v dip" k sf... .3153 "320'! v - “‘ “6' £§$Qf“‘;:q_q ‘ “er? This is to certify that the thesis entitled A STUDY OF THE EFFECTS OF ISOACIDS UREA AND SULFUR ON THE RATE OF FER- MENTATION IN THE RUMEN. presented by . Maria Esperanza Quispe Salas has been accepted towards fulfillment of the requirements for M. S. degree in _An1maLS.cience Wm. M Major professor Date—{mm 0-7639 MS U is an Affirmative Action/Equal Opportunity Institution MSU RETURNING MATERIALS: PIace in book drop to LJBRARJES remove this checkout from mun-n. your record. FINES win be charged if book is returned after the date stamped below. - A. I. . .. * M A :3?!" w‘“ . #5:) 1,: EEE dféfii‘ w~ q.,~ .«=~memseaw~ L .. ‘ . ' ~ '1 5' A STUDY OF THE EFFECTS OF ISOACIDS, UREA, AND SULFUR ON THE RATE OF FERMENTATION IN THE RUMEN By Maria Esperanza Quispe Salas A THESIS Submitted to Michigan State University . in partial fulfillment of the requirements for the degree of MASTER OF SCIENCE Department of Animal Science 1982 ABSTRACT A STUDY OF THE EFFECTS OF ISOACIDS, UREA AND SULFUR ON THE RATE OF FERMENTATION IN THE RUMEN By Maria Esperanza Quispe Salas Agricultural by-products such as pineapple tops are available as a new alternative ruminant feed. In order to find ways to enhance their fermentation in the rumen, a 23 factorial crossover experiment was con- ducted in Two 4x4 quasi-Latin squares, to study the effects of isoacids (isobutyrate, 2-methyl butyrate, iso-valerate and valerate), urea and 'l sulfur on the rate of fermentation in the rumen. Eight fistulated Tabas— co rams divided by body weight into two groups of four. Each ram re- ceived four of the diets with different combinations of supplementation with the 3 factors.“ The levels of supplementation used were: 0.07 g and 0.14 g of iso- acids/Kg body weight; 0 and 0.43 g of urea/Kg body weight, and 0 and 0.086 g of sulfur/Kg body weight. After each of the 8 experimental weekly periods, rumen acetate production was measured using an isotope dilution procedure. Increases in acetate production were found when di- ets contained higher levels of isoacids (0.14 g/Kg of body weight), in combination with urea and sulfur supplementation (0.43 g and 0.086 g/Kg' body weight, respectively). DEDICATED TO My parents, whose many sacrifices and encouragement so meaningfully enriched my life. and My husband, -Paco, whose pacience and understanding made this work posible. ii ACKNOWLEDGMENTS The author expresses her gratitude to her major professor Dr. Robert M. Cook for his guidance and support throughtout her graduate program. Also she would like to recognize and express her appreciation to Dr. Eri- berto Roman Ponce, staff and workers at "La Posta" Paso del Toro, Vera— cruz in Mexico for their willingness to be of service and for the use of their facilities. Special thanks to Dr. John Gill for his help throughout his courses and guidance on the statistical component of this work. A very sincere gratitude is also extended to her friends: Donald Kirsh and Manuel Villarreal for their valuable suggestions. Mimi Gjorup and Edgardo Cardozo for their moral support during her academic program. 111 TABLE OF CONTENTS LIST OF TABLES. . . . . . . . . . . . . . . . . . . . . . . . . LIST OF FIGURES . . . . . . . . . . . . . . . . . . . . . . . . LIST OF ABREVIATIONS. . . . . . . . . . . . . . . . . . . . . . INTRODUCTION. . . . . . . . . . . . . . . . . . . . . . . . . . REVIEW OF LITERATURE. . . . . . . . . . . . . . . . . . . . . . Pineapple By-Products Use in Livestock Rations . . . . . . VFA Production in the Rumen. . . . . . . . . . . . . . . . Discovery of Rumen Volatile Fatty Acids . . . . . . . Techniques of Measuring VFA Production. . . . . . . . Factors that Affect VFA Production. . . . . . . . . . Quantitative Data on VFA. . . . . . . . . . . . . . . Nutrition of The Rumen Microbiota. . . . . . . . . . . . . Isoacid Requirements. . . . . . . . . . . . . . . . . Ammonia Requirements. . . . . . . . . . . . . . . . . Sulfur Requirements . . . . . . . . . . . . . . . . . Dietary Nitrogen to Sulfur Ratios. . . . . . . . . . . . . Practical Feeding Trials Using Isoacids. . . . . . . . . . MTERIALS MD mmons C O O O O O O O O O O I O O I O O O I 0 Animals and Management . . . . . . . . . . . . . . . . . . Ration Formulations. . . . . . . . . . . . . . . . . . . . Experimental Design. . . . . . . . . . . . . . . . . . . . Chemical Analysis. . . . . . . . . . . . . . . . . . . . Determination of Rumen Volume . . . . . . . Analysis for Volatile Fatty Acid Concentrations . . . Determination of Specific Activity of Acetate .-. . . Determination of Rumen Hydrogen Sulfide Concentration Determination of Ruminal Ammonia Concentration. . . . Analysis of Urea and Glucose. . . . . . . . . . . . . Determination of Blood Glucose. . . . . . . . . . . . Determination of Urea Nitrogen. . . . . . . . . . . . Statistical Analysis . . . . . . . . . . . . . . . . . . . iv Page viii ix b H NmNN-F‘ ‘14 15 15 20 22 22 24 26 26 28 28 .32 33 33 35 41 41 42 43 RESULTS AND bIscussxou . . CONCLUSIONS. . . APPENDIX . . . . . . . . BIBLIOGRAPHY . . . . . 44 68 69 75 10. 11. 12. 13. 14. 15. 16. 17. LIST OF TABLES Page Chemical Composition of Pineapple Canning By-Products.........5 In-Vivo Production of Ruminal VFA by sheep....................9 Net Production Rate of VFA...................................10 Rates of Acetate Production in the Rumen of Sheep............11 Some Functions of the Mbin Nutritional Groups of Rumen Bacteria based on Energy Sources.............................16 Some Functions of the Main Nutritional Groups of Rumen Bacteria based on Nitrogen and Carbon Sources................17 Volatile Fatty Acids and Other Acids Required for Growth Of certain Rmn Bacteria...O...0.0.0.0...0.0.0.000000000000018 Chemical Composition of Treatment Rations Fed to Sheep.......29 Proximate Analysis of Pineapple Tops used in Feeding Trials..30 Experimental Design for Rations..............................31 Rumen Fluid Volume of Experimental Sheep.....................45 Analysis of Variance for VFA Concentration in Rumen Fluid....47 Effects of Treatment Combinations on VFA Concentrations in the Rumno.OOOIOOOOOOOOOOOOOOOOOO0.0.0...0.00.00.00.00000048 Pooled Treatment Comparisons of The Effects of Isoacids (A) and Urea (B) on Butyrate Concentration in the Rumen..........49 Paired Treatment Comparisons of 2-Methyl Butyrate Concen- tration in me RmnOOOOOOOOOOOOOOOOOIO0.0.0.00.000000000000051 Pooled Treatment Comparisons of the Effects of Isoacids (A) and Urea (B) on Isovalerate Concentration in the Rumen.......52 Pooled Treatment Comparisons of the Effects of Isoacids (A) and Sulfur (C) on Valerate Concentration in the Rumen........53 Vi 18. 19. 20. 21. 22. 23. 24. 25. 26. 27. 28. 29. 30. Effects of Treatment Combinations on Acetate Turnover Time in the RmnOCOOOOOIOOOIOOCOOOOOO0.0.0.000...0.00.00.00.54 Analysis of Variance of Acetate Production, Turover Time, Hydrogen Sulfide Concentration, and Ammonia in the Rumen.....55 Effects of Isoacids (A), Urea (B) and Sulfur (C) on Acetate Production in the Rumen (4 lightest animals).........59 Effects of Isoacids (A), Urea (B) and Sulfur (C) on Acetate Production in the Rumen (4 heaviest animals).........60 Pooled Treatment Comparisons of The Effects of Isoacids (A) and Sulfur (C), and N:S Ratios on Acetate Production in the RmnOOOCOOOOOOOOCOOOOOOOIOOOOOCOOOOIOOOOOOOOOOOOOOOOOOOOOOO 61 Effects of Treatment Combinations on Hydrogen Sulfide and Ammonia Concentration in the Rumen...........................63 N:S Ratios in The Rumen and Rations......................... 64 Effects of Treatment Combinations on Urea and Glucose Concentration in Blood...0.0.0.000...OOOOOOOOOOOOOOOOOOOO0..65 Analysis of Variance of Urea and Glucose Concentrations ’ in BIOOdO...0.0.0.000...0.0...00......OOOOOOOOOOOOOOOIOOOOOO 66 Effects of Isoacids, Urea and Sulfur on Volatile Fatty Acid Concentration in the Rumen............................. 69 Rumen Ammonia Concentrations in Sheep Fed Diets with and Without Urea swplemntationOOOOOOO0.00000000000000000000000 72 Rumen Hydrogen Sulfide Concentration in Sheep Fed Diets with and without Sulfur Supplementation......................73 Effects of Treatment Combinations on Urea and Glucose concentrations in BlOOdoo-oooooooooooooooooooooooo .......... 74 vii LIST OF FIGURES Figure ’ Page 1. Generalized Scheme for Ruminal Degradation and Fermentation of Carbohydrates. . . . . . . . . . . . . . .13 2. Fate of Proteins in the Rumen. . . . . . . . . . . . . . .19 3. Separation of VFA Standards by High Pressure Liquid mromtography O O O I O O O 0 O O O 0 O I O O O O O I 0 O 36 4. Quantitation of Acetate in Rumen Fluid by High Pressure Liqu1d Chromatography O O I O O C O O O O O I O O O O O O O 38 viii LIST OF ABREVIATIONS VFA Volatile Fatty Acids MJ Mega Joules l4C Carbon-14 N Nitrogen PEG Polyethylene Glycol ix INTRODUCTION Cellulosic by-product materials, such as pineapple tops,are abun- dant in the Mexican tropics. Since 1905, pineapple by-products have been used to replace traditional forages. These materials can be an important feed for ruminants. For optimum use of pineapple by-products in cattle rations, it is necessary to study factors that limit their fermentation in the rumen. Many tropical pastures have a high yield of dry matter, but annual animal production is seriously limited by the seasonal nature of this production. The main factor limiting pasture growth is the ladk of soil moisture for long periods of the year. During the rainy season there is abundant high quality forage available for grazing. When pasture growth ceases at the end of the rainy season, the pasture consists of a large bulk of mature feed from which some of the leafier parts have al- ready been removed. Severe weight and production loss of cattle during