Objective: Selection for growth-related traits in commercial chicken flocks has been associated with suppressed immune efficiency, increased susceptibility to various pathogens and stressors, and higher mortality rates. This study was conducted to investigate the potential effects of in-ovo injection of Lactobacillus rhamnosus during late embryonic development on hatchability, mortality, growth performance, selected blood parameters, relative weights of immune-related organs, immune response, malondialdehyde concentration, and the expression of genes associated with inflammatory responses in the jejunum of Arian chickens.

Methods: In this study, one hundred 18-day-old Arian embryonated eggs were randomly assigned to either the probiotic or the control group. Probiotic treated eggs were injected with appropriate amount of Lactobacillus rhamnosus (ATCC 7469) suspension solution, while eggs of the control group were injected with same volume of sterile phosphate-buffered saline. Each treatment consists of five replicates, with 10 chickens in each replicate. Resulting chicks were reared under uniform management conditions for 42 days. Performance metrics, including hatchability, cumulative mortality, and body weight at hatch, 21, and 42 days, were recorded. At 42 days, four representative birds per experimental group were euthanized for blood and jejunal tissue collection. Evaluated parameters included relative weights of the bursa of Fabricius and spleen, Newcastle disease virus (NDV) antibody titers, and serum biochemical indices (total protein, albumin, and globulin). Additionally, cecal Lactobacillus and Escherichia coli populations, jejunal lipid peroxidation (MDA), and the mRNA expression of TNF-α, IL-6, and IL-10 were determined.

Results: Statistical analysis revealed that in ovo delivery of Lactobacillus rhamnosus exerted no significant influence on hatchability, overall mortality, or body weight at hatch, 21, and 42 days of age (P > 0.05). Similarly, systemic physiological indices—including serum total protein and albumin levels—and the relative weights of the Bursa of Fabricius and Spleen remained unaffected by the intervention (P > 0.05). Furthermore, no significant alterations were observed in cecal Escherichia coli colonization or jejunal IL-10 gene expression (P > 0.05). Conversely, the treatment effectively modulated oxidative stress and inflammatory pathways; L. rhamnosus supplementation significantly attenuated malondialdehyde (MDA) concentrations and downregulated the mRNA expression of pro-inflammatory IL-6 and TNF-α in the jejunum (P < 0.05). Additionally, the intervention enhanced humoral immunity and gut health, as evidenced by significantly higher Newcastle disease vaccine antibody titers, elevated serum globulin concentrations, and a marked increase in cecal Lactobacillus populations compared to the control group (P < 0.05)

Conclusion: The findings of this study suggest that in-ovo injection of Lactobacillus rhamnosus during late embryonic development effectively enhances the immune function of broiler chickens. This improvement is likely mediated through multiple mechanisms, including the modulation of cecal microbiota composition and the regulation of signal transduction pathways that control the expression of inflammatory markers in the gastrointestinal tract. Investigating maternal-to-egg microbial seeding provides a novel framework for understanding how maternal-derived microbiomes drives early immune maturation in broiler chicks through epigenetic remodeling and metabolic rewiring.