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Growth and liver enzyme response to dietary levels of sulphur amino acids in growing rats and pigs receiving… Ngwira, Timothy Nyamayanji 1978

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GROWTH AND LIVER ENZYME RESPONSE TO DIETARY LEVELS OF SULPHUR AMINO ACIDS IN GROWING RATS AND PIGS RECEIVING BARLEY-BASED DIETS by TIMOTHY NYAMAYANJI NGWIRA B.Sc, University of Malawi, 1969 M.Sc, University of Wales (Aberystwyth), 1972 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY in THE FACULTY OF GRADUATE STUDIES Department of ANIMAL SCIENCE We accept t h i s thesis as conforming to the required standard UNIVERSITY OF BRITISH COLUMBIA July 1978 Timothy Nyamayanji Ngwira, 1978 - 1 1 -In presenting this thesis in partial fulfilment of the requirements for an advanced degree at the University of Brit ish Columbia, I agree that the Library shall make it freely available for reference and study. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the Head of my Department or by his representatives. It is understood that copying or publication of this thesis for financial gain shall not be allowed without my written permission. Depa rtment The University of British Columbia 20 75 Wesbrook Place Vancouver, Canada V6T 1W5 Date i i i ABSTRACT The growth of weanling rats receiving varying lev e l s of methionine plus cystine i n barley-based d i e t s showed that 0.23-0.40% dry matter (DM) basis l e v e l s could not support optimal growth. No s i g n i f i c a n t differences were detected i n carcass composition or growth of rats receiving 0.45-0.70% DM methionine plus cystine dietary l e v e l s when either the cystine l e v e l was held constant at 0.20% DM i n a l l d i e t s or the methionine:cystine r a t i o was held constant at 2:1 and 1:1 i n a l l d i e t s . When either the dietary cystine concentration was held constant at 0.20% DM or the methionine:cystine r a t i o was held constant at 2:1 i n a l l d i e t s , the re s u l t s indicated that cystathionine synthase a c t i v i t y was constant between 0.35 and 0.50% DM dietary methionine plus cystine. The a c t i v i t y of the enzyme was then i n h i b i t e d , reaching minimum a c t i v i t y at the 0.60% DM dietary methionine plus cystine l e v e l . Thereafter, the a c t i v i t y increased to l e v e l s higher than the a c t i v i t y l e v e l s obtained between 0.35 and 0.50% DM dietary methionine plus cystine l e v e l s . These r e s u l t s indicate that 0.50% DM dietary methionine plus cystine i s the l i m i t for normal methionine metabolism. When either the dietary cystine concentration was held constant at 0.20% DM or the methionine:cystine r a t i o was held - i v --constant at 2:1 i n a l l d i e t s , the a c t i v i t y of N 5-methyltetra-hydrofolate-homocysteine-methyltransferase (mTHF Enz.) was constant between 0.35 and 0.50% DM methionine plus cystine. The enzyme a c t i v i t y then increased, reaching maximum l e v e l s at the 0.60% DM methionine plus cystine l e v e l . Thereafter the enzyme a c t i v i t y was i n h i b i t e d to l e v e l s corresponding to a c t i v i t i e s obtained between 0.35-0.50% DM dietary methionine plus cystine. The r e s u l t s indicate that normal methionine metabolism occurs up to the 0.50% DM methionine plus cystine i n the d i e t . When the methionine:cystine r a t i o was held constant at 1:1 i n a l l d i e t s i n which the methionine plus cystine concentration varied between 0.35 and 0.70% DM, cystathionine synthase a c t i v i t y did not respond to the varying l e v e l s of methionine plus cystine, whereas mTHF Enz. a c t i v i t y was i n h i b i t e d progressively with increasing l e v e l s of dietary methionine plus cystine. The same results showed that increasing the diet a r y serine l e v e l from 0.38 to 0.58% DM depressed feed intake and the a c t i v i t i e s of both cystathionine synthase and mTHF Enz. i n rats fed the 0.35-0.70% DM dietary methionine plus cystine range. The i n t e r a c t i o n between choline and methionine plus cystine when the methionine: cystine r a t i o was held at 1:1 i n a l l d i e t s showed that the 1200 mg choline chloride/kg DM die t i n h i b i t e d mTHF Enz. a c t i v i t y and activated cystathionine synthase more than the 1000 mg choline chloride/kg DM d i e t . - v -The pig t r i a l , i n which g i l t s were fed barley-based d i e t s containing varying l e v e l s of methionine plus cystine, showed that the change i n urinary urea-nitrogen excretion of pigs on test d i e t s from the p o s i t i v e control d i e t , could be used as an indicator of methionine plus cystine requirements for optimal growth. Using t h i s parameter, the requirement for methionine plus cystine for the 32.6±0.6 kg g i l t was 0.55% DM on barley-based d i e t s where cystine was held constant at 0.20% DM l e v e l . - v i -TABLE OF CONTENTS Page ABSTRACT . i i i TABLE OF CONTENTS v i LIST OF TABLES x LIST OF FIGURES xiv LIST OF APPENDICES x v i ACKNOWLEDGEMENTS x v i i INTRODUCTION 1 LITERATURE REVIEW 7 Protein digestion 7 Transport and interactions of amino acids during absorption 9 Absorption and transportation of peptides 15 Contribution of endogenous nitrogen secretion . . . 17 Protein q u a l i t y 19 Factors a f f e c t i n g protein q u a l i t y 20 Amino acid requirements 25 Factors a f f e c t i n g amino acid requirements . . . . 27 Individual amino acid requirements 3 0 Methionine metabolic pathway 3 0 Inborn error diseases of methionine metabolism . . 39 Sulphur amino acids i n monogastric animal n u t r i t i o n 43 Rats 43 Poultry 47 Pigs 51 - v i i -Page Methods for determining the t o t a l amino acids, a v a i l a b l e amino acids and protein quality of feeds 53 \ A. Total amino acid analysis 53 B. Methods for measuring available amino acid l e v e l s and protein q u a l i t y 56 Chemical methods 56 B i o l o g i c a l methods 60 Mi c r o b i o l o g i c a l methods • . . 67 Enzymatic assays . 71 EXPERIMENTS 7 4 RAT TRIAL 1 74 Growth response of weanling rats to graded l e v e l s of methionine plus cystine in barley-based d i e t s f o r t i f i e d with synthetic amino acids 74 Introduction and aim 74 Materials and methods 75 Results 81 Discussion 87 Conclusion 90 RAT TRIAL 2 91 E f f e c t of two l e v e l s of dietary protein and two le v e l s of methionine plus cystine on the growth of weanling rats fed barley-based diets 91 Introduction and aim 91 Materials and methods 91 Results 95 - v i i i -Page Di scussion 103 Conclusion 105 RAT TRIAL 3 106 Growth and l i v e r enzyme response i n growing rats to graded lev e l s of methionine plus cystine i n f o r t i f i e d - b a r l e y diets 106 1. - Response at constant cystine concentration i n the die t 106 Introduction and aim 106 Materials and methods . 107 Results 114 Discussion 124 Conclusion 131 RAT TRIAL 4 133 Growth and l i v e r enzyme response i n growing rats to graded l e v e l s of methionine plus cystine i n f o r t i f i e d - b a r l e y d i e t s 133 2. Response at a constant methionine:cystine r a t i o 133 Introduction and aim 133 Materials and methods . . . 134 Results 138 Discussion 148 Conclusion 152 RAT TRIAL 5 . . . ' 154 Interaction between serine, choline and methionine plus cystine in weanling rats fed f o r t i f i e d barley-based diets at a constant methionine:cystine r a t i o 154 - i x -Page Introduction and aim 154 Materials and methods 155 Results 160 Discussion 177 Conclusion . 181 PIG TRIAL 183 The e f f e c t of varying lev e l s of dietary methionine plus cystine on urinary urea excretion i n growing female pig 183 Introduction and aim 183 Materials and methods 184 Results 190 Discussion 192 Conclusion 197 GENERAL DISCUSSION AND CONCLUSION 198 LITERATURE CITED 205 APPENDIX 1 230 APPENDIX 2 231 APPENDIX 3 : . . . . 232 APPENDIX 4 233 - x -LIST OF TABLES Page I. Formulation (% DM) of diets for Rat T r i a l 1 76 II . E s s e n t i a l amino acid composition (% DM) of die t s for Rat T r i a l 1 78 I I I . Average d a i l y body-weight gain (g), food consumption (g) and food conversion e f f i c i e n c y (g weight gain/g feed consumed) of rats i n Rat T r i a l 1 82 IV. The e f f e c t of dietary methionine plus cystine l e v e l s on some selected variables of rat carcass composition i n Rat T r i a l 1 85 V. Results of single degree of freedom contrasts of variables i n Tables III and IV 86 VI. Composition (% DM) of diets in Rat T r i a l 2 93 VII. Amino acid composition (% DM) of di e t s i n Rat T r i a l 2 94 VIII. Average d a i l y body-weight gain (g), food consumption (g) and conversion e f f i c i e n c y (g weight gain/g food consumed) of rats on d i e t s i n Rat T r i a l 2 96 IX. E f f e c t of dietary protein l e v e l s and dietary methionine plus cystine l e v e l s on rat carcass composition i n Rat T r i a l 2 100 X. Results of contrasts of variables presented i n Tables VIII and IX of Rat T r i a l 2 101 XI. Composition (% DM) of rat diets with constant cystine content of 0.2% DM i n Rat T r i a l 3 109 XII. Amino acid, protein (% DM) and energy (kcal/kg) content of diets for Rat T r i a l 3 111 - x i - Page XIII. Average d a i l y body-weight gain (g) food consumption (g) and food conversion e f f i c i e n c y (g weight gain/g food consumed) of rats fed on d i e t s containing varying l e v e l s of methionine plus cystine i n Rat T r i a l 3 115 XIV. The e f f e c t of varying l e v e l s of dietary methionine plus cystine on some selected variables of rat carcass composition i n Rat T r i a l 3 120 XV. Results' of orthogonal contrasts of variables presented in Tables XIII and XIV. 121 XVI. Composition (% DM) of rat diets containing a constant methionine:cystine r a t i o of 2:1 i n Rat T r i a l 4 135 XVII. Amino acid, protein (% DM) and energy (kcal/kg) content of d i e t s i n Rat T r i a l 4 137 XVIII. Average d a i l y body-weight gain, feed consumption and feed conversion e f f i c i e n c y of rats fed on di e t s containing varying methionine plus cystine l e v e l s i n Rat T r i a l 4 139 XIX. The e f f e c t of varying l e v e l s of dietary methionine plus cystine on r a t carcass composition i n T r i a l 4 142 XX. E f f e c t of varying l e v e l s of dietary methionine plus cystine on the a c t i v i t i e s of l i v e r cystathionine synthase and N^-methyltetrahydrofolate-homocysteine-methyltransf erase (mTHF Enz.) and urinary urea-nitrogen excretion of rats i n T r i a l 4 144 XXI. Results of contrasts of variables that showed s i g n i f i c a n t differences between die t s i n Tables XVIII, XIX and XX of Rat T r i a l 4 145 XXII. Percentage composition of di e t s i n Rat T r i a l 5 157 - x i i -Page XXIII. Amino acid (% DM), choline chloride (mg/kg DM), protein (% DM) and gross energy (kcal/kg DM) content of d i e t s i n Rat T r i a l 5 159 XXIV. Average d a i l y body-weight gain (g), feed consumption (g) and feed conversion e f f i c i e n c y (g weight gain/g feed consumed) of ra t s fed varying l e v e l s of methionine plus cystine i n Rat T r i a l 5 XXV. The e f f e c t of varying l e v e l s of dietary serine on feed consumption of rats in Rat T r i a l 5 162 163 XXVI. The e f f e c t of varying l e v e l s of dietary methionine plus cystine on the carcass composition of rats i n Rat T r i a l 5 XXVII. The e f f e c t of varying l e v e l s of dietary methionine plus cystine on the a c t i v i t i e s of l i v e r cystathionine synthase and N 5-methyltetrahydrofolate-homocysteine-methyltransf erase of rats i n Rat T r i a l 5 XXVIII. E f f e c t of dietary choline chloride l e v e l s on the a c t i v i t i e s of l i v e r cystathionine synthase and N5-methyltetrahydrofolate-homocysteine-methyltransferase i n Rat T r i a l 5 165 167 169 XXIX. E f f e c t of the i n t e r a c t i o n between dietary methionine plus cystine and serine on the a c t i v i t i e s of l i v e r cystathionine synthase and N^-methyltetrahydrofolate-homocysteine-methyltransf erase i n Rat T r i a l 5 173 XXX. E f f e c t of the i n t e r a c t i o n between dietary methionine plus cystine and choline chloride on the a c t i v i t i e s of cystathionine synthase and N5-methyltetrahydrofolate-homocysteine-methyltransf erase i n Rat T r i a l 5 176 XXXI. The ingredients (% DM), crude protein (% DM) and d i g e s t i b l e energy content (kcal/kg DM) of d i e t s used i n the Pig T r i a l 186 - x i i i -Page XXXII. Amino acid composition of d i e t s i n the Pig T r i a l , calculated from components 188 XXXIII. Apparent nitrogen d i g e s t i b i l i t y (% DM), average d a i l y feed consumption (kg DM), average d a i l y urea-nitrogen excretion (g), urea-nitrogen excretion/kg feed consumption (g/kg DM) and change i n urea excretion per feed consumed r e l a t i v e to the p o s i t i v e control d i e t i n pigs of 32.6±0.6 kg l i v e -weight fed experimental d i e t s varying in methionine plus cystine l e v e l s 193 XXXIV. Single degree of freedom contrasts of parameters that showed s i g n i f i c a n t differences between d i e t s i n Table XXXIII. 194 - xiv -LIST OF FIGURES Page 1. Methionine metabolic pathway 31 2. The e f f e c t of varying l e v e l s of dietary methionine plus cystine on the feed conversion e f f i c i e n c y of rats i n T r i a l 3 118 3. The e f f e c t of varying l e v e l s of dietary methionine plus cystine on the urinary urea-excretion by rats in T r i a l 3 123 4. The e f f e c t of varying l e v e l s of dietary methionine plus cystine on the a c t i v i t y of l i v e r cystathionine synthase of rats i n T r i a l 3 125 5. E f f e c t of varying l e v e l s of dietary methionine plus cystine on the a c t i v i t y of l i v e r N^-methyltetrahydrofolate-homocysteine-methyl-transferase of rats i n T r i a l 3 126 6. The e f f e c t of varying l e v e l s of die t a r y methionine plus cystine on the a c t i v i t y of l i v e r cystathionine synthase i n Rat T r i a l 4 143 7. The e f f e c t of varying l e v e l s of dietary methionine plus cystine on the a c t i v i t y of l i v e r N^-methyltetrahydrofolate-homocysteine-methyltransf erase i n Rat T r i a l 4 147 8. E f f e c t of the i n t e r a c t i o n between dietary lev e l s of methionine plus cystine and serine on the a c t i v i t y of rat l i v e r cystathionine synthase i n T r i a l 5 168 9. E f f e c t of varying l e v e l s of dietary methionine plus cystine on the a c t i v i t y of ISP-methyl tetrahydrofolat e-homocysteine-me thy 1-transferase i n the l i v e r s of rats i n T r i a l 5 170 10. The e f f e c t of the i n t e r a c t i o n between dietary l e v e l s of methionine plus cystine and serine on the a c t i v i t y of N^-methyltetrahydrofolate-homocysteine-methyltransferase i n l i v e r s of rats i n T r i a l 5 171 - XV -Page 11. The e f f e c t of the i n t e r a c t i o n between dietary l e v e l s of methionine plus cystine and choline on the a c t i v i t y of N^-methyltetrahydrofolate-homocysteine-inethyltransferase i n l i v e r s of rats i n T r i a l 5 174 12. The e f f e c t of varying dietary l e v e l s of methionine plus cystine on the change i n urea-nitrogen excretion per kg DM feed consumed when the pigs were fed a p o s i t i v e control d i e t followed by t e s t diets 191 - xvi -LIST OF APPENDICES Page E s s e n t i a l amino acid requirements (% DM diet) of the growing r at 230 Es s e n t i a l amino acid requirements (% DM diet) of the growing pig (NRC, 1973) 231 Amino acid requirements (% DM diet) of growing meat-producing poultry of 0-6 weeks of age 232 Amino acid requirements of the 6 month - 1 year growing infant (mg/kg body-weight/day) 233 -' x v i i -ACKNOWLEDGEMENTS I wish to express my gratitude to Dr. R.M. Beames for the enthusiastic guidance throughout the course of these studies and e s p e c i a l l y during the preparation of the manuscript. I would l i k e to thank Paul W i l l i n g , Aleck Ostry and Dr. B. Douglas for t h e i r help during the pig t r i a l . The patience of my family and the help of my wife i s g r a t e f u l l y acknowledged. Thanks to Mrs. M. S t r i k e r for s t a t i s t i c a l help and to Mrs. R. Gerstmar for typing the manuscript. I would l i k e to thank the Canadian Commonwealth Scholarship and Fellowship Committee and the National Research Council for t h e i r f i n a n c i a l support. - I -INTRODUCTION A l l animals and humans require dietary protein as a source of e s s e n t i a l amino acids plus a dietary source of nitrogen for the synthesis of non-essential.amino acids. For optimal growth, the d i e t L~90% dry matter (DM)] should contain- 12% crude protein for growing rats (NRC, 1972); 22-12% crude protein for pigs 5-100 kg respectively (NRC, 1973); 1.2 g/kg/day crude protein for the- 1 year old infant (FAQ, 1973) and 23% crude protein for the growing chick (NRC, 1971). When these l e v e l s are not met, defic i e n c y symptoms such as stunted growth occur. It- has been observed that the quantity of protein i s not the only l i m i t i n g factor i n requirements but that a c e r t a i n minimum q u a l i t y i s also necessary. This q u a l i t y has been shown by Rose et a l . (1942) to be controlled by "e s s e n t i a l amino acids" and t h e i r a v a i l a b i l i t y i n the d i e t . Thus the NRC (1972) l i s t s as e s s e n t i a l amino acids for the growing rat: arginine, h i s t i d i n e , i s o l e u c i n e , leucine, methionine plus cystine, phenylalanine plus tyrosine, threonine, tryptophan, valine and cystine. In order to promote optimal growth, these amino acids should each be present above a stated minimal l e v e l . I f , however, the a v a i l a b i l i t y of one of these e s s e n t i a l amino acids i s l i m i t i n g , the u t i l i z a t i o n of the t o t a l dietary protein i s lowered and def i c i e n c y symptoms may r e s u l t . Such a deficiency can be corrected either by supplementing - 2 -the d i e t with the l i m i t i n g amino acids or by adding a protein source which incorporates the desired l e v e l of missing amino acids i n an av a i l a b l e form. It has, however, been found that c e r t a i n amino acids, when incorporated i n the die t i n excess amounts, are toxi c . T o x i c i t y r e s u l t s i n the disruption of normal c e l l u l a r metabolism. The most toxic amino acid observed to date i s methionine (Benevenga, 1974) . There has been much work on defining the l e v e l s of various e s s e n t i a l amino acids required for optimal growth. For each animal species and for the chick, figures for a l l the e s s e n t i a l amino acids, with the exception of methionine plus cystine, seem to be well established. The figures given for methionine plus cystine are c o n f l i c t i n g . This c o n f l i c t i s alarming i n view of i t having been stated that most t h i r d world diets are l i m i t i n g i n sulphur amino acids (Allaway, 1969) and that methionine plus cystine are very toxic when incorporated at high dietary l e v e l s (Benevenga, 1974) . This, therefore, would indicate a need for a closer look at the l e v e l s of methionine plus cystine recommended as dietary requirements not only for humans but for animals and poultry. The methionine plus cystine requirement for the growing rat has been reported by the NRC (1972) as 0.67% of the dry feed or 0.60% of a 90% DM feed. It should be noted that no - 3 -a-llowance has been made for a decreasing requirement with age. Sowers e t a l . (1972), using 50 g male albino r a t s , and Stockland et a l . (1973), using 50 g male Sprague-Dawley ra t s , gave require-ments of 0.47% and 0.41-0.49% methionine plus cystine r e s p e c t i v e l y in 90% DM synthetic d i e t s . Both these figures d i f f e r from those given by NRC (1972). In the growing pig, the l i s t e d requirements (NRC, 1973) for a l l e s s e n t i a l amino acids, including methionine plus cystine, vary with age and therefore with l i v e weight. Requirements for methionine plus cystine, as a proportion of a 90% DM d i e t , are as follows: 0.69% for 5-10 kg weight; 0.56% for the 10-20 kg weight; 0.50% f o r the 20-35 kg weight; 0.45% for the 35-60 kg weight and 0.41% for the 60-100 kg weight. These figures d i f f e r from those published by many researchers. Esnaola (1972) published a range of 0.40-0.45% for the 22-62 kg weight while Keith e t a l . (1972) published a figu r e of 0.46% for the 20-40 kg weight pig when methionine was added to a casein-based semi-p u r i f i e d d i e t . Esnaola (1972) reviewed many papers l i s t i n g methionine plus cystine requirements for the growing pig and found a great v a r i a t i o n among the r e s u l t s . Esnaola also speculated that the NRC figures were overestimates since Oestemer et a l . (1970), P f i r t e r (1970) and Stockland et a l . (1971) obtained no response to methionine supplementation of d i e t s that contained l e s s - A -than the NRC (1968) recommended amounts of t o t a l methionine plus cystine. The objective of the r a t t r i a l s reported i n t h i s thesis was to monitor growth and nitrogen responses, as well as responses of two l i v e r enzymes, cystathionine synthase (EC 4.2.1.22) and N^-methyltetrahydrofolate-homocysteine-methyltransferase (EC 2.1.1.13) to dietary methionine plus cystine at l e v e l s s l i g h t l y above and s l i g h t l y below the NRC (1972) recommended values. The two enzymes have been shown by Laster e t a l . (1965), F i n k e l s t e i n et a l . (1971) and F i n k e l s t e i n (1974) to be among those responsible for methionine metabolism i n rats and humans. The transsulphuration and remethylation pathways have been shown by F i n k e l s t e i n (1974) to share the common intermediate, homocysteine. A f t e r homo-cysteine i s formed, i t can be converted to either cystathionine by cystathionine synthase (EC 4.2.1.22) or back to methionine by two enzymes; betaine-homocysteine-methyltransferase (EC 2.1.1.5) and N -methyltetrahydrofolate-homocysteine-methyltransf erase (EC 2.1.1.13). The reaction producing cystathionine has been shown (Finkelstein, 1962) to be the only channel i r r e v e r s i b l y transforming methionine to other sulphur compounds or body protein i n the form of cysti n e . F i n k e l s t e i n (1967) showed that cystathionine synthase a c t i v i t y decreased when rats were fed low protein diets and increased when they were fed high protein d i e t s , but in neither instance - 5 -was the dietary l e v e l of methionine plus cystine stated. Methionine can be conserved through the remethylation reactions when diets are low i n either methionine or methionine plus cystine ( F i n k e l s t e i n , 1967; F i n k e l s t e i n et a l . , 1971, 1974). F i n k e l s t e i n e t a l . (1971, 1974) indicated that during the remethylation process N^-methyltetrahydrofolate-homocysteine-methyltransferase (mTHF Enz.) i s a more important enzyme than the betaine-homocysteine-methyltransferase (BH Enz.) when methionine i s low i n the d i e t . However, when methionine i s i n excess, the BH Enz. may become dominant i n order to counteract methionine t o x i c i t y (Benevenga, 1974), because of the requirement for three molecules of methionine for the formation of each molecule of choline. The choline can be u t i l i z e d i n the resynthesis of methionine. The present t r i a l s were designed to determine whether information obtained from the a c t i v i t i e s of cystathionine synthase and mTHF Enz. could be used to e s t a b l i s h the optimal requirements of methionine plus cystine for the growing r at and to compare such estimates with those based on growth, urinary urea-nitrogen excretion, blood amino acid l e v e l s and plasma urea-nitrogen l e v e l s . The range of dietary methionine plus cystine used i n the rat experiments was repeated also i n a pig t r i a l , i n which parameters were nitrogen balance and urinary urea-nitrogen excretion. - 6 -Diets used i n both r a t and pig t r i a l s were based on barley grain supplemented with amino acid mixtures which varied i n methionine and cystine content. It was considered that the r e s u l t s obtained on such diets would be of more value i n determining recommendations than those obtained on synthetic d i e t s . - 7 -LITERATURE REVIEW Protein digestion For an animal to benefit from the ingested food, the food must break down to constituents which can be absorbed and transported to the s i t e s of metabolism. This breakdown i s i n i t i a t e d by mastication, followed by enzymatic hydrolysis. The various p r o t e o l y t i c enzymes eventually give r i s e to free amino acids as well as t r i - and dipeptides which are absorbed and metabolized. Digestion of the proteins s t a r t s i n the stomach where the d i l u t e hydrochloric acid (HC1) a l t e r s the s p a t i a l configuration of the proteins i n order to prepare them for hydrolysis by the enzymes. The HCl i n the stomach also aids i n the conversion of the i n a c t i v e forms of g a s t r i c j u i c e pepsinogen, parapepsinogen I and parapepsinogen II into t h e i r a c t i v e forms of pepsin, parapepsin I and parapepsin II r e s p e c t i v e l y (Taylor 1968) . These three enzvmes have been shown to be the f i r s t to i n i t i a t e p r o t e o l y s i s of the proteins that are moistened by stomach j u i c e s . Subsequent protein hydrolysis, however, does not take place i n the stomach but i n the duodenum, jejunum and throughout the small i n t e s t i n e where absorption also occurs. This means that the stomach acts as a mixing compartment, storage chamber and also as a - 8 -place for the i n i t i a t i o n of enzymatic proteolysis of dietary protein. The stomach controls the rate at which the peptides enter the duodenum. The rate of passage of peptides from the stomach depends on the amount and type of the protein ingested but not on i t s q u a l i t y . Vegetable proteins are digested more slowly than animal proteins and the rate of stomach emptying i s also slower (Peraino and Harper, 1963). Mild heat treatment decreases the rate of stomach emptying, but severe heat damage of proteins increases the rate of stomach emptying as well as lowering d i g e s t i b i l i t y . G i t l e r (1964) showed that the rate of stomach emptying i n rats was most rapid when a low protein d i e t was fed and that the rate decreased as the l e v e l of protein increased up to 30%. In man, meals with large amounts of free amino acids are emptied more slowly than those containing lower l e v e l s of free amino acids. In both man and r a t , the rate of protein emptying by the stomach i s reduced by the presence of carbohydrates (Johansson, 1973; Mehnert and Forster, 1968). Once the peptides enter the small intestine, they are subjected to t r y p s i n action. Trypsin has been shown by Gray and Cooper (1971), to convert the procarboxypeptidases i n the pancreatic j u i c e to carboxypeptidase A, which cleaves the terminal aromatic or branched-chain amino acids of the peptide chains; and carboxypeptidase B, which cleaves the terminal - 9 -arginine and lysi n e when t h e i r reactive amino groups i n the side chain are free. The peptides are also subjected to the hydrolytic action of elastase which cleaves neutral amino acids. Elastase i s the enzyme from proelastase i n the pancreatic j u i c e . The pancreatic juice i s also known to produce chymotrypsin from i t s i n a c t i v e form, chymotrypsinogen. Chymotrypsin cleaves aromatic amino acids i n the peptides. The succus entericus i s known to produce t r y p s i n and other aminopeptidases that further breakdown the peptides into i n d i v i d u a l free amino acids. Both stomach emptying and p r o t e o l y t i c enzyme a c t i v i t i e s respond to dietary protein and amino acid l e v e l s . Snook and Meyer (1964) showed an increase i n trypsin and/or chymotrypsin a c t i v i t y i n the i n t e s t i n e when the protein content of r a t di e t s was increased. Dietary protein regulates p r o t e o l y s i s i n the small i n t e s t i n e by infl u e n c i n g pancreatic enzyme synthesis as well as i t s secretion. Dietary protein also retards the rate of i n a c t i v a t i o n of enzymes during digestion (Snook, 1965). Transportation and in t e r a c t i o n s of amino acids during absorption I t i s currently accepted that the whole of the e p i t h e l i a l l i n i n g of the small i n t e s t i n e i s being continuously renewed. It i s t h i s l i n i n g that i s responsible for the absorption of the exogenous and endogenous free amino acids as well as dipeptices. - 10 --.The functional c h a r a c t e r i s t i c s of absorption are confined to the v i l l i of the epithelium whereas the crypt epithelium acts as a secretory structure. The epithelium has two bordering membranes through which amino acids must be transported; the luminal membrane, i . e . the brush border, and the basolateral membrane (Hendrix and Bayless, 1970) . The presence of these membranes and the fact that, normally, amino acid absorption occurs against a concentration gradient, led researchers to look for possible energy-coupled transport systems. A l l amino acids, except aspartic acid and glutamic acid, have been shown to be a c t i v e l y transported through a carrier-mediated system (Wiseman, 1968). The rate of transport has been shown to diminish when energy reactions are i n h i b i t e d (Randall and Evered, 1964). The transport system also requires pyridoxal phosphate. Finch and Hird (1960) observed that the rate of transport of amino acids at high concentrations was inversely related to the a f f i n i t y for the transport system, whereas at low amino acid concentrations, the transport was i n the same order as the a f f i n i t y for the transport system. This means that at high amino acid concentrations, the c a r r i e r s i t e i s saturated and the rate of transport depends on the v e l o c i t y of d i s s o c i a t i o n of the amino a c i d - c a r r i e r complex. However, at low amino acid concentrations the rate depends on the degree - 11 -of saturation of the c a r r i e r by the amino acids, being proportional to the a f f i n i t y for the transport system. I t follows, therefore, that the highest a f f i n i t y for the transport system should be shown by the neutral amino acids possessing l i p o p h i l i c side chains, except arginine, l y s i n e and proline, which are transported by a d i f f e r e n t system (Wilson, 1962). The rate of absorption of free amino acids diminishes with increasing charge: amino acids with non-polar side chains are r a p i d l y absorbed, such as isole-ucine, leucine, phenylalanine, tryptophan and v a l i n e . By contrast, aspartic acid, glycine, glutamic acid and arginine are slowly absorbed. Using everted pieces of r a t i n t e s t i n e , Wiseman (1964) showed that L-amino acids are more a c t i v e l y transported than t h e i r D-isomers. Matthews and Laster (1965) showed that absorption i s maximal between the jejunum and the mid-ileum. Cole et a l . (1976) reported that at l e a s t four transport mechanisms for amino acids e x i s t : 1. for a-amino-monocarboxylic amino acids, 2. f o r basic amino acids, 3. for imino acids and 4. for a-amino d i c a r b o x y l i c acids. 1. Transport mechanism for a-amino-monocarboxylic (neutral) amino acids In t h i s mechanism, the c a r r i e r accepts amino acids with an unsubstituted amino group i n the a-position to the carboxyl group except for proline and hydroxyproline. The c a r r i e r does not accept amino acids with e l e c t r i c a l l y - c h a r g e d side chains, and requires the presence of both the amino and the carboxyl groups. Amino acids transported by th i s c a r r i e r mechanism are alanine, arginine, cysteine, glutamine, glycine, h i s t i d i n e , i s oleucine, leucine, methionine, phenylalanine, p r o l i n e , hydroxyproline, serine, threonine, tryptophan, tyrosine and v a l i n e . This l i s t includes c y c l i c and a l i p h a t i c amino acids (Hajjar and Curran, 1 9 7 0 ) . In t h i s mechanism, the transport rate f a l l s with increasing l i p o p h i l i a of the amino acids, i . e . the less l i p o p h i l i c the amino acid, the higher the rate of transport. Munck ( 1 9 6 8 ) showed that l y s i n e i n h i b i t s the neutral amino acid transport system. Reiser and Christiansen ( 1 9 7 3 ) showed that l y s i n e i n h i b i t s the uptake of alanine but alanine stimulates l y s i n e uptake. From t h i s data i t i s evident that l y s i n e does not use the c a r r i e r of neutral amino acids. However, Rerat e t a l . ( 1 9 7 6 ) concluded that alanine, leucine, methionine, phenylalanine, serine and threonine are also transported by the c a r r i e r of basic amino acids. In r a t s , tryptophan, isoleucine and valine i n h i b i t l y s i n e uptake but lysin e does not i n h i b i t the uptake of these amino acids (Cole et a l . , 1 9 7 6 ) . In hamsters, methionine i n h i b i t s the - 13 -uptake of l y s i n e . In chicks, arginine i n h i b i t s the uptake of methionine; leucine antagonizes isoleucine and valine absorption (Allen and Baker, 1972; D'Mello and Lewis, 1970a). D'Mello and Lewis (1970) also showed that threonine antagonizes tryptophan uptake i n chicks. Tasaki and Takahashi (1966) showed that absorption of leucine and phenylalanine was i n h i b i t e d by methionine while the opposite e f f e c t was weaker i n hens. Methionine also i n h i b i t s glutamic acid while glutamic acid accelerates methionine absorption. The o v e r a l l picture emerging from a l l these interactions seems to be that although i n h i b i t i o n between neutral amino acids occurs during t h e i r transportation, the most dramatic e f f e c t s are between amino acids transported by d i f f e r e n t transport systems, e.g. l y s i n e transported by the basic amino acid system and glutamic acid transported by the a-amino d i c a r b o x y l i c acid transport system. 2. Transport mechanism for basic amino acids This transport mechanism i s also used by many of the neutral amino acids. The c a r r i e r mainly transports arginine, cystine, h i s t i d i n e , l y s i n e and ornithine. In t h i s mechanism, leucine has been shown to accelerate l y s i n e transport (Munck and Schultz, 1969) . However, the best stimulant of lys i n e uptake has been shown to be alanine (Munck, 1972) . The - 14 -• c a r r i e r mechanism depends on sodium during i t s energy -requiring transportation process. 3 . Transport mechanism for imino acids Munck (1966a)confirmed e a r l i e r studies which showed that a separate transport mechanism e x i s t s for proli n e , hydroxy-p r o l i n e , sarcosine, betaine and alanine. Daniels et al. (1969) showed that g-alanine, and S and ct-aminobutyric acid were i n h i b i t o r s of the c a r r i e r . Proline transport i s also i n h i b i t e d by methionine and sarcosine. 4. Transport mechanism for a-amino dic a r b o x y l i c amino acids This transport mechanism has only been studied by Schultz et al. (1970). The mechanism i s said to be responsible for the transportation of aspartic acid and glutamic acid. The system i s sodium and energy dependent. Schultz et al. (1970) point out the f a c t that the two amino acids have mutual i n h i b i t i o n properties. Further work needs to be done on t h i s transport mechanism, e s p e c i a l l y on possible interactions with other amino acids, i n view of the fa c t that absorbed aspartic acid and glutamic acid seem to be r a p i d l y transaminated to give alanine. - 15 -Absorption and transportation of peptides Newey and Smyth (1960) suggested that peptides were absorbed by the i n t e s t i n a l epithelium independent of the transport mechanism for amino acids. Matthews et a l . (1968) and Cook (1972) showed that the absorbing capacity for amino acids i s higher when amino acids are presented as d i - and tri p e p t i d e s than when presented as free amino acids. However, Navab and Asatoor (1970) have pointed out the fact that not a l l d i - and tr i p e p t i d e s are absorbed. Crampton et a l . (1973) showed that the s i t e s of maximal absorption are d i f f e r e n t l y located for free amino acids and for peptides. So f a r , maximal peptide absorption seems to be i n the jejunum and at high concentrations. This i s true of the dipeptide Met-Met which i s absorbed i n the jejunum whereas free methionine i s absorbed i n the ileum (Liz et a l . 