In Vitro Evaluation of Enzymatically Transformed Alfalfa Saponins on Methane Reduction, Rumen Microbes and Metabolomics in Goats

Alfalfa (<i>Medicago sativa</i> L.) saponins (AS), primarily pentacyclic triterpenoids, may reduce methane emissions from goats (<i>Capra hircus</i> L.). This study evaluated the methane-suppressing potential of <i>Aspergillus niger</i> β-glucosidase-modified AS using in vitro rumen fermentation (0.10 mg/mL inoculum, 24 h incubation, gas chromatography detection). Among the 21 alfalfa cultivars, Pegasis (fall dormancy 9) exhibited the highest antioxidant efficacy (half maximum effective concentration 2.13 mg/mL) and the lowest ferric-reducing activity (0.32 μM Fe²⁺/g) (<i>p</i> < 0.05). Fresh/silage AS reduced methane proportions to 4.50-5.21% of total gas, while enzymatic biotransformation further decreased it to 3.34-3.48% (<i>p</i> < 0.05). Methanogen abundance declined by 20.10-44.93%, and general anaerobic fungi declined by 34.22-44.66% compared to untreated AS (<i>p</i> < 0.05). Metabolomics linked methane suppression to six pathways, including zeatin biosynthesis (via nucleotide metabolites accumulation) and prolactin signaling pathway (via bioactive molecules downregulation), suggesting impaired methanogen energy metabolism and hydrogen flux redirection as mechanisms. Enzymatic AS also enhanced volatile fatty acid production, indicating improved fiber digestion. These in vitro findings demonstrate that enzyme-treated AS modulates rumen fermentation through dual methane mitigation and nutrient utilization enhancement, offering a sustainable feed additive strategy for livestock.