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Effect of abomasal methionine infusion on methionine metabolism in the lamb Strath, Robert Adam 1977

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THE EFFECT OF ABOMASAL METHIONINE INFUSION ON METHIONINE METABOLISM IN THE LAMB by ROBERT ADAM STRATH B . S c , U n i v e r s i t y of B r i t i s h Columbia, 1973 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE i n THE FACULTY OF GRADUATE STUDIES (Dept. of Animal Science)' We accept t h i s t h e s i s as conforming to the r e q u i r e d s t a n d a r d THE UNIVERSITY OF BRITISH COLUMBIA December 1976 (c) Robert Adam S t r a t h , 1976 In p r e s e n t i n g t h i s t h e s i s i n p a r t i a l f u l f i l l m e n t of the requirements f o r an advanced degree at the U n i v e r s i t y of B r i t i s h Columbia, I agree t h a t the L i b r a r y s h a l l make i t f r e e l y a v a i l a b l e f o r r e f e r e n c e and study. I f u r t h e r agree t h a t p e r m i s s i o n f o r e x t e n s i v e copying.of t h i s t h e s i s f o r s c h o l a r l y purposes may be g r a t e d by the Head of my Department or by h i s r e p r e s e n t a t i v e s . I t i s understood t h a t c o p y i n g or p u b l i c a t i o n of t h i s t h e s i s f o r f i n a n c i a l g a i n s h a l l not be allowed without my w r i t t e n p e r m i s s i o n . Robert Adam S t r a t h Department of ANIMAL SCIENCE The U n i v e r s i t y of B r i t i s h Columbia Vancouver, Canada i i ABSTRACT The ruminant with i t s e x t e n s i v e s y n t h e s i s of amino a c i d s by s y m b i o t i c microorganisms presents unique problems to s t u d i e s of amino a c i d n u t r i t i o n . P r o t e i n q u a l i t y i s dependent upon the a v a i l a b l e amino a c i d s l e a v i n g the rumen, r a t h e r than those i n the i n g e s t e d d i e t . P r o t e c t e d p r o t e i n s or amino a c i d s may be f e d to the animals or a l t e r n a t i v e l y n i t r o g e n o u s compounds may be a d m i n i s t e r e d p o s t r u m i n a l l y to study ruminant amino a c i d n u t r i t i o n . There i s both a t h e o r e t i c a l and p r a c t i c a l i n t e r e s t i n d e f i n i n g ruminant amino a c i d requirements under v a r i o u s p r o d u c t i o n c o n d i t i o n s . The r e l a t i v e l y h i g h content of the s u l p h u r amino a c i d s i n ruminant products compared to t h a t p r e s e n t i n the rumen microorganisms suggests that these amino a c i d s may be a l i m i t i n g f a c t o r i n ruminant p r o d u c t i o n . In t h i s study the e s s e n t i a l s u l phur c o n t a i n i n g amino a c i d , D,L-methionine, was i n f u s e d i n t o the abomasum of two growing lambs. Graded l e v e l s of methionine were i n f u s e d f o r t h r e e days at each l e v e l . A j u g u l a r blood sample was c o l l e c t e d on the t h i r d day of each i n f u s i o n l e v e l . Plasma f r e e amino a c i d s were determined f o r each i n f u s i o n l e v e l . One lamb I n i t i a l l y had an i n c r e a s e i n plasma methionine c o n c e n t r a t i o n w i t h each i n c r e a s e i n the l e v e l of abomasal methionine. T h i s i n d i c a t e s t h a t methionine was not l i m i t i n g f o r t h i s lamb. For the other lamb an i n f l e c t i o n p o i n t on the methionine response curve was observed j u s t below 2.0 gm of i n f u s e d abomasal methionine. T h i s suggested t h a t methionine may have been l i m i t i n g f o r t h i s lamb. One lamb developed d i a r r h o e a at the higher i n f u s i o n i i i l e v e l s so a comparison of the two lambs was not p e r m i t t e d . T a u r i n e i n c r e a s e d with i n c r e a s i n g methionine i n f u s i o n s and c y s t i n e rose to a cons t a n t l e v e l . At t h i s i n f u s i o n l e v e l the n o n - e s s e n t i a l amino a c i d s - s e r i n e , g l y c i n e and a l a n i n e - f e l l t o c o n s t a n t l e v e l s . T h i s suggests t h a t methionine c o n v e r s i o n to c y s t i n e was impaired due to a decreased c o n c e n t r a t i o n of s e r i n e . G e n e r a l l y , h i g h methionine c o n c e n t r a t i o n s depressed the plasma c o n c e n t r a t i o n s of most amino a c i d s . 35 A t r a c e r dose of S-L-methionine was i n j e c t e d i n t o a j u g u l a r v e i n of one lamb at l e v e l s below and above the l i m i t i n g methionine i n f u s i o n . S e r i a l j u g u l a r b l o o d samples were c o l l e c t e d at v a r i o u s i n t e r v a l s to 24 hour p o s t - i n j e c t i o n . T o t a l u r i n e was c o l l e c t e d once d a i l y ' f o r f o u r days p o s t - i n j e c t i o n . The r a d i o a c t i v i t y a s s o c i a t e d with the plasma p r o t e i n s and other s u l p h u r c o n t a i n i n g compounds of plasma were expressed as per-c e n t a g e s . A plasma methionine s p e c i f i c a c t i v i t y curve i n d i c a t e d some k i n e t i c parameters of plasma methionine. When methionine was suspected of b e i n g l i m i t i n g , a l l of the a c t i v i t y of plasma was i n the plasma p r o t e i n f r a c t i o n a f t e r 24 hours. There was no d e t e c t a b l e c o n v e r s i o n of methionine s u l p h u r to o t h e r sulphur c o n t a i n i n g compounds i n d e p r o t e i n i z e d plasma. U r i n a r y e x c r e t i o n of the a d m i n i s t e r e d l a b e l was 13% a f t e r f o u r days. During the i n f u s i o n l e v e l when methionine was not l i m i t i n g , o n l y one h a l f of the a c t i v i t y of plasma was i n the p r o t e i n f r a c t i o n a f t e r 24 hours. At t h i s l e v e l t h e r e was r a d i o a c t i v i t y i n methionine, t a u r i n e , c y s t i n e , c y s t a t h i o n i n e and methionine i v s u l f o x i d e s o f f r e e plasma. The u r i n a r y e x c r e t i o n o f r a d i o -a c t i v i t y a f t e r f o u r days was o n e - h a l f o f the i n j e c t e d dose. The plasma m e t h i o n i n e k i n e t i c s i n d i c a t e d t h a t t h e e l e v a t e d m e t h i o n i n e p o o l s i z e was not due t o an i n c r e a s e i n t o t a l e n t r y r a t e but due t o a s m a l l e r i n c r e a s e i n i r r e v e r s i b l e l o s s . T h i s a g r e e s f a v o u r a b l y w i t h i m p a i r e d m e t h i o n i n e m e t a b o l i s m . I t was c o n c l u d e d t h a t a t r a c e r dose of m e t h i o n i n e gave a dynamic p i c t u r e o f m e t h i o n i n e m e t a b o l i s m . Below i t s l i m i t i n g l e v e l , m e t h i o n i n e i s m a i n l y i n v o l v e d i n a n a b o l i c p r o c e s s e s . Above the l e v e l o f l i m i t i n g m e t h i o n i n e , c a t a b o l i c m e t a b o l i s m of m e t h i o n i n e becomes i n c r e a s i n g l y i m p o r t a n t . T h e r e f o r e , i t 35 i s s u g g e s t e d t h a t S-methiomne t r a c e r t e c h n i q u e s may p r o v i d e a r e l a t i v e l y q u i c k and r e l i a b l e t o o l f o r e v a l u a t i o n o f t h e s u l p h u r amino a c i d n u t r i t i o n o f r u m i n a n t s . V TABLE OF CONTENTS Page ABSTRACT i i TABLE OF CONTENTS . v LIST OF TABLES v i i LIST OF FIGURES . v i i i INTRODUCTION 1 I. SULPHUR IN RUMINANT NUTRITION 3 Rumen sulphur metabolism 5 I n t e r r e l a t i o n s h i p s of sulphur w i t h other m i n e r a l s 8 P o s t - r u m i n a l s u l p h u r metabolism 11 E f f e c t s of su l p h u r d e f i c i e n c y and excess . . . 15 Sulphur supplementation . 18 I I . NITROGEN IN RUMINANT NUTRITION 21 I I I . PLASMA AMINO ACIDS AND RUMINANT NUTRITION . . . . 27 IV. PLASMA UREA AND RUMINANT NUTRITION 35 EXPERIMENTS 37 I. E f f e c t of abomasal methionine i n f u s i o n on plasma amino a c i d p r o f i l e s and plasma urea c o n c e n t r a t i o n s 37 a) M a t e r i a l s and methods 37 b) R e s u l t s 39 c) D i s c u s s i o n 48 I I . Abomasal methionine i n f u s i o n and r a d i o a c t i v e methionine t r a c e r 52 a) M a t e r i a l s and methods 52 b) R e s u l t s 53 c) D i s c u s s i o n 61 v i P a g e S U M M A R Y 6 7 B I B L I O G R A P H Y 7 0 v i i LIST OF TABLES T a b l e Page 1. E f f e c t of abomasal i n f u s i o n of methionine on the c o n c e n t r a t i o n of amino a c i d s i n plasma i n lamb #1 . 40 2. E f f e c t of abomasal i n f u s i o n of methionine on the c o n c e n t r a t i o n of amino a c i d s i n plasma i n lamb #2 41 3. E f f e c t of abomasal i n f u s i o n of methionine on the c o n c e n t r a t i o n of urea i n plasma „ 47 35 4. S a c t i v i t y of the s u l p h u r - c o n t a i n i n g amino a c i d s , expressed as a percentage of the t o t a l a c t i v i t y i n the s u l p h u r - c o n t a i n i n g amino a c i d s at 5.0 gm abomasal methionine 57 5. Methionine plasma k i n e t i c s 60 v i i i LIST OF FIGURES F i g u r e Page 1. E f f e c t s of abomasal methionine i n f u s i o n s on the c o n c e n t r a t i o n of sulphur amino a c i d s i n plasma i n lamb #1 43 2. E f f e c t s of abomasal methionine i n f u s i o n s on the c o n c e n t r a t i o n of sulphur amino a c i d s i n plasma i n lamb #2 44 3. E f f e c t s of abomasal methionine i n f u s i o n s on the c o n c e n t r a t i o n s of some n o n - e s s e n t i a l amino a c i d s i n plasma i n lamb #2 45 4. E f f e c t s of abomasal methionine i n f u s i o n s on the c o n c e n t r a t i o n of the branch-chained amino a c i d s i n plasma i n lamb #2 46 35 5. Percentage of S a c t i v i t y of plasma l o c a t e d i n the plasma p r o t e i n s at two abomasal methionine i n f u s i o n l e v e l s . . 54 6. Percentage of a d m i n i s t e r e d dose d e t e c t e d i n u r i n e 56 7. Plasma methionine s p e c i f i c a c t i v i t y curve a t 0.0 gm abomasal methionine i n f u s i o n 58 8. Plasma methionine s p e c i f i c a c t i v i t y curve at 5.0 gm abomasal methionine i n f u s i o n 59 1 INTRODUCTION The m i c r o b i a l c e l l s i n the rumen have a m o d i f y i n g a f f e c t on the d i e t of the ruminant. The sources of n u t r i e n t s a v a i l a b l e to the host animal i n c l u d e u n a l t e r e d f@ed, m i c r o b i a l c e l l s and m i c r o b i a l m e t a b o l i t e s . T h e r e f o r e , the c l a s s i c a l methods used i n n u t r i t i o n a l experiments w i t h the simple stomached animal may not be a p p l i c a b l e f o r s t u d i e s with the ruminant. Methionine has been shown to be l i m i t i n g f o r both sheep and c a t t l e t i s s u e s y n t h e s i s . Changes i n PAA p a t t e r n s due to p o s t - r u m i n a l i n f u s i o n s are the techniques used f o r the e s t i m a -t i o n of the methionine requirement. Changes i n PAA l e v e l s are m o d i f i e d by both the l i v e r and muscle t i s s u e s . A r e l a t i v e l y s m a l l change i n PAA l e v e l s r e p r e s e n t s a much g r e a t e r change i n t i s s u e l e v e l s . A dynamic p i c t u r e of amino a c i d n u t r i t i o n can be s t u d i e d by u s i n g r a d i o a c t i v e amino a c i d s . The o b j e c t of t h i s t h e s i s i s to study the e f f e c t s of abomasally i n f u s e d methionine i n the growing lamb. The lambs w i l l be f e d a standard d i e t of hay and b a r l e y . J u g u l a r blood samples w i l l be c o l l e c t e d at i n c r e a s i n g l e v e l s of abomasal methionine i n f u s i o n s . The changes i n the PAA p r o f i l e s w i l l be measured at the v a r i o u s methionine i n f u s i o n l e v e l s . The plasma methionine response curve s h o u l d i n d i c a t e i f methionine i s l i m i t i n g f o r these lambs. Another o b j e c t i v e of t h i s r e s e a r c h i s to o b t a i n a dynamic p i c t u r e o f • m e t h i o n i n e metabolism by employing a r a d i o a c t i v e t r a c e r . Rather than u s i n g a carbon l a b e l l e d t r a c e r t h i s study w i l l use a t r a c e r c o n t a i n i n g r a d i o -a c t i v e s u l p h u r . C o l l e c t i o n of the o x i d a t i v e products i n t h i s 2 case w i l l be from u r i n e which w i l l e l i m i n a t e the problems of e x p i r e d CO^ c o l l e c t i o n . Some plasma parameters w i l l be e s t i m a t e d from the plasma methionine s p e c i f i c a c t i v i t y c u r v e s . The i n c o r p o r a t i o n of r a d i o a c t i v i t y i n t o plasma p r o t e i n s as w e l l as o t h e r s u l p h u r c o n t a i n i n g compounds of plasma should i n d i c a t e the g e n e r a l m e t a b o l i c f a t e of methionine. I t i s hoped t h a t changes i n the PAA p r o f i l e s and the k i n e t i c s of the r a d i o -a c t i v e s u lphur c o n t a i n i n g t r a c e r w i l l i n d i c a t e the m e t a b o l i c f a t e of methionine i n the lamb. 3 I. SULPHUR IN RUMINANT NUTRITION The amount and p o s s i b l e forms of p l a n t sulphur t h a t may u l t i m a t e l y be i n g e s t e d by ruminant animals have been docu-mented ( G a r r i g u s , 1970; Moir, 1970; and Playne, 1975). I n f o r m a t i o n on s u l p h u r c o n t e n t of f e e d s , on sulphur r e q u i r e -ments of l i v e s t o c k and on s u l p h u r metabolism i n ruminants has been l i m i t e d u n t i l r e c e n t y e a r s , l a r g e l y because of problems and u n c e r t a i n t i e s i n the d e t e r m i n a t i o n of sulphur (Beaton et a l . , 1968). Of the t o t a l s u l phur i n p l a n t m a t e r i a l most i s o r g a n i c i n c o m p o s i t i o n (Moir, 1970). Sulphur c o n c e n t r a t i o n s i n whole p l a n t tops cannot be used to make r e l i a b l e e s t i m a t e s of sulphur i n t a k e by animals g r a z i n g p a s t u r e s because they graze s e l e c t i v e l y . Playne (1975) has shown that s p e c i e s , season and f e r t i l i z a t i o n a f f e c t the s u l p h u r content of p l a n t m a t e r i a l . F e rmentation i n the rumen, on p r o t e i n - r i c h d i e t s , may l e a d to n i t r o g e n and sulphur waste v i a the p r o d u c t i o n and a b s o r p t i o n of ammonia and s u l p h i d e . However, the s y n t h e s i s of m i c r o b i a l p r o t e i n from n o n - p r o t e i n n i t r o g e n and s u l p h u r may enhance the n u t r i t i v e v a l u e of poor q u a l i t y d i e t s . In a d d i t i o n to the d i e t a r y sources of sulphur t h e r e i s some r e c y c l i n g of sulphur w i t h i n the body of the ruminant. Bray (1969) was able to demonstrate t h i s phenomenon i n sheep by i n t r a v e n o u s i n j e c t i o n s of S - l a b e l l e d s u l p h a t e f o l l o w e d by the r e c o v e r y of the l a b e l l e d s u l p h a t e i n rumen. He showed t h a t the l a b e l l e d s u l p h a t e c r o s s e d the rumen w a l l from blood to the rumen. I t was a l s o shown t h a t some sulphate i s 4 r e c y c l e d v i a the s a l i v a . Moir (1970) r e p o r t e d a high c o r r e l a t i o n between l e v e l s of blood s u l p h a t e and s a l i v a r y s u l p h a t e . E f f i c i e n t r e c y c l i n g of s u l p h u r through p a n c r e a t i c and b i l e s e c r e t i o n s a l s o conserves s u l p h u r i n sheep ( B i r d , 1972). The s u l p h a t e r e c y c l e d to the rumen i s s u b j e c t e d to r e d u c t i o n and i s then a v a i l a b l e f o r p r o t e i n s y n t h e s i s , i n much the same way as ammonia i s r e l e a s e d from r e c y c l e d urea, thus p r o v i d i n g a v a l u a b l e c o n s e r v a t i o n mechanism when fe e d i s low i n s u l p h u r . S u l p h i d e and s u l p h a t e form a r e c y c l i n g system t h a t i s i n many ways s i m i l a r to the ammonia-urea systems (Bray and T i l l , 1975). Even though sulphur i s r e c y c l e d to the r e t i c u l o - r u m e n the N:S r a t i o of r e c y c l e d n i t r o g e n to. r e c y c l e d sulphur may be very wide so that, m i c r o b i a l growth i s l i m i t e d due to i n s u f f i c i e n t s u l p h u r (Moir, 1970). Kennedy et. a_l. (1975 ) q u a n t i t a t e d the r e c y c l i n g of s u l p h a t e to the rumen by employing r a d i o t r a c e r t e c h n i q u e s . They concluded t h a t s u l p h a t e r e c y c l i n g to the rumen of sheep i s c o n s i d e r a b l y l e s s than t h a t of c a t t l e due mainly to r e l a t i v e l y low c o n c e n t r a t i o n of i n o r g a n i c s u l p h a t e i n b l o o d . They suggest t h a t the d i f f e r e n c e i n the i n o r g a n i c s u l p h a t e i n b l o o d r e f l e c t s the high s u l p h u r content of wool i n sheep. The gut can be d i v i d e d i n t o two main systems i n terms of sulphur metabolism i n the ruminant. The f i r s t i s the r e t i c u l o -rumen where microbes reduce d i e t a r y and r e c y c l e d s u l p h u r and c o n v e r t t h i s i n t o m i c r o b i a l p r o t e i n . The second major system i s p o s t - r u m i n a l and concerns the o v e r a l l process of d i g e s t i o n and a b s o r p t i o n . Each system has an e f f e c t on the o t h e r and has i t s own n u t r i e n t requirement. 