Near-complete inhibition of rumen methanogenesis via microbial and enzymatic modulation using a low dose of Asparagopsis taxiformis combined with 3-nitrooxypropanol

<h4>Background</h4>Enteric methane (CH₄) from ruminants represents a major contributor to agricultural greenhouse gas emissions. The red seaweed Asparagopsis taxiformis (A. taxiformis) is a highly effective CH₄ emission inhibitor, but its large-scale application is restricted by limited biomass availability. This study evaluated whether reducing the inclusion level of A. taxiformis (0.32% dry matter, DM) combined with 3-nitrooxypropanol (3-NOP; 0.05% DM) could maintain a high inhibitory efficacy, and elucidated the underlying microbial mechanisms through in vitro fermentation and metagenomics analysis.<h4>Results</h4>The combined treatment decreased CH₄ production by 98.21% (P < 0.01) without impairing DM degradation, and markedly shifted rumen fermentation towards propionate, lowering the acetate-to-propionate ratio (1.59 vs. 2.65; P < 0.01). Metagenomic profiling revealed substantial reductions in the abundance of Methanobrevibacter and Ruminococcus, along with increased levels of propionate-associated bacteria such as Prevotella, Treponema, Eubacterium, and Selenomonas (P < 0.01). Functionally, the combined treatment downregulated key enzymes in hydrogenotrophic and methylotrophic methanogenesis, including methyl-coenzyme M reductase (EC:2.8.4.1) and tetrahydromethanopterin S-methyltransferase (EC:2.1.1.86), thereby blocking terminal methanogenic steps.<h4>Conclusions</h4>Collectively, these results demonstrate that co-supplementation with A. taxiformis and 3-NOP achieves near-complete methanogenesis inhibition at drastically reduced seaweed dosage through coordinated changes in fermentation patterns, microbial community structure, and methanogenic enzymatic pathways. This approach provides a practical strategy to overcome biomass limitations of A. taxiformis and warrants validation in long-term in vivo trials.