Moisture-driven shifts in the fermentation characteristics and microbial community of alfalfa silage treated with different additives

Understanding the changes in the bacterial ecosystem during anaerobic fermentation of alfalfa (<i>Medicago sativa</i> L.) could provide clearer insight into how moisture content and additives affect the fermentation characteristics and chemical composition of alfalfa silage. Alfalfa was harvested at the budding stage, with moisture contents of 78, 68, and 58%. The treatments included a control (CK), commercial <i>Lactiplantibacillus plantarum</i> (CL), screened <i>L. plantarum</i> (LP, 1 × 10⁶ cfu/g FW), and propionic acid (P, 6 mL/kg FW). The results showed that the addition of CL, LP, and P significantly reduced pH and NH₃-N content and decreased the relative abundance of <i>Enterococcus</i> at moisture contents of 78 and 68%. These treatments also increased LA and dry matter (DM) content and decreased the relative abundance of <i>Lactobacillus</i>. The addition of CL and LP significantly reduced pH, NH₃-N content, and the relative abundance of <i>Enterococcus</i>, while increasing the relative abundance of <i>Lactobacillus</i> at a moisture content of 58%. Functional prediction analysis suggested that additives improved metabolism functions in alfalfa silage. Carbohydrate metabolism, specifically starch and sucrose metabolism, accounted for the highest proportion in the LP treatment group. <i>Lactobacillus</i> and <i>Sphingomonas</i> were negatively correlated with pH and positively correlated with LA and Flieg's score, whereas <i>Enterococcus</i>, <i>Weissella</i>, <i>Leuconostoc</i>, and <i>Enterobacter</i> were positively correlated with pH. In conclusion, the absence of wilting was not conducive to the anaerobic fermentation of alfalfa. Appropriately reducing the moisture content was beneficial for enhancing the effectiveness of additives in promoting alfalfa fermentation.