Effect of ciliate protozoa on the activity of polysaccharide-degrading enzymes and fibre breakdown in the rumen ecosystem.

The effect of ciliate protozoa on the activity of polysaccharide-degrading enzymes in microbial populations from the digesta solids and liquor fractions of rumen contents was examined after the refaunation of ciliate-free sheep with an A-type rumen protozoal population. Although the culturable rumen bacterial population was reduced after refaunation the number of fibrolytic micro-organisms detected was higher; the xylanolytic bacterial population and numbers of fungal zoospores were increased after refaunation. The proportion of propionic acid was lower in the refaunated animals, whereas the concentration of ammonia and the acidic metabolites acetate, butyrate and valerate were all increased. The range of enzyme activities present in the digesta subpopulations were the same in defaunated and refaunated animals. The activities of the polysaccharide-degrading enzymes, however, were increased in the microbial populations associated with the digesta solids after refaunation, and at 16 h after feeding the activities were 4-8 times (beta-D-xylosidase 20 times) higher than the levels detected in the adherent population from defaunated sheep. The protozoa, either directly through their own enzymes or indirectly as a consequence of their effects on the population size and activity of the other fibrolytic micro-organisms present, have an important role in determining the level of activity of polysaccharide-degrading enzymes in the rumen ecosystem. Although the extent of ryegrass (Lolium perenne) hay digestion was similar after 24 h in the absence or presence of protozoa, the initial ruminal degradation was higher in refaunated sheep.     

Postprandial variations in the activity of polysaccharide-degrading enzymes in microbial populations from the digesta solids and liquor fractions of rumen contents

The diurnal variations in the specific activities of polysaccharide-degrading enzymes after feeding were monitored in adherent and non-adherent microbial populations separated from bovine rumen liquor and digesta solids. There were marked differences in the activity profiles of the enzymes within the subpopulations. Enzymes involved in the degradation of soluble carbohydrates were more active in the non-adherent populations, and in the liquor phase subpopulation activities increased in the 1–2 h post-feed period. The muralytic enzymes were most active in the adherent population. Specific activities increased by up to 20-fold over the 24 h period, with an initial five-fold increase occurring between 8 h and 12 h after feeding. Enzyme levels in the three non-adherent populations were similar at the end of the postprandial period. In the population recovered from the liquid associated with the digesta particles, however, the activities did not increase until the latter stages of the period, whereas in the non-adherent population from the digesta solids the activities varied little during the diurnal cycle. The numbers of micro-organisms associated with the digesta solids were similar at 2 h and 20 h after feeding; the variations in enzyme levels did not occur as a result of a population increase but were due to increased activities in an established population. The plant cell wall structural polysaccharides were degraded at different rates. There was no appreciable cellulose digestion during the first 8 h of the postprandial period and although hemicellulosic constituents were removed continuously the rate of loss of both polymers was increased in the later stages of the diurnal cycle when enzyme activities were maximal.     

Postprandial variations in the activity of polysaccharide-degrading enzymes in microbial populations from the digesta solids and liquor fractions of rumen contents

The diurnal variations in the specific activities of polysaccharide-degrading enzymes after feeding were monitored in adherent and non-adherent microbial populations separated from bovine rumen liquor and digesta solids. There were marked differences in the activity profiles of the enzymes within the subpopulations. Enzymes involved in the degradation of soluble carbohydrates were more active in the non-adherent populations, and in the liquor phase subpopulation activities increased in the 1-2 h post-feed period. The muralytic enzymes were most active in the adherent population. Specific activities increased by up to 20-fold over the 24 h period, with an initial five-fold increase occurring between 8 h and 12 h after feeding. Enzyme levels in the three non-adherent populations were similar at the end of the postprandial period. In the population recovered from the liquid associated with the digesta particles, however, the activities did not increase until the latter stages of the period, whereas in the non-adherent population from the digesta solids the activities varied little during the diurnal cycle. The numbers of micro-organisms associated with the digesta solids were similar at 2 h and 20 h after feeding; the variations in enzyme levels did not occur as a result of a population increase but were due to increased activities in an established population. The plant cell wall structural polysaccharides were degraded at different rates. There was no appreciable cellulose digestion during the first 8 h of the postprandial period and although hemicellulosic constituents were removed continuously the rate of loss of both polymers was increased in the later stages of the diurnal cycle when enzyme activities were maximal.     