1972). In the case of the dipeptide Glu-Glu, absorption i s rapid at low concentrations and less rapid at high concentrations, i n d i c a t i n g that amino acids and peptides have separate uptake mechanisms (Burston e t a l . , 1972). More convincing evidence for peptide absorption has come from studies involving patients with Hartnup disease (Navab and Asatoor, 1970) who cannot absorb phenylalanine, and patients with C y s t i n u r i a ( H e l l i e r e t a l . , 1972) who cannot absorb l y s i n e . These patients have been shown to absorb - 16 -amino acids from dipeptides Phe-Phe and Lys-Lys respectively, normally. Uptake of dipeptides may be i n h i b i t e d by other dipeptides but not by free amino acids (Matthews et a l . , 1971). The phenomenon of faster amino acid absorption from dipeptides than from equivalent free amino acids was f i r s t shown i n man by tolerance tests (Craft and Matthews, 1968; Craft e t a l . , 1968) and by i n t e s t i n a l perfusions (Adibi and P h i l l i p s , 1968). In humans, the dipeptide Gly-Gly causes a more rapid and extensive increment of p o r t a l free glycine than ingestion of a s i m i l a r amount of free glycine (Craft et a l . , 1968). Hydroxyproline peptides, however, are absorbed and transported i n t a c t (Prockop and Sjoerdsma, 1961). Newey and Smyth (1962) showed that hydrolysis of g l y c y l -glycine and other dipeptides did not take place i n the i n t e s t i n a l lumen but was i n t r a c e l l u l a r . At the present stage of research, i t i s believed that the dipeptides are absorbed by some as yet undiscovered mechanism and are then subjected to hydrolysis within the i n t e s t i n a l c e l l s , and free amino acids are lib e r a t e d into the po r t a l blood (Cole e i a l . , 1976). Not only do the i n t e s t i n a l c e l l s hydrolyze the peptides, they also have been shown by Neame and Wiseman (19 58) and Wiseman (1964) to carry out transamination of some of the free - 17 --.amino acids before they are released into the po r t a l blood. Thus aspartic and glutamic acids which are found i n high concentrations i n dietary ingredients were not recovered among the products released into the p o r t a l blood whereas alanine concentration increased s i g n i f i c a n t l y (Wiseman, 1964). If t h i s transamination occurs on a major scale, measurement of p o r t a l blood amino acid concentrations cannot be used to measure a v a i l a b i l i t y of dietary amino acids to the animal. Contribution of endogenous nitrogen secretion Digestive enzymes, mucopolysaccharides, desquamated i n t e s t i n a l c e l l s , urea and amino acids produced by the c e l l u l a r catabolism are constantly being released into the gastro-i n t e s t i n a l t r a c t . These secretions are mixed with the exogenous nitrogen compounds and are thus subjected to p r o t e o l y s i s and absorption i n the same way that the exogenous nitrogen compounds are hydrolysed and absorbed. Nasset (1965) calculated that endogenous nitrogen secretion into the i n t e s t i n a l t r a c t i n humans amounts to 64-263 g protein per day. Nasset and Ju (19*61) and Twombly and Meyer (1961) calculated that at c e r t a i n periods a f t e r protein ingestion, the endogenous nitrogen secretion can d i l u t e the exogenous protein by up to 7-9 times i n the dog and the rat. However, the exact d i l u t i o n factor seems to depend on the - 18 -dietary protein content and i t s d i g e s t i b i l i t y (Rerat and Lougnon, 1963). Nasset (1965) also showed that the spectrum of free amino acids i n the small i n t e s t i n e s i s more constant than that of dietary protein. The f a c t that the free amino acid composition i s constant i n the endogenous secretions, coupled with the f a c t that the exogenous protein i s markedly d i l u t e d by the endogenous nitrogen compounds means vthat the spectrum of amino acids i n the i n t e s t i n e s approaches constancy. Thus Zebrowska (1973) showed that i n the duodenum, the amino acid content was s i m i l a r to that of dietary protein while i n the ileum the amino acid content was related to that of a protein-free d i e t due to the s t a b i l i z a t i o n by endogenous nitrogen secretions. Digestion of endogenous protein occurs along the entire length of the small i n t e s t i n e but i s prominent in the ileum and probably i n the caecum. This digestion i s continuous throughout the day. Twombly and Meyer (1961) showed that only 10% of the endogenous nitrogen secretions appears i n the faeces, thus implying that digestion of these endogenous compounds i s extensive. The most common enzymes that hydrolyze these nitrogenous compounds, apart from the normal p r o t e o l y t i c ones already mentioned, are the cathepsins which are l i b e r a t e d from desquamated i n t e s t i n a l c e l l s . Most of the normal p r o t e o l y t i c enzymes escape autodigestion and are unusually stable, i n that - 1 9 -i n t e s t i n a l f l o r a have l i t t l e action on them (Lepkovsky et a l . , 1 9 6 6 ) . Since digestion of endogenous nitrogen i s considerable in areas where t r a n s i t i s sluggish and where f l o r a l a c t i v i t y i s s i g n i f i c a n t , some of the e s s e n t i a l amino acids are l o s t through b a c t e r i a l action and therefore there i s a net e s s e n t i a l amino acid loss by the body. Compensation for t h i s loss must come from dietary sources i f the animal i s to maintain a p o s i t i v e nitrogen balance. By synthesizing the endogenous nitrogen compounds discontinuously, and by the continuous digestion of the compounds i n the i n t e s t i n e s , the digestive t r a c t thus regulates protein metabolism. In t h i s way i t presents some e s s e n t i a l amino acids for absorption and d i s t r i b u t i o n to tiss u e s and helps ensure stable amino acid concentration i n the blood of the fa s t i n g animal. Protein q u a l i t y The a b i l i t y of a protein to s a t i s f y a given p h y s i o l o g i c a l requirement i s mainly a function of the a v a i l a b i l i t y and balance of i t s amino acids i n r e l a t i o n to the renewal and synthesis of tis s u e proteins. The b i o l o g i c a l e f f i c i e n c y of dietary protein u t i l i z a t i o n , however, depends not only on the balance of av a i l a b l e amino acids but also on the nitrogen and energy intake. It varies from species to species and i s affected b y the p h y s i o l o g i c a l state of the animal. - 20 -The t o t a l quantity of amino acids present i n a protein can be determined by chemical analysis a f t e r acid or a l k a l i hydrolysis. However such values should not be used a d d i t i v e l y i n feed formulation because the amino acids are only p a r t i a l l y and v a r i a b l y digested and absorbed by the l i v i n g organism. It i s not the proportion of amino acids i n a protein but t h e i r s t r u c t u r a l arrangement that determines the protein's s u s c e p t i b i l i t y to enzymatic digestion and absorption. When a protein contains a v a i l a b l e amino acids in quantities that meet the requirements of an animal i n a p a r t i c u l a r p h y s i o l o g i c a l state, the q u a l i t y of the protein i s said to be high. However, when one of the e s s e n t i a l amino acids i s l i m i t i n g , e i t h e r because of low concentration, or because i t i s unavailable, the protein q u a l i t y i s lowered (Carpenter, 1960). The amino acids which are absorbed are said to be a v a i l a b l e . Factors a f f e c t i n g protein q u a l i t y 1. Protease inhibitors in raw materials The improvement i n protein q u a l i t y as a r e s u l t of properly heating soybean meal was c l a s s i c a l l y shown by Osborne and Mendel (1917). With the discovery of a heat l a b i l e trypsin i n h i b i t o r i n soybeans (Bowman, 1944) i t was shown that the low n u t r i t i v e q u a l i t y of raw soybean protein was due to retarded digestive release of amino acids (Hou et a l . , 1949 ), - 21 -p a r t i c u l a r l y methionine (Melnick et a l . , 1946). The heating of raw soybeans destroys the i n h i b i t o r , thus rendering methionine available for absorption, and thereby improving the protein q u a l i t y . Apparently, a n t i b i o t i c s have the same e f f e c t as heat treatment (Guggenheim and Goldberg, 1964), by counteracting the f a l l i n t r y p t i c and amylotic a c t i v i t i e s of the pancreas. Booth et a l . (1960) showed that pancreatic hypertrophy caused by trypsin i n h i b i t o r s leads to an excessive loss of endogenous protein secreted by the pancreas. Since t h i s protein i s r i c h i n cystine, i t represents a net loss of sulphur amino acids from the body. This, coupled with the fact that methionine absorption i s impaired, explains why the apparent a v a i l a b l e methionine i n raw soybean meal i s so low. Most other legumes, including peanuts, navy beans and lima beans contain t r y p s i n i n h i b i t o r s . Other protein i n h i b i t o r s are phytohaemagglutinins i n legumes; lathyrogens i n c e r t a i n v a r i e t i e s of peas; goitrogens i n soybeans, cabbage, cauliflower, turnip, kale, brussel sprouts, b r o c c o l i , rapeseed and mustards; cyanogens i n lima beans, sorghum and cassava; gossypol i n cottonseed meal (Liener, 1973). Heat treatment and/or soaking before feeding, i s important i n order to destroy these protein i n h i b i t o r s and - 22 -therefore improve the protein q u a l i t y of the food (Liener, 1973) . 2. Food processing I t i s now well established that heat damage of proteins i s increased i n the presence of carbohydrates. However, heat treatment i n the absence of carbohydrates also leads to a lowering of protein q u a l i t y by the formation of new linkages r e s i s t a n t to hydrolysis by the proteases in the g a s t r o i n t e s t i n a l t r a c t (Bjarnason and Carpenter, 1970). Heat damage leads to losses of amino nitrogen, and decreases s o l u b i l i t y and d i g e s t i b i l i t y due to loss of polar groups through i n t e r n a l formation of amides and esters (Ford, 1973). Ester linkages are produced between the carboxyl groups of aspartic acid or glutamic acid,and the hydroxyl groups of hydroxyamino acids such as hydroxyproline. There may also be linkages between aspartic acid or glutamic acid and the t h i o l group of cysteine (Ford, 1973). A l t e r n -a t i v e l y there may be an amide l i n k between asparagine and aspartic acid and between glutamine and glutamic acid. Such linkages render the amino acids involved unavailable for absorption (Bjarnason and Carpenter, 1970). These same authors indicate that the e-amino group of lysi n e does not react with the carboxyl groups but with the amide groups of - 23 --asparagine and glutamine, rendering l y s i n e unavailable too. Heat may produce damaged proteins whose peptides are not e a s i l y hydrolyzed by proteases. In t h i s way, some of the amino acids reaching absorption s i t e s may s t i l l be locked i n i n d i g e s t i b l e peptides (Ford and Shorrock, 1971), the commonly involved amino acids being aspartic acid, glutamic acid and l y s i n e . In most cases of heat damage, ly s i n e and methionine are rendered unavailable while cystine i s completely destroyed; and the release of leucine, l y s i n e and methionine as free amino acids i s eithe r blocked or delayed (Shorrock, 1972). Cystine and Cysteine are degraded to H2S upon heating (Bjarnason and Carpenter, 1970). Cysteic acid, l i b e r a t e d during oxidative reactions, i s unavailable for absorption (Miller e t a l . , 19 70). Methionine may be converted to methionine sulphoxides, the a v a i l a b i l i t y of which i s s t i l l being disputed, . or into methionine sulphone which i s said to be unavailable for u t i l i z a t i o n ( M i l l e r et a l . , 1970; E l l i n g e r and Palmer, 1969). By f a r the most quoted outcome of heat>damage i s the lysine i n t e r a c t i o n with carbohydrates i n the M a i l l a r d reaction. Hodge (1953) described the f i r s t steps of the reactions between amino acids and aldoses. The reactions give r i s e to deoxy-ketosyl d e r i v a t i v e s v i a S c h i f f bases and a l d o s y l -amines. The f i n a l products are normally keto acids and amino sugars. Thus,fructose reacts with l y s i n e to form f r u c t o s e l y s i n e . Raffinose has been shown to react with the e-amino group of l y s i n e i n cottonseed meals (El-Nokrashy and Frampton, 1967) . Such amino sugars render the amino acids involved, mainly l y s i n e , unavailable for u t i l i z a t i o n by the animal, lowering the protein q u a l i t y of the food. Overheated milk produces lys i n e d e r i v a t i v e s such as l a c t u l o s e l y s i n e , furosine and pyridosine (Finot et a l . , 1969) rendering l y s i n e unavailable. I t i s often argued that some of the unavailable l y s i n e i n overheated milk i s i n an aldosylamine or S c h i f f base form and the r e s t i s i n the deoxy-ketosyl form. 3. Amino acid d e f i c i e n c i e s in protein The two most quoted examples of amino acid d e f i c i e n c i e s are those of tryptophan and lys i n e i n the ordinary v a r i e t i e s of maize, compared with the opaque-2 v a r i e t i e s (Coppock, 1970) and tryptophan, cystine and tyrosine i n g e l a t i n , demonstrated by Kauffman (Maynard and L o o s l i , 1969). Lysine i s generally known to be the f i r s t l i m i t i n g amino acid i n most grains (Carpenter and E l l i n g e r , 1955; Carpenter, 1958; and Martinez e t a l . , 1961) followed by threonine (Aw-Yong and Beames, 1975) i n winter cereals and tryptophan then threonine i n maize (Bressani, 1971). Sulphur amino acids are said to be l i m i t i n g i n most legumes (M i l l e r and Donosa, 1963). - 25 -Amino acid requirements decrease with age, with the r e s u l t that a product that appears to be of low q u a l i t y i n a young animal may be p e r f e c t l y adequate for an older animal (Arroyave, 1973). The other problem with evaluating a protein l i e s i n the v a r i a t i o n i n the r e s u l t s obtained from d i f f e r e n t methods. Chemical methods, e.g. Carpenter's (1960) for available l y s i n e , do not necessarily agree with microbiological or growth methods (Ford, 1962) e s p e c i a l l y when the q u a l i t y i s p a r t i c u l a r l y poor. There i s , thus, a need to standardize the methods used in protein q u a l i t y determinations. Amino acid requirements The form of amino acids that i s normally u t i l i z e d by animals i s the L-isomer. The chick, however, has been shown to u t i l i z e the D-isomer as e f f i c i e n t l y as the L-isomer (Marrett e t a l . , 1964; Sugahara et a l . , 1967). Amino acids that are u t i l i z e d by the animals are normally divided into two groups: (1) e s s e n t i a l amino acids and (2) non-essential amino acids. 1. By " e s s e n t i a l amino acid" i s meant the amino acid that cannot be synthesized by the animal or, when synthesized, cannot be synthesized i n quantities enough to meet the requirements of that p a r t i c u l a r species. The following amino acids have been designated as e s s e n t i a l for normal growth of r a t s , pigs and humans: arginine, h i s t i d i n e , i s o l e u c i n e , leucine, l y s i n e , methionine, phenylalanine, threonine, tryptophan and v a l i n e . These amino acids have to be supplied i n the d i e t in amounts that meet the requirements of the animal under consideration. 2. By "non-essential amino acid" i s meant an amino acid that can e a s i l y be synthesized by the animal i n amounts enough to meet requirements. In order to synthesize these amino acids, the animal must have a source of nitrogen and a source of keto acids. Non-essential amino acids include alanine, aspartic acid, c i t r u l l i n e , glutamic acid, glycine, hydroxy-pro l i n e , p r o l i n e and serine. Although t h i s d i s t i n c t i o n has been made, i t must'be emphasized that the c l a s s i f i c a t i o n i s not completely accurate. For example, glycine i s e s s e n t i a l for the growth of chicks but i t i s not e s s e n t i a l for adult chickens. H i s t i d i n e i s e s s e n t i a l for infants but not for adults (Arroyave, 1973). Two amino acids, cystine and tyrosine, can replace part of methionine and phenylalanine respectively. These amino acids are i n a group by themselves i n that they are neither completely e s s e n t i a l nor non-essential. - 2 7 -Factors a f f e c t i n g amino acid requirements There are four main factors a f f e c t i n g amino acid requirements: (1) genetic f a c t o r s , (2) age and p h y s i o l o g i c a l state, (3) dietary protein and energy, and (4) food intake. 1. Genetic factors These are more evident i n the case of poultry where the high producing st r a i n s (HA) reported by Nesheim (1968) required higher concentration of arginine i n the d i e t than the low producing (LA) s t r a i n s . These same birds were shown (Nesheim et a l . , 1971) to have d i f f e r e n t l y s i n e requirements. I t i s generally accepted that at the same ph y s i o l o g i c a l age, the absolute amino acid requirements by chicks are d i f f e r e n t from those of pigs, rats and humans, although the determination of equivalent p h y s i o l o g i c a l ages i s not something which can be done with any great degree of accuracy. 2. Age and p h y s i o l o g i c a l state Requirements for e s s e n t i a l amino acids, i n terms of dietary concentration, decrease with age. In chickens (Combs, 1967), i t has been calculated that amino acid requirements at 7 weeks of age are 7 5-8 5% of the requirements at 5 weeks of age. However, t h i s p r i n c i p l e cannot be applied to a l l e s s e n t i a l amino acids since recent findings point out that l y s i n e and methionine requirements do not decrease as - 28 -f a s t as they were o r i g i n a l l y considered to decrease in chicks (Boomgaardt and Baker, 1973; Chung e t a l . , 1973; Graber et a l . , 1971). In the growing pig, l y s i n e requirements decline faster than methionine plus cystine requirements (Homb, 1976). Newly born pigs respond more dramatically to protein q u a l i t y than older pigs and Wohlbier (1928) demonstrated a higher degree of sow's milk u t i l i z a t i o n by new-born pigs than by older pigs of 20-50 kg l i v e weight. In humans, a one-year old c h i l d requires 1.2 g/kg/day of amino acids, 4 0% of which are e s s e n t i a l amino acids (Arroyave, 1973; FAO, 1973) , whereas an adult requires 0.57 g/kg/day amino acids, 20% of which are e s s e n t i a l amino acids. Kielanowski (1972.) using balance studies showed va r i a t i o n s between boars, castrates and g i l t s , i n the rate of protein deposition and, therefore, amino acid and protein requirements. The NRC (1971) shows the amino acid requirements of b r o i l e r s , i n terms of dietary concentration, to be higher than those of replacement p u l l e t s of a s i m i l a r age. In humans, the require-ments of the adult female (FAO, 1973; RDA, 1974) are lower than those of the adult male. It i s generally accepted that amino acid requirements increase during pregnancy, l a c t a t i o n and egg laying. Increases also occur when environmental factors such as heat stress, injury or i n f e c t i o n are imposed on the animal. - 29 -3. Dietary protein and energy factors In animals, amino acid requirements, as a percentage of the d i e t , vary with protein and energy content of the d i e t (Kielanowski, 1976). Under mild methionine deficiency, chicks have an impaired food conversion r a t i o (Solberg et a l . , 1971) although there i s l i t t l e e f f e c t on weight gain. This means that with a mild deficiency, extra energy consumption occurs to promote maximal rate of growth. Lysine requirement, as a percentage of the d i e t , has been shown to increase with the protein content of chick d i e t s . However when expressed as a proportion of protein content, l y s i n e requirement i s independent of protein (Boomgaardt and Baker, 1973) and energy (Boomgaardt and Baker, 197 3) concentration. 4. Food intake Amino acid requirements, i n terms of g/day, are known to depend on food intake, and thus on a l l factors which a f f e c t food consumption, such as energy content of the d i e t (March and B i e l y , 1972) and ambient temperature (Kubena e t a l . , 1972) . As pointed out by March and B i e l y (1971), there i s also an i n t e r a c t i o n between protein requirement, environment and breed, with the r e s u l t that i t i s d i f f i c u l t to define requirements p r e c i s e l y . - 30 -Individual amino acid requirements The i n d i v i d u a l amino acid requirements for optimal growth are presented i n Appendix I for the growing r a t , Appendix II for the growing p i g , Appendix III for meat-producing poultry and Appendix IV for the 6 months to 1 year human in f a n t . The requirements for methionine plus cystine w i l l be discussed i n the section on sulphur amino acids i n monogastric animal n u t r i t i o n . Methionine metabolic pathway F i n k e l s t e i n (1971) stated that there are three major functions of methionine i n the body: a. U t i l i z a t i o n for protein synthesis, b. Conversion to s-adenosyl methionine which i s the primary b i o l o g i c a l methyl-group donor, c. Conversion to cystathionine, cysteine and further derivatives of cysteine. This l a s t function serves as the major route i n methionine catabolism i n mammals, whereby excess dietary methionine i s excreted i n the urine as inorganic sulphate (Laster et a l . , 1965) . Once methionine i s absorbed from dietary sources, i t s main function i s to be incorporated into tissue protein (Fig. 1). However, since other methionine derivatives are also b i o l o g i c a l l y important, methionine i s acted upon by the 31 Protein 4 N,N-DJJT£thyl-Glycine Betaine Choline i r •Metliionine 1© . . S-Adenosyl-L-Methionine I © S-Adenosyl-L-Homocysteine : Homocysteine Serine Cystathioniiie Cysteainjne Cysteine + Homoserine ©• Cysteine Sulphinate Hypotaurine Pyruvate + Sulphate B-Sulphinyl Pyruvate Taurocholic Acid S-Sulpho-Cysteine Figure 1. Methionine metatolic pathway. FIGURE 1. - 32 -Key to abbreviations: mTHF THF 5,10-MeTHF [CI] Key to enzymes: l 2 3 k 5 6 7 8 9 1 0 11 1 2 1 3 1 4 1 5 NJ-methyltetrahydrofolic acid Tetrahydrofolic acid 5,10-Methylene tetrahydrofolic acid One-carbon metabolic pathway L-methionine-S-adenosyl transferase Unnamed transmethylase S-adenosyl-L-homocysteine hydrolase Betaine-homocysteine-methyl transferase 5 N -methyltetrahydrofolate-homocysteirie-methyltrans fera se Cystathionine synthase Cystathiona se Cysteine oxidase Cysteine disulphydrase Cysteine decarboxylase Cysteamine oxygenase Cysteine sylphinate decarbosylase Cysteine sulphinate dehydrogenase . Serine transhydroxy methylase 5,10-methylene tetrahydrofolate reductase - 33 -enzyme ATP:L-methionine-s-adenosyltransferase (MAE; EC 2.5.1.6) to form s-adenosyl-L-methionine (sAMe). The ++ reaction requires ATP as well as Mg ions. The enzyme i s also capable of catalyzing the conversion to methionine, analogs such as ethionine and trifluoromethionine. The sAMe i s said to be the primary methyl-group donor i n mammalian metabolism. Pegg and Williams-Ashman (1969) observed that an enzyme c a l l e d s-adenosyl-L-methionine decarboxylase decarboxylates sAMe and transfers the propylamine group to putrescine to form spermidine which i s a precursor of spermine. This synthesis of polyamines may be a major function of sAMe,particularly i n rapidl y growing tissue i n which polyamine synthesis may contribute necessary cations i n de novo protein synthesis (Russell and Lombardini, 1971). However the bulk of sAMe i s converted to s-adenosyl-L-homocysteine (sAH) i n a transmethylation reaction. The sAH formed i s cleaved by a s p e c i f i c hydrolase, s-adenosyl-L-homocysteine hydrolase (EC_ 3.3.1.1) to y i e l d L-homocysteine and adenosine (de l a Haba and Cantoni, 1959). This reaction i s re v e r s i b l e and the equilibrium strongly favors s-adenosyl-L-homocysteine in v i t r o . However, in v i v o , the reaction s h i f t s to hydrolysis because homo-cysteine i s constantly being u t i l i z e d to such an extent that there i s no observable tissue homocysteine (Salvatone et a l . , - 34 -1968) . sAH i n h i b i t s transmethylation reactions and the tis s u e accumulation of t h i s compound has been shown to disrupt many normal biochemical processes (Zappia et a l . , 1969) , e s p e c i a l l y when methionine i s incorporated at toxic l e v e l s i n food (Benevenga, 1974). It has been suggested that sAH may be deaminated to s-adenosyl-y-thio-a-ketobutyrate (Duerre et a l . , 1969). In addition to s-adenosyl-L-homocysteine hydrolase, three other enzymes u t i l i z e L-homocyst'eine as a substrate. The f i r s t enzyme i s betaine-homocysteine-methyltransferase (BH Enz.; EC 2.1.1.5). This enzyme requires betaine, a choline derivative,as a methyl donor and produces methionine. This reaction does not r e s u l t i n any net increase i n the methyl pool. In the absence of dietary choline, there i s a net loss i n methyl groups since three methionine molecules are u t i l i z e d to synthesize a single choline (or betaine) molecule. Because of t h i s requirement, F i n k e l s t e i n et a l . (1974) have concluded that BH Enz. i s an important enzyme when dietary methionine i s i n excess,but not when methionine i s l i m i t i n g . BH Enz. i s i n h i b i t e d by i t s reaction products, dimethylglycine and methionine (Ericson, 1960), and such i n h i b i t i o n may be important in the regulation of the pathway. The Michaelis Constant for BH Enz. i s 4 x 10 5 mol/L. - 35 -The second enzyme u t i l i z i n g homocysteine uses c N -methyltetrahydrofolic acid of the one-carbon metabolic 5 pathway. The enzyme i s c a l l e d N -methyltetrahydrofolate-L-homocysteine-methyltransferase (mTHF Enz.; EC 2.1.1.13). The enzyme requires trace amounts of sAMe, Vitamin Bg and Vitamin B.^* T n e transferred methyl group i s derived from the "one carbon pool" and represents a "newly formed" methyl group ( F i n k e l s t e i n , 1971). Thus mTHF Enz. represents an i n t e r - d i g i t a t i o n of f o l i c acid, Vitamin ^-^2' methionine and one-carbon metabolism. The enzyme i s i n h i b i t e d by both s-adenosyl-homocysteine and L-homocysteine. F i n k e l s t e i n (1971) found that a low protein d i e t increased the a c t i v i t y of the enzyme while a high protein d i e t decreased the a c t i v i t y . F i n k e l s t e i n et a l . (1971, 1974) ,have come to the conclusion that mTHF Enz. i s important i n the regulation of methionine metabolism when methionine i s l i m i t i n g , i n that i t conserves methionine better than BH Enz. This i s supported by the lower Michaelis Constant, 6 x 10 ^ mol/L compared with that of _3 cystathionine synthase (1.2 x 10 mol/L) which catabolizes homocysteine i r r e v e r s i b l y . The t h i r d enzyme that u t i l i z e s L-homocysteine i s cystathionine synthase (CS; EC 4.2.1.22). The enzyme catalyzes the reaction between serine and homocysteine, in the presence of pyridoxal phosphate, to y i e l d cystathionine. - 3 6 -The reaction i s i r r e v e r s i b l e , so that any L-homocysteine which i s converted to cystathionine can no longer serve as a methionine precursor. CS i s i n h i b i t e d by sulphydryl compounds and by disulphides. These i n h i b i t o r s form disulphide bonds with homocysteine and reduce i t s concen-tration within the animal. The enzyme i s also i n h i b i t e d by threonine, glycine and alanine, a l l of which in part, act by competing with serine. High l e v e l s of dietary methionine increase the a c t i v i t y of the enzyme, whereas low lev e l s of dietary methionine decrease i t s a c t i v i t y ( Finkelstein, 1967). -3 The Michaelis Constant of the enzyme a c t i v i t y i s 1.2 x 10 mol/L. Once formed, cystathionine i s cleaved by the pyridoxal phosphate-dependent enzyme cystathionase (EC_ 4.4.1.1), to form cysteine and a-ketobutyrate. This reaction i s reversible, but removal of the products s h i f t s the reaction in the d i r e c t i o n of hydrolysis. Like CS, cystathionase a c t i v i t y increases with an increasing dietary methionine l e v e l , thus cat a b o l i z i n g most of the excess methionine i n the d i e t . The Michaelis Constant of the enzyme a c t i v i t y i s 3 x 10 ^ mol/L. The Km's for MAE, CS and cystathionase are a l l of the _3 magnitude 10 mol/L, while the Km's for BH Enz. and mTHF Enz. - 5 are approximately 10 mol/L. This means that at a low dietary methionine concentration, L-homocysteine remethylation predominates over the transsulphuration pathway. On the other hand, Km for methionine during protein synthesis i s - 4 - 6 i n the range of 10 -10 mol/L. This means that at low dietary methionine concentrations, protein synthesis proceeds despite a marked f a l l i n s-adenosyl methionine synthesis ( F i n k e l s t e i n , 1971). Studies on methionine metabolic pathway i n rats have produced f i v e general conclusions (Fin k e l s t e i n , 1971): 1. That BH Enz. i s r e s t r i c t e d to the l i v e r . The l i v e r has been found to possess a l l the f i v e important enzymes i n the methionine metabolic pathway. This has led to the gen e r a l i z a t i o n that the mTHF Enz. i s the major homocysteine remethylating enzyme i n extrahepatic t i s s u e s . Species differences occur, as has been shown by Mudd et a l . (1969), who found BH Enz. i n human kidneys. 2. That every tissue has a mechanism for u t i l i z i n g homocysteine, which explains why homocysteine i s absent from normal blood. 3. That high l e v e l s of cystathionine synthase are accompanied by high lev e l s of cystathionase, except in the brain, where cystathionine accumulates for an unknown reason (Tallan e t a l . , 1958). - 3 8 -- 4 . That the heart, lungs and testes are r e l a t i v e l y d e f i c i e n t i n cystathionine synthase, which means that methionine metabolism i s v i a remethylation by mTHF Enz. 5. That i n t e s t i n a l mucosa lacks both L-homocysteine methylases and,therefore/methionine metabolism i s v i a the transsulphuration pathway. In these c e l l s , t r y p s i n i n h i b i t s BH Enz. by digesting i t . S p e c i f i c a c t i v i t i e s of MAE, BH Enz. and mTHF Enz. decline with age, while a c t i v i t i e s of CS and cystathionase increase with age ( F i n k e l s t e i n , 1967; Volpe and Laster, 1972). It was pointed out e a r l i e r that the products of cystathionine breakdown by cystathionase are cysteine and a-ketobutyrate. In the c e l l s , cysteine i s e a s i l y converted to cystine for incorporation into proteins. On the other hand, cystine i s also e a s i l y converted into cysteine for further breakdown into inorganic sulphate for excretion, and into compounds such as taurine, taurocholic acid and pyruvate. Cysteine i s also a precursor of coenzyme A. Catabolism of cysteine i n the ra t has been shown to occur i n two main pathways: the pyruvate (or sulphate) pathway and the taurine pathway. The pyruvate pathway can i t s e l f take two d i f f e r e n t forms. The f i r s t i s a d i r e c t conversion of cysteine into pyruvate, NH^ and H-,S,catalyzed by the enzyme cysteine - 39 -desulphydrase (EC 4.4.1.1), having the same c a t a l y t i c functions as cystathionase. The second i s an i n d i r e c t formation of pyruvate v i a cysteine sulphinate, which i s produced from cysteine by the enzyme cysteine oxidase (EC_ 1.13.11.20). Therefore the f i r s t steps i n cysteine catabolism are cont r o l l e d by these two hepatic enzymes. Since the products of t h i s catabolism are necessary for the normal c e l l u l a r metabolism, a supply of cystine (cysteine) and methionine i n the d i e t i s important. Inborn error diseases of methionine metabolism Most of the findings reported i n t h i s area have been obtained with humans but very few with r a t s . Most of the c l i n i c a l syndromes are acquired or genetically-determined errors in the methionine-metabolic pathway. Some studies have linked n u t r i t i o n a l l i v e r disease with a d e f i c i e n t dietary intake of methionine and choline. 1. Homocystinuria due to cystathionine synthase insufficiency The disease was f i r s t described as a c l i n i c a l e n t i t y by Carson et a l . (1963) as a r e s u l t of a deficiency of cystathionine synthase i n the l i v e r . This deficiency occurs in the presence of normal l e v e l s of the methionine-activatirig enzyme, MAE, and cystathionase. The patients excrete large amounts of homocysteine i n t h e i r urine, and have a reduced - 40 -capacity to form inorganic sulphate from methionine. Both methionine and homocysteine accumulate i n the plasma and cerebrospinal f l u i d , and overflow into the urine (Laster et a l . , 1965). Treatment of the disease involves feeding of a low-methionine high-cystine d i e t i n conjunction with massive doses of Vitamin Bg. The vitamin Bg stimulates the synthesis of cystathionine synthase and s t a b i l i z e s any r e s i d u a l CS a c t i v i t y i n the c e l l . 2. Homocystinuria due to N~*-methyltetrahydrofolate-L-homocysteine-methyltransferase (mTHF Enz.) deficiency The disease i s characterized by a deficiency in the a c t i v i t y of l i v e r , kidney and skin mTHF Enz. i n the presence of normal l e v e l s of BH Enz., MAE, CS and cystathionase. Methyl malonyl-CoA isomerase i s also low i n the l i v e r . It has been postulated (Fleisher and Gaull, 1974) that a defect i n the conversion of Vitamin B ^ t o i t s coenzymatically active deoxy-adenosyl and methyl B ^ forms, account for the loss of a c t i v i t y . The l e v e l of methionine in the plasma i s either low or normal but methylmalonic acid i s excreted i n t h e i r urine ( F i n k e l s t e i n , 1975). 3. Homocystinuria due to deficiency of 5, 10-methylene tetrahydrofolate reductase a c t i v i t y The disease was f i r s t described by Mudd et a l . (1972). In t h i s disease, patients have very low l e v e l s of 5, - 41 -10-methylene tetrahydrofolate reductase,while l e v e l s of mTHF Enz. and CS are normal. The reductase enzyme deficiency r e s u l t s i n an i n a b i l i t y to synthesize 5-methyltetrahydrofolate, the major form of fo l a t e i n blood and l i v e r , i n amounts s u f f i c i e n t for the remethylation of homocysteine to methionine. Homocysteine accumulates i n the plasma, while plasma methionine concentration i s reduced. Cystathionine i s increased i n the blood, and overflows into urine because the capacity for transsulphuration i s not adequate to cope with the excess cystathionine synthesized. 4. Cystathioninuria The disease was described by Harris et al. (1959), whereas i t s enzymatic etiology was established by Frimpter (1965). There i s reduced cystathionase a c t i v i t y i n the l i v e r . However the a c t i v i t y can be stimulated by adding pyridoxal phosphate (PLP) which s t a b i l i z e s small amounts of the enzyme. The disorder i s characterized by an increased homocysteine excretion as well as the excretion of N-acetyl cystathionine. There i s cystathionaemia, despite a low renal threshold of cystathionine,while cystathionine accumulates i n various ti s s u e s . 5. Sulphite oxidase deficiency This enzyme converts sulphite into sulphate before i t i s excreted. During i t s deficiency, excretion of large amounts of - 4 2 --s-sulpho-L-cystine, sulphite and thiosulphate occur, whereas excretion of inorganic sulphate i s reduced, and does not increase with cystine supplementation. There are large amounts of s-sulpho-L-cystine i n plasma. Since approximately 70% of the ingested methionine i s converted into inorganic sulphate, and since dietary inorganic sulphate may be important in r a t s , such an enzyme de f i c i e n c y may be detrimental to the growth of the affected animals (Mudd et a l . , 1967; Byington et al. , 1972) . 