chelates in thefeed had a positive effect on feed conversions. It took6.45% less feed per Kg of growth in group 4 compared togroup 1. Group 3 consumed 6.05% 1ess feed per Kg ofgrowth compared to group 1.

At sixty days, all of the birds were weighed(Table 7). Fifty chickens from each group were randomlyselected, fasted for twelve hours, and then sacrificed.The carcasses were examined and the percentage of meat,fat, bone, skin and subcutaneous flesh, head and neck,claws, gizzard, homogenized liver, spleen, heart,testicles, and lungs, feathers, and homogenizedintestines and pancreas were measured. The results ofthis examination are shown in Table 8 and include ananalyses of variances.


I Table 8 ICarcass Weight as a Percentage of Uve Weight

UverSpleen Intes-

Head Heart tine &Car- & Giz- Testicles Fea- Pan-cass Meat Fat Bone Skin Neck Claws zard Lungs thers creas

% % % % % % % % % % % % % % %p.v. car- p.v.- car- p.v. car- p.v. car- p.v. p.v. p.v. p.v. p.v. p.v. p.v.

Group cass cass cass cass

67.10 54.80 36.77 3.79 2.54 32.28 21.66 9.13 6.13 7.21 5.32 2.88 3.71 7.52 2.881 ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

1.13 0.80 0.59 0.29 0.20 0.81 0.61 0.60 0.32 0.43 0.29 0.16 0.25 0.71 0.27

67.30 54.92 36.93 3.46 2.33 32.70 22.01 8.92 6.20 7.00 2.18 2.94 3.49 7.31 2.702 ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

1.26 0.72 0.63 0.22 0.15 0.67 0.54 0.71 0.40 0.52 0.38 0.24 0.33 0.66 0.22

68.28 54.98 37.54 2.64 1.80 33.00 22.53 9.38 6.40 6.59 5.10 2.67 3.50 7.42 2.583 ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

1.03 0.91 0.70 0.34 0.25 0.72 0.48 0.48 0.29 0.63 0.41 0.18 0.20 0.58 0.30

69.09 56.29 39.62 2.27 1.57 32.80 22.66 8.64 5.97 6.44 4.87 2.43 3.67 6.83 2.444 ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

1.18 0.86 0.52 0.36 0.17 0.79 0.63 0.59 0.51 0.44 0.33 0.19 0.28 0.60 0.32

L.S.D. 0.98 1.07 1.10 0.65 0.44 --- --- --- --- --- --- --- --- --- ---

(P<0.05)

*p. v. = analysis of variance

As can be seen from Table 8, the warm killingpercentage without the heads, necks, claws and internalorgans varied from 67.10% for the controls (group 1), to68.28% for group 3, and 69.09% for group 4. These are1.76% and 3.10% increases, respectively. Theseincreases were not statistically significant, however.

There were also differences in the amount of meaton the carcasses in the four groups. Group 4 had 2.72%more fl esh than group 1, and group 3 had .18% more.When compared to the carcass percentages, it is obviousthat the amino acid chelates not only contributed to an


increase in the meat, but also in the bones to supportthat increased weight.

The percentages of fat in groups 3 and 4 aredec idedl y lower than in group 1. These decreases of29.13% and 38.19% are both statistically significant atP < 0.05. There were no other statistical variations inthe other tissues and organs measured and reported inTable 8. The general health of the four groups ofbroilers was good during the trial period. This isreflected, to a degree, by the mortality rates reportedin Table 7.

From these data it is reasonable to conclude thatwhen the amino acid chelates of copper, iron, zinc,manganese, and cobalt are included at the rates of 1000ppm (100 ppm metal) or 1500 ppm (150 ppm metal) in thediets of broilers during the growing period, there is apositive effect on growth. This increase is between 6%and 7%. There is also an increase in marketable meat(approximately 8%) containing significantly less fat.


References

1. Lenkeit, W., Einfuhrung in die Ernahrungsphysiologie der Haustiere (Stuttgart: F. Enke) 23, 75,79, 1952.

2. Bruggermann, J. and Kirsch, W., Futter undFutterung, N. 17, 1952.

3. Scharrer,(Berlin:

K., Bischemie der Spuren ElementsPaul Parey) 1955.

4. Underwood, E., Trace Elements in Human and AnimalNutrition (New York: Academic Press) 1956.

5. Masoero, P., et ~., Rivista di Zootecnia, 29:911, 1956.

6. Abrams, J., Animal Nutrition and VeterinaryDietetics (Edinburough: Green & Son) 1961.

7. Mitchel, H., Comparative Nutrition of Man andDomestic Animals (New York: Academic Press)1962.

8. Worden, A., et ~., Animal Health, Production andPasture (London: Longmans) 1963.

9. Beaton, G. and McHenry, E., Nutri t ion Macronutrients and Nutrient Elements (London: AcademicPress) 1964.

10. Bonsembiante, M., Alimentazione Animale, 9:391,1965.

11. Masoero, P., et ~., Fisiologio della Nutrizione,(Rome: UTET) 1980.


12. Masoero, P., Rivista di Zootecnia, 42:342, 1969.

13. Masoero, P. and Bosticco, A., Annali Fac. Vet. diTorino, V. 4, 1954.

14. Thompson, D., Trace Elements in Animal Nutrition(Skokie: Int. Minerals and Chemical Corp.) 1970.

15. Ashmead, D., "Why Organic Chelated TraceMinerals?", Western Dairy J., 26:20, April, 1972.

16. Maymone, B., Alimentazione Animale, 7:1-15,1963.

17. Jones, L., Veterinary Pharmacology andTherapeutics (Ames: Iowa State University Press)381, 1965.

18. Cantarow, A. and Schepartz, B., Biochemistry(London: John Wiley &Sons, Inc.) 654, 1962.

19. Nettler, A., "Mighty Minerals," Paper presentedat a Miller Pharmacal Seminar, Fall, 1972.

20. Manis, J. and Schachter, D., "Fe59 Amino acidcomplexes: Are they intermediate in Fe59

absorption across intestinal mucosa?", Proc. Soc.Exp. Biol. Med., 119: 1185, 1965.

21. Peeler, H., "Trace elements requirements andinterrelationships," Paper presented at Minn.Nutri. Conference for Feed Mfg. and Dealers,1963.

22. Bjerrum, J., et li., "Stabi 1i ty constants ofmetal-ion complexes with solubility products ofinorganic substances. II. Inorganic ligands withsolubility products of inorganic substances," TheChemical Society, Spec. Pub. No.7, 1958.


23. Scott, M., "Organic chelation of mineral elementscall ed maj or absorpt ion-contro11 i ng factor, "Feedstuffs, 43:40, February 26, 1973.

24. Graff, D., et ,g!., "Absorption of mineralscompared with chelates made from various proteinsources into rat jejunal slices in vitro," Paperpresented to Utah Academy Arts, Letters, andSciences, April, 1970.

25. Ashmead, D., "The need for chelated traceminerals," Vet. Med. Small Animal Cline., 69:476,1974.

26. Cunha, T., Swine Feeding and Nutrition (New York:Interscience Publishing) 62, 1957.

27. Ashmead, H., et li., "Chelation does notguarantee mineral metabolism," J. App. Nutri.,26:5, 1976.

28. Hopson, J. and Ashmead, D., "Iron deficienciesand their relationship to infectious diseases,"Vet. Med. Small Animal Cline., 71:809, 1976.

29. Underwood, E., Trace Elements in Human and AnimalNutrition (New York: Academic Press), 1971.

30. Svajar, A., "Getting more iron into the nursingpig," Feedstuffs, 46:34, March 8, 1976.

31. Brady, P., et li., "Evaluation of an amino acidiron chelate hematinic," J. Animal Sci., 41:308,1975.

32. Herbein, J. and Martin, R., "Response to insulinin the fetal and newborn pig," J. Animal Sci.,41:316, 1975.


33. Spruill, D., et .li., "Effect of dietary proteinand iron on reproduction and iron related bloodconstituents in swine," J. Animal Sci., 33:376,1971.

34. Hinze, P., et .li., "Effects of Metalosates onpiglet growth,1I Mod. Vet. Pract., 49:79, 1968.

35. Brady, P., et .li., "A continued evaluation ofiron proteinate," Proc. Am. Soc. Animal Sci.,1351, June, 1976.

36. Miller, D., IIIndirect prevention of baby piganemia,1I Hog Farm Mgmt., 13:44, April, 1976.

37. Ashmead, D., "Prevention of baby pig anemia withamino acid chelates," Vet. Med. Small AnimalClinc., 70:607, 1975.

38. Bonomi, A., et .li., Annali Fac. Med. Vet. diParma, V. 1, 1981.

39. Bonomi, A., et .li., "I complessi 01 igodinamicichelati nell'alimentazione dei uifelli in fuse disuezzamento, " 11 Nuovo Progresso Veterinario,38:281, June, 1983.

40. Bonomi, A., et .li., "I complessi oligodinamicichelati nell'alimentazione dei vitellion," AnnaliFac. Med. Vet. di Parma, 3:318, 1983.

41. Bonomi, A., et .li., Rivista di Coniglicoltura,19:37, Nov., 1982.

42. Bonomi, A., et .li., "I complessi oligodinamicichelati nell'alimentazione del tacchino dacarne," Rivista di Avicoltura, 51:23, June, 1982.

43. Bonomi, A., et .li., "I complessi oligodinamicichelati nell'alimentazione delle galline


ovaiole," Rivista di Avicoltura, 51:51, Sept,1982.

44. Simmonds, P., et li., "Design of a nickel-63electron absorption detector and analyticalsignificance of high temperature operation,"Analytical Chemistry 39:1431, 1967.

45. Mondino, A., "A new system of automatic aminoacid analysis," J. Chromatog., 30:100, 1967.

46. Fiske, C. and Sabbarow, Y., Official Chemists,(Washington D.C.: A.O.A.C.) 1970.

47. Post, J., Anlellung zur Planung und Auswertungvon Fledenversuchen mit Hilfe der Varianalyse(Berlin: Springer Verlay), 1952.

48. Fischer, R., La Programmazione degli Espermental(Pisa: Nistri-Lischi), 1954.

49. Pompily, G. and Napolitani, D., "Piano degliesperimenti ed elaborazione probabilistica deirisultati," La Ricerca Scientifica, 24, 1954.

50. Fischer, R. and Yates, F., Statistical Tables forBiological Agricultrual and Medical Research(London: Oliver and Boyd), 1953.

51. Bonomi, A., et li., "I complessi oligodinamie;chelati nell' alimenazione dei broilers," Rivistadi Avicoltura, 9:35, Sept., 1983.

Chapter 19

GROWTH RATES AND FEED CONVERSION INBROILER CHICKS FED AMINO ACID CHELATES

Louis Cuitun and Eduardo GuillenProductos Quimico Agropecuarios, S.A.

The patterns of growth rates in poultry have beenwell documented. There is a nonlinear relationshipbetween weight gains and the age of the bird. Thegrowth rate follows a sigmoidal curve and includes threephases of post hatch growth: (1) a period of relativelyslow weight increase, (2) a period of rapid growth, and(3) a second period of slow ~rowth which occurs afterthe time of sexual maturity. ) These average growthrates are summarized in Table 1.(2)

I Table 1 IWhole Body Weights and Rates of Gain

Body Weight (gm) Daily Rate of Gain (gm/day)Cockere1

Age Mean S.E. Mean S.E.

Day 1 36.4 :t 1.41 --- ---Week 1 51.2 ± 1.00 2.46 :to.39Week 2 108.6 :t 1.41 8.21 :to. 071Week 3 191.9 ± 1.73 11.9 :to. 16Week 4 229.2 + 6.16 5.32 :t1 . 14Week 5 335.9 :t 23.7 15.3 :t1 .94Week 6 434.9 :t 47.3 14.2 ±1.05Week 7 540.2 ± 32.5 15.0 ±3.59Week 8 699.9 ± 2.62 22.8 ±0.26Week 10 911.7 ± 4.52 15.1 ±0.89Week 12 1143.3 0 16.5 ±0.29Week 14 1256.3 ± 5.00 8.07 ±0.35Week 16 1451.9 ± 40.8 14.0 :t2. 55Week 18 1647.5 ± 2.73 14.0 ±2.70Week 20 1773.5 :t 34.4 8.98 :t2. 63Week 22 1759.8 ±138.3 -9.50 -0. 97:t9 . 7

318

Growth Rates and Feed Conversion in Broiler Chicks Fed 319Amino Acid Chelates

Excluding disease and husbandry, there are threebasic factors that will influence growth rates: diet,envi ronmental temperatures, and hormones. The 1attertwo are outside the scope of this discussion. Sufficeit to say that the growth and sexual deve1opment ofbirds are under hormonal control, and that environmentaltemperatures can affect both the growth of the thyroid,as well as chick survival.

Studies conducted in several parts of the worldwith varying environmental temperatures, in which aminoacid chelates have been included in the diets of growinganimals and birds, have indicated a superior growth ratewhen compared to non-chelated metal salts. Such studieshave been conducted at both private institutions and atthe experimental stations of several universities. Somestudies have concentrated on a single mineral nutrient,such as zinc, and measured its effect on growth rates. (3)

Other stud ies have looked at the effects of severalmi nera1s on growth rates. (4)

This current study was initiated for the purposeof comparing the effect of several amino acid chelateson growth rates and other parameters of economicimportance in broiler production. Since there is adifference ingrowth rates of bi rds ra i sed on floorhousing compared to those raised on five levelbatteries, both methods were compared in this study.Both males and females (50:50) were used in each trialbecause of potential growth rate differences based onthe sex of the bi rd. Furthermore, because economi cswere a factor, the study looked at the effects of dietsin which the source of its minerals was 100%, 40%, 20%or no amino acid chelates with the balances beingobtained from mineral salts. The experimental designcan be seen in Table 2.


I Table 2 IExperimental Design to Measure Growth Rates with Mineral Supplementation

Treat Given Name Description No. Birds/Rep. No. Repli- No.Birds/-ment cations Treatment

1 Chelate 100% amino 50 (floor) 3 150acid chelates 10 (battery) 3 30

2 Inorganic 100% inorganic 50 (floor) 3 150trace elements 10 (battery) 3 30

3 80/20 80% inorganic 50 (floor) 3 150trace elements/ 10 (battery) 3 3020% amino acidchelates

4 60/40 60% inorganic 50 (floor) 3 150trace elements/ 10 (battery) 3 3040% amino acidchelates

The trace elements included in this study andtheir levels per treatment are shown in Table 3. All ofthe levels are expressed as ppm of metal in the feed.In each case, the iodine was provided as KI, sinceiodine cannot be chelated.

I Table 3 IMineral Treatments (mg/kg feed)

__1_ __2_ _3_ _4_

Amino Acid Chelate Inorganic 80/20 60/40

Manganese 35 50 40/4 30/8Copper 3 5 4/0.4 3/0.8Iodine 0.10 0.15 0.12/0.01 0.09/0.02Iron 33 50 40/4 30/8Cobalt -- 0.05 0.04/- 0.03/-Zinc 30 50 40/4 30/8


Two feed rations were provided to the chicks asseen in Table 4. The first, a starter feed, was givenfrom day 1 to day 35. The finisher feed was providedfrom day 36 to day 56, at which time the studyconcluded. The levels of minerals indicated in Table 3were maintained regardless of the feed formulation beingfed and were added after the feed had been manufactured.

Table 4

Starter and Finisher Feed Formulations

MiloRock phosphateAlfalfa, 22%Sesame, 43%

Cottonseed meal, 43%Fish meal, 65%Brewer's dry yeastSoybean meal, 47%

Meat and bone meal, 49%Cromofi lora.;)Vi tami n mi xb

Monocalcium phosphate

Sodium chloridedl-MethionineCholine chloride, 25%Lysine-HCl

TOTAL

Starter1-35 days

635201018.33

503010

193.33

201.553

2.61.3640.300

1000.427 Kg

Finisher36-56 days

713.33151015

5030

148.33

4.3353

2.61.3620.4561.145

999.556 Kg

.;)A commercial source of natural xanthophylls.

bThe calculated analysis per dose of 5 kg is as follows: Vitamin A,8,000,000 IU; vitamin 03 , 3,000,000 IU; vitamin E, 5,000 IU; vitamin K,2.2 gm; vitamin B12 , 15 mg; vitamin"B2 , 5 gm; pantothenic acid, 6.75 gm;niacin, 25 gm; choline chloride-25%, 300 gm; Coxistac (coccidiostat),500 gm; ETQ-10%, 500 gm; Cromofil oro (pigmenting agent), 1,500 gm; andcarrier to 5,000 gm.


The analyses of the feed formulations in Table 4are provided in the following table.

I Table 5 IStarter and Finisher Feed Analyses

Nutrient Starter Feed Finisher Feed

Crude protein, % 21 18Ether extract, % 1.9 1.84Crude fiber% 4.2 4.3Calcium, % 1.1 0.73Total phosphorus, % 0.73 0.57Available phosphorus, % 0.48 0.34Metabolizable energy, Mcal 2819 2891Choline, mg/kg 1450 1350Xanthophylls, mg/kg 20 54Arginine, % 1.39 1.16Lysine, % 1.06 0.95Methionine, % 0.45 0.40Methionine plus cysteine, % 0.76 0.69Tryptophan, % 0.23 0.20

The above feeds were provided free choice to thebirds. They also had water provided free choice.

Except for bei ng caged in ei ther the battery1eve1s or on-floor hous i ng, the management pract ices forall of the birds were the same. Veterinary practices,including vaccinations, etc., were also the same for allof the chicks.

The fi ve 1evel battery tri a1 produced greaterweight gains than the floor trial. Because the resultswere different, they are reported separately below. Theaverage weekly body weights for the two trials are seenin Tables 6 and 7.


I Table 6 IWeight Gains for Battery Trial

Treatment Number*

Week 1 2 3 4

1 102 102 106 1042 283 261 278 2783 404 379 405 3964 643 599 622 6275 917 780 816 9246 1216 817 836 11807 1545 958 988 15328 1837 1062 1090 1911

Increase5-8 weeks 920 282 274 987

*1, 100% Amino acid chelate (AAC); 2, Inorganic (I); 3, 80/20 (1/AAC) ;4, 60/40 (I/AAC).

I Table 7 IWeight Gains for Floor Trials

Treatment Number*

Week 1 2 3 4

1 100 98 102 1032 232 209 224 2223 389 376 390 4014 602 607 612 5995 821 801 790 7986 1033 787 (-14) 762 (-28) 10577 1208 881 889 12288 1481 966 1033 1492

Increase5-8 weeks 660 165 243 694

*1, 100% Amino acid chelate (AAe); 2, 100% Inorganic (I); 3, 80/20(I/AAC); 4, 60/40 (I/AAC).


For the fi rst fi ve weeks of 1i fe there is anapparent normal growth curve for the battery and floorhousing groups, with the battery housing group growingmore rapidly than the floor housing. It should be notedthat in both cases these chicks grew more rapidly thanthe published growth rates in Table 1. There are datathat suggest that the amount of space per bird in floorhousing will influence growth rates.(S) No analysis ofthis type was conducted in this current study other thanto note the differences in growth rates.

At six weeks of age, when the starter feed wasreplaced by the finishing feed, there was a significantchange in the growth rates of all of the groups. Again,the growth rates were greater than the published data inTable 1.

During the six to eight week period, thedifferences in the groups which received the amino acidchel ates became more apparent. In both the batterytrials and floor trials, the chicks receiving 100% aminoacid chelate grew much faster than those birds receiving100% inorganic minerals. In both cases, the increaseresulting from the chelates was approximately triple theincreases from the inorganic source of minerals (920 gmversus 282 gm and 660 gm versus 165 gm - treatmentversus control).

Furthermore the total 8 week gain, as well as theone week to five week gains, were greater for the aminoac id che1ate groups. From these data alone, it i sobvious that inclusion of the amino acid chelates in thediet enhances growth rates.

The above observat ions were al so confi rmed byexamining Groups 4 and 3 in that order. As the levelsof amino acid chelates decreased in the diet (40% ~ 20%)the growth rates decl i ned. It is obvi ous from thesedata that the greater bioavailability of the minerals inthe amino acid chelate group influenced growth rates.


Based on the data in Tables 6 and 7, it would appearthat when the amino acid chelates are added to the feedration at 60%, or more, of the trace mineral package, upto a undetermined level of less than 100%, there is amarked improvement in the growth rates of the chicks.An amount lower than 60%, perhaps 50%, may also providemost of the nutritonal requiremetns for the ballisticgrowth rates observed.

The average weekly cumulative feed consumptionsper bi rd are shown in Tab1es 8 and 9. The data areshown for the battery trial as well as the floor trial.

I Table 8 IAverage Per Bird Cumulative Feed Consumption for Battery Trial (grams)

Treatment Number*

Week 1 2 3 4

1 82 78 87 862 307 322 297 3043 748 769 748 7734 1248 1288 1233 12895 1748 1370 1523 16876 2533 1670 1889 24827 3307 1995 2348 32878 4085 2320 2755 3978

Increase5-8 weeks 2337 950 1232 2291

*1, 100% Amino acid chelate (AAC); 2, 100% Inorganic (I); 3, 80/20(I/AAC); 4, 60/40 (I/AAC).


I Table 9 IAverage Per Bird Cumulative Feed Consumption for Floor Trial (grams)

Treatment Number*

Week 1 2 3 4

1 107 101 110 1022 346 323 345 3343 709 657 702 6714 1195 1032 1075 10645 1443 1348 1431 13396 1946 1602 1696 17637 2483 1798 1966 22448 3094 1978 2282 2812

Increase5-8 weeks 1651 630 851 1473

*1, 100% Amino acid chelate (AAC); 2, 100% Inorganic (I); 3, 80/20(I/AAC); 4, 60/40 (I/AAC).

Although feed consumption was normal for both thebattery and floor tri a1s, 1ess feed was consumed perbird in the floor trial than in the battery. That isunderstandable since the growth of the battery group wasgreater and it more feed per bi rd was requ i red tosupport greater growth. The feed conversions ratios areshown in Table 10.


I Table 10 IFeed Conversions

Treatment Number*

1 2 3 4

BatteryTotal weight (g) 1837 1062 1090 1911Total feed (g) 4085 2320 2755 3978Feed conversion 2.22 2.18 2.53 2.08

FloorTotal weight (g) 1481 966 1033 1492Total feed (g) 3094 1978 2282 2812Feed conversion 2.09 2.05 2.21 2.88

*1, 100% Amino acid chelate (AAC); 2, Inorganic (I); 3, 80/20 (I/AAC);4, 60/40 (I/AAC).

Feed conversion was better in the floor trial.The feed conversions were slightly better for the totalinorganic mineral when compared to the total amino acidchelate, but the differences were not statisticallysignificant. The major difference in feed conversionwas in group 4 (60/40). This was statisticallysignificant (P < 0.05). Since the only variable amongthe treatments was the sources of the trace minerals, itwould appear that when the inorganic trace minerals arereplaced totally, or up to 60%, by the amino acidchelates, the chelates tend to overcome whatevershortcomings were or could be present with inorganicminerals in the feed rations.

By comparing feed conversion data in Table 10 tothe publ i shed data in Fi gure 1(6), it is easy to see thesuperior feed conversion resulting from the chelates.


4,.....-----------------------.

3.5

~ 3&::

.!! 2.5u

~ 2'1:JCD 1.5CDLL

1 ------------------------------

0.5

O~-----------'-----~----"------I2 4 6

Age In Weeks8 10

Figure 1. The relation of age to feed conversion.

Although this investigation did not examine thereasons for the improved feed conversion, Malleto, etli., (7) have presented data developed at the Universityof Turin that demonstrate that the amino acid chelatessignificantly enhance protein synthesis in the body fromdietary protein and also make more efficient use of thedi etary carbohydrates and protei ns in the feedstuffsthrough greater enzymatic activity within the lumen.Schutte has reported that many of these enzymes aremineral dependant,(8) making the greater bioavailabilityof the minerals in the amino acid chelated form moreavailable to activate those enzymes. Since mineralnutrition also plays a direct role in somatic growth,the greater availability of the amino acid chelates maycontribute directly to enhanced growth rates.


References

1. Patrick, H. and Schaible, P., Poultry: Feeds andNutrition (Westport: AVI Publishing co.) 55,1980.

2. Ibid, 60.

3. Ashmead, H., "Growth regulating effect of zincproteinate [amino acid chelate] on chicks," Proc.49 of Am. Assoc. Advancement of Sci. AnnualMeeting, Logan, Utah, 1968.

4. Bonomi, A., et li., "I complessi oligodinamie:Chelati nell' alimenazione dei broilers," Rivistadi Avicoltura, 9:35, Sept. 1983.

5. Ewing, W. R., Poultry Nutrition (Pasadena: TheRay Ewing Company) 133, 1963.

6. Patrick and Schaible, op. cit., 426.

7. Malleto, S., et li., "Studies on the nourishingaction of the amino acid chelates," Unpublished,1983.

8. Schutte, K., The Biology of the Trace Element(Philadelphia: J. B. Lippincott Co.) 17, 1964.

Chapter 20

THE USE OF AMINO ACID CHELATES INGROWING TURKEYS

Alberto Bonomi, Afro Quarantelli,Paola Superchi, Alberto Sabbiono, and Danielle 801si

University of Parma

Adetailed discussion concerning the importance ofminerals to turkey nutrition is not necessary. It iswell established that certain minerals are required toparticipate in the vital processes that allow the birdsto make efficient use of their feeds and maintain goodhealth.

In spite of this general knowledge, there is stillmuch to be learned. More precise information isrequired concerning the optimum balance of energy,protein (amino acids), vitamins, minerals, antibiotics,enzymes and hormones. More information is needed on thedigestibility of feedstuffs.(1) Furthermore, there arestill many unknowns concerning the absorption andmetabolism of trace elements introduced into the turkeyfeeds.

As a frequent result of this lack of knowledge,certain mineral formulations or different sources oftrace elements will cause declines in turkey production.Thus, many studies in the past few years haveconcentrated on trying to determine the proper minerallevels and integrate them into the feed sufficient tocover the biological needs of the turkey and enhance itsproduction.

Tabl e 1 demonstrates the extent of manynutritionists' knowledge of the mineral requirements inturkeys.(2) In conjunction with this table, it has beenreported that "no good evidence is available concerningthe requirements of turkeys for the trace minerals.