the dry season is a common phenomenon in the Mexican tropics. Yet, while cattle starve, there are in the same area millions of tons of by—products from the agri- cultural industry that are wasted. These by-products, as in the case of pineapple residues, could be used as cattle feed. The feeding practices in the Mexican tropics are based on princi- ples established mainly in the temperate zones using European breeds of cattle. In Mexico only 20% of the cattle are European breeds. The rest Zebu (22.52) and other breeds (57.52). The production potential of the cattle in the tropics cannot be achieved until their!nutrient require- ments are known. In order to improve cattle production in the Mexican tropics basic research is needed that will lead to methods to increase the digestibility of agricultural by-products. In Mexico 560,000 metric tons of pineapple fruit were processed in 1978 from six states (25). A great part of the annual production is processed at the food plants. However, these factories can utilize on- ly 15 to 25% of the fruit. The rest is waste which constitutes a pol- lution problem. If these residues were usable as livestock feed, they would be equivalent to several thousand tons of forage. Numerous research trials have been conducted in Hawaii, India and Mexico (55, 56, 61, 69, 72) on the use of pineapple plant or pineapple by-products as ruminant feed. Most of the studies dealt with its util- ization in combination with the rest of the pineapple residues (stems, leaves and pulp) as silages. However, pineapple tops have not been fed alone because of the high fiber content. They can be categorized as high-fiber, low nitrogen by- products. The low nitrogen content limits the intake or digestibility Therefore in order to utilize them in a diet a source or non-protein ni- trogen should be added to correct the microbial deficit. All studies that have been reported so far are short-term trials. Long term feeding trials and more research are needed to determine ef- fects on production, reproduction and general health. This study investigated several factors which might benefit the utili- zation of cellulosic by-products -by ruminants, and which could aid in developing more effective systems for their utilization. REVIEW OF LITERATURE Pineapple By-Products Use in Livestock Rations Pineapple by-products show promise as a source of roughage for cat- tle. There is an abundant supply throughout the year (48, 50,54) and it is a good source of nutrients (21, SS). The nutritive value of the pineapple residues for bovine feed expressed in dry matter is equivalent to that of cereal grain by-products. These pineapple residues resulted in better milk yields than any other tropical forages due to the high level of total digestible nutrientes (65 - 74%). Most tropical forages are lower than 552 in total digestible nutrients (48). The two types of residues resulting from pineapple processing are non-pulp (tops, leaves and inner cores), and pulp. The chemical come position of pineapple residues fractions are presented in Table 1. These fractions vary considerably according to the fruit variety, degree of maturity and technology used in the cannery. All contribute to the great variation observed in chemical composition. Many factors must be taken into account when considering the nutri- tional value of by products. They often vary in chemical composition, are strictly seasonal. local in.production and~often contain' unde- sirable contaminants of organic or inorganic origin (61). In the case of pineapple by-products, all of these disadvantages should be considered. However, if a permanent market could be developed, many tropical areas 4 .Awev “mafia: "mounom no is 2.2 S: cm; a; m 35 an o.~ m¢.mm ~.o~ ~m.o w.o N mmeaeefiua mu m.~ em.mo n.ma no.0 H.n m muoo cc ~.¢ o~.mo n.NN qw.o o.n ma mm>ooA mm ~.m mm.~o «.mm um.o N.m mm mace Ne ~.< mw.Hn ~.o~ mm.o «.0 cm mafia: IN Hounds £m< uoouuxo meow woman uoouuxo namuoue uaaum HouOu mo uuooouelzn mun emwouufiz moouu Hanna moouo ooauuoeoum mo mama Amwmmm gonna: hunv muosooumlhm meaeeoo waeeooeewm mo coauamoeaou HoufiEmnu .H manna areas would benefit. In Mexico, only 20% of the cattle are European breeds. The rest are Zebu (22.52) and other breeds (57.52). The production potential of the cattle in the tropics cannot be achieved until their nutrient requires ments are known. In order to improve cattle production in the Mexican tropics basic research is needed on methods to increase the digestibili- ty of agricultural by-products. In Mexico, 560,000 metric tons of pine- apple .fruit were processed in 1978 from six states (25). A great part of the annual production is processed at the food plants. However , these factories can utilize only 15 to 25% of the fruit. The remainder is was- te, which constitutes a pollution problem. If these residues were usa- ble as livestock feed, they would be equivalent to several thousand tons of forage. Numerous research trials have been conducted in Hawaii, India.and Mexico (55, 56, 61, 69, 72) on the use of the pineapple plant or pine- apple by-products as ruminant feeds. Most studies dealt with utili- zation of pineapple stems, leaves and pulp as silage. However, pineap- ple tops have not been fed alone because of the high fiber content. They can be categorized as high-fiber, low nitrogen by—products. The low nitrogen content limits the intake or digestability. Therefore, in order to utilize then in a diet a source of non-protein nitrogen should be added to correct any microbial deficit of ammonia. All the studies that have been reported, thus far, were short-term trials. Long-term feeding trials and more research are needed to deter- mine effects on animal production, reproduction and general health. Two studies have shown that pineapple green chop ensiles without problems, and because of its succulent nature, it is a good localy grown roughage 7 for both beef and dairy cattle. Yearling dairy heifers weighing approxi- mately 300 Kg will consume 14-16 Kg of pineapple silage daily. These an- imals produced an average of 24 Kg, (72). Beef cattle weighing an.a~er- age of 320 Kg consumed 16 to 20 Kg of silage a day plus 2.2 Kg of molas- ses and 2.2 Kg of protein supplement (81).- In both studies (72, 81) si- ' lages were low in dry matter and animals would need to consume larger amounts to meet their requirements. Confirming the variability in the product, a range of 0.05 to 0.78 Kg in daily body weight gains was ob- tained (72). VFA Production in the Rumen Discovery of Rumen Volatile Fatty Acids In 1944, Barcroft, et a1 (8) demostrated that volatile fatty acids are absorbed from the rumen. Elsden (23) later confirmed that these ac- ids were acetate, propionate and butyrate. Walter (1970) and Hulton ‘ (1972), cited by Naga and Harmeyer (51), suggested that micrdbial yield could be affected by VFA production in the rumen. The reactions occuring in the rumen and the effects of the end prod- ucts of these reactions on the metabolism of the ruminant is well estab- lished (7). The role of the volatile fatty acids; acetate, propionate and butyrate in the productive processes of domestic animals have been the subject of several studies (10, 27, 82). These acids are the prin- cipal source of energy, as well as biosynthetic substrates, for various ruminant tissues. Production rates of these acids are studied to deter— mine the efficiency of utilization of plant materials by ruminants. They also estimate the availability of energy to the animal under certain conditions and diets. 8 Several studies have examined the relationship in the rumen between pro duction of VFA , synthesis of microbial protein, and carbohydrate fer- mentation (15, 24, 34). The significance of fermntation of carbohy- drates to VFA's was soon recognized (34). It was also shown that ud- crobial protein can replace dietary protein as the main source of amino acids for ruminant tissues (35). Techiniques of Measuring VFA Production. Many techniques have been used to measure the production rates of fermentation products. Gray et a1 (32) described two main approaches for conducting such studies. These are: 1) 3111359 techniques and (2) I_n E19. techniques. Several $31112 studies are summarized in Tables 2, 3 and 4. As a result of various experimental techniques, there are different ways of ezqaressing production rates of rumen volatile fatty acids. They include ( 1) methods based on changes in rumen VFA concentrations after feeding, (2) production of VFA in inoculated rumen fluid (in E13), (3) analysis of the blood draining the rumen (£11132), (4) isotope dilution techniques applied to either‘the rumen or the whole animal. The latter probably offers the most accurate means of measuring production of or— ganic acids in the rumen, since the measurements may be made $2M with minimum disturbance to the animal. Both Anison (1965) and Wagner (1964), cited by Lang (42), discussed the inherent difficulties of obtaining representative samples of rumen contents and suggest that the rates of production of acids are not uni- form throughout the rumen. Total VFA production in the rumen has been measured by an isotope dilution procedure based on the infusion of 9 .Aoev :Haxm one odnmoux "ouuoom. I‘, mdmaa some zoo sag ‘N@ofi has” @Noc anoc 8.” o o gfigucfi :0Uaux? «vuddunfimq HUI-o. oN meqozsa neuron: I I oc.o mm.c n~.~ . . seasoned :ouauxue ou-ono~ 0:.: .~ undue use xoo xaoaoc_u:ou muse: on no» ~o>uoueu autos: no as. an; azuoaqu ax ~.~ ' am." I I ' O O I O I O O O O I O sqmuchn ”fish” No .I QNQQV .I. .I .l o o o o 0 on. o o o o :NQUOMQ any-ah” RNA Amomdv :1ao—— Ocean”: II Na.“ In ' ' o o o o o o o o o o o =Hflvuoun— Q‘SHU NNN anauwmau noun: .oaoc *NOQV ' I I o o o o o o o o o o o . m~w>nfl~u=fl HF- N- “nQDc NQIQ ' I - O O I O O O O O I O O O a~a>uuucd h- N Inca—V ._a .9 auto «ae.