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UBC Theses and Dissertations

Study of some techniques for evaluating protein quality Goudie , Carol 1970

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A STUDY OF SOME TECHNIQUES FOR EVALUATING PROTEIN QUALITY b y CAROL GOUDIE B.Sc. University of B r i t i s h Columbia, 1958 A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE i n the Department, of POULTRY SCIENCE We accept this thesis as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA December, 1970. In presenting t h i s t h e s i s in p a r t i a l f u l f i l m e n t of the requirements f o r an advanced degree at the U n i v e r s i t y of B r i t i s h Columbia, I agree that the L i b r a r y s h a l l make i t f r e e l y a v a i l a b l e f o r reference and study. I f u r t h e r agree that permission for e x t e n s i v e copying of t h i s t h e s i s f o r s c h o l a r l y purposes may be granted by the Head of my Department or by h i s r e p r e s e n t a t i v e s . It i s understood that copying or p u b l i c a t i o n of t h i s t h e s i s f o r f i n a n c i a l gain s h a l l not be allowed without my w r i t t e n p e rmission. Depa rtment The U n i v e r s i t y of B r i t i s h Columbia Vancouver 8, Canada Date ^ J U c 2 - f / 9 7Q Abstract The present study consisted of three sections eaoh of which was undertaken to obtain information on some aspect of protein q u a l i t y evaluation by b i o l o g i c a l t e s t i n g . The f i r s t study consisted of two experiments i n which the free amino acids were determined i n the plasma of birds fed cereal d i e t s to determine i f t h i s method would indicate the amino acid adequacy of the d i e t s . The second study was an investigation of the use of chicken muscle meal as a reference supplementary protein. The t h i r d study consisted of an investigation of the f r a c t i o n a t i o n of i n t e s t i n a l nitrogen as a method for studying protein q u a l i t y . In the f i r s t series of experiments i n which plasma free amino acids were determined to investigate protein q u a l i t y two groups of White Leghorn chicks each were fed a control diet consisting of a high protein wheat diet supple-mented with 0.24% lysine and 0.06% methionine and the control diet further supplemented with 0.1% L-arginine and glycine and 0.045% L-leucine and L-threonine. Blood samples were collected from birds on each diet a f t e r a 14 hour f a s t and a f t e r 3 hours and 6 hours of ad libitum feeding following the f a s t . The PFAA patterns obtained at both 3 and 6 hours a f t e r commence-ment of feeding from chicks which had been fed these two diets for three weeks indicated a lysine deficiency. The 6 hour plasma pattern from chicks fed the supplemented diet showed a greater percentage of threonine and l e s s of the other EAA except arginine i n comparison with the pattern seen i n the plasma from birds fed the oontrol d i e t . i Lysine was shown to be the f i r s t l i m i t i n g amino acid i n diets containing 10.7$ crude protein (N x 6.£5) from wheat and 4$ crude protein from rapeseed or soy bean meal by growth test and PFAA patterns. After 3 hours of ad l i b i t u m feeding d i f f e r e n t l e v e l s of c i r c u l a t i n g lysine were observed i n the plasma from birds fed the • raj>eseed diets supplemented with 0.15$ l y s i n e . The levels of c i r c u l a t i n g lysine were not s i g -n i f i c a n t l y d i f f e r e n t due to sample v a r i a t i o n , but i n general the higher l e v e l s of plasma lys i n e were seen i n birds with the heavier body weights. A single l e v e l of supplementation was inadequate to determine the next l i m i t i n g amino acid i i n these d i e t s . In an investigation of the use of chicken muscle meals as a reference supplementary protein various chicken muscle meals were compared to an isol a t e d soy protein and methionine reference supplement. The meals were prepared by freeze drying either cooked or raw t i s s u e . One meal was treated with antioxidant. Diets containing 8$ protein from the protein supplement and 10$ pro*©* 1 1 from the wheat basal di e t supported as good growth as that obtained with a good q u a l i t y commercial fishmeal when tested at the same l e v e l of supplementation. A l l the chicken meals and the fishmeal supported better growth than the ISP and methionine r e f e r -ence c o n t r o l . A few small t r i a l s to investigate the use of gut n i t r o -gen l e v e l s for evaluating protein q u a l i t y were carried out. Preliminary t r i a l s included the determination of the nitrogen recoverable a f t e r the administration of d i f f e r e n t test meals with d i f f e r e n t protein sources, carbohydrates and f i b e r . Results obtained a f t e r administering single test meals were not considered r e l i a b l e . A method was used where by the i n t e s t i n a l contents were sampled one-half hour a f t e r several meals had been administered and the nitrogen fractionated into TCA soluble and insoluble portions. ¥hen the amount of recoverable nitrogen insoluble i n TCA was taken as the c r i t e r i o n of quality, differences were demonstrated between a sample of good qua l i t y protein supplement and one of extremely poor q u a l i t y . Less of the nitrogen recovered from birds fed a high percentage of f i b e r i n the test meals was soluble i n TCA than when the protein was fed with corn starch or glucose mono-hydrate. The amount of l i q u i d i n the s l u r r y was found to affept the amount of nitrogen recoverable i f several feedings were ad--ministered i n a r e l a t i v e l y short period of time. Once the bio-l o g i c a l method was established preliminary determinations of the amounts of recoverable free basic amino acids from the i n t e s t i n a l segments were carried out. Generally a greater number of micromoles of basic amino acid per segment were found i n the jejunum than i n the ileum. In some cases there was l e s s of these amino acids present i n the gut of birds fed protein with glucose monohydrate than from the gut of birds receiving other treatments but t h i s may only hatoe been due to the e f f e c t of the l i q u i d i n the s l u r r y . I t was recommended that p e l l e t s would be a more b i o l o g i c a l l y accurate method to administer the test meals. Acknowledgement The writer wishes to express her gratitude to Dr. Jacob B i e l y of the Department of Poultry Science f o r his constant encouragement and i n t e r e s t during this study. Acknowledgement i s extended to Professor B.E. March of the Department of Poultry Science f o r suggesting the ex-periments and f o r help i n organizing the thesis material. Thanks are also given to Dr. D.B. Bragg of the same department f o r his valuable advice concerning amino acid analysis, and to Drs. P.M. Townsley of the Department of Food Science and J?'. Ford of the Department of Zoology f o r reading the manuscript and f o r t h e i r advice. TABLE OF CONTENTS Page Introduction 1 Part 1. Plasma free amino acids (PFAA as a c r i t e r i o n of protein quality-L i t e r a t u r e Review 3 Experimental Experiment I 22 Results and Discussion 30 Summa ry 36 Experiment I I 37 Results and Discussion 42 Summary 46 Part 2. Chicken muscle as Reference Protein Introduoti on 47 Preparation of chicken meals 48 Chemical Analysis 51 B i o l o g i c a l evaluation Experiment I I I 55 Experiment IV 60 Results and Discussion 64 Summary 70 Part 3. T r i c h l o r a c e t i c aoid (TCA) f r a c t i o n a t i o n of gut nitrogen as a method for studying protein q u a l i t y . Introducti on 71 General experimental procedures 76 General discussion 95 Summary 99 Bibliography 100 Introduction The primary function of dietary protein i s to supply amino acids f o r the synthesis of body proteins. The a b i l i t y 1 of a protein to f u l f i l l t h i s function i s a measure of i t s q u a l i t y . Several factors influence the q u a l i t y of a protein per se. The f i r s t of these i s i t s amino acid composition, more s p e c i f i c a l l y , the balance of the eseential amino acids i n the protein with respect to a p a r t i c u l a r animal*s require-ment. The second faotor influencing q u a l i t y and more d i f f i c u l t to measure i s the d i g e s t i b i l i t y and a v a i l a b i l i t y of the essen-t i a l amino acids i n a protein source. D i g e s t i b i l i t y and a v a i l a b i l i t y are influenced by the treatment of the protein material p r i o r to processing, the processing i t s e l f , and storage con-d i t i o n s . Most proteins used as supplementary sources of es s e n t i a l amino acids i n poultry diets must undergo some pro-cessing p r i o r to use. Chemical analysis of a protein can provide information ©n the t o t a l amino acid composition. I t does not provide i n -formation on the a v a i l a b i l i t y of the amino acids. Standard b i o l o g i c a l methods such as b i o l o g i c a l value, net protein u t i l i z a t i o n and net protein r a t i o provide information about the o v e r a l l n u t r i t i v e value of a protein source. These t e s t s , how-ever do not indicate which amino aoid i s l i m i t i n g nor d© they give information about the other amino aoids. Beoause of t h i s the effeot of mixing protein souroes cannot be forecast,;. Three aspects of protein q u a l i t y evaluation by bio-l o g i c a l t esting were chosen as requring some preliminary study: 1) Plasma free amino acids (.PFAA) as a c r i t e r i o n of protein q u a l i t y . 2) The need f o r a protein source to serve as a p r a c t i c a l reference standard. 5} T r i c h l o r a c e t i c acid (TCA) f r a c t i o n a t i o n of gut nitrogen as a method for studying protein q u a l i t y . This study, therefore, i s composed of three sections each dealing with one of the above aspects of the b i o l o g i c a l evalua-t i o n of protein q u a l i t y . 3 P a r t 1. P l a s m a f r e e amino a o i d s (PFAA) a s a o r i t e r i o n o f p r o t e i n q u a l i t y . L i t e r a t u r e R e view I n 1906 H o w e l l r e p o r t e d t h a t t h e c o n c e n t r a t i o n o f amino a c i d i n t h e p o r t a l v e i n was g r e a t e r t h a n t h a t l n t h e j u g u l a r v e i n a f t e r i n g e s t i o n of a p r o t e i n m e a l . F o l l o w i n g t h i s Van S l y k e and M e y e r (1912) r e p o r t e d t h e p r e s e n c e o f amino n i t r o g e n I n t h e n o n - p r o t e i n f r a c t i o n o f t h e b l o o d o f dogs and n o t e d t h a t i t s c o n c e n t r a t i o n i n c r e a s e d a f t e r f e e d i n g meat. T h a t t h e f r e e amino a o i d s a r e t r a n s p o r t e d a s s u c h i n t h e b l o o d was o b s e r v e d by A b e l , Rowntree and T u r n e r ( 1 9 1 3 ) . S i n c e t h i s e a r l y work many s t u d i e s have been done r e l a t i n g d i e t t© t h e c o n c e n t r a t i o n o f f r e e amino a c i d s i n b o t h p o r t a l and s y s t e m i c b l o o d . The l i t e r a t u r e on t h i s s u b j e c t c o v e r s s t u d i e s w i t h human s u b j e c t s , d o g s , r a t s and c h i c k e n s and i s c o n c e r n e d m a i n l y w i t h t h e p h y s i o l o g y o f d i g e s t i o n and a b s o r p t i o n , p a t h o l o g i o a l c o n d i t i o n s and amino a c i d n u t r i t i o n . I n r e v i e w i n g r e s e a r c h r e l a t i n g p l a s m a f r e e amino a c i d (PFAA) l e v e l s and p r o t e i n q u a l i t y M c L a u g h l a n (1963) p l a c e s s t u d i e s i n t h i s f i e l d i n t o t h r e e m a j o r c l a s s e s : (1) d e t e r m i n a t i o n o f p e a k c o n c e n t r a t i o n o f amino a c i d i n p o r t a l b l o o d . (2) q u a n t i t a t i v e r e l a t i o n s h i p s between t h e p l a s m a amino a c i d s i n s y s t e m i c b l o o d a n d c o m p o s i t i o n o f the i n g e s t e d p r o t e i n . (3) methods f o r p r e d i c t i n g t h e l i m i t i n g amino a c i d i n a f o o d . D e n t o n and E l v e h j e m (1954) f a s t e d dogs w i t h p o r t a l v e i n c a n n u l a e 18 h o u r s t h e n f e d them 200 gm. of a c o m p l e t e r a t i o n (16$ protein) containing either beef, casein or zein, ' Determination of the PFAA showed that the concentration of a l l 10 amino acids i n the plasma increased i n both the p o r t a l and r a d i a l veins a f t e r feeding beef and casein. Those i n the p o r t a l vein increased i n 0 - 2 hours and those i n the r a d i a l vein much'slower peaking around 4 - 6 hours. The amino acids were detected i n much larger quantities i n the p o r t a l vein than i n the r a d i a l vein. The concentrations of most of the e s s e n t i a l amino acids ^decreased i n dogs fed z e i n . Wheeler and Morgan (1958) studied changes i n the con-centration of p o r t a l blood amino acids as a measure of the rate of digestion of protein by r a t s . The rats were trained to^ eat 5.0 gm. of either raw or overheated pork and samples of portal blood v/ere taken at i n t e r v a l s from 0.5 to 5 hours a f t e r the test meals. Peak concentrations of PFAA were ob-tained approximately 45 min. a f t e r the meals i n adult animals fed pork, but not u n t i l 5 hours a f t e r feeding i n animals given overheated pork. Th i r t y minutes a f t e r the meal, free amino acid l e v e l s i n plasma of young rats fed raw pork varied from 101 to 340$ of those i n animals given overheated pork. The average weight gain of rats fed overheated pork was 22$ 'less than that of rats fed raw pork. They interpreted the r e s u l t s as supporting t h e i r theory that decreases i n b i o l o g i -c a l value caused by overheating proteins are at le a s t i n part due to f a i l u r e of digestive enzymes to hydrolyse the over-heated proteins so as to make available simultaneously to the animal an assortment of amino acids favourable to good growth or maintenance. 5 In an attempt to correlate the b i o l o g i c a l a v a i l a b i l i t y of the most l i m i t i n g amino acids i n some food proteins with t h e i r concentration i n the respective proteins Guggenheim, Halevy and Friedmann (1960) measured the free lysine and methionine i n the vena cave of rats fed wheat gluten, zein, soya protein, casein and lactalbumin. The concentrations of these amino acids increased i n amounts apparently related to the amino acid composition of the protein. Moderate heating of raw soybean protein increased plasma amino acid l e v e l s whereas over-heating reduced them. Various carbohydrates taken with the soy-bean protein also influenced amino acid l e v e l s i n the plasma. Goldberg and Guggenheim (1962) reported much higher l y s i n e and methionine l e v e l s i n rats fed casein rather than lactalbumin although the concentrations of these amino acids were approximately the same i n the two proteins. They con-cluded that there was not a close r e l a t i o n s h i p between the rate and extent of the release of amino acids during digestion and the amino acid composition of dietary proteins. The r e l a t i o n s h i p between plasma amino acid l e v e l s of systemic blood and protein q u a l i t y has been studied i n several species of animals. Charkey et a l . (1953) and Richardson, Blaylock and Lyman (1953) measured arginine,. l y s i n e , methionine, tryptophan and ,sj^line i n the plasma of chicks fed diets con-tai n i n g soybean meal and peanut ©eal. The concentration of each of these amino acids correlated reasonably well with i t s concentration i n the dietary protein. In reviewing these experiments Almquist (1954) drew attention to the f a c t that a dietary deficiency of an amino acid r e s u l t s i n inordinately 6 low levels of the amino acid in the plasma. Low plasma arginine levels were observed in animals given a soybean protein-casein diet whereas lysine was markedly depressed in the plasma of chicks fed peanut meal. A l l of these findings were consistent with the theory that an abnormally low level of a particular amino acid in the plasma indicated a deficiency of that am,ino acid in the dietary protein. This relationship has been demonstrated for lysine in human subjects (Albanese and Orto, 1960) as well as in dogs (Denton and Elvehjem, 1954), rats (MoLaughlan et a l . 1961) and chicks (Richardson et a l . 1953; H i l l and Olsen, 1963a); and for methionine in rats (Wagle, Mitbander and Sreenivasan, 1962), and pigs (Puohal et a l . 1962); and for tryptophan In dogs (Longeneoker and Hause, 1959) and chicks ( H i l l , Mclndoo and Olsen, 1961). It has been reported that the concentrations of plasma amino acids i n -cluding that of an amino acid which is deficient in the diet, may be elevated when growth i s limited by suboptimal amounts of the B-complex vitamins in the diet. (Charkey et a l . 1950; Wagle et. a l . 1962). Ohilds and Combs (1964) assessed the availability of the methionine in fishmeals known to dif f e r in their protein quality as measured by chick assay. Higher levels of methionine were observed in the plasma when the higher quality fishmeals were fed. However i t was stressed that the test ingredients must be consumed in essentially the same amounts during the feeding period i f the results were to be valid* In an attempt to develop a bioassay using plasma free amino acids (PFAA) levels in young growing chicks as a measure of protein q u a l i t y Smith and Soott (1955 a, b); .conducted a series of experiments. In the feeding phase of the assay ohioks having undergone a 4 hour fas t were fed 12 meals (0.8 gm. of diet/meal) spaoed 30 minutes apart over a period of 6 hours. Blood f o r analysis was taken 30 minutes a f t e r the ingestion of the terminal meal. This feeding procedure afforded an opportunity f o r the plasma pattern to reach a "steady state" i n response to a given di e t (Dean, 1963). PFAA t i t r e s obtained from chicks fed the standard c r y s t a l l i n e amino acid diet (equivalent to 18.34% crude protein) were compared with those obtained from ohicks fed an equivalent amount of i n t a c t protein In place of the c r y s t a l l i n e mixture. The percentage ohange calculations f o r each amino acid were as follows: Test protein plasma oono. - reference plasma cone, x 100 Referenoe plasma concentration. The referenoe plasma concentration f o r most comparisons was that l e v e l of amino acid noted i n the plasma through the feeding of the standard reference d i e t , although i n desired comparisons as i n heat treatment effeots the unheated protein plasma l e v e l s were used as the referenoe concentration. The magnitude of the percentage ohange was to indicate the r e l a t i v e adequacy of an amino acid to that of the referenoe d i e t , where inadequacies or amino acid d e f i c i e n c i e s were i n -dicated hy increasing negative values. The test proteins included various fishmeals, some overheated, sesame meal fed with and without l y s i n e supplementation and raw, heated and overheated soybean meals fed with and without methionine supplementation. 