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Amino acid supplementation of Peace River barley for growing-finishing pigs 1973

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C I AMINO ACID SUPPLEMENTATION OF PEACE RIVER BARLEY FOR GROWING-FINISHING PIGS by AN SIK CHUNG B.S.A., Kon-kuk University, 1966 A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE i n the Department of ANIMAL SCIENCE We accept t h i s thesis as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA March, 1973 In presenting t h i s thesis i n p a r t i a l f u l f i l m e n t of the require- ments f o r an advanced degree at the University of B r i t i s h Columbia, I agree that the Library s h a l l make i t fr e e l y available f o r reference and study. I further agree that permission f o r extensive copying of t h i s thesis f o r scholarly purposes may be granted by the Head of my Department or by h i s representatives. I t i s understood that copying or publication of t h i s thesis f o r f i n a n c i a l gain s h a l l not be allowed without my written permission. Department of Animal Science The University of B r i t i s h Columbia Vancouver 8, Canada Date M^e^J <?J ^ /fyS ABSTRACT The work reported i n t h i s thesis comprised two sections, each of which compared s i x rations i n pigs i n the body weight range 16-85 kg. A l l rations were based on barley. A supplement of L-lysine HC1 was added to provide a t o t a l l ysine content of 0.90% and,0.75%, with the l a t t e r l e v e l also tested with 0.05% added L-threonine, alone or with 0.10% DL- methionine or 0.10% DL-methionine plus 0.10% L-isoleucine. A control ra t i o n consisted e s s e n t i a l l y of barley and soybean meal to provide 0.75% t o t a l lysine. These rations were used f o r growth and nitrogen balance experiments. The supplementation of barley with 0.50% L-lysine HC1 (0.75% t o t a l lysine) tended to improve nitrogen u t i l i z a t i o n more than a supplemental l e v e l of 0.69% L-lysine HC1 (0.90% t o t a l l y s i n e ) . The b i o l o g i c a l value of the r a t i o n containing the lower l e v e l of lysine was s i g n i f i c a n t l y higher than that of the ration containing the higher l e v e l of l y s i n e , which was r e f l e c t e d i n a trend i n d i c a t i n g that pigs fed the lower l e v e l of lysine were leaner and had larger eye muscle areas than those fed the higher l e v e l of ly s i n e . Adding 0.05% L-threonine to the lower l e v e l of lysine improved growth performance but not carcass quality. The addition of L-lysine and L-threonine to the barley tended to improve nitrogen balance above that obtained by the addition of L-lysine alone. The further addition of 0.10% DL-methionine with or without 0.10% L-isoleucine did not improve growth performance but gave a further s l i g h t non-significant improvement i n nitrogen metabolism measurements. Therefore, both the feeding experiment and nitrogen balance experiment indicated that threonine was the second l i m i t i n g amino acid a f t e r l y s i n e . Supplementation of barley with amino acids regardless of levels or combinations, gave s i g n i f i c a n t l y poorer d a i l y gain, feed e f f i c i e n c y and carcass q u a l i t y results but s i g n i f i c a n t l y higher b i o l o g i c a l value figures than the barley-soybean control r a t i o n . Thus, i t would appear that the amino acid-supplemented rations s t i l l were d e f i c i e n t i n some way, although the b i o l o g i c a l value data would tend to contradict the suggestion that t h i s i s associated with an amino acid imbalance. TABLE OF CONTENTS Page ACKNOWLEDGEMENTS i LIST OF TABLES i i LIST OF FIGURES i i i I. INTRODUCTION 1 I I . REVIEW OF LITERATURE 2 A. ESSENTIAL AMINO ACIDS AND NONESSENTIAL AMINO ACIDS 2 B. AVAILABILITY OF AMINO ACIDS IN THE RATION 3 C. AMINO ACID DEFICIENCY, EXCESS AND INTERACTION 5 D. METHODS OF DETERMINING AMINO ACID REQUIREMENT 7 E. THE USE OF AMINO ACIDS IN PIG NUTRITION 11 F. AMINO ACID REQUIREMENT FOR GROWING-FINISHING PIGS 13 a. The Requirement f o r Lysine 14 b. The Requirement f o r Methionine 16 c. The Requirement f o r Tryptophan 18 d. The Requirement f o r Threonine 19 e. The Requirement f o r Isoleucine . 20 G. SUPPLEMENTATION WITH AMINO ACIDS OF GRAIN-PROTEIN CONCENTRATE RATIONS AND GRAIN ONLY 21 H. FACTORS AFFECTING AMINO ACIDS SUPPLEMENTATION OF FEEDS 25 a. Energy Content of the Rations 25 b. Protein Content of the Rations 28 c. Age and Sex of the Species Consuming the Rations 31 I I I . EXPERIMENT I 33 A. EXPERIMENTAL PROCEDURE 33 a. General 33 Page b. Animals 33 c. Rations 34 d. Management 34 ( i ) Housing ' 34 ( i i ) Feeding and Watering 35 ( i i i ) Feed Storage and Mixing 35 e. Records 35 f. Chemical Analysis 35 g. Carcass Measurements 36 h. Calculations 37 i . S t a t i s t i c a l Analysis of Data 37 B. RESULTS 41 C. DISCUSSION 45 D. CONCLUSION 48 IV. EXPERIMENT I I 59 A. EXPERIMENTAL PROCEDURE 59 a. General 59 b. Animals 59 c. Rations 59 d. Management 59 ( i ) Housing 59 ( i i ) Feeding and Watering 60 ( i i i ) Mixing Rations 60 e. Feces and Urine Collection 60 f. S t a t i s t i c a l Analysis of Data 61 B. . RESULTS 64 C. DISCUSSION 67 D. CONCLUSION 71 V. GENERAL CONCLUSIONS AND RECOMMENDATIONS 73 VI. APPENDIX 86 i ACKMOWL£TJGEMENT The w r i t e r wishes to express his gratitude to Dr. R.M. Beames of the Department of Animal Science f o r his constant encouragement and guidance throughout the course of t h i s study. Thanks are also extended to Dr. W.D. K i t t s , Chairman of the Department of Animal Science, f o r the use of necessary f a c i l i t i e s f o r t h i s study. I also wish to express my gratitude to Mrs. L. Mather f o r her assistance i n carrying out the amino ac i d analyses. LIST OF TABLES i i Table Page I. Composition of rations used i n Experiments I and I I . . . . . 38 I I . Content of es s e n t a i l amino acids and proximate constituents of barley and soybean meal on a dry matter basis 39 I I I . Content of es s e n t a i l amino acids of each ration (g amino acid/lOOg mixed feed) on an air-dry basis 40 IV. Summary of effects of the addition of amino acids to barley on body weight gain, feed consumption, feed conversion r a t i o and carcass measurements i n the feeding t r i a l 43 V. Comparison of the effects of supplementation with amino acids on d a i l y weight gains and carcass measurements of g i l t s and barrows 44 VI. A l l o c a t i o n of pigs within replicates and periods f o r Experiment I I 62 VII. Summary of the effects of supplementation of barley with amino acid on apparent dry matter d i g e s t i b i l i t y , nitrogen balance, apparent nitrogen d i g e s t i b i l i t y , and on nitrogen retained as a percentage of nitrogen intake, and as a percentage of nitrogen absorbed (average 7-day figures) . . . 66 LIST OF FIGURES i i i Figure Page I. The effe c t of supplementation with lysine and threonine of a barley ra t i o n (Treatment 4) compared to a barley-soybean control r a t i o n (Treatment 1) on mean body weight from the s t a r t u n t i l f i r s t pigs sent f o r slaughter , 55 I I . The e f f e c t of supplementation with the higher l e v e l of lysine (Treatment.2) and the lower l e v e l of lysine (Treatment 3) on mean weight gain from s t a r t u n t i l f i r s t pigs sent f o r slaughter 56 I I I . The ef f e c t of supplementation with the higher l e v e l of lysine (Treatment 2), the lower l e v e l of lysine (Treatment 3), lysine plus threonine (Treatment 4) and soybean control r a t i o n (Treatment 1) on feed e f f i c i e n c y r a t i o from s t a r t u n t i l f i r s t pigs sent f o r slaughter 57 IV. General view of the metabolism cage 63 V. Pig shown i n the cage which could be adjusted f o r the size of the p i g 63 1 I. INTRODUCTION I t i s w e l l known that cereal protein has a low n u t r i t i o n a l value, p r i m a r i l y due to a r e l a t i v e l y low l e v e l of the es s e n t i a l amino acids, l y s i n e , methionine, threonine, tryptophan and isoleucine. Pig rations are normally based on these cereals. I t i s therefore necessary that a supplement provide adequate levels of these l i m i t i n g amino acids. T r a d i t i o n a l l y , the protein of grain i s supplemented with high quality protein meal of plant or animal o r i g i n to increase the t o t a l protein content and to counteract any ess e n t i a l amino acid deficiency i n the basal rat i o n of the growing pig. The t o t a l or p a r t i a l replacement of protein supplement by synthetic amino acids could reduce the cost of a ration. The importance of the lysine content of the diet i s w e l l estab- l i s h e d and there are many examples i n the l i t e r a t u r e of the improvement i n performance that can be brought about by lysine supplementation of cereal rations. I t has been reported that supplementing cereal mixtures with l y s i n e , threonine, tryptophan and i n some experiments also with isoleucine results i n performance equal to that obtained with rations of grain plus protein concentrates. The present study was undertaken to examine the effects of adding two levels of lysine with or without threonine, methionine and isoleucine to a basal ra t i o n of Peace River barley i n terms of d a i l y gain, feed con- version effeciency, carcass q u a l i t y and nitrogen balance. 2 I I . REVIEW OF LITERATURE A. ESSENTIAL AMINO ACIDS AND NONESSENTIAL AMINO ACIDS Amino acid supplementation of feeds i s based on the results of over s i x t y years of research on the n u t r i t i o n a l q u a l i t y of proteins and t h e i r constituent amino acids. Much of the fundamental work was carried out by Osborne and Mendel (1919, 1920) who studied the growth-promoting q u a l i t i e s of i s o l a t e d proteins and recognized that s p e c i f i c amino acids were missing or present i n low concentrations i n those proteins which did not support adequate growth of the animal. Rose. (1938) divided the amino acids found i n proteins into two main categories, the e s s e n t i a l and the nonessential amino acids. Essential amino acids are those which cannot be synthesized by the organism at a rate adequate to meet metabolic require- ments and must be supplied i n the diet. Nonessential i s the term applied to those andno acids which the body can synthesize from normal food con- stituents v i a transamination reactions. I t should be emphasized that t h i s c l a s s i f i c a t i o n i s based on growth studies i n the rat (Rose 1938). Class- i f i c a t i o n of the amino acids with respect to t h e i r growth ef f e c t i n the white rat are given below. Glycine i s not e s s e n t i a l f o r the growing pig but i s e s s e n t i a l f o r the growing chick. The requirement f o r arginine and glycine appears more acute i n the more rapidly feathering breeds of birds (Hegsted et a l . 1941). ESSENTIAL NONESSENTIAL Arginine* Methionine Alanine Hydroxy glutamic acid Hi s t i d i n e Phenylalanine Aspartic acid Hydroxy proline Isoleucine Threonine C i t r u l l i n e Norleucine Leucine ' Tryptophan Cystine Proline Lysine Valine Glutamic acid Serine Glycine Tyrosine * Arginine can be synthesized by the r a t but not at a s u f f i c i e n t l y rapid rate to meet the demands of normal growth. 3 The proportion of e s s e n t i a l amino acids provided by various proteins i s the major factor influencing the b i o l o g i c a l value. The f a i l u r e of young animals to grow on a d i e t deficient i n one or more of the e s s e n t i a l amino acids i s a r e f l e c t i o n of i t s i n a b i l i t y to synthesize adequate quantities of body protein under these experimental conditions. The r e s u l t of an amino acid deficiency i n the d i e t i s that the tissues do not synthesize protein requiring that p a r t i c u l a r amino acid. The terms es s e n t i a l and nonessential relate mainly to dietary requirements and have l i t t l e meaning with respect to the r e l a t i v e impor- tance which amino acids may have i n metabolism. The amino acids which are e s s e n t i a l i n the diet are compounds with carbon skeletons which can not r e a d i l y be synthesized by the body. In a r e a l sense, the so-called nonessential amino acids are of equal significance f o r the economy of the organism which i s p a r t i c i p a t i n g i n diverse c e l l u l a r reactions and functions, and provide precursors f o r the synthesis of many important c e l l u l a r c onsti- tuents. Indeed, certain of the nonessential amino acids, e.g., glutamic a c i d , have so many important metabolic roles that, were a mammal to lose suddenly i t s capacity to synthesize glutamic a c i d , serious disorganization of key reactions of metabolism might r e s u l t as the animal may be unable to wait u n t i l the next meal to replenish i t s supply. B. AVAILABILITY OF AMINO ACID IN THE RATION I t has been stressed that the content of amino acids i n a protein may not accurately r e f l e c t i t s n u t r i t i v e value, since some of the amino acids may not be available to synthesize the tissue of the animal. Problems involved i n determining a v a i l a b i l i t y of amino acids have been discussed. by Grau and C a r r o l l (1958). A considerable number of b i o l o g i c a l and a few chemical procedures have been developed. A satisfactory chemical procedure 4 f o r measuring lysine a v a i l a b i l i t y has been described by Carpenter (1960) which depends upon the reaction of dinitroflurobenzene with the e-amino group of lysin e . The reaction produces a colored lysine derivative and i t can, therefore, be used t o estimate the lysine with the free e-amino group. The results obtained with animal proteins correlate closely with gross protein value determined i n the chick under conditions where the lysine content of the materials i s emphasized. With vegetable protein and high carbohydrate d i e t s , the method i s not as s a t i s f a c t o r y , the f i g - ures obtained being too low because of the destruction of the colored lysine derivative. Moran et a l . (1963) used the reaction of the dye Orange G with basic amino acids to determine the a v a i l a b i l i t y of free amino, amidazole, and guanidyl group of proteins. The dye binding capac- i t y of soybean meal heated f o r varying periods of time was closely related to growth of chicks fed the meal. The most widely used b i o l o g i c a l procedures f o r determining a v a i l a b i l i t y of amino acid are those based on growth of rats (Calhoun et a l . 1960). In t h i s method, increments of the pure amino acid to be tested are added to a basal diet deficient i n the amino acid and a growth response curve i s obtained. The response found with protein-bound amino acids i s then compared with that obtained with the pure amino acid to determine a v a i l a b i l i t y . Oh e t a l . (1972) determined b i o l o g i c a l a v a i l a b i l i t y of methionine i n various protein supplements. Four procedures of bioassay were studied on the basis of slope-ratio.^technique: (a) weight gain versus levels of dietary methionine; (b) e f f i c i e n c y of feed u t i l i z a t i o n versus levels of dietary methionine; (c) weight gain versus t o t a l available methionine consumed; and (d) gain i n weight attributable to intake of t e s t protein methionine versus methionine intake from the test protein. 5 Method (d) was the most precise and r e l i a b l e method as judged by the usual s t a t i s t i c a l c r i t e r i a . Using t h i s method ,the available methionine content of meat, f i s h , blood, rapeseed, soybean and feather meal was estimated to be 91.1, 89.9, 66.3, 87.2, 94.7 and 35.0% respectively. Nielson (1971) showed that the d i g e s t i b i l i t y of amino acids from d i f f e r e n t balanced rations was related to the d i g e s t i b i l i t y of n i t r o - gen i n growing pigs. The results indicated that the d i g e s t i b i l i t y of lysine was not s i g n i f i c a n t l y d ifferent from the d i g e s t i b i l i t y of nitrogen. Lower d i g e s t i b i l i t y c o e f f i c i e n t s were found for methionine, threonine and alanine, whereas the d i g e s t i b i l i t y c o e f f i c i e n t s f o r cystine, v a l i n e , leucine, isoleucine, phenylalanine, tyrosine, h i s t i d i n e , arginine, glutamic acid and aspartic acid were higher than f o r nitrogen. There i s much var i a t i o n i n amino acid a v a i l a b i l i t y from various protein supplements. Amino acid contents should be l i s t e d as available amino acid rather than t o t a l . However, i t i s d i f f i c u l t to show available figures because a v a i l a b i l i t y of amino acids i n many ration components varies from batch to batch, and the method of determining amino acid a v a i l a b i l i t y i s d i f f i c u l t and time consuming. C. AMINO ACID DEFICIENCY, EXCESSES AND INTERACTION Deficiencies, interactions and excesses can reduce growth rate although excesses generally have to be at higher levels than those found i n normal rations before they interfere with growth. The question of amino acid imbalance and antagonism has arisen f a i r l y recently. To explain the term imbalance i t might be appropriate to give the d e f i n i t i o n of a well-balanced protein, which i s "a protein i n which the es s e n t i a l amino acids occur i n i d e a l proportions, and are thus required i n minimal quantities to s a t i s f y the amino acid requirements of the animal". Amino acid imbalance may be defined as a depression i n growth (or any other adverse effect) which results from an a l t e r a t i o n of amino acid balance of the r a t i o n , produced by adding a mixture of amino acids or a protein that lacks one of the es s e n t i a l amino acids. The adverse effect can be counteracted by feeding a small supplement of the most l i m i t i n g amino acid. For example, Harper (1959) mentioned that when gelati n i s added to a casein d i e t , growth may decrease unless tryptothan i s also added. I t i s t y p i c a l of an imbalance that the requirement f o r the most l i m i t i n g amino ac i d i s increased. Antagonism i s described as an excess of a single amino acid that may cause a growth depression and r e s u l t i n an increased need for one or more of the other amino acids. An excess of leucine causes an increase i n the requirement f o r isoleucine. This recognition i s based upon the observation that dietary additions of incom- plete mixtures of amino acids cause a severe growth depression which i s prevented by supplementation of the diet with the amino acids that are most l i m i t i n g (Harper 1958; Deshpande et a l . 