Evidence for Stratification of Rumen Wall Microbial Communities Revealed By 16S rRNA Based Amplicon Sequencing

Rumen microbes have been characterized as two distinct groups; feed-associated and luminal microbes and epithelial rumen wall microbes. The rumen wall environment has been relatively understudied regarding microbial taxonomy and function. Rumen microbial fermentations provide key metabolic products for the host such as short chain fatty acids, which are absorbed through rumen epithelial tissues. Rumen wall microorganisms are thus positioned in a prime location to interact with the host, possibly assisting in nutrient exchange across the rumen wall, barrier function and signaling to the host. In addition, the rumen content naturally stratifies based on particle size and density. This stratification is always present, and shifts from large, dry forages nearer to the dorsal portion of the rumen (mat) to the liquid phase in the ventral section of the rumen. Constant interaction with the stratification of the rumen contents led us to hypothesize that the rumen wall microbial communities organize in a similar stratified manner mirroring the stratification of the rumen content, adapting to the different nutrient conditions. Five fistulated, milking Holstein cows of similar age and management conditions were sampled for this project. Three different rumen epithelial biopsies were taken through the fistula, corresponding to the stratification of the rumen content: one aligned with the dorsal portion of the mat (A), another ten centimeters ventral to the first (B) and the last 10 centimeters ventral to the second site (C). We sampled each of these cows twice to gain insight into the temporal stability of the rumen wall microbial populations. DNA was extracted using the Qiagen Powerlyzer Powersoil kit and used for 16S rRNA gene Illumina MiSeq sequencing. Sequences were clustered into operational taxonomic units (OTU) based on a 97% similarity cutoff using MOTHUR. After quality control, 1,079,735 reads remained for 30 samples which were clustered into 3,520 OTUs with 10 or more reads. 96% of the reads were bacterial, whereas 4% affiliated to Archaea. Statistical analysis revealed that among the 20 most abundant OTUs, phylotypes classified as Prevotella, Brachymonas, Fibrobacter, and Suttonella were significantly more abundant at the sample site A compared to C. On a whole community level, Analysis of molecular variance (AMOVA) revealed differences between groups A and C, although not statistically significant (P=0.07). Initial data from this project reveal first evidence that a stratification of rumen wall microbes is present in dairy cattle. Currently, additional rumen wall samples are being sequenced to provide a larger dataset for more robust statistical analyses.