Shell gland RNA-seq reveals key genes regulating eggshell quality and potential links between eggshell quality and hatchability across different laying stages.

The quality of hatching eggs and their hatchability are key factors affecting the production efficiency of breeding flocks. The quality of hatching eggs is commonly graded directly based on the eggshell quality. Our study monitored the eggshell quality and hatchability in Hy-Line Brown laying hens aged 23 to 73 weeks, to determine core eigenvectors that influence hatchability from multiple eggshell quality indicators. Hatching performance indicators, including fertile egg rate, hatchability of fertile eggs and hatchability of eggs set, were found to exhibit a trend of low level in early laying stage, and increased to a peak at 39 weeks of age during mid-laying stage, followed by a continuous decline in late laying stage. Similar trend also observed in eggshell quality indicators, such as eggshell strength, eggshell thickness, eggshell weight, eggshell rate and eggshell microstructure. Canonical correlation analysis revealed egg weight, equatorial eggshell thickness and pointed end eggshell thickness showed strong correlation with the main indicators affecting hatchability of fertile eggs (r = 0.987, P = 0.013). Further, shell gland RNA sequencing was applied to explore candidate genes or pathways that might affect eggshell thickness. Short time series expression miner (STEM) analysis identified several key genes (ATP2C2, CA9, CDC20B, WNT4, WNT7A and CACNB3) that were enriched in pathways related to calcium signaling, bicarbonate metabolism, vesicle transport and Wnt signaling, thereby highlighting their potential critical roles in eggshell thickness regulation. Our findings preliminarily delineate the relationship between easily measurable eggshell quality and hatchability, which provides a theoretical basis and candidate gene targets for improving hatching performance.