Mitochondrial-targeted antioxidant supplementation is promising for in vitro culture of mammalian embryos. Elamipretide (SS-31) is a synthetic tetrapeptide that binds to the inner mitochondrial membrane, contributing to the prevention of oxidative stress. In this study, the effects of SS-31 supplementation in maturation medium on the developmental competence of porcine oocytes and its protective function were evaluated. Porcine oocytes were matured in maturation medium with SS-31 at 0.001, 0.01, 0.1, 0.5, 1, 1.5, 2.5, and 5 µM, with 0 µM and DMSO-treated groups established as controls. In vitro fertilization and embryo culture were performed to analyze developmental potential. Oocytes cultured in medium with 1 µM SS-31 exhibited higher maturation and blastocyst formation rates than the control (0 µM) (78.3 ± 3.8% vs. 55.2 ± 4.1% and 7.6 ± 1.6% vs. 2.8 ± 1.8%, respectively). Oocytes treated with 1 µM SS-31 showed significantly lower reactive oxygen species, higher glutathione content, and improved mitochondrial membrane potential than those without treatment. DNA fragmentation in oocytes after in vitro maturation was significantly lower in the 1 µM SS-31 supplemented group than in the control. This study demonstrates that SS-31 exerts beneficial effects on the in vitro production of porcine embryos via the enhancement of the mitochondrial antioxidant capacity.

Background: Oxidative stress is a critical factor affecting the maturation and developmental competence of porcine oocytes during in vitro culture, reducing the efficiency of blastocyst formation. Antioxidant supplementation in the culture medium has been proposed to mitigate oxidative stress and improve developmental outcomes. This study aimed to evaluate the effects of indole-3-propionic acid (IPA), a potent antioxidant, on porcine oocyte maturation, fertilization, and subsequent blastocyst development under in vitro conditions. Methods: Porcine cumulus–oocyte complexes (COCs) were matured for 44 h in maturation medium supplemented with IPA at various concentrations (0.5, 1, 5, and 10 µM). As a control, COCs were cultured in maturation medium containing the IPA dilution vehicle (ethanol) without IPA. After maturation culture, in vitro fertilization and embryo culture were performed to evaluate fertilization rate and developmental competence. Meiotic competence, fertilization status, blastocyst formation, and DNA integrity of oocytes and blastocysts were evaluated. To further investigate the protective effect of IPA against oxidative stress, COCs were exposed to 1 mM H ₂ O ₂ in a maturation medium supplemented with or without IPA (0.5 µM) during maturation culture. After maturation, intracellular reactive oxygen species (ROS) and glutathione (GSH) levels in oocytes were assessed. Results: Supplementation with 0.5 μM IPA in the in vitro maturation medium significantly enhanced the rates of oocyte maturation, fertilization, and blastocyst formation compared with the control group ( p < 0.05). In addition, IPA supplementation at concentrations ranging from 0.5 to 5 µM significantly reduced DNA fragmentation in both matured oocytes and blastocysts. Furthermore, IPA supplementation effectively reduced the intracellular ROS levels elevated by H ₂ O ₂ while significantly increasing intracellular GSH levels ( p < 0.05). These findings indicate that IPA supplementation protects porcine oocytes from oxidative stress during in vitro maturation by reducing ROS generation and enhancing GSH levels. Consequently, IPA improves oocyte quality, fertilization potential, and developmental competence of embryos. Conclusions: This study demonstrates the potential of IPA as a beneficial supplement to in vitro maturation medium for improving the efficiency of porcine embryo production by enhancing oocyte quality and protecting against oxidative damage.

CRISPR/Cas9-based multiplex genome editing via electroporation is relatively efficient; however, lipofection is versatile because of its ease of use and low cost. Here, we aimed to determine the efficiency of lipofection in CRISPR/Cas9-based multiplex genome editing using growth hormone receptor (GHR) and glycoprotein alpha-galactosyltransferase 1 (GGTA1)-targeting guide RNAs (gRNAs) in pig zygotes. Zona pellucida-free zygotes were collected 10 h after in vitro fertilization and incubated with Cas9, gRNAs, and Lipofectamine 2000 (LP2000) for 5 h. In Experiment 1, we evaluated the mutation efficiency of gRNAs targeting either GHR or GGTA1 in zygotes transfected using LP2000 and cultured in 4-well plates. In Experiment 2, we examined the effects of the culture method on the development, mutation rate, and mutation efficiency of zygotes with simultaneouslydouble-edited GHR and GGTA1, cultured using 4-well (group culture) and 25-well plates (individual culture). In Experiment 3, we assessed the effect of additional GHR-targeted lipofection before and after simultaneous double gRNA-targeted lipofection on the mutation efficiency of edited embryos cultured in 25-well plates. No significant differences in mutation rates were observed between the zygotes edited with either gRNA. Moreover, the formation rate of blastocysts derived from GHR and GGTA1 double-edited zygotes was significantly increased in the 25-well plate culture compared to that in the 4-well plate culture. However, mutations were only observed in GGTA1 when zygotes were transfected with both gRNAs, irrespective of the culture method used. GHR mutations were detected only in blastocysts derived from zygotes subjected to GHR-targeted lipofection before simultaneous double gRNA-targeted lipofection. Overall, our results suggest that additional lipofection before simultaneous double gRNA-targeted lipofection induces additional mutations in the zygotes.

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.