Metabolism of α-linolenic acid during incubations with strained bovine rumen contents: products and mechanisms.

Description of α-linolenic acid (cis-9,cis-12,cis-15-18 : 3, ALA) metabolism in the rumen is incomplete. Ruminal digesta samples were incubated with ALA and buffer containing water or deuterium oxide to investigate the products and mechanisms of ALA biohydrogenation. Geometric Δ9,11,15-18 : 3 isomers were the main intermediates formed from ALA. An increase in the n+1 isotopomers of Δ9,11,15-18 : 3 was due to 2H labelling at C-13. Isomers of Δ9,11,13-18 : 3, cis-7,cis-12,cis-15-18 : 3 and cis-8,cis-12,cis-15-18 : 3 were also formed. No increase in n+1 isotopomers of Δ7,12,15-18 : 3 or Δ8,12,15-18 : 3 was detected. Enrichment in n+2 isotopomers of 18 : 2 products indicated that ALA metabolism continued via the reduction of 18 : 3 intermediates. Isomers of Δ9,11,15-18 : 3 were reduced to Δ11,15-18 : 2 labelled at C-9 and C-13. ALA resulted in the formation of Δ11,13-18 : 2 and Δ12,14-18 : 2 containing multiple 2H labels. Enrichment of the n+3 isotopomer of Δ12,15-18 : 2 was also detected. Metabolism of ALA during incubations with rumen contents occurs by one of three distinct pathways. Formation of Δ9,11,15-18 : 3 appears to be initiated by H abstraction on C-13. Octadecatrienoic intermediates containing cis-12 and cis-15 double bonds are formed without an apparent H exchange with water. Labelling of Δ9,11,13-18 : 3 was inconclusive, suggesting formation by an alternative mechanism. These findings explain the appearance of several bioactive fatty acids in muscle and milk that influence the nutritional value of ruminant-derived foods.

Oral Samples as Non-Invasive Proxies for Assessing the Composition of the Rumen Microbial Community.

Microbial community analysis was carried out on ruminal digesta obtained directly via rumen fistula and buccal fluid, regurgitated digesta (bolus) and faeces of dairy cattle to assess if non-invasive samples could be used as proxies for ruminal digesta. Samples were collected from five cows receiving grass silage based diets containing no additional lipid or four different lipid supplements in a 5 x 5 Latin square design. Extracted DNA was analysed by qPCR and by sequencing 16S and 18S rRNA genes or the fungal ITS1 amplicons. Faeces contained few protozoa, and bacterial, fungal and archaeal communities were substantially different to ruminal digesta. Buccal and bolus samples gave much more similar profiles to ruminal digesta, although fewer archaea were detected in buccal and bolus samples. Bolus samples overall were most similar to ruminal samples. The differences between both buccal and bolus samples and ruminal digesta were consistent across all treatments. It can be concluded that either proxy sample type could be used as a predictor of the rumen microbial community, thereby enabling more convenient large-scale animal sampling for phenotyping and possible use in future animal breeding programs aimed at selecting cattle with a lower environmental footprint.

Bovine Host Genetic Variation Influences Rumen Microbial Methane Production with Best Selection Criterion for Low Methane Emitting and Efficiently Feed Converting Hosts Based on Metagenomic Gene Abundance.

