STUDISS ON THE AMINO ACID COMFOSITION OF A MICROBIAL FRACTION CF BOVINE RUMEN INGLSTA by Nancy-Lee Stobbs nN ABSTRACT Submitted to the School of Graduate Studies of Michigan State University of Agriculture and Applied Science in partial fulfillment of the requirements for the degree of MASTER OF SCIENCE Department of Microbiology Year 1955 (I \ Approved CTE\YO%CRj%jkfYY\KX/VVOOJ v-‘CJ-Im Innmzz: Nancy-Lee Stobbs The use of urea as a source of non-protein nitrogen in ruminant nutrition has received concentrated attention dur- ing the last decade. The question concerning the value of such a compound has been approached in various ways, and is found to be a complex problem. The investigation here re- corded is concerned primarily with the amino acid compo- sition of the microbial protein material synthesized from a ration supplemented with urea as against a ration supple- mented with a natural protein nitrogen source. Microbiological assay determinations for the ten es- sential amino acids were carried out on the liquid ingesta and the isolated protein sediment. Synthesis was_accomplished using the in yitgg technique. Since the data obtained indicated no sizable increase in the amount of amino acids as a result of synthesis, com- parisons between the two supplements were made on the basis of an amino acid ratio. The author found the quality of the microbial fraction to be of a relatively constant nature. When these ratios were compared with the ratios of some purified proteins and natural feedstuffs, flue quality appeared to be fair. Although urea failed to show any superiority over the natural protein along the line of synthesizing ca- pacity, it promoted synthesis equally as well. The data observed led to the conclusion that the mi- crobial fraction of rumen ingesta is of a constant compo- Nancy—Lee ptotbs sition and provides an adequate amount of building materials to the ruminant. It was also concluded that urea affords a good source of nitrogen for the flora of the rumen, with which they are capable of synthesizing a portion of the protein necessary for the general health and welfare of the animal. The use of urea and such related compounds as nitro- gen sources can prove an be very beneficial and economical to the farmers of today and those of the future. STUDIES ON THE AMINO ACID COMFOSITION OF A MICROBIAL FRACTION OF BOVINE RUMnN INGESTA by Nancy-Lee Stobbs A THESIS Submitted to the School of Graduate Studies of Michigan State University of Agriculture and Applied Science in partial fulfillment of the requirements for the degree of MASTER OF SCIENCE Department of Microbiology 1955 ACKNOWLEDGMENTS The author wishes to eXpress her sincere appreciation to Dr. W. L. Mallmann, under whose constant SUpervision and unfailing interest this investigation was undertaken. Grateful acknowledgment is also extended to Dr. R. W. Luecke of the Department of Agricultural Chemistry for his valuable guidance and advice throughout this course of study. Thanks are also due to Dr. R. L. Salsbury of the De- partment of Agricultural Chemistry for his kind assistance and sincere interest in this problem, particularly in respect to the laboratory procedure. Appreciation is also extended to all instructors who made this course of graduate study so enjoyable and in- formative. TABLE OF CONTENTS Pag Purpose of Study..................................l Introduction....... ..... ..........................3 Revfiaw of Literature...... ....... ... ........ ......5 Experimental Procedures.................. Presentation of Data.............................3L Analysis of Data.................. ..... ..........hO Discussion.......................................h3 Summary....................................., ...46 Appendix................. o.0.OOOOOOOOOOOOOOOOOOOOhB BibliograthOOOOOOO0.00.00.00.00.0000000000000000SO PURPOSE OF STUDY The review of literature which is to follow indicates the great scepe of the problem of ruminant nutrition as re- gards the use of N.P.N. sources as supplements to dietary ni- trogen. The greatest quantity of eXperimental work has been devoted to the study of urea as a partial replacement for ration protein nitrogen. The main interest along this line has concerned an estimation of the amount of microbial pro- tein synthesis occurring and the actual value of urea supple- mentation as concerns growth, milk production, etc. Few in- vestigations have been made regarding amino acid utilization by the microorganisms involved and the amino acid composition of the mixed rumen contents. One investigator isolated the synthesized protein as a sediment. However, little work has been conducted on the amino acid composition of this synthe- sized protein from the standpoint of the relative increase of the essential amino acids and the comparison of the pro- tein formed from an N.P.N. source and that synthesized from a natural ration. Therefore, the problem concerned in the following experimental work involved an in zitgg study of protein synthesis from a soybean-oil meal ration and a urea- supplemented ration with subsequent amino acid analysis of the protein formed. Analysis was made on both the liquid and the dried sediment prior to and following the incubation -1- period and a comparison made between the two methods. In the first series of trials the liquor was supplemented with urea, a readily available carbohydrate, and yeast extract. In the second series, conducted on the isolated sediment, the additives were urea and glucose with cellulose serving as the substrate. The author shall attempt to describe the composition of the microbial protein along with an estimation of the increase (or decrease) of each essential amino acid and subsequently estimate the value of the protein synthesized from a urea- supplemented ration as compared with that formed from a natural ration. A comparison will also be made with some feedstuffs, bacterial cells, and purified proteins to demon- strate the relative nutritive value of the synthesized protein. INTRODUCTION The complex study of living organisms revolves about many types of relationships which generally involve what is termed the host and the parasite. These relationships may be beneficial or harmful, depending upon the prevailing con- ditions. The relationship in which both the host and para- site derive benefit is termed symbiosis, and each unit con- cerned is necessary for the natural continued existence of the associated unit. The symbiotic association of primary rank in.the