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I swine-157$”. . . . .. 1 ., . a flu -‘ "a 3 i 9-H} ciaigan atme University i UM“ iii Milli}! ul/ I mwMAru 1.4 - This is to certify that the thesis entitled EVALUATION OF THE CONTRIBUTION OF RECYCLED UREA TO THE SYNTHESIS OF THE MICROBIAL PROTEII IN THE RUMEN USING N-LABELLED UREA presented by Adnan M. Al-Dehneh has been accepted towards fulfillment of the requirements for M. S . degreein Anlmal Sc1ence m; 17/7” 475%; Major professor Date 11/14/86 0-7639 MS U I: an Affrmative Action/Equal Opportunity Institution MSU LIBRARIES RETURNING MATERIALS: place in book drop to remove this checkout from your record. FINES wiII be charged if book is returned after the date stamped below. EVALUATION OF THE CONTRIBUTION OF RECYCLED UREA TO THE SYNTHESIS OF THE MICROBIAL PROTEIN IN THE RUMEN USING 15N-LABELLED UREA by Adnan M. AL-DEHNEH A THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree of MASTER OF SCIENCE Department of Animal Science 1 9 8 6 ABSTRACT EVALUATION OF THE CONTRIBUTION OF RECYCLED UREA TO THE SYNTHESIS OF THE MICROBIAL PROTEig IN THE RUMEN USING N-LABELLED UREA by Adnan M. AL—DEHNEH Urea-lsN was continually infused into the jugular vein for 3d in two duodenally cannulated cows fed diets of 1:2 and 2:1 foragezconcentrate. Duodenal digesta samples were taken every 3h, and coccygeal blood and milk were sampled twice daily. Urine was collected for 5d starting 1d before infusion and total feces for 3d during infusion. Fecal samples were also taken twice daily during the 5d of collection. 15 Urinary excretion of N accounted for about 90 percent of that which exited from the body; whereas, feces and milk each accounted for about 5 percent. Recovery of 15N during the infusion period ranged from 30 to 50 percent 15N ratios, as percent of of that infused. Estimates using the total N passing into the duodenum, that was bacterial N, were 50 to 90 percent and appeared directly pr0portiona1 to DM intake of cows. Recycled-N incorporated into rumen microbes was greater (24 vs. 14% of N in bacteria passing into the duodenum) in cows fed the high concentrate than the high forage diet. Also, incorporation of recycled N into rumen microbes was higher in the lactating than the dry cow (24 vs. 14%) and the flow of nitrogen from the rumen to the small intestine was greater for the concentrate than the forage diet (122.0 vs. 101.0% of nitrogen intake). In summary, more recycled-N in duodenal digesta and more N flow from the rumen to the small intestine were ob- served in cows on the concentrate than the forage diet. ACKNOWLEDGEMENTS The author would like to express his extended ap- preciation to his advisor, Dr. J. T. Huber for his excel— lent counsel, patience, and assistance in the development and completion of this research study, and for the provi- sion of his invaluable knowledge and guidance throughout my graduate program. Also, special thanks are due to the mem- bers of the committee, Dr. W. T. Magee and Dr. D. E.Ullrey for their patience and understanding of the drastic changes in my life. Special thanks are extended to Dr. C. B. Theurer, Dr. W. H. Brown and Dr. M. C. Young for their participation in serving on the committee at the University of Arizona. The author also expresses sincere thanks to Dr. R. C. Wanderley for his generous help during this experiment. Finally, the author is especially grateful to his family for their unlimited patience, love, and continuous support throughout the completion of this study. iv TABLE OF CONTENTS LI ST OF TABLES C O O O O O O O O O O 0 LIST OF FIGURES . . . . . . . . . . . INTRODUCTION 0 O O O O O O O O O O O 0 Importance of NPN to Protein Synthesis LITERATURE REVIEW . . . . . . . . . . Quantitative Model for N—Kinetics . Detoxication of NH3 by Liver . . . N-Excretion . . . . . . . . . . . . Urea-N in Saliva . . . . . . . . . Use of Recycled Nitrogen . . . . . Lower Threshold Value for Ammonia . Urea Turnover in Blood . . . . . . Other N Compounds Recycled into the Ammonia Use at Various Sites . . . Urea Diffusion into the Rumen . . . Urea Flux Throughout the Body . . . Carbon Dioxide Effect on Urea Diffusion Mechanism of Blood Urea Transfer into the Rumen O O O O O O O O O O O O O O 0 Amount of Urea Transfer from Blood or Saliva Recycling of Nitrogen in the Digestive Tract Blood—Urea Transfer to the Abomasum and Intestine O O O O 0 O O O O O O O O Page vii ix WWI-HP 11 12 12 14 15 16 17 17 20 21 23 24 25 26 27 vi TABLE OF CONTENTS (continued) Microbial Protein Synthesis . . . Using 15 Excretion'of 15N-Urea . . . . . . Conclusion . . . . . . . . . . . MATERIALS AND METHODS . . . . . . . Animals . . . . . . . . . . . . . Sample Collections . . . . . . . Sample Preparation . . . . . . . Analytical Procedures . . . . . . RESULTS . . . . . . . . . . . . . . Nitrogen Enrichment in Biological Fate and Recovery of 15 Into the Blood . . . . . . . . . Duodenal and Bacterial Protein-N Originating from Blood Urea (Recycled-N) . . DISCUSSION 0 O O O O O O O O O O O O Duodenal Digesta N-Flow . . . . . Rate of Nitrogen Enrichment in Duodenal Digesta, N-Urea in Tracing N Metabolism . . Samplings N Which was Infused Duodenal Bacteria, and Rumen Bacteria . . . Nitrogen Enrichment in Urine, Feces, and Milk Fate of 15 SUMMARY AND CONCLUSIONS . . . . . . APPENDIX 0 O O O O O O O O O O O O O BIBLIOGRAPHY O O O O O O O O O O O O N (Excretion vs. Body Retention) Page 28 30 33 34 35 35 36 37 38 40 42 44 45 51 51 53 54 55 57 59 82 LIST OF TABLES Table 1. Composition of Concentrate Diet . . . . . . . 2. DM Intake, Duodenal Flow and Fractionation and Fecal Output of Duodenal DM of Cows Fed High Grain or High Forage Diets . . . . . . . 3. Nitrogen Intake, Duodenal Flow and Fraction- ation, and Fecal Output of Duodenal-N of Cows Fed High Grain or High Forage Diets . . . 4. Average of 3 Peak Values for 15N-Enrichment of Each Cow in Duodenal Digesta, Duodenal Bacteria, Urine, Feces, Milk, and Blood . . . 5. Fate of 15N Infused into Blood . . . . . . . . 6. Example of Calculation, Cow No. 6 on Grain Diet 0 O 0 O O O O O O I O O O O O O O O C 0 O 7. Percent of N Which Originated from Blood Urea (or Recycled N) . . . . . . . . . . . . . Appendix Table 8. Duodenal N Flow as Percent of Intake, and {gactions of Duodenal N from Cow Infused with N urea O O O O O O O O O O O O O O O O O O O 9. Average of 3 Peak Values for 15N-Enrichment of Each Cow in Rumen Bacteria . . . . . . . . 10. PH, Dry Matter, Nitrogen and Cr203 in DUOdenal DigeSta O O O O O O O O O O O O O O 0 11. Fecal-DM Output, Percent of Fecal-DM, Percent of Cr203 in Fecal-DM . . . . . . . . . 12. Percent of N in Fecal-DM, Fecal-DM Output, and N-Output in Fecal-DH o o o o o o o o o o o 13. Fecal-Cr203 Output, and Percent of Fecal Cr203 Recovery 0 O O O O O O O O O O O O O O O O O 0 vii Page 35 41 42 43 45 46 46 75 75 76 76 77 77 viii LIST OF TABLES (continued) Appendix Table Page 14. Total Urine Output, Percent of N in Urine, and Total N-Output in Urine o o o o o o o o o 78 15. Total Milk Output, Percent of N in Milk, and Total N-Output in Milk . . . . . . . . . . . . 78 16. Percent of Nitrogen in Blood . . . . . . . . . 79 17. Balance of Nitrogen . . . . . . . . . . . . . 79 18. Apparent and True Rumen-DM Disappearance, and Undegraded RumEH-N o o o o o o o o o o o o o t 80 19. Total Tract-BM and N Digestibility o o o u o o 80 20. Intestinal-BM and N Digestibility o o o o o o 81 Figure 1. | ,,;-€§L, LIST OF FIGURES A quantitative, whole—animal model of nitrogen transactions in the sheep (from NOlan’ 1974) I I I I I I I I I I I I I I I I 2. A general three-pool, open—compartment model for nitrogen transactions associated with rumen fluid ammonia, plasma urea, and cecum fluid NH3 in sheep (from Nolan et_al., 1976) I I I I I I I I I I I I I I I I I I I I 3. A model of nitrogen metabolism in sheep (from Nolan and Leng, 1972) . . . . . . . . 4. A quantitative sub-model of nitrogen transactions in the stomach of a sheep (from Nolan, 1974) . . . . . . . . . . . . . 5. Diagram of recirculation and utilization in ruminant of blood urea and other N compounds (from Boda and Havassy, 1975) . . . . . . . 6. 15N—Enrichment in duodenal digesta and duodenal bacteria of cow (6) on grain diet . 7. 15N-Enrichment in urine of cow (6) on grain diet I I I I I I I I I I I I I I I I I 8. 15N-Enrichment in feces of cow (6) on grain diet I I I I I I I I I I I I I I I I I 9. 15N-Enrichment in milk of cow (6) on grain diet I I I I I I I I I I I I I I I I I Appendix Figure 10. 15N-Enrichment in duodenal digesta and duodenal bacteria of cow (958) on grain diet 11. 15N-Enrichment in duodenal digesta and duodenal bacteria of cow (958) on grain diet 12. 15N—Enrichment in duodenal digesta and duodenal bacteria of cow (6) on forage diet. ix Page 18 47 48 49 50 59 60 61 X , "I 1:537 1.2;” LIST OF FIGURES (continued) Appendix Figure Page 13. 15N-Enrichment in urine of cow (958) on grain diet I I I I I I I I I I I I I I I I I I 62 14. 15N-Enrichment in urine of cow (958) on forage diet I I I I I I I I I I I I I I I I I 63 15. 15N-Enrichment in urine of cow (6) on forage diet I I I I I I I I I I I I I I I I I 64 16. 15N—Enrichment in feces of cow (958) on grain diet I I I I I I I I I I I I I I I I I I 65 17. 15N-Enrichment in feces of cow (958) on forage diet I I I I I I I I I I I I I I I I I 66 18. 15N—Enrichment in feces of cow (6) on forage diet I I I I I I I I I I I I I I I I I 67 19. 15N-Enrichment in milk of cow (968) on forage dietI I I I I I I I I I I I I I I I I I 68 20. 