Open Collections

UBC Theses and Dissertations

UBC Theses Logo

UBC Theses and Dissertations

Degradability and utilization of dietary nitrogen sources commonly fed to dairy cows Snider, Jeffrey Allan 1983

Your browser doesn't seem to have a PDF viewer, please download the PDF to view this item.

Item Metadata

Download

Media
831-UBC_1983_A6_7 S65.pdf [ 3.73MB ]
Metadata
JSON: 831-1.0095817.json
JSON-LD: 831-1.0095817-ld.json
RDF/XML (Pretty): 831-1.0095817-rdf.xml
RDF/JSON: 831-1.0095817-rdf.json
Turtle: 831-1.0095817-turtle.txt
N-Triples: 831-1.0095817-rdf-ntriples.txt
Original Record: 831-1.0095817-source.json
Full Text
831-1.0095817-fulltext.txt
Citation
831-1.0095817.ris

Full Text

DEGRADABILITY AND UTILIZATION OF DIETARY NITROGEN SOURCES COMMONLY FED TO DAIRY COWS by JEFFREY ALLAN SNIDER B.Sc. (Agric) The University of B r i t i s h Columbia, 1977 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE i n THE FACULTY OF GRADUATE STUDIES (Department of Animal Science) We accept this thesis as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA February 1983 © J e f f r e y A l l a n Snider, 1983 I n p r e s e n t i n g t h i s t h e s i s i n p a r t i a l f u l f i l m e n t o f t h e r e q u i r e m e n t s f o r an advanced degree a t the U n i v e r s i t y o f B r i t i s h C o l u m b i a , I agree t h a t t h e L i b r a r y s h a l l make i t f r e e l y a v a i l a b l e f o r r e f e r e n c e and s t u d y . I f u r t h e r agree t h a t p e r m i s s i o n f o r e x t e n s i v e c o p y i n g o f t h i s t h e s i s f o r s c h o l a r l y purposes may be g r a n t e d by t h e head o f my department o r by h i s o r her r e p r e s e n t a t i v e s . I t i s u n d e r s t o o d t h a t c o p y i n g o r p u b l i c a t i o n o f t h i s t h e s i s f o r f i n a n c i a l g a i n s h a l l n o t be a l l o w e d w i t h o u t my w r i t t e n p e r m i s s i o n . Department o f frft/lM^U C (6W C6" The U n i v e r s i t y o f B r i t i s h Columbia 1956 Main Mall Vancouver, Canada V6T 1Y3 DE-6 (3/81) ABSTRACT Various forages commonly provided to dairy cattle were fed to cannulated steers in order to study dietary nitrogen breakdown and ut i l i z a t i o n . Feed intake and apparent dige s t i b i l i t y of dry matter, fibre and nitrogen were monitored as well as microbial nitrogen production in the rumen. Two Holstein and two Ayrshire steers, each fitted with a re-entrant duodenal cannula were used. Two of the steers were also used before the cannulae were fi t t e d . Five different forages were investigated: low quality grass hay (1.7%N), a l f a l f a cubes (2.3%N), high quality grass hay (2.7%N), Italian ryegrass (1.7%N) and orchard grass (2.1%N). For each forage, each steer was allowed a two-week adaptation period followed by a four-day digestiblity collection period. Fecal samples were obtained daily, and digesta samples were collected at the duodenum three times daily. Unfortunately, due to illnesses, only one steer was able to complete a l l of the diges t i b i l i t y t r i a l s with a l l of the forages. When low quality hay and al f a l f a cubes were given, cannulation had no effect (p>.05) on feed intake or apparent dige s t i b i l i t y of fibre and nitrogen. Only apparent digestiblity of dry matter was higher (p<.05) for the intact steers. Feed intake, and apparent di g e s t i b i l i t y of dry matter and nitrogen were greater (p<.05) for the higher nitrogen (al f a l f a cube) diet. Forage type had no effect (p>.05) on feed intake, and apparent dige s t i b i l i t y of dry matter and fibre when any of the forages were given to cannulated steers. Nitrogen apparent - i i i -dige s t i b i l i t y was greater (p<.05) for the higher nitrogen forages. The amount of feed offered (maintenance or ad libitum) had no effect (p>.05) on feed intake, dry matter, fibre and nitrogen d i g e s t i b i l i t i e s . In respect to apparent d i g e s t i b i l i t i e s and quantities of microbial nitrogen obtained at the duodenum, forage type had a significant effect on a l l parameters. The proportion of apparent dig e s t i b i l i t y of dry matter and fibre occurring in the rumen were greater (p<.05) for the lower nitrogen forages. The proportion of nitrogen dig e s t i b i l i t y occurring in the rumen was greater (p<.05) for the higher nitrogen forages. The ratio of microbial nitrogen: total nitrogen was greater (p<.05) for the lower nitrogen forages. The particular day of the diges t i b i l i t y t r i a l or time of duodenal sample collection had no effect (p>.05) on apparent d i g e s t i b i l i t i e s or total microbial nitrogen. Feeding level had a significant effect on some of the parameters at the duodenum. Apparent di g e s t i b i l i t y of fibre and nitrogen and total microbial nitrogen were higher (p<.05) when the forage was fed ad libitum. The quantity of bypass nitrogen measured at the duodenum, and the apparent dige s t i b i l i t y of the bypass nitrogen was much lower for the lower nitrogen forages. - i v -TABLE OF CONTENTS Page ABSTRACT i i TABLE OF CONTENTS i v LIST OF TABLES .' v LIST OF FIGURES v i ACKNOWLEDGEMENTS v i i INTRODUCTION 1 LITERATURE REVIEW 4 1. Nitrogen Digestion and Absorption 4 1.1 Rumen 4 1.2 Rumen M i c r o b i a l P r o t e i n 5 1.3 Post Rumen 6 2. Bypass Protein 7 3. Ammonia Utilization 9 4. Metabolizable Protein 12 4.1 Assumptions 13 5. Nitrogen and Energy Relationships 17 5.1 Carbohydrates 17 5.2 YATP 20 5.3 Fibre and VFA 21 6 . Estimating Microbial Protein Synthesis 23 6.1 DAP 23 6.2 RNA 24 6.3 3 5 S 24 6.4 1 5N 25 6.5 3 2 P 25 6.6 Amino Acids 26 6.7 ATP 27 7. Cannulation 27 MATERIALS AND METHODS 31 1. Cannulation 31 2. D i g e s t i b i l i t y Trials 33 3. Laboratory Analysis 34 4. S t a t i s t i c a l Analysis 37 RESULTS AND DISCUSSION 41 SUMMARY BIBLIOGRAPHY 60 63 - v -LIST OF TABLES Page Table 1 Forage Composition 42 Table 2 E f f e c t of Status and Forage Type on Intake and Apparent D i g e s t i b i l i t y 43 Table 3 E f f e c t of Forage Type on Intake and Apparent D i g e s t i b i l i t y (Total C o l l e c t i o n and Chromic Oxide Methods) 45 Table 4 E f f e c t of Forage Type and Feeding Level on Intake and Apparent D i g e s t i b i l i t y 48 Table 5 E f f e c t of Forage Type, Day and Time on Apparent D i g e s t i b i l i t y (Stomach) and Mi c r o b i a l Nitrogen 49-50 Table 6 E f f e c t of Forage Type, Feeding Level, Day and Time on Apparent D i g e s t i b i l i t y (Stomach) and Mic r o b i a l Nitrogen 57-58 Table 7 U t i l i z a t i o n of Bypass Nitrogen 59 - v i -LIST OF FIGURES Page Figure 1 Summary of Metabolizable Protein Calculations 14 Figure 2 The Two Major Reactions of Ruminal Fermentation 19 Figure 3 Design of the Re-Entrant Cannula 28 - v i i -ACKNOWLEDGEMENTS The author would like to extend appreciation to Dr. J.A. Shelford for his guidance and assistance throughout this project. He also wishes to acknowledge the help and cooperation of B i l l Slack and Paul Willing of the U.B.C. South Campus Dairy Unit. Thanks are also due to the laboratory technicians in the Animal Science Department who kindly offered help and advice whenever It was needed. - I -INTRODUCTION The provision of protein for the high producing dairy cow i s v i t a l l y important for optimum production. E f f e c t i v e nitrogen u t i l i z a t i o n involves supplying the small i n t e s t i n e with adequate amino acids to s a t i s f y the productive requirements of the animal. In the rumen, many nitrogenous compounds can be both degraded and synthesized, and a proper d e s c r i p t i o n of the nitrogen reaching the duodenum can aid i n determining diets of maximum e f f i c i e n c y to the animal. Numerous recent reviews on nitrogen metabolism have pointed out the importance of the supply of synthesized microbial protein, and dietary protein that has escaped degradation i n the rumen (Roy et a l . , 1977; Satter and R o f f l e r , 1975; Smith 1975; Tamminga, 1979). Carbohydrates, p a r t i c u l a r l y c e l l u l o s e and starch, make up the lar g e s t percentage of most dairy c a t t l e d i e t s . Most of th i s portion of the diet i s fermented by the microbes to v o l a t i l e f a t t y acids (VFA), the p r i n c i p a l ones being a c e t i c , propionic and butyric acids. To ensure a good supply of microbial c e l l s and a high degree of digestion of the f i b r e component of the die t , optimum fermentation i s c r i t i c a l i n the high producing dairy cow. Diets which contain high proportions of forages and other roughage-type feeds favour the production of acetate i n the rumen. Without proper f i b r e digestion, the VFA r a t i o s can s h i f t towards propionic acid and r e s u l t i n depressed milk f a t production. The supply of nutrients for the microbes i n the rumen i s one of the main factors that can influence the rate and amount of f i b r e digestion. Rumen microbial protein synthesis requires an adequate supply of nitrogen to achieve maximum e f f i c i e n c y . Low microbial growth rates may - 2 -a f f e c t amino acid a v a i l a b i l i t y to the animal as well as influence fermentation rate. In many cases, the protein content of the feedstuff i s i n s u f f i c i e n t , thereby l i m i t i n g the amount of ava i l a b l e nitrogen with a resultant decrease i n f i b r e digestion. In other cases, incorrect timing of nitrogen reduces the rate of fermentation. There are other factors which may also influence microbial growth and nitrogen a v a i l a b i l i t y to the animal. The chief source of energy (ATP) i n the rumen i s derived from the fermentation of carbohydrates to VFA, and microbial growth i s d i r e c t l y proportional to the amount of ATP generation (Bauchop & Elsden, 1960). Maeng et a l . (1976) suggested that an adequate supply of ammonia-nitrogen, carbon skeletons, free amino acids and sulphur i s also needed for optimum microbial c e l l synthesis. Closely linked to the concept of optimum fermentation i s the concept of by-pass protein. In the high producing cow, microbial protein alone w i l l not meet the cow's requirements and by-pass protein becomes important. This escaped dietary protein supplements microbial protein and provides the host animal with a source of amino acids for digestion and absorption i n the small i n t e s t i n e . Although many experiments have been conducted and numerous a r t i c l e s written on the subject, knowledge i s s t i l l l i m i t i n g on the degradab i l i t y i n the rumen of protein i n common fee d s t u f f s . The major areas explored i n the current study were the aspects of protein degradation by microbial action, the measurement of protein breakdown by microbes i n the rumen, and the contributing factors influencing t h i s breakdown. The main purpose of this study was to determine the extent of ruminal breakdown of the protein of forages used i n dairy c a t t l e diets - 3 -(degradability). Variation between feeds and between animals was measured. Analysis of the protein source and i t s efficiency of ut i l i z a t i o n to the animal was also undertaken. The effect of cannulation and its possible influence on dig e s t i b i l i t y in the animal were considered. - 4 -LITERATURE REVIEW 1. Nitrogen Digestion and Absorption 1.1 Rumen Proteins are the most common nitrogenous components found i n forages and provide the major part of the nitrogen used by rumen microbes and the host animal. Dietary proteins entering the rumen are often extensively degraded by both bacteria and protozoa, each with a somewhat d i f f e r e n t mechanism of protein breakdown. Bacteria hydrolyse some or a l l of the peptide bonds to break the protein chain into smaller parts. This process takes place outside the b a c t e r i a l c e l l and the r e s u l t i n g peptides and amino acids are transported inside the c e l l where the peptides are further hydrolysed to amino acids (Tamminga, 1979). In turn, the amino acids are e i t h e r incorporated into b a c t e r i a l protein or broken down to v o l a t i l e f a t t y acids, ammonia, carbon dioxide, methane, and some heat of fermentation. The end products r e s u l t i n g from this degradation are excreted back into the surrounding medium (Tamminga, 1979). The role of protozoa i n the rumen and the extent to which the protozoa u t i l i z e nitrogenous components of the feed has not been well established. Protozoa are capable of engulfing small feed p a r t i c l e s and b a c t e r i a , and p r o t e o l y s i s of dietary proteins occurs inside the protozoal c e l l . Any of the r e s u l t i n g amino acids which are not incorporated into protozoal protein are often expelled into the surrounding medium rather than being further degraded (Hungate, 1966; Coleman, 1975). - 5 -The microorganisms i n the rumen use the end products of protein digestion as buil d i n g blocks for the i r own c e l l s . Eventually a proportion of the microbes are passed down the i n t e s t i n a l t r a c t , digested, and used as a protein source by the animal. Therefore, regardless of the source of protein the ruminant i s fed, much of i t Is converted to b a c t e r i a l and protozoal protein before actual u t i l i z a t i o n by the animal. 