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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 EFFECT OF FORMALDEHYDE TREATMENT OF THE FORAGE PORTION OF THE DIET, THE ADDITION OF BRANCHED-CHAIN VOLATILE FATTY ACIDS AND/OR SULPHUR ON THE UTILIZATION OF NITROGEN AND CARBOHYDRATE BY SHEEP by AMBROSE KWAME /TUAH B.Sc.(Agric.) Kumasi, 1968 M.Sc.(Anim.Sci.) Kumasi, 1970 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY THE FACULTY OF GRADUATE STUDIES in the Department of Animal Science We accept this thesis as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA in June, 1978 In presenting th is thes is in p a r t i a l fu l f i lment of the requirements for an advanced degree at the Univers i ty of B r i t i s h Columbia, I agree that the L ibrary sha l l make it f ree l y ava i lab le for reference and study. I fur ther agree that permission for extensive copying of th i s thes is for scho lar l y purposes may be granted by the Head of my Department or by h is representat ives . It is understood that copying or pub l i ca t ion of th is thes is for f i n a n c i a l gain sha l l not be allowed without my wri t ten permission. ' Department of Animal Science The Univers i ty of B r i t i s h Columbia 2075 Wesbrook Place Vancouver, Canada V6T 1W5 Date June 29, 1978 - i i -ABSTRACT Formaldehyde treatment of dietary protein to reduce i t s degradation i n the rumen has been reported to 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%, 1.0% and 1.2% on an a i r dry basis) were applied to a dehydrated and hammermilled grass-clover forage. In v i t r o nitrogen d i g e s t i b i l i t y and ammonia-nitrogen production at *the microbial stage of incubation were reduced s i g n i f i c a n t l y (p ^0.05) as the l e v e l of formaldehyde was increased. Nitrogen d i g e s t i b i l i t y f o r the combined microbial and acid-pepsin stages of incubation was s i g n i f i c a n t l y (p ^0.05) reduced only at the 1.2% l e v e l of formaldehyde a p p l i c a t i o n compared to the untreated forage. Ram lambs ranging i n body weights of 29kg to 36kg were then used i n studies of nitrogen and carbohydrate metabolism. One percent formaldehyde was applied to the grass-clover forage. Each of the f i v e d i e t s (14% C P . on D.M. basis) contained 50% grass-clover forage, 38% cassava, 11% barley and 1% sheep mineral premix on a dry matter basi s . Diet one contained the untreated forage while the others contained the formaldehyde treated forage. Diets three and f i v e were supplemented with i s o v a l e r i c acid (3.0g/Kg diet) and i s o b u t y r i c acid (2.3g/Kg d i e t ) . Diets four and f i v e were supplemented with sulphur i n the form of sodium sulphate. - i i i -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 acid-detergent f i b r e and c e l l u l o s e were increased s i g n i f i c a n t l y (p ^0.05) by formaldehyde treatment of the forage. 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 of nitrogen was s i g n i f i c a n t l y (p <0.05) depressed by formaldehyde treatment of the forage except f o r the die t supplemented with VFAS (diet three). 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 dry matter and organic matter were not affected s i g n i f i c a n t l y (p./' 0.05). Ruminal f l u i 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 acids were s i g n i f i c a n t l y (p<C0.05) higher f o r animals fed d i e t three than f o r animals fed di e t s two and four. Formaldehyde treatment of the forage resulted i n s i g n i f i c a n t l y (p <0.05) higher l e v e l s of v a l e r i c a c i d and lower l e v e l s of ammonia i n ruminal f l u i d . Ruminal f l u i d l e v e l s of t o t a l v o l a t i l e f a t t y acids, a c e t i c , propionic and b u t y r i c acids and rumen pH were not affected s i g n i f i c a n t l y (p y 0.05) by formaldehyde treatment of the forage. The r a t i o of microbial protein-nitrogen (estimated from RNA-N) to t o t a l abomasal digesta nitrogen was s i g n i f i c a n t l y (p ^  0.05) decreased by formaldehyde treatment of the forage except f o r the die t supplemented with VFAS (diet three). The concentration of non-protein-nitrogen i n abomasal digesta was s i g n i f i c a n t l y (p <^0.05) reduced by formaldehyde treatment of the forage. Abomasal digesta pH, concentration of t o t a l nitrogen, RNA-N, microbial protein-nitrogen, acid-detergent f i b r e , and c e l l u l o s e were not affected. - i v -The reduction i n the r a t i o of microbial protein-nitrogen to t o t a l abomasal digesta nitrogen and non-protein-nitrogen by formaldehyde treatment of the forage suggests that the treatment reduced microbial degradation of dietary protein except perhaps f o r the di e t supplemented with VFAS (diet three). Nitrogen balance was s i g n i f i c a n t l y (p .^0.05) improved by formaldehyde treatment of the forage except f o r the sulphur supplemented diets.. Sulphur supplementation tended to o f f s e t the b e n e f i c i a l e f f e c t s of formaldehyde protection of the forage protein. Supplementation with VFAS did not further enhance nitrogen u t i l i z a t i o n . Formaldehyde treatment of the forage s i g n i f i c a n t l y (p <C0.05) improved sulphur balance except f o r the di e t supplemented with both sulphur and VFAS (diet f i v e ) . D a i l y feed intake and urine output per u n i t metabolic body s i z e and growth rate over a seventeen-day period were not s i g n i f i c a n t l y (p~? 0.05) affected by formaldehyde treatment of the forage. The flow of t o t a l digesta, organic matter, dry matter, acid-detergent f i b r e , c e l l u l o s e and t o t a l nitrogen through the duodenum for a 24-hour period was markedly higher f o r the d i e t s containing the formaldehyde treated forage. The flow of microbial protein-nitrogen and non-protein-nitrogen however was markedly depressed by formaldehyde treatment of the forage. A sheep f i t t e d with a duodenal re-entrant cannula was used f o r t h i s study. - v -TABLE OF CONTENTS Page ABSTRACT . . i i TABLE OF CONTENTS v LIST OF TABLES ; i x LIST OF FIGURES x LIST OF APPENDIX TABLES x i ACKNOWLEDGEMENTS x i i i INTRODUCTION 1 LITERATURE REVIEW 4 Rumen microbiology 4 Rumen ba c t e r i a 4 Rumen Protozoa 7 Other rumen micro-organisms. . 8 Factors a f f e c t i n g populations of rumen micro-organisms 8 Mic r o b i a l protein-nitrogen composition and microbial protein synthesis 12 Metabolism of nitrogen 16 Degradation of proteins 16 Protein anabolism by rumen b a c t e r i a 23 E f f e c t of formaldehyde treatment on the digestion of proteins i n the rumen 24 Possible reasons f o r the v a r i a b l e responses to formaldehyde treatment of d i f f e r e n t types of p r o t e i n s . . 29 Digestion and absorption of nitrogen i n the small i n t e s t i n e . . .32 Digestion of protein i n the large i n t e s t i n e 38 - v i -Carbohydrate metabolism i n the rumen • • • 39 E f f e c t s of formaldehyde treatment on carbohydrate metabolism and v o l a t i l e f a t t y acid production i n the rumen 42 OBJECTIVES 43a MATERIALS AND METHODS 44 Introduction 44 Experiment I 44 Treatment of the forage with formaldehyde 44 In v i t r o incubation 45 Treatment of grass-clover forage f o r i n vivo t r i a l s 46 Experiment II 46 Nitrogen, carbohydrate and sulphur metabolism and feed intake studies • 46 Experiment I I I 49 Rumen and abomasal digesta metabolites studies .• 49 Experiment IV • • • 51 Duodenal digesta flow studies 51 C o l l e c t i o n and sampling of digesta 51 CHEMICAL ANALYSES 52 EXPERIMENTAL DESIGNS AND STATISTICAL ANALYSIS 55 RESULTS . . . . . . . . . . . . . 57 Experiment I • 57 In v i t r o digestion t r i a l s 57 Experiment II 59 Chemical composition of die t s and ingredients 59 Dail y feed intake, d a i l y urine output, metabolic body sizes of animals (kg) at the beginning of the metabolism study period, and the d a i l y gain i n weight during the pre-metabolism assay period 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 of dry matter, organic matter, nitrogen, acid-detergent f i b r e and c e l l u l o s e 61 - v i i -Nitrogen metabolism 64 Sulphur metabolism £6 Experiment I I I 6 8 Rumen parameters (Data c o l l e c t e d from slaughtered animals) . • . • . ^8 Abomasal parameters (Data c o l l e c t e d from slaughtered animals) . . . 7 ^ Experiment IV 7 0 Chemical composition of die t s used for duodenal flow rate measurements 3® 7 3 Feed intake during the duodenal flow measurements 73 Duodenal flow parameters Some chemical f r a c t i o n s of duodenal digesta, t o t a l d a i l y intake and d a i l y digesta flow through the duodenum of these f r a c t i o n s . • 7 ^ Dai l y nitrogen intake, nitrogen components of duodenal digesta and the d a i l y flow of these components through the 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 of some chemical f r a c t i o n s i n the pre-duodenum portion of dige s t i v e t r a c t ( a l l compartments of stomach) and the change i n quantity of nitrogen entering the duodenum d a i l y compared with intake 7 ^ DISCUSSION 8 1 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 • Feed intake 8 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 nitrogen, acid-detergent f i b r e , c e l l u l o s e , dry matter and organic matter 8 8 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 of nitrogen 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 ADF and Ce 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 of dry matter and organic matter 97 Rumen parameters 97 Rumen pH Q O Rumen ammonia-nitrogen 1 no Rumen t o t a l v o l a t i l e f a t t y a c i d concentration x y j - v i i i -Acet i c , Propionic and Butyric a c i d proportions i n the rumen f l u i d 105 Is o v a l e r i c and Isobutyric acid proportions i n the rumens 107 V a l e r i c a c i d proportions • -109 Abomasal and duodenal digesta parameters 110 Abomasal pH 110 Abomasal digesta N%, duodenal digesta N%, arid t o t a l d a i l y flow of nitrogen from the abomasum into the duodenum I l l Abomasal and duodenal digesta non-protein nitrogen l e v e l s 114 Abomasal and duodenal digesta RNA-N, microbial N, %RNA-N: % t o t a l digesta N, % microbial N: % t o t a l digesta N 115 Abomasal ADF%, duodenal ADF%, c e l l u l o s e % , and quantities of ADF and c e l l u l o s e a r r i v i n g at the duodenum 118 Duodenal digesta flow 119 Sulphur metabolism 123 Nitrogen metabolism 129 'Growth rate 132 SUMMARY AND CONCLUSIONS 135 LITERATURE CITED 142 APPENDIX 162 - i x -LIST OF TABLES Table Page 1 Composition of Rations 47 2 In v i t r o Digestion T r i a l s 58 3 Chemical Composition of Diets and Ingredients (D.M. basis) . 60 4 Dail y Feed Intake, Da i l y Urine Output, Metabolic Body Sizes of Animals at the Beginning of the Metabolism Study Period and the Daily Gain i n Weight During the Pre-metabolism Assay Period 62 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 Fractions (%) 63 6 Nitrogen Metabolism 65 7 Sulphur Metabolism 67 8 Rumen parameters 69 9 Abomasal parameters 71 10 Chemical Composition of Diets Used f o r Duodenal Flow Rate Measurements 72 11 Feed Intake During the Duodenal Flow Measurements. .74 12 Duodenal Digesta Flow Parameter^ 75 13 Some Chemical Fractions of Duodenal Digesta, T o t a l D a i l y Intake and Dail y Digesta Flow Through the Duodenum of These Fractions 77 14 Dai l y Nitrogen Intake, Nitrogen Components of Duodenal Digesta and the Daily Flow of These Components Through the Duodenum 78 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 of Some Chemical Fractions i n the Pre-duodenum Portion of Digestive Tract ( A l l Compartments of Stomach) and the Change i n Quantity of Nitrogen Entering the Duodenum Dai l y Compared with Intake. . 79 - x -LIST OF FIGURES Figure Page 1 Schematic representation of the protein regeneration cycle i n ruminants 16a 2 An o u t l i n e of the pathways of fermentation of the major carbohydrate constituents to 3C units i n the rumen 39a 3 An o u t l i n e of the pathways of degradation of 3C units i n the rumen 39b - x i -LIST OF APPENDIX TABLES Table Page I ANOVA percent N d i g e s t i b i l i t y , 1st stage of i n v i t r o digestion of 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 II ANOVA percent N d i g e s t i b i l i t y 2nd stage of i n v i t r o digestion of rye-grass-clover forage with the d i f f e r e n t l e v e l s of formaldehyde treatment 164 III ANOVA i n v i t r o ammonia-nitrogen production (ppm) per gram dry matter of rye-grass-clover forage with the d i f f e r e n t l e v e l s of formaldehyde treatment 165 IV ANOVA metabolic body s i z e of animals at the beginning of the metabolism studies (kg) . . . . 166 V 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 s of nitrogen (%) 167 VI 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 of acid-detergent f i b r e (%) 168 VII 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 of c e l l u l o s e (%) 169 VIII ANOVA nitrogen excreted i n urine per unit of metabolic body s i z e per day (g) 170 IX ANOVA % nitrogen excreted i n urine over intake. . . 171 X ANOVA % nitrogen excreted i n urine over digested. . 172 XI ANOVA % nitrogen retained over intake 173 XII ANOVA % nitrogen retained over digested 174 XIII ANOVA nitrogen balance g/day 175 XIV ANOVA nitrogen balance (g) per unit of metabolic body siz e per day. 176 - x i i -XV ANOVA sulphur intake per day (g) 177 XVI ANOVA sulphur intake per unit of metabolic body s i z e per day (g) 178 XVII ANOVA sulphur excreted un urine per day per uni t of metabolic body s i z e (g) .179 XVIII ANOVA % sulphur excreted i n urine over intake . . . .180 XIX ANOVA t o t a l amount of sulphur l o s t i n urine and faeces as a percentage of intake 181 XX ANOVA sulphur balance per day (g) 182 XXI ANOVA sulphur balance per day per unit of metabolic body s i z e (g) 183 XXII ANOVA molar proportion of a c e t i c a c i d i n rumen f l u i d (%) 184 XXIII ANOVA molar proportion of propionic acid i n rumen f l u i d (%) 185 XXIV ANOVA molar proportion of i s o b u t y r i c a c i d i n rumen f l u i d (%) 186 XXV ANOVA molar proportions]of 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 XXVI ANOVA molar proportion of v a l e r i c acid i n rumen f l u i d (%) 188 XXVII ANOVA rumen ammonia-nitrogen l e v e l s (ppm). 189 XXVIII ANOVA abomasal digesta non-protein-nitrogen concentration (%) . . . 190 XXIX ANOVA % RNA-N: % t o t a l N i n abomasal digesta . . . . 191 XXX ANOVA % microbial-protein-nitrogen: % t o t a l digesta nitrogen f o r abomasal digesta (x:l) 192 - x i i i -ACKNOWLEDGEMENTS The author i s extremely pleased to acknowledge the assistance and invaluable advice of h i s supervisor, Dr. R.M. T a i t of the Department of Animal Science i n the planning and execution of the research and the preparation of t h i s t h e s i s . The assistance and advice offered i n the preparation of t h i s thesis by the other members of the author's graduate committee, Dr. W.D. K i t t s , Dean of the Faculty of A g r i c u l t u r a l Sciences, Dr. D.B. Bragg, Chairman of the Department of Poultry Science, Dr. V.C. Brink of the Department of Plant Science and Dr. H.C. Nordan of the Department of Zoology i s greatly appreciated. The cooperation of the Chairman of the Department of Animal Science, Dr. B.D. Owen, f o r the use of f a c i l i t i e s i s appreciated. Dr. CR. Krishnarmuti, Messrs David K i t t s and Robert Pratt of the Department of Animal Science are thanked for the s u r g i c a l preparation of the sheep f i t t e d with the re-entrant cannula. Mr. G. Galzy, Ms. Frances Newsome and Miss Mariana Kung are thanked for t h e i r t e c h n i c a l assistance. A l l members of s t a f f and fa c u l t y of the Department of Animal Science are thanked for t h e i r assistance. The help of Messrs Paul W i l l i n g and John Ciok of the Uni v e r s i t y Farm i s greatly appreciated. The author i s pleased to acknowledge the f i n a n c i a l assistance of C.I.D.A. His employers, the U n i v e r s i t y of Science and Technology, Kumasi, Ghana are also thanked for granting him leave of absence to - x i v -undertake t h i s study. The family and friends of the author are thanked f o r t h e i r moral support. - 1 -INTRODUCTION Studies on the digestion of feedstuffs by some workers have revealed that most of the e s s e n t i a l amino acids being made a v a i l a b l e to the ruminant are from microbial sources (Weller et a l . , 1958; Bergen et a l . , 1967; Leibholz, 1972; P h i l l i p s o n , 1972). The rumen micro-organisms u t i l i z e dietary nitrogen f o r the synthesis of t h e i r c e l l u l a r proteins. They generally prefer de novo synthesis of amino acids from simpler nitrogen sources such as ammonia, and also carbon skeletons, rather than making use of amino acids i n the feed (Saeur et a l . , 1975; Umuna et_ al_. , 1975). The micro-organisms hydrolyze the proteins to amino acids with enzymes, de-aminate some portions, and u t i l i z e part of the ammonia released to resynthesize amino acids f o r incorporation into microbial protein (Church, 1975c). A portion of the released ammonia i s l o s t to the animal. The degree of dietary nitrogen loss depends p a r t l y on the rate of protein breakdown which i n turn depends on protein s o l u b i l i t y i n the ruminal f l u i d (Church, 1975c). This also depends on the rate of amino acid synthesis by the rumen micro-organisms. The loss of dietary protein i s more than compensated for i f the dietary protein i s of poor q u a l i t y , as the microbial protein so formed i s of medium q u a l i t y . The micro-organisms are k i l l e d by the abomasal acid. Then the microbial protein i s made a v a i l a b l e to the animal a f t e r digestion i n the lower parts of the dige s t i v e t r a c t . - 2 -I f , however, the dietary protein i s of high q u a l i t y , there i s a loss to the ruminant due to the above processes. Therefore, attempts have been made to by-pass rumen digestion of protein when high q u a l i t y d ietary protein i s fed. The by-pass can be i n the form of abomasal infusions ( L i t t l e and Mitchel, 1967; Sc h e l l i n g and H a t f i e l d , 1968) or by reducing the s o l u b i l i t y of the protein i n the rumen (Ferguson et al., 1967). The common treatments applied to reduce s o l u b i l i t y of di e t a r y proteins i n the rumen are heat or aldehydes ( P h i l l i p s o n , 1972), although tannic acid and v o l a t i l e f a t t y acid treatments have been attempted suc c e s s f u l l y (Nishimuta et a l . , 1973; Barker et a l . , 1973; Candlish et a l . , 1973; Atwal et a l . , 1974). Protection of protein contained i n casein from attack by rumen micro-organisms has been reported to cons i s t e n t l y give s i g n i f i c a n t l y p o s i t i v e responses of nitrogen balance,wool growth and growth rate ( L i t t l e and M i t c h e l l , 1967; Sc h e l l i n g and H a t f i e l d , 1968; Reis and Tunks, 1969; P h i l l i p s o n , 1972). This has been a t t r i b u t e d to the higher q u a l i t y of casein protein compared with rumen microbial protein. Faichney and Davies (1972) treated groundnut (peanut) meal with formaldehyde and obtained a s l i g h t but non-significant response i n nitrogen balance compared to untreated groundnut meal. Treatment of soybeam meal has been reported to give v a r i a b l e responses. Some workers (Schmidt <et a l . , 1971; Schmidt et a l . , 1974) obtained negative responses i n nitrogen balance while Peter et a l . (1970), Peter et a l . (1971), Nimrick et a l . (1972) and Amos et a l . (1974) obtained 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) obtained increased nitrogen retention when soybean was infused abomasally. There have been l i m i t e d reports on formaldehyde treatment of dehydrated forages (Hemsley et a l . , 1970; Dinius et: a l . , 1975; Beever et a l . , 1976). Experiments, reported l a t e r , were conducted to study the e f f e c t of formaldehyde treatment of the grass-legume forage portion of the d i e t on nitrogen and carbohydrate u t i l i z a t i o n by sheep. I s o v a l e r i c and i s o b u t y r i c acids were added to some of the d i e t s containing the formaldehyde treated forage. Protection of the dietary protein may r e s u l t i n d e f i c i e n c y of these acids i n the ruminal f l u i d . el-Shazly (1952a and 1952b) reported that these v o l a t i l e f a t t y acids r e s u l t from the deamination of v a l i n e and leucine. A l l i s o n el; a l . (1962) reported that Ruminococcus fl a v e f a c i e n s s t r a i n C94, a rumen c e l l u l o l y t i c micro-organism requires i s o b u t y r i c and i s o v a l e r i c acids f o r growth. Sulphur was also supplemented to some of the d i e t s . Kennedy et a l . (1975) reported that the extent of incorporation of recycled urea-nitrogen into microbial protein may be l i m i t e d by the quantity of recycled sulphur. Recycling of sulphur into the rumen was reported by these workers to be le s s than r e c y c l i n g of nitrogen into the rumen. Sulphur de f i c i e n c y may become more acute with the protection of dietary protein. About ninety percent (90%) of the sulphur i n most plants was reported to be present i n the sulphur-containing amino acids (Beaton et a l . , 1968). Nitrogen, on the other hand, may not be l i m i t i n g i n the rumen with the protection of dietary protein. Langlands (1973b) and Faichney (1974) reported adequate amounts of recycled urea-nitrogen into the rumen with formaldehyde treatment of d i e t s for sheep. - 4 -LITERATURE REVIEW Rumen microbiology The ruminant i s able to subsist on roughages because of the micro-organisms present i n the reticulorumen that can ferment feeds. There are many types of micro-organisms i n the rumen, the major ones being b a c t e r i a and c i l i a t e d protozoa. Other types observed at times i n the rumen are y e a s t - l i k e organisms, phages and f l a g e l l a t e d protozoa (Church, 1975b). Rumen ba c t e r i a There are a number of ways rumen b a c t e r i a are c l a s s i f i e d . Methods of c l a s s i f i c a t i o n include morphology, gram-stain reaction and various products of metabolism. The use of any one of these methods alone has l i m i t a t i o n s . Rumen ba c t e r i a are too s i m i l a r to i d e n t i f y s o l e l y on the basis of morphology. Some st r a i n s within the same species give both p o s i t i v e and negative reactions to the gram s t a i n . There i s usually a great deal of overlapping with d i f f e r e n t species of b a c t e r i a with regard to source of energy, substrates attacked and fermentation end-products and by-products (Church, 1975b). In view of the problems l i s t e d above concerning i d e n t i f i c a t i o n of rumen ba 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 c l a s s i f i c a t i o n used by Hungate (1966), based p a r t i a l l y on substrates and p a r t i a l l y on the end-products i n i n v i t r o culture systems, has been used i n t h i s l i t e r a t u r e review. - 5 -There are about ten (10) c l a s s i f i c a t i o n groups known using t h i s system. (a) C e l l u l o l y t i c b a c t e r i a : Have the enzyme c e l l u l a s e and may also be able to degrade c e l l o b i o s e . These types are i n greatest concentration when animals are fed fibrous r a t i o n s . The most important of these c e l l u l o l y t i c species include Bacteroides succinogenes, Ruminococcus f l a v e f a c i e n s , Ruminococcus albus, Clostridium loch h e a d i i , and Cillobacterium c e l l u l o s o l v e n s . (b) Hemicellulose digesting b a c t e r i a : Most organisms which can hydrolyze c e l l u l o s e are also able to u t i l i z e hemicellulose. A number of organisms which can u t i l i z e hemicellulose, however, cannot u t i l i z e c e l l u l o s e . Some of the species which u t i l i z e hemicellulose are 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 Bacteroides  ruminicola. (c) Amylolytic b a c t e r i a : Some c e l l u l o l y t i c organisms can digest starch; however, there are some organisms which are purely amylolytic and cannot digest c e l l u l o s e . Amylolytic b a c t e r i a are i n the greatest concentrations when rations containing large amounts of starch are fed. Examples of amylolytic organisms are: Bacteroides amylophilus, Succinimonas amylolytica, 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. alactacidigens,  Bacteroides ruminicola, Selenomonas ruminantium, Selenomonas l a c t i l y t i c a and Streptococcus bovis. (d) Bacteria u t i l i z i n g sugars: Most of the b a c t e r i a u t i l i z i n g polysaccharides are also able to u t i l i z e sugars. The sugars may o r i g i n a t e from plants, dead and l y s i n g b a c t e r i a c e l l s , or from capsular material - 6 -(mostly carbohydrates of b a c t e r i a l c e l l w a l l s ) . High concentrations of micro-organisms dependent on lactose for energy are present i n the rumens of young animals. An example of b a c t e r i a u t i l i z i n g sugars i s Eubacterium ruminantium. (e) Bacteria u t i l i z i n g organic acids: The organic acids which are probably u t i l i z e d to the greatest extent are l a c t i c , s u c c i n i c , malic and fumaric. Others including formic, a c e t i c and o x a l i c are also u t i l i z e d by some organisms. The l a t t e r may not be the only source of energy to the micro-organisms u t i l i z i n g them. Some l a c t i c acid u t i l i z i n g organisms are: V e i l l o n e l l a gazogenes, V. alacalescens, Peptostreptoccus  e l s d e n i i , Propionibacterium sp., Desulphovibrio sp., and Selenomonas  l a c t i l y t i c a . (f) P r o t e o l y t i c b a c t e r i a : Bacteria that u t i l i z e amino acids as the primary energy sources. Bacteroides amylophilus, Clostridium  sporogenes and B a c i l l u s l i c h e n i f o r m i s are the best known to have p r o t e o l y t i c c a p a b i l i t y . (g) Ammonia producing b a c t e r i a : These produce ammonia from various sources. Examples of these are Selenomonas ruminantium, Peptostreptococcus e l s d e n i i , Bacteroides ruminicola, and c e r t a i n s t r a i n s of B u t y r i v i b r i o sp. (h) Methanogenic b a c t e r i a : These produce methane gas. They are obligate anaerobes and are therefore d i f f i c u l t to culture. Since normally about 25% of the gas i n the rumen i s methane (Church, 1975b), a large number of them are indicated. Important examples of these are Methanobacterium ruminantium, and M. formicium. Other species of l e s s importance are Methanobacterium sohngenii, M. suboxydans and Methanosarcina sp. - 7 -( i ) L i p o l y t i c b a c t e r i a : These u t i l i z e g l y c e r o l and hydrolyze g l y c e r o l from l i p i d s . Some l y p o l y t i c micro-organisms hydrogenate unsaturated f a t t y acids and may produce p o s i t i o n a l isomerization i n f a t t y acids. Certain l i p o l y t i c micro-organisms may metabolize long chain f a t t y acids i n t o ketones. V i v i a n i (1970) reported, of one l y p o l y t i c bacterium, Anaerovibrio l y p o l y t i c a . Hobson and Mann (1961) reported Selenomonas ruminantium s t r a i n l a c t i l y t i c a n s as a g l y c e r o l fermenter. (j) Vitamin synthesizers: Some rumen b a c t e r i a are able to synthesize B- complex vitamins inc l u d i n g cobalamin ( v i t . B12) provided they are supplied with adequate cobalt ( P h i l l i p s o n , 1975). The vitamin synthesizers have not been studied extensively. Rumen Protozoa Rumen protozoa are c l a s s i f i e d according to morphology. They are easier to c l a s s i f y by that method compared to b a c t e r i a because of t h e i r l a rger s i z e . Counts of protozoa i n the rumen vary from none to as high as f i v e (5) m i l l i o n per ml of rumen f l u i d . The s i z e of rumen protozoa va r i e s from t h i r t y eight microns i n length and f i f t e e n microns i n width for Charon equi to one hundred and ninety f i v e microns i n length and one hundred and nine microns i n width for Metadinium medium. Rumen protozoa are mainly c i l i a t e s although at times such f l a g e l l a t e s as Monocercomonas ruminantium, 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 bovis and Chilomastix sp. are observed. - 8 -The most important rumen c i l i a t e s belong to subclasses h o l o t r i c h i a and s p i r o t r i c h i a . Examples of the h o l o t r i c h i a are Dasytricha ruminantium, I s o t r i c h a i n t e s t i n a l i s , I s o t r i c h a prostoma and also Charon equi, which i s rare. Examples of the s p i r o t r i c h i a subclass belonging to the order entodinimorpha ( o l i g o t r i c h s ) are Diplodinium spp., Eudiplodinium spp., Polyplastron spp., E l y s t r o p l a s t r o n spp., Ostracodinicum spp., and Enoploplastron spp. Rumen protozoa are obligate anaerobes. Other rumen micro-organisms Other rumen micro-organisms include bacteriophages ( b a c t e r i a l viruses) y e a s t - l i k e organisms, and f a c u l t a t i v e l y anaerobic fungi. There are about one hundred and twenty f i v e (125) morphologically d i s t i n c t types of bacteriophages observed i n the rumen. Some have been observed inside rumen ba c t e r i a . The bacteriophages may exceed rumen b a c t e r i a i n numbers by two to ten times. Pun and Satter (1975) reported the existence of nitrogen f i x i n g b a c t e r i a i n the rumen but believed these b a c t e r i a were not of any s i g n i f i c a n t value. Factors a f f e c t i n g populations of rumen micro-organisms Many kinds of micro-organisms are present i n the rumen. There are many ways by which micro-organisms may a r r i v e i n the rumen. Methods of entering the rumen include being c a r r i e d by food, water, l i c k i n g and drenching with drugs (Church, 1975b). Micro-organisms that are normally - 9 -not part of the rumen microbial population may be present at times. G a l l and Hubtanen (1950) l i s t e d the following as c r i t e r i a to be s a t i s f i e d i f micro-organisms are to be considered t y p i c a l of the rumen environment: (a) the organism must be able to l i v e anaerobically; (b) i t should be able to produce the types of end-products c h a r a c t e r i s t i c of the rumen, and (c) the rumen should contain not l e s s than one m i l l i o n per gram of the organisms. This l a s t c r i t e r i o n applies to b a c t e r i a but not protozoa. It should also be noted that 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 microbial populations. One of the factors which can a f f e c t type and number of micro-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 . I n h i b i t i o n by v o l a t i l e f a t t y acids on the growth of some others such as Escherichia c o l i has been reported. R e s t r i c t i o n i n growth of one type of microbe by toxins or a n t i b i o t i c s produced by another type has been postulated. Those micro-organisms producing the toxins and a n t i b i o t i c s are not affected (Church, 1975b). Hungate (1970) reported that mycoplasma which can k i l l b a c t e r i a and protozoa have been observed free i n the rumen. The mycoplasma secretes an enzyme or enzymes capable of digesting B u t y r i v i b r i o sp., Ruminococcus sp., and Escherichia c o l i but not the gram-positive Streptococcus bovis. - 10 -Diet can a f f e c t m i c r o b i a l populations i n the rumen. The d i e t may also be influenced by geographic l o c a t i o n or season. Some types of micro-organisms may be present i n some ruminant species but not others. For instance a large oval form of b a c t e r i a (Quinn's oval) i s common i n sheep but not c a t t l e . Perhaps the most important