Bacillus megaterium supplementation improves growth performance, immunity, antioxidant capacity, and gut microbiota in cold-stressed neonatal calves

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.