TMT-Based quantitative proteomic analysis reveals age-related changes in eggshell matrix proteins and their correlation with eggshell quality in Xinyang blue-shelled laying hens.

The decline in eggshell quality with increasing hen age may be related to changes in ultrastructure and chemical composition, with matrix proteins playing key roles in these changes. However, research on blue-shelled eggs remains limited. This study investigated the effects of hen age (35, 55, 75, and 85 weeks) on the physical, mechanical, and chemical properties of eggshells in the Xinyang blue-shelled laying hens, as well as their ultrastructural and nanostructural characteristics. Subsequently, a comparative proteomic analysis was performed to elucidate the differential protein profiles in eggshells from hens at 35 and 85 weeks of age. Results showed that egg weight, eggshell weight, and eggshell surface area increased with hen age, whereas eggshell stiffness decreased (p < 0.05). As the age advanced, the eggshell organic matter content declined (p < 0.05). The effective layer ratio, mammillae density, as well as the porosity and total pore area in the mammillary layer also decreased with age, whereas the ratio of the mammillary layer increased (p < 0.05). Compared to eggshells collected from 35-week-old hens, those from 85-week-old hens showed increases in egg weight, eggshell weight, surface area, and both the ratio and thickness of the mammillary layer (p < 0.05). However, significant decreases were observed in eggshell stiffness, organic matter content, phosphorus content, effective layer ratio, mammillae density, as well as the porosity and total pore area in the mammillary layer (p < 0.05). Additionally, eggshell stiffness, phosphorus content, and organic matter content were significantly correlated with each other (p < 0.05). Proteomic analysis identified 37 downregulated and 68 upregulated differentially expressed proteins (DEPs, FC > 1.2 or < 0.83, with a p-value < 0.05) in eggshells from 85-week-old hens compared to those from 35-week-old hens. These DEPs are associated with functions such as biomineralization, calcium transport, immunity, and proteases and protease inhibitors. Mantel and Pearson correlations suggest that these functions may be involved in regulating eggshell stiffness, phosphorus content, and organic matter content. Overall, the eggshell stiffness decreased from 35 to 85 weeks of age, which may be attributed to the reductions in eggshell organic matter and phosphorus contents, as well as the deteriorations in eggshell ultrastructure. The proteins associated with biomineralization, calcium transport, immunity, and proteases and protease inhibitors may contribute to these changes.