. .... . , ... .. ‘... . . V ‘..q . . . ‘. . ‘. . a n. < M~d¢o._.aot.._.. . . . . . . . . . .4 o... .l.. .V.. c 4 on. . . . .. n.... .. ..A. ...) 3.. . . . . ,.. .. ......Ir ..‘. ', ... . . . . . . . .‘ . .‘ ‘.... ..,. ..... .. ‘0‘ _. ..... ... _. . .. . . . . . . ......n ........o I .... . . . .... .‘ ...V.<.. ... o. .. _. . . ‘ .v . ,.‘.¢ . 4 ..... ._ u. . _ . . o . v . . , _ . . . nu. . _ . . .. . . .. . . . . .. _ . . . . . . . . . . ..a . — n . . . ... .. . u . . .. . . . .. . . . . - . a . » .. _ ....r. ‘ . . ... . . A, ... _\ I . p. n .. . . . . . . . _ ... _ .. . . . . . . V ., . .... . _ . . _ . ‘ _. . . . o _ . _ .. . g .- , . o . ,_.... . .. . ... A , . .. . . _ o . .. . . . .r . . v . _ ..... . . . > . . ’_ . .4 A . . . ... .‘_. c . . y 0 n . . _ . _ .. .. . . v . — .. .0 . . ’. o.....l-.... .. ‘ . . 4......5..... _...... . ‘ ... .. ....o'.. 1.. .Aw .t,.o4' —rtl.. . 4.. _,_. ..r..lo.} . . . . . .. A .r. . .. . . . . _ . _ . A ‘ ‘ . . . .. o . . . . .— . . < .‘ ... . .. .. p . . - u 4 . . t . .. -. _ _. . , . . _ .. .. SHEEP NDERSON t . . . . . I III- .. . . . 0. E El . .. . _ e ...... . . _ _ B 8. E, EL 2 . A 9 . E .hu no . ._ . M ... ..L . . . r N . ... .. ...I . N , P .mm H .N _ . h I . _ . M _ ... . ... ~. . . ...... ‘ ..u . _ . . 7.. ...; .. ..... a... . ... 11...?” o. _ _ .. p. . ._.‘.... .o. .. ‘..o... ... .. . ., . . l“.....‘.9k .. . . .... .. ... ...: .....J...“: ... . H. . ‘ . . . .. .... _.‘ ....1..A .. . , . C» ..... . ...r. ..HXK...» ...... éwfiflwdrw bormomunvug¥ “in... ... c .0. .. . . _. .. . ... . .x... o n. ... ...; x; . .0... . .....¢.‘o“ .W. . ‘.w”......w..._..nnp.?8. ..a r..;3.%....1..,€. .3... dwelt. ...... .... .....,o........t: 5......) o . . . .. ... s. . . . .. a ... . .. . ...... 1 ,... o - . e; I. . r I -Iu-..f..l - pr! . ...] W U Yw mm“ RM? mum” LM ABSTRACT TRYPTOPHAN METABOLISM IN SHEEP By Constantine Llewellyn Fenderson Since the essential amino acid, tryptophan, has been ignored in previous amino acid studies in ruminants, two experiments were designed to study the role of tryptophan in protein metabolism in sheep. Experiment 1 was a 4 x 4 Latin Square in which four rations containing different levels and sources of crude protein were fed to four sheep of approximately equal body weight. Each experimental period consisted of a 21-day ration adjustment which was followed by two sample collec- tion days. On day 22 blood and rumen samples were taken before feeding and at 3, 6, 9, 12, 16, 20 and 24 hours post feeding. Blood samples were analyzed for plasma free tryptophan, total free amino nitrogen and blood urea nitrogen concentrations. Rumen samples were analyzed for rumen ammonia nitrogen and volatile fatty acid concentra- tion. On day 24 of each period, a 2 : l starch-glucose mixture in water was instilled into the rumen of each sheep. Blood and rumen samples were secured before and 4 hours after instilling the starch-glucose. Amino acid Constantine Llewellyn Fenderson levels were determined in the pre-energy instillation (To) and post energy instillation (T4) blood samples. The resulting T4/T0 ratio of plasma amino acid levels was used as an indicator of amino acid deficiencies. Plasma trypto- phan levels were not affected by dietary treatments in sheep. The range of mean daily tryptophan levels was from 1.86 to 2.11 mg per 100 ml plasma. The finding that rapid availability of energy (VFA) to sheep did not induce a marked depression of plasma free tryptophan suggested that tryptophan was not limiting or deficient in sheep fed the four rations used in this study. The T4/T0 ratios of tryptophan were 87%, 93%, 98% and 91% for rations l, 2, 3 and 4 respectively. The total free amino acid nitrogen concentration was not influenced by dietary treatments. The range of mean daily total free amino acid level was from 2.38 to 2.81 micromoles of a—NHZ-N (as citrulline) per m1 plasma. Increased rumen ammonia and blood urea nitrogen concentrations were associated with increased levels of dietary crude protein. The second experiment was conducted primarily to determine the constancy or variability of rumen microbial tryptophan concentrations. Rumen bacteria and protozoa were isolated from sheep (fed the four rations from experi- ment 1) before feeding, at l l/Z hours and 4 hours post feeding. Samples were analyzed for tryptophan and other amino acids. The results indicated that tryptophan Constantine Llewellyn Fenderson content of rumen bacterial and protozoal preparations was quite constant and was not affected by dietary treatments or time after feeding. The average tryptophan levels in bacteria and protozoa were 1.38 and 1.00 gm tryptophan per 100 gm protein respectively. The bulk amino acid composi- tion of rumen bacterial and protozoal preparations was constant and was not affected by dietary treatments or time after feeding. TRYPTOPHAN METABOLISM IN SHEEP By Constantine Llewellyn Fenderson A THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree of MASTER OF SCIENCE Department of Animal Husbandry 1972 "1‘ ACKNOWLEDGEMENTS I would like to express my sincere thanks and apprecia- tion to the following people whose efforts have helped me in obtaining my degree and the preparation of this manuscript. Dr. Werner G. Bergen for his invaluable help and his constant and vigilant supervision of my graduate program and the preparation of this manuscript. Dr. I. E. Ullrey and Dr. D. Narins for their correc- tions and suggestions which increased the clarity of this manuscript and their participation in my graduate program. Dr. W. T. Magee for his invaluable help in the prepara- tion and interpretation of the statistical analysis of the data. Dr. R. H. Nelson for making the facilities at Michigan State University available for this research. Elaine Pink for her laboratory assistance and Lurline Walker, Joice Adams, Carolyn Edwards, Susan Steiner and Cheryl Smith for their typing assistance. My wife Una and Son John whose constant love and devotion I always relied on after a hard day's work. ii TABLE OF CONTENTS LIST OF TABLES LIST OF FIGURES I. II. III. IV. INTRODUCTION LITERATURE REVIEW The Importance of Micro- -organisms in Supplying Protein to the Ruminant Effects of Dietary Protein on PIasma Amino Acids . . . . . . . Effects of Energy on Plasma .Amino Acids . . . The Role of Tryptophan in Mammalian Metabolism . MATERIALS AND METHODS Experiment 1 . General Design of. Experiment Sample Collection and Preparation Tryptophan Determination . 0- -Amino Nitrogen Determination Rumen Ammonia and Blood Urea Nitrogen Determination Volatile Fatty Acids Determination Experiment 2 . . . Sample Preparation Tryptophan Analysis Statistical Analysis RESULTS Experiment 1 . Plasma Free Tryptophan Plasma a— -Amino Nitrogen Rumen Ammonia Nitrogen . Blood Urea Nitrogen . Rumen Volatile Fatty Acids Plasma Amino Acid Concentration Experiment 2 . . Rumen Microbial Amino Acid Concentration iii 10 17 19 19 19 23 25 26 27 28 29 29 30 32 33 33 33 36 4o 44 49 54 59 59 "5 ‘...__._.__ VI. VII. VIII. TABLE OF CONTENTS (Continued) Page DISCUSSION . . . . . . . . . . . . . . . . . . . 64 GENERAL CONCLUSIONS . . . . . . . . . . . . . . 72 BIBLIOGRAPHY . . . . . . . . . . . . . . . . . . 73 APPENDIX . . . . . . . . . . . . . . . . . . . . 80 iv Table 10 ll 12 l3 14 15 LIST OF TABLES Experimental Design for Experiment 1 Rations Crude Protein and Dry Matter Content of Rations Plasma Free Tryptophan Concentration During 24 Hour Period . . . . . Plasma Tryptophan Concentration Before and After Starch-Glucose Infusion . . . Plasma a-Amino Nitrogen Concentration During 24 Hour Period . . . . . . . . Plasma o-Amino Nitrogen Concentration Before and After Starch-Glucose Infusion Rumen Ammonia Nitrogen Concentration During 24 Hour Period . Rumen Ammonia Nitrogen Concentration Before and After Starch-Glucose Infusion Blood Urea Nitrogen Concentration During 24 Hour Period . Blood Urea Concentration Before and After Starch-Glucose Infusion Rumen Acetate Concentration During 24 Hour Period . . . . . . . . Rumen Acetate Concentration Before and After Starch-Glucose Infusion Rumen Proprionate Concentration During 24 Hour Period . . . . . . . . . Rumen Proprionate Concentration Before and After Starch-Glucose Infusion Page 20 21 22 34 37 38 41 42 45 46 48 50 51 52 53 Tab16 16 17 18 I9 20 21 22 Table 16 17 18 19 20 21 22 LIST OF TABLES (Continued) Rumen Butyrate Concentration During 24 Hour Period . Rumen Butyrate Concentration Before and After Starch-Glucose Infusion . . . . . . . Plasma Amino Acid Concentration Before and After Starch-Glucose Infusion Tryptophan Levels in Isolated Ruminal Bacteria Tryptophan Levels in Isolated Ruminal Protozoa Amino Acid Levels in Isolated Ruminal Bacteria Amino Acid Levels in Isolated Ruminal Protozoa vi Page 55 56 57 6O 6O 61 62 Figure 7b Figure LIST OF FIGURES Diurnal pattern of plasma free tryptophan concentrations . . . . . . . . . . . . Diurnal pattern of plasma a-amino nitrogen concentrations . . . . . . . . . . . Diurnal pattern of rumen ammonia concentrations . . . . . . . . . . . . Diurnal pattern of blood urea nitrogen concentrations vii Page 35 39 43 47 INTRODUCTION The present unchecked trend in human population growth is toward eventual, if not already existing over—population. It is therefore obvious that adequate food must be pro- vided to cope with the demand of such rapidly increasing population if its growth rate cannot be checked. One way in which this can be achieved is to constantly increase agricultural production by the application of new scien- tific research knowledge to current systems of agriculture. To reach the present standard of productive efficiency of animals, researchers all over the world have delved deeply into the science of nutrition, breeding and management of farm animals. Farm animals are important to man in that they provide a source of income for many farmers and high quality animal protein in the human diet. Ruminants form an important class of farm animals which through their rumen micro- organisms, possess the unique ability to convert forages and nonprotein nitrogen into animal protein. Proteins form an important class of dietary essential nutrients which are required in every metabolic reaction of the animal's body. Their importance is manifested in indispensable functions such as cellular construction, catalysis, metabolic regula- tion and the defense mechanism against pathogens (Mahler and Cordes, 1966). Partial or complete absence of protein from the diet over a period of time leads to severe nutri- tional disease such as kwashiorkor and marasmus. Nutritionists have divided the amino acids into essential amino acids and nonessential amino acids based on the ability of the animal's tissues to synthesize these amino acids. Essential amino acids are those amino acids which cannot be synthesized, in sufficient amount by the animal's tissues, to meet the requirement of the animal, whereas nonessential amino acids can be synthesized by the animal's tissues as long as the precursors for their synthesis are supplied in the diet. Thus essential amino acids must be supplied in the diet. The absence of any one essential amino acid from the dietary protein produces no growth. However, this does not hold true for maintenance. The amino acid supply to ruminants consists of a mixture of undegraded dietary and ruminally synthesized microbial protein. Consequently, the amino acids which are limiting in these proteins will limit the productive performance of the animal. The limiting amino acid may be defined as that essential amino acid that is available in the least amount in relation to the requirement of the animal. T becaus few pt The p1 protei pool f 3 TryptOphan is an important essential amino acid, but because of difficulties in its determination there are few published data regarding