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The effect of formaldehyde treatment of the forage portion of the diet, the addition of branched-chain… Tuah, Ambrose Kwame 1978

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THE OF  E F F E C T OF FORMALDEHYDE  TREATMENT OF T H E FORAGE PORTION  T H E D I E T , T H E ADDITION OF BRANCHED-CHAIN V O L A T I L E FATTY A C I D S  AND/OR SULPHUR ON T H E U T I L I Z A T I O N OF NITROGEN AND CARBOHYDRATE BY SHEEP  by  AMBROSE KWAME /TUAH B.Sc.(Agric.) Kumasi, 1 9 6 8 M.Sc.(Anim.Sci.) Kumasi, 1 9 7 0 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY in THE FACULTY OF GRADUATE STUDIES i n the Department of Animal Science We accept t h i s thesis as conforming to the required  standard  THE UNIVERSITY OF BRITISH COLUMBIA June, 1978  In p r e s e n t i n g t h i s  thesis  an advanced degree at the L i b r a r y I  further  for  of  freely  available  thesis for  Department of  of  the  requirements  Columbia,  I agree  reference and copying o f  this  for  that  study. thesis  purposes may be granted by the Head of my Department or It  i s understood that  f i n a n c i a l gain s h a l l  written permission. '  Animal  The U n i v e r s i t y o f B r i t i s h 2075 Wesbrook P l a c e V a n c o u v e r , Canada V6T 1W5  Date  for  agree t h a t p e r m i s s i o n for e x t e n s i v e  representatives.  this  fulfilment  the U n i v e r s i t y of B r i t i s h  s h a l l make it  scholarly  by h i s  in p a r t i a l  June 29, 1978  Science Columbia  not  copying or  publication  be allowed without my  - i i -  ABSTRACT  Formaldehyde treatment degradation  of d i e t a r y p r o t e i n to reduce i t s  i n the rumen has been r e p o r t e d t o be b e n e f i c i a l  i n some  instances. Four l e v e l s  of formaldehyde (0.0%,  0.8%,  an a i r dry b a s i s ) were a p p l i e d to a dehydrated grass-clover forage.  1.0% and  and  1.2%  on  hammermilled  In v i t r o n i t r o g e n d i g e s t i b i l i t y and  ammonia-  n i t r o g e n p r o d u c t i o n at *the m i c r o b i a l stage of i n c u b a t i o n were significantly  (p ^ 0 . 0 5 ) as the l e v e l of formaldehyde was  N i t r o g e n d i g e s t i b i l i t y f o r the combined m i c r o b i a l and stages of i n c u b a t i o n was 1.2%  significantly  increased.  acid-pepsin  (p ^ 0 . 0 5 ) reduced  o n l y at  l e v e l of formaldehyde a p p l i c a t i o n compared to the u n t r e a t e d Ram  formaldehyde was diets  (14% C P .  cassava,  basis.  carbohydrate  metabolism.  a p p l i e d to the g r a s s - c l o v e r f o r a g e . on D.M.  b a s i s ) contained  11% b a r l e y and  D i e t one  contained  the forage.  lambs r a n g i n g i n body weights of 29kg to 36kg were then used  i n s t u d i e s of n i t r o g e n and  38%  reduced  contained  50%  the u n t r e a t e d  supplemented w i t h i s o v a l e r i c  acid  D i e t s f o u r and  i n the form of sodium  percent  Each of the  five  grass-clover forage,  1% sheep m i n e r a l premix on a dry matter f o r a g e w h i l e the  the formaldehyde t r e a t e d f o r a g e .  (2.3g/Kg d i e t ) .  One  sulphate.  others  D i e t s t h r e e and  (3.0g/Kg d i e t ) and  f i v e were  isobutyric  f i v e were supplemented w i t h  acid  sulphur  - iii  The  apparent d i g e s t i b i l i t y c o e f f i c i e n t s  f i b r e and c e l l u l o s e  coefficient  of acid-detergent  were i n c r e a s e d s i g n i f i c a n t l y  formaldehyde treatment  of the forage.  ^ 0 . 0 5 ) by  (p <0.05) depressed by  o f t h e f o r a g e except  (diet three).  (p  The apparent d i g e s t i b i l i t y  o f n i t r o g e n was s i g n i f i c a n t l y  formaldehyde treatment w i t h VFAS  -  f o r the diet  supplemented  The apparent d i g e s t i b i l i t y c o e f f i c i e n t s o f  dry matter and o r g a n i c matter were n o t a f f e c t e d s i g n i f i c a n t l y Ruminal f l u i d significantly animals  levels  and i s o b u t y r i c  fed diets  lower l e v e l s  two and f o u r .  Formaldehyde treatment  (p <0.05) h i g h e r l e v e l s  o f ammonia i n r u m i n a l f l u i d .  of t o t a l v o l a t i l e f a t t y a c i d s , a c e t i c ,  The  of v a l e r i c  Ruminal f l u i d  r a t i o o f m i c r o b i a l p r o t e i n - n i t r o g e n ( e s t i m a t e d from RNA-N)  (diet three).  of t h e f o r a g e except  of the forage.  (p ^ 0.05)  f o r the d i e t  (p <^0.05) reduced  supplemented  by formaldehyde  Abomasal d i g e s t a pH, c o n c e n t r a t i o n of t o t a l  n i t r o g e n , RNA-N, m i c r o b i a l p r o t e i n - n i t r o g e n , a c i d - d e t e r g e n t cellulose  decreased  The c o n c e n t r a t i o n o f n o n - p r o t e i n - n i t r o g e n  i n abomasal d i g e s t a was s i g n i f i c a n t l y  and  levels  of the forage.  by formaldehyde treatment  treatment  acid  (p y 0.05) by formaldehyde  to t o t a l abomasal d i g e s t a n i t r o g e n was s i g n i f i c a n t l y  w i t h VFAS  of the forage  p r o p i o n i c and b u t y r i c a c i d s  and rumen pH were n o t a f f e c t e d s i g n i f i c a n t l y treatment  a c i d s were  (p<C0.05) h i g h e r f o r animals f e d d i e t t h r e e than f o r  resulted i n significantly and  of i s o v a l e r i c  (p./' 0.05).  were n o t a f f e c t e d .  fibre,  - iv -  The r e d u c t i o n i n t h e r a t i o o f m i c r o b i a l p r o t e i n - n i t r o g e n to t o t a l abomasal d i g e s t a n i t r o g e n and n o n - p r o t e i n - n i t r o g e n by formaldehyde  treatment o f the f o r a g e suggests t h a t the treatment  reduced m i c r o b i a l d e g r a d a t i o n o f d i e t a r y p r o t e i n except f o r the d i e t supplemented  w i t h VFAS ( d i e t  N i t r o g e n b a l a n c e was s i g n i f i c a n t l y  perhaps  three). (p .^0.05) improved by  formaldehyde  treatment o f t h e f o r a g e except f o r the s u l p h u r  supplemented  diets..  Sulphur supplementation tended t o o f f s e t t h e  b e n e f i c i a l e f f e c t s o f formaldehyde  p r o t e c t i o n of the forage p r o t e i n .  Supplementation w i t h VFAS d i d n o t f u r t h e r enhance n i t r o g e n u t i l i z a t i o n . Formaldehyde treatment o f t h e f o r a g e s i g n i f i c a n t l y improved  s u l p h u r b a l a n c e except f o r t h e d i e t supplemented  s u l p h u r and VFAS ( d i e t  (p <C0.05) w i t h both  five).  D a i l y f e e d i n t a k e and u r i n e output p e r u n i t m e t a b o l i c body s i z e and growth r a t e over a seventeen-day (p~?  0.05) a f f e c t e d by formaldehyde  p e r i o d were n o t s i g n i f i c a n t l y treatment o f t h e f o r a g e .  The f l o w o f t o t a l d i g e s t a , o r g a n i c matter, d r y matter, a c i d - d e t e r g e n t f i b r e , c e l l u l o s e and t o t a l n i t r o g e n through the duodenum f o r a 24-hour p e r i o d was markedly treated forage.  h i g h e r f o r t h e d i e t s c o n t a i n i n g t h e formaldehyde  The f l o w o f m i c r o b i a l p r o t e i n - n i t r o g e n and n o n - p r o t e i n -  n i t r o g e n however was markedly the f o r a g e . for this  depressed by formaldehyde  treatment o f  A sheep f i t t e d w i t h a duodenal r e - e n t r a n t cannula was used  study.  - v -  TABLE OF CONTENTS  ABSTRACT  Page  . . i i  TABLE OF CONTENTS  v  LIST OF TABLES  ;  LIST OF FIGURES  ix x  LIST OF APPENDIX TABLES  xi  ACKNOWLEDGEMENTS  xiii  INTRODUCTION  1  LITERATURE REVIEW  4  Rumen m i c r o b i o l o g y  4  Rumen b a c t e r i a  4  Rumen P r o t o z o a  7  Other rumen m i c r o - o r g a n i s m s . Factors  a f f e c t i n g populations  Microbial protein-nitrogen synthesis  .  8  o f rumen micro-organisms  8  c o m p o s i t i o n and m i c r o b i a l p r o t e i n 12  Metabolism o f n i t r o g e n  16  Degradation of proteins  16  P r o t e i n anabolism by rumen b a c t e r i a  23  E f f e c t o f formaldehyde treatment on t h e d i g e s t i o n o f p r o t e i n s i n t h e rumen  24  P o s s i b l e reasons f o r t h e v a r i a b l e responses t o formaldehyde treatment o f d i f f e r e n t types o f p r o t e i n s . .  29  D i g e s t i o n and a b s o r p t i o n  of nitrogen  i n t h e s m a l l i n t e s t i n e . . .32  Digestion of p r o t e i n i n the large i n t e s t i n e  38  - vi -  Carbohydrate metabolism i n t h e rumen  • • • 39  E f f e c t s o f formaldehyde treatment on c a r b o h y d r a t e metabolism and v o l a t i l e f a t t y a c i d p r o d u c t i o n  i n t h e rumen  42  OBJECTIVES  43a  MATERIALS AND METHODS  44  Introduction  44  Experiment I  44  Treatment o f the f o r a g e w i t h  formaldehyde  44  In v i t r o i n c u b a t i o n  45  Treatment o f g r a s s - c l o v e r f o r a g e  f o r i n vivo t r i a l s  46  Experiment I I Nitrogen,  46  c a r b o h y d r a t e and s u l p h u r metabolism and feed  intake studies  •  46  Experiment I I I  49  Rumen and abomasal d i g e s t a m e t a b o l i t e s  studies  .• 49  Experiment IV • • •  51  Duodenal d i g e s t a f l o w s t u d i e s  51  C o l l e c t i o n and sampling o f d i g e s t a  51  CHEMICAL ANALYSES  52  EXPERIMENTAL DESIGNS AND STATISTICAL ANALYSIS  55  RESULTS . . .  . . . . . . . . . .  Experiment I •  57  In v i t r o d i g e s t i o n t r i a l s Experiment I I Chemical composition  57  57 59  o f d i e t s and i n g r e d i e n t s  59  D a i l y feed i n t a k e , d a i l y u r i n e output, m e t a b o l i c body s i z e s of animals (kg) a t t h e b e g i n n i n g o f the metabolism study p e r i o d , and t h e d a i l y g a i n i n weight d u r i n g t h e p r e metabolism assay p e r i o d  59  Apparent d i g e s t i b i l i t y c o e f f i c i e n t s o f d r y m a t t e r , o r g a n i c m a t t e r , n i t r o g e n , a c i d - d e t e r g e n t f i b r e and c e l l u l o s e  61  - vii -  N i t r o g e n metabolism  64  Sulphur metabolism  £6  Experiment I I I Rumen parameters  6  (Data c o l l e c t e d  Abomasal parameters  8  from s l a u g h t e r e d animals) . • . • . ^8  (Data c o l l e c t e d  from s l a u g h t e r e d animals) . . . ^ 7  Experiment IV  7  Chemical c o m p o s i t i o n o f d i e t s used f o r duodenal f l o w r a t e measurements  0  3® 73  Feed i n t a k e d u r i n g the duodenal f l o w measurements 73 Duodenal f l o w parameters Some c h e m i c a l f r a c t i o n s o f duodenal d i g e s t a , t o t a l d a i l y i n t a k e and d a i l y d i g e s t a f l o w through t h e duodenum o f t h e s e f r a c t i o n s . • ^ 7  D a i l y n i t r o g e n i n t a k e , n i t r o g e n components o f duodenal d i g e s t a and t h e d a i l y f l o w o f t h e s e components through t h e duodenum . . . . ^ 7  Apparent d i g e s t i b i l i t y c o e f f i c i e n t s o f some c h e m i c a l f r a c t i o n s i n t h e pre-duodenum p o r t i o n o f d i g e s t i v e t r a c t ( a l l compartments of stomach) and t h e change i n q u a n t i t y o f n i t r o g e n e n t e r i n g the duodenum d a i l y compared w i t h i n t a k e DISCUSSION  ^  7  8  In v i t r o d i g e s t i b i l i t y t r i a l s  1  •  Feed i n t a k e Apparent d i g e s t i b i l i t y c o e f f i c i e n t s o f n i t r o g e n , f i b r e , c e l l u l o s e , d r y m a t t e r and o r g a n i c matter  8  5  8  8  acid-detergent Q Q  Apparent d i g e s t i b i l i t y c o e f f i c i e n t s o f n i t r o g e n Apparent d i g e s t i b i l i t y c o e f f i c i e n t s  o f ADF and C e l l u l o s e  Apparent d i g e s t i b i l i t y c o e f f i c i e n t s o f d r y matter and o r g a n i c matter Rumen parameters  97 97  Rumen pH QO  Rumen ammonia-nitrogen Rumen t o t a l v o l a t i l e f a t t y a c i d  concentration  1 no x y j  - viii -  A c e t i c , P r o p i o n i c and B u t y r i c a c i d p r o p o r t i o n s  i n the  rumen f l u i d I s o v a l e r i c and I s o b u t y r i c a c i d p r o p o r t i o n s  105 i n t h e rumens  V a l e r i c acid proportions  • -109  Abomasal and duodenal d i g e s t a parameters  110  Abomasal pH  110  Abomasal d i g e s t a N%, duodenal d i g e s t a N%, arid t o t a l  daily  flow o f n i t r o g e n from t h e abomasum i n t o t h e duodenum Abomasal and duodenal d i g e s t a n o n - p r o t e i n  107  I l l  nitrogen levels  114  Abomasal and duodenal d i g e s t a RNA-N, m i c r o b i a l N, %RNA-N: % t o t a l d i g e s t a N, % m i c r o b i a l N: % t o t a l d i g e s t a N Abomasal ADF%, duodenal ADF%, c e l l u l o s e % , and q u a n t i t i e s  115  of ADF and c e l l u l o s e a r r i v i n g a t t h e duodenum  118  Duodenal d i g e s t a f l o w  119  Sulphur metabolism  123  N i t r o g e n metabolism  129  'Growth r a t e  132  SUMMARY AND CONCLUSIONS  135  LITERATURE CITED  142  APPENDIX  162  - ix-  LIST OF TABLES Table  Page  1  Composition o f R a t i o n s  47  2  In v i t r o D i g e s t i o n T r i a l s  58  3  Chemical Composition o f D i e t s and I n g r e d i e n t s (D.M. b a s i s ) .  60  D a i l y Feed I n t a k e , D a i l y U r i n e Output, M e t a b o l i c Body S i z e s o f Animals a t t h e B e g i n n i n g o f t h e Metabolism Study P e r i o d and t h e D a i l y G a i n i n Weight D u r i n g t h e Pre-metabolism Assay P e r i o d  62  4  5  Apparent D i g e s t i b i l i t y C o e f f i c i e n t s o f Some Chemical F r a c t i o n s  (%)  63  6  N i t r o g e n Metabolism  65  7  Sulphur Metabolism  67  8  Rumen parameters  69  9  Abomasal parameters  71  10  Chemical Composition o f D i e t s Used f o r Duodenal Flow Rate Measurements  72  11  Feed Intake D u r i n g t h e Duodenal Flow Measurements. .74  12  Duodenal  13  Some Chemical F r a c t i o n s o f Duodenal D i g e s t a , T o t a l D a i l y Intake and D a i l y D i g e s t a Flow Through t h e Duodenum o f These F r a c t i o n s  77  14  D a i l y N i t r o g e n I n t a k e , N i t r o g e n Components o f Duodenal D i g e s t a and t h e D a i l y Flow o f These Components Through t h e Duodenum  78  15  D i g e s t a Flow Parameter^  75  Apparent D i g e s t i b i l i t y C o e f f i c i e n t o f Some Chemical F r a c t i o n s i n t h e Pre-duodenum P o r t i o n of D i g e s t i v e T r a c t ( A l l Compartments o f Stomach) and t h e Change i n Q u a n t i t y o f N i t r o g e n E n t e r i n g t h e Duodenum D a i l y Compared w i t h I n t a k e . . 79  - x -  LIST OF FIGURES  Figure  1  2  3  Page  Schematic r e p r e s e n t a t i o n o f the p r o t e i n r e g e n e r a t i o n c y c l e i n ruminants  16a  An o u t l i n e o f the pathways o f f e r m e n t a t i o n of the major c a r b o h y d r a t e c o n s t i t u e n t s to 3C u n i t s i n the rumen  39a  An o u t l i n e o f the pathways of d e g r a d a t i o n of 3C u n i t s i n t h e rumen  39b  - x i-  LIST OF APPENDIX TABLES  Table  I  II  III  IV  V  VI  VII  VIII  Page  ANOVA p e r c e n t N d i g e s t i b i l i t y , 1 s t stage o f i n v i t r o d i g e s t i o n o f rye-grass-clover forage with.the d i f f e r e n t l e v e l s of formaldehyde treatments  163  ANOVA p e r c e n t N d i g e s t i b i l i t y 2nd stage o f i n v i t r o d i g e s t i o n of r y e - g r a s s - c l o v e r forage with the d i f f e r e n t l e v e l s of formaldehyde treatment  164  ANOVA i n v i t r o ammonia-nitrogen p r o d u c t i o n (ppm) p e r gram d r y matter o f r y e - g r a s s c l o v e r forage w i t h t h e d i f f e r e n t l e v e l s o f formaldehyde treatment  165  ANOVA m e t a b o l i c body s i z e o f animals a t the b e g i n n i n g o f t h e metabolism s t u d i e s (kg) . . . .  166  ANOVA apparent d i g e s t i b i l i t y of n i t r o g e n (%)  coefficients 167  ANOVA apparent d i g e s t i b i l i t y of a c i d - d e t e r g e n t f i b r e (%)  coefficient  ANOVA apparent d i g e s t i b i l i t y of c e l l u l o s e (%)  coefficient  168  169  ANOVA n i t r o g e n e x c r e t e d i n u r i n e p e r u n i t o f m e t a b o l i c body s i z e p e r day (g)  170  IX  ANOVA % n i t r o g e n e x c r e t e d  i n u r i n e over i n t a k e .  . . 171  X  ANOVA % n i t r o g e n e x c r e t e d  i n u r i n e over d i g e s t e d .  XI  ANOVA % n i t r o g e n r e t a i n e d over i n t a k e  173  XII  ANOVA % n i t r o g e n r e t a i n e d over d i g e s t e d  174  XIII  ANOVA n i t r o g e n b a l a n c e  g/day  175  XIV  ANOVA n i t r o g e n b a l a n c e body s i z e p e r day.  (g) p e r u n i t o f m e t a b o l i c  . 172  176  - xii-  XV  ANOVA s u l p h u r  XVI  ANOVA s u l p h u r i n t a k e p e r u n i t o f m e t a b o l i c body s i z e p e r day (g)  XVII  ANOVA s u l p h u r  i n t a k e p e r day (g)  excreted  u n i t of metabolic  178  un u r i n e p e r day p e r  body s i z e  excreted  (g)  XVIII  ANOVA % s u l p h u r  XIX  ANOVA t o t a l amount o f s u l p h u r and  177  .179  i n u r i n e over i n t a k e . . . .180 lost i n urine  f a e c e s as a p e r c e n t a g e o f i n t a k e  181  XX  ANOVA s u l p h u r b a l a n c e p e r day (g)  182  XXI  ANOVA s u l p h u r b a l a n c e p e r day p e r u n i t o f m e t a b o l i c body s i z e (g) ANOVA molar p r o p o r t i o n o f a c e t i c a c i d i n rumen f l u i d (%)  183  XXII XXIII  184  ANOVA molar p r o p o r t i o n o f p r o p i o n i c a c i d i n rumen f l u i d (%)  185  ANOVA molar p r o p o r t i o n o f i s o b u t y r i c a c i d i n rumen f l u i d (%)  186  ANOVA molar p r o p o r t i o n s ] o f i s o v a l e r i c a c i d p_r.opor.tion i n rumen f l u i d (%)  187  ANOVA molar p r o p o r t i o n o f v a l e r i c a c i d i n rumen f l u i d (%)  188  XXVII  ANOVA rumen ammonia-nitrogen l e v e l s  189  XXVIII  ANOVA abomasal d i g e s t a n o n - p r o t e i n - n i t r o g e n c o n c e n t r a t i o n (%) . . .  XXIV  XXV  XXVI  (ppm).  190  XXIX  ANOVA % RNA-N: % t o t a l N i n abomasal d i g e s t a . . . . 191  XXX  ANOVA % m i c r o b i a l - p r o t e i n - n i t r o g e n : % t o t a l d i g e s t a n i t r o g e n f o r abomasal d i g e s t a ( x : l )  192  - xiii -  ACKNOWLEDGEMENTS  The and  author  i s extremely  p l e a s e d t o acknowledge t h e a s s i s t a n c e  i n v a l u a b l e a d v i c e o f h i s s u p e r v i s o r , Dr. R.M. T a i t o f t h e  Department o f Animal S c i e n c e i n t h e p l a n n i n g and e x e c u t i o n o f t h e r e s e a r c h and t h e p r e p a r a t i o n o f t h i s t h e s i s .  The a s s i s t a n c e and a d v i c e  o f f e r e d i n t h e p r e p a r a t i o n o f t h i s t h e s i s by t h e o t h e r members o f t h e author's  graduate committee, Dr. W.D. K i t t s , Dean o f t h e F a c u l t y o f  A g r i c u l t u r a l S c i e n c e s , Dr. D.B. Bragg, Chairman o f t h e Department o f P o u l t r y S c i e n c e , Dr. V.C. B r i n k o f t h e Department o f P l a n t S c i e n c e and Dr. H.C. Nordan o f t h e Department o f Zoology i s g r e a t l y a p p r e c i a t e d . The Dr.  c o o p e r a t i o n o f t h e Chairman o f t h e Department o f Animal  B.D. Owen, f o r t h e use o f f a c i l i t i e s i s a p p r e c i a t e d .  Science,  Dr. C R .  K r i s h n a r m u t i , Messrs David K i t t s and Robert P r a t t o f t h e Department o f Animal S c i e n c e a r e thanked f o r t h e s u r g i c a l p r e p a r a t i o n of t h e sheep f i t t e d with the re-entrant Mr.  cannula.  G. Galzy, Ms. Frances  Newsome and M i s s Mariana Kung a r e  thanked f o r t h e i r t e c h n i c a l a s s i s t a n c e .  A l l members o f s t a f f and  f a c u l t y o f t h e Department o f Animal S c i e n c e a r e thanked f o r t h e i r assistance.  The h e l p o f Messrs P a u l W i l l i n g and John Ciok o f t h e  U n i v e r s i t y Farm i s g r e a t l y a p p r e c i a t e d . The of C.I.D.A.  author  i s pleased  t o acknowledge t h e f i n a n c i a l a s s i s t a n c e  H i s employers, t h e U n i v e r s i t y o f S c i e n c e and Technology,  Kumasi, Ghana a r e a l s o thanked f o r g r a n t i n g him l e a v e o f absence t o  - xiv -  undertake t h i s study.  The f a m i l y and f r i e n d s  f o r t h e i r moral s u p p o r t .  of the author a r e thanked  - 1 -  INTRODUCTION  S t u d i e s on the d i g e s t i o n o f f e e d s t u f f s by some workers have revealed  t h a t most o f the e s s e n t i a l amino a c i d s b e i n g made a v a i l a b l e  to t h e ruminant a r e from m i c r o b i a l sources (Weller  e t a l . , 1958;  Bergen e t a l . , 1967; L e i b h o l z , 1972; P h i l l i p s o n , 1972).  The rumen  micro-organisms u t i l i z e d i e t a r y n i t r o g e n f o r t h e s y n t h e s i s o f t h e i r c e l l u l a r proteins.  They g e n e r a l l y p r e f e r de novo s y n t h e s i s  a c i d s from s i m p l e r n i t r o g e n skeletons,  s o u r c e s such as ammonia, and a l s o carbon  r a t h e r than making use o f amino a c i d s i n t h e feed  et a l . , 1975; Umuna et_ al_. , 1975).  (Saeur  The micro-organisms h y d r o l y z e the  p r o t e i n s t o amino a c i d s w i t h enzymes, de-aminate  some p o r t i o n s , and  u t i l i z e p a r t o f t h e ammonia r e l e a s e d  to resynthesize  incorporation into microbial protein  (Church, 1975c).  r e l e a s e d ammonia i s l o s t  o f amino  to the animal.  amino a c i d s f o r A p o r t i o n o f the  The degree o f d i e t a r y  nitrogen  l o s s depends p a r t l y on t h e r a t e of p r o t e i n breakdown which i n t u r n depends on p r o t e i n s o l u b i l i t y i n t h e r u m i n a l f l u i d  (Church, 1975c).  T h i s a l s o depends on t h e r a t e o f amino a c i d s y n t h e s i s by t h e rumen micro-organisms.  The l o s s o f d i e t a r y p r o t e i n i s more than compensated  f o r i f t h e d i e t a r y p r o t e i n i s o f poor q u a l i t y , as t h e m i c r o b i a l p r o t e i n so formed i s o f medium q u a l i t y . abomasal  acid.  The micro-organisms a r e k i l l e d by the  Then t h e m i c r o b i a l p r o t e i n i s made a v a i l a b l e t o t h e  animal a f t e r d i g e s t i o n i n t h e lower p a r t s of the d i g e s t i v e t r a c t .  - 2-  I f , however, t h e d i e t a r y p r o t e i n i s o f h i g h q u a l i t y , t h e r e i s a l o s s t o t h e ruminant due t o t h e above p r o c e s s e s .  Therefore,  have been made t o by-pass rumen d i g e s t i o n o f p r o t e i n when h i g h dietary protein i s fed. infusions  attempts quality  The by-pass can be i n t h e form o f abomasal  ( L i t t l e and M i t c h e l , 1967; S c h e l l i n g and H a t f i e l d , 1968) o r  by r e d u c i n g t h e s o l u b i l i t y o f t h e p r o t e i n i n t h e rumen (Ferguson 1967).  The common treatments  a p p l i e d t o reduce s o l u b i l i t y o f d i e t a r y  p r o t e i n s i n t h e rumen a r e heat o r aldehydes  ( P h i l l i p s o n , 1972),  t a n n i c a c i d and v o l a t i l e f a t t y a c i d treatments successfully 1973;  (Nishimuta  e t al.,  e t a l . , 1973; Barker  although  have been attempted  e t a l . , 1973; C a n d l i s h e t a l . ,  Atwal e t a l . , 1974). P r o t e c t i o n o f p r o t e i n c o n t a i n e d i n c a s e i n from a t t a c k by rumen  micro-organisms has been r e p o r t e d t o c o n s i s t e n t l y g i v e p o s i t i v e responses  significantly  o f n i t r o g e n balance,wool growth and growth r a t e  ( L i t t l e and M i t c h e l l , 1967; S c h e l l i n g and H a t f i e l d , 1968; R e i s and Tunks, 1969;  P h i l l i p s o n , 1972).  T h i s has been a t t r i b u t e d t o t h e h i g h e r  quality  of c a s e i n p r o t e i n compared w i t h rumen m i c r o b i a l p r o t e i n . Faichney  and Davies  (1972) t r e a t e d groundnut  (peanut) meal w i t h  formaldehyde and o b t a i n e d a s l i g h t but n o n - s i g n i f i c a n t response balance  compared to u n t r e a t e d groundnut meal.  has been r e p o r t e d t o g i v e v a r i a b l e responses.  i n nitrogen  Treatment o f soybeam meal Some workers  (Schmidt  <et a l . , 1971; Schmidt e t a l . , 1974) o b t a i n e d n e g a t i v e responses  i n nitrogen  b a l a n c e w h i l e P e t e r e t a l . (1970), P e t e r e t a l . (1971), N i m r i c k  et a l .  (1972) and Amos e t a l . (1974) o b t a i n e d p o s i t i v e responses  i n nitrogen  - 3 -  balance.  L i t t l e and M i t c h e l l (1967) o b t a i n e d  r e t e n t i o n when soybean was  infused  increased  nitrogen  abomasally.  There have been l i m i t e d r e p o r t s on formaldehyde treatment dehydrated f o r a g e s  (Hemsley et a l . , 1970;  Dinius  et: a l . ,  of  1975;  Beever et a l . , 1976). Experiments, r e p o r t e d  l a t e r , were conducted to study the e f f e c t  of formaldehyde treatment of the grass-legume forage on n i t r o g e n and  c a r b o h y d r a t e u t i l i z a t i o n by  I s o v a l e r i c and containing p r o t e i n may el-Shazly  p o r t i o n of the d i e t  sheep.  i s o b u t y r i c a c i d s were added to some of the d i e t s  the formaldehyde t r e a t e d f o r a g e .  P r o t e c t i o n of the d i e t a r y  r e s u l t i n d e f i c i e n c y of these a c i d s i n the r u m i n a l (1952a and  1952b) r e p o r t e d  t h a t these v o l a t i l e f a t t y  r e s u l t from the deamination of v a l i n e and  leucine.  r e p o r t e d t h a t Ruminococcus f l a v e f a c i e n s s t r a i n C94, micro-organism r e q u i r e s Sulphur was (1975) r e p o r t e d  i s o b u t y r i c and  i n t o m i c r o b i a l p r o t e i n may R e c y c l i n g of s u l p h u r  be  a rumen c e l l u l o l y t i c  i s o v a l e r i c a c i d s f o r growth.  of i n c o r p o r a t i o n of r e c y c l e d  i n t o the rumen was  reported  i n t o the rumen.  by  of the s u l p h u r  i n the s u l p h u r - c o n t a i n i n g on the other hand, may of d i e t a r y p r o t e i n .  i n most p l a n t s was  amino a c i d s  not be  sulphur.  Sulphur d e f i c i e n c y  reported  About to be  be may  ninety  present  (Beaton e t a l . , 1968).  Nitrogen,  l i m i t i n g i n the rumen w i t h the  protection  Langlands (1973b) and  adequate amounts of r e c y c l e d u r e a - n i t r o g e n treatment of d i e t s f o r sheep.  urea-nitrogen  these workers to  become more acute w i t h 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 . (90%)  Kennedy et a l .  l i m i t e d by the q u a n t i t y of r e c y c l e d  l e s s than r e c y c l i n g of n i t r o g e n  percent  acids  A l l i s o n el; a l . (1962)  a l s o supplemented to some of the d i e t s .  t h a t the extent  fluid.  Faichney  (1974) r e p o r t e d  i n t o the rumen w i t h formaldehyde  - 4 -  LITERATURE REVIEW  Rumen m i c r o b i o l o g y  The ruminant i s a b l e t o s u b s i s t on roughages because o f the micro-organisms p r e s e n t i n the r e t i c u l o r u m e n t h a t can ferment  feeds.  There a r e many types o f micro-organisms i n the rumen, the major b e i n g b a c t e r i a and c i l i a t e d p r o t o z o a .  ones  Other types observed a t times  i n the rumen a r e y e a s t - l i k e organisms, phages and f l a g e l l a t e d p r o t o z o a (Church,  1975b).  Rumen b a c t e r i a  There a r e a number o f ways rumen b a c t e r i a a r e c l a s s i f i e d . of c l a s s i f i c a t i o n i n c l u d e morphology, p r o d u c t s of metabolism. limitations.  Methods  g r a m - s t a i n r e a c t i o n and v a r i o u s  The use o f any one of t h e s e methods a l o n e has  Rumen b a c t e r i a a r e too s i m i l a r t o i d e n t i f y s o l e l y on the  b a s i s o f morphology.  Some s t r a i n s w i t h i n the same s p e c i e s g i v e b o t h  p o s i t i v e and n e g a t i v e r e a c t i o n s t o the gram s t a i n .  There i s u s u a l l y a  g r e a t d e a l o f o v e r l a p p i n g w i t h d i f f e r e n t s p e c i e s of b a c t e r i a w i t h r e g a r d to source of energy, s u b s t r a t e s a t t a c k e d and f e r m e n t a t i o n end-products and b y - p r o d u c t s (Church, 1975b). In view o f the problems l i s t e d above c o n c e r n i n g i d e n t i f i c a t i o n of rumen b a c t e r i a , c l a s s i f i c a t i o n has been d i f f i c u l t .  The  classification  used by Hungate (1966), based p a r t i a l l y on s u b s t r a t e s and p a r t i a l l y the end-products i n i n v i t r o c u l t u r e systems, has been used i n t h i s l i t e r a t u r e review.  on  - 5 -  There are about t e n this  (10)  c l a s s i f i c a t i o n groups known u s i n g  system.  (a) a l s o be  Cellulolytic bacteria:  a b l e to degrade c e l l o b i o s e .  concentration  Have the enzyme c e l l u l a s e and These types a r e i n  when animals are f e d f i b r o u s r a t i o n s .  of these c e l l u l o l y t i c  species  include Bacteroides  Ruminococcus f l a v e f a c i e n s , Ruminococcus a l b u s , and  The  may  greatest most important  succinogenes,  Clostridium loch headii,  Cillobacterium cellulosolvens. (b)  Hemicellulose  digesting bacteria:  Most organisms which  can h y d r o l y z e c e l l u l o s e are a l s o a b l e to u t i l i z e h e m i c e l l u l o s e . of organisms which can u t i l i z e h e m i c e l l u l o s e , cellulose.  Some of the  however, cannot  s p e c i e s which u t i l i z e h e m i c e l l u l o s e  B u t y r i v i b r i o f i b r i s o l v e n s , Lachnospira multiparus,  and  A number utilize  are  Bacteroides  ruminicola. (c) digest  Amylolytic  bacteria:  s t a r c h ; however, there  amylolytic greatest  and  cannot d i g e s t  concentrations  are f e d .  and  ruminicola,  Streptococcus (d)  cellulose.  organisms a r e :  can  purely  b a c t e r i a are i n  l a r g e amounts of Bacteroides  the  starch  amylophilus,  B u t y r i v i b r i o f i b r i s o l v e n s , J3. a l a c t a c i d i g e n s ,  Selenomonas ruminantium, Selenomonas  lactilytica  bovis.  Bacteria u t i l i z i n g  polysaccharides  Amylolytic  when r a t i o n s c o n t a i n i n g  Succinimonas a m y l o l y t i c a ,  organisms  are some organisms which are  Examples of a m y l o l y t i c  Bacteroides  Some c e l l u l o l y t i c  are a l s o a b l e  from p l a n t s , dead and  sugars:  to u t i l i z e  Most of the b a c t e r i a sugars.  lysing bacteria cells,  The  utilizing  sugars may  or from c a p s u l a r  originate  material  -  (mostly carbohydrates  6  -  of b a c t e r i a l c e l l w a l l s ) .  High c o n c e n t r a t i o n s  of micro-organisms dependent on l a c t o s e f o r energy are p r e s e n t rumens of young animals. Eubacterium  An example of b a c t e r i a u t i l i z i n g  i n the  sugars i s  ruminantium.  (e)  Bacteria u t i l i z i n g  organic acids:  The  o r g a n i c a c i d s which  a r e p r o b a b l y u t i l i z e d to the g r e a t e s t extent a r e l a c t i c , and f u m a r i c .  Others  i n c l u d i n g f o r m i c , a c e t i c and o x a l i c are a l s o  u t i l i z e d by some organisms.  The  l a t t e r may  energy to the micro-organisms u t i l i z i n g organisms a r e :  succinic, malic  not be the o n l y source of  them.  Some l a c t i c  acid  utilizing  V e i l l o n e l l a gazogenes, V. a l a c a l e s c e n s , P e p t o s t r e p t o c c u s  e l s d e n i i , P r o p i o n i b a c t e r i u m sp., D e s u l p h o v i b r i o sp., and  Selenomonas  lactilytica. (f)  Proteolytic bacteria:  as the primary  energy s o u r c e s .  B a c t e r i a that u t i l i z e  amino a c i d s  Bacteroides amylophilus, C l o s t r i d i u m  sporogenes and B a c i l l u s l i c h e n i f o r m i s a r e the b e s t known to have proteolytic (g)  capability. Ammonia p r o d u c i n g b a c t e r i a :  various sources.  Examples of these a r e Selenomonas ruminantium,  Peptostreptococcus of  Butyrivibrio (h)  These produce ammonia from  e l s d e n i i , B a c t e r o i d e s r u m i n i c o l a , and  certain  strains  sp.  Methanogenic b a c t e r i a :  These produce methane gas.  a r e o b l i g a t e anaerobes and a r e t h e r e f o r e d i f f i c u l t t o c u l t u r e . n o r m a l l y about 25% of the gas  i n the rumen i s methane (Church,  a l a r g e number of them are i n d i c a t e d . Methanobacterium ruminantium, and M.  They Since  1975b),  Important examples of these  formicium.  importance a r e Methanobacterium s o h n g e n i i , M.  Other s p e c i e s of  are less  suboxydans and Methanosarcina  sp.  - 7 -  (i)  Lipolytic bacteria:  hydrolyze glycerol  from l i p i d s .  These u t i l i z e g l y c e r o l Some l y p o l y t i c  hydrogenate u n s a t u r a t e d f a t t y a c i d s and may isomerization i n fatty acids.  micro-organisms  produce  Viviani  one l y p o l y t i c b a c t e r i u m , A n a e r o v i b r i o l y p o l y t i c a .  (1961) r e p o r t e d Selenomonas ruminantium glycerol  positional  C e r t a i n l i p o l y t i c micro-organisms  metabolize long chain f a t t y a c i d s i n t o ketones. of  and  may  (1970) r e p o r t e d ,  Hobson and Mann  s t r a i n l a c t i l y t i c a n s as a  fermenter.  (j) synthesize  Vitamin synthesizers:  Some rumen b a c t e r i a a r e a b l e to  B- complex v i t a m i n s i n c l u d i n g cobalamin ( v i t . B12) p r o v i d e d  they a r e s u p p l i e d w i t h adequate c o b a l t s y n t h e s i z e r s have not been s t u d i e d  (Phillipson,  1975).  The  vitamin  extensively.  Rumen P r o t o z o a  Rumen p r o t o z o a a r e c l a s s i f i e d a c c o r d i n g to morphology.  They a r e  e a s i e r t o c l a s s i f y by t h a t method compared to b a c t e r i a because of larger size. as f i v e  their  Counts o f p r o t o z o a i n the rumen v a r y from none t o as h i g h  (5) m i l l i o n per ml o f rumen f l u i d .  The s i z e o f rumen p r o t o z o a  v a r i e s from t h i r t y e i g h t microns i n l e n g t h and f i f t e e n microns i n w i d t h f o r Charon e q u i one hundred  t o one hundred  and n i n e t y f i v e microns i n l e n g t h and  and n i n e microns i n w i d t h f o r Metadinium medium.  p r o t o z o a a r e m a i n l y c i l i a t e s a l t h o u g h a t times such f l a g e l l a t e s Monocercomonas ruminantium,  Rumen as  C a l l i m a s t i x f r o n t a l i s , Tetratichomonas sp.,  Pentatrichomonas hominis, Monocercomonas b o v i s and C h i l o m a s t i x sp. a r e observed.  - 8 -  The most important rumen c i l i a t e s b e l o n g t o s u b c l a s s e s h o l o t r i c h i a and s p i r o t r i c h i a . D a s y t r i c h a ruminantium, and a l s o Charon e q u i ,  Examples o f t h e h o l o t r i c h i a a r e  Isotricha intestinalis, which i s r a r e .  I s o t r i c h a prostoma  Examples o f t h e s p i r o t r i c h i a  s u b c l a s s b e l o n g i n g t o t h e o r d e r entodinimorpha  (oligotrichs) are  D i p l o d i n i u m spp., E u d i p l o d i n i u m spp., P o l y p l a s t r o n spp., E l y s t r o p l a s t r o n spp., O s t r a c o d i n i c u m spp., and E n o p l o p l a s t r o n spp. are o b l i g a t e  Other rumen  Rumen p r o t o z o a  anaerobes.  micro-organisms  Other rumen micro-organisms v i r u s e s ) y e a s t - l i k e organisms, a r e about one hundred  include bacteriophages  and f a c u l t a t i v e l y a n a e r o b i c f u n g i .  and twenty  five  There  (125) m o r p h o l o g i c a l l y d i s t i n c t  types o f b a c t e r i o p h a g e s observed i n t h e rumen. i n s i d e rumen b a c t e r i a .  (bacterial  Some have been observed  The b a c t e r i o p h a g e s may exceed rumen b a c t e r i a i n  numbers by two t o t e n t i m e s .  Pun and S a t t e r  (1975) r e p o r t e d t h e e x i s t e n c e  of n i t r o g e n f i x i n g b a c t e r i a i n t h e rumen b u t b e l i e v e d these b a c t e r i a were n o t o f any s i g n i f i c a n t v a l u e .  F a c t o r s a f f e c t i n g p o p u l a t i o n s o f rumen  Many k i n d s o f micro-organisms many ways by which micro-organisms  micro-organisms  a r e p r e s e n t i n t h e rumen.  may a r r i v e i n t h e rumen.  e n t e r i n g t h e rumen i n c l u d e b e i n g c a r r i e d by food, water, d r e n c h i n g w i t h drugs  (Church, 1975b). Micro-organisms  There a r e  Methods o f  l i c k i n g and  that are normally  - 9 -  not p a r t of the rumen m i c r o b i a l p o p u l a t i o n G a l l and Hubtanen (1950) l i s t e d  may  be p r e s e n t  at t i m e s .  the f o l l o w i n g as c r i t e r i a to  s a t i s f i e d i f micro-organisms a r e to be  considered  be  t y p i c a l o f the rumen  environment:  (a)  the organism must be  a b l e to l i v e  (b)  i t s h o u l d be a b l e t o produce the t y p e s of end-products c h a r a c t e r i s t i c of the rumen,  (c)  the rumen should gram of the  c o n t a i n not  and l e s s than one  protozoa.  populations.  o f t h e f a c t o r s which can a f f e c t t y p e and number o f m i c r o -  organisms i n the rumen i s c y c l i c a l v a r i a t i o n s . f a t t y a c i d s on the growth of some o t h e r s reported.  R e s t r i c t i o n i n growth o f one  I n h i b i t i o n by  organisms p r o d u c i n g the t o x i n s and  and  Hungate (1970) r e p o r t e d  volatile  such as E s c h e r i c h i a c o l i has type of microbe by  a n t i b i o t i c s produced by another t y p e has been p o s t u l a t e d .  1975b).  per  a l s o be noted t h a t animals on the same or s i m i l a r d i e t s  might have d i f f e r e n t m i c r o b i a l One  million  organisms.  T h i s l a s t c r i t e r i o n a p p l i e s to b a c t e r i a but not I t should  anaerobically;  a n t i b i o t i c s are not  toxins  The  or  Those m i c r o -  affected  t h a t mycoplasma which can k i l l  p r o t o z o a have been observed f r e e i n the rumen.  (Church, bacteria  mycoplasma  s e c r e t e s an enzyme or enzymes c a p a b l e of d i g e s t i n g B u t y r i v i b r i o sp., Ruminococcus sp.,  and  Streptococcus bovis.  E s c h e r i c h i a c o l i but not  the  been  gram-positive  - 10  -  D i e t can a f f e c t m i c r o b i a l p o p u l a t i o n s i n the rumen. may  a l s o be i n f l u e n c e d by geographic  micro-organisms may  be present  l o c a t i o n or season.  The  diet  Some types  i n some ruminant s p e c i e s but not  of  others.  For i n s t a n c e a l a r g e o v a l form of b a c t e r i a (Quinn's o v a l ) i s common i n sheep but not c a t t l e . i s the d i e t . of  Perhaps the most important  influencing factor  Church (1975b) i n d i c a t e d t h a t the optimum pH f o r growth  rumen micro-organisms i s between 5.5  and  7 w h i l e the optimum  temperature range i s 39-41°C. E a d i e and Mann (1970) r e p o r t e d t h a t h i g h s o l u b l e  carbohydrate  l e v e l s i n the d i e t r a p i d l y r e s u l t s i n low pH from the p r o d u c t i o n excess  acid.  