i n f l u e n c i n g f a c t o r i s the d i e t . Church (1975b) indicated that the optimum pH f o r growth of rumen micro-organisms i s between 5.5 and 7 while the optimum temperature range i s 39-41°C. Eadie and Mann (1970) reported that high soluble 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 production of excess a c i d . This i n turn changes the m i c r o - f l o r a and fauna. Under such conditions, the only c i l i a t e s found are Entodinia sp. (Eadie and Mann, 1970). Hobson (1971) however was of the opinion that protozoa are absent below pH 6. Church (1975b) also reported that d i e t s high i n soluble carbohydrates generally r e s u l t i n a depression i n the numbers of c e l l u l o l y t i c organisms. Eadie and Mann (1970) however reported that with d i e t s high i n soluble carbohydrates v i a b l e b a c t e r i a counts may be higher than with roughage d i e t s . These workers also reported f l a g e l l a t e protozoa and large b a c t e r i a such as Selenomonas sp. to be present i n high concentration i n animals fed high carbohydrate d i e t s . S l y t e r et a l . (1970) also reported that the l e v e l of feeding of high carbohydrate d i e t s affected the types of rumen microbes. F u l l feeding of a high carbohydrate diet reduced the number of protozoa while r e s t r i c t e d feeding d i d not. These workers also reported that feeding of high carbohydrate d i e t s caused more unstable rumen conditions than feeding - 11 -roughage d i e t s . These unstable conditions resulted i n greater animal v a r i a t i o n s even with i d e n t i c a l twins with respect to s t a b i l i t y of pH, and v o l a t i l e f a t t y acid production. They also indicated that animals with l e a s t a b i l i t y to s t a b i l i z e rumen conditions were the f i r s t to lose the c i l i a t e protozoa. Certain rumen micro-organisms also require some factors which are produced by others. Factors which i n h i b i t the growth of those producing these factors i n h i b i t the population of those requiring these factors (Church, 1975b). Bacteroides succinogenes, R. albus, 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 require B vitamins produced by other 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 Bacteroides  succinogenes and also R. f l a v e f a c i e n s require 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 acids, f o r t h e i r growth ( A l l i s o n et a l . , 1962; A l l i s o n and Bryant, 1963; Hemsley and Moir, 1963; A l l i s o n et a l . , 1966). A l l i s o n and Bryant (1963) thought that the mechanism for the synthesis of the isopropyl moiety i n the branched-chain f a t t y acids was inadequate i n these micro-organisms. el-Shazly (1952a and 1952b) reported that the branched-chain f a t t y acids resulted from the de-amination of proteins. Sulphur i s also required by some micro-organisms (Kennedy et a l . , 1975). Hobson (1971) claimed that f a c u l t a t i v e anaerobes such as Streptococcus bovis and V e i l l o n e l l a gazogenes take up the small amount of oxygen e x i s t i n g i n the rumen, t h i s enables the methanogenic bacteria.and protozoa which are completely anaerobic to survive. This also helps Bacteroides amylophilus and Selenomonas ruminantium which cannot t o l e r a t e oxidation-reduction p o t e n t i a l of even - 45 mv. Methane i n h i b i t o r s such as bromochloromethane and unsaturated f a t t y acids have been used to - 12 -suppress methane production (Johnson at a l . , 1972; Sawyer et a l . , 1974). These may also adversely a f f e c t the populations of 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 protozoal numbers are more pronounced than b a c t e r i a l numbers. Highest protozoal concentrations have been recorded two hours a f t e r feeding (Clarke, 1965) and b a c t e r i a numbers are highest about 4-8 hours a f t e r feeding. (Moir and Sommers, 1956; Bryant and Robinson, 1968). Seasonal v a r i a t i o n s are due mainly to changes i n feed. Nitrogen d e f i c i e n c y or mineral d e f i c i e n c y due to seasonal e f f e c t s on d i e t , can l i m i t growth (Schwartz and G i l c h r i s t , 1975; Church,1975b; Amos et a l . , 1976a). M i c r o b i a l protein-nitrogen composition and m i c r o b i a l protein synthesis The nitrogen content of rumen b a c t e r i a i s about 10.5% of the c e l l dry matter ( i . e . about 65% CP). Amino nitrogen i s about 75% of the t o t a l nitrogen i n mixed organisms from the rumen (bacteria, protozoa and others). Rumen ba c t e r i a contain about 86% of t h e i r t o t a l nitrogen as amino nitrogen ( A l l i s o n , 1970). The non-amino-nitrogen content of rumen b a c t e r i a i s mainly n u c l e i c acids, DNA and RNA. Nucleic a c i d may account for 14-19% of the t o t a l nitrogen of rumen micro-organisms. Most of t h i s i s RNA since DNA accounts f o r 2.2 - 4.1% of the t o t a l nitrogen ( A l l i s o n , 1970). L i t t l e has been done to characterize the walls of rumen b a c t e r i a . The c e l l - w a l l of several non-rumen b a c t e r i a accounts for 10-20% of the mass ofthe c e l l and the c e l l - w a l l m a t erial i n these organisms i s 5-10% N ( A l l i s o n , 1970). I f rumen ba c t e r i a have s i m i l a r proportions of walls - 13 -and w a l l nitrogen and i f the t o t a l c e l l s have a nitrogen content of 10.5% then c e l l - w a l l nitrogen would be 5-19% of the t o t a l c e l l nitrogen, ( A l l i s o n , 1970). The b a c t e r i a l c e l l walls may have peptidoglycan or mucoprotein as major components. These are r e s i s t a n t to t r y p s i n and pepsin attack and may not be useful to the host. Non-amino-nitrogen i n the c e l l w a l l i s mainly i n the form of neucleic acids. Muramic acid and °C-; ^ -diaminopimelic acids are unique to b a c t e r i a l c e l l walls while 2-aminoethylphosphonic acid i s also unique to protozoal c e l l walls (Work, 1951; Work and Dewey, 1958; A l l i s o n , 1970). Bergen et a l . (1967) reported that the l i m i t i n g amino acid i n pooled c e l l u l o l y t i c s t r a i n s was methionine and for n o n - c e l l u l o l y t i c s t r a i n s was leucine. Protozoal proteins are more d i g e s t i b l e than b a c t e r i a l proteins and protozoa have a higher l y s i n e content than b a c t e r i a (Church, 1975b). Hogan and Weston (1970) indicated that about 10-12g dry weight of microbes are synthesized per mole of ATP but DM mi c r o b i a l weight synthesis could increase to about 20g per mole ATP, with an increase i n -1 -1 d i l u t i o n rate from 0.1 hr to 0.3 to 0.5 hr . They indicated that 2.37g of b a c t e r i a nitrogen are produced per mole of v o l a t i l e f a t t y acid produced. Walker and Nader (1968), reported 13-14g dry weight of microbial c e l l s synthesized per mole of ATP. Hume (1970) reported an increase i n microbial protein synthesis with the addition of 2-methylbutyric, i s o v a l e r i c and n - v a l e r i c acids to d i e t s containing non-protein-nitrogen. Other workers (Hemsley and Moir, 1963; Umuna et a l . , 1975) have also reported increases i n m i c r o b i a l protein synthesis when some branched-chain f a t t y acids and v a l e r i c acid were added to d i e t s containing urea. - 14 -Beever et a l . (1977) reported microbial protein synthesis per lOOg O.M. digested i n the rumen of 16.7g for a grass s i l a g e d i e t and 6.6g for a formaldehyde treated grass s i l a g e d i e t . Hume (1970) reported a negative c o r r e l a t i o n between a c e t i c acid proportions i n rumen v o l a t i l e f a t t y acids and protein synthesis (r = -0.62, p 0.025). He concluded that the e f f i c i e n c y with which energy was used f o r microbial growth was diminished as a c e t i c a c i d proportions i n rumen f l u i d increased. Schwartz and G i l c h r i s t (1975) and Ishaque et: a l . (1971) also reported that propionic a c i d fermentation was more e f f i c i e n t f or microbial protein synthesis than a c e t i c or bu t y r i c acid fermentation. Harrison et a l . (1976) however reported a c e t i c a c i d fermentation to be more e f f i c i e n t f o r m i c r o b i a l protein synthesis than propionic a c i d fermentation. These workers infused a r t i f i c i a l s a l i v a at the rate of four l i t r e s per day to a l t e r propionic acid fermentation to a c e t i c acid fermentation. The e f f e c t of the increased flow rate was not delineated. A number of methods have been used to assess m i c r o b i a l protein synthesis. These include measuring diaminopimelic acid (DAP) and aminoethylphosphonic acid (AEP) i n the sample (Hogan and Weston, 1970; Amos, et a l . , 1976a); p r e c i p i t a t i o n of microbial protein 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 and'tungistic acids and measuring the amount of the p r e c i p i t a t e d protein (Hemsley and Moir, 1963; Hume, 1970; Barr et a l . , 1975); l a b e l l i n g the rumen pool with radioactive 35 c sulphur, . ?, and measuring r a d i o a c t i v i t y l e v e l of sulphur i n sulphur-containing amino acids (Walker and Nader, 1975; Hume, 1974); and the measurement of r i b o n u c l e i c acid-nitrogen (Smith, 1975). - 15 -A l l the methods other than the RNA-N, l a b e l l i n g with J J S and the combination of DAP and AEP measure mainly rumen b a c t e r i a l p rotein synthesis. The DAP measures b a c t e r i a l p r o t e i n synthesis and AEP measures protozoal protein synthesis (Work, 1951; Work and Dewey, 1958; Church, 1975b). The RNA-N method measures both b a c t e r i a l and protozoa protein synthesis without apportioning f r a c t i o n s to each type 35 of organism (Smith, 1975). With the S method, problems of i n f u s i o n to maintain steady state conditions e x i s t . Smith (1975) believes that the DAP method has disadvantages. One of these i s that DAP 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 degradation and thus increases measured protein synthesis. The AEP method according to Smith (1975) i s u n r e l i a b l e and unsa t i s f a c t o r y . The te c h n i c a l b u l l e t i n of the International Atomic Energy Agency (IAEA) (1970) indi c a t e s that DAP i s r e s t r i c t e d to gram-negative b a c t e r i a and b a c t e r i a contents of DAP vary greatly. Hogan and Weston (1970) estimated that b a c t e r i a l contents of DAP vary from 35-46 mg/g DM b a c t e r i a l nitrogen with an average of 41 mg/g. Smith (1975) believes that the RNA-N method i s more r e l i a b l e since the r a t i o of RNA-N to t o t a l nitrogen i s about 0.075 + 0.010 and for DAP-N to t o t a l nitrogen i s 0.5 - 1.1 f o r d i f f e r e n t b a c t e r i a l species. RNA i s associated with ribosomes and the quantity of 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 correlated with the rate of protein synthesis (Church, 1975b). Although there i s about 30% r e c y c l i n g of RNA i n the rumen, RNA i s broken down quickly and may not a f f e c t measured protein synthesis (McAllan and Smith, 1973; Smith, 1975; Smith and Smith, 1977). Dietary RNA and other sources of RNA introduced into the rumen are also quickly broken down (Smith, 1975; Smith and Smith, 1977). - 16 -Metabolism of nitrogen There i s quite a v a r i a t i o n i n the nitrogenous compounds presented to the rumen micro-organisms. The most important are: proteins, n u c l e i c acids and non-protein nitrogen c o n s i s t i n g of amino acids, peptides, 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 , urea and at times biuret i n t e n t i o n a l l y put into the d i e t . The non-protein-nitrogen i n many natural feedstuffs ranges from 4 to 5% of t o t a l nitrogen i n some seeds and 60-75% of t o t a l nitrogen i n unwilted s i l a g e s . (Church, 1975c). A l l i s o n (1970) indicated that about 5-10% of the t o t a l nitrogen may be bound with l i g n i n i n the c e l l w all and i s l a r g e l y i n d i g e s t i b l e . Yu and Thomas (1976) estimated that about 7% of t o t a l nitrogen i s present i n the acid-detergent i n s o l u b l e nitrogen i n normal forages. The d i v e r s i t y of nitrogenous compounds presented to rumen microbes r e s u l t s i n considerable v a r i a t i o n i n the nitrogen metabolism i n the reticulo-rumen. Nitrogen metabolism i n the ruminant i s described by the diagram of Houpt (1970) reproduced as Figure 1. Degradation of Proteins Many researchers ( A l l i s o n and Peel, 1971; Mathison and M i l l i g a n , 1971; Nolan and Leng, 1972; Umuna et a l . , 1975) have indicated that rumen micro-organisms prefer de novo synthesis of proteins. Dietary proteins, except globulins i n young animals, may be degraded before - 16a -F i g . 1. Schematic representation of the protein regeneration cycle i n ruminants. (Adapted from Houpt, 1970) - 17 -being u t i l i z e d by the microbes. The end-products of protein degradation i n the rumen are carbon dioxide, hydrogen sulphide a c e t i c , propionic and b u t y r i c acids, higher branched-chain f a t t y acids containing s i x carbon atoms and ammonia (0-130 mg%, as NH_,-N) (Church, 1975c). Some intermediary products in c l u d i n g free amino acids (0.1-1.5 mg%), d i f f u s i b l e peptides (0.2-1.0 mg%), nucleotide - N (1.5-40 mg%), and protein-N(100-400 mg%) have been shown (el-Shazly, 1952a and b; Annison, 1956; E l l i s and Pfander, 1965; A l l i s o n , 1970; Smith and McAllan, 1970; A l l i s o n and Peel, 1971). M i c r o b i a l nitrogen as a proportion of the t o t a l nitrogen i n the rumen i s quite v a r i a b l e - about 63-81% (Weller et a l . , 1958; Blackburn and Hobson, 1960). Blackburn and Hobson (1960) estimated that about 47-77% of the nitrogen i n the rumen was contained i n protozoa and b a c t e r i a , while 54-74% was present i n the rumen f l u i d . The proportion of dietary protein escaping degradation i n the rumen i s affected by many factors which a f f e c t retention times i n the reticulo-rumen. These include s p e c i f i c gravity, p a r t i c l e s i z e of d i e t , and high water consumption r e s u l t i n g from high s a l t intake (Chalupa, 1975). S o l u b i l i t y of the protein also a f f e c t s dietary protein degradation i n the rumen (Hemsley et a l . , 1970; Chalupa, 1975). Chalupa (1975) also indicated that feeding high l e v e l s of soluble carbohydrates, which decreases rumen pH, decreased degradation of protein i n the rumen. However, Tagari et a l . (1964) reported greater degradation of protein i n the rumen by s u b s t i t u t i n g r e a d i l y soluble carbohydrates for roughages. Chalupa (1975) reported that about 40-80% of dietary protein may be degraded i n the rumen. Satter and R o f f l e r (1975) gave the following as the percentage of p r o t e i n - 18 -escaping degradation f o r various feedstuffs fed i n basal r a t i o n s : barley 10%, cottonseed meal and peanut meal 20%, sunflower meal 25%, soybean meal 45%, dried grass and white f i s h meal 50%, and Peruvian f i s h meal 70%. Ferguson (1975) indicated that 61% of soybean meal, 56% of zein, and 9% of casein proteins escaped degradation. Nolan and Leng (1972) reported that about 59% of the protein entering the rumen was degraded there; while 29% of the degraded protein was u t i l i z e d as amino acids, 71% was further degraded to ammonia. Nolan (1975) reported that about 30% of dietary protein intake i s generally degraded to ammonia with dried and processed forage d i e t s . He estimated that about 70% of the dietary protein e i t h e r passes i n t a c t from the rumen or i s assimilated by rumen micro-organisms i n the form of compounds other than ammonia, such as peptides, amino acids or n u c l e i c a c i d bases. P i l g r i m et a l . (1969) estimated that 23-27% of dietary protein i n a l f a l f a hay was converted to ammonia as compared to 17% from a l f a l f a p e l l e t s . The rate of deamination of amino acids v a r i e s with the amino acids i n question. Isaacs and Owens (1971) reported that a s p a r t i c and glutamic acids, and arginine were degraded i n the rumen to the extent of about 90%; v a l i n e , leucine, i s o l e u c i n e , methionine, alanine and glycine appeared to be r e l a t i v e l y stable toward microbial action. The phenolic amino acids were degraded to about 50%. Some of the p r o t e o l y t i c and ammonia producing b a c t e r i a have been l i s t e d previously under rumen microbiology. Blackburn and Hobson (1960) indicated that p r o t e o l y t i c a c t i v i t y was maximum between pH 6 and 7 and i s not dependent on d i e t . Blackburn (1968) also suggested that proteases - 19 -obtained from B_. amylophilus had a broad area of a c t i v i t y between pH 5.5-9.5. Isaac and Owens (1971) reported that e x t r a - c e l l u l a r enzymes other than amino peptidase could possibly help i n hydrolyzing proteins i n the rumen. A l l i s o n (1970) indicated that although p r o t e o l y t i c enzymes may be mainly cell-bound, some gram p o s i t i v e c o c c i such as Clostridium sp., Eubacterium sp., and Lachnospira multiparus produce e x t r a - c e l l u l a r enzymes. A l l i s o n (1970) also reported that i n B_. amylophilus the protease i s a c o n s t i t u t i v e enzyme. Abou Akkada and Blackburn (1963) reported that proteases i n some p r o t e o l y t i c microbes possessed both exo- and endo-peptidase a c t i v i t i e s . P r o t e o l y t i c a c t i v i t y of rumen protozoa i s not well understood. Abou Akkada and Howard (1962) reported rumen protozoa to hydrolyze casein to peptides and amino acids, as p r i n c i p a l end-products. Ammonia was formed as a r e s u l t of the hydrolysis of the amide groups i n the casein but not from de-amination of amino acids. Warner (1956) thought that ammonia was the end-product of nitrogen metabolism i n mixed suspensions of protozoa. Purser and Moir (1966) observed high concentrations of ammonia i n the rumen of sheep containing protozoa compared to defaunated animals. Blackburn (1965) noted that though many p r o t e o l y t i c enzymes have been extracted from rumen protozoa, i t i s d i f f i c u l t to claim with c e r t a i n t y that they were produced by the protozoa and not by b a c t e r i a found i n them. C i l i a t e s are noted to take up only small quantities of "^N-labelled amino acids and may therefore depend greatly on rumen b a c t e r i a f o r amino acids and should most probably have p r o t e o l y t i c enzymes ( A l l i s o n , 1970). - 20 -The concentration of ammonia i n the rumen f l u i d i s affected by time of feeding and type of d i e t (Church, 1975c). Ammonia-nitrogen concentration i s highest at 90-130 minutes a f t e r feeding (Church, 1975c). The addition of starch to roughage basal d i e t s containing e i t h e r casein or urea resulted, i n increased e f f i c i e n c y of ammonia u t i l i z a t i o n and con-sequently lowered rumen ammonia l e v e l s (Barej el: a l . , 1970). In contrast, Tagari et a l . (1964) and Schwartz and G i l c h r i s t (1975) indicated that protein breakdown i n the rumen could be enhanced and ammonia l e v e l s increased when high l e v e l s of starch are fed since the most p r o t e o l y t i c species, 15. amylophilus, S^. ruminantium and M. e l s d e n i i could increase i n numbers ten f o l d . Satter and R o f f l e r (1975) reported that maintenance of ruminal ammonia-nitrogen l e v e l s i n excess of 5mg/100 ml rumen f l u i d does not improve microbial p r o t e i n synthesis. They calculated that rumen ammonia-nitrogen l e v e l s i n dairy c a t t l e fed normal dairy d i e t s may vary from l-5mg/100ml depending on the energy content of the di e t with the highest l e v e l s associated with lowest energy content d i e t . Studies on the degradation of proteins i n the rumen are complicated by secretion of urea into the rumen v i a s a l i v a , and of ammonia through the rumen epithelium, absorption of ammonia and other nitrogenous compounds by the rumen epithelium and r e c y c l i n g of microbial p r o t e i n within the rumen (Church, 1975c). M i c r o b i a l protein i s r e a d i l y hydrolyzed and deaminated. M i c r o b i a l protein r e c y c l i n g and microbial protein synthesis from urea and/or ammonia and free amino acids may be r e l a t i v e l y more important than degradation of ingested protein to be used f o r mi c r o b i a l protein synthesis (Church, 1975c). Nolan and Leng (1972), estimated - 21 -that about 4.3gm/N might be recycled per day within the rumen of sheep. They also suggested that about 30% of ammonia co n t i n u a l l y being incorporated into ruminal microbial protein may have been recycled through the amino acid and ammonia pools. Chalupa (1975), reported that about 30% of b a c t e r i a l protein i s degraded i n the rumen. Hume (1970), was also of the opinion that desquamation of rumen epithelium though small, could be a source of nitrogen to rumen micro-organisms. Urea trans f e r into the rumen ammonia pool from blood (dependent on rumen ammonia concentration) i s one source of nitrogen to rumen microbes (Church, 1975c). Nolan (1975) reported that about l-2g of urea was transferred d a i l y i n sheep fed lucerne chaff d i e t s . Houpt (1970) reported that on low protein d i e t s about 92% of endogenous urea enters the alimentary canal and about 84% i s converted to complex nitrogenous compounds. Hemler and Bartley (1971) were of the opinion that more urea enters the rumen from blood than from s a l i v a . Hogan (1975) however thought that more urea entered the rumen from s a l i v a than from plasma. Hogan el: a l . (1969) and Hogan (1975) estimated urea entering the rumen of sheep to be about 2-5g N per day and thought almost a l l came from s a l i v a since about 2-5g, N/day could be present i n s a l i v a secreted (about 10 l i t r e s containing 28mg N/100 ml on a roughage d i e t ) . A l l e n and M i l l e r (1976) reported urea entry into rumen to be an active process rather than by simple d i f f u s i o n . Houpt (1970) considers that' the major portion of the urea entering the rumen from blood i s converted to ammonia by rumen b a c t e r i a l urease within the c o r n i f i e d layers before entering the rumen. - 22 -Urea from s a l i v a , blood and i n the di e t i s hydrolyzed by u r e o l y t i c enzymes probably produced by several b a c t e r i a ( A l l i s o n , 1970). The enzymes from the mixed b a c t e r i a were stimulated by Mn, Mg, Ca, Sr, Ba, but i n h i b i t e d by Na, K and Co. An enzyme from a s i n g l e bacteria s t r a i n was not stimulated by a l l the divalent ions. Ammonia produced i n the rumen could be used f o r (1) microbial amino acid synthesis or (2) could be absorbed into the blood stream from the reticulo-rumen and omasum or (3) could pass i n t o the abomasum and consequently the duodenum, since there i s v i r t u a l l y no absorption of ammonia from the abomasum. Part of the ammonia absorbed from the forestomach could be l o s t through urine a f t e r conversion to urea or the urea could be recycled. Part of the ammonia could also be used f o r the synthesis of amino acids i n the l i v e r (Houpt, 1970; Hembry et^ a l . , 1975). The absorption of ammonia from the rumen i s dependent on the rate of microbial protein synthesis which i n turn i s dependent on the rate of ammonia formation and energy made a v a i l a b l e to the microbes (Hembry ej: a]L., 1975). Hemler and Bartley (1971) and Hogan (1961) also reported that ammonia absorption from the rumen depends on the concentration gradient at pH 6.5. I t was n e g l i g i b l e at pH 4.5. Estimates of ammonia u t i l i z e d f o r any of the three functions have been v a r i a b l e . Nolan and Leng (1972) showed that about 80% of the microbial nitrogen was derived from ammonia and only about 20% of microbial nitrogen came d i r e c t l y from amino acids. Mathison and M i l l i g a n (1971) estimated that 50-65% of b a c t e r i a l nitrogen and 31-55% of protozoal nitrogen were derived from rumen ammonia. They also indicated that 17-54% of the ammonia derived from dietary protein (about 60-92% of dietary protein transferred to ammonia by t h e i r - 23 -estimation) of chopped hay or barley plus chopped hay, was absorbed from the rumen. Nolan (1975) reported that b a c t e r i a l nitrogen derived from ammonia was about 30-80% and protozoal nitrogen derived from ammonia 25-64%. Hogan and Weston (1967) reported that i n sheep fed high protein d i e t s (C.P. 19.8%) up to 31% of the nitrogen disappeared between the ingested feed and duodenal digestion. P i l g r i m e^ t a l . (1969) estimated that 57-66% of the ammonia-nitrogen was absorbed from the rumen or u t i l i z e d for m i c r o b i a l p r o t e i n synthesis and the remainder passed on in t o the omasum. Protein anabolism by rumen b a c t e r i a Rumen microbes are able to synthesize both e s s e n t i a l and non-e s s e n t i a l amino acids. Sauer et a l . (1975) reported that rumen microbes 14 r e a d i l y u t i l i z e d C-labelled-HCO_, and acetate f o r the synthesis of the carbon skeletons of amino acids and subsequently the amino acids. In : 14 contrast, C-labelled propionate was u t i l i z e d f o r i s o l e u c i n e b i o -14 synthesis but l a b e l l e d C from propionate f a i l e d to appear i n other amino acids to any s i g n i f i c a n t extent. Forward t r i c a r b o x y l i c acid cycle reactions only proceeded to keto- or 2-oxo- glutarate. Acetate was carboxylated to pyruvate which was then carboxylated to oxaloacetate. Oxaloacetate then e q u i l i b r a t e d with fumarate and thereby carbon atoms 1 and 4 as well as 2 and 3 became randomized. Most of the 2-oxo precursors of amino acids appeared to be formed v i a ferrodoxin dependent reductive carboxylation. Of the amino acid precursors investigated, - 24 -only 3-hydroxypyruvate, the precursor of serine, appeared to be synthesized v i a an oxidative step, ( i . e . 3-phosphoglyceric acid to 3-phosphohydroxypyruvic a c i d ) . Rumen microbes r e u t i l i z e d benzene rings i n the biosynthesis of phenylalanine and tyrosine. De novo synthesis of the benzene r i n g was of minor importance. Kristensen (1974) was also of the opinion that reductive carboxylation of phenylacetate and indole-3-acetic a c i d to form phenylalanine and tryptophan r e s p e c t i v e l y was of greater importance than de novo synthesis of the carbon skeletons. Reductive carboxylation of preformed isobutyrate to form v a l i n e was reported by Sauer et al. (1975) to be of minor importance to de novo synthesis of the carbon skeleton from acetate. There was however a considerable degree of reductive carboxylation of preformed i s o v a l e r i c acid to form leucine as compared to de novo synthesis of the carbon skeleton. A l l i s o n et a l . (1962), A l l i s o n and Bryant (1963), and A l l i s o n et a l . (1966) suggested that biosynthesis of the isopropyl moiety was inadequate because a considerable quantity of materials containing t h i s group i s present i n c e l l u l a r l i p i d where isopentadecanoic acid i s a major f a t t y acid, a f t e r f i n d i n g branched-chain f a t t y acids as requirements for R. f l a v e f a c i e n s s t r a i n C94. E f f e c t of formaldehyde treatment on the digestion of proteins i n the rumen The digestion of dietary proteins i n the rumen by rumen micro-organisms could e i t h e r be b e n e f i c i a l or wasteful to the host ( P h i l l i p s o n , 1972). The extent of m i c r o b i a l degradation i s dependent on protein - 25 -solubility. One way to reduce protein solubility and then digestion in 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 ^ - X - CH OH (1) 1 oL °c i R - X - CH„0H + R - NHr- fcR - X - CH - NH - R + H„0 (2) z z * 2 2 (Methlylene compound) R - XH + R 1 - NH - CH2OH ^R - X - CH - NH - R1 + H20 (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 histidine may take part in 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 in the near-neutral pH of the rumen but the H + ions in the abomasum break down the linkage with the - 26 -release of formaldehyde (Rattray and Joyce, 1970). Formaldehyde i s not harmful to rumen microbes at the low concentrations used to treat feeds. M i l l s et^ a l . (1972) using 14 C-labelled formaldehyde reported that ruminants e f f e c t i v e l y metabolized formaldehyde and there was no accumulation of i t i n the carcass or milk. Sixty to eighty percent (60-80%) of the formaldehyde was metabolized to carbon dioxide and methane, 11-27% was voided i n the faeces and 5-6% excreted i n the urine. Small a c t i v i t i e s of "^C from l a b e l l e d formaldehyde were detected i n milk and body tissues but not as formaldehyde. The pathways whereby formaldehyde i s converted to methane are obscure, but three mechanisms have been proposed by M i l l s et a l . (1972). (a) Formaldehyde i s converted to formate and i t i s subsequently metabolized to carbon dioxide and methane. (b) Formaldehyde i s successively reduced to methanol and methane. (c) There i s an a c y l o i n condensation of formaldehyde with ribose-5-phosphate to form allulose-6-phosphate which i s further metabolized v i a the g l y c o l y t i c sequences to produce methane and carbon dioxide. Dinius e_t a l . (1974) also reported that treatment of protein per se with formaldehyde did not i n t e r f e r e with rumen microbial a c t i v i t y . - 27 -Feedstuffs that have been treated with formaldehyde can be c l a s s i f i e d into o i l seed meals, f i s h meal, casein, dried forages, s i l a g e s , hays and cereal grains. The response with formaldehyde treatment of proteins has been v a r i a b l e . Treatment of casein has been reported to cons i s t e n t l y y i e l d p o s i t i v e responses of nitrogen balance j growth rate and wool growth ( L i t t l e and M i t c h e l l , 1967; Sc h e l l i n g and H a t f i e l d , 1968; Reis and Tunks, 1969; Faichney, 1971; Faichney and Weston, 1971; P h i l l i p s o n , 1972; Sharma and I n g a l l s , 1974). Treatment of o i l seed meals such as peanut, soybean and rapeseed meals at times gives p o s i t i v e response and at times no response or at times negative response. Schmidt et a l . (1971) and Schmidt et a l . (1974) obtained negative responses while Peter ^ t a l . (1970), Peter et a l . (1971), Nimrick et a l . (1972) and Amos et a l . (1974) obtained p o s i t i v e responses of nitrogen balance growth rate and wool growth with treatment of soybean meal. Sharma and Ingalls (1974) and Sharma et a l . (1972) did not get a response with the treatment of rapeseed meal. Faichney and Davies (1972) and Faichney and Davies (1973) reported that the growth rate of calves improved when formaldehyde treated groundnut (peanut) meal was used i n d i e t s containing e i t h e r 12% or 13.4% crude protein. However, formaldehyde treatment did not improve gains when the d i e t s contained e i t h e r 15% or 20.5% crude protein. Faichney (1972) however reported that with higher crude protein d i e t s ( C P . 