Multi-omics reveals key cell types and gene families regulating eggshell strength in chicken uteri

Eggs represent an accessible and nutrient-dense source of high-quality animal protein, and decades of selective breeding have markedly elevated reproductive output in commercial laying hens. However, sustaining elevated productivity while improving eggshell integrity presents a critical challenge, as the molecular mechanisms of eggshell strength remain unclear. In this study, phenotypic assessment of eggshell strength was combined with single-cell transcriptomic profiling of the uterus from high- and low-strength groups, transcriptomic analysis of multiple tissues, and quantitative proteomic analysis of uterine fluid. Serum calcium and phosphorus levels did not differ significantly between groups. A single-cell atlas of the Rhode Island Red uterus was successfully generated for the first time, identifying nine distinct cell populations encompassing smooth muscle, epithelial, endothelial, and immune subsets. Integration of transcriptomic and proteomic datasets revealed that genes encoding collagens ( COL4A1/ 2, COL1A1/ 2, COL5A1, and COL6A1/ 2/ 3), solute carriers ( SLC4A4/ 7, SLC6A4, SLC9A2/ 9, and SLC38A2), ATPases ( ATP1A1, ATP1B1, ATP2B1/ 2, ATP2A2/ 3, and ATP2C1), calcium voltage-gated channels ( CACNB2, CACNA1C, and CACNA2D1), annexins ( ANXA5 and ANXA6), and integrins ( ITGB1 and ITGA9) were key molecular determinants associated with variation in eggshell strength. These genes were primarily enriched in signaling cascades involved in focal adhesion, actin cytoskeleton regulation, extracellular matrix (ECM)-receptor interactions, and calcium signaling. Notably, collagen family genes were predominantly localized to smooth muscle cells, consistent with the tissue remodeling and uterine inversion that occur during shell calcification, which may enhance spatial proximity between calcium ions and matrix proteins. These findings establish a multi-omics framework for understanding the uterine regulatory mechanisms underlying eggshell formation and offer a molecular foundation for breeding strategies aimed at prolonging laying cycles while preserving shell quality.