6. Hypermethioninaemia This i s a transient neonatal disease (Levy et a l . , 1969). In most cases the abnormality disappears when the amount of protein i n the d i e t i s reduced. The increase i n plasma methionine r e s u l t s from a delay i n the maturation of the capacity for transsulphuration. Gaull et a l . (1972) showed that s-adenosyl-methionine synthase and CS i n f e t a l and neo-natal human l i v e r are 25% of the values of mature control subjects. F e t a l l i v e r lacks cystathionase, but l i v e r s of babies have a c t i v i t i e s approximating those of adults. The s p e c i f i c a c t i v i t y of BH Enz. i n neonatal l i v e r i s the same as that of adults,but mTHF Enz. a c t i v i t y i n neonatal l i v e r i s higher than that of adults (Gaull et a l . , 1973). These enzyme patterns suggest that methionine conservation, rather than transsulphuration, i s the predominant pathway i n infants. This means that cystine may be e s s e n t i a l at t h i s early age i n l i f e , whereas methionine intake needs to be low (Gaull et al. , 1973). Other sulphur amino acid metabolic diseases that have not yet been f u l l y explained include methionine malabsorption, c y s t i n u r i a , c y s t i n o s i s and B-mercaptolactate-cysteinuria (Fi n k e l s t e i n , 1975) . Sulphur amino acids i n monogastric animal n u t r i t i o n Sulphur i n the form of sulphur amino acids i s important for the normal growth of a l l animals. Suboptimal intakes of sulphur amino acids produce i n f e r i o r growth. In monogastric animals and humans, sulphur i s u t i l i z e d most e f f i c i e n t l y i n the organic form of methionine and cystine. Other sulphur compounds that are also e s s e n t i a l for metabolism i n organisms include b i o t i n , l i p o i c acid and glutathione. These products are required i n such minute quantities that they a r e , c l a s s i f i e d as either enzymes (Glutathione i s a precursor of coenzyme A) or vitamins. Rats Early research by Womack and Rose (1941) showed require-ments of the growing rat for DL-methionine to be 0.5% when 0.4% L-cystine was present and 0.6% when no cystine was present - 4 4 -i n the d i e t . This early work established that there i s an optimal requirement for the sulphur amino acids, and that cystine could replace part, but not a l l , of the methionine requirement. Rama Rao et a l . (1959) reported that 0.49% methionine plus cystine i n the d i e t was the requirement for sulphur amino acids i n growing r a t s . Rama Rao et a l . (1961) found 0.16% to be the methionine requirement for growing rats when cystine was i n excess, and 0.34% to be the cystine requirement for the growing r a t . Sowers et a l . (1972) obtained 0.47% as the requirement for sulphur amino acids in the growing r a t . On a molecular basis, they calculated the requirement to be 0.50% when t o t a l sulphur amino acid requirements were met by methionine alone, but only 0.43% when methionine was present at 0.17% and cystine was present at a maximum l e v e l of 0.26% i n the d i e t . Stockland et a l . (1973) obtained sulphur amino acid requirements of 0.49% methionine when cystine was absent from the d i e t ; 0.44% when cystine was present at a 0.19% l e v e l i n the d i e t , and 0.41% when cystine was present at a 0.24% l e v e l i n the d i e t . Apart from defining the requirements for sulphur amino acids i n the growing r a t , these figures also show that requirements decrease as the proportion of cystine i n the d i e t increases. Realizing that requirements decrease with increasing dietary cystine l e v e l , Sowers et a l . (1972) - 4 5 -and Stockland et a l . (1973) expressed the requirements i n terms of the t o t a l number of moles of sulphur i n the d i e t . Thus Stockland e t a l . (1973) presented the t o t a l sulphur requirements by the growing r a t as 3.12-3.28 m moles per 100 g d i e t . The va r i a t i o n s from t h i s range are minimal, and such an expression of requirement i s more meaningful i n view of the variable r a t i o of methionine to cystine i n most d i e t s . Perhaps a s t i l l more accurate expression would be the number of moles of t o t a l sulphur i n the d i e t per 100 g of protein. Womack and Rose (1941) indicated that cystine could supply 6 4 % of the sulphur amino acid requirement. Rama Rao e t a l . (1961) gave figures that indicate that cystine could supply 68% of t o t a l sulphur requirements f o r growth. However, Byington and Howe (1972) and Byington et a l . (1972) recommended that 70% of the t o t a l sulphur requirement should come from methionine and 30% from cystine i n order to obtain optimal growth. They also showed that 50% methionine, 50% cystine i n the total sulphur amino acid requirement was optimal for growth, in the presence of an adequate l e v e l of choline. The NRC (1972) .methionine plus cystine requirement (0.60% on 90% dry matter basis) for the growing r a t indicates that cystine could supply at lea s t 50% of the requirement. In view of the fact that methionine plus cystine requirements decrease with increasing age of the rat, and since, at maturity, only maintenance and replacement of tissues takes place, i t should be argued that the proportion of - 46 --of cystine i n the t o t a l sulphur amino acid requirement could increase with advancing age. Wellers et a l . (I960) argued that inorganic sulphate could supply a l l the sulphur needs except those required for protein synthesis,in adult r a t s . Wellers (1962) calculated that inorganic sulphate could supply 33% of the t o t a l sulphur needs of the adult r a t . Brown and Gamatero (19 70) showed that inorganic sulphur i n the r a t d i e t improved growth and protein e f f i c i e n c y . Smith (1973) obtained an inorganic sulphur requirement of 0.02% of the dry d i e t for adult r a t s . This means that inorganic sulphur can, therefore, spare the need for sulphur amino acids i n adult r a t s , which agrees with the findings of Waldschmidt (1962) and Houniven and Gustafsson (1967) who showed that SC>4 was converted to cysteine and that t h i s could not be ascribed to i n t e s t i n a l f l o r a . Whittle and Smith (1974) found that S0 4~ did not conserve sulphur amino acids but decreased taurine excretion. This finding may i n d i r e c t l y mean the sparing of cysteine catabolism (Figure 1). For the growing r a t , inorganic sulphate i s without e f f e c t (Byington et a l . , 1972). This lack of response i s expected since, during growth, there i s a heavier demand for methionine and cystine i n protein synthesis and also because the transsulphuration pathway has not reached i t s t o t a l capacity. Wretlind and Rose (1950) showed that when dietary cystine was fixed at 0.2% of the dry d i e t , L-methionine was superior - 47 -for growth compared with D-methionine. I t i s now accepted that rats do not have high a c t i v i t y of the enzyme that converts the D-isomer into the L-form. N u t r i t i o n a l studies with Weanling rats have indicated that methionine sulphoxide& but not methionine sulphone,can replace methionine ( M i l l e r et a l . , 1970; Njaa, 1962). However, E l l i n g e r and Palmer (1969) found that methionine sulphoxides were "unavailable" to the very young rat, a conclusion also reached by Cuq et a l . (1973). Benevenga and Harper (1970) showed that 3% dietary glycine and 4.2% dietary serine could a l l e v i a t e the growth depressant e f f e c t s of methionine when incorporated at lev e l s above 1.5% in d i e t s . These two amino acids probably a l l e v i a t e the t o x i c i t y of methionine by increasing decarboxylation of methionine to CC>2 (Case e t a l . , 1976), and conversion of the methionine to cys t e i c acid, taurine and other excretory products. The two amino acids, serine and glycine, have also been shown (Benevenga and Harper, 1970) to increase the rate of stomach emptying, thereby reducing the amount of methionine being absorbed. Poultry The chicken, unlike the rat, has a very high a c t i v i t y of D-amino oxidase, an enzyme that converts D-methionine into L-methionine. This may explain why Sugahara et a l . (19 67) found the u t i l i z a t i o n of D-methionine by the chick to be the same as the u t i l i z a t i o n of L-methionine. - 48 -Graber and Baker (1971) observed that dietary cystine supplementation caused an increase in food intake in chicks. However, an increase i n the methionine content of the d i e t was shown by Boomgaardt and Baker (197 3) to have l i t t l e or no e f f e c t on feed intake but to increase the lean content of the chickens. The r e l a t i o n s h i p between cystine and methionine in higher animals i s well known. In poultry, i t i s obvious that high l e v e l s of cystine must be either fed or derived from methionine, since t h i s amino acid i s needed for the growth of feathers. When cystine content i s low i n the d i e t , methionine i s transformed into cystine, but Fisher (1976) claimed that methionine can replace cystine with an e f f i c i e n c y of only 79%. No reasons were given for t h i s claim. Graber and Baker (1971), Graber et al. (1971) and Boomgaardt and Baker (1973) estimated methionine requirements for the growing chick to be i n the range 0.63-0.70% when cystine was absent, and 0.52% when cystine was present. The lowering of requirements i n the presence of cystine has already been mentioned i n ra t s . When expressed on the basis of t o t a l sulphur amino acid intake, these values become 475 and 495 mg/day, of t o t a l sulphur amino acids. On the molecular basis, where one mole methionine equals 0.5 moles cystine, the requirements for sulphur amino acids, i n terms of t o t a l dietary sulphur, become equal. As i n ra t s , therefore, the expression of requirements in terms of the t o t a l number of moles of sulphur per 1 0 0 g d i e t i s better than i n terms of the p r o p o r t i o n of sulphur amino acids - 49 -in the d i e t , which i s the method currently being used. This i s because when cystine replaces methionine on an egual weight basis, the replacement on a sulphur basis i s i n the r a t i o of 1.25:1, i . e . for each 1 g cystine added, t h e o r e t i c a l l y , there should be a reduction of 1.25 g methionine i f methionine were qu a n t i t a t i v e l y transformed to cystine on a s t r a i g h t molecular basis. Cystine requirements, when expressed as a proportion of t o t a l sulphur amino acid requirements, change with the age of the chicken (Graber et a l . , 1971). The proportions have been estimated as 56, 67 and 87% at 2 weeks, 7 weeks and maturity resp e c t i v e l y . In growing p u l l e t s , maximum growth was estimated to occur when dietary methionine l e v e l was at 0.25-0.28% l e v e l , but the content of cystine was not published (Fisher, 1976). Kim and McGinnis (1972) obtained a requirement of 0.44-0.46% dietary methionine plus cystine for the same type of b i r d s . In the laying hen, egg production was shown by Fisher (1976) to increase with the increasing l e v e l of dietary methionine. Fisher (1976) calculated the e f f i c i e n c y of methionine u t i l i z a t i o n i n egg production as 86%. There i s no d i r e c t estimate given for the amount of cystine that i s u t i l i z e d by the lay i n g hen. The various experiments have only used methionine and reported the findings in terms of t o t a l - 50 -sulphur amino acid requirements (Reid and Weber, 1973). The NRC (1971) suggests that 47% of the t o t a l sulphur amino acids could be supplied by cystine whereas the ARC (1963) recommends 60% replacement value. Harms and Damron (1969) showed that cystine could supply 50% of the t o t a l sulphur amino acid requirements. In most of these replacement values, cystine replaces methionine on equal weight basis. Machlin et al. (1953) and Mason et al. (1965) showed that the hen could produce small and q u a n t i t a t i v e l y i n s i g n i f i c a n t amounts of cystine from inorganic sulphur without the involvement of i n t e s t i n a l microbes. Damron and Harms (1973) obtained numerically large, but not s i g n i f i c a n t , responses i n egg production by adding sodium sulphate to a methionine d e f i c i e n t d i e t . Jensen (1972), however, f a i l e d to show such a response. The chicken embryo and v i t e l l e n sac have been shown to produce cysteine from serine and (Chapeville, and Fromageot, 1963; Sentenac and Fromageot, 1964). The cysteine forms cys t e i c acid which i s used i n obligatory production of taurine. Du'ring the formation of cysteic acid, H2S i s also l i b e r a t e d . Thus, i n the chicken embryo and yolk sac, there i s a cysteine regenerative cycle not found i n the hatched chicken or i n mammals. - 5 1 -Pigs Work on sulphur requirements i n pigs has not been as extensive as that reported for rats and chickens. However, because of the pig's rapid growth, i t i s the only animal to date whose methionine plus cystine requirements have been given i n such minute d e t a i l s as to r e f l e c t the decrease with age (NRC, 197 3) . In pigs, as i n rats, D-methionine oxidase a c t i v i t y i s low, and, therefore, the conversion of D-methionine to L-methionine i s slow. In r a t s , Edwards et a l . (1963) and Tasaki and Takahashi (196 6) showed that L-methionine was more completely absorbed and u t i l i z e d than D-methionine. These r e s u l t s agree with the findings of K n i p f e l et a l . (1972) but not with the findings of Rerat (Rerat and Laugnon, 1965) who showed that D-methionine was u t i l i z e d as e f f i c i e n t l y as L-methionine by the growing pig. Early research by Shelton et a l . (1951) showed that cystine could supply 50% of the t o t a l sulphur amino acid requirements of the growing pig. Later, Becker et a l . (1955) showed that cystine could supply 4 0% of sulphur amino acid requirements. These figures are lower than those of M i t c h e l l et a l . (1968) of 70% for the weanling pig. Baker et a l . (1969) showed that cystine could supply 56% of sulphur amino acid requirements for the 15 kg liveweight pig without a f f e c t i n g the feed intake or weight gain. However, - 52 -they showed that above 56% replacement value, weight gain as well as feed intake was reduced. Using the same l e v e l of feed intake, they showed that cystine could supply up to 66% of the t o t a l sulphur amino acid requirements for the growing pig. Kroening et al. (1965) showed that young pigs needed 0.5% dietary methionine plus cystine when the crude protein l e v e l was 12%; 0.6% methionine plus cystine when the dietary crude protein l e v e l was 18%; and 0.7% when crude protein l e v e l was 25%. Homb (1976) reported that the requirement of methionine plus cystine for the weanling pig should be 0.6-0.7% of the dry d i e t . The ARC (1967) figure for the same body weight range of pigs i s 0.5-0.6% dietary methionine plus cystine. The NRC (1973) figures are 0.69, 0.56, 0.50 and 0.44% for the 5-10, 10-20, 20-35 and 35-60 kg l i v e weight pigs respectively, i n di e t s containing 22, 18, 16 and 14% crude protein respectively. Both the NRC (1973) and ARC (1967) assume that 50% of the t o t a l sulphur amino acid requirements can be met by cystine. Keith et al. (1972), using serum amino acid l e v e l s as a parameter, obtained a methionine plus cystine requirement of 0.46% i n a d i e t containing 16% crude protein, for pigs of 20-40 kg l i v e weight. Braude and Esnaola (1973) obtained a methionine plus cystine requirement of 0.40-0.45% i n a 16% crude protein d i e t for pigs of 22-62 kg l i v e weight. They suggested that the nitrogen balance technique i s not a good ind i c a t o r of the methionine plus cystine requirements. - 53 -Poppe (1976) reported r e s u l t s from Europe and Russia of the requirements of methionine plus cystine by the growing pig and drew attention to the large v a r i a t i o n s within each l i v e weight category. Methods used for determining the t o t a l amino acids, a v a i l a b l e  amino acids,and protein q u a l i t y of feed A. Total amino acid analysis Acid hydrolysis Acid hydrolysis of feed p r i o r to amino acid analysis i s achieved by the treatment of the feed with 6 N HC1 at 110°C for 24 hr i n a tube flushed with nitrogen, placed under vacuum and sealed (Blackburn, 1968). The tube i s then cooled, opened and the acid removed under reduced pressure. The r e s u l t i n g residue i s then dissolved i n a c i t r a t e buffer, pH 2.0 and the i n d i v i d u a l amino acids analyzed by ion-exchange -chromatography (Spackman et a l . , 1958). With t h i s analysis, some of the amino acids are completely destroyed. Others, which are p a r t i a l l y destroyed or l i b e r a t e d slowly must be estimated by measuring the k i n e t i c s of destruction or release and a correction factor used to extrapolate back to the o r i g i n a l amino acid content ( H i l l , 1965). During hydrolysis, asparagine and glutamine are converted to a s p a r t i c acid :and glutamic acid, and are quantitated on the - 54 -chromatogram. The amide groups are released into the solution as ammonia. Tryptophan and cystine are destroyed and have to be analyzed by separate procedures (Hugli and Moore, 1972; Moore, 1963). Serine and threonine are subjected to p a r t i a l destruction of about 5-10% depending on the duration of hydrolysis (Rees, 1946). Arginine, aspartic acid, glutamic acid, l y s i n e , p r o l i n e and tyrosine are also p a r t i a l l y destroyed (Sanger and Thompson, 1963). The bonds formed by valine, leucine and isoleucine are s l i g h t l y r e s i s t a n t to acid hydrolysis and low y i e l d s of these amino acids are often encountered, but maximum yi e l d s occur a f t e r 72 hr of hydrolysis (Mahowald e t al. , 1962). Because the rates of destruction and release of amino acids vary with time of hydrolysis and d i f f e r from one feed to another, i t i s desirable to determine the k i n e t i c s of destruction and release of each amino acid i n the feed under examination. By t h i s procedure, correc t i o n factors can be applied to each amino acid of the feed under standardized conditions. Ion-exchange chromatography gives no i n d i c a t i o n of the d i g e s t i b i l i t y of a protein, nor does i t indicate the a v a i l a b i l i t y of amino acids i n the feed. The method cannot be used to estimate heat damage due to processing. A l k a l i n e hydrolysis (Tryptophan determination) Because tryptophan i s destroyed during acid hydrolysis, i t s l e v e l in a feed has to be determined by a s p e c i f i c method. The - 55 -tec h n i q u e s which have been used i n c l u d e the s p e c t r o p h o t o m e t r i c method of Goodwin and Morton (194 6) , the c o l o r i m e t r i c method of S p i e s and Chambers (1959), and the p-toluene s u l p h o n i c a c i d method of L i u and Chang (19 71). A l l these methods have i n h e r e n t problems which r e s u l t i n a low tryptophan r e c o v e r y ( H u g l i and Moore, 1972). The most common methods f o r tryptophan d e t e r m i n a t i o n i n c o r p o r a t e a p r e p a r a t o r y a l k a l i n e h y d r o l y s i s (Dreze, 1960; I n g l i s and Leaver, 1964; M i l l e r , 1967; Knox et al. , 1970, and H u g l i and Moore, 1972). The o r i g i n a l method by Dreze (1960) used Ba(OH) 2 as the c a t a l y s t i n the p r o t e i n h y d r o l y s i s . The method lead s to the d e s t r u c t i o n of almost a l l o t h e r amino a c i d s except tryptophan. However, the y i e l d of try p t o p h a n i s low because the amino a c i d i s adsorbed on BaSO^ or BaCO^ formed d u r i n g the r e a c t i o n ( H u g l i and Moore, 1972). A more pro m i s i n g method uses NaOH i n s t e a d of B a t O H ^ ( H u g l i and Moore, 1972). In t h i s method a feed sample i s put i n t o a pyrex tube and NaOH i s added. The tube i s then p l a c e d under vacuum, s e a l e d and h y d r o l y z e d a t 110°C f o r 22 h r . The tube i s then c o o l e d , opened, and the sample i s d i l u t e d . The pH of the r e s u l t i n g s o l u t i o n i s then changed to 4.25. Tryptophan c o n t e n t i s then estimated by ion-exchange chromatography (Spackman et al., 1958). Recovery of tryptophan i n the presence of s t a r c h i s 98-100% (Hugli and Moore, 1 9 7 2 ) . The above method measures t o t a l tryptophan content of the feed but g i v e s no i n d i c a t i o n of i t s a v a i l a b i l i t y to the animal. - 56 -Total methionine and cystine When protein i s subjected to acid hydrolysis, a l l the cystine i s destroyed. Moore (1963) developed a method i n which the feed sample i s i n i t i a l l y subjected to performic acid oxidation p r i o r to the 6 N HCl acid hydrolysis. The performic acid changes methionine into methionine sulphone and cystine into c y s t e i c acid, both of which are r e s i s t a n t to the 6 N HCl acid h y d r o l y s i s . The procedure measures the t o t a l cystine and methionine content of the feed, but does not predict the a v a i l a b i l i t y of these amino acids to the animal during the digestion process. B. Methods for measuring available amino acid levels and protein quality Chemical methods a. Dye binding capacity (DBC) The most commonly used dyes are the pthaleine and the acid azo dyes. Among the acid azo dyes, Orange 12 and acrilane Orange 6, are the ones most commonly used. Acid azo dyes were f i r s t used by Fraenkel-Conrat and Cooper (1944). The method that i s currently being used for protein q u a l i t y analysis was described by Mossberg (1969). In t h i s method, a solution of the acid diazo dye i s mixed with a f i n e l y ground feed sample. The dye i s bound q u a n t i t a t i v e l y by the free basic imidazole, - 57 -guanido and e-amino groups of the protein which occur in the polypeptide chains on h i s t i d i n e , arginine and l y s i n e respectively, and the free a-amino terminal groups. The unbound dye i n the s o l u t i o n i s measured c o l o r i m e t r i c a l l y a f t e r f i l t r a t i o n or c e n t r i f u g a t i o n . There i s a d i r e c t stoichiometric equivalence between the amount of dye bound and the number of free basic groups of the protein i n the sample. If the amino acid composition of the protein i n the feed remains constant, the amount of the dye bound w i l l be a function of t o t a l protein content. The method i s inexpensive, rapid, highly reproducible and can be automated (Udy, 1971; Lakin, 1973). I t i s necessary to c a l i b r a t e the method separately for d i f f e r e n t feed materials since each feed has i t s own character-i s t i c amino acid pattern. Fine grinding of the sample i s recommended. Oily samples need f a t extraction f i r s t because l i p i d s i n h i b i t dye binding (Udy, 1971). Calcium, c h l o r o p h y l l , f i b e r and tannins have been shown to i n t e r f e r e with dye binding (Mossberg, 1969). Hurrel and Carpenter (1975) showed that Remazol blue and Acid Orange 12 were better dyes than c r e s o l red i n protein q u a l i t y determinations, and that Remazol blue detected mild heat damage better than Orange 12. However, the procedure for Orange 12 was much simpler than that of Remazol blue. The method has been used i n high-lysine cereal breeding programmes (Munck et at. , 1969), i n the evaluation of factors - 58 -a f f e c t i n g harvesting and storage of grains (Mossberg, 1970) and i n the routine measurement of protein q u a l i t y of wheat (Udy, 1956), milk (Ashworth and Chaudry, 1962), pulses, m i l l e t s and cereals (Kaul et al. , 1970). The method has also been used i n monitoring heat damage due to processing. Mild heat treatment, i n which the basic amino acids are not destroyed, does not lead to changes i n DBC. Severe heat treatment destroys basic amino acids, e s p e c i a l l y l y s i n e , when carbohydrates are present (Bjarnason and Carpenter, 1970). In such cases the DBC values are reduced (Udy, 1971) . When the b i o l o g i c a l value of a feed i s li m i t e d by i t s content of a basic amino ac i d , i t s DBC correlates very well with b i o l o g i c a l t ests for protein q u a l i t y (Lakin, 1973). b. Carpenter's method (1960) for " a v a i l a b l e l y s i n e " This method was used i n i t i a l l y for evaluating heat damage during processing of protein meals derived from animal and f i s h t i s s u e s . Heat damage in these feeds occurs when the E-amino group of ly s i n e reacts with either the carbohydrates i n the M a i l l a r d reaction (Hodge, 1953), or the amide groups of asparagine and glutamine (Bjarnason and Carpenter, 1970), giving r i s e to linkages that r e s i s t enzymatic hydrolysis. The reacted lys i n e thus becomes unavailable to the animal. The method i s based on the Sanger reaction between l - f l u o r o - 2 , 4 - d i n i t r o benzene (FDNB) and the free e-amino group of lys i n e , the guanido group of - 59 -arginine, and the imidazole group of h i s t i d i n e i n the poly-peptide chain. A f t e r t h i s i n i t i a l reaction, the products are acid hydrolyzed to l i b e r a t e DNP-lysine, together with the i n t e r f e r i n g compounds such as DNP-arginine and DNP-histidine. These i n t e r f e r i n g products are removed p r i o r to the colorimetric determination of the DNP-lysine at the 435 nm wavelength. This figure i s an estimate of the "available l y s i n e " i n the feed since any e-amino groups that had previously reacted with either carbohydrates or amide groups would not have been free to react with FDNB. The major disadvantage of t h i s method i s that, during hydrolysis, any carbohydrates present may react with the e-amino groups of lysine, thus reducing the DNP-lysine values (Matheson, 1968) . This problem makes the method of l i m i t e d value for vegetable proteins (Roach et a l . , 1967). Consequently, modified techniques were developed by Nielson and Weidner (1966), Roach et a l . (1967) , Matheson (1968) and Booth (1971) . The FDNB method has been used to measure the protein q u a l i t y of food (Duckworth et a l . , 1961; Pritchard et a l . , 1964; and Erbersdobler, 197 0) , and the extent of heat damage due to processing (Boyne et a l . , 1967; and Booth, 1971) . There i s a high c o r r e l a t i o n between ly s i n e a v a i l a b i l i t y measured with FDNB and Protein E f f i c i e n c y Ratio (PER) obtained with rats (Bruggemann et a l . , 1969). The major disadvantage of the method i s that i t cannot be used to assess the absorption of the amino acid from the gut. - 60 -A l t e r n a t i v e chemical methods include those u s i n g trinitrobenzene sulphonic acid (TNBS) (Kakade and Liener, 1969) ; 0-methylisourea (Mauron and Bujard, 1964^ and acrylo-n i t r i l e (Riehm and Scheraga, 1966). However, these a l t e r n a t i v e methods do not give values of a v a i l a b l e l y s i n e that correlate with growth methods i n the same way as the FDNB method (Carpenter and Booth, 1973). B i o l o g i c a l methods a . Protein e f f i c i e n c y r a t i o (PER) The method was originated by Osborne et al. (1919). They defined PER as the gain i n body weight, i n grams, per gram of protein consumed by weanling r a t s . PER = Weight increase of experimental animals Weight of protein consumed Osborne et al. (1919) r e a l i z e d that the r e s u l t s varied with the l e v e l of protein i n the diet,and recommended assay at a 10% dietary crude protein l e v e l . In t h i s method, a group of rats i s fed the t e s t protein d i e t and another group i s fed a standard protein (casein) diet ad libitum. Feed consumption and weight gain are recorded over a period of 14 days. The method implies that the gain of body weight by the growing rat i s constant regardless of age and s i z e of the animal. M i t c h e l l and Carman (1926) , M i t c h e l l (1944), and Bender (19 56) , however, pointed out that: - 61 -1. the assumption that weight increase i s an index of protein synthesis i s not necessarily v a l i d . 2. r e s u l t s vary with the l e v e l of protein i n the d i e t . 3. proteins that do not provide good growth cannot be evaluated. Despite these drawbacks, the method i s commonly used to evaluate protein q u a l i t y of feed. Eggum (1969) reported a good c o r r e l a t i o n between weight gain and nitrogen gain. Also, the method has been found to c l a s s i f y proteins i n the same order as that from mi c r o b i o l o g i c a l assays (Ford, 1960). McLaughlan and Keith (1975) have modified the method to produce a close c o r r e l a t i o n with values obtained with the Net Protein Retention (NPR) method. Lactalbumin was used as the standard protein i n both methods. b. Net protein retention (NPR) In order to solve some of the problems^inherent i n the PER method, Bender and Doell (1957) introduced the Net Protein Retention (NPR) method. Two groups, each of four weanling r a t s , balanced as regards l i t t e r and t o t a l body weight are used to test each protein. One group i s fed a protein-free d i e t while the other i s fed a d i e t containing the t e s t protein at a 10% le v e l on dry matter basis. At the end of 7 or 10 days, the - 62 -animals are weighed and the p r o t e i n i n t a k e d u r i n g the p e r i o d i s determined. NPR = Weight gain of test protein fed group + Weight loss of non-protein fed group ~~ Weight of protein consumed In t h i s e q u a t i o n , p r o t e i n t h a t i s used f o r both growth and maintenance i s i n c l u d e d . NPR v a l u e s are much b e t t e r i n d i c a t o r s of p r o t e i n q u a l i t y i n c e r e a l g r a i n s than PER v a l u e s (Eggum, 1969). The NPR v a l u e s can be measured r e l a t i v e to e i t h e r c a s e i n (Eggum, 1969) or l a c t a l b u m i n (McLaughlan and K e i t h , 1975). c. Relative protein value (RPV) T h i s method was suggested by Hegsted (1974). Here, the r a t e of body weight change of weanling r a t s fed on v a r i o u s l e v e l s of the t e s t p r o t e i n , i s compared w i t h t h a t o b t a i n e d w i t h animals fed on a r e f e r e n c e p r o t e i n , such as c a s e i n . The t e s t r e q u i r e s t h a t the body weight change be l i n e a r over the range of p r o t e i n i n t a k e s s t u d i e d , and t h a t such a range be wide enough to produce an a c c u r a t e l i n e a r r e g r e s s i o n l i n e (McLaughlan and K e i t h , 1975). The method uses s i x groups, each of fo u r weanling r a t s . The r a t s are f e d a r e f e r e n c e p r o t e i n d i e t c o n t a i n i n g 1.3% n i t r o g e n (N) f o r 2 days. One group i s then l e f t on the r e f e r e n c e p r o t e i n d i e t w h i l e the other groups are a s s i g n e d to the t e s t p r o t e i n and r e f e r e n c e p r o t e i n d i e t s c o n t a i n i n g 0.3% N, 0.8% N and 1.3% N. The f e e d i n g i s ad l i b i t u m f o r 14 days. Weight g a i n and f o o d consumption a r e r e c o r d e d over the t r i a l p e r i o d . F o r each p r o t e i n s o u r c e and for each protein l e v e l , the weight gain i s plotted against protein intake and the slope calculated. R p V _ Slope obtained with test feed  Slope obtained with reference protein and Relative U t i l i z a b l e Protein = RPV x Protein Concentration. It should be pointed out that unusually high l e v e l s of protein intake r e s u l t i n non-linear body weight responses, thus d i s t o r t i n g the slope of the curve downward and underestimating the protein q u a l i t y . If the response curve cuts the weight change axis below the weight loss of the animals fed the protein-free basal diet, then the assay i s i n v a l i d . McLaughlan and Keith (1975) found that food proteins d e f i c i e n t i n threonine, i . e . those i n which threonine i s the f i r s t l i m i t i n g amino acid, gave slopes s i m i l a r to those obtained with the reference protein, yet growth on the former food was s t i l l i n f e r i o r to that obtained with the reference protein. Thus although the method correlates well with the PER method (McLaughlan and Keith, 1975), the method i s not v a l i d for foods where threonine i s the f i r s t l i m i t i n g amino acid. d.. B i o l o g i c a l value (BV) This nitrogen balance method was originated by Thomas (1909) and improved by M i t c h e l l (1924), hence the present name, Thomas-Mi t c h e l l Method. The method measures the percentage of absorbed - 64 -nitrogen which i s retained i n the body. In i t s simplest form, t h i s i s obtained from the difference between intake and excretion (Schelling, 1975; A l l i s o n , 1964): B V = Nitrogen retained Nitrogen absorbed Nxtrogen intake - [Faecal + Urinary nitrogen] , n n or BV - 2-r— =—T—= = . , J x 10 0 Nitrogen intake - Faecal nitrogen The above equations, as proposed by Thomas (1909) took no account of endogenous nitrogen excretion. To correct for t h i s , M i t c h e l l (1924) modified the formula to: N intake - [Faecal N - Metabolic faecal N] -B V _ [Urinary N - Endogenous Urinary N] x 10 0 N intake - [Faecal N - Metabolic N] The numerator measures nitrogen retained and the denominator, nitrogen absorbed. Metabolic faecal nitrogen (MFN) includes nitrogen from various juices secreted into the i n t e s t i n e , sloughed off e p i t h e l i a l c e l l s , and probably dietary nitrogen which has been hydrolyzed but unabsorbed. Endogenous urinary nitrogen (EUN) represents the basal nitrogen loss of an animal receiving either a nitrogen-free d i e t or a die t containing a low l e v e l of high q u a l i t y (100% BV) protein. - 65 -Direct measurements of MFN and EUN are d i f f i c u l t , and, i n order to use the equation, MFN and EUN should be measured independently. The following methods have been used to determine the MFN and EUN values ( A l l i s o n , 1964): 1. Measurements obtained with animals on a nitrogen free d i e t : some d i f f i c u l t i e s may occur i n making the animals eat such a d i e t . To avoid t h i s , a known amount of a completely u t i l i z a b l e protein i s added, and the r e s u l t s adjusted accordingly. 2. Using values obtained previously, e.g. MFN i s approximately 0.1 g per 100 g dry matter intake for the rat and man. 3. P l o t t i n g nitrogen intake against nitrogen excretion, for a constant amount of food varying i n i t s content of a constant source of protein. Extrapolation to zero nitrogen intake provides a value for EUN. The BV method i s the most valuable method for determining the protein q u a l i t y of food (Von Der Decken et al. , 1975) but i s laborious and time consuming. Also, there are errors i n the determination of MFN and EUN. Further the method requires that the protein intake be r e s t r i c t e d to a l e v e l which produces a nitrogen balance which i s s l i g h t l y negative or at equilibrium. The method i s affected by the protein intake i n such a way that the estimated BV decreases as nitrogen absorption increases - 66 -beyond the maintenance requirements (Von Der Decken et al. , 1975). It i s also affected by age, with adult animals giving higher values than growing animals. The method i s also affected by the energy l e v e l i n the d i e t . e. Net protein u t i l i z a t i o n (NPU) The method was introduced by M i l l e r and Bender (1955) to replace the longer procedures required for measuring BV. Two groups, each of 4 weanling r a t s , are normally used. One group i s fed the te s t protein d i e t containing 10% crude protein, and the other group i s fed a protein-free d i e t . The nitrogen intake for both groups i s measured,and the animals slaughtered at the end of a 10-day feeding period. Carcass nitrogen i s determined either by the kjeldahl method or by measuring the body water content, since the nitrogen/water r a t i o has been shown to be constant i n rats over a short assay period (Bender and M i l l e r , 1953), at a s p e c i f i c age. P e l l e t (1973) does not recommend the body water method for Sprague-Dawley r a t s . Body N of test group - [body N of non-protein te s t N p u _ group - N consumed by non-protein group*]  N-consumed by test group *a correc t i o n i s applied for the small amount of nitrogen i n e v i t a b l y present i n p u r i f i e d food constituents. - 67 -NPU assumes that carcass amino acid composition i s unaffected by the d i e t (Pellet and Kaba, 1 9 7 2 ) . However, Barnes e t at. ( 1 9 4 6 ) showed that the percentage of protein retained f e l l with increasing protein concentration i n the diet,such that NPU was not constant for any s p e c i f i c protein. I t has been proposed, therefore, ( M i l l e r and Payne, 1 9 6 1 ) that two terms to used: NPU ^, and NPU . The NPU _^ , i s obtained at or about std op standard maintenance l e v e l of protein intake whereas NPU . i s c operative obtained at any other stated protein l e v e l . Thus N P U s t d 1 S a measure of protein q u a l i t y and NPUQp i s a measure of the protein for a p a r t i c u l a r requirement. In theory the N P U s t c j should c l o s e l y approximate the product of B i o l o g i c a l Value and true nitrogen d i g e s t i b i l i t y . M i c r o b i o l o g i c a l methods a. Tetrahymen pyriformis The organism i s a c i l i a t e d protozoa which can be grown in pure culture on a chemically defined medium. I t was f i r s t used by Rockland and Dunn ( 1 9 4 6) to measure protein q u a l i t y of food. In t h e i r f i r s t t r i a l , Rockland and Dunn ( 1 9 4 6 ) used acid production over a 41 day period as an index of protein q u a l i t y . Kidder and Dewey ( 1 9 5 1 ) showed that the organism was strongly prot e o l y t i c , and that i t s n u t r i t i o n a l requirements were similar - 68 -- to those of a growing rat with the exception that serine was also required. F e r n e l l and Rosen (1956) modified the method whereby ammonia production was measured over a 4-day period. Stott and Smith (1966) further developed the method to assay av a i l a b l e amino acids. The samples were suspended i n water to give varying nitrogen l e v e l s . Growth was assessed aft e r 4 days of incubation by counting the number of organisms per ml of culture f l u i d . In t h i s early method, counting was made d i f f i c u l t by the f a c t that food p a r t i c l e s looked the same as the organisms. The i n i t i a l r e s u l t s showed that the method underestimated the a v a i l a b i l i t y of ly s i n e when values were compared with those obtained by Carpenter's (1960) method for avail a b l e l y s i n e , due to incomplete digestion of the protein by the microbes (Boyne et a l . , 1967). The method was improved when the sample was predigested with papain (Boyne et a l . , 1967). Shorrock (1972) modified the counting by photographing the organisms on the haemocytometer, and counting the organisms on the photograph using a d i g i t a l counter. Predigestion also had the advantage of clearing the protein solution thereby making counting easier. The c l a r i t y of the solution a f t e r incubation made i t possible to follow growth response by absorbance measurement which was done at the 580 nm wavelength. Results obtained aft e r predigestion with enzymes have correlated well with those obtained using growth tests such as PER and NPU andalso with Carpenter's (1960) av a i l a b l e lysine t e s t and chick assay for proteins f i r s t l i m i t i n g i n lysine (Boyne et a l . , 1967; Shorrock and Ford, 1973; Shepperd ez a l . 