330

The Use of Amino Acid Chelates in Growing Turkeys 331

Therefore it might be wise to add small amounts of theseto practical rations, since experience has shown thatthis will do no harm. Research at Cornell and Texas A& M has demonstrated that chicks and poults require amineral (or minerals) not listed in Table 1. Thisunidentified nutrient is required both for growth andfor proper bone formation. It is known to be mineral innature, because it is present in the ash of suchmaterials as distillers, dried solubles, fish solubles,and dri ed whey." (2)

I Table 1 IThe Mineral Requirements of Turkeys (Per Kg of Diet)

A~proximate Amount Required

0-12 12-20 20-26Minerals Weeks Weeks Weeks Breeders

Calcium % 1.8-2.0 1.5-1.7 1.0-1.2 2.5Phosphorus, total, % 0.9-1.2 0.7-0.8 0.6-0.7 0.8Phosphorus, inorganic, % 0.65-0.75 0.5-0.6 0.4-0.5 0.6Salt, % 0.5 0.5 0.5 0.5Manganese, mg 55 55 33 33Potassium* ? ? ? ?Iodine* ? ? ? ?Magnesium* ? ? ? ?Iron* ? ? ? ?Copper* ? ? ? ?Zinc* ? ? ? ?Molybdenum ? ? ? ?

*"These minerals are undoubtedly required. There is no evidence,however, that good rations require special supplementation with any ofthem, except possibly with iodine, iron and/or copper in certain areaswhere the soi 1 is defi ci ent in one or more of these el ements. "(2)

Not only is there a need to establish theessential nature of certain trace elements for thegrowing turkey, but also, to enhance their absorption.It is specul ated that perhaps some of the confus ionarising out of the data in Table 1 relates to thebioavailability of the sources of minerals studied. If


they were more biologically available, as has beenreported with the amino acid chel ates, (3) the metabol icresponse in growing turkeys might be more dramatic.

To illustrate this, a study involving over 12,000rna1e turkeys was recent1y comp1eted. (11) The poul ts wereplaced into two houses. One house, containing 6,696birds, was designated as the control group, and theother house, which held 6,697 birds, was the treatmentgroup. The experiment commenced at one day of life andcontinued for 115 days. Both groups received the samefree choice feed, water, and had the same diseasecontrol program.

The poults were given a starter feed until theyreached the 49th day of age (7 weeks). At this point,their feed was changed to a grower feed. In addition tothe feeds described above, the treated group alsoreceived the mineral supplement shown in Table 2 forseven days. It was mixed in the feed at the rate of 500ppm.

I Table 2 IMi nera1 Supplement Given to Growing Turkeys

Magnes i urn" 20 %Potass i umb 12 %Carrier 68 %

.. Metal supplied as an amino acid chelate.b Metal supplied as an amino acid complex.

At the end of seven days the chelate was removedfrom the treated group's diet, and the treated groupsubsequently received the same feed as the control groupfor the remainder of the experiment. At 115 days of age(16.4 weeks) both groups were marketed. During the

The Use of Amino Acid Che/ates in Growing Turkeys 333

experiment, data on weight gains, feed conversions,mortality, etc. were recorded, which are summarized inTable 3.

I Table 3 IEffects of Amino Acid Chelates on Growing Turkeys

Control Treated

7 weeks 16.4 weeks 7 weeks 16.4 weeks

Condemnation --- 10.62 % 1.7 % 3.95 %Mortality 1.9 % 12.69 % --- 5.02 %Weight 20.07 lbs 22.84 lbsFeed conversion 2.79 2.50

These data demonstrated that with only seven daysof supplementation of two minerals, one of which (K) isnot considered essential in turkeys according to Table1, total mortality was reduced 60.4% with a 13.8%increase in weight and 10.4% improved feed conversion.The return to the turkey farmer was increased 14.3% perbird. Based on the findings of the above experiment,there was justification to further investigate theeffects of other amino acid chelates on growing turkeys.

The following study was conducted using 1,600 maleturkey poults that were all one-day old. The breed wasNicholas Cuddy. The poults were divided into eightgroups of 200 birds each.

The feeding program was divided into threeperi ods, the fi rst of wh i ch was from day one to dayfifty. The second period went from the 51st day to the100th day, and the third period commenced on day 101 andcontinued until the 150th day.

Groups 1, 3, 5, and 7, of the eight groups ofturkeys, were considered control groups. Groups 2, 4,


6 and 8 were the treated groups and received the aminoacid chelates at the rate of 1000 ppm of the total feed(100 ppm of metal). (The amino acid chelates used inthis study contained 10% metals.) Excluding the aminoacid chelates, the feed formulation for groups 1 and 2;s shown in Table 4. The vitamin and mineral supplementused in the feed formula is shown in Table 5.

I Table 4 IGroup 1 and 2 Diets (Kg/100 Kg)

Period 1 Period 2 Period 3

Soy flour (50% protein) 13 10 7Sunflower flour (45% protein) 9 7 5Meat scraps 8 6 4Fish scraps 8 6 4Antibiotic supplement 3 3 3Corn flour 40 50 60Ground wheat 11 10 9Dried yeast 1 1 1Corn gluten 2 2 2Calcium carbonate 1 1 1Dicalcium phosphate 1 1 1Sa1t (NaCl) 0.5 0.5 0.5Vitamin and mineral supplement 0.5 0.5 0.5Fat 2 2 2


I Table 5 IVitamin and Mineral Supplement (Per Kg)

Vitamin i\ 3,000,000 IUVitamin D 400,000 IUVitamin B1 500 mgVitamin B2 2,000 mgVi tami n B6 700 mgVitamin 812 6 mgPyridoxine 7,000 mgPantothenic acid 4,000 mgFolic acid 500 mgCholine chloride 100,000 mgdl-Methionine 50,000 mgZinc bacitracin 4,000 mgManganese 25,000 mgCobalt 200 mgCopper 500 mgZinc 10,000 mgIron 5,000 mgIodine 300 mg

Groups 3 and 4 received the same feed, except thatGroup 4 also received amino acid chelates. Their feedsfor the three feeding periods, excluding the amino acidchelates are shown in Table 6. The vitamin formulationwas the same as in Table 5. The amino acid chelatesformulation is again as shown in Table 7.


I Table 6 IGroup 3 and 4 Diets (Kg/100 Kg)


Soy flour (50% protein) 14.3 10.7 7.3Sunflower flour (45% protein) 9.2 7.1 5.1Meat scraps 7.6 5.8 3.9Fish scraps 7.6 5.8 3.9Antibiotic supplement 3 3 3Corn flour 40 50 60Ground wheat 10.4 9.6 8.7Dried yeast 1 1 1Corn gluten 2 2 2Calcium carbonate 1.03 1.02 1.03Dicalcium phosphate 1.05 1.03 1.03Salt (NaCl) 0.5 0.5 0.5Vitamin and mineral supplement 0.5 0.5 0.5Fat 2.14 2.1 2.01Amino acid chelate supplement .1 .1 .1

I Table 7 IAmino Acid Chelate Supplement (g/Kg)

Iron 9.00Copper 2.15Zinc 3.00Manganese 1.20Cobalt 0.08

Excluding the amino acid chelates in group 6, thefeed formulas for groups 5 and 6 are shown in Table 8.The vitamin and mineral supplements are the same as inTable 5, and the amino acid chelate formula given togroup 6 is the same as shown in Table 7.


I Table 8 IGroup 5 and 6 Treatment Diet (Kg/100 Kg)


Soy flour (50% protein) 14.8 11 7.4Sunflower flour (45% protein) 9.4 7.3 5.2Meat scraps 7.1 5.7 3.8Fish scraps 7.1 5.7 3.8Antibiotic supplement 3 3 3Corn flour 40 50 60Ground wheat 10.1 9.3 8.6Dried yeast 1 1 1Corn gluten 2 2 2Calcium carbonate 1.6 1.5 1.7Dicalcium phosphate 1.8 1.6 1.5Salt (NaC1) 0.5 0.5 0.5Vitamin and mineral supplement 0.5 0.5 0.5Fat 2.2 2.02 2.1Amino acid chelate supplement 0.1 0.1 0.1

Except for the inclusion of the amino acid chelatein group 8, the diets of groups 7 and 8 are shown inTable 9. As with the other groups, the vitamin andmineral supplement is as shown in Table 5, and the aminoacid chelate formula given to group 8 is as shown inTable 7.


i Table 9 IGroup 7 and 8 Treatment Diet (Kg/lOO Kg)


Soy flour (50% protein) 15.3 11.3 7.4Sunflower flour (45% protein) 9.5 7.4 5.3Meat scraps 7.6 5.6 3.8Fish scraps 7.6 5.6 3.8Antibiotic supplement 3 3 3Corn flour 40 50 60Ground wheat 9.8 9.1 8.5Dried yeast 1 1 1Corn gluten 2 2 2Calcium carbonate 1.01 1.01 1.01Dicalcium phosphate 1.01 1.01 1.01Salt (NaC1) 0.5 0.5 0.5Vitamin and mineral supplement 0.5 0.5 0.5Fat 2.03 2.02 2.02Amino acid chelate supplement .1 .1 .1

The chemical analysis of the four feeds withoutthe inclusion of the amino acid chelates in the feeds ofthe experimental groups for Peri ods 1, 2, and 3 areshown in Tables 10, 11, and 12, respectively.

I Table 10 IChemical Analysis of Period 1 Feeds (%)

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

Moisture 12.10 12.18 12.09 12.15Ash 7.57 7.68 7.49 7.62Crude protein 28.15 28.22 28.17 28.12Crude fat 5.78 5.73 5.72 5.76Crude fiber 3.31 3.35 3.39 3.42Inactive ingredients 43.09 42.84 43.14 42.93


Table 11

Chemical Analysis of Period 2 Feeds (%)

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


I Table 12 IChemical Analysis of Period 3 Feeds (%)

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


From these tables it can be seen that the feedswere relatively similar from group to group with onlyslight modifications. As a result, it was hoped thatthe experimental data would not only provide proof ofgreater efficacy of the amino acid chelates, but also anindication of any synergism between the amino acidchelates and the various sources of feedstuffs.

Each turkey was individually weighed at 50, 100,and 150 days. The results are reported in Table 13.The statistical analyses(4-7) are included in the sametable. As can be seen, in the first fifty days, thoseturkeys which received 16% animal proteins had greaterwei ght ga ins than the turkeys recei vi ng protei n from


vegetable sources (group 1 versus groups 3, 5, and 7).Group 1 grew 4.11% more than group 3, 5.21% more thangroup 5, and 6.1% more than group 7. However, theaddition of the amino acid chelates to the diets ofgroups 4, 6 and 8 increased the growth rates of groups4 and 6 giving similar weight gains as reported forgroup 1.

At 100 days, the turkeys in groups 3, 5, and 7which had received the partial substitutions of thevegetable proteins for meat protein grew more slowlythan group 1 which received meat protein. Group 1 grew4.84%, 6.32%, and 7.00% better than groups 3, 5, and 7,respectively. With the addition of the amino acidche1ates to the feed, groups 4 and 6 grew 1. 59% and2.31% better than group 1, even though groups 4 and 6had partial substitution of the meat source of protein.Although increases were noted, the differences were notsignificant.

At 150 days, the differences between group 1 andgroups 3, 5, and 7 were even more marked: 5.32%, 6.88%,and 7.59%. The addition of the amino acid chelateshelped to recover the losses incurred through thereduction of meat proteins. The differences betweengroup 1 and 4, 6, and 8 were 1.88%, 5.63%, and 6.38%which were less decrements than seen in groups 3, 5, and7 above.


I Table 13 IWeight Gains and Feed Conversions

50 Days 100 Days 150 Days

Group Mean Uve Weight No. of Mean Uve No. of Mean Uve No. of FeedBirds Weight Birds Weight Birds Conversion

1 2,580.30±64.29 190 8,113.28±153.40 185 13,420.81 ±212.81 182 3.032 2.713.95±71.35 192 8.563.56±169.23 187 14.350.87±200.32 185 2.853 2,474.25±80.23 189 7,720.60±148.72 186 12,706.82±246.58 184 3.144 2,565.29±68.74 191 7,984.28±179.36 187 13,168.50±231.79 185 3.055 2.445.87±81.80 188 7.600.52±161.59 185 12,497.46±250.30 185 3.216 2,521.64±76.39 189 7.925.87±180.20 188 12,665.22±218.16 187 3.127 2,422.90±83.42 190 7,545.35±173.14 188 12,402.17±246.31 182 3.278 2,437.32±91.64 189 7.616.75±158.38 187 12.564.56±238.74 183 3.16

85.36 231.42 293.48

Least Square Difference (P<0.05).

It is interesting to note from Table 13 that ineach feed formul a, the add it i on of the ami no ac idchelates improved feed conversion. It took 5.94% lessfeed (group 1 versus group 2), 2.87% less feed (group 3versus group 4), 2.8% less feed (group 5 versus group6), and 3.63% less feed (group 7 versus group 8) toachieve a kilogram of weight when the amino acidchelates were included in the diets.

At 150 days, twenty turkeys from each group wererandomly selected and sacrificed after being fasted fortwelve hours. The carcass, meat, fat, bone, skin, headand neck, feet, gizzard and stomach, internal organs,feathers and intestine and pancreas were examined andrecorded as a percentage of live weight. Muscle, fat,skin and subcutaneous flesh are also shown aspercentages of carcass weight. These data aresummarized in Table 14.


I Table 14 IOrgan Weights as a Percentage of Uve Weight

Uver Intes-Spleen tine

Skin & Head Heart &Car- Subcutane- & Giz- Testicles Fea- Pan-cass Muscle Fat Bone ous Flesh Neck Feet zard Lungs thers creas

% % % % % % % % % % % % % % %Group ± car- ± car- ± car- ± car- ± ± ± ± ± ± ±

p.v. cass p.v.* cass p.v. cass p.v. cass p.v. p.v. p.v. p.v. p.v. p.v. p.v.±p.v. ± ± ±

p.v. p.v. p.v.

79.52 71.13 56.56 3.04 2.42 18.31 14.56 7.52 5.98 4.80 2.38 1.53 3.62 4.85 2.501 ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

3.16 2.28 1.82 0.31 0.30 1.51 1.18 1.00 0.81 0.52 0.23 0.23 0.46 0.54 0.25

81.60 73.26 59.78 2.36 1.92 17.57 14.38 6.81 5.55 4.40 2.17 1.48 3.50 4.33 2.212 ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

3.00 2.15 1.96 0.45 0.37 1.42 1.23 1.08 0.90 0.71 0.16 0.14 0.38 0.49 0.23

77.49 69.20 53.62 3.49 2.70 19.40 15.03 7.91 6.13 4.98 2.76 1.61 3.88 5.12 2.883 ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

3.29 2.31 2.08 0.30 0.25 1.38 1.20 1.04 0.76 0.63 0.27 0.16 0.41 0.62 0.21

79.20 70.83 56.09 3.00 2.37 18.50 14.65 7.67 6.07 4.67 2.45 1.72 3.77 4.75 2.464 ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

2.93 2.06 2.00 0.51 0.34 1.56 1.29 0.95 0.65 0.58 0.15 0.15 0.40 0.71 0.26

77.12 69.12 53.30 3.38 2.61 19.51 15.04 7.99 6.16 5.10 2.63 1.80 4.00 5.28 2.945 ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

2.85 2.18 1.75 0.42 0.30 1.25 1.10 1.16 0.84 0.80 0.21 0.15 0.29 0.60 0.20

77.74 69.51 54.04 3.36 2.62 19.74 14.96 7.89 6.13 5.06 2.49 1.98 3.95 4.92 2.706 ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

3.04 2.46 1.68 0.58 0.36 1.31 1.20 1.12 0.82 0.67 0.19 0.17 0.27 0.59 0.18

76.53 68.67 52.55 3.84 2.94 19.68 15.06 7.81 5.97 5.17 2.70 1.85 4.00 5.19 2.807 ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

3.22 2.32 1.93 0.49 0.40 1.40 1.27 0.99 0.61 0.74 0.19 0.19 0.32 0.63 0.22

77.28 69.00 53.32 3.53 2.73 19.37 14.97 8.10 6.26 4.91 2.83 1.93 3.91 5.10 2.968 ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

3.10 2.49 2.11 0.43 0.33 1.47 1.32 1.03 0.70 0.55 0.24 0.16 0.38 0.68 0.19

LS.D. 1.25 1.10 1.38 -- --- -- --- --- --- --- -- --- --- --- ---(P<0.05)

*p.v. =- analysis of variance

As a percent of the totalcarcass ranged from 76.53% to

live body weight, the81.60%. The hi gher


percentages came from the turkeys wh i ch rece i ved theamino acid chelates in their diets. Group 2, whencompared to group 1, had 2.62% more carcass. Group 4had 2.21% more than group 3. Group 6 had 0.08% morecarcass than group 5. And finally, group 8 had 1.00%more carcass than group 7. All of these di fferencesexcept groups 5 and 6 were statistically significant atP<O.05.

Even though group 4 received a higher amount ofvegetable protein than meat protein, the carcass weightapproximated the weight in group 1, the control group.It is believed that this weight increase is a directresult of the influence of the amino acid chelates onincreasing protein utilization from the diet.

The production of meat was also influenced by theamino acid chelates. Group 2 had 5.69% more muscle meatthan group 1 on a live weight percentage basis. Group4 increased 4.61% over group 3. These data were alsostatistically significant (P<O.OS).

There were no statistical differences in the fat,bones, skin, and other tissues and organs examined andweighed.

After homogenization, dehydration and ashing, thebreast meat was chemically analyzed for its mineralcontents. The breasts were also analyzed for moisture,protein and fat content. Tables 15 and 16 present thesedata. There was very little variation between thegroups.


I Table 15 IAnalysis of Breast Meat

Group Dry Weight % Ash % Protein % Fat %

1 72.91 ± 0.24 1.26 ± 0.04 23.82 ± 0.69 1.35 ± 0.552 73.11 ± 0.68 1.25 ± 0.07 23.30 ± 0.90 1.32 ± 0.293 73.20 ± 1.30 1.27 ± 0.11 22.78 ± 0.58 1.50 ± 1.274 73.45 ± 0.58 1.17 ± 0.03 22.80 ± 0.81 1.36 ± 0.755 73.40 ± 0.79 1.14 ± 0.07 22.73 ± 0.79 1.95 ± 0.936 72.95 ± 0.82 1.14 ± 0.06 23.40 ± 1.06 1.71 ± 0.887 72.79 ± 0.73 1.11 ± 0.05 23.81 ± 0.43 1.58 ± 0.768 73.15 ± 0.68 1.14 ± 0.06 23.58 ± 1.23 1.52 ± 0.49

I Table 16 IMineral Analyses of Breast Meat (per 100 gm Net Weight)

Group Ca P Mg K

1 6.00 ± 0.01 225.00± 7.62 28.40 ± 1.14 379.00 ± 26.372 6.30 ± 0.54 230.40 ± 4.04 28.20± 2.49 356.20 ± 15.193 6.40 ± 1.14 238.60 ± 9.66 30.20 ± 1.30 367.80 ± 17.684 6.40 ± 0.54 221.80± 5.72 30.00± 0.71 352.40 ± 13.24.5 6.60 ± 1.34 225.00 ± 2.23 30.60 ± 1.14 360.60 ± 28.006 6.00 ± 1.22 231.80 ± 4.49 29.80 ± 1.30 361.00± 11.117 5.80 ± 0.83 230.20 ± 9.01 29.20 ± 0.84 368.60 ± 13.728 7.00 ± 0.70 224.25± 12.25 28.80± 2.17 354.00± 29.61

Table 16 continued

Mineral Analyses of Breast Meat (per 100 gm Net Weight)

Group Na Fe Zn Cu

1 69.00 ± 6.52 0.93 ± 0.26 1.02 ± 0.09 0.41 ± 0.162 72.20 ± 8.76 1.02 ± 0.11 1.05 ± 0.03 0.46 ± 0.203 79.00 ± 7.70 0.83 ± 0.10 1.09 ± 0.12 0.34 ± 0.234 77.40 ± 6.80 0.77 ± 0.17 1.05 ± 0.15 0.43 ± 0.145 66.60 ± 5.68 0.77 ± 0.26 0.78 ± 0.05 0.26 ± 0.146 58.20 ± 3.11 0.81 ± 0.30 1.03 ± 0.32 0.22 ± 0.097 56.00 ± 4.58 1.09 ± 0.30 0.89 ± 0.09 0.24 ± 0.098 58.40 ± 8.11 0.89 ± 0.10 0.86 ± 0.08 0.30 ± 0.06


The meat from the breasts were diced, the fatremoved and then the digestibility determined accordingto the Bjoll ema-Wedemeyer method. The resul ts forprotein, digestibility, and the coefficients ofdigestibility are shown in Table 17. There were nostatistically significant differences in the groups.

I Table 17 IDigestibility of the Breast Meat

Coefficient ofGroup Total Protein % Nondigestible % Digestible Digestibility

1 23.82 ± 0.69 0.39 ± 0.04 23.43 98.362 23.30± 0.90 0.35± 0.03 22.95 98.493 22.78 ± 0.58 0.56 ± 0.15 22.22 97.544 22.80± 0.81 0.57 ± 0.14 22.23 97.505 22.73 ± 0.79 0.49 ± 0.06 22.24 97.846 23.40 ± 1.06 0.58 ± 0.14 22.82 97.527 23.81 ± 0.43 0.36 ± 0.06 23.45 98.488 23.58 ± 1.23 0.32 ± 0.10 23.26 98.64

The last table, Table 18, summarizes thetenderness of the meat. Using the Schonberg andLochmann(·) method wh i ch has been elaborated by Kruger, (9)

in which the meat was exposed to trypsin for 96 hoursand the percent of undigested material measured, it wasdetermined that there were no statistically significantvariations between the groups.


i Table 18 ITenderness of Breast Meat

Group Percent of Nondigested Material

1 3.41 ± 0.392 3.89 ± 0.443 3.09 ± 1.354 2.85 ± 0.215 3.10 ± 1.346 3.20 ± 0.997 3.46 ± 0.558 3.89 ± 0.37

In summary, it was found that when the amino acidchelate formula (shown in Table 7) was included in theturkey feed at the rate of 1000 ppm (100 ppm metal) ofthe total feed, feed growth rates were increased andmore efficient use made of the protein sources found inthe feed. Feed convers i on was a1so enhanced. Whenbutchered, the carcass weight of the turkeys receivingthe amino acid chelates was greater, with a 2.61%greater ki 11 i ng percentage and as. 69% greater meatyield, comparing groups 1 and 2 (the mean increase forall groups was 3.29%).

These tests demonstrated that growth rates inturkeys will vary based on the source of the protein fedto them. Animal sources provide greater growth ratesthan vegetable sources. Nevertheless, the amino acidchelates improved the utilization of protein from thevegetable sources in the feed, and, in some instances,allowed the turkeys to make as effi cient use of avegetable protein as they normally would have made witha meat protein. Since vegetable proteins are generallyless expensive than meat proteins, this has an economic


impact. This research demonstrated that by includingthe amino acid chelates in the feed at 1000 ppm (100 ppmof metal) meat protein sources can be reduced by about5 to 7% under the constra i nts of the tested rat ionswithout having a negative impact on the production ofthe birds. oO

)


References

1. Patri ck, H. and Scha i bl e, P., Poul try: Feeds andNutrition (Westport: AVI Publishing Co.) 553,1980.

2. Ewing, W., Poultry Nutrition (Pasadena: The RayEwing Company) 1044, 1963.


4. Post, J., Anleitung zur Planung und Auswertungvon Feldenversuchen mit Hilfe der Varianzanalyse(Berlin: Springer Verlag) 1952.

5. Fischer, R., La programmazione degli Esperimenti(Pisa: Nistri-Lischi) 1954.

6. Pompily, G. and Napolitani, D., "Piano degliesperimenti ed eleourazione probablistisca deirisultati," La Ricerca Scientifica, 24, 1954.

7. Fischer, R. and Yates, F., Statistical Tables forBiological, Agricultural and Medical Research(London: Oliver and Boyd) 1953.

8. Schonberg, F. and Lochmann,Lebensmittelhyg, 8:11, 1957.

E. , Arch.

9. Kruger, H., Ein Beitrag zur obiektiven Bestimmungder Fleischgualitat von Jungmastrindern (Hemburg:Verlag Paul Parey) 1965.

10. Bonomi, A., et ~., "I complessi oligodinamicichelati nell'alimentazione del tacchino dacarne," Rivista di Avicoltura, 51:23, June, 1982.

Chapter 21

THE ROLE OF AMINO ACID CHELATES INOVERCOMING THE MALABSORPTION

SYNDROME IN POULTRY

Angelo FerrariZoopropylaetie Institute of Piedmont

Liguria and Valle d'Aosta, ItalyGermano Cagliero

Teevet, S.P.A., Turin, Italy

Since 1976, a syndrome resulting from defectiveintestinal absorption of either a nutrient and/orelectrolytes and fluid leading to clinicalmanifestations and less efficient productivity has beennoted in many broiler breeding farms as well as inlaying houses. Although this problem has become apopular topic of discussion in recent times, it is by nomeans a recent disease. Occasional occurrences of thesame or an analogous syndrome have been described in thepast.

There are many different names and idiomaticexpressions that have been used to describe thisdigestive disturbance. Among the most common names are:Malabsorption syndrome, pale bird syndrome, femoral headnecrosis, brittle bone disease, osteoporosis, andinfectious stunting (or runting) syndrome. Apart fromcommon names above, it is considered by someveteri nari ans as II infect i ous proventri cul us ", a1though,because the etiology of the principal syndrome has notbeen fully understood, this could be a different aspectof the same disease. For the purpose of this discussionthe problem will be called "malabsorption syndrome."

The malabsorption syndrome generally affects meatproducing broilers. It rarel{ strikes the young heavyproducers(1) or young hens. () Neverthe1ess, it isreadily transmitted to young poults and chicks.

349


Broiler operations affected by this malabsorptionsyndrome regularly relapse into the same symptoms.Frequently, the problem remains in a particular area ofthe poultry house and recurs only in that specific area.The syndrome can be more severe during certain seasonsthan others. (3) All of the above suggests that there i san external cause for the disease rather than being acongenital defect.

