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Quercetin (QUE) is a component of the flavonoid family that shows various therapeutic properties, such as antioxidant effects. However, whether QUE affects porcine oocyte in vitro aging has not yet been investigated. Therefore, in this study, we applied various doses of QUE to freshly isolated porcine oocytes and found that 10 µM QUE improved the oocyte maturation rate in vitro, as reflected by the increased degree of cumulus cell expansion and first polar body extrusion. More importantly, we found that QUE reduced in vitro aging and improved the maturity level of porcine oocytes after another 24 h of culturing, accompanied by the upregulated expression levels of bone morphogenetic protein 15, growth differentiation factor 9, Moloney sarcoma oncogene, and cyclin-dependent kinase 2. In addition, we found that QUE treatment significantly reduced the intracellular reactive oxygen species levels, apoptosis, and autophagy and upregulated the expression levels of superoxide dismutase 2 and catalase in aged porcine oocytes. In addition, QUE restored impaired mitochondrial membrane potential and spindle assembly in aged porcine oocytes. Our findings demonstrate that QUE can protect porcine oocytes from in vitro aging by reducing oxidative stress and maintaining mitochondrial function.

The dominant white phenotype in pigs is thought to be mainly due to a structural mutation in the KIT gene, a splice mutation (G > A) at the first base in intron 17 which leads to the deletion of exon 17 in the mature KIT mRNA. However, this hypothesis has not yet been validated by functional studies. Here, we created two mouse models, KIT D17/+ to mimic the splice mutation, and KIT Dup/+ to partially mimic the duplication mutation of KIT gene in dominant white pigs using CRISPR/Cas9 technology. We found that the splice mutation homozygote is lethal and the heterozygous mice have a piebald coat. Slightly increased expression of KIT in KIT Dup/+mice did not confer the patched phenotype and had no obvious impact on coat color. Interestingly, the combination of these two mutations reduced the phosphorylation of PI3K and MAPK pathway associated proteins, which may be related to the impaired migration of melanoblasts observed during embryonic development that eventually leads to the dominant white phenotype.

Bone morphogenetic protein 15 (BMP15) is strongly associated with animal reproduction and woman reproductive disease. As a multifunctional oocyte-specific secret factor, BMP15 controls female fertility and follicular development in both species-specific and dosage-sensitive manners. Previous studies found that BMP15 played a critical role in follicular development and ovulation rate in mono-ovulatory mammalian species, especially in sheep and human, but study on knockout mouse model implied that BMP15 possibly has minimal impact on female fertility of poly-ovulatory species. However, this needs to be validated in other poly-ovulatory species. To investigate the regulatory role of BMP15 on porcine female fertility, we generated a BMP15-knockdown pig model through somatic nuclear transfer technology. The BMP15-knockdown gilts showed markedly reduced fertility accompanied by phenotype of dysplastic ovaries containing significantly declined number of follicles, increased number of abnormal follicles, and abnormally enlarged antral follicles resulting in disordered ovulation, which is remarkably different from the unchanged fertility observed in BMP15 knockout mice. Molecular and transcriptome analysis revealed that the knockdown of BMP15 significantly affected both granulosa cells (GCs) and oocytes development, including suppression of cell proliferation, differentiation, and follicle stimulating hormone receptor (Fshr) expression, leading to premature luteinization and reduced estradiol (E2) production in GCs, and simultaneously decreased quality and meiotic maturation of oocyte. Our results provide in vivo evidence of the essential role of BMP15 in porcine ovarian and follicular development, and new insight into the complicated regulatory function of BMP15 in female fertility of poly-ovulatory species.

Porcine reproductive and respiratory syndrome virus (PRRSV) 1 and 2 differ in their recognition of CD163. Substitution of porcine CD163 SRCR5 domain with a human CD163-like SRCR8 confers resistance to PRRSV 1 but not PRRSV 2. The deletion of CD163 SRCR5 has been shown to confer resistance to PRRSV 1 in vivo and both PRRSV 1 and 2 in vitro. However, the anti-PRRSV 2 activity of modifying the CD163 SRCR5 domain has not yet been reported. Here, we describe the highly efficient generation of two pig breeds (Liang Guang Small Spotted and Large White pigs) lacking a short region of CD163 SRCR5, including the ligand-binding pocket. We generated a large number of gene-edited Large White pigs of the F0 generation for use in viral challenge studies. The results of this study show that these pigs are completely resistant to infection by species 2 PRRSV, JXA1, and MY strains. There were no clinical symptoms, pathological abnormalities, viremia, or anti-PRRSV antibodies in the CD163 SRCR5-edited pigs compared to wild-type controls after viral challenge. Porcine alveolar macrophages (PAMs) isolated from CD163 SRCR5-edited Large White pigs also displayed resistance to PRRSV in vitro. In addition, CD163 SRCR5-edited PAMs still exhibited a cytokine response to PRRSV infection, and no significant difference was observed in cytokine expression compared to wild-type PAMs. Taken together, these data suggest that CD163 SRCR5-edited pigs are resistant to PRRSV 2, providing a basis for the establishment of PRRSV-resistant pig lines for commercial application and further investigation of the essential region of SRCR5 involved in virus infection.