Stilbene compounds reduce ruminal methane production and are associated with shifts in methanogenesis-related microbial functions

Plant-derived stilbenes are potential antimethanogenic compounds, but whether structurally related stilbenes differ in their methane mitigating effects and associated rumen microbial responses remains unclear. Here, we compared pterostilbene (PTE) and pinostilbene (PIN) to determine their effects on ruminal methane production and metagenomic signatures related to methanogenesis and hydrogen metabolism. Molecular docking predicted that PTE and PIN could occupy the active site pocket of methyl-coenzyme M reductase. At 200 mg/kg substrate dry matter, PTE and PIN reduced methane production by 13% and 19%, respectively, and methane proportion by 17% and 21%, without decreasing total volatile fatty acid concentration or significantly increasing gaseous H 2 . Both treatments were associated with shifts in bacterial and archaeal communities, and methanogenesis-related metagenomic functional potential. PTE showed mainly nominal decreases in several methanogenesis-associated functional features, whereas PIN produced broader and more pronounced changes across methanogenesis-related functions. PIN was also associated with more extensive changes in CAZyme and hydrogenase profiles and affected more metagenome-assembled genomes than PTE. PTE and PIN reduced ruminal methane production without broadly suppressing the measured fermentation, with PIN eliciting broader metagenomic responses. These findings support stilbenes as candidate antimethanogenic compounds and warrant further biochemical and in vivo validation.