Integrative multi-omics analysis suggests regulatory mechanisms of sperm storage capability in aged laying hens

Sperm storage capability is the female reproductive tract’s ability to maintain sperm viability and fertility over prolonged periods. This trait declines with age, especially during the late laying period, reducing production efficiency. However, the underlying mechanisms remain unclear. This study assessed sperm storage traits, hormone profiles, uterovaginal junction (UVJ) histomorphology, and performed multi-omics (single-cell transcriptomics, metabolomics, metagenomics) in 90-week White Leghorn hens. Compared to the low sperm storage capability group, hens with high capability showed higher serum levels of prostaglandin F2α, testosterone, and triiodothyronine, as well as a larger cross-sectional area of sperm storage tubules (SST). Single-cell transcriptomic profiling of the UVJ revealed nine major cell types. Pseudotime trajectory analysis identified SST cells as a terminally differentiated cell type, originating from ciliated epithelial precursors via a transitional stage of proliferating epithelial cells. SST cells from the high group showed upregulation of hypoxia-responsive genes (HIF1A and EIF2AK3) and glycolysis. Meanwhile, immune cells exhibited downregulation of immune-related genes, indicating a local immunosuppressive microenvironment that may facilitate sperm survival. Integrating metabolomic and metagenomic data further suggested a possible link between Ligilactobacillus aviarius (L. aviarius) activity and enhanced glycolysis, potentially contributes to lactate accumulation. Collectively, we propose that a hypoxic SST niche drives host anaerobic glycolysis, and may be reinforced by L. aviarius. This synergistic lactate accumulation may induce cytoplasmic acidification to suppress sperm activation, thereby contributing to long-term storage. These findings provide a theoretical foundation for targeted manipulation of the UVJ microenvironment to enhance reproductive efficiency in aging hen populations.