5 Rumen Sulphur Metabolism Sulphur i s r e q u i r e d f o r m i c r o b i a l p r o t e i n s y n t h e s i s i n the rumen ( B i r d , 1972). With r e s p e c t to sulphur metabolism t h r e e groups of b a c t e r i a can be d i s t i n g u i s h e d i n the rumen. Those b a c t e r i a t h a t cannot meet t h e i r own needs f o r reduced s u l p h u r form the f i r s t group. At l e a s t two s t r a i n s of S t r e p - tococcus b o v i s are unable to use s u l p h a t e or s u l p h i t e f o r growth (Bray and T i l l , 1975). The second group of micro-organisms which reduce s u l p h a t e to the s u l p h i d e l e v e l and i n c o r p o r a t e the sulphur i n t o c e l l u l a r m a t e r i a l s without the p r o d u c t i o n of any f r e e d e t e c t a b l e s u l p h i d e s and are termed a s s i m i l a t o r y s u l p h a t e - r e d u c i n g microorganisms (Bray and T i l l , 1975). The i n i t i a l a c t i v a t i o n of s u l p h a t e r e q u i r e s adenosine t r i p h o s p h a t e (ATP) from which adenosine-5'-phospho-sulphate (APS) i s formed. APS i s then p h o s p h o r y l a t e d i n the 3' p o s i t i o n by ATP to y i e l d 3'-phosphoadenosine-5'-phosphosulphate (PAPS), which i s the a c t i v a t e d form reduced to the s u l p h i d e l e v e l . A t h i r d group of microorganisms u t i l i z e s s u l p h a t e as the t e r m i n a l e l e c t r o n a c c e p t o r and produces massive amounts of hydrogen s u l p h i d e (H^S), and these are- termed d i s s i m i l a t o r y s u l p h a t e r e d u c i n g organisms. APS, r a t h e r than PAPS, i s the a c t i v a t e d form of s u l p h a t e reduced by these microorganisms ( S i e g e l , 1975). D i s s i m i l a t o r y s u l p h a t e r e d u c e r s have been found i n con-2 8 c e n t r a t i o n s of 10 -10 /ml of rumen l i q u o r ( Huisingh, 1973; c i t e d by Bray and T i l l , 1975). H u i s i n g h has a l s o shown that D e s u l f o v i b r i o ruminus can reduce s u l p h a t e at the r a t e of 9.4 1 0 micromoles/hr/10 c e l l s . 6 R e c e n t l y M e r r i c k s and S a l s b u r g (1976) have shown t h a t the rumen p r o t o z o a a l s o have an e f f e c t on m e t h i o n i n e - s u l p h u r meta-b o l i s m . A compartmental model of sulphur metabolism i n the rumen has been proposed by Bray and T i l l (1975). Sulphide i s the c e n t r a l i n t e r m e d i a t e between the incoming sulphur from d i e t a r y and r e c y c l e d sources and the outgoing s u l p h u r made up of s u l p h u r i n c o r p o r a t e d i n t o m i c r o b i a l c e l l s and o t h e r l o s s e s from the system. R e c e n t l y Gawthorne and Nader (1976) have shown i n d i r e c t l y t h a t methionine s u l p h u r can be i n c o r p o r a t e d i n t o m i c r o b i a l c e l l s ( 4 3 - 4 8 % ) without e n t e r i n g the s u l p h i d e p o o l . Thus, the extent of d i r e c t i n c o r p o r a t i o n of s u l p h u r amino a c i d s by rumen microorganisms appears to be g r e a t e r than g e n e r a l l y b e l i e v e d (Whanger, 1972). In v i t r o and _in v i v o a v a i l a b i l i t y s t u d i e s of d i f f e r e n t o r g a n i c and i n o r g a n i c forms of sulphur (Kahdon e t a_l. , 1975a and 1975b) have shown t h a t they are a l l a v a i l a b l e i n v a r i o u s p r o p o r t i o n s to the microbes f o r i n c o r p o r a t i o n i n t o m i c r o b i a l c e l l s . L-methionine was b e s t f o r f i x a t i o n i n t o m i c r o b i a l p r o t e i n and the hydroxy analogs of methionine were p o o r l y u t i l i z e d . Lambs were f e d s e m i - p u r i f i e d d i e t s and the a d d i t i o n of any of the s u l p h u r forms r e s u l t e d i n a f a s t e r r a t e of g a i n , i n c r e a s e d consumption of dry matter and g r e a t e r r e t e n t i o n of both n i t r o g e n and sulphur (Katilon, e t al. , 1975b). Sulphur supplementation i n c r e a s e d i_n v i t r o d i g e s t i o n of f o r a g e c e l l u l o s e by rumen microorganisms (Spears e t a l . , 1976). They found e l e m e n t a l s u l p h u r and methionine were e q u a l l y e f f e c t i v e f o r i n c r e a s e d c e l l u l o s e d i g e s t i o n of Ky 31 and Kenby t a l l f e s c u e . 7 Due to the metabolism of the microbes i n the forestomach of the ruminant the d i g e s t a p a s s i n g to the abomasum i s p r e s e n t e d to the host i n an a l t e r e d form from.that o r i g i n a l l y consumed. In a d d i t i o n to s y n t h e s i z i n g s u l p h u r amino a c i d s ( i . e . m i c r o b i a l c e l l p r o t e i n ) , the microorganisms a l s o s y n t h e s i z e s u l p h u r c o n t a i n i n g v i t a m i n s (thiamine and b i o t i n ) which have n u t r i t i o n a l v a l u e f o r the host ( G a r r i g u s , 1970). S i n c e v i t a m i n s are produced i n t r a c e amounts r e l a t i v e to the s y n t h e s i s of p r o t e i n , they do not q u a n t i t a t i v e l y p l a y a l a r g e r o l e i n the s u l p h u r balance of the p r o d u c i n g ruminant. I n c r e a s e s i n both thiamine and b i o t i n l e v e l s have been observed i n rumen samples when d i e t s have been supplemented w i t h e l e m e n t a l s u l p h u r ( A l b e r t , 1954). D e f i c i e n c i e s of thiamine i n ruminants have not been thought to e x i s t under f i e l d c o n d i t i o n s but the N u t r i t i o n Foundation, Inc. (1969) have r e p o r t e d t h a t c l i n i c a l symptoms of thiamine d e f i c i e n c y i n young c a t t l e and sheep were cured a f t e r treatment with t h i a m i n e . When c o n s i d e r i n g the s u l p h u r requirements of the ruminant animal and the d i f f e r e n t sources and c h e m i c a l forms of sulphur i n the d i e t i t i s d i f f i c u l t to e s t i m a t e the exact l e v e l of d i e t a r y s u l p h u r - c o n t a i n i n g compounds r e q u i r e d f o r both 'the microbes and the host. S i n c e most of the n a t u r a l s u l p h u r c o n t a i n i n g compounds e n t e r the " s u l p h i d e p o o l " (due to the rumen m i c r o b i a l a c t i v i t y ) i t suggests t h a t only the t o t a l s u l p h u r l e v e l i n the d i e t be c o n s i d e r e d f o r n u t r i t i o n a l s t u d i e s . The o v e r a l l amino a c i d c o m p o s i t i o n of rumen b a c t e r i a or mixed b a c t e r i a and p r o t o z o a i s not g r e a t l y a f f e c t e d by the 8 f e e d consumed (Purser and B u e c h l e r , 1966; L e i b h o l z , 1972). However, these workers have shown t h a t the sulphur c o n t a i n i n g amino a c i d s (methionine and c y s t i n e ) of the rumen b a c t e r i a v a r y to a g r e a t e r degree than the o t h e r amino a c i d s . The a d d i t i o n of methionine to the b a s a l r a t i o n f o r sheep brought about a h i g h e r content of a r g i n i n e , a s p a r t i c a c i d and s e r i n e i n b a c t e r i a l p r o t e i n , and a s l i g h t d e crease i n i s o l e u c i n e and l e u c i n e c o n t e n t s ( K u r i l o v et. al_. , 1976). The h i n d gut i n sheep may a l s o p l a y a r o l e i n s u l p h u r metabolism (Bray and T i l l , 1975). Judson et. a_l. (1975 ) s t u d i e d 35 the d i g e s t i o n and u t i l i z a t i o n of S - l a b e l l e d b a c t e r i a t h at were p l a c e d i n the caecum of sheep. Most of the a c t i v i t y was e x c r e t e d i n the f e c e s (70%) or the u r i n e (21%). Approximately 10% of the a c t i v i t y was r e t a i n e d of which 3% was d e t e c t e d i n the f l e e c e . I n t e r r e l a t i o n s h i p s of Sulphur with o t h e r M i n e r a l s The i n t e r r e l a t i o n s h i p s of sulphur w i t h other m i n e r a l s have been reviewed (Muth and O l d f i e l d , 1970; Whanger, 1972). T h e r e f o r e , o n l y more r e c e n t evidence w i l l be d i s c u s s e d here. The i n t e r a c t i o n s of copper, molybdenum, selenium and s u l p h u r have been s t u d i e d by many workers. S u t t l e (1975) observed t h a t molybdenum and sulphur i n h i b i t the r e p l e t i o n of plasma copper l e v e l s . Sulphur alone i n h i b i t e d r e p l e t i o n s l i g h t l y w h i l e molybdenum alone had no e f f e c t . H u i s i n g h et. a l . (1973) proposed t h a t copper becomes u n a v a i l a b l e v i a two r o u t e s : (1) the f o r m a t i o n of c u p r i c molybdate which i s absorbed and e x c r e t e d r e n d e r i n g both copper and molybdate l e s s a v a i l a b l e ; 9 and (2) the f o r m a t i o n of an i n s o l u b l e c u p r i c s u l p h i d e i n the rumen, i n t e s t i n e s or t i s s u e s . Regarding the i n t e r a c t i o n of s u l p h a t e and molybdate they proposed t h a t s e v e r a l s i t e s are i n v o l v e d with d i f f e r e n t e f f e c t s . Dick e t a_l. (1975 ) have suggested t h e r e may be a b l o c k i n g of copper t r a n s p o r t a c r o s s membranes which i s c o n t r o l l e d by the molybdenum i n t a k e . A c c o r d i n g to them the e l e v a t e d blood-copper v a l u e s which are observed with high molybdenum d i e t s are a r e s u l t of m o b i l i z e d t i s s u e copper and not due to a b s o r p t i o n from the rumen. Gawthorne and Nader (1976) r e p o r t e d t h a t molybdenum decreased r e d u c t i o n of s u l p h a t e to s u l p h i d e i n the rumen by competing f o r the f i r s t enzyme, A T P - s u l p h u r y l a s e . Under these c o n d i t i o n s t h e r e was an i n c r e a s e i n c o n c e n t r a t i o n of s u l p h i d e i n the rumen which was due to r e d u c t i o n i n a b s o r p t i o n of s u l p h i d e from the rumen. Bryden and Bray (1972) observed t h a t molybdenum s e v e r e l y depress rumen s u l p h i d e l e v e l s . H u i s i n g h e t a l . (1975) r e p o r t e d t h a t 50 ppm of molybdenum i n the d i e t s i g n i f i c a n t l y i n h i b i t e d s u l p h i d e p r o d u c t i o n from s u l p h a t e but enhanced s i g n i f i c a n t l y the p r o d u c t i o n of s u l p h i d e from methionine. The reasons f o r t h i s o b s e r v a t i o n are not known. Selenium may compete with sulphur i n the s y n t h e s i s of sul p h u r c o n t a i n i n g amino a c i d s and p o s s i b l e other s u l p h u r c o n t a i n i n g compounds i n the rumen. In p o u l t r y Cantor e_t a l . (1975) found s e l e n o m e t h i o n i n e to have low a v a i l a b i l i t y when i n c o r p o r a t e d i n t o a p o u l t r y r a t i o n . Selenomethionine may be produced by the same pathway as methionine and i f i n c o r p o r a t e d i n t o p r o t e i n s may a f f e c t systems where -SH i s r e q u i r e d . 75 —2 Godwin et _al_. (1971) found i n o r g a n i c SeO^ gi v e n as Na 0 10 7 R 75 SeO^ had about 3% of Se i n c o r p o r a t e d i n t o milk of sheep as s e l e n o m e t h i o n i n e . Selenium competes wi t h sulphur i n the f o l l o w i n g r e a c t i o n s (De Meiro, 1975 ) : ATP + S 0 4 ~ 2 APS A T P - s u l p h u r y l a s e or — > or SeO^" 4 APSe ATP + APS PAPS or — — — > or APSe PAPSe Selenium i n PAPSe can take the p l a c e of s u l p h u r i n a l l r e a c t i o n s i n v o l v i n g s u l p h u r y l a t i o n . R e c e n t l y Fuss and Godwin (1975) s t u d i e d the f a t e of 75 75 selenium, g i v e n as Na s SeO, or Se s e l e n o m e t h i o n i n e , admini-s t e r e d I n t r a v e n o u s l y to ewes and lambs. Small, though 75 s i g n i f i c a n t amounts of selenium, d e r i v e d from Na^ SeO^, were i n c o r p o r a t e d as selenoamino a c i d s i n t o the p r o t e i n s of l i v e r , k i d n e y and pancreas, as w e l l as i n t o the p r o t e i n s of milk and plasma. The a c t i v i t y c o u l d be d e t e c t e d i n both selenomethio-nine and s e l e n o c y s t i n e chromatographic f r a c t i o n s . P reston e_t a_l. (1974) r e p o r t e d an i n t e r a c t i o n between su l p h u r and potassium i n c a t t l e d i e t s . Rate of g a i n i n s t e e r s was depressed by i n c r e a s i n g the i n o r g a n i c sulphur c o n t e n t of the d i e t from 0.10 to 0.14%, but the a d d i t i o n of potassium appeared to overcome t h i s d e p r e s s i o n . 