Eggshell Membrane and Chick Gastrointestinal Microbiota Interaction in Late-Stage Incubation of White Leghorn and Broiler Hatching Chicks

Hatching eggs possess multiple physical and chemical barriers that limit microbial invasion; however, the role of the eggshell membrane in shaping late-stage embryonic and early post-hatch gastrointestinal (GI) microbiota remains poorly understood. This study aimed to (i) validate a reproducible eggshell membrane extraction method, (ii) assess whether microbial loads differ between nest- and floor-laid eggs, (iii) examine relationships between eggshell membrane-associated microbiota and embryonic intestinal microbiota, and (iv) determine whether microbial blooms align with key stages of the hatching process. In a preliminary experiment using White Leghorn hatching eggs, no significant differences were observed in aerobic, anaerobic, or fungal membrane counts between nest- and floor-laid eggs. In a commercial hatchery study using Ross 708 broiler eggs, membrane and GI microbial populations were evaluated across days 18–20 of incubation, corresponding to pre-pipping, internal pipping, and external pipping/post-hatch stages. Significant, day-dependent shifts in microbial counts were observed, with strong interactions between sampling day and location (membrane vs. GI) for most bacterial groups. Enterococci and anaerobic bacteria were enriched in the GI tract prior to hatch, whereas aerobic, Gram-negative, and Staphylococcus populations were more abundant on membranes during late incubation. Post-hatch chicks exhibited markedly higher GI microbial loads compared to embryos, indicating rapid colonization during the hatch transition. Collectively, these findings demonstrate that the pipping and hatching process represents a critical window for microbial redistribution from eggshell membranes to the developing chick gut, highlighting the hatchery as a key control point for early-life microbial exposure and intervention strategies.