THE UTILIZATION OF VARIOUS NON-PROTEIN NITROGEN SOURCES BY RUMEN MICROORGANISMS IN A CONTINUOUS FLOW IN VITRO FERMENTATION SYSTEM BY \ Benny E1 Brent A THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Animal Husbandry I966 Benny E. Brent candidate for the degree of Doctor of Philosophy DISSERTATION: The Utilization of Various Non-Protein Nitrogen Sources by Rumen Microorganisms in a Continuous Flow lfl.!i££2 Fermentation System OUTLINE OF STUDIES: Main Area: Animal Husbandry (Animal Nutrition) Minor Areas: Biochemistry, Physiology BIOGRAPHICAL ITEMS: Born: July 3, 1937. Alton, Kansas Undergraduate studies: Kansas State University, l955-l959 Graduate studies: Kansas State University, l959-l960 Michigan State University, l960-l966 EXPERIENCE: Graduate Research Assistant, Michigan State University, l960-l963 Graduate Council Fellow, l963-l96h Principal Chemist, Department of Animal Husbandry, Michigan State University, l96h-l966 MEMBER: American Society of Animal Science Society of the Sigma Xi Phi Kappa Phi Gamma Sigma Delta Alpha Zeta American Society for the Advancement of Science ABSTRACT THE UTILIZATION OF VARIOUS NON-PROTEIN NITROGEN SOURCES BYRUMEN MICROORGANISMS IN A CONTINUOUS FLOW IN VITRO FERMENTATION SYSTEM by Benny E. Brent A novel l2.¥l££2 rumen fermentation systemhas been designed which duplicates closely in quality and quantity, ifl.¥l¥2 reactions and end products. The system was Used in a study of the utilization of several non-protein nitrogen compounds. The in vitrg system was of the dialyzing, continuous flow type and was designed to automatically sample both fermentation liquor and dialyzate. The fermentation liquor volume was 250 ml. and the volume of dialyiing solution was llOO ml. Original inoculum was 250 ml. of rumen fluid strained through A layers of cheesecloth. The fluid was obtained from a donor cow maintained on a constant high concentrate corn-hay diet. Removal of two 5 ml. samples per hour from the fermentation liquor and two l0 ml. samples from the dialyzing solution along with replacement of equal amounts of fresh dialyzing solution in each compartment gave turnover times of 25 hours and 55 hours for fermentation liquor and dialyzate, respectively. Dialyzing solution was a mineral buffer adjusted to pH7.5. The substrate consisted of 5 gm. of the same diet that was fed the donor cow. Five gm. of the substrate plus lOO mg. of nitrogen from the nitrogen source under study were introduced into the fermentation liquor each l2 hours. Benny E. Brent Substrates studied were: (i) control, to which no nitrOgen was added, (2) urea, (3) soy protein, (A) l, 3 - dimethylurea, (5) biuret, (6) biurea, (7) gUanidine hydrochloride, (8) guanylurea sulfate, and (9) thiocarbanalide. Precipitate nitrogen (32,000 X g) was determined on the fermentation liquor, and total nitrogen, ammonia, and volatile fatty acids were determined in both the fermentation liquor supernatant and dialyzate. These measurements showed that of the compounds studied, only urea was hydrolyzed to ammonia to an amount approaching completion. Biurea and thiocarbanalide were largely insoluble under the conditions of the system. However, biurea released significantly (P < .05) more ammonia than was released from the control substrate. Soy protein, l, 3 - dimethylurea, biuret and biurea all released more ammonia (P4< .05) than the control. However, of these nitrogen sources, only soy protein and l, 3 - dimethylurea appear to be hydrolyzed to an extent sufficient to be useful to the ruminant. Although differences between substrates in the amount and pro- . portions of volatile fatty acids produced were difficult to interpret, the similarity between these values and those from intact animals indicate that the la vitro system closely duplicated l vivo con- ditions. The validity of this observation is further strengthened by the fact that the system maintained an active protozoal population throughout the 5 day fermentation periods. ACKNOWLEDGEMENTS \ The author wishes to express his sincere appreciation to Dr. H. H. Newland for guidance during his graduate program and for critical evaluation of this manuscript, to Dr. D. E. Ullrey, Dr. E. R. Miller, Dr. R. H. Luecke, and Dr. E. P. Reineke for serving on the guidance committee, and to Dr. R. H. Nelson, Chairman of the Animal Husbandry Department, for the financial aid in the form of a graduate assistantship during 1960, 1961, and l962. The author's appreciation goes to the Graduate Council for financial support in the form of a fellowship during the academic years of l963 and l96h. Special thanks for encouragement, support, and cooperation in completion of the thesis study are due Dr. E. R. Miller and Dr. D. E. Ullrey, in whose laboratory the author served as principal chemist during the past two years. Mrs. Betty Bradley, M. T. (A.S.C.P.) and Mr. John Schoepke carried out many of the chemical determinations. Their contribution is deeply appreciated. Special thanks go to Mr- Kenneth Kemp for computer programming and help with the statistical analysis. The author is indeed grateful to his parents, Mr. and Mrs. H. A. Brent, for constant encouragement throughout his graduate study. Finally, for typing the rough draft, proofreading, and for con- tinued support, sacrifice, encouragement, and eternal patience, the author thanks his wife, Eleanor, who made it all worthwhile. I. II. TABLE OF CONTENTS INTRODUCTION . REVIEW OF LITERATURE . A. Relationship between Dietary Carbohydrates and Non- protein Nitrogen . Relationship of Dietary Nitrogen Sources to Ruminal Nitrogen Metabolism. The Central Role of Ammonia in Catabolic Rumen Reactions. . l. Amino Acid Catabolism . fifl'Ooaa—Wb-°3'O manna-m L.- -°3'ID"MO0.0U'@ Glycine . Alanine . Valine Leucine . Isoleucine. Serine . . . Threonine . Phenylalanine . Tyrosine. . . Tryptophan. Cystine-cysteine. Methionine. Proline and Hydroxyproline. Aspartic Acid . Glutamic Acid . Histidine . Arginine. Lysine. . Amide Catabolism. Urea. Biuret. Urea-formaldehyde Complex . Ammoniated By-products. Ammonium Salts. Guanidine . . Creatine and Creatinine . Dicyandiamide . . . Amides of Organic Acids . Urea Derivatives. III. IV. VI. VII. 0. E. F. TABLE OF CONTENTS (Continued) Ammonia Absorption from the Rumen . The Adaptation Response . In Vitro Rumen Fermentation Systems . EXPERIMENTAL PROCEDURES . A. F0 General . Design of the Ig_!l££2 System . Operation of the In Vitro System Experimental Design and Sampling Procedures . Analytical Methods. Ammonia . . . Nitrogen by Semimicro Kjeldahl Analysis . #WN— Volatile Fatty Acids by Gas Chromatography. .Statistical Treatment . RESULTS AND DISCUSSION. A. Precipitate Nitrogen. B. Fermentation Soluble Nitrogen Fractions . C. Dialyzate Nitrogen Fractions. D. Fermentation Liquor Volatile Fatty Acids. E. Dialyzate Volatile Fatty Acids. F. Performance of the Continuous lg 11559 System . SUMMARY . LITERATURE CITED. APPENDIX. Microkjeldahl Quantitated by Direct Nesslerization. Pa l20 l26 128 lQZ LIST OF TABLES Table l \n-F'w Protein synthesis in mg. N/lOO gm. rumen fluid stimulated by various energy sources. . . . . . . . . . . . . . .1. Nitrogen digestibility and biological values on urea and casein diets . . . . . . . Buffer composition . . . . . . . . . . . . . . . . . . . . Diet of fiStualted donor cow . . . . . . . . . . . . . . Composition of basal substrate for in vitro fermentation . Mean dialyzate total nitrogen, ammonia nitrogen, and non- ammonia nitrogen levels with various nitrogen sources. Mean weight in mg. of total nitrogen, ammonia nitrogen, and non-ammonia nitrogen transferred out of the in vitro system in the dialyzate per l2 hour period. Molar percentages of acetate, propionate, and butyrate in fermentation liquor. Mean dialyzate volatile fatty acids on various substrates. Acetate/propionate ratios for fermentation liquor and dialyzate. 16 73 75 77 l08 ll2 117 119 121 Figure 7a,b 8a,b 9a,b l0a,b lla,b LIST OF FIGURES Page Diagram of the fermentation and dialysis chambers of the 13 vitro system. . . . . . . . . . . . . . . 66 Photograph of the Transferator modified to operate two syringes. . . . . . . . . . . . . . . . . . . . 67 Photograph of the Transferator-activating microswitch mounted in the fraction collector head. . . . . . . 69 Diagram of the flow pattern of the in vitro system . . . 7] Photograph of the complete 12 vitro system in operation. 72 Structural formulas of substrates examined in the in vitro system. . . . . . . . . . . . . . . . . . . . 79 Changes in fermentation liquor precipitate nitrogen with time after substrate addition . . . . . . . . . . . 93-9h Changes in fermentation liquor soluble nitrogen with 97~98 time after substrate addition . . . . . . . . . Changes in fermentation liquor ammonia nitrogen with time after substrate addition . . . . . . . . . . . 99-100 Changes in fermentation liquor non-ammonia nitrogen with time after substrate addition. . . . . . . . . l0l-l02 Changes in fermentation liquor total principal volatile fatty acid with time after substrate addition . . . llSall6 iv Table la 2a 2b 3a 3b ha hb 5a 5b 6a 6b 7a 7b 8a 8b 9a 9b lOa lDb lla llb l2a l2b LIST OF APPENDIX TABLES Fermentation liquor precipitate nitrogen, mcg./m. Analysis of variance (Table la) . . Fermentation liquor soluble nitrogen, mcg./ml. Analysis of variance (Table 2a) . . Fermentation liquor ammonia nitrogen, mcg./ml. Analysis of variance (Table 3a) . . Fermentation liquor non-ammonia nitrOgen, mcg./ml. Analysis of variance (Table ha) . . Dialyzate soluble nitrogen, mcg./ml. Analysis of variance (Table 5a) . . Dialyzate ammonia nitrogen, mcg./ml. Analysis of variance (Table 6a) . . Dialyzate non-ammonia nitrogen, mcg./ml. Analysis of variance (Table 7a) . . . . . . . . Fermentation liquor acetate, micromoles/ml. Analysis of variance (Table 8a) . . Fermentation liquor propionate, micromoles/ml. Analysis of variance (Table 9a) . . . Fermentation liquor butyrate, micromoles/ml. Analysis of variance (Table IOa) Fermentation liquor total fatty acid, micromoles/ml. Analysis of variance (Table lla), , , Fermentation liquor acetate/propionate ratio. Analysis of variance (Table lZa). . . V Page 1A2 1113 Ihh 1115 1N6 160 1.61 l62 1(3 161* 165 IEELS l3a l3b lha lhb l5a 15b l6a l6b l7a l7b lBa l8b l9a l9b 20a 20b 21a Zlb 22a 22b 233 23b LIST OF APPENDIX TABLES (Continued) Fermentation liquor molar percent acetate . Analysis of variance (Table 13a). . . Fermentation liquor molar percent propionate. Analysis of variance (Table lha). . . Fermentation liquor molar percent butyrate. Analysis of variance (Table ISa). . Dialyzate acetate, micromoles/ml. Analysis of variance (Table l6a). . . Dialyzate molar percent propionate. Analysis of variance (Table l7a). Dialyzate molar percent butyrate. Analysis of variance (Table l8a). Dialyzate total volatile fatty acid, micromoles/ml. Analysis of variance (Table l9a). . . Dialyzate molar percent acetate . Analysis of variance (Table 20a). . . Dialyzate molar percent propionate. Analysis of variance (Table 21a). . . Dialyzate molar percent butyrate. Analysis of variance (Table 22a). . Dialyzate acetate/propionate ratio. Analysis of variance (Table 233). . . vi 170 171 172 173 17h 175 177 178 1.79 180 181 182 183 TEL 185 186 187 I. INTRODUCTION Throughout its evolution, the ruminant's digestive system has made use of at least a certain amount of non-protein nitrOgen (NPN). This nitrogen has come not only from nitrates and other sources found in plants, but it has entered the rumen as urea via the salivary secretion (Hirose gtflgl., l960; Houpt, l959; Decker _£._l., 1960; McDougall, I948; Sommers, l96la) and by direct diffusion from the blood (Houpt, 1959; Decker, l960).i Several authors have further noted that on low protein rations, urea is retained in the body and recirculated by saliva to a greater extent than when protein is sufficient (Hirose, I960; Sommers, l96lb; Schmidt-Nielsen._£._l., l957; Livingston‘_£‘_l., I962). Thus, urea conservation may have some survival value to the animal. Most of the early work on NPN utilization by ruminants was done in Germany. Paramount among the early papers, which were reviewed and referenced by Stangel (1963), is one by Zuntz (l89l) who pointed out the protein-sparing action cf asparagine and amides in ruminants and postulated that the mechanism might operate via the rumen micro- organisms. A number of sources of NPN have been examined for usefulness in supporting microbial protein synthesis in the rumen. Several of these have been studied by 13 vitro or ”artificial rumen” techniques (Belasco, l95h). In the study at hand, a new in x1352 procedure has been developed making possible the continuous culture of rumen microorganisms and the examination of the end-products of their metabolism at intervals following substrate addition. The purpose of this study was to examine various NPN sources in this continuous flow system with respect to their utilization by rumen microorganisms in vitro and to elucidate more completely the variables and possibly the metabolic pathways involved in NPN utilization. II. REVIEW OF LITERATURE Although the process of non-protein nitrogen (NPN) utilization has been studied extensively, there are Still a number of areas of uncertainty. The process itself cannot be isolated for study since it is intimately concerned with growth and multiplication of the microorganisms. Consequently, any factor which limits or enhances microbial growth will also limit or enhance non-protein nitrogen utilization. The process is further complicated by the metabolism of NPN in the animal's body tissue and by the translocation of it in the circulaa tory system. If urea is fed to an animal, the concentration of urea in the rumen at any given time is the net result of (I) feed urea entering the rumen, (2) urea entering the rumen via saliva, (3) urea diffusing from the blood into the rumen across the rumen mucosa, (h) urea being hydrolyzed by urease to ammonia, (5) urea diffusing across the rumen mucoSa into the blood stream, and finally (6) urea passing from the rUmen into the more distal parts of the gut. In an attempt to simplify the system for conversion of non- proteln nitrogen to bacterial protein, many workers have turned to in vitro fermentations, the ”artificial rumen.” The various types of'lg vitro systems have attempted to duplicate as closely as possible the conditions in the intact rumen. The various factors influencing NPN utilization in the rumen, the various pathways of ruminal nitrogen metabolism, and the construc» tion and operation of various lfl.¥l££2 fermentation systems will be treated separately in the discussion that follows. A. Relationship between Dietary Carbohydrates and Non-protein Nitrogen Zuntz (l89l) noted in his classic paper that excess soluble carbohydrate produced a decrease in cellulose digestion, supposedly because the microorganisms attacked the sugars in preference to cellulose. This insight into rumen metabolism is rather remarkable considering the state of development of the science of ruminant nutrition and the analytical tools with which he worked. Hart gtflgl. (l939) noted that the most efficient utilization of non-protein nitrogen occurred when some soluble sugar such as corn molasses was fed in the ration. Three years later, Johnson gtwgl. (l9h2) confirmed the work of both Hart 35,21. (1939) and Zuntz (l89l) when they showed that corn molasses decreased cellulose digestion ‘ but increased nitrogen utilization. They postulated, as had Zuntz (l89l), that the cellulose digestibility depression may have been due to diversion of the bacteria from less to more soluble carbo- hydrate substrates. Mills gt‘gl. (l9h2) observed an increased utilization of urea when starch was added to a basal diet of timothy hay and urea. They also observed an increase in rumen protein concentration from 7.7 percent on a urea plus timonthy hay ration to 9.3 percent on this same ration with added molasses. Starch addition caused additional urea utilization, as did l36 gm. of casein. The authors concluded that a ration composed of roughage, molasses and urea must be sup- plemented with a readily fermentable but insoluble carbohydrate source (such as starch) for maximum urea utilization. They observed that this supplementation need not exceed 3 to 5 percent of the ration. It should be pointed out that these rations were non-isonitrogenous and this fact could have biased the outcome of the experiment. The effect of excess soluble carbohydrate was investigated by Willett‘gtigl. (l9h6). They fed a ration based on molasses, pine- apple bran and concentrates and showed that although milk production in dairy cattle was lower with urea than with natural protein, molasses in amounts up to 25 percent of the concentrate portion of the ration had no detrimental effect upon protein synthesis from urea. Prior to the work of Arias _£ El. (l95l) the study of carbohydrate and energy requirements for NPN utilization was limited to only a few sources; namely cellulose, starch and molasses. Their work, however, concerned six different carbohydrate sources; dextrose, cane molasses, sucrose, starch, cellulose, and ground corn cobs. These energy sources were added to‘ig'vltrg fermentation flasks and observations were made on their influence on conversion of urea to microbial nitrogen. All compounds enhanced urea utilization, providing they underwent digestion. Small amounts of readily fermentable carbo- hydrate (0.25 percent of the fermentation mixture) aided cellulose digestion, and consequently, urea utilization, while large amounts (l percent of the fermentation mixture) inhibited cellulose digestion. Inhibition was greatest with sucrose, Starch and dextrose, and least with molasses and cellulose. Much of the work reported to this point concerned rather crude, or at least poorly defined carbohydrate sources. McNaught (l95l) carried out an extensive 12.xitrg fermentation study in which protozoaw free rumen fluid was incubated for h hours with 0.05 percent urea and l.0 percent of the carbohydrate source in question. Maltose was usually used as a reference and activity was expressed in terms of NPN converted to protein. In McNaught's study, purified carbohydrates produced the responses shown in Table I. From the results of this study, the author concluded that for conversion of urea to protein, a sugar was required with a primary alcohol group and a reducing group. One should observe the sizable variability in control values. In this experiment a rumen collection was split into aliquots and the same inoculum used for the treatments and their respective controls. This variability is often character- istic of the _i_rl £552 technique and is probably the reason for the conspicuous lack of statistical analysis in many such studies. Hunt _£_gl, (l954), using an in x1352 technique, showed that starch addition (9 gm. to a 500 ml. fermentation flask) increased (Pic .0l) syntheSis of riboflavin and utilization of urea. Starch depressed cellulose digestion (P < .Dl) and had no significant effect upon vitamin BIZ syntheSis. ‘ Several of the authors quoted (Johnson t l., l9h2; Burroughs _£,_l,, l950a; Hunt _£H_l., l95h) have pointed out the antagonistic relationship between the starch content of the diet and cellulose digestion. Belasco (I956) used an in vitro technique to study the effects of various carbohydrates and the relationship between starch TABLE 1. Protein Synthesis in mg. N/lOO gm. Rumen Fluid Stimulated ”by Various Energy750urces. Control Experimental Maltose 8.9 Raw potato starch 2.9 Boiled potato 15.5 Raw maize starch 5.5 Boiled maize starch 15.8 Maltose 9.5 Raffinose 7.h Inulin 9.2 Maltose 13.0 Fructose lh.0 Cellobiose l3.5 Maltose 5.7 Mannose 5.5 Sorbose -3.0 Maltose l7.6 L(+) Arabinose 2l.h No addition 32.0 D(-) Arabinose 3.1 D(+) Zylose 18.7 Maltose 7.0 Glucuronic acid -0.6 Glucose 7.l Gluconic acid -l.0 Glucosaccharic acid -0.2 Maltose 5.7 Mannitol 0.6 Sorbitol 0.6 Maltose 