Seaweed extraction and fractionation as a strategy to identify bioactive compounds with potential methane inhibition in dairy cows

This study investigated the anti-methanogenic potential of bioactive compounds extracted from Dictyota dichotoma , Sargassum muticum , and Delesseria sanguinea using maceration with solvents of varying polarity and tested in an in vitro fermentation system simulating rumen fermentation in dairy cows. Seaweed was extracted with 75 % methanol (MeOH), ethyl acetate, and heptane. The 75 % MeOH extract was further subjected to solid-phase fractionation to obtain fractions with increasing concentrations of acetonitrile (15 %, 45 %, 65 %). Extracts and fractions were reconstituted in dimethyl sulfoxide and tested in vitro using maize silage as a standard substrate. Total gas (TGP) and methane (CH 4 ) production were measured at 24 and 48 h, while volatile fatty acids (VFA) and dry matter degradability (DMD) were assessed at 48 h. Untargeted metabolomics of extracts and fractions revealed species-specific metabolite profiles, with D. dichotoma characterized by prenol lipids, tentatively identified as diterpenoids and retinoids. Sargassum muticum was characterized by a high abundance of sulphated medium chain fatty acids and other sulphated compounds. In vitro rumen fermentation showed that highly lipophilic extracts of D. dichotoma (ethyl acetate and heptane), significantly reduced TGP at 48 h (115–123 vs control 157 mL/g DM) ( P < 0.05). A lower CH 4 production at 24 h was also observed (6.34–7.14 vs control 9.83 mL/g DM). However, a reduced total VFA production ( P < 0.05) and DMD (P < 0.05) was also observed. A shift in fermentation towards propionate formation when using lipophilic extracts of D. dichotoma suggests a specific inhibition of methanogenic archaea. In contrast, non-fractionated methanolic extracts exhibited no anti-methanogenic effect and only a decrease in TGP was observed for the 65 % ACN fraction of D. dichotoma , highlighting the importance of targeted extraction. The study provides evidence that bioactive compounds in D. dichotoma may hold promise for methane mitigation in ruminants, but further fractionation is necessary to separate archaeal inhibition from adverse effects on fermentation. • Lipophilic extracts of D. dichotoma reduced methane in an in vitro rumen system. • Anti-methanogenic activity linked compounds such as terpenoids, diterpenoids. • Extraction and fractionation improved bioactive compound identification and potency. • Ethyl acetate and heptane were effective solvents for lipophilic bioactives. • Sustainable optimization of extraction methods is crucial for practical applications.