15N-Enrichment in blood of cow (958) on grain diet I I I I I I I I I I I I I I I I I I 69 21. 15N—Enrichment in blood of cow (6) on grain diet I I I I I I I I I I I I I I I I I I 70 22. 15N—Enrichment in blood of cow (958) on forage diet I I I I I I I I I I I I I I I I I 71 23. 15N—Enrichment in blood of cow (6) on forage dietI I I I I I I I I I I I I I I I I I 72 24. 15N—Enrichment in rumen bacteria of cow (958) on forage diet . . . . . . . . . . . . . 73 25. 15N-Enrichment in rumen bacteria of cow (6) on forage diet . . . . . . . . . . . . . . 74 INTRODUCTION 1W The importance of dietary non-protein nitrogen to ruminants stems from the presence of microorganisms in the rumen which synthesize the essential amino acids for the host from non-protein nitrogen sources (Virtanen, 1963). These microorganisms, in addition to fermenting fibrous plant material, synthesize their own protoplasm from the end-products of protein and carbohydrate catabolism together with non-protein nitrogen from both endogenous and dietary sources. Microbial protein is mainly digested in the abomasum and small intestine, and resultant amino acids are absorbed from the small intestine. The amount of microbial protein synthesized depends on several factors, including the amount of readily available carbohydrate present in the rumen (Pearson and Smith, 1943; Annison at al., 1954; Belasco, 1956; Lewis and McDonald, 1958). Urea is recycled to the rumen from the blood, either across the rumen wall (Houpt, 1959; Decker, gt_al., 1960; Engelhardt and Nickel, 1965; Houpt and Houpt, 1968) or in saliva (McDonald, 1948; Somers, 1961). Starch, the main component of grain, is regarded as one of the most suitable carbohydrate sources for maximum microbial protein synthesis because of its rate of breakdown in the rumen 1 2 (Chalmers and Synge, 1954). Often soluble carbohydrates, such as glucose, sucrose, and maltose, may be broken down too rapidly (Belasco, 1956) and cellulose too slowly (Lewis, 1957) to be as useful as starch in providing energy for microbial protein synthesis. However, a combination of the several sources will generally result in a good fermen- tation. This thesis deals with quantifying the amount of urea recycled into the rumen which is used for synthesis of microbial protein on a high grain vs. high forage diet fed to dairy cows. LITERATURE REVI EW D l'l l' H i 1 E H-K' I' Nolan (1974) described a quantitative whole animal model which doesn't contain definitive values but serves as a hypothesis for N kinetics (Figure 1). Recently, a whole-animal model incorporating values for N transfer by pathways that appear biologically impor- tant was deve10ped from results of isot0pe dilution studies with 14C— and 15N-labelled compounds for sheep given dif- ferent diets (Nolan and Leng, 1972). This model indicated that transfer of urea from blood to the rumen was quantita- tively less important as a mechanism for providing N to the rumen than had been previously suggested, and the results indicated that there was an important site of endogenous urea degradation in the lower digestive tract. These earlier studies have been extended by Nolan et_al. (1976) to include direct measurements of the exchanges of N be- tween pools in the blood, cecum and rumen (Figure 2). The pathways of ammonia, amino acid and urea meta- bolism have been incorporated into a model described by Nolan and Leng (1972). For convenience, the digestive tract has been divided into three areas: (1) the anterior area of microbial colonization (i.e., the reticulo-rumen), (2) the abomasum, duodenum, jejunum and anterior ileum, and 3 4 GUT BLOOD TWSSUES D I I ue 20 f— A ‘fi 20 4‘ 0.5 Endogenous Non - men N r *T v --------H------b Fromm NPN I4 ’1’ I RIVICU'O ‘ Ann“: 9 “1“ Ci) q 2“ Rumm ands A 4 x . 2 \f {$3 ’3 x f ‘ - Mucrobu 4-5 A y 20 Omosum l l jg 2'3 8 I I; -- .Nlchr .N- J: 1 £ 1 C 5 I II - ‘h Abomcwm L I . I 1"] i ’8 ; gL6 5 I03 ‘1 [.5 : E' cellular 255 I'cululor ' f ; ’Ammo orads Ammo and: Ima '5-------, ' ‘ 50 Uuodenum 7 ’ " 1 I I I [57 b"? ‘- ' ' - . + ] ‘dllfi N Body I Ammo 7N?“ $21 Prolcns 1 ._°. "(‘l‘. - ~ g. . I ”cum : ; ,7‘~-- K. -19 --°.'°S".'.‘". .. - _ __ --, , ‘ _ "(Iv-om Ht 9 I 5 6' 1’0 2 5 , " 1 § ; Mum a ° 0 COCCum I g "' "U"--- ‘ NAN )5 I : no “"1: :: ._.4 7 A I ' ? 49-2 f 15 t5 ' MKIODQ$ -«M. __ _ _ .— Ul'uc' ' : 05 Col-m ' I t 5 9 3- 5! MUCus Non -crco LOwev ---- "‘2 20 N Calcn I L J L-—: «LS—.4 Y 02 8 - ’ *— Rectum 30 1; 1 r Focces Urine 53 2 Wool. 5km NC. 2 3 FIGURE 1. A quantitative, whole-animal model of nitrogen transactions in the sheep (from Nolan, 1974) 5 9-9 6-1 (a) 7., (b) v k) :1 ‘ Rumen NH,—N 4 Blood urn-N 14 I k (d) 04 \ l :4 I }J - ' 10 13 $6 12‘ (a) (I) (x) (h) (a) 0) § 1 F 2-1 (k) 2-4 (I) —-—-> Canal NH,-N --—->- FIGURE 2. A general three—pool, open-compartment model for nitrogen transactions associated with rumen fluid ammonia, plasma urea, and cecal fluid NH in sheep (frOm Nolan et_al., 1976) 3 6 (3) the major areas of microbial colonization in the lower digestive tract (i.e., lower ileum, cecum and large bowel). The pools of N in the digestive tract were considered separately from the pools of ammonia, urea and amino acids in the body, which were considered separately from the larger pools with slower turn-over rates such as the muscle proteins and structural protein pathways which were be- lieved to be the major routes for conversion of one nitro- genous compound into another (Figure 3). The N of compounds digested in the rumen which do not enter the ruminal ammonia pool are changed in the rumen to nitrogenous compounds more complex than ammonia. This could occur by uptake by microbes of peptides and amino acids (Portugal, 1963; Wright and Hungate, 1967; Coleman, 1967) or by absorption of free amino acids across the rumen wall (Demaux et_al., 1961; Cook et_al., 1965), but the latter is assumed to be quantitatively insignificant. Ammonia is lost from the rumen (a) by incorporation into microbial cells which pass to the abomasum, (b) by direct absorption through the rumen wall into portal blood, and (c) by loss in fluid entering the abomasum. Ammonia entering the abomasum is probably absorbed when it reaches the small intestine (Smith, 1969). Nolan (1974) suggested a sub-model of nitrOgen metabolism in the reticulo-rumen, omasum, and abomasum (Figure 4). A rumen sub-model which adequately described processes of N metabolism in the reticulo-rumen, omasum, Cu III] "M. Dunn N N. fin".- t I I ‘Ilahbh N H 1| Hoe no“! N "u’\-.l~b-I l----.-- -00.‘ 1c r 1' - V ~--.----..—-.. .- ulv 'I'I "-(I' N haul N N] Inn" Una. FIGURE 3 . (from Nolan and Leng, 1972) fluo- ally-hm “mu-Hutu j.“ and N \\ ‘3 W08 “at A model of nitrogen metabolism in sheep men" a _.-- '1 2| 1 (6351 : o s 1 V 1 i cmxnnwms u [Laurent "1 ,L m 1 #- RETICULO - / "‘ ‘ , ” ; annouu I , ’ our: ACID N 4 _ A 1 3 5 4 \{O 90 /‘ o unmouu fi UlCfloalAL N ] j n 7 [ man 09 OMASUM 0'“ ~ , ' , I o 3 s II A ¢ gun a SOIUBLE CElL = 09 U E O u ‘ 7! (2:31 $"0 £(ICOI‘ : l r J . NAN AHMOMA u I 'OUOdENUM ‘ I m! . 2 ' J for“; n ‘9 3 (327) FIGURE 4. A quantitative sub-model of nitrogen transactions in the stomach of a sheep (from Nolan, 1974) 9 and abomasum might include quantitative assessment of the following: 1. The quantities of soluble and insoluble protein and non-protein nitrogen from the diet. Amount of endo— genous urea and other endogenous N compounds, and their contribution to the total pool of N compounds which are available for fermentation in the rumen. 2. The extent to which proteins are degraded to simpler compounds (i.e., petides, amino acids and ammonia) or pass undegraded from the rumen. 3. The extent to which peptides, amino acids, ammonia and nucleic acids are assimilated by bacteria and protozoa, and the net rate of efflux of microbial N to the small intestine. 4. The quantities of soluble N compounds absorbed through the walls of the forestomachs. 5. The quantities of N recycled through pools within the rumen itself (e.g., as a result of ingestion of other microorganisms by protozoa and lysis of bacteria). In contrast to the rumen, the large intestine has received little attention and there is little quantitative information for which a steady-state sub-model can be determined. Firstly, a conceptual model of N transitions is required. In general, digestive processes appear to be similar to those in the rumen. Cecal contents exhibit cellulase, protease, deaminase and urease activities (Becker, 1967), and products of fermentation include 10 volatile fatty acids, ammonia and microbial protein (Williams, 1965). Proteolytic activity appears to be greater in the contents of the large intestine than in the rumen (Becker, 1971) and isobutyric and isovaleric acids occur in the cecum in proportions higher than those in the rumen. Orskov gt_al. (1970) indicated extensive breakdown of pro- tein in the rumen. Although the rumen is the major site of digestion of dietary organic matter (OM), fermentation in the hind gut can play a significant role in overall digestion of structural carbohydrates; for example, in sheep given rations containing high levels of cereals, as much as 30 percent of the cellulose in a ration may be fermented by microorganisms in the hind gut (MacRae and Armstrong, 1969). Nitrogenous compounds enter the cecum from the upper digestive tract in feed residues, undigested rumen microorganisms and endogenous materials (e.g., mucus) (Hecker, 1973). In addition, substantial quantities of urea-N enter the cecum from the blood. Hence, the total input of N into the cecum from a sheep's blood ranges between 4-15 g/day. There is generally a net absorption of N between the ileum and rectum. It seems probably that hind gut fermentation is energy-limiting, and absorption of N from the hind gut is largely as ammonia (McDonald, 1948). Absorption from the cecum and colon of other nitrogenous compounds, including amino acids (Demaux §t_al., 1961), 11 does not appear quantitatively important, but is still an open question. 