Comparative and combined effects of in ovo probiotic injection and early feeding on growth development, intestinal morphology, ileum microbiota, digestive enzymes, and immune responses in broiler chickens

The present study was conducted to evaluate the effects of in-ovo injection and early feeding on performance, intestinal morphology, ileum microbiota, digestive enzyme activities, and immune system development in broiler chickens. 1,800 Ross 308 eggs were divided into 6 treatment groups with 10 replicates of 30 eggs in a completely randomized 3 × 2 factorial design. The experimental treatments included six groups as follows: NI-NF: Non-injected eggs and as hatches chicks with 48 h delayed access to feed (negative control); NI-YF: Non-injected eggs and as hatched chicks with immediate nutrient gel access; SL-NF: In-ovo saline injection and delayed feed access; SL-YF: In-ovo saline injection and as hatched chicks with immediate nutrient gel access; PB-NF: In-ovo probiotic injection (100 μL per egg) and delayed feed access; PB-YF: In-ovo probiotic injection and as hatched chicks with immediate nutrient gel access. Hatchability did not differ among treatments (P = 0.721). Chicks from probiotic-injected eggs (PB-NF and PB-YF) exhibited higher body weight at hatch than those from non-injected (NI-NF, NI-YF) and saline-injected (SL-NF, SL-YF) eggs (P = 0.043). At 48 h post-hatch, the PB-YF group recorded the greatest body weight (P = 0.004). During 11-24 days, PB-YF birds showed the highest BWG, differing from all other groups (P = 0.004). From 25-42 days, PB-YF, SL-YF, and NI-YF maintained higher BWG values compared with NI-NF and SL-NF (P = 0.017). At all ages, chicks with early feeding access (NI-YF, SL-YF, and PB-YF) exhibited significantly greater villus height compared with their delayed-fed counterparts (NI-NF, SL-NF, and PB-NF) (P < 0.05). At 10 days, the PB-NF group exhibited the lowest C. perfringens and E. coli population, significantly lower than NI-NF and SL-NF (P = 0.034; P = 0.001). At 10 days, PB-YF group chicks exhibited the highest amylase, lipase and protease activity (P < 0.05). A significant interaction between in ovo probiotic injection and early feeding was detected for IgM, IgA, IL-10, and IL-6 at 3 and 10 days of age (P < 0.05). At day 3, the PB-YF group showed the lowest corticosterone level (P = 0.001), while no significant difference was detected at day 10 (P = 0.128). In conclusion, in ovo probiotic injection improved early survival, immunity, and initial growth, whereas early feeding provided more persistent advantages throughout the production period; together, these strategies acted synergistically to optimize broiler health, gut development, and overall performance.