Effects of wilting and additives on fermentation characteristics, microbial composition, metabolome, and ruminal degradation properties of mulberry silage

BACKGROUND: Optimizing the silage processing technology for mulberry is essential to improve the utilization efficiency of this feed resource. This study investigated the effects of a wilting pretreatment and silage additives on fermentation dynamics, microbial community structure, metabolites, and in situ ruminal degradation characteristics of whole-plant mulberry silage. A 2 × 3 factorial arrangement with two conditions (62% vs. 73% moisture content) and three silage additives (control, Lactiplantibacillus plantarum (LP), and organic acids (OA)) was applied in a completely randomized design with 6 replications. All samples were ensiled for 60 days before analysis. RESULTS: The wilting procedure increased lactic acid and crude protein (CP) contents while lowering pH (P < 0.05). Both OA and LP additive treatments reduced pH and increased CP content in mulberry silage (P < 0.05). The LP treatment specifically reduced ammonia nitrogen and pH and improved lactic acid content (P < 0.05). The interaction between wilting and additive led to decreases in acetic acid and neutral detergent fiber contents (P < 0.05). 16S rRNA sequence revealed that LP inoculation enriched the relative abundance of Lactiplantibacillus while suppressing that of Enterococcus (P < 0.05). Lactiplantibacillus abundance was positively correlated with contents of lactic acid, CP, and beneficial metabolites L-arginine and salicin (P < 0.05). These two differential metabolites were enriched in phosphotransferase system and arginine biosynthesis pathways (P < 0.05). The in situ ruminal study further confirmed that wilting improved DM digestibility while reducing methane and ammonia nitrogen concentration. The LP treatment also reduced ruminal ammonia nitrogen level (P < 0.05). CONCLUSION: The combined application of a wilting pretreatment and LP inoculant presents a validated and effective approach to comprehensively improve the fermentation quality and nutritive value of mulberry silage.