8 Smith and Scott (1965 b) found that the PFAA assay-described could be of considerable value when a q u a l i t a t i v e comparison of the amino acid a v a i l a b i l i t y of s i m i l a r proteins was desired. A low l e v e l of lysine i n the plasma of sesame meal fed chicks showed that l y s i n e i s the f i r s t l i m i t i n g amino aoid i n t h i s protein. S i m i l a r l y , methionine was observed to be the f i r s t l i m i t i n g amino acid i n a l l the soybean meals studied. Comparisons of meals with and without supplemental t i o n of t h e i r f i r s t l i m i t i n g amino acid showed an increased u t i l i z a t i o n of a l l amino acids f o r protein synthesis. This was demonstrated by a marked reduction of a l l the other amino acids i n the plasma. An o v e r a l l apparent loss of d i g e s t i b i l i t y r e s u l t s from severe heat treatment of proteins and the amino aoids of raw soybean meal were shown to be l e s s available to chicks than those of properly heated soybean meal. However the use of this technique to predict the order and degree of amino aoid l i m i t a t i o n of i n t a c t proteins f o r chiok growth was shown to be dependant upon theextent to which the standard reference diet provided the amino aoid require-ments f o r chiok growth. As an i l l u s t r a t i o n of t h i s pointy i n addition to the reduction i n the plasma concentration of the l i m i t i n g amino acid there was a continual "apparent" deficiency of h i s t i d i n e , threonine and methionine i n proteins where no deficiency oould be demonstrated by amino acid supplementation and growth t r i a l s . Modifications i n the amino acid composition of the c r y s t a l l i n e amino aoid reference diet used by Smith and Soott (1965 a) were e a s i l y detected i n the plasma of chicks when 9 oompared to that obtained from chicks fed the standard referenoe diet (Dean and Scott, 1966). In t h i s case the PAS t i t r e was a sensitive indicator of amino acid adequacy. Diets containing suboptimal amounts of either lysine or valine resulted i n a marked lowering of the l i m i t i n g amino acid and an increase i n most of the other amino acids i n the plasma. A diet i n which lysine and valine were demonstrated by growth assay to be f i r s t and second l i m i t i n g , respectively, resulted i n a decrease i n these two amino acids i n plasma with lys i n e showing the greater decline. Most of the other amino aoids i n plasma increased as a r e s u l t of t h i s treatment. A dietary exoess of l y s i n e resulted i n a marked increase i n plasma l y s i n e and r e l a t i v e l y small changes i n other amino acids. A diet containing suboptimal amounts of valine and threonine and superoptimal amounts of l y s i n e , tyrosine and isoleuoine resulted In marked changes i n the corresponding amino aoids l n plasma, the former two decreased and the l a t t e r three increased. R e l a t i v e l y small ohanges were observed i n the other amino acids i n plasma. In the development of the synthetic reference diet used i n these experiments Dean and Soott (196£) were able to define t h e i r amino acid d i e t , which supports good chick growth, by determining i n turn the l e v e l ofeach amino acid required to maximize weight gain. L i t t l e attention was paid to amino acid l e v e l s i n exoess of requirement unless obvious growfch depressions resulted. In a follow up on the basic problem of the r e l a t i v e amounts of the c r y s t a l l i n e amino acids i n a reference standard f o r comparison with plasma t i t r e s obtained 10 from intact protein Smith (1966 a) used two reference diets. Both were developed by Dean and Scott (1962, 1965). Sunflower meal was the test protein. Both these crystalline amino acid mixtures were shown to support equal rates of growth but one of the amino aoid mixtures had five essential amino aoids at levels greater than the others and henoe could be considered in excess of the ohicks* requirement for growth. Chlola growth t r i a l s were con-ducted whioh established that lysine was the only limiting amino acid in sunflower meal. Plasma amino acid t i t r e s were determined on chicks fed sunflower meal and on chicks fed the two crystalline amino aoid mixtures! A lower amino acid t i t r e in the plasma of chicks fed the sunflower meal than in ohicks fed the reference diet was taken as an indication of a deficiency of this amino aoid in the intaot proteins. Using the amino acid mixture containing the excess amino acids as a reference, sunflower meal appeared to be limiting in several amino acids. When the other mixture was used as a reference, oniy lysine appeared to be limiting. Smith (1966 a) points out that the use of plasma t i t r e s to evaluate proteins by this method therefore depends on a referenoe diet which contains neither deficient nor excessive levels of amino acids. This important aspect was also pointed out by Smith and Scott (1965 b) - wthe selection of an intact protein or some other amino aoid mixture as a control diet merely because i t promotes good growth of this animal can lead to serious misinterpreta-tions when used to measure the amino acid adequacy of various other proteins by this technique." 11 In their earlier reports on plasma amino acid levels as indicators of protein quality Smith and Scott (1965 a) em-phasized that valid comparisons between the referenoe and test diets could be achieved only under conditions of equal feed intake and after absorption had reached a "steady state". To ensure these conditions an elaborate feeding practice was followed (Dean, 1963). In subsequent investigations both these workers and Smith (1966 a) observed that diets with amino acid deficiencies tended to aooumulate in the crop in greater quantities than balanced diets. This was taken as an indica-tion that the diets were not reaching the absorption areas of the intestinal tract at equal rates. To improve the precision of the PFAA assay Smith (1966 b) suggested the use of an internal standard/ aminoisobutyric acid (AIBA), which would be added to each diet. Its level in the plasma would provide a relative measure of the amounts of feed reaching the small intestine. Absorption of AIBA into the blood stream was re-ported to be at a rate which was proportional to dietary levels. Chioks fed imbalanced diets exhibited lower AIBA levels in the plasma than those fed the complete reference diet. Conversion of the plasma t i t r e s to an equi-AIBA ratio helped to reduce the number of "apparent" deficiencies by yielding; PAA t i t r e s which were more or less proportional to dietary intake. However a possible arglnine-sparing effect has been demonstrated both in plasma and muscle free amino acids from chicks fed either a ossein or a wheat gluten based alet supplemented with arginine and to which this compound was incorporated (Tsang-Cheng Shao and H i l l , 1969). Several methods for assessing PFAA changes i n order to predict the l i m i t i n g amino acid i n a food have been suggested. The s u i t a b i l i t y of the reference PAA pattern i n some of the methods for chicks i s reviewed. In investigating the r e l a t i o n -ship between PAA and the composition of the ingested protein Longenecker and Hause (1959) proposed the plasma amino acid r a t i o which was calculated: PAA r a t i o - ^ B " A * X 100 Amino acid requirement of the adult dog where B was the average of 5 hourly l e v e l s of PAA concentration i n mg/100 ml plasma a f t e r a single large test meal. A was the PAA concentration a f t e r an 18 hour f a s t immediately before the meal. The PAA changes i n the adult dog a f t e r a meal were found to be d i r e c t l y dependent upon the amino acid composition of the protein ingested i f i t were postulated that the i n d i v i d u a l e s s e n t i a l amino acids were removed from the blood by the body tissues at rates proportional to the amino acid requirements of the dog. By this method l y s i n e was found to be f i r s t l i m i t i n g amino acid i n wheat gluten, tryptophan i n g e l a t i n and arginine i n casein. In r i c e , the PAA r a t i o indicated ly s i n e to be the f i r s t l i m i t i n g amino acid and threonine the second. These data were i n agreement with rat growth studies (Longeneoker, 1963). McLaughlan (1964) investigated various procedures for expressing the PAA data obtained from rats fed p r a c t i c a l d i e t s . The f i r s t procedure evaluated was a modified PAA r a t i o method. PAA r a t i o s were calculated from the equation: Mean PAA l e v e l , - Mean PAA l e v e l , * * * * J T>.» - t e s t group fasted group (12 Hr.) X 100 Modified PAA r a t i o = ;— a K B e  Animals requirement f o r amino acids By t h i s method l y s i n e was the l i m i t i n g amino acid i n both egg and 13 oatmeal d i e t s and trytophan was l i m i t i n g i n a corn f i s h f l o u r d i e t , but t h i s method also indicated that tryptophan was l i m i t i n g i n peas which are def i c i e n t i n methionine by growth t r i a l s . Therefore the r a t i o method gave a misleading impression of the l i m i t i n g amino acid i n certa i n foods. The seoond method tested was designated as the PAA score (egg pattern). The PAA pattern of r a t s fed an egg d i e t was used as a reference pattern with which to compare PAA patterns obtained with other d i e t s . PAA l e v e l s found with egg were set at 100. Most of the foods appeared to be l i m i t i n g i n methionine as judged by egg scores. The s l i g h t deficiency of lysi n e and exoess of methionine i n egg were r e f l e c t e d to an exaggerated degree i n the plasma l e v e l s of these amino acids thereby making PAA egg pattern unsuitable as a reference standard. The t h i r d method tested, the PAA score ( f a s t i n g pattern), was defined as the r a t i o of the concentration of an amino acid i n the plasma of rats i n the test group to the concentration of the amino acid i n the plasma of fasted r a t s , expressed as a percentage: Mean PAA l e v e l , test group PAA soore = X 100 Mean PAA l e v e l , fasted group As with the other methods tested the lowest soore indicated the l i m i t i n g amino aoid i n the d i e t . Deficiency of an amino acid i n the plasma was re f l e c t e d to an exaggerated degree i n the PAA scores. Lysine was the l i m i t i n g amino acid i n both egg and oatmeal diets while methionine appeared to be l i m i t i n g i n the pea d i e t . On the corn f i s h f l o u r d i e t the PAA score f o r trypto-phan was very low, i n agreement with the calculated value. Thus, McLaughlan (1964) considered the PAA score (fasting pattern) to 14 be the most s a t i s f a c t o r y method for detecting the l i m i t i n g amino acid i n a d i e t . Rao, Noel and McLaughlin (1968) tested the PAA score and PAA r a t i o method f o r predicting the l i m i t i n g amino acids for rats i n p r a c t i o a l diets containing mixtures of cereals and protein supplements. The cereals consisted of r i c e , wheat and corn and the supplements included peanut, soy, cottonseed and chick pea f l o u r s . Levels of plasma l y s i n e , methionine, threonine, tryptophan, leucine, and isoleueine were determined. Growth tests were carr i e d out with the d i e t s , with or without a supplement of the amino acids Indicated to be l i m i t i n g by these two PAA methods. PAA r a t i o s , PAA scores, and protein e f f i c i e n c y r a t i o data of rats indicated that l y s i n e and threonine were equally l i m i t i n g i n rice-peanut, rice-soy, rice-cottonseed, rioe-ohiok pea, wheat-peanut, and wheat-cottonseed d i e t s . The plasma methods were also correct i n showing that l y s i n e was the l i m i t i n g amino acid i n corn-peanut, corn-soy and corn-cottonseed mixtures. Plasma methods did not indicate a methionine deficiency when this amino acid was co-l i m i t i n g with lysine and threonine. Chemical scores f a i l e d to deteot the threonine deficiency present i n 8 of 12 d i e t s . Limiting amino acids were not established for the corn-chick-pea d i e t . Rab et a l . (1968) concluded that these PAA methods were e f f e c t i v e for predicting a deficiency of a single amino acid or of two c o - l i m i t i n g amino acids, but les s s a t i s f a c t o r y f o r predicting multiple amino acid d e f i c i e n c i e s . As noted by H i l l and Glsen (1963 b) the s u i t a b i l i t y 15 of the f a s t i n g PAA pattern as a reference f o r the chick must be questioned. A marked inorease i n the l e v e l s of free lysine and threonine i n the blood plasma of fasted chicks was reported by Charkey et a l . (1953, 1954) and confirmed by Gray et a l . (1960). That this increase i s a progressive one has been shown for plasma lysi n e ( H i l l and Olsen, 1963 bi Zimmerman and Scott, 1967 b; Boomgaardt and McDonald, 1969) and threonine ( H i l l and Olsen, 1963 b; Boomgaardt and McDonald, 1969) l e v e l s with increase i n length of the fasting period . A l l three groups of workers found considerable v a r i a t i o n i n the fasting pattern f o r Individual amino acids. Zimmerman and Scott (1967 b) remarked that i t would make a considerable difference i n data interpre-t a t i o n whether a 3 or 24 hour fasting. PAA pattern was used as a referenoe point. To overoome the accumulation of some amino acids i n the plasma of f a s t i n g chicks H i l l and Olsen (1963 b) proposed a modification i n the PAA r a t i o method of Longenecker and Hause (1959). They replaced the fasting plasma pattern with one exhibited by chicks fed a non-protein nitrogen (NPN) d i e t . When chioks received the NPN portion of the diet f o r 24 hours, rather than being completely deprived of feed, the concentration of a l l jamino acids investigated, including l y s i n e and threonine were below values obtained when the complete diet was fed. In a more detailed investigation of the e f f e c t of feeding a NPN diet on plasma amino aoid l e v e l s i n the chiok, Zimmerman and Soott (1967 b) found that t h i s lowered the con-oentration of e s s e n t i a l amino acids i n the blood plasma below the l e v e l noted when chicks were fasted over a comparable 16 period of time (5, 6, 12, or 24 hours). But plasma l y s i n e always increased with time even when the chicks were fed a NPN d i e t . Plasma threonine decreased and the other amino aoids were l a r g e l y unaffeoted. The accumulation of lysine i n the plasma of both the fasted and NPN fed chicks indicates a li m i t e d use of these PAA patterns as referenoe points. Another method of expressing amino acid adequacy, already mentioned, was that used by Smith and Soott (1965 a,b) i n which the reference pattern was that obtained from chicks fed a complete c r y s t a l l i n e amino acid standard d i e t . This synthetic diet was chosen to avoid using an intaot protein as a referenoe standard i n which the a v a i l a b i l i t i e s of the con-stituent amino aoids would be unknown. There are detractions from using a synthetic diet as a referenoe standard i n this manner. One i s the determination of the exact amino aoid require-ments i n the reference standard. This point has already been noted i n the review of the development of the chick PFAA assay. Zimmerman and Scott (1965) have reported that the shape of the plasma amino acid curve oould be used to determine the amino aoid requirement of the chick. They demonstrated a d e f i n i t e r e l a t i o n s h i p between PFAA l e v e l s and the amino acid adequacy of a synthetio d i e t . The f i r s t l i m i t i n g amino aoid remains at a very low l e v e l i n the blood i r r e s p e c t i v e of the s e v e r i t y of the amino aoid deficiency^ for example, the l y s i n e p r o f i l e indicated that this amino aoid did not aooumulate i n the plasma so long as the oonoentration of dietary lysine was l e s s than that needed (0.83%) to maximize weight gain. Not u n t i l l y s i n e l e v e l s i n exoess of 0.94%, i . e . approximately 10% i n i n exoess, were fed did t h i s amino aoid s t a r t to aooumulate i n the plasma. After t h i s l e v e l had been reached lysi n e accumulated very r a p i d l y i n the plasma. Similar r e s u l t s were obtained with valine and arginine. Since the point at which the amino acid s t a r t s to accumulate i n the plasma coincides with the break i n the growth curve they concluded that the plasma technique could be used to determine the chicks re-quirement f o r an amino a c i d . I t must be emphasized that these conclusions were drawn from long-term (7 day) assays. Data reported by Zimmerman and Scott (1967 a) indicate that the length of the experimental feeding period w i l l a l t e r the shape of the plasma amino acid curve when diets containing graded l e v e l s of single amino aoids are fed. In both a 50 minute and a 6 hour assay lysine accumulated i n the plasma even though the d i e t a r y concentration of t h i s amino aoid was l e s s than the amount needed to maximize growth. This accumulation of plasma lysine i n a 30 minute assay has been reported to be highly correlated with l y s i n e intake and to be l i n e a r (Kelly and Scott, 1968). A seoond d i f f i c u l t y i s the problem of comparing re-su l t s obtained from feeding a c r y s t a l l i n e amino aoid diet to those obtained from feeding a diet containing i n t a c t p r o t e i n . There are at least two aspects to thi s problem, the r e l a t i v e growth rate and degree of PAA response. The complex problem of comparing the n u t r i t i v e values of amino acid mixtures with those of whole proteins has been b r i e f l y disoussed by Bender (1969). There i s a difference between the * i d e a l * amino acid mixture f o r maximum nitrogen retention (Bender, 1961, 1965) 18 and one for maximum growth. Work with rats has shown that the addition of asparagine to an amino aoid mixture (Breuer et a l . 