11 P o s t - r u m i n a l Sulphur Metabolism Sulphur i s r e q u i r e d by the animal because i t i s a v i t a l c o n s t i t u e n t of c e r t a i n amino a c i d s ( i . e . methionine and c y s t i n e ) and t h e r e f o r e of p r o t e i n s . In a d d i t i o n , sulphur i s a c o n s t i -tuent of c e r t a i n v i t a m i n s ( i . e . thiamine and b i o t i n ) . Thiamine i s i n v o l v e d i n the "one carbon p o o l " and s p e c i f i c a l l y carbon d i o x i d e metabolism (Ledger, 1975). B i o t i n i s a l s o i n v o l v e d i n the "one carbon p o o l " and i s i n v o l v e d i n d e c a r b o x y l a t i o n r e a c t i o n s ( E i s e n b e r g , 1975). Sulphur i s a l s o a component of c e r t a i n co-enzymes (Co-enzyme A, Abiko, 1975; and l i p o i c a c i d , Koike and Koike, 1975). G l u t a t h i o n e ( M e i s t e r , 1975) i s a l s o a s u l p h u r c o n t a i n i n g compound and i s important i n mammalian metabolism. The s u l p h u r amino a c i d s found i n p r o t e i n are L-methionine, L - c y s t i n e , and L - c y s t e i n e . Methionine i s c o n s i d e r e d the most important i n the d i e t of a monogastric animal, s i n c e the others can be s y n t h e s i z e d from methionine (Maynard and L o o s l i , 1969). Methionine, l i k e most amino a c i d s can be i n c o r p o r a t e d i n t o p r o t e i n but a l s o has o t h e r f u n c t i o n s . I t s c o n v e r s i o n to c y s t a t h i o n i n e , c y s t i n e and c y s t e i n e can supply the animal's needs f o r these sulphur c o n t a i n i n g amino a c i d s . M e t h i o n i n e may form S-adenosylmethionine (SAM), which i s a u n i v e r s a l b i o l o g i c a l t r a n s m e t h y l a t i n g agent. Abundant evidence has accumulated t h a t p r o t e i n s y n t h e s i s i s i n i t i a t e d by the formu-l a t i o n of methionine to N-formylmethionine (Lehninger, 1972). The m u l t i f u n c t i o n a l nature of the methionine requirement makes i f d i f f i c u l t t o determine the exact l e v e l r e q u i r e d i n the 12 d i e t . Other d i e t a r y compounds may spare the methionine from some of the above r o l e s ( M o l i t o s i s and Baker, 1976). I n o r g a n i c sulphur i n i t s o x i d i z e d form, s u l p h a t e , p l a y s a r o l e i n d e t o x i f y i n g and e x c r e t i o n of many compounds (De Meio, 1975). Sulphate i s the end product of su l p h u r amino a c i d o x i d a t i o n and i s e x c r e t e d i n the u r i n e . When r e v i e w i n g the p u b l i s h e d r e s u l t s of the s u l p h u r requirements of the ruminant one i s impressed with the wide range and l a c k of agreement. Some of the d i f f e r e n c e c o u l d be a t t r i b u t e d to the type of animals b e i n g s t u d i e d . I t seems ve r y p o s s i b l e t h a t sheep, beef and d a i r y c a t t l e may have d i f f e r e n t r e q u i r e m e n t s . A l s o the p h y s i o l o g i c a l s t a t u s or type of p r o d u c t i o n c r i t e r i a b e i n g measured ( i . e . wool growth or r a t e of ga i n i n sheep) may a f f e c t the r e q u i r e d d i e t a r y sulphur l e v e l . Other c o n s i d e r a t i o n s which may have an e f f e c t on the e s t i m a t i o n of the sulphur requirement are type of d i e t ( n a t u r a l or s e m i - p u r i f i e d ) , source of sulphur ( o r g a n i c or i n o r g a n i c ) , source of n i t r o g e n ( n a t u r a l or n o n p r o t e i n n i t r o g e n ) and the type of e x p e r i m e n t a l d e s i g n . Much of the e a r l y r e s e a r c h attempting to e s t a b l i s h the su l p h u r requirement of the ruminant was conducted w i t h sheep. One of the f i r s t s t u d i e s r e p o r t e d (Whiting e_t _ a l . , 1954) used mature range ewes f e d v a r i o u s scources of supplemental sulphur at d i f f e r e n t l e v e l s added to a d i e t low i n s u l p h u r . They concluded t h a t the su l p h u r requirement was l e s s than 0.1% of the d i e t , which i s lower than l e v e l s which have been p u b l i s h e d s u b s e q u e n t l y . A l b e r t et al_. (1956) used growing lambs and concluded t h a t the sulphur requirement was met when 0.138% 13 s u l p h u r was added as m ethionine. Perhaps the h i g h e s t sulphur requirement was r e p o r t e d by Evans and Davis (1961) who demon-s t r a t e d t h a t on the b a s i s of c e l l u l o s e d i g e s t i o n i n the rumen the optimum l e v e l of s u l p h u r i n the d i e t was 0.29%. T h i s h i g h e r l e v e l based upon iri v i v o r e s u l t s i s s i m i l a r to some of the optimum l e v e l s r e p o r t e d from iri v i t r o r e s u l t s (Barton e_t a l . , 1971; B u l l and V a n d e r s a l l , 1973). Bray (1965) concluded t h a t a s u l p h u r l e v e l of 0.14% was adequate f o r maximum n i t r o g e n r e t e n t i o n insheep. B i r d (1972) has shown t h a t the r a t i o of N:S i n sheep t i s s u e i s about 13.5:1, and c o n s i d e r e d t h a t a narrower r a t i o than t h i s was necessary f o r o p t i m a l usage of d i e t a r y n i t r o g e n by sheep. B i r d (1973) observed the r a t i o of n i t r o g e n to s u l p h u r i n rumen b a c t e r i a to be about 20:1; he concluded t h a t the microbes were d e f i c i e n t i n s u l p h u r amino a c i d s with r e s p e c t to the host's t i s s u e s y n t h e s i s . Moir ejt _al_. (1967) conc l u d e d t h a t the optimum n i t r o g e n to s u l p h u r r a t i o i s about 10:1. Kennedy et. a l . (1975) a l s o showed t h a t a N:S of 10:1 i s optimum f o r sheep, and animals on low q u a l i t y roughages may have N:S r a t i o s as wide as 50:1 and at best 12:1 which i n c l u d e s the r e c y c l e d s u l p h u r . T h i s r e l a t i o n s h i p between d i e t a r y n i t r o g e n and s u l p h u r i s p r e f e r r e d by many workers and c o n s i d e r s the sulphur requirement as a f u n c t i o n of the n i t r o g e n content of the d i e t . Kennedy and S i e b e r t (1975), suggest f e e d i n g molasses with urea supplemented r a t i o n to decrease the N:S r a t i o towards the optimum. Molasses c o n t a i n s approximately 10 gm S/Kg DM and may have c o n t r i b u t e d to the p o s i t i v e r e s u l t s i n molasses-urea r a t i o n s r e p o r t e d throughout the l i t e r a t u r e . 14 Some s t u d i e s u s i n g d a i r y c a t t l e have f a i l e d to show any improvement when n a t u r a l d i e t s were supplemented wi t h s u l p h u r . Jacobson e_t a_l. (1969) r e p o r t e d no improvement i n d a i r y cows when n a t u r a l d i e t s c o n t a i n i n g about 0.10% sulphur were supple-mented with sodium s u l p h a t e to r a i s e s u l p h u r to 0.18%. Grieve e t a l . (1973a) observed no improvement i n feed i n t a k e or milk y i e l d when sodium s u l p h a t e was added to c o r n s i l a g e d i e t s c o n t a i n i n g between 0.11 and 0.13% s u l p h u r . N i t r o g e n u t i l i z a -t i o n was not improved by the a d d i t i o n of sodium s u l p h a t e (G r i e v e e_t _al_. , 1973b). In these s t u d i e s i t appeared t h a t the s u l p h u r requirement was met by 0.11-0.13% sulphur p r e s e n t i n the b a s a l d i e t . Bouchard and Conrad (1973) found t h a t the a d d i t i o n of s u l p h u r to a low sulphur d i e t Improved the p r o d u c t i o n of d a i r y cows as measured by v a r i o u s c r i t e r i a . Based upon r e g r e s s i o n a n a l y s i s they concluded t h a t sulphur b a l a n c e c o u l d be accom-p l i s h e d by 0.12% d i e t a r y s u l p h u r and t h a t 0.18% s u l p h u r would a l l o w f o r a p o s i t i v e s u l p h u r balance of 4.0 gm d a i l y i n producing cows. Chalupa ejt a_l. (1973 ) r e p o r t e d an improved growth r a t e i n Angus s t e e r s when sodium s u l p h a t e or e l e m e n t a l s u l p h u r was added to b r i n g d i e t a r y s u l p h u r from 0.05 to 0.13%, but there was no improvement measured at h i g h e r s u l p h u r l e v e l s . Thus, the 0.13% d i e t a r y sulphur appeared to meet the s t e e r s ' r e q u i r e -ment. The i n c o r p o r a t i o n of i n o r g a n i c s u l p h u r c o n t a i n i n g compounds i n t o ruminant r a t i o n s as w e l l as absorbed and 15 r e c y c l e d i n o r g a n i c s u l p h a t e may have "sulphur-amino a c i d s p a r i n g a c t i o n " . T h i s e f f e c t has been observed i n p o u l t r y (Ross and Harms, 1970; and M a r t i n , 1972) i n which supplemental s u l p h a t e gave p o s i t i v e responses i n growth r a t e . H i n t o n and Harms (1972) i d e n t i f i e d s u l p h a t e as the u n i d e n t i f i e d growth f a c t o r i n f i s h s o l u b l e s i n p o u l t r y d i e t s . In man d i e t a r y i n o r g a n i c s u l p h a t e has been shown to i n c r e a s e n i t r o g e n r e t e n t i o n ( Z e z u l k a and Calloway, 1976). Sulphate e f f e c t i v e l y r e p l a c e d up to 50% of the d i e t a r y methionine requirement as measured by c o l l a g e n metabolism and growth i n the young p i g (Robel, 1976). T h e r e f o r e , the b e n e f i c i a l r e p o r t s of s u l p h a t e supplementation i n the d i e t may not be co m p l e t e l y due to rum i n a l b i o s y n t h e s i s of s u l p h u r amino a c i d s but may be a t t r i b u t e d t o the "s u l p h u r - a m i n o - a c i d s p a r i n g a c t i o n " of s u l p h a t e f o r the. s y n t h e s i s of v a r i o u s s u l p h a t e d compounds. The NRC e s t i m a t e s of s u l p h u r requirements of sheep (1975), beef c a t t l e (1976) and d a i r y c a t t l e (1971) are 0.14-0.18%, 0.10% and 0.20% r e s p e c t i v e l y . Elam (1975) has reviewed the su l p h u r requirements of the ruminant and supports the NRC recommendations. E f f e c t s of Sulphur D e f i c i e n c y and Excess The e f f e c t s of s u l p h u r d e f i c i e n c y i n sheep have been reviewed by Whanger and Matrone (1970). Symptoms of a sulphur d e f i c i e n c y are not s p e c i f i c and may be d i f f i c u l t to i d e n t i f y . Some of the symptoms i n c l u d e l o s s of a p p e t i t e , l o s s of weight, e x c e s s i v e l a c r i m a t i o n , weakness, d u l l n e s s , e m a c i a t i o n and death. 16 Gram p o s i t i v e organisms are predominant i n the rumen of s u l p h u r - d e f i c i e n t sheep (Whanger and Matrone, 1970). Hume and B i r d (1970) found decreased m i c r o b i a l p r o t e i n s y n t h e s i s i n the rumen when sulphur was d e f i c i e n t . S l y t e r e t aJL. (1971) r e p o r t e d lower t o t a l rumen b a c t e r i a counts f o r c a l v e s , f e d low (0.04%) or h i g h (1.72%) l e v e l s of s u l p h u r compared to c a l v e s f e d 0.34% s u l p h u r . F u r t h e r evidence of the c r i t i c a l n a ture of s u l p h u r i n the rumen i s the r e s u l t of v a r i o u s workers t h a t the d i g e s t i -b i l i t y of c e r t a i n r a t i o n c o n s t i t u e n t s are reduced when sulphur i s l i m i t i n g i n the d i e t . S t a r c h d i g e s t i o n by rumen microbes was i n c r e a s e d by the a d d i t i o n of v a r i o u s forms of s u l p h u r (Kennedy e t a l . , 1968). M a r t i n et a_l. (1964) r e p o r t e d t h a t i n s t e e r s c e l l u l o s e d i g e s t i o n was lower f o r r a t i o n s t h a t were d e f i c i e n t i n s u l p h u r . S i m i l a r e f f e c t s were r e p o r t e d i n sheep by Bray and Hemsley (1969). Barton e t a l . (1971) used i n  v i t r o t e c h n i q u e s to show .the marked i n f l u e n c e of s u l p h u r con-c e n t r a t i o n s on c e l l u l o s e d i g e s t i o n by rumen microbes. A d d i t i o n a l i_n v i t r o s t u d i e s by B u l l and V a n d e r s a l l (1973) i n d i c a t e the a d d i t i o n of sodium s u l p h a t e , c a l c i u m s u l p h a t e , and D,L-methionine,- were equal at equal sulphur c o n t e n t i n promoting c e l l u l o s e d i g e s t i o n . The optimum sulphur l e v e l was 0.16 to 0.24%. A d d i t i o n a l ruminal e f f e c t s to s u l p h u r d e f i c i e n t d i e t s have been found i n the form of end p r o d u c t s of m i c r o b i a l a c t i o n . Rumen microorganisms from sheep fed a s u l p h u r - d e f i c i e n t d i e t formed more a c e t a t e and p r o p i o n a t e on a p u r i f i e d d i e t than those from sheep fed the d i e t with s u l p h u r added (Whanger and 17 Matrone, 1965). F u r t h e r , t h e r e was an accumulation of l a c t a t e i n the rumen of the s u l p h u r d e f i c i e n t sheep. The cause of these e f f e c t s on l a c t a t e and VFA p r o d u c t i o n has not been f u l l y e l u c i d a t e d . One of the common i n d i c a t i o n s of a s u l p h u r - d e f i c i e n c y i s reduced feed consumption. I t i s suspected t h a t the reduced feed i n t a k e i s the r e s u l t of a reduced m i c r o b i a l p o p u l a t i o n and the subsequent r e d u c t i o n i n r a t e of d i g e s t i o n of feed components i n the rumen. A lower r a t e of d i g e s t i o n would be expected to reduce r a t e of passage of f e e d through the d i g e s t i v e t r a c t and thereby cause a r e d u c t i o n i n f e e d i n t a k e by the a n i m a l . Reports of' lower feed i n t a k e by animals f e d d i e t s low i n s u l p h u r have been made f o r sheep (Kahlon e_t a l . , 1973) beef c a t t l e (Chalupa e t a l . , 1973) and d a i r y c a t t l e (Chalupa e t al_. , 1971; L e i b h o l z and Kang, 1973). There i s abundant e v i d e n c e t h a t the sulphur c o n t a i n i n g amino a c i d methionine i s one of the most t o x i c amino a c i d s . L a r g e l y on the b a s i s of experiments with r a t s , Harper e_t a l . (1970) concluded t h a t consumption ...of methionine at f o u r times i t s requirement r e s u l t s i n growth d e p r e s s i o n and t i s s u e damage when i t i s i n c o r p o r a t e d i n t o a d i e t low i n p r o t e i n . Numerous attempts have been made to overcome the growth-depressing e f f e c t s of a methionine-induced t o x i c i t y . Katz and Baker (1975) s t u d i e d the e f f e c t s of adding supplemental g l y c i n e or t h r e o n i n e to a methionine excess r a t i o n i n young c h i c k s . They found t h a t g l y c i n e was p a r t i a l l y e f f e c t i v e i n a l l e v i a t i n g the growth d e p r e s s i o n caused by excess methionine. The a d d i t i o n of t h r e o n i n e t o g e t h e r with g l y c i n e improved performance s t i l l f u r t h e r . 