1.2 Rumen M i c r o b i a l Protein The protein content of ruminal microbes has been reported i n a number of papers. McNaught et a l . (1950, 1954) found that rumen ba c t e r i a had approximately 42% crude protein as compared to 26% for protozoa, and stated this was due, i n part, to a higher polysaccharide content of the rumen protozoa. Weller (1957), using a v a r i e t y of d i e t s , reported crude protein l e v e l s of rumen bac t e r i a from 58-77% and that of protozoa from 24-49%, with the highest values being for microbes from animals on grass. Abdo et a l . (1964) found the crude protein content of mixed organisms to be from 38-49% when they were either freeze dried or acetone dried. The appreciable differences indicated from these reports were probably due to contamination of the microbial preparations and differences i n chemical technique and ra t i o n s . Since microbial protein constitutes a major part of the ruminant's nitrogenous food, i t s value for the synthesis of tissue becomes important. The food value has been determined by feeding rumen microbes to mice or r a t s . This has been done because of the d i f f i c u l t y i n obtaining s u f f i c i e n t microbial protein to feed a ruminant, and also since the use of non-ruminants avoids the complications of rumen action - 6 -on feed protein. In early studies done by McNaught et a l . (1954), i t was indicated that the true d i g e s t i b i l i t y of bacterial crude protein fed to rats was 74%, and that protozoal crude protein was 91% digestible. Mason and Palmer (1971) isolated rumen bacteria from cattle given a barley-soybean diet, and they concluded that approximately 80% of bacterial nitrogen was absorbed by rats. 1.3 Post-Rumen After rumen fermentation of ingested protein, microbial and undegraded dietary, protein (bypass protein), free amino acids, ammonia, and nucleic acids flow with the digesta through the omasum and abomasum to the small intestine. Some enzymatic digestion occurs in the abomasum by the digestive protease pepsin, but the majority takes place in the small intestine. In the small intestine, protein is broken down to free amino acids and peptides by the pancreatic proteases trypsin, chymotrypsin, and carboxypeptidase A, and the major portion of amino acid absorption occurs here (Bergen, 1978). Dietary nucleic acids are extensively degraded in the rumen and rumen microbes contain about 20% of their nitrogen content as nucleic acids (Hungate, 1966). The nucleic acids passing to the small intestine are of microbial origin, and they are broken down by digestive nucleases DNase, RNases, phosphodiesterases, and phosphomonesterases (Bergen, 1978). From the microbial nucleic acids arise primarily nucleotides and nucleosides, and these are absorbed by the mucosa in the small intestine. The digesta that pass from the small intestine into the cecum contain some undigested protein, as well as material derived from - 7 -endogenous sources such as digestive enzymes and e p i t h e l i a l c e l l s . Proteins that reach the cecum are degraded r a p i d l y with a further rapid deamination of amino acids, and they are not involved d i r e c t l y with the amino acid metabolism of the animal (Hecker, 1971). Only i n s i g n i f i c a n t q u antities of e s s e n t i a l amino acids are absorbed from the large i n t e s t i n e . 2. Bypass P ro te in The microbial synthesis of protein i n the rumen provides a mechanism for u t i l i z a t i o n of nonprotein nitrogenous (NPN) compounds. However, at a given l e v e l of energy consumption the quantities are not always adequate to provide s u f f i c i e n t amino acids for production. As a r e s u l t , except where low l e v e l s of production can be tolerated, proteins that escape ruminal degradation are needed. The outflow of amino acids from the rumen can be increased by "optimizing rumen microbial protein production, s e l e c t i n g feed ingredients that are r e s i s t a n t to rumen microbial degradation, or using methods to decrease rumen microbial degradation of proteins and amino acids" (Chalupa, 1978). When dietary f i b r e and NPN are subjected to ruminal fermentation, th e i r n u t r i t i o n a l value to the animal i s enhanced. However, production i n ruminants i s often improved when feeds low i n f i b r e and high i n protein escape degradation i n the rumen and are digested post-ruminally (Orskov et a l . , 1970; Hedde et a l . , 1974). U t i l i z a t i o n of nutrients post-ruminally eliminates energy losses associated with fermentation and protein losses r e s u l t i n g from the transformation of dietary protein to microbial protein (Black, 1971; Chalupa, 1974). Diets high i n soluble - 8 -protein and low in soluble carbohydrates have excessive ammonia production coupled with insufficient energy supplied for protein synthesis. As a result, a large amount of nitrogen leaves the rumen as ammonia and is not ut i l i z e d . Normal procedures in the manufacture of feed ingredients can influence the magnitude of protein breakdown in the rumen. Hale (1973) demonstrated that grain processing procedures can influence ruminal fermentation. Increased ruminal degradation can result from the disruption of the protein matrix, whereas heat generated during grain processing can decrease ruminal protein fermentation. Some procedures w i l l increase microbial protein production by increasing the amount of starch fermented in the rumen (Waldo, 1973). Chemical treatment of proteins also provides a potential mechanism for controlling the quantity of bypass protein. Certain chemical agents, such as tannins and aldehydes, form reversible cross linkages with amino and amide groups which decrease solubility of proteins at the pH of the rumen. As a result, the treated proteins are made available to the host by degradation in the acidic environment of the abomasum (Hatfield, 1973). Formaldehyde has been the most widely used chemical agent. Treatment of casein with formaldehyde has generally resulted in increased nitrogen retention, wool growth, and muscle growth (Faichney, 1971; Barry, 1972; MacRae et a l . , 1972). Procedures have been devised to protect free amino acids from ruminal degradation by using encapsulation methods. Neudorffer et a l . (1971) have shown the technique could be used to increase availability of methionine to the lower gut by coating methionine with tristearin and - 9 -a l i q u i d unsaturated f a t t y acid. This would reduce the chance of ,limited response by the animal r e s u l t i n g from an inadequate supply of absorbable amino acids. Under most conditions, approximately 40% of dietary protein escapes fermentation i n the rumen, and the animal i s normally supplied with a mixture of microbial and bypass protein ( M i l l e r , 1973). Chalupa et a l . (1973) and Chalupa (1974) f e l t t h i s mixture alone may be i n s u f f i c i e n t a f t e r observing higher production responses from animals given increased quantities of abomasal supplements of proteins and amino acids. However, Hungate (1966) reported that i f preformed proteins are the sole nitrogen source i n ruminant d i e t s , excessive bypass w i l l l i m i t microbial growth and v o l a t i l e f a t t y acid production. To provide for rumen bypass of i n t a c t proteins or amino acids w i l l depend upon whether shortages of amino acids, i n the combination of b a c t e r i a l and rumen bypass nitrogen, are l i m i t i n g production, and on whether the ones i n short supply can be i d e n t i f i e d . The attainment of optimum protein production i n ruminants must involve: 1. use of NPN for rumen protein production 2. optimization of rumen bypass protein 3. possible supplementation with rumen non-degradable amino acids 3. Ammonia U t i l i z a t i o n Since microorganisms play such a v i t a l r ole at the s t a r t of the digestive process i n the ruminant, the amino-acid n u t r i t u r e of the animal i s highly influenced. As a r e s u l t , according to Satter and - 10 -Roffler (1975), ruminants are able to: "1. u t i l i z e dietary nonprotein nitrogen effi c i e n t l y 2. upgrade low quality dietary protein to high quality microbial protein 3. survive on low intakes of dietary nitrogen by efficiently u t i l i z i n g nitrogen recycled into the reticulo-rumen via saliva." However, a major drawback to these advantages may arise when high protein diets are fed, resulting in more dietary protein loss than synthesis and a net loss of protein. This is caused by excessive ammonia production, with nitrogen leaving the rumen as ammonia and eventually being excreted in the urine as urea. This w i l l be further discussed later. After a feedstuff has been ingested by the ruminant, i t may either be degraded by rumen microbes or may escape breakdown and pass to the lower gut to be digested or excreted in the feces. Satter and Roffler (1975) agreed with Miller (1973) in a 40% average escape rate for dietary protein, with the remaining 60% of dietary protein degraded almost entirely to ammonia (NH3). Dietary NPN, salivary nitrogen, and possibly some urea entering across the rumen wall are converted almost entirely to NH3 as well. The amount of ammonia that can be utilized by the microbes depends on the size of the population and their growth rate. Feeds that are high in total digestible nutrients are usually more fermentable, w i l l support increased bacterial numbers, and as a result more ammonia (NPN) can be uti l i z e d . According to Satter and Roffler (1975), i t does - 11 -not make much difference to the host whether dietary true protein is degraded i f the rumen bacteria can u t i l i z e a l l the N H 3 produced. Either way, dietary or converted nitrogen ends up as protein presented to the intestine for absorption. However, when rumen bacteria are unable to convert a l l the N H 3 produced to microbial protein, only dietary bypass protein and some microbial protein w i l l contribute to the amino acid nutrition of the host. The N H 3 derived from NPN and degraded true protein w i l l not be of any value. Satter and Slyter (1974) studied the effect of N H 3 concentration on microbial protein production in steers fed a variety of diets. Under in vitro conditions, 5 mg. N H 3 - N/100 ml. rumen f l u i d was found to be the point of N H 3 accummulation, and nothing was gained by further supplementation with NPN. Also, dietary true protein degraded to NH3 was not utilized when rumen N H 3 - N exceeded 5 mg./lOO ml. This value agrees closely with the findings of Mercer and Annison (1976). Satter and Slyter (1974) did not discuss levels of NH3 where toxicity may occur. Chalmers et a l . (1976) reported that N H 3 absorption is pH dependent and as pH in the rumen rises, there is increased NH3 absorption by diffusion through the rumen wall. If N H 3 absorption exceeds the threshold value for N H 3 in the liver, N H 3 w i l l pass into the peripheral blood and may cause toxicity. Roffler and Satter (1975 a) studied the influence of diet composition on mean rumen-NH3 concentration, and collected over 1000 ingesta samples from 211 cattle fed a variety of typical diets. Mean rumen-NH3 concentration reached 5 mg. NH3-N/IOO ml. at approximately 13% - 12 -dietary crude protein (dry matter b a s i s ) , and above t h i s concentration rumen-NH3 increased r a p i d l y with increasing p r o t e i n . In another series of experiments involving 406 cows on l a c t a t i o n t r i a l s , R o f f l e r and Satter (1975 b) examined comparisons of NPN supplemented diets to unsupplemented control d i e t s . Addition of NPN to low protein diets caused a large increase i n milk production, but response i n milk production declined progressively as protein content of the diet was increased p r i o r to NPN supplementation. When the diets contained about 12.5% crude protein, p r i o r to supplementation, the point of zero response i n milk production to NPN supplementation occurred. They also looked at the r e l a t i o n s h i p between predicted rumen-NH3 concentration and response i n milk production, and found zero response with rumen-NH3 l e v e l s i n excess of 4 mg./lOO ml. This value agrees c l o s e l y with 5 mg. N H 3 - N/100 ml. shown to be adequate for maximal microbial growth rates. 4. Metabollzable P r o t e i n Burroughs et a l . (1972, 1973; c i t e d by Satter and R o f f l e r , 1975) used the term "metabollzable protein" to describe the amount of protein absorbed from the g a s t r o i n t e s t i n a l t r a c t . I t represents the portion of the dietary crude protein intake that i s available i n a - amino form for metabolism by the host. Satter and R o f f l e r (1975) looked at the amount of metabollzable protein per unit of crude protein intake. When rumen-NH3 was u t i l i z e d completely for microbial protein production, the proportion of metabollzable protein was much higher than when rumen-NH3 was i n excess. From a given amount of crude protein, they concluded - 13 -that proportionately there was more metabolizable protein when low protein diets were fed compared to high protein d i e t s . In t h e i r a r t i c l e , Satter and R o f f l e r (1975) attempted to r e l a t e absorbable (metabolizable) protein to r e a d i l y measured features of a feed or d i e t , such as crude protein or t o t a l d i g e s t i b l e nutrient (TDN) content. They came up with a very concise schematic summary, making many assumptions along the way to form the basis for metabolizable protein (See Figure 1). 4.1 Assumptions a. The amount of nitrogen recycled i n the rumen i s equal to 12% of the dietary nitrogen. The major source of recycled nitrogen to the rumen i s s a l i v a r y nitrogen. Bailey and Balch (1961) examined the composition and rate of secretion of s a l i v a i n cows at rest and found that a quantity of nitrogen equivalent to 10% of the dietary nitrogen i s recycled. This was l a t e r confirmed by Bailey (1961) who examined s a l i v a composition and secretion rate i n cows during eating. The amount of nitrogen recycled into the rumen, expressed as a percent of dietary intake, can vary according to the protein content of d i e t s . However, Satter and R o f f l e r (1975) f e l t that with t y p i c a l dairy and feedlot beef d i e t s , a value of 10 to 15% seemed to be a reasonable estimate. b. With t y p i c a l ruminant d i e t s , 85% of the dietary nitrogen intake i s i n true protein form and 15% i n NPN form. According to Waldo (1968),the seeds of most plants such as corn, wheat and barley contain approximately 95% of the nitrogen i n the form of true protein. Forages such as a l f a l f a have about 80% of the - 14 -FIGURE 1. Summary of Metabolizable Protein Calculations 1000 g NPN DIETARY CP True Protein i Ruminal NH3 Pool Degraded to NH 3 850 Converted to Mi c r o b i a l N ! Urea Pool i "1 i 700 / / / / / f / / / M i c r o b i a l / M i c r o b i a l NPN / True 140 X Protein N v . i Escapes Ruminal Degradation Pro t e i n Metabolism Excreted i n Urine 340 560 110 , 4 0 Excreted i n Feces { 4 1 450g M i c r o b i a l O r i g i n 750g Metabolizable Protein 300g Dietary O r i g i n (Satter and R o f f l e r , 1975) - 15 -nitrogen as true protein. However, Jorgensen and Crowley (1972) showed that ensiled forages may have as l i t t l e as 50% of the nitrogen in true protein form. Satter and Roffler (1975) fel t that the most readily available protein was broken down during the ensiling process, and that this protein would be most vulnerable to degradation in the rumen at feeding. As a result, ensiled feeds had no visible effect on rumen-NH3 concentration compared to non-ensiled feeds except what could be due to crude protein and TDN content. This was in agreement Roffler and Satter (1975 a). c. 40% of the true dietary protein escapes degradation in the rumen. A l l of the dietary NPN and recycled nitrogen passes through the rumen-NH3 pool. Satter and Roffler (1975) chose the 40% figure as a suitable average from Miller (1973). The extent of protein degradation varies with the nature of the protein fed and the amount of time spent in the rumen. Rumination time is highly influenced by the physical form of the diet and by feed intake. Experiments were undertaken to study the effect of feed intake on the quantity of non-NH3 