1975 and Shorrock, 1976). Modifications to the method have been made by Shorrock (1976) and Shorrock and Ford (1973). Shepperd et a l . (1977) predigested the te s t protein with pronase and determined tetrahymanol, a pentacyclic pentene s p e c i f i c a l l y produced by microbe (Mallory et a l . , 1963; Thompson et a l . , 1971) by gas-l i q u i d chromatography. The method was based on the l i n e a r r e l a t i o n s h i p that existed between c e l l numbers and tetrahyman content of the culture extract (Shepperd et a l . , 1975). The method can be used to measure the a v a i l a b l e l e v e l s of several amino acids v i z . arginine, h i s t i d i n e , i s o l e u c i n e , leucine, l y s i n e , methionine and tryptophan. The fa c t that Tetrahymena p y r i f ormis requires 4 days for assay whereas Streptococcus zymogenes requires only 2 days, makes the former method less popular. The other problem i s that the method has not been standardized ye£. The microbe can be used to assay available lysine whereas ly s i n e i s not required for growth of Streptococcus zymogenes with the resul that i t cannot be"'used to measure av a i l a b l e l y s i n e . - 70 -b. Streptococcus zymogenes Ford (1966) u t i l i z e d t h i s organism for the assessment of protein q u a l i t y . The organism requires the same exogenous amino acids as Tetrahymena pyviformis with the exception of threonine, phenylalanine and lys i n e and the addition of glutamic acid. It i s powerfully p r o t e o l y t i c , and grows quickly, allowing a 48 hr test when there i s an adequate supply of i n t a c t protein as the main source of nitrogen. The organism, i n routine work, i s grown i n a chemically-defined medium in which other nutrients are incorporated at the same l e v e l as the requirements of the growing rat (Ford, 1960). In order to obtain r e s u l t s that correlate well with those obtained by growth methods, predigestion of the food protein with papain has been recommended (Ford, 1960, 1962). Growth response i s measured a f t e r 48 hr by determining the absorbance of the culture medium at 580 nm wavelength. Casein i s normally used as a standard protein. Protein q u a l i t y values obtained with t h i s organism correlate well with those obtained with rats (Ford, 1960, 1962). The method has been used to measure available arginine, h i s t i d i n e , i s o leucine, leucine, methionine, tryptophan and v a l i n e but not l y s i n e , phenylalanine or threonine. The organism has been used to measure heat damage due to processing of meat meals, f i s h meals and legume-based meals (Ford, 1962; Boyne et a t . , 1967). - 71 -Modifications of the method have been made to allow for automation (Ford, 1 9 6 4 ; Kennedy, 1965 and Boyne et al. , 1 9 7 5 ) . The method has been standardized and i s faster than that u t i l i z i n g Tetrahymena p y r i f ' o r m i s . Enzymatic assays Methods for assessing the content of available amino acids and for determining protein q u a l i t y have been reviewed by Mauron (1970) . a . Available amino acids Melnick et al. (1946), i n t h e i r experiments,digested food protein with pancreatin, and were able to follow the degree of hydrolysis of the protein by periodic withdrawal of samples and measurement of i n d i v i d u a l amino acids. Riesen et al. (1947) and Sheffner et al. (1956), using pepsin followed by pancreatin digestion, observed that amino acid l i b e r a t i o n by the enzymes was much higher when milk proteins were f i r s t treated with mild heat. Mauron et al. (1955) devised a procedure for monitoring the q u a l i t y of heat-processed milk by using pepsin followed by pancreatin digestion. The method involves the i n i t i a l d i a l y s i s of the protein, followed by pepsin digestion for 15 hr. The r e s u l t i n g solution i s then subjected to pancreatin hydrolysis for 24 hr. The liberated amino acids are analyzed by either column chromatography (Spackman et a l . , 1958) or i n d i v i d u a l techniques. Results which were obtained (Mauron, 1970) for avai l a b l e l y s i n e using t h i s method correlated well with those derived by rat and chick growth methods. Ford and Salter (1966) modified the method by passing the enzyme digest through Sephadex G25 to remove products that i n h i b i t e d the enzyme a c t i v i t y . Cuq et a l . (1973) hydrolyzed processed casein to monitor methionine a v a i l a b i l i t y . Pieniazek e t a l . (1975) used pancreato peptidase, leucine amino peptidase and prolidase to monitor methionine a v a i l a b i l i t y i n heat-treated and normal casein. b. Protein quality evaluation The r e l a t i o n s h i p between the pattern of amino acids released by the digestive enzymes and the b i o l o g i c a l value of food proteins was f i r s t studied by Sheffner et a l . (1956). Amino acid patterns r e s u l t i n g from in v i t r o pepsin digestion showed incomplete hydrolysis of food proteins by the enzyme. The combination of pepsin, trypsin and erepsin during the hydrolysis procedure improved the quantity of amino acids li b e r a t e d from the food proteins. Sheffner et a l . (1956) correlated the amino acid pattern obtained during pepsin digestion of a food protein with that of whole egg protein. This c o r r e l a t i o n was c a l l e d the - 73 -Pepsin Digest-residue (PDR) amino acid index. Sheffner et al. (1956) showed that t h i s PDR index represented a net protein u t i l i z a t i o n (NPU) of the food protein. However, the c o r r e l a t i o n of t h i s index with NPU was poor since pepsin digestion of the food protein was incomplete. Modifications to t h i s index have included the pepsin pancreatin digest (PPD) index of Akeson and Stahmann (1964). This modification uses both pepsin and pancreatin i n the hydrol-y s i s of the food protein. The c o r r e l a t i o n between the PPD index and BV i s higher than that of the PDR index (Mauron, 1970). A s l i g h t modification of the PPD index was proposed by Mauron (1970). This involves the d i a l y s i s of the food against tap water followed by 0 .'1 N HCl i n order to remove enzyme i n h i b i t o r s . This method has been termed the Pepsin Pancreatin Digest Dialysate index (PPDD) (Mauron, 1970). Enzymatic methods generally are less expensive and less time consuming than r a t feeding t e s t s . The methods can be repeated e a s i l y and with minimum v a r i a t i o n . They indicate r e l a t i v e amounts of each e s s e n t i a l amino acid i n the protein and thus give protein q u a l i t y as well as amino acid a v a i l a b i l i t y . The only problems appear to be that digestion i s not complete, and that no i n d i c a t i o n i s given of the presence of possible toxins i n the food (Stahmann and Woldegiorgis, 1975). - 74 -EXPERIMENTS RAT TRIAL 1 Growth response of weanling r a t s t o graded l e v e l s of methionine  p l u s c y s t i n e i n b a r l e y - b a s e d d i e t s f o r t i f i e d w i t h s y n t h e t i c  amino a c i d s Introduction and aim I t was c o n s i d e r e d d e s i r a b l e i n an i n i t i a l t r i a l with r a t s to i n v e s t i g a t e two p o i n t s : (a) the comparative response of the r a t s to a d i e t of barley-soybean meal and to a d i e t of b a r l e y supplemented w i t h s y n t h e t i c e s s e n t i a l amino a c i d s at the NRC (1972) requirements, (b) the response of the r a t s to methionine supplementation of a d i e t complete i n a l l e s s e n t i a l amino a c i d s , but c o n t a i n i n g o n l y 0.23%, dry matter b a s i s (DM), methionine p l u s c y s t i n e . In p r e v i o u s experiments i n t h i s l a b o r a t o r y , a barley-soybean meal combination has been used as a p o s i t i v e c o n t r o l d i e t (Chung and Beames, 1974; Aw-Yong and Beames, 1975) . An equal performance w i t h a g r a i n p l u s amino a c i d s d i e t would s i m p l i f y i n t e r p r e t a t i o n of responses to v a r y i n g l e v e l s of amino a c i d supplementation. The reason f o r t e s t i n g a l e v e l of 0.23% DM methionine p l u s c y s t i n e i n an otherwise complete d i e t was to e s t a b l i s h a l o w e r l i m i t f o r subsequent i n v e s t i g a t i o n s . In a r e c e n t r e s e a r c h r e p o r t (Oesterner et a l . , 1970), t h i s l e v e l o f s u l p h u r amino a c i d s had b e e n suggested as adequate f o r t h e 21 kg l i v e w e i g h t p i g . - 75 -Materials and methods E x p e r i m e n t a l d e s i g n There were s i x d i e t a r y treatments, with each d i e t p r o v i d e d to e i g h t i n d i v i d u a l l y - f e d r a t s . Both the d i e t a r y treatments and r e p l i c a t e s were randomly d i s t r i b u t e d among the 4 8 cages i n a room w i t h temperature maintained a t 27°C. Animals and cages F o r t y - e i g h t male r a t s (Woodlyn/Wistar s t r a i n , Woodlyn L a b o r a t o r i e s , Guelph, O n t a r i o ) , 27 days of age a t the s t a r t of the 21-day f e e d i n g p e r i o d were used. The s t a i n l e s s s t e e l cages had wire s c r e e n f l o o r s o f 12 mm spacing, and were f i t t e d w i t h t r a y s covered w i t h paper towels f o r the c o l l e c t i o n of faeces and u r i n e . Trays were emptied and towels renewed d a i l y . D i e t s D i e t s (Table I) were based on b a r l e y g r a i n which was ground i n a 2.00 mm l a b o r a t o r y hammer m i l l ( C h r i s t y and N o r r i s L t d . , Chelmsford, England) f i t t e d w i t h a 0.75 mm screen. The d i e t s i n c l u d e d a p o s i t i v e c o n t r o l ( d i e t 3) of b a r l e y p l u s soybean meal. The d i e t s were formulated as f o l l o w s : TABLE I . F o r m u l a t i o n (% DM) o f D i e t s f o r Rat T r i a l 1 I n g r e d i e n t s and C o m p o s i t i o n D i e t No. 1 2 3 4 5 6 B a r l e y 96.82 48.43 77.49 48.43 48.43 48 .43 Soybean meal - - 19 . 37 - - -M a i z e s t a r c h — 43.24 — 43.24 43.24 43.24 D e f l u o r i n a t e d r o c k phosphate* 1.64 1. 64 1.52 1.64 1.64 1.64 C a l c i u m carbonate"*" _ . 54 .54 . 57 .54 .54 . 54 I o d i z e d salt"!' f i ) . 50 . 50 . 50 . 50 .50 . 50 Tr a c e m i n e r a l + V i t a m i n premix^-/ .50 .50 . 50 .50 .50 .50 Threonine" 1" 1" - - - .36 .36 . 36 V a l i n e " - - .34 .34 .34 I s o l e u c i n e " - - - .35 . 35 . 35 L e u c i n e " - - - - .37 .37 .37 P h e n y l a l a n i n e + t y r o s i n e " - - - . 34 .34 .34 H i s t i d i n e " - - - .18 .18 .18 L y s i n e " - - - . 70 .70 .70 A r g i n i n e " - - - .35 .35 . 35 Tr y p t o p h a n " - - - . 08 . 08 .08 M e t h i o n i n e + C y s t i n e " - - - - .22 .37 A l a + Asp + G l y + G l u (1:1:1:1)** - 5.15 - 2 .03 1. 81 1.66 % Crude p r o t e i n 10.30 10 .30 17 .10 10. 30 10.30 10 . 30 GE (K c a l / k g ) 3624.0 3718.0 3803.9 3718.0 3718.0 3718.0 *Estimated to c o n t a i n 300 g/kg calcium and 140 g/kg phosphorus. + E s t i m a t e d to co n t a i n 400 g/kg Ca and O g/kg P. ^Estimated to provide 0.15 mg iodine/kg dry d i e t . TABLE I: (continued) §Provided (1 kg dry diet) 44 mg manganese as MnSC^.H^O, 100 mg zinc as ZnSC>4.7H20, 500 mg butylated hydroxy toluene; 20 yg cyanocobalamin, 2.9 mg r i b o f l a v i n , 11 mg n i c o t i n i c a c i d , 5 mg calcium pantothenate, 3.6 mg pyridoxine, 0.2 mg D-biotin, 925 pg r e t i n o l , 10 yg e r g o c a l c i f e r o l and 1 g choline c h l o r i d e . " A l l amino acids were incorporated as L-isomers (Ajinomoto Co., Tokyo, Japan) to meet the NRC (1972) recommendations. **Incorporated according to Abernathy and M i l l e r (1965) and Womack (1969) to bring the crude protein l e v e l to 10.30% of the dry d i e t . + + I n c o r p o r a t e d according to Aw-Yong and Beames (1975). ( TABLE I I . E s s e n t i a l amino a c i d composition (% DM) of d i e t s f o r Rat T r i a l 1 Amino A c i d Threonine D i e t No. 0.40 0.40 0.69 0.57 0.57 0.57 V a l i n e 0.51 0.26 0.82 0.60 0.60 0.60 I s o l e u c i n e L e u c i n e 0.40 0.20 0.72 0.55 0.55 0.55 0.75 0.38 1.27 0.75 0.75 0.75 CO P h e n y l a l a n i n e + t y r o s i n e 0.92 0.46 1.43 0.80 0.80 0.80 H i s t i d i n e 0.23 0.12 0.43 0.30 0.30 0.30 L y s i n e 0.39 0.20 0.90 0.90 0.90 0.90 A r q i n i n e 0.49 0.25 0.97 0.60 0.60 0.60 T r y p t o p h a n 0.18 0.09 0.29 0.17 0.17 0.17 M e t h i o n i n e + c y s t i n e 0.45 0.23 0.60 0.23 0.45 • 0.60 D i e t No. - 79 -I n g r e d i e n t s % Methionine + c y s t i n e (DM) 1 B a r l e y 0.45 2 B a r l e y + s t a r c h + n o n - e s s e n t i a l amino a c i d s (NEAA) 0.23 3 B a r l e y + soybean meal ( p o s i t i v e c o n t r o l ) 0 .60 4 B a r l e y + s t a r c h + e s s e n t i a l amino a c i d s (EAA) + NEAA 0.23 5 B a r l e y + s t a r c h + EAA + NEAA + methionine 0.45 6 B a r l e y + s t a r c h + EAA + NEAA + methionine 0 .60 A l l the above d i e t s c o n t a i n e d m i n e r a l s and v i t a m i n s a t l e v e l s to meet the NRC (1972) requirements f o r the growing r a t . Where e s s e n t i a l amino a c i d s were added, the t o t a l l e v e l s e q u a l l e d the NRC (197 2) requirements f o r the growing r a t except f o r methionine and c y s t i n e . Table I I shows the amino a c i d composition of the d i e t s . Food and water were p r o v i d e d ad l i b i t u m . Water was renewed d a i l y . Body weight and food i n t a k e f o r each r a t were reco r d e d d a i l y . At the c o n c l u s i o n of the 21-day f e e d i n g p e r i o d , the r a t s were k i l l e d by a blow on the head. A f t e r f l u s h i n g the i n t e s t i n a l t r a c t with water to remove food p a r t i c l e s and f a e c a l m a t e r i a l s , the c a r c a s s e s were s t o r e d a t -40°C f o r l a t e r carcass chemical a n a l y s i s . - 80 -A n a l y t i c a l methods Both the b a r l e y and the soybean meal were analyzed f o r t h e i r t o t a l amino a c i d c ontent. H y d r o l y s a t e s were prepared by the method of Kohler and P a l t e r (1967) . C y s t i n e was determined as c y s t e i c a c i d and methionine as methionine sulphone a f t e r o x i d a t i v e h y d r o l y s i s w i t h p e r f o r m i c a c i d (Moore, 1963). The samples were analyzed u s i n g an amino a c i d a n a l y z e r (Durrum Model D500; Durrum, Palo A l t o , C a l i f o r n i a ) . Tryptophan was determined a f t e r a l k a l i n e h y d r o l y s i s ( H u g l i and Moore, 1972) u s i n g a Beckman-Spinco A n a l y z e r (Model 120B; Beckman Instruments Inc., P a l o A l t o , C a l i f o r n i a ) . Food and c a r c a s s crude p r o t e i n were determined by the macro-k j e l d a h l method, w h i l e ash was analyzed by dry ashing i n a m u f f l e furnace a t 550°C ( A s s o c i a t i o n of O f f i c i a l A g r i c u l t u r a l Chemists, 1975) . Gross energy content o f d i e t a r y components and d i e t s were determined u s i n g an a d i a b a t i c bomb c a l o r i m e t e r (A. Gallenkamp and Co. L t d . , London, England). Body composition of the r a t s was determined a f t e r d r y i n g the thawed c a r c a s s e s a t 95°C f o r 72 h r . The d r i e d c a r c a s s e s were weighed and f a t was e x t r a c t e d w i t h petroleum s p i r i t ( b o i l i n g p o i n t 30-60°C) i n a So x h l e t apparatus u s i n g 43 x 120 mm thimbles ( A s s o c i a t i o n of O f f i c i a l A g r i c u l t u r a l Chemists, 1975). The r e s i d u e was d r i e d i n an oven a t 95°C f o r 5 hr, weighed and ground u s i n g a 200 mm l a b o r a t o r y hammer m i l l f i t t e d w i t h 0.75 mm screen. The r e s u l t i n g samples were " b a l l - m i l l e d " f o r 24 hr and the uniform powder was used i n the analyses f o r crude p r o t e i n and ash. - 81 -S t a t i s t i c a l procedures A l l the r e s u l t s were s u b j e c t e d to a n a l y s i s of v a r i a n c e and a l l the d i f f e r e n c e s between means were t e s t e d a t the 5% p r o b a b i l i t y l e v e l , u s i n g the Newman K e u l 1 s M u l t i p l e Range T e s t a c c o r d i n g to the programme of Halm and Lee (1975), UBC-MFAV. The same programme was a l s o used i n the s i n g l e degree of freedom c o n t r a s t s between means-Results Growth, feed i n t a k e and feed c o n v e r s i o n e f f i c i e n c y (FCE) r e s u l t s are p r e s e n t e d i n T a b l e I I I ; c a r c a s s composition r e s u l t s are i n Table IV;and s e l e c t e d c o n t r a s t s f o r the parameters i n both T a b l e I I I and Table IV t h a t show s i g n i f i c a n t d i f f e r e n c e s between means, are presented i n T a b l e V. Average d a i l y body-weight g a i n The g a i n by r a t s on the p o s i t i v e c o n t r o l d i e t ( d i e t 3) was s i g n i f i c i a n t l y h i g h e r (P<0.05) than on any of the other d i e t s d u r i n g the f i r s t week of the t r i a l p e r i o d as w e l l as f o r the o v e r a l l t r i a l . D uring the second and t h i r d weeks o f t h e t r i a l , g a i n on both d i e t s 5 and 6 (0.45 and 0.60% DM methionine p l u s c y s t i n e r e s p e c t i v e l y ) was not d i f f e r e n t from t h a t on the p o s i t i v e c o n t r o l d i e t . Apart from the f i r s t week, d u r i n g which r a t s on 0.60% DM d i e t a r y methionine p l u s c y s t i n e ( d i e t 6) g a i n e d s i g n i f i c a n t l y TABLE I I I . Average d a i l y body-weight g a i n (g), food consumption (g) and food <\J c o n v e r s i o n e f f i c i e n c y (g weight gain/g food consumed) of r a t s i n 0 0 Rat T r a i l 1 S t a t i s t i c a l significance of difference Diet No. 1 2 3 4 5 6 between diets j - , ^ o f Newman Keul's Methionine + cystine (% DM) 0. 45 0. 23 0. 60 0.23 0. 45 0. 60 Mean F-Test Test I n i t i a l body-weight (g) 105 .1 105 .0 105 .5 104.0 102 .6 106 .4 0 .79 NS Week 1 2. 28 -0. 67 5. 52 -0.16 2. 15 3. 92 0 .29 * 2 4 5 1 6. 3 Average d a i l y body weight g a i n (g) Week 2 Week 3 2. 3. 92 67 -0. 0. 16 28 6. 5. 04 67 0.42 0.44 5. 5. 79 05 5. 4. 65 93 0 0 .38 .35 * * 2 2 4 4 1 1 6 6 5 5 3 3 O v e r a l l 2. 92 -0. 31 5. 78 0.29 4 . 18 4. 69 0 . 33 * 2 4 1 6 5 3 Week 1 14. 53 10. 05 15. 79 11.28 14. 16 13. 68 0 .37 * 2 4 6 5 1 3 Average d a i l y food consumption Week 2 17. 79 9. 94 18 . 30 11.41 18 . 28 18. 78 0 .60 * 2 4 1 5 3 6 Week 3 19. 67 9. 81 20. 06 10.90 18. 80 20 . 40 0 .72 * 2 4 5 1 3 6 (g) O v e r a l l 17. 40 10. 16 18. 05 11.16 17. 15 17 . 28 0 .54 * 2 4 5 6 1 3 Average food Week 1 0. 17 . -0. 11 0. 35 -0.04 0. 16 0. 30 0 .03 * 2 4 5 1 6 3 c o n v e r s i o n e f f i c i e n c y g g a i n / g Week 2 Week 3 0. 0. 18 20 -0. 0. 04 05 0. 0. 34 30 0.07 0.09 0. 0. 33 29 0. 0. 31 26 0 0 .02 .02 * * 2 2 4 4 1 1 6 6 5 5 3 3 feed consumed (DM) O v e r a l l 0. 19 -0. 03 0 . 33 0.05 0. 27 0. 29 0 .02 * 2 4 1 5_ J5 3 * F - t e s t s i g n i f i c a n t at the 5 % p r o b a b i l i t y l e v e l . NS - Not s i g n i f i c a n t (P > 0.05) - 83 -(P<0.05) more than those on 0.45% DM d i e t a r y methionine plus c y s t i n e , ( d i e t 5 ) , both d i e t s produced a s i m i l a r r a t e of growth. Both d i e t s 2 and 4, c o n t a i n i n g 0.23% DM d i e t a r y methionine p l u s c y s t i n e , produced weight l o s s e s during the f i r s t week of the t r i a l , w i t h l o s s e s on d i e t 2 c o n t i n u i n g during the second week of the t r i a l . However both d i e t s 2 and 4 promoted minimal r a t e s of body weight g a i n , t h a t could not be s t a t i s t i c a l l y d i f f e r e n t i a t e d over the t r i a l p e r i o d . Gains on both these d i e t s were s i g n i f i c -a n t l y (P<0.05) l e s s than those on d i e t s 5 and 6 over the whole t r i a l p e r i o d . D i e t 1 promoted s i g n i f i c a n t l y (P<0.05) higher gain than d i e t s 2 and 4, but s i g n i f i c a n t l y lower gain than d i e t s 5 and 6 f o r a l l p e r i o d s . Average d a i l y feed consumption Feed consumption of r a t s on d i e t s 2 and 4 was s i g n i f i c a n t l y (P<0.05) lower than on d i e t s 1, 3 5 and 6 f o r each of the three weeks of t r i a l as w e l l as f o r the o v e r a l l t r i a l p e r i o d . There was no s i g n i f i c a n t d i f f e r e n c e i n feed consumption between d i e t s 1, 3, 5 and 6 during the second and t h i r d weeks of t r i a l nor d u r i n g the o v e r a l l t r i a l p e r i o d . However, during the f i r s t week of the t r i a l , feed consumption of the r a t s on d i e t 3 was s i g n i f i c a n t l y (P<0.05) higher than that of r a t s on d i e t s 1, 5 and 6. - 84 -Average feed c o n v e r s i o n e f f i c i e n c y (FCE) Feed c o n v e r s i o n e f f i c i e n c y of r a t s on d i e t 2 was s i g n i f i c -a n t l y (P<0.05) lower than t h a t on d i e t 4 d u r i n g the f i r s t and t h i r d week of the t r i a l , but the two d i e t s showed no s i g n i f i c a n t d i f f e r e n c e i n FCE d u r i n g e i t h e r the second week of the t r i a l or d u r i n g the o v e r a l l t r i a l p e r i o d . FCE on both d i e t s 2 and 4 was s i g n i f i c a n t l y lower (P<0.05) than on d i e t s 1, 3 5 and 6 d u r i n g each week of the t r i a l and du r i n g the o v e r a l l t r i a l p e r i o d . Feed c o n v e r s i o n e f f i c i e n c y of r a t s on d i e t 1 was s i g n i f i c -a n t l y (P<0.05) h i g h e r than on d i e t s 2 and 4 but s i g n i f i c a n t l y (P<0.05) lower than on d i e t s 3, 5 and 6 d u r i n g the second and t h i r d weeks of the t r i a l and d u r i n g the o v e r a l l t r i a l p e r i o d . FCE of r a t s on d i e t 5 was the same as t h a t on d i e t 6. The FCE of the r a t s on the p o s i t i v e c o n t r o l d i e t , d i e t 3, was s i g n i f i c a n t l y (P<0.05) hi g h e r than t h a t produced on d i e t s 5 and 6 d u r i n g the f i r s t week of the t r i a l as w e l l as d u r i n g the o v e r a l l t r i a l p e r i o d . However the FCE produced on the p o s i t i v e c o n t r o l d i e t d u r i n g the second week of the t r i a l was the same as t h a t produced on d i e t 5, and the same as FCE on d i e t s 5 and 6 d u r i n g the t h i r d week of the t r i a l . C arcass composition The r a t s on d i e t s 2 and 4 produced s i g n i f i c a n t l y (P<0.05) lower c a r c a s s dry weight, t o t a l c a r c a s s f a t , percentage crude p r o t e i n i n f a t - f r e e c a r c a s s , t o t a l c a r c a s s p r o t e i n and t o t a l TABLE IV. The e f f e c t o f d i e t a r y methionine p l u s c y s t i n e l e v e l s on some s e l e c t e d v a r i a b l e s of r a t c a r c a s s composition i n Rat T r i a l 1 D i e t No. SE Methionine + cystine (% DM) Carca s s measurements 45 23 60 23 • 1 45 60 of Mean Carcass dry weight (g) 53 .7 28 .1 61 .3 31 .4 54 .8 59 .4 2.14 % f a t i n dry c a r c a s s 37 .1 23 .3 20 .2 19 .8 24 .7 24 .6 1.01 T o t a l c a r c a s s f a t (g) 20 .1 6 .3 12 .3 6 .2 13 .6 15 .3 0.88 % crude p r o t e i n i n f a t - f r e e dry c a r c a s s 77 .4 75 .5 79 .3 75 .9 80 .1 79 .4 0.35 % crude p r o t e i n i n whole dry c a r c a s s 48 .2 57 .4 62 .8 60 .4 59 . 8 59 .2 0.81 T o t a l c a r c a s s p r o t e i n (g) 25 .6 16 . 2 38 . 9 19 .0 32 .9 35 .3 1.30 % ash i n f a t - f r e e c a r c a s s (DM) 15 . 5 17 . 8 13 . 9 17 . 4 14 . 3 14 .0 0 . 25 % ash i n whole c a r c a s s (DM) 9 .7 13 .5 10 .9 13 .9 10 .6 10 .5 0.27 T o t a l c a r c a s s ash (g) 4 . 9 3 .6 6 .4 4 .2 5 . 6 6 .1 0 .17 S t a t i s t i c a l significance of difference between diets  Newman-Keul1s F-Test Test 2 4 1 5 6 3 4 3 2 6 5 1 4 2 3 5 6 1 2 4 1 3 6 5 1 2 6 5 4 3 2 4 1 5 6 3 3 6 5 1 4 2 1 6 5 3 2 4 2 4 1 5 6 3 • F - t e s t s i g n i f i c a n t a t the 5% p r o b a b i l i t y l e v e l . 86 -TABLE V. Results of single degree of freedom contrasts of variables i n Tables III and IV. Comparisons of diets nos. 1V6 2V4 3V5 3V6 4V5 4V6 5V6 Average d a i l y body-weight Week 1 * NS * * * * * gain (g) Week 2 * NS NS NS * * NS Week 3 * NS NS NS * * NS Overall * NS * * * NS Average d a i l y feed Week 1 NS NS * * * NS consumption (g) Week 2 NS NS NS NS * * NS Week 3 • NS NS NS NS * * NS Overall NS NS NS NS * * NS Average feed conversion Week 1 * * * * * * * e f f i c i e n c y Week 2 * NS NS * * * NS Week 3 * * NS NS * * NS Overall * NS * * * * NS Carcass dry weight NS • NS * NS * * NS % f a t i n dry carcass * NS * * NS Total carcass f a t * NS NS NS * * NS % crude protein i n f a t - f r e e carcass * NS NS NS * * NS % crude protein i n whole carcass * NS NS * NS NS NS Total carcass protein * NS * * * * NS % ash i n f a t - f r e e carcass * NS NS NS * * NS % ash in whole carcass NS NS NS NS * NS Total carcass ash * * * NS * NS *P<0.05; NS not s i g n i f i c a n t (P>0.05). - 87 -c a r c a s s ash; s i g n i f i c a n t l y h i g h e r (P<0.05) percentage ash i n f a t - f r e e c a r c a s s and percentage ash i n the whole c a r c a s s than d i e t s 1, 3, 5 and 6. There was no s i g n i f i c a n t d i f f e r e n c e between d i e t s 2 and 4 and between d i e t s 5 and 6 i n any of the c a r c a s s parameters a n a l y z e d . D i e t 1 was s i g n i f i c a n t l y (P<0.05) d i f f e r e n t from d i e t s 3, 5 and 6 i n percentage f a t i n the dry c a r c a s s , t o t a l c a r c a s s f a t , per-centage crude p r o t e i n i n the whole dry c a r c a s s , t o t a l c a r c a s s p r o t e i n , percentage ash i n f a t - f r e e c a r c a s s and t o t a l c a r c a s s ash. Dis cu s s i on D i e t 2, c o n t a i n i n g very low l e v e l s of a l l the e s s e n t i a l amino a c i d s , produced very poor growth. S i m i l a r l y , d i e t 4 produced very poor growth even though a l l the e s s e n t i a l amino a c i d s , w i t h the e x c e p t i o n of methionine p l u s c y s t i n e , had been added to equal the NRC (1972) requirements f o r the growing r a t . In d i e t 4, the 0.23% DM methionine p l u s c y s t i n e l e v e l was the o n l y l i m i t i n g f a c t o r . It t h e r e f o r e can be concluded t h a t a methionine p l u s c y s t i n e l e v e l of 0.23% DM i n the d i e t does not promote o p t i m a l growth i n the growing r a t . T h i s i n t e r p r e t a t i o n , based on growth r a t e , was supported by c a r c a s s a n a l y s i s data w i t h the low crude p r o t e i n c o n t e n t , expressed as a percentage of f a t - f r e e dry c a r c a s s , i n d i c a t i n g a reduced muscle growth i n r e l a t i o n to s k e l e t a l growth. The f a c t that performance of the - 88 -rats on d i e t 2 was not s i g n i f i c a n t l y d i f f e r e n t from that on d i e t 4 indicated that 0.23% DM methionine plus cystine was extremely growth l i m i t i n g . This finding i s contrary to the report by Oestemer et al. (1970) who found that 0.227-0.279% DM d i e t a r y methionine plus cystine supported optimal growth i n 21 kg. pigs fed on opaque-2 corns and that the growth rate was not a l t e r e d by supplementing the d i e t s with 0.07, 0.14, 0.21 or 0.2 8% DM DL-methionine. Diet 1, containing only barley as a major component, contained only 0.45% DM methionine plus cystine. The rats on t h i s d i e t performed poorly, compared with those on d i e t 5, which had a 0.45% DM methionine plus cystine l e v e l and a l l other e s s e n t i a l amino acids supplemented to meet the NRC (1972) requirements for the growing r a t . I t follows that i n d i e t 1, some e s s e n t i a l amino acids other than methionine plus cystine were l i m i t i n g . These could probably be l y s i n e and threonine (Aw-Yong and Beames, 1975). Both growth and carcass parameters support the conclusion that r a t performance on d i e t 5, containing ,0.45% DM methionine plus cystine, was the same as that on d i e t 6, containing 0.60% DM methionine plus cystine. I t would appear from t h i s , that 0.45 and 0.60% DM dietary methionine plus cystine, i n the presence of optimal l e v e l s of other nutrients for growth, support the same rate of growth i n young rat s . This finding - 89 -means t h a t the NRC (1972) recommended methionine p l u s c y s t i n e l e v e l of 0.67% DM more than c o v e r s the r a t requirements f o r growth. Rats on both d i e t s 5 and 6, which produced growth r a t e s t h a t were not s i g n i f i c a n t l y d i f f e r e n t , had s i g n i f i c a n t l y (P<0.05) lower growth than r a t s on the p o s i t i v e c o n t r o l d i e t ( d i e t 3). However, c a r c a s s a n a l y s e s d i d not r e f l e c t these d i f f e r e n c e s . I t should be p o i n t e d out t h a t d i e t 6 had the same l e v e l of methionine p l u s c y s t i n e as the p o s i t i v e c o n t r o l d i e t . The major d i f f e r e n c e between these two d i e t s was i n p r o t e i n c o n t e n t ( d i e t 3, 17.10 g/100 g DM; d i e t 6,.10.30 g/100 g DM). A l s o , t h e r e was a small d i f f e r e n c e i n gross energy c o n t e n t . A l t h o u g h d i e t 3 c o n t a i n e d h i g h e r l e v e l s of many of the e s s e n t i a l amino a c i d s , t h i s would not e x p l a i n the d i f f e r e n c e i n FCE and growth r a t e i n the f i r s t week of the experiment as w e l l as d u r i n g the o v e r a l l p e r i o d i f the NRC (1972) requirement l e v e l s as p r o v i d e d i n d i e t 6 i n f a c t were adequate to meet requirements a n d . p r o v i d i n g no i n t e r a c t i o n s were r e d u c i n g amino a c i d a v a i l a b i l i t y . T h i s l a t t e r p o s s i b i l i t y , however, cannot be d i s c o u n t e d (Harper, 1964). I t i s a l s o p o s s i b l e t h a t the l e v e l of p r o t e i n , 10.3% DM i n d i e t 6 compared with 17.1% i n the p o s i t i v e c o n t r o l d i e t , was l i m i t i n g . T h i s l a s t p o s s i b i l i t y was f u r t h e r e x p l o r e d i n Rat T r i a l 2. - 90 -Conclusion The t r i a l has shown t h a t 0.23% DM d i e t a r y methionine p l u s c y s t i n e i s inadequate f o r s a t i s f a c t o r y growth i n young r a t s . The performance of r a t s g i v e n e i t h e r 0.45 or 0.60% DM d i e t a r y methionine p l u s c y s t i n e , i n the presence of o p t i m a l l e v e l s of a l l other n u t r i e n t s , c o u l d not be d i f f e r e n t i a t e d u s i n g growth and c a r c a s s parameters. I t i s suggested, t h e r e f o r e , t h a t a requirement of 0.67% DM methionine p l u s c y s t i n e as suggested by the NRC (1972) may be an o v e r e s t i m a t e . I t i s a l s o c o n s i d e r e d t h a t the l e v e l of p r o t e i n i n the p r e s e n t t r i a l should be i n v e s t i g a t e d , as the performance of r a t s on a l l d i e t s c o n t a i n i n g 10.3% DM crude p r o t e i n was poorer than t h a t of r a t s r e c e i v i n g the p o s i t i v e c o n t r o l d i e t which c o n t a i n e d 17.10% DM crude p r o t e i n . - 91 -RAT TRIAL 2 E f f e c t of two l e v e l s of. d i e t a r y p r o t e i n and two l e v e l s of  methionine p l u s c y s t i n e on the growth of weanling r a t s fed  b a r l e y - b a s e d d i e t s Introduction and aim In Rat T r i a l 1, i t was shown t h a t the growth r a t e , FCE and c a r c a s s c o m p o s i t i o n parameters c o u l d not d i f f e r e n t i a t e the growth of r a t s fed on 0.45 and 0.60% DM d i e t a r y methionine p l u s c y s t i n e . I t was a l s o shown t h a t the growth of r a t s on the e x p e r i m e n t a l d i e t s c o n t a i n i n g 0.23, 0.45, and 0.60% DM d i e t a r y methionine p l u s c y s t i n e was s i g n i f i c a n t l y lower than t h a t on a barley-soybean meal p o s i t i v e c o n t r o l d i e t . I t was suggested t h a t the l e v e l of crude p r o t e i n i n the e x p e r i m e n t a l d i e t s , 10.3% DM, c o u l d be l i m i t i n g . I t was c o n s i d e r e d d e s i r a b l e , t h e r e f o r e , to r a i s e the crude p r o t e i n l e v e l from 10.3% DM to the 12.0% DM l e v e l recommended by NRC (1972) f o r the growing r a t and to compare the two l e v e l s , both a t 0.45 and 0.60% DM d i e t a r y methionine p l u s c y s t i n e . Materials and methods Experimental d e s i g n There were e i g h t d i e t a r y t r e a t m e n t s w i t h each d i e t p r o v i d e d t o f i v e i n d i v i d u a l l y - f e d r a t s . B o t h " t he d i e t a r y t r e a t m e n t s and - 92 -r e p l i c a t e s were randomly d i s t r i b u t e d among 40 cages i n a room w i t h temperature maintained a t 27°C. Animals and cages F o r t y male r a t s (Woodlyn/Wistar s t r a i n , Woodlyn L a b o r a t o r i e s , Guelph, O n t a r i o ) , 23 days of age a t the s t a r t o f the 21-day f e e d i n g p e r i o d were used. The cages and procedures were the same as those i n Rat T r i a l 1. D i e t s B a r l e y and soybean meal were ground i n the same way as i n Rat T r i a l 1. The b a r l e y - b a s e d d i e t s (Table VI), which i n c l u d e d a p o s i t i v e c o n t r o l d i e t ( d i e t 4), were formulated as f o l l o w s : % crude % Methionine D i e t No. I n g r e d i e n t s p r o t e i n + c y s t i n e 1 B a r l e y 10.30 0.45 2 B a r l e y + s t a r c h + e s s e n t i a l amino a c i d s (EAA) to equal 1 + n o n - e s s e n t i a l amino a c i d s (NEAA) 10.30 0.45 3 B a r l e y + EAA to equal 1 + NEAA 12.00 0.45 4 B a r l e y + soybean meal ( p o s i t i v e c o n t r o l ) 17.10 0.60 5 B a r l e y + EAA to equal NRC (1972) requirements except f o r meth-i o n i n e and c y s t i n e + NEAA 10 . 3 0 0.45 6 B a r l e y + EAA as i n 5 + NEAA 1 2 . 0 0 0.45 7 B a r l e y + EAA as i n 5 + NEAA + methionine 1 0 . 3 0 0 . 6 0 8 B a r l e y + EAA as i n 5 + NEAA -f methionine 1 2 . 0 0 0 . 6 0 TABLE V I . C o m p o s i t i o n (% DM) o f d i e t s i n Rat T r i a l 2 D i e t No. I n g r e d i e n t s and C o m p o s i t i o n 1 2 3 4 5 6 7 8 B a r l e y 96.82 88.71 88.71 77.49 80 . 21 80. 21 80.21 80.21 Soybean meal - - - 19.37 - - - -S t a r c h - 7.24 6.54 - 14.84 13.14 14.77 12.94 D e f l u o r i n a t e d r o c k phosphate* 1.64 1.64 1.64 1. 52 1.64 1.64 1.64 1.64 C a l c i u m carbonate" 1" .54 .54 . 54 .57 .54 .54 .54 . 54 I o d i z e d s a l t + .50 .50 .50 . 50 .50 . 50 .50 . 50 T r a c e m i n e r a l + V i t a m i n p r e m i x § . 50 . 50 . 50 . 50 . 50 .50 .50 . 50 Threonine"1""1" - .04 .04 - .17 .17 .17 .17 V a l i n e " - .04 .04 - .17 .17 . 17 . 17 I s o l e u c i n e " - .04 . 04 - . 22 .22 .22 .22 L e u c i n e " - . 07 . 07 - .13 .13 .13 .13 P h e n y l a l a n i n e + t y r o s i n e " - . 08 ' . 08 - .04 .04 . 04 . 04 H i s t i d i n e " - . 02 . 02 - .11 .11 .11 . 11 L y s i n e " - . 04 .04 - .58 . 58 . 58 .58 A r g i n i n e " - . 04 .04 - .19 .19 .19 . 19 '1.' ryptophnn" - . 01 . 01 - - - - -M e t h i o n i n e + C y s t i n e " - .04 .04 - . 08 .08 . 23 . 23 A l a -I- Asp + G l y + C l u (1:1:1:1)** - .45 2 .15 - . 08 1.78 — 1.6 3 Crude p r o t e i n 10 . 30 10.30 12.00 17 .10 10 . 30 12. 00 10 . 30 12. 00 Gl:: ( k c a l / k g ) 3624.0 3625.7 3625.4 3803.9 3625.7 3625.7 3625.7 3625.7 .For footnotes, see Table I, Rat T r i a l 1 TABLE VII. Amino acid composition (%DM) of diets for Rat T r i a l 2 Diet No. — ( T h r e o n i n e 0.40 0.40 0.40 0.69 0.57 0.57 0.57 0.57 V a l i n e 0.51 0.51 0.51 0.82 0.60 0.60 0.60 0.60 I s o l e u c i n e L e u c i n e 0.40 0.40 0.40 0.72 0.55 0.55 0.55 0.55 0.75 0.75 0.75 1.27 0.75 0.75 0.75 0.75 vo P h e n y l a l a n i n e + T y r o s i n e 0.92 0.92 0.92 1.43 0.80 0.80 0.80 0.80 l i s t i d i n e 0.23 0.23 0.23 0.43 0.30 0.30 0.30 0.30 •Lysine A r g i n i n e T r y p t o p h a n 0.39 0.39 0.39 0.90 0.90 0.90 0.90 0.90 0.49 0.49 0.49 0.97 0.60 0.60 0.60 0.60 0.18 0.18 0.18 0.29 0.17 0.17 0.17 0.17 M e t h i o n i n e + C y s t i n e 0.45 0.45 0.45 0.60 0.45 0.45 0.60 0.60 iO.'b 17 J '0,1 1^0 'O.h U-- 95 -A l l the d i e t s contained minerals and vitamins at levels recommended by NRC (1972) for growing r a t s . Table VII shows the amino acid composition of the d i e t s . The procedures for feeding, data recording and carcass preparation a f t e r slaughter have already been outlined i n Rat T r i a l 1. A n a l y t i c a l methods Methods used in the analysis of both barley and soybean meal for amino acid content, and i n the preparation of carcasses and chemical analyses, were the same as those outlined i n Rat T r i a l 1. S t a t i s t i c a l procedures The same UBC:MFAV programme by Halm and Lee (1975) that was used i n Rat T r i a l 1, was used for the analysis of data in the present t r i a l . Results Table VIII gives the results of the average d a i l y body-weight gain, feed consumption and feed conversion e f f i c i e n c y of rats on each d i e t . Carcass composition r e s u l t s are presented in Table 9. The relevant contrasts between diets presented i n Tables 8 and 9 are l i s t e d in Table 10. TABLE V I I I . Average d a i l y body-weight gain (g), food consumption (g) and food conversion e f f i c i e n c y (g weight gain/g food consumed) of rats on diets i n Rat T r i a l 2 Diet No, Crude Protein 10.30 10r30 12.00 17.10 10.30 12.00 10.30 12.00 Methionine + Cystine 0.45 0.45 0.45 0.60 0.45 0.45 0.60 0.60 (%DM) I n i t i a l weight 69.8 63.0 69.8 70.2 71.2 64.8 68.4 64.4 SE of Mean 1.03 Statistical significance of difference between diets  F-Test Newman Keul's Test NS Average d a i l y body-weight gain (g) Average d a i l y food consumption (g) Average food conversion e f f i c i e n c y (q gain/g feed consumed Week 1 2. 45 2. 24 2.07 5.43 3.43 4.06 4.63 3.83 0. 