1966) and the i n c l u s i o n of several of the non-essential amino acids rather than the larger quantities of only a few of these improve the growth rate (Salmon, 1964; Rogers and Harper, 1965) . The degree and time of PAA response to the administra-t i o n of free amino acids i s d i f f e r e n t from that shown by intact p r o t e i n . Kratzer (1944) observed that the o r a l administration of a hydrolysed protein ( g e l a t i n and casein) caused a greater increase i n the blood amino nitrogen of a chick than does the administration of the i n t a c t p rotein. When 75 mg. of lysine was force-fed as a s l u r r y i n a c r y s t a l l i n e amino acid mixture plasma l y s i n e increased r a p i d l y and reached a peak 20 to 30 minutes a f t e r the c r y s t a l l i n e amino acid meal had been ingested and decreased sharply thereafter. When an equivalent amount of lysine i n dried egg white was fed only minor differences i n plasma l y s i n e concentrations were noted over the range of 0 - 4 hours. A much greater concentration of plasma lysine was associated with the c r y s t a l l i n e lysine meal than with the dried egg white meal. When a constant doee of lysine (25 mg./ meal) was administered i n a series of multiple meals, plasma l y s i n e increased i n a l i n e a r fashion up to the sixteenth and f i n a l meal when the c r y s t a l l i n e amino acid mixture was used, whereas plasma l y s i n e plateaued at the eighth meal of dried egg white. However, when the c r y s t a l l i n e amino acid mixture was a component of the complete d i e t , the concentration of plasma lysine plateaued at the eighth meal also (Kelly and Scott, 1968) . A t h i r d factor related to comparing synthetic diets to those containing intact protein i s the e f f e c t on appetite 19 which may be i n part due to the osmotic e f f e c t of d i f f e r e n t carbohydrates i n synthetic diets (Harper and Spivey, 1958). By feeding rats a c r y s t a l l i n e amino acid diet i n gel form Rogers and Harper (1965) found an increased weight gain over that of feeding the,same diet dry. The increased weight gain was attributed p r i m a r i l y to increased food intake. They sugges-ted that t h i s was due to the g e l preventing the severe osmotic e f f e c t , namely, preventing water from being drawn into the stomach and a c t i v a t i n g the stre t c h receptors and thereby re-ducing food intake. They added that the b e n e f i c i a l e f f e c t of feeding'diets i n g e l form appear to be greatest when the die t i s inadequate i n some respect. In birds the vagus nerve does not have branches i n the crop therefore the same mechanism i s not involved. I t has also been reported that neither gum acacia (2$) nor agar (5%) exerted any influence on the growth promoting a b i l i t y of a c r y s t a l l i n e amino acid d i e t fed to chicks (Soott, K e l l y , and Huston, 1966). In e a r l y studies on the extent of absorption of amino aoid from d i f f e r e n t proteins Denton and Elvehjem (1954) found that the concentration of amino acids i n the por t a l v e i n inoreased r a p i d l y a f t e r feeding dogs beef or casein but i t decreased before showing an increase a f t e r feeding z e i n . The reason f o r the decrease i n the concentration of amino acids a f t e r feeding zein was believed to be due i n part to the absorption of carbohydrates before the amino aoids were li b e r a t e d from the proteins. Feeding oarbohydrates to fast i n g human subjects has been reported by many workers to decrease plasma amino aoid nitrogen. In addition Harris and Harris 20 (1947} and Munro and Thomson (1952.} noted that the i n d i v i d u a l amino acids were not a l l depressed to the same extent. Mnnro and Thomson (1952) reported that the drop was not a constant percentage of the amount i n c i r c u l a t i o n but varied from 8% f o r h i s t i d i n e to 36$ f o r isoleucine. Nor did the amino acids disappear from the plasma i n equimolar quantities when the reductions were expressed i n millimoles, the depressions i n the various amino aoids were highly s i g n i f i c a n t l y d i f f e r e n t (P^O.01). The administration of f a t did not show the same eff e c t as that of glucose, the l e v e l of amino acids f e l l very s l i g h t l y , Guggenheim et a l . (1960) reported that the carbohydrate accompanying the protein has a marked e f f e c t on the response of plasma leve l s of l y s i n e and methionine. A f t e r a single test meal of soya protein given simultaneously with d i f f e r e n t oarbohydrates they found that lactose induced the highest lysi n e peak, fructose the longest duration of increase, whereas dextrose depressed the usual r i s e of l y s i n e . Staroh, laotose and fructose caused a higlier r i s e of methionine than sucrose and dextrose. MoLaughlan et a l . (1963) found plasma methionine and threonine were s i g n i f i c a n t l y lower when f i s h was taken with butter. In other studies (unpublished data) they found that higher responses f o r l y s i n e and methionine were obtained a f t e r oasein was fed than a f t e r milk was fed despite the fa c t that the concentrations of these amino acids were s i m i l a r i n the two foods. They oonoluded that since butter lowers plasma amino acid responses and higher plasma amino acid l e v e l s were found with oaseln than with milk i t did not appear p r a c t i c a l to use the magnitude of the plasma amino aoid response as an i n d i c a t i o n of protein q u a l i t y . They suggested however to use t h i s d i r e c t approach to study amino acid a v a i l a b i l i t y i n a food before and a f t e r processing:. Experimental 22 Experiment I. This experiment was set up to investigate the protein adequacy of a high protein wheat d i e t f o r the young chick. A comparison between body weight, feed conversion and plasma free amino aoid patterns exhibited by chicks receiving two d i f f e r e n t l y supplemented diets was made. Seventy-two day-old-White Leghorn chicks (males and females) were d i s t r i b u t e d a t random into four l o t s of 18 ohioks each i n an e l e c t r i c a l l y heated battery brooder. Two l o t s were assigned to di e t A and the other two l o t s diet B shown i n Table 1. Feed and water were supplied ad li b i t u m . Average body weighte and feed conversion are summarized i n Table 3. At 25 days of age a l l chicks were subjected to an over-night (14 hr.) f a s t . Water was available at a l l times i n t h i s and i n the following experiments where birds were fasted. Blood samples were co l l e c t e d at three separate times from d i f f e r e n t i n d i v i d u a l s , selected at random from each d i e t . Samples of blood f o r a representative f a s t i n g pattern from chicks on each diet were taken. A l l l o t s were then fed t h e i r respective diets ad l i b i t u m f o r either 3 or 6 hours a f t e r which time blood samples were c o l l e c t e d . Three birds per l o t were bled from the jugular vein (6 ml./bird). Blood was collected i n dry heparin-ized tubes and prepared f o r amino acid analysis as described i n the procedure f o r blood samples. There were two r e p l i c a t e samples of plasma for amino acid analysis for each di e t per c o l l e c t i o n . Data are presented i n Tables 3 and 4. 23 Procedure f o r Blood Samples, The i n d i v i d u a l blood samples were held i n crushed ice u n t i l they could be centrifuged 8 minutes at 1200 r.p.m. An aliquot of plasma was taken from each sample (2 ml. plasma per bird i n Experiment I; 1 ml.plasma per bird i n Experiment II) to make a 6 ml. pooled sample of plasma contained i n a 40 ml. centrifuge tube.. Immediately a f t e r pooling the plasma was deproteinized with p i c r i c a c i d . T h i r t y ml. of 1% p i c r i c acid was added to each 6 ml. of plasma very slowly and with constant vigorous a g i t a t i o n to prevent lumping of the p r e c i p i t a t e . As a precaution against loss by breakage each sample of 36 ml. was then divided between two heavy walled centrifuge tubes and the p r e c i p i t a t e removed by eentrifugation at 2500 r.p.m. f o r 20 minutes. T h i r t y m i l l i l i t r e s of the supernatant were passed through a Dowex"1" column to remove the p i c r i c a c i d . The piorate-free eluent and washings were concentrated under vacuum i n ia rotary evaporator at 45 - 47°C almost to dryness. The residue was taken up i n a small amount of 0.01 NHC1, the pH adjusted to 7 - 8 and the samples l e f t 4 hours at room temperature. The pH was adjusted to 2.2 and the t o t a l volume made up to 5 ml. with pH 2.2 c i t r a t e buffer. A l l samples were stored at -20°G u n t i l the amino aoids were analysed by ion exchange chromatography. An i n t e r n a l standard of 0.05 mioromoles of norleuoine was added to the Dowex column i n Experiment I I . However, this amino acid was not separated from what was probably glucosamine and con-sequently was of no value f o r monitoring the r e l i a b i l i t y of anal y s i s . The chromatographic conditions are desoribed under a n a l y t i c a l conditions for phy s i o l o g i c a l f l u i d s . 1. Dowex 2 - x 8 200 - 400 mesh chloride form. Bio-Rad Laboratories, 32nd & G r i f f i n , Richmond, C a l i f . 24 A n a l y t i c a l conditions f o r physiological f l u i d  analysis (Hitaohi manual with modifications) A n a l y t i c a l Conditions 1. Object of analysis 2. Ion exchange r e s i n 3. Packed Resin Height 4. Analysis time 5. Buffer flow rate 6. Ninhydrin flow rate 7. Reaction time 8. Column temperature 9. Buffers 10. Programming: Buffer change Column temperature 11. Sample aliquot 12. Recorder scale 1(0D) 13. Regeneration 9 x 500 mm column Neutral & a c i d i c amino acid analogues Hitachi Spherical #3105 540 mm. 5:30 hr. 60 ml/hr 30 ml/hr 100°C, 7.5 min. 32.8°C*0.5°G 65°C 2 0.5°C pH 3.25 2$ EtOH (95$) -*pH 3.25*0.6ml H C l / l -*pH 4.25 0:15 min. 3:45 hr. 3:20 hr. 1 ml 0 —O 0-2:30 hr. 0-0.2 2:30 hr.-end 0.2N NaOH f o r 45 min. at 2.5 Kg/cm2 pH 3.25 for 2 hr. at 2.5 _ kg/cm2 @ 55° or 65 C 9 x 150 mm column Basic amino acid analogues Same 130 mm. 4:35 hr. 60 ml/hr 30 ml/hr Same 32.8°C ±0.5°C — » 65 QC£0.5°C pH 4.267 2:20 hr. 1 ml 0-0.2 0.2N NaOH f o r 15 min. at 2.5 Kg/cm pH 4.267 for 30 min. at 60 ml/hr @ 55° or 65°C 1. Recorder f u l l scale normal 0 - *C 0D expanded 0 - 0.2 0D In using the instrument, a standard mixture of amino a c i d s 1 was analysed with each new batch of ninhydrin. 1. AA - 5, Standard amino acid s o l u t i o n . C<albiochem, Box 54262, Los Angeles, C a l i f . 90054 Urea, methionine D L sulphoxide and oreatlne were included i n the standard for l o c a t i o n . 26 Table 1» Composition of diets used i n experiment 1. Diet A Diet B Ingredients % % Ground wheat 75 . 75. Wheat shorts 15. 15. CaBP0 4 . 2H0H 1.85 1.85 Limestone 1. 1. Iodized s a l t 0.35 0.35 Corn o i l 3. 3. Choline chloride 50% 0.25 0.25 Micronutrients"*" 0.5 0.5 L-Lysine 0.24 0.24 DL-Methionine 0.06 0.06 L-Arginine - 0.1 Glycine - 0.1 L-Threonine ( a l i o free) - 0.045 L-Leuoine - 0.045 Wheat 2.75 2.46 1. Hioronutrients supplied per kilogram of d i e t : Vitamin A 11,000 IU.; Vitamin D 3 1,100 IU.; vitamin E 22 IU.; vitamin Big 0.018 mg.;menadione 1.0 mg.; r i b o f l a v i n 7.2 mg.; ni a c i n 54.mg.; D-caloium pantothenate 20.mg.; pyridoxine HC1 6.0 mg.; b i o t i n 0.18 mg.; thiamine HCL 3.6 mg.; f o l a o i n 2.4 mg.; zinc baoitraoin 27.5 mg.; oleandomycin 11.0 mg.; Amprol 499.4 mg.; manganese 50.0 mg.; zino b a c i t r a c i n 22.0 mg. 27 Table 2. Average body weights of chicks and e f f i c i e n c y of feed u t i l i z a t i o n i n experiment 1.  Days on test 1 7 14 21 24 Diet gm. gm. gm. :< gm. gm. A 41.9 42.,0 , 59.2 58.2 96.5 90.8-143.2 137.5 169.9 165.6 Av. 42 59 94 140 168 B 43.0 41.8 61*6 61.4 105.8 106 .3 166.1 164.6 197.5 194.8 Av. 42 62 106 165 196 B/A x 100 105 113 118 117 Feed conversion Feed/Gain  > Days on test...... Q ^ 1 4 . 0 -t 21 Diet A 2.61 2.78 B 2.39 2.45 Table 3. Free amino acids i n the plasma of chicks fed d i f f e r e n t Average mioromoles/lOO ml, plasma 3 hour feeding Chloks/ Starved 14 hours die t s i n experiment I. 6 hour feeding M e t Amino Acid Taurine A7 7.0 ±0.0^ ,7.0 +2.2 A 12.4+ 2.4 fi 18.6 +1.5 A ,9.6 ± 0.4 B *8.9± 1.0 Threonine 11.4 ±2.8 19.0 ±1.8 53.1+ 8.8 57.0 +4.6 35.0 ± 8.4 54.4 +1.4 Glycine 40.7 ±1.6 35.4 ±0.4 77.4+ 2.6 75.7+10.2 75.2 ± 4.2 69.6+ 6.4 Alanine 43.6 ±0.2 48.0 ±4.7 71.0+ 3.0 62.8 ±0.7 66.3 ± 5.5 58.0± 1.8 Valine 10.3 ± 0.7 11.8 ±1.8 29.0+ 2.6 31.7 ±1.1 26.4 ± 1.4 26.2± 3.2 Cystine 3.1 ± 0.6 4.0 ±0.6 10.0+ 0.5 12.7 ±0.9 10.2 ± 1.0 9.0+ 0.3 Methionine 3.7 ± 0.2 5.8 +0.2 11,6+ 0.4 16.0 ±2.6 12.4 ± 2.4 12..0± 1.1 Isoleucine 5.0 ± 0.3 7.8 ±1.6 14.0+ 1.2 17.0 ± 2.4 11.8± 0.6 12 .2± 1.0 Leuoine 9.6 ± 0.1 15,0 ±3.8 21.1+ 0.9 30.4 ±0.1 25.0 ± 1.8 23.2± 1.4 Tyrosine 22.0 ± 1.'4 20.0 ± 2.2 32.2 ±2*5 41.6 ± 2.7 27.7± 3.8 28.2 ±2.8 Phenylalanine 7.4± 0.3 8.2 ±0.6 18.4 ±3.6; 20.8 ±0.6 13.8 ± 9.6 14.7 ±0.7 Ornithine - mm 2.4+ 0.4 2.3 ±0.3 2.2± 0.1 3.0 ±0.3 Lysine 13.0± 0.8 .22.8 ± 4.1 8.7 ±0.5 8.6 ± 1.4 6.0± 1.5 9.2 ±0.8 His t i d i n e 6.2± 0.8 6.8+ 0.6 22.2 ±2.6 22.2 ± 5.8 24 .6± 1.2 20.2 ± 2.0 Arginine 6.5 ±0.7 Total E s s e n t i a l Amino Acids 98.3± 3.2 10.8± 1.0 132.0+16.2 23.6 ±3.6 243.8± 21.0 29 .3 ±0*6 287.4 ±3.8 21.7± 214.4± 0.3 5.6 36.0 ±1.8 245.4 ±7.2 1. Mean ±S E 29 Table 4. Free e s s e n t i a l amino acids i n the plasma of chicks fed d i f f e r e n t d iets i n experiment 1 expressed as percentages of the t o t a l 1 e s s e n t i a l amino acids present i n the plasma. Diet A Diet B Plasma sample fasted 3 hr. 6 hr. fasted 3 hr. 6 hr. . . . 14 hr. feeding feeding 14.hr... feeding: feeding Amino aoid % % % % %> <$> Threonine 11.6 21.7 16.5 14.4 19.9 21.0 Valine 10.5 11.9 12.3 8.9 11.1 10.1 Cystine 3.1 4.2 4.8 3.1 4.5 3.5 Methionine 3.8 4.8 5.7 4.4 5.6 4.6 Isoleuoine 5.1 5.8 5.5 . 5.8 5.9 4.7 Leucine 9.8 8.7 11.7 11.2 10.6 8.9 Tyrosine 22.5 13.2 12.9 15.2 14.5 10.8 Phenylalanine 7.5 7.5 6.4 6.3 7.2 5.7 Lysine 13.2 3.6 2.8 17.2 3.0 3.6 H i s t i d i n e 6.3 9.3 11.5 5.2 7.7 5.3 Arginine 6.6 9.7 10.1 8.5 10.3 14.0 l.Excluding tryptophan, includes cystine and tyrosine. 30 Results and Disoussion. This test was preliminary to a more detailed study of the a b i l i t y of the protein i n high protein wheat t o support the growth of the young chiok. More s p e o i f i c a l l y , i t was designed to ascertain whether amino acids other than lysine and methionine are required supplements f o r the early growth of chicles on wheat d i e t s . There was no mortality on e i t h e r d i e t . The appearance of the birds receiving d i e t B with arginine, glycine, leuoine and threonine supplementation was muoh better than those receiving diet A (Table 1). The feathering was more advanced, smoother and glossy. Also, there was much l e s s cannibalism on the d i e t with the extra supplementation. In the formulation of the basal d i e t , because It was an a l l - p l a n t d i e t , a factor of 5.7 was used to c a l c u l a t e the crude protein content. This crude protein l e v e l was then used to determine the amount of each i n d i v i d u a l amino aoid required i n t h i s diet at t h i s protein l e v e l . On a calculated basis only, the e s s e n t i a l amino aoid (EAA) pattern i n the basal diet shows arginine, glycine, leucine, threonine and methionine to be somewhat below the 1969 C o r n e l l recommendations f o r s t a r t i n g replacement p u l l e t s . Supplementing the basal d i e t with 0.1$ of arginine and glycine and with 0.045$ each of threonine and leuoine corresponded to an inorease of approximately 0.35$ crude protein on a t o t a l nitrogen x 5.7 basis. Chemioal analysis showed these two diets to contain 14.3 and 14.4$ crude protein res p e c t i v e l y . The wheat and wheat shorts analysed at 14.6 and 14.9% orude protein respectively. The a d d i t i o n a l amino aoid supplementation resulted i n an increase of 17% i n ohiofc growth over that on the control d i e t at 24 days of age. This difference i n body weight was highly s i g n i f i c a n t (P<0.01}. I t i s not possible to asoertain which amino acid was respons-r Ible f o r the increased growth on diet B. Subsequent work i n our laboratory has shown that White Leghorn p u l l e t s fed 14% crude protein (N x 5.7) wheat diets supplemented with 0.5% l y s i n e and 0.1% methionine showed no response to the addition of 0.1% arginine, 0.05% leucine, 0.05% isoleucine or 0.075% valine, nor to a combination of 0.3% glutamio aoid and 0.1% g l y c i n e . An apparent response to 0.05% threonine was demon-strated at two weeks (Bragg, Biely, and Goudie, 1970). Anderson and Warniok (1969) found l y s i n e , threonine, and the sulphur amino aoids to be the most l i m i t i n g i n a high protein wheat f o r b r o i l e r chicks. Table 3 shows the amino acid l e v e l s i n the plasma of the ohioks fed the two d i e t s . A l l values are presented on a molar basis. The chromatographic conditions used did not separate serine from asparagine and glutamine and were i n -adequate for the quantitation of proline, glutamic aoid and 3-methyl h i s t i d i n e . Accordingly, the l e v e l s of these com-pounds were not included i n the a n a l y t i c a l data presented. I t was observed that the plasma from fasted birds previously fed the "better? 1 diet (diet B, Table 1) contained more c i r c u l a t i n g PFAA than from those previously fed the conr-t r o i d i e t (diet A, Table 1), A s i m i l a r s i t u a t i o n was found by H i l l and Olsen (1963 b) who fed a protein-free diet to 32 several groups of birds which had previously reoeived various proteins with and without amino acid supplementation. In most oases the l e v e l of each amino aoid measured, 24 hours a f t e r commencement of feeding the protein-free d i e t , was higher i n the plasma of birds which had been fed the amino acid-supplemented diet than i n plasma of birds which had received the same protein without supplementation. Beoause there was a difference i n the t o t a l amount of c i r c u l a t i n g EAA between the two d i e t s , the i n d i v i d u a l EAA (including cystine and tyrosine, excluding tryptophan) are also expressed as percentages of the t o t a l mioromoles of EAA f o r each diet (Table 4). A f t e r either three or s i x hours of ad l i b i t u m feeding either d i e t , l y s i n e i s present i n the lowest concentration when i t i s expressed as e i t h e r mlcro-moles/lOO ml. plasma or as a percentage of the t o t a l micro-* moles of EAA. A f t e r six hours of feeding, the plasma from those birds receiving d i e t B shows no difference i n the amount of l y s i n e and oystine. In this experiment the cystine deter-minations can be questioned for at l e a s t two reasons, (1) the samples had been stored several months before analysis and (2) Downes (1961) has reported that oystine ooprecipitates with the proteins i n sheep blood i f the samples are not deproteinized immediately. These samples were not deprotein^ ized f o r several hours a f t e r c o l l e c t i o n . Also, i t has been observed i n growth studies i n t h i s laboratory, but not reported that no improvement i n growth occured when 0.1% DL methionine was added to a wheat di e t supplemented with only 6.24% l y s i n e . 35 I t has been found that a diet containing 94% of a high protein wheat (oaloulated dietary orude protein approx-imately 14% as N x 5.7) required about 0.5% of supplementary lysi n e f o r maximum growth response to l y s i n e of White Leghorn p u l l e t s (Bragg et a l . 