Mulberry has high crude protein and biologically active compounds but is difficult to be ensiled due to the lack of adequate epiphytic LAB. This study aimed to investigate the effects of inoculation with Lactiplantibacillus plantarum and Pediococcus pentosaceus isolated from mulberry with higher antioxidant capacity alone or in combination with Streptococcus bovis on chemical characteristics, antioxidant capacity, bacterial community, and metabolite composition of mulberry silage. The results showed that all inoculation groups had higher dry matter and lower pH than the control group, particularly in LP (dry matter, DM, 32.03% and pH = 4.44) and LP_PP_SB (DM, 31.68% and pH = 4.26) after 60 days of ensiling. Ammonia nitrogen (AN) content was the lowest in both LP_SB and LP_PP_SB groups, which were 1.86 g/kg FM and 1.05 g/kg FM, respectively, (P < 0.05). Only the LP_PP_SB group showed increased polyunsaturated fatty acids (PUFA, 1.2851 g/kg DM, P < 0.05) than the control group. Ferric-reducing antioxidant power (FRAP) values were increased in all inoculation-treated groups compared with the control group (P < 0.05). 2,2-Diphenyl-1-picrylhydrazyl (DDPH), 3-ethylbenzothiazoline-6-sulfonic acid (ABTS), and FRAP exhibited the highest levels in the LP_PP- and LP_PP_SB-treated groups. Enterobacter was dominant in both the control and SB-treated groups, and the relative abundance was 41.18% and 32.35%, respectively (P < 0.05). The relative abundance of Lactiplantibacillus was higher in the LP-, LP_PP-, and LP_SB-treated groups (81.84%−82.69%). Relative abundance of Pediococcus was higher in the PP-, PP_SB-, and LP_PP_SB-treated groups (74.27%−85.27%). Untargeted metabolomics analysis results showed that five flavonoids (apigenin, eriodictyol, quercetin-3-glucoside, rutin, and kaempferol-3-O-rutinoside)were upregulated in all inoculation groups (except for the SB-treated groups). Among them, eriodictyol was both positively correlated with ABTS and FRAP and also showed the highest relative abundance in the LP_PP- and LP_PP_SB-treated groups. To the best of our knowledge, this study was the first to investigate the relationship between inoculants of epiphytic lactic acid bacteria and antioxidant capacity by 16s rRNA Illumina sequencing technology and untargeted metabolomics analysis, respectively. Consequently, inoculated L. plantarum, P. pentosaceus alone, respectively, or in combination with S. bovis increased the relative abundance of Lactiplantibacillus and Pediococcus and decreased the relative abundance of Enterobacter, particularly in the LP_PP_SB-treated group. In addition, inoculants could increase the relative abundance of five flavonoids (apigenin, eriodictyol, quercetin-3-glucoside, rutin, and kaempferol-3-O-rutinoside), especially eriodictyol to improve the antioxidant capacity of mulberry silage.

Introduction: Neonatal calves exhibit immature digestive and immune systems, rendering them susceptible to environmental stressors such as cold temperatures, which exacerbate gastrointestinal dysfunction and diarrhea incidence. Antibiotic use for mitigation poses risks, including microbiota disruption and resistance development, necessitating safe probiotic alternatives. Methods: This study evaluated the effects of Bacillus megaterium supplementation on growth performance, diarrhea occurrence, serum biochemical, immune, and antioxidant parameters, and rectal microbiota composition in neonatal calves under Xinjiang’s cold climate. Fifty crossbred calves were randomly assigned to five groups ( n = 10): basal diet (Group I), basal plus 50 mg/day gentamicin (Group II), or basal plus 250, 500, or 1,000 mg/day B. megaterium (Groups III–V). Supplementation occurred via milk over 28 days, with assessments of growth performance, fecal scores, serum indices, and rectal microbiota. Results and discussion: The 500 mg/day B. megaterium treatment (Group IV) significantly increased average daily gain (ADG) and reduced feed-to-gain ratio (F/G) and diarrhea frequency compared to control ( p < 0.05). Serum IgG increased, whereas pro-inflammatory cytokines (IL-1β, TNF-α, IFN-γ) decreased in the probiotic group compared with controls ( p < 0.05). Antioxidant capacity improved significantly, with GSH-Px and CAT elevated and MDA reduced ( p < 0.05). Rectal microbiota Shannon index was significantly higher in Group IV compared to the Group II (median: 2.7 vs. 3.8; p < 0.05). The relative abundance of Firmicutes increased, and beneficial genera ( Lactobacillus , Faecalibacterium , Ruminococcaceae_UCG-014 ) were enriched, whereas Escherichia–Shigella decreased in Group IV ( p < 0.05). Beneficial taxa were positively associated with immune and antioxidant markers and negatively associated with pro-inflammatory cytokines. Overall, these findings suggest that B. megaterium is a promising antibiotic alternative for promoting calf health, productivity, and beneficial gut microbiota under cold stress, with implications for more sustainable ruminant production systems.