18 In d a i r y c a t t l e excess methionine and methionine hydroxy analog have both been shown to reduce feed i n t a k e ( S a t t e r et. a l . , 1975). In sheep, excess methionine has reduced both feed i n t a k e ( K e l l y and Thomas, 1975) and wool growth (Reis e t a l . , 1973a). In c o n t r a s t to t h i s e f f e c t of methionine, excess c y s t i n e d i d not appear to a f f e c t wool growth a d v e r s e l y . Reis e t a l . (1973a) concluded t h a t the e f f e c t s of high l e v e l s of methionine on wool growth are due s p e c i f i c a l l y to the i n a b i l i t y of the animal to metabolize t h i s amino a c i d r a p i d l y enough or to s t o r e i t i n t i s s u e by a mechanism such as t h a t p o s s i b l e w i t h c y s t i n e , namely t h i o l - d i s u l p h i d e r e a c t i o n s with p r o t e i n s . T h i s c o n c l u s i o n i s supported by plasma amino a c i d data (Reis e t a l . , 1973b) which showed t h a t plasma methionine l e v e l s i n c r e a s e s t e e p l y w i t h i n c r e a s i n g amounts of abomasally i n f u s e d methionine whereas equimolar amounts of c y s t i n e caused o n l y a s l i g h t r i s e i n plasma c y s t i n e . Benevenga (1974) r e p o r t e d methionine t o x i c i t y i s not due to methionine per se but i n v o l v e s the metabolism of the methionine methyl group. H i s work does not support the s u g g e s t i o n by Reis e_t a_l. (1973b) that the t o x i c e f f e c t s of e x c e s s i v e d i e t a r y methionine can be a s c r i b e d to i t s e f f e c t on t r a n s p o r t of other amino a c i d s . Sulphur Supplementation S i n c e i t appears that ruminant d i e t s may be l e s s than optimum i n t h e i r s u l phur c o n t e n t , the a d d i t i o n of supplemental s u l p h u r may p r o v i d e b e n e f i c i a l i n c r e a s e s i n p r o d u c t i o n . I t has been shown t h a t low s u l p h u r d i e t s may depress both the 19 growth .rate of the rumen microbes and the ho s t . On o c c a s i o n s the rumen microbes* s u l p h u r requirement has been met y e t the host requirement i s s t i l l suboptimal (Fenderson and Bergen, 1975). T h e r e f o r e i n any r a t i o n f o r m u l a t i o n the c h o i c e of supplementation of a s u l p h u r c o n t a i n i n g compound should be determined by e i t h e r the microbes, the host or both of t h e i r requirement f o r s u l p h u r . I f s u l p h u r i s d e f i c i e n t f o r m i c r o b i a l growth the c h o i c e should be a s u l p h u r c o n t a i n i n g compound t h a t i s r e a d i l y a v a i l a b l e to the micro-organisms. T h i s then should i n c r e a s e the b i o l o g i c a l v a l u e of the d i g e s t a e n t e r i n g the abomasum. I f o n l y the host requirement f o r sulphur c o n t a i n i n g compounds i s not met then the p r e f e r r e d c h o i c e of supplementation w i l l be compounds which are p o o r l y u t i l i z e d by the microbes but have hig h b i o l o g i c a l v alue to the ho s t . Compounds t h a t are a v a i l a b l e to the microbes i n c l u d e L-methionine, DL-methionine, sodium s u l p h a t e , ammonia s u l p h a t e , c a l c i u m s u l p h a t e and el e m e n t a l sulphur (Kahlon et. al_. , 1975a). I t has been suggested t h a t ammonia s u l p h a t e may have broader a p p l i c a t i o n f o r i t can supply both s u l p h u r and n i t r o g e n (Elam, 1975). The number of compounds t h a t bypass rumen f e r m e n t a t i o n and p r o v i d e the sulphur requirement of the animal i s i n c r e a s i n g . Methionine hydroxy analog i s the most popular (Baker, 1975) and has a p o s i t i v e e f f e c t on milk y i e l d and compo s i t i o n which was equal to abomasal methionine (Olson and Grubough, 1974). Other compounds i n c l u d e N-hydroxyrnethy 1-DL-methionine (Cheeke and Whanger, 1976), DL-homocysteine t h i o l a c t o n e (Amos e t a l . , 20 1974), N-acetyl-DL-methionine and S-methyl t h i o b u t a n e - 1 , 2 - d i o l (Steadman e_t a_l. , 1975). Rees e_t al_. (1974) have compared the m e r i t s of s u l p h u r f e r t i l i z a t i o n of pasture with sulphur supplementation of the animal and have found t h a t i n t a k e s of d i g e s t i b l e energy by sheep were h i g h e r with s u l p h u r - f e r t i l i z e d grass than with d i r e c t s u p p l ementation. With sulphur f e r t i l i z a t i o n , more DM d i g e s t i o n took p l a c e i n the rumen. However, i n most pas t u r e c o n d i t i o n s o n l y low l e v e l s of f e r t i l i z e r w i l l be used, and t h a t a d d i t i o n a l n u t r i e n t requirements s h o u l d be met by d i r e c t supplementation to the g r a z i n g animal. 21 I I . NITROGEN IN RUMINANT NUTRITION The p r o t e i n n u t r i t i o n of ruminant animals must be con-s i d e r e d i n terms of amino a c i d s absorbed from the g a s t r o i n -t e s t i n a l t r a c t i n r e l a t i o n to. amino a c i d s r e q u i r e d f o r p r o d u c t i v e purposes. Amino a c i d s a v a i l a b l e f o r a b s o r p t i o n are of a d i f f e r e n t spectrum than those pre s e n t i n the feed p r o -t e i n s . The p r o t e o l y t i c a c t i v i t y of the rumen microorganisms and the s y n t h e s i s of m i c r o b i a l p r o t e i n g r e a t l y a l t e r the d i e t a r y p r o t e i n , so that the p r o t e i n p r e s e n t i n the d i g e s t a p o s t - r u m i n a l l y i s made up of undegraded f e e d p r o t e i n , m i c r o -b i a l p r o t e i n and endogenous p r o t e i n s e c r e t i o n s . Another c h a r a c t e r i s t i c of ruminant p r o t e i n n u t r i t i o n i s the b e n e f i c i a l e f f e c t s of n o n - p r o t e i n n i t r o g e n (NPN) i n the d i e t . T h i s c a p a b i l i t y of changing NPN i n t o p r o t e i n n i t r o g e n i s a l s o a f u n c t i o n a t t r i b u t e d to the rumen microorganisms. These c h a r a c t e r i s t i c s a l l o w the ruminant animal to be an i n t e g r a l l i n k i n the human food c h a i n because poor q u a l i t y p r o t e i n s o u r c e s can be f e d to ruminants and c o n v e r t e d i n t o animal p r o t e i n of high b i o l o g i c a l v a l u e . The e f f e c t s of the rumen microbes on d i e t a r y p r o t e i n have been reviewed (Bla c k b u r n , 1965; Hutton, 1972; Church, 1974). The e x t e n t of d e g r a d a t i o n of d i e t a r y p r o t e i n i s l a r g e l y dependent on the s o l u b i l i t y of the m a t e r i a l i n rumen l i q u o r (Wohlt e t a l . , 1973) and any p rocess which reduces s o l u b i l i t y w i l l r e s u l t i n a decreased r u m i n a l breakdown of food p r o t e i n . Such processes i n c l u d e h e a t i n g ( T a g a r i e_t a_l. , 1965), t r e a t -ment with v e g e t a b l e tannins (Wohlt, e t aJL. , 1973) and t r e a t -22 ment with formaldehyde (Reis and Tunks, 1969; F aichney, 1975). Some d i e t a r y p r o t e i n u s u a l l y escapes rumen breakdown and there i s evidence t h a t both feed i n t a k e (Orskov e t a l . , 1971) and p r o c e s s i n g of the feed (Coelho da S i l v a et, a l . , 1972 ) i n f l u e n c e t h i s p r o p o r t i o n . S a t t e r and R o f f l e r (1974) f e e l t h a t 40% of the d i e t a r y p r o t e i n escapes rumen d e g r a d a t i o n under most d a i r y and f e e d l o t management systems. T h i s v a l u e of rumen by- p a s s i n g of p r o t e i n i s much higher than most workers have o b t a i n e d (Smith, 1975) and a more r e a l i s t i c range i s from 15 to 35%. Since a l a r g e p r o p o r t i o n of the n i t r o g e n e n t e r i n g the rumen i s i n c o r p o r a t e d i n t o m i c r o b i a l c e l l s , the p r o d u c t i o n or growth r a t e of rumen microorganisms i s of great n u t r i t i o n a l i n t e r e s t . U l t i m a t e l y the m i c r o b i a l p r o t e i n w i l l be the major source of p r o t e i n to the h o s t . To q u a n t i t a t e the amount of m i c r o b i a l p r o t e i n being produced, workers are employing many methods such as the b a c t e r i a l markers diamino p i m e l i c a c i d (DAP) and amino e t h y l p h o s p h o r i c a c i d (AEP) (Ibrahim and I n g a l l s , 1972) and a l s o m i c r o b i a l n u c l e i c a c i d s (Smith and M c A l l a n , 1970). 15 The more r e c e n t i_n v i v o methods are employing isotopes,. N (Mathison and M i l l i g a n , 1971), 3 5 S (Walker e t a l . , 1975) and 14 C (Singh e_t a l . , 1974) have been r e p o r t e d to be s u c c e s s f u l . Thomas (1973) has reviewed m i c r o b i a l p r o t e i n s y n t h e s i s and d i s c u s s e d the s i g n i f i c a n c e and l i m i t a t i o n s of o p t i m a l m i c r o b i a l growth. He c oncludes t h a t ruminants have an important r o l e i n the p r o d u c t i o n of p r o t e i n of high b i o l o g i c a l v a l u e . Another c h a r a c t e r i s t i c of ruminant n i t r o g e n metabolism i s the r e c y c l i n g of n i t r o g e n . I t i s w e l l accepted t h a t blood urea n i t r o g e n e n t e r s the rumen. I t was f e l t t h a t both s a l i v a 23 and d i r e c t t r a n s p o r t of urea a c r o s s the rumen w a l l c o n t r i b u t e d to t h i s n i t r o g e n r e c y c l i n g . I s o l a t e d p r e p a r a t i o n s have shown t h a t urea from blo o d can e n t e r the rumen through the e p i t h e l i u m ( T h o r l a c i u s e t a l . , 1971). Nolan e t a l . (1973) suggest that s a l i v a i s the main rou t e i n the i n t a c t animal and t r a n s p o r t d i r e c t l y from b l o o d o n l y o c c u r s at n o n - p h y s i o l o g i c a l b l o o d urea l e v e l s . The p o s t - r u m i n a l d i g e s t i o n of n i t r o g e n o u s compounds has not been s t u d i e d i n g r e a t d e t a i l . Church (1974) showed t h a t i n t e s t i n a l p r o t e i n d i g e s t i o n i s s i m i l a r i n a l l mammalian s p e c i e s . Armstrong and Button (1975) d i s c u s s p r o t e i n d i g e s t i o n by comparing the ruminant to the monogastric animal. The ruminant appears to possess the same complement of p r o t e a s e s which are p r e s e n t i n the monogastric. There i s a d i f f e r e n c e i n the a c i d c o n d i t i o n s of the upper p a r t of the s m a l l i n t e s t i n e . In the ruminant e x t e n s i o n of the a c i d i c c o n d i t i o n s e x i s t due i n p a r t to the c o p i o u s s e c r e t i o n of a c i d by the abornasum and i n p a r t to the weakly a l k a l i n e nature of the b i l e and the p a n c r e a t i c s e c r e t i o n s (Kay, 1969). The use of r e - e n t r a n t cannulas i n sheep by Ben-Ghedalia e t a l . (1974) has i n d i c a t e d t h a t the s i t e s of d i g e s t i o n and a b s o r p t i o n of p r o t e i n s are the small i n t e s t i n e . In c a l v e s the f l o w of d i g e s t a p r o t e i n and the p a n c r e a t i c s e c r e t i o n s has been s t u d i e d with v a r i o u s a r t i f i c i a l m i l k s (Ternouth e_t a l _ . , 1975). L i k e the monogastic, amino a c i d s and s m a l l p e p t i d e s are the end products of p r o t e o l y t i c d i g e s t i o n . There i s a l s o i n c r e a s i n g e v i d e n c e to suggest t h a t q u a n t i t a t i v e l y a b s o r p t i o n of d i p e p t i d e s i s more important than i n d i v i d u a l amino a c i d s 24 (Mathews, 1974; Kim et _ a l . , 1974). Under normal c o n d i t i o n s n u t r i t i o n a l l y important amounts of amino a c i d s are not absorbed from the rumen ( L e i b h o l z , 1971). An area which has o n l y r e c e n t l y been s t u d i e d i s the f u n c t i o n of the l a r g e i n t e s t i n e i n the ruminant. The r o l e of the caecum i n ruminant n i t r o g e n metabolism i s now b e i n g a p p r e c i a t e d f o r i t appears t h a t i t may be the major s i t e of urea d e g r a d a t i o n i n sheep (Nolan e_t al_. , 1973). Ruminal b a c t e r i a 35 t h a t were l a b e l l e d with S and i n j e c t e d i n t o the caecum of sheep and 3% of the i n j e c t e d a c t i v i t y was r e t a i n e d i n the f l e e c e (Judson e t a_l. , 1975 ). Recent t r a c e r s t u d i e s of n i t r o g e n metabolism i n ruminants 15 u s i n g the s t a b l e i s o t o p e N have demonstrated the p o t e n t i a l of i s o t o p e t r a c e r techniques as a means of s t u d y i n g q u a n t i t a -t i v e n i t r o g e n t r a n s a c t i o n s i n the body of the normal f e e d i n g a n i m a l . Q u a n t i t a t i v e models of n i t r o g e n metabolism i n sheep are c o n s i d e r i n g d i e t a r y p r o t e i n , m i c r o b i a l a c t i v i t y , r e c y c l i n g of n i t r o g e n , p o s t - r u m i n a l n i t r o g e n metabolism as w e l l as the host p r o t e i n requirement (Nolan and Leng, 1974). As more i n f o r m a t i o n i s being c o l l e c t e d these models are becoming more complete (Nolan, 1975; Mazanov and Nolan, 1976). These models d e s c r i b e both the b i o c h e m i s t r y and the s i g n i f i c a n c e of rumen m i c r o b i a l n i t r o g e n metabolism i n terms of the host n i t r o g e n metabolism. The amount of m a t e r i a l a v a i l a b l e on ruminant n i t r o g e n n u t r i t i o n i s v e r y l a r g e and the r e s u l t i s that c e r t a i n f i e l d s o f ruminant n i t r o g e n n u t r i t i o n are emerging. Thomas (1976) and van Es (1975) have both reviewed the e f f e c t of d i e t on 25 milk p r o t e i n p r o d u c t i o n . They i n d i c a t e t h a t milk p r o t e i n i s v e r y important i n human n u t r i t i o n , e s p e c i a l l y of growing c h i l d r e n . The p r o t e c t i o n of d i e t a r y p r o t e i n s and amino a c i d s a g a i n s t rumen m i c r o b i a l f e r m e n t a t i o n has a l s o r e c e n t l y been reviewed ( P h i l l i p s o n , 1972; Ferguson, 1975). They concluded t h a t the p r o t e c t i o n of d i e t a r y components can i n c r e a s e p r o d u c t i o n under c e r t a i n c o n d i t i o n s . The u t i l i z a t i o n of non-p r o t e i n n i t r o g e n i n ruminant r a t i o n s i s one of the most bene-f i c i a l a p p l i c a t i o n s of a p p l i e d n u t r i t i o n . Again t h i s area has been e x t e n s i v e l y reviewed (Chalupa, 1973; B u l l , 1973; Huber, 1975; K e r t z and E v e r e t t , 1975; S a t t e r and R o f f l e r , 1974). The a d d i t i o n of NPN i s of g r e a t e s t b e n e f i t when p r o t e i n i s the l i m i t i n g n u t r i e n t . Due to the s p e c i a l c h a r a c t e r i s t i c s of ruminant metabolism i t appears t h a t the n u t r i t i o n a l methods used f o r monogastrics may not be a p p r o p r i a t e . B l a x t e r (1975) suggests t h a t an a l t e r n a t i v e method f o r e s t i m a t i n g p r o t e i n requirements be e s t a b l i s h e d u t i l i z i n g the e n e r g y - p r o t e i n r e l a t i o n s h i p s . M i l l e r (1973) has d e v i s e d a system f o r the e v a l u a t i o n of f e e d s as sources of n i t r o g e n and amino a c i d s . H i s scheme takes i n t o account the metabolism and growth r a t e of the rumen micro-organisms. Another approach i s the b i o l o g i c a l v a l u e of p r o t e i n i n ruminants (Black and Colebrook, 1976) which compares f a v o u r a b l y w i t h the more c o n v e n t i o n a l methods. The most p r a c t i c a l method f o r e s t i m a t i n g d a i r y c a t t l e n i t r o g e n r e q u i r e -ments i s the m e t a b o l i z a b l e p r o t e i n (MP) f e e d i n g s t a n d a r d (Burroughs e t _ a l . , 1975). The MP f e e d i n g standard f o r l a c t a t i n g cows r e p r e s e n t s a balance between animal requirements f o r m e t a b o l i z a b l e amino a c i d s (MAA) and t h e i r f u l f i l l m e n t by d i e t s composed of a wide v a r i e t y of f e e d s t u f f s a f t e r rumen m i c r o b i a l a c t i v i t y . 