its metabolism in the ruminant. The present study was designed to determine the effect of protein source and level in rations on the plasma tryptophan pool in sheep. §T*:;" LITERATURE REVIEW The Importance of Micro-organisms in Supplying Protein to the Ruminant The ruminant animal, because of its importance in supplying food for human needs and because of its symbiotic relationship with its rumen microbiota, has captured the interest of a large number of researchers. The discovery of rumen protozoa by Gruby and Delfond (1843) was the first identification of micro-organism in the ruminant stomach. Pasteur (1863) discovered the role of bacteria in the fermentation of plant materials. Zunt (1879) inferred that rumen micro-organisms ferment fiber anaerobically and thus form acids and gas. One of the unique capabilities of rumen micro-organisms is the conversion of nonprotein nitro- gen and B-linked cellulose to microbial protein which is digested and absorbed in the lower gastrointestinal tract of the host. El-Shazly and Hungate (1966) using the diaminopimelic acid concentration of bacteria procedure, found that 69-90 percent of the total nitrogen passing to the small intestine was of microbial origin. Ammonia produced by rumen micro-organisms from both protein and nonprotein nitrogen is the most important nitrog for ru studio 1969) showed bacter ingest K and se centra lambs. greate thus c animal ammoni that I faUnat When f ration retent in fan thEre defaun that p USe it betwee1 5 nitrogen source for rumen bacteria but is less important for rumen protozoa (Allison, 1969). In vivo isotope studies involving 15N labeled ammonia or urea (Allison, 1969) and 15 N labeled ammonium citrate and urea (Abe, 1968) showed that ammonia nitrogen is first incorporated into bacterial cells and then into protozoal cells following the ingestion of the bacteria by protozoa. Klopfenstein 35 31. (1966), Males and Purser'(1970) and several other workers reported that rumen ammonia con- centration was higher in faunated lambs than defaunated lambs. This can be explained by the fact that there is a greater bacterial concentration in the defaunated animal thus causing greater ammonia utilization than in the faunated animal in which protozoa are extremely poor utilizers of ammonia (Males and Purser, 1970). Barringer (1968) reported that rations, supplemented with soymeal and zein, fed to faunated lambs had higher digestibility coefficient than when fed to defaunated lambs; dry matter intake for all rations used was 9.2 percent higher; higher nitrogen retention and lower urinary nitrogen excretion were noted in faunated lambs on the same rations. On the other hand, there were higher viable bacterial concentrations in the defaunated lambs on semipurified rations, further suggesting that protozoa ingest a portion of the bacterial cells and use it as a nitrogen source and a high degree of competition between protozoa and bacteria for substrate per s3, On natu1 perce semi; 1968: rats bacte diges tein that zoal limiw of r1 Prep: 1968: intes dIEt; Sizec reP01 compc neit} miCTc and E COmPo 6 natural type rations microfauna apparently produce a higher percentage of microbial protein than microflora whereas on semipurified rations the converse is true (Bergen gt_gl,, 1968). Bergen gt gt. (1968b) fed rumen microbial protein to rats and observed a slightly higher biological value of bacterial protein than protozoal protein; a higher true digestibility for protozoal protein and a higher net pro- tein utilization in protozoal protein. He also reported that histidine was the first limiting amino acid in proto- zoal protein while cystine (sulfur amino acid) was the limiting amino acid in bacterial protein. Lysine content of rumen protozoal preparation is higher than bacterial preparation (Purser and Buechler, 1966; Bergen gt gt., 1968). The amount and quality of protein reaching the small intestine of the ruminant depends on the degradation of dietary protein and the amount of microbial protein synthe- sized in the rumen (Smith, 1969). Bergen gt gt. (1968a) reported that within a microbial preparation the amino acid composition is not affected significantly by rations, and neither is the bulk amino acid composition and quality of microbial preparation affected by ration change. Purser and Buechler (1966) also reported a constant amino acid composition in rumen bacterial preparation. esse1 mien is a micr of r Down repo phen boli Synt essc tryr bee: mine amir rumf has 5311 and (19c 2911 dige Was lamb 7 The unique ability of the ruminant to provide its own essential amino acid supply is strictly a function of rumen micro—organisms. Thomas gt gt. (1949) reported that there is a remarkable synthesis of all amino acids by rumen micro-organisms and that the essential amino acid content of rumen material was similar for urea and casein diets. Downes (1961) working with both growing and mature sheep reported that isoleucine, leucine, lysine, methionine, phenylalanine, threonine, valine and histidine were meta- bolically essential to ruminants since they cannot be synthesized by their tissues; but arginine was not an essential amino acid for the sheep. However, whether tryptophan was an essential amino acid was not assessed because of the difficulties in accurate tryptophan deter— mination. Downes (1961) therefore concluded that tissue amino acid metabolism of sheep is similar to that of non- ruminants. The rate of digestion of various proteins in the rumen has been correlated with the solubility of the protein in salt solutions or rumen fluid (El-Shazly, 1958; Blackburn and Hobson, 1960; Hendricks and Martin, 1963). Ely gt gt. (1967) after estimating the extent of conversion of dietary zein to microbial protein, concluded that the low apparent digestibility of zein indicated that dietary zein nitrogen was not efficiently utilized. They also reported that all lambs fed zein protein diets showed a negative nitrogen bala the and not equi used a di tion vert a la mlCI thrc to L (195 the and and thre IEgt fI‘on Eras repC Cent fed Cont 8 balance. Thus because most of the zein protein bypassed the rumen resulting in depressed microbial protein synthesis and because lysine is severely limiting in zein, there were not enough utilizable amino acids to maintain nitrogen equilibrium. Plasma urea levels were higher when urea was used as dietary nitrogen source than when zein was used as a dietary nitrogen source (Ely gt gt., 1969). The explana- tion for this lies in the fact that urea is rapidly con— verted to ammonia by rumen micro-organisms, consequently, a large amount of the ammonia which is not converted to microbial protein by rumen micro-organisms is absorbed through the rumen walls into the ruminal vein and converted to urea by the liver (Lewis, 1961). According to McDonald (1954) only about 40% of the dietary zein is degraded in the rumen and converted to microbial protein. Abou Akkada and Osman (1967) observed that changes in ruminal ammonia and blood urea concentrations were considerably higher three hours after feeding leguminous forages than non- leguminous forages and that most of the nitrogen excretion from legume-fed animals was urinary nitrogen whereas for grasses it was fecal nitrogen. Tagari gt gt. (1964) reported an increase in rumen ammonia and blood urea con- centrations with an increase in protein level in the ration fed to sheep. A significant correlation between protein content of the ration, rumen ammonia and blood urea concen- tration has been reported by several workers (Preston gt gt., 9 1965; Lewis, 1957; Tagarigt_gt3, 1964). Lewis (1957) reported that the major factor controlling blood urea con- centration is the concentration of rumen ammonia in that a change in rumen ammonia concentration is usually accompanied by a change in blood urea concentration. Rumen ammonia‘ concentration is dependent on the type of ration, that is, the amount and type of energy source and the quantity and solubility of nitrogenous material in the ration. Conse- quently, blood urea concentration is indirectly dependent on the type of ration fed to the animal. McDonald (1947) reported that urea occurs in significant quantities in the saliva of sheep. According to Lewis (1957) as rumen ammonia increases there is a greater loss of urea in the urine and a greater return of nitrogen to the rumen via saliva. Lewis and McDonald (1958) reported that rumen ammonia concentra~ tion dropped to a low level between eight and sixteen hours after feeding and increased during the last eight hours of the 24 hour period after feeding. They therefore claimed that the increase during the last eight hours may be due to continued uptake of nitrogen through saliva, slowing down of bacterial growth and autolysis of some of the micro- organisms. flrskov and Fraser (1969), studying the effects of protein on nitrogen retention in lambs, observed that liquid protein supplement given directly to the abomasum, using nipple bottle, resulted in decreased urinary nitrogen loss and increased nitrogen retention, whereas the dry protei urinar micro- N absorp tion 0 those : rumen c is absc supplie and con In resp sheep a Cycle e Th the pas informa Ples of Working Plasma ; Protein the amoL someWhat Who W0 I‘k 10 protein supplement fed in the normal way produced increased urinary nitrogen due to extensive deamination by rumen micro-organism. Nitrogen for urea synthesis thus arises from ammonia absorption from the rumen but may also arise from deamina- tion of amino acids in body tissues. However, except in those rare cases where a poor quality protein bypasses rumen degradation and a poorly balanced amino acid mixture 4 is absorbed from the small intestine, the ruminal ammonia supplies the major portion of nitrogen for urea synthesis and completely overshadows tissue amino acid deamination._ In response to the extra nitrogen load for urea synthesis, sheep appear to have higher activities of hepatic urea cycle enzymes (Payne and Morris, 1969). Effects of Dietary Protein on Plasma Amino Acids The study of plasma amino acid in several species over the past two decades has provided a wide range of valuable information which explains some of the fundamental princi- ples of mammalian protein metabolism. McLaughlan (1963) working with rats, reported that the concentration of plasma amino acids increased after a meal of good quality protein and the duration of this increase was related to the amount and composition of the protein fed. This was somewhat substantiated by the work of Hogan gt gt. (1968) who working with sheep, observed an increase in total plasma ll essential amino acid concentration in response to each successive casein infusion in the abomasum, but the increase after the third infusion was less than that of the preceding two. Oltjen and Putman (1966) after feeding urea diets to steers, observed an increase in serine and glycine and a decrease in valine, isoleucine, leucine, and phenyla- lanine in blood plasma. Such a decrease in the branched chain amino acids may be attributed to less rumen micro- bial protein synthesis, consequently, a limited amount of protein is available to the animal, thus with adequate available energy such condition resulted in a typical kwashiorkor type of protein calorie malnutrition. Accord— ing to Klopfenstein gt gt. (1966) a lower than normal individual plasma amino acid concentration in faunated lambs suggested a greater amino acid utilization rather than deamination. McLaughlan (1963) in his review of pro- tein quality, pointed out that there is usually a good correspondence between the amount of amino acid in plasma and the protein fed, but since other dietary constituents such as glucose or butter may influence plasma amino acid concentration, plasma amino acid ggt gg should not be used to compare protein qualities. Fasting or imbalanced amino acid diets has a marked effect on plasma amino acid concentration. Ganapathy and Nasset (1962) working with dogs, reported that ingestion of protein may cause an increase or decrease in plasma 12 amino acid levels. The work of Leibholz and Cook (1967) showed a lower concentration of free CE-amino acid nitrogen, a significantly lower urea concentration and a significantly higher concentration of lysine in blood plasma of starved lambs than lambs fed maintenance ration. According to Brown gt gt. (1961), extended fasting of 88 hours in cattle caused plasma glycine level to increase approxi- mately twofold, a significant increase in the aromatic amino acids, lysine, valine, threonine, leucine, and iso- leucine, a decrease in serine and alanine and no signifi- cant changes in the plasma concentrations of glutamic acid, cystine, histidine and arginine. Zimmerman and Scott (1967) working with chickens, observed that plasma lysine, methionine, isoleucine, leucine, tyrosine, phenylalanine and histidine concentrations increased with each extension of fasting period whereas plasma cystine decreased and proline, glutamic acid and arginine were unaffected by fasting. These workers also observed that feeding a non- protein diet to chickens caused a lower plasma amino acid concentration as compared to the plasma amino acid levels of fasted chickens. Feeding nitrogen free diets to rats, Bergen and Purser (1968c) observed a lower total plasma essential amino acid levels than other treatments although the individual histidine levels were higher. Denton and Elvehjem (1954) fed nonprotein diets to dogs and observed it. ...