1970). Therefore, these two di e t s were probably d e f i c i e n t i n l y s i n e . The response to the ad d i t i o n a l amino aoids i n diet B could be attributed to the fact that the extra supplementation provides a s l i g h t l y better balanced diet at thi s l e v e l of l y s i n e . The decrease i n mioromoles of ly s i n e between the f a s t i n g and three-hour pattern i s considered to r e s u l t from the re-establishment of protein synthesis. This decrease i n l y s i n e has been observed to occur within an hour of i n i t i a l feeding (K e l l y and Scott, 1968) provided the l e v e l of dietary l y s i n e does not exceed the requirement. A notable change between the three- and six-hour patterns from the two diets i s the d i f f e r -ence i n the percentage of threonine which decreased with diet A and remained the same i n the oase of die t B. Zimmerman and Soott (1965) have shown that severe d e f i c i e n c i e s of either l y s i n e or. arginine markedly increase plasma threonine when a synthetic d i e t i s fed to chicks. In t h e i r experiments, each increment of the f i r s t - l i m i t i n g amino acid (lysine or arginine) resulted i n a progressive deoline i n plasma threonine. The point where plasma threonine reached i t s lowest concentration did not, however, coincide with the di e t a r y concentration of the f i r s t - l i m i t i n g . a m i n o acid that maximized weight gain. For th i s reason, Zimmerman and,Scott (1965) did not consider that th i s rapid decline i n plasma threonine indicated that threonine 34 i s the next l i m i t i n g amino aoid. As a d d i t i o n a l evidence for t h i s statement i t was found that a large excess of dietary l y s i n e or valine also increased the concentration of plasma threonine. On the other hand, Swendseid, Villalofoos, and F r i e d r i c h (1963) have interpreted t h i s marked reduction i n threonine with supplementation of the f i r s t l i m i t i n g amino acid i n both an 18% wheat gluten and an 8% casein d i e t as i n d i c a t i n g that i t i s the next limiting- amino acid i n these diets f o r r a t s . This i n t e r p r e t a t i o n i s supported by the f a c t that threonine i s the next l i m i t i n g amino acid a f t e r l y s i n e i n bread protein (Rosenberg, Rohdenburg, and Eckert, 1960). Lysine i s known also to be the next l i m i t i n g a f t e r the sulphur amino acids i n a 6% casein diet (Harper, 1959) f o r r a t s . For the chick, as mentioned e a r l i e r , threonine appears to be the next l i m i t i n g i n wheat di e t s either a f t e r l y s i n e (Anderson and Warnicki 1969) or a f t e r l y s i n e and methionine (Bragg, et a l . 1970). Also of possible i n t e r e s t i s the s l i g h t s h i f t i n the proportion of i s o l e u c i n e i l e u c i n e . This r a t i o i s the same i n the fasted pattern from both d i e t s . A f t e r three hours of feeding there i s a reduction i n the proportion with both rations. After s i r hours feeding the proportion i n plasma from chicks fed diet A i s 1:2.13 while that from chicks fed diet B i s 1:1.89, The difference i n r a t i o could be interpreted as evidence f o r increased protein biosynthesis i n chicks on diet B promoted by the addition of threonine. There was a d e f i n i t e response to the supplemental leucine a f t e r three hours of feeding, but a f t e r s i x hours there was no difference i n the- number of 35 micromoles of leucine c i r c u l a t i n g i n the plasma of chicks fed the respective d i e t s . I t i s i n t e r e s t i n g to note that the addition of glycine to diet A was not r e f l e c t e d i n an increase i n the amount of c i r c u l a t i n g glyoine despite the f a c t that the d i e t was c a l -culated to be suboptimal i n this amino aoid. Afte r s i x hours of feeding, the only amino acids added to diet B over those supplementing diet A which were r e f l e c t e d i n the ohicks plasma were threonine and arginine. These two amino acids oan account fo r the increased number of micromoles of c i r c u l a t i n g EA\A between these two d i e t s . Arginine was not l i m i t i n g a t t h i s l e v e l of l y s i n e supplementation (Bragg, et a l . 1970) and was therefore c i r c u l a t i n g i n exoess. (Increased amounts of c i r c u l a t i n g ornithine appear to be associated with a l y s i n e deficiency and arginine exoess - see Experiment II.) From these observations, i t i s not possible to prediot the next l i m i t i n g amino aoid a f t e r l y s i n e i n the two d i e t s used. The PFAA method used does indicate the lysine deficiency. I t would be i n t e r e s t i n g to pursue the l y s i n e , threonine and possibly isoleucine:leucine relationships further i n wheat d i e t s . 36 Summary PFAA l e v e l s as a c r i t e r i o n of protein q u a l i t y i n -dicated a ly s i n e deficiency i n birds fed high protein wheat diets supplemented with 0.24$ lysine and 0.06$ methionine. Supplementation of the control diet with 0.1$ of arginine and glyoine and 0.045$ of both leucine and threonine improved the growth rate of White Leghorn chicks over that of the chicks fed the control d i e t . A comparison of plasma patterns obtained from birds fed the c o n t r o l and supplemented diets showed a higher l e v e l of threonine, when i t was expressed as a percentage of the t o t a l plasma EAA present, i n the plasma of birds fed the supplemental d i e t at s i x hours a f t e r commence-ment of feeding and a comparative drop i n the percentage of a l l the other EAA (except arginine) and e s p e c i a l l y h i s t i d i n e . 37 Experiment I I . At the end of a three week growth t e s t , a deter-mination was made of the PFAA patterns from birds fed d i e t s containing a protein supplement of e i t h e r a soybean meal or one of f i v e market samples of rapeseed meal. The soybean and rapeseed meals supplied 4$ of crude protein to a basal di e t composed of 84.4$ ground wheat, 2$ bonemeal, 1$ lime^-stone, 0.5$ iodized s a l t and 0.5$ of a vitamin and mineral premix. The t o t a l crude protein content of the d i e t s was calculated to be approximately 14.7$. At this l e v e l of protein supplementation, the rapeseed and wheat mixture would be p r i m a r i l y d e f i c i e n t i n l y s i n e , (evidence f o r t h i s can be seen i n Table 5) aHa therefore, a l l the rapeseed diets were supplemented with 0.15$ l y s i n e . Two rapeseed meal d i e t s without lys i n e supplementation were a l s o included i n the t e s t . The average body weights at three weeks of age of four l o t s of 12 male b r o i l e r chicks are shown i n Table 5. Body weight data were subjected to an analysis of variance and the d i f f e r -ences among meatus tested by multiple range analysis (Duncan, 1955). Blood samples were obtained from chicks on a l l d i e t s including the soya diet on two consecutive days. Six birds were bled each day. On the f i r s t day blood was obtained from the b r a c h i a l vein; on the second day from the heart. This gave two r e p l i c a t e samples of pooled plasma f o r each d i e t . Samples were prepared f o r analysis and analysed for PFAA as described i n Experiment I i n the procedure f o r blood samples. On the days that blood was taken, each l o t of birds was fasted for two hours and then offered their respective diets ad li b i t u m . Blood samples were taken three hours a f t e r the feed was offered. The PFAA data are presented, i n Tables 6 and 7. The ly s i n e and threonine values were subjected to an analysis of varianoe. Table 5. Average body weights of chioks i n experiment I I fed d i f f e r e n t sources of supplementary protein Protein supplement Av. body weight at three wk. grams Soyabean meal 262 de Rapeseed meal A + L-lysine 350 e Rapeseed meal E + L-lysine 374 c Rapeseed meal P + L - l y s i n e 315 Rapeseed meal H 177* Rapeseed meal H + L-lysine 335 b Rapeseed meal F 232 e Rapeseed meal F + L-lysine 380 1. Body weights followed by the same superscript are not s i g n i f i c a n t l y d i f f e r e n t . Table 6. Free amino aoids i n the plasma of chicks „fed d i f f e r e n t d i e t s i n experiment I I expressed as average mioromoles/lOO ml plasma. Protein supplement S,oya (Rapeseed Rapeseed Rapeseed Rapeseed Rapeseed Rapeseed Rapeseed Amino aoid ' F 1£ ly s i n e 3 H H lysine JA l y s i n e 3£ lysine iP l y s i n e taurine 18.3 + 4.3 22.2 t9.7 12.6 + 1.8 22.6 ±13.2 •15.4 + 2 .6 16.8 ± 2.2 k18.3±1.5 23.7 Threonine 62.8 + 7.8 69.6 ±1.0 39.8 + 7.0 98.2± 2.0 35.2 + 9 .7 32.2 ± 1.9 42.2*3.4 33.6 Glycine 77.0 + 2.2 82.6 ±4.6 75.4 + 7.0 113.4 + 0.1 79 .6 ±13 .4 65.6 ± 1.2 66.0±3.4 71.2 Alanine 75.8 + 4.4 65.3 ±3.4 88.8 +16.8 69.6 + 2.6 83.9 ± 5 .4 81.4 ±13.0 83.8+0.7 74.8 Valine 28.8 + 1.4 28.4 ±4.9 26.6 + 8.8 33.0± 0.8 22.1 ± 1 .9 26 .4 ± 0.3 23.2±0.6 21.7 Cystine 8.4 + 0.0 6.6 ±0.4 8.4 + 0.9 9.5 + 1.0 7.8± 1 .0 6.1± 0.8 6.6 ±0.6 7.9 Methionine 5.3 + 0.5 6.5 ±0.9 7.7 + 2.2 8.2 + 0.2 6.8± 1 .0 6.4± 0.8 7.2±1.0 7.8 Isoleucine 13.6 + 1.0 12.1 ±0.7 11.4 + 5.6 16 .4 ± 0.2 9.9 ± 1 .2 9.8± 2.Q 10.2 ±0.8 10.6 Leucine 19.5 + 2.4 17.6 ±1.2 20.8 + 8.0 22.2± 0.6 18.0 ± 0 .8 19.0 ± 3.0 19.8 ±0.6 19.2 Tyrosine 35.4 + 0.4 30.3 ±5.7 25.2 + 4.4 38.9 + 2.8 31.2± 2 .8 29 .6 ± 3.8 29.8+0.2 31.0 Phenylalanine 17.1 + 0.1 14.6 ±2.0 15.1 + 4.2 20.5 ± 2.1 14.5 ± 1 .2 15.3 ± 1.2 15.4+1.4 15.6 Ornithine 4.0 + 0.7 2.9 ±0.3 1.6 + 0.0 4.2± 1,2 1.2± 0 .0 1.6* 0.2 1.6±0.0 1.4+0 .2 Lysine 10.4 + 2.4 12.6 ±7.0 21.7 + 4.7 6.1± 1.5 13.8 ± 1 .0 18.0± 4.0 18.0±0.6 13.4±1 .4 Hi s t i d i n e 32.6 + 2.6 23.4 ±5.2 24.2 + 4.7 32.1± 2.5 26.1± 0 .7 29.8± 2.0 23.8±1.4 26 .6 ±2 .8 Arginine 44.2 + 4.1 32.6 ±3*. 4 20.6 + 7.2 42.6 + 6.4 18.5± 2 .0 22.6 ± 2.9 22.2±2.4 20.7+0 .8 Total e s s e n t i a l 278.2 ± 6.0 254.3±16.3 221.6±57.8 327.8+20.2 203.8±21.2 215.2± 5.3 218.2±0.6 206.0 amino acids 1. Mean ± S E 41 Table 7. Free e s s e n t i a l amino acids i n the plasma of chicks fed d i f f e r e n t sources of supplementary protein i n experiment I I expressed as percentages of the t o t a l e s s e n t i a l amino acids present i n the plasma. Protein supplement Soya / Rapeseed meal bean F F H H A E P meal l y s l y s l y s l y s l y s Amino acid % % Threonine 22.5 27.5 18.4 30.1 17.0 15.0 19.3 16.3 Valine 10.3 11.1 11.7 10.1 10.9 12.3 10.6 10.5 Cystine 3.0 2.6 4.0 2.9 3.8 2.8 3.0 3.8 Methionine 1.9 2.5 3.4 2.5 3.3 3.0 3.3 3.8 Isoleucine 4.8 4.8 4.8 5.0 4.8 4.6 4.6 5.1 Leucine 7.0 6.9 9.1 6.8 8.9 8.8 9.0 9.3 Tyrosine 12.8 11.8 11.7 11.9 15.3 13.7 13.7 15.0 Phenylalanine 6.1 5.7 6.8 6.2 7.1 7.1 7.1 7.6 Lysine 3.8 5.2 9.9 1.8 6.9 8.4 8;3 7.2 H i s t i d i n e 11.7 ,9.1 11.1 9.8 12.9 13.8 10.9 11.6 Arginine 15.9 12.8 9.112.9 9.1 10.5 10.1 9.7 .1.Excluding tryptophan, including oystine and tyrosine 42 Results and Discussion. I t can be seen from the difference i n growth rates (Table 5) between diets containing rapeseed meals F and H with and without lysine supplementation that lysine i s the f i r s t l i m i t i n g amino aoid i n these d i e t s . Table 6 presents the PFAA quantitated (see Experiment I.) and expressed as mioromoles/lOO ml plasma. Considerable v a r i a t i o n was observed i n the l e v e l s of many of the amino acids from r e p l i c a t e plasma samples. Some of this v a r i a t i o n may be explained by the f a c t that blood from r e p l i c a t e l o t s was sampled on two consecutive days and on which d i f f e r e n t methods of blood c o l l e c t i o n were employed. On the f i r s t day blood was c o l l e c t e d from the braohial vein. Because t h i s method was slow the time of blood sampling varied from three to four hours aft e r the commencement of feeding. On the second day blood samples were c o l l e c t e d by cardiac puncture exactly three hours a f t e r commencement of feeding each group of bird s . The routine f o r preparing the protein-free amino aoids had been established by the second day. A l l blood samples were oentrifuged within an hour of c o l l e c t i o n and the plasma samples Ldepr oteinized . within 5 hours from the start of the f i r s t blood c o l l e c t i o n . Elapsed time on the f i r s t day was probably closer to ten hours. I t has been demonstrated that the cystine i n sheep blood i s not stable for t h i s length of time (Downes, 1961). There seems no reasonable explanation fo r the large discrepancy i n the number of micromoles of c i r -c u l a t i n g PFAA between the, r e p l i c a t e samples of plasma from birds fed the diet containing rapeseed meal F supplemented with 43 l y s i n e . Because of the v a r i a t i o n i n the concentration of amino a d d s between the replioate plasma samples the i n d i v i d u a l EAA were expressed as a percentage of the t o t a l mioromoles of EAA present for eaoh day (see Experiment I ) . Table 7 presents the averages of the percentages. Plasma obtained from birds fed the soya diet shows a low percentage of l y s i n e and methionine. In t h i s diet lysine would be the limiting, amino a c i d . With the exception of the plasma lysine l e v e l i n birds fed the di e t s containing rapeseed meals A and H supplemented with l y s i n e , the l e v e l of plasma lysine was very s i m i l a r i n birds from l o t s whose average body weights were not s l g n i f i -oantjly d i f f e r e n t . However there was no s i g n i f i c a n t difference between the plasma l y s i n e l e v e l s whether they were expressed as mioromoles/lOO ml. or as percentages of the t o t a l EAA present i n the sample. Of the rapeseed meals investigated and using the PFAA values obtained i t would appear that meal F contained the most, and meals H and P the l e a s t , available l y s i n e . As i n Experiment I, the PFAA method used r e f l e c t e d a ly s i n e deficiency i n the d i e t . The addition of a single l e v e l Of l y s i n e was i n s u f f i c i e n t to demonstrate whether t h i s was the only l i m i t i n g amino aoid at this l e v e l of protein. Although there was no s i g n i f i c a n t difference between the l e v e l s of plasma i t i s evident that t h i s PFAA method oan d i s t i n g u i s h between meals containing d i f f e r e n t amounts of available l y s i n e . The addition of 0.15$ l y s i n e to diets containing rapeseed meals F and H was r e f l e c t e d i n an inorease i n the concentration of plasma l y s i n e . 44 A comparison of the PFAA patterns obtained from ohlcks fed diets containing rapeseed meals F and E, with and without l y s i n e supplementation, showed some changes. Lysine a addition to the d i e t s produced a marked decrease i n the percent-age of plasma threonine (see Experiment I) and an increased percentage of most of the other amino acids except arginine. Jones (1964) demonstrated the existence of a lysine-arginine i n t e r r e l a t i o n s h i p i n the tissue of chicks fed casein g e l a t i n diets with and without the addition of exoess l y s i n e . Plasma from chicks fed the lysine supplemented diets also contained fewer micromoles of glycine and an increased number of micro-moles of alanine i n comparison with the unsupplemented d i e t s . In general, a higher l e v e l of ornithine was seen i n the plasma samples containing both a comparatively low l e v e l of l y s i n e and a comparatively high l e v e l of arginine. A l l the plasma samples obtained from birds fed the rapeseed meals exhibit low l e v e l s of cystine and methionine. However, no growth response was observed when these same diets were supplied with 0.1% of DL-methionine (March, unpublished data). A f t e r oystine and methionine the next amino acid present i n low concentration was isoleucine. In some oases the con-centration of t h i s amino acid was l e s s than that of methionine and cystine together. Phenylalanine was present i n amounts not much greater than i s o l e u c i n e . These observations cannot be interpreted as i n d i c a t i n g the order of l i m i t i n g amino aoids as i t has been shown that t h i s must be a stepwise procedure when th i s method of determination is employed (Smith, 1966 a ) . 45 I t was observed that there was a s h i f t i n the pro-portion of isoleucine:leucine between the lysine-unsupple-mented and the supplemented d i e t s . The proportions of these two amino acids i n the plasma from birds fed diets containing rapeseed meals F and H were 1:1.45 and 1:1.35 respectively. With l y s i n e supplementation of the d i e t s the proportions became 1:1.8£ and 1:1.81 respectively. The proportions i n the plasma from birds fed the other d i e t s were 1:1.43 (soy d i e t ) , 1:1.93 (meal A supplemented with l y s i n e ) , 1:1.94 (meal E supplemented with lysine) and 1:1.81 (meal P supplemented with l y s i n e ) . In Experiment I the s h i f t i n the proportion of these amino acids from the three-to -six-hour feeding pattern i n the plasma of birds fed d i e t B was thought to be due to increased u t i l i z a t i o n of leuoine i n r e l a t i o n to isole u c i n e . In the present experiment the s h i f t with the addition of lysine seems to be due to increased u t i l i z a t i o n of isoleuoine i n proportion to leuoine. I t would be in t e r e s t i n g to examine the PFAA patterns of birds fed the above diets with d i f f e r e n t l e v e l s of supplementary l y s i n e . From comparison of the patterns i t might be possible to determine the next l i m i t i n g amino aoid i n these d i e t s . 46 Summary Growth studies showed a diet containing approximately 14.7$ crude protein (N x 6.25) 10.7$ from wheat and 4$ from either of two rapeseed meals and a soybean meal supplement to be f i r s t l i m i t i n g i n l y s i n e . The l y s i n e deficiency was also indicated by the PFAA pattern. A supplement of 0.15$ lysine to the wheat basal diet and a supplement of one of 5 market samples of rapeseed meal resulted i n d i f f e r e n t l e v e l s of c i r c u l a t i n g plasma l y s i n e i n the blood of male b r o i l e r chicks fed the d i e t s containing the various meals. The differences were not s i g n i f i c a n t . When the concentration of plasma l y s i n e was expressed as micromoles per 100 ml. plasma, a higher concentration of l y s i n e was found i n the plasma of birds with the heavier body weights. A single l e v e l of supplemental lysine was inadequate to indicate the next l i m i t i n g aminoacid(s) i n these diets. Part 2. Chicken muscle as Referenoe Protein. 47 Introduction Several protein sources have been used as standards i n the b i o l o g i c a l evaluation of protein concentrates for chick d i e t s . In p r a c t i c a l a p p l i c a t i o n i t has been considered a more sensitive test i f the control d i e t would support optimum growth of the ohiok. It was decided to investigate the use of chicken muscle as the protein supplement i n the control diet for chick assay of protein concentrates. This project i s divided i n t o three sections: (1) the preparation of the various meals. (2) t h e i r chemical analysis and (3) the b i o l o g i c a l evaluation of the various preparations of the meals. 48 Preparation of chicken meals. 1 The meals were prepared from e i t h e r raw or cooked chicken t i s s u e . A l l samples were freeze dried. In the case of the "raw** samples, the peotoral muscles were dissected from the bones and frozen at -ZQ°C i n blocks. Immediately-p r i o r to freeze drying about 1.5 - 2.0 kg of frozen tissue was chipped from a block and freeze dried i n bulk. The meat was considered dry when the temperature of the tissue approached o room temperature (23 G) and was c r i s p to the touch. For the "cooked" samples, unless otherwise stated, the tissue was autoolaved on the bones at 15 l b / i n pressure for the times indicated f o r each preparation. The material was then deboned, chopped, frozen i n polyethylene bags and freeze-dried i n bulk. Afte r drying a l l samples were ground i n a Wiley m i l l to pass a 2 mm. sieve, mixed under nitrogen and stored i n a i r - t i g h t containers under nitrogen at room temperature (23°C). The approximate age of each preparation at the time of bioassay i s given i n Tables 10 and 13. A b r i e f description of each preparation follows: Meal A. was prepared from the pectoral muscles of adult White Leghorn cockerels about 24 months old . The material was auto-claved 15 minutes the same day as i t was dissected. (These birds were from a vitamin A experiment) . Meal B. was a "raw* preparation from the same source as meal A. Meal C. was a "raw" preparation composed of the pectoral muscles dissected from White Leghorn cockerels 5 weeks of age. (These birds were from an SPV test on Cannery Waste water recovery aluminum precipitate A l 0.44$). The meat was frozen i n t h i n layers as dissected. 