Dietary bile acid supplementation improves eggshell calcium content and structural integrity in late-laying hens

The objective of this study was to investigate whether dietary bile acid (BA) supplementation affects production performance and improves eggshell quality in late-laying hens by transcriptionally upregulating shell gland biomineralization genes ( CALB1 , CA2 , OC-116 ), thereby producing calcium-dense eggshells for functional food applications. In this study, a total of 72 laying hens were assigned to three groups: peak laying hens at 161 days of age (Peak lay), late laying hens at 462 days of age (Late lay), and BA-supplemented late laying hens at 462 days of age (Late lay + BA). BA supplementation did not significantly affect laying rate or feed efficiency but showed a numerical reduction in eggshell breakage and increased calcium concentration in shell gland fluid, while improving eggshell ultrastructure as evidenced by increased mammillary density and effective eggshell thickness. Serum calcium levels remained unchanged across groups, whereas calcium concentration in shell gland fluid increased. Transcriptomic analysis of shell gland tissue revealed distinct gene expression profiles associated with both laying stage and BA supplementation. Key genes involved in calcium transport and eggshell mineralization (e.g., CALB1 , CA2 , and OC-116 ) were downregulated during late lay but significantly restored by BA supplementation, as confirmed by qRT-PCR. Expression levels of these genes were positively correlated with effective eggshell thickness and mammillary density. Collectively, these findings suggest that BA supplementation improves eggshell quality in late-laying hens through upregulation of key biomineralization genes in the shell gland, thereby enhancing local calcium transport and mineralization efficiency. This nutritional strategy produces calcium-rich eggshells that have the potential to serve as a high-quality raw material for functional eggshell powder, thereby supporting by-product valorization.