Maximum susceptibility to the malabsorptionsyndrome is usually recorded within the first seven daysafter hatching. In the second week of life andcontinuing through the next five weeks, thissusceptibil ity rapidly decl ines. (4) The infection seemsto be transmitted by way of the digestive system byeither direct or indirect contagion. Incubatorinfections and flock contagion seem very similar, buthave not yet been scientifically documented.Insufficient environmental hygiene, incorrectapplication of the Hall full-all empty" system,excessive crowding, unbalanced diet, and someintercurrent infections all seem to favor the emergenceof the malabsorption syndrome and aggravate itseffects. (5)

After 4 to 6 days of incubation followingexposure, approximately 20% to 30% of the chicksinfected begin to present vague signs of the syndromewhich then seem to follow a systematic progression ofillness. During the second week of life their growthrates decelerate when compared to normal chicks. Theirlimbs weaken. Their small intestines may becomeinflamed, although the intensity of the enteritis willvary from chick to chick. This enteritis appears to bethe basic cause of the malabsorption.

By the third week of life, skeletal defects, suchas enlargement of the tarsi, and scarce calcification ofthe long bones, will develop. The affected birds willbegin to limp and exhibit signs of rickets and

The Role of Amino Acid Chelates in Overcoming the 351Malabsorption Syndrome in Poultry

osteoporosis. Frequently, there is also depigmentationof the skin, possibly due to malabsorption of vitaminsA and E. It is this latter clinical manifestation whichgives the syndrome another name: "Pale Bird Syndrome."Feathering defects, such as ruffling, begin to developat this stage, thus providing still another name, forthe syndrome: "Helicopter Birds." Enteritis usuallypersists in the infected birds.

At the fourth week of life, there is continuedprogression of the bone alterations cited above.Femoral incurvation appears, as well as tibial rotationdeviation. Radiological examinations of isolated thighbones genera11 y reveal very th in cort i cal s where thelongitudinal bending and external convexity take place.The cortical from the medial side is thickened. Themedullar duct is dilated.(6) It is during this fourthweek that the enteric symptoms and feathering defectsseem to regress. Even though the diarrhea appears todecl ine, .L. col i infections tend to increase, and insome way appear to be re1ated to osteomye1it is. Thebone inflammation may spread, or it may remainlocalized.

By the fi fth week of 1i fe all of the aboveclinical symptoms seem to regress, except the skeletalalterations which may become accentuated. Corticals arethin and fine, and sometimes discontinuous, radiopaquelines run parallel to them in the medullar duct. Insome of the bended th igh-bones, the cort icalor theexternal side, appears thicker than on the medial side.The width of the medullar duct increases. Inside, thetrabecular structure appears normal, but there is anirregular trend with the presence of severaltransversal, radiopaque thin lines. This probablyresults from a demineralization of pre-existent loosergrooves. Di aphysary nutri ent hol es, when they can befound, have varying diameters, from small to normal, inconnection to the bone radius size. Epiphyses are


normally formed, but the trabecular shape is irregular.Subchondral bone density changes so that in some thighbones it is normal, while others contain a structurewith opaque transversal tracts which vary to transparentones, all parallel to each other and to the articularsurface. Some articular surfaces have no incrustationcart i1age. (5) There may be a part i a1 detachment of thefemoral head. (5)

Because of the malabsorption of the nutrients, theweight of the affected birds is considerably lower thanbi rds of the same age i n heal thy flocks. The feedconversion index of infected flocks rises significantly.The mortality rate from the malabsorption syndrome isusually 3% to 4%, but among the more severe cases, itcan go as high as 12% to 15%.

Pathological examination of the diseased birdsreveals inflammation of intestinal pouches (catarrhalentrotyphlitis) of varying intensities. Undigested foodis usually present along with a yellow-orange mucus inthe small distal region. There is atrophy and/orfibrosis of the pancreas accompanied by atrophy of thelymphatic organs. Occasionally there is atrophy of thegizzard musculature with poorly defined hepatic lesions.Myocarditis with slight hydropericardium is also noted.

Hematochemical and physiopathological examinationsreveal a reduction of carotenoid plasma and of some ofthe fat soluble vitamins and alkaline phosphatases.Blood Ca, P, Na, K, and C1 are occasionally withinnormal levels, but in most cases, Ca, P, glucose, totalprotein, albumin, urea and uric acid appearsignificantly decreased.(7) These clinical findingsregarding the blood differences are seen in Table 1.(6)


I Table 1 IBlood Chemistry Differences Between Healthy Birds and BirdsInfected with the Malabsorption Syndrome

Healthy Group Malabsorption SyndromeGroup

Chlorine 105 mEq/L 107 mEq/LCreatinine 0.38 mg/dl (100 ml) a.33 mg/dl (100 m1)Total proteins 3.45 gm/dl (100 ml) 2. 73 gm/d1 (10a m1)Albumin 1.23 gm/dl (100 ml) 0.9 gm/dl (100 ml)Calcium 11.7 mg/dl (100 ml) 9.7 mg/dl (100 ml)Inorganic phosphorus 9.85 mg/dl (100 ml) 7.7 mg/dl (100 ml)Cholesterol 123 mg/dl (100 ml) 112 mg/dl (100 ml)Glucose 238 mg/dl (100 ml) 165 mg/dl (100 ml)Urea 4 mg/dl 3 mg/dlBilirubin 0.35 mg/dl 0.30 mg/dlAlkaline phosphatase 700 I.U. 500 I . U.

In brief, Table 1 shows that many bloodnutritional parameters are reduced in the malabsorptiongroup. This includes proteins, glucose a~d minerals.The mean glucose levels in the infected birds are only69% of that of the healthy birds, and thus, can havesevere consequences for bird development. Similarly,the reduced alkaline phosphatase activity coupled withlower circulating levels of calcium and phosphorus areindicative of a sub-optimal level of bone activity. Thenet effect is a severely curtailed source of nutrientswhich is reflected in poor growth rates, reduced feedefficiencies, and elevated mortality rates.

As a result of an increasing world-wide diffusionof the disease with its associated economic losses dueto poorer feed conversions, higher mortality rates, andmore waste at slaughter, numerous studies have beenconducted to ascertain its cause. Initially, it wasbelieved that certain forms of mycoses (fungi) were thecausative agents. Some researchers still maintain this


hypothesis is valid; however, there are numerousindications of Escherichia coli infections. Pasteurellaanatipestifer has also been noted, and Staphylococcusinfections may also be present. Lately, however, mostof the etiological studies conducted on themalabsorption syndrome, and analogous forms of it, haverevealed the presence of a reovirus in the gastroenterictracts of infected chicks.

Reovirus stock has been isolated from thepancreas, the intestines, liver, the spinal cord andfemoral bone marrows of birds suffering frommalabsorption syndrome. ().5.'.9.10) Almost all of thereovirus under observation was serologically correlatedto the tenosynovitis virus (TVS), such as S-1133. Itwas found, however, that at least one varying serotypeshowed a one-way crossed reaction with the S-1133serotype.

Rosenberger isolated and infected the intestinalansae of some chicks with the reovirus suspected to beresponsible for the malabsorption syndrome anddiscovered a reduced capacity of the intestine to absorbl-methionine and d-glucose.(')

There has been some evidence of the presence ofcalcivirus in the diseased chicks' intestines, whichneither grow on the chicken embryo as a substrate mediumnor in various types of cellular cultures. In the samebirds, however, a reovirus has been isolated from thepancreas which was capable of reproducing the typicalmanifestations of the syndrome including a pancreaticfailure to secrete digestive enzymes.

It has been possible to reproduce gastroenteric,pancreatic, and occasionally, hepatic lesions by usingmany of the isolated reovirus even though the resultsare not always consistent. Nevertheless, these lesions,however they arise, seem to explain the digestivetrouble and absorption defects of the birds. Secondary


deficiency phenomena, such as the inability to absorbfat soluble vitamins, in particular, vitamins A, OJ' andE, (5) and some of the trace element salts, (11) result fromthe infection.

It still remains to be clarified whether: (1) theviral agents at issue are capable of blocking orretarding the affected chicks' growth solely throughmalabsorption mechanisms; or (2) acting directly on theendocrine system; or (3) whether alterations of thelymphatic system intervene in the complex chain ofpathological events which then become the basis of themalabsorption syndrome and correlated forms. One thingis certa in: the mal absorpt i on syndrome may not beconsidered as simply a viral infection. It appears toresult from a great number of factors including viral,bacterial, nutritional" and even management. (11,15)

It has been observed in broilers that injectionsof the parents with vaccines prepared with reovirusavi ary seem to be useful in prevent i ng "femur-headnecrosis" in the chicks. When vaccination with apreparat ion conta in i ng thorough ly attenuated reovi ruseS)were carried out on one-day old chicks, variable resultswere produced. To date, since no vaccine has beendeveloped that will prevent the malabsorption syndromefrom occurring, other alternatives should be considered.These alternatives rest primarily in the field ofnutrition.

There has been some success in the use of vitaminsupplements fed to chicks immediately upon arrival inthe coop. For example, the addition of 2.5% Brewer'syeast to the diets of the ch icks has resul ted inconsiderable receding of the symptoms. (10) Brewersyeast, of course, contains high amounts of riboflavin,pantothenic acid, niacin, and chol ine. (13)


The inability of the chick's body to producevitamin 0] appears to be one of the nutritional aspectsof this syndrome. Vitamin 0] by itself is not able topromote reabsorption of calcium by the bones unless itis first transformed into 25-hydroxy-0] in the liver andthen into 1,25-dihydroxy-D) in the kidney. The latterof these metabo1i tes i s produced when ca1ci urn bloodlevels decrease. It is at this point that theparathyroi d hormone (PTH) intervenes and provokes anincreased renal phosphate secretion which induces theconversion of 25-hydroxy-D] into 1,25-dihydroxy-D).This conversion process is controlled by the calciumblood level. If the level of calcium in the bloodrises, the PTH activity diminishes and the production of1, 25-d i hydroxy-D] decreases. (11.18.19)

The chicks do not seem able to synthesize 1,25dihydroxy-D) during their first ten days of life.Therefore, they must make use of the calcium reserveobta i ned from wi th in the egg pri or to hatch ing. (19)

In addition to the vitamin D, amino acid chelateshave been found to aid in certain aspects of themalabsorption syndrome. Because the latter appear to beabsorbed through a pathway in the small intestine, whichis different from that employed by metal salts,(16) theycircumvent the interferi ng factors in the i ntest i newh i ch the rna1absorpt ion syndrome creates. (12)

Looking only at mineral nutrition as it relates tothe rna1absorpt i on syndrome, and based on the aboverational of greater mineral absorption through the useof amino acid chelated minerals which utilize adifferent absorption pathway in the intestine, thefollowing experiment with baby chicks was conducted toascertain if the clinical symptoms of the syndrome couldbe reduced by increasing mineral uptake.

Five hundred, one-day old chicks were infectedwith the reovirus and arbitrarily divided on an odd/even


basis into two groups of 250 birds each. Both groupsreceived identical feed, housing conditions, etc.,except the experi mental group recei ved a formul at ioncontaining iron (9%), zinc (3%), manganese (1.2%),cobalt (.08%), and copper (2.2%) as amino acid chelatesmixed with the feed at a rate of 100 ppm. The controlgroup received equivalent amounts of these minerals aselemental salts. Within four to six days afterexposure, the control group began presenting the usualclinical signs associated with the syndrome. Skeletalalterations occurred. Some enteritis in varying degreeswas also present. Growth rates were reduced. As shownin Table 2, the total cumulative mortality in thecontrol group was 3.2% greater than the experimentalgroup. After exhibiting some initial symptoms of thedisease, the chicks which had received the amino acidchelates appeared to return to normal health and grew atequivalent or better rates than generally expected forhealthy non-infected chicks, even though clinical dataindicated that the reovirus remained present.

I Table 2 IPercent of Mortalitl Rates in Reovirus Infected Chicks

Week 1 Week 2 Week 3 Week 4

Control 3.4 1.8 0.4 5.6

Experimental 1.4 0.6 0.4 2.4

IDifference I - 2.0 I - 1.2 I -- I - 3.2 I

This treatment with the amino acid chelates gavehighly satisfactory results by supplying supplementalamounts of biologically available essential traceelements which are usually almost congenitally deficientat the time of arrival into the coop. The higherabsorption of the minerals from this particular type of


chelate through a secondary pathway in the intestinesappears to favor the development of specific immunityfactors, the formation of which apparently require thesesupplemental minerals. When developed, these immunityfactors appear to aid the ch icks in overcomi ng thenegative effects of the reovirus.

Having obtained success with the chicks, it wasdecided to conduct a second experiment this time usingbreeder hens in an attempt to prevent the offspring fromsuccumbing to the malabsorption syndrome. The rationalefor this approach was based on the fact that immunitymust be developed from the mi nera1s supp1i ed to thechick before hatching or consume them in a highlyavailable amino acid chelated form shortly afterhatching. Other research has shown that an increaseddeposition of minerals into the egg is possible when thelaying hen is fed metal amino acid chelates,(l4) so itwas believed that a similar phenomenon, (the greatertransfer of metals to the embryo before hatching) couldhe1p prevent the rna1absorpt i on syndrome from man i fest i ngitself in the prenatally supplemented chicks infectedwith the reovirus. This was particularly important,since the above work with the amino acid chelatesupplemented chicks indicated that they could functionequally well when compared to normal chicks even thoughthey were infected with the reovirus.

In the experiment with the breeder hens, 1,000hens, of similar age, from five commercial breeder hencoops were used. Each coop had a past history ofrepeated malabsorption syndrome within their respectiveflocks. The breeder hens in each coop were divided intotwo groups and maintained in wire cages. The hens inthe experimental group received the amino acid chelateformula shown in Table 3 mixed in their feed at the rateof 0.10% chelates in the total feed. This amino acidchelate formula was fed to the experimental groupthroughout the entire period of the experiment. It wasbelieved that the hens in the experimental group would

The Role of Amino Acid CheJates in Overcoming the 359Malabsorption Syndrome in Poultry

transfer a significant portion of these biologicallyavailable minerals to the egg to help build immunity intheir chicks. The control group was fed an inorganicm; nera1 supp1ement w; th an equ ; val ent amount of mi nera1sas contained in the amino acid chelate formula.

Table 3

Mineral Composition of Amino Acid Chelate SupplementGiven to Broiler Hens with a History ofMalabsorption Syndrome

Iron amino acid chelateZinc amino acid chelateCopper amino acid chelateManganese amino acid chelateCobalt amino acid chelate

58.1%19.4%14.2%7.8%0.5%

During the 20 week trial period, both groups ofhens laid about the same total number of eggs - 46,950for the experimental group and 46,500 for the controlgroup. The eggs were hatched under heat 1amps. Thepercent of hatchabi 1i ty was about the same for bothgroups. The experimental group hatched slightly moreeggs, but the increase was not statisticallysignificant.

There were no differences in the treatment of thechicks after hatching.

When the baby chicks were hatched they wereclosely monitored for the first three weeks of life,which, as indicated earlier, is the most critical periodfor the occurrence of the malabsorption syndrome. Thedata which emerged from this experiment are shown inTable 4 and Table 5.


These tabl es show the mortal i ty rates of thechicks hatched from the control and experimental groups,respectively. During the first week of life, themortality of the control group was 2.39 times that ofthe experimental group. In the second week of life themortal ity rate for the control group was 1.05 timesgreater than the experimental group. In the controlgroup a total of 5.54% of the chicks died compared to2.40% of the ch i cks in the experi mental group. Th i smeans that approximately twice as many chicks died whenthe breeder hens did not receive amino acid chelatesduring the laying period. A post mortem of a samplingof the dead chicks indicated they had died as a resultof having the malabsorption syndrome.

I Table 4 IMortality Rates of Chicks from Control Hens

Number of Mortality Mortality Mortality Mortalityof Chicks 1st Week 2nd Week 3rd Week %

10,000 328 334 36 6.989,500 331 201 40 6.029,000 409 80 36 5.839,000 354 73 38 5.169,450 172 138 35 3.65

46,950· 1,594· 826" 185" 5.48b

.. Totals per categoryb Average percent mortality


I Table 5 IMortality Rates of Chicks from Experimental (Amino Acid Chelate) Hens

Number of Mortality Mortality Mortality Mortalityof Chicks 1st Week 2nd Week 3rd Week %

9,000 130 61 35 2.179,500 128 58 37 2.359,500 152 57 34 2.559,500 150 59 38 2.609,000 101 42 36 1.98

46,500" 661" 277" 180" 2.28b

~ Totals per categoryb Average percent mortality

Statistical analyses of mortality rates using thestudents t test indicated that the survival rate of theexperimental group was statistically significant(P<O.OOl) compared to the control group. The mainbenefit, therefore, of supplementing the parent stockhens was to predi spose thei r progeny to an improvedimmuno log ical response to subsequent infect ion fo 11 owi nghatching. While it is likely that the breeder hens alsobenefitted from the metal amino acid chelatesupplementation, no measurements were taken. This wasnot the focus of the study.

The data appear to confi rm the hypothes is that thepoor ability to absorb specific trace elements isassociated with the malabsorption syndrome, which, inturn, may result in poor development of the immunesystem. Whether this lower absorption is a result ofthe malabsorption or one of the causes was notascertained. The problem does appear, however, to becontrollable by feeding the breeder hens amino acidchelates during their laying period. The chicks mayalso be supplemented, directly, following hatching. Thehigher absorption of the amino acid chelates compared to


mineral salts is due to the chelation of the mineralswith amino acids which allows them to be absorbed asdipeptide-like analogs in the jejunum rather than theduodenum, as is the case wi th metal cat ions. Therna1absorpt i on syndrome apparent1y causes mod i fi cat i on ofthe duodenum, which then interferes with cation uptake.The percentage of increase in uptake from the amino acidchelates was not assessed as part of this study, theclinical data indicate that increased absorption of theamino acid chelates does occur(l2) with correspondingimprovements in immunity.

In summary, the feeding of amino acid chelatesappeared to aid in the prevention of certain clinicalsymptoms of the malabsorption syndrome, although theminerals did not curtail the reovirus infection. Such anapproach would, therefore, be expected to providebeneficial results in larger scale applications inproduction breeding. Reversals for most of the semicongenital deficiencies of trace elements that babychicks normally exhibit were seen. The amino acidchelates appeared to be an effective prophylactic to theeffects of the malabsorption syndrome when fed to thechick or to the breeder hen prior to and during the layperiod.


References

1. Good, R. E., "Problem Di Zampe Nei Polli,"Zootecnica Avicunicola, 45 March, 1981.

2. Marchi, R., and Zanella, A., "CaratterizzazioneDi Reovirus Isolati Dall'intestino Di Polli DaCarne Affetti Dalla Cosiddetta Sindome CrescitaStenatat e Debolezza Degli Arti", Clinica Vet.,105:44,1982.

3. McFerran A., "Rotavirus," Clinica Vet. 105:1,1982.

4. Vertonnen, M. H., et li, "Infectious stunting andleg weakness in Broiler: Pathology andbiochemical changes in blood plasma," AvianPathology, 9:133, 1980.

5. Van Der, Heide L., et li., "Isolation of Avianreovirus as a possible etiological agent ofosteoporosis in broiler chickens," Avian Disease,6:1982.

6. Ferrari, A., et li., "Research on a case ofmalabsorption syndrome (Pale Bird) in broilers,"Unpublished study, Experimental ZooprophylaticInstitute of Piedmont Liguria and ValleD'Aosta, Turin, Italy, 1981.

7. Ferrari, A., Barale, G., Beccaria, E., Guarda,F., Micheletto, B., Pozzi, L., "Ricerche su unepisodio della sindrome di malassorbimento (PaleBird) nei broilers." Clin., Vet., 105:29, 1982.


8. Rosenberger, J. K., "Characterization of severalpathogenic and apathogenic avian reovirusisolated," Proc. 11 Ann. Meet. AVMA, 1981.

9. Ruff, M.D., "Nutrient absorption and changes inblood plasma of broilers with malabsorptionsyndrome," Proc. 12 Ann. Meet. AVMA, 1982.

10. Ferrari, A., "Mal absorption syndrome inbroilers," Paper given at Albion Meeting, Rennes,France, 1982.

11. Ferrari, A., "The use of Metalosates in theprevention of malabsorption syndrome inbroi 1ers," Paper gi ven at Avi co Meet., Athens,Greece, 1982.

12. Christy, H., and Ashmead, D., "The use of aminoacid chelated minerals in egg quality," inAshmead, D., ed., Chelated Mineral Nutrition inPlants, Animals, and Man, (Charles C Thomas:Springfield) 191, 1982.

13. Patrick, H., and Schaible, P., Poultry: Feedsand Nutrition (Westport: AVI Publishing Co.,Inc.) 333, 1980.

14. Ashmead, D., and Christy, H., "Factores queinterferem com a absorcao intestinal de mineraise uma solucao par esse problems," I simposiosobre Nudricao Mineral, San Paulo, Brazil, May19, 1984.

15. Rosenberg, I. H., et li., "Malabsorptionassociated with diarrhea and intestinalinfections," Am. J. Clin. Nutr., 30:1248, 1977.

16. Ashmead, H. D., et li., Intestinal Absorption ofMetal Ions and Chelates (Charles C Thomas:Springfield) 1985.


17. Hurwitz, S., and Bar, A., "Intestinal calcium inthe laying fowl and its importance in calciumhomeostasis," Am. J. Clin. Nutr., 22:391, 1969.

18. Norman, A., et li., "Basic studies on themechanism of action of vitamin D," Am. J. Clin.Nutr., 22:396, 1969.

19. Harms, R., and Damron, B., "Calcium in BroilerNutrition," in Harms, R., et li., eds., Calciumin Broiler, Layer and Turkey Nutrition (West DesMoines: National Feed Ingredients Association)13, 1976.

Chapter 22

THE VALUE OF AMINO ACID CHELATES INEGG PRODUCTION

Alberto Bonomi, Afro Quarantelli, Paoli Superchi,and Alberto Sabbiono

University of Parma

In order for a chicken to produce an egg, numerousmineral s such as calcium, (1) phosphorus

t(1) manganese, (1)

copper, (2) iron, (3) zinc, (It) and cobalt, (5) must be presentin the feed. The absorption, transport, and metabolismof these essential minerals are controlled by one ormore enzymat ic systems and these mi nera1s are in anactive state of movement in and out of their variousmolecular configurations within the chicken's body.Some of this movement occurs upon the death anddigestion of body cells, while ~ther movement is theresult of molecular exchange. ( ) In either case,however, a constant supply of biologically availableminerals is required if optimum egg production is to beobtained.

Realizing the importance of the above traceelements and the fact that they are usually not as wellabsorbed as calcium and phosphorus, it was decided todesign an experiment utilizing 2,000 layers to test theeffects of minerals with chelated amino acids on eggproduction.

The hens selected were Hubbard Golden Comets. Atthe beginning of the study they were 5.5 months of ageand had a mean weight of 1,950 grams each. Theexperiment lasted for twelve months, at which time thechickens were almost eighteen months old.

The hens were di vided into four groups of 500each. They were rna i nta i ned in wi re cages wi th threebirds to a cage. Group 1 was the control group. Groups

366

The Value of Amino Acid Chelates in Egg Production 367

2, 3, and 4 were the treated groups. All birds receivedthe same feed, except group 2 also received an aminoacid chelate formula shown in Table 1 at the rate of 50ppm of the total feed, group 3 received the amino acidchelate formulation in Table 1 at the rate of 100 ppm ofthe total feefJ, and group 4 received the amino acidchelate formula in Table 1 at the rate of 150 ppm of thetotal feed.

The basic feed formula given to all four groups isshown in Table 2. It was provided as a flour and fed ab1ibitum.

Table 1

Metal Composition of Amino Acid Chelate Formula(% metal/Kg)

Iron*ZincManganeseCopperCobalt

9.003.001.202.150.08

* Percentages are equivalent amounts of metals only,although all metals were supplied as amino acidchelates.


Table 2

Basic Feed Formula (Kg/100 Kg)

Soy flour (44% protein)Sunflower flour (45% protein)Meat mealFi sh mealMedicated mealCorn flourBarley flourWheat germGround wheatCorn glutenFatCalcium carbonateDicalcium carbonateSodium chlorideVitamin and mineral supplementVitamin AVi tami n D3

Vitamin EVitamin 82

Vitamin 812

PyridoxineVitamin KPantothenic acidFolic acidCholine chlorideDL MethionineB.H.T.CobaltIronManganeseCopperZincQ.S. with soya flour

8.03.05.01.03.0

55.05.03.06.02.01.04.51.50.50.5

3,500,000 IU/Kg200,000 IU/Kg

1,500 mg/Kg500 mg/Kg

5 mg/Kg5,000 mg/Kg

400 mg/Kg3,000 mg/Kg

300 mg/Kg120,000 mg/Kg30,000 mg/Kg1,000 mg/Kg

150 mg/Kg8,000 mg/Kg

12,000 mg/Kg300 mg/Kg

9,000 mg/Kg


Records were rna i nta i ned on the da i1y number ofeggs laid and the weights of those eggs. Feedconsumption was also recorded daily. Weight gains ofthe birds were measured monthly.

The average numbers of eggs produced per hen permonth in each of the groups are seen in Table 3 andcompared graphically in Figure 1.

I Table 3 IMean Eggs/Hen/Month

Groups Mean squaredifference

Month 1 2 3 4 P<0.05

June 48.32 ~ 1.80 48.63 + 2.03 49.28 ~ 1.71 48.75 + 1.93 ---July 71.56 ~ 1.61 71.80 ~ 1.80 72.68 ~ 1.93 71.94 ~ 2.40 ---August 76.23 ± 1.52 76.69:t 1.78 80.12:t 2.12 79.46 ± 2.04 1.48September 79.48 ± 2.10 79.74 + 2.21 84.00 + 2.42 83.39 + 3.00 1.36October 80.67 ± 2.49 81.07 ±2.30 85.13 ±1.93 84.85 ±3.16 1.51November 81.19 ± 1.98 82.96 ± 1.74 85.17 ± 2.16 83.18 + 2.87 1.29December 75.37 ± 2.31 78.04 ± 2.25 81.58 ± 2.48 80.73 ±2.15 1.27January 74.59 ± 2.63 76.83 + 2.39 79.75 ± 2.50 78.52 1: 2.90 1.68February 74.62 ± 2.40 76.57 ~ 2.58 78.38 ± 2.76 78.001: 3. 13 1.30March 71.121: 3.01 71.01 1: 2.74 75.161: 2.81 73.89 1: 3.25 1.70Apr; 1 67.101: 2.86 67.94 ± 2.00 72.00 ± 2.29 71.15 + 2.44 1.42May 63.10 ± 2.58 63.81 1: 2.24 70.231: 2. 72 69.19 ~ 2.58 1.54


100

en(!] 80C!lWLL 600a:w 40 -m~::J 20 -Z

oJun Jul Aug Sep Oct Nov Dec Jan Feb Mar Apr May

MONTHS• Control • Group 2 • Group 3 • Group 4

Fi gure 1. The mean egg product i on per hen permonth.

