Alfalfa (Medicago sativa L.) saponins (AS), primarily pentacyclic triterpenoids, may reduce methane emissions from goats (Capra hircus L.). This study evaluated the methane-suppressing potential of Aspergillus niger 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) (p < 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% (p < 0.05). Methanogen abundance declined by 20.10–44.93%, and general anaerobic fungi declined by 34.22–44.66% compared to untreated AS (p < 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.

The scarcity of effective selective herbicides has made weed management a limiting factor for alfalfa yield and large-scale cultivation. This study aimed to preliminarily screen selective herbicides with acceptable safety to alfalfa and establish an effective weed management program based on sequential pre- and post-emergence herbicide applications through greenhouse screening and multi-year field trials. The greenhouse herbicide safety evaluation showed that, among the 22 herbicides tested, the post-application of 2 pre-emergence herbicides (s-metolachlor and prodiamine) and 5 post-emergence herbicides (benazolin, bentazon, fluazifop-p, imazethapyr, and nicosulfuron) showed a relatively higher survival rate (~4× the standard dose) on two alfalfa cultivars through assessing visual efficacy, plant height, and dry biomass per plant. However, even herbicides that result in relatively high plant survival can produce induce significant growth inhibition and biomass reduction when applied at high doses. Those herbicides were further tested under field conditions (2020–2023) for evaluation of weed control efficiency and alfalfa plant biomass yield potential and nutritive values. Among the herbicide application regimes, the pre-application of prodiamine followed by post-application of bentazon or fluazifop-p showed the most effective weed control efficacy (70–85% reduction of weed biomass in the alfalfa plot), which was greater than the reductions achieved with all single-herbicide applications (24–48%) and sequential application of s-metolachlor followed by the five post-emergence herbicides (25–46%). Additionally, the optimized combinations had no significant effect on the 1st and subsequent alfalfa plant height compared to that of alfalfa in weed-free control. No significant reductions in alfalfa yield or nutritive value were observed in the herbicide-treated plots treated with prodiamine followed by bentazon or fluazifop-p compared with the weed-free control. Collectively, sequential application combining pre- (prodiamine) and post-emergence (bentazon or fluazifop-p) herbicides shows effective weed control in alfalfa under the tested conditions, which provides a useful weed management program to enhance alfalfa forage production and nutritive values.