nitrogen reaching the abomasum in sheep (Hogan and Weston, 1967; Coelho da Silva et a l . , 1972 a). They found that increased feed intake decreased protein degradation, (thereby increasing bypass protein) and decreased carbohydrate fermentation in the rumen, thereby compensating with decreased microbial protein production. When feed intake was altered there was minimal effect on the amount of non-NH3 digesta flow to the lower gut. Hogan (1974) - 16 -reported that microbial c e l l yield depends on retention time of the substrate in the rumen. For maximum yields, sufficient time must be allowed for the organisms to reproduce and develop. However, microbial cells must be removed before they begin to use up substrate for maintenance purposes, which w i l l decrease microbial c e l l yield. d. 90% of a l l NH3 produced is incorporated into microbial nitrogen when the ration fed does not exceed the crude protein upper limit value. Using an NH3 u t i l i z a t i o n curve, Roffler and Satter (1975 a) examined the entire range from no dietary crude protein to the protein level where rumen-NH3 nitrogen reaches 5 mg./lOO ml. They found a mean value of about 90% NH3 u t i l i z a t i o n . e. None of the rumen-NH3 is incorporated into microbes when the ration fed exceeds the crude protein upper limit value. This was discussed earlier - Satter and Slyter (1974), Roffler and Satter (1975 b). f. 80% of microbial nitrogen is in true protein form, and 20% in NPN form. Hungate (1966) and Smith (1969) both found that about 80% of microbial crude protein is true protein, and the remaining 20% is NPN, mainly nucleic acids. g. 80% of microbial true protein is absorbed in the small intestine. Discussed earlier - Mason and Palmer (1971). h. 87% of dietary true protein that escapes rumen breakdown is absorbed in the small intestine. True dige s t i b i l i t y of protein is probably greater than 90% in grain, and less in forages. Digestiblity of bypass protein may be - 17 -s l i g h t l y less than dietary true protein since the more available proteins would have been degraded i n the rumen. Hogan (1974) stated that at l e a s t 80% of amino acids of microbial and dietary o r i g i n are absorbed from the i n t e s t i n e . Since microbial amino acids predominate i n the i n t e s t i n e and a value of 80% d i g e s t i b i l i t y was given to microbial protein, a value of 87% d i g e s t i b i l i t y for bypass protein was chosen by Satter & R o f f l e r (1975). Due to the number of assumptions made by Satter and R o f f l e r (1975) some shortcomings can arise when N requirements are calculated. There i s no d i f f e r e n t i a t i o n between the absorption of the d i f f e r e n t forms of NPN - i . e . amino acids, NH3, nucleic acids - to allow for differences i n response. Also, N requirements are not d i r e c t l y r e l a t e d to the t o t a l energy intake and concentration i n the d i e t . As a r e s u l t , Roy et a l . (1977) proposed a system based on an estimate of the t o t a l amino acid-N absorbed from the small i n t e s t i n e , and i t s e f f i c i e n c y of u t i l i z a t i o n at a p a r t i c u l a r energy input i n the d i e t . However, i n t h i s system as well values have been adopted from the l i t e r a t u r e to complete c a l c u l a t i o n s . 5. Nitrogen and Energy Relationships 5.1 Carbohydrates Quantitatively, carbohydrates are very important to the ruminant. Plant tissues contain about 75% carbohydrates, with the amount and d i s t r i b u t i o n depending on age and species of the plant. As a r e s u l t , carbohydrates provide the primary source of energy for both the rumen microbes and the host animal. The carbohydrates found i n plant - 18 -tissues are primarily polysaccharides - cellulose, hemicellulose, pectins, fructans and starches. Of these, cellulose is by far the most abundant in forages as opposed to cereal grains. Nitrogen-carbohydrate interrelationships are very important to overall rumen metabolism since dietary carbohydrate provides the energy and dietary nitrogen provides the building blocks for microbial growth. The two major reactions of ruminal fermentation are the degradation of carbohydrates to volatile fatty acids (VFA) as a method of energy (ATP) generation, and the accompanying production of microbial cells (see Figure 2). The capacity for microbial growth depends primarily on the extent of ATP production. Other factors such as growth rate can influence the maintenance needs for microbial c e l l s . Marr et a l . (1963) found that the major maintenance energy needs by microbes were for motility, replacement of lysed cel l s , intracellular ionic concentrations, active transport, and turnover of intracellular components. It is only after these requirements have been met that net microbial growth can occur from which the animal can benefit. For optimal c e l l synthesis, precursors other than ATP are needed as well. These include a sufficient supply of NH3-N, carbon skeletons, sulphur, free amino acids, and undefined cofactors (Maeng et a l . 1976). VFA production and cellular growth are highly coupled processes, and an imbalance of any component in the overall flow can affect ruminal fermentation, VFA and microbial c e l l production. For maximum efficiency of microbial growth to occur, nitrogen and energy availability in the rumen must be balanced. If the nitrogen FIGURE 2. The Two Major R e a c t i o n s o f Ruminal F e r m e n t a t i o n VFA P r o t e i n Carbon S k e l e t o n s Amino A c i d s P e p t i d e s phur . M i c r o b i a l Ce l I s O t h e r ( c o ) f a c t o r s (Bergen and Yokoyama,I 977) - 20 -l e v e l Is I n s u f f i c i e n t , fermentation without useful ATP production may occur. In contrast, i f the nitrogen l e v e l i s excessive, energy may be the l i m i t i n g factor for e f f i c i e n t nitrogen u t i l i z a t i o n . In general, an increase i n the proportion of r e a d i l y fermentable carbohydrate i n the r a t i o n Increases the uptake of nitrogen by the microbes i n the rumen. This i s due to an increase i n a v a i l a b i l i t y of energy for growth and a decrease i n the energy needed for maintenance by the organisms. Stern et a l . (1978) concluded that a major factor a f f e c t i n g the u t i l i z a t i o n of degraded dietary nitrogen was the type and rate of a v a i l a b i l i t y of carbohydrates. Lewis and MacDonald (1958) found that the greatest u t i l i z a t i o n of protein i n the rumen occurred when a carbohydrate was present that could be fermented at a comparable rate to the protein. Robertson and Hawke (1965) noted that i n f u s i o n of starch into the rumen of cows on high-nitrogen pasture resulted i n c o n s i s t e n t l y lower rumen-NH.3 l e v e l s as compared to control cows. Bergen and Yokoyama (1977) reported that substrate disappearance i n the rumen depended upon the rate of degradation and the rate of passage. Coarse, poorly digested roughage w i l l be retained longer i n the rumen to achieve breakdown. 5.2 YATP Bauchop and Elsden (1960) defined the r e l a t i o n s h i p between microbial c e l l growth and ATP as YATP (molar growth y i e l d per mole ATP or g. microbes (dry weight) per mole ATP). From t h e i r work and others (Forrest and Walker, 1971; Stouthamer, 1969), It was concluded that the YATP was a constant value for microbes, 10.5. However, Stouthamer and Bettenhausen (1973) proposed that YATP was not constant and that the - 21 -growth e f f i c i e n c y of the microorganisms depended on t h e i r s p e c i f i c growth rate and maintenance requirements. They found that YATP was low (<10) i n slow growing cultures where maintenance c o e f f i c i e n t s were higher, but would approach the YATP maximum of 25 at high growth rates. From the formulation of the YATP concept, many workers attempted to c a l c u l a t e the quantity of microbial protein synthesized i n the rumen for each 100 g. of organic matter fermented. Using ingesta passage studies with dried forage d i e t s , i t was shown that the microbial protein synthesis rate was from 15 to 23 g. per 100 g. organic matter fermented i n sheep (Hogan and Weston, 1970; Lindsay and Hogan, 1972), and i n c a t t l e (Hagemeister et a l . , 1976; Walker et a l . , 1975). 5.3 Fibre and VFA As mentioned e a r l i e r , carbohydrates i n the rumen are fermented by the microbes to VFA's, p r i n c i p a l l y acetate, propionate and butyrate (ionized form). When high forage diets are fed, the r a t i o s among the three f a t t y acids w i l l be approximately 50-65% acetate, 18-25% propionate, and 12-20% butyrate (Foley et a l . , 1972). Fermentation of c e l l u l o s e i n the rumen i s regulated by the a c t i v i t i e s of the f i b r e - d i g e s t i n g microbes, since these l a r g e l y determine the rate of disappearance of digesta and, through t h i s , the rate of food intake by the animal. The most active c e l l u l o l y t i c species i n the rumen are Bacteroides succinogenes, Ruminococcus albus, and R. Flavefaciens (Dehority, 1973). The growth of the f i b r e - d i g e s t i n g b acteria i n the rumen i s most commonly lim i t e d by a shortage of nitrogen. S l y t e r et a l . (1971) and - 22 -Van Gylswyck (1970) found reduced numbers of c e l l u l o l y t i c b a c t e r i a when low protein diets were fed to steers and sheep r e s p e c t i v e l y . Minson and Pigden (1961) noted that when mature sheep were fed diets below .74% nitrogen they showed negative nitrogen balance, reduced crude f i b r e d i g e s t i o n and dry matter intake. At t h i s percentage nitrogen, the rumen NH3-N was about 1-2 mg.%. Glover and Dougall (1960) showed a reduction i n f i b r e d i g e s t i b i l i t y i n rumen contents associated with diets containing less than .96 mg.% NH3-N. Hume et a l . (1970) fed sheep protein free, p u r i f i e d diets containing .54, .95, 1.82, and 3.29% nitrogen as urea, and observed increased microbial population as the nitrogen l e v e l rose. Low nitrogen l e v e l s led to reduced carbohydrate d i g e s t i b i l i t y which i n turn led to decreased energy generation. This resulted i n decreased microbial growth. Inadequate nitrogen l e v e l s , leading to reduced f i b r e digestion, can s h i f t VFA r a t i o s i n favour of propionate and r e s u l t i n depressed milk fat production. Grinding and p e l l e t i n g the r a t i o n , or feeding a d i e t high i n concentrates also tends to reduce the proportion of acetate and increase propionate. In addition, there w i l l be higher microbial c e l l synthesis and flow rate of ingesta to the duodenum, and more nitrogen w i l l be incorporated when the fermentation y i e l d s a high proportion of propionate (Hume, 1970). This increase i n nitrogen u t i l i z a t i o n means that the diet must supply more degradable nitrogen i n the rumen to prevent l i m i t a t i o n of b a c t e r i a l growth. Inadequate nitrogen l e v e l s can also reduce carbohydrate use and energy output, which w i l l i n turn reduce microbial growth. - 23 -6. Estimating M i c r o b i a l P r o t e i n Synthesis An adequate description of the nitrogen reaching the duodenum requires measurement of the contributions made by undegraded dietary, bacterial, protozoal and endogenous fractions. Research in this area has mainly been concerned with estimating the dietary and microbial fractions of digesta. The methods used have generally relied upon the measurement of the concentration of some microbial component (or marker) in the digesta. By relating this to the marker concentration in the actual microbes, an estimate of the proportion of microbial material in the digesta is obtained. Diaminopimelic acid (DAP), aminoethylphosphonic acid (AEP), ribonucleic acid (RNA), and isotopes ( 3 5S, 1 5N, 3 2P) incorporated into protein in the rumen have been used as microbial markers. Differences in the amino acid profiles of individual components reaching the duodenum have also been used. More recently, ATP was proposed as a marker of microbial activity. 6.1 DAP Weller et a l . (1958) used DAP to estimate the rate of bacterial protein synthesis. This technique takes advantage of the fact that DAP is present in the c e l l membrane of many types of rumen bacteria, but is absent from plant material. The DAP method involves estimating the ratio of DAP: nitrogen in mixed rumen bacteria and the amount of DAP in digesta. From these values, the amount of bacterial nitrogen in digesta can be calculated (Hogan and Weston, 1970). - 24 -Ibrahim and Ingalls ( 1 9 7 2 ) used DAP to measure bacterial protein synthesis and AEP, which is found in the l i p i d fraction of protozoa, to measure protozoal protein synthesis. In theory, DAP and AEP together can be used to estimate total rumen microbial protein synthesis. However, there is some doubt as to the accuracy of the DAP method, since i t assumes the DAP: nitrogen ratio to be constant among various microbial species. Work and Dewey ( 1 9 5 3 ) have shown this ratio to vary among species. 6.2 RNA Smith and McAllan ( 1 9 7 0 ) used the RNA: total nitrogen ratio in rumen fluid and microbes to estimate the extent of conversion of dietary nitrogen to bacterial and protozoal nitrogen. This technique assumes that nearly a l l dietary RNA is degraded in the rumen. However, more recently Smith et a l . ( 1 9 7 8 ) indicated that the RNA method slightly overestimated the microbial contribution at the duodenum. Another problem with the RNA method is the effect of diet and environment on the va r i a b i l i t y of the RNA: total nitrogen ratio of mixed bacteria (Smith and McAllan, 1 9 7 4 ) . McAllan and Smith ( 1 9 7 4 ) compared RNA and DAP as markers for estimating microbial nitrogen in duodenal digesta. Using a protozoa-free calf, they found good agreement between the two methods. However, when a faunated cow was used the DAP method underestimated microbial contribution in the cow because i t did not account for the presence of protozoa. 6.3 f f s 35 S is the most common of the radioisotopes used as tracers to distinguish between microbial and dietary protein. Walker and Nader - 25 -(1975) described an i n vivo method for measuring rumen microbial protein 35 synthesis. S-labelled inorganic sulphate was infused continuously into the rumen and the incorporation of sulphur into microbial protein was measured. 3 5 The predominant method of using S as a microbial marker has 35 been to measure differences i n the r a t i o of s p e c i f i c a c t i v i t i e s of S i n either the cystine (Leibholz, 1972), methionine (Beever et a l . , 1974), or t o t a l sulphur amino acids of duodenal digesta and a separated 3 5 microbial f r a c t i o n (Hume, 1974). A possible l i m i t a t i o n of the S method i s the error introduced due to the d i r e c t j o i n i n g of dietary sulphur amino acids into the microbial f r a c t i o n . Also, the method i s quite expensive and tedious. However, i t does have the advantages of not r e q u i r i n g quantitative recovery of sulphur, and can determine t o t a l m icrobial protein synthesis rather than just b a c t e r i a l . 