Metformin Prevents Follicular Atresia in Aging Laying Chickens through Activation of PI3K/AKT and Calcium Signaling Pathways: Increased follicular atresia occurs with aging and results in reduced fecundity in laying chickens. Therefore, relieving follicular atresia of aging poultry is a crucial measure to maintain sustained high laying performance. As an antiaging agent, metformin was reported to play important roles in preventing aging in diverse animals. In this study, the physiological state of the prehierarchical follicles in the peak-laying hens (D280) and aged hens (D580) was compared, followed with exploration for the possible capacity of metformin in delaying atresia of the prehierarchical follicles in the aged D580 hens. Results showed that the capacity of yolk deposition within follicles declined with aging, and the point of endoplasmic reticulum- (ER-) mitochondrion contact decreased in the ultrastructure of the follicular cells. Meanwhile, the expression of apoptosis signaling genes was increased in the atretic small white follicles. Subsequently, the H₂O₂-induced follicular atresia model was established to evaluate the enhancing capacity of metformin on yolk deposition and inhibition of apoptosis in the atretic small white follicles. Metformin inhibited apoptosis through regulating cooperation of the mitochondrion-associated ER membranes and the insulin (PI3K/AKT) signaling pathway. Furthermore, metformin regulated calcium ion homeostasis to relieve ER-stress and inhibited release of mitochondrion apoptosis factors (BAD and caspase). Additionally, metformin activated PI3K/AKT that suppressed activation of BAD (downstream of the insulin signaling pathway) in the atretic follicles. Further, serum estrogen level and liver estrogen receptor-α expression were increased after dietary metformin supplementation in D580 hens. These results indicated that administration of dietary metformin activated the PI3K/AKT and calcium signaling pathway and enhanced yolk deposition to prevent chicken follicular atresia.

In the poultry, only less than 5% primordial follicles in the ovary can develop into the prehierarchical follicles (PHFs) leading to progressive development, ovulation, and egg formation. This low rate of recruitment indicates a huge potential for improvement of the laying performance. A great reduction in egg production is caused by aging with extensive follicular atresia. In this study, age-related changes in the laying performance and ovarian status were compared between the peak-lay (D280) and aged (D580) chickens. Subsequently, a cross coculture of PHFs and granulosa cells (GCs) from D280 or D580 hens was adopted to reveal the mechanism of declined follicle development. Results showed that persistent endoplasmic reticulum (ER) stress in GCs of the aged hens was accompanied with intensified apoptosis. Bone morphogenetic protein 4 (BMP4) secreted by GCs of PHFs in D280 hens was capable of relieving ER stress and improving follicular dominance for selection in D580 hens. During this action, BMP4 reduced free calreticulin (CALR, an ER marker) content and attenuated cell apoptosis in PHFs of D580 hens via the PERK-CHOP-BCL2/caspase3 or CALR-Ca²⁺-BCL2-caspase12 pathway. Furthermore, BMP4 prevented follicular atresia by promoting production of steroid hormones to improve survival of GCs in PHFs from the aged hens. In conclusion, intensified ER stress and apoptosis occurred in GCs of PHFs in aged chickens, while BMP4 secreted by GCs was capable of improving follicular viability by alleviating ER stress to promote follicular development.

The decline in laying performance during the late laying period of poultry causes significant economic losses. This study aimed to investigate the differences in production performance-related parameters between peak (D120) and late (D330) laying phases in Korean quail (Coturnix japonica). A total of 200 D120 quails and 200 D330 quails were randomly divided into 8 groups, with 50 quails per group. The laying rate and egg weight in each group were recorded daily for 8 weeks. At the 8th week, egg quality traits, serum biochemical indices, and the morphology of both ovarian tissue and eggshell gland were evaluated. Compared with D120 quails, the D330 quails exhibited the following changes: (i) Ovarian development deteriorated, with fewer follicles, leading to a reduction in laying rate. (ii) Albumen height, Haugh unit (HU), and eggshell thickness were significantly decreased. Eggshell strength was reduced by 10.05 %, and eggs became more elongated. However, there was no significant differences in egg weight, yolk weight, or yolk color. (iii) The length and density of eggshell gland villi decreased significantly, accompanied by increased lesions, resulting in functional impairment of the eggshell gland. (iv) Serum hormone levels changed significantly: 17β-estradiol (E₂), progesterone (P₄), and calcitonin (CT) levels decreased, whereas follicle-stimulating hormone (FSH), luteinizing hormone (LH), and parathyroid hormone (PTH) levels increased. (v) Total antioxidant capacity (T-AOC) and the activities of catalase (CAT), superoxide dismutase (SOD), and glutathione peroxidase (GSH-Px) decreased significantly in serum, whereas malondialdehyde (MDA) levels increased significantly. (vi) Serum calcium (Ca) levels showed a nonsignificant decreased trend (P = 0.35), whereas serum phosphorus (P) levels increased significantly (P < 0.001). In conclusion, decreased serum E₂ and P₄ levels, along with reduced antioxidant capacity, contributed to ovarian and eggshell gland deterioration, ultimately impairing laying performance and egg quality in late laying quails.

The egg laying process of poultry is determined by follicular development. In the present study, hens with lower egg production rates had significantly fewer small white follicles than those with higher rates. In order to investigate the regulatory mechanisms governing the development from white follicles to yellow follicles, transcriptome sequencing was performed on small white follicles (SWFs), small yellow follicles (SYFs) and F6 (the smallest hierarchical follicles) of high-yield laying hens. Pathways enrichment analysis revealed that the vascular endothelial growth factor (VEGF) signaling, peroxisome proliferator-activated receptor (PPAR) signaling, tight junction and gap junction pathways, as well as steroid hormone biosynthesis were involved in follicular development. Compared with the transition from SYFs to F6, the transition from SWFs to SYFs showed more pronounced changes in gene expression profiles. This was validated by Western blot analysis, which revealed significantly upregulated expression of angiogenesis-related proteins (VEGF, VEGFR1, VEGFR2, ANGPT1 and ANGPT2) and downregulated expression of pigment epithelium derived factor (PEDF) in SYFs compared to SWFs. Immunofluorescence staining (IF) confirmed a markedly higher CD31-positive staining area in the theca layer of SYFs than in that of SWFs. Meanwhile, the estradiol (E₂) and progesterone (P₄) levels in SYFs increased by 131.6% and 22.2%, respectively, compared to SWFs. At the transcript level, SYFs exhibited upregulation of apolipoprotein D (ApoD), apolipoprotein A1 (APOA1), apolipoprotein L (APOLD1), PPAR and very low-density apolipoprotein (VLDL) mRNA, alongside downregulation of occludin, which was further confirmed by increased VLDLR and PPAR-γ protein abundance and decreased occludin levels. In summary, our results revealed that the SWFs to SYFs transition proceeds with pronounced changes in transcriptional profiles associated with angiogenesis, sex steroid biosynthesis and yolk deposition. The increased steroid hormone production likely enhances the capacity for yolk synthesis. Furthermore, the elevated vascularization in the theca layer of SYFs in coupled with a reduction in tight junctions may facilitate the transport of yolk precursors to access the follicles.