¢ -.e I I I . . . . . . . . . . . . mqa>uouca u; a an new cameo caucus: new as: aueaceo «on no we a Aeom~v .qo no nudge: «cm.n I an.c om.o mm.n . . . . . . a~o>umu=« o; «a vow ouqouno use use cease >ae.:ouao;3 use so me e Aooo~v .mo no hope «oo.n an.c I I I . . . . . . o~a>uoueu u: «H no can muo:o so: canoes: now new ou—ou~o Now we as a nobody uuuua .wema ma.a o~.~ ~¢.o 4e.m nn.m . . . . . . . . . nu: co.m~ on mu: co.mc aonu oao>uoucu Adena: on com undouqe a com Anoafiv ouoeoua .aeoa oa.n on.m H~.c «a.o c~.n . . . . . . on: oc.m« cu an: oc.wo souw m~o>uoucu Adana: no new «Logo oudouue m cam uncououux oxoueu nook fiance ouuxuon oueoueoum ouuoo< Co mx\~auoe Aaoav coauuaooue Aqua: .emonm he Hoofiaom mo coauooooum o>fi> eH becauuoeoo .~ «Home. 10 Table13 . Net Production Rate of VFA (Average of two sheep) Production Mol/kg dry Caloric Name of Acid of acid matter g/day value mol/day consumed MJ Infusion of luc-labelled individual VFA Acetic Acid . . . . . 2.750 2.790 165.25 2.41 Propionic Acid . . . 0.529 0.535 39.15 0.82 Butyric Acid . . . . 0.464 0.470 40.83 1.02 Infusion of lL‘C-labelled VFA mixture Acetic Acid . . . . . 2.996 3.085 179.76 2.62 Propionic Acid . . . 0.591 0.598 43.73 0.91 Butyric Acid . . . . 0.285 0.288 25.08 0.62 Source: Krishna 5 Ekern (40)- 11 Age one On .oHv scum voodoo< “mouoom .omeoaunma uoz « eowuaaao Ammoumnnu nonmaonmv uncuomH om.~ monoumom. Ne chmHv uumonou a wood coamomefi unmumooo oq.~ ems echoes; Nooa scamsmoa No.H mom meuooog Noe “53:8 an: + $22 Noe «I too: .H... on .85 aoamomeu om.~ zm: echoes; Now oomumnoo eon + uoonz Now soumnmea Amefivmmw moooofiueoov 3328 26 mnmnm e38 3 33$ 1.. no 5533 unmamuommma Aeomnm\hoo\aozv Ava uo vocab: nofiuonooue ououoo< pooh unwwo3 mmmum>¢ mo Honeaz mwoum>< monouommm .eomsw mo amend unu 6H cowuooooum muouoo¢ mo mouom .q manna 12 14 individually C-labelled acids (82). When 14 C—labelled VFA were intro- duced into the rumen, the results tended to support the hypothesis of non uniform VFA production. Samples of rumen contents obtained from various sites, after insufficient time for complete mixing, varied up to 202 in specific activity. Such large differences could represent a sampling error. The mean specific activity (after mixing) is related to the mean production rate of the acid in the rumen. If production rates vary throughtout the rumen, local variations may occur in amount or type of VFA, or in numbers and types of organisms responsible for fermentations. Also, there any be layering of the food materials. Any attempt to mix rumen fluid uniformly by circulation pumps disturbs the normal milieu within the rumen and affects rates and patterns of fermentation (76). Factors that Affect VFA Production It has been established that the VFA, produced in the digestive tract of the ruminant by microbial fermentation processes, represent an important source of energy to the host (74). The amounts and propor- tions of VFA produced are available depending on the nature of the diet, the time after feeding and the age of the animal (74). Other factors, according to Beeson (1965) cited by J. J. O'Conor, et a1 (54) affecting molar concentration of VFA's could be: -Roughage to concentrate ratio -Physical form of feed -Buffers -Salivary output dKind and amount of protein 13 -Frequency of feeding -Balance of nutrients Still others might include, level of dry matter intake, and rate of ab- sorption of volatile fatty acids from.the rumen (46). A reason for ‘ these changes is the intensive microbial degradation of plants, which are primarily composed of carbohydrate polymers, taking place within the rumen. These polymers initially are hydrolyzed in the rumen to oligosac- charides, which are subsequently fermented to VFA carbon dioxide and methane. (Fig.1) GENERALIZED SCHEME FOR RUMINAL DEGRADATION AND FERMENTATION OF CARBOHYDRATES Carbohydrate polymers Oligo-saccharides (Acetate? O Butyrate Formate< Pyruvate-————-9Acetyl—CoA---9fi Caproate H: + C00 . ' LValerate Oxaloacetate Lactate \ / \ Prop ionate 0 CH4 Succinate Source: Russell and Hespell (65). 14 AQuantitative Data on VFA Numerous studies have quantitated production of VFA in the rumen. In 1958 Stewart (74) by means of an in 3132:33H2i552. technique, meas- ured the VFA production rates, and found concentrations increased from 2 to 6 hours after feeding, while the rates of VFA production were greatest during the first two hours. An average of 2.9 g. were pro- duced per steer/day. Leng and Leonard (43) found that molar proportions of VFA in the rumen remained constant throughout a 24 hour period, that VFA concentrations increased after feeding and reached a plateau around 16 hours post-feeding, but during the subsequent 6 hours a steady de- cline occurred. Later studies of simultaneous measurements of the rates of produc- tion of VFA in the rumen of sheep suggested that interconversions of the: main acids were possible (44). Conversion of acetic acid into butyric accounted for between 40-502 of the butyrate produced, and conversion of butyrate into acetate accounted for 6-132 of the acetic. However, the interconversion between propionate into acetate and butyrate was small. Weller et a1. (83) also measured the rates of production of indi- vidual and total fatty acids by infusion of a mixture of 14C-Labelled acids. These studies showed that the proportion of acids produced in the rumen was similar throughout the feeding cycle. The infusion of certain mixtures of labelled fatty acids showed that the molar composition of the total VFA initially formed in the rumen was a- cetic 77-832, propionic 15-181, and butyric 1-7Z. Mean rates of VFA production determined by Weller et al in 1967, (82) in seven sheep dur ing fourteen 3-day periods throughout:winter, spring and summer months ranged from 3.4 to 5.3 moles total VFAis per day. Naga and Harmeyer(31), 15 studied in vitro VFA production at different rates of rumen, microbial protein synthesis, and generally found negative correlations between microbial growth and volatile fatty acid production. Nutrition of the Rumen Microbiota Isoacid Requirements A major problem in ruminant nutrition is to define the nutrients required by rumen microorganisms for maximum fermentation of feedstuffs, particularly for low protein, highly fibrous plant materials. Informa- tion is developing on the nutritional requirements of some of the pre- dominant groups of rumenbacteria. Tables 5 and 6 show rumen bacteria grouped on the basis of some nutrient requirements. These organisms are unique in that they synthesize the a-keto acid analogue of several amino acids by direct carboxylation of the corresponding acid (6). For example, isobutyrate is carboxylated to form the a-keto acid analogue of valine. Table 7 shows the VFA required by rumen bacteria, and also 3 other acids that are believed to stimulate the growth of certain rue men bacteria. When high quality protein is fed to ruminants, the isoacids may be produced in sufficient quantities to satisfy the nutritional require- ments of the rumen cullulolytic bacteria. However, when highly fibrous plant materials containing low amounts of poor quality protein are fed, an isoacid (isobutyrate, 2-methyl butyrate, isovalerate and N-valerate) deficiency is undoubtedly a major factor limiting the growth of the rue uen bacteria and consequent digestion of cellulose. Fermentation end products, such as formate, lactate or ethanol may appear in the rumen. Ammonia, carbon dioxide and either short straight, l6 TableLS . Some Functions of the Main Nutritional Groups of Rumen Bacteria Based on Energy Sources ENERGY SOURCES (One or more of the important species in the nutritional group have this function) Group 1 Group 2 Group 3 Group 4 Group 5 Cellulose Cellulose* Pentosans Pentosans Pentosans Pentosans Starch Starch Starch Starch Starch Lactate A A A A A B B B *Cellulose digestion by butyrivibrio included in this group (A) Proteolitic ability (B) Aminoacid catabolism Source: Adapted from (13). 17 Table 6 . Some Functions of the Main Nutritional Groups of Rumen Bacteria based on Nitrogen and Carbon Sources Essential nitrogen and carbon sources other than energy. Group 1 Group 2 Group 3 Group 4 Group 5 VFA* Peptides A.a. - Peptides & Aa. VFA (stimulate) 19.1% 31.5% 24.7% 5.6% 5.6% *Require one or more of the acids-Isobutyrate, 2-methyl butyrate or isovalerate; sometimes require N-valerate or longer chain fatty acids. Source: Adapted from (13). 18 Table 7 . Volatile fatty acids and other acids required for growth of certain rumen bacteria Acid Source* N-valeric CHZO, proline arginine, lysine Isovaleric Leucine 2-methylbutyric Isoleucine Isobutyric Valine Phenylacetic Phenylalanine Indoleacetic Tryptophan Imidazoleacetic Histidine *These source compounds are catabolized by other bacteria to produce the acids. Source: Adapted table from ( 4, 5, 6 and 77 ). 19 branched-chain or aromatic.fatty acids areuformed from.protein~degra- dation as shown in Fig. 2 and are used in the production of microbial protein. Figure 2. FATE OF PROTEINS IN THE RUMEN Dietary and other proteins I Polypeptides V/ Amino acids + Short peptides + NH3 + C02 I-——9Acetate, isobutyratefi -—%2—methyl butyrate >-—-—-—>Microbia1 growth *--9Isovalerate -—9phenylacetic \ NH + CO2 >Anrino acids Source: Rusell and Hespell (65). 