Elucidating the mechanism by which stage-specific microbial succession drives directed metabolomic transformation to maintain long-term stable ensiling of alfalfa

This study investigated changes in fermentation characteristics, nutritional composition, microbial communities, and metabolite profiles during 180 days of anaerobic storage of alfalfa silage. Fresh alfalfa and samples ensiled for 60, 120, and 180 days were analyzed using physicochemical measurements, microbial community sequencing, and metabolomic profiling. Compared with fresh alfalfa, ensiled samples exhibited lower pH and water-soluble carbohydrate concentrations and higher lactic acid concentrations. Crude protein and other nitrogen-containing components showed changes during storage, while NH₃-N/TN increased between days 0 and 180. Bacterial community composition shifted from the predominance of Weizmannia acidiproducens and Enterococcus mundtii at day 60 to greater relative abundances of Rhabdanaerobium thermarum and Lactiplantibacillus plantarum at days 120 and 180. Fungal alpha-diversity indices did not differ significantly among sampling times. Metabolomic analysis revealed differences in metabolites and pathways associated with carbohydrate metabolism, amino acid metabolism, and redox regulation. Although pH and lactic acid concentrations were relatively stable between days 120 and 180, changes in nutritional and microbial characteristics were still observed. These findings indicate that prolonged anaerobic storage can maintain relatively stable fermentation acidity without necessarily ensuring equivalent preservation of all nutritional components.