. v .I. “A .. ., H . u‘ ‘ ..V . V . . . . . ‘ ‘ a .a-i.., .y. t ., . .n x \ , ‘ ‘ . . ‘ ‘ , ‘ A . , . y y I. u . . . ‘ v . .l .. . y . .n v lllllllllllllllllIIIIIIIIIUllllllllllllllllIIIlIIIIllllIlllll 3 1293 10503 765 I H895 LIBRARIES MICHIGAN STATE UNIVERSITY gAST LANSING, MIcH. 48824 This is to certify that the thesis entitled THE EFFECT OF MONENSIN AND PROTEIN LEVEL ON RUMINAL PROTEASE AND DEAMINASE ACTIVITY AND THE PHYSIOLOGICAL STATE OF UNBOUND RUMEN BACTERIA presented by SCOTT MICHAEL BARAO has been accepted towards fulfillment of the requirements for M.S. degree in Animal Science 4W6 @499“, Major professor Date7I/fd /7IJ9§‘ 0-7639 MS U i: an Affirmative Action/Equal Opportunity Institution MSU LIBRARIES RETURNING MATERIALS: Place in book drop to remove this checkout from your record. FINES will be charged if book is returned after the date stamped below. 5. Lfi ”'3’ 2;) c? 9 67 THE EFFECT OF MONENSIN AND PROTEIN LEVEL ON RUMINAL PROTEASE AND DEAMINASE ACTIVITY AND THE PHYSIOLOGICAL STATE OF UNBOUND = RUMEN BACTERIA BY Scott Michael Barao A THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree of MASTER OF SCIENCE Department of Animal Science 1983 ABSTRACT THE EFFECT OF MONENSIN AND PROTEIN LEVEL ON RUMINAL PROTEASE AND DEAMINASE ACTIVITY AND THE PHYSIOLOGICAL STATE OF UNBOUND RUMEN BACTERIA BY Scott Michael Barao Four 300 kg ruminally-cannulated steers were used in a 4x4 latin square design trial to study the effects of monensin and nitrogen level on ruminal protease and deaminase activity and the physiological state of unbound rumen bacteria. Experimental diets consisted of 80% corn (75% DM) and 20% corn silage. Diets were supplemented with or without monensin (250mg/hd/day) and urea at either 12% or 16% calculated crude protein. Serial rumen samples were taken and bacteria were harvested. RNA/protein values increased after feeding reaching peak values by 6 hours. Monensin increased RNA/protein values while urea addition had little effect. Cell- associated protease increased immediately after feeding followed by a subsequent decrease in supernatant values. Deaminase increased rapidly after feeding and as with Scott Michael Barao protease, showed greatest activity with high-protein treatments. Monensin depressed both protease and deaminase activity. DEDICATION This thesis is dedicated to my grandfather, Leo, who passed away before the completion of this work. I thank him for his love and support and for my many very happy memories. ii ACKNOWLEDGEMENTS I would like to extend my deepest appreciation to Dr. Werner G. Bergen for his guidence and encouragement throughout the preparation of this thesis. I also extend my thanks to Mr. Douglas Bates for his expert assistance and guidence in the many laboratory analyses involved in this research. I also thank him for our many interesting and thought-provoking discussions. My appreciation is also extended to Dr Bennink, Dr.Waller and Dr.Yokoyama for their participation on my guidence committee and to Dr. Nelson for making the facilities needed for this research, available to me. To my wife Janet, my parents, Frank and Deanna and my grandmother Clara, I thank you for your love and support throughout my college career and throughout the preparation of this thesis. iii TABLE OF CONTENTS PAGE LIST OF TABLES.OOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOO Vi LI ST OF FIGURES O O O O O O O O O O O O O O O O O O O O I O O O O O O O O O O O 0 Vi 1 INTRODUCTION 0 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 1 LITERATURE REVIEW 0 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 4 Overall Rumen Fermentation................... 4 Carbohydrate Metabolism...................... 5 Nitrogen Metabolism.......................... 7 Growth Yields of Rumen Microbes.............. 10 Microbial Protein Synthesis.................. 13 Concentrate Feeding.......................... 16 Rumen Ammonia................................ 17 RNA to Protein Ratios........................ 19 Monensin Effects on Rumen Fermentation....... 21 Monensin Effects on Microbial Populations.... 22 Monensin and Microbial Growth................ 23 Monensin Effects on Protease Enzymes......... 26 Mode of Action of Monensin................... 27 MATERIALS AND METHODSOOOOOOOOOOOOOOOOOOOOOOOOOOO 30 Experimental Design.......................... 30 Sample Collection and Preparation............ 33 Rumen Ammonia Determination.................. 38 RNA to Protein Determination................. 39 Protease Determination....................... 41 Deaminase Determination...................... 42 Statistical Analysis......................... 43 iv PAGE RESULTSOOOOOOOOOOOOOOOOOOOOOOOOOOOOOO0.0.0.0.... 44 Rumen pH and Volatile Fatty Acid and Ammonia Concentrations....................... 44 Bacterial Cell and Supernatant Protease Activity..................................... 58 Rumen Fluid Deaminase Activity............... 64 RNA to Protein Ratios of Free Bacteria....... 68 DISCUSSIONOOO0.0...00......OOOOOOOOOOOOOOOOOOOOO 73 Rumen Ammonia, pH and Volatile Fatty Acids... 73 Protease, Deaminase and RNA to Protein values...OOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOO 75 SUWYOOCOOOOOOOOOOOOO0.00000000000000000000CCO 85 LITERATURE CITEDOOOOOOOOOOOOOOO00.000.000.000... 87 TABLE 1. LIST OF TABLES Experimental Diet Composition................ Rumen Rumen Rumen Rumen Rumen Rumen Rumen Rumen Total pH Values.............................. Ammonia Concentration.................. Acetate Concentration.................. Propionate Concentration............... Butyrate Concentration................. Isobutyrate Concentration.............. Valerate Concentration................. Isovalerate Concentration.............. Volatile Fatty Acids and A/P Ratios.... Average Twelve Hour Volatile Fatty Acids..... Cell Protease ActiVitYOOOCOOOOOOOOOOOOOOOOCOO Supernatant Protease Activity................ Ratios of Supernatant to Cell Protease....... Rumen Fluid Deaminase Activity............... RNA to Protein Ratios of Free Rumen Bacteria. vi PAGE 31 45 47 49 51 52 53 S4 55 56 57 59 62 65 67 70 LIST OF FIGURES FIGURE PAGE 1. Sampling Procedure for RNA and Protein........ 34 2. Sampling Procedure for Protease and Deaminase. 36 3. Rumen Ammonia Concentrations.................. 48 4. Dry Cell Protease Activity.................... 60 5. Cell Supernatant Protease Activity............ 63 6. Supernatant to Cell Protease Ratios........... 66 7. Deaminase Activity............................ 69 8. RNA to Pretein RatiOSOOOOOOOOOOOOOOOOOO0...... 72 vii INTRODUCTION The past fifty years of animal related research have produced great advances in the fields of animal nutrition, genetics and health. These advancements and. improvements have allowed food pmoduction to increase rapidly. It seems likely, however, that future advancements will be slower and less spectacular than those obtained in the past (Blaxter, 1970). As for the field of nutrition, very few new essential nutrients will be discovered. The major thrust of research will shift from one of discovery to one of thorough understanding of chemical and physiological processes, and how ‘they are affected by various factors. This type of research will lead to methods of controlling metabolism to improve the efficiency of food utilization by the animal, and ultimately increase the production of animal product. Extensive investigation into the area of rumen fermentation will be conducted and these investigations will center upon the aspects of rumen fermentation which, if modified, have the potential to increase animal efficiency. The ruminant, through its symbiotic relationship with anaerobic microorganisms, has the unique ability to digest and utilize roughage feeds producing high quality animal protein from low quality plant protein. It is within the reticulorumen that this symbiotic relationship is important. Food material consumed by the ruminant is initially fermented by the rumen microorganisms and used to meet their own growth requirements (Hungate, 1966). High quality protein in the form of microbial cells as well as undigested and unfermented feed will then flow out of the rumen and into the lower digestive tract. The lower gastrointestinal tract provides further digestion and utilization of the feedstuffs through the production of hydrolytic enzymes and absorption of nutrients along the tract. It is this post ruminal digestion that becomes increasingly important as ruminants are fed a larger percentage of high quality feeds which can be more efficiently converted to meat protein when they are digested and absorbed postruminally. The trend towards more efficient animal production has led to the inclusion of various high quality feedstuffs, many of which do not require initial digestion by rumen microorganisms, and may be of greater benefit to the animal when utilized in the lower digestive tract. Much work has been done to increase the rate of passage of feed from the rumen to the lower tract through feed processing and feed additives that would alter rumen fermentation and digestion. Monensin is a biologically active compound with the chemical formula, C36H62011' and is produced by a strain of Streptomyces cinnamonensis (Haney and Hoehn, 1967). In vitro, this compound exibits moderate activity against gram-positive ndcroorganisms (Haney and Hoehn, 1967). Monensin has received wide use as a coccidiostat throughout the poultry industry and has been marketed under the trade name Cbban. Monensin is marketed under the trade name Rumensin for use in cattle feeds. Monensin has been found to improve the efficiency with which ruminants convert feed tn) animal carcass through reduction of feed intake and improvement in efficiency of gain (Potter et al., 1976a; Raun et al., 1976). In vitro studies conducted by Richardson and co-workers (1976) found that monensin caused a shift in the molar proportions of the volatile fatty‘ acids produced in the rumen, with the largest shift being in favor of propionate. Dinius et a1. (1976) studied rumen anunonia concentrations and reported that these values were decreased with monensin. It is