1:! . l' E11113 , It is well established (McIntyre, 1971; Oltjen at 31., 1962) that the capacity of the liver to detoxify ammo- nia increases with increasing N intake. High protein intake resulted in a significantly greater activity of liver ornithine transcarbamylase, arginine synthetase and arginase. Chalupa gt_al. (1970) found an increase of three urea cycle enzymes of the liver, carbamylphosphate synthe- tase, ornithine transcarbamylase and arginase, when the animals were given urea. He suggested that urea-fed ani- mals were able to detoxify more ammonia than protein-fed animals since the rate-limiting enzyme of the cycle did not change, and the average urea production rate of the liver in animals fed urea as the sole nitrogen source was signi— ficantly higher than in protein-fed animals. It is likely that during short periods after feeding the ammonia load of the liver is higher in urea-fed animals than those which receive an equal amount of protein-N. This, however, is still uncertain. A decrease of total turnover of urea with time was noticed after urea-feeding, and the total urea production was very low from 5-10 hours after feeding. Experiments were also carried out to study the nature of ammonia toxicosis. The changes of ammonia concentration in the cerebrospinal fluid along with the 12 rise of the blood ammonia levels were examined. The pro- duction of cerebrospinal fluid, and also its pressure, de- creased notably in the state of ammonia intoxication. Rise and fall of blood ammonia are followed by similar changes in ammonia content in the cerebrospinal fluid, but with some delay. This accounts for the observation that ammonia level in the nervous symptoms during ammonia toxicosis cannot always be explained by the level of ammonia in venous blood. Clarification of the mechanism of ammonia toxicity might be facilitated by use of 15N (Juhasz, 1972). Nzflxcretion Nitrogen is eliminated from the organs partly through feces and partly through urine. Ninety percent of N-compounds originating in the organs in the course of in- termediatary metabolism are excreted in the urine. Schmidt-Nielsen and Osaki (1958) found that in sheep, urea clearance and excretion of urea suddenly de- creased with low protein intake. Therefore, tubular active rediffusion of urea is the mechanism by which the organisms save urea in case of low protein supply. H -H . S 1' It is known that ruminants produce a large quantity of basic saliva. Ruminant saliva has been known, since 1921, to contain N (Kehar and Mukherjee, 1949). Later, the presence of salivary urea was demonstrated (Krober and . Gibbons, 1962), and its concentration increased when higher 13 rumen ammonia levels occurred (Lewis, 1955). In cattle, urea-N represented an average of 77 percent of the total N in mixed saliva (Lewis, 1957). Higher salivary concentra- tions of total N resulted in higher percentages of urea-N. The urea content of mixed saliva was about 65 percent of the plasma urea concentration, which ranged from 4-19 mg/dl urea-N. Some data on the sheep partoid-N shows that urea represented 60-70 percent of total N in both mixed or parotid saliva (Lewis, 1962). Sheep differed from cattle in that the percentage of salivary-N as urea N was not affected by concentration of total-N (Lewis et_al., 1957). Somers (1961) found that the concentration of urea—N in mixed saliva increased with increasing rate of saliva secretion, and suggested maximum concentrations of approxi- mately 30 mg/dl for total N in saliva. After intake of urea, secretion rate of saliva decreased, blood ammonia concentrations immediately increased, and blood urea level rose after a slight delay (Juhasz, 1972). The rise of urea concentration in the blood was followed by a rise in saliva. The quantity of urea excreted per unit of time, nevertheless, fell considerably during the first hour due to a significant decrease of the salivary secretion rates. These observations show that both the composition and the secretion rate of saliva are influenced by the quantity of ingested nitrogen. If N-uptake is high, less saliva is produced, and the total quantity of urea recycled into the rumen is reduced for some time. Factors responsible for 14 those phenomena should probably be sought in the rumen (Juhasz, 1972). Urea flowing from the blood into the forestomach through the wall of the rumen is considered to be an impor- tant source of ruminal nitrogen. Urea passes from the blood into the rumen by diffusion across the rumen wall and is split to ammonia by the enzyme urease. The quantity of nitrogen recycled into the rumen by diffusion from blood is about 10 times that secreted through saliva, but this varies with type of diet. At urea concentrations of 20-60 mg/dl of blood, 6-169 urea will be recycled daily in sheep. The passage of urea across the rumen epithelium from blood to the rumen is not yet fully understood and deserves further study, perhaps by radioisotope techniques (Juhasz, 1972). In summary, secretion of nitrogen in saliva has an upper limit. Weston and Hogan (1967) showed that following a single intraparotid infusion of urea, the secretion of total nitrogen or urea in parotid saliva reached the limit when blood urea nitrogen was approximately 30 mg/dl and did not increase with further increase in blood urea. Maximum transfer of urea through the rumen wall was found when blood urea nitrogen was about 20 mg/dl (Gartner, 1962; 1963). Wen It is now well established that ruminants use endogenous urea for protein synthesis (Decker et_al., 1960; 15 Houpt, 1959; Simonnet gt_al., 1957). This special nitrogen conservation cycle involves: 1) transfer of endogenous urea from the blood into the rumen, 2) hydrolysis of this urea to ammonia and carbon dioxide by bacterial urease, 3) use of ammonia nitrogen by rumen microbes for protein synthe- sis, and 4) digestion and absorption of the microbial pro- teins from the small intestine. All of the amino acids commonly found in proteins, essential and nonessential, are synthesized by rumen microorganisms. In addition, a por- tion of the ammonia formed in the rumen is absorbed into the portal blood. This ammonia can be used for hepatic synthesis of nonessential amino acids, similar to what occurs in nonruminants (Rose and Decker, 1956). Through these processes considerable metabolic urea nitrogen may be reclaimed as amino acid nitrogen instead of being excreted in the urine (Houpt and Houpt, 1968). Win Hydrolysis of endogenous urea to carbon dioxide and ammonia occurs in the digestive tract of all animals and depends on urease of bacterial origin (Levenson gt_al., 1959). In the ruminant, N33 is available for microbial cell growth, and microbial protein synthesized from recy- cled urea-nitrogen provides the animal with an additional source of available protein. Studies by Juhasz (1972) indicated the effect of the ruminal ammonia concentrations on its absorption and that the differences between the ammonia levels of portal and hepatic blood are much greater 16 in ruminants than nonruminants. The liver is able to extract almost all ammonia from the blood; thus, the maxi- mum ammonia concentrations in rumen liquor at which an in- crease in the ammonia levels of the peripheral blood occurs is the "liver threshold value." Toxic signs are observed at high ammonia levels in the peripheral blood. Juhasz (1972) indicated that the role of the "liver threshold value” as well as the hepatic utilization or detoxication of ammonia in the synthesis of urea and probably in the synthesis of protein warrant further clarification, perhaps by isotOpe techniques. Recently Payne and Morris (1969) and Chalupa et_al. (1970) have shown that on ingestion of large doses of urea, hepatic enzymes involved in urea synthesis increase. In sheep, as well as in cattle, aug- mentation of ammonia concentration in rumen liquor resulted in an increase of N in the blood (Juhasz and Kiraly, 1961). Concentration of ammonia in rumen liquor depends on quan- tity and quality of ingested N (Juhasz, 1972). W Short-term fluctuations in plasma urea concentra— tions after urea feeding are associated with a simultaneous change in total turnover. Coccimano and Leng (1967) repor- ted a positive linear regression between plasma urea con- centration and total urea turnover. Harmeyer et_al. (1967) showed that no simple relation exists between plasma urea and endogenous urea turnover rate when considered under various feeding conditions and that variation increases 17 greatly when plasma urea exceeds 11 mM/ml. Also, they indicated that endogenous urea turnover is influenced by factors other than plasma urea concentrations. These factors are still unknown, and primarily affect long-term changes. Urease activity of the rumen wall (Houpt and Houpt, 1968) and C02 concentrations (Thorlacius gt_al., 1971) have been shown to influence urea permeability of the rumen wall. The mechanisms involved in these changes are yet to be investigated. Qther_N_QQmpQnnds_Bes¥£lsd_into_the_3nmen Boda and Havassy (1975) think that N, apart from urea-N, can return to the rumen in the following nitro- genous compounds (Figure 5): 1) As glutamic acid, synthe- sized from ammonia and - ketoglutaric acid in the rumen wall and tissues; 2) As glutamine, synthesized from gluta- mic acid and ammonia in the rumen wall and tissues; 3) As ammonia, eliminated from glutamine by glutaminase in the rumen wall or from other amides by amidase (glutaminase) activity in the rumen wall. For example, after intravenous administration of acetamide, NH3 concentration in the rumen was increased (Hoshino et_al., 1966; Chomyszynet gt_al., 1970); 4) As amino acids formed by transamination of the corresponding keto acids with glutamic acid. E . H I y . 