rela- tive importance to man would undoubtedly be daat existing between the ruminant and.the associated microflora and micro- fauna of its alimentary tract. The ruminant is a polygastric mammal, i.e. the digestive tract is composed of four stomachs, each with its particular digestive process. The first of these stomachs is a large sack, found at the lower part of the esophagus,and is termed the rumen. Large quantities of ingesta may be retained in this sacculation for various peri- ods of time. In the case of the bovine, food may be held for some 2h hours. The purpose of food retention is to facili- tate pre-digestion of bulk ingesta so that it can ultimately be absorbed from the alimentary tract and utilized by the animal. Any remaining bulk material is returned to the mouth for further mastication and die process repeated. Hence, -3- it can be seentflmn;the ruminant is able to utilize certain types of energy-containing compounds to a much greater degree than the non-ruminating herbivore. The microorganisms contained in the rumen are the primary agents in this pre-digestian process and it is of interest to examine briefly the conditiais which prevail and contribute to thereontinual proliferation of these microorganisms and, also, to show a general View of tn; Chemis- try involved in rumen digestion. The temperature in the rumen is approximately 39°C and the normal reaction is just below pH 7.0. Since a large prOportion of the ingesta is carbohydrate material the acids produced by bacterial action predominate over the alkaline substances formed during fermentation and the reaction tends to become extremely acid in nature. However, this acid condition is neutralized dur- ing fermentation by the saliva.which has a reactionmapproximate- 1y pH 8.2. The environment is controlled throughout by the con- stant mixing action caused by the muscular action of the rumen walls and also the passage of gas, formed during fermentation, to the upper part of the rumen. Hence, no stagnation is allowed to occur in any part of the contents. All these factors aid the maintenance and proliferation of a particular type of microbial population. The bacteria and protozoa here encountered are re- presentative of a population the density of which is never met ,ig‘xitgg and probably not elsewhere in nature. Such a popula- tion arises from the ingesta. When filled, the rumen contents constitute about one-fifth of the total body weight. As an example, with an animal weigh- ing 1,000 pounds the rumen holds about 25 gallons. REVIEW OF LITERATURE In a series of studies on the chemistry of rumen di- gestion during a 12-hour period Hale (19) found that dry matter in general was digested at an even rate throughout. The predominant action wiring the first 6 hours was the rapid disappearance of more soluble nutrients, viz. proteins and carbohydrates, from the rumen. Cellulose was slightly dis- integrated while lignin and crude fiber remained untouched. The second 6 hours exhibited rapid digestion of cellulose which was paralleled by digestion of both carbohydrate and protein. However, the fact that lignin digestion did not go beyond a few percent was evidence fliat the chemical compo- sition of the plant ingesta imposes certain limitations upon the ruminant microbial activity. The variable digestion of lignin in the caecum, after its passage from the rumen, ex- poses cellulose and protein to further disintegration, proba- bly by the iodophile microflora. Herein lies the major function of this organ in the ruminant. It is not as yet clear whether or not the fatty acids observed in the rumen are intermediate or end-products of digestion. Hale (18) also concluded that during due course of a 12—hour digestion period between feedings passage of nutrients from the rumen is affected by (l) the passage of the concentrated rumen contents which remain from previous digestion periods, and -5- f (2) the separation of nutrients from recent plant ingesta with subsequent washing from the rumen. Therefore, it is seen that many nutrients pass from the rumen in both the digestible and indigestible form. Such studies are suggestive of the fact that maximum limits of rumen digestion are normally reached within the 12-hour digestion period. The reaction in the ru- men reach es a maximum acidity approximately 6 hours after feeding, after which time the values increase gradually to the preceding level of near neutrality. It is interesting to note here that Monroe and Perkins (32) reported higher acid in ingesta from cattle on pasture than when roughages, corn and silage, were fed. The claim that non-protein nitrogenous substances could serve as a replacement for a portion of the dietary protein required for maintenance of normal health and growth has frequently been considered during the last half-century. How- ever, this subject did not receive any attention until re- cently when there arose the possibility of feed ration short- age during the war, and until non-protein nitrogenous com- pounds such as urea were manufactured in great quantities from atmOSpheric nitrogen. The belief is that the rumen mi- cPoorganisms are capable of utilizing the N.P.N. source of nitrogen in the synthesis of their cellular protein which is ultimately utilized by the animal when digestion is completed in the glandular stomach. In other words, the ruminant may be said to live upon the mass of microorganisms formed from the ingested material rather than on the material per se. It may thus be seen dust the substitution of some N.P.N. com- pound may be a more economical supply of nitrogen for the ruminant, provided the microbial utilization and conversion is efficient both qualitatively and quantitatively. The question of the value of urea as a N.P.N. source for ruminants has received perhaps the most serious con- sideration during the latter years of study on this problem. It has been observed that rumen microorganisms can utilize this source of nitrogen provided a readily available carbo- hydrate is present as an energy source. Urea is believed to be hydrolyzed to ammonia by the inherent microbial enzyme urease,and theeammonia nitrogen in turn utilized by the organ- isms in the synthesis of their cellular protein. The value of substituting urea for a portion of dietary protein is sub- stantiated by the literature. However, when urea is em- ployed as the sole source of dietary nitrogen, the efficiency is known to be markedly reduced. Agrawala 23 al. (1) found that calves could not