Nest McKain

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 1H was incorporated in preference to 2H. 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.

Ammonia Production by Ruminal Microorganisms and Enumeration, Isolation, and Characterization of Bacteria Capable of Growth on Peptides and Amino Acids from the Sheep Rumen

ABSTRACT Excessive NH3 production in the rumen is a major nutritional inefficiency in ruminant animals. Experiments were undertaken to compare the rates of NH3 production from different substrates in ruminal fluid in vitro and to assess the role of asaccharolytic bacteria in NH3 production. Ruminal fluid was taken from four rumen-fistulated sheep receiving a mixed hay-concentrate diet. The calculated rate of NH3 production from Trypticase varied from 1.8 to 19.7 nmol mg of protein−1 min−1 depending on the substrate, its concentration, and the method used. Monensin (5 μM) inhibited NH3 production from proteins, peptides, and amino acids by an average of 28% with substrate at 2 mg/ml, compared to 48% with substrate at 20 mg/ml (P = 0.011). Of the total bacterial population, 1.4% grew on Trypticase alone, of which 93% was eliminated by 5 μM monensin. Many fewer bacteria (0.002% of the total) grew on amino acids alone. Nineteen isolates capable of growth on Trypticase were obtained from four sheep. 16S ribosomal DNA and traditional identification methods indicated the bacteria fell into six groups. All were sensitive to monensin, and all except one group (group III, similar to Atopobium minutum), produced NH3 at >250 nmol min−1 mg of protein−1, depending on the medium, as determined by a batch culture method. All isolates had exopeptidase activity, but only group III had an apparent dipeptidyl peptidase I activity. Groups I, II, and IV were most closely related to asaccharolytic ruminal and oral Clostridium and Eubacterium spp. Group V comprised one isolate, similar to Desulfomonas piger (formerly Desulfovibrio pigra). Group VI was 95% similar to Acidaminococcus fermentans. Growth of the Atopobium- and Desulfomonas-like isolates was enhanced by sugars, while growth of groups I, II, and V was significantly depressed by sugars. This study therefore demonstrates that different methodologies and different substrate concentrations provide an explanation for different apparent rates of ruminal NH3 production reported in different studies and identifies a diverse range of hyper-ammonia-producing bacteria in the rumen of sheep.

A survey of peptidase activity in rumen bacteria

Twenty-nine strains of 14 species of rumen bacteria were screened for their ability to hydrolyse Ala2, Ala5, GlyArg-4-methoxy-2-naphthylamide (GlyArg-MNA) and Leu-MNA. Several species, notably Megasphaera elsdenii, were active against Ala2, and a smaller number, including Bacteroides ruminicola, Butyrivibrio fibrisolvens, Ruminococcus flavefaciens, Lachnospira multipara and Ruminobacter amylophilus, broke down Ala5. Streptococcus bovis had an exceptionally high leucine arylamidase activity. However, only Ba. ruminicola hydrolysed GlyArg-MNA. Further investigation revealed that only Ba. ruminicola and Bu. fibrisolvens hydrolysed Ala5 to Ala3 and Ala2, with little ALa4 being produced, in a manner similar to rumen fluid. The activity of Ba. ruminicola against synthetic peptidase substrates, including GlyArg-MNA, LysAla-MNA, ArgArg-MNA, GlyPro-MNA, LeuVal-MNA, and Ala3-p-nitroanilide, was similar to that of rumen fluid, whereas the activity of Bu. fibrisolvens was quite different. Since the main mechanism by which peptides are broken down in the rumen is similar to dipeptidyl aminopeptidase type I, for which GlyArg-MNA is a diagnostic substrate, it was concluded that Ba. ruminicola was the most important single species in peptide breakdown in the rumen.

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.

Selective isolation of bacteria with dipeptidyl aminopeptidase type I activity from the sheep rumen

Five-hundred-and-six fresh isolates of rumen bacteria were tested for their ability to hydrolyse the synthetic substrate for dipeptidyl aminopeptidase type I, GlyArg-4-methoxy-2-naphthylamide (GlyArg-MNA), using a gel overlay technique. Twelve positive isolates were small Gram-negative rods which resembled Bacteroides ruminicola in their biochemical and morphological properties. SDS-PAGE of whole cell extracts indicated that two were similar to B. ruminicola strain B14, six resembled B. ruminicola strain M384, and four were similar to B. ruminicola GA33. All hydrolysed GlyArg-MNA, Ala2 and Ala5, and showed no activity against Leu-MNA. Ala3 and Ala2, but no Ala4, was produced from Ala5. The different groups had different, distinctive activity profiles. The two remaining positive isolates were Lactobacillus spp. with an exceptionally high Leu-MNA activity. It was concluded that, although different strains may only be distantly related, B. ruminicola forms the most important group of bacteria in the rumen to possess a dipeptidyl aminopeptidase type I activity.

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.

Breakdown of different peptides by Prevotella (Bacteroides) ruminicola and mixed microorganisms from the sheep rumen

Several di-, tri-, and oligopeptides were incubated individually in vitro with rumen fluid from two sheep receiving a mixed grass hay/concentrate diet and with washed cells ofPrevotella (formerlyBacteroides)ruminicola M384 andP. ruminicola B14. The rates of breakdown of most peptides were similar in the rumen fluid from the two sheep. Acidic and proline-containing peptides tended to be more slowly degraded than neutral or basic peptides. The dipeptide at the N-terminus of higher peptides was observed as an early product of hydrolysis, confirming that a dipeptidyl aminopeptidase type of activity was present. The relative rates of breakdown of dipeptides byP. ruminicola were different from that of rumen fluid, but the hydrolysis of higher peptides followed a similar pattern, and dipeptides from the N-terminus were detected as early products.

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.

A note on the destruction of porcine enteroviruses in anaerobic digestions

Abstract Porcine enteroviruses serotypes F17, F26 and F78, suspended in Earles balanced salt solution, were placed in dialysis tubing in stirred-tank, continuous-flow piggery waste digesters operating at 35 and 60°C. In the mesophilic digester there was a rapid decrease in titre in the first few hours and the virus was largely deactivated in 1 to 2 days. However, some virus persisted for about 9 days. In the thermophilic digester the virus was totally inactivated in 1 h. Virus was also added to the liquid of a batch digester at 35°C. Inactivation was similar to that in the continuous digester. Temperature was probably the principal factor involved in inactivation of the virus, but comparison with temperature control tests showed that other factors probably played a part.