The layer hen oviduct undergoes functional shifts in immunometabolism-associated microbiota over the production cycle

ABSTRACT Age-related decline in egg production represents a major limitation in the productive lifespan of commercial layer hens, yet the contribution of the oviductal microbiome to this process remains poorly understood. Here, we characterized microbial diversity and composition along the hen reproductive tract from High-Lay into Post-Lay, leveraging species-level resolution and an expanded temporal framework spanning High-Lay, Late-Lay, and Post-Lay phases. From a flock of 250 animals, 8 to 14 were sampled at each time point for analysis. Alpha and beta diversity differed significantly between the oviduct and ceca; however, species-level analysis also revealed a marked decrease in alpha diversity in the ovary as compared to the isthmus-uterus junction, a distinction that was pronounced in High-Lay hens but diminished with age. Beta-diversity analyses revealed that the Late- and Post-Lay microbiomes are more similar to each other than the High-Lay microbiome, suggesting a shift toward a lay-unsupportive microbiome in the latter half of the productive lifecycle. Predictive and hypothesized functional interpretation of differentially abundant taxa suggests a potential transition from a “lay-supportive” microbiome in High-Lay hens, possibly enriched for xenobiotics handling, antimicrobial production, and estrogen-responsive taxa to a more “lay-unsupportive” microbiome in Late- and Post-Lay hens, which may be characterized by pathways associated with inflammation, apoptosis, bile acid metabolism, and non-sex-steroid hormone biosynthesis. Collectively, these data suggest that age-associated restructuring of the oviductal microbiome may precede reproductive senescence and reflect a potential shift in microbial function from reproductive support toward stress adaptation and immune maintenance. These findings identify the Late-Lay period as a critical window for microbiome-based interventions aimed at sustaining reproductive performance. IMPORTANCE Examination of the aging layer hen reproductive microbiome and its association with hen health and egg-lay revealed a “lay-supportive” microbiome in the High-Lay period that appears to be replaced by a “lay-unsupportive” microbiome as early as the Late-Lay period that more closely resembles the Post-Lay microbiome. Elucidating the specific taxa behind these shifts identified short-chain fatty acid producers and bile acid metabolizers that may be involved in immunomodulation, which could inform probiotic supplementation strategies that support aged-hen health. Orally supplementing aged hens to extend the lay period will allow for longer and more sustainable production cycles along with reducing bird resources, an outcome which is also economically desirable.