Megumi Nagahara

The CRISPR/Cas9 system has been widely used for gene editing in various species; however, mosaicism remains a significant challenge. This study aimed to improve gene editing efficiency and reduce mosaicism in porcine embryos by exploring double electroporation pre- and post-in vitro fertilization combined with zona pellucida (ZP) removal. We evaluated the effects of these treatments on the development and mutation rates of oocytes/zygotes edited with guide RNAs (gRNAs) targeting GGTA1, CMAH, or B4GALNT2 genes. Double electroporation significantly increased the total and biallelic mutation rates in ZP-intact zygotes but not in ZP-free zygotes edited using GGTA1-targeted gRNAs. All blastocysts from ZP-free zygotes exhibited biallelic mutations following double electroporation. For the CMAH gene, all blastocysts exhibited mutations (biallelic mutations ≥ 80%); however, double electroporation and ZP removal did not affect their mutation rates or efficiency. For the B4GALNT2 gene, double electroporation significantly increased total mutation rates in ZP-intact zygotes, whereas all blastocysts from ZP-free zygotes showed biallelic mutation. These findings suggest that double electroporation, particularly with ZP removal, may enhance gene-editing efficiency, reduce mosaicism and improve the success of genetic modifications.

Background and Aim: Electroporation-mediated delivery of Clustered-Regularly Interspaced Short Palindromic Repeats (CRISPR)/CRISPR-associated protein 9 components is an effective approach for genome editing in porcine embryos; however, mosaicism remains a major limitation. Selecting normally fertilized zygotes with two pronuclei (2PN) before electroporation may improve editing uniformity and reduce mosaicism. Therefore, this study investigated the effects of 2PN selection, optimized timing of pronuclear observation, and Hoechst 33342 staining conditions on embryo development and genome editing efficiency in porcine embryos. Materials and Methods: In the first experiment, pronuclear dynamics after in vitro fertilization (IVF) were evaluated by collecting zygotes at 8–24 h post-fertilization, followed by centrifugation, fixation, and Hoechst 33342 staining. In the second experiment, the effects of Hoechst 33342 concentration (5 and 10 μg/mL) and exposure duration (10 and 30 min) on embryo development were assessed using live zygotes collected 20 h after fertilization. In the third experiment, zygotes with visible 2PN selected after centrifugation and Hoechst staining were electroporated with guide RNA targeting the TP53 gene. Developmental competence, mutation efficiency, and mosaicism were compared with non-selected control zygotes. Results: The proportion of zygotes with 2PN gradually increased after fertilization and reached the highest level (47.4%) at 20 h post-IVF before significantly declining at 24 h (p < 0.05). Among the staining conditions tested, treatment with 5 μg/mL Hoechst 33342 for 30 min resulted in the highest blastocyst formation rate (14.3%, p < 0.05). Following electroporation, blastocyst development rates did not differ significantly between the 2PN-selected group (6.5%) and the control group (10.8%). Similarly, the biallelic mutation rate and total mutation efficiency were comparable between selected zygotes (78.6% and 85.5%, respectively) and controls (81.8% and 92.5%, respectively). Conclusion: Selection of porcine zygotes with 2PN before electroporation did not improve genome editing efficiency or reduce mosaicism despite optimization of pronuclear observation timing and Hoechst staining conditions. The persistence of mosaicism may be associated with asynchronous DNA replication between maternal and paternal pronuclei. Although 2PN selection was safe for embryo development, additional strategies targeting pronuclear synchronization and replication timing are required to achieve more uniform genome editing in porcine embryos. Keywords: blastocyst development, CRISPR/Cas9, electroporation, genome editing, Hoechst 33342, mosaicism, porcine embryos, two pronuclei.

