Current knowledge about the relationships between ruminal bacterial communities and metabolite profiles in the yak rumen is limited. This is due to differences in the nutritional and metabolic features between yak and other ordinary cattle combined with difficulties associated with farm-based research and a lack of technical guidance. A comprehensive analysis of the composition and alterations in ruminal metabolites is required to advance the development of modern yak husbandry. In the current study, we characterized the effect of feed type on the ruminal fluid microbiota and metabolites in yak using 16S rRNA gene sequencing and liquid chromatography-mass spectrometry (LC-MS). Bacteroidetes and Firmicutes were the predominant bacterial phyla in the yak rumen. At the genus level, the relative abundance of Bacteroidales BS11 gut group, Prevotellaceae UCG-003, Ruminococcaceae UCG-011, Bacteroidales RF16 group and Ruminococcaceae UCG-010 was significantly ( P < 0.01) higher in the forage group compared to that in the concentrate group, while the concentrate group harbored higher proportions of Bacteroidales S24-7 group, Ruminococcaceae NK4A214, Succiniclasticum and Ruminococcus 2 . Yak rumen metabolomics analysis combined with enrichment analysis revealed that feed type altered the concentrations of ruminal metabolites as well as the metabolic pattern, and significantly ( P < 0.01) affected the concentrations of ruminal metabolites involved in protein digestion and absorption (e.g., L-arginine, ornithine, L-threonine, L-proline and β-alanine), purine metabolism (e.g., xanthine, hypoxanthine, deoxyadenosine and deoxyadenosine monophosphate) and fatty acid biosynthesis (e.g., stearic acid, myristic acid and arachidonic acid). Correlation analysis of the association of microorganisms with metabolite features provides us with a comprehensive understanding of the composition and function of microbial communities. Associations between utilization or production were widely identified between affected microbiota and certain metabolites, and these findings will contribute to the direction of future research in yak.

BackgroundHerbivores rely on digestive tract lignocellulolytic microorganisms, including bacteria, fungi and protozoa, to derive energy and carbon from plant cell wall polysaccharides. Culture independent metagenomic studies have been used to reveal the genetic content of the bacterial species within gut microbiomes. However, the nature of the genes encoded by eukaryotic protozoa and fungi within these environments has not been explored using metagenomic or metatranscriptomic approaches.Methodology/Principal FindingsIn this study, a metatranscriptomic approach was used to investigate the functional diversity of the eukaryotic microorganisms within the rumen of muskoxen (Ovibos moschatus), with a focus on plant cell wall degrading enzymes. Polyadenylated RNA (mRNA) was sequenced on the Illumina Genome Analyzer II system and 2.8 gigabases of sequences were obtained and 59129 contigs assembled. Plant cell wall degrading enzyme modules including glycoside hydrolases, carbohydrate esterases and polysaccharide lyases were identified from over 2500 contigs. These included a number of glycoside hydrolase family 6 (GH6), GH48 and swollenin modules, which have rarely been described in previous gut metagenomic studies.Conclusions/SignificanceThe muskoxen rumen metatranscriptome demonstrates a much higher percentage of cellulase enzyme discovery and an 8.7x higher rate of total carbohydrate active enzyme discovery per gigabase of sequence than previous rumen metagenomes. This study provides a snapshot of eukaryotic gene expression in the muskoxen rumen, and identifies a number of candidate genes coding for potentially valuable lignocellulolytic enzymes.

The objective of this study was to systematically evaluate and compare the effects of select antimethanogen compounds on methane production, feed digestion and fermentation, and populations of ruminal bacteria and methanogens using in vitro cultures. Seven compounds, including 2-bromoethanesulphonate (BES), propynoic acid (PA), nitroethane (NE), ethyl trans-2-butenoate (ETB), 2-nitroethanol (2NEOH), sodium nitrate (SN), and ethyl-2-butynote (EB), were tested at a final concentration of 12 mM. Ground alfalfa hay was included as the only substrate to simulate daily forage intake. Compared to no-inhibitor controls, PA, 2NEOH, and SN greatly reduced the production of methane (70 to 99%), volatile fatty acids (VFAs; 46 to 66%), acetate (30 to 60%), and propionate (79 to 82%), with 2NEOH reducing the most. EB reduced methane production by 23% without a significant effect on total VFAs, acetate, or propionate. BES significantly reduced the propionate concentration but not the production of methane, total VFAs, or acetate. ETB or NE had no significant effect on any of the above-mentioned measurements. Specific quantitative-PCR (qPCR) assays showed that none of the inhibitors significantly affected total bacterial populations but that they did reduce the Fibrobacter succinogenes population. SN reduced the Ruminococcus albus population, while PA and 2NEOH increased the populations of both R. albus and Ruminococcus flavefaciens. Archaeon-specific PCR-denaturing gradient gel electrophoresis (DGGE) showed that all the inhibitors affected the methanogen population structure, while archaeon-specific qPCR revealed a significant decrease in methanogen population in all treatments. These results showed that EB, ETB, NE, and BES can effectively reduce the total population of methanogens but that they reduce methane production to a lesser extent. The results may guide future in vivo studies to develop effective mitigation of methane emission from ruminants.Methane (CH 4 ) emissions from ruminants can result in a significant loss of feed efficiency: up to a 12% loss of gross energy intake for forage-fed cattle and 4% for concentrate-fed cattle (14). Because methane is 25 times more potent than carbon dioxide as a greenhouse gas (11), methane emitted from ruminants amounted to 141 teragrams of CO 2 equivalents (Tg CO 2 eq), accounting for 25% of total methane emissions from anthropogenic activities in the United States in 2008 (26). To mitigate the negative impact on climate change and to improve feed efficiency, numerous strategies for reducing methane emission from ruminant livestock have been tested. Plant extracts (7, 9), vaccines (28), ionophores (27), and dietary strategies (21) have been evaluated for their efficacy in reducing ruminal methane emission. However, only monensin has been used in animal-feeding operations, and it typically achieves only transient reductions in methane production (12). More importantly, the monensin-driven reduction in methane reduction is largely attributable to decreased feed digestibility (4,19...