3.6 Alginic acid 0.9 Glucose 7.0 Glucosamine 0.7 Maltose 7.2 Sodium acetate 0.4 No addition -l.6 Sodium butyrate ~l.3 Maltose 9.6 Sodium propionate -l.7 No addition -3.7 Maltose 9.6 Sodium betahydroxybutyrate 3.5 No addition 1.6 Maltose 9.2 Lactic acid (0.5%) -0.5 Maltose lh.1 Sodium citrate -2.7 No addition -2.2 Sodium hydrogen malate -l.2 Sodium potassium tartarate -l.3 7 Fumaric acid -0.5 Haltose (0.3%) 2.3 Succinic acid (0.3%) -2.li and cellulose on urea utilization. Each flask contained 900 ml. of solution. In this size fermentation flask, a combination of 9 gm. starch and 9 gm. cellulose produced better urea utilization than 18 gm. of either carbohydrate. In general, starch provided better urea utilization than cellulose at comparable levels. In the same system, 2 gm. starch increased urea utilization 33 percent. The same amount of xylan and pectin increased urea utilization 16 and IB percent respectively. When total carbohydrate was maintained at 9 gm. (1 percent of the fermentation medium), the best combination of cellulose digestion and urea utilization was reached with a cellulose:starch ratio of l:h. Starch and dextrose were compared in the final part of this study. At low levels both carbohydrates performed in a comparable manner. However, at high levels (.66 to 1 percent of the fermentation mixture) dextrose depressed both urea utilization and cellulose digestion.l Belasco (1956) used corn starch in all his comparisons. However, Bloomfield gtigl. (1958) reported a considerable difference in percent urea utilization under the influence of starches from different sources. In their 13 £1552 study, corn starch supported 98 percent utilization of urea while wheat starch, potato starch, and soluble starch supported 81, 63, and 55 percent urea utilization respectively. Similar results had been reported for potato starch by McNaught (1951), who showed that this carbohydrate supported increased urea utilization after it was cooked. Bloomfield 5£_21, (1958) examined several energy sources including valeric, succinic, propionic, lactic and pyruvlc acids, and alanine. Since only the latter three stimulated urea utilization, the authors speculated on the necessity for a functional group on the alpha carbon. Lactic acid did not support urea utilization in the study of McNaught (1951). From her work she postulated the need for a primary alcohol and a reducing group. Starch was more active in stimulating urea utilization than simple sugars but fructose was the most active of the sugars. Lactose was consistently inferior. The need of soluble carbohydrate for efficient urea utilization has been referred to previously. The depressing effect of large amounts of starch on cellulose digestion has also been documented (Johnson gt 31., 19102; Burroughs 2331., 1950a; Hunt e_t__a_l_., 1951+). Ina further investigation of this problem, El-Shazly‘gtngl. (I961) showed that this depressing effect may not be due to starch,-but to a low level of available nitrogen In the ration, producing competition between starch-fermenting and cellulose-fermenting organisms. The depressing effect of starch on cellulose utilization was decreased with urea levels up to 177 m9. urea nitrogen per 100 m1. of fermentation medium. At this point, urea itself became inhibitory. In no case, however, was restoration of cellulose digestion complete, and high starch levels still permitted less cellulose digestion than low starch levels. By careful experimental design, it was shown that neither buildup of end products (volatile fatty acids and lactic acid) or decreased pH were the factors depressing cellulose digestion. lO By use of a nylon bag technique, the ameliorating effect of ureawas shown to operate lg 2119 as long as the hay:corn ratio in the diet was greater than l:l. . From the previously reviewed papers one might conclude that the necessity of a readily fermentable carbohydrate source is a simple requirement of the bacteria for energy. Elliott and Topps (196“) noted that nitrogen balance could be maintained only when the level. of total digestible nutrients was adequate for maintenance. This was true even when total crude protein was about 10 percent. On an all roughage ration, both digestible nitrogen and total digestible nutrient intake required for nitrogen equilibrium were much higher than when some concentrate was added to the ration. Chalupa'g£.gl. (196“) added a small amount of ethanol to'lg 31552 rumen fermentations and obServed an increased rate of cellulose digestion. No advantage was observed, however, when ethanol was fed to fistulated steers on an otherwise normal diet. AdditiOn of ethanol to a semipurified diet (25 ml./kg. of diet) resulted in small but nonslgniflcant increases in nitrogen utilizati6n. These authors theorized that ethanol acted simply as a source of energy in the rumen. Combe and Tribe (1962) obtained similar results with ethanol. Although the requirement for energy alone in NPN utilization is an attractive theory, the addition of a soluble and readily available form of energy may not increase nitrogen utilization. Faichney (1965) added sucrose to the diet of sheep maintained on "straw and urea: He showed no significant change in rate of cellulose ll digestion, nitrogen balance, weight gain, or rumen ammonia.” Sucrose wes given at the rate of O, 45, and 90 gm. per animal per day. Dry matter intake was about 700 gm. per animal per day. The results of Faichney (1965) are essentially in agreement with Campling Ethel. (1962) and Hemsley and Moir (1963). The latter authors concluded that there is no support for the widely held view that the addition of a readily available energy source pgr‘gg is necessary for the enhanced utilization of urea-supplemented diets. McNaught and Smith (1947), in a review of the early literature comparing starch and molasses, postulated that the disadvantage of molasses and other soluble forms may be due to either its rapid "wash-out" or absorption from the rumen. A Because of the abundance of carbohydrates in the diet, it is obvioUs that they must furnish considerable energy to the rumen fermentational syStem. Nevertheless, McNaught (1951) and Bloomfield (I958) speculated on the necessity of specific Structural configura- tions for the support of non-protein nitrogen utilization. Probably the truth lies somewhere in the nebulous middle ground. That is, readily available energy is probably necessary, but in controlled amounts. Possibly, soluble forms such as sucrose tend to flood the system. Further experimentation in this problem might include the con- tinuoUs instion of soluble energy forms, thus removing the effects of large amounts of energy becoming suddenly available. If the requirement is for energy Eg£.§g, then production of bacterial protein should proceed in the artificial rumen under the influence of 12 addition of adenoslne triphosphate, or with other high energy phosphate compounds. Combe and Tribe (I962) observed no effect from adding phosphoric acid to a low quality roughage diet supplemented with urea and molas- ses or starch. Finally, It should be remembered that microbial protein synthesis is a function of microbial growth. Consequently, the energy require- ment from carbohydrates is certainly not destined exclusively for driving the pathways of amino acid and protein synthesis, but for all the other energy-requiring mechanisms of the microorganism as well. B. Relationship of Dietary Nitrogen Sources to Ruminal Nitrogen Metabolism Protein metabolism in the rumen is a dynamic phenomenon. New amino acids can be synthesized from non-protein nitrogen (NPN); natural proteins can undergo proteolysis in the rumen; and amino acids from all sources can be deaminated. The ammonia so derived can be used in synthesis of new amino acids, incorporated into microbial protein, or absorbed through the rumen wall. In addition, a certain amount of protein can pass through the rumen unchanged. These factors all complicate the study of rumen nitrogen metabolism since the rumen level of any nitrogen containing compound at any given time is the algebraic sum of these processes. Finally, the integrity of the rumen microorganisms, and consequently their syn- thetic capabilities, depend on their energy supply. l3 Urea and several other NPN sources can be utilized by rumen microorganisms. Factors which determine whether or not a particular chemical entity can be utilized as a nutrient have been discussed by Oginsky and Umbreit (1959), who set forth the following criteria: (I) it must be capable of either crossing the cell membrane by dif- fusion or transport, or be decomposed by extracellular enzymes into fragments that can penetrate the cell, and (2) the cell must contain enzymes that can incorporate the compound without alteration into protoplasm, or that can chemically transform it into other molecules suitable for incorporation into protoplasm, or that can liberate energy by metabolizing the compound. These same authors (Oginisky and Umbreit, 1959) point out that ammonia is the form of nitrogen used by bacteria fof amino acid production. The only pathways available for conversion of ammonia to alpha-amino groups are, according to these authors, amlnation of alpha keto glutaric acid to glutamic acid, and amination of fumarlc acid to aspartic acid. The enzymes involved here are glutamic dehydrogenase and aspartase, respectively. Both are operative in anaerobic conditions and, consequently, should be available in rumen microorganisms. Mortenson (1962), on the other hand, notes that although aspartase . and glutamic dehydrogenase have long been considered the only pathways of ammonia incorporation, the mechanism of action of aspartase is unknown, and glutamic dehydrogenase is absent from enough micro- organisms to ferce a reevaluation of its importance to bacterial metabolism. In addition, Mortenson (1962) discusses an enzyme, 1h alanine dehydrogenase, which catalyzes the formation of alanine from pyruvate via a series of reactions involving iminopropionate. The final mechanism is not yet clear. Pathways are presented concerning amide formation. These further pathways indicate that many more mechanisms for NPN incorporation into amino acids are available than was formerly believed. However, ammonia still appears to‘be the intermediate in non-protein nitrogen metabolism. Following incorporation of ammonia into alanine, aspartic acid and glutamic acids, and several of the amides, various interconversions occur leading to formation of the other amino acids necessary for synthesis of complete proteins. These individual pathways are dis- cussed in considerable detail in a review by Umbarger and Davis (1962). Paramount among the interconversions mentioned by these authors are transaminations involving alpha-keto acids. The variety of species of rumen microorganisms and their symbiotic relation, not only among themselves, but also with the host animal, make it seem logical that while the pathways for metabolism of a given nutrient may be limited in a single microbial species, necessary intermediates may be available, and metabolic. blocks bypassed, through the synthetic capabilities of other species. Very early researchers assumed that if NPN supported growth A on an otherwise inadequate diet, then amino acids must necessarily be synthesized to support this growth (Bartlett and Cotton, 1938; Hart._£‘_l.,’l939). Bartlett and Cotton (1938) reviewed early German work and observed that natural proteins in theSe rations may have been high enough to meet the animal's requirements. Their own experiments 15 ran for 1&2 days using a basal ration of meadow hay, oat straw, H mangles, tapioca flour and flaked maize. The authors assumed that ' any growth response on this low protein diet was from urea nitrogen. Later, digestibility and nitrogen balance studies were carried out, and biological values computed. Classic among these studies were those by Harris and Mitchell (l9hla, b). They determined biolOgical value, digestibility and nitrogen balance for sheep under conditions of both growth and maintenance. Their results include metabolic and endogenous nitrogen determinations on nitrogen free diets. The semi- purified diets for maintenance were composed of wheat straw, wood pulp, sugar, corn oil, citric acid, and a salt mixture. They also examined the effect of a natural protein (casein) on the same parameters. Their results are shown in Table 2., Silage was the principal ingredient of the basal diet during the growth phase. It supported no growth and would not consistently maintain nitrogen balance. Biological value of nitrogen in the basal ration was 82 while the addition of urea to produce a calculated protein level of 8, 11, and 15 percent supported biological values of 7h, 60, and NA, respectively. This phenomenon of decreased biological value of nitrogen should be expected when the protein requirement of the animal is exceeded, as was probably the case at least in the 15 percent protein ration. Harris g£.gl. (l9h3) carried out a similar experiment with steers comparing soybean meal with urea. When corrected for metabolic nitrogen, digestion coefficients of both nitrogen sources were 9h. Biological values were 3“ for urea and 60 for soybean meal. 16 TABLE 2. Nitrogen Digestibility and Biological Values on Urea and Casein Diets. True nitrogen Biological value digestibility,1 at nitr0gen guilibrium Basal + Urea 88.8 62 Basal + Casein 86.9 69 17 Apparently, both were fed at levels higher than the requirement. At slaughter, the rumen contents of animals fed on urea rations had more true protein than that from unsupplemented cattle, giving added evidence for protein synthesis from NPN. Krebs (1937), in a review, was reluctant to believe any useful amount of protein synthesis occurred in the rumen, or if it did occur, that the microbial protein so produced was of any appreciable value to the host. Biological value studies on NPN sources have been numerous. W However, their reSUIts have been, at best, variable. Johnson _£h_l. (I9h2), using lambs, fed crude protein levels of approximately 12 percent and obtained biological values of near 60, irrespective of whether the protein source was soybean meal, casein, or urea. From these results, he advanced the theory that most of the nitrogen in the ration, whatever its source, is transformed to microbial protein whose biological value is near 60. Later, Johnson._t._1. (l9hh) added additional evidence to this theory when, using rat feeding experiments, they observed bioIOQIcal values for ruminal bacteria and protozoal fractions of 66 and 68 respectively. Loosli and Harris (l9h5) supplemented a 6.55 percent crude protein ration with urea alone, urea plus sulfate and/or methionine, and linseed meal. Results were expressed as percent of absorbed nitrogen retained. This measure apparently does not take into account urinary endogenous and metabolic fecal nitrogen losses. They noted that urea plus methionine increased percent of absorbed nitrogen retained more than did urea plus sulfate. Urea plus methionine gave 18 about the same value as did linseed meal, and urea alone gave values slightly lower than the basal. These data indicate at least a partial requirement for methionine and cast some doubt on the theory of Johnson £3 21. (l9h2). Somewhat the same results were reported by Lofgreen gt 21. (19h7). Nitrogen retention was improved (P .05) by adding 0.2 percent methionine to a 10 percent crude protein ration in which #0 percent of the total nitrogen was from urea. The same lambs utilized egg protein better than linseed meal protein (P .05) or protein synthesized by rumen microorganisms from urea (P .01). In this experiment lambs indeed appeared to have some amino acid rec quirement not met by the microorganism's synthetic pathways. C. The Central Role of Ammonia in Catabolic Rumen Reactions Although many of the early German papers published from 1879 to 1911 discuss the use of non-protein nitrogen (NPN) in ruminants, little is said about the central role of ammonia in NPN metabolism (Stangel, 1963). During this period such compounds as asparagine, various amides, and numerous ammonium salts were fed. Armsby (1911), in a review of the nutritive value of NPN, made several observations on the subject and, for the most part, discounted the contribution of NPN to the over-all economy of the animal. Apparently, the first reference to ruminal ammonia levels was by Linkeit and Becker (1938). They noted that when urea was given to ruminants, its decomposition was completed in 30 minutes and at the same time, ammonia concentration in the rumen was markedly increased. 19 Until 19h0, most of the proof for NPN utilization had rested on feeding experiments. In these studies, growth was promoted and/or nitrogen balance was maintained by adding NPN to rations which, when fed alone, would not support growth or nitrogen balance. Then, in an attempt to further clarify the question of whether or not rumen micro- organisms actually did produce protein from NPN, Wegner._£‘gl. (19A0) adopted a revolutionary new approach when they incubated rumen contents with feeds to which urea was added. Although this early fermentation work suffered from many difficulties, one of which was pH control,»” the authors observed that shortly after starting the fermentation, the urea was converted to ammonia. Furthermore, as the fermentation cons tinued, the level of ammonia in the fluid fell. They domonstrated quite conclusively that this decrease in ammonia could be accounted for as an increase in protein. Although there could be many critia cisms of the technique, especially in light of later, more sophisticated 12.xitrg methods, these authors showed, for the first time, a con- nection between ruminal ammonia levels and ruminal protein metabolism. The following year, these same workers (Wagner _£'_l., l9hl) confirmed their ill 11.32 observation by use of a fistulated heifer in which both ammonia and urea were largely absent from the rumen by h to 6 hours after feeding. Evidence was given for protein production in the rumen. In I9h3, the relationship was more firmly established in a group of three classic papers (Pearson and Smith, I9h3a, b, c). In the second paper of the series Pearson and Smith (l953b) poStulated that the first step in the utilization of urea was its conversion to ammonia. 20 Hydrolysis rates were calculated, and the authors observed that the urease of the rumen was similar in chemical properties and specificity to that of jackbean meal. McDonald (1952) and El-Shazly (I952a, b) were among the first to show that ruminal ammonia arose not only from NPN metabolism, but from natural proteins as well. McDonald (1952) showed that ammonia was the main soluble form of nitrogen in the rumen and found that rumen micro- organisms rapidly deaminated soluble casein and gelatin with a conse- quent increase in ammonia. Zein was not thus affected. Warner (1956) showed that ammonia liberation in 12 xltrg fermentations was due to . amino acid deamination, and not to protein hydrolysis 335,35, Toluene-treated cultures hydrolyzed protein only to the amino acid stage, and ammonia was not released. Protein hydrolysis to amino acids has been treated extensively”in reviews by McLaren (l96h) and Blackburn (1965). ' El-Shazly (l952b) found that during deamination of amino acids by rumen microorganisms in vitro, the amounts of ammonia, carbon dioxide, and fatty acids produced were roughly equivalent to the amount of amino acid metabolized. Possible metabolic pathways were discussed. The previoUs references point out the importance and central role of ammonia in ruminal protein metabolism. That is, ammonia may arise from catabollsm of protein and subsequent deamination of amino acids. This ammonia may then be either absorbed into the circulatory system or incorporated by bacteria in g£_ggxg synthesis of amino acids. Ammonia metabolism in reference to individual amino acids will be treated in a subsequent section. 