27 I I I . PLASMA AMINO ACIDS AND RUMINANT NUTRITION The plasma f r e e amino a c i d s r e p r e s e n t a balance between i n p u t from i n t e s t i n a l a b s o r p t i o n and body p r o t e i n c a t a b o l i s m . and output by p r o t e i n s y n t h e s i s and amino a c i d c a t a b o l i s m . Munro (1970) has suggested t h a t f r e e amino a c i d s are the c u r r e n c y of p r o t e i n metabolism. He a l s o suggests t h a t changes i n plasma amino a c i d ( P A A ) c o n c e n t r a t i o n s may 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 metabolism. The use of f r e e amino a c i d s as an i n d i c a t o r of p r o t e i n n u t r i t i o n s t a t u s has been reviewed (McLaughlan, 1975; S h e l f o r d , 1974) and i t appears t h a t , as y e t , t h e r e i s not c o n v i n c i n g e v i d e n c e that PAA l e v e l s are u s e f u l f o r e v a l u a t i n g p r o t e i n q u a l i t y per se. However, many i n v e s t i g a t o r s have r e p o r t e d that f e e d i n g d i e t s d e f i c i e n t i n one e s s e n t i a l amino a c i d r e s u l t s i n a r e l a t i v e l y low l e v e l of t h a t amino a c i d i n the plasma. T h i s f a c e t of PAA l e v e l s i s u s e f u l as a n u t r i -t i o n a l i n d i c a t o r . S e v e r a l workers have shown t h a t i n monogastrics a subop-t i m a l d i e t a r y c o n t e n t of an e s s e n t i a l amino a c i d r e s u l t s i n a low plasma c o n c e n t r a t i o n , and t h a t a d i e t a r y excess r e s u l t s i n a hi g h plasma l e v e l (Zimmerman and S c o t t , 1965; Boomgaardt and Baker, 1973; I t o h e t aJL. , 1974). When i n c r e a s i n g amounts of the f i r s t l i m i t i n g amino a c i d were f e d , the plasma c o n c e n t r a -t i o n s remained low u n t i l the amount r e q u i r e d f o r maximum growth was exceeded, then a sharp and l i n e a r i n c r e a s e i n c o n c e n t r a t i o n was observed. The p o i n t of i n t e r s e c t i o n of the two l i n e s o b t a i n e d when amino a c i d i n t a k e was p l o t t e d a g a i n s t PAA con-28 c e n t r a t i o n has been accepted as a measure of amino a c i d requirement under the o v e r a l l c o n d i t i o n s of the experiment. F a v o u r a b l e comparisons of animal performance i n the p i g ( K e i t h e t a l . , 1972) and the c h i c k (Zimmerman and S c o t t , 1965) p r o v i d e support f o r the v a l i d i t y of the procedure. The use of PAA p r o f i l e s to determine the q u a l i t y of d i e t a r y p r o t e i n has been employed i n numerous s p e c i e s . I t has been used to a s s e s s the amino a c i d requirement of p o u l t r y (Zimmerman and S c o t t , 1965), swine (Davey e_t al_. , 1973 ), man (I r w i n and Hegsted, 1971), r a t ( S t o c k l a n d e t a l . , 1970), c r o -c o d i l i a (Herbert and Coulson, 1975), sheep (Reis et. a l . , 1973b), and c a t t l e ( D e r r i g e t a l . , 1974). The ruminant with i t s e x t e n s i v e s y n t h e s i s of amino a c i d s by s y m b i o t i c microorganisms p r e s e n t s unique problems t o s t u d i e s of amino a c i d n u t r i t i o n . P r o t e i n q u a l i t y i s dependent upon the a v a i l a b l e amino a c i d s l e a v i n g the rumen, r a t h e r than those i n the i n g e s t e d d i e t . P r o t e c t e d p r o t e i n s or amino a c i d s may be f e d to the animals or a l t e r n a t i v e l y n i t r o g e n o u s compounds may be a d m i n i s t e r e d p o s t r u m i n a l l y to study ruminant amino a c i d n u t r i t i o n . P r o t e c t e d p r o t e i n s ( B r o d e r i c k e_t al_. , 1974) and p o s t - r u m i n a l i n f u s i o n s of amino a c i d s ( B u r r i s et. _a l . , 1976) have both been used i n c o n j u n c t i o n with PAA l e v e l s . Other r o u t e s of a d m i n i s t r a t i o n f o r the d e t e r m i n a t i o n of ruminant amino a c i d n u t r i t i o n are i n t r a p e r i t o n e a l (Barry, 1976) and i n t r a v e n o u s i n f u s i o n s ( K e l l y and Thomas, 1975). However, these a d m i n i s t r a t i o n r o u t e s by-pass the i n t e s t i n a l mucosa and l i v e r so t h a t t h e i r n u t r i t i o n a l s i g n i f i c a n c e i s l i m i t e d . The s p e c i a l f e a t u r e s of n i t r o g e n metabolism i n ruminants, 29 which stem from the e x t e n s i v e breakdown of d i e t a r y p r o t e i n and the s y n t h e s i s of m i c r o b i a l p r o t e i n imply the absence of s p e c i f i c d i e t a r y requirements f o r e s s e n t i a l amino a c i d s . How-ever, there i s c o n s i d e r a b l e t h e o r e t i c a l and p r a c t i c a l i n t e r e s t i n d e f i n i n g the amino a c i d requirements o f ruminant t i s s u e s i n animals of known n u t r i t i o n a l and p h y s i o l o g i c a l s t a t u s . Wake-l i n g et a l . (1970) were the f i r s t i n v e s t i g a t o r s who u t i l i z e d PAA l e v e l s i n ruminant amino a c i d n u t r i t i o n . They conducted a s e r i e s of experiments i n which graded l e v e l s of L-methionine were i n f u s e d i n t o the duodenum of sheep; the plasma methionine l e v e l s responded i n a sigmoid manner. S i m i l a r experiments wi t h l y s i n e showed t h a t plasma l y s i n e c o n c e n t r a t i o n i n c r e a s e d l i n e a r l y with i n c r e a s i n g passage of l y s i n e i n t o the duodenum over the whole range of i n f u s i o n . These r e s u l t s suggest t h a t methionine, but not l y s i n e , was l i m i t i n g under the imposed e x p e r i m e n t a l c o n d i t i o n s . The high c o n t e n t of su l p h u r c o n t a i n i n g amino a c i d s i n wool suggests t h a t methionine may be l i m i t i n g f o r maximum p r o d u c t i o n i n sheep. The e f f e c t of abomasal methionine i n f u -s i o n s on the PAA response c u r v e (Reis e t a_l. , 1973a) and wool growth (Reis e_t a_l. , 1973b) c o n f i r m t h a t s u l p h u r c o n t a i n i n g amino a c i d s may be l i m i t i n g . K e l l y and Thomas (1975) c o n c l u -ded t h a t methionine may be l i m i t i n g f o r p r o t e i n s y n t h e s i s i n the t i s s u e s but i s not i n v o l v e d i n the c o n t r o l of feed i n t a k e . B a r r y (1976) has confirmed'the i n c r e a s e d wool growth response to supplementary methionine, but i n d i c a t e d ; t h a t methionine may have a r o l e i n the c o n t r o l of v o l u n t a r y i n t a k e . The i n f l u e n c e of abomasal supplements of z e i n and l y s i n e on wool growth r a t e 30 and PAA p r o f i l e s suggests t h a t i n some c o n d i t i o n s l y s i n e may be the l i m i t i n g amino a c i d f o r sheep pr o d u c t i o n (Reis and Tunks, 1976). In the growing c a l f methionine has been shown to be the f i r s t l i m i t i n g amino a c i d ( W i l l i a m s and Smith, 1974). In the preruminant c a l f graded l e v e l s of methionine i n the d i e t caused a sigmoid PAA curve i n d i c a t i n g that methionine may be l i m i t i n g ( W i l l i a m s and Smith, 1975). Only methionine i n f u s i o n r e s u l t e d i n a two-phase PAA response curve and the q u a n t i t y of threonine, l y s i n e and t r y p -tophan a v a i l a b l e from d i g e s t a met the requirement f o r growing s t e e r s (Fenderson and Bergen, 1975). The improved amino a c i d balance of opaque-2 corn d i d not a f f e c t the PAA c o n c e n t r a t i o n s i n s t e e r s (Redd e_t al_. , 1975). This l a t t e r i n f o r m a t i o n suggests th a t PAA p r o f i l e s may be of value f o r the e v a l u a t i o n of new feeds f o r ruminants. The use of PAA p r o f i l e s f o r the determination of n u t r i -t i o n a l s t a t u s i s considered to be most s e n s i t i v e f o r the growing animal. For the high milk producing ruminant PAA l e v e l s a l s o provide a s e n s i t i v e measurement of amino a c i d n u t r i t i o n . L i n z e l l and Mepham (1974) suggest that milk p r o t e i n s y n t h e s i s may be l i m i t e d by the a v a i l a b i l i t y of e i t h e r methionine or tryptophan i n the l a c t a t i n g goat. Vik-Mo e_t a_l. (1974) found methionine to be the f i r s t l i m i t i n g amino a c i d f o l l o w e d by phenylalanine and l y s i n e . Methionine has been shown to be l i m i t i n g f o r d a i r y cows by Broderick et a l . (1974) and D e r r i g et a l . (1974). Broderick et a l . (1974) suggests that v a l i n e and l y s i n e are the next l i m i t i n g amino a c i d s . Methionine 31 and l y s i n e have r e c e n t l y been shown to l i m i t milk p r o t e i n ' s y n t h e s i s i n l a c t a t i n g cows i n t h a t o r d e r ( S p i r e s e_t a l . , 1975). Kellaway e t a_l. (1974) have r e p o r t e d r e s u l t s i n d i c a t i n g t h a t t h r e o n i n e i s the f i r s t l i m i t i n g amino a c i d f o l l o w e d by-p h e n y l a l a n i n e and methionine i n the l a c t a t i n g cow. S h e l f o r d (1974) has shown th a t e s s e n t i a l amino a c i d s may be l i m i t i n g milk p r o t e i n s y n t h e s i s . Of the amino a c i d s , abomasal l y s i n e i n f u s i o n was observed to cause g r e a t e s t i n c r e a s e i n milk p r o t e i n p r o d u c t i o n . When comparing PAA l e v e l s r e p o r t e d by v a r i o u s i n v e s t i g a t o r s many f a c t o r s should be c o n s i d e r e d . Time of sampling has been shown to have a marked e f f e c t on PAA l e v e l s i n swine (Davey e_t a_l. , 1973), man (Marrs, 1975), sheep (Cross e t _ a l . , 1975) and the l a c t a t i n g goat (Mepham and L i n z e l l , 1974). The l o c a t i o n of the sampling s i t e i s i m p o r t a n t . Hormonal a f f e c t s on PAA l e v e l s has been reviewed by Munro (1970). Davis (1972) and Tao e t a l . (1974) have i n d i c a t e d t h a t p r o l a c t i n , growth hormone and i n s u l i n are i n v o l v e d i n sheep amino a c i d metabolism and are r e f l e c t e d by changes i n PAA l e v e l s . In the l a c t a t i n g d a i r y cow PAA c o n c e n t r a t i o n s have been shown to be a f f e c t e d by the e s t r o u s c y c l e (Mason et a l . , 1973) and the stage of l a c t a t i o n ( F i s h e r e t a l . , 1975). Other c o n s i d e r a t i o n s are the p a r t i c i p a t i o n of muscle t i s s u e i n m a i n t a i n i n g PAA homeostasis i n ruminants (Cross e_t a l . , 1974) and acute changes i n environmental temperature ( B e l l e t a l . , 1975). The d i e t a r y l e v e l s of p r o t e i n and energy a f f e c t PAA 32 l e v e l s i n the c a l f ( L e i b h o l z , 1975). Eskeland et a l . (1974) have compared d i f f e r e n t sources of energy f o r p r o t e i n f o r m a t i o n i n the lamb. By measuring changes i n PAA l e v e l s they c o n c l u d e d t h a t g l u c o s e and p r o p i o n a t e are the best energy s o u r c e s . Con-t i n u o u s l y f e d s t e e r s had a d i f f e r e n t PAA p r o f i l e than those f e d twice a day ( B o i l a and D e v l i n , 1975). High f i b e r - u r e a d i e t s a l s o a f f e c t PAA p r o f i l e s i n d a i r y c a t t l e (Bouchard and Conrad, 1975). Wolfrom e t a_l. (1976) suggest t h a t f r e e amino a c i d s i n whole blood as w e l l as plasma should be determined i n f u t u r e s t u d i e s of amino a c i d metabolism i n sheep. They found s m a l l but s i g n i f i c a n t d i f f e r e n c e i n methionine l e v e l i n whole blo o d and plasma. They a l s o i n d i c a t e d t h a t the r o u t e of i n t r a v e n o u s i n f u s i o n i s very important. An a l t e r n a t i v e approach t o the measurement of amino a c i d requirements u t i l i z e s r a d i o a c t i v e amino a c i d s . The b a s i s f o r t h i s method i s s i m i l a r t o t h a t p r e v i o u s l y d i s c u s s e d u s i n g PAA l e v e l s to e s t i m a t e amino a c i d r e q u i r e m e n t s . S i n c e amino a c i d s absorbed from the d i g e s t i v e t r a c t can-not be s t o r e d i n major q u a n t i t i e s , - they are d i s t r i b u t e d as f r e e amino a c i d s i n b l o o d and t i s s u e s . The r a i s e d amino a c i d con-c e n t r a t i o n i n b l o o d and t i s s u e s a u t o m a t i c a l l y causes an i n c r e a s e d r a t e o f amino a c i d d e g r a d a t i o n . T h i s i s because the K v a l u e s of the enzymes i n i t i a t i n g amino a c i d d e g r a d a t i o n are i n g e n e r a l h i g h , much high e r than the c o n c e n t r a t i o n of amino a c i d s i n the t i s s u e s (Krebs, 1972). I f the ext e n t of o x i d a -t i o n of e s s e n t i a l amino a c i d s i s s i g n i f i c a n t l y i n c r e a s e d when t h e i r supply exceeds t i s s u e r equirements, the p a t t e r n of r e -33 14 l e a s e of CO^ f o l l o w i n g the a d m i n i s t r a t i o n of a l a b e l l e d amino a c i d to an animal should p r o v i d e an a l t e r n a t i v e procedure f o r the e s t i m a t i o n of e s s e n t i a l amino a c i d r e q u i r e m e n t s . The use of r a d i o a c t i v e amino a c i d s f o r n u t r i t i o n a l s t u d i e s has mainly been conducted on m o n a g a s t r i c s . Benevenqa e t a l . (1968) 14 c o n c l u d e d t h a t the o x i d a t i o n o f C - e s s e n t i a l amino a c i d s i s a r e l i a b l e t e chnique and compares f a v o u r a b l y with growth r a t e and c a r c a s s a n a l y s i s . Neale and Waterlow (1974a) found no d i f f e r e n c e i n o x i d a t i o n r a t e between i n t r a g a s t r i c and i n t r a -venous a d m i n i s t r a t i o n . However, they d i d suggest t h a t i n t e r -p r e t a t i o n of the r e s u l t s p r e s e n t s many p i t f a l l s and t h a t the s k i n of the r a t has a s i g n i f i c a n t r o l e i n the p r o t e i n meta-b o l i s m of the whole body. Neale and Waterlow (1974b) c r i t i -c a l l y e v a l u a t e d the amino a c i d o x i d a t i o n method f o r d e t e r m i n i n g the amino a c i d requirement. The c h o i c e and p o s i t i o n of the l a b e l are very important f o r the e s t i m a t i o n of endogenous l o s s e s of amino a c i d s (Neale, 1976). 14 Newport e t a_l. (1976) r e p o r t e d t h a t the CO^ output from amino a c i d o x i d a t i o n i s r e l i a b l e f o r the e s t i m a t i o n of amino a c i d requirements i n small p i g s . They found f a v o u r a b l e r e s u l t s f o r both l y s i n e and methionine. Perry (1975) has shown t h a t the breed of p i g should be c o n s i d e r e d when c a l c u l a t i n g the p r o p o r t i o n of the l a b e l i n c o r p o r a t e d i n t o the muscle p r o t e i n . Armstrong and Annison (1973) compared the amino a c i d 14 requirements of sheep measured by PAA p r o f i l e s and C 0 ? o u t -put procedures. Comparable r e s u l t s with both methods were o b t a i n e d f o r t h r e o n i n e and methionine. Cross e t aJL. (1975) s t u d i e d the uptake of r a d i o a c t i v i t y from l a b e l l e d l e u c i n e 34 i n j e c t e d i n t r a v e n o u s l y i n t o wethers. They c a l c u l a t e d the plasma h a l f - t i m e and the p r o p o r t i o n of the a c t i v i t y i n c o r p o r a -ted i n t o plasma p r o t e i n . F a s t i n g d i d not have a s i g n i f i c a n t e f f e c t on plasma h a l f - t i m e v a l u e s . In ruminants the c o l l e c -t i o n of CC>2 i s c o m p l i c a t e d because i t o r i g i n a t e s from the rumen and the r e s p i r a t o r y system. Cross e_t a_l. (1975 ) suggest t h a t plasma parameters be s t u d i e d i n ruminants when r a d i o -a c t i v e amino a c i d s are a d m i n i s t e r e d , so t h a t the d i f f i c u l t i e s i n v o l v e d i n c o l l e c t i o n of r e s p i r e d CO^ from l a r g e animals be e l i m i n a t e d . The experiments of B i r t and C l a r k (1976) i n d i c a t e t h a t r a d i o a c t i v e amino a c i d s w i t h r a t s may a s s i s t i n i n t e r p r e t i n g the r o l e of the l i v e r and muscle i n m o d i f y i n g the PAA response to d i e t a r y amino a c i d s . 