1958). In three papers by D'Mello and Lewis (1970 a, b, c ) , evidence has been presented to demonstrate the unique interaction between lysine and arginine, between leucine, isoleucine and valine * and between threonine and tryptophan i n the chick. The re s u l t s indicate that excess dietary lysine increases the arginine requirement of the young chick, and excess threonine increases tryptophan requirement qua n t i t a t i v e l y , while excess leucine increases the quantities of isoleucine and valine required to sustain normal growth. The results of these experiments permit the general conclusion that the requirements of amino acids are interdependent. The unique interaction between lysine and arginine i n chick n u t r i t i o n supports the observations of other authors (O'Dell et a l . 1958; Jones 1964; Smith 7 and Lewis 1966). The interactions between leucine, isoleucine and valine also have been observed i n experiments with rats (Harper et a l . 1954; Benton et a l . 1956; Rogers et a l . 1967). The interaction or i n t e r r e l a t i o n - ship between threonine and tryptophan has been known to occur i n the r a t (Salmon 1954; Morrison et al.. 1960; Florentino et a l . 1962). D. METHOD OF DETERMINING AMINO ACID REQUIREMENT Determination of the n u t r i t i o n a l value of protein i s inherently non-specific. In the case of vitamins or e s s e n t i a l minerals, one can evaluate a single essential nutrient at a time. Conversely, i n the assay of protein value, one must be concerned with, the q u a l i t a t i v e and quanti- t a t i v e adequacy of at least nine amino acids. There are also problems of amino acid balance and u t i l i z a t i o n to be considered. I t i s inevitable that d i f f e r e n t assay methods w i l l d i f f e r i n the c l a s s i f i c a t i o n of proteins as to n u t r i t i v e value. The d i f f e r e n t assay methods have d i f f e r i n g parameters of measurement. Despite the vagaries inherent i n assay procedures, i t i s of interest to note that most methods have c l a s s i f i e d most food proteins i n the same general order of adequacy. The balanced, proteins of meat, milk and eggs have been used as standards of excellence. The major grain proteins are low i n one or more ess e n t i a l amino acids and are generally improved i n feeding value by appropriate amino acid f o r t i f i c a t i o n . Balance can also be achieved, as i n the case of manufactured feeds f o r poultry and swine, by f o r t i f i c a t i o n with a single protein source such as soybean meal. The approximate amino acid needs of several species are known. By a knowledge of the e s s e n t i a l amino acid composition of a protein, one can predict i t s feeding value with a f a i r degree of accuracy. One cannot predict, however, i n e f f i c i e n c i e s i n d i g e s t i b i l i t y or the effects of pro- 8 cessing, which are not generally revealed by composition analyses. Protein evaluation methods, which r e f l e c t true feeding value, continue, therefore, to provide the ultimate i n b i o l o g i c a l evaluation. Indirect methods of protein evaluation confirm and complement the direct feeding methods. Most of the experimental work referred to i n t h i s review has been carried out on the growing animal, mostly with an ad l i b i t u m feeding regime. Growth r a t e , feed conversion and i n many cases carcass quality have acted as the c r i t e r i a i n p i g production f o r the evaluation of r a t i o n adequacy. The performance and carcass q u a l i t y of pigs are influenced not only by the q u a l i t y of the d i e t , but also by the system of feeding. Clausen et a l . (195.9-1964)' i n t r i a l s with amino acids, have used both ad l i b i t u m feeding and r e s t r i c t e d feeding. An interpretation of t h e i r •results i s that the response to both treatments has been about the same. I t i s recommended that any program f o r the evaluation of amino amino acid supplementation include an evaluation of the e f f e c t on the growth of an animal. Carcass measurements have i n some cases added v a l - uable information i n growth experiments. Backfat thickness, eye muscle area and fat-lean r a t i o are a l l correlated to the percentage of lean (or protein) i n the carcass, with a correlation c o e f f i c i e n t of about 0.7 (Clausen et a l . 1959-1964; Marcum et a l . 1961). Although performance i s important i n p r a c t i c a l animal production, weight gain i s an inaccurate c r i t e r i o n of nitrogen retention. Nitrogen balance values are of much greater value i n assessing protein quality. The concept of " b i o l o g i c a l value" was f i r s t introduced by Thomas (1909) i n terms of percent- of digestible nitrogen from a t e s t food which was retained by the human adult. M i t c h e l l et a l . (1945) brought the method to a greater state of precision by using the growing r a t . B i o l o g i c a l 9 value i s a dir e c t measure of the proportion of dietary protein which can be u t i l i z e d by body tissues and may be defined as the percentage of the absorbed nitrogen which i s retained by the animal. A balance t r i a l i s conducted i n which nitrogen intake and urinary and f e c a l excretions of nitrogen are measured. The res u l t s are used to calculate the b i o l o g i c a l value as follows: n„ N intake-(Fecal N + Urinary N) , n n B V = ! A T • -4- i =5 n — M X 1 0 0 N intake-Fecal N The metabolic f e c a l nitrogen and the endogenous urinary nitrogen are not d i r e c t l y derived from immediate dietary nitrogen. The existence i n both feces and urine of nitrogen f r a c t i o n s , the excretion of which i s inde- pendent of dietary nitrogen, i s most convincingly demonstrated by the fact that some nitrogen i s excreted when the animal i s given a nitrogen- free diet. The revised formula i s R y N intake-(Fecal N-MFN)-(Urinary N-EUN) , n n N intake-(Fecal N-MFN) X where MFN = metabolic f e c a l nitrogen and EUN = endogenous urinary nitrogen. In determining b i o l o g i c a l value, the greatest amount possible of the dietary protein should be provided by the protein under t e s t . Protein intake must be s u f f i c i e n t to allow adequate nitrogen retention, but must not be i n excess of that required f o r maximum retention; i f the l a t t e r l e v e l were exceeded,the r e s u l t i n g general amino acid catabolism would depress the estimate of b i o l o g i c a l value. For t h i s reason, s u f f i c i e n t non-nitrogenous nutrients must be given t o prevent catabolism of protein to provide energy. The amino acid mixture absorbed by the animal i s required f o r the synthesis of body proteins. The e f f i c i e n c y with which t h i s synthesis can be effected depends p a r t l y on how clos e l y the amino acid proportions of the absorbed mixture resemble those of body proteins, and pa r t l y on 10 the extent to which these proportions can be modified. The b i o l o g i c a l value of a food protein depends upon the number and kind of amino acids present i n the molecule. Since es s e n t i a l amino acids cannot be effec- r i v e l y synthesized i n the animal body, an imbalance of these i n the diet leads to a wastage. Food protein with a deficiency of any p a r t i c u l a r e s s e n t i a l amino acid w i l l tend to have a low b i o l o g i c a l value. Since b i o l o g i c a l value i s dependent primarily upon essential amino acid make-up, i t would seem l o g i c a l to' assess the n u t r i t i v e value of a protein by a quantitative estimation of i t s e s s e n t i a l amino acid constitution and then compare t h i s with the known amino acid requirement of a p a r t i c u l a r class of animal. To overcome the economic problems associated with b i o l o g i c a l methods f o r describing protein q u a l i t y , two schemes have been proposed, both of which arrive at a numerical value f o r q u a l i t y by considering the r e l a t i v e amount of the amino acids present i n the protein as determined by chemical (or i n some cases b i o l o g i c a l ) analysis. Block and M i t c h e l l (1946) suggested that poor protein q u a l i t y was caused prima r i l y by a r e l a t i v e shortage of some one ess e n t i a l amino acid. They took the composition of whole egg protein as the standard or i d e a l , and determined the percentage by which the amino acid i n greatest d e f i c i t was considered l i m i t i n g . The complement of the percentage d e f i c i t of t h i s amino acid was the chemical score given to that protein. Such values correlate w e l l with the b i o l o g i c a l values f o r the r a t and human beings but not for poultry. They are useful f o r grouping proteins i n t o categories, but suffer a serious disadvantage i n that no account i s taken of the deficiencies of amino acids other than the amino acid i n greatest d e f i c i t . 11 Oser (1951), although approving the general p r i n c i p l e of chemical score as outlined by Block and M i t c h e l l (1946), indicated that a l l essen- t i a l amino acids should be considered rather than the one which i s most deficient with respect to some standard. He developed the essential amino acid index which may be defined as the geometric mean of the esse n t i a l amino acids when each i s expressed as a percentage of the l e v e l of the same amino acid i n egg protein and i s calculated as ^..-r _ n/-100a . 100b , 100c , , 100J / ae be ce je , where a, b, c, ... j = percentage of the esse n t i a l amino acids i n the food protein, ae, be, ce, ... je = percentage of the same essential amino acids i n egg protein, and n = the number of amino acids entering i n t o the calculation. The index has the advantage of predicting the effects of supplemen- ta t i o n i n combinations of proteins. I t has the disadvantage that proteins of very d i f f e r e n t amino acid composition may have the same or a very s i m i l a r index. E. THE USE OF AMINO ACIDS IN PIG NUTRITION In the 1930's the ten amino acids es s e n t i a l f o r promoting normal growth i n the r a t were v e r i f i e d (Rose 1938), and i n the 1940's the quanti- t a t i v e requirements of the 12 ajirino acids needed i n chick growth were v i r t u a l l y established. The f i r s t serious attempt to f i n d the requirements f o r amino acids i n p i g n u t r i t i o n was probably made by Beeson et a l . (1948, 1949), who used a p u r i f i e d r a t i o n i n combination with hydrolyzed f i s h protein f o r young pigs. The ra t i o n was p r a c t i c a l l y tryptophan-free (0.01%). Addition of 0.4% DL-tryptophan caused a marked response i n growth rat e , as the control pigs had no weight gain and pigs fed tryptophan gained 634 g a day during a four-week period. This c l e a r l y demonstrates the 12 significance of tryptophan i n the rat i o n of the growing pig. Work was continued by Beeson and h i s associates at Purdue. More precise than the f i r s t attempt was the next experiment, where graded levels- of DL-tryptophan from 0.1 to 0.4 were added to the basal diet containing 0.01% tryptophan (Shelton 1951a). In t h i s case zein and gelatin were used i n the basal d i e t , which contained 24.5% protein. Although there were only two pigs per treatment, the res u l t s were very consistant, 0.2% DL-tryptophan being s u f f i c i e n t f o r normal growth. At the same time as the Purdue group (Beeson et a l . 1948, Mertz et a l . 1949; Beeson et a l . 1953, Mertz et a l . 1955) were investigating the requirements f o r other amino acids, L o o s l i and h i s co- workers at Cornell ( B e l l et a l . 1950; Brinegar et a l . 1950a and 1950b; Kroening et a l . 1962) performed,extensive studies along the same l i n e s . The t h i r d group of workers that has done much work i n evaluating the quan- t i t a t i v e needs of amino acids i n the growing pigs, i s Becker and h i s associates at I l l i n o i s (Becker et a l . 1955; Becker, 1959 and Becker et a l . 1963). Several other s c i e n t i s t have also carried out such studies. In recent years, f o r instance, comprehensive experiments have been undertaken at Cambridge (Evans, 1958, 1960, 1962, 1963) and i n Copenhagen (Clausen et a l . 1959-1964). Most experiments have been done on weanling pigs (eight weeks o l d ) , but suckling pigs have also been used to some extent. The method frequently used i s to make up a basal ra t i o n of p u r i f i e d carbohydrate-rich ingredients plus a protein that i s de f i c i e n t i n one or more of the naturally occurring amino acids. I t has not been too d i f f i c u l t to f i n d protein low i n tryptophan (hydrolyzed feather meal, z e i n , g e l a t i n ) , lysine (zein, l i n - seed meal, wheat protein ) , isoleucine (blood f l o u r ) , methionine (expeller soybean meal) and threonine (isolated soybean protein, r i c e protein). I f 13 the protein used i s a poor source of other amino acids i n addition to the one i n question, a proper supplement of the others must be given. The sca r c i t y of proteins deficient i n the amino acids not mentioned above has li m i t e d the use of t h i s technique. Consequently, a diet with a l l ten ess e n t i a l amino acids (for the rat) as the sole source of nitrogen, except ammonium c i t r a t e , was successfully developed as early as 1950 by Shelton et a l . for weanling pigs. Experiments with addition of one or more amino acids.to p r a c t i c a l type diets have also been b e n e f i c i a l to our present knowledge of the quan- t i t a t i v e requirement of the dif f e r e n t amino acids (Clausen et a l . 1959- 1964). Another approach to the problem was proposed by Curtin et a l . (1952c). This was based on amino acid composition of the carcasses of the pigs. The lysine requirement, established e a r l i e r , was used as the basis while the requirement of the other e s s e n t i a l amino acids was established i n proportion to the content of lysine i n the body. Studying the amino acid content of pigs at various ages, Durtin et a l . (1952c) found a surprisingly constant l e v e l of the i n d i v i d u a l amino acids, when they were expressed i n percentagesof the t o t a l protein i n the body. In 1952 Mertz et a l . concluded, on the basis of t h e i r own experiments as w e l l as those of others, that the growing p i g , l i k e the growing r a t , needs ten amino acids. The p i g also resembles the rat i n the a b i l i t y to synthesize part, but not a l l of the arginine required f o r normal growth and performance. F. AMINO ACID REQUIREMENT FOR GROWING-FINISHING PIGS I t has been known f o r many years that the protein requirement of animals can be stated more precisely i n terms of amino acid requirements. Pigs require f o r normal growth ten e s s e n t i a l amino acids. Also they should be given additional nitrogen so that t h e i r bodies can synthesize the non- 14 ess e n t i a l amino acids. Amino acid .needs increase as protein l e v e l and c a l o r i c density increase (Bowland 1962; Becker et a l . 1963; VanLoen 1966). Hence, some calculations and interpolations are required (Becker et a l . 1963; VanLoen 1966). a. The Requirement f o r Lysine. Mertz et a l . (1949) were the f i r s t to o f f e r experimental evidence that lysine was an es s e n t i a l factor i n the growing pig. Brinegar et a l . (1949) concluded that 0.58% lysine was the requirement of weanling pigs when the protein content of the ra t i o n was 10.6%. In l a t e r studies (Brin- egar et a l . 1950a,b), a basal r a t i o n containing 22% protein was fed to weanling pigs and the growth data showed a requirement of 1.20% lysine . According to these studies there seemed to e x i s t a proportionality between the lysine requirement of 5.5% - 5.7% of the dietary protein when the pro- t e i n content ranged from 10.6 - 22%, meaning that the growing animal's lysine requirement could be stated as a percentage of the t o t a l dietary protein. McWard et a l . (1959) found that the requirement of lysine i n a 12.8% protein diet was 0.71% and i n the 21.7% protein r a t i o n , 0.95%. Change et a l . (1958) f a i l e d t o achieve consistent r e s u l t s . In one experiment the lysine requirement, expressed as a percentage of the d i e t , was not influenced by the protein l e v e l , while i n a second experiment they found a lysine requirement of 0.7% when the ra t i o n contained 10 or 15% protein, and 0.9% with a r a t i o n of 20% protein. Becker (1959) arrived at the conclusion, based on chick and p i g experiments, that the require- ment of each amino acid i s l i n e a r , but not proportionally related to the protein content of the ra t i o n . His recommended allowances of amino acids i n the n u t r i t i o n of weanling pigs are given f o r seven di f f e r e n t protein l e v e l s , varying from 12 to 24% protein i n the dry ra t i o n . The need f o r 15 each amino a c i d , expressed as a percentage of the t o t a l dietary protein, i s assumed to decrease by a certain percentage as the protein l e v e l of ratio n i s increased. Most of the lysine work mentioned above points t o - ward a requirement i n weanling pigs of about 0.6 - 0.7% of the rati o n when the t o t a l dietary protein content i s 12 - 15%. Becker (1963) recom- mends 0.74% lysine f o r weanling pigs fed a 16% protein r a t i o n . I t i s suggested by the I l l i n o i s workers that c a l o r i c density of the ra t i o n may also influence the amino acid requirement. This can par t l y be hidden by the fact that the pigs eat more of a die t low i n calories than of a diet of high c a l o r i c density (McWard et a l . 