survive on such a purified ration be- cause the organisms could not convert the large quantity of ammonia to protein-nitrogen with sufficient rapidity to re- duce the toxic effect of ammonia upon the microbial pepu- lation. Therefore, while urea is suitable as a feed in- gredient because of its economy, lack of odor, high nitro- gen content (h6.6% N}, and biological availability, the amount which can be substituted is limited to a certain de- gree by the rapidity of its conversion to ammonia in the rumen. It is interesting to note the fact that lambs appear to possess greater efficiency in utilizing urea-nitrogen than do steers. The first type of study carried out in an effort to ex- amine the question of utilization of N.P.N. compounds, with emphasis upon urea, was feed experiments involving sheep and steers. Hart gt 31. (23) considered that the best type of experiment for such a problem was one involving long term growth, milk production, wool production, etc. They ran four calves, one receiving a basal ration of 6 percent protein, one receiving an addition of urea, the third receiving an ad- dition of ammonium bicarbonate ami the fourth receiving an addition of casein. Each supplement was of such an amount that the total protein was brought up to 18 percent. The study ran for forty weeks. While the animal receiving casein showed more growth, the rate was at certain times approached by the animal receiving urea and bicarbonate. The increment of weight was constant in composition. They concluded with reasonable certainty that the growth record so obtained was proof of the whole or partial utilization of ammonia and urea for synthesis of protein via the intervention of rumen microbes. They found definitely that urea and sodium bi- carbonate nitrogen can be utilized by growing heifers when grains or timothy hay supply part of the protein. It was also apparent that bacterial multiplication could be enhanced by the ingestion of some soluble carbohydrate. Wegner (35) explained the ability of dairy calves to utilize the inor- ganic nitrogen from urea and bicarbonate for part of the ration protein through the production of protein from this nitrogen by growth of bacteria in.the rumen and subsequent digestion of these cells in the fourth stomach and intestines. Smith and Baker (3h) conducted an experimental study on milk production and found that while no significant decrease occurred when urea replaced the nitrogen equivalent of blood meal, the yield immediately fell when urea was removed. Simi- lar studies were run with meat and wool production. As a re- sult of these feeding trials, it was concluded that under favorable conditions urea can be utilized by ruminants for meat, wool and milk production, provided the proportion of nitrogen supplied as urea did not exceed L0 percent. The workers also theorized that urea may prove particularly valuable in areas where grain is especially rich in carbo- hydrate and poor in protein, since an adequate amount of readily available carbohydrate must be present to balance all nitrogen in the diet. Hart g§,§l. (23) likewise concluded that maximum growth response to urea occurred when the sup- ply of urea-nitrogen was no more than.40 percent of the total dietary nitrogen. Agrawala gt 31. (1) conducted studies on the use of urea as sole source of dietary nitrogen. However, as mentioned previously, the conversion of urea to ammonia was too rapid -10- and became toxic for the microbial population. The purified ration consisted of cornstarch, glucose, cellophane, lard, mineral mix and urea. The nitrogen equivalent (N x 6.25) was 12 percent. CellOphane was found to be an unsuitable sub- stitute for natural roughage because cellulose derivatives are more resistant to microbiological attack and degradation than is the cellulose moleoile. In addition, since ruminants are somewhat specific in their natural protein requirements (amino acids) a ration low in this form of protein could lead to the loss of specific ruminal species of microorganisms. Although approximately 90 percent of the N.P.N. disappeared within 6 hours, the indication was that only a small portion of the urea-nitrogen was utilized for synthesis. This was due to the presence of more elementary nitrogen in the puri- fied ratflan. However, the amount of true protein did in- crease when the purified ration was fed (33-109 gms.). In this vein, Pearson and Smith, in l9h2, calculated from 33 11232 synthesis that in a 75 kg. rumen #50 gms. of protein could be synthesized in 2a hours. Johnson and co-workers (27) found that upon addition of urea in amounts to produce the equivalent of 12 percent crude protein in a basal ration the result was a nitrogen-retention that was not enhanced by further urea addition, although in- creased effect was encountered by raising the true protein content of the ration. Wegner gt 31. (36) found that urea, added to a basal -11- ration with 1 percent dry matter, completely disappeared within one hour after feeding, having been hydrolyzed to ammonia-nitrogen and/or converted to protein. Although the percent of ammonia-nitrogen in the supplemented basal ration was initially high due to hydrolysis of the added urea, it decreased in h to 6 hours to a level approximating that in the basal ration, and in the same time. Protein synthesis was offered as the cause for this disappearance of ammonia— nitrogen. Growth of rumen microorganisms utilizing the added urea was also evidence of an increased protein content. These workers concluded that urea-nitrogen utilization must occur within h-6 hours after feeding as urea-nitrogen and ammonia- nitrogen are found to be negligible after that time. Duncan 23 al. (1h) showed the mixed proteins synthesized from urea to be similar'to those found in the rumen of a calf receiving natural ration. Harris and Mitchell (22) demonstrated that.the addition of urea to a low-nitrogen ration enhanced cellulose digestion and was itself digested as much as 88.8 percent. Their stud- ies showed that sheep fed on rations containing urea and minimal amounts of protein providing only 10 percent of the nitrogen required for equilibrium could be maintained in body and nitrogen equilibrium for more than 100 days. The bio- logical value of urea-nitrogen at N-equilibrium is equal to 62 percent (Casein N 2-7952) . In an attempt to demonstrate more conclusive positive -12- evidence of microbial protein synthesis, Wegner (36) supple- mented a low-protein ration of silage and starch (N x 