Background and Aim: Porcine follicular fluid (pFF) is frequently used to mimic the follicular microenvironment during in vitro maturation (IVM) of oocytes. However, the influence of oxidative stress levels within pFF on oocyte quality and embryo development remains unclear. This study aimed to investigate how varying oxidative stress index (OSI) of pFF affect porcine oocyte meiotic progression, fertilization, and embryonic development during IVM. Materials and Methods: Oocytes were matured in IVM media supplemented with 30% pFF classified into low (OSI 19), medium (OSI 22), and high (OSI 25) oxidative stress groups, based on the ratio of diacron-reactive oxygen metabolites to biological antioxidant potential. Post-IVM, oocytes were assessed for meiotic stage, DNA fragmentation, reactive oxygen species (ROS), and glutathione (GSH) levels. Fertilization and embryo development outcomes were monitored following in vitro fertilization and culture. Results: The OSI 19 group showed significantly higher maturation to the metaphase II stage and improved fertilization and blastocyst formation rates compared to OSI 22 and OSI 25 groups (p < 0.05). ROS and GSH levels were also significantly elevated in OSI 19 oocytes, without an increase in DNA fragmentation. Blastocysts from the OSI 25 group exhibited significantly higher DNA fragmentation index than those from the OSI 19 group (p < 0.05). Conclusion: The OSI of pFF modulates porcine oocyte competence and embryonic outcomes. Lower OSI is associated with enhanced antioxidant balance, meiotic maturation, and embryo quality. Monitoring pFF oxidative status may improve assisted reproductive outcomes in swine. Keywords: embryo development, glutathione, in vitro maturation, oxidative stress index, porcine follicular fluid, reactive oxygen species.

Background and Aim: Mosaicism, which is characterized by the presence of wild-type and more than one mutant allele, poses a serious problem in zygotic gene modification through the clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein 9 system. Therefore, we used pig embryos to compare the gene editing efficiencies achieved by combining electroporation and lipofection using different aminopeptidase N (APN)-targeting guide RNA (gRNA) sequences. Materials and Methods: Six gRNAs (gRNA1–6) with different target sequences were designed to target APN. Zona pellucida (ZP)-intact zygotes collected 10 h after the start of in vitro fertilization (IVF) were electroporated with each gRNA to compare their gene editing efficiency. The gRNA sequences that achieved the lowest and highest mutation rates (gRNA4 and gRNA6, respectively) were selected for additional lipofection to assess gene editing efficiency following combined treatment. As ZP removal is essential for lipofection, ZP-free zygotes were electroporated with gRNA4 or gRNA6 10 h after IVF initiation, followed by lipofection with the same gRNAs 24 or 29 h after IVF initiation. The electroporated ZP-intact and ZP-free zygotes were used as controls. Results: gRNA4 and gRNA6 exhibited the lowest and highest mutation rates, respectively. gRNA4-targeted ZP-free embryos subjected to additional lipofection 29 h after IVF initiation exhibited significantly higher total and biallelic mutation rates than ZP-intact embryos that received only electroporation. Additional lipofection of gRNA6-targeted embryos had no obvious effect on mutation rates. Conclusion: Electroporation combined with lipofection using gRNAs with low mutation rates may improve gene editing efficiency in pig embryos. However, the effects may vary based on the timing of gene editing. Keywords: electroporation, guide RNA sequence, lipofection, pig embryo.

Background and Aim: The antioxidant effects of ergothioneine (EGT) on in vitro culture of porcine zygote are not well established. The study investigated the impact of EGT supplementation on meiotic competence and development of porcine oocytes. Materials and Methods: The impact of EGT concentrations (0, 5, 10, 25, 50, and 100 μM) during in vitro maturation (IVM) on the progression of meiotic maturation, fertilization, and blastocyst formation in porcine oocytes was assessed. The DNA fragmentation level was evaluated to determine oocyte and blastocyst quality. Results: The proportion of metaphase II oocytes was significantly greater (p < 0.05) in EGT-cultured oocytes than in control oocytes, regardless of the EGT concentration, and those oocytes with 10 μM or more EGT had fewer DNA-fragmented nuclei (p < 0.05). Blastocysts derived from oocytes cultured with 10 μM EGT had the highest proportion (p < 0.05), while those from control oocytes or oocytes cultured with 50 μM or less EGT had significantly higher proportions. Despite EGT supplementation, there were no noticeable differences in total cell numbers and DNA fragmentation indices in the derived blastocysts. Conclusion: Supplementing with EGT during IVM leads to better oocyte maturation, quality, and embryonic development due to decreased DNA fragmentation. The present study failed to elucidate the mechanism of DNA fragmentation reduction by EGT. More research needs to be conducted to explore the antioxidant mechanism of EGT. Keywords: antioxidant, DNA fragmentation, ergothioneine, maturation, porcine oocytes.