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

## TL;DR

Changing the light cycle to 28 hours improves eggshell quality in older hens by altering gut microbes and metabolites, which helps with calcium absorption and uterine cell growth.

## Contribution

The study reveals how a 28-h light cycle improves eggshell quality in aged hens by altering gut microbiota and metabolite rhythms.

## Key findings

- The 28-h light cycle increased eggshell thickness and strength in aged hens.
- The 28-h cycle altered gut microbiota and metabolite rhythms, enhancing calcium absorption and uterine cell growth.
- Key microbes like Weissella, YRC22, and Paludibacter were linked to improved eggshell formation through synergistic interactions.

## Abstract

This study investigated how altering the light cycle affects eggshell quality in older laying hens. We compared a standard 24 h light cycle (16L:8D) with a 28 h light cycle (16L:12D), and found that the 28 h light cycle improved eggshell strength and thickness. Through analysis of gut bacteria and blood metabolites, the study showed that the 28 h light cycle increased microbial diversity and altered daily rhythms of both microbes and metabolites. These changes were linked to improved calcium absorption and support for uterine cell growth, both critical for strong eggshell formation. The findings suggest that modifying light schedules can be a practical strategy to enhance egg quality in aging hens.

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. Weissella promotes calcium deposition in eggshells through synergistic interactions with calcium chelators such as gluconic acid and threonine acid. Meanwhile, YRC22 and Paludibacter 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.