T h i s i n t u r n changes the m i c r o - f l o r a and  such c o n d i t i o n s , the o n l y c i l i a t e s Mann, 1970). a r e absent  below pH 6.  cellulolytic  organisms.  of the o p i n i o n t h a t  and  protozoa high  E a d i e and Mann (1970) however r e p o r t e d t h a t v i a b l e b a c t e r i a counts may  These workers a l s o r e p o r t e d  be  flagellate  l a r g e b a c t e r i a such as Selenomonas sp. to be p r e s e n t i n  h i g h c o n c e n t r a t i o n i n animals  f e d h i g h carbohydrate  diets.  (1970) a l s o r e p o r t e d t h a t the l e v e l of f e e d i n g of h i g h d i e t s a f f e c t e d the types of rumen microbes. d i e t reduced  f e e d i n g d i d not. carbohydrate  (Eadie  g e n e r a l l y r e s u l t i n a d e p r e s s i o n i n the numbers  h i g h e r than w i t h roughage d i e t s .  carbohydrate  Under  Church (1975b) a l s o r e p o r t e d t h a t d i e t s  w i t h d i e t s h i g h i n s o l u b l e carbohydrates  p r o t o z o a and  a r e E n t o d i n i a sp.  Hobson (1971) however was  i n s o l u b l e carbohydrates of  found  fauna.  of  Full  S l y t e r et a l .  carbohydrate  f e e d i n g of a h i g h  the number of p r o t o z o a w h i l e  restricted  These workers a l s o r e p o r t e d t h a t f e e d i n g of h i g h  d i e t s caused more u n s t a b l e rumen c o n d i t i o n s than  feeding  - 11 -  roughage d i e t s .  These u n s t a b l e c o n d i t i o n s r e s u l t e d i n g r e a t e r  animal v a r i a t i o n s even w i t h i d e n t i c a l twins w i t h r e s p e c t t o of pH,  and v o l a t i l e f a t t y a c i d p r o d u c t i o n .  animals w i t h l e a s t a b i l i t y first  stability  They a l s o i n d i c a t e d  that  t o s t a b i l i z e rumen c o n d i t i o n s were the  t o l o s e the c i l i a t e p r o t o z o a . C e r t a i n rumen micro-organisms  a r e produced by o t h e r s .  F a c t o r s which i n h i b i t  p r o d u c i n g these f a c t o r s i n h i b i t factors  a l s o r e q u i r e some f a c t o r s which  (Church, 1975b).  the growth of those  the p o p u l a t i o n o f those r e q u i r i n g  these  B a c t e r o i d e s succinogenes, R. a l b u s , and  B u t y r i v i b r i o f i b r i s o l v e n s r e q u i r e B v i t a m i n s produced by o t h e r microbes (Church, 1975b).  Some ammonia u t i l i z i n g b a c t e r i a , e s p e c i a l l y B a c t e r o i d e s  succinogenes and a l s o R. f l a v e f a c i e n s r e q u i r e i s o v a l e r i c ,  N-valeric  i s o - b u t y r i c and 2-methyl b u t y r i c a c i d s , f o r t h e i r growth ( A l l i s o n e t a l . , 1962;  A l l i s o n and B r y a n t , 1963;  1966).  A l l i s o n and Bryant  Hemsley and M o i r , 1963;  (1963) thought  A l l i s o n et a l . ,  t h a t the mechanism  s y n t h e s i s o f the i s o p r o p y l moiety i n the b r a n c h e d - c h a i n f a t t y was  inadequate i n these micro-organisms.  el-Shazly  f o r the acids  (1952a and 1952b)  r e p o r t e d t h a t the b r a n c h e d - c h a i n f a t t y a c i d s r e s u l t e d from the  de-amination  of p r o t e i n s .  (Kennedy  et a l . ,  1975).  Sulphur i s a l s o r e q u i r e d by some micro-organisms  Hobson (1971) c l a i m e d t h a t f a c u l t a t i v e anaerobes  such  as S t r e p t o c o c c u s b o v i s and V e i l l o n e l l a gazogenes take up the s m a l l amount of oxygen e x i s t i n g i n the rumen, t h i s enables the methanogenic b a c t e r i a . a n d p r o t o z o a which a r e c o m p l e t e l y a n a e r o b i c t o s u r v i v e . B a c t e r o i d e s a m y l o p h i l u s and Selenomonas ruminantium o x i d a t i o n - r e d u c t i o n p o t e n t i a l of even - 45 mv.  This also helps which cannot  tolerate  Methane i n h i b i t o r s  as bromochloromethane and u n s a t u r a t e d f a t t y a c i d s have been used t o  such  - 12  suppress methane p r o d u c t i o n These may  -  (Johnson at a l . , 1972;  Sawyer e t a l . , 1974).  a l s o a d v e r s e l y a f f e c t the p o p u l a t i o n s o f methanogenic b a c t e r i a .  C y c l i c a l v a r i a t i o n s i n p r o t o z o a l numbers a r e more pronounced than b a c t e r i a l numbers. two  Highest  hours a f t e r f e e d i n g  about 4-8 1968).  p r o t o z o a l c o n c e n t r a t i o n s have been  ( C l a r k e , 1965)  hours a f t e r f e e d i n g .  recorded  and b a c t e r i a numbers are  (Moir and  Sommers, 1956;  Bryant  highest and  Seasonal v a r i a t i o n s a r e due m a i n l y to changes i n f e e d .  d e f i c i e n c y or m i n e r a l  d e f i c i e n c y due  l i m i t growth (Schwartz and  to s e a s o n a l  Gilchrist,  1975;  Robinson,  Nitrogen  e f f e c t s on d i e t ,  can  Church,1975b; Amos e t a l . ,  1976a).  M i c r o b i a l p r o t e i n - n i t r o g e n composition  The dry matter  n i t r o g e n content ( i . e . about 65%  and 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  of rumen b a c t e r i a i s about 10.5% CP).  Amino n i t r o g e n i s about 75%  of the  cell  of the  total  n i t r o g e n i n mixed organisms from the rumen ( b a c t e r i a , p r o t o z o a Rumen b a c t e r i a c o n t a i n about 86% ( A l l i s o n , 1970).  The  and RNA.  accounts f o r 2.2  - 4.1%  of rumen b a c t e r i a i s  N u c l e i c a c i d may  o f the t o t a l n i t r o g e n of rumen micro-organisms. s i n c e DNA  others).  of t h e i r t o t a l n i t r o g e n as amino n i t r o g e n  non-amino-nitrogen content  m a i n l y n u c l e i c a c i d s , DNA  and  account f o r 14-19%  Most of t h i s i s  of the t o t a l n i t r o g e n  (Allison,  RNA 1970).  L i t t l e has been done t o c h a r a c t e r i z e the w a l l s of rumen b a c t e r i a .  The  c e l l - w a l l of s e v e r a l non-rumen b a c t e r i a accounts f o r 10-20% of the mass o f t h e c e l l and  the c e l l - w a l l m a t e r i a l i n these organisms i s 5-10%  ( A l l i s o n , 1970).  N  I f rumen b a c t e r i a have s i m i l a r p r o p o r t i o n s of w a l l s  - 13 -  and w a l l n i t r o g e n and i f the t o t a l c e l l s have a n i t r o g e n content o f 10.5%  then c e l l - w a l l n i t r o g e n would be 5-19% o f t h e t o t a l  nitrogen,  ( A l l i s o n , 1970).  peptidoglycan  cell  The b a c t e r i a l c e l l w a l l s may have  o r mucoprotein as major components.  These a r e r e s i s t a n t  to t r y p s i n and p e p s i n a t t a c k and may n o t be u s e f u l t o t h e h o s t .  Non-  amino-nitrogen i n the c e l l w a l l i s m a i n l y i n t h e form o f n e u c l e i c a c i d s . Muramic a c i d and c e l l walls while c e l l walls et  al.  2-aminoethylphosphonic a c i d i s a l s o unique t o p r o t o z o a l  (1967) r e p o r t e d  leucine.  a c i d s a r e unique t o b a c t e r i a l  (Work, 1951; Work and Dewey, 1958; A l l i s o n ,  cellulolytic  and  °C-; ^ - d i a m i n o p i m e l i c  t h a t t h e l i m i t i n g amino a c i d  1970).  Bergen  i n pooled  s t r a i n s was m e t h i o n i n e and f o r n o n - c e l l u l o l y t i c s t r a i n s was  P r o t o z o a l p r o t e i n s a r e more d i g e s t i b l e than b a c t e r i a l  protozoa  have a h i g h e r  l y s i n e content  proteins  than b a c t e r i a (Church, 1975b).  Hogan and Weston (1970) i n d i c a t e d t h a t about 10-12g d r y weight o f microbes a r e s y n t h e s i z e d  p e r mole o f ATP but DM m i c r o b i a l weight  s y n t h e s i s c o u l d i n c r e a s e t o about 20g p e r mole ATP, w i t h an i n c r e a s e i n -1 d i l u t i o n r a t e from 0.1 h r  -1 t o 0.3 t o 0.5 h r .  They i n d i c a t e d t h a t 2.37g  of b a c t e r i a n i t r o g e n are produced p e r mole o f v o l a t i l e f a t t y a c i d produced. Walker and Nader (1968), r e p o r t e d synthesized  p e r mole o f ATP.  13-14g d r y weight o f m i c r o b i a l  cells  Hume (1970) r e p o r t e d an i n c r e a s e i n  microbial protein synthesis with  the a d d i t i o n o f  i s o v a l e r i c and n - v a l e r i c a c i d s t o d i e t s c o n t a i n i n g  2-methylbutyric, non-protein-nitrogen.  Other workers (Hemsley and M o i r , 1963; Umuna e t a l . , 1975) have a l s o r e p o r t e d i n c r e a s e s i n 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 when some branchedc h a i n f a t t y a c i d s and v a l e r i c a c i d were added t o d i e t s c o n t a i n i n g  urea.  - 14 -  Beever e t a l . (1977) O.M. d i g e s t e d for  r e p o r t e d 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 p e r lOOg  i n t h e rumen o f 16.7g f o r a grass  a formaldehyde t r e a t e d grass  a negative  silage diet.  Hume (1970) r e p o r t e d  c o r r e l a t i o n between a c e t i c a c i d p r o p o r t i o n s  v o l a t i l e f a t t y a c i d s and p r o t e i n s y n t h e s i s He  s i l a g e d i e t and 6.6g  i n rumen  ( r = -0.62, p  0.025).  concluded t h a t t h e e f f i c i e n c y w i t h which energy was used f o r m i c r o b i a l  growth was d i m i n i s h e d increased.  as a c e t i c a c i d p r o p o r t i o n s  Schwartz and G i l c h r i s t  also reported  fluid  (1975) and Ishaque et: a l . (1971)  that propionic a c i d fermentation  microbial protein synthesis  i n rumen  was more e f f i c i e n t f o r  than a c e t i c o r b u t y r i c a c i d  H a r r i s o n e t a l . (1976) however r e p o r t e d  fermentation.  a c e t i c a c i d fermentation than p r o p i o n i c  t o be  more e f f i c i e n t  f o r microbial protein synthesis  fermentation.  These workers i n f u s e d a r t i f i c i a l s a l i v a a t t h e r a t e o f  f o u r l i t r e s p e r day t o a l t e r p r o p i o n i c a c i d f e r m e n t a t i o n fermentation.  The e f f e c t o f t h e i n c r e a s e d  acid  to a c e t i c acid  flow r a t e was n o t d e l i n e a t e d .  A number o f methods have been used t o a s s e s s m i c r o b i a l p r o t e i n synthesis.  These i n c l u d e measuring d i a m i n o p i m e l i c  aminoethylphosphonic Amos, e t a l . , 1976a);  acid  acid  (DAP) and  (AEP) i n t h e sample (Hogan and Weston, 1970;  p r e c i p i t a t i o n of m i c r o b i a l p r o t e i n with  t r i c h l o r o a c e t i c , p e r c h l o r i c , p i c r i c a n d ' t u n g i s t i c a c i d s and measuring the amount o f t h e p r e c i p i t a t e d p r o t e i n 1970;  Barr  e t a l . , 1975);  35 sulphur,  (Hemsley and M o i r , 1963; Hume,  l a b e l l i n g t h e rumen p o o l w i t h r a d i o a c t i v e  c  . ?, and measuring r a d i o a c t i v i t y l e v e l o f s u l p h u r  c o n t a i n i n g amino a c i d s  i n sulphur-  (Walker and Nader, 1975; Hume, 1974); and t h e  measurement o f r i b o n u c l e i c a c i d - n i t r o g e n  (Smith, 1975).  - 15 -  All  the methods o t h e r than the RNA-N, l a b e l l i n g w i t h  and the combination of DAP protein synthesis.  S  and AEP measure m a i n l y rumen b a c t e r i a l  The DAP measures b a c t e r i a l p r o t e i n s y n t h e s i s and  AEP measures p r o t o z o a l p r o t e i n s y n t h e s i s 1958;  J J  Church, 1975b).  (Work, 1951; Work and Dewey,  The RNA-N method measures b o t h b a c t e r i a l and  p r o t o z o a p r o t e i n s y n t h e s i s w i t h o u t a p p o r t i o n i n g f r a c t i o n s to each type 35 of organism (Smith, 1975).  With the  S  method, problems o f i n f u s i o n  to m a i n t a i n steady s t a t e c o n d i t i o n s e x i s t . the DAP  method has d i s a d v a n t a g e s .  One  Smith (1975) b e l i e v e s  of these i s t h a t DAP  that  released  into  the rumen from c e l l l y s i s due to r e c y c l i n g of b a c t e r i a i s r e s i s t a n t to d e g r a d a t i o n and thus i n c r e a s e s measured p r o t e i n s y n t h e s i s .  The  AEP  method a c c o r d i n g to Smith (1975) i s u n r e l i a b l e and u n s a t i s f a c t o r y .  The  t e c h n i c a l b u l l e t i n o f the I n t e r n a t i o n a l Atomic Energy Agency (IAEA)  (1970)  i n d i c a t e s t h a t DAP  i s r e s t r i c t e d t o gram-negative b a c t e r i a and b a c t e r i a  c o n t e n t s o f DAP v a r y g r e a t l y . b a c t e r i a l c o n t e n t s o f DAP w i t h an average o f 41  Hogan and Weston (1970) e s t i m a t e d t h a t  v a r y from 35-46 mg/g  DM b a c t e r i a l n i t r o g e n  mg/g.  Smith (1975) b e l i e v e s t h a t the RNA-N method i s more r e l i a b l e the r a t i o of RNA-N to t o t a l n i t r o g e n i s about 0.075 + 0.010 to t o t a l n i t r o g e n i s 0.5  - 1.1  since  and f o r DAP-N  f o r different b a c t e r i a l species.  RNA i s  a s s o c i a t e d w i t h ribosomes and the q u a n t i t y o f ribosomes i n b a c t e r i a l c e l l s i s p o s i t i v e l y c o r r e l a t e d w i t h the r a t e of p r o t e i n (Church, 1975b). rumen, RNA synthesis  A l t h o u g h t h e r e i s about 30% r e c y c l i n g o f RNA  i s broken down q u i c k l y and may (McAllan and Smith, 1973;  D i e t a r y RNA  synthesis  and o t h e r sources of RNA  q u i c k l y broken down (Smith, 1975;  i n the  not a f f e c t measured p r o t e i n  Smith, 1975;  Smith and Smith, 1977).  i n t r o d u c e d i n t o the rumen a r e a l s o  Smith and Smith, 1977).  - 16  -  Metabolism of n i t r o g e n  There i s q u i t e a v a r i a t i o n i n the n i t r o g e n o u s presented  to the rumen micro-organisms.  p r o t e i n s , n u c l e i c a c i d s and n o n - p r o t e i n acids, peptides,  The most important  n i t r o g e n c o n s i s t i n g of amino  at times b i u r e t i n t e n t i o n a l l y put  The n o n - p r o t e i n - n i t r o g e n  nitrogen i n unwilted  c e l l w a l l and  s i l a g e s . (Church, 1975c).  of the t o t a l n i t r o g e n may  i s largely indigestible.  presented  Nitrogen  (1970) i n d i c a t e d  Thomas (1976)  estimated  i n the a c i d - d e t e r g e n t  d i v e r s i t y of nitrogenous  insoluble  compounds  reticulo-rumen.  metabolism i n the ruminant i s d e s c r i b e d by the diagram  (1970) reproduced as F i g u r e  Degradation of  Nolan and  1.  Proteins  Many r e s e a r c h e r s 1971;  Allison  total  to rumen microbes r e s u l t s i n c o n s i d e r a b l e v a r i a t i o n i n the  n i t r o g e n metabolism i n the  of Houpt  The  60-75% of  be bound w i t h l i g n i n i n the  Yu and  t h a t about 7% of t o t a l n i t r o g e n i s p r e s e n t n i t r o g e n i n normal f o r a g e s .  into  i n many n a t u r a l f e e d s t u f f s ranges  from 4 to 5% of t o t a l n i t r o g e n i n some seeds and  t h a t about 5-10%  are:  amides, amines, v o l a t i l e amines, ammonium s a l t s ,  n i t r a t e s , n i t r i t e s , u r e a and the d i e t .  compounds  ( A l l i s o n and  Leng, 1972;  P e e l , 1971;  Umuna et a l . , 1975)  Mathison and  Milligan,  have i n d i c a t e d t h a t  rumen micro-organisms p r e f e r de novo s y n t h e s i s of p r o t e i n s .  Dietary  p r o t e i n s , except g l o b u l i n s i n young animals, may  before  be degraded  - 16a -  Fig.  1.  Schematic r e p r e s e n t a t i o n o f the p r o t e i n r e g e n e r a t i o n i n ruminants. (Adapted from Houpt, 1970)  cycle  - 17  b e i n g u t i l i z e d by  the microbes.  -  The  end-products of  protein  d e g r a d a t i o n i n the rumen are carbon d i o x i d e , hydrogen acetic, propionic acids containing  Some i n t e r m e d i a r y  d i f f u s i b l e peptides  (1.5-40 mg%), and 1952a and  b u t y r i c a c i d s , higher branched-chain f a t t y  s i x carbon atoms and  (Church, 1975c). (0.1-1.5 mg%),  and  Smith and M c A l l a n , 1970;  Ellis  (0.2-1.0 mg%),  and  A l l i s o n and  et a l . , 1958;  as NH_,-N)  p r o d u c t s i n c l u d i n g f r e e amino a c i d s  of the t o t a l n i t r o g e n  - about 63-81% (Weller B l a c k b u r n and  ammonia (0-130 mg%,  protein-N(100-400 mg%)  b; Annison, 1956;  as a p r o p o r t i o n  sulphide  nucleotide  have been shown Pfander, 1965;  P e e l , 1971).  - N  (el-Shazly,  Allison,  Microbial  1970; nitrogen  i n the rumen i s q u i t e v a r i a b l e B l a c k b u r n and  Hobson, 1960).  Hobson (1960) e s t i m a t e d t h a t about 47-77% of the  i n the rumen was  contained  i n p r o t o z o a and  nitrogen  b a c t e r i a , w h i l e 54-74%  was  p r e s e n t i n the rumen f l u i d . The  proportion  of d i e t a r y p r o t e i n e s c a p i n g d e g r a d a t i o n i n  rumen i s a f f e c t e d by many f a c t o r s which a f f e c t r e t e n t i o n times i n reticulo-rumen. and  the  the  These i n c l u d e s p e c i f i c g r a v i t y , p a r t i c l e s i z e of d i e t ,  h i g h water consumption r e s u l t i n g from h i g h  salt  intake  (Chalupa, 1975).  S o l u b i l i t y of the p r o t e i n a l s o a f f e c t s d i e t a r y p r o t e i n d e g r a d a t i o n i n rumen (Hemsley et a l . , 1970; that feeding pH,  high  Chalupa, 1975).  indicated  l e v e l s of s o l u b l e c a r b o h y d r a t e s , which d e c r e a s e s rumen  decreased d e g r a d a t i o n of p r o t e i n i n the rumen.  (1964) r e p o r t e d  greater  However, T a g a r i  d e g r a d a t i o n of p r o t e i n i n the rumen by  r e a d i l y s o l u b l e c a r b o h y d r a t e s f o r roughages. t h a t about 40-80% of d i e t a r y p r o t e i n may and  Chalupa (1975) a l s o  the  Chalupa (1975)  substituting reported  be degraded i n the rumen.  R o f f l e r (1975) gave the f o l l o w i n g as the percentage of  et a l .  protein  Satter  - 18  escaping  degradation f o r various  b a r l e y 10%,  cottonseed  soybean meal 45%, f i s h meal 70%. 56%  d r i e d grass  f e e d s t u f f s fed i n b a s a l r a t i o n s : peanut meal 20%,  sunflower meal  and w h i t e f i s h meal 50%,  Ferguson (1975) i n d i c a t e d t h a t 61%  of z e i n , and  9%  Leng (1972) r e p o r t e d was  meal and  -  Peruvian  of soybean meal,  of c a s e i n p r o t e i n s escaped d e g r a d a t i o n . t h a t about 59%  degraded t h e r e ; w h i l e 29%  amino a c i d s , 71% was  and  25%,  Nolan  and  of the p r o t e i n e n t e r i n g the rumen  of the degraded p r o t e i n was  f u r t h e r degraded to ammonia.  utilized  as  Nolan (1975) r e p o r t e d  t h a t about 30%  of d i e t a r y p r o t e i n i n t a k e i s g e n e r a l l y degraded to ammonia  w i t h d r i e d and  processed forage  diets.  He  e s t i m a t e d t h a t about  70%  of the d i e t a r y p r o t e i n e i t h e r passes i n t a c t from the rumen or i s a s s i m i l a t e d by rumen micro-organisms i n the form of compounds o t h e r ammonia, such as p e p t i d e s , et was  amino a c i d s or n u c l e i c a c i d bases.  Pilgrim  a l . (1969) e s t i m a t e d t h a t 23-27% of d i e t a r y p r o t e i n i n a l f a l f a c o n v e r t e d to ammonia as compared to 17% The  i n question. a c i d s , and  from a l f a l f a  than  hay  pellets.  r a t e o f deamination of amino a c i d s v a r i e s w i t h the amino a c i d s Isaacs  and  Owens (1971) r e p o r t e d  t h a t a s p a r t i c and  a r g i n i n e were degraded i n the rumen to the extent  v a l i n e , l e u c i n e , i s o l e u c i n e , m e t h i o n i n e , a l a n i n e and be r e l a t i v e l y s t a b l e toward m i c r o b i a l a c t i o n . were degraded to about  The  glutamic  of about  90%;  g l y c i n e appeared to  phenolic  amino a c i d s  50%.  Some of the p r o t e o l y t i c and  ammonia p r o d u c i n g b a c t e r i a have been  l i s t e d p r e v i o u s l y under rumen m i c r o b i o l o g y . i n d i c a t e d that p r o t e o l y t i c a c t i v i t y i s not dependent on d i e t .  was  B l a c k b u r n and  maximum between pH  Hobson (1960) 6 and  B l a c k b u r n (1968) a l s o suggested t h a t  7  and  proteases  - 19 -  o b t a i n e d from B_. a m y l o p h i l u s had a broad a r e a o f a c t i v i t y between pH 5.5-9.5.  Isaac and Owens (1971) r e p o r t e d t h a t  extra-cellular  enzymes o t h e r than amino p e p t i d a s e c o u l d p o s s i b l y h e l p i n h y d r o l y z i n g p r o t e i n s i n the rumen.  Allison  (1970) i n d i c a t e d t h a t  although  p r o t e o l y t i c enzymes may be m a i n l y c e l l - b o u n d , some gram p o s i t i v e such as C l o s t r i d i u m sp., Eubacterium produce  e x t r a - c e l l u l a r enzymes.  sp., and L a c h n o s p i r a m u l t i p a r u s  Allison  (1970) a l s o r e p o r t e d t h a t i n  B_. a m y l o p h i l u s the p r o t e a s e i s a c o n s t i t u t i v e enzyme. Blackburn  Abou Akkada and  (1963) r e p o r t e d t h a t p r o t e a s e s i n some p r o t e o l y t i c  possessed both exo- and endo-peptidase  microbes  activities.  P r o t e o l y t i c a c t i v i t y o f rumen p r o t o z o a i s not w e l l Abou Akkada and Howard  cocci  understood.  (1962) r e p o r t e d rumen p r o t o z o a t o h y d r o l y z e  c a s e i n to p e p t i d e s and amino a c i d s , as p r i n c i p a l end-products.  Ammonia  was formed as a r e s u l t o f the h y d r o l y s i s of the amide groups i n the c a s e i n but not from de-amination  o f amino a c i d s .  t h a t ammonia was the end-product  of n i t r o g e n metabolism  of p r o t o z o a .  P u r s e r and M o i r  Warner (1956) thought i n mixed suspensions  (1966) observed h i g h c o n c e n t r a t i o n s of  ammonia i n the rumen o f sheep c o n t a i n i n g p r o t o z o a compared to defaunated animals. B l a c k b u r n  (1965) noted t h a t though many p r o t e o l y t i c  have been e x t r a c t e d from rumen p r o t o z o a , i t i s d i f f i c u l t c e r t a i n t y t h a t they were produced found i n them.  enzymes  to claim with  by the p r o t o z o a and not by b a c t e r i a  C i l i a t e s a r e noted t o take up o n l y s m a l l q u a n t i t i e s of  " ^ N - l a b e l l e d amino a c i d s and may t h e r e f o r e depend g r e a t l y on rumen b a c t e r i a f o r amino a c i d s and s h o u l d most p r o b a b l y have p r o t e o l y t i c enzymes  (Allison,  1970).  - 20 -  The by  concentration  time o f f e e d i n g and type o f d i e t (Church, 1975c).  concentration The  o f ammonia i n the rumen f l u i d  i s highest  i s affected  Ammonia-nitrogen  a t 90-130 minutes a f t e r f e e d i n g  (Church, 1975c).  a d d i t i o n o f s t a r c h t o roughage b a s a l d i e t s c o n t a i n i n g  or u r e a r e s u l t e d , i n i n c r e a s e d sequently  either casein  e f f i c i e n c y o f ammonia u t i l i z a t i o n and con-  lowered rumen ammonia l e v e l s (Barej  el: a l . , 1970).  T a g a r i e t a l . (1964) and Schwartz and G i l c h r i s t  In c o n t r a s t ,  (1975) i n d i c a t e d  p r o t e i n breakdown i n t h e rumen c o u l d be enhanced  that  and ammonia l e v e l s  i n c r e a s e d when h i g h l e v e l s o f s t a r c h a r e f e d s i n c e t h e most p r o t e o l y t i c s p e c i e s , 15. a m y l o p h i l u s , S^. ruminantium and M. e l s d e n i i c o u l d i n numbers t e n f o l d .  S a t t e r and R o f f l e r (1975) r e p o r t e d  increase  t h a t maintenance  of r u m i n a l ammonia-nitrogen l e v e l s i n excess o f 5mg/100 ml rumen does not improve 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 . ammonia-nitrogen l e v e l s  fluid  They c a l c u l a t e d t h a t rumen  i n d a i r y c a t t l e f e d normal d a i r y d i e t s may  vary  from l-5mg/100ml depending on t h e energy content o f t h e d i e t w i t h the highest  l e v e l s a s s o c i a t e d w i t h lowest energy content d i e t . Studies  by  on t h e d e g r a d a t i o n o f p r o t e i n s i n t h e rumen a r e  s e c r e t i o n o f u r e a i n t o t h e rumen v i a s a l i v a , and o f ammonia  the rumen e p i t h e l i u m , by  absorption  complicated through  o f ammonia and o t h e r n i t r o g e n o u s compounds  t h e rumen e p i t h e l i u m and r e c y c l i n g o f m i c r o b i a l p r o t e i n w i t h i n t h e  rumen (Church, 1975c). deaminated.  M i c r o b i a l p r o t e i n i s r e a d i l y hydrolyzed  M i c r o b i a l p r o t e i n r e c y c l i n g and m i c r o b i a l p r o t e i n  and synthesis  from u r e a and/or ammonia and f r e e amino a c i d s may be r e l a t i v e l y more important than d e g r a d a t i o n o f i n g e s t e d protein synthesis  (Church, 1975c).  p r o t e i n t o be used f o r m i c r o b i a l  Nolan and Leng (1972), e s t i m a t e d  - 21  -  t h a t about 4.3gm/N might be r e c y c l e d per day w i t h i n the rumen of sheep. being  They a l s o suggested t h a t about 30%  of ammonia c o n t i n u a l l y  i n c o r p o r a t e d i n t o r u m i n a l m i c r o b i a l p r o t e i n may  r e c y c l e d through the amino a c i d and r e p o r t e d t h a t about 30% Hume (1970), was  ammonia p o o l s .  have been  Chalupa  (1975),  of b a c t e r i a l p r o t e i n i s degraded i n the rumen.  a l s o of the o p i n i o n t h a t desquamation of rumen  e p i t h e l i u m though s m a l l , c o u l d be a source  of n i t r o g e n to rumen m i c r o -  organisms. Urea t r a n s f e r i n t o the rumen ammonia p o o l from b l o o d on rumen ammonia c o n c e n t r a t i o n ) microbes (Church, 1975c). urea was  Nolan  i s one  source  (dependent  of n i t r o g e n to rumen  (1975) r e p o r t e d  t h a t about l - 2 g of  t r a n s f e r r e d d a i l y i n sheep f e d l u c e r n e c h a f f d i e t s .  (1970) r e p o r t e d  t h a t on low p r o t e i n d i e t s about 92%  e n t e r s the a l i m e n t a r y nitrogenous  compounds.  c a n a l and  about 84%  Hemler and  Houpt  of endogenous urea  i s converted  to complex  B a r t l e y (1971) were of the  t h a t more urea e n t e r s the rumen from b l o o d than from s a l i v a . however thought t h a t more urea e n t e r e d plasma.  Hogan el: a l . (1969)  from s a l i v a s i n c e about 2-5g, (about  N per day and  and M i l l e r  urea  in saliva  ml on a roughage d i e t ) .  secreted Allen  (1976) r e p o r t e d u r e a e n t r y i n t o rumen to be an a c t i v e  r a t h e r than by simple  diffusion.  process  Houpt (1970) c o n s i d e r s that' the major  p o r t i o n of the u r e a e n t e r i n g the rumen from b l o o d i s converted by rumen b a c t e r i a l urease w i t h i n the c o r n i f i e d l a y e r s b e f o r e the rumen.  entering  thought almost a l l came  N/day c o u l d be p r e s e n t  10 l i t r e s c o n t a i n i n g 28mg N/100  Hogan (1975)  the rumen from s a l i v a than from  and Hogan (1975) e s t i m a t e d  the rumen o f sheep to be about 2-5g  opinion  to ammonia  entering  - 22 -  Urea from s a l i v a , b l o o d and i n t h e d i e t i s h y d r o l y z e d u r e o l y t i c enzymes p r o b a b l y 1970).  by  produced by s e v e r a l b a c t e r i a ( A l l i s o n ,  The enzymes from t h e mixed b a c t e r i a were s t i m u l a t e d by Mn,  Mg, Ca, S r , Ba, b u t i n h i b i t e d by Na, K and Co.  An enzyme from a s i n g l e  b a c t e r i a s t r a i n was n o t s t i m u l a t e d by a l l t h e d i v a l e n t i o n s . Ammonia produced i n t h e rumen c o u l d be used f o r (1) m i c r o b i a l amino a c i d s y n t h e s i s o r (2) c o u l d be absorbed i n t o t h e b l o o d the r e t i c u l o - r u m e n  stream from  and omasum o r (3) c o u l d pass i n t o the abomasum and  consequently the duodenum, s i n c e t h e r e i s v i r t u a l l y no a b s o r p t i o n o f ammonia from t h e abomasum.  P a r t o f the ammonia absorbed from t h e  forestomach c o u l d be l o s t through u r i n e a f t e r c o n v e r s i o n u r e a c o u l d be r e c y c l e d .  P a r t o f t h e ammonia c o u l d a l s o be used f o r t h e  s y n t h e s i s o f amino a c i d s i n the l i v e r  (Houpt, 1970; Hembry et^ a l . ,  The a b s o r p t i o n o f ammonia from the rumen i s dependent 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 which i n t u r n i s dependent ammonia f o r m a t i o n 1975).  t o u r e a o r the  on the r a t e o f on the r a t e o f  and energy made a v a i l a b l e t o t h e microbes  (Hembry ej: a]L.,  Hemler and B a r t l e y (1971) and Hogan (1961) a l s o r e p o r t e d  ammonia a b s o r p t i o n from t h e rumen depends on t h e c o n c e n t r a t i o n at pH 6.5.  I t was n e g l i g i b l e a t pH 4.5.  Estimates  f o r any o f the t h r e e f u n c t i o n s have been v a r i a b l e .  1975).  that  gradient  o f ammonia u t i l i z e d Nolan and Leng  (1972)  showed t h a t about 80% of the m i c r o b i a l n i t r o g e n was d e r i v e d from ammonia and o n l y about 20% of m i c r o b i a l n i t r o g e n came d i r e c t l y from amino a c i d s . Mathison and M i l l i g a n  (1971) e s t i m a t e d  t h a t 50-65% o f b a c t e r i a l  nitrogen  and 31-55% o f p r o t o z o a l n i t r o g e n were d e r i v e d from rumen ammonia.  They  a l s o i n d i c a t e d t h a t 17-54% o f t h e ammonia d e r i v e d from d i e t a r y p r o t e i n (about 60-92% of d i e t a r y p r o t e i n t r a n s f e r r e d to ammonia by t h e i r  - 23 -  estimation)  o f chopped hay o r b a r l e y p l u s chopped hay, was absorbed  from t h e rumen.  Nolan (1975) r e p o r t e d  that b a c t e r i a l  nitrogen  d e r i v e d from ammonia was about 30-80% and p r o t o z o a l n i t r o g e n from ammonia 25-64%.  Hogan and Weston (1967) r e p o r t e d  that  h i g h p r o t e i n d i e t s (C.P. 19.8%) up t o 31% o f the n i t r o g e n between the i n g e s t e d estimated  feed and duodenal d i g e s t i o n .  derived i n sheep f e d  disappeared  P i l g r i m e^t a l . (1969)  t h a t 57-66% o f t h e ammonia-nitrogen was absorbed from the  rumen o r u t i l i z 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 and t h e remainder  passed on i n t o t h e omasum.  P r o t e i n anabolism by rumen b a c t e r i a  Rumen microbes a r e a b l e t o s y n t h e s i z e b o t h e s s e n t i a l and none s s e n t i a l amino a c i d s .  Sauer e t a l . (1975) r e p o r t e d  t h a t rumen microbes  14 readily utilized carbon s k e l e t o n s  C-labelled-HCO_, and a c e t a t e  f o r the synthesis of the  o f amino a c i d s and subsequently t h e amino a c i d s .  In :  14 contrast,  C - l a b e l l e d propionate 14  synthesis but l a b e l l e d  was u t i l i z e d  C from p r o p i o n a t e  amino a c i d s t o any s i g n i f i c a n t e x t e n t .  for isoleucine bio-  failed  t o appear i n other  Forward t r i c a r b o x y l i c a c i d  c y c l e r e a c t i o n s o n l y proceeded t o k e t o - o r 2-oxo- g l u t a r a t e . was  carboxylated  Oxaloacetate  t o p y r u v a t e which was then c a r b o x y l a t e d  then e q u i l i b r a t e d w i t h  to oxaloacetate.  fumarate and t h e r e b y carbon atoms  1 and 4 as w e l l as 2 and 3 became randomized. precursors  Acetate  Most o f t h e 2-oxo  o f amino a c i d s appeared t o be formed v i a f e r r o d o x i n dependent  reductive carboxylation.  Of t h e amino a c i d p r e c u r s o r s  investigated,  - 24  -  o n l y 3-hydroxypyruvate, the p r e c u r s o r synthesized  v i a an o x i d a t i v e s t e p ,  3-phosphohydroxypyruvic a c i d ) .  o f s e r i n e , appeared to  ( i . e . 3 - p h o s p h o g l y c e r i c a c i d to  Rumen microbes r e u t i l i z e d benzene  r i n g s i n the b i o s y n t h e s i s o f p h e n y l a l a n i n e s y n t h e s i s of the benzene r i n g was (1974) was  be  and  tyrosine.  of minor importance.  De  novo  Kristensen  a l s o of the o p i n i o n t h a t r e d u c t i v e c a r b o x y l a t i o n of  and  i n d o l e - 3 - a c e t i c a c i d t o form p h e n y l a l a n i n e  and  was  of g r e a t e r importance than de novo s y n t h e s i s of the carbon  phenylacetate  tryptophan r e s p e c t i v e l y skeletons.  R e d u c t i v e c a r b o x y l a t i o n of preformed i s o b u t y r a t e to form v a l i n e was  r e p o r t e d by  Sauer et al.  (1975) to be of minor importance to de novo  s y n t h e s i s of the carbon s k e l e t o n from a c e t a t e . considerable  There was  however a  degree of r e d u c t i v e c a r b o x y l a t i o n of preformed  a c i d to form l e u c i n e as compared t o de novo s y n t h e s i s of the skeleton.  A l l i s o n et a l . (1962), A l l i s o n and  Bryant  isovaleric carbon  (1963), and  Allison  et a l . (1966) suggested t h a t b i o s y n t h e s i s of the i s o p r o p y l moiety  was  inadequate because a c o n s i d e r a b l e q u a n t i t y of m a t e r i a l s c o n t a i n i n g group i s p r e s e n t  i n c e l l u l a r l i p i d where i s o p e n t a d e c a n o i c  this  a c i d i s a major  f a t t y a c i d , a f t e r f i n d i n g b r a n c h e d - c h a i n f a t t y a c i d s as requirements f o r R.  flavefaciens strain  C94.  E f f e c t of formaldehyde treatment on the d i g e s t i o n o f p r o t e i n s i n the rumen  The  d i g e s t i o n of d i e t a r y p r o t e i n s i n the rumen by rumen m i c r o -  organisms c o u l d e i t h e r be b e n e f i c i a l o r w a s t e f u l 1972).  The  extent  of m i c r o b i a l d e g r a d a t i o n  to the host  (Phillipson,  i s dependent on p r o t e i n  - 25 -  solubility.  One way to reduce protein s o l u b i l i t y and then digestion  i n the rumen i s to treat the feedstuff with formaldehyde  (Ferguson  et a l . , 1967; Ferguson, 1975). Barry (1976a) described the reaction of formaldehyde with proteins by three equations:  R - XH + n m n  ^  N  R - XH + R  1  -  (1)  °c i NHr- fcR - X - CH - NH - R + H„0 z * 2 2 (Methlylene compound)  oL  1  R - X - CH„0H + R z  - X - CH OH  -  NH - CH OH 2  ^R - X - CH  -  NH - R  1  + H0 2  (2)  (3)  The f i r s t reaction i s said to be rapid and occurs at neutral pH and room temperature.  -XH can be a terminal amino group, the primary  amide groups of asparagine and glutamine and the epsilon amino and guanidyl groups of lysine, and arginine respectively.  The phenol group  of tyrosine and phenyl group from phenylalanine, the indole group of tryptophan and the imadazole group of h i s t i d i n e may take part i n the reactions under conditions other than neutral pH and room temperature, such as high temperature.  After the formation of the methylol compounds,  condensation reactions then take place slowly over time, with the formation of methylene cross-linkages between protein chains (Equations 2 and 3). These methylene cross-linkages are stable i n the near-neutral pH of the rumen but the H  +  ions i n the abomasum break down the linkage with the  - 26 -  r e l e a s e o f formaldehyde Formaldehyde  ( R a t t r a y and J o y c e , 1970).  i s not h a r m f u l t o rumen microbes a t the low  c o n c e n t r a t i o n s used to t r e a t f e e d s .  M i l l s et^ a l .  (1972) u s i n g  14 C - l a b e l l e d formaldehyde r e p o r t e d t h a t ruminants m e t a b o l i z e d formaldehyde and t h e r e was carcass or milk.  effectively  no a c c u m u l a t i o n o f i t i n the  S i x t y t o e i g h t y p e r c e n t (60-80%) o f t h e formaldehyde  was m e t a b o l i z e d t o carbon d i o x i d e and methane, 11-27% was v o i d e d i n the f a e c e s and 5-6%  e x c r e t e d i n the u r i n e .  Small a c t i v i t i e s of  "^C  from l a b e l l e d formaldehyde were d e t e c t e d i n m i l k and body t i s s u e s but not as formaldehyde.  The pathways whereby formaldehyde i s c o n v e r t e d  to methane a r e obscure, but t h r e e mechanisms have been proposed by M i l l s et a l . (1972).  (a)  Formaldehyde  i s c o n v e r t e d t o formate and i t i s  subsequently m e t a b o l i z e d t o carbon d i o x i d e and methane. (b)  Formaldehyde  i s s u c c e s s i v e l y reduced t o methanol  and methane. (c)  There i s an a c y l o i n c o n d e n s a t i o n of formaldehyde w i t h r i b o s e - 5 - p h o s p h a t e t o form a l l u l o s e - 6 - p h o s p h a t e which i s f u r t h e r m e t a b o l i z e d v i a the g l y c o l y t i c sequences t o produce methane and carbon d i o x i d e .  D i n i u s e_t a l . (1974)  a l s o r e p o r t e d t h a t treatment o f p r o t e i n per se  w i t h formaldehyde d i d not i n t e r f e r e w i t h rumen m i c r o b i a l  activity.  - 27 -  F e e d s t u f f s t h a t have been t r e a t e d w i t h formaldehyde classified  i n t o o i l seed meals,  can be  f i s h meal, c a s e i n , d r i e d f o r a g e s ,  s i l a g e s , hays and c e r e a l g r a i n s . The response w i t h treatment o f p r o t e i n s has been v a r i a b l e .  Treatment  formaldehyde o f c a s e i n has been  r e p o r t e d t o c o n s i s t e n t l y y i e l d p o s i t i v e responses o f n i t r o g e n b a l a n c e j growth r a t e and wool growth ( L i t t l e and M i t c h e l l , 1967; S c h e l l i n g and Hatfield,  1968; R e i s and Tunks, 1969; F a i c h n e y , 1971; F a i c h n e y and  Weston, 1971; P h i l l i p s o n , 1972; Sharma and I n g a l l s , 1974). of o i l seed meals such as peanut,  Treatment  soybean and rapeseed meals a t times  g i v e s p o s i t i v e response and a t times no response o r a t times n e g a t i v e response.  Schmidt  et a l . (1971) and Schmidt  e t a l . (1974) o b t a i n e d  n e g a t i v e responses w h i l e P e t e r ^ t a l . (1970), P e t e r e t a l . (1971), N i m r i c k e t a l . (1972) and Amos e t a l . (1974) o b t a i n e d p o s i t i v e  responses  of n i t r o g e n b a l a n c e growth r a t e and wool growth w i t h treatment o f soybean meal.  Sharma and I n g a l l s  (1974) and Sharma e t a l . (1972) d i d n o t get  a response w i t h t h e treatment o f rapeseed F a i c h n e y and Davies  meal.  (1972) and F a i c h n e y and D a v i e s  t h a t t h e growth r a t e of c a l v e s improved when formaldehyde  (1973) r e p o r t e d treated  groundnut  (peanut) meal was used i n d i e t s c o n t a i n i n g e i t h e r 12% o r 13.4% crude p r o t e i n . However, formaldehyde  treatment d i d n o t improve  g a i n s when the d i e t s  c o n t a i n e d e i t h e r 15% o r 20.5% crude p r o t e i n .  F a i c h n e y (1972) however  r e p o r t e d t h a t w i t h h i g h e r crude p r o t e i n d i e t s  ( C P . 20%) i n which 50%  o f t h e crude p r o t e i n was from t r e a t e d o r u n t r e a t e d peanut meal,  treatment  increased the proportion o f p r o t e i n digested i n the small i n t e s t i n e , compared w i t h a d i e t o f lower crude p r o t e i n content (13% CP.;).  - 28 -  Langlands (1971b) and S a v i l l e et: a l . (1971) observed no response w i t h treatment o f c o t t o n s e e d  meal.  