20 Ammonia requirements In 1948 McDonald (49) demonstrated that ammonia is produced from the ruminal degradatation of dietary protein. Ammonia can be produced by rumen microbes from.both protein and non-protein nitrogeneous subs stances, and it is probably the most important source of nitrogen for ruminants. Ammonia (NH3 and/or NH4) appears to be incorporated rapidly into rumen bacteria in the form of amide or amino groups and used for amino acid synthesis (62). Ammonia is obligatory for the synthesis of rumen mi- crobial protein as many pure culture studies have shown; the fact that ammonia is the major source of nitrogen for microbial growth also has been confirmed by many ignyigg studies (16, 52). Generally speaking, bacteria can grow in media with ammonia levels as low as 1 mg/dl. How- ever, according to Satter and Slyter (77), ammonia concentrations of 2 to 5 mg/dl in the rumen are needed for maximum microbial yield. Illiecf nois workers found that it is possible for organisms to grow in a me- dium.of 1.7 mg/dl, however, this level might not give maximum yield cf bacterial cells. When ruminants are fed straw, the rumen ammonia concentration is 1-3 mg NH3-N/dl (26). _Ammonia can be absorbed into the blood from the rumen and converted to urea in the liver. Blood urea can then enter the rumen by diffusion through the rumen wall and by secretion of saliva. This phenomenon is now recognized as the "nitrogen cycle" in ruminants. Urea can be used to supply ammonia when natural protein is not pre- sent in the diet. There is little doubt that entry of plasma urea into the rumen can provide a significant source of nitrogen for microbial growth and enhance survival where dietary nitrogen intake is low. For 21 sheep and cattle fed low quality hay, endogenous urea may provide 25% of nitrogen available in the rumen. One way to determine the amount of urea that can be utilized with a particular diet is to monitor rumen ammonia concentration while in- cresing dietary urea addition. The point at which rumen ammonia accur mulates signifies the point of maximum urea utilization (70). This cat- egorization of feed ingredients as to siutability for use with urea still requires more quantitative information. Reported values for. rumen ammonia levels that give maximum micro- bial growth have ranged from 1 to 25 mg/dl. Satter and Roffler (70) re- ported that an ammonia level of 5 mg NH3-N/d1 is the upper limit for ammonia utilization by the rumen microbiota. They proposed that needs above this value must come from supplementary feed protein that bypasses rumen fermentation. However, Miller (47) and Orskov et al (58) reported that 23 mg NH3-N/dl of rumen fluid is the upper limit for ammonia utili- zation by the rumen microbiota. These studies have not considered the need for other intermediates for amino acid biosynthesis by rumen microbiota, such as isoacids and sulfur, eventhough feeding trials have shown an increase in nitrogen re- tention when isoacids were added to diets (17, 26, 57). The benefits of using both urea and natural protein to-supplement corn silage for balancing rations for high producing cows have been studied extensively (Huber and Thomas, 1971; Conrad and Mugerwa, 1970; cited by Felix, 1976). However, the optimum level of urea which can be successfully fed, espe- cially if the ration is comprised of highly fibrous roughages, is still controversial and requires more investigation. 22 Sulfur requirements In addition to ammonia and isoacids, sulfur is an essential nutri- ent for the synthesis of rumen microbial protein, and thus for optimum fermentation of substrates. Practically all of the sulfur present in protein is in S-containing amino acids cystine, methionine and cysts- thionine, or in tissues as metabolic derivatives which accounts for : about 1% of the total S. In the rumen, dietary sulfur is converted to hydrogen sulfide. Sulfide is the key intermediate between breakdown of ingested or re- cycled sulfur and subsequent utilization of sulfur by rumen microbiota. Based on findings of many workers (9, ll, 29), it is obvious that ru- minant animals require sulfur for systemic metabolism. However, if nor- mal rumen function is to take place, rumen microorganisms must also be supplied with adequate sulfur. Without adequate sulfur, rumen microbes have a reduced ability to function normally, thus digestibilities and nitrogen retention are de- creased (76). In sheep, dry matter digestibility increased as sulfur in rumen fluid increased from 0.07 to 4.2 ug/dl. The precise level at which rumen sulfide concentrations limit rumen fermentation has not been determined, particularly if urea is a major source of supplemental nitrogen. Dietary Nitrogen to Sulfur Ratios Bray et a1, (12) found that sulphate supplementation of a sheep in? creased crude fiber digestility and nitrogen and sulfur retention. Also, Bray (12) showed that inorganic 35$ (sulphate) was transferred to 23 the rumen by passage of sulphate was apparently increased by influx of water into the rumen. Only 0.3 to 1.4 % of the injected sulphate used this route over a 4 h. period. Therefore, it was suggested that the utilization of recycled urea nitrogen may be severely limited in sheep on low sulfur and low nitrogen intakes, unless sulfur is recycled to '- the rumen by routes other than across the rumen wall or in other forms. In most temperate zone forages, the sulfur is in the protein com- ponent which has an average N:S ratio of about 15:1. However, the total N:S ratio of the fodder can vary from 4:1 to 50:1. The desirable N:S is reported to be 10-13.5:l for sheep and 13.5-15:1 for cattle in the tem- perate zones (45). A number of researchers (9, 16, 20, 29, 45, 84) have questioned the meaning of dietary nitrogen to sulfur ratios. Potential- ly, there are a multitude of correct dietary nitrogen to sulfur ratios depending on the availabilities of dietary nitrogen and sulfur (45). When the diet consists of low protein, fibrous plant materials, supplementation with urea requires simultaneous supplementation with sulfur. Sulfur supplements used are elemental sulfur, various sulphate salts, and, in some cases, s-amino acids and methionine hydroxy analogue. A dietary sulfur deficiency restricts dry matter digestibility. The effects of sulfur on the fermentation of carbohydrates has been re- viewed (84). Jones and Haag (37) observed a growth response in dairy heifers fed a basal ration of low sulfur hay plus grain when 3% urea and 1% sodium.sulphate were added. Lassiter, et a1 (41) and Brown et a1 (14) also observed a growth response to a sulfur supplementation of ra- tions for dairy heifers. Other studies with sulfur supplementation have given inconsistent results. This probably indicates that levels of sul- fur in the basal diets were sufficient for the production levels achieved. 24 Based upon published values (53) for nitrogen and sulfur content of feed, it can be shown that the use of 0.5% ureaenitrogen and 0.5% sulfur in simple concentrate mixes of practical diets for dairy cattle can re-‘ sult in N:S of 18-20:l . The National Research Council (53) reports that the sulfur requirement of lactating cows is 0.2% of the total diet, which implies a N:S ratio of 12:1 for medium producing cows (15% protein in the total diet dry matter). ‘Moreover, Rending and weir (64) studied effects of S fertilization of a S-deficient soil on the nutritional quality of forage produced for lambs and showed consistent,though not always significant, trend towards higher gains in lambs when S fertili- zation was practiced. In S-fertilization experiments in New Zealand, McNaught and Chriss- toffels (50) reported N:S ratios of 17 to 18.5 for white clover and 11 to 12 in grasses gave maximum yields. Pumphrey and Moore (63) found a N:S ratio of 11 or less indicated an adequate S supply for digestibility and growth of alfalfa. Thus the N:S ratios found desirable for optimum growth of plants are slightly higher than the N:S ratio of 10:1 to 15:1 suggested as optimum for ruminants (20). Moreover, plants growing at an optimum rate may not always be of ideal nutritional quality for ru- minants (1). Practical Feeding:Trials Using_Iso—acids Metabolic studies have been conducted to determine the effect that short-chain VFA have on the utilization of various dietary components. A conventional balance trial was conducted with 8 lambs consuming a P“- rified diet (39% cellulose and urea as the sole N source). The addition 9 of a short chain VFA mixture significantly increased the apparent N 25 digestibility (l7). Umunna et al (80) showed an increase in nitrogen retention and decrease in urinary nitrogen loss when animals on urea and high roughage rations were ruminally infused with isobutyric and/or iso-valeric acids. Infusion of these acids did not affect dry matter or protein digestibility. Oltjen et a1 (55) studied the influence of branched-chain VFA on the~rumen microbial population and fermentation patterns as well as nitrogen utilization by steers fed urea or isolated protein supplemented-diets, they found no difference in rumen protozoa numbers, but nitrogen retention was greater with isoacid supplementae.. tion, but most of this change was observed with isolated soy protein, suggesting the importance of dietary amino acid balance. An 39:3izg experiment with dairy cows and heifers showed a posi- tive effect on milk production, body weight, feed intake and nitrogen balance, when isoacids were added to a urearbased diet (27, 28). ' In the present study , an igggigg rumen fermentation trial with Tabasco rams was carried out in order to evaluate fibrous materi- als as a potential feed source for ruminants. The effects of urea, sul fur and isoacid supplementation on the rate of rumen fermentation of chopped dried pineapple tops was studied. Our specific objective was to determine the levels of rumen ammonia, rumen sulfur and rumen isoacids (iso—butyrate, 2-methyl butyrate, iso-valerate and valerate ) that yields maximum fermentation of pineapple tops. MATERIALS AND METHODS The experiment was conducted at Centro Experimental Pecuario "La Posta", Paso del Toro, Veracruz, Mexico during the months of January, February and March of 1980. The state of Veracruz is located on the east coast of Mexico, between 17 08' and 22 28' north latitude, and stretches along the coastline of the Gulf of Mexico. The experimental procedure was carried out in Mexico at the station and the chemical analysis and supportive work at Michigan State Univer- sity. Animals and Management Eight Tabasco sheep were sorted by weight into 2 groups. Four lighter sheep, each weighing approximately 25 Kg, were separated and identified (82, S S , S ). A second group of four heavier sheep 3’ 4 5 weighing 35 Kg were also identified (86, S7, 88’ $10). All sheep were fitted with rumen cannulae and housed in individual metabolic cages. Sheep were fed basal high fiber diet consisting of pineapple tops plus normal minerals. A daily dry-matter intake of approximately 1 Kg was maintained during the experiment. The animals were gradually adapted to urea. Water was provided 3d libitum. 