As can be seen from Table 3, there was nostatistical difference in the egg production of thegroups during the first two months of the laying period.However, at the end of the th i rd, fourth and fi fthmonths, there were statistical differences (P < 0.05)between groups 3, 1, and 2, and between groups 4, 1, and2. The hens receiving the amino acid chelate formula,shown in Table 1, at the rates of 100, ppm and 150 ppmof the feed produced more eggs at the rates of 5.1% and4.24% for August, 5.69% and 4.92% for September, and5.53% and 5.18% for October, respectively, than did thecontrol group (group 1).

At the end of six months of experimentation(November) the increased egg production for groups 2, 3,and 4 were significantly greater (P < 0.05) than group1. There were a1so sign i fi cant d; fferences between


groups 2 and 1 and 3, between groups 3 and 1, 2, and 4,and groups 4 and 1, and 3. groups 2, 3, and 4 had eggproduction increases over the control group of 2.18%,4.90%, and 2.45% respectively.

During the months of December, January, andFebruary (months 7,8, and 9) egg production declined inall groups. Nevertheless, the decline was not as greatin the groups that were receiving the amino acidchelates. The least amount of decline was seen ingroups 3 and 4 which were receiving 100 ppm and 150 ppm,respectively, of the amino acid chelates. The greateregg production of groups 2, 3, and 4 were statisticallysignificant (P < 0.05) when compared to the controlgroup. There were also significant differences betweengroups 2 and 1, 3 and 4, groups 3, and 1 and 2, andgroups 4, and 1 and 2.

At the end of the tenth, eleventh, and twelfthmonths of the experiment, there were statisticallydifferences (P < 0.05) between groups 1 and 3 and 4,groups 2 and 3 and 4 (group 2 laid about the same totalnumber of eggs as the control group), groups 3 and 1 and2, and groups 4 and 1 and 2.

As stated above, the eggs were weighed each day.These results were summarized, monthly, and thenstatistically analyzed. They are presented in Table 4.


Table 4

Mean Weight Per Egg (g)

Groups MeanSquare

Month 1 2 3 4 DifferenceP<O.OS

June 54.381:t0.204 54.428:t0.222 54.402:t0.208 54. 297:t0.194 ---July 55.221±0.212 55.372:t0.219 55. 396:t0.249 55. 348:t0.207 ---August 56.680:t0. 261 56.631±0.245 56.742±0.262 56.594±0.300 ---September 56.521±0.206 57.710±0.228 57.930±0.308 57.895±0.263 0.153October 57.700±0.243 58.930±0.297 59 .183±0 .300 59 . 152±0 .281 0.172November 59. 630:t0.309 59.751±0.229 61.000±0.239 61.931±0.340 0.196December 60.818±0.315 60.901±0.274 62.780±0.225 62.639±0.311 0.245January 62.960±0.288 63.103±0.319 64.208±0.260 64.103±0.307 0.288February 64.429±0.263 64.618±0.332 66.149±0.336 65.121±0.263 0.311March 65.000:t0.3OO 65.143±0.306 66.848±0.290 65.458±0.304 0.229Apri 1 65.622±0.321 65.702±0.280 66.957±0.271 66.108±0.339 0.261May 66.337±0.303 66.551±0.271 67. 524±0. 259 66.983±0.288 0.293

As seen in Table 4, the weight of eggs increasedas the laying period progressed. There were nostatistically significant differences until the fourthand fifth months of lay, at which time groups 3 and 4produced heavier eggs (.71% and .65%, respectively, inSeptember and .82% and .77%, respectively, in October).This trend continued in the sixth, seventh, and eighthmonths as well. In this period groups 3 and 4 laidheavier eggs than the control group by 2.29% and 2.18%,respectively, in November; 3.32% and 3.00%,respectively, in December; and 1.98% and 1.81%,respectively, in January. In the last period, theninth, tenth, eleventh, and twelfth months, thesituation was not particularly different than thepreceding period. The increased weights of the eggsfrom groups 3 and 4 were statistically significant (P <0.05) when compared to groups 1 and 2. Over all, theheaviest eggs were produced from group 3 which received100 ppm of the amino acid chelate formula in its feed.


Feed consumption for the production of twelve eggsis shown in Table 5 and Figure 2. When compared to thecontrol group, groups 3 and 4 consumed 4.9% and 4.58%less feed for each dozen eggs produced. group 2 and thecontrol group ate about the same amount of feed perdozen eggs produced.

I Table 5 IFeed Consumption Per Dozen Eggs Produced (Kg)

Group

Month 1 2 3 4

June 2.148 2.170 2.102 2.109July 2.230 2.240 2.163 2.171August 2.295 2.278 2.210 2.218September 2.373 2.365 2.270 2.274October 2.442 2.450 2.346 2.361November 2.510 2.490 2.400 2.417December 2.628 2.600 2.478 2.486January 2.715 2.683 2.560 2.541February 2.829 2.795 2.610 2.622March 2.864 2.852 2.700 2.708Apr; 1 2.903 2.907 2.738 2.749May 2.932 2.919 2.781 2.793


3.1

0)

~uCDE::::JCDc:oouCDCDu..

1.9JlIl Jul Aug Sep Oct Nov Dec Jan Feb Uar Apr May

MONTHS

• Control • Group 2 • Group 3 • Group 4

Figure 2. Agraphic protrayal of the feed consumedper dozen eggs produced.

The 1ast measurement in th is experi ment deal t wi ththe weight gains of the hens during the year they wereinvolved in the study. It should be remembered that atthe commencement of this experiment, the birds were 5.5months old and commencing the first laying period oftheir lives. Table 6 and Figure 3 summarize the datafrom these monthly weighings.


I Table 6 IMean Monthly Weights of Hens (g)

Group MeanSquare

2 3 4Difference

Month 1P<0.05

Initial wgt. 1938.29:!.114.36 1955.46:!.122. 10 1927.72:!.111.29 1949.58:!.131.60 --June 1974.12:.129.17 1983.13;:101.30 1964.59:.106.48 1981.33:,125.19 --July 2038.27:,143.70 2080.15:.129.11 2088.63:,138.16 2132.25:!.136.68 27.52August 2081.15:!.110.21 2136.49!.130.16 2151.18!.117.49 2198.62!.112.36 34.29September 2124.61:.121.57 2175.38;:115.23 2182.49:.119.80 2239.23:.128.72 29.72October 2170.10:.139.40 2189.17!,132.55 2274.21!,124.12 2288.13:,131.27 48.36November 2214.25:,118.62 2212.80:,114.23 2303.65:,134.62 2310.74:,140.30 41.28December 2278.56:,125.90 2301.09:,119.92 2378.41!,116.83 2359. 28!,1 09.54 55.16January 2321.85!,102.68 2336.37:.113.33 2424.10:.125.50 2406.59:,118.64 39.14February 2369.14:!.101.75 2370.81:!.119.80 2487.08:!.138.63 2418.96:141.22 37.19March 2392.70:,150.23 2403.94:.123.36 2536.11:.113.89 2460.73:.116.02 30.15April 2426.58:,128.70 2448.16:,115.84 2584.20:,138.45 2503.68:127.94 33.30May 2465.39:!.116.68 2480.32:.124.37 2638.91:.141.76 2550.60:.136.27 44.26

~r-----------------------------,

2500

2000

1500

500

oJim ~I Aug Sep Oct Nov Dec Jan Feb Uar Apr May

MONTHS

• Control • Group 1 • GrQl4) 2 • Gr'rq) 3

Figure 3. Weight gains of the hens during thelaying period.


Even though their feed consumption per dozen eggsproduced was less, the hens in groups 3 and 4, whichwere receiving 100 ppm and 150 ppm, respectively, of theamino acid chelates, grew more rapidly and were largerthan their counterparts in groups 1 and 2. There wereno statistically significant differences in the weightsunt i 1 the th i rd month at wh i ch time group 4 was theheaviest (P<0.05). This trend continued through thefirst 8 months. However, in the last four months, thehens in group 3 overtook those in group 4. At the endof the study, both groups were significantly heavierthan groups 1 and 2 (P<0.05).

From the above data, it was obvious that when theamino acid chelates shown in Table 1 are included at therate of 100 ppm in the feed, they have a very positiveeffect on egg production and growth rates. This groupproduced approximately 5.8% more eggs than the controlgroup. The difference is statistically significant(P<0.05). The effectiveness of the amino acid chelatewas especially prominent during molting and during thewinter months.

The beneficial effects of improved feedutilization from supplying amino acid chelated tracemi nera1sin 1ayer feeds is obvi ous. At the abovedosages, the hens consumed 4.9% less feed and achieveda 5.8% increase in egg production when compared to thecontrol group. Furthermore, the eggs in group 3 were1.62% heavier than in group 1 which improved theirgrading. These data were all statistically significant(P<0.05).

The total weight gain for group 3 was 7% greaterthan for the controls.

When the 1eve1 of the ami no ac id che1ates wasincreased from 100 ppm of to 150 ppm, favorable resultswere also obtained. These improvements were alsostatistically significant when compared to group 1.


Nevertheless, the increased differences between group 3(100 ppm) and group 4 (150 ppm) were negligible. At alesser dose (50 ppm), there were no meaningfulimprovements over the control group. Thus, the 100 ppmin the ration not only results in a significantproduction improvement, but is also more economical thanthe higher dose, particularly when considering the feedconsumption per dozen eggs.

As stated in the beginning, the trace elementsselected for this study are known to influence eggproduction. When these minerals are supplied as aminoacid chelates, it appears that they are absorbed morecompletely. With their higher absorption, they seem tofavorably influence the physiological systems in thehen's body that are responsible for egg production. Thenet result is more and larger eggs at less cost.


References

1. Patrick, H. and Schaible, P., Poultry: Feeds andNutrition (Westport: A.V.I. Publishing Co.) 157183, 1980.

2. Davis, G. and Mertz, W., "Copper" in Mertz, W.,ed., Trace Elements in Human and Animal Nutrition(London: Academic Press) 34, 1987.

3. Morris, E., "Iron", in Mertz, W., ed., TraceElements in Human and Animal Nutrition (London:Academic Press) 118, 1987.

4. O'Dell, B., "Copper and Zinc in PoultryNutrition" in O'Dell, et li., eds., Copper andZinc in Animal Nutrition (West Des Moines:N.F.I.A.) 44, 1979.

5. Ewing, W., Poultry Nutrition (Pasadena: The RayEwing Co.) 871, 1963.

6. Post, J., An1ei tung zur Pl anung und Auswertungvon Feldenversuchen mit Hilfe der Varianzanalyse,(Berlin: Springer Verlag) 1952.

7. Fischer, R., La programmazione degli esperimenti(Pisa: Nistr Lischi) 1954.

8. Pompily, G. and Napol itani, D., "Piano degl iesperimenti ed elaborazione probabilistica deirisulati," La Ricerca Scientifica, 24, 1954.

9. Fischer R. and Yates, F., Statistical Tables forBiological, Agricultural and Medical Research(London: Oliver and Boyd) 1954.


10. Bonomi, A., et li., "I complessi oligodinamicichelati nell' alimentazione delle gallineovaiole," Rivista di Avicoltura, 9:51, September19, 1983.

Chapter 23

THE ROLE OF AMINO ACID CHELATEDMAGNESIUM IN EGG PRODUCTION

Davi d Atherton, Thomson and Joseph Limited

Magnes i urn was fi rst shown to be an essent i a1nutrient in 1926. (1) By 1942, research was publ ishedwhich proved it to be an essential nutrient inpoultry.(2) In 1967, Sell, et ~., induced a magnesiumdeficiency in ~hite Leghorn layers by using semipuri fi ed diets. () They observed a sharp decrease inegg production within 9 days after starting theexperiment. Production ceased entirely by the 21st day.At the same time, the magnesium content of the eggsrapidly declined. The hatchability of the fertile eggsdecreased to zero within fifteen days after starting thetreatment. When the magnes i urn 1eve1s were increasedfrom 50 ppm to 500 ppm, egg production commenced again,and hatchabi 1i ty of fert i1e eggs returned to norma1within six days.

In spi te of these data, there is no generalagreement regarding optimum magnesium requirements forlaying hens. Sell, et ~. recommended 250-350 ppm. (3)Edwards and Nugara believe between 490 and 900 ppm ofmagnesium is essential.(4) Hajj and Sell later revisedtheir earlier estimate(3) and reported 355 ppm isrequired.(S) Currently (1984) the National ResearchCouncil in the United States recommends 500 ppm ofmagnes i urn for 1ayers. (6)

Part of the controversy is due to the fact that nodefinitive data exist on the "true" intestinal absorption of magnesium by chickens. It is believed thatapproximately 45 to 55% of the ingested magnesium may beabsorbed,(7) but absorption probably declines withincreasing dietary levels. Using 28magnesium, Edwards,et ~. found 51% of a dose in the feces within four daysafter injection.(8) Very little of this magnesium was

380

The Role of Amino Acid Chelated Magnesium in Egg Production 381

reabsorbed after it entered the jejunum. Thi s wasprobably due to the precipitation of the magnesium inthe alkaline pH of the intestines as it moved throughthe jejunum and ileum.(9)

In addition to a high pH, dietary phosphorus orcalcium will affect magnesium absorption.(·) Vitamin 0)may infl uence uptake of magnes i um(lO) by changi ng ratesof accretion or mobilization of bone salts.(ll) Dietaryfat wi 11 reduce magnes i urn absorpt ion. (12) Lactose mayincrease magnesium blood levels by stimulating increasedfeed consumption.(l) And finally, the level of dietaryprotei n has been reported to affect magnes i urnabsorpt ion. (14)

Because increased protein has a positive effect onmagnesium absorption, it was decided to test the effectsof feeding magnesium that had been chelated with aminoacids on egg production. Previous research hasindicated that in this chelated state, the magnesium isnot only absorbed in greater quantities, (15) but inconjunction with other amino acid chelated minerals, ithas resulted in increasing egg production when comparedto equivalent intake of inorganic metal salts.(16)

For thi s current study 360 Hi sex Brown 1ayers,with a mean age of sixty weeks, were arbitrarilyass igned to one of four feed treatments. There wereninety birds in each treatment. Each treatment hadthree replicates with thirty birds per replicate.

The three replicates were designated as A, B, andC for each treatment. Each replicate group of thirtybirds was accommodated in a single row of 6 X 5 birdcages:

Birds per replicateBirds per treatmentBirds per tierTotal birds in study

3090

120360


The random distribution of the treatments amongthe cage tiers is shown in Table 1.

I Table 1 IRandom Distribution of Tiers

Tier A Group 3 Group 2 Group 4 Group 1

Tier B Group 2 Group 1 Group 3 Group 4

Tier C Group 4 Group 3 Group 1 Group 2

All of the groups received the same layer feedexcept for the addition of the various magnesiumsupplements. Table 2 summarizes the magnesiumsupplements provided and the quantity of magnesiumsupplied per group.

I Table 2 IMagnesium Supplement (g/1000 Kg)

Treatment MgO Mg Amino Acid Chelate Total Mg

1 (Control) --- --- ---2 177 23 2003 --- 50 504 200 --- 200

The chemical analysis of the four feeds is shownin Table 3. Four samples were taken from the feed foreach treatment and the results averaged.


I Table 3 IChemical Analysis of Feeds (%)

Treatment Protein Ca P Na Mg

1 17.67 5.00 0.70 0.18 0.162 16.42 4.95 0.66 0.19 0.193 16.80 4.85 0.62 0.18 0.174 16.85 5.36 0.59 0.18 0.18

The experiment lasted from August to October, fora total of twelve weeks (84 days), during which time thenumber of eggs produced, the weights of the eggs, thequal i ty of the eggs, the feed convers; ons, and thelaying hen mortality were measured.

Table 4 presents a summary of those results forthe egg production with their statistical significance.


I Table 4 ISummary of Results on Egg Production

Treatment Numbers 1 2 3 4

Original hen numbers 90 90 90~(P<O.01)Total eggs laid 55283 59S7b 5792b

Eggs/hen housed 61.42 66.19 64.36 65.18% egg production (hen housed) 73.10 78.80 76.62 n.59

Egg size (as % of all sampled)

1 21.44 14.65 15.11 20.902 31.50 32.61 28.81 30.983 29.33 32.14 34.52 30.034 9.89 11.57 12.24 9.445 .81 2.17 1.94 1.506 .00 .00 .53 .007 .00 .00 .04 .00

On farm seconds 7.03 6.87 6.81 7.15

Average egg weight (grams) 67.12 66.37 65.89 67.37

Total egg mass/hen housed (kg) 4.12 4.39 4.24 4.39

Average speci iic gravity 1.08083

1.08043 1.0828b 1.0s0r(p<D.01)Average haugh unit n.D7 76.75 76.75 75.39Average shell color 39.00 38.~ 38.62 ~OOAverage shell texture 2.143 2.04 2.05b 2.03 (P<O.Ol)Average yolk color 10.14 10.25 10.23 10.41

Inclusions per sample of 480 eggsMeat spots 55 3~ 38 32Blood spots 103 4 133 163 (P<O.05)Total Inclusions (meat and blood 65 43 51 48spots)

The inclusion of magnesium in the diets of thelaying hens had a positive effect on production. Diets2, 3, and 4 all produced more eggs than the control.The increase in each case was statistically significant(P<O.Ol) from the control. The fact that treatment 3(which only provided 50 ppm of magnesium as the amino


acid chelate) was still statistically equivalent to theMgO supplement (which provided 200 ppm of magnesium) isan indication of the greater bioavailability of themagnesium. This observation is further borne out bynoting that diet 2 (which had a combination of MgO andthe amino acid chelate) had a higher production levelthan diet 4 (which had an equivalent amount of magnesiumthat was totally from MgO.)

The mean specific gravity of the eggs from diet 3(magnesium amino acid chelate) was statistically higherthan the other groups (P<O.Ol).

The shell texture was significantly improved(P<O.Ol) by the inclusion of magnesium in the diet. Theeggs that were graded as seconds averaged about 7% ofthe total production. This amount is considered low fora flock at this stage of lay. The magnesium amino acidchelate treatments (diets 2 and 3) had slightly reducedpercentages of seconds on the average.

Finally, there were significantly less (P<0.05)blood spots in the eggs from the hens receiving themagnesium amino acid chelate and MgO in combination(treatment 2) when compared to the control group and theother diets. Aga in these data suggest the greaterbioavailability of the amino acid chelate. When thehens received only 50 ppm of magnesium as the amino acidchelate, they produced eggs that had almost 19% lessblood spots than eggs from the hens receiving 200 ppm ofmagnesium as MgO.

Blood spots occur when one or more of the bloodvessels in the follicle rupture, and the blood becomesincorporated into the yolk during its formation. Meatspots are degenerated blood spots. Both lower thequa1i ty of the egg and reduce its marketabi 1i ty. (17)

When the incidence of meat and blood spots are addedtogether, diet 1 had 65 (13.5%), diet 2 had 43 (8.9%),diet 3 had 58 (10.6%), and diet 4 had 48 (10%)


incidences. Clearly the inclusion of amino acidchelated magnesium had a positive effect on what wasonce thought to be a problem of genetics that would notrespond to nutri t ion. (17)

The data on feed consumption and hen condition areshown in Table 5. It is clear that feed conversion isaffected by the inclusion of magnesium to the diet. Theinclusion of magnesium significantly (P<O.05) reducedthe amount of feed consumed to produce a dozen eggs.None of the other parameters were statisticallysignificant, although it should be noted that there wasno hen mortality in the two groups receiving themagnesium amino acid chelates whereas there was in thecontrol group and group 4 which received its magnesiumas MgO.

I Table 5 IFeed Conversion and Hen Condition

Treatment 1 2 3 4

Original hen numbers 90 90 90 90Total feed fed (kgs) 956.80 956.85 962.65 950.88Food fed/hen housed (kg) 10.63 10.63 10.70 10.57

(Kg feed/Kg eggs) 2.58· 2.42b 2.52· 2.42b (P<0.05)(Kg feed/dozen eggs) 2.08 1.93 1.99 1.99

Ave body wgt at start (kg) 2.07 2.14 2.02 2.01Ave body wgt at midway (kg) 2.09 2.12 2.03 2.05Ave body wgt at end (kg) 2.10 2.14 2.05 2.06

Total mortality 3 0 0 1Mortality (% of hen housed) 3.33 .00 .00 1.11

In summary, there is disagreement on the level ofmagnesium that should be included in layer feeds. Thesedata demonstrate a positive effect from its inclusion,but do not indicate the optimum level to include. The


amount of magnes i urn used in th i s study is be low theN.R.C. recommendations, and this may have skewed theresul ts. Sell has suggested that there is greatermagnesium absorption, regardless of the source, whenchickens are given a magnesium deficient diet. oe

) Thereis some indication of synergism between the MgO and themagnesium amino acid chelate in diet 2. Even thoughdiets 2 and 4 both contained 200 ppm of magnesium, diet2 with the amino acid chelate, out performed theinorganic MgO diet 4. If dietary levels of magnesiumhad been increased in all of the diets, perhaps therewould have been an even greater difference between theeffects of the ami no ac id che1ate and the inorgan icsource of magnesium on egg production.


References

1. Leroy, J., "Necessite du magnesium pour lacroissance de la souris," Cempt. Rend. Soc. deBiol., 94:431, 1926.

2. Almquist, H., "Magnesium requirement of thechick," Proc. Soc. Exp. Biol. Med., 49:544, 1942.

3. Sell, J., et li., "Effect of magnesium deficiencyin the hen on egg production and hatchability ofeggs," Brit. Poultry Sci., 8:55, 1967.

4. Edwards, H. and Nugara, D., "Magnesiumrequirement of the laying hen," Poultry Sci.,47:963, 1968.

s. Hajj, R. and Sell, J., "Magnesium requirement ofthe laying hen for reproduction," J. Nutr.,97:441, 1969.

6. National Research Council, Nutrient Requirementsof Poultry (Washington D.C.: National Academy ofSciences) 1984.

7. Guenter, W. and Sell, J., "A method fordetermining "true" availability of magnesium fromfoodstuffs using chickens," J. Nutr., 104:1446,1974.

8. Edwards, H., et li., "Effect of calcium andphosphorus levels in the diet on magnesiummetabolism in chickens," in International AtomicEnergy Agency, ed., The Use of Radioisotopes inAnimal Biology and Medical Sciences (New York:Academic Press) V 2, 83, 1962.


9. Guenter, W. and Sell, J., "Magnesium binding inthe digesta of the chicken," Can. J. Physiol.Pharmacol., 53:311, 1975.

10. Worker, N. and Migicousky, B., "Effect of vitaminD on the ut i 1i zat i on of beryl 1i urn, rnagnes i urn,calcium, strontium, and barium in the chick," J.Nutr. 74:490, 1961.

11. Richardson, J. and Welt, L., "The hypomagnesemiaof vitamin D administration," Proc. Soc. Exp.Bi 0 1. Med., 118 : 512, 1965.

12. Hakansson, J., "The effect of fat on calcium,phosphorus and magnesium balances in chicks,"Swedish J. Agr. Res., 5:145, 1975.

13. Scholz, R. and Featherstone, W., "Influence oflactose and glucose on magnesium - 28 retentionin the chick," J. Nutr., 91:231, 1967.

14. Sell, J., "Magnesium in Nonruminant Nutrition,"in Sell, J. and Fontenot, J., eds., Magnesium inAnimal Nutrition (West Des Moines: National FeedIngredients Assoc.) 9, 1980.

15. Ashmead, H., "Tissue transportation of organictrace minerals," J. App. Nutr., 22:42, Spring,1970.

16. Hinze, P., "Metalosates: Their electromotivepotential in laying hens," Feed Management,20:28, March, 1969.

17. Ewing, W., Poultry Nutrition (Pasadena: The RayEwing Co.) 22, 1963.

18 . Sell, op. cit., 5.

Section 5. HORSES

391

Chapter 24

THE EFFECTS OF AMINO ACID CHELATES ONTHOROUGHBRED MARES

Martin G. Robl* and Richard J. Forfa**University of Maryland

Individual or multiple factors can affect thereproductive efficacy of individual mares, but thegeneral management techniques are an equally importantpart of any successful brood mare operat ion. Theaverage foaling rate in hand-bred mares is onlyapproximately 60%, and this has not improved much overthe last thirty years. (1) Early pregnancy loss thatoccurs between fertilization and 150 days post-ovulationranks as a major cause of the infert i1i ty. (2) Theincidence of these abzrtions has been reported to bebetween 11%(3) and 13%( ).

Barren mares need to be evaluated for reproductivesoundness at the end of their unsuccessful breedingseason because most mares will be actively cycling atthis time, and a true estimation of their futurebreeding potential is possible. (1)

Malnutrition in mares may be associat~d with earlyembryonic loss as reported by Van Nickerk.( ) Kenney hassuggested a close association between nutritionalinsufficiencies and embryonic losses in mares.(6)However, there is a sparsity of reports indicatingspecific mineral relationships with ovulation and earlyembryonic loss in the mare. A recent study reportedthat ami no ac id che1ated mi nera1s had a benefi cia1effect on improving conception rates and reducing earlyembryonic loss in the bovine.(7) This current study was

* Currently Veterinary Specialty Diagnostics, Inc.**Currently Monoacy Equine Associates

393


conducted as a field trial in an attempt to study thepossible effects of these amino acid chelates on theconception rates and embryonic loss of a group of barrenthoroughbred mares.

Twelve barren mares from a Maryland thoroughbredbrood mare farm wi th good management techn iques wereselected for this study. All of the mares had beenbarren for one or two previous years while the farm hada foaling rate of approximately 65% using the hand-bredtechnique. Each mare was evaluated during September forthe following breeding season. This evaluation includeda gross examination, palpation of the reproductivetract, uterine swabs for aerobic, anaerobic, and fungalcultures, and three uterine biopsies (right and lefthorn, and body) for light microscopic evaluation. Bloodsamples were taken for routine evaluation of the red andwhite blood cell components.

A portion of each blood sample and each uterinebiopsy was also used for a mineral content evaluation byX-ray dispersive microanalysis with a scanning electronmicroscope probe using a technique that was adapted froma method to evaluate mineral levels in plant tissues. c

.)