Rumen enzyme profile and fermentation characteristics in sheep as affected by treatment with sodium lauryl sulfate as defaunating agent and presence of ciliate protozoa

The efficiency of sodium lauryl sulfate as a defaunating agent and effect of rumen protozoa on nutrient utilization, fermentation characteristics and enzyme profile were evaluated in adult sheep maintained on a mixed ration containing 65:35% Pala (Ziziphus numularia) leaf: concentrate. Twenty-one adult Malpura sheep divided into three equal groups (DF, RF and F) were either defaunated by oral administration of sodium lauryl sulfate at the rate of 8 g/100 kg body weight (DF), or defaunated and again refaunated (RF), or maintained faunated (F). Daily dry matter intake was similar in defaunated, refaunated and faunated sheep. However, digestibility of cell wall and cell wall contents (NDF, ADF and cellulose) were lower (P < 0.01) in defaunated than refaunated and faunated sheep. Irrespective of the presence or absence of rumen protozoa, daily intake of DCP and DE were similar in the three experimental groups. Even with similar DM, DCP and DE intake, N-retention, blood glucose level, ruminal concentration of total VFA and total-N were higher (P < 0.01), while rumen pH and NH₃-N concentration were lower (P < 0.01) in defaunated sheep. Ruminal activity of amylase, xylanase, protease and urease enzymes were not influenced by presence or absence of ciliate protozoa. However, carboxymethyl cellulase enzyme activity was lower (P < 0.01) in the rumen of defaunated sheep. The total and differential counts of rumen protozoa were similar in refaunated and faunated sheep indicating lack of residual toxic effect of sodium lauryl sulfate. It is concluded that absence of ciliate protozoa increased ruminal TVFA, total-N with lower NH₃-N concentration and fibre digestibility in sheep. Moreover, sodium lauryl sulfate was fully effective for complete removal of rumen ciliate protozoa and successfully defaunated the sheep.     

The effect of rumen ciliates on chitinolytic activity,chitin content and the number of fungal zoospores in the rumen fluid of sheep

The objective of this study was to investigate the effect of selected protozoa on the degradation and concentration of chitin and the numbers of fungal zoospores in the rumen fluid of sheep. Three adult ewes were fed a hay-concentrate diet, defaunated, then monofaunated with Entodinium caudatum or Diploplastron affine alone and refaunated with natural rumen fauna. The average density of the protozoa population varied from 6.1 · 10⁴ (D. affine) to 42.2 · 10⁴ cells/ml rumen fluid (natural rumen fauna). The inoculation of protozoa in the rumen of defaunated sheep increased the total activity of chitinolytic enzymes from 2.9 to 3.6 μmol N-acetylglucosamine/g dry matter (DM) of rumen fluid per min, the chitin concentration from 6.3 to 7.2 mg/g DM of rumen fluid and the number of fungal zoospores from 8.1 to 10.9 · 10⁵ cells/ml rumen fluid. All examined indices showed diurnal variations. Ciliate population density was highest immediately prior to feeding and lowest at 4 h thereafter. The opposite effects were observed for the numbers of fungal zoospores, the chitin concentration and chitinolytic activity. Furthermore, it was found that chitin from zoospores may account for up to 95% of total microbial chitin in the rumen fluid of sheep. In summary, the examined ciliate species showed the ability of chitin degradation as well as a positive influence on the development of the ruminal fungal population.     

Influence of ciliate protozoa on biochemical changes and hydrolytic enzyme profile in the rumen ecosystem

AIMS: To assess the effect of presence or absence of rumen protozoa on fermentation characteristics and enzyme profile in growing lambs. METHODS AND RESULTS: Weaner lambs (G1, G2, G3, G4, G5 and G6 groups) were defaunated by oral administration of sodium laurel sulphate (at 8 g 100 kg(-1) body weight). The lambs of G4, G5 and G6 groups were refaunated. The roughage and concentrate ratio in the diet of G1 and G4, G2 and G5, and G3 and G6 were 50:50 (R1), 65:35 (R2) and 80:20 (R3), respectively. Daily dry matter intake was similar in defaunated and faunated lambs. However, digestibility of organic matter (OM), cellulose and gross energy were lower in defaunated lambs while crude protein (CP) digestibility was similar in both defaunated and faunated lambs. The rumen pH and NH3-N were lower (P < 0.01) while TVFA, total-N and TCA-ppt-N were higher (P < 0.01), in defaunated lambs. Ruminal activity of carboxymethyl cellulase was lower (P < 0.01) in defaunated lambs and amylase, xylanase, protease and urease were similar in faunated and defaunated lambs. Nutrient utilization, rumen metabolites and ciliate protozoal count were higher, whereas digestibility of fibre fractions was lower in high rather than low concentrate fed lambs. The rumen protozoa present before defaunation were B-type and the protozoa which re-established on refaunation were also B-type. CONCLUSIONS: Absence of ciliate protozoa decreased nutrient digestibility and increased ruminal TVFA and total-N with lower NH3-N concentration, indicating better energy and protein utilization in defaunated lambs. SIGNIFICANCE AND IMPACT OF THE STUDY: Defaunation improved energy and protein utilization in lambs.     