R a t t r a y and Joyce (1970)  r e p o r t e d responses w i t h treatment o f l i n s e e d meal but n o t meat meal. Many workers have t r e a t e d crops w i t h formaldehyde b e f o r e  ensiling  (Barry and Fennessy, 1972; Brown and V a l e n t i n e , 1972; Barker e t a l . , 1973; V a l e n t i n e and Brown, 1973; B a r r y , Barry,  1976b; B i n n i e and B a r r y ,  1975; V a l e n t i n e and R a d c l i f f e , 1975;  1976).  Some workers have r e p o r t e d  p o s i t i v e responses such as i n c r e a s e d feed i n t a k e , feed e f f i c i e n c y , g a i n i n weight and p r o d u c t i o n  of m i l k , b u t t e r f a t ,  p r o t e i n and SNF w i t h formaldehyde treatment o f s i l a g e s Brown, 1973; Barry, Barry,  conversion milk  ( V a l e n t i n e and  1975; V a l e n t i n e and R a d c l i f f e , 1975; B i n n i e and  1976). Barry  (1973a) r e p o r t e d  t h a t formaldehyde treatment o f r y e - g r a s s  c l o v e r hay s i g n i f i c a n t l y reduced n i t r o g e n , energy and o r g a n i c matter digestibilities.  Barry  (1973b) r e p o r t e d r e d u c t i o n i n l i v e w e i g h t l o s s e s  when formaldehyde t r e a t e d r y e - g r a s s - c l o v e r hay was f e d a t maintenance and h a l f - m a i n t e n a n c e l e v e l s t o sheep.  Amos e_t a l . (1976b) observed no  s i g n i f i c a n t differences i n nitrogen-balance grass hay o r u n t r e a t e d  when formaldehyde t r e a t e d  hay were f e d a t 600g DM t o 18kg sheep.  775g DM were f e d d a i l y , t h e r e were s i g n i f i c a n t  When  improvements i n t h e flow  of 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 , when t r e a t e d hay was f e d compared t o t h e u n t r e a t e d .  Langlands  (1973a and 1973b) and E n t w i s t l e  (1973) r e p o r t e d improvement i n n i t r o g e n u t i l i z a t i o n w i t h treatment o f wheat.  -  2 9 -  There has been l i m i t e d work r e p o r t e d on formaldehyde treatment of a r t i f i c i a l l y d r i e d forages.  D i n i u s et a l . (1975) u s i n g  diets  (16% C . F . ) n t a i n i n g 75% a l f a l f a meal on a DM b a s i s showed t h a t C O  formaldehyde  treatment o f t h e a l f a l f a a t 1% and 2% l e v e l s s i g n i f i c a n t l y reduced energy, d r y matter, crude p r o t e i n , a c i d - d e t e r g e n t f i b r e  digestibilities  and n i t r o g e n r e t e n t i o n . However, Hemsley et a l . (1970) r e p o r t e d t h a t 1% treatment of d r i e d f o r a g e c o n t a i n i n g about 25% by 15%.  CP.  formaldehyde  improved wool  growth  D i g e s t i b i l i t y of f i b r e was n o t a f f e c t e d but n i t r o g e n  d i g e s t i b i l i t y was  reduced.  Beever et a l . (1976) r e p o r t e d decreased  n i t r o g e n d i g e s t i b i l i t y w i t h formaldehyde treatment o f d r i e d f o r a g e but c e l l u l o s e d i g e s t i o n was  improved over u n t r e a t e d .  P o s s i b l e r e a s o n s f o r t h e v a r i b l e responses t o formaldehyde treatment of d i f f e r e n t types o f p r o t e i n s  Treatment of c a s e i n w i t h formaldehyde has been a s s o c i a t e d w i t h p o s i t i v e responses o f n i t r o g e n r e t e n t i o n , m i l k p r o d u c t i o n , wool  growth  or growth r a t e because t h i s source of p r o t e i n i s of h i g h e r q u a l i t y than microbial protein.  The h o s t t h e r e f o r e b e n e f i t s from p r o t e c t i o n  m i c r o b i a l d e g r a d a t i o n (Reis and Tunks, 1969; Langlands, 1971a; «2t al.,  1974;  P h i l l i p s o n , 1972).  against Amos  Treatment a l s o reduces d i g e s t i o n of  p r o t e i n s i n the rumen which a l s o reduces l o s s e s o f n i t r o g e n i n t h e form of ammonia.  Ammonia l e v e l s i n rumen f l u i d have been reduced by  formaldehyde treatment (Hemsley et al_. , 1970; Hogan and Weston, Sharma e t a l . ,  1972;  Sharma and I n g a l l s , 1973;  1970;  Bhargava and Ranjhan,  1974;  - 30  Sharma and N i c h o l s o n ,  1975b).  -  Other workers however d i d not  reduced rumen ammonia-nitrogen l e v e l s w i t h treatment 1975;  Sharma and N i c h o l s o n ,  1975a).  reduced plasma u r e a - n i t r o g e n  l e v e l s w i t h treatment of rapeseed meal  i n sheep b e i n g untreated  I n g a l l s , 1974)  l e v e l s i n the b l o o d by treatment.  (1973) r e p o r t e d t h a t b e f o r e  (Dinius et a l . ,  Sharma e t a l . (1972) r e p o r t e d  but i n the subsequent work (Sharma and on u r e a - n i t r b g e n  observe  t h e r e was  B a r r y and  no  effect  Fennessy  f e e d i n g , rumen ammonia l e v e l s were h i g h e r  f e d formaldehyde t r e a t e d s i l a g e s compared to those b e i n g  silages.  Sharma and N i c h o l s o n  rumen ammonia n i t r o g e n l e v e l s w i t h meal one hour a f t e r f e e d i n g but not  (1975b) observed  decreased  formaldehyde treatment of faba bean f o u r hours a f t e r  Hemsley et a l . (1970) p o s t u l a t e d as one  reason  feeding. for positive  responses t o formaldehyde treatment, t h a t a g r e a t e r amount of p r o t e i n was d i g e s t e d i n the i n t e s t i n e .  They a l s o p o s t u l a t e d t h a t i n t h e i r  experiment the p l a n t p r o t e i n s might have been more d i g e s t i b l e than b a c t e r i a l p r o t e i n , i n the i n t e s t i n e s .  According  to these workers,  the  d i g e s t i o n of the p l a n t p r o t e i n s i n the i n t e s t i n e s might have i n c r e a s e d a v a i l a b l e energy from amino a c i d s t o the animal.  Barry  (1973a) a l s o  r e p o r t e d i n c r e a s e d d i g e s t i o n of n i t r o g e n i n the s m a l l i n t e s t i n e  with  formaldehyde treatment. Langlands  (1973a, 1973b) r e p o r t e d improvement i n the  of p r o t e i n of t r e a t e d wheat.  He  utilization  a t t r i b u t e d t h i s improvement t o  g r e a t e r amount of n i t r o g e n e s c a p i n g  fermentation  i n the rumen.  the  fed  - 31 -  Faichney and Weston (1971) r e p o r t e d t h a t the i n c r e a s e d amount o f p r o t e i n d i g e s t e d i n the i n t e s t i n e as a r e s u l t o f  formaldehyde  treatment s t i m u l a t e d the s e c r e t i o n of i n s u l i n .  The i n s u l i n  the  I t a l s o has an e f f e c t  e n t r y r a t e o f amino a c i d s i n t o the c e l l s .  on the s e c r e t i o n of growth hormone. fully  increased  T h i s hormonal a s p e c t has not been  investigated. Ferguson  (1975) (without g i v i n g data) a l s o p o s t u l a t e d  treatment w i t h formaldehyde might performance.  that  i n c r e a s e feed i n t a k e and thus  improve  Davies and F a i c h n e y (1973) however r e p o r t e d formaldehyde  treatment o f b a r l e y t o d e c r e a s e feed i n t a k e and performance  of s t e e r s .  With s i l a g e s p o s i t i v e responses of m i l k and SNF p r o d u c t i o n have been a t t r i b u t e d t o the p r e v e n t i o n o f breakdown of p r o t e i n d u r i n g e n s i l i n g and i n the rumen (Brown and V a l e n t i n e , ' 1 9 7 2 ; V a l e n t i n e and Brown, V a l e n t i n e and R a d c l i f f e ,  1973;  1975).  In experiments where no responses or n e g a t i v e responses had been o b t a i n e d , v a r i o u s reasons have been g i v e n .  Schmidt  t h a t overtreatment o f soybean meal w i t h formaldehyde n e g a t i v e response. t r e a t e d hay.  meal.  thought  r e s u l t e d i n the  Sharkey ej: aJ_. (1972) observed no response w i t h  They a t t r i b u t e d t h i s t o l o s s e s of formaldehyde  hay making p r o c e s s and thus the p r o t e i n was Langlands  e t a l . (1974)  d u r i n g the  not a d e q u a t e l y p r o t e c t e d .  (1971b) observed no response w i t h treatment of c o t t o n s e e d  He a t t r i b u t e d t h i s t o extreme d e p r e s s i o n i n the d i g e s t i b i l i t y  of n i t r o g e n and o r g a n i c m a t t e r as a r e s u l t of b o t h heat a p p l i e d d u r i n g e x t r a c t i o n of the o i l and the a p p l i c a t i o n o f the f o r m a l i n .  S a v i l l e et a l .  (1971) a t t r i b u t e d no response t o treatment o f c o t t o n s e e d meal to .  - 32 -  under- or o v e r - p r o t e c t i o n of the meal  and  depressed rumen pH  as a r e s u l t of f e e d i n g o f g r a i n which might have broken the bridges.  methylene  Sharma and I n g a l l s (1974) however had pH v a l u e s o f about  f o r formaldehyde  t r e a t e d c a s e i n and 5.7  rapeseed meal and s t i l l had adequate  f o r formaldehyde  5.5  treated  p r o t e c t i o n of the p r o t e i n s .  Sharma e t a l . (1974) i n d i c a t e d t h a t d i e t s c o n t a i n i n g about 15% CP f o r s t e e r s had p r o t e i n l e v e l s i n excess o f the amounts r e q u i r e d and was  the cause of no response t o the rapeseed meal treatment.  suggested crude p r o t e i n l e v e l s o f about 11-12%.  this  They  I t should however  be noted t h a t i n the same paper, they had a response t o treatment of c a s e i n i n d i e t s w i t h s i m i l a r crude p r o t e i n l e v e l s as the rapeseed meal diets. Rattray  and Joyce  (1970) observed a response i n n i t r o g e n r e t e n t i o n  but not wool growth or growth r a t e w i t h treatment of l i n s e e d meal. e x p e r i m e n t a l p e r i o d o f f i v e weeks, a c c o r d i n g  t o them, may  s h o r t f o r a response o f growth r a t e t o be demonstrated.  have been too They a l s o  a n e g a t i v e response w i t h treatment of meat meal u s i n g any o f the parameters. overprotected  They suggested t h a t a l e v e l o f 2.5%  Their  reported  three  formaldehyde a p p l i c a t i o n  the p r o t e i n i n the meat-meal.  D i g e s t i o n and a b s o r p t i o n  of n i t r o g e n i n the s m a l l i n t e s t i n e  The main p r o t e o l y t i c enzyme i n the abomasum of ruminants i s pepsin.  Rennin i s a l s o p r e s e n t i n the young pre-ruminant.  s e c r e t e s h y d r o c h l o r i c a c i d which k i l l s rumen micro-organisms  The abomasum (Maynard  - 33  -  and L o o s l i , 1969).  Maximal p r o t e o l y s i s by abomasal c o n t e n t s was  t o occur between pH  2 and  (1975a) claimed  3 (Hill,  i t occurred  a t pH  1961; 2.1.  m o s t l y i n the i n t e s t i n e s , a f t e r the  P h i l l i p s o n , 1975)  but  D i g e s t i o n of p r o t e i n s  shown  Church occurs  reticulo-rumen.  M a t e r i a l s e n t e r i n g the s m a l l i n t e s t i n e of ruminants i n c l u d e endogenous n i t r o g e n s e c r e t e d i n t o the abomasum and microbial nitrogen, non-protein-nitrogen fermentation  i n the r e t i c u l o - r u m e n  small  intestine,  r e s u l t i n g from m i c r o b i a l  and n i t r o g e n o u s  or endogenous o r i g i n t h a t have escaped r u m i n a l  components of d i e t a r y  fermentation„(Armstrong  and Hutton, 1975). There i s no q u a n t i t a t i v e i n f o r m a t i o n on the endogenous s e c r e t i o n i n t o the s m a l l i n t e s t i n e 29%  (Armstrong and  Hutton, 1975).  Approximately  of the ammonia-nitrogen i n duodenal f l o w i s d e r i v e d from ammonia  i n rumen f l u i d .  I t i s hypothesized  t h a t the o t h e r  71%  i s d e r i v e d from urea  t h a t passes i n t o the abomasum w i t h g a s t r i c s e c r e t i o n s (Nolan, Other n o n - p r o t e i n - n i t r o g e n the rumen has (Nolan,  1975).  t o sheep 7.1g, was  up  might be n u c l e i c a c i d s s i n c e the d i g e s t a from  to 20% of m i c r o b i a l n i t r o g e n i n the form of RNA Nolan (1975) r e p o r t e d t h a t when 21g  11.Og  and  1.2g  of n i t r o g e n was  DNA fed  ammonia r e s p e c t i v e l y .  The p r o t e i n e n t e r i n g the s m a l l i n t e s t i n e i s h y d r o l y z e d  Gray and  and  of the n i t r o g e n a r r i v i n g at the duodenum  from d i e t a r y p r o t e i n , m i c r o b i a l p r o t e i n and  proteases  1975).  i n p a n c r e a t i c j u i c e and p e p t i d a s e s  from i n t e s t i n a l s e c r e t i o n s .  Cooper (1971) r e p o r t e d t h a t p a n c r e a t i c j u i c e c o n t a i n s  ( a t t a c k i n g c e n t r a l l y l o c a t e d p e p t i d e bonds) and o n l y t e r m i n a l bonds of p r o t e i n s o r p e p t i d e s ) .  by  endopeptidases  exopeptidase ( c l e a v i n g The  endopeptidases a r e :  - 34 -  (a)  T r y p s i n - t h i s a t t a c k s p r o t e i n s a t l o c a t i o n s o f the b a s i c amino a c i d s y i e l d i n g a r g i n i n e and l y s i n e t e r m i n a l peptides.  (b)  Chymotrypsin - t h i s a c t s i n t e r i o r l y a t aromatic amino a c i d s i t e s t o produce C - t e r m i n a l tryptophan  (c)  phenylalanine,  t y r o s i n e , and  peptides.  Pancreopeptidase E (elastase) - attacks  aliphatic  (non-polar) amino a c i d - c o n t a i n i n g p o r t i o n o f p r o t e i n t o produce a l i p h a t i c carbon t e r m i n a l  The  peptides.  exopeptidases a r e : Carboxypeptidase A: Attacks and  peptides  r e s u l t i n g from a c t i o n s o f chymotrypsin  p a n c r e o p e p t i d a s e , y i e l d i n g n e u t r a l amino a c i d s and  small  peptides.  Carboxypeptidase B: Attacks  peptides  r e s u l t i n g from a c t i o n o f t r y p s i n t o g i v e  b a s i c amino a c i d s and s m a l l  peptides.  These enzymes a r e s e c r e t e d as zymogens. amount o f t r y p s i n o g e n  i s by e n t e r o k i n a s e .  a c t i v a t e s the r e s t of the trypsinogen a l l the other pancreatic proteases  A c t i v a t i o n of a small  Then the t r y p s i n produced  to t r y p s i n .  T r y p s i n then a c t i v a t e s  (Gray and Cooper, 1971).  s e c r e t i o n i s under hormonal and nervous c o n t r o l .  Pancreatic  The nervous p a r t i s  through t h e vagus nerve which can be s t i m u l a t e d by d i s t e n s i o n of the abomasum.  T h i s i s t h e i n i t i a l f l o w known as t h e c e p h a l i c phase.  more r a p i d flow i s under t h e c o n t r o l o f hormones.  The  Pancreozymin i s a  - 35  hormone which has  -  an e f f e c t on p a n c r e a t i c flow and  s i m i l a r t o the v a g a l r e f l e x . flow i s s e c r e t i n .  The  hormone which induces  The most powerful  s e c r e t i n i s . a c i d i n g e s t a present  stimulus  i n the duodenum.  Other f a c t o r s which  amino.acids ( H i l l ,  1975).  a c i d c o n d i t i o n s from the abomasum extend i n t o the upper p a r t  of the s m a l l i n t e s t i n e due the abomasum and  i n p a r t t o the copious  s e c r e t i o n of a c i d by  p a r t l y to the weakly a l k a l i n e n a t u r e  p a n c r e a t i c s e c r e t i o n s of the ruminant. proximal  the more r a p i d  f o r the r e l e a s e of  a f f e c t s e c r e t i n r e l e a s e a r e peptone, soaps and The  i t s effect i s  The  p a r t of the s m a l l i n t e s t i n e may  of the b i l e  slow r i s e i n pH  i n the  extend the a c t i v i t y of  abomasal p e p s i n but d e l a y t h a t of the p a n c r e a t i c p r o t e a s e s .  the  The  a c t i v a t i o n of p a n c r e a t i c zymogens r e q u i r e a pH above 5 (Armstrong Hutton, 1975).  Ben  - Ghedalia  e t al.  and  and  (1974) working w i t h sheep r e p o r t e d  t h a t the s e c t i o n of the i n t e s t i n e s betwen 1 and  3 metres and  3. and  7  metres from the p y l o r u s were s i t e s of g r e a t p r o t e o l y s i s but poor f o r absorption.  They r e p o r t e d t h a t the s e c t i o n 7 to 15 metres d i s t a n t  from the p y l o r u s showed the g r e a t e s t a b s o r p t i o n of d i g e s t i v e of n i t r o g e n .  The  products  lower s e c t i o n o f the i n t e s t i n e from 15 t o 25 metres  d i s t a n t from the p y l o r u s had  low net a b s o r p t i o n .  In a subsequent  paper, Ben-Ghedalia e t a l . (1976) r e p o r t e d t h a t i f p r o t e i n was a t the lower s e c t i o n (15 to 25 metres d i s t a n t from the p y l o r u s ) was  a c o n s i d e r a b l e degree of d i g e s t i o n and  The  e a r l i e r work i n d i c a t i n g low net a b s o r p t i o n was  most p r o t e i n s had  been d i g e s t e d and  there  a b s o r p t i o n of p r o t e i n s .  products  due  to the f a c t  that  absorbed i n the upper s e c t i o n s  of the s m a l l i n t e s t i n e i f p r o t e i n s were s u p p l i e d at maintenance The  administered  lower s e c t i o n t h e r e f o r e d i d not have d i g e s t i v e and  levels.  absorptive  limitations.  - 36 -  Peptidases are present i n t e s t i n a l secretions  i n t r a c e amounts i n p a n c r e a t i c o r  (Gray and Cooper, 1971).  i n t e s t i n a l dipeptidase  About 10-20% o f  a c t i v i t y i s known t o o c c u r i n t h e m i c r o v i l l a r  membrane ( b r u s h - b o r d e r ) . of t h e mucosal p e p t i d a s e  I n t r a c e l l u l a r peptidases activity.  account f o r 80%  There a r e about 4 t o 8  peptidases  which a r e h y d r o l a s e s . The major end-products absorbed, a f t e r p r o t e i n d i g e s t i o n a r e f r e e amino a c i d s and s m a l l p e p t i d e s , 1972;  w i t h t h e l a t t e r predominating  Armstrong and Hutton, 1975).  since g l y c i n e peptides  Gray and Cooper (1971) suggested  p r o b a b l y entered  the c e l l s at rates  comparable t o t h e amino a c i d , presumably a t t h e same e n t r y oligopeptides  that  were more r e a d i l y absorbed than g l y c i n e , i t  indicated that the small peptides  If  (Matthews,  sites.  a r e absorbed and t h e e n t r y r a t e i s the same as t h e f r e e  amino a c i d e n t r y r a t e , more amino a c i d s a r e p r e s e n t f u r t h e r h y d r o l y s i s by i n t r a c e l l u l a r p e p t i d a s e s a c i d s a r e absorbed.  Some o f t h e p e p t i d e s  b o r d e r by t h e brush-border enzymes b e f o r e  i n t h e c e l l on  than when t h e f r e e amino  are also digested  a t the brush-  entering the c e l l .  O x i d a t i v e metabolism i s l i n k e d up w i t h sodium movement out o f the cell  i n t h e t r a n s p o r t o f amino a c i d s i n t o the c e l l s .  of Che amino a c i d , N a  +  complex  and a membrane compound, presumably a p r o t e i n ,  i s formed t h e r e b y p e r m i t t i n g barrier,  A ternary  e n t r y and r e l e a s e beyond t h e o u t e r  (Gray and Cooper, 1971; Matthews, 1972).  Since  cell  the oxidative  metabolism i s n o t l i n k e d d i r e c t l y w i t h the amino a c i d e n t r y , t h e system i s n o t a c t i v e t r a n s p o r t but r a t h e r termed secondary a c t i v e (Gray and Cooper, 1971).  transport  There a r e some amino a c i d s , t h e dependence  - 37 -  of whose t r a n s p o r t on sodium i s n o t f u l l y known (e.g. t h e d i c a r b o x y l i c amino a c i d s ) .  Williams  (1969) r e p o r t e d t h a t t h e r e were c o n s i d e r a b l e  v a r i a t i o n s i n a b s o r p t i o n between and w i t h i n s p e c i e s . of amino a c i d s presented in different  The p r o p o r t i o n s  t o t h e l i v e r were n o t n e c e s s a r i l y t h e same  s u b j e c t s o f the same s p e c i e s when t h e p r o p o r t i o n s o f  a v a i l a b l e amino a c i d s f o r a b s o r p t i o n from t h e s m a l l i n t e s t i n e s were the same.  He a l s o grouped amino a c i d s i n t o s i x (6) c a t e g o r i e s  to r a t e o f a b s o r p t i o n i n sheep.  In order of decreasing  (a)  I s o l e u c i n e , a r g i n i n e , methionine, v a l i n e  (b)  Leucine,  (c)  Aspartic acid, serine, tyrosine, alanine  (d)  Alanine, p r o l i n e , threonine  (e)  P r o l i n e , threonine,  (f)  Glycine  lysine,  according  r a t e these a r e :  phenylalanine  glutamic  acid, histidine  Some amino a c i d s have an e f f e c t on t h e t r a n s p o r t o f o t h e r amino acids  (Gray and Cooper, 1971; Matthews, 1972).  m e t h i o n i n e may i n h i b i t Leucine  F o r i n s t a n c e l e u c i n e and  t h e t r a n s p o r t o f o t h e r amino a c i d s  (Matthews, 1972).  augments the t r a n s p o r t o f l y s i n e and a r g i n i n e but the reason f o r  t h i s i s unknown (Gray and Cooper, 1971). The  i n t e r a c t i o n o f one k i n d o f amino a c i d w i t h another may be  a l l o s t e r i c - due t o attachment o f an amino a c i d t o one c a r r i e r i n d u c i n g c o n f i g u r a t i o n a l changes i n another a d j a c e n t due  to competition  the same c a r r i e r  f o r energy supply.  (Matthews, 1972).  site  c a r r i e r - o r even  They may n o t n e c e s s a r i l y be s h a r i n g  Hexoses (D-glucose and D - g a l a c t o s e )  - 38 -  reduce  a b s o r p t i o n of amino a c i d s .  T h i s i s perhaps because they  share the same c a r r i e r mechanism o r compete f o r the l i m i t e d of energy  source  (Gray and Cooper, 1971).  D i g e s t i o n of p r o t e i n i n the l a r g e i n t e s t i n e .  Undigested  and unabsorbed r e s i d u e s of n i t r o g e n o u s  compounds  l e a v i n g the s m a l l i n t e s t i n e s e n t e r the caecum and t h e l a r g e i n t e s t i n e . These a r e m a i n l y f e e d r e s i d u e s , u n d i g e s t e d rumen micro-organisms endogenous m a t e r i a l s .  and  In a d d i t i o n , s u b s t a n t i a l q u a n t i t i e s of u r e a -  n i t r o g e n e n t e r the caecum from the b l o o d .  The t o t a l i n p u t of n i t r o g e n  i n t o the caecum and the l a r g e i n t e s t i n e may range between 4 and 15g per day f o r sheep.  The micro-organisms  proteolytic activities.  p r e s e n t i n the caecum and c o l o n c a r r y out  I n f a c t , p r o t e o l y t i c a c t i v i t y appears  t o be  g r e a t e r i n the c o n t e n t s o f the l a r g e i n t e s t i n e and caecum than i n the c o n t e n t s of the r e t i c u l o - r u m e n .  The presence  o f i s o - b u t y r i c and  i s o - v a l e r i c a c i d s i n the caecum i n p r o p o r t i o n s h i g h e r than those o c c u r i n g i n the rumen i n d i c a t e s t h e r e i s e x t e n s i v e breakdown o f p r o t e i n i n t h i s region  (Nolan, 1975). A b s o r p t i o n o f n i t r o g e n from the hindgut  of ammonia, about 43% o f which i s used amino a c i d i n t h e l i v e r  (Nolan, 1975).  i s p r o b a b l y i n the form  f o r the s y n t h e s i s o f n o n - e s s e n t i a l S i n c e the microbes  present are  not k i l l e d by a c i d s and l a t e r s u b j e c t e d t o p r o t e a s e s as o c c u r s w i t h rumen microbes,  they a r e perhaps of l i t t l e n u t r i t i v e v a l u e , i n terms of  n i t r o g e n , to the h o s t  (Nolan, 1975).  - 39 -  Carbohydrate metabolism i n the rumen  The metabolism o f c a r b o h y d r a t e s by rumen micro-organisms i s i l l u s t r a t e d by the pathways i n d i c a t e d i n F i g u r e s 2 and 3.  The  end-  p r o d u c t s o f c a r b o h y d r a t e metabolism i n the rumen a r e g e n e r a l l y v o l a t i l e f a t t y a c i d s , methane, hydrogen (Hobson,  1971).  gas, and carbon d i o x i d e  E t h a n o l has been produced by rumen microbes i n  i n v i t r o c u l t u r e s but i n i n v i v o o n l y t r a c e s have been d e t e c t e d . I t i s p o s s i b l e t h a t i n i n v i v o , e t h a n o l i s produced but may  be absorbed  through the rumen w a l l , s i n c e i t has the a b i l i t y t o absorb e t h a n o l if  infused i n large quantities.  a c i d and methane.  I t may  a l s o be m e t a b o l i z e d t o a c e t i c  T h i s has been observed when s m a l l q u a n t i t i e s o f  a l c o h o l a r e i n t r o d u c e d i n t o the rumen a t a time.  I t c o u l d a l s o be  m e t a b o l i z e d w i t h a c e t a t e t o produce b u t y r a t e and h i g h e r v o l a t i l e acids  (Hobson,  fatty  1971).  In the pathways shown by Leng  (1970), methane i s produced  from  f o r m i c a c i d but Hungate e_t a l . (1970) and Hungate (1967) i n d i c a t e d t h i s pathway may  not be q u a n t i t a t i v e l y important.  Those workers  that  indicated  t h a t methane i s produced from the r e d u c t i o n o f carbon d i o x i d e w i t h hydrogen.  M i l l e r and Wblin  even f o r m i c a c i d i t s e l f  (1973) working w i t h R. a l b u s r e p o r t e d  that  i s produced from the r e d u c t i o n of carbon d i o x i d e  but not from f o r m a t e - p r o d u c i n g p y r u v a t e l y a s e r e a c t i o n .  They r e p o r t e d  t h a t v e r y s m a l l amounts o f carbon d i o x i d e were produced from f o r m i c a c i d breakdown and t h i s o c c u r s o n l y d u r i n g the growth o f t h i s organism. pathway i s not y e t u n d e r s t o o d .  The  - 39a -  Hemicellulose  Pectins  Cellulose  Starch  I  j  anhydroglucose chains  pectic acid (polygalacturonic acid)  xylooligosaccharide  xylobiose xylose (and other pentoses)  dihydroxyacetone  P  glyceraldehyde-3-P • ^[2H]  T 1,3-di-P-glycerate 3-P-glycerate  I  2-P-glycerate  1  i  phosphoenolpyruvate  F i g . 2.  pyruvate An o u t l i n e of the pathways of f e r m e n t a t i o n of t h e major carbohydrate c o n s t i t u e n t s of p l a n t s to 3C u n i t s i n the rumen. (Adapted from Leng, 1970).  [2H] Pyruvate  +  Lactate / ^ A c e t y l CoA  C0  2  + H  ^•Acetate  2  ^  Oxaloacetate  • A c e t y l CoA.  A c e t y l CoA1^2  [4H].  •CoA Malonyl CoA ^ ^ A c e t o a c e t y l CoA Acetyl  CoA  L a c t y l CoA H 0 2  A c r y l y l CoA  Propionyl CoA  CoA  [2H]  Succinate [2H;  <  3 - H y d r o x y b u t y r y l CoA S  ^ H  2  Propionyl  Propionate  CoA  4  S u c c i n y l CoA  0  ,Acetate  (  C r o t o n y l CoA  ^ A c e t y l CoA  [2H] Methyl malonyl CoA  Propionate  B u t y r y l CoA •Acetate • A c e t y l CoA  J  Butyrate  3.  An o u t l i n e o f the pathways o f d e g r a d a t i o n (Adapted from Leng, 1970).  o f 3C u n i t s  i n the  rumen.  - 40  -  The major end-products of carbohydrate  metabolism i n the rumen  a r e perhaps the s h o r t c h a i n v o l a t i l e f a t t y a c i d s , a c e t i c , p r o p i o n i c and b u t y r i c .  The  c o n c e n t r a t i o n of t o t a l s h o r t c h a i n f a t t y a c i d s  been observed to i n c r e a s e w i t h i n c r e a s i n g l e v e l s of f e e d i n g , the amount of f e e d i n the rumen (Hodgson e t d . ,  1976).  has  reflecting  The  average  p r o p o r t i o n s of the major v o l a t i l e f a t t y a c i d s when f o r a g e s a r e f e d are:  a c e t i c 65%,  p r o p i o n i c 20%,  and b u t y r i c 9%,  (Leng and B r e t t , 1966).  There a r e a number of f a c t o r s which a f f e c t the p r o p o r t i o n s of the v o l a t i l e fatty acids.  The  three  changes i n the p r o p o r t i o n s of the v a r i o u s  fatty  a c i d s a r e mostly brought about by changes i n rumen pH which i n t u r n a f f e c t s microbial populations. Roughage: diet.  concentrate  The most important  fermentable'carbohydrate.  Some of these  f a c t o r s are d i s c u s s e d below.  r a t i o or a c o n c e n t r a t e v e r s u s  aspect  of t h i s i s the content  ,0rskov (1975),. N i c h o l s o n and  of  a roughage readily  Sutton  (1969),  Wilke and Merwe (1976) i n d i c a t e d t h a t h i g h l e v e l s of c o n c e n t r a t e d i e t can r e s u l t i n i n c r e a s e d p r o p i o n i c a c i d p r o p o r t i o n and acetic acid proportion.  0rskov  fermentable  i n young f o r a g e s can a l s o r e s u l t  carbohydrate  p r o p i o n i c a c i d and and N i c h o l s o n  and  g i v e n d i e t s below f u l l with a corresponding e t a l . (1976)  and  decrease i n  (1975) i n d i c a t e d t h a t h i g h l e v e l s of  low b u t y r i c a c i d l e v e l s . Sutton  i n the  i n high  Whitelaw at a l . (1970)  (1969) r e p o r t e d t h a t when sheep f e d b a r l e y  are  f e e d i n g the b u t y r i c a c i d p o r t i o n i s i n c r e a s e d  decrease i n the p r o p i o n i c a c i d f r a c t i o n .  Hodgson  0rskov (1975) a l s o r e p o r t e d h i g h e r p r o p o r t i o n s  p r o p i o n i c a c i d at the expense of a c e t i c a c i d , on h i g h c o n c e n t r a t e  of diets.  - 41 -  The p a r t i c l e s i z e o f roughage can patterns.  G r i n d i n g o f t h e f e e d decreases  also affect  fermentation  t h e s i z e o f p a r t i c l e s and  t h i s r e s u l t s i n t h e exposure o f a g r e a t e r s u r f a c e a r e a , i n c r e a s e s the p r o p o r t i o n o f p r o p i o n i c a c i d and a l t e r s rumen pH ((7Jrskov, 1975) . Feed p r o c e s s i n g such as p e l l e t i n g o f c e r e a l g r a i n s may i n c r e a s e t h e p r o p o r t i o n o f p r o p i o n i c a c i d compared t o t h e f e e d i n g o f  unprocessed  whole g r a i n s (Gfrskov, 1975). Feeding fatty acids.  frequency  a l s o a f f e c t s t h e p r o p o r t i o n s o f the v o l a t i l e  High frequency  o f f e e d i n g of h i g h c o n c e n t r a t e  diets  a b o l i s h e s t h e g r e a t f l u c t u a t i o n i n rumen pH and i n c r e a s e s b u t y r i c p r o p o r t i o n s because o f t h e s u r v i v a l o f p r o t o z o a The Glucose  type of carbohydrate  acid  (0rskov, 1975).  a l s o a f f e c t s end-products o f f e r m e n t a t i o n .  or sucrose r e s u l t s i n higher b u t y r i c a c i d while high proportions  of s t a r c h r e s u l t s i n p r o p i o n i c a c i d type o f f e r m e n t a t i o n  (0rskov, 1975).  B u f f e r s added t o t h e rumen can r e s u l t i n changes i n f e r m e n t a t i o n pattern.  H a r r i s o n e t a l . (1976) by i n f u s i n g f o u r l i t r e s o f a r t i f i c i a l  s a l i v a a day a l t e r e d p r o p i o n a t e f e r m e n t a t i o n t o a c e t a t e A l h a s s a n e t a l . (1969)  and K r a b i l e t a l . (1969) by f e e d i n g sodium s u l p h i t e  i n c r e a s e d the p r o p o r t i o n o f p r o p i o n i c a c i d . the a d d i t i o n o f one a c i d may suppress  Hobson (1972) r e p o r t e d t h a t  the production of that p a r t i c u l a r  a c i d - t h i s i s r e f e r r e d t o as f e r m e n t a t i o n product The  fermentation.  suppression.  other v o l a t i l e f a t t y acids normally reported are i s o v a l e r i c ,  i s o b u t y r i c and v a l e r i c a c i d s . r e s u l t from deamination 1952a and 1952b).  I s o v a l e r i c and i s o b u t y r i c a c i d s n o r m a l l y  o f l e u c i n e and v a l i n e r e s p e c t i v e l y ( e l - S h a z l y ,  L a c t i c a c i d may accumulate i n t h e rumen under abnormal  c o n d i t i o n s of l a c t i c a c i d o s i s  (Dunlop, 1972).  - 42  -  E f f e c t s o f formaldehyde treatment on carbohydrate v o l a t i l e f a t t y a c i d p r o d u c t i o n i n the rumen  The  e f f e c t s of formaldehyde treatment  f a t t y a c i d p r o d u c t i o n have been v a r i a b l e .  metabolism  of f e e d s t u f f s on  Sharma and  Beever et a l . (1976), and Beever e t a l . (1977),  r e p o r t e d no d i f f e r e n c e s  Sharma et a l . (1972) r e p o r t e d decreased  a c i d p r o d u c t i o n w i t h treatment i n one  experiment observed  of rapeseed  meal.  individual  volatile  Langlands  fatty  (1973b)  no d i f f e r e n c e s i n t o t a l v o l a t i l e f a t t y  p r o d u c t i o n and p r o p o r t i o n s of the major f a t t y a c i d s . v o l a t i l e f a t t y a c i d p r o d u c t i o n to be reduced  He  acid  reported  significantly in  experiment r e p o r t e d i n the same paper (Langlands, Barry  volatile  I n g a l l s (1973),  i n v o l a t i l e f a t t y a c i d p r o d u c t i o n or p r o p o r t i o n s of the fatty acids.  and  total  another  1973b).  (1973a) r e p o r t e d v o l a t i l e f a t t y a c i d c o n c e n t r a t i o n to  decrease w i t h formaldehyde treatment n - v a l e r i c a c i d were reduced and Fennesy, 1973).  of hay.  The  l e v e l s of i s o - and  by formaldehyde treatment  of s i l a g e s  (Barry  In t h i s experiment, they r e p o r t e d v o l a t i l e f a t t y  acid  l e v e l s to be h i g h e r i n sheep f e d t r e a t e d s i l a g e about one hour b e f o r e feeding.  Barry  (1972) r e p o r t e d s i m i l a r p r e f e e d i n g c o n d i t i o n s w i t h  treatment  of c a s e i n .  These two workers thought t h a t the h i g h l e v e l s of  v o l a t i l e f a t t y a c i d and ammonia i n the rumen b e f o r e f e e d i n g i n sheep f e d t r e a t e d s i l a g e i n d i c a t e d g r e a t e r s t a b i l i t y of c o n d i t i o n s i n the rumen for fermentation.  Barry  (1976c) r e p o r t e d v a r i a b l e r e s u l t s w i t h  f a t t y a c i d p r o d u c t i o n i n t h r e e s e r i e s of experiments.  volatile  In experiment  t o t a l v o l a t i l e f a t t y a c i d , i s o - and n - v a l e r i c a c i d s were reduced  but  2,  - 43 -  not  isobutyric.  total volatile  I n experiment 3, f o r m a l i n treatment d i d not a f f e c t f a t t y acid production.  D i n i u s e_t a l . (1975) r e p o r t e d reduced apparent c o e f f i c i e n t s of acid-detergent  digestibility  f i b r e , n e u t r a l detergent  h e m i c e l l u l o s e w i t h treatment of d r i e d a l f a l f a . i n rumen pH, f i v e hours a f t e r f e e d i n g .  f i b r e and  There was no d i f f e r e n c e  Beever e t a l . (1976), however  observed enhanced d i g e s t i b i l i t y o f c e l l u l o s e w i t h treatment of d r i e d grass. but  There was a d e p r e s s i o n  i n d i g e s t i b i l i t y o f c e l l u l o s e i n the rumen  subsequent i n c r e a s e d d i g e s t i o n o f c e l l u l o s e i n the caecum r e s u l t e d  i n higher  c e l l u l o s e d i g e s t i b i l i t y with  f o r m a l i n treatment of d r i e d g r a s s .  - 43a -  OBJECTIVES  These s t u d i e s r e p o r t e d i n t h i s t h e s i s were undertaken w i t h the f o l l o w i n g  1)  objectives:  To determine by i n v i t r o s t u d i e s the optimum l e v e l of  formaldehyde treatment of grass-legume f o r a g e p r o t e i n t o o b t a i n p r o t e c t i o n from rumen m i c r o b i a l d e g r a d a t i o n w i t h o u t subsequent decrease i n enzymatic d i g e s t i o n . 2)  To a s s e s s t h e e f f e c t s of formaldehyde treatment of  the f o r a g e p r o t e i n i n terms o f n i t r o g e n and f i b r e u t i l i z a t i o n i n v i v o . 3)  To determine i f d i e t a r y s u p p l e m e n t a t i o n w i t h s u l p h u r  and/or branched c h a i n f a t t y a c i d s i s n e c e s s a r y when a p o r t i o n of the dietary protein i s protected  from m i c r o b i a l d e g r a d a t i o n .  - 44 -  MATERIALS AND METHODS  Introduction  The e x p e r i m e n t a l work was conducted i n f o u r phases. phase employed  i n v i t r o p r o c e d u r e s t o determine t h e optimum l e v e l o f  formaldehyde treatment o f dehydrated and hammermilled forage.  The f i r s t  grass-clover  T h i s was f o l l o w e d by i n v i v o experiments i n which apparent  d i g e s t i b i l i t i e s of dry matter, organic matter, n i t r o g e n , f i b r e and c e l l u l o s e sulphur balances.  acid-detergent  were determined as w e l l as feed i n t a k e , n i t r o g e n and A t t h e end o f t h e above d e t e r m i n a t i o n s t h e animals  were s l a u g h t e r e d f o r t h e purpose o f measuring rumen pH, rumen d r y matter c o n t e n t , and t h e rumen l e v e l s acids.  o f ammonia-nitrogen  Abomasal pH and abomasal d i g e s t a  and v o l a t i l e  contents of acid-detergent  f i b r e , t o t a l n i t r o g e n and RNA-N were a l s o measured. of  fatty  The f o u r t h  phase  t h e study i n v o l v e d t h e use o f a sheep f i t t e d w i t h a duodenal r e -  e n t r a n t cannula f o r measuring d i g e s t a f l o w .  EXPERIMENT I  Treatment o f t h e f o r a g e w i t h formaldehyde  The dehydrated and hammermilled  r y e - g r a s s - l a d i n o c l o v e r f o r a g e Was  t r e a t e d w i t h 10% formaldehyde s t o c k s o l u t i o n mixer s i m i l a r was  (formalin) i n a small upright  t o t h e method used by Schmidt e t a l . (1974).  sprayed on t h e f o r a g e , u s i n g  b e i n g t u r n e d around i n t h e mixer.  The f o r m a l i n  an a e r o s o l spray gun, as i t was T e n - k i l o g r a m (10 kg) batches o f t h e  - 45 -  f o r a g e were t r e a t e d a t a time. sprayed  on the/forage,  The treatment r e s u l t e d i n formaldehyde  on an a i r d r y b a s i s , of 0.0%, 0.8%, 1.0% and 1.2%  w i t h the same q u a n t i t i e s o f f l u i d added sealed i n polythene by  (12 ml/100 g ) .  The f o r a g e  was  bags f o r one week a f t e r t h e treatment as d e s c r i b e d  S a v i l l e e t a l . (1971).  In v i t r o  incubation  lg  samples (DM b a s i s ) o f t h e t r e a t e d g r a s s - c l o v e r were  i n rumen f l u i d p l u s a r t i f i c i a l Troelsen  (1969).  s a l i v a , according  (Technicon  w i t h 50 ml o f artificial  through a s h l e s s f i l t e r paper.  was used f o r d e t e r m i n i n g  autoanalyzer  d e i o n i z e d " water and f i l t e r e d  was determined by the m a c r o - K j e l d a h l  The  ammonia-nitrogen u s i n g the  model EDP 910 120-45-5).  s a l i v a served as b l a n k .  digestibility  t o the method of  A f t e r 48 hours of i n c u b a t i o n , the m i x t u r e was  c e n t r i f u g e d a t 1500 x g and f i l t e r e d supernatant  incubated  again.  The r e s i d u e was then washed Rumen f l u i d  The n i t r o g e n content  o f the r e s i d u e  method (AOAC, 1970).  was c a l c u l a t e d by the method o f B a r r y  plus  Nitrogen  (1972).  There were  t h r e e r e p l i c a t i o n s f o r each treatment and b l a n k . Three r e p l i c a t e s o f each treatment and b l a n k were a l s o taken through the two-stage d i g e s t i o n procedure.  A f t e r the rumen inoculum . p l u s b u f f e r  d i g e s t i o n , each r e p l i c a t e was c e n t r i f u g e d and the supernatant for  ammonia-nitrogen d e t e r m i n a t i o n  as d e s c r i b e d above.  then d i g e s t e d w i t h a c i d - p e p s i n f o r 48 hours. r e s i d u e was then c e n t r i f u g e d , f i l t e r e d first  stage  incubation.  was used  The r e s i d u e was  The a c i d - p e p s i n  digested  and washed as d e s c r i b e d f o r the  The n i t r o g e n content  of the r e s i d u e was determined  - 46  by  -  the m a c r o - K j e l d a h l method (AOAC, 1970).  n i t r o g e n was  digestibility  of  then c a l c u l a t e d .  Treatment of g r a s s - c l o v e r f o r a g e  The  The  for i n vivo  g r a s s - c l o v e r f o r a g e was  i n the same manner as d e s c r i b e d t r e a t e d forage was  trials  t r e a t e d w i t h 1% l e v e l of formaldehyde  f o r the i n v i t r o t r i a l s above.  