1959). This point has e a r l i e r been stressed by Sewell et a l . (1956) and Abernathy et a l . (1958), but i s hardly clear enough to j u s t i f y a d e f i n i t e conclusion. B e l l et a l . (1968) concluded that the lysine requirement f o r 23 to 57 kg pigs fed a ra t i o n containing 3,330 k c a l . digestible energy/kg was about 0.7%. Evidence concerning the lysine and protein requirement of f i n i s h i n g pigs (57 - 90 kg approximately) likewise remains controversial. B e l l (1965) demonstrated that-by increasing the dietary lysine l e v e l from 0.55 to 0.67% was as e f f e c t i v e as increasing the protein l e v e l from 13 to 16%, when the response c r i t e r i a were growth rate and e f f i c i e n c y of feed u t i l i z a t i o n . The reverse was the case when indices of lean meat content were assessed. The B r i t i s h A.R.C. publication (1967) suggested that young pigs would give satisfactory performance i f , up to about 50 kg liveweight, the diet contains about 0.75 - 0.8% lysine and thereafter about 0.6 - 0.65% (0.9 - 0.95 and 0.7 - 0.75% of the dry matter i n the die t respectively) compared with the N.A.S. - N.R.C. (1968) recommendation of 0.7% to 20 - 35 kg pigs and 0.5% f o r f i n i s h i n g pigs. 16 b. The Requirement f o r Methionine. B e l l et a l . (1950) demonstrated the need f o r methionine i n swine rations. Curtin (1952a, 1952b, 1952c) using soybean plus p u r i f i e d diets containing 22% protein as feed f o r weanling pigs, found no improvement i n growth rat e , feed conversion or nitrogen balance by adding DL-methionine. He stated that the requirement did not exceed 0.31% methionine, when the cystine content was 0.38%. Shelton (1951a) used oxidized casein, ge l a t i n and tryptophan as amino acid sources i n a p u r i f i e d diet to which an animal- protein-factor-supplement, l i v e r extract, and choline were also added. Using weanling pigs i n growth studies, they concluded that the methionine requirement i n the absence of cystine amounted to 0.60%, but the need of S-containing amino acids could also be met by using 0.30% methionine plus 0.30% cystine. Using weanling pigs, graded levels of DL-methionine and L-cytine were added to a p u r i f i e d r a t i o n containing 12% i s o l a t e d soybean protein. Becker et a l . (1955) concluded that a l e v e l of 0.25% methionine i n the presence of 0.17% cystine supported a sa t i s f a c t o r y rate of growth. Expressed as a percentage of the dietary protein, the combined methionine + cystine requirment was calculated t o be 3.33%. Cystine can apparently provide about 40% of the need f o r S-bearing amino acids. A considerable proportion of the t r i a l s conducted t o determine the methionine requirement of pigs have been carried out with corn-soybean meal rations. However, the method f o r extracting the o i l meal i s relevant as the heat treatment w i l l influence protein q u a l i t y (Becker et a l . 1953). This may at least be one of the explanations f o r the experiments on sup- plementation of methionine to soybean meal rations having f a i l e d t o give consistent r e s u l t s . There may be other factors. Berry et a l . (1962) have shown that methionine i s d e f i n i t e l y the f i r s t l i m i t i n g amino acid i n soy- 17 bean meal, as might be expected from the amino acid composition. In t h e i r experiment with soybean meal added to a p u r i f i e d r a t i o n , a s i g n i f i - cant growth-promoting value was found by adding methionine. As the grains are also r e l a t i v e l y poor sources of methionine, one could expect a response i n the growth rate of pigs when adding methionine to a grain-soybean meal ration. Experimental evidence f o r t h i s has been produced by some workers ( B e l l et a l . 1950; Pfander et a l . 1953; Bayley et a l . 1968), while others have reported no growth response (Catron et a l . 1953; Sewell et a l . 1958; Arcker et a l . 1959; Change et a l . 1960; Beames et a l . 1969; Oestemer et a l . 1970). Other rather commonly used protein feeds, such as peanut meal, cottonseed meal and sesame meal, have about the same methionine content as soybean meal. In some cases (Whitehair et a l . 1952), addition of DL- methionine to rations containing these meals as protein concentrates has improved feed u t i l i z a t i o n . Becker et a l . (1966) indicated a requirement of 0.50% methionine + cystine f o r growing pigs with an i n i t i a l weight of 20 kg fed a d i e t con- t a i n i n g 16% protein; they also indicated that cystine could supply 40% of the t o t a l need f o r the su l f u r bearing amino acids. The N.A.S.-N.R.C. (1968) indicated a methionine requirement of 0.50% of the d i e t f o r pigs i n the weight range 20 - 30 kg. Oestemer et a l . (1970) suggested that the methionine + cystine requirement of growing pigs from 21 to about 40 kg was somewhat less than the figure of 0.42% to 0.50% of the diet as reported by investigators previous cit e d . These t r i a l s were conducted i n a series of experiments with growing swine to determine the capacity of opaque-2 corn to provide adequate dietary methionine. The corn rations (crude protein 10.85%) contained 0.275, 0.279, 0.227% methionine + cystine. Neither 18 rate of gain, gain/feed nor protein evaluation r a t i o (PER) was s i g n i f i - cantly improved by supplementing the basal corn d i e t with 0.07, 0.14, 0.21 or 0.28% DL-methionine. Keith et a l . (1972) indicated that when Yorkshire g i l t s averag- ing 18 kg body weight were fed a semipurified diet containing s i x graded levels of methionine during s i x 4-day feeding periods, p l o t t i n g serum methionine concentration against dietary methionine intake showed that the methionine requirement was 0.46% of the rat i o n . This estimate was substantiated by animal performance data (protein 18.39%). H i l l (1965) has pointed out that methionine and cystine are largely destroyed by hydrochloric acid during acid hydrolysis and must, therefore,.be converted to stable derivatives. I f methionine and cystine were under-estimated i n the basal r a t i o n or cereal diet as the r e s u l t of destruction 'by ac i d , t h e i r calculated requirement would be variable. Therefore, the method used i n determining the amino acid contents i n various experiments should be taken into account when interpreting r e s u l t s , c. The Requirement of Tryptophan. Beeson et a l . (1948, 1949) were the f i r s t to investigate the need f o r tryptophan. Shelton et a l . (1951b) working with weanling pigs, used a p u r i f i e d 24.5% protein r a t i o n containing zein and ge l a t i n plus some amino acids. They concluded that the growing p i g needed 0.2% tryptophan i n the diet or. 0.8% of the t o t a l dietary protein. By adding graded levels to a basal rat i o n with 0.09% tryptophan (17% protein), the Purdue workers found an optimum supplement of 0.06% DL-tryptophan. The requirement was set at 0.12% L-tryptophan, considering L-tryptophan as the only form to be u t i l i z e d . The finding that tryptophan can be converted i n t o n i a c i n i n pigs (Luecke et a l . 1946) and confirmed by Powick et a l . (1948) i s con- 19 sidered important i n corn ra t i o n s , as the corn i s a very poor source of available n i a c i n . Becker et a l . (1954a) suggested 0.13% tryptophan to be adequate f o r weanling pigs. This agrees with nitrogen balance studies made by Meade (1956), s t a r t i n g with pigs of 32 kg liveweight and using a 15.9% protein corn-soybean meal ra t i o n . In t h i s t r i a l n i a c i n was added to the basal rat i o n . The A.R.C. (1967) suggests that 0.15% to 0.20% L-tryptophan i n the dry matter i n the d i e t should be adequate i n the presence of adequate amounts of n i c o t i n i c acid. The N.A.S.-N.R.C. (1968) recommends that 0.13% tryptophan i s adequate f o r growing pigs weighing 20 - 35 kg and that the l e v e l can be reduced t o 0.09% f o r f i n i s h i n g pigs, d. The Requirement f o r Threonine. The threonine requirement has been studied i n weanling pigs by Shelton et a l . (1950c), and i n suckling pigs by Sewell et a l . (1952), using p u r i f i e d or semi-purified diets including casein and washed i s o l a t e d soybean protein. In a report from I l l i n o i s (Berry et a l . 1962) i t i s suggested that the protein i n soybean meal has threonine as the t h i r d l i m i t i n g amino acid (after methionine and l y s i n e ) . Experimental evidence (Berry et a l . 1962) with rats and pigs indicates that threonine might be second l i m i t i n g and lysine t h i r d . Beeson et a l . (1953) gave young pigs a diet based on corn and including nine es s e n t i a l amino acids. The diet provided 13.2% protein and 0.2% threonine. When a supplement of L-threonine was given, maximum weight gain and best feed conversion e f f i c i e n c y were with 0.4% dietary threonine (3.0% of the dietary crude protein). Mertz et a l . (1952) also estimated the requirement of L-threonine to be 0.4% of the diet. With providing 12% CP. based on dried skim milk, Becker et a l . (1954b) calculated that 0.61% L-threonine was a satisfactory dietary con- 20 centration. These workers calculated the threonine content of a diet which had produced satisfactory growth, so that t h e i r estimate of requirement might w e l l be excessive. In a s i m i l a r way, Evans (1958) calculated the threonine content of diets which had been proven to give sa t i s f a c t o r y performance, namely diets containing e i t h e r 7% f i s h meal, 20% ground nut meal, or 15% soybean meal, to be 0.52, 0.55 and 0.55% respectively. In further experimental work, the same author (1963) gave a diet containing 6% soybean meal, sup- plemented with lysine and methionine which provided 0.43% threonine to pigs up to 36 kg liveweight and obtained a s i g n i f i c a n t response i n terms of growth rate and feed conversion e f f i c i e n c y when a supplement of 0.15% L-threonine was added. Robinson et a l . (1963) fed an a l l - c e r e a l d i e t supplemented with lysine and methionine, and providing 0.46% threonine was supplemented with isoleucine, threonine and tryptophan and a s i g n i f i c a n t improvement i n per- formance was noted. An appraisal of these data, however, indicates that the response was more probably due to the isoleucine and/or the tryptophan, since these amino acids were more severely deficient with respect to assumed requirements. The A.R.C. (1967) suggests that the requirement f o r L-threonine f o r weanling pigs i s about 0.45 - 0.5% of the ration (0.5 - 0.6%':of the dry matter i n the ra t i o n ) . This compares w e l l with the l e v e l of 0.45% f o r growing pigs given by the N.A.S.-N.R.C. (1968). e. The Requirement f o r Isoleucine. Brinegar et a l . (1950c) fed a semi-purified diet of starch, glucose, blood f l o u r and DL-methionine to growing pigs. Maximum weight gain (630 g/day) was obtained when L-isoleucine was added to give a l e v e l 21 of 0.7% of the ration equal to. 3.2% of the t o t a l dietary protein. Becker et a l . (1963) reported that pigs weighing-5 kg required 0.76% dietary i s o - leucine, 3.45% of the dietary protein, while pigs weighing 45 kg required 0.35% dietary isoleucine, 2.63% of the dietary protein. Bravo et a l . (1970) reported that pigs weighing from 20 - 40 kg require from 0.27 to 0.32% isoleucine i n the diet when average d a i l y gain, feed conversion r a t i o and levels of plasma free isoleucine were the response c r i t e r i a . Using p r a c t i c a l type r a t i o n s , Evans (1962) found no improvement ei t h e r i n growth or nitrogen retention from the addition of isoleucine to a diet containing 0.59% isoleucine. The A.R.C. (1967) suggests that 0.65% L-isoleucine i n the di e t (0.75% of the dry matter i n the diet) i s adequate f o r pigs between 14 and 45 kg liveweight and that the requirement might w e l l be less f o r pigs of over 45 kg liveweight. This compares with a recommendation of 0.5% f o r pigs weighing 20 - 35. kg by N.A.S.-N.R.C. (1968). G. SUPPLEMENTATION WITH AMINO ACID OF GRAIN-PROTEIN CONCENTRATE RATIONS AND GRAIN ONLY. For p i g diets normally based on cereals, protein concentrates are added to increase the t o t a l protein content and to counteract any essen t i a l amino acid deficiencies i n the basal r a t i o n . The importance of the lysine content of the ra t i o n i s w e l l established and there are many examples i n the l i t e r a t u r e showing the improvement i n performance that can be brought about by lysine supplementation of rations containing poor-quality protein (Evans 1960; Jones et a l . 1962; Rozman et a l . 1968; Ostrowski 1969; Braude et a l . 1972). I t has also been reported that a small improvement i n performance can be obtained when lysine i s added to diets containing white f i s h meal, but economic appraisal of the results 22 showed that t h i s was not a viable proposition at p r e v a i l i n g prices (Braude and Lerman 1970). However, the greatest use of lysine and other amino acids as supplements may l i e i n the possible replacement of protein r concentrates, since the l a t t e r are by f a r the most expensive dietary com- ponents. I t has been reported that the feeding of cereal mixtures sup- plemented with l y s i n e , threonine, tryptophan, methionine and i n some experiments also isoleucine, results i n performance equal to that obtained with rations containing protein concentrates (Robinson and Lewis 1963; MUller et a l . 1967b). Weanling pigs were fed a cereal diet with different supplements of amino acids. The cereal d i e t consisted of wheat, barley and oats. The t r i a l was conducted from 14 - 18 kg body weight to 50 kg body weight. The combination of lysine and threonine had a marked eff e c t upon gains and feed conversion. Supplementing with lysine alone enhanced gains i n one case by 19% and i n another case by 21%. The combination of lysine and threonine increased gains by 59% i n the f i r s t t r i a l and by 92% i n the second t r i a l . Feed conversion improved by 31 - 34%. In the combination of lysine and tryptophan, 0.02% added tryptophan showed a pos i t i v e influence, whereas a tryptophan addition of 0.04% caused a depression i n a l l t r i a l s . The supplement of lysine alone increased weight gain by 26 - 28%, whereas the combination of lysine and tryptophan increased gain by 35 - 42%. In t r i a l s with a combination of two methionine levels and l y s i n e , a s l i g h t increase was recorded i n comparison with a supplement of lysine alone. How^ ever, differences were not s t a t i s t i c a l l y s i g n i f i c a n t . A supplement of 0.03% methionine gave better r e s u l t s than a supplement of 0.05% methionine (MUller et a l . 1967a). I t appears that threonine and tryptophan are both equal as the second l i m i t i n g amino acids. 23 Since the report by Jensen et a l . (1965) on grain plus amino acid d i e t s , there has been considerable e f f o r t put into finding n u t r i t i o n a l l y adequate and econimical rations using these two ingredients as the major components. From a series of papers (Muller and MaMk 1967a, 1967b, 1967c; M i l l e r et a l . 1967a, 1967b, 1967c; Muller and Rozman 1968; Rozman et a l . 1968) i t i s evident that Czechoslovakian s c i e n t i s t s are greatly interested i n t h i s type of rati o n . In the paper by Jensen et a l . (1965) i t was reasoned on the basis of the University of I l l i n o i s feeding standards (Becker et a l . 1963) that lysine and possibly methionine were a l l that were required as a supplement to sorghum f o r pigs from 45 to 90 kg body weight, whereas lysine and tryp- tophan were the required amino acids to supplement com. Their results showed a growth response to the addition of 0.25% lysine to sorghum grain but no response to the further addition of methionine. In a s i m i l a r experi- ment reported by Beames et a l . (1968) up to 0.2% lysine improved growth rate and feed e f f i c i e n c y of fattening pigs, but the improvement i n carcass q u a l i t y was s l i g h t . Ericson et a l . (1962) also improved growth rate and feed e f f i c i e n c y i n a wheat barley-amino acid r a t i o n fed from 32 kg body weight to 90 kg with lysine added to the grain to a l e v e l of 0.86% of the ration. In the supplementation of several grain-protein concentrate rations with amino acids, responses have been obtained to incremental increases of lysine up to levels i n excess of most of the suggested require- ments (Ericson et a l . 1962), but even though such supplemented diets have often given - sub-optimal performance, the further addition of methionine generally gives no response (Jensen et a l . 