__-.. 13 a decrease in all plasma amino acid concentrations of portal blood except tryptophan. The removal of an essential amino acid from the diet will cause a severe deficiency and consequently, a low plasma concentration of that particular essential amino acid, whereas there will be an increase in plasma levels of all other amino acids. Such increase in plasma level L of all the other amino acids can be interpreted as the . 1; result of very poor utilization of these amino acids due to the absence of the essential amino acid which impedes protein synthesis. Consequently, high plasma amino acid concentration may indicate proper amino acid absorption but not efficient amino acid utilization. This was sub- stantiated by Purser (1970) who pointed out that the plasma concentration of a specific amino acid does not always reflect nutritional status unless the amino acid is markedly limiting. Zimmerman and Scott (1965), working on the interrelationship of plasma amino levels and weight gain in chicken, reported that the first limiting amino acid in a diet remains at very low and constant level in the blood regardless of the severity of the deficiency and that increments in excess of the amount required to maximize weight gain resulted in a rapid and linear accumulation of that amino acid in the blood. Kumta and Harper (1962) working with rats suggested that if the influx of amino acid to the blood stimulates cellular uptake or protein SW 5110 ami ind (MC 196 aci 35 add int tin ami the lin lin imt grc lin 01‘ C35 the and C01 and the l4 synthesis then the concentration of essential amino acid in short supply should fall much lower than the other essential amino acid. Plasma amino acid may thus serve as a sensitive indication of the limiting or deficient amino acid in diets (McLaughlan, 1964; McLaughlan gt gt., 1967; and Rao gt gt., 1968; Purser gt gt., 1966; Potter gt gt., 1968). Harper and Rogers (1965) in their discussion on amino -‘... . ‘v acid imbalance simply defined an amino acid imbalanced diet A- as one which is a low protein diet and in which there are additional amounts of essential amino acids which cause an intake depression. Two further important conditions dis- tinguish an amino acid imbalance from a simple essential amino acid deficiency. First, both the control diet and the imbalanced diet contain exactly the same amount of the limiting amino acid and second, the concentration of the limiting amino acid (or acids) must be increased in the imbalanced diet to overcome the appetite depression or growth retardation. On the other hand, addition of the limiting amino acid to the control diet produces very little or no additional growth response. An imbalanced diet of casein-gelatin or oxidized casein in which tryptophan was the limiting amino acid produced severe growth depression and marked lowering of plasma tryptophan level which was corrected by addition of tryptophan to the diet (Sauberlich and Salmon, 1955). According to Harper and Rogers (1965), the major effect of amino acid imbalanced diets is a drastic 15 decrease in intake of the imbalanced diet, so much that rats will eat a nonprotein diet which will not support life instead of eating the imbalanced diet which will support life. After reviewing several of their previous experi- mental data and data of several other workers, Harper and Rogers (1965) concluded that growth can be improved in rats on amino acid imbalanced diets as long as food intake can be maintained. Effects of Energy on Plasma Amino Acids The utilization of dietary protein by an animal depends on several closely integrated factors and one of 14C labeled these factors is available energy. Using glycine, Munro gt gt. (1962) observed that in diets con- taining adequate amounts of protein a high caloric intake caused an increase in total amount of glycine taken up by liver protein, but with protein free diets the dietary caloric level had no influence on incorporation. They therefore concluded that the major effect of the caloric content of the diet in influencing protein metabolism is on the utilization of circulating amino acids (plus tissue free amino acid pools) between meals and not during the active phase of amino acid absorption after meals. A similar conclusion was drawn by Knipfel gt gt. (1969) from amino acids incorporation data when fasted rats were in- 14 jected with C lysine and subsequently fed glucose. Rao and McLaughlan (1967) studied the effect of time on the 16 nitrogen sparing effect of dietary carbohydrates in rats and observed no significant difference in weight gain between rats fed carbohydrates with protein or rats fed casein in the morning and carbohydrates eight hours after. However, rats fed casein alone had an elevated plasma amino acid level until carbohydrates were fed eight hours later. Rats fed carbohydrate with casein had a signifi- cantly greater nitrogen retention on the first day but not on the following days, than rats fed protein and carbo- hydrate separately. They concluded that the effect of time factor on the nitrogen sparing effect of dietary carbo- hydrates was only transient. Munro (1964) reviewed several reports on the difference in actions of carbohydrate and fat on protein metabolism in several species and then concluded that fat either fails to reduce nitrogen output in fasting animals or is less effective than carbohydrate under comparable circumstances. He hypothesized that the apparent depression of plasma amino acids and their incor- poration into muscle protein after a carbohydrate meal is influenced by insulin secretion. Christensen (1964) pointed out that body tissue can absorb amino acids without immediately metabolizing them and that the increase in amino acid transport into tissues caused by insulin secre- tion in response to glucose ingestion might not necessarily accelerate tissue protein synthesis. Dror gt gt. (1969) reported that the positive effect of vfiiition of starch on 17 protein utilization depends on the physiological status of the animal and the composition of the diet, particularly the energy protein ratio. Infusion of energy, in man (Crofford gt gt., 1964) in sheep (Potter gt gt., 1968) in the lactating dairy cow, (Halfpenny gt gt., 1969) and in rats (Knipfel gt gt., 1969) caused a significant depres- sion of plasma free essential amino acids. The relative depression or depression pattern of essential amino acids depends on the source of energy infused (Potter gt gt., 1968). Potter gt gt. (1968) reported more cellular uptake of plasma free amino acid from proprionic acid and glucose than from butyrate and acetate infusion. The Role of Trygtophan in Mammalian Metabolism The role of tryptophan in mammalian metabolism seems to be very important, but unfortunately, enough work has not been done to pinpoint a specific role which is unique to tryptophan, other than for protein synthesis, although several attempts have been made. Munro (1968) summarized all reports on the loss of heavy polysomes and an accumu- lation of monosomes and oliogosomes which was somewhat unique to the absence of dietary tryptophan alone. He concluded that tryptophan is the essential amino acid which normally determines polysome aggregation in liver cells of intact animals, but not because of some specific or unique role but because tryptophan is least abundant in free amino acid pool and in the protein synthesized from such pool. l8 Tryptophan is uniquely low in tissue free amino acid pool, body tissue protein and dietary protein (Munro, 1970). Scott gt gt. (1969) reported that collagen which represents more than 1/2 of the animal's body protein contains abso- lutely no tryptophan. The absence of dietary tryptophan will more severely limit the charging of tryptophanyl t-RNA than the absence of any other amino acid (Munro, 1970). The availability of niacin in foods or feed stuffs is often extremely low, consequently, excess dietary tryptophan plays a vital role in satisfying the animal's requirement for niacin if niacin is not supplemented in the diet. The requirement for niacin is dependent on the level of dietary tryptophan (Harmon gt gt., 1969). Murata and Kimura (1969) feeding threonine free diet, tryptophan free diet or complete amino acid diet to rats concluded that dietary. tryptOphan which is normally used for protein synthesis is converted to nicotinic acid when protein formulation is limited by omission of threonine. Tryptophan also seems to be important in the mainten- ance of pregnancy. Lojkin (1962) feeding different levels of tryptophan in diets to pregnant rats, reported that 100 percent of the rats fed 0.136 percent tryptOphan diet had healthy and normal fetuses on the 20th day of gestation but in all rats fed .096 percent diet there was 100 percent resorption of fetus. She therefore, concluded that approxi- mately 0.136 percent tryptophan is required for normal pregnancy to proceed in rats. MATERIALS AND METHODS A. Experiment 1 1. General Design of Experiment In this study, four mature, rumen fistulated wethers ___ __ -3 __ _.._____ I were randomly allotted to a 4 x 4 Latin Square experiment (Table 1), involving four rations (Table 2), which con- tained different levels of protein (Table 3), and different energy sources. These rations were selected because they were used in previous experiments in this laboratory (Purser gt gt., 1966; Bergen gt gt., 1968a,b). However, rations 3 and 4 which were previously isonitrogenous are not isonitrogenous in the present study, because a 50% crude protein soybean meal was used instead of the original 40% crude protein soybean meal. Wethers were housed indoors singly in 4 feet x 6 feet metal stalls and given free access to fresh water. Each period consisted of a 21 day ration-adjustment period and a 3 day sampling period. The sheep were fed 1/2 of the dietary allowance at each feeding, 8 a.m. and 8 p.m., at the rate of 7% of their metabolic body weight (B.W. '75) (Kleiber, 1961). On each sampling day the animals were fed only in the morning immediately after the first sample was taken. On 19 20 TABLE 1 Experimental Design for Experiment 1 S h e e p N o. Period 94 990 32 28 l A B C D 2 B C D A- 3 D A B C 4 C D A B Treatments: A = Ration 1 B = Ration 2 C = Ration 3 D = Ration 4 21 TABLE 2 Rations Ingredients ta 23 Sb 4b Alfalfa meal _ 38.0 50.0 - Ground corn cobs 31.0 8.0 - - Ground corn 47.0 42.0 - 70.0 Cerelose 3.0 5.0 - - Starch 7.0 - - - Solka Floc 5.0 - - - Urea 0.5 - - 1.0 Mineral-Vitamin mixC 2.0 2.0 2.0 2.0 Soybean meal - - - 15.0 Basal Pelletsd - - 43.0 7.0 Molasses 5.0 ' 5.0 5.0 5.0 aPurser gt gt. (1966). bBergen et al. (1968a). CMineral-Vitamin mix contained in percent: Dicalcium phosphate, 47. 