Following an evaluation of the gross andmicroscopic examinations of the tissues and the resultsof the microbial cultures, the barren mares were placedinto two groups (control and treated) and paired asclosely as possible on the basis of age, the length ofnon-reproductivity, and comparable microscopic tissuechanges seen in the uterine biopsy examinations.

A basal ration used on the farm, consisting of amixture of Timothy grass hay, alfalfa, and a preparedcommercial supplement, was fed to both groups of mares.The treated group of six mares was fed an additionalthree ounces per day of the amino acid chelateformulation shown in Table 1 for approximately 45 to 60days prior to breeding and 45 to 60 days after breeding.

The Effects of Amino Acid Chelates on Thoroughbred Mares 395

Table 1

Amino Acid Chelate Formula Fed to Mares

Prtassiurn*M~!gnes i urnIronr~anganese

CopperZincCobalt

4.565 %2.480 %1.000 %0.700 %0.400 %1.423 %0.008 %

*Potassium was supplied as an amino acid complex.All of the other minerals were supplied as aminoacid chelates.

An artificial light regimen was used on some maresin both groups. The breeding dates, number of breedingsper conception, and confirmed pregnancies through fivemonths of gestat i on were compared . Several mares ofeach group were sold at this time, and thus all of themares could not be monitored throughout the entiregestation period. The study was terminated at the fivemonth gestation point.

Blood samples for routine evaluation of the redand white blood cell components were taken at the end ofthe five month gestation period. Additional bloodsamples were also taken at that time for analysis oftheir mineral contents.

The reproductive performance of the two groups ofbarren mares is presented in Table 2.


I Table 2 IReproductive Performance of Thoroughbred Mares

Number Uterine Days to Breeding ServicesAge Years Received Biopsy after : after PerYrs Barren Ughts Category Culture lights: Jan. 1 Conception

Control Group

20 2 No II Cervixl - 167 414 2 No II - 101 18 3 No II - 101 18 1 Yes II - 61 14 2 No I - 128 34 maiden No I - 98 1

Mean 9.7 2 1.7 109.3 1.83

(5 Mares)

Treatment Group

21 1 Yes II Cervix2 76 45 116 1 Yes II Uterine3 77 46 116 1 Yes II 92 61 111 2 Yes II 83 52 15 1 Yes I 100 69 15 maiden Yes I 138 107 5

Mean 12.3 1.2 1.7 94.3 63.3 1.67

(5 Mares)

Difference (treated - control)

+2.6 -0.8 - - -46* -0.16

IE. coli, 2Aetinobacilluseguuli, 3veast *P<0.02

The age of the barren mares in the two groupsranged from 4 to 21 years. The mean age of the controlgroup of barren mares was 9.7 years, while the averageage of the treated group of barren mares was 12.3 years2.6 years older, on the average, than the control group.


The microscopic evaluation and categoryspecification was made utilizing the proceduresdescribed by Kenney.(6) The microscopic evaluationplaced four mares from each group into Category II, andtwo mares into Category I. The average rat i ng wasconsidered to be 1.7 for each group.

The days to breed i ng and concept ion fo 11 owi ngJanuary 1st are also presented in Table 2. Four of thecontrol and five of the treated mares conceived on thefirst service. The mares in the treated group conceivedan average of 46 days earlier than the control group.This decrease in the number of days to conception wassignificant (P<O.02). In the control group, one marehad to be bred three times before conceiving, whileanother was bred four times before conceiving near theend of the observation period. One mare in the treatedgroup was bred five times before she became pregnant,but all of the others were bred after one service.

The results of the microbial cultures indicatedthe presence of the following microorganisms from thevarious locations: No.1 (control) had I. coli from thecervix; No.7 (treated) had Actinobacillus eguuli fromthe cervix; and No.8 had a yeast organism (possibly acontaminant) obtained by uterine swab. These organismsdid not appear to alter the conception rates of any ofthe mares in this study.

The mean values of the various blood parametersincluding red and white blood cell components indicatedno dramatic abnormalities for any of the mares in eitherthe beginning or ending samplings.

The blood samples (taken initially and at the endof the study) and uterine tissue samples taken at theinitiation of the study were examined by the scanningelectron microscope probe method adapted from the planttissue techn ique. (8) However, there were inherentproblems in making the transition from plant to animal.


It became apparent that the results were not readilyreproducible. Consequently, these data were notconsidered in the overall evaluation of this study.

The results of this field study utilizing a smallgroup of mares suggested that the amino acid chelatesshown in Table 1 are beneficial in inducing ovulationand earlier conception rates, since the treated groupaveraged 46 days earlier conception than the controlgroup which was significant (P<O.02).

These findings are similar to the results obtainedin a study previously conducted in cattle. (7) Themechanism for this improvement in reproductiveefficiency has not been determined. However, themi nera1 components in the ami no ac id che1ate used inboth studies are primarily involved with enzymaticprocesses in the body. These results suggest that theprocess of chelating minerals with amino acids producescompounds which, after ingestion, are more effective atthe tissue or cellular level than equavalent amounts ofthe inorganic forms of similar minerals.

The results of this field study in mares shouldprovide initial data for designing larger studies todetermine the mechanisms involved in improvingfertility. Any beneficial effects made towardsimproving reproductive efficiency in mares should beconsidered to be economically helpful to the equineindustry.


References

1. Honey, W. G., "Management factors affectingequine fertility," in Morrow, D.A., ed., CurrentTherapy in Theriogenology (Philadelphia: W. B.Saunders) 737, 1986.

2. Wood, G. L., et li., "A field study of earlypregnancy loss in standardbred and thoroughbredmares," 5(5):264, 1985.

3. Chevalier, F., and Palmer, E., "Ultrasoundechography in the mare," J. Reprod. Fert. Suppl.,32:423, 1982.

4. Bain, A. M., "Foetal losses during pregnancy inthe thoroughbred mare: A record of 2,562pregnancies," N. A. Vet. J., 17:155, 1969.

5. Van Niekerk, D. H., "Early embryonic resorptionin mares," J. S. Afr. Vet. Assoc., 36(1):61,1965.

6. Kenney, R. M., "Cyclic and pathologic changes inthe mare endometrium as detected by biopsy with anote on early embryonic death," J.A.V.M.A.,172:241, 1978.

7. Manspeaker, J. E., et li., "Prevention of earlyembryonic mortality in the bovine fedMetalosates®," Abst. IVth Inter. Symp. of Vet.Lab. Diag. Amsterdam, The Netherlands, 253, 1986.

8. Gardner, J., 1985, Personal Communication,Brigham Young Univ., Provo, Utah.

Chapter 25

COPPER-RESPONSIVE EPIPHYSITIS ANDTENDON CONTRACTURE IN A FOAL

Susan HildebranWapakoneta, OH

John HuntSugar Creek Veterinary Service, Greenfield, IN

Editor's Note: The following case study represents thebenefits of copper supplementation for the reversal ofepi physi t is and tendon contracture. Interruptedsupplementation in the study affirmed the positiveeffects of dietary copper. The paper is reprinted herethrough the kind permission of Modern VeterinaryPractice Magazine. It originally appeared in Volume 67,March, 1986, pages 268 to 270. Tables 1 and 2, whichwere not part of the original text, present the formulasfor Equine Organic Iron Supplement and Replamin ExtraBreeder Pac, respectively, which are mentioned in thetext.

A Thoroughbred filly exhibited firm swellingsproximal and distal to the front fetlocks followingperiodic lameness and general stiffness (Fig 1). Thefilly had always looked "not quite right", but nevermanifested definitive clinical signs until 4 months ofage. The filly was slightly underweight and lethargic,and had a poor appetite and a history of injuries andrespiratory infections. Radiographs confirmed extensiveepiphysitis, especially of the left front distal thirdmetacarpal bone (Fig 2).

400

Copper-Responsive Epiphysitis and Tendon Contracture in a Foal 401

Figure 1. Characteristicfetlocks in epiphysitis.

appearance of the


Fi gure 2. Dorsopa1mar radi ograph of the 1eft frontfetlock of a 4-month-old filly shows epiphyseallipping (arrow) on the medial aspect of the distalend of the third metacarpal bone.


Copper deficiency was suspected because the fillyhad subsisted largely on mare's milk during recentillnesses. Treatment consisted of 42 mg chelated copperfrom Equine Organic Iron Supplement (Albion), fed on 30g/day, with 50 9 dicalciurn phosphate daily. [Table 1]

I Table 1 IEquine Organic Iron Supplement

Per Kg Per Lb

Iron- 7.04 gm 3.2 gmCopper- 1.40 grn 636.0 mgMagnes i urn" 3.30 gm 1.5 gmZi nc" 8.80 gm 4.0 gmPhosphorusb 49.94 gm 22.7 gmCa1ci urn" 49.94 gm 22.7 gmPotass i umb 20.02 gm 9.1 gmIodinec 59.40 mg 27.0 mgVitamin E 2,200 IU 1,000 IUThiamine HCl 660.00 mg 300.0 mgRiboflavin 704.00 mg 320.0 mgNiacin 660.00 mg 300.0 mgPantothenic acid 880.00 mg 400.0 mgCholine chloride 8.80 gm 4.0 gmVi tami n 812 11.00 mg 5.0 mgFolic acid 220.00 mg 100.0 mg

- Mineral given as amino acid chelate.b Mineral given as amino acid complex.C Mineral given as potassium iodide.

The filly's appetite improved in several days, andthe animal became less stiff and more energetic. Afterone month, the pasterns were more naturally angled, and


all soreness was gone. Following 2 months ofsupp1ementat ion, the fet lock shape was great1y improved,and the pastern angl es were normal. The fi 11 y wasenergetic and completely normal by the third month.

Feeding of the copper supplement was discontinuedafter 4 months, dropping the daily copper intake from175 ppm to 23 ppm. Other minerals were available adlibitum, but the filly did not eat them. Three weeksafter discontinuation of copper supplementation, thepasterns became unnatura11 y upri ght. After 4 weekswithout extra copper, the filly suffered a sudden onsetof musculoskeletal pain, inapptance and lethargy.

Supplementation was resumed with a multivitaminmineral preparation (Replamin Extra Breeder Pac:Albion) at 30 g/day. [Table 2]

Table 2

Replamin Extra Breeder Pac

Crude ProteinCrude FatCrude FiberMagnes i umA

Potass i umb

Zi ncA

Iron A

ManganeseA

Copper"Coba1til

10.00 %2.50 %

11.00 %5.60 %4.00 %1.42 %1.00 %0.70 %0.40 %0.008 %

.. Mineral given as amino acid chelate.b Mineral given as amino acid complex.


There was some improvement, but less thananticipated. Because of inadequate labeling andmi scommun i cat i on wi th the manufacturers' representat i ve,the dose of copper was actually only 10% of the intendedtherapeutic dose. Realizing the error, we reinstituteduse of the original Equine Organic Iron Supplement at 60g/day to furnish 84 mg chelated copper.

The response was dramat i c. The pastern angl esdropped 50 in 2 days and the filly was sound in 2 weeks.After one month, the pasterns were normal and moreflexible than ever. At present, the filly appearscompletely normal, though large for 15 months, and willcont i nue recei vi ng the copper suppl ement unt i 1 fullymatured.

Copper is essential to many tissue functions,especially formation of collagen, elastin, cartilage andbone. Thus, growing animals are most susceptible todeficiency. Whole-body analysis of horses shows thatcopper content decreases wi th age. (I) Hepat i c copperlevels vary widely, but fetal livers contain an averageof 319 ppm, neonatal livers 406 ppm, livers of olderfoals 172 ppm, and adult livers 31 ppm.(2) Because fetalhepatic stores are high early in gestation, adequatemineral nutrition throughout pregnancy is important.

Copper is an essential cofactor needed for normalcross-link formation in collagen and elastin.(3) Inother species, impaired cross-linking in collagen andelastin associated with copper deficiency has causedbone di sorders, 1ameness and weak arteri es. Reducedserum copper levels have been correlated with fataluteri ne artery ruptures. (4) A defi nit i ve study of copperstatus of horses dying of aortic rupture is needed.

Horses are less susceptible to copper deficiencyand taxi city than are other spec i es, so few reportsexist in the literature. Toxicity was not produced inhorses fed copper at 791 ppm. (5) On 1and known to be


copper deficient for cattle, mature horses showed nosigns of copper deficiency; however, foals had enlargedjoints and reduced pastern and fetlock angulation.(6)

The recommended level of dietary copper is 9 ppm;however, growing horses may have higher requirements.The actual amount of available copper depends on thesource and on interaction with other elements, such aszinc, iron, cadmium, molybdenum and sulfur. Highdietary levels of molybdenum enhance copper excretion,thus raising the requirement. Rachitis in foals andyearl ings was 1inked to a high molybdenum level (5-25ppm) in herbage that interfered with utilization of bothcopper and phosphorus.(7) Zinc toxicity also causessecondary copper deficiency that does not respond tocopper therapy. Advanced osteochondrosis lesions werefound in foals with low serum copper and high serum zinc1eve1s. (I) Other invest igators have reported cases ofosteodysgenesis in foals that responded to calciumcopper edetate. (9.10)

Epiphysitis and tendon contracture are seenfrequently in horses in the midwestern u.s. Soils inthe area are low in copper, in addition to selenium andiodine. Analysis of soil samples in this case had "low"zinc levels but "good" copper levels. However, thoseresults were misleading. The soil copper level was 0.9ppm, which is well below the worldwide average of 20ppm, but slightly above the critical 0.5 ppm level.According to one investigator, soil copper levels shouldbe between 5 and 150 ppm. (11)

Several factors likely contributed to copperdeficiency in this filly. Considering the low soilcopper level, grazing would have supplied minimalamounts, espec i all yin a dry year, as was the caseduring the dam's pregnancy. The scarce hay supply wasstretched with alfalfa pellets which may have been highin molybdenum. In that copper absorption and retentionin ponies were inversely proportional to molybdenum


intake, the filly may have been born with low hepaticcopper reserves. (12) The mare leaked colostrum for 2days before foaling, which may have diminished thesupply of copper to the neonate. Colostrum containscopper at 0.8 - 1.2 ppm, while copper levels in mare'smilk ranges from 0.15 to 0.4 ppm. (13)

The filly's subsistence on milk for 6 weeks whilesick and injured would have depleted its marginalhepatic stores and precipitated pathologic changes inthe bones and tendons, with consequent clinical signs.The filly exhibited pica, eating rotting wood and tracemineral salt. Standard trace mineral salt contains0.035% copper, which is insufficient to be therapeutic.Generally, if a trace mineral salt is safe for sheep, itcontains too little copper for horses. There is a needfor mineral salt specifically formulated for horses.

In this case, the basic diet supplied copper at17.5 ppm. With supplementation, it was 23 ppm initiallyand 29 ppm during the second episode. It is importantto note that the chelated forms of minerals have muchhigher absorption rates than do their inorganic salts;copper absorption is improved 3-4 times with an aminoacid chelate.(14)

Epiphysitis can be caused by many factors, most ofwhich are not fully understood. As under-mineralizedbones are most likely to be deformed by compression,mineral nutrition of the patient must be investigated,especially for copper, calcium and phosphorus. Anexcess, deficit or imbalance of calcium and/orphosphorus can cause epiphysitis, and today with alfalfabeing fed, it is often difficult to supply sufficientphosphorus.

Supplementation of dicalcium phosphate wasprobably important to the filly's recovery, though noattempt was made to differentiate its effect from thatof copper supplementation. Tendon contracture seems to


respond to copper alone. In the past year over 20horses I have treated have responded favorably tovarious supplements of copper. This filly responded inproportion to the amount of copper administered.

Copper supplementation for tendon contracture isindicated before resorting to surgery. The rapidamelioration of tendon contracture gives an indicationof response to dietary copper, often within a week.Progress should be monitored through a series ofphotographs and close observat ion. Naturally, earlytreatment improves the chances of revers i ng damage.Younger animals are more responsive than those nearingmaturity.

Research should define the copper requirement forgrowing horses, so feeds can be more adequatelyfortified with absorbable micronutrients, especially inareas with deficient soils.


References

1. Schryver, H., et li., "Mineral composition of thewhole body, liver, and bone of young horses," J.Nutr., 104:126, 1974.

2. Egan, D. and Murri n, M., "Copper concentrat ionand distribution in the livers of equine fetuses,neonates, and foals," Res. Vet. Sci., 15:147,1973.

3. Go1d, A. and Bl ockman, D., "Pept i de sequences andrelative reactivity of the reactive sulfhydrylgroups of rabbi t muscl e phosphoryl ase," Bi ochem. ,9:4480, 1970.

4. Stowe, H. , "Effects of age and impend i ngparturi t i on upon serum copper of thoroughbredmares," J. Nutr., 95:179, 1968.

5. Smith, J., et li., "Tolerance of ponies to highlevels of dietary copper," J. Anim. Sci.,41:1645-1649, 1975.

6. Univ. Fl. Ext. Bull. #513R, 1957.

7. Walsh, T. and O'Moore, L., "Excess of molybdenumin herbages as a possible contributory factor inequine osteostrophia," Nature, 171:1166, 1953.

8. Bridges, C., et li., "Considerations of coppermetabo1ism in osteochondros i s of suckl i ng foal s, II

JAVMA, 185:175, 1984.

9. Carbery, J. , "Osteodysgenes is ina foalassociated with copper deficiency," N. Zeal Vet.J., 26:279, 1978.


10. Egan, D. and Murrin, M., "Copper-responsiveosteodysgenes is ina thoroughbred foal," Iri shVet. J., 27:61, 1973.

11. Buckman, H. and Brady, N., The Nature andProperties of Soils, (New York: MacMillan), 23and 489, 1974.

12. Cyrnbaluk, N., et li., "Influence of dietarymo1ybdenum on copper metabo1i sm in pon i es," J.Nutr., 111:96, 1981.

13. Lewis, L., Feeding and Care of the Horse,(Philadelphia: Lea & Febiger) 25-26, 1982.

14. Ashmead, D. and Christy, H., "Factors interferingwith intestinal absorption of minerals," Anim.Nutr. Health, 40:10, Aug, 1985.

Section 6. FISH

411

Chapter 26

THE USE OF AMINO ACID CHELATES INRAINBOW TROUT OPEN-FORMULA DIETS

Harvey H. AshmeadAlbion Laboratories, Inc.

Paul CuplinIdaho State Department of Fish and Game

The major goal in a fish feeding program is tomatch feed availability to the fish in such a way thatgrowth is maximized with a minimum of feed waste.Because fish are fed in a water diet, the value of thefeed deteriorates rapidly as a function of the amount oftime the feed is in the water. Unconsumed feed in thewater is not only a loss of feed, but it also encouragesthe growth of algae and bacteria. This reduces waterquality which will negatively affect the liveability ofthe fish. TI )

Open-formula dry fish feed diets have been used inthe Idaho State Department of Fish and Game fishhatcheries for many years. 2 Numerous diets have beentested at the hatcheri es through vari ous i ngred ientsubstitutions by production personnel. The purpose ofeach modification has been to increase feed efficiencyand maximize the problems of feeding fish as citedabove.

Knowledge of the exact trace mineral requirementsfor trout is far from complete, although advances havebeen made. Trying to ascertain the exact requirementsis difficult for a variety of reasons including thesources of the mineral. Certain sources of essentialminerals, such as zinc from galvanized metal, can bevery toxic to trout. Because minerals chelated to aminoacids have been reported to be less toxic thanequivalent amounts of the same metals as salts,(3) itwas decided to incorporate them in some of the standard

413


trout diets being used and measure any resulting changesin fish performance.

Previous research(2) has demonstrated that rainbowtrout which are raised in extremely soft water requireadditional mineral supplementation in order to performas well as trout raised in hard water. Some of the softwater from Idaho fisheries used to raise these troutcontains only 14 ppm of dissolved solids. Previous tothese studies, manganese sulphate, manganous oxide, zincoxi de and copper oxi de had been added to soft waterdiets with varying degrees of success.

Previ ous experi ments wi th Cutthroat trout frydiets have shown good results. Their diets included theamino acid chelate formula shown in Table 1, at the rateof 0.25% of the total feed. Thus, it was decided to trythis same chelate formula in this current study.

Table 1

Amino Acid Chelate Formula

Calcium'CoboltCopperIronMagnesiumManganeseZinc

41.15 %0.20 %0.62 %8.23 %

41.15 %.42 %

8.23 %

. Percentages are equivalent amounts of metals only,although all all of the metals were supplied asamino acid chelates.

Seven fish hatcheries were ultimately involved inthe study. During the first year of the study, however,the diet of only one hatchery was modified to includethe above amino acid chelates. During the first year

The Use of Amino Acid Chelates in Rainbow Trout 415Open-Formula Diets

the diet appeared to perform very well, but no testswere conducted that would evaluate the contribution ofthe amino acid chelated to the feed. It was determined,however, that the amount of chelates supplemented wassafe. This was an important consideration because ofthe potential for toxicity evident with so may forms ofmineral supplementation in rainbow trout.

The fo 11 owi ng year, seven hatcheri es were se1ectedto test the effects of the amino acid chel ates onperformance. The assessment cri teri a used were feedconversion, mortality and chemical analysis of the fishto determine absorption and metabolism of the minerals.Each hatchery was divided into an experimental and acontrol group. The diets of the two groups were thesame (Table 2) except that the experimental groupreceived the amino acid chelate formula (Table 1) at therate of 0.25% of the total feed.


Table 2

Test Diet Incredient Formula

Percent

Ingredient

Herring mealBlood flour (spray dried)Soybean flour mealWheyWheat middlingsBrewer's yeastKelp mealCondensed fish solublesHerri ng oi 1Salt (iodized)Amino acid chelates (Table 1)Lignin sulfonateVi tami n concentrate(a)

Control

3110158195312

3.5

2.0.5

Experimental

3110158195312

3.250.25

20.5

(a)(Analysis per 454 grams of vitamin concentrate)Vitamin A 20,000 USP UnitsVitamin D3 3,600 IUAscorbic acid 200,000 milligramsVi tami n B12 20 mi 11 i gramsCholine chloride 250,000 milligramsBiotin 600 milligramD-Calcium pantothenate 50,000 IUVitamin E 60,000 milligramsFolic Acid (without zinc folate) 3,000 milligramsNiacin 100,000 milligramsPyridoxine hydrochloride 20,000 milligramsRiboflavin 90,000 milligramsThiamine hydrochloride 90,000 milligrams

The chemical analysis for the total mineralcontent of the feeds is shown in Table 3. The analysiswas determined by atomic absorption spectrophotometry.

The Use of Amino Acid Chelates in Rainbow Trout 41 7Open-Formula Diets

I Table 3 IMineral Contents of Control and Experimental Diets

Milligrams/100 grams

Diet Fe Mg Cu Mn Co Zn Ca

Control 8.5 278.0 1.0 2.8 0.06 5.5 2300(actual)

Amino acid 2.0 10.0 0.015 0.010 0.005 2.0 10.0chelates

(calculated)Experimental 11.3 306.0 1.1 3.3 0.10 6.8 2030

(actual)

Equal numbers of rainbow trout were used in theexperimental and control groups as the starting numbersof fish in each hatchery. The fish numbers were reducedwhen the density, due to growth, exceeded one pound offish per cubic foot of water.

All of the fish were started on the fry feed shownin Table 4. The quantity of the ration which was fed tothe fish daily was calculated by percent of body weightand water temperature. As noted above, when excessamounts of feed are provi ded, the unconsumed excessquickly looses its food value. In order to avoid thisproblem and obtain an accurate concept of the amount offeed actually consumed, these calculations wereessential.


I Table 4 IFry Feed Fornula

Ingredient Percent

Herring meal 69.0Blood flour meal 7.0Brewer's yeast 5.0Oat flour 4.0Lecithin 5.02250A - 300D feeding oil 3.0Salt (NaCl iodized 0.01% potassium 3.0

iodide)Condensed fish solubles 1.0Vi tam; n prem; x(a) 0.5

(,J)See Table 2 for the vitamin formula

The feeding of the test diets, shown in Table 2,commenced when the fish were large enough to eat size 2fry feed.

It is reasonable to believe that the fish arecapable of absorbing minerals from the surrounding waterthrough their skin and gills as well as from the diet.Consequently, the water from each hatchery was assayedfor its m; nera1 contents by atom; c absorpt; onspectrophotometry. The results are shown in Table 5.


I Table 5 IMineral Content of Fish Hatchery Water

Milligrams/100 Grams

Hatchery Fe Mg Cu Mn Co Zn Ca

(A) 0 1.40 0 0 0 0.010 4.5(B) 0 0.19 0 0 0 0.010 1.6(C) 0 4.30 0.01 0 0 0.026 5.0(D) 0 0.82 0 0 0 0.002 2.3( E) 0 9.60 0 0 0 0.400 3.3(F) 0 0.48 0 0 0 0.010 2.0(G) 0 2.10 0 0 0 0.006 4.6

During the course of the experiment, fish sampleswere co11 ected and anal yzed on a month1y bas is. Thefish were each weighed, sacrificed, and then dissolvedin a mixture of 85% nitric acid and 15% perchloric acid.The dissolved fish were then made up to volumes whichprovided a standard concentration of each sample. Afterappropriate dilutions with distilled and deionizedwater, the samples were assayed for their mineralcontents by atomic absorption spectrophotometry. Themean results of these fish assays are shown in Table 6.


Table 6

Mean Minerai Composition of Fish Samples

Milligrams/100 Grams

Hat- Diet Feeding Fe Mg Cu Mn Zn Cachery Period in

Months

(A) Control 5 1.0 39.0 0.1 0.1 1.2 521Experimental 5 1.0 37.0 0.15 0.1 1.6 537

(8) Control 5 1.5 32.0 0.07 0.1 1.3 490Experimental 5 1.2 46.0 0.09 0.1 2.0 510

(C) Control 7 1.0 43.0 0.08 0.1 1.1 660Experimental 7 1.5 51.0 0.11 0.3 1.1 553

(D) Control 1 3.0 33.0 0.1 0.1 1.12 560Experimental 1 3.0 34.0 0.4 0.0 1.16 500

(E) Control 4 2.0 32.0 0.25 0.033 1.05 558Experimental 4 1.66 32.0 0.26 0.050 1.31 571

(F) Control 4 1.2 38.0 0.04 0.3 1.0 820Experimental 4 1.0 18.0 0.02 0.1 1.1 1,000

(G) Control 5 7.7 55.0 0.11 0.4 1.3 525Experimental 5 2.1 55.0 0.11 0.2 1.3 495

Mean 2.5 38.8 0.11 0.16 1.15 590.57± Control ± ± :t :t :t :t

S.D. 2.4 8.2 .06 .13 .10 114.53

Mean 1.6 39.0 0.16 0.12 1.37 595.14:t Experimental :t :t :t :t :t :t

S.D. 0.7 12.7 .12 .09 .32 180.72

The analyses of these samples did not indicate anystatistical trend in metal deposition in the fish.Furthermore, there does not appear to be a relationshipbetween the size of the fish and the mineral deposition.