21 * Week 2 3. 05 3. 05 3.64 6.44 4.62 4.44 5.24 5.04 0. 20 * Week 3 4. 03 2. 84 4.23 6.04 5.05 5.04 5.24 4.83 0. 19 * Overall 3. 23 2. 65 2.87 5.85 4.25 4.82 5.05 4.63 0. 19 * Week 1 11. 04 10. 05 10.86 11.61 10.86 11.64 12.44 10.65 0. 24 NS Week 2 15. 42 14. 24 15.46 17.04 15.43 16.45 17.82 16.05 0. 30 NS Week 3 18. 83 15. 85 19.24 19.28 19.24 19.05 20.02 19.44 0. 33 NS Overall 15. 04 13. 46 14.45 16.25 15.06 15.83 16.65 15.26 0. 27 NS Week 1 0. 26 0. 25 0.24 0.47 0.33 0.39 0.39 0.38 0. 02 * Week 2 0. 22 0. 23 0.26 0.39 0.30 0.29 0.32 0.33 0. 01 * Week 3 0. 22 0. 20 0.24 0.32 0.28 0.27 0.28 0.26 0. .01 * Overall 0.23 0.22 0.23 0.38 0.30 0.31 0.32 0.31 0.01 3 2 1 5 8 6 7 4 2 1 3 6 5 8 7 4 2 1 3 8 6 5 7 4 2 3 1 5 8 6 7 4 3 2 1 5 8 6 7 4 1 2 3 6 5 7 8 4 2 1 3 8 6 7 5 4 2 3 1 5 6 8 7 4 T'-fl.O'i; NS n o t s i g n i f i c a n t - 97 -Average d a i l y body-weight g a i n During the f i r s t week of the t r i a l , there was no s i g n i f i c a n t d i f f e r e n c e i n average d a i l y body-weight g a i n between d i e t s 1,2 and 3 (Table V I I I ) . However g a i n on d i e t s 1, 2 and 3 was s i g n i f i c a n t l y (P<0.05) lower than g a i n on the other d i e t s , whose e s s e n t i a l amino a c i d content, except f o r methionine p l u s c y s t i n e , met the NRC (1972) requirements f o r growing r a t s . There were no s i g n i f i c a n t d i f f e r e n c e s between d i e t s 5, 8 and 6 nor between d i e t s 8, 6 and 7 (Table V I I I ) . The g a i n on the p o s i t i v e b a r l e y -soybean meal c o n t r o l d i e t ( d i e t 4) was the same as t h a t on d i e t 7, but s i g n i f i c a n t l y (P<0.05) g r e a t e r than the g a i n on a l l the o t h e r d i e t s . There was no d i f f e r e n c e between d i e t s 2 and 3; d i e t s 5 and 6; 7 and 8; i . e . both the 10.30 and 12.00% p r o t e i n d i e t s produced the same r a t e of g a i n d u r i n g the f i r s t week of the t r i a l (Table X ) . R e s u l t s f o r body-weight g a i n d u r i n g the second week of the t r i a l f o l l o w e d the same p a t t e r n as d u r i n g the f i r s t week except t h a t g a i n on the p o s i t i v e c o n t r o l d i e t ( d i e t 4) was s i g n i f i c a n t l y (P<0.05) g r e a t e r than on d i e t 7 and t h a t the d i f f e r e n c e between gai n s on d i e t s 3 and 6 were markedly reduced and were not s i g n i f i c a n t l y d i f f e r e n t . D uring the t h i r d week of t h e . t r i a l , the r a t e s of g a i n on d i e t s 5, 6, 7 and 8 were the same. Gain on d i e t 8 was s i g n i f i c a n t l y (P<0.05) lower than on the p o s i t i v e c o n t r o l d i e t - 93 -( d i e t 4). However g a i n s on d i e t s 4, 5, 6 and 7 were the same (Table V I I I ) . The average d a i l y body-weight g a i n on d i e t 2 was s i g n i f i c a n t l y (P<0.05) lower than on a l l the other d i e t s . For the o v e r a l l t r i a l p e r i o d , g a i n on d i e t s 1, 2 and 3 was the same. Gain on the p o s i t i v e c o n t r o l d i e t ( d i e t 4) was the same as t h a t on d i e t 7 c o n t a i n i n g 0.60% DM methionine p l u s c y s t i n e w i t h a crude p r o t e i n content of 10.30% DM, but was s i g n i f i c a n t l y (P<0.05) g r e a t e r than t h a t on d i e t s 5, 6 and 8. Average d a i l y feed consumption There was no d i f f e r e n c e between any of the treatments i n the average d a i l y feed consumption f o r any of the t h r e e t r i a l weeks or f o r the o v e r a l l t r i a l p e r i o d . However i n t a k e of r a t s on d i e t 2 was c o n s i s t e n t l y , although not s i g n i f i c a n t l y , lower than the i n t a k e on the other d i e t s . Average feed c o n v e r s i o n e f f i c i e n c y (FCE) During the f i r s t week of the t r i a l , r a t s on d i e t s 1, 2, 3, 5 and 8 produced s i g n i f i c a n t l y (P<0.05) lower FCE v a l u e s than those on the p o s i t i v e c o n t r o l d i e t ( d i e t 4). The r a t s on d i e t s 6 and 7 had the same FCE valu e s as those on the p o s i t i v e c o n t r o l d i e t . D i f f e r e n c e s i n FCE v a l u e s of r a t s on d i e t s 5, 6, 7 and"8 were not s i g n i f i c a n t (Table V I I I ) . During the second week of the t r i a l , the p o s i t i v e c o n t r o l d i e t ( d i e t 4) p r o v i d e d s i g n i f i c a n t l y (P<0.05) h i g h e r FCE v a l u e s than any other d i e t . There was no s i g n i f i c a n t d i f f e r e n c e between FCE v a l u e s o b t a i n e d on d i e t s 5, 6, 7 and 8 (Table X), nor between d i e t s 1, 2, 3 and d i e t s 5 and 6 u s i n g the s i n g l e decree of freedom comparisons. During the t h i r d week of the t r i a l , the FCE v a l u e s of r a t s on' d i e t 4 were the same as those on d i e t s 5, 6 and 7. There was no d i f f e r e n c e i n FCE v a l u e s of r a t s on d i e t s 5, 6, 7 and 8; n e i t h e r were t h e r e d i f f e r e n c e s between FCE v a l u e s produced on d i e t s 1, 2 and 3. For the o v e r a l l t r i a l p e r i o d , the p o s i t i v e c o n t r o l d i e t ( d i e t 4) r e s u l t e d i n FCE v a l u e s t h a t were s i g n i f i c a n t l y (P<0.05) g r e a t e r than any o b t a i n e d on the ot h e r d i e t s (Tables V I I I and X) . The FCE v a l u e s produced by r a t s on d i e t s 1, 2 and 3 were the same, and were s i g n i f i c a n t l y (P<0.05) lower than on d i e t s 5, 6, 7 and 8. The FCE v a l u e s produced by r a t s on d i e t s 5, 6, 7 and 8 were the same. Carca s s composition 'Percentage ash, t o t a l ash and t o t a l f a t i n the c a r c a s s showed no s i g n i f i c a n t d i f f e r e n c e s between treatments. Dry carcas weight of r a t s on d i e t 2 was s i g n i f i c a n t l y (P<0.05) lower than t h a t produced on any of the other d i e t s (Table IX). Dry c a r c a s s weight of the r a t s on the p o s i t i v e c o n t r o l d i e t (diet 4) was TABLE IX. E f f e c t o f d i e t a r y p r o t e i n l e v e l s and d i e t a r y methionine plu s c y s t i n e l e v e l s on r a t c a r c a s s composition i n Rat T r i a l 2 Diet No. SE of Mean S t a t i s t i c a l significance of difference between diets  F-Test Newman Keul's Test Carcass dry weight (g) 41. 45 36. 26 42. 47 51. 04 46 .24 46. 05 49 .64 44. 86 0 .95 % fat i n dry carcass 34. 83 33. 24 37. 64 21. 44 29 .24 29. 67 29 .03 29. 65 0 .94 Total carcass fat (g) 14. 64 12. 04 16. 05 10. 66 13 .26 13. 84 14 .07 13. 07 0 .47 % crude protein i n fat-free carcass (DM) 78. 84 79. 46 79. 23 83. 43 81 .04 81. 85 82 .85 82. 23 0 .39 % crude protein i n whole carcass (DM) 51. 03 52. 27 48. 67 64. 86 57 .03 56. 85 58 .44 57. 24 0 .89 Total dry carcass protein (g) 20. 86 19. 05 20. 84 33. 06 26 .43 26. 06 29 .22 25. 43 0 .79 % ash i n fat-free carcass (DM) 15. 65 16. 05 15. 65 12. 63 14 .64 15. 73 14 .74 14. 55 0 .21 % ash i n whole carcass (DM) 10. 05 10. 64 9. 64 9. 65 10 .24 10. 23 9 .65 9. 45 0 .14 Total carcass ash (g) 3. 86 3. 64 3. 85 4. 84 4 .45 4. 45 4 .46 3. .87 0 .10 2 1 3 8 6 5 7 4 NS NS NS 4 7 5 8 6 2 1 3 1 3 2 5 6 8 7 4 3 1 2 6 5 8 7 4 2 3 1 8 6 5 7 4 4 8 5 7 3 1 6 2 *P<0.05; NS not significant (P>0.05) TABLE X. Results of contrasts of variables presented i n Tables VIII and IX lv2 lv3 lv4 2v3 3v5 3v6 4v5 4v6 4v7 4v8 5v6 5v7 5v8 6v7 6v8 7v8 Average Week 1 NS NS * NS * * * * NS * NS * NS NS NS NS daily Week 2 NS NS * NS * NS * * * * NS NS NS NS NS NS body weight Week 3 * NS * * NS NS NS NS NS * NS NS NS NS NS NS gain Overall NS NS * NS * * * * NS * NS NS NS NS NS NS Average Week 1 NS NS * NS NS * * NS NS * NS NS NS NS NS NS food Week 2 NS NS * NS NS NS * * * * NS NS NS NS NS NS conversion efficiency Week 3 NS NS * NS * NS NS * * * NS NS NS NS NS NS Overall NS NS * NS * * * * * * NS NS NS NS NS NS Carcass dry weight * NS * * NS NS NS * NS * NS NS NS NS NS NS % fat in dry carcass NS NS * NS * * * * * * NS NS NS NS NS NS % crude protein in fat-free carcass NS NS * NS NS * NS NS NS NS NS NS NS NS NS NS % crude protein in whole carcass NS NS * NS * * * * * * NS NS NS NS NS NS Total carcass protein NS NS * NS * * * * * * NS * NS * NS % ash in fat--free carcass NS NS * NS NS NS * * * * NS NS NS NS NS NS *P<0.05; NS not significant (P>0.05) - 102 -s i g n i f i c a n t l y (P<0.05) g r e a t e r than t h a t of r a t s on d i e t s 1, 2 and 3 but the same as t h a t produced by r a t s on d i e t s 5, 6, 7 and 8. The percentage crude p r o t e i n i n the f a t - f r e e c a r c a s s was s i g n i f i c a n t l y (P<0.05) higher i n r a t s on the p o s i t i v e c o n t r o l d i e t ( d i e t 4) than on d i e t s 1, 2 and 3. Rats on d i e t 1 co n t a i n e d a s i g n i f i c a n t l y (P<0.05) lower percentage crude p r o t e i n i n the f a t - f r e e c a r c a s s than r a t s on d i e t s 4 and 7. Values on d i e t s 2, 3, 4, 5, 6, 7 and 8 (Table X) showed no s i g n i f i c a n t d i f f e r e n c e s . The percentage crude p r o t e i n i n the whole c a r c a s s of r a t s on the p o s i t i v e c o n t r o l d i e t ( d i e t 4) was s i g n i f i c a n t l y (P<0.05) highe r than t h a t i n c a r c a s s e s of r a t s on a l l the other d i e t s . The percentages f o r d i e t s 1 and 3 were s i g n i f i c a n t l y (P<0.05) lower than f o r d i e t s 4 and 7 w h i l e t h e r e were no s i g n i f i c a n t d i f f e r e n c e s between d i e t s 2, 5, 6, 7 and 8 i n the percentage crude p r o t e i n i n the whole c a r c a s s . The t o t a l c a r c a s s p r o t e i n o f r a t s on d i e t s 1, 2 and 3 d i d not d i f f e r s i g n i f i c a n t l y but a l l were s i g n i f i c a n t l y (P<0.05) lower than on d i e t s 4, 5, 6, 7 and 8 (Tables IX and X). Carcass p r o t e i n of r a t s on d i e t 8 was s i g n i f i c a n t l y (P<0.05) lower than t h a t of r a t s on d i e t 7. However, t o t a l c a r c a s s p r o t e i n of r a t s on d i e t 4 ( p o s i t i v e c o n t r o l d i e t ) was s i g n i f i c a n t l y (P<0.05) higher than t h a t produced on any of the o t h e r d i e t s . The percentage ash i n the f a t - f r e e - 103 -carcass of rats on d i e t 4 was s i g n i f i c a n t l y (P<0.05) lower than on a l l the other d i e t s . Discussion In almost a l l the parameters used to assess the differences between rats fed on the various d i e t s , i t was evident that, providing a l l other nutrients were the same, addition of non-e s s e n t i a l amino acids to increase the protein content of the diets from 10.30 to 12.00% did not r e s u l t i n any differences i n growth. However, i t should be pointed out that the presence of optimal l e v e l s , for growth, of a l l the other e s s e n t i a l amino acids, as i n the comparison between diets 2 and 5, improved growth rate s i g n i f i c a n t l y . The lack of response to the increase in t o t a l protein l e v e l beyond the 10.30% (DM basis) indicated t h i s l e v e l to be adequate when a l l amino acids were present at the NRC (1972) requirement l e v e l s for growing r a t s . Since the r e s u l t s are unable to demonstrate a s i g n i f i c a n t difference i n rate of gain or FCE on diets 1, 2 and 3, i t could be infe r r e d that amino acid a v a i l a b i l i t y on these three diets was also s i m i l a r , i . e . synthetic amino acids i n d i e t 2 were of equal value to those provided completely by the barley i n di e t 1. This picture for diets containing amino acid l e v e l s lower than those recommended by NRC (1972) did not apply when diets met these nutrient requirements, with feed e f f i c i e n c y for the whole experimental period being s i g n i f i c a n t l y lower on diets 7 and 8 that on the barley-soybean meal p o s i t i v e c o n t r o l d i e t . T h i s would i n d i c a t e a m o d i f i c a t i o n i n amino a c i d a b s o r p t i o n when the h i g h e r l e v e l s of s y n t h e t i c amino a c i d s are added to meet the NRC (1972) requirements f o r growing r a t s . Since the p o s i t i v e c o n t r o l d i e t was not composed of f r e e amino a c i d s , i t c o u l d be p o s s i b l e t h a t p e p t i d e a b s o r p t i o n i n t h i s d i e t a l l e v i a t e d i n t e r -a c t i o n s between f r e e amino a c i d s (Matthews et al .•, 1973), thereby promoting a h i g h e r r a t e o f growth. R e s u l t s i n t h i s t r i a l a l s o i n d i c a t e d t h a t none of the parameters used c o u l d d i s t i n g u i s h between the r a t e of growth of weanling r a t s on d i e t s c o n t a i n i n g 0.45% (DM b a s i s ) ( d i e t s 5 and 6) and d i e t s c o n t a i n i n g 0.60% (DM b a s i s ) ( d i e t s 7 and 8) methionine p l u s c y s t i n e . These r e s u l t s are i n agreement w i t h those o b t a i n e d i n Rat T r i a l 1, but have the added advantage i n t h a t the same r e s u l t s were o b t a i n e d a t both the 10.30% and the 12.00% DM d i e t a r y p r o t e i n l e v e l s . As was argued i n Rat T r i a l 1, t h i s l a c k of d i s t i n c t i o n between the two l e v e l s of methionine p l u s c y s t i n e q u e s t i o n s the v a l i d i t y of the NRC (1972) recommended l e v e l of 0.60% methionine p l u s c y s t i n e (90% DM b a s i s ) or 0.67% (DM b a s i s ) as the o p t i m a l l e v e l f o r growth i n r a t s . There i s a need, t h e r e f o r e , to f i n d o t h e r parameters f o r d e t e r m i n i n g the d i e t a r y methionine p l u s c y s t i n e o p t i m a l r e q u i r e -ments f o r growth i n r a t s . These new parameters, used i n c o n j u n c t i o n w i t h d i e t s c o n t a i n i n g s m a l l i n c r e m e n t a l i n c r e a s e s i n - 105 -the l e v e l of methionine p l u s c y s t i n e , perhaps c o u l d help i n d e f i n i n g , the o p t i m a l requirements f o r these amino a c i d s i n growing r a t s . Conclusi on For a l l the d i e t a r y f o r m u l a t i o n s , a 10.30% (DM b a s i s ) p r o t e i n l e v e l promoted a r a t e of growth s i m i l a r to t h a t obtained with 12.00% (DM b a s i s ) p r o t e i n l e v e l . Thus i t c o u l d be concluded t h a t the p r o t e i n was not l i m i t i n g i n Rat T r i a l 1. B a r l e y , d i l u t e d w i t h s t a r c h , and supplemented w i t h s y n t h e t i c amino a c i d s promoted the same r a t e of growth as a b a r l e y - o n l y d i e t when the e s s e n t i a l amino a c i d l e v e l s i n both d i e t s were equal, but lower than,the o p t i m a l l e v e l s recommended by NRC (1972). However, when the e s s e n t i a l amino a c i d s were i n c o r p o r a t e d i n t o d i e t s to the NRC (1972) recommended l e v e l s f o r o p t i m a l growth i n r a t s , the s y n t h e t i c amino acid-supplemented d i e t s promoted lower growth than a p o s i t i v e c o n t r o l d i e t based on b a r l e y and soybean meal. I t i s suggested t h a t f r e e amino a c i d i n t e r a c t i o n s may have c o n t r i b u t e d towards the lower growth r a t e i n the s y n t h e t i c amino acid-supplemented d i e t s . I t was shown t h a t 0.45% (DM b a s i s ) methionine p l u s c y s t i n e i n the d i e t promoted the same r a t e of growth as 0.60% (DM b a s i s ) methionine p l u s cystine-. I t was suggested, t h e r e f o r e , t h a t the NRC (1972) recommended l e v e l of 0.67% (DM b a s i s ) f o r growth i n r a t s , be s c r u t i n i z e d c l o s e l y u s i n g some parameters other than those used i n Rat T r i a l s 1 and 2. - 106 -RAT TRIAL 3 Growth and l i v e r enzyme response i n growing rats to graded  l e v e l s of methionine plus cystine i n f o r t i f i e d - b a r l e y diets 1. Response at constant cystine concentration i n the d i e t Introduction and aim I t was observed i n Rat T r i a l s 1 and 2 that the growth and carcass composition parameters could not d i f f e r e n t i a t e the growth of rats receiving barley-based diets containing 0.45 and 0.60% dry matter (DM) basis, methionine plus cystine. I t was suggested that some other parameters should be used to d i f f e r e n t i a t e these l e v e l s of dietary methionine plus cystine i n order to c l e a r l y define the optimal requirement range for the growth of the weanling r a t s . One such parameter could be the a c t i v i t i e s of some of the l i v e r enzymes that metabolize methionine, as shown i n Figure 1. The enzyme cystathionine synthase (CS) has been shown (Finkelstein, 1967) to catalyze the reaction that i s the only channel i n methionine metabolic pathway, leading to methionine catabolism. Cystathionine synthase a c t i v i t y has been observed (Finkelstein, 1967) to increase with increasing dietary methionine content but the pattern of t h i s increase has not been shown. The other enzymes are the transferases that convert homocysteine back to methionine. The a c t i v i t y of the f i r s t of these enzymes, - 107 -N 5 - m e t h y l t e t r a h y d r o f o l a t e - h o m o c y s t e i n e - m e t h y 1 t r a n s f e r a s e , (mTHF Enz.) has been observed to decrease w i t h i n c r e a s i n g d i e t a r y methionine content. The second enzyme, b e t a i n e -h o m o c y s t e i n e - m e t h y l t r a n s f e r a s e (BH E n z . ) . i s s a i d to be important f o r the c o n t r o l of methionine metabolism when the d i e t a r y meth-i o n i n e l e v e l i s h i g h because i t r e q u i r e s t h r e e molecules of homocysteine, and t h e r e f o r e methionine, i n order to regenerate one molecule o f methionine ( F i n k e l s t e i n et al. , 1974). However, F i n k e l s t e i n et al. argue t h a t at low and normal l e v e l s of d i e t a r y methionine c o n t e n t , mTHF Enz. i s more important f o r the c o n t r o l of methionine metabolism than BH Enz. Another p o s s i b l e parameter i s the u r i n a r y u r e a - n i t r o g e n e x c r e t i o n t h a t Brown and C l i n e (19 74) showed to be an i n d i c a t o r of amino a c i d requirements i n growing p i g s . The t h i r d r a t t r i a l was t h e r e f o r e designed to determine whether the i n f o r m a t i o n obtained from the a c t i v i t i e s o f c y s t a t h i o n i n e synthase and mTHF Enz., and the e x c r e t i o n o f urea n i t r o g e n c o u l d be used to e s t a b l i s h the o p t i m a l requirements of methionine p l u s c y s t i n e f o r the growth of weanling r a t s . Materials and methods Experimental d e s i g n The d e s i g n i n c o r p o r a t e d i n d i v i d u a l l y - h o u s e d r a t s per nine d i e t a r y treatments with four d i e t . Treatments and r e p l i c a t e s • - 108 -were randomly assigned. The temperature of the rat room was kept constant at 27°C. Animals and Cages T h i r t y - s i x male rats (Woodlyn/Wistar s t r a i n , Woodlyn Laboratories, Guelph, Ontario) 27 days of age at the s t a r t of the 21-day feeding period were used. The rats were randomly assigned to the s t a i n l e s s - s t e e l cages with wire screen f l o o r s of 12 mm spacing. Twelve cages were equipped with urine c o l l e c t i n g funnels while the others were f i t t e d with trays covered with paper towels beneath the wire screen. Rats were randomly allocated to the u r i n e - c o l l e c t i n g cages and urine was c o l l e c t e d from each r at for a t o t a l period of 4 days, with c o l l e c t i o n s t a r t i n g on the seventh day of the t r i a l . Diets The diets (Table I I ) were based on barley, prepared i n the same way as i n Rat T r i a l 1. The diets included a "positive c o n t r o l " (diet 1) which was prepared i n the same way as i n Rat T r i a l s 1 and 2. The eight other d i e t s were composed of barley and synthetic amino acids, with varying lev e l s of methionine. The basal d i e t provided (% DM) 0.2% cystine and 0.15% methionine. Cystine content was held constant i n a l l the eight amino acid-supplemented diets with methionine increments of 0.05% DM added to a maximum l e v e l of 0.70% DM methionine plus cystine i n diet 9. TABLE X I . C o m p o s i t i o n (% DM) o f r a t d i e t s w i t h c o n s t a n t c y s t i n e c o n t e n t o f 0.2% DM i n R a t T r i a l 3 D i e t No. M e t h i o n i n e + C y s t i n e * 0.60 0.35 0.40 0.45 0.50 0.55 0.60 0.65 0.70 B a r l e y 77.49 76.06 76.06 76.06 76.06 76.06 76.06 76.06 76.06 S o y a - b e a n m e a l 19.37 - - - - - - - -M a i z e s t a r c h - 16.80 16.80 16.80 16.80 16.80 16.80 16.80 16.80 M e t h i o n i n e + - - 0.05 0.10 0.15 0.20 0.25 0.30 0.35 NEAA - 2.01 1.96 1.91 1.86 1.81 1.76 1.71 1.66 Threonine-!- - 0.26 0.26 0.26 0.26 0.26 0.26 0.26 0.26 V a l i n e 5 - 0.18 0.18 0.18 0.18 0.18 0.18 0.18 0.18 I s o l e u c i n e 5 - 0.24 0.24 0.24 0.24 0.24 0.24 0.24 0.24 L e u c i n e s - 0.17 0.17 0.17 0.17 0.17 0-17 0.17 0.17 P h e n y l a l a n i n e + t y r o s i n e 5 - 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 H i s t i d i n e 5 - 0.12 0.12 0.12 0.12 0.12 0.12 0.12 0.12 L y s i n e 5 - 0.60 0.60 0.60 0.60 0.60 0.60 0.60 0.60 A r g i n i n e § - 0.22 0.22 0.22 0.22 0.22 0.22 0.22 0.22 T r y p t o p h a n s - 0.03 0.03 0.03 0.03 0.03 0.03 0.03 0.03 D e f l u o r i n a t e d r o c k p h o s p h a t e " 1.52 1.64 1.64 1.64 1.64 1.64 1.64 1.64 1.64 C a l c i u m c a r b o n a t e 1 1 0.62 0.57 0.57 0.57 0.57 0.57 0.57 0.57 0.57 I o d i z e d s a l t * * 0.50 0.50 0.50 0.50 0.50 0.50 0.50 0.50 0.50 T r a c e m i n e r a l + V i t a m i n p r e m i x + + 0.50 0.50 0.50 0.50 0.50 0.50 0.50 0.50 0.50 NEAA N o n - e s s e n t i a l amino a c i d s were i n c o r p o r a t e d as a glutam i c a c i d (Abernathy & M i l l e r , 1965; Womack, dry d i e t . * E x p r e s s e d a s t o t a l amino a c i d s , not r e s i d u e s 1:1:1:1 mixture of a l a n i n e - a s p a r t i c a c i d - g l y c i n e -1969) t o b r i n g the crude p r o t e i n l e v e l t o 12.0% TABLE XI. Continued + Methionine was supplied as L-^nethionine (Ajinomoto Co., Tokyo, Japan) . ''' Incorporated according to Aw-Yong and Beames (1975) . § A l l amino acids were incorporated as L-isomers (A j inomoto Co.) to meet the (US) National Research Council (1972) recommendations. " Estimated to contain 30% calcium and 14% phosphorus. 11 Estimated to contain 40% Ca and 0% P. ** Estimated to provide 0.15 mg iodine/kg dry diet. ++ Provided (/kg dry diet) 44 mg manganese as MnSC^ .I^ O, 110 mg zinc as ZnSO4.7H.2O, 500 mg butylated hydroxy toluene, 20 ug cyanocobalamin, 2.9 mg riboflavin, 11 mg nicrotinic acid, 5 mg calcium pantothenate, 3.6 mg pyridoxine, 0.2 mg D-biotin, 925 ugm retinol, 10 ug ergocalciferol, 1 g choline chloride. TABLE X I I . Amino a c i d , p r o t e i n (% DM) and energy ( k c a l / k g ) c o n t e n t o f d i e t s f o r Rat T r i a l 3. D i e t No. 1 2 3 4 5 6 7 8 9 A r g i n i n e 0.97 0.60 0.60 0.60 0 .60 .0.60 0 . 60 0 .60 0 .60 H i s t i d i n e 0.43 0.30 0.30 0.30 0.30 0 .30 0 .30 0 .30 0.30 I s o l e u c i n e 0.72 0.55 0.55 0.55 0.55 0.55 0.55 0.55 0 . 55 L e u c i n e 1.27 0.75 0.75 0.75 0.75 0.75 0.75 0.75 0.75 L y s i n e 0.90 0.90 0.90 0.90 0.90 0.90 0.90 0.90 0.90 M e t h i o n i n e + c y s t i n e ( c y s t i n e 0.2% DM) 0.60 0.35 0.40 0.45 0.50 0.55 0.60 0.65 0.70 P h e n y l a l a n i n e + t y r o s i n e 1.43 0 .80 0.80 0.80 0.80 0.80 0.80 0 .80 0.80 T h r e o n i n e 0 .69 0. 57 0.57 0.57 0 . 57 0 . 57 0 . 57 0 .57 0 . 57 T r y p t o p h a n 0.29 0.17 0.17 0.17 0 .17 0.17 0.17 0.17 0 .17 V a l i n e 0.82 0. 68 0.68 0.68 0.68 0.68 0 .68 0. 68 0 .68 Crude P r o t e i n (% N x 6.25) 17.10 12.00 12.00 12.00 12.00 12.00 12.00 12.00 12.00 Gross energy ( k c a l / k g DM) 3804 3780 3780 3780 3780 3780 3780 3780 3780 - 112 -The d i e t s were i s o n i t r o g e n o u s at 12% crude p r o t e i n c ontent (% N x 6.25) and i s o c a l o r i c w i t h 3780 k c a l gross energy/kg DM d i e t , except f o r the " p o s i t i v e c o n t r o l " d i e t 1, which c o n t a i n e d 3804 k c a l / k g DM d i e t . Threonine was i n c o r p o r a t e d a t a l e v e l based on net requirements e s t i m a t e d by Aw-Yong and Beames (1975). The requirements of a l l o t h e r n u t r i e n t s were based on NRC (1972) recommended valu e s f o r the growing r a t . Amino a c i d s were added as the L-isomers (Ajinomoto Co., Tokyo, Japan) to meet these requirements. Table XII shows the amino a c i d composition of the d i e t s and of the d i e t a r y components. Food was p r o v i d e d ad l i b i t u m and water was renewed d a i l y . Body weights and feed i n t a k e s were recorded d a i l y . At the end of the 21-day f e e d i n g p e r i o d , the r a t s were f a s t e d f o r 18 hr. Each r a t was then stunned by a blow on the head, the l i v e r r a p i d l y removed, and a p o r t i o n s u b j e c t e d immediately to each of the two enzyme-extraction methods. A f t e r f l u s h i n g the i n t e s t i n a l t r a c t w i t h water to remove fe e d p a r t i c l e s and f a e c a l m a t e r i a l s , the c a r c a s s e s were s t o r e d a t -40°C u n t i l l a t e r c a r c a s s a n a l y s e s . A n a l y t i c a l methods C y s t a t h i o n i n e synthase was e x t r a c t e d from the l i v e r u s i n g the method of Kashiwamata and Greenberg (1970). A f t e r p r e c i p i t -a t i o n w i t h ammonium s u l p h a t e (40% s a t u r a t i o n ) the enzyme was d i a l y z e d a g a i n s t 0.1 M potassium phosphate b u f f e r , pH 7.5, and the r e s u l t i n g s o l u t i o n used f o r the enzyme assay. The p r o t e i n - 113 -content of the enzyme e x t r a c t was determined by the B i u r e t Method (Wooton, 197 4) . The assay medium i n c o r p o r a t e d copper s u l p h a t e to i n h i b i t c y s t a t h i o n a s e . Enzyme a c t i v i t y was monitored by d e t e r m i n i n g the a b s o r p t i o n of the product of c y s t a t h i o n i n e and n i n h y d r i n a t 455 nM wavelength (Stasar, G i l f o r d Instrument L a b o r a t o r i e s Inc., O b e r l i n , O h i o ) . The enzyme a c t i v i t y was expressed as n moles c y s t a t h i o n i n e formed per mg p r o t e i n per 60 minutes. N - m e t h y l t e t r a h y d r o f o l a t e - h o m o c y s t e i n e - m e t h y 1 t r a n s f e r a s e was e x t r a c t e d from the l i v e r by the method of Mudd et al. (1970). A f t e r c e n t r i f u g i n g a t 50,000 g f o r 120 minutes, the supernatant f r a c t i o n was d i a l y z e d f o r 3 hr a g a i n s t three changes of 0.1 M potassium phosphate b u f f e r , pH 7.5, c o n t a i n i n g 0.003 M 14 reduced g l u t a t h i o n e . The assay medium c o n t a i n e d 5-( C ) -m e t h y l t e t r a h y d r o f o l i c a c i d (Amersham Corp., O a k v i l l e , O n t a r i o ) . A f t e r paper chromatography i n n - b u t y l alcohol-ammonia-water (75:5:20, by volume, upper phase), the areas p o s i t i v e to n i n h y d r i n were removed, s o l u b i l i z e d and counted i n PCS l i q u i d -c o u n t i n g c o c k t a i l (Amersham Corp.) u s i n g a l i q u i d s c i n t i l l a t i o n counter (Isocap 300, N u c l e a r - C h i c a g o ) . The p r o t e i n content of the enzyme e x t r a c t was determined, by the B i u r e t Method (Wootton, 1974). The enzyme a c t i v i t y was expressed as n moles methionine formed per mg p r o t e i n per 60 minutes. U r i n a r y urea-nitrogen was determined using the method of Brcwn (1971). Results were expressed asg N/kg DM d i e t consumed. Carcass analyses, and analyses f o r amino acids i n the ba r l e y and the soybean meal were performed as o u t l i n e d i n Rat T r i a l 1. S t a t i s t i c a l procedures A l l the r e s u l t s were subjected to a n a l y s i s of vari a n c e and orthogonal c o n t r a s t s as o u t l i n e d i n the U n i v e r s i t y of B r i t i s h Columbia BMD:10V ( B j e r r i n g et al. , 1975). A l l d i f f e r e n c e s between means were t e s t e d a t the 5% p r o b a b i l i t y l e v e l , using the Newman-Keul 1s M u l t i p l e Range Test according to the programme of B j e r r i n g et al. (1975). Results The r a t e of growth, feed i n t a k e and feed conversion e f f i c i e n c y (FCE) r e s u l t s are presented i n Table XIII, carcass composition r e s u l t s i n Table XIV, and r e s u l t s of orthogonal c o n t r a s t s i n Table XV. Average d a i l y body-weight gai n The average i n i t i a l weight of the r a t s at the s t a r t of the t r i a l was 100.3+1.0 g wit h no s i g n i f i c a n t d i f f e r e n c e between treatment groups. At the end of the f i r s t week, the average d a i l y body-weight gain of r a t s on the p o s i t i v e c o n t r o l d i e t , d i e t 1, was s i g n i f i c a n t l y (P<0.05) higher than that of r a t s on TABLE X I I I . Average d a i l y body-weight g a i n (g), food consumption (g) and food c o n v e r s i o n e f f i c i e n c y (g weight gain/g food consumed) of r a t s f e d on diets''' c o n t a i n i n g v a r y i n g l e v e l s of methionine p l u s c y s t i n e i n Rat T r i a l 3 Diet No. St a t i s t i c a l significance of SE difference of between diets: Mean F test Methionine + cystine (% DM) Average daily body-weight gain (g) Average daily food consumption Food conversion efficiency ; 0. 60 0. 35 0. 40 0. 45 0. 50 0. 55 0. 60 0. 65 0. .70 Week 1 5. 13 2. 23 3. 58 3. 53 4. 62 4. 50 4. 28 3. 47 4 .48 0 .18 Week 2 5. 83 3. 85 5. 23 5. 35 5. 38 5. 45 5. 43 5. 18 5 .60 0 .15 Week 3 5. 65 4. 30 4. 78 5. 48 5. 60 5. 30 5. 95 4. 95 5 .73 0 .15 Overall 5. 55 3. 48 4. 50 4. 78 5. 35 5. 08 5. 23 4. 50 5 .30 0 .13 Week 1 15. 07 14. 25 15. 97 14. 45 15. 42 15. 82 14. 07 13. 72 14 .45 0 .23 Week 2 17. 65 15. 70 18. 17 16. 82 18. 40 17. 07 16. 17 15. 50 16 .65 0 .30 Week 3 19. 40 16. 47 17. 22 18. 75 19. 22 18. 57 18. 35 17. 00 18 .50 0 .33 Overall 17. 35 15. 47 17. 25 16. 80 17. 67 17. 17 16. 17 15. 42 16 .52 0 .26 Week 1 0. 34 0. .16 0. 22 0. .25 0. 30 0. 29 0. 30 0. .25 0 .31 0 .01 VJeek 2 0. 33 0. 25 0. 29 0. 32 0. 32 0. 32 0. 33 0. 34 0 .34 0 .01 Week 3 0. 29 0. 26 0. 27 0. 28 0. 29 0. ,29 0. 33 0. ,29 0 .31 0 .01 Overall 0. 32 0. 22 0. 26 0. .29 0. 31 0. 30 0. 32 0. ,30 0 .33 0 .01 NS * NS NS NS NS * NS * *P<0.05; MS not significant (P>0.05); t Methionine was supplied as L-methionine (Ajinomoto Co., Tokyo, Japan) - 1 1 6 -a l l the other d i e t s (Tables XIII and XV). The g a i n on d i e t s c o n t a i n i n g 0.40 and 0.45% DM methionine p l u s c y s t i n e was the same but the g a i n on both these d i e t s was s i g n i f i c a n t l y (P<0.05) lower than on the d i e t s c o n t a i n i n g 0.50-0.70% DM methionine plu s c y s t i n e . However, the or t h o g o n a l c o n t r a s t t a b l e shows t h a t g a i n on 0.45% DM methionine p l u s c y s t i n e was the same as t h a t on the 0.50% DM l e v e l . For the second week, o r t h o g o n a l c o n t r a s t s (Table XV) i n d i c -a ted t h a t 0.4 0% DM methionine p l u s c y s t i n e met requirements, v/hereas 0.45% DM l e v e l was r e q u i r e d f o r maximal growth i n the t h i r d week. For the o v e r a l l t r i a l p e r i o d , 0.40% DM methionine p l u s c y s t i n e again appeared adequate. Average d a i l y feed consumption There was no s i g n i f i c a n t d i f f e r e n c e between treatments i n the amount of feed consumed d u r i n g each of the f i r s t two weeks nor d u r i n g the o v e r a l l t r i a l p e r i o d . However, fe e d i n t a k e of r a t s on the p o s i t i v e c o n t r o l d i e t d u r i n g the t h i r d week was s i g n i f i c a n t l y (P<0.05) h i g h e r than on the d i e t s c o n t a i n i n g 0.35 and 0.40% DM methionine p l u s c y s t i n e . Average feed c o n v e r s i o n e f f i c i e n c y (FCE) During the f i r s t week of the t r i a l , FCE of r a t s on the p o s i t i v e c o n t r o l d i e t was s i g n i f i c a n t l y (P<0.05) h i g h e r than on a l l the experimental d i e t s . The FCE of r a t s on 0.35 and - 117 -0.40% DM methionine plus_ cystine diets was s i g n i f i c a n t l y (P<0.05). lower than that of rats on 0.45-0.70% DM methionine plus cystine d i e t s . The orthogonal contrasts, Table XV, also show that the FCE of rats receiving 0.40 and 0.45% DM methionine plus cystine diets was s i g n i f i c a n t l y (P<0.05) lower than that of rats on 0.50-0.70% DM l e v e l s . These differences p e r s i s t e d i n the second week of the t r i a l but the performance of rats on the 0.35% DM methionine plus cystine d i e t had improved to equal that of rats on the 0.40% DM l e v e l . During the t h i r d week of the t r i a l , the rats receiving 0.35 and 0.40% DM dietary methionine plus cystine produced an FCE that was s i g n i f i c a n t l y (P<0.05) lower than that of rats on 0.45-0.70% DM methionine plus cystine l e v e l s . For the o v e r a l l t r i a l period, the FCE of rats on 0.35% DM dietary methionine plus cystine was s i g n i f i c a n t l y (P<0.05) lower than that of rats on the 0.40% DM l e v e l . The lack of difference between the 0.40 and 0.45% DM dietary l e v e l but the greater and uniform FCE values of rats receiving 0.50-0.70% DM l e v e l s of methionine plus cystine indicated the requirements to be between 0.45 and 0.50% DM methionine plus cystine. The FCE values are plotted i n Figure 2. Values were tested for " f i t " i n the ecuations: v = ab X; y = a + —; and x 2 y = a + bx + cx where y i s the FCE (g gain/g feed consumed), x i s the dietary methionione plus cystine l e v e l (% DM) and a, I r Calculated from the equation Y = -0.095 + 1.271X - 0.98X':  \-~==\ Calculated from the equation Y = 0.4209 - 0.06501/X | | Observed values 1 % ] P o s i t i v e c o n t r o l value I I I > / / K i / / v. i 0. 35 0.40 0.45 0. 50 0.55 0 .60 0.65 0.70 D i e t a r y m e t h i o n i n e + c y s t i n e (% DM) 2 The e f f e c t o f v a r y i n g l e v e l s o f d i e t a r y m e t h i o n i n e p l u s c y s t i n e on t h e f e c o n v e r s i o n e f f i c i e n c y o f r a t s i n T r i a l 3. Each p o i n t d e p i c t s a mean and s t a n d a r d e r r o r o f f o u r r a t s p e r d i e t . - 119 -b and c are c o n s t a n t s . The f o l l o w i n g r e l a t i o n s h i p s were developed: y = 0.1749 (1.094) X (R 2 0.56; P<0.05) y = 0.4209 - ° ' 0 6 5 0 1 ( R 2 0.65; P<0.05) y = -0.095 + 1.271x - 0.98x 2 (R 2 0.66; P<0.05) The r e g r e s s i o n curves d e r i v e d from the l a t t e r two equations are presented i n F i g u r e 2. Carca s s composition V a r i a b l e s t h a t showed no s i g n i f i c a n t d i f f e r e n c e s between treatments, both by Newman-Keul's M u l t i p l e Range T e s t and by the o r t h o g o n a l c o n t r a s t s were: percentage crude p r o t e i n i n f a t - f r e e c a r c a s s , percentage crude p r o t e i n i n the whole c a r c a s s , percentage ash i n the f a t - f r e e c a r c a s s , percentage ash i n the whole c a r c a s s and the t o t a l c a r c a s s ash. Rats on 0.35% DM d i e t a r y methionine p l u s c y s t i n e had a s i g n i f i c a n t l y (P<0.05) lower f i n a l dry c a r c a s s weight than r a t s on a l l the other d i e t s . The percentage f a t of c a r c a s s e s of r a t s r e c e i v i n g the p o s i t i v e c o n t r o l d i e t was s i g n i f i c a n t l y (P<0.05) lower than t h a t of r a t s r e c e i v i n g the 0.35 and 0.4 0% DM d i e t a r y methionine p l u s c y s t i n e . The r a t s r e c e i v i n g 0.35 and 0.40% DM d i e t a r y methionine p l u s c y s t i n e c o n t a i n e d s i g n i f i c a n t l y (P<0.05) g r e a t e r f a t c o n c e n t r a t i o n than c a r c a s s e s of r a t s TABLE X I V . The e f f e c t o f v a r y i n g l e v e l s o f d i e t a r y m e t h i o n i n e p l u s c y s t i n e o n some s e l e c t e d v a r i a b l e s o f r a t c a r c a s s c o m p o s i t i o n i n R a t T r i a l 3 D i e t No. Methionine + c y s t i n e (% DM) 0.60 0.35 0.40 0.45 0.50 0.55 0.60 0.65 0.70 S t a t i s t i c a l s i g n i f i c a n c e o f SE d i f f e r e n c e of between d i e t s : Mean F t e s t Dry c a r c a s s weight (g) 50.17 41.78 52.83 46.72 54.85 51.35 53.08 46.86 52.30 1.23 NS Fat content o f dry ca r c a s s (%) 15.57 19.92 24.61 14.81 19.19 17.92 17.53 15.38 15.97 0.85 NS T o t a l c a r c a s s f a t (g) 7.84 8.34 11.81 6.94 10.50 9.13 9.46 7.21 8.38 0.46 NS T o t a l carcass p r o t e i n (Nitrogen x 6.25) (g) 33.91 26.88 32.94 31.68 36.04 33.17 37.79 32.43 35.32 0.86 NS NS not s i g n i f i c a n t (P>0.05); TABLE XV. R e s u l t s of o r t h o g o n a l c o n t r a s t s of v a r i a b l e s presented i n Tables X I I I and XIV Comparisons of d i e t nos. 1 'v 2-9 1 v 2&3 1 v 4-9 2 v 3 ,2&3 v 4-9 3 v 4 3&4 v 5-9 4 v Average Week 1 * * * * \ V * NS * NS d a i l y body Week 2 NS * NS * * NS NS NS weight Week 3 NS * NS NS * NS NS NS g a i n (g) O v e r a l l * * NS * * NS NS NS Average Week 1 NS NS NS NS NS NS NS NS d a i l y food Week 2 NS NS NS NS NS NS NS NS consumption Week 3 NS * NS NS NS NS NS NS (g) O v e r a l l NS NS NS NS NS NS NS NS Food conver-• Week 1 * * * * * NS * NS s i o n e f f i c - Week 2 NS * NS NS * NS * NS ie n c y (g wt Week 3 NS NS NS NS * NS NS NS gain/g food O v e r a l l * NS * * NS * NS consumed) Dry c a r c a s s weight (g) NS NS NS * NS NS NS NS Fat c o n c e n t r a t i o n of dry c a r c a s s (%) NS * NS NS * * NS NS T o t a l c a r c a s s f a t (g) NS NS NS NS NS * NS NS T o t a l c a r c a s s crude p r o t e i n (Nitrogen ;-: 6.25) (g) NS NS NS NS * NS NS NS *P<0.05; NS not s i g n i f i c a n t (P>0.05) - 122 -r e c e i v i n g 0.45-0.70% DM d i e t a r y methionine plu s c y s t i n e . The r e s u l t s f o r the t o t a l c a r c a s s p r o t e i n i n d i c a t e t h a t the r a t s r e c e i v i n g 0.35 and 0.40% DM d i e t a r y methionine p l u s c y s t i n e had s i g n i f i c a n t l y (P<0.05) lower t o t a l c a r c a s s p r o t e i n than those r e c e i v i n g d i e t s c o n t a i n i n g 0.45-0.70% DM methionine p l u s c y s t i n e . U r i n a r y u r e a - n i t r o g e n e x c r e t i o n F i g u r e 3, r e l a t i n g d i e t a r y methionine p l u s c y s t i n e to u r i n a r y u r e a - n i t r o g e n e x c r e t i o n , shows a sharp decrease from 2.78 g urea N/kg DM feed consumed a t 0.35% DM methionine p l u s c y s t i n e l e v e l to 1.40 g urea N/kg DM feed consumed a t 0.45% DM methionine p l u s c y s t i n e l e v e l . The r e d u c t i o n i n u r i n a r y urea e x r e t i o n then d i m i n i s h e s t o a slow l i n e a r decrease as the c o n c e n t r a t i o n of d i e t a r y methionine p l u s c y s t i n e i n c r e a s e s to the 0.70% DM l e v e l . The u r i n a r y u r e a - n i t r o g e n e x c r e t i o n v a l u e s were t e s t e d f o r x b 2 " f i t " i n the eq u a t i o n s : y = ab ; y = a + —; y = a + bx + cx , where y i s the u r i n a r y u r e a - n i t r o g e n e x c r e t i o n (g/kg DM feed consumed); x i s the d i e t a r y methionine plu s c y s t i n e l e v e l (% DM) and a, b, and c are c o n s t a n t s . The f o l l o w i n g r e l a t i o n s h i p s were developed: y = 4.087 (0.8 2 ) X (R 2 0.77; P<0.05) 0 9149 2 y = -0.3277 + v ' y ± ^ (R 0.73; P<0.05) y = 8.174 - 22.99x + 18.7x 2 (R 2 0.83; P<0.05) Although the polynomial curve provided the c l o s e s t f i t , e x p l a i n i n g 0 . 