35 IV. PLASMA UREA AND RUMINANT NUTRITION Mercer and M i l l e r (1973) suggested t h a t the response of plasma urea (PU) to abomasal i n f u s i o n of methionine may be of v a l u e i n e s t i m a t i n g the methionine requirements of sheep. In c o n t r a s t , W i l l i a m s and Smith (1974) found t h a t PU c o n c e n t r a -t i o n and abomasal i n f u s i o n s of amino a c i d s are not r e l i a b l e methods f o r e s t i m a t i n g the amino a c i d requirements i n the c a l f . Young e t a_l. (1973) has shown t h a t PU i n c r e a s e d w i t h i n c r e a s i n g p r o t e i n i n t a k e f o r a given p r o t e i n , but i s dependent on the nature of the p r o t e i n . They i n d i c a t e d t h a t t h i s dependence i s r e l a t e d to the extent to which ammonia i s formed from a p r o t e i n i n the rumen r a t h e r than the q u a l i t y of the p r o t e i n . In the monogastric animals e l e v a t e d PU c o n c e n t r a t i o n s are due to i n c r e a s e d amino a c i d c a t a b o l i s m (Lewis and Speer, 1973). T h i s s u g g e s t i o n t h a t e l e v a t e d PU c o n c e n t r a t i o n s i n sheep are due to h i g h e r ruminal ammonia l e v e l s i s f u r t h e r supported by Pfander e t a l . (1975) and Wohlt e t al. (1976) who measured PU w i t h v a r y i n g p r o t e i n a l l o w a n c e s . The e l e v a t e d plasma urea c o n c e n t r a t i o n s i n s t e e r s f e d a high p r o t e i n d i e t were due to h i g h ruminal ammonia l e v e l s (Fenderson and Bergen, 1976). W i l l i a m s and Smith (1975) s t u d i e d PAA and PU c o n c e n t r a t i o n s i n the pre-ruminant c a l f . The PAA and PU c o n c e n t r a t i o n s de-c r e a s e d soon a f t e r f e e d i n g , which s u r p r i s i n g l y i s i n c o n t r a s t to the simple-stomached a n i m a l s . They d i d show that PU con-c e n t r a t i o n may be of value i n d e t e r m i n i n g the methionine requirement, f o r these c a l v e s had t h e i r lowest PU c o n c e n t r a t i o n at the optimum d i e t a r y methionine l e v e l . Mehra (1976) noted t h a t the b l o o d urea c o n c e n t r a t i o n of b u f f a l o and d a i r y c a l v e s 36 responds to v a r y i n g p r o t e i n l e v e l s i n a s i m i l a r manner. T o r e l l e t j a l . (1974) have d i s c u s s e d the f a c t o r s a f f e c t i n g PU and i t s use as a n u t r i t i o n a l index f o r sheep. They concluded t h a t n e i t h e r ages nor sampling time had any s i g n i f i c a n t e f f e c t on PU l e v e l but v a r i a t i o n between animals with age c l a s s was s i g -n i f i c a n t . K i r k and Walker (1976a) suggest t h a t PU can be a r e l i a b l e i n d i c a t o r of p r o t e i n q u a l i t y i n the preruminant lamb. P r o t e i n q u a l i t y and u r i n a r y n i t r o g e n c o n s t i t u e n t s have r e c e n t l y been d i s c u s s e d i n terms of PU l e v e l s i n sheep ( K i r k and Walker, 1976b). They concluded t h a t PU i s a v a l u a b l e c r i t e r i a to e s t i m a t e p r o t e i n q u a l i t y but has l i m i t e d use f o r e s t i m a t i n g ruminant amino a c i d r e q u i r e m e n t s . 37 EXPERIMENTS Experiment I - E f f e c t of Abomasal Methionine I n f u s i o n on Plasma Amino A c i d P r o f i l e s and Plasma Urea C o n c e n t r a t i o n s In t h i s experiment graded l e v e l s of methionine w i l l be i n f u s e d i n t o the abomasum of two growing lambs. J u g u l a r blood samples w i l l be o b t a i n e d at each i n f u s i o n l e v e l and plasma f r e e amino a c i d s and plasma urea w i l l be determined by an amino a c i d a n a l y s e r . The plasma methionine response curve should i n d i c a t e i f methionine i s l i m i t i n g p r o d u c t i o n for. these lambs. The changes i n the PAA p r o f i l e w i l l h o p e f u l l y i n d i c a t e the e f f e c t of i n c r e a s i n g methionine i n f u s i o n s . (A) M a t e r i a l s and Methods Two D o r s e t - S u f f o l k ewe lambs, #13 (lamb #1) and #31 (lamb #2), weighed 28 and 34 kg r e s p e c t i v e l y d u r i n g the e x p e r i -mental p e r i o d . Each lamb was s u r g i c a l l y f i t t e d with an aboma-s a l cannula d u r i n g the f i r s t week post-weaning. The cannula (Medical-Grade S i l a s t i c Tubing, Dow-Corning) had an i n t e r n a l diameter of 2.6 mm. At one end a s i l a s t i c c o l l a r was a t t a c h e d to the c a n n u l a and s u t u r e d i n t o the abomasum. The abomasum was sutured to the abdominal w a l l where the cannula was passed under the s k i n to themidback. The s u t u r e s were removed one week a f t e r the o p e r a t i o n . The e x p e r i m e n t a l work commenced two weeks p o s t - o p e r a t i o n . The animals were f e d 400 gm of hay (Timothy-orchard grass mixture) and 165 gm b a r l e y twice d a i l y . Both animals were accustomed to b e i n g handled and had been on the above d i e t f o r 38 over two weeks b e f o r e the commencement of the experiment. During the e x p e r i m e n t a l p e r i o d the animals were p l a c e d i n m e t a b o l i c cages. Water and a c o l b a l t - i o d i z e d s a l t l i c k were a v a i l a b l e throughout the e x p e r i m e n t a l p e r i o d . The lambs remained i n the m e t a b o l i c cages u n t i l the f i n a l b l o o d sample was c o l l e c t e d at the h i g h e s t i n f u s i o n l e v e l . The percentage n i t r o g e n (Technicon, 1974a) and phosphorus (Technicon, 1974b) of the d i e t were determined by an auto-a n a l y s e r c o l o u r i r n e t r i c method. The percentage s u l p h u r was determined a f t e r wet a s h i n g with magnesium a c e t a t e ( P i p e r , 1950) and by a c o l o u r i r n e t r i c method (Tabataba, 1974). Each animal was abomasally i n f u s e d with graded l e v e l s (0.0, 1.0, 2.0, 3.0, 4.0 and 5.0 gm/day) of D,L-met'hionine ( N u t r i t i o n a l B i o c h e m i c a l C o r p o r a t i o n ) i n p h y s i o l o g i c a l s a l i n e at an i n f u s i o n r a t e of 250 ml/day. On the t h i r d day of each i n f u s i o n l e v e l j u g u l a r b l o o d was c o l l e c t e d i n t o h e p a r i n i z e d v a c u t a i n e r tubes (Becton, D i c k i n s o n & Co..Canada L t d . ) at 12:00 noon. J u g u l a r b l o o d plasma was o b t a i n e d from the c e n t r i f u g a t i o n of whole b l o o d at 5,000 rpm f o r 10 minutes. The plasma was d e p r o t e i n i z e d with s u l f o s a l i c y l i c a c i d (50 mg/ml plasma) at 15,000 rpm f o r 20 minutes at 4°C. The supernatant was f r o z e n u n t i l a n a l y z e d . One ml of t h i s s o l u t i o n was a p p l i e d d i r e c t l y to the column of a H i t a c h i KLA3B amino a c i d a n a l y z e r f o r the d e t e r m i n a t i o n of the n e u t r a l and a c i d i c amino a c i d s (Spackman e t a l . , 1958 ). Plasma urea was a l s o determined i n t h i s a n a l y s i s . The b a s i c amino a c i d s were not determined as workers i n p r e v i o u s s t u d i e s have i n d i c a t e d t h a t the e f f e c t of 39 methionine i n f u s i o n on these amino a c i d s was not of m e t a b o l i c s i g n i f i c a n c e . (B) R e s u l t s The f e e d was consumed at a l l the i n f u s i o n l e v e l s of both lambs. The percentage n i t r o g e n , phosphorus and s u l p h u r of the grass (1.22%, 0.26% and 0.13%) and the b a r l e y (2.16%, 0.77% and 0.44%) were expressed on dry matter b a s i s r e s p e c t i v e l y . The plasma f r e e amino a c i d s were expressed as micromoles per 100 ml of b l o o d plasma. Lamb #1 developed d i a r r h o e a a f t e r the 3.0 gm D,L-methionine i n f u s i o n per day. I t was observed t h a t t h i s lamb was consuming a l l of i t s feed and the i n f u s i o n was c o n t i n u e d . The amino a c i d p r o f i l e f o r lamb #1 (Table 1) i n d i c a t e s t h a t the d i a r r h o e a d r a s t i c a l l y a f f e c t e d a l l plasma amino a c i d s . T h e r e f o r e , t h i s prevented a comparison to be made between the two e x p e r i m e n t a l lambs a t the 4.0 and 5.0 gm per day of methionine i n f u s i o n . The plasma methionine response c u r v e f o r lamb #1 ( F i g . 1) i n c r e a s e d at i n c r e a s i n g c o n c e n t r a t i o n s of the methionine i n f u s i o n . A f t e r t h i s lamb developed d i a r r h o e a the plasma methionine dropped to a lower l e v e l . The l i n e a r response at the f i r s t i n f u s i o n l e v e l s suggests t h a t methionine was not l i m i t i n g i n the d i g e s t a a v a i l a b l e to t h i s lamb. Lamb #2 had a d i f f e r e n t plasma methionine response curve ( F i g . 2 ) . An i n f l e c t i o n p o i n t was p r e s e n t j u s t below the 2.0 gm methionine l e v e l . T h i s response i s t y p i c a l of a l i m i t i n g amino a c i d and suggests t h a t methionine was below i t s o p t i m a l l e v e l i n the d i g e s t a a v a i l a b l e to t h i s lamb. TABLE 1. EFFECT OF ABOMASAL INFUSION OF METHIONINE ON THE CONCENTRATION OF AMINO ACIDS IN PLASMA IN LAMB #1 PLASMA AMINO A C I D CONCENTRATIONS MOLES/100 ML.) FOR DL - M E T H I O N I N E SUPPLEMENTATION (G/DA Y ) O F : 0.0 1.0 2.0 3.0 4.0 5.0 TAURINE 0.84 1.57 2.70 5.80 11.57 4.76 THREONINE 7.81 7.12 6.47 6.27 6?82 6.57 SERINE 7.10 4.88 4.03 4.75 3.94 4.22 GLUTAMIC ACID 7.63 7.87 7.61 7.83 8.40 6.50 GLYCINE 45.60 34.50 27.60 21.40 36.60 29.70 ALANINE 24.30 18.40 12.30 10.30 16.50 15.70 CYSTINE 4.80 10.26 9.61 9.70 6.21 2.37 METHIONINE 1.20 3.24 6.15 8.29 4.68 4.47 ISOLEUCINE 6.13 5.96 5.75 6.99 9.96 14.02 LEUCINE 4.85 4.95 5.19 6.32 8.02 15.06 TYROSINE 5.58 5.41 6.71 6.74 9.35 12.76 PHENYLALANINE 3.80 3.21 3.68 4.41 5.90 7.77 CYSTATHIONINE TRACE 0.42 0.67 0.81 0.14 0.16 o TABLE 2. EFFECT OF ABOMASAL INFUSION OF METHIONINE ON THE CONCENTRATION OF AMINO ACIDS IN PLASMA IN LAMB #2. P L A S M A A M I N O A C I D C O N C E N T R A T I O N S ( j U / M O L E S / 1 0 0 M L ) F O R D L - M E T H I O N I N E S U P P L E M E N T A T I O N ( G / D A Y ) O F : 0.0 1.0 2.0 3.0 4.0 5.0 TAURINE 1.46 2.20 3.11 3.99 5.27 9.49 THREONINE 12.00 10.32 3.83 2.58 4.51 6.48 SERINE 10.31 5.60 2.84 2.85 2.30 3.04 PROLINE 3.30 2.57 2.63 3.81 3.70 2.66 GLUTAMIC ACID 11.90 14.31 3.88 3.01 2.78 3.61 GLYCINE 48.90 41.54 19.45 24.36 17.20 21.77 ALANINE 27.00 21.61 10.45 10.04 10.45 13.84 VALINE 21.20 20.85 9.72 11.25 8.90 11.96 CYSTINE 1.48 5.32 6.35 6.89 7.42 7.04 METHIONINE 3.30 4.14 8.19 17.85 22.58 40.64 ISOLEUCINE 10.70 11.55 4.92 5.86 4.70 5.57 LEUCINE 19.60 8.90 7.92 7.16 4.90 5.45 TYROSINE 3.10 4.26 5.03 6.25 2.75 6.91 PHENYLALANINE 7.00 7.30 6.89 6.71 2.81 7.84 42 The o t h e r sulphur c o n t a i n i n g compounds-cystine, c y s t a -t h i o n i n e and taurine-were a f f e c t e d by the i n f u s i o n . Plasma t a u r i n e i n c r e a s e d with each i n c r e a s e i n the methionine i n f u s i o n l e v e l f o r lamb #2 and u n t i l 4.0 gm/day methionine i n f u s i o n f o r lamb #1. Plasma c y s t i n e i n c r e a s e d with the f i r s t i n f u s i o n s but l e v e l e d o f f with f u r t h e r i n c r e a s e s i n methionine i n f u s i o n s . T h i s was observed i n lamb #2 ( F i g . 2) and lamb #1 ( F i g . 1; 3.0 gm/day m e t h i o n i n e ) . C y s t a t h i o n i n e was o n l y measured i n the plasma f o r lamb.#l. I t i n c r e a s e d through a l l i n c r e a s i n g i n f u s i o n l e v e l s u n t i l the d i a r r h o e a developed, whereafter i t f e l l to t r a c e amounts. Methionine s u l f o x i d e s were not i n c l u d e d i n the standard so i t was not p o s s i b l e to q u a n t i f y t h i s amino a c i d . In both lambs a peak was observed where the methionine s u l f o x i d e s were expected to o c c u r . T h i s peak i n c r e a s e d with i n c r e a s i n g methionine i n f u s i o n s . The n o n - e s s e n t i a l amino a c i d s - a l a n i n e , g l y c i n e and s e r i n e - tended to decrease with i n c r e a s i n g methionine i n f u s i o n l e v e l s i n both lambs. Glutamic a c i d was observed to decrease i n lamb #2, but not i n lamb #1, with i n c r e a s i n g methionine i n f u s i o n s . Plasma g l u t a m i c a c i d l e v e l s were c o n s t a n t i n lamb #1. The branch-chained amino a c i d s - i s o l e u c i n e and l e u c i n e -decreased w i t h i n c r e a s i n g methionine i n f u s i o n s f o r both lambs. V a l i n e was d e t e c t e d i n t r a c e amounts i n the plasma of lamb #1. In lamb #2 plasma v a l i n e decreased with i n c r e a s i n g methionine i n f u s i o n s . The plasma c o n c e n t r a t i o n of p h e n y l a l a n i n e and t y r o s i n e 43 INFUSION L E V E L S I gm methionine a y) Fig. 1. Effects of abomasal methionine infusions on the concentration of sulphur amino acids in plasma i n lamb #1. 44 Figv-2-.- Effects of abomasal methionine infusions on the concentration of sulphur amino acids i n plasma i n lamb #2. . 45 IN FUSION L E V E L S ( g m m e t h i o n i n e / (J a y ) Fig. 3. Effects of abomasal methionine infusions on the concentrations of some non-essential amino acids i n plasma i n lamb #2. 46 Q E «o o a. INFUSION LEVELS ( qm methionine / d a y ) Fig. 4. Effects of abomasal methionine infusions on the concentration of the branch-chained amino acids i n plasma i n lamb #2. 4 7 TABLE 3. EFFECT OF ABOMASAL INFUSION OF METHIONINE ON THE CONCENTRATION OF UREA IN PLASMA INFUSION LEVEL PLASMA UREA CONCENTRATION / , x ( p i MOLE/100 ML) - < G M / D A Y ) _ L A W # 1 ' LAMB_#2 °'° 171.0 123,0 L 0 568.0 1 5 8 I 0 2 , 0 255.0 241.0 3 , 0 215.0 2 5 1 . 0 4 , 0 2 9 2 . 0 226.0 5 ' ° 2 4 3 . 0 U 6 l 0 48 appeared to have i n c r e a s e d w i t h i n c r e a s i n g i n f u s i o n l e v e l s . Threonine decreased i n plasma c o n c e n t r a t i o n s f o r both lambs at the i n c r e a s i n g i n f u s i o n l e v e l s . P r o l i n e was o n l y measured i n the plasma of lamb #2 and was c o n s t a n t . The plasma urea c o n c e n t r a t i o n f o r lamb #2 (Table 3) peaked at the 3.0 gm/day methionine i n f u s i o n l e v e l a f t e r which the plasma urea c o n c e n t r a t i o n d e c r e a s e d . T h i s peak i n plasma urea was a l s o observed f o r lamb #1 at the 1.0 gm/day methionine i n f u s i o n l e v e l . (C) D i s c u s s i o n I t has been proposed t h a t the requirement f o r a l i m i t i n g amino a c i d can be determined from the i n f l e x i o n p o i n t of a curve r e l a t i n g the plasma c o n c e n t r a t i o n of an amino a c i d to the i n t e s t i n a l s u p p l y . The plasma methionine curve f o r lamb #1 ( F i g . 1) i n c r e a s e d l i n e a r l y f o r the f i r s t i n f u s i o n l e v e l s and suggests t h a t methionine was not the l i m i t i n g amino a c i d . The plasma methionine curve f o r lamb #2 ( F i g . 2) had an i n f l e x i o n p o i n t j u s t below the 2.0 gm methionine i n f u s i o n l e v e l . T h i s i n d i c a t e d t h a t methionine was l i m i t i n g f o r t h i s lamb. The reason f o r the d i f f e r e n t plasma methionine curves i s not c l e a r . The lambs were of the same sex, same breed, s i m i l a r weight and age, and r e c e i v e d the same q u a n t i t y and type of f e e d s . Weight gains s i n c e b i r t h i n d i c a t e t hat lamb #2 had a hig h e r growth r a t e than lamb #1. T h i s h i g h e r growth r a t e of lamb #2 may e x p l a i n the d i f f e r e n c e i n the plasma response c u r v e . 