1965; Beames and Pepper 1969). Optimal growth and feed e f f i c i e n c y , however, were obtained by Muller and 24 Rozman (1968) with maize, barley and wheat when each was supplemented with lysine (0.41 - 0.61%), threonine (0.1 - 0.27%), tryptophan (0.04 - 0.15%) and methionine (0.14 - 0.25%) from weanling pigs of 30 kg body weight and with reduced levels thereafter. Cystine can s a t i s f y 40 percent of the need f o r methionine (N.A.S — N.R.C. 1968). Although the methionine + cystine requirements of growing pigs are widely taken as being 0.5 - 0.6% of the r a t i o n , there are some experiments which indicate that 0.3 - 0.42% i s adequate f o r optimal growth (Beames and Pepper 1969; Oestemer et al._ 1970). I t could be suggested that lack of response to methionine supplementation under some circumstances was due to other factors i n the ra t i o n being l i m i t i n g . However, the r e s u l t of Oestemer et a l . (1970) would tend to refute t h i s as they obtained good performance with a t o t a l methionine + cystine l e v e l of 0.23 - 0.28% of the ratio n . Berry et a l . (1966), using corn-soybean and corn-sugar-soybean d i e t s , found that the response to supplementation with lysine and methionine depended on the addition of threonine, i n d i c a t i n g that t h i s amino acid i s l i m i t i n g i n low protein, corn-soybean meal diets. Pecora and Hundley (1951) obtained a large improvement i n growth rate of rats fed r i c e by supplementing the diet with lysine and threonine. Sure (1954) demonstrated that wheat and rye were deficient i n lysine and threonine and further, that rye was deficient i n valine. Pond et a l . (1958) indicated that lysine and threonine were probably the most l i m i t i n g amino acids i n milo f o r growth and that the l i v e r f a t content of rats receiving the basal ra t i o n was s i g n i f i c a n t l y reduced by addition of 0.5% L-lysine and 0.2% DL-threonine. Rosenberg et a l . (1959) established lysine and threonine as the f i r s t and second l i m i t i n g amino acids i n r i c e protein f o r 25 the growth of the weanling r a t . Methionine, isoleucine and tryptophan were next l i m i t i n g amino acids i n t h i s order. Pick et a l . (1971) showed that excellent d a i l y gain, gain/feed and protein e f f i c i e n c y r a t i o (nitrogen retention) could be obtained when a d i e t containing 89.5% opaque-2 was supplemented with 0.1% L-isoleucine, 0.18 t o 0.35% L-lysine, 0.2% DL-methionine, 0.12% L-phenylalanine, 0.14% L-threonine and 0.08% L-valine. F i n a l dietary levels of 0.36% isoleucine, 0.7% lysine or l e s s , 0.40% methionine + cystine, 0.59. t o 0.65% phenylalanine + tyrosine, 0.43% threonine and 0.54% valine appeared to be adequate f o r the growing r a t . H. FACTORS AFFECTING AMINO ACIDS SUPPLEMENTATION OF FEEDS a. Energy Content of the Ration. I t has long been r e a l i z e d that a protein can be u t i l i z e d at maximum e f f i c i e n c y only i f there i s s u f f i c i e n t energy i n the diet from nonprotein sources t o s a t i s f y the requirement of the organism f o r c a l o r i e s . That the available energy i n a die t can be of c r i t i c a l importance f o r the successful amino acid supplementation of animal feeds was not rea l i z e d u n t i l a d i r e c t relationship between the energy and protein content was demonstrated i n b r o i l e r feeds. The system f o r expressing the energy value i s that digestible or metabolizable energy are used almost universally f o r pigs and poultry. When protein-energy relationships are considered, i d e a l l y only the completely balanced portion of the protein should be c a l - culated as protein and the res t as energy. Using a corn-soybean meal d i e t (Rosenberg et a l . 1955) l i t t l e or no response was obtained i n the chick when a small amount of methionine was added t o the ra t i o n i n spite of the fact that the f i r s t l i m i t i n g amino acid i n the d i e t was considered to be methionine. However, when fat was added to the diet the chicks 26 responded to the supplemental dietary methionine with improved growth and feed e f f i c i e n c y . This e f f e c t was also shown when carbohydrates were used to replace the f a t ( B a l d i n i and Rosenberg 1957). These studies suggest a d i r e c t r e l a t i o n between c a l o r i c density and the l e v e l of amino acids necessary i n the d i e t . The combined results of two such experiments (B a l d i n i and Rosenberg 1955; B a l d i n i and Rosenberg 1957) indicated that three different calorie l e v e l s , 1960, 1985 and 2205 calories of productive energy per kg, required different levels of methionine f o r optimum performance of chicks. In poultry, the energy-methionine relationship has been explained on the . basis that birds eat primarily to s a t i s f y t h e i r energy requirements, although t h e i r food intake i s governed also by a number of other factors including protein concentration and composition of the protein. When the energy con- tent of a diet i s increased,birds eat less per unit of gain. An increase i n energy content of the diet r e s u l t s , therefore, i n an increase i n the e f f i c i e n c y of feed u t i l i z a t i o n . An increase of the f a t content of the diet increases the c a l o r i c density and may cause a drop i n food intake. I f protein content i s not increased i n a r a t i o n with added f a t , the l e v e l of protein per c a l o r i e i s decreased, r e s u l t i n g i n a f a l l i n protein intake even though the calorie intake may be unchanged. In b r o i l e r rations con- t a i n i n g d i f f e r e n t levels of added f a t , t h i s question i s an important one, and has been stressed by Waddel (1959). B a l d i n i et a l . (1957), Rosenberg and Culik (1955), and Williams and Grau (1956) showed that the c a l o r i c density i s one of the factors governing the amino acid requirements of growing turkeys, rats and chicks. Sewell et a l . (1956), Abernathy et a l . (1958) and McWard et a l . (1959) observed that pigs eat more of a diet low i n calories than of a 27 diet of high c a l r o i c density. Whether or not a l l of the supplemented amino acids - l y s i n e , threonine, methionine, etc. - needed to effe c t amino acid balance can be u t i l i z e d w i l l depend largely, upon the energy content of the diet. This concept of amino acid u t i l i z a t i o n , of course, does affect the way i n which the amino acid requirements are formulated, but naturally t h i s c a l l s f o r proper knowledge of the requirement of both essential amino acids and energy. Henry (1968) found that ad l i b i t u m fed growing-finishing pigs achieved maximum energy intake on diets having 3.25 Mcal/kg digestible energy. Robinson etlal. (1964) " concluded that the best growth performance and carcass lean content of pigs receiving a di e t of low energy content was recorded when the r a t i o of Kcal D.E. to kg dietary lysine was approximately 3500. At the higher dietary energy l e v e l the best performance i n terms of carcass q u a l i t y was also recorded at a s i m i l a r r a t i o of energy to lysine. Overall carcass q u a l i t y was adversely affected by high energy levels i n the f i n i s h i n g diets but was improved as the protein l e v e l was raised. The res- ponse of g i l t s to increasing protein and lysine levels i n the diet continue beyond the l e v e l where the response i n barrows creases. Robinson's experi- ment showed no s i g n i f i c a n t sex differences i n terms of liveweight gain or ef f i c i e n c y of feed u t i l i z a t i o n but g i l t s were much superior to barrows i n terms of carcass quality. With pigs averaging 16 to 22 kg liveweight i n i - t i a l l y , the rate of gain and gain/feed r a t i o were influenced by the dietary lysine l e v e l s . There was no evidence of interaction between lysine l e v e l and energy l e v e l of the d i e t on rate of gain, but there was a s i g n i f i c a n t i nteraction f o r gain feed r a t i o i n one experiment. The calculated lysine needs, expressed as percentage of the d i e t , were 0.65, 0.77 and 0.80 f o r 28 maximum rate of gain and 0.66, 0.71 and 0.85 f o r maximum feed e f f i c i e n c y i n diets containing 2926, 3267 and 3718 kca l . ME. per kg respectively. These values were equivalent to an average lysine need i n the diet equal to 0.23% per 1000 kca l . ME. (Mit c h e l l et al.1965). b. Protein Content of the Ration. One of the major points of disagreement i n establishing the amino acid requirement i s whether i t should be given as a percentage of the diet or as a percentage of the t o t a l dietary protein. Arguments have been pro- duced f o r the v a l i d i t y of the concept of a certain percentage of the dietary protein at a l l l e v e ls of protein i n the rat i o n (Brinegar et a l . 1950a) while other research work has produced r e s u l t s i n disagreement with t h i s f i n d i n g (Hutchinson et a l . 1957). Altogether, much evidence has been pro- duced i n favor of the concept that the requirement should be stated as a percentage of the d i e t , but i n order to interpret these data.it i s ; necessary that the protein l e v e l of the diets be known. Harper (1959), and Harper and Kumta (1959) are of the opinion that the increase i n the requirement of an amino acid with increasing pro- t e i n percentage i s rather small and of doubtful significance over the normal range of protein l e v e l s . I f the requirement of an amino acid i s expressed i n percentage of the t o t a l dietary protein, t h i s percentage w i l l decrease as the protein l e v e l of the r a t i o n i s raised. Barnes and Bosshardt (1946) have emphasized that the i d e a l evaluation of protein should be made at the l e v e l of protein that gives maximum e f f i c i e n c y i n converting food protein to body protein. This protein l e v e l w i l l be somewhat lower than the protein allowance recommended f o r farm animals. The frequently used technique to test the supplementation by a certain amino acid of a p r a c t i c a l type diet at two or three different protein levels around the recommended standard 29 may be a useful means of detecting responses both as to protein l e v e l and amino acid supplementation at the same time The report of Grau (1948) i s frequently referred to i n order to i l l u s t r a t e the' connection between the requirements of lysine and protein. As the protein percentage of the rat i o n was increased from 10 to 30, the calculated lysine requirement was doubled. Becker (1963) states that i s o - leucine and lysine requirements of pigs, expressed as a percentage of the t o t a l dietary protein, decreases with increasing protein l e v e l i n the rati o n . Harper (1959) mentions that i f there are two l i m i t i n g factors such as protein and l y s i n e , the lysine need w i l l increase u n t i l protein, as such, becomes l i m i t i n g . This i s not actually an increase i n the require- ment. Harper (1959) adds; "Nevertheless, there does come a point at which i t i s possible to demonstrate that an adequate diet i s made less adequate i f the o v e r a l l protein i s increased while the l e v e l of one amino acid i s held constant". Boomgaardt and Baker (1970, 1971) indicated that the lysine and tryptophan requirements of young chicks are a constant percentage of d i e t - ary crude protein. Expressed as a percentage of diet, the requirement increased l i n e a r l y as dietary crude protein l e v e l increased. I t was estab- li s h e d that the lysine requirement remained constant at 4.59% of the protein, and tryptophan requirement was 0.87% of the protein. These res u l t s with tryptophan and lysine do not support the generally-accepted concept that the requirement f o r an essentail amino acid decreases l i n e a r l y as a percent- age of the protein and increases c u r v i l i n e a r l y as a percentage of the diet as dietary protein l e v e l increases from deficiency to adequacy. This experimentally established relationship may be expressed i n a di f f e r e n t manner when the r a t i o of calories to protein i s held constant, the amount 30 of amino acids required by the body f o r best performance and expressed as percent of protein remains r e l a t i v e l y constant when the concentration of protein i n the diet i s increased. When the r a t i o of calories to pro- t e i n i s increased,the amino acid requirement as a percentage of protein increases also. When the r a t i o of calories to protein decreases, the amino acid requirement, as a percentage of protein, decreases (Rosenberg 1959). The effect of change i n protein l e v e l i s a problem of consider- able p r a c t i c a l importance as feeds f o r domestic animals range from 10% protein f o r the fattening p i g t o 30% protein f o r the turkey poult (Rosen- berg 1959). Percent protein i n the diet i s plotted against percent of amino acid in. the protein f o r l y s i n e , methionine, and the combination of methionine and cystine i n corn-soy diets. As the l e v e l of protein i s increased, the r e l a t i v e amount of lysine i n the diet increases, while the relative amounts of the sulfur-bearing amino acids decrease. Therefore, as the protein l e v e l increases, methionine deficiency i s l i k e l y t o occur. Conversely, as the protein l e v e l decreases, lysine deficiency i s l i k e l y to occur (Rosenberg et a l . 1959). Lawrence (1971) indicated that barley d i e t s , compared with maize d i e t s , gave better growth rates and energetic conversion'efficiencies, l i g h t e r carcasses, lower k i l l i n g out percentage and smaller eye muscles. The narrow ca l o r i e protein/lysine (CPL) r a t i o . 1 . 2 diets when compared with wide CPL r a t i o diets gave better growth rates and energetic conversion e f f i c i e n c i e s , l i g h t e r cold carcass weights, smaller backfat deposits and greater percentage of lean and bone but smaller percentage of f a t i n the carcass. 1. Digestible energy: crude protein = 198:1, Crude protein: lysine = 19.8:1. 2. Digestible energy: crude protein = 263:1 or 273:1, crude protein: lysine = 27.6:1 or 26.9:1. 31 c. Age and Sex of. the Species Consuming the Rations. The unique functions i n the animal body served by the amino acids r e s u l t i n g from protein digestion are a l l anabolic i n character. They relate to the replacement of ess e n t i a l tissue constituents that have been degraded i n catabolic reactions, or to the formation of new tissue constituents i n growth. In the rapidly growing animal, the l a t t e r func- tions dominate the body's requirements f o r amino acids. In the mature animal, the replacement functions may dominate the amino acid requirements, but the growth functions s t i l l p e r s i s t , since some tissues continue to grow throughout l i f e ( M itchell 1949). Among adult animals of di f f e r e n t species, the r e l a t i v e importance of the growth functions i n determinging amino acid requirement w i l l depend mainly on the rate of growth of the epidermal structures, such as h a i r , n a i l s and claws. Species differences i n amino a c i d requirements during growth would be expected not so much on the basis of differences i n the nature of the anabolic reactions, as on the basis of differences i n the rate of growth of new tissue i n comparison with the weight of tissue to be main- tained, that i s , the percentage rate of growth. The higher the percentage growth rat e , the greater the extent to which the growth of new tissue dominates the t o t a l amino acid requirements (M i t c h e l l 1959). I t seems clear that as f a r as growth rate and feed conversion e f f i c i e n c y i n pigs are concerned, the crude protein requirements as a percentage of the diet f a l l s with increasing liveweight. Considering the period up to about 45 - 50 kg liveweight, the requirement as a percentage of the dry matter i n the diet appears to be about 16 - 17% crude protein, and from 50 kg to 90 kg liveweight about 13 - 14% crude protein. A.R.C. (1967) indicated that the presence of a n t i b i o t i c s reduced the requirement f o r crude protein, 32 at least as f a r as growth rate i s concerned. Less attention has been paid to a difference between sexes with respect to t h e i r amino acid requirements. A c t u a l l y , there i s a difference although t h i s i s r e l a t i v e l y small. Maximum growth was obtained with about 0.65% lysine i n the d i e t of male rats while only about 0.57% lysine was needed f o r maximum growth of the females. Interestingly enough, t h i s difference i n the requirement disappears i f the amount of lysine required per gram of gain i s calculated (Rosenberg and Rohdenberg, 1952). A s i m i l a r difference has been observed i n the growing chick (Rosenber 1959). Accord- i n g l y , i t has been proposed to feed male and female b r o i l e r s and poults separate die t s . Bayley et a l . (1968) indicated that there was a s i g n f i c i a n t i nteraction between the s t r a i n of p i g and protein l e v e l . Lacombe and Yorkshire pigs responded to an increase i n the dietary protein l e v e l from 13 to 16% by increasing gain, whereas the crossbred and Landrace pigs did not. Smith et a l . (1967) showed that g i l t s were more responsive to an improvement i n the l e v e l and type of protein than barrows, while Bay ley et a l . (1968) indicated that boars were even more responsive than g i l t s to the adequacy of the protein content of the diet. Robinson et a l . (1964) indicated that the response of g i l t s continued with increasing protein and lysine l e v els i n the diet while that of barrows ceased at a much lower l e v e l . This w i l l influence the l e v e l of lysine or other amino acids to be recommended and w i l l be different f o r barrows and g i l t s . Robinson (1964) therefore concluded no s i g n i f i c a n t sex differences i n terms of liveweight gain or e f f i c i e n c y of feed u t i l i z a t i o n but g i l t s were superior to barrows i n terms of carcass quality. 