38; high zinc trace mineral salt, 47.38; NaZSO 4. 78; Vitamin A (10,000 IU/g), 0.32; Vitamin D ,008 IU/g), 0.10. dBasal Pellets contained in percent (air dry): Ground hay, 50.0; ground corn cobs, 30.0; Molasses, 5.0; Urea, 1.1; Mineral mix, 2.0; Soybean meal, 11.9. 22 TABLE 3 Crude Protein and Dry Matter Content of Rations Ration 1 Ration 2 Ration 3 Ration 4 % % % % Crude Protein 6.9 11.1 15.8 20.2 Dry matter 93.1 93.0 94.4 92.3 8 — ..- ..1‘ 4n._‘ . l . AIL-1:4; 23 each occasiOn sampling began at 8 a.m. On day 22 of the period blood and rumen samples were taken immediately before feeding (To) and sequentially at 3, 6, 9, 12, 16, 20 and 24 hours post feeding. On day 24 both blood and rumen samples were similarly taken immediately before feeding, but instead of the a.m. feeding a 2 : l starch- glucose mixture was instilled in the rumen at the rate of 1.5% of the metabolic body weight in 500 ml distilled water. Blood and rumen samples were again taken at 4 hours after dosing. 2. Sample Collection and Preparation a. Blood samples At each sampling time 10 ml of blood were collected from the right jugular vein of each sheep using a heparin- ized syringe. Immediately after collection, samples were centrifuged at 4,080 xg for 10 minutes to separate the liquid plasma from the solid portion of red cells. The plasma was then transferred into small test tubes with disposable pasteur pipettes. Two ml of the plasma were stored in the freezer (-70°) for later use in the spectro- flurometric determination of tryptophan (see Section 3 below). To the rest of the plasma 1 mM norleucine was added as an internal standard, at the rate of 0.1 ml norleucine per ml of plasma. This was followed by the addition of 50% (w/v) sulfasalycylic acid at the rate of 0.1 ml sulfasalycylic acid per ml of plasma and the mixture I. . M “..."... re fo ac Be ta} col wer of tub Sat: of ‘ fur1 frOn 1 m1 SOlu for‘ miHat 24 placed in an ice bath for 30 minutes. The sulfasalycylic acid was added to precipitate plasma protein. After 30 minutes the mixture was centrifuged at 47,000 xg for 15 minutes. The supernatant (protein free filtrate) was removed with a pasteur pipette and stored in the freezer for determination of a-amino nitrogen and complete amino acid analysis (Purser gt gt., 1966; Makdani gt gt., 1971; Bergen and Potter, 1971). b. Rumen samples A representative sample (50 m1) of rumen content was taken from the rumen through the rumen cannula with a collection tube at each sampling time. Rumen contents were squeezed through two layers of cheese cloth and 19 ml of the liquid portion (rumen liquor) transferred to‘a test tube containing 1 ml of saturated mercuric chloride. Saturated mercuric chloride prevents further degradation of the included substrates by rumen microbes and stops any further chemical reactions. A 5 ml sample was then taken from the 20 m1 rumen liquor-mercuric chloride mixture and 1 m1 of 25% metaphosphoric acid added to precipitate soluble protein and the mixture centrifuged at 12,100 xg for 15 minutes. The supernatant was saved for the deter- mination of short chain volatile fatty acids by gas liquid chromatography (see Section 6 below). 25 3. Tryptophan Determination Tryptophan was assayed using the "Simplified Spectro- fluorometric Micromethod” of Wapnir and Stevenson (1969).. Tryptophan standards were made by dissolving 100 mg of L-tryptophan in 100 ml of deionized distilled water to form a stock solution. Aliquots of 1 m1, 2 ml, 3 nfl., 4 m1 and 5 ml were taken from the stock solution and deionized distilled water added to each to make tryptophan standard solutions of l, 2, 3, 4, and S mg/100 ml respectively. Maximum recovery of tryptophan in plasma was obtained during trial runs, consequently, no conversion factor was necessary. A 20 ul portion of the sample or standard was placed in the center of a printed circle on filter paper cards (S a S 903-C, Schleicher 8 Schuell, Inc.) and air dried. A water filled circle was used as blank. Each sample or standard was run in triplicate. The dried circle was cut out, sectioned in four pieces and placed in clean, dry, labeled test tubes. To each test tube 1 ml of 78% (v/v) ethanol was added to elute the free tryptophan from the sectioned circle. Each test tube was stoppered immediately to avoid any evaporation of the ethanolic solution. Occasionally the test tube racks were gently shaken, making sure that each piece of paper was properly covered by the solvent at all times. After not less than 30 minutes 0.5 ml of the ethanolic extract was rapidly transferred to 26 another small test tube and 3 m1 of 0.02 M tris base solution added. The contents were mixed and immediately read on the Aminco-Bowman Spectrophotofluorometer (at an excitation wave length of 287 nm, emission wave length of 357 nm, excitation slit width of 2, emission slit width of 5, photomultiplier slit width of 4, meter-multiplier of 0.01 and a sensitivity of 50, using quartz cuvettes). To prevent quenching all glassware was acid washed, rinsed once with deionized distilled water, rinsed twice with absolute ethanol and rinsed twice with 78% ethanol. 4. a-Amino Nitrogen Determination (total free amino aciditest) The method of Palmer and Peters (1969) was used. A citrulline standard was made by dissolving 0.3504 g Citrul- line (M. W. 175.19) in deionized distilled water to form a stock solution of 10 uM/ml. From this stock solution 0.2, 0.4, 0.6, 0.8, 1.0 and 2.0 uM/ml citrulline standard solutions were made by diluting the appropriate aliquots to 100 ml with deionized distilled water. Fresh 0.25% 2, 4, 6, trinitrobenzene sulfonic acid (TNBS) (0.25 g/100 ml H2O) and 0.05 M Na2B4O7 - 10 H20 (19.07 g sodium borate per liter of water, buffered at pH 9.2) solutions were made weekly. To each test tube 0.2 m1 of either the citrulline standard or protein free filtrate, 1.6 ml of the 0.05 M sodium borate solution and 0.2 ml of the 0.25% TNBS reagent 27 were added and mixed. The mixture was incubated at 37°C for 20 minutes. At the expiration of this period, 2 ml of 1 N hydrochloric acid were added to each tube (to stop the reaction), and the content mixed. The optical density (O.D.) was read immediately on the Coleman Spectrophoto- meter, model 620, at a wave length of 420 nm using a reagent blank. 5. Rumen Ammonia and Blood Urea Nitrogen Determination a. Rumen NHS-N Rumen NHS-N was determined by the micro-diffusion method of Conway (1960). All dishes were prepared by placing 1 ml glycerol in the outer well, 1 ml boric acid solution (.04 N) to the inner well, 0.5 m1 of the original rumen liquor and 0.5 ml of distilled water to one side of the middle well and 1 ml potassium carbonate (KZCO3) solution (100% w/v) to the other side of the middle well, making sure that there was no dripping into the other ' compartments or on the sample. The lid was quickly placed on the plate and gently rotated in the glycerol a number of times to provide a seal, thus preventing the escape of the liberated volatile nitrogenous base. A water blank was prepared for each batch of samples run, in a similar manner, except that instead of the diluted sample or standard 1 ml distilled water was added. Each sample was run in duplicate. The dishes were swirled gently in order 28 to carefully mix the KZCO3 solution with the sample, and placed on a rotator for one hour. At the end of this period the content of the inner well (green in color due to the trapped nitrogen) of each dish was titrated with a standard solution of 0.04 N HCl until the color matched that of the water blank (light pinkish red) and burette readings recorded. El 1 b. Blood urea nitrogen L4 Blood urea nitrogen was determined by the micro- diffusion technique of Conway (1960). The procedure is the same as that used for the determination of rumen NHS-N, except that before the KZCO3 solution was added, 0.5 m1 urease solution (20 mg/ml) was added to the middle well to hydrolyze urea to NH3 and C02. After urea hydrolysis was completed (45 minutes) 1 ml KZCO3 solution was then added to the middle well and the procedure continued in the usual NH —N determination manner. 3 NH3-N was not determined in the plasma samples since a prior experiment had shown that the amount of NHS-N in normal plasma of peripheral blood was too low to determine with any degree of precision. 6. Volatile Fatty Acids Determination Rumen volatile fatty acid concentrations were deter- mined by Gas Liquid Chromatography using Packard instru- ments (model 840). The column (6 ft glass) was packed 29 with chromosorb 101, the flow rate of carrier gas (N2) was 70 ml/min., and the column temperature was maintained at 195°C. Volatile fatty acids in the rumen fluid were identified qualitatively and quantitatively by comparison with a standard solution of volatile fatty acids. B. Experiment 2 Four wethers, fitted with rumen cannulae, of approxi- LL mately equal body weight were fed each at 8 a.m. and 8 p.m. one of the four rations used in experiment 1, for 21 days at the rate of 7% of their metabolic body weight. On day 22 approximately 250 ml rumen content were taken from each sheep immediately before feeding at 8 a.m. and 1-1/2 and 4 hours after feeding and cooled to 3°C in ice. 1. Sample Preparatiqg Rumen protozoa and bacteria were separated from each rumen sample as follows: rumen contents were squeezed through two layers of cheesecloth and the rumen liquor (I) collected. The residue was resuspended in an ample amount of saline solution (0.9% NaCl) and squeezed through two layers of cheesecloth and the liquid portion (rumen liquor (2)) collected and mixed with rumen liquor (1). To the mixture of rumen liquors an equal volume of 37% formaldehyde solution was added to fix and preserve the rumen micro-organisms. The fixed solution was centrifuged at 250 xg for 15 minutes. The supernatant was saved for 30 subsequent isolation of bacteria and the pellets (protozoa) were washed and then freeze-dried. Supernatant from the first spin was centrifuged at 2,500 xg for 15 minutes after which the residue (feed particles) was discarded and the supernatant centrifuged again at 40,000 xg for 15 minutes. This time the supernatant was discarded and the pellets . ‘1 (bacteria) resuspended in a large amount of saline solution “—— and again centrifuged at 40,000 xg for 15 minutes. The supernatant was discarded and the pellets were freeze- dried. Samples were kept at 3°C throughout the entire sample preparation procedure. 