£54. I! o --‘ ".n‘ In fi‘_.—flf .uwu‘vwwan ’¢'- :1 Q5 '21 9". fl 0 9‘ 1.3%: ‘.‘....‘....._..--._. L nu v \.e .431o:;! This is to certify that the dissertation entitled A Study on the Action of Teichomycin A2, Avoparcin, and Isoacids in Sheep presented by Altair V. Brondani has been accepted towards fulfillment of the requirements for Ph.D. degreein Animal Science IOWA M Major professor Date W65, MS U is an Affirmative Action/Equal Opportunity Institution 0-12T71 MSU RETURNING MATERIALS: Place in book drop to remove this checkout from LIBRARIES - your record. FINES will be charged if book is , returned after the date stamped below. egg 7 tags; $2745.45 00:) H170 AH;:]~15‘Qflw 355’2 ci’fl ”3 11% A STUDY ON THE ACTION OF TEICHOMYCIN A2, AVOPARCIN, AND ISOACIDS IN SHEEP BY Altair V. Brondani A DISSERTATION Submitted to Michigan State University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Animal Science 1986 ABSTRACT A STUDY ON THE ACTION OF TEICHOMYCIN A2, AVOPARCIN, AND ISOACIDS IN SHEEP BY Altair V. Brondani The objective of this study was to investigate the action of glycopeptides and isoacids in the rumen in an attempt to further explain how those compounds affect productive performance in ruminants. Production rates of rumen VFA were measured by a single injection radioisotope technique. In the first experiment, twelve rumen-cannulated crossbred ewes were fed either high roughage (HR) or low roughage (LR) diets supplemented with 0 (CONTROL), 30 ppm TE-Az, or 30 ppm AVG. In both diets, TE-Az and AVO increased propionate (p<.10) without significantly affecting acetate (p>mlO) and butyrate (p>10) production. Protozoal, bacterial, and soluble protein fractions were not altered by glycopeptides in either diet. Concentrations of ammonia-N were decreased (HR and LR, p<.05)land of alpha-amino-N were increased (HR, p<.05; LR, p<.10) by TE-Az and AVO. The effects of isoacids, urea, and sulfur on rumen fermentation rates in sheep fed high fiber rations were studied in two trials. ZRates of acetate production were taken as a measure of rumen fermentation“ Overall, feeding r . . 4 L t . T . .. . ,. . . . .. a . to..- Altair V. Brondani isoacids at 0.2 g/kg bw/day increased (p<.05) production rates of acetate in both trials. This effect was dependent on urea and sulfur supplementation. Six rumen-cannulated crossbred ewes (BW=40 kg) were fed 1200 g alfalfa hay/day supplemented with 0 (CONTROL), 0.1 (1501), or 0.2 (1502) g of isoacids/kg bw/day; In each of three 15-day periods, one level of isoacids was administered to all animals. Blood samples (30-min intervals for 12 hr) and rumen fluid samples (hourly intervals for 8 hr) were collected at the end of each period. Plasma concentrations of growth hormone and cortisol were not affected (p>.lO) by isoacids. Animals receiving 1802 had lower mean plasma insulin (p<.10), higher rumen acetate (p<.10) and lower rumen propionate (p<.10) than controls. It is proposed that decreased propionate production in the I302 group resulted in lower stimulus for insulin secretion. These results may explain why isoacids consistently increase milk production in lactating cows fed a variety of diets. They also suggest that glycopeptides and isoacids affect performance of ruminants through overlapping mechanisms, in which propionate plays the central role. I'l . .. n .t _ .5 _ . ’ . u a Q h . . . a I ~( . . . a.. ha I, . . ... a \v o I o a.. . . e v .. vb .. . .f ., ... . .0 I. ‘ I It . t. i, , . . o l. a . .. . a . a e u s v a. O . . 3... i 2 ...u L. .4. , . ACKNOWLEDGEMENTS The author expresses sincere gratitude to his major professor, Dr; Robert M. Cook, for his guidance, ecouragement, and high interest during the development of this work. Gratitude is also extended to the other members of the graduate committee, Dr. Werner Bergen, Dr. Dale Romsos and DrulMelvin Yokoyama for their assistance during the course of this program. Thanks are extended to Dr. John Gill for statistical advice, and to Mr. Jim Liesman and Dr. John Walter for their invaluable assistance with the computer analysis. Special thanks are expressed to Mr; George Good, from the Sheep Research Center, for his assistance with the experimental animals, and to Mr; Steve Roof, from Dow Chemical Co., Midland, MI, for his help during the study with glycopeptides. Appreciation is extended to the fellow graduate students, faculty and staff of the Animal Science Department for their help and friendship during the author's stay at Michigan State. Special acknowledgements are expressed to the Instituto de Zootecnia, SP, Brazil, fer granting the author‘s leave of absence and to EMBRAPA.(Empresa Brasileira de Pesquisa Agropecuaria) for the financial support. ii TABLE OF CONTENTS Page LIST OF TABLES C O O O O O O O O O O O O O O O O O O O O O O O O O O ........... V LIST OF FIGURES ... .................................. vii I. Introduction ... ..... . ........ ... ............ 1 I I 0 Literature ReView O O O O O O O O O O O O O O O O O O O O ....... 3 Ionophores O O O O I O O O O O O O O O O O O I O O O O O O O O O O O O O O O O 3 Effect of monensin on rumen volatile fatty aCids O O O ,. O O I O O O O O O O O O O O O O O O O O O O O O O O O O O O 0 4 Effect of monensin on nitrogen metabolism in the men 0 O I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 7 Effect of monensin on rumen microorganisms . 9 Glycopeptides O O O O C O O I O O O O O O O O O O O O O O O ........ 1 2 AvoparCin O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 12 TaiChomYCin A2 0 O O O O O O O O O O O O O O O O O 0000000000 1 5 IsoaCids O O O O O O O O I O O O O O O O I O O O O O O O O O O O O O O O O 0 O O 16 Sources of branched-chain VFA in the rumen .. 17 Effects of isoacids on rumen microbial protein synthesis ........................ 19 Effects of isoacids on DM digestibility .... 21 Effects of isoacids on nitrogen retention .. 23 Effects of isoacids on plasma hormone concentrations .................. ......... 24 III. Effects of teichomycin A2 and av0parcin on in vivo production rates of rumen volatile fatty acids in sheep fed high and low roughage diets 31 Introduction ....... ........................ 31 Materials and Methods .......... . ........... 32 Volatile fatty acids ............ ......... 34 Protein fractions ............... ........ . 35 Soluble protein and alpha-amino-N ....... 36 iii 9 'n :- -»_ .- a. “ Insoluble protein ....................... Ammonia nitrogen .... ..... ..... .......... Rumen volume and rumen fluid turnover rate Production rates of acetate, propionate, and butyrate ...... Statystical analysis ......... . .......... Results and discussion .................... IV. Effects of isoacids, urea, and sulfur on rumen fermentation in sheep fed high fiber diets ..... ..... Introduction ......... ........... . .......... Materials and methods ..... .... ............. Results and discussion ..................... V. Effects of isoacids on plasma concentrations of growth hormone, insulin, and cortisol in Sheep O......OOOOOOOOOOOOOOOOOO0.00.00...O ..... Introduction ........................ ....... Materials and methods ............ .......... Results and discussion ........... ...... .... VI. smary ......OOOOOOO......OOOOOOOOO .......... LIST OF REFERENCES iv 36 37 37 37 4O 40 58 58 59 63 79 79 80 82 93 98 Table 10. LIST OF TABLES Effects of teichomycin A2 and avoparcin on concentrations of rumen VFA in sheep fed high and low roughage diets u.u.n.u. Effects of teichomycin A2 and avoparcin on the molar percent distribution of rumen VFA in sheep fed high or low roughage diets ...... . Effects of teichomycin A2 and avoparcin on acetate production and rumen fluid variables in sheep fed high and low roughage diets ...................... ....... Effects of teichomycin A2 and avoparcin on propionate production, rumen volume, and rumen fluid turnover rate in sheep fed high and low roughage diets. ............ Effects of teichomycin A2 and avoparcin on butyrate production, rumen volume, and rumen fluid turnover rate in sheep fed high and low roughage diets .u.. ....... Effects of teichomycin A2 and avoparcin on rumen nitrogen constituents in sheep fed high and low roughage diets u.u.u.u. Composition and analysis of basal diets ..... Effects of urea and sulfur on feed intake and rumen fermentation variables in sheep fed high fiber diets .n.u.u.u.. Effects of isoacids and urea on feed intake and on rumen fermentation variables in sheep fed high fiber diets ....... Effects of isoacids and urea on concentrations of rumen volatile fatty acids in sheep fed high fiber diets ...... n Page 42 44 46 48 50 54 60 64 67 69 11. 12. 13. 14. Effects of isoacids and urea on the molar percent distribution of rumen volatile fatty acids in sheep fed high fiber diets Effects of isoacids and sulfur on feed intake and on rumen fermentation variables in sheep fed high fiber diets ...... . ......... Effects of isoacids on rumen volatile fatty acids in sheep fed a high roughage diet OO......OOOOOO.........OOOOOO0.00...... Effects of isoacids on plasma concentrations of insulin, growth hormone, cortisol, and glucose in sheep fed a high roughage diet vi 7O 74 83 87 Figure 10. LIST OF FIGURES Effects of isoacids on rumen VFA in sheep fed high fiber, high NPN diets. Values are relative concentrations with respect to control (control=100) .................. Effects of isoacids on rumen VFA in sheep fed diets containing preformed protein. Values are relative concentrations with respect to control (control=100) ..... ..... Acetate production rates as affected by isoacids + sulfur (ISOZ-S), isoacids + urea (ISOZ-U), or urea + sulfur (U + S) in sheep fed high fiber diets (Trial 1). Values are relative rates of production (ISOZ-S=100) .. Acetate production rates as affected by isoacids + sulfur (ISOZ-S), isoacids + urea (ISOZ-U), or urea + sulfur (U + S) in sheep fed high fiber diets (Trial 2). Values are relative rates of production (ISOZ-S=100) .. Effect of isoacids on plasma concentrations of growth hormone in sheep fed a high roughage diet (n36) ......OOOOOOOOOOOOOOOOO Effect of isoacids on plasma concentrations of insulin in sheep fed a high roughage diet (n36) ......OOOOOOOOOOOOOO0.0...0...... Effect of isoacids on plasma concentrations of cortisol in sheep fed a high roughage diet (n=6) ......OOOOOOOO... ..... 00.0.00... Summary of the effects of isoacids in ruminants fed a) high fiber, high NPN diets or b) diets containing preformed protein.... Summary of the effects of glycopeptides (teichomycin A2 and avoparcin) in ruminants ........................ ......... Action of isoacids and glycopeptides in ruminants ................................. vii Page 71 72 75 76 85 88 9O 94 96 96 I. INTRODUCTION The potential for chemical manipulation as a means of improving productive performance in ruminants is enormous. Currently, the direct effect of hormonal growth promotants and antibiotics accounts for 18% of meat production per unit of input in the United States (Rumsey, 1983). Chemical agents known to be effective in ruminants can be divided into two broad classes according to their mode of action (Cook, 1985). One class of compounds acts to increase the quantity or quality of nutrients made available for absorption. Examples of this type of products are ionophores (monensin, lasalocid, narasin), glycopeptides (avoparcin, teichomycin A2), and isoacids. The second group increases the efficiency with which the absorbed nutrients are utilized for productive purposes. These include hormones (growth hormone, diethylstilbestrol), beta-agonists (clenbuterol, cimaterol), and anabolic agents such as zeranol (Cook, 1985). Because of the complexities associated with every metabolic reaction, in which a single change may result in a cascade of interrelated responses, the two mechanisms may not be completely independent. For instance, recent reports indicate that isoacids, in addition to their known effect on the rumen fermentation, can also affect the plasma endocrine profile of lactating cows (Towns and Cook, 1984; Fieo et al., 1984). In the studies reported here, some of the aspects involved in the action of rumen additives are addressed. In Chapter III, the effects of the glycopeptides teichomycin A2 and avoparcin on the rate of production of volatile fatty acids in yiyg are reported. Chapters IV and V describe the effects of isoacids on rumen fermentation and on plasma hormone hormone concentrations in sheep. Results are discussed in terms of the relationship between final products of fermentation and regulation of nutrient utilization by the host animal. II. LITERATURE REVIEW IONOPHORES Ionophores constitute a group of compounds that have the ability to mediate the transport of cations across biologicaltmembranes. Several of these compounds, including monensin, lasalocid, narasin and salinomycin have been used successfully as feed additives for ruminants. In this review, monensin, the most widely used ionophore, is used in the discussion of the possible mechanisms of action of this class of compounds in the