Inhibition by 1,10-phenanthroline of the breakdown of peptides by rumen bacteria and protozoa

The rate of peptide breakdown in the rumen frequently exceeds the rate at which the amino acids released can be used for microbial growth. The final step in this often wasteful process involves the cleavage of dipeptides. The main rumen bacterial species with high dipeptidase activity, Prevotella ruminicola, Fibrobacter succinogenes, Lachnospira multipara and Megasphaera elsdenii , had activities which were inhibited >95% by 1,10-phenanthroline, a chelator of divalent metal ions and metalloprotease inhibitor. Dipeptidase activity in digesta taken from the rumen of sheep decreased by 33% in the presence of 1,10-phenanthroline, while mixed bacteria from the same samples were inhibited by 80% and the activity of mixed protozoa decreased by only 15%. Thus a substantial amount of dipeptide breakdown appears to be due to ciliate protozoa in the mixed population. Extensive washing of the protozoa increased the sensitivity of protozoal dipeptidase activity to 1,10-phenanthroline, suggesting that protozoa too have a metallo-dipeptidase activity but that it is normally protected from inhibition by 1,10-phenanthroline. Breakdown of the pentapeptide, Ala₅, was also inhibited 27% by 1,10-phenanthroline in the mixed population, and when Trypticase, a pancreatic casein hydrolysate containing a mixture of oligopeptides, dipeptides and amino acids, was incubated with rumen fluid, the production of ammonia and free amino groups was inhibited 71% by 1,10-phenanthroline. It was concluded that metal ion chelation inhibits oligopeptidase and dipeptidase activities of rumen micro-organisms and may be a means of controlling ammonia production from peptides in the rumen.     

Stability and stabilization of potential feed additive enzymes in rumen fluid*

Four commercial preparations of fibrolytic enzymes, from Irpex lacteus, Trichoderma viride, Aspergillus niger, and a mixture designed to be similar to the I. lacteus extract, were incubated in vitro with digesta taken from the rumen of sheep receiving a grass hay/concentrate diet, and the survival of major enzyme activities was measured. Some activities, including the beta-1,4-endoglucanase and xylanase from the extract derived from Aspergillus niger, were stable for at least 6 h in rumen fluid. The same activities in the other extracts also retained substantial activity for several hours. beta-Glucosidase and beta-xylosidase activities were much more labile, most being almost completely destroyed after 1 h, and sodium dodecyl sulfate-polyacrylamide gel electrophoresis indicated that most proteins in the extracts were digested extensively after up to 7 h of incubation. Adding bovine serum albumin (0.5 g/l) to the incubation increased the half-life of Trichoderma viride beta-glucosidase activity from less than 0.5 h to 3 h. Proteins extracted from plant materials, particularly the soybean 7S globulin fraction, also conferred protection from proteolytic breakdown, but none was as effective as bovine serum albumin. It was concluded that the stability of most fibrolytic enzymes in rumen fluid is not likely to be a limiting factor in the use of enzymes as feed additives for ruminants; but if the enzymes are not stable, means can be found for their stabilization.     

The importance of methanogens associated with ciliate protozoa in ruminal methane production in vitro

The importance of methanogenic bacteria associated with ciliate protozoa was estimated either by removing protozoa from whole rumen fluid (using defaunated rumen fluid to correct for the effects of centrifugation on bacteria) or by isolating the protozoa. Rumen fluid was withdrawn from sheep inoculated with either Polyplastron multivesiculatum , a co-culture of Isotricha prostoma plus Entodinium spp. or a mixed type B fauna of Entodinium, Eudiplodinium and Epidinium spp. Methanogenesis was highest in rumen fluid containing a mixed protozoal population of the following genera: Entodinium, Eudiplodinium and Epidinium , was lower in defaunated rumen fluid and lowest in rumen fluid containing either I. prostoma plus Entodinium or P. multivesiculatum . Methanogenic bacteria associated with rumen ciliates were apparently responsible for between 9 and 25% of methanogenesis in rumen fluid.     