26 27 Ration Formulations Pineapple tops were obtained from a regional canning plant located at the small village of "Los Robles" near the experimental station. The pineapple tops were processed through a silage chopper (1-1% in. long ) at the station and then spread on the ground to dry to 20% moisture. Part of this material was further dried and finely ground for use in a premix to prepare the chemical supplements. Eight different ra- tions were prepared to provide combinations of isoacids, crude protein- and sulfur, each at 2 levels. On the basis of prior cattle experimen- tation, isoacids were administered at 0.14 g/kg body weight. Thus, at the high level 3.5 g and 4.9 g of isoacids were fed to the lightest and heaviest groups of sheep, respectively. One half of this amount was fed at the low level. In order to achieve two levels of ammonia in the rumen (about 5 and 15 mg/ 100 ml), pineapple tops were fed alone or supplemented with urea. For both groups, urea was offered at 0.43 g/Kg body weight (11 g for the light sheep and 15 g for the heavy sheep). Sulfur addition to the diets was determined on the basis of N:S ra- tios. Four different nitrogen/sulfur treatment combinations were used: low nitrogen/high sulfur, high nitrogen/ high sulfur, low nitrogen/low sulfur, and high nitrogen/ low sulfur, yielding N:S ratios of 3:1, 5:1, 8:1 and 12:1. For the low sulfur rations, no additional sulfur was provided, but for the high sulfur level 0.086 g /Kg body weight of added sulfur re- ; sulted in intakes of 2.17 g for the lightest group and 3.01 g for the heaviest group. Mineral supplementation was estimated at 0.8 g/Kg body weight, therefore 20 g and 27 g were supplied to the respective groups. 28 Rations and proximate analysis of pineapple tops are described in Tables 8 and 9. Experimental Design_ A three-factor 23 crossover experiment was performed in 2-4 x 4 quasi-Latin squares (ABC interaction confounded with squares) . Each experimental diet was fed for a one week period. After this period the last day was devoted to experimental and sampling procedures. The 4 animals in each square were given a different diet for each of 4 weeks as shown in Table 10. Treatment effects were estimated by measuring the rate of acetate production in the rumen on the last day of each ex- perimental period. Chemical Analysis On the sampling day, all 4 animals were fed rations ad libitum, plus water free choice. Thirty minutes after feeding, rumen fluid sam- ples were taken every 20 minutes and blood samples at 1, 2 and 3 hours. Rumen fluid dilution rate was determined by measuring the rate of disappearance from the rumen of Polyethylene glycol, a.water soluble marker (36). This method consisted of adding 10 g of PEG (M. W. 4000, Sigma Chemical Company) dissolved in 150 ml of water to the rumen through a perforated plastic tube (15 mm x 20 cm long), which achieved a better distribution of the solution throughout the rumen. This plastic tube was fitted to one end of a dosing syringe which allowed infusion of liquid solutions into the rumen as well as collection of the samples. Igugigg_acetate production rates were determined by the single in- jection radioisotope technique. Each animal received approximately 100 uCi of Na (1-140) acetate (New England Nuclear Corp. Boston, MA) 29 .oofiuou one ou venom Random no new: oz a o.n~ o.n~ o.am o.h~ o.o~ o.o~ o.o~ o.o~ Awe uanuuaez o.m I I o.« ~.~ ~.N I I Ame unoesm o.mi I o.mi I o.HH I o.HH «I any nun: m.~ m.k m.m a.m m.~ e.m . e.m m.~ Aeav nnaunonH coke ache coke cone news news «awe mama Amy neon manduueam we my we we «a me NH Ha muomfiomumoH nadaen< unuo>uum nauaana undemea muomauomua .emmnm ou mom mnOfiumm uomSunoue mo ooauwmoeaoo HmofiEoso aw manna 30 Table 9. Proximate Analysis of Pineapple Tops Used in Feeding Trials Wet basis Dry basis (Z) (7.) Moisture1 83.6 0 Crude Protein 1.0 6.6 Sulfur 0.021 0.133 ’ lDetermined by forced aire oven drying at 100-110 0 for 24 hours. 31 49:: :3: u: Ho>oH sea ”A mommm< "we a a m a o m a u e aommn< use moaned "me as we me we cam o9 me we as am my we as o9 am me he ea me om Hoaaoa mm so on no moOfiuom unwanm mo Ho>oq no own: mo Ho>ma um .mouooomfi mo Ho>oa n< : m A e o m < u e m a m .m o m d u a nommm< "Ne moaned use ooeuedsooo usuauodues me me as ea om N9 is o9 me mm as on me we mm on me we in mm Hoaee< on mm me so mooauom influence unus>oos so N powwow anemones nonsense ov museum meofiuom How owwmon Hmucuafiuoexm .o~ manna 32 intraruminally, which was infused with 10 g of PEG. The radio-labeled acetate was dissolved in a 0.001 M solution of sodium acetate and in- fused into the rumen of each animal after the morning feeding. Rumen fluid samples were collected every 20 minutes throughout a 3 hour period to study changes in dilution rates, volatile fatty acid concentrations, specific radioactivity of acetate, ammonia concentrar tions and hydrogen sulfide concentrations. At each time a 30 ml sample was obtained from the rumen, strained through 3 layers of cheesecloth, inmediately acidified with 50% sulfuric acid (v/v), frozen at -16 OIC and stored for subsequent analysis. Determination of Rumen Volume A 4 ml aloquot of strained rumen fluid was assayed for PEG, using a modified version of the method of Smith (68), which was as follows: 1. Replicate 2 ml samples of strained rumen fluid were diluted with 1.5 m1 of distilled water in 12-15 m1 centrifuge tubes. 2. Two m1 of 0.3 N Ba(0H)2 was added to the centrifuge tubes followed by 2 ml of 5% FeSOa' 7 H20 (w/v) and mixing through. 3. Subsequently, (15 ml of 10 % BaCl f2H20 (w/v) solution was added to 2 the centrifuge tubes and mixed. 4. After 5 min., the mixture was centrifuged at 3,600 x G for 5 minutes. 5. 0.25 ml of the resulting supernatant was combined with 2.25 ml of dis tilled water in a spectrophotometer cuvette to a total volume of 2.5 ml. (This final dilution was obtained from preliminary tests in order to adjust absorbsnce at a measurable range). 6. 2.5 ml solution containing 30% TCA95.9% BaCl -2H20 was rapidly added 2 to the sample using a finn pipette (H. Thomas Co, No 7734—W05). The 33 sample was inmediately mixed using a vortex mixer. Finally, the reaction was allowed to proceed for 3 minutes, after which absorbance was measured at 500 nm using a Beckman Model 6/20 spectrophotometer. A standard curve for polyethylene glycol was prepared using a range of 0.10 to 0.50 absorbance. Analysis for Volatile Fatty Acid Concentrations 1. Six ml of strained rumen fluid were centrifuged at 3,600 x G for 10 min. and a 1 ml aliquot of supernatant was used for analysis. The aliquot was acidified with 200 pl of redistilled 88% Formic Acid and 0.1 molar solution of phosphoric acid. The VFA analysis was performed on a.Hewlett-Packard 5730 A gas chro- matograph equipped with a flame ionization detector, a 7671 A auto- sampler and a 3380 A integrator. Temperature of 125 C was set for the column and flow rate at 40 ml/min. A silanized glass column (approximately 6 foot x 2 mm ID) was packed with Carbopack c/O. 3%-CW 20W/0.1 % H3PO4 (Supelco 1-1825). Phos- phoric acid-treated glass wool was used in the column ends. Data output from.this equipment was given in mmoles/100 ml of rumen fluid. Determination of Specific Activity of Acetate 1. 2. Three ml of strained rumen fluid was used for determining the spe- cific activity of acetic acid. This amount of sample was centrifuged at 39,400 x 0 for 20 minutes in a Sorvall, Model RC 2-8, in order to remove microbial protein and feed particles. - A 2 ml aliquot was deprotenized by the addition of 2 ml of 0.3 N . 7. 10. 34 Barium Hydroxide (Sigma # 14-3)* and 2 ml of 0.3 N Zinc Sulfate (Sigma # l4-4)*, and centrifuging for 20 minutes at 39,400 x G. This second supernatant was filtered through a filter paper (Mhnktells' # 52-80150). The clear filtrate from.3 was mixed with 50 ul of 10 M NaOH (final pH of filtrate was approximately 10 ) in order to prevent volatilization of the volatile fatty acids during the freeze-drying process. Acetate recoveries from rumen fluid were of approximately 90295%. Dehydration of the sample was performed in a Lyophilizer (Vertis, Mod- el 25 SRC) until sample was completely dry. Each sample was reconstituted with 1.9 ml of a 1% solution of H3P04 in deionized water and the pH was adjusted to 2.0 using 100 ul of 18 M H2804. A 500 ul. aliquot was taken for analysis using a high pressure liquid chromatograph apparatus which consisted of a mini pump (Laboratory Data Control, Model N51-33R), a Ryodine sample injector (Ryodine, Mod- e1 7120), a pressure dispenser (Laboratory Data Control, Model 110), a UV detector (Laboratory Data Control, Model 1203) with a 214 filter, and a chart recorder (Perkin—Elmer, Model 0-23) providing 0.018 ab- sorbance units full scale. Individual volatile fatty acids were separated using a C8 4.6 mm x 25 cm LiChromasorb, 10 umlHibar II analytical column (Egu. Reagents # A00/2/04) and a 4.6 nm x 70 mm.Perisorb RP-8 guard column (E4M..Re- agents # 910436-94) fitted in front of the analytical column. The mobile phase used was 1% H3P04 in double distilled deionized water which was degassed under vacuum just before use. The flow rate was set at 2.0 ml/min, requiring a pressure of 1500 to 11. 