rumen. The basic mode of action of ionophores in the rumen, including their effects on microbial metabolism has been reviewed (Bergen and Bates, 1984). Monensin is the major component in a complex of four closely related, biologically active compounds produced by a strain of Streptomyces cinnamonensis (Haney and Hoehn, 1968). Preliminary studies indicated the ability of monensin to alter the normal patterns of rumen fermentation under both 12 ytttg and 12 gtgg conditions (Richardson gt g;,, 1976). Results of subsequent feeding trials, in which monensin was fed to cattle under a variety of environmental conditions, clearly demonstrated its positive effects on cattle performance (Goodrich gt gt,, 1976; Potter gt g;,, 1976; Raun gt g;,, 1976). Monensin has been shown to improve feed efficiency either by reducing intake, 4 increasing average daily gain, or both, depending on the feeding regimen (Short, 1978). In animals on a roughage diet, where rumen size limits intake, monensin increases rate of gain without altering feed intake. In concentrate- fed animals, where intake is controlled by chemostatic mechanisms (Conrad, 1966), monensin decreases feed intake without altering the rate of gain (Short, 1978). Studies on the mechanisms of action of monensin have been intense during the past few years. Ruminal changes observed in monensin-treated animals include a shift in the VFA molar proportions favoring propionate, a decrease in methane production and a decrease in rumen protein breakdown and deamination, resulting in lower levels of ammonia-N (Bergen and Bates, 1984). Additionally, changes in digestibility, rate of protein utilization, rumen fill and rate of passage have also been reported (Bergen and Bates, 1984: Schelling, 1984). The information currently available indicates that monensin causes important changes in the distribution of bacterial species in the rumen. The ultimate result is the generation of final products of fermentation which are highly favorable to the host animal. In the following review, some of these changes are discussed. Effects gt monensin gt rumen volatile fatty acids The effect of monensin on rumen fermentation is well established. Its most consistent effect is the increase in the molar proportion of propionic acid with a concomitant decline in the molar proportion of acetate and butyrate, but with minor effects upon total VFA in the rumen (Richardson et al, 1976; Chalupa, 1980; Bergen and Bates, 1984). Usually, this shift in the VFA molar proportion is associated with a decrease in methane production without accumulation of gaseous hydrogen (Van Nevel and Demeyer, 1977; Chalupa, 1980). Increased propionate production at the expense of acetate and methane should improve the efficiency of rumen fermentation due to improved retention of carbon and energy during the fermentative process. However, it is unlikely that this effect accounts for all the improvement in performance measured.in monensin-treated animals. Assuming that all VFA arise from hexose (672 kcal/mole), Hungate (1966) has estimated the efficiency of conversion of hexose to acetate, propionate, and butyrate. One mole of hexose yields 2 moles of acetate (420 kcal), with an efficiency of 62%. One mole of butyrate (524 kcal) is produced per mole of hexose, with an efficiency of 78%. In propionate synthesis, however, all the energy of the hexose plus the energy corresponding to the two electrons required for the conversion of pyruvate to propionate, is retained in the product. It is evident, therefore, that a high-propionate type of fermentation is energetically more efficient than i one yielding more acetate or more butyrate. Based on the shift in the molar proportion of VFA associated with monensin (from 60:30:10 to 52:40a8 for acetic, propionic and butyric, respectively), Richardson et a1. (1976) calculated a theoretical energy savings to the animal of 5.6%. Therefore, fermentation efficiency was improved by monensin because of increased recovery of metabolic hydrogen in the rumen" The figures obtained by Richardson gt gt. (1976) are slightly higher than the 3% increase in efficiency estimated by Prange gt gt. (1978). The theoretical increase in energy savings by monensin via the increase in propionate production evidently assumes the capacity of the host to use efficiently the extra propionate available. Results of short term studies involving intraruminal infusions of VFA.(Armstrong gt gt” 1958: Armstrong and Blaxter, 1961) indicated a higher efficiency of utilization for propionic acid than for acetic acid. Blaxter and Wainman (1964) gave further support to this hypothesis by relating the higher molar proportion of propionate in concentrate diets to the increase in efficiency of metabolizable energy utilization by the animal. This evidence has been used frequently to explain part of the improvement in animal performance obtained with monensin. 1However, the hypothesis of higher efficiency of utilization for propionate has been challenged. Results obtained in long-term infusion studies (Bull et. al, 1970; Poole and Allen, 1970) have indicated that the major VFA are used with equal efficiencies. 'The main reason for the discrepancies between results seems to be the length of time involved in different experiments. Longer periods would be 7 required for the animals to adapt to the sudden increase in acetic acid in studies involving acetate infusion (Bull gt gt,, 1970). This adaptation would involve the increase in the synthesis of pentosephosphate pathway enzymes responsible for NADPH generation. In short-term studies, the shortage of NADPH molecules would channel acetate to oxidation rather than to fatty acid synthesis, resulting in a higher heat increment (Bull gt gt,, 1970). Raun gt gt. (1976) have indicated that a shift toward more ruminal propionate production at the expense of acetate and methane could not account for all the improvement in performance obtained in feedlot studies. This observation led them to suggest that monensin is responsible for energy savings beyond those accounted for by the shift in VFA. These other potential savings would include a lower heat increment (Smith, 1971), a protein sparing effect involving amino acids normally used for gluconeogenesis (Leng gt gt” 1967; Reilly and Ford, 1971) and stimulation of protein synthesis in the host (Eskeland gt gt,, 1974). Effect gt Monensin gt Nitrogen Metabolism tg the Rumen Results of several experiments have indicated that at least part of the increase in efficiency of growth caused by monensin is associated with a decrease in ruminal losses of nitrogen. In tg ttttg studies in which casein was used as sole substrate, monensin depressed protein degradation (Van Nevel and Demeyer, 1977). In the same study, monensin resulted in a slightly higher accumulation of alpha-amino nitrogen and lower levels of ammonia-N. tg ytxg studies have confirmed the above observations. Dinius gt gt. (1976) reported significantly lower rumen ammonia-N levels in monensin-treated animals. These values were in line with the higher nitrogen retention measured in monensin-treated animals. Poos gt gt. (1979) fed lambs diets containing either brewers dried grain (BDG) or urea. Addition of monensin decreased rumen ammonia-N regardless of the nitrogen source. When the same diet was fed to steers, monensin increased total-N and non-ammonia-N’(NAN) flow to the lower intestine. Also, monensin supplementation increased plant nitrogen passage by 55% and 37%, for the grain- and urea-supplemented diets, respectively. The amounts of both essential and non- essential amino acids reaching the duodenun were higher for the combination BOG-monensin relative to control. The above results clearly indicate an effect of monensin in depressing protein degradation in the rumen. The resulting increase in ruminal escape of dietary protein should constitute an additional factor contributing to the increase in performance of monensin-treated animals. However, several studies have indicated that this process may be coupled with a decrease in efficiency of rumen microbial growth (Van Nevel and Demeyer, 1977; Poos gt gt., 1979; Isichei, 1980: Bergen and Bates, 1984). 9 Van Nevel and Demeyer (1977) reported a severe depression of total and net microbial growth yields by monensin under tg vitro conditions. I vivo studies have shown that monensin causes a significant decrease in the turnover of rumen contents (Dinius gt gt,, 1976; Lamenager et al, 1978). Based on these observations, Bergen and Bates (1984) have suggested that the lower efficiency of microbial growth caused by monensin might be attributed to an increase in maintenance requirements of the rumen microorganismsm Similar observations were reported by Allen and Harrison.(1979). In this study, lambs were fed a diet based on grass hay and ground maize, supplemented with monensin. The YATP values were 14.9 and 11,8 respectively for control and monensin-treated animals. The authors suggested that these values might be associated with the lower dilution rate measured in the treated group UL071 vs. (L941/h). This lower turnover rate might have resulted in a higher energy requirement for maintenance of the microbes (Stouthamer and Bettenhousen, 1973; Isaacson gt gt, 1975; Hespell and Bryant, 1979). Effects gt monensin gg rumen microorganisms Although considerable research has been devoted to determine how rumen fermentation is affected by monensin, the mechanism by which the microbes and their activities are controlled by the antibiotic has not been elucidated. The l0 following review will cover the work on species sensitivity to ionophores. Monensin is a member of the carboxylic class of ionophore antibiotics (Short, 1978). These compounds have the ability to form complexes with certain monovalent cations, rendering them permeable in biological membranes (Pressman, 1976). Therefore, dissipation of the normal cation or proton gradients across bacterial cell membranes seem to be the central event in monensin action on rumen microorganisms (Romatowski, 1979; Bergen and Bates, 1984). The effect of monensin on specific rumen bacteria species has been examined in only a few studies. The observation that lower methane production is obtained with monensin treatment has raised the question as to whether this might be due to a direct effect of the antibiotic upon methanogens. tg-gtttg studies carried out by Van Nevel and Demeyer (1977) discarded this possibility. Methane formation from H2 and C02 by washed-cell suspensions of mixed rumen bacteria was not inhibit by monensin. However, when formate was used as the sole substrate, considerable inhibition was observed. These results led the authors to suggest that the methane-depressing property of monensin is due to an inhibition of organisms decomposing formate into C02 and H2, rather than to a direct toxic effect on the microbes. Additional studies have confirmed the hypothesis that methanogens may not be the primary site of monensin action. Chen and Wolin (1979) reported that while the sensitivity ll of methanogens to monensin is dependent upon the specific organism, none of the species tested were completely inhibited. Similar results were reported by Henderson gt gt. (1981). The effect of monensin on carbohydrate-fermenting rumen bacteria have also been assessed. Chen and Wolin (1979) found that Ruminococcus albus, Ruminococcus flavefaciens and Butyrivibrio fibrisolvens, are highly sensitive to monensin. Succinate-producing bacteria, such as g. succinogenes and g. ruminicola were less sensitive to the antibiotic. Selenomonas ruminantium, which decarboxylates succinate to propionate, was found to be highly resistant to monensin. Similar results were observed in studies conducted by Dennis gt gt. (1981). Major succinate producers (Bacteroides, Selenomonas, Succinivibrio) were not inhibited by monensin. Most of the lactate-producing species were inhibited by levels of the antibiotic ranging from 0.38 to 3.0 ug/ml. However, major lactate fermenters were not sensitive at these levels. The authors proposed that monensin could be effective in preventing lactic acidosis in ruminants, which was subsequently confirmed in studies by Nagaraja gt gt_(1981). Attempts have been made to explain the changes in the final products of rumen fermentation of monensin-treated animals based on the sensitivity of important rumen bacteria species to the antibiotic (Chen and Wolin, 1979; Henderson gt gt., 1981). These changes are likely to result from the l2 selective inhibition of bacteria species which do not contribute significantly to the propionate pool in the rumen. Consequently, growth of organisms such as Selenomonas ruminantium and Bacteroides ruminicola are favored. Inhibition of growth of major acetate and hydrogen producers in the rumen (Ruminococci and g. fibrisolvens) would result in a decrease in those end-products. This would therefore contribute to the decrease in the acetate to propionate ratio and to a reduced availability of hydrogen to the methanogens (Chen and Wolin, 1979; Henderson gt gt” 1981). GLYCOPEPTIDES Avoparcin Avoparcin, a biologically active compound produced by a strain of Streptomyces candidus (Kunstmann gt gt., 1968), has both tg ytgg and tg ytttg activity against Gram positive bacteria (Redin and Dornbush, 1969). Its structure is similar to those of vancomycin and ristocentin, although substituents on the phenolic rings of the seven amino acids are slightly different (Hlavka gt gt,, 1974). Most of the preliminary work with avoparcin.in animal nutrition was conducted in Europe, where it has been marketed largely as growth promoter for poultry and swine. Studies with ruminants carried out in the United States have shown promising results. Johnson gt gt. (1979) conducted a 13 llz-day trial to evaluate the effect of 0, 16.5, 33, and 66 ppm avoparcin and 33 ppm monensin on the performance of steers fed a high-barley ration. Avoparcin-treated animals showed improved feed efficiency and higher weight gain relative to controls or monensin-treated steers. Both avoparcin and monensin caused a shift in the VFA molar proportions towards propionate. Essentially the same results were obtained with finishing heifers (Dyer gt gt, 1980). All avoparcin-treated animals consumed less feed per unit gain than controls. Carcass characteristics were not affected by avoparcin. DeLay gt gt. (1978) fed 0, 16.5, 33 or 66 ppm avoparcin to steers. They reported 1.5, 2.7, and 4.9% reduction in average daily feed intake and 1.9, 5.7, and 11.6% improvement in feed efficiency for the three avoparcin levels relative to control. . The mechanisms by which avoparcin exerts its positive effects on the performance of ruminants still remain to be completely elucidated. .As in the case of monensin, improved efficiency in treated animals has been associated with the action of avoparcin in the gastrointestinal tract. Chalupa gt gt. (1981), measured the effect of avoparcin on rumen environment and fermentation. 'Pwo rumen-cannulated heifers were maintained on a 25% corn silage plus 75% concentrate diet and supplemented with 0 or 75 ppm avoparcin in a reversal experiment. Quantities of rumen ingesta (47.1 vs 31.6 kg), dry matter (3.8 vs 2.8 kg) and liquid (43.3 vs l4 28.8 kg) were increased by avoparcin. However, slower rates (%V/h) of dry matter disappearence (7.6 vs 10.5) and of liquid turnover (4.8 vs 6.7) resulted in similar values of dry matter disappearence (6.92 vs 6.93 kg/day) and flow of liquid (49.8 vs 46.5 kg/day). Molar percent of both propionate and butyrate were increased by avoparcin, whereas acetate was decreased.‘Total VFA concentrations were not affected by the antibiotic. Higher rumen ammonia and lower alpha-amino nitrogen levels were also reported for the avoparcin-treated group. tg ttttg incubation of ingesta from the treated group generated less methane. When corrected for the larger ingesta volume, however, production was the same (Chalupa e_t_al. 1981). Froetschel gt gt. (1983) fed sheep either high or low fiber diets with or without addition of 50 ppm avoparcin. Compared to control, avoparcin decreased total VFA“ concentration (100 mM vs. 118 mM) and increased the molar percent of propionate (23.5% vs. 19.3%). Avoparcin significantly increased propionate production relative to control (2.07 vs. 1.68 moles/day). In the low fiber diet, avoparcin decreased ammonia concentration.tglgttg as well as amino acid degradation tg ytttg. As in the case of monensin, attempts have been made to relate the changes in final products of fermentation to the sensitivity of important rumen bacteria to the antibiotic. Stewart gt gt. (1983) tested the effect of avoparcin on pure and mixed cultures of rumen bacteria. Of the Gram negative species tested, all except g. succinogenes were 15 able to grow in the presence of 200 ug avoparcin/ml. Conversely, most Gram positive bacteria could not grow in the presence of 8 ug avoparcin/ml, even after adaptation. .Among the cellulolytic species, 3. flavefaciens was most vulnerable to growth inhibition by avoparcin. Incubation of rumen contents tg ytttg with the addition of 5 and 10 ug avoparcin/ml had virtually no effect on the digestion of dried grass and straw. It appears, therefore, that avoparcin is able to inhibit the growth of many Gram positive bacteria, with little undesirable effects on the Gram negative species (Stewart gt gt,, 1983). This might explain the similarity between avoparcin and monensin in terms of changes in final products of fermentation. Teichomxc in g; Teichomycin A2 is one of the major components of a complex of antibiotics produced by Actinoplanes teichomyceticus nov. sp. ATCC 31121. Properties such as antibacterial spectrum, absence of activity on bacteria lacking cell wall, and chemical structure qualify teichomycin A2 as a member of the glycopeptide class of antibiotics. This class also include vancomycin and the ristocetins (Bardone gt gt,, 1977). The main features distinguishing teichomycin A2 from the other glycopeptide antibiotics are the ocurrence of glycosamine as the basic sugar and the presence of aliphatic acid residues (Somma gt gt, 1984). l6 Teichomycin A2 is active tg vitro and tg_vivo against medically important Gram positive bacteria such as Streptococci, Staphylococci, and Diplococci. Its antimicrobial properties are derived from its ability to interfere with cell wall biosynthesis by inhibiting polymerization of peptidoglycan (Parenti gt gt., 1978). tg ytttg and tg ytyg studies with teichomycin A2 (Phillips and Tadman, 1980; Brondani, 1983) have shown that the antibiotic was able to alter the normal patterns of rumen fermentation. IMost importantly, those changes were of the same magnitude of those reported for avoparcin. Decreased methane production, shift in the molar proportion of volatile fatty acids towards propionate, and decrease in ammonia production were among the changes caused by teichomycin A2 in the rumen. ISOACIDS The branched-chain fatty acids (isobutyric acid, 2- methylbutyric acid, and isovaleric acid), and the straight chain 5-carbon valeric acid are important growth factors for certain cellulolytic bacteria (Bryant and Doetsch, 1955; Allison gt gt., 1962; Allison, 1969; Dehority gt gt., 1967) In the rumen, these acids are primarily derived from degradation of dietary proteins, but can also come from the recycling of bacterial protein (Annison, 1954; Pittnann and Bryant, 1964). Low availability of isoacids limits rumen l7 fermentation, especially with high roughage diets, or in situations of high feed intake and high energy demand as in lactation (Cook, 1985). E! ytgg studies have demonstrated that C4 and C5 VFA can positively affect growth rates (Lassiter gt gt., 1958; Felix gt gt,, 1980a), feed intake (Hemsley and Moir, 1963), nitrogen retention (Cline gt gt, 1966; Umunna gt gt., 1975; Felix, 1976), and milk production (Felix, 1976; Felix et al., 1980b; Papas gt gt., 1984). The mechanisms by which isoacids exert their positive effects on animal performance have not been completely elucidated. However, several changes have been observed in animals fed isoacids. These include increased microbial protein synthesis, increase dry matter digestibility, increased nitrogen retention, as well as changes in the plasma concentration of growth hormone and insulin. In this review, the possible mechanisms of action of isoacids in ruminants are addressed. .Aspects of the involvement of branched-chain VFA on amino acid synthesis by rumen bacteria .have been reviewed (Allison, 1969, 1970; Bryant, 1973: Felix, 1976). Sources gt branched-chain VFA tg the rumen The requirement of branched-chain VFA by rumen microorganisms is well established. Although recycling of bacterial protein in the rumen may produce some C4 and C5 acids (Annison, 1954; Miura gt gt., 1980 ), they are l8 primarily derived from the deamination of amino acids from dietary protein. Hydrolysis of dietary proteins by mixed rumen microorganisms is quite rapid. The rate of hydrolysis appears to be directly related to the degree of solubility of the protein (Smith, 1975). The intermediate products of protein hydrolysis, that is amino acids and peptides, remain in the medium for a short period of time after feeding, indicating rapid degradation into ammonia and carbon skeletons or rapid fixation into microbial cells (Bryant, 1973). Amino acid degradation in the rumen is accomplished primarily via oxidative decarboxylation. Final products include ammonia, carbon dioxide, and the carbon skeleton corresponding to the parent amino acid. The carbon skeletons can be further metabolized, giving rise to acetic, propionic, butyric, and valeric acids (Bryant, 1973). Additionally, isobutyric, isovaleric,and 2-methylbutyric acids (El-Shazly, 1952; Annison, 1954) can be derived from protein catabolism by mixed rumen bacteria. Final products of aromatic amino acid degradation include phenylacetic, phenylpropionic, and indole acetic acids (Scott gt gt” 1964). Proline undergoes reductive ring cleavage and deamination generating valeric acid (Dehority gt gt., 1958). The mechanism of synthesis of several amino acids by rumen microorganisms involve the reversal of the oxidative decarboxylation pathway previously described. Isobutyrate, 2-methylbutyrate, isovalerate, acetate, and phenylacetate 19 can be carboxylated into their corresponding keto acid and then aminated to produce valine, isoleucine, leucine, alanine, and phenylalanine, respectively. Although the majority of branched-chain VFA in the rumen originates from deamination of amino acids, alternative sources have been reported (Annison, 1954; Miura gt gt,, 1980). Several groups of bacteria are able to synthesize the carbon blocks required for amino acids synthesis. Precursors include intermediates of carbohydrate fermentation (mainly phosphoenolpyruvate), as well as fermentation end products, such as acetate, propionate, butyrate, valerate, and carbon dioxide (Otagaki gt gt., 1955; Kay and Hobson, 1963; Allison, 1969; 1970; Dehority, 1971). Lysis of these bacteria in the rumen can therefore provide extra sources of branched-chain VFA. Quantification of branched-chain VFA generated either from amino acids or from gg‘ggyg synthesis is difficult, because of the dynamic nature of protein metabolism in the rumen. However, the available information seems to indicate that the vast majoriy of the branched-chain VFA is in fact derived from dietary protein (Allison, 19 69; 1970) . Effects gt isoacids gg rumen microbial protein synthesis The main function of branched-chain fatty acids in the rumen is to serve as carbon skeletons for the synthesis of amino acids by the bacteria. In addition, they can be utilized in the biosynthesis of long-chain fatty acids and 20 aldehydes (Allison gt gt., 1961; Dehority gt gt., 1967). It would be expected, therefore, that isoacid supplementation would result in increased bacterial protein synthesis. This has in fact been confirmed. Russell and Sniffen (1983) studied the effect of isoacids on growth of mixed rumen bacteria mantained on different substrates. Supplementation of isobutyrate, 2-methylbutyrate, isovalerate, and valerate when bacteria were grown on mixed carbohydrates had little effect on synthesis of bacterial dry weight, DNA, RNA, or carbohydrates. When timothy hay was used, isovalerate and 2-methylbutyrate increased protein synthesis by 11.2 and 16.4 % respectively. Isobutyrate and valerate alone had no effect. Combination of the four acids increased bacterial protein synthesis by 18.7%. Responses to an inoculum of 60% concentrate and timothy hay were not significant. Russell (1984), incubated mixed rumen bacteria in artificial media with mixed carbohydrates as substrate to evaluate the effect of isoacids on bacterial protein synthesis. When all four acids were present at 2 mM each, bacterial protein synthesis was increased by 22%. Tests with individual acids indicated that only isovalerate and 2- methylbutyrate were effective. Hemsley and Moir (1963) tested the influence of isoacids on microbial protein synthesis in sheep. The addition of 0.56% of a mixture of isobutyrate, isovalerate, and n-valerate to a diet of milled oaten hay increased total rumen microbial protein by 70 percent. When sheep being fed low-protein teff hay were supplemented with urea plus 21 isoacids, total concentration of cellulolytic bacteria doubled relative to control, and feed intake increased significantly (Van Gylswyk, 1970). Hume (1970) fed sheep a low protein purified diet supplemented with a mixture of isobutyric acid (27.0%), 2- methylbutyric acid (26.3%), isovaleric acid (29.9%), and n- valeric acid (16.8%). Rumen microbial protein production was significantly higher in the treated group. Also, the amounts of total nitrogen and TCA-nitrogen flowing out of the rumen were greater in those sheep receiving the VFA mixture than in those on the basal diet. Similar results were reported by Kay and Phillipson (1964). Sheep receiving a poor quality hay and infused with a urea plus isoacids supplement showed a 10 to 