Role and mechanisms of moisture regulation in enhancing alfalfa silage quality

Managing moisture content is crucial for optimizing the fermentation quality of alfalfa silage; however, the underlying mechanisms of water spraying following wilting remain poorly understood. This study aimed to elucidate the microbial mechanisms through which this rehydration process enhances fermentation quality by modulating key microorganisms and metabolic pathways. In this study, alfalfa was subjected to four moisture treatments: ND50 (wilted to 50% moisture content), ND65 (wilted to 65% moisture content), PS50 (wilted and water-sprayed to 50% moisture content), and PS65 (wilted and water-sprayed to 65% moisture content). Following 90 days of ensiling, changes in nutritional quality, fermentation characteristics, microbial communities, and metabolites were analyzed. Results showed that the PS65 treatment exhibited significantly lower dry matter (DM), water-soluble carbohydrates (WSC), pH, ammonia nitrogen content (NH 3 -N), amino acids (AA), and butyric acid (BA) than the ND65 group ( P < 0.05). Similarly, DM, crude protein (CP), WSC, and pH were significantly lower in the PS50 treatment than in the ND50 group ( P < 0.05). Lactic acid bacteria (LAB) exceeded 97% relative abundance across all treatments, with Lactobacillus predominating. The key differential microorganisms among the treatments were primarily Weissella and Exiguobacterium ( P < 0.05). Furthermore, yeast counts in the water-sprayed treatments (PS65 and PS50) were significantly lower than those in the wilted treatments (ND65 and ND50) ( P < 0.05). Compared with the ND50 group, both up and down-regulated metabolites in the PS50 treatment were predominantly phenolic acids. In contrast, the comparison between the PS65 and ND65 groups revealed 81 significantly up-regulated and 89 down-regulated metabolites, with lipids and flavonoids being the primary components, respectively. Furthermore, multiple metabolic pathways, including ascorbate and aldarate metabolism as well as phenylpropanoid biosynthesis, were significantly enriched in the water-sprayed treatment (PS65). Microbial traceability analysis revealed that the differential metabolites were mainly derived from Lactiplantibacillus , Lentilactobacillus , and Weissella . Within the appropriate moisture range for alfalfa silage, the PS65 treatment not only significantly altered the relative abundances of key bacteria, including Weissella and Exiguobacterium , and fungi, including Coprinellus and Cladosporium ( P < 0.05), but also significantly decreased silage pH ( P < 0.05) and increased Lactic acid (LA)content ( P < 0.05) by modulating metabolic pathways such as phenylpropanoid biosynthesis.