33 I I I . EXPERIMENT I • A. EXPERIMENTAL PROCEDURE a. General. Protein i s one of the more costly nutrients i n p r a c t i c a l swine rations. Barley i s the most common energy source f o r Western Canadian swine growers, while soybean meal i s widely used as a protein source. Consequently, swine rations generally include these two feedstuffs as major ingredients. In t h i s experiment, barley and soybean meal were the sole major ingredients i n the control r a t i o n . Swine do not require protein per se but require the contained amino acids f o r the synthesis of body tissue. The ess e n t i a l amino acids f o r optimum growth of swine which are generally deficient i n barley accord- ing t o N.A.S.-N.R.C. (1968) recommendations are l y s i n e , threonine, methio- nine and isoleucine. The following experiment was designed to study the eff e c t of supplementation of Peace River barley with two levels of lysine i n combination with threonine, methionine and isoleucine on the performance of growing-finishing pigs. The experimental design consisted of s i x treatments with three r e p l i c a t i o n s . Each of the s i x treatments within replicates comprised a pen of s i x pigs (three male castrates and three females) which were group-fed. b. Animals. A t o t a l of 108 Yorkshire and Yorkshire x Landrace pigs were used i n t h i s experiment. Pigs were commenced on t r i a l at an average body weight of 16.9 kg, weighed weekly and sent f o r slaughter at a body weight of 84 kg or over. The two l a s t pigs i n the pen were withdrawn from t r i a l when the body weight of the heavier one exceeded 84 kg. Rations and pens were allocated on a random basis. 34 c. Rations. The ingredients f o r the s i x rations are shown i n Table I. The t r i a l consisted of s i x treatments as follows: 1. Barley-soybean meal to supply 0.75% t o t a l lysine (control ration) 2. Barley + ,69% L-lysine HC1 to supply 0.90% t o t a l l ysine 3. Barley + .50% L-lysine HC1 to supply 0.75% t o t a l l ysine 4. Barley + .50% L-lysine HC1 to supply 0.75% t o t a l lysine + 0.05% L-threonine 5. Barley + .50% L-lysine HC1 to supply 0.75% t o t a l l ysine + 0.05% L-threonine + 0.10% DL-methionine 6. Barley + .50% L-lysine HC1 to supply 0.75% t o t a l lysine + 0.05% L-threonine + 0.10% DL-methionine + 0.10% L-isoleucine. The amino acidbcontent and chemical composition of the barley and the soybean meal are shown i n Table I I . The amino acid composition of each rat i o n i s l i s t e d i n Table I I I . A l l rations were computed to con- t a i n adequate amounts of a l l nutrients known to be required f o r growing- f i n i s h i n g pigs except t o t a l protein and aniino acid content. d. Management. ( i ) Housing. The experiment was conducted at the Swine Research Unit, the University of B r i t i s h Columbia. The building was insulated, with a i r exhaust fans f i t t e d with thermostatic controls set at 18.5°C. The housing consisted of concrete floored p a r t i a l l y 35 2 sl a t t e d pens with an'area per p i g of 1.2 m . ( i i ) Feeding and Watering. Pigs were fed t h e i r rations twice d a i l y at 8:00 a.m. and 1:00 p.m. Feeding was i n troughs on an ad l i b i t u m basis with maximum allowance l i m i t e d to 2.7 kg/day. Daily records of feed consumption were kept. Water was supplied ad l i b i t u m by nipple. ( i i i ) Feed Storage and Mixing. A l l feed ingredients were purchased and stored p r i o r to the commencement of the experiment to ensure a uniform rati o n composition throughout. The barley was stored i n the whole form. As required, 450 kg batches of each rat i o n were prepared by hammer m i l l i n g the barley and then mixing i t with the other ingredients i n a v e r t i c a l mixer* f o r 4 - 5 minutes. On the f i r s t r e p l i c a t e the grain was ground i n a medium screen (10 mm diameter) but a fi n e screen (7 mm diameter) was used f o r replicates two and three to reduce scattering of feed which was a problem with the coarser grind. e. Records. A l l pigs were weighed i n i t i a l l y and thereafter at weekly i n t e r - vals throughout the experiment. Weekly feed consumption, corrected f o r s p i l t feed, was also recorded. f. Chemical Analysis. A l l feed was analyzed f o r moisture, ash, crude protein and ether * K e l l y Duplex M i l l Machinary, size 220, s e r i a l 57364. The Duplex M i l l Mfg. Co., S p r i n g f i e l d , Ohio. 36 extract according to A.O.A.C. methods (1960) and acid detergent f i b r e was determined according to VanSoest method (1963). Hydrolyzates of the barley and the soybean meal were analyzed f o r a l l e s s e n t i a l amino acids except tryptophan on a Phoenix* model 7800 amino acid analyzer equipped with a VARIPUMP according to the method described by Piez and Morris (1960). The hydrolyzates were prepared by acid hydrolysis (Kohler et a l . 1967) except f o r cystine, methionine and tryptophan analysis. Cystine was deter- mined as cysteic acid and methionine as methionine sulfone by oxidative hydrolysis (moore 1963). The foregoing analysis was done because of the losses of methionine and cystine associated with acid hydrolysis. For tryptophan analysis, the barley were hydrolyzed with the enzyme pronase and tryptophan was determined by spectrophotometical method (Spies et a l . 1968). g. Carcass Measurements. Dressed weight was measured on the hot carcass while the follow- ing measurements were made on the c h i l l e d carcass. "A", "B" . and "c" measurements were taken according to the procedure of Buck et a l . (1962). The other carcass measurements were obtained according to Canadian Record of Performance f o r Swine (1967). Maximum shoulder f a t Minimum middle f a t Maximum l o i n f a t Eye muscle Area "A" measurement of l o i n eye (width) " Phoenix Precision Instrument Co., 3083-05 North 5 Street, Philadelphia, Pa. 19140, U.S.A. 37 "B" measurement of l o i n eye (depth) "C" measurement of f a t at cut not including s k i n , i . e . , 40 mm from mid l i n e Carcass length h. Calculations. Average d a i l y gain was determined as the b value i n the equation derived from weekly body weights y = a + bx, where "y" i s body weight, "a" i s i n i t i a l weight, and "x" days on t r i a l . The average da i l y feed con- sumption and feed conversion r a t i o ( t o t a l feed eaten divided by t o t a l body weight gain, both values expressed i n the same units) were calculated f o r each pen. Carcass measurements were determined f o r each pig. i . S t a t i s t i c a l Analysis of Data. The data were subjected to an analysis of variance using,^least squares technique as shown by Harvey (1960) which i s suitable f o r comparing unequal sub-groups and hence relevant i n t h i s experiment with missing values. Means from comparisons showing a s i g n i f i c a n t "F 1 value were tested according to the multiple range test of Duncan (1955). Daily weight gain was analyzed with s t a r t i n g weight as the covariable. Dressing percentage, carcass length, maximum shoulder f a t , mid backfat, maximum l o i n f a t , backfat 40 mm from midline, l o i n area and l o i n area index were analyzed with dressed weight as the covariable. 38 Table I. Composition of rations used i n experiments I and I I (Ai r dry basis) Treatment 1 2 3 4 5 6 Ingredients •t % % % % % Barley 83.10 96.05 96.24 96.19 .96.09 95.99 Soybean meal 13.76 L-lysine HC1* 0.69 0.50 0.50 0.50 0.50 L-threonine* 0.05 0.05 0.05 DL-methionine* 0.102 0.102 L-isoleucine* 0.10 Defluorinated rock phosphate 1.49 1.64 1.64 1.64 1.64 1.64 . Limestone 0.57 0.54 0.54 0.54 0.54 . 0.54 Iodized s a l t 0.5 0.5 0.5 0.5 0.5 0.5 Copper sulfate 0.078 0.078 0.078 0.078 0.078 0.078 Trace mineral and vitamin premix 0.5 0.5 0.5. 0.5 0.5 0.5 The trace mineral and vitamin premix provided the following per kg of r a t i o n : manganese 44 mg as MnSO^.H 0, Zinc 110 mg as ZnSO .7H 0, B.H.T. 500 mg, Vitamin A 3085 I.U., Vitamin Dg 440 I.U., Vitamin B±2 20 yg, r i b o f l a v i n 2.9 mg, n i a c i n 11 mg, calcium pantothenate 11 mg (45% equivalent). * . The L-lysine HC1 was fed grade (Ajinomoto Co. , Japan) and wa's 98% pure, containing 78% L-lysine. The L-threonine and L-isoleucine were pure. The DL-methionine was 98% pure. 39 Table I I . Content of es s e n t i a l amino acids and proximate constituents of barley and soybean meal on a dry matter basis. Component Concentration Barley Soybean Meal Arginine (g/lOOg d.m.) Hist i d i n e (g/lOOg d.m.) Isoleucine (g/lOOg d.m.) Leucine (g/lOOg d.m.) Lysine (g/lOOg d.m.) Methionine (g/lOOg d.m.) (Cystine) (g/lOOg d.m.) Phenylalanine (g/lOOg d.m.) (Tyrosine) (g/lOOg d.m.) Threonine (g/lOOg d.m.) Tryptophan (g/lOOg d.m.) Valine (g/lOOg d.m.) 0.529 0.219 0.345 0.696 0.434 0.141 0.178 0.617 0.301 0.293 0.210 0.848 1.526 1.383 2.135 3.600 3.578 0.632 0.614 3.025 1.636 2.010 0.900 4.245 Crude Protein '(.%) (N x 6.25) Ash (%) Crude f i b r e (%) Crude f a t (%) 10.50 3.19 5.87 3.39 53.78 6.30 6.50 3.95 40 Table I I I . Content of es s e n t i a l amino acids of each rat i o n (g amino acid/lOOg mixed feed) on an air- d r y basis. Ration Amino Acid 1 2 3 4 5 6 Arginine 0. 56 0. ,44 0. 44 0. 44 0. 44 0. 44 Histidine 0. 33 0. .18 0. 18 0. 18 0. 18 0. 18 Isoleucine 0. 51 0. .28 0. 28 0. 28 0. 28 0. 39 Leucine 0. 90 0. ,57 0. 57 0. 57 0. 57 0. 57 Lysine 0. 75 0. .90 0. 75 0. 75 0. 75 0. 75 Methionine 0. 18 0. ,12 0. 12 0. 12 0. 17 0. 17 (Cystine) 0. 20 0. .15 0. 15 0. 15 0. 15 0. 15 Phyenylalanine 0. 81 0. ,51 0. 51 0. 51 0. 51 0. 51 (Tyrosine) 0. 31 0. .25 0. 25 0. 25 0. 25 0. 25 Threonine 0. 46 0. .24 0. 24 0. 29 0. 29 0. 29 Tryptophan 0. 27 0. .17 0. 17 0. 17 0. 17 0. 17 Valine 1. 13 0. .70 0. 70 0. 70 0. 70 0. 70 Dry- matter content % 89. 95 88. .85 89. 12 88. 93 89. 16 89. 36 Crude protein % 15. 81 10. .15 10. 18 10. 12 10. 25 10. 31 (N x 6.25) 41 B. RESULTS The experimental data are summerized i n Table IV. There was a s i g n i f i c a n t difference between amino acid supplemented rations and the control r a t i o n i n feed e f f i c i e n c y and d a i l y weight gain but not i n carcass quality. Adding 0.05% L-threonine to the lower l e v e l of lysine s i g n i f i c a n t l y * improved d a i l y gain and feed conversion r a t i o compared t o the lower l e v e l of lysin e . Further addition of 0.10% DL-methionine e i t h e r alone or i n com- bination with 0.10% L-isoleucine did not improve performance but gave s i g - n i f i c a n t l y higher d a i l y gain and a better feed conversion r a t i o : than the lower l e v e l of lysine alone. Average d a i l y gain and feed e f f i c i e n c y f o r pigs fed the lower l e v e l of lysine were s i g n i f i c a n t l y lower than those fed the higher l e v e l of lysin e . The lower l e v e l of lysine gave s i g n i f i c a n t l y less mid backfat than the higher l e v e l of lysine but there was no s i g n i f i c a n t difference between the two levels of lysine i n other carcass measurements. However, the trend showed that the lower l e v e l of lysine produced better carcass q u a l i t y than the higher l e v e l . The addition of 0.1% DL-methionine to the lysine and threonine supplemented r a t i o n reduced feed intake r e s u l t i n g i n a lower d a i l y gain but did not s i g n i f i c a n t l y affect feed conversion ratio;. The barley-soybean control r a t i o n gave a s i g n i f i c a n t l y higher weight gain, better feed conversion r a t i o and measurements indicating lean- er carcasses than the barley-amino acid rations. There was no signficant difference between treatments i n dressing percentage and maximum shoulder f a t . The barley-soybean control r a t i o n produced less backfat , and larger * Where significance i s indicated, i t refers t o significance at the 5% l e v e l . 42 eye muscle areas and indices. The lower l e v e l of lysine and supplementation gave a s i g n i f i c a n t l y greater dressing percentage than the rat i o n to which both lysine and threonine were added. The minimum mid f a t measurement of pigs receiving the barley-soybean control r a t i o n was s i g n i f i c a n t l y less than that of pigs receiving the higher l e v e l of lysine or the lower l e v e l of lysine with threonine and methionine. The barley-soybean control r a t i o n produced less maximum l o i n f a t and less backfat at 40 mm from the mid l i n e than the higher l e v e l of lysine . The barley-soybean control ration produced a s i g n i f i c a n t l y greater eye muscle area and eye muscle index than other treatments except f o r the lower l e v e l of lysine . Adding threonine, methionine and isoleucine to the lower l e v e l of lysine did not af f e c t backfat or eye muscle measure- ments. Barrows gave s i g n i f i c a n t l y higher d a i l y weight gain than g i l t s while g i l t s had consistently less backfat, higher dressing percentage and larger eye muscle area. Treatment and sex interaction was obtained f o r d a i l y weight gain and f o r carcass quality. G i l t s fed the higher l e v e l of lysine gave a s i g n i f i c a n t l y higher d a i l y weight gain than those fed the lower l e v e l of lysine. Supplementation with l y s i n e , lysine and threonine, and l y s i n e , threonine, methionine and isoleucine tended to show higher d a i l y weight gain f o r barrows than g i l t s but supplementation with l y s i n e , threonine and methionine produced an adverse ef f e c t . 43 Table IV. Summary of the ef f e c t of the addition of amino acids t o barley on body weight gain, feed consumption (D.M.), feed conversion rat i o n (D.M.) and carcass measurements i n feeding t r i a l . Treatment 1 2 . 3 4 5 6 Results of Soy 0.9% 0.75% lys + lys+threo lys +threo Signi f i c a n t Control lys l y s threo + meth +meth+isol Tests * Starting 1 6 8 3 1 6 > 6 3 i y > 3 7 g g 2 Wt. (kg) 83.20 82.83 80.53 84.53 82.34 84.05 F i n a l Wt. (kg) ^ f 1 ??ily .609 .461 .413 .525 .498 .501 1 4 6 5 2 3 gam (kg) _ D.M. Feed(kg) 2.954 3.533 3.790 3.320 3.300 3.330 1 5 4 6 2 3 Wt. gain (kg) Mean d a i l y ± ^ 7 7 1 > 7 3 g 1 > 6 Q g 1 > g 5 3 1 4 6 5 2. 3 feed intake (kg) — — (mm?) Experimental 1 Q 1 > 1 + 1 4 6 _ 2 1 4 6 _ 8 1 3 5 > 3 period (days 136.2 Dressing % 78.36 78.67 79.46 78.05 78.44 78.52 3 2 6 5 1 4 Length cm 79.15 77.10 74.25 78.07 78.49 77.41 1 5 4 6 2 3 Max shoulder 3 7 > Q 3 Q ^ 3 J ^ 3 ? _ 5 g > 7 3 Q ^ 5 2 6 4 3 1 rat (mm) fat'Sm) 1 6 2 1 > 1 2 5 - 4 2 1- 9 2 3' 7 2 3 ' X 2 4' 3 2 6 4 5 3 1 Max l o i n 2 g > y 3 3 > 5 ± 2 4 5 6 3 1 f a t (mm) ? h ? f a w ^ 1 8-8 22.6 20.5 21.2 20.5 20.8 2 4 6 3 5 1 (40 mm) (mm) 1 4 3 5 2 6 Eye muscle index A x B 3714 3312 3382 3415 3376 3286 (mm2) Eye Muscle 2 7 1 g 2 4 y 6 2 5 3 2 2 ^ 1 3 6 2 4 5 area — Treatment means not underscored by the same l i n e are s i g n i f i c a n t l y d i f f e r e n t at 5% l e v e l of p r o b a b i l i t y (Duncan, 1955). 44 Table V. Comparison of the effects of supplementation with amino acids on d a i l y weight gains and carcass measurements of g i l t s and barrows. Barrows ( V G i l t s S i g n i f i c a n t F Test (s2) 1 Daily Wt. Gain 517 486 Carcass Measurement Dressing % 78. 01 79. ,16 A A Carcass length (cm) 77. 99 76. ,83 N.S. Max. shoulder f a t (mm) 39. ,44 36. ,19 A A rfk Min. middle f a t (mm) 24. 69 21. ,80 Max. l o i n f a t (mm) 32. 92 30. ,85 A Backfat 40 mm from mid (mm) 22. ,44 18. .99 2 Eye muscle index (mm ) 3243 3586 A A 2 Eye muscle area (mm ) 2374 2658 A A Treatment and sex interaction Treat. x Sex Daily Wt. Gain (g) Treat. x Sex Daily Wt. i 1. T 1 S 1 601 7. T 1 S 2 618 2. T 2 S 1 476 8. T 2S 2 446 3. T 3 S 1 467 9. T3 S2 359 4. T 4 S 1 546 10. T4 S2 505 5. T 5 S 1 483 11. T5 S2 513 6. V l - 530 12. T6 S2 473 Duncan's te s t of Treat, x Sex effe c t on weight gain at 5% l e v e l of prob- a b i l i t y . 7 1 4 6 11 10 5 2 12 3 8 9 * P < 0.05 ** P < 0.01 Treatment means not underscored by the same l i n e are s i g n i f i c a n t l y d ifferent. 45 C. DISCUSSION I t might be concluded from the re s u l t s obtained i n t h i s feeding t r i a l , that the growing-finishing p i g could perform reasonably w e l l on a basal r a t i o n of Peace River barley supplemented with lysine and threonine, providing additional vitamins and minerals were given. However, the weight gain and feed e f f i c i e n c y were somewhat less with the lysine and threonine supplemented r a t i o n than with the soybean supplemented control ra t i o n . The addition of methionine to the ra t i o n alone or i n combination with isoleucine did not improve performance beyond that obtained with the additional lysine and threonine. Mean values indicated a s i g n f i c a h t l y better performance with 0.90% t o t a l lysine than with 0.75% t o t a l l y s i n e . Obtained by the addition of lysine alone was i n f e r i o r to that obtained when other amino acids were also added, ind i c a t i n g that lysine was not the only l i m i t i n g amino acid i n the barley. L-lysine supplementation to a high barley d i e t (75% barley meal, 23% wheat o f f a l ) f o r growing pigs has been shown elsewhere to improve per- formance s i g n i f i c a n t l y (Braude et a l . 1972). This r a t i o n contained 0.57% t o t a l lysine compared with 0.45% l y s i n e i n the unsupplemented high barley r a t i o n , thus approximating the lysine content of a standard B r i t i s h barley meal-wheat o f f a l r a t i o n supplemented with 7% white f i s h meal. Many workers (Evans 1960; Jones et a l . 1962; Ericson et a l . 