6.25='A%) with urea equivalent to 5 percent dry matter. The protein level from the supplemented ration was found to be about 20 percent greater when determined several hours after feeding. Since tests showed the filtrate nitrogen level always to re— turn to the Same low level as in the basal ration, this in- creased total nitrogen was due to protein formation. Hart 33 3;. concluded that it is entirely possible to improve low- protein rations of poor biological value through the use of molasses and/or urea, and that the microbial protein subse- quently formed may be of greater value as a ration supplement than some of the protein concentrates in use at the present time. I It is now a well established practice both in America Iand abroad to employ urea as a partial protein source in a properly balanced ration for ruminants (7). McDonald re- ported,in 195h, that about to percent of the zein used in his studies, contributing about 94 percent of the total dietary nitrogen, was utilized for synthesis of rumen microbial pro- tein. Loosli, in 19h9, maintained growth in lambs when urea supplied essentially all the nitrogen, and also demonstrated synthesis of the ten essential amino acids. Hamilton gt 3;. (20) found urea to be satisfactory as a nitrogen source for .lambs with the provision.that at least 25 percent of the .feed-nitrogen be in the form of preformed protein and, fur- thery that the total protein equivalent be under or equal to -13- 12 percent. In comparing utilization of urea and soybean oil meal nitrogen, Harris and co-workers (21) found the biological value of urea-nitrogen to be 34 while that of soybean oil meal nitrogen was 60 when fed at 12 and 1h percent protein equiva- lent levels. However, a greater amount of true protein was detected in the rumen of steers receiving urea than in those steers receiving only the low protein ration. The poor urea utilization was attributed to the feeding of a level exceed- ing that of maximum conversion to true protein by the micro- organisms. It has been noted that Smith (33) reported utili- zation provided the proportion of nitrogen supplied as urea did not exceed 40 percent of the total nitrogen. In con- nection with this, Belasco (7) assumed it probable that at a protein equivalent level of #3 percent the hydrolysis of urea to ammonia and carbon dioxide exceeded the rate of am- monia utilization by the organisms resulting in decreased synthesis. As a result of research conducted in 1949, McDon- ald was lead to believe that ammonia, escaping fixation in the rumen, is absorbed into the venous circulation by which it is tranSported to the liver and subsequently converted to urea. A large portion of this urea is then returned to the rumen as a normal saliva constituent. In an effort to obtain a more rapid and convenient method for the study of urea utilization and synthesis, the lg vitro technique was adopted. Essentially this method in- .14- volves the removal of rumen contents from a rumen-fistulated steer. Gross material is removed and the resulting liquor is supplemented with the desired substance to be tested. Us- ing conditions which approximate those occurring naturally in the rumen, i.e. 39°C, anaerobic atmosphere, and agitation, the samples are incubated for a period of time and the final sample tested as required. Pearson and Smith, in l9h3, were some of the first workers to conduct investigations on urea utilization by means of this technique. They incubated the more liquid portion of the rumen contents for 6 hours with urea and a suitable carbohydrate energy source under a car- bon dioxide gas phase in an effort to determine whether one could detect the synthesis of protein from urea. Results of a typical experiment may have been as follows (33): (l) the total nitrogen remained constant (2) urea was rapidly converted to ammonia with a subsequent decrease in N.P.N. which oc— curred mainly, if not entire- ly, in the ammonia fraction the value obtained by subtracting die N.P.N. values from those for total nitrogen indicated that the protein synthe- sis appeared to occur most rapidly during the first 3 hours of incubation. Baker, in 1943, showed that during this three- hour period the microbiological conditions in the rumen liquid ‘were very comparable to those of the initial sample as it came from the rumen. However, fliese conditions showed a marked difference after six hours due to the autolytic effect pro— .15. duced. Wegner (35) demonstrated a negligible urea level oc- curring after 2h hours incubation. An increase in ammonia- nitrogen comparable to the decrease in urea-nitrogen seemed to indicate an initial hydrolysis of urea to ammonia which was followed by the disappearance of the ammonia-nitrogen. Belasco (8), in studying the effect of adding increasing amounts of urea to this artificial rumen, found that a steady increase in free ammonia resulted with increasing urea con- centration. Metabolism of both urea and cellulose increased with increasing levels up to and inclifling the 35 percent protein equivalent level. However, a sharp decrease bean in utilization and digestion was noted.at the A5 percent protein equivalent level. Burroughs 23 31., in 1951, noted an increased cellulose digestion.and urea utilization upon the addition of urea to purified protein and protein.meals (7). In an 13 33359 study involving the comparison of urea and protein meals (soybean, linseed, cotton seed, and corn gluten) at comparable nitrogen Llevels, Belasco (7) demonstrated tme superiority of urea as a nitrogen source in promoting cellulytic digestion. In 1:1 urea-protein meal mixtures thepercent of urea utilization was consistently higher than in mixtures employing urea alone at a similar total nitrogen level , although the rates of cellu- lose digestion were similar to those obtained with only urea. Belasco (8) explained this superiority of urea by the fact that urea, being hydrolyzed by rumen bacterial urease, is a -16- form of readily available nitrogenwwhile the nitrogen from the protein fraction of a meal, which represents‘a complex polypeptide, is essentially unavailable until cleavage and/or deamination occurs. The increased cellulytic response demon- strated widu urea indicates the importance of the availability of ammonia-nitrogen frmn either protein or non-protein sources in the efficient digestion of roughage. Huffman (25) re- ported that nitrate can apparently be advantageously and safely substituted for urea, provflied sufficient fermentable sugar is