5.! Boda and Havassy (1975) assumed that blood urea-N utilization takes place during nitrogen recirculation 18 UMAM LIVER mrrnnraunr < CYCLE ) mm..- HITABUUSH on. &- (IIOCIUURIC ACID ”NI'CDJ'AIP "d. UREA . All/N0 ACIDS 0° Iranummuu a UYMMC ‘ OUR/NI 61 06094 MINI umnmnr unuBLOOD VESS E L \ :1 ”‘9 ‘I ‘ § . 3 : P E \\ r4 3, g 5‘ 2 . (‘0; .t" " Cb): 6‘ IC \\ 'l 50““ \ l 5 ‘UD l ,m‘ \\\\ O“. 6 art's. N H -——— unu 3 RUMEN INTER/0R 1 SAL/VA .n Mlcroblal proun N H4 _’ synthesis FIGURE 5. Diagram of recirculation and utilization of blood urea and other N compounds in ruminants (from Boda and Havassy, 1975) 19 between the liver, blood pool and digestive tract (mainly the forestomachs), in such a way that part of the ammonia produced by urea hydrolysis in the digestive tract is not taken up by the rumen microflora and is used in other parts of the digestive tract. This may occur immediately after ammonia resorption by the rumen wall and tissues, with a small portion of ammonia again converted to urea in the rumen wall (Kosarovet et_al., 1972), but urea synthesis occurs mostly in the liver. Certain nitrogenous compounds, whose precursors are urea, circulate between the digestive tract and blood pool. Urea-N utilization for protein synthesis by microbes in the rumen may therefore be based on less urea synthesized from the resorbed ammonia during nitrogen recirculation than that hydrolyzed and assimilated in the forestomachs. During 24 hours, less than 50 percent of the administered 15 N dose may be excreted and more than 90 percent of that retained is utilized by microbes. The amount of the blood urea-N utilized daily is quantitatively significant, particularly on low protein diets (Boda and Havassy, 1975). Since a substantial part of blood urea-N may be utilized in the rumen wall and tissues, a clear understand- ing of the process of this utilization is necessary for clarification of the different aspects of nitrogen metabo— lism of ruminants. Besides amino—acid synthesis in the rumen epithelium, glutamine synthesis in particular might be quantitatively important because its amidic-N is needed 20 for synthesis of purine bases (Straub, 1965; Campbell, 1970) and glucosamine (Gottschalk, 1966). These all con- tribute to the body stores of nucleic acid and glyc0pro- teins. Q E'EE . . I I] B The mechanism of endogenous urea influx into the rumen has been studied in several investigations. Gartner claimed in 1962 that the flow of urea from the blood through the rumen wall has to be interpreted as an active transport. Data obtained from in yitrg experiments showed that the amount of urea which appears at the lumen of the mucosa is not a linear function of blood urea concentra- tion. When the urea concentrations exceeded 10 mg/dl, no further increase in ammonia concentrations at the lumen side could be detected. It was concluded that a flux of urea against a concentration gradient was not observed. These findings of Gartner have not been confirmed by subsequent investigators. Engelhardt and Nickel (1965), as well as Houpt and Houpt (1968), reinvestigated the prob- lem and described the transfer of urea from the blood into the rumen as a diffusion process. In 11119 experiments with sheep's mucosa by Engelhardt and Nickel (1965) showed a linear relationship between urea flux from the blood to the lumen, and they interpreted that flux as a diffusion process. Experiments of Engelhardt and Nickel (1965) also indicated that the flux of urea into the rumen of goats was 21 by diffusion. When blood urea concentration was elevated by an intravenous injection of urea, a corresponding in- crease of urea influx into the rumen was observed. Houpt and Houpt (1968) worked with sheep and goats using a rumen pouch and studied the changes of urea concentrations in the pouch solution at various initial pouch concentrations. They found that the direction of net flux depended on the concentration gradient, which also suggests a diffusion process. They also found that large differences existed in urea + ammonia fluxes between different animals. Under the experiemtnal conditions the pouch of goats exhibited much greater permeability than that of sheep. 11W Weston and Hogan (1967) conducted similar experi- ments. They administered increasing amounts of urea into the abomasum of sheep and produced a corresponding increase in blood urea and ruminal ammonia. When blood urea reached about 6.1 uM/ml, further increases failed to increase rumi- nal ammonia concentrations. This may be interpreted as the "saturation phenomenon" commonly associated with active transport. McIntyre (1971) found that plasma urea concen- trations affected urinary urea excretion. He postulated that the kidney increased its rate of urea excretion after plasma concentrations reached about 10.7 uM/ml. Little doubt remains that the transfer of urea nitrogen from the blood into the forestomach of ruminants occurs through a 22 diffusion process. The data of Engelhardt and Nickel (1965) and Houpt and Houpt (1968) clearly show that the amount of urea nitrogen which enters the rumen is deter- mined mainly by the blood urea concentrations. Urea influx into the rumen is of quantitative im- portance, and it may contribute more than 50 percent of the total nitrogen utilized by rumen microbes. Starvation may be regarded as the starting point for varying food and nitrogen intakes (Varady and Harmeyer, 1972). It was shown by Varady et_al. (1967 and 1970) that blood urea concen- tration in ruminants increases during starvation. In goats, but not in sheep, Varady and Harmeyer (1972) also found a slight increase in plasma urea 3-4 hours after feeding. Plasma urea almost doubles in sheep after 30 hours of starvation; but thereafter, concentrations con- tinously decrease. Varady and Harmeyer (1972) suggested that the accumulation of body urea takes place because of a pronounced diminution of endogenous turnover and excretion. It is somewhat surprising that endogenous urea hydrolysis decreases when blood urea level increases. As mentioned earlier, movement of urea in the alimentary tract is regarded as a diffusion process. Its dependency on blood urea concentration has been shown repeatedly. How- ever, the decrease in endogenous turnover concommitantly with an increase in blood urea level may be due to a change in blood flow through the ruminal mucosa with factors other than blood urea supply being important, e.g., a change in 23 permeability. This idea is supported by the difference in urea space between fed and starved animals (Varady and Harmeyer, 1972). McIntyre and Williams (1970) showed that when 12 g of urea was infused intravenously for 8 hours daily for 8 days, there was an improvement in the nitrogen balance of sheep fed low protein rations. This was attributed to in- creased microbial protein synthesis in the rumen resulting from recycled urea nitrogen. Concentrations of urea nitrogen in plasma are pro- portional to the amount of urea nitrogen infused intraven- eously or intraruminally in ruminants (Weston and Hogan, 1967). Rumen ammonia concentrations in ruminants also were linearly related to the amount of urea infused intrarumi- nally. However, McIntyre and Williams (1970) suggested that a plateau in plasma urea nitrogen concentrations of about 30 mg/dl is reached during intravenous urea infusions. 