6.4 1 5N The technique of u t i l i z i n g 1 5N as a marker i s expensive and quite complicated, and as a r e s u l t has not been widely used. I t involves 15 15 quantitating N incorporation into microbes from either ( NHi+)2S0i t ( P i l g r i m et a l . , 1970) or ^NH^Cl (Mathison and M i l l i g a n , 1971). The methods are based on the a s s i m i l a t i o n of nitrogen from ammonia, and do not account for microbial protein d i r e c t l y synthesized from amino acids or peptides. 6.5 ^ P Bucholtz and Bergen (1973) proposed an i n v i t r o method for estimating microbial protein production based on the incorporation of P into microbial phospholipids. This approach was l a t e r expanded to - 26 -include the combination of 0 iP-labelled extracellular phosphate in the total microbial phosphorus as the measure of microbial growth (Van Nevel and Demeyer, 1977). However, this method involved many basic assumptions, and later results proved some of them to be invalid. More recently, Smith et a l . (1978) described a technique based on 32 the incorporation of P-labelled inorganic phosphate into rumen 3 2 bacterial nucleic acids. They compared P: non-ammonia nitrogen ratios in samples of rumen bacteria and duodenal contents with similar estimates using DAP and RNA as bacterial markers. Their estimates based 32 upon P~labelled RNA nucleotides were approximately 85% of those based upon total RNA. This difference was attributed to the presence of small amounts of dietary RNA adding to total RNA. Ling and Buttery (1978) assessed the use of RNA, 3 5S, DAP and AEP as microbial nitrogen markers in duodenal digesta. They suggested the use of AEP as a protozoal nitrogen marker was invalid since they found AEP present in large quantities in dietary and bacterial material. They concluded that even though DAP did not account for protozoal contribution to duodenal digesta nitrogen, i t would s t i l l be widely used since the majority of microbial nitrogen is bacterial in origin. Where total microbial values are required, the choice of method becomes either 3 5 35 RNA or S. They concluded that the use of S is more appropriate where precise estimates are required, and the use of RNA where more general microbial nitrogen estimates are needed. 6.6 Amino Acids Evans et a l . (1975) proposed a method for estimating proportions of microbial and dietary proteins in duodenal digesta. It was based - 27 -upon differences i n the amino acid p r o f i l e s of the proteins reaching the duodenum. The method assumed that microbial protein had a constant composition, protein i n each dietary component behaved as a single unit, and the p r o f i l e of the digesta was equal to the sum of a l l the p r o f i l e s c o n t r i b u t i n g to i t . Buttery and Cole (1977) suggested that with more development this technique would y i e l d answers with the least bias, since i t r e l i e d on the analysis of several d i f f e r e n t amino acids. 6.7 ATP The use of ATP as a rumen microbial marker was studied by Forsberg and Lam (1977). They found that extraction of ATP from rumen contents varied i n e f f i c i e n c y , and also observed differences i n the concentration of ATP i n rumen microbes. Wolstrup and Jensen (1978) used s i m i l a r methods to extract ATP and reported less v a r i a t i o n to that found by Forsberg and Lam (1977). Also, they showed the ATP concentration i n the rumen contents was dependent upon the nitrogen source fed to the animals. Wolstrup and Jensen (1978) indicated, however, that more in v e s t i g a t i o n i s necessary before the ATP method could be used r o u t i n e l y . 7. C annulation The measurement of the extent of digestion i n d i f f e r e n t sections of the g a s t r o - i n t e s t i n a l t r a c t has added greatly to the knowledge of ruminant n u t r i t i o n . In the l a t e f i f t i e s and early s i x t i e s methods were developed for the s u r g i c a l preparation of cannulae at various points along the g a s t r o - i n t e s t i n a l t r a c t . These techniques have allowed researchers to make prolonged and repeated quantitative c o l l e c t i o n s of F I G U R E 3. T h e D e s i g n of t h e R e - E n t r a n t C a n n u l a n t e s f i n a l T r a c t Digesta Flow ( A s h , 1 9 6 2 ) - 29 -digesta passing out of the reticulo-rumen and along the i n t e s t i n a l t r a c t . The techniques have been described for sheep by P h i l l i p s o n (1952), Hogan and P h i l l i p s o n (I960), Singleton (1961), Goodall and Kay (1965) and Brown et a l . (1968), and for c a t t l e by Horney et a l . (1972). The p r i n c i p l e of the re-entrant cannula i s i l l u s t r a t e d i n Figure 3 from the design by Ash (1962). Cannulae are usually "homemade", processed most often from materials such as po l y v i n y l c h l o r i d e p l a s t i s o l s (Brown et a l . , 1968). They should be somewhat f l e x i b l e , and the angle of the curved b a r r e l onto the flange should be made to f a c i l i t a t e optimum flow of digesta when i t i s diverted outside the animal. Homey et a l . (1972) found i n t e s t i n a l malfunction leading to general metabolic disorders to be the most common cause for temporary loss of cannulated animals from t h e i r experiment. This was a re s u l t of continuous ingesta loss due to cannula leakage, or from acute depletion due to cannula expulsion. Also, blockage of the cannulae led to serious reduction of i n t e s t i n a l flow r e s u l t i n g i n loss of appetite. However, aft e r the leakage or block was corrected and e l e c t r o l y t e solutions were administered, the symptoms soon disappeared. Egan and Tudor (1976) commented that s u r g i c a l procedures used i n c a t t l e seemed less e f f e c t i v e than comparable ones used i n sheep. They found cannula leakage and early breakdown to be the common experience with c a t t l e , possibly due to greater s t r a i n put on the cannula by the heavier musculature and by the weight of the abomasum and i n t e s t i n e s with t h e i r digesta f i l l . - 30 -MacRae and Wilson (1977) conducted an experiment to examine whether sheep given medium qu a l i t y hay diets might exhibit any chronic stress associated with s u r g i c a l preparation. They found that voluntary feed intake, d i g e s t i b i l i t y and rate of passage of digesta were not s i g n i f i c a n t l y affected by establishment of ei t h e r re-entrant cannulae or simple T-shaped cannulae at both the duodenum and ileum. Several other workers have shown that s u r g i c a l preparation of various cannulae i n calves (Putnam and Davis, 1965) and i n steers (Drori and L o o s l i , 1959; Hayes et a l . , 1964) had l i t t l e e f f e c t on r a t i o n d i g e s t i b i l i t y . - 31 -MATERIALS AND METHODS 1. Cannulation Two Holstein and two Ayrshire steers, each approximately one year old, were used in this study. A l l four had re-entrant cannulas inserted at the duodenum employing the technique described by Homey et a l . (1972). The steers were prepared for surgery by withholding the two previous feedings, and both pentathal and nembutal were administered as anaesthetics - dosage 6 mg./kg. body weight. The surgical incision was made to expose the right side of the abdominal cavity and done and in a vertical cranial direction. After the abdominal cavity was entered, the pylorus and the duodenum were identified. The midpoint of the exposed duodenum was immobilized using intestinal clamps, then transected, and the proximal and distal portions of the duodenum were sutured closed. To prevent any flow of ingesta into the closed segments of the intestine, intestinal compression forceps were used. A small opening was made in each segment in order to insert both ends of the cannula, and then a purse-string suture was used to secure them. The suture ends were l e f t long so that the intestinal (duodenal) portions could be joined to the abdominal wall. Once both sections of the cannula were in place, the ends of the cannula were plugged to prevent contamination of the abdominal cavity. Stab incisions were then made in the abdominal wall, and the cannula and long suture ends were brought through to the outside and secured. A piece of plastic tubing from the barrel of a syringe was used to join the two cannula ends and - 32 -allow Ingesta to flow. After surgery was completed, antibiotics were administered throughout the recovery period in order to prevent infection. After the operations, daily record sheets were kept with information on temperature, appetite, bowel movements, cannula leakage, and any treatment the steer received (electrolyte or antibiotic). Other things closely watched were ingesta flow, cud chewing, urination, breathing and general alertness. The area around the cannula was kept clean with frequent washings and iodine spray. If there was a cannula blockage, rubber tubing and Lactated Ringer's solution were used to clear the blockage and rehydrate the animal. Homey et a l . (1972) reported that after surgical operation, animals were able to undergo metabolic experiments for long periods, and their l i f e expectancy did not seem to be reduced. However, they also stated that repeated expulsion of cannulae, blockages, and excessive gastro-intestinal leakage could markedly reduce animal longevity. Unfortunately, in this experiment, these problems were encountered on numerous occasions, resulting in dehydration and electrolyte imbalance in the animals. Also, internal contamination and infection were problems, resulting in elevated temperatures and loss of appetite. Infection probably resulted from abrasion to the abdominal wall and intestine, due to the expulsion and subsequent replacement of the cannulae. Regretfully, three of the four steers died prematurely and as a result, the data from these animals are limited. Weight gain data was d i f f i c u l t to compile since there were many fluctuations due to illness. - 33 -When cannula problems were minimal, a l l the steers gained weight normally and seemed well adapted to their cannulae. A l l of the data compiled in the tables and subsequently used in the s t a t i s i t i c a l analysis were collected when the cannulae were functioning with minimal problems. Data obtained when the cannulae were malfunctioning (blockage, leakage) were not used. 2. Digestibility Trials Each steer was subjected to a four-day collection period in order to estimate the digestiblity of the ration. The five forages that were tested on these t r i a l s were low quality grass hay, a l f a l f a cubes, high quality grass hay, Italian ryegrass and orchard grass. The low and high quality hays were made up of mixed grasses, and the ryegrass and orchard grass were from pure stands. A l l the hays and grasses were put through a hay buster so the forages given to the steers were of uniform size. Each steer was gradually adapted to a new forage with a two-week changeover period. Data was collected from two steers before cannulation in order to compare the results with subsequent cannulation data. The steers were fed twice daily, at 1 p.m. and 3 a.m., in two equal portions at either a maintenance or ad libitum level. The maintenance level of feeding was 15% above NRC maintenance requirements to ensure a steady gain for the steers. The feeding level at ad libitum was restricted by 10% below ad libitum to help encourage cleanup by the steers. A mineral mix was added routinely at each feeding. Salt was offered free choice. A metabolism crate was built to house the steers during the - 34 -feeding t r a i l s but, unfortunately, the animals did not perform well inside the crate. They were very nervous and refused to l i e down at a l l , and as a result fatigue and loss of appetite were both factors. It was decided to move the steers into a free-stall area for the dig e s t i b i l i t y t r i a l s where they could be more relaxed and perform normally. This turned out to be more successful but made urine collection impossible. Feces were collected in a box fitted into the gutter behind each steer, and then were shovelled into a large, plastic bucket and kept covered. The collection days always started at 9.00 a.m., and the amounts of feces were weighed, recorded, mixed often and sampled daily. A 500 g. fecal grab sample was obtained daily from each steer for component analysis, put in an aluminum tray, covered and frozen immediately. At the end of the t r i a l these samples were thawed, and a composite sample was analysed for dry matter by oven drying at 65°C for 72 hours. Chromic oxide In gelatin capsules were used as an indigestible marker in order to obtain di g e s t i b i l i t y data at the duodenum. The chromic oxide was Incorporated in the t r i a l at a level of .5% of the forage fed, and one capsule was given at each feeding. Duodenal samples were collected three times daily, 10 a.m., 12 p.m., and 2 p.m., with no relation to time of feeding. Approximately 100 ml. of digesta was collected into a whirl-pak bag and then each sample was frozen to be used for analysis later. 3. Laboratory Analysis In the laboratory, oven-dried forage and fecal samples, and freeze-dried duodenal samples were ground and analysed in duplicate for - 35 -dry-matter, ADF, nitrogen and ADF-nitrogen. Forage samples were also analysed for organic matter. For f i b r e and ADF-N determination, procedures of Goering and Van Soest (1970) were followed. For nitrogen determination, procedures i n the A.O.A.C. (1975) were followed. The duodenal samples were thawed and freeze-dried rather than oven-dried i n order to minimize protein loss or damage, and were analysed for chromic oxide and microbial nitrogen. Chromic oxide analysis was done using an atomic absorption spectrophotometer, employing the technique reported by Williams et a l . (1962). In order to d i s t i n g u i s h between dietary and microbial nitrogen c o n t r i b u t i o n i n the duodenal samples, i t was necessary to determine the quantity of microbial nitrogen present. Smith and McAllan (1970, 1971) reported that dietary nucleic acids are rapidly degraded i n the rumen and do not contribute appreciably to nucleic acid concentrations i n the digesta. Since nucleic acids form a q u a n t i t a t i v e l y important part of the nitrogen presented to the ruminant duodenum, i t was assumed that these nucleic acids were derived almost e n t i r e l y from microbial o r i g i n . M i c r o b i a l nitrogen determination was done by using a modified technique of the one described by McAllan and Smith (1969). In t h e i r study, they examined i n d e t a i l the extraction and estimation of n u c l e i c acids, s p e c i f i c a l l y RNA and DNA, i n bovine digesta. Once the concentration of RNA In the digesta was determined, an estimate of the amount of microbial nitrogen was obtained. Dried 100 mg. duodenal samples were hydrolysed with 10 ml. N-K0H for 18 hours at 37°C. Af t e r centrifugation, the supernatant f r a c t i o n - 36 -containing RNA which had been hydrolysed to acid soluble nucleotides was decanted. 