15% increase in the flow of nitrogen to the duodenun. .A similar infusion of the straight-chain VFA had little effect on nitrogen flow rate. Effects gt isoacids gg dry matter digestibility There is an enormous amount of information indicating that the predominant rumen cellulolytic bacteria, as well as certain noncellulolytic bacteria, either require or are stimulated by branched-chain fatty acids (Bryant and Doetsch, 1955; Robinson and Allison, 1967; Allison, 1969, 1970; Slyter and Weaver, 1971). Allison gt gt. (1958) reported an absolute requirement of branched-chain VFA by several strains of Ruminococci. Bentley gt gt. (1954), 22 Dehority gt gt. (1957; 1967) and Allison and Bryant (1963) reported that isoacids are required for the growth of several rumen cellulolytic species, including strains of Ruminococci, Butyrivibrio and Bacteroides. These results coupled with the increase in bacterial protein synthesis discussed previously would suggest that isoacids would invariably cause improvement in dry matter digestibility. Studies conducted tg ytttg have in fact confirmed that prediction. However, tg ttyg results have not been as consistent. Burroughs gt gt. (1951) reported that cellulose digestion by mixed cultures of rumen bacteria occurs even when urea or ammonia are used as the sole nitrogen source. Bentley gt gt. (1955) reported that rumen microorganisms grown.under 12.21252 conditions had a higher rate of cellulose digestion and of urea nitrogen incorporation into protein when isoacids or their amino acid precursors were added to the medium. Soofi gt gt. (1982) reported an increase in digestibility of soybean stover when isoacids were supplemented to an artificial medium. Addition of starch to the medium had a depressing effect on digestibility, indicating either a negative action of starch on isoacid utilization or a higher utilization of isoacids and starch at the expense of fiber. Gorosito gt gt. (1985) incubated mixed ruminal bacteria in an artificial medium to which an equimolar mix (<.30 mM) of C4 and C5 acids was added. Cell wall digestion of both 23 isolated cell walls and of intact forages was increased by isobutyric, 2-methy1butyric, and isovaleric acids, but not by nrvaleric. An increase in cellulose digestion by lambs fed urea and isoacids has been reported by Cline gt gt. (1966). Hungate and Dyer (1966) reported that steers fed wheat straw and urea supplemented with valeric and isovaleric acid had higher intake, probably due to higher fiber digestion. Hefner gt gt. (1985) conducted extensive studies on isoacids supplementation of corn crop residue diets. tg gttg cotton thread disappearence was increased by isoacids. However, results were dependent on the source of dietary nitrogen. Lambs fed natural protein supplements had higher dry matter and fiber digestibilities than isoacids and urea supplements. These results give support to the findings of Hemsley and Moir (1963) and Hume (1970). It is worth pointing out that despite the lack of evident changes in digestibility, isoacids increased nitrogen retention and decreased urinary nitrogen loss in the aforementioned experiments. Effects gt isoacids gg nitrogen retention One of the most consistent effects observed when isoacids are fed to ruminants is increased nitrogen retention. Oltjen gt gt. (1971) fed steers either a protein-free or a protein-containing diet supplemented with 0.27% 2-methylbutyrate, 0.31% isovalerate, 0.25% 24 isobutyrate, and 0.20% phenylacetate. Sources of nitrogen were either urea or isolated soy protein. Isoacid supplementation resulted in higher nitrogen retention and lower urinary nitrogen loss. Most of these changes were found in steers fed isolated soy protein. Oltjen gt gt. (1970) reported similar results. Felix (1976) supplemented two mixtures of isoacids to lactating cows fed a corn silage plus concentrate diet. Percent of absorbed nitrogen retained was higher and loss of urinary nitrogen was lower in cows fed isoacids. No differences between the two isoacid mixtures were shown. 'Umuna gt gt. (1970) fed lambs a high roughage diet supplemented with urea plus isobutyric and isovaleric acids. Treated animals had higher nitrogen retention and lower urinary nitrogen loss. Neither rumen ammonia nor plasma urea were affected by isoacid supplementation. Similarly, Cline gt gt. (1966) reported a significant increase in nitrogen retention by lambs supplemented with.4.18.10) but increased propionate concentrations. Increases relative to control were 26.0% and 30.8% respectively for teichomycin A2 and.avoparcin (p<.10). There was a tendency for lower butyrate concentrations with glycopeptide supplementation, but differences were not significant (p>.10). Total VFA concentrations were not significantly changed by the treatments (p>.10). In general, concentrations of isoacids tended to be lower in teichomycin A2 and avoparcin treated animals. Teichomycin A2 decreased the concentrations of isobutyrate and isovalerate (p<.05), whereas avoparcin decreased isovalerate concentrations relative to control (p<.05). Consequently, total isoacids were lower in both teichomycin A2 and avoparcin groups (p<.10). The molar percent of acetate and butyrate tended to be lower for the glycopeptide-treated groups in the high roughage diet (Table 2). However, differences relative to control were not significant (p>4un. Propionate molar percent was increased by the glycopeptides relative to control (p<.05). .As a result, the acetate to propionate ratio was lower for the treated groups in the high roughage diet (p<.05). In the low roughage ration, the trends for VFA concentrations were similar to those in the high roughage ration. Propionate concentrations increased (p<.10) and butyrate concentrations decreased (p<.10) due to glycopeptide supplementation. Concentrations of acetate and total VFA were not affected by treatments (p>gun. The lower concentrations of isoacids caused by the glycopeptides 42 .cowuot 8 :_;owz act 8 :_ cams zoom we Lotto utmocmumm .Ao_.vavcm .Amo.vavuo utmcewu pawtomtmaam cossou 8 unoauwz caveat 8 swaps: so; 8 :_ memo: a .ceoe can mcomu8>tmmao emu 88.8 k8.8 88.8 8N.8 88.8 0.888 t88.8 888.8 .8.888.. .838» m... 8k.8 88.8 84.8 88.8 k8.8 8_.8_ 8~.8_ .838. 88.8 8_.8 _~.8 8_.8 88.8 8_.8 8P.8 N_.8 auatmpa> 88.8 m..8 81.8 8_.8 _8.8 8.8.8 888.8 88—.8 888.8.8>8m. 88.8 8_.8 e..8 8_.8 88.8 m_.8 ...8 8_.8 anarsu=8-z-m No.8 t_e.8 t88.8 8.8.8 8_.8 88.8 me.8 .8.8 . asmtspsm 88.8 e_.8 8_.8 _N.8 N8.8 888..8 8p_.8 88_.8 msmtsuzaoms m_.8 t8k.~ .88.N 88k.p NF.8 t.8.~ t8~.~ aek._ 8.888_a8aa 88.8 N..8 88.8 .8.8 em.8 _8.8 mk.8 88.8 asahau< 8288 8>e N<-8e _8rh=88 8:88 8>< ~<-8. F8t3e88 8,E8_r8> ommamzom 384 mmmgmzom mu“: 8 —u\mmpossv mauve bonanza; 38_ 8:8 58.; no» ammsm cw coast 48 m:o_a8ta:ou=8o =8 :Fotm 8>8 8:8 N< cpossosowmu to abooecu up mam<~ 43 in the high roughage ration were not detected in the low ' roughage ration. Teichomycin A2 and avoparcin increased propionate (p<.01) and decreased butyrate (p<.05) molar percent with respect to control. Acetate molar percent was slightly lower in the treated groups but differences were not significant. This ultimately resulted in lower acetate to propionate ratios in the treated groups than in the control group (p<.01). The effect of teichomycin A2 and avoparcin on the rates of production of acetate, propionate and butyrate in sheep fed high and low fiber diets are shown in Tables 3, 4, and 5, respectively. Since measurements for each acid were carried out on separate days, rumen fluid volumes and dilution rates corresponding to each acid are also provided. Pool fractional turnover time for each acid was derived by regressing the ln SA of the respective acid against time. Pool size was obtained by multiplying the rumen volume in liters by the average concentration of the acid on testing day. Daily production was obtained by dividing the pool size of the acid by its fractional turnover time (Cook, 1966; Knox et al, 1967; Froetschel gt gt,, 1983). Rumen volume and fractional turnover rate of rumen fluid were obtained by using the slope and intercept of the best fit line generated by regressing PEG concentrations against time (Bauman gt gt., 1969). The average correlation coefficients for the line of best fit for each acid and its corresponding PEG data were as follows: acetate, 0.969 and .=o_u8t 8 :_;u_3 38L 8 :_ c885 3888 $8 totem ct8uc8umw .Amo.vavem .Apo.v8vuo ”Lmecmv pawtumtmazm 885586 8 ozoaumz :o_a8t 8 =_;a_3 so; 8 =8 888828 .8885 can m=o_u8>emmao oM8 44 m~.o om.m om.m um.m m—.o em.m o.m mm.m owamc mu< m~.c m.~ N.N m._ ~—.o N.P ¢.~ m.~ 898sop8> N—.o m.~ m.— o._ m_.o m.o m.o ¢.~ mu8hmp8>omm mo.o m.~ m.p w.— mp.o m.p p._ c.~ mu8hxu=muza~ _m.o wm.u $0.0 mm.m m¢.o m.o «.8 m.m mumexuam op.o m.p o.N —.N ——.o m.— p.~ a.” opmexuznOmH mm.o o_.mm um.¢~ um.mp «c.o om.m~ oo.- um.m~ mu8comaoea mm.o m.~m ¢.Pm m.¢m mw.o N.mm m.oo o.mo wu8umo< mimm o>< N< N om8smzom so; 8m8gm=om zm_: a8mumwc 8m8zm=ot so_ to :88: new 888:8 cw <1> swear to :o_o:nwtum_c gamutma c8pos one :8 :Pue8ao>8 8:8 ~< cpoxsosuwmu co muomeem .N mam.10). Although acetate pools were not affected by the treatments (p>.10), the lower rate of acetate production due to avoparcin resulted in significantly higher acetate pool turnover time relative to control (p<.10). In the low roughage ration, trends were basically the same as those found in the high roughage ration. Acetate production and acetate pool sizes tended to be lower in the treated groups, but differences 46 .:o.88: 8 :.:u.: 28: 8 :. :882 :888 88 88::8 8:88:8888 ..o..vav :8.8.8 88.:omtmazm :8ssoo 8 888:8.3 :o.u8: 8 :.:u.3 38: 8 :. 8:88: 88 .:8oe :88 8:8.u8>:8888 88 88.8 ... 8.8 ..8 88.. 8... 8.8. .... .8\8 .8888 88.8888. 88.8 ..8. ..8. 8.8. 88.. 8.8 8.8 ..8 .8 .88.. 88>8888. .8.8 8.8 8.8 8.8 .8.8 8.8 8.8 ..8 8.8... .8e=.8> 8.8.. 88888 88.8 8..8 88.8 88.8 .8.8 8..8 .8.8 8..8 8.8.88.8: .88..8=8888 ..8 8.88. 8..8 8.88. 8.8 8..88. 88.8.. 88.8.. 8.: .88.. .888888. 88.8 88.8 .8.8 .8.8 88.8 88.8 88.8 88.8 88.88 .88.. .888 888aoo< 8288 8.8 88-8. .88.:88 8:88 888 88-8. .8.8888 8.88.88. :. 88.88.:8> 8.8.8 coast mm8am=om 388 88888888 88.: 8888.8 888gm=ot 38. 8:8 :8.: 888 888:8 8:8 :o.uo:8o:: 8888888 :8 :.o:888>8 8:8 ~< :.oaso:o.mu 88 888888“ .m mam<8 47 were not significant (p>.10). Propionate production rates were significantly increased by glycopeptide supplementation, in both rations (Table 6). In the high roughage diet, increases in propionate production relative to control were 48% (p<.05) and 38.7% (p<.05) respectively for teichomycin A2 and avoparcin. In the low-roughage ration, increases in propionate production due to teichomycin A2 and avoparcin were 34.8% (p<.10) and 36.8% (p<.10), respectively. The increase in propionate production resulted in higher pool sizes for both antibiotics in both rations. However, significant differences from control were detected only for avoparcin in the low roughage diet (p<.10). Butyrate production (Table 7) was not affected by treatments in either ration.(p>gun. The small numerical decrease in butyrate production measured for the treated groups in the high roughage ration was also detected in the butyrate pool sizes within that ration. However, differences were not significant (p>.10). In general, the changes in the rate of production were in good agreement with the changes in VFA concentrations. The only exception was butyrate, in which there was a trend for decrease in the concentration despite the lack of change in the production rates. The reasons for the results for butyrate are unclear. One possibility would be a change in rumen volume. ‘If the rate of production of butyrate is unchanged but the rumen volume is increased by the 48 .:8.u8t 8 :.:..3 :8: 8 :. :885 :888 .8 88:88 8.88:8.m8 88 .8885 :88 8:8.u8>:8888 88 ..8..v8v 88.8.8 .8.888t8888 :85588 8 888:..3 :8.b8: 8 :.:..z 38. 8 :. 8:882 8..8 8.8 8.. 8.8 .8.8 ..8 ..N. m.o. ::\a .8.8: :8>8::=. 88.8 8.8. 8.8. N.m. 8N.. ..o. 8.8 m.m a: .85.. L.8655. «8.8 8.8 m.m 8.8 8m.o ..8 ..m 8.8 8.8... .ms=.8> 8.8.. :8sam 8..8 om..~ omo.~ 888.. N..o cm... 888.. 88~.. 888\88.8s.:8.88=88:8 N... 88.8.. 88.8.. 8...~. m..a ~.m.. m.m~. m.mm. :.E .85.8 :8>8:::. mo.o 8.8.8 88...o 8m..o No.8 8..8 8..8 «..8 88.82 .8N.8 .888 888:8.88:8 8288 o>< N<-m. .88.:88 828m o>< ~<-u. .8gu:8u 8.88.:8> mm8zmsom 388 888:8888 :m.: 88.8.8 888:8:8: :8. 8:8 :8.: 88. 