Methane produced by methanogenic archaea in ruminants contributes significantly to anthropogenic greenhouse gas emissions. The host genetic link controlling microbial methane production is unknown and appropriate genetic selection strategies are not developed. We used sire progeny group differences to estimate the host genetic influence on rumen microbial methane production in a factorial experiment consisting of crossbred breed types and diets. Rumen metagenomic profiling was undertaken to investigate links between microbial genes and methane emissions or feed conversion efficiency. Sire progeny groups differed significantly in their methane emissions measured in respiration chambers. Ranking of the sire progeny groups based on methane emissions or relative archaeal abundance was consistent overall and within diet, suggesting that archaeal abundance in ruminal digesta is under host genetic control and can be used to genetically select animals without measuring methane directly. In the metagenomic analysis of rumen contents, we identified 3970 microbial genes of which 20 and 49 genes were significantly associated with methane emissions and feed conversion efficiency respectively. These explained 81% and 86% of the respective variation and were clustered in distinct functional gene networks. Methanogenesis genes (e.g. mcrA and fmdB) were associated with methane emissions, whilst host-microbiome cross talk genes (e.g. TSTA3 and FucI) were associated with feed conversion efficiency. These results strengthen the idea that the host animal controls its own microbiota to a significant extent and open up the implementation of effective breeding strategies using rumen microbial gene abundance as a predictor for difficult-to-measure traits on a large number of hosts. Generally, the results provide a proof of principle to use the relative abundance of microbial genes in the gastrointestinal tract of different species to predict their influence on traits e.g. human metabolism, health and behaviour, as well as to understand the genetic link between host and microbiome.

The rumen microbial metagenome associated with high methane production in cattle.

BACKGROUND: Methane represents 16 % of total anthropogenic greenhouse gas emissions. It has been estimated that ruminant livestock produce ca. 29 % of this methane. As individual animals produce consistently different quantities of methane, understanding the basis for these differences may lead to new opportunities for mitigating ruminal methane emissions. Metagenomics is a powerful new tool for understanding the composition and function of complex microbial communities. Here we have applied metagenomics to the rumen microbial community to identify differences in the microbiota and metagenome that lead to high- and low-methane-emitting cattle phenotypes. METHODS: Four pairs of beef cattle were selected for extreme high and low methane emissions from 72 animals, matched for breed (Aberdeen-Angus or Limousin cross) and diet (high or medium concentrate). Community analysis was carried out by qPCR of 16S and 18S rRNA genes and by alignment of Illumina HiSeq reads to the GREENGENES database. Total genomic reads were aligned to the KEGG genes databasefor functional analysis. RESULTS: Deep sequencing produced on average 11.3 Gb per sample. 16S rRNA gene abundances indicated that archaea, predominantly Methanobrevibacter, were 2.5× more numerous (P = 0.026) in high emitters, whereas among bacteria Proteobacteria, predominantly Succinivibrionaceae, were 4-fold less abundant (2.7 vs. 11.2 %; P = 0.002). KEGG analysis revealed that archaeal genes leading directly or indirectly to methane production were 2.7-fold more abundant in high emitters. Genes less abundant in high emitters included acetate kinase, electron transport complex proteins RnfC and RnfD and glucose-6-phosphate isomerase. Sequence data were assembled de novo and over 1.5 million proteins were annotated on the subsequent metagenome scaffolds. Less than half of the predicted genes matched matched a domain within Pfam. Amongst 2774 identified proteins of the 20 KEGG orthologues that correlated with methane emissions, only 16 showed 100 % identity with a publicly available protein sequence. CONCLUSIONS: The abundance of archaeal genes in ruminal digesta correlated strongly with differing methane emissions from individual animals, a finding useful for genetic screening purposes. Lower emissions were accompanied by higher Succinovibrionaceae abundance and changes in acetate and hydrogen production leading to less methanogenesis, as similarly postulated for Australian macropods. Large numbers of predicted protein sequences differed between high- and low-methane-emitting cattle. Ninety-nine percent were unknown, indicating a fertile area for future exploitation.

Diversity and community composition of methanogenic archaea in the rumen of Scottish upland sheep assessed by different methods.