Embryo recovery and transfer techniques efficiently assist superior cow selection for beef and milk production. In this study, we evaluated the use of vulvar blood flow volume (BFV) measurements obtained with a portable blood flow meter in predicting the follicle number. We explored the relationship between vulvar BFV and follicular hormone dynamics in cows. Eleven Japanese Black cows underwent superovulation treatment, after which vulvar BFV, serum estradiol (E ₂ ) and progesterone levels, and follicle number were measured. All measurements were repeated in the luteal phase (7 days before estrus), 1 and 2 days before estrus, and 2 and 7 days after estrus. The vulvar BFV was significantly elevated 1 day before estrus compared with that during the luteal phase. Furthermore, the vulvar BFV dynamics mirrored the follicle number and serum E ₂ level, both of which were significantly elevated 1 day before estrus compared with those during the luteal phase. In addition, positive correlations were observed between vulvar BFV and follicle number, between serum E ₂ level and follicle number, and between vulvar BFV and E ₂ level. Thus, vulvar BFV measurements could serve as a practical and non‐invasive strategy for predicting follicle number and may provide a valuable tool for farmers.

Background and Aim: Genetically engineered pigs are invaluable biomedical models for xenotransplantation and the study of human diseases. Although electroporation (EP) and lipofection are individually effective for clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) ribonucleoprotein (RNP) delivery, their combined application in porcine embryos has not been systematically evaluated. This study aimed to determine whether packaging Cas9-guided RNA complexes in cationic lipids enhances EP-mediated gene editing efficiency without compromising embryonic development. Materials and Methods: Porcine zygotes with their zona pellucida removed were edited using RNPs targeting beta-1,4-N-acetyl-galactosaminyl transferase 2 (B4GALNT2) or growth hormone receptor (GHR) genes. Four treatment groups were tested: (1) EP with RNPs (EP), (2) EP with lipofectamine-packaged RNPs (EPL), (3) transfection with lipofectamine-packaged RNPs before EP (TL + EPL), and (4) EP followed by lipofection (EPL + TL). Blastocyst formation was evaluated morphologically, and mutation rates were assessed by Sanger sequencing followed by tracking of indels by decomposition (TIDE) analysis. Results: Blastocyst formation rates were comparable across all treatments, indicating that lipofectamine packaging and EP caused no detectable cytotoxicity. For B4GALNT2, no mutations were induced by EP alone, whereas TL + EPL treatment significantly increased total and mosaic mutation rates (p < 0.05). For GHR, the total mutation and mosaic mutation rates were likewise higher in TL + EPL compared with EP, although mutation efficiency (indel percentage per edited embryo) remained unchanged. These results suggest that pre-EP lipofection promotes RNP uptake by facilitating lipid-membrane interactions that are potentiated by subsequent membrane destabilization through EP. Conclusion: Packaging RNPs in cationic lipids and applying sequential lipofection followed by EP significantly enhances CRISPR/Cas9-mediated mutagenesis in porcine zygotes without affecting developmental competence. This dual-delivery approach provides a simple, reproducible, and low-toxicity workflow for generating gene-edited embryos, with potential applicability to large-animal biomedical models. Keywords: cationic lipid, CRISPR/Cas9, electroporation, genome-editing efficiency, lipofectamine, porcine zygote, xenotransplantation.

Since individual embryos cannot be evaluated in group culture, establishing a single culture from in vitro maturation to in vitro culture may provide new insights into oocyte and embryo quality. This study aimed to develop a single culture system for individual oocytes, from in vitro maturation through fertilization to embryo development. The effects of curcumin supplementation during in vitro maturation on oocyte maturation, embryo development, and embryo quality were examined in single and group culture systems. Porcine oocytes were cultured individually in 20 µL microdroplets, with one oocyte per droplet, or in groups of 50 oocytes per 500 µL. The maturation medium contained curcumin at concentrations of 20 µM or less. Supplementation with 10 µM curcumin increased oocyte maturation in both systems compared to the controls. The fertilization rates and oocyte/embryo quality did not differ among the treatment groups. Oocytes matured with 10 µM curcumin in a single culture showed a higher blastocyst formation rate (7.0%) than the control (2.3%). In the group culture, 10 µM curcumin increased cleavage rates compared to the control (75.2% vs. 63.0%), but blastocyst formation rates did not differ. Blastocyst formation rates were similar between single and group cultures under control (2.3% and 4.3%, respectively) or 10 µM curcumin (7.0% and 11.4%, respectively) conditions. Therefore, porcine oocytes can develop to the blastocyst stage in a single culture system. Incorporating antioxidants during in vitro maturation may be an effective condition for in vitro embryo culture that can be implemented in a single oocyte.

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.

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 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.