2. Tryptophan Analygis Tryptophan determination on freeze-dried preparations of protozoa and bacteria were done by a modified BaOH2 protein hydrolysis procedure, followed by a modified colorimetric determination of tryptophan (Miller, 1967; Spies, 1967; Knox gt gt., 1970). To a screw capped glass test tube (approximately 25 x 150 mm) containing 5 g Ba(OH)2 - 8 H20, 300 mg of the finely ground dry protozoa or bacteria sample were added and the content was properly mixed. The walls of the tube were washed down with 4 m1 deionized-distilled water and the tube was saturated with nitrogen gas, tightly capped and then autoclaved at 15 1b/ in.2 for 7 hours after which the tube was left to cool overnight. In the following morning the tube was opened, 31 4 ml 6 N HCl added to the sample and the mixture trans- ferred to a 50 ml volumetric flask. The tube was finally washed twice into the flask with 1 ml portions of 6 N HCl, and 25 m1 of 17.5% NaZSO4 solution were then added to the flask to precipitate the barium. The resulting suspension was mixed and the volume made up to 50 ml with deionized distilled water. Ten ml of the well mixed sus- pension were centrifuged for 30 minutes at 14,000 xg. Two ml aliquots of the supernatant were transferred to three stoppered test tubes. To the first two tubes, 5 ml of 0.5% (w/v) p-dimethylaminobenzaldehyde in con- centrated HCl were added and mixed. After 20 minutes 0.2 ml aqueous 0.2% (w/v) sodium nitrite solution was added and properly mixed. The resulting solution was filtered through Whatman #2 filter paper after 25 minutes and the O.D. read on a Gilford spectrophotometer at 590 nm within 40 minutes after filtration. To the third tube (blank) 5 m1 concentrated HCl were added, followed by the addition of 0.2 ml of 0.2% sodium nitrite, filtration and reading on the Gilford spectrophotometer. A standard tryptophan curve was prepared by determining the O.D. of 2 ml aliquots of L-tryptophan solutions containing 0.5 to 4 mg tryptophan per 100 ml water. A preliminary experiment, using Lysozyme (7.5% tryptophan), revealed a 98.8% tryptophan recovery, consequently, a recovery factor was ignored. 32 C. Statistical Analysis Data were statistically analyzed for Analysis of Variance (Appendix 1) on a CDC, 3600 computer at the Michigan State University Computer Laboratory. Differ- ences between means were determined by the'Duncan's New Multiple Range Test” on the CDC, 3600 computer. #2221111 RESULTS Experiment 1 Plasma Free Tryptophag Plasma free tryptophan concentrations of sheep fed the four rations are shown in Table 4. The range of mean daily plasma tryptophan levels was 1.86 to 2.11 mg per 100 m1 plasma. There were no significant differences in plasma free tryptophan concentrations of sheep fed the four rations before feeding and at 3, 6, 12, 16, 20 and 24 hours post feeding. However, there was a significant difference (P<.05) between plasma concentrations of sheep fed ration 3 and sheep fed the other three rations at 9 hours after feeding. The diurnal patterns of plasma free tryptophan concentrations are shown in Figure l. Tryptophan concentration increased to its maximum level at 9 hours after feeding, then declined to its minimum level at 16 hours after feeding for sheep fed rations l and 2 and at 20 hours after feeding for sheep fed rations 3 and 4. Sheep fed ration 3 were the only ones which had a signifi- cant difference (P<.05) in tryptophan concentration between time after feeding (between T9 and T20). Although ration 4 had the highest level of crude protein (Table 3) the plasma 33 34 TABLE 4 Plasma Free Tryptophan Concentrationa’b During 24 Hour Period (Experiment 1) TimeC Ration l Ration 2 Ration 3 Ration 4 TO 1.82:.07 1.84:.05 2.05:.07 1.78:.06 T3 1.761.10 2.01:.05 2.08:.11 1.83:.04 T6 1.83:.04 2.04:.06 1.96:.10 1.83:.09 T9 2.07:.07 2.25:.09 2.46:.06 2.10:.10 T12 1.89:.04 2.05:.10 2.41:.08 1.98:.14 T16 1.65:.09 1.68:.09 1.96:.08 1.971.16 T20 1,911.11 l.87i.13 1.82:.09 1.71:.10 T24 1.901.15 1.87:.03 2.11:.05 1.86:.11 a‘ bMean and standard error for 4 sheep. CHours after feeding. Mg tryptophan per 100 m1 plasma. 35 3.0 Ration lH—H Ration 2‘—‘—-‘—' Ration 354-4-4 flu Ration 4r-o--o-—o 3: 1' 2.5 N g / \\ E / 1 \ f O q 2 d \/ __ no / x E x 0—-—— r \y/ / 1.5 fi> f 1 I 4 e I t 3 6 9 12 16 20 24 Time in hours Figure 1. Diurnal pattern of plasma free tryptophan concentrations 36 free tryptophan levels of sheep fed this ration were lower (although not statistically significant) than the corres- ponding levels of sheep fed rations 2 and 3 and approxi- mately equal to the corresponding levels of ration l which is roughly three times lower in crude protein content. Plasma free tryptophan concentrations before and after starch-glucose infusion are shown in Table 5. There were no significant differences between tryptophan levels of sheep fed the four rations. The T4/TO ratios were 87%, 93%, 98% and 91% for sheep fed rations l, 2, 3 and 4 respectively. Plasma d-amino Nitrogen Plasma a-amino nitrogen concentrations of sheep fed the four rations are presented in Table 6. There were no significant differences in plasma a-amino nitrogen concen- trations of sheep fed the four rations before feeding and at 3, 6, 9, 20 and 24 hours post feeding. However, there were significant differences between plasma a-amino nitrogen levels of sheep fed rations at 12 and 16 hours after feed- ing (P<.005). Plasma a-amino nitrogen concentrations of sheep fed the four rations decreased during the first six hours after feeding (Figure 2) and then increased between 9 and 12 hours after feeding; decreased at sixteen hours and then increased between 20 and 24 hours after feeding. Only sheep fed ration 3 had a significant difference (P<.05) in a-amino nitrogen concentration at various intervals after 37 TABLE 5 Plasma Tryptophan Concentrationa’b Before and After Starch—glucose Infusion (Experiment 1) TimeC Ration l Ration 2 Ration 3 Ration 4 T0 1.55:.08 1.62:.10 1.88:.15 1.613.15 T4 1.35:.09 1.51:.16 1.85:.17 1.46i.18 T /T Ratio 870 93% 980 910 aMg tryptophan per 100 ml plasma. bMean and standard error for 4 sheep. CHours after feeding. 38 TABLE 6 Plasma d-amino Nitrogen Concentrationa’b During 24 Hour Period (Experiment 1) TimeC Ration 1 Ration 2 Ration 3 Ration 4 T0 2.95:.16 2.91:.06 3.05:.10 2.64:.08 T3 2.59:.06 2.40:.09 2.59:.07 2.33:.11 T6 2.56:.12 2,681.04 2.57:.03 2.13:.10 T9 2.80t.07 2.70:.05 2.63:.05 2.18:.13 T12 2.81:.10 2.98:.05 2.86:.07 2.44:.16 T16 2.79:.10 2.76:.05 2.43i.05 2.29:.09 T20 2.82:.13 2.85:.06 2,651.08 2.31:.17 T24 3.16:.10 2.80:.12 2.86:.07 2.73:.12 aMicromoles of a-NHz-N (as citrulline) per ml plasma. bMean and standard error of 4 sheep. CHours after feeding. 39 4.04 Ration Ration Ration Ration fir 2.01- a-amino nitrogen micromoles per m1 plasma q— - J_ l I 3 6 £9 12 1'6 1- Time in hours .htnuna x_.u_.p_4s °—-O——¢-—0 h >- 2'0 74 Figure 2. Diurnal pattern of plasma a-amino nitrogen concentrations 40 feeding (between T0 and T16)' Plasma a-amino nitrogen concentrations of sheep fed ration 4 were lower than the corresponding values of sheep fed the other three rations throughout the 24 hour period. Plasma a-amino nitrogen concentrations before and after starch—glucose infusion are shown in Table 7. There was a decline in the a-amino nitrogen levels of sheep fed if all rations due to the infusion of energy. The T4/T0 ratios were 83%, 88%, 84% and 80% for sheep fed rations l, 2, 3 and 4 respectively. Rumen Ammonia Nitrogen Rumen ammonia nitrogen concentrations of sheep fed the four rations throughout the 24 hour period are shown in Table 8. There were significant differences between rumen ammonia concentrations of sheep fed the four rations 0, P<.001; T3, T6, T9, T12, 16’ P<.0005). Higher rumen ammonia nitrogen con- throughout the period (T T T and T 20 24’ centrations were associated with the higher crude protein rations. The diurnal patterns of rumen ammonia nitrogen concen- trations are presented in Figure 3. Rumen ammonia nitrogen concentration increased rapidly to its maximum level at three hours after feeding, then gradually decreased to its minimum level at nine hours, then gradually increased from 16 through 24 hours post feeding. 41 TABLE 7 Plasma d-amino Nitrogen Concentrationa’b Before and After Starch—glucose Infusion ! IHA Ration 4 e Time Ration 1 Ration 2 Ration 3 ‘ I TO 2.97:.12 3.03:.12 3.07:.03 2.78:.07 T4 2.46:.08 2.67:.1 2.59:.09 2.22:.06 9 9 9 9 T4/T0 83a 88. 840 80. aMicromoles of a-NHZ-N (as citrulline) per ml plasma. bMean and standard error for 4 sheep. CHours after feeding. . . . a b Rumen Ammonia N1trogen Concentrat1on ’ TAB 42 LE 8 Hour Period (Experiment 1) During 24 TimeC Ration l Ration 2 Ration Ration 4 TO 5.91:2.2 l4.76:1.6 21.0: 1 43.87: 3.5 T3 7.70:2.1 19.04:2.3 22.68:l 66.17: 3.4 T6 2.94:1 1 11.51:2.5 13.34:l 60.05:l4.5 T9 3.85:1 l 12.15:2.0 13.2 :1. 37.10: 1.1 T12 3.84:1 2 l4.06:2.0 16.51:0 39.16: 2.6 T16 5.85:0 8 15.Sl:2.l l6.77:0 42.65: 3.1 T20 10.57:l.0 18.84:2.2 19.02:l 44.09: 2.1 T24 13.31:l.0 19.15:2.1 l6.28:0 47.39: 1.1 aMg nitrogen per 100 ml rumen content. bMean and standard error for 4 sheep. CHours after feeding. 43 70» Ration l H—x—u Ration Z H—o—O Ration 3 k+—K-—F /\ Ration 4 o-—o-—o-—° \ 60> / \A \ / \ 40*, \t//°// 30L mg nitrogen per 100 ml rumen content J __l L I r I 3 6 0 12 16 20 24 Time in hours Figure 3. Diurnal pattern of rumen ammonia concentrations 44 Rumen ammonia nitrogen concentrations before and after starch-glucose infusion are shown in Table 9. There was a significant depression in rumen ammonia concentration four hours after starch-glucose infusion. The T4/T0 ratios were 25%, 67%, 29% and 44% for sheep fed rations l, 2, 3 and 4 respectively. Blood Urea Nitrogen Blood urea nitrogen concentrations of sheep fed the four rations are shown in Table 10. There were significant differences in blood urea nitrogen levels of sheep fed the? four rations throughout the 24 hour period (TO, P<.O4; T T T T T and T T P<.005). As in the case 3’ 6’ 9’ 12’ 16’ 20 24’ of rumen ammonia, higher blood urea nitrogen concentrations were associated with the higher crude protein rations. The diurnal patterns of blood urea nitrogen are shown in Figure 4. Blood urea nitrogen concentrations rapidly increased at three hours after feeding then gradually decreased to their minimum levels at 9 hours in the case of sheep fed rations l and 2 and 12 hours after feeding for sheep fed rations 3 and 4, then gradually increased through to 24 hours post feeding. Blood urea nitrogen concentrations before and after starch-glucose infusion are presented in Table 11. Blood urea nitrogen concentrations were not greatly depressed by energy infusion, when compared with the significant . 'F‘T‘T‘T_‘ ‘1 45 TABLE 9 Rumen Ammonia Nitrogen Concentrationa’b Before and After Starch-glucose Infusion (Experiment 1) Time Ration l Ration 2 Ration 3 Ration 4 TO 9.07:1.9 20.18:2.7 24.14:1.6 45.40:5.8 T4 2.30:0.8 l3.54:2.7 7.11:1.3 l9.99:4.4 T4/T0 25% 67% 29% 44% aMg nitrogen per 100 ml rumen content. bMean and standard error of 4 sheep. CHours after feeding. 46 TABLE 10 Blood Urea Nitrogen Concentrationa’b During 24 Hour Period (Experiment 1) Time Ration l Ration 2 Ration 3 Ration 4 T0 4.45:1 9.90:1.1 l7.56:l.0 25.04:2.0 T3 5.35:1 9.67:1.6 19.15:2.6 29.58:3.1 T6 5.17:1 7.31:0.6 19.12:l.4 29.33:2.3 T9 4.12:0 8.69:1.0 l7.60:l.4 25.85:l.3 T12 4.28:0 9.76:1.0 17.36:1.4 24.80:l.8 T16 4.23:0. 11.13:0.7 18.80:1.1 21.87:0.6 T20 6.03:0. 14.21:O.7 18.85:1.1 23.39:1.l TZ4 8.48:1. l7.86:0.6 23.31:l.2 24.75:l.0 aMg nitrogen per 100 ml blood plasma. b CHours after feeding. Mean and standard error for 4 sheep. 47 501+ Ration.l.x—*—&—* Ration 2 H—o—a Ration 3 any...“ Ration 4 o-—o--o--c 404 mg nitrogen per 100 m1 plasma 044- C‘ 69 H I N H 0‘ N O 24 Time in hours Figure 4. Diurnal pattern of blood urea nitrogen concentrations Cl. 48 TABLE 11 Blood Urea Concentrationa’b Before and After Starch-glucose Infusion (Experiment 1) Timec Ration l Ration 2 Ration 3 Ration 4 T0 4.85:0.2 12.01:l.4 l6.55:0.9 21.38:l.2 T4 3.71:0.1 9.49:1.1 15.07:0.8 16.66:l.4 T4/TO 76% 79% 91% 78% aMg nitrogen per 100 ml blood plasma. bMean and standard error for 4 sheep. CHours after feeding. 