Ruminal archaeomes of two mature sheep grazing in the Scottish uplands were analysed by different sequencing and analysis methods in order to compare the apparent archaeal communities. All methods revealed that the majority of methanogens belonged to the Methanobacteriales order containing the Methanobrevibacter, Methanosphaera and Methanobacteria genera. Sanger sequenced 1.3 kb 16S rRNA gene amplicons identified the main species of Methanobrevibacter present to be a SGMT Clade member Mbb. millerae (≥ 91% of OTUs); Methanosphaera comprised the remainder of the OTUs. The primers did not amplify ruminal Thermoplasmatales-related 16S rRNA genes. Illumina sequenced V6-V8 16S rRNA gene amplicons identified similar Methanobrevibacter spp. and Methanosphaera clades and also identified the Thermoplasmatales-related order as 13% of total archaea. Unusually, both methods concluded that Mbb. ruminantium and relatives from the same clade (RO) were almost absent. Sequences mapping to rumen 16S rRNA and mcrA gene references were extracted from Illumina metagenome data. Mapping of the metagenome data to 16S rRNA gene references produced taxonomic identification to Order level including 2-3% Thermoplasmatales, but was unable to discriminate to species level. Mapping of the metagenome data to mcrA gene references resolved 69% to unclassified Methanobacteriales. Only 30% of sequences were assigned to species level clades: of the sequences assigned to Methanobrevibacter, most mapped to SGMT (16%) and RO (10%) clades. The Sanger 16S amplicon and Illumina metagenome mcrA analyses showed similar species richness (Chao1 Index 19-35), while Illumina metagenome and amplicon 16S rRNA analysis gave lower richness estimates (10-18). The values of the Shannon Index were low in all methods, indicating low richness and uneven species distribution. Thus, although much information may be extracted from the other methods, Illumina amplicon sequencing of the V6-V8 16S rRNA gene would be the method of choice for studying rumen archaeal communities.

Archaeal abundance in post-mortem ruminal digesta may help predict methane emissions from beef cattle.

Methane produced from 35 Aberdeen-Angus and 33 Limousin cross steers was measured in respiration chambers. Each group was split to receive either a medium- or high-concentrate diet. Ruminal digesta samples were subsequently removed to investigate correlations between methane emissions and the rumen microbial community, as measured by qPCR of 16S or 18S rRNA genes. Diet had the greatest influence on methane emissions. The high-concentrate diet resulted in lower methane emissions (P < 0.001) than the medium-concentrate diet. Methane was correlated, irrespective of breed, with the abundance of archaea (R = 0.39), bacteria (-0.47), protozoa (0.45), Bacteroidetes (-0.37) and Clostridium Cluster XIVa (-0.35). The archaea:bacteria ratio provided a stronger correlation (0.49). A similar correlation was found with digesta samples taken 2-3 weeks later at slaughter. This finding could help enable greenhouse gas emissions of large animal cohorts to be predicted from samples taken conveniently in the abattoir.

Hydrogen and methane emissions from beef cattle and their rumen microbial community vary with diet, time after feeding and genotype.

The aims of the present study were to quantify hydrogen (H2) and methane (CH4) emissions from beef cattle under different dietary conditions and to assess how cattle genotype and rumen microbial community affected these emissions. A total of thirty-six Aberdeen Angus-sired (AAx) and thirty-six Limousin-sired (LIMx) steers were fed two diets with forage:concentrate ratios (DM basis) of either 8:92 (concentrate) or 52:48 (mixed). Each diet was fed to eighteen animals of each genotype. Methane (CH4) and H2 emissions were measured individually in indirect respiration chambers. H2 emissions (mmol/min) varied greatly throughout the day, being highest after feed consumption, and averaged about 0·10 mol H2/mol CH4. Higher H2 emissions (mol/kg DM intake) were recorded in steers fed the mixed diet. Higher CH4 emissions (mol/d and mol/kg DM intake) were recorded in steers fed the mixed diet (P< 0·001); the AAx steers produced more CH4 on a daily basis (mol/d, P< 0·05) but not on a DM intake basis (mol/kg DM intake). Archaea (P= 0·002) and protozoa (P< 0·001) were found to be more abundant and total bacteria (P< 0·001) less abundant (P< 0·001) on feeding the mixed diet. The relative abundance of Clostridium cluster IV was found to be greater (P< 0·001) and that of cluster XIVa (P= 0·025) lower on feeding the mixed diet. The relative abundance of Bacteroides plus Prevotella was greater (P= 0·018) and that of Clostridium cluster IV lower (P= 0·031) in the LIMx steers. There were no significant relationships between H2 emissions and microbial abundance. In conclusion, the rate of H2 production immediately after feeding may lead to transient overloading of methanogenic archaea capacity to use H2, resulting in peaks in H2 emissions from beef cattle.

