Enhanced Macrophage and Granulocytic Recruitment with Increased Neo-Angiogenesis in Chicken Embryo Yolk Sac Following In Ovo Probiotic Blend Administration

The yolk sac (YS) plays a pivotal role in avian embryonic development, contributing to both haematopoiesis and immune maturation. This study aimed to evaluate the effects of in ovo administration of a commercial probiotic blend (Slab51^®) on YS cellular dynamics in chicken embryos. At embryonic day (ED) 18, Ross308 broiler eggs were injected with either the probiotic suspension (P) or sterile saline solution (C). YS tissues were sampled at 8, 12, 24 and 36 h post-inoculation for histological and immunohistochemical analysis. Probiotic-treated embryos exhibited a significant reduction in granulocytic foci within the YS, potentially reflecting enhanced peripheral migration of mature granulocytes. Concurrently, a progressive increase in Iba-1+ macrophages was observed in the probiotic group, suggesting accelerated macrophage differentiation. Immunophenotyping revealed a predominance of M1-iNOS+ macrophages across all timepoints, although a significant increase in M2-CD204+ macrophages was detected at 36 h in probiotic-treated embryos, indicating a possible anti-inflammatory shift. Furthermore, a marked increase in CD31+ endothelial cells in the probiotic group supports an associated rise in neo-angiogenesis. These findings suggest that in ovo probiotic administration modulates the YS microenvironment by promoting early macrophage recruitment, macrophage polarization and vascular remodeling. To our knowledge, this is the first report to demonstrate probiotic-induced structural and immunological alterations in the chicken embryo YS. These results provide novel insights into the early immunomodulatory effects of probiotics and highlight the potential of the YS as a key mediator of host-probiotic interaction during embryogenesis.