given simultaneously. Hart (23) stated the theory of urea utilization as follows: "The bacteria in the rumen find the medium of simple nitrogenous salts and sugar an excellent one in which to grow. Through dieir multiplica- tion they build proteins which would contain the amino acids necessary for supplements to the proteins of the ration. These bacterial cells pass from the rumen to the fourth stomach where they are digested and become just so much protein for the animal." Johnson gt 31. (28) found that all fmod nitrogen, up to the maximum amount of nitrogen capable of bacterial utilization, would exhibit a biological value characteristic of the mixed microorganisms reaching the glandular stomach. The value appears to be about 60. The consumption of any nitrogen above the required amount should then possess a biological value comparable to that of a non—ruminant of similar require- ments. .17- The question of ruminant nutrition may be regarded as a question of the balancing of nutrients required by the mi- crobes harbored in the digestive tract. The rumen is well suited to the maintenance of a large, prolific population, which is capable of digesting plant uanstituents and which can further synthesize many nutrient compounds for the host. The microorganisms have readily available access to the nutri- ents consumed by the host due to their'location near'the anterior portion of the digestive tract. Further, certain nutrients are transported from the animal body to the omasum by the continuous flow of saliva. This flow also aids in the maintenance of a high water level in die rumen, promoting fermentation. The main contributors to synthesis are the micro-iodo- philes. Macro-iodophiles contribute only very little to the total protein synthesis. Factors such as the volume of the organ, period of retention, and the extent to which Optimal conditions prevail, determine the total output of microbial products. Fluids are retained in circulation, affording a permanent medium for microbial activity, by changes taking place between the rumen and reticulum. Ingested plant material is seen to be the natural habitat of this microbial population as well as the functional link between it and the host animal. Decomposition is throughout accompanied by synthesis since the maintenance of the pOpulation is a direct consequence of proliferation (5). A source of nitrogen is .13- essential for this synthesis. This cellular digestion is, therefore, initially bound up with Una nitrogen requirements of the microorganisms. Since the medium in which the micro- organisms grow is determined by the ration fed, it becomes probable that a varying ration could effect a change in the microflora and thereby a change in the synthetic reactions induced. Bentley (9) demonstrated that this was true by showing the depression of cellulose digestion in steers by feeding;starch. On the other hand, B-vitamin synthesis and urea utilization were improved by rations rich in readily a- vailable carbohydrate. A natural difference is known to exist between the microflora of animals fed on roughage and those receiving grain-rich rations. Along this line, Gall (16) found the winter'and summer ration effect to be the only variable which seemed to influence bacterial pOpulation, and the changes were more quantitative than qualitative. The presence or absence of certain minerals is known to have an effect on ruminant nutrition. Since calcium is found in abundance in roughage, a deficiency is uncommon under natural conditions. Cobalt is the most prevalent deficiency found among ruminants, in many instances being attributed to a phosphorus deficiency. Call (16) exhibited an increase in bacterial counts of sheep with cobalt supplementation, as well as a marked bacterial type alteration when deficient. Using diminution of N.P.N., when incubated $9 11359, as an index of bacterial growth, McNaught (31) observed that rumi- "19.. nant bacteria could tolerate 100 p.p.m. of iron, 10 p.p.m. of cepper, somewhat less than 10 p.p.m. of cobalt and 100-l,000 p.p.m. of molydenum. Definite inhibition occurred in the presence of 1,000 p.p.m. of iron, 25 p.p.m. of cepper, 1,000 p.p.m. of cobalt, and 2,000 p.p.m. molydenum. The amount of iron associated with microorganisms was found to increase Vwith in 11332 incubation. Certain antibiotics may enhance growth of these organisms‘aS‘well. Knodt (29) found that aureomycin increased the rate of growth of dairy calves and did not apparently effect rumen flora. No salivary enzyme is possessed by the bovine for the degradation of cellulose, nor is there present any such se- cretion in the rumen. It is up to the flora alone to per- form the function of degradation. any feeds are known to exert an influence upon these organisms and fine ability to digest cellulose. Dried distillers solubles, soybean oil meal, and linseed oil meal appear to be fine most helpful, followed by corn molasses, corn, wheat bran, and cottonseed meal (13). Belasco (8) noted fluat urea gave greater cellulose digestion than did soybean oil meal at equivalent nitrogen levels, and furthermore, urea maintained higher levels of digestion than did any of the protein meals tested at compar- able nitrogen levels, especially at the lower levels. Balch (2) suggested a decreased rate ofjpassage through the reticu- lar rumen to be associated with the increased digestion of crude fiber. Certain unidentifiable "cellulytic factors" -20.. have been known to stimulate microbiological activity 13 yitrg and have a marked effect on cellulose digestion (9). The factors are apparently present in autoclaved rumen juice, ex- tracts of various plant materials, molasses and yeast extract. A considerable portion of the crude protein ingested by the ruminant is converted to ammonia by microbial proteolytic enzymes and the ammonia synthesized into microbial protein. As a consequence, a large portion of die protein ultimately used by the ruminant appears to be microorganismal, regard- less of the nature of the nitrogenous compounds contained in the ration consumed (27). The active rumen flora will uti- lize ammonia rapidly as a source of nitrogen in the presence of sufficient readily available carbohydrate and prevents its accumulation. Moir and Williams (25) estimated the conversion of ingested nitrogen to microbial protein to be about 50 per- cent in sheep. Protein digestion in.the rumen can be due only to proteolytic enzymes contained in the food or produced by the microbes. Sym (1938) demonstrated.a highly active pro- teinase, considered of microbial origin, present in the rumen contents (30). McNaught (25) found the dehydrated rumen bacterial cells to contain hh.h percent crude protein with a digestibility value of 73 and a biological value of 88. However, different nitrogen sources varied markedly in their biological value and in their capacity to promote bacterial growth. She also noted an increase of lysine in incubated samples of from 9.3 to 11.6 mg/100 ml. of rumen fluid, and considered this as evidence of protein synthesis (13). Using -2L- the 1g y__i_t_1:g method of study, Smith (33) found that about 8 mg. N/lOO g. of rumen liquid was being converted to protein during incubation. He calculated that if synthesis WUJld proceed at this rate in UK? intact rumen, about 300 g. of protein, or roughly one-third.of the protein requirements of a cow yielding 2 or 3 gallons of milk daily, would be synthe- sized in one day. He also found the optimum temperature for maximum synthesis to be between 30°ard l.O°C, with hydrolysis predominating above LOOC. Therefore, synthesis and hydrolysis of protein undoubtedly proceed simultaneously, predominance depending upon prevailing1nimmi conditions. In an effort to isolate this protein and estimate the‘amount of true protein, Smith (3h) separated a sediment containing the synthesized protein by centrifuging the liquid at 3,000 r.p.m. fer one hour. He noted that the weight of sediment and total protein increased in the presence of carbohydrate while N.P.N. de- creased, and that there was an increased number of iodophiles accompanied by synthesis of a starch-like polysaccharide. Conversely, in the.absence of carbohydrate protein hydrolysis predominated and total protein-nitrogen and bacterial count decreased while N.P.N. increased, thus pointing out the im- portance of adequate available carbohydrate in the conversion of N.P.N. to pnotein, and also duet an increased iodophile count accompanies synthesis. Typical analytical figures for this sediment as found by Smith are: 0.5 percent moisture, 36.3 percent protein, h6.6 percent polysaccharide, 9.5 per- cent lipoid material, and 6.2 percent ash. These values are -22.. very similar to correSponding values for feeding_stuffs such as linseed cakes. Wagner (35) criticizes the comparability of results secured by the 13 gitgg method, however, since in the rumen a maximum flora is always present While in ig liggg eXperiments the flora must first develop. During this inter- vening time, chemical changes, such as proteolysis, Which do not have time to occur naturally may be taking place. This may be diagrammed in such a manner: Bacterial Growth NH3H.‘ ‘ Protein Bacterial Proteolytic Enzymes The crude protein of roughages contain from 10 to 15 percent N.P.N. as free amino acids, nucleic acids, purine and pyrimidine bases, etc. However, rumen microorganisms have been considered by some investigators to be relatively simple in the nature of their requirements, and not in need of com- plex forms such as amino acids. Hamilton gt §l° (20) found urea satisfactory as a nitrogen source for growing lambs, provided at least 25 percent of the feed nitrogen was in the form of preformed protein and, further, that the total pro- tein equivalent in the ration was not in excess of 12 per- cent. This latter fact was confirmed by Wegner (37). How- ever, although simple nitrogenous compounds such as urea are undoubtedly utilized for microbial protein synthesis, most workers have discovered that such compounds are not as ef- fective as the nitrogen from natural proteins. This may be -23- due to the rapid conversion to ammonia, the excess of which becomes unavailable to bacteria for conversion since it is absorbed through the rumen wall. When one is considering;the pmotein synthesizing power of rumen microorganisms, it is well to remember that microbi- al proteins are not fixed structures but exist in a dynamic steady state (10). This fact is probably very advantageous to the organism. Since a cell must respond to varying con- ditions such as growth, infection, etc., the lability of the structures offer an easier ability to adapt to such changes. Protein metabolism involves a steady dynamic state of con- tinuous, equal synthesis and breakdown. Protein fimino Acids:Catabolic Products A continuous breakdown and reconstitution of peptide bonds occurs, and synthesis takes place both when amino acids are supplied in the diet and when due animal is fasting. The major initial reaction occurring during nitrogen xnetabolism is the loss of the alpha-amino group, due to either oxidation or transamination. While the alpha-amino group ultimately appears in mammals, mainly in urea, the residual cartnm.skeleton may be reaminated or converted to other pro- ducts. It is a generally accepted fact that urea arises znainly by the action of arginase on arginine to yield urea euki ornithine (10). Glutamic acid, being an important link 'between carbohydrate and protein metabolism, probably repre- -24- sents the most significant pathway for the formation of alpha- amino groups in higher animals via the conversion of ammonia. Since the first step in the catabolism of most amino acids is usually deamination, most of the nitrogen of the alpha- amino group appears in urea, uric acid, or allantoin. The chief amino acid involved in transamination is 1-g1utamic acid. Although certain d-amino acids occur in the natural state, their presence remains to be proved in protein and their biological significance is doubtful. Protein break- down accounts for the formation of ammonia in the rumen and may also account for the origination of the volatile fatty acids. Although only low concentrations of amino acids exist in the rumen liquor, there is evidence of some protease ac- tivity by microorganisms. Duncan gt 2l° (1h) exhibited the ability of rumen microorganisms to utilize urea-nitrogen for synthesis of amino acids, and found the amino acid pat- tern from the purified ration to be basically similar to that obtained from a natural ration, with the exception of histi- dine. The average percent increase of amino acids from the mixed rumen proteins over a 6-hour period was found to be as follows: Arginine......h3.35fi Histidine.....39.57 Isoleucine....h3.66 Leucine.......h1.07 Lysine........hh.83 Methionine....hh.07 Phenylalanine.hl.96 Threonine.....39.h3 Tryptophan....h0.h5 Valine........h1.0a -25- 110 consistent difference was observed between 0 and 6—hours (of incubation