Validation of in sacco method: influence of sampling site,nylon bag or rumen contents,on fibrolytic activity of solid-associated microorganisms

Three ruminally cannulated cows, fed twice daily with a 70:30 forage:concentrate diet, were used to investigate the differences in fibrolytic activity of solid-associated microorganisms between nylon bags and rumen contents. Two different grass hays (regrowth and late harvested) were incubated in ruminal nylon bags. After 2 h or 23 h incubation time, pH was measured in bags and rumen contents, and enzymes of solid-associated microorganisms were extracted from bag residues and surrounding digesta by grinding, freezing, defrosting and sonication. Xylanase, avicelase, β-D-xylosidase and β-D-glucosidase activities were measured. Activities were lower in bag residues than in rumen digesta, and differences were greater after 2 h than after 23 h incubation time. Causes of these differences are discussed. For each incubation time and each enzyme, the differences in solid-associated microorganisms activities between rumen and bags contents were independent of the quality of hay in the bag. Thus the lower fibrolytic activity inside the bags may account for an underestimation of in vivo ruminal fiber degradation by the in sacco method, but this underestimation may be similar whatever the nature and content of forage cell walls.     

The dynamics of major fibrolytic microbes and enzyme activity in the rumen in response to short- and long-term feeding of Sapindus rarak saponins

AIMS: To investigate the short- and long-term effects of an extract of Sapindus rarak saponins (SE) on the rumen fibrolytic enzyme activity and the major fibrolytic micro-organisms. METHODS AND RESULTS: Two feeding trials were conducted. In the short-term trial, four fistulated goats were fed a basal diet containing sugar cane tops and wheat pollard (65:35, w/w) and were supplemented for 7 days with SE at a level of 0.6 g kg(-1) body weight. Rumen liquor was taken before, during and after SE feeding. In the long-term trial, 28 sheep were fed the same basal diet as the goats and were supplemented for 105 days with 0.24, 0.48 and 0.72 g kg(-1) body mass of the extract. Rumen liquor was taken on days 98 and 100. Protozoal numbers were counted under the microscope. Cell wall degradation was determined by enzyme assays and the major fibrolytic micro-organisms were quantified by dot blot hybridization. Sapindus extract significantly depressed rumen xylanase activity in both trials and carboxymethylcellulase activity in the long-term trial (P < 0.01). Fibrobacter sp. were not affected by the SE in both trials, while ruminococci and the anaerobic fungi showed a short-term response to the application of saponins. Protozoal counts were decreased only in the long-term trial with sheep. CONCLUSION: These data suggest that there is an adaptation of Ruminococcus albus, Ruminococcus flavefaciens and Chytridiomycetes (fungi) to saponin when fed over a long period. The fact that no correlation between the cell wall degrading enzyme activities and the cell wall degrading micro-organisms was observed suggests that the organisms tracked in this experiment are not the only key players in ruminal cell wall degradation. SIGNIFICANCE AND IMPACT OF THE STUDY: Sapindus rarak saponins partially defaunate the rumen flora. Their negative effect on cell wall degradation, however, is not related to rumen organisms currently recognized as the major cell wall degrading species. The adaptation of microbes in the long-term feeding experiment suggests that the results from short-term trial on the ruminal microbial community have to be interpreted carefully.     

Contribution of protozoa to lysine synthesis in the in vitro rumen microbial ecosystem

Isotopic tracer experiments were conducted in vitro to determine contribution of protozoa toward the biosynthesis of lysine in the rumen microbial ecosystem. The presence of protozoa in a rumen microbial suspension always increased lysine synthesis from aspartate. Rumen contents from a faunated goat produced a higher amount of lysine than did those from a defaunated one.     

Contribution of protozoa to lysine synthesis in the in vitro rumen microbial ecosystem.

