Changes in the gut microbiome, metabolic pathways, and intestinal gene expression during the peak, mid, and decline egg production phases in laying hens

The decline in egg production due to aging poses a significant challenge for the egg industry. This study investigated the cecal microbiota, predicted metabolic pathways, and ileal gene expression to clarify how gut health, nutrient availability, and physiological status influence egg production in laying hens across different ages. Cecal digesta and ileal tissues were collected from Hy-Line-W36 laying hens at peak-production (37-weeks of age), mid-decline (67-weeks of age), and decline-phase (87-weeks of age) (n = 15/ group). DNA from cecal digesta (8/group) was used for 16S rRNA gene sequencing with the Illumina and QIIME2 platforms to refine the raw reads. Ileal tissues were used for RNA extraction, and gene expression was quantified through qPCR. R, PICRUSt2, and STAMP were used for statistical analysis. Despite similar richness, hens in the mid-decline and decline-phases of egg production exhibited reduced microbial diversity. At peak production, the microbiome was dominated by carbohydrate-fermenting genera such as Prevotella, Megasphaera, and Anaerotignum; mid-decline hens acquired more Limosilactobacillus and butyrate-producing Acutalibacteraceae, while the decline-phase hens were enriched with maintenance-oriented taxa, including Peptostreptococcaceae. Functional predictions mirrored these shifts, where peak-production hens' profiles favored glycolysis, nucleotide synthesis, and rapid acetyl-CoA turnover; mid-decline hens showed enhanced amino acid biosynthesis, iron scavenging, and SCFA production; and decline-phase hens emphasized nucleotide/cofactor repair and stress-response pathways while central-carbon routes declined. Additionally, age-related changes in enzymatic functions suggested reduced metabolic efficiency. In decline-phase, the expression of ileal antioxidant (SOD1, SOD2, GPX1) and tight-junction genes (CLDN1, OCLN) significantly decreased. The higher expression of Gadd45b at the decline-phase suggests cellular damage and leakage at the ileum. Moreover, decline-phase hens exhibited decreased levels of TLR4 and NF-κB, along with higher levels of IL-4 and IL-12, indicating a compromised immune response. Additionally, reduced expression of solute carrier transporter SLC1A1, SLC5A1 (amino acid and peptide), SLC7A6 (L-lysine, L-arginine), SLC19A2 (Thiamin), and FATP4 (Fatty acid) during the decline-phase, indicating insufficient energy availability for the hens, ultimately leading to a decrease in egg production. These findings reveal age-related changes in gut microbiota, nutrient transport, and immunity, contributing to decreased egg production in hens. This information could be pivotal for developing dietary and management strategies to enhance flock health and productivity.