49 Plasmas c y s t i n e i n c r e a s e d with i n c r e a s i n g methionine i n f u s i o n l e v e l s but p l a t e a u e d at the h i g h e r i n f u s i o n l e v e l s . T h i s e f f e c t of methionine on plasma c y s t i n e has been r e p o r t e d by Tao et a l . (1974). Reis et a l . (1973b) found t h a t plasma c y s t i n e i n c r e a s e d at each i n c r e a s i n g l e v e l of methionine i n f u s i o n . Tao e_t al_. (1974) suggest t h a t the rou t e of i n f u s i o n p l a y s a key r o l e i n amino a c i d metabolism. They used the in t r a v e n o u s i n f u s i o n method and Reis e_t a_l. (1973b) used p o s t -r u m i n a l i n f u s i o n s . Tao e_t a_l. (1974) suggest t h a t the gut t i s s u e and l i v e r , which were by-passed when the n u t r i e n t s o l u t i o n was a d m i n i s t e r e d p a r e n t e r a l l y v i a j u g u l a r v e i n s , p l a y an important r o l e i n the b i o s y n t h e s i s of c y s t i n e . In growing p i g s K e i t h e_t al_. (1972) found t h a t plasma c y s t i n e l e v e l e d o f f at the higher d i e t a r y methionine l e v e l s . T h e r e f o r e , i t appears t h a t the plasma c y s t i n e responses may i n f a c t be e x p l a i n e d by i n f u s i o n r o u t e . The i n c r e a s i n g c o n c e n t r a t i o n s of both t a u r i n e and c y s t a -t h i o n i n e i n plasma when excess amounts of methionine are i n f u s e d i n d i c a t e t h a t the lambs are a b l e to co n v e r t a propor-t i o n of the i n f l u x of methionine to c y s t e i n e . Reis 'et a l . (1973a) suggest that t h i s c o n v e r s i o n of methionine t o c y s t e i n e i s inadequate i n sheep and c o n t r i b u t e s to the e l e v a t e d plasma c o n c e n t r a t i o n of methionine. The d ecrease i n plasma l e v e l s of some of the n o n - e s s e n t i a l amino a c i d s ( s e r i n e , g l y c i n e and a l a n i n e ) compares f a v o u r a b l y w i t h the r e s u l t s of Reis e t al. (1973b). Tao et a l . (1974) found that plasma s e r i n e and g l y c i n e decreased but plasma a l a n i n e i n c r e a s e d i n sheep i n f u s e d with graded l e v e l s of 50 methionine. S e r i n e i s i n v o l v e d i n c y s t a t h i o n i n e s y n t h e s i s and a decreased plasma l e v e l may e x p l a i n the i n a b i l i t y of sheep to c o n v e r t a l a r g e i n f l u x of methionine to c y s t e i n e . C y s t e i n e can r e a d i l y be removed and i s not as t o x i c as methionine. T o x i c amounts of methionine have caused lowered plasma c o n c e n t r a t i o n s of g l y c i n e and s e r i n e i n r a t s (Benevenga and Harper, 1970), swine ( K e i t h et a l . , 1972) and sheep ( S c h e l l i n g e t a l . , 1973). Katz and Baker (1975) found that g l y c i n e was p a r t i a l l y e f f e c t i v e i n a l l e v i a t i n g the growth d e p r e s s i o n caused by excess methionine i n c h i c k s . The t o x i c e f f e c t s of excess methionine have been d i s c u s s e d i n terms of g l y c i n e , s e r i n e and c y s t e i n e metabolism (Benevenga, 1974). He suggests t h a t g l y c i n e and s e r i n e supply are important f o r the metabolism of methionine and t h a t low l e v e l s may i m p a i r i t s c o n v e r s i o n to c y s t e i n e . These amino a c i d s ( s e r i n e , g l y c i n e and a l a n i n e ) are key p r e c u r s o r s f o r g l u c o s e s y n t h e s i s i n the ruminant (Bergman, 1973), which p l a c e s a f u r t h e r demand on t h e i r r e q u i r e m e n t . The changes i n the plasma urea c o n c e n t r a t i o n w i t h the graded i n f u s i o n l e v e l s of methionine p r o b a b l y r e f l e c t changes i n the s u b s t r a t e supply ( i . e . amino a c i d s ) f o r urea s y n t h e s i s . G e n e r a l l y t h e r e was a d e p r e s s i o n i n a l l of the plasma amino a c i d s e x c e p t . f o r the s u l p h u r c o n t a i n i n g amino a c i d s which i n c r e a s e d i n plasma c o n c e n t r a t i o n . Plasma urea i n c r e a s e d i n c o n c e n t r a t i o n up to 3.0 gm/day methionine i n f u s i o n (lamb #2). The plasma c y s t i n e c o n c e n t r a t i o n p l a t e a u e d at t h i s i n f u s i o n l e v e l and the plasma c o n c e n t r a t i o n s of the n o n - e s s e n t i a l amino a c i d s ( a l a n i n e , s e r i n e and g l y c i n e ) appeared to have reached a 51 c o n s t a n t lower c o n c e n t r a t i o n ( F i g . 3 ) . A f t e r t h i s p o i n t amino a c i d supply may have been c r i t i c a l and urea s y n t h e s i s was d e p ressed. Methionine metabolism was a l s o impaired at the h i g h e r i n f u s i o n l e v e l s so t h a t i t s c o n v e r s i o n to u r e a would a l s o be reduced. T h e r e f o r e , the d e c r e a s i n g plasma urea c o n c e n t r a t i o n s a f t e r the plasma urea c o n c e n t r a t i o n peak r e f l e c t s d e c r e a s i n g s u b s t r a t e f o r urea s y n t h e s i s . The r e d u c t i o n i n the c o n c e n t r a t i o n of the branch chained e s s e n t i a l amino a c i d s ( v a l i n e , l e u c i n e and i s o l e u c i n e ) with methionine i n f u s i o n i n sheep has been r e p o r t e d by Reis et a l . (1973b), Tao e t a l . (1974) and S c h e l l i n g e t a l . (1973). They suggest t h a t s u l p h u r c o n t a i n i n g amino a c i d s may be l i m i t i n g wool growth and a r e d u c t i o n i n e s s e n t i a l amino a c i d s i s c o n s i s t e n t w i t h the expected e f f e c t s of s u p p l y i n g a l i m i t i n g amino a c i d . T h i s i s o n l y v a l i d up u n t i l the requirement i s met f o r the l i m i t i n g amino a c i d . I n c r e a s i n g an amino a c i d beyond i t s requirement a l s o r e s u l t s i n a r e d u c t i o n i n plasma l e v e l s f o r most e s s e n t i a l and n o n - e s s e n t i a l amino a c i d s . T h i s appears to be t r u e f o r sheep but i n p i g s the plasma l e v e l of some of the e s s e n t i a l amino a c i d s i n c r e a s e d when methionine was i n c l u d e d i n the d i e t beyond i t s requirement ( K e i t h e_t _ a l . , 1972). S c h e l l i n g e t a_l. (1973 ) have shown by n i t r o g e n b a l a n c e e x p e r i -ments that p r o t e i n s y n t h e s i s can s t i l l be i n c r e a s i n g even a f t e r the plasma l e v e l of a l i m i t i n g amino a c i d begins to r i s e . In g e n e r a l , methionine i n excess of i t s requirement depresses most plasma amino a c i d c o n c e n t r a t i o n s . 52 E x p e r i m e n t I I - Abomasal M e t h i o n i n e I n f u s i o n and R a d i o a c t i v e M e t h i o n i n e T r a c e r The use o f r a d i o a c t i v e t r a c e r s g i v e s a dynamic p i c t u r e o f amino a c i d m e t a b o l i s m . I n t h i s e x p e r i m e n t a lamb w i l l r e c e i v e a t r a c e dose o f r a d i o a c t i v e s u l p h u r c o n t a i n i n g m e t h i o n i n e under two c o n d i t i o n s . One c o n d i t i o n w i l l be t h a t m e t h i o n i n e i s l i m i t i n g and the o t h e r w i l l be t h a t m e t h i o n i n e i s i n e x c e s s o f the l i m i t i n g r e q u i r e m e n t . U r i n a r y e x c r e t i o n o f r a d i o a c t i v i t y , i n c o r p o r a t i o n o f r a d i o a c t i v i t y i n t o plasma p r o t e i n s and o t h e r compounds, a n d p l a s m a k i n e t i c s w i l l b e e s t i m a t e d . i t i s hoped t h a t a c o m p a r i s o n o f t h e s e p a r a m e t e r s under the above c o n d i t i o n s w i l l i n d i c a t e the m e t a b o l i c f a t e o f m e t h i o n i n e . (A) M a t e r i a l s and Methods Lamb #2 was p l a c e d i n a m e t a b o l i c cage and r e c e i v e d two c o n t i n u o u s i n f u s i o n s . The f i r s t was 250 ml o f p h y s i o l o g i c a l s a l i n e per day ( z e r o i n f u s i o n l e v e l ) . The o t h e r c o n t a i n 5.0 gm o f D L - m e t h i o n i n e i n f u s e d i n 250 ml o f p h y s i o l o g i c a l s a l i n e per day ( h i g h i n f u s i o n l e v e l ) . The lamb remained i n t h e m e t a b o l i c cage f o r one week d u r i n g each i n f u s i o n . The d i e t has a l r e a d y been d e s c r i b e d i n E x p e r i m e n t I . Two i n d w e l l i n g j u g u l a r c a n n u l a s were i n s e r t e d and f l u s h e d w i t h h e p a r i n i z e d s a l i n e (100 u n i t s o f h e p a r i n / m l ) . At each o f 35 the two i n f u s i o n l e v e l s a t r a c e r dose o f S - L - m e t h i o n i n e (Amersham/Searle SJ.123) was i n j e c t e d v i a a c a n n u l a i n t o the j u g u l a r v e i n . The doses were 93.8 and 83.9 jdCi a t 0.0 and 5.0 gm/methionine i n f u s i o n s r e s p e c t i v e l y . F o l l o w i n g t h e i n j e c t i o n t h e c a n n u l a was i m m e d i a t e l y f l u s h e d w i t h 10 ml o f 53 s t e r i l e s a l i n e . S e r i a l b l o o d samples (7 ml) were c o l l e c t e d v i a t h e o t h e r j u g u l a r c a n n u l a . B l o o d was c o l l e c t e d r e g u l a r l y f o r t h e f i r s t f o u r hours p o s t - i n j e c t i o n . A 24 hour b l o o d sample was a l s o o c o l l e c t e d . B l o o d was s t o r e d i n the c o l d (4 C ) . Plasma was o b t a i n e d and d e p r o t e i n i z e d as i n E x p e r i m e n t I . T o t a l u r i n e was c o l l e c t e d once d a i l y f o r f o u r days p o s t - i n j e c t i o n . . Plasma, d e p r o t e i n i z e d plasma and u r i n e were c o u n t e d f o r 35 S r a d i o a c t i v i t y as soon as p o s s i b l e i n an Isocap/300 l i q u i d s c i n t i l l a t i o n c o u n t e r . The c h a n n e l r a t i o c o u n t i n g method was u t i l i z e d and t h e s c i n t i l l a t i o n c o c k t a i l was PCS (Amersham/ S e a r l e ) . The d e p r o t e i n i z e d plasma was f r o z e n and s t o r e d f o r amino a c i d a n a l y s i s . A H i t a c h i KLA3B amino a c i d a n a l y z e r was used t o d e t e r m i n e t h e n e u t r a l and a c i d i c amino a c i d s . A s t r e a m s p l i t t i n g d e v i c e c o n n e c t e d a t the base o f the r e s i n column removed one q u a r t e r o f t h e volume l e a v i n g the column. T h i s p o r t i o n was c o l l e c t e d by a f r a c t i o n c o l l e c t o r (KBR-Bromma, 7000 U l t r o r a c F r a c t i o n C o l l e c t o r ) . The f r a c t i o n s were c o u n t e d 35 f o r S a c t i v i t y as p r e v i o u s l y d e s c r i b e d . (B) R e s u l t s 35 The c o u n t i n g e f f i c i e n c y o f S r a n g e d from 68% t o 76%. U r i n e had t h e l o w e s t and d e p r o t e i n i z e d plasma had t h e h i g h e s t c o u n t i n g e f f i c i e n c i e s . The d i f f e r e n c e between plasma and d e p r o t e i n i z e d plasma r a d i o a c t i v i t y was c o n s i d e r e d t o r e p r e s e n t the r a d i o a c t i v i t y a s s o c i a t e d w i t h the plasma p r o t e i n s . 54 F i g . 5. Percentage of S a c t i v i t y of plasma located i n the plasma proteins at two abomasal methionine infusion l e v e l s . 55 The p r o p o r t i o n of the plasma a c t i v i t y d e t e c t e d i n the plasma p r o t e i n s ( F i g . 5) i n d i c a t e d t h a t more of the a c t i v i t y i s a s s o c i a t e d with the plasma p r o t e i n s at the zero i n f u s i o n l e v e l . A f t e r 24 hours a l l the a c t i v i t y i n plasma was d e t e c t e d i n the p r o t e i n f r a c t i o n at the zero i n f u s i o n l e v e l . At the high i n f u s i o n l e v e l only one h a l f of the plasma a c t i v i t y was a s s o c i a t e d w i t h plasma p r o t e i n s a f t e r 24 hours. 35 The c u m u l a t i v e r e c o v e r y of S i n u r i n e expressed as a percentage of a d m i n i s t e r e d dose ( F i g . 6) i n d i c a t e d a d i f f e r e n c e 35 of S e x c r e t i o n a t the two i n f u s i o n l e v e l s . At the h i g h i n f u s i o n l e v e l over 50% of the i n j e c t e d a c t i v i t y was p r e s e n t i n the u r i n e a f t e r f o u r days. At the z e r o i n f u s i o n l e v e l only 13% of the i n j e c t e d dose c o u l d be accounted f o r i n the u r i n e a f t e r f o u r days. The S- c o n t a i n i n g amino a c i d f r a c t i o n s were counted f o r 35 S a c t i v i t y . At the zero i n f u s i o n l e v e l a l l the a c t i v i t y d e t e c t e d i n the d e p r o t e i n i z e d plasma was l o c a t e d i n the 35 methionine f r a c t i o n . At the high i n f u s i o n l e v e l S a c t i v i t y was d e t e c t e d i n plasma methionine, c y s t i n e , t a u r i n e , c y s t a -t h i o n i n e and the f r a c t i o n c o l l e c t e d at the expected l o c a t i o n of methionine s u l f o x i d e s ( T a b l e 4 ) . 35 The percentage of the S a c t i v i t y l o c a t e d i n the methionine f r a c t i o n decreased with time at the high i n f u s i o n l e v e l . Due to the c l o s e n e s s of the methionine and c y s t a -t h i o n i n e peaks I t was d i f f i c u l t to s e p a r a t e these f r a c t i o n s c o m p l e t e l y . Even so, t h e r e was metabolism of methionine su l p h u r to c y s t e i n e , t a u r i n e , and the methionine s u l f o x i d e s . T h i s was apparent f o r a f t e r ten minutes o n l y 53% of the a c t i v i t y 80 6 Ql 0.0 g m / d a 0 1 2 3 4 T I M E ( d a y s ) 6. Percentage of administered dose detected i n urine. TABLE 4. 3 5 S ACTIVITY OF THE SULPHUR-CONTAINING AMINO ACIDS, EXPRESSED AS A PERCENTAGE OF THE TOTAL 3 5 S ACTIVITY IN THE SULPHUR-CONTAINING AMINO ACIDS AT 5.0 GM ABOMASAL METHIONINE. AMINO ACIDS (Z) METHIONINE TIME (MIN) METHIONINE CYSTINE TAURINE SULFOXIDES CYSTATHIONINE 0 100 0 0 0 0 10 53 9 13 18 7 60 35 2 30 22 11 120 26 TRACE 31 17 26 180 22 TRACE 27 23 28 240 14 TRACE 28 21 34 7. Plasma methionine specific activity curve at 0.0 gm abomasal methionine infusion. Fig. 8.. Plasma methionine specific activity curve at 5.0 gm abomasal methionine infusion. TABLE 5 METHIONINE PLASMA KINETICS INFUSION POOL IRREVERSIBLE TOTAL ENTRY LEVEL SIZE LOSS RATE (5 METHIONINE/DAY) (}A MOLES) MOLES/MIN) l M MOLES/MIN) 0.0 81.4 3.85 12.3 5.0 1290.0 8.4 36.1 61 d e t e c t e d i n the p r o t e i n f r e e plasma was l o c a t e d i n the methionine f r a c t i o n . The amount of the a c t i v i t y i n the c y s t i n e f r a c t i o n was r e l a t i v e l y low. The p r o p o r t i o n of the a c t i v i t y i n t a u r i n e and the methionine s u l f o x i d e s appeared to l e v e l o f f at about 30% and 20% r e s p e c t i v e l y . The p r o p o r t i o n of the a c t i v i t y i n the c y s t a t h i o n i n e f r a c t i o n i n c r e a s e d w i t h each s e r i a l b l o o d sample. One t h i r d of the a c t i v i t y of p r o t e i n f r e e plasma was d e t e c t e d i n c y s t a t h i o n i n e a f t e r f o u r hours. The s p e c i f i c a c t i v i t y c u r ves f o r methionine (zero i n f u s i o n l e v e l , F i g . 7; high i n f u s i o n l e v e l , F i g . 