Project description:Investigation of whole genome gene expression level changes in rumen epithelium of dairy cattle at different stages of rumen development and on different diets.
Project description:Protozoa comprise a major fraction of the microbial biomass in the rumen microbiome, of which the entodiniomorphs (order: Entodiniomorphida) and holotrichs (order: Vestibuliferida) are consistently observed to be dominant across a diverse genetic and geographical range of ruminant hosts. Despite the apparent core role that protozoal species exert, their major biological and metabolic contributions to rumen function remain largely undescribed in vivo. Here, we have leveraged (meta)genome27 centric metaproteomes from rumen fluid samples originating from both cattle and goats fed diets with varying inclusion levels of lipids and starch, to detail the specific metabolic niches that protozoa occupy in the context of their microbial co-habitants. Initial proteome estimations via total protein counts and label-free quantification highlight that entodiniomorph species Entodinium and Epidinium as well as the holotrichs Dasytricha and Isotricha comprises an extensive fraction of the total rumen metaproteome. Proteomic detection of protozoal metabolism such as hydrogenases (Dasytricha, Isotricha, Epidinium, Enoploplastron), carbohydrate-active enzymes (Epidinium, Diplodinium, Enoploplastron, Polyplastron), microbial predation (Entodinium) and volatile fatty acid production (Entodinium and Epidinium) was observed at increased levels in high methane-emitting animals. Despite certain protozoal species having well-established reputations for digesting starch, they were unexpectedly less detectable in low methane emitting- 37 animals fed high starch diets, which were instead dominated by propionate/succinate-producing bacterial populations suspected of being resistant to predation irrespective of host. Finally, we reaffirmed our abovementioned observations in geographically independent datasets, thus illuminating the substantial metabolic influence that under-explored eukaryotic populations have in the rumen, with greater implications for both digestion and methane metabolism.
Project description:Protozoa comprise a major fraction of the microbial biomass in the rumen microbiome, of which the entodiniomorphs (order: Entodiniomorphida) and holotrichs (order: Vestibuliferida) are consistently observed to be dominant across a diverse genetic and geographical range of ruminant hosts. Despite the apparent core role that protozoal species exert, their major biological and metabolic contributions to rumen function remain largely undescribed in vivo. Here, we have leveraged (meta)genome27 centric metaproteomes from rumen fluid samples originating from both cattle and goats fed diets with varying inclusion levels of lipids and starch, to detail the specific metabolic niches that protozoa occupy in the context of their microbial co-habitants. Initial proteome estimations via total protein counts and label-free quantification highlight that entodiniomorph species Entodinium and Epidinium as well as the holotrichs Dasytricha and Isotricha comprises an extensive fraction of the total rumen metaproteome. Proteomic detection of protozoal metabolism such as hydrogenases (Dasytricha, Isotricha, Epidinium, Enoploplastron), carbohydrate-active enzymes (Epidinium, Diplodinium, Enoploplastron, Polyplastron), microbial predation (Entodinium) and volatile fatty acid production (Entodinium and Epidinium) was observed at increased levels in high methane-emitting animals. Despite certain protozoal species having well-established reputations for digesting starch, they were unexpectedly less detectable in low methane emitting-animals fed high starch diets, which were instead dominated by propionate/succinate-producing bacterial populations suspected of being resistant to predation irrespective of host. Finally, we reaffirmed our abovementioned observations in geographically independent datasets, thus illuminating the substantial metabolic influence that under-explored eukaryotic populations have in the rumen, with greater implications for both digestion and methane metabolism.
Project description:The structure and function of the microbiome inhabiting the rumen are, amongst other factors, mainly shaped by the animal’s feed intake. Describing the influence of different diets on the inherent community arrangement and associated metabolic activities of the most active ruminal fractions (bacteria and archaea) is of great interest for animal nutrition, biotechnology and climatology. Samples were obtained from three fistulated Jersey cows rotationally fed with corn silage, grass silage or hay, each supplemented with a concentrate mixture. Samples were fractionated into ruminal fluid, squeezed solid and solid matter. DNA, proteins and metabolites were analyzed subsequently. DNA extracts were used for Illumina sequencing of the 16S rRNA gene and the metabolomes of rumen fluids were determined by 500MHz-NMR spectroscopy. Tryptic peptides derived from protein extracts were measured by LC-ESI-MS/MS and spectra were processed by a two-step database search for quantitative metaproteome characterization. Protein- and DNA-based datasets revealed significant differences between sample fractions and diets and affirmed similar trends concerning shifts in phylogenetic composition. Ribosomal genes and proteins belonging to the phylum of Proteobacteria, particularly Succinivibrionaceae, exhibited a higher abundance in corn silage-based samples while fiber-degraders of the Lachnospiraceae family emerged in great quantities throughout the solid phase fractions. The analysis of 8163 quantified bacterial proteins revealed the presence of 166 carbohydrate active enzymes in varying abundance. Cellulosome affiliated proteins were less expressed in the grass silage, glycoside hydrolases appeared in slightest numbers in the corn silage. Most expressed glycoside hydrolases belonged to families 57 and 2. Enzymes analogous to ABC transporters for amino acids and monosaccharides were more abundant in the corn silage whereas oligosaccharide transporters showed a higher abundance in the fiber-rich diets. Proteins involved in carbon metabolism were detected in high numbers and identification of metabolites like short-chain fatty acids, methylamines and phenylpropionate by NMR enabled linkage between producers and products. This study forms a solid basis to retrieve deeper insight into the complex network of gut microbial adaptation.
