Multidomain metataxonomic profiling of 16 S rRNA, 18 S rRNA and mcrA reveal rumen microbiota remodelling linked to parity in commercial Holstein dairy cows

Although parity affects dairy cow physiology, feed intake, productivity and methane emissions, little is known about how it affects rumen microbial symbioses under commercial management. Parity reconfigures the composition and roles of rumen microbes, with measurable consequences on production parameters in Holstein cows. Multi-marker metataxonomic profiling of rumen fluid was conducted in first (P1) and third (P3) parity cows using a full-length and long-read 16 S rRNA (V1-V9) for bacteria, short read 18 S rRNA (V4) for eukaryotes, and short read mcr A gene sequencing for methanogens. Compared to P1, P3 cows had significantly greater milk output and individual cow cell count (ICCC). Differential abundance analyses revealed taxon-level reorganisation in P3, including enrichment of Firmicutes related taxa ( Kandleria , UBA1067 ( Kiritimatiellia ) and UBA3789 ( Bacilli ), a 61% decrease in the fibrolytic Ruminococcus flavefaciens , and a > 3-fold increase in Selenomonas ruminantium. Eukaryotic profiling revealed a significant restructuring of the ciliate community with unclassified Trichostomatia dominating in P1, but were nearly completely replaced by Charonina spp. in P3. Lower methanogen richness in P3 ( P = 0.04) and a compositional shift from hydrogenotrophic Methanobrevibacter dominance toward methylotrophic lineages ( Methanosphaera ) were observed by the mcr A profiling. Metabolic pathway analysis of the rumen microbiome revealed that P3 cows possessed greater predicted abundance of microbial functions associated with carbohydrate fermentation, propionate and succinate metabolism and amino acid metabolism, whereas P1 cows showed relatively greater enrichment of methane related pathways, biofilm formation, and bacterial secretion systems. The findings demonstrate that parity is associated with distinct restructuring of the rumen microbiome and its predicted metabolic functions in Holstein cows under commercial conditions. Multiparous cows exhibited a more specialised, metabolically efficient rumen microbiota characterised by enrichment of secondary fermenter bacterial consortia, Charonina spp. (Ciliate protozoa) and a shift towards methylotrophic methanogens ( Methanosphaera ). These compositional and functional changes coincided with significantly improved milk production. This in vivo field experiment improves our understanding of microbiomeassociated metabolic adaptations across parity and highlights the importance of considering parity when developing rumen targeted nutritional and methane mitigation strategies in dairy cattle.