clear that monensin has effects on rumen fermentation and metabolism, and the study described in this thesis was undertaken to investigate the effect of monensin on the protease and. deaminase activity' and. physiological status of rumen bacteria in the liquid, small particle, pool. REVIEW OF LITERATURE OVERALL RUMEN FERMENTATION Rumen fermentation is a coupled process between carbohydrate degradation, volatile fatty acid production, ATP generation and microbial cell synthesis from nitrogenous precursors, carbon skeletons and other needed substrates (Bergen and Yokoyama, 1977). The rumen is a near ideal fermentation site. In most ruminant species the rumen is approximately one-seventh of the mass of the animal, is maintained at a relatively constant temperature (390C), is buffered well by salivary secretions and, compared to many other microbial ecosystems, is well supplied with nutrients. The microflora inhabiting the rumen is dense and contains approximately 1 x 109 to 1 x 1010 bacteria and 1 x105 protozoa per milliliter of rumen fluid (Hungate, 1966). Diversity within this population is extensive and approximatley 200 species of bacteria and 20 species of protozoa have been isolated (Bryant, 1959). During rumen fermentation short chain fatty acids and microbial cells are synthesized from the digested feed and these products serve as sources of energy and protein respectively for the ruminant. Methane, heat and ammonia are evolved and these products can represent a significant loss of energy and nitrogen for the animal. The efficiency of utilization of nutrients by ruminants is determined largely by the balance of these fermentation products and this balance is ultimately controlled by the types of microorganisms present in the rumen (Russell and Hespell, 1981). CARBOHYDRATE METABOLISM Degradation and fermentation of polysaccarides can be grouped into three stages. The initial stage includes attachment of the microorganisms to plant particles and disassociation of carbohydrate polymers from structural plant materials. Studies by Amos and Akin (1979) and Cheng et a1. (1979) have indicated that both bacteria and protozoa are involved at this stage. The second stage, the hydrolysis of released polymers and small saccharides, is catalized by numerous extracellular enzymes of which cellulases are the predominant type (King, 1961). The final stage, the intracellular fermentation of small saccharides is relatively well ‘understood, primarily because of the use of pure culture studies over the last two decades. Pure and mixed culture studies have established that the major biochemical pathway employed by the rumen bacteria for hexose degradation is the Embden-Meyerhof— Parnas pathway (Joyner and Baldwin, 1966; Wallnofer, 1966). For the degradation of pentose and deoxyhexose, there is less information available, but Turner and Roberton (1979) have demonstrated that the most likely pathway is a combination of the pentose cycle plus glycolosis. The major intracellular products of hexose or pentose deradation are pyruvate and phosphoenolpyruvate. These products are further metabolized by various pathways to produce fermentation products such as the volatile fatty acids. Other fermentation products include ethanol, succinate and lactate but these compounds rarely accumulate in the rumen due to further fermentation by other species of rumen bacteria (Hungate, 1966). For example, in the rumen most of the propionate is derived from succinate (Blackburn and Hungate, 1963) which is decarboxylated to propionate by organisms such as Selenomonas ruminantium (Scheifinger and Wolin, 1973). Another major factor regulating fermentation products formed in the rumen is interspecies hydrogen transfer. The turnover of hydrogen in the rumen is high because of its rapid utilization by methane producing bacteria (Hungate, 1966). These bacteria combine carbon dioxide and hydrogen to produce methane and water. Within the rumen the partial pressure of hydrogen is extremely low and thus the formation of hydrogen gas from pyridine nucleotides by non-methanogenic bacteria is thermodynamically feasible (Wolin, 1974). In the absence of methanogens hydrogen accumulates and less reduced fermentation products result (Latham and Wolin, 1977). The shifts in electron disposal in the presence of the methanogenic organisms result in a decrease in the flows of carbon atoms into single culture fermentation products such as ethanol and lactate (Taylor, 1982). Acetate produced from pyruvate yields ATP whereas lactate and ethanol production from pyruvate yields no ATP. Thus, more energy is made available for bacterial growth when the methanogenic bacteria are present (Bergen and Yokoyama, 1977). NITROGEN METABOLI SM The metabolism of nitrogenous compounds within the rumen is very complex owing to the number of compounds and the number of microbial species involved. Ammonia is the principle source of nitrogen for microbial growth, and most nitrogenous compounds entering the rumen such as protein and urea are degraded to ammonia before the nitrogen is assimilated by the microorganisms (Bryant, 1977). In the case of protein, free amino acids are intermediates in the process of proteolysis but, as they are rapidly deaminated, their concentration in then rumen fluid is very low (Robson and Wallace, 1982) . Overall nitrogen metabolism in the rumen is inefficient in that energy is required for the resynthesis of protein by microbial cells. Resynthesis of protein from ammonia may be incomplete so that nitrogen is lost to the host and excreted as urea. Part of the protein nitrogen will form bacterial cell walls and nucleic acids which are largely unavailable to the host (Owens and Bergen, 1983). It is important to understand that the ability of rumen microbes to convert ammonia nitrogen to protein is extremely valuable. Animals on a. low' protein diet. may conserve nitrogen by recycling urea to the rumen in the saliva and by diffusion from blood across the rumen wall. Hydrolysis of the recycled urea to ammonia makes nitrogen available to the microbes for protein synthesis (Blackburn, 1965). It has been known for many years that the proteolytic activity in the rumen is associated with the particulate fraction of the rumen fluid (Blackburn and Hobson, 1960; Wright, 1967). Activity was found in protozoa and both large and small bacteria. Nugent and Mangan (1981) presented evidence using isotope-labeled protein which suggests that even soluble proteins are adsorbed onto nucrobial surfaces while they undergo hydrolysis. Protein solubility has usually been cited as the main determining factor in the rate of degradation of a protein by rumen microorganisms, with less soluble proteins being degraded more slowly than those that were highly soluble (Bergen and Yokoyama, 1977). Tamminga (1979) has given estimates of ruminal degradation of protein insoluble in ruminal buffers and these values range from 35 to 50 percent. These findings suggest that solubility alone is a poor indicator of the extent of ruminal degradation across a variety of diets and feeding conditions (Owens and Bergen, 1983). The number of disulfide bridges and the tertiary structure of the protein are important factors for consideration in determining the proteins degradability (Mahadevan et al., 1980). One of the most important proteolytic populations of bacteria in the rumen are found associated with the rumen epithelium (Cheng et al., 1979). These bacteria are tightly bound to the epithelial tissue and can be seen to invade epithelial cells. When sheep are maintained entirely by infusion of volatile fatty acids and bicarbonate into the rumen and casein into the abomasum, the epithelial associated bacteria survive while the rumen fluid population disappears (Blackburn and Hobson, 1960). These wall adherent bacteria actively digest epithelial cells and may have a role in the entry' of endogenous nitrogen into the lower tract. Protease activity in the rumen fluid of infused steers was shown to increase as the level of nitrogen nutrition increased, but it is unclear whether this was due to heavier colonization of the rumen wall or to an increase in sloughing of epithelial tissue (Cheng et al., 1979). 10 Protozoa within the rumen contribute to nitrogen metabolism but do not use ammonia directly as a nitrogen source. It has been determined in vitro, using nitrogen-15 labeled ammonia, that 31 to 64 percent of the protozoal nitrogen in sheeps' rumen was derived from ammonia (Pilgrim et al., 1970; Mathison and Milligan, 1971). Uptake of the labeled ammonia by the rumen protozoa was probably a result of engulfment of labeled rumen bacteria. Coleman and Laurie (1974) reported that free amino acids and protein nitrogen can be utilized by protozoa found in the rumen. Pilgrim (1974) studied the passage of protozoa out of the rumen and found that only 6 to 29 percent of the number expected to flow out of the rumen, actually did. Through the use of passage study techniques, Pilgrim (1974) concluded that the contribution of protozoa to the flow of protein into the lower tract is small. Recently, Steinhour et al. (1982), using passage study techniques, estimated that the total non-ammonia-nitrogen flow to the lower tract in steers consisted of between 22 and 41 percent protozoal nitrogen. These values represent the highest estimates to date. GROWTH YIELDS OF RUMEN MICROBES Growth yield is usually defined as the dry weight of cells produced per mole of substrate fermented (Ysubstrate), or weight of cells produced per mole of ATP 11 formed in the fermentation pathway used by the organism (YATP) (Bergen, 1981) . In the calculation and interpretation of growth yields we can identify two problems: 1. Measured growth yields of bacteria are virtually always lower than the yield which would be expected by consideration of the ATP thought to be consumed by known biosynthetic pathways,and are usually substantially less than this value, the theoretical growth yield (Hespell and Bryant, 1979). 