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Comparative analysis of binding of intact glucose-grown Fibrobacter succinogenes strain S85 cells and adhesion-defective mutants AD1 and AD4 to crystalline and acid-swollen (amorphous) cellulose showed that strain S85 bound efficiently to both forms of cellulose while mutant Ad1 bound to acid-swollen cellulose, but not to crystalline cellulose, and mutant Ad4 did not bind to either. One-and two-dimensional electrophoresis (2-DE) of outer membrane cellulose binding proteins and of outer membranes, respectively, of strain S85 and adhesion-defective mutant strains in conjunction with mass spectrometry analysis of tryptic peptides was used to identify proteins with roles in adhesion to and digestion of cellulose. Examination of the binding to cellulose of detergent-solubilized outer membrane proteins from S85 and mutant strains revealed six proteins in S85 that bound to crystalline cellulose that were absent from the mutants and five proteins in Ad1 that bound to acid-swollen cellulose that were absent from Ad4. Twenty-five proteins from the outer membrane fraction of cellulose-grown F. succinogenes were identified by 2-DE, and 16 of these were up-regulated by growth on cellulose compared to results with growth on glucose. A protein identified as a Cl-stimulated cellobiosidase was repressed in S85 cells growing on glucose and further repressed in the mutants, while a cellulose-binding protein identified as pilin was unchanged in S85 grown on glucose but was not produced by the mutants. The candidate differential cellulose binding proteins of S85 and the mutants and the proteins induced by growth of S85 on cellulose provide the basis for dissecting essential components of the cellulase system of F. succinogenes.Fibrobacter succinogenes S85 is a rod-shaped, gram-negative, strictly anaerobic ruminal bacterium that is one of the major cellulolytic bacteria within the rumen (2, 24). Because of the ability of this bacterium to efficiently adhere to and degrade plant cell walls, it has been extensively studied to elucidate the mechanism involved in adhesion and digestion of plant cell walls. As a result, five endoglucanases (31, 33-35), one cellobiosidase (17), one cellodextrinase (15), four xylanases (21, 52), two acetyl xylan esterases (22), and two cellulose-binding proteins (11) have been purified or cloned and purified and the catalytic properties of the enzymes determined. Although those studies have provided valuable information about many of the cellulases and hemicellulases of F. succinogenes, the mechanism of binding of cells to cellulose and hydrolysis of cellulose has not been solved.Similar to the case with other anaerobic cellulolytic bacteria, adhesion of F. succinogenes cells to cellulose appears to be a prerequisite for rapid and efficient cellulose hydrolysis by this organism (30). This claim is supported by the observation that carboxymethylcellulose, which blocks adhesion to cellulose, also blocked cellulose degradation (25). Gong and Forsberg (12) isolated nonadherent mutants of F. succinogenes S85 tha...

This study examined changes of rumen fermentation, ruminal bacteria biodiversity and abundance caused by nitrate addition with Ion Torrent sequencing and real-time polymerase chain reaction. Three rumen-fistulated steers were fed diets supplemented with 0%, 1%, and 2% nitrate (dry matter %) in succession. Nitrate supplementation linearly increased total volatile fatty acids and acetate concentration obviously (p = 0.02; p = 0.02; p<0.01), butyrate and isovalerate concentration numerically (p = 0.07). The alpha (p>0.05) and beta biodiversity of ruminal bacteria were not affected by nitrate. Nitrate increased typical efficient cellulolytic bacteria species (Ruminococcus flavefaciens, Ruminococcus ablus, and Fibrobacter succinogenes) (p<0.01; p = 0.06; p = 0.02). Ruminobactr, Sphaerochaeta, CF231, and BF311 genus were increased by 1% nitrate. Campylobacter fetus, Selenomonas ruminantium, and Mannheimia succiniciproducens were core nitrate reducing bacteria in steers and their abundance increased linearly along with nitrate addition level (p<0.01; p = 0.02; p = 0.04). Potential nitrate reducers in the rumen, Campylobacter genus and Cyanobacteria phyla were significantly increased by nitrate (p<0.01; p = 0.01). To the best of our knowledge, this was the first detailed view of changes in ruminal microbiota by nitrate. This finding would provide useful information on nitrate utilization and nitrate reducer exploration in the rumen.

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