49 . Meal.D. one-half of each f r e s h l y dried batch of meal C was treated with 500 ppm (0.05%) of ethoxyquin (E^). This a n t i -oxidant was sprayed on the f r e s h l y ground meal. The meal was mixed thoroughly during and afte r antioxidant treatment. Meal E. a "raw** sample prepared from the pectoral muscles of White Leghorn p u l l e t s 40 weeks of age. (These p u l l e t s were from an experiment i n which the cocoidiostat Buquinolate was being tested). As these birds were very f a t i t was necessary to trim the muscle of f a t before freezing. Meal F. A "cooked" sample autoclaved 15 minutes prepared from one-half of the fresh tissue obtained f o r meal E. I t was intended that meals E and F would serve as fresh meals for comparison with meals A and B which had been stored 6 months at the time of bioassay. Meal G. a "cooked" meal autoclaved 10 minutes. Meal B*. a "cooked" meal autoclaved 30 minutes. These two meals were made from raw muscle of the same o r i g i n as meals A and B which had been st.ored for 6 months at o -20 G. The material was thawed overnight at room temperature and then autoclaved i n layers 1 to 2 inohes th i c k . Meal I. a "raw" sample prepared from the pectoral muscles of adult White Leghorn cockerels. (These birds were from a Vitamin A experiment). The muscle was frozen at -20°C as i t was dissected. Meal J . a "cooked" sample autoclaved 15 minutes prepared from muscle from the same source as meal I . Meal K. This meal was a composite of the thigh (leg) musole( hearts, l i v e r s , gizzards and blood from the same birds from which the tissues f o r meals I and J" were obtained. Meals A - F were tested i n experiment III Meals G - K were tested i n experiment IV 51 Chemical analysis: a l l analyses were performed immediately p r i o r to b i o l o g i c a l evaluation. a) Proximate analysis: ( i ) t o t a l nitrogen was determined by the Gunning method; ( i i ) moisture was determined by vacuum drying the samples at 95 - 10G°C for 4 hours; ( i i i ) crude ether ex-tr a c t s were obtained from samples which were extracted 48 hours with anhydrous d i e t h y l ether and which had been dried previously fo r 4 hours i n vacuum 95 - 100°C; (iv) the ash content was determined on samples which had been Ignited 1£ - 14 hours at 700 - 750°C. b) Creatine: aqueous extracts from the meals were prepared according to A.O.A.C. (1960) para;. 23.018 and 23.019. The o p t i c a l densities were read at 520 myu. The concentration of creatinine i n each extract was determined by r e l a t i n g the observed density to that obtained with a known concentration of very si m i l a r o p t i c a l density (Oser, 1965 p.1040). c) Albuminoid nitrogen: The 'true* protein content was deter-mined according to A.O.A.C. para. 22.020 and 22.021. d) Amino acid composition: A. Hydrolysis; ( i ) aoid hydro-lysates f o r the determination of a l l amino acids except tryptophan, cystine and methionine were prepared according to the method of Kohler and Palter (1967) procedure B with some modifications. Hydrolysis was carried out i n an a i r oven at 110°+ 2°C for 24 hours, ( i i ) cystine and methionine were determined according to the prooedure of Moore (196®) with the exceptions that (a) standards of cysteic acid and methionine sulfone (Calbiochem, Box 54282, Los Angeles, C a l i f . 90054) were used and (b) no correction for recovery of cys t e i c acid was made. 52 B. Chromatography: amino acid analysis was carried out by the Spaokman, Stein and Moore (1958) protein hydrolysate method with an -Hitachi Perkin and Elmer model KLA - 3B automatic amino acid analyser using a spherical r e s i n (Hitachi 3105). The a n a l y t i c a l data are reported i n Tables 8 and 9. The precision of determining the t o t a l amino acid content i n r e p l i c a t e hydrolysates range from 100-0.2 to 100:£l.5$. Individual amino acid duplication has been found to be within 100-2.4$. Recovery feased on nor leucine ranged from 98.1 to 99.5$. No recovery connections were made. Table 8. Chemical analysis of the chicken meals, i s o l a t e d herring meal used i n experiments I I I and IV. Average $ Album- $ albumin- , Meal Description T o t a l inoid old N of ' P r o t e i n ^ Mois N t o t a l -N ture A. *cooked* stored 15.12 91.94 4.96 B. (raw* stored 14.99 89.98 3.89 G. •raw*EQ,2control 14.59 87.57 3.84 D. *raw*EQ, 500 ppm 14.57 87.53 4.06 E. *raw* fresh 14.72 88.45 3.91 F. *oooked* f r e s h 14.64 89.54 4.27 G. 'cooked* 10 min.15.27 13 .93 91.18 93.53 4.10 H. *cooked* 30 min.15.64 14.11 90.22 95.78 1.87 H. *raw'white* 15.10 12.76 84152 90.63 2.63 J . 'cooked white* 15.27 13.50 89.08 92.99 2.42 K. 'cooked red* 13.35 12.15 91.03 82.09 2.46 ISP experiment I I I 13.35 3 experiment IV 13.70 4 13.52 98.72 Herring meal 11.67 5 10.29 88.21 soy protein (ISP) and Crude ether Ash Crea- To t a l extract tine Analysis 1.47 2.38 1.03 101.78 1.38 4.14 1.84 101.23 4.01 4.66 1.81 101.89 4.04 4.59 1.76 101.98 3.34 4.27 1.78 101.75 5.42 2.86 0.97 103 .06 2.58 0.95 2.67 0.97 4.42 1.86 3.14 1.21 3.50 0.65 1. ( t o t a l N -i oreatine N) x 6.25 2. Ethoxyquin 3. 83.47$ crude p r o t e i n 4. 85.61$ crude protein 5. 72.91$ orude protein_ Table 9. Amino acids i n some of the supplementary proteins fed i n experiment II/V expressed as gm/16 gm. nitrogen. Meal I •raw whit e * Meal J * cooked white' Meal K 'cooked red* Herring meal ISP Aspartio acid 8.97 ±0.02 9.56 +0.07 9138 ±0.09 9.61 ±0.18 12.93 ±0.15 Threonine 4.36 +0.03 4.42 +0.00 4.34 +0.08 4.45 ±0.10 4.03 ± 0,14 Serine 3.66 ±0.01 3.77 ±0.04 3.80 ±0.02 3.97 + 0.00 5.39 ± 0.03 Glutamic acid 15.48 ±0.37 15.98 ±0.28 15.81 ±0.63 14.82 ± 0.19 22.66 ± 0.27 Proline 3.35 ±0.02 3.30 ±0.05 4.27 +0.00 3.79 ± 0.06 5.42 + 0.16 Glycine 4.53 ±0.22 4.55 +0.18 5.78 ±0.25 5.99 ± 0.23 4.37 + 0.08 Alanine 5.56 ±0.11 5.74 +0.00 5.78 ± 0 .22 6.15 ±0.02 4.28 ± 0.03 Cystine 1.00 +0.01 1.04 ±0.01 1.04 ±0.04 0.84 ±0.01 1.04 ± 0.06 Valine 5.67 5.84 5.74 5.00 5.75 Methionine 3.00 2.86 ± 0.12. 2.57 + 0.04 2.82 ±0.00 1.36 ± 0.06 Isoleucine 4.82 ±0.10 5.03 ± 0,01 4.71 + 0.07 4.55 ± 0.10 5.42± 0.04 leucine 7.79 ±0.07 8.26 ± 0.16 7.93 + 0.11 7.83 ± 0.08 8.66 + 0.05 Tyrosine 2.89 + 0.04 3.34+ 0.16 2.88+ 0.02 2.83 ± 0.03 3.86± 0.12 Phenylalanine 3.86 ± 0.03 4.01+ 0.00 3.95 +0.04 3.95 f 0.06 5.68* 0.04 Lysine 7.44 +0.10 8.14 ±0.34 6 .91 +0.03 7.20+ 0.30 5.53± 0.03 Hi s t i d i n e 1-methyl h i s t i d i n e Ammonia 3.84 +0.07 2.68 + 0.20 1.14 + 0.03 3.51± 0.03 1.81 ± 0.12 0.95+ 0.00 2.67 ±0.11 0.33 +0.09 1.30 ± 0.01 2.12± 0.22 1.46± 0.02 2.74± 1.67± 0.02 0.07 Arginine gm N recovery 5.20 + 0.09 14.25 ±0.11 5.67+ 0.09 14.38± 0.11 5.93 + 0.07 14.07 + 0.20 5.24± 0.08 13.70± 0.08 6.95± 15.49+ 0.01 0.08 55 B i o l o g i c a l evaluation. Experiment III.. Five hundred day-old White Leghorn cockerels were d i s -tributed at random i n t o e l e c t r i c a l l y heated battery brooders and fed a ohiok s t a r t e r diet f o r 9 days. At 9 days of age a l l the chicks were weighed. A population having a body weight range of 72 - 84 grams was selected. Chicks from t h i s seleoted population were then distributed at random Into 21 l o t s of ten chicks each, wing-banded and reweighed. Six experimental d i e t s 1 and a control diet were assigned at random to three l o t s of chicks each. Feed and water were supplied ad l i b i t u m . The ohioks were weighed i n d i v i d u a l l y and feed consumption measured on the days indicated i n Tables 10 and 11 which show summaries of the average body weights and feed conversion for each period. Diet #5 was exhausted aft e r the eleventh day of experiment. The supplementary protein values (SPV) (March, et a l . 1966) for the various supplements are included i n .Table 10. The average body weights were subjedted to an analysis of varianoe and Dunoans New Multiple Range Test (1955). Spy - Average body weight on te s t protein diet x 1 0 0 average body weight on control d i e t 1. The experimental d i e t s and control d i e t were oomposed of a cereal basal diet and a protein supplement. The t e s t protein supplements consisted of various chicken meals (Table 8) and i n the case of the c o n t r o l d i e t of isol a t e d soy protein (ISP) which supplied 8% of crude protein to the f i n a l d i e t . The ISP diet was supplemented with 0.3% DL-methionine. The basal diet con-si s t e d of a 12% crude protein cereal mixture of ground wheat and ground yellow corn, 85 pounds of which contributed 10.2$ orude protein to the t o t a l experimental d i e t ; 2.45$ bonemeal; 1.0$ limestone; 0.5$ iodized s a l t ; and a vitamin and mineral premix . The t o t a l weight of each d i e t was adjusted with glucose monohydrate. 2. Minerals and vitamins supplied per kilogram of d i e t : Manganese sulphate 132 mg.; menadione 0.48 mg.; r i b o f l a v i n 3.96 mg.; D-oalcium pantothenate 9.24 mg.; f o l a c i n 0.55 mg.; pyridoxine H01 2.86 mg.; b i o t i n 0.088 mg.; choline chloride 1320. mg.; vitamin E 9 I.U.: vitamin A 4400 I.U.; vitamin B 3 396 I.U.; vitamin B^ 2 13.2 mog.; AmprolT-25 499.4 mg.; zino ba c i t r a c i n 27*5 mg.; oleandomycin 11.0 mg. > 57 Table 10. Summaries of the average body weights of the chioks fed the experimental d i e t s i n experiment I I I and the Supplementary protein values of the poultry meals tested. Average body weights Supplementary Protein Age of protein values supple-: supple- I n i t i a l Days on te s t Days on te s t Diet ment ment height gm. 7 gm. 11 gmi 1 1 3 gm.l 7 gm! 11 gm. 13 gm 1. Meal A 24 77 135 177 196 102 103 102 2. Meal B 24 77 133 173 193 100 101 101 3. Meal C 2 78 133 170 190 100 99 99 4. Meal D 2 77 132 170 189 99 99 98 5. Meal E 2 77 130 165 - 98 96 ?• 6. Meal F 2 77 134 174 197 101 101 103 7. ISP*-*-methionine 77 133 172 192 1. There were no s i g n i f i c a n t differences between the body weights of birds receiving any of the d i f f e r e n t treatments. x. Isolated soyabean protein 58 Table 11. Summary of the feed conversion (feed/gain) obtained on the diets fed i n experiment I I I . Protein Diet supple- Days o'n test ment 0 - 7 0- 11 0 - 1 3 1 1.99 a 1. Meal A a. 90 1.9®*' 2. Meal B 1.93 1.98 a b 2.04 a b 3. Meal C 2.04 2.09 a b 2.17 b 4. Meal D 2.02 2 . i o a b 2.14 b 5. Meal E 2.05 2.14 b mm 6. Meal F 1.87 1.94a 1.97a 7. ISP^-f 2.02 2.05 a b 2.11 a b methionine 1. Values followed by the same superscript are not s i g n i f i c a n t l y d i f f e r e n t x. Isolated soyabean protein Table IB. Chemical analysis of diets fed - experiment III Diet % Ca % Total P. 1 1.05 0.730 2 1.03 0.722 3 1.09 0.736 . 4 1.00 0.784 5 1.08 0.742 6 1.04 0.722 7 0.99 0.722 60 Experiment IV. Four hundred day-old White Leghorn cockerels were d i s -tributed at random into 15 l o t s i n e l e c t r i c a l l y heated battery brooders. They were supplied with water and a commercial chick s t a r t e r diet ad l i b i t u m . At 3 days of age the chicks were i n d i v i d u a l l y weighed i n order to select a standardized popula-t i o n . Chicks whose body weight ranged from 46 - 52 grams were dis t r i b u t e d at random into 21 compartments. There were 10 ohloks per compartment. Six experimental d i e t s 1 and a oontrol diet were assigned to compartments at random. Each d i e t was assigned to t r i p l i c a t e l o t s of ohioks. The chicks were then wing banded, reweighed and offered the t e s t diets ad l i b i t u m . Individual body weights and feed consumption were measured at weekly i n t e r v a l s f o r three weeks. The summaries are presented i n Tables 13 and 14 with the supplementary protein value for each period. At the end of three weeks two of the diets (#1 and #2) were exhausted. The remaining f i v e were continued on test to the end of the fourth week. S t a t i s t i c a l analysis was as for Experiment I I I . 1. The experimental d i e t s and control d i e t were composed of a wheat basal d i e t which contributed 10.0$ crude protein and a protein supplement which supplied 8$ crude protein to the f i n a l d i e t . The protein supplements included 5 samples of chicken meal, a commercial sample of herring meal and i n the control i s o l a t e d soy protein supplemented with 0.37$ DL-meth-ionine. In addition to ground wheat the basal diet consisted of 0.5$ iodized s a l t , 10$ corn o i l , and a mineral and vitamin premix . Calcium and phosphorus were adjusted to 1.0 and 0.6$ 61 respectively with limestone, bonemeal and trio a l c i u m phosphate. The t o t a l weight of each d i e t was adjusted with gluoose mono-hydrate . 2. Minerals and vitamins supplied per kilogram of d i e t : manganese sulphate 220 mg.; menadione 1.06 mg.; r i b o f l a v i n 7.2 mg.; D-calcium pantothenate 20.0 mg.; f o l a o i n 2.4 mg.; pyridoxine HC1 6.0 mg.; b i o t i n 0.18 mg.; choline chloride 2600 mg.; thiamine HC1 1.8 mg.; ni a c i n 27.0 mg.; vitamin E 22 I . T J . ; vitamin A 8800 I.U.; vitamin D 3 396 I.U.; vitamin B^g 0.018 mg.; Amprol 49914 mg.; zinc b a c i t r a c i n 27.5 mg.; oleandomycin 11.0 mg. 62 Table 13. Summaries of the average body weights of the chioks fed the experimental diets i n experiment 17 and the supplementary protein values of the protein supple-ments tested. Average body weights 1 Supplementary protein value Protein Age of I n i t i a l supple-Diet ment supple -ment wk. weight gm. Weeks on 1 2 gm. gm. tes t 3 gm. 4 gm Weeks on • 1 2 t e s t 3 4 1. Meal G 30 48 94 150 221 a - 108 115 121 2. Meal H 30 48 95 153 221 a - 109 118 121 3. Meal I 4.5 48 96 154 223 a 309 110 118 123 123 4. Meal J 4.5 49 96 153 223 a 308 110 118 123 123 5. Meal K 4.5 49 96 154 223 a 311 110 118 123 123 6. X H.M. 49 93 147 214 a 298 107 113 118 118 7. ISP-J-methionlne 49 87 130 182 b 252 1. Body weights followed by the same superscript are not s i g n i f i c a n t l y d i f f e r e n t . x. Herring meal Tatole 14. Summary of the feed conversion (feed/gain) obtained on the diets fed i n experiment 17. Average feed conversion Pretein Diet supple-ment Weeks 0 - 1 on test 0 - 2 0 - 3 0 - 4 1. Meal G 1.75 1.78 1.88 -2. Meal H 1.72 1.78 1.89 -3. Meal I 1.72 1.77 1.90 2.05 4. Meal J 1.72 1.79 1,90 2.06 5 Meal K 1.70 1.78 1.89 2.02 6. E-M* 1.84 1.89 1.96 2.09 7. ISP + 2.00 2.05 2.10 2.21 methionine 64 Results and Disoussion. Preparation of Poultry Meals; Obtaining tissue from adult male birds was r e l a t i v e l y easy. For bulk preparation i t is d e f i n i t e l y not e f f i c i e n t to use young birds as too much time i s involved i n obtaining a very small amount of t i s s u e . Since there are n u t r i t i o n a l problems associated with fat r a n c i d i t y i t i s not reoommended that material from p u l l e t s i n l a y be used. On a cooked or raw wet-weight basis the dry matter content of the o r i g i n a l material ranged from 41 - 44% and 25 - 28% respectively. I f the raw tissue were chipped i n the frozen state and freeze-dried immediately i t retained a pink f l e s h colour. Chemical Analysis: The i n i t i a l proximate analysis ( t o t a l nitrogen x 6.25, moisture, orude ether extract and ash) t o t a l l e d w e l l over 100%. Myosin from chicken muscle has been reported to contain 16.6% nitrogen (Block and B o i l i n g , 1951) but as muscle i s known to contain creatine, about 0.3 - 0.5% of wet weight (Oser, 1965) which i s 32.05% nitrogen i t was f e l t that the protein as t o t a l nitrogen x 6.25 was overestimated. A rough determination of the t o t a l creatine and creatinine content (the picrate does not always follow Beers Law, Oser, 1965) of the freeze-dried meals showed them to contain approximately 0.2 - 0.6% creatine nitrogen. Correcting the t o t a l nitrogen f o r creatine nitrogen before estimating the protein oontent reduced the "protein" content but a l l the samples for which a oomplete proximate analysis was available s t i l l t o t a l l e d over 100%. The creatine corrected protein values were used i n c a l c u l a t i n g the amount of 65 protein used to supplement the experimental d i e t s . This would not make the d i e t s isonitrogenous but rather "Lsoproteinaceous". Albuminoid nitrogen was determined on the second set of meals, G - K, a sample of commercial herring meal and the i s o l a t e d soy protein (ISP) used i n the oontrol d i e t . The raw freeze-dried samples would contain a l l the residue of the ti s s u e f l u i d s whereas muoh of t h i s would have been l o s t i n the cooked meals, even though they were not pressed, and the soluble mater-i a l was not evaporated and added to the cooked meal before dry-ing. Consequently, the cooked samples had a higher percentage of albuminoid nitrogen and lower ash and creatine oontents than the freeze-dried meals. Amino Acid Analysis: There i s var i a t i o n i n the. reported values for herring meal and iso l a t e d soy protein (ISP) so comparison with the values obtained i s d i f f i c u l t . Although not s t r i c t l y compar-able there i s reasonable agreement between the values obtained and those reported by Anderson and Warnick (1969) for l i g h t and dark chicken meat with the exception of alanine which i s almost twice their reported value for t h i s amino acid . A l l arginine values are low compared to the reported values. The amino acid content of the chicken meals i s quite s i m i l a r to that of herring meal except f o r a greater amount of h i s t i d i n e i n the chicken meals. Considerable amounts of 1-methyl h i s t i d i n e have been reported i n muscle extracts (Tallon, Moore and Stein, 1954). There was no detectable amount of ornithine i n the raw freeze-dried meal. Unfortunately the presence of this amino acid i n the meal composed of the red meat, etc. was not determined. 66 Some d i f f i c u l t y was encountered i n preparing the samples fo r determination of oystine and methionine as cys t e i c aoid and methionine sulfone. The meals did not dissolve i n the performio aoid and so were l e f t over night as recommended by Moore (1963) f o r proteins not s o l u b i l i z e d by t h i s a c i d . Some samples foamed badly when the HBr was removed. The resolution of methionine sulfone from aspartic acid by the conditions described was adequate f o r the quantitation of the sulfone, however, i t has been recommended that improved resolution could be re a l i z e d by the addition of alcohol (up to 6% of either methyl or e t h y l ) , increased column size (approximately 54 - 55 cm.) and about a 5°G temperature increase (Redman, 1970; Donohue, personal communication). Various combinations of these parameters would have to be t r i e d to f i n d the optimal conditions for resolution. An improved method for the determination of the t o t a l cystine-cysteine i n proteins has been reported (Spencer and Wold, 1969) but unfortunately does not include the determination of methionine. 