20%) i n which 50% of the crude protein was from treated or untreated peanut meal, treatment increased the proportion of protein digested i n the small i n t e s t i n e , compared with a d i e t of lower crude protein content (13% CP.;). - 28 -Langlands (1971b) and S a v i l l e et: a l . (1971) observed no response with treatment of cottonseed meal. Rattray and Joyce (1970) reported responses with treatment of linseed meal but not meat meal. Many workers have treated crops with formaldehyde before e n s i l i n g (Barry and Fennessy, 1972; Brown and Valentine, 1972; Barker et a l . , 1973; Valentine and Brown, 1973; Barry, 1975; Valentine and R a d c l i f f e , 1975; Barry, 1976b; Binnie and Barry, 1976). Some workers have reported p o s i t i v e responses such as increased feed intake, feed conversion e f f i c i e n c y , gain i n weight and production of milk, b u t t e r f a t , milk protein and SNF with formaldehyde treatment of sil a g e s (Valentine and Brown, 1973; Barry, 1975; Valentine and R a d c l i f f e , 1975; Binnie and Barry, 1976). Barry (1973a) reported that formaldehyde treatment of rye-grass clover hay s i g n i f i c a n t l y reduced nitrogen, energy and organic matter d i g e s t i b i l i t i e s . Barry (1973b) reported reduction i n liveweight losses when formaldehyde treated rye-grass-clover hay was fed at maintenance and half-maintenance l e v e l s to 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 when formaldehyde treated grass hay or untreated hay were fed at 600g DM to 18kg sheep. When 775g DM were fed d a i l y , there were s i g n i f i c a n t improvements i n the flow of e s s e n t i a l and non-essential amino acids, when treated hay was fed compared to the untreated. Langlands (1973a and 1973b) and Entwistle (1973) reported improvement i n nitrogen u t i l i z a t i o n with treatment of wheat. - 2 9 -There has been l i m i t e d work reported on formaldehyde treatment of a r t i f i c i a l l y dried forages. Dinius et a l . (1975) using d i e t s (16% C.F.) C Ontaining 75% a l f a l f a meal on a DM basis showed that formaldehyde treatment of the a l f a l f a at 1% and 2% l e v e l s s i g n i f i c a n t l y reduced energy, dry matter, crude protein, acid-detergent f i b r e d i g e s t i b i l i t i e s and nitrogen retention. However, Hemsley et a l . (1970) reported that 1% formaldehyde treatment of dried forage containing about 25% C P . improved wool growth by 15%. D i g e s t i b i l i t y of f i b r e was not affected but nitrogen d i g e s t i b i l i t y was reduced. Beever et a l . (1976) reported decreased nitrogen d i g e s t i b i l i t y with formaldehyde treatment of dried forage but c e l l u l o s e d i g e s t i o n was improved over untreated. Possible reasons f o r the v a r i b l e responses to formaldehyde treatment of d i f f e r e n t types of proteins  Treatment of casein with formaldehyde has been associated with p o s i t i v e responses of nitrogen retention, milk production, wool growth or growth rate because t h i s source of protein i s of higher q u a l i t y than microbial protein. The host therefore benefits from protection against microbial degradation (Reis and Tunks, 1969; Langlands, 1971a; Amos «2t al., 1974; P h i l l i p s o n , 1972). Treatment also reduces digestion of proteins i n the rumen which also reduces losses of nitrogen i n the 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, 1970; Sharma et a l . , 1972; Sharma and I n g a l l s , 1973; Bhargava and Ranjhan, 1974; - 30 -Sharma and Nicholson, 1975b). Other workers however did not observe reduced rumen ammonia-nitrogen l e v e l s with treatment (Dinius et a l . , 1975; Sharma and Nicholson, 1975a). Sharma et a l . (1972) reported reduced plasma urea-nitrogen l e v e l s with treatment of rapeseed meal but i n the subsequent work (Sharma and I n g a l l s , 1974) there was no e f f e c t on urea-nitrbgen l e v e l s i n the blood by treatment. Barry and Fennessy (1973) reported that before feeding, rumen ammonia l e v e l s were higher i n sheep being fed formaldehyde treated s i l a g e s compared to those being fed untreated s i l a g e s . Sharma and Nicholson (1975b) observed decreased rumen ammonia nitrogen l e v e l s with formaldehyde treatment of faba bean meal one hour a f t e r feeding but not four hours a f t e r feeding. Hemsley et a l . (1970) postulated as one reason f o r p o s i t i v e responses to formaldehyde treatment, that a greater amount of protein was digested i n the i n t e s t i n e . They also postulated that i n t h e i r experiment the plant proteins might have been more d i g e s t i b l e than b a c t e r i a l protein, i n the i n t e s t i n e s . According to these workers, the digestion of the plant proteins i n the i n t e s t i n e s might have increased a v a i l a b l e energy from amino acids to the animal. Barry (1973a) also reported increased digestion of nitrogen i n the small i n t e s t i n e with formaldehyde treatment. Langlands (1973a, 1973b) reported improvement i n the u t i l i z a t i o n of protein of treated wheat. He a t t r i b u t e d t h i s improvement to the greater amount of nitrogen escaping fermentation i n the rumen. - 31 -Faichney and Weston (1971) reported that the increased amount of protein digested i n the i n t e s t i n e as a r e s u l t of formaldehyde treatment stimulated the secretion of i n s u l i n . The i n s u l i n increased the entry rate of amino acids into the c e l l s . I t also has an e f f e c t on the secretion of growth hormone. This hormonal aspect has not been f u l l y investigated. Ferguson (1975) (without giving data) also postulated that treatment with formaldehyde might increase feed intake and thus improve performance. Davies and Faichney (1973) however reported formaldehyde treatment of barley to decrease feed intake and performance of steers. With s i l a g e s p o s i t i v e responses of milk and SNF production have been a t t r i b u t e d to the prevention of breakdown of protein during e n s i l i n g and i n the rumen (Brown and Valentine,'1972; Valentine and Brown, 1973; Valentine and R a d c l i f f e , 1975). In experiments where no responses or negative responses had been obtained, various reasons have been given. Schmidt et a l . (1974) thought that overtreatment of soybean meal with formaldehyde resulted i n the negative response. Sharkey ej: aJ_. (1972) observed no response with treated hay. They a t t r i b u t e d t h i s to losses of formaldehyde during the hay making process and thus the p r o t e i n was not adequately protected. Langlands (1971b) observed no response with treatment of cottonseed meal. He a t t r i b u t e d t h i s to extreme depression i n the d i g e s t i b i l i t y of nitrogen and organic matter as a r e s u l t of both heat applied during extraction of the o i l and the a p p l i c a t i o n of the formalin. S a v i l l e et a l . (1971) a t t r i b u t e d no response to treatment of cottonseed meal to . - 32 -under- or over-protection of the meal and depressed rumen pH as a r e s u l t of feeding of grain which might have broken the methylene bridges. Sharma and Ingalls (1974) however had pH values of about 5.5 for formaldehyde treated casein and 5.7 for formaldehyde treated rapeseed meal and s t i l l had adequate protection of the proteins. Sharma et a l . (1974) indicated that d i e t s containing about 15% CP for steers had protein l e v e l s i n excess of the amounts required and t h i s was the cause of no response to the rapeseed meal treatment. They suggested crude protein l e v e l s of about 11-12%. I t should however be noted that i n the same paper, they had a response to treatment of casein i n d i e t s with s i m i l a r crude protein l e v e l s as the rapeseed meal d i e t s . Rattray and Joyce (1970) observed a response i n nitrogen retention but not wool growth or growth rate with treatment of linseed meal. Their experimental period of f i v e weeks, according to them, may have been too short f or a response of growth rate to be demonstrated. They also reported a negative response with treatment of meat meal using any of the three parameters. They suggested that a l e v e l of 2.5% formaldehyde a p p l i c a t i o n overprotected the protein i n the meat-meal. Digestion and absorption of nitrogen i n the small 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 also present i n the young pre-ruminant. The abomasum secretes hydrochloric acid which k i l l s rumen micro-organisms (Maynard - 33 -and L o o s l i , 1969). Maximal p r o t e o l y s i s by abomasal contents was shown to occur between pH 2 and 3 ( H i l l , 1961; P h i l l i p s o n , 1975) but Church (1975a) claimed i t occurred at pH 2.1. Digestion of proteins occurs mostly i n the i n t e s t i n e s , a f t e r the reticulo-rumen. Materials entering the small i n t e s t i n e of ruminants include endogenous nitrogen secreted into the abomasum and small i n t e s t i n e , m i c r o b i a l nitrogen, non-protein-nitrogen r e s u l t i n g from m i c r o b i a l fermentation i n the reticulo-rumen and nitrogenous components of dietary or endogenous o r i g i n that have escaped ruminal fermentation„(Armstrong and Hutton, 1975). There i s no quantitative information on the endogenous secretion into the small i n t e s t i n e (Armstrong and Hutton, 1975). Approximately 29% of the ammonia-nitrogen i n duodenal flow i s derived from ammonia i n rumen f l u i d . I t i s hypothesized that the other 71% i s derived from urea that passes into the abomasum with g a s t r i c secretions (Nolan, 1975). Other non-protein-nitrogen might be n u c l e i c acids since the digesta from the rumen has up to 20% of m i c r o b i a l nitrogen i n the form of RNA and DNA (Nolan, 1975). Nolan (1975) reported that when 21g of nitrogen was fed to sheep 7.1g, 11.Og and 1.2g of the nitrogen a r r i v i n g at the duodenum was from dietary protein, microbial protein and ammonia r e s p e c t i v e l y . The protein entering the small i n t e s t i n e i s hydrolyzed by proteases i n pancreatic j u i c e and peptidases from i n t e s t i n a l secretions. Gray and Cooper (1971) reported that pancreatic j u i c e contains endopeptidases (attacking c e n t r a l l y located peptide bonds) and exopeptidase (cleaving only terminal bonds of proteins or peptides). The endopeptidases are: - 34 -(a) Trypsin - t h i s attacks proteins at locations of the basic amino acids y i e l d i n g arginine and l y s i n e terminal peptides. (b) Chymotrypsin - t h i s acts i n t e r i o r l y at aromatic amino acid s i t e s to produce C-terminal phenylalanine, tyrosine, and tryptophan peptides. (c) Pancreopeptidase E (elastase) - attacks a l i p h a t i c (non-polar) amino acid-containing portion of protein to produce a l i p h a t i c carbon terminal peptides. The exopeptidases are: Carboxypeptidase A: Attacks peptides r e s u l t i n g from actions of chymotrypsin and pancreopeptidase, y i e l d i n g neutral amino acids and small peptides. Carboxypeptidase B: Attacks peptides r e s u l t i n g from a c t i o n of t r y p s i n to give basic amino acids and small peptides. These enzymes are secreted as zymogens. A c t i v a t i o n of a small amount of trypsinogen i s by enterokinase. Then the t r y p s i n produced activates the re s t of the trypsinogen to t r y p s i n . Trypsin then activates a l l the other pancreatic proteases (Gray and Cooper, 1971). Pancreatic secretion i s under hormonal and nervous c o n t r o l . The nervous part i s through the vagus nerve which can be stimulated by distension of the abomasum. This i s the i n i t i a l flow known as the cephalic phase. The more rapid flow i s under the con t r o l of hormones. Pancreozymin i s a - 35 -hormone which has an e f f e c t on pancreatic flow and i t s e f f e c t i s s i m i l a r to the vagal r e f l e x . The hormone which induces the more rapid flow i s s e c r e t i n . The most powerful stimulus for the release of s e c r e t i n is.acid ingesta present i n the duodenum. Other f a c t o r s which a f f e c t s e c r e t i n release are peptone, soaps and amino.acids ( H i l l , 1975). The acid conditions from the abomasum extend i n t o the upper part of the small i n t e s t i n e due i n part to the copious secretion of acid by the abomasum and p a r t l y to the weakly a l k a l i n e nature of the b i l e and pancreatic secretions of the ruminant. The slow r i s e i n pH i n the proximal part of the small i n t e s t i n e may extend the a c t i v i t y of the abomasal pepsin but delay that of the pancreatic proteases. The a c t i v a t i o n of pancreatic zymogens require a pH above 5 (Armstrong and Hutton, 1975). Ben - Ghedalia et al. (1974) working with sheep reported that the section of the i n t e s t i n e s betwen 1 and 3 metres and 3. and 7 metres from the pylorus were s i t e s of great p r o t e o l y s i s but poor for absorption. They reported that the section 7 to 15 metres d i s t a n t from the pylorus showed the greatest absorption of d i g e s t i v e products of nitrogen. The lower section of the i n t e s t i n e from 15 to 25 metres distant from the pylorus had low net absorption. In a subsequent paper, Ben-Ghedalia et a l . (1976) reported that i f p r o t e i n was administered at the lower section (15 to 25 metres distant from the pylorus) there was a considerable degree of digestion and absorption of proteins. The e a r l i e r work i n d i c a t i n g low net absorption was due to the f a c t that most proteins had been digested and products absorbed i n the upper sections of the small i n t e s t i n e i f proteins were supplied at maintenance l e v e l s . The lower section therefore d i d not have dig e s t i v e and absorptive l i m i t a t i o n s . - 36 -Peptidases are present i n trace amounts i n pancreatic or i n t e s t i n a l secretions (Gray and Cooper, 1971). About 10-20% of i n t e s t i n a l dipeptidase a c t i v i t y i s known to occur i n the m i c r o v i l l a r membrane (brush-border). I n t r a c e l l u l a r peptidases account f o r 80% of the mucosal peptidase a c t i v i t y . There are about 4 to 8 peptidases which are hydrolases. The major end-products absorbed, a f t e r protein d i g e s t i o n are free amino acids and small peptides, with the l a t t e r predominating (Matthews, 1972; Armstrong and Hutton, 1975). Gray and Cooper (1971) suggested that since glycine peptides were more r e a d i l y absorbed than glycine, i t indicated that the small peptides probably entered the c e l l s at rates comparable to the amino acid, presumably at the same entry s i t e s . If oligopeptides are absorbed and the entry rate i s the same as the free amino acid entry rate, more amino acids are present i n the c e l l on further hydrolysis by i n t r a c e l l u l a r peptidases than when the free amino acids are absorbed. Some of the peptides are also digested at the brush-border by the brush-border enzymes before entering the c e l l . Oxidative metabolism i s linked up with sodium movement out of the c e l l i n the transport of amino acids i n t o the c e l l s . A ternary complex of Che amino acid, Na + and a membrane compound, presumably a protein, i s formed thereby permitting entry and release beyond the outer c e l l b a r r i e r , (Gray and Cooper, 1971; Matthews, 1972). Since the oxidative metabolism i s not linked d i r e c t l y with the amino acid entry, the system i s not act i v e transport but rather termed secondary a c t i v e transport (Gray and Cooper, 1971). There are some amino acids, the dependence - 37 -of whose transport on sodium i s not f u l l y known (e.g. the d i c a r b o x y l i c amino a c i d s ) . Williams (1969) reported that there were considerable v a r i a t i o n s i n absorption between and within species. The proportions of amino acids presented to the l i v e r were not ne c e s s a r i l y the same i n d i f f e r e n t subjects of the same species when the proportions of av a i l a b l e amino acids f o r absorption from the small i n t e s t i n e s were the same. He also grouped amino acids into s i x (6) categories according to rate of absorption i n sheep. In order of decreasing rate these are: (a) Isoleucine, arginine, methionine, v a l i n e (b) Leucine, l y s i n e , phenylalanine (c) Aspartic a c i d , serine, tyrosine, alanine (d) Alanine, p r o l i n e , threonine (e) P r o l i n e , threonine, glutamic a c i d , h i s t i d i n e (f) Glycine Some amino acids have an e f f e c t on the transport of other amino acids (Gray and Cooper, 1971; Matthews, 1972). For instance leucine and methionine may i n h i b i t the transport of other amino acids (Matthews, 1972). Leucine augments the transport of l y s i n e and arginine 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 of one kind of amino acid with another may be a l l o s t e r i c - due to attachment of an amino acid to one c a r r i e r s i t e inducing configurational changes i n another adjacent c a r r i e r - or even due to competition for energy supply. They may not n e c e s s a r i l y be sharing the same c a r r i e r (Matthews, 1972). Hexoses (D-glucose and D-galactose) - 38 -reduce absorption of amino acids. This i s perhaps because they share the same c a r r i e r mechanism or compete f o r the l i m i t e d source of energy (Gray and Cooper, 1971). Digestion of protein i n the large intestine. Undigested and unabsorbed residues of nitrogenous compounds leaving the small i n t e s t i n e s enter the caecum and the large i n t e s t i n e . These are mainly feed residues, undigested rumen micro-organisms and endogenous materials. In addition, s u b s t a n t i a l quantities of urea-nitrogen enter the caecum from the blood. The t o t a l input of nitrogen into the caecum and the large i n t e s t i n e may range between 4 and 15g per day f o r sheep. The micro-organisms present i n the caecum and colon carry out p r o t e o l y t i c a c t i v i t i e s . In f a c t , p r o t e o l y t i c a c t i v i t y appears to be greater i n the contents of the large i n t e s t i n e and caecum than i n the contents of the reticulo-rumen. The presence of i s o - b u t y r i c and i s o - v a l e r i c acids i n the caecum i n proportions higher than those occuring i n the rumen indicates there i s extensive breakdown of protein i n t h i s region (Nolan, 1975). Absorption of nitrogen from the hindgut i s probably i n the form of ammonia, about 43% of which i s used f o r the synthesis of non-essential amino acid i n the l i v e r (Nolan, 1975). Since the microbes present are not k i l l e d by acids and l a t e r subjected to proteases as occurs with rumen microbes, they are perhaps of l i t t l e n u t r i t i v e value, i n terms of nitrogen, to the host (Nolan, 1975). - 39 -Carbohydrate metabolism i n the rumen The metabolism of carbohydrates by rumen micro-organisms i s i l l u s t r a t e d by the pathways indicated i n Figures 2 and 3. The end-products of carbohydrate metabolism i n the rumen are generally v o l a t i l e f a t t y acids, methane, hydrogen gas, and carbon dioxide (Hobson, 1971). Ethanol has been produced by rumen microbes i n i n v i t r o cultures but i n i n vivo only traces have been detected. It i s po s s i b l e that i n i n vivo, ethanol i s produced but may be absorbed through the rumen w a l l , since i t has the a b i l i t y to absorb ethanol i f infused i n large q u a n t i t i e s . I t may also be metabolized to a c e t i c ac i d and methane. This has been observed when small quantities of alcohol are introduced into the rumen at a time. I t could also be metabolized with acetate to produce butyrate and higher v o l a t i l e f a t t y acids (Hobson, 1971). In the pathways shown by Leng (1970), methane i s produced from formic acid but Hungate e_t a l . (1970) and Hungate (1967) indicated that 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 indicated that methane i s produced from the reduction of carbon dioxide with hydrogen. M i l l e r and Wblin (1973) working with R. albus reported that even formic acid i t s e l f i s produced from the reduction of carbon dioxide but not from formate-producing pyruvate lyase reac t i o n . They reported that very small amounts of carbon dioxide were produced from formic acid breakdown and t h i s occurs only during the growth of t h i s organism. The pathway i s not yet understood. - 39a -Starch Cellulose I anhydroglucose chains Pectins p e c t i c acid (polygalacturonic acid) Hemicellulose j 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 phosphoenolpyruvate i pyruvate F i g . 2. An outline of the pathways of fermentation of the major carbohydrate constituents of plants to 3C units i n the rumen. (Adapted from Leng, 1970). Pyruvate [2H] C 0 2 + H 2 A c e t y l CoA- • A c e t y l CoA. •CoA ^ 1^2 Malonyl CoA ^^Aceto a c e t y l CoA A c e t y l CoA CoA < [2H; 3-Hydroxybutyry l CoA S ^ H 2 0 Cro tony l CoA [2H] B u t y r y l CoA •Acetate •Ace ty l CoA Bu ty ra t e Oxaloaceta te [4H]. P rop iony l CoA Succ ina te Propionate S u c c i n y l CoA Methyl malonyl CoA J + Lac ta te / ^ A c e t y l CoA ^ • A c e t a t e L a c t y l CoA A c r y l y l CoA H 2 0 [2H] P rop iony l CoA 4 ( ,Ace ta te ^Acetyl CoA Propiona te 3. An o u t l i n e o f the pathways o f degradat ion o f 3C un i t s i n the rumen. (Adapted from Leng, 1970). - 40 -The major end-products of carbohydrate metabolism i n the rumen are perhaps the short chain v o l a t i l e f a t t y acids, a c e t i c , propionic and b u t y r i c . The concentration of t o t a l short chain f a t t y acids has been observed to increase with increasing l e v e l s of feeding, r e f l e c t i n g the amount of feed i n the rumen (Hodgson et d . , 1976). The average proportions of the major v o l a t i l e f a t t y acids when forages are fed are: a c e t i c 65%, propionic 20%, and b u t y r i c 9%, (Leng and B r e t t , 1966). There are a number of factors which a f f e c t the proportions of the three v o l a t i l e f a t t y acids. The changes i n the proportions of the various f a t t y acids are mostly brought about by changes i n rumen pH which i n turn a f f e c t s microbial populations. Some of these factors are discussed below. Roughage: concentrate r a t i o or a concentrate versus a roughage d i e t . The most important aspect of t h i s i s the content of r e a d i l y fermentable'carbohydrate. ,0rskov (1975),. Nicholson and Sutton (1969), Wilke and Merwe (1976) indicated that high l e v e l s of concentrate i n the d i e t can r e s u l t i n increased propionic a c i d proportion and decrease i n a c e t i c a c i d proportion. 0rskov (1975) indicated that high l e v e l s of fermentable carbohydrate i n young forages can also r e s u l t i n high propionic acid and low b u t y r i c a c i d l e v e l s . Whitelaw at a l . (1970) and Nicholson and Sutton (1969) reported that when sheep fed barley are given diets below f u l l feeding the b u t y r i c a c i d portion i s increased with a corresponding decrease i n the propionic acid f r a c t i o n . Hodgson et a l . (1976) and 0rskov (1975) also reported higher proportions of propionic acid at the expense of a c e t i c acid, on high concentrate d i e t s . - 41 -The p a r t i c l e s i z e of roughage can also a f f e c t fermentation patterns. Grinding of the feed decreases the s i z e of p a r t i c l e s and t h i s r e s u l t s i n the exposure of a greater surface area, increases the proportion of propionic acid and a l t e r s rumen pH ((7Jrskov, 1975) . Feed processing such as p e l l e t i n g of cereal grains may increase the proportion of propionic acid compared to the feeding of unprocessed whole grains (Gfrskov, 1975). Feeding frequency also a f f e c t s the proportions of the v o l a t i l e f a t t y acids. High frequency of feeding of high concentrate d i e t s abolishes the great f l u c t u a t i o n i n rumen pH and increases b u t y r i c acid proportions because of the s u r v i v a l of protozoa (0rskov, 1975). The type of carbohydrate also a f f e c t s end-products of fermentation. Glucose or sucrose r e s u l t s i n higher b u t y r i c acid while high proportions of starch r e s u l t s i n propionic acid type of fermentation (0rskov, 1975). Buffers added to the rumen can r e s u l t i n changes i n fermentation pattern. Harrison et a l . (1976) by i n f u s i n g four l i t r e s of 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 propionate fermentation to acetate fermentation. Alhassan et a l . (1969) and K r a b i l et a l . (1969) by feeding sodium sulphite increased the proportion of propionic acid. Hobson (1972) reported that the addition of one acid may suppress the production of that p a r t i c u l a r a c i d - t h i s i s re f e r r e d to as fermentation product suppression. The 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 acids. I s o v a l e r i c and i s o b u t y r i c acids normally r e s u l t from deamination of leucine and v a l i n e r e s p e c t i v e l y (el-Shazly, 1952a and 1952b). L a c t i c a c i d may accumulate i n the rumen under abnormal conditions 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 of formaldehyde treatment on carbohydrate metabolism and v o l a t i l e f a t t y acid production i n the rumen  The e f f e c t s of formaldehyde treatment of feedstuffs on v o l a t i l e f a t t y acid production have been v a r i a b l e . Sharma and Ingalls (1973), Beever et a l . (1976), and Beever et a l . (1977), reported no differences i n v o l a t i l e f a t t y acid production or proportions of the i n d i v i d u a l f a t t y acids. Sharma et a l . (1972) reported decreased v o l a t i l e f a t t y acid production with treatment of rapeseed meal. Langlands (1973b) i n one experiment observed no differences i n t o t a l v o l a t i l e f a t t y acid production and proportions of the major f a t t y acids. He reported t o t a l v o l a t i l e f a t t y a c i d production to be reduced s i g n i f i c a n t l y i n another experiment reported i n the same paper (Langlands, 1973b). Barry (1973a) reported v o l a t i l e f a t t y a c i d concentration to decrease with formaldehyde treatment of hay. The l e v e l s of i s o - and n - v a l e r i c acid were reduced by formaldehyde treatment of s i l a g e s (Barry and Fennesy, 1973). In t h i s experiment, they reported v o l a t i l e f a t t y acid l e v e l s to be higher i n sheep fed treated s i l a g e about one hour before feeding. Barry (1972) reported s i m i l a r prefeeding conditions with treatment of casein. These two workers thought that the high l e v e l s of v o l a t i l e f a t t y acid and ammonia i n the rumen before feeding i n sheep fed treated s i l a g e indicated greater s t a b i l i t y of conditions i n the rumen for fermentation. Barry (1976c) reported v a r i a b l e r e s u l t s with v o l a t i l e f a t t y a c i d production i n three ser i e s of experiments. In experiment 2, t o t a l v o l a t i l e f a t t y acid, i s o - and n - v a l e r i c acids were reduced but - 43 -not i s o b u t y r i c . In experiment 3, formalin treatment did not a f f e c t t o t a l v o l a t i l e f a t t y a c i d production. Dinius e_t a l . (1975) reported reduced 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 acid-detergent f i b r e , neutral detergent f i b r e and hemicellulose with treatment of drie d a l f a l f a . There was no d i f f e r e n c e i n rumen pH, f i v e hours a f t e r feeding. Beever et a l . (1976), however observed enhanced d i g e s t i b i l i t y of c e l l u l o s e with treatment of dried grass. There was a depression i n d i g e s t i b i l i t y of c e l l u l o s e i n the rumen but subsequent increased digestion of c e l l u l o s e i n the caecum resulted 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 formalin treatment of dried grass. - 43a -OBJECTIVES These studies reported i n t h i s thesis were undertaken with the following objectives: 1) To determine by i n v i t r o studies the optimum l e v e l of formaldehyde treatment of grass-legume forage protein to obtain protection from rumen microbial degradation without subsequent decrease i n enzymatic digestion. 