rawdata.xlsx

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 days. 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 supplementation slightly mitigated Cp-induced growth suppression, it significantly increased the jejunal villus height and inhibited epithelial cell apoptosis (P < 0.05). It also downregulated pro-inflammatory genes including NOD1, MyD88, NF-κB, and JNK, and inhibited the M1-type macrophage polarization in the jejunum compared to Cp challenge. Cp challenge altered β-diversity and enriched Clostridium perfringens, while LR21 in ovo injection increased Roseburia, Lactobacillus, and specifically Lactobacillus reuteri. Furthermore, LR21 in ovo injection increased the production of its signature metabolite, reuterin, in Cp-challenged broilers. In vitro, reuterin suppressed pro-inflammatory cytokines in macrophages and protected intestinal organoids from Cp-induced damage. Mechanistically, reuterin inhibited the TLR4/MAPK/NF-κB signaling pathway and activated the Nrf2/HO-1 pathway thereby alleviating inflammation response in Cp-infected macrophages. Additionally, reuterin maintained iron homeostasis of enteroids by upregulating FTH1 and SLC40A1 (P < 0.05) and enhanced antioxidant capacity via SLC7A11-mediated cystine uptake (P < 0.05), thereby protecting against ferroptosis.