Background This study evaluated the effects of growth stage and storage time on fermentation characteristics, bacterial communities and their functionality in alfalfa (Medicago sativa L.) silage. Alfalfa was harvested at initial flowering (10–20% bloom, AL1) and full flowering (> 80% bloom, AL2) stages, respectively. The harvested alfalfa was ensiled in 15 L plastic silos. Triplicate silos were sampled after 1, 3, 7, 15, 30 and 60 days of ensiling, respectively. Fermentation products were analyzed on each sampling day. The bacterial communities and their functional potential after 3 and 60 days were analyzed by high-throughput sequencing technique and PICRUSt2 method. Results AL2 had better fermentation quality than AL1 with lower pH, ammonia nitrogen and butyric acid concentrations and higher lactic acid concentrations on day 60. AL2 had higher abundances of Weissella and Lactobacillus after 3 days, and lower abundances of Enterobacter and Enterobacteriaceae on day 60 compared to AL1. In metabolic pathway analysis, ensiling promoted the carbohydrate and amino acid metabolism, and inhibited the signal transduction and membrane transport. In enzyme analysis, AL2 had lower abundances of nitrite reductase (NADH) and ornithine decarboxylase than AL1 on day 60. In phenotype analysis, AL2 had higher proportions of facultatively anaerobic and lower proportions of anaerobic, potentially pathogenic and gram negative than AL1 on day 60. Conclusions High throughput sequencing technique combined with PICRUSt2 can be successfully used to describe the changes of bacterial communities and their functionality in silage. This approach can improve our understanding of the silage microbiology to further regulate the fermentation products. Graphical Abstract

This study aimed to investigate the impacts of three organic acid salts and two bacterial additives on the fermentation profile, in vitro digestibility and gas production, and aerobic stability of total mixed ration (TMR) silage prepared with 10% fresh weight (FW) of wet hulless barley distillers’ grains (WHDG), 35% FW of common vetch, 15% FW of whole-crop oat, 10% FW of hulless barley straw, and 30% FW of mixed concentrate. The total mixed ration (555 g kg−1 FW) was ensiled with six treatments: (1) no additives (control); (2) calcium propionate (CAP; 0.5% FW); (3) potassium sorbate (POS; 0.1% FW); (4) sodium diacetate (SDA; 0.5% FW); (5) Lactobacillus buchneri (LB; 1 × 106 cfu g−1 FW); and (6) Lactobacillus casei (LAC; 1 × 106 cfu g−1 FW). All silos (20 L) were opened for the fermentation profile and in vitro digestibility analysis after 95 days of fermentation, and then subjected to an aerobic stability experiment for 14 days. All the TMR silage was well preserved with a low pH (4.32~4.51), acceptable levels of butyric acid (1.34~1.56 g kg−1 dry matter), and ammonia nitrogen (69.1~87.1 g kg−1 total nitrogen). All the groups were steady during aerobic exposure, while the SDA treatment was more stable with lower populations of undesirable microorganisms on day 14. The CAP, POS, and SDA treatments evidently (p < 0.05) enhanced the in vitro digestibility of dry matter (54.8~57.5 vs. 48.4%) and neutral detergent fiber (48.4~51.6 vs. 41.1%) compared to the control. By comprehensive consideration, SDA is recommended as additive to enhance fermentation quality, aerobic stability, and in vitro digestibility of TMR silage containing 10% FW of WHDG.