Integrating proteomics and metabolomics to elucidate the regulatory mechanisms of pimpled egg production in chickens

Eggshells not only protect the contents of the egg from external damage but are also a key factor influencing consumer choice, second only to price. In the later stages of egg production, the incidence of pimpled eggs significantly increases, severely affecting the hatchability and food safety of the eggs. This study compares the differences in the uterine proteomes and metabolomes of hens producing pimpled eggs and those producing normal eggs, aiming to identify the proteins and metabolites that may play a crucial role in the formation of pimpled eggs. A total of 242 differentially expressed proteins (DEPs) were identified in uterine tissue, of which 116 were upregulated and 126 were downregulated. Enrichment analysis revealed that the DEPs were enriched in pathways related to ion transport, energy metabolism, and immune responses. The study found that in the normal eggs (NE) group, HCO₃⁻ was predominantly transported via SLC4A1, although other transport pathways may also play a role. In contrast, in the pimpled eggs (PE) group, bicarbonate ions (HCO₃⁻) was primarily transported through SLC4A4. Additionally, a total of 44 differentially metabolites (DMs) were identified in the uterus, with 5′-Adenylic acid (ATP) being significantly downregulated in the PE group. The ions and matrix proteins required for eggshell formation are transported from uterine cells to the uterine fluid against a concentration gradient, a process that consumes a substantial amount of energy. The decrease in ATP concentration in the PE group may be a significant factor influencing the formation of pimpled eggs. Subsequently, we found that the DEPs and DMs were jointly enriched in several signaling pathways, including the FoxO signaling pathway related to energy metabolism, nicotinate and nicotinamide metabolism, and tryptophan metabolism associated with immune response. Notably, the DMs involved in these signaling pathways were all downregulated in the PE group. Our research findings indicate that SLC4A1, SLC4A2, and ATP2B4 (DEPs), along with 5′-adenylic acid and trigonelline (DMs), influence the formation of eggshells through mechanisms related to energy metabolism, ion transport, and immune response. These DEPs and DMs may serve as potential biomarkers for the genetic improvement of eggshell quality.