8) can be r e p r e s e n t e d by two e x p o n e n t i a l components. The p o o l s i z e , i r r e v e r s i b l e l o s s , and t o t a l e n t r y r a t e were determined by the mathematical treatment of i s o t o p e d i l u t i o n data (Leng, 1970). These data (Table 5) were c a l c u l a t e d at both i n f u s i o n l e v e l s . The methionine pool s i z e s were 81.4 and 1290.0 umoles r e s p e c t i v e l y at the low and high i n f u s i o n l e v e l s . The t o t a l e n t r y r a t e at the high i n f u s i o n l e v e l was three f o l d l a r g e r than the lower l e v e l . The d i f f e r e n c e f o r t o t a l e n t r y r a t e at the high and z e r o i n f u s i o n l e v e l s was 23 .8 umoles/min. I r r e -v e r s i b l e l o s s at the h i g h e r l e v e l was over twice the s i z e of the lower l e v e l . The d i f f e r e n c e f o r i r r e v e r s i b l e l o s s at the high and zero l e v e l s was 4.5 umoles/min. (C) D i s c u s s i o n The c h o i c e of the l a b e l f o r amino a c i d metabolism a i d s 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 . Reeds (1974), and Neale and Waterlow (1974b) have i n d i c a t e d t h a t the l o c a t i o n of the 62 14 C - l a b e l i n e s s e n t i a l ammo a c i d s i s important m the e s t i m a t i o n of amino a c i d o x i d a t i o n . Carbon l a b e l l i n g of non-e s s e n t i a l amino a c i d s i s u n s u i t a b l e f o r s t u d i e s of p r o t e i n t u r n o v e r and the c h o i c e of the p o s i t i o n of the l a b e l on the molecule i s important when l a b e l l e d e s s e n t i a l amino a c i d s are employed (James et. a_l. , 1975 ). Short term changes i n amino a c i d metabolism are e v a l u a t e d b e t t e r w i t h amino a c i d s with a sma l l pool s i z e ; the e q u i l i b r a t i o n time i n the e x c r e t o r y b i c a r b o n a t e pool i s a l s o s h o r t e r than i n the urea pool so that 15 N i s l e s s u s e f u l than carbon l a b e l l i n g . M ethionine i s an e s s e n t i a l amino a c i d that c o n t a i n s s u l p h u r , carbon, hydrogen, n i t r o g e n and oxygen atoms. The f a t e of these atoms d i f f e r s with the metabolism of methionine. When methionine i s i n c o r p o r a t e d i n t o p r o t e i n a water molecule i s l o s t . The r o l e of methionine i n the one carbon pool i s to donate a methyl group so t h a t carbon and hydrogen atoms are removed. Only the sulphur atom of methionine i s i n c o r p o r a t e d i n t o c y s t e i n e and c y s t i n e the other s u l p h u r c o n t a i n i n g amino a c i d s of p r o t e i n . When methionine i s o x i d i z e d the carbon i s e x p i r e d as CO2 and the n i t r o g e n i s e x c r e t e d as urea i n the u r i n e . The o x i d a t i o n of the sulphur c o n t a i n i n g amino a c i d s r e s u l t s i n i n o r g a n i c s u l p h a t e being e x c r e t e d i n the u r i n e . T h e r e f o r e , the r o l e of methionine i n p r o t e i n metabolism and 35 c y s t e i n e s y n t h e s i s i s f o l l o w e d best by S - l a b e l l e d methionine. Cross e_t al_. (1975) were, able to determine the h a l f time of l e u c i n e i n plasma of wethers. F r a c t i o n a t i o n of the plasma was not r e q u i r e d f o r t h i s amino a c i d . With methionine, i t i s necessary to f r a c t i o n a t e plasma to account f o r the c o n v e r s i o n 63 of methionine to i t s m e t a b o l i t i e s . B i r t and C l a r k (1976) 14 a d m i n i s t e r e d (U- C ) - L - a l a n i n e to r a t s and c o u l d account f o r 30% to 50% of the a c t i v i t y i n f r e e t a u r i n e i n the t i s s u e . T h i s i s not s u r p r i s i n g s i n c e the primary s y n t h e s i s of t a u r i n e i s from- c y s t e i n e , and the carbon c h a i n of c y s t e i n e comes from s e r i n e . T h i s p o t e n t i a l c o n v e r s i o n of methionine carbon to the 14 n o n - e s s e n t i a l amino a c i d s l i m i t s the use of C - l a b e l l e d methionine as a s p e c i f i c t r a c e r f o r s u l p h u r amino a c i d meta-b o l i s m . 35 The t r a c e r used i n t h i s experiment was S-L-methionine and the abomasal supplement was DL-methionine. In the r a t (Shannon e t _ a l . , (1975) and man (Kies e t _ a l . , 1975; and Z e z u l k a and Calloway, 1976) the metabolism of the D- and L~ methionine molecules i s d i f f e r e n t . L - m e t h i o n i n e i s p r e f e r e n -t i a l l y i n c o r p o r a t e d i n t o p r o t e i n w h i l e more D-methionine i s e x c r e t e d i n the u r i n e . D-methionine had equal n u t r i t i o n a l v a l u e of i t s L - i s o m e r f o r the c h i c k (Sugahara jet al_. , 1967). Tr i e b w a s s e r e_t _a_l. (1976 ) have shown t h a t the dog o x i d i z e d L - t r y p t o p h a n to a g r e a t e r degree than D - t r y p t o p h a n . The u r i n a r y e x c r e t i o n p a t t e r n of the D- and L - t r y p t o p h a n was d i f f e r e n t . The dog e x c r e t e s D-tryptophan i n t a c t w h i l e L -tryptophan i s e x c r e t e d mainly as t r y p t o p h a n m e t a b o l i t i e s . T h e r e f o r e , the metabolism of amino a c i d s d i f f e r s not o n l y with s p e c i e s and p h y s i o l o g i c a l s t a t e s but a l s o with the isomer of the amino a c i d . In t h i s experiment w i t h the lamb the t r a c e r was L - m e t h i o n i n e ; the e f f e c t of DL-methionine supplementation on L-methionine metabolism was s t u d i e d . 64 35 The i n c o r p o r a t i o n of the S l a b e l of methionine i n t o 35 plasma p r o t e i n s and u r i n a r y e x c r e t i o n of S i n d i c a t e s the m e t a b o l i c pathways f o r methionine. At the low i n f u s i o n l e v e l a l l the a c t i v i t y of plasma was l o c a t e d i n plasma p r o t e i n s a f t e r 24 hours and methionine was the o n l y f r a c t i o n which c o n t a i n e d any d e t e c t a b l e a c t i v i t y i n the p r o t e i n f r e e plasma. A f t e r f o u r days 13% of the i n j e c t e d a c t i v i t y c o u l d be accounted f o r t h a t e x c r e t e d i n the u r i n e . T h i s suggests that methionine was u t i l i z e d f o r a n a b o l i c p r o c e s s e s which i n c l u d e s p r o t e i n synthe-s i s . At the high i n f u s i o n l e v e l one h a l f of the a c t i v i t y of plasma was i n the p r o t e i n f r a c t i o n a f t e r 24 hours. The a c t i v i t y i n the d e p r o t e i n i z e d plasma was l o c a t e d i n t a u r i n e , c y s t a t h i o n i n e , c y s t i n e , methionine s u l f o x i d e s and methionine. T h i s i n d i c a t e d a c a t a b o l i s m of methionine f o r t a u r i n e and methionine s u l f o x i d e s are both c a t a b l i c products of s u l p h u r c o n t a i n i n g amino a c i d s . T h i s i s supported by the h i g h e r a c t i v i t y p r e s e n t i n the u r i n e . U r i n a r y e x c r e t i o n of the a c t i v i t y accounted f o r over one h a l f of the i n j e c t e d dose. The amount of methionine e n t e r i n g the plasma p o o l per day was c a l c u l a t e d as 2.53 gm and 7.60 gm on the zero and high i n f u s i o n l e v e l s r e s p e c t i v e l y . The d i f f e r e n c e between these v a l u e s agrees f a v o u r a b l y with the supplemental 5.0 gm/day of DL-methionine at the high i n f u s i o n l e v e l . The b a s i c assump-t i o n f o r these c a l c u l a t i o n s i s t h a t i n t e s t i n a l amino a c i d s are absorbed at a c o n s t a n t r a t e . Since amino a c i d a b s o r p t i o n i s never 100% and the L-amino a c i d s are absorbed more e f f i c i e n t l y than the D-amino a c i d s 65 (Mathews, 1974) i t i s apparent t h a t the t o t a l e n t r y r a t e has a n o n - i n t e s t i n a l component. T h i s suggests that at the high i n f u s i o n l e v e l p r o t e i n t u r n o v e r i s g r e a t e r than the lower l e v e l . T h e r e f o r e , more methionine would be e n t e r i n g the pool by t i s s u e p r o t e i n breakdown. Reis e_t a_l_. (1973a) s t a t e d t h a t t i s s u e p r o t e i n s y n t h e s i s i n c r e a s e d at the h i g h e r methionine l e v e l s but wool s y n t h e s i s decreased. Waterlow (1975) has shown t h a t p r o t e i n t u r n o v e r i n the whole body i s h i g h e r i n the animal which i s s y n t h e s i z i n g more t i s s u e p r o t e i n . T h e r e f o r e , at the high i n f u s i o n l e v e l t i s s u e p r o t e i n s y n t h e s i s and p r o t e i n t u r n -over may be g r e a t e r than those at the low i n f u s i o n l e v e l . The r a t i o of the methionine pool s i z e at the high to the low i n f u s i o n l e v e l s i s 15.8:1. The methionine c o n c e n t r a t i o n i n plasma r a t i o i s 12.3:1 f o r the high to low i n f u s i o n l e v e l s . The d i f f e r e n c e between these v a l u e s may be a t t r i b u t e d to the t i s s u e c o n c e n t r a t i o n of methionine. Amino a c i d s are f r e e i n both plasma and t i s s u e and t h e i r c o n c e n t r a t i o n s vary (Munro, 1970). Plasma methionine c o n c e n t r a t i o n was determined on j u g u l a r b l o o d which may not r e f l e c t the methionine c o n c e n t r a -t i o n throughout the c i r c u l a t o r y system. These c h a r a c t e r i s t i c s may account f o r the d i f f e r e n c e between the r a t i o s of pool s i z e and plasma c o n c e n t r a t i o n . The higher r a t i o of methionine pool s i z e compared to the r a t i o of plasma methionine c o n c e n t r a t i o n supports the s u g g e s t i o n by Munro (1970) t h a t small changes i n PAA c o n c e n t r a t i o n s r e f l e c t s l a r g e r changes i n body amino a c i d metabolism. C r o s s e_t a_l. (1974) have i n d i c a t e d t h a t muscle t i s s u e i s v e r y important i n PAA homeostasis i n the ruminant. The l a r g e r p r o p o r t i o n of the i n j e c t e d a c t i v i t y e x c r e t e d 66 i n the u r i n e and the l a r g e r i r r e v e r s i b l e l o s s at the high i n f u s i o n l e v e l i n d i c a t e s c a t a b o l i s m of methionine at t h i s l e v e l . M e t h i o n i n e was i n excess and methionine s u l p h u r i s conver t e d to methionine s u l f o x i d e s , c y s t e i n e and.to t a u r i n e . Sulphur i s e x c r e t e d i n the u r i n e i n t h r e e forms: i n o r g a n i c s u l p h a t e , e t h e r e a l s u l p h a t e and n e u t r a l s u l p h u r . The i n o r g a n i c s u l p h a t e of the u r i n e i s d e r i v e d almost e n t i r e l y from the o x i d a t i o n of the p r o t e i n molecule. T h e r e f o r e , the a c t i v i t y d e t e c t e d i n the u r i n e i n d i c a t e s sulphur amino a c i d o x i d a t i o n . The magnitude of the i n c r e a s e i n i r r e v e r s i b l e l o s s (4.5 u moles/min.) i s much s m a l l e r than the i n c r e a s e i n t o t a l e n t r y r a t e (23.8 u moles/min.). T h i s d i f f e r e n c e may account f o r the e l e v a t e d plasma methionine c o n c e n t r a t i o n and methionine pool s i z e at the high i n f u s i o n l e v e l . U r i n a r y l o s s of methionine s u l p h u r c o n t r i b u t e s to the i r r e v e r s i b l e l o s s d i f f e r e n c e . At the high i n f u s i o n l e v e l f o u r times as much of the i n j e c t e d dose appeared i n the u r i n e compared to the low i n f u s i o n l e v e l which supports the s u g g e s t i o n of i n c r e a s e d c a t a b o l i s m of methionine. In Experiment I the 0.0 gm methionine i n f u s i o n per day was shown to be l i m i t i n g f o r lamb #2. The e x c r e t i o n of a c t i v i t y i n the u r i n e at t h i s l e v e l may i n d i c a t e t h a t methionine a l s o has a r o l e to supply s u l p h a t e i n sheep. Moir (1975) d i s c u s s e s the c a t a b o l i s m of sulphur amino a c i d s f o r s u l p h a t i o n purposes. The q u a n t i t a t i v e requirements of s u l p h a t e are not known but undoubtedly methionine has "sulphur-amino a c i d s p a r i n g a c t i o n " . 6 7 SUMMARY The PAA p r o f i l e at the graded abomasal methionine i n f u s i o n s i n d i c a t e s t h a t methionine was not l i m i t i n g i n the d i g e s t a a v a i l a b l e to lamb #1. The plasma methionine curve f o r lamb #2 had an i n f l e c t i o n p o i n t j u s t below the 2.0 gm per day of abomasal methionine. The d i f f e r e n c e between these lambs may be a r e s u l t of d i f f e r e n t growth r a t e s . Lamb #2 had a h i g h e r growth r a t e than that of lamb #1. T h e r e f o r e , one would expect lamb #2 to have a h i g h e r methionine requirement compared to lamb #1. The i n c r e a s e d plasma c o n c e n t r a t i o n s of t a u r i n e and methionine s u l f o x i d e s at the h i g h e r i n f u s i o n l e v e l s i n d i c a t e s a c a t a b o l i s m of sulphur amino a c i d s . The p l a t e a u i n the plasma c y s t i n e c o n c e n t r a t i o n and the lower plasma l e v e l s of s e r i n e , g l y c i n e and a l a n i n e suggest t h a t the metabolism of methionine may be i m p a i r e d . The c o n v e r s i o n of methionine to c y s t a t h i o n i n e i s l i m i t e d by the supply of s e r i n e . The e l e v a t e d plasma con-c e n t r a t i o n of methionine at the h i g h e r i n f u s i o n l e v e l s may have been due i n p a r t to t h i s l i m i t e d c o n v e r s i o n of methionine to c y s t e i n e . The i n j e c t i o n of the l a b e l l e d methionine g i v e s a dynamic p i c t u r e of methionine metabolism. Methionine sulphur can be c o n v e r t e d to c y s t e i n e , t a u r i n e , c y s t a t h i o n i n e and methionine s u l f o x i d e s . At the zero methionine i n f u s i o n no d e t e c t a b l e c o n v e r s i o n was observed. At the high i n f u s i o n l e v e l a c t i v i t y was d e t e c t e d i n the o t h e r s u l p h u r c o n t a i n i n g compounds. T h i s suggests t h a t at the zero i n f u s i o n l e v e l methionine was 68 i n v o l v e d i n a n a b o l i c p r o c e s s e s ; t h i s i s supported by the s m a l l e x c r e t i o n of l a b e l and the l a r g e p r o p o r t i o n of the a c t i v i t y a s s o c i a t e d with the p r o t e i n s of plasma. At the high i n f u s i o n l e v e l methionine i s being c a t a b o l i z e d . A l a r g e p r o p o r t i o n of the i n j e c t e d dose was r e c o v e r e d i n the u r i n e and l e s s of the a c t i v i t y of plasma was l o c a t e d i n the p r o t e i n f r a c t i o n . The plasma parameters e s t i m a t e d from the d i l u t i o n data a l s o i n d i c a t e the pathway of methionine metabolism. The r a t i o of methionine pool s i z e and plasma c o n c e n t r a t i o n at the zero and high i n f u s i o n l e v e l s supports the s u g g e s t i o n t h a t changes i n plasma c o n c e n t r a t i o n s r e f l e c t s l a r g e r changes i n t i s s u e l e v e l s . The r a t i o was h i g h e r f o r the methionine pool s i z e than f o r the plasma c o n c e n t r a t i o n . The e l e v a t e d methionine plasma c o n c e n t r a t i o n at the higher i n f u s i o n l e v e l was not due to an i n c r e a s e d e n t r y r a t e but to a much s m a l l e r i n c r e a s e i n i r r e v e r s i b l e l o s s . T h i s supports the su g g e s t i o n of impaired methionine metabolism at high plasma c o n c e n t r a t i o n of methionine. The i n j e c t i o n of l a b e l l e d methionine at d i f f e r e n t l e v e l s of sulphur supplementation and the c o l l e c t i o n of s e r i a l blood and u r i n e samples may prove to be a r e l a t i v e l y quick and r e l i a b l e method f o r the e s t i m a t i o n of the sulphur requirement of the ruminant. When methionine i s i n excess, l e s s o f the l a b e l w i l l be a s s o c i a t e d with the p r o t e i n f r a c t i o n of plasma and more of the i n j e c t e d dose w i l l appear i n the u r i n e . When methionine i s l i m i t i n g i n the d i g e s t a a v a i l a b l e to the animal more of the a c t i v i t y of plasma w i l l be l o c a t e d i n the p r o t e i n f r a c t i o n and a s m a l l p r o p o r t i o n of the i n j e c t e d dose w i l l be e x c r e t e d i n the u r i n e . 70 BIBLIOGRAPHY Abiko, Y. (1975) Metabolism of Coenzyme A iri M e t a b o l i c Path-ways. V o l . I I I . Metabolism of S u l f u r Compounds, ed. D.M. 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