67 B i o l o g i c a l evaluation: The o r i g i n a l intention i n designing Experiment I I I was to test methods of meal preparation, i . e . oooked vs. raw freeze-dried (meals A and F vs. B and E); the e f f e c t s of storage, samples of both cooked and raw meals stored six months were compared to fresh preparations (meals A and -B vs. F and E) and the e f f e c t of antioxidant treatment i n a fresh sample (meal c vs. D) for the production of a meal which would promote the best growth. By using an 18$ protein chiok s t a r t e r r a t i o n which Is considered a sub-optimal l e v e l of protein f o r maximum chiok growth i t was therefore intended to stress protein adequacy. Once i t was determined which preparation gave the best growth more meal would be prepared i n t h i s manner and tested at a much lower l e v e l of supple-mentation. The SPV basal d i e t and standard have been used i n t h i s laboratory f o r ten years. The r e s u l t s of Experiment I I I were most disappointing and can most l i k e l y be attributed to p a l e t a b i l i t y and mechanical problems. The texture of the experimental diets was poor because of the low fat content of the chiefs® meals and t h e i r powdery consistency. In consequenoe severe beak necrosis was encountered. Interestingly this problem has never appeared in the soya protein control d i e t . Two other p o s s i b i l i t i e s for poor growth that were considered possible were the calcium and phosphorus content and the amount of l i n o l e i c acid present i n the d i e t s . The determination of calcium and phosphorus (Table 12) indicated no problem. A rough estimate of the 68 l i n o l e l o acid oontent of the experimental diets and the soy protein control showed them a l l to contain approximately 1.1% l i n o l e i o a c i d . There was no s i g n i f i c a n t difference between the body weights of chicks fed the d i f f e r e n t diets but there seemed some difference between the cooked and raw samples i n feed conver-sion (feed/gain) at thirteen days on t e s t . Because of these reasons the test was repeated with d i f f e r e n t meals and with modifications to the basal d i e t . In Experiment IV ten percent of corn o i l was included i n the basal diet at the expense of weight i n wheat but not of t o t a l protein. (A high protein wheat was used.) The vitamin supplementation was increased to cover any deorease i n feed intake associated with the higher energy oontent. Chicks three weeks on test i n Experiment IV had "bands'* of missing barbules i n the wing feathers mainly i n the secondary feathers. Some feather t i p s were broken o f f at these "bands". There was no obvious difference i n the incidence of these "bands" between the treatments. There was cannibalism (toe p i picking) i n some l o t s but not associated with any specif io d i e t . The supplementary protein values obtained for a l l the chicSfren meals are the maximum that have been obtained with White Leghorn cockerels using a 12% protein cereal mixture (wheat and oorn or wheat alone) to supply 10.2% protein to the d i e t and a fishmeal supplement supplying 8% protein. In this t e s t the energy oontent had been increased to stress the protein adequacy. The difference inbody weight and feed conversion between the assay protein c o n t r o l and a l l the other 69 preparations including fishmeal was highly s i g n i f i c a n t (P<0.01). There were no differences between the chicken and fishmeal. nor between any of the d i f f e r e n t methods of preparing the chicken meal. When the supplementary protein value assay procedure1, was f i r s t employed i n thi s laboratory (March, et a l . 1966) a range i n SPV from 107 - 140 was obtained. The better the protein q u a l i t y of the meals the higher the SPY. O r i g i n a l l y the test bi r d had been the New Hampshire cockerel. Several years l a t e r , for p r a c t i c a l reasons, t h i s breed was replaced by White Leghorns. Preliminary tests had indicated that t h i s breed was just as sensitive a test bird as the New Hampshire. However, since White Leghorns have been used the highest SPY obtained has been 122 at three or four weeks on t e s t . Since the r e s u l t s of the present test have been obtained the s e n s i t i v i t y of thi s method has been greatly improved by the use of male b r o i l e r chicks and the incorporation of 1% f a t i n the basal diet to improve p a l a t a b i l i t y and to prevent beak necrosis. From Experiment IV i t would appear that chicken meal could be a s a t i s f a c t o r y reference protein supplement.' Further te s t i n g at lower l e v e l s of supplementation and with f a s t e r growing stock would be required to determine which method of preparation consistently promoted maximum chick growth. 70 Summary-Diets containing various preparations of freeze-dried raw and cooked chicken muscle which supplied 8% of protein to a 10% crude protein wheat basal d i e t supported better growth (P4.0.01) than the is o l a t e d soy protein (ISP) and methionine reference standard used when the diets Contained 10% of corn o i l . There was no difference between the body weights of birds fed the chicken meals and those fed a commercial sample of herring meal. Further t e s t i n g of these meals with f a s t e r growing stock and lower l e v e l s of supplemental protein would be required to determine th e i r value for use as reference meals. Part 3. T r i c h l o r a c e t i c acid (TGA) f r a c t i o n a t i o n of gut nitrogen as a method for studying protein q u a l i t y . Introduction The extent to whioh dietary protein can be hydrolysed i n and absorbed from the d i g e s t i v e t r a c t determines the "usefulness** of a protein source for a p a r t i c u l a r animal. Preliminary studies were car r i e d out to determine whether the t r i c h l o r a c e t i c acid (TCA) f r a c t i o n a t i o n of gut nitrogen could be used as a bioassay to evaluate protein q u a l i t y . The studies conducted included determining l e v e l s of gut nitrogen remaining a f t e r feeding various amounts of protein material, the e f f e c t d i f f e r e n t carbohydrates and f i b r e had on the extent of digestion and the l e v e l s of free amino acids present i n the gut segments following various protein meals. A bioassay based on the recovery of i l e a l amino acids has been suggested by Payne et a l . (1968) to des-cribe the a v a i l a b i l i t y of amino acids i n proteins. Souroes of nitrogen i n the gut include (1) the d i e t (2) enzymes secreted into the i n t e s t i n a l lumen (3) mucin (4) c e l l u l a r debris from erosion of i n t e s t i n a l epitheleiua (5) baoteria. The secretions of nitrogenous material Into the lumen of the duodenum have been found to r a i s e the t o t a l nitrogen content to approximately ten times the amount ingested (Bolton, 1961). Examination of the oontent of the duodenum has shown.that about 30 percent of the t o t a l nitrogen i s present as mucin (Bolton, 1964), Much of the remainder may 7S represent c e l l u l a r debris due to erosion of the l i n i n g of the gut. Bacteria, which contain twenty percent crude protein i n t h e i r dry matter, thrive i n the crop and oaeca (Bolton, 1969). Bolton (1964) presented data which indicated that, with d i e t s using c e l l u l o s e as a marker, most protein disappeared from the i n t e s t i n e posterior to the duodenum. Imondi (1965) obtained data from male b r o i l e r type chickens of 4, 6, 8 and 10 weeks of age with respect to the disappearance of dietary protein from various l e v e l s of the small i n t e s t i n e . Chromic oxide was fed as a marker and the r a t i o of crude nitrogen to chromic oxide was determined for each area. A p r a c t i c a l type diet was used i n these studies. The duodenum was defined as that portion of the inte s t i n e from the gizzard to the d i s t a l attachment of the pancreas; the jejunum from the duodenum to Meckels diverticulum; the ileum from the diverticulum to the i l e a l - e a e o a l - c o l i o junction. The r a t i o of nitrogen to chromic oxide i n the duodenum was four to f i v e times that i n the feed. In a l l age groups the r a t i o increased or remained the same on going from the upper to the lower duodenum. The increase i n nitrogen was attributed to the pancreatic juice which empties into the d i s t a l portion of the duodenum. The upper half of the jejunum was found to be the most active s i t e of absorption as measured by the d i s -appearance of nitrogen from the lumen; 79.7 peroent of the nitrogen presented to the jejunum by the duodenum disappeared i n the upper half of t h i s segment. The lower jejunum accounted f o r 9.8 percent of the nitrogen disappearance. The ileum served to play a minor role i n nitrogen absorption. 73 Bird (1968) has pointed out that the true a b i l i t y of a region of the gut to absorb nitrogen was not determined i n th i s study. I t was a l s o noted that the various segments did not have the same material a v a i l a b l e for absorption. Masset (1964) considers that the presence of the en-dogenous nitrogen secretions i n the lumen of the gastro i n t e s t -i n a l t r a c t acts as a homeostatic device which prevents wide fluctuations i n the amino ac i d mixture available f o r absorption. The effectiveness of such hpmeostasis probably depended upon the presence of a large and mobile reserve i n the alimentary t r a c t i t s e l f . Data presented by Snook and Meyer (1964) indicated that endogenous nitrogen secretions and digestive enzymes markedly inorease i n response to protein feeding. They also estimated that about 90 percent of the endogenous nitrogen was digested and absorbed. . In causing protein to p r e c i p i t a t e t r i c h l o r a c e t i c acid (TCA) apparently acts both by the formation of insoluble protein s a l t s and by a l t e r a t i o n of the pH of the aqueous medium causing an a l t e r a t i o n of the intramolecular shape. I t i s suited f o r those determinations i n which the removal only of proteins i s desired as i t does not a f f e c t the s o l u b i l i t y of intermediate weight polypeptides and amino acids. None of the nonprotein nitrogenous material i s removed from solution by 5 percent TCA. However, i t has been shown that as the concen-t r a t i o n of the a c i d i s increased the proportion of intermediate si z e peptides i n the precipitate increases (Oser, 1965). An example of a method using TCA to fractionate gut 74 nitrogen was that used by B i e l o r a i and Bondi (1963) to study the r e l a t i o n s h i p between * a n t i t r y p t i c f a c t o r s • of some plant protein feeds and the products of proteolysis preoipitable by TCA. A f t e r a two hour feeding period the i n t e s t i n a l contents of chicks were separated, weighed and thoroughly mixed. Four gram samples of the contents were suspended i n 15 ml. of citrate-phosphate buffer, pH8. The well-mixed suspension was centrifuged at 4000 - 5000 g, f i l t e r e d and centrifuged again at 12,000 g. Aliquots of the f i l t r a t e s were used fo r deter-mination of amino groups and f o r examination of the presence of a TCA - precipitable f r a c t i o n . For the l a t t e r purpose an equal volume of 10 percent w/v TCA was added to part of the f i l t r a t e . A f t e r being kept at room temperature for 1 - 2 hours the pre-c i p i t a t e obtained was separated by oentrifugation at 4000 -5000 g, and f i n a l l y the supernatant was c l a r i f i e d by o e n t r i -fuging at 12,000 g. Nitrogen was determined i n the s o l u t i o n by mioro-Kjeldahl before and a f t e r the addition to the TCA and the TCA p r e c i p i t a b l e nitrogen calculated by difference. Beoause of the possible effeot of the caeca and i n t e s t i n a l microflora i n a l t e r i n g apparent amino aoid d i g e s t i -b i l i t y valves Payne et a l . (1968) investigated the release of amino acids by normal digest i v e processes within the duodenum and jejunum. Diets containing either a good-quality f i s h meal, a good q u a l i t y f i s h meal autoclaved with sucrose, or c r y s t a l -l i n e amino acids were fed to five-week old nitrogen-depleted b i r d s . A protein free basal d i e t was fed ad l i b i t u m and i n g e l a t i n capsules, referred t o as protein free diet ( g e l a t i n ) . A l l the test d i e t s were force fed i n g e l a t i n capsules at 4 hour i n t e r v a l s for a t o t a l of seven feedings. T h i r t y minutes 75 af t e r the l a s t feeding, a l l chicks were s a c r i f i c e d and the duodenal and jejunal sections removed separately. The contents of these sections were washed into separate v i a l s with 5$ TCA. The contents of two birds were pooled for each segment. Each sample was f i l t e r e d , and the f i l t r a t e f r a c t i o n was brought to 50 ml. with d i s t i l l e d water. Amino acids were determined for the f i l t r a t e and p r e c i p i t a t e f r a c t i o n s separately. The amino acids soluble i n 5$ TCA were expressed as a percent of the t o t a l amino acids recovered from each section. The proportion of amino acids soluble i n 5$ TCA was taken as an index of the rate of protein digestion. No difference was observed i n the TCA-soluble amino acids recovered from the duodenal contents. The feeding of f i s h meal resulted i n a reduced quantity of amino acids TCA-soluble r e l a t i v e to the basal treatments (protein free g e l a t i n and protein free basal d i e t ) . While the feeding of free amino acids i n g e l a t i n capsules was found to increase the proportion of TCA-soluble amino acids i n the jejunum. The feeding of f i s h meal autoclaved with an equal quantity of sucrose resulted i n a s i g n i f i c a n t reduction i n the quantity of amino acids soluble i n 5$ TCA. Heat treatment of f i s h meal was found by analysis to r e s u l t i n destruction of arginine, l y s i n e , and h i s t i d i n e by 85, 60 and 39$ respectively. S i g n i f i c a n t quantities of the amino acids although soluble i n TCA remained i n the jejunal contents. The amino acids recovered from jejunal contents following the feeding of a d e l i b e r a t e l y damaged f i s h meal were found to be less soluble i n 5$ TCA than when the f i s h meal was fed undamaged. This was taken to indicate that the amino acids were released to a 76 greater extent from the undamaged protein thus suggesting that the d i f f e r e n t i a l release of i n t e s t i n a l amino acids i s a sensi-t i v e index of protein q u a l i t y . In further work these same researohers fed d i e t s con-t a i n i n g various proteins to supply 30% crude protein plus one percent chromic oxide to nitrogen depleted b i r d s . The percent of amino aoids soluble i n 5% TCA and the percent of consumed amino aoids recovered from the jejunal and i l e a l contents were calculated. The r e s i d u a l amino aoids i n the i l e a l contents were taken as an index of protein q u a l i t y . The amino acids recovered from the i l e a l contents of chioks fed casein were present l a r g e l y i n the TCA-soluble f r a c -t i o n , however, most of the amino aoids consumed were absorbed. E s s e n t i a l l y a l l of the amino acids were recovered from the i l e a l contents of chicks fed autoclaved f i s h meal, which was confirmed by the low TGA-solubility of amino acids following the feeding of the poor q u a l i t y f i s h meal. A marked difference was observed i n the recovery of amino acids fron the jejunal and i l e a l contents. Most of the amino acids from f i s h meal were absorbed from the ohick's ileum, while those from casein were absorbed from the jejunum. 76 General experimental procedures. White Leghorn cockerels were used i n a l l the studies ca r r i e d out, their ages d i f f e r for the various tests and there-fore are given i n each oase. In an examination of the digestive t r a c t s of three s i x -week old birds which had been fasted 24 hours i t was noticed that fibrous material and seed p a r t i c l e s were contained i n the gizzard. I t was found that the same type of material was s t i l l retained i n the gizzard of birds fasted f o r 50 hours. For t h i s reason a l l the birds used i n the following experiments were placed on a low f i b r e diet based on corn and herring meal. Even with a low f i b r e diet i t was s t i l l found necessary to f a s t the birds f o r long periods to clear the digestive t r a c t of most of the fibrous material. With a l l age birds this was complic-ated by feather picking with more than 16 hours of f a s t i n g . Grinding the feed to pass a 1.0 mm. mesh sieve and o f f e r i n g g r i t both helped to reduce the amount of material the birds would r e t a i n i n t h e i r gizzards. Once the method f o r the administration of the t e s t meals was established the general procedure f o r the c o l l e c t i o n of the i n t e s t i n a l contents was as follows. At the predetermined time a f t e r the l a s t meal each bird was given an over dose of Nembutal by intravenous i n j e c t i o n . Birds s a c r i f i c e d i n t h i s manner did not undergo the agonal spasms t y p i c a l of those following c e r v i c a l d i s l o c a t i o n and thus there was l e s s l i k e l i -hood of a disturbance of the contents i n the digestive t r a c t . The abdominal cavity was opened and the i n t e s t i n a l t r a c t quickly but gently exposed. Haemostats and/or sorew clamps 77 were located at Meckels diverticulum, one at the posterior t i p of the duodenum and one s l i g h t l y proximal to the ileooaecal junotion. Once as many clamps were i n place as possible the intestine was completely removed from the body cav i t y and the in d i v i d u a l sections emptied (and washed i f speoified) into beakers of appropriate size to permit d i l u t i o n or the addition of TCA i n the proportion of one volume of i n t e s t i n a l contents to f i v e volumes of TCA of a spec i f i e d concentration. Following the addition of TCA the method of handling the mixture varied somewhat and i s indioated i n each case where modifications to the previous test have been made. Usually a f t e r centrifuging eaoh mixture the supernatant was decanted into a volumetric f l a s k and the residue washed on to #2 Whatman f i l t e r paper. The washings from the residue were added to the supernatant which was then made up to volume. Total nitrogen (macro-Kjeldehl) was determined on the entire residue and on an aliquot of the supernatant. Nitrogen recovered for each segment was the sum of the residue nitrogen and supernatant nitrogen. I t must be pointed out that reference to the soluble or insoluble fractions as, for example, 5 percent TCA soluble or insoluble, means the supernatant or residue obtained from treating a sample of i n t e s -t i n a l contents i n the manner