3 l I' .3 EEE I Q E'EE . Thorlacius et_al., (1971) studied the effect of carbon dioxide on urea diffusion through ruminal epithelium and found that the temporary substitution of 100 percent CO2 for N2 by gassing the rumen initiated a marked rise in the flux of urea from the blood to the rumen. This res- ponse could be divided into three phases. Initially, there was a lag period during which the flux of urea was un- changed, followed by a phase in which the urea flux rose 24 sharply to a maximum. The third phase was a slow decline toward the prestimulatory level. It was also reported that a graded response could be observed if the CO2 was diluted with N2 and the maximum observed was greatly reduced. Moreover, the length of time that flux was enhanced was considerably shorter. H l . E E] i H I E . I I] E Houpt and Houpt (1968) hypothesized that the normal mechanism for blood urea nitrogen transfer across the rumen wall involved a close association of bacterial urease with the rumen epithelium and penetration by urease of the epi- thelial layers for an unknown distance, with the cornified layers constituting the major barrier to the physical move- ment of small molecules across the rumen wall. The ammonia molecule is smaller and more lipid soluble than urea and should penetrate cell layers much more rapidly than urea. The essence of this hypothesis is that urea molecules pass by diffusion from the blood vessels to the basal epithelial layers, the site of the bacterial urease action and are hydrolyzed to ammonia and carbon dioxide. The ammonia molecules penetrate the cornified cell barrier by diffusion and enter the rumen interior, thus enhancing urea nitrogen transfer from the blood to the rumen. Transfer of blood urea nitrogen into the rumen is most important when the animal is on a low-protein ration. Under these conditions, rumen ammonia is extensively uti- lized for microbial protein synthesis, and rumen ammonia 25 concentrations are comparatively low. By the proposed hypothesis, the final movement of ammonia from the epi- thelium into the rumen would depend on a concentration gra- dient from the site of urea hydrolysis to the rumen inter- ior. A low rumen ammonia concentration would incresae urea nitrogen transfer. Conversely, if concentrations were high, less urea would transfer and more of the ammonia in the epithelium would diffuse back into the blood. This relationship between rumen ammonia concentration and urea nitrogen transfer from the blood into the rumen contributes to the control of rumen ammonia concentrations (Houpt and Houpt, 1968). Although evidence has been reported (Cocimano and Leng, 1967) that intact urea is transferred from blood to sites of microbial degradation in the digestive tract, there is controversy about the extent of transfer to the rumen and the means by which transfer to the rumen occurs (Allen and Miller, 1976). Weston and Hogan (1967) sugges- ted a maximal transfer of blood urea to the rumen of sheep of approximately 5 g N/d, while Nolan and Leng (1972) concluded that only 1.2 g N/d was transferred. WWW Secretion of urea in saliva contributes to the transfer of blood urea to the rumen (Somers, 1961), but Houpt (1959) estimated that transport of urea across the rumen epithelium could account for up to 95 percent of the total N transfer. Nolan and Leng (1972) suggested, how- 26 ever, that virtually all transfer of intact urea occured via the saliva, and dietary factors might explain observed differences in the importance of transfer sites. Weston and Hogan (1967) showed that sheep on a low nitrogen diet had limited transfer of urea from blood to the rumen at plasma urea concentrations lower than 16-18 mg N/dl, but at higher plasma urea concentrations (caused by infusing urea into the abomasum) rumen ammonia could not be increased above 8-10 mg N/dl. Nolan and Leng (1972) reported that recycling of ammonia takes place largely within the rumen itself, namely, ammonia -> other nitrogenous compounds -> ammonia. This could result from lysis of bacteria in the rumen due to bacteriophage activity (Hoogenrand et_al., 1967), en- gulfment of bacteria by protozoa which utilize bacterial amino acids (Coleman, 1967) and produce ammonia as an end- product of their intermediary metabolism (McDonald, 1968), or death of bacteria (Hungate, 1967). It was suggested that 30 percent of the ammonia incorporated into rumen microbial protein may have recycled through the amino acid and ammonia pools. One estimate has indicated that 40 per- cent of ruminal bacteria is engulfed by protozoa (Abe and Kandatsu, 1969). It was assumed that most of the ammonia-N was recycled through the amino acid pool (Nolan and Leng, 1972). If this occurs on all diets, it may be an important process in supplying the requirements for amino acids and 27 branched-chain fatty acids for some species of bacteria in the rumen. However, if rumen degradation of microorganisms could be prevented, it would increase the quantity of protein leaving the rumen which is available for digestion in the small intestine. WW Concerning the transfer of blood urea into the abomasum via gastric secretions, Nolan (1974) indicated that in sheep, only 29 percent of the ammonia N in duodenal fluid was derived from ammonia in rumen fluid. It is hypo- thesized that the other 71 percent is derived from blood urea that passes into the abomasum with gastric secretions (Harrop, 1974). The transfer of blood urea into the intestines has been studied by many researchers. There is evidence that urea enters the small intestine, and concentrations of urea in intestinal digesta approach those occurring in the blood (Hecker, 1967). Towards the ileal-cecal junction, in an area where bacterial activity occurs (Ben-Gheldalia et_al., 1974), urea concentrations in digesta decline (Becker, 1967), presumably because of microbial urease. The result- ing ammonia must either be absorbed from the ileum or pass into the large intestine. Infusion of labelled ammonia into the cecum indicated that urea either passes through the cecal wall or enters the gut just anterior to the cecum. Urea also enters the colon or rectum through the gut wall as indicated by the rapid labelling of ammonia in 28 feces (Nolan et_al., 1976). Other experiments showed that most of the endogenous N passing into the duodenum origi- nates in the forestomachs and not in the saliva. Sources of endogenous N include urea, salivary proteins, mucus from the respiratory tract, and epithelial cells sloughed from the mucosa of the buccal cavity, the esophagus and the stomach itself. Arginine, glutamate and glutamine and other amino acids may also be transferred to the fore— stomachs (Boda and Havassy, 1975; Harmeyer et_al., 1967). Proteins, urea and ammonia are present in gastric secre- tions and may contribute up to 2.4 g N/d in sheep (HarrOp, 1974). H' l' J E I . S I] . The urease activity of the rumen is sufficient to convert rapidly to NH3 all the urea likely to be included in the diet as a partial substitute for protein. The uti- lization of urea by the ruminant takes place in two stages, first the conversion of urea to NH3 and then the incorpora- tion of NH3 into protein. Pearson and Smith (1943) found that about 100 g of rumen contents converts 100 mg urea to NH3 per hour. This hydrolysis is affected by many factors, such as tempera- ture, pH, concentration of urea, nature of gases present and presence of starch or certain inhibitory substances. Ammonia, the end product of urea hydrolysis is the prefer- red source of nitrogen for a large majority of rumen bac- teria (Brown et_al., 1958, 1960; Browning and Lusk, 1966). 29 Low contributions of ammonia may limit growth of bacteria. At dietary levels less than 1.2 percent nitro- gen, rumen functions were impaired, feed intake was reduced and growth of the host animal stopped or was markedly limited (Bryant and Robinson, 1962; Burroughs et_al., 1951a, 1951b; Caffrey £L_a1., 1967a). Phosphorus is probably the most important mineral for stimulating microbial growth, but others are also ef- fective (Brown gt_al., 1958; Caffrey et_al., 1967b; Colovos et_al., 1963, 1967; Conrad and Hibbs, 1961). Sulfur is apparently needed in largest amounts for optimal utiliza- tion of urea and other forms of NPN (Colovos et_al., 1967) and should be considered when formulating rations rela- tively high in NPN. Portugal (1963) used carbon-labelled amino acids and found that only about 10 percent of the carbon in amino acids was incorporated into microbial protein in the rumen; whereas, Weller et_al. (1962) concluded that up to 80 per- cent of the dietary plant N is incorporated into microbial cells. These findings indicate that the carbon and N of dietary protein are separated during metabolism in the rumen before incorporation into microbial cells. Bryant and Robinson (1962) showed both a requirement and prefer- ence of certain rumen organisms for ammonia compared to amino acids. Hobson et_al. (1968) found that one rumen isolate formed 93 percent of its cellular N from ammonia. 30 Clearly, rumen ammonia is an important source of N for rumen microbes. Investigations using heterotroPhic bacteria have suggested increased incorporation of amino acids and pep- tides when these were readily available (Warner, 1956). Since there is usually a positive correlation between non- ammonia and ammonia-N in the rumen (Blackburn and Hobson, 1960), ammonia increases are likely to be accompanied by enhanced amounts of amino acids and peptides available to the rumen microbes. Of the calculated incorporation of N into microbial cells, it has been generally assumed that 80 percent was derived from ammonia and 20 percent from amino acids. Pilgrim gt_al. (1970) estimated in sheep given lucerne that 62-64 percent of the N in bacterial protein and 35-41 per- cent in protozoal protein was derived from ruminal ammonia. The presence of dietary sources of readily avail- able carbohydrates in the form of starch or sugar enhanced the microbial synthesis of protein, and nitrogen balances in sheep fed such rations were greater than on all-roughage rations (McIntyre and Williams, 1970). [1' 15IHI . I . 1H“ 1 J. On the first and third days when 15N-urea was ad- ministered intravenously, more than 50 percent was retained and only a small amount (0-6%) appeared in rumen fluid (McIntyre and Williams, 1970). The net passage into the rumen of nitrogenous compounds, whose precursor was blood 31 urea, was increased when the capacity to retain blood urea was lowered, i.e., when dietary nitrogen intake was high. In other studies by Boda and Havassy (1975) with high and 15 low N diets, blood N-urea incorporation into TCA preci- pitable protein in plasma was slower on low N and blood 15N decreased more rapidly on high N. On the 4th and 5th day 15N into the rumen was markedly lower for the low-N diet, incorporation of 15N after infusion, when net passage of into plasma protein had increased. 