8 3 of the v a r i a n c e , i t d e v i a t e d c o n s i d e r a b l y from 2.8 2.5 I C 8 < 2.0 c o •H 4J CU u CJ c 81 nj 3 1.5 1.0 r-1 to Hh rri rii 0.35 0.40 0.45 0.50 0.55 0.60 D i e t a r y methionine + c y s t i n e (% DM) 0.65 0.70 F i g u r e 3. The e f f e c t of v a r y i n g l e v e l s of d i e t a r y methionine plus c y s t i n e on the u r i n a r y urea e x c r e t i o n by r a t s i n T r i a l 3. Each p o i n t d e p i c t s a mean and standard e r r o r f o r four r a t s per d i e t . - 124 -the observed pattern between the level s 0.6 0 and 0.7 0% DM dietary methionine plus cystine. Consequently neither i t nor any of the other curves were considered to be of any aid i n the i n t e r p r e t -ation of the r e s u l t s , and consequently were not plotted i n Figure 3. A c t i v i t i e s of the l i v e r enzymes The a c t i v i t y of l i v e r cystathionine synthase (Figure 4) shows an almost constant l e v e l of 24 u mole/mg protein/60 minutes between 0.35 and 0.50% DM dietary methionine plus cystine. The a c t i v i t y thereafter decreased, reaching an average minimum value of 16.12 u mole/mg protein/60 minutes at the 0.60% DM l e v e l , subsequently r i s i n g to an average l e v e l of 23.69 u mole/mg protein/ 6 0 minutes at the 0.7 0% DM methionine plus cystine l e v e l . The a c t i v i t y of mTHF Enz. (Figure 5) was constant at 0.64 n mole/mg protein/60 minutes (units) between the l e v e l s of 0.35 and 0.50% DM methionine plus cystine. It then increased to an average of 2.92 units at the 0.60% DM methionine plus cystine l e v e l , thereafter decreasing to 0.48 units at the 0.70% DM dietary methionine plus cystine. Discussion During the 3 weeks of the t r i a l , weight gain was lower on diets containing 0.35 and 0.40% DM than on the 0.45-0.70% DM Ml • fO n • rt o rh y-in c H fD M rt H fj 3* 3" O f0 (5 CD ro TJ O rh O rt H- P-3 < rt p-rt ft) TJ O P- rh < O B> rt M r( cn p-K < P-0) (5 3 M *a 3 ro o OJ K 3 tn rt o a H 3 rt cn a 3* P-cn O rt 3 0 P-3 3 Ci ro PJ M tn a > < 3 K rt 3- 3 0> fO in rt fO 3* P-rh O O O rh 3 rt P-l-t 3 rh CU fD O rt C tn TJ rt r-1 P- C 3 tn Cystathionine synthase a c t i v i t y (u rrole/mg protein/60 minutes) ro fD • O rh • a P-Q rt W r| fD i-l r( o ri ft r t co TJ ro rt CD ft •-a  rt ^< p- cn a n W P-3 U> O o 3 K> Ul o LH o o a P-fD rt 0) rt o 3 ro rt 3* P-0 3 P-3 o fD + o 0 <^ ' tn rt P-3 o ro Ul .—. Ul o s: — o CTl O O o a. p* a 3< - s z i -mTKF Enz. a c t i v i t y (n rrole/irg protein/60 minutes) c n ra 3 3 rt- M ro ro 3- i-n Cu r r ro H i rt O rt (5 P-3 rt I "O o c a 3 in ro o T J o o o «i 01 rt rt rt co ro p -P- 3 3 ro 1 ro O o Ul 3 D - ro 0 >< a> 0 ci H n ft 3 it rt 0 a. M P- 0 U < H i 11 3 F -rt 01 r t <J 0 cs H » > < 01 p -3 ro H ro a i-l 0 K rt Si fl) hn H- S) M C/> 3 G. ro P".Q >< P-ro O < M 3 1-1 H I ro ro ro 1-1 M < rr 0 1-1 ro 3* M CJ Z P- P-r t aicrt 0 0 l-ti C/l 1 3 • 0 - 3 0 P-1-1 p - ro Mi 3 3 r t ro Hi & 0 i-3 >< P- + c M H-1 ro 0 h P- rt r t cu ro w << h P- rt H. in 0 ai 1-! ^ rr Ul Ul r t ui ft) p -o • 3-3 3 K ro ro T) Di rt ro M M 3" —^  H P O P-O Ml 0 a D- 0 3 • 2 p . ^ H - 0 ro 13 01 3 . • o o I—I—I I — i — I 3-^  - 9 Z T -- 127 -d i e t a r y methionine p l u s c y s t i n e . The weight gained by r a t s r e c e i v i n g 0.45-0.70% DM methionine p l u s c y s t i n e was the same as t h a t on the p o s i t i v e c o n t r o l d i e t . I t should be noted t h a t as the r a t s matured, the weight g a i n on both the 0.35 and 0.40% DM d i e t a r y methionine p l u s c y s t i n e more c l o s e l y approached the g a i n o b t a i n e d on the other d i e t s , presumably as a r e s u l t of the r a p i d l y d e c r e a s i n g requirements w i t h i n c r e a s i n g age (Hartsook and M i t c h e l l , 1956). The FCE r e s u l t s i n d i c a t e d t h a t l e v e l s of 0.35 and 0.40% DM d i e t a r y methionine p l u s c y s t i n e were i n s u f f i c i e n t f o r the o p t i m a l growth of the r a t s . L i k e the weight g a i n r e s u l t s , there was no d i s t i n c t i o n between d i e t s w i t h i n the methionine p l u s c y s t i n e range of 0.45-0.70% DM. F i g u r e 2 shows a l e v e l l i n g o f f of the growth r a t e between 0.4 5 and 0.50% DM d i e t a r y methionine p l u s c y s t i n e although the mean va l u e s beyond the 0.50% DM l e v e l d i d not show a c o n s i s t e n t p a t t e r n . .The " f i t t i n g " o f the r e c i p r o c a l and p o l y n o m i a l equations o n l y p a r t i a l l y e x p l a i n e d the v a r i a n c e . The " f i t t i n g " of the curves d i d not a i d i n the i n t e r p r e t a t i o n of the r e s u l t s , e s p e c i a l l y beyond the 0.50% DM methionine p l u s c y s t i n e l e v e l where the FCE v a l u e s were e r r a t i c f o r no apparent reason. On the b a s i s of growth and FCE, r e s u l t s so f a r c o u l d be i n t e r p r e t e d to i n d i c a t e t h a t 0.45-0.50% DM methionine p l u s c y s t i n e i s adequate f o r o t p i m a l growth i n weanling r a t s . T h i s f i n d i n g would e x p l a i n why no d i f f e r e n c e s were o b t a i n e d , i n T r i a l s 1 and 2, between the growth r a t e s of r a t s on 0.45% DM - 128 -and those on 0.60% DM d i e t a r y methionine p l u s c y s t i n e . The range, 0.45-0.50% DM methionine p l u s c y s t i n e , found i n t h i s t r i a l , when expressed as 3.34-3.67 m moles t o t a l sulphur from methionine p l u s c y s t i n e per 100 g d i e t , i s i n agreement with v a l u e s r e p o r t e d by Sowers et al. (1972) and by Stockland et al. (197 3) but d i f f e r e n t from the requirements suggested by Womack and Rose (1941) and by the NRC (1970) who both g i v e the requirements of 0.67% DM methionine p l u s c y s t i n e , with c y s t i n e able to c o n t r i b u t e o n e - t h i r d t o h a l f the t o t a l sulphur i n t a k e from methionine p l u s c y s t i n e . R e s u l t s o b t a i n e d from the c a r c a s s a n a l y s e s c l e a r l y i n d i c a t e t h a t these c r i t e r i a cannot be used i n the assessment of methionine p l u s c y s t i n e requirements of the growing r a t , e s p e c i a l l y w i t h i n the range covered i n the p r e s e n t t r i a l . The few s i g n i f i c a n t d i f f e r e n c e s shown by the o r t h o g o n a l comparisons i n dry c a r c a s s weight, percentage f a t i n the c a r c a s s and t o t a l c a r c a s s p r o t e i n , although g i v i n g some support to the weight g a i n and FCE r e s u l t s , are of l i m i t e d u s e f u l n e s s as they o f t e n appear c o n t r a d i c t o r y . T h i s same l a c k of u s e f u l n e s s of c a r c a s s analyses was shown i n Rat T r i a l s 1 and 2 between the 0.45 and 0.50% DM d i e t a r y methionine p l u s c y s t i n e . U r i n a r y urea e x c r e t i o n r e s u l t s i n d i c a t e d t h a t the o p t i m a l d i e t a r y requirement of methionine p l u s c y s t i n e may be 0.45% DM or 3.34 n moles t o t a l sulphur per 100 g of d i e t from methionine p l u s c y s t i n e l e v e l s . However, the gradual decrease in - 129 -u r i n a r y urea e x c r e t i o n between 0.4 5 and 0.7 0% DM d i e t a r y methionine p l u s c y s t i n e i n d i c a t e s a p o s s i b l e c o n t i n u i n g improvement i n the t o t a l amino a c i d u t i l i z a t i o n . The u r i n a r y urea e x c r e t i o n r e s u l t s are i n agreement wi t h those of FCE whereby 0.45% DM d i e t a r y methionine p l u s c y s t i n e was the p o i n t where maximum change i n response o c c u r r e d . However, urea e x c r e t i o n has the added advantage of being a l e s s v a r i a b l e i n d i c a t o r beyond t h i s l e v e l . Other workers (Brown and C l i n e , 1974; F u l l e r et al. , 1975) a l s o have found u r i n a r y urea e x c r e t i o n to be a s e n s i t i v e i n d i c a t o r of amino a c i d requirements i n p i g s . R e s u l t s o b t a i n e d w i t h the enzymes i n d i c a t e d t h a t normal enzyme a c t i v i t y was d i s t u r b e d when d i e t a r y methionine p l u s c y s t i n e was i n c r e a s e d beyond the 0.50% DM l e v e l . C y s t a t h i o n i n e synthase a c t i v i t y i n h i b i t i o n s t a r t e d a t 0.50% DM methionine p l u s c y s t i n e and reached maximum i n h i b i t i o n a t the 0.60% DM dietary methionine p l u s c y s t i n e l e v e l . T h i s i n h i b i t i o n c o u l d have a r i s e n from the accumulation of methionine, N, N - d i m e t h y l g l y c i n e and p o s s i b l y s-adenosyl methionine, the immediate products of the r e m e t h y l a t i o n r e a c t i o n (Figure 1), or by c y s t a t h i o n i n e , the end product of the r e a c t i o n u t i l i z i n g t h i s enzyme. Accumulation of s-adenosyl-homocysteine (sAH) would not l i k e l y be the.cause of the i n h i b i t i o n s i n c e F i n k e l s t e i n et al. (1974) have observed t h a t sAH a c t i v a t e s c y s t a t h i o n i n e synthase at a i l c o n c e n t r a t i o n s of s u b s t r a t e s e r i n e and homocysteine. One other p o s s i b l e f a c t o r - 130 -which may have i n h i b i t e d the a c t i v i t y of c y s t a t h i o n i n e synthase i s the methionine to c y s t i n e r a t i o which was shown by Shannon et al. (1972) to a f f e c t the a c t i v i t y o f the enzyme. Shannon et al. (1972) observed maximum i n h i b i t i o n of the enzyme when c y s t i n e accounted f o r 50% or more of the t o t a l sulphur amino a c i d s i n the d i e t s , whereas i n the p r e s e n t t r i a l , maximum i n h i b i t i o n o c c u r r e d at the p o i n t where c y s t i n e accounted f o r 33% of the t o t a l sulphur amino a c i d s . To e l u c i d a t e t h i s p o i n t , i t would be d e s i r a b l e to r e p e a t t h i s t r i a l , but with the methionine t o c y s t i n e r a t i o h e l d c o n s t a n t . Such was done i n Rat T r i a l 4. The prese n t t r i a l a l s o showed a d e f i n i t e i n c r e a s e i n the a c t i v i t y of c y s t a t h i o n i n e synthase as the d i e t a r y l e v e l of methionine p l u s c y s t i n e i n c r e a s e d from 0.60 to 0.70% DM. I t i s u n l i k e l y t h a t t h i s i n c r e a s e was due to a c t i v a t i o n of the enzyme by s e r i n e s i n c e d i e t a r y s e r i n e was the same i n a l l the e x p e r i m e n t a l d i e t s . However, t h i s i n c r e a s e d a c t i v i t y c o u l d have been p a r t l y due to i n c r e a s e d c o n c e n t r a t i o n of the r e m e t h y l a t i o n products p r i o r t o the f o r m a t i o n of c y s t a t h i o n i n e . C y s t a t h i o n i n e synthase a c t i v i t y i n h i b i t i o n o c c u r r e d a t the same l e v e l of d i e t a r y methionine p l u s c y s t i n e where mTHF Enz. a c t i v i t y was i n c r e a s e d i . e . at 0.50% DM d i e t a r y methionine p l u s c y s t i n e . • From t h i s o b s e r v a t i o n i t may be suggested t h a t whatever product i n h i b i t e d c y s t a t h i o n i n e synthase a c t i v i t y must have a c t i v a t e d the r e m e t h y l a t i o n p r o c e s s v i a the one-carbon - 131 -met a b o l i c pathway i n which mTHF Enz. takes p a r t . When the d i e t a r y l e v e l of methionine p l u s c y s t i n e i n c r e a s e d from 0.60 to 0.70% DM, the a c t i v i t y of mTHF Enz. decreased, i n agreement w i t h o b s e r v a t i o n s by F i n k e l s t e i n et al. (1971). T h i s i n h i b i t i o n , p r o b a b l y , was due to product i n h i b i t i o n as suggested by Burke et al. (1971). Cone 1usion R e s u l t s from weight g a i n and FCE i n d i c a t e 0.45-0.50% DM d i e t a r y methionine p l u s c y s t i n e to be the op t i m a l range f o r growth of weanling r a t s . The same v a r i a b l e s show t h a t 0.35 and 0.40% DM d i e t a r y methionine p l u s c y s t i n e are unable to support o p t i m a l growth i n young r a t s . U r i n a r y u r e a - n i t r o g e n e x c r e t i o n r e s u l t s i n d i c a t e 0.42-0.47% DM d i e t a r y methionine p l u s c y s t i n e t o be the op t i m a l range f o r growth, although maximal u t i l i z a t i o n o f the added methionine was s t i l l improving s l o w l y , even at ,0.70% DM methionine p l u s c y s t i n e . Enzyme a c t i v i t i e s i n d i c a t e t h a t normal metabolism oc c u r s between 0.35 and 0.50% DM methionine p l u s c y s t i n e and t h a t beyond the 0.50% DM l e v e l , enzyme a c t i v i t y d i s t u r b a n c e s occur. A l l these r e s u l t s l e a d to the c o n c l u s i o n t h a t 0.4 5-0.50% DM d i e t a r y methionine plu s c y s t i n e i s the o p t i m a l range of requirements when c y s t i n e i s present at a l e v e l of 0.20% i n the d i e t . However, i t should be p o i n t e d out t h a t the gr o s s energy of the d i e t s used i n the present t r i a l , 3780 k c a l / k g DM feed, i s lower than t h a t of NRC (1972) of 44 00 k c a l / k g DM feed. The gross energy used i n the present - 132 -t r i a l , t h e r e f o r e , r e p r e s e n t s 86% of the recommended NRC (1972) requirements. Based on t h i s f i g u r e , the o p t i m a l requirements of methionine p l u s c y s t i n e f o r growth of weanling r a t s i n the p r e s e n t t r i a l would be 0.52-0.58% DM d i e t i f the energy l e v e l were comparable to the NRC (1972) l e v e l . T h i s r e s u l t i s s t i l l lower than the NRC (1972) recommended requirements of 0.67% DM methionine p l u s c y s t i n e f o r the growing r a t . - 133 -RAT TRIAL 4 Growth and l i v e r enzyme response i n growing r a t s to graded  l e v e l s of methionine p l u s c y s t i n e i n f o r t i f i e d b a r l e y - b a s e d  d i e t s 2. Response a t a con s t a n t m e t h i o n i n e : c y s t i n e r a t i o Introduction and aim Rat T r i a l 3 showed t h a t the o p t i m a l requirements f o r d i e t a r y methionine p l u s c y s t i n e i n weanling r a t s was 0.45-0.50% dry matter (DM) b a s i s when the l e v e l of c y s t i n e i n the d i e t s was h e l d c o n s t a n t a t 0.20% DM. The a c t i v i t i e s of l i v e r c y s t a t h i o n i n e 5 synthase and of N - m e t h y l t e t r a h y d r o f o l a t e - h o m o c y s t e m e -m e t h y l t r a n s f e r a s e (mTHF Enz.) were c o n s t a n t between the 0.35-0.50% DM d i e t a r y methionine p l u s c y s t i n e l e v e l s . The a c t i v i t i e s of both enzymes showed d i s t u r b a n c e s when the d i e t a r y methionine p l u s c y s t i n e l e v e l was i n c r e a s e d to l e v e l s h i g h e r than 0.50% DM. Byington et al. (1972) showed t h a t growth of young r a t s was o p t i m a l when the r a t i o of methionine to c y s t i n e was 70:30 but not when t h i s r a t i o was l e s s than 50:50. The methionine to c y s t i n e r a t i o i n the d i e t s g i v e n t o the r a t s i n T r i a l 3 was not c o n s t a n t , and i t has been shown t h a t c y s t a t h i o n i n e synthase i s i n h i b i t e d by low p r o p o r t i o n s of methionine to c y s t i n e ( F i n k e l s t e i n , 1967; Shannon et al. , 1972). I t was t h e r e f o r e - 134 -d e c i d e d e s s e n t i a l l y t o rep e a t Rat T r i a l 3 but w i t h a constant methionine to c y s t i n e r a t i o of 2:1, wh.ich i s c l o s e to the r a t i o recommended by Byington et al. (1972). Materials and methods E x p e r i m e n t a l d e s i g n The d e s i g n i n c o r p o r a t e d s i x d i e t a r y treatments w i t h f o u r i n d i v i d u a l l y - h o u s e d weanling r a t s per d i e t . The treatments and r e p l i c a t e s were randomly a s s i g n e d to i n d i v i d u a l cages. The temperature of the r a t room was kept constant a t 27°C. Animals and cages Twenty-four male r a t s (Woodlyn/Wistar s t r a i n , Woodlyn L a b o r a t o r i e s , Guelph, Ontario) 27 days of age a t the s t a r t of the 21-day f e e d i n g p e r i o d were used. The r a t s were randomly a s s i g n e d to the s t a i n l e s s - s t e e l cages as d e s c r i b e d i n Rat T r i a l 3. U r i n e c o l l e c t i o n was c a r r i e d out i n the same way as i n Rat T r i a l 3. D i e t s The d i e t s (Table XVI) were based on b a r l e y grain, prepared i n the same way as o u t l i n e d i n Rat T r i a l 1. D i e t s c o n t a i n e d a m e t h i o n i n e : c y s t i n e r a t i o o f 2:1. A minimum l e v e l of d i e t a r y methionine p l u s c y s t i n e of 0.45% DM ( d i e t 1) was chosen f o r TABLE XVI. Composition (% DM) of rat diets containing a constant methionine cystine r a t i o of 2:1 i n Rat T r i a l 4 Ingredients Diet No. Methionine 4" NEAA Barley Maize starch Threonine 1' Valine§ Isoleucine§ Leucine 5 Phenylalanine + t y r o s i n e 5 H i s t i d i n e 5 . Lysine 5 Arginine 5 Tryptophan 5 Defluorinated rock phosphate" Calcium carbonated Iodized s a l t * * Trace mineral + Vitamin premix++ 0.19 0.24 0.29 3.04 2.99 2.94 0.34 2.89 0.39 2.84 0.44 2.79 55.56] 35.14! - 0.34! - 0.38! - 0.32! - 0.32! - 0.27! - 0.17! - 0.68! - 0.32! - 0.06c - 1.64! - 0.57! - 0.50! - 0.50° For footnote, see Table 1, Rat T r i a l 1 Indicates a l e v e l constant i n a l l diets - 136 -t h i s t r i a l , because any f u r t h e r lowering v/ould have r e q u i r e d t h i s b a s a l d i e t and a l l other d i e t s t o be v i r t u a l l y completely s y n t h e t i c . T h i s would have been i n c o n f l i c t with the b a s i c aim of these experiments which was to t e s t r e l a t i o n s h i p s on b a r l e y -based d i e t s . • The d i e t s were i s o n i t r o g e n o u s a t 12% crude p r o t e i n and i s o c a l o r i c at 3780 k c a l / k g DM g r o s s energy. M i n e r a l s and v i t a m i n s were added to a l l the d i e t s to meet the NRC (1972) requirements f o r the growing r a t . Amino a c i d s were added as the L-isomers (Ajinomoto Co., Tokyo, Japan) to meet the NRC (1972) requirements f o r the growing r a t . Threonine was i n c o r p o r a t e d at a l e v e l based on the net requirements estimated by Aw-Yong and Beames (1972). T a b l e XVII shows the amino a c i d (% DM), p r o t e i n (% DM) and gross energy (kcal/kg DM) content of the d i e t s . Feed was p r o v i d e d ad l i b i t u m , and water renewed d a i l y . Body weights and feed i n t a k e s were reco r d e d d a i l y . At the end of the 21-day f e e d i n g p e r i o d , the r a t s were f a s t e d f o r 18 hr. Each r a t was then stunned by a blow to the head, the l i v e r was r a p i d l y removed and a p o r t i o n was immediately s u b j e c t e d to each of the two enzyme e x t r a c t i o n procedures. The remaining r a t c a r c a s s e s were prepared and s t o r e d as o u t l i n e d i n Rat T r i a l 3. A n a l y t i c a l methods C y s t a t h i o n i n e synthase a c t i v i t y was assayed u s i n g the procedures o u t l i n e d i n Rat T r i a l 3. The a c t i v i t y of TABLE XVII. Amino acid, protein (% DM) and energy (kcal/kg) content of diets in Rat T r i a l 4 Diet No, Arginine H i s t i d i n e Isoleucine Leucine Lysine Methionine + cystine Phenylalanine + tyrosine Threonine Tryptophan Valine 0.45 0.50 0.60 0.30* 0.55* 0.75* 0.9 0' 0.55 0.8 0* 0.57* 0.17' 0.68* 0.60 0.65 0.70 U> Crude protein (% N x 6.25) Gross energy (kcal/kg DM) 12. 00c 3780c Indicates a l e v e l constant in a l l diets, - 138 -N - m e t h y l t e t r a h y d r o f o l a t e - h o m o c y s t e i n e - m e t h y 1 t r a n s f e r a s e was measured u s i n g the method of Mudd ei al. (1970) as o u t l i n e d i n Rat T r i a l 3. U r i n a r y u r e a - n i t r o g e n e x c r e t i o n was determined u s i n g the method of Brown (1971) and the r e s u l t s were expressed as gN/kg DM d i e t consumed. Carcass a n a l y s e s , and a n a l y s i s of amino a c i d s i n b a r l e y were performed as o u t l i n e d i n Rat T r i a l 1. S t a t i s t i c a l procedures A l l the r e s u l t s were s u b j e c t e d to a n a l y s i s of v a r i a n c e and s i n g l e degree of freedom c o n t r a s t s as o u t l i n e d i n the U n i v e r s i t y of B r i t i s h Columbia programme MFAV (Halm and Le, 1975). A l l the d i f f e r e n c e s between means were t e s t e d a t the 5% p r o b a b i l i t y l e v e l u s i n g the Newman-Keul 1s M u l t i p l e Range T e s t (Halm and Le, 1975). Results Average d a i l y body weight g a i n , feed consumption and feed c o n v e r s i o n e f f i c i e n c y (FCE) r e s u l t s are pre s e n t e d i n Table XVIII, c a r c a s s c o m p o s i t i o n r e s u l t s i n Table XIX, l i v e r enzyme a c t i v i t i e s and u r i n a r y u r e a - n i t r o g e n e x c r e t i o n i n Ta b l e XX, and s i n g l e degree of freedom c o n t r a s t s f o r parameters t h a t showed s i g n i f i c a n t d i f f e r e n c e s between t h e i r means, i n Table XXI. T A B L E X V I I I . Average d a i l y body-weight g a i n , feed consumption and feed c o n v e r s i o n e f f i c i e n c y of r a t s f e d on d i e t s c o n t a i n i n g v a r y i n g methionine p l u s c y s t i n e l e v e l s i n Rat T r i a l 4 Diet No. SE of Mean St a t i s t i c a l significance of difference between diets F-Test Newman Keul's Test Methionine + cystine (% DM) 0.45 0.50 0.55 0.60 0.65 0.70 Week 1 3. 30 4.05 3.79 4.53 3.29 4.32 0.20 NS Average d a i l y Week 2 5. 30 6.04 4.54 4.80 5.34 5.30 0.19 NS Ixxiy-weight gain (g) Week 3 6. 28 5.81 4.31 5.77 4.27 5.80 0.30 NS Overall 4. 81 5.30 4.06 5.27 4.29 5.06 0.17 NS Week 1 14. 54 14.28 14.77 15.03 13.28 14.30 0.25 NS Average d a i l y Week 2 18. 30 18.82 16.28 17.29 16.05 17.81 0.43 NS feed Week 3 21. 80 20.54 16.81 17.81 16.55 19.02 0.59 * consumption (g) Overall 18. 29 18.03 15.79 16.33 15.52 17.04 0.37 NS Average feed Week 1 0. 25 0.31 0.38 0.32 0.27 0.34 0.01 NS conversion Week 2 0. 31 0.33 0.30 0.30 0-37 0.31 0.01 * e f f i c i e n c y (g gain/g feed VJeek 3 0. 30 0.30 0.28 0.32 0.27 0.32 0.01 NS DM consumed) Overall 0. 29 0.31 0.29 0.31 0.30 0.32 0.01 NS 5 3 4 6 2 1 4 3 1 6 2 5 *P<0.05; NS not significant (P>0.05) - 140 -Average d a i l y body-weight g a i n T h i s parameter showed no d i f f e r e n c e between the v a r y i n g l e v e l s of d i e t a r y methionine p l u s c y s t i n e e i t h e r d u r i n g each week of the t r i a l or f o r the o v e r a l l t r i a l p e r i o d . Average d a i l y f e e d consumption D i e t a r y methionine p l u s c y s t i n e l e v e l had no e f f e c t on feed consumption d u r i n g the f i r s t and second weeks of the t r i a l or f o r the o v e r a l l t r i a l p e r i o d . However, d u r i n g the t h i r d week of the t r i a l (Table X V I I I ) , r a t s r e c e i v i n g 0.45% DM d i e t a r y methionine p l u s c y s t i n e consumed s i g n i f i c a n t l y (P<0.05) more feed than r a t s on 0.55, 0.60 and 0.65% DM d i e t a r y methionine p l u s c y s t i n e w h i l e the 0.50% DM d i e t a r y methionine p l u s c y s t i n e , l e v e l r e s u l t e d i n i n t a k e s s i g n i f i c a n t l y (P<0.05) g r e a t e r than those achieved on the 0.55 and 0.65% DM methionine p l u s c y s t i n e l e v e l s . S i n g l e degree of freedom comparisons (Table XXI) i n d i c a t e d feed i n t a k e o f the r a t s r e c e i v i n g the 0.45% DM methionine p l u s c y s t i n e d i e t to be g r e a t e r a l s o than the i n t a k e on the 0.70% DM methionine p l u s c y s t i n e d i e t . Average feed c o n v e r s i o n e f f i c i e n c y (FCE) During the f i r s t and t h i r d weeks of the t r i a l , the d i e t s had no s i g n i f i c a n t e f f e c t on FCE. However, d u r i n g the second week of the t r i a l , the FCE of r a t s on 0.65% DM methionine p l u s - 141 -cystine was s i g n i f i c a n t l y (P<0.05) higher than that of rats re c e i v i n g 0.55 and 0.60% DM dietary methionine plus cystine (Table XVIII). Single degree of freedom comparisons (Table XXI) extended the s i g n i f i c a n t (P<0.05) su p e r i o r i t y of the 0.65% DM methionine plus cystine over the 0.55 and 0.60% DM l e v e l s as shown by the Newman Keul's Test, to include the 0.45 and 0.70% DM l e v e l s also. Carcass composition None of the carcass parameters, except the percentage ash in f a t - f r e e carcass, were influenced by dietary treatments (Table XIX). Rats on 0.65% DM dietary methionine plus cystine produced a s i g n i f i c a n t l y (P<0.05) lower percentage ash i n the f a t - f r e e carcass than rats on 0.55% DM methionine plus cystine. Single degree of freedom comparisons (Table XXI) indicated rats receiving 0.45, 0.60 and 0.65% DM dietary methionine plus cystine to have a lower percentage ash in the f a t - f r e e carcass than rats on 0.55% DM methionine plus cystine. Liver enzyme a c t i v i t i e s The a c t i v i t y of l i v e r cystathionine synthase was s i g n i f i c a n t l y (P<0.05) lower on 0.60% DM dietary methionine plus cystine than on any of the other diets except for the 0.4 5% DM methionine plus cystine d i e t (Tables XX and X X I ) . The a c t i v i t y o f the enzyme was highest on the 0.70% methionine p l u s TABLE XIX. The e f f e c t o f v a r y i n g l e v e l s o f d i e t a r y m e t h i o n i n e p l u s c y s t i n e on r a t c a r c a s s c o m p o s i t i o n i n T r i a l 4 Diet No. SE of Mean S t a t i s t i c a l significance of difference between d i e t s  Newman-Keul1s F-test Test Methionine + cystine (% DM) 0. 45 0. 50 0. 55 0. 60 0. 65 0 .70 Dry carcass weight (g) 57. 29 54. 27 51. 02 53. 80 55. 03 55 .29 0 .76 NS ?-, f a t i n dry carcass 23. 03 19. 53 18. 54 17. 57 17. 57 19 .54 0 .70 NS Total carcass f a t (g) 13. 04 10. 30 9. 29 9. 09 , 9. 56 10 .54 0 .46 NS % crude protein i n f a t - f r e e carcass 81. 81 81. 05 80. 03 81. 55 82. 55 82 .79 0 .47 NS % crude protein i n whole carcass 62. 80 64. 81 64. 54 68. 29 67. 32 66 .07 0 .80 NS To! .a i carcass crude protein 35. 81 35. 06 32. 81 36. 80 37. 05 36 .56 0 .64 NS ash in f a t - f r e e carcass 14. 09 14. 55 16. 30 13. 79 12. 56 14 .30 0 .34 * ?. ash in whole carcass 11. 04 11. 09 13. 06 11. 29 10. 52 11 .52 0 .29 NS Total carcass ash 6. 06 6. 27 6. 55 5. 81 5. 31 6 .28 0 .17 NS 5 4 1 6 2 3 *l?-'0.05; MS not significant (P>0.05) Cystathionine synthase activity (y mole/mg protein/60 minutes) |_i p< N J N> CO UJ O Ul O Ul O Ul O Ul I 1 1 1 I i I I "1 P-c H Q a. cu 1-3 (0 0 3" 13 r t a p - P -o < ro rr p - i n in it ^ K ra p o O rt 1-tl B P M l 3 P -< < ti ro CJ 3 M I-I a K o P-w K 3 rt in n3 CS r t 3 O P a r t ro CJ 3" < i-i p - ra a o 3 0 (0 tn a o r t i-l CO O 3 P\ rt 3" 0) P I 0, i-i 0 co K c ro M 3 p - ra M 3 r t CJ 3" rt S3 P-01 o o rt 3 TJ P-» H 3 M «. ro p -a. OJ "d P H H ra c rt Ul 0 K oi Cu rt o p-3" 3 ra O 0 a r t a p- O ra .!> r t Ul OJ t l •< 3 ro r t 3" o P- Ul O o 3 P-3 ro + 0 o Ul <n Ul rt p -3 ro . . o o CTt o o Ul o o 3" ro - Zvl -TABLE XX. E f f e c t o f v a r y i n g l e v e l s o f d i e t a r y methionine p l u s c y s t i n e on the a c t i v i t i e s of l i v e r c y s t a t h i o n i n e synthase and N^- m e t h y l t e t r a h y d r o f o l a t e - h o m o c y s t e i n e -m e t h y l t r a n s f e r a s e (mTHF Enz.) and u r i n a r y u r e a - n i t r o g e n e x c r e t i o n of r a t s i n T r i a l 4 SF Diet No. St a t i s t i c a l significance of difference between diets of Newman-Keul's Mean F-Test Test Methionine + cystine (% DM) 0.45 0.50 0.55 0.60 0.65 0.70 Cystathionine Synthase (l.i niole/ing protein/ 60 minutes) 12.81 18.53 17.54 9.80 19.55 29.55 1.43 * 4 1 3 2 5 6 n f l L ' J J F E n z . (n mole/mg protein/ 60 minutes)' 0.70 0.74 1.06 2.03 0.81 0.70 0.16 * 6 1 2 5 3 4 Urinary urea-nitrogen (g/kg DM feed) 1.07 1.05 1.01 1.01 1.00 1.00 0.06 * 6 5 4 3 2 1 TABLE XXI. R e s u l t s of c o n t r a s t s of v a r i a b l e s t h a t showed s i g n i f i c a n t d i f f e r e n c e s between d i e t s i n Tables XVIII, XIX and XX of Rat T r i a l 4 lv2 lv3 lv4 lv5 lv6 2v3 2v4 2v5 2v6 3v4 3v5 3v6 4v5 4v6 5v6 Average daily feed consumption, week 3 NS * * * * * NS • * NS NS NS NS NS NS NS FCE, week 2 NS NS NS * NS NS NS NS NS NS * NS * NS * % ash i n fat-free carcass NS * NS NS NS NS NS NS NS * * NS NS NS NS Cystathionine synthase activity * NS NS * * NS * NS * * NS * * * * IIITIII.' Unz. ac t i v i t y NS * * NS NS * * NS NS * * * * * NS Urinary urea-nitrogen * * * * * * * * * NS NS NS NS NS NS *P<0-05; NS not significant (P>0.05) - 146 -c y s t i n e d i e t . F i g u r e 6 shows that the a c t i v i t y of the enzyme was low at 0.45% d i e t a r y methionine plus c y s t i n e (12 u mole/ mg protein/60 minutes) and then increased to a value of 18.53 u mole/mg protein/60 minutes at the 0.50% DM d i e t a r y methionine p l u s c y s t i n e l e v e l . Above t h i s d i e t a r y l e v e l , the c y s t a t h i o n i n e synthase a c t i v i t y was i n h i b i t e d , reaching a minimum l e v e l of 9.80 y mole/mg protein/60 minutes on the 0.60% DM methionine pl u s c y s t i n e d i e t . T h e r e a f t e r the enzyme a c t i v i t y increased and reached a maximum l e v e l of 29.55 u mole/mg protein/60 minutes at 0.70% DM d i e t a r y methionine plus c y s t i n e . The a c t i v i t y of mTHF Enz. (Tables XX and XXI) d i d not vary s i g n i f i c a n t l y at the 0.45, 0.50, 0.65 and 0.70% DM d i e t a r y methionine plus c y s t i n e l e v e l s . The a c t i v i t y i n r a t s r e c e i v i n g the 0.55% DM methionine p l u s c y s t i n e d i e t was s i g n i f i c a n t l y (P<0.05) higher than th a t of r a t s r e c e i v i n g the 0.45, 0.50, 0.65 and 0.70% DM methionine plus c y s t i n e d i e t s . However, the a c t i v i t y of the enzyme was s i g n i f i c a n t l y (P<0.05) higher, 2.03 n mole/mg protein/60 minutes, i n r a t s r e c e i v i n g the 0.60% DM methionine plus c y s t i n e d i e t than i n r a t s r e c e i v i n g the 0.55% DM methionine plus c y s t i n e d i e t (1.06 n mole/mg p r o t e i n / 60 minutes). The a c t i v i t y of mTHF Enz. (Figure 7) was constant i n r a t s fed on 0.4 5 and 0.50% DM d i e t a r y methionine plus c y s t i n e , then rose to a maximum value of 2.03 n mole/mg protein/60 minutes at the 0.60% DM methionine plus c y s t i n e d i e t a r y l e v e l . The a c t i v i t y then diminished markedly at the 0.70% DM methionine 3. O r o <? QJ •L) O u cu tl) 2.Or rh b > l l U ro N fi 1.0 fTl rh th —3 0.45 0.50 0.55 0.60 0.65 D i e t a r y methionine plus c y s t i n e ( % DM) 0.70 F i g u r e 7. The e f f e c t of v a r y i n g l e v e l s of d i e t a r y methionine plus c y s t i n e on the a c t i v i t y of l i v e r N 5-methyltetrahydrofolate-homocysteine-methyltransferase i n Rat T r i a l 4. Each p o i n t d e p i c t s a mean a standard e r r o r of four r a t s per d i e t . - 148 -p l u s c y s t i n e d i e t a r y l e v e l to 0.70-n mole/mg p r o t e i n / 6 0 minutes, which a c t i v i t y was the same as t h a t p r e v a i l i n g on the 0.4 5% DM methionine p l u s c y s t i n e d i e t . U r i n a r y u r e a - n i t r o g e n e x c r e t i o n Urea e x c r e t i o n by r a t s r e c e i v i n g the 0.45 and 0.50% DM methionine p l u s c y s t i n e d i e t s was s i g n i f i c a n t l y (P<0.05) h i g h e r (Tables XX and XXI) than t h a t of r a t s r e c e i v i n g 0.55-0.70% DM methionine p l u s c y s t i n e . However, u r i n a r y urea e x c r e t i o n by r a t s r e c e i v i n g 0.45% DM d i e t a r y methionine p l u s c y s t i n e was s i g n i f i c a n t l y (P<0.05) h i g h e r than t h a t of r a t s r e c e i v i n g 0.50% DM methionine p l u s c y s t i n e . These r e s u l t s would i n d i c a t e a methionine p l u s c y s t i n e requirement of 0.50 to 0.55% DM d i e t . Discussion The r e s u l t s o b t a i n e d i n t h i s t r i a l show t h a t none of the growth parameters (average d a i l y body-weight g a i n , feed consumption and FCE) nor any of the c a r c a s s composition v a l u e s were s u f f i c i e n t l y s e n s i t i v e to d i f f e r e n t i a t e the performance of r a t s r e c e i v i n g 0.45 to 0.70% DM methionine p l u s c y s t i n e i n the d i e t s . The few s i g n i f i c a n t r e s u l t s a s s o c i a t e d w i t h these parameters gave p a t t e r n s t h a t tended to c o n f l i c t with each o t h e r . T h i s o b s e r v a t i o n i s i n agreement with the f i n d i n g s r e p o r t e d i n Rat T r i a l 3 where the d i e t a r y l e v e l of c y s t i n e was he l d c o n s t a n t at 0.20% DM i n a l l the d i e t s . - 149 -The a c t i v i t y of l i v e r c y s t a t h i o n i n e synthase f o l l o w e d the same p a t t e r n as t h a t p r e v a i l i n g i n Rat T r i a l 3 i n which c y s t i n e i n c o r p o r a t i o n i n the d i e t s w a s h e l d c o n s t a n t a t the 0.20% DM l e v e l . However, two s m a l l d i f f e r e n c e s should be p o i n t e d out. F i r s t l y , the a c t i v i t y l e v e l of c y s t a t h i o n i n e synthase a t the 0.45% DM d i e t a r y methionine p l u s c y s t i n e was lower than t h a t a t the 0.50% DM l e v e l (Figure 6). T h i s a c t i v i t y was a l s o lower than t h a t o b t a i n e d at the same l e v e l , 0.4 5% DM methionine p l u s c y s t i n e , i n Rat T r i a l 3 i n which the d i e t a r y c y s t i n e content was h e l d c o n s t a n t at 0.20% DM i n a l l the d i e t s . The f i n d i n g c o u l d i n d i c a t e t h a t a higher l e v e l of d i e t a r y c y s t i n e i s r e q u i r e d when the methionine l e v e l i s lower than the 0.35% DM d i e t . Presumably t h i s requirement i s f o r enzyme s y n t h e s i s s i n c e c y s t i n e does not c a t a l y z e the c y s t a t h i o n i n e synthase r e a c t i o n . The second d i f f e r e n c e between the r e s u l t s of t h i s t r i a l and those of Rat T r i a l 3 was the r a p i d i n c r e a s e i n the enzyme a c t i v i t y a t l e v e l s above 0.60% DM methionine p l u s c y s t i n e , where maximum i n h i b i t i o n o c c u r r e d . The c y s t i n e content at-0.60% DM methionine p l u s c y s t i n e i n the d i e t was 0.20% DM i n both Rat T r i a l 3 and i n the p r e s e n t t r i a l where the r a t i o of 2:1 methionine to c y s t i n e i n the d i e t was used. However, at 0.70% DM d i e t a r y methionine p l u s c y s t i n e , t h e l e v e l of c y s t i n e i n the d i e t i n Rat T r i a l 3 was s t i l l 0.20% DM while t h a t i n the p r e s e n t t r i a l was 0.23% DM d i e t . T h e r e f o r e , t h i s r a p i d i n c r e a s e i n c y s t a t h i o n i n e synthase a c t i v i t y could be due - 150 -to the increased cystine content when the present r e s u l t s are compared with those of Rat T r i a l 3 i n which cystine was held constant at 0.20% DM d i e t . Thus at these l e v e l s , 0.65 and 0.70% DM methionine plus cystine i n the present t r i a l , cystine had not reached the l e v e l s where i t s t a r t s to i n h i b i t cysta-thionine synthase a c t i v i t y (Finkelstein, 1967). Such a l e v e l , however, was not stated by F i n k e l s t e i n (1967). 