Host genetic regulation of rumen 6-hydroxymelatonin reduces methane emissions in dairy cattle

The mechanisms linking host genetics to ruminal methane emissions remain unclear. Here, we integrated multiomics data from 304 lactating cows and demonstrated that methane emission per dry matter intake (M/D) exhibitd higher heritability (h² = 0.42) than microbiability (m² = 0.19), highlighting the predominant role of host genetics. Mendelian randomization (MR) analysis identified three heritable Prevotella species (including Prevotella_bryantii) that causally reduce methane emissions. Network suggested that Prevotella_bryantii, which harbors the [NiFe]_Group_1d hydrogenase, exerts this effect by competing with methanogens for H₂. Furthermore, the methane-reducing effect of Prevotella _bryantii was confirmed by in vitro fermentation experiments. To trace the host regulation upstream, the host-derived metabolite 6-hydroxymelatonin was identified as a key regulator that positively influences these Prevotella species by MR analysis, which was further validated by in vitro fermentation and pure bacterial culture experiments. Genome-wide association studies linked ruminal 6-hydroxymelatonin levels to host genetic variants (e.g., 5:106926534) near candidate genes including ITFG2. Functional studies in bovine hepatocytes revealed that ITFG2 knockdown activated the mTORC1 pathway, upregulated CYP1A2 expression, and increased 6-hydroxymelatonin synthesis. Furthermore, cattle carrying the TA genotype at 5:106926534 exhibited significantly lower predicted and measured methane emissions. Collectively, this study unveils a pathway whereby host genetics (via ITFG2/mTORC1) modulate hepatic 6-hydroxymelatonin synthesis, which enriches specific rumen Prevotella that compete with methanogens for hydrogen, thereby reducing methane.