Proteomic changes in extracellular vesicle and uterine fluid proteins correlated with eggshell quality in aged laying hens

Extracellular vesicles (EVs) in uterine fluid (UF) are thought to regulate eggshell calcification, yet their composition and functions remain unclear. This study compared EVs and EV-deleted UF (UF^EV-) between high and low eggshell breaking strength groups to explore their contributions to eggshell calcification and quality decline in aged hens. The results show that the low strength group exhibited significant decreases across multiple parameters, including eggshell breaking strength, eggshell percentage (eggshell weight/egg weight), thickness, and fracture toughness, compared to the high breaking strength group (P < 0.05). Additionally, eggshells with low breaking strength also displayed increased type B mammillae, along with reduced effective layer ratio and decreased early fusion of mammillae (P < 0.05). There were no significant differences on particle concentration, size distribution, and calcium concentration of EVs, while the protein concentration of EVs at 7 h post-oviposition (PO) was lower in the low breaking strength group than in the high breaking strength group (P < 0.05). In the high breaking strength group, the particle concentration, the percentage of large particles, and the protein concentration of EVs were reduced at 17 h PO compared to 7 h PO (P < 0.05). At 7 h PO, the intersectional proteins between UF^EV- and EVs were mainly involved in endocytosis, nucleocytoplasmic transport, proteasome, and metabolism. In contrast, proteins unique to UF^EV- were enriched in other glycan degradation and the spliceosome, while EV-specific proteins were associated with ribosome, apelin signaling pathway, regulation of actin cytoskeleton, endocytosis, and progesterone-mediated oocyte maturation pathways. Additionally, 290 differentially expressed proteins (DEPs) were identified in UF^EV-, while 183 DEPs were detected in EVs. The DEPs in both UF^EV- and EVs were primarily associated with vesicular transport, matrix protein, ion transport, and protein synthesis. Thus, declined eggshell quality is closely associated with altered protein composition in both UF^EV- and EVs, particularly those involved in vesicle function, matrix deposition, and ion transport.