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

Eggs are an affordable and nutritious source of high-quality animal protein. The production levels and reproductive performance of laying hens have been significantly improved over the past several decades. Improving eggshell quality while maintaining high productivity poses a new challenge for breeders. Eggshell strength is an important productive trait in poultry and of great economic significance, yet its genetic mechanism remains unclear. In this study, we conducted analyses of the eggshell strength phenome, single-cell transcriptomes of the uterus from the high and low eggshell strength groups, multi-tissue transcriptomes, and the proteome of the uterine fluid to explore the regulatory mechanisms that affect eggshell strength. There were no significant differences in the concentrations of serum calcium and phosphorus between the high and low eggshell strength groups. The first single cell transcriptome atlas of the uterus of Rhode Island Red chickens were successfully constructed, and nine cell types were identified in this atlas. These cell types can be divided into four categories: smooth muscle cells, epithelial cells, endothelial cells, and immune cells. Through integrating multi-omics results, the roles of collagen family genes (COL4A1/2, COL1A1/2, COL5A1, and COL6A1/2/3), solute carrier family genes (SLC4A4/7, SLC6A4, SLC9A2/9, and SLC38A2), ATPase family genes (ATP1A1, ATP1B1, ATP2B1/2, ATP2A2/3, and ATP2C1), calcium voltage - gated channel family genes (CACNB2, CACNA1C, and CACNA2D1), annexin family genes (ANXA5 and ANXA6), and integrin subunit family genes (ITGB1 and ITGA9) in determining eggshell strength were clarified. These genes associated with eggshell strength were mainly closely related to focal adhesion, regulation of actin cytoskeleton, ECM-receptor interaction, and the calcium signaling pathway. In addition, collagen family genes were found to be mainly expressed in smooth muscle cells. This might be related to the inversion that occurs in the uterine region during eggshell calcification, which enables a closer combination of calcium ions and matrix proteins. The insights derived from this study provide a theoretical basis for understanding the regulatory mechanisms underlying the potential for enhancing eggshell strength. Additionally, they lay a theoretical foundation for achieving an ultra-long laying period in laying hens in the future.