4.4 ml. 4N -HC10i t was added to the supernatant, l e f t for 30 minutes, and then the mixture was centrifuged. The supernatant fraction was neutralized with 3.5 N KOH, and fin a l l y N-KOH, and the mixture was then fil t e r e d and the f i l t r a t e was saved. Dowex-1 x 8, 20-50 mesh, resin was used to purify and collect the RNA molecules from the f i l t r a t e . A large quantity of Dowex was poured into a beaker, washed with demineralized water, the fines were poured off, and the beaker was then covered. A small beaker was used for each f i l t r a t e sample and a quantity of the resin was poured into each beaker. The water was then poured off, leaving the resin s t i l l moist, and the f i l t r a t e was then added. The mixture was left to stand for 30 minutes, occasionally stirring with a glass rod. The f i l t r a t e was then poured off, again making sure the resin was s t i l l moist and the RNA molecules were l e f t attached to the Dowex resin. The resin was then washed with .025M-tris, and the supernatant was decanted and saved to check the RNA level ( i t should be 0). 50 ml. of .5N-HC1 was then added which acted to remove the RNA molecules from the resin. The quantity of RNA i n the solution was determined by ultraviolet absorption at 260 my. Several constants were used in the calculation of microbial nitrogen from the quantity of RNA. As a result, a brief description is given showing the constants and steps used in the conversion. McAllan and Smith (1969) expressed their results in terms of highly-polymerized yeast RNA, 14% nitrogen. - 37 -Pure RNA Yeast = 20 units absorbance/mg. (Handbook of Bio-Chemistry) g. RNA/100 g. Duodenal sample g. RNA at Duodenum/Day Yeast RNA = 14% Nitrogen (McAllan & Smith, 1969) .*., = g. RNA-N at Duodenum/Day .114 g. RNA-N/g. Total Microbial Nitrogen (McAllan & Smith, 1972) ."., = g. Total Microbial N at Duodenum/Day 4. S t a t i s t i c a l Analys is In this study, forage type, status (intact or cannulated), feeding level (maintenance or ad libitum), day and time were the various treatment parameters analysed. A l l observations recorded during the digestiblity t r i a l s were subjected to an analysis of variance (ANOVA), using the package program UBC BMD10V adapted by Bjerring et a l . (1975). The Newman-Keuls test was used for the comparison of means. The differences between treatments were tested at a significance level of 5% throughout the entire experiment. Results from interactions between the various treatments were not significant and were, therefore, deleted from the data tables. - 38 -The models used to determine the effects on apparent digestiblity and microbial nitrogen were: TABLE 2 Y i j = u + Si + Fj + SFij + E i j Where u = overall mean common to a l l samples Si = the effect of the i * " * 1 status th Fj = the effect of the j forage t i l SFij = the interaction of the j forage within the I status E i j = the unexplained residual error associated with each sample TABLE 3 Yi = u + Fi + E i Where u = overall mean common to a l l samples th F i = the effect of the i forage Ei = the unexplained residual error associated with each sample - 39 -Y i j = Where TABLE 4 u + Fi + Lj + FLij + E i j = overall mean common to a l l samples th F i = the effect of the i forage th Lj = the effect of the j level t i l FLij = the interaction of the j feeding level th within the i forage E i j = the unexplained residual error associated with each sample Where TABLE 5 Yijk = u + Fi + Dj + Tk + F Dij + FTik + DTjk + Eijk u = overall mean common to a l l samples F i = the effect of the i forage Dj = the effect of the j day Tk FDij FTik DT jk th the effect of the k time the interaction of the j*"*1 day within the i* " forage the interaction of the k*"*1 time within the i ^ forage th th , the interaction of the k time within the j day - 4 0 -TABLE 6 Y i j k l = u + Fi + Lj + Dk + TI + FLij + FDik + F T i l + LDjk + DTkl + E i j k l Where u — overall mean common to a l l samples Fi = the effect of the i * " * 1 forage Lj = the th effect of the j feeding level Dk = the th effect of the k day TI = the effect of the l * " * 1 time FLij the interaction of the j ^ feeding level within the i FDik = the interaction of the th k day within the .th . i forage F T i l = the interaction of the th 1 time within the i* " * 1 forage LDjk = the interaction of the th k day within the j*"** feeding L t j l = the interaction of the th 1 time within the jf ckfeeding DTkl = the interaction of the l^time within the k*"*1 day th E i j k l = the unexplained residual error associated with each sample - 41 -RESULTS AND DISCUSSION The composition of the various forages is presented in Table 1. The organic matter, dry matter and fibre contents differed only slightly between forages, with the main difference being in the nitrogen content. The low quality hay and Italian ryegrass had much lower nitrogen percentages than the other three forages. The high quality hay clearly had the highest nitrogen content. In Table 2, the effects of cannulation and forage type on animal performance are examined. Cannulation did not affect (p>.05) feed intake, fibre and nitrogen d i g e s t i b i l i t i e s and only dry matter dig e s t i b i l i t y was found to be significantly different (p<.05). These results are in agreement with several authors. Harris and Phillipson (1962) observed that duodenal re-entrant cannulation in five ewes did not adversely affect feed consumption and di g e s t i b i l i t y of a hay diet. MacRae (1974) reported l i t t l e difference between di g e s t i b i l i t y coefficients for hay-plus-barley diets in intact and cannulated sheep (duodenal and i l e a l ) . Also, MacRae and Wilson (1977) found that feed intake and digestiblity in sheep were not significantly affected by duodenal and i l e a l cannulation. Similar intact versus cannulated experiments with cattle were found to be more scarce in the literature, probably due to the greater expense and risk of loss. Hayes et a l . (1964) examined the influence of ruminal, abomasal, and intestinal fistulation on digestion in steers. Both intact and fistulated steers were tested and i t was found that fistulation had no significant effect on apparent digestion of dry matter, energy, crude fibre or crude protein. TABLE 1. Forage Composition (D.M. basis) Sample O.M.% D.M.% A.D.F.% NITR.% A.D.I.N.% A.D.F. - N (% of Total) Low Quality Grass Hay 91.5 85.5 34.3 1.7 0.88 17.7 A l f a l f a Cubes 90.7 88.4 36.2 2.3 0.93 14.7 High Quality Grass Hay 90.1 86.5 33.7 2.7 1.03 12.9 I t a l i a n Ryegrass 92.3 85.6 32.9 1.7 0.88 17.0 Orchard Grass 91.7 85.8 30.9 2.1 0.73 10.7 ADIN % x ADF % % * Total HI X 1 0 0 TABLE 2. E f f e c t of Status and Forage Type on Intake and Apparent D i g e s t i b i l i t y (Total C o l l e c t i o n Method) D.M.INT./DAY (kg.) D.M.DIG. (%) FIBRE DIG. (%) NITR. DIG. (%) e mean st.dev. e mean st.dev. e mean st.dev. e mean st . dev. Status 1 5.20 1.14 61.82 1.19 51.15 4.79 60.22 9.32 Status 2 5.43 1.70 59.95 1.93 47.27 3.50 58.00 14.04 S i g n i f . ns ns ns Forage 1 4.22 0.81 59.62 1.58 46.67 3.50 49.47 5.44 Forage 2 6.40 0.66 62.15 0.85 51.75 3.98 68.75 3.16 S i g n i f . * * ns * * = s i g n i f i c a n t difference at p<.05 ns = not s i g n i f i c a n t n = 4 Status 1 = intact (#2, 4) Status 2 = cannulated (#1, 2, 3) Forage 1 = low quality hay (#1, 2, 3, 4) (at maintenance l e v e l ) Forage 2 = a l f a l f a cubes (#1, 2, 4) no s i g n i f i c a n t interactions Animal I d e n t i f i c a t i o n s H o l s t e i n A WL H o l s t e i n B #2 Ayrshire A #3 Ayshire B #4 - 44 -As shown in Table 2, the intakes of the two forages were significantly different. In every case, each steer consumed more al f a l f a cubes per day than low quality hay. This may have been partly due to the greater palatability of the cubes and a preference by the steers to the physical form. Decreased feed intake of the coarse hay ration may also have been due to a lower nitrogen level in the feed. Weston and Hogan (1967) and Liebholz and Hartmann (1972) found that sheep given the low nitrogen hay diet had greatly reduced feed intake and apparent digestiblity of dry matter. It is generally accepted that the digestiblity of nutrients tends to decrease as the percentage of protein in the diet decreases, especially at the lower levels of protein content. In ruminants, the addition of protein or of nitrogen compounds util i z e d by microorganisms to a ration having a wide nutritive ratio increases the breakdown of crude fibre, and in turn makes other nutrients more digestible (Schneider & Flatt, 1975). In Table 2, the dry matter dige s t i b i l i t y of the low quality hay forage was significantly lower than that of the a l f a l f a cubes. Weston and Hogan (1967) and Liebholz and Hartmann (1972) also reported a marked decrease in nitrogen digestiblity when the sheep were fed the low nitrogen diet. Table 2 shows a similar significant decrease in nitrogen dig e s t i b i l i t y for the lower nitrogen, low quality hay diet. This is also in agreement with Bratzler et a l . (1959) and Markley et a l . (1959). The effect of forage type on intake and dig e s t i b i l i t y using two collection methods is presented in Table 3. Chromic oxide capsules were used to obtain digestiblity data at the duodenum and also to provide a check against the total collection method. Since cannula leakage or TABLE 3. E f f e c t of Forage Type on Intake and Apparent D i g e s t i b i l i t y (Total C o l l e c t i o n and Chromic Oxide Methods - Digesta Values not Adjusted for 100% C r 2 0 3 Recovery) Forage 1 Forage 2 Forage 3 Forage 4 Forage 5 S i g n i f . D.M.INT/DAY(kg.) 4.10 6.75 5.90 4.80 5.95 st.dev. 1.13 0.64 0.75 0.42 0.21 TOT.D.M. DIG (%) 58.40 61.50 62.70 60.40 61.30 ns st.dev. 1.13 0.57 0.48 0.57 1.41 T0T.FIBR.DIG.(%) mean 45.35 49.20 54.30 47.75 52.05 st.dev. 4.03 2.40 1.22 0.64 0.92 T0T.NITR.DIG.(%) 46.10 69.908 69.60 56.901 60.45 ab ab st.dev. 4.95 0.42 0.51 2.26 2.62 CR.D.M.DIG.(%) mean 49.30 52.60 55.90 53.10 53.75 st.dev. 5.37 0.28 0.77 0.28 0.50 CR.FIBR.DIG.(Z) 33.50 37.30 46.20 48.30 42.80 ns st.dev. 9.90 1.84 2.31 0.28 0.28 CR.NITR.DIG.(%) 34.35 62.85J 64.10* 49.001 52.651 st.dev. 10.82 0.21 3.40 1.70 2.05 * - s i g n i f i c a n t difference at p<.05 ns - not s i g n i f i c a n t Forages: 1 - low qua l i t y hay (#1, 3) 2 " a l f a l f a cubes (#1, 2) 3 - high quality hay (#1, 2) 4 - I t a l i a n ryegrass (01 - maint. and ad l i b ) 5 = orchard grass (#1 - maint. and ad l i b ) A l l steers cannulated - 46 -expulsion could have seriously affected digesta flow and hence overall d i g e s t i b i l i t y results, the recoveries of Cr£03 provided a good indication of the accuracy of the collection procedures. The recovery rates of Cr2C>3 in the digestiblity t r a i l s averaged about 83% (range 78.4 - 85.3%), which was good considering some of the problems encountered. Harris and Phillipson (1962), using twelve-hour collection periods, reported Cr£03 recovery rates at the duodenum of 86-90%, with sheep fed on dried grass diets. MacRae et a l . (1972), using twenty-four collection periods, found recoveries of ( ^ 0 3 at the duodenum averaged 82%, with sheep fed on dried grass diets. Forage type had no significant effect (p>.05) on dry matter intake, and dry matter and fibre d i g e s t i b i l i t y . For the higher nitrogen forages, one would have expected the dig e s t i b i l i t y values for dry matter and fibre to be greater due to the increased dietary nitrogen level and increased soluble carbohydrates. These values were indeed greater but not significantly. The dry matter digestiblity values appear to be consistent with literature values when the nitrogen content of the forage is similar. Topps et a l . (1968 a), in experiments with cannulated ewes fed grass hay diets, reported a mean of 61.8% dry matter dig e s t i b i l i t y with a hay diet containing 1.6% nitrogen. Topps et a l . (1968 b), using two cannulated steers on hay diets, observed dry matter digestiblity values of 65.6% for the 1.9% nitrogen diet, and 61.8% for the 1.5% nitrogen diet. Nitrogen dig e s t i b i l i t y of each forage proved to be significantly different. Dry matter intake did not have a significant effect, but overall nitrogen intake probably was the influencing factor. The total - 47 -nitrogen d i g e s t i b l i t y values i n Table 3 seem to be i n agreement with the range of l i t e r a t u r e values. Coelho da S i l v a et a l . (1972 a) offered a chopped, dried lucerne diet containing 2.55% nitrogen to cannulated sheep and observed a mean apparent nitrogen d i g e s t i b l i t y of 71.1%. MacRae et a l . (1972), using cannulated sheep on a dried grass diet containing 2.3% nitrogen, reported a mean nitrogen d i g e s t i b i l i t y value of 69.4%. As shown i n Table 4, feeding l e v e l had no s i g n i f i c a n t e f f e c t on any parameter except t o t a l nitrogen d i g e s t i b l i t y . Even though a greater quantity of forage was offered to the steers at the ad libi t u m l e v e l , dry matter intake for the two forages was not s i g n i f i c a n t l y d i f f e r e n t at the maintenance or ad li b i t u m l e v e l s . Again, i t was l i k e l y that increased o v e r a l l nitrogen intake was the cause for the higher nitrogen d i g e s t i b l i t y value. With regards to forage type, orchard grass containing a higher nitrogen concentration showed a s i g n i f i c a n t l y higher f i b r e and nitrogen d i g e s t i b l i t y than the I t a l i a n ryegrass. The r e s u l t s i n Table 5 show that forage type had a s i g n i f i c a n t e f f e c t on a l l d i g e s t i b l i t y parameters i n samples c o l l e c t e d at the duodenum. The lower nitrogen forages, I t a l i a n ryegrass and low q u a l i t y hay, had the highest values for proportion of d i g e s t i b l i t y of dry matter and f i b r e i n the stomach. (Stomach refers to reticulo-rumen, omasum, and abomasum). The highest nitrogen forage, high q u a l i t y hay, had the l e a s t proportion of d i g e s t i b i l i t y of dry matter and f i b r e occur i n the stomach. These re s u l t s show that the lower nitrogen forages, containing a higher proportion of lower q u a l i t y protein and c e l l u l o s e , had a slower degradation rate i n the rumen, slower rate of passage, and hence more 4. Eff e c t of Forage Type and Feeding Level on Intake and Apparent D i g e s t i b i l i t y D.M.INT/DAY(kg.) T0T.D.M. DIG (%) T0T.FIBR.DIG.