2) To assess the e f f e c t s of formaldehyde treatment of the forage protein i n terms of nitrogen and f i b r e u t i l i z a t i o n i n vivo. 3) To determine i f dietary supplementation with sulphur and/or branched chain f a t t y acids i s necessary when a portion of the dietary protein i s protected from microbial degradation. - 44 -MATERIALS AND METHODS Introduction The experimental work was conducted i n four phases. The f i r s t phase employed i n v i t r o procedures to determine the optimum l e v e l of formaldehyde treatment of dehydrated and hammermilled grass-clover forage. This was followed by i n vivo 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, nitrogen, acid-detergent f i b r e and c e l l u l o s e were determined as we l l as feed intake, nitrogen and sulphur balances. At the end of the above determinations the animals were slaughtered f o r the purpose of measuring rumen pH, rumen dry matter content, and the rumen l e v e l s of ammonia-nitrogen and v o l a t i l e f a t t y acids. Abomasal pH and abomasal digesta contents of acid-detergent f i b r e , t o t a l nitrogen and RNA-N were also measured. The fourth phase of the study involved the use of a sheep f i t t e d with a duodenal r e -entrant cannula for measuring digesta flow. EXPERIMENT I Treatment of the forage with formaldehyde The dehydrated and hammermilled rye-grass-ladino clover forage Was treated with 10% formaldehyde stock s o l u t i o n (formalin) i n a small upright mixer s i m i l a r to the method used by Schmidt et a l . (1974). The formalin was sprayed on the forage, using an aerosol spray gun, as i t was being turned around i n the mixer. Ten-kilogram (10 kg) batches of the - 45 -forage were treated at a time. The treatment resulted i n formaldehyde sprayed on the/forage, on an a i r dry basis, of 0.0%, 0.8%, 1.0% and 1.2% with the same quantities of f l u i d added (12 ml/100 g). The forage was sealed i n polythene bags for one week a f t e r the treatment as described by S a v i l l e et a l . (1971). In v i t r o incubation l g samples (DM basis) of the treated grass-clover were incubated i n rumen f l u i d plus a r t i f i c i a l s a l i v a , according to the method of Troelsen (1969). A f t e r 48 hours of incubation, the mixture was centrifuged at 1500 x g and f i l t e r e d through ashless f i l t e r paper. The supernatant was used for determining ammonia-nitrogen using the autoanalyzer (Technicon model EDP 910 120-45-5). The residue was then washed with 50 ml of deionized" water and f i l t e r e d again. Rumen f l u i d plus a r t i f i c i a l s a l i v a served as blank. The nitrogen content of the residue was determined by the macro-Kjeldahl method (AOAC, 1970). Nitrogen d i g e s t i b i l i t y was calculated by the method of Barry (1972). There were three r e p l i c a t i o n s for each treatment and blank. Three r e p l i c a t e s of each treatment and blank were also taken through the two-stage digestion procedure. A f t e r the rumen inoculum . plus buffer digestion, each r e p l i c a t e was centrifuged and the supernatant was used for ammonia-nitrogen determination as described above. The residue was then digested with acid-pepsin f o r 48 hours. The acid-pepsin digested residue was then centrifuged, f i l t e r e d and washed as described f o r the f i r s t stage incubation. The nitrogen content of the residue was determined - 46 -by the macro-Kjeldahl method (AOAC, 1970). The d i g e s t i b i l i t y of nitrogen was then cal c u l a t e d . Treatment of grass-clover forage f o r i n vivo t r i a l s The grass-clover forage was treated with 1% l e v e l of formaldehyde i n the same manner as described f o r the i n v i t r o t r i a l s above. The treated forage was then used i n r a t i o n s for animal feeding t r i a l s . EXPERIMENT II Nitrogen, carbohydrate and sulphur metabolism and feed intake studies The d i e t s (Table 1) contained grass-clover forage, cassava, barley and a mineral premix. Sodium sulphate was added to d i e t s 4 and 5. The d i e t s were p e l l e t e d (9mm) a f t e r mixing i n a h o r i z o n t a l mixer. I s o v a l e r i c and i s o b u t y r i c acids were sprayed on d i e t s 3 and 5 at the rate of 3.0g/kg die t for i s o v a l e r i c and 2.3g/kg d i e t f or i s o b u t y r i c immediately before feeding. A f t e r measuring the required volumes of the acids f o r Ikg d i e t i n t o a measuring c y l i n d e r , i t was d i l u t e d to 10ml and sprayed using an aerosol spray gun. The d i e t s contained approximately 14% crude protein as reported by Bryant (1973) to meet the requirement of confined lambs weighing 29kg up to slaughter. Table 1. Composition of Rations. Ingredient (% DM) Diet 1 Diet 2 Diet 3 Diet 4 Diet 5 Cassava 38 38 38 37.33 37.33 Barley 11 11 11 11 11 Untreated grass 50 — — Treated grass — 50 50 50 50 Sodium sulphate — — — 0.67 0.67 Mineral premix* 1 1 1 1 1 The composition of the sheep mineral premix, given by the manufacturers was as follows 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 of Fluorine was 0.2% while the minimum l e v e l s of Vitamins A and D^ were 551,150 Iu/kg and 110,230 Iu/kg respectively. - 48 -Twenty-five young ram lambs of the Dorset breed were used f o r the metabolism t r i a l s , at South campus Univ e r s i t y of B r i t i s h Columbia sheep un i t . Five animals were randomly assigned to each d i e t . There was a pre-experimental period of twenty days and a c o l l e c t i o n period of 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 fed i n d i v i d u a l l y ( f i v e animals at a time one on each d i e t ) during seventeen days of the pre-experimental period of twenty days. A f t e r seventeen days i n the i n d i v i d u a l pens, they were transferred to the cages, allowed a three-day adjustment period, before the seven-day c o l l e c t i o n period. The pre-experimental period of seventeen days was also used f o r feed intake assays. During the feed intake assay, there was a ten-day pre-conditioning period and a seven-day measurement of feed intake, as recommended by Heaney et: a l . (1968). During the ten-day pre-conditioning period, the animals were brought to maximum feed intake. They were fed ad l i b i t u m and residues weighed back. I f they l e f t l e s s than 10% of feed offered, the feed allowance was increased by 25%. The minimum l e v e l of feeding during the seven-day feed intake assay period was the same as the maximum l e v e l of feed intake during the 10-day pre-conditioning period. The amount of feed l e f t o v e r was weighed d a i l y . I f the animals l e f t l e s s than 10% of the feed offered, the feed allowance was increased by 25%. During the metabolism t r i a l s , the animals were fed ad l i b i t u m based on the l e v e l determined during the feed intake assay period. The animals were fed twice d a i l y at 8:30 a.m. and 3:00 p.m., throughout the twenty-seven-day period. - 49 -T o t a l amount of faeces voided were c o l l e c t e d and weighed d a i l y during the seven-day metabolism t r i a l . Samples of faeces were dried i n a forced d r a f t oven at 80°C f o r 24 hr s i m i l a r to the procedure of Hume et a l . (1970). Urine was c o l l e c t e d over 100 ml of 6N HC1 i n p l a s t i c - c o n t a i n e r s with narrow necks into which were f i t t e d the rubber tubes draining the urine from the urine compartment of the metabolism cage. This l i m i t e d exposure of the urine to the atmosphere. Samples of urine were taken d a i l y a f t e r measuring the volume. The samples were kept i n the freezer compartment of a r e f r i g e r a t o r . The addition of 100 ml of 6N HC1 reduced pH of urine to between 2 and 3. This pH l e v e l (between 2 and 3) was recommended by Martin (1966) to reduce to 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 urine. At the end of the t r i a l s , the urine samples were thawed and f i l t e r e d through layers of cheese c l o t h as was c a r r i e d out by Bryant (1973). The f i l t r a t e was used f o r nitrogen and sulphur analyses. The animals were weighed on the 1st, 10th, 17th and 27th days of the t r i a l . EXPERIMENT I I I Rumen and abomasal digesta metabolites studies The animals used i n the previous experiment were slaughtered for these studies. The animals were fed at about 2:00 a.m. on the day of slaughter. They were then transported to the slaughter house and slaughtered 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 t r a c t - 50 -was removed i n t a c t immediately a f t e r slaughter placed . i n buckets containing i c e and transported' d i r e c t l y to the laboratory f o r the various measurements. The pH of the rumen f l u i d was determined immediately a f t e r s t r a i n i n g rumen digesta. The ammonia-nitrogen l e v e l s were determined using the autoanalyzer used i n experiment I. The rumen f l u i d f o r rumen ammonia-nitrogen determination was a c i d i f i e d as described by Chalmers et: a l . (1954). Samples of rumen f l u i d were c o l l e c t e d as described by Alhassan et^ a l . (1969) and the measurements of 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 acids were c a r r i e d out as described by Ross and K i t t s (1971). The t o t a l rumen contents were weighed. Samples of the rumen content were freeze-dried f o r storage. Samples of the freeze-dried material were oven dried (105°C for 24 hours) for dry matter determination. This was s i m i l a r to the method of MacRae and Ulyatt (1974). Abomasal f l u i d was also strained f o r pH measurement. The contents of the abomasum were freeze dried. Samples of the freeze-dried abomasal contents were used for ash, acid-detergent f i b r e , c e l l u l o s e , t o t a l nitrogen and RNA-N analyses as described under chemical a n a l y s i s . Samples of the freeze-dried materials were further dried at 105°C for 24 hours to determine dry matter content of the abomasum (MacRae and .Ulyatt, 1974). - 51 -EXPERIMENT IV Duodenal digesta flow studies A ram f i t t e d with a duodenal re-entrant cannula was used f o r t h i s experiment. The re-entrant cannula was f i t t e d i n a s u r g i c a l operation as described by Brown et al.(1968). This animal was used for studies of duodenal flow 6 weeks a f t e r the operation. The animal was fed each of the d i e t s i n turn f o r ten days. On the tenth day, a l l the materials flowing from the abomasum into the duodenum were c o l l e c t e d over a 24-hour period. The animal was fed l,500g ( a i r dry basis) of each d i e t i n two equal portions twice d a i l y at 8:30 a.m. and 3:00 p.m. C o l l e c t i o n and sampling of digesta The piece of tube j o i n i n g the two ends of the cannula was removed during the day of c o l l e c t i o n . Separate lengths of rubber tubings were attached to each end. The tubing attached to the e x i t cannula was arranged so that the digesta were delivered into a glass c o n i c a l f l a s k (2,000 ml capacity) placed i n a bucket. The top of the f l a s k was sealed with laboratory parafilm. The f l a s k was maintained at ambient temperature. The tubing attached to the return cannula was fastened to the crate, about 40-50 cm above the cannula, allowing return of digesta to the duodenum. Aft e r every one and a h a l f hours (1% h r ) , the f l a s k was replaced. The volume of the c o l l e c t e d digesta was measured and the weight recorded. A ten percent (10%) sample was taken a f t e r vigorous shaking. The sample taken was immediately placed i n a capped p l a s t i c container and kept i n the freezer section 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 digesta was returned - 52 -manually into the i n t e s t i n e through the return cannula,, after adding donor sample c o l l e c t e d previously to make up f o r the "quantity taken for analyses. The digesta was warmed i n a water bath (39.°C). and returned in;small quantities over a period of one and a h a l f hours. This method of sample c o l l e c t i o n was s i m i l a r to that of Thompson and Lamming (1972). The amount of feed l e f t a f t e r every one and a h a l f hours was weighed. The frozen duodenal samples were thawed i n a r e f r i g e r a t o r before further analysis. Weighed samples were freeze-dried and analyzed for nitrogen, acid-detergent f i b r e , RNA-N, ash and TCA-N. A sample of the freeze-dried material was further dried i n the oven for 24 hours, for dry matter determination, as described by MacRae and Ulyatt (1974). Chemical analyses The chemical analyses of a l l samples, unless otherwise described, were c a r r i e d out as outlined below. (a) Nitrogen: Nitrogen contents of feedstuf f s , r a t i o n s , faeces, digesta and urine were determined by macro-Kjeldahl method of the AOAC (1970). (b) Ash: Ash contents of the ra t i o n s , faeces and digesta were determined by the AOAC (1970) method. (c) Moisture: Moisture content of the rations and faeces were determined using the method of AOAC (1970). Moisture content of rumen, abomasal and duodenal digesta samples was determined i n two stages: freeze-drying followed by oven-drying as described by MacRae and Ulyatt (1974). - 53 -(d) Acid-detergent f i b r e and c e l l u l o s e : Acid-detergent f i b r e and c e l l u l o s e contents of r a t i o n s , faeces and digesta were determined according to the method of Van Soest and Wine (1968) as modified by Waldern (1971). (e) Acid-detergent f i b r e i n s o l u b l e nitrogen: Acid-detergent f i b r e i n s o l u b l e nitrogen content of the grass-clover forage was determined using the method of Yu and V e i r a (1977). (f) Sulphur: Ashing of urine, faeces, r a t i o n and feedstuff samples f o r sulphur determinations, was done according to the method of Bird and Fountain (1970). For the feedstuff, r a t i o n , and f a e c a l samples, 2g dry matter were weighed in t o an ashing c r u c i b l e . This was mixed thoroughly with 2.5 g of mixture of sodium bicarbonate-silver oxide (25:1) as an o x i d i z i n g agent. The mixture was ashed at 550°C f o r f i v e (5) hours i n a muffle furnace. With theurine samplesJ_ml-samples were pipetted into the c r u c i b l e s , then dried at 55°C i n an oven. The dried urine sample was then ashed. The ash i n each case was dissolved i n 20 ml 6N HC1, a f t e r cooling, following the method of Johnson et a l . (1970). The s o l u t i o n was then d i l u t e d to 1000 ml with d i s t i l l e d water. A 10 ml al i q u o t was combined with 10 ml of g e l a t i n barium chloride s o l u t i o n containing 0.3g, BaC^. The g e l a t i n barium chloride s o l u t i o n was prepared following the method of Tabatabi (1974). The suspension of the ash s o l u t i o n and g e l a t i n -barium-chloride 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. The t u r b i d i t y of the sulphate p r e c i p i t a t e d was determined using spectrophotometer (Spectronic 20 at wavelength of 500 mu) following the method of Johnson at a l . (1970). - 54 -The extractable sulphate-sulphur i n the grass-clover forage was determined using the procedure outlined i n the b u l l e t i n of the 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 Food, London, Technical B u l l e t i n 27 (1973). The weighed sample was shaken with 0.12N hydrochloric acid and activated charcoal. A f t e r shaking for 30 min, i t was f i l t e r e d through Whatman No.2 f i l t e r paper and the f i l t r a t e used for sulphate-sulphur a n a l y s i s . Standards of sulphate-sulphur were prepared according to the procedure outlined 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. The only modification was that the sulphate was dissolved i n 0.12N HC1. (g) RNA: Ribonucleic a c i d contents of the abomasal and duodenal digesta were determined using the method of Guinn (1966). The homogenized samples were, treated with ethanol-NaCl (4:1 r a t i o of 95% ethanol and 10% NaCl), 95% ethanol, and ethanol-10% NaCl to remove chlo r o p h y l l and other pigments. The n u c l e i c acids were then extracted from the moist samples with 10% NaCl at 100°C. The tubes containing the samples were stoppered. This method was used by Ling and Buttery (1975), for extracting n u c l e i c acids from rumen b a c t e r i a and duodenal digesta. RNA extracted from the samples was determined with spectro-photometer (Unicam SP800B), U.V., wavelength 260 mu. Standard solutions of RNA were prepared and a standard curve obtained as described by Akinwande (1973). The nitrogen content of the RNA extracted from the duodenal digesta was assumed to be of the same magnitude as the t o r u l a yeast RNA used for preparing the standards. This assumption was also - 55 -made by Smith and McAllan (1970). The microbial protein content of the digesta was calculated by assuming RNA-N: t o t a l N i n rumen microbes to be 0.075 as was reported by Smith (1975). Sutton et a l . (1975) made a s i m i l a r assumption. (h) NPN: The non-protein-nitrogen f r a c t i o n s of the grass-legume forage, abomasal and duodenal samples were determined using the method of Goshtasbpour-Parsi et a l . (1977). One gram (1 g) sample of the freeze-dried duodenal or abomasal digesta or grass-legume forage was mixed with 10 ml deionized water. Ten m i l l i l i t r e s (10 ml) of 20% TCA so l u t i o n was then added. I t was shaken f o r ten minutes mechanically, heated to 90°C i n a water bath, and further shaken i n the hot water bath for 10 minutes. The tube was then centrifuged at 15,000 xg. The supernatant plus second washing of the residue, was analyzed f o r nitrogen by the macro-Kjeldahl method (AOAC, 1970). This was the non-protein-nitrogen f r a c t i o n . Experimental designs and s t a t i s t i c a l analysis Results from the t r i a l s were subjected to s t a t i s t i c a l a n a l y sis. In the i n v i t r o d i g e s t i b i l i t y determinations, a completely randomized design (CRD) was used. The F-test was applied and s i g n i f i c a n t differences between means were determined using Tukey's W values (Steel and T o r r i e , 1960). - 56 -Randomized block designs were used i n the other t r i a l s . The block i n t h i s case was the group or batch. The F-test was applied and s i g n i f i c a n t differences between means were determined using Tukey's W values as above (Steel and T o r r i e , 1960). No s t a t i s t i c a l analysis was applied to data c o l l e c t e d from the studies using the cannulated animal. - 57 -RESULTS In V i t r o digestion t r i a l s The r e s u l t s of the nitrogen d i g e s t i b i l i t y t r i a l s at the f i r s t stage i n v i t r o digestion of the rye-grass-clover with d i f f e r e n t l e v e l s of formaldehyde treatment are shown i n Table 2. The average values fo r nitrogen d i g e s t i b i l i t y f o r the treatments were 31.88%, 15.72%, 6.87%, and 5.69% for 0%, 0.8%, 1.0%, and 1.2% l e v e l s of formaldehyde treatment re 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) differences between the treatments at 0% level,formaldehyde and the rest of the treatments(0.8%, 1.0%, and 1.2% l e v e l s of formaldehyde). Treatment at 0.8% l e v e l of formaldehyde was s i g n i f i c a n t l y (p<0.05) d i f f e r e n t from 1.0% and 1.2% l e v e l s of formaldehyde. Differences between 1% and 1.2% l e v e l s of formaldehyde treatments were not s i g n i f i c a n t ( p ^ 0.05). The r e s u l t s of nitrogen d i g e s t i b i l i t y at the end of the second stage of i n v i t r o digestion (combined microbial and acid-pepsin stages) of the forage at the four l e v e l s of formaldehyde treatment are shown i n Table 2. The average values f or nitrogen d i g e s t i b i l i t y f o r the treatments are 80.95%, 79.76%, 75.85% and 71.01% for the 0.0%, 0.8%, 1.0% and 1.2% l e v e l s of formaldehyde re s p e c t i v e l y . Treatment at 1.2% s i g n i f i c a n t l y (p Z.0.05) reduced nitrogen d i g e s t i b i l i t y compared with treatments at 0.0% or 0.8%. A l l other differences were not s i g n i f i c a n t (p -^ 0.05). The mean differences between the second stage (combined microbial and acid-pepsin) and f i r s t stages of i n v i t r o nitrogen d i g e s t i b i l i t y are 49.07, 64.04, 68.98 and 65.32 percentage units f o r 0.0%, 0.8%, 1.0%, and 1.2% l e v e l s of formaldehyde treatment r e s p e c t i v e l y . Table 2. In V i t r o Digestion T r i a l s . Parameter 0.0% Formaldehyde Treatment 0.8% 1.0% 1.2% No. of Replicates S.E. 1st Stage N dig e s t i o n % 31.88a 2nd Stage N dig e s t i o n % 80.95a NH^-N production (ppm) 228.79a Calculated N dig e s t i o n from NH3~N% 30.26 15.72b 79.76a 78.58b 10.39 6.87c 75.85ab 65.27b 8.63 5.69c 71.01b 30.04c 3.97 + 1.22 + 1.41 + 7.60 Ul oo Means on the same l i n e bearing 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). - 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 production during the microbial stage of incubation (Table 2). The mean values for i n v i t r o ammonia-nitrogen production at 0.0%, 0.8%, 1.0% and 1.2% l e v e l s of formaldehyde treatment are 228.79 ppm, 78.58 ppm, 65.27 ppm and 30.04 ppm r e s p e c t i v e l y . Ammonia-nitrogen productions at 0.8% and 1.0% were not s i g n i f i c a n t l y (p "y 0.05) d i f f e r e n t . The differences between any other 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 calculated using the ammonia-nitrogen production figures are shown i n Table 2. Chemical composition of d i e t s and ingredients The chemical composition of the feed ingredients and the d i e t s fed to the animals f o r the feed intake and metabolism t r i a l s are shown i n Table 3. D a i l y feed intake, d a i l y urine output, metabolic body sizes of animals (kg) at the beginning of the metabolism study period, and the d a i l y gain i n weight during the pre-metabolism assay period  There were no s i g n i f i c a n t (p^-0.05) differences between di e t s with respect to d a i l y feed intake (g D.M.) per u n i t of metabolic body s i z e both during the pre-metabolism and the metabolism assay periods (Table 4). D a i l y nitrogen intake (g/Wkg^*^), urine output per u n i t of metabolic body s i z e per day and the growth rates of the animals over seventeen Table 3. Chemical Composition of Diets and Ingredients (D.M. b a s i s ) . Chemical Fr a c t i o n % (D.M. Basis) Diet/Ingredient N S ADF C e l l . Ash Ext.S ADF-IN NPN 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 r 8.11 Diet 5 2. 29 0.27 17.33 13.38 8.48 Grass-legume forage 3. 78 0.25 0.07 0.20 0.66 Cassava 0. 42 0.05 Barley 1. 77 0.12 - 61 -day period were also not 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 (Table 4). The metabolic body sizes of the animals at the beginning of the metabolism assay period were not s i g n i f i c a n t l y (p^O.05) d i f f e r e n t (Table 4). However, the animals used i n the f i r s t group were s i g n i f i c a n t l y (p <0.05) heavier than animals used i n the t h i r d group. The mean metabolic body sizes of animals used i n the f i v e groups were 14.37kg, 14.11kg, 12.74kg, 13.13kg, and 14.19kg f or group one, two, three, four and f i v e r e s p e c t i v e l y . 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 dry matter, organic matter, nitrogen, acid-detergent 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 of dry matter, (D.M.), organic matter (O.M.), nitrogen (N), acid-detergent f i b r e (A.D.F.) and c e l l u l o s e are presented i n Table 5. 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 dry matter, and organic matter were not 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 between dietary treatments. 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 of nitrogen was s i g n i f i c a n t l y (p < 0.05) higher for d i e t one than f o r d i e t s two, four and f i v e . The differ e n c e between die t s one and three was not s i g n i f i c a n t (p > 0.05). The di f f e r e n c e between d i e t s three and four was s i g n i f i c a n t (p < 0.05), with d i e t three having the 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 of acid-detergent f i b r e and c e l l u l o s e were higher f o r a l l the d i e t s containing the treated forage than f o r die t one (p ^0.05). Table 4. Dail y Feed Intake, D a i l y Urine Output, Metabolic Body Sizes of Animals at the Beginning of the Metabolism Study Period and the Dai l y Gain i n Weight During the Pre-metabolism Assay Period. Diet containing Diets containing formaldehyde treated forage Daily Intake untreated forage + VFAS + SO" + + VFAS + SO (g/Wkg 0 , 7 5) 1 2 3 4 5 ; S.E. D.M. during pre-metabolism 91.33 * 100.62 101.93 98.02 96.47 + 4.70 assay period D.M. during the metabolism 90.17 96.65 104.29 90.88 94.25 + 4.52 study period N during the metabolism 2.07 2.18 2.38 2.08 2.18 +0.10 study period Growth rate during 154.76 170.77 170.77 160.10 149.42 +21.31 1st 17 days (g/day) Metabolic body s i z e at the 14.42 13.17 14.02 13:43" 13.50 + 0.36 beginning of metabolism studies (kg) Urine output (mis/day/Wkg°' 7 5) 80.32 77.02 61.44 79.81 78.86 +12.29 * Each value represents the mean of f i v e determinations, t Sulphur was added as Na^SO^ Table 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 Fractions (%). Diet containing Diets containing formaldehyde treated forage untreated forage + VFAS + -SC^t • + VFAS + SO Apparent Dig.Coeff. 1 2 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 Cell u l o s e % 42.95a 49.10b 49.04b 49.33b 48.76b + 1.33 * Each value represents the mean of f i v e determinations, t Sulphur was added as Na^SO^ Means on the same l i n e bearing d i f f e r e n t l e t t e r s (a, b, c, d) are s i g n i f i c a n t l y d i f f e r e n t (p <0.05). - 64 -Nitrogen metabolism S t a t i s t i c a l analysis of the nitrogen metabolism study indicated a s i g n i f i c a n t (p £ 0.05) period e f f e c t f o r nitrogen excretion per unit of metabolic s i z e . The animals used i n the second group excreted a s i g n i f i c a n t l y (p<.0.05) greater amount of nitrogen (g/day/Wkg^" 7~^) i n the urine than animals used i n groups four and f i v e . The values were 0.595, 0.618, 0.543, 0.461 and 0.448 (g/day/Wkg°' 7 5) for groups one, two, three, four and f i v e r e s p e c t i v e l y . The parameters associated with nitrogen metabolism-are presented i n Table 6. The d a i l y nitrogen excreted i n urine per u n i t of metabolic body s i z e was s i g n i f i c a n t l y (p<0.05) higher f o r di e t one than for the rest of the d i e t s . Urinary excretion of nitrogen as a percentage of intake was s i g n i f i c a n t l y (p 0.05) greater f o r di e t one than f o r di e t s two, three and four but there was no s i g n i f i c a n t (-p_> 0.05) differ e n c e between d i e t s one and f i v e . The percentage of digested nitrogen excreted i n urine was s i g n i f i c a n t l y (p < 0.05) higher f o r di e t one than f o r the rest of the d i e t s . Nitrogen retained as a percentage of intake was s i g n i f i c a n t l y (p 0.05) increased by formaldehyde treatment of the forage. On d i e t three the animals also retained a s i g n i f i c a n t l y . ( p ^. 0.05) greater amount of nitrogen as a percentage of intake than the animals on diet f i v e . Nitrogen retained as a percentage of digested was s i g n i f i c a n t l y (p < 0.05) improved by formaldehyde treatment of the forage portion of the d i e t s . D a i l y nitrogen balance (g/day/Wkg^*7~*) was s i g n i f i c a n t l y (p<.0.05) improved f or animals on di e t s two and three compared to animals on di e t one. Nitrogen balance (g/day) was s i g n i f i c a n t l y (p <. 0.05) d i f f e r e n t between animals on d i e t one and di e t three.. Nitrogen intake (g/day) was not - s i g n i f i c a n t l y ;(p ^  0.05) affected by treatments. Table 6. Nitrogen Metabolism. Diet containing Diets containing formaldehyde treated forage untreated forage + VFAS + SO f + VFAS + SO 1 2 3 4 5 S.E. Nitrogen intake (g/day) 29.81* 28.64 33.44 27.80 29.51 + 1.65 Dail y N intake (g/Wkg^*7^) 2.07 2.18 2.38 2.08 2.18 + 0.10 App.dig.coeff. of N % 54.13a 47.06bc 51.25ac 44.90bd 47.24bc + 1.17 Dail y N excreted i n urine (g/Wkg 0 , 0) 0.805a 0.461b 0.467b 0.417b 0.518b .0.03 N excreted i n urine, .% of intake 39.07a 21.07b 19.97b 19.86b 23.94ab + 3.69 N excreted i n urine, % of digested 72.10a 44.65b 39.11b 44.25b 50.39b + 3.42 N retained» % of intake 15.06a 26.00bc 30.24bc 25.04bc 23.30bd + 1.48 N retained, % of digested 27.90a 55.39b 58.93b 55.75b 49.61b + 3.41 Nitrogen balance (g/day) 4.49a 7.43ab 10.19b 6.99ab 6.86ab + 0.86 Nitrogen balance (g/day/Wkg^*^) 0.313a 0.567b 0.726b 0.525ab 0.508ab + 0.05 * Each value represents the mean of f i v e determinations, t Sulphur was added as Na^SO, Means on the same l i n e bearing 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 associated with sulphur metabolism are presented i n Table 7. Animals on die t s four and f i v e consumed s i g n i f i c a n t l y (p K. 0.05) greater amounts of sulphur per day than animals on d i e t s one and two but not more than animals on di e t three. Animals fed the sulphur supplemented di e t s consumed s i g n i f i c a n t l y (p 4.0.05) a greater amount of sulphur per day per unit of metabolic body s i z e than animals on the other d i e t s . Urinary excretion of sulphur as a percentage of intake was s i g n i f i c a n t l y (p < 0.05) greater f o r animals on die t s four and f i v e than for animals on die t s two and three. Faecal and urinary sulphur losses expressed as a percentage of intake was s i g n i f i c a n t l y (p <C 0.05) greater for animals on d i e t s one and f i v e than animals on d i e t s two and three. Animals on di e t four l o s t s i g n i f i c a n t l y (p < 0.05) a greater percentage of consumed sulphur i n urine and faeces than animals on di e t two but not animals on di e t three. The animals fed the sulphur supplemented d i e t s excreted a s i g n i f i c a n t l y (p^.0.05) greater amount of sulphur i n the urine per day than the animals fed the other di e t s (g/Wkg^*^^). Animals on d i e t s two, three and four retained s i g n i f i c a n t l y (p <. 0.05) a greater amount of sulphur (g) per day than animals on d i e t one. Sulphur balance per day per un i t of metabolic body s i z e was s i g n i f i c a n t l y (p 4^0.05) greater f o r animals on die t s two, three and four than f o r animals on di e t one. Table 7. Sulphur Metabolism. Diet containing untreated forage Diets containing formaldehyde treated forage + VFAS + S0.+ + VFAS + SO. 4 ' 4 S.E. Da i l y sulphur intake (g) Da i l y sulphur intake ' (g/wkg°-7!)) Sulphur excreted i n u r i n e , % of intake Sulphur excreted i n urine d a i l y (g/Wkg 0- 7 5) Sulphur balance (g/day) Sulphur balance per day (g/wkg 0- 7 5) Ratio of retained sulphur to retained (nitrogen (1:X) Sulphur l o s t i n urine and faeces>% of intake 2.26a* 0.157a 31.38a 0.050a 0.459a 0.032a 10.13 79.67a 2.20a 2.64ab 0.168a 0.188a 14.17b 17.41b 0.024a 0.034a 0.802b 0.843b 0.061b 0.061b 3.30b 3.39b + 0.18 9.78 12.26 63.69b 67.57bc 0.247b 0.251b + 0.01 41.43a 42.23a + 2.76 0.102b 0.105b + 0.007 0.752b 0.672ab + 0.064 0.056b 0.050ab + 0.005 9.73 10.36 + 1.26 76.89ac 80.28ad +2.37 * Each value represents the mean of f i v e determinations, t Sulphur was added as Na2S0, Means on the same l i n e bearing 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 of sulphur retained to nitrogen retained was not 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 f o r any of the d i e t s . Rumen parameters (Data c o l l e c t e d from slaughtered animals) The rumen pH, rumen ammonia-nitrogen concentration (ppm), dry matter content of the rumen at the time of slaughter of the animals (g), t o t a l v o l a t i l e f a t t y acid concentration (u-mole/ml) and molar proporations of a c e t i c , propionic, 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 acids are presented i n Table 8. There were no s i g n i f i c a n t (p> 0.05) differences between any of the treatments with respect to rumen pH, rumen dry matter content at the time of slaughter of the animals, and t o t a l v o l a t i l e f a t t y acid concentrations. There were no s i g n i f i c a n t (p > 0.05) differences between treatments with respect to a c e t i c , propionic, and b u t y r i c acid proportions. There were however s i g n i f i c a n t (p < 0.05) differences between the animals used i n the second and fourth groups with respect to a c e t i c acid and propionic acid concentration. The average values of a c e t i c a c i d proportions f o r the f i v e groups (blocks) of animals were 40.73%, 46.69%, 41.98%, 37.61%, and 42.81% for f i r s t , second, t h i r d , fourth and f i f t h groups re s p e c t i v e l y . The average values of propionic acid proportions f o r the f i v e groups (blocks) of animals were 31.24%, 22.66%, 26.49%, 35.26% and 29.35% f o r the f i r s t , second, t h i r d , fourth and f i f t h groups r e s p e c t i v e l y . The proportions of i s o v a l e r i c acid and i s o b u t y r i c acid were s i g n i f i c a n t l y (p ^ 0.05) lower f o r di e t s two and four compared with d i e t three. The other treatments were not 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 . Table 8. Rumen parameters. Diet containing Diets containing formaldehyde treated forage untreated forage + VFAS .. + SC^t + VFAS + S0 4 1 2 3 4 5 S.E. Rumen pH 5.50* 5.11 5.25 5.22 5.26 0.16 Rumen NH^-N (ppm) 21.14a 14.36b 14.30b 12.90b 13.54b 1.54 Dry matter i n rumen (g) 574.93 643.73 447.89 449.04 478.15 109.43 Tot a l VFA cone. ( ^ -mole/ml) 166.12 186.03 165.44 166.81 150.06 18.17 Molar proportions of VFA: 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 Isobutyric % 1.54a 0.71ab 2.11ac 0.66a 1.73a 0.27 I s o v a l e r i c % 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 value represents the mean of f i v e determinations. T Sulphur was added as Na^SO^ Means on the same l i n e bearing 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 resulted i n s i g n i f i c a n t l y (p ^  0.05) higher l e v e l s of n - v a l e r i c a c i d . Abomasal parameters (Data c o l l e c t e d from slaughtered animals) i Abomasal pH, percent nitrogen, acid-detergent f i b r e , c e l l u l o s e , non-protein nitrogen, r.ibonucleic-acid-nitrogen,and microbial-nitrogen contents of the abomasal digesta are presented i n Table 9. The r a t i o s of % RNA-N: % t o t a l abomasal digesta-N and % microbial-N: % t o t a l abomasal digesta-N are also shown i n Table 9. Formaldehyde treatment of the forage s i g n i f i c a n t l y (p<0.05) reduced the non-protein-nitrogen of the abomsasl digesta. The r a t i o s of % RNA-N: % t o t a l abomasal digesta N and % microbial-N: % t o t a l abomasal digesta N were greater (p<0.05) f o r digesta from animals fed die t one than for digesta from animals fed die t s two, four and f i v e but not for digesta from animals fed d i e t three. A l l the other parameters measured were not s i g n f i c a n t l y (p> 0.05) affected by d i e t . Chemical composition of die t s used f o r duodenal flow rate measurements The chemical composition of the batch of die t s used for duodenal i, flow rate measurements i s given i n Table 10. Table 9. Abomasal parameters. Diet containing Diets containing formaldehyde treated forage untreated forage + VFAS + SO.t + VFAS + SO. 4 4 S.E. Abomasal pH 3.84* 3.94 3.88 3.83 3.70 0.22 Abomasal digesta (D.M. b a s i s ) : N% 2.27 3.01 2.80 3.16 3.06 0.23 ADF' % 17.31 18.28 17.40 17.77 17.29 0.40 Cell u l o s e % 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 % M i c r o b i a l 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 r a t i o (x:l) 0.786a 0.480b 0.583ab 0.451b 0.439b 0.068 * Each value represents the mean of f i v e determinations, t Sulphur was added as Na2S0^ Means on the same l i n e bearing d i f f e r e n t l e t t e r s (a, b) d i f f e r s i g n i f i c a n t l y (p < 0.05), Table 10. Chemical composition of diets