Some reports demonstrated successful genome editing in pigs by one-step zygote microinjection of mRNA of CRISPR/Cas9-related components. Given the relatively long gestation periods and the high cost of housing, the establishment of a single blastocyst-based assay for rapid optimization of the above system is required. As a proof-of-concept, we attempted to disrupt a gene (GGTA1) encoding the α-1,3-galactosyltransferase that synthesizes the α-Gal epitope using parthenogenetically activated porcine oocytes. The lack of α-Gal epitope expression can be monitored by staining with fluorescently labeled isolectin BS-I-B₄ (IB4), which binds specifically to the α-Gal epitope. When oocytes were injected with guide RNA specific to GGTA1 together with enhanced green fluorescent protein (EGFP) and human Cas9 mRNAs, 65% (24/37) of the developing blastocysts exhibited green fluorescence, although almost all (96%, 23/24) showed a mosaic fluorescent pattern. Staining with IB4 revealed that the green fluorescent area often had a reduced binding activity to IB4. Of the 16 samples tested, six (five fluorescent and one non-fluorescent blastocysts) had indel mutations, suggesting a correlation between EGFP expression and mutation induction. Furthermore, it is suggested that zygote microinjection of mRNAs might lead to the production of piglets with cells harboring various mutation types.

The present study was carried out to examine the effects of valproic acid (VPA), a histone deacetylase inhibitor, on in vitro development of miniature pig somatic cell nuclear transfer (SCNT) embryos and on expression of a mouse Oct-3/4 promoter-driven enhanced green fluorescent protein (EGFP) gene (EGFP expression only detected in Oct-3/4-expressing cells) introduced into donor cells for SCNT during their development. The addition of 4 mM VPA to embryo culture medium for 48 h after activation significantly ( p < 0.01) increased the blastocyst formation rate of SCNT embryos compared with the control, whereas VPA did not affect their cleavage rate. The rate of SCNT embryos expressing EGFP at 5 days of culture was not affected by the presence or absence of VPA treatment. At 7 days of culture, however, the addition of 4 mM VPA to embryo culture medium for 48 h after activation significantly ( p < 0.05) increased the rate of SCNT embryos expressing EGFP compared with the control. The results indicate that VPA enhances the ability of miniature pig SCNT embryos to develop into blastocysts and maintains the ability of them to express Oct-3/4 gene.

The effects of mechanical vibration during in vitro maturation and/or in vitro culture after artificial activation of pig oocytes on maturation and development were examined. In addition, the optimal conditions were applied to in vitro production of blastocysts derived from miniature pig somatic cell nuclear transfer (SCNT) embryos. Mechanical vibration during in vitro maturation did not affect the rates (60.5 ± 1.9-69.5 ± 2.2%) of oocytes reaching the metaphase-II stage. However, the blastocyst formation rates after activation of oocytes matured with mechanical vibration for 5 sec at intervals of 30-60 min or for 10 sec at intervals of 60 min were significantly (P<0.05) higher than those of oocytes matured without mechanical vibration (25.7 ± 2.0-28.1 ± 2.7% vs. 12.3 ± 1.4% and 25.8 ± 1.8% vs. 15.7 ± 1.9%, respectively). In contrast, mechanical vibration during in vitro culture after activation did not affect the blastocyst formation (11.6 ± 5.2-16.5 ± 3.0%) of oocytes. Mechanical vibration for 5 sec at intervals of 60 min during in vitro maturation of oocytes did not affect fusion (66.8 ± 3.5-72.1 ± 3.1%) with miniature pig somatic cells after enucleation. However, the blastocyst formation rate of SCNT embryos was improved (P<0.05) by mechanically vibrating recipient oocytes for 5 sec at intervals of 60 min during in vitro maturation, regardless of the presence or absence of the same treatment during in vitro culture (17.6 ± 2.5% vs. 9.4 ± 0.9% and 13.0 ± 0.3% vs. 7.4 ± 0.9%, respectively). The results indicated that mechanical vibration enhances the cytoplasmic maturation of in vitro-matured pig oocytes, resulting in improvement of their parthenogenetic development. In addition, it was shown that in vitro maturation of oocytes with mechanical vibration can be applied to efficient production of blastocysts derived from miniature pig SCNT embryos.

The present study was carried out to examine the activation and development of cloned embryos produced by transferring miniature pig somatic cells into enucleated farm pig oocytes after exposing to ultrasound. The rates of the pronucleus-like structure formation and polar body-like structure extrusion in embryos exposed to ultrasound did not differ from those applied electric pulses. Although there was no significant difference in the blastocyst formation rates between different activation methods, the mean number of cells in the blastocysts developed from embryos activated by exposing to ultrasound was significantly (p < 0.05) higher than that obtained by applying electric pulses. The results of the present study showed that ultrasound stimulation can induce the activation and in vitro development of cloned embryos derived from miniature pig somatic cells.

The present study was carried out to examine the activation and parthenogenetic development of pig oocytes after exposure to ultrasound in sorbitol media supplemented with different concentrations of Ca2+. The activation rates (68.8-75.6%) of oocytes exposed to ultrasound in media containing 0.1-1.0 mM Ca2+ were significantly (P<0.05) higher than those (54.3-58.3%) of oocytes exposed to ultrasound in media containing 0-0.05 mM Ca2+. When oocytes were exposed to ultrasound in media containing 0.1-0.5 mM Ca2+, the blastocyst formation rates (20.5-21.3%) were significantly (P<0.05) higher than those (3.3-6.0%) of oocytes exposed to ultrasound in media containing 0, 0.05 or 1.0 mM Ca2+. The results of the present study showed that the concentration of Ca2+ in the medium used for exposure to ultrasound affects the activation and parthenogenetic development of pig oocytes and showed that the optimal Ca2+ concentration is 0.1-0.5 mM.

The effects of Ca²⁺ concentration in activation medium and cytochalasin B treatment after activation on the parthenogenetic development of pig oocytes were examined. In addition, cloned embryos derived from miniature pig somatic cells were activated under optimal conditions and the effects of Ca²⁺ in fusion medium on the development of embryos after activation was examined. When oocytes were activated in 0.1 mM Ca²⁺ and then treated with cytochalasin B, the blastocyst formation rate (28.6%) was significantly higher than those activated in 0-0.05 or 1.0 mM (11.0-18.3%). Treatment with cytochalasin B decreased the second polar body extrusion rate of activated oocytes. The presence or absence of Ca²⁺ in fusion medium did not affect the fusion rate of miniature pig somatic cells with recipient oocytes. A few cloned embryos developed to the blastocyst stage (2.7-9.0%) without an additional activation treatment. On the other hand, significantly more embryos developed to the blastocyst stage after activation treatment when they were fused in the absence (28.9%) of Ca²⁺ rather than the presence (16.5%) of it. These results show that the highest blastocyst formation rate for miniature pig cloned embryos is obtained when donor cells and recipient oocytes are fused in the absence of Ca²⁺ and then activated in 0.1 mM Ca²⁺ and treated with cytochalasin B.