Differential recovery of bacterial and archaeal 16S rRNA genes from ruminal digesta in response to glycerol as cryoprotectant.

Bacteria and archaea in frozen (-20°C) ruminal digesta were analysed by qPCR and cloning/sequencing of 16S rRNA genes. Samples frozen with and without glycerol as cryoprotectant indicated a major loss of Bacteroidetes in unprotected samples, resulting in higher proportions of Firmicutes. Archaeal numbers and diversity were unaffected.

Ammonia production by human faecal bacteria, and the enumeration, isolation and characterization of bacteria capable of growth on peptides and amino acids.

BACKGROUND: The products of protein breakdown in the human colon are considered to be detrimental to gut health. Amino acid catabolism leads to the formation of sulfides, phenolic compounds and amines, which are inflammatory and/or precursors to the formation of carcinogens, including N-nitroso compounds. The aim of this study was to investigate the kinetics of protein breakdown and the bacterial species involved. RESULTS: Casein, pancreatic casein hydrolysate (mainly short-chain peptides) or amino acids were incubated in vitro with suspensions of faecal bacteria from 3 omnivorous and 3 vegetarian human donors. Results from the two donor groups were similar. Ammonia production was highest from peptides, followed by casein and amino acids, which were similar. The amino acids metabolized most extensively were Asp, Ser, Lys and Glu. Monensin inhibited the rate of ammonia production from amino acids by 60% (P = 0.001), indicating the involvement of Gram-positive bacteria. Enrichment cultures were carried out to investigate if, by analogy with the rumen, there was a significant population of asaccharolytic, obligately amino acid-fermenting bacteria ('hyper-ammonia-producing' bacteria; HAP) in the colon. Numbers of bacteria capable of growth on peptides or amino acids alone averaged 3.5% of the total viable count, somewhat higher than the rumen. None of these were HAP, however. The species enriched included Clostridium spp., one of which was C. perfringens, Enterococcus, Shigella and Escherichia coli. CONCLUSIONS: Protein fermentation by human faecal bacteria in the absence of sugars not only leads to the formation of hazardous metabolic products, but also to the possible proliferation of harmful bacteria. The kinetics of protein metabolism were similar to the rumen, but HAP bacteria were not found.

Toxicity of unsaturated fatty acids to the biohydrogenating ruminal bacterium, Butyrivibrio fibrisolvens.

BACKGROUND: Health-promoting polyunsaturated fatty acids (PUFA) are abundant in forages grazed by ruminants and in vegetable and fish oils used as dietary supplements, but only a small proportion of PUFA finds its way into meat and milk, because of biohydrogenation in the rumen. Butyrivibrio fibrisolvens plays a major role in this activity. The aim of this study was to investigate the mechanisms by which PUFA affect the growth of B. fibrisolvens, how PUFA are metabolized and the metabolic response to growth in the presence of PUFA. RESULTS: Linoleic acid (LA; cis-9, cis-12-18:2) and alpha-linolenic acid (LNA; cis-9, cis-12, cis-15-18:3) increased the lag phase of B. fibrisolvens JW11, LNA having the greater effect. Growth was initiated only when the PUFA had been converted to vaccenic acid (VA; trans-11-18:1). The major fish oil fatty acids, eicosapentaenoic acid (EPA; 20:5(n-3)) and docosahexaenoic acid (DHA; 22:6(n-3)), were not metabolized and prevented growth. Cellular integrity, as determined fluorimetrically by propidium iodide (PI) ingression, was affected as much by 18:1 fatty acids, including VA, as 18:2 fatty acids. The methyl esters of LNA, LA, EPA and DHA had no effect on growth or other measurements. The ATP pool decreased by 2/3 when LA was added to growing bacteria, whereas most acyl CoA pools decreased by >96%. CONCLUSIONS: It was concluded that biohydrogenation occurs to enable B. fibrisolvens to survive the bacteriostatic effects of PUFA, and that the toxicity of PUFA is probably mediated via a metabolic effect rather than disruption of membrane integrity.

