Rumen metagenomics reveals the mechanism by which the combined supplementation of Asparagopsis taxiformis and 3-nitrooxypropanol efficiently alleviates metahne emissions

Enteric fermentation in ruminants is a major source of anthropogenic greenhouse gas emissions.The seaweed Asparagopsis taxiformis (A. taxiformis) is a potent methane inhibitor, but its sustainable application is limited by supply constraints, necessitating strategies to enhance its efficacy at lower doses.This study evaluated the effect of reducing the level of A. taxiformis supplementation (0.32% DM) in combination with the addition of 3-nitrooxypropanol (3-NOP, 0.25% DM) on rumen fermentation (CH4 production, fermentation parameters, and microbial communities) for 48 h in vitro.The combined treatment achieved a 98.21% CH4 emissions (P<0.01) and significantly decreased the A:P ratio compared to the control group. Microbiome analysis revealed that decreased the relative abundances of Methanobrevibacter and Ruminococcus (P<0.01), alongside increased abundances of specific propionate-producing bacteria (Prevotella, Eubacterium, Treponema, Clostridium, Selenomonas) and the methylotrophic methanogen Methanosphaera (P<0.01), after the supplementation of A. taxiformis and 3-NOP.At the functional level, the combination inhibited critical enzymatic reactions, including those catalyzed by methyl-coenzyme M reductase alpha subunit (EC 2.8.4.1) and methyl-coenzyme M reductase (MCR) (P<0.01), effectively blocking the terminal step of methanogenesis. These results suggest that combining supplementation reduces the level of supplementation A. taxiformis, in the naturally biomass-limited, in mitigating rumen CH4 emissions, while also potentially enhancing the ability of single use 3-NOP to mitigating CH4 emissions. This strategy offers a promising approach for mitigating CH4 emissions in ruminants.