Genetic and metabolic regulation of eggshell quality in aging hens: Integrated multi-omics insights into CYP7A1, CALM1, and cholic/stearic acid metabolism.

While breeding improvements have prolonged the laying cycle for greater productivity, a concurrent deterioration in eggshell quality is commonly observed in aged hens. To investigate the genetic basis of this deterioration, we conducted the multi-omics analyses on the hypothalamus, liver, duodenum, and uterus from 100-week-old Rhode Island Red hens. We identified significant genomic regions associated with total eggshell weight during 99-100 weeks (TESW99-100) and eggshell brownness at 100 week (ESB100). Transcriptomic profiling revealed distinct gene expression patterns across tissues, indicating that the uterus is the primary site that influences eggshell quality. Further single-cell analysis highlighted the critical role of unterine epithelial cells in the uterus in modulating these traits. Metabolomic profiling revealed strong associations between specific metabolites and eggshell quality phenotypes; cholic acid and taurocholic acid were tightly linked with TESW99-100, while stearic acid and alpha-linolenic acid levels were associated with ESB100. By integrating these multi-omics datasets, we identified six significant SNPs that not only regulated expression of two functional candidate genes (CYP7A1 and CALM1). These interactions were ultimately consolidated into a SNP-gene-metabolite ternary network. Together, these findings provide a multi-layered molecular framework for understanding the age-related decline in eggshell quality and offer new insights into its genetic regulation.