2. ATP produced by the breakdown of the energy source is derived not only from substrate-level phosphorylation, which is of known stoichiometry, but also from processes associated with electron transfer reactions and transmembrane vectorial metabolism of usually unknown stoichiometry. It is thus impossible to be certain of the amount of ATP synthesized per unit substrate metabolized in growing cells (Gunsalus and Shuster, 1961). Hespell and Bryant (1979), among others, have calculated the theoretical YATP for bacterial growth . It has been shown that this value is not constant, but depends on the composition of the cell, the energy source, and the availabilty of cell monomers (amino acids, fatty acids and nucleotide bases) as well as the maintainence requirement and specific growth rate of the organism (Stouthhamer, 1973). Some of these factors would be expected to influence the yields of rumen bacteria as well. Rumen 12 bacteria tend to accumulate intracellular polysaccarides at certain periods during the feeding cycle, and since this costs less in terms of ATP than synthesis of macromolecules, theoretical YATP would be increased at these times (Hespell and Bryant, 1979). In contrast, the lactate fermenters would be expected to have very low theoretical YATP values because of the high energy cost of conversion of lactate to cell material. The concept of YATP, the grams of bacterial dry-matter synthesized per mole of ATP available, was introduced in 1960 by Bauchop and Elsden after finding that the yields of different species of microorganisms on different substrates were comparable when calculated in terms of ATP produced through the metabolism of these substrates. The range of observed YATP values found by Bauchop and Elsden (1960) and most of the values found subsequently, was in the region of 10 grams per mole (Stouthhamer, 1977), much lower than the theoretical YATP yield. For a population of mixed rumen bacteria, estimates of YATP MAX range from 21 to 33 grams per mole (Hespell and Bryant, 1979). To approach maximum efficiency of microbial growth, all nutrients required for growth must be present simultaneously and in adequate amounts (Hespell and Bryant, 1979). If these criteria are not met, maximal efficiency will be reduced. Additional factors which by influencing molar growth yields would influence the efficiency of microbial growth include, the pathway for the degradation of energy 13 substrates and a possible anaerobic electron transfer during fermentation. Cytochrome systems have been isolated in anaerobic bacteria such as those of the cytochrome b type functioning in oxidative phosphorylation and found in Selenomonas ruminantium and Propionibacteria. Stouthhamer (1976) has reported other membrane-bound electron transport systems in anaerobic bacteria which may allow additional ATP to be produced. MI CROBIAL PROTEIN SYNTHESI S Protein synthesis in the rumen will be influenced by factors such as nutrient supply, microbial population and growth condition such as rumen pH and temperature. Nutrient requirements for microbial protein synthesis vary with the microbial species, and the types and numbers of organisms are influenced by the diet and by conditions in the rumen (Hobson, 1972). With diets containing preformed protein and adequate vitamins and minerals for the host animal, the elements to be the most likely deficient for the microbes are nitrogen and sulfur. As the crude protein content of the diet is reduced or when the nitrogen is largely non-protein, microbial synthesis may be limited by amino acids or fatty acids, the carbons skeletons of which are needed for the synthesis of valine, leucine, isoleucine, phenylalanine and tryptophan and cell walls (Chalupa, 1968). The concentrations at which l4 nutrients become limiting is not clearly known and this may be due to changes in the microflora. which occurs with changes in the diet. Since all nutrients are not released from feedstuffs at the same rate and since waste-products of one organism may be the substrates for others, nutrient supply to a particular microbial species may vary widely after feeding. Maximum use of ammonia derived from non-protein nitrogen sources occurs when carbohydrate fermentation takes place at the same rate as the ammonia production (Johnson, 1976). Storage of polysaccarides by bacteria and protozoa plays an important role in the syncronization of energy supplies and other nutrients, especially nitrogen which may be absorbed from the rumen and then recycled (Blackburn, 1965). The quantitative estimation of the extent of microbial synthesis in the rumen has been studied in great detail. The amount of microbial crude protein in the form of microbial cells has most often been estimated and expressed in relation to either organic matter apparently digested in the rumen (digestible organic matter) or fermentable organic matter, that amount of organic matter truly fermented in the rumen. The use of fermentable organic matter has proven to be more accurate while allowing for the calculation of ATP production such that the yield of microbial cells or the total amount of microbial protein synthesized would be equal to the product of the digestible 15 energy intake, the percent digestion in the rumen and the efficiency of microbial protein synthesis. Microbial dry-matter yields are effected by nitrogen insufficiency (Hume et al., 1970), composition of the ration (Cole et al., 1976; Orskov et al., 1972) and the starch and sugar content of the feed (McAllan and Smith, 1977). Microbial crude protein yields are effected by high concentrate feeds. Prigge et a1. (1978) demonstrated that microbial protein yields were lower per 100 grams of digestible organic matter for high concentrate diets than for diets with high roughage content and this has been related to the rate of rumen digesta flow. In vitro continuous culture studies have shown that growth rate or efficiency of ndcrobial cell synthesis increases with rate of turnover or dilution rate (Isaacson et al., 1975). Stouthhamer (1976) explained this phenomona as a decrease in maintenance energy required by the faster growing organisms. Goetsch and Galyean (1982) have shown that rumen liquid turnover rates are slower for concentrates than for forage diets and thus it is likely that microbial yields are effected in a similar manner within the rumen. Estimates of YATP for slow growing cultures are generally low (less than 10) but may approach a YATP-MAX of 25 at high growth rates (Bergen and Yokoyama, 1977). CONCENTRATE FEEDING AND RUMEN FERMENTATION Rations with a high content of readily fermentable material (high concentrate rations), in general, stimulate the production of volatile fatty acids in the rumen and cause a decrease in the pH of the rumen contents. Dramatic changes in the rumen microbial population have been shown to result from the addition of large amounts of readily fermentable carbohydrate in the ruminant diet (Mackie et al., 1978). Recent evidence suggests that the increased proportion of fermented starch metabolized to propionate results from an adaptive balance between amylolytic and lactate utilizing bacteria with high concentrate diets (Mackie and Gilchrist, 1979). Mackie et a1. (1978) studied the progression of changes which occured in the rumen of animals adapted to high concentrate diets and found a variety of chemical and microbial adaptations. Mackie et a1.(1978) showed progressive decreases in the rumen pH as grain content was increased, transient increases in lactic acid, little change in total concentrations of volatile fatty acids but some shift in molar proportions of the acids with increased propionate production, little change in glucose concentration of the rumen fluid and little change in the ammonia concentration of the rumen fluid. As for microbial changes, the number of total culturable bacteria generally increased with increased concentrate feeding. As expected, the number of amylolytic bacteria 16 17 increased with an increase in grain and the increase in the number of lactate-utilizing bacteria such as Streptococcus bggi§_ followed that of the amylolytic. Mackie and co-workers (1978) also showed an increase in protozoal numbers in proportion to the increase in starch and sugar in the diet. Oltjen and Davis (1965) demonstrated that the rate at which grain can be introduced into the ration without upsetting the balance of the rumen population was dependent on the rate at which protozoa can increase their numbers in response to the added starch. The numbers and types of microorganisms and the pattern of fermentation in the rumen is therefore greatly influenced by the level of concentrate feeds included in the diets of ruminants. RUMEN AMMONIA CONCENTRATION The effect of rumen ammonia concentration on microbial efficiency and dry-matter digestion have been studied both in vivo and in vitro (Satter and Slyter, 1974; Satter and Roffler, 1977; Orskov et al., 1972; Mehrez et al., 1977; Wallace, 1979). The amount of ammonia that can be assimilated by rumen bacteria and the subsequent effect on digestion and microbial efficiency will depend on the amount of energy available to the microbes in the form of fermentable Organic matter (Johnson, 1976). Diets which are highly 18 digestible, such as high concentrate diets, will have more readily fermented organic matter than diets high in roughage. Bryant (1959) demonstrated that a mixed population of rumen bacteria could assimilate ammonia from dilute solutions in vitro. Subsequent nutritional models were based on the principle that urea supplementation should be adjusted to minimize accumulation of ammonia in the rumen and thereby maximize the efficiency of microbial nitrogen production and nitrogen retention by the ruminant (Satter and Roffler, 1977; Roffler and Satter, 1975). In contrast, Mehrez et a1. (1977) recommended a high ruminal ammonia concentration for sheep fed a barley based diet, as they found that a considerable excess of ammonia in the rumen resulted in increased degradation of feed in nylon bags and so increased the efficiency at which the feed was used. The recommended ammonia concentration of Mehrez et a1. (1977) of 14mM (23mg/100m1) is much higher than that of Satter and Slyter (1974) at 3.5mM (5mg/100m1). Wallace (1979) found the hydrolytic activity of rumen bacteria of barley-fed sheep increased when ammonia concentration in the rumen fluid was increased to a level considered by some workers (e.g. Satter and Slyter, 1974) to be in excess of the requirements of rumen bacteria. The ammonia level maintained by Wallace ( 1979) was approximately 13.5mM, similar to that used by Mehrez et a1. (1977). In view of these contrasting recommendations for 19 optimum rumen ammonia concentration, further investigation is required. RNA TO PROTEIN RATIOS IN RUMEN BACTERIA Estimation of the physiological status of rumen bacteria at various times after feding and with the addition of various chemical agents is extremely important as part of the process of studying bacterial changes related to exogenous inputs into the microbial environment. The determination of ribonucleic acid (RNA) content and protein content of the bacterial cell and the ratio which exists between the two is a valuable tool in the estimation of this physiological state. The characteristic physiological growth states of bacteria may be described as states of balanced growth at which, over any time interval, the whole system increases by the same factor (Campbell, 1957). The growth rate of a microbial population with a constant chemical composition may be determined by measuring the changes in any cell constituents such as RNA content or protein content. In bacteria, the cellular mass and the amounts of deoxyribonucleic acid (DNA), ribonucleic acid (RNA) and protein were observed by several workers to increase with increasing bacterial growth rate (Maaloe and Kjeldgaard, 1966; Schaechter et al., 1958). Cell mass and RNA were 20 found to increase more rapidly such that the ratio of RNA to protein increased exponentially with the growth rate. It has also been observed that the ratios of RNA to protein and RNA to DNA are linear functions of growth rate while the ratio of DNA to protein is independent of rate of growth (Kjeldgaard, 1967). Ribosomal efficiency is an important feature in the application of the RNA to protein ratio to the growth state of a bacterial cell. The ribosomal efficiency is the average rate of protein synthesis per ribosome .and is a function of the number of ribosomes actively synthesizing protein as well as the peptide chain elongation rate. When bacterial cells are moved to a higher nutritional state (shift-up) there is a slow change in RNA synthesis (Nierlich, 1978). Eventually, an increase in stable RNA is obtained due to a shift in the functioning RNA polymerase enzymes. It was concluded (Maaloe and Kjeldgaard, 1966) that new RNA must be synthesized in order to achieve the increase in protein synthesis observed after a shift-up transition. Work by Bates (1980) in which he grew rumen bacterial strains at various specific growth rates and stationary phase clearly showed a variation in RNA to protein ratios with changes in specific growth rate. A high positive correlation (.79) was demonstrated by this worker (Bates, 1980) between the RNA to protein ratio and specific growth rate for five strains of anaerobic rumen bacteria. Gillett 21 et a1. (1982) demonstrated that RNA/Protein values increased with time after feeding and that this ratio (RNA/Protein) can be used to assess the physiological or growth state of bacterial cells. MONENSIN EFFECTS ON RUMEN FERMENTATION Monensin (Rumensin, Eli Lilly & Co.) is a polyether antibiotic produced by a strain of Streptomyces cinnamonensis which increases the molar proportion of propionic acid when added to the ruminant diet (Richardson et al., 1974; Potter et al., 1976; Sakauchi et al., 1979). An improvement in feed efficiency which is probably associated with the alteration in rumen fermentation has been observed when monensin is fed to feedlot cattle (Raun et al., 1976). Dinius et a1. (1976) reported that monensin at the level of 33 parts per million (ppm) had no influence on the number of protozoa, total bacteria or cellulolytic bacteria in rumen fluid. Inhibitory effects of monensin on methanogenisis in the rumen were reported by Van Nevel and Demeyer (1979) and Chen and Wolin (1979). Additionally, Sakauchi and Hoshino (1981) have studied the effect of monensin on fermentation and populations of rumen microbes in bloated ruminants and found a significant decrease in the incidence of feedlot bloat related to a decrease in rumen fluid viscosity in cattle fed monensin. The volatile fatty acid patterns remained the same in both bloated and 22 non-bloated animals studied. MONENSIN AND VOLATILE FATTY ACID PRODUCTION An estimate of the theoretical energy savings from adding monensin to the diet of a ruminant can be made on the basis of its ability to change the distribution of volitile fatty acids produced in the rumen. The efficiency of ruminal fermentation may be limited by the relative inefficiency of the acetic and butyric acid fermentations (Van Nevel, 1969). The propionic acid fermentation is more energetically efficient and theoretically reduces the large losses of methane associated with the production of acetic and butyric acids (Wolin, 1960). As well, the utilization of propionic acid in ruminant tissue may be higher than that of acetic acid (Blaxter, 1962). Eskeland and co-workers (1974) reported that propionic acid increased nitrogen retention in the ruminant more so than acetic or butyric acids. Leng and co—workers (1967) have theorized that this nitrogen retetion occurs through a sparing of protein from gluconeogenisis and that propionate may be utilized better as an energy source for protein synthesis. Studies of the shift in molar proportions of volatile fatty acids in favor of propionate when monensin was fed to ruminants were conducted by many workers (Richardson et al., 1974; Richardson et al., 1976; Dinus et al., 1976; 23 Perry et al., 1976). In vitro studies conducted by Richardson et a1. (1974) demonstrated that the addition of 1.0 ppm of monensin increased propionate production by 45 percent but had no effect on total volatile fatty acid production. Similar results were obtained when rumen fluid from both high grain and high roughage fed cattle was used. Richardson et a1. (1976) further investigated monensin to determine an optimum. dose-response level for the drug. Monensin was added at six different concentrations to incubation mixes containing rumen innocula from cattle. Results of these workers (Richardson et al.,1976) indicated that at the 1.0 ppm level monensin reduced acetic, valeric and isovaleric acids and produced a 50 percent increase in the propinoic acid production. Total volatile fatty acid production was unchanged. In feeding trials conducted by Richardson et a1. (1974) monensin was fed to cattle at varying levels from 200 to 500 ndlligrams (mg) per day. A 52 percent increase in propionic acid over controls was observed at the 200 mg per day dose, results which would confirm similar findings in vitro. MONENSIN EFFECTS ON MICROBIAL POPULATIONS Haney and Hoehn (1967) demonstrated the inhibitory effects of monensin on the growth of gram-positive 24 microorganisms, and many workers have since studied the effects of this ionophore on rumen bacterial populations both in vivo and in vitro. Work by Van Nevel and Demeyer (1977) showed in vitro, that monensin had no direct toxic effect on methanogenic bacteria commonly found in the rumen. Monensin did not inhibit methane formation from hydrogen and carbon dioxide but was shown to inhibit formate decomposition during fermentation. Earlier work by Dinius et al. (1976) in continuous culture has shown small decreases in methane production associated with the addition of monensin to the culture media. Later, Chen and WOlin (1979) demonstrated that the sensitivity of methanogens to monensin was variable and depended on the organism in question. A delayed growth response was the most often observed condition in these bacteria. In the study of specific rumen organisms Chen and Wolin (1979) found Bacteroides succinogenes and Bacteroides ruminocola , both succinate producers in the rumen, to be less sensitive to monensin than the previously studied methanogenic bacteria. Important cellulolytic bacteria such as Ruminococci and Bugyvibrio fibrosolvens were highly sensitive to monensin. It was hypothesized by these workers (Chen and Wolin, 1979) that the selection of a rumen microbial population capable of producing greater proportions of propionic acid such as the gram-negative organisms, was influenced by monensin. Henderson et a1. (1981) studied the effects of 25 monensin on pure and mixed cultures of rumen bacteria and suggested that monensin addition to the rumen results in the selective inhibition of the growth of rumen bacteria that are not important producers of propionate. Growth of Selenonmonas ruminantium, Megasphaera elsdenii and Bacteroides ruminicola was shown to be favored. This work was in agreement with earlier work done by Chen and Wolin (1979) . MONENSIN AND EFFICIENCY OF MICROBIAL GROWTH Studies on the efficiency of microbial growth and the effects of monensin on this rumen parameter has been investigated by many workers (Richardson et al., 1976; Chalupa, 1977; Van Nevel and Demeyer, 1977; Wallace et al., 1981; Isichei, 1980). In vitro studies by Richardson et al. (1976) showed no change in microbial cell yield or efficieny due to monensin, with similar results being reported by Chalupa (1977). Other workers showed significant reductions in microbial efficiency when monensin was added (Van Nevel and Demeyer, 1977). Poos et a1. (1979) reported significant decreases in bacterial-nitrogen flow to the abomasum with monensin fed animals. Similar findings were reported by Isichei (1980) while studying the effects of monensin on protein metabolism in steers using passage study 26 techniques. Recent investigation by Wallace and co-workers (1981) looking at the effects of monensin on rumen fermentation using the rumen simulation technique reported an increase in the efficiency of microbial growth