Early-life supplementation of poultry-derived lactobacilli drives microbial succession and gut immune modulation in broiler chickens.

While probiotic supplementation via feed or drinking water is well known to improve poultry gut health by modulating the microbiota and enhancing immune function, the effects of in ovo supplementation remain largely unexplored. In this study, we investigated the effects of administering a lactobacilli cocktail in ovo (embryonic day 18), post-hatch, and in combination on gut immunity and the succession of the cecal microbiota in broilers over 5 weeks. 16S rRNA gene-based sequencing of cecal contents revealed a steady increase in Shannon diversity during the first 2 weeks (PERMANOVA, p < 0.005), with community structure stabilizing by week 3 across all groups. In ovo administration of lactobacilli improved early hatch rates and modulated microbial composition during early succession, including reductions in Klebsiella and Enterococcus, and enrichment of Lactobacillus, during the first two weeks (MaAsLin2, q < 0.25). These microbiome shifts were accompanied by a reduction in the expression of the pro-inflammatory cytokines, including interferon gamma (IFN-γ), interleukin-1β (IL-1β), and IL-8 in the cecal tonsils. These findings highlight the transient yet critical role of early-life probiotic interventions in shaping gut microbial colonization and immune response in broiler chickens. More importantly, a single in ovo lactobacilli dose yielded effects comparable to weekly oral or combined administration.