Metabolism of conjugated linoleic acids and 18 : 1 fatty acids by ruminal bacteria: products and mechanisms.

Cultures of ruminal bacteria known to metabolize unsaturated fatty acids were grown in medium containing 50 microg ml(-1) of geometric and positional isomers of conjugated linoleic acid (CLA) or 18 : 1 fatty acids and 37.4 % deuterium oxide to investigate the mechanisms responsible for fatty acid metabolism. Butyrivibrio fibrisolvens JW11 converted cis-9,trans-11-18 : 2 and trans-9,trans-11-18 : 2 to trans-11-18 : 1 as the main product, labelled at C-9, and metabolized trans-10,cis-12-18 : 2 to trans-10-18 : 1, labelled at C-13, and smaller amounts of trans-12-18 : 1 and cis-12-18 : 1. Butyrivibrio proteoclasticus P-18 did not grow in the presence of cis-9,trans-11-18 : 2 or trans-10,cis-12-18 : 2, but grew in medium containing trans-9,trans-11-18 : 2, forming 18 : 0. Propionibacterium acnes, a ruminal species that isomerizes linoleic acid to trans-10,cis-12-18 : 2, did not metabolize CLA isomers further. B. fibrisolvens metabolized small amounts of trans-10-18 : 1, trans-11-18 : 1 and cis-9-18 : 1, but the products formed were not detected. B. proteoclasticus, on the other hand, carried out substantial conversion of 18 : 1 substrates to 18 : 0. P. acnes hydrated cis-9-18 : 1 and trans-11-18 : 1 to 10-OH-18 : 0, which was further oxidized to yield 10-O-18 : 0. The deuterium enrichment in the intermediates formed during incubations with 9,11 geometric isomers of CLA was about half that of the products from trans-10,cis-12 CLA and 18 : 1 isomers, suggesting that the reduction of 9,11 geometric isomers CLA by ruminal bacteria occurs via different mechanisms compared with the metabolism of other unsaturated fatty acids.

Isomers of conjugated linoleic acids are synthesized via different mechanisms in ruminal digesta and bacteria.

Digesta samples from the ovine rumen and pure ruminal bacteria were incubated with linoleic acid (LA) in deuterium oxide-containing buffer to investigate the mechanisms of the formation of conjugated linoleic acids (CLAs). Rumenic acid (RA; cis-9,trans-11-18:2), trans-9,trans-11-18:2, and trans-10,cis-12-18:2 were the major CLA intermediates formed from LA in ruminal digesta, with traces of trans-9,cis-11-18:2, cis-9,cis-11-18:2, and cis-10,cis-12-18:2. Mass spectrometry indicated an increase in the n+1 isotopomers of RA and other 9,11-CLA isomers, as a result of labeling at C-13, whereas 10,12 isomers contained minimal enrichment. In pure culture, Butyrivibrio fibrisolvens and Clostridium proteoclasticum produced mostly RA with minor amounts of other 9,11 isomers, all labeled at C-13. Increasing the deuterium enrichment in water led to an isotope effect, whereby (1)H was incorporated in preference to (2)H. In contrast, the type strain and a ruminal isolate of Propionibacterium acnes produced trans-10,cis-12-18:2 and other 10,12 isomers that were minimally labeled. Incubations with ruminal digesta provided no support for ricinoleic acid (12-OH,cis-9-18:1) as an intermediate of RA synthesis. We conclude that geometric isomers of 10,12-CLA are synthesized by a mechanism that differs from the synthesis of 9,11 isomers, the latter possibly initiated by hydrogen abstraction on C-11 catalyzed by a radical intermediate enzyme.