(%) T0T.NITR.DIG.(%) CR.D.M.D IG.(%) CR.FIBR. DIG.(%) CR.NITR. DIG.(%) e mean st.dev. e mean st.dev. e mean st.dev. e mean st.dev. e mean st.dev. e mean st.dev. e mean st.dev. Forage 4 4.80 0.42 60.40 0.57 47.75 0.64 56.90 2.26 53.10 0.28 38.30 0.28 49.00 1.70 Forage 5 5.95 0.21 61.30 1.41 52.05 0.92 60.45 2.62 53.75 0.50 42.80 0.58 52.65 2.05 * * ns ns S i g n i f . ns ns Feeding Level 1 5.15 0.92 60.15 0.21 49.35 2.90 56.95 2.33 53.35 0.07 40.75 3.18 49.50 2.40 2 5.60 0.71 61.55 1.06 50.45 3.18 60.40 2.69 53.50 0.85 40.35 3.42 52.15 2.76 ns * ns ns ns S i g n i f . ns ns 1 * - s i g n i f i c a n t difference at p<.05 ns • not s i g n i f i c a n t e n - 2 Forages: 4 - I t a l i a n ryegrass (#1) 5 - Orchard grass (#1) Feeding Level: 1 " Maintenance + 15% 2 - Ad libitum - 10% ( *• 0 0 (Stomach = Rettculo-Rumen, Omasum, Abomasum) (Digesta Values Adjusted for 100% C r 2 0 3 recovery) STOMACH D.M. DIG (%) Forage 1 Forage 2 Forage 3 Forage 4 Forage 5 S i g n i f . Day 1 [Day 2 | Day 3 Day 4 Si g n i f . STOMACH FIBR. DIG.(Z) mean 69.55" 63.26C 62.84° 68.47° 65.82 b * 66.14 65.72 65.33 65.72 at.dev. STOMACH NITR. DIG.(%) mean 5.54 89.54 a 4.88 1.10 82.31 b 1.05 0.94 74.88c 1.21 0.85 87.66 a 0.84 1.06 81.55 b 0.88 * 3.12 83.15 3.91 4.05 82.84 4.46 3.95 82.49 4.52 3.33 82.75 3.89 ns st.dev. -30.35" 5.80 a 7.34a -22.07 c -14.28 b * -12.16 -13.04 -12.76 -12.91 ns st.dev. 5.40 2.22 1.86 2.78 3.61 15.58 14.74 15.11 15.27 MTCROB. NITR. (g.) (AT DU0D.) mean 70.79° 81.42 b 75.83c 77.25C 92.75* 79.70 79.96 80.33 80.11 st.dev. 5.79 5.42 2.44 4.58 3.55 9.40 9.07 9.01 8.99 * = s i g n i f i c a n t difference at p<.05 ns - not s i g n i f i c a n t e n - 24 (Forage) (2 forage x 4 day x 3 time) e n = 30 (Day) (10 forage x 3 time) MICR0B N: TOTAL N (AT DU0D.) mean 0.80" 0.56C 0.51d 0.77s 0.651 0.67 0.67 0.68 0.67 ns Forages: 1 - low quality hay (01, 3) 2 - a l f a l f a cubes (#1, 2) 3 = high qu a l i t y hay (#1, 2) 4 = I t a l i a n ryegrass 01 - maint. and ad l i b ) 5 = Orchard grass (ffl - maint. and ad l i b ) st.dev. 0.07 0.06 0.02 0.03 0.02 0.11 0.13 0.12 0.12 (contd.) ( vo I TABLE 5: (Cont.) STOMACH D.M. DIG. (%) STOMACH FIBR. DIG.(%) STOMACH N i l •R. DIG.(%) MICROB. NI (AT DI Tn. (g.) 0D.) MICROB N: (AT DI TOTAL N OD.) e mean st.dev. e mean st.dev. e mean st.dev. e mean st.dev. e mean st.dev. Time 1 Time 2 Time 3 S i g n i f . 65.58 65.66 65.39 ns 3.71 3.69 3.46 83.07 82.84 82.43 ns 4.08 4.35 4.12 -12.86 -12.76 -12.53 ns 14.81 15.07 15.45 80.00 80.14 79.94 ns 8.66 9.24 9.33 0.67 0.68 0.68 ns 0.12 0.12 0.12 * - s i g n i f i c a n t difference at p<.05 ns «* not s i g n i f i c a n t e n - 40 (10 forage x 4 day) Time: 1 - 10 a.m. 2 - 1 2 p.m. 3 = 3 p.m. no s i g n i f i c a n t interactions O I - 51 -rumen digestion. The higher nitrogen forages, containing a higher proportion of soluble protein and soluble carbohydrates, had a faster degradation rate in the rumen, shorter retention time, and hence less rumen digestion. Hungate (1966) reported that the rate of microbial degradation of soluble carbohydrates exceeds the rate of passage so very few soluble carbohydrates escape ruminal fermentation. Van Soest (1975) found that increased rate of passage in the rumen w i l l decrease the extent of roughage digestion. In their experiments, Cloete (1964) and Bruce et a l . (1966) found that the addition of protein to diets composed of low quality hay or hay plus maize, increased the relative absorption of dry matter and organic matter from the small intestine. For the higher nitrogen forages, the disappearance of nitrogen from the rumen in the form of NPN compounds (eg. urea, NH3) was also an influential factor. The range of dry matter digestiblity values in Table 5 agrees well with values found in the literature. Hogan and Phillipson (1960), using sheep with duodenal re-entrant cannulas on hay plus cereal diets, estimated approximately 70% of the dry matter disappeared in the stomach. Ridges and Singleton (1962), using duodenal cannulated goats on a variety of diets, found that 58% of the dry matter digestiblity occurred in the stomach. In their experiments with cannulated sheep on a hay diet, Topps et a l . (1968a) reported 67% of the digestible dry matter disappeared in the stomach. Beever et a l . (1972) reported 64.4% digestiblity in the stomach of a ryegrass forage fed to sheep cannulated at the duodenum. The range of fibre d i g e s t i b i l i t y values in Table 5 agrees well with values found in the literature. From their experiments, Ridges and - 52 -Singleton (1962) reported 93% f i b r e d i g e s t i b i l i t y i n the stomach, and Beever et a l . (1972) found 94.3% c e l l u l o s e d i g e s t i b l i t y . MacRae and Armstrong (1969), using cannulated sheep on a hay d i e t , found c e l l u l o s e to be 91% d i g e s t i b l e i n the stomach. Watson et a l . (1972), using cannulated l a c t a t i n g cows on a dried grass, flaked corn d i e t , found 88.9% c e l l u l o s e d i g e s t i b l i t y i n the stomach. In Table 5, the lower nitrogen forages showed s i g n i f i c a n t l y lower proportion of nitrogen d i g e s t i b l i t y i n the stomach than the higher nitrogen forages. This was due to the substantial net addition of nitrogen i n the duodenal samples c o l l e c t e d for the lower nitrogen forages. In f a c t , the higher nitrogen forages showed only s l i g h t nitrogen digestion i n the stomach. This would agree with the findings of Bergen (1978), mentioned i n an e a r l i e r section, who found the majority of protein digestion and amino acid absorption takes place i n the small i n t e s t i n e . Also, one would expect excessive N H 3 production and nitrogen disappearance from the rumen from the high nitrogen forages. As mentioned e a r l i e r , R o f f l e r and Satter (1975 a) found that above 13% dietary crude protein the rumen-NH3 concentration increased r a p i d l y with increasing protein, and none of rumen-NH3 i s incorporated into microbes. When rumen-NH3 was u t i l i z e d completely for microbial protein production, the proportion of metabollzable protein was much higher than when rumen-NH3 was i n excess. They concluded that low protein diets had a greater proportion of metabollzable protein than high protein d i e t s . S i milar changes i n the nitrogen content of the digesta were reported by Harris and P h i l l i p s o n (1962) and Clarke et a l . (1966). This - 53 -effect of nitrogen addition is of great importance to the ruminant in helping i t overcome a deficiency of dietary nitrogen. Because of the wide variety of diets used and the further complication of net addition of nitrogen to digesta, i t was d i f f i c u l t to find an exact range of values in the literature. However, the basic trends of nitrogen dig e s t i b i l i t y in the stomach were easy to identify. Ridges and Singleton (1962) reported 11% crude protein digestibility, and Hogan and Phillipson (1960) found 25% nitrogen digestiblity in the stomach. Topps et a l . (1968 a, b) reported substantial net additions of nitrogen to the stomach contents when both sheep and steers were fed hay diets. Van't Klooster and Rogers (1969), using cannulated cows on a variety of hay and hay plus concentrate diets, found the amount of nitrogen flowing through the proximal duodenum was greater than the amount ingested. Watson et a l . (1972), reported a 15.5% increase in nitrogen at the duodenum, and 88.9% di g e s t i b i l i t y of nitrogen in the small intestine. Beever et a l . (1972) and Coelho da Silva et a l . (1972 b), reported net nitrogen increases of 7.3% and 12.5% on two different hay diets, with corresponding nitrogen dig e s t i b i l i t y in the small intestine of 97.4% and 89.9% respectively. At the duodenum, forage type had a significant effect on the amount of microbial nitrogen and the ratio of microbial nitrogen: total nitrogen. Table 5 shows that the higher nitrogen forages had greater quantities of microbial nitrogen present in the duodenal samples. This was to be expected, and the more meaningful parameter would be the proportion of total nitrogen at the duodenum that is microbial nitrogen. These values clearly indicate that the lower nitrogen forages - 54 -had the highest ratio of microbial nitrogen: total nitrogen. This was like l y due to the lower dietary nitrogen intake and the relatively increased production of microbial protein in the rumen. The lower nitrogen forages, containing a greater proportion of lower quality protein and cellulose, had a longer retention time in the rumen and thereby produced higher yields of microbial c e l l s . Recycled dietary nitrogen into the rumen via the saliva was probably an influencing factor as well. Leibholz (1972) concluded that the percentage of microbial protein In the rumen and duodenum is inversely related to the dietary nitrogen intake. Literature values for the ratio of microbial nitrogen: total nitrogen were quite varied due to the wide variety of diets and the dietary source of nitrogen. However, the trend and the range of values in Table 5 were in good agreement with many authors. Smith et a l . (1968), feeding cannulated calves a variety of hay and cereal diets, reported an average of 65% of total rumen nitrogen to be microbial nitrogen. They also found that the proportion of microbial nitrogen: total nitrogen in digesta entering the duodenum paralleled that in rumen flu i d but was consistently slightly lower. They attributed this difference to be due to the addition of endogenous nitrogen in the abomasal secretions. Smith and McAllan (1970) fed cannulated calves several different diets and found 55-80% of the total nitrogen in rumen flu i d was of microbial origin, with the higher percentages corresponding to lower dietary nitrogen intake. Smith and McAllan (1971) fed cannulated calves various hay plus maize diets and reported 40-55% of - 55 -the t o t a l nitrogen i n duodenal contents was of microbial o r i g i n . Leibholz (1972) fed cannulated sheep a low nitrogen and high nitrogen lucerne d i e t and found 96.9% and 53.3% microbial nitrogen i n the rumen i n the low and high nitrogen diets r e s p e c t i v e l y . Smith et a l . (1978), using cannulated steers fed a v a r i e t y of d i e t s , reported that 53-83% of the t o t a l nitrogen at the duodenum was m i c r o b i a l . Again, the diets with the lowest d a i l y nitrogen intakes had the highest r a t i o s of microbial nitrogen: t o t a l nitrogen. The day of the d i g e s t i b l i t y t r i a l and the time of day of the sample c o l l e c t i o n had no s i g n i f i c a n t e f f e c t on the d i g e s t i b l i t y parameters and microbial nitrogen. In f a c t , the r e s u l t s i n Table 5 show that the values were almost i d e n t i c a l for each p a r t i c u l a r day and time. Experiments i n the l i t e r a t u r e that used longer term c o l l e c t i o n s were li m i t e d , however Thompson and Lamming (1972) obtained l i t t l e day-to-day v a r i a t i o n i n observed flows of duodenal digesta during seventy-two hour continuous c o l l e c t i o n s . The r e s u l t s i n Table 6 show that forage type had a s i g n i f i c a n t e f f e c t on a l l d i g e s t i b i l i t y parameters and microbial nitrogen at the duodenum. (Values taken from Table 5). Feeding l e v e l had a s i g n i f i c a n t e f f e c t on f i b r e and nitrogen d i g e s t i b l i t y , and t o t a l microbial nitrogen. As was shown i n Table 4, dry matter intake for the two forages was not s i g n i f i c a n t l y d i f f e r e n t at the maintenance or ad l i b i t u m l e v e l s . As a r e s u l t , the higher l e v e l of nitrogen i n the orchard grass forage, and hence the increased nitrogen intake, was the major cause of the higher l e v e l s of d i g e s t i b l i t y and microbial nitrogen. Feeding l e v e l did not s i g n i f i c a n t l y a f f e c t the r a t i o of microbial nitrogen: t o t a l - 56 -nitrogen of the two forages. Day and time had no significant effect on the di g e s t i b i l i t y parameters and microbial nitrogen. Table 7 shows the determination and ut i l i z a t i o n of bypass nitrogen for the various forages. The lower nitrogen forages, with the highest ratios of microbial nitrogen: total nitrogen at the duodenum, had the lowest quantities of bypass nitrogen. Apparent dige s t i b i l i t y of the bypass nitrogen on these lower quality protein diets was very low, thereby providing l i t t l e benefit from the bypass nitrogen to the animal. In contrast, the bypass nitrogen from the higher nitrogen forages was highly digestible and hence more beneficial. TABLE 6. E f f e c t of Forage Type, Feeding Level, Day and Time on Proportion of Apparent D i g e s t i b i l i t y Occurlng in the Stomach and on Microbial Nitrogen STOMACH D.M. DIG. (%) Forage 4 Forage 5 S i g n i f . Feeding Level 1 2 S i g n i f . STOMACH FIBR. DIG.(%) mean 68.47 65.82 * 66.53 67.22 st.dev. 0.85 1.06 0.94 1.37 STOMACH NITR. DIG.(%) mean 87.66 81.55 * 84.59 85.94 * - s i g n i f i c a n t difference at p<.05 ns = not s i g n i f i c a n t 6 n ~ 24 (2 forage x 4 day x 3 time) Forages: 4 ™ I t a l i a n ryegrass (#1) 5 • orchard grass (#1) Feeding Level: 1 = maintenance + 15% 2 - ad libitum - 10% st.dev. 0.84 0.88 2.41 2.42 -22.07 -14.28 * -19.34 -17.01 st.dev. 2.78 3.61 3.22 6.26 MICROB. NITR. (g.) (AT DU0D.) 77.25 92.75 * 82.08 87^92 st.dev. 4.58 3.55 9.06 7.68 MICROB N: TOTAL N (AT DUOD.) 0.77 0.65 * 0.71 0.71 st.dev. 0.03 0.02 0.08 0.05 (contd.) TABLE 6: (Cont.) STOMACH D.M. DIG. (%) STOMACH FIBR. DIG.(%) STOMACH NITR. DIG.(%) MICROB. NI (AT Dl TR. (g.) I0D.) MICROB N: (AT Dl TOTAL N I0D.) e mean st.dev. e mean st.dev. e mean st.dev. e mean st.dev. e mean st.dev. Day 1 68.19 0.91 85.75 . 