15 When N-urea was introduced through a fistula into the anterior part of the jejunum, 63 percent of the dose 15 was retained by the animal. Hence, excretion of N in urine and feces totalled 37 percent (Boda et_al., 1976). About 81 percent of the intra-jejunally administered 15N participated in metabolic processes (retained N plus uri— nary excreted N). Apart from the rumino-hepatical circula— tion, the entgrg;hapitigal circulation of nitrogenous sub- stances, including endogenous urea-nitrogen, plays a quantitative role in recycling of blood urea nitrogen recycling. Boda gt_al. (1976) also showed that all parts of the digestive tract take part in blood urea-N utiliza- tion, and nitrogen compounds synthesized from blood urea- 15N were recycled into the alimentary tract. Nitrogen compounds were secreted from blood mainly into the fore- stomachs, abomasum and duodenum, but were absorbed from the entire intestinal tract. 32 In other studies, Boda et_al. (1976) attempted to confirm the hypothesis that blood urea in ruminants is a qualitatively significant nitrogen source by administering 15N-urea intravenously. It was shown that in the nitrogen 1 pool of sheep, considerable 5N (44-76% from a given dose) was retained in the body and the greater part of the unre- tained portion was excreted in urine within 24 hours. Ex- 15 cretion in feces amounted to 1.35-2.37 percent of the N 15 dose. After low nitrOgen intake, more N from a given dose was retained in the nitrogen-pool than after high 15 nitrogen intake. The daily N excretion in urine and the 15N ammonia level in rumen fluid were parallel. The 15 N from the labelled urea transported in blood passes into the sheep's rumen relatively quickly with maxi- mum enrichment 3 hours after a single infusion. Boda at al. (1976) supposed that considerable amounts of ammonia produced in the rumen wall by means of urea hydrolysis could be used to direct the enzymatic synthesis of amino acids and other nitrogenous substances in the rumen wall, liver or other organs. Most of these presynthesized nitro- genous substances pass into the rumen and probably into the other parts of the alimentary tract, and serve as nitrogen sources in digestive processes. The atom-percent 15N en- 15N from a richment in blood as well as the percentage of given dose decreased in the course of 24 hours and then increased again during the next two days. It may be pre- sumed that for the first 3 hours after a single infusion of 33 15 15 N-urea into the blood, incorporation of N into the blood plasma proteins takes place via synthesis of amino acids from ammonia in the liver after urea hydrolysis in the alimentary tract. This synthesis is realized predomi- nantly by the enzyme glutamate which catalyzes the synthe- sis of glumatic acid and its subsequent transamination. From the second to the third day, the labelled urea nitro- gen was probably incorporated into the blood plasma pro- teins by two routes: amino acids absorbed from synthesis of microbial proteins and synthesis from non-microbial nitro- gen compounds such as amide-N for synthesis of purine bases and histidine (Campbell, 1970), as well as glucosamine (Gottschalk, 1966), which are all necessary for synthesis of nucleic acids and prosthetic groups of glyc0proteins. E I . E 15H_H Boda et_al. (1976) found that of the 15 N-urea ex- creted after a single infusion, most appears on the same day the labelled urea was administered. On the following 15 day, only small amounts of the N (1.2-4.2%) were excre- ted, and on the 3rd-9th day, 15 15 N excretion was impercep- tible. The amount of N excreted in urine was extremely variable (24-56% of that infused). At the low nitrogen in- take, blood urea-N retention was higher (70-76% of infused) than at higher nitrogen intake (44-62% of infused). Lower nitrogen intake was associated with lower ammonia concen- trations in the rumen, lower blood urea levels and lower nitrogen excretion in urine. 34 15 Excretion of N in feces was less than 1 percent of the dose, and was negligible in comparison with that in 1 . ' . . . . 5N urinary excretion of intravenously adminis- urine. The tered urea practically ceases two days after infusion of a single dose. This indicates that the retained blood urea-N is incorporated into other nitrogen metabolites used for biosynthetic processes. The percentage of the retained 15 N decreased pro- portionally with increased N excreted in urine in 24 hours, with increased ammonia concentration in the rumen, and with urea in blood plasma before feeding (Boda and Havassy, 1975). However, many metabolic pathways associated with blood-N utilization are still unknown. CQannSiQn Based on the literature mentioned, nitrogen recyc- ling in dairy cows has been demonstrated on both high and low-N diets. Some challenges which remain for further in- vestigation are: 1) To develop more precise methods to quantitatively predict nitrogen recycling; 2) To establish the role of energy level of the diet in affecting the amount of nitrogen recycling that occurs. The following study was designed to use 15 N-labelled urea as a precise method to estimate the urea movement from the blood into the rumen microorganisms pool and its contribution to the microbial protein synthesis. MATERIALS AND METHODS Animals Two dairy cows fitted with flexible (T) cannulas in the proximal duodenum were used for two periods (28 days each). During the first period, cows were fed a diet (C) of 1:2 forage:concentrate, while a 2:1 forage:concentrate diet (F) was fed during the second period. The forage was a mixture of 70 percent of alfalfa cubes and 30 percent of cotton seed hulls. Composition of the concentrate mixture is shown in Table 1. Table 1. Compositiog of Concentrate Mixture Fed in Both Diets . Component Percent Flaked milo 87.0 Soybean meal 7.0 Molasses 4.0 Dical 1.5 Salt .5 Vit A ___ a) The concentrate mixture was fed at 1:2 forage:concen- trate or 2:1 forage:concentrate. 35 36 Diets were calculated to be isonitrogenous, but crude protein was determined to be 14 percent for the C diet and 13 percent for the F diet. I Chromium oxide (Cr203) was given as a marker in gelatin capsules at feeding (12h intervals). Each capsule contained 129 of Cr203 powder. 15 Urea enriched with N at 10 atom percent was dis- solved in 3,000 ml of sterile saline solution, and continu- 15N isotope per day for 3d using infu- ally infused at 2g sion tubing placed in the jugular vein which was attached to a bag containing infusate. Bags were placed directly above each cow. DrOps of infusate were adjusted regularly 15N urea was infused at a constant rate over the so that entire period. The infusion apparatus allowed cows full comfort and normal behavior in eating, drinking, and movement. Cows were adjusted to their shaded, cement pens equipped with rubber mats for several weeks before infusion started so as to retain normal habits in eating and drinking. W Duodenal digesta were collected every 3h starting 1d prior to infusion, and sampling continued for a total of 5d. Samples were collected in clean plastic jars (400 m1 digesta each time), pooled for 3, 6, 8, 12, 24 and 30h , and frozen at -20C until analyzed. Coccygeal blood was' sampled twice daily using heparinized sterile.vacutainer tubes (10ml) and spun immediately in a refrigerated 37 centrifuge at 4500 rpm. Plasma was then separated and frozen at -20C until analyzed. Milk samples were taken every milking during the 5d collection period in sterile plastic bags‘and frozen at -20C until analyzed. Fecal samples were taken from the rectum twice daily during col- lection and frozen in plastic bags at -20C until analyzed during the 3d of infusion. Total feces were collected in clean covered buckets for 3d. The daily fecal production was thoroughly mixed, sampled and frozen at -20C until analysis. Total collection of urine was at 12, 24, and 36h by use of a sterile 24" french urinary catheter equipped with 75 baloon, which was inserted into the cows bladder and then inflated. The catheter was connected to a 2 l collection bag and then to a 20 1 plastic bucket. Hydro- chloric acid was added daily to the urine collection bucket to prevent escape of ammonia. Samples of urine were col- lect daily and refrigerated until analysis. W Duodenal digesta samples were thawed at room tem- perature and oven-dried at 50C to avoid heat damage. Fecal samples were prepared similarly. For obtaining bacteria from duodenal and rumen fluid, unfrozen samples of digesta were strained through 3 layers of cheesecloth and wool pyrex, differentially centrifuged at 3,000 and 18,000 9 (Smith and McAllan, 1974), and then washed with a buffer solution. 38 W Duodenal digesta, feces, milk, blood, urine and bacteria from the rumen and duodenum were digested with sulfuric acid using a block digestor (Goering and VanSoest, 1970). In preparation for 15 N analysis, sample aliquots were steam-distilled (Barker and Volk, 1964), the liberated ammonia was collected into 0.05 MHCL, and the resultant solution was dried on a drying block at 95C for 24 hours. 15 Samples were then analyzed for N using mass spectrometry (Consolidated Electrodynamics Corporation model 21-621). The atom percent 15 N in the samples was calculated from the N—28 to N-29 mass ratio following the methods of Bremner (1965) and Frota and Tucker (1972, 1978). The main steps of these procedures were summarized as follows by Mohammed and Tucker (1981): 1. About 2 m1 of solution were brought to complete dryness. 