49 depressions noted for rumen ammonia. The T4/TO ratios were 76%, 79%, 91% and 78% for sheep fed rations l, 2, 3 and 4 respectively. Rumen Volatile Fatty Acids Rumen acetate concentrations of sheep fed the four rations are shown in Table 12. There were no significant differences in rumen acetate concentrations of sheep fed the four rations throughout the 24 hour period. Rumen acetate concentration increased to its maximum level at three hours after feeding and then gradually decreased throughout the rest of the 24 hour period. The rumen acetate concentrations before and after starch-glucose infusion are presented in Table 13. The data indicate that the starch glucose infusion caused a slight depression in rumen acetate concentration. The T4/TO ratios were 75%, 94%, 66% and 80% for sheep fed rations l, 2, 3 and 4 respectively. Rumen prOprionate concentrations of sheep fed the four rations are shown in Table 14. There were no signifi- cant differences between rations in the production of rumen proprionate. Rumen proprionate concentrations reached their maximum levels at three hours then steadily de- creased throughout the rest of the 24 hour period after feeding. Rumen proprionate concentration before and after starch—glucose infusion are shown in Table 15. 50 TABLE 12 Rumen Acetate Concentrationa’b During 24 Hour Period (Experiment 1) TimeC Ration l Ration 2 Ration 3 Ration 4 TO 60.0:5 47.0: 5 55.0: 7 56.0:6 T3 67.0:5 64.0:12 84.0:12 75.0:7 T6 60.0:7 67.0: 8 79.0: 9 58.0:3 T9 59.0:4 56.0: 6 67.0:11 60.0:4 T12 43.0:4 43.0: 8 58.0: 4 60.0:4 T16 44.0:2 40.0: 9 44.0: 1 56.0:6 T20 40.0:3 29.0: 3 30.0: 2 42.0:3 T24 28.0:3 22.0: 2 20.0: 1 31.0:5 aMicromoles acetate per ml rumen liquor. bMean and standard error for 4 sheep. CHours after feeding. 51 TABLE 13 Rumen Acetate Concentrationa’b Before and After .—1 Starch-glucose Infusion (Experiment 1) TimeC Ration 1 Ration 2 Ration 3 Ration 4 T0 58.0:4 53.0:10 58.0:10 62.0:10 T4 44.0:4 50.0: 9 38.0: 6 50.0: 4 9 4 9 T4/T0 75% 940 660 800 'aMicromoles acetate per 100 ml rumen liquor. bMean and standard error for 4 sheep. CHours after feeding. 52 TABLE 14 Rumen Proprionate Concentrationa’b 24 Hour Period (Experiment 1) During TimeC Ration l Ration 2 Ration 3 Ration 4 TO 11.0:2 12.0:3 12.0:2 13.0:2 T3 13.0:2 21.0:6 24.0:2, 23.0:1 T6 10.0:2 19.0:4 19.0:2 17.0:2 T9 10.0:2 14.0:3 14.0:2 16.0:2 T12 10.0:3 11.0:3 12.0:1 13.0:2 T16 7.0:1 10.0:4 10.011 ‘ 14.011 T20 6.0:0 6.0:2 7 0:0 9.041 T24 5.0:0 5.0:1 4.0:0 7.0:1 aMicromoles proprionate per 100 ml rumen liquor. bMean and standard error for 4 sheep. CHours after feeding. 53 TABLE 15 Rumen Proprionate Concentrationa’b Before and After Starch-glucose Infusion (Experiment 1) TimeC Ration l Ration 2 Ration 3 Ration 4 T0 10.0:1 14.0:4 13.0:2 16.0:4 T4 15.0:3 22.0:7 12.0:1 28.0:5 T4/TO 150% 157% 92% 175% aMicromoles proprionate per 100 ml rumen liquor. bMean and standard error for 4 sheep. CHours after feeding. 54 The starch-glucose infusion caused a substantial increase in rumen proprionate concentrations of sheep fed rations l, 2 and 4 and a slight decrease in sheep fed ration 3. The T4/TO ratios were 150%, 157%, 92% and 175% for sheep fed rations 1, 2, 3 and 4 respectively. Rumen butyrate concentrations of sheep fed the four rations are shown in Table 16. There were no significant differences between rations in rumen butyrate concentrations at 6, 9 and 12 hours after feeding. However, rumen butyrate was significantly higher in sheep fed ration 4 than sheep fed the other three rations before feeding and at 3, 16, 20 and 24 hours after feeding (P<.05). Rumen butyrate concen- trations before and after starch-glucose infusion are presented in Table 17. Starch-glucose infusion caused a slight decrease in rumen butyrate concentrations of sheep fed rations l and 4 and a slight increase for sheep fed rations 2 and 3. The T4/TO rations were 88%, 118%, 142% and 92% for sheep fed rations l, 2, 3 and 4 respectively. Plasma Amino Acid Concentration Plasma amino acid concentrations before and after starch-glucose infusion are presented in Table 18. There was a substantial depression in the total plasma free essential amino acid concentrations of sheep fed the four rations after the intraruminal infusion of the starch- glucose solution. However, individually, there was an increase in lysine and histidine concentrations in sheep 55 TABLE 16 Rumen Butyrate Concentrationa’b During 24 Hour Period (Experiment 1) TimeC Ration 1 Ration 2 Ration 3 Ration 4 r0 8 0:0 7.0:1 6 0:1 12.0:2 T3 9.0:2 9.0:1 9.0:2 14.0:3 T6 9.0:3 10.0:1 9.0:2 11.0:1 T9 9.0:2 9.0:1 7.0:1 12.0:2 T12 8.0:1 8.0:3 7.0:1 12.0:3 T16 6.0:1 6.0:2 5.0:0 13.0:3 T20 5 0:1 4 0:0 3.0:0 9 0:2 T24 3 0:0 3 0:1 2 0:0 7 0:2 aMicromoles butyrate per 100 ml rumen liquor. bMean and standard error for 4 sheep. CHours after feeding. 56 TABLE 17 Rumen Butyrate Concentrationa’b Before and After Starch—glucose Infusion (Experiment 1) 1 Jill. 7F . _. 11.. . s Time Ration l Ration 2 Ration 3 Ration 4 T0 8.0:0 11.0:3 7.0:2 12.0:1 T4 7.0:0 13.0:4 10.0:2 11.0:1 9 9 9 9 T4/T0 88. 118. 142. 920 aMicromoles butyrate per 100 m1 rumen liquor. bMean and standard error for 4 sheep. CHours after feeding. 57 '. 1:1} . . :44 66.N 4N.N N6.N 6N.N 66\.6.z New rm 64464.66N 6 6646N.6N6 66. 6. 6N N4N4. 66N 6. N646N. NN6 N446N 64. 6.N 446.NN 6.4N4N6.6N NN. 6. N 466.4N 6. N 46N.6N 66N66464 NN. 4.6 46N.6 4.N 466.4 66. N. 6 466.6 6. 6 46N.6 6644666 66. 4.6 466.N4 6.4N4N6.66 46. 6. N 466.66 6. 6 466.66 66N66N< 66.N 6.6N466.46 N.4N446.N6 6N.N 6. 6N4N6.6NN 4. 6N46N.66N meNusNu 66. N.N 446.6N 6.4 466.4N 66. 6. 6 466.NN 4. 6 4N6.6N oeNN646 6N. 6.6 466.4N 6.4N46N.46 66. N. 6 466.NN N. 6 466.N6 6646446N6 66. 6.6 4N6.6N 6.6 466.6N N6. 6. N 46N.6N N. 4 466.6N 6N66 6N6646N6 66. 6.N 466.6 6.N 46N.6N N6. 6. N 466.6 4. N 466.NN 664644666< 6N. 6.4 46N.NN N.4 4NN.NN 46. 6. N 4NN.6N 6. N 4N6.NN 66N466 66. 6.6 466.N 6.6 466.N 6N.N N. 6 4NN.N N. 6 46N.N 6N64 6N44466< 66. 6.6 46N.NN N.6 466.6N 66. 4. 4 4NN.6N 6. N 4N6.NN oemeN-N66465-z mvNu< ocNE< NmNuco mmocoz 66w 6.6N464.N6N 6.N6466.QNN 66w N.6N4N6.NNN 6.66464.mNN N44ON 66. N.N 464.N N.6 466.NN NN. N.N 4NN.N 6.N 4N6.6 6:464N4N66666 N6. 6.6 46N.6N 6.6 46N.4N NN. N.N 4NN.6N 6.4 466.NN meNusoN 66. 6.N 4N6.6 6.6 464.4N 6N. 6.6 466.6N 6.6 466.6N 66N666N66N 6N. 6.6 46N.4 6.N 4NN.6 66. N.N 466.4 6.N 4N6.6 66N664646z 66. N.6 446.NN N.N 466.66 N6. 6.6 4N6.6N 6.6 466.6N 66NN4> 66. 4.N 4N6.6N 4.6 4NN.4N 6N. 4.6 466.NN 6.6 446.NN 66N66646N NN. 6.6 4NN.NN N.N 4N6.6N 66. 4.N 46N.6N 6.N 466.NN 66N6464< N6. 6.6 446.6 4.N 466.NN 66. N.6 46N.6N 6.N 466.6N 66N6N46N: 66. N.N 466.4 6.N 466.N 66. 6.N 446.6 N.N 466.6N 66N666 mwNo< ocNE< NmNucommm 6N\4N 4N 6N 6NN4N 4N 6N 646< oeNE< N :6N466 . N :6N466 . mN udoENNoame cONmsmcH omoost-nonum Nopw< use opowom :oNumNucoocou pNo< ocNE< «EmmNm 6.6 6N 66646 58 .meNom ocNEm NmNucommo ou NmNucommoco: mo oNNmm .mooam Nsow Now NONNo wumwcmum one :66: .6566Na NE OON Non moNOEONosz U 44.N 4N.N N4.N 66.N 66\.6.z 66. 6. N646N. 6NN 6.6 46N..666 6N. 6. NN466.66N 6 .N6 46N.66N N646N 6N. 6.6 446. NN 6.6 466. NN 6N. 6. N 464.6N 6. N 446.6N chNONNN 66. 6.6 46N.4 N.N 466.4 66. N. 6 466.N 6. N 4N6.6 6:446N6 66. 6.6 4N6 64 N.N 4N6.66 6N. 4. 4 4N6.N4 6. NN466 66 66N64N< N6. N.6 466.N6N 6. 4 466. 6NN 6N. N. 6 466.N6 6. 6N46N 46 chUNNu 66. 6.4 46N.6N 6.N 446. 6N 66. 4. 6 4N6.6N 6. N 466.6N 66NN646 NN. N.6 466.6N 6.6 464.N6 N6. 4. 6 446.6N 6. 6 466.6N oeNeaNsNo N6. 6.6 466.NN 6.N 466. NN 6N. 6. 6 466.NN 6. 6 4NN. 6N 6N66 6N6446N6 66. 6.N 4N6.6 6.N 466. 6 6N. N. 6 4N6.6 N. N 46Y N 66N6N4666< 66. N.4 4N6.6N 6.N 4N6.NN 6N. 6. 6 466.NN 6. N 466.6N 66N466 66.N 6.6 46N.N N.6 4N6. N 66. 6. 6 4NN.N 6. 6 4N6.N 6N66 6N44466< N6. 6.N 4N6.NN N.4 466. 6N 66.N 6. 6 446.6N 6. 6 466.6N 66N6NN-NN646E-2 mwNo< ocNE< NmNuc om mocoz 66. 4.NN466.6NN 6. @6466. 64N 66. 6.46N4N6466N 6. 6N466. 46N NNNON 6N. 4.N 4NN.6N 6.N 446. 6N 66. Y 6 46N.6 N. N 446. 6N 66N66N6NN6666 N6. 6.4 464.NN 6.4 46N. 6N NN. N. N 466.6N N.6 4N6. 6N 66N666N NN. N.N 46N.6 6.N 466. 6N NN. N.6 466.NN 4.N 466.4N 66N666N66N 46.N 6.N 446.6 6.6 4NN.6 66. 6. N 46N.6 6.N 4NN.6 66N664646z 6N. 6.4 4N6.NN N.6 4N6.6N 4N. N. 6 466.66 N.N 4N6.N4 66NN6> 66. 6.N 466.6N 6.6 46N.6N NN. 4. 4 446 6N N.6 4N6.66 66N66646N N6. 4.N 446.NN N.4 466.4N N6. 6. N 4N6.6N N.4 466.6N 66N6N64< N6. N.N 466.6N N.6 466.NN 6N.N 6. N 466.6 6.6 4N4.N 66N6N4646 66. 6.N 466.N 6.N 466.6 66.N 4. N 466.6 6.N 4N6.6 66N6NN mwNu< ocNE< NwNuco :6 m 6NN4N 4N 64 6NN4N 4N 6N 6N6< oeNe< (H :oNNmm m :oNNmm N6666N46666 6N 666H0Hw>n ogp cfi wfium o:HEm wo>H0mop ma OOH Hog wfiom ocflEm wo>H0moh wzm Hm.m mN.v mm.v mm.m NH.m m~.m Nw.m mm.v mm.v Hm.v mm.m .mzm BN.HH mm.m mo.OH Hm.w ma.m Hv.m no.w mm.w OH.OH NN.w no.w .564 mm.“ mm.w mm.n wH.m ww.n mm.w mm.w 5N.m nm.u NH.m HN.w .SoHH oo.H vm.o 00.0 wH.H mo.H v~.H HN.H mm.H mm.H mH.H mm.o .poz No.0 efi.c Ho.m v0.0 wH.o NN.o HH.o mm.m NH.~ mm.m mm.m .Hm> mv.n ov.o va.o mo.o av.m ow.m wn.m Hm.m mN.w mm.o em.m .mH< mN.m Nv.m No.0 wm.o mv.m om.m om.m mN.m oo.m Nv.m um.m .xao Hm.m N~.m em.m oa.m mw.¢ ¢N.m mm.e Hw.¢ wo.m em.v mm.v .oum Nv.mH mo.HN NH.NN wm.mH Nw.wH oo.mH mm.o~ mm.o~ ev.ma mn.HN om.HN .HSHU HH.m mo.m na.m mm.m wN.m n<.m vm.m Nm.m mN.¢ wm.m mm.m .Hom on.¢ mm.¢ om.v mo.m mm.v mm.m o~.m oo.m m<.m mo.m mm.m .opna mm.oH Hm.oa Hm.m om.oH wo.m «v.0H mn.OH oo.HH 0H.HH oo.oa mm.OH .Qm< mo.v mm.m mm.m mv.m mv.© vw.v mo.v wH.v nm.~ vm.m No.¢ .my< mN.H mo.H ww.o ou.H mm.m mm.H no.H vo.H om.H om.H Nv.H .mflz mo.m om.m mN.m NN.w mw.m mm.m mm.w Nw.m mm.n nw.m wv.n .m%A we N\H ah ah we N\H HH ob N\H He o9 «H N\H HP o9 uflo< v coHumm m cowumm N :owumm H :owumm ocfle< NN mqmoq wfio< o:wE< 63 e-Formyl-lysine and also a formyl derivative of histidine. Tyrosine was completely destroyed. Thus the data for lysine and histidine are of little value for interpretation and comparison to previous results. DISCUSSION Results from this study indicated that levels of plasma free tryptophan in sheep were not affected by the type of ration or the quantity of crude protein fed. In humans, Young 33 31. (1971) found little change in fasting or postprandial plasma tryptOphan concentrations at tryptophan intakes below 3 mg per kilogram body weight per day or at tryptophan intakes above 5 mg per kilogram body weight per day. However, they reported that the concen- tration of tryptophan in fasting plasma increased linearly between intakes of 3 and 5 mg tryptophan per kilogram body weight per day. In ruminants the amount and quality of protein reaching the small intestine depends on the extent of dietary protein degradation and the extent of resynthesis of microbial protein (Smith, 1969). When feeding practical type rations (dietary protein which is readily degradable), the quantity of protein reaching the lower gut is not markedly affected by dietary nitrogen intake in ruminants. This levelling effect has been ascribed to the activity of the ruminal microbes (Hungate, 1966). Hogan and Weston (1969) summarized data showing that the amount of protein reaching the lower gut in sheep did not vary between 64 I '1 llllill I ll ‘1' ll. " 'all’ II. . I'll II I I III I IIIII \llllll ' I lllllllu l I 6S dietary intakes of 8 to 20% crude protein. According to Purser and Buechler (1966), Bergen gt 31. (1968) and Purser (1970) the bulk amino acid composition of rumen microbial protein reaching the lower gut is constant and is not affected by change of ration. The results of the second experiment of the present study indicated that the content of tryptophan in rumen bacterial and protozoal protein was fairly constant and was not affected by dietary treatments or time after feeding. Since the quantity of . rumen microbial protein and the bulk amino acid and trypto- phan content of rumen microbial protein reaching the lower gut was quite constant at the levels of dietary protein intakes used in this study, it can be postulated that the amount of tryptophan reaching the small intestine and available for absorption was pretty much constant among the 4 rations used. The above postulation is only correct if little or no protein bypassed the rumen and if there were no large differences in tryptophan digestibility or availa- bility from the microbial protein (Bergen gt 31., 1967). A considerable proportion of the dietary proteins in rations l, 2 and 4 were in the form of zein protein (a protein which is not degraded extensively in