Isotopic tracer experiments were conducted in vitro to determine contribution of protozoa toward the biosynthesis of lysine in the rumen microbial ecosystem. The presence of protozoa in a rumen microbial suspension always increased lysine synthesis from aspartate. Rumen contents from a faunated goat produced a higher amount of lysine than did those from a defaunated one.     

Effect of the tropical forage calliandra on microbial protein synthesis and ecology in the rumen

AIMS: To determine the effect of condensed tannins in Calliandra calothyrsus (calliandra) on rumen microbial function. METHODS AND RESULTS: Microbial populations, ruminal protein synthesis and fermentation end-products were measured in sheep fed roughage hay supplemented with calliandra (30%), with and without inclusions of polyethylene glycol (PEG) to counteract the effect of tannin. Molecular and conventional enumeration techniques were used to quantify rumen bacteria, fungi and protozoa, and protein synthesis was predicted from estimates of urinary purine excretion. The total number of cellulolytic bacteria, including populations of Fibrobacter succinogenes and Ruminococcus spp., was significantly lower in sheep supplemented with calliandra and these populations increased when animals were treated with PEG. By contrast, protozoa and fungi and the microbial group containing Bacteroides-Porphyromonas-Prevotella bacteria appeared to be less affected. The efficiency of microbial protein synthesis in the rumen was not altered significantly. CONCLUSION: Calliandra caused significant shifts in rumen microbial populations without changing the efficiency of protein synthesis. SIGNIFICANCE AND IMPACT OF THE STUDY: The effect of calliandra tannins on rumen digestion may result more from complexing with nutrients than direct inhibition of micro-organisms.     

Studies on the Defaunation of the Ovine Rumen Using Dioctyl Sodium Sulphosuccinate

The lethal concentrations of dioctyl sodium Sulphosuccinate (DSS) to nine species of rumen ciliate protozoa were determined. The quantity of DSS required to remove these protozoa from the rumen of sheep was approximately 30 times the lethal concentration. This was caused by interaction of the DSS with rumen particulate material, and the protozoa were killed only after the particulate fraction was saturated with DSS. Defaunation was conducted most efficiently after withdrawal of food for 24 h followed by a period (1–4 d) of reduced food intake by the host animal. In toxic concentrations of DSS the cilia of both holotrich and entodiniomorphid protozoa became detached, followed by leakage of the cell contents via the gullet or anus (in the entodiniomorphids) or at variable points in the cell wall (in the holotrichs). The population densities of most of the other rumen micro-organisms examined decreased during defaunation, but regained their original population density within 11–21 d. After this time the population densities of these organisms exceeded that shown before defaunation, with the exception of Oscillospira guilliermondii which was eliminated.     

Effect of the tropical forage calliandra on microbial protein synthesis and ecology in the rumen

C.S. MCSWEENEY, B. PALMER, R. BUNCH AND D.O. KRAUSE. 2001 .
Aims: To determine the effect of condensed tannins in Calliandra calothyrsus (calliandra) on rumen microbial function.
Methods and Results: Microbial populations, ruminal protein synthesis and fermentation end-products were measured in sheep fed roughage hay supplemented with calliandra (30%), with and without inclusions of polyethylene glycol (PEG) to counteract the effect of tannin. Molecular and conventional enumeration techniques were used to quantify rumen bacteria, fungi and protozoa, and protein synthesis was predicted from estimates of urinary purine excretion. The total number of cellulolytic bacteria, including populations of Fibrobacter succinogenes and Ruminococcus spp., was significantly lower in sheep supplemented with calliandra and these populations increased when animals were treated with PEG. By contrast, protozoa and fungi and the microbial group containing Bacteroides - Porphyromonas - Prevotella bacteria appeared to be less affected. The efficiency of microbial protein synthesis in the rumen was not altered significantly.
Conclusions: Calliandra caused significant shifts in rumen microbial populations without changing the efficiency of protein synthesis.
Significance and Impact of the Study: The effect of calliandra tannins on rumen digestion may result more from complexing with nutrients than direct inhibition of micro-organisms.     