2.44 -17.55 5.24 84.25 9.67 0.71 0.06 Day 2 67.67 1.36 85.36 2.21 -17.63 5.04 85.00 8.89 0.72 0.08 Day 3 66.91 0.86 84.95 2.58 -19.11 5.47 86.17 8.22 0.72 0.07 Day 4 67.88 1.31 85.72 2.70 -18.40 4.99 84.58 9.48 0.71 0.06 S i g n i f . ns ns ns ns ns Time 1 67.35 1.36 85.36 2.25 -18.67 4.75 85.06 8.21 0.71 0.07 Time 2 67.58 1.22 85.62 2.61 -18.21 5.83 85.19 8.96 0.71 0.06 Time 3 67.37 0.99 85.05 2.57 -17.64 4.82 84.75 9.79 0.71 0.07 Si g n i f . ns ns ns ns ns * » s i g n i f i c a n t difference at p<.05 Time: 1 = 10 a.m. 2 - 2 p.m. ns » not s i g n i f i c a n t 3 = 2 p.m. no s i g n i f i c a n t interactions e n - 12 (Day) (2 feeding l e v e l x 2 forage x 3 time) e n ~ 16 (Time ) (2 feeding l e v e l x 2 forage x 4 day) I Ln oo I - 59 -TABLE 7. U t i l i z a t i o n of Bypass Nitrogen *FECAL FECAL SAMPLE TOT. NITR. INTAKE(g.) « T R . DIG. (%) DIG. NITR. (gO FECAL NITR. (g.) MICROB. NITR. (g) BYPASS NITR. (g.) Forage 1 69.70 46.10 32.13 37.57 24.42 13.15 Forage 2 155.25 69.90 108.52 46.73 30.37 16.36 Forage 3 159.30 69.60 110.87 48.43 31.48 16.95 Forage 4 81.60 56.90 46.43 35.17 22.86 12.31 Forage 5 124.95 60.45 75.53 49.42 32.12 17.30 SAMPLE TOT. NITR. (g.) (AT DUOD.) MICROB. NITR. (g.) (AT DUOD.) BYPASS NITR. (g.) (AT DUOD) FECAL BYPASS NITR. (g.) DIG. BYPASS NITR. (g.) DIG. BYPASS NITR. (%) Forage 1 88.49 70.79 17.70 13.15 4.55 25.71 Forage 2 145.39 81.42 63.97 16.36 47.61 74.43 Forage 3 148.68 75.83 72.85 16.95 55.90 76.73 Forage 4 100.32 77.25 23.07 12.31 10.76 46.76 Forage 5 142.69 ._ 92.75 49.94 17.30 32.64 65.36 Forage 1 » Low quality grass hay Forage 2 - A l f a l f a cubes Forage 3 - High quality grass hay Forage 4 - I t a l i a n ryegrass Forage 5 - Orchard grass •assume 65% d i g e s t i b i l i t y of microbial - 60 -SUMMARY The purpose of this study was to determine the extent of ruminal breakdown and the efficiency of ut i l i z a t i o n of the protein source of various typical forages fed to dairy cattle. Conclusions were d i f f i c u l t to make from this experiment because of the limited number of animals involved, the animal mortality, and the resulting incomplete data obtained. However, one can see definite trends appearing from the digestiblity t r i a l s and the subsequent s t a t i s t i c a l analysis of the results. The effects of cannulation were examined and shown to have no negative effect on feed intake or digestiblity parameters of the forage. When cannula problems (blockage, leakage) were minimal, a l l the steers gained weight normally and had no problem adapting to their cannulas. The benefits of the cannulation procedure are obvious as i t provides a highly functional and informative method for determining protein breakdown and uti l i z a t i o n along the gastrointestinal tract. With regard to total d i g e s t i b i l i t y data, no significant differences (p>.05) were observed between forage types except in terms of nitrogen digestiblity, which was higher for the higher nitrogen forages. One might expect a l l parameters to be higher because increased dietary nitrogen intake and increased soluble carbohydrates is usually followed by increased fibre breakdown and digestiblity of other nutrients. Since the majority of the data is limited basically to one animal, this would probably provide an explanation. However, the contribution of the rumen microbes to protein production cannot be - 61 -discounted, since the digestiblity values for the higher nitrogen forages are higher but not significantly higher than the lower nitrogen forages. Overall, i t would seem that the ab i l i t y of the rumen microbes to upgrade low quality protein and the importance of recycled nitrogen is quite evident. The data collected at the duodenum showed the extent of digestiblity confined to the upper gastrointestinal tract (basically the rumen), and the contribution of the rumen microbes. Forage type had a significant effect (p<.05) on a l l dig e s t i b i l i t y parameters and the quantity of microbial nitrogen presented at the duodenum. The higher nitrogen forages had significantly lower dry matter and fibre digestiblities in the rumen. This was due to an increased proportion of soluble protein and soluble carbohydrates, which led to a shorter rumen retention time and decreased rumen digestion. Nitrogen dig e s t i b i l i t y in the rumen was only slight for the higher nitrogen forages, and there were substantial net additions of nitrogen for the lower nitrogen forages. This was to be expected since the majority of nitrogen absorption takes place in the small intestine. Nitrogen recycling via the saliva into the rumen plays a large role when intakes of dietary nitrogen are low. The ratios of microbial nitrogen: total nitrogen at the duodenum were significantly higher (p<.05) for the lower nitrogen forages. This was due to increased NH3 u t i l i z a t i o n at the lower nitrogen level, resulting in increased microbial protein production. Again, a higher rumen retention time resulting in an increased proportion of microbial cells was also a factor. - 62 -The higher ratios of microbial nitrogen: total nitrogen at the duodenum for the lower nitrogen forages resulted In expected lower quantities of bypass nitrogen. The apparent di g e s t i b i l i t y of the bypass nitrogen for these forages was also much lower, thereby greatly reducing the potential benefits of bypass nitrogen to the animal. The data would seem to indicate that dietary protein can be indigestible and hence unavailable to the animal, and also can be overfed resulting in waste and inefficient u t i l i z a t i o n . As a result, care should be taken to achieve maximum ut i l i z a t i o n of the protein source. - 63 -BIBLIOGRAPHY Abdo, K.M., King, K.W. and Engel, R.W. 1964. Prot e i n q u a l i t y of rumen microorganisms. J . Anim. S c i . 23:734 - 736. Ash, R.W. 1962. G a s t r o - i n t e s t i n a l re-entrant cannulae for studies of digestion i n sheep. Anim. Prod. 4:309 - 312. Associ a t i o n of O f f i c i a l A n a l y t i c a l Chemists, O f f i c i a l Methods. 12th Ed. 1975. Bailey, C.B. and Balch, C C . 1961. S a l i v a secretion and i t s r e l a t i o n to feeding i n c a t t l e . 2. The composition and rate of secretion of mixed s a l i v a i n the cow during r e s t . B r i t . J . Nutr. 15:383 - 402. Bailey, C.B. 1961. Sa l i v a secretion and i t s r e l a t i o n to feeding i n c a t t l e . 3. The rate of secretion of mixed s a l i v a i n the cow during eating, with an estimate of the magnitude of the t o t a l d a i l y secretion of mixed s a l i v a . B r i t . J . Nutr. 15:443 -451. Barry, T.N. 1972. The e f f e c t of feeding formaldehyde-treated casein to sheep on nitrogen retention and wool growth. N.J. Agric. Res. 5:107 -116. Bauchop, T. and Elsden, S.R. 1960. The growth of microorganisms i n r e l a t i o n to th e i r energy supply. J . Gen. M i c r o b i a l . 23:457 - 469. Beever, D.E., Coelho da S i l v a , J.F., Prescott, J.H.D. and Armstrong, D.G. 1972. The e f f e c t i n sheep of physical form and stage of growth on the s i t e s of digestion of a dried grass. 1. Sites of digestion of organic matter, energy, and carbohydrate. B r i t . J . Nutr. 28:347 - 356. Beever, D.E., Harrison, D.G., Thomson, D.J., Cammell, S.B. and Osbourn, D.F. 1974. The e f f e c t of drying and e n s i l i n g grass on i t s digestion i n sheep. Sites of energy and carbohydrate digestion. B r i t . J . Nutr. 32:99 - 112. Bergen, W.G. and Yokoyama, M.T. 1977. Productive l i m i t s to rumen fermentation. J . Anim. S c i . 46:573 - 584. Bergen, W.G. 1978. Postruminal digestion and absorption of nitrogenous components. Fed. Proc. Fed. Am. Soc. Exp. B i o l . 37:1223 - 1227. Black, J.L. 1971. A t h e o r e t i c a l consideration of the e f f e c t of preventing rumen fermentation on the e f f i c i e n c y of u t i l i z a t i o n of dietary energy and protein i n lambs. B r i t . J . Nutr. 25:31 - 55. - 64 -Br a t z l e r , J.W., Keck, E., Marriott, L.F., and Washko, J.B. 1959. N u r t r i t i v e value of orchard grass as affected by l e v e l of nitrogen f e r t i l i z a t i o n and stage of maturity. J . Dairy S c i . 42:934 -935. Brown, G.F., Armstrong, D.G. and MacRae, J.C. 1968. The establishment i n one operation of a cannula into the rumen and re-entrant cannulae into the duodenum and ileum of the sheep. B r i t . Vet. J . 124:78 -81. Bruce, J . , Goodall, E.D., Kay, R.N.B., P h i l l i p s o n , A.T. and Vowles, L.E. 1966. The flow of organic and inorganic materials through the alimentary t r a c t of the sheep. Proc. Royal Soc. 166:46 - 62. Bucholtz, H.F. and Bergen, W.G. 1973. M i c r o b i a l phospholipid synthesis as a marker for microbial protein synthesis i n the rumen. Appl. Microbi. 25:504 - 513. Buttery, P.J. and Cole, D.J.A. 1977. Chemical analysis: sources of error. Proc. Nutr. Soc. 36:211 - 218. Bje r r i n g , J.H., Grieg, M. and Halm, J . 1975. UBC BMD10V. Computing Centre, U n i v e r s i t y of B r i t i s h Colombia. Chalmers, M.J., Grant, I. and White, F. 1976. Nitrogen passing through the wall of the ruminant digestive t r a c t . In. Protein Metabolism and N u t r i t i o n . Ed. D.J.A. Cole, Butterworth, Boston. Chalupa, W., Chandler, J.E. and Brown, R.E. 1973. Abomasal i n f u s i o n of mixtures of amino acids to growing c a t t l e . J . Anim. S c i . 37:339 - 340. Chalupa, W. 1974. Amino-acid n u t r i t i o n of growing c a t t l e . Tracer Studies on Non-protein Nitrogen for Ruminants. Int. Atomic Energy Agency, Vienna. Chalupa, W. 1978. Postruminal fate of nitrogenous components. Symposium introductory remarks. Fed. Proc. Fed. Am. Soc. Exp. B i o l . 37:1222. Clarke, E.M.W., E l l i n g e r , G.M. and P h i l l i p s o n , A.T. 1966. The influence of diet on the nitrogenous components passing to the duodenum and through the lower ileum of sheep. Proc. Royal Soc. 166:63. Cloete, J.G. 1964. Studies on metabolic f e c a l nitrogen excretion i n sheep. Thesis - University of Stellenbosch from J . Cloete. 1966. S. A f r . J . Agric. S c i . 9:379. Coelho da S i l v a , J.F., Seeley, R.C, Thomson, D.J., Beever, D.E. and Armstrong, D.G. 1972 (a). The e f f e c t i n sheep of physical form on the s i t e s of digestion of a dried lucerne d i e t . 2. Sites of nitrogen digestion. B r i t . J . Nutr. 28:43 - 61. - 65 -Coelho da S i l v a , J.F., Seeley, R.C, Beever, D.E., Prescott, J.H.D. and Armstrong, D.C 1972 (b). The e f f e c t i n sheep of physical form and stage of growth on the s i t e s of digestion of a dried grass. 2. Sites of nitrogen digestion. B r i t . J . Nutr. 28:357 - 371. Coleman, G.S. 1975. The i n t e r r e l a t i o n s h i p between rumen c i l i a t e protozoa and bacteria. In Digestion and Metabolism i n the Ruminant. Proceedings of the IV International Symposium on Ruminant Physiology, Sydney, A u s t r a i l i a . Ed. I.W. MacDonald and A.C.I. Warner, Uni v e r s i t y of New England Publishing Unit, Armidale. Dehority, B.A. 1973. Hermicellulose degradation by rumen b a c t e r i a . Fed. Proc. Fed. Am. Soc. Exp. B i o l . 32:1819 - 1825. D r o r i , D. and L o o s l i , J.K. 1959. Influence of f i s t u l a t i o n on the d i g e s t i b l i t y of feeds by steers. J . Anim. S c i . 18:206 - 210. Egan, A.R. and Tudor, CD. 1976. Establishment of re-entrant cannulas into the duodenum of c a t t l e . Aust. Vet. J . 52:99. Evans, R.S., Axford, R.F.E. and Offer, N.W. 1975. A method for estimating the quantities of microbial and dietary proteins flowing i n the duodenal digesta of ruminants. Proc. Nutr. Soc. 34:65 A. Faichney, G.J. 1971. The e f f e c t of formaldehyde-treated casein on the growth of ruminant lambs. Aust. J . Agric. Res. 22:453 - 460. Foley, R.C. Dairy C a t t l e : P r i n c i p l e s , P r a c t i c e s , Problems, P r o f i t s . Lea & Febiger, Philadelphia, 1972 . p. 197. Forrest, W.W. and Walker, D.J. 1971. The generation and u t i l i z a t i o n of energy during growth. In. Advances i n M i c r o b i a l Physiology. Ed. A.H. Rose and J.F. Wilkinson, Academic Press, New York. Forsberg, C H . and Lam, K. 1977. Use of Adenosine 5'-triphosphate as an i n d i c a t o r of the microbiota biomass i n rumen contents. Appl. Microbi. 33:528 - 537. Glover, J . and Dougall, H.W. 1960. The apparent d i g e s t i b i l i t y of the non-nitrogenous components of ruminant feeds. J . A g r i c . S c i . 5:391 - 394. Goering, H.K. and Van Soest, P.J. 1970. Forage f i b r e analysis. U.S.D.A. Handbook, No. 379. Goodall, E.D. and Kay, R.N.B. 1965. Digestion and absorption i n the large i n t e s t i n e of the sheep. J . P h y s i o l . 176:12 - 23. Hagemeister, H., Kaufmann, W. and P f e f f e r , E. 1976. Factors infl u e n c i n g the supply of nitrogen and amino acids to the i n t e s t i n e of dairy cows. In. Protein Metabolism and N u t r i t i o n . Ed. J . A. Cole, K.N. Boorman, P.J. Buttery, D. Lewis, R.J. Neale and H. Swan, Butterworth, Boston. - 66 -Hale, W.H. 1973. Influence of processing on the u t i l i z a t i o n of grains (starch) by ruminants. J . Anim. S c i . 37:1075 - 1080. Handbook of Biochemistry. Ed. C. Long. D. Van Nostrand Company Inc., Princeton, New Jersey. 1968. Har r i s , L.E. and P h i l l i p s o n , A.T. 1962. The measurement of the flow of food to the duodenum of sheep. Anim. Prod. 4:97 - 116. H a t f i e l d , E.E. 1973. Treating proteins with tannins and aldehydes. In. E f f e c t of processing on the N u t r i t i v e Value of Feeds. Nat. Acad. S c i . , Washington, D.C. Hayes, B.W., L i t t l e , CO. and M i t c h e l l , J r . , G.E. 1964. Influence of ruminal, abomasal, and i n t e s t i n a l f i s t u l a t i o n on digestion i n steers. J . Anim. S c i . 23:764 -766. Hecker, J.F. 1971. Metabolism of nitrogenous compounds i n the large i n t e s t i n e of sheep. B r i t . J . Nutr. 25:85 - 96. Hedde, R.D., Knox, K.L., Johnson, D.E. and Ward, G.M. 1974. Energy and protein u t i l i z a t i o n i n calves fed v i a rumen by-pass. J . Anim. S c i . 39:114. Hogan, J.P. 1974. Quantitative aspects of nitrogen u t i l i z a t i o n i n ruminants. J . Dairy S c i . Ed. 284. Hogan, J.P. and P h i l l i p s o n , A.T. 1960. The rate of flow of digesta and the i r removal along the digestive t r a c t of the sheep. B r i t . J . Nutr. 14:147 - 155. Hogan, J.P. and Weston, R.H. 1967. The digestion of two diets of d i f f e r i n g protein content but with s i m i l a r capacities to sustain wool growth. Aust. J . Agric. Res. 18:973 - 981. Hogan, J.P. and Weston, R.H. 1970. Quantitative aspects of microbial protein synthesis i n the rumen. In. Physiology of Digestion and vMetabolism i n the Ruminant. Proceedings of the Third International Symposium, Cambridge, England. Ed. A.T. P h i l l i p s o n , O r i e l Press, Newcastle. Homey, F.D., Leadbeater, P.A. and Neudoerffer, T.S. 1972. Re-entrant cannulation and postoperative therapy i n c a t t l e . Am. J . Vet. Res. 33:1385 - 1390. Hume, I.D. 1970. Synthesis of microbial protein i n the rumen. I I . A response to higher v o l a t i l e f a t t y acids. Aust. J . Agric. Res. 21:297 - 304. Hume, I.D. 1974. The proportion of dietary protein escaping degradation i n the rumen of sheep fed on various protein concentrates. Aust. J . Agric. Res. 25:155 - 165. - 67 -Hungate, R.E. The Rumen and i t s Microbes. Academic Press, New York. 1966. Ibrahim, E.A. and I n g a l l s , J.R. 1972. M i c r o b i a l protein biosynthesis i n the rumen. J . Dairy S c i . 