used for duodenal flow rate measurements. Chemical F r a c t i o n Diet containing Untreated forage Diets containing formaldehyde treated forage + VFAS + SO.f + VFAS + SO, D.M. % ,N % (D.M. basis) ADF % (D.M. basis) Ce l l u l o s e % (D.M. basis) Ash % (D.M. basis) 86.71 2.21 16.80 12.64 8.12 86.95 86.95 2.24 2.24 17.10 17.10 13.01 13.01 8.16 8.16 86.95 86.95 2.20 2.20 16.92 16.92 13.01 13.01 8.20 8.20 ^ Sulphur was added as Na^SO^ - 73 -Feed intake during the duodenal flow measurements The d a i l y t o t a l feed intake, the feed intake during f i r s t , second, t h i r d and fourth six-hour periods are also given i n Table 11. Feed intake during the f i r s t , second, t h i r d and fourth six-hour periods as a percentage of t o t a l feed intake i s also given i n Table 11. The mean feed intake as a percentage of t o t a l f o r a l l of the d i e t s during the f i r s t , second, t h i r d and fourth six-hour periods and f i r s t and second twelve-hour periods were 40.72%, 17.01%, 22.52%, 20.20%, 57.28% and 42.72% respectively. Duodenal flow parameters The d a i l y digesta flow (ml and g), the average hourly digesta flow (ml) during the f i r s t , second, t h i r d and fourth six-hour periods and f i r s t and second twelve-hour periods are given i n Table 12. The percentage of t o t a l d a i l y flow during the f i r s t , second, t h i r d and fourth six-hour periods and also during the f i r s t and second twelve-hour periods are also shown i n Table 12. The mean flow as a percentage of t o t a l f o r a l l of the die t s during the f i r s t , second, t h i r d and fourth six-hour periods and f i r s t and second twelve-hour periods were: 25.20%, 24.96%, 24.37%, 24.35%, 50.13% and 49.87% respectively. Table 11. Feed intake during the duodenal flow measurements: Diet containing Diets containing formaldehyde treated forage Untreated forage + VFAS + S0,t + VFAS + SO, 4 4 Feed Intake (g): (D.M. basis) Daily t o t a l 1300.65 1304.25 1304.25 1304.25 1304.25 Intake 1st 6 hrs 515.06 429.53 436.49 646.91 596.48 Intake 2nd 6 hrs 86.71 198.25 365.19 173.90 285.20 Intake 3rd 6 hrs 477.77 285.20 189.55 309.54 206.20 Intake 4th 6 hrs 221.11 391.28 313.02 173.96 217.38 % Intake of 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 hrs 17.00 30.00 24.00 13.33 16.67 ^ Sulphur was added as Na2S0^ Table 12. Duodenal flow parameters. Diet containing untreated forage Diets containing formaldehyde treated + VFAS + SO.t + VFAS + 4 forage SO. 4 Duodenal digesta flow 1 2 3 4 5 Total (ml) 15533 16760 17019 16615 16340 To t a l (g) 15576 16790 17053 16720 16338 Hourly (ml) 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 hrs (ml) 7385 7985 9734 8225 7990 2nd 12 hrs (ml) 8168 8775 7285 8390 8350 Flow % of t o t a l : 1st 6 hrs 20.28 24.31 29.99 25.43 25.98 2nd 6 hrs 27.26 23.33 27.20 24.07 22.92 3rd 6 hrs 26.33 28.70 21.33 26.30 24.94 4 th 6 hrs 26.25 23.66 21.48 24.20 26.16 1st 12 hrs 47.54 47.64 57.19 49.50 48.78 2nd 12 hrs 52.58 52.36 42.81 50.50 51.10 Sulphur was added as Na„S0 - 76 -Some chemical f r a c t i o n s of duodenal digesta, t o t a l d a i l y intake and d a i l y digesta flow through the duodenum of these f r a c t i o n s  The d a i l y dry matter, organic matter, acid-detergent-fibre and c e l l u l o s e intakes (g) are given i n Table 13. The dry-matter, organic matter, acid-detergent-fibre and c e l l u l o s e composition (%) of the duodenal digesta i s shown i n Table 13. The t o t a l d a i l y flow through the duodenum (g) of dry matter, organic matter, acid-detergent f i b r e and c e l l u l o s e are also shown i n Table 13. Dai l y nitrogen intake, nitrogen components of duodenal digesta and the d a i l y flow of these components through the duodenum  The t o t a l d a i l y nitrogen intake (g) on each of the d i e t s i s shown i n Table 14. The nitrogen, non-protein-nitrogen, r i b o n u c l e i c acid-nitrogen and microbial-nitrogen composition of the duodenal digesta i s shown i n Table 14. Table 14 also contains the % RNA-N: % t o t a l N and % microbial-N: % t o t a l N r a t i o s , 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 d a i l y microbial N: t o t a l d a i l y N r a t i o s . The t o t a l d a i l y nitrogen, non-protein-nitrogen, true protein nitrogen, r i b o n u c l e i c - a c i d - n i t r o g e n and microbial nitrogen flowing through the duodenum are also given i n Table 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 of some chemical f r a c t i o n s i n the pre-duodenum portion of digestive t r a c t ( a l l compartments of stomach) and the change i n quantity of nitrogen entering the duodenum d a i l y compared with intake  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 (%) i n thepreduodenum of the digestive t r a c t ( a l l compartments of stomach) of dry matter, organic matter, acid-detergent-fibre and c e l l u l o s e are shown i n Table 15. The Table 13. Some chemical f r a c t i o n s of duodenal digesta, t o t a l d a i l y intake and d a i l y digesta flow through the duodenum of these f r a c t i o n s . Diet containing Diets containing formaldehyde treated forage untreated forage + VFAS + S0 4+ + VFAS + S0 4 1 2 3 4 5 Tot a l d a i l y 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 Cell u l o s e 164.40 169.68 169.68 169.68 169.68 Duodenal digesta: (Chemical f r a c t i o n ) D.M. % 5.68 5.97 5.96 5.95 5.99 O.M. % (D.M. basis) 86.16 87.24 86.98 88.44 87.96 A.D.F.% (D.M. basis) 17.10 18.25 17.65 18.10 17.80 Cell u l o s e % (D.M. basis) 12.03 13.10 12.80 13.12 12.95 Tot a l D a i l y Flow (g): 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 Cel l u l o s e 106.27 131.07 129.83 129.70 126.75 t Sulphur was added as Na SO, 2 4 Table 14. Dai l y nitrogen intake, nitrogen components of duodenal digesta and the d a i l y flow of these components through the duodenum. Diet containing Diets containing formaldehyde treated forage untreated forage + VFAS + SO.t + VFAS + SO, _ 4 4 To t a l d a i l y nitrogen intake (g) 28.74 Duodenal digesta:(D.M.basis) N % 2.40 NPN % 0.842 RNA-N % 0.145 Microbial-N % 1.93 % RNA-N:% T o t a l N r a t i o 0.060 % Microbial-N:% T o t a l N 0.804 To t a l d a i l y flow (g): N 21.20 NPN 7.44 True protein N 13.76 RNA-N 1.28 Microbial-N 17.05 Tot a l d a i l y RNA-N: T o t a l d a i l y N 0.060 To t a l d a i l y Microbial-N: 0.804 Total d i a l y N 29.22 3.10 0.614 0.111 1.48 0.036 0.477 31.02 6.10 24.92 1.11 14.81 0.036 0.477 29.22 2.96 0.701 0.122 1.63 0.041 0.551 30.02 7.10 22.92 1.24 16.53 0.041 0.551 28.69 3.15 0.662 0.094 1.25 0.030 0.397 31.14 6.52 24.62 0.929 12.36 0.030 0.397 28.69 2.98 0.681 0.093 1.24 0.031 0.416 29.16 6.66 22.50 0.910 12.14 0.031 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 of some chemical f r a c t i o n s i n the pre-duodenum portion of digestive t r a c t ( a l l compartments of stomach) and the change i n quantity of nitrogen entering the duodenum d a i l y compared with intake. Diet containing Diets containing formaldehyde treated forage untreated forage + VFAS + SO^t + VFAS + S0 4 1 2 3 4 5 % App. Dig. Coeff. of: 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 Ce l l u l o s e 35.35 22.75 23.48 23.56 25.30 Change i n quantity of N -7.54 +1.88 +0.80 +2.45 +0.47 entering duodenum d a i l y compared with intake (g) t Sulphur was added as Na oS0 - 80 -change i n quantity of nitrogen entering duodenum d a i l y compared with intake i s also given i n Table 15. - 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% decreased i n v i t r o nitrogen d i g e s t i b i l i t y during the microbial stage (Table 2). The values i n t h i s experiment (31.88%, 15.72%, 6.87% and 5.69% "for 0.0%,0.8%,1.0% and 1.2% l e v e l of formaldehyde treatment respectively) were lower than the values reported by Barry (1973a) and Barry (1976c). In the e a r l i e r study by Barry (1973a), he reported nitrogen d i g e s t i b i l i t y at the microbial stage of digestion to be l e s s than 30% for the treated and about 42% for the untreated, using grass-legume hay. The figures he reported i n the l a t e r experiment were 59.5% and 39.9% for the untreated and the treated grass-legume hay r e s p e c t i v e l y (Barry, 1976c). There are a number of factors which possibly contributed to the rather lower figures obtained i n t h i s t r i a l compared to those of Barry (1973a and 1976c). One possible reason f o r the low figures obtained i n the present study i s that the heat applied during the drying of the forage reduced s o l u b i l i t y of protein. However, i t was u n l i k e l y that the heat applied caused great heat-damage to the forage as the acid-detergent f i b r e i n s o l u b l e nitrogen l e v e l was l e s s than 7% of t o t a l nitrogen, which was the maximum l e v e l reported by Yu and Thomas (1976) for undamaged forages. If heat-damage had occurred, d i g e s t i b i l i t y of nitrogen at the second stage of incubation (combined microbial and acid-pepsin) would also have been reduced, but t h i s did not happen. In f a c t , the d i g e s t i b i l i t y of nitrogen at the second stage of incubation reported l a t e r on i n t h i s - 82 -discussion compared favourably with those reported by Barry (1976c). His figures ranged from 77.2% to 82.5% for the treated material and the f i g u r e for untreated was 84.8%. Another possible reason for the lower figures i n the present t r i a l f o r the f i r s t stage incubation compared to those of Barry (1973a and 1976c) was that tannins i n the forage might have protected the proteins. It was not l i k e l y that the tannins i n the forage greatly protected the proteins as, i f that had occurred, d i g e s t i b i l i t y of nitrogen at the second stage of incubation would have been reduced greatly. This i s because Ferguson (1975) and McLeod (1974) reported that forage tannins which are of the condensed type, protect proteins permanently and the bonds are not broken i n a c i d i c medium. The hydrolyzable types of tannins, which form r e v e r s i b l e bonding with proteins are present only i n seeds. A t h i r d p o ssible reason for the lower figures of f i r s t stage i n v i t r o incubation nitrogen d i g e s t i b i l i t y compared with figures of Barry (1973a and 1976c) i s that a great amount of soluble nitrogen was converted to microbial protein-nitrogen and thus was not measured. Annison j i t _al. (1954) reported that the extent of accumulation of degraded products of amino-acids i n the rumen could be reduced when r e a d i l y fermentable carbohydrates were present i n great amounts. The rumen micro-organisms grew very f a s t and u t i l i z e d nitrogen from the breakdown of proteins. In the present t r i a l , although the r e a d i l y fermentable carbohydrate l e v e l of the forage was not measured, i t was most l i k e l y high as the forage was immature, as indicated by the high nitrogen l e v e l . - 83 -There was a compensatory stepwise increase i n the d i g e s t i b i l i t y of nitrogen at the second stage of incubation with the formaldehyde treated samples up to 1% l e v e l of treatment. The nitrogen d i g e s t i b i l i t y figures f o r the second stage i n v i t r o t r i a l (combined m i c r o b i a l and acid-pepsin) were 80.95%, 79.76%, 75.85%, and 71.01% for 0.0%, 0.8%, -1.0%, and 1.2% l e v e l s of formaldehyde treatment r e s p e c t i v e l y . The differences between the o v e r a l l (combined microbial and acid-pepsin) and the f i r s t stage incubation nitrogen d i g e s t i b i l i t y figures were 49.07, 64.04, 68.98, and 65.32 percentage units f o r 0.0%, 0.8%, 1.0%, and 1.2% l e v e l s of formaldehyde treatment r e s p e c t i v e l y . Barry (1976c) also reported a compensatory increase i n nitrogen d i g e s t i b i l i t y at the second stage of incubation with formaldehyde treatment. The compensatory increase i n nitrogen d i g e s t i b i l i t y f o r the treated samples perhaps occurred because a l l the d i g e s t i b l e nitrogen which was not i n s o l u t i o n at the f i r s t stage of incubation, was digested by the acid-pepsin a f t e r the a c i d i t y broke down the bonds between the formaldehyde and the protein molecules. Ammonia-nitrogen production was s i g n i f i c a n t l y reduced with increasing l e v e l s of formaldehyde treatment up to the l e v e l of 1.2% whereas nitrogen d i g e s t i b i l i t y decreased only up to the 1.0% l e v e l . The ammonia-nitrogen l e v e l s per gram of dry matter incubated, as shown i n Table 2, were 228.79 ppm, 78.58 ppm, 65.27 ppm, and 30.04 ppm f o r 0.0%, 0.8%, 1.0% and 1.2% l e v e l of formaldehyde treatment re s p e c t i v e l y . The reason for the further decrease i n ammonia-nitrogen production beyond the 1.0% l e v e l of formaldehyde treatment i s not c l e a r . 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 stage of incubation) obtained from c a l c u l a t i o n s based on the means of the ammonia-nitrogen figures were 30.26%, 10.39%, 8.63% and 3.97% for 0.0%, 0.8%, 1.0% and 1.2% l e v e l s of formaldehyde treatment respectively. These figures were s l i g h t l y lower, except for the 1.0% l e v e l formaldehyde treatment, than those obtained from the actual measurements of d i g e s t i b i l i t y . The reason for the s l i g h t increase i n the fi g u r e f o r the 1.0% l e v e l of formaldehyde a p p l i c a t i o n compared to the actual measured value i s not obvious. Barry (1976c) also reported lower figures of nitrogen d i g e s t i b i l i t y at the f i r s t stage of incubation using ammonia-nitrogen figures (28% and 17% for untreated and formaldehyde treated hay respectively) compared with figures obtained from actual measurements (59.5% and 39.9% for untreated and formaldehyde treated hay r e s p e c t i v e l y ) . Sharkey et a l . (1972) reported that the nitrogen d i g e s t i b i l i t y figures obtained from actual measurements includes a l l soluble nitrogen compounds released during fermentation while ammonia-nitrogen takes into account only nitrogen fermented to ammonia. I t should also be noted that dipeptides, amino acids and n u c l e i c acids could be u t i l i z e d by rumen microbes without f i r s t being broken down to ammonia (Nolan, 1975). These compounds would not be measured i f they are i n so l u t i o n when only ammonia-nitrogen l e v e l i s measured and therefore the actual l e v e l of u t i l i z a t i o n of protein i n the feed by microbes would not be c o r r e c t l y assessed by measuring only the ammonia-nitrogen l e v e l . It seems therefore that ammonia-nitrogen production may not be the best in d i c a t o r of the optimum l e v e l of formaldehyde treatment. It also gives no i n d i c a t i o n of the e f f e c t of formaldehyde treatment on the digestion - 85 -of nitrogen by enzymes i n the i n t e s t i n e . Barry (1976a) reported that nitrogen d i g e s t i b i l i t y measurement using both microbial and acid-pepsin stages of incubation was a more r e l i a b l e method of determining optimum l e v e l s of formaldehyde treatment than ammonia-nitrogen l e v e l s . Feed Intake The d a i l y dry matter intake per unit of metabolic bodysize both during the pre-metabolism and metabolism study periods was not affected s i g n i f i c a n t l y (p ^ -0.05) by treatments (Table 4). The values for the pre-metabolism assay period were 91.33, 100.62, 101.93, 98.02 and 96.47 g/ Wkg^"7^ per day for d i e t s one, two, three, four and f i v e r e s p e c t i v e l y ; the values f o r the metabolism assay period were 90.17, 96.65, 104.29, 90.88 and 94.25 g/Wkg d a i l y , f or d i e t s one, two, three, four and f i v e r e s p e c t i v e l y . Feed intake i s an important fa c t or a f f e c t i n g the n u t r i t i v e value of die t s of ruminants (Crampton and Har r i s , 1969). Any differences i n the performances of the animals on the d i f f e r e n t d i e t s i n t h i s experiment could therefore not be a t t r i b u t e d to v a r i a t i o n s i n feed intake. Kempton et a l . (1977) and Ferguson (1975) reported that response to formaldehyde treatment of die t s was mediated mainly through increased feed intake compared to untreated d i e t s . Davies and Faichney (1973) reported decreased feed intake with formaldehyde treatment of barley r a t i o n s . I t i s noteworthy that the addition of the pure v o l a t i l e f a t t y acids to some of the d i e t s containing the formaldehyde treated forage (diets three and f i v e ) did not depress feed intake. Hume (1970) postulated that the addition of the pure v o l a t i l e f a t t y acids to d i e t s - 86 -could decrease p a l a t i b i l i t y and therefore i n h i s studies sodium s a l t s of these acids were used. However, Hume (1970) did not i n d i c a t e that the hypothesis that the pure v o l a t i l e f a t t y acids reduced p a l a t i b i l i t y was tested. Animals were fed ad l i b i t u m i n t h i s experiment as, was done by Hemsley and Moir (1963) and Adeleye (1972). Some workers have r e s t r i c t e d feed intake during metabolism studies (Barry, 1973b, Barry and Andrews, 1973; and Amos et a l . , 1976b). The argument f or r e s t r i c t i n g feed intake i s to reduce v a r i a b i l i t y i n intake of dry matter and nitrogen, e s p e c i a l l y nitrogen, which could a f f e c t nitrogen balance. One reason f o r feeding these animals ad l i b i t u m i n the present t r i a l was that i f r e s t r i c t e d feeding was c a r r i e d out, r e s u l t s from such experiments could not be applied, without l i m i t a t i o n s , to p r a c t i c a l farm conditions where animals are fed ad li b i t u m . Nimrick et a l . (1972) noted that when animals are fed ad li b i t u m , performance may not be the same as when r e s t r i c t i o n of feed intake i s practi s e d . With ad l i b i t u m feeding low rumen pH occurs, e s p e c i a l l y when high l e v e l s of r e a d i l y fermentable carbohydrate are fed. Protein 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 feeding i s c a r r i e d out compared to r e s t r i c t e d feeding, when protein breakdown i n the rumen may be great thus reducing i t s u t i l i z a t i o n . Eadie and Mann (1970) observed that the types of b a c t e r i a i n the rumen may also be d i f f e r e n t when high carbohydrate d i e t s are fed ad l i b i t u m as compared to r e s t r i c t e d feeding. Amos et_ a l . (1976b) r e s t r i c t e d feed intake and observed no difference i n nitrogen-balance between formaldehyde treated and untreated coastal bermudagrass hay. - 87 -When feed intake was increased from 600g dry matter to 775g dry matter, the e s s e n t i a l and non-essential amino acids a r r i v i n g at the duodenum were increased s i g n i f i c a n t l y f or the formaldehyde treated material. Nitrogen intake per day per u n i t of metabolic body s i z e (2.07, 2.18, 2.38, 2.08 and 2.18 g f o r d i e t s one, two, three, four and f i v e r e s p e c t i v e l y , Tables 4 and 6) was also not affected by treatment. This i s understandable since nitrogen l e v e l s i n the d i e t s (Table 3), were almost i d e n t i c a l (2.29%, 2.24%, 2.25%, 2.28% and 2.29% for d i e t s one, two, three, four and f i v e respectively) and dry matter intake, as discussed above, was not affected. With nitrogen intake being almost i d e n t i c a l on a l l d i e t s , d i etary sources of nitrogen, for microbial a c t i v i t i e s i n the rumen, could be l i m i t e d for the d i e t s containing the formaldehyde treated forage. The non-protein-nitrogen from the grass-legume forage and the nitrogen from the barley and cassava t o t a l l e d about 0.681g for every lOOg of the r a t i o n s . There could be a supply of nitrogen to the rumen micro-organisms with the d i e t s containing the formaldehyde treated forage through r e c y c l i n g . It seemed that nitrogen was l i m i t i n g i n the rumens of the animals fed the d i e t s containing formaldehyde forage as rumen ammonia-nitrogen, and microbial protein content of abomasal digesta were reduced (these r e s u l t s are discussed l a t e r ) . The concentration of v a l e r i c acid i n the rumens of animals fed the d i e t s containing the formaldehyde treated forage was lower compared to the c o n t r o l . This indicated a l i m i t a t i o n to rumen microbial growth most l i k e l y by nitrogen. I t i s therefore s u r p r i s i n g that feed intake was - 88 -not depressed for the diets containing the formaldehyde treated forage 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 microbial 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 nitrogen, acid-detergent f i b r e , c e l l u l o s e , dry matter and organic 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 of nitrogen The values 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 nitrogen obtained i n t h i s experiment were generally lower than the values reported by Beever et: a l . (1976). The values f o r t h i s experiment were 54.13, 47.06, 51.25, 44.90 and 47.24% for d i e t s one, two, three, four and f i v e respectively, while figures reported by Beever et a l . (1976) were 69.8% for untreated and 61.8% for treated rye grass. The values obtained i n t h i s experiment were however s i m i l a r to those reported by Dinius at a l . (1975) and Barry (1973a). Dinius e_t a l . (1975) reported apparent nitrogen 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 63.1, 41.1 and 29.8% for 0.0, 1.0 and 2.0% formaldehyde treatment of a l f a l f a meal. Barry (1973a) reported values of 55.2, 50.6, and 51.1% for 0.0, 2.4 and 4.8% l e v e l s of a p p l i c a t i o n of formaldehyde to rye-grass-clover during the process of hay making. The rather low value obtained for the untreated d i e t (diet one) i n t h i s experiment compared with the r e s u l t s of Beever at a l . (1976) and Dinius at a l . (1975) could possibly be a t t r i b u t e d to a number of factors reducing digestion 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 rations has been reported to increase protection of proteins as a r e s u l t of heat generated during the p e l l e t i n g process (Kempton et a l . , 1977). - 89 -(b) High l e v e l s of soluble carbohydrates. Cassava and barley constituted about 50% of the r a t i o n . High l e v e l s of soluble carbohydrate i n d i e t s have been reported to decrease rumen pH and thus reduce s o l u b i l i t y of protein i n the rumen f l u i d (0rskov, 1975; Kempton et a l . , 1977). However, Tagari et a l . (1964) reported increased nitrogen d i g e s t i b i l i t y i n the rumen with the addition of soluble carbohydrates to hay d i e t s . It i s i n t e r e s t i n g to note that the most p r o t e o l y t i c microbes i n the rumen are the amylolytic ones (Hungate, 1967; Church, 1975b). It i s therefore s u r p r i s i n g that lowered digestion of nitrogen occurs i n the rumen when high l e v e l s of soluble carbohydrate are fed, which should promote the growth of the amylolytic types of rumen microbes. Perhaps nitrogen d i g e s t i b i l i t y of the d i e t s containing the formaldehyde treated forage (except d i e t three) was reduced as a r e s u l t of l e s s digestion of nitrogen taking place i n the rumen. Although the heat treatment of the grass-clover forage, the p e l l e t i n g of the r a t i o n s , and the high l e v e l s of soluble carbohydrates could have caused reduction i n the d i g e s t i b i l i t y of nitrogen i n the rumen as discussed above for the c o n t r o l untreated d i e t , formaldehyde a p p l i c a t i o n further reduced nitrogen d i g e s t i b i l i t y i n the rumen. Dinius et: a l . (1974) reported that heating of protein had additive e f f e c t s to formaldehyde treatment. The lowered l e v e l s of ammonia-nitrogen i n the rumens of the animals fed the d i e t s containing the treated forage compared with the d i e t s containing the untreated forage supports the contention that there was generally reduced di g e s t i o n of nitrogen i n the rumen. - 90 -The reason f o r the rather high degree of digestion of nitrogen for d i e t three compared to the other d i e t s containing the formaldehyde treated forage i s not c l e a r . The ammonia-nitrogen l e v e l s i n the rumens of the animals fed d i e t three were s t i l l lower than the l e v e l s i n the rumens of animals fed d i e t one although nitrogen d i g e s t i b i l i t y was not 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 . It was possible that the addition of v o l a t i l e f a t t y acids to d i e t three promoted the growth of some microbes which could u t i l i z e nitrogen e f f i c i e n t l y i n the rumen. Hemsley trt a l . (1970) reported that forage protein which by-passed the rumen was more d i g e s t i b l e than b a c t e r i a l protein. In t h i s experiment, a greater amount of protein from the forage by-passed the rumen for the d i e t s containing the formaldehyde treated forage as indicated by the duodenal flow data (Table 14). This protein by-passing the rumen was s t i l l not digested to a greater extent i n the i n t e s t i n e than the protein i n d i e t one. H i l l (1975) and Horn and Huber (1975) observed that the most powerful stimulus for the release of s e c r e t i n , which also has an e f f e c t on the secretion of pancreatic enzymes, i s acid ingesta i n the duodenum. In t h i s experiment abomasal pH was not d i f f e r e n t for any of the dietary treatments. It was thought that with treatment of the forage portion of the d i e t s with formaldehyde, abomasal pH would be high as hydrogen ions would be required to break the bond between the protein and formaldehyde as indicated by Barry (1976a). It seems from t h i s t r i a l that there was a possible compensatory increase i n the production of hydrochloric acid i n the abomasum with formaldehyde - 91 -treatment of the forage portion of the d i e t s sinee abomasal pH values were not affected (Table 9)• The time a f t e r feeding when pH measurements were c a r r i e d out was perhaps not optimum to demonstrate di f f e r e n c e s . Knight et a l . (1972) observed that the greatest f a l l i n abomasal pH occurred i n the f i r s t hour a f t e r feeding. In the present t r i a l , the animals were slaughtered about four to s i x hours (4-6 hrs) a f t e r feeding. The r e s u l t s of the experiments of Knight et a l . (1972) indicated 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 feeding with pH l e v e l s reaching pre-prandial conditions about s i x hours (6 hrs) a f t e r feeding. H i l l (1975) reported that other factors which also a f f e c t the release of s e c r e t i n are peptones, soaps and amino acids a r r i v i n g at the duodenum. Since these parameters were not measured, i t i s not possible 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 nitrogen for the d i e t s containing the formaldehyde treated forage. Abomasal non-protein-nitrogen l e v e l s determined with slaughtered animals were lower with the d i e t s containing the treated forage (Table 9). The t o t a l d a i l y flow of non-protein-nitrogen through duodenum measured with the cannulated sheep tended to be somewhat lower f o r the d i e t s containing the treated forage than for the diet containing the untreated forage (7.44, 6.10, 7.10, 6.52 and 6.6.6 g/day for d i e t s one, two, three, four and f i v e r e s p e c t i v e l y ) . - 92 -The addition of sulphate alone to one of the die t s containing formaldehyde treated forage (diet four) somehow reduced nitrogen d i g e s t i b i l i t y . I t i s not clear how the sulphate adversely affected nitrogen d i g e s t i b i l i t y . Umuna and Woods (1975) reported reduction i n nitrogen d i g e s t i b i l i t y when urea i n d i e t s was coated with 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 of nitrogen obtained i n the i n vivo t r i a l s were generally lower than the nitrogen d i g e s t i b i l i t y obtained i n the i n v i t r o trial's (combined microbial and acid-pepsin stages). Barry (1976c) reported i n v i t r o nitrogen d i g e s t i b i l i t y (both m i c r o b i al and acid-pepsin stages of digestion) to be about 72-84% while i n the i n vivo t r i a l s , values obtained ranged from 52% to 68%. The discrepancy between the i n v i t r o and the i n vivo nitrogen d i g e s t i b i l i t y i n the present t r i a l could perhaps be explained as follows: the i n v i t r o measurement was true d i g e s t i b i l i t y of the grass-legume forage while the i n vivo was apparent, not taking into account endogenous secretions of nitrogen. It must also be noted that during the i n v i t r o measurement, only the grass-legume forage was used, while with the i n vivo cassava and barley formed part of 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 of ADF and Ce l l u l o s e There was a s i g n i f i c a n t (p < 0.05) increase i n the i n vivo d i g e s t i b i l i t y of acid-detergent f i b r e and c e l l u l o s e with formaldehyde treatment of the forage (Table 5, ADF values: 32.57, 36.97, 36.91, 36.45 and 36.59% for d i e t s one, two, three, four 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 or die t s one, two, three, four and f i v e r e s p e c t i v e l y ) . Dinius et al". (1975) reported a s i g n i f i c a n t - 93 -reduction i n the d i g e s t i b i l i t y of acid-detergent f i b r e (38.8%, 33.8%, and 26.4% for 0.0%, 1.0%, and 2.0% l e v e l s of formaldehyde treatment r e s p e c t i v e l y ) . Beever et a l . (1976) reported higher values 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 treated high temperature (900°C) dried grass forage (83.9% vs 89.5% for the untreated and treated r e s p e c t i v e l y ) . The l e v e l s of acid-detergent f i b r e and c e l l u l o s e i n the abomasal digesta samples (Table 9) were not s i g n i f i c a n t l y (p y 0.05) d i f f e r e n t . The d i g e s t i b i l i t y of acid-detergent-fibre i n the stomach (pre-duodenum) from the animal f i t t e d with re-entrant cannula (Table 15) was markedly greater f o r di e t one than f or the re s t (30.87%, 18.12%, 19.72%, 18.92% and 21.05% for d i e t s one, two, three, four and f i v e r e s p e c t i v e l y ) . The same trend 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% for d i e t s one, two, three, four and f i v e r e s p e c t i v e l y ) . I t could be argued that with the d i e t s containing the formaldehyde treated forage most of the digestion of acid-detergent-fibre and c e l l u l o s e took place outside the reticulo-rumen. A great quantity of nitrogen might have reached the caecum and the colon as there was a reduction i n the digestion of nitrogen i n the reticulo-rumen when the d i e t s containing the formaldehyde treated forage were fed. The micro-organisms i n the caecum and colon having access to a great amount of nitrogen might have had greater a c t i v i t i e s than those i n animals fed the di e t containing the untreated forage, with l e s s nitrogen reaching the caecum and colon. Nolan (1975) reviewing l i t e r a t u r e on digestion i n the hindgut reported that p r o t e o l y t i c a c t i v i t y appears to be greater i n the contents of the large i n t e s t i n e than i n the contents of the rumen. He also observed - 94 -that a great deal of fermentation of s t r u c t u r a l carbohydrates occurs i n the hindgut e s p e c i a l l y with sheep fed d i e t s containing high l e v e l s of cereals. The sources of nitrogen i n the hindgut f o r such fermentation a c t i v i t i e s of the micro-organisms are: nitrogeneous compounds a r r i v i n g from the small i n t e s t i n e i n the form of feed residues, undigested rumen micro-organisms and endogenous secretions; and entry of urea-nitrogen from the blood. In the case of d i e t three, though nitrogen d i g e s t i b i l i t y was not s i g n i f i c a n t l y (p y 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 was s i g n i f i c a n t l y (p <0.05) higher. The microbes i n the hindguts of animals fed di e t three were probably supplied with a greater amount of nitrogen from the small i n t e s t i n e than f or di e t one. I t must be noted that the concentration of nitrogen i n the abomasal digesta samples was not affected by d i e t a r y treatment (Table 9) and the nitrogen concentration of the duodenal digesta was not markedly d i f f e r e n t . It was s t i l l p ossible that the animals fed the di e t s containing the formaldehyde treated forage had a greater amount of nitrogen a r r i v i n g i n the duodenum as flow of dry matter could be d i f f e r e n t . From the data obtained with the animal f i t t e d with re-entrant cannula, the d a i l y flow of nitrogen was markedly higher f o r the di e t s containing the formaldehyde treated forage than for the d i e t s containing untreated material (Table 14, 21.20g, 31.02g, 30.02g, 31.14g and 29.16g for di e t s one, two, three, four and f i v e r e s p e c t i v e l y ) . Nitrogen a r r i v i n g i n the duodenum was greater f o r di e t three compared with d i e t one. Enough nitrogen was - 95 -probably l e f t f o r m i c r o b i a l a c t i v i t y i n the hindgut even i f a greater portion of i t was digested and absorbed i n the small i n t e s t i n e with d i e t three compared to d i e t one. With a greater amount of nitrogen a r r i v i n g i n the hindgut for d i e t three 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 greater and therefore d i g e s t i o n of f i b r e i n the hind gut would be greater f o r d i e t three than f o r d i e t one. Beever et: a l . (1976) observed a greater amount of d i g e s t i o n of f i b r e i n the hindgut for the treated compared to the untreated d i e t . In t h e i r study, 95% of the d i g e s t i o n of f i b r e occurred i n the r e t i c u l o -rumen for the untreated d i e t and 70% for the formaldehyde treated high temperature dried grass ( 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 being 83.9%, and 89.5% f o r untreated and treated r e s p e c t i v e l y ) . In the present study, the s i t e s for the digestion of f i b r e were d i f f e r e n t f or the d i e t containing the untreated forage and the d i e t s containing the formaldehyde treated forage. Nitrogen was 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 degradation of f i b r e i n the rumen when the d i e t s containing the formaldehyde treated forage were fed (rumen ammonia-nitrogen l e v e l s were lower for d i e t s two, three, four and f i v e than f o r d i e t one, Table 8). A greater proportion of f i b r e was digested i n the hindgut where nitrogen might not have l i m i t e d microbial a c t i v i t i e s when the d i e t s containing the formaldehyde treated forage were fed (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 A.D.F. and c e l l u l o s e i n the four compartments of the stomach were observed to be higher f o r d i e t one than f o r the others using the sheep f i t t e d with the duodenal re-entrant cannula, Table 15). However, when the d i e t containing the untreated forage was fed, nitrogen was most l i k e l y not a l i m i t i n g factor i n the rumen but was most probably - 96 -l i m i t i n g i n the hindgut. There were high l e v e l s of r e a d i l y fermentable carbohydrate present i n the rumen. Therefore, the digestion of f i b r e could not be c a r r i e d out to the maximum extent. 