Characterization of daily patterns within the rumen metaproteome of holstein dairy cattle

Our understanding of rumen microbial diversity and the biochemical pathways performed by specific microbe populations is expanding; however, there is a distinct lack in the use of proteomic techniques to progress our knowledge of shifts in magnitude and pattern of these protein-mediated pathways. It was hypothesized that within a 24-h period, there would be fluctuations of rumen microbial protein abundances due to feed intake-mediated nutrient availability. This study investigated the fluctuations of bovine rumen metaproteome over a 24-h period utilizing three mid to late-lactation Holsteins (DIM= 207 ± 53.5) that were fed the same TMR ad libitum. Rumen fluid was collected on three days within a five-day period at three time points throughout the day relative to their first offering of TMR (0h, 4h, and 6h). 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. A total of 242 proteins were characterized across 12 microbial species, with 21 proteins identified from a variety of 7 species affected by time of collection. From 4 of these species, 8 50S ribosomal protein subunits were affected by hour of sampling, with 7 out of 8 of these subunits increased in abundance over the three timepoints. Ruminococcus flavefaciens 007c had lower abundance of a nitrogen fixing protein (P = 0.04) as hour of sampling increased, while three other species expressed a decrease in abundance of gluconeogenesis-involved proteins GAPDH (P = 0.04) and PPDK (P = 0.003 and P = 0.026) as hour of sampling increased. Protein abundances of cysteine synthase (P = 0.033; F. succinogenes) and O-acetylhomoserine sulfhydrolase (P = 0.037; T. saccharophilum), two proteins involved in amino acid synthesis, were negatively 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. Proteomics, LC-MS/M