88888 c. 8.8: :8>8:t:p 8.8.. :85:: 8:8 .ms:.8> :8szt .:8.88:88:8 888:8.88L8 :8 :.8t888>8 8:8 ~< :.8858:8.8u .8 8888.88 .8 mam<~ 49 treatments, a tendency would exist for lower concentrations of butyrate in the rumen. Increases in rumen volume have in fact been reported for avoparcin (Froetschel gt gl,, 1983) and for monensin (Lamenager gt gt., 1978; Prange gt gt” 1978). However, such an effect would necessarily result in decreased concentrations for the other acids as well, and this was not observed in the present study. One of the main objectives of the present study was to determine if the changes in rumen VFA concentrations ' elicited by teichomycin A2 (Phillips and Tadman, 1980; Brondani, 1983) and by avoparcin (Johnson et al., 1979: Chalupa gt gt., 1981; Froetschel et al., 1983) could be explained on the basis of changes in production rates. Changes in VFA found in the present study followed similar trends for both glycopeptides. In general, supplementation with teichomycin A2 and avoparcin resulted in significant increases in propionate concentrations and production rates. These results are in line with previous reports for teichomycin A2 (Phillips and Tadman, 1980; Brondani, 1983) and for avoparcin (Johnson gt gl,, 1979; Chalupa gt gt” 1981: Froetschel gt gt., 1983; Lindsey e_t gt., 1985). Acetate and butyrate, on the other hand, failed to show significant changes despite the trends toward lower concentrations and production rates. This is in sharp contrast with results of rumen metabolism studies in both sheep (Froetschel gt gl,, 1983) and cattle (Chalupa gt gl,, 1981). Froetschel gt gt. (1983) reported higher propionate but lower acetate, butyrate, and total VFA concentrations in 50 ..o..A8v 88.888 8888.8 8. 8888...:8.m 88: 888 888288888 .8 8888..8 .88.888 8 8.88.: 388 8588 888 c. 8885 8888 .8 88888 888888888 8 .888e 888 888.88>88m88 88 ...o 8.8 8.8 8.8 88.8 8.8. 8.8. 8.8. 8m.o 8.8 ..8 8.8 88.8 888.8 888.0 888.0 m... 8.88. 8.8.8 «.8.8 888.0 888.0 ..o.o .mo.o 88.8 .88.o 888.8 888.8 808.8 888.8 880.8 .mo.o .8.8 8.8. 8... ..8. 88\8 .8888 88>8c88. 8... 8.8 8.8 ..8 88 .85.» 88>8888. 88.8 ..8 8.8 8.8 m88... .8e:.8> 8.3.. 888:8 888\m8.8e .88.88=8888 ..m. 8.88. 8.88. 8.88. 8.5 .85.» 88>8888. 88.85 .8~.m .888 88888888 8:88 o>< 88-8. .888888 88888888 388 8:88 o>< ~ 88888888 88.: 88888.8 88888888 :8. 888 88.8 88. 88888 8. 8888 88>88888 8.8.. 88288 888 .8e:.8> 885:8 .88.88=8888 88888888 88 8.88888>8 8:8 88 8.885888.88 .8 8888..8 .8 8888. 5] sheep fed high and low fiber diets supplemented with avoparcin. Chalupa gt gt. (1981) reported that avoparcin supplementation to cattle increased propionate and butyrate and decreased acetate. Total VFA concentrations were not affected by the antibiotic. The changes in VFA patterns found in the present study, however, are in line with those obtained in feedlot trials (Johnson gt gl,, 1979; Dyer gt gt., 1980). Under those conditions, avoparcin consistently increased propionate without affecting significantly acetate or butyrate concentrations. The reasons for such discrepancies are unclear. Evidently the conditions under which each experiment was carried out may have affected the results. Differences in diet composition, time of exposure to the antibiotic, and type of animal all must have contributed to the differences. However, based on the mode of action of glycopeptides on rumen microorganisms (Stewart gt gt” 1383) it would be expected that such differences for acetate and butyrate would be in magnitude rather than in trend" One point to be considered is the time required for the patterns of rumen VFA to return to baseline values when feeding of antibiotics is dicontinued. While return to baseline following withdrawal seems to be almost instantaneous when ionophores are fed (W.G. Bergen, personal communication), the effects of glycopeptides seem to be more persistent (Phillips and Tadman, 1980; Brondani, 1983). In the present study, the persistency of glycopeptide effects on rumen VFA following 52 withdrawal were checked. Ten days after glycopeptide supplementation had been discontinued, rumen VFA were measured in animals fed the high roughage diet. Molar percent distribution of rumen VFA in animals previously belonging to control, teichomycin A2 and avoparcin groups were as follows: acetate, 69.2, 64.0, and 66.4 (p>.10); propionate 18.9, 23.7, and 22.5 (p<.10); butyrate, 7.9, 7.3, and 7.1 (p>.1oy. These results question the validity of using experimental designs such as latin squares or reversal designs to study the effects of glycopeptides on rumen metabolism. If these designs are to be used, it is absolutely crucial that long adaptation periods to the new treatment be observed to avoid carryover of residual effects from one period to the next. It is possible, therefore, that occurrence of carryover could explain the differences in results between the present study and those of Froetschel gt gt.(l983). In the study by Froetschel gt g;.(1983), the design used was a 4x4 latin square, with sheep and period serving as blocking criteria. Despite the fact that each treatment period lasted for 28 days, measurements started on the 10th day of each period. This might have affected the VFA concentration results. In the same study (Froetschel gt_g;,, 1983), propionate production rates were measured at the end of each period (days 21 and 25). The authors reported an increase in propionate production due to avoparcin. This is in line with the results of the present study. 53 Table 6 summarizes the effects of teichomycin A2 and avoparcin on rumen nitrogen constituents in sheep fed high and low fiber diets. Soluble protein, bacterial protein, and protozoa protein fractions were not affected by the treatments (p>dun. Values for the protozoal protein fraction were markedly different between the two rations. Mean protozoal protein concentrations were 66.2 and 250.1 mg/dl respectively for high and low roughage diets. Several factors may explain this difference (Brondani, 1983). The first possibility involves the rumen fluid fractionation process. Since the protozoa remain primarily associated with coarse feed particles, a possibility exists that most of the protozoa would be discarded with the feed residue. The resulting smaller pellet would therefore underestimate the protozoa protein fraction for the high roughage ration. The second possibility is related to the fact that the proportion of protozoa to bacteria in the rumen fluid is a function of the composition of the diet (Coleman, 1975). Eadie £5.2L-(1970) reported a protozoa to bacteria ratio of 4:1, on a volume basis, for animals on a high grain ration. In animals receiving a high roughage diet, however, this ratio was only 1:1. In the present study, differences in protozoal protein concentration between the two diets are in good agreement with the four-fold difference in protozoa numbers favoring the low roughage diet reported by Eadie et a1. (1970). A third possibility is related to the mechanism of protein degradation by protozoa. In these organisms, 54 .88.888 8 8.88.3 388 8 8. 8888 8888 .8 88888 88888888 . ..8o.v8. 88...8 88.88888888 888588 8 88888.3 88.888 8 8.88.3 388 8 8. 8888288 ..88.v8. 88...8 88.88888888 888888 8 88888.3 88.888 8 8.88.3 388 8 8. 88882 88 .8888 888 888.88>88m88 888 88.. 888... 8.8.8. 888... m... 88.... 88.8.8. 888.8. 2-8.88888 88.8 8.... 8.8.8 8m..m 88.8 U.8.8 888.8 888.8 2-88.88-888.< 88.8. N...8N 88.888 8..888 ...8 88.88 8..8. 88..8 8.88888 .8o~o.o88 88.. 8..88 88.88 «..88 8..8 88..8 8.88 88.88 8.8.888 .8.888888 .... 8..88 88.88 88.88 8..8 88.88 ..8. 8..88 8.88888 8.83.88 .888 8.8 88-8. .888888 .888 8.8 88-8. .88.:88 8.88.88. 88888888 388 88888888 88.: 88.8 88. 88888 8. 888888.88888 888888.: 8..8\8sv 888.8 88888888 38. 888 88888 88 8.88888>8 888 88 8.888888.88 .8 8888..8 .8 8888. 55 protein degradation takes place within the cell, requiring that small feed particles be absorbed before protein degradation can occur (Coleman, 1975). Since the particle size of the low roughage ration was much smaller it is possible that the protozoal protein fraction from animals on that diet included both protozoal and feed protein. The alpha-amino-N and ammonia-N fractions were significantly affected by glycopeptide supplementation in both diets. In the high roughage ration, increases in alpha-amino-N relative to control were 81.6% (p<.05) and 118% (p<.05) respectively for teichomycin A2 and avoparcin. In the low roughage diet, teichomycin A2 and avoparcin increased alpha-amino-N concentrations by 50.1% QxLOS) and 48.6% (p<.05), respectively. Ammonia-N in the high roughage diet was decreased by 10% (p>.05) and 23.8% (p<.05) by teichomycin A2 and avoparcin, respectively. In the low roughage diet, decreases in ammonia-N due to teichomycin A2 and avoparcin were 33.2% and 22.7%, respectively (Table 6). The overall results for ammonia-N and alpha-amino-N indicate that teichomycin A2 and avoparcin exert a marked effect in the nitrogen metabolism in the rumen. These results are in good agreement with in yigrg results for teichomycin A2 (Brondani, 1983) and in 1119 results for avoparcin (Chalupa gt al,, 1981; Froetschel at al,, 1983). The decrease in ammonia-N concentration concomitantly with the increase in the alpha-amino nitrogen suggest that teichomycin A2 and avoparcin decrease deamination. 56 Additional evidence is provided by the decline in the concentration of isoacids in treated animals (Table 1). Although some rumen bacterial species are able to synthesize carbon skeletons g3 ggyg (Bryant and Doestch, 1955; Miura et alq 1380), the majority of isoacids in the rumen come from the degradation of dietary protein. Therefore, the decline in concentration of isoacids suggests that amino acid breakdown was depressed by the glycopeptides. The mechanism by which this is accomplished is not clear. Because utilization of most amino acids occurs intracellularly, it has been proposed that prevention of transport into bacterial cells might be the process involved (Stuart e3 al., 1977). This mechanism has in fact been associated with the action of diaryliodonium chemicals, which also suppress ruminal degradation of amino acids (Chalupa, 1976; Broderick and Balthrop, 1979). However, because of the mode of action of glycopeptides on bacterial cells, a mechanism involving a selective effect against specific microbial species (Stewart g5 al,, 1983) would appear more plausible. Results of this study have confirmed in 23533 results indicating the striking similarity between avoparcin and teichomycin A2 in their ability to alter the metabolism of carbon and nitrogen in the rumen. Both compounds increase propionate production but do not have a major effect on acetate and butyrate production rates. Additionally, both glycopeptides increase alpha-amino-N and decrease ammonia-N concentrations in rumen fluid, suggesting a depressing 57 effect of these compounds on protein and/or amino acid degradation in the rumen. Effects of glycopeptides were more pronounced in the high roughage ration. IV. Effects of isoacids, urea, and sulfur on rumen fermentation in sheep fed high fiber diets. Introduction The ruminal fermentation is a coupled process between carbohydrate degradation and microbial cell synthesis (Bergen, 1979). .Ammonia-N and other factors such as carbon skeletons and sulfur are required for this process. A deficiency in any of these substrates will decrease the efficiency of microbial growth and consequently reduce the availability of volatile fatty acids and microbial protein to the host animal (Bergen and Yokoyama, 1977). Therefore, a major concern in ruminant nutrition is to define the nutrients required by rumen microorganisms for maximum fermentation of feedstuffs, particularly of low protein, highly fibrous plant material (Cook, 1985). There is a considerable amount of information in the literature indicating the advantages of supplementing isoacids, urea, and sulfur to ruminants (for reviews, see Bray and Till, 1975; Huber and Kung, 1981; Orskov, 1982; Cook, 1985). However, very little information is available concerning the interaction among those supplements, particularly when high fiber diets are fed. The present study was designed to evaluate the effects isoacids, urea, and su fur, fed alone or in combination, on the rate of rumen fermentation in sheep fed high fiber diets. By varying the level of each supplement in the diet, it was possible to obtain different concentrations of 58 59 isoacids, ammonia-N, and hydrogen sulfide in the rumen fluid. The rate of acetate production, measured by a radioisotope technique, was taken as a measure of the rate of rumen fermentation (Cook, 1966; Davis, 1967; Rogers and Davis, 1981). The changes in acetate production rates as affected by the interaction between the three main factors are discussed. Materials and methods Two trials were carried out. The experimental design for both trials consisted of a 2x2x2 factorial crossover conducted in two 4x4 quasi-latin squares (Gill, 1978b). Double blocking criteria were animals and time (non-random repeated measurements were obtained from each subject assigned to a sequence of treatment combinations). In each trial, 8 rumen-cannulated adult sheep were used. .Animals were divided in two groups of four animals according to body weight, and randomly assigned to rows of one square, corresponding to a predetermined sequence of tratment combinations (Gill, 1978b). Composition of the basal diets for trials 1 and 2 is given in Table 7. In each trial, eight different rations were prepared from the basal diet, to provide combinations of isoacids, nitrogen, and sulfur, each at two levels. 