described above. The f i n a l concen-t r a t i o n of a mixture of one volume of i n t e s t i n a l contents and f i v e volumes of 5 percent TCA i s approximately 4.2 percent but for convenience these fractions are referred to as 5, 15, e t c percent soluble or insoluble. Also the insoluble f r a c t i o n con-tains both the undigested material and the TCA insoluble mater-i a l which together are referred to as the residue i n the following experiment s. 78 The ease and accuracy of administering protein material i n g e l a t i n capsules and i n s l u r r y form to young birds was i n -vestigated. When approximately 1.0, £.0 and 3.0 grams of f i n e l y ground dogfish meal were administered to i n d i v i d u a l birds i n g e l a t i n capsules i t was found that the capsules did not dissolve r e a d i l y i n the crop. Each capsule was made of approximately 0.1 gram of g e l a t i n . Two hours a f t e r administer-ing the test material, i n t a c t capsules were s t i l l i n the crop. This method of administering material while i t could be made very accurate and was very easy had two obvious disadvantages (1) the abnormally slow release of meal and (£) of contributing 0.1 gram of protein per capsule to the system. This would be a d e f i n i t e disadvantage i f blood samples were to be^  taken as the amino acids of g e l a t i n would most l i k e l y be the f i r s t absorbed. Pip e t t i n g given quantities of protein i n s l u r r y form was investigated next. A sample of dogfish meal alone and with a c a r r i e r i n the r a t i o of 30:70 dogfish to corn starch, gluoose monohydrate or ground ce l l u l o s e was mixed to a soupy consistency with water and kept homogeneous with a magnetic s t i r r e r . T r i p l i c a t e aliquots could be pipetted with a./ precision of 100^*0.9$. This method of administering protein material was easy and accurate enough to be used for f u r t h e r t e s t i n g . To test t h i s method with birds, 5 ml. aliquots of these same mixtures representing £l-3£ mg. nitrogen were pipetted into the crops of eight week old birds which had been fasted 50 hours, two per treatment. The method appeared quick and 79 simple provided the material was f i n e l y ground, the pipette was of the fast transfer type, and the birds were not too small. These birds were used to follow the nitrogen into the i n t e s t i n -a l phase. The birds were s a c r i f i c e d 2 - 3 hours l a t e r and the i n t e s t i n a l contents from the duodenum to the ileocaeoal junction from each p a i r of birds was expressed out, pooled and blended i n 100 - 200 ml. of water. Aliquots were taken f o r t o t a l nitrogen (macro-Kjeldahl). The approximate amount of nitrogen recovered from the intestine was very small averaging 25, 10, 9 and 4 mg./bird respectively from the four d i f f e r e n t treatments. In the following experiments much smaller d i l u t i o n s were made. In a study of the e f f e c t of ground c e l l u l o s e on the amount of gut nitrogen present during digestion, herring meal alone and i n the r a t i o s of 30:70 and 60:40 to ground c e l l u l o s e was administered i n s l u r r y form to nine week old birds which had been starved 48 hours (one bird/treatment). The birds were s a c r i f i c e d two hours a f t e r feeding a single meal and the con-tents from the duodenum to the ileocaeoal junction were washed out with water, blended and aliquots taken f o r nitrogen deter-mination. Increasing the amount of nitrogen to c e l l u l o s e i n -creased the amount of nitrogen recovered i n most cases but feeding double the amount of a given mixture did not resu l t i n double the amount of nitrogen recovered except at the lower, r a t i o of nitrogen:cellulose where 27 and 54 mg. nitrogen fed resulted i n 30 and 69 mg. nitrogen reoovered. Feeding one portion of f i s h meal:cellulose (60:40) resulted i n a nitrogen recovery s i m i l a r to nitrogen recovered from feeding two portions of f i s h meal:cellulose (30:70) i . e . i n both cases the same amount of nitrogen was fed. A greater amount of nitrogen was recovered from the intestines of b i r d s fed the herring meal alone than when i t was fed with a c a r r i e r , i . e . when 107 mg. nitrogen was fed with c e l l u l o s e approximately 70 mg. nitrogen were recovered but when 93 mg. herring meal nitrogen was fed alone 103 mg. nitrogen was recovered. As these were a l l single determinations no conclusions can be drawn from these observations. Monson et a l . (1950) related the time of t r a n s i t of the gut oontents to the carbohydrate source i n the d i e t . They found that lactose based diets passed down the gut most rapid-l y , those based on dextrin were slowest and tho'se based on sucrose were intermediate. I t i s not known whether the ex-cre t i o n time of chicks as affected by d i f f e r e n t carbohydrates a f f e c t s protein d i g e s t i b i l i t y and therefore a v a i l a b i l i t y of the constituent amino acids. This would be a consideration where information obtained from synthetic systems were related to p r a c t i c a l s i t u a t i o n s . To f i n d i f any difference i n the degree of digestion could be deteoted with d i f f e r e n t carbohydrates, a small t r i a l was conducted i n which d i f f e r e n t l e v e l s of TCA were used to fractionate the nitrogen i n the gut. Previously i t had been found that about 38% of the nitrogen present i n a pooled sample of i n t e s t i n a l material from two 16 week old birds remained insoluble when i t was treated with 5 percent TCA and l e f t 0.5 hours before f i l t r a -t i o n . In the present t e s t dogfish meal was fed i n the r a t i o of 50:50 with corn starch, glucose monohydrate and ground c e l l u l o s e to ten week old birds, two per treatment. A single 81 meal containing 174 mg. of crude nitrogen was fed to each bird i n each case. Two hours a f t e r being fed the birds were s a c r i -f i c e d and the i n t e s t i n a l contents of each p a i r of birds ex-pressed out and blended i n a minimum of water. Total nitrogen was determined i n each sample. An average of 93, 67 and 74 mg. of nitrogen per bird was reoovered when the protein source was fed with corn staroh, glucose monohydrate and ground c e l l u l o s e respectively. About the same percentage of the t o t a l nitrogen was insoluble i n 5 and 15 percent TCA, 36, 34 and 37 percent respectively for the three d i f f e r e n t treatments. ( A l l the 15 percent TCA mixtures jammed on the f i l t e r papers.) When 30 percent TCA was used 38, 38 and 40 percent of the t o t a l n i t r o -gen was recovered i n the residue. An i n i t i a l survey of the eff e c t of protein q u a l i t y on the percentage of 5 percent TCA insoluble nitrogen of t o t a l nitrogen recoverable from the jejunum and ileum of chicks was oonduoted next. Two heated meals were prepared from a good qu a l i t y herring meal: treatment (1) a severely damaged meal, a herring meal and glucose monohydrate (1:3) mixture was moistened with water and heated i n an a i r oven 7 hours at 100°C; treatment (S) a moderately damaged meal, herring meal alone was heated as treatment (1); treatment (3) was the o r i g i n a l meal. This herring meal had been evaluated b i o l o g i -c a l l y and found to be of good q u a l i t y . The two heat-damaged meals prepared from this meal were not tested b i o l o g i c a l l y but th e i r charred appearanoe indicated meals of poor q u a l i t y . Six 15 week old birds which had been starved 30 hours and without crater about 3 hours were each given 101, 72 or 72 mg. of 82 nitrogen of each of the three meals, two birds were given one treatment. Birds receiving treatments (2) and (3) were fed the herring meals i n the r a t i o of 1 plus 6 with glucose mono-hydrate. An error i n d i l u t i o n s made the amount fed birds receiving treatment (1) incomparable with the other two t r e a t -ments on an absolute nitrogen recovery basis. They can be compared on a percentage basis. Two hours a f t e r the or a l ad-ministration of a single meal of the d i f f e r e n t meals the birds were s a c r i f i c e d and the i n t e s t i n a l contents from the birds on eaoh treatment were pooled and treated with 5 percent TCA. Af t e r approximately one hour the mixtures were centrifuged at 2400 r.p.m., and nitrogen determined on the supernatants and residues. The percentage of 5 percent TCA insoluble nitrogen of the t o t a l nitrogen recovered from the i n t e s t i n a l t r a c t s of the pooled i n t e s t i n a l contents of each treatment were 57.8, 48.5 and 65.4% respectively for treatments (1), (2) and (3). In comparing the three treatments a greater percentage of the nitrogen was recovered i n the insoluble f r a c t i o n from both the jejunum and ileum of birds fed treatment (.3), the o r i g i n a l meal, than from the other two treatments. In comparing t r e a t -ments (2) and (3) more nitrogen was recovered from the intes-* t i n a l t r a c t of birds fed treatment (3) than those fed treatment (2). This would indicate that the meals fed were not at com-parable stages i n the digestive t r a c t . E i ther the moderately damaged meal had passed through the entire t r a c t , which seems u n l i k e l y i n birds of t h i s age, or because the material had not yet arrived i n the i n t e s t i n a l t r a c t s of the birds fed treatment 83 (2). I t has been reported that poor q u a l i t y meals do not pass through the crop as r e a d i l y as good qua l i t y ones (Smith, 1966 b) . This indicated a disadvantage i n feeding a single meal and sam-i p l i n g the i n t e s t i n a l contents at a r e l a t i v e l y short time a f t e r -wards. I f treatments (2) and (3) are compared the r e s u l t s can be interpreted i n d i f f e r e n t ways. One inte r p r e t a t i o n based on the percentage of nitrogen recovered i s that the s l i g h t l y damaged meal (treatment (2)) was s o l u b i l i z e d but had not been absorbed to the same extent as treatment (3) meal or the r e s u l t s could be interpreted to mean that more of the t o t a l nitrogen had been s o l u b i l i z e d and absorbed from treatment (2) and that treatment (3) meal was not s o l u b l i z e d . I t was concluded that r e s u l t s from a single feeding were not meaningful. At t h i s point i t was decided to examine the fr e e amino acid content i n the soluble f r a c t i o n s from the d i f f e r e n t t r e a t -ments. Aliquots of each sample were analysed f o r free amino acids as described i n part 2. A greater quantity of the free basic amino acids were present i n the soluble f r a c t i o n from birds fed the heat damaged meals. There were no marked d i f -ferences between the amounts present i n the jejunum and ileum In any of the soluble fractions from any of the three t r e a t -ments. When the a c i d i c and neutral amino acids were expressed as a percentage of the leucine present i n each sample a greater quantity of ninhydrin p o s i t i v e compounds eluted before aspartio acid i n the heat damaged meals than i n the o r i g i n a l samples. I t has been found that the ninhydrin p o s i t i v e browning reaotion intermediates from f r i e d mixtures of amino acids and sugars increase to a maximum and then decrease with time (Talley and 84 Porter, 1968). As a percentage of leucine there was less as-p a r t i c acid and cystine present i n the samples from the birds fed the heat damaged meals than i n the sample from those fed the o r i g i n a l meal. Also a smaller percentage of serine was present i n the sample from birds fed the severely damaged meal than from the other two samples. Methionine was present i n a greater percentage i n the heated samples than i n the unheated sample. This seems pe c u l i a r . The pattern of the other amino aoids i n r e l a t i o n to leucine i s quite s i m i l a r . -These observations come from single determinations therefore no conclusions can be drawn from these values. Another test was carried out to investigate the amounts of nitrogen present, the percentage of the t o t a l nitrogen i n -soluble i n 2 percent TCA and the amounts of free basic amino aoids i n the TCA soluble f r a c t i o n i n the i n t e s t i n a l contents of birds fed several meals, spaced at regular i n t e r v a l s , of herring meal with d i f f e r e n t c a r r i e r s . I t was f e l t that sampl-ing gut contents which were undergoing normal passage through the gut would represent a more natural and therefore more accurate measure of the s o l u b i l i t y of proteins and the r e l a t i v e amounts of various amino acids present at a given time. In most of the following experiments the percentage of protein i n the mixtures fed was that which would most l i k e l y be found i n a p r a c t i c a l d i e t . The amount fed i n a given period was c a l -culated from the d a i l y feed consumption determined immediately p r i o r to each p a r t i c u l a r study. Eight 16 week old birds starved 24 hours were eaoh given 3 meals (Table 15) of 10 - 11 grams each consisting of 85 approximately 300 mg. of nitrogen. In four hours each b i r d reoeived about 30 grams of feed which contained approximately 900 mg. of nitrogen. -Herring meal was fed as the protein source and was combined 25:75 with d i f f e r e n t o a r r i e r s : -treatment (1) herring meal (HM) plus glucose monohydrate^ treatment (2) HM plus corn staroh; treatment (3) HM plus corn meal; treatment (4) HM plus ground c e l l u l o s e . Eaoh mixture was given to two birds. Each b i r d was s a c r i f i c e d 0.5 hours a f t e r i t s l a s t meal. The i n t e s t i n a l contents of both birds fed the same treatment were divided into jejunum and ileum as described, mixed with 2$ TCA and the mixtures l e f t at room temperature 1 - 1 . 5 hours then centrifuged at 2400 r.p.m. 10 minutes. Nitrogen was determined i n the f r a c t i o n s as described previously. In both cases where the protein was fed with a oarbo-hydrate (corn starch or oorn meal) and where i t was fed with oellulose f a r more nitrogen was recovered i n both gut sections than when HM was fed with glucose monohydrate (Table 15). A smaller proportion of the t o t a l nitrogen was insoluble i n 2 percent TCA from the contents of birds from both segments fed HM and glucose monohydrate than any of the other treatments. More insoluble nitrogen was found i n the gut contents of birds fed the c e l l u l o s e . Of the nitrogen present i n each segment a smaller percentage was insoluble i n 2 percent TCA from the jejunum than from the ileum except i n the case of the birds reoeiving treatment (4). There was no difference i n the percent-age of insoluble nitrogen recovered from either segment i n the oellulose fed birds. Adding 5 ml. of 50$ TCA to 10 ml. of the 86 2 percent TCA supernatant did not change the amount of soluble nitrogen. Aliquots from the 2 percent TCA soluble f r a c t i o n s were chromatographed for the basic amino acids. Whether the amino acids present i n the segments were expressed as micro-moles per segment or as micromoles per mg. of soluble n i t r o -gen the amounts decreased from the jejunum to the ileum i n the samples from birds fed the f i r s t three treatments. The con-tents from birds fed treatment 4 did not r e a l l y show t h i s change. The most probable explanation i s that the i n t e s t i n a l contents took much longer to c o l l e c t than was allowed f o r i n the schedule consequently the i n t e s t i n a l material from these birds which had been s a c r i f i c e d on time were not collected u n t i l about f o r t y minutes l a t e r . The enzymatic release of amino acids had continued but the absorption of released amino acids had not occured. I t was observed that lower amounts of l y s i n e , h i s t i d i n e and arginine were present i n the gut of birds fed HM with glucose monohydrate than i n those fed the other three mixtures. Table 15. Amounts of nitrogen present i n the gut and the percentage of nitrogen i n the TGA fract i o n s recovered from the i n t e s t i n e of birds fed Herring Meal (HM) combined with d i f f e r e n t " c a r r i e r s " . Average gut nitrogen recovered/bird x t 2$ TCA t o t a l f TCA TCA Treatment Segment Residue soluble segment "soluble 'insoluble of t o t a l N of t o t a l N mg. * mg. mg. 1. 1. jejunum 6.5 15.5 22.0 70.5 29.5 HM gluoose monohydrate ileum 8.4 12.5 20.9 59 .8 40.2 Total 14.9 28.0 42.9 65.3 34.7 2. jejunum 50.4 41.0 91.4 44.8 55.2 HM corn starch ileum 27.7 16.5 44.2 37.3 62.7 Total 78.1 57.5 135.6 42.4 57.6 3. jejunum 40.3 31.0 71.3 43.4 56.6 HM corn meal ileum 20.9 14,5 35.4 41.0 59.0 Total 61.2 45.5 106.7 42.6 57.4 4. jejunum 51.9 31.5 83.4 37.8 62.2 HM ground c e l l u l o s e ileum 37.7 25.0 62.7 39.9 60.1 Total 89.6 56 .5 146 .1 38.7 61.3 1. Individual birds were given a meal every 2 hours. In 4 hours birds receiving: Treatment 1 received 883 mg. nitrogen Treatment 2 reoeived 963 mg. nitrogen. Treatment 3 received 917 mg. nitrogen Treatment 4 received 842 mg. nitrogen 88 Two small experiments were carried out t o determine what effect the amount of l i q u i d i n the s l u r r y had on the amount of gut nitrogen recovered. In the previous experiment, for convenience i n administration, a l l test materials had been administered i n the same t o t a l volume of s l u r r y . A l l the s l u r r i e s had been brought to the same volume as that which the f i s h meal and ground c e l l u l o s e could be pipetted i n . In the f i r s t experiment birds weighing approximately 227 grams and starved 27 hours were given a t o t a l of approximately 50 mg. nitrogen i n three feedings during two hours (5 birds per t r e a t -ment). _ Herring meal and a c a r r i e r either glucose monohydrate or c e l l u l o s e were combined 28 plus 72. Treatments 1 and 2 were the same, HM plus glucose monohydrate, except that the amount of feed fed to birds receiving treatment 1 was adminis-tered i n three times the s l u r r y volume that treatment 2 was fed i n . Treatment 3 consisted of HM and ground c e l l u l o s e . The t e s t was conducted as described previously. There was no r e a l difference between the amounts of nitrogen recovered nor between the percentage of nitrogen recovered int-the 2$ TCA i n -soluble f r a c t i o n i n the gut contents from birds receiving treatments 1 and 2. As noted before the amounts of recoverable nitrogen were muoh greater from birds fed the c e l l u l o s e than from those fed the gluoose monohydrate and the percentage of insoluble nitrogen i n each segment was greater than that In the same segments from birds receiving the other two t r e a t -ments. The addition of 5 ml. of 50% TCA to 10 ml. of the 2% TCA soluble f r a c t i o n did not change the amount of soluble n i t r o -gen. There appeared no marked difference between the amount of 89 basic amino aoids i n the 5$ TCA soluble f r a c t i o n recovered from any of the three treatments from either segment. The previous experiment was repeated with another group of birds six weeks old, three per treatment, only the number of feedings was increased to a t o t a l of f i v e i n 4 hours and another treatment was included, that of feeding herring meal alone. Data from t h i s second experiment i s summarized i n Table 16. The percentage of nitrogen i n the 5% TCA i n -soluble f r a c t i o n was the same i n the two i n t e s t i n a l segments whether the feed was fed i n the same volume or not but the t o t a l amount of nitrogen recovered from birds receiving the more d i l u t e s l u r r y was one-half of that recovered from the l e s s d i l u t e s l u r r y . More nitrogen was recovered from the intestine of birds fed the HM alone than from any of the other treatments. As noted i n the previous two experiments the percentage of TCA insoluble nitrogen was greater i n the gut of the birds receiving c e l l u l o s e than i n any of the other treatments. 