Abstract
Probiotic supplementation supports poultry gut health by modulating microbiome and promoting immune development, yet limited information is known about the effects of early, particularly embryonic, supplementation. In this study, we investigated the effects of administering a lactobacilli cocktail in ovo (embryonic day 18), post-hatch, or both on gut immunity and the succession of the cecal microbiota in broilers over five weeks. 16S rRNA gene-based sequencing of cecal contents revealed a steady increase in Shannon diversity during the first three weeks (PERMANOVA, p < 0.005), with community structure stabilizing by week 3 across all groups. In ovo lactobacilli administration 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 reduced expression of pro-inflammatory cytokines (IFN-γ, IL-1β, and IL-8) in 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.