The results of the fish performance are presentedin Table 7 along with feed conversions, mortality andblood hematocri ts. The vari at ions between hatcheri eswere too great to evaluate statistically.


I Table 7 IFeed Corversion, Mortality and Hematocrlts of Raimbow Trout In Each Testby Hatchery and Duration of Test for Control and Experimental Diets

No. of Fish No. Feed Lbs Fish Mortal-Hat- at Beginning Months Conver- Feed Per Ity in Hema-chery Diet afTest Tested sion* Lb Fish Percent tocrit

(A) Control 7,840 5 .84 1.85 6.4 30Experimental 7,840 5 .73 1.60 4.5 32

(B) Control 16,000 5 .81 1.79 3.1 --Experimental 16,000 5 .62 1.36 3.6 --

(C) Control 10,160 7 .60 1.33 0.4 31Experimental 10,160 7 .65 1.43 0.5 33

(D) Control 14,658 5 .72 1.58 5.0 37Experimental 14,658 5 .67 1.48 5.5 32

(E) Control 15,030 7 .55 1.22 0.6 33Experi mental 15,030 7 .59 1.30 0.8 35

(F) Control 15,000 4 .84 1.86 1.9 36Experimental 15,000 4 .86 1.89 2.1 30

(G) Control 1,400 6 .66 1.45 2.0 35Experimental 1,400 6 .64 1.42 2.2 36

Mean Control .72 1.58 2.n 33.67± ± ± ± ±

S.D. .12 .25 2.20 2.80

Mean Experimental .68 1.49 2.74 33.00± ± ± ± ±

S.D. .09 .19 1.80 2.10

*Kg of feed per Kg of fish.

Mortality rates for the fish recelvlng the aminoacid chelates were slightly less, but not significantlyso. There was a1so no sign i fi cant difference in thehematocrit percentages between the two groups of trout.Feed conversion, on the other hand, was improved 5.9% bythe inclusion of the amino acid chelates in the diet.It took 5.7% less feed to produce a pound of fish.

From the data in Table 6, it was evident that morezinc could be deposited in the bodies of the fish after


a few months of feeding. Several of the other metalswere a1so increased, but not as much. There was norelationship between the metals in the water and themetals deposited in the fish. The deposition of zincis, neverthe1ess, an important factor in the resul tsshown in Table 7.

In a rapidly growing fish, the metal content inthe tissues, may be manifested as a lowering of metallevels (rather than an increase) due to cell division,water intake, and generally more active body metabolism.A relationship between a greater amount of zincdeposition in the fish and more efficient conversion ofpounds of feed to pounds of fish appears to be evident.Zinc is essential for growth. Its level in the tissueswi 11, to a degree, regul ate growth rates, (4) becausezinc is essential for protein synthesis.(S)

The addition of amino acid chelates, andparticularly zinc amino acid chelate, to the diets ofrainbow trout promoted greater growth rates and betterfeed conversions.

The Use of Amino Acid Che/ates in Rainbow Trout 423Open-Formula Diets

References

1. Powless, T., "Feeding on demand," Feed Mgmt.,40:6, Nov., 1989.

2. Cuplin, P., "Performance evaluation of chelatedminerals in Idaho open - formula diets," Paperpresented at Am. Fishery Convention, Tucson, Az.,1968.

3. Larson, A., "L.D. 50 studies with chelatedminerals," in Ashmead, D., ed. ,Chelated MineralNutrition in Plants. Animals and Man(Springfield: Charles C Thomas) 163, 1982.

4. Ashmead, H., "Growth regulating effect of zincproteinate," Proc. Pacific Division of Am. Assoc.Advancement of Sci., 1968.

5. Odell, B., "Introduction, II in Pories, W., et li.,eds., Clinical Applications of Zinc Metabolism(Springfield: Charles C Thomas) 5, 1974.

Chapter 27

THE USE OF ZINC AMINO ACID CHELATES INHIGH CALCIUM AND PHOSPHORUS DIETS OF

RAINBOW TROUT

Ronald W. Hardy and Karl D. ShearerUniversity of Washington

The dietary zinc requirement of rainbow trout fedpurifie? fiets has been reported to range between 15 and30 ppm. 1 It has been reported that zinc deficienciesin rainbow trout may result in poor growth, highmortality, lens fata)acts, and reduced zinc levels incertain tissues. 1,2,3 It has been demonstrated thatdietary zinc availability can be reduced by otherdietary ingredients or nutrients in fish diets. Forexample, Ketola found that in a practical dietcontaining high-ash white fish meal, 60 ppm of dietaryzinc was insufficient to prevent poor growth andcataracts in ra i nbow trout. He a1so found that byadding extra calcium, phosphorus, sodium, and potassiumto this diet, he increased the incidence and severity ofthose cataracts. When dietary supplementationcontaining 150 ppm of zinc was included in the whit~

fish meal diet, these problems were overcome. (4

Spinelli, et li., have reported that the availability ofdietary zinc in purified rainbow trout diets was reducedwhen diet~rt levels of calcium and magnesium wereincreased. 5

There is a trend toward increased incorporation ofcertain fish that were previously used to make fish mealinto human food products, such as surimi. One result ofthis trend is greater use of white fish meal in troutfeeds in place of fish meals made from whole fish, suchas herri ng. As noted above, the concentrat ions ofcalcium and phosphorus are higher in the high-ash whitefish meals. This is of concern because high dietary ashlevels have been linked with the high incidence ofcataracts in salmon smelts, Q. Kisutch and Q.

424

The Use of Zinc Amino Acid Chelates in High Calcium 425and Phosphorus Diets of Rainbow Trout

Tshawytscha, in Pacific Northwest salmon hatcheries.(6)Therefore, this study was designed to examine theeffects of dietary supplementation with Ca3 (P04)2 (boneash) on whole body zinc levels of rainbow trout fedpurified diets. Three additional sources of zinc,ZnS04 , zinc amino acid chelate and ZnS04-EDTA were usedat each dietary level of calcium phosphatesupplementation.

Groups of 125 rainbow trout, which had a meanweight of 1.1 grams, were randomly distributed intosixteen circular, polypropylene, indoor, 200-L tanks.Each tank was serrated and supplied with 2.4 liters perminute of constant temperature (15°C) dechlorinated citywater. A natural photoperiod was used. The fish ineach tank were bulk weighed and counted every 21 daysfor a period of 84 days. At the conclusion of theexperiment, the fish in each tank were individuallyweighed.

Twelve experimental diets based on NRCrequirements{]) were formulated and fed in a 3 x 4factorial design. The basal diet, shown in Table 1, wasmodified by adding Ca3 (P04)2' at the expense of dextrinto produce diets containing 1% calcium and 0.9%phosphorus (low calcium phosphate, 2% calcium and 1.5%phosphorus (medium calcium phosphate), and 4% calciumand 2.4% phosphorus (high calcium phosphate). Nine ofthe diets were then fortified with 20 ppm of zinc aseither ZnS04 + 1,000 ppm of ethylenediaminetetraaceticacid (EDTA) as seen in Table 2. Each diet was mixed,pelleted by extrusion, and stored frozen at -20°C untilit was fed.


Table 1

Composition of Experimental Diet

Ingredient

Vitamin-free caseinGelatinPre-gelatinized wheat

starchCarboxylmenthylcelluloseDext ri na

Vi tami n premi xb

70% Choline chlorideSoybean oi 1l-Methioninel-TryptophanCa3 (PO.)/Trace mineral solutiond

Zinc supplement fl

Water

a Dextrin replaced as required by CaJ(PO.)2.

g/kg Diet

260.052.523.5

5.074.0, 62.0, 38.0

10.05.052.04.02.0

12.0, 24.0, 48.0100 ml

Various (see footnote)400 ml

(See footnote C below.)

b Vitamin premix supplied the following per kg diet: 141 mg O-calciumpantothenate; 41 mg pyridoxine-HC1; 111 mg riboflavin; 293 mgniacinamide; 17 mg folic acid; 57 mg thiamine mononitrate; 0.79 mgbiotin; 0.08 mg vitamin B12 ; 15 mg menadione sodium bisulfite; 668 mgalpha tocopherol acetate; 8,800 I.U. vitamin A palmitate or acetate;352 mg myo-inositol; 1,188 mg ascorbic acid; and 660 I.U. Vitamin OJ.

C Ca 3 (P04 )2: 2.4% added to diets 1-4, 4.8% to diets 5-8, 9.6% to diets9-12

d Trace mineral solution supplied the following per kg diet: 27 mg Mn;265 mg Fe; 9.7 mg Cu; 0.9 mg Co; 2,934 mg Mg; 10 mg I; and 5,995 mg Na

fl 20 9 Zn/g diet as: ZnS04 - diets 2, 6, 10; zinc amino acid chelate diets 3, 7, 11; ZnSO. + 1,000 9 EOTA/g diet - diets 4, 8, 12


Table 2

Experimental Design and Diet Number for the 3 x 4 Factorial

Dietary Calcium PhosphateLevel (%)

Dietary Zinc Source 2.4 4.8 9.6

No supplemental zinc a 1 5 9ZnSo4 (20 ppm zinc) 2 6 10Zinc amino acid chelate (20 ppm) 3 7 11ZnS04 + EDTA (20 ppm l;nc)b 4 8 12

a Basal diet contained 35 ppm Zn

b EDTA added at 1,000 ppm

The amount of feed which was supplied to the fishwas determined by the method of Buterbaugh andWilloughby(S) using a hatchery constant of sixteen. Thetrout were hand-fed four times per day with the feedingrate being adjusted weekly based on projected growth.Each week, the fish received seven days' rations in fivedays.

The proximate and elemental compositions of thesamples of each diet and of the individual fish fromeach dietary treatment were determined. This wasaccomplished by collecting diet samples at the beginningand again at the end of the experiment. At thetermination of the experiment (day 84), ten fish,weighing within one standard deviation of the mean, werese1ected from each treatment for e1ementa1 analys is.Proximate composition was determined by usingconvent iona1 procedures. (9) El ementa1 compos it i on wasdetermined using procedures described by Hardy, et ~.(10) The digestible energy values were calculated usingvalues of 16.7, 33.5, and 8.4 KJ/g for protein, fat, andcarbohydrates, respectively (11)


The analysis of variance was performed on finalweight and whole body zinc concentration data.(12)Scheffe's test and Duncan's new multiple range test wereused to test for statistical significance (P<O.05)between treatment means for the final weights and thewhole body zinc concentrations, respectively.(13)

The dietary supplementation with Ca3 (P04 )2increased the ash, calcium, and phosphorus levels of thediets and slightly decreased the calculated digestibleenergy contents of the diets which are shown in Table 3.

Table 3

Ash, Calcium, Phosphorus, and Zinc Concentrations ofExperimental Di ets~,b

Dry Weight Basis

Diet % Ash % Calcium % Phosphorus Zinc (ppm)

1 5.7 1.1 0.9 362 5.3 1.1 0.9 653 5.1 1. 1 0.9 614 5.3 1.1 0.9 585 7.8 2.1 1.5 356 7.7 2.1 1.4 557 7.8 2.2 1.5 628 7.7 2.2 1.5 669 12.9 4.1 2.4 3410 12.4 4.0 2.4 5311 12.4 4.0 2.4 6112 12.6 4.2 2.4 55

a Calculated digestible energy contents of the diets were: Diets 1-4,14,677 KJ/kg; Diets 5-8, 14,477 KG/kg; and Diets 9-12, 14,075 KJ/kg.Calculated values are presented for comparative purposes only

b All diets contained 50% moisture; however, 60% protein and 10.5% fatwere represented on dry weight basis, by analysis

The wei ght ga ins of the fi sh were notsignificantly (P<O.05) affected by either the dietarycalcium phosphate level or the zinc supplementation,


although there was a trend toward reduced growth ingroups of fish fed diets containing high calciumphosphate. The mean weights of the fish fed dietscontaining low, medium, or high calcium phosphate were8.75 gm, 7.65 gm, and 7.30 gm, respectively. Thedietary zinc supplementation did not significantlyaffect final fish weight (P<0.05). The mean values were7.47 gm for fish fed diets supplemented with zincsulfate, and 8.50 gm for fish fed diets supplementedwith ZnS04-EDTA. Food conversion values averaged 0.89(dry fOOd fed/wet fish weight gain) and were notsignificantly affected by any dietary treatment. Theapparent zinc retention was reduced in a stepwise mannerat each level of dietary calcium phosphatesupplementation from 36% to 30% to 20% for low, medium,and high cal ci urn phosphate supp1ementat; on. At eachlevel of dietary calcium phosphate supplementation,similar apparent zinc retention values between dietaryzinc treatments were observed. These data are seen inTable 4.


Table 4

Final Weight, Feed Conversion Rates and Apparent ZincRetention of Experimental Fish After 84 Days

Final Food conversion Apparent ZincDiet ± SEM Weight a Rateb RetentionC (%)

1 8.7 ± 0.5 ab 0.97 362 8.5 ± 0.4 ab 0.93 353 8.8 ± 0.3 ab 0.77 474 9.0 ± 0.4 a 0.81 365 7.3 ± 0.3 ab 0.80 326 8.1 ± 0.4 ab 0.82 297 6.6 ± 0.3 ab 1.00 308 8.6 ± 0.4 ab 0.80 299 6.4 ± 0.3 b 0.95 2210 7.4 ± 0.4 ab 0.92 1911 7.6 ± 0.3 ab 0.85 1812 7.8 ± 0.3 ab 1.08 19

a Initial weight 1.1 grams

b Food conversion rate - Food fed (Dry)Weight gain (wet)

C Apparent zi nc retent ion = Final Zn/fish (gm)-initial Zn/fish (gm) x 100(gm) Zn fed

Proximate composition of the rainbow trout wassimilar between dietary treatments as shown in Table 5.


Table 5

Proximate Composition and Calcium and Zinc Concentrations ofthe Rainbow Trout at the End of the Experiment

Wet Weight Basisa

Diet %Moisture %Protein % Fat % Ash Ca In(ppm) (ppm)

1 74.3 14.0 9.43 1.90 4,260 14.82 73.3 13.9 9.69 1.82 4,230 21.63 73.5 14.3 9.30 1.75 4,080 23.24 73.8 13.8 9.55 1.78 3,680 20.45 74.7 13.4 9.11 1.95 4,810 11.66 73.0 14.0 10.15 1.89 4,400 14.97 75.2 14.4 8.16 2.18 5,580 19.78 75.6 12.8 9.17 2.27 4,260 14.59 74.3 14.1 9.20 2.29 5,840 10.610 74.3 14.2 9.17 2.08 5,340 11.411 74.6 13.7 8.66 2.03 4,520 11.512 74.2 14.3 8.86 1.96 5,300 12.4

a Promixate values represent single analysis from pooled samples of tenfish. Calcium and zinc concentrations are means of individual valuesof ten fish.

Whole body zinc concentrations were significantlyaffected by the dietary source of the zinc, as shown inTable 6. Fish fed diets containing low calciumphosphate without zinc supplementation had significantlylower whole body zinc levels (P<O.05) than fish fed zincsupplemented diets. The source of supplemental zinc didnot affect whole body zinc levels among fish fed dietscontaining low calcium phosphates. Increasing thedietary calcium phosphate levels appeared to reducewhole body zinc levels in all the dietary groups exceptin the group fed the diet supplemented with zinc aminoacid chelate. The feeding of the zinc amino acidchelate resulted in greater zinc deposition in bodytissues than the other sources of zinc. Somedi fferences were noted between groups recei vi ng zincsupplementation in the medium calcium phosphate diets.


The zinc content of the fi sh wh i ch were fed dietscontaining high calcium phosphate was not significantlyaffected by dietary zinc supplementation, regardless ofthe source. In trout fed diets which were supplementedwith zinc, the whole body levels were significantlyreduced (P<O.05) in those groups fed the high calciumphosphate diets compared to the treatment groups of fishfed diets containing low or medium calcium phosphate.

Table 8

Whole Body Zinc Concentration IJ./g Dry Weight 1: SEM) of the Rainbow Trout atthe End of the Experiment

Dietary Calcium Phosphate Levels (%)

Dietary zinc sources of(20 p.g Zn/gm diet) 2.4 4.8 9.6

No supplemental zinc 57.4 :!: 3.5 bc 46.0 :!: 3.5 ed 41.4:!: 4.6 fznso4 80.9 :!: 3.7 a 55.3 :!: 2.4 cde 44.5 :!: 3.0 fzn amino acid chelate 87.6 :!: 2.0 a 79.5 :!: 3.2 a 45.3 :!: 1.4 e1Znso4 + EDTA n.81: 3.0 a 59.3 1: 3.6 bc 47.9 1: 1.5 def

Values with the same letter are not significantly different (P <0.05)

Contrary to anticipation, there were no cataractsobserved in fish from any of the experimental treatmentgroups.

Numerous studies on a variety of animal shaveshown that the biological availability of dietary zincinclusion of calcium to practical diets reduces theabsorption of zinc in swine(14,1S) and poultry.(16)Pensack, et ~. reported that in purified diets, theaddition of calcium did not reduce zinc availability inchicks,(17) but subsequent work showed that there was aninteraction between calcium and the phytic acid presentin the diet which resulted in decreased zincavailability.(20,21) This reduction was believed to be


due to absorption of zinc onto an insoluble calciumphosphate complex which was then carried through the gutand excreted. In support of this proposed model,calcium phosphates have been shown in vitro toprecipitate from solutions when the solution pH isincreased from three to six. This pH change takes zincout of the solution, as well.(22)

Certain chelating agents have been shown toimprove zinc availability in diets containing zinccomplexing compounds. (23) For example, Vohra and Kratzerfound that chelates with zinc stability constantsbetween thirteen and seventeen were effective in turkeypoult diets.(24) The addition of EDTA to the diet hasincreased zinc availability in eX8erimental dietscontaining isolated soybean protein 25,26) and phyticac id(27) and a1so in pract i cal ch i cken diets. (28)

Data on trace element requirements and dietaryfactors affecting their availability in fish arebeginning to accumulate. It was previously noted thatthe zinc dietary requirement of rainbow trout wasbetween fifteen and thirty ppm in a purified diet. Itwas also noted that the dietary zinc levels necessary toprevent deficiency signs in rainbow trout was muchhigher in diets which contained high-ash white fishmeal. There is a reduction in rainbow trout growth whenphytates are added to the diet, although there are nodata relating to their effects on zinc availability infish diets.

The analyses of the results of this present studyshow that whole body levels of zinc were significantlyreduced in rainbow trout fed a purified diet containinghigh levels of calcium and phosphate. This reduction ispresumably due to reduced zinc availability in the highcalcium phosphate diets. Efforts to increase zincavailability by chelation were only partiallysuccessful. Adding 1,000 ppm EDTA to the diets


supplemented with ZnS04 did not result in anydifferences in whole body zinc concentrations at anydietary calcium phosphate level when compared to theaddition of ZnS04 alone. Weight gains were slightlyincreased in the ZnSO~,-EDTA supplemented diets. Thiswas perhaps due to a oeneficial effect of EDTA on theavailability of some trace element other than zinc,because our studies have been unable to measure anyeffect on whole body zinc levels of EDTA supplementationto rainbow trout diets containing soybean meal. On theother hand, dietary supplementation with zinc which hadbeen chelated to amino acid chelates proved effective atmaintaining whole body zinc levels when the dietarycalcium phosphate level was increased from low tomedium. An additional increase in dietary calciumphosphate to the high level significantly reduced wholebody zinc levels in all treatments, including the zincamino acid chelate. This suggests under that conditionthere i s the need for higher 1eve1s of zincsupplementation particularly in the more biologicallyavailable amino acid chelate form. Under normalcircumstances, supplementing diets with a biologicallyavailable amino acid chelate should insure an adequatelevel of absorption of the zinc. If the zinc amino acidchelate were 100% available, the whole body zinc levelsof rainbow trout fed diets containing high calciumphosphate should have been higher in fish fed diet 11compared to diets 10 or 12. Our observations of wholebody zinc concentrations and apparent zinc retentionindicate that the zinc absorption from zinc amino acidchelate is reduced at a high level of dietary calciumphosphate, or that the higher calcium-phosphorus levelscauses the absorbed zinc to be removed from the tissuesafter deposition. More likely the latter scenario isthe case since it appears that there is littleinterference of zinc absorpt i on and retent i on at thelower dietary levels of calcium phosphate.

Despite the significantly lower whole body zincconcentrations in the trout which were fed diets


containing the high level of calcium phosphate, nodeficiency signs were observed. Ogino and Yang reportedthat trout exhibiting zinc deficiency signs had wholebody zinc levels of between 28 to 32 ~g/g, dry weight,compared to whole body zinc levels of 72 to 77 ~g/g infish which had been fed diets containing adequateamounts of zinc,(1) and which were the levels that wereobtained with the zinc amino acid chelate except in thecase of high dietary calcium phosphate. It is possiblethat the length of this present study was insufficientto reduce whole body zinc levels in fish fed the highcalcium phosphate diets to the levels at which overtdeficiency sign would appear. The dietary calcium andphosphorus 1evel s used in thi s experiment generallycovered the range of values reported to occur incommercial fish feeds. Tacon and DeSilva have foundthat the ranges of calcium and phosphorus in trout feedsin Europe were 1.3 to 3.7%, and 0.9 to 2.8%,respectively.(23) Commercial salmonid feeds in NorthAmerica typically have calcium and phosphorus levelswhich range from 2.2% to 4.2% and 1.2% to 2.6%,respectively. Thus, the deleterious effect of highdietary calcium and phosphorus on the whole body zincconcentration observed in this present study may alsooccur in practical salmonid diets which contain high-ashfish meal. In addition to high calcium and phosphorus,many commercial fish feeds use ingredients which containsignificant amounts of phytic acid, and some forms offiber which may further reduce zinc bioavailability.Therefore, the potent i a1 for reduct i on of zincavailability in practical salmonid diets may be evengreater than was seen in this experiment. The generalapproach to this problem in the past has been toestabl ish ingredient specifications for trout and salmondiets that restrict the use of high-ash fish meal.Practical considerations may force this approach to bere-examined and allow the use of high-ash fish meals informulations as long as the trace element fortificationcan be safely increased.


References

1. Ogino, C., and C. Y. Yang, "Requirements ofrainbow trout for dietary zinc," Bull. Jpn. Soc.Sci, Fish., 44:1015-1018, 1978.

2. Wekell, J., etli., "High-zinc supplementation ofrainbow trout diets," Prog, Fish. Cult., 45(3) : 144 , 1983.

3. Satoh, S., et li., "Effects of deletion ofseveral trace elements from fish meal diets ongrowth and mineral composition of rainbow troutfingerlings," Bull. Jpn. Soc. Sci. Fish.,49:1909-1916, 1983.

4. Ketola, H. G., "Influence of dietary zinc 0

cataracts in rainbow trout (Salmo Gairdneri), J.Nutr., 109:965-969, 1979.

5. Spinelli, J., Houle, C. R., and Wekell, J. C.,"The effect of phytates on the growth of rainbowtrout (Salmo Gairdneri) fed pure diets containingvarying quantities of calcium and magnesium,"Aquaculture, 30:71-83, 1983.

6. Roley, D., Personal Communication, 1981.

7. National Research Council, Nutrient Requirementsof Trout, Salmon, and Catfish, (Washington, D.C.:National Academy of Sciences) 57, 1973.

8. Buterbaugh, G. L. and Willoughby, H., "A feedingguide for brook, brown, and rainbow trout," Prog.Fish. Cult., 77:210-215, 1967.


9. Association of Official Analytical Chemists,Official Methods of Analysis (Washington, D.C.:Assoc. Off. Anal. Chern.) 1904, 1975.

10. Hardy, R. W., et li., "Fish silage in aquaculturediets," World Maricult. Soc. J., 1983.

11. Jobl ing, M., "A short review and critique ofmethodology used in fish growth and nutritionstudies," J. Fish. Biol., 23:685, 1983.

12. Steel, R. G. D. and Torrie, J. H., Principles andProcedures of Statistics (New York: McGraw-Hill)481,1960.

13. Zar, J. H., Biostatistical Analysis (EnglewoodCliffs: Prentice-Hall Inc.) 620, 1974.

14. Lewi s, P. , et li., "The effect of certa innutritional factors including calcium,phosphorus, and zinc in parakeratosis in swine,"J. Animal Sci., 15:741, 1956.

15. Hoesktra, W. G., "Recent observations on mineralinterrelationships," Fed. Proc., 23:1068, 1964.

16. Roberson, R. H. and Schaible, P. J., "The effectof elevated calcium and phosphorus levels on thezinc requirement of the chick," Poultry Sci.,39:837, 1960.

17. Pensack, J., et li., "The effects of calcium andphosphorus on the zinc requ i rements of growi ngchickens," Poultry Sci., 37:1232, 1958.

18. O'Dell, B. L. "line availability in the chick asaffected by phytate, calcium, andethylenediaminetetraacetate," Poultry Sci.,43:415, 1964.


19. Likuski, H. J. A. and Forbes, R. M., "Mineralutilization of the rat, IV. Effects of calciumand phytic acid on the utilization of dietaryzinc," J. Nutr., 84:145, 1964.

20. Cabell, C. A. and Earle, I. P., "Additive effectsof calcium and phosphorus in utilization ofdietary zinc," J. Animal Sci., 24:800, 1965.

21. Heth, D., et li., "Effect of calcium, phosphorus,and zinc or zi nc-65 absorpt i on and turnover i nrats fed semi-purified diets," J. Nutr., 88:331,1966.

22. Lew; s, P. K. et li., "The effect of method offeeding upon the susceptibility of the pig toparakeratosis," J. Animal Sci., 16:927, 1957.

23. O'Dell, B. L., "Dietary factors that affectbiological availability of trace elements,"Annals New York Acad. Sci., 199:70, 1972.

24. Vohra, P. and Kratzer, F. H., "Influence ofvarious chelating agents on the availability ofzinc,1I J. Nutr., 82:249, 1964.

25. Kratzer, F. et li., "The effect of autoclavingsoybean protein and the addition ofethylenediaminetetraacetic acid on the biologicalavailability of dietary zinc for turkey poults,"J. Nutr., 68:313, 1959.