Relation between phylogenetic position, lipid metabolism and butyrate production by different Butyrivibrio-like bacteria from the rumen.

The Butyrivibrio group comprises Butyrivibrio fibrisolvens and related Gram-positive bacteria isolated mainly from the rumen of cattle and sheep. The aim of this study was to investigate phenotypic characteristics that discriminate between different phylotypes. The phylogenetic position, derived from 16S rDNA sequence data, of 45 isolates from different species and different countries was compared with their fermentation products, mechanism of butyrate formation, lipid metabolism and sensitivity to growth inhibition by linoleic acid (LA). Three clear sub-groups were evident, both phylogenetically and metabolically. Group VA1 typified most Butyrivibrio and Pseudobutyrivibrio isolates, while Groups VA2 and SA comprised Butyrivibrio hungatei and Clostridium proteoclasticum, respectively. All produced butyrate but strains of group VA1 had a butyrate kinase activity <40 U (mg protein)(-1), while strains in groups VA2 and SA all exhibited activities >600 U (mg protein)(-1). The butyrate kinase gene was present in all VA2 and SA bacteria tested but not in strains of group VA1, all of which were positive for the butyryl-CoA CoA-transferase gene. None of the bacteria tested possessed both genes. Lipase activity, measured by tributyrin hydrolysis, was high in group VA2 and SA strains and low in Group VA1 strains. Only the SA group formed stearic acid from LA. Linoleate isomerase activity, on the other hand, did not correspond with phylogenetic position. Group VA1 bacteria all grew in the presence of 200 microg LA ml(-1), while members of Groups VA2 and SA were inhibited by lower concentrations, some as low as 5 microg ml(-1). This information provides strong links between phenotypic and phylogenetic properties of this group of clostridial cluster XIVa Gram-positive bacteria.

Clostridium proteoclasticum: A ruminal bacterium that forms stearic acid from linoleic acid.

The aim of this study was to identify ruminal bacteria that form stearic acid (18 : 0) from linoleic acid (cis-9,cis-12-18 : 2). One 18 : 0-producing isolate, P-18, isolated from the sheep rumen was similar in morphology and metabolic properties to 'Fusocillus' spp. isolated many years ago. Phylogenetic analysis based on nearly full-length 16S rRNA gene sequence (>1300 bp) analysis indicated that the stearate producer was most closely related to Clostridium proteoclasticum B316(T). Clostridium proteoclasticum B316(T) was also found to form 18 : 0, as were other bacteria isolated elsewhere, which occurred in the same family subclass of the low G+C% Gram-positive bacteria, related to Butyrivibrio fibrisolvens. These bacteria are not clostridia, and the ability to form 18 : 0 was present in all strains in contrast to proteolytic activity, which was variable. Production of 18 : 0 occurred in growing, but not in stationary-phase, bacteria, which made detection of biohydrogenating activity difficult, because of the inhibitory effects of linoleic acid on growth.

Influence of flavomycin on microbial numbers, microbial metabolism and gut tissue protein turnover in the digestive tract of sheep.

