Host identity dominates over intra-ruminal spatial variation in shaping the rumen microbiome of Holstein-Friesian dairy cows.

The rumen is a structurally complex fermentation chamber with distinct physicochemical zones, resulting in intra-ruminal biogeography and raising the question whether microbial communities vary spatially within the rumen. We investigated the extent of intra-ruminal microbial variation by analyzing rumen fluid collected from six rumen cannulated Holstein-Friesian dairy cows. Rumen fluid samples were obtained in duplicate from three anatomical rumen regions: the cranial sac (Front), the front ventral sac (Middle), and the middle ventral sac (Back).and subjected to 16S rRNA gene amplicon sequencing. Hierarchical clustering, ordination, and redundancy analyses revealed a strong host-specific signature, with individual cow explaining 37 to 43% of the total community variation (p = 0.001). In contrast, neither rumen sampling location nor technical duplicate significantly affected alpha- or beta-diversity metrics. Only a small subset of low-abundance taxa (<1% of the total community) differed between the rumen sampling locations, whereas core genera such as Prevotella, Christensenellaceae R-7 group, Lachnospiraceae NK3A20 group, Rikenellaceae RC9 gut group, and Methanobrevibacter were consistently dominant across sites. Differential abundance analysis revealed significantly different ASVs across rumen sampling locations, despite no major shifts in overall community structure. These results demonstrate that the rumen microbiome is stable within individual cows with negligible spatial structuring across the horizontal plane, although caution is warranted before extrapolating this uniformity to vertical stratification layers or alternative sampling techniques.