1962-; Ostrowski 1969) have confirmed that lysine supplementation to poor-quality protein rations im- proved performance. Sure (1954) demonstrated that wheat and rye were def i c i e n t i n lysine and threonine and further that rye was d e f i c i e n t i n valine f o r the growth of r a t . Pond et a l . (1958) indicated that lysine and threonine were 46 probably the most l i m i t i n g amino acids i n milo f o r the growing r a t . Rosen- berg et a l . (1959) established lysine and threonine as the f i r s t and the second l i m i t i n g amino acids i n r i c e protein f o r the growth of the weanling rat. Berry et_ a l . (1966) using a corn-soybean combination reported a response to supplementation with lysine and methionine that was dependent on the addition of threonine, i n d i c a t i n g that t h i s amino acid was l i m i t i n g i n low-protein com soybean meal rations f o r weanling pigs. MUller et a l . (1967a) showed that lysine and threonine supplementation to a basal cereal diet had an extraordinarily favourable ef f e c t upon gains and feed e f f i c i e n c y . The supplementation of lysine alone enhanced gain i n one case by 19% and i n another case by 22%. The combination of lysine and threonine increased gain by 59% i n the f i r s t t r i a l and by 92% i n the second, and the feed e f f i - ciency was improved by 31 - 34% over that obtained with the unsupplemented control r a t i o n . They considered from the viewpoint of n u t r i t i o n that lysine was the f i r s t , threonine the second, tryptophan the t h i r d and methionine the fourth l i m i t i n g amino ac i d ' i n the cereal diet f o r the growing pigs. The cereal consisted of wheat, barley and oats. In the present experiment,the mean d a i l y gain and feed e f f i c i e n c y values obtained with the lower l e v e l of lysine were markedly improved when 0.95% L-threonine was added to the diet. This indicated that threonine was the second l i m i t i n g amino acid i n the basal ra t i o n of barley which was i n agreement with Sure (1954), Pond et a l . (1958), Rosenberg et a l . (1959), Berry et a l . (1966) and MUller et a l . (1967a). The experiment (Table I I I ) showed that the basal ra t i o n contained :annsufficient l y s i n e , methionine, threonine and isoleucine f o r growing pigs according to the N.A.S.-N.R.C. standards (1968) but that there was adequate tryptophan. 47 Although the methionine + cystine requirements of growing pigs are widely accepted as being 0.5 - 0.6% of the ra t i o n (A.R.C. 1967), there are some experiments which indicate that 0.3 - 0.42% i s adequate f o r optimal growth (Beames and Pepper 1969, Oestemer e t a l . 1970). I t could be suggested that the lack of response to methionine supplementation under some circumstances was due to other factors i n the ra t i o n being l i m i t i n g . However, the results of Oestemer et a l . (1970) would tend to refute t h i s because they obtained good performance with rations i n which opaque-2 corn provided a l l the sulphur-containing amino acids. The corn contained 0.23 - 0.28% methionine + cystine. Neither the rate of gain, gain/feed nor pro- t e i n e f f i c i e n c y r a t i o were s i g n i f i c a n t l y improved by supplementing the basal corn rations (Crude protein 10.85%) with 0.07, 0.14, 0.21 or 0.28% DL-methionine. Jensen et a l . (1965) indicated that the addition 0.20 or 0.25% l y s i n e to the approximately 8.0% crude protein milo provided a balance of es s e n t i a l amino acids which supported gains equal to that of f i n i s h i n g pigs fed a corn-soybean 12.0% crude protein d i e t . They were unable to obtain a response to 0.10% DL-methionine i n addition to the lysine supplementation even though the methionine plus cystine content of the milo r a t i o n was only 0.25%. Muller et a l . (1967c) showed that supplementing with methionine and lysine was not as eff e c t i v e as. adding lysine alone to cereal rations containing 0.31% methionine plus cystine, and that the enrichment of cereal diets with the combination of lysine plus threonine l e d to an increase i n gain above the l e v e l attained by lys i n e supplementation alone. This agrees with the present study i n which lysine and threonine supplementation of barley improved performance whereas the further addition of methionine had no ef f e c t . I t has been reported that feeding cereal mixtures supplemented 48 with l y s i n e , threonine, tryptophan, methionine and i n some experiments also isoleucine resulted i n performance equal to that obtained with diets containing protein concentrate (Robinson et a l . 1963; MUller et a l . 1967b). MUller and Male*k (1967a, 1967b, 1967c) showed that 'monodiets', i . e . , single cereal diets (wheat, barley or com) with added amino acids could be used successfully to rear pigs from weaning (average liveweight about 20 kg)^ Beames and Pepper (1969) indicated that the use of l y s i n e , either with or without methionine, was not completely successful as a replacement of h a l f the soybean meal i n a grain plus 15 percent soybean rat i o n f o r pigs of less than 45 kg body weight. From a body weight of 45 kg to the slaughter weight of approximately 90 kg, the above authors showed that h a l f of 15% soybean meal could be replaced by amino acids without reducing the growth of the pigs , although results with t h i s type of supplement i n combination with sorghum over the body weight range (20 - 89 kg) were not as satisfactory as f o r wheat. In the present experiment, the replacement of protein con- centrates such as soybean meal by l y s i n e , lysine plus threonine, lysine plus threonine plus methionine plus isoleucine did not improve the perform- ance to equal that of the barley-soybean control r a t i o n , but supplementation with these amino acids s i g n i f i c a n t l y improved performance above that obtain- ed by adding ly s i n e alone. Performance of animals on amino acid supplemented barley rations was i n f e r i o r to the barley-soybean control r a t i o n . This i s i n agreement with Beames et a l . (1968), Beames and Pepper (1969), and Barber et a l . (1969), but i n disagreement with Robinson et a l . (1963) and MUller et a l . (1967b). I t i s possible that the response to the addition of lysine and threonine to the barley ra t i o n was li m i t e d by either the l e v e l of t o t a l N i n the r a t i o n , which was markedly lower than the l e v e l i n the barley-soybean control r a t i o n , or a lower b i o l o g i c a l a v a i l a b i l i t y of lysine and threonine 49 or other e s s e n t i a l amino acids. Bayley et a l . (1968) indicated that the supplementation of a p r a c t i c a l corn-soybean rat i o n with e i t h e r 0.1% lysine or 0.05% methionine had no b e n e f i c i a l e f f e c t on growth rate or feed e f f i c i e n c y , but with the levels used there was a p o s i t i v e effect on gain when both amino acids were added together. The e f f e c t was marked f o r the low-protein r a t i o n , but making up the difference i n lysine, and s u l f u r amino acids did not overcome the difference i n gains between the two dietary protein l e v e l s . Ericson et a l . (1962) showed that supplementation with L-lysine HC1 markedly increased d a i l y gain (40 - 60%) and decreased the amount of feed per kg of gain (25 - 30%), when the basal r a t i o n consisted of 27% and 63% barley protein 9and, that there was some response due to the L-lysine HC1 supplementation i n spite of the r e l a t i v e l y high l e v e l of added f i s h meal and soybean meal. This experiment also indicated that supplementation with protein concentrates gave f a r better gain and feed e f f i c i e n c y than supplementation with amino acids. B l a i r et a l . (1969) showed that liveweight gain was not improved s i g n i f i c a n t l y by increasing the protein l e v e l above 16, 14 and 12% respec- t i v e l y , f o r the 2 3 - 4 5 , 45 - 68 and 68 - 90 kg body weight categories. However, feed conversion e f f i c i e n c y was improved s i g n i f i c a n t l y during the 23 - 45 kg range by increasing the protein l e v e l to 18%. Lean meat produc- t i o n and the e f f i c i e n c y of conversion of feed to lean meat were improved by increasing the above protein levels s l i g h t l y . Liveweight gain was not improved s i g n i f i c a n t l y by increasing the lysine l e v e l above 1.04, 0.74 and 0.70%, respectively, f o r the 2 3 - 4 5 , 45 - 68 and 68 - 90 kg ranges. However, feed conversion e f f i c i e n c y was improved s i g n i f i c a n t l y during the 23 - 45 kg stage by increasing the lysine l e v e l to 1.22%. Raising the 50 lysine l e v e l at each l e v e l of protein had no signficant effect on the rate and e f f i c i e n c y of lean meat gain. Generally animals require more amino acids, especially l y s i n e i n early growth and gradually decrease t h e i r requirement with age. In the present experiment,higher levels of lysine (0.90%) gave a s i g n i f i c a n t l y higher mean d a i l y weight gain and better feed e f f i c i e n c y than the lower l e v e l of lysine (0.75%). P i g performance showed that there was not much difference between the two levels of lysine i n the early stages but the higher l e v e l of lysine gave improved mean d a i l y weight gain and feed e f f i - ciency than the lower l e v e l i n the l a t e r stages (Figure 2, Figure 3). I t i s possible that the response to the higher l e v e l of lysine was l i m i t e d during early growth by the l e v e l of crude protein i n the rat i o n . In t h i s experiment, threonine was the second l i m i t i n g amino acid f o r pigs since supplements of lysine and threonine to the barley produced performance next to that of pigs fed the barley-soybean control ration. Braude et a l . (1972) and Morrison et a l . (1961) suggested t h a t l l y s i n e supplementation re s u l t s i n a progressive decrease i n plasma threonine concentration. The higher l e v e l of lysine was perhaps more imbalanced i n the early stage than i n l a t e r stages. A l e v e l of 0.29% threonine i n these rations was consider- ably lower than the published threonine requirement (0.45%) according to N.A.S.-N.R.C. (1968) but i s adequate i n the l a t e r stages since the require- ment of threonine i s 0.27% (crude protein 12%) by calculations which are based upon the assumption that the requirements f o r a l l amino acids express- ed as a percentage of the protein, decrease l i n e a r l y as the dietary protein increases (Becker et a l . 1963). Therefore, i n the l a t e r stage, pigs can u t i l i z e more lysine than i n the early stage. Pierce and Bowland (1972) showed that the addition of 0.2% L-lysine HC1 alone; to the low protein diet 51 (CP. 14%, lysine 0.57%) improved feed intake i n the growing and f i n i s h i n g period and the o v e r a l l period but not i n the s t a r t i n g period compared with the low protein unsupplemented diet. B l a i r et a l . (1969) showed that feed e f f i c i e n c y improved s i g n i f i c a n t l y during the 23 - 45 kg stage by increasing the l y s i n e l e v e l to 1.22%. In the present experiment pigs fed the higher l e v e l of l y s i n e gave better feed intake and growth rate than those receiving the lower l e v e l of lysine i n the l a t e r stage but not i n the early stage (Figure 2), but the higher l e v e l gave s l i g h t l y better feed e f f i c i e n c y than the lower l e v e l of l y s i n e i n the early stage. These results were i n agree- ment with B l a i r e t a l . (1969), and Pierce and Bowland (1972). In p a r t i c u l a r , attention may be drawn to the carcass q u a l i t y i n t h i s experiment. I t i s quite understandable that an energy excess w i l l lead to f a t t e r carcasses. In the present experiment d a i l y feed intake was 1.55 - 1.78 kg over a l l period. This figure showed that the t o t a l energy intake would not be i n excess since barley was used as a basal r a t i o n . I t i s worthwhile i n t h i s respect to determine not only nrinimum but i n p a r t i c u l a r optimum requirements, i f quality meat i s to be obtained economically from heavy pigs. Backfat measurements of pigs given the lower l e v e l of l y s i n e were of lower magnitude than those of pigs receiving the higher l e v e l of l y s i n e . Mid backfat was s i g n i f i c a n t l y less but maximum shoulder f a t , maximum l o i n f a t and backfat at 40 mm from the mid l i n e were not s i g n i f i c a n t l y less. Dressing percentage, eye muscle area and eye muscle index were not s i g n i f i c a n t l y affected by levels of l y s i n e supplementation but the treiid showed that the lower l e v e l of lysine gave a higher dressing percentage, a larger eye muscle area and a larger eye muscle index. This trend might come from e i t h e r amino acid imbalance at given protein levels or other l i m i t i n g factors. The higher l e v e l of l y s i n e (0.90%) at approxi- 52 mately 11% protein would be i n excess of the growing pigs requirements compared to 0.58% lysine at 11.6% protein (Brinegar 1949), 0.71% lysine at 12.8% protein diet (McWard 1959) and 0.70% lysine when the r a t i o n con- tained 10 or 15% protein (Chance et a l . 1958). This excess of lysine might cause an amino acid imbalance since performance showed that threonine was li m i t e d . Low-protein rations based on barley and/or wheat or corn properly balanced by supplementation with lysine produced leaner carcasses than unsupplemented low-protein r a t i o n , as shown by Brooks et a l . (1959), Bow- land (1962), C a h i l l y et a l . (1963), Nielsen et a l . (1963), Braude et a l . (19720. Adding threonine to the lower l e v e l of lysine had no s i g n i f i c a n t e f f e c t on backfat, eye muscle area and dressing percentage. This would have been'partly explained by a difference i n feed intake as the feed intake on the lower l e v e l of l y s i n e was 1.55 kg per day while further addition of threonine increased t h i s to 1.74 kg per day f o r the whole period. The further addition of methionine, alone or i n combination with isoleucine, d i d not affect backfat, eye muscle area, eye muscle index or dressing per- centage. This indicated that there was no response from adding methionine and isoluecine on carcass qu a l i t y as w e l l as performance of pigs. Clausen (1959-1964) collected a large quantity of data showing that amino ac i d supplementation of protein concentrates fed as supplements to barley diets have also effected a marked improvement i n the carcass leanness of the Danish pigs. In the present experiment, the barley-soybean control r a t i o n (about 17% CP.) gave the lowest backfat thickness, larger eye muscle area and higher eye muscle index. Work by Bowland et a l . (1959), Ashton et a l . (1955), Becker et a l . (1956) and re s u l t s summarized by A.R.C. (1967), a l l support the 53 view that lowering the crude protein l e v e l below about 16-17%, at least i n the i n i t i a l stages of the growing period, w i l l produce adverse effects on carcass quality. These results are i n agreement with the observations f o r t h i s study that pigs fed a barley-soybean control ration produced better carcass q u a l i t y than pigs fed lower protein (about 11%) amino acid supple- mented barley but Clawson (1967), Meade et a l . (1966) and Pierce and Bow- land (1972) reported no influence of dietary protein on l o i n area and back- f a t thickness. Pierce and Bowland (1972) suggested that lack of treatment effects on t o t a l backfat thickness and on l o i n area was due to a wide tolerance l e v e l f o r dietary protein levels before gross carcass changes were noted (protein l e v e l was 14, 17 and 20%). Barrows gave s i g n i f i c a n t l y higher d a i l y weight gain than g i l t s which i s i n agreement with Tjong-A-Hung et a l . (1972) but which i s i n disagreement with Newell and Bowland (1972) and Pierce and Bowland (1972). G i l t s were superior t o barrows i n a l l carcass measurements except carcass length. These results are i n agreement with Wong et a l . (1968), Newell and Bowland (1972) .and Pierce and Bowland (1972), and Tjong-A-Hung et a l . (1972) who showed that g i l t s had lower backfat measurements and larger eye muscle area. There was not much difference i n weight gain between the two levels of lysine f o r barrows, but g i l t s had a s i g n i f i c a n t l y d ifferent res- ponse to lysine, l e v e l s . This was i n agreement with Robinson (1964) who showed that the response of g i l t s continued with increasing protein and lysine levels i n the diets while that of barrows ceased at a much lower l e v e l . Adding lysine and threonine resulted i n a s i m i l a r improvement i n the growth rate of both barrows and g i l t s when compared with the addition of lysine alone. Further adding methionine with or without isoleucine did 54 not give consistant r e s u l t s . These res u l t s may be caused by a dif f e r e n t amino acid requirement of sex. Therefore, further study on sex and sup- plementation with amino acids interaction would give more r e l i a b l e r e s u l t s when a separated feeding system according to sex i s adopted. 4 5 6 Weeks on trial 10 n 12 13 14 15 Figure 1. The effect of supplementation with lysine and threonine of basal ration of barley (Treat. 4) compared to the control ration (Treat. 1) on mean body weight from the start until f i r s t pigs sent for slaughter. cn cn  a . 3 Treat. 3 Treat. 2 Treat. 4 Treat. 1 6 7 8 Weeks on trial 10 11 12 13 14 15 Figure 111. The effect of supplementation with lower level of lysine (Treat. 3), higher level of lysine (Treat. 2), lysine and threonine (Treat. 4) and control ration (Treat. 1) on Feed efficiency ratio from start until f i r s t pigs sent for slaughter. 