90 Table 16. Amounts of nitrogen present i n the gut and the percentage of t o t a l nitrogen i n the TCA fraotions reoovered from the int e s t i n e s of birds fed herring meal (HM) alone and combined with d i f f e r e n t ^ c a r r i e r s " . Gut nitrogen recovered/bird x 5% TCA -Total % TCA £ TCA Treatment Segment Residue soluble segment soluble insoluble mg. mg. mg. mg. of t o t a l N of t o t a l N 1. jejunum 8.2 7.0 15.2 46.0 54.0 HM ileum 11.2 11.1 22.3 49.8 50.2 Total 19.4 18.1 37.5 48.3 51.7 2. jejunum 1.6 2.1 3.7 56 .8 43.2 HM glucose ileum 5.8 5.7 11.5 49.6 50 .4 monohydrate 48*7 •Total 7.4 7.8 15.2 51.3 3. jejunum 3.2 4.1 7.3 56.2 43.8 HM glucose ileum 9 .9 10.7 20.6 51.9 48.1 monohydrate Tot a l 13 .1 14.8 27.9 53.0 47.0 4. jejunum 9.1 4.6 13 .7 33. e 66.4 HM ground ileum 8.7 5.4 14.1 38.3 61.7 c e l l u l o s e Total 17.8 10.0 27 .8 36.0 64 .0 1. A l l birds received 5 meals of 90 mg. N each or a t o t a l of 450 mg. N i n 4 hours. Each meal was contained i n 5.6 ml. f o r birds receiving treatments l a n d 3. For those receiving treatments 2 and 4 each mear was eentained i n 14 ml. 91 The l a s t test i n the series was designed to compare two d i f f e r e n t protein concentrates, dogfish frame meal and chicken muscle meal, for the amounts of recoverable gut nitrogen. These two meals represented a very poor and a very good qua l i t y meal respectively when they were evaluated b i o l o g i c a l l y by supple-mentary protein value. In t h i s t e s t adult birds were used. Since they are d i f f i c u l t to manage alone only one bird per digestion time could be scheduled. They were fed every four hours and the i n d i v i d u a l birds were s a c r i f i c e d either two or four hours a f t e r the l a s t meal. Two feedings of equivalent amounts of protein nitrogen were given to two birds per t r e a t -ment. Eighteen percent of each feeding consisted of protein and the rest corn starch. In each case more insoluble n i t r o -gen was recovered from the b i r d s fed the dogfish frame meal than those fed the chicken musqle; The t o t a l amount of n i t r o -gen recovered from birds fed the same protein souroe and s a c r i f i c e d at either 6 or 8 hours a f t e r the commencement of the test were very s i m i l a r , 63 and 87 mg. of nitrogen from those fed the dog f i s h and 32 and 87 mg. of nitrogen from those fed the ohloken muscle (E9 mg. of nitrogen was recovered from a bird included i n the chicken muscle test and s a c r i f i c e d at 4 hours a f t e r the commencement of the test*) There was a decrease i n the amount of nitrogen i n the supernatant when 5 ml. of 50% TCA was added to 10 ml. of the supernatant from the i n t e s t i n a l contents of the birds fed the dog f i s h frame meal. This was the only case i n which increasing the con-centration of TCA ab©v^ 5% made any appreciable e f f e c t . 92 I t had been observed i n 3 separate experiments that the percentage of soluble nitrogen was le s s i n the i n t e s t i n a l con-tents of birds which had been fed c e l l u l o s e with the protein rather than glucose monohydrate or corn starch. To determine i f t h i s were due to the adsorption of some of the nitrogen con-t a i n i n g oompounds on to the c e l l u l o s e several small tests were carri e d out'. In two tests casein hydrolysate was used as a source of free amino a c i d . The hydrolysate was incubated one o hour at 25 or 42 C with or without ground c e l l u l o s e at the pH encountered i n the d i f f e r e n t segments of the i n t e s t i n e . The mixtures were treated with 5% TCA i n the same way that the i n t e s t i n a l contents were. No difference i n the amount of 5% TCA soluble nitrogen or free basic amino aoid oontent was observed between the supernatants of the control and ground c e l l u l o s e treated mixtures. As there seemed to be no difference i n the percentage of supernatant nitrogen when the nitrogen was i n the form of free amino aoids, other protein degradation products were tested. These products included peptone, proteose peptone, (Baoto #3) and peptonized milk. Four aliquots of each were incubated, two without ground c e l l u l o s e and two with, f o r 3.5 hours. At the end of t h i s inoubation 5% TCA was added to one r e p l i c a t e of eaoh mixture and the prooedure continued as for the i n t e s t i n a l contents. In eaoh case where c e l l u l o s e was included i n the incubation mixture a s l i g h t l y smaller amount of nitrogen was reoovered but not enough to show the same difference as that observed i n the gut. I t was observed that the proteose peptone plus TCA 93 supernatant was oloudy while the supernatant from the same mixture but with ground oellulose was c l e a r . Consequently the ef f e c t of a combination of longer peptides and c e l l u l o s e was investigated. Equivalent amounts of protein from f i s h meal and f i s h meal plus ground c e l l u l o s e were incubated with 0.2$ pepsin i n HC1 (6.1 ml./l) at 42°C. At the i n t e r v a l s i n d i c a -ted i n Table 17 aliquots were taken and mixed with 5$ TCA and treated i n the same way as the i n t e s t i n a l oontents. A f t e r the addition of TCA to the herring meal incubation mixture the solution stayed '•murky*'. I f c e l l u l o s e were present the super-natants were much les s "murky". At the end of three hours of Incubation the mixtures were neutralized with sodium bicarbon-ate to p 6.6 - 7.1 and four ml. of a solution of 0.5 grams of tr y p s i n 1:250 i n 10 ml. were added. Samples from this incuba-t i o n mixture were taken 20 and 40 minutes l a t e r . The r e s u l t s are shown i n Table 17 i n which the percentages of TCA soluble nitrogen i n the mixture containing herring meal and c e l l u l o s e are expressed as a percentage of that soluble i n the mixture containing herring meal alone. These percentages show that, except f o r the 0.5 hour samples, one possible explanation f o r the lower percentage of soluble nitrogen i n the supernatant of birds fed c e l l u l o s e with the protein than from those fed glucose monohydrate or staroh was that when the TCA was added to the mixtures some nitrogen containing material was not c e n t r i -fuged out of solution but i f c e l l u l o s e were present t h i s material beoame attached to the ground material and was therefore removed from s o l u t i o n . I t can be seen from Table 17 that as the peptide length becomes shorter with the increase i n incubation time and the addition of more enzyme that the ef f e c t of the c e l l u l o s e becomes l e s s . 94 Table 17. Comparison of the percentages of TCA soluble nitrogen of t o t a l nitrogen from incubation mixtures of herring meal and herring meal-f c e l l u l o s e . f Incubation $5 TCA % of soluble N Enzyme Substrate time soluble of substrate 2 x -,0Q hr. nitrogen substrate 1 of t o t a l N Pepsin 1. HM 0.5 29.2 128. 2. HM 0.5 37.4 c e l l u l o s e 1. HM 1.0 40.7 Pepsin 79.5 2. HM 1.0 32.4 c e l l u l o s e 1. HM 2.0 38.3 Pepsin 85.9 2. HM 2.0 32.9 c e l l u l o s e Pepsin 1. HM 3:20 68.1 Trypsin 90.2 2.. HM c e l l u l o s e 3:20 61.4 Pepsin 1. HM 3.40 76.3 Trypsin 94.1 2. HM 3.40 71.8 c e l l u l o s e 95 General Discussion. The work with protein digestion i n which the intestine was t i e d off was not continued. I t was f e l t that preliminary information oould be obtained from normal b i r d s . The steps taken to reduce the content of fibrous material i n the digest-ive t r a c t a f t e r a f a s t seemed s a t i s f a c t o r y . Some material did remain i n the t r a c t no matter what procedures were taken. No r e p l i c a t i o n i n which a nonprotein diet was fed to i n d i v i d u a l birds was done therefore the a c t u a l 'error' induced by t h i s material between d i f f e r e n t birds was not determined. Obtaining well mixed samples from birds whose digestive system was f u l l of feathers was d i f f i o u l t . I f the test d i e t s were t o be fed for a period of time long enough to pass through the entire t r a c t a f a s t i n g period would probably not be necessary. Gelatin capsules were not used for the reasons given. This observation was made on only three birds, i t may not be the general case. Under natural feeding conditions, when a bird has been deprived of food long enough to empty the crop and gizzard, the f i r s t beakfuls of food pass d i r e c t l y to the gizzard, thereafter food not required to keep the gizzard f u l l remains i n the crop (Bolton, 1969). The administration of material i n s l u r r y was easy and f a i r l y acourate so long as the materials were f i n e l y ground, a sapid transfer pipette was used and the t e s t birds were of a size that they could be fed rapidly and the i n t e s t i n a l contents removed quickly (approximately 8 - 16 weeks of age for White Leghorns). The amount of l i q u i d i n the s l u r r y had to be adjusted f o r d e l i v e r y depending on the protein source and 96 " c a r r i e r " involved. The amount of l i q u i d i n the s l u r r y was found to a f f e c t the amount of nitrogen recovered from the in t e s t i n e i f several meals were administered within a f a i r l y short period of t i p s , i . e . the digestive system could l i t e r -a l l y be washed out, but the percent of nitrogen soluble i n TCA appeared to remain about the same. The material has to have a higher l i q u i d oontent to be pipetted than the l i q u i d content normally found i n the crop. While p e l l e t i n g some materials i s d i f f i c u l t and force feeding p e l l e t s to birds i s . not as easy as the other two methods mentioned i t i s f e l t that the material would be acted upon under more normal conditions i n the gut. The amounts of f i b r e fed with the protein material was not l o g i c a l , t h i s amount would never exist i n p r a c t i c a l feeds. A more reasonable combination would be a mixture of carbohydrate and ground c e l l u l o s e which would contribute about 3 - 4 % f i b r e to each meal. In most of the t r i a l s i n which ground c e l l u l o s e was Incorporated with a protein the amount of residue (undigested material plus that insoluble i n TCA) was greater and the percent of TCA soluble nitrogen l e s s than recovered i n the other experi-ments. In most of the tests i n which the free basic amino acids present were determined those present i n the c e l l u l o s e t r e a t -ment were more. There may be several explanations for the difference i n soluble and insoluble nitrogen between the c e l l u -lose containing diets and those with l i t t l e i f any f i b r e . (1) the lumen was completely f i l l e d with very dry material. This would suggest that considerable erosion of the i n t e s t i n a l e p i -thelium could have taken place and contributed to the insoluble 97 f r a c t i o n . The occurence of a good deal of erosion i s suggested by the f a c t that when a highly digestible diet i s fed to birds, the c e l l s l i n i n g the duodenum are long and have pointed apices, but i f the food i s fibrous and of low d i g e s t i b i l i t y the c e l l s are short and have blunted apices (Hallsworth and Coates 1962). (2) Bolton (1961) has reported a considerable increase i n the nitrogen content of the food during i t s passage along the t r a c t . On a normal layers mash, using c e l l u l o s e as marker, approximate-l y a ten f o l d increase i n nitrogen was shown by the time the material reached the end of the duodenum. Using the same feed with oat h u l l s added to give a c e l l u l o s e content of 27$, a 50 f o l d inorease i n the r a t i o of protein present i n the duodenum was found. Of th i s 30$ could be isola t e d as mucus. This accelerated increase would accord completely with the greatly enhanced erosion of the mucosal l i n i n g shown by the high f i b r e d i e t . (3) The presenoe of the c e l l u l o s e i n the TCA mixtures tended to ."clear" the supernatants of nitrogen material thus increasing the amount of residual nitrogen and decreasing the percent of soluble nitrogen. Most of these studies were carried out only to develop a routine procedure. No nonprotein diets were fed as r e l a t i v e rather than absolute recoveries of nitrogen were considered more meaningful. Several d i f f e r e n t areas of investigation were included f o r preliminary information. There was no r e p l i c a t i o n of any of the treatments i n any of the t e s t s . Because of t h i s no conclusions can be drawn from the values obtained. It was found that one person can complete four treatments involving 12 - 16 birds i n d i v i d u a l l y treated (White Leghorns approximately 98 8 - 1 6 weeks of age) per day. More treatments and or more birds might require assistanoe i f the same p r e c i s e l y timed method; were used as was developed here. Times f o r the b i o l o g i c a l part of the test varied depending upon the size of the b i r d s . This was found to be an important consideration where more than t o t a l nitrogen was to be determined on the gut contents. The gut contents were treated with TCA immediately a f t e r c o l l e c t i o n to stop enzyme a c t i v i t y rather than separating the soluble and insoluble materials with no p r i o r treatment. ( P o s s i b i l i t y of c o - p r e c i p i t a t i o n of other material?). With a normal diet there was very l i t t l e difference i n the super-natant nitrogen when 5 or 30% TCA w/v was added to al i q u o t s i n the r a t i o of gut contents : TCA of 1:5 w/v. However, the percent of TCA i n some of the supernatants was increased to determine i f any of the intermediate products i n proteolysis could be p r e c i p i t a t e d . The supernatants obtained from the gut contents of birds fed dogfish frame meal was the only test i n which some products could be precipitated with 10 ml. 5% TCA supernatant plus 5 ml. of 50% TCA (w/v). But i t was observed i n some of the tests where the 5% TCA supernatants were stored overnight i n the fridge that a fine p r e c i p i t a t e had formed i n some of the supernatants. I t has been demonstrated that employing 5% TCA i n the proportions of one volume of sample to f i v e volumes of 5% TCA removes not only the protein but also a l l the peptides from samples of albumin digested 16 hours with 0.01% pepsin, i n v i t r o (IL-Lakany, personal communication). I t has been recommended that no higher proportions should be used of 5% TCA than a 99 1 : 1 volume to volume i f d i f f e r e n t peptides are to be l e f t i n solution. For the determination of f r e e amino acids i n the soluble portions of the gut contents a TGA f r a c t i o n a t i o n i s unsuitable i f the amino acids are to be determined by automatic ion ex-change chromatography as the TCA breaks down i n the reaction bath. Summary Several small t r i a l s were conducted to obtain prelim-inary information on l e v e l s of gut nitrogen recoverable a f t e r various dietary treatments. The technique employed was adequate to d i s t i n g u i s h between an extremely poor and a good q u a l i t y protein concentrate when the amount of recoverable TCA insoluble nitrogen was the c r i t e r i o n . Administration of test meals i n s l u r r y form was found to be easy and f a i r l y accurate but attention must be given to the volume of l i q u i d i n the s l u r r y . Results obtained from a single feeding a f t e r an extended f a s t were not considered r e l i a b l e . Because of the action of f i b r e i n the i n t e s t i n a l t r a c t and the i n t e s t i n a l response to protein fed with and without a oa r r i e r only test meals and materials of sim i l a r type and consistency should be assayed together and compared i f thi s method of protein evaluation i s considered. Much lower l e v e l s of TCA should have been used i n the f r a c t i o n -ation of the i n t e s t i n a l nitrogen. 100 Bibliography-Abel, J . J . , Rowntree, L.G., and B.B. Turner. 1913. On the removal of d i f f u s i b l e substanoes from the c i r c u l a t i n g blood by means of d i a l y s i s . Ass. Amer. Phys., Trans. 28:51 r-54. Albanese, A."A., and L.A*.0rto. 1960. S c i . Exhibit 154 t h Amer. Convention Med. Soc. State of New York, May 9-13. oited by J.M.MoLaughlan. Fed. Proc. 22:1182-1125. Almquist, H.«T. 1954. U t i l i z a t i o n of amino acids by chicks. Arch. Biochem. Biophys-, 52:197-202. Anderson, J.O., and R.E. Warnick. 1969. Available amino aoids i n poultry feedstuffs. Feedstuffs 41(13):32-37. Association of O f f i c i a l A g r i c u l t u r a l Chemists (A.O.A.C), 1960. O f f i c i a l Methods of Analysis, 9 t n E d i t i o n . Fashington.D.C. Bender, A.E. 1961. Natl .Acad ..Sci .-Natl. Res.Council Publ. 843 Meeting protein needs, p.407-421. Bender, A.E. 1965. The balancing of amino acid mixtures and proteins. Nutr.Soc.,Proo. 24:190-196. Bender, A.E. 1969. Newer methods of assessing protein q u a l i t y . Chemistry and Industry (London) No. 27:5 July,1969:1. B l e l o r a i , R., and A.Bondl. 1963. Relationship between ' a n t i -oryptic factors* of some plant protein feeds and products of proteolysis precipitable by t r i c h l o r a c e t i c a c i d . J.Sci.Fd.Agrio. 14:124-132. Bird, F.H. 1968. Role of the avian small intestine i n amino aoid metabolism. Fed.Proc. 27:1194-1198. Block, R.J., and B o i l i n g . 1951. The amino acid composition of proteins and foods. Second e d i t i o n . C.C.Thomas, Sp r i n g f i e l d , 111. Bolton, W. 1961. The absorption of food from the gut of the fowl. Nutr.Soc., Proc. 20:xxv. Bolton, W. 1964. The n u t r i t i o n a l significance of the endogenous nitrogenous secretions i n poultry, p.117-122. In: .H.N. Munro ed. The role of the g a s t r o i n t e s t i n a l t r a c t i n protein metabolism. Blaokwell, Oxford. Bolton, .¥. 1969. Digestion, absorption and metabolism i n poultry, p.183-203. In: S i r David Cuthbertson ed. N u t r i t i o n of Animals of A g r i c u l t u r a l Importance. International Encyclopaedia of Food and N u t r i t i o n . Part 1. The Science of N u t r i t i o n of Farm Livestock. 101 Boomgaardt, J . , and B.E. McDonald, 1969. Comparison of fa s t i n g v plasma amino acid patterns i n the pig, rat and chicken, Can. J . Physiol. Pharmacol. 47:392-395. Bragg, D.B., J . 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