26. Nielson, F., et li., IIEffect of some dietarysynthetic and natural chelating agents on thezinc deficiency syndrome in the chick," J. Nutr.,89:35,1966.


27. O'Dell, B. L. and Savage, J. E., "Effect ofphytic acid on zinc availability," Proc. Soc.Exp. 8iol. Med., 103:304, 1960.

28. Suso, F. A. and Edwards, Jr., H. M., "Influenceof various chelating agents on absorption of 60CO ,59Fe, 54Mn , and 65Zinc by chickens," Poultry Sci.,47:1417, 1968.

29. Tacon, A. G. J. and DeSilva, S. S., "Mineralcomposition of some commercial fish feedsavailable in Europe," Aquaculture, 31:11, 1983.

Chapter 28

THE EFFECTS OF IRON AMINO ACID CHELATEIN CULTURED EELS

Katsuhiro Suzuki, Yoshito Iwahasi, andTakeharu Takatsuka

Shizuoka Prefectural Fisheries Experimental StationLake Hamanako Branch, Japan

Takaaki WakabayashiEisai, Co., Ltd., Japan

Iron amino acid chelate is a complex of iron froma ferrous salt and amino acids from hydrolyzed protein.Data from numerous sources have reported t~at iron inthis form is easily absorbed and util ized. 1) It hasbeen added to the feed of domestic animals withexcellent success. There are no data, however, relatingto its use in eels. Thus a study was devised to measurethe affects of iron amino acid chelate in the eel.

The eels were divided into five groups and placedin tanks measuring 1.8 meters in width by 4.2 meters in1ength and havi ng a depth of 0.6 meters. The totalvolume of water in each tank was 4.5 cubic meters.

A stagnant water system was employed for theculture in which the water temperature was maintainedbetween 24.6°C and 29.4°C.

All of the eels received the same standard feedshown in Table 1 which assayed at 47.6% crude protein,3.8% crude fat, 14.5% ash, and 8.4 %water.

440

The Effects of Iron Amino Acid Chelate in Cultured Eels 441

I Table 1 IStandard Eel Feed Formula

White fish meal 67.00 %Brewer's yeast 5.00 %Potato starch 22.00 %Skim milk 3.00 %Ethoxyquin 0.015%Vi tami n supplement (a) 2.00 %Mineral supplement (b) 1.00 %

Crude protein 47.6 %Ash 14.5 %Crude fat 3.8 %Loss on drying 8.4 %

(a) Vitamin supplement 3.0 gmVitamin B1-HCl 10.0 gmVi tami n 62 2.0 gmVi tami n B6 -HCl 40.0 gmNicotinic acid 14.0 gmO-pantothenic acid calcium 200.0 gmInositol 0.3 gmD-Biotin 0.75 gmFolic acid 4.5 mgVi tami n B12 100.0 gmVitamin C 0.6 gmB-Carotine (Food Additive) 2.25mgVitamin 0] oil (1 mg=40, 000 IU) 20.0 gmVitamin E 2.0 gmVitamin K] (menadione) 20.0 gmp-Aminobenzoic acid 400.0 gmCholine chloride Q-Sd-Cellulose 1,000 gm

(b) Mineral SupplementSodium Chloride (NaCl) 0.0 gmCa-Lactate (C 6 HlO06Ca. 5H2 O) 35.0 gmMgS04 .7H2 O 150.0 gmFerric Citrate (FeC 6 Hs07 • 5H2 O) 25.0 gmNaH 2 P04 .2H2 O 250.0 gmKH 2 P04 320.0 gmCa (H2 P04 ) 2' H2 O 200.0 gm

990 gm

As stated above the eels were divided into fivegroups. Group 1, the control group, received the


standard feed shown in Tabl e 1. Group 2, the torul agroup, received the same standard feed except torulayeast was subst i tuted for brewer's yeast. Group 3received 60 ppm of iron as the amino acid chelate addedto the standard formula in Table 1. Group 4 receivedthe standard feed plus 150 ppm of iron as the amino acidchelate. And finally, group 5 received the standardfeed formula shown in Table 1 plus 250 ppm of iron asiron amino acid chelate.

The test period lasted 83 days, from June 28th toSeptember 19th. During that time measurements were madeevery 21 days except the final measurement which wasmade 20 days after the previ ous measurement. Thepercentage of internal organs (liver and internal organsexcept for kidney and heart) to the whole weight werecalculated. Blood tests were conducted for hematocrit,hemoglobin, plasma proteins, and red blood cells.Growth rates, feed efficiency and average body weightsas well as individual body weights and lengths weredetermined.

The resul ts for feed effi ci ency and changes inbody weights are shown in Table 2. The groups receivingthe iron amino acid chelate generally grew faster,weighed more and had better feed conversion/utilizationthan the groups deprived of this amino acid chelate.


I Table 2 IResults of Culture (For The Entire Test Period)

1 2 3 4 5

Mean beginning body weight 49.6 49.6 49.6 50.1 50.1(gm) (101) (101) (101) (100) (100)

Mean ending body weight 132.0 130.9 134.7 132.6 141.2(gm) (95) (98) (97) (98) (100)

Growth rate (gm/day) 1.18 1.17 1.18 1.17 1.25

Total initial weight (gm) 5015 5012 5008 5011 5006

Total final weight (gm) 12882 13124 13476 13185 14119

Total weight increase (gm) 7867 8112 8468 8174 9113

Weight of feed given (gm) 11709.8 11311.3 11554.1 11565.4 11798.7

Feed conversion 1.61 1.53 1.56 1.55 1.49

Feed efficiency (%) 67.2 71.7 73.3 70.6 77.9

Protein efficiency (%) 135.0 142.2 147.1 144.6 162.2

Taking data from Table 1, Figure 1 shows thechanges in feed efficiency. Figure 2 looks at thechanges in total feed efficiency. Finally, Figure 3examines the changes in average body weight.


100.....--------------------,*GROUP 5

~r,--.-•• -••••••••GROUP A .

. GROUP 360 . GROUP·2············

GROUP 1

>(,)zG 80u:LLW

owWu.

g, 40.....z~ 20a:wa..

o 2 3 4 5 6

PERIOD OF MEASUREMENT

Fi gure 1. Changes in feed effi c; ency brought aboutby the iron amino acid chelate.

100.....-------------------,

..................................................................

20 0 ••••• 0 •••••••••••••••••••••••••••••••••

>u1:5 80 ROUP 5

U L_--::::===-"'--:I::::::;;;=::::=.:~===:=:::~===:=-"==1~:GROUPS 3 & 4u:: GROUP 2lli 60 GROUP 1

o~ I

~ 40oI-ZWUa:wa..

OL-------L---------1-----~

0-21 0-42 Q-63

DAYS

Figure 2. Total feed efficiency of the fivegroups.


145 r-----------------_ GROUP I

125

...J:CJW105~

>caQJ

w 85 .CJ<a:w>c(

65 .

. GROUPS 3 .4

GROUPS 1 & 2

45~----"""'--------'-------J21 42 DAYS 63 83

Figure 3. Changes in the mean body weight of theeels.

Group 5, which received 250 ppm of iron from theamino acid chel ate performed better than the othergroups. The feed efficiency of Group 5 was 13% higherthan the control group. The improved weight gain fromthat same group was 15.8%. When these data are coupledwi th a 7. 5% improvement in feed convers i on the ironamino acid chelate becomes more important.

By taking the data which generated Table 1, thegrowth curve equation for each group can be calculatedfrom the mean body weights and are as follows:

Group 1: BW (t) = 48.9 e x p (0.0123t), (r2 = 0.992)

2: BW (t) = 49.2 e x p (0.0122t), (r2 = 0.990)

3: BW (t) 49.4 e x p (0.0123t), (r2 = 0.996)

4: BW (t) 49.5 e x p (O.0121 t), (r2 = 0.996)

5: BW (t) 49.6 e x p (O.0129t), (r2 = 0.996)


The regression analyses of the above growth curveequat ions were cal cul ated. It was found that thedifferences among the groups were not significant(according to the t-test). The reason may be that thefeed intake rate was different in each group. Eventhough the feed intake rate in group 5 was lower thanthat of the other groups, the growth rate was almost ashigh as the other groups, which means that the feedefficiency was commensurately higher.

Furthermore, since the feed intake and the numberof ee1s are somewhat different in each group, thecumulative feed intake and cumulative increase of bodyweight per eel up to the time of measurement are shownin Table 3, and their relation is shown in Figure 4.

I Table 3 IThe cumulative feed intake (x) and the cumulative increaseof body weight (y).

1 2 3 4 5

x y x y x y x y x y

0 21 18.3 11.1 17.4 10.6 18.8 12.5 18.6 11.8 18.9 12.80 42 50.1 34.7 49.7 34.3 50.4 36.1 50.2 33.1 51.0 36.20 63 83.4 57.6 81.1 59.1 82.4 58.1 83.3 57.2 85.7 64.20 83 116.7 76.7 114.1 79.4 115.0 81.3 116.3 80.6 118.0 91.1


§ 100 ,....---------------------,.....:::r::~ GROUPS~ 80·· . . " "" " "......... ... 'GROUP "3

w GROUP 2~ GROUPS & 4LL 60owen

~ 40 .u~

w~ 20:5::>~ O~-----"'--------J"-------'----"'---"""""""'-------"------I<..> 20 40 60 80 100 120 140

CUMULATIVE FEED INTAKE (9)

Fi gure 4. The re1at i onsh i p between cumul at i ve feedintake and cumulative increase of body weight (pereel).

The following regression equations were obtainedfrom the data generated in Figure 4.

Group 1: y = 0.669 + 0.149 x (r = 0.998)2: y = 0.719 - 1.287 x (r = 0.999)3: y = 0.712 0.479 x (r = 0.999)4: y = 0.707 1.747 x (r = 0.999)5: y = 0.792 0.308 x (r = 0.999)

A regression analysis of these equations weremade, and the regression coefficients were compared.These data indicated that the slope of the regressionline of group 5 was significantly steeper than those ofthe other groups. Thisslope represents the feedefficiency, iron amino acid chelate containing 250 ppm


and resulted in significantly improving the feedefficiency (P<O.OS). Figure 4 suggests that thisbeneficial effect appears early and continues to have aneffect as the iron amino acid chelate is fed over thecourse of the production schedule.

The individual eels were weighed on August 9th,30th, and September 18th. Their body weights are shownbelow in Table 4.

I Table 4 IThe Mean Body Weight of Each Eel

Mean body we; ght ± s. D. (gm) (Coefficient of Variation)

August 9 1 83.94± 22.01 26.2 %2 84.97 ± 20.96 24.7 %3 85. 50 ± 25.88 30.3 %4 83.19 ± 21.87 26.3 %5 85.67 ± 23.84 27.8 %

August 30 1 110.67 ± 24.24 21.9 %2 110.61 ± 28.98 26.2 %3 109.43 ± 28.16 25.7 %4 108.95± 23.74 21.8 %5 114.21 ± 26.34 20.4 %

Sept. 18 1 132.97 ± 27.63 20.9 %2 130.93± 27.64 21.1 %3 134.91 ± 31.09 23.0 %4 132 . 58 ± 25. 45 19.2 %5 141.2 ± 29.43 20.8 %

The mean body weight of group 5 was found to begreater than the mean weight of the eels in the othergroups at the final measurement on September 18th. Thecoefficient of variation tended to reduce as the groupsmatured. Thus, differences in somatic growth in theindividual eels became more uniform over time.

The amount of fat, the percent of weight of theorgans compared to the whole body, and the results of


the blood tests are shown in Table 4. This tablecompares those data at the beginning of the experimentto the data obtained at the conclusion. All of the datain Table 4 are the mean values plus or minus arestandard deviation.

I Table 5 IFat, Weight of Organs and Blood Tests (Mean ~ Standard Deviation)

IBeginning I 1 I 2 I 3 I 4 I 5

Body weight- (gm) 69.7 ~ 10.6 160.2 ~ 12.4 162.2 ~ 18.5 167.7 ~ 26.2 165.8 ~ 16.5 163.9 ~ 18.6

Overall length- (em) 36.2 :!: 1.5 46.5 :!: 1.6 46.7 :!: 2.1 47.8 :!: 2.9 47.4 :!: 1.5 47.5 ~ 1.78

Fat 1.46 ~ 0.09 1.59 ~ 1.0 1.6 :. 0.05 1.5 :. 0.1 1.6 :. 0.06 1.6 :. 0.06

Liver weight (gm) 1.14!:. 0.27 2.71!:. 0.37 2.4 !:. o.~ 2.6 !:. 0.5 2.5 !. 0.5 2.50 !:. 0.30

lWeight percent COlo) of 1.64:. 0.28 1.69:. 0.17 1.49 ~ 0.27 1.54 ~ 0.29 1.48 :. 0.17 1.54 ~ 0.24liver

Weight of internal 2.19:. 0.33 4.39:. 0.88 3.96:, 0.36 4.22:!: 0.67· 3.84:!: 0.62· 3.49:!: 0.54a

organs (gm)

Weight percent rIo) of 3.10 ~ 0.52 2.73! 0.« 2.46! 0.20 2.46! 0.35 2.32! 0.33 2.15 ~ 0.37internal organs

Hematocrit (%) 32.9 :: 2.38 31.4 :: 3.4 32.4 :: 2.0 34.7 :: 3.71 34.4:: 6.19 33.6:: 5.01

Hemoglobin (gm/dl) 7.8 ! 0.66 7.6 ! 0.6 8.4 ! 0.6 9.4 :. 1.04 9.3 :. 1.2 9.5 :. 1.1

Plasma proteins 6.6 :. 1.0 7.5 ! 1.1 7.4 ! 0.9 8.0 ! 0.6 7.9:. 0.6 7.5 ! 1.0gm/dl)

- Mean values of eels tested.Ia Significant at P<0.05

To illustrate the differences among the groups,95% confidence intervals of each measurement are shownin Fi gures 4 - 9. As can be seen in the fi gures,significant differences occurred in the weights of theinternal organs and in the hemoglobin levels. Theweight percentages of the internal organs was smaller ingroups 4 and 5 than in group 1. Th is means that theeels given feed with higher concentrations of iron aminoacid chelate tended to have lower intestinal organ as


compared to total body weight. Muscul ar meat weightwould, therefore, be higher.

The hemoglobin levels of groups 3, 4 and 5, thegroups given iron amino acid chelate, were significantlyhigher than groups 1 and 2, indicating that besidesaffecting iron amino acid chelate promoted hemoglobinsynthesis in addition to the previously mentionedeffects.

2--r-------------------,

1.5

~

~u..

0.5

oGROUP 1 GROUP 2 GROUP 3 GROUP 4 GROUP 5

TEST GROUPS

Figure 5. 95% Confidence interval of average bodyfat over the course of three successive weighingperiods.

The Effects of Iron Amino Acid Chelate in Cultured Eels 4518.......---------------------,

enz<{ 5 .

~~O~

-J W 4 - .<3=Z>-ffio1-0 3zmu:u..0°~ ~ 2 .:I:cnCJ<{W~


TEST GROUPS

Figure 6. 95% Confidence interval of average ofweight of the internal organs as a percent of thetotal body weight over the course of threesuccessive weighing periods.

2,...---------------~r--

""7" r-- ...--- r-:- .---

- 1.5\ . . r---

.~ . .~ . 1\~ \ \\ ~ .. ~~, \(1)

\' \ \ ~ ..:>

~::J

\ \'~'0 0.5 \. \. '"

~ ••C\ 0' I~\

~:(1) \. r\\ .0 0~

(1)

a.

t-o.s'(i)

~ -1

Group 1 Group 2 Group 3 Group 4 Group 5

Test Groups

Figure 7. 95% Confidence interval of average ofweight percent of liver over the course of threesuccessive weighing periods.


40.....------------------,

! 30t:a::uo 20~~w:r::

10


TEST GROUPS

Figure 8. 95% Confidence interval of average ofhematocri t over the course of three success; vesampling periods.

12~--------------_,

10 .

~ 8- .

~ziiio 6...JC)o~

~ 4

2

GROUP 1 GROUP 2 GROUP 3 GROUP. GROUP 5

TEST GROUPS

Figure 9. 95% Confidence interval of average ofhemoglobin over the course of three successivesampling periods.


10~---------------------.

CfJZ 6WI-oa: 4a..«~ 2CfJ«--.Ja.. 0

GROUP 1 GROUP 2 GROUP 3 GROUP 4 GROUP 5

TEST GROUPS

Figure 10. 95% Confidence interval of average ofpl asma proteins over the course of three successivesampling periods.

These data demonstrate the val ue of provi di nggrowing eel 250 ppm of iron daily as the amino acidchelate. Growth rates, body weights, and feedefficiency were all statistically improved. Hemoglobinlevels were also improved.


References


2. Ashmead, D., ed., Chelated Mineral Nutrition inPlants, Animals and Man (Springfield: Charles CThomas) 1982.

Section 7. SUMMARY AND CONCLUSION

455

Chapter 29

SUMMARY AND CONCLUSION


When a chelate is formed, a polyvalent metal (sucnas Ca, Mg, Mn, Fe, Zn, Cu, or Co) forms a complex witha ligand. In this complex, the ligand becomes theelectron donating molecule while the metal ion is theelectron acceptor. The term "chelate" was suggested byMorgan and Drew(1) to describe these metal complexes inwhich the metal atom is held by the ligand at more thanone point with the metal atom occupying a centralposition within the complex as seen in the examples inFigure 1. (2)

8I

CHs- C?Ha I/ I H.-CH 8-CHa H

zn ,/ I I 'd'N..._CH_COO·z'O-c ~o- / ~o ~o RI CYlnINI MIIND ACID CHB.ATD or DE 0c~

/ CH. H 00, 0 I I /~ 0,elett. N HO---..C~C Cu \ ~o\o 7 'cw. I T C

>C:O~N/~ H II ° I r0'c~ I /C, / -VO...... M

/ " _ CH" ItO c~ ~ '"", '0o c COPPa 'b ~. /'

U CHB.ATID TO IRON 'cCOIAI1 ~Iq AICOIIBIC ACID CHELATED ,COORDINATED WITH EDTA (VITAilI. C) TO CITRIC ACID 0

Figure 1. Metals chelated with various ligands.

457


Mellor has established two criteria that must bemet by the ligand in order to form a chelate: (1) theligand must possess at least two functional groups, oneof which is capable of donating a pair of electrons tobond with the metal; and (2) the functional groupdonating the electrons must be located in such a waythat a ring structure is forme~ rith the metal ion asthe closing member of the ring. 3

The quantitative measurement of the affinity of aparticular metal ion to complex with a l~~and is calledits formation or stability constant. Stabilityconstants can increase or decrease based on a variety ofconditions including, the valence of the metal, thenumber of members formi ng the ri ng, the number ofligands chelated to a single metal ion, the nature ofthe ligand, the metal ion involved, etc. Table 1 showsthe electron, to the stability constant of the chelate.A ligand with a higher stability constant will displacea metal from a ligand with a lower stability constant.For this reason EOTA chelates, as an example, arepoisoning because the EOTA, with its higher stabilityconstant, removes the heavy metal from the naturallyoccurring chelates found in the body and which havebound the intruding metal with lower stabilityconstants. (5)

Summary and Conclusion 459

I Table 1 IThe Influence of Donating Electrons of a Ligand to theGeneral Strength of the Coordination Complexing

Complex Bonding Character Relativestrength

~O++Metal

f..- OThe oxygen is mostly ionic. Generally

weak

~OThe nitrogen is covalent in nature, Relatively++Metal

f...- O and the oxygen is mostly ionic strong

~O Both of the bonds are covalent in Very++Metal

f...- Onature with sulfur displaying the strongstrongest bonding character.

As noted above, the nature of the ligand'sdonating electrons will affect the stability constant ofa chelate. The metal ion being chelated will alsoinfluence the bonding strength of the resultant chelatebecause certa i n metal s preferent i all y choose certa indonor atoms from the ligand. Consequently, depending onthe ligand and the metal ion, a chelate or a metalcomplex can be formed with varying stability constants.Table 2 summarizes these observations.(18)


I Table 2 IGeneral Role of Metal ions in Complexes and Chelates inBiological Processes

Na. K Mg,Ca, Zn,Cd, CU,Fe,Mo,(Mn)* (Co) (Mn)

Bond Strength Weak Moderate Strong Strong

Biological Charge transfer, Trigger Hydrolysis, Oxidation-Function ion gradients, reactions, pH control reduction

nerve conduction phosphate reactionstransfer

Preferred DonorAtom in Ugand 0 0 N&S N&S

*Parenthesis indicate cations with dual or multiple characteristics

Vohra and Kratzer, (6) in their study of zinc,proposed that the ability of a chelating agent toenhance mineral availability depended upon the chelatehaving a higher stability constant for the metal thanthe metal binding substances in the animal's feed. theytheorized that if a highly stable chelate were formed,then the mineral was protected from gastrointestinalreactions and absorbed. Following absorption the metalreleased from the metal-chelate complex. This impliedthat the natural ligands in the body tissues must havehigher stability constants than the ingested chelatingagent in order to remove the metals.

When tested in vivo, the model of Vohra andKratzer as cited above(6J(ffd not work as reported by R.F. Miller at a Delmarva Nutrition Short Course.'?) Hedemonstrated that synthetic chelated forming highstability constants that protected minerals in the gutdid not optimize mineral metabolism.(?) The formationof such a chelate could be too stable making subsequentdegradation and release of the metal in the body forphysiological usage only occurred with inordinate


difficulty. Frequently, an absorbed chelate havingthese properties was excreted into the urine and lowerbowel still intact as the original molecule.

The late John Miller proposed that the role of thechelate in nutrition was to protect the cation in thegastrointestinal tract until it reached the site ofmineral absorption. At that point, he bel ieved thechelate released the metal to the intestinal mucosaltissue, at which time the metal was absorbed.(8) Usingiron as an example of the mineral and amino acids asexample ligands, this concept is illustrated in Figure2. (9)

LUMEN

AMINO ACIDS

(CHELATING AGENTS)

<;.FE+· FE· CHELATE --.-

~PRECIPITATING LIGANDS

FE PRECIPITAT1~~ow>~<owZ

INTESTlNAL CELL

FERRITIN

l' \FE·· --+

(CH~TION) ~

APOFERRITI N

BLOOD

RED CELLS

DEPOT ..CELLS ,

~

(CHELATION)

'-:.. TRANSFERRIN

.. URINE

Figure 2. Equilibrium binding and chelationmechanism for intestinal regulation and control ofiron uptake with a mucosal directed, activetransport systems, assumi n9 that free metal s arereleased at the mucosal membrane to luminal bindingagents.


This model also has the inherent drawbacks whichwere discussed above relating to the previous models.It assumes that the binding proteins on the mucosal cellmembrane have higher stability constants than theingested chelate sources. Miller's model dictates thisto be an essential criterion for the removal of thosecations from the ingested chelates. The difficulty withthis model arises in the additional requirement that, ifthey are to protect the minerals, the ingested chelatesmust have higher stability constants than potentialprecipitating ligands, which exist in the normal diet.If the precipitating ligands were to have higherstabi 1i ty constants than the ingested chel ates, thenthey would naturally replace the chelating ligands. Ifthat were to occur, then the "protective" benefitassociated with the chelate would be lost. (Withcertain chelating agents such as ascorbic acid, citricacid and gluconic acid this removal of the metal byligands in the gastrointestinal which result inprecipitation of the metal-l igand product tract canoccur.) If the diagram in Figure 2 were an accuratedepiction of the intestinal absorption of amino acidchelates of metals for the purpose of protecting themeta1, then the stabi 1i ty constants of the bi nd i ngproteins on the mucosal cell membrane would have to havethe potential of forming higher stability constants thanthe precipitating ligands because the chelate has to beprotected form the precipitation ligands but stillrelease the metal to the binding proteins on the mucosalmembrane. If this were the case, there would be no needfor the "protective" action of the chelate because thebinding proteins on the mucosal membrane would becapable of scavenging all necessary minerals from anysource and should form complexes that had higher stability constants than the precipitating ligands.Absorption would occur with no difference between thechelates and the non-chelated minerals.

The proceeding chapters throughout this book havenot supported such a hypothetical mechanism. It has


been shown that there is a defi ni te metal absorpt ionbenefit when the respective polyvalent cations arechelated with amino acids. The cumulative resultsreported support an enhanced absorption of the intactamino acid chelate over that of metal salts when bothare subjected to normal precipitating ligands in thediet. The following in vivo data in Table 3 which werereported by Ashmead using radioisotopes in amino acidchelates and salts fed to experimental animals furthersustain this conclusion.(10)

I Table 3 IMineral Deposition in Animal Tissues Following ingestion ofMinerals from Various Sources (corrected counts/minute)

Tissue 45Ca1ci um 65Zi nc 59! ron 54Manganese

CaC1 2 AAC* ZnC1 2 AAC* FeC1 2 AAC* MnC1 2 AAC*

Bone 3682 5772 350 780Muscle 614 1206 2.41 3.88 2 54 800 660Heart 642 932 6.42 6.32 63 151 370 190Liver 664 742 5.15 8.65 136 243 760 070Brain 698 804 1.22 2.41 31 130 620 170Kidney 686 730 5.45 8.55 2 327 470 600Lung 676 648 720 330Blood Serum 8 31 700 1797RBC 18 13 742 2076Whole Blood 27 44 .90 1.64 1335 4215

*Amino Acid Chelate

Because of the problems with the previous models,Ashmead, et gl., proposed a different modfl to describethe absorption of an amino acid chelate. 2 This modeloperates via the identical intestinal pathway utilizedfor the uptake of small pept ides. In the ami no ac idchelates which have been properly formed, the moleculebypasses the digestive actions of the acid and alkalineenvironments of the stomach and intestines. Thegastrointestinal peptidase enzymes do not appreciablyaffect it, and the stability constants of the amino acid


chelates are higher than those of precipitating ligandssuch as phytates, phosphates, etc. Thus, instead ofbeing degraded in the gastrointestinal tract or on themicrovilli of the mucosal cell, the chelate is presentedto the mucosa1 membrane and absorbed i ntact fro~ )thelumen into the mucosal cell as shown in Figure 3. 2

,tM I NJ .Ale I 0 O'ELATE

Fi gure 3. The i ntest ina1 absorpt i on of an ami noacid chelate.