in terms of dry-matter produced per unit dry-matter digested. MONENSIN EFFECTS ON PROTEASE ENZYMES Little information is presently available on the effects of nwmensin on the activity of the protease enzyme within the rumen. One of the few reports of research in this area is from a study by Wallace and co-workers (1981). These workers reported an increase in the specific activity of the protease enzyme in the rumen fluid studied. Other work by Dinus et a1. (1976) showed reductions in overall rumen ammonia concentration and the rate of protein degradation seen with monensin. These findings suggest that the rumen protease and deaminase activity within the rumen, may be inhibited. The investigation described fir: this thesis was partially designed to Igive further information. into the effects of monensin on the protease and deaminase activity of bacteria found unbound in the rumen contents. MODE OF ACTION OF MONENSIN The mechanism of action of the antibiotic monensin is somewhat unclear. Shifts in the molar proportions of volatile fatty acids in favor of propionate and the energetic benefits obtained from these shifts has been offered as a partial explanatirni of the drug's action. Studies using [l4C]-glucose suggested that the increase propionate production was due to the acrylate pathway, but data from in vitro batch culture work indicated‘ that monensin increased the succinate decarboxylating capacity of the rumen bacteria (Romatowski, 1979). Other changes associated with the effects of monensin include decreased rumen ammonia concentrations, decreased methane production, depressed protease and deaminase activity and a decrease in the efficiency of microbial protein production (Chalupa, 1980). Monensin has been identified as a sodium ionophore and as such has a biological activity related to its ability to regulate the movement of cations such as sodium, potassium and. calciunl across cell. membranes (Pressman, 1976). Monensin mediates primarily Na+/H+ exchange through the antiport system because the affinity of monensin for Na+ is ten times that for K+ (Pressman, 1976). In order for ionophores to transport ions across cell membranes efficiently, certain kinetic criteria must be met. At the 27 28 region of high—dielectric charge at the cell membrane interface, complexation and decomplexation reactions must be rapid. The exchange of the ion solvation shell from the ionophore oxygen system must be a concerted reaction in order for the energy of activation of transport not to rise excessively. Once the complex leaves the cell interface and enters the low—dielectric region of the membrane interior where it is immune from attack, it may attain high stability (Haynes et al., 1969). When these criteria are met within a cell, the ionophore may then cause an alteration in membrane permeability and hence effect transport of material in and out of the cell. Monensin actively dissipates proton gradients within the cell as long as the potential stored in the proton gradient exceeds that stored in the sodium gradient. When the gradients are reversed, monensin will disipate the sodium gradient at the expense of the Na+ coupled symport system for required cellular substrates (Bergen and Bates, 1983). The equilibrium affinities between ions and ionophores in any given in vitro system may not provide a definitive guide as to the relative transport reactions that would occur in a given biological system. Ionophores other than monensin are less well documented. Lasolocid, ‘a potassium ionophore recently approved for use with feedlot cattle, produces changes in rumen fermentation similar to monensin (Bartley et al., 29 1979). The mode of action of this new antibiotic has yet to be elucidated but its action may be similar to the lipophilic, cation-binding, ionophore monensin. MATERIALS AND METHODS GENERAL EXPERIMENTAL DESIGN Four Holstein steers weighing approximately 300 kilograms each were fitted with permanent rumen cannulas. The steers were housed in individual pens (approximately 1.8 x 2.5 meters) inside the metabolism unit at the Michigan State University Beef Cattle Research Center. The metabolism unit contains 20 pens and 8 collection crates with individual feed and water sources. The temperature in the unit is controlled between 10 and 18 C. A 4x4 Latin Square design metabolism trial was conducted using these animals to determine the effect of the feed additive monensin sodium fed in high concentrate diets on protease and deaminase activity of rumen bacteria, rumen ammonia concentration, volatile fatty acid . PIOduction, free amino acid levels in the rumen and the Physiological state of the rumen bacteria. The level of fee(iing was set at a constant 2.3% of average body weight and the animals were fed twice daily at 12 hour intervals. All animals were allowed free access to water. Animals 30 321 .uumn Hauocfifi moons H wH 0mm MN. on. m.~ m~ 05. NH omN MN. HA. mo. NH MN. Hm. ow. IIIIIIIIII:o nose co a IIIIIIIII .aouadsomao. «Ow Gdflugh Ova—NU “aac\ea\ae. asaeom equcoco: Madam omounorm aha m «um .00 and oumzmnosn Esau~60ao awonuouw ZEN .mu own .ccuo .ocounoswq OFOImOIm 2.: no ammN can: voouvoum 2mm couomuuxu aco>~om duo: . ovoom . unconaom anImOIm ZhH to COM aOm x: kudnco «sonar: .umolz manna guano: cuoo Hnmn~ouv zmu m: N no sedge» acme .omaoz so“: .cooo v m N A IIIIIIIIIIIII . emHoIIIIIIIIIIII FZMHDmmUzH mBHHD dezmlnmumxm ho ZOHBHmOAZOU .u mAm mm ZWZDM .N mamdfi 46 Rumen ammonia concentrations are presented in Table 3. Rumen ammonia concentration increased rapidly after feeding and was generally at its highest level by two hours post-feeding. A steady decline in the ammonia concentration was observed with pre-feeding levels being reached between six and twelve hours later. As expected, urea feeding significantly increased ruminal ammonia concentrations at all times after feeding. The addition of monensin to the experimental diets decreased overall rumen ammonia concentration of both the high and low protein levels. Rumen ammonia values for the soy plus urea diet showed the fastest rise after feeding and had the highest overall ammonia levels (Figure 3). The rumen ammonia values from the soy diet were observed to have the smallest change across time of the four diets studied. Rumen volatile fatty acid concentrations are presented in Tables 4 through 8a. Millimolar amounts of acetate were observed to be lower overall in diets containing monensin and this trend coencided with overall higher propionate concentration in those diets. All volatile fatty acid concentrations were at their highest level by four to six hours post-feeding after which a decline to pre-feeding levels was observed. Decreased acetate levels observed in monensin diets were found to be significant at 4 hours after feeding for the soy versus the soy plus urea plus monensin diets an at 8 and 12 hours after feeding for the 47 .Aao.vmv umwwflc mumfluomuomsm DCOHOMMHG nufl3 mammE CESHOU o.Q .mamEch H500 mo mace: o mmfi. «NH. mam. was. HHm. 0mm. 2mm szzmzoz + 00m.va Omv.ma owm.0a 0mm.mH 0mm.ma OHN.NH emms + wow Dam.na om0.ma OHN.mH omw.0m oam.>m oav.>H dumb + wow an.v nmo.v neo.v nvm.v nmv.m has.v szzmzoz + sow nmoé DES 03K nomd OBS omens wow IIIIIIIIIIIIIIIIIIIIIIIIIIII AO\02IIIIIIIIIIIIIIIIIIIIIIIII emHo was we we as me 09 ozHammm muses mmsom mmBmHD mmomod mZOHfideZMUZOU dHZOZSd zmzbm .m mqmdB mew—a : mwom0< mzo_h<¢bzmuzou <_zo==< zmzsz ”ZR—um... But: MBDOI N.— 0 0 Q IIIIIJ _ _ _ 48 z_mzmzo:Icm om z_mzmzozu>om >ow 0000009.. - - - .".‘ 10’9“ i 380915 49 .Amo.vm0 Hemmec mumwuomuomsm uconommwv Suez mCMOE QESHOU I o.n .mameflcm u500 mo memo: m mm.m mv.m 50v. H0.N mv.H 00.H 2mm szzmzoz + nnm.ma Omv.mm cm.av omv.mm vo.mm vm.mH mmma + wow oon.~m hom.vq oo.mm “Ozunn.am mm.mm om.om ammo + wow noo.ea omo.nm mm.mm “O;L>H.Vm mH.mm mm.mH szzmzoz + wow “u;uvm.mm va.mv mo.Nv Qmm.0v h0.0m vm.vm wow IIIIIIIIIIIIIIIIIIIIIIIIIIIII ZEIIIIIIIIIIITIIIIIIIIIIIIIII emHa was me we we me as quommm mmemm mmaom mmBmHD mmomod mZOHfideszZOU MBQBQU< zmzam .v mqmde 50 four diets (Table 4). Millimolar propionate concentrations were shown to be significantly higher in diets containing monensin at all times after feeding with the exception of the 12 hour values (Table 5). Rumen butyrate concentrations were at their highest levels by 4 hours after feeding and levels of this volatile fatty acid showed no significant differences across experimental diets (Table 6). Millimolar isobutyrate, isovalerate and valerate levels showed similar inceases across time with the highest levels being reached between 4 and 6 hours after feeding (Tables 6a, 7, 7a). No significant differences across diets were observed with the exception of the 2 hour soy plus urea versus soy plus urea plus monensin values for valeric acid (Table 7) and the 2 and 4 hour values across all diets for the isovalerate concentrations (Table 7a). Isovalerate concentrations were also significantly different at 8 hours after feeding for the soy versus soy plus monensin diets and at 12 hours after feeding for the soy versus soy plus urea plus monensin diets. Level of protein was shown to have no significant effect on any of the volatile fatty acids studied. Total volatile fatty acid concentration and acetate to propionate ratios are presented in Table 8. Total volatile acid concentration was highest between 6 and 8 hours after feeding for all diets and there were no significant effects of monensin or level of protein on the total volatile fatty acid production. Overall acetate to propionate ratios were 51 .AHo.vmv umwwflo mumfluomuomsm UCOHOMMHU £0H3 mcmoe :EDHOU 0.0 .Amo.vmv Mommav mumfluomuomsm ucouommwv sues mcmoa :Edaou o.n .mameflcm H500 mo mace: o hv.a hm.N 0H.v mm.N hm.N mh.m 2mm mo.mm 0mm.vv moea.mv nmm.em omv.am Dam.mm szzmzoz + flmmD + WOm vm.mm O.QHH.0N Uflmv.vm 00v.0m QHB.0H £50.0H flmmD + Now mm.mm nmm.vm o.eéumv.vv nmm.ov OMH.Hm ooa.mm szzmzoz + wow 00.0N th.mm O.U.QH0.mm OCQmNJHM Qmm.ma Qmm.hH wow IIIIIIIIIIIIIIIIIIIIIIIIIIIII EEIIIIIIIIIIIIIIIIIIIIIIIIIII smHo NHB 08 0B VB NE 09 UZHQmmh mmfihd mmDOm memHQ mmOfiUfl WZOHfigBZMUZOU mBOm oe.HH oa.va nm.mH mm.ma OH.m va.m «mm: + AOm Hq.HH mm.w GH.NH Ho.va ma.m mm.oH szzmzoz + wow vo.oa nm.aa Rm.e mn.va mm.ma m¢.mH wow IIIIIIIIIIIIIIIIIIIIIIIIIIII ZEIIIIIIIIIIII III- emHo was we we we me 09 ozHammm mmema mmaom mmBmHD mmOmU/w mZOHB/NKBZQUZOU mfigwgm szDm .0 @493. 