Project description:The time-resolved impact of monensin on the active rumen microbiome in a rumen-simulating technique (Rusitec) was studied with metaproteomic and metabolomic approaches. Upon monensin treatment, decreased catabolism linked to fiber degradation was observed by the reduced abundance of proteins assigned to fibrolytic bacteria and glycoside hydrolases, sugar transporters and carbohydrate metabolism. Reduced amounts of ammonium as well as branched-chain fatty acids pointed towards a decreased proteolytic activity. The family Prevotellaceae exhibited increased resilience in the presence of monensin, with a switch of metabolism from acetate to succinate production. Prevotella species harbor a membrane bound electron transfer complex, which drives the reduction of fumarate to succinate, the substrate for propionate production in the rumen habitat. Besides the increased succinate production, a concomitant depletion of methane concentration was observed upon monensin exposure. Our study demonstrates that Prevotella sp. shifts its metabolism successfully in response to monensin exposure and Prevotellaceae represents the key bacterial family stabilizing the rumen microbiota during exposure to monensin.
Project description:We evaluated the effects of pure glyphosate and the formulations Durano TF and Roundup® LB plus in different concentrations on rumen microbiota bioreactor model using the Rumen Simulation Technique (RUSITEC). Application of the compounds in three concentrations (0.1 mg/L, 1.0 mg/L or 10 mg/L, n = 4 each).
Project description:A healthy rumen is crucial for normal growth and improved production performance of ruminant animals. Rumen microbes participate in and regulate rumen epithelial function, and the diverse metabolites produced by rumen microbes are important participants in rumen microbe-host interactions. SCFAs, as metabolites of rumen microbes, have been widely studied, and propionate and butyrate have been proven to promote rumen epithelial cell proliferation. Succinate, as an intermediate metabolite in the citric acid cycle, is a final product in the metabolism of certain rumen microbes, and is also an intermediate product in the microbial synthesis pathway of propionate. However, its effect on rumen microbes and rumen epithelial function has not been studied. It is unclear whether succinate can stimulate rumen epithelial development. Therefore, in this experiment, Chinese Tan sheep were used as experimental animals to conduct a comprehensive analysis of the rumen microbiota community structure and rumen epithelial transcriptome, to explore the role of adding succinate to the diet in the interaction between the rumen microbiota and host.
Project description:Microbiome DNA from the adhering fraction of a sheep rumen. The RSTs were generated using an improved version of SARST (referred to as iSARST) from the microbiome DNA extracted from the adhering fraction of the rumen content taken from a sheep. The iSARST method is going to be submitted to Nature Biotechnology for publication. Keywords: other
Project description:The rumen is a major the site of digestion and absorption of energy predominantly in the form of short chain fatty acids as well as amino acids and simple carbohydrates. The value of crops such as perennial wheat (PW) for grain and grazing compared to conventional wheat (W), or the addition of lucerne to PW (PWL) is still being determined. The research sought to determine if these diets representing high potassium (K): low sodium (Na) ratio (W, PW and PWL) was associated with changes in the membrane bound proteins that transport nutrients in the rumen epithelium (RE). Information about these proteins and the influence of different diets is scarce due to difficulties developing a procedure to examine them using quantitative proteomics. The research focuses on proteins extracted using an enzymatic procedure (Bond et al. 2019) from the rumen epithelium layer of the sheep’s’ forestomach. A membrane protein enriched fraction was then extracted and prepared for TMT-MS. The comprehensive assignment of proteins (>4000) to cellular location and quantification of differences in protein abundance will progress the annotation and understanding of protein functions in the ruminant genomes.