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 dry matter and organic 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 of dry matter (64.78%, 65.69%, 65.82%, 64.80%, and 63.71% for d i e t s one, two, three, four and f i v e r espectively) and organic matter (65.35%, 65.91%, 66.01%, 65.17% and 63.83% for d i e t s one, two, three, four, and f i v e respectively) were not affected s i g n i f i c a n t l y (p y 0.05) by treatments. The values obtained were s i m i l a r to those reported by other workers (Amos at a l . , 1976b; Sharma and Nicholson, 1975b). Amos at a l . (1976b) reported dry matter d i g e s t i b i l i t y figures of 64.4%, 63.4%, 61.3%, and 56.3% for 0.0%, 0.5%, 1.0% and 1.5% l e v e l s of formaldehyde treatment of coastal bermuda grass r e s p e c t i v e l y . Sharma and Nicholson (1975b) reported dry matter d i g e s t i b i l i t i e s of 65.4% and 63.9%, for rations containing untreated and formaldehyde treated faba bean meal re s p e c t i v e l y . They also reported dry matter d i g e s t i b i l i t i e s of 53.5% and 55.2% f o r rations containing untreated and treated rapeseed meal r e s p e c t i v e l y (Sharma and Nicholson, 1975a). In some of the reports c i t e d above (Sharma and Nicholson, 1975a, 1975b; Barry 1976c) dry 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 affected by formaldehyde treatment as was the case i n the present experiment. Sharma and Nicholson (1975a and 1975b) did not observe a s i g n i f i c a n t reduction i n nitrogen d i g e s t i b i l i t i e s i n the two t r i a l s , with formaldehyde treatments of faba bean and rapeseed meal. Barry (1976c) reported no s i g n i f i c a n t reduction i n dry matter d i g e s t i b i l i t y of d i e t s treated with formaldehyde although there 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 . Dinius e_t a l . (1975) however reported a s i g n i f i c a n t reduction i n dry matter, nitrogen and f i b r e d i g e s t i b i l i t i e s with formaldehyde treatment. In the present experiment, although nitrogen 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 reduced (except for d i e t three) by treatment of the forage portion of the diet s with formaldehyde, dry matter and organic matter d i g e s t i b i l i t i e s were not affe c t e d because of an increase i n the diges t i o n of some other f r a c t i o n s , e s p e c i a l l y f i b r e . For d i e t three, nitrogen d i g e s t i b i l i t y was not s i g n i f i c a n t l y l e s s than for 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) greater than for d i e t one. I t was expected that dry matter and organic 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 for the two d i e t s . However, there were s l i g h t but non-significant differences between die t s one and three with respect to nitrogen and dry matter or organic matter d i g e s t i b i l i t i e s . The nitrogen d i g e s t i b i l i t y was s l i g h t l y higher for d i e t one than for d i e t three (54.13% for d i e t one and 51.25% for d i e t three); dry matter d i g e s t i b i l i t y was s l i g h t l y higher for d i e t three 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 three); organic matter d i g e s t i b i l i t y was also s l i g h t l y higher for d i e t three than f o r diet one (65.35% for die t one and 66.01% for d i e t three). Rumen Parameters  Rumen pH The pH value of the rumen f l u i d ranged from 5.11 to 5.50 f o r a l l the d i e t s . Rumen pH was not s i g n i f i c a n t l y (p> 0.05) affected by - 98 -the treatments. Dinius et a l . (1975) also observed no differences i n rumen pH with treatment of a l f a l f a meal with formaldehyde. The pH values obtained i n the present t r i a l were s i m i l a r to the values reported by Sharma and Ingalls (1974). They reported average pH values of about 5.6. They also reported no differences i n pH with formaldehyde treatment of d i e t s . In the present experiment, the samples for pH measurements were taken 4-6 hours a f t e r feeding. Hodgson et a l . (1976) reported that as v o l a t i l e f a t t y acid concentration increased, the rumen pH decreased. In the present experiment, as the pH was measured at the time when concentration of v o l a t i l e f a t t y acids was expected to be highest i t i s not s u r p r i s i n g that the pH values were r e l a t i v e l y low. S a v i l l e et a l . (1971) postulated that i f rumen pH values were low, i t was possible that the bonds between formaldehyde and proteins could be broken. Sharma and In g a l l s (1974) reported low pH values around 5.6 but s t i l l no breakdown i n the bonding occurred. There i s also a c y c l i c a l v a r i a t i o n i n rumen pH and the values obtained i n t h i s experiment could have been at the lowest f o r the day. The animals also di d not refuse feed and there were no signs of rumenitis. Rumen ammonia-nitrogen Rumen ammonia-nitrogen l e v e l s (Table 8) were s i g n i f c a n t l y (p 0.05) reduced by formaldehyde treatment of the forage portion of the die t s (21.14, 14.36, 14.30, 12.90 and 13.54 ppm for d i e t s one, two, three, four and f i v e r e s p e c t i v e l y ) . Reports by some workers indicated reduced.rumen ammonia-nitrogen l e v e l s with treatment of die t s with formaldehyde (Sharma and Nicholson, 1975b; Bhargava and Ranjhan, 1974; - 99 -Sharma and I n g a l l s , 1973; Sharma et a l . , 1972; Hogan and Weston, 1970; Hemsley et: a l . , 1970). Other workers however observed no decrease i n rumen ammonia nitrogen l e v e l s with formaldehyde treatment of d i e t s (Dinius et a l . , 1975; Sharma and Nicholson, 1975a). Ammonia present i n the rumen could be from a v a r i e t y of sources: recycled urea-nitrogen e i t h e r from blood or s a l i v a ; r from the breakdown of protein or amino acids, and reduction of n i t r a t e s ; from the breakdown of microbial protein during r e c y c l i n g of nitrogen within the rumen (Houpt, 1970). Ammonia-nitrogen could also be l o s t through the ruminal w a l l . Such losses have been reported to be dependent on pH with maximum losses at pH 6.5, and n e g l i g i b l e at pH 4.5 (Hemler and Bartley, 1971; Hogan, 1961). Ammonia-nitrogen could be passed on to the abomasum and subsequently to the i n t e s t i n e as there i s l i t t l e , absorption of ammonia-nitrogen from the abomasum (Houpt, 1970; Hembry et a l . , 1975). In the present experiment, rumen pH values 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 therefore losses of ammonia-nitrogen through the ruminal w a l l are u n l i k e l y to have contributed to the v a r i a t i o n s i n rumen ammonia-nitrogen with formaldehyde treatment. I f anything, losses of nitrogen i n the rumen were possibly greater f o r di e t one than f o r the other four d i e t s . This i s because the quantity of nitrogen leaving the abomasum was greater f o r a l l the four d i e t s containing the treated forage compared to the co n t r o l , using the animal f i t t e d with re-entrant cannula (Table 14). The greater amounts of nitrogen a r r i v i n g at the duodenum with the di e t s containing the formaldehyde treated forage compared with d i e t one were u n l i k e l y to be due to differences i n the losses of ammonia i n the abomasum as such losses - 100 -are reported to be n e g l i g i b l e (Houpt, 1970; Hembry et a l . , 1975). Nitrogen r e c y c l i n g i n t o the rumen i s reported to be highest when ammonia-nitrogen l e v e l s i n the rumen are low (Church, 1975c, Houpt, 1970). It i s therefore also u n l i k e l y that the higher ammonia-nitrogen l e v e l s i n the rumens of animals fed d i e t one compared to the r e s t , was due to greater r e c y c l i n g of nitrogen into the rumens from s a l i v a and blood urea-nitrogen. One possible reason for the higher' l e v e l s of rumen ammonia-nitrogen i n the rumens of animals fed d i e t one compared with the rest was a greater degree of r e c y c l i n g of microbial nitrogen within the rumen.. Perhaps the most probable explanation f o r the higher l e v e l s of ammonia-nitrogen i n the rumens of animals fed d i e t one compared with the rest i s a greater degree of digestion of nitrogen i n the rumens of the animals fed t h i s d i e t . This i s supported by the lower quantity of nitrogen a r r i v i n g i n the duodenum when die t one was fed to the sheep f i t t e d with the re-entrant cannula, compared to the other d i e t s . The quantity of non-protein-nitrogen a r r i v i n g at the duodenum was also s l i g h t l y greater for d i e t one than for the r e s t . The concentration of non-protein-nitrogen i n the abomasal digesta samples from the slaughtered animals was also s i g n i f i c a n t l y (p < 0.05)higher for d i e t one than for the rest (Table 9). Generally, the l e v e l s of ammonia i n the rumens for a l l the d i e t s were low compared with figures reported by Barry (1973a) and Sharma and Nicholson (1975a and 1975b). The ammonia-nitrogen l e v e l s reported by Sharma and Nicholson (1975a) were 8.99mg/100ml and 5.47mg/100ml for d i e t s containing untreated and formaldehyde treated 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 one hour a f t e r feeding. These workers (Sharma and Nicholson, 1975b) reported rumen ammonia-nitrogen l e v e l s of 24.25mg/100ml and 13.66mg/100ml for diet s containing untreated and formaldehyde treated faba bean meal resp e c t i v e l y . These measurements were also c a r r i e d out on samples c o l l e c t e d one hour a f t e r feeding. Measurements c a r r i e d out on samples c o l l e c t e d four hours a f t e r feeding i n the same experiment gave r e s u l t s of 15.40mg/100ml and 6.96mg/100ml for die t s containing untreated and formaldehyde treated faba bean d i e t s . Barry (1973a) taking measurements on samples c o l l e c t e d four hours a f t e r feeding, reported figures of 27.0mg/100ml for untreated and 14.8mg/100ml for formaldehyde treated lucerne"* hay. Dinius et^ a l . (1975) however reported low ammonia-nitrogen l e v e l s of 3 to 7 mg/lOOml. They took samples f o r ammonia-nitrogen measurements j u s t before feeding and at one hour i n t e r v a l s f o r eight hours and combined the r e s u l t s to a r r i v e at the values reported above. The rather low l e v e l s of ammonia-nitrogen observed i n the present experiment could perhaps be a t t r i b u t e d to the time of sampling. Samples were taken four to s i x hours a f t e r feeding. Rumen ammonia-nitrogen l e v e l i s reported to reach peak l e v e l s about 90 to 130 minutes a f t e r feeding (Church, 1975c). Hembry et a l . (1975) reported rumen ammonia l e v e l s of 12 to 30 ppm s i x hours^after feeding and 16 to 46 ppm four hours a f t e r feeding d i e t s containing urea, soybean meal and casein. - 102 -Rumen t o t a l v o l a t i l e f a t t y acid concentration There were no s i g n i f i c a n t (py 0.05) differences •between treatments with respect to the production of v o l a t i l e f a t t y acids 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, three, four and f i v e r e s p e c t i v e l y ) . Several workers have also reported no s i g n i f i c a n t l y differences i n the production of v o l a t i l e f a t t y acids i n the rumen with formaldehyde treatment. (Beever et a l . , 1976; Beever et a l . , 1977; Sharma and I n g a l l s , 1973). Other workers however have observed decreased concentrations of v o l a t i l e f a t t y acids with treated d i e t s (Sharma e_t a l . , 1972; Barry, 1973a). M i c r o b i a l protein-nitrogen i n the abomasal digesta samples (measured by the r a t i o of % microbial - N : % t o t a l digesta N) was decreased s i g n i f i c a n t l y with a l l the d i e t s containing the formaldehyde treated forage, except f o r d i e t three (0.768, 0.480, 0.583, 0.451, and 0.439 for die t s one, two, three, four and f i v e r e s p e c t i v e l y ) . The r a t i o s for duodenal digesta, using the sheep f i t t e d with re-entrant cannula (0.804, 0.477, 0.551, 0.397, and 0.416 for d i e t s one, two, three, four and f i v e respectively) and the t o t a l amounts of microbial protein a r r i v i n g at the duodenum per day (17.05, 14.81, 16.53, 12.36, and 12.14g for d i e t s one, two, three, four and f i v e respectively) were also reduced by treatment of the forage portion of the di e t s with formaldehyde. No s t a t i s t i c a l analysis was performed however with the duodenal digesta as only one animal was used but i t s t i l l indicated the trend shown by the abomasal digesta samples. Smith et a l . (1968) reported that the proportion of r i b o n u c l e i c -acid nitrogen to t o t a l nitrogen i n duodenal digesta p a r a l l e l e d the proportion of r i b o n u c l e i c acid-nitrogen to t o t a l nitrogen i n rumen f l u i d - 103 -except that the l e v e l s i n the duodenal digesta were c o n s i s t e n t l y lower because of the addition of endogenous secretions of nitrogen i n the abomasum. I t i s therefore i n l i n e to extrapolate measurements of microbial protein-nitrogen i n duodenal digesta to what happened i n the rumen. From the data reported above f or the present t r i a l , i t could therefore be concluded that m i c r o b i a l protein synthesis i n the rumen was reduced by formaldehyde treatment of the forage portion of the d i e t , except f o r d i e t three. However, v o l a t i l e f a t t y acid production was not affected. Beever et a l . (1977) reported that rumen microbial populations were capable of adapting t h e i r metabolic pathways to make maximum use.of carbohydrates while microbial protein synthesis may be depressed. This phenomenon was termed by these workers as uncoupled fermentation. I t i s probable that t h i s occurred i n the present study. Hodgson et: a l . (1976) reported that the amount of dry matter i n the rumen could a f f e c t the concentration of short chain f a t t y acids i n the rumen. The quantities of dry matter i n the rumens of the sheep (Table 8) were not 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 with d i e t a r y treatment (574.93, 643.73, 447.89, 449.04, and 478.15g f o r d i e t s one, two, three, four, and f i v e r e s p e c t i v e l y ) . Therefore, possibly t h i s parameter had no e f f e c t on the production of v o l a t i l e f a t t y acids i n the rumen. Dinius elt a l . (1975) reported that time of sampling had some e f f e c t s on the concentration of v o l a t i l e f a t t y acids i n the rumen. In t h e i r study, the v o l a t i l e f a t t y acid concentration at two, three, and four hours a f t e r feeding was s i g n i f i c a n t l y reduced by formaldehyde treatment of the lucerne portion of the die t but at s i x hours there were no s i g n i f i c a n t differences between the formaldehyde - 104 -treated and the untreated. Sharma et a l . (1972) and Barry (1973a) who observed differences i n concentration of v o l a t i l e f a t t y acids i n the rumen due to formaldehyde treatment took t h e i r samples three to four hours a f t e r feeding. Beever et a l . (1976), and Beever et^ a l . (1977) who reported no s i g n i f i c a n t reduction i n t o t a l v o l a t i l e f a t t y acid production i n the rumen measured i t s production over twenty four hours. Sharma and Ingalls (1973) who also reported no s i g n i f i c a n t reduction i n the production of 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 three to four hours a f t e r feeding. Therefore, i t i s possible that the equality of production of v o l a t i l e f a t t y acids reported i n the present study was not due to the time of sampling. The l e v e l s of v o l a t i l e f a t t y acids i n the rumens were high compared with figures reported by Dinius et a l . (1975) and Nicholson and Sutton (1969). Dinius et a l . (1975) reported figures (at four hours a f t e r feeding) of about 100 , 86 , and 50 /i-moles/ml f o r d i e t s containing lucerne meal treated with formaldehyde at l e v e l s of 0, 1 and 2% r e s p e c t i v e l y . The corresponding figures f o r s i x hours a f t e r feeding were about 80 , 72 , and 60 ^ i-moles/ml r e s p e c t i v e l y . In the case of t h e i r experiments the l e v e l of lucerne i n the d i e t s was about 75% while i n the present experiment the l e v e l of forage was only about 50% with the remainder of the rati o n s made up of r e a d i l y fermentable carbohydrates. This high l e v e l of r e a d i l y fermentable carbohydrate might have produced the high l e v e l of t o t a l v o l a t i l e f a t t y a c i d observed i n the present experiment. Nicholson and Sutton (1969) reported t o t a l v o l a t i l e f a t t y acid l e v e l s of about 82.1 to 97.0 / l - m o l e s / l i t r e when they fed d i e t s containing 20% hay and 80% concentrate to sheep. The l e v e l s of r e a d i l y soluble carbohydrate were high i n t h e i r d i e t s . They however, composited - 105 -samples taken at three, s i x , nine, and eleven hours a f t e r feeding f o r the v o l a t i l e f a t t y acid measurements. Therefore, the high l e v e l s of v o l a t i l e f a t t y acid observed i n the present experiment compared to the l e v e l s reported by Nicholson and Sutton (1969) could most l i k e l y be due to the time of sampling. Ace t i c , propionic, and b u t y r i c acid proportions i n the rumen f l u i d The molar proportions of a c e t i c , propionic and b u t y r i c acids i n the rumen f l u i d were not affected by dietary treatment (Table 8). The second group of animals however had s i g n i f i c a n t l y (p < 0.05) higher molar proportions of a c e t i c acid than the fourth group, while the fourth group had s i g n i f i c a n t l y (p < 0.05) higher molar proportions of propionic acid than the second group (Results, page 68 ). Differences i n the molar proportions of a c e t i c and propionic acids i n the rumens of the second and fourth groups (blocks) of animals could have occurred 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 et a l . (1970) reported v a r i a t i o n s i n types of rumen microbes with f u l l feeding, even with i d e n t i c a l twins. These v a r i a t i o n s i n populations of types of microbes could also a f f e c t proportions of the various short-chain f a t t y acids. In the present experiment the proportion of soluble carbohydrates i n the d i e t s fed to the d i f f e r e n t groups (blocks) of animals was not d i f f e r e n t . Therefore, the higher l e v e l s of propionic acid and the lower l e v e l s of a c e t i c acid i n the rumens of sheep i n the fourth group compared to those i n the second group could not be due to d i f f e r e n t l e v e l s of soluble carbohydrates. - 106 -Wilke and Merwe (1976) reported that high l e v e l s of concentrate i n the diet could r e s u l t i n high molar proportions of propionic acid at the expense of the a c e t i c acid proportion. The average proportions of the various f a t t y acids for a l l the d i e t s (acetic 41.96%, propionic 29.00%, n-butyric 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 (except f o r a c e t i c and butyric) to the values reported by Sutton (1969). He reported that when a cow was fed'a diet containing flaked maize (5kg per day) and hay (1.0kg per'day) the proportions of the various acids were: a c e t i c 54.8%, propionic 25.1%, b u t y r i c 14.0%, i s o b u t y r i c 1.4%, i s o v a l e r i c 2.3%, and v a l e r i c 2.4%. 0rskov et a l . (1970) reported the following l e v e l s of the various acids i n the rumen of sheep fed chopped dried grass: a c e t i c 68.6%, propionic 20.2%, b u t y r i c 6.0%, i s o b u t y r i c 2.5%, i s o v a l e r i c 2.0%, and v a l e r i c 0.8%. In the experiment of 0rskov et a l . (1970) the sheep were slaughtered one hour a f t e r feeding and then samples taken. The same workers i n the same paper, reported the following l e v e l s of various acids when barley was fed (animals slaughtered one hour a f t e r feeding and samples taken): a c e t i c 53.6%, propionic 20.6%, b u t y r i c 16.2%, i s o b u t y r i c 1.7%, i s o v a l e r i c 1.4%, n - v a l e r i c 5.4%, and caproic 1.1%. Nicholson and Sutton (1969) and Whitelaw est a l . (1970) reported that when sheep on barley d i e t s are fed below f u l l feeding the proportion of b u t y r i c a c i d i s increased with a corresponding decrease i n the propionic acid f r a c t i o n . In the present experiment, both b u t y r i c a c i d and propionic acid proportions were very high. There was rather a decrease i n a c e t i c acid proportion which i s usually reported above 50% even when animals are fed d i e t s containing - 107 -great amounts of r e a d i l y fermentable carbohydrate (Sutton, 1969; and 0rskov et a l . , 1970). The animals were fed ad l i b i t u m . The lowered a c e t i c a c i d l e v e l was therefore probably due to the high l e v e l of soluble carbohydrates i n the r a t i o n s . I s o v a l e r i c and Isobutyric acid proportions i n the rumens The molar proportions of i s o v a l e r i c and i s o b u t y r i c acids were higher f o r d i e t three than for d i e t s two and four (p*£ 0.05). The i s o v a l e r i c a c i d proportions were 2.53, 0.60, 2.58, 0.39 and 2.18% for d i e t s one, two, three, four and f i v e r e s p e c t i v e l y . The i s o b u t y r i c acid proportions were 1.54, 0.71, 2.11, 0.66 and 1.73% r e s p e c t i v e l y for di e t s one, two, three, four and f i v e . I t was expected that with formaldehyde treatment of the forage portion of the d i e t s molar proportions of i s o b u t y r i c and i s o v a l e r i c acids would be reduced. These acids are derived from branched-chain amino acids (el-Shazly, 1952a and 1952b). A l l i s o n and Bryant (1963) reported that the mechanism for the synthesis of the isopropyl moiety i n the branched-chain f a t t y acids was inadequate i n some micro-organisms. Langlands (1973a) reported reduction (not s i g n i f i c a n t ) i n the l e v e l s of i s o b u t y r i c and i s o v a l e r i c acids following formaldehyde treatment of wheat. Faichney and White (1977a) reported s i g n i f i c a n t reductions of i s o b u t y r i c and i s o v a l e r i c acid l e v e l s with formaldehyde treatment of concentrate d i e t s . Barry and Fennessy (1973) reported 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 acids combined with formaldehyde treated s i l a g e s . Barry (1976c) reported - 108 -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 a c i d combined, but not is o b u t y r i c acid, with treatment of d i e t s with.formaldehyde. Hobson (1971) reported that the addition of one acid may suppress i t s production. This phenomenon was not observed by Hume (1970) who reported increased l e v e l s of branched-chain f a t t y acids i n the rumens of animals fed d i e t s supplemented with the sodium s a l t s of these acids. The present r e s u l t s seem to conform with those of Hume (1970). I t i s su r p r i s i n g that the 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 acids were not s i g n i f i c a n t l y higher for d i e t f i v e which was supplemented with v o l a t i l e f a t t y acids than f o r d i e t s two and four. I t i s to be noted that three animals on d i e t two, and one on d i e t four had l e v e l s of i s o b u t y r i c and i s o v a l e r i c acids which could not be measured. Other sources of protein, which could supply branched-chain amino acids to be deaminated to branched-chain f a t t y acids are (a) recycled m i c r o b i a l protein, (b) desquamated rumen epithelium, and (c) protein present i n s a l i v a . For d i e t three, i t i s d i f f i c u l t to speculate which of the three sources mentioned above contributed to the higher l e v e l s of the branched-chain f a t t y acids i n addition to what was supplemented and what came from the breakdown of dietary protein i n the rumen. The high l e v e l s f o r d i e t f i v e , though not s i g n i f i c a n t l y higher than d i e t s two and four, could be due mainly to the supplemented qu 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 that, unlike d i e t three, the d i g e s t i b i l i t y of nitrogen was not - 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 or d i e t four {diet three had s i g n i f i c a n t l y (p < 0.05) higher nitrogen d i g e s t i b i l i t y than d i e t four). The amounts of microbial protein nitrogen a r r i v i n g at the duodenum were not markedly d i f f e r e n t with d i e t f i v e compared with d i e t s two and four. The high l e v e l s of the branched-chain f a t t y acids i n the rumens of animals fed d i e t one, although not s i g n i f i c a n t l y d i f f e r e n t , compared with di e t s two and four, could be a t t r i b u t e d to greater breakdown of dietary protein i n the rumens. V a l e r i c a c i d proportions The v a l e r i c acid l e v e l s i n the rumens of animals fed the d i e t s containing formaldehyde treated forage were s i g n i f i c a n t l y ( p 0 . 0 5 ) higher than the l e v e l s i n the rumens of animals fed d i e t one (1.82, 3.30, 3.19, 3.34 and 3.15% for d i e t s one, two, three, four and f i v e r e s p e c t i v e l y ) . Cline et a l . (1958) reported that with a decrease i n a v a i l a b l e nitrogen i n the rumen, v a l e r i c acid l e v e l s increased. In the present t r i a l , m icrobial protein synthesis, measured by % RNA-N: % t o t a l abomasal digesta N or % microbial-N: % t o t a l abomasal digesta N r a t i o , was decreased with the other four d i e t s compared to d i e t one (Table 9), although not s i g n i f i c a n t l y f o r d i e t three. The amount of microbial nitrogen a r r i v i n g at the duodenum d a i l y was markedly les s for the formaldehyde treated d i e t s compared to d i e t one using the sheep f i t t e d with re-entrant cannula. I t i s l i k e l y that there was a l i m i t a t i o n to microbial protein synthesis by nitrogen and hence the higher l e v e l s of v a l e r i c acid with di e t s two, three, four, and f i v e compared with d i e t one. - 110 -Barry (1973a and 1976c) and Barry and Fennessy (1973) however reported 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 acids combined. Since the two acids were combined i n these reports, i t i s d i f f i c u l t to say which of these acids a c t u a l l y contributed to the lowered l e v e l s with formaldehyde treatment. Langlands (1973a) however reported a reduction i n the l e v e l s of n - v a l e r i c a c i d , with formaldehyde treatment of d i e t s . However, Faichney and White (1977a) reported an increase i n the l e v e l of n - v a l e r i c a c i d with formaldehyde treatment of concentrate d i e t s . The values of n - v a l e r i c a c i d for the untreated and the treated d i e t s (4.10% and 4.98% for untreated and formaldehyde treated'dietsrespectively) were however not s i g n i f i c a n t . The increased l e v e l of v a l e r i c acid i n the case of animals fed d i e t three, compared with d i e t one could 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 protein i n the rumen for the former d i e t , although o v e r a l l d i g e s t i b i l i t y was not s i g n i f i c a n t l y d i f f e r e n t for the two d i e t s . 