then used i n r a t i o n s f o r animal f e e d i n g  The  trials.  EXPERIMENT I I  Nitrogen,  The and  c a r b o h y d r a t e and  d i e t s (Table 1) c o n t a i n e d  a m i n e r a l premix.  feeding.  up  to  5.  The  5 at the r a t e of 3.0g/kg  2.3g/kg d i e t f o r i s o b u t y r i c immediately  d i l u t e d to 10ml  barley  Isovaleric  before  A f t e r measuring the r e q u i r e d volumes o f the a c i d s f o r Ikg  an a e r o s o l spray  by Bryant  cassava,  a f t e r m i x i n g i n a h o r i z o n t a l mixer.  i n t o a measuring c y l i n d e r , i t was  The  studies  added to d i e t s 4 and  i s o b u t y r i c a c i d s were sprayed on d i e t s 3 and  d i e t f o r i s o v a l e r i c and  feed i n t a k e  grass-clover forage,  Sodium s u l p h a t e was  d i e t s were p e l l e t e d (9mm) and  s u l p h u r metabolism and  and  sprayed  diet  using  gun.  d i e t s contained  a p p r o x i m a t e l y 14%  crude p r o t e i n as  reported  (1973) to meet the requirement of c o n f i n e d lambs weighing 29kg  slaughter.  T a b l e 1.  Composition  Ingredient  (% DM)  of Rations.  Diet 1  Diet 2  Diet 3  Diet 4  Diet 5  Cassava  38  38  38  37.33  37.33  Barley  11  11  11  11  11  50  —  Treated grass  —  50  50  50  50  Sodium s u l p h a t e  —  —  —  0.67  0.67  Untreated  grass  M i n e r a l premix*  The  1  1  —  1  1  composition o f t h e sheep m i n e r a l premix, g i v e n by the manufacturers  1  was as f o l l o w s  Ca 20%, P 19%, Mn 0.15%, Zn 0.60%, Fe 0.20%, I 0.01%, Co 0.008%, Cu 0.015%; the maximum l e v e l s o f F l u o r i n e was 0.2% w h i l e the minimum l e v e l s o f Vitamins A and D^ were 551,150 Iu/kg and 110,230 Iu/kg  respectively.  - 48 -  T w e n t y - f i v e young ram lambs o f the D o r s e t breed were used f o r the metabolism sheep u n i t .  trials,  a t South campus U n i v e r s i t y o f B r i t i s h  F i v e animals were randomly a s s i g n e d t o each  Columbia  diet.  There was a p r e - e x p e r i m e n t a l p e r i o d of twenty days and a c o l l e c t i o n p e r i o d o f seven  days.  The animals were kept i n s l a t t e d f l o o r i n d i v i d u a l pens and f e d individually  ( f i v e animals a t a time one on each d i e t ) d u r i n g seventeen  days o f the p r e - e x p e r i m e n t a l p e r i o d o f twenty days i n the i n d i v i d u a l pens, a three-day adjustment  days.  After  seventeen  they were t r a n s f e r r e d t o t h e cages, a l l o w e d  p e r i o d , b e f o r e the seven-day c o l l e c t i o n  period.  The p r e - e x p e r i m e n t a l p e r i o d of seventeen days was a l s o used f o r feed i n t a k e a s s a y s .  D u r i n g t h e f e e d i n t a k e assay, t h e r e was a ten-day  p r e - c o n d i t i o n i n g p e r i o d and a seven-day measurement o f f e e d i n t a k e , as recommended by Heaney et: a l . (1968). p e r i o d , the animals were brought  D u r i n g the ten-day  t o maximum f e e d i n t a k e .  ad l i b i t u m and r e s i d u e s weighed back.  I f they l e f t  o f f e r e d , the f e e d allowance was i n c r e a s e d by 25%.  pre-conditioning They were f e d  l e s s than 10% of feed The minimum l e v e l of  f e e d i n g d u r i n g the seven-day f e e d i n t a k e assay p e r i o d was the same as the maximum l e v e l o f f e e d i n t a k e d u r i n g the 10-day p r e - c o n d i t i o n i n g The amount o f f e e d l e f t o v e r was weighed d a i l y .  period.  I f the animals l e f t  less  than 10% of t h e f e e d o f f e r e d , the f e e d allowance was i n c r e a s e d by 25%. D u r i n g the metabolism based on t h e l e v e l determined  trials,  the animals were f e d ad l i b i t u m  d u r i n g t h e f e e d i n t a k e assay p e r i o d .  animals were f e d t w i c e d a i l y a t 8:30 a.m. twenty-seven-day  period.  and 3:00 p.m.,  The  throughout t h e  - 49 -  T o t a l amount of f a e c e s v o i d e d were c o l l e c t e d and weighed d u r i n g the seven-day metabolism  trial.  daily  Samples of f a e c e s were d r i e d i n  a f o r c e d d r a f t oven at 80°C f o r 24 h r s i m i l a r t o the procedure o f Hume e t a l . (1970). U r i n e was  c o l l e c t e d over 100 ml of 6N HC1  w i t h narrow necks i n t o which were f i t t e d  i n plastic-containers  the rubber tubes d r a i n i n g the  u r i n e from the u r i n e compartment of the metabolism exposure o f the u r i n e t o the atmosphere. d a i l y a f t e r measuring  the volume.  cage.  This  Samples of u r i n e were taken  The samples were kept i n the f r e e z e r  compartment of a r e f r i g e r a t o r . The a d d i t i o n of 100 ml of 6N HC1 pH of u r i n e t o between 2 and 3.  limited  T h i s pH l e v e l  (between  reduced  2 and 3)  was  recommended by M a r t i n (1966) t o reduce t o i n s i g n i f i c a n t l e v e l s the ammonia l o s t from the u r i n e . At filtered  the end of the t r i a l s , the u r i n e samples were thawed and through l a y e r s of cheese c l o t h as was  The f i l t r a t e was  c a r r i e d out by Bryant  (1973).  used f o r n i t r o g e n and s u l p h u r a n a l y s e s .  The animals were weighed on the 1 s t , 10th, 17th and 27th days of the  trial.  EXPERIMENT I I I  Rumen and abomasal d i g e s t a m e t a b o l i t e s s t u d i e s  The animals used i n the p r e v i o u s experiment were s l a u g h t e r e d f o r these s t u d i e s . slaughter.  The animals were f e d a t about 2:00  a.m.  on the day of  They were then t r a n s p o r t e d t o the s l a u g h t e r house and  s l a u g h t e r e d between 7:00  a.m.  - 8:00  a.m.  The g a s t r o - i n t e s t i n a l  tract  -  50 -  was removed i n t a c t immediately a f t e r s l a u g h t e r p l a c e d c o n t a i n i n g i c e and t r a n s p o r t e d ' v a r i o u s measurements.  . i n buckets  d i r e c t l y t o the l a b o r a t o r y f o r the  The pH o f t h e rumen f l u i d was determined  immediately a f t e r s t r a i n i n g rumen d i g e s t a .  The ammonia-nitrogen  were determined u s i n g the a u t o a n a l y z e r used i n experiment I . fluid  (1954).  Samples of rumen f l u i d were  c o l l e c t e d as d e s c r i b e d by A l h a s s a n et^ a l .  (1969) and the measurements  t o t a l and i n d i v i d u a l v o l a t i l e f a t t y a c i d s were c a r r i e d out as  d e s c r i b e d by Ross and K i t t s weighed.  (1971).  The t o t a l rumen c o n t e n t s were  Samples o f t h e rumen c o n t e n t were f r e e z e - d r i e d f o r s t o r a g e .  Samples o f the f r e e z e - d r i e d m a t e r i a l were oven d r i e d for  dry matter d e t e r m i n a t i o n .  and  Ulyatt  (105°C f o r 24 hours)  T h i s was s i m i l a r t o the method o f MacRae  (1974).  Abomasal f l u i d was a l s o s t r a i n e d f o r pH measurement. of  The rumen  f o r rumen ammonia-nitrogen d e t e r m i n a t i o n was a c i d i f i e d as  d e s c r i b e d by Chalmers et: a l .  of  levels  the abomasum were f r e e z e d r i e d .  The c o n t e n t s  Samples o f the f r e e z e - d r i e d abomasal  c o n t e n t s were used f o r ash, a c i d - d e t e r g e n t f i b r e , c e l l u l o s e ,  total  n i t r o g e n and RNA-N a n a l y s e s as d e s c r i b e d under c h e m i c a l a n a l y s i s . Samples o f the f r e e z e - d r i e d m a t e r i a l s were f u r t h e r d r i e d a t 105°C f o r 24 hours to determine d r y matter content of the abomasum (MacRae and . U l y a t t , 1974).  - 51  -  EXPERIMENT IV  Duodenal d i g e s t a f l o w s t u d i e s  A ram  f i t t e d w i t h a duodenal r e - e n t r a n t cannula was  t h i s experiment.  The  r e - e n t r a n t cannula was  fitted  o p e r a t i o n as d e s c r i b e d by Brown et a l . ( 1 9 6 8 ) .  in a  On  surgical  T h i s animal was  s t u d i e s of duodenal flow 6 weeks a f t e r the o p e r a t i o n . each of the d i e t s i n t u r n f o r t e n days.  used f o r  The  the t e n t h day,  used f o r  animal was  fed  a l l the  m a t e r i a l s f l o w i n g from the abomasum i n t o the duodenum were c o l l e c t e d  over  a 24-hour p e r i o d . The  animal was  f e d l,500g  e q u a l p o r t i o n s twice d a i l y at 8:30  C o l l e c t i o n and  The  ( a i r dry b a s i s ) of each d i e t i n a.m.  and  3:00  p.m.  sampling of d i g e s t a  p i e c e of tube j o i n i n g the two  d u r i n g the day  two  of c o l l e c t i o n . The  ends of the cannula was  removed  Separate l e n g t h s of rubber t u b i n g s were  attached  t o each end.  tubing attached  to the e x i t cannula  arranged  so t h a t the d i g e s t a were d e l i v e r e d i n t o a g l a s s c o n i c a l f l a s k  (2,000 ml c a p a c i t y ) p l a c e d i n a bucket.  The  with laboratory parafilm.  maintained  The  The  tubing attached  f l a s k was  top of the f l a s k was  A f t e r every one  replaced.  The volume of the c o l l e c t e d d i g e s t a was  shaking.  The  and  sample taken was  temperature.  fastened  a h a l f hours (1% h r ) , the f l a s k  A ten percent  (10%)  sealed  to  the  a l l o w i n g r e t u r n o f d i g e s t a to  duodenum.  weight r e c o r d e d .  at ambient  t o the r e t u r n cannula was  c r a t e , about 40-50 cm above the cannula,  was  sample was  was  measured and  taken a f t e r  immediately p l a c e d i n a capped  the  vigorous plastic  c o n t a i n e r and kept i n the f r e e z e r s e c t i o n of a r e f r i g e r a t o r .  Following  the c o l l e c t i o n of the sample the remainder of the d i g e s t a was  returned  the  - 52 -  manually  i n t o t h e i n t e s t i n e through t h e r e t u r n cannula,,  after  adding  donor sample c o l l e c t e d p r e v i o u s l y t o make up f o r t h e "quantity taken f o r analyses.  The d i g e s t a was warmed i n a water b a t h  (39.°C). and r e t u r n e d  i n ; s m a l l q u a n t i t i e s over a p e r i o d o f one and a h a l f hours.  T h i s method  of sample c o l l e c t i o n was s i m i l a r t o t h a t o f Thompson and Lamming  (1972).  The amount o f f e e d l e f t a f t e r every one and a h a l f hours was weighed. The f r o z e n duodenal further analysis.  samples were thawed i n a r e f r i g e r a t o r b e f o r e  Weighed samples were f r e e z e - d r i e d and a n a l y z e d f o r  n i t r o g e n , a c i d - d e t e r g e n t f i b r e , RNA-N, a s h and TCA-N.  A sample o f t h e  f r e e z e - d r i e d m a t e r i a l was f u r t h e r d r i e d i n t h e oven f o r 24 hours, f o r dry matter d e t e r m i n a t i o n , as d e s c r i b e d by MacRae and U l y a t t  Chemical  (1974).  analyses  The  c h e m i c a l a n a l y s e s o f a l l samples,  unless otherwise d e s c r i b e d ,  were c a r r i e d out as o u t l i n e d below.  (a)  Nitrogen:  Nitrogen contents of f e e d s t u f f s ,  f a e c e s , d i g e s t a and u r i n e were determined AOAC  rations,  by m a c r o - K j e l d a h l method o f the  (1970). (b)  Ash: Ash c o n t e n t s o f t h e r a t i o n s , f a e c e s and d i g e s t a were  determined by t h e AOAC (1970) method. (c) determined  Moisture:  M o i s t u r e content o f t h e r a t i o n s and f a e c e s were  u s i n g the method o f AOAC (1970).  abomasal and duodenal  M o i s t u r e content of rumen,  d i g e s t a samples was determined  i n two s t a g e s :  f r e e z e - d r y i n g f o l l o w e d by o v e n - d r y i n g as d e s c r i b e d by MacRae and U l y a t t (1974).  - 53 -  (d) and  A c i d - d e t e r g e n t f i b r e and c e l l u l o s e :  Acid-detergent  c e l l u l o s e c o n t e n t s of r a t i o n s , f a e c e s and d i g e s t a were  fibre  determined  a c c o r d i n g t o the method o f Van Soest and Wine (1968) as m o d i f i e d by Waldern  (1971). (e)  Acid-detergent f i b r e insoluble nitrogen:  Acid-detergent  f i b r e i n s o l u b l e n i t r o g e n content o f the g r a s s - c l o v e r f o r a g e was u s i n g the method o f Yu and V e i r a (f)  Sulphur:  determined  (1977).  A s h i n g o f u r i n e , f a e c e s , r a t i o n and f e e d s t u f f  samples f o r s u l p h u r d e t e r m i n a t i o n s , was done a c c o r d i n g to the method o f B i r d and F o u n t a i n  (1970).  F o r the f e e d s t u f f , r a t i o n , and f a e c a l  2g d r y matter were weighed i n t o an a s h i n g c r u c i b l e . t h o r o u g h l y w i t h 2.5 g  of m i x t u r e  (25:1) as an o x i d i z i n g agent.  samples,  T h i s was mixed  o f sodium b i c a r b o n a t e - s i l v e r  oxide  The m i x t u r e was ashed a t 550°C f o r f i v e (5)  hours i n a m u f f l e f u r n a c e . With t h e u r i n e samplesJ_ml-samples were p i p e t t e d i n t o t h e c r u c i b l e s , then d r i e d a t 55°C i n an oven.  The d r i e d u r i n e  sample was then ashed. The ash i n each case was d i s s o l v e d i n 20 ml 6N HC1, a f t e r f o l l o w i n g the method o f Johnson e t a l . (1970). The s o l u t i o n was d i l u t e d to 1000 ml w i t h d i s t i l l e d water. w i t h 10 ml o f g e l a t i n barium The g e l a t i n barium of T a b a t a b i  (1974).  cooling, then  A 10 ml a l i q u o t was combined  c h l o r i d e s o l u t i o n c o n t a i n i n g 0.3g, B a C ^ .  c h l o r i d e s o l u t i o n was prepared  f o l l o w i n g the method  The suspension of the ash s o l u t i o n and g e l a t i n -  b a r i u m - c h l o r i d e s o l u t i o n was shaken f o r f i v e minutes and allowed to stand f o r one hour. determined  The t u r b i d i t y o f the s u l p h a t e p r e c i p i t a t e d  u s i n g spectrophotometer  was  ( S p e c t r o n i c 20 a t wavelength o f 500 mu)  f o l l o w i n g the method o f Johnson a t a l . (1970).  - 54  The was  -  extractable sulphate-sulphur  i n the g r a s s - c l o v e r  forage  determined u s i n g the procedure o u t l i n e d i n the b u l l e t i n of  M i n i s t r y of A g r i c u l t u r e , F i s h e r i e s , and B u l l e t i n 27 a c i d and  (1973).  Food, London, T e c h n i c a l  The weighed sample was  activated charcoal.  through Whatman No.2  A f t e r shaking  f i l t e r paper and  the  shaken w i t h 0.12N f o r 30 min,  hydrochloric  i t was  the f i l t r a t e used f o r  filtered  sulphate-  sulphur a n a l y s i s . Standards of s u l p h a t e - s u l p h u r  were p r e p a r e d a c c o r d i n g  to  the  procedure o u t l i n e d i n the t e c h n i c a l b u l l e t i n r e f e r r e d to above. o n l y m o d i f i c a t i o n was  (g)  RNA:  t h a t the s u l p h a t e was  d i s s o l v e d i n 0.12N  R i b o n u c l e i c a c i d contents  10% N a C l ) ,  95%  ethanol,  o t h e r pigments. samples w i t h  ethanol-NaCl  and  (4:1 r a t i o of 95%  T h i s method was  extracted  The  tubes c o n t a i n i n g the  used by L i n g and  from the samples was  photometer (Unicam SP800B), U.V., of RNA  were p r e p a r e d and  Akinwande (1973).  Buttery  ethanol  and  a standard  samples (1975), f o r  duodenal d i g e s t a . determined w i t h  wavelength 260 mu. curve o b t a i n e d  The n i t r o g e n content  duodenal d i g e s t a was y e a s t RNA  homogenized  The n u c l e i c a c i d s were then e x t r a c t e d from the m o i s t  e x t r a c t i n g n u c l e i c a c i d s from rumen b a c t e r i a and RNA  The  duodenal  e t h a n o l - 1 0 % NaCl to remove c h l o r o p h y l l and  10% NaCl a t 100°C.  were stoppered.  HC1.  of the abomasal and  d i g e s t a were determined u s i n g the method of Guinn (1966). samples were, t r e a t e d w i t h  The  of the RNA  spectro-  Standard s o l u t i o n s  as d e s c r i b e d  by  e x t r a c t e d from  the  assumed to be of the same magnitude as the t o r u l a  used f o r p r e p a r i n g  the s t a n d a r d s .  T h i s assumption was  also  - 55 -  made by Smith and M c A l l a n  (1970).  The m i c r o b i a l p r o t e i n content  of the d i g e s t a was c a l c u l a t e d by assuming RNA-N: t o t a l N i n rumen microbes  t o be 0.075 as was r e p o r t e d by Smith  (1975) made a s i m i l a r  (h)  NPN:  (1975).  Sutton e t a l .  assumption.  The n o n - p r o t e i n - n i t r o g e n f r a c t i o n s o f the grass-legume  f o r a g e , abomasal and duodenal  samples were determined  of G o s h t a s b p o u r - P a r s i e t a l . (1977). f r e e z e - d r i e d duodenal  u s i n g t h e method  One gram (1 g) sample o f t h e  o r abomasal d i g e s t a o r grass-legume  mixed w i t h 10 ml d e i o n i z e d water. s o l u t i o n was then added.  Ten m i l l i l i t r e s  f o r a g e was  (10 ml) o f 20% TCA  I t was shaken f o r t e n minutes m e c h a n i c a l l y ,  heated t o 90°C i n a water b a t h , and f u r t h e r shaken i n t h e h o t water b a t h f o r 10 minutes.  The tube was then c e n t r i f u g e d a t 15,000 xg. The  supernatant p l u s second washing o f the r e s i d u e , was a n a l y z e d f o r n i t r o g e n by t h e m a c r o - K j e l d a h l method (AOAC, 1970). nitrogen  T h i s was t h e n o n - p r o t e i n -  fraction.  E x p e r i m e n t a l d e s i g n s and s t a t i s t i c a l  analysis  R e s u l t s from the t r i a l s were s u b j e c t e d t o s t a t i s t i c a l In the i n v i t r o d i g e s t i b i l i t y d e t e r m i n a t i o n s , a c o m p l e t e l y design  (CRD) was used.  analysis. randomized  The F - t e s t was a p p l i e d and s i g n i f i c a n t  between means were determined u s i n g  Tukey's W v a l u e s  differences  ( S t e e l and T o r r i e ,  1960).  - 56 -  Randomized b l o c k d e s i g n s were used b l o c k i n t h i s case was and  i n the o t h e r t r i a l s .  the group o r b a t c h .  The F - t e s t was  s i g n i f i c a n t d i f f e r e n c e s between means were determined  W v a l u e s as above ( S t e e l and T o r r i e , No  statistical  a n a l y s i s was  s t u d i e s u s i n g the c a n n u l a t e d  The  applied u s i n g Tukey's  1960).  a p p l i e d to d a t a c o l l e c t e d from  animal.  the  - 57 -  RESULTS  In V i t r o d i g e s t i o n  trials  The r e s u l t s o f t h e n i t r o g e n d i g e s t i b i l i t y t r i a l s a t the f i r s t stage i n v i t r o d i g e s t i o n o f t h e r y e - g r a s s - c l o v e r w i t h d i f f e r e n t of for  formaldehyde treatment a r e shown i n T a b l e 2.  levels  The average v a l u e s  n i t r o g e n d i g e s t i b i l i t y f o r t h e treatments were 31.88%, 15.72%,  6.87%, and 5.69% f o r 0%, 0.8%, 1.0%, and 1.2% l e v e l s o f formaldehyde treatment r e s p e c t i v e l y .  There were s i g n i f i c a n t  (p 4 . 0.05) d i f f e r e n c e s  between t h e treatments a t 0% l e v e l , f o r m a l d e h y d e and t h e r e s t o f t h e treatments(0.8%, 1.0%, and 1.2% l e v e l s o f formaldehyde). 0.8% l e v e l o f formaldehyde was s i g n i f i c a n t l y 1.0% and 1.2% l e v e l s o f formaldehyde.  Treatment a t  (p<0.05) d i f f e r e n t  from  D i f f e r e n c e s between 1% and 1.2%  l e v e l s o f formaldehyde treatments were n o t s i g n i f i c a n t  ( p ^ 0.05).  The r e s u l t s of n i t r o g e n d i g e s t i b i l i t y a t t h e end o f the second stage o f i n v i t r o of  (combined m i c r o b i a l and a c i d - p e p s i n s t a g e s )  t h e f o r a g e a t t h e f o u r l e v e l s o f formaldehyde treatment a r e shown i n  T a b l e 2. are  digestion  The average v a l u e s f o r n i t r o g e n d i g e s t i b i l i t y f o r t h e treatments  80.95%, 79.76%, 75.85% and 71.01% f o r t h e 0.0%, 0.8%, 1.0% and 1.2%  l e v e l s o f formaldehyde r e s p e c t i v e l y .  Treatment a t 1.2% s i g n i f i c a n t l y  (p Z.0.05) reduced n i t r o g e n d i g e s t i b i l i t y 0.0% o r 0.8%.  compared w i t h treatments a t  A l l o t h e r d i f f e r e n c e s were n o t s i g n i f i c a n t  ( p ^ 0.05). -  The mean d i f f e r e n c e s between the second stage (combined and a c i d - p e p s i n ) and f i r s t  microbial  stages of i n v i t r o n i t r o g e n d i g e s t i b i l i t y are  49.07, 64.04, 68.98 and 65.32 percentage u n i t s f o r 0.0%, 0.8%, 1.0%, and 1.2% l e v e l s o f formaldehyde treatment r e s p e c t i v e l y .  T a b l e 2.  In V i t r o D i g e s t i o n  Trials.  Formaldehyde Treatment Parameter  No. of  0.0%  0.8%  1.0%  1.2%  Stage N d i g e s t i o n %  31.88a  15.72b  6.87c  5.69c  +  1.22  2nd Stage N d i g e s t i o n %  80.95a  79.76a  75.85ab  71.01b  +  1.41  228.79a  78.58b  65.27b  30.04c  +  7.60  8.63  3.97  1st  NH^-N  production  (ppm)  C a l c u l a t e d N d i g e s t i o n from NH ~N% 3  30.26  Means on the same l i n e b e a r i n g d i f f e r e n t  letters  10.39  ( a , b, c, d) d i f f e r s i g n i f i c a n t l y  Replicates  S.E.  Ul  oo  (p < 0 . 0 5 ) .  - 59 -  Formaldehyde treatment s i g n i f i c a n t l y  (p < 0.05)  reduced i n v i t r o  ammonia-nitrogen p r o d u c t i o n d u r i n g the m i c r o b i a l stage of i n c u b a t i o n (Table 2). 0.8%,  1.0%  The mean v a l u e s f o r i n v i t r o ammonia-nitrogen p r o d u c t i o n a t and  78.58 ppm,  1.2%  l e v e l s of formaldehyde treatment are 228.79  65.27 ppm  and and  30.04 ppm 1.0%  respectively.  productions  a t 0.8%  different.  The d i f f e r e n c e s between any  0.0%,  ppm,  Ammonia-nitrogen  were not s i g n i f i c a n t l y  (p  "y  0.05)  o t h e r p a i r were s i g n i f i c a n t  (p 4.0.05). Nitrogen d i g e s t i b i l i t i e s  c a l c u l a t e d u s i n g the  p r o d u c t i o n f i g u r e s are shown i n T a b l e  Chemical composition  The  chemical  of d i e t s and  composition  ammonia-nitrogen  2.  ingredients  of the feed i n g r e d i e n t s and  the d i e t s  f e d to the animals f o r the f e e d i n t a k e and metabolism t r i a l s are shown i n Table  3.  D a i l y feed i n t a k e , d a i l y u r i n e output, m e t a b o l i c body s i z e s of animals (kg) at the b e g i n n i n g of the metabolism study p e r i o d , and the d a i l y g a i n i n weight d u r i n g the pre-metabolism assay p e r i o d  There were no  significant  r e s p e c t t o d a i l y feed i n t a k e  (p^-0.05) d i f f e r e n c e s between d i e t s w i t h  (g D.M.)  b o t h d u r i n g the pre-metabolism and  per u n i t of m e t a b o l i c  body s i z e  the metabolism assay p e r i o d s per u n i t of  (Table 4 ) .  Daily nitrogen intake  (g/Wkg^*^), u r i n e output  metabolic  body s i z e per day and  the growth r a t e s of the animals over seventeen  T a b l e 3.  Chemical Composition o f D i e t s and I n g r e d i e n t s (D.M.  basis).  Chemical F r a c t i o n % (D.M. Diet/Ingredient  N  S  ADF  Cell.  Ash  Diet 1  2. 29  0.17  16.87  13.03  7.47  Diet 2  2. 24  0.18  16.98  13.28  7.76  Diet 3  2. 25  0.18  16.83  12.95  7.53  Diet  4  2. 28  0.27  17.12  13.19  8.11  Diet 5  2. 29  0.27  17.33  13.38  8.48  Grass-legume f o r a g e  3. 78  0.25  Cassava  0. 42  0.05  Barley  1. 77  0.12  r  Basis) Ext.S  ADF-IN  0.07  0.20  NPN  0.66  - 61 -  day p e r i o d were a l s o n o t s i g n i f i c a n t l y (p"^ 0.05) d i f f e r e n t The m e t a b o l i c body s i z e s  o f t h e animals  at the beginning  metabolism assay p e r i o d were n o t s i g n i f i c a n t l y (p^O.05) (Table 4 ) .  However, t h e animals  significantly  used i n the f i r s t  (p <0.05) h e a v i e r than animals  The mean m e t a b o l i c body s i z e s  o f animals  (Table 4 ) .  of the different  group were  used i n t h e t h i r d group.  used i n t h e f i v e groups were  14.37kg, 14.11kg, 12.74kg, 13.13kg, and 14.19kg f o r group one, two, t h r e e , f o u r and f i v e  respectively.  Apparent d i g e s t i b i l i t y c o e f f i c i e n t s o f d r y matter, o r g a n i c matter, n i t r o g e n , a c i d - d e t e r g e n t f i b r e and c e l l u l o s e .  The  apparent d i g e s t i b i l i t y c o e f f i c i e n t s  o r g a n i c matter cellulose  (O.M.), n i t r o g e n  are presented  o f d r y m a t t e r , (D.M.),  (N), a c i d - d e t e r g e n t  i n T a b l e 5.  fibre  (A.D.F.) and  The apparent d i g e s t i b i l i t y c o e f f i c i e n t s  of d r y matter, and o r g a n i c m a t t e r were n o t s i g n i f i c a n t l y (p> d i f f e r e n t between d i e t a r y treatments.  0.05)  The apparent d i g e s t i b i l i t y  c o e f f i c i e n t o f n i t r o g e n was s i g n i f i c a n t l y (p < 0.05) h i g h e r f o r d i e t one than f o r d i e t s and  two, f o u r and f i v e .  t h r e e was n o t s i g n i f i c a n t  t h r e e and f o u r was s i g n i f i c a n t  The d i f f e r e n c e between d i e t s one  (p > 0.05).  The d i f f e r e n c e between d i e t s  (p < 0.05), w i t h d i e t t h r e e h a v i n g t h e  greater value. The  apparent d i g e s t i b i l i t y c o e f f i c i e n t s  and c e l l u l o s e were h i g h e r f o r a l l t h e d i e t s than f o r d i e t one (p ^ 0 . 0 5 ) .  of acid-detergent  fibre  containing the t r e a t e d forage  T a b l e 4.  D a i l y Feed Intake, D a i l y U r i n e Output, M e t a b o l i c Body S i z e s o f Animals a t t h e B e g i n n i n g o f the Metabolism Study P e r i o d and the D a i l y Gain i n Weight D u r i n g the Pre-metabolism Assay P e r i o d .  Diet containing u n t r e a t e d forage  D a i l y Intake (g/Wkg  0 , 7 5  )  1  D i e t s c o n t a i n i n g formaldehyde t r e a t e d forage + VFAS + SO" + + VFAS + SO 2  3  4  5 ;  S.E.  100.62  101.93  98.02  96.47  + 4.70  D.M. d u r i n g pre-metabolism assay p e r i o d  91.33 *  D.M. d u r i n g t h e metabolism study p e r i o d  90.17  96.65  104.29  90.88  94.25  + 4.52  2.07  2.18  2.38  2.08  2.18  +0.10  154.76  170.77  170.77  160.10  149.42  +21.31  14.42  13.17  14.02  13:43"  13.50  + 0.36  80.32  77.02  61.44  79.81  78.86  +12.29  N d u r i n g the metabolism study p e r i o d Growth r a t e d u r i n g 1st 17 days (g/day) M e t a b o l i c body s i z e a t the b e g i n n i n g o f metabolism s t u d i e s (kg) U r i n e output  (mis/day/Wkg°' ) 75  * Each v a l u e r e p r e s e n t s the mean o f f i v e t Sulphur was added as Na^SO^  determinations,  T a b l e 5.  Apparent D i g e s t i b i l i t y  C o e f f i c i e n t s of Some Chemical  Diet containing u n t r e a t e d forage Apparent  Dig.Coeff.  D i e t s c o n t a i n i n g formaldehyde t r e a t e d f o r a g e + VFAS + -SC^t • + VFAS + SO 2  1  F r a c t i o n s (%).  3  4  5  S.E.  D.M.  %  64.78*  65.69  65.82  64.80  63.71  +  1.02  O.M.  %  65.35  65.91  66.01  65.17  63.83  +  0.95  N %  54.13a  47.06bc  51.25ac  44.90bd  47.24bc  +  1.17  A.D.F. %  32.57a  36.97b  36.91b  36.45b  36.59b  + 0.89  Cellulose %  42.95a  49.10b  49.04b  49.33b  48.76b  + 1.33  *  Each v a l u e r e p r e s e n t s the mean o f f i v e  t  Sulphur was added as Na^SO^  Means on the same l i n e b e a r i n g d i f f e r e n t  determinations,  letters  (a, b, c, d) a r e s i g n i f i c a n t l y  different  (p < 0 . 0 5 ) .  - 64 -  Nitrogen  metabolism  S t a t i s t i c a l a n a l y s i s o f t h e n i t r o g e n metabolism study i n d i c a t e d a significant of m e t a b o l i c significantly  (p £ size.  0.05) p e r i o d e f f e c t f o r n i t r o g e n e x c r e t i o n p e r u n i t The animals used i n t h e second group e x c r e t e d a  (p<.0.05) g r e a t e r amount o f n i t r o g e n  u r i n e than animals used i n groups f o u r and f i v e .  (g/day/Wkg^"~^) 7  i n the  The v a l u e s were 0.595,  0.618, 0.543, 0.461 and 0.448 ( g / d a y / W k g ° ' ) f o r groups one, two, t h r e e , 75  f o u r and f i v e r e s p e c t i v e l y . The i n T a b l e 6.  parameters a s s o c i a t e d w i t h n i t r o g e n m e t a b o l i s m - a r e The d a i l y n i t r o g e n e x c r e t e d  body s i z e was s i g n i f i c a n t l y r e s t o f the d i e t s . was s i g n i f i c a n t l y  i n u r i n e per u n i t of metabolic  (p<0.05) h i g h e r f o r d i e t one than f o r t h e  U r i n a r y e x c r e t i o n o f n i t r o g e n as a percentage o f i n t a k e (p  0.05) g r e a t e r f o r d i e t one than f o r d i e t s two, t h r e e  and  f o u r but t h e r e was no s i g n i f i c a n t  one  and f i v e .  significantly  presented  (-p_> 0.05) d i f f e r e n c e between d i e t s  The p e r c e n t a g e o f d i g e s t e d n i t r o g e n e x c r e t e d (p < 0.05) h i g h e r  i n u r i n e was  f o r d i e t one than f o r t h e r e s t o f t h e d i e t s .  N i t r o g e n r e t a i n e d as a percentage o f i n t a k e was s i g n i f i c a n t l y i n c r e a s e d by formaldehyde treatment of t h e f o r a g e .  (p  0.05)  On d i e t t h r e e t h e  animals a l s o r e t a i n e d a s i g n i f i c a n t l y . ( p ^. 0.05) g r e a t e r amount o f n i t r o g e n as a percentage o f i n t a k e than t h e animals on d i e t  five.  N i t r o g e n r e t a i n e d as a percentage o f d i g e s t e d was  significantly  (p < 0.05) improved by formaldehyde treatment of t h e f o r a g e p o r t i o n o f the diets.  D a i l y n i t r o g e n balance  (g/day/Wkg^* ~*) was s i g n i f i c a n t l y 7  (p<.0.05)  improved f o r animals on d i e t s two and t h r e e compared t o animals on d i e t one.  Nitrogen balance  (g/day) was s i g n i f i c a n t l y  between animals on d i e t one and d i e t three.. not  -significantly  ;(p ^  0.05) a f f e c t e d by  (p <. 0.05) d i f f e r e n t  Nitrogen treatments.  intake  (g/day) was  Table  N i t r o g e n Metabolism.  6.  Diet containing u n t r e a t e d forage  Nitrogen intake  (g/day)  D a i l y N i n t a k e (g/Wkg^* ^) 7  D i e t s c o n t a i n i n g formaldehyde t r e a t e d f o r a g e + VFAS + SO f + VFAS + SO S.E.  1  2  3  4  5  29.81*  28.64  33.44  27.80  29.51  + 1.65  2.18  2.38  2.08  2.18  + 0.10  2.07  + 1.17  54.13a  47.06bc  51.25ac  44.90bd  47.24bc  D a i l y N excreted i n urine (g/Wkg )  0.805a  0.461b  0.467b  0.417b  0.518b  N e x c r e t e d i n u r i n e , .% of i n t a k e  39.07a  21.07b  19.97b  19.86b  23.94ab  + 3.69  N e x c r e t e d i n u r i n e , % of digested  72.10a  44.65b  39.11b  44.25b  50.39b  + 3.42  N retained» % of i n t a k e  15.06a  26.00bc  30.24bc  25.04bc  23.30bd  + 1.48  N r e t a i n e d , % of d i g e s t e d  27.90a  55.39b  58.93b  55.75b  49.61b  + 3.41  App.dig.coeff.  of N %  .0.03  0 , 0  Nitrogen balance  (g/day)  Nitrogen balance  (g/day/Wkg^*^)  4.49a 0.313a  7.43ab 0.567b  10.19b  6.99ab  6.86ab  + 0.86  0.726b  0.525ab  0.508ab  + 0.05  * Each v a l u e r e p r e s e n t s the mean o f f i v e d e t e r m i n a t i o n s , t Sulphur was added as Na^SO, Means on the same l i n e b e a r i n g d i f f e r e n t l e t t e r s (a, b, c, d) d i f f e r s i g n i f i c a n t l y  (p < 0.05).  - 66 -  Sulphur  metabolism  Thelparameters a s s o c i a t e d w i t h s u l p h u r metabolism  are presented  i n T a b l e 7. Animals on d i e t s f o u r and f i v e consumed s i g n i f i c a n t l y (p K. 0.05) g r e a t e r amounts of s u l p h u r p e r day than animals on d i e t s one and two but not more than animals on d i e t t h r e e .  Animals  f e d the s u l p h u r  supplemented d i e t s consumed s i g n i f i c a n t l y (p 4.0.05) a g r e a t e r amount of s u l p h u r p e r day p e r u n i t o f m e t a b o l i c body s i z e than animals on the other d i e t s . U r i n a r y e x c r e t i o n o f s u l p h u r as a percentage o f i n t a k e was significantly  (p < 0.05) g r e a t e r f o r animals on d i e t s f o u r and f i v e than  f o r animals on d i e t s two and t h r e e .  F a e c a l and u r i n a r y s u l p h u r l o s s e s  expressed as a percentage o f i n t a k e was s i g n i f i c a n t l y (p <C 0.05) g r e a t e r f o r animals on d i e t s one and f i v e than animals on d i e t s two and t h r e e . Animals on d i e t f o u r l o s t s i g n i f i c a n t l y (p < 0.05) a g r e a t e r percentage of consumed s u l p h u r i n u r i n e and f a e c e s than animals on d i e t two but not animals on d i e t t h r e e . excreted  The animals f e d the s u l p h u r supplemented  diets  a s i g n i f i c a n t l y (p^.0.05) g r e a t e r amount o f s u l p h u r i n the u r i n e  per day than the animals f e d the o t h e r d i e t s  (g/Wkg^*^^).  Animals on d i e t s two, t h r e e and f o u r r e t a i n e d s i g n i f i c a n t l y (p <. 0.05) a g r e a t e r amount o f s u l p h u r (g) per day than animals on d i e t one.  Sulphur b a l a n c e p e r day p e r u n i t o f m e t a b o l i c body s i z e was  significantly  (p 4^0.05) g r e a t e r f o r animals on d i e t s two, t h r e e and f o u r  than f o r animals on d i e t one.  Table  Sulphur Metabolism.  7.  Diet containing u n t r e a t e d forage  D i e t s c o n t a i n i n g formaldehyde t r e a t e d f o r a g e + VFAS + S0.+ + VFAS + SO. 4 ' 4 S.E.  D a i l y sulphur  i n t a k e (g)  D a i l y sulphur  intake '  2.26a*  2.20a  2.64ab  3.30b  3.39b  + 0.18  0.157a  0.168a  0.188a  0.247b  0.251b  + 0.01  31.38a  14.17b  17.41b  41.43a  42.23a  + 2.76  Sulphur e x c r e t e d i n u r i n e d a i l y (g/Wkg - )  0.050a  0.024a  0.034a  0.102b  0.105b  + 0.007  Sulphur b a l a n c e  (g/day)  0.459a  0.802b  0.843b  0.752b  0.672ab  + 0.064  Sulphur b a l a n c e (g/wkg - )  p e r day  0.032a  0.061b  0.061b  0.056b  0.050ab  + 0.005  10.36  + 1.26  80.28ad  +2.37  ( /wkg°- ) 7!)  g  Sulphur e x c r e t e d % of i n t a k e  0  0  i n urine,  75  75  Ratio of r e t a i n e d sulphur to r e t a i n e d ( n i t r o g e n (1:X)  10.13  Sulphur l o s t i n u r i n e and faeces>% of intake  79.67a  9.78  63.69b  12.26  67.57bc  9.73  76.89ac  * Each v a l u e r e p r e s e n t s t h e mean o f f i v e d e t e r m i n a t i o n s , t Sulphur was added as Na2S0, Means on t h e same l i n e b e a r i n g d i f f e r e n t l e t t e r s (a, b, c, d) d i f f e r s i g n i f i c a n t l y  (p <  0.05).  - 68 -  The r a t i o o f s u l p h u r r e t a i n e d t o n i t r o g e n r e t a i n e d was not significantly  ( p ^ 0 . 0 5 ) d i f f e r e n t f o r any o f the d i e t s .  Rumen parameters  (Data c o l l e c t e d from s l a u g h t e r e d  animals)  The rumen pH, rumen ammonia-nitrogen c o n c e n t r a t i o n matter content  (ppm), d r y  o f the rumen a t the time o f s l a u g h t e r o f the animals ( g ) ,  t o t a l v o l a t i l e fatty acid concentration  (u-mole/ml) and molar  proporations  of a c e t i c , p r o p i o n i c , b u t y r i c , i s o b u t y r i c , i s o v a l e r i c and v a l e r i c a r e presented  i n T a b l e 8.  There were no s i g n i f i c a n t  (p>  acids  0.05) d i f f e r e n c e s  between any o f the treatments w i t h r e s p e c t t o rumen pH, rumen d r y matter content  a t the time of s l a u g h t e r of the animals,  f a t t y acid concentrations.  and t o t a l  There were no s i g n i f i c a n t  (p >  volatile 0.05)  d i f f e r e n c e s between treatments w i t h r e s p e c t t o a c e t i c , p r o p i o n i c , and butyric acid proportions.  There were however s i g n i f i c a n t  (p <  0.05)  d i f f e r e n c e s between the animals used i n the second and f o u r t h groups w i t h r e s p e c t t o a c e t i c a c i d and p r o p i o n i c a c i d c o n c e n t r a t i o n . of a c e t i c a c i d proportions  f o r t h e f i v e groups ( b l o c k s ) of animals were  40.73%, 46.69%, 41.98%, 37.61%, and 42.81% f o r f i r s t , f o u r t h and f i f t h  The average v a l u e s  groups r e s p e c t i v e l y .  second,  third,  The average v a l u e s of p r o p i o n i c  a c i d p r o p o r t i o n s f o r the f i v e groups ( b l o c k s ) of animals were 31.24%, 22.66%, 26.49%, 35.26% and 29.35% f o r the f i r s t , and  fifth  second, t h i r d ,  fourth  groups r e s p e c t i v e l y .  The p r o p o r t i o n s of i s o v a l e r i c a c i d and i s o b u t y r i c a c i d were significantly three.  (p ^ 0.05) lower f o r d i e t s two and f o u r compared w i t h  The other treatments were n o t s i g n i f i c a n t l y  ( p > 0.05)  diet  different.  T a b l e 8.  Rumen parameters.  Diet containing u n t r e a t e d forage 1 Rumen pH  5.50*  Rumen NH^-N (ppm)  21.14a  D i e t s c o n t a i n i n g formaldehyde t r e a t e d f o r a g e + VFAS .. + SC^t + VFAS + S 0 2  3  4  4  S.E.  5  5.11  5.25  5.22  5.26  0.16  14.36b  14.30b  12.90b  13.54b  1.54  Dry m a t t e r i n rumen (g)  574.93  643.73  447.89  449.04  478.15  109.43  T o t a l VFA cone.  166.12  186.03  165.44  166.81  150.06  18.17  Acetic %  42.04  45.24  39.03  42.74  40.77  1.57  Propionic %  29.82  27.91  28.39  31.91  26.97  2.13  n-Butyric %  22.26  23.02  24.70  21.17  25.20  2.62  ( ^-mole/ml)  Molar p r o p o r t i o n s o f VFA:  Isobutyric %  1.54a  0.71ab  2.11ac  0.66a  1.73a  0.27  Isovaleric %  2.53a  0.60ab  2.58ac  0.39a  2.18a  0.44  n-Valeric %  1.82a  3.30b  3.19b  3.34b  3.15b  0.28  * Each v a l u e r e p r e s e n t s t h e mean o f f i v e d e t e r m i n a t i o n s . T Sulphur was added as Na^SO^ Means on t h e same l i n e b e a r i n g d i f f e r e n t l e t t e r s (a, b, c, d) d i f f e r s i g n i f i c a n t l y  (p ^ 0.05).  - 70 -  Formaldehyde treatment higher l e v e l s of n - v a l e r i c  Abomasal parameters  resulted i n significantly  (p ^ 0.05)  acid.  (Data c o l l e c t e d from s l a u g h t e r e d  animals)  i Abomasal pH, p e r c e n t n i t r o g e n , a c i d - d e t e r g e n t f i b r e ,  cellulose,  non-protein n i t r o g e n , r.ibonucleic-acid-nitrogen,and m i c r o b i a l - n i t r o g e n c o n t e n t s o f t h e abomasal d i g e s t a a r e p r e s e n t e d  i n T a b l e 9.  % RNA-N: % t o t a l abomasal d i g e s t a - N and % m i c r o b i a l - N :  The r a t i o s o f  % t o t a l abomasal  d i g e s t a - N a r e a l s o shown i n T a b l e 9. Formaldehyde reduced  treatment  of the forage s i g n i f i c a n t l y  t h e n o n - p r o t e i n - n i t r o g e n o f the abomsasl d i g e s t a .  (p<0.05) The r a t i o s o f  % RNA-N: % t o t a l abomasal d i g e s t a N and % m i c r o b i a l - N : % t o t a l abomasal d i g e s t a N were g r e a t e r (p<0.05) f o r d i g e s t a from animals than f o r d i g e s t a from animals d i g e s t a from animals were n o t s i g n f i c a n t l y  Chemical  composition  The  f e d d i e t one  f e d d i e t s two, f o u r and f i v e b u t n o t f o r  fed d i e t three.  A l l t h e o t h e r parameters measured  ( p > 0.05) a f f e c t e d by d i e t .  o f d i e t s used f o r duodenal f l o w r a t e measurements  c h e m i c a l composition  o f t h e b a t c h o f d i e t s used f o r duodenal i,  f l o w r a t e measurements i s g i v e n i n T a b l e 10.  T a b l e 9.  Abomasal parameters.  Diet containing u n t r e a t e d forage  D i e t s c o n t a i n i n g formaldehyde t r e a t e d forage + VFAS + SO.t + VFAS + SO. 4 4 S.E.  