The absorption of an intact amino acid chelate, asshown in Figure 3, is, to a degree, dependent upon themolecular weight of the chelate. To assume that achelate of any size could be absorbed, intact, throughthe mucosal membrane is, of course, ludicrous. Thatwould be akin to thinking that an undigested portion ofliver could be absorbed into the intestinal cells,i~tact because it contained chelated iron. The amino


acid chelate must have a molecular weight of less than1,500 da110ns to initially comply for intactabsorption. 2) Similarity to molecules recognized byspecific carriers also plays a part in the improvedbioavailability of the amino acid chelates as in thecase of dipeptide and tripeptide-like chelates beingsimilar to the small peptides which the mucosal cell isalready accustomed to. Larger molecules requiredigestion before absorption, and digestion results in aloss of the chelate benefits.

Once absorbed, the fate of the amino acid chelateis varied. Because the amino acid chelate is a selfcontained and protected organic molecule which iscompatible with biological processes, it can betransferred, intact, into tissues and enter into manymetabolic processes without further degradation. Thisis very evident in cases of amino acid chelates beinggiven to gestating animals. The amino acid chelatedmineral are transferred across the placenta and into thefetus as intact molecules, because their size and formis compatible with other small molecules which arelikewise, not impeded (i.e. sugars, amino acids, etc.).The result is the offspring are born with higher tissuemineral levels, including(hjgher hemoglobi? levels, wheniron amino acid chelates 13 are provided. 11)

The delivery of greater quantities of a particularmineral to specific sites in the body is also evidenceof this intact absorption and translocation of the aminoacid chelates. Since minerals are utilized as essentialenzyme cofactors in many necessary systems of the body,the resul ts of thi s predi spos it ion to greater absorpt ionare evident in the marked improvements of those systems.To illustrate, an enhanced immune response has beendemonstrated when an~mrls diets were supplemented withamino acid chelates. 12 In another study, it was notedthat problems with infertility were significantlyreduced by providin~ pietary supplements containingamino acid chelates. 13 In all of these above cited


cases as well as others included in this book, thehigher deposition of the supplemented minerals withinthe targeted tissue necess i tated that the ami no ac idchelates be maintained as intact molecules in order toeffect the transfer rather then being broken and havingtheir metal components bound by standard transfer agentsin the body. The exact mechanism for the transfer of alarge quantity of a specific amino acid chelate to atarget tissue has not yet been defined. Nevertheless,when equivalent amounts of metal salts are administered,that portion which is absorbed is diffused throughoutthe body and not concentrated in a specific tissue ascan occur with the amino acid chelate. The amino acidche1ate, therefore responds to a separate and un iquemetabolism which is different from that of absorbed freemetal cations.

Besides being utilized as an intact amino acidchelate, degradation of the chelate to the amino acidligands and the metal can occur in the tissue of usage.Anderson demonstrated, while working with iron chelates,that the iron binding capacity in~ was considerablydifferent from that in vitro. Consequentlydegradation models developed in vitro may not beapplicable in the animal. For example, in vitro certainche1ates do no degrade. But once absorbed they aremetabolized and function differently than in vitromodels would predict. This concept is clearlydemonstrated in numerous studies where feed conversionrates are enhanced, and where greater weight gains areachieved. In these cases, the amino acid chelate wasabsorbed and subsequently delivered the mineral into thebody. Degradation occurred at the point where needed,with the mineral from the absorbed amino acid chelatebei ng incorporated into enzyme systems essent i a1 toeffect move immediate changes in the animal'smetabolism. These metabolic changes in the animal couldnot have occurred if the chelate had not degraded thusallowing the metal to become a co-factor in a specificenzyme necessary to accomplish that metabolic function.


The degradation of the absorbed amino acid chelate isprobably due to a higher stabi 1i ty constant for theligand portion of the enzyme which requires the metalcation that the stability constant of the originalabsorbed amino acid chelate. In the cells of the body,the stability constant of the amino acid chelate isfrequent1y mod i fi ed by changes in pH as the che1atemigrates a1rRss the membranes of the cell and itsorganelles. 22 A pH change in the environment where aspecific enzyme ligand requires a cation could inducethe amino acid ligand to donate its cation and degradeintracellularly.

There are millions of ligand molecules in ananimal's body. The absorbed minerals are found attachedto one, two, or many ligands (within the limits ofsterochemistry) in order to create the biologicalsystems essent i al for the 1i fe process. Whenfunctioning in enzymes, which is generally where thenutritionist sees the most radical changes in animalperformance as a result of mineral supplementation, theabsorbed mi nera1s become part of meta11 oenzymes andmetal-act i vated enzymes. The metal and the protei n(which functions as the ligand) are covalently combinedin the metalloenzymes. Enzymatic activity is lost orretarded when the metal is experimentally removed fromthe protei n. In the case of the metal act i vatedenzymes, the metal and the protein ligand are reversiblycombined. In that situation, one metal may be replacedby certain other metals which ~il~ block, accelerate orretard the enzymatic activity. 15

Since these metal-conta in i ng enzymes representultra-efficient biological catalysts that are producedwithin living systems, the feeding of highly availableamino acid chelates generally results in greater enzrm~

synthesis and, hence greater enzymatic activity. 16

This results in significantly enhanced performance fromthe animals.


The bodies of animals also contain millions ofother unique protein molecules, besides those used forenzymes, all of which are excellent ligands for metalions. The primary structure is probably thepolypeptide. These protein molecules form ternarycompl exes wi th the metal s then conferri ng enzymat i cactivity and becoming involved in the transport andstorage of other biologically active molecules.Increased superoxide di smutase act i vi ty is one suchbenefit rTs~lting from the intake of certain amino acidchelates. 17

Normally, one would anticipate that greaterabsorption of the mineral equates to greater potentialfor toxicity. Toxicity is, to a degree, a function ofthe 1igand that bi nds the mi nera1. In 1etha1 dosestudies, it was found that the amino acid chelates weresignificantly less toxic than 1o~responding amounts ofmetals in the form of salts. 19 Long term, multigeneration histopathological studies with amino acidchelates produced ?o)abnormalities in the tissues of theanimals examined. 20 Thus, within the context ofnutritive practices, the amino acid chelates areconsidered a safe source of highly available minerals.

In summary, Kratzer and Vohra wrote, "the dietaryrequirement for a mineral may be greatly reduced by theaddition of a chelating agent to a diet. A practicalquest ion ari ses about whether one shoul d correct amarginal mineral deficiency in a diet by adding themineral [as a salt], a chelate [as a ligand], or achelated mineral. While it might be possible to use thechelate, or chelated mineral, it is a matter of relativecosts that will influence the decision. In most cases,the cost of the mineral supplement itself( i~ less thanthat of the che1ate or che1ated mi nera1." 21

The observations of these two professors iscorrect, and as the data contained within this bookdemonstrate, if the decision to use amino acid chelates


or metal salts in animal feeds is based on a completeassessment of economics, the decision must result inchoosing the amino acid chelates. Based on thestatistically analyzed animal performances, reported inthe pages of this book, the return on investment is fargreater wi th the ami no ac id che1ates when than forcomparable metal salts. Supplementing with amino acidchelates is more cost effective, yielding greaterprofits per unit spent.


References

1. Morgan, G. and Drew, H., "Researches on residualaffinity and coordination. II Acetylacetones ofselenium and tellurium," J. Chern. Soc., 117:1456,1920.


3. Mellor, D., "Historical background andfundamental concepts," in Dweyer, F. and Mellor,D., eds., Chelating Agents and Metal Chelates(New York: Academic Press) 1-50, 1964.

4. Kragten, J., Atlas of Metal - Ligand Equilibriain Aqueous Solution (Chichester: Ellis HorwoodLtd) 1978.

5. Seven, J., ed., Metal Binding in Medicine(Philadelphia: J. B. Lippincott Co) 1960.

6. Vohra, P. and Kratzer, F., "Influence of variouschelating agents on the availability of zinc," J.Nutr., 82:249, 1964.

7. Miller, R., "Chelating agents in poultrynutrition," Presented at Delmarva Nutrition ShortCourse, 1968.

8. Miller, J., "Chelation", privately printed, 1960.

9. Ashmead, H., et ~., "Chel ation does notguarantee mineral metabolism," J. Appl. Nutr.,26:7, Summer, 1974.


10. Ashmead, H. D., IIA peptide dependent intestinalpathway for the absorption of essentialminerals,1I in Southgate, D., et li., eds.,Nutrient Availability: Chemical and BiologicalAspects (Cambridge: The Royal Chemical Society)123, 1989.

11. Ashmead, D. and Graff, D., "Placental transportof chelated iron,1I Proc. Int. Pig Vet. Soc.Congress, Mexico, 207, 1982.

12. Coffey, R., "Predisposition to disease: Theinter-relationship of the bovine immune responseand trace element physiology," Paper given atNational Cattleman Assoc., 1986.

13. Manspeaker, J., et li., "Chelated minerals: Theirrole in bovine fertility," Vet. Med., 82:951,1987.

14. Anderson, W., IIIron chelation in the treatment ofCsoley's anemia," in Martell, A., ed., InorganicChemistry in Biology and Medicine (WashingtonD.C.: American Chemical Society) 140, 1980.

15. Schuette, K., The Biology of the Trace Elements(Philadelphia: J. B. Lippincott Co.) 17, 1964.

16. Maletto, S., "Studies on the nourishing actionmodel in the protalosates," Unpublished, 1982.

17. Coffey, R., et li., "Clinical data onimmunoglobulin serum levels, trace elements infeedstuffs and liver copper levels in clinicallyill cattle," Unpublished, 1982-1985.

18. Williams, D., The Metals of Life (London: VanNostrand Reinhold) 1971.


19. Larson, A., "L.D. 50 Studies with ChelatedMinerals," in Ashmead, D., ed., Chelated MineralNutrition in Plants, Animals, and Man(Springfield: Charles C Thomas) 163, 1982.

20. Jeppsen, R., "Assessment of long-term feeding ofchelated amino acid minerals (Metalosates@) insows), Unpublished, 1987.

21. Kratzer, F. and Vohra, P., Chelates in Nutrition(Boca Raton: CRC Press, Inc.) 158, 1986.

NAME INDEX

A

~bion Laboratories, Inc., X, 86, 106,107, 207, 457Ash~,H.DeVVayne,21,47,207,457

Ash~, Harvey H., 413~,~, 269, 291, 380

B

Biti, R Ricci, 243Boling, James A, 187BoIsi, DanielIe, 330Bonomi, Alberto, 302, 330, 365Brigham Young University, 142

c

Gagliero, Germano, 76, 86, 349Coffee, Robert T., 117Cornell University,331Corradi, Fulvia, 170Cuitun, Louis, 318Cuplin, Paul, 413

D

Dameley, A.H., DVM, 251

F

Ferrari, Angelo, 349Forfa, Richard J., 393Formigoni, Andrea, 170

G

Guillen, Eduardo, 318

H

Hardy, Fbnald W., 424Herrick, John B., 3Hildebran, Susan, 400Hunt, John, 400

473

Iowa Sta1e ~iversity, 216, 217Iwahasi, Yoshito, 440

J

J. Bibby Agricultural LTD., 274Jeppsen, Robert B., 106

K

Kropp, J. Fbbert, 153

L

Lucchelli, Luigina, 302

M

MaIetto, Silvana, 76, 86Manspeaker, Joseph E., 140Michigan State University, 212, 213,236Miller, John, 461Miller, R F., 460Ming Uan, Feng, 231

o

Oklahoma State University, 150, 159

p

Parisini, Paoli, 170, 243

a

Quarantelli, Afro, 330, 365

R

Fbbl, Martin G., 140, 393


s

Sabbiono, Alberto, 302, 330, 365Sacchi, C., 243Shearer, Karl D., 424Superchi, Paola, 302, 330, 365Suzuki, Katsuhio, 440

T

Takatsuka, Takehuru, 440

u

University of Bologna, 170, 243University of Chile, 21University of Ilinois, 225University of Kentucky, 187, 197University of Maryland, 140, 144, 393

University of Parma, 302, 330University of Perugia, 191, 192UnNersityofTurin,76,86,328University of Washington, 424

v

Volpelli, LA, 243

w

Wakabayashi. Takaaki, 440Werner, Alfred, IX

x

Xian-Ming, Cao, 231

v

Van Ping, Zhou, 231

z

ZUnino, Hugo, 21

INDEX

A

Absorption, (see Intestinal Absorption)~kaline Phosphatase, 353Aluminum, 125, 141, 126, 142, 190American Association of Feed Control

Officials (MFCO), X, XI, 51Amino Acid OleIate (see Chelation)Amino Acid Complex (see Complex)Amylase, 192Anemia, 28-30, 223-225, 231,236,239Arsenic, 190Ascorbic Acid, (see Vitamin C)ATP, 3, 24, 80, 190Average Daily Gain, (see Growth)

B

Backfat, (see Carcass)Bioavailability, (see Intestinal Absorption)Biotin, 33, 416, 426Birth Weight, 207, 213, 215, 218Blood~um, 352, 356, 406, 428, 432Erythrocyte, 164Hemoglobin, 8, 29, 30, 70, 71, 110,

162, 190,213, 231,232, 234, 235,236, 237, 239, 449, 450, 453, 465

Hemosiderin, 223lymphocytes, 124Phosphorus, 352Plasma Iron, 213Potassium, 352Serum, 155, 158Sodium, 9-16, 352Transferrin, 8, 29, 171, 212

Boron,36Breeding~ption (rates), 142, 146, 159,

166-167, 182, 246, 248, 262, 393,395, 397, 398

Embryonic MortaJity, 147, 150,393EndometriaJ Scarring, 140, 141, 149End~s, 148, 150

475

Estrus, 141, 144, 146-148, 159, 165-167, 243, 245-249

Fertility, 140, 153, 249Gestation, 216~ 29, 421, 449Inseminations, 183Periglandular Rbrosis, 141, 149, 150Pregnancy, 141, 159, 177, 182-185,

209,211,227,243,249,252-253,265,393, 405

Reproduction/Reproductive, 170, 176,182-185, 244, 246, 266, 393, 396

Retained Placentas, 184Brittle Bone Disease, 349Butterfat, (see Milk)

c

Cadmium, 10-16, 26, 406~um, 4, 9-16, 22, 24, 26-28, 32-36,

49, 67-68, 96, 154, 158, 171, 188-189,197,200,208,293,305,331,381,424425, 433-435~um Amino Acid Olelate, 67-68,292,

403, 414, 417, 463~ydrates, 22, 47, 49, 76, 187, 197Carcass, 20Q

Backfat, 201, 259, 260-261, 266, 269270, 274, 280-282Fat Over Rib, 201Grade, 201liver Copper, 405, 407liver Iron, 214Marbling, 201Quality, 194-195

Cartilage, 405Catalase, 162Cataracts, 424, 432Catarrhal Entrotyphlitis, 352Cellobiose, 82-83Cellulase, (see Fiber)Chelation, VH, 6-7, 49-51, 106, 457

Amino Acid Chelate(s), VIII, Xl, 51-59,


82-83,86,93-99, 103, 106-111, 133,145, 149-150, 157, 159-161, 165167, 171, 180-185, 191-197, 201,223, 251, 291, 299, 305, 311, 319,324, 346, 361, 370, 376, 398, 407,457, 462-468Minerals CheIated to knino Acids,

86, 413Amino Acid CheIated Mineral, 244Diethytenetriaminepentraacetic Acid

(DTPA), 303EDTA, IX, 8, 106,303,425,433-434,

457, 458Molar Ratio, Xl, 51Molecular Weight, XI, 17, 51,54,70,

212, 251, 464-465Stability Constant, 17, 55, 106,433,

458-464, 467Choline Q-aoride, (see Vitamins)Chromium, 3, 190Cobalt, 4,8-16,25-26,30, 100, 142,

170,190,302,305,308,335,365,368Cobalt Amino Acid Chelate, 81,87,90,

107-108, 158, 176, 192, 197-198,245,302,312,320,336,357,359,367,395,404, 414, 417

Collagen, 405Complex, Amino Acid, 108Conception (Rates), (see Breeding)Copper, 9-16, 23-26, 29, 32, 36, 52-53,

100, 119, 126, 135, 142-143, 150, 153155,160-165,170-171,190,302,305,308, 330, 335, 365, 368, 403-406

Copper Amino Acid Chelate, 38,40,81,87, 90, 107-108, 132-134, 144, 158,161-162, 165, 167, 176, 192, 197-198,245,292,294,302,312,320,336,357,359,367,395, 403-405, 414, 417

o

D-Calcium Pantothenate, (see Vitamins)Deficiency

Copper Deficiency, 405Iron Deficiencies, 405

Diethytenetriaminepentaacetlc Acid(DTPA), (see Chelation)

Digestion,201

~, 76, 79~, 76, 79DNA, 188

E

EeIs,440,EDTA, (see Chelation)Egg~), 300, 358, 384, 372Egg Production, 365, 380, 384Bastin, 405Embryonic Mortality, (see Breeding)Endometrial Scarring, (see Breeding)Endometritis, (see Breeding)Energy, 188, 247

Metabolizable Energy, 92-97, 101-102Enteritis, 350Epiphysitis, 400Equine Organic Iron Supplement, 403,

405Erysipelas, 224Erythrocyte Superoxide Dismutase, 164,

468Estrus, (see Breeding)Ethylenediamine tetraacetic Acid (EDTA),

(see Chelation)

F

Farrowing, 215-216, 251-252Fat Over Rib, (see Carcass)Fatty Acids, 274Feed Conversion(s), 193-197, 279-281,

303, 309-310, 318, 326-327, 333, 341,352, 373, 383, 386, 415, 421-422, 430,443, 445, 448, 453

Feed~, 225, 274, 413FemoraJ Head Necrosis, 349Fertility, (see Breeding)Fiber, 34, 49, 53

Cellulase, 192Auorine, 22, 190Folic Acid, (see Vitamins)

G

Gestation, (see Breeding)

Index 477

Growth (rates), 188, 194, 196,207,227,232, 237-239, 279, 303, 309, 318, 340,346, 376, 424, 443, 453Average Daily Gain, 199Weaning weight, 166,219weight Gain, 193, 199, 323, 369, 375,

445Glucose, 353Glutathionine Peroxidase, 162

H

Hatch Weight, 293Hematocrit, (see Blood)Hemoglobin, (see Blood)Hemosiderin, (see Blood)HstopathoIogy, 110Hyperplasia, 187Hypertrophy, 187

Immunity, 358, 358, 359Immune Response, 117-118, 122,

125-126, 130, 135, 361,465Immune System, 117Immunoglobulin, 122-127IgA, 123, 126IgO, 123, 125-126IgE, 123, 126IgG, 123, 125-126, 129-13119M, 123, 125-126, 129-131V~nation, 119, 121, 122, 128

Infectious Stunting, 349Inositol, 441Insulin, 3Intestinal Absorption

Absorption, 6, 21,28,47,51, 59, 171172, 184, 201, 208-209, 212, 227,285, 293, 299, 303, 357, 361-362,365, 377, 380-381, 407, 415, 433434, 461-466

Availability, 282, 432, 460Bioavailability, 157, 299, 302, 324,

328, 385, 435Iodine, 100, 158, 190, 305, 330, 335,

403

Iron, 8, 9-16, 24-29, 32, 36, 40, 52-53,59, 70, 96, 100, 126, 143, 170-171, 190,207-212,216,222-227,231-235,246-248,293, 302, 305, 330, 335, 365, 406

Iron Amino Acid 0leIate, 38,58,69-71,81,87, 90, 107-108, 144, 176, 192, 197198, 207-221, 225-227, 231, 233, 239,245, 249, 251, 266, 292, 302, 312, 320,336, 357, 359, 367, 395, 403-404, 414,417, 440, 463

~on Dexban, 215, 218, 219, 224

L

Lactase, 82, 83~, 134, 179, 180, 181, 184, 185

Milk Production, 170, 178, 180, 183,185

LO-SO, (see Toxicity)Lead, 8-16, 30, 119, 126Upid(s), 22, 25, 33, 47, 49, 53, 187,381Uver Copper, (see Carcass)Uver Iron, (see Carcass)Lymphocytes, (see Blood)

M

Magnesium, 9-16, 22-27, 34-36, 40, 5253, 80, 90, 142-154, 161-162, 189, 195,197,212,275,305,331,380,381,424

Magnesium Amino Acid Chelate, 38, 87,107-108, 144, 158, 161-162, 197-198,245, ~385, 395, 403-404, 414, 417

Malabsorption Syndrome, 349Maltase, 82-83Manganese, 9-16,24,26-27,30,36, 66

67,100, 126, 135, 142-143, 149-150, 159162, 170, 190, 197, 274, 276, 283, 285,293,302,305,308,331,335,365,368

Manganese Amino Acid Chelate, 66-67,81,87,90, 107-108, 132-134, 144, 158,161-162, 176, 192, 196-198, 245, 274283, 292, 294, 302, 312, 320, 336, 357,359, 367, 395, 403-404, 414, 417, 463

Manganese Gluconate, 278Marbling, (see Carcass)Metabolism, 201, 227Metabolizable Energy, (see Energy)


Metal Proteinate(s), lX, 51, 54Metalloenzyme, 9, 23-24, 30, 135, 467Milk, 173, 175, 177, 179, 181

Butterfat, 1n, 180, 181, 182Milk Production, (see Lactation)~urn, 15, 24, 26, 32, 36, 159-

160, 190,331,406Monosaccharides, 76, 79Morbidity, 132, 135,221-222Mortality, 132, 135,217-221,223,227,

247-258,266, 291,293,295,298-299,309,310,333,352,357,360,383,386,415,421,424Stillbirth, 246-255

Mucosal Cell, 54-58, 63, 66, 461, 462,464

Muscle Iron, 213Myocarditis, 352

N

Niacin, (see Vitamins)Nickel, 143, 190

o

Osteochondrosis lesions, 406Osteodysgenesis, 406Osteogenesis, 200Osteomyelitis, 351Osteoporosis, 349Oxalic Acid, 5, 34, 172

p

Pale Bird Syndrome, 349Pantothenic Acid, (see Vitamins)Pastern, 403, 405Pathology, 72Periglandular Fibrosis, (see Breeding)Peroxidoxine, (see Vitamins)Phosphorus, 3, 4, 22, 24-26, 34, 36,

142, 153, 188-189, 197,200,209,330,365,381,403,424-425,428,434

Phosphorus Amino Acid Complex, 403Phytic Acid, 5, 27,34, 171-172, 432

433, 435Placenta, 236, 465

Placental Barrier, 231PIacentaIT~, 69, 219, 223, 232Placental Transport, 70, 211

Plasma Iron, (see Blood)~, 76, 78, 189Potassium, 9, 26, 33-36, 80, 108, 143,

161, 189, 197,223,305,331,424Potassium Amino Acid Complex, 144,

158, 161,245, 395, ~404Pregnancy, (see Breeding)Propionic Acid, 102~n, 22, 25, 47, 49, 187, 214, 244Protein Metabolism, 88, 90, 196, 343

Protein Sparing, 88-89, 93-94, 97, 101102, 199-201, 346

Protein Synthesis, 103, 104, 188Proteinase, 90Purine, 214, 225Pyridoxine, (see Vitamins)

R

Rachitis, 406~rus, 354, 356, 358, 358, 362Replamin Extra Breeder Pac, 404Reproduction, (see Breeding)Retained Placentas, (see Breeding)Rboflavin, (see Vitamins)RNA, 190, 191Rumen Bypass, 145, 171, 463Runt Pigs, 216,

s

Saccharase, 82, 83Selenium, 24, 154, 158, 161, 191Silicon, 22SOD

Copper Superoxide Dismutase, 162165

Zinc Superoxide Dismutase, 162-165Sodium, 9-16, 26, 35, 80, 142, 189, 305,

424Soil Copper, 406Spleen Iron, 213Stability Constant, (see Chelation)Stillbirth, (see Mortality)Strontium, 190

Index 479

Sulfur, 22, 32, 36, 142, 189, 197, 406Superoxide Dismutase, 164

T

Tendon Contracture, 400Tenosynovitis, 354Teratogenic Bfects, (see Toxicity)Thiamine, (see Vitamins)Thyro~n, 223, 283, 284Tin, 190To~, 71, 145,406,413,415,468

Copper Toxicity, 405LD-SO Studies, 72Teratogenic Bfects, 107, 111

Transferrin, (see Blood)Trehalase, 82-83

v

Vaccination, (see Immunity)Vanadium, 23, 24, 191Vitamins, 22, 47, 187

Ascorbic Acid, 32, 142,416,426,441Choline Chloride, 96, 308, 321, 335,

355, 368, 403, 416D-Calcium Pantothenate, 416, 426Folic Acid, 33, 335, 368, 403, 426, 441Niacin, 32, 99-100, 158, 244-245, 308,

321,355,368,403,416,426Pantothenic Acid, 33, 96, 244, 308,

321,335,355,368,403,441Peroxidoxine, 308Pyridoxine, 33, 96, 99, 308, 335, 368,

416, 426, 441Riboflavin, 33, 96, 99, 244, 308, 321,

335, 355, 368, 403, 416, 426, 441Thiamine, 33, 96, 99, 188, 308, 335,

403, 416, 426, 441Vrtamin A, 96, 99-100, 142, 158, 160

161, 245-246, 308, 321, 335, 355,368,416,441

Vitamin 81, (see Vitamin, Thiamine)Vrtamin B2, (see Vitamin, Riboflavin)Vrtamin 86, (see Vitamin, Pyridoxine)Vitamin 812, 4, 33, 96, 99, 188, 190,

321,335,368,403,416,426,441

Vitamin C, (see Vitamin, Ascorbic Acid)Vitamin 0, 32, 96, 160-161, 171, 335,

355, 356, 381Vitamin 03, (see Vitamin, Niacin)Vitamin E, 99-100, 142, 158, 160-161,

245,321,355,368,403,416,441Vitamin K, 96, 99, 245, 321, 368, 441

w

Weaning Weights, (see Growth Rates)weight, 208, 333, 341, 352, 443

z

Zinc, 8-16, 26-27, 30, 34, 36, 52-53, 6065, 100, 119, 126, 133, 135, 143, 150,154, 158-162, 191, 197, 222, 235, 302,305, 308,331, 335, 368, 406, 424, 427428,431-435

Zinc Amino Acid Chelate, 38, 40, 60-65,81, 87, 90, 107-108, 132-134, 144, 158,161-162, 176, 197-198, 245, 274, 294,302, 312, 320, 336, 357, 359, 367, 395,404, 414, 417, 425-427, 431-435, 463

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