the rumen, McDonald, 1954) and thus a large amount of this protein will pass out of the rumen undegraded to the lower gut. Since zein contains low levels of tryptophan, intestinal digestion of this protein would depress the level of 66 tryptophan being absorbed from the small intestine. The constant level of tryptophan in the plasma circulating pool and other tissue pools may also be attributed to the fact that any excess in the absorbed tryptophan which is not immediately used for protein synthesis or other cellular metabolic processes will be rapidly deaminated and cata- oolyzed, establishing an equilibrium between the entry and the removal of tryptophan from the plasma pool. The diurnal pattern of plasma tryptophan levels for sheep was similar to the patterns reported for nonruminants (Wurtman, 1970). When compared to the fasting level, the concentration of tryptophan in the plasma pool increased gradually (although such a change was not statistically significant) to its highest level by 9 hours postprandial, declined to its lowest level between 15 and 20 hours post- prandial and then rose back to the fasting level by 24 hours postprandial. Chester (1971) reported that diet induced alterations observed in plasma amino acid concen- trations vary with time after feeding and that the plasma amino acid pattern reflects the incoming dietary amino acid more than the animal's amino acid requirements. Feigin gt gt. (1971) reported a definite and constant pattern of blood amino acid finmhmicity in humans despite variations in the amino acid concentrations. A 12 hour shift in the sleep and wakefulness cycle resulted in a rapid reversal of such diurnal rhythmicity. The diurnal 67 ’pattern of plasma tryptophan throughout the 24 hour period of this study indicated that tryptophan concentration reached its maximum level at nine hours after feeding whereas the bUlk amino acid concentration, as indicated by the a-amino nitrogen concentration, reached its maximum level at 12 hours after feeding. The differences in the diurnal pat- terns of tryptophan and a-NHZ-N may be related to the fact that tryptophan is involved in some form of metabolic process or processes such as polysome aggregation (Munro, 1970) or acts as the precursor for tryptamine, serotonin, melatonin and nicotinamide adenine dinucleotide synthesis (Sourkes, 1971) which precede protein synthesis or that tryptophan is more rapidly absorbed and degraded than the other amino acids. It has been claimed by Potter gt gt. (1968) and shown by Purser gt gt. (1966) and Munro (1964) that administra- tion of a dose of energy to a fasted animal will cause a depression in plasma free essential amino acids, in par- ticular those low in the diet and also the branched chain amino acids. Purser gt gt. (1966) stated that the limiting amino acid for the ruminant may be indicated by the essen- tial amino acid which showed the largest percent decline after the administration of readily available energy into the rumen. Such a method for determining the limiting amino acid may not be precise, however, if the level of an essential amino acid is not greatly depressed by the sudden '1‘! 68 influx of energy. This finding may suggest that such an essential amino acid was not among those acids which were low in supply. Based on this assumption the T4/T0 ratio of plasma tryptophan concentrations (Table 5) indicated that tryptophan was not limiting in the lower gut for sheep fed the four rations in question. Using Purser's gt gt. (1966) T4/TO ratio method to determine the limiting amino acid (see Table 18) it would appear that lysine was the most limiting amino acid for sheep fed ration 1 and second limiting for sheep fed ration 2. Leucine on the other hand seemed to be the limiting amino acid for sheep fed ration 2 and the second limiting for sheep fed ration 4. Phenyla— lanine seemed to be the limiting amino acid for sheep fed ration 3 and threonine for sheep fed ration 4. The T4/T0 ratio of rations l and 2 compared well with those reported by Klopfenstein gt gt. (1966) who used the same two rations in their studies. The a-amino nitrogen data presented in Table 6 indi- cated that the total plasma amino acid concentration was not influenced by dietary treatments. Depending on the quantity of dietary protein fed and the quality of protein reaching the abomasum of ruminants there may or may not be differences in the total amino acid concentration due to dietary treatments (Purser, 1970). One could argue that in ruminants, if the dietary protein sources are such that there is little or no rumen bypass, then the plasma amino 69 acids should not be influenced by dietary treatments. However, if a large proportion of the protein bypaSses the rumen then one may expect to find variations in plasma amino acid levels due to quality and quantity of protein fed. Qrskov gt gt. (1971a), using soybean meal as a protein supplement, observed an increase in total amino acids reaching the small intestine with each increasing level of dietary soy protein up to 19% crude protein, beyond which there was no further increase. In another study using fish meal and urea as dietary crude protein supplements, wrskov gt gt. (1971b) reported an increase in total amino acids reaching the small intestine with each increasing level of dietary fish meal but no increase when increments of urea supplement were used. They concluded that the increase in amino acids in the small intestine when fish meal supplement was used was due to the fact that most of this protein bypassed the rumen. Increased levels of urea did not increase microbial protein synthesis hence the total amino acid reaching the small intestines when urea supplements were used was constant (Hogan and Weston, 1969). Hume gt gt. (1970) reported a linear increase in flow rate of protein (i.e., tungstic acid precipitable nitrogen x 6.25) out of the rumen with increasing levels of nitrogen intake between 2 and 9 grams nitrogen per day, no further increase between 9 and 16 grams nitrogen per day and a net nitrogen loss (as a percent of intake) at intakes greater than 16 grams 70 nitrogen per day. In the present study the lack of signifi- cant differences in plasma total free amino acid nitrogen between sheep fed the four rations can be attributed to the_ fact that the supplies of amino acids to the lower digestive tract were the same for sheep fed the four rations. Thus the changes observed in the diurnal pattern of total amino acid nitrogen were mainly changes in protein reaching the intestine due to time after feeding and metabolic rhythmicity. Sheep fed ration 4 which contained the highest level of crude protein and digestible energy had the lowest levels of total plasma amino acid nitrogen. This can be inter- preted to mean that there was greater protein synthesis or cellular uptake of amino acids in sheep when ration 4 was fed (Halfpenny gt gt., 1969). The observed levels of rumen ammonia and blood urea nitrOgen which were associated with the higher crude pro- tein rations in this study were in agreement with the report of Lewis (1957); Tagari gt gt. (1964) and Preston gt gt. (1965) who reported a significant correlation between the crude protein content of the ration, rumen ammonia and blood urea concentrations. The drop in rumen ammonia and blood urea concentrations to a low level between 9 and 16 hours after feeding and the subsequent increase during the last 8 hours of the 24 hour period was in accordance with the work of Lewis and McDonald (1958) who claimed that the increase in rumen ammonia and blood urea levels during the llll'fllll‘llull‘lllllll ' 'I 71 last 8 hours may be due to continued ruminal uptake of nitrogen through salivary secretion, slowing down of bacterial growth and autolysis of some micro-organisms. Purser and Buechler (1966), Bergen gt gt. (1968) and Purser (1970) reported that the amino acid composition of rumen microbial preparations was constant. The amino acid data of rumen bacterial and protozoal preparations presented in Tables 21 and 22 respectively, indicated not only a constancy of the amino acid composition of rumen microbial protein but that the amino acid composition was not in- fluenced by levels of dietary nitrogen intake or the time after feeding (at least during the first four hours after feeding). These findings further strengthen the suggestion made earlier in this discussion that lack of significant differences in plasma total free amino acid nitrogen among sheep fed the four rations was due to a constant supply of amino acid to the lower gut for digestion and absorption. 1:111- O GENERAL CONCLUSIONS Plasma free tryptophan concentration in sheep was not affected by dietary treatments or levels of crude pro- tein fed. Tryptophan did not appear to be deficient or limiting in sheep when fed four rations containing different sources and levels of nitrogen. Tryptophan content of rumen bacterial and protozoal preparations was constant and was not affected by dietary treatments or time after feeding. The diurnal rhythmicity of plasma tryptophan was the same for sheep fed the four rations and was only slightly influenced by time after feeding. The bulk amino acid composition of rumen bacterial and protozoal preparations was constant and was not affected by dietary treatments or time after feeding. Normal rations such as those Used in the present study will not markedly influence the total plasma amino acid nitrogen concentration in sheep. Increased rumen ammonia and blood urea nitrogen concen- trations were associated with increased levels of dietary crude protein. 72 RP. BIBLIOGRAPHY BIBLIOGRAPHY Abe, M. and M. Kandastu. 1968. Utilization of nonprotein nitrogen compounds by ruminants. Arch. Tiernenahrung 18:247-263 (Nutr. Abstr. and Rev., 1969). Abou Akkada, A. R. and H. E. S. Osman. 1967. The use of ruminal ammonia and blood urea as an index of the nutritive value of protein in some foodstuff. J. Agric. Sci. 69:25. Allison, M. J. 1969. Biosynthesis of amino acids by ruminal micro organisms. J. Anim. Sci. 29:797. Barringer, R. D. 1968. Influence of rumen protozoa on nitrogen metabolism in sheep. Dissertation Abstr. (B) 28:4364B-4365-B. Bergen, W. G. 1967. Studies on the effect of dietary and physiological factors on the nutritive quality and utilization of rumen microbial protein. Ph.D. Thesis. Ohio State University. Bergen, W. G., D. G. Purser and J. H. Cline. 1968a. Effect of ration on the nutritive quality of microbial pro- tein. J. Anim. Sci. 27:5. Bergen, W. G., D. B. Purser and J. H. Cline. 1968b. Deter- mination of limiting amino acids of rumen isolated microbial protein fed to rat. J. Dairy Sci. 51:1698- 1700. Bergen, W. G. and D. B. Purser. 1968c. Effect of feeding different protein sources on plasma and gut amino acids in the growing rat. J. Nutr. 95:333. Bergen, W. G. and E. L. Potter. 1971. c-N-methyl lysine metabolism in sheep. J. Anim. Sci. 32:1245. Blackburn, F. H. and P. N. Hobson. 1960. Proteolysis in the sheep rumen by whole and fractionated rumen con- tents. J. Gen. Microbiol. 22:272-281. 73 1:11;! 74 Brown, H. E., H. O. Kunkel and J. M. Prescott. 1961. Free amino acids of bovine plasma. Identification and effect of fasting. J. Anim. Sci. 20:967. Chesters, J. K. 1971. Problems caused by variation of food intake in experiments on protein and nucleic acid metabolism. Proc. Nutr. Soc. 30:1. Christensen, H. N. 1964. In: Mammalian Protein Metabolism, Vol. 1, Ed. by H. N. MfiKro and J. B. Allison. Academic Press Inc., New York. Conway, E. J. 1960. Ammonia. Biological determinations. IN In: Microdiffusion Analysis and Volumetric Error. Ed. ET J. Conway. Chemical Publishing Co., Inc., New York. Denton, A. E. and C. A. Elvehjem. 1954a. Availability of 4 amino acid tg_vivo. J. Biol. Chem. 206:449. Denton, A. E. and C. A. Elvehjem. 