60 TABLE 7. Composition and analysis of basal dietsa Ingredient, % Corn stover (IFN 1-02-776) ---- 25.0 Corn cobs (IFN 1-02—782) ---- 25.0 Sugarcane bagasse (IFN 1-04-686) 44.0 ---- Sorghum grain (IFN 4-08-139) 55.0 49.0 Bone meal (IFN 6-00-400) 0.5 0.5 Trace mineral salt 0.5 0.5 AnalysisC Crude protein, % 7.22 7.71 Crude fiber, % 22.0 18.2 Digestible energy, Meal/kg 2.57 2.89 Total nitrogen, % 1.155 1.233 Total sulphur, % 0.144 0.181 N/S 8.0 6.81 gDry basis. TMS containing a guaranteed minimun of: 0.35% Zn, 0.20% Mn, 0.20% Fe, 0.03% Cu, 0.005% Co, 0.007% I, and 96% NaCl. CCalculated NRC values. Based on results of previous experiments (Felix, 1976: Quispe-Salas, 1982), isoacids were administered at 0.1 (1501) and 0.2 (1802) g/kg bw/day. In order to achieve two levels of ammonia in the rumen (about 5 and 15 mg/dl) , the basal ration was fed either alone or supplemented with urea at.1.5% of the dry matter. Sulfur supplementation was designed to provide four different nitrogen to sulfur ratios (Bray and Hemsley, 1969, Bray and Till, 1975; Orskov, 1982). Elemental sulfur at 0.2% of the dry matter was used as supplement. Final N:S ratios were aproximately 3:1, 5:1, 8:1, and 12:1 (Quispe-Salas, 1982). Isoacids, urea and sulfur were first pre-mixed with part of the sorghun and 61 then incorporated into the totally mixed ration. Daily ration was offered at 0700 hours and feed intake was recorded daily for each animal. Water was provided ad libitum. Animals were adapted to a given diet for 14 days prior to measurements. In yiyg production rates of acetate were measured by a single injection radioisotope technique. On day 15, three hours after the morning feeding, each animal received an intraruminal injection of 100 uCi of Na-[l-C14Jacetate, along with 100 ml of a 10% polyethylene glycol solution (PEG, M.W. 4000, Sigma Chemical Company). To ensure a more homogeneous distribution, solutions were infused at several different locations in the rumen. This was performed with the aid of a dosing syringe attached to a perforated plastic tube (15 mm dia.x 30.5 cm long). A similar device was used for collection of rumen fluid samples. Following infusion, rumen fluid samples were collected every 20 minutes for the next three hours. Samples were strained through four layers of surgical gauze, immediately acidified with 50% sulfuric acid, frozen at -20°C and stored for subsequent analyses. Concentrations of hydrogen sulfide in the rumen were determined as described by Quispe-Salas (1982). Procedures for the determination of acetate production, rumen fluid dilution rates, rumen ammonia-N and concentrations of rumen VFA were described in chapter III. Overall significance of treatment effects was determined by analysis of variance (Gill, 1978a; 1978b; 1978c) according to the model in (II): (II) Yijklmn'= + Cn + (AC) 1n + (BC) Inn 4' (ABC)lmn + E(ijklmn) where, Yijklmn (AB)lm (AC)1n (Bc)mn (ABC)1mn 62 is the observed value for the jth sheep within the ith square under the 1th level of urea, the mth level of isoacids, and the nth level of sulfur during the kth period: is the overall mean; is the fixed effect of the ith square: is the random effect of the jth sheep within the ith square; is the fixed effect of the kth period within the ith square; is the fixed effect of the 1th level of urea; is the fixed effect of the mth level of isoacids; is the fixed effect of the nth level of sulfur; is the fixed effect of the interaction between level of urea and level of isoacids; is the fixed effect of the interaction between level of urea and level of sulfur; is the fixed effect of the interaction between level of isoacids and level of sulfur: is the fixed effect of the interaction among level of urea, level of isoacids, and level of sulfur; in this case, non- separable from the effect of squares; E(ijklmn) is the random residual error. Specific differences between treatment means within each two-way interaction were determined by Bonferroni-t tests (Gill, 1978a; 1978b). 63 Results and discussion The objective of these studies was to evaluate the effects of isoacids, urea, and sulfur on rumen fermentation in sheep fed high fiber diets. Results are presented according to the effect of the three two-way interactions (Gill, 1978b), ime, the interactions between urea and sulfur, urea and isoacids, and between isoacids and sulfur. Rumen volumes and rumen fluid turnover rates were not affected by treatments in either trial. Mean rumen fluid volumes averaged across treatments and respective standard errors were 3.2 (0.29) and 3.8 (0.26) liters, respectively for trials 1 and.2. Mean rumen fluid turnover rates for trials 1 and 2 were 13.1 (0.74) and 11.8 (0.81) percent/houry respectively. The effects of urea and sulfur on feed intake, rumen ammonia-N, rumen sulfide-S and acetate production rates are presented in Table 8. Average daily dry matter intake did not differ among treatment groups in either trial (p>JUD. Addition of urea increased the concentrations of rumen ammonia-N in both trials (ptmmno eon .cmms Log meowum>gmmno we FN.o ac.m e_m.N www.m mem.~ eN.o e_.m em~.N eee._ em_.m see\me_os .=o_eueeore e t ~.m e.,e_ m.ee_ ~..mp e.me_ ..8 N.ee_ N.ee_ N._e_ e.em_ e_s .eewp te>octee mo.o mm.o 8N.o em.o km.o No.o _m.o mm.o NN.o e~.o mmpoe .QNem .ooa mmomumo< m~.o _m.e c~.m em.m Ne.m mm.o _m.m em.m ep.N me.~ eem-me_e_em _~.o _p.m_ mm.e m_.e_ m~.m em.o __.m_ e..m e_.N_ __.m uaz-e_=oaa< ---- N.e N.m N.o_ o.“ ---- o.m _.m N.~_ _.m m\z __.o ow._ mm._ ek.o km.o mP.o 88.” me.. me.o mm.o eesee\e .mxee=_ tee_=m mm.o om.m mm.m we.“ ee.m ee.o mm.m __.m mN.m Ne.e unsee\e .mxmac_ cmmoguwz e.e_ Nee Pee ~_e one m.m_ Nee Nee eme “mm esee\e .mxoucw coupes syn Ame Nev Ame Rev eZNW m + a tet_em eat: _Otecou exam m + = tetpem eel: _OLpeou e_ee_te> N Peete he levee . weave tenet saw; no» ammgm :_ mopnawtm> coeumucosgmw cuss; new excuse come :0 gzwpzm new not: yo muomwem .m mom.10). When the two factors were fed in combination, acetate production rates relative to the other groups increased (p<.10). While the lack of response in the sulfur group can be explained on the basis of the low level of rumen ammonia—N (Table:8), the results in the urea group were somewhat unexpected. The nitrogen to sulfur ratio (Table 8) and the percentage of sulfur in the basal diets (Table 7) were within the values commonly recommended for adult sheep (Bray and Hemsley, 1969; Bray and Till, 1975; Orskov, 1982). It seems that the total sulfur intake for the animals in the urea group may have been limiting. Hume and Bird (1969) reported that a total sulphur intake of 1.95 g/day supported maximum rumen microbial protein synthesis in sheep. In the present study, intake of total sulfur in the urea group averaged 0.65 g/day and 0:74 g/day, respectively for trials 1 and 2. For the Urea + Sulfur group, however, average daily intake of total sulfur for trials 1 and 2 were 1-66 and 1.80, respectively. Apparently the sulfide-S concentrations elicited by the low sulfur intake were limiting. The precise level at which rumen sulfide concentration limits microbial growth or fermentation has 66 not been clearly defined (Bray and Till, 1975), but a limiting level of 1 ug sulfide-S/ml of rumen fluid has been suggested (Harrison and McAllan, 1980). In the present study, sulfide-S concentrations in the urea group were at least twice that value, but evidently they were not high enough to allow for maximun fermentation. These results clearly indicate that sulfur supplementation based solely on the nitrogen to sulfur ratio or on the percentage of sulfur in dry matter is not adequate when high fiber diets containing urea are fed. The total amount of sulfur that would allow for maximum microbial protein synthesis should be considered first. Once that is provided, supplementing urea to attain a N/S ratio of about 10 (Orskov, 1981) should result in maximun fermentation efficiency. Effects of urea and isoacids on feed intake, ammonia-N and total isoacid concentrations, and on the rate of acetate production are shown in Table 9. Concentrations of ammonia-N were significantly increased by the addition of urea in trial 1 (p<.01) and trial 2 (p<.01). Similarly, increasing the level of isoacids in the diet resulted in higher concentration of these acids in the rumen in both trials (p<.01). In trial 1, the rate of acetate production was higher when urea was supplemented along with isoacids. Increasing the amount of supplemental isoacids in the diet (I801 vs I802) did not affect the rate of acetate production (p>JUD. However, urea supplementation to the low level of isoacids 67 ._m_tu m :_;u_3 3o; 8 c_ some game we gotta cgmucmum; .Amo.vav gmew_u “e_tomtmasm cossoo m aaoza_3 _mwto m :_;u_z so; a c_ memomem .Apo.vav mueomome to acmeem acmo_e_:m_mu .Aeo.vav not: we pummem acmo_w_:m_mo .:665 Log mcoeam>gmmno eon .2665 Log m=o_um>gmmno mm _N.o mm.m mmw.~ mmm.m mem.w up.o mem.m mmm.~ cm.~ mpm.P xmc\mopos .comuozuogm e e mm.~ o.om_ e.mm_ ~.mme w.mmp N_.m ~.e._ m.mmp m.mm_ a.~m_ cps .oseu em>oct=h mo.o mm.o mN.o AN.o eN.o Po.o UP36 mm_.o me~.o mmp.o mmeoe .mN_m _ooa mmumumo< No.o me.o me.o m~.o c~.o mo.o mm.o me.o mN.o mm.o camvwooome peach _N.o .N.mp em.“ _P.m_ Pm.m em.o om.e_ ep.~ —.m_ mm.m. unzumwcoss< m.ep wee eke ome Pme ~.m_ N—e mme ewe ewm mamc\m .mxmu=_ gouaas sea szmm anmomH NemH zupomH FomH czmm :-~om~ ~0m_ :upomH _omH menowtm> m Paste P Pm_gh mee_e tenet new; eat ammcm cw mopam_tm> :omumucmsgmw cuss; co new excuse tome :c not: can mcwomomm eo muoowwm .m mom<~ “Us I C. 1' n‘b &L L» 68 (ISOl-U) resulted in 49% increase in the rate of acetate production relative to 1801 (p<.05). When both factors were present in the higher level (ISOZ-U), acetate production was increased by 30% relative to the ISOl-U group (p<.05). This increase in acetate production rate was responsible for the increase in both concentration (Table 10) and pool size (Table 9) of acetate in the ISOZ-U group. These results clearly illustrate the fact that rates of fermentation in the rumen can be only as high as the availability of the most limiting nutrient. In trial 1, ammonia-N provided by the basal diet was more limiting than isoacids. Once nitrogen supply was corrected by the addition of urea to the diet, availability of carbon skeletons for amino acid synthesis became limiting. Evidence for this assertion is given by the further increase in acetate production in the 1502-0 group relative to the ISOl-U group. Trends in acetate production found in trial 2 were basicaly the same as in trial 1. However, significant increases in acetate production were found only in the ISOZ‘U treatment.(p<.05). .Apparently the levels of ammonia-N provided by the basal diet in trial 2 (Table ) was sufficient to support microbial growth as efficiently as the one elicited by urea (ISOl-U) supplementation (Huber and Kung, 1981; Orskov, 1982). It was not sufficiently high, however, to allow the utilization of the higher amounts of isoacids supplied by the 1502 treatment. The increases in the rate of acetate production were accompanied by changes in concentrations of individual VFA 69 .Powtu 8 :p:u_3 so; 8 :8 came 5888 we Lotto ugmuc8amm .A_o.vav 8888888_ 88 uomeem 8:8088_888mn 48.x: 6:28 88.788.88.838 89.58 8 8:55? 28.75 8 8.85.; 38.8 8 5 8:88:88 .8885 Log m:o_u8>tmmno 8mm 88.8 8e.8 8e.8 88.8 88.8 88.8 88.8 8e.8 88.8 88.8 888_8888_ _888t .e.8 888.8 8888.8 88e8.8 888.8 e8.8 88e.8 8e8.8 8e8.8 888.8 81> .8888 888.8 e..8 8..8 88.8 88.8 888.8 8_.8 8..8 88.8 88.8 888888.88 888.8 8_.8 8..8 88.8 88.8 888.8 8_.8 ._.8 88.8 88.8 888888_8>888 e88.8 8_.8 88.8 88.8 88.8 888.8 88.8 8..8 88.8 88.8 888888888-z-8 88.8 e8.8 88.8 88.8 _8.8 e8.8 .8.8 ee.8 8e.8 8e.8 88888888 888.8 e_.8 8..8 88.8 88.8 888.8 8..8 e_.8 88.8 88.8 888888888888 __.8 _8._ _8._ 88.. .e._ 8..8 88.. 88._ 88.. 8_._ 88888.8888 .8.8 888.8 8888.8 8888.8 88e.8 88.8 888.8 888.e 88e.8 U_8.e 888888< 8288 8.888. 8888 8-_88_ .888 8288 8-888. 8888 8-_888 .888 8_88_88> 8 .8_8t _ _8_8e 8A_u\mm_ossv mumwu 88888 ;m_; 888 88858 :8 88808 zuumm 8—888_8> 885:; to 8:8_88tu=mo=ou :8 888: 8:8 mueumom_ mo muummeu .o_ m4m888ao vw8 e..8 e.e 8.e 8.8 8.8 8..8 e.e 8.8 8.e 8.8 8.88. 8.8 e8.8 8.. 8.. 8.8 8.. 88.8 8.8 ..8 8.. 8.. 888888.88 88.8 ... 8.. 8.8 8.8 e8.8 8.. 8.. 8.8 8.. 888888.8>88. 88.8 ... 8.. 8.8 8.8 88.8 8.. 8.. ..8 8.. 888888888-2-8 8..8 8.8 8.8 8.8 ..8 8..8 e.8 8.8 8.8 8.8 888.8888 88.8 8.. 8.. ... 8.8 88.8 8.. ...8 8.. 8.. 88888888888. 88.8 88.8. 88... 88.8. 88.8. 88.8 8e.8. 88... 88.8. 88.8. 8.888.888. .e.8 88.8. 88.8. 88... 8... .8.8 88.8. 8..88 888.8. 88.8. 8888888 .288 8-888. 888. 8-.88. .88. .288 8-888. 888. 8-.88. .88. 8.88.888 8 .8.8. . .8... 8888.8 888.. 88.8 88. 88888 8. 88.88 88.8. 8..88.o> c8588 .8 :88888..u8.u 8888888 88.85 8:8 :8 888: 8:8 88.8888. we 88888$m ... m4mwumHmu who 88:.8> .888.8 282 88.: .88888 :88: 888 @8828 :8 coach co moflomomm mo muowumm to . F) .8 musmflm 88.208 NM. 0 N0 . WW \\\\\\\\\\\\\\ \ \\\\\\\\\\\\\ WW Io; .ION .100 ..OQ .-0m .-00 .-Ob .500 .-00 ..OO— 103. ..ONp ..Oflp ..Ofip 00p 72 /////////////////// k\\\\\\\\\\\\\\\V /////////W W W 7W \\\\\\\\\\\\\\\\\\\\. W 1 ”T 1 I r 1* T I I r 1’ 1 O <3 (3 C3 C3 CI 0 ‘O ‘O O ‘O O O ‘O OOOOOOOOOOOOO pppp TOD“. C4 [m ISOACIDS IZZI comam. 