when amino acid composition of microbial pro- ‘tein was expressed as percent of sample. However, total amounts present indicated extensive synthesis. Black, in 1952, concluded that the essential or "indes- pensible" amino acids are synthesized by the microorganisms umile the tissues of the cow furnish the non-essential amino acids. Ruminants are the least definite in Uneir amino acid requirements of all herbivores. Therefore, even N.P.N. sources of annbined nitrogen, such as urea and ammonia, have a pronounced protein replacing value in ruminant nutrition. The only exception to the non-critical problem of ruminant protein requirement is the young of the Species in which the synthetic ability is not well developed and to which high quality proteins must be supplied.ir1the ration. One can see, therefore, that the term "non-essential amino acids" is sig- nificant only when qualified as to species and age period of the particular animal (3). The rate of amino acid exchange is different in dif- ferent tissues, but, in general, the rate of incorporation into intact cells 13 332:9 is of the same order of magnitude as ;g v1vo. Conditions were found by el-Shazly (15) for us- ing washed suspensions of mixed rumen microorganisms which attacked amino acids present in mixtures in much the same relative rates as those attacking U16 whole rumen liquor. The rapidity with which ammonia is accumulated in the rumen fol- -26- lxowing feeding reflects the high microbial activity. From tflie fact that amino acids do not accumulate it is apparent tfloat the rate of amino acid uptake exceeds proteolysis caused by the microbial proteinase, or that in addition they may be deaminated free amino acids (30). Even When the source of dietary nitrogen is wholly pro- vs- tein, an appreciable amount is converted to bacterial protein. This bacterial protein is relatively constant in composition for different conditions in the rumen according to Holmes II]... - ' -. (2h). In comparing it with whole egg protein, it is well supplied with arginine, histidine, valine, and tryptophan, but deficient in leucine, threonine, and phenylalamine, and although moderately rich in methionine as compared with other bacterial protein, it is very deficient in this as well as isoleucine. The value of supplanenting a ration with methi- onine has been shown by Gallup (17) who found the addition of 1.6, 2, and 3 g/day to a urea supplemented low-protein ration to increase the average digestibility of nutrients and nitro- gen utilization. Johanson gt 3;. (26) concluded that the fact that rumen bacterial protein is rich in cystine and methionine is of considerable importance as evidence to the theory of protein synthesis prior to digestion and absorption. They also concluded that in these animals the value of the N.P.N. substance, i.e. urea, is greatly enhanced by supple- mentation with methionine. Sulfur requirements are interesting to note, since rumi- -27- rrants utilize inorganic sulfur in the synthesis of cystine and methionine. Alexander )2) found that a low-protein diet is necessary for conversion of ammonia into protein. The power to deami- nate amino-acids depends upon the diet (power of rumen micro— organisms) and the rate of microbial attack is determined by the solubility of the protein. While the biological value_ 5.." 'LI. ' of this microbial protein is fair and may logically be ex— pected to depend upon its amino-acid composition, since it is hydrolyzed to the component amino acids, its inferiority may perhaps be related to the methionine deficiency of "food yeast." EXPERIMENTAL PROCEDURE Two rumen fistulated steers were employed in the study. fSteer A received a ration composed of 25 pounds of corn si- re] lage1 plus 2 pounds of soybean-oil meal2 per day. Steer B ‘ :received a ration of 25 pounds of corn silage plus 2 pounds E of a urea-corn mixture3 per day. Both were fed an additional 25 pounds of mineral mixl+ per day. i Rumen ingesta was collected from each steer about 13 hours after feeding to facilitate removal of ingesta by elimi- nating much gross material. The fresh sample was filtered through cotton guaze to remove solid material, and the re- sulting liquor was centrifuged in an International centrifuge to rid the liquor of the smaller food particles and larger masses of protozoa. In the first series of trials the liquor was centri- fuged at 2,000 r.p.m. fbr 10 minutes. The resulting super- natant was prepared for incubation by the addition of 0.3 per- cent urea, 1 percent glucose, and 0.5 percent Difco yeast extract. Duplicate 20 m1. portions were removed for the preparation of the amino acids filtrate and duplicate 10 ml. Corn Silage...........2.72% protein, 6h.03% water" Soybean-oil Meal.....h8.hb% protein Corn-Urea Mixture....1h.79% urea, t6.13% protein Mineral Mix..........50% trace mineral salt-+50% dicalcium phosphate P4»toh4 O O 0 -23- -29- portions removed for preparation of the tryptophan filtrate. Duplicate 25 ml. portions were withdrawn for the protein ni- trogen determination and duplicate 1 ml. samples removed for determination of dry matter content. The initial sample was then incubated in a water bath at 390 C under 002 for a period of 6 hours, after which time test portions were removed F a s detailed above . v- L18! . Amino acid hydrolysates were prepared by adding 20 ml. of concentrated HCl and autoclaving at 15/}: pressure (1210 C.) for 8 hours. The hydrolysate was cooled and adjusted to pfli 6.8 with 19 N NaOH, diluted to 100 ml. with distilled water and filtered through 51.0 Whatman filter paper. The resulting :filtrate was a 1-5 dilution of the original sample. Storage inas made under toluene at refrigeration temperature. Trypto- jphan.hydrolysates were prepared by adding 40 ml. of 3 N NaOH and autoclaving as for the amino acid samples. The hydroly- Sate was adjusted with concentrated HCl to pH 7.0,diluted to ICKlnfld and filtered through #AO Whatman filter paper. The ffilnal filtrate represented a 1.