Abomasal pH  3.84*  3.94  3.88  3.83  3.70  0.22  2.27  3.01  2.80  3.16  3.06  0.23  Abomasal d i g e s t a (D.M. b a s i s ) :  N%  * t  ADF' %  17.31  18.28  17.40  17.77  17.29  0.40  Cellulose %  12.27  13.47  12.84  12.96  12.73  0.35  NPN %  0.864a  0.676b  0.643b  0.650b  0.656b  0.043  RNA-N %  0.132  0.108  0.120  0.102  0.101  0.012  % RNA-N: % T o t a l N r a t i o ( x : l )  0.059a  0.036b  0.044ab  0.034b  0.033b  0.005  % Microbial N  1.76  1.44  1.60  1.36  1.35  0.16  % M i c r o b i a l N: % T o t a l N ratio (x:l)  0.786a  0.480b  0.583ab  0.451b  0.439b  0.068  Each v a l u e r e p r e s e n t s t h e mean o f f i v e Sulphur was added as Na2S0^  Means on the same l i n e b e a r i n g d i f f e r e n t  determinations,  letters  (a, b) d i f f e r s i g n i f i c a n t l y  (p < 0.05),  T a b l e 10.  Chemical  c o m p o s i t i o n o f d i e t s used  Diet containing Untreated forage Chemical  D.M.  (D.M. b a s i s )  ADF %  (D.M. b a s i s )  Cellulose % Ash %  ^  D i e t s c o n t a i n i n g formaldehyde t r e a t e d f o r a g e + VFAS + SO.f + VFAS + SO,  Fraction  %  ,N %  f o r duodenal f l o w r a t e measurements.  (D.M. b a s i s )  (D.M. b a s i s )  Sulphur was added as Na^SO^  86.71  86.95  86.95  86.95  86.95  2.21  2.24  2.24  2.20  2.20  16.80  17.10  17.10  16.92  16.92  12.64  13.01  13.01  13.01  13.01  8.12  8.16  8.16  8.20  8.20  - 73 -  Feed i n t a k e d u r i n g t h e duodenal f l o w measurements  The d a i l y  t o t a l f e e d i n t a k e , the f e e d i n t a k e d u r i n g  first,  second, t h i r d and f o u r t h s i x - h o u r p e r i o d s a r e a l s o g i v e n i n T a b l e 11. Feed i n t a k e d u r i n g t h e f i r s t ,  second, t h i r d and f o u r t h s i x - h o u r  periods  as a percentage o f t o t a l f e e d i n t a k e i s a l s o g i v e n i n T a b l e 11. mean f e e d i n t a k e as a percentage o f t o t a l f o r a l l o f t h e d i e t s the f i r s t ,  The  during  second, t h i r d and f o u r t h s i x - h o u r p e r i o d s and f i r s t and  second twelve-hour p e r i o d s were 40.72%, 17.01%, 22.52%, 20.20%, 57.28% and 42.72% r e s p e c t i v e l y .  Duodenal f l o w parameters  The d a i l y  digesta flow  (ml) d u r i n g t h e f i r s t , first of  (ml and g ) , the average h o u r l y d i g e s t a flow  second, t h i r d and f o u r t h s i x - h o u r p e r i o d s and  and second twelve-hour p e r i o d s a r e g i v e n i n T a b l e 12.  total daily  flow during the f i r s t ,  p e r i o d s and a l s o d u r i n g the f i r s t  The percentage  second, t h i r d and f o u r t h s i x - h o u r  and second twelve-hour p e r i o d s a r e  a l s o shown i n T a b l e 12. The mean f l o w as a p e r c e n t a g e o f t o t a l f o r a l l o f the d i e t s the f i r s t ,  second, t h i r d and f o u r t h s i x - h o u r p e r i o d s and f i r s t  during  and second  twelve-hour p e r i o d s were: 25.20%, 24.96%, 24.37%, 24.35%, 50.13% and 49.87% r e s p e c t i v e l y .  T a b l e 11.  Feed i n t a k e d u r i n g the duodenal f l o w measurements:  Diet containing Untreated forage  Feed Intake  D i e t s c o n t a i n i n g formaldehyde t r e a t e d f o r a g e + VFAS + S0,t + VFAS + SO, 4 4  ( g ) : (D.M. b a s i s )  Daily total  1300.65  1304.25  1304.25  1304.25  1304.25  Intake 1 s t 6 h r s  515.06  429.53  436.49  646.91  596.48  Intake 2nd 6 h r s  86.71  198.25  365.19  173.90  285.20  Intake 3 r d 6 h r s  477.77  285.20  189.55  309.54  206.20  Intake 4 t h 6 h r s  221.11  391.28  313.02  173.96  217.38  % Intake o f t o t a l :  ^  1st  6 hrs  39.60  32.93  33.47  49.60  45.73  2nd  6 hrs  6.67  15.20  28.00  13.33  21.87  3rd  6 hrs  36.73  21.87  14.53  23.73  15.73  4th 6 h r s  17.00  30.00  24.00  13.33  16.67  Sulphur was added as Na2S0^  T a b l e 12.  Duodenal f l o w parameters. Diet containing u n t r e a t e d forage  Duodenal d i g e s t a f l o w  D i e t s c o n t a i n i n g formaldehyde t r e a t e d f o r a g e + VFAS + SO.t + VFAS + SO. 4 4  1  2  3  4  5  Total  (ml)  15533  16760  17019  16615  16340  Total  (g)  15576  16790  17053  16720  16338  647.2  698.3  709.1  692.3  680.8  1st 6 hrs (ml)  3150  4075  5104  4225  4245  2nd 6 hrs (ml)  4235  3910  4630  4000  3745  3rd 6 hrs (ml)  4090  4810  3630  4370  4075  4th 6 hrs (ml)  4078  3965  3655  4020  4275  1st 12 h r s (ml)  7385  7985  9734  8225  7990  2nd 12 h r s (ml)  8168  8775  7285  8390  8350  1st 6 h r s  20.28  24.31  29.99  25.43  25.98  2nd 6 h r s  27.26  23.33  27.20  24.07  22.92  3rd 6 h r s  26.33  28.70  21.33  26.30  24.94  4 th 6 h r s  26.25  23.66  21.48  24.20  26.16  1st 12 h r s  47.54  47.64  57.19  49.50  48.78  2nd 12 h r s  52.58  52.36  42.81  50.50  51.10  Hourly  (ml)  Flow % of  total:  Sulphur was added as Na„S0  - 76 -  Some chemical f r a c t i o n s o f duodenal d i g e s t a , t o t a l d a i l y i n t a k e and d a i l y d i g e s t a f l o w through t h e duodenum o f these f r a c t i o n s  The d a i l y d r y m a t t e r , o r g a n i c matter, a c i d - d e t e r g e n t - f i b r e and c e l l u l o s e intakes  (g) a r e g i v e n i n T a b l e  13.  The dry-matter,  m a t t e r , a c i d - d e t e r g e n t - f i b r e and c e l l u l o s e c o m p o s i t i o n d i g e s t a i s shown i n T a b l e  13.  organic  (%) o f t h e duodenal  The t o t a l d a i l y flow through t h e duodenum  (g) o f d r y matter, o r g a n i c m a t t e r , a c i d - d e t e r g e n t  f i b r e and c e l l u l o s e  a r e a l s o shown i n T a b l e 13.  D a i l y n i t r o g e n i n t a k e , n i t r o g e n components o f duodenal d i g e s t a and the d a i l y f l o w o f these components through t h e duodenum  The t o t a l d a i l y n i t r o g e n i n t a k e i n T a b l e 14.  The n i t r o g e n , n o n - p r o t e i n - n i t r o g e n ,  and m i c r o b i a l - n i t r o g e n c o m p o s i t i o n T a b l e 14.  T a b l e 14 a l s o c o n t a i n s  % total N ratios,  ribonucleic acid-nitrogen  o f t h e duodenal d i g e s t a i s shown i n t h e % RNA-N: % t o t a l N and % m i c r o b i a l - N :  t o t a l d a i l y RNA-N: t o t a l d a i l y N and t o t a l  m i c r o b i a l N: t o t a l d a i l y N r a t i o s . nitrogen,  (g) on each o f t h e d i e t s i s shown  The t o t a l d a i l y n i t r o g e n ,  daily non-protein-  t r u e p r o t e i n n i t r o g e n , r i b o n u c l e i c - a c i d - n i t r o g e n and m i c r o b i a l  n i t r o g e n f l o w i n g through t h e duodenum a r e a l s o g i v e n i n T a b l e 14.  Apparent d i g e s t i b i l i t y c o e f f i c i e n t s o f some chemical f r a c t i o n s i n t h e pre-duodenum p o r t i o n o f d i g e s t i v e t r a c t ( a l l compartments o f stomach) and t h e change i n q u a n t i t y o f n i t r o g e n e n t e r i n g t h e duodenum d a i l y compared w i t h i n t a k e  The apparent d i g e s t i b i l i t y c o e f f i c i e n t s the d i g e s t i v e t r a c t  (%) i n thepreduodenum of  ( a l l compartments o f stomach) o f d r y m a t t e r ,  matter, a c i d - d e t e r g e n t - f i b r e and c e l l u l o s e a r e shown i n T a b l e  organic  15. The  Some c h e m i c a l f r a c t i o n s of duodenal d i g e s t a , t o t a l d a i l y i n t a k e and d a i l y d i g e s t a flow through t h e duodenum of these f r a c t i o n s .  T a b l e 13.  Diet containing u n t r e a t e d forage 1 Total daily  Diets containing + VFAS 2  3  formaldehyde t r e a t e d forage + S0 + + VFAS + S0 4  4  4  5  intake(g):  D.M.  1300.65  1304.25  1304.25  1304.25  1304.25  O.M.  1195.04  1197.82  1197.82  1197.30  1197.30  A.D.F.  218.51  223.03  223.03  220.68  220.68  Cellulose  164.40  169.68  169.68  169.68  169.68  5.68  5.97  5.96  5.95  5.99  86.16  87.24  86.98  88.44  87.96  17.10  18.25  17.65  18.10  17.80  12.03  13.10  12.80  13.12  12.95  D.M.  883.41  1000.57  1014.33  988.59  978.77  O.M.  761.15  872.90  882.27  874.31  860.92  A.D.F.  151.06  182.60  179.03  178.94  174.22  Cellulose  106.27  131.07  129.83  129.70  126.75  Duodenal d i g e s t a : (Chemical f r a c t i o n ) D.M.  %  O.M.  %  (D.M.  A.D.F.%  (D.M.  basis) basis)  C e l l u l o s e % (D.M.  basis)  T o t a l D a i l y Flow ( g ) :  t  Sulphur was added as Na SO, 2 4  Table  14.  D a i l y n i t r o g e n i n t a k e , n i t r o g e n components of duodenal d i g e s t a and the d a i l y flow o f t h e s e components through the duodenum. Diet containing u n t r e a t e d forage  D i e t s c o n t a i n i n g formaldehyde t r e a t e d forage + VFAS + SO.t + VFAS + SO, _ 4 4  28.74  29.22  29.22  28.69  28.69  N %  2.40  3.10  2.96  3.15  2.98  NPN %  0.842  0.614  0.701  0.662  0.681  RNA-N %  0.145  0.111  0.122  0.094  0.093  1.93  1.48  1.63  1.25  1.24  % RNA-N:% T o t a l N r a t i o  0.060  0.036  0.041  0.030  0.031  % Microbial-N:% Total N  0.804  0.477  0.551  0.397  0.416  Total daily nitrogen intake  (g)  Duodenal d i g e s t a : ( D . M . b a s i s )  Microbial-N  %  T o t a l d a i l y flow (g): N NPN True p r o t e i n N RNA-N Microbial-N  29.16  21.20  31.02  30.02  31.14  7.44  6.10  7.10  6.52  6.66  13.76  24.92  22.92  24.62  22.50  1.28  1.11  1.24  17.05  14.81  16.53  0.929 12.36  0.910 12.14  T o t a l d a i l y RNA-N: T o t a l d a i l y N  0.060  0.036  0.041  0.030  0.031  Total daily Microbial-N: Total dialy N  0.804  0.477  0.551  0.397  0.416  Sulphur was added as Na2S0^  Table  15.  Apparent d i g e s t i b i l i t y c o e f f i c i e n t o f some chemical f r a c t i o n s i n the pre-duodenum p o r t i o n o f d i g e s t i v e t r a c t ( a l l compartments of stomach) and t h e change i n q u a n t i t y o f n i t r o g e n e n t e r i n g the duodenum d a i l y compared w i t h i n t a k e .  Diet containing untreated forage  D i e t s c o n t a i n i n g formaldehyde t r e a t e d + VFAS + SO^t + VFAS +  1  2  3  4  5  D.M.  32.08  23.28  22.23  24.20  24.96  O.M.  36.31  27.12  26.34  26.98  28.09  A.D.F.  30.87  18.12  19.72  18.92  21.05  Cellulose  35.35  22.75  23.48  23.56  25.30  -7.54  +1.88  +0.80  +2.45  +0.47  % App. D i g . C o e f f . o f :  Change i n q u a n t i t y o f N e n t e r i n g duodenum d a i l y compared w i t h i n t a k e (g)  t  Sulphur was added as Na S0 o  forage S0 4  - 80 -  change i n q u a n t i t y of n i t r o g e n e n t e r i n g duodenum d a i l y with  intake i s also given i n Table  15.  compared  - 81  -  DISCUSSION  In v i t r o d i g e s t i b i l i t y t r i a l s  Formaldehyde treatment up  to a l e v e l of 1.0%  n i t r o g e n d i g e s t i b i l i t y d u r i n g the m i c r o b i a l stage i n t h i s experiment 1.2%  (31.88%, 15.72%, 6.87%  in vitro  (Table 2).  The  values  and 5.69% "for 0.0%,0.8%,1.0% and  l e v e l of formaldehyde treatment r e s p e c t i v e l y ) were lower than  v a l u e s r e p o r t e d by B a r r y study by B a r r y  (1973a) and  about 42%  Barry  (1976c).  In the  f o r the u n t r e a t e d ,  l e s s than 30%  r e p o r t e d i n the l a t e r experiment were 59.5% the t r e a t e d grass-legume hay  and  the  f o r the t r e a t e d  u s i n g grass-legume hay. 39.9%  The  the  earlier  (1973a), he r e p o r t e d n i t r o g e n d i g e s t i b i l i t y at  m i c r o b i a l stage of d i g e s t i o n to be  and  decreased  and  f i g u r e s he  f o r the  r e s p e c t i v e l y (Barry, 1976c).  untreated There  a r e a number o f f a c t o r s which p o s s i b l y c o n t r i b u t e d to the r a t h e r lower f i g u r e s obtained 1976c). study  One  in this t r i a l  p o s s i b l e reason  compared to those of B a r r y  f o r the low  f i g u r e s obtained  (1973a  and  i n the  present  i s t h a t the heat a p p l i e d d u r i n g the d r y i n g o f the forage  s o l u b i l i t y of p r o t e i n .  However, i t was  u n l i k e l y t h a t the heat a p p l i e d  caused g r e a t heat-damage to the f o r a g e as the a c i d - d e t e r g e n t i n s o l u b l e n i t r o g e n l e v e l was  reduced  fibre  l e s s than 7% of t o t a l n i t r o g e n , which  the maximum l e v e l r e p o r t e d by Yu and Thomas (1976) f o r undamaged I f heat-damage had stage of i n c u b a t i o n  occurred,  d i g e s t i b i l i t y of n i t r o g e n at the  (combined m i c r o b i a l and  have been reduced, but t h i s d i d not happen.  was  forages.  second  a c i d - p e p s i n ) would a l s o In f a c t ,  the d i g e s t i b i l i t y  of n i t r o g e n at the second stage o f i n c u b a t i o n r e p o r t e d l a t e r on i n t h i s  - 82  -  d i s c u s s i o n compared f a v o u r a b l y w i t h those H i s f i g u r e s ranged from 77.2%  to 82.5%  r e p o r t e d by B a r r y  f o r the t r e a t e d m a t e r i a l  the f i g u r e f o r u n t r e a t e d was  84.8%.  lower f i g u r e s i n the present  t r i a l f o r the f i r s t  compared to those of B a r r y  (1976c).  Another p o s s i b l e reason  (1973a and  stage  1976c) was  f o r a g e might have p r o t e c t e d the p r o t e i n s .  f o r the  incubation  t h a t t a n n i n s i n the  I t was  not l i k e l y t h a t  t a n n i n s i n the f o r a g e g r e a t l y p r o t e c t e d the p r o t e i n s as, i f occurred,  d i g e s t i b i l i t y of n i t r o g e n a t the second stage of  would have been reduced g r e a t l y . and McLeod  T h i s i s because Ferguson  broken i n a c i d i c  medium.  The  hydrolyzable  incubation (1975)  the bonds a r e  stage  n i t r o g e n d i g e s t i b i l i t y compared w i t h f i g u r e s of B a r r y i s t h a t a g r e a t amount of s o l u b l e n i t r o g e n was thus was  r e p o r t e d t h a t the extent  not measured.  of accumulation  the  o n l y i n seeds.  f o r the lower f i g u r e s of f i r s t  p r o t e i n - n i t r o g e n and  A  i n vitro  fast  and  present  i n great amounts.  The  the f o r a g e was  converted  1976c)  to m i c r o b i a l  Annison j i t _ a l . (1954)  of degraded p r o d u c t s  of aminocarbohydrates  rumen m i c r o - o r g a n i s m s grew v e r y  the r e a d i l y fermentable  not measured, i t was  third  (1973a and  u t i l i z e d n i t r o g e n from the breakdown of p r o t e i n s . t r i a l , although  not  incubation  a c i d s i n the rumen c o u l d be reduced when r e a d i l y fermentable were p r e s e n t  had  types of t a n n i n s , which form  r e v e r s i b l e bonding w i t h p r o t e i n s a r e p r e s e n t p o s s i b l e reason  the  that  (1974) r e p o r t e d t h a t f o r a g e t a n n i n s which a r e of  condensed type, p r o t e c t p r o t e i n s permanently and  and  carbohydrate  In  the  level  most l i k e l y h i g h as the f o r a g e  immature, as i n d i c a t e d by the h i g h n i t r o g e n  level.  of was  - 83 -  There was  a compensatory  s t e p w i s e i n c r e a s e i n the d i g e s t i b i l i t y  of n i t r o g e n a t the second s t a g e o f i n c u b a t i o n w i t h the formaldehyde t r e a t e d samples up t o 1% l e v e l  o f treatment.  f i g u r e s f o r t h e second s t a g e i n v i t r o  trial  The n i t r o g e n  digestibility  (combined m i c r o b i a l and  a c i d - p e p s i n ) were 80.95%, 79.76%, 75.85%, and 71.01% f o r 0.0%,  0.8%,  1.0%,  The  and 1.2%  l e v e l s of formaldehyde treatment r e s p e c t i v e l y .  d i f f e r e n c e s between the o v e r a l l and the f i r s t  (combined m i c r o b i a l and a c i d - p e p s i n )  stage i n c u b a t i o n n i t r o g e n d i g e s t i b i l i t y f i g u r e s were  49.07, 64.04, 68.98, and 65.32 percentage u n i t s f o r 0.0%, and 1.2%  l e v e l s of formaldehyde treatment r e s p e c t i v e l y .  a l s o r e p o r t e d a compensatory  0.8%, Barry  The  i n c r e a s e i n n i t r o g e n d i g e s t i b i l i t y f o r the t r e a t e d samples o c c u r r e d because a l l the d i g e s t i b l e n i t r o g e n which was  the  the f i r s t  1.0%, (1976c)  increase i n n i t r o g e n d i g e s t i b i l i t y at the  second stage of i n c u b a t i o n w i t h formaldehyde treatment.  at  -  s t a g e of i n c u b a t i o n , was  compensatory  perhaps  not i n s o l u t i o n  d i g e s t e d by the a c i d - p e p s i n  after  a c i d i t y broke down the bonds between the formaldehyde and the p r o t e i n  molecules. Ammonia-nitrogen  p r o d u c t i o n was  significantly  l e v e l s of formaldehyde treatment up t o the l e v e l d i g e s t i b i l i t y decreased o n l y up t o the 1.0%  reduced w i t h i n c r e a s i n g  of 1.2% whereas n i t r o g e n  level.  The  ammonia-nitrogen  l e v e l s per gram o f d r y matter i n c u b a t e d , as shown i n T a b l e 2, were 228.79 ppm, and 1.2% the  78.58 ppm,  65.27 ppm,  and 30.04 ppm  l e v e l o f formaldehyde treatment r e s p e c t i v e l y .  f u r t h e r decrease i n ammonia-nitrogen  level  f o r 0.0%,  1.0%  The r e a s o n f o r  p r o d u c t i o n beyond  of formaldehyde treatment i s n o t c l e a r .  0.8%,  the  1.0%  Nitrogen d i g e s t i b i l i t y  - 84 -  f i g u r e s ' ( a t the f i r s t  s t a g e o f i n c u b a t i o n ) o b t a i n e d from c a l c u l a t i o n s  based on the means o f the ammonia-nitrogen 8.63%  and 3.97%  f o r 0.0%,  treatment r e s p e c t i v e l y . the  1.0%  level  0.8%,  1.0%  and 1.2%  l e v e l s o f formaldehyde  These f i g u r e s were s l i g h t l y  lower, except f o r  formaldehyde treatment, than those o b t a i n e d from the  a c t u a l measurements o f d i g e s t i b i l i t y . i n the f i g u r e f o r the 1.0% to  f i g u r e s were 30.26%, 10.39%,  level  The r e a s o n f o r t h e s l i g h t  of formaldehyde a p p l i c a t i o n compared  the a c t u a l measured v a l u e i s not o b v i o u s .  B a r r y (1976c) a l s o  lower f i g u r e s o f n i t r o g e n d i g e s t i b i l i t y a t the f i r s t u s i n g ammonia-nitrogen  figures  increase  reported  stage of i n c u b a t i o n  (28% and 17% f o r u n t r e a t e d and  formaldehyde  t r e a t e d hay r e s p e c t i v e l y ) compared w i t h f i g u r e s o b t a i n e d from a c t u a l measurements (59.5% and 39.9% respectively).  f o r u n t r e a t e d and formaldehyde t r e a t e d  hay  Sharkey e t a l . (1972) r e p o r t e d t h a t t h e n i t r o g e n  d i g e s t i b i l i t y f i g u r e s o b t a i n e d from a c t u a l measurements i n c l u d e s a l l s o l u b l e n i t r o g e n compounds r e l e a s e d d u r i n g f e r m e n t a t i o n w h i l e ammonian i t r o g e n takes i n t o account o n l y n i t r o g e n fermented t o ammonia.  I t should  a l s o be noted t h a t d i p e p t i d e s , amino a c i d s and n u c l e i c a c i d s c o u l d  be  u t i l i z e d by rumen microbes w i t h o u t f i r s t b e i n g broken down t o ammonia (Nolan, 1975).  These compounds would not be measured i f they a r e i n  s o l u t i o n when o n l y ammonia-nitrogen  level  i s measured and t h e r e f o r e the  a c t u a l l e v e l o f u t i l i z a t i o n of p r o t e i n i n the feed by microbes would not be c o r r e c t l y a s s e s s e d by measuring o n l y the ammonia-nitrogen It  seems t h e r e f o r e t h a t ammonia-nitrogen  i n d i c a t o r o f the optimum l e v e l  p r o d u c t i o n may  level.  not be the b e s t  of formaldehyde treatment.  It also  gives  no i n d i c a t i o n o f t h e e f f e c t o f formaldehyde treatment on the d i g e s t i o n  - 85 -  of n i t r o g e n by enzymes i n t h e i n t e s t i n e .  Barry  (1976a) r e p o r t e d  t h a t n i t r o g e n d i g e s t i b i l i t y measurement u s i n g both m i c r o b i a l and acid-pepsin  stages  determining  optimum l e v e l s of formaldehyde treatment than ammonia-  nitrogen  o f i n c u b a t i o n was a more r e l i a b l e method o f  levels.  Feed Intake  The d a i l y d r y matter i n t a k e p e r u n i t o f m e t a b o l i c during  bodysize  both  t h e pre-metabolism and metabolism study p e r i o d s was n o t a f f e c t e d  significantly  (p ^-0.05) by treatments  (Table 4 ) .  The v a l u e s  f o r the  pre-metabolism assay p e r i o d were 91.33, 100.62, 101.93, 98.02 and 96.47 g/ Wkg^" ^ p e r day f o r d i e t s one, two, t h r e e , f o u r and f i v e r e s p e c t i v e l y ; 7  the v a l u e s  f o r the metabolism assay p e r i o d were 90.17, 96.65, 104.29,  90.88 and 94.25 g/Wkg five respectively.  d a i l y , f or d i e t s one, two, t h r e e , f o u r and  Feed i n t a k e i s an important f a c t or a f f e c t i n g t h e  n u t r i t i v e v a l u e o f d i e t s o f ruminants  (Crampton and H a r r i s , 1969).  Any  d i f f e r e n c e s i n t h e performances o f t h e animals on t h e d i f f e r e n t d i e t s i n t h i s experiment c o u l d t h e r e f o r e n o t be a t t r i b u t e d t o v a r i a t i o n s i n feed i n t a k e .  Kempton e t a l . (1977) and Ferguson (1975) r e p o r t e d  that  response t o formaldehyde treatment o f d i e t s was mediated m a i n l y through increased  feed i n t a k e compared t o u n t r e a t e d  (1973) r e p o r t e d decreased feed i n t a k e w i t h barley rations.  diets.  D a v i e s and F a i c h n e y  formaldehyde treatment o f  I t i s noteworthy t h a t t h e a d d i t i o n o f t h e pure v o l a t i l e  f a t t y a c i d s t o some o f t h e d i e t s c o n t a i n i n g t h e formaldehyde t r e a t e d forage  ( d i e t s t h r e e and f i v e ) d i d n o t depress feed i n t a k e .  postulated  Hume (1970)  t h a t t h e a d d i t i o n o f the pure v o l a t i l e f a t t y a c i d s t o d i e t s  - 86 -  c o u l d decrease p a l a t i b i l i t y and t h e r e f o r e i n h i s s t u d i e s sodium of these a c i d s were used. the h y p o t h e s i s  salts  However, Hume (1970) d i d n o t i n d i c a t e t h a t  t h a t t h e pure v o l a t i l e f a t t y a c i d s reduced  palatibility  was t e s t e d . Animals were f e d ad l i b i t u m i n t h i s experiment Hemsley and M o i r  (1963) and A d e l e y e (1972).  f e e d i n t a k e d u r i n g metabolism s t u d i e s 1973;  and Amos e t a l . , 1976b).  as, was done by  Some workers have r e s t r i c t e d  (Barry, 1973b, B a r r y and Andrews,  The argument f o r r e s t r i c t i n g feed  intake  i s t o reduce v a r i a b i l i t y i n i n t a k e o f d r y matter and n i t r o g e n , e s p e c i a l l y n i t r o g e n , which c o u l d a f f e c t n i t r o g e n b a l a n c e . these animals ad l i b i t u m i n t h e p r e s e n t f e e d i n g was c a r r i e d out,  One r e a s o n f o r f e e d i n g  t r i a l was t h a t i f r e s t r i c t e d  r e s u l t s from such experiments c o u l d n o t be  applied, without l i m i t a t i o n s ,  t o p r a c t i c a l farm c o n d i t i o n s where animals  are f e d ad l i b i t u m .  N i m r i c k e t a l . (1972) noted t h a t when animals  are f e d ad l i b i t u m ,  performance may n o t be t h e same as when r e s t r i c t i o n  of feed i n t a k e i s p r a c t i s e d .  With ad l i b i t u m f e e d i n g low rumen pH o c c u r s ,  e s p e c i a l l y when h i g h l e v e l s o f r e a d i l y f e r m e n t a b l e c a r b o h y d r a t e a r e f e d . P r o t e i n s o l u b i l i t y i s reduced and i t s u t i l i z a t i o n i s improved when ad l i b i t u m f e e d i n g i s c a r r i e d out compared t o r e s t r i c t e d f e e d i n g , when p r o t e i n breakdown i n the rumen may be g r e a t utilization.  thus r e d u c i n g i t s  E a d i e and Mann (1970) observed t h a t t h e types o f b a c t e r i a  i n t h e rumen may a l s o be d i f f e r e n t when h i g h c a r b o h y d r a t e d i e t s a r e f e d ad l i b i t u m as compared t o r e s t r i c t e d f e e d i n g .  Amos et_ a l . (1976b)  r e s t r i c t e d feed i n t a k e and observed no d i f f e r e n c e i n n i t r o g e n - b a l a n c e between formaldehyde t r e a t e d and u n t r e a t e d  c o a s t a l bermudagrass hay.  - 87  When feed i n t a k e was  increased  -  from 600g dry matter to 775g dry  m a t t e r , the e s s e n t i a l and n o n - e s s e n t i a l duodenum were i n c r e a s e d  amino a c i d s a r r i v i n g at  the  s i g n i f i c a n t l y f o r the formaldehyde t r e a t e d  material. Nitrogen 2.18,  2.38,  i n t a k e per day  2.08  and  2.18  r e s p e c t i v e l y , Tables  per u n i t of m e t a b o l i c  g f o r d i e t s one,  4 and  6) was  two,  body s i z e  (2.07,  t h r e e , f o u r and  a l s o not a f f e c t e d by  five  treatment.  T h i s i s u n d e r s t a n d a b l e s i n c e n i t r o g e n l e v e l s i n the d i e t s (Table were almost i d e n t i c a l one,  two,  discussed  (2.29%, 2.24%, 2.25%, 2.28%  t h r e e , f o u r and above, was  f i v e r e s p e c t i v e l y ) and  not a f f e c t e d .  and  2.29%  3),  for diets  d r y matter i n t a k e ,  With n i t r o g e n i n t a k e b e i n g  i d e n t i c a l on a l l d i e t s , d i e t a r y sources of n i t r o g e n ,  almost  for microbial  a c t i v i t i e s i n the rumen, c o u l d be  l i m i t e d f o r the d i e t s c o n t a i n i n g  formaldehyde t r e a t e d f o r a g e .  non-protein-nitrogen  legume f o r a g e and  The  the n i t r o g e n from the b a r l e y and  about 0.681g f o r e v e r y lOOg of the r a t i o n s .  from the  cassava  was  the  grass-  totalled  There c o u l d be a supply  n i t r o g e n to the rumen micro-organisms w i t h the d i e t s c o n t a i n i n g formaldehyde t r e a t e d f o r a g e  as  through r e c y c l i n g .  I t seemed t h a t  of  the nitrogen  l i m i t i n g i n the rumens of the animals f e d the d i e t s c o n t a i n i n g  formaldehyde f o r a g e content later).  as rumen ammonia-nitrogen, and m i c r o b i a l p r o t e i n  of abomasal d i g e s t a were reduced The  concentration  (these r e s u l t s are  of v a l e r i c a c i d i n the rumens of animals f e d  the d i e t s c o n t a i n i n g the formaldehyde t r e a t e d f o r a g e was t o the c o n t r o l .  discussed  lower compared  T h i s i n d i c a t e d a l i m i t a t i o n to rumen m i c r o b i a l growth  most l i k e l y by n i t r o g e n .  I t i s t h e r e f o r e s u r p r i s i n g t h a t feed i n t a k e  was  - 88  not depressed f o r the d i e t s  -  c o n t a i n i n g the formaldehyde t r e a t e d  f o r a g e as a r e s u l t of the l i m i t a t i o n of a v a i l a b l e  nitrogen for  m i c r o b i a l growth.  Apparent d i g e s t i b i l i t y c o e f f i c i e n t s of n i t r o g e n , a c i d - d e t e r g e n t c e l l u l o s e , dry m a t t e r and o r g a n i c matter  Apparent d i g e s t i b i l i t y c o e f f i c i e n t s  fibre,  of n i t r o g e n  The v a l u e s of apparent d i g e s t i b i l i t y c o e f f i c i e n t s of n i t r o g e n obtained  i n t h i s experiment were g e n e r a l l y lower than the v a l u e s  by Beever et: a l . (1976). 51.25, 44.90 and  The  values  47.24% f o r d i e t s  respectively,  while figures  for untreated  and  61.8%  Barry  (1973a).  f o r t h i s experiment were 54.13, 47.06,  one,  two,  f o r treated rye grass.  and  The  values obtained  of 63.1,  41.1  and  51.1%  f o r 0.0,  2.4  29.8%  Barry  and  4.8%  f o r 0.0,  r a t h e r low v a l u e o b t a i n e d  f o r the u n t r e a t e d  experiment compared w i t h the r e s u l t s  1.0  (1973a) r e p o r t e d  in this  and  values  diet  of hay  of  making.  ( d i e t one)  i n this  of Beever at a l . (1976) and  at a l . (1975) c o u l d p o s s i b l y be a t t r i b u t e d  2.0%  l e v e l s of a p p l i c a t i o n  formaldehyde t o r y e - g r a s s - c l o v e r d u r i n g the p r o c e s s The  69.8%  t o those r e p o r t e d by D i n i u s at a l . (1975)  formaldehyde treatment of a l f a l f a meal. 50.6,  five  D i n i u s e_t a l . (1975) r e p o r t e d apparent n i t r o g e n  digestibility coefficients  of 55.2,  t h r e e , f o u r and  r e p o r t e d by Beever et a l . (1976) were  experiment were however s i m i l a r and  reported  Dinius  to a number of f a c t o r s  reducing  d i g e s t i o n i n the rumen: (a)  p e l l e t i n g of the r a t i o n .  P e l l e t i n g of r a t i o n s has  r e p o r t e d to i n c r e a s e p r o t e c t i o n of p r o t e i n s as a r e s u l t of heat d u r i n g the p e l l e t i n g p r o c e s s  (Kempton e t a l . , 1977).  been generated  - 89 -  (b)  High l e v e l s of s o l u b l e c a r b o h y d r a t e s .  b a r l e y c o n s t i t u t e d about 50%  of the r a t i o n .  c a r b o h y d r a t e i n d i e t s have been r e p o r t e d  Cassava  High l e v e l s of  soluble  to decrease rumen pH  thus reduce s o l u b i l i t y of p r o t e i n i n the rumen f l u i d Kempton et a l . , 1977).  and  and  (0rskov,  1975;  However, T a g a r i et a l . (1964) r e p o r t e d  i n c r e a s e d n i t r o g e n d i g e s t i b i l i t y i n the rumen w i t h the a d d i t i o n of s o l u b l e c a r b o h y d r a t e s to hay  diets.  I t i s i n t e r e s t i n g to note t h a t  the most p r o t e o l y t i c microbes i n the rumen are the a m y l o l y t i c ones (Hungate, 1967;  Church, 1975b).  I t i s therefore s u r p r i s i n g that  lowered d i g e s t i o n of n i t r o g e n occurs  i n the rumen when h i g h  of s o l u b l e c a r b o h y d r a t e are f e d , which should the a m y l o l y t i c types of rumen microbes.  promote the growth of  Perhaps n i t r o g e n  of the d i e t s c o n t a i n i n g the formaldehyde t r e a t e d f o r a g e three) was  forage,  digestibility  (except  reduced as a r e s u l t of l e s s d i g e s t i o n of n i t r o g e n  p l a c e i n the rumen.  A l t h o u g h the heat treatment of the  the p e l l e t i n g of the r a t i o n s , and  levels  taking  grass-clover  the h i g h l e v e l s of  soluble  c a r b o h y d r a t e s c o u l d have caused r e d u c t i o n i n the d i g e s t i b i l i t y n i t r o g e n i n the rumen as d i s c u s s e d  diet  of  above f o r the c o n t r o l u n t r e a t e d  diet,  formaldehyde a p p l i c a t i o n f u r t h e r reduced n i t r o g e n d i g e s t i b i l i t y i n the rumen.  D i n i u s et: a l . (1974) r e p o r t e d  e f f e c t s to formaldehyde treatment.  that heating  The  of p r o t e i n had  lowered l e v e l s of ammonia-  n i t r o g e n i n the rumens of the animals f e d the d i e t s c o n t a i n i n g treated forage  compared w i t h the d i e t s c o n t a i n i n g the u n t r e a t e d  supports the c o n t e n t i o n n i t r o g e n i n the rumen.  t h a t t h e r e was  additive  the forage  g e n e r a l l y reduced d i g e s t i o n of  - 90 -  The for  r e a s o n f o r the r a t h e r h i g h degree of d i g e s t i o n of  d i e t t h r e e compared to the o t h e r d i e t s c o n t a i n i n g the  treated forage  i s not  clear.  The  significantly  (p >  0.05)  formaldehyde  ammonia-nitrogen l e v e l s i n the rumens  of the animals f e d d i e t t h r e e were s t i l l rumens of animals f e d d i e t one  nitrogen  lower than the l e v e l s i n the  a l t h o u g h n i t r o g e n d i g e s t i b i l i t y was  different.  I t was  not  p o s s i b l e t h a t the a d d i t i o n  of v o l a t i l e f a t t y a c i d s to d i e t t h r e e promoted the growth of some microbes which c o u l d u t i l i z e n i t r o g e n e f f i c i e n t l y i n the rumen. Hemsley trt a l . (1970) r e p o r t e d the rumen was  t h a t f o r a g e p r o t e i n which by-passed  more d i g e s t i b l e than b a c t e r i a l p r o t e i n .  In t h i s experiment,  a g r e a t e r amount of p r o t e i n from the f o r a g e by-passed the rumen f o r the d i e t s c o n t a i n i n g the formaldehyde t r e a t e d f o r a g e duodenal f l o w data  (Table 14).  s t i l l not d i g e s t e d  to a g r e a t e r e x t e n t  i n d i e t one.  (1975) and  Hill  most p o w e r f u l s t i m u l u s  as i n d i c a t e d by  This p r o t e i n by-passing  the  the rumen  was  i n the i n t e s t i n e than the p r o t e i n  Horn and Huber (1975) observed t h a t  the  f o r the r e l e a s e of s e c r e t i n , which a l s o has  an  e f f e c t on the s e c r e t i o n of p a n c r e a t i c enzymes, i s a c i d i n g e s t a i n the duodenum.  In t h i s experiment abomasal pH was  the d i e t a r y t r e a t m e n t s .  I t was  f o r a g e p o r t i o n of the d i e t s w i t h  not d i f f e r e n t  thought t h a t w i t h  f o r any  treatment of  the  formaldehyde, abomasal pH would  h i g h as hydrogen i o n s would be r e q u i r e d t o break the bond between p r o t e i n and  formaldehyde as i n d i c a t e d by B a r r y  t h i s t r i a l t h a t t h e r e was production  (1976a).  be the  I t seems from  a p o s s i b l e compensatory i n c r e a s e i n the  o f h y d r o c h l o r i c a c i d i n the abomasum w i t h  of  formaldehyde  - 91 -  treatment  of the f o r a g e p o r t i o n of the d i e t s s i n e e abomasal pH  were not a f f e c t e d The  (Table 9)•  time a f t e r f e e d i n g when pH measurements were c a r r i e d out  perhaps not optimum to demonstrate d i f f e r e n c e s . observed  values  K n i g h t et a l . (1972)  t h a t the g r e a t e s t f a l l i n abomasal pH o c c u r r e d i n the  hour a f t e r f e e d i n g .  was  first  In the p r e s e n t t r i a l , the animals were s l a u g h t e r e d  about f o u r to s i x hours (4-6 h r s ) a f t e r f e e d i n g . experiments of Knight  The  r e s u l t s of the  et a l . (1972) i n d i c a t e d a r i s e i n abomasal pH  a f t e r the f i r s t hour a f t e r f e e d i n g w i t h pH  l e v e l s reaching pre-  p r a n d i a l c o n d i t i o n s about s i x hours (6 h r s ) a f t e r f e e d i n g .  Hill  r e p o r t e d t h a t o t h e r f a c t o r s which a l s o a f f e c t the r e l e a s e of  (1975)  secretin  a r e peptones, soaps and amino a c i d s a r r i v i n g at the duodenum.  Since  these parameters were not measured, i t i s not p o s s i b l e to comment  on  t h e i r e f f e c t s on the d i g e s t i b i l i t y of n i t r o g e n f o r the d i e t s c o n t a i n i n g the formaldehyde t r e a t e d f o r a g e . Abomasal n o n - p r o t e i n - n i t r o g e n l e v e l s determined  with  slaughtered  animals were lower w i t h the d i e t s c o n t a i n i n g the t r e a t e d f o r a g e The  t o t a l d a i l y flow of n o n - p r o t e i n - n i t r o g e n through  w i t h the c a n n u l a t e d  sheep tended  to be somewhat lower  (Table 9 ) .  duodenum measured f o r the  diets  c o n t a i n i n g the t r e a t e d f o r a g e than f o r the d i e t c o n t a i n i n g the u n t r e a t e d forage  (7.44, 6.10,  f o u r and f i v e  7.10,  6.52  respectively).  and  6.6.6  g/day f o r d i e t s one,  two,  three,  - 92 -  The a d d i t i o n o f s u l p h a t e alone formaldehyde t r e a t e d f o r a g e digestibility.  t o one of the d i e t s c o n t a i n i n g  ( d i e t f o u r ) somehow reduced n i t r o g e n  I t i s n o t c l e a r how the s u l p h a t e a d v e r s e l y a f f e c t e d  nitrogen d i g e s t i b i l i t y .  Umuna and Woods (1975) r e p o r t e d r e d u c t i o n i n  n i t r o g e n d i g e s t i b i l i t y when u r e a i n d i e t s was coated w i t h  sulphur.  The apparent d i g e s t i b i l i t y c o e f f i c i e n t s o f n i t r o g e n o b t a i n e d i n the i n v i v o t r i a l s were g e n e r a l l y lower than t h e n i t r o g e n obtained stages).  i n the i n v i t r o t r i a l ' s (combined m i c r o b i a l and a c i d - p e p s i n Barry  (1976c) r e p o r t e d i n v i t r o n i t r o g e n  (both m i c r o b i a l and a c i d - p e p s i n stages while  digestibility  o f d i g e s t i o n ) to be about 72-84%  i n the i n v i v o t r i a l s , v a l u e s o b t a i n e d  The d i s c r e p a n c y i n the present  digestibility  ranged from 52% t o 68%.  between t h e i n v i t r o and t h e i n v i v o n i t r o g e n trial  c o u l d perhaps be e x p l a i n e d  digestibility  as f o l l o w s : the i n v i t r o  measurement was t r u e d i g e s t i b i l i t y o f t h e grass-legume f o r a g e w h i l e the i n v i v o was apparent, n o t t a k i n g i n t o account endogenous s e c r e t i o n s o f nitrogen.  I t must a l s o be noted t h a t d u r i n g t h e i n v i t r o measurement,  o n l y t h e grass-legume f o r a g e was used, w h i l e w i t h  the i n v i v o  cassava  and b a r l e y formed p a r t o f the r a t i o n .  Apparent d i g e s t i b i l i t y c o e f f i c i e n t s o f ADF and C e l l u l o s e  There was a s i g n i f i c a n t d i g e s t i b i l i t y of acid-detergent treatment o f t h e f o r a g e  (p < 0.05) i n c r e a s e i n the i n v i v o f i b r e and c e l l u l o s e w i t h  formaldehyde  (Table 5, ADF v a l u e s : 32.57, 36.97, 36.91, 36.45 and  36.59% f o r d i e t s one, two, t h r e e , f o u r and f i v e r e s p e c t i v e l y ; c e l l u l o s e : 42.95, 49.10, 49.04, 49.33 and 48.76% f o r d i e t s one, two, t h r e e , f o u r and f i v e r e s p e c t i v e l y ) . D i n i u s e t al". (1975) r e p o r t e d a s i g n i f i c a n t  - 93 -  r e d u c t i o n i n the d i g e s t i b i l i t y o f a c i d - d e t e r g e n t and  fibre  (38.8%, 33.8%,  26.4% f o r 0.0%, 1.0%, and 2.0% l e v e l s of formaldehyde treatment  respectively).  Beever e t a l . (1976) r e p o r t e d h i g h e r v a l u e s of  c e l l u l o s e d i g e s t i b i l i t y f o r formaldehyde t r e a t e d h i g h (900°C) d r i e d g r a s s f o r a g e  (83.9% vs  temperature  89.5% f o r the u n t r e a t e d  and  treated r e s p e c t i v e l y ) . The l e v e l s o f a c i d - d e t e r g e n t  f i b r e and c e l l u l o s e i n the abomasal  d i g e s t a samples (Table 9) were not s i g n i f i c a n t l y  (p y 0.05)  different.  The d i g e s t i b i l i t y o f a c i d - d e t e r g e n t - f i b r e i n the stomach (pre-duodenum) from the animal f i t t e d w i t h r e - e n t r a n t cannula g r e a t e r f o r d i e t one than f o r the r e s t  (Table 15) was markedly  (30.87%, 18.12%, 19.72%, 18.92% and  21.05% f o r d i e t s one, two, t h r e e , f o u r and f i v e r e s p e c t i v e l y ) . The same t r e n d was observed f o r c e l l u l o s e  (35.35%, 22.75%, 23.48%, 23.56% and  25.30% f o r d i e t s one, two, t h r e e , f o u r and f i v e r e s p e c t i v e l y ) . be argued t h a t w i t h  I t could  the d i e t s c o n t a i n i n g the formaldehyde t r e a t e d  forage  most o f the d i g e s t i o n of a c i d - d e t e r g e n t - f i b r e and c e l l u l o s e took p l a c e o u t s i d e the r e t i c u l o - r u m e n .  