As an antioxidant, procyanidin B1(PB1) can improve the development of somatic cell nuclear transfer (SCNT) embryos; PB1 reduces the level of oxidative stress (OS) during the in vitro development of SCNT embryos by decreasing the level of reactive oxygen species (ROS) and increasing the level of glutathione (GSH) and mitochondrial membrane potential (MMP). Metabolite hydrogen peroxide (H₂O₂) produces OS. Catalase (CAT) can degrade hydrogen peroxide so that it produces less toxic water (H₂O) and oxygen (O₂) in order to reduce the harm caused by H₂O₂. Therefore, we tested the CAT level in the in vitro development of SCNT embryos; it was found that PB1 can increase the expression of CAT, indicating that PB1 can offset the harm caused by oxidative stress by increasing the level of CAT. Moreover, if H₂O₂ accumulates excessively, it produces radical-(HO-) through Fe^2+/3+ and damage to DNA. The damage caused to the DNA is mainly repaired by the protein encoded by the DNA damage repair gene. Therefore, we tested the expression of the DNA damage repair gene, OGG1. It was found that PB1 can increase the expression of OGG1 and increase the expression of protein. Through the above test, we proved that PB1 can improve the repairability of DNA damage. DNA damage can lead to cell apoptosis; therefore, we also tested the level of apoptosis of blastocysts, and we found that PB1 reduced the level of apoptosis. In summary, our results show that PB1 reduces the accumulation of H₂O₂ by decreasing the level of OS during the in vitro development of SCNT embryos and improves the repairability of DNA damage to reduce cell apoptosis. Our results have important significance for the improvement of the development of SCNT embryos in vitro and provide important reference significance for diseases that can be treated using SCNT technology.

Salidroside (Sal) possesses several pharmacological activities, such as antiaging, and anti-inflammatory, antioxidant, anticancer activities, and proliferation-promoting activities, but the effects of Sal on oocytes have rarely been reported. In the present study, we evaluated the beneficial effects of Sal, which is mainly found in the roots of Rhodiola. Porcine cumulus oocyte complexes were cultured in IVM medium supplemented (with 250 μmol/L) with Sal or not supplemented with Sal. The maturation rate in the Sal group increased from 88.34 ± 4.32% to 94.12 ± 2.29%, and the blastocyst rate in the Sal group increased from 30.35 ± 3.20% to 52.14 ± 7.32% compared with that in the control group. The experimental groups showed significant improvements in the cumulus expansion area. Sal reduced oocyte levels of reactive oxygen species (ROS) and enhanced intracellular GSH levels. Sal supplementation enhanced the mitochondrial membrane potential (MMP), ATP level, and mtDNA copy number, which shows that Sal enhances the cytoplasmic maturation of oocytes. Oocytes in the Sal group exhibited slowed apoptosis and reduced DNA breakage. Cell cycle signals and oocyte meiosis play important roles in oocyte maturation. The mRNA expressions of the MAPK pathway and MAPK phosphorylation increased significantly in the Sal group. The mRNA expression of the oocyte meiosis gene also increased significantly. These results show that Sal enhances the nuclear maturation of oocytes. Moreover, Sal increased the number of blastocyst cells, the proliferation of blastocysts, and the expressions of pluripotency genes. Sal down-regulated apoptosis-related genes and the apoptotic cell rate of blastocysts. In summary, our results demonstrate that Sal is helpful to improving the quality of porcine oocytes in vitro, and their subsequent embryonic development.

M⁶A is one of the most common and abundant modifications of RNA molecules present in eukaryotes. The methyltransferase complex, consisting of methyltransferase-like 3 (METTL3), METTL14, and WTAP, is responsible for the m⁶A modification of RNA. WTAP was identified as an mRNA splicing regulator. Its role as a regulatory subunit of the m⁶A methyltransferase complex in embryonic development remains largely unknown. To investigate the role of WTAP in porcine early embryonic development, si-WTAP was microinjected into porcine parthenogenetic zygotes. WTAP knockdown significantly reduced the blastocyst rate and global m⁶A levels, but did not affect the cleavage rate. Betaine was supplemented into the in vitro culture (IVC) to increase the m⁶A levels. Betaine significantly increased the global m⁶A levels but did not affect the blastocyst rate. Furthermore, the pluripotency genes, including OCT4, SOX2, and NANOG, were downregulated following WTAP knockdown. The apoptotic genes BAX and CASPASE 3 were upregulated, while the anti-apoptotic gene BCL2 was downregulated in WTAP knockdown blastocysts. TUNEL staining revealed that the number of apoptotic cells was significantly increased following WTAP knockdown. Our study indicated that WTAP has an indispensable role in porcine early embryonic development.

Feline mesenchymal stem cells (MSCs) isolated from adipose (A-MSCs) and uterine (U-MSCs) tissues during routine ovariohysterectomy represent valuable therapeutic sources in veterinary regenerative medicine. While quantitative real-time PCR (qRT-PCR) remains the gold standard for transcriptional profiling in these cells, its analytical reliability relies on empirically validated reference genes. In this study, we assessed the expression stability of nine candidate reference genes (ACTB, B2M, GAPDH, GUSB, HMBS, HPRT1, RPL7, TBP, and YWHAZ) in feline A-MSCs and U-MSCs using the geNorm and NormFinder algorithms. Both computational approaches identified TBP and HMBS as the most stably expressed genes across both cell types, whereas the historically popular GAPDH was the least stable. All geNorm pairwise variation values were below the commonly used threshold of 0.15 in both A-MSCs and U-MSCs. To illustrate the impact of reference gene choice, we evaluated the pluripotency marker OCT4. Normalizing using TBP and HMBS revealed significant differences in OCT4 expression between A-MSCs and U-MSCs (p < 0.05). In contrast, applying the unstable GAPDH masked this difference. These findings identify TBP and HMBS as promising reference gene candidates for qRT-PCR normalization in P3 undifferentiated feline A-MSCs and U-MSCs.