2. Effects of isoac P shee ed protein. Figure ntaini fed diets co ng preform ntrations w ntrol (control=100). ith Values are ct to co 73 in the rumen (Table 10). The overall increase in fermentation in the ISOZ—U group in both trials resulted in higher concentrations for both acetate and propionate. On a molar percent basis, however, this increase favored acetate (Table 11). These results plus the results reported in Chapter V (Table 13) indicate that the effects of isoacids on VFA production has two components. In diets low in preformed protein, where the contribution by the diet to the ruminal isoacid pool is small, addition of isoacids will result in an increase in the production of all major VFA and consequently of total VFA (Table 10). In this situation, changes in the acetate to propionate ratios are less pronounced (Table 11). Similar results have been obtained in yiyg_(Quispe-Salas, 1982) and in yitrg (Machado gt 3;” 1985). When preformed protein is fed, on the other hand, the increases in total VFA due to isoacid supplementation are usually small (Hume, 1970; Oltjen et al, 1971: Felix, 1976). However, because isoacids select for cellulolytic species of bacteria (Van Gylswyk, 1969; Oltjen gt a1” 1971), addition of these acids to the diet would tend to divert the flux of carbon towards acetate production, decreasing production of propionate. This is evident from the results shown in Table 13. Addition of isoacids increased acetate and decreased propionate but did not change significantly the concentrations of total VFA in sheep fed an all-alfalfa diet. Similar shifts have been reported when isoacids were added to diets with low fiber ..8... 8 8.08.: 38. 8 c. 0885 .888 .8 .8..8 0.8008888 ...0.v0. 80.8888. .8 .88..8 8088...:0.m0 ...0.vav .8..=8 .8 888..8 8088...:0.m8 .0885 .80 8:8..8>.88no 00 n .0885 .80 8:8..8>.88ao 08 74 88.0 00.0 ...m 00.8 80.8 0..0 m..~ 00.8 .~.N me.~ .80\88.os .co..8:0o.¢ N..w N.0N. ..wm. 0..m. m..m. ...0 0.0m. 0.0m. 0.0.. ..mm. 0.5.85.“ .8>o:.:. 80.0 08.0 00.0 .8.0 e~.0 80.0 08.0 mm.0 0..0 .~.0 88.85.8~.8 .888 88.8.88< 80.0 88.0 88.0 88.0 mm.0 00.0 88.0 0m.0 .N.0 .m.0 0080.8888. .8.8. mm.0 .m.0 00.e no.8 08.m 88.0 .m.0 0..m 80.. m..e 888-80...:m 8.0. eme 808 088 8.8 8.0. 088 0.e ..e eme 8880\0 .8.8.8. .8.885 ..0 8288 8.808. 808. m-.0m. .08. 8288 8-808. ~08. m-.0m. .08. 8.88..8> N .8... . . .8... 8.8.8 .88.. 88.8 88. 08828 :. 88.08..8> 08.88.085.8. 085:. :0 008 8.88:. 088. :o .8..:8 0:8 80.8888. .8 8.88..“ .N. m.0<. 75 ..88Hu8-888.. 88.888088. 88 8888. 8>.»8.8. 8.8 88:.8> ... .8.880 888.0 .88.. 88.: 88. 88808 a. .m + 8. .28.:m + 88.: no ..DumomH. 88.: + mcfiomom. ..msmomH. “28.5m + mowomom. >8 Gmuomuum m8 mmumu :ofluoscoum mumumo¢ .m 8.36.. DINOm. mINOw. . \\\\\\\\\‘ 008 \\\\\\\\\\\‘g Opp &\\\\\\\\\\\\\\‘ fin— no; ..Ou ..On ..0* .uon ..Om ..Oh ,sOO .uoo ..OOp to;— ..ON. ..Onp ..Otp 1.008 ..Oop Oh. 76 ..88Hu8u888HV :o.uosco.8 .8 8888. 8>.88.8. mum mm:.8> ..8 .mfluev mum.0 umnwu 30.: 00. @8828 c. .m + D. h:u.:m + 80.: yo ..Dlmomm. 88.: + 80.0808. ..mlmomH. us..:m + 80.08088 >3 cmuomuum m8 mmumu sawuozcoum mumumo< w+D DINOm. MINOm. - . .8 mhsmfim \\\\\\\V 10' ION ran 10* low 100 ..Oh ..00 10G §\\\\\\\\\\\\\\ \\ @N— \. §\\\\\\\\\\\\\\‘ 1 00— I088 ION. Ton. 1O¢p loop 100.. th 77 (Oltjen gt al,, 1971; Felix, 1976) and high fiber (Hume, 1970). The overall effects of isoacids on rumen VFA as related to the source of dietary nitrogen is summarized in Figure 1 (NPN) and Figure 2 (preformed protein). The possible implications of the shifts in rumen VFA with respect to the intermediary metabolism of the host animal are adressed in Chapter V. Effects of isoacids and sulfur on feed intake, total isoacids, sulfide-S concentrations and rate of acetate production are summarized in Table 12. Feed intake was not affected by treatments in either trial (p>.10y. Increasing the level of isoacids in the diet resulted in higher concentrations of these acids in the rumen.(p<.01y. Similarly, sulfide-S was higher in the treatment groups receiving elemental sulfur in the diet (p.10). On the average, therefore, providing supplemental sulfur to diets containing two levels of isoacids did not improve feed utilization relative to isoacids fed alone. It should be noted, however, that acetate production values in the 1802-8 group (Table 12) were lower than those obtained with the ISOZ-U group (Table 9) and Urea+Sulfur group (Table 8) in both trials. This suggests that in the 1802-3 group fermentation was being limited by the level of ammonia-N in the rumen. .A summary of these results is presented in Figures 3 and 4. As pointed out by Bergen and Yokoyama (1977), the production of VFA from carbohydrates in the rumen is coupled 78 with microbial growth. Maximum microbial yield can be attained only if precursors for protein synthesis are made available to the microbiota simultaneously and in adequate quantities. In this study it has been demonstrated that high fiber diets low in nitrogen are not well utilized unless urea, isoacids and sulfur are supplemented. Addition of these factors to high fiber diets should improve performance of ruminants. \L Effects of isoacids on plasma concentrations of growth hormone, insulin, and cortisol in sheep. Introduction Branched-chain volatile fatty acids (isobutyric, 2- methylbutyric, isovaleric) and the straight-chain valeric acid are either required by or enhance the growth of major rumen cellulolytic bacteria (Bryant and Robinson, 1962; Dehority gt gt,, 1967; Slyter and Weaver, 1969; Bryant, 1973). 22 ytzg studies have demonstrated that supplementation of isoacids to ruminants positively affects growth rates (Lassiter gt gt., 1958; Felix gt gt., 1980a), and milk production (Felix gt_gl,, 1980b; Papas gt gt” 1984). The mechanisms by which isoacids improve performance of ruminants are not completely understood. Changes observed in animals fed isoacids include increases in microbial protein synthesis (Hume, 1970; Russel, 1984), in dry matter digestibility (Cline gt gt., 1966; Soofi gt gl,, 1962), and in nitrogen retention (Cline gt gt., 1966; Umunna gt gt” 1975; Felix, 1976). Recent reports have indicated that lactating cows fed isoacids have lower plasma concentrations of insulin (Towns and Cook, 1984) and higher plasma concentrations of growth hormone (Towns and Cook, 1984; Fieo gt gt., 1984). Since low insulin and high growth hormone concentrations in plasma 79 80 have been associated with higher milk production (Hart, 1983; McDowell, 1983), it has been proposed that the changes in plasma hormone profile might further explain the positive effects of isoacids in lactating cows (Towns and Cook, 1984; Fieo gt gt,, 1984; Cook, 1985). However, it is unclear whether the changes in plasma hormone found in lactating cows were caused by isoacids directly or were a consequence of variations in metabolic demands in the treated cows (Hart, 1983; Bauman and McCutcheon, 1986). The objective of the present study was to determine the effect of isoacids on plasma hormone concentrations in adult sheep fed at maintenance level. Changes in rumen volatile fatty acid and in plasma concentrations of growth hormone, insulin, and cortisol due to treatments are discussed. Materials and Methods Six crossbred ewes (average BW= 41.2 kg) were fitted with rumen cannulas and placed in individual pens in an environmentally controlled room for the entire experiment. Animals were fed 1200 g of alfalfa hay per day offered in equal portions, at 0800 and 1600 hr. The experiment was divided into three periods of two weeks each. In each period, one of the treatments was administered to all six animals. Treatments consisted of 0 (CONTROL), ogl (1801), and 0.2 (1502) gram of isoacids/kg bw/day. The isoacid mixtures containing equal amounts of isobutyric, 2- methylbutyric, isovaleric and valeric acids were injected 81 directly into the rumen through the cannulas. At the end of each period, measurements were performed. In order to prevent stress due to excessive manipulation of the animals, rumen and blood samples were collected in separate days. On day 14 of each period, rumen fluid samples were collected at 0800 hr and then at hourly intervals for the next eight hours. After the last rumen sample, animals were fitted with jugular catheters and prepared for the next day bleeding; Blood samples were collected 30 minutes prior to morning feeding and then every 30 minutes for the next 12 hours. Preparation of rumen samples and VFA determination were performed as described in in.Chapter III. Hormone concentrations in plasma were analized by radioimunoassay procedures. Insulin and cortisol were determined using commercial kits (Micromedic System, Inc., Horsham, PA). Determination of growth hormone was according to the procedure validated by the National Hormone and Pituitary Program. Plasma glucose was determined by the coupled system of glucose oxidase and peroxidase (Sigma Chemical Company, St. Louis, MO). Concentrations of rumen VFA and plasma variables were averaged across the 12-hr sampling period. Means were submitted to anlysis of variance (Gill, 1978a) for the determination of overall significance. Specific differences among treatment means were tested by Bonferroni t-tests (Gill, 1978a; 1978c). 82 Results and discussion Concentrations and molar percent distributions of rumen volatile fatty acids are presented in Table 13. Concentrations of isoacids in rumen fluid were higher in the isoacid-treated groups. *Increases in mean total isoacid concentrations relative to control group 55.9% (p<.001) and 108% (p<.001) respectively for I501 and 1502 groups (Table 13). Animals in the 1802 group had higher acetate and lower propionate concentrations relative to control animals (p<.10). Similar trends were observed in animals receiving the lower levels of isoacids (I801). However, differences from control were not significant (p>aun. Aspects of the effect of isoacids on rumen VFA have been adressed in Chapter IV. Plasma concentrations of growth hormone, insulin, and cortisol as affected by isoacids are depicted in Figures 5, 6, and 7, respectively. Results averaged across the 12-hr sampling period are summarized in Table 14. Growth hormone, cortisol, and glucose concentrations were not affected by the treatments (p>.10). Mean concentration of plasma insulin was 26.4% lower in animals of the ISOZ group than in the control animals (p<.10). One of the most consistent responses obtained in animals treated with isoacids is increased nitrogen retention. Similar responses have been found in animals 83 TABLE 13. Effects of isoacids on rumen volatile fatty acids in sheep fed a high roughage dieta Isoacids Varlable “235.23?"“';;;I““";;5;""’ smm VFA, mmoles/dl Acetate 6.73i 7.04ij 7.55j 0.24 Propionate 1.95i 1.80ij 1.56j 0.11 Butyrate 0.75 0.71 0.84 0.08 Total VFAb 9.43 9.55 9.95 0.41 Total isoacids 0.34C 0.53d 0.716 0.03 cz/c3 3.44f 3.92fg 4.349 0.21 VFA, molar % Acetate 71.3f 73.6fg 75.39 1.25 Propionate 20.5f 13.9fg 15.89 0.92 Butyrate 7.9 7.4 8.3 0.54 aAnimals in each group (n=6) were sampled at 0800 hr and Ehen at hourly intervals for the next 8 hours. casoacids are not included. eMeans in a row without common superscripts differ(p<. 001) fgMeans in a row without common superscripts differ(p<. 05) leeans in a row without common superscripts differ(p<. 