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 of nitrogen for d i e t one was s l i g h t l y higher than that of d i e t three. Abomasal and duodenal digesta parameters  Abomasal pH The pH values of the abomasal f l u i d were generally higher than what have been reported by some workers (Knight et a l . , 1972; Wheeler and N o l l e r , 1977). The values obtained i n t h i s experiment were 3.84, 3.94, 3.88, 3.83 and 3.70 f o r d i e t s one, two, three, four and'five r e s p e c t i v e l y . wheeler and N o l l e r (1977) reported abomasal pH - I l l -values of 2.74 + 0.185 for sheep fed d i e t s containing about 80% corn. Knight et a l . (1972) reported that there was a considerable c y c l i c a l v a r i a t i o n i n abomasal pH. The lowest abomasal pH, about 2.05, occurred about one hour a f t e r feeding. The prefeeding pH l e v e l was approximately 2.90. There was a gradual increase i n the abomasal pH from one hour a f t e r feeding, with l e v e l s s i x to eight hours a f t e r feeding, approaching the pre-feeding l e v e l s . The rather high pH l e v e l s observed i n t h i s experiment cannot be explained f u l l y . The samples f o r pH measurements were taken about four to s i x hours a f t e r feeding and t h i s timing e f f e c t could have contributed to the high values. I t i s i n t e r e s t i n g to note that Lee (1977) also reported high abomasal pH values f o r sheep fed die t s of e i t h e r wheat or lucerne (3.20 + 0.20 f o r wheat di e t s and 3.49 +0.33 for lucerne d i e t s ) . Lee (1977) did not in d i c a t e the length of time a f t e r feeding when the animals were slaughtered for the measurements. Abomasal digesta N%, duodenal digesta N%, and t o t a l d a i l y flow of nitrogen from the abomasum into the duodenum  The abomasal nitrogen concentration (N%) was not s i g n i f i c a n t l y (p y 0.05) affected by formaldehyde treatment of the forage portion of the d i e t s . The values, on a dry matter basis, were 2.27, 3.01, 2.80, 3.16 and 3.06% f o r d i e t s one, two, three, four and f i v e r e s p e c t i v e l y . Sharma and Nicholson (1975a) also observed no s i g n i f i c a n t increase i n the concentration of nitrogen i n abomasal digesta with d i e t s containing formaldehyde treated rapeseed meal (3.13% f o r untreated and 3.20% for - 112 -formaldehyde treated). However, the t o t a l d a l l y amount of nitrogen flowing through the duodenum was s i g n i f i c a n t l y higher f o r the d i e t containing formaldehyde treated rapeseed meal than f o r the d i e t containing the untreated rapeseed meal. The nitrogen content of duodenal digesta, on a dry matter b a s i s , i n the present experiment was 2.40, 3.10, 2.96, 3.15 and 2.98% for d i e t s one, two, three, four and f i v e r e s p e c t i v e l y . Since only one animal was used i t i s not possible to say the differences between the d i e t s containing the formaldehyde treated forages and the d i e t containing the untreated forage were s i g n i f i c a n t . They were most l i k e l y not s i g n i f i c a n t as they were s i m i l a r to the abomasal digesta nitrogen concentration reported above. The t o t a l quantities of nitrogen a r r i v i n g at the duodenum d a i l y were 21.20, 31.02, 30.02, 31.14, and 29.16g for d i e t s one, two, three, four and f i v e r e s p e c t i v e l y (Table 14). The figures above i n d i c a t e that the formaldehyde treatment of the forage portion of the d i e t greatly increased the d a i l y flow of nitrogen from the abomasum to the duodenum, whereas the concentration of nitrogen i n the duodenal digesta was not markedly affected. The increase i n the quantity of nitrogen flowing through the duodenum d a i l y with formaldehyde treatment of the forage portion of the diet was possibly because of the increase i n the d a i l y flow of dry matter with formaldehyde treatment of the forage. Sharma and Nicholson (1975a) who reported no s i g n i f i c a n t increase i n the nitrogen concentration of abomasal digesta but s i g n i f i c a n t increase i n the t o t a l d a i l y flow of nitrogen through the duodenum with formaldehyde treatment of the rapeseed meal portion of d i e t , also observed an increase i n the flow of dry matter through the duodenum with formaldehyde treatment. - 113 -The d a i l y q uantities of nitrogen flowing through the duodenum on a l l the di e t s containing the formaldehyde treated forage were greater than the t o t a l quantities of nitrogen consumed d a i l y (-7.54g, +1.88g, +0.8g, +2.45g, and +0.47g f o r di e t s one, two, three, four and f i v e r e s p e c t i v e l y ) . The increase i n the amount of nitrogen a r r i v i n g d a i l y at the duodenum compared to the t o t a l d a i l y intake could be due to (a) increased r e c y c l i n g of urea-nitrogen into the four compartments of the stomach, (b) reduction i n the amount of loss of nitrogen through the ruminal w a l l . Beever e_t a l . (1976) and Hemsley et a l . (1970) reported a greater amount of nitrogen flowing through the duodenum compared with intake with d i e t s containing formaldehyde treated forages. Hemsley £t a l . (1970) reported lower quantities of nitrogen flowing through the duodenum per day than consumed with untreated forage d i e t . Faichney and White (1977b) reported d a i l y net gain i n nitrogen i n the four compartments of the stomach when four concentrate d i e t s , both untreated and formaldehyde treated were fed (except f o r one untreated d i e t where there was l o s s ) . The d a i l y net gain of nitrogen was greater f o r the treated d i e t s than • for the untreated. Sharma and Nicholson (1975a) reported greater quantities of nitrogen flowing through the duodenum d a i l y than consumed on d i e t s containing both formaldehyde treated and untreated rapeseed meal. The same workers (Sharma and Nicholson,1975b) however reported lower quantities of nitrogen flowing through the duodenum than consumed d a i l y f o r d i e t s - 114 -containing untreated and formaldehyde treated faba beans (net gain i n nitrogen d a i l y : -6g and -3g f o r d i e t s containing untreated and formaldehyde treated faba beans r e s p e c t i v e l y ) . Abomasal and duodenal digesta non-protein nitrogen l e v e l s The abomasal digesta concentration of non-protein-nitrogen was s i g n i f i c a n t l y (p 4. 0.05) higher for animals fed d i e t one than for animals fed the other d i e t s . The values, on a dry matter basis , were 0.864, 0.676, 0.643, 0.650 and 0.656% for d i e t s one, two, three, four and f i v e r e s p e c t i v e l y . Sharma and Nicholson (1975a) reported abomasal digesta non-protein-nitrogen concentrations of 0.61% and 0.64% on a dry matter basis for d i e t s containing untreated and formaldehyde treated rapeseed meal res p e c t i v e l y . These were not s i g n i f i c a n t l y d i f f e r e n t . The non-protein-nitrogen i n the abomasal digesta could come from dietary sources or from intermediary products of protein breakdown i n the reticulo-rumen. Since, i n the present study, the ingredient composition of a l l the d i e t s was the same, the most l i k e l y source for the increase i n abomasal digesta non-protein-nitrogen with the d i e t s containing the untreated forage was intermediary products of the breakdown of protein i n the reticulo-rumen. The concentration of non-protein-nitrogen i n the duodenal digesta (Table 14) was also greater with the d i e t containing untreated forage compared with the other four d i e t s (0.842, 0.614, 0.701, 0.662 and 0.681 on a dry matter basis , for d i e t s one, two, three, four and f i v e r e s p e c t i v e l y ) . - 115 -The t o t a l q uantities of non-protein-nitrogen flowing through the duodenum per day was also s l i g h t l y greater with the di e t containing untreated forage compared with the others (7.44, 6.10, 7.10, 6.52 and 6.66g f o r d i e t s one, two, three, four and f i v e i n that order). The t o t a l q uantities of true-protein-nitrogen a r r i v i n g at the duodenum d a i l y was however markedly greater with the die t s containing the formaldehyde treated forage than f o r the d i e t containing the untreated forage (13.76, 24.92, 22.92, 24.62 and 22.50g fo r d i e t s one, two, three, four, and f i v e r e s p e c t i v e l y ) . Some workers reported s i g n i f i c a n t l y lower quantities of non-ammonia-nitrogen flowing through the duodenum d a i l y f o r untreated d i e t s compared with formaldehyde treated d i e t s (Williams and Smith, 1976; Hemsley et a l . , 1970; Faichney and White, 1977a). Sharma and Nicholson (1975a) reported s i g n i f i c a n t l y greater quantities of true-protein-nitrogen a r r i v i n g at the duodenum with a diet containing formaldehyde treated rapeseed meal compared with a di e t containing untreated rapeseed meal. Abomasal and duodenal digesta RNA-N, microbial N, %RNA-N: % t o t a l digesta N, % microbial N: % t o t a l digesta N  The abomasal digesta concentration of r i b o n u c l e i c acid-nitrogen and therefore microbial protein-nitrogen were not affected s i g n i f i c a n t l y (p > 0.05) by treatment (Table 9). The r a t i o s of % r i b o n u c l e i c acid-nitrogen to % t o t a l abomasal digesta nitrogen and % microbial protein-nitrogen to % t o t a l abomasal digesta nitrogen were s i g n i f i c a n t l y (p ^ 0.05) higher f o r d i e t one than - 116 -f o r d i e t s two, four, and f i v e . Ling and Buttery (1975) used the r a t i o of microbial nitrogen to t o t a l digesta nitrogen to measure the contribution of m i c r o b ial protein-N to t o t a l duodenal digesta N. The mean values reported by those workers were 0.58, 1.01, and 0.63 for d i e t s containing f i s h meal, urea and soybean meal r e s p e c t i v e l y . Their values f o r "diets containing f i s h meal and soybean were s i m i l a r to the values obtained f o r the untreated d i e t i n the present experiment. (0.786, 0.480, 0.583, 0.451 and 0.439 f o r d i e t s one, two, three, four and f i v e r e s p e c t i v e l y ) . McAllan and Smith (1974) also reported microbial nitrogen to t o t a l digesta non-ammonia-nitrogen r a t i o s to be 0.60, 0.58, 0.79, and 0.59 for d i e t s containing flaked maize, crushed oats, r o l l e d barley and flaked maize plus urea r e s p e c t i v e l y . For d i e t s containing dairy cubes, the r a t i o was reported to be 0.78. The r e s u l t s of the present experiment i n d i c a t e that the contribution of microbial protein-nitrogen to the nitrogen a r r i v i n g at the abomasum (and subsequently the duodenum) could not be measured by the concentration of microbial protein-nitrogen (microbial-protein-nitrogen %) as t h i s was not affected by treatment but the r a t i o of % m i c r o bial protein nitrogen to % t o t a l nitrogen was affected. With the duodenal digesta, the r a t i o obtained using % microbial protein nitrogen: % t o t a l digesta nitrogen was the same as t o t a l m i c r o b i a l protein nitrogen: t o t a l digesta nitrogen (Table 14). Therefore, i t seems v a l i d to use % microbial protein nitrogen: % t o t a l nitrogen r a t i o to c a l c u l a t e the microbial protein nitrogen contribution to t o t a l N a r r i v i n g at the duodenum. The c a l c u l a t i o n s for the abomasal samples are therefore v a l i d . - 117 -In the experiments of McAllan and Smith (1974) RNA-N was determined and converted to mic r o b i a l nitrogen by d i v i d i n g the RNA-N by 0.075 and mul t i p l y i n g the product by 100. The 0.075 f i g u r e was supposed to be the r a t i o of RNA-N to t o t a l N i n microbial protein (Smith, 1975). Sutton et a l . (1975) also reported a r a t i o of 0.076:1. In the experiment of Ling and Buttery (1975) the r a t i o used was 0.095. Kropp et^ a l . (1977) reported that the percentage of RNA-N i n rumen b a c t e r i a l p rotein was 10% or a r a t i o of 0.10. A l l i s o n (1970) reviewing l i t e r a t u r e on the composition of b a c t e r i a l nitrogen reported that n u c l e i c acids accounted for 14-19% of t o t a l microbial nitrogen with most of i t coming from RNA since DNA accounts f o r only 2.2-4.1%. In experiments where the r a t i o has been determined, usually b a c t e r i a samples are used and the r a t i o may not apply to mixed rumen microbes containing protozoa. It i s therefore suggested here that i n experiments where the r a t i o i s not determined on mixed rumen microbes containing protozoa, and where figures f o r absolute microbial p r o t e i n synthesis are not required but only comparisons between treatments are to be made, i t may be better to use a r a t i o of RNA-N to t o t a l digesta nitrogen. In t h i s experiment t h i s r a t i o , though lower than that of microbial protein-N: t o t a l N, p a r a l l e l l e d i t (Tables 9 and 14). The r a t i o s (RNA-N: t o t a l N or microbial protein N: t o t a l N obtained from duodenal digesta were s i m i l a r to those of the abomasal digesta. Diets one and three had the highest quantities of microbial protein nitrogen a r r i v i n g at the duodenum. While with d i e t one i t can - 118 -be said that there was a great deal of dietary protein converted to microbial protein with some losses of nitrogen, with d i e t three there was also a great deal of conversion of dietary protein to mi c r o b i a l protein but with l i t t l e or no losses of dietary protein. I t i s not clear why the addition of the v o l a t i l e f a t t y acids alone had those e f f e c t s . Abomasal ADF%, c e l l u l o s e % , duodenal ADF%, c e l l u l o s e % , and quantities of 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 differences between the treatments with respect to concentration of c e l l u l o s e and acid detergent f i b r e i n the abomasal digesta (Table 9). Beever et a l . (1976) reported c e l l u l o s e concentrations of 13.62%, and 19.67% on an organic matter basis, f o r untreated and treated d i e t s . Beever et a l . (1977) reported duodenal digesta concentrations of 10.43%, 10.78%, and 11.97% for c o n t r o l , formaldehyde treated s i l a g e and formaldehyde treated dried s i l a g e (on OM b a s i s ) . In the two experiments of (Beever, et a l . , 1976; Beever, et a l . , 1977) the quantities of c e l l u l o s e a r r i v i n g at the duodenum were higher f o r the treated than the untreated materials. In the f i r s t experiment i t could be argued that the concentration was> also higher f o r the treated than the untreated. In the second experiment the l e v e l s were almost i d e n t i c a l . The differences i n the quantities a r r i v i n g at the duodenum were affected i n t h i s case by increased flow of organic matter through the duodenum. In the present t r i a l , i t i s therefore possible that even though the abomasal digesta concentrations 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 quantities escaping d i g e s t i o n i n the four compartments of the stomach may have been greater for the di e t s containing the formaldehyde treated forage than for the d i e t containing the untreated forage. In f a c t , the quantities of a c i d -detergent f i b r e and 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 with the re-entrant cannula were higher for the d i e t s containing the treated forage than the d i e t containing the untreated forage (Table 13). The concentrations of c e l l u l o s e and acid-detergent f i b r e i n the duodenal digesta were not markedly d i f f e r e n t between treatments (Table 13). 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 these f r a c t i o n s i n the four compartments of the stomach were higher for the d i e t containing the untreated forage than for the d i e t s containing the formaldehyde treated forage (Table 15). Duodenal digesta flow The experiment of MacRae and Wilson (1978) demonstrated that there were no differences between i n t a c t sheep and sheep f i t t e d with duodenal re-entrant cannulae with respect to feed intake, dry matter d i g e s t i b i l i t y and nitrogen balance. It i s therefore assumed that the r e s u l t s obtained from the animal f i t t e d with the re-entrant cannula i n t h i s experiment could apply to i n t a c t sheep. - 120 -Oldham and Ling (1977) and Leibholz and Hartman (1972) reported that the duodenal digesta flow measured over twenty four hours without a marker corr e c t i o n , gave v a l i d estimates of duodenal flow through the duodenum. They further claimed that correcting flow f o r 100% recovery of a marker was of doubtful value for reducing v a r i a b i l i t y i n parameters measured. Therefore, i n the present t r i a l no such marker corrections were made. The duodenal digesta flow rate (ml/hr), t o t a l duodenal digesta flow (ml) per 24 hrs, t o t a l duodenal digesta dry matter flow per 24 hr (g) and t o t a l duodenal digesta organic matter flow per 24 hrs (g), (Tables 12 and 13) were greater f o r the d i e t s containing the formaldehyde treated forage than for d i e t one. This was expected as there was greater digestion of dry matter or organic matter i n the four compartments of the stomach when d i e t one was fed compared to the other d i e t s (% D.M. d i g e s t i o n i n the four compartments of the stomach: 32.08, 23.28, 22.23, 24.20 and 24.96 for d i e t s one, two, three,, four and f i v e r e s p e c t i v e l y ; % O.M. digestion i n the four compartments of the stomach: 36.31, 27.12, 26.34, 26.98, and 28.09 for d i e t s one, two, three, four and f i v e r e s p e c t i v e l y ) . The dry matter and organic matter concentrations of the duodenal digesta were not markedly d i f f e r e n t for treatments. Sharma and Nicholson (1975a) reported dry matter d i g e s t i b i l i t i e s of 33.6% for d i e t s containing untreated rapeseed and 22.0% f o r the d i e t s containing formaldehyde treated rapeseed meal i n the four compartments of the stomach. The values of t h i s experiment therefore are s i m i l a r to the values of Sharma and Nicholson (1975a). - 121 -The average hourly digesta flow rates were 647.20, 698.33, 709.135, 692.29 and 680.83 ml for d i e t s one, two, three, four and f i v e r e s p e c t i v e l y . P h i l l i p s and Dyck (1964) reported values of 672, 739, 651 and 622 ml/hr f o r d a i l y dry matter intakes of 900, 850, 800, and 900g re s p e c t i v e l y . Duodenal digesta flow rates of 720 to 773 ml/hr and 861 ml/hr were reported by Van't Klooster et^ al_. (1969) f o r some of t h e i r experiments. Thompson and Lamming (1972) however reported digesta flow rates of 267.65, 299.84 and 290.64 ml/hr for d i e t s containing 30% long, chopped and ground barley straw r e s p e c t i v e l y . Feed intake was about 900g/day (D.M.) and ground maize accounted for 54.7% of the rations. P h i l l i p s and Dyck (1964) reported that there was a d i u r n a l c y c l i c a l pattern of flow. The highest flow occurred at feeding with the lowest occuring 6-12 hrs a f t e r feeding. In t h e i r experiments, the animals were fed once a day and a l l the feed was consumed within a short time a f t e r feeding. This pattern was not observed i n the present experiment. Leibholz and Hartman (1972) also did not observe a consistent d i u r n a l c y c l i c a l pattern of flow. In t h i s t r i a l , with d i e t one there was a r i s e at 12 hrs and s l i g h t f a l l i n flow rate at 18 hrs and almost a l e v e l l i n g of f e f f e c t up to 24 hrs. With d i e t s two, four and f i v e , there were s l i g h t decreases i n the flow rate at 12 hrs and r i s e s at 18 hrs and f a l l s at 24 hrs. With d i e t three, there was a continuous f a l l throughout the twenty-four hour period. The flow rates did not follow feed intake patterns e i t h e r . With a l l the d i e t s , there were decreases i n feed intake at 12 hrs. With d i e t s three and f i v e , the decline i n feed intake continued up to the 18 hr period and from there, there was an increase. - 122 -With di e t two, there was a r i s e i n feed intake from 12 hrs up to twenty four hours. There were increases i n feed intake at 18 hrs and declines up to twenty four hours with d i e t s one and four (Table 11). Perhaps there were no c y c l i c a l patterns of flow i n t h i s experiment because feed intake was spread almost over the twenty-four hour period. Leibholz and Hartman (1972) fed t h e i r animals hourly from automatic continuous feeders. Perhaps one of the reasons f o r the use of very small numbers of animals f o r duodenal flow measurements where automation does not e x i s t , i s the tendiousness of c o l l e c t i n g data over very long periods of time. Some workers have therefore reduced the time period over which measurements are made (Harris and P h i l l i p s o n , 1962). Harris and P h i l l i p s o n (1962) observed that the accumulated recovery i n duodenal contents, c o l l e c t e d for s i x separate 12-hr periods from each of four sheep was only 86-90% of the expected value. When t h e i r observed values were corrected f o r 100% recovery of C^O^, the flow rates approximated expected values. In the present t r i a l the average percentage flow for each 6-hr period of the t o t a l flow for'the f i v e d i e t s approximated 25% (Table 12) and f o r a 12-hr period, approximated 50% (Table 12) although there were considerable v a r i a t i o n s for each d i e t . I t seems therefore that i n the present t r i a l , i f f i v e separate measurements had been taken for each d i e t f or 6-hr or 12-hr period, the average values could have been extrapolated to a 24-hr c o l l e c t i o n period, although no marker c o r r e c t i o n was made. It must be pointed out here however that, since only one animal was used the e f f e c t s of animal v a r i a t i o n s cannot be accounted f o r . - 123 -One other problem with duodenal digesta flow rate measurement i s feed intake. Feed intake i s usually r e s t r i c t e d i n most experiments to l e v e l s below ad l i b i t u m intake. This i s because there i s a d i u r n a l v a r i a t i o n i n feed intake with animals fed 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 rate could occur. Therefore, i f sampling i s done once over a 24-hr period when there i s v a r i a t i o n i n feed intake, figures obtained might not be e a s i l y interpreted. With automation, i f the period of measurement was extended, say to seven days, as i s done with d i g e s t i b i l i t y t r i a l s , then animals could be fed ad l i b i t u m . In the present experiment feed intake was r e s t r i c t e d as measurement was c a r r i e d out once over a 24-hr period for each d i e t . The l e v e l of feeding was fixed at average d a i l y ad l i b i t u m intake for d i e t two at the beginning of the t r i a l . Only one d i e t was used to f i x the intake l e v e l as r e s u l t s from feed intake assays e a r l i e r indicated there were no s i g n i f i c a n t (p > 0.05) differences between the d i e t s (Table 4). I t i s possible however that at the actual time of measurement of flow rate for each d i e t , the l e v e l of feed intake determined was not a c t u a l l y the ad l i b i t u m intake as the animal continued growing throughout the experimental period. Sulphur metabolism Sulphur intake, i f expressed as grams intake per unit of metabolic body s i z e per day, was s i g n i f i c a n t l y higher for d i e t s four and f i v e than for d i e t s one, two, and three (p < 0.05). I t was not s u r p r i s i n g that - 124 -animals fed d i e t s four and f i v e consumed greater amounts of sulphur per day compared to the others (g) as sulphur concentration i n those di e t s was higher and feed intake was not s i g n i f i c a n t l y d i f f e r e n t for a l l the d i e t s (Tables 3 and 4). When sulphur intake was expressed i n absolute terms (g/day) the animals on d i e t s four and f i v e consumed more sulphur per day (p ^ 0.05) than the animals on d i e t s one and two but not animals on d i e t three. I t must be noted that dry matter intake (g/day) of animals on d i e t three tended to be greater than intake of animals on diets four and f i v e . Converting the sulphur intakes to metabolic body s i z e b a s i s , removed, to some extent, the e f f e c t s of the intake, and the s l i g h t l y larger s i z e of animals on d i e t three compared to di e t s four and f i v e . The amounts of sulphur excreted i n the urine per day expressed as grams per unit of metabolic body s i z e were greater for d i e t s four and f i v e than for the other d i e t s (Table 7, p < 0.05). The higher excretion of sulphur i n the urine by animals on the two sulphur-supplemented d i e t s , compared to the others could p a r t l y be due to the higher intakes. The sulphur added was most l i k e l y not u t i l i z e d greatly by the rumen micro-organisms for the synthesis of sulphur-containing amino acids- The low u t i l i z a t i o n of the added sulphur was possibly not due to non-adaptation of the rumen microbes to the supplement. Bird and Moir (1971) postulated that micro-organisms might adapt to sulphur supplementation within twenty four to twenty seven hours while Bird (1972b) claimed that a period of nine days was required. The preliminary period i n the present t r i a l - 125 -before the s t a r t of the metabolism studies was twenty days. Kahlon et a l . (1975a) reported that i n i n v i t r o culture systems, the a v a i l a b i l i t y of sulphur from sodium sulphate for m i c r o b i a l protein synthesis was 55.4%. These workers also observed that a sulphur concentration of 21.5 ug/ml of rumen innoculum was not adequate to meet the needs of rumen microbes i n an i n v i t r o system while concentrations of 86.7 pg/ml and 130 pg/ml were apparently i n h i b i t o r y to microbial protein synthesis. A sulphur concentration of 43.3 ug/ml was reported by them to r e s u l t i n the greatest microbial protein synthesis. Hume and Bi r d (1970) reported that there were no differences i n microbial protein synthesis due to source of sulphur. Johnson et a l . (1970) however reported that losses of sulphur to rumen microbes due to i t s excretion i n the faeces were 20.39%, 21.77%, and 63.32% for methionine, sodium sulphate 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 of the added sulphate i n the present study was not l i k e l y due to i t s low a v a i l a b i l i t y for microbial protein synthesis. The t o t a l amount of sulphur l o s t i n the urine and faeces expressed as a percentage of intake was not s i g n i f i c a n t l y higher (p > 0.05) for die t s four and f i v e than for d i e t one and not higher for die t four than for diet three; (79.67%, 63.70%, 67.57%, 76.89%, 80.28% for die t s one, two, three, four, and f i v e ) . There was better sulphur u t i l i z a t i o n only for diet two compared to die t s four and f i v e (p < 0.05). I t could be inferred from data on the contribution of microbial protein-nitrogen to .total abomasal or duodenal digesta nitrogen (Tables 9 and 14) that the added sulphate did not i n h i b i t m i c r o b i a l protein synthesis to a great extent - 126 -and therefore i t s low u t i l i z a t i o n could not be at t r i b u t e d to that. The added sulphate supplied about 0.15% sulphur which was below the 0.2% l e v e l B i r d (1972b) suggested was the maximum l e v e l for supplementation. Above t h i s l e v e l according to Bird (1972b) hydrogen sulphide t o x i c i t y could occur e s p e c i a l l y i f energy or nitrogen was l i m i t i n g . The hydrogen sulphide r e s u l t s from the reduction of sulphate to sulphide. Sulphate i s converted to sulphide before i t i s incorporated into sulphur containing amino acids by the rumen microbes (Bird, 1971; Saeur et a l . , 1975; Dodgson and Rose, 1966). Sulphur supplementation was not required i n the present t r i a l . Sulphur was most l i k e l y not l i m i t i n g i n the die t s containing formaldehyde treated forage without sulphate supplementation. There were high l e v e l s of extractable sulphate-sulphur i n the grass-legume forage (Table 3).Beaton et a l . (1968) and Martin (1972) reported that sulphur i n sulphur containing amino acids accounts for about ninety percent of the t o t a l sulphur i n plants. Sulphate-sulphur from t h i s estimate, would account for a maximum of ten-percent of t o t a l sulphur i n plants, not taking into account sulphur present i n organic compounds other than amino acids. The l e v e l of extractable sulphate-sulphur i n the grass-legume forage (about 29%) was higher than the fi g u r e of Beaton et a l . (1968) and Martin (1972). Sulphate-sulphur l e v e l s i n plants could increase with f e r t i l i z e r a p p l i c a t i o n (Bray and Hemsley, 1969). Jones and Quagliato (1973) applying f e r t i l i z e r sulphur to some t r o p i c a l forages reported marked increases i n sulphate-sulphur compared to t o t a l sulphur content. - 127 -With formaldehyde treatment even i f a l l the organic sulphur from the grass-legume forage was not a v a i l a b l e , the t o t a l amounts of sulphur from the sulphate-sulphur i n the grass-legume forage, and the cassava and barley would be about 0.069%. The t o t a l amount of nitrogen from the non-protein nitrogen of the grass-legume forage (assuming a l l the protein nitrogen was protected) the nitrogen i n the cassava and barley would be 0.68%. The r a t i o of sulphur to nitrogen would be about 1:10 which i s the same as the optimum reported by Kennedy jst a l . (1975), for low q u a l i t y forage. They took into account the fa c t that recycled nitrogen into the rumen was greater than recycled sulphur, when a r r i v i n g at the above r a t i o . Whanger and Matrone (1966) reported that with sulphur deficiency i n the rumen, there was an accumulation of l a c t i c a c i d . The accumulation of l a c t i c acid was due to the non-functioning of the a c r y l a t e pathway for conversion of l a c t i c a c i d to propionic acid (Whanger and Matrone, 1967). These workers, previously reported reduced l e v e l s of propionic acid, butyric and higher f a t t y acids i n rumens of animals fed sulphur-deficient d i e t s compared to animals fed d i e t s containing adequate l e v e l s of sulphur (Whanger and Matrone, 1965). In the present t r i a l propionic and butyric acid l e v e l s were not lower f o r the d i e t s containing the formaldehyde treated forage without sulphur supplementation than the d i e t s containing the formaldehyde treated forage with sulphur supplementation. The percentages of sulphur intake excreted i n the urine were higher for d i e t s four and f i v e than for d i e t s two and three (p ^0.05) - 128 -but not for d i e t one (p > 0.05). The percentage of sulphur intake excreted i n the urine was higher for d i e t one than was for d i e t s two and three (p < 0.05). The reasons for the higher;percentages of excretion of sulphur i n the urine for di e t s four and f i v e compared to die t s two and three are s i m i l a r to those given already above. There was a greater percentage of sulphur excreted i n urine with d i e t one compared to d i e t s two and three although the amounts excreted per unit of metabolic body s i z e were s i m i l a r because when calculated on a percentage basis, differences i n intake are not taken into account. Sulphur balance, expressed either as grams per unit of metabolic body s i z e or i n absolute terms was better for di e t s two, three and four than f o r d i e t one (p < 0.