Improved Eggshell Quality in Aged Hens Through Circadian Gut Microbiota and Metabolite Changes Induced by a 28-h Ahemeral Light Cycle

The decline in eggshell quality of aged laying hens represents a major economic challenge in poultry production. While a 28 h ahemeral light cycle has been shown to improve eggshell quality, its underlying mechanism remains unclear. This study randomly assigned 260 74-week-old Hy-Line Brown laying hens to two light cycle groups, a normal 24 h cycle group (16L:8D) and a 28 h ahemeral cycle group (16L:12D). Each treatment comprised 130 hens divided into two replicate groups. The trial lasted 16 weeks. We systematically analyzed circadian rhythms of gut microbiota and serum metabolites using 16S rRNA sequencing and untargeted metabolomics. Compared with the 24 h cycle, the 28 h cycle significantly enhanced eggshell thickness by 0.04 mm and 0.02 mm, and eggshell strength by 4.19 N and 4.76 N at 79 and 84 wk, respectively. Mechanistically, the 28 h light cycle remodeled the circadian rhythms of gut microbiota, increasing their richness and diversity, and altered the rhythmic patterns of serum metabolites. We identified nine microbial genera and three hundred seventy metabolites that exhibited opposite rhythmic patterns under the two light cycles. These changes were primarily enriched in pathways related to amino acid, carbohydrate, lipid, and energy metabolism. Correlation analysis further revealed strong associations between key microbes and functional metabolites. <i>Weissella</i> promotes calcium deposition in eggshells through synergistic interactions with calcium chelators such as gluconic acid and threonine acid. Meanwhile, <i>YRC22</i> and <i>Paludibacter</i> synergistically support membrane formation substances, thereby promoting the proliferation of uterine epithelial cells and eggshell formation. Our findings indicate that the 28 h ahemeral light cycle improved eggshell quality in aged hens by remodeling the circadian rhythms of gut microbiota and metabolites, thereby synergistically enhancing calcium ion absorption and uterine tissue health. This provides a novel theoretical basis and practical direction for improving late-phase egg quality through light management strategies.