12. 13. 14. 15. 35 2000 psi. After each injection, the injector was placed in the load position - so that the guard column could be washed with the following sequence of solvents: 10 m1 of 65% aqueous acetonitrile, 10 ml of deionized water and 10 ml of 1% (v/v)-aqueous phosphoric acid in order to pre- vent VFA carryover to the analytical column. 500 pl. aliquots from standards containing similar amounts and pro- portions of acetic acid as found in the samples to be essayed were injected onto the column to prepare a standard curve. See Figs. 3 and 4. The 0.8 ml fractions from the column were collected in 7 ml scintil- lation vials for determination of specific radioactivity using a fraction collector (Isco, Model 328). Five ml of Aqueous Scintillation counting cocktail, (ACS Amershan Corporation)* were added to each vial and then assayed for radioac- tivity using a Liquid Scintillation Spectrophotometer (Searle Ana- lytic Inc, ISOCAP/ 300 Model 68708) with data output to a Model 8491 teletype. Samples were counted for 10 minutes using the channel ratio method, 14CSCR), to determine efficiency. Recovery of radio- (Program.2, activity from the column was essentially 100% for radioactive acetic acid . No appreciable radioactivity was detected after running non radioactive acetic through the column inmediately following a radio- active sample. This shows that there was not a carryover between samples. Determination of Rumen Hydrgggn Sulfide Concentration Ruminal hydrogen sulfide concentration was determined with a sulfide #Amershan Corporation, 2636 S. Clearbrook Dr., Arlington Heights, 1160005. Figure 3. 36 Separation of VFA Standards by High Pressure Liquid Chromatography Column: 4.6 mm x 25 cm Li-Chromasorb C , eluent 1% phosphoric acid in double distilled deionized water, flow rate 2 ml/min, injection of a mixture of VFA standards ( formic: 0.7 mM, acetic: 7 nM, and propio- nic: lmM )in 250 pl, sample pH 2. VFA concentrations were determined by absorbance at 210 nm. 37 Acetic Propionic Formic l L J L l l 1 1 0 LI 2 3‘ 4. ,5 6 Elution time, min. Figure 4 . 38 Quantitation of.Acetate in Rumen Fluid by High Pressure Chromatography Column: 4.6 mm x 25 cm Li-Chromasorb C3, eluent 1% phosphoric acid in double distilled deionized water, flow rate 2 ml/min, inection of 250 ul of sample (7 mM acetate), sample pH 2. Acetic acid concentration was determined by absorbance at 210 nm. 39 Acetic Formic Propionic Elution time, min. 40 hydrogen sulfide sensing electrode, Lazar Model GS-136 connected to a Lazar Model digital potentiometer. 1. 3. An aliquot of 5 ml of strained rumen fluid was combined with 2.5 ml of an antioxidant buffer* and 2.5 ml of deionized water. The standarization procedure was carried out using a series of dilu- tions in a range of 0.5 to 10 ppm obtained from primary solution**. The diluent was a mixture of 25% (v/v) antioxidant buffer and 75% (v/v) distilled water. The electrode was equilibrated in a 10 ppm sulfide solution (NaSO4) for 30 minutes before being used. Standards were read by immersing the electrode to a depth of 2 cm and stirring at a constant rate until a constant reading was achieved. The electrode was allowed to stabilize for a few minutes before mili volt readings were recorded. Rinsing of the electrode with distilled water between samples was found to be critical. Potential readings (millivolts) were plotted vs. sulfide concentra- tion on semilog paper, (concentration was plotted on logarithmic axis) in order to obtain a linear standard curve. Sample readings proceeded in a similar manner and values were con- verted to mg of sulfur/100 ml of rumen fluid. Determination of Ruminal Ammonia Concentration Determination of ruminal ammonia concentration was performed with an ammonia sensing electrode (Orion, Model 95-10) attached to an Orion Model potentiometer *Buffer: 250 g of sodium salicilate, 65 g of ascorbic acid, 85 g of NaOH, and distilled water up to 600 ml. 41 1. Two ml of previously strained rumen fluid were centrifuged at 27,000 x G for 5 minutes. 2. One ml of the resulting supernatant was mixed with 9 ml of deior nized.water and 50 ul of 40% NaOH and read. 3. Standards were prepared in a range between 2-20 mg of ammonia/100 ml of solution. To obtain best results, samples and standard were ana- lyzed at the same temperature (22°C). 4. The electrode was allowed to stabilize for 3 minutes between samples. The potential values (mv) were compared against the ones of the standard curve in order to determine the ammonia concentration (mg ammonia/ 100 ml rumen fluid). Analysis of Urea and Glucose Blood samples were taken from the jugular vein at 1, 2 and 3 hours after the beginning of the sampling period. Samples were collected in heparinized blood collection tubes for urea and glucose analysis. Determination of Blood Glucose Based on an enzymatic celdrimetric procedure (Sigma Technical Bulle- tin No 510 ). 1. Samples were first deprotenized by mixing 0.5 ml of blood with 5.5 ml of of water, 2 ml of Zinc Sulfate (Sigma No. 14-3) and 2 ml of Barium Hydroxide (Sigma No. 14-3). 2. The mixture was then centrifuged at 39,400 x G for 15 minutes. 3. Duplicate 0.5 ml subsamples of the clear filtrate were analyzed for **Primary sulfide standard solution: 7.5 g of sodium sulfide crystal to 250 ml of buffer and enough distilled water to make 1 liter. This makes 1000 ppm standard. 42 glucose content, along with a reagent blank (6 ml of distilled wa- ter) and 0.5 ml of a glucose standard (Sigma, stock solution No 635-100).5 ml of Combined solution Reagent "A" were added to each tube. Then all tubes were incubated at 3790 in a.water bath for 30 i;5 minutes or at room temperature for 45 minutes. At the end of the incubation period, readings of absorbance at 450 nm of the standard and sample were determined using a spectrophotg meter (Coleman Junior I, Model 6/20). Test values were calculated using the following formula: Serum.Glucose(mg/lOO ml) = A test x 100 Astandard Determination of Urea Nitrogen Quantitation of urea nitrogen in blood was carried out using a highly sensitive, colorimetric procedure. 1. Whole blood samples were deprotenized by mixing 1.8 ml of cold 3 % (w/v) Trichloroacetic acid with 0.2 ml of whole blood followed by vortex mixing. Samples were then centrifuged at 39,400 x G for 15 minutes. A reagent blank (0.2 m1 of 3% (w/v) TCA), a urea nitrogen standard (0.2 ml of 1:10 dilution of urea standard stock solution # 535-30 in 3% TCA) and duplicate 0.2 ml subsamples of the clear supernatant were subjected to the analytical procedure. A premix consisting of 7 parts of BUN Acid Reagent (Sigma, Stock # 535-3)a in 5 parts of BUN Color Reagent (Sigma, Stock # 535-5)b was prepared and 4.8 ml were added to each tube simultaneously. *SIGMA Chem. C. 3500 De Dekab St., St. Louis, Missouri 63118 43 5. All tubes were placed in a boiling water bath for exactly 10 minutes- 6. .Then,they were removed and placed in a container of cold water for 3-5 minutes. 7. The contents were transferred to cuvettes and absorbance was measured at 525 nm using a Coleman Junior, Model 6/20 Spectophotometer. 8. Urea concentrations were determined directly from a standard curve pre- pared following the described procedure using different dilutions (1:1, 1:2, 1:4, 1:6, 1:8, 1:10) of urea standard in water. Values were expressed in mg urea-nitrogen/IOO ml of solution. (a) Contains Ferric chloride, phosphoric and sulfuric acids. (b) Contains 0.18% (w/v) Diacetyl monoxide and thiosemicarbazide. Statistical Analysis Analysis of variance and single degree of freedom comparisons (28) were used to test for differences between treatment means of rumen and blood parameters. Comparisons of the ruminal response were made over the effects of isoacids (Factor A), nitrogen (Factor B), and sulfur (Fac- tor C), or within various combinations, depending on which interactions were significant. For each variable measured, Bonferroni-t tests were used to compare the differences between treatments. RESULTS AND DISCUSSION In this investigation, the production rate of acetate is defined as the rate at which acetate enters the pool in the rumen as determined by an isotope, dilution technique. To calculate the total amount of acetar tate, the rumen fluid volume of each animal was determined using the PEG method. Differences in rumen fluid volume were found between individual sheep on the same ration and between sheep on different rations. The range of values was from 4.2 to 8.5 liters. Also, heavier animals showed a slight increase in the rumen fluid volume compared to lighter ani- mals See Table 11. Variations in rumen fluid volume of the same animals under differ- ent treatments might be due to variations in the eating or drinking pat- terns in response to that treatment or to the level of each treatment factor (isoacids, sulfur or urea) in the diet. It was observed that during the experiment animals drank rather large amounts 6f water. This could have definetely contributed to changes in the dilution rate of the marker. Measurements on sheep of a Swedish native breed, weighing approxi- mately 50 Kg and fitted with permanent rumen fistulae, showed a mean rumen fluid volume of 4.5 L (36). Additional studies have shown rumen volume values in a range of 4.7 to 6.0 L. in cross-bred wethers (10). 