Multi-omics analysis reveals gut microbiota-host metabolism-shell gland axis underlying eggshell deterioration in aged laying hens.

Eggshell quality is an important indicator of the nutritional status, health, and welfare of laying hens and plays a key role in minimizing economic losses and maintaining egg marketability during extended laying cycles. During the late laying period, aged hens commonly exhibit progressive eggshell thinning and reduced shell strength, which increase egg breakage and limit productive lifespan. Although shell gland dysfunction has been implicated in eggshell deterioration, how intestinal microbial remodeling, host metabolism, and shell gland function are coordinated during this process remains unclear. In this study, we integrated eggshell phenotyping, shell gland histology, cecal microbiota profiling, metabolomics, and transcriptomics in Guizhou Recessive White-feather chickens at peak and late laying stages. We found that late-laying hens displayed impaired eggshell ultrastructure and shell gland mucosal degeneration, accompanied by a shift from Lactobacillus- and Limosilactobacillus-enriched microbiota to a dysbiotic community characterized by increased Escherichia-Shigella. Multi-omics analyses revealed disrupted tryptophan metabolism, altered indole-3-pyruvic acid and indoleacetic acid levels, and extensive remodeling of shell gland energy metabolism, including oxidative phosphorylation and the tricarboxylic acid cycle. Key genes related to mitochondrial function and calcium transport, including MFN1, MDH1, ATP2A3, and SLC8A1, were dysregulated, suggesting impaired mitochondrial homeostasis and calcium handling in the shell gland. Integrated analysis further linked microbial genera, tryptophan-related metabolites, shell gland genes, and eggshell quality traits, supporting a coordinated gut microbiota-host metabolism-shell gland axis. These findings provide a systems-level view of eggshell deterioration in aged laying hens and identify potential microbial and metabolic targets for improving eggshell quality during extended laying cycles.