-05) . Sulphur retention was c l o s e l y linked with nitrogen retention. Since animals on di e t s two and three had greater ( s i g n i f i c a n t ) retention of nitrogen than animals on d i e t one, i t was not s u r p r i s i n g that sulphur retention was better on these two d i e t s than for d i e t one. With d i e t four the animals retained more nitrogen (approaching 5% s i g n i f i c a n c e ) than animals on d i e t one. The sulphur-nitrogen r a t i o also tended to be better, though not s i g n i i f i c a n t , f o r animals on d i e t four than animals on d i e t one. The sulphur to nitrogen r a t i o s were not affected by treatments (10.13, 9.78, 12.26, 9.73, and 10.36 for d i e t s one, two, three, four and f i v e r e s p e c t i v e l y ) . Bird (1972a) reported a somewhat higher r a t i o of about 13.5+0.58. It i s not clear why the r a t i o s were lower i n t h i s experiment except that perhaps sulphur u t i l i z a t i o n might have been better - 129 -than the experiments of Bird (1972a). It must also be noted that there i s considerable v a r i a t i o n i n the reported r a t i o s as Bird (1973) reported the r a t i o i n sheep tissues to be 15 and Kahlon et^ al. (1975b) reported r a t i o s of 1:8.14 to 1:27.16 of retained sulphur to retained nitrogen i n sheep. Nitrogen Metabolism The d a i l y nitrogen intake, expressed i n absolute terms (29.81, 28.64, 33.44, 27.80 and 29.51g for d i e t s one, two, three, four and f i v e respectively) or expressed as intake/day/unit of metabolic body s i z e (2.07, 2.18, 2.38, 2.08 and 2.18g,for d i e t s one, two, three, four and f i v e respectively) were not affected by treatments (p } 0.05, Table 6). This i s perhaps due to the f a c t that dry matter intake per unit of metabolic body siz e (Table 4), the concentration of nitrogen i n the diets (Table 3), and the metabolic body sizes of the animals (Table 4) were not s i g n i f i c a n t l y d i f f e r e n t (p > 0.05). The nitrogen balance expressed as g/day (4.49, 7.43, 10.19, 6.99, and 6.86g for d i e t s one, two, three, four and f i v e respectively) was only greater for d i e t three than for d i e t one (p 0.05). Nitrogen balance expressed as grams per day per unit of metabolic body s i z e (0.313, 0.567, 0.726, 0.522, and 0.508g for d i e t s one, two, three, four and f i v e respectively) was however greater for d i e t s two and three than for d i e t one (p 0.05). There was an improvement i n nitrogen balance for d i e t two when expressed as retained nitrogen per unit of metabolic body s i z e perhaps because of the s l i g h t l y larger weight of animals on - 130 -d i e t one than on d i e t two (Table 4). Animals on d i e t three were however about the same weight as animals on. d i e t one and retained a s i g n i f i c a n t l y greater amount of nitrogen i n each case and hence s i z e per se was not a factor contributing to the greater retention of nitrogen by animals on diet three than animals on d i e t one. The greater retention of nitrogen of animals on d i e t s two and three was due mainly to the greater urinary losses of nitrogen by animals on d i e t one than the animals on d i e t s two and three. M i c r o b i a l degradation of protein i n animals fed d i e t one was greater than animals on d i e t s two and three as ammonia concentration i n the rumens of animals fed d i e t one was greater than the ammonia concentration i n the rumens of animals fed d i e t s two and three (Table 8). The synthesis of microbial protein from the dietary sources was also greater for d i e t one than for d i e t two (Tables 9 and 14). With the sheep f i t t e d with re-entrant cannula there were losses of nitrogen i n the four compartments of the stomach for d i e t one while there were net gains for d i e t s two and three (Table 15). Ammonia could be l o s t through the ruminal w a l l . When such ammonia a r r i v e s i n the l i v e r , i t may be converted to urea which may be l o s t i n the urine as described by Houpt (1970). With d i e t three, although nitrogen d i g e s t i b i l i t y was not decreased s i g n i f i c a n t l y (p 0.05) compared to d i e t one, there was no net loss of nitrogen i n the four compartments of the stomach. This 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 nitrogen by the rumen microbes i n the rumens of animals fed d i e t three compared to animals fed d i e t one. In f a c t , microbial protein N contribution to duodenal digesta N was not s i g n i f i c a n t l y (p y. 0.05) lower for d i e t three than for d i e t one (Table 9). - 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 is not clear although in the case of animals on diet four, i t was mediated partly through reduced d i g e s t i b i l i t y of nitrogen. In the case of diet five, there was an abnormally high loss of nitrogen in 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 in calves by 20%, feed intake by 9% and feed e f f i c i by 12%. Brown and Arlyne (1970) reported that sulphate added to rat diets improved performances only up to 0.10% level of supplementation. Above that level, performance was decreased although not significantly. Measuring nitrogen retention as a percentage of digested, all.the diets containing the formaldehyde treated forage were significantly (p ^0.05) superior to the diet containing the untreated forage. This was because a greater amount of the nitrogen digested was lost in the urine with diet one compared to the others. Similarly, using nitrogen retained as a percentage of intake as an index of nitrogen retention, diets two, three, four and five were better than diet one (p ^ 0.05). Diet three was also superior to diet five (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 closely nitrogen balance expressed as grams per day per unit ;of metabolic body size. This was because those measures do not take into account variations in nitrogen intake, and nitrogen d i g e s t i b i l i t y with the animals on the different diets. They - 132 -also did not take into account the va r i a t i o n s i n the sizes of animals used. Amos et_ al. (1974) and Driedger and H a t f i e l d (1972) also reported that nitrogen retained as a percentage of intake did not follow nitrogen balance with d i f f e r e n t d i e t s . Nitrogen losses i n the urine (g/Wkg^'7~Vday or percentage of digested) did not follow c l o s e l y the nitrogen balance f i g u r e s . This was because the measured values did not take into account differences i n intake and i n d i g e s t i b i l i t y . The differences i n nitrogen excretion were not due to differences i n output of urine. D a i l y excretion of urine (ml/Wkg^ * 7~*) was not affected by treatment (p>0.05, Table 4). Growth rate The growth rates of the animals during the f i r s t seventeen days (pre-metabolism study period) were not s i g n i f i c a n t l y (p > 0.05) affected by dietary treatment (Table 4). The growth rate figures were 154.76, 170.77, 170.77, 160.10 and 149.42g/day for d i e t s one, two, three, four and f i v e r e s p e c t i v e l y . Rattray and Joyce (1970) reported a p o s i t i v e response of nitrogen retention but not wool growth or growth rate with formaldehyde treatment of t h e i r d i e t s . Their experimental period was f i v e weeks. It i s possible that i n the present experiment, as i n the t r i a l of Rattray and Joyce (1970), the period of the experiment was too short for a growth rate response to be demonstrated. Ames and Brink (1977) however measured growth rates of sheep at d i f f e r e n t environmental temperatures - 133 -for only twelve days and differences i n responses could be assessed. Driedger and H a t f i e l d (1972) also used a sixteen-day preliminary period and a six-day metabolism study period. They observed differences i n responses to nitrogen retention during the metabolism study period and also growth rate during the sixteen-day preliminary period with tannin treatment of soybean meal. The growth rates of t h e i r animals for the sixteen-day period were 277g/day f or the d i e t containing the tannin treated soybean meal and 177g/day for the d i e t containing the untreated soybean meal. The growth rate f i g u r e s reported i n the present t r i a l were lower than those reported by T a i t (1972) f or male lambs. His figures were 237g/day, 239g/day and 233g/day for animals fed 100% dried grass, 50% barley plus 50% dried grass and 100% barley rations r e s p e c t i v e l y . The animals used by T a i t (1972) weighed about (average) 19.2kg at the s t a r t of the experiment and were fed to a t t a i n the weight of about 45kg (average). In the present experiment the animals ranged i n bodyweight of 29kg to 36kg at the s t a r t of the experiment. The greater growth rates of the animals used by T a i t (1972) compared to those of the animals used i n the present t r i a l could most l i k e l y be accounted 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 at the s t a r t of the experiments. Adeleye (1972) using animals with average bodyweight of 30.65kg at the s t a r t of the experiment reported d a i l y weight gain of llO g , 70g, 90g, 130g, and 70g when d i e t s containing soybean meal, urea, biu r e t , poultry droppings and poultry l i t t e r were fed. A l l these t r i a l s ( T a i t , 1972; Adeleye, 1972; and present t r i a l ) were c a r r i e d out on the same farm. The same breed of sheep (Dorset) was used. The other factor - 134 -which could cause v a r i a t i o n s i n the growth rates of the animals i n a the experiments apart from the stage of growth at which measurements were c a r r i e d out , was type of feed and l e v e l of intake. - 135 -SUMMARY AND CONCLUSIONS Di f f e r e n t l e v e l s of formaldehyde (0.0%, 0.8%, 1.0% and 1.2% on,an a i r dry basis) were applied to a grass-clover forage to determine the optimum l e v e l for protection of the protein. In v i t r o nitrogen d i g e s t i b i l i t y was reduced s i g n i f i c a n t l y (p < 0.05) as l e v e l of formaldehyde a p p l i c a t i o n was increased except between 1.0% and 1.2%, at the microbial stage of incubation (31.88%, 15.72%, 6.87%, and 5.69% for 0.0%, 0.8%, 1.0% and 1.2% l e v e l s of formaldehyde treatment). The ammonia nitrogen production, at the microbial stage of incubation, was also reduced s i g n i f i c a n t l y (p <0.05) as l e v e l of formaldehyde a p p l i c a t i o n increased except between 0.8%, and 1.0% (228.79 ppm, 78.58 ppm, 65.27 ppm, and 30.04 ppm for 0.0%, 0.8%, 1.0% and 1.2% l e v e l s of formaldehyde treatment). Nitrogen d i g e s t i b i l i t y f o r the combined microbial and acid-pepsin stages of incubation, was reduced s i g n i f i c a n t l y (p<,0.05) only at the 1.2% l e v e l of formaldehyde a p p l i c a t i o n (80.95%, 79.76%, 75.85%, and 71.01% f o r 0.0%, 0.8%, 1.0% and 1.2% l e v e l s of formaldehyde a p p l i c a t i o n ) . The optimum l e v e l chosen was 1.0% since nitrogen 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) reduced at the microbial stage but not s i g n i f i c a n t l y (p y 0.05) reduced at the combined microbial and acid-pepsin stages, compared to the untreated. Ram lambs ranging i n body weights of 29kg to 36kg were then used i n i n vivo studies of nitrogen and carbohydrate u t i l i z a t i o n with treatment of the grass-clover forage at 1% l e v e l of formaldehyde. The e f f e c t s of supplementation with i s o v a l e r i c and i s o b u t y r i c acids - 136 -and/or sulphur were also studied. The die t s (14% CP on D.M.' basis) contained 50% grass-clover forage, 38% cassava, 11% barley and 1% sheep mineral premix on dry matter basis. Sodium sulphate was added at 0.67% replacing an equal portion of the cassava i n the d i e t s supplemented with sulphur. Diet one contained the untreated forage while the others contained the formaldehyde treated forage. Diets three and f i v e were supplemented with i s o v a l e r i c a c i d (3.0g/kg diet) and i s o b u t y r i c acid (2.3g/kg diet) and d i e t s four and f i v e were supplemented with sulphur. The v o l a t i l e f a t t y acids were sprayed onto the d i e t s j u s t before feeding. Dry matter intake (g/Wkg^"7"Vday), 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 dry matter and organic matter were not s i g n i f i c a n t l y (p y 0.05) affected by dietary treatments. Formaldehyde treatment of the forage s i g n i f i c a n t l y (p 0.05) increased 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 acid-detergent f i b r e and c e l l u l o s e . (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% for d i e t s one, two, three, four 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% for d i e t s one, two, three, four and f i v e r e s p ectively. Greater amounts of the acid-detergent f i b r e and c e l l u l o s e were digested i n the hindgut with formaldehyde treatment of the forage. Rumen pH, dry matter 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 acids, propionic, b u t y r i c and a c e t i c acids were not affected s i g n i f i c a n t l y ( p ^ 0.05) by the dietary treatments. There were however, s i g n i f i c a n t l y (p < 0.05) increased 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 acids i n the rumens of animals fed d i e t three compared to those fed diet s two and four. 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 acid l e v e l s - 137 -tended to decrease with the diets containing the formaldehyde treated forage without v o l a t i l e f a t t y a c i d supplementation compared to the d i e t containing the untreated material, most l i k e l y because of greater degradation of proteins i n the rumen with the l a t t e r :than with the former ( i s o v a l e r i c : 2.53, 0.60, 2.58, 0.39 and 2.18% for d i e t s one, two, three, four and f i v e respectively; i s o b u t y r i c : 1.54, 0.71, 2.11, 0.66 and 1.73% for d i e t s .one, two, three, four, and f i v e r e s p e c t i v e l y ) . Formaldehyde treatment of the grass-clover forage resulted i n s i g n i f i c a n t (p < 0.05) increases i n n - v a l e r i c acid l e v e l s i n the rumen and t h i s could be due to reduced microbial growth due to l i m i t a t i o n of nitrogen. (1.82, 3.30, 3.19, 3.34 and 3.15 for d i e t s one, two, three, four and f i v e r e s p e c t i v e l y ) . Rumen ammonia-nitrogen l e v e l s were s i g n i f i c a n t l y (p^0.05) higher for animals fed d i e t s containing the untreated forage than for animals on the other d i e t s (21.14, 14.36, 14.30, 12.90 and 13.54 ppm for d i e t s one, two, three, four and f i v e r e s p e c t i v e l y ) . The higher l e v e l s of rumen ammonia-nitrogen i n the case of animals fed the die t containing the untreated forage compared to the others was most l i k e l y due to a greater rate of protein 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 of the former compared to the l a t t e r . Abomasal pH, abomasal digesta concentrations of acid-detergent f i b r e , c e l l u l o s e , t o t a l nitrogen, r i b o n u c l e i c acid nitrogen, and microbial protein nitrogen were not affected by treatment. The r a t i o s of % RNA-N: % t o t a l abomasal digesta nitrogen (0.060, 0.036, 0.044, 0.034 and 0.033 for d i e t s one, two, three, four and f i v e r espectively) and % microbial nitrogen: % t o t a l abomasal digesta nitrogen (0.786, 0.480, 0.583, 0.451 and 0.439 for d i e t s one, two, three, four and f i v e - 138 -respectively) were reduced s i g n i f i c a n t l y (p <_ 0.05) except f o r d i e t three by the formaldehyde treatment of the forage. The abomasal digesta concentration of non-protein-nitrogen was s i g n i f i c a n t l y (p<0.05) higher f o r the d i e t containing the untreated forage compared to the others. The higher r a t i o of microbial-nitrogen: t o t a l abomasal digesta nitrogen (except f o r d i e t three) and the higher l e v e l s of non-protein-nitrogen i n the abomasal digesta for the d i e t containing the untreated forage compared to the others indicated that there was a greater degree of degradation of dietary protein by rumen microbes. For d i e t three, there might have been a greater degree of degradation of dietary protein as for die t one but i n the case of di e t three the conversion of the degraded protein to microbial protein was more e f f i c i e n t . Sulphur balance (g/Wkg^*7"Vday) was s i g n i f i c a n t l y (p <_ 0.05) improved by the formaldehyde treatment of the forage except f o r the d i e t supplemented with both sulphur and VFAS (diet f i v e ) . The values were 0.032, 0.061, 0.061, 0.056 and 0.050g for die t s one, two, three, four and f i v e r e s p e c t i v e l y . Sulphur intake per unit of metabolic body s i z e per day was s i g n i f i c a n t l y (p < 0.05) higher f o r the d i e t s supplemented with sulphur than f o r the other d i e t s . The values were 0.157, 0.168, 0.188, 0.247 and 0.251g for d i e t s one, two, three, four and f i v e r e s p e c t i v e l y . Sulphur retained to nitrogen retained r a t i o s were however not affected s i g n i f i c a n t l y (p> 0.05) by dietary treatments. The amount of sulphur excreted i n urine per day (g/Wkg^'7^/day) was s i g n i f i c a n t l y (p < 0.05) higher f o r the sulphur supplemented d i e t s than f o r the rest (0.050, 0.024, 0.034, 0.102, and 0.103g for die t s one, two, three, four and f i v e r e s p e c t i v e l y ) . - 139 -Sulphur excreted i n urine as a percentage of intake was s i g n i f i c a n t l y (p ^0.05) higher f o r di e t s one, four and f i v e than for die t s two and three. The values were 31.38, 14.17, 17.41, 41.43 and 42.23% for d i e t s one, two, three, four and f i v e r e s p e c t i v e l y . The loss of sulphur i n urine and faeces as a percentage of intake was s i g n i f i c a n t l y (p 4. 0.05) higher for d i e t s one, four and f i v e than f or d i e t two and for die t s one and f i v e than f o r d i e t three. The values were 79.67, 63.69, 67.57, 76.89 and 80.28% for diets one, two, three, four and f i v e r e s p e c t i v e l y . Nitrogen intake (g/day or g/Wkg^"7"Vday) was not s i g n i f i c a n t l y (p> 0.05) affected by dietary treatments. 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 of nitrogen was s i g n i f i c a n t l y (p < 0.05) higher for d i e t one than for di e t s two, four, and f i v e and for di e t three than f or d i e t four. The values were 54.13, 47.06, 51.25, 44.90, and 47.24% for d i e t s one, two,, three, four and f i v e r e s p e c t i v e l y . Nitrogen balance (gN/Wkg^'7"Vday) was s i g n i f i c a n t l y (p < 0.05) better f o r d i e t s two and three than for d i e t one. The nitrogen balance values were (g/Wkg , / J / d a y ) 0.459, 0.802,0.843, 0.752 and 0.672g f o r di e t s one, two, three, four and f i v e r e s p e c t i v e l y . Formaldehyde treatment of the forage s i g n i f i c a n t l y (p < 0.05) reduced the amount of nitrogen excreted i n the urine per day per unit of metabolic body s i z e (0.805, 0.461, 0.467, 0.417 and 0.518g for d i e t s one, two, three, four and f i v e r e s p e c t i v e l y ) . Nitrogen excreted i n urine as a percentage of digested and nitrogen excreted i n urine as a percentage of intake were also reduced s i g n i f i c a n t l y (p < 0.05) by formaldehyde treatment of the forage. The values f o r nitrogen excreted i n urine as a percentage of digested were 72.10, 44.65, 39.11, 44.25 and 50.39% f or diet s one, two, three, four and f i v e r e s p e c t i v e l y . The values f o r - 140 -nitrogen excreted i n the urine as a percentage of intake were 39.07, 21.07, 19.97, 19.86 and 23.94% for d i e t s one, two, three, four, and f i v e r e s p e c t i v e l y . Nitrogen retained as a percentage of digested (27.90, 55.39, 58.93, 55.75, and 49.61% for d i e t s one, two, three, four and f i v e respectively) and nitrogen retained as a percentage of intake (15.06, 26.00, 30.24, 25.04 and 23.30% f o r d i e t s one, two, three, four and f i v e i n that order) were s i g n i f i c a n t l y (p < 0.05) greater for the d i e t s containing the formaldehyde treated.forage compared to the diet containing the untreated forage. Nitrogen u t i l i z a t i o n was improved for a l l the d i e t s containing the formaldehyde treated forage as a r e s u l t of lower urinary nitrogen losses. The addition of sulphur to the d i e t s containing the formaldehyde treated forage tended to o f f s e t the b e n e f i c i a l e f f e c t s of the formaldehyde treatment. The addition of the v o l a t i l e f a t t y acids to the d i e t s containing the formaldehyde treated forage did not further enhance nitrogen u t i l i z a t i o n . Duodenal flow was measured over a twenty-four-hour period using a sheep f i t t e d with a re-entrant cannula. The quantities of t o t a l digesta, dry matter, organic matter, nitrogen, acid-detergent f i b r e , and c e l l u l o s e flowing through the duodenum d a i l y were markedly higher for the d i e t s containing the formaldehyde treated forage than for the d i e t containing the untreated forage. The d a i l y amounts of microbial protein and non-protein-nitrogen a r r i v i n g at the duodenum were markedly higher f o r the d i e t containing the untreated forage than for the r e s t . Growth rate of the animals during the seventeen day pre-metabolism period and t h e i r metabolic body sizes at the beginning of the metabolism studies were not 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 f or dietary treatments. The lack of a response i n growth rate may have been due to the r e l a t i v e l y - 141 -short period of measurement. Formaldehyde treatment of the forage portion of the di 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 nitrogen economy of the animal. The treatment of the forage portion of the di e t with formaldehyde resulted i n changes i n the s i t e s of digestion of both protein and f i b r e . The digestion of these f r a c t i o n s was depressed i n the rumen but increased i n the lower sections of the dige s t i v e t r a c t . This study indicated that supplementation of the d i e t s containing the formaldehyde treated grass-legume forage with either sulphur and/or branched chain v o l a t i l e f a t t y acids was not necessary as no b e n e f i c i a l e f f e c t s were observed. There was an i n d i c a t i o n from t h i s study that formaldehyde treatment of the forage portion of the di e t might r e s u l t i n improved e f f i c i e n c y of u t i l i z a t i o n of dietary protein by ruminants. 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Estimation of the extent of heat damage i n a l f a l f a haylage by laboratory measurement. J . Anim. S c i . 42:766-774. Yu, Yu and D.M. Veira. 1977. E f f e c t of a r t i f i c i a l heating of a l f a l f a haylage on chemical composition and sheep performance. J. Anim. S c i . 44:1112-1118. - 162 -APPENDIX TABLES I - XXX Table I. ANOVA percent N d i g e s t i b i l i t y , 1st stage of i n v i t r o digestion of rye-grass-clover forage with the d i f f e r e n t l e v e l s of formaldehyde treatments. Source SS df Variance Fc a l T o t a l Treatment Error 1350.47 1314.8181 35.6519 11 3 8 438.2727 4.46 98.27** ON LO ** p < 0.01 S.E. + 1.219 Table I I . ANOVA percent N d i g e s t i b i l i t y 2nd stage of i n v i t r o digestion of rye-grass-clover forage with the d i f f e r e n t l e v e l s of formaldehyde treatment. Source SS df Variance F c a l Total Treatment Error 228.83062 181.22628 47.60434 11 3 8 60.40876 5.9505425 10.1518** ** p < 0.01 S.E. + 1.408 Table I I I . ANOVA i n v i t r o ammonia-nitrogen production (ppm) per gram dry matter of rye-grass-clover forage with the d i f f e r e n t l e v e l s of formaldehyde treatment. Source SS df Variance F c a l T o t a l 145794.2618 23 i Treatment 138869.9399 3 46289.98 133.70** M Error 6924.3219 20 346.22 i ** p < 0.01 S.E. + 7.596 Table IV. ANOVA metabolic body sizes of animals at the beginning of the metabolism studies (kg). Source SS df Variance F c a l Total Treatment Block Error 26.157944 5.108064 10.493344 10.556536 24 4 4 16 1.277016 2.623336 0.6597835 1.93 3.98* p < 0.05 S.E. + 0.3632584478 Table V. 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 of nitrogen (%) . Source SS df Variance Fc a l T o t a l Treatment Block Error 465.15122 275.4159 79.90234 109.83298 24 4 4 16 68.853975 19.975585 6.86456125 10.03** 2.91 ** p < 0.01 S.E. + 1.171713382 Table VI. 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 of acid-detergent f i b r e (%). Source SS df Variance F c a l T o t a l Treatment Block Error 169.55554 70.09646 35.91918 63.5399 24 4 4 16 17.524115 8.979795 3.97124375 4.41* 2.26 p C 0.05 S.E. + 0.8912063454 Table VII. 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 of c e l l u l o s e (%). Source SS df Variance F c a l T o t a l Treatment Block Error 312.12734 149.96314 19.5969 142.5673 24 4 4 16 37.490785 4.899225 8.91045625 4.21* 0.55 p < 0.05 S.E. + 1.334949905 Table VIII. ANOVA nitrogen excreted body s i z e per day (g). i n urine per unit metabolic Source SS df Variance F c a l T o t a l 0.71007224 24 Treatment 0.48606944 4 0.12151736 18.94** Block 0.12136904 4 0.03034226 4.73* Error 0.10263376 16 0.00641461 ** p < 0.01 * p < 0.05 S.E. for treatment and block means + 0.0358179005 Table IX. ANOVA % nitrogen excreted i n urine over intake. Source SS df Variance F c a l Total 2712.62046 24 Treatment 1329.91614 4 332.479035 4.88** i Block 293.50034 4 73.375085 1.08 H i — 1 Error 1089.20398 16 68.07524875 • ** p < 0.01 S.E. + 3.689857687 Table X. ANOVA % nitrogen excreted i n urine over digested. Source SS df Variance F c a l T o t a l Treatment Block Error 4826.9737 3343.26462 547.19962 936.50946 24 4 4 16 835.816155 136.799905 58.53184125 14.28** 2.34 ** p < 0.01 S.E. + 3.421457036 Table XI. ANOVA % nitrogen retained over intake. Source SS df Variance F c a l T o t a l Treatment Block Error 885.5855 622.11626 87.91154 175.5577 24 4 4 16 155.529065 21.977885 10.97235625 14.17** 2.00 p < 0.01 S.E. + 1.481374784 Table XII. ANOVA % nitrogen retained over digested. Source SS df Variance F c a l T o t a l Treatment Block Error 4354.29162 2945.51022 480.9983 927.7831 24 4 4 16 736.377555 120.249575 57.98644375 12.70** 2.07 ** p < 0.01 S.E. + 3.405479225 Table XIII. ANOVA nitrogen balance g/day. Source SS df Tot a l 141.9019326 24 Treatment 82.6064218 4 Block 6.731593 4 Error 59.2955108 16 Variance F c a l 20.65160545 5.57** ( 1.68289825 0.45 ^ 3.70596425 ' ** p < 0.01 S.E. + 0.8609255775 Table XIV. ANOVA nitrogen balance body s i z e per day. (g) per unit of metabolic Source SS df Variance F c a l Total 0.72847696 24 Treatment 0.43781416 4 0.10945354 8.20** i Block 0.07698256 4 0.01924564 1.44 1 Error 0.21368024 16 0.013355015 ** p < 0.01 S.E. + 0.0516817472 Table XV. ANOVA sulphur intake per day (g) . Variance F c a l 1.584074 0.123694 0.1693015 9.36** 0.73 Source SS df Total Treatment Block Error 9.539896 6.336296 0.494776 2.708824 24 4 4 16 ** p < 0.01 S.E. + 0.1840116844 Table XVI. ANOVA sulphur intake per unit of metabolic body s i z e per day (g). Source SS df Variance F c a l Total Treatment Block Error 0.05090784 0.03885544 0.00239544 0.00965696 24 4 4 16 0.00971386 0.00059886 0.00060356 16.09** 0.99 ** p <. 0.01 S.E. + 0.0109869012 Table XVII. ANOVA sulphur excreted i n urine per day per unit of metabolic body siz e (g). Source SS df Variance F c a l T o t a l Treatment Block Error 0.03454384 0.02941184 0.00086824 0.00426376 24 4 4 16 0.00735296 0.00021706 0.000266485 27.59** 0.81 p <. 0.01 S.E. + 0.0073004794 Table XVIII. ANOVA % sulphur excreted i n urine over intake. Source SS df Variance F c a l T o t a l Treatment Block Error 4392.9128 3443.66224 335.63716 613.6134 24 4 4 16 860.91556 83.90929 38.3508375 22.45** 2.19 p < 0.01 S.E. + 2.769506725 Table XIX. ANOVA t o t a l amount of sulphur l o s t i n urine and faeces as a percentage of intake. Source SS df- Variance F c a l Total Treatment Block Error 1884.5519 1134.6117 299.1533 450.7869 24 4 4 16 283.652925 74.788325 28.17418125 10.07** 2.65 i oo m i < 0.01 S.E. + 2.373781003 Table XX. ANOVA sulphur balance per day (g). Source SS df Variance F c a l T o t a l Treatment Block Error 0.99126424 0.46094104 0.20486304 0.32546016 24 4 4 16 0.11523526 0.05121576 0.02034126 5.67** p < 0.01 S.E. + 0.0637828503 Table XXI. ANOVA sulphur balance per day per unit of metabolic body s i z e (g). Source SS df Variance F c a l Total Treatment Block Error 0.005612 0.0028912 0.00102 0.0017008 24 4 4 16 0.0007228 0.000255 0.0001063 6.80** 2.40 p < 0.01 S.E. + 0.0046108567 Table XXII. ANOVA molar proportion of acetic acid i n rumen f l u i d (%). Source SS df Variance Fcal T o t a l Treatment Block Error 521.26974 106.9635 217.39594 196.9103 24 4 4 16 26.740875 54.348985 12.30689375 2.17 4.42* p < 0.05 S.E. + 1.568878182 Table XXIII. ANOVA molar proportion of propionic acid i n rumen f l u i d (%). Source SS df Variance F c a l Total Treatment Block Error 891.38178 74.0105 454.64618 362.7251 24 4 4 16 18.502625 113.661545 22.67031875 0.82 5.01** p < 0.01 S.E. + 2.12933411 Table XXIV. ANOVA molar proportion of is o b u t y r i c acid i n rumen f l u i d (%). Source SS df Variance F c a l Total Treatment Block Error 13.77958095 6.18916095 .1.814484283 5.775935717 24 4 4 16 1.547290238 0.4536210708 0.3609959823 4.29* 1.26 * p < 0.05 S.E. + 1.163467023 Table XXV. ANOVA molar proportions of i s o v a l e r i c acid proportion i n rumen f l u i d (%). Source SS df Variance F c a l T o t a l Treatment Block Error 40.99798095 17.13082595 8.272439283 15.59471572 24 4 4 16 4.282706488 2.068108921 0.9746697323 4.39* 2.12 * p < 0.05 S.E. + 0.4415132461 Table XXVI. ANOVA molar proportion of v a l e r i c acid i n rumen f l u i d (%). Source SS df Variance F c a l Total Treatment Block Error 15.930784 8.315384 1.445024 6.170376 24 4 4 16 2.078846 0.361256 0.3856485 5.39** 0.94 00 00 p < 0.01 S.E. + 0.2777223434 Table XXVII. ANOVA rumen ammonia-nitrogen l e v e l s (ppm). Source SS df Variance F c a l T o t a l Treatment Block Error 537.0824 224.1664 122.8504 190.0656 24 4 4 16 56.0416 30.7126 11.8791 4.7176* 2.585 p < 0.05 S.E. + 1.541369521 Table XXVIII. ANOVA abomasal digesta non-protein-nitrogen concentration (%). Source SS df Variance F c a l T o t a l Treatment Block Error 0.33877336 0.17597416 0.01710296 0.14569624 24 4 4 16 0.04399354 0.00427574 0.009106015 4.83** 0.47 ** p < 0.01 S.E. + 0.042675555 Table XXIX. ANOVA % RNA-N: % t o t a l N i n abomasal digesta. Source SS df Variance F c a l T o t a l Treatment Block Erro r 0.005168 0.0025188 0.0003084 0.0023408 24 4 4 16 0.0006297 0.0000771 0.0001463 4.30* 0.53 i h-1 h-1 I * p < 0.05 S.E. + 0.0054092513 Table XXX. ANOVA % microbial-protein-nitrogen: % t o t a l digesta nitrogen for abomasal digesta ( x : l ) . Source SS df Total 0.850612 24 Treatment 0.4185084 4 Block 0.0633512 4 Error 0.3687524 16 Variance F c a l 0.1046271 4.53* 0.0158378 0.69 ^ 0.023047025 * p " < 0.05 S.E. + 0.067892599 PUBLICATIONS TUAH, A.K. 1971. The preparation of s i l a g e i n small-sized s i l o s i n Ghana. Proc. Ghana Anim. S c i . Symp. 4:68-71. TUAH, A.K. and Tetteh J . Adinku. 1972. The e f f e c t of feeding molasses-urea supplement on the performance of confined 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 various forages f o r s i l a g e i n three types of s i l o s i n Ghana. Ghana J. S c i . 13(2): 203-209. Abstracted i n : Herbage Abstracts (1975) 45:73; Microb. Abstracts (1975) 10:28. Also paper read at West A f r i c a n Science A s s o c i a t i o n b i e n n i a l meeting (1972) held at U n i v e r s i t y of Ghana, Legon, Accra. TUAH, A.K. and 0. Okyere. 1974. Preliminary studies on the ensilage of some species of t r o p i c a l grasses i n the Ashanti forest b e l t of Ghana. Ghana J . . Agric. S c i . 7:81-87. Abstracted i n : Herbage Abstracts (1975) 45:329; Abst. Trop. Agric. 1975 1(5):53. Also paper read at Ghana Science Association meeting (1973) held at U.S.T., Kumasi, Ghana. TUAH, A.K. 1974. Prospects 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 Ashanti Region of Ghana. Review of the s i t u a t i o n . Ghana JT Agric. S c i . 7:157-164. Abstracted i n : Herbage Abstracts (1976) 46:59. TUAH, A.K. and K. Boa-Amponsem. 1974. Rice bran ( r i c e - m i l l feed) i n the d i e t s of growing-finishing Large White pigs. Ghana.Anim. S c i . Symp. 6:8pages. TUAH, A.K. RvM. T a i t . 1977. Nitrogen metabolism i n sheep fed formaldehyde-treated grass-clover forage supplemented with branched-chain f a t t y acids and/or sulphur. Can.J.Anim.Sci. 57:844 (Abstract). Paper read at CSAS (Western Branch) meeting held at Winnipeg, Manitoba. June 16-18, 1977. 

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