Abstract
Bacillus species represent promising alternatives to antimicrobial growth promoters, offering potential benefits for productivity and gut health in broilers. This study aimed to isolate Bacillus strains with inhibitory activity against Clostridium perfringens and evaluate their probiotic potential through in vitro and in vivo approaches. In Experiment 1 (in vitro), five strains-B. siamensis C66, B. tequilensis Y7, B. velezensis L15, B. amyloliquefaciens C271, and B. siamensis C377-were isolated and assessed for stress tolerance, digestive enzyme production, and antimicrobial activity. All strains demonstrated high survival rates under acid and bile stress, produced multiple digestive enzymes, and significantly inhibited the growth of C. perfringens. In Experiment 2 (in vivo), 630 day-old male Arbor Acres broilers were randomly assigned to one of seven dietary treatments for 42 days: a negative control (CON, basal diet), a positive control (ANfT, basal diet supplemented with 6.4 g/t virginiamycin), and five groups receiving basal diet supplemented with one of the Bacillus strains at 1 × 1011 CFU/kg. Among these, B. amyloliquefaciens C271 significantly increased breast muscle yield (p < 0.05), improved jejunal morphology-evidenced by increased villus height and villus height-to-crypt depth ratio (p < 0.05)-and positively modulated cecal microbiota composition compared to the CON group. These findings demonstrate that the newly isolated B. amyloliquefaciens C271 possesses strong probiotic properties in vitro and promotes growth performance and gut health in broilers, suggesting its viability as an antibiotic growth promoter substitute.