44 ‘45 Table 11. Rumen Fluid Volume of Experimental-Sheep Sheep No. Sheep Wt. Treatments* Rumen Fluid Volume (k8) (liter) 55 27.6 T1 5.410 T 5.989 T; 7.57. T4 4.544 Average: 5.879 T, 6.644 T3 5.617 ’1'4 4.935 Average: 5.757 53 25 5 T1 6.456 T 4.700 2 T3 7.636 T4 6.890 Average: 6.420 36 27.5 Tl 5.688 T2 4.509 T3 8.174 T4 6.872 Average: 6.310 3 34.5 T 5.652 6 T2 5.324 T7 4.700 T8 8.628 Average: 6.070 87 34.5 T5 5.150 T; 7.052 T8 7.764 Average: 6.225 S 35.0 T 4.351 8 mg 7.896 T7 5.105 T8 6.946 Average: 6.074 S 36.0 I 5.806 10 5 I 4.140 I? 8.471 T8 5.989 Average: 6.100 *Details of treatments are given on page 31.5heep were fed ad libitum during the sampling period. 46 Similar results were obtained in a study using Merino ewes and wethers; values in the study of Leng, ranged from 4 to 5.4 L (43). Another study on the absorption of volatile fatty acids from the reticulo-rumen showed a.higher rumen volume ranging from 5.9 to 7.2 L for mature Merino sheep (44), and ours from 4.2 to 8.5 L. Therefore, the rumen volume results obtained were similar to those, but slightly higher than others (10, 36, 43). The effects of isoacids, urea nitrogen and sulfur on the concentra- tions of volatile fatty acids are summarized in Table 13. There were four observations per treatment(see Table 27). The statistiCal analysis of variance for VFA production is presented in Table 12. There were no significant differences among treatments in the ruminal acetate, propio- nate and iso-butyrate concentrations ( P< 0.05 ). Mean ruminal butyrate conCentrations are shown in Table 13. Iso- acids and urea interacted as shown in Table 12. Ruminal butyrate con- centration was slightly increased with joint supplementation of high leg als of isoacids and urea. However, this effect was not significant (_p,,o.1o’ Table 14 ). Increase in butyrate concentration with increasing dietary urea concentration could be caused by a stimulus of the rumen cellulolitic bacterial population in response to increasing ammonia levels Treatment effects on ruminal 2-methy1 butyrate concentrations are presented in Table 13. The concentration of this branched acid was in- creased when high isoacid was given instead of low isoacid in combina- tion with urea and no sulfur ( P< 0.01 ), as shown on paired treatment comparisons in Table 15. This might be due to degradation of this acid tutu-nun chat-.uv- an! .eq.--Hu-..-.u:..uv <1.) .hg-h acre-e-s-ees) h.- I‘ewsasg-h‘ .\~ .-§.-E.P 47 AHeévmv «a A 3.3 a v s e e ouaooHuHcmHm cmcac.c o~Hoo.o unocc.o c¢noc.c oocco.c aHnoc.o c~o¢c.o Hmoco.o omcco.o newco.c Quezon and: NH o o H H H H H H H eopoauu uo moouwefi oumuoHo> I z. 8:322»; mHocc.c ocooo.c «Hoco.c ~o~oo.c «coco.o Hoooo.o oeHcc.o nooco.o coooc.o o~ooc.o nuance coo: NH 0 m H H H H H H H sopoouu no moouwo: . ououoHe>uomH as a . as e oucmuHuchHm mmooo.o mmooo.c Hooco.o choo.o echo.o cmcoo.o Howoo.c NHHc¢.o Hmooc.o memoc.c Quezon new: ~H o o H H H H H H H Eopoouu mo newsman . ououhunmuHasno: N e oozooHuHcmHm qqcmo.o mmHma.c canco.o ¢¢ sou ooonuo> uo oHoaHoc< .N- oHnea I1 ' ‘1IIY. I11 I I. Iv. 1‘... . a Al‘l‘~I~e fi-u—--..- -.J.C..v- wad -—8\u¢..-V De. uh§.h oumuon> oumukunm oumuxunm mumuzunm mumsoHeoum mumumo< :OHumanEoo IomH Hmnumalm IomH mamaummue H353 spasm mo H395 «mz CO WCOHUQCHQHEOU “Cg—HUQUHH. MO mUUQMNm oMH ”HQQH. 49 Table 14. Pooled Treatment Comparisons. of The Effects of Isoacids (A) and Urea (B) on Butyrate Concentration in The Rumen. Mean Difference* Comparison Treatment Contrasts (mg/ d1 of Rumen Fluid) . Significance A B -C A B C H L H L L H A/BL + vs + 0.148 P> 0.10 AHBLCL ALBLCL BHCL CH A/BH AH+ vs ALB-P 0.186 P>-0.10 AHBHCH ALBHCL B B C B/AL ALECH vs AL} L 0.124 P> 0.10 ALBHCL ALBLCH AHBHCL AHBLCH B/Au + vs + 0.209 P>0.10 ‘ AHBHCH AHBLCL *SED =- 1“ 0.093 50 when there is low ammonia in the rumen. Sulfur showed a negative effect on the concentration of this acid ( P <0.01 ), as indicated in the last comparison in Table 15. Iso-valerate concentrations for various treatments are shown on Table 13. For this acid 4 contrasts were made from the interaction AB (isoacids-nitrogen) which was found to be significant ( P< 0.01 ), Ta- ble 12. When isoacids were increased in combination with urea, the con- centration of iso-valerate increased ( P<:0.001). In the absence of urea the effect of isoacids was minimal (Table 16). This result was supported by the work of other investigators which reported pronounced depressions in iso-valeric acid concentration when ruminants were fed diets essentially free of protein. Moreover these comparisons ‘agree with the results of Cline et a1 (17), in the sense that the decrease in rumen iso-valerate indicates that when supplementa ry urea was added to the diet more of the available ammonia was being converted into microbial protein resulting in anincrease of this acid. Mean differences of ruminal valerate concentrations are presented in Table 17. When rations were supplemented with sulfur, there was a positive response to increased isoacids ( P3<0.05 ). The effects of the dietary parameters on liquid turn over time were estimated by the regression analysis of l/turn over rate. Average turn over times for each treatment are shown in Table 18, and the analysis of variance for this variable is presented in Table 19. There were not sig- nificant interactions between treatment factors. Some decrease in turn over time was noticed with increasing levels of isoacids and with sulfur in the ration, but the differences were not significant ( P>'0.10 ), be- cause of massive error variance. 51 Table 15. Paired treatment Comparisons of 2-Methy1 Butyrate Concen- tration in the Rumen Mean Difference? Treatment Contrasts (mg/d1 of Rumen Fluid) Significance A/BLCL: ALBLCL vs AHBLCL 0.012 ** A/BLCH: AHBLCH vs ALBLCH 0.017 ** (positive effect of iso- A/BHCL: AHBHCL vs ALBHCL 0-071 acids, urea present, sul- fur absent, P< 0.01) : B A/BHCH ALBHCH vs AH HCH 0.008 ** : B mm 6.3.0. vs 6. .6. 0-020 ** may ALBHCH vs ALBLCH o-om .. (positive effect of urea, B/AHCL: AHBHCL vs AHBLCL 0.063 high level of isoacids, no sulfur, P< 0.01) B/AHCH: AHBLCH vs AHBHCH 0.010 ** C/ALBH‘ ALBHCH VS ALBHCL 0°°06 ** C/AHBL: AHBLCH vs AHBLCL 0.016 ** (negative effect of sulfur, C/AHBH: AHBHCL vs AHBHCH 0.057 high level of isoacids, urea present, P< 0.10) * SED - 0.011 **( P>0.10) 52 Table 16. Pooled Treatment Comparisons of the Effects of Isoacids (A) and Urea (B) on Isovalerate Concentration in the Rumen Mean Difference* Comparison Treatment Contrasts (mg/d1 of Rumen Fluid) Significance AHBLCH ALBLCH A/BL -+ vs .+ 0.004 P >0.10 AHBLCL ALBLCL AHBHCL ALBHCH (positive effect of 139 A/BH .+ vs .+ 0.025 acid, when urea high, AHBHCH ALBHCL P < 0 . 00 1) ALBHCH B c B/AL + vs AL} L 0.014 P >0.10 ALBHCL ALBLCH A B C A B C H H L H L H B/AH + vs 1 0.015 P >0.10 AHBHCH AHBLCL *SED - i 0.006 Table 17 o 53 Pooled Treatment Comparisons of the Effects of Isoacids (A) and Sulfur (C) on Valerate Concentration in the Rumen. Mean Difference* Comparison Treatment Combination (mg/d1 Of Rumen FIUid) Significance A/CL A/ CH C/AL c/AH AHBHCL + AHBLCL AHBLCH + AHBHCH ALBHCH A B C L L H AHBLCH + ‘v AHBHCH ALBLCL + ALBHCL ALBHCH + ALBLCH ALBLCL A B C L H H AHBHCL + AHBLCL 0.0180 0.0375 0.0125 0.0245 P>'0.10 (positive effect of isg acids, with sulfur, P< 0.05) P> 0.10 P>I0.10 *SED - i 0.011 54 Table 18. Effects of Treatment Combinations on Acetate'Turnover Time in the Rumen. Treatment Combination (mggfigggs T1: ALIBLCL ' 125.480 T2: AHBHCL . 114.898 T3: AHBLCH 86.805 T4: ALBHCH 75.961 T5: ALBLCH ‘ 83.134 T6: ALBHCL 148.609 T7:‘AHBLCL 94.040 I8: AHBHCH 78.017 * sax - 1 29.90 A Ho.c vk veg An0.0v._ V K 55 «a , mocmoHeHcmHm sch.o an.~H mmo.oH coc.o noH.cH mmH.n~ noH.H ~mn.o mHm.am¢ Hmo.H amazon can: «H o o H H H H H H H someone mo museums :oMouUHz :onoas< e e «a a «e oocouHchmHm avn.H omH.n oHH.m Hon.m Hoo.H mmo.o oo~.o HnH.Ho~ coo.m oom.o~ unseen coo: NH o o H H H H H H H aopoouu mo avenue: aeHCHam =6m608_: poneuHqumHm QOm chH sewn meH a menu amHN scan mam cams phenom coo: NH 0 o H H H H H H H eovoouu mo newsman uo>ocuze e e oocooHuHcmHm nooHc.o eunHo.c «coco.c mHHHc.c cannc.c NowHo.o onnoc.c omnmo.o «moHo.o amooo.o phonon can: NH c o H H H H H . H H someone Ho newsman :oHuusvcum pumuou< monsoon nouns ouoavm om< u< on m< usuHamuo mou=un .omoeHI< moo:m apoHuom mcoHuoouousH mmoeoo=uah .onuoapoum ououou< no oucaHun> mo aHmzHoc< .mH oHaok 56 Means of acetate production in the rumen are shown in Tables 20 and 21. A11 mean acetate production values appear normal and found to be consistant except Treatment 5 (ALBLCH) which was 0.172 moles/hr x sheep and less than Treatment 1 (ALBLCL) value which was 0.20 moles/hr x sheep. This resulting low mean could have been caused by the lower dry matter intake shown by 2 of the sheep at 2 of the sampling times. This resul- ted in a decrease of VFA production. Because of significant interaction AC ( P< 0.01 ), Bonferroni-t tests were made on low isoacids/sulfur supplemented treatments compared with treatments with the same level of sulfur but higher isoacid levels. Results from these analyses indicated that acetate production was in— creased when isoacids were increased with sulfur present ( P< 0.05 ). The same was found when sulfur was added in presence of high isoacids ( P< 0.10 ) See Table 22. When treatments with different N:S ratios were compared, a tenden- cy of increasing acetate production in the rumen was found with 5:1 ra- tios, compared to ratios of 3:1, 8:1, or 12:1. This effect is clearly seen in Figure 3. Ruminal ammonia concentration and nitrogen to sulfur ratios relationships with acetate production were compared. It was found that ruminal ammonia concentration had a very low correlation with res- pect to acetate production (0.18) as presented in Figure 4. Moreover there is evidence that N:S ratios are more important in predicting the production of acetate ( R2 = 14.6% ), as compared to absolute ammonia concentrations in the rumen ( r2: 3.2% ) See Figures 3 and 4. The curve predicting acetate production from N:S ratio