Combination of In Ovo Nutritional Components Improves Embryonic Development, Growth Performance, and Gut Health in Broiler Chickens

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Abstract
This study evaluated the effects of intra‐amniotic administration of combined energy, amino acid, and vitamin sources on embryonic development, post‐hatch growth performance, and intestinal health in broiler chickens. On Day 18 of incubation, 460 fertile Ross 308 eggs were allocated to four groups: non‐injected negative control (NC), saline‐injected positive control (PC), nutrient solution I (NS‐I; glucose, lysine, and ascorbic acid), and nutrient solution II (NS‐II; creatine monohydrate, glutamine, and ascorbic acid). At hatch, chick quality traits and intestinal histomorphometric and immunohistochemical parameters were assessed, and post‐hatch performance was monitored for 35 days. Intra‐amniotic administration, particularly NS‐II, significantly increased hatch weight, chick length, and total intestinal length ( p < 0.05). Both nutrient solutions improved jejunal morphology at hatch by increasing villus height, the villus height‐to‐crypt depth ratio, and absorptive surface area, while reducing crypt depth ( p < 0.05). The proliferation index and PCNA expression were significantly higher in the nutrient‐injected groups, indicating enhanced epithelial cell proliferation. During the 35‐day rearing period, NS‐II significantly improved body weight gain, feed intake, carcass weight, and the European Production Efficiency Factor (EPEF) ( p < 0.05), without adversely affecting the feed conversion ratio. Cecal microbiota composition and overall meat quality parameters were not negatively affected; however, lower coliform and mesophilic bacterial counts were observed in meat samples from the NS‐II group during refrigerated storage on Days 5 9. In conclusion, intra‐amniotic supplementation with combined energy, amino acid, and vitamin sources, particularly creatine monohydrate and glutamine combined with ascorbic acid, positively modulated early intestinal development and improved growth performance in broilers without compromising meat quality.

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