A g r e a t q u a n t i t y of n i t r o g e n might have  reached the caecum and the c o l o n as t h e r e was a r e d u c t i o n i n the d i g e s t i o n of n i t r o g e n i n the r e t i c u l o - r u m e n when the d i e t s c o n t a i n i n g t h e formaldehyde t r e a t e d forage were f e d . and  colon having  access  t o a great amount o f n i t r o g e n might have had  g r e a t e r a c t i v i t i e s than those untreated Nolan  The micro-organisms i n the caecum  i n animals f e d the d i e t c o n t a i n i n g the  f o r a g e , w i t h l e s s n i t r o g e n r e a c h i n g the caecum and c o l o n .  (1975) r e v i e w i n g  l i t e r a t u r e on d i g e s t i o n i n the hindgut  reported  t h a t p r o t e o l y t i c a c t i v i t y appears t o be g r e a t e r i n the c o n t e n t s l a r g e i n t e s t i n e than i n the c o n t e n t s  of the rumen.  of the  He a l s o observed  - 94 -  t h a t a g r e a t d e a l of f e r m e n t a t i o n i n the hindgut  of s t r u c t u r a l carbohydrates  occurs  e s p e c i a l l y w i t h sheep f e d d i e t s c o n t a i n i n g h i g h  of c e r e a l s .  The sources  o f n i t r o g e n i n the h i n d g u t  fermentation  a c t i v i t i e s of the micro-organisms a r e :  levels  f o r such nitrogeneous  compounds a r r i v i n g from the s m a l l i n t e s t i n e i n the form of feed  residues,  undigested  entry of  rumen micro-organisms and endogenous s e c r e t i o n s ; and  urea-nitrogen  from t h e b l o o d .  In the case o f d i e t t h r e e , though n i t r o g e n d i g e s t i b i l i t y was not significantly  (p y  significantly  (p <0.05) h i g h e r .  0.05) lower than d i e t one f i b r e d i g e s t i b i l i t y The microbes i n t h e h i n d g u t s  animals f e d d i e t t h r e e were p r o b a b l y  was of  s u p p l i e d w i t h a g r e a t e r amount of  n i t r o g e n from the s m a l l i n t e s t i n e than f o r d i e t one.  I t must be noted  t h a t the c o n c e n t r a t i o n o f n i t r o g e n i n the abomasal d i g e s t a samples was n o t a f f e c t e d by d i e t a r y treatment  (Table 9) and t h e n i t r o g e n  c o n c e n t r a t i o n o f the duodenal d i g e s t a was not markedly d i f f e r e n t .  It  was s t i l l p o s s i b l e t h a t the animals f e d the d i e t s c o n t a i n i n g the formaldehyde t r e a t e d f o r a g e had a g r e a t e r amount o f n i t r o g e n i n the duodenum  as f l o w of d r y matter c o u l d be d i f f e r e n t .  o b t a i n e d w i t h the animal f i t t e d w i t h r e - e n t r a n t cannula, o f n i t r o g e n was markedly h i g h e r  arriving  From the d a t a the d a i l y  flow  f o r the d i e t s c o n t a i n i n g t h e formaldehyde  t r e a t e d f o r a g e than f o r the d i e t s c o n t a i n i n g u n t r e a t e d m a t e r i a l  (Table  14, 21.20g, 31.02g, 30.02g, 31.14g and 29.16g f o r d i e t s one, two, t h r e e , f o u r and f i v e r e s p e c t i v e l y ) . N i t r o g e n a r r i v i n g i n the duodenum g r e a t e r f o r d i e t t h r e e compared w i t h d i e t one.  was  Enough n i t r o g e n was  - 95 -  probably  left  f o r m i c r o b i a l a c t i v i t y i n the hindgut  g r e a t e r p o r t i o n of i t was  d i g e s t e d and  w i t h d i e t t h r e e compared to d i e t one. a r r i v i n g i n the hindgut  absorbed  i n the s m a l l  With a g r e a t e r amount of n i t r o g e n  t h e r e f o r e d i g e s t i o n of f i b r e i n the h i n d  gut would be g r e a t e r f o r d i e t t h r e e than f o r d i e t Beever et: a l . (1976) observed  In t h e i r study,  a g r e a t e r amount of d i g e s t i o n of  95% of the d i g e s t i o n of f i b r e o c c u r r e d i n the  temperature d r i e d grass 89.5%  one.  f o r the t r e a t e d compared to the u n t r e a t e d  rumen f o r the u n t r e a t e d d i e t and  and  70%  N i t r o g e n was  reticulo-  ( o v e r a l l d i g e s t i b i l i t y of f i b r e b e i n g 83.9%,  f o r u n t r e a t e d and  the u n t r e a t e d f o r a g e and  diet.  f o r the formaldehyde t r e a t e d h i g h  treated respectively).  In the p r e s e n t  the s i t e s f o r the d i g e s t i o n of f i b r e were d i f f e r e n t  forage.  intestine  f o r d i e t t h r e e than f o r d i e t one, m i c r o b i a l  a c t i v i t y would be g r e a t e r and  f i b r e i n the hindgut  even i f a  f o r the d i e t c o n t a i n i n g  the d i e t s c o n t a i n i n g the formaldehyde t r e a t e d  perhaps the main l i m i t i n g f a c t o r f o r m i c r o b i a l  d e g r a d a t i o n of f i b r e i n the rumen when the d i e t s c o n t a i n i n g the  formaldehyde  t r e a t e d f o r a g e were f e d (rumen ammonia-nitrogen l e v e l s were lower two,  t h r e e , f o u r and  study,  f i v e than f o r d i e t one,  p r o p o r t i o n of f i b r e was  Table 8).  d i g e s t e d i n the hindgut  for diets  A greater  where n i t r o g e n might not  have l i m i t e d m i c r o b i a l a c t i v i t i e s when the d i e t s c o n t a i n i n g the formaldehyde t r e a t e d f o r a g e were f e d (the apparent c o e f f i c i e n t s of A.D.F. and were observed  digestibility  c e l l u l o s e i n the f o u r compartments of the stomach  t o be h i g h e r f o r d i e t one  than f o r the o t h e r s u s i n g  sheep f i t t e d w i t h the duodenal r e - e n t r a n t cannula, T a b l e 15). when the d i e t c o n t a i n i n g the u n t r e a t e d f o r a g e was  fed, nitrogen  most l i k e l y not a l i m i t i n g f a c t o r i n the rumen but was  most  the  However, was  probably  - 96 -  l i m i t i n g i n t h e hindgut. fermentable  carbohydrate  There were h i g h l e v e l s o f r e a d i l y p r e s e n t i n t h e rumen.  Therefore, the  d i g e s t i o n o f f i b r e c o u l d n o t be c a r r i e d out t o the maximum e x t e n t .  Apparent d i g e s t i b i l i t y c o e f f i c i e n t s o f d r y matter and o r g a n i c matter  The  apparent  d i g e s t i b i l i t y c o e f f i c i e n t s o f d r y matter  (64.78%, 65.69%,  65.82%, 64.80%, and 63.71% f o r d i e t s one, two, t h r e e , f o u r and f i v e r e s p e c t i v e l y ) and o r g a n i c matter  (65.35%, 65.91%, 66.01%, 65.17% and  63.83% f o r d i e t s one, two, t h r e e , f o u r , and f i v e r e s p e c t i v e l y ) were not a f f e c t e d s i g n i f i c a n t l y  (p y  0.05) by treatments.  The v a l u e s  o b t a i n e d were s i m i l a r t o those r e p o r t e d by o t h e r workers (Amos a t a l . , 1976b; Sharma and N i c h o l s o n , 1975b).  Amos a t a l . (1976b) r e p o r t e d d r y  matter d i g e s t i b i l i t y f i g u r e s o f 64.4%, 63.4%, 61.3%, and 56.3% f o r 0.0%,  0.5%, 1.0% and 1.5% l e v e l s o f formaldehyde treatment  bermuda g r a s s r e s p e c t i v e l y .  Sharma and N i c h o l s o n  of coastal  (1975b) r e p o r t e d d r y  matter d i g e s t i b i l i t i e s o f 65.4% and 63.9%, f o r r a t i o n s c o n t a i n i n g u n t r e a t e d and formaldehyde t r e a t e d f a b a bean meal r e s p e c t i v e l y .  They  a l s o r e p o r t e d d r y m a t t e r d i g e s t i b i l i t i e s o f 53.5% and 55.2% f o r r a t i o n s c o n t a i n i n g u n t r e a t e d and t r e a t e d rapeseed and N i c h o l s o n , 1975a).  meal r e s p e c t i v e l y  (Sharma  I n some o f t h e r e p o r t s c i t e d above (Sharma and  N i c h o l s o n , 1975a, 1975b; B a r r y 1976c) d r y matter d i g e s t i b i l i t i e s were not s i g n i f i c a n t l y a f f e c t e d by formaldehyde treatment the p r e s e n t experiment. observe  Sharma and N i c h o l s o n  and  1975b) d i d n o t  a s i g n i f i c a n t r e d u c t i o n i n n i t r o g e n d i g e s t i b i l i t i e s i n t h e two  t r i a l s , w i t h formaldehyde treatments Barry  (1975a  as was t h e case i n  o f faba bean and rapeseed  (1976c) r e p o r t e d no s i g n i f i c a n t r e d u c t i o n i n d r y matter  meal. digestibility  o f d i e t s t r e a t e d w i t h formaldehyde a l t h o u g h t h e r e was a s i g n i f i c a n t  - 97 -  reduction i n nitrogen d i g e s t i b i l i t y .  D i n i u s e_t a l . (1975) however  r e p o r t e d a s i g n i f i c a n t r e d u c t i o n i n d r y m a t t e r , n i t r o g e n and f i b r e d i g e s t i b i l i t i e s with In t h e p r e s e n t s i g n i f i c a n t l y reduced  formaldehyde treatment. experiment, although (except  n i t r o g e n d i g e s t i b i l i t y was  f o r d i e t t h r e e ) by treatment o f the f o r a g e  p o r t i o n o f the d i e t s w i t h formaldehyde, d r y matter and o r g a n i c matter d i g e s t i b i l i t i e s were n o t a f f e c t e d because o f an i n c r e a s e i n t h e d i g e s t i o n o f some o t h e r f r a c t i o n s , For d i e t  especially  fibre.  t h r e e , n i t r o g e n d i g e s t i b i l i t y was n o t s i g n i f i c a n t l y  l e s s than f o r d i e t one and f i b r e d i g e s t i b i l i t y was s i g n i f i c a n t l y (p<,0.05) g r e a t e r than f o r d i e t one.  I t was expected t h a t d r y matter and o r g a n i c  matter d i g e s t i b i l i t i e s would have been s i g n i f i c a n t l y d i f f e r e n t two  diets.  f o r the  However, t h e r e were s l i g h t but n o n - s i g n i f i c a n t d i f f e r e n c e s  between d i e t s  one and t h r e e w i t h r e s p e c t t o n i t r o g e n and d r y m a t t e r o r  o r g a n i c matter d i g e s t i b i l i t i e s . The n i t r o g e n d i g e s t i b i l i t y was s l i g h t l y h i g h e r f o r d i e t one than f o r d i e t t h r e e  (54.13% f o r d i e t one and 51.25%  f o r d i e t t h r e e ) ; d r y matter d i g e s t i b i l i t y was s l i g h t l y h i g h e r  for diet  t h r e e than f o r d i e t one (64.78% f o r d i e t one, and 65.82% f o r d i e t o r g a n i c matter d i g e s t i b i l i t y was a l s o s l i g h t l y h i g h e r  for diet  three);  three  f o r d i e t one (65.35% f o r d i e t one and 66.01% f o r d i e t t h r e e ) .  Rumen Parameters  Rumen pH  The  pH v a l u e o f t h e rumen f l u i d ranged from 5.11 t o 5.50 f o r  a l l the d i e t s .  Rumen pH was n o t s i g n i f i c a n t l y (p> 0.05) a f f e c t e d by  than  - 98 -  the treatments.  D i n i u s e t a l . (1975) a l s o observed no d i f f e r e n c e s  i n rumen pH w i t h treatment o f a l f a l f a meal w i t h formaldehyde. pH v a l u e s  obtained  i n the present  t r i a l were s i m i l a r t o t h e v a l u e s  r e p o r t e d by Sharma and I n g a l l s (1974). v a l u e s o f about 5.6.  The  They r e p o r t e d average pH  They a l s o r e p o r t e d no d i f f e r e n c e s i n pH w i t h  formaldehyde treatment o f d i e t s .  In t h e p r e s e n t  experiment, the samples  f o r pH measurements were taken 4-6 hours a f t e r f e e d i n g .  Hodgson e t a l .  (1976) r e p o r t e d t h a t as v o l a t i l e f a t t y a c i d c o n c e n t r a t i o n the rumen pH d e c r e a s e d .  In t h e p r e s e n t  increased,  experiment, as the pH was  measured a t the time when c o n c e n t r a t i o n o f v o l a t i l e f a t t y a c i d s was expected t o be h i g h e s t i t i s n o t s u r p r i s i n g t h a t the pH v a l u e s were r e l a t i v e l y low.  S a v i l l e et a l .  (1971) p o s t u l a t e d t h a t i f rumen  pH  v a l u e s were low, i t was p o s s i b l e t h a t t h e bonds between formaldehyde and p r o t e i n s c o u l d be broken.  Sharma and I n g a l l s (1974) r e p o r t e d low pH  v a l u e s around 5.6 b u t s t i l l no breakdown i n the bonding o c c u r r e d . i s a l s o a c y c l i c a l v a r i a t i o n i n rumen pH and the v a l u e s o b t a i n e d experiment c o u l d have been a t the lowest  f o r t h e day.  There i n this  The animals a l s o  d i d n o t r e f u s e feed and t h e r e were no s i g n s o f r u m e n i t i s .  Rumen ammonia-nitrogen  Rumen ammonia-nitrogen l e v e l s (p  (Table 8) were s i g n i f c a n t l y  0.05) reduced by formaldehyde treatment o f the f o r a g e p o r t i o n  of t h e d i e t s (21.14, 14.36, 14.30, 12.90 and 13.54 ppm f o r d i e t s one, two,  t h r e e , f o u r and f i v e r e s p e c t i v e l y ) .  Reports by some workers i n d i c a t e d  reduced.rumen ammonia-nitrogen l e v e l s w i t h treatment o f d i e t s w i t h formaldehyde (Sharma and N i c h o l s o n ,  1975b; Bhargava and Ranjhan, 1974;  - 99 -  Sharma and I n g a l l s , 1973; Sharma e t a l . , 1972; Hogan and Weston, 1970; Hemsley et: a l . , 1970).  Other workers however observed no d e c r e a s e i n  rumen ammonia n i t r o g e n l e v e l s w i t h formaldehyde treatment o f d i e t s ( D i n i u s e t a l . , 1975; Sharma and N i c h o l s o n , Ammonia p r e s e n t  1975a).  i n the rumen c o u l d be from a v a r i e t y o f s o u r c e s :  recycled urea-nitrogen  e i t h e r from b l o o d o r s a l i v a ;  r from t h e  breakdown o f p r o t e i n or amino a c i d s , and r e d u c t i o n o f n i t r a t e s ;  from  the breakdown o f m i c r o b i a l p r o t e i n d u r i n g r e c y c l i n g o f n i t r o g e n w i t h i n the rumen (Houpt, 1970). Ammonia-nitrogen c o u l d a l s o be l o s t through t h e r u m i n a l w a l l . Such l o s s e s have been r e p o r t e d t o be dependent on pH w i t h maximum l o s s e s at pH 6.5, and n e g l i g i b l e a t pH 4.5  (Hemler and B a r t l e y , 1971; Hogan, 1961).  Ammonia-nitrogen c o u l d be passed on t o t h e abomasum and subsequently the i n t e s t i n e as t h e r e i s l i t t l e ,  to  a b s o r p t i o n o f ammonia-nitrogen from the  abomasum (Houpt, 1970; Hembry e t a l . , 1975).  In t h e p r e s e n t  experiment,  rumen pH v a l u e s were not s i g n i f i c a n t l y d i f f e r e n t , and t h e r e f o r e l o s s e s o f ammonia-nitrogen through t h e r u m i n a l w a l l a r e u n l i k e l y t o have c o n t r i b u t e d t o the v a r i a t i o n s i n rumen ammonia-nitrogen w i t h treatment.  I f anything,  formaldehyde  l o s s e s o f n i t r o g e n i n the rumen were p o s s i b l y  g r e a t e r f o r d i e t one than f o r t h e o t h e r  four d i e t s .  T h i s i s because  the q u a n t i t y o f n i t r o g e n l e a v i n g t h e abomasum was g r e a t e r f o r a l l t h e f o u r d i e t s c o n t a i n i n g the t r e a t e d f o r a g e compared t o t h e c o n t r o l , u s i n g the animal f i t t e d w i t h r e - e n t r a n t cannula  ( T a b l e 14).  The g r e a t e r amounts  of n i t r o g e n a r r i v i n g a t t h e duodenum w i t h  the d i e t s c o n t a i n i n g the  formaldehyde t r e a t e d f o r a g e compared w i t h d i e t one were u n l i k e l y t o be due to d i f f e r e n c e s i n the l o s s e s o f ammonia i n the abomasum as such l o s s e s  - 100  a r e r e p o r t e d t o be n e g l i g i b l e  -  (Houpt, 1970;  Hembry e t a l . , 1975).  N i t r o g e n r e c y c l i n g i n t o the rumen i s r e p o r t e d to be h i g h e s t when ammonia-nitrogen l e v e l s i n the rumen a r e low  (Church, 1975c, Houpt, 1970).  I t i s t h e r e f o r e a l s o u n l i k e l y t h a t the h i g h e r ammonia-nitrogen i n the rumens of animals f e d d i e t one  compared to the r e s t , was  g r e a t e r r e c y c l i n g of n i t r o g e n i n t o the rumens from s a l i v a and urea-nitrogen.  One  p o s s i b l e reason  due  to  blood  f o r the higher' l e v e l s of rumen  ammonia-nitrogen i n the rumens of animals f e d d i e t one r e s t was  levels  compared w i t h  a g r e a t e r degree of r e c y c l i n g of m i c r o b i a l n i t r o g e n w i t h i n  rumen.. Perhaps the most p r o b a b l e  e x p l a n a t i o n f o r the h i g h e r  ammonia-nitrogen i n the rumens of animals f e d d i e t one  the  levels  compared  the  of  with  the r e s t i s a g r e a t e r degree of d i g e s t i o n of n i t r o g e n i n the rumens of the animals f e d t h i s d i e t .  T h i s i s supported  by  the lower q u a n t i t y of  n i t r o g e n a r r i v i n g i n the duodenum when d i e t one was f i t t e d w i t h the r e - e n t r a n t cannula, q u a n t i t y of n o n - p r o t e i n - n i t r o g e n s l i g h t l y g r e a t e r f o r d i e t one non-protein-nitrogen animals was the r e s t  f e d to the sheep  compared to the o t h e r d i e t s .  a r r i v i n g at the duodenum was  than f o r the r e s t .  The  also  concentration  i n the abomasal d i g e s t a samples from the  also s i g n i f i c a n t l y  The  (p < 0 . 0 5 ) h i g h e r f o r d i e t one  of  slaughtered than f o r  (Table 9 ) .  G e n e r a l l y , the l e v e l s of ammonia i n the rumens f o r a l l the d i e t s were low Nicholson  compared w i t h (1975a and  f i g u r e s r e p o r t e d by B a r r y  1975b).  The  (1973a) and  Sharma  ammonia-nitrogen l e v e l s r e p o r t e d  Sharma and N i c h o l s o n  (1975a) were 8.99mg/100ml and  containing untreated  and  and by  5.47mg/100ml f o r d i e t s  formaldehyde t r e a t e d rapeseed meal r e s p e c t i v e l y .  - 101 -  These measurements were c a r r i e d out on samples c o l l e c t e d a f t e r feeding.  These workers (Sharma and N i c h o l s o n ,  one hour  1975b) r e p o r t e d  rumen ammonia-nitrogen l e v e l s o f 24.25mg/100ml and 13.66mg/100ml f o r diets  containing untreated  respectively.  and formaldehyde t r e a t e d f a b a bean meal  These measurements were a l s o c a r r i e d out on samples  collected  one hour a f t e r f e e d i n g .  Measurements  collected  f o u r hours a f t e r f e e d i n g i n t h e same experiment gave  of 15.40mg/100ml and 6.96mg/100ml f o r d i e t s formaldehyde t r e a t e d faba bean d i e t s . on samples c o l l e c t e d  c a r r i e d out on samples  c o n t a i n i n g u n t r e a t e d and  Barry  (1973a) t a k i n g measurements  f o u r hours a f t e r f e e d i n g , r e p o r t e d  27.0mg/100ml f o r u n t r e a t e d  results  f i g u r e s of  and 14.8mg/100ml f o r formaldehyde t r e a t e d  lucerne"* hay. D i n i u s et^ a l . (1975) however r e p o r t e d of 3 t o 7 mg/lOOml. j u s t before combined  low ammonia-nitrogen  levels  They took samples f o r ammonia-nitrogen measurements  f e e d i n g and a t one hour i n t e r v a l s f o r e i g h t hours and  the r e s u l t s  to a r r i v e a t the values  reported  above.  The r a t h e r low l e v e l s o f ammonia-nitrogen observed i n t h e p r e s e n t experiment c o u l d perhaps be a t t r i b u t e d  t o t h e time o f sampling.  were taken f o u r to s i x hours a f t e r f e e d i n g . l e v e l i s reported feeding  Samples  Rumen ammonia-nitrogen  t o reach peak l e v e l s about 90 t o 130 minutes a f t e r  (Church, 1975c).  Hembry e t a l . (1975) r e p o r t e d  l e v e l s o f 12 t o 30 ppm s i x h o u r s ^ a f t e r  rumen ammonia  f e e d i n g and 16 t o 46 ppm f o u r  hours a f t e r f e e d i n g d i e t s c o n t a i n i n g u r e a ,  soybean meal and c a s e i n .  - 102 -  Rumen t o t a l v o l a t i l e f a t t y a c i d  There were no s i g n i f i c a n t •between  treatments  with  concentration  (py  respect  0.05) d i f f e r e n c e s t o the p r o d u c t i o n of  v o l a t i l e f a t t y a c i d s i n the rumen (166.12, 186.03, 165.44, 166.81 and  150.06 /i-moles/ml f o r d i e t s one, two, t h r e e , f o u r and f i v e  respectively).  S e v e r a l workers have a l s o r e p o r t e d no  d i f f e r e n c e s i n the production formaldehyde treatment. and  I n g a l l s , 1973).  concentrations 1972;  Barry,  o f v o l a t i l e f a t t y a c i d s i n the rumen w i t h  (Beever e t a l . , 1976; Beever e t a l . , 1977; Sharma  Other workers however have observed decreased  of v o l a t i l e f a t t y acids with  1973a).  significantly  t r e a t e d d i e t s (Sharma e_t a l . ,  M i c r o b i a l p r o t e i n - n i t r o g e n i n the abomasal d i g e s t a  samples (measured by the r a t i o o f % m i c r o b i a l - N : % t o t a l d i g e s t a N) was decreased s i g n i f i c a n t l y w i t h a l l the d i e t s c o n t a i n i n g t h e formaldehyde treated forage,  except f o r d i e t  three  (0.768, 0.480, 0.583, 0.451, and  0.439 f o r d i e t s one, two, t h r e e , f o u r and f i v e r e s p e c t i v e l y ) . f o r duodenal d i g e s t a , u s i n g t h e sheep f i t t e d w i t h r e - e n t r a n t  The r a t i o s  cannula  (0.804, 0.477, 0.551, 0.397, and 0.416 f o r d i e t s one, two, t h r e e ,  four  and f i v e r e s p e c t i v e l y ) and t h e t o t a l amounts o f m i c r o b i a l p r o t e i n a r r i v i n g at the duodenum p e r day (17.05, 14.81, 16.53, 12.36, and 12.14g f o r d i e t s one,  two, t h r e e , f o u r and f i v e r e s p e c t i v e l y ) were a l s o reduced by  treatment o f t h e f o r a g e p o r t i o n o f t h e d i e t s w i t h formaldehyde. s t a t i s t i c a l a n a l y s i s was performed however w i t h only one animal was used but i t s t i l l  No  the duodenal d i g e s t a as  i n d i c a t e d t h e t r e n d shown by t h e  abomasal d i g e s t a samples. Smith et a l . (1968) r e p o r t e d  that the p r o p o r t i o n of r i b o n u c l e i c -  a c i d n i t r o g e n t o t o t a l n i t r o g e n i n duodenal d i g e s t a p a r a l l e l e d the p r o p o r t i o n o f r i b o n u c l e i c a c i d - n i t r o g e n t o t o t a l n i t r o g e n i n rumen  fluid  - 103 -  except t h a t t h e l e v e l s i n the duodenal d i g e s t a were c o n s i s t e n t l y lower because o f t h e a d d i t i o n o f endogenous s e c r e t i o n s o f n i t r o g e n i n t h e abomasum.  I t i s t h e r e f o r e i n l i n e t o e x t r a p o l a t e measurements  of m i c r o b i a l p r o t e i n - n i t r o g e n i n duodenal d i g e s t a t o what happened i n t h e rumen. From t h e data r e p o r t e d above f o r t h e p r e s e n t t h e r e f o r e be concluded  trial,  i t could  t h a t 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  was reduced by formaldehyde treatment o f t h e f o r a g e p o r t i o n o f the d i e t , except f o r d i e t t h r e e . affected.  However, v o l a t i l e f a t t y a c i d p r o d u c t i o n was n o t  Beever e t a l . (1977)  were capable carbohydrates  of adapting  r e p o r t e d t h a t rumen m i c r o b i a l  t h e i r metabolic  populations  pathways t o make maximum use.of  w h i l e 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 may be depressed.  phenomenon was termed by these workers as uncoupled f e r m e n t a t i o n . probable  that t h i s occurred  i n the present  study.  This It i s  Hodgson et: a l . (1976)  r e p o r t e d t h a t the amount o f d r y matter i n t h e rumen c o u l d a f f e c t the c o n c e n t r a t i o n o f s h o r t c h a i n f a t t y a c i d s i n t h e rumen.  The q u a n t i t i e s  of d r y matter i n the rumens o f t h e sheep (Table 8) were n o t s i g n i f i c a n t l y (p> and  0.05) d i f f e r e n t w i t h d i e t a r y treatment  (574.93, 643.73, 447.89, 449.04,  478.15g f o r d i e t s one, two, t h r e e , f o u r , and f i v e r e s p e c t i v e l y ) .  Therefore,  p o s s i b l y t h i s parameter had no e f f e c t on t h e p r o d u c t i o n o f  v o l a t i l e f a t t y a c i d s i n the rumen.  D i n i u s elt a l . (1975) r e p o r t e d  time o f sampling had some e f f e c t s on t h e c o n c e n t r a t i o n o f v o l a t i l e a c i d s i n t h e rumen.  I n t h e i r study,  the v o l a t i l e f a t t y a c i d  that fatty  concentration  at two, t h r e e , and f o u r hours a f t e r f e e d i n g was s i g n i f i c a n t l y reduced by formaldehyde treatment o f t h e l u c e r n e p o r t i o n o f t h e d i e t but a t s i x hours t h e r e were no s i g n i f i c a n t d i f f e r e n c e s between t h e formaldehyde  - 104 -  t r e a t e d and the u n t r e a t e d . who observed  Sharma e t a l . (1972) and B a r r y  (1973a)  differences i n concentration of v o l a t i l e f a t t y acids  i n the rumen due t o formaldehyde treatment to f o u r hours a f t e r f e e d i n g .  took t h e i r samples  three  Beever et a l . (1976), and Beever et^ a l .  (1977) who r e p o r t e d no s i g n i f i c a n t r e d u c t i o n i n t o t a l v o l a t i l e  fatty  a c i d p r o d u c t i o n i n t h e rumen measured i t s p r o d u c t i o n over twenty f o u r hours.  Sharma and I n g a l l s (1973) who a l s o r e p o r t e d no s i g n i f i c a n t  r e d u c t i o n i n t h e p r o d u c t i o n o f t o t a l v o l a t i l e f a t t y a c i d , took t h e i r samples t h r e e t o f o u r hours a f t e r f e e d i n g .  Therefore,  i t i s possible  t h a t the e q u a l i t y o f p r o d u c t i o n o f v o l a t i l e f a t t y a c i d s r e p o r t e d i n the p r e s e n t The  study was n o t due t o t h e time  o f sampling.  l e v e l s o f v o l a t i l e f a t t y a c i d s i n t h e rumens were h i g h  compared w i t h f i g u r e s r e p o r t e d by D i n i u s e t a l . (1975) and N i c h o l s o n and  Sutton  (1969).  D i n i u s e t a l . (1975)  hours a f t e r f e e d i n g ) o f about 100 ,  86 ,  reported f i g u r e s (at four and 50 /i-moles/ml  for diets  c o n t a i n i n g l u c e r n e meal t r e a t e d w i t h formaldehyde a t l e v e l s o f 0, 1 and 2% r e s p e c t i v e l y . were about 80 ,  The c o r r e s p o n d i n g 72 ,  f i g u r e s f o r s i x hours a f t e r  and 60 ^i-moles/ml  respectively.  feeding  I n the case o f  t h e i r experiments the l e v e l o f l u c e r n e i n the d i e t s was about 75% w h i l e i n the p r e s e n t experiment the l e v e l o f f o r a g e was o n l y about 50% w i t h the remainder o f the r a t i o n s made up of r e a d i l y fermentable T h i s h i g h l e v e l o f r e a d i l y fermentable  carbohydrate  the h i g h l e v e l o f t o t a l v o l a t i l e f a t t y a c i d observed experiment.  N i c h o l s o n and Sutton  carbohydrates.  might have produced i n the p r e s e n t  (1969) r e p o r t e d t o t a l v o l a t i l e  fatty  a c i d l e v e l s o f about 82.1 t o 97.0 / l - m o l e s / l i t r e when they f e d d i e t s c o n t a i n i n g 20% hay and 80% c o n c e n t r a t e s o l u b l e carbohydrate  t o sheep.  were h i g h i n t h e i r d i e t s .  The l e v e l s o f r e a d i l y They however, composited  - 105  -  samples taken a t t h r e e , s i x , n i n e , and  e l e v e n hours a f t e r  f o r the v o l a t i l e f a t t y a c i d measurements.  Therefore,  of v o l a t i l e f a t t y a c i d observed i n the p r e s e n t the l e v e l s r e p o r t e d by N i c h o l s o n be due  t o the time of  and  Sutton  feeding  the h i g h  levels  experiment compared to  (1969) c o u l d most  likely  sampling.  A c e t i c , p r o p i o n i c , and b u t y r i c a c i d p r o p o r t i o n s i n the rumen f l u i d  The molar p r o p o r t i o n s o f a c e t i c , p r o p i o n i c and b u t y r i c a c i d s i n the rumen f l u i d were not a f f e c t e d by d i e t a r y treatment  (Table 8 ) .  second group of animals however had  0.05)  significantly  (p <  The  h i g h e r molar  p r o p o r t i o n s of a c e t i c a c i d than the f o u r t h group, w h i l e the f o u r t h group had  significantly  (p < 0.05)  h i g h e r molar p r o p o r t i o n s of p r o p i o n i c  than the second group ( R e s u l t s , page 68 p r o p o r t i o n s of a c e t i c and and  ).  acid  D i f f e r e n c e s i n the molar  p r o p i o n i c a c i d s i n the rumens of the  second  f o u r t h groups ( b l o c k s ) of animals c o u l d have o c c u r r e d because of  i n d i v i d u a l animal d i f f e r e n c e s .  S l y t e r e t a l . (1970) r e p o r t e d v a r i a t i o n s  i n types of rumen microbes w i t h f u l l These v a r i a t i o n s i n p o p u l a t i o n s  f e e d i n g , even w i t h i d e n t i c a l  o f types of microbes c o u l d a l s o a f f e c t  p r o p o r t i o n s o f the v a r i o u s s h o r t - c h a i n f a t t y a c i d s . experiment the p r o p o r t i o n of s o l u b l e carbohydrates the d i f f e r e n t groups ( b l o c k s ) of animals was the h i g h e r  l e v e l s of p r o p i o n i c a c i d and  not  In the  present  i n the d i e t s f e d to  different.  Therefore,  the lower l e v e l s of a c e t i c a c i d  i n the rumens of sheep i n the f o u r t h group compared to those group c o u l d not be due  twins.  to d i f f e r e n t l e v e l s of s o l u b l e  i n the  carbohydrates.  second  - 106  -  Wilke and Merwe (1976) r e p o r t e d t h a t h i g h l e v e l s concentrate  of  i n the d i e t c o u l d r e s u l t i n h i g h molar p r o p o r t i o n s  of  p r o p i o n i c a c i d a t the expense of the a c e t i c a c i d p r o p o r t i o n . The  average p r o p o r t i o n s  of the v a r i o u s f a t t y a c i d s f o r a l l the d i e t s  ( a c e t i c 41.96%, p r o p i o n i c 29.00%, n - b u t y r i c 23.27%, i s o b u t y r i c 1.35%, i s o v a l e r i c 1.66%, and n - v a l e r i c 2.96%) were s i m i l a r and b u t y r i c ) to the v a l u e s r e p o r t e d by Sutton when a cow  (except  (1969).  was fed'a d i e t c o n t a i n i n g f l a k e d maize (5kg  He  for acetic  reported  per day)  and  that hay  (1.0kg per'day) the p r o p o r t i o n s o f the v a r i o u s a c i d s were: a c e t i c 54.8%, p r o p i o n i c 25.1%, b u t y r i c 14.0%, i s o b u t y r i c 1.4%, v a l e r i c 2.4%.  0rskov e t a l . (1970) r e p o r t e d  i s o v a l e r i c 2.3%,  the f o l l o w i n g l e v e l s of  v a r i o u s a c i d s i n the rumen of sheep f e d chopped d r i e d g r a s s : p r o p i o n i c 20.2%, b u t y r i c 6.0%, v a l e r i c 0.8%. slaughtered  and  i s o b u t y r i c 2.5%,  the  a c e t i c 68.6%,  i s o v a l e r i c 2.0%,  and  In the experiment of 0rskov e t a l . (1970) the sheep were one hour a f t e r f e e d i n g and  then samples taken.  The  same workers  i n the same paper, r e p o r t e d the f o l l o w i n g l e v e l s of v a r i o u s a c i d s when b a r l e y was  f e d (animals  slaughtered  one hour a f t e r f e e d i n g and  samples  t a k e n ) : a c e t i c 53.6%, p r o p i o n i c 20.6%, b u t y r i c 16.2%, i s o b u t y r i c i s o v a l e r i c 1.4%,  n - v a l e r i c 5.4%,  and  c a p r o i c 1.1%.  N i c h o l s o n and  1.7%, Sutton  (1969) and Whitelaw est a l . (1970) r e p o r t e d t h a t when sheep on b a r l e y d i e t s a r e f e d below f u l l a corresponding  f e e d i n g the p r o p o r t i o n of b u t y r i c a c i d i s i n c r e a s e d  d e c r e a s e i n the p r o p i o n i c a c i d f r a c t i o n .  experiment, b o t h b u t y r i c a c i d and high.  There was  In the  present  p r o p i o n i c a c i d p r o p o r t i o n s were v e r y  r a t h e r a decrease i n a c e t i c a c i d p r o p o r t i o n which i s  u s u a l l y r e p o r t e d above 50%  with  even when animals a r e f e d d i e t s c o n t a i n i n g  - 107  -  g r e a t amounts o f r e a d i l y fermentable 0rskov et a l . , 1970). a c e t i c a c i d l e v e l was carbohydrates  The  carbohydrate  animals were f e d ad l i b i t u m .  therefore probably  due  Isobutyric acid proportions  f o r d i e t t h r e e than f o r d i e t s two  i s o v a l e r i c a c i d p r o p o r t i o n s were 2.53, two,  t h r e e , f o u r and  a c i d p r o p o r t i o n s were 1.54, d i e t s one,  lowered  to the h i g h l e v e l of s o l u b l e  i n the rumens  The molar p r o p o r t i o n s of i s o v a l e r i c and  d i e t s one,  The  and  i n the r a t i o n s .  I s o v a l e r i c and  higher  (Sutton, 1969;  two,  0.71,  t h r e e , f o u r and  and  0.60,  i s o b u t y r i c a c i d s were  four  (p*£  2.58,  0.39  five respectively. 2.11, five.  0.66 I t was  0.05). and  The  2.18%  for  The i s o b u t y r i c  and 1.73%  respectively for  expected t h a t w i t h  formaldehyde treatment of the f o r a g e p o r t i o n of the d i e t s molar p r o p o r t i o n s of i s o b u t y r i c and  i s o v a l e r i c a c i d s would be reduced.  a c i d s a r e d e r i v e d from b r a n c h e d - c h a i n amino a c i d s 1952b).  A l l i s o n and  Bryant  ( e l - S h a z l y , 1952a and  (1963) r e p o r t e d t h a t the mechanism f o r the  s y n t h e s i s of the i s o p r o p y l moiety i n the b r a n c h e d - c h a i n f a t t y was  inadequate i n some micro-organisms.  reduction  acids  Langlands (1973a) r e p o r t e d  (not s i g n i f i c a n t ) i n the l e v e l s o f i s o b u t y r i c and  a c i d s f o l l o w i n g formaldehyde treatment of wheat. (1977a) r e p o r t e d  These  Faichney  s i g n i f i c a n t r e d u c t i o n s of i s o b u t y r i c and  a c i d l e v e l s w i t h formaldehyde treatment of c o n c e n t r a t e  isovaleric and  White  isovaleric  diets.  Barry  Fennessy (1973) r e p o r t e d reduced l e v e l s of i s o v a l e r i c and v a l e r i c combined w i t h formaldehyde t r e a t e d s i l a g e s .  Barry  and  acids  (1976c) r e p o r t e d  - 108 -  reduced l e v e l s o f i s o v a l e r i c and v a l e r i c a c i d combined, b u t n o t i s o b u t y r i c a c i d , w i t h treatment o f d i e t s with.formaldehyde. (1971) r e p o r t e d production. reported  t h a t t h e a d d i t i o n o f one a c i d may suppress i t s  T h i s phenomenon was n o t observed by Hume (1970) who  i n c r e a s e d l e v e l s o f b r a n c h e d - c h a i n f a t t y a c i d s i n t h e rumens  of animals f e d d i e t s supplemented w i t h The  Hobson  present  t h e sodium s a l t s o f these a c i d s .  r e s u l t s seem t o conform w i t h  those o f Hume (1970).  It i s  s u r p r i s i n g t h a t t h e l e v e l s o f i s o v a l e r i c and i s o b u t y r i c a c i d s were n o t s i g n i f i c a n t l y higher  f o r d i e t f i v e which was supplemented w i t h  f a t t y a c i d s than f o r d i e t s two and f o u r . animals on d i e t two, and one on d i e t  volatile  I t i s t o be noted t h a t  three  f o u r had l e v e l s o f i s o b u t y r i c and  i s o v a l e r i c a c i d s which c o u l d n o t be measured.  Other s o u r c e s o f p r o t e i n ,  which c o u l d supply b r a n c h e d - c h a i n amino a c i d s t o be deaminated t o branched-chain f a t t y a c i d s a r e  (a)  recycled microbial protein,  (b)  desquamated rumen e p i t h e l i u m , and  (c)  p r o t e i n present  i n saliva.  For d i e t t h r e e , i t i s d i f f i c u l t  t o s p e c u l a t e which o f t h e t h r e e  sources  mentioned above c o n t r i b u t e d t o t h e h i g h e r l e v e l s of t h e b r a n c h e d - c h a i n f a t t y a c i d s i n a d d i t i o n t o what was supplemented and what came from t h e breakdown o f d i e t a r y p r o t e i n i n t h e rumen. f i v e , though n o t s i g n i f i c a n t l y h i g h e r due  The h i g h l e v e l s f o r d i e t  than d i e t s two and f o u r , c o u l d be  m a i n l y t o t h e supplemented q u a n t i t i e s .  I t i s i n t e r e s t i n g to note  t h a t , u n l i k e d i e t t h r e e , t h e d i g e s t i b i l i t y o f n i t r o g e n was n o t  - 109  -  s i g n i f i c a n t l y d i f f e r e n t from e i t h e r  d i e t two  t h r e e had  higher nitrogen  s i g n i f i c a n t l y (p  than d i e t f o u r ) . a t the  The  two  and  f a t t y a c i d s i n the  0.05)  amounts of m i c r o b i a l  duodenum were not  with diets  <  or d i e t f o u r  protein  four.  The  h i g h l e v e l s of the  The  v a l e r i c a c i d l e v e l s i n the  h i g h e r than the 3.19,  3.34  respectively). available the  arriving  two  although  and  protein  four,  i n the  not could  be  rumens.  proportions  c o n t a i n i n g formaldehyde t r e a t e d  3.30,  nitrogen  branched-chain  rumens of animals f e d d i e t one,  to g r e a t e r breakdown of d i e t a r y  Valeric acid  digestibility  markedly d i f f e r e n t w i t h d i e t f i v e compared  s i g n i f i c a n t l y d i f f e r e n t , compared w i t h d i e t s attributed  {diet  l e v e l s i n the and Cline  3.15%  rumens of animals f e d  f o r a g e were s i g n i f i c a n t l y  the  (p0.05)  rumens of animals f e d d i e t one  f o r d i e t s one,  two,  diets  (1.82,  t h r e e , f o u r and  five  et a l . (1958) r e p o r t e d that w i t h a decrease i n  n i t r o g e n i n the  rumen, v a l e r i c a c i d l e v e l s i n c r e a s e d .  present t r i a l , m i c r o b i a l  protein  s y n t h e s i s , measured by  In  % RNA-N:  % t o t a l abomasal d i g e s t a N o r % m i c r o b i a l - N : % t o t a l abomasal d i g e s t a N r a t i o , was  decreased w i t h the  (Table 9), a l t h o u g h not The was  was  significantly for diet  amount of m i c r o b i a l  markedly l e s s f o r the  u s i n g the  other four d i e t s  diets  sheep f i t t e d w i t h r e - e n t r a n t c a n n u l a . protein  f i v e compared w i t h d i e t  one.  two,  duodenum d a i l y  compared to d i e t  It i s l i k e l y that  s y n t h e s i s by  the h i g h e r l e v e l s of v a l e r i c a c i d w i t h d i e t s  one  three.  n i t r o g e n a r r i v i n g a t the  formaldehyde t r e a t e d  a l i m i t a t i o n to m i c r o b i a l  compared to d i e t  n i t r o g e n and three, four,  there  hence and  one  - 110  Barry  (1973a and  -  1976c) and  B a r r y and  Fennessy  (1973) however  r e p o r t e d lowered l e v e l s of i s o v a l e r i c and n - v a l e r i c a c i d s combined. Since the two to  a c i d s were combined i n these r e p o r t s , i t i s d i f f i c u l t  say which of these a c i d s a c t u a l l y c o n t r i b u t e d to the lowered  l e v e l s w i t h formaldehyde treatment.  Langlands (1973a) however r e p o r t e d  a r e d u c t i o n i n the l e v e l s of n - v a l e r i c a c i d , w i t h treatment of d i e t s .  However, Faichney  formaldehyde  and White (1977a) r e p o r t e d  an  i n c r e a s e i n the l e v e l of n - v a l e r i c a c i d w i t h formaldehyde treatment of concentrate and  diets.  The v a l u e s of n - v a l e r i c a c i d f o r the  the t r e a t e d d i e t s (4.10% and  4.98%  f o r untreated  t r e a t e d ' d i e t s r e s p e c t i v e l y ) were however not The  and  formaldehyde  significant.  i n c r e a s e d l e v e l of v a l e r i c a c i d i n the case of animals f e d  d i e t t h r e e , compared w i t h d i e t one  c o u l d be a t t r i b u t e d to l e s s d i g e s t i o n  of p r o t e i n i n the rumen f o r the former d i e t , a l t h o u g h d i g e s t i b i l i t y was The  untreated  not  overall  s i g n i f i c a n t l y d i f f e r e n t f o r the two  diets.  