58 D. CONCLUSION Pigs fed the lower l e v e l of lysine had a s i g n i f i c a n t l y lower growth rate and poorer feed e f f i c i e n c y than those fed the higher l e v e l of lysine. The trend that pigs fed the lower l e v e l of lysine were leaner and with larger eye muscle areas than those fed the higher l e v e l of lysine. These res u l t s could be explained on the basis of a slower growth r a t e , everything else being equal, producing a leaner carcass. Adding 0.05% L-threonine t o the lower l e v e l of ly s i n e improved feed intake, d a i l y weight gain and feed e f f i c i e n c y but not carcass q u a l i t y above the improvement r e s u l t i n g from the addition of lysine alone to the basal ra t i o n of barley. The further addition of 0.10% DL-methionine with or without 0.10% L-isoleucine did not improve feed intake, d a i l y weight gain, feed e f f i c i e n c y or carcass quality. Nor did supplementation with these amino acids to the lower l e v e l of lysine improve carcass quality. In t h i s experiment threonine was the second l i m i t i n g amino acid i n Peace River barley a f t e r l y s i n e , which i s w e l l known as the f i r s t l i m i t i n g amino acid i n barley f o r growing pigs. By adding 0.05% L-threonine to the lower l e v e l of lysine mean d a i l y gain improved by 27.1% and feed e f f i c i e n c y by 14%. How- ever, supplementation with lysine and threonine resulted i n a considerably i n f e r i o r performance than that obtained with barley-soybean control r a t i o n , with d a i l y weight gain, feed e f f i c i e n c y and carcass q u a l i t y being s i g n i f i - cantly poorer. I t was possible that the barley diet even with the addition of the four amino acids, was l i m t i n g e ither i n the l e v e l of t o t a l N or as a r e s u l t of an imbalance of amino acids was due to a low threonine l e v e l since addition of 0.05% L-threonine was deficient i n the rati o n f o r grow- ing pigs by N.A.S.-N.R.C. (1968). Either adding nitrogen or adding more threonine, or both may improve performance compared to that of the present experiment. 59 IV. EXPERIMENT I I A. EXPERIMENTAL PROCEDURE a. General. Although growth performance i s an important parameter i n p r a c t i c a l animal production, weight gain and feed conversion r a t i o give only an i n - accurate measure of nitrogen u t i l i z a t i o n . Nitrogen balance values are of much greater value i n assessing protein quality. The following experiment was done to obtain nitrogen absorption and u t i l i z a t i o n data to compliment the results obtained i n the main feeding experiment (experiment I) using the same rations. The experiment u t i l i z e d three groups each of s i x pigs, with each group maintained i n d i g e s t i b i l i t y crates f o r a three-week period. Within groups the s i x experimental rations were randomly allocated, with each pig receiving the same ra t i o n f o r the three-week period (Table VI). The f i r s t week was an acclimatization period. The second and t h i r d weeks were divided into the two one-week c o l l e c t i o n periods. b. Animals. A t o t a l of 18 male castrate Yorkshire pigs between 30 kg and 45 kg i n three groups of s i x were used. c. Rations. Rations were the same as those used i n experiment I. d. Management. ( i ) Housing The metabolism room was insulated and supplied with space heaters which kept the temperature at approximately 21 ± 1° C. The metabolism crates were 60 a modification of the S h i n f i e l d design (Frape et a l . 1968). The urine tray which was in c l i n e d from front to reat at an angle of approximately 30° from the horizontal was made of f i b r e glass. The feces tray was made of p l a s t i c . In order to prevent some pigs from turning i n the crates i n the acclimatization period a sheet of plywood board was attached to both side of the crates. A fi n e wire screen was placed on the urine tray i n order to re t a i n the small amount of feces which f e l l anterior to the rear of the f l o o r (Figure IV and V). ( i i ) Feeding and watering Pigs were fed twice d a i l y ad l i b i t u m at 9:00 a.m. and 2:00 p.m. f o r 40 minutes on rati o n which was mixed with water (feed:water = 1:1) just before feeding. Water was given a f t e r the afternoon feeding ad li b i t u m f o r 20 minutes. Feed residues were removed, put in t o p l a s t i c bags and stored at 3° C. At the end of each one-week c o l l e c t i o n period, the residues f o r each pig were pooled and dried at 60° C for 72 hours. ( i i i ) Mixing rations Each rat i o n was prepared by passing the barley through the fine screen (7 mm diameter) of a hammer m i l l . Feed samples were taken f o r determination of moisture and nitrogen at the time of feeding the r a t i o n , e. Feces and Urine Collection A l l feces from each p i g were collected i n p l a s t i c bags and stored 61 at 3 C u n t i l completion of the seven-day t r i a l . The t o t a l f e c a l output of each p i g was then pooled and weighed, and a 400 g aliquot dried at 60°C f o r 72 hours (Saben and Bowland 1971). The dried feces were ground and stored f o r l a t e r measure of nitrogen and moisture. Urine was c o l l e c t - ed i n a p l a s t i c j a r to which was added 50 ml dil u t e d sulphuric acid (50% V/V) each day. Each day urine volume was measured and a 10% aliquote re- tained at 3°C f o r pooling at the end of the experiment. The pooled urine was used f o r subsequent nitrogen determination, f. S t a t i s t i c a l Analysis of Data. The data f o r nitrogen balance, apparent nitrogen d i g e s t i b i l i t y , nitrogen retained as a percentage of nitrogen absorbed, nitrogen retained as a percentage of nitrogen intake and dry matter d i g e s t i b i l i t y were sub- jected to an analysis of variance (UBC-LSA8) and means from comparison showing s i g n i f i c a n t F values were tested according to the multiple range tes t of Duncan (1955). Nitrogen balance data were analyzed by covariance (UBC-LSA8) with feed intake because of .highly s i g n i f i c a n t covariance. 62 Table VI. A l l o c a t i o n of pigs within replicates and periods f o r Experiment I I Rations 1 2 3 4 5 6 Soy lys+threo lys+threo Period Control 0.9% l y s 0.75% l y s lys+threo +meth +meth+isoleu (Weeks) ,T Pig No. 1 1 2 3 4 5 6 2 1 2 3 4 .5 6 3 1 2 3 4 5 6 4 7 8 9 10 11 12 5 7 8 9 10 11 12 6 7 8 9 10 11 12 7 13 14 15 16 17 18 8 13 14 15 16 17 18 9 13 14 15 16 17 18 Figure V. Pig shown i n the cage which could be adjusted f o r the size of pig. 64 B. RESULTS The res u l t s of the metabolism t r i a l are shown i n Table VII. The addition of 0.50% L-lysine HC1 (0.75% t o t a l lysine) to the basal ration of barley produced a s i g n i f i c a n t l y better nitrogen balance than the addition of 0.69% L-lysine HC1 (0.90% t o t a l l y s i n e ) . However, when nitrogen balance was adjusted f o r feed intake, there was no s i g n i f i c a n t difference between two levels of L-lysine. The addition of 0.05% L-threonine to the lower l e v e l of lysine did not s i g n i f i c a n t l y improve nitrogen balance and also d i d not when nitrogen balance was adjusted -for feed intake. No further improve- ment of nitrogen balance was achieved by the addition of 0.10% DL-methionine to the lower l e v e l of lysine. However, when nitrogen retention was adjust- ed f o r feed intake, adding 0.10% DL-methionine with or without 0.10% L- isoleucine further improved the qu a l i t y of barley protein above that observed with lysine alone. Adding 0.10% L-isoleucine to the above rat i o n improved nitrogen balance when nitrogen balance was adjusted by feed intake. Nitrogen absorbed as a percentage of N intake tended to be improved more by the addition of 0.05% L-threonine to the lower l e v e l of lysine but t h i s increase was not s i g n i f i c a n t . Further improvement of N absorbed/N intake was obtained from the addition of methionine e i t h e r alone or i n combin- ation with isoleucine. When nitrogen absorbed was expressed as a percentage of nitrogen consumed, the barley-soybean control r a t i o n was s i g n i f i c a n t l y higher than the barley-amino acids rations. Nitrogen retained as a percentage of N absorbed was improved more by the 'addition. ..of 0.05% L-threonine to the lower l e v e l of lysine than by adding the lysine alone to the barley ration. The lower l e v e l of lysine gave a better value f o r N retained as a percentage of N absorbed than the higher l e v e l of ly s i n e . The barley-soybean control r a t i o n gave the second 65 lowest N retained as a percentage of N absorbed. The lowest value was obtained with the barley plus the higher l e v e l of ly s i n e . Further addition of methionine and isoleucine gave s i g n i f i c a n t l y higher b i o l o g i c a l value than the barley-soybean control r a t i o n and adding lysine alone to the barley. Nitrogen retained, when expressed as a percentage of nitrogen consumed was not s i g n i f i c a n t l y d i f f e r e n t f o r the grain-soybean control r a t i o n and the rations containing threonine, with or without methionine and isoleucine, but a l l except the grain-threonine rat i o n gave a higher retention than the grain-lysine combination. The res u l t s showed that the major e f f e c t of improving protein q u a l i t y was due to the threonine addition to the lower l e v e l of l y s i n e . The addition of methionine and isoleucine gave non-significant improvement i n N balance which was adjusted f o r feed intake, N d i g e s t i b i l i t y , b i o l o g i c a l value and N retained as a percentage of N consumed, but a s l i g h t response. 66 Table VII. Summary of the effects of supplementation of barley with amino acids on apparent dry matter d i g e s t i b i l i t y , nitrogen balance, apparent nitrogen d i g e s t i b i l i t y and on nitrogen retained as a percentage of nitrogen absorbed (average 7-day figures). Treatment 1 2 3 4 5 6 Results of Soy 0.9% 0.75% ly s + lys+threo lys+threo Sign i f i c a n t Control lys lys threo + + meth +meth+isol Tests * Total feed intake (g) Total N intake (g) N balance (g) N balance a 9801 8660 10437 99841 8404 267.03 150.70 179.221168888 144.33 120.37 55.16 68.56 70.84 63.61 SrS^e 1 1 7 , 3 7 6 0 , 4 5 6 1 , 6 7 5 8 , 0 3 l 7 0 , 6 7 .(g) N absorbed 0 N intake "° N retained 0 N intake "° 76.05 64.58 61.95 64.43 66.01 45.10 36.61 38.26 42.24 44.Oi N absorbed % 5 9 , 3 9 5 6 , 6 2 6 1 , 7 6 6 5 , 4 3 6 6 , 8 0 D.M. d i g e s t i b i l i t y 78.70 77.32 76.45 77.11 78.18 10199 171.17 70.50 69.52 66.18 43.50 64.60 78.22 1 4 6 3 5 2 1 5 6 4 3 2 1 6 5 2 4 3 1 5 6 4 3 2 5 4 6 3 1 2 1 6 5 2 4 3 Treatment means not underscored by the same l i n e are s i g n i f i c a n t l y d ifferent at 5% l e v e l of pr o b a b i l i t y (Duncan, 1955). 67 C. DISCUSSION Although there were s l i g h t variations i n the r e l a t i v e effects of treatments, the values f o r N balance, N absorbed/N intake, N retained/N absorbed and N retained/N intake were i n reasonably close agreement i n showing a progressive improvement i n nitrogen retention by the addition of L-threonine, DL-methionine and isoleucine to the barley plus lysin e . The r e s u l t s indicated that the threonine content of barley was deficient f o r growing pigs since the main improvement i n protein quality was associated with the addition of threonine and lysine i n contrast t o the addition of lysine alone. Further addition of methionine with or without isoleucine did not s i g n i f i c a n t l y improve N balance, N d i g e s t i b i l i t y , b i o l o g i c a l value and N retained/N absorbed above that obtained lysine and threonine only. An improvement i n nitrogen balance by the addition of L-lysine HC1 to grain has been reported by many authors (Ericson et a l . 1962; Bow- land and Grimson 1969; Pick et a l . 1971). Braude et a l . (1972) indicated that N retention as a percentage of N intake tended to be higher with lysine supplementation to barley than with unsupplemented barley f o r growing- f i n i s h i n g pigs. Solberg (1971) showed that compared with a moderately lys i n e - d e f i c i e n t d i e t , lysine additions had no s i g n i f i c a n t e f f e c t on ei t h e r the d i g e s t i b i l i t y or the metabolizable energy content of the d i e t but' a lysine deficiency caused a decreased nitrogen retention, an increased u r i c acid biosynthesis and an increased feed intake i n chicks. Pick et a l . (1971) showed that rats fed diets containing 0.73% lysine had s i g n i f i c a n t l y (p <_ 0.01) greater gain/feed r a t i o and retained more nitrogen than those fed 0.54% lysine diets. However, increasing the dietary lysine t o 0.92% did not e f f e c t further improvements i n any of the response c r i t e r i a . Also these results indicated that the l e v e l of dietary 68 isoleucine did not exert a s i g n i f i c a n t e f f e c t on d a i l y gain, gain/feed, N balance and N retained as a percentage of N absorbed. In the present experiment,the lower l e v e l of lysine gave better N balance and N retained/N absorbed than the higher l e v e l of l y s i n e , and the lower l e v e l of lysine had a s l i g h t l y better N retained/N intake value. Therefore, the lower l e v e l of lysine (0.75% t o t a l lysine) was more suitable than the higher l e v e l of lysine (0.90% t o t a l lysine) i n the barley r a t i o n f o r growing pigs which was supported by Pick et a l . (1971) who showed that 0.73% lysine was adequate i n an opaque-2 corn r a t i o n f o r growing ra t s . The lower le v e l , of lysine gave a s i g n i f i c a n t l y higher b i o l o g i c a l value and s l i g h t l y better net protein u t i l i z a t i o n than the higher l e v e l of lysine although d i g e s t i b i l i t y of nitrogen did not d i f f e r . Therefore, the different r e s u l t s of the two levels of lysine seemed to be derived from N retention but not N absorption. I t i s possible that the response to the higher l e v e l of lysine was due to e i t h e r an excessive amount of lysine which would give r i s e to an imbalance of amino acids because the threonine was deficient i n the ration according to N.A.S.-N.R.C. standard (1968) or to a lower amount of nitrogen intake than that of the lower l e v e l of lysine since nitrogen retention increases as nitrogen intake increases. Bressani (1971) indicated that adding lysine and threonine.to r o l l e d oats improved the nitrogen balance i n humans above that obtained with l y s i n e . He obtained no further improvement i n nitrogen balance by further addition of methionine. These res u l t s agree with the present experi- ment which showed a marked response to threonine when added to barley plus l y s i n e . Muller and Rozman (1968) showed that l y s i n e , threonine or tryptophan and methionine supplementation of barley f o r growing-finishing pigs improved N retained/N intake and N retained/N digested as w e l l as d a i l y gain and 69 feed e f f i c i e n c y . This agrees with the present experiment i n which adding l y s i n e , threonine and methionine with or without isoleucine gave an improve- ment i n N balance, N retained/N intake percentage i n t h i s order. Bowland and Grimson (1969) indicated that a better N retained/N absorbed value was obtained with a lysine-methionine supplemented low pro- t e i n d i e t (14% CP.) than with a higher protein or an amino acid unsupple- mented low protein diet f o r early weaned pigs. There was an increase i n d a i l y nitrogen retention when L-lysine was added to diets containing three percent urea but t h i s did not occur where both L-lysine and DL-methionine was added. This observation suggested that f o r pigs fed the low protein diet plus urea, methionine was not the second l i m i t i n g amino acid a f t e r l y s i n e . This agreed with our experiment i n which adding methionine pro- duced only a s l i g h t response since threonine was the second l i m i t i n g amino acid a f t e r lysine. The barley-soybean control ra t i o n gave the highest N balance, the best opponent N d i g e s t i b i l i t y and the highest value f o r N retained as a percentage of N intake but the second lowest value f o r b i o l o g i c a l value, a f t e r the higher l e v e l of lysin e . I t appears that the N of the barley- soybean rat i o n was better digested but of a lower b i o l o g i c a l value than the N of the l y s i n e plus threonine, lysine plus threonine plus methionine and lysine plus threonine plus methionine plus isoleucine rations. These results were i n agreement with those of Bowland and Grimson (1969) who showed that apparent nitrogen d i g e s t i b i l i t y was low on low protein diets but increased with urea or with protein supplementation of the low-protein diet and the highest apparent b i o l o g i c a l value was obtained with supplemen- tat i o n by lysine and methionine to low protein diet. I t i s possible that the response to the lower b i o l o g i c a l value of barley-soybean control ra t i o n 70 was due to e i t h e r a higher protein concentration since the barley-soybean rati o n was about 17% crude protein and the other rations were about 11% or a higher protein intake which was approximately one and h a l f times more protein than those of the other rations. These res u l t s were i n agreement with Metta and M i t c h e l l (1956), and Rippon (1959) who found that the b i o l - o g i cal value of d i f f e r e n t proteins decreased l i n e a r l y as the protein con- centration or intake increased. The results of the present study showed that barley protein could be improved by supplementation with four amino acids. However, barley-amino acids rations gave lower values f o r nitrogen retention than the barley- soybean control ration. Therefore, further studies might solve the problems of supplementation of cereal proteins with synthetic amino acids. There was a p o s s i b i l i t y of nitrogen losses i n feces and urine. Saben and Bowland (1971) suggested that either wet or dry p i g f e c a l material may be used f o r nitrogen and energy determinations i n p i g digestion t r i a l s where feces i s dried at 60°C f o r 72 hours. Martin (1966) indicated that losses of NHg from feces were ne g l i g i b l e and the losses from urine depended on the temperature and pH at which i t was collected. The average loss of nitrogen on c o l l e c t i o n of urine at a pH value below 2.0 was 1.33% when the ambient temperature was between 25 and 28°C and 0.97% when i t was 15 and 18°C. In the present experiment, urine was collected i n strong sulphuric acid so that the pH of urine was below 2 even though samples were stored f o r quite long periods i n a freezer. Therefore losses of nitrogen from feces and urine would be negl i g i b l e i n t h i s experiment. 