2. The completely dried sample (now as a salt) was cooled and the air was replaced with argon gas. 3. About .3 ml of argon-saturated deionized water was added to each sample which was held on ice. The water was frozen immediately, forming an ice layer over the sample to keep it free of air. The mixture of ice and sample were then connected to the argon gas system. 39 4. A similar amount (.3 m1) of argon-saturated sodium hypobromite was then added to each sample and allowed to freeze. 5. The sample tubes were attached to the mass spec- trometer. This was followed by a diffusion pump- to insure an air-free condition. Samples were thawed to permit sodium hypobromite to react with ammonia and convert it into N gas. The reaction involved is generally represented as follows: 2 NH3 + 3 NaOBr-—-->3 Na Br + 3 H20 + N2 (Bremner, 1965) 6. Finally, the N2 was analyzed for peak heights of N-28 and N—29 mass from which the atom percent of 15N was calculated as follows: Total atom percent of 15N = 100/2R + 1 Where R = N-28 peak / N-29 peak All samples were analyzed for nitrogen using the Technicon Auto Analyzer and for dry matter by.oven-drying at 100 C according to AOAC (1980). Sample aliquots were prepared for chromium assay by initial digestion with H2504 and redigestion using periodic acid and then analyzed by atomic absorption spectropho- tometry. RESULTS Dry matter intakes were similar for both diets with the lactating cow (no. 6) consuming about 60 percent more than the non-lactating cow (no. 958) (Table 2). Uncorrec- ted dry matter disappearance (across diets) in the rumen averaged (27 and 46%) on the grain and forage diet, res- pectively. Whereas, 53 and 68 percent of diet dry matter was fermented in the rumen on the grain and forage diet respectively, after correction for de novo synthesis of microbial cellular dry matter (Appendix Table 18). Dry matter fermented in the rumen of the lactating cow was similar with both diets, while the dry cow showed unexpectedly low values when fed the grain diet, especially for uncorrected DM (Table 2). Total tract digestibility of DM was slightly higher for the grain diet (76 vs. 73%), thus, resulting in more intestinal degradation on this diet (Appendix Table 19). Bacterial synthesis values (gcp/ngMc digested in rumen) were (71, 54% on the grain diet and 74, 74% on the forage diet) (Appendix Table 8). Even though slightly more dry matter disappeared on the forage diet which was predominantly fiber, that used on the grain diet was mostly starch which might result in a higher efficiency of microbial synthesis. 40 41 Table 2. Intake, Duodenal Flow and Fecal Output of DM of Cows Fed High Grain or High Forage Diets. Diet High Grain High Forage COW 958 6 Ave 958 6 Ave Dry matter (Kg/d) Intake 8.1 13.7 10.9 8.5 14.1 11.3 Duodenal Flow (Total) 7.5 7.4 7.5 4.6 7.8 6.2 From Feed 4.58 5.14 4.86 2.45 4.84 3.64 From Bacteria 2.87 2.30 2.59 2.13 2.92 2.52 Fecal Output 1.62 3.72 2.67 2.05 4.34 3.20 Digesteda In Rumen 3.52 8.56 6.04 6.05 9.26 7.66 Post Duodenum 5.88 3.68 4.83 2.55 3.46 3.00 a) Corrected for de novo synthesis of microbial cells. Nitrogen intakes were similar for both diets, with the lactating cow (no. 6) consuming about 62 percent more than the non-lactating (no. 958) (Table 3). More duodenal-N flow was observed in cows fed the high grain than high forage diet (122 vs. 101% of intake) (Appendix Table 8). 286 g/d on high grain and 233 g/d on high forage. Comparing the diets, nitrogen flow was Total tract N digestibility was similar for the two diets (73.4 vs. 73.2%) (Appendix Table 19). 42 Table 3. Nitrogen Intake, Duodenal Flow and Fecal Output of Cows Fed High Grain or High Forage Diets. Diet High Grain High Forage COW 958 6 Ave 958 6 Ave Nitrogen (g/d) Intake 191.2 295.0 243.1 174.0 294.0 234.0 Duodenal Flow (Total) 269.0 303.0 286.0 184.0 282.0 233.0 From Feed 79.2 139.6 109.4 48.4 74.5 61.5 From Bacteria 189.8 163.4 176.6 135.6 207.5 171.5 Post Duodenum 226.6 213.4 220.0 141.1 196.8 168.9 Fecal Output 42.4 89.6 66.0 42.9 85.2 64.1 H'! E . l I . E' J . J S 1' Nitrogen enrichment of all samplings gradually in- creased with time after initiation of urea 15 In duodenal digesta, 15 N appeared as early as 8h after N infusion. beginning of infusion, and gradually increased with time and peaked at 60-70h. The average of the highest 3 enrich- ment values (Table 4) were .071 and 0.74 atom—percent for the two cows on the grain diet (Figures 6 and 10 in Appendix) and .064 and .043 atom-percent for the two cows on the forage diet (Figures 11 and 12 in Appendix). In duodenal bacteria, 15 N also appeared at about 8h after initiation of infusion, and, as expected, the pattern paralleled that of duodenal digesta. The average of the 3 highest values for bacterial enrichment were .092 and .14 43 atom-percent for the two cows on the grain diet (Figures 6 and 10 in Appendix) and .075 and .077 atom-percent for the two cows on the forage diet (Figures 11 and 12 in appendix). Table 4. Average of 3 Peak Values for 15N Enrichment of Each Cow in Duodenal Digesta, Duodengl Bacteria, Urine, Feces, Milk, and Blood . Enrichment (Atom-Percent) DIET COW Digesta Bacteria Urine Feces Milk Blood Grain 958 .071 .092 .62 .066 ---- .015 6 .074 .14 .44 .072 .062 .017 Avg .073 .12 .53 .069 .062 .016 Forage 958 .064 .075 .59 .060 ---- .017 6 .043 .077 .49 .053 .060 .023 Avg .054 .076 .54 .057 .060 .020 15 a) For calculation of N enrichment, observed values were fgbtracted from a standard value of .361 which is the N concentration of non-enriched nitrogen that {i.e., for cow 958 on grain diet: .432 (observed value) - .361 = .071 (enrichment value)}. NitrOgen enrichment in urine appeared as early as 12h after initiation of infusion, rapidly increased with time, and peaked at 60-70h after infusion began. 15 15 ment. cycled N were based on urinary blood nitrogen components, so plasma. 15 Hence, all calculations for 15 15 N enrichment was assumed to equal blood urea Urinary N was measured in blood N enrich- N enrichment of re- N in blood urea from other The average of the 3 highest peak values for uri- nary enrichment were .62 and .44 atom-percent for the two 44 cows on the grain diet (Figures 7 and 13 in Appendix) and .59 and .49 atom-percent for the two cows on the forage diet (Figures 14 and 15 in Appendix). Most of the 15 N excreted from the body exited through the urine with uri— nary 15N enrichment values 10 times as high as those in bacteria, feces or milk (Table 4). Nitrogen enrichment in feces and milk appeared as early as 10 to 12h after initial infusion and gradually increased with time to peak at 50-80h (Figures 8, 9, and 16-19 in Appendix). About 15 percent of the fecal and milk-N originated from blood urea (Table 4). E I i B E'isH H]. l I E i I I E] i Nitrogen-15 retained in the different tissues and organs of the body was higher in the two cows fed the for- age than the grain diets (70.1 vs. 56.9%) (Table 5). These results are in agreement with the work of Smith et_al. (1982) who showed that 40-70 percent of 15 N dose was re- tained in the body of cows fed corn silage treated with ammonia-ISN. 45 15 Table 5. Fate of N Infused into Blooda. Urinary Duodenal Excretion Passage Output (g/d) Retained DIET cow (g/d) (g/d) in Milk in Feces (%) Grain 958 .62 .188 --- .05 60.9 6 .82 .224 .06 .05 52.9 Avg .72 .206 .06 .05 56.9 Forage 958 .71 .070 --- .03 74.5 6 .79 .074 .04 .05 65.7 Avg .75 .072 .04 .04 70.1 15 a) Amount of N infused was 2 g/d. E i J i E I . J l I . N : . . I' E E] 3_ Urea_lBac¥slsd:N1 As shown in Table 7, more recycled nitrogen appear- ed in duodenal and bacterial protein of cows fed the grain than the forage diet. It was assumed that essentially all the 15 N passing into the duodenum was as bacterial protein. An example of the method used in calculating the amount of duodenal and bacteria N originating from blood urea is given in Table 6. As was mentioned, urinary enrichment which was measured directly, was assumed equal to blood urea enrichment because urinary urea came directly from blood urea with only minimal dilution of other N source. Percent lsN enrichment in bacteria isolated from the duodenal digesta of cows fed the forage diet was similar to that of bacteria isolated from the rumen. 46 Also given in Table 7 are measurements of the per- cent of the total duodenal N as bacterial N, which was cal- 15N enrichment between duodenal and culated as the ratio of bacterial N., Table 6. Example of Calculation, Cow No. 6 on Grain Diet Item Percent 15 Urinary enrichment in N .44 Blood urea enrichment in 15N (assumed) .44 Digesta enrichment in 15N .07 Percent of N in digesta coming from blood urea (.07/.44) 17 15 Bacteria enrichment in N .14 Percent of N in bacteria (exiting rumen) coming from blood urea (.14/.44) 32 Table 7. Percent of N Which Originated From Blood Urea (or Recycled N) Recovered in Duodenal or Bacterial N. Recycled Recycled Total N as Duodenal N Bacterial N Bacteria (total N (total in digesta) bacterial N) DIET COW Percent Percent Percent Grain 958 11.9 15.2 78.3 6 17.3 32.6 53.1 Avg 14.6 23.9 61.1 Forage 958 11.2 12.6 88.8 6 8.7 14.9 58.4 AVg 9.9 13.8 71.7 SEM 2.79 5.34 47 uoflp Cadum so Amy zoo mo mfiumuomn Hmcmposp was mumomflp Hmcwposp cfi ucmenofiuchZmH .w mmDuHm ZOHmszH mo Bm<9m 20mm Any mSHB Tmop —.nm 9mm ndm N.nm m.mn m.wN min— 0 (EH—95 .le (hmmoa (ml. .4 mpwn. .. mmmmn. I nwmov. .. nonwv. 