apparent d i g e s t i b i l i t y c o e f f i c i e n t of n i t r o g e n f o r d i e t one  s l i g h t l y h i g h e r than t h a t of d i e t  Abomasal and  Abomasal  was  three.  duodenal d i g e s t a parameters  pH  The  pH v a l u e s of the abomasal f l u i d were g e n e r a l l y h i g h e r  what have been r e p o r t e d by some workers (Knight Wheeler and N o l l e r , 1977).  The v a l u e s o b t a i n e d  were 3.84,  and  3.94,  3.88,  and'five r e s p e c t i v e l y .  3.83  3.70  et  than  a l . , 1972;  i n t h i s experiment  f o r d i e t s one,  two,  three,  four  wheeler and N o l l e r (1977) r e p o r t e d abomasal  pH  - Ill  -  v a l u e s o f 2.74 + 0.185 f o r sheep f e d d i e t s c o n t a i n i n g about 80% c o r n . Knight  e t a l . (1972) r e p o r t e d t h a t t h e r e was a c o n s i d e r a b l e  v a r i a t i o n i n abomasal pH.  The lowest  about one hour a f t e r f e e d i n g . 2.90.  There was a g r a d u a l  abomasal pH, about 2.05, o c c u r r e d  The p r e f e e d i n g pH l e v e l was  approaching  The r a t h e r h i g h pH l e v e l s observed i n t h i s  experiment cannot be e x p l a i n e d The  approximately  i n c r e a s e i n t h e abomasal pH from one hour  a f t e r f e e d i n g , w i t h l e v e l s s i x t o e i g h t hours a f t e r f e e d i n g , the p r e - f e e d i n g l e v e l s .  cyclical  fully.  samples f o r pH measurements were taken about f o u r t o s i x  hours a f t e r f e e d i n g and t h i s t i m i n g e f f e c t c o u l d have c o n t r i b u t e d t o the high values.  I t i s i n t e r e s t i n g t o n o t e t h a t Lee (1977) a l s o r e p o r t e d  h i g h abomasal pH v a l u e s f o r sheep f e d d i e t s o f e i t h e r wheat o r l u c e r n e (3.20  + 0.20 f o r wheat d i e t s and 3.49 + 0 . 3 3 f o r l u c e r n e d i e t s ) .  Lee  (1977) d i d n o t i n d i c a t e the l e n g t h o f time a f t e r f e e d i n g when t h e animals were s l a u g h t e r e d  f o r t h e measurements.  Abomasal d i g e s t a N%, duodenal d i g e s t a N%, and t o t a l d a i l y f l o w o f n i t r o g e n from t h e abomasum i n t o the duodenum  The (p y  abomasal n i t r o g e n c o n c e n t r a t i o n  (N%) was n o t s i g n i f i c a n t l y  0.05) a f f e c t e d by formaldehyde treatment o f t h e f o r a g e p o r t i o n o f  the d i e t s .  The v a l u e s , on a d r y matter b a s i s , were 2.27, 3.01, 2.80,  3.16 and 3.06% f o r d i e t s one, two, t h r e e , f o u r and f i v e r e s p e c t i v e l y . Sharma and N i c h o l s o n  (1975a) a l s o observed no s i g n i f i c a n t i n c r e a s e i n  the c o n c e n t r a t i o n o f n i t r o g e n i n abomasal d i g e s t a w i t h d i e t s c o n t a i n i n g formaldehyde t r e a t e d rapeseed meal (3.13% f o r u n t r e a t e d  and 3.20% f o r  - 112  formaldehyde t r e a t e d ) .  -  However, the t o t a l d a l l y amount of n i t r o g e n  f l o w i n g through the duodenum was  s i g n i f i c a n t l y higher  f o r the  c o n t a i n i n g formaldehyde t r e a t e d rapeseed meal than f o r the c o n t a i n i n g the u n t r e a t e d  rapeseed meal.  The  3.10,  2.96,  3.15  five respectively.  and  2.98%  f o r d i e t s one,  S i n c e o n l y one  animal was  two,  significant.  experiment  was  and  used i t i s not p o s s i b l e  the d i e t c o n t a i n i n g the u n t r e a t e d  They were most l i k e l y not  of  three, four  to say the d i f f e r e n c e s between the d i e t s c o n t a i n i n g the t r e a t e d f o r a g e s and  diet  n i t r o g e n content  duodenal d i g e s t a , on a dry matter b a s i s , i n the p r e s e n t 2.40,  diet  formaldehyde  f o r a g e were  s i g n i f i c a n t as they were s i m i l a r  to the abomasal d i g e s t a n i t r o g e n c o n c e n t r a t i o n r e p o r t e d above.  The  total  q u a n t i t i e s o f n i t r o g e n a r r i v i n g a t the duodenum d a i l y were 21.20, 31.02, 30.02, 31.14, and respectively  29.16g f o r d i e t s one,  (Table 14).  The  two,  t h r e e , f o u r and  five  f i g u r e s above i n d i c a t e t h a t the formaldehyde  treatment of the f o r a g e p o r t i o n of the d i e t g r e a t l y i n c r e a s e d the  daily  f l o w of n i t r o g e n from the abomasum t o the duodenum, whereas the c o n c e n t r a t i o n of n i t r o g e n i n the duodenal d i g e s t a was affected.  i n c r e a s e i n the q u a n t i t y of n i t r o g e n f l o w i n g through  the  duodenum d a i l y w i t h formaldehyde treatment o f the f o r a g e p o r t i o n of  the  d i e t was  The  not markedly  p o s s i b l y because of the i n c r e a s e i n the d a i l y flow of dry matter  w i t h formaldehyde treatment of the f o r a g e . who  r e p o r t e d no  Sharma and N i c h o l s o n  (1975a)  s i g n i f i c a n t i n c r e a s e i n the n i t r o g e n c o n c e n t r a t i o n  abomasal d i g e s t a but  s i g n i f i c a n t i n c r e a s e i n the t o t a l d a i l y flow  of of  n i t r o g e n through the duodenum w i t h formaldehyde treatment of the rapeseed meal p o r t i o n of d i e t , a l s o observed an i n c r e a s e i n the f l o w of dry matter through the duodenum w i t h formaldehyde treatment.  - 113 -  The d a i l y q u a n t i t i e s o f n i t r o g e n f l o w i n g through the duodenum on a l l the d i e t s c o n t a i n i n g t h e formaldehyde t r e a t e d f o r a g e g r e a t e r than the t o t a l q u a n t i t i e s o f n i t r o g e n consumed  were  daily  (-7.54g,  +1.88g, +0.8g, +2.45g, and +0.47g f o r d i e t s one, two, t h r e e , f o u r and five respectively).  The i n c r e a s e i n the amount of n i t r o g e n  arriving  d a i l y at t h e duodenum compared to the t o t a l d a i l y i n t a k e c o u l d be due to  (a)  increased r e c y c l i n g of urea-nitrogen  i n t o the f o u r  compartments o f the stomach, (b)  r e d u c t i o n i n the amount o f l o s s o f n i t r o g e n through the  ruminal w a l l .  Beever e_t a l . (1976) and Hemsley e t a l . (1970) r e p o r t e d a g r e a t e r amount o f n i t r o g e n f l o w i n g through the duodenum compared w i t h w i t h d i e t s c o n t a i n i n g formaldehyde t r e a t e d f o r a g e s .  intake  Hemsley £t a l . (1970)  r e p o r t e d lower q u a n t i t i e s o f n i t r o g e n f l o w i n g through the duodenum per day  than consumed w i t h u n t r e a t e d  forage d i e t .  Faichney  and White  (1977b)  r e p o r t e d d a i l y n e t g a i n i n n i t r o g e n i n the f o u r compartments of the stomach when f o u r c o n c e n t r a t e t r e a t e d were f e d (except  d i e t s , both untreated  f o r one u n t r e a t e d  and formaldehyde  d i e t where t h e r e was  loss).  The d a i l y n e t g a i n of n i t r o g e n was g r e a t e r f o r the t r e a t e d d i e t s than • f o r the u n t r e a t e d .  Sharma and N i c h o l s o n  (1975a) r e p o r t e d g r e a t e r q u a n t i t i e s  of n i t r o g e n f l o w i n g through the duodenum d a i l y than consumed c o n t a i n i n g b o t h formaldehyde t r e a t e d and u n t r e a t e d  on d i e t s  rapeseed meal.  The  same workers (Sharma and Nicholson,1975b) however r e p o r t e d lower q u a n t i t i e s of n i t r o g e n f l o w i n g through the duodenum than consumed  daily for diets  - 114  c o n t a i n i n g u n t r e a t e d and  -  formaldehyde t r e a t e d f a b a beans (net g a i n  i n n i t r o g e n d a i l y : -6g and -3g  f o r d i e t s containing untreated  and  formaldehyde t r e a t e d f a b a beans r e s p e c t i v e l y ) .  Abomasal and  The  duodenal d i g e s t a n o n - p r o t e i n n i t r o g e n l e v e l s  abomasal d i g e s t a c o n c e n t r a t i o n of n o n - p r o t e i n - n i t r o g e n  significantly  (p 4.  0.05)  h i g h e r f o r animals f e d d i e t one  animals f e d the other d i e t s .  five respectively.  0.656% f o r d i e t s one,  Sharma and N i c h o l s o n  two,  three, four  (1975a) r e p o r t e d abomasal  d i g e s t a n o n - p r o t e i n - n i t r o g e n c o n c e n t r a t i o n s of 0.61% matter b a s i s f o r d i e t s c o n t a i n i n g u n t r e a t e d and rapeseed meal r e s p e c t i v e l y .  than f o r  The v a l u e s , on a d r y matter b a s i s , were  0.864, 0.676, 0.643, 0.650 and and  was  and  0.64%  on a dry  formaldehyde t r e a t e d  These were not s i g n i f i c a n t l y  different.  The n o n - p r o t e i n - n i t r o g e n i n the abomasal d i g e s t a c o u l d come from d i e t a r y sources o r from i n t e r m e d i a r y p r o d u c t s of p r o t e i n breakdown i n the r e t i c u l o - r u m e n . composition  S i n c e , i n the p r e s e n t study, the i n g r e d i e n t  of a l l the d i e t s was  the same, the most l i k e l y  i n c r e a s e i n abomasal d i g e s t a n o n - p r o t e i n - n i t r o g e n w i t h the c o n t a i n i n g the u n t r e a t e d f o r a g e was o f p r o t e i n i n the The  intermediary products  source f o r the diets of the breakdown  reticulo-rumen.  c o n c e n t r a t i o n of n o n - p r o t e i n - n i t r o g e n i n the duodenal d i g e s t a  (Table 14) was  a l s o g r e a t e r w i t h the d i e t c o n t a i n i n g u n t r e a t e d  compared w i t h the other f o u r d i e t s  forage  (0.842, 0.614, 0.701, 0.662 and  on a dry matter b a s i s , f o r d i e t s one,  two,  t h r e e , f o u r and  five  0.681  respectively).  - 115 -  The t o t a l q u a n t i t i e s o f n o n - p r o t e i n - n i t r o g e n  flowing  through t h e duodenum p e r day was a l s o s l i g h t l y g r e a t e r w i t h t h e d i e t containing untreated 6.10,  forage  compared w i t h t h e o t h e r s  7.10, 6.52 and 6.66g f o r d i e t s one, two, t h r e e ,  i n that order).  (7.44,  f o u r and f i v e  The t o t a l q u a n t i t i e s o f t r u e - p r o t e i n - n i t r o g e n  a r r i v i n g a t t h e duodenum d a i l y was however markedly g r e a t e r w i t h t h e diets containing containing  t h e formaldehyde t r e a t e d f o r a g e  the untreated  forage  than f o r t h e d i e t  (13.76, 24.92, 22.92, 24.62 and 22.50g  f o r d i e t s one, two, t h r e e , f o u r , and f i v e r e s p e c t i v e l y ) . reported  s i g n i f i c a n t l y lower q u a n t i t i e s o f non-ammonia-nitrogen  through t h e duodenum d a i l y f o r u n t r e a t e d t r e a t e d d i e t s (Williams and White, 1977a). greater  Some workers  d i e t s compared w i t h formaldehyde  and Smith, 1976; Hemsley e t a l . , 1970;  Sharma and N i c h o l s o n  (1975a) r e p o r t e d  quantities of true-protein-nitrogen  a diet containing  flowing  Faichney  significantly  a r r i v i n g a t t h e duodenum w i t h  formaldehyde t r e a t e d rapeseed meal compared w i t h a  d i e t containing untreated  rapeseed meal.  Abomasal and duodenal d i g e s t a RNA-N, m i c r o b i a l N, %RNA-N: % t o t a l d i g e s t a N, % m i c r o b i a l N: % t o t a l d i g e s t a N  The abomasal d i g e s t a c o n c e n t r a t i o n  of r i b o n u c l e i c  acid-nitrogen  and t h e r e f o r e m i c r o b i a l p r o t e i n - n i t r o g e n were n o t a f f e c t e d (p > 0.05) by treatment  significantly  (Table 9 ) .  The r a t i o s o f % r i b o n u c l e i c a c i d - n i t r o g e n t o % t o t a l d i g e s t a n i t r o g e n and % m i c r o b i a l p r o t e i n - n i t r o g e n d i g e s t a n i t r o g e n were s i g n i f i c a n t l y  to % t o t a l  (p ^ 0.05) h i g h e r  abomasal abomasal  f o r d i e t one than  - 116  f o r d i e t s two,  f o u r , and  five.  -  L i n g and  Buttery  o f m i c r o b i a l n i t r o g e n to t o t a l d i g e s t a n i t r o g e n c o n t r i b u t i o n of m i c r o b i a l p r o t e i n - N The mean v a l u e s  reported  diets containing  by  f o r "diets c o n t a i n i n g  values  obtained  (0.786, 0.480, 0.583, 0.451 and  five respectively).  nitrogen 0.79,  those workers were 0.58,  and  and  0.59  The  i n the p r e s e n t  reported  r e s u l t s of the p r e s e n t  of m i c r o b i a l p r o t e i n - n i t r o g e n a f f e c t e d by  n i t r o g e n was  three,  four microbial  f l a k e d maize, crushed o a t s ,  to be  For d i e t s  contribution  at the abomasum the  concentration  ( m i c r o b i a l - p r o t e i n - n i t r o g e n %) as  this  the r a t i o of % m i c r o b i a l p r o t e i n affected.  With the duodenal d i g e s t a ,  using % microbial protein nitrogen: % t o t a l  (Table 14).  rolled  containing  experiment i n d i c a t e t h a t the  treatment but  0.58,  0.78.  the same as t o t a l m i c r o b i a l p r o t e i n n i t r o g e n :  protein nitrogen:  Therefore,  i t seems v a l i d to use  digesta  total  digesta  % microbial  % t o t a l n i t r o g e n r a t i o to c a l c u l a t e the  p r o t e i n n i t r o g e n c o n t r i b u t i o n to t o t a l N a r r i v i n g The  two,  to the n i t r o g e n a r r i v i n g  to % t o t a l n i t r o g e n was  the r a t i o o b t a i n e d  nitrogen  the  experiment.  subsequently the duodenum) c o u l d not be measured by  nitrogen  for  Their  Smith (1974) a l s o r e p o r t e d  f l a k e d maize p l u s u r e a r e s p e c t i v e l y .  of m i c r o b i a l p r o t e i n - n i t r o g e n  not  0.63  soybean were s i m i l a r to  0.439 f o r d i e t s one,  for diets containing  d a i r y cubes, the r a t i o was  was  and  to t o t a l d i g e s t a non-ammonia-nitrogen r a t i o s to be 0.60,  b a r l e y and  (and  diet  M c A l l a n and  1.01,  N.  soybean meal r e s p e c t i v e l y .  f i s h meal and  f o r the u n t r e a t e d  ratio  to measure the  to t o t a l duodenal d i g e s t a  f i s h meal, u r e a and  values  (1975) used the  microbial  at the duodenum.  c a l c u l a t i o n s f o r the abomasal samples are t h e r e f o r e  valid.  - 117 -  In t h e experiments o f M c A l l a n and Smith (1974)  RNA-N was  determined and c o n v e r t e d t o m i c r o b i a l n i t r o g e n by d i v i d i n g t h e RNA-N by 0.075 and m u l t i p l y i n g t h e p r o d u c t by 100. was  The 0.075 f i g u r e  supposed t o be t h e r a t i o o f RNA-N t o t o t a l N i n m i c r o b i a l p r o t e i n  (Smith, 1975).  Sutton e t a l . (1975) a l s o r e p o r t e d  In t h e experiment o f L i n g and B u t t e r y Kropp et^ a l . (1977) r e p o r t e d bacterial  a r a t i o o f 0.076:1.  (1975) t h e r a t i o used was 0.095.  t h a t t h e percentage o f RNA-N i n rumen  p r o t e i n was 10% or a r a t i o o f 0.10.  l i t e r a t u r e on t h e c o m p o s i t i o n o f b a c t e r i a l  Allison  (1970)  nitrogen reported  reviewing that n u c l e i c  a c i d s accounted f o r 14-19% of t o t a l m i c r o b i a l n i t r o g e n w i t h most o f i t coming from RNA s i n c e DNA accounts f o r o n l y 2.2-4.1%.  I n experiments  where t h e r a t i o has been determined, u s u a l l y b a c t e r i a samples a r e used and  t h e r a t i o may n o t a p p l y  t o mixed rumen microbes c o n t a i n i n g  protozoa.  I t i s t h e r e f o r e suggested here t h a t i n experiments where t h e r a t i o i s not  determined on mixed rumen microbes c o n t a i n i n g p r o t o z o a , and where  f i g u r e s f o r absolute m i c r o b i a l p r o t e i n synthesis  are not required but  o n l y comparisons between treatments a r e t o be made, i t may be b e t t e r t o use  a r a t i o o f RNA-N t o t o t a l d i g e s t a n i t r o g e n .  In t h i s  experiment  this ratio,  though lower than t h a t of m i c r o b i a l p r o t e i n - N :  parallelled  i t (Tables 9 and 1 4 ) .  The obtained digesta.  ratios  t o t a l N,  (RNA-N: t o t a l N o r m i c r o b i a l p r o t e i n N: t o t a l N  from duodenal d i g e s t a were s i m i l a r  t o those o f t h e abomasal  D i e t s one and t h r e e had t h e h i g h e s t  protein nitrogen arriving  a t t h e duodenum.  q u a n t i t i e s of m i c r o b i a l  While w i t h d i e t  one i t c a n  - 118 -  be s a i d t h a t t h e r e was a g r e a t  d e a l of d i e t a r y p r o t e i n c o n v e r t e d t o  m i c r o b i a l p r o t e i n w i t h some l o s s e s of n i t r o g e n , was a l s o a g r e a t  deal of conversion  with d i e t three  there  o f d i e t a r y p r o t e i n to m i c r o b i a l  p r o t e i n but w i t h l i t t l e o r no l o s s e s o f d i e t a r y p r o t e i n .  I t i s not  c l e a r why the a d d i t i o n o f t h e v o l a t i l e f a t t y a c i d s alone had those effects.  Abomasal ADF%, c e l l u l o s e % , duodenal ADF%, c e l l u l o s e % , and q u a n t i t i e s o f ADF and c e l l u l o s e a r r i v i n g at the duodenum  There were no d i f f e r e n c e s between the treatments w i t h r e s p e c t to concentration digesta  (Table  o f c e l l u l o s e and a c i d d e t e r g e n t f i b r e i n the abomasal 9).  Beever  e t a l . (1976) r e p o r t e d  of 13.62%, and 19.67% on an o r g a n i c treated d i e t s . concentrations  cellulose  concentrations  matter b a s i s , f o r u n t r e a t e d  Beever e t a l . (1977) r e p o r t e d  duodenal  and  digesta  o f 10.43%, 10.78%, and 11.97% f o r c o n t r o l , formaldehyde  t r e a t e d s i l a g e and formaldehyde t r e a t e d d r i e d s i l a g e (on OM b a s i s ) . In t h e two experiments o f (Beever, e t a l . , 1976; Beever, e t a l . , 1977) the q u a n t i t i e s o f c e l l u l o s e a r r i v i n g a t the duodenum were h i g h e r f o r the t r e a t e d than the u n t r e a t e d  materials.  be argued t h a t the c o n c e n t r a t i o n the u n t r e a t e d .  I n the f i r s t  experiment i t c o u l d  was> a l s o h i g h e r f o r the t r e a t e d  I n the second experiment the l e v e l s were almost  than identical.  The d i f f e r e n c e s i n the q u a n t i t i e s a r r i v i n g a t the duodenum were a f f e c t e d in  t h i s case by i n c r e a s e d  In the p r e s e n t t r i a l ,  flow of organic  i t i s therefore  abomasal d i g e s t a c o n c e n t r a t i o n s  matter through the duodenum.  p o s s i b l e t h a t even though the  of acid-detergent  f i b r e and c e l l u l o s e  - 119  -  were s i m i l a r on a l l the d i e t s , the q u a n t i t i e s e s c a p i n g d i g e s t i o n i n the f o u r compartments o f the stomach may  have been g r e a t e r f o r the  d i e t s c o n t a i n i n g the formaldehyde t r e a t e d forage than f o r the c o n t a i n i n g the u n t r e a t e d f o r a g e . detergent  f i b r e and  diet  In f a c t , the q u a n t i t i e s of a c i d -  c e l l u l o s e a r r i v i n g at the duodenum of the sheep  f i t t e d w i t h the r e - e n t r a n t cannula were h i g h e r f o r the d i e t s c o n t a i n i n g the t r e a t e d f o r a g e than the d i e t c o n t a i n i n g the u n t r e a t e d (Table 13).  The  forage  c o n c e n t r a t i o n s of c e l l u l o s e and a c i d - d e t e r g e n t  i n the duodenal d i g e s t a were not markedly d i f f e r e n t between  fibre  treatments  ( T a b l e 13). The  apparent  digestibility  c o e f f i c i e n t s of these f r a c t i o n s i n the  f o u r compartments of the stomach were h i g h e r f o r the d i e t c o n t a i n i n g the u n t r e a t e d f o r a g e than f o r the d i e t s c o n t a i n i n g the formaldehyde t r e a t e d f o r a g e ( T a b l e 15).  Duodenal d i g e s t a flow  The  experiment of MacRae and W i l s o n  (1978) demonstrated t h a t  t h e r e were no d i f f e r e n c e s between i n t a c t sheep and  sheep f i t t e d  with  duodenal r e - e n t r a n t cannulae w i t h r e s p e c t to feed i n t a k e , dry matter digestibility  and n i t r o g e n b a l a n c e .  r e s u l t s o b t a i n e d from the animal  I t i s t h e r e f o r e assumed t h a t the  f i t t e d w i t h the r e - e n t r a n t cannula i n  t h i s experiment c o u l d a p p l y to i n t a c t  sheep.  - 120  -  Oldham and L i n g (1977) and L e i b h o l z and Hartman (1972) r e p o r t e d t h a t the duodenal d i g e s t a f l o w measured over twenty f o u r hours w i t h o u t a marker c o r r e c t i o n , gave v a l i d e s t i m a t e s of duodenal f l o w the duodenum.  through  They f u r t h e r c l a i m e d t h a t c o r r e c t i n g f l o w f o r  r e c o v e r y of a marker was  of d o u b t f u l v a l u e f o r r e d u c i n g  i n parameters measured.  T h e r e f o r e , i n the p r e s e n t  c o r r e c t i o n s were made.  100%  variability  t r i a l no such marker  The duodenal d i g e s t a f l o w r a t e ( m l / h r ) ,  total  duodenal d i g e s t a f l o w (ml) per 24 h r s , t o t a l duodenal d i g e s t a d r y matter  f l o w p e r 24 h r  f l o w p e r 24 h r s  (g) and  t o t a l duodenal d i g e s t a o r g a n i c matter  (g), (Tables 12 and  13) were g r e a t e r f o r the  diets  c o n t a i n i n g the formaldehyde t r e a t e d f o r a g e than f o r d i e t one. expected  as t h e r e was  (% D.M.  f i v e r e s p e c t i v e l y ; % O.M.  of the stomach: t h r e e , f o u r and The  24.96 f o r d i e t s one,  Nicholson  two,  d r y matter and o r g a n i c matter c o n c e n t r a t i o n s of the duodenal  u n t r e a t e d rapeseed  this  three,,  28.09 f o r d i e t s one,  f o r treatments.  (1975a) r e p o r t e d d r y matter d i g e s t i b i l i t i e s of 33.6%  of  two,  respectively).  d i g e s t a were not markedly d i f f e r e n t  rapeseed  the  d i g e s t i o n i n the f o u r compartments  36.31, 27.12, 26.34, 26.98, and five  f e d compared  d i g e s t i o n i n the f o u r compartments of  stomach: 32.08, 23.28, 22.23, 24.20 and f o u r and  was  g r e a t e r d i g e s t i o n of dry matter or o r g a n i c matter  i n the f o u r compartments of the stomach when d i e t one was to the o t h e r d i e t s  This  and  22.0%  Sharma and  for diets containing  f o r the d i e t s c o n t a i n i n g formaldehyde t r e a t e d  meal i n the f o u r compartments of the stomach. experiment (1975a).  Nicholson  The  values  t h e r e f o r e a r e s i m i l a r to the v a l u e s of Sharma and  - 121  The  -  average h o u r l y d i g e s t a f l o w r a t e s were 647.20, 698.33,  709.135, 692.29 and  680.83 ml  five respectively.  P h i l l i p s and Dyck (1964) r e p o r t e d v a l u e s  672,  739,  800,  and  and  651  and  f o r d i e t s one,  two,  three, four  of t h e i r experiments.  (1969)  ml/hr  f o r some  l o n g , chopped and  290.64 ml/hr f o r d i e t s  ground b a r l e y straw r e s p e c t i v e l y .  about 900g/day (D.M.) and  ground maize accounted f o r  of the r a t i o n s . P h i l l i p s and Dyck (1964) r e p o r t e d t h a t t h e r e was  p a t t e r n of flow. o c c u r i n g 6-12 fed  to 773  Thompson and Lamming (1972) however r e p o r t e d  d i g e s t a f l o w r a t e s of 267.65, 299.84 and  54.7%  850,  Duodenal d i g e s t a f l o w r a t e s of 720  861 ml/hr were r e p o r t e d by Van't K l o o s t e r et^ al_.  Feed i n t a k e was  of  622 ml/hr f o r d a i l y dry matter i n t a k e s of 900,  900g r e s p e c t i v e l y .  c o n t a i n i n g 30%  and  highest  flow occurred  hrs a f t e r feeding.  once a day  feeding.  The  a t f e e d i n g w i t h the  lowest  In t h e i r experiments, the animals were  and a l l the feed was  T h i s p a t t e r n was  a diurnal cyclical  consumed w i t h i n a s h o r t time a f t e r  not observed i n the p r e s e n t  experiment.  L e i b h o l z and Hartman (1972) a l s o d i d not observe a c o n s i s t e n t d i u r n a l c y c l i c a l p a t t e r n of flow. at off  12 h r s and e f f e c t up  In t h i s t r i a l ,  w i t h d i e t one  slight f a l l  i n f l o w r a t e a t 18 h r s and  to 24 h r s .  With d i e t s two,  f o u r and  t h e r e was  almost a l e v e l l i n g  five,  t h e r e were  s l i g h t decreases i n the f l o w r a t e a t 12 h r s and r i s e s a t 18 h r s f a l l s at 24 h r s .  With d i e t t h r e e , t h e r e was  the twenty-four hour p e r i o d . patterns e i t h e r . i n t a k e a t 12 h r s . continued  up  The  a continuous  f l o w r a t e s d i d not  a rise  fall  and throughout  f o l l o w feed  intake  With a l l the d i e t s , t h e r e were d e c r e a s e s i n feed With d i e t s t h r e e and  to the 18 hr p e r i o d and  f i v e , the d e c l i n e i n feed  from t h e r e , t h e r e was  an  intake  increase.  - 122 -  With d i e t two, t h e r e was a r i s e i n feed i n t a k e from 12 h r s up t o twenty f o u r hours.  There were i n c r e a s e s i n feed i n t a k e a t 18 h r s and  d e c l i n e s up t o twenty f o u r hours w i t h d i e t s  one and f o u r  (Table 1 1 ) .  Perhaps t h e r e were no c y c l i c a l p a t t e r n s of flow i n t h i s experiment because feed i n t a k e was spread  almost over t h e twenty-four hour p e r i o d .  L e i b h o l z and Hartman (1972) f e d t h e i r animals h o u r l y from automatic continuous  feeders.  Perhaps one o f t h e reasons f o r t h e use o f v e r y s m a l l numbers o f animals f o r duodenal flow measurements where is  t h e tendiousness  automation does n o t e x i s t ,  o f c o l l e c t i n g data over v e r y l o n g p e r i o d s of time.  Some workers have t h e r e f o r e reduced t h e time p e r i o d over which measurements a r e made ( H a r r i s and P h i l l i p s o n ,  1962).  observed t h a t t h e accumulated r e c o v e r y f o r s i x separate  recovery  In t h e p r e s e n t  i n duodenal c o n t e n t s ,  collected  When t h e i r observed v a l u e s were c o r r e c t e d f o r  o f C^O^, t h e f l o w r a t e s approximated expected v a l u e s . t r i a l t h e average percentage flow f o r each 6-hr p e r i o d  of t h e t o t a l f l o w f o r ' t h e f i v e d i e t s  approximated 25% ( T a b l e 12) and f o r  a 12-hr p e r i o d , approximated 50% (Table 12) a l t h o u g h v a r i a t i o n s f o r each d i e t . if  (1962)  12-hr p e r i o d s from each o f f o u r sheep was o n l y 86-90%  of t h e expected v a l u e . 100%  H a r r i s and P h i l l i p s o n  t h e r e were c o n s i d e r a b l e  I t seems t h e r e f o r e t h a t i n t h e p r e s e n t  f i v e s e p a r a t e measurements had been taken f o r each d i e t  trial,  f o r 6-hr  or 12-hr p e r i o d , t h e average v a l u e s  c o u l d have been e x t r a p o l a t e d t o  a 24-hr c o l l e c t i o n p e r i o d , a l t h o u g h  no marker c o r r e c t i o n was made.  I t must be p o i n t e d out here however t h a t , s i n c e o n l y one animal used t h e e f f e c t s o f animal v a r i a t i o n s cannot be accounted f o r .  was  - 123  One  other  i s feed intake. levels  -  problem w i t h duodenal d i g e s t a flow r a t e measurement Feed i n t a k e i s u s u a l l y r e s t r i c t e d  below ad l i b i t u m i n t a k e .  i n most experiments to  T h i s i s because t h e r e i s a d i u r n a l  v a r i a t i o n i n feed i n t a k e w i t h animals f e d ad l i b i t u m and  considerable  d i u r n a l v a r i a t i o n s i n flow r a t e c o u l d occur.  i f sampling  Therefore,  i s done once over a 24-hr p e r i o d when t h e r e i s v a r i a t i o n i n feed f i g u r e s obtained  might not be e a s i l y i n t e r p r e t e d .  p e r i o d of measurement was digestibility present  trials,  With automation, i f the  extended, say to seven days, as i s done w i t h  then animals c o u l d be f e d ad l i b i t u m .  experiment feed  i n t a k e was  The  from feed (p >  0.05)  d i e t was  i n t a k e assays e a r l i e r  the carried  l e v e l of f e e d i n g  f i x e d at average d a i l y ad l i b i t u m i n t a k e f o r d i e t two Only one  In  r e s t r i c t e d as measurement was  out once over a 24-hr p e r i o d f o r each d i e t .  of the t r i a l .  intake,  at the  was  beginning  used to f i x the i n t a k e l e v e l as r e s u l t s i n d i c a t e d t h e r e were no  d i f f e r e n c e s between the d i e t s  (Table 4 ) .  significant  It i s possible  however t h a t a t the a c t u a l time of measurement of flow r a t e f o r each d i e t , the l e v e l of feed  i n t a k e determined was  i n t a k e as the animal continued  not a c t u a l l y the ad l i b i t u m  growing throughout the experimental  period.  Sulphur metabolism  Sulphur i n t a k e , i f expressed as grams i n t a k e per u n i t of body s i z e per day, f o r d i e t s one,  two,  was and  s i g n i f i c a n t l y higher three  (p < 0.05).  f o r d i e t s f o u r and I t was  not  metabolic five  than  s u r p r i s i n g that  - 124  animals f e d d i e t s f o u r and per day  f i v e consumed g r e a t e r amounts of  compared to the o t h e r s  d i e t s was  h i g h e r and  -  (g) as sulphur  feed i n t a k e was  f o r a l l the d i e t s ( T a b l e s 3 and  4).  not  c o n c e n t r a t i o n i n those  significantly  When sulphur  different  i n t a k e was  i n a b s o l u t e terms (g/day) the animals on d i e t s f o u r and more s u l p h u r  per day  (p ^  0.05)  but not animals on d i e t t h r e e . (g/day) of animals on d i e t animals on d i e t s f o u r and metabolic  The  I t must be noted t h a t dry matter  intake  t h r e e tended to be g r e a t e r than i n t a k e of five.  Converting  the sulphur  i n t a k e s to  the e f f e c t s of  amounts of s u l p h u r  excreted  t h r e e compared to  i n the u r i n e per day  expressed  body s i z e were g r e a t e r f o r d i e t s f o u r  f i v e than f o r the other d i e t s (Table 7, p  < 0.05).  i n the u r i n e by animals on the two  The  to the h i g h e r  s u l p h u r added was  g r e a t l y by  most l i k e l y not u t i l i z e d  higher  intakes.  p o s s i b l y not due  of the rumen microbes to the supplement.  f o u r to twenty seven hours w h i l e B i r d required.  The  diets, The  low  to n o n - a d a p t a t i o n  B i r d and Moir  t h a t micro-organisms might adapt to sulphur  and  the rumen m i c r o -  organisms f o r the s y n t h e s i s of s u l p h u r - c o n t a i n i n g amino a c i d s - The u t i l i z a t i o n of the added sulphur was  as  excretion  sulphur-supplemented  compared to the o t h e r s c o u l d p a r t l y be due  n i n e days was  the  five.  grams per u n i t of m e t a b o l i c  of sulphur  f i v e consumed two  the s l i g h t l y l a r g e r s i z e of animals on d i e t  d i e t s f o u r and  expressed  than the animals on d i e t s one and  body s i z e b a s i s , removed, to some extent,  i n t a k e , and  sulphur  (1971) p o s t u l a t e d  supplementation w i t h i n twenty  (1972b) claimed  that a p e r i o d of  p r e l i m i n a r y p e r i o d i n the p r e s e n t  trial  - 125 -  before  t h e s t a r t o f t h e metabolism s t u d i e s was twenty days.  Kahlon  et a l . (1975a) r e p o r t e d  that i n i n v i t r o  a v a i l a b i l i t y of sulphur  from sodium s u l p h a t e 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 was 55.4%.  c u l t u r e systems, the  These workers a l s o observed t h a t a sulphur  c o n c e n t r a t i o n of 21.5 ug/ml o f rumen innoculum was not adequate t o meet t h e needs o f rumen microbes i n an i n v i t r o system w h i l e of 86.7 pg/ml and 130 pg/ml were a p p a r e n t l y protein synthesis.  A sulphur  concentrations  i n h i b i t o r y to m i c r o b i a l  c o n c e n t r a t i o n o f 43.3 ug/ml was r e p o r t e d  by them t o r e s u l t i n the g r e a t e s t 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  Bird  (1970) r e p o r t e d  t h a t t h e r e were no d i f f e r e n c e s i n 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 due t o source however r e p o r t e d  Hume  of sulphur.  that l o s s e s of sulphur  Johnson et a l . (1970)  t o rumen microbes due to i t s  e x c r e t i o n i n the f a e c e s were 20.39%, 21.77%, and 63.32% f o r methionine, sodium s u l p h a t e and elemental sulphur,supplementory sources r e s p e c t i v e l y . The  low u t i l i z a t i o n o f t h e added s u l p h a t e  i n the p r e s e n t  study was not  l i k e l y due t o i t s low a v a i l a b i l i t y 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 . The  t o t a l amount o f s u l p h u r  lost  i n t h e u r i n e and f a e c e s expressed  p e r c e n t a g e o f i n t a k e was not s i g n i f i c a n t l y h i g h e r f o u r and f i v e than f o r d i e t one and not h i g h e r d i e t three;  0.05) f o r d i e t s  f o r d i e t f o u r than f o r  (79.67%, 63.70%, 67.57%, 76.89%, 80.28% f o r d i e t s one, two,  t h r e e , f o u r , and f i v e ) . diet  (p >  as a  There was b e t t e r sulphur  two compared t o d i e t s f o u r and f i v e  (p < 0.05).  from data on the c o n t r i b u t i o n o f m i c r o b i a l abomasal or duodenal d i g e s t a n i t r o g e n  u t i l i z a t i o n only f o r I t c o u l d be i n f e r r e d  p r o t e i n - n i t r o g e n to . t o t a l  (Tables 9 and 14) t h a t the added  sulphate d i d not i n h i b i t m i c r o b i a l p r o t e i n synthesis to a great  extent  - 126 -  and  t h e r e f o r e i t s low u t i l i z a t i o n c o u l d not be a t t r i b u t e d t o t h a t .  The added s u l p h a t e s u p p l i e d about 0.15% sulphur which was below the 0.2%  level Bird  (1972b) suggested  was the maximum l e v e l f o r supplementation.  Above t h i s l e v e l a c c o r d i n g t o B i r d c o u l d occur e s p e c i a l l y i f energy  (1972b) hydrogen s u l p h i d e t o x i c i t y  or n i t r o g e n was l i m i t i n g .  The hydrogen  s u l p h i d e r e s u l t s from t h e r e d u c t i o n o f s u l p h a t e t o s u l p h i d e .  Sulphate  i s converted to s u l p h i d e b e f o r e i t i s i n c o r p o r a t e d i n t o s u l p h u r c o n t a i n i n g amino a c i d s by t h e rumen microbes  ( B i r d , 1971; Saeur e t a l . , 1975;  Dodgson and Rose, 1966). Sulphur  supplementation  was n o t r e q u i r e d i n the p r e s e n t  trial.  Sulphur was most l i k e l y n o t l i m i t i n g i n the d i e t s c o n t a i n i n g formaldehyde t r e a t e d f o r a g e without  s u l p h a t e supplementation.  There were h i g h  of e x t r a c t a b l e s u l p h a t e - s u l p h u r i n the grass-legume f o r a g e  (Table  levels 3).Beaton  et a l . (1968) and M a r t i n (1972) r e p o r t e d t h a t s u l p h u r i n sulphur c o n t a i n i n g amino a c i d s accounts plants.  f o r about n i n e t y p e r c e n t of the t o t a l s u l p h u r i n  S u l p h a t e - s u l p h u r from t h i s e s t i m a t e , would account  of t e n - p e r c e n t of t o t a l s u l p h u r i n p l a n t s , not t a k i n g i n t o  f o r a maximum account  s u l p h u r p r e s e n t i n o r g a n i c compounds o t h e r than amino a c i d s .  The l e v e l  of e x t r a c t a b l e s u l p h a t e - s u l p h u r i n the grass-legume f o r a g e (about h i g h e r than the f i g u r e of Beaton e t a l . (1968) and M a r t i n Sulphate-sulphur l e v e l s i n p l a n t s could increase with application  (Bray and Hemsley, 1969).  applying f e r t i l i z e r  29%) was  (1972).  fertilizer  Jones and Q u a g l i a t o  (1973)  s u l p h u r t o some t r o p i c a l f o r a g e s r e p o r t e d marked  i n c r e a s e s i n s u l p h a t e - s u l p h u r compared t o t o t a l s u l p h u r  content.  - 127  -  With formaldehyde treatment from the grass-legume f o r a g e was  even i f a l l the o r g a n i c  not a v a i l a b l e , the t o t a l amounts  of sulphur from the s u l p h a t e - s u l p h u r and  sulphur  i n the grass-legume f o r a g e ,  the cassava and b a r l e y would be about 0.069%.  The  t o t a l amount  of n i t r o g e n from the n o n - p r o t e i n n i t r o g e n of the grass-legume f o r a g e (assuming a l l the p r o t e i n n i t r o g e n was cassava and  b a r l e y would be 0.68%.  would be about 1:10  The  p r o t e c t e d ) the n i t r o g e n i n the r a t i o of sulphur to n i t r o g e n  which i s the same as the optimum r e p o r t e d by  Kennedy jst a l . (1975),  f o r low q u a l i t y f o r a g e .  They took i n t o  the f a c t t h a t r e c y c l e d n i t r o g e n i n t o the rumen was s u l p h u r , when a r r i v i n g a t t h e above r a t i o .  g r e a t e r than r e c y c l e d  Whanger and Matrone (1966)  r e p o r t e d t h a t w i t h s u l p h u r d e f i c i e n c y i n the rumen, t h e r e was of l a c t i c a c i d .  The a c c u m u l a t i o n  account  of l a c t i c a c i d was  due  an  to the non-  f u n c t i o n i n g of the a c r y l a t e pathway f o r c o n v e r s i o n of l a c t i c a c i d propionic acid  (Whanger and Matrone, 1967).  r e p o r t e d reduced  to  These workers, p r e v i o u s l y  l e v e l s of p r o p i o n i c a c i d , b u t y r i c and h i g h e r f a t t y a c i d s  i n rumens of animals  f e d s u l p h u r - d e f i c i e n t d i e t s compared to  f e d d i e t s c o n t a i n i n g adequate l e v e l s of sulphur 1965).  accumulation  In the p r e s e n t  animals  (Whanger and Matrone,  t r i a l p r o p i o n i c and b u t y r i c a c i d  l e v e l s were not  lower f o r the d i e t s c o n t a i n i n g the formaldehyde t r e a t e d f o r a g e sulphur supplementation forage with sulphur  without  than the d i e t s c o n t a i n i n g the formaldehyde t r e a t e d  supplementation.  The percentages  of s u l p h u r i n t a k e e x c r e t e d i n the u r i n e were  h i g h e r f o r d i e t s f o u r and  f i v e than f o r d i e t s two  and  t h r e e (p  ^0.05)  - 128  but not f o r d i e t one  (p >  0.05).  e x c r e t e d i n the u r i n e was and t h r e e (p < 0.05).  -  The percentage of sulphur i n t a k e  h i g h e r f o r d i e t one than was  for diets  two  The reasons f o r the h i g h e r ; p e r c e n t a g e s of  e x c r e t i o n o f s u l p h u r i n the u r i n e f o r d i e t s f o u r and f i v e compared to d i e t s two and t h r e e a r e s i m i l a r to those g i v e n a l r e a d y above. was  There  a g r e a t e r percentage of s u l p h u r e x c r e t e d i n u r i n e w i t h d i e t one  compared  to d i e t s two and t h r e e a l t h o u g h the amounts e x c r e t e d per u n i t o f m e t a b o l i c body s i z e were s i m i l a r because when c a l c u l a t e d on a percentage d i f f e r e n c e s i n i n t a k e a r e not taken i n t o  basis,  account.  Sulphur b a l a n c e , expressed e i t h e r as grams per u n i t of m e t a b o l i c body s i z e or i n a b s o l u t e terms was than f o r d i e t one  (p < 0.-05) .  nitrogen retention.  