Integrated in vivo and in vitro evidence reveals that dietary chlorogenic acid improves eggshell quality in H 2 O 2 ‑challenged laying hens by preserving shell gland health and modulating calcium metabolism

This study examined chlorogenic acid (CGA) effects on eggshell quality, gland integrity, and calcium metabolism in laying hens under hydrogen peroxide (H2O2)-induced oxidative stress. In Experiment 1, 240 43–week-old Hy-Line Brown hens were randomly assigned to four groups 0, 400, 600, 800 mg/kg CGA for 12 weeks. Experiment 2 assigned 240 hens to control, H2O2, CGA (600 mg/kg), and CGA+H2O2 groups. H2O2 was intraperitoneally injected at 2.96 mmol/kg body weight on days 64 and 78. In vivo, CGA linearly increased eggshell thickness at all measured sites (p < 0.05). The thickness of the palisade and mamillary layer was improved, accompanied by upregulated expression of carbonic anhydrase (CA), ovocalyxin-32 (OCX32), and progesterone receptor (PGR) (p < 0.05). CGA alleviated oxidative stress by decreasing malondialdehyde content and increasing glutathione peroxidase (GSH-Px) activity (p < 0.05). H2O2 exposure reduced palisade layer thickness, induced mucosal atrophy and inflammatory infiltration, and increased H2O2 and total superoxide dismutase levels while decreasing GSH-Px activity (p < 0.05). It also downregulated OCX32 and proliferating cell nuclear antigen (PCNA) expression (p < 0.05). All these effects were reversed by CGA. In vitro, H2O2 decreased shell gland epithelial cell viability, reduced mitochondrial membrane potential, and elevated intracellular and mitochondrial Ca2+ levels. It also upregulated sodium-calcium exchanger-1 (NCX1), interleukin-1β (IL-1β), and IL-6, while downregulating sarco/endoplasmic reticulum Ca2+-ATPase 3 (ATP2A3), CA, and OCX32 (p < 0.05). CGA pre-treatment attenuated all these alterations. Collectively, CGA protects shell gland function and improves eggshell quality through maintaining redox balance, calcium homeostasis, and mitochondrial integrity.