Longitudinal analysis reveals associations of rumen microbial fermentation and serum metabolic indices with milk-yield variation in periparturient Holstein cows

Periparturient dairy cows face the combined metabolic challenges of parturition, lactogenesis, and negative energy balance. The rumen microbiome supports energy homeostasis through volatile fatty acid (VFA) production, yet most studies have compared healthy and diseased animals, leaving milk-yield variation among healthy cows largely unexplained. We therefore compared rumen microbial, fermentation, and metabolic characteristics between healthy high- and low-yielding periparturient cows. Eighty clinically healthy Holstein cows were stratified based on average milk yield across days 3 to 21 postpartum. Cows falling within the upper and lower quartiles were assigned to the high-yield (HY) and low-yield (LY) groups, respectively ( n = 20), with milk yield serving as a descriptive grouping variable. Blood, ruminal digesta, and postpartum milk samples were collected at − 21, 1, 3, 7, 14, and 21 d relative to calving. HY cows exhibited higher milk lactose concentrations, while ruminal VFA concentrations showed no intergroup differences following parturition ( q = 0.85–0.93). Prepartum HY cows displayed elevated ruminal microbial alpha diversity and greater relative abundances of Bacteroidota and Prevotella , although these distinctions became marginal following multiple-testing correction ( q = 0.059 and q = 0.21–0.94); prepartum VFA concentrations were comparable between groups. Rumen microbial community structure differed pre-calving. The number of microbe-metabolite associations retained following Benjamini–Hochberg correction rose from 2 out of 280 prepartum pairs to 32 out of 280 postpartum pairs, and 10 out of 280 associations persisted after further adjustment for sampling day; subsequent interpretations were built upon these retained associations. Across all experimental animals, higher prepartum Shannon diversity and greater F082 relative abundance correlated with elevated subsequent milk yield ( q = 0.007 and 0.045, respectively). Group-specific temporal patterns were observed for postpartum non-esterified fatty acid (NEFA) and β-hydroxybutyrate (BHBA), and these kinetic profiles are presented descriptively. Prepartum differences in rumen community structure were associated with subsequent differences in serum metabolic trajectories and with the selected milk-yield phenotype, whereas measured VFA concentrations did not differ significantly. We hypothesize that these prepartum microbial differences contribute to the observed postpartum patterns, but this requires validation in independent cohorts before any causal or management interpretation.