21 1. Amino Acid Catabolism Since this review concerns 12.21352 fermentation using mixed rumen populations, literature will be limited, in so far as is practical, to these cases. The discussion will include end products of rumen microbial metabolism, and will, where possible, indicate the specific mechanisms concerned. An extensive and complete review on amino acid catabolism has been prepared by Lewis (1961) and covers work from 1907 up to 1961. It deals with anaerobic and facultative anaerobic species as well as mixed rumen populations. a. Glycine This amino acid is deaminated only very slowly by rumen micro- organisms. Lewis and Emery (l962b) found only 10 percent deamination of glycine in 10 hours by a cheesecloth strained mixture of rumen inoculum. Under the same conditions, washed cell suspensions de- aminated only 2 percent of the original amino acid. Earlier ig_xi££g work by Sirotnak'gt‘gl. (1953) had shown no deamination by rumen bacteria. VanDenHende.g£'gl. (l963b, c) incubated glycine with sheep rumen microorganisms and obtained only limited deamination. End products were ammonia and acetic acid. An in vivo study by Looper ._£.gl. (1959) showed no deamination of glycine even when the amino acid was added in an amount to supply more than half of the total dietary nitrogen. Lewis (1955) added glycine to the rumen of a sheep at the rate of 100 mg. of amino nitrogen per kg. of body weight and observed that ammonia levels from the amino acid were only slightly higher than when hay alone was fed. 22 Metabolic studies involving the intact animal are, at best, difficult to interpret since there is always some ammonia present in the rumen and the origin of this ammonia is in doubt. However, in both i vivo studies noted here, the level of the amino acid was high enough that an increase in ruminal ammonia should have occurred if the amino acid were, in fact, deaminated. Ifl,vitrg fermentations, especially those involving washed cell suspensions, may suffer from shortages of cofactors or ”raw materials” necessary for normal metabolic pathway operation. However, it would appear from the work of Lewis and Emery (1962) and VanDenHende _£_gl. (1963) that glycine is only slowly deaminated in the rumen. The specific reactions of glycine degradation are in doubt. However, its conversion to serine can be eliminated. The glycine- serine interconversion which functions in many microorganisms (Oginsky and Umbreit, 1959) clearly does not occur since serine is deaminated rapidly (Lewis and Emery, 1962; Sirotnak _£ 21., 1953) and would thus pull the reaction to completion. The Strickland fe- action, proposed by El-Shazly (l952b), apparently does not operate since in the previously mentioned i vivo studies and the experiment by Lewis and Emery (1962) other amino acids of the ”Strickland pairs" are present. Oglnsky and Umbreit (1959) note that the Strickland reaction apparently operates via a rather poorly defined enzyme system unique to the proteolytic clostridia. Lewis and Emery (I963) incubated not only ”Strickland pairs” but also three or four different amino acids at the same time, without increased deamination except in the case of proline and alanine. This furnishes fUrther evidence againSt glycine deamination via the Strickland reaction. 23 b. Alanine VanDenHende (l963b, c) and Lewis and Emery (1962) found alanine to be deaminated only slightly more rapidly in 12.Xl££2 systems than was glycine, while Sirotnak _£ 21, (1953) observed neither deamination nor decarboxylation of the amino acid. Hueter et 1. (1958) studied a combination of glycine and DL-alanine both < ivo and in vitro and found that although there was a large ammonia production, no volatile fatty acids, and only a small amount of lactic acid were produced. This is difficult to understand since neither glycine nor alanine are deaminated singly in lfl.ll££2 systems. They do, however, constitute a ”Strickland pair,” with alanine being the hydrogen donor and glycine the acceptor. If a Strickland reaction had occurred, glycine should have been reductively deaminated to acetic acid. Alanine fed alone gave rise to ammonia in a steer (Looper t 1., 1959) and sheep (Lewis, 1955). It should be noted, however, that Looper _£'gl. (1959) observed peak ammonia concentrations from alanine at 8 hours after feeding. More readily deaminated amino acids produced peaks in rumen ammonia much sconer. Lewis (1955) found peak ammonia production with all amino acids at about 3 heurs after feeding. The significance of this difference is not clear. Unfortunately, in neither case was the identity of the resulting metabolite determined. T. R. Lewis (I961), in reviewing bacterial decomposition of alanine, concluded that it undergoes, for the most part, reductive deamination to propionate. If alanine acts as a hydrogen donor in a Strickland reaction, however, the corresponding alpha-keto acid 2h should be the end product. Since Hueter 33,21. (I958) detected no volatile fatty acid production, the reductive deamination pathway can probably be ruled out. c. Valine Early studies by Sirotnak gt'gl. (1953) indicated that valine was neither decarboxylated nor deaminated by rumen microorganisms in 31:52. Lewis and Emery (1962), however, showed that strained rumen fluid deaminated 28 percent of the amino acid in 10 hours while washed rumen bacteria deaminated 11 percent in the same time. A number of inc vestigators (Dehority g£_gl,, I958; Menahan and Schultz, 1963, l96h; VanDenHende _£‘_l., l963b) have shown that the end product of valine metabolism in the rumen is isobutyric acid. Greenberg (1961) showed the pathway from valine to isobutyric acid involved firSt, oxidative deamination to alpha-keto isovaleric acid and decarboxylation to isobutyric acid. The reason for accumulation of isobutyric acid in rumen contents following valine administration is not clear. Supm posedly, further degradation of isobutyric acid is possible. t al. (1958) and El-Shazly (l952a) discussed the Dehority possibility of valine metabolism by way of the Strickland reaction, with valine becoming oxidized. This explanation would fit into the step in which valine is oxidatively deaminated to alpha-ketoisovalerate. Later studies by Lewis and Emery (1962) showed that although some deamination of valine occurred when incubated alone with washed rumen bacteria, no increase in its breakdown was observed when it was incubated with a hydrogen-accepting member of a Strickland pair. 25 d. Leucine This amino acid has a deamination rate very similar to valine (see previous section), with 2h percent of a dose of the amino acid being deaminated by strained rumen fluid in 10 hours, and 11 percent being deaminated in the same time by a washed cell suspension (Lewis and Emery, 1962). Although El-Shazly (l952b) had earlier postulated that leucine acted as a hydrogen donor in Strickland reactions, Sirotnak._t.gl. (1953) found little evidence for its deamination. VanDenHende‘gt‘gl. (l963b) concurred with the finding of Lewis and Emery (1962) that the amino acid was rather slowly deaminated and that it was probably not metabolized via the Strickland reaction. The metabolism of leucine appears to proceed in a manner very much like that of valine. Isovalerate has been reported as the principal and produce of leucine metabolism by VanDenHende 33 31. (l963b), and Menahan and Schultz (l963, l96h). Dehority (1958) had postulated that the growth response of rumen bacteria to leucine was due to its metabolism to the corresponding branched chain fatty acid. Of special significance in leucine metabolism by rumen micro~ t al. (1955) who studied the organisms was the research by Otagaki metabolism of leucine labeled with carbon-1h in the 3 position. They found that 50 percent of the isotope was recovered in the volatile fatty acid fraction, 10 percent in cellular material, and almost none in the carbon dioxide fraction. From these data, they proposed a pathway in which leucine underwent first, oxidative deamination to the keto acid, and second, decarboxylation to isovalerate labeled in the 2 position. They further proposed the possibility of beta-oxidation of isovalerate to a three-carbon fragment and acetate labeled in the 2 position. 26 Unfortunately, modern methods for separation of volatile fatty acids were not available and these authors could not narrow the activity in the yoke- tile fatty acid fraction down to a specific acid. Nevertheless, this study agrees with the isovalerate production from leucine noted by earlier authors. e. Isoleucine El-Shazly (1952b) proposed metabolism of isoleucine via the Strickland reaction, with the amino acid acting as a hydrOgen donor. Sirotnak (1953) could show no evidence of isoleucine deamination by rumen microorganisms £3 31352, Dehority (1958) postulated that the growth response to isoleucine by rumen microorganisms was due to its metabolism to a branched chain fatty acid. Lewis and Emery (1962) and VanDenHende _£Hgl. (l963b, c) observed slow deamination of isoleucine that was not increased by incubation with a Strickland hydrogen donor, thus casting doubt upon the Strickland react on as'a metabolic route. VanDenHende (l963c) showed that isoleucine was oxidatively deaminated to the corresponding keto acid and then decarm boxylated, as was the case with alanine, valine, and leucine. VanDenHende (l963b) noted that the product of the decarboxylation step was methyim ethylacetic acid. The metabolism of isoleucine has been reviewed by Greenberg (196?). He concurred with VanDenHende (l963c) in both the oxidative deamination and decarboxylation steps, but concluded that the branched chain den rivative of coenzyme A is formed. These mechanisms were largely deduced through work with mammalian tissue slices, but appear compatible with reactions in the rumen since they can apparently proceed without the aid of the cytochrome system. 27 The lack of information on ruminal metabolism of this amino acid suggeets a need for intensive study using modern techniques for radios active carbon tracing and sophisticated fatty acid separation. f. Serine Serine is rapidly deaminated by rumen microorganisms (Sirotnak I_£._l., 1953; VanDenHende, l963c). Lewis and Emery (1962) showed complete deamination of this amino acid in 10 hours by both strained rumen fluid and washed cell suspensions. Walker (1958) studied an enzyme from a sheep rumen isolate, LC 1, which appeared to oxidatively deaminate both serine and threonine to the corresponding alpha-keto acid. Optimum pH for the enZyme was 9.5. but the system still operated at 87 percent of optimum velocity at pH 7.5. Pyridoxal phosphate and glutathione were necessary cofactors fer the purified enzyme. Serine was metabolized to ammonia, carbon dioxide, and acetic acid in an experiment by VanDenHende (l963a). When the washed cells were aged at 3° C for 48 hours in vaccuo, serine yielded pyruvic acid. Optimum pH in this system was 9. Although it would appear quite dangerous to propose a pathway for rumen metabolism of any particular compound on the basis of a pathway or enzyme found in one organism, the similarity in the results of VanDenHende (l963a) and Walker (1958) give good evidence that a specific deaminase for serine and threonine does, in reality, operate in the rumen. The apparent disagreement between the end products from these two amino acids can be resolved by noting that the acetic acid observed by VanDenHende (1963a) could arise as a 28 decarboxylation product of pyruvate, an alpha-keto acid corresponding to oxidative deamination of serine (Walker, 1958). Although other pathways cannot be ruled out, the deamination rate of serine, when compared with other amino acids (Lewis and Emery, 1962), make it appear that rumen microbiota are well equipped to handle this amino acid. 9. Threonine The metabolism of this amino acid appears to be very similar to serine. However, deamination proceeds at a slower rate (Lewis and Emery, I962; VanDenHende, l963c). Walker (1958) found that the Dmisomer inhibited the activity of the purified deaminase discussed under serine metabolism. End products found by VanDenHende (I963a) were ammonia, carbon dioxide, acetic acid and propionic acid.‘ Walker (1958) showed that alpha-keto butyric acid was an intermediate. This acid could presumably undergo decarboxylation to the propionic acid found by VanDenHende (I963a) but the origin of the acetate is not clear. One further interesting aspect of threonine metabolism is that, although threonine is apparently deaminated in the rumen more slowly than serine, the deaminase isolated by Walker (1958) acts more rapidly on threonine. This phenomenon might indicate a second path» way of serine metabolism. h. Phenylalanine The subject of phenylalanine metabolism by rumen microorganisms has been treated only briefly. Sirotnak._£‘gl. (1953) found no dea amination or decarboxylation of the amino acid, while Lewis (196!) showed that it was deaminated rather rapidly by strained rumen fluid. 29 However, deamination was much lower with washed cell suspensions, indicating the possibility that some sort of accessory factors are needed for phenylalanine metabolism. VanDenHende (I964) showed that phenylalanine was metabolized to phenylacetic acid, with phenylpyruvic acid arising as an intermediate. This scheme is interesting because of its similarity to systems already discussed. For example, phenylpyruvic is the keto acid arising from the oxidative deamination of phenylalanine. Phenylacetic acid, then, arises from the oxidative decarboxylation of phenylpyruvate. The conversion of phenylalanine to tyrosine apparently has not been examined. The production of tyrosine must occur in the ruminant because of the ability of the animal to thrive on a diet containing no tyrosine. i. Tyrosine There is an almost complete lack of information concerning tyrosine metabolism by rumen microorganisms. Apparently, the only work in which tyrosine was studied is in the paper by Sirotnak (l953) which showed, at least under the conditions of his study, that it was not metabolized. Greenberg (1961) reviewed the catabolism of tyrosine and showed that certain homogenates under anaerobic conditions carry out a transamination involving alpha-keto glutaric acid, with the production of para-hydroxyphenylpyruvic acid-ma situation very close to that for phenylalanine. Since so many amino acids in the rumen undergo oxidative deamination to the corresponding keto acid, it appears that unless the enzyme system is extremely specific, tyrosine could give rise to a number of other amino acids. 30 The further decomposition of the products of phenylalanine and tyrosine metabolism merit additional investigation. Phenylacetic acid metabolism is not discussed. Once tyrosine is metabolized to para-hydroxyphenylpyruvate, there seems little reason that it shouid not continue to parauhydroxyphenylacetate. The further breakdown of these compounds to homogentisic acid has been shown in mammals but apparently not in rumen microorganisms. j. Tryptophan Tryptophan metabolism by rumen microorganisms has received relatively little study. Although Sirotnak (l953) found no deamination of the amino acid, Lewis and Emery (l962a) showed 35 percent of a test dose was decomposed within l0 hours by strained rumen fluid. Only 15 percent of a similar dose was metabolized by washed cell suspensions. Indole and skatole were shown as intermediates in tryptophan metabolism by Lewis and Emery (l962b). These two compounds may, according to the review by Greenberg (l96l), arise by way of (l) deamination of tryptophan to its alpha-keto acid, (é) oxidative decarboxylation to indclacetic acid, (3) decarboxylation of indolacetic acid to skatole, and finaiiy, (h) oxidative decarboxylation of skatole to indole. Further metaboiism of these compounds has apparently not been studied. k. Cystine-cysteine Sirotnak (I953) reported decarboxylation and deamination of botn of these amino acids at similar rates. Lewis and Emery (l962a), on the other hand, noted that anystine was decomposed at a rate only half that of cysteine. It is interesting that in the work of Lewis and Emery (l962a) the decomposition rates for these amino acids in 3] washed cell suspensions were very near those for strained rumen liquor. No rumen end products of cystine or cysteine metabolism have been reported. Greenberg (l96l) discussed the formation of pyruvate from cysteine or cystine. ‘In this pathway, once cystine is split to two cysteine molecules, the sulfur is removed and appears as hydrogen sulfide. Then oxidative deamination occurs, yielding ammonia and pyruvic acid. This pathway, however, does not agree with the observation of Sirotnak g£_2l, (l953). In their study, nearly a mole for mole relationship occurred between ammonia and carbon dioxide. The possibility exists, however, that the pyruvate had been decarboxylated to acetate in some other metabolic step. I. Methionine Lewis and Emery (1962) found that 32 percent of this amino acid was decomposed by strained rumen fluid in lo hours, while washed cell suspensions metabolized the amino acid at a rate only about half this. No information on the end products of methionine metabolism in the rumen was found. Lewis (l96l) reviewed work showing that Pseudomonas produced ammonia, alpha-ketobutyrate,‘and methylmercaptan. 