-lO dilution of the original Samuflxh Storage was made as above. Protein nitrogen was de- isermined by the Kjeldahl method. The 25 ml. sample was pre- <=ipitated by the addition of 15 ml. of 10% sodium tungstate Eind.60 m1. of 0.33 N H280“. The resulting precipate was Separated out by filtering through Reeve Angel #812 fluted f7:11ter paper, and the determination run on the precipitate plus the paper. Calculation was then based upon the amount -30- or protein nitrogen per 100 ml. of liquor (N x 6.25). Per- cent dry matter was ascertained by drying the liquid samples of known weight in a vacuum oven and calculating the dry xnatter by loss of moisture. The amino acid filtrate was assayed microbiologically .for dl-methionine,1 l-lysine,2 l-ar,inine, dl-threonine and F' .l-histidine,3 dl-leucine, dl-valine, dl-phenylalanine, and i dlyisoleucine,h as well as l-glutamic acid5 and dl-aspartic {acid.2 TryptOphan assay was carried out according to the 6 method of Keuken, Lyman and Hale. Values were calculated 5 .as mg. of l-amino acid per gram of dried rumen material. Any increase in amount was assumed.to be the result of microbial synthesis. The assay organisms used were Lactobacillus arabi- ggggig 17-5 (#801h), Streptococcus fecalis (#9790), and £3229- ;Qgcillus mesenteroides P-60 (#80h2), obtained from the Ameri- cainype Culture Collection. In trial II, determination.of total N.P.N. was made in corder to ascertain whether the N.P.N. content was decreased as a result of incubation.7 The second series of trials involved the isolation of Synthesized protein material and analysis of this dried sedi- Inent. Since trouble was encountered with steer A and samples Lyman 23 al., J. Biochem., 166:161 (19h6). McMahon, J: R., and Snell, ET—E., J. Biochem., 152: 83 (19hh). ——— Creenhut, I. T., Schweigert, B. 8., and Eluehjem, C. A., J. Biochem., 1§3:69 (19h6). Schweigert, SE 31., J. Biochem., 155:183 (l9hh). Lyman 23 al., J. Biochem., 151:395—T19h5). Lyman 22 31., J. Biochem., 11; (1947). . See Appendix I for method. \)O\\nb w NH -31- were not withdrawn, the results of these latter trials are based upon the rumen ingesta obtained from the urea supplemented steer only. In an attempt to promote greater microbial activi- tar 12 gitgg, cellulose was incorporated as a substrate for the iJicubated samples. The substance employed was Solka-floc.l lflie rumen sample was removed as previously described. How- F-J- ever, it was used in both the centrifuged and uncentrifuged state. Greater activity was expected from the uncentrifuged hill“. . liquor due to the additional number of microorganisms. The sample used for incubation in trial IV was uncen- trifuged. The nitrogen source was 0.05 percent urea, the Ineadily available carbohydrate was 0.01 percent glucose and 31.0 percent Solka-floc served as the substrate. The sample unis run in duplicate. An initial O-hour sample was prepared IRDr sediment isolation by centrifuging in the International Centrifuge for 10 minutes at 2,000 r.p.m. Following the ad- <1ition of the above mentioned substances to the uncentrifuged Samples, a 25 m1. portion was removed for cellulose determi- nation.2 The samples were then incubated for 21. hours under 002 at 39°C. At the end cf this incubation period one-half of the inoculum was removed for sediment isolation and a Sample removed for cellulose determination. The remaining hEilf was again brought to volume with an artificial complex3 afui half quantities of urea and glucose added. A cellulose \_ 1‘- w- 1. Purified wood cellulose (SW ADA). 2. Crampton, E. W., and Maynard, L. A., J. Nutr., 12 (1938). 3. McDougall's Modified Synthetic Saliva with trace elements. -32- sample was withdrawn and incubation carried on for another 24 hours, after which a cellulose sample was again removed. Isolation of due sediment was accomplished by centri- :Fuging off the gross material at 2,000 r.p.m. for 10 minutes ill the International centrifuge and running the supernatant tfldrough the Sharples Super Centrifuge for 20 - 30 minutes. ‘The sediment thus obtained was resuspended in a 50/50 absolute alcohol-water mixture and spun down in a Sorval centrifuge at. 60,000 r.p.m. for 20 - 30 minutes. The sediment was washed four times in this manner, alternating with absolute alcohol and the alcohol-water mixture. The final sediment was washed with ether and ai r-dried. The dried material was crushed and tfim resulting powdery material hydrolyzed for assay. Amino acid hydrolysates were prepared by adding 10 m1. of concentrated HCl per 0.1 gram of sample and autoclaving for 8 hours as described previously. Tryptophan samples were Prepared by adding 5 m1. of l. N NaOH per 0.2 gram of sample and hydrolyzing as above. The hydrolysates were adjusted to the desired pH, diluted to 100 m1. and filtered as before. Microbiological assays were carried out as for the liquid, and calculations made accordingly on the basis of mg/gm. of Sediment. The feeding ration was changed fnom corn silage to poor quality timothy hay plus the mineral mix for trials V and ‘V]:. The samples were neither supplemented nor incubated, but merely strained and precentrifuged in the International centri- fuge. The time of centrifugation for samples 6 and 7 was 10 minutes at 1,500 r.p.m. while for samples 8 and 9 due time ‘was 5 minutes at 1,000 r.p.m.. Isolation of the sediment and :prepsration_of the filtrates were as described. The sediment .from.samples 6 and 9 was very dark in color. The supernatant lgiquid from the Sharples centrifugation was used.for'the as- say of any free amino acids which might have been present. The ration was again altered for trial VI by changing l and tile corn-urea mixture Us a corn-biuret (crude) mixture feeding this in the amount of 1% pounds per day along with time poor quality timothy hay. The sample was centrifuged at 1, 500 r.p.m. for 5 minutes and a sample set aside for sedi- ment isolation (sample 10). Two samples were then prepared for incubation as follows: Sample 1 Sample 2 Substrate..2% Solka-floc 2% Solka-floc Com lex....1 1. McDermits 1 1. McDermits (é strength) ( stgength)ftrace ele- .ftrace elements ments Inoculum...l liter centri- 1 liter centrifuged rumen fuged rumen liquor 11 nor N-source...0.2% urea 0.65m Dragkett Assay Protein Carbohy....0.08% glucose 0.08% glucose Dujplicate 25 ml. portions were withdrawn from each sample for determination of Protein-N and triplicate 25 ml. portions re- mo‘ved for cellulose determination. Incubation was carried \ A 1. 14.7% biuret-urea, ca. 42.4% crude protein. 2. See Appendix I for formulation. 3. Standardized protein from soybeans, Drackett Pro- ducts Co. out over a 24-hour period and test pprtions again withdrawn. Gross matter was removed from the incubated sample and the supernatant spun down in the Sorval centrifuge for 30 minutes. The sediment obtained was washed as previously described. Filtrates were prepared as before and analysis made in the manner already outlined. 'n in‘v‘j 0!.) 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