53 mameflcm H500 00 05002 n owo. Hmo. wmo. an. mwo. mac. 2mm mw.a em.m mo.m ma.a wo.~ ww.H szzmzoz + «mm: + uOm vm.a wm.H mm.H mo.m mh.H «R.H ammo + >Om Av.H mm.H mm.~ Hw.a HR.H wv.a szzmzos + 50m mm.H wa.m mm.m av.m wo.m mm.H 50m IIIIIIIIIIIIIIIIIIIIIIIIIIIIII SEIIIIIIIIII I I I .mmmmm may we we we we we ozHOmmm mnemm mmaom mmBmHD mmomod mZOHBANmBZWUZOU mfigngmH ZMSDm .00 Sada. 54 .A00.vmv wawHC mumfiuomuom5m ucouowwHO nuflz mcmwe CE5HOU 0.3 .mHmEHCM M500 mo mammz m 0mm. vow. wwm. mow. nmm. mmv. zmm Hm.m ww.w wm.e wm.m onm.w wm.m szzmzoz + «mm: + wow vo.v mm.m mm.m ww.v DHw.m mm.m ammo + wow an.v vo.m om.> mm.n o.hmm.w ma.v szzmzoz + sew em.v Rm.m av.m mH.e o.nmo.w mm.w wow IIIIIIIIIIIIIIIIIIIIIIIIIIIII zsIIIIIIIIIIIIIIIIIIIIIIIIIII emHo was we we as me 09 wzHammm mmem< mmoom mmBmHQ mmomU< mZOHBgBZmHUZOU m9¢m§> ZWEDm .h mamas. 55 .Amo.vmv 000000 mumfluomumm5m 050000000 £003 mcwme :E5HOU 0.0 .mHMEflcm H500 00 0:00: 0 NRA. NNN. woo. mNm. «NH. Nww. 2mm ova.a 6.5mm.5 NN.H ova.N DNH.N «N.N szzmzoz + ammo + Now U.nmo N new N No N new N ems N we N ammo + Now 6.50m.N omN.H mo.N ONN.N oow.5 ww.N szzmzoz + Now nov.N an.N wN.N nao.m nav.N Nm.N wow IIIIIIIIIIIIIIIIIIIIIIIIIIIIIII ZEIIIIIIIIIIIIIIIIIIIIIIIIII .wmmwm NHe we we we Ne 09 ozHommm amend mmaom mmemHo mmomoa monaamezmozoo meammqa>omH zmzam .an mamas Ln 94hr VIN They _ r_.<- n._.—.<~LA ~— . netv— .. A v.—» ”L.-.<.~.H.—. v< A_Z< «Ty—- .v< >.~..—.<..~ n.—.~ I.— .~.<. nu ~> . u <.~CV.H. v~ u.~.~. ~<.~. 56 .chEHcm H500 00 memo: m HN.N ww.v no.m wn.m mm.H wm.H 2mm om. ov.ww RN.voa Nw.NNN mo.om Nw.vn No.mw szzmzoz + «mm: + wow Nv.H NH.wN mw.wm mw.mw wv.Nw Nv.ww mm.wm «mm: + wow NR. mo.ww mm.mn mn.woa mm.HoH NN.NN vm.ww szzmzoz + wow mN.H Nm.ww Nv.wm ww.mm on.wm mn.on wN.mw wow IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII ZEIIIIIIIIIIIIIIIIIIIIIIIIII emHO Au< NNB we we 49 Ne 09 ozHommm mmema mmsom mmBmHQ mmOMUfl won—”Ham WB¢ZOHmOmm OH. mBfiwMUfi QZfl QHUfl Edam m3HB§O> €43.09 .0 Emma. 57 .Amo.vmv newmflw mumfluomummsm DcouomwNw EDA: memos casaoo 0.0 .0008005 5500 00 name: 0 mN.m NNN. wmm. ooa. NN.H wo.N mm.N 2mm szzmzoz + mm.mw owN.H NN.w mm.N om.NH OHN.wm wm.wN OmH mfi mfiflmeDmOmH MB¢MWBDm mB¢ZOHmomm mB mmBmHD mmomo< mZOHfideZmUZOU DHUfl MfiBfim MAHB¢AO> “Dom NH mw¢mm>< .mm m4m¢fi 58 decreased in diets containing monensin with level of protein having no apparent effect on this parameter. Average twelve hour volatile fatty acid concentration is presented in Table 8a. Significant differences were noted. in propionate and isovalerate concentration across diets with total values for the six volatile acids studied remaining similar. BACTERIAL CELL AND SUPERNATANT PROTEASE ACTIVITY Cell-associated protease activity' of free: rumen bacteria is presented in Table 9 on a dry cell basis. Protease activity in milliunits of activity per milligram of dry cells was observed to be at its lowest level immediately before feeding after which the dry cell protease activity increased with time after feeding. Highest observed levels of activity was at 6 hours after feeding for the soy plus urea diet and at 8 hours after feeding for the three other diets as shown in Figure 4. Monensin tended to decrease the protease activity present in the bacterial cells and this depression was found to be significant at 6 hours after feeding between the soy plus urea and soy plus urea plus monensin diets. Level of protein tended to change cell protease activity with overall higher protease activity being observed in diets containing urea. The level of protein effect was found to 59 .Aao.vmv M00000 000000000050 050000000 5003 0:008 555000 0.0 .mamaecm H500 00 05002 0 mo. mo. No. No. No. zmm szzmzoz + Ome. oNo.H mm. owe. NH. «mm: + wow new 5mm. mN. nNN. e0. szzmzoz + wow Dom. th. mN. 0.50m. ma. 00m IIIIIIIIIIII mqqmo emu oz\meHZDEIIITIIIIIIIIIII emeo we we we Ne oe ozHomme mmeRN mmoom m.mBmHQ mmOmUAN flHmmfiodm ZMZDm mmmm .00 NBH>HBU¢ mmdmrhOmm «HAMU .m mqmdh. mew—n : mmozu< <_z000h_>_00< mmm= 60 z_mzmzozIom z_mzmzozI>ow 6000.000. --0.. SIIZIE GZ-Guuh Cuba—‘0 $2.03 I! o ‘ 0' Allv .3 iIZIIIISIIZIIIV .3 a) ...... \.IIIII.I I!) 41 38091:! '1 W0 SW ISLINfl ITIIW C) o 61 be significant at 6 hours after feeding between the soy and the soy’ plus urea. diets. An extremely rapid and large increase in dry cell protease activity was noted between 6 and 8 hours after feeding for the soy-only diet. The protease activity present in the cell-free rumen fluid of unbound rumen bacteria is presented in Table 10 and Figure 5. Cell-free values are expressed as milliunits of protease activity per milliliter of cell supernatant. In contrast to the dry cell protease activity, the supernatant protease activity was observed to be at it lowest level at 2 hours post-feeding. Pre-feeding levels of supernatant protease activity were, on the average, depressed by fifty percent within 2 hours after feeding. Following this decline was a slow increase in protease values until 4 hours post-feeding after which enzyme activity increased rapidly in diets containing urea. In the diet containing soy-only, a more gradual increase in supernatant protease activity was noted. Protease activity from the diets containing soy plus monensin remained relatively constant after reaching its lowest level at 2 hours post—feeding. Monensin tended to depress supernatant protease activity but no significant differences were obtained with the exception of the 0 hour valves between the soy plus urea and soy plus urea plus monensin diets. Diets containing a higher level of protein also tended to have higher overall supernatant protease activity but this observation was found not to be significant. 62 .A00.vmv 000000 000000000050 000000000 0003 00005 085000 0.0 .0006000 0500 00 00002 0 on. wmm. 00m. Nvm. mmq. 200 2002020: + mm.N0 OmN.00 00.00 oN.e omw.00 0005 + 000 cm.00 oov.wN mN.00 mw.m 000.e <00: + 000 me.00 000.0 00.0 mN.m o.nm0.00 2002020: + 000 cm.00 o.nwv.00 me.m mv.w 0.0mm.m0 >00 IIIIIIIIIIIIIIIIIII 02\mestEIIIIIIIIIIIIIIIIII e000 we we we Ne oe 0200000 m0e0< 00500 mmBmHD mmOfiUd flHmmBU¢m ZflZDfi mmmm mo BZMdemmmDm Aqmu ZH >9H>HBU¢ mmdm90mm .00 qudfi 63 000_= : mmoz0< >__>_00< 00(00000 hz<0om om z _ mzwzoz->om >8 00:00. 7'... S 38091:! 64 Table 11 contains the ratios of relative supernatant protease activity to dry cell protease activity. This presentation of protease activity takes into account the relative proportions of cell supernatant and dry cell mass as represented in the typical bacterial cell (Hungate, 1966), approximately 95% supernatant and 5% dry cell mass. Across all diets, the supernatant to cell (S:C) ratio was observed to be greatest immediately before feeding. A 75% decline in S:C ratio produced the lowest measured values by 2 hours after feeding and these values remained low up to 8 hours after feeding (Figure 6). Addition of monensin and level of protein appeared to have no significant effect on S:C ratios. RUMEN FLUID DEAMINASE ACTIVITY Rumen fluid deaminase activity is presented as micromoles of enzyme activity (substrate hydrolysed or deaminated) per milliliter of strained rumen fluid in Table 12. Deaminase activity in rumen fluid from all diets was observed to be relatively low prior to feeding and remained low until 2 hours post-feeding. Deaminase activity increased rapidly at 2 hours after feeding and reached peak values by 4 hours after feeding for all diets with the exception of the soy plus urea plus monensin combination. For all diets, the deaminase activity had returned to 65 .0005000 0500 00 00005 EOHM mHm mOHHMNm M N0m. O0N. m0m. N00. 00m. 2mm ZHmZMZOE + oh.m 00.0 vm.m mb.v mw.h0 dmmD + wow mo.m vm.v 0m.m mm.m mm.m0 flmmD + 00m No.0 00.0 vw.© mm.v 0®.N0 ZHmzmzoz + wow mm.m mh.© ©m.n m0.v ov.w0 NOm a we we 09 NE 09 0209mmm mMBmd mmDOm 0.WBH>HBU¢ mmflmfiomm 00m0 wma OB mO0 x wBH>HBU¢ mmflmBOmm Bzmfiflzmmmbm m>HB¢0mm m0 mOHB00>000< 00000000 00 00000 0000 >00 00 02000200000 623mm."— flmhu< ”~50: Q w .v N In 3 z _ mzmzoz-om .3333: & om I .0 0 I. z_ mzmzoz->om :2!!! >9” .I..--..-.--. 1130 I 83dn$ 9 380915 67 .AHo.vmv Hmwwfiv .Amo.vmv memww mumfluomummsm ucmummmflc £uw3 mammE :EsHoo m.© mumfluomummsm ucoumwwfiv zuMB mammE CEDHOU 0.3 .mHmecm know mo mammz m ooo. moo. ooo. moo. moo. 2mm szzmzoz + o.nnmo. Hvo. mmoo. moo. oomo. «mm: + yew oomo. moo. ovam. ooo. ooo. HBU€ mm¢ZH24mD DHDAm zmzsm .NH wqmfifi 68 pre-feeding levels by 6 hours after feeding. Deaminase values for the soy-urea-monensin diet rose slightly after feeding but remained at a constant and low level across all times after feeding (Figure 7). Monensin addition generally depressed deaminase activity across all times and at both protein levels. This depression in enzyme activity with monensin was found to be significant between the soy plus urea and the soy plus urea plus monensin diets at 4 hours post-feeding. RNA TO PROTEIN RATIOS OF FREE RUMEN BACTERIA RNA to protein ratios from free rumen bacteria across diets and time after feeding are presented in Table 13. RNA 1x3 protein values were at their lowest levels immediately before feeding across all diets. Monensin addition and level of protein had no significant effect on RNA to protein ratios at any time after feeding although the ratios observed in diets containing monensin did tend to be somewhat higher than those diets not containing monensin. RNA to protein ratios (R:P) within diet but across time after feeding did show a significant increase in all diets with the exception of the soy plus urea diet. R:P ratios were highest at 4 hours after feeding for the soy, soy-monensin and soy-urea-monensin diets and followed a 69 00000 0 000000 00000 00000 00000000 00 00000000 0000_0000 GZEUHL zmt< ”:30: a w t N ”mung OS-SSSSSS-uooogd. z_mzm_zo:-om 33339 om 1.1-1. z_mzmzoz->om II!!! >om 339.33... O N 0 ID N 0 1w Isa-loud l annals 70 .Amo.vmv Hommflo mumfluomummsm usmuwMMMo nu03 mzou mo mammz o.m.m.o.o .gcmHmMMHo waucmoHMHcmflm no: momma CESHOU Q .mameflco knew mo mammz o NHo. o0o. 00o. oHo. 00o. zoo ZHozmzoz + 00o. m0o0. ovoq. mom0. oomm. oomm. ammo + 000 mmo. onm. omHM. omOm. onom. Umom. «was + 00m oqo. mmom. 0m00. mooo. oo00. umvm. szzmzoz + mom moo. m.n000. 