0n the basis of the metabolism of other amino acids, a pathway involving cleavage of the methylmercaptan, followed by oxidative deamination, seems quite likely. m. Proline and Hydroxyproline Metabolism of these two amino acids by strained rumen fluid was found by Lewis and Emery (I962) to be very slow. Metabolism by washed cell suspensions was almost nil. Much the same results were found for proline alone by VanDenHende (l963c). El-Shazly (l952b) found proline 32 fermentation yielded delta-amino valeric acid, and postulated a mechanism involving the Strickland reaction. Dehority _£‘_l. (I958) also found evidence for the Strickland reaction but noted that an end product was valeric acid, indicating that deamination had taken place following ring cleavage. VanDenHende, on the other hand, found no evidence for a Strickland reaction involving proline. Delta-amino valeric acid metabolism was studied by Lewis and Emery (I962). Although only 2] percent disappeared in ID hours, there was no difm ference in the action of strained rumen fluid and washed cell suspensm ions. This observation might be useful in evaluating the differences in strained fluid and washed cells, since apparently all necessary cofactors were present in the washed cell fraction. It appears from these studies that the first step in proline and hydroxyproline catabolism is ring cleavage yielding delta-amino valeric acid (or its gamma-hydroxy analog). This is followed by deamination. Since the work of Lewis and Emery (I962) shows more rapid metabolism of deltao amino valerate than proline, it would appear that the rate limiting reaction is concerned with ring cleavage. n. Aspartic Acid The end products of ruminal aspartate metabolism have been fairly well elucidated. Aspartic acid is metabolized quite rapidly (Lewis and Emery, I962). Looper g£_gl, (I959) administered aspartic acid I vivo and measured peak rumen ammonia levels after eight hours. Hueter t I. (I958) observed that DL aspartate was fermented to volatile fatty acids. Sirotnak‘gt‘gl. (I953) noted that aspartate A 33 metabolism yielded almost equal molar amounts of carbon dioxide and ammonia. In I95“, Sirotnak t al. postulated a pathway involving dew amination of aspartate to succinate and decarboxylation of succinate to propionate, which underwent further oxidative decarboxylation to acetate. This pathway was based upon studies using a number of possible inter- mediate compounds. VanDenHende._t._l. (I959) confirmed this work, noting that one mole of aspartate was metabolized to one mole of carbon dioxide, one mole of ammonia, and one mole of propionic acid. Optimum pH was 7.5. VanDenHende _£Hgl. (I959) discussed a number of other pathways and alternate intermediates. Thus, according to the available literature, aspartate is fermented to propionate (or to acetate) with succinate as an intermediate, as was first proposed by Sirotnak (I953). o. Glutamic Acid Several workers (Lewis and Emery, I962; Lewis, l963c; VanDenHende t al., I963c) have reported fairly rapid deamination of glutamicacid by rumen microorganisms. Looper t 21° (I959) noted that following I i vivo administration of the amino acid, the peak ammonia concentration 3 in the rumen occurred at 8 hours after dosing. Otagaki (I955) incubated glutamic acid labeled in the C-l position and found that-the O'“ that was not incorporated into protein was found in both the carbon dioxide and volatile fatty acid fractions. Unfortunately, this study was rather inconclusive since only about ho percent of the total radio~ activity was recovered. The authors speculated on the operation of a 'speclfic decarboxylase and noted the possibility that the carbon dioxide originated after deamination. They did not attempt to explain the appearance of the activity in the volatile fatty acid fraction, since 3% carbon one should be removed prior to any alteration of the original carbon chain. Studies with labeled carbonate indicated that the activity probably was not due to incorporation of carbon dioxide from glutamic acid metabolism back into volatile fatty acids. VanDenHende (l963c) proposed two alternate pathways for glutamate metabolism.) In the first, glutamic acid undergoes oxidative de- amination to alpha-ketoglutarate, decarboxylation to succinic acid, and further decarboxylation to propionic acid. The other pathway involves mesaconic and citramalic acids, a pathway which seems rather unlikely on the basis of the molecular rearrangements necessary. Greenberg (1961) points out that the enzyme, L-glutamic dehydro- genase, which is responsible for conversion of glutamate to alpha- ketoglutarate, is widely distributed in bacteria, yeast, plants, and mammalian tissues. Consequently, its presence in the rumen is quite likely. In addition, oxidative deamination to an alpha-keto acid is a well documented amino acid pathway in the rumen. Alpha-ketoglutarate is a part of the citric acid cycle and undergoes decarboxylation to succinate. Finally, the conversion of succinate to propionate in the rumen is well known. This pathway would appear, then, to be the most likely in the rumen. However, this pathway does not explain the appearance of activity in the volatile fatty acid fraction following fermentation of C-1 labeled glutamate (Otagaki, 1955). p. Histidine ‘ ' Lewis and Emery (1963) found that strained rumen liquor released 33 percent of the theoretically available ammonia from histidine over a 10 hour period. In the same time, washed cells released 1h percent. 35 Under the conditions imposed by VanDenHende (l963d) 200 micromols of DL-histidine yielded 150 micromols of ammonia. This study utilized washed cell suspensions. In a study on the mechanisms of histidine fermentation, VanDenHende (l963a) noted that end products were ammonia, and propionic, butyric, and acetic acids. Formic acid may have been present, but was probably metabolized to carbon dioxide and hydrOgen and did not appear. He found the intermediates included urocanic acid, formamidinoglutaric acid, formamid, and glutamic acid. According to literature reviewed by Greenberg (1961) urocanic acid arises from deamination of histidine, resulting in a double bond between the alpha and beta carbons. Glutamic acid is also an end product. Formamid was discussed as a metabolite in some organisms. Although VanDenHende (I963a) has worked out a number of the intermediates in the degradation of histidine, knowledge of the enzymes concerned, their cofactors, mode of action, and optimal con- ditions is far from complete. Nevertheless, there is sufficient similarity between the intermediates described by VanDenHende (1963a) and the intermediates concerned with mammalian histidine metabolism - that the two processes are probably not too different. q. Arginine Lewis and Emery (1962) found arginine yielded 80 percent of its theoretical ammonia ih 10 hours of incubation with strained rumen contents. VanDenHende 35,21. (l963d), using washed cells, showed 100 micromols of L-arginine yielded iso micromols of ammonia. Thus, in both of these experiments more ammonia was released than could be 36 accounted for by removal of the alpha-amino group. Lewis and Emery (l962b) showed the metabolic products to be ornithine, delta-amino valeric acid, and in some cases, putriscene. Ornithine may arise via cleavage of urea from arginine. Urea, of course, should be rapidly hydrolyzed, and appear only as ammonia and carbon dioxide. Deltawamino valeric acid may arise from ornithine by deamination at the alpha carbon. Lewis and Emery (l962b) discuss the ruminal importance and metabolic aspects of this compound. Putriscene, which was noted only in a few cases, could arise simply from decarboxylation of ornithine. Since it did arise in only a few cases, the metabolic significance of putriscene is open to question. r. Lysine Sirotnak._£._l, (1953) observed no deamination or decarboxylation of lysine in an in 21:59 rumen system. VanDenHende (1963b) showed only deamination. On the other hand, Lewis and Emery (l962a) noted 5h percent disappearance of the amino acid when incubated with strained rumen fluid. In a later study, Lewis and Emery (l962b) showed lysine was converted to delta-amino valeric acid and cadaverine. No other amines were found. Hueter _£‘gl. (1958) found that volatile fatty acids were not producedduring lysine metabolism. Both Looper _£Mgl. (1959) and Lewis and Emery (I962c) noted very rapid increases in rumihal ammonia. They (Lewis ahd Emery, l962c) also observed a rapid increase in blood lysine shortly following its administration, pointing out the possibility of lysine absorption as such from the rumen. 37 Greenberg (1961), in a review paper, notes that delta~aminovaleric acid is an intermediate in lysine metabolism of certain bacteria, but normally does not occur in the rumen, and that when delta-aminovalerate does occur, it is by way of oxidative deamination and subsequent de~ carboxylation with the intermediate compound being alpha-keto-epsilon- aminocaproic acid. Cadaverine, reported by Lewis and Emery (l962b), could arise from decarboxylation of lysine. The appearance of delta;aminovaleric acid in the reaction mixture makes it appear that, contrary to Greenbergis (I961) observation, a pathway involving this intermediate is certainly functional in the rumen. Its catabolism was slow in in 21352 incubations. However, paper chromatograms showed glutaric acid was not produced. In fact, glutaric acid was not metabolized in this study by rumen micro» organisms. The advent of gas chromatOgraphy and its ability to separate the various five-carbon branched and straight-chained acids could probably be used to advantage In the study of delta-aminovalerate, Quantitative data is badly needed on lysine metabolism. Apparently, the alpha-amino groups are removed rapidly, as evidenced by the rapid increase in rumen ammonia. However, appearance of lysine in the blood shortly after lysine feeding leads one to believe that only part of the amino acid goes through the deamination pathway. 2. Amide Catabolism Numerous non-protein nitrogen (NPN) sources have been examined in the literature, chiefly by way of feeding trials. Relatively little has been done concerning biochemical pathways of metabolism of NPN compounds. Consequently, evidence for utilization of many 38 NPN compounds depends upon growth, maintenance or production data. One may reason, then, that if an animal grows on a certain NPN compound, and this growth cannot be attributed to another component of the diet, then it follows that the compound has undergone biochemical change, making it available to either the rumen bacteria, or the animal's body per 35. a. Urea The conversion of urea to ammonia via the enzyme urease has been reported repeatedly. Pearson and Smith (l9h3b) have shown that ammonia release is the principal step in its use, and Oglnsky and Umbreit (1959) have dlscussed the incorporation of ammonia into various amino acids. - Urea feeding to ruminants has been recently reviewed by Satapathy (I963). The most complete work on urea is a collection of abstracts hy Stangel (I963). The utilization of urea is well documented and is often the standard against which other NPN compounds are compared. b. Biuret Biuret is a condensation product of two molecules of urea via the removal of ammonia. Probably its popularity as an experimental subject is due mainly to its lack of toxicity (Berry g£_gl., 1956; Repp g£__ls, 1955; Hatfield _£._l., 1959). Its performance in animal growth and/or production has been found equal to urea by Hatfield et al. (1959) and Heiske (1955). On the other hand, growth response lower than that from urea was observed by Campbell £3 21, (I963). 39 Ewan._£.gl. (1958) noted that urea supported higher nitrogen balance than biuret and that nitrogen balance increased when lambs received inoculum from lambs previously fed biuret. Welch _£‘_l. (1956) studied both a crude and a purified biuret. The crude product depressed nitrogen digestion but had no effect on nitrogen utilization. With pure biuret, both measures were reduced. Hatfield gt‘gl. (1955) showed that biuret, when compared to urea (l) lowered nitrogen di- ’ gestibility (P < .01), (2) lowered nitrogeh balance (P < .02), and (3) increased the amount of biuret excreted in the urine (P Q .01). Anderson gt al. (1959) used a semipurified lamb diet with all nitrogen coming from urea; When biuret replaced 50 percent of the urea, no depression occurred in nitrogen utilization or nitrogen balance. However, when biuret replaced all the urea, significant depression occurred in both measurements. ; Biuret has been studied in regard to the so-called adaptation response. Several workers have noted that biuret utilization in- creased with time on feed. This subject will be treated in more detail In another section. The extent to which biuret is used by the animal, If indeed it is used at all, remains unresolved. Much of the early work suffered from doubts concerning the purity of the biuret used. In fact, one crude biuret (Campbell g£_gl., 1956) was shown to contain #0 percent biuret, h5.5 percent urea, 6.7 percent triuret, and 7.6 percent cyanuric acid-~hardly a valid test of the usefulness of biuret. to Little has been done concerning biuret in lg_xl£;g systems. The appearance of rather large amounts of biuret in the urine of steers following biuret feeding (Hatfield'_£.gl., 1955) indicates that certainly a large amount of biuret was leaving the rumen un- changed and causes speculation as to whether or not biuret utilization, if it does occur, might occur outside the rumen. c. Urea-formaldehyde Complex This compound was studied in lambs by Anderson £3 21. (I959). Digestion and nitrogen metabolism studies showed it to decrease ‘ apparent digestibility of crude protein and reduce crude fiber digestion. Nitrogen from this complex was used inefficiently. d. Ammoniated By-products A number of ammoniated industrial by-products have been ex- amined as nitrogen sources for ruminants. Pinck gt‘gl. (1935) and Millar (l9hl) described ammoniation processes for various plant products including peat, lignin, dextrose, beet pulp, and starch. Hillar (l9hl) found some nitrogen addition could be accomplished without heat or pressure. This fraction was water soluble. Ammonia added under conditions of high pressure and temperature was largely water insoluble. Pinck g£.gl. (1935) showed that most of the water soluble fraction was in the form of ammonium salts and urea. However, upon use of heat and pressure, ammoniation yielded a complex polymeriz- ation product which was insoluble in the usual solvents, or In strong acid and alkali. hl Millar (l9hh) showed that ruminants utilized the nitrogen from ammoniated beet pulp, but not at the same rate as soybean meal nitrogen. Knodt S£.§Un (1951), however, studied ammoniated cane molasses, ammoni- ated inverted-cane molasses, and ammoniated condensed distillers molasses solubles in calves and found that after 15 to 16 weeks of age nitrogen from these sources was as digestible as soy or oats nitrogen. Apparently, nitrogen balance was not determined. Tillman and Kidwell (1951) fed ammoniated condensed distillers molasses solubles to beef cattle and noted slightly lower growth than when cotton seed meal was used. Tillman and Swift (I953) carried out digestion trials using ammoniated by-products and found that the products, when compared with urea or soybean meal depressed digestibility of all ration constituents except ether extract. In addition, the ammoniated products promoted lower nitrogen balances. Much of the poor response to ammoniated by-products was explained by an lg_xl££g study by Hershberger et al. (1959). They studied ammoniated invert cane molasses, ammoniated corn cobs, ammoniated furfural residue, ammoniated bagassee, and ammoniated bagassee ex- tract and found that only the ammonia in these products was utilized by the microorganisms. The so-called "bound nitrogen" (Millar, l9hl; Pinck _£._l., 1935) in ammoniated invert cane molasses was not utilized even after an adaptation period of l23 days. The ig_xi££g study of Hershberger 35.31, (1959) confirms much of the work on practical feeding trials. Apparently, a large proportion of the total nitrogen is in the bound form and is unavailable to the rumen microorganisms. Nevertheless, the previously discussed work 42 of Knodt 35.31. (1951) raises some questions. If only free ammonia were available for bacterial use, the bound nitrogen should have been either excreted in the feces, thus lowering the digestibility, or should have been utilized further down the digestive tract. e. Ammonium Salts Belasco (l95h) studied a wide range of inorganic ammonium salts and ammonium salts of organic acids. Each salt was compared to urea by an I vitro technique. Salts tested included ammonium salts of succinic, lactic, alpha-keto glutaric, formic, malic, pyruvic, fumaric, citric, adipic, acetic, sulfuric, carbonic, sulfamic, nitrilotri- sulfonlc, triamidodiphosphoric, and nitric acids. Ammonia utilization was within a few percentage points of that for urea except in the case of ammonium nitrate, with which there was no nitrogen utilization and an excessive level of ammonia developed. With ammonium nitrate there was no bacterial growth. This work suffers from lack of sufficient replication. The reason for the 13.11359 toxicity to the bacteria of ammonium nitrate is not apparent, since diets for the in £1552 system were isonitro- genous. In a rumen, where the potential is slightly on the reducing side, the nitrate could probably be converted to nitrite and finally to ammonia, which is useful to the cells. Repp £5 21. (l955b) fed ammonium formate, propionate, and acetate to lambs and found them all to be about equal in feeding value to urea. The speed rate of ammonia release in the rumen from ammonium salts was studied by Repp gt‘gl. (l955a). Urea, ammonium a formate, ammonium propionate, and ammonium acetate were much less #3 toxic than ammonium succinate, glycine, propionamide, formamide, and biuret. The authors do not speculate on the lack of toxicity of ammonium succinate. That ammonia is the form of nitrogen utilized by rumen bacteria is pointed out in work by Wolin‘gt‘gl. (1959) on an isolated strain of Streptococcus bovis. Rumen bacteria were grown on media In which only ammonia served as a nitrogen source. Several of the isolates were strains of Streptococcus bovis. Asparagine or a combination of glutamic acid and arginine supported growth while glutamic acid, urea, biuret, and nitrate did not. Thus, this particular isolate must have pathways capable of ammonia incorporation into amino acids, but not for urea or biuret hydrolysis to ammonia. f. Guanidine Various salts of guanidine were examined by Belasco (l95h) in an 12_xi££g system. When compared with urea by amount of cellulose digestion supported, guanidine acetate, carbonate, and hydrochloride proved to be almost as efficient as the urea control, and at the same time supported considerably lower ammonia levels. Belasco (l95h) postulated that guanidine breakdown occurs viama two-step ehzymatic breakdown. In the first, guanidine is split to yield ammonia and urea. Then the urea is hydrolyzed to ammonia in the usual manner. Thus, depending upon the velocity of the first reaction, the ammonia may become available at a rate commensurate with the synthetic capabilities of the microflora. Lu. Repp gt‘gl. (I955a) have reported on the toxicity to lambs of guanidine carbonate. Drenching with this compound produced no change in either blood ammonia or blood urea. Dosage was equivalent to the nitrogen in 15 gm. urea per 45.h kg. animal weight. Although this dose caused rapid death in animals given urea and ammonium salts, death followed guanidine administration by about 8 hours. Presumably, the cause was not related to rumen function. g. Creatine and Creatinine Belasco (1954) found that both creatine and creatinine supported lower cellulose hydrolysis in ifl.li££2 systems than did urea or guanidine salts. Creatinine supported twice the ammonia level of creatine. Since the substrates were added in isonitrogenous amounts, the discrepancy in ammonia yield is difficult to explain. Welch _£.gl. (1956) found creatine, when supplied to lambs to the extent of 33 percent of the ration nitrogen, enhanced nitrogen utilization over urea, and compared favorably with soy protein in increasing digestibility of protein and organic matter. In a later study, Welch _£'gl. (1957) found added vitamin 8'2 increased both the apparent digestibility of nitr09en and nitrogen utilization of creatine. When studied 12.xitrg, using volatile fatty acid pro- duction as a criterion, vitamin 312 showed no effect. However, ad- dition of homocystine or methionine increased the action of creatine. The two latter compounds gave no response with urea substrate (McLaren t l., 1958). “5 The utilization of creatine was compared with urea, soy protein and biuret by Anderson (1959). A mixture of 50 percent creatine and 50 percent urea, compared to urea alone in semipurified diets, in- creased digestion of organic matter (P < .05) and crude fiber (P < .01). The percent of absorbed nitrogen retained was also increased over urea‘ diets (P < .05). It is interesting that the only nitrogen source supporting greater nitrOgen balance than creatine was soy protein. The discrepancy between the action of vitamin 8'2 jg_vitro and i£_vivo is difficult to explain. Presumably, 812 is sufficient in the rumen, although the animals in question were on a purified diet. Campbell t 21- (I959) in i vivo studies, found vitamin BIZ markedly improved digestibility of organic matter (P <:.05), crude protein (Pi< .01), and crude fiber (P < .05). Essentially, the same results were seen with sarcosine, one of the products of metabolism of creatine. Since the early screening study by Belasco (195h) no further study has been made of creatinine. Since this compouhd was apparently utilized to a greater extent than creatine, it would appear that this compound should receive attention as an NPN source. h. Dicyandiamide This compound first appeared in the rumen nutrition literature in l95h (Nagruder and Knodt, l95h). When fed to dairy heifers at a level to furnish #7 percent of the total concentrate nitrogen, daily gain and nitrogen digestibility were very similar to urea and soybean meal rations. Davis 35,21. (1956) found the compound supported milk A production equal to urea or soybean meal over a 5k day period. No as differences were shown in nitrogen