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. • Multi-omics analysis reveals key regulators of eggshell quality in aged laying hens. • Uterine epithelial cells play a central role in eggshell quality modulation. • Cholic and taurocholic acids are strongly associated with late-stage eggshell weight. • Stearic and alpha-linolenic acids correlate with eggshell brownness in aged hens. • C YP7A1 and CALM1 are functional candidate genes linked to key metabolic pathways. • A SNP–gene–metabolite regulatory network explains age-related eggshell deterioration.