In ovo injection of Lactobacillus reuteri 21 modulates gut microbiota and attenuates Clostridium perfringens infection via reuterin-mediated immunomodulation and antioxidant defense in Meiling yellow-feather broilers

1. In ovo injection of Lactobacillus reuteri 21 enables probiotic pioneer colonization LR21 inoculation increases intestinal reuterin content 2. Reuterin suppresses macrophage inflammation in Clostridium perfringens (Cp) infection 3. Reuterin regulates glutathione metabolism and antioxidant defense during Cp infection Clostridium perfringens (Cp) is a major enteric pathogen in poultry, threatening both animal health and food safety. This study investigated the protective effects of Lactobacillus reuteri 21 (LR21) administered via in ovo injection against Cp infection in broilers. A total of 360 chicks, previously injected in ovo on embryonic day 18, were randomly allocated to four groups ( n =6 replicates, 15 birds each): CON (PBS), Cp (PBS+Cp), IOF (LR21), and IOF-Cp (LR21+Cp). Birds were reared for 21 d. A two-way ANOVA was applied to determine the main and interaction effects for in vivo outcomes and one-way ANOVA for in vitro assays. Significant findings were followed by Tukey's HSD for pairwise comparisons. Although in ovo injection of LR21 slightly mitigated Cp-induced growth suppression, it significantly increased the jejunal villus height and reduced epithelial apoptosis ( P