digestibility. In a study ex- t 31. (I956) observed the same lactational tending over 196 days, Rust results. However, animals on dicyandiamide lost 39 kg. over the 196 day period in comparison to 13 kg. 1055 for urea and a 1.3 kg. gain , for soybean meal. Dicyandiamide, although shown to be utilized as a non-protein nitrogen source, has apparently not been evaluated bY.DE.!l££2 techniques nor by the use of fistulated animals. i. Amides of Organic Acids The work of Belasco (195“) evaluated many of these compounds as to their usefulness to rumen microorganisms. When evaluated on the basis of cellulose digestion response, the amides of the monocarboxylic acids-~propionamide, butyramlde, glycinamide, and formamide-~compared fairly favorably with urea. On the other hand, the amides of di- carboxylic acids such as succinamide, oxamide, malonamide, glutaramide, diglycolamide and adlpamide were apparently unavailable as nitrogen sources. The author points out that the latter group of compounds is insoluble in water. The use of formamide by Repp £5.21. (l955b) produced not only slow gains in lambs but also appeared to be responsible for a paralysis of the rear quarters which the authors found peculiar to this par- ticular amide. Propionamide, when fed to lambs to supply 50 percent of the nitrOgen in the ration, produced rather slow gains during the early part of the trial, but gains increased in the later period. In an lfl.!l££2 study more ammonia was released from propionamide by inocula from lambs ”adapted” to the compound than by rumen contents of control lambs. “7 The inability of propionamide or formamide to yield ammonia is shown by work of Repp g; 21' £1955a). When animals were drenched with these compounds, no toxicity 8ccurred and blood ammonia and urea levels did not increase. j. Urea Derivatives Several urea derivatives have been studied by Belasco (l95h). Included were methylenediurea, n-butylurea, hydrazoformamide, ethylurea, ethylenediurea, acetylurea, methylurea, allylurea, ethyleneurea, dimethylurea, and semicarbazide HCI. None of these compounds promoted cellulose digestion, and several actually appeared bacteriostatic. Apparently, enzymes able to operate on these compounds are not available. Although no apparent response occurred, these compounds should be examined further, using critera other than simple cellulose digestion. In fact, the apparent toxicity of these compounds to rumen bacteria should be studied with the possibility of gaining further information on the rumen fermentation. Since the earliest studies with urea for ruminants, attempts have been made to tailor the rate of ammonia release to the needs of the microorganisms. A number of nitrogen sources have been tried in hopes of finding one with a limited rate of ammonia release. However, examination of the foregoing material will show that ammonia release from the compounds is necessary in order for microbial inn corporation of the ammonia into amino acids (and finally microbial protein) to occur. Study of the section of this work concerning amino acid catabolism by rumen microorganisms shows that: (I) there should be available from amino acids a fairly large supply of ammonia us for synthetic reactions, and (2) there are a number of metabolic opportunities for ammonia fixation to occur. Perhaps foremost in these opportunities is transamination. Many amino acids are degraded by rumen microorganisms by deamination and the production of a keto acid. The kinetics of ammonia incorporation into amino acids need to be studied extensively. Further studies need tobe made on the rate of ammonia loss from the rumen under the influence of high and low ammonia concentrations. Work on this problem has been carried out by Bloomfield _£._l. (1960) who found that urea was hydrolyzed four times more rapidly than the corresponding uptake of ammonia nitrogen by rumen bacteria. The excess nitrogen was absorbed from the rumen, and part was finally rem cycled and used in protein synthesis. Knowledge of the mechanism of ammonia absorption is, at best, rather primitive. If indeed ammonia release must be regulated, a fresh look needs to be taken at methods for accomplishing this end. Several novel approaches have been tried. Barbituric acid was used by Harbers _£'gl. (1962) in an attempt to depress urease activity. Although this action was detrimental to digestibility, growth, and feed utilization, the idea merits further study. Johnson _£‘gl. (1962) studied the effect of coating urea prills or adding capper sulfate. Few of the coating materials showed promise. Copper concentratlons high enough to slow enzymatic hydrolysis were t al. (1966) studied several urease also bacteriocidal. Loper inhibitors both in vitro and I vivo. Cobalt and copper compounds were inhibitory to urease, but were also toxic to other rumen functions. 1.9 Immunization of sheep against urease has been shown by Gllmp and Tillman (1965). They found that ammonia levels In the ruminal vein were higher in the control sheep than in those which had been lmmunlzed. They concluded that the ureolytlc action of the rumen had been decreased. Owing to the fact that; (l) urease ls largely of microbial origin in the rumen, (2) urease is normally present In extremely high levels, (3) observations were made on only two lambs per treatment, and (h) rumen fluid Itself was not examined, make these conclusions seem extreemely dangerous. D. Ammonia Absorption from the Rumen The literature contains several references concerning the rate and magnitude of ammonia absorption from the rumen, but there is little information concerning the precise mode of operation of this important phenomenon. McDonald (l9h8) first showed that ammonia absorption from the rumen did, In fact, occur, made calculations on the magnitude of this transfer, and noted that part of the nitrogen loss was compensated for by addition of salivary urea to the rumen contents. Much of this work was conflrmed by Dinning (l9h8), who studied absorption of ammonia from the rumen and its appearance in hepatic circulation. Ammonia absorption was shown by Coombe £3 21. (1960) to be re- lated to the rumen pH. This was confirmed by Yoshlda and Nakamura (1963). Since absorption was much more rapid at a higher pH, it was assumed that It was more rapidly absorbed In its unionized form. SO Hogan (l96l) showed that at pH 6.5, ammonia absorption from the rumen of an anesthetized sheep was related to its concentration, with higher absorptions being noted in animals showing highest transport rates of acetate. At this pH there appeared to be no relation between ammonia transport rate and changing rumen concentrations of sodium, potassium, chloride, carbon dioxide, lactate, or net water movement. At pH “.5, ammonia transfer was not related to its concentration or to volatile fatty acid transport. Several of these observations were confirmed by Yoshida (l963). He found that both ammonia and acetic acid in ruminal vein blood of goats increased when ammonium acetate was placed in the rumen. If rumen pH was increased with potassium hydroxide, ammonia in the ruminal vein increased while acetate decreased. The author postulated that the unionized form of both ammonia and acetic acid were absorbed at a more rapid rate than the ionized form. Ammonia absorbed from the rumen into the bloodstream is, at least partially, lost to the animal. However, various pathways for the conversion and/or further use of this absorbed nitrOgen are available. Lardy and Feldott (l949) and Rose E£.El- (l9h9) and later, Hfiiler g; 31. (lébh), showed that non-protein nitrogen could be used outside the rumen for synthesis of non-essential amino acids. This can occur, according to McLaren (l96h), by reductive amination of alpha-ketoglutaric acid followed by various transaminations. McLaren (l96h) postulated several other pathways for the metabolic conservation of ammonia nitrOgen. The reutrn of urea via the saliva has been noted previously (McDonald, l9h8). The transfer of urea from the bloodstream to the rumen contents Si has been studied by Decker 53.21. (l960) and Houpt (l959). The phenomenon of conservation of urea by ruminants under stress conditions has been studied by Schmidt-Nielsen g£,gl, (1958). Although ammonia loss through the rumen wall appears wasteful of nitrogen, quantitative data relating this loss to net nitrogen economy is incomplete. Early investigators assumed that once ammonia left the rumen, it was lost to the animal. Newer techniques, especially those concerning radioisotopes, have proven this theory to be at least partially false. Quantitative studies are needed to measure not only the magnitude of ammonia loss from the rumen, but also the magnitude of incorporation of this "lost" ammonia into non-essential amino acids and other nitrogen—containing compounds by the body proper. Finally, the lack of information on mechanisms of ammonia ab- sorption attests to the grave need for sophisticated experimentation in this field. Although knowledge on the subject is, at best, sketchy, references quoted here cast serious doubt upon the following state- ment from Annison and Lewis (l959): ”There is no evidence for any mechanism regulating the absorption of ammonia from the rumen into the portal blood and it seems probable that ammonia transference is effected by simple diffusion through the rumen wall." E. The Adaptation Response Several authors have reported increased non-protein nitrogen (NPN) utilization as the length of time increases following addition of the source to the ration. Reppigtlgl. (l955a) found 2 to 3 weeks were required for animals to become adapted to the NPN sources urea, ammonium formate, ammonium acetate, ammonium propionate, and propionamide. 52 In addition, rumen fluid from lambs adapted to propionamide released significantly more ammonia from propionamide than unadapted lambs. Urea and biuret adaptation was studied by Ewan ££.21. (l958). They found that when a sheep fed a biuret ration received rumen inoculum from a sheep maintained on biuret, nitrogen balance was greater (P«( .05) than when sheep were not inoculated or inoculated from urea;fed sheep. When a urea-adapted sheep served as inoculum donor for a sheep started on a urea ration, there was no increase in nitrogen balance. Welch‘gt‘gl. (l957) and McLaren.g£'gl. (l959) noted that when diethylstilbestrol was added to the diet, the maximum biological value for crude biuret nitrogen occurred within l0 days after the start of biuret feeding. However, this measure had not reached a maximum value after 50 days when diethylstilbestrol was absent. In a subsequent study, McLaren‘_£‘gl. (l960) measured metabolic fecal and urinary endogenous nitrogen, creatine, allantoin, protein- bound iodine, and serum urea and ammonia on lambs "adapting" to urea and biuret with and without diethylstilbestrol. Their results led them to conclude the action of the hormone was directly upon the tissues to promote better utilization of NPN in the synthesis of non- essential amino acids. Smith.g£.gl. (I960) measured the regression of nitrogen utilization upon time and published the equation: Y a lll.93 - I.093x, - l.065X2 + .20lX3, where Y I percent of absorbed nitrogen retained, Xl- percent urea nitrogen in the diet X2- percent nitrogen in the diet X3- length of time (days) urea was fed. 53 Thus, the percent of absorbed nitrogen retained was shown to increase 0.2 percent for each day the animal was on the urea ration. It should be noted, however, that the other two terms of the equation each account for several times as much of the biological value as days on urea. The adaptation of cattle to biuret was studied by Campbell‘g£._l. (l963). Biuret-supplemented rations became more efficient with time. Furthermore, these workers observed an increasing postprandial rumen ammonia concentration up to 5 weeks, indicating, according to the authors, an increased biuret hydrolysis. Campbell (I962) had observed the emergence of a bacterial species capable of utilizing biuret. However, Campbell gtwél. (l963) state that: ”Studies of the bacterium failed to show that biuret was being utilized via hydrolysis to ammonia, although alternate pathways appeared difficult to conceive.” In a study by Johnson and McClure (l963, l96h) using sheep, there appeared to be an adaptation to biuret but not urea when apparent digestibility was considered. No differences appeared with nitrogen retention or biological value. Inoculum taken from a sheep adapted to biuret for 9h days could release no ammonia from biuret lg'xlggg. According to the authors: ”The results suggest that if ruminants do utilize biuret as a source of nitrogen, it is not through direct utilization of biuret nitrogen.” The preceding papers point out the state of confusion concerning the so-called adaptation response. Although Smith g£_gl, (l960) have calculated a regression of biological value of urea nitrogen upon time, no such work has been done with biuret. Sh Much of the work on biuret by the west Virginia group (Helchygg‘gl., 1957; McLaren gt _l., l959) was carried out with a crude product that contained as much as ho percent urea, causing one to speculate whether or not some offthe reported adaptation response to biuret might not, in reality, be adaptation to urea. McLaren'gt‘gl. (l959, l960) noted more rapid adaptation to biuret when diethylstilbestrol was added to the ration, and claimed that the adaptation to biuret occurred in the tissues.“ Yet, when Karr‘g£,gl. (l963) implanted lambs with diethylstilbestrol, they found it had a greater effect on urea nitrogen utilization than on soybean meal or biuret utilization. Similar results were found later in a comparable experiment (Karr 5_t_ al., l96h). Oral diethylstilbestrol increased protozoal population on both high concentrate and hay rations, indicating some influence on the rumen fermentation (Christiansen _£'_l,, l9b“). Lewis (l960) noted a decrease in peak rumen ammonia concentrations following ammonium salt drenching and stated that ”the organisms within the rumen reach a maximum level of adaptation (7 days) to handle large quantities of ammonia by synthetic pathways." 0n the other hand, Barth‘gt‘gl. (196l) found that lEHXLEEQ protein synthesis by rumen bacteria did not in- ' .crease as a function of time, even though the utilization of absorbed nitrogen increased from 36 percent to 51 percent in five weeks. The term "adaptation response” has come to have quite a varied meaning, and since it has appeared in the literature in so many con- texts, will probably escape precise definition. However, it is clear that the anatomical, physiological and biochemical site of the 55 adaptation response is as elusive now as when it was first studied. In addition, it appears that there may be separate "adaptation responses” for biuret and urea. This statement may, in all probability, be extended to include separate responses on a number of other non-protein nitrogen sources. It must be pointed out that there is a good possibility that the adaptation response(s) occurs in more than one location and in more than one physiological function. Some authors have tried to locate the response in the rumen while others have data to show it is in the tissues. Both may be partially right. F. In Vitro Rumen Fermentation Systems The 12.x1552 method for rumen metabolism studies, popularly known as the "artificial rumen" technique, was apparently first used in America by Hegner g£_gl, (l9h0). From their introdUCtion: "Preliminary trials were inaugurated in which we attempted to duplicate the con- ‘ ditions found in the rumen. These experiments consisted in adding urea to rumen contents and following the fate of the inorganic nitrogen. All samples were incubated at 39° C. Results in this trial were negative since the level of inorganic nitrogen did not decrease.” From this point, the authors went to strained rumen fluid as inoculum, and finally added pH control by buffering. Earlier work with ig_xl££g fermentations had been carried out by Woodman and Evans (l938), who studied the end products of cellulose ' digestion with rumen microorganisms but made little attempt at duplicating the conditions in the rumen. 56 Excellent reviews on the lg_xltggbmethods of rumen fermentation study have been written by Johnson (l963) and Bentley (l959). Both of these reviewers discussed the various systems used, the methods of inoculum preparation, and the interpretation of information from‘lg £53.52 results. The earliest type of lfl.!l££2 system, in which rumen contents were incubated in a closed vessel, has already been discussed. The work of Hegner‘gt‘gl. (l9h0) pointed out the requirement for pH control, since in their work, the pH of the closed system decreased rapidly due to the production of organic acids. An important development concerned with the _ig M technique was the determination of the composition of sheep saliva by HcDougall (l9h8). A buffer-salt solution based upon this information has been used in most subsequent lg 2.13.59. rumen studies. The salt should not, however, be used without sight of the fact that in the intact animal, the composition of the rumen fluid is not the sum of the salivary com- ponents and the metabolic end products, but also includes solutes entering the rumen by diffusion from the circulation. The all-glass, impermeable system allows for the accumulation of metabolic end-products, and may adversely affect results. However, this system has been used extensively. Hegner‘g£_gfl, (ISHO) strained rumen fluid through cheesecloth and used this strained-fluid as inoculum. This cheesecloth straining has become almost universal in subsequent work. 57 Harston (l9h8) addedgentle stirring to his sytem, and in addition to buffers, added the trace minerals, iron, copper, zinc, and cobalt. However, the greatest change over other systems was his source of inoculum. Instead of using strained rumen liquor, he centrifuged the bacteria from strained liquor, discarded the supernatant, and resuse pended the bacteria in the buffer medium, thus introducing the ”washed cell‘technique." The first departure from the all-glass system was made by Lou ‘gg‘gl. (l9h9). He compared the fermentation in an all-glass system with that carried out in a dialysis bag suspended in a tank of buffer medium. More cellulose digestion occurred in the dialyzing system, but there was little difference in the two systems in their levels of volatile fatty acids. McNaught g£_gl, (I950) used the all-glass system with strained inoculum to study the effect of minerals on rumen bacteria. Copper depressed bacterial growth at a concentration of l0 ppm. Cobalt and molybdenum were less toxic. Iron was not toxic at a level of IOOO ppm, and complexing of the iron by 2,2'-dipyridyi depressed bacterial activity. Serial culture of rumen microorganisms was performed by Arias _£_gl, (i95l). Each fermentation ran for h to 2k hours, and then flask contents were used as the inoculum for the succeeding flask. The f‘VSt.lfl.Xl££2 studies on the dependence of rumen bacteria upon substances in cell-free rumen liquor was carried out by Bentley t l. (l95h). Rumen bacteria alone suspended in a phosphate buffer a A 58 solution containing cellulose, urea, glucose, and mineral salts could not support cellulose digestion. Addition of a cell-free rumen fluid allowed cellulose digestion to proceed. The missing nutrients were found to be biotin, vitamin BIZ and para-aminobenzoic acid. Cheng gtwgl. (I955) used basically the same technique as Bentley _£_gl. (l954), but obtained active fermentation of cellulose without any addition of rumen fluid supernatant. The discrepancy here is unexplained. This same method (Cheng g£,_l., l955) was used successfully by Anderson‘gt.gl. (l956) to evaluate phosphorus availabilitonf feed supplements to rumen microorganisms. The washed cell technique was used by Dehority (l96i) and Dehority and Johnson (l96l) to study the effects of particle size of cellulose and feed materiai on microbial digestion‘lg.xl££g, They added biotin, para-aminobenzoic acid, and valeric acid to their medium. Valeric acid had been shown to be essential to microbial activity and one source of cellulolytlc activity of cell-free rumen fluid by Bentley gt‘gl. (l955). One of the problems of 19.11552 studies has always been the variability of the various criteria between replications even on the same sub-sample of rumen fluid. Bowden and Church (l962a) found much of this variability could be decreased by placing strained rumen fluid in a tall vessel at 39° C, allowing the coarse material to come to the top, and utilizing the bottom layer as inoculum. The usefulness of the ig‘vltgg technique for the evaluation of forages was shown by Bowden and Church (l962b). Using an all-glass "rumen," strained rumen fluid inoculum, and a buffered mineral 59 mixture (Bowden and Church, l962a), they obtained highly significant correlations with'lg‘vltgg cellulose and dry matter digestibility, and a number of‘ig‘leg digestibility measures. The first indication of the importance of protozoa in inuxltrg systems was shown by Yoder 55 31. (l963). When a protozoa fraction of rumen fluid was added to the bacterial fraction, volatile fatty acid production increased 70 percent and cellulose digestion increased by 80 percent. The study points out the necessity of the difficult task of preservation of protozoal populations in‘ig'xltgg fermentation systems. Yoder‘gt‘gl. (l96h) fractionated protozoal cells and noted water and/or alcohol extracts of the cells stimulated cellulose digestion in the presence or absence of B vitamins. In an attempt to more closely duplicate the conditions existing within the viable rumen, several investigators have used continuous flow systems. Harbers and Tillman (l962) used an 12.xltrg technique in which a nutrient medium,mineral-solution, or cell-free rumen fluid was pumped through a culture of either mixed rumen microorganisms or a pure culture. The bacteria were imprisoned by a bacteriological filter at each end of the growth chamber. Cellulose hydrolysis was inversely related to flow rate when the turnover number (the number of hours required for a volume of flow equal to the volume of the growth chambers) was less than about 13.5 hours. Cell-free rumen fluid supported greater cellulose hydrolysis than did either the nutrient broth or mineral solution. 