10) mStandard error of each mean in the same row. 84 treated with the anabolic agents trenbolone (Sharpe gt gt" 1984) and zeranol (Sinnet-Smith gt gt., 1983). The increase in nitrogen retention by the anabolic agents has been associated with changes in the circulating levels of cortisol (Sharpe gt gt., 1984). The fact that supplementation with isoacids in the present study had no effect on plasma cortisol concentrations (Table 14, Figure 7) suggests that this hormone is not involved in the mechanism of action of isoacids in ruminants. Plasma concentrations of growth hormone were not affected byisoacid supplementation (Figure 5, Table 14). Towns and Cook (1984) and Fieo gt gt. (1984) reported that lactating cows fed isoacids had higher plasma levels of growth hormone. The reasons for those changes are unclear. The primary role of growth hormone is to preserve body protein, particularly during periods of energy deficit. This is accomplished by diverting glucose and fatty acids away from tissue deposition while inhibiting proteolysis and stimulating protein incorporation into muscle (Hart, 1983; McDowell, 1983; Bauman and McCutcheon, 1986). ‘During lactation, growth hormone plays an important role in partitioning nutrients towards milk production and away from tissue deposition (Bauman and McCutcheon, 1986). Therefore, it is difficult to establish if the changes in plasma growth hormone measured in lactating cows fed isoacids were caused by the acids directly or were a consequence of increased metabolic demands. ‘The inverse relationship 85 .Aousv uwwo mmozwsou so“: a com mmmnm :fi mCOEuo: zuzouw mo mcowuouucoocoo osmoam so mowomomfi mo nommmm .m Gunmen NOW. 0 — Om. i 4015.200 0 OOON COO p CON p 006 p . A — b P n p p p o (um/6°) 86 between plasma levels of growth hormone and nutrient availability is well established (Hertelendy and Kipnis, 1973; Hart, 1983; McDowell, 1983). Since isoacid supplementation increases milk production in lactating cows, the higher demand for nutrients in the isoacid-treated group might have triggered a higher response in growth hormone release (Hart, 1983; McDowell, 1983). However, in one of the experiments (Fieo gt gt., 1984), changes in hormone concentration were found despite the lack of response in milk production. The possibility that isoacids affected growth hormone secretion directly in the studies with lactating cows cannot be ruled out. However, it should be kept in mind that because of the almost complete removal of VFA with more than three carbons from the blood by the liver (Young, 1977; Ricks and Cook, 1981), the concentration of isoacids in peripheral blood is usually low. Therefore, a direct interaction of these acids with the pituitary would be rather unlikely. Towns and Cook (1984) suggested that the increase in plasma levels of growth hormone might be governed by a reflex mechanism mediated by chemoreceptors specific for isoacids in the rumen wall. A similar mechanism, mediated by stretch receptors, is known to participate in the inhibitory action of feeding on growth hormone secretion in goats (Tindall, 1982). In the present experiment, changes in plasma growth hormone due to feeding were detected (Figure 5), but they did not differ among treatments. It would be expected that if isoacids actually 87 TABLE 14. Effects of isoacids on plasma concentrations of insulin, growth hormone, cortisol, and glucose in sheep fed a high roughage dieta Isoacids Variable "261333116?""EESZ"'"E§SS" SEMd Insulin, uU/ml 24.9b 23.1bc 19.7C 1.36 Growth hormone, ng/ml 3.84 4.11 3.51 0.23 Cortisol, ng/ml 5.42 4.71 5.12 0.44 Glucose, mg/dl 52.6 47.8 49.3 2.11 aAnimals in each group (n=6) were sampled at 0730 hr and then at 30-minute intervals for the next 12 hours. bcMeans in a row without a common superscript differ (p<.10) dStandard error of each mean in the same row. had a direct effect on growth hormone secretion through a reflex mechanism, changes in pattern should have been apparent. Another point to be considered is the relationship between growth hormone, insulin, and glucose concentrations in plasma. High levels of growth hormone tend to inhibit the effect of insulin in promoting peripheral glucose utilization (McDowell, 1983) resulting in augmentation of plasma levels of insulin and glucose. In cows fed isoacids, Zhowever, this relashionship was not apparent- Towns and Cook (1984) reported that during the eight-hour sampling period, isoacid-treated cows had higher growth hormone but lower glucose and lower insulin than control cows. 88 .Aeucc amen momzmsou now: a own @0050 :w seasmsfl mo m:o~umpucmocoo mEmMHo :0 mowomomw mo uomuum 4015.200 OOON CON 9 pup-b-nP-pL p p L .o musmflm 000 A . ... y “Anu'v AA’V o. IN. r3 -3 ,_ a. row .3 uwm 10.4. you ..on ..«n 13... {on ..on 0V (I‘M/n") 89 Based on the above considerations and on the results of the present experiment, it seems that isoacids do not affect the plasma levels of growth hormone directly. The reasons for the differences in result from those of Towns and Cook (1984) and Fieo gt g;.(1984) cannot be elucidated at this point, indicating that further research in this area is necessary. In additions to the changes in growth hormone, Towns and Cook (1984) reported lower levels of plasma insulin in lactating cows fed isoacids. In the present study, supplementation of 0.2 g of isoacids/kg bw/day to sheep fed an all-alfalfa ration also resulted in lower levels of plasma insulin (Table 14, Figure 6). ‘The mechanism by which such a decrease took place is unclear. As in the case of growth hormone, the possibility that isoacids absorbed from the rumen might have a direct effect on the beta-cells of pancreas should be considered. However, because most of the VFA with more than three carbons are removed from the blood by the liver (Young, 1977; Ricks and Cook, 1981), a direct effect of isoacids is very unlikely. Arguably, a direct interaction with the islets would not be a requirement for the action of isoacids on insulin secretion. As discussed previously, the presence of specific chemoreceptors in the rumen wall or in the portal system (Leek, 1986) could allow isoacids to decrease insulin secretion through reflex mechanisms. From a physiologycal standpoint, however, the necessity for such an intervention is difficult to justify. Although there are differences in emphasis to accomodate the 90 ..oucv amen moocmsou 2m“: 0 com mmmcm aw Homwuuoo mo mcofiumuusmosoo osmoam co mowomomw mo pomuum .h musmflm Now. 0 wow. + JOKbZOU 0 0:5. OOON 009 p CON p 000 b . (p p . . . . . . . . . . . p p . . . p N I n I v __ a M 0 I 0 r. h (Wu/5°) 91 ruminant mode of digestion and intermediary metabolism (Basset, 1978; Hart, 1983; Brockman, 1986), insulin performs the same anabolic actions in ruminants as in nonruminants, iue., it stimulates allocation of substrates such as fatty acids, amino acids and glucose into body tissues (Basset, 1978; Hart, 1983). The four components of the isoacid mixture used in the present study are final products of microbial fermentation in the rumen. Their concentration in rumen fluid and in portal blood are higher in the hours following a meal, coinciding with the peak in substrate availability in plasma (Huntington, 1983). It is reasonable to expect, therefore, that if isoacids were to participate in the mechanisms governing insulin secretion in ruminants, their action would be stimulatory rather than inhibitory. Support for this contention is given by studies in which isobutyrate (Ross and Kitts, 1972) or valerate (Hertelendy gt gt., 1968) were injected intravenously in sheep. The increase of these acids in peripheral circulation resulted in higher plasma level of insulin. Several final products of fermentation have been proposed to increase insulin secretion in ruminants (Brockman, 1978; Trenkle, 1978; Basset, 1980). However, considerable evidence suggests that propionate is the only major metabolite involved directly in the process (Emmanuel and Kennelly, 1984; Bines and Hart, 1984; Istasse and Orskov, 1984). In the present experiment, isoacid supplementation significantly decreased the concentrations 92 of propionate in the rumen (Table 13). It is proposed, therefore, that the lower plasma levels of insulin found in this study were due to the decrease in propionate production in the rumen, which resulted in lower stimulus for insulin secretion (Emmanuel and Kennelly, 1984; Bines and Hart, 1984; Istasse and Orskov, 1984). As indicated previously, Towns and Cook (1984) reported that lactating cows fed high grain diets supplemented with isoacids have lower concentrations of plasma insulin. Despite the fact that in that study measurements of VFA concentrations were not made, results reported by others (Hume, 1970; Oltjen gt gt., 1971; Felix, 1976; Hefner gt gg,, 1985) clearly indicate that supplementation of isoacids to ruminants fed high concentrate diets results in a shift in the molar proportion of rumen VFA favoring acetate. Therefore, it is very likely that the decrease in plasma insulin found by Towns and Cook (1984) were also caused by lower propionate production in the rumen. In the present study it has been demonstrated that isoacids decrease insulin but do not change concentrations of growth hormone and cortisol in plasma. It has also been proposed that the decrease in insulin in ruminants fed isoacids is due to lower propionate production in the rumen. The significance of these results with respect to changes in animal productivity is addressed in the following section. VI. SUMMARY Attempts to explain the mechanisms by which isoacids improve performance in ruminants have consistently implicated the increases in digestion and in microbial yield as the major factors. Towns and Cook (1984) were the first to report that isoacids alter the plasma endocrine profile in lactating cows. Cook (1985) suggested that those changes would be an additional factor to explain the positive effects of isoacids on milk production. In the present study it has been proposed that the decrease in plasma insulin caused by isoacids is due to lower propionate production in the rumen. Based on the results reported in this dissertation and on literature data, it is possible to predict that the efficiency with which isoacids improve performance in ruminants will vary according to the physiological stage of the animal (iJL, lactation or growth) as well as on the type of diet animals are fed (Figure 8). In lactating cows, where low levels of plasma insulin are necessary if adequate supply of substrates to the mammary gland are to be mantained (Hart, 1983; McDowell, 1983), isoacid supplementation should increase milk production regardless of the diet (Figures 8a and 8b). This 93 94 Barnum. maummu mnmunwms + tummms —. i mu m + mum “mummy i mean-m. mm A NITROGEN mamas Qamnnu “p § mu monument b) 4.4:?" __ f mama imam mama 8 mm mu: imam sermon 1:3“ch J Figure 8. Summary of the effects of isoacids in ruminants fed a) high fiber, high NPN diets or b) diets containing preformed protein. 95 explains why milk production in cows fed either low quality, high NPN diets (Felix, 1976; Felix gt gt., 1980) or high quality diets containing preformed protein (Felix, 1976; Papas gt gt., 1984) is consistently increased by isoacids. In growing animals, however, the degree of efficacy of isoacids is diet-dependent. Ideally, animals in the growing stage should have high levels of plasma insulin so that partition of nutrients towards deposition is maximized (McDowell, 1983L. Since plasma insulin is lower when isoacids are fed, isoacid supplementation to growing animals fed high quality diets should be less effective or even non- existent. Any possible positive effect that isoacids might have in the rumen would be offset by the decrease in insulin secretion, resulting in lower rates of uptake of amino acids (Bergen, 1978)-and other substrates by peripheral tissues (Figure 8b). This may explain why feedlot cattle have shown little (Deetz gt g_l_., 1985) or no response (Owens gt g_l_., 1983) to isoacid supplementation. In growing animals fed high roughage, high NPN diets (Figure 8a) the increase in feed utilization (See Chapter IV; Cline, 1966; Soofi gt gt., 1982) and in microbial cell yield (Chalupa and Bloch, 1983; Russel, 1983) by isoacids should increase the rate of growth and feed efficiency (Felix gt gt., 1981; Cook and Barradas, 1985). This discussion has centered on the proposition that part of the beneficial effects of isoacids can be explained on the basis of the shift in rumen fermentation towards the 96 HUMINM. META!" INPUTS .4. sarcomas: A moment: (IETHNIE Aflflflllflrfl f A ammo-n f EXTRA- RUMINAL A Pusan msuuu i mean nvuumun i NITROBEH amen-Ion i cam ? mu: pmnmou Figure 9. Summary of the effects of glycopeptides (teichomycin A2 and avoparcin) in ruminants. aummnwnmu Figure 10. Action of isoacids and glycopeptides in ruminants. "' Isnacxns 97 production of more acetate and less propionate. The similarity of this concept to the concept proposed to explain the effect of glycopeptides and ionophores on animal growth is readily apparent. As demonstrated in Chapter III, glycopeptides increase propionate production and decrease amino acid degradation in the rumen. Therefore, in addition to the savings in energy during fermentation, the extra propionate would also promote, via stimulation of insulin secretion, the utilization by the host animal of amino acids spared in the rumen. These actions in combination should account for the positive effects of glycopeptides on animal growth. The effects of these compounds on milk production are yet to be determined (Figure 9). The present study is the first to offer an explanation as to why isoacids consistently increase milk production in lactating cows fed a variety of diets. Also, it is the first study to demonstrate that isoacids and glycopeptides affect animal performance through overlapping mechanisms, in which propionate plays the central role (Figure 10). The possible interactions between these two classes of compounds should receive further attention. LIST OF REFERENCES LIST OF REFERENCES Allen, J. D. and D. G. Harrison. 1979. The effect of the dietary addition of monensin upon digestion in the stomachs of sheep. (Proc. Nutr. Soc. 38:32a. Allison, M.J. 1969. Biosynthesis of amino acids by ruminal microorganisms. J. Anim. Sci. 29:797. Allison, M.J. 1970. Nitrogen metabolism of ruminal microorganisms. gt: Physiology of Digestion and Metabolism in the Ruminant. A.T. Phillipson (ed). Oriel, New Casttle Upon Tyne, UK. p. 456. Allison, M.J. and M.P. Bryant. 1963. Biosynthesis of branched-chain fatty acids by rumen bacteria. Arch. Biochem. Biophys. 101:269. Allison, M.J., M.P. Bryant and R.N. Doetsch. 1958. 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