1¢n¢¢. I mung... I 03.3. .. nnwom. .. mvwm. 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SI (%WOLV) XTIW NI LNSWHDIHNH-N DISCUSSION WM These data show greater flow of N into the duodenum than there was intake of feed N, and agree with studies by Wanderley and Theurer (1983) in beef steers who showed that the flow of nitrogen reaching the small intestine was greater than that consumed. Based on Cr203 flow estimates, Wanderley and Theurer (1983) reported that the average N reaching the small intestine was 140 percent of that consumed in steers fed the grain diet and 101 percent for the forage diet. The flow of all the amino acids, especially DAP, into the small intestine was greater on grain than forage diet. Our data also show greater N flow on the grain diet. In another study, Wanderley gt_al. (1986) showed that the crude protein flow into the duodenum in beef steers was about 33 percent greater for a grain than forage diet, although protein intake was about 10 percent less on grain. Duodenal bacterial protein, rather than feed pro- tein escaping ruminal degradation, accounted for most of this difference, suggesting that greater amounts of bacter- ial protein were synthesized in the rumen of the grain-fed than forage fed steers and might be attributed to higher 51 52 intake of rumen available energy (Oldham and Tamminga, 1980). In the studies of Wanderley et_alL (1986), micro- bial-N production exceeded the amount of feed N degraded in the rumen, with the grain diet, presumably by utilizing large amounts of recycled N for ruminal bacterial synthe- sis. Dietary grain to forage ratios showed a marked effect on ruminal protein digestion and duodenal flow of protein. Our data support those of Wanderley and Theurer (1983) and Wanderley et_al. (1986) in that a mean of 21 percent more N reached the duodenum of cows fed the grain diet than was consumed. Whereas, passage of N into the duodenum on the forage diet equalled N intake. Our data contrast with those of Wanderley et_al. (1986) in that we show more passage of feed N on the grain diet but little difference in bacterial flow. However, due to less feed N degraded in the rumen on the grain diet, there was less NH3 from the feed N available for microbial synthesis. This difference in NH3 availability for growth of bacteria was apparently compen- sated for by greater recycling of blood urea into the rumen. The 15 N data suggest that about twice as much re- cycled nitrogen was captured in bacterial protein when the lactating cow (6) was fed the grain diet as compared to the forage diet, perhaps suggesting a different extent in re- cycling between diets. 53 RaLa_Q£_NiLLQg3n_EnIiQhmBnL_in_DnQdfinal_DisfifiLai_DnQd§nal Bacteria; and Rumen Baghe:iao The N enrichment of duodenal digesta, and duodenal bacteria appeared as early as 8h after infusion. The en- richment in duodenal digesta and bacteria, was greater in cows fed the grain than the forage diet (Figure 6 and 10-12 in Appendix). The average 3 peak values considered the steady state were reached after 60-70h of infusion. Concentration of 15 N then decreased gradually for about 72h when infusion stopped. The enrichment curves of duodenal digesta and bacteria had similar trends in peaking, but the bacterial curves took a longer time to decrease after infusion stopped in comparison with the digesta curve. Oldham et_al. (1980) showed that the enrichment of rumen ammonia N and plasma urea N gradually increased after a continuous infusion of 15NH4C1 into the rumen for 29h and then started decreasing after infusion stopped. The en- richment of rumen ammonia N increased much faster than the plasma urea N, and the 15 N enrichment in NH3 N remained constant for the last 10h of infusion. These data illus- trate the difficulties in achieving good mixing of isotope and of taking "representative" samples of rumen fluid from dairy cows. The plateau of their calculated enrichment was the arithmetic mean of the abundances measured in the last nine samples taken before the infusion was stopped. The 15 plateau urea- N abundance was the arithmetic mean of the 54 last six samples taken before the infusion st0pped. This 15N is likely to be a minimum transfer coefficient, as urea- abundance will probably rise slowly for a long time. Oldham et_al. (1980) showed very rapid equilibra- tion of bacterial N with rumen NH3N. The prOportions of bacterial N derived from rumen NH3N ranged between 0.7 and 0.8. H'l E . l I . u . E i ”'1! Enrichment of urine appeared as early as 12h after infusion and was greater in cows fed the grain than the forage diet. The steady state estimated in our study was the average 3 peak values the animal reached between 60 and 72h after initiation of infusion. A sharp increase was noticed in the urine enrich- ment curves compared to enrichment curves for duodenal digesta, bacteria, rumen bacteria, feces, and milk. Also, a sharper decrease was noticed in urinary enrichment after infusion stopped. This was probably due to a much more rapid turnover of urea in blood than NH3 in the rumen. 15 Most of the N dose which was excreted, exited in the 15N concentrations about 10 times as high urine, which had as those in bacteria, feces, or milk (Figure 7 and 13-15 in Appendix). Feces enrichment appeared as early as 10h after infusion started and was greater in cows fed the grain than the forage diet. The 3 peak values were considered as the steady state and were observed 50-80h after initial 55 infusion. Milk and feces together accounted for only about 15 10 percent of the total N excreted in this study. Smith et_al. (1982) showed that more than one-third of the 15N excreted was voided in feces and a similar amounts in urine 15 and milk of cows fed corn silage treated with ammonia N (Figures 8 and 16-18 in Appendix). The higher fecal and lower urinary excretion in 15 their study was a result of NH3 being incorporated into the diet and going directly to the rumen; whereas, 15N only entered into the rumen through recycling in our study. The milk enrichment appeared as early as 12h after infusion and was greater in cows fed the grain than the forage diet (Figures 9 and 19 in Appendix). The steady 15 state for N in milk was the avearge of 3 peak values reached 50-70h after initial infusion. Smith gt_al. (1982) showed that 15 15 N in milk accounted for 25.5 percent of total N excretion in cows fed corn silage treated with ammonia, 15 again reflecting direct entry of N into the rumen. E l E 15n [E I. E 3 E I I. 1 15N-duodenal passage, 15N-output in feces and milk were greater in cows fed the grain than the forage diet. However, none of the cows excreted over 50 percent of the infused 15N during the entire sampling period. In fact, 53-75 percent of the urea-lsN dose was retained in the body. Smith et_al. (1982) showed that 40-70 percent of 15 the fed N was retained after 3 weeks in the body of cows 56 fed corn silage treated with ammonia-ISN. However, most had been eliminated in one cow sampled at 6 weeks. Boda et_al. (1976) showed that after a single ad- 15 ministration, 44-76 percent of N was retained in the nitrogen pool when sheep were fed 7.6-24g/d of N using an intravenous injection of 15N-urea. SUMMARY AND CONCLUSIONS Two duodenally cannulated cows were used to study the recycled-N from blood urea utilized by rumen bacteria. For 28 days prior to and during infusion, cows were fed diets of 1:2 or 2:1 forage:concentrate. Urea-lsN solution was continuously infused into the jugular vein for 3d. Samples of duodenal digesta were taken every 3h for 5d. Blood and milk were sampled twice daily. Urine was collec- ted for 5d starting 1d before infusion and feces were col- lected for the 3d during infusion. Fecal samples were taken twice daily during the 5d of collection. Recycled nitrogen incorporated into rumen microbes apeared to be greater in cows fed the grain than the forage diet (23 vs. 14% of microbial-N-passing into the duodenum). Also, it was higher in the lactating than the dry cow. The flow of nitrogen from the rumen to the small intestine was greater in cows fed the concentrate than in cows fed the forage diet (122.0 vs. 101.0% of intake). 15 Within 10-12h after starting N infusion, labelled nitrogen appeared in milk and feces with peaks at 50-80h. About 15 percent of the milk-N and fecal-N originated from 15N was 10 times that for 15 blood-urea. Urine enrichment of bacteria, feces or milk. Most of the N excretion was in urine. 57 58 These results suggest that the N-recycling process in dairy cows can contribute significantly to the animal's nitrogen pool, especially when "normal" diets high in grain or forage are fed. More research is suggested to investi- gate in great depth the factors which control recycling of N into the rumen. APPENDIX 59 5&5». Ix) 5.890 ...ml " 817°0’Pl pomp casum co Ammmv 300 no mfiumuomn Hmcwposp can mumwmac Haemaogc an namenoaucm-2mfl .OH mmscHa onmamzH so smasm 20mm and mzHa - zezz: - 9: 90: ~ 9'19 F :Z'oz. - 99 zs - 21's: - 991: 'J mpmn. :mvmhn. Inc—mm. unmmov. :Nowv. umnvn¢. zmnmvv. Inmnmv. rmmhv. (%WO$V) VIHSIOVH TVNHQODG ONV VISSOIU TVNSQODG NI LNBNHDIHMQ-N SI 60 35.5.3 (XI 5805 IT) [- S’I’ZH umac ommuom co Ammmv 300 no mwumuomn accoposp can mummmac Hmcocoac ca nameaofiuem-2ma .HH mmsoHa )- wt'zr onmamzH so amasm 20mm Ago msz " 82819 ' lSS'HQ " 933 l? “ 616'09.‘ " €19 OZ ‘ 90): Ol .\ J m—on. .. nnnhn. x moon. 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