60 A culture system in which both the fluid and the bacteria were mobile was used by Adler _£_gl, (l958). The culture periods were lO hours in length. In contrast to-the work of Harbers and Tillman (1962), growth rate was positively related with flow rate. Unfortunately, few- measures were made which would indicate the normalcy of the fermentation. A complex continuous-flow system which maintained volatile fatty acid and protozoa levels at near those observed 12_leg was described by Stewart gt 21- (l96l). The culture volume was 5.5 l. and the flow rate of substrate was #50 ml. per hour for a turnover number of about ll hours. According to the authors, the system seldom worked more than 2h hours due to mechanical malfunctions which easily may have been complicated by the fact that their substrate consisted of ground feed suspended in HcDougall's (l958) artificial saliva. RufenerH_£.gl. (1963) developed a continuous-flow system which maintained an "essentially normal 12 21:2 fermentatiow' for periods of from 3 to l0 days. The 325 ml. culture was changed atna rate of l.h3 volumes per day, or a “turnover number" of l6.8 hours. The fluid added to the system was 60 parts of HcDougall's (l9h8) artificial saliva diluted with #0 parts tap water.7 Both grain and hay-fed animals were used as inoculum donors and hay and concentrates were added separately to each culture. Volatile fatty acid was of the same order of magnitude as that found in vivo, and a single culture responded to changes from hay to grain or the reverse by a change in molar per- centage of volatile fatty acid. This type rumen presents several interesting innovations. First, gas was collected from each fermentation system. Second, the instrument was constructed with six 6l fermentation units operating in the same frame, enabling a number of experiments to go on at once. Finally, pH control was achieved by the use of ion exchange resin enclosed in a dialysis bag. This resin was generated to a bicarbonate phase and was replaced when feed was placed in the chamber. A continuous-flow system with automatic pH control was used by Bowie (l962). The culture media was fed into and removed from the system with a peristaltic pump at a continuous rate. A pH meter was combined with a servo-mechanism and solenoid valve to control pH within 0.01 pH units. The system was generally operated for 8 hours. ’Turn- over number was approximately l2.9 hours. Perhaps the most advanced 12_xi££g system yet reported was that of Davey _£‘gl. (l960) in which the rumen inoculum was placed in a dialysis bag and dialyzed against saline. Provisions were made for adding material to or removing it from the dialysis flask. The instrument operated for periods of up to lh days. When bacteriological and biochemical parameters of the system and a cow maintained on the same feed were compared, amazing similarity was found. Slyter £5.21, (l965) cultured rumen microorganisms for up to lg days. On the l7th day, samples were taken for bacteriological examination and found that most of the strains cultured were typical of what one would expect in an intact animal on the same ration. Gray gt‘gl. (l962) developed an ifl.¥l££2 system in which both the rumen inoculum and the dialyzing fluid were subjected to addition and withdrawal. The dialyzing fluid was pumped through 6.h mm. dialyzing tubing wound into the rumen inoculum. In order to determine if the 62 fermentation adequately duplicated that in the intact animals, radio- active volatile fatty acids were introduced into both a sheeps rumen and the ”artificial rumen," and the rate of dilution of the radioactivity by synthesized acids was followed in both. Experiments of only 2 to 3 hours were carried out with this system. Results were very comparable over this short duration. No reason is given for not extending the fermentations over longer periods of time. Advantages and disadvantages are numerous for each of the various 12_xl££g systems. In addition, different types of information can be obtained from each system. In the past, rates of cellulose digestion have been compared with the all-glass systemlmainly because of its, physical arrangement. Studies requiring long-term fermentations must generally be carried out in either dialyzing or continuous-flow systems. Certainly, any study in which population dynamics were studied would require this type. El-Shazly 35 al. (l960) compared the all-glass, semipermeable membrane, and continuous-flow'lg.xi££g systems in regard to cellulose digestion, total volatile fatty acid production, ammonia level, and bacteriology, and found no major differences in the various types for periods of up to 30 hours. They concluded that because of its simplicity the all-glass system was to be preferred. They ob- served, however, that for longer fermentations, the continuous-flow system may be advisable. Harner (l956a) published a set of criteria which should be met by I vitro rumen fermentation systems: 63 l. The maintenance of normal numbers, appearance, and proportions of the bacteria, selenomonads, and protozoa. 2. The maintenance of normal rates of digestion of cellulose, starch, and protein, and the normal interaction between these. 3. The ability to predict quantitative results.1_ vivo. It would appear from examination of the literature that another criterion should be added, especially for the longer period ferment; ations: that of a steady population in a continuous-flow system. If a decrease in microbial population occurs during a continuouseflow ‘ fermentation, either the flow rate is too great, the bacteria and/or protozoa are not multiplying in a normal manner, or both. If bacteria and protozoa are not growing actively, then certainly the conversion of non-protein nitrogen cannot occur, and the fermentation is not normal. III. EXPERIMENTAL PROCEDURES A. General The purpose of this study was two-fold: (l) to study the ruminal metabolic fate of several non-protein nitrogen compounds, and (2) to simplify the study of these compounds through design and construction of an _i_g 119:2 fermentation system which would closely approximate j}; xlxg conditions. Urea, ammonium salts, and various ammoniated by-products have been used extensively in the past as ruminal non-protein nitrogen (NPN) sources. In all these compounds, ammonia release is quite rapid, and much of the nitrogen is apparently lost to the fermentation system. Furthermore, rapid release of high ammonia levels has, in a few cases, led to toxicity symptoms. For these reasons, NPN compounds which are decomposed more slowly should be more efficiently used. In additian, little is known of the process of initial release of ammonia in the rumen. Urea is decomposed by the extremely substrate-specific enzyme, urease. Its high substrate specificity precludes its action on other compounds. Consequently, any other NPN compounds would, necessarily, be decomposed by some other enzyme system. These investigations were designed in part to study the possible presence of enzyme systems other than urease capable of hydrolysis of nitrogen-containing compounds. ’ i The criteria of Oglnski and Umbreit (l959) for usefulness of a chemical entity as a nutrient, were discussed in a previous section. lAs was first shown by Hegner g£.gl, (l9h0) and Pearson and Smith ‘(l9h3b) ammonia is probably the central intermediate in NPN metabolism. “ 61+ 65 Thus, a system was needed for the detection of small, and possibly quite transient, increases in ammonia. Further, in order to study specific chemical entities, it was necessary to use substances which were, in as far as commercially available, chemically pure. These considerations made the use of fistulated animals impractical and necessitated the design and construction of a".lfl.!i££2 fermentation system which would, on a small scale, simulate rather closely jg_xlxg conditions. With these considerations in mind, a continuous-flow dialyzing system was designed and built which closely approximated rumen cone ditions. The instrument was adapted for automatic periodic sampling because previous studies with fistulated steers (Newland 33.21., l963) had shown the necessity of developing concentration vs. time plots of, rumen metabolites, particularly during early phases of digestion. 8. Design of the in vitro system A diagram of the dialysis and fermentation chamber is shown in Figure I. This chamber was mounted on the column of a C.M.E. volumetric fraction collector.l A G.M.E. Transferator2 was modified for operating two syringes as shown in Figure 2. The microswitch activating cam of the Transferator was reprogramed to hesitate at the extreme rearward '0. M. E. Fractionator Model Vl5 , Gilson Medical Electronics, Piiddleton, Wisconsin. 26.M.E. Transferator, Gilson Medical Electronics, Middleton, filisconsin. 66 Fermentation liquor /—‘/—-‘filulier ill /_\Coz ill _. A“: not '- Substrate ill latter in _ .J -. 52mm o.d. plexiglass . -, -:;- . . . I w" :I Dialysis tlllllllg I? 5’}: I l : :' .a. i ‘I 1' an ’2 I '. '. 3: | | : . :0 ' ._ 5' .- l i ' a T I i 1 :2 l ' .C' : r i :r- O 5 l .‘J: l , V ' l ‘. 1: I D l ‘ 'I‘ | '1‘ V E l .' 1': I 5' g ' : i:1 l E V l ' a 5 vv ' l 3' i. ' 1-' E l ' :-: w l L l | I '.l I l ". i I Z- \ I a x [I /lonntlng Halos ~_, _-; 9/ 100 mesh stainless stool scroon ;: 95-- Lil. ploxiglass -_'. VOLUMES: ." Fermentation liqnor 250 mi .5._J Dialyzate llOOml 'l'F—" Stirring bar lnftor ant C Fig. l.--Diagram of the fermentation and dialysis chambers of the E vitro system. Fig. 2.--Photograph of the Transferator modified to operate two syringes. 68 position(syringes full). Activation of the Transferator.was automatic via a microswitch clamped in the head unit of the fraction collector in such a way that it was depressed momentarily during cycling of the fraction collector (Figure 3). The switch, connected in the "normally open” position, activated the Transferator by way of the plug generally used for external foot-switch operation. BUffer solution was fed to the dialyzing chamber by a peristaltic pump.‘I The pump was set to deliver 20 ml. per hour, and was operated with 1.59 mm. id. plastic tubing.2 The dialyzing chamber, with fermentation bag in place, held ll00 ml. of buffer solution. Overflow from the chamber was carried to the fraction collector head. This flow rate served to time the sampling in the entire system. The Transferator and double syringe system was equipped with threeeway silicone rubber sleeve-type valves3 and arranged so that on the withdrawal stroke, 5 ml. of fermentation liquor were drawn into the upper syringe and buffer solution was drawn into the lower one. On the compression stroke, the fermentation liquor was transferred to the fraction collector table, and buffer was added to the fermentation to replace the 5 ml. sample that was removed. At the bottom of the lSigmamotor, Model Tm 20-2, Sigmamotor, Inc., 3 North Main Street, Middleport, New York. . 2Tygon No. R-3603, Plastics and Synthetics Division, U. S. Stoneware Co., Akron, Ohio. . 3ClayeAdams Aupette Valve No. A-2703, Clay-Adams Inc., New York, New York. . {I'll} Fig. 3.--Photograph of the Transferator-activating microswitch mounted in the fraction collector head. 70 sampling system for the fermentation liquor was interposed a 100 mesh stainless steel screen.1 This screen was coarse enough to allow protozoa to pass unharmed, but held most of the feed particles back. A diagrammatic flow sheet of the system is shown in Figure h. A photograph of the complete operating system is shown in Figure 5. With these flow rates into and out of the system, turnover time (defined here as hours required for a complete change of liquor in the chamber in question) was 55 hours for the dialyzing chamber and 25 hours for the material inside the fermentation bag. C. Operation of the in vitro system The entire system was operated at 39' C in a walk-in incubator. Prior to the inoculation of the system with rumen contents, llOO ml. of the buffer solution were added to the dialyzing Ehamber. In order to prevent a serious drop in the volatile fatty acid (VFA) level of the fermentation system, “.25 ml. glacial acetic acidA(Th:9 millimoles), l.50 ml. propionic acid (l9.l millimoles), and l.l5 ml: butyric acid ’ (i2.5 millimoles) were added, producing volatile fatty acid concentra- tions in the diaiyzing solution of 68.l, l7.h, and ll.0 micromoles per ml. of acetic, propionic, and butyric acids, respectively. The composition of the buffer solution is shown in Table 3. 1Small Parts Inc., Box 792, Biscayne Annex, Miami, Florida. Syringo Co; 3 Out . Co, h f Syringo J» i f} ‘* j Foristaltlo f roar L ¢ in .H” —-i—l-i-q.—l-P-- I t g l f i ,' Fornontation liquor l (' Dialyzing flail J Dialysato vi Fornoatation liquor i Hill MD Fraction oollootor Fig. 4.--Diagram of the flow pattern of the in vitro system. ystem in operation. in vitro 5 Fig. 5.--Photograph of the complete TABLE 3. Buffer composition. 73 Disodium hydrogen phosphate Sodium bicarbonate Sodium carbonate Urea Water to make #000 ml. Adjust to pH 7.5 with cone. HCl. NazHP04 Nch03 NachB 3.15 gm. 16.68 gm. l6.32 gm. l.288 gm. 7# The dialysis bag was prepared from #9 mm. diameter dialyzing H tubing... Since this tubing is made of regenerated cellulose, it was necessary to use it in double thickness. This was accomplished by cutting off approximately 65 cm. of dialysis tubing, soaking it in tap water, and turning it inside out far enough that the two out ends just met. At this point, air was forced from between the inner and outer bag by rolling toward the cut ends with a heavy photographic print roller. A knot was tied at the cut end at a point that would allow the bag, when installed on the support, to extend about 2 cm. beyond the various tubes inside the bag. The knot was then trimmed of excess material and the bag was inverted, leaving the knot on the inside. 'The bag was attached to the support unit, secured with a tight string, and the assembly lowered into the dialyzing chamber. Rumen fluid was obtained from a fistulated cow receiving the diet shown in Table #. The fluid was transferred from the cow to a large vacuum bottle, and taken immediately to the laboratory where it was filtered through # layers of cheesecloth. Two hundred fifty ml. of the filtrate were added to the inside of the fermentation bag. All supporting electrical equipment was turned on and carbon dioxide was started bubbling through the fermentation medium. Each l2 hours, 5 gm. of finely ground substrate were added to the system. The basal substrate was made up of the same ingredients lSargent No. S-25275-C, E. H. Sargent s C0,, 8560 West Chicago Avenue, Detroit, Michigan. 75 TABLE #. Diet of fistulated donor cow. Ingredient gm./dayg Alfalfa hay l360 Ground shelled corn 3#90 50 ‘1 soybean meal 73 Trace mineral salt . 36 Dicalcium phosphate 36 76 received by the donor cow. Composition of the basal substrate is shown in Table 5. In addition to the basal substrate, thenon-protein nitrogen source under study was added in an amount equal to l00 m. of nitrogen. ~ Following inoculation of the system and the original substrate'.. addition, the system was allowed to equilibrate for l2 hours, at which time, substrate was again added and sample collections were started. 0. Experimental design and sampling procedures Each non-protein nitrogen compound was studied in the‘lg‘gltgg system for a total of 5 collection periods over a period of 5 days. (Following the initial l2 hour equilibration period (see previous section), gamples acre collected for 9 hours out of alternate 12 hour periods, so that one set of collections was being made per day. In this manner there were 5 curves of each metabolite concentration versus time after feeding for each substrate. This design allowed for observation of any "adaptation" effects that might have occurred. In order that the samples collected be representative of the time at which they were removed from the system, 0.5 ml. of 50% Y/V sulfuric acid was added to each collection tube in the fraction collector. A volume correction was made for this 0.5 mi. of acid on all fermentation liquor samples but not for dialyzato samples. In fermentation liquor samples, carbon dioxide bubbles frequently 2 become inadvertently trapped in the sampling system causing smaller than normal samples, so that the acid made up a larger part of these particular samples. 77 TABLE 5. Composition of basal substrate for'ig vitro fermentation. Ingredient ,fgm. Alfalfa hay 1.667 Ground shelled corn 3.200 50 %.soybean meal .067 Trace mineral salt .033 Dicalcium phosphate _;233 Total 5.000 gm. 78 “M Unfortunately, time, equipment, and analytical capacity were not great enough to allow for replicate fermentations on each noneprotein nitrogen compound. However, the donor cow was maintained on a constant diet so that comparisons between compounds are valid. The experimental treatments and structural formulas of non-protein nitrogen sources used are shown in Figure 6. Rumen fermentation liquor was centrifuged at 32,000 x g for l5‘ minutes, the supernatant saved for later analysis, and the precipitate resuspended in about 5 mi. of distilled water. The sample was re- centrifuged, and the supernatant discarded. The resulting pellet was analyzed for total nitrogen by a modification of the Kjeldahl method. This pellet was considered to be composed of microbial material, although certainly, contamination from the substrate was present to some extent. Hourly samples of fermentation liquor supernatants and dialysates were examined for ammonia nitrogen by the Conway (l963) method as modified by abrink (l955) and for total nitrogen by the direct Nesslerization method of Minari and Zilversmit (l963). Alternate hour samples of both fermentation liquor supernatant and dialyzate were examined for volatile fatty acids by gas chromatography. E. Analytical methods l. Ammonia Ammonia was determined by the method of Conway (l963) as modified by abrink (l955). The method depends upon the fact that un-ionized ammonia (NH3) is volatile and acts as a base when in contact with 79 SUBSTRATES EXAMINED IN THE IN VITRO SYSTEM CONTROL - No added nitrogen. O liliEA - llzll - c - Nliz PlililflEO SOY PROTEIN '.' 