The proliferation and apoptosis of ovarian granulosa cells (GC) is critical for follicular development and ovulation, especially for egg-laying performance in female birds. In geese, follicular atrophy is accompanied by follicular GC apoptosis during brooding stages. MicroRNAs are involved in follicular development, atrophy, ovulation, and degeneration. Our previous high-throughput sequencing study of ovaries from laying and brooding geese revealed that miR-192-2 may be involved in follicle growth and development, as well as follicle cell proliferation and apoptosis in geese. To further investigate the effect of miR-192-2 on GC in geese, we screened the target gene of miR-192-2 (IGFBP2) and constructed a miR-192-2 overexpression and interference vector, synthesized a small interfering RNA for IGFBP2. The results showed that after miR-192-2 overexpression, the mRNA and protein expression of proliferation-related genes (PCNA, CDK2, CCND1, and CCND2) were significantly decreased, the mRNA and protein expression of apoptosis-related genes (Caspase3, Caspase8, and Caspase9) were significantly increased, and the mRNA and protein expression of anti-apoptosis gene Bcl-2 were significantly decreased. While the mRNA and protein expressions of PCNA, CDK2, CCND1, and CCND2 were significantly decreased after the downregulation of IGFBP2 expression, the mRNA and protein expressions of Caspase3, Caspase8, and Caspase9 were significantly increased, and the mRNA and protein expression of Bcl-2 was significantly decreased. In conclusion, miR-192-2 inhibited proliferation and promoted apoptosis of follicular GC by targeting IGFBP2 in Zhedong White geese. Apoptosis of GC leads to follicular atresia, which in turn leads to brooding behavior in female geese. Therefore, it is possible to explore inhibiting the expression of miR-192-2 in production to alter the brooding behavior of Zhedong White Geese, thereby improving egg production and economic returns for producers.

The Zi goose is an excellent local goose breed in China, characterized by strong tolerance to roughage, high cold resistance, and high egg production. Anthocyanins are natural water-soluble pigments with numerous biological functions such as growth promotion, antioxidant activity, and immune regulation. However, there are few reports on whether anthocyanins have an improving effect on Zi goose semen. This study aimed to explore the effects of anthocyanins on semen quality, semen antioxidant capacity, and sperm apoptosis of Zi geese, so as to provide references for the large-scale breeding and industrial utilization of Zi geese. Sixty 12-month-old Zi geese were selected for the experiment, and their semen was collected by the massage method. Semen diluent containing different concentrations of anthocyanins was added to the mixed semen, which was then stored at 37 °C for detections. The results showed that the sperm survival rate of Zi geese was the highest when 30 mg/L of anthocyanins was added to the diluent. Compared with the control group, the anthocyanin group showed significantly higher sperm survival rate, sperm motility, plasma membrane integrity rate, acrosome integrity rate, mitochondrial activity, and DNA integrity (p < 0.05), while the sperm mortality rate was significantly decreased (p < 0.05). There were no significant differences in semen pH value, sperm density, and sperm abnormality rate (p > 0.05). The contents of superoxide dismutase and glutathione peroxidase were significantly increased (p < 0.05), while the levels of reactive oxygen species and malondialdehyde were significantly decreased (p < 0.05) in the anthocyanin group. Anthocyanins significantly increased the mRNA and protein expression of Bcl-2 (p < 0.05), and significantly decreased the mRNA and protein expression of Bax, Caspase-3, and P53 (p < 0.05). In conclusion, adding 30 mg/L anthocyanins to semen diluent can improve semen quality in Zi geese.

The dynamic balance between proliferation and apoptosis of follicular granulosa cells (GCs) is crucial for follicular development in poultry. microRNAs play important roles in ovarian development and follicular function. Previous transcriptome analyses showed that miR-194-3 was significantly differentially expressed in the ovaries of Zhedong white geese during the laying and brooding stages. Therefore, the aim of this study was to investigate the regulatory role and molecular mechanism of miR-194-3 on the proliferation and apoptosis of follicular GCs in Zhedong white geese. We first screened the target gene CHD4 of miR-194-3 and constructed the miR-194-3 mimic and inhibitor, a small interfering RNA of target gene CHD4. The experimental results showed that the overexpression of miR-194-3 significantly down-regulated the mRNA and protein expression of GC proliferation genes (PCNA, CDK-2, and CCND-1), reduced the proportion of EdU-labeled positive cells, blocked cell cycle progression, simultaneously up-regulated the mRNA and protein expression of Caspase-3 and Caspase-9, and significantly increased the rate of apoptosis. In contrast, the inhibition of miR-194-3 expression promoted the proliferation of goose follicular GCs, accelerated cell cycle progression, and decreased the apoptosis rate. Bioinformatics prediction combined with the results of the dual luciferase reporter assay confirmed that CHD4 was a direct target gene of miR-194-3. The knockdown of CHD4 expression resulted in the down-regulation of PCNA, CDK-2 and CCND-1 expression; blockage of cell cycle progression; attenuation of cell proliferation; an up-regulation of Caspase-3 and Caspase-9 expression and a significant increase in apoptotic cell death. In summary, both miR-194-3 overexpression and CHD4 knockdown produced similar effects on goose follicular GC proliferation and apoptosis, suggesting that CHD4 may partially mediate the regulatory effects of miR-194-3; however, additional targets or pathways cannot be excluded.