0.noov. m.nmom. o.noom. o.nmom. woo BmHQ 200 me oh 09 me o9 oZHommm mmemo mmoo: m.UZHQth KWBhd MSHB 02¢ mBMHD mmomod demfivdm ZmZDm mmmm m0 mOHfiflm ZHmfiomm OB 42m .MH mam<9 71 slow decline between 4 and 8 hours post-feeding. R:P values from the soy-urea diets reached its highest level by 2 hours after feeding and remained extremely constant out ‘UD 8 hours after feeding (Figure 8). RNA to protein values at 8 hours post-feeding remained above pro-feeding levels but were on the decline. 00000 0 000000 00000000 00000 0000 00 0000000 0000000 00 000 "cl—Gum..— flmt< mflq—O: a w G N 72 a z _ mzwzoznsm: ->om .3333. 0000-000 .00.... z _ mzmzo:->om ill... >Dw non-=3..- J . f NI 31.08:! IVNH 8 38091:! DISCUSSION RUMEN AMMONIA, pH AND VOLATILE FATTY ACIDS The results obtained from the parameters of rumen pH, volatile fatty acid concentration and rumen ammonia concentration were consistant with the work of other investigators (Richardson et al., 1976b, Potter et .al., 1976) using similar diets and experimental conditions. As expected, rumen pH tended to be slightly acidic as a result of the level of concentrate included in the experimental diets. There were no significant changes in rumen pH as a result of the addition of monensin or the (change in protein level in the experimental diets. The relatively constant pH levels observed are an indication of a well maintained rumen fermentation. Rumen ammonia concentrations were observed to rise quickly immediately after feeding but as ammonia was assimilated by the rumen organisms and was lost across the rumen wall, the ammonia levels were decreased by 4 to 6 hours after feeding. As expected, the addition of urea to the experimental diets caused an increase in overall rumen ammonia levels but the patterns of ammonia loss with time after feeding were 73 74 relatively similar. Monensin was shown to depress ammonia concentrations across all times after feeding when included in experimental diets. This is in agreement with the effect of monensin on rumen fermentation reported by several workers (Dinius et al., 1976, Van Nevel and Demeyer, 1977, Sakauchi and Hoshino, 1981). Using an in vitro incubation system, Van Nevel and Demeyer (1977) demonstrated that monensin addition resulted in lowered protein degradation along with lowered ammonia production and suggested that the protein-sparing effect of monensin resulted from the inhibition of microbial deamination. The effect of monensin on microbial deamination will be discussed later in this text. Rumen volatile fatty acid concentration was highest in all diets between 4 and 6 hours after feeding indicating the greatest rumen fermentation activity at or near these times. If these rumen VFA levels are considered indicative of rate of fermentation in the rumen then this would suggest a highly active bacterial population at the times of maximum volatile acid production. If RNA to protein values can be accepted as a means of assessing the physiological state and hence the growth rate of rumen microorganisms (Bates et al., 1984) we can than compare these values of RNA/protein to the rumen fermentation pattern as indicated by volatile fatty acid production. The results of this work would show a positive correlation between peak RNA to protein ratios and highest volatile 7S fatty acid concentration in the diets studied. In agreement with the results of Richardson et a1. (1976), Potter et al. (1976) and Raun et al. (1976) monensin addition shifted the proportionate amounts of volatile fatty acids produced in the rumen in favor of propionate. The shift towards increased propionate concentration has been explained. by 'the change in microflora present in the rumen after addition of monensin. An increased number of gram-negative bacterial strains such as Selenomonas ruminantium, Bacteroides ruminicola, Bacteroides amylophilus and Butyrivibrio fibrisolvens will populate the rumen and these organisms tend to produce higher amounts of propionate than their gram-positive counterparts. In changing the molar proportions of the rumen volatile acids towards increased propionate and less acetate and butyrate, monensin theoretically increases the efficiency of converting feed energy to energy in the acid end-products available for absorption (Richardson et al., 1976). Total volatile fatty acid production and average 12 hour production was unchanged by the addition of monensin or by increased protein content in the diet however. PROTEASE, DEAMINASE AND RNA TO PROTEIN RATIOS Early work by Blackburn and Hobson (1960a) using whole and fractionated sheep rumen contents indicated that active 76 proteolysis is caused by whole rumen fluid and is not necessarily connected with active growth of the bacteria. These workers (Blackburn and Hobson, 1960a) also found little free protease in the rumen fluid and concluded that the main activity was associated with the microorganisms. Further work by Blackburn and Hobson (1960b) on the isolation of the proteolytic bacteria in the sheep rumen resulted in the isolation of only a small portion of the proteolytic bacteria and since proteolysis was found to be always extensive in mixed culture, it was concluded that the total breakdown of protein in the rumen was caused by several kinds of bacteria acting together. Assuming this to be the case, any change in the physiological status of these bacteria could theoretically effect the rate and or extent of proteolysis in the rumen. These same workers (Blackburn and Hobson, 1960c) studied rumen proteolysis in relation to time after feeding. The results of the determination of proteolytic activity showed that although there was some increase in activity after feeding, the rapid breakdown of foodstuff protein was due more to there being a relatively constant proteolytic activity in the rumen rather than to a sudden increase in enzyme concentration. Although these workers (Blackburn and Hobson, 1960c and Blackburn and Hobson, 1960) studied the extent of proteolysis in the rumen with time after feeding, they used unfractionated strained rumen fluid for their 77 determinations which may have prevented a clear discrimination between any increase in protease activity in relation cellular location of such activity, an important aspect especially when consideration of bacterial stage of growth is coupled to protease production. The results of the bacterial cell protease activity from the work described in this thesis showed an interesting trend in the partition of the enzyme activity with time after feeding between the cell bound fraction and the supernatant free fraction of the unattached rumen bacteria. Immediately prior to feeding, the greatest protease activity was found to be located in the supernatant fraction of the bacterial cells. These levels were observed to decrease rapidly after feeding while cell bound protease activity was shown to increase. Similar results have been obtained by Hazelwood et a1. (1981) working with cultures of Bacteroides ruminicola R8/4 in which they observed that proteolytic activity was largely cell-associated and that the extracellular activity only reached significant levels after stationary phase had been reached. Additional work by Nugent and Mangan (1981) showed that proteolytic activity in the rumen is almost entirely associated with the bacterial cells and that rumen fluid and protozoa have little proteolytic activity. Brock and Forsberg (1980) and Brock et a1. (1982) studying the proteolytic activity of rumen microorganisms and the effect of proteinase 78 inhibitors, concluded that approximately 25% of the protease activity of rumen contents could be recovered in the strained rumen fluid fraction and the balance of the activity was associated with the particulate fraction. It was determined that the specific activity of the protease from the bacterial fraction was 6 to 10 times higher than that from the protozoal fraction such that the proteolysis of soluble protein in the rumen was affected primarily by cell-associated enzymes of the rumen bacterial fraction. The change in location of proteolytic activity observed in this study may be affected by the time after feeding in view of the influx of fermentable substrates into the rumen and the availability of soluble materials to the bacterial population in the rumen. This protease activity response may be correlated with a change in physiological status of the bacteria in the rumen observed during this period. RNA to protein ratios may be used as an indicator of the physiological status of rumen bacteria as it has been shown that RNA content as well as RNA to protein ratios vary with the physiological state or specific growth rate of bacteria studied (Nierlich, 1978). Bergen et a1. (1982) showed increased RNA/protein values with increased growth rate of rumen bacteria grown in pure culture. RNA/protein values of .2 to .3 are usually obtained for enteric organisms growing at or near stationary phase 79 (.06-.07 doublings/hour) (Bergen et al., 1982). It is apparent that the total amount of RNA synthesized in the rumen depends largely on the extent of bacterial growth. The results of RNA to protein ratios observed in this study indicate a: shift in physiological state of the rumen bacteria immediately after feeding. Greatest RNA to protein values were observed between 4 and 6 hours after feeding (.31 to .50) which suggests greatest bacterial growth at these times when examined in relation to RNA/protein values obtained by Bates et a1. (1983) for rumen bacteria grown in pmre culture and extrapolated back to zero growth rate. Correspondingly, observed cell bound protease activity, deaminase activity' and volatile fatty acid concentrations attained their highest levels at these times. As time post-feeding increased, RNA/protein ratios of the free rumen bacteria studied began to decline indicating a return to the stationary