b e t t e r f o r d i e t s two,  Sulphur r e t e n t i o n was  S i n c e animals on d i e t s two and  t h r e e and  closely linked with t h r e e had g r e a t e r  ( s i g n i f i c a n t ) r e t e n t i o n of n i t r o g e n than animals on d i e t one, not s u r p r i s i n g t h a t s u l p h u r r e t e n t i o n was d i e t one.  four  i t was  b e t t e r on these two d i e t s than f o r  With d i e t f o u r the animals r e t a i n e d more n i t r o g e n (approaching  5% s i g n i f i c a n c e ) than animals on d i e t one.  The s u l p h u r - n i t r o g e n r a t i o  a l s o tended to be b e t t e r , though not s i g n i i f i c a n t , f o r animals on d i e t f o u r than animals on d i e t  one.  The s u l p h u r to n i t r o g e n r a t i o s were not a f f e c t e d by (10.13, 9.78,  12.26, 9.73,  and f i v e r e s p e c t i v e l y ) . of about 1 3 . 5 + 0 . 5 8 . experiment  and 10.36  Bird  f o r d i e t s one,  three, four  (1972a) r e p o r t e d a somewhat h i g h e r r a t i o  I t i s not c l e a r why  except t h a t perhaps  two,  treatments  the r a t i o s were lower i n t h i s  s u l p h u r u t i l i z a t i o n might  have been b e t t e r  - 129  than the experiments of B i r d  -  (1972a).  I t must a l s o be noted t h a t  i s c o n s i d e r a b l e v a r i a t i o n i n the r e p o r t e d r a t i o s as B i r d the r a t i o i n sheep t i s s u e s to be 15 and r a t i o s of 1:8.14 to 1:27.16  Kahlon et^ al.  of r e t a i n e d sulphur  there  (1973) r e p o r t e d  (1975b) r e p o r t e d  to r e t a i n e d n i t r o g e n i n  sheep.  Nitrogen  Metabolism  The  d a i l y n i t r o g e n i n t a k e , expressed  28.64, 33.44, 27.80 and  29.51g f o r d i e t s one,  f i v e r e s p e c t i v e l y ) or expressed size and  (2.07, 2.18,  2.38,  2.08  body s i z e  as i n t a k e / d a y / u n i t  and  three, four of m e t a b o l i c  2.18g,for d i e t s one,  two,  The  body s i z e s of the animals (p >  n i t r o g e n balance  6.86g f o r d i e t s one,  only greater f o r d i e t  body  three,  four  0.05,  Table  6).  metabolic  expressed  expressed  two,  t h r e e , f o u r and  as grams per day  0.05).  as g/day (4.49, 7.43,  t h r e e than f o r d i e t one  f i v e r e s p e c t i v e l y ) was (p  (Table 4) were not  0.05).  (0.313, 0.567, 0.726, 0.522, and  d i e t one  (p }  and  ( T a b l e 4), the c o n c e n t r a t i o n of n i t r o g e n i n the d i e t s (Table 3),  the m e t a b o l i c  balance  terms (29.81,  to the f a c t t h a t dry matter i n t a k e per u n i t of  significantly different  and  two,  f i v e r e s p e c t i v e l y ) were not a f f e c t e d by treatments  T h i s i s perhaps due  and  i n absolute  d i e t two when expressed  6.99,  f i v e r e s p e c t i v e l y ) was (p  0.05).  per u n i t of m e t a b o l i c  0.508g f o r d i e t s one,  however g r e a t e r f o r d i e t s two There was  10.19,  Nitrogen body s i z e  two,  and  three, four  t h r e e than f o r  an improvement i n n i t r o g e n b a l a n c e  as r e t a i n e d n i t r o g e n per u n i t of  for  metabolic  body s i z e perhaps because of the s l i g h t l y l a r g e r weight of animals  on  and  - 130  d i e t one  than on d i e t  two  -  (Table 4 ) .  Animals on d i e t  however about the same weight as animals on. d i e t one  t h r e e were and  retained a  s i g n i f i c a n t l y g r e a t e r amount of n i t r o g e n i n each case and per se was  hence s i z e  not a f a c t o r c o n t r i b u t i n g to the g r e a t e r r e t e n t i o n of  n i t r o g e n by animals on d i e t  t h r e e than animals  r e t e n t i o n of n i t r o g e n of animals on d i e t s two  on d i e t one. and  The  t h r e e was  greater  due  mainly  to the g r e a t e r u r i n a r y l o s s e s of n i t r o g e n by animals on d i e t one the animals on d i e t s two  and  i n animals f e d d i e t one was as ammonia c o n c e n t r a t i o n  three.  M i c r o b i a l degradation  than  of p r o t e i n  g r e a t e r than animals on d i e t s two  and  i n the rumens of animals f e d d i e t one  three  was  g r e a t e r than the ammonia c o n c e n t r a t i o n i n the rumens of animals f e d d i e t s two  and  three  (Table 8).  d i e t a r y sources and  14).  was  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  a l s o g r e a t e r f o r d i e t one  than f o r d i e t two  With the sheep f i t t e d w i t h r e - e n t r a n t cannula  the  (Tables 9  t h e r e were l o s s e s  of n i t r o g e n i n the f o u r compartments of the stomach f o r d i e t one w h i l e were net g a i n s f o r d i e t s two lost  and  through the r u m i n a l w a l l .  i t may  be converted  by Houpt  (1970).  not decreased  three  significantly  be l o s t  t h r e e , although (p  Ammonia c o u l d  When such ammonia a r r i v e s i n the  to urea which may  With d i e t  (Table 15).  0.05)  i n the u r i n e as  be liver,  described  nitrogen d i g e s t i b i l i t y  compared to d i e t one,  there  no net l o s s of n i t r o g e n i n the f o u r compartments of the stomach. i s an i n d i c a t i o n of more e f f i c i e n t u t i l i z a t i o n of n i t r o g e n by  there  was was This  the rumen  microbes i n the rumens of animals f e d d i e t t h r e e compared to animals f e d d i e t one.  In f a c t , m i c r o b i a l p r o t e i n N c o n t r i b u t i o n to duodenal d i g e s t a  N  significantly  one  was  not  (Table 9).  (p y.  0.05)  lower f o r d i e t t h r e e than f o r d i e t  - 131 -  Within the diets containing the formaldehyde treated forage sulphur supplementation tended to reduce nitrogen retention (diets 4 and 5 vs diet 2 and 3 ).  The cause of this effect i s not clear although  in the case of animals on diet four, i t was mediated p a r t l y through reduced d i g e s t i b i l i t y of nitrogen. was  In the case of diet f i v e ,  there  an abnormally high loss of nitrogen i n the urine compared to the  other diets containing the formaldehyde treated forage.  Winter (1976)  reported that the addition of sulphur to starter diets containing biuret reduced weight gains i n calves by 20%, feed intake by 9% and feed by 12%.  effici  Brown and Arlyne (1970) reported that sulphate added to rat  diets improved performances only up to 0.10% Above that l e v e l , performance was  l e v e l of supplementation.  decreased although not  significantly.  Measuring nitrogen retention as a percentage of digested, a l l . t h e diets containing the formaldehyde treated forage were s i g n i f i c a n t l y (p ^0.05) superior to the diet containing the untreated was because a greater amount of the nitrogen digested was urine with diet one compared to the others.  forage.  This  lost i n the  S i m i l a r l y , using nitrogen  retained as a percentage of intake as an index of nitrogen retention, diets two,  three, four and f i v e were better than diet one  Diet three was  (p ^ 0.05).  also superior to diet f i v e (p <0.05) using nitrogen  retained as a percentage of intake as an index of nitrogen u t i l i z a t i o n . These measures did not follow c l o s e l y nitrogen balance expressed as grams per day per unit ;of metabolic body size. This was because those measures do not take into account variations i n nitrogen intake, and nitrogen d i g e s t i b i l i t y with the animals on the d i f f e r e n t d i e t s .  They  - 132 -  a l s o d i d not take i n t o account the v a r i a t i o n s i n t h e s i z e s of animals used.  Amos et_ al. (1974) and D r i e d g e r and H a t f i e l d (1972) a l s o  reported  t h a t n i t r o g e n r e t a i n e d as a percentage o f i n t a k e d i d n o t f o l l o w  nitrogen balance with d i f f e r e n t d i e t s .  Nitrogen  l o s s e s i n the u r i n e  (g/Wkg^' ~Vday or percentage o f d i g e s t e d ) d i d not f o l l o w c l o s e l y the 7  nitrogen balance f i g u r e s .  T h i s was because the measured v a l u e s  take i n t o account d i f f e r e n c e s i n i n t a k e and i n d i g e s t i b i l i t y .  d i d not The  d i f f e r e n c e s i n n i t r o g e n e x c r e t i o n were n o t due t o d i f f e r e n c e s i n output of u r i n e .  D a i l y e x c r e t i o n of u r i n e  treatment  (p>0.05, T a b l e 4 ) .  (ml/Wkg^ * ~*) was not a f f e c t e d by 7  Growth r a t e  The  growth r a t e s of t h e animals d u r i n g  (pre-metabolism study p e r i o d ) by d i e t a r y treatment  the f i r s t  were not s i g n i f i c a n t l y  (Table 4 ) .  seventeen days  (p >  0.05) a f f e c t e d  The growth r a t e f i g u r e s were 154.76, 170.77,  170.77, 160.10 and 149.42g/day f o r d i e t s one, two, t h r e e , f o u r and f i v e respectively.  Rattray  and Joyce (1970) r e p o r t e d  a p o s i t i v e response of  n i t r o g e n r e t e n t i o n but not wool growth or growth r a t e w i t h formaldehyde treatment of t h e i r d i e t s . It and  T h e i r e x p e r i m e n t a l p e r i o d was f i v e weeks.  i s p o s s i b l e t h a t i n t h e present Joyce (1970),  experiment, as i n t h e t r i a l of R a t t r a y  t h e p e r i o d o f t h e experiment was too s h o r t f o r a  growth r a t e response t o be demonstrated.  Ames and B r i n k  (1977) however  measured growth r a t e s of sheep a t d i f f e r e n t environmental temperatures  - 133 -  for  o n l y twelve days and d i f f e r e n c e s i n responses c o u l d be a s s e s s e d .  D r i e d g e r and H a t f i e l d  (1972) a l s o used a s i x t e e n - d a y p r e l i m i n a r y  and a s i x - d a y metabolism  study p e r i o d .  period  They observed d i f f e r e n c e s i n  responses t o n i t r o g e n r e t e n t i o n d u r i n g t h e metabolism  study p e r i o d and  a l s o growth r a t e d u r i n g the s i x t e e n - d a y p r e l i m i n a r y p e r i o d w i t h t a n n i n treatment o f soybean meal.  The growth r a t e s of t h e i r animals f o r the  s i x t e e n - d a y p e r i o d were 277g/day f o r t h e d i e t c o n t a i n i n g the t a n n i n t r e a t e d soybean meal and 177g/day f o r the d i e t c o n t a i n i n g the u n t r e a t e d soybean  meal.  The growth r a t e f i g u r e s r e p o r t e d i n the p r e s e n t t r i a l were lower than those r e p o r t e d by T a i t  (1972) f o r male lambs.  H i s f i g u r e s were  237g/day, 239g/day and 233g/day f o r animals f e d 100% d r i e d g r a s s , 50% b a r l e y p l u s 50% d r i e d g r a s s and 100% b a r l e y r a t i o n s r e s p e c t i v e l y . animals used by T a i t the experiment  (1972) weighed about  (average) 19.2kg a t the s t a r t of  and were f e d t o a t t a i n t h e weight  In  the p r e s e n t experiment  at  t h e s t a r t of the experiment.  t h e animals ranged  of about  45kg ( a v e r a g e ) .  i n bodyweight of 29kg t o 36kg  The g r e a t e r growth r a t e s of the animals used by T a i t compared t o those of t h e animals used be accounted experiments.  i n the present t r i a l  (1972) c o u l d most  likely  f o r by the v a r i a t i o n s i n t h e i r s i z e s a t t h e s t a r t of the Adeleye  (1972) u s i n g animals w i t h average bodyweight of  30.65kg a t t h e s t a r t o f t h e experiment llOg,  The  r e p o r t e d d a i l y weight  g a i n of  70g, 90g, 130g, and 70g when d i e t s c o n t a i n i n g soybean meal, u r e a ,  b i u r e t , p o u l t r y droppings and p o u l t r y l i t t e r  were f e d .  A l l these  trials  ( T a i t , 1972; Adeleye, 1972; and p r e s e n t t r i a l ) were c a r r i e d out on t h e same farm.  The same breed o f sheep (Dorset) was used.  The o t h e r f a c t o r  - 134 -  which c o u l d cause v a r i a t i o n s i n the growth r a t e s of the animals i n a the experiments a p a r t  from the s t a g e of growth a t which measurements  were c a r r i e d out , was  type of feed and l e v e l of i n t a k e .  - 135  SUMMARY AND  -  CONCLUSIONS  D i f f e r e n t l e v e l s of formaldehyde (0.0%, 0.8%, dry b a s i s ) were a p p l i e d to a g r a s s - c l o v e r f o r a g e l e v e l f o r p r o t e c t i o n o f the p r o t e i n . reduced s i g n i f i c a n t l y was  (p  <  0.05)  i n c r e a s e d except between 1.0%  incubation 1.2%  1.0%  and  to determine the optimum  and  1.2%,  (31.88%, 15.72%, 6.87%, and  at the m i c r o b i a l stage of  5.69% The  f o r 0.0%,  0.8%,  0.8%,  1.0%  and  (228.79 ppm, 1.2%  78.58 ppm,  a l s o reduced  65.27 ppm,  i n c u b a t i o n , was  and  reduced s i g n i f i c a n t l y  1.0%  and  1.2%  l e v e l chosen was  except between  acid-pepsin  stages  s i n c e n i t r o g e n d i g e s t i b i l i t y was  The  0.0%,  optimum  significantly  reduced at the combined m i c r o b i a l and  stages,  (p y  0.05)  compared to  untreated. Ram  lambs r a n g i n g  i n body weights of 29kg to 36kg were then used  i n i n v i v o s t u d i e s of n i t r o g e n and  carbohydrate u t i l i z a t i o n  treatment o f the g r a s s - c l o v e r f o r a g e The  level  71.01% f o r  significantly  acid-pepsin  0.0%,  of  (p<,0.05) o n l y at the 1.2%  (p ^ 0 . 0 5 ) reduced a t the m i c r o b i a l stage but not  the  for  Nitrogen  l e v e l s of formaldehyde a p p l i c a t i o n ) .  1.0%  production,  30.04 ppm  of formaldehyde a p p l i c a t i o n (80.95%, 79.76%, 75.85%, and 0.8%,  and  significantly  l e v e l s o f formaldehyde t r e a t m e n t ) .  d i g e s t i b i l i t y f o r the combined m i c r o b i a l and  1.0%  ammonia n i t r o g e n  (p <0.05) as l e v e l of formaldehyde a p p l i c a t i o n i n c r e a s e d 1.0%  was  as l e v e l o f formaldehyde a p p l i c a t i o n  a t the m i c r o b i a l stage of i n c u b a t i o n , was  and  on,an a i r  In v i t r o n i t r o g e n d i g e s t i b i l i t y  l e v e l s of formaldehyde t r e a t m e n t ) .  0.8%,  1.2%  e f f e c t s of supplementation w i t h  with  a t 1% l e v e l of formaldehyde. i s o v a l e r i c and  isobutyric acids  - 136 -  and/or s u l p h u r were a l s o s t u d i e d .  The d i e t s  (14% CP on D.M.' b a s i s )  c o n t a i n e d 50% g r a s s - c l o v e r f o r a g e , 38% cassava, 11% b a r l e y and 1% sheep m i n e r a l premix on d r y matter b a s i s .  Sodium s u l p h a t e was added  a t 0.67% r e p l a c i n g an equal p o r t i o n of the cassava i n t h e d i e t s supplemented w i t h s u l p h u r .  D i e t one c o n t a i n e d the u n t r e a t e d  w h i l e the o t h e r s c o n t a i n e d the formaldehyde t r e a t e d f o r a g e . t h r e e and f i v e were supplemented w i t h i s o v a l e r i c a c i d and i s o b u t y r i c a c i d  forage Diets  (3.0g/kg d i e t )  (2.3g/kg d i e t ) and d i e t s f o u r and f i v e were  supplemented w i t h s u l p h u r .  The v o l a t i l e f a t t y a c i d s were sprayed  onto  the d i e t s j u s t b e f o r e f e e d i n g . Dry matter  i n t a k e (g/Wkg^" "Vday), apparent 7  c o e f f i c i e n t s of d r y matter  and o r g a n i c matter were not s i g n i f i c a n t l y  (p y 0.05) a f f e c t e d by d i e t a r y treatments. the forage s i g n i f i c a n t l y  digestibility  (p  Formaldehyde treatment o f  0.05) i n c r e a s e d the apparent  c o e f f i c i e n t s o f a c i d - d e t e r g e n t f i b r e and c e l l u l o s e .  digestibility  (ADF D i g e s t i b i l i t i e s :  32.57%, 36.97%, 36.91%, 36.45% and 36.59% f o r d i e t s one, two, t h r e e , f o u r and f i v e r e s p e c t i v e l y ; C e l l u l o s e d i g e s t i b i l i t i e s :  42.95%, 49.10%,  49.04%, 49.33% and 48.76% f o r d i e t s one, two, t h r e e , f o u r and f i v e respectively.  G r e a t e r amounts o f the a c i d - d e t e r g e n t f i b r e and c e l l u l o s e  were d i g e s t e d i n the hindgut Rumen pH, d r y matter  w i t h formaldehyde treatment  o f the forage.  content i n the rumen, rumen l e v e l s of v o l a t i l e  f a t t y a c i d s , p r o p i o n i c , b u t y r i c and a c e t i c a c i d s were n o t a f f e c t e d significantly  ( p ^ 0.05) by t h e d i e t a r y treatments.  significantly  (p < 0.05) i n c r e a s e d l e v e l s of i s o v a l e r i c and i s o b u t y r i c  a c i d s i n the rumens o f animals d i e t s two and f o u r .  There were however,  f e d d i e t t h r e e compared t o those f e d  The i s o v a l e r i c a c i d and i s o b u t y r i c a c i d  levels  - 137 -  tended t o decrease w i t h t h e d i e t s c o n t a i n i n g t h e formaldehyde t r e a t e d f o r a g e w i t h o u t v o l a t i l e f a t t y a c i d supplementation compared t o t h e d i e t c o n t a i n i n g the u n t r e a t e d degradation  m a t e r i a l , most l i k e l y because o f g r e a t e r  o f p r o t e i n s i n t h e rumen w i t h  ( i s o v a l e r i c : 2.53,  the l a t t e r :than w i t h t h e former  0.60, 2.58, 0.39 and 2.18% f o r d i e t s one, two, t h r e e ,  f o u r and f i v e r e s p e c t i v e l y ; i s o b u t y r i c : 1.54, 0.71, 2.11, 0.66 and 1.73% f o r d i e t s .one,  two, t h r e e , f o u r , and f i v e r e s p e c t i v e l y ) .  treatment o f t h e g r a s s - c l o v e r f o r a g e  Formaldehyde  resulted i n significant  (p < 0.05)  i n c r e a s e s i n n - v a l e r i c a c i d l e v e l s i n t h e rumen and t h i s c o u l d be due t o reduced m i c r o b i a l growth due t o l i m i t a t i o n o f n i t r o g e n . (1.82, 3.30, 3.19, 3.34 and  3.15 f o r d i e t s one, two, t h r e e , f o u r and f i v e r e s p e c t i v e l y ) .  ammonia-nitrogen l e v e l s were s i g n i f i c a n t l y f e d d i e t s c o n t a i n i n g the u n t r e a t e d diets  forage  (p^0.05) higher  Rumen  f o r animals  than f o r animals on t h e o t h e r  (21.14, 14.36, 14.30, 12.90 and 13.54 ppm f o r d i e t s one, two, t h r e e ,  f o u r and f i v e r e s p e c t i v e l y ) .  The h i g h e r  l e v e l s o f rumen ammonia-nitrogen  i n the case o f animals f e d the d i e t c o n t a i n i n g t h e u n t r e a t e d  forage  compared t o t h e o t h e r s was most l i k e l y due t o a g r e a t e r r a t e o f p r o t e i n breakdown unaccompanied by e f f i ' c i e n t i u t i l i z a t i o n i n the case o f t h e former compared t o t h e l a t t e r . Abomasal pH, abomasal d i g e s t a c o n c e n t r a t i o n s  of acid-detergent  f i b r e , c e l l u l o s e , t o t a l n i t r o g e n , r i b o n u c l e i c a c i d n i t r o g e n , and m i c r o b i a l p r o t e i n n i t r o g e n were n o t a f f e c t e d by treatment. of % RNA-N: % t o t a l abomasal d i g e s t a n i t r o g e n  The r a t i o s  (0.060, 0.036, 0.044,  0.034 and 0.033 f o r d i e t s one, two, t h r e e , f o u r and f i v e r e s p e c t i v e l y ) and  % m i c r o b i a l n i t r o g e n : % t o t a l abomasal d i g e s t a n i t r o g e n  (0.786,  0.480, 0.583, 0.451 and 0.439 f o r d i e t s one, two, t h r e e , f o u r and f i v e  - 138 -  r e s p e c t i v e l y ) were reduced  significantly  t h r e e by the formaldehyde treatment  (p <_ 0.05) except  o f the f o r a g e .  for diet  The abomasal d i g e s t a  c o n c e n t r a t i o n o f n o n - p r o t e i n - n i t r o g e n was s i g n i f i c a n t l y  (p<0.05) h i g h e r  f o r the d i e t c o n t a i n i n g the u n t r e a t e d f o r a g e compared to the o t h e r s . The h i g h e r r a t i o o f m i c r o b i a l - n i t r o g e n : t o t a l abomasal d i g e s t a n i t r o g e n (except f o r d i e t t h r e e ) and the h i g h e r l e v e l s o f n o n - p r o t e i n - n i t r o g e n i n the abomasal d i g e s t a f o r t h e d i e t c o n t a i n i n g the u n t r e a t e d  forage  compared t o t h e o t h e r s i n d i c a t e d t h a t t h e r e was a g r e a t e r degree 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 by rumen microbes.  For d i e t three, there  might have been a g r e a t e r degree o f 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 as f o r d i e t one b u t i n t h e case o f d i e t t h r e e the c o n v e r s i o n of the degraded p r o t e i n t o m i c r o b i a l p r o t e i n was more e f f i c i e n t . Sulphur b a l a n c e  (g/Wkg^* "Vday) was s i g n i f i c a n t l y  (p <_ 0.05) improved  7  by the formaldehyde treatment  of the f o r a g e except  w i t h both sulphur and VFAS ( d i e t f i v e ) .  f o r the d i e t  The v a l u e s were 0.032, 0.061, 0.061,  0.056 and 0.050g f o r d i e t s one, two, t h r e e , f o u r and f i v e Sulphur  supplemented  i n t a k e per u n i t o f m e t a b o l i c body s i z e p e r day was  respectively. significantly  (p < 0.05) h i g h e r f o r the d i e t s supplemented w i t h s u l p h u r than f o r the other d i e t s .  The v a l u e s were 0.157, 0.168, 0.188, 0.247 and 0.251g  f o r d i e t s one, two, t h r e e , f o u r and f i v e r e s p e c t i v e l y . to n i t r o g e n r e t a i n e d r a t i o s were however n o t a f f e c t e d (p>  0.05) by d i e t a r y treatments.  Sulphur r e t a i n e d significantly  The amount o f s u l p h u r e x c r e t e d i n  u r i n e p e r day (g/Wkg^' ^/day) was s i g n i f i c a n t l y 7  the sulphur supplemented d i e t s than f o r the r e s t  (p < 0.05) h i g h e r f o r (0.050, 0.024, 0.034,  0.102, and 0.103g f o r d i e t s one, two, t h r e e , f o u r and f i v e  respectively).  - 139 -  Sulphur e x c r e t e d  i n u r i n e as a percentage o f i n t a k e was s i g n i f i c a n t l y  (p ^0.05) h i g h e r f o r d i e t s one, f o u r and f i v e than f o r d i e t s two and three. one, and  The v a l u e s were 31.38, 14.17, 17.41, 41.43 and 42.23% f o r d i e t s  two, t h r e e , f o u r and f i v e r e s p e c t i v e l y .  The l o s s o f s u l p h u r  faeces as a percentage o f i n t a k e was s i g n i f i c a n t l y  i n urine  (p 4. 0.05) h i g h e r  f o r d i e t s one, f o u r and f i v e than f o r d i e t two and f o r d i e t s one and f i v e than f o r d i e t t h r e e .  The v a l u e s were 79.67, 63.69, 67.57, 76.89 and  80.28% f o r d i e t s one, two, t h r e e , f o u r and f i v e r e s p e c t i v e l y . Nitrogen (p>  intake  (g/day o r g/Wkg^" "Vday) was n o t s i g n i f i c a n t l y 7  0.05) a f f e c t e d by d i e t a r y treatments.  c o e f f i c i e n t o f n i t r o g e n was s i g n i f i c a n t l y  The apparent (p <  digestibility  0.05) h i g h e r f o r d i e t one  than f o r d i e t s two, f o u r , and f i v e and f o r d i e t t h r e e than f o r d i e t  four.  The v a l u e s were 54.13, 47.06, 51.25, 44.90, and 47.24% f o r d i e t s one, two,, t h r e e , f o u r and f i v e r e s p e c t i v e l y . significantly  Nitrogen balance  (gN/Wkg^' "Vday) was 7  (p < 0.05) b e t t e r f o r d i e t s two and t h r e e than f o r d i e t one.  The n i t r o g e n b a l a n c e v a l u e s were (g/Wkg  , / J  / d a y ) 0.459,  0.802,0.843,  0.752 and 0.672g f o r d i e t s one, two, t h r e e , f o u r and f i v e r e s p e c t i v e l y . Formaldehyde treatment o f the f o r a g e s i g n i f i c a n t l y the amount o f n i t r o g e n e x c r e t e d metabolic one,  body s i z e  (p < 0.05) reduced  i n t h e u r i n e p e r day p e r u n i t o f  (0.805, 0.461, 0.467, 0.417 and 0.518g f o r d i e t s  two, t h r e e , f o u r and f i v e r e s p e c t i v e l y ) . N i t r o g e n e x c r e t e d  as a percentage o f d i g e s t e d and n i t r o g e n e x c r e t e d of i n t a k e were a l s o reduced s i g n i f i c a n t l y treatment o f t h e f o r a g e .  i n urine  i n u r i n e as a percentage  (p < 0.05) by formaldehyde  The v a l u e s f o r n i t r o g e n e x c r e t e d  i n u r i n e as  a percentage o f d i g e s t e d were 72.10, 44.65, 39.11, 44.25 and 50.39% f o r d i e t s one, two, t h r e e , f o u r and f i v e r e s p e c t i v e l y .  The v a l u e s f o r  - 140  n i t r o g e n excreted  i n the u r i n e as a percentage of i n t a k e were  39.07, 21.07, 19.97, 19.86 f o u r , and digested  -  and  five respectively.  23.94% f o r d i e t s one, Nitrogen  of i n t a k e  f i v e r e s p e c t i v e l y ) and  49.61% f o r d i e t s one,  23.30% f o r d i e t s one,  f i v e i n t h a t o r d e r ) were s i g n i f i c a n t l y  (p <  f o r the d i e t s c o n t a i n i n g the formaldehyde t r e a t e d . f o r a g e the d i e t c o n t a i n i n g the u n t r e a t e d  two,  n i t r o g e n r e t a i n e d as a percentage  (15.06, 26.00, 30.24, 25.04 and  t h r e e , f o u r and  three,  r e t a i n e d as a percentage of  (27.90, 55.39, 58.93, 55.75, and  t h r e e , f o u r and  two,  forage.  Nitrogen  two,  0.05)  greater  compared to  utilization  was  improved f o r a l l the d i e t s c o n t a i n i n g the formaldehyde t r e a t e d  forage  as a r e s u l t of lower u r i n a r y n i t r o g e n l o s s e s .  sulphur  The  a d d i t i o n of  to the d i e t s c o n t a i n i n g the formaldehyde t r e a t e d f o r a g e the b e n e f i c i a l e f f e c t s of the formaldehyde treatment.  tended to The  offset  a d d i t i o n of  the v o l a t i l e f a t t y a c i d s t o the d i e t s c o n t a i n i n g the formaldehyde t r e a t e d f o r a g e d i d not  f u r t h e r enhance n i t r o g e n  Duodenal f l o w was  utilization.  measured over a twenty-four-hour p e r i o d  a sheep f i t t e d w i t h a r e - e n t r a n t  cannula.  The  q u a n t i t i e s of  using  total  d i g e s t a , dry matter, o r g a n i c matter, n i t r o g e n , a c i d - d e t e r g e n t  fibre,  and  c e l l u l o s e f l o w i n g through the duodenum d a i l y were markedly h i g h e r  for  the d i e t s c o n t a i n i n g the formaldehyde t r e a t e d f o r a g e  diet  The  than f o r the  c o n t a i n i n g the u n t r e a t e d  forage.  and n o n - p r o t e i n - n i t r o g e n  a r r i v i n g at the duodenum were markedly  f o r the d i e t c o n t a i n i n g the u n t r e a t e d  d a i l y amounts of m i c r o b i a l p r o t e i n  f o r a g e than f o r the  Growth r a t e o f the animals d u r i n g p e r i o d and  t h e i r metabolic  s t u d i e s were not The  the seventeen day  body s i z e s at the b e g i n n i n g  significantly  ( p ) 0.05)  l a c k of a response i n growth r a t e may  higher  rest. pre-metabolism  of the metabolism  d i f f e r e n t f o r d i e t a r y treatments. have been due  to the  relatively  - 141 -  s h o r t p e r i o d o f measurement. Formaldehyde treatment of the f o r a g e p o r t i o n o f the d i e t had b e n e f i c i a l e f f e c t s i n terms of the n i t r o g e n economy o f the a n i m a l . The treatment o f the f o r a g e p o r t i o n o f the d i e t w i t h formaldehyde r e s u l t e d i n changes i n the s i t e s o f d i g e s t i o n o f b o t h p r o t e i n and fibre.  The d i g e s t i o n of these f r a c t i o n s was d e p r e s s e d i n t h e rumen  but i n c r e a s e d i n the lower s e c t i o n s o f the d i g e s t i v e  tract.  T h i s study i n d i c a t e d t h a t s u p p l e m e n t a t i o n o f t h e d i e t s c o n t a i n i n g the formaldehyde t r e a t e d grass-legume f o r a g e w i t h e i t h e r s u l p h u r and/or branched c h a i n v o l a t i l e f a t t y a c i d s was n o t n e c e s s a r y as no b e n e f i c i a l e f f e c t s were observed. 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S c i . 44:1112-1118.  - 162 -  APPENDIX TABLES I - XXX  ANOVA p e r c e n t N d i g e s t i b i l i t y , 1st stage o f i n v i t r o d i g e s t i o n of r y e - g r a s s - c l o v e r forage w i t h the d i f f e r e n t l e v e l s of formaldehyde treatments.  T a b l e I.  df  SS  Source  Variance  11  Total  1350.47  Treatment  1314.8181  3  438.2727  35.6519  8  4.46  Error  **  p  <  0.01  Fcal  S.E.  + 1.219  98.27** ON LO  Table I I .  ANOVA p e r c e n t N d i g e s t i b i l i t y 2nd stage o f i n v i t r o d i g e s t i o n o f r y e - g r a s s - c l o v e r forage w i t h the d i f f e r e n t l e v e l s of formaldehyde treatment.  df  SS  Source  Total  228.83062  11  Treatment  181.22628  3  47.60434  8  Error  **  p  <  0.01  S.E. + 1.408  Variance  Fcal  60.40876  10.1518**  5.9505425  ANOVA i n v i t r o ammonia-nitrogen p r o d u c t i o n (ppm) per gram d r y matter o f r y e - g r a s s - c l o v e r f o r a g e w i t h the d i f f e r e n t l e v e l s o f formaldehyde treatment.  Table I I I .  Source  SS  Total  df  145794.2618  Variance  Fcal  23 i  Treatment Error  **  p  <  0.01  138869.9399  3  46289.98  6924.3219  20  346.22  S.E. + 7.596  133.70**  M  i  Table IV.  ANOVA m e t a b o l i c body s i z e s o f animals a t the b e g i n n i n g of t h e metabolism s t u d i e s ( k g ) .  df  SS  Source  Variance  Fcal  26.157944  24  5.108064  4  1.277016  1.93  Block  10.493344  4  2.623336  3.98*  Error  10.556536  16  Total Treatment  p  <  0.05  S.E.  + 0.3632584478  0.6597835  ANOVA apparent d i g e s t i b i l i t y  T a b l e V.  SS  Source  Total  465.15122  Treatment  275.4159  Fcal  4  68.853975  10.03**  19.975585  24  79.90234  4  Error  109.83298  16  p  <  0.01  o f n i t r o g e n (%) .  Variance  df  Block  **  coefficient  S.E. + 1.171713382  6.86456125  2.91  ANOVA apparent d i g e s t i b i l i t y c o e f f i c i e n t  Table VI.  SS  Source  df  of a c i d - d e t e r g e n t f i b r e (%).  Variance  Fcal  4.41*  169.55554  24  Treatment  70.09646  4  17.524115  Block  35.91918  4  8.979795  Error  63.5399  Total  p  C  0.05  16  S.E. + 0.8912063454  3.97124375  2.26  ANOVA apparent d i g e s t i b i l i t y  Table VII.  df  SS  Source  coefficient  o f c e l l u l o s e (%).  Variance  Fcal  4.21*  Total  312.12734  24  Treatment  149.96314  4  37.490785  Block  19.5969  4  4.899225  Error  142.5673  16  p  <  0.05  S.E. +  1.334949905  8.91045625  0.55  Table VIII.  ANOVA n i t r o g e n e x c r e t e d i n u r i n e per u n i t body s i z e p e r day ( g ) .  Variance  Fcal  4  0.12151736  18.94**  0.12136904  4  0.03034226  4.73*  0.10263376  16  0.00641461  SS  df  Total  0.71007224  24  Treatment  0.48606944  Block Error  Source  **  p < 0.01  metabolic  *  p  <  0.05  S.E.  f o r treatment and b l o c k means + 0.0358179005  ANOVA % n i t r o g e n e x c r e t e d i n u r i n e over i n t a k e .  T a b l e IX.  Source  SS  df  Variance  Fcal  4.88**  Total  2712.62046  24  Treatment  1329.91614  4  332.479035  293.50034  4  73.375085  Block Error  **  1089.20398  p  <  0.01  S.E. +  16  3.689857687  68.07524875  1.08  i  H  i—  •  1  ANOVA % n i t r o g e n e x c r e t e d i n u r i n e over d i g e s t e d .  T a b l e X.  Source  SS  df  Total  4826.9737  24  Treatment  3343.26462  Block Error  **  p  <  0.01  Variance  Fcal  4  835.816155  14.28**  547.19962  4  136.799905  936.50946  16  S.E. +  3.421457036  58.53184125  2.34  ANOVA % n i t r o g e n r e t a i n e d over i n t a k e .  T a b l e XI.  SS  Source  df  Variance  Fcal  14.17**  24  Total  885.5855  Treatment  622.11626  4  155.529065  87.91154  4  21.977885  Block  16  175.5577  Error  p  <  0.01  S.E. +  1.481374784  10.97235625  2.00  ANOVA % n i t r o g e n r e t a i n e d over d i g e s t e d .  T a b l e XII.  Source  SS  df  Variance  Fcal  12.70**  Total  4354.29162  24  Treatment  2945.51022  4  736.377555  Block  480.9983  4  120.249575  Error  927.7831  16  **  p  <  0.01  S.E.  +  3.405479225  57.98644375  2.07  ANOVA n i t r o g e n balance g/day.  Table XIII.  Source  SS  Total  df  Variance  Fcal  5.57**  141.9019326  24  82.6064218  4  20.65160545  Block  6.731593  4  1.68289825  Error  59.2955108  16  3.70596425  Treatment  **  p <  0.01  S.E.  + 0.8609255775  0.45  (  ^ '  T a b l e XIV.  ANOVA n i t r o g e n balance (g) p e r u n i t of m e t a b o l i c body s i z e p e r day.  df  SS  Source  Variance  Fcal  Total  0.72847696  24  Treatment  0.43781416  4  0.10945354  8.20**  Block  0.07698256  4  0.01924564  1.44  Error  0.21368024  16  **  p  <  0.01  S.E. + 0.0516817472  0.013355015  i  1  T a b l e XV.  ANOVA s u l p h u r i n t a k e p e r day (g) .  Variance  Fcal  4  1.584074  9.36**  0.494776  4  0.123694  0.73  2.708824  16  SS  df  Total  9.539896  24  Treatment  6.336296  Block Error  Source  **  p <  0.01  S.E. +  0.1840116844  0.1693015  T a b l e XVI.  Source  ANOVA s u l p h u r i n t a k e p e r u n i t o f m e t a b o l i c body s i z e per day ( g ) .  SS  df  Variance  Fcal  16.09**  Total  0.05090784  24  Treatment  0.03885544  4  0.00971386  Block  0.00239544  4  0.00059886  Error  0.00965696  16  0.00060356  **  p <. 0.01  S.E. + 0.0109869012  0.99  T a b l e XVII.  ANOVA s u l p h u r e x c r e t e d i n u r i n e p e r day p e r u n i t o f m e t a b o l i c body s i z e ( g ) .  SS  Source  df  Variance  Fcal  27.59**  Total  0.03454384  24  Treatment  0.02941184  4  0.00735296  Block  0.00086824  4  0.00021706  Error  0.00426376  16  p  <. 0.01  S.E. + 0.0073004794  0.000266485  0.81  ANOVA % s u l p h u r e x c r e t e d i n u r i n e over i n t a k e .  Table XVIII.  SS  Source  df  Variance  Fcal  22.45**  24  Total  4392.9128  Treatment  3443.66224  4  860.91556  Block  335.63716  4  83.90929  Error  613.6134  p  <  0.01  S.E.  16  + 2.769506725  38.3508375  2.19  ANOVA t o t a l amount o f s u l p h u r l o s t i n u r i n e and f a e c e s as a percentage o f i n t a k e .  T a b l e XIX.  Variance  SS  df-  Total  1884.5519  24  Treatment  1134.6117  4  283.652925  Block  299.1533  4  74.788325  Error  450.7869  16  Source  <  0.01  S.E. + 2.373781003  Fcal  i  28.17418125  10.07** 2.65  oo m i  ANOVA s u l p h u r balance p e r day ( g ) .  T a b l e XX.  df  SS  Source  Variance  Fcal  5.67**  Total  0.99126424  24  Treatment  0.46094104  4  0.11523526  Block  0.20486304  4  0.05121576  Error  0.32546016  16  0.02034126  p  <  0.01  S.E. + 0.0637828503  ANOVA s u l p h u r b a l a n c e p e r day p e r u n i t o f m e t a b o l i c body s i z e ( g ) .  T a b l e XXI.  SS  Source  df  Variance  Fcal  24  Total  0.005612  Treatment  0.0028912  4  0.0007228  6.80**  Block  0.00102  4  0.000255  2.40  Error  0.0017008  p  <  0.01  16  S.E. + 0.0046108567  0.0001063  T a b l e XXII.  ANOVA molar p r o p o r t i o n o f a c e t i c a c i d i n rumen f l u i d (%).  SS  Source  df  Variance  Fcal  24  Total  521.26974  Treatment  106.9635  4  26.740875  2.17  Block  217.39594  4  54.348985  4.42*  Error  196.9103  p  <  0.05  16  S.E. + 1.568878182  12.30689375  ANOVA molar p r o p o r t i o n o f p r o p i o n i c a c i d i n rumen f l u i d (%).  Table XXIII.  SS  Source  891.38178  Total  df  Variance  Fcal  0.82  24  74.0105  4  18.502625  Block  454.64618  4  113.661545  Error  362.7251  Treatment  p  <  0.01  16  S.E. + 2.12933411  22.67031875  5.01**  T a b l e XXIV.  Source  ANOVA molar p r o p o r t i o n o f i s o b u t y r i c a c i d i n rumen f l u i d (%).  SS  df  Variance  Fcal  13.77958095  24  Treatment  6.18916095  4  1.547290238  4.29*  Block  .1.814484283  4  0.4536210708  1.26  Error  5.775935717  16  0.3609959823  Total  *  p  <  0.05  S.E. + 1.163467023  T a b l e XXV.  Source  ANOVA molar p r o p o r t i o n s o f i s o v a l e r i c a c i d p r o p o r t i o n i n rumen f l u i d (%).  SS  df  Variance  Fcal  Total  40.99798095  24  Treatment  17.13082595  4  4.282706488  4.39*  4  2.068108921  2.12  Block  8.272439283  Error  *  p  15.59471572  <  0.05  16  S.E. + 0.4415132461  0.9746697323  ANOVA molar p r o p o r t i o n o f v a l e r i c a c i d i n rumen f l u i d (%).  T a b l e XXVI.  Variance  Fcal  4  2.078846  5.39**  1.445024  4  0.361256  0.94  6.170376  16  SS  df  15.930784  24  Treatment  8.315384  Block Error  Source  Total  p  <  0.01  S.E.  +  0.2777223434  00 00  0.3856485  T a b l e XXVII.  ANOVA rumen ammonia-nitrogen l e v e l s  Variance  Fcal  4  56.0416  4.7176*  122.8504  4  30.7126  2.585  190.0656  16  11.8791  SS  df  Total  537.0824  24  Treatment  224.1664  Block Error  Source  p  <  0.05  (ppm).  S.E. + 1.541369521  T a b l e XXVIII.  ANOVA abomasal  Source  SS  digesta non-protein-nitrogen  df  c o n c e n t r a t i o n (%).  Variance  Fcal  Total  0.33877336  24  Treatment  0.17597416  4  0.04399354  4.83**  Block  0.01710296  4  0.00427574  0.47  Error  0.14569624  16  **  p <  0.01  S.E. + 0.042675555  0.009106015  T a b l e XXIX.  ANOVA % RNA-N: % t o t a l N i n abomasal  Source  SS  df  Total  0.005168  Treatment  0.0025188 0.0003084  Block  0.0023408  Error  *  p  <  0.05  digesta.  Variance  Fcal  0.0006297  4.30*  24 4 4 16  S.E. + 0.0054092513  0.0000771  0.53  i  hh-  1 1  0.0001463  I  ANOVA % m i c r o b i a l - p r o t e i n - n i t r o g e n : % t o t a l n i t r o g e n f o r abomasal d i g e s t a ( x : l ) .  T a b l e XXX.  Source  Variance  Fcal  4  0.1046271  4.53*  0.0633512  4  0.0158378  0.69  0.3687524  16  SS  df  Total  0.850612  24  Treatment  0.4185084  Block Error  *  p " <  0.05  digesta  S.E. + 0.067892599  0.023047025  ^  PUBLICATIONS  TUAH, A.K. 1971. i n Ghana.  The p r e p a r a t i o n of s i l a g e i n s m a l l - s i z e d s i l o s P r o c . Ghana Anim. S c i . Symp. 4:68-71.  TUAH, A.K. and T e t t e h J . Adinku. 1972. The e f f e c t of f e e d i n g molassesurea supplement on the performance o f c o n f i n e d West A f r i c a n Dwarf sheep. Proc. Ghana Anim. S c i . Symp. 5:83-93. TUAH, A.K. and C.W. Cameron. 1973. The use of v a r i o u s f o r a g e s f o r s i l a g e i n t h r e e types of s i l o s i n Ghana. Ghana J . S c i . 13(2): 203-209. A b s t r a c t e d i n : Herbage A b s t r a c t s (1975) 45:73; M i c r o b . A b s t r a c t s (1975) 10:28. A l s o paper read at West A f r i c a n S c i e n c e A s s o c i a t i o n b i e n n i a l meeting (1972) h e l d at U n i v e r s i t y of Ghana, Legon, A c c r a . TUAH, A.K. and 0. Okyere. 1974. P r e l i m i n a r y s t u d i e s on the e n s i l a g e o f some s p e c i e s of t r o p i c a l g r a s s e s i n the A s h a n t i f o r e s t b e l t of Ghana. Ghana J . . A g r i c . S c i . 7:81-87. A b s t r a c t e d i n : Herbage A b s t r a c t s A g r i c . 1975 1(5):53.  (1975) 45:329; A b s t . Trop.  A l s o paper read at Ghana S c i e n c e A s s o c i a t i o n meeting h e l d a t U.S.T., Kumasi, Ghana.  (1973)  TUAH, A.K. 1974. P r o s p e c t s f o r d a i r y i n g i n the "Kumasi D i s t r i c t " of the A s h a n t i Region o f Ghana. Review o f the s i t u a t i o n . Ghana JT A g r i c . S c i . 7:157-164. A b s t r a c t e d i n : Herbage A b s t r a c t s (1976) 46:59. TUAH, A.K. and K. Boa-Amponsem. 1974. R i c e b r a n ( r i c e - m i l l feed) i n the d i e t s o f g r o w i n g - f i n i s h i n g Large White p i g s . Ghana.Anim. S c i . Symp. 6:8pages. TUAH, A.K. RvM. T a i t . 1977. N i t r o g e n metabolism i n sheep f e d formaldehydet r e a t e d g r a s s - c l o v e r f o r a g e supplemented w i t h b r a n c h e d - c h a i n f a t t y a c i d s and/or s u l p h u r . Can.J.Anim.Sci. 57:844 ( A b s t r a c t ) . Paper read at CSAS (Western Branch) meeting h e l d a t Winnipeg, Manitoba. June 16-18, 1977.  

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