Integrated metatranscriptomics identifies Lachnospiraceae as keystone taxa regulating rumen biohydrogenation and milk ω-6/ω-3 polyunsaturated fatty acids ratio in dairy cows

Enhancing milk nutritional quality through increased ω-3 polyunsaturated fatty acid (PUFA) content and a reduced ω-6/ω-3 PUFA ratio represents a significant opportunity for improving dairy products. While ruminal biohydrogenation substantially influences milk fatty acid (FA) composition, the specific microbial mechanisms regulating the milk fat ω-6/ω-3 PUFA ratio remain poorly characterized. This study aimed to identify key microbial taxa and metabolic pathways controlling this nutritionally relevant parameter, thereby establishing a foundation for targeted microbiome interventions to optimize milk FA profiles. Analysis of 95 Holstein cows revealed that rumen bacterial community composition explained 41. 0% of the variation in the milk ω-6/ω-3 PUFA ratio. Comparative analysis of cows with contrasting phenotypes (high-ratio, HFR; low-ratio, LFR) demonstrated distinct FA profiles across rumen fluid, serum, and milk, with α-linolenic acid (ALA, C18: 3 C9, 12, 15) and linoleic acid (LA, C18: 2 C9, 12) emerging as critical determinants. Integrated metatranscriptomic and amplicon sequencing identified members of the family Lachnospiraceae, particularly Butyrivibrio and Eubacterium genera, as central regulators of PUFA metabolism. Notably, HFR-associated microbiomes showed enrichment of FA isomerase gene transcripts. Experimental validation using isolated strains demonstrated that B. hungatei preferentially hydrogenated ALA, while EubacteriumI efficiently metabolized LA, establishing a mechanistic basis for differential substrate biohydrogenation that influences the final ω-6/ω-3 PUFA ratio. Collectively, these results indicate that rumen microbial community structure and transcriptional activity are closely associated with variation in the milk ω-6/ω-3 PUFA ratio. Members of Lachnospiraceae appear to contribute to substrate-specific biohydrogenation processes that may influence downstream milk FA composition. These findings provide a multi-omics framework for understanding microbiome–lipid interactions and support future efforts to develop microbiome-targeted strategies for improving dairy nutritional quality.