55:971 -978. Jorgensen, N.A. and Crowley, J.W. 1972. Corn sil a g e for Wisconsin c a t t l e . U n i v e r s i t y WI Ext. B u l l . A 1178. Madison. Klooster, A. Th. van't and Rogers, P.A.M. 1969. Observations on the Digestion and Absorption of Food Along the G a s t r o - i n t e s t i n a l Tract of F i s t u l a t e d Cows. Dept. of Animal Physiology, A g r i c u l t u r a l U n i v e r s i t y , Wageningen, The Netherlands. V o l . 11:3 - 19. Leibholz, J . and Hartmann, P.E. 1972. Nitrogen metabolism i n sheep. 1. The e f f e c t of protein and energy intake i n the flow of digesta into the duodenum and on the digestion and absorption of nutrie n t s . Aust. J . A g r i c . Res. 23:1059 - 1071. Leibholz, J . 1972. Nitrogen metabolism i n sheep. I I . The flow of amino acids into the duodenum from dietary and microbial sources. Aust. J . Ag r i c . Res. 23:1073 - 1083. Lewis, D. and McDonald, I.W. 1958. The i n t e r - r e l a t i o n s h i p s of i n d i v i d u a l proteins and carbohydrates during fermentation i n the rumen of the sheep. 1. The fermentation of casein i n the presence of starch or other carbohydrate materials. J . A g r i c . S c i . 51:108 - 118. Lindsay, J.R. and Hogan, J.P. 1972. Digestion of two legumes and rumen b a c t e r i a l growth i n defaunted sheep. Aust. J . Agric. Res. 23:321 -330. Ling, J.R. and Buttery, P.J. 1978. The simultaneous use of r i b o n u c l e i c acid, S, 2, 6 - diaminopimelic acid and 2-aminoethylphosphonic acid as markers of microbial nitrogen entering the duodenum of sheep. B r i t . J . Nutr. 39:165 - 179. MacRae, J.C. and Armstrong, D.G. 1969. Studies on i n t e s t i n a l digestion i n the sheep. 2. Digestion of some carbohydrate constituents i n hay, cereal and hay-cereal r a t i o n s . B r i t . J . Nutr. 23:377 - 387. MacRae, J . C , Ulyatt, M.J., Pearce, P.D. and Hendtlass, J . 1972. Quantitative i n t e s t i n a l digestion of nitrogen i n sheep given formaldehyde-treated and untreated casein supplements. B r i t . J . Nutr. 27:39 - 50. MacRae, J.C. 1974. The use of re-entrant cannulae to p a r t i t i o n d igestive function within the g a s t r o - i n t e s t i n a l t r a c t of ruminants. In. Digestion and Metabolism i n the Ruminant. Proceedings of the IV International Symposium on Ruminant Physiology, Sydney, A u s t r a l i a . Ed. I.W. MacDonald and A.C.I. Warner, Uni v e r s i t y of New England Publishing Unit, Armidale. - 68 -MacRae, J.C. and Wilson, S. 1977. The e f f e c t s of various forms of g a s t r o - i n t e s t i n a l cannulation on digestive measurements i n sheep. B r i t . J . Nutr. 38:65 71. Maeng, W.J., Van Nevel, C.J., Baldwin, R.L. and Morris, J.G. 1976. Rumen microbial growth rates and y i e l d s : e f f e c t of amino acids and protein. J . Dairy S c i . 59:68 - 79. Markley, R.A., Cason, J.L. and Baumgardt, B.R. 1959. E f f e c t of nitrogen f e r t i l i z a t i o n or urea supplementation upon the d i g e s t i b i l i t y of grass hays. J . Dairy S c i . 42:144 - 152. Marr, A.G., Nilson, E.H. and Clark, D.J. 1963. The maintenance requirements of Escherichia c o l i . Ann. NY Acad. S c i . 102:536. Mason, V.C. and Palmer, R. 1971. Studies on the d i g e s t i b i l i t y and u t i l i z a t i o n of the nitrogen of i r r a d i a t e d rumen bacteria by ra t s . J . A g r i c . S c i . 76:567 - 572. Mathison, G.W. and M i l l i g a n , L.P. 1971. Nitrogen metabolism i n sheep. B r i t . J . Nutr. 25:351 - 366. McAllan, A.B. and Smith, R.H. 1969. Nucleic acid metabolism i n the ruminant. Determination of nucleic acids i n digesta. B r i t . J . Nutr. 23:671 - 682. McAllan, A.B. and Smith, R.H. 1972. Nucleic acids i n ruminant digesta as indices of microbial nitrogen. Proc. Nutr. Soc. 31:24A. McAllan, A.B. and Smith, R.H. 1974. Contribution of microbial nitrogen to duodenal digesta i n the ruminant. Proc. Nutr. Soc. 33:41 - 42A. McAllan, A.B. and Smith, R.H. 1976. E f f e c t of dietary nitrogen source on carbohydrate mstcibolism i n the iruni6n of tTIG young s t GGIT • B i r i t • J . Nutr. 36:511 - 522 McNaught, M.L., Smith, J.A.B., Henry, K.M. and Kon, S.K. 1950. The u t i l i z a t i o n of non-protein nitrogen i n the bovine rumen. 5. The i s o l a t i o n and n u t r i t i v e value of a preparation of dried rumen ba c t e r i a . Biochem. J . 46:32 - 36. McNaught, M.L., Owen, E.C., Henry, K.M. and Kon, S.K. 1954. The u t i l i z a t i o n of non-protein nitrogen i n the bovine rumen. 8. The n u t r i t i v e value of the proteins of preparations of dried rumen bac t e r i a , rumen protozoa, and brewer's yeast for r a t s . Biochem. J . 56:151 - 156. Mercer, J.R. and Annison, E.F. 1976. U t i l i z a t i o n of nitrogen i n ruminants. In. Protein Metabolism and N u t r i t i o n . Ed. D.J.A. Cole, Butterworth, Boston. - 69 -M i l l e r , E.L. 1973. Symposium on nitrogen u t i l i z a t i o n by the ruminant. Evaluation of foods as sources of nitrogen and amino acids. Proc. Nutr. Soc. 32:79 - 84. Minson, D.J. and Pigden, W.J. 1961. E f f e c t of a continuous supply of urea on u t i l i z a t i o n of low q u a l i t y forages. J . Anim. S c i . 20:962A. Neudorffer, T.S., Duncan, D.B. and Homey, F.D. 1971. The extent of release of encapsulated methionine i n the i n t e s t i n e of c a t t l e . B r i t . J . Nutr. 25:333 - 341. Orskov, E.R., Fraser, C. and Corse, E.L. 1970. The ef f e c t on protein u t i l i z a t i o n of feeding d i f f e r e n t protein supplements v i a the rumen or v i a the abomasum i n young growing sheep. B r i t . J . Nutr. 24:803 -809. P h i l l i p s o n , A.T. 1952. The passage of digesta from the abomasum of sheep. J . P h y s i o l . 116:84 - 97. Pi l g r i m , A.F., Gray, F.V., Weller, R.A. and B e l l i n g , C.B. 1970. Synthesis of microbial protein from ammonia i n the sheep's rumen and the proportion of dietary nitrogen converted into microbial nitrogen. B r i t . J . Nutr. 24:589 - 598. Putnam, P.A. and Davis, R.E. 1965. Postruminal f i b r e d i g e s t i b l i t y . J . Anim. S c i . 24:826 - 829. Ridges, A.P. and Singleton, A.G. 1962. Some quantitative aspects of digestion i n goats. J . P h y s i o l . 161:1 - 9. Robertson, J . A. and Hawke, J . C. 1965. Studies on rumen metabolism. IV. E f f e c t of carbohydrate on ammonia l e v e l s i n the rumen of pasture-fed cows and i n rumen liqu o r s incubated with ryegrass extracts. J . S c i . Fd. Agr i c . 16:268 - 276. R o f f l e r , R.E. and Satter, L.D. 1975(a). Relationship between ruminal ammonia and nonprotein nitrogen u t i l i z a t i o n by c a t t l e . 1. Development of a model for pred i c t i n g nonprotein nitrogen u t i l i z a t i o n by c a t t l e . J . Dairy S c i . Ed. 264. R o f f l e r , R.E. and Satter, L.D. 1975(b). Relationship between ruminal ammonia and nonprotein nitrogen u t i l i z a t i o n by c a t t l e . I I . Ap p l i c a t i o n of published evidence to the development of a t h e o r e t i c a l model for predicting nonprotein nitrogen u t i l i z a t i o n . J . Dairy S c i . Ed. 265. Roy, J.H.B, Balch, CC., M i l l e r , E.L., Orskov, E.R. and Smith, R.H. 1977. C a l c u l a t i o n of the N-requirement for ruminants from nitrogen metabolism studies. In. Protein Metabolism and N u t r i t i o n , Proceedings of the Second International Symposium, Flevohof, Netherlands. Centre for A g r i c u l t u r a l Publishing and Documentation, Wageningen. - 70 -Satter, L.D. and Sl y t e r , L.L. 1974. E f f e c t of ammonia concentration on rumen microbial protein production i n v i t r o . B r i t . J . Nutr. 32:199 -208. Satter, L.D. and R o f f l e r , R.E. 1975. Nitrogen requirement and u t i l i z a t i o n i n dairy c a t t l e . J . Dairy S c i . 58:1219 - 1237. Schneider, B.H. and F l a t t , W.P. The Evaluation of Feeds through D i g e s t i b i l i t y Experiments. Un i v e r s i t y of Georgia Press, 1975. p. 233-272. Singleton, A.G. 1961. The electromagnetic measurement of the flow of digesta through the duodenum of the goat and the sheep. J . Physiol. 155:134 - 147. S l y t e r , L.L., Oltjen, R.R., Williams, J r . , E.E. and Wilson, R.L. 1971. Influence of urea, biuret and starch on amino acid patterns i n ruminal bacteria and blood plasma and on nitrogen balance of steers fed high f i b r e p u r i f i e d d i e t s . J . Nutr. 101:839 - 846. Smith, R.H., McAllan, A.B. and H i l l , W.B. 1968. Nucleic acids i n bovine n u t r i t i o n . 2. Production of microbial nucleic acids i n the rumen. Proc. Nutr. Soc. 27:48A. Smith, R.H. 1969. Nitrogen metabolism and the rumen. J . Dairy Res. 36:313 - 331. Smith, R.H. and McAllan, A.B. 1970. Nucleic acid metabolism i n the ruminant. 2. Formation of microbial nucleic acids i n the rumen i n r e l a t i o n to the digestion of food nitrogen, and the fate of dietary n u c l e i c acids. B r i t . J . Nutr. 24:545 - 556. Smith, R.H. and McAllan, A.B. 1971. Nucleic acid metabolism i n the ruminant. 3. Amounts of nucleic acids and t o t a l and ammonia nitrogen i n digesta from the rumen, duodenum and ileum of calves. B r i t . J . Nutr. 25:181 - 190. Smith, R.H. and McAllan, A.B. 1974. Some factors influencing the chemical composition of mixed rumen bac t e r i a . B r i t . J . Nutr. 31:27 - 34. Smith, R. H. 1975. Nitrogen metabolism i n the rumen and the composition and n u t r i t i v e value of nitrogen compounds entering the duodenum. In. Digestion and Metabolism i n the Ruminant. Proceedings of the IV International Symposium on Ruminant Physiology, Sydney, A u s t r a l i a . Ed. I. W. MacDonald and A.C.I. Warner, Un i v e r s i t y of New England Publishing Unit, Armidale. Smith, R.H., McAllan, A.B., Hewitt, D. and Lewis, P.E. 1978. Estimation of amounts of microbial and dietary nitrogen compounds entering the duodenum of c a t t l e . J . Agric. S c i . 90:557 - 568. - 71 -Stern, M.D., Hoover, H., Sniffen, C.J., Crooker, B.A. and Knowlton, P.H. 1978. E f f e c t s of nonstructural carbohydrate, urea and soluble protein l e v e l s on microbial protein synthesis i n continuous culture of rumen contents. J . Anim. S c i . 47:944 - 956. Stouthamer, A.H. 1969. Determination and s i g n i f i c a n c e of growth y i e l d s . In. Methods i n Microbiology, Vol. 1. Ed. J.R. Norris and D.W. Ribboxs, Academic Press, New York. Stouthamer, A.H. and Bettenhaussen, C. 1973. U t i l i z a t i o n of energy f o r growth and maintenance i n continuous and batch cultures of microorganisms. Biochim. Biophys. Acta. 301:53 - 70. Tamminga, S. 1979. Protein degradation i n the forestomachs of ruminants. J . Anim. S c i . 49:1615 - 1630. Thompson, F. and Lamming, G.E. 1972. The flow of digesta, dry matter and starch to the duodenum i n sheep given rations containing straw of varying p a r t i c l e s i z e . B r i t . J . Nutr. 28:391 - 403. Topps, J.H., Kay, R.N.B. and Goodall, E.D. 1968(a). Digestion of concentrate and of hay diets i n the stomach and in t e s t i n e s of ruminants. 1. Sheep. B r i t . J . Nutr. 22:261 - 280. Topps, J.H., Kay, R.N.B., Goodall, E.D., Whitelaw, F.G. and Reid, R.S. 1968(b). Digestion of concentrate and of hay diets i n the stomach and i n t e s t i n e s of ruminants. 2. Young steers. B r i t . J . Nutr. 22:281 - 290. Van Gylswyck, N.0. 1970. The e f f e c t of supplementing a low-protein hay on the c e l l u l o l y t i c b a cteria i n the rumen of sheep and on the d i g e s t i b l i t y of c e l l u l o s e and hemicellulose. J . Agric. S c i . 74:169 - 180. Van Nevel, C.J. and Demeyer, D.I. 1977. Determination of rumen microbial growth i n v i t r o from B i t . J . Nutr. 38:101. Van Soest, P.J. 1975. Physio-chemical aspects of f i b e r digestion. In. Digestion and Metabolism i n the Ruminant. Ed. I.W. MacDonald and A.C.I. Warner, Uni v e r s i t y of New England Publishing Unit, Armidale. Waldo, D.R. 1968. Symposium: Nitrogen U t i l i z a t i o n by the Ruminant. Nitrogen metabolism i n the ruminant. J . Dairy S c i . 51:265 - 275. Waldo, D.R. 1973. Extent and p a r t i t i o n of cereal grain starch digestion i n ruminants. J . Anim. S c i . 37:1062 - 1074. Walker, D.J. and Nader, C.J. 1975. Measurement i n vivo of rumen microbial protein synthesis. Aust. J . Agric. Res. 26:689 - 698. - 72 -Walker, D.J., Egan, A.R., Nader, C.J., Ulyatt, M.J. and Storer, G.B. 1975. Rumen microbial protein synthesis and proportions of microbial and non-microbial nitrogen flowing to the i n t e s t i n e s of sheep. Aust. J . Agric. Res. 26:699 - 708. Warner, A.C.I. 1956. Pr o t e o l y s i s by rumen micro-organisms. J.Gen. M i c r o b i o l . 14:749 - 762. Watson, M.J., Savage, G.P. and Armstrong, D.G. 1972. Sites of disappearance of apparently d i g e s t i b l e energy and apparently d i g e s t i b l e nitrogen i n the digestive t r a c t of cows receiving dried grass-concentrate d i e t s . Proc. Nutr. Soc. 31:98A. Weller, R.A. 1957. The amino acid composition of hydrolysates of microbial preparations from the rumen of sheep. Aust. J . B i o l . S c i . 10:384 - 389. Weller, R.A., Gray, F.V. and P i l g r i m , A.F. 1958. The conversion of plant nitrogen to microbial nitrogen i n the rumen of sheep. B r i t . J . Nutr. 12:421 - 429. Weston, R.H. and Hogan, J.P. 1967. The digestion of chopped and ground roughages by sheep. 1. The movement of digesta through the stomach. Aust. J . Agric. Res. 18:789 - 801. Williams, C.H., David, D.J. and lismaa, 0. 1962. The determination of chromic oxide i n faeces samples by atomic absorption spectrophotometry. J . Agric. S c i . 59:381 - 385. Wolstrup, J . and Jensen, K. 1978. Adenosine triphosphate and deoxyribonucleic acid i n the alimentary t r a c t of c a t t l e fed d i f f e r e n t nitrogen sources. J . Appl. Bact. 45:49 - 56. Work, E. and Dewey, D.L. 1953. The d i s t r i b u t i o n of a, e -diaminopimelic acid among various micro-organisms. J . Gen. M i c r o b i o l . 9:394 - 409. 

Cite

Citation Scheme:

        

Citations by CSL (citeproc-js)

Usage Statistics

Share

Embed

Customize your widget with the following options, then copy and paste the code below into the HTML of your page to embed this item in your website.
                        
                            <div id="ubcOpenCollectionsWidgetDisplay">
                            <script id="ubcOpenCollectionsWidget"
                            src="{[{embed.src}]}"
                            data-item="{[{embed.item}]}"
                            data-collection="{[{embed.collection}]}"
                            data-metadata="{[{embed.showMetadata}]}"
                            data-width="{[{embed.width}]}"
                            async >
                            </script>
                            </div>
                        
                    
IIIF logo Our image viewer uses the IIIF 2.0 standard. To load this item in other compatible viewers, use this url:
http://iiif.library.ubc.ca/presentation/dsp.831.1-0095817/manifest

Comment

Related Items