Characterization of variations within the rumen metaproteome of Holstein dairy cattle relative to morning feed offering

Abstract

few studies have utilized proteomic techniques to progress our knowledge of protein-mediated pathways within the rumen microbial community, and no previous research has used these techniques to investigate the patterns or variations of these proteins within this community. it was hypothesized that there would be fluctuations of rumen microbial protein abundances due to feed intake-mediated nutrient availability and that these could be identified using non gel-based proteomic techniques. This study investigated the fluctuations of bovine rumen metaproteome utilizing three mid to late-lactation Holsteins. Rumen fluid was collected at three timepoints on three days relative to their first morning feed offering (0 h, 4 h, and 6 h). Samples were pooled within timepoint within cow across day, analyzed using Lc-MS/MS techniques, and analyzed for variations across hour of sampling using pRoc MiXeD of SAS with orthogonal contrasts to determine linear and quadratic effects. A total of 658 proteins were characterized across 19 microbial species, with 68 proteins identified from a variety of 15 species affected by time of collection. Translation-related proteins such as 50S and 30S ribosomal protein subunit variants and elongation factors were positively correlated with hour of sampling. Results suggest that as nutrients become more readily available, microbes shift from conversion-focused biosynthetic routes to more encompassing DnA-driven pathways.The pregastric rumen is the dominant site of microbial colonization and microbe-mediated fermentation within the ruminant digestive tract, and functionality of this chamber is a key factor that dictates the animal's efficiency of nutrient utilization and production 1-4 . Feed and production efficiency, ruminant animal health, and environmental emissions are all affected by rumen ecology, hence there is growing interest within the ruminant livestock sector to understand the in situ or vivo functionality within and among rumen microbes. Current knowledge of the rumen microbiome is cross-disciplinary and rapidly expanding, with novel research emerging that is focused on diversity analysis and community structures of the microbiota 5-8 , as well as metabolic pathway analysis and metatranscriptomics 9-12 .Despite advances in our understanding, there is still a gap in knowledge regarding the undercurrents and interplay of microbe-specific metabolic pathways because of their dynamicity, adaptability, and complexity. Utilizing a variety of approaches to characterize the rumen in terms of microbial ecology and pathway dynamics appears to be necessary. For instance, microbial diversity analysis has revealed that basal diet and diurnal rumen pH patterns can be somewhat independent of bacterial community profile 13 , while more recently, Söllinger et al. 14 paired metabolomics with quantitative metatranscriptomics to assess the diurnal fluctuations of individual rumen microorganisms and also reported a dissociation between the functional microbial transcripts and microbiome pathway products ...

Metaproteome plasticity sheds light on the ecology of the rumen microbiome and its connection to host traits

“…In the current work, we optimize the procedure for extraction and identification of rumen proteins that, when compared to previous studies, allowed us to substantially improve the number of proteins identified [23,[46][47][48]. We investigated the rumen microbiome state through its produced proteome profile and its link to the host feed efficiency phenotype, previously Fig.…”

Differentiating siliceous particulate matter in the diets of mammalian herbivores

“…Every sample was collected ~5 hr after the onset of morning feeding on a single day. Access to the rumen via the cannula is described by Honan and Greenwood (2020) and approved by the Institutional Animal Care and Use Committee of the University of Vermont (protocol no. 201900019).…”

Influence of dietary carbohydrate profile on the dairy cow rumen meta-proteome

“…Protein identification and quantification were performed on a Q-Exactive Plus MS coupled to an EASY-nLC 1200 (Thermo Scientific), similar to the workflow described by Honan and Greenwood (2020). Briefly, the fractionated peptide samples were dissolved in 10 μL of 2.5% formic acid (FA) and 2.5% acetonitrile (ACH) in water and then loaded onto a 100-μm × 500-mm capillary column packed with Halo C18 (2.7-μm particle size, 90-nm pore size, Michrom Bioresources) at a flow rate of 300 nL/min.…”

“…Previous research by Snelling and Wallace (2017) and Hart et al (2018) provided the first glimpses of microbe-specific proteins in the rumen using gel-based proteomic techniques. Recent modifications by Honan and Greenwood (2020) eliminated the use of gels to expand the breadth of proteins identified in the rumen meta-proteome. This modified approach was applied in this study as a tool to characterize shifts in the meta-proteome in response to dietary carbohydrate profile changes.…”