Flavomycin is an antibiotic that promotes growth in ruminant and non-ruminant livestock. The aim of this study was to determine the mechanism of action of flavomycin in sheep by measuring microbial numbers, microbial metabolism and gut tissue protein turnover at different sites in the digestive tract. Two weight-matched groups (n 5) of male castrate lambs (30 kg) received 800 g grass cubes/d for 6 weeks, with one group receiving 20 mg/d flavomycin during the last 2 weeks. Samples of digesta and gut tissue segments were obtained immediately post mortem, 90 min after a flood-dose of [ring-D5]phenylalanine. Viable bacterial counts and volatile fatty acid concentrations were highest in ruminal digesta, followed by the colon and caecum, then the duodenum and ileum. The only effect of flavomycin was an increased bacterial count in the rumen (3.5 v. 1.2 x 10(9) per g; P=0.04). Acetate was proportionally greater and propionate and butyrate were lower in the caecum and colon than the rumen. Flavomycin had no effect on volatile fatty acid proportions or ammonia concentrations. Bacteria growing on peptides as sole C source were not affected by flavomycin. Proteolytic, peptidolytic and amino acid deamination activities were similar in the rumen, caecum and colon; they tended to be lower in animals receiving flavomycin. Protein turnover in ruminal wall and duodenal tissues, measured by a flood-dose technique, decreased with flavomycin (P=0.075 and 0.027, respectively). Thus, flavomycin differs from ionophores in its mode of action. It may influence protein metabolism of both digesta and tissue throughout the ruminant digestive tract.

Influence of flavomycin on ruminal fermentation and microbial populations in sheep.

Flavomycin is a phosphoglycolipid antibiotic that promotes growth in ruminants. The aim of this study was to characterize the effects of flavomycin on ruminal micro-organisms and their metabolic consequences. In sheep receiving a mixed grass hay/concentrate diet, inclusion of 20 mg flavomycin day(-1) decreased ruminal ammonia and total volatile fatty acid concentrations (P<0.001), but the acetate : propionate ratio was unchanged. Ruminal pH tended to be lower with flavomycin, and ammonia-production rates of ruminal digesta from control animals measured in vitro tended to be inhibited by flavomycin. Pure-culture studies indicated that anaerobic fungi, protozoa and most bacterial species were insensitive to flavomycin. Fusobacterium necrophorum was the most sensitive species tested, along with some high-activity ammonia-producing (HAP) species. Effects on F. necrophorum in vivo were inconsistent due to large inter-animal variation. HAP numbers appeared to be decreased. Changes in the rumen bacterial-community structure were assessed by using denaturing-gradient gel electrophoresis (DGGE) analysis of rumen digesta 16S rRNA. DGGE profiles differed from animal to animal, but remained consistent from day to day. The community structure changed when flavomycin was introduced. The roles of F. necrophorum and HAP species in ammonia formation and of F. necrophorum in the invasion of wall tissue are consistent with the observed effects of flavomycin on ruminal ammonia formation and, in other studies, on decreasing tissue-turnover rates.

Influence of dipeptidyl peptidase inhibitors on growth, peptidase activity, and ammonia production by ruminal microorganisms.

The aim was to investigate known and potential new inhibitiors of dipeptidyl peptidases (DPP) for their effects on ruminal microorganisms. Gly-Phe diazomethylketone (GPD), Ala-Ala chloromethylketone (AAC), benserazide (DL-serine 2-(2,3,4- trihydroxybenzyl) hydrazide), and diprotin A (Ile-Pro-Ile) inhibited DPP activities of Prevotella albensis, P. ruminicola, P. bryantii, P. brevis, and mixed ruminal microorganisms, though incompletely and, except for diprotin A, without absolute specificity for any of the peptidases. Leucine aminopeptidase activity of Streptococcus bovis was also inhibited by GPD and benserazide. The inhibitors had no effect on the growth of the bacteria, except for GPD, which inhibited growth of P. albensis when only peptides were available for growth. Benserazide had some inhibitory effects on the growth of Megasphaera elsdenii and Prevotella spp., even in the absence of peptides. The predatory activity of ciliate protozoa on bacteria was unaffected by DPP inhibitors. Ammonia production from casein by mixed ruminal microorganisms was inhibited significantly (P < 0.05) by AAC (29% inhibition) and benserazide (33%). It was concluded that DPP inhibitors can influence the rate of NH3 production in the rumen and may form the basis for developing protein-sparing feed additives for ruminants.