Apigenin Prevents Ovarian Aging by Regulating Ca<sup>2+</sup>-Mediated Endoplasmic Reticulum Stress in Laying Chickens.

Abstract

The sustainability of egg production in the poultry industry is frequently challenged by the progressive decline in ovarian function as laying chickens age. A primary driver of this reproductive transition is the functional deterioration of small white follicles (SWFs), which constitute the vital pre-hierarchical follicular reserve necessary for sustained egg production. However, the molecular mechanisms underlying age-associated SWF atresia remain poorly understood. In this study, we investigated the protective efficacy of apigenin (AP), a natural bioactive flavonoid, in mitigating follicular senescence by targeting calcium ion (Ca²⁺)-mediated endoplasmic reticulum stress (ERS) in D-galactose (D-gal)-induced SWFs and in naturally aged chickens. Our results revealed that AP treatment effectively rebalanced the D-gal-induced disruption of cell proliferation and survival. Molecular analysis of SWFs revealed that AP treatment promoted the coordinated restoration of transcriptional profiles of key Ca²⁺-handling genes, effectively counteracting the age-related disruption of ionic regulation. In addition, AP suppressed the aberrant upregulation of IP₃R and modulated the expression of other key Ca²⁺-regulatory genes, including <i>CACNA1C</i>, <i>CACNA1D</i>, <i>CAMKII</i>, <i>MCU</i>, and <i>ATP2B1</i>. This restoration of intracellular Ca²⁺ homeostasis was associated with attenuation of the ERS response, as evidenced by the decreased levels of GRP78 and CHOP, and the suppression of Caspase-3-mediated apoptotic signaling. The biological relevance of these findings was further validated in vivo using naturally aged chickens. Dietary supplementation with AP significantly enhanced pre-hierarchical follicle recruitment in aged laying chickens, and improved egg production and eggshell quality in aged laying chickens. Collectively, these findings indicate that AP can modulate ERS signaling in laying chickens by maintaining intracellular Ca²⁺ homeostasis, thereby enhancing laying performance. These results highlight AP as a promising nutritional intervention to enhance reproductive performance and extend productive longevity in poultry.