5 The a c t i v i t y of N -methyltetrahydrofolate-homocysteme-methyltransferase showed a s i m i l a r pattern i n t h i s t r i a l as in Rat T r i a l 3,where cystine concentration was held constant at 0.20% DM d i e t . This means that the pattern and presumably the rate of remethylation of homocysteine i n rats receiving either the fixed 0.20% DM cystine i n Rat T r i a l 3 or the 2:1 methionine to cystine r a t i o i n Rat T r i a l 4 are the same. The r e s u l t s of the a c t i v i t i e s of both cystathionine synthase and mTHF Enz. indicate 0.55% DM methionine plus cystine to be the l e v e l beyond which disturbances i n enzyme a c t i v i t y occur. It could be concluded, therefore, that 0.50-0.55% DM dietary methionine plus cystine i s the range for optimal methionine plus cystine intake for growing r a t s . Urinary urea excretion i s greater at 0.45 and 0.50% DM dietary methionine plus cystine than at the 0.55-0.70% DM l e v e l s . Numerically, the differences i n urea excretion between 1.07 and 1.00 gN/kg DM feed consumed at 0.45 and 0.70% DM dietary methionine plus cystine respectively, found i n the - 151 -present t r i a l , are not as pronounced as those between 2.68 and 0.90 gN/kg DM feed consumed at 0.35 and 0.70% DM methionine plus c y s t i n e r e s p e c t i v e l y , i n Rat T r i a l 3,in which c y s t i n e was held constant at 0.20% DM i n a l l d i e t s . The f a c t t h a t the 0.35 and 0.40% DM d i e t a r y methionine plus c y s t i n e l e v e l s were not in c l u d e d i n the present t r i a l (because i t was d e s i r e d that the d i e t s not approximate s y n t h e t i c diets)., meant th a t the sharp urea e x c r e t i o n d e c l i n e observed between the 0.35 and 0.45% DM d i e t a r y methionine plus c y s t i n e l e v e l s i n Rat T r i a l 3, could not be observed i n the present t r i a l . As c a l c u l a t e d from t h i s parameter, 0.50-0.55% DM methionine plus c y s t i n e would be the range f o r optimal growth of r a t s i n the present t r i a l . With b a r l e y at 55.56% DM i n c l u s i o n , i f given an energy d i g e s t i b i l i t y of 80% [ d e r i v e d from b a r l e y DM d i g e s t i b i l i t y v alues of swine, (Beames and Ngwira, 1978)] and the remainder of the energy y i e l d i n g p u r i f i e d components a s c r i b e d a d i g e s t -i b i l i t y of 100%, the d i g e s t i b l e energy (DE) content of the d i e t s would be 3973.3 k c a l / k g DM d i e t , and the ME value (Morgan e t al. , 1975) would be 97% of t h i s , at 3854.1 kc a l / k g DM d i e t . Taking the NRC (1972) ME requirement of 4000 kca l / k g DM d i e t f o r growing r a t s , the c a l c u l a t e d energy content of the r a t d i e t s (ME) would be 96.4% of the NRC (1972) requirements. I f , then, methionine plus c y s t i n e requirement f o r the optimal growth of r a t s found i n the present t r i a l , 0.50-0.55% DM d i e t , were to be based on the NRC (1972) ME requirement of - 152 -4000 kcal/kg DM d i e t , then the methionine plus cystine requirement would be 0.52-0.57% DM d i e t . I t should be pointed out that these c a l c u l a t i o n s have been based on figures from swine feeding r e s u l t s and therefore the f i n a l ME values may not be necessarily correct. However, these c a l c u l a t i o n s bring out the fact that the gross energy values obtained i n a l l the rat t r i a l s were lower than the NRC (1972) requirements, and lower than the expected gross energy values when calculated from the dietary components. Conclusion Growth rate and carcass composition parameters cannot be used to define p r e c i s e l y the requirements of methionine plus cystine for growth i n weanling r a t s . The a c t i v i t i e s of l i v e r cystathionine synthase and N^-methyltetrahydrofolate-homocysteine-methyltransferase show that 0.50% DM dietary methionine plus cystine l e v e l supports normal a c t i v i t i e s but l e v e l s beyond 0.50% DM lead to enzyme disturbances. The patterns of l i v e r enzyme a c t i v i t i e s obtained using diets containing 2:1 methionine to cystine r a t i o are almost the same as those i n Rat T r i a l 3, i n which cystine was held contant at 0.20% DM, which resulted in the methionine to cystine r a t i o ranging from 0.75:1 at the 0.35% DM l e v e l of methionine plus cystine to 2.5:1 at the 0.70% DM l e v e l . - 1 5 3 -The r e s u l t s i n d i c a t e 0.45-0.50% DM to be the o p t i m a l range f o r methionine p l u s c y s t i n e d i e t a r y c o n c e n t r a t i o n f o r the weanling r a t s when the d i e t a r y gross- energy c o n c e n t r a t i o n i s 37 8 0 k c a l / k g DM d i e t . - 154 -RAT TRIAL 5 Interaction between serine, choline and methionine plus cystine  in weanling rats fed f o r t i f i e d barley-based d i e t s at a constant  methionine:cystine r a t i o Introduction and aim In Rat T r i a l 3,in which the cystine l e v e l i n a l l the diets was held constant at 0.20% DM, i t was found that the rate of growth of weanling rats could be used as a r e l i a b l e parameter to monitor the requirements of.methionine plus cystine when these amino acids were incorporated into diets at l e v e l s between 0.45 and 0.70% DM d i e t . However, the a c t i v i t i e s of cystathionine 5 synthase and N -methyltetrahydrofolate-homocysteine-methyltrans-ferase (mTHF Enz.), which were almost constant at l e v e l s between 0.35 and 0.50% DM methionine plus cystine, were altered when the dietary methionine plus cystine content was increased beyond the 0.50% DM l e v e l . Rat T r i a l 4, i n which the methionine to cystine r a t i o was held constant at 2:1, confirmed the same pattern of enzyme response towards dietary l e v e l s of methionine plus cystine. Since Shannon et al. (1972) showed that cystathionine synthase a c t i v i t y was altered s t a r t i n g at the point where methionine to cystine r a t i o approached or exceeded 1:1, i t was desired that, i n the present rat t r i a l , a methionine to cystine r a t i o be used to f i n d out whether cystathionine - 155 -synthase and mTHF Enz. a c t i v i t i e s c o u l d respond i n the same way they responded i n Rat T r i a l s 3 and 4. S e r i n e has been observed to a l l e v i a t e methionine t o x i c i t y (Benevenga and Harper, 1970). I t was f e l t t h a t some of t h i s a l l e v i a t i o n might be through the a c t i v i t i e s o f c y s t a t h i o n i n e synthase and mTHF Enz. s i n c e s e r i n e i s connected w i t h the enzyme r e a c t i o n s (Figure 1 ) . Choline has been shown by Byington et al. (1972) to be r e q u i r e d f o r the growth of weanling r a t s o n l y when d i e t a r y methionine to c y s t i n e r a t i o approaches 1:1. However the e f f e c t of c h o l i n e on c y s t a t h i o n i n e synthase and mTHF Enz. i s unknown, e s p e c i a l l y over the methionine p l u s c y s t i n e range covered i n Rat T r i a l s 3 and 4. The aim of the pr e s e n t r a t t r i a l was to determine the e f f e c t on growth and l i v e r c y s t a t h i o n i n e synthase and mTHF Enz. a c t i v i t y i n weanling r a t s of v a r y i n g l e v e l s of d i e t a r y s e r i n e and c h o l i n e , and the i n t e r a c t i o n between these components a t d i f f e r e n t l e v e l s of d i e t a r y methionine p l u s c y s t i n e . Materials and methods Experimental d e s i g n There were twenty d i e t a r y treatments. The treatments were made up of f i v e l e v e l s of d i e t a r y methionine p l u s c y s t i n e (1:1 r a t i o ) , two l e v e l s of s e r i n e and two l e v e l s of c h o l i n e (a 5x2x2 f a c t o r i a l ) . Each treatment contained f o u r r e p l i c a t e s . Both dietary treatments and r e p l i c a t e s were randomly d i s t r i b u t e d among 80 cages i n a room with temperature maintained at 27°C. Animals and cages Eighty male rats (Woodlyn/Wistar s t r a i n , Woodlyn Laboratories, Guelph, Ontario) 27 days of age at the s t a r t of the 21-day feeding period were used. The s t a i n l e s s - s t e e l cages had wire screen f l o o r s of 12 mm spacing and were f i t t e d with trays covered with paper towels for the c o l l e c t i o n of faeces and urine. Trays were emptied and towels renewed d a i l y . Diets Diets (Table XXII) were based on barley grain prepared i n the same way as outlined i n Rat T r i a l 1. There were 5 l e v e l s of methionine plus cystine, 2 l e v e l s of serine and 2 l e v e l s of choline chloride. The barley was d i l u t e d with corn starch and supplemented with synthetic L-amino acids (Ajinomoto Co., Tokyo, Japan) such that e s s e n t i a l amino acid l e v e l s met the NRC (1972) recommendations for growing r a t s , whereas threonine was incorporated according to Aw-Yong and Beames (1975). Non-es s e n t i a l amino acids brought the crude protein content to 12% DM. A l l the diets contained minerals and vitamins supplemented at l e v e l s to meet the NRC (1972) requirements for the growing ra t . Table XXIII shows the amino acid, gross energy and choline chloride content of the d i e t s . TABLE XXII. Percentage composition of d i e t s i n Rat T r i a l 5 Die t No. 10 11 12 13 14 15 16 17 18 20 Barley Starch 76.09c fethicnine + cys tine 1,2 Serins Cholin 3 Threonine -2 Ala: Asp: Giy: Glu' 2 ine Valine 2,4 T=oleucin=-" Phenylalanine + tyrosine 2 Histidine^ ^ " i n l r e 2 c Defiuorinafced rock phosphate" Trace rrdneral prendx^ Iodized sait^ 15.92 15.92 15.90 15.90 15.92 15.92 15.90 15.90 15.92 15.92 15.90 15.90 15.92 15.92 15.90 15.90 15.92 15.92 15.90 15.90 - - - - 0.10 0.10 0.10 0-10 0.15 0.15 0.15 0.15 0.25 0.25 0.25 0.25 0.35 0.35 0.35 0.35 0.20 - 0.20 - 0.20 - 0.20 - 0.20 - 0.20 - 0.20 - 0.20 - 0.20 - 0.20 0.02 0.02 - - 0.02 0.02 - - 0.02 0.02 - - 0.02 0.02 - - 0.02 0.02 2.01 1.81 2.01 1.81 1.91 1.71 1.91 1.71 1.86 1.66 1.86 1.66 1.76 1.56 1.76 1.56 1.66 1.46 1.66 1.46 0.26. 0.18! 0.24! 0.17! 0.08! 0.12c 0.60c 0.22c 0.02c 1.64£ 0.54c 0.40° 1.00c 0.50° •" Indicates a l e v e l constant i n a l l d i e t s . - 158 -TABLE XXII. Continued Contains a methionine to c y s t i n e r a t i o of 1:1. S u p p l i e d as pure grade L-amino a c i d s (Ajinomoto Co., Tokyo, Japan) Estimated to have a 50% c h o l i n e c h l o r i d e a c t i v i t y . E s t imated to have a Ala:Gly:Glu:Asp r a t i o of 1:1:1:1 (Womack, 1969; Abernathy and M i l l e r , 1965). Estimated to c o n t a i n 30% Ca and 14% P (DM) Estimated to c o n t a i n 40% Ca, no P (DM) Estimated to c o n t a i n , per kg d i e t : 3600 I.U.. V i t a m i n A; 400 I.U. V i t a m i n D3; 20 mg a - t o c o p h e r o l ; 180 mg A s c o r b i c a c i d ; 20 mg I n o s i t o l ; 1 g C h o l i n e c h l o r i d e ; 9 mg Menadione; 20 mg p-aminobenzoic a c i d ; 18 mg N i a c i n ; 4 mg R i b o f l a v i n ; 4 mg P y r i d o x i n e HCl; 4 mg Thiamine HCl; 12 mg Calcium pantothenate; 80 ug B i o t i n ; 360 ug F o l i c a c i d and 5.4 ug V i t a m i n B^ 0. Estimated to c o n t a i n , per kg d i e t : 4.47 g ^CO^; 1.73 g MgC0 3.H 20; 330 mg FeS0 4.7H 20; 60 mg MnS0 4.H 20; 100 mg ZnC0 3; 8.0 mg HaF; 17.5 mg CuSC>4.5H20; 6.0 mg C o C l 2 ; i n 3.279 g corn s t a r c h . E s timated to p r o v i d e 0.15 mg I 9 / k g DM d i e t . TABLE XXIII. Amino a c i d (% DM), c h o l i n e c h l o r i d e mg/kg DM), p r o t e i n (% DM) and gross energy (kcal/kg DM) content of d i e t s i n Rat T r i a l 5. Diet No. Choline chloride (rrn/kg DM) Methionine + cystine (%) Sarine (%) Threonine (%) Valine (%) Isoleucine (%) Leucine (%) Phenylalanine + tyrosine (%) Histidine (%) Lysine (%) Arginine (%) Tryptophan (%) Protein (%) Gross energy ( k c a l A g DM) 8 10 11 12 13 14 15 16 17 18 • 19 20 1000 1000 1200 1200 1000 1000 1200 1200 1000 .1000 1200 1200 1000 1000 1200 1200 1000 1000 1200 1200 0.35 0.35 0.35 0.35 0.45 0.45 0.45 0.45 0.50 0.50 0.50 0.50 0.60 0.60 0.60 0.60 0.70 0.70 0.70 0.70 0.35 0.58 0.38 0.58 0.38 0.58 0.38 0.58 0.38 0.58 0.38 0.58 0.38 0.58 0.38 0.58 0.38 0.58 0.38 0.58 0.57' 0.68 0.55 0.75 0.80 0.30 0.90 0.60 0.17 12.00 3800c a ,, -" I n d i c a t e s a l e v e l constant i n a l l d i e t s . - 160 -The procedures used for feeding, data recording and carcass preparation af t e r slaughter, have already been outlined in Rat T r i a l 3. A n a l y t i c a l methods Methods used i n the analysis of both barley and soybean meal for amino acid content, in the preparation of carcasses, and i n chemical analyses were the same as those outlined i n Rat T r i a l 1. Assay methods for both cystathionine synthase and mTHF Enz. have been outlined i n Rat T r i a l 3. S t a t i s t i c a l procedures A l l the results' were subjected to analysis of variance and a l l differences between means were tested at the 5% p r o b a b i l i t y l e v e l , using the Newman Keul's Multiple Range Test according to the University of B r i t i s h Columbia MFAV Programme (Halm and Le, 1975). . Results Tables XXIV to XXX give growth, carcass composition and l i v e r enzyme responses to varying l e v e l s of methionine, serine, choline and t h e i r i n t e r a c t i o n s . - 161 -Average d a i l y body-weight gain The average d a i l y body-weight gain of rats on 0.35% dietary methionine plus cystine was s i g n i f i c a n t l y (P<0.05) lower than on the other l e v e l s during a l l the three weeks of the t r i a l period as well as for the o v e r a l l t r i a l period (Table XXIV). Dietary methionine plus cystine l e v e l s of 0.45-0.70% DM gave the same average d a i l y body-weight gain throughout the t r i a l . Levels of serine, choline and t h e i r i nteractions with each other as well as with methionine (Tables XXV-XXX) had no influence on the average d a i l y body-weight gain throughout the t r i a l period. Average d a i l y feed consumption The average d a i l y feed consumption (Table XXIV) of rats receiving 0.35% DM dietary methionine plus cystine was s i g n i f i c a n t l y (P<0.05) lower than values obtained on other levels of dietary methionine plus cystine during the second and t h i r d weeks of the t r i a l as well as for the o v e r a l l t r i a l period. Feed consumption on 0.45-0.70% DM dietary methionine plus cystine did not vary s i g n i f i c a n t l y . Table XXV shows that the higher l e v e l of dietary serine s i g n i f i c a n t l y (P<0.05) lowered the average d a i l y feed consumption during the second and t h i r d weeks of the t r i a l as well as for the o v e r a l l t r i a l period. TABLE XXIV. Average d a i l y body-weight gain (g), feed consumption (g) and feed conversion e f f i c i e n c y (g weight gain/g feed consumed) of rats fed varying l e v e l s of methionine plus cystine i n Rat T r i a l 5 Dietary methionine + cystine (% DM) 0 .35 0 .45 0 .50 0 . 60 0 .70 SE of Level No. 1 2 3 4 5 Mean Week 1 2 .29 3 .66 3 .99 4 .22 4 .10 0.13 Average d a i l y body-weight gain (g) Week 2 Week 3 2 3 .91 .49 5 6 .10 .54 5 6 .30 .68 5 6 .92 .60 5 7 .48 .23 0.15 0.18 Overall 2 .97 4 .99 5 .42 5 .54 • 5 .55 0.13 Week 1 13 .47 14 .72 14 .41 14 . 04 13 .73 0.16 Average d a i l y Week 2 15 .98 18 .29 18 .80 . 18 .73 18 . 54 0.21 feed consumption W g e k 3' 16 . 85 20 .81 20 .99 20 .86 20 .91 0.29 Overall 15 .54 17 .92 18 .16 17 .61 17 .56 0.20 Average feed Week 1 0 .19 0 . 27 0 .30 0 . 32 0 .31 0.01 conversion e f f i c i e n c y (g gain/g Week 2 Week 3 0 0 .20 .23 0 0 .29 .32 0 0 .29 .34 0 0 .33 . 33 0 0 .31 .36 0.01 0.01 feed consumed) Overall 0 .21 0 . 30 0 .31 0 .33 0 .33 0.01 Statistical significance of difference between diets F-test NS * Newman Keul's Test for Level No. 1 2 3 5 4 1 2 4 3 5 1 2 3 4 5 1 2 5 4 3 1 2 4 5 3 1 5 4 2 3 1 2_3_ 5 4 1- 2 3 5 4 1 2 4 3 5 1 2 3 5 4 *P<0.05; NS not s i g n i f i c a n t (P>0.05) - 163 -TABLE XXV. The e f f e c t of v a r y i n g l e v e l s of d i e t a r y s e r i n e on feed consumption of r a t s i n Rat T r i a l 5 Dietary serine (% DM) 0.38 0.58 SE of L e v e l No. 1 2 Mean Average d a i l y feed consumption (g) Week 1 Week 2 Week 3 O v e r a l l 14.22 13.93 18.49 17.64 20.64 19.52 17.80 16.92 0.16 . 0.21 0.29 0.20 S t a t i s t i c a l significance of difference between diets F-test NS *P<0.05; NS not s i g n i f i c a n t (P>0.05) NB. The two d i e t a r y s e r i n e l e v e l s d i d not s i g n i f i c a n t l y a l t e r the average d a i l y body-weight g a i n , feed c o n v e r s i o n e f f i c i e n c y , c a r c a s s c o m p o s i t i o n and l i v e r enzyme a c t i v i t i e s i n growing r a t s . - 164 -Choline l e v e l s and serine x methionine plus cystine, choline x methionine plus cystine, choline x serine and methionine plus cystine x serine x choline i n t e r a c t i o n s had no s i g n i f i c a n t e f f e c t on feed consumption. Feed conversion e f f i c i e n c y (FCE) There was no e f f e c t of serine or choline on the FCE of the r a t s . However, the FCE of rats on 0.35% DM methionine (Table XXIV) was s i g n i f i c a n t l y (P<0.05) lower than on any of the other d i e t a r y l e v e l s during each of the t r i a l weeks.as well as for the o v e r a l l t r i a l period. For the o v e r a l l t r i a l period, 0.45% DM methionine plus cystine resulted i n a s i g n i f i c a n t l y (P<0.05) lower FCE value than those obtained on the 0.50-0.70% DM methionine plus cystine l e v e l s . Carcass composition Neither serine nor choline l e v e l s affected carcass composition. Table XXVI shows that the percentages of f a t and of crude protein i n the whole carcass were not affected by l e v e l s of methionine plus cystine. Dry carcass weight, t o t a l carcass crude protein and t o t a l carcass ash of rats on 0.35% DM dietary methionine plus cystine were s i g n i f i c a n t l y (P<0.05) lower than those of rats receiving 0.45-0.70% DM dietary methionine plus TABLE XXVI. The e f f e c t of varying l e v e l s of dietary methionine plus cystine on the carcass composition of rats i n Rat T r i a l 5 Dietary methionine + cystine (% DM) Statistical significant of difference between diets Level No. 0.35 1 0.45 2 0.50 3 0.60 4 0.70 5 Of Mean F-test Newman Keul's Test for Level No. Dry carcass weight (g) 40.50 51.80 53.55 53.36 54.61 0.77 * 1 2 4 3 5 % fat in the dry carcass 18.69 20.41 17.99 17.99 19.42 0.45 NS Total carcass fat (g) 7.67 10.43 9.67 9.50 10.49 0.31 * 1 4 3 2 5 % crude protein in fat-free carcass 80.97 81.56 81.74 83.35 82.92 0.23 * 1 2 3 5 4 % crude protein in the whole carcass 65.23 64.67 66.61 67.92 66.30 0.42 NS Total carcass protein (g) 26.48 33.37 35.61 36.23 36.30 0.50 * 1 2 3 4 5 % ash in fat-free carcass 15.73 15.04 14.25 13.48 13.98 0.17 * 4 5 3 2 1 % ash in the whole carcass 12.61 11.68 11.56 10.97 11.11 0.16 * 4 5 3 2 1 Total carcass ash (g) 4.92 5.92 5.99 5.56 5.98 0.09 1 4 2 5 3 *P<0.05; NS not significant (P>0.05 -166 -cystine. The t o t a l carcass crude protein of rats receiving 0.45% DM methionine plus cystine was s i g n i f i c a n t l y (P<0.05) lower than that of rats receiving 0.50-0.70% DM methionine plus cystine. Percentage crude protein i n f a t - f r e e carcass, ash i n fa t - f r e e carcass and ash i n whole carcass showed that rats receiving 0.35% DM dietary methionine plus cystine grew poorly compared with those receiving 0.50-0.70% DM methionine plus cystine. Enzyme a c t i v i t i e s The a c t i v i t y of l i v e r cystathionine synthase was not affected by the dietary l e v e l s of methionine plus cystine (Table XXVII), serine, methionine plus cystine x choline, serine x choline and methioninine plus cystine x serine x choline. However, the a c t i v i t y of the enzyme was s i g n i f i c a n t l y (P<0.05) higher (19.77 u mole/mg protein/60 minutes) at 1200 mg choline chloride per kg DM than at 1000 mg choline chloride per kg DM d i e t (16.95 u mole/mg protein/60 minutes) as shown i n Table XXVIII. The i n t e r a c t i o n between dietary serine and methionine plus cystine s i g n i f i c a n t l y (P<0.05) affected the l i v e r cystathionine synthase (Table XXIX and Figure 3). The a c t i v i t y of the enzyme at the 0.35% DM dietary methionine plus cystine l e v e l when serine l e v e l was 0.38% DM d i e t , was 16.55 \i mole mg/mg protein/60 minutes. The l e v e l of a c t i v i t y on t h i s serine concentration was increased to 20.54 u mole/mg protein TABLE XXVII. The e f f e c t of varying le v e l s of dietary methionine plus cystine on t h e ' a c t i v i t i e s of l i v e r cystathionine synthase and N5-methyltetra-hydrofolate-homocystine-methyl transferase of rats i n Rat T r i a l 5 Dietary methionine + cystine (% DM) Statistical significance 0.35 0.45 0.50 0.60 0.70 SE of difference between diets of Newman'Keul's Test Level No. 1 2 3 4 5 Mean F-test for Levels 1,2,3,4,5 Cystathionine synthase activity (y mole/mg protein/60 minutes) 19.06 17.60 18.93 19.36 16.86 0.47 NS mTHF Enz. activity (n mole/mg protein/ 60 minutes) 5.54 4.31 3.04 2.87 3.43 0.27 * 4 3 5 2 1 *P<0.05; NS not significant (P>0.05) c CysUthicrvLnc synthase activity (y mole/mg protein/60 minutes) t l CD PI 0 3 M, 0 O, rn 3* ro tn 0 13 O rr 0 H- H- 0 3 3 rn rr ro rr a 0 3 0 3 to TJ H- rr y-O 3" 3 rr 0 rr tn (5 0 rt Cu o a-rr 0 3 y- rr ro < y-cu y- 0 3 ty 3 & tr 3 o ID a. rh tw tn rt ra rr CD ra ti rr 3 3 D. p a B) y- y-rt < ra 0. rr rt Bl ra i-i >-i 0 <^ H <^ 0 H y> H rr ra P < 0 IT- ra rh S' y y- tn rh o 0 3 0 C H rh h 3 ra 3 H ra SD tn rr rr^< 3 tn 3 H-rr 0 T> 3" 3 0) CD H-i-1 cn 3 ra ro & y ft 3 I-1 rr c • tn H o CD t-J CO rr Ul M-ro O CD M 3 ra rr 3* M-O 3 y-3 ra cn rr 3 CD D > > n n cn rr 0) rr ro H* ro n < r( < H-3 rr 3 rr ro ro v; O 0 ro rr ro rt <; <: ro O ro O r-* i—1 Ln U) cn Ct) >-! a a - 091 -TABLE XXVIII, E f f e c t of dietary choline chloride l e v e l s on the a c t i v i t i e s of l i v e r cystathionine synthase and N 5-methyltetrahydrofolate-homocystine-methyltransferase i n Rat T r i a l 5 Level No. Dietary choline chloride l e v e l (mg/kg di e t DM) 1000 1 1200 2 Statistical significance SE of difference of between diets Mean F-test Cystathionine synthase a c t i v i t y (p mole/mg protein/60 minutes) 16.95 19.77 0.47 m THF Enz. a c t i v i t y (N mole/mg protein/60 minutes) 4.50 3.17 0.27 *P<0.05 NB. The two l e v e l s of dietary choline did not a f f e c t the average d a i l y body-weight gain, feed conversion e f f i c i e n c y , feed consumption and carcass composition of the rats 6.o r OJ XI 3 o S-l a 4.0 3.0 r 'ij 2.0 1.0 o 0.35 0.45 0.50 0.60 D i e t a r y methionine + c y s t i n e (% DM) 0.70 F i g u r e 9. E f f e c t of v a r y i n g l e v e l s of d i e t a r y methionine plus c y s t i n e on the a c t i v i t y of N 5 - m e t h y l t e t r a h y d r o f o l a t e - h o m o c y s t e i n e - m e t h y l t r a n s f e r a s e i n the l i v e r s of r a t s i n T r i a l 5. Each p o i n t d e p i c t s a mean and standard e r r o r f o r four r a t s per d i e t . nmiF Eti2. a c t i v i t y (n mole/mg protein/60 minutes c a Pu 3- o ro o x> 3 P- O 0 o r r K 01 Ul rr 3 P-fO D rn o 3 1 3 cu ro 3 rr Cb 3 ui ro cn ro rr r r P-CD fi 0 3 3 CD 3 rr a 3 ro CD 01 r r l i 1-1 rn 3* CD a ro ro 0 M rr ro CD CD P" CO n 0 Ii (D rr 3 0 p-Ii P- < tr 3 P- (0 0 rr r r rh P" P- ro rh < 0 ro 0 ro rn 3 C ii Ii cn a a U1P* 0 1 ro CD rn 3 rt rr ro CD cn ii rr >i CD 3 - K T) rr ro cn M r-* Ii rr ro P- ro < a 3 r t ro p ' i r-1 ro >J CD 01 rr i i 3 • P ^ 0 CD a rn p" M 0 3 Ul rn ro * 0 r r r- 3 CD P-M rr 0 CD ro 3 O 1 p-Tjr 3 ro '0 0 TJ P pj 3 c rr U) O O Ul o cn o CD ii <^ 3 ro o 3 p-3 ro w rr p-3 O cn o ID > o 0 rr rt P-< •4 H- P-rt rr ^ CD CD rr rr O O Ln CO ca s-3 i~3 0 O K -x Cl Ul ro 111 rt ii p-3 3 ro ro P* P-ro ro < < ro ro P-- TAT -- 172 -60 minutes a t 0.50% DM and 20.19 u mole/mg protein/60 minutes at 0.60% DM d i e t a r y methionine p l u s c y s t i n e , and t h e r e a f t e r decreased to 17.67 u mole/mg protein/60 minutes a t 0.70% DM d i e t a r y methionine p l u s c y s t i n e . However, a t the l e v e l of 0.58% d i e t a r y s e r i n e , the a c t i v i t y of the enzyme when d i e t a r y methionine p l u s c y s t i n e was 0.35% DM d i e t was 21.56 u mole/ mg protein/60 minutes, but was i n h i b i t e d to l e v e l s below those o b t a i n e d w i t h s e r i n e i n c o r p o r a t e d a t 0.38% DM d i e t , when the d i e t a r y methionine p l u s c y s t i n e l e v e l was i n c r e a s e d to the 0.70% DM l e v e l (Figure 8). The l e v e l s o f d i e t a r y methionine p l u s c y s t i n e , and of c h o l i n e c h l o r i d e , a s w e l l as the i n t e r a c t i o n s between methionine p l u s c y s t i n e and both s e r i n e and c h o l i n e c h l o r i d e , s i g n i f i c a n t l y a f f e c t e d the a c t i v i t y of l i v e r m THF Enz. Table XXVII and F i g u r e 9 show t h a t the a c t i v i t y of m THF Enz. was h i g h e s t a t 0.35% DM d i e t a r y methionine p l u s c y s t i n e (5.54 n mole/mg p r o t e i n / 60 minutes) but was g r a d u a l l y i n h i b i t e d as the d i e t a r y l e v e l of methionine p l u s c y s t i n e i n c r e a s e d , r e a c h i n g a minimum of 2.8 n moles/mg protein/60 minutes at 0.60% DM methionine p l u s c y s t i n e , t h e r e a f t e r r i s i n g to 3.43 n mole/mg protein/60 minutes at 0.70% DM d i e t a r y methionine p l u s c y s t i n e l e v e l . Table XXVIII shows t h a t the hi g h e r d i e t a r y c h o l i n e c h l o r i d e l e v e l i n h i b i t s the a c t i v i t y of m THF Enz. i n the r a t l i v e r . The r e s u l t s presented i n Tab l e XXIX on the a c t i v i t y of mTHF Enz., when p l o t t e d (Figure 10) show t h a t when d i e t a r y TABLE XXIX. E f f e c t of. the i n t e r a c t i o n between d i e t a r y methionine plus c y s t i n e and serine on the a c t i v i t i e s of l i v e r c y s t a t h i o n i n e synthase and N^-methyitetrahydrofolate-hcmocysteine-methyltransferase i n Rat T r i a l 5 SE of Nes-.—>an-Xeul1 s Test Mean F-test based on level no. Dietarv rrsthionine + cystine (% DM) 0.35 0.45 0.50 0.60 0.70 Dietary serine (% DM) 0.38 0.58 0.38 0.58 0.38 0.58 0.38 0.58 0.38 .0.58 I,.r."-1 No. 1 2_ 3 4_ 5 6 7 8_ £ ' 1° Cystathionine synthase activity (;J rrole/rng prctein/50 minutes) 16.55 21.56 18.92 16.24 20.54 17.31 20.19 18.54 17.67 16.05 0.47 * 10 4 1 6 9 8 3 7 5 2 m " Enz. activity (n rele/mc rroteir/ i'J minutesf 4.92 6.17 2.57 6.06 4.18 1.91 2.81 2.93 3.68 3.17 0.27 * 6 3 7 S 10 9 5 1 2 "?<0.05 ?3. The i n t e r a c t i o n between d i e t a r y methionine plus c y s t i n e and serine d i d not a f f e c t the average d a i l y body-weight gain, feed consumption, feed conversion e f f i c i e n c y or carcass composition of the r a t s . 6.Of A c t i v i t y at 1000 mg choline chloride/kg DM d i e t A c t i v i t y at 1200 Eg choline chloride/kg DM d i e t 4.0 2.0 0.35 0.45 0.50 0.60 0.70 D i e t a r y methionine + c y s t i n e (% DM) F i g u r e 11. The e f f e c t of the i n t e r a c t i o n between d i e t a r y l e v e l s of methionine p l u c y s t i n e and c h o l i n e on the a c t i v i t y of N 5 - m e t h y l t c t r a h y d r o f o l a t e -homocysteine-methyltransferase i n l i v e r s of r a t s i n T r i a l 5. Each p o i d e p i c t s a mean and standard e r r o r of four r a t s per d i e t . - 175 -s e r i n e l e v e l i s at 0.38% DM, the a c t i v i t y of the enzyme f o l l o w s a random p a t t e r n as the l e v e l of methionine plus c y s t i n e i n c r e a s e s . However, at 0.58% DM d i e t a r y s e r i n e , the a c t i v i t y of mTHF .Enz. i s constant at 6.17-6.06 n mole/mg protein/60 minutes between 0.35 and 0.45% DM d i e t a r y methionine plus c y s t i n e but t h e r e a f t e r i s d r a s t i c a l l y i n h i b i t e d to a minimum of 1.91 n mole/mg protein/60 minutes at 0.50% DM d i e t a r y methionine plus c y s t i n e . The a c t i v i t y then g r a d u a l l y r i s e s to a l e v e l of 3.17 n mole/mg protein/60 minutes at 0.70% DM d i e t a r y methionine plus c y s t i n e . Table XXX and Fig u r e 11 show the e f f e c t of the i n t e r a c t i o n between d i e t a r y methionine plus c y s t i n e and c h o l i n e c h l o r i d e on mTHF Enz. a c t i v i t y . The a c t i v i t y of the enzyme when c h o l i n e c h l o r i d e was present at 1000 mg/kg DM d i e t showed a s l i g h t n o n - s i g n i f i c a n t i n h i b i t i o n as the l e v e l of the d i e t a r y methionine plus c y s t i n e i n c r e a s e d . However, t h i s i n h i b i t i o n was more pronounced when the d i e t a r y c h o l i n e c h l o r i d e l e v e l was increased to 1200 mg/kg DM. At t h i s c h o l i n e c h l o r i d e l e v e l , a c t i v i t y of mTHF Enz. decreased s i g n i f i c a n t l y (P<0.05) from 5.66 n mole/mg protein/60 minutes at 0.35% DM d i e t a r y methionine plus c y s t i n e to 1.53 n mole/mg protein/60 minutes at 0.50% DM d i e t a r y methionine plus c y s t i n e . Thereafter the a c t i v i t y increased s l i g h t l y but not s i g n i f i c a n t l y to a l e v e l of 2.42 n mole/mg protein/60 minutes at the 0.70% DM d i e t a r y methionine plus c y s t i n e l e v e l . •TABLE XXX. E f f e c t - o f the i n t e r a c t i o n between d i e t a r y methionine plus c y s t i n e and ch o l i n e c h l o r i d e on the a c t i v i t i e s of c y s t a t h i o n i n e synthase and IP-methyltetrahydrofolate-homocysteine-methyltrar.s-fe r a s e i n Rat T r i a l 5 cf Statistical significance of difference between diets -r cystine (% DM) Dietary choline chloride Level No. Cystathionine synthase activity (v rrcie/mg prctein/60 minutes 0.35 0.45 0.50 0.60 0.70 1000 1200 1000 1200 1000 1200 1000 1200 1000 1200 1 2 3 4 5 6 7 8 9 10 17.55 14.81 17.31 19.81 15.29 20.56 20.40 20.55 18.92 18.43 0.47 mTHF L.nz. activity (n rcle/mg protein/ 60 minutes) 5.43 5.66 3.93 4.68 4.55 1.53 4.18 1.56 4.43 2.42 0.27 68 10 3 7 9 5 4 1 2 *P<0.05; KS not significant (P>0.05) NB. The i n t e r a c t i o n between d i e t a r y methionine plus c y s t i n e and c h o l i n e c h l o r i d e d i d not a f f e c t the average d a i l y body-weight gain, feed consumption, feed conversion e f f i c i e n c y or carcass composition of the r a t s - 177 -Discussion The r e s u l t s on average d a i l y body-weight gain, feed consumption and FCE have indicated that 0.35% DM dietary methionine plus cystine does not support optimal growth of wean-l i n g r a t s . This finding i s i n agreement with the r e s u l t s obtained i n Rat T r i a l 3 i n which cystine was held constant at 0.20% DM i n a l l d i e t s . The FCE r e s u l t s obtained i n the present t r i a l i n dicate that 0.45% DM dietary methionine plus cystine does not promote growth as e f f i c i e n t l y as 0.50-0.70% DM methionine plus cystine, although e f f i c i e n c y on the 0.45% DM l e v e l was s i g n i f i c a n t l y (P<0.05) better than at the 0.35% DM l e v e l . This f i n d i n g d i f f e r s s l i g h t l y from the findings i n Rat T r i a l s 3 and 4 where the 0.45% DM l e v e l of dietary methionine plus cystine could not be d i f f e r e n t i a t e d from higher l e v e l s of methionine plus cystine i n the promotion of growth. I t appears, therefore, that a higher proportion of cystine to methionine s l i g h t l y increases the methionine plus cystine requirements for optimal growth. This r e s u l t contradicts the findings of Stockland et a l . (1973) who showed that the requirement of methionine plus cystine decreases with increasing cystine content i n rats fed on casein-based d i e t s . On a purely molecular basis t h i s i s d i f f i c u l t to explain as 1 g cystine requires the catabolism of 1.125 g methionine for i t s formation. Carcass composition parameters only managed to i d e n t i f y 0.35% DM methionine plus cystine as being below requirements - 178 -f o r o p t i m a l growth of the r a t s . The other l e v e l s of d i e t a r y methionine p l u s c y s t i n e c o u l d not be d i f f e r e n t i a t e d u s i n g c a r c a s s composition, which r e s u l t s are i n agreement wi t h those o b t a i n e d i n Rat T r i a l s 3 and 4. I n c o r p o r a t i o n of s e r i n e i n t o the d i e t s a t the 0.58% DM l e v e l reduced feed i n t a k e . T h i s f i n d i n g i s c o n t r a r y to those, of Benevenga and Harper (1970) who showed t h a t g l y c i n e and s e r i n e improved feed i n t a k e of r a t s f e d d i e t a r y l e v e l s of 3.0% DM methionine i n casein-based d i e t s . O b v i o u s l y , the l e v e l s used by Benevenga and Harper (1970) are not p h y s i o l o g i c a l and t h e r e f o r e t h e i r f i n d i n g s cannot be a p p l i e d t o the p r e s e n t r e s u l t s . The a d d i t i o n of s e r i n e d i d not improve growth i n the r a t s even when the d i e t a r y methionine p l u s c y s t i n e l e v e l was 0.35% DM. T h i s i s c o n t r a r y to f i n d i n g s i n chickens where Baker and- Sugahara (1970) found a combination of s e r i n e and c h o l i n e improved the growth of chi c k e n s f ed on c r y s t a l l i n e amino acid-based d i e t s . T h i s d i f f e r e n c e l i e s i n the f a c t t h a t c h i c k e n s r e q u i r e g l y c i n e which can e a s i l y be r e p l a c e d by s e r i n e whereas r a t s do not r e q u i r e g l y c i n e f o r growth. Another reason may be the f a c t t h a t where growth improvement o c c u r r e d , the d i e t s d i d not have any methionine or c y s t i n e i n them whereas i n the pr e s e n t t r i a l , a l l d i e t s c o n t a i n e d some methionine and c y s t i n e . However, no e x p l a n a t i o n can be advanced f o r the feed i n t a k e r e d u c t i o n brought about by the high e r s e r i n e l e v e l i n the d i e t s used i n the pr e s e n t t r i a l . - 179 -N e i t h e r 1000 and 1200 mg/kg DM d i e t c h o l i n e c h l o r i d e nor 0.38 and 0.58% DM d i e t a r y s e r i n e a f f e c t e d growth r a t e of the r a t s or c a r c a s s composition at any of the l e v e l s of d i e t a r y methionine p l u s c y s t i n e . T h i s would suggest t h a t these two compounds are not r e q u i r e d at the l e v e l s used i n t h i s study i n order to improve growth. The enzyme p a t t e r n s o b t a i n e d u s i n g the 1:1 r a t i o of meth-i o n i n e t o c y s t i n e were d i f f e r e n t from those o b t a i n e d i n Rat T r i a l 3, i n which c y s t i n e was h e l d constant a t 0.20% DM i n a l l d i e t s , o r i n Rat T r i a l 4, i n which the methionine to c y s t i n e r a t i o was h e l d c o n s t a n t at 2:1 i n a l l d i e t s . In the pr e s e n t t r i a l , c y s t a t h i o n i n e synthase a c t i v i t y d i d not vary as the d i e t a r y l e v e l s of methionine p l u s c y s t i n e i n c r e a s e d from 0.35 to 0.70% DM, a f i n d i n g t h a t i s c o n t r a r y to the f i n d i n g s i n Rat T r i a l s 3 and 4, where the a c t i v i t y of the enzyme was i n h i b i t e d , s t a r t i n g at 0.50% DM, but r i s i n g a g a i n , s t a r t i n g a t the 0.60% DM methionine p l u s c y s t i n e d i e t a r y l e v e l . I t c o u l d be concluded, t h e r e f o r e , t h a t the high e r c y s t i n e p r o p o r t i o n a b o l i s h e d c y s t a t h i o n i n e synthase responses to i n c r e a s i n g l e v e l s of methionine, probably through s u p p r e s s i o n of enzyme s y n t h e s i s , s i n c e the l e v e l s o b t a i n e d i n the present t r i a l were not as high as those i n Rat T r i a l 3, i n which c y s t i n e was he l d constant at 0.20% DM i n a l l d i e t s , or i n Rat T r i a l 4, i n which the methionine to c y s t i n e r a t i o was he l d c o n s t a n t a t 2:1 i n a l l d i e t s . In the i n t e r a c t i o n between s e r i n e and methionine p l u s - 1 8 0 -c y s t i n e , h i g h l e v e l ( 0 . 5 8 % DM) o f d i e t a r y s e r i n e i n h i b i t e d t h e a c t i v i t y o f the enzyme s l i g h t l y more t h a n t h e low l e v e l ( 0 . 3 8 % DM), e s p e c i a l l y a t h i g h e r d i e t a r y m e t h i o n i n e p l u s c y s t i n e c o n t e n t s , f o r no a p p a r e n t r e a s o n . 5 N - m e t h y l t e t r a h y d r o f o l a t e - h o m o c y s t e i n e - m e t h y 1 t r a n s f e r a s e has been shown t o be i n h i b i t e d by h i g h l e v e l s o f b o t h s e r i n e and c h o l i n e , i n a d d i t i o n t o t h e i n h i b i t i o n by h i g h e r l e v e l s o f d i e t a r y m e t h i o n i n e p l u s c y s t i n e i n t h e p r e s e n t t r i a l . The most pronounced and p r o l o n g e d i n h i b i t i o n o c c u r r e d w i t h t h e h i g h e r c h o l i n e c h l o r i d e c o n t e n t ( 1 2 0 0 mg/kg. DM d i e t ) . T h i s means t h a t as the d i e t a r y c o n t e n t o f m e t h i o n i n e i n c r e a s e d , t h e BH Enz. ( F i g u r e 1) became more i m p o r t a n t i n t h e removal o f a c c u m u l a t i n g homocysteine t h a n t h e mTHF Enz., a f i n d i n g v/hich i s i n agreement w i t h t h e r e p o r t by F i n k e l s t e i n ( 1 9 7 4) . The i n h i b i t i o n o f mTHF Enz. by t h e h i g h l e v e l o f s e r i n e ( 0 . 5 8 % DM) was not e x p e c t e d s i n c e t h i s amino a c i d does n o t p a r t i c i p a t e i n t h e enzyme r e a c t i o n i n a d i r e c t way, and t h e r e f o r e no r e a s o n can be advanced f o r i t . I n h i b i t i o n o f mTHF Enz. w i t h an i n c r e a s i n g m e t h i o n i n e p l u s c y s t i n e c o n c e n t r a t i o n i n t h e d i e t has been documented by F i n k e l s t e i n (19 6 7 ) . However, a l t h o u g h such an i n h i b i t i o n was a l s o o b s e r v e d i n Rat T r i a l 3, i n whi c h c y s t i n e was h e l d c o n s t a n t a t 0 . 