9 '.' 1-3 lllllilllmlllill - n3c-ll- -ll-cll3 2 '.' 9 BilliiET - llzli-c-ll-C-llilz ‘i '.' '.' 2 BillliEA - Nllzc-ll-ll-c-Nllz ".." cumulus llcl - llilzc-llilz- llcl uuuo GIMllYlllllEA sm- lilllziE-i'l-i-ullszlzl; also, Tllloclllmululli - Qt - 3 ' i‘ Fig. 6.--Structural formulas of substrates examined in the in vitro system. 80 water, while ammonia in its salt form (NH#+) is not volatile. _Samples were preserved with sulfuric acid, thus assuring that all the ammonia was in the salt form. The samples were placed in the sealed Obrink (l955) modified Conway (l963) microdiffusion chamber, and were mixed with an alkali (K2C03) which converted the ammonia to its un-ionized volatile form. -This ammonia was absorbed in a boric acid solution con- taining an indicator. The ammonia, acting as an alkali, changed the indicator to its basic color. Titration was carried out with a micro- buret to return the indicator to its end-point. The amount of titrant required to change the indicator back to its original color was directly proportional to the amount of ammonia in the sample. Because the determination required several steps, one of which was a diffusion of released nitrogen from the sample into boric acid, standards were carried through with each set of samples. This avoided errors due to incompleteness of the reaction. Actually, the transfer of ammonia from the sample to the indicator was almost quantitative since the boric acid was in excess, pulling the reaction to completion. 3. Reagents l) Boric acid indicator. Dissolve 5 gm. boric acid in 700 ml. deionized-water and 200 ml. ethanol. Add l0 ml. of mixed methyl reda- bromcresol green indicator (0.066 gm. methyl red and 0.033 gm. bromcresol green dissolved in 70 ml. ethanol and brought to l00 ml. with deionized water). Adjust the indicator very slightly into its acid (red) range with NaOH and/or HCl. Add 0.25 gm. Tergitol NPx' Union Carbide Chemicals Co., 270 Park Avenue, New York l7, N. Y. 81 bring to one l. with deionized water, and mix. 2) Potassium carbonate, #5%. Bring #50 gm. (or l 1b. jar) of potassium carbonate and 0.25‘gm. Tergitol NPX to one 1. and boil for 15 minutes to drive off ammonia.. 3) Standard acid. Make up approximately 0.05 N hydrochloric or sulfuric acid. This acid does not need to be accurately standardized provided standards are run with each set of samples. #) Standard ammonia solutions. Dry reagent-grade ammonium chloride to a constant weight at about 102° C. Then make up solutions of .3821, .76#2, and 1.l#6# gm. per 1. dry ammonium chloride in 0.2N hydrochloric acid. This makes standards of 100, 200, and 300 mcg. per ml. respectively of ammonia nitrogen. b. Procedure Place about 1 m1. of boric acid indicator solution (just enough to cover the surface) into the center well of an abrink (l955) modified 68 mm Conway microdiffusion unit.‘ Place 1 ml. #91 potassium carbonate solution in one side of the sample chamber and 1.5 ml. of the same solution in the sealing ring. Introduce a measured amount of sample containing 50 to 150 mcg. of ammonia nitrogen on the opposite side of the sample chamber from the potassium carbonate. Seal the lid in the sealing groove and mix the sample with the carbonate. Set aside until ammonia transfer to the indicator is complete (about 1 hour at 39° C). Titrate the indicator to its original eolor using a microtitrator and standard acid. IObrink-modified Conway microdiffusion diSh,'Fisher #8-76#, Fisher Scientific Company, 1#58 N. Lamon Avenue, Chicago,-Illinois. - 82 c. Quantitation Each set of samples should contain one or more blanks to which no ammonia is added and one or more of each standard. After titration of the standards, and correction for any blank titer (generally nil), the mcg. of ammonia nitrogen for each standard is divided by its ‘ irespective corrected titer to yield mcg. of ammonia nitrogen per ml. of titrant. In practice, 5 ml. volumes of samples and standards were used. Since volumes were identical, dividing mcg. of ammonia N per ml. of standard by its respective titer yielded results in terms of mcg. ammonia N per ml. per m1. of titer. Reliability of the system was checked automatically because the factor should be the same for each standard, and any departure indicated incomplete recovery or cone tamination. The mean factor obtained by averaging the factor for all standards was used as a constant by which all sample titers were multiplied. 2. Nitrogen by Semimicro Kjeldahl Analysis This method was a modification of the classic Kjeldahl method. Its only unique features are adaptation to rather small samples, and subsequent savings of time, reagents, and equipment. As described here, it is applicable not only to analysis of rumen microorganisms but also to any biological samples, either liquid or solid in which less than about .6 gm. of dry matter can constitute a representative sample. Using 0.1 N standard acid and a 10 ml. buret, samples cone taining from 1.# to 1# mg. of nitrogen can be analyzed to an accuracy of three significant figures. 83 a. Egulgmgnt Basic equipment for the determination was built by AmincoI and consisted of twelve-place rotary digestion units, 100 ml. digestion flasks with expansion bulbs and ground glass joints, and compatible steam distillation and condensation equipment. The obvious advantage of this sytem over most others is that the digestion flask is applied directly to the distillation apparatus by a ground glass joint, thus doing away with transfer of the digest and consequent inherent errors. Titrations were carried out with a Sargent spectro-electro titrator operated in the spectrophotometric mode at 575 millimicrons. b. Reagents l) Concentrated sulfuric 2313; 2) Sodium sulfate, anhydrous granular. 3) 22222; sulfate: 10% solution in water. #) §2212E hydroxide: 50% solution in water. 5) Boric acid: 2% in water. 6) Bromcresol ngfl: 1% in water. 7) Standard ggigi_ 0.1N hydrochloric acid accurately standardized against primary standard grade tris (hydroxymethyl) aminomethane. .. ' c. Procedure Digest the sample containing l.# to 1# mg. N with 1 gm. anhydrous sodium sulfate, 1 ml. 10% copper sulfate, and 3 to 7 m1. concentrated ISargent Model SE Spectrophotometric-Electrometric Automatic Malmstadt Derivative Titrator, No. S-29700. E. H. Sargent 8 Co., 8560 West Chicago Avenue, Detroit, Michigan , 8# sulfuric acid (depending upon amount of organic matter that must be _ digested). Continue the digestion about 30 minutes after all organic matter is decomposed. Let the samples cool, add 25 ml. water, cool again, and connect the flasks to the distillation apparatus. Add sufe ficient sodium hydroxide to make the sample strongly alkaline and steam distill into 10 ml. 2% boric acid with 2 drops bromcresol green indicator for 7.5 minutes. Titrate to bromcresol green endpoint, and subtract appropriate blank value. d. Calculation mg. N - (m1. HCl) (normality)(l#). 3. Microkjeldahl Quantitated by Direct Nesslerization The necessity of nitrogen determination on a large number of very small samples made the standard semimicrokjeldahl determination impractical. Thus, a procedure was developed which utilized digestion of the sample in a test tube, followed by development of color in the same tube by direct Nesslerization. The procedure was a modification of the method of Minari and Zilversmit (1963). According to Hawk.g£’gl. (195#), color development during Nesslerization depends upon the reaction: NHQOH + 2(KI)2 H912 + 3KOH NHgZI + 7K1 +#H20. The actual color is due to NHgZI (dimercuric ammonium iodide), a colloid, and is consequently sensitive to conditions under which the color is developed. One of the most troublesome aspects of direct Nesslerization is the development of turbidity following color development. This has been attributed to a number of different variables, including high temperature, excess alkali, excess soidum sulfate, and presence of 85 inorganic salts which will react with mercury. These variables have been discussed by Thompson and Morrison (1951). Acetone in very small quantities was found by James _£'_l. (19#1) to produce turbidity. Williams (196#) also notes that, "Extraneous materials such as traces of organic solvents produce turbidity ....” In our laboratory, traces of atmospheric acetone fumes were found to produce not only turbidity, but also a heavy precipitate which completely destroyed the usefulness of the determination. The same effect was found with methyl isobutyl ketone. Addition of only one , drop of liquid acetone following development of the precipitate caused the precipitate to dissolve, and the original color returned. However, in only a few seconds, this color faded out completely. Although development of turbidity due to organic solvent fumes in the atmos- phere has apparently been discussed little in the literature (James _£.gl., l9#1), the presence of these fumes in many laboratories may well be the cause of much unexplained difficulty. The cause of turbidity from organic solvent fumes is open to question but might be in some way associated with surface tension. Several procedures utilize gum arabic or some other protective colloid to prevent turbidity development. In the procedure used here, potassium cyanide was used to prevent turbidity, but had no preventive effect on that caused by solvent fumes. a.~ Reagents 1) Nessler regggnt (Koch and McMeekin, l92#): Mix 30 gm. potassium iodide, 20 ml. water and 22.5 gm. iodine. Add 30 gm. metallic mercury and shake while cooling with tap water until the 86 solution is a pale yellow, or a turbid pale green. Allow the precipitate to settle and decent and save the supernatant. Add one drop of super» natant to 1% starch solution. If no blue color develops (indicating free iodine), make up a solution of l gm. potassium iodide, l ml. water, and l gm. iodine, and add dropwise to the supernatant (with continued testing in starch solution) until free iodine is noted. Mix the supernatant with 975 ml. of IO% sodium hydroxide. Store the solution in the cold in a brown bottle. According to Minari and Zilversmit (1963) color is stabilized, and development of some turbidity prevented by adding .977 gm. potassium cyanide to each i. of Nessler reagent. '2) Digestion mixture. To A gm. potassium sulfate and .5 ml. selenium oxychloride, add 125 ml. water. Then add slowly, with cooling, 125 ml. concentrated sulfuric acid. 3) Standards. (See Ammonia procedure.) Prepare standards containing lOO, 200, 300, and 500 mcg. ammonia nitrogen per ml. by bringing 0.382l, 0.7692, l.lh6h, and l.9|06 gm. pure dried ammonium chloride to l l. with O.lN hydrochloric acid. b. Procedure Transfer a sample containing less than 50 mcg. of nitrogen to a l80 mm. X 25 mm. Pyrex culture tube. Add 0.2 ml. digestion mixture and heat until clear. Allow sulfuric acid to reflux up the side of the tube about 2 to 3 cm. for l5-20 minutes. Let cool and add 3 ml. water. Let cool again and add 3 ml. Nessler's reagent. Allow to stand for 30 minutes and read optical density using an appropriate blank at #20 millimicrons. For maximum accuracy, a reliable 87 spectrophotometer should be used which is capable of reading optical densities of l to 1.5, since 50 mcg. of nitrogen produces optical densities in this range. c. Calculation In practice, standards containing 50, l00, 200, 300, and 500 mcg. of nitrogen per ml. were carried through the entire procedure. In this way, the same volume of samples and all standards could be digested, making possible the use of automatic diluting equipment. Micrograms nitrogen per ml. were divided by respective optical densities for each standard. Thus, a factor was obtained in terms of mcg. N per ml. per optical density unit. Ideally, this factor was the same for all standards, and this indicated a linear relationship between mcg. N and optical density. The average factor obtained from factors for individual standards above was multiplied by sample optical density values to yield mcg. N per ml. h. Volatile fatty acids by gas chromatography Volatile fatty acids were separated and quantitated by gas chromatography. Since acetic, propionic, and butyric acids are already volatile, it was not necessary to prepare methyl esters as in the separation of longer chained fatty acids. Separations were carried out on an FFAPI stationary phase in a 3.l8 mm. i.d. teflon column 2.78 meters long. Parameters of operation were as follows: 'FFAP, Cat. No. 82-1350, Wilkins Instrument and Research, Inc., 2700 Mitchell Drive, Walnut Creek, California 88 Carrier gas: Nitrogen Oven temperature: lh08 C isothermal Detector: Hydrogen flame ionization. Input impedence, output sensitivity, and signal attenuation were adjusted to produce an electrometer output of about 0.75 mv. from a 3 microliter injection of a standard solution containing 80 micromoles per ml. of acetic acid. Contamination of the column with residual acids was prevented by saturating the nitrogen carrier gas with water at 70° C. a. Sample_preparation With this method of volatile fatty acid determination, sample preparation needs to meet only two criteria: (1) The sample must be free of extraneous debris, and (2) the acids must be in the acid, and not the salt form. To meet these requirements, samples were collected in sufficient sulfuric acid to bring the pH to near l, and were centrifuged at 32,000 x g to clear the solution of any sediment. b. Standard solution A standard solution containing 80 micromoles acetic acid, 25 micromoles propionic acid, and 25 micromoles butyric acid per ml., respectively, was prepared as follows: l0.2180 gm. pure barium acetate, 2.90l6 gm. sodium propionate, and 2.7522 gm. sodium butyrate were weighed into a one liter volumetric flask, taken up in 500 ml. water, adjusted to a pH of below 3 with sulfuric acid to change all the acids to their acid form, and brought to one i. with water. This standard was stored under refrigeration in a glass-stoppered bottle. 89 c. Quantitation Injection size for both samples and standard solution was 3 microliters. Previous studies had shown that peak widths for each acid were quite consistent in both samples and standards. This allowed quantitation on the basis of peak height alone, since peak height was then directly proportional to the area under the various curves. Thus, peak heights were determined for the standard solution9 and samples were quantitated by simple proportionality. Since sample size was the same with both standards and unknowns, volatile fatty acids in the unknowns were expressed as micromoles per ml. (the units of the standard solution) without further calculation. F. Statistical treatment Variables examined directly were: l) mcg./ml. precipitate nitrogen 2) mcg./ml. fermentation liquor soluble nitrogen 3) mcg./ml. fermentation liquor ammonia nitrogen h) mcg./ml. dialyzate nitrogen S) mcg./ml. dialyzate ammonia nitrogen 6) micromoles/mi. fermentation liquor acetate, propionate, and butyrate 7) micromoles/ml. dialyzate acetate, propionate, and butyrate. In addition, appropriate values from these variables were used to compute: 90 l) mcg./ml. fermentation liquor non-ammonia nitrOgen 2) mcg./ml. dialyzate non-ammonia nitrogen 3) micromoles/ml. total volatile fatty acid (VFA) in the fermentation liquor h) micromoles/ml. total VFA in the dialyzate 5) Fermentation acetate/propionate ratio 6) Dialyzate acetate/propionate ratio 7) micromoles/ml. difference between the fermentation liquor and dialyzate in acetate, propionate, and butyrate 8) fermentation liquor molar percent acetate, propionate and butyrate 9) dialyzate molar percent acetate, propionate and butyrate. Two-way analysis of variance was carried out by computer. The analysis separated effects of the various NPN compounds, effects of hours after substrate introduction, and interaction between the two. The computer was used to calculate the transformations from the raw data listed above, and to run analysis of variance on all variables, both raw and transformed. Since twouway analysis of variance demands that no data be missing, subclass means were entered in place of missing data Then the number of missing observations was subtracted from the degrees of freedom in the error term, producing a larger mean square for error, which in turn decreased the F statistic for compounds, hours, and interaction. Duncan's (l955) multiple range test was applied to all data, including treatment means, hour means, and subclass means within 9] both hours and compounds. In all cases, this analysis was carried out at a 95 percent confidence level. Tables of subclass means, means for substrates, and means for hours are presented in the appendix along with their respective analysis of variance tables. IV. RESULTS AND DISCUSSION A. Precipitate Nitrogen Results of precipitate nitrogen determinations on 32,000 x g. fermentation liquor precipitates are presented graphically in Figure 7 and in tabular form in Appendix Table l. Analysis of variance showed highly significant differences (P < .Ol) between both substrates and hours. Purified soy protein yielded a higher mean level of precipitate nitrogen than did any other compound (P < .05) and was higher in this parameter at all hours except hours 0, 2, and 9. At all these hours thiocarbanalide yielded statistically similar values, and at hour 2, urea was also similar. The high mean square for error in the analysis of variance (Appendix Table l) attests to the high degree of variability in this measure. Inspection of Figure shows that much of this variability was due to the values for thiocarbanalide and soy protein. Thiocarbanalide was insoluble, and soy protein only partially soluble, and were perhaps not completely dispersed throughout the fermentation liquor. Since these values are the means for five observations, a large amount of the variability from this source has already been removed. Biurea, on the other hand, was also insoluble, but produced average precipitate nitrogen values that were significantly (P < .05) lower than for the control. The author noted that when the two compounds were placed in water, thiocarbanalide floated, while biurea settled out. Thus, biurea may have been heavy enough to 92 93 fermentation liquor Precipitate Nitrogen (meg/ml.) 1'3 Dimethylurea Soy protein mam.” Control — 1200 Biuret II-l-l-l- .‘k u r e a n“““s ‘ ‘\ AK faea_ 1m 8) \ I". \ ’l’l x‘ Q \"s " “ " \ 800 \l g \‘vl/M ‘ § . \n \ 1' .l i! i «2’ ' ' l“...tlloooalu..,"":: — o — I — | C , § 0 - a - |" \ ‘ I," 0 2M uh"llllllllllllllflonu' . 0"“...“II '0', '0 "' 0123456789 Hours Fig. 7a.--Changes in fermentation liquor precipitate nitrogen with time after substrate addition. 9h Fermentation liquor Precipitate Nitrogen (mg. mi.) 1200 / Thiocarbanalide 1000 Guanylurea 804 — i Guanidine IiCI n“... Biurea --—.-.-.. Q : C a ‘ .5 :"s 5 ’s 5 ‘3 : g. : 9 3 ‘ Q E S ’s, s ’o. a Q ' Q ..,, .- 'o. «as: s. 5 ll : : “‘ g h - A‘ ’ ‘ ’ 'll"'. é $4. 400 , ~11» ~ I C. ' t ‘ - 4’ .IIIIIIIl-IIIII "" o‘ . ..'.IIIIII"" 7' 01234 5 0 7 8 9 Hours Fig. 7b.--Changes in fermentation liquor precipitate nitrOgen with time after substrate addition. 