Intricate microbiome differences observed in lactating cows across methane intensity phenotypes.

Methane emissions from ruminants can be expressed through several metrics as total production, yield, or intensity, each reflecting distinct aspects of energy utilisation. Among these, methane intensity defined as grams of methane emitted per kilograms of energy-corrected milk, directly links emissions to productive efficiency; however, the microbial mechanisms underlying variation in this trait remain unclear. Here, we applied genome-resolved metagenomics and metatranscriptomics to characterise rumen microbial identity, functional potential, and transcriptional activity in lactating cows differing in methane intensity while sharing breed and diet. Microbial community composition and diversity were comparable across phenotypes. However, rumen microbial gene expression revealed marked functional divergence. The rumen content of low-methane-intensity cows showed enriched transcription of fructan-degrading carbohydrate-active enzymes and butyrate-forming pathways, primarily encoded by RUG440 (Atopobiaceae) metagenome-assembled genomes. These functions suggest a fructan-butyrate metabolic axis supported by potential cross-feeding between primary degraders and butyrate producers. Conversely, the high-methane intensity rumen exhibited greater transcription of pectin-degrading carbohydrate-active enzymes, mainly carried by Prevotella metagenome-assembled genomes, suggesting methyl-ester hydrolysis and methanol release. Despite higher methanogenesis transcript levels in high-methane intensity cows, total methane production did not differ between groups. Together, these findings reveal two contrasting functional configurations of the rumen microbiome in Norwegian Red dairy cattle: a fructan-butyrate-oriented metabolism in low-methane-intensity cows and a pectin-methanol-oriented metabolism in high-methane-intensity counterparts. This study provides genome-resolved, multi-omic evidence that microbial carbohydrate specialization and fermentation routing contribute to methane intensity phenotypes in dairy cows, offering mechanistic insights for improving ruminant climate efficiency.