indicates an optimum ratio of 5:1. The decrease in acetate production shown at 10:1 was not repre- sented by data and resulted fromethe curve being forced low by required 57 mOHHHHm muz oHumu muz EaaHumo «.0 oneoaoomm me0.10 A1.315311 Annaca ALBLCL A1.1311311 8:1 vs 5:1 ‘+ V! + 0.100 P >0.10 AHBLCL AaBaCa ABBHCL A1.311611 12:1 v3 5:1 + v; + 0.103 P>0.10 A1.131101. A11311511 * sen - i 0.011 ** N:S Ratios 62 Means of hydrogen sulfide concentration are presented in Table 23. When sheep were fed sulfur supplemented rations, the concentrations of hydrogen sulfide in the rumen were approximately 7 to 9 ug/ml of rumen fluid; compared to 0.7 to 4 ug/ml of rumen fluid when no sulfur was a added. When mean N:S ratios of rations were compared with N:S ratios of rumen contents, the same general pattern was found (Table 24). Ratios of low N:S ratios, such as 3:1 and 5:1 resulted in nitrogen to hydrogen sulfide ration in the rumen of 9 to 10:1 and 16 to 18:1 respectively. Ratios of high N:S ratios, such as 8:1 and 12:1 gave ratios of 14:1 and 36 to 54:1 respectively. A ratio of 90:1 on treatment 1, found to be rather wide and resulted from a very low hydrogen sulfide value found in the rumen. For the rest of the treatments, this difference in N:S rae tios between rations and rumen could be due to a decrease in ruminal H28 production during the experiment. In general, it was found that addition of sulfur to the diet resul- ted in higher rumen hydrogen sulfide concentrations ( P1<0.01 ), Table 19. Mean blood urea and glucose concentration in blood are represented in Table 25. Analysis of these two variables is shown in Table 26. From these analysis, significant effects.of Urea (B), ( P< 0.01 ) and sulfur (C), ( P< 0.05 ) were found in blood urea. Blood urea in- creased greatly ( P< 0.01 ) when diet was supplemented with urea, even when ratios were low in isoacids and without addition of sulfur. Blood glucose levels are shown in Table 25. There was no effect of treatments on the concentration of glucose in blood. 63 Table 23. -Effects of Treatment.Combinations-on Hydrogen Sulfide and Ammonia Concentration in the Rumen. Means* Treatment Combination Sulfur Ammonia ug/ml- .. _ w mg/dl‘ T1: 111}chL .o.738 6.709 12: AHBHCL 4.075 14.820 T3: AHBLCH 7.813 7.406 T 4: ALBHCH 7.384 12.842 T5: ALBLCH 7-043 7.035 T6: ALBHCL 2-950 15.961 T7: AHBLCL 3.520 4.819 T8: 1138ch 9-110 13.998 *SEM 10.661 11.298 641 Hun Hum HuwH Hun Ham Ham HuNH Hum HucH Hn¢H Huqn HuoH HHGH Hua Huon Huoa 2.6.5.5 Sonnet H.635 1.0.545 Hausa—.5 Eadie Hausa—=5 Sugar: 3 .H. s .H. OH. nu. ca. ma. NH. H .H. oueuaueoub coHumz. ages: meoHumx was span: was :H moHusm an: .«N oHan 65 Table 25. Effects of Treatment Comabinations on Urea and Glucose Con- Centration in Blood. Means‘:_SEM* Treatment Combination Urea Glucose (mg/d1 of Whole Blood) 11: ALBLCL 4.710 37.965 12: AHBHCL 7.458 43.429 T3: AHBLCH 7.166 43.130 T4: ALBHCH 8.498 40.330 T5: ALBLCH 7.457 33.276 15: ALBHCi 11.207 31.767 T7: AHBLCL 5.623 38.461 T8: 638303 9.998 40.330 *szu “' 4 30.929 15.852 66 A Hc.c vm we « H85 2: 1. a «e moaooHqumHm nnc.n ccH.n Hem.H Non.s n~M.H ~0~.H nec.c mae.H~ ~w~.c¢ eHn.o season coo: NH 0 c H H H H H H H souoouu no assumes sou: sonoonchHm smH N.na c.an n.0mu oH man n.o o.c c.H m.HH~ season can: «H o c H H H H H H, H sovuoum mo saunas: umoosHu souancm nouuu oueacm museum om< u< on m< uawHamlc sous-m .oeoaHl< moosm ocoHuom acoHuoouoqu mwoeo mo anszc< .ow oHnae 67 Values found were in the range of 37 to 44 mg/dl of whole blood. This compares with reported ranges in sheep of 30 to 50 mg/dl (78). These values were only determined to find out the levels of glucose in animals in the tropics (See Table 30). CONCLUSIONS Ruminal concentrations of acetate, propionate and iso-butyrate were not affected by treatments. Butyrate, 2-methy1 butyrate, iso-valerate and valerate levels in the rumen fluid were affected by level of iso- acids fed, or interactions of isoacids with urea and sulfur. Isovalerate and 2-methy1 butyrate increased with high level of isoacids in the pres- ence of urea, and valerate was increased by isoacids in the presence of sulfur. Turn over time of acetate was not affected by treatments, but ace- tate production rates were increased when the high level of isoacids was fed (0.14 g/kg of body weight), in combination with sulfur supplemené tation. Rumen ammonia and blood urea were greater when animals were fed urea. Ruminal hydrogen sulfide concentrations were increased when sulfur was added. Blood glucose levels were not affected by any treatment. The results of this study suggest that N:S ratios are more important than absolute concentrations of ammonia in the rumen. The N:S for an op- timum fermentation as measured by acetate production was 5:1. Amount of 0.43 g of urea/Kg body weight and 0.086 g of sulfur/Kg body weight gave this ratio. Therefore, it is concluded from this study that for optimum rumen fermentation of pineapple tops by sheep the ration should be supplemented with 0.14 g of isoacids, 0.43 g of urea and 0.086 g of sulfur per Kg of body weight. 68 APPENDIX 69 osmo.c stmo.c nnso.o «Nao.o asso.c saso.o osoH.o saac.o ssssss< Hemo.o naso.c Hoso.c nnmc.o own ssmc.c Hmuo.c smoo.c masc.c as ssmo.c Henc.c ~sm¢.¢ mano.o 8m nsmc.c seno.c ansc.o Asn~.c m ~s~o.c asso.o cHso.o soHH.c em Noso.o cHsc.c mnse.o Hmnc.a Mm HsHH.o ancH.o chH.c essc.c mm sans.o «HsH.o AHsH.o nnao.c m sassuss-s oass.H HNon.H snmn.H osmH.H nmcH.H sAsH.H emas.H ns~n.H sess6>< ~3n¢.~ senaeH scam.~ asnn.H sum nmo~.H o-~.c anon.H ~ns~.~ as News.H cNHs.H oHeH.H 8Hm~.H m sssn.H ssmH.H seo~.H mosH.~ 8m Hans.c coss.o «was.H sHsH.H mm smHo.H Assc.H smss.H ~44H.H Am HHN¢.H cHnn.H ccsM.H oosc.H mm meno.H ssHs.H anH.H uses.H s sasosaoss ones.e onse.8 Hnsn.e snsn.a sens.n cosn.o 8AHN.H seen.o ossss>< smsm.a sNHH.s cess.s ocse.s ems mass.a AsHo.s messes asso.o as sss~.a naas.e Amen.a moss.s em oaaa.s cmsm.e seen.s Neon.s , sm sans.s ~mss.n sn¢s.o susa.o mm ens~.s on~s.o ess~.s Assn.m A name.“ osmo.s caNs.s s¢ns.n Mm scan.m snen.e csan.a oss~.s m suu< ussso< =o=e=< sosn=< Hoens< =osas< =o=mu< =oea=< so=n=< soans< Hugo: munoaumoue Hoch< muHaaHuo> «spasm as. :H onususcoocoo uHo< zuuom oHHusHo> :o usuHsm can sou: .anomomH Ho ouoouum 70 chc.c nsao.o Nasc.c soHo.c Aaao.o «sic.o ssmc.c cho.c sssss>< mnmc.c Hch.a nMHc.o cHsc.c emu ssso.c cch.o . ano.o m emac.c sono.c nsHo.c mcHo.c as msHo.o ano.o H~H8.o smsc.o mm - nn~c.c ammo.o ammo.o mm snwc.o ammo.c osno.o ss~c.c NA mHNo.o Nano.c HO~H.¢ Hch.o mm masseuse nch.c -no.c mmnH.c sHso.c s Haeusx-~ woos.o omen.c smmm.o snmn.c ~sms.c ossn.c amne.o Hons.o 6s6t8>< H_se.c ssmc.o assm.o csos.c ems mean.o nss~.c Nom8.o Hane.o m Hens.c cha.c sNos.o a8~4.¢ Mm smcs.o ssos.c secs.o chA.o sm asn~.o ns~8.c «som.c -~m.¢ Hm . oHcs.c Hens.c cane.c Hmsm.c m Hosm.c stm.o n~mm.o s~nm.c mm Necm.o o~nn.c osmm.c «Has.c m usssusm =o=n=< sosm=< sues < moans< =o:ea< soan=< so=n=< soans< Hsssu .H.v.u:oov 5N oHnme 71 .vHaHh :95: mo 2.1:: « sssc.c Hsuc.o sumc.o ssuc.c oHNc.c s~so.c osmo.e Hsmc.c smsssss somo.c emwo.o snmo.o ~sn¢.¢ ems onso.o - sHNc.o Nisc.o as neso.c saso.c - - 8m Hsmc.c asso.o nasc.o mmso.o em . mHso.s Hmso.o meno.o mm nsnc.c NHsc.o asso.c HHac.o Na sanc.c osso.c snmc.c ssso.o mm cNHa.o smmH.c omnc.o Homo.o m susssass Hsso.o asca.o snoo.o ano.c HnHo.o sawc.o moso.o some.c sssuo>< anso.c HsHo.o soo.c ssoo.o own Hsso.o . . «Hmc.o Hm maso.o Haso.c ssso.o . 8m snoo.o . . ssHo.o m 4 1 cH~o.c ss~o.o ~s~¢.o em sHNo.o soHo.o Hesc.o Hsso.c mm msHo.c Hnso.o ncnc.c Hssc.c mm c-c.o ss~c.c mosc.c msnc.o m susssss>166H zone—=3. nose? so:ns< =osms< zone: noun? soap—.54 soon? HssH6 46.2.63. 3 935 72 Table 28. . Runen Ammonia Concentrations in Sheep Fed Diets with and .without Urea Supplementation Treatments Non Supplemented Supplemented Sheep NO- ALBLCL AHBLCH “,8ch AgBLCL AHBHCL ALBHCH ALBHCL 438.503 ---------- mg/dl ------------- 35 8.944 5.968 20.921 13.580 32 6.780 6.560 15.030 9.480 53 4.737 9.769 13.500 12.826 54 6.378 7.329 9.832 15.482 56 8.753 4.430 14.710 12.836 57 5.352 4.937 13.695 14.498 58 8.635 6.262 19.069 22.010 310 5.403 3.848 16.373 6.650 Average 6.709 7.406 7.035 4.819 14.820 12.842 15.961 . 13.998 ‘73 13b1,. 29, . Rumen Hydrogen Sulfide Concentration in Sheep Fed Diets with and without Sulfur Supplementation. Treatmts Non Supplemented Supplemented Sheep NO- ALgLCL 583851 ALBHCL A113191. AHBLCH ALBHCH ALBLCH 'AHBHCH """"""""" ppm""""'"""‘ 35 1.401 5.145 12.770 6.180 82 0.150 3.417 7.737 10.185 53 0.615 2.161 . 4.450 7.161 54 0.787 5.580 6.297 6.012 85 _ 2.840 1.803 4.421 8.163 57 4.140 6.297 8.122 9.350 58 1.833 4.428 10.141 12.345 310 2.985 1.589 5.490 6.588 Average 0.738 4.075 2.950 31529 7.813 7.384 7.043 9.111 74- Table 30- Effects of Treatment Combinations on Urea and Glucose Concen- trations in Blood. Blood Urea Blood Glucose Treatment Animal (mg/d1) (mg/d1) 1 S 6 83 33.30 1 s? 4.33 56.02 s; 3.00 31.35 34 5.00 31.19 Means 4.79 37.96 T2 55 6.16 56.02 52 9.66 38.88 S3 9.00 43.90 54 5.00 34.91 Means 7.45 43.42 T3 85 5.00 31.35 52 10.00 38.53 83 7.00 56.73 S4 6.66, 45.90 Means 7.16 43.12 T4 S5 10.33 31.19 52 6.33 57.71 S3 9.00 44.10 54 8.33 28.32 Means 8.49 40.33 T5 56 7.55 28.66 87 8.33 50.33 58 6.00 27.11 510 8.00 27.00 Mean! 7.47 33.27 T6 56 8.33 31.89 87 8166 34.07 38 16.33 36.11 310 11.50 25.00 Means 11.20 31.76 T7 56 4.16 35.33 57 6.33 35.17 58 4.66 30.44 510 7.33 52.90 Means 5.62 38.46 T8 86 12.00 31.11 S7 9.33 31.69 58 11.33 33.88 1 7.33 57.57 Means 9.99 38.56 BIBLIOGRAPHY 10. 11. BIBLIOGRAPHY Allaway, W. H. and J. F. Thompson. 1965. Soil Science. 101:4. Alexander, C., R. Meyer and E. Bartley. 1969. Effect of quan- tity of rumen dry-matter and other factors on determinations of rumen fluid volume with polyethylene glycol. J. Animal Sci. 29:1. Allison, M. J. 1969. 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