1954b. Amino acid con- centration in the portal vein after ingestion of amino acid. J. Biol. Chem. 206:455. Downes, A. M. 1961. On the amino acid essential for the tissue of sheep. Australian J. Biol. Sci. 14:254. Dror, Y., A. Mayevsky and A. Bondi. 1969. Some effects of starch on protein utilization by sheep. Brit. J. Nutr. 23:727. el-Shazly, K. 1958. Studies on the nutritive value of some common feedingstuffs. I. Nitrogen retention and ruminal ammonia curve. J. Agri. Sci. 51:149. el-Shazly, K. and R. E. Hungate. 1966. Method of measuring diaminopimelic acid in total rumen contents and its application to the estimation of bacterial growth. Appl. Microbiol. 14:27. Ely, D. G., C. 0. Little, P. G. Wolfolk and G. E. Mitchell, Jr. 1967. Estimation of the extent of conversion of dietary zein to microbial protein in rumen of lambs. J. Nutr. 91:314. Ely, D. G., C. 0. Little and G. E. Mitchell, Jr. 1969. Amino and urea nitrogen levels in lambs receiving different sources and injections of lysine and methion- ine. Can. J. Physiol. and Pharmacol. 47:929. Feigin, R. D., R. Beisel and R. W. Wannemacher, Jr. 1971. Rhythmicity of plasma acids and relation to dietary intake. Amer. J. Clin. Nutr. 24:329-341. 11... C... . H351 .- 75 Ganapathy, S. N. and E. S. Nasset. 1962. Free amino acid in dog blood and gut contents after feeding meat. J. Nutr. 78:241. Grouby, D., and O. Delfond. 1843. Compt. Rend. 17:1305- 1308. (Cited by R. E. Hungate tg: The Rumen and Its Microbes. 1966. Academic Press, New York and London). Halfpenny, A. F., J. A. F. Rook and G. H. Smith. 1969. Variation with energy nutrition in the concentrations of amino acids of blood plasma in the dairy cow. Brit. J. Nutr. 23:547. f i]? Harmon, B. G., D. E. Becker, A. H. Jensen and D. H. Baker. y 1969. Nicotinic acid-tryptophan relationship in the 1 nutrition of the weaning pig. J. Anim. Sci. 28:848. A Harper, A. E. and Q. R. Rogers. 1965. Amino acid imbalance. ‘ Proc. Nutr. Soc. 24:173. i Hendricks, H. and J. Martin. 1963. In vitro study of the nitrogen metabolism in the rumen?” Compus. Rendus de Researches Verslagen over Navorsigen. 31:9. (Cited by R. E. Hungate. In: Rumen and Its Microbes. 1966. Academic Press, NEw York and London). Hogan, J. P., R. H. Weston and J. R. Linsday. 1968. Influ- ence of protein digestion of plasma amino acid levels in sheep. Aust. J. Biol. Sci. 21:1263-1375. Hogan, J. P. and R. H. Weston. 1969. Quantitative aspect of microbial protein synthesis. In: Physiology of Digestion and Metabolism in the Raminant by A. T. Phillipson. Oriel Press. Newcastle, Eng. Hume, I. D., R. J. Moir and M. Somers. 1970. Synthesis of microbial protein in the rumen. 1. Influence of level of nitrogen intake. Austr. J. Agri. Res. 21:283-296. Hungate, R. E. 1966. The Rumen and Its Microbes. Academic Press, New York. Kay, R. N. B. 1969. Digestion of protein in the intestine of adult ruminant. Proc. Nutr. Soc. 28:140. Kleiber, M. 1961. The Fire of Life. John Wiley and Sons, Inc. p. 384. ‘ Klopfenstein, T. J., D. B. Purser and W. J. Tyznik. 1966. Effects of defaunation of feed digestlbility. Rumen metabolism and blood metabolites. J. Anim. Sci. 25:765. [Iillllfiltlllalil ‘l.[ I II III‘ I! ill! III]. A 76 Knipfel, J. E., H. G. Botting, R. J. Noel and J. M. McLaughlan. 1969. Amino acids in blood plasma and tissues of rats following glucose force feeding. Can. J. Biochem. 47:323. Knox, R , G. D. Kohler, R. Palter and H. C. Walker. 1970. Determination of tryptophan in feeds. Anal. Biochem. 36:136-143. Kumta, U. S. and A. E. Harper. 1962. Amino acid balance and imbalance. IX. Effect of amino acid imbalance on blood amino acid pattern. Proc. Soc. Expt. Biol. Med. 110:512. Leibholz, Jane and C. F. Cook. 1967. Free amino acids, ammonia and urea concentration in blood plasma of starved lambs. J. Nutr. 93:561. Lewis, D. 1957. Blood urea concentration in relation to protein utilization in the ruminant. J. Agri. Sci. 48:438-446. Lewis, D. and I. W. McDonald. 1958. The interrelations of individual proteins and carbohydrates during fermenta- tion in the rumen of sheep. Lewis, D. 1961. The fate of nitrogenous compounds in the rumen. pp. 127. t2; Digestive Physiology and Nutri— tion of the Ruminant. Butterworths, Ed. D. Lewis. London. J. Agri. Sci. 51:108. Little, 0. C., G. E. Mitchell, Jr. and G. D. Potter. 1968. Nitrogen in the abomasum of wethers fed different protein sources. J. Anim. Sci. 27:1722. Lojkin, Mary E. 1962. Tryptophan-niacin metabolism. 1. Pregnancy, ovarian hormones and levels of tryptophan intake as factors affecting the tryptophan-niacin metabolism of rat. J. Nutr. 78:287. and E. H. Cords. 1966. Biological Chemistry. Harper and Row, New York and London. Mahler, H. R. Makdani, D. D., J. T. Huber and W. G. Bergen. 1971. Effect of histidine and methionine supplementation in nutrition quality of commercially prepared fish protein concen- tration in rat diets. J. Nutr. 101:367. Males, J. R. and D. B. Purser. 1970. Relationship between rumen ammonia levels and the microbial population and volatile fatty acid proportions in faunated and defaunated sheep. Applied. Microbiol. 19:485-490. IIIIII‘I‘IllllII-Ili‘.‘ l'll'lII'I-‘ll III I 77 McDonald, 1. W. 1947. The absorption of ammonia from the rumen of sheep. vBiochem. J. 42:584. McDonald, I. W. 1954. The extent of conversion of food protein to microbial protein in the rumen of sheep. Biochem. J. 56:120-125. McLaughlan, J. M. 1963. Relationship between protein quality and plasma amino acid levels. Fed. Proc. 22:1122. McLaughlan, J. M. 1964. Blood amino acid studies. V. Determination of the limiting amino acid in diets. Can. J. Biochem. 42:1353. McLaughlan, J. M., S. Venkat Rao and F. F. Noel. 1967. Blood amino acid studies. VI. Use of plasma amino acid score for predicting limiting amino acids in dietary proteins. Can. J. Biochem. 45:31. Miller, E. L. 1967. Determination of the tryptophan con- tent of feedstuffs with particular reference to cereals. J. Sci. Fd. Agri. 18:381-386. Munro, H. N., J. Chisholm and D. J. Neismith. 1962. The influence of the calorie content of the diet on the uptake of labeled amono acids by tissue protein. Brit. J. Nutr. 16:245. Munro, H. N. 1964. Regulation of protein metabolism. tg; Mammalian Protein Metabolism. Vol. 1, p. 381. H. N. Munro and J. B. Allison, Eds. Academic Press, New York. Munro, H. N. 1968. Role of amino acid supply in regulating ribosome function. Fed. Proc. 27:1231. Murate, Kiku and Toshizd Rimura. 1969. TryptOphan metabo- lism of rats fed a threonine-free amino acid diet. J. Nutr. 98:437. Oltjen, R. R. and P. A. Putman. 1966. Plasma amino acid nitrogen retention by steers fed purified diets con- taining urea or isolated soy protein. J. Nutr. 89:385. Oltjen, R. R., A. S. Kozae, P. A. Putman and R. P. Lehmann. 1967. Metabolism, plasma amino acid and salivary studies with steers fed corn, wheat, barley and milo all concentrate rations. J. Anim. Sci. 26:1415. Orskov, E. R. and C. Fraser. 1969. The effect on nitrogen retention in lambs of feeding protein supplement direct to abomasum. Comparison of liquid and dry feeding and of various sources of protein. J. Agr. Sci. 73:469. ( I l. . ii‘l‘r‘l x||tln [.I II III I111 I14 11... 78 Qrskov, E. R., C. Fraser and I. McDonald. 1971a. The effect of increasing the concentration of soybean in a barley diet on apparent disappearance of feed constituents along the digestive tract. Brit. J. Nutr. 25:225. Qrskov, E. R., C. Fraser and I. McDonald. 1971b. The effect of urea or fishmeal supplementation on barley diets on the apparent digestion of protein, fat, starch and ash in the rumen, the small intestine and the large intestine and calculation of volatile fatty acid pro- duction. Brit. J. Nutr. 25:243. Palmer, D. W. and T. Peters, Jr. 1969. Automated determina- tion of free amino groups in serum and plasma using 2, 4, 6-Trinitrobenzene sulfonate. Cln. Chem. 15:891. Pasteur, L. 1863. Compt. Rend. 52:1260-1264. (Cited by R. E. Hungate. 1966. Rumen and Its Microbes. Academic Press, New York and London). Payne, E. and J. G. Morris. 1969. The effect of protein content in the diet on the rate of urea formation in sheep liver. Biochem. J. 113:659. Potter, E. L., D. B. Purser and J. H. Cline. 1968. Effect of various energy sources upon plasma free amino acids in sheep. J. Nutr. 95:655. Preston, R. L., D. D. Schnakenberg and W. H. Pfander. 1965. Protein utilization in ruminants. I. Blood urea nitrogen as affected by protein intake. J. Nutr. 86: 281. Purser, D. B. and S. M. Buechler. 1966. Amino acid compo- sition of rumen microorganisms. J. Dairy Sci. 49:81. Purser, D. B., T. J. Klopfenstein and W. J. Tyznik. 1966. Effects of defaunation on feed digestibility, rumen metabolism and blood metabolites. J. Anim. Sci. 25:765. Purser, D. G. 1970. Amino acid requirements of ruminants. Fed. Proc. 29:51-54. Rao, M. N. and J. M. McLaughlan. 1967. Effect of time factor on the nitrogen sparing effect of dietary carbo- hydrate. Can. J. Biochem. 45:1653. Rao, V. S., F. J. Noel and J. M. McLaughlan. 1968. Blood amino acid studies. Prediction of limiting amino acids in mixtures of dietary proteins. Can. J. Physiol. and Pharmacol. 46:707. _7"*. r___—_.___ . ll | III‘ I I. Ill [ .l: {A II" II. II [AI l I. It all: 1;- 79 Sauberlich, H. E. and W. D. Salmon. 1955. ‘Amino acid imbalance as related to tryptOphan requirement of the rat. J. Biol. Chem. 214:463. ' Smith, R. H. 1969. Reviews of the progress of dairy sicence. Section G. Nitrogen metabolism and the rumen. J. Dairy Res. 36:313. Sourkes, T. L. 1971. Alpha-methyltryptophan and its actions on tryptophan metabolism. Fed. Proc. 30:897. Spies, J. R. 1967. Determination of tryptophan in proteins. Anal. Chem. 39:1412. — -* An. Tagari, H., Y. Dror, I. Ascarelli and A. Bondi. 1964. The influence of levels of protein and starch in rations of sheep on the utilization of protein. Brit. J. Nutr. 18:333. Thomas, W. E., J. K. Loosli, H. K. Williams, F. H. Ferris and L. A. Maynard. 1949. Synthesis of amino acid in the rumen. Fed. Proc. 8:398-399. Wapnir, R. A. and J. H. Stevenson. 1969. Estimation of free tryptophan in plasma. A simplified spectrofluoro- metric micromethod. Clinica Chemica Acta. 26:203-206. Wurtman, R. J. 1970. Diurnal rhythms in mammalian protein metabolism. In: Mammalian Protein Metabolism. Ed. H. N. Munro. —Vbl. IV. Academic Press, New York and London. Young, V. R., M. A. Hussein, E. Murray and N. S. Scrimshaw. 1971. Plasma tryptophan response curve and its rela- tion to tryptOphan requirements in young adult men. J. Nutr. 101:45-60. Zimmerman, R. A. and H. M. Scott. 1965. Interrelationship of plasma amino acid levels weight gain in chicks as influenced by suboptimal and superoptimal dietary concentrations of single amino acids. J. Nutr. 87:13. Zimmerman, R. A. and H. M. Scott. 1967. Effect of fasting and feeding a nonprotein diet on plasma amino acid levels in the chick. J. Nutr. 91:507. Zunt, N. 1879. Landwirtsch. Jahrb. 8:65-117. (Cited by R. E. Hungate. 1966. Rumen and Its Microbes. Academic Press, New York and London). ll .Il II .I ‘ ! { f...‘|‘|\l I (.1 fl‘ l: lull. II I III 4 ‘A APPENDIX ANALYSIS OF VARIANCE “-4- 8O mH.VH nw.mn~ 0H.H0 0 poppm m . 0 No.0 0v.n NOMMvoN 0000.0 00MNNIYN0.0N0N m ucoswmouh H choEE< amend 40H choee< :oEDm we choEE< coEsm 00.nv 0H.0 NH.0 0 Howum H00.o Hm.wm v~.vvoH wwwp 0n.H 0H.o 00.0 nm.o no.0 m psoEumohH oh choEE< 205:0 Homavvh cowopqu ocHE<-avaovoh comopqu ocHE<-d no.0 mH.o No.0 0 Hogpm mH.0 nm.m HN.0 Ibn.o mth mm.0 th m acoEumoHH VNH somehqu ocHE<-a 0N9 comopsz ocHE<-a 0 H :0 Ohqu ocHE<-a No.0 00.0 00.0 0 Achpm Hoo.0 00.NN 00.0 00.0. o0.m 0N.b MH.0 0m.N mNno m psoEpmoHH mHH smwogqu ocHE<-5 09 comopsz ocHE<-d oh ammopqu ocHE<-e 00.0 no.0 0H.o 0 Honhm 0N.o 0m.H no.0 00.0 WM.H .HHMD NN.D 00.H 0H.0 m ucoEumopb mo comopsz ocHE<-d oh :omopqu ocHE mo mHmnqu< H mqm wo oopsom Hwoscmu:000 H mqm mo oopsom H0oscfiucouo H mgm