95 settle out below the reach of the bubling carbon dioxide, and was thus not included in the samples. In most cases, peak precipitate nitrogen was observed either at hour 2 or 3. This effect was noted with the control substrate, as well as substrates to which nitrogen was added. When hourly values for all compounds were averaged, however, hours A and 7, as well as hours 2 and 3 were significantly (P < .05) higher than hour zero. If no microbial (synthesis were occurring, precipitate nitrOgen should obey the equation Y = voékt , where Yo a concentration of precipitate nitrogen immediately after substrate addition Y = concentration of precipitate nitrogen at time t e - base of natural logarithms ' k a proportion of the original material removed per unit time. If bacteria and protozoa were multiplying in the medium, the precipitate nitrogen should have fallen at a rate slower than that predicted by the equation. Since 10 ml. of fermentation liquor were removed from the 250 ml. system per hour, k in the equation is l0/250 x l00 or 4 percent per hour. If observed mean precipitate nitrogen values for hour 3 through hour 9 are plotted on semimlog paper and the best straight line fitted to these points, the resulting line is essentially flat, indicating production of microbial protoplasm, Precipitate nitrogen values include all microbial cellular protein, but unfortunately, were contaminated with large amounts of feed protein, The difference for the theoretical k (k 8 .Oh) and the observed k (k = .00) is certainly not large. However, microbial synthesis was, 96 in fact, occurring at a rate commensurate with the flow rate of the system, since after 5 days of operation, protozoa were generally still abundant and motile, and end-product production indicated competence of the various metabolic pathways, Several methods for the assay of synthesized microbial protein have been attempted. In a system in which all nutrients were soluble, 32,000 x g. centrifugation and nitrogen estimation should give a good indication of cellular synthesis. However, in the presence of particulate feed protein, the problem is complicated. Wegner _£.gl. (l9h0) and Pearson and Smith (l9h3a, b, c) quantitated nitrogen, precipitated by traditional protein precipitation methods such as trichloroacetate and phosphotungstic acids. Bowie (l962) attempted to assay microbial growth on the basis of changes in the DNAzRNA ratio The traditional protein precipitants suffer from not only contaminating microbial protein with feed protein, but contaminating the fraction with any soluble proteins in the rumen fluid as well. Bowie's (l962) method offers some promise, but again, is not too specific. The accurate quantitation of Synthesized microbial protein in the presence of feed proteins must await further experimentation. B. Fermentation soluble nitrogen fractions Fermentation liquor soluble nitrogen, ammonia nitrogen, and non» ammonia nitr09en levels are shown in Figures 8, 9, and l0, and Appendix Tables 2, 3, and h, respectively. Analysis of variance showed highly significant (P < .Ol) differences between hour means Fermengiation liquor Soluble Nitrogen (meg/ml.) l ' 3 n illlet hYI II re a "um-"mum"- Soy protein !' in, S "‘—. ' Control Biuret . I, . 5:1, I 9‘ """W. = o ‘ A '0. 4‘1”“: ~. ‘97" I " [1 'O'o \\ o " /’/ o'c.‘ .’ s, i 'r‘ '1 ! ‘0‘ .‘ x'flu' 1'! '~,.‘ ‘.\" \‘t‘ .‘ 0“" Hours Fig. 8a.--Changes in fermentation liquor soluble nitrogen with 01234 50739 time after substrate addition. Fermentgation liquor Soluble Nitrogen 900 (mg/ml.) Thiocarbanalide (iuanylurea 804 —— 100 Guanidine llOl n...“ Biurea ---....-.. _: ' \ . ooo \ \ V’ ‘~ '2.“ ‘~, 2 500 w m $."~.‘.'$é$ {t'~ zoo ’W’ """‘~7.:~-—-—'---“" "'o«‘.|““*’l," 012 3 4 5 678 9 Hours Fig. 8b.--Changes in fermentation liquor soluble nitrogen with t Ime after substrate addition. 99 Fermentation liquor Nll3-N (meg/ml.) l ' 3 ll imet llyl u rea so! III’OI e i ll "."/l/I/A Control — 5°" ‘ Biuret --------- i . u I ‘ r e a n“““w I K 4' 1' 4’ 4' E 350 ,1 ~~~ \ I ““\ ‘ ‘ib EN'.' is G: I 1. III/. l" 15. "s. o h , ~ . 'luflluuuu.“u“OI-mun“unannounu. 0 ~ . h . ~ I ~ . l” - ' - . ’ ‘ ’ 0’ Fig. 93.--Changes in fermentation liquor ammonia nitrogen with t”he after substrate addition. 100 Fermentation liquor Nli3-N (meg/ml.) 35° ' Thiocarbanalide 300 Cuanylurea 904 —- 250 Guanidine IlCl -----__ Biurea --—....-.. ‘l‘l‘l ’Ia., "flca!IHluIHIfiflhflkaiflflggflhnfl::::::::z 012 3 45 67 8 9 Fig. 9b.--Changes in fermentation liquor ammonia nitrogen with tlme after substrate addition. IOI Fermentation liquor Non-ammonia Nitrogen (Neg/ml.) l ' 3 n i Ill ethyl II re a "um-mummi- a,“ Soy protein IND g" 5 a Control Biuret ..-.-.-.. ‘°° llrea .-...._. g ~' 3""...9." 3!! ‘3'?" "9., 5 I o "a," 5M :3 ’ I ‘ o .0. ‘ I" s" A. 1'3"" 3‘3"“!4” \. I .3 ‘ 0..."..u. - 3 ’~. \ i m "a. ‘5 ‘I I) “*3 o I 4 s, :IE ‘30", "' ‘k a kid“ 3tv.0‘\ \‘ 0'. 100 12 3 4 5 618 9 llours Fig. l0a.--Changes in fermentation liquor non-ammonia nitrogen with time after substrate addition. l02 Fermentation liquor Non-ammonia Nitrogen (Neg/ml.) 11' i Dcal’llalla I i d0 "Hum-Immu- Cuanylurea $0. — 000 Cuanidine NCI ....... o“~ Biurea Il-I-I-II \. I! 400 ‘~ \. ‘ND 5’ $ 3M “‘.\sss a. 2‘ s 'o. = 'o. .5 'fiuuuouullfl'~o,' g: g - I -’ ’. 0‘ 'o'l' ‘9 2” s’ ‘ ‘ 'Nu-fml—‘-'-o" 0" luau"... ""lulunu 100 01234 5618 9 Hours Fig. lOb.--Changes in fermentation liquor non-ammonia nitrogen with time after substrate addition. l03 and means for the various substrates in soluble nitrogen, ammonia nitrogen, and nitrogen not accounted for as ammonia., In addition, . highly significant (P < .Ol) interactions were found between hours and compounds in soluble nitrogen and non-ammonia nitrogen. The average soluble nitrogen was higher on i, 3-diemehylurea than any other compound studied (P < .05). The control substrate (to which_ no nitrogen was added) and thiocarbanalide yielded statistically similar soluble nitrogen levels. Biurea, although apparently insoluble in water, produced a soluble nitrogen level higher than the controlw (P < .05). Biuret, guanidine hydrochloride, and guanylurea sulfate yielded statistically similar results, all higher (P < .05) than the control. Urea yielded soluble nitrogen levels statistically similar (P < .05) to biuret, and guanylurea sulfate. Figure shows that soluble nitrogen values are divided into two groups; those from the control substrate and compounds which were insoluble, and those compounds which were soluble under the conditions of the fermentation. Guanylurea sulfate is interesting in this aspect because its solubility in water appeared quite low, although it did dissolve in an acid pH. Since it produced a fairly high soluble nitrOQen level, it was evidently soluble under the conditions imposed here With the insoluble compounds (biurea and thiocarbanalide) soluble nitrogen levels were not statistically different during any part of the fermentation period. However, soluble nitrogen for the control substrate tended to decrease‘from hour l to hour 9, while biurea remained essentially the same. 10h Peak soluble nitrogen concentrations were reached either 2 or . 3 hours following substrate addition. A similar pattern occurred with precipitate nitrogen. The fact that peak levels did not occur at hour I is probably a result of instrumental dead space. Significant dif- ferences between hours on each substrate are noted in Appendix Table 2. Fermentation liquor ammonia can be used as an indication of whether or not a compound is hydrolized by rumen microorganisms. These values are presented in Appendix Table 3 and Figure 9. As expected, urea yielded the highest average ammonia levels of any compound studied (P < .05). The next highest level was produced by purified soy protein (P < .05). Guanidine hydrochloride and thiocarbanalide gave rise to similar mean ammonia values that were both lower (P < .05) than the ammonia released by the control substrate. Biuret, biurea, and guanylurea sulfate substrates produced statistically similar mean ammonia levels, and ammonia levels for biuret and biurea were higher (P < .05) than for the control substrate. It would appear from Figure 9 that urea was the only compound which released high amounts of ammonia. However, close inspection of the ammonia curve for the control substrate shows that it apparently de» creased from the time of substrate addition to about four hours and then started to increase. The only significant (P‘< .05) difference in control ammonia levels was between hour 4 and hour 9. Soy protein, l.3-dimethylurea, biurea, guanidine hydrochloride, and guanylurea sulfate showed no significant differences in ammonia levels with time after substrate addition, indicating that amnonia levels were 105 essentially constant during each fermentation period. Ammonia nitrogen from biuret tended to first increase, then reach a low point at 7 hours and start to increase again. If the fermentation were being carried out in a closed chamber and no nitrogen were being converted to ammonia, the ammonia nitrogen level should fall according to the equation Y 8 Yea. t, which was dis- cussed in section A. Since the system used in this study contained a. dialyzing chamber of l,l00 ml., the calculations would be considerably more complex. A concentration gradient existed between the fermentation chamber and dialyzing medium which removed ammonia from the fermentation medium. Since the magnitude of this gradient changed with time after substrate addition, accurate calculation of the amount of ammonia removed by this route was almostimpossible. However, it can be seen that the rate of ammonia removal from the fermentation liquor should have been A percent per hour (l0 ml. removed per hour from a 250 ml. fermentation chamber) plus the rate at which ammonia was transferred by dialysis. From these considerations, if nitrogen-containing materials were not being hydrolyzed, ammonia should have been washed out of the system rather rapidly. The fact that, in general, there were few significant decreases in ammonia levels following substrate addition indicates that some nitrogen-containing substance might have been converted to ammonia, The source of the ammonia was probably feed nitrogen. In any case, the maintenance of a steady nitrogen level during fermentation attests to the metabolic competence of the rumen microorganisms and the ability of the in xltgg system used here to pro- vide the microorganisms with the necessary environment to maintain this competence . l06 Anabolic reactions concerning the incorporation of ammonia into microbial protoplasm have not been considered in the above discussion. Removal of ammonia from the fermentation system by this route would be an additional depressing factor on ammonia levels. The fact that) metabolic activity, as measured by end product production, remained essentially normal, shows that microbial synthesis was taking place at least at a rate commensurate with the flow rate of the system.' Various anabolic and catabolic reactions concerning ammonia and amino acids are treated in some detail in the review of literature. All ammonia nitrogen values were subtracted from their respective soluble nitrogen values. These values were classified as non-ammonia nitrogen, and are found in Appendix Table h and Figure l0. This nitrOgen fraction should include all nitrogen contained in compounds which were not hydrolyzed to ammonia in the rumen. Figure l0 shows that these curves fell into a fairly well defined pattern. The low group in- cluded the control, urea, biurea, and thiocarbanalide substrates. The high non-ammonia nitrOgen group included i, 3-dimethylurea, soy protein, biuret, guanidine hydrochloride, and guanylurea sulfate. Biurea and thiocarbanalide were insoluble in water, explaining their presence in the low group. Urea was rapidly hydrolyzed to ammonia, and the control substrate had no added nitrogen. The high non-ammonia nitrogen group included the remaining compounds, which apparently were either not converted to ammonia or were converted slowly. When the means of the non-ammonia nitrogen levels for each substrate were compared (Appendix Table 9), only the mean for biurea was found to be statistically l07 similar to the control mean. Means for thiocarbanalide and urea were higher (P < .05). In the high group, means for soy protein, biuret and guanylurea sulfate were not significantly different, and means for guanidine hydrochloride and guanylurea sulfate were similar. The mean for l, 3-dimethylurea was significantly (P < .05) higher than the mean for any of the other substrates. Non-ammonia nitrogen levels for the urea substrate consistently remained above the control value. This would indicate incomplete hydrolysis of urea to ammonia. Unfortunately, direct urea determinations were not carried out to clarify this observation. However, the rapid increase in ammonia nitrogen indicates that urease was quite active. Since peak ammonia levels and peak soluble nitrogen levels on the urea substrate both occurred simultaneously at the second hour following substrate addition, hydrolysis of urea was almost instantaneous; a situation seen in intact animals. Unfortunately, this shows only that urea hydrolysis occurred early, and does not indicate the completeness of that hydrolysis. C. Dialyzate nitrogen fractions Values for dialyzate total nitrogen, ammonia nitrogen, and non- ammonia nitrogen are presented, along with appropriate analysis of variance tables, in Appendix Tables 5, 6, and 7. In all three parameters there were highly significant (Pr< .Ol) differences between substrates, but in no case were there significant differences between hours or significant interactions. In this case, data is not presented in graph form since there were no changes in any of the three parameters with time after substrate addition. Means for each substrate are presented in Table 6. l08 TABLE 6. Mean dialyzate total nitrOgen, ammonia nitrogen, and non- ammonia nitrogen levels with various nitrogen substrates. mcg./ml. mcg./ml. mcg./ml. Substrate total N ammonia N non-ammonia N Control 1213* 973 24° Urea 307c 250d S7b Soy protein l95b '53c 37a l,3-Dimethylurea 35ld l32b 219d Biuret 346d H7b 229d Biurea l83b l2lb 62b Guanidine HCl 344d 883 256e Guanylurea SO“ 293c thb l69c Thiocarbanalide 109a 88a 21a *Values within columns with like superscripts are not significantly ‘different (P < .05). '09 Composition of the buffered dialyzing solution was shown in Table 3. The solution contained 322 mcg. urea or ISO mcg. nitrogen per ml. Thus, any difference in total nitrogen from lSO mcg./ml. represents either diffusion from the fermentation liquor to the dialyzate or the reverse. Examination of total nitrogen values in Table 6 indicates that on the control and thiocarbanalide substrates, nitrogen was diffusing from the dialyzing solution back into the fermentation liquor. There was no significant difference between total soluble nitrogenvalues in dialyzing fluid for these two substrates. Comparison of the control nitrogen level with that for biurea indicates that although biurea was apprently in- soluble, diffusion of nitrogen out of the fermentation liquor was occurring. The difference between the control total nitrogen level and that for biurea was significant (P < .05). Total dialyzate nitrogen on the soy protein substrate was higher (P < .05) than for the control, but was lower than the soluble nitrogen level for this substrate in the fermentation liquor. This indicates that the soluble nitrogen was largely in the form of water soluble proteins which remained inside the dialysis sac. Guanylurea sulfate produced dialyzate nitrogen levels significantly (Pg< .05) lower than guanidine hydrochloride. Since the two compounds gave rise to very similar soluble nitrogen levels in the fermentation liquor, this difference is difficult to explain. Biuret, l, 3- idimethylurea, and guanidine hydrochloride all gave use to statistically similar total nitrogen levels. llO Ammonia nitrogen levels for control, guanidine hydrochloride, and thiocarbanalide substrates were all statistically similar, and lower than would be expected on the basis of the urea present in the buffer solution. Early in each S-day experiment, ammonia levels in the dialyzing solution were low and the non-ammonia nitrogen fraction was sufficient to account for most of the urea present in the buffer. However, as the fermentation progressed, the dialyzing solution invari- ably became contaminated with bacteria, which hydrolyzed the urea by way of urease. Thus, the mean dialyzate ammonia levels for substrates which did not release ammonia were lower than would be predicted on the basis of urea present in the dialyzing solution. Inspection of the ammonia and non-ammonia nitrogen values indicates that while biuret and l, 3-dimethylurea may have been hydrolyzed to some extent, hydrolysis of guanidine hydrochloride appears doubtful. The dialyzate non-ammonia nitrogen level from guanylurea sulfate was lower (P (.05) than for guanidine hydrochloride. Ammonia levels indicate that guanidine hydrochloride may have been partially hydrolyzed. However, the reason for the low rate of transfer of this compound across the dialyzing membrane is open to question. If the compound were solublized, it should have transferred across the membrane. Soluble nitrogen values in the fermentation liquor indicate that it was at least as soluble as guanidine hydrochloride. The lack of change in the dialyzate nitrogenfractions with time after substrate addition indicates that transfer of these compounds from the fermentation liquor to the dialyzate was about equal to the rate of transfer of the solution from the dialyzing chamber to the lll collection system. If the values on Table 6 are multiplied by the volume of liquid flow into and out of the dialyzing system in each l2 hour period (2ho ml.), an indication of the amount of each fraction transferred from the system in the dialyzate can be calculated. These valuesgare shown in Table 7. If the mg. of non-ammonia nitrogen for the control substrate is subtracted from the remaining values, it appears that l, 3-dimethylurea, biuret, guanidine hydrochloride, and guanylurea sulfate are transferred into the dialyzing medium in an amount not too different from half of the lOO mg. nitrogen originally added. From examination of values for the various nitrogen fractions in both fermentation liquor and dialyzate, it appears that urea was the only non-protein nitrogen (NPN) source which was hydrolyzed to ammonia to an amount approaching completion. Biuret, biurea, l, 3-dlmethylurea, and guanylurea sulfate all yielded fermentation liquor nitrogen levels higher (P < .05) than the control substrate. Dialyzate ammonia nitrogen was higher (P < .05) than the control for the same substrates. Interpretation of these results is very difficult because rumen ammonia is a highly dinamlc entity. Although flow rate of material into and out of the fermentation bag and dialyzing chamber were known, the amount of ammonia being converted to bacterial protein is unknown. When the rate of ammonia removal from the fermentation system is known, or at least held constant, then ammonia metabolism can be represented by the formula. ll2 TABLE 7. Mean weight in mg. of total nitrogen, ammonia nitrogen, and non-ammonia nitrogen transferred out of the in vitro system in the dialyzate per 12 hour period. mg. total mg. ammonia mg. non Substrate nitrggen nitrogen ammonia nitroggg_ Control 29.0 23.3 5.7 ‘ Urea 73.7 60.0 l3.7 Soy protein h6.8 37.9 8.9 l,3-Dimethylurea 8h.2 3l.7 52.5 Biuret 83.0 28.l 54.9 Biurea “3.9 29.0 lh.9 Guanidine HCl 82.6 Zl.l 6|.5 Guanylurea 50“ 70.3 29.8 #0.5 Thiocarbanalide 26.2 21.1 5.1 ll3 Substrate nitrogen NH microbial protein. Here, substrate nitrogen includes both feed nitrogen and nitrogen from NPN sources. The problem is further complicated by the fact that microbial protein can be degraded to ammonia as well as synthesized from ammonia. Non-ammonia nitrogen serves as an indication of the amount of the nitrogen-containing substrate remaining unmetabolized. 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