Abstract:

The study aimed to explore the regulatory pathways and important candidate genes that affect the differences in bone metabolism before and after egg laying in laying hens, providing a theoretical basis for further research on the regulatory mechanisms for maintaining bone health in laying hens. A total of 15 at 15-week-old and 12 at 22-week-old Hyline Grey laying hens with similar body weights and reared under identical conditions were selected. Blood and tibia samples were collected to measure plasma levels of calcium, phosphorus, estrogen, and other biochemical markers related to bone metabolism. Based on estrogen levels, tibia tissues from 6 hens within each age group were selected to construct 12 transcriptome libraries. Differentially expressed genes (DEGs) were then screened from these libraries, and GO functional annotation and KEGG pathway enrichment analyses were performed with DEGs. Additionally, 6 randomly selected DEGs were validated for their relative expression levels using qRT-PCR. When comparing 15-week-old hens to 22-week-old hens, significant changes (P < 0.05) were observed in plasma bone metabolism markers and estrogen levels in the laying hens. The clean reads of 40 082 240-186 154 554 were obtained from the 12 constructed tibial cDNA libraries, with a minimum Q30 value of 92.18% and a contrast rate between 76.52% and 90.55%. A total of 1 832 DEGs were screened from the tibia of laying hens at 15 and 22 weeks of age. Among them, 914 genes were downregulated and 918 genes were upregulated. GO functional annotation revealed that differentially expressed genes were significantly enriched in protein processing, response to endoplasmic reticulum stress, and collagen fibril organization (P < 0.05). KEGG enrichment analysis revealed significant enrichment pathways related to bone metabolism, including steroid biosynthesis, thyroid hormone synthesis, and TGF- β signal pathway, MAPK signaling pathway, PI3K-Akt signaling pathway. Screening out 18 genes were shared by these pathways as key candidate genes for regulating bone metabolism. This study revealed that there are differences in gene expression in the tibia tissue of laying hens, and multiple DEGs and pathways affecting bone metabolism at pre- and post-laying stages were screened in laying hens. These results provide theoretical basis for further research on the mechanism of bone metabolism changes in laying hens before and after egg laying.

Key words: laying hens, transcriptome, differentially expressed genes, physiological stages, tibia, bone metabolism