phase of growth (RNA/Protein = .2-.3) in these bacteria. With this drop in RNA/protein values was observed. an increase in ‘protease enzyme activity in the cell-free supernatant and a decrease in cell bound protease activity. This is in agreement with work by Hazelwood et al. (1981) who reported significant extracellular protease activity only after stationary phase had been reached in the Bacteroides ruminicola R8/4 strain studied. These workers (Hazelwood et al., 1981) found cell-associated protease activity to be highest (90% 80 of total) during the mid-log phase of growth and lowest (50% of total) during the late-declining stage of growth in the bacteria studied using Fraction 1 protein as the sole nitrogen source. Enzyme activity remained almost entirely cell bound during the exponential phase of growth after which progressive cell lysis was accompanied by the release of an increasing proportion of the protease into the culture medium (Hazelwood et al., 1981). Increased cell lysis in response to substrate limitations may be responsible for the largest proportionate change in location of enzyme activity in view of this work by Hazelwood et al. (1981) and the work of Nolan and Leng (1972) using lS-N to estimate bacterial turnover, in which they showed that approximately 50% of the bacterial cells lysed as when substrate became depleted. Early results of Nugent and Mangan (1981) indicate as well that this may be the case. The change in dietary nitrogen level with the addition of urea was shown to increase the overall protease activity. This finding may be a result of relieving a possible nitrogen, but not energy, limitation in the rumen bacteria. Similar results may have been expected with RNA to protein values if nitrogen was limiting bacterial growth although this may not have been the case at either of the dietary crude protein levels used, and if under nitrogen limiting conditions these RNA/protein values are truley 81 indicative of bacterial growth. Under conditions of nitrogen limitation the intracellular proteins may be degraded at a fast rate which would skew the RNA/protein values in favor of RNA resulting in higher RNA/protein values when dietary crude protein (available nitrogen) was at a low level. WOrk by Bates (1983), in which higher RNA to protein ratios were observed when cells studied in pure culture were sent into stationary phase due to a nitrogen limitation, helps to confirm this hypothesis. The conditions of nitrogen limitation may be simulated by monensin through a depression in amino acid transport in treated cells and this in part would explain the higher RNA/protein values observed with monensin treatment. The increase in rumen ammonia concentrations which resulted from the elevated dietary nitrogen content when urea was included in the experimental diets was found to correlate with a rise in overall protease and deaminase activity. These findings are in ;partial agreement with Mehrez et al. (1977) and Wallace (1979) who found that the hydrolytic activity of rumen bacteria from barley-fed sheep increased when the concentration of ammonia in the rumen fluid increased. In contrast, recent work by Hanlon et al. (1982) showed a depression in protease activity in a strain of non-ruminal bacteria (Bacillus licheniformis) in response to elevated ammoniunl chloride concentrations in glucose-exhausted cultures. 82 The shifts in ndcrobial populations in the rumen upon addition of nmmensin are well documented as being in favor of the typically gram-negative bacterial strains (Haney and Hoehn, 1967, Richardson et al., 1976. Dinius et al., 1976). Research done in the early 1960's indicated that the primary proteolytic organisms in the rumen are gram negative rods such as Bacteroides ruminicola, Bacteroides amylophilus, Selenomonas ruminantium and Butyrivibrio species (Blackburn. and. Hobson, 1962). Some gram positive bacterial strains may also contribute to the proteolytic activity in the rumen but any such species would be inhibited by monensin. It would be logical to assume that if population shifts alone could explain the effect of monensin on rumen fermentation then protease activity in the rumen should actually increase when monensin is fed. Work by Van Nevel and Demeyer (1977) as well as the work described in this thesis has shown that this in fact is not the case as monensin actually depressed the proteolytic activity present in the rumen. This monensin effect may result from the compound's having a direct action on the physiological status of the rumen bacteria and not be entirely due to a population shift. Monensin was observed to depress protease activity across all times after feeding. At the same time, RNA/protein ratios tended to be increased with the monensin treatments but these results were not significant. This 83 result may explain to some extent the depressed ruminal ammonia levels observed in animals fed diets containing monensin especially in view of the similar depression observed in deaminase activity. This depression of proteolysis with monensin addition may be the result of the inhibitory effect of monensin on peptide transport into microbial cells as it is known that some bacteria can use only ammonia or peptides as nitrogen sources (Bryant, 1970). Monensin may also depress total cell numbers resulting in a depression of available proteolytic and deaminative enzymes. Recent work by Hazelwood et al. (1983) in which Streptococcus bovis and Butyvibrio spp. were grown in culture in the absence of ammonia indicates that these species of rumen bacteria may make a substantial contribution to total proteolytic activity when present in mixed culture. In view of these findings and the fact that monensin would selectively inhibit these organisms it may be speculated that the depressed protease activity observed with monensin addition may be in part due to the loss of the proteolytic contribution by these bacteria. Monensin may as well have a direct effect on both protease and deaminase activity (Van Nevel and Demeyer, 1977). Deaminase activity in samples of strained rumen fluid was observed to increase immediately after feeding across all diets, corresponding to an influx of nitrogenous substrate. The deaminase activity measured in these "v... \‘5' .yu. 9., a ».¥ ~. 0., 0.5 'S‘r. ‘WL'. b a ll. 'fll ‘~' 84 samples was depressed when monensin was added to the diet. This depression in deaminase activity may be coupled to a depression in amino acid transport in monensin treated cells (Ando et al., 1982) if in fact there is a link between the rate of amino acid transport and deamination (Scheifinger, 1983). Data from Dickerson (1983) working with monensin addition to in vitro fermentations of fractionated rumen fluid indicates a total supression of deaminative activity in response to monensin addition. Van Nevel and Demeyer (1977) observed a similar depression in feed protein degradation in the rumen of sheep and suggested an altering of the final site of digestion of the dietary protein in the animal. The beneficial effect of monensin on animal performance was thus attributed to both the change in rumen fermentation patterns as well as to this shift in dietary protein site of digestion due to depressed proteolysis and deamination (Poos et al., 1979, Isichei, 1980). 7"!" I .It« A flfl‘t u va1 ”ETC-C now-‘~ 1113‘ C.” I u a) La ii . ‘1; ' 1 SUMMARY In summary, monensin was shown to shift the proportionate amounts of the volatile fatty acids in the rumen in favor of propionic acid and at the expense of acetic and butyric acids. Neither monensin or protein level had any significant effect on total VFA concentrations in the rumen. Ammonia concentrations in the rumen were depressed by monensin at both protein levels and at all times after feeding. Urea resulted in overall higher rumen ammonia concentrations when included in the experimental diets. Rumen pH was somewhat acidic but was unaffected by either monensin or protein level. Regardless of treatment, protease enzyme concentration was shown to change with respect to its cellular location with time after feeding. Immediately prior to feeding protease concentrations were highest in the cell-free supernatent fraction with only a small amount of activity being cell-associated. Within two hours after feeding the cell-associated protease activity increased markedly with a concomitant decrease in supernatent values. Monensin depressed protease activity at all times after feeding regardless of protein level and in both locations examined. 85 86 Deaminase activity increased immediately after feeding andwas highest in the urea-no monensin treatment. Monensin depressed deaminase activity at all times after feeding. RNA/protein levels of the unbound bacteria studied increased from ratios indicative of stationary phase at zero time to ratios indicative of active growth by six hours post-feeding. After approximately six hours post-feeding, RNA/protein levels began to decline indicating a return to stationary phase of growth in the bacteria studied. Peak RNA/protein levels were positively correlated with highest cell-associated protease activity, highest deaminase activity and peak volatile fatty acid concentrations. Monensin increased the RNA/protein values when included in the diet but this increase was less dramatic at the higher protein level. LITERATURE CITED LI TERATURE CITED Amos, H. E. and D. E. Akin. 1979. Rumen protozoal degradation of structurally intact forage tissue. Appl. Enviro. Micro. 36:513. Ando, A., I. Kusaka and S. Fukui. 1982. Sodium dependent active transport systems for organic solutes in an alkalophilic bacillus. J. Gen. Micro. 128:1057. Bartley, E. E., E. L. Herrod, R. M. 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Metabolic interaction amoung intestinal microorganisms . Am. J. Clin . Nutr . 27:1320. Wright, D. E. 1967. Metabolism of peptides by rumen microorganisms. Appl. Micro. 15:547. .. _ ___.- _..__——.-0~—“- _—.. . ,v‘ ' "‘31"? I. ' I 5300' V3,, .n V": at. . .0». ‘1 Wm"; STRTE UN MICHIGAN WW" I 13112 IV. LIBRARIES IHI”NIH\IIHIIWIWHI 37653 1'. lllllllll 110 £355