2 0 % DM i n a l l d i e t s , i n Rat T r i a l 4 , i n w h i c h the m e t h i o n i n e t o c y s t i n e r a t i o was h e l d c o n s t a n t a t 2 : 1 i n a l l d i e t s , a n d i n the p r e s e n t t r i a l s , i t must be p o i n t e d o u t t h a t t h e a c t i v i t i e s - 181 -o f the enzyme obtained, i n t h e p r e s e n t t r i a l a r e h i g h e r t h a n t h o s e o b t a i n e d i n Rat T r i a l s 3 and 4. I t i s p o s s i b l e t h a t t h e h i g h e r p r e p o r t i o n o f c y s t i n e i n the d i e t s o f t h e p r e s e n t t r i a l , w h i c h a b o l i s h e d c y s t a t h i o n i n e s y n t h a s e response to. h i g h d i e t a r y m e t h i o n i n e p l u s c y s t i n e c o n t e n t , must have promoted h i g h e r mTHF Enz. s y n t h e s i s and a c t i v i t y t h a n i n d i e t s c o n t a i n i n g either a c o n s t a n t 0.20% DM c y s t i n e o r i n whi c h t h e m e t h i o n i n e t o c y s t i n e r a t i o was h e l d c o n s t a n t a t 2:1. Conclusion When t h e c y s t i n e t o m e t h i o n i n e r a t i o i n a d i e t i s 1:1 and the g r o s s energy l e v e l i s 3300 k c a l / k g DM d i e t , the o p t i m a l r e q u i r e m e n t o f m e t h i o n i n e p l u s c y s t i n e f o r growth o f w e a n l i n g r a t s i s c l o s e t o 0.50% DM u s i n g w e i g h t g a i n , f e e d consumption and f e e d c o n v e r s i o n e f f i c i e n c y as p a r a m e t e r s . H i g h l e v e l s o f s e r i n e (0.58% DM) and c h o l i n e c h l o r i d e (1200 mg/kg DM) a f f e c t n e i t h e r the r a t e o f growth nor c a r c a s s c o m p o s i t i o n . H i g h l e v e l s o f d i e t a r y s e r i n e (0.58% DM), however, d e p r e s s f e e d consumption of the gr o w i n g r a t . The a c t i v i t i e s o f b o t h c y s t a t h i o n i n e s y n t h a s e and 5 N - m e t h y l t e t r a h y d r o f o l a t e - h o m o c y s t e m e - m e t h y 1 t r a n s f e r a s e i n l i v e r s o f r a t s r e c e i v i n g a 1:1 r a t i o o f m e t h i o n i n e t o c y s t i n e f o l l o w d i f f e r e n t p a t t e r n s o f response t o t h o s e o f r a t s r e c e i v i n g e i t h e r a c o n s t a n t 0.2 0% DM l e v e l o f d i e t a r y c y s t i n e {Rat T r i a l 3) o r a c o n s t a n t m e t h i o n i n e t o c y s t i n e r a t i o o f 2:1 - 182 -(Rat T r i a l 4) when the d i e t a r y methionine plus c y s t i n e l e v e l s vary between 0.3 5 and. 0.70% DM l e v e l s . In the present t r i a l , both enzymes are i n h i b i t e d by i n c r e a s i n g l e v e l s of d i e t a r y methionine plus cystine, but i n h i b i t i o n of mTHF Enz. i s more pronounced than t h a t of c y s t a t h i o n i n e synthase. Choline c h l o r i d e does not i n h i b i t the a c t i v i t y of c y s t a t h i o n i n e synthase when the d i e t a r y content i s at 1200 mg/kg DM compared wi t h the d i e t a r y content of 1000 mg/kg DM. However, the i n t e r a c t i o n between d i e t a r y c h o l i n e c h l o r i d e and methionine plus c y s t i n e s i g n i f i c a n t l y i n h i b i t s the a c t i v i t y of mTHF Enz. i n r a t s r e c e i v i n g 1200 mg c h o l i n e c h l o r i d e / k g DM d i e t compared wit h those r e c e i v i n g 100 mg c h o l i n e c h l o r i d e / k g DM d i e t between the 0.50-0.70% DM range of d i e t a r y methionine plus c y s t i n e . Rats r e c e i v i n g 0.58% DM d i e t a r y s e r i n e had the same level ' of cystathionine synthase activite as those r e c e i v i n g 0.3 8% DM d i e t a r y s e r i n e at a l l l e v e l s of d i e t a r y methionine plus c y s t i n e . However, r a t s r e c e i v i n g 0.58% DM d i e t a r y s e r i n e had s i g n i f i c a n t l y lower l e v e l s of mTHF Enz. a c t i v i t y than those r e c e i v i n g 0.38% DM. d i e t a r y s e r i n e , e s p e c i a l l y between 0.50 and 0.70% DM d i e t a r y methionine plus c y s t i n e l e v e l s . Thus, se r i n e i n h i b i t s mTHF Enz. a c t i v i t y when present at high d i e t a r y l e v e l s , e s p e c i a l l y at high d i e t a r y methionine plus cystine, l e v e l s when the methionine to c y s t i n e r a t i o i s held constant at 1:1 i n the d i e t s . - 183 -P I G T R I A L The e f f e c t o f v a r y i n g l e v e l s o f d i e t a r y m e t h i o n i n e p l u s c y s t i n e  on u r i n a r y u r e a e x c r e t i o n i n t h e g r o w i n g f e m a l e p i g Introduction and aim Brown and C l i n e (1974) showed t h a t u r i n a r y u r e a - n i t r o g e n e x c r e t i o n c o u l d be u s e d as an i n d i c a t o r o f a m i n o a c i d r e q u i r e m e n t s i n m a l e p i g s r e c e i v i n g c o r n - s o y b e a n m e a l d i e t s . I n t h e i r r e p o r t s , d i e t a r y l y s i n e a n d t r y p t o p h a n c o n c e n t r a t i o n s w e r e t h e v a r i a b l e s . F u l l e r et al. (1974) showed, u s i n g u r i n a r y u r e a - n i t r o g e n e x c r e t i o n i n g r o w i n g f e m a l e p i g s f i t t e d w i t h u r e t h r a l c a t h e t e r s , t h a t l y s i n e and t h r e o n i n e s u p p l e m e n t a t i o n o f b a r l e y - b a s e d d i e t s i m p r o v e d t h e u t i l i z a t i o n o f t h e . b a r l e y p r o t e i n . F u l l e r et al. (1975) u s i n g t h e same t e c h n i q u e as F u l l e r et al. (1974) demon-s t r a t e d t h a t l y s i n e was t h e f i r s t - l i m i t i n g a m i n o a c i d i n b a r l e y -b a s e d d i e t s a n d t h a t t h r e o n i n e was t h e s e c o n d l i m i t i n g a m i n o a c i d . S i n c e t h e s e r e p o r t s h a v e i n d i c a t e d t h a t u r i n a r y u r e a n i t r o g e n e x c r e t i o n c a n be u s e d t o m e a s u r e t h e r e q u i r e m e n t s o f s p e c i f i c a m i no a c i d s f o r t h e g r o w i n g p i g , t h e p r e s e n t p i g t r i a l was d e s i g n e d t o u s e t h i s t e c h n i q u e t o e s t i m a t e t h e m e t h i o n i n e p l u s c y s t i n e r e q u i r e m e n t s f o r t h e g r o w t h o f g i l t s . - 184 -Materials and methods Experimental d e s i g n The d e s i g n i n c o r p o r a t e d s i x treatments and t h r e e p e r i o d s . W i t h i n p e r i o d s , one g i l t was p l a c e d on each of the s i x treatments. The g i l t s were used i n three groups of s i x i n metabolism c r a t e s , i n a room kept at a c o n s t a n t temperature of 27°C. Each animal r e c e i v e d a f o r t i f i e d barley-soybean meal p o s i t i v e c o n t r o l d i e t f o r the i n i t i a l 7 days. The animals were then g i v e n the t e s t d i e t s f o r a p e r i o d of 7 days. The s i x d i e t s were a l l o c a t e d at random w i t h i n each p e r i o d . Animals and cages E i g h t e e n g i l t s (pure-bred Y o r k s h i r e ) of 32.610.6 kg l i v e -weight a t the s t a r t of the f e e d i n g p e r i o d were used. The p i g s were f i t t e d w i t h s i z e 10 F o l e y u r e t h r a l c a t h e t e r s , coated w i t h t e f l o n (Bardex, C.R. Bard I n t e r n a t i o n a l L t d . , England). The c a t h e t e r s , which had a 5 ml b a l l o o n and a l u e r v a l v e were i n s e r t e d w i t h the p i g s under f l u o r o t h a n e a n a e s t h e s i a a t the s t a r t of the t r i a l . The c a t h e t e r s were secured to the back of the p i g above the t a i l by an adhesive canvas pad. P l a s t i c t u b i n g f i t t e d t o the open end of the c a t h e t e r d i r e c t e d the u r i n e to a 9 - l i t r e narrow-neck p o l y e t h y l e n e b o t t l e c o n t a i n i n g 50 ml 0.1 N I^SO^. The mouth of the b o t t l e was covered to a v o i d contamination of the u r i n e . - 185 -The c a g e s , o f t h e S h i n f i e l d d e s i g n (Frape et a l . , 1968) were f i t t e d w i t h f e e d e r s , f e e d s p i l l a g e t r a y s , d r i n k i n g w a t e r n i p p l e s and t r a y s f o r f a e c e s c o l l e c t i o n . D i e t s The p o s i t i v e c o n t r o l d i e t (Table XXXI) was based on 81.94% DM b a r l e y and 15.00% DM soybean meal. The s i x e x p e r i m e n t a l d i e t s (Table XXXI) were based on 35.33% DM b a r l e y and 15.00% DM soybeen meal d i l u t e d w i t h 41.58% DM s t a r c h t o produce a b a s a l d i e t c o n t a i n i n g 0.35% DM m e t h i o n i n e p l u s c y s t i n e . T h i s l e v e l o f d i e t a r y m e t h i o n i n e p l u s c y s t i n e p r o v i d e d a m e t h i o n i n e l e v e l o f 0.15% DM and a c y s t i n e l e v e l o f 0.20% DM d i e t . T h i s c y s t i n e l e v e l o f 0.20% DM was h e l d c o n s t a n t i n a l l t h e e x p e r i m e n t a l d i e t s . The s i x e x p e r i m e n t a l d i e t s were i s o n i t r o g e n o u s a t 16% DM crude p r o t e i n and i s o c a l o r i c a t 3476.1 k c a l measured d i g e s t i b l e energy (DE)/kg DM d i e t . A l l o t h e r n u t r i e n t s and amino a c i d s i n a l l t h e d i e t s were added t o meet t h e NRC (1973) r e q u i r e m e n t s f o r the 20-35 kg l i v e w e i g h t p i g . T h r e o n i n e was i n c o r p o r a t e d a c c o r d i n g t o t h e net r e q u i r e m e n t s e s t i m a t e d by Aw-Yong and Beanies (1975). The m e t h i o n i n e p l u s c y s t i n e l e v e l s o f the d i e t s were 0.35, 0.45, 0.55, 0.65, 0.75 and 0.85% DM d i e t . T a b l e XXXII shows the e s s e n t i a l amino a c i d c o m p o s i t i o n o f the d i e t s c a l c u l a t e d from t h e a n a l y s i s o f i n d i v i d u a l components. TABLE XXXI. The i n g r e d i e n t s (% DM) crude p r o t e i n (% DM) and d i g e s t i b l e energy-c o n t e n t (kcal/kg DM) of d i e t s used i n the P i g T r i a l P o s i t i v e c o n t r o l Methionine + c y s t i n e * (constant c y s t i n e l e v e l of 0.2 0% DM) N o n - e s s e n t i a l amino a c i d s " Threonine*** I s o l e u c i n e * Lysine" 1" B a r l e y Soybean meal Corn s t a r c h D e f l u o r i n a t e d rock phosphate** Limestone ++ 81. 94 15. 00 1.49 0.57 0.50 Io d i z e d s a l t t Trace mineral + Vitamin premix§ 0.50 % crude p r o t e i n 17.10 DE (kcal/kg DM) 3756 D i e t No. 0.10 0.20 0.30 0.40 0.50 0.65 4.55 4.45 4.35 4.25 0 .13 0.04c 0.13 3 5.33 15. 00 41.58c 1. 50 0.54 0.5 0 0.50 a 16.00 3476 . l c - 187 -TABLE X X X I . c o n t i n u e d * S u p p l e m e n t e d a s p u r e g r a d e L - i s o m e r s ( A j i n o m o t o Co., T o k y o , J a p a n ) + S u p p l i e d as f e e d g r a d e L - l y s i n e H C l ** S u p p l e m e n t e d as L - T h r e o n i n e _ ( A j i n o m o t o C o ., T o k y o , J a p a n ) a c c o r d i n g t o Aw-Yong and Beames (1975) ** E s t i m a t e d t o c o n t a i n 30% Ca and 14% P ++ E s t i m a t e d t o c o n t a i n 40% C a , no P t E s t i m a t e d t o s u p p l y 0.15 mg p e r k g d i e t § E s t i m a t e d t o c o n t a i n (/kg d i e t ) 44 mg Mn a s MnSO.-H-O; 110 mg Zn a s ZnSO.^HoO; 500 mg B.H.T.; 3085 I.U. V i t a m i n A, 400 I.U. V i t a m i n D3; 20 pg V i t a m i n B]_2; 2.9 mg r i b o f l a v i n ; 11 mg n i a c i n ; 5 mg c a l c i u m p a n t o -t h e n a t e " I n c o r p o r a t e d a s a 1:1:1: m i x t u r e o f g l u t a m i c a c i d : g l y c i n e : a l a n i n e : a s p a r t i c a c i d (Womack,. 1969 ; A b e r n a t h y and M i l l e r , 1965) a •>-" I n d i c a t e s a l e v e l c o n s t a n t i n a l l d i e t s . TABLE XXXII. Amino a c i d c o m p o s i t i o n of d i e t s i n the P i g T r i a l , c a l c u l a t e d from components Amino a c i d P o s i t i v e c o n t r o l * D i e t No. C y s t i n e Methionine Threonine V a l i n e I s o l e u c i n e L eucine P h e n y l a l a n i n e + T y r o s i n e H i s t i d i n e L y s i n e A r g i n i n e Tryptophan 0 . 27 0.24 0.57 0.98 0 . 58 1.18 1.28 0 .40 0.75 0.82 0.19 0.20 0.15 0.25 0.35 0.45 0.55 0.65 , 0 . 5 7 a , « 0 . 5 i a >--< — 0.50 a >-0 . 7 9 0.87 3 0.27' 0.70 0.62 0.19 * P o s i t i v e c o n t r o l amino a c i d composition was c a l c u l a t e d a c c o r d i n g to Aw-Yong and Beames (1975) -v" I n d i c a t e s a l e v e l c o n s t a n t i n a l l d i e t s - ' 1 8 9 -A l l the p i g s were g i v e n 3 days i n which to r e c o v e r from the a n a e s t h e t i c . They were then fed on the p o s i t i v e c o n t r o l d i e t f o r seven days. A f t e r 7 days, the p i g s were then put on the t e s t d i e t s f o r an a d d i t i o n a l seven days. The c a t h e t e r s were then removed and the p i g s r e l e a s e d . Feed was g i v e n ad libitum d u r i n g the 14 days of t h e ' t r i a l p e r i o d . Water was c o n s t a n t l y a v a i l a b l e . U r i n e and faeces were c o l l e c t e d f o r a n a l y s i s f o r the l a s t f o u r days on both the p o s i t i v e c o n t r o l d i e t and the t e s t d i e t as i t has been shown by F u l l e r et al. (1974) t h a t d a i l y urea e x c r e t i o n w i l l s t a b i l i z e a f t e r f o u r days. A n a l y t i c a l methods Dry matter d i g e s t i b i l i t y of the d i e t s was determined by u s i n g the i n t e r n a l marker, 4 N HC1 i n s o l u b l e ash (McCarthy et al. , 1974). D a i l y u r i n a r y urea e x c r e t i o n was measured by complete c o l l e c t i o n and the u r e a - n i t r o g e n content was measured by the method of Brown (1971) . Because of the i n h e r e n t v a r i a t i o n between p i g s i n urea e x c r e t i o n , the d i f f e r e n c e between a standard e x c r e t i o n on a b a s a l d i e t and t h a t on a t e s t d i e t , f o r each p i g , i s a more accu r a t e method of comparing urea e x c r e t i o n on t e s t d i e t s than i s the t o t a l e x c r e t i o n comparison between p i g s on each t e s t d i e t ( F u l l e r et al. , 1974). T h i s method was used i n the p r e s e n t t r i a l . - 190 -Amino a c i d content of the ba r l e y and soybean meal was determined as o u t l i n e d i n Rat T r i a l 1. S t a t i s t i c a l procedures A l l data were subjected to a n a l y s i s of var i a n c e and a l l d i f f e r e n c e s between means were t e s t e d at the 5% p r o b a b i l i t y l e v e l using the Newmem-Keul's M u l t i p l e Range Test, as w e l l as the s i n g l e degree of freedom c o n t r a s t s according to the U n i v e r s i t y of B r i t i s h Columbia. MFAV programme of Halm and Le (1975) . • Results During the i n i t i a l 4-day c o l l e c t i o n p e r i o d when a l l pigs were r e c e i v i n g the p o s i t i v e c o n t r o l f o r t i f i e d barley-soybean meal d i e t , mean values obtained f o r the v a r i o u s parameters were: 1. average d a i l y feed consumption, 1.27+0.10 kg DM 2. apparent n i t r o g e n d i g e s t i b i l i t y , 85.9+0.19% 3. d a i l y urea-nitrogen e x c r e t i o n , 7.6 0±0.3 6g 4. d a i l y urea-nitrogen e x c r e t i o n / k g DM feed consumed, 4.44±0.26 g/kg DM. When the pigs were given the t e s t d i e t s , no s i g n i f i c a n t d i f f e r e n c e s were recorded, i n the average d a i l y feed consumption, average d a i l y urea e x c r e t i o n or average urea-nitrogen e x c r e t i o n 2.0 1.5h ! 8 1 1.0 c o Tl 0.5r I .5 .5 -0.5 -1.0 CD i — ' -1.5 -2.01 0.30 0.35 0.45 0.55 0. G5 0.75 0.85 Dietary methionine + cystine (% DM) Figure 12. The effect of varying dietary .levels of methionine plus cystine on the change of urea-nitrogen excretion per kg DM feed consumed when the pigs were fed a positive control diet followed by test diets. Each point depicts ?. mean and standard error for three pigs per diet. - 192 -p e r k g d r y f e e d consumed. However, t h e c h a n g e i n u r i n a r y u r e a -n i t r o g e n e x c r e t i o n p e r u n i t w e i g h t o f f e e d consumed f r o m t h a t o f e a c h a n i m a l on t h e p o s i t i v e c o n t r o l d i e t showed s i g n i f i c a n t (P<0.05) d i f f e r e n c e s b e t w e e n d i e t s . T h i s d i f f e r e n c e , by S t u d e n t N e w m a n - K e u l 1 s T e s t , was s i g n i f i c a n t o n l y b e t w e e n d i e t 1 and d i e t 3. Ho w e v e r , t h e s i n g l e d e g r e e o f f r e e d o m c o m p a r i s o n s , T a b l e X X X I V , show t h a t t h e c h a n g e , f r o m t h a t o n t h e b a s a l d i e t , i n u r e a e x c r e t i o n p e r k g DM d i e t by p i g s r e c e i v i n g 0.35% DM m e t h i o n i n e p l u s c y s t i n e was s i g n i f i c a n t l y (P<0.05) h i g h e r t h a n on a l l o t h e r d i e t s . F i g u r e 12 shows a minimum n i t r o g e n w a s t a g e w i t h a d i e t a r y l e v e l o f m e t h i o n i n e p l u s c y s t i n e o f 0.55% DM b a s i s . The o t h e r p a r a m e t e r s h o w i n g s i g n i f i c a n t d i f f e r e n c e s b e t w e e n t h e t e s t d i e t s was t h e a p p a r e n t n i t r o g e n d i g e s t i b i l i t y o b t a i n e d by t h e a c i d i n s o l u b l e a s h m e t h o d ( T a b l e X X X I I I ) . H owever, as c a n be s e e n i n T a b l e X X X I V , t h e r e i s no c l e a r p a t t e r n i n t h e s e d i f f e r e n c e s . T h e r e w e r e no s i g n i f i c a n t d i f f e r e n c e s b e t w e e n r e p l i c a t e s i n a ny o f t h e p a r a m e t e r s t e s t e d . Discussion The r e s u l t s shown i n F i g u r e 12 i n d i c a t e t h a t t h e 0.55% DM m e t h i o n i n e p l u s c y s t i n e l e v e l p r o m o t e d t h e g r e a t e s t n i t r o g e n r e t e n t i o n i n t h e g i l t s . H o w e v e r , n e i t h e r t h e s t u d e n t Newman-K e u l ' s T e s t n o r t h e s i n g l e d e g r e e o f f r e e d o m c o m p a r i s o n s c o u l d TABLE XXXIII. Apparent n i t r o g e n d i g e s t i b i l i t y (%), average d a i l y feed consumption (kg), average d a i l y u r e a - n i t r o g e n e x c r e t i o n (g), urea n i t r o g e n e x c r e t i o n / k g feed consumption (a/kg DM) and change i n urea e x c r e t i o n per feed consumea r e l a t i v e to p o s i t i v e c o n t r o l d i e t i n pi g s of 32.6+0.60 kg l i v e w e i g n t ted experimental d i e t s v a r y i n g i n methionine plus c y s t i n e l e v e l s S t a t i s t i c a l significance S^ of difference between diets of — — Mean F-test Newman-Keul's Test Dietary methionine + cystine (% DM) Diet No. Average daily feed, consumption (kg) Apparent nitrogen " d i g e s t i b i l i t y (%) Average daily-feed consumption on the positive control diet (kg) Average daily urea-nitrogen excretion (g) Urea-nitrogen excretion (g)/kg feed consumption Urea-nitrogen excretion (g)/kg feed consumption on positive control diet Change i n urea-nitrogen excretion/kg feed consumption relative to positive control 0.35 0.45 0.55 0.65 0.75 0.85 1 2 3 4 5 6 1.06 1.09 1.07 1.68 1.37 1.04 0.09 86.70 84.40 87.07 89.69 87.04 87.06 0.46 1.40 1.38 1.36 1.05 1.38 1.06 0.10 7.41 6.76 7.72 6.73 7.72 7.07 0.28 5.05 3.41 4.38 4.02 4.40 5.06 0.27 3.06 4.04 5.04 4.72 5.05 4.70 0.26 1.39 -0.30 -1.05 -0.41 -0.05 0.56 0.22 NS NS NS NS NS 2 1 5 6 3 4 3 4 2 5 6 1 TABLE XXXIV. S i n g l e degree of freedom c o n t r a s t s of parameters t h a t showed s i g n i f i c a n t d i f f e r e n c e s between d i e t s i n Table XXXIII Apparent n i t r o g e n d i g e s t i b i l i t y (%) Change i n urea-n i t r o g e n e x c r e t i o n r e l a t i v e to c o n t r o l d i e t (g/kg feed DM) lv2 lv3 lv4 lv5 lv6 NS * NS NS 2v3 2v4 2v5 2v6 3v4 * * * * * NS NS NS NS NS 3v5 3v6 4v5 4v6 5v6 NS NS * * NS NS NS NS NS NS *P<0.05; NS not s i g n i f i c a n t (P>0.05 - 195 -d i f f e r e n t i a t e the means i n the range 0.4 5-0.7 5 DM d i e t a r y methionine p l u s c y s t i n e . T h i s must have been p a r t l y due to the l a r g e s t a n d a r d e r r o r s a s s o c i a t e d w i t h the mean v a l u e s o b t a i n e d f o r the 0.45, 0.55 and 0.65% l e v e l s . The r e g u l a r p a t t e r n of these r e s u l t s , w i t h a hig h n i t r o g e n e x c r e t i o n a t a d e f i c i e n t methionine p l u s c y s t i n e l e v e l , r e d u c i n g to a minimum l e v e l when methionine p l u s c y s t i n e was adequate, then i n c r e a s i n g when excess methionine p l u s c y s t i n e was i n c o r p o r a t e d , show the d i f f e r e n c e i n urea e x c r e t i o n t o be a more meaningful parameter than t o t a l urea e x c r e t i o n per day or per k i l o g r a m of feed consumed, both of which produced an e r r a t i c p a t t e r n . Measurements of urea e x c r e t i o n per day can o n l y be v a l i d where feed consumption i s r e s t r i c t e d and, t h e r e f o r e , uniform. In the presen t t r i a l , feed was g i v e n ad l i b i t u m , and, although s t a t i s t i c a l t e s t s showed no s i g n i f i c a n t d i f f e r e n c e s i n feed consumption between experimental d i e t s , feed i n t a k e may s t i l l have c o n t r i b u t e d to the v a r i a t i o n s i n urea e x c r e t i o n of i n d i v i d u a l pigs, p a r t i c u l a r l y o n . d i e t 4 (0.65% DM methionine p l u s c y s t i n e ) , d i e t 5 (0.75% DM methionine p l u s c y s t i n e ) and d i e t 6 (0.85% DM methionine p l u s c y s t i n e ) . However, the f a c t t h a t the mean value s f o r change i n urea e x c r e t i o n at the 0.65 and 0.75% DM d i e t a r y methionine p l u s c y s t i n e l e v e l s were approximately on the same s t r a i g h t l i n e between v a l u e s on e i t h e r e x t r e m i t y would suggest t h i s modifying e f f e c t to be s m a l l . - 19 6 -The optimal requirements, 0.55% DM methionine plus c y s t i n e , obtained i n the present t r i a l f o r g i l t s of 32.610.6 kg l i v e -weight may be s l i g h t l y i n e r r o r because of the l a r g e increments of 0.10% methionine used i n t h i s t r i a l . Consequently, the present value does not n e c e s s a r i l y d i f f e r from the NRC (197 3) requirement of 0.50% DM f o r the 20-35 kg l i v e w e i g h t range of p i g s . The present r e s u l t s are higher than those of K e i t h e t al. (1972), using Y o r k s h i r e g i l t s averaging 18 kg l i v e w e i g h t , who obtained a methionine plus c y s t i n e requirement of 0.46-0.48% DM d i e t . K e i t h e t al. (1972) used casein-based d i e t s i n t h e i r t r i a l and the requirements were obtained using serum f r e e amino acids as a parameter. The r e s u l t s obtained i n the present t r i a l , 0.55% DM methionine plus c y s t i n e , f o r the 32.6±0.6 kg l i v e w e i g h t g i l t s , a l s o d i f f e r from those of Braude and Esnaola (1973), of 0.40-0.45% DM d i e t , f o r the optimal growth of. 20-60 kg l i v e w e i g h t c a s t r a t e d male pigs r e c e i v i n g s e m i - p u r i f i e d d i e t s based on groundnut meal, c a s e i n or soybean p r o t e i n . However, the la r g e l i v e w e i g h t range of 20-60 kg covered i n the experiments of Braude and Esnaola (1973) may mask the higher requirements f o r methionine plus c y s t i n e at the lower s e c t i o n of t h i s l i v e w e i g h t range,and t h e r e f o r e e x p l a i n the d i f f e r e n c e between t h e i r value and the present t r i a l value of 0.55% DM methionine plus c y s t i n e . Another p o s s i b l e reason f o r the d i f f e r e n c e may l i e i n the f a c t that Braude and Esnaola (1973) used c a s t r a t e d male pigs and - 197 -the present t r i a l used g i l t s s ince P i e r c e and Bowland (1972) have i n d i c a t e d that g i l t s produce leaner carcasses than barrows and, t h e r e f o r e , i t could be p o s s i b l e • t h a t the methionine plus c y s t i n e requirement of. g i l t s i s higher than that of barrows i n order to depo s i t the lean. Conclusion The change i n u r i n a r y urea e x c r e t i o n per kg feed consumed was a b e t t e r i n d i c a t o r of methionine plus c y s t i n e requirements f o r the growing g i l t s than the average d a i l y urea e x c r e t i o n or the average d a i l y urea e x c r e t i o n per kg feed consumed. Using t h i s i n d i c a t o r , the methionine plus c y s t i n e requirements f o r optimal growth of 32.6±0.6 kg l i v e w e i g h t g i l t s (purebred Yorkshire) was found to be 0.55% DM d i e t . Since t h i s value i s s l i g h t l y higher than the NRC (1973) value, and higher than those given by Braude and Esnaola (1973) and K e i t h e t al. (1972) , the a d d i t i o n of even smaller increments of methionine plus c y s t i n e may be a b e t t e r approach i n t h a t the graph may have a sharper end-point than the one obtained here. - 198 -GENERAL DISCUSSION AND CONCLUSION There i s a wide v a r i a t i o n i n the estimated methionine plus c y s t i n e requirements f o r growth i n both pigs and r a t s . Oestemer e t al. (1970) observed that 0.23% DM methionine plus c y s t i n e could promote optimal growth i n 21 kg l i v e w e i g h t g i l t s r e c e i v i n g opaque-2 corn-based d i e t s , whereas the NRC (1973) recommend 0.50% DM methionine plus c y s t i n e f o r the 20-35 kg l i v e w e i g h t p i g . Stockland e t al. (1973) showed the methionine plus c y s t i n e requirement f o r optimal growth i n weanling r a t s r e c e i v i n g a casein-based d i e t to be 0.41-0.49% i n a 90% DM d i e t , whereas the NRC (1972) recommend a l e v e l of 0.60% i n a 90% DM d i e t . With these wide v a r i a t i o n s i n the e s t i m a t i o n of methionine plus c y s t i n e requirements, i t i s apparent t h a t both d i e t a r y f a c t o r s and experimental techniques must a f f e c t the assessment of the requirements. Because of t h i s , the present t r i a l s were designed to t e s t the e f f e c t i v e n e s s of va r i o u s methods i n d e f i n i n g methionine plus cystine, optimal requirements f o r growth of r a t s and p i g s . They i n v e s t i g a t e d a range of methionine plus c y s t i n e concentrations i n f o r t i f i e d barley-based d i e t s , i n order to defi n e the requirements f o r optimal metabolism of r a t s , using growth, e f f i c i e n c y of feed u t i l i z a t i o n , u r i n a r y urea e x c r e t i o n and the a c t i v i t i e s of the two l i v e r enzymes that take p a r t . i n methionine metabolism: c y s t a t h i o n i n e synthase and - 199 -5 N - m e t h y l t e t r a h y d r o f o l a t e - h o i n o c y s t e i n e ~ m e t h y l t r a n s f e r a . s e . The methionine p l u s c y s t i n e requirements of g i l t s were assessed on d i e t s of v a r y i n g methionine p l u s c y s t i n e c o n c e n t r a t i o n s u s i n g u r i n a r y urea e x c r e t i o n as the major i n d i c a t o r . The r a t t r i a l s showed t h a t 0.23-0.40% DM d i e t a r y methionine p l u s c y s t i n e does not support o p t i m a l growth i n weanling r a t s . They a l s o showed t h a t once the l e v e l of d i e t a r y methionine p l u s c y s t i n e r i s e s above 0.45% DM, c a r c a s s composition and growth parameters are not s u f f i c i e n t l y s e n s i t i v e to d i f f e r e n t i a t e the performance of the r a t s on the v a r y i n g methionine p l u s c y s t i n e d i e t a r y l e v e l s . Thus, growth at 0.4 5% DM was the same as t h a t at 0.7 0% DM d i e t a r y methionine p l u s c y s t i n e , i n d i e t s c o n t a i n i n g a constant c y s t i n e c o n c e n t r a t i o n (0.2 0% DM, Rat T r i a l 3) and constant methionine to c y s t i n e r a t i o (2:1, Rat T r i a l 4 and 1:1, Rat T r i a l 5). The f a c t t h a t o p t i m a l growth was achieved at 0.45-0.50% DM methionine p l u s c y s t i n e d i e t a r y l e v e l s q u e s t i o n s the v a l i d i t y of the 0.67% DM methionine p l u s c y s t i n e requirement f o r growth i n r a t s recommended by the NRC (1972) . The use of u r i n a r y u r e a - n i t r o g e n e x c r e t i o n as a parameter i n the d e t e r m i n a t i o n of methionine plu s c y s t i n e requirements of weanling r a t s i n d i c a t e s 0.4 2-0.47% DM to be adequate f o r o p t i m a l growth. The u r i n a r y urea e x c r e t i o n curve formed an i n v e r t e d image of the FCE curve at the v a r y i n g d i e t a r y methionine p l u s c y s t i n e l e v e l s , w i t h both parameters i n d i c a t i n g 0.4 2-0.50% DM methionine p l u s c y s t i n e to be the range at which o p t i m a l - 200 performance i s reached. From both these parameters i t can therefore be concluded that the requirement of dietary methionine plus cystine for the optimal growth of rats i s 0.42-0.50% i n the dry d i e t . The r e s u l t s from l i v e r enzyme a c t i v i t i e s showed that, when the dietary cystine l e v e l was held constant at 0.20% DM (Rat T r i a l 3), or when the methionine to cystine r a t i o was held constant at 2:1 (Rat T r i a l 4), both cystathionine synthase and mTHF Enz. exhibit constant a c t i v i t y when the dietary l e v e l s of methionine plus cystine are i n the range 0.35-0.50% DM. The disturbances that occurred at l e v e l s higher than 0.50% DM dietary methionine plus cystine i n the a c t i v i t y of both enzymes, indicate abnormal methionine metabolism, probably r e s u l t i n g from an accumulation of reactants such as methionine, s-adenosyl methionine and homocysteine. These findings could be interpreted to mean that 0.50% DM i s the maximum l e v e l of methionine plus cystine incorporation i n the d i e t s before metabolic disturbances occur i n the methionine metabolic pathway. These r e s u l t s , together with the FCE and urinary urea-nitrogen excretion, emphasize the fact that the range 0.42-0.50% DM methionine plus cystine i s optimal for the growth of rat , either when the cystine l e v e l i s held constant at 0.20% DM ( T r i a l 3), or when the methionine to cystine r a t i o i s constant at 2:1 ( T r i a l 4). The dietary methionine plus cystine requirement range for the optimal growth of weanling r a t s , 0.42-0.50% DM, may also - 201 -be expressed as 3.31-3.67 m moles t o t a l sulphur per 100 g DM d i e t . This range i s i n agreement with the t o t a l amino acid sulphur requirement obtained by Sowers et al. (1972) and Stockland et al. (1973), and i s more meaningful, as i t i s not confounded by the methionine to cystine r a t i o . However, the methionine plus cystine requirements estimated i n the present t r i a l s may be lower than those found by other workers, purely because of the low energy levels of the diets which were used i n the present t r i a l s . A l l the diets approximated an average gross energy concentration of 3780 kcal/kg DM, which i s 86% of the 4400 kcal/kg DM recommended by the NRC (1972). I f the estimated dietary methionine plus cystine requirements were adjusted to the higher energy concentration, 0.42-0.50% DM would increase to 0.49-0.58% DM d i e t . This fi g u r e i s s t i l l lower than the NRC (1972) recommended requirement of 0.67% DM dietary methionine plus cystine for the growing r a t . I t was shown i n the rat t r i a l s that when the dietary cystine l e v e l was held constant at 0.20% DM (Rat T r i a l 3), growth and enzyme patterns, in response to varying lev e l s of dietary methionine were the same as when the dietary methionine, to cystine r a t i o was held constant at 2:1 (Rat T r i a l 4). When the dietary methionine to cystine r a t i o was held constant at 1:1 (Rat T r i a l 5), although the rate of growth of the rats was the same as on both a constant cystine l e v e l of 0.20% DM (Rat T r i a l 3), and a constant methionine to cystine - 202 -r a t i o of 2:1 (Rat T r i a l 4), the enzyme activity patterns were altered. Thus, c y s t a t h i o n i n e synthase a c t i v i t y was g e n e r a l l y lower i n r a t s r e c e i v i n g the 1:1 methionine to c y s t i n e r a t i o d i e t s (Rat T r i a l 5) than i n r a t s r e c e i v i n g the 2:1 methionine to c y s t i n e r a t i o d i e t s (Rat T r i a l 4), i n agreement wi t h o b s e r v a t i o n s of Shannon et al. (1972). The a c t i v i t y of mTHF Enz. decreased with an i n c r e a s i n g d i e t a r y methionine p l u s c y s t i n e l e v e l when the methionine t o c y s t i n e r a t i o was 1:1 (Rat T r i a l 5), but the a c t i v i t y was g e n e r a l l y h i g h e r than when the methionine to c y s t i n e r a t i o was 2:1 (Rat T r i a l 4 ) . T h i s means t h a t i n h i b i t i o n of the c y s t a t h i o n i n e synthase g e n e r a l l y corresponded t o the i n c r e a s e d mTHF Enz. a c t i v i t y , presumably i n order to remove the i n c r e a s i n g l e v e l s o f homocysteine o r i t s p r e c u r s o r s . These r e s u l t s c o u l d be i n t e r p r e t e d t o mean t h a t , although the methionine t o c y s t i n e r a t i o may not a f f e c t the growth r a t e of the weanling r a t s , the enzymes are d e f i n i t e l y a f f e c t e d . Byington et a l . (1972) i n d i c a t e d t h a t the 70:30 methionine to c y s t i n e r a t i o i s o p t i m a l f o r the growth of r a t s . Using a s i m i l a r methionine to c y s t i n e r a t i o , Rat T r i a l 4 of the presen t experiments showed the p a t t e r n s o f enzyme a c t i v i t i e s when the d i e t a r y methionine p l u s c y s t i n e v a r i e d between 0.45 and 0.70% DM d i e t . I f t h i s p a t t e r n of enzymes (Rat T r i a l 4) were accepted as the standard p a t t e r n of enzyme a c t i v i t i e s when the methionine to c y s t i n e r a t i o i s op t i m a l f o r growth i n r a t s , then the 1:1 methionine to c y s t i n e r a t i o , which was a completely - 203 -d i f f e r e n t enzyme p a t t e r n (Rat T r i a l 5) , i s not satisfactory for the optimal.growth of r a t s . T h i s means t h a t the s t a t e d maximum c y s t i n e replacement l e v e l of 50-68% of methionine requirements (NRC, 197 2: Rama Rao et al., 19 61) may not be c o r r e c t . I t i s suggested t h a t these r a t i o s be i n v e s t i g a t e d f u r t h e r u s i n g the enzymes of the methionine m e t a b o l i c pathway i n order to d e f i n e more a c c u r a t e l y the maximum l e v e l f o r c y s t i n e replacement of methionine. The r e s u l t s o b t a i n e d i n the p i g t r i a l u s i n g the change i n the u r i n a r y u r e a - n i t r o g e n e x c e r t i o n per kg DM feed consumed, u s i n g g i l t s , showed t h a t u r i n a r y urea e x c r e t i o n can be used as an i n d i c a t o r of amino a c i d requirements f o r growth i n p i g s . T h i s f i n d i n g i s i n agreement w i t h the c o n c l u s i o n s of Brown and C l i n e (1974), F u l l e r et al. (1974) and F u l l e r et al. (1975). Using t h i s parameter, the requirements of d i e t a r y methionine p l u s c y s t i n e f o r o p t i m a l n i t r o g e n r e t e n t i o n of 32.610.6 kg l i v e w e i g h t g i l t s r e c e i v i n g b a r l e y - b a s e d d i e t s has been found to be 0.55% DM d i e t . However, because of the l a r g e increments (0.10% DM) of d i e t a r y methionine used i n t h i s experiment, t h i s observed requirement i s not p r e c i s e . The p r e s e n t requirement, 0.55% DM methionine p l u s c y s t i n e , i s s l i g h t l y h igher than the NRC (1973) recommended l e v e l of 0.50% DM f o r the 20-35 kg l i v e w e i g h t p i g , and i s much high e r than the 0.23% DM methionine p l u s c y s t i n e i n d i c a t e d by Oestemer et al. (1970). - 2 0 4 -With the urea excretion of each pig on a standardized basal d i e t used to correct i t s excretion value on the test d i e t , the method appears to be quite sensitive,and o f f e r s the advantage of producing r e s u l t s i n a much shorter time than would be available from a growth and slaughter t r i a l . The present t r i a l s showed that both l i v e r cystathionine 5 synthase and N -methyltetrahydrofolate-homocysteme-methyltrans-ferase could be used to assess the optimal requirement of dietary methionine plus cystine for normal metabolism and, therefore, growth of weanling rats i n conjunction with the growth parameters. 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(1972) (1959) Arginine 0. 67 H i s t i d i n e 0. 33 0. 21 Isoleucine 0. 61 0. .55 Leucine 0. .81 0. . 69 Lysine 1. . 00 0. .90 Methionine + cystine 0. . 67 0. .49 Phenylalanine + tyrosine 0. . 89 0 . 72 Threonine 0. . 56 0, . 51 Tryptophan 0, .17 0, .11 Valine 0 . 67 0. .56 - 231 -APPENDIX 2 E s s e n t i a l amino a c i d requirements (% DM d i e t ) of the growing p i g (NRC, 197 3) Liveweight (kg) Amino a c i d 5--10 10--20 20--35 35--60 60--100 Ar g i n i n e 0 .28 0 . 23 0. 20 0 . 18 0 . 16 H i s t i d i n e 0 .25 0. 20 0. 18 0 . 16 0 .15 I s o l e u c i n e 0 .69 0. 56 0. 50 0. .44 0 .41 Leucine 0 .83 0. 68 0. 60 0 . 52 0 .48 Lysine 0 . 96 0. 79 0. 70 0 . 61 0 . 57 Methionine + c y s t i n e 0 .69 0 . 56 0. 50 0 . 44 0 .41 Phenylalanine + t y r o s i n e 0 . 69 0 . 56 0. 50 0 . 44 0 .41 Threonine 0 . 62 0. 51 0. 45 0, . 39 0 .37 Tryptophan 0 .18 0. 15 0. 13 0. .11 0 . 11 V a l i n e 0 .69 0-. 56 0. 50 0 .44 0 .41 - 232 -APPENDIX 3 Amino acid requirements (% DM diet) of growing meat producing poultry of 0-6 weeks of age NRC Hewitt and Lewis (1972) (1972)  Arginine 1.20 0.85 Glycine 1.00 0.61 H i s t i d i n e 0.40 0.40 Isoleucine 0.75 0.61 Leucine 1.4 0 1.3 4 Lysine 1.10 0.85 Methionine + cystine 0.75 0.79 Phenylalanine + tyrosine 1.30 1.27 Threonine 0.7 0 0.5 3 Tryptophan 0.20 0.17 Valine 0.85 0.79 - 233 -APPENDIX 4 Amino acid requirements of the 6 months - 1 year growing infant (mg/kg body-weight/day) NAS-RDA FAO/WHO (1974) (1973) H i s t i d i n e 33 34 Isoleucine 80 70 Leucine 128 161 Lysine 97 103 Methionine + cystine 45 58 Phenylalanine + tryosine 132 125 Threonine 63 87 Tryptophan 19 17 Valine 89 93 

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