Correlation between microbial enzyme activities in the rumen fluid of sheep under different treatments

Five total mixed rations prepared from finger millet (Eleusine Coracana) straw as a roughage (48%) and mixed concentrate (52%), supplemented with a 1% isoacid mixture (i-C4, i-C5, C5 and phenylacetic acid in equal proportions) or oil (groundnut oil, 5% more than the control) or urea (5% more nitrogen than the control), and protein (groundnut cake, 5% more nitrogen than the control) were given in a Latin square experiment to sheep. Enzymatic activities were estimated for urease, cellulase, protease, amylase, and lipase in various fractions of rumen fluid on the one hand and rumen microbial biomass on the other hand. Rumen samples were taken 3-4 hours after feeding and mixed rumen bacteria were separated as a strained rumen fluid without protozoa (SRFWP), cell free rumen fluid (CFRF) and enzymes associated with the bacteria cell (EABC). Samples of SRFWP and EABC contained higher enzyme activities than CFRF. Depending on the type of enzymes in each fraction, some significant coefficient of determination (r2) was seen. These values showed very close cooperative action between proteolytic and amylolytic enzymes under the experimental conditions, or perhaps the presence of some species of bacteria with both activities. Lipolytic bacteria are completely specialized for lipase production only (P < 0.05). The results showed oil, isoacid and crude protein enhanced microbial production (P < 0.05) and this can change the pattern of enzymes in the rumen of sheep.     

Pros and Cons of Ion-Torrent Next Generation Sequencing versus Terminal Restriction Fragment Length Polymorphism T-RFLP for Studying the Rumen Bacterial Community

The development of next generation sequencing has challenged the use of other molecular fingerprinting methods used to study microbial diversity. We analysed the bacterial diversity in the rumen of defaunated sheep following the introduction of different protozoal populations, using both next generation sequencing (NGS: Ion Torrent PGM) and terminal restriction fragment length polymorphism (T-RFLP). Although absolute number differed, there was a high correlation between NGS and T-RFLP in terms of richness and diversity with R values of 0.836 and 0.781 for richness and Shannon-Wiener index, respectively. Dendrograms for both datasets were also highly correlated (Mantel test = 0.742). Eighteen OTUs and ten genera were significantly impacted by the addition of rumen protozoa, with an increase in the relative abundance of Prevotella, Bacteroides and Ruminobacter, related to an increase in free ammonia levels in the rumen. Our findings suggest that classic fingerprinting methods are still valuable tools to study microbial diversity and structure in complex environments but that NGS techniques now provide cost effect alternatives that provide a far greater level of information on the individual members of the microbial population.     

The distribution of polysaccharide-degrading enzymes in the bovine rumen digesta ecosystem

The location and level of activity of the principal polysaccharidases and glycoside hydrolases involved in the degradation of plant structural and storage polysaccharides were monitored in microbial populations isolated from liquid and particulate phases of bovine rumen digesta. The three principal subpopulations, and their constituent subgroups studied, all contained polysaccharide depolymerizing enzymes; however, the specific activities of the enzymes that degraded the plant cell wall structural polymers were highest within the adherent particle-associated populations. Separate functional groups of organisms could be recongnized in the particle-associated population by their distinctive enzyme profiles.     

Influence of dietary acetylated peptides on fermentation and peptidase activities in the sheep rumen

The predominant mechanism of peptide breakdown by rumen micro-organisms is aminopeptidase. Thus acetylation of the N-terminus of peptides inhibits their degradation by rumen micro-organisms in short-term incubations with rumen fluid in vitro . An experiment was undertaken to determine if adaptation of the rumen microbial population would take place when acetylated peptides were fed for a prolonged period, which would enable the microbial population to break down the protected peptides and thus decrease their nutritive value. Three adult sheep, fitted with permanent rumen cannulae, received a maintenance hay/concentrate diet to which was added, at each meal, 20 g of casein enzymic hydrolysate ('peptides') or 20 g of peptides previously treated with acetic anhydride. The diets were fed for 28 d in a 3 × 3 latin square and samples were taken during the last 7 d. Fermentation products and NH₃ concentrations indicated that acetylated peptides remained less degradable than untreated peptides. There was a trend towards increased proteolytic activity with acetylated peptides, and dipeptidase activity increased by 18% and 28%, respectively, compared with untreated peptides and control treatments. Activity against N-acetyl-Ala₂ also increased when acetylated peptides were fed, but it remained only 13% of the rate of Ala₂ hydrolysis. No increase was found in the rate of ammonia production from acetylated peptides in animals receiving acetylated peptides–this rate was 26% of that found with untreated peptides–and acetylated peptides continued to persist for longer in the rumen than untreated peptides after feeding. Thus it was concluded that the rumen microbial population did not adapt to utilize acetylated peptides.