71 D. CONCLUSION The metabolism t r i a l was conducted to determine the effe c t of amino ac i d supplementation of Peace River barley on nitrogen balance and apparent b i o l o g i c a l value. The addition of L—stysine and L-threonine to the barley tended to improve nitrogen balance when adjusted f o r feed intake, apparent nitrogen d i g e s t i b i l i t y , b i o l o g i c a l value and N retained/N intake above that obtained by the addition of lysine alone but the difference was not s i g n i f i c a n t . Further addition of methionine with or without isoleucine markedly improved the above c r i t e r i a although not a l l improvements were s i g n i f i c a n t above the addition of lysine alone. The results showed that the major e f f e c t of improving protein q u a l i t y appeared to be due to the threonine supplementa- t i o n to the lower l e v e l of lysine since further supplementation with methio- nine and with or without isoleucine gave further s l i g h t l y non-significant improvement i n nitrogen metabolism measurements. Whereas, isoleucine d i d not a f f e c t the protein q u a l i t y so much as methionine. Therefore, threonine was the second l i m i t i n g amino acid a f t e r l y s i n e i n t h i s experiment and methio- nine and Isoleucine were probably l i m i t i n g too. The difference i n apparent d i g e s t i b i l i t y of protein between two levels of lys i n e was not s i g n i f i c a n t but the b i o l o g i c a l value of the lower l e v e l of lys i n e r a t i o n was s i g n i f i c a n t l y higher than that of the higher l e v e l of lys i n e r a t i o n . This indicated that N retention varied with the l e v e l of lys i n e but N absorption was not influenced by the diff e r e n t lysine l e v e l . Therefore, 0.75% lysine seemed to be adequate f o r growing pigs i n th i s experiment. Further studies i n amino acid d i g e s t i b i l i t y and blood amino ac i d patterns may explain these r e s u l t s . Also l a b e l l e d amino acids would a i d i n determining amino acid absorption and"retention. 72 Supplementation with lysine and threonine with and without methionine, and also isoleucine resulted i n a s i g n i f i c a n t l y lower N balance, apparent N d i g e s t i b i l i t y but a s i g n i f i c a n t l y higher b i o l o g i c a l value than the barley-soybean control ration. I t was concluded that both protein and amino acid supplementation to the basal rat i o n of barley could affect nitrogen balance and b i o l o g i c a l value. Either adding nitrogen or adding more threonine, or both to the barley may improve nitrogen balance and b i o l o g i c a l value but not d i g e s t i b i l i t y . 73 V. GENERAL CONCLUSIONS AND RECOMMENDATIONS The supplementation with 0.50% L-lysine HC1 (0.75% t o t a l lysine) to a basal rat i o n of barley seemed to improve protein q u a l i t y compared to the supplementation with 0.69% L-lysine HC1 (0.90% t o t a l l y s i n e ) . The b i o l o g i c a l value of the lower l e v e l of lysine r a t i o n (0.75% t o t a l lysine) was s i g n i f i c a n t l y higher than that of the higher l e v e l of lysine (0.90% t o t a l l y s i n e ) , and the trend showed that pigs fed the lower lysine were leaner and had larger eye muscle areas than those fed the higher l e v e l of lysine even though the lower l e v e l of lysine gave a s i g n f i c a n t l y lower growth rate and poorer feed e f f i c i e n c y than the higher l e v e l of ly s i n e . Both the feeding experiment and nitrogen balance experiment indicated that threonine was the second l i m i t i n g amino acid since supple- mentation with lysine and threonine to the barley improved growth perform- ance s i g n i f i c a n t l y and tended to improve nitrogen retention and b i o l o g i c a l value to a greater extent than adding lysine alone. However, supplementa- t i o n of barley with lysine and threonine resulted i n s i g n i f i c a n t l y poorer d a i l y gain, feed e f f i c i e n c y , carcass qu a l i t y and nitrogen retention but s i g n i f i c a n t l y higher b i o l o g i c a l value than the barley-soybean control ration. Therefore, these treatments were unable to produce the results equivalent to that from normally supplemented soybean rati o n . The r e s u l t of the present study showed that barley protein could be markedly improved by supplementation with lysine and threonine and they demonstrated the importance of taking these into account i n a ration. I t i s w e l l known that one of the roles of protein concentrates i s to counter- act amino acid deficiencies and t o t a l nitrogen i n the cereal components of a ration and t h i s almost in e v i t a b l y gives r i s e to an excess of the other amino acids which are not needed f o r protein synthesis. In order to reduce 74 t h i s excess as much as possible, i t i s necessary to supplement these l i m i t i n g amino acids such as lysine and threonine and also to have a thorough knowledge of dietary amino acid content and a v a i l a b i l i t y at given components. The shortage and higher cost of protein supplements f o r use i n feed i s increasing. 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A.A. Abanese protein and amino acid n u t r i t i o n . 1949. Academic Press, New York and London. Arch. Anat.. Physiol. Abt. 21l). Van Loen, A. 1966. Amino acids i n animal n u t r i t i o n . 2 1 . Dutch State Mines, Heerlen, Holland. Van Soest, P.J. 1963. Use of detergents i n the analysis of fibrous feed. I I . A rapid method f o r the determination of f i b r e and l i g n i n . J.A.O.A.C. 46, 829. Waddel, J. 1958. In "Processed plant protein foodstuff". A.M. A l t s c h u l , ed., p. 307-351. Academic press, New York. Whitehair, C.K. and Macvicar, K. 1952. The value of amino acid supplemented to a low protein; a l l - p l a n t r a t i o n f o r swine. Oklahoma Agr. Exp. Sta. Misc. Publ. 27. Williams, M.A. and Grau, C.R. 1956. Food intake and u t i l i z a t i o n of ly s i n e - d e f i c i e n t protein by chick i n r e l a t i o n to the digestible energy concentration of the diet. J . Nutr. 59, 243. Wong, W.C, Bcylan^j W.J. and Stothers, S.C 1968. Effects of dietary protein l e v e l and sex on swine performance and carcass t r a i t s . Can. J . Anim. S c i . 48, 383. 86 APPENDIX Table Page IA. Average d a i l y body weight gain (kg) of the 107 pigs 87 IB. Analysis of variance of the average d a i l y body weight gain of 107 pigs. (Covariable s t a r t i n g weight) 87 IIA. Average feed intake (kg) per p i g per day f o r each ra t i o n 88 IIB. Analysis variance of average d a i l y feed intake 88 IIIA. Average feed conversion e f f i c i e n c y r a t i o per p i g . (D.M. feed intake (kg)/weight gain (kg)) 89 IIIB. Analysis of variance of feed conversion e f f i c i e n c y r a t i o n .89 IVA. Minimum middle f a t (mm) 90 IVB. Analysis of variance of minimum middle f a t 90 2 VA. Eye muscle (width x depth) (mm ) of the 103 pigs 91 VB. Analysis of variance of eye muscle (width x depth) . . . ~ . . .91 VIA. Nitrogen retention (g/week) 92 VIB. Analysis of variance f o r nitrogen retention 92 VIC. Analysis of co-variance f o r nitrogen retention with feed intake as the covariable 93 VID.. Analysis of co-variance f o r nitrogen retention with nitrogen intake as the covariable 93 VILA. Nitrogen retained as a percentage of nitrogen intake 94 VIIB. Analysis of variance f o r nitrogen retained as a percentage of nitrogen intake 94 VIIIA. Apparent nitrogen d i g e s t i b i l i t y (%) 95 VIIIB. Analysis of variance f o r apparent nitrogen d i g e s t i b i l i t y 95 IXA. Nitrogen retained as a percentage of nitrogen absorbed . . . . 96 IXB. Analysis of variance f o r nitrogen retained as a percentage of nitrogen absorbed 96 87 Table IA. Average d a i l y body weight gain (kg) of the 107 pigs. Treatment Rep 1 Male Female Rep 2 Male Female Rep 3 Male Female 1. Barley + Soybean (Control) 2. Barley + L-lysine HC1 (0.9% t o t a l lysine) 3. Barley + L-lysine HC1 (0.75% t o t a l lysine) 4. Treatment 3 + 0.05% L-threonine 5. Treatment 4 + 0.10% DL-methionine 6. Treatment 5 + 0.10% LIT isoleucine 0.78 0.70 0.61 0.50 0.44 0.52 0.46 0.47 0.44 0.58 0.56 0.57 0.39 0.62 0.45 0.49 0.58 0.49 0.56 0.62 0.50 0.46 0.39 0.24 0.42 0.44 0.44 0.56 0.46 0.59 0.40 0.46 0.44 0.48 0.49 0.61 0.65 0.37 0.45 0.50 0.53 0.44 0.54 0.5.0 0.56 0.54 0.50 0.52 0.50 0.57 0.52 0.55 0.49 6.0 0.65 0.62 0.41 0.51 0.41 0.43 0.32 0.39 0.51 0.57 0.41 0.50 0.52 0.44 0.50 0.50 0.59 0.60 0.45 0.66 0.47 0.53 0.35 0.40 0.56 0.46 0.55 0.56 0.56 0.57 0.56 0.54 0.53 0.56 0.45 0.63 0.64 0.62 0.51 0.41 0.37 0.35 0.33 0.32 0.49 0.49 0.59 0.48 0.44 0.35 0.50 0.39 0.47 Table IB. Analysis of variance of the average d a i l y body'weight gain of 107 pigs. (Covariable s t a r t i n g weight). Variance Ratio Source D.F. S.S. M.S. F-value F(nec.) p = 0.05 p = 0.01 Total 106 0. 859 Treatment (a) 5 0. 374 0. 0748 Block (b) 2 0. 0094 0. 0047 Sex (c) 1 0. 0257 0. 0257 a x b 10 0. 0231 0. 0023 a x e 5 0. 0561 0. 0112 b x c 2 0. 0102 0. 0051 a x b x c 10 0. 0451 0. 0045 Covariable 1 0. 0333 0. 0333 Residual 70 0. 263 0. 0038 19.939** 2.368 3.339 1.254 3.150 4.977 6.864* 4.001 7.077 0.616 1.993 2.632 2.992* 2.368 3.339 1.363 3.150 4.977 1.202 1.993 2.632 8.871** 4.001 7.077 * P < 0.01 ** P < 0.05 88 Table IIA. Average Feed intake (kg) per p i g per day f o r each rat i o n . Treatment . Rep 1 Rep 2 Rep 3 1. Barley + Soybean (Control) 1.79 1.79 1.75 2. Barley + L-lysine HC1 (0.9% t o t a l lysine) 1.64 1.61 1.55 3. Barley + L-lysine HC1 (0.75% t o t a l lysine) 1.51 1.59 1.56 4. Treat. 3 + 0.05% ^threonine 1.74 1.67 1.81 5. Treat. 4 + 0.10% DL-methionine 1.61 1.62 1.59 6. Treat. 5 + 0.10% L-isoleucine 1.66 1.67 1.63 Table IIB. Analysis variance of average d a i l y feed intake. Source D'.F. S.S. M.S. F-value Variance Ratio F(nec.) P=0.05 P=0.01 Total 17 1.3012 Treatment 5 1.1086 0.2217 23.9738** 3.2039 5.3160 Covariable s t a r t i n g weight 1 0.0253 0.0253 2.7382 4.8443 9.6460 Residual 11 0.1017 0.00925 ** P < 0.01 * P < 0.05 89 Table IIIA. Average feed conversion e f f i c i e n c y r a t i o per pig. (D.M. feed intake (kg)/weight gain (kg). Treatment Rep 1 Rep 2 Rep 3 1. Barley + Soybean (Control) 2.83 3.07 2.94 2. Barley + 0.69% L-lysine HC1 (0.9% t o t a l lysine) 3.55 3.46 3.59 3. Barley + 0.50% L-lysine HC1 (0.75% t o t a l lysine) 3.72 3.70 3.95 4. Treat. 3 + 0.05% L-threonine 3.33 3.25 3.37 5. Treat. 4 + 0.10% DL-methionine 3.40 3.20 3.30 6. Treat. 5 + 0.10% L-isoleucine 3.38 3.21 3.41 Table IIIB. Analysis of variance of feed conversion e f f i c i e n c y r a t i o Variance Ratio Source D.F. S.S. M.S. F-value F(nec.) p=0.05 p=0.01 Total 17 0. 1328 Treatment 5 0. 1128 0. 0226 12.72114** 3.2039 5.3160 Covariable s t a r t i n g 1 0. 000157 0. 000157 0.0886 4.8443 9.6460 weight Residual 11 0. 0195 0. 00177 * P < 0.05 P < 0.01 90 Table IVA. Minimum middle f a t (mm) Treatment Rep 1 Rep 2 Rep 3 Male Female . Male Female Male Female 1. Barley + Soybean 18 20 24 29 20 20 (Control) — 25 23 24 18 20 — — 19 18 21 23 2. Barley + 0.69% L-lysine 32 32 32 21 21 24 ( t o t a l 0.90% lysine) 30 28 33 24 28 18 29 22 27 19 17 21 3. Barley + 0.50% L-lysine 25 10 29 28 16 14 ( t o t a l 0.75% lysine) 24 23 24 17 25 15 22 18 26 22 30 19 4. Treatment 3 + 0.05% 33 26 25 18 20 21 L-threonine 26 24 21 25 24 25 26 22 27 15 33 21 5. Treatment 4 + 0.10% 26 22 23 18 26 18 DL-methionine 17 26 18 17 23 29 29 — 28 22 16 29 6. Treatment 5 + 0.10% 32 24 27 25 24 19 L-isoleucine 25 25 28 18 25 18 — 28 30 20 26 22 Table IVB. Analysis of variance of minimum middle f a t . Variance Ratio Source D.F. S.S. M.S. F-value F(nec.) p = 0.05 p = 0.01 Total 102 2344.427 Treatment (a) 5 197.5564 39. 5113 2.7410* 2.3683 3.3389 Rep (b) 2 74.9634 37. 4817 2.6002 3.1504 4.9774 Sex (c) 1 206.0355 206. 0355 14.29337** 4.0012 7.0771 a x b 10 270.2762 27. 0276 1.8750 1.9926 2.6318 a x e 5 116.5186 23. 3037 1.6167 2.3683 3.3389 b x c 2 44.5127 22. 2564 1.5440 3.1804 4.9774 a x b x c 10 78.6352 7. 8635 0.5455 1.9926 2.6318 Covariable 1 162.9588 162. 9588 11.3050** 4.0012 7.0771 * P < 0.05 ** P < 0.01 91 Table VA. Eye muscle (width x depth) 2 (mm ) of the 103 pigs • Treatment Rep 1 Rep 2 Rep 3 Male Female Male Female Male Female 1. Barley + Soybean 3496 3195 4080 3818 4131 (Control) 3666 3876 3744 3978 3456 3478 — — 3647 4131 4346 3773 2. Barley + L-lysine HC1 2451 3360 3216 3128 3818 3225 (0.9% t o t a l lysine) 2940 3408 2655 4080 3332 4480 2280 3024 3168 3311 3848 3900 3. Barley + L-lysine HC1 3256 3724 3015 4272 3888 3654 (0.75% t o t a l lysine) 2747 3713 2418 3444 3240 3200 2911 3621 3034 3698 3395 3096 4. Treatment 3 + 0.05% 3174 3330 2967 4437 3648 3036 L- threonine 3036 4374 3358 3420 3626 3266 3034 3519 3036 3588 4000 3430 5. Treatment 4 + 0.10% 3600 3476 3266 4080 3182 3266 DL-methionine 2856 3360 2829 3080 3036 3901 3696 — 3384 4042 3060 3219 6. Treatment 5 + 0.10% 3010 3042 3066 2970 2856 3358 L-isoleucine 3420 3124 3330 3680 3195 3975 — 3285 3287 2775 3900 3640 Table VB. Analysis of variance of eye muscle (width x depth). Variance Ratio Source D.F. S.S. M.S. F-value F(nec.) p=0.05 p=0.01 Total 102 21357320 Treatment (a) 5 1628173 325634.6 2.457* 2.368 3.339 Block (b) 2 882920 441460.0 3.332* 3.150 4.977 Sex (c) 1 2889814 288981.4 21.808** 4.001 7.077 a x b 10 2019311 201931.1 1.524 1.993 2.632 a x e „5 968194 193638.7 1.461 2.368 3.339 b x c 2 1949199 974599.5 7.355** 3.150 4.977 a x b x c 10 1050038 105003.8 0.792 1.993 2.632 Covariable 1 997188 997188.4 7.525** 4.001 7.077 Residual 66 8745643 132509.7 * P < 0.05 ** p < 0.01 92 Table VIA. Nitrogen retention (g/week). Treatment Rep 1 Rep 2 Rep 3 Rep 4 Rep 5 Rep 6 •1. Barley + Soybean (Control) 123.8 126. 5 115.1 116. 0 2. Barley + 0.69% L-lysine HC1 (0.9% t o t a l lysine) 56.0 59. 7 64.3 55. 3 42. 47 53.19 3. Barley + 0.50% L-lysine HC1 57.9 60. 5 72.4 67. 0 73. 01 80.61 4. Treatment 3 + 0.05% L-threonine 74.2 70. 2 88.2 73. 0 53. 43 65.98 5. Treatment 4 + 0.10% DL-methionine 56.2 63. 0 64.7 76. 4 56. 42 64.80 6. Treatment 5 +.0.10% L-isoleucine 41.3 60. 6 86.6 79. 5 70. 79 84.22 Table VIB. Analysis of variance f o r nitrogen retention. Variance r a t i o Source D.F. S.S. M.S. F-value F(nec.) P=0.05 P=0.01 Total 33 14685.70 Treatment 5 11565.06 2313.011 20.7535** 2.5336 3.6990 Residual 28 3120.642 111.4515 ** P < 0.01 93 Table VIC. Analysis of co-variance f o r nitrogen retention with feed intake as the covariable. Source D.F. S.S. M.S. F-value Variance r a t i o F(nec.) p=0.05 p=0.01 Total 33 14697.28 Treatment 5 9943.194 1988.639 63.4828** 2.5336 3.6990 Covariable s t a r t i n g weight 1 2275.892 2275.892 72.6526** 4.1709 7.5625 Residual 27 845.7928 31.3257 Table VID. Analysis of covvariance with nitrogen intake f o r nitrogen retention as the covariable. Source D.F. . S.S. M.S. F-value Variance r a t i o F(nec.) p=0.05 p=0.01 Total 33 14685.70 Treatment 5 945.2005 189.0401 5.2763** 2.5336 3.6990 Covariable s t a r t i n g weight 1 2153.281 2153.281 60.1002** Residual 27 967.3605 35.8282 ** P = <0.01 94 Table VIIA. Nitrogen retained as a percentage of nitrogen intake. Treatment Rep 1 Rep 2 Rep 3 Rep 4 Rep 5 Rep 6 46.64 30.64 37.09 39.37 35.78 38.89 40.14 37.41 36.12 38.90 46.15 42.00 41.74 40.95 41.11 46.86 Table VIIB. Analysis of variance f o r nitrogen retained as a percentage of nitrogen intake. Variance r a t i o Source D.F. S.S. M.S. F-value F(nec.) p=0.05 p=0.01 Total 33 0.0668 Treatment 5 0.0325 0.0065 5.3117** 2.5336 3.6990 Residual 28 0.0343 0.0012 ** P = < 0.01 ±. earxey + ioyDean (Control) 2. Barley + 0.69% L-lysine (0.90% t o t a l lysine) 3. Barley + 0.50% L-lysine (0.75% t o t a l lysine) 4. Treatment 3 + 0.05% L-threonine 5. Treatment 4 + 0.10% DL-methionine 6. Treatment 5 + 0.10% L-isoleucine 45.54 42.77 45.43 38.07 35.94 38.57 38.03 35.15 41.57 49.95 45.35 45.73 45.27 43.50 45.81 37.99 45.81 48.26 95 Table VIIIA. Apparent nitrogen d i g e s t i b i l i t y (%). Treatment Rep 1 Rep 2 Rep 3 Rep 4 Rep 5 Rep 6 1. Barley + Soybean (Control) 72.85 72.55 81.30 77.47 — — 2. Barley + 0.69% L-lysine HC1 (0.90% t o t a l lysine) 65.25 65.98 66.42 57.49 65.74 66.60 3. Barley + 0.50% L-lysine HC1 (0.75% t o t a l lysine) 59.84 57.50 66.69 62.44 62.17 63.03 4. Treatment 3 + 0.05% L- threonine 69.88 67.31 63.99 59.14 63.40 62.84 5. Treatment 4 + 0.10% DL-methionine 66.35 68.82 68.72 65.37 63.52 63.26 6. Treatment 5 + 0.10% L-isoleucine 65.24 68.70 . 71.27 65.23 63.86 69.08 Table VIIIB. Analysis of variance f o r apparent nitrogen d i g e s t i b i l i t y . Source D.F. S.S. M.S. F-value Variance r a t i o F(nec.) p=0.05 p=0.01 Total 33 0.0840 Treatment 5 0.0537 0 .0107 9.957* * 2.5336 3.6990 Residual 28 0.0302 0 .0011 ** P < 0.01 96 Table IXA. Nitrogen retained as a percentage of nitrogen absorbed. Treatment Rep 1 Rep 2 Rep 3 Rep 4 Rep 5 Rep 6 1. Barley + Soybean (Control) 62.51 58.96 55.88 60.20 — — 2. Barley + 0.69% L-lysine (0.90% t o t a l lysine) 58.35 54.50 58.07 53.30 56.42 59.11 3. Barley + 0.50% L-lysine (0.75% t o t a l lysine) 63.55 61.13 62.23 57.31 62.56 63.68 4. Treatment 3 + 0.05% L-threonine 71.47 67.37 71.46 63.26 56.86 61.90 5. Treatment 4 + 0.10% DL-methionine 68.23 63.21 66.66 70.59 66.13 65.97 6. Treatment 5 + 0.10% L-isoleucine 58.22 66.67 67.71 62.83 64.38 67.84 Table IXB. Analysis of variance f o r nitrogen retained as a percentage of nitrogen absorbed. Source D.F. S.S. M.S. F-value Variance r a t i o F(nec.) p=0.05 p=0.01 Total Treatment Residual 33 5 28 0.0779 0.0437 0.0342 0.0087 0.012 7.153** 2.5336 3.6990 ** P <0.01

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