In porcine embryo culture, one of reactive oxygen species (ROS) is harmful factors that are made during in vitro culture. To decrease the detrimental effect of ROS on embryo development, superoxide dismutase, catalase and glutathione peroxidase could be used in the embryo culture. Out of these antioxidants, 7,8-dihydroxyflavone (7,8-DHF) was reported its antioxidant effects to prevent the glutamine-triggered apoptosis. Therefore, this study was performed to investigate the most appropriate concentration of 7,8-DHF in porcine embryonic development. For that, 5 different concentration (0, 0.1, 0.5, 1, 2 uM) of 7,8-DHF was supplemented in the porcine IVM media and then maturation and blastocyst formation rates were compared among 5 groups. In maturation rates of porcine oocytes, significant higher maturation rates was shown in the 1.0 uM group compared with another 4 groups (83.3 ± 2.1 vs. 80.7 ± 1.4, 79.8 ± 1.4, 78.3 ± 1.2, 79.4 ± 1.6), respectively (P<0.05). In the embryo culture, 1.0 uM group also showed the significant higher cleavage rates (76.8 ± 3.1 vs. 62.1 ± 5.0, 65.7 ± 4.0, 68.6 ± 3.7, 64.6 ± 4.0%) and blastocyst formation rates - (39.6 ± 4.0% vs. 28.6 ± 3.3, 31.1 ± 3.9, 29.3 ± 2.5, 39.6 ± 4.0, 26.4 ± 3.2%), respectively (P<0.05). There was no significant difference among 5 groups in the cell number of blastocyst (P<0.05). In conclusion, supplement of 1.0 uM of 7,8-DHF was effective to increase the porcine embryonic development competence as antioxidant to ROS.

Transcription activator like effector nucleases (TALENs) are artificial restriction enzymes generated by fusing a TALE DNA binding domain to a DNA cleavage domain which remove and introduce specific genes to produce transgenic animals. To investigate the efficient laboratory techniques for the injection of TALEN mRNA, pEGFP-N1 commercial plasmid were microinjected into porcine parthenogenetic and in vitro fertilization (IVF). In Experiment 1, to investigate injection time, compared 4 different time durations (2 hr, 4 hrs, 6 hrs & 8 hrs) after post activation of parthenogenetic embryos and after 6 hrs of co-incubation with sperms in IVF embryos. There were significant difference (P<0.05) in development to the blastocysts (4.4, 8.9, 3.9, 0.6%), GFP expression in blastocysts (1.3, 5.7, 2.3, 0.0%) which injected after post activation of 4 hrs compared with other 3 groups. IVF embryos after 2 hrs and 4 hrs injected were expressed GFP significantly higher than rest of two groups (P<0.05). In Experiment 2, compared development of 2 different concentrations (20 ng/μl and 50 ng/μl) of EGFP injection. There were significant difference (P<0.05) between two treatments which has higher cleavage (58.8 vs 41.9%), blastocysts development rate (13.0 vs 11.1%) and GFP expressed blastocysts (5.7 vs 0.0%) in 20 ng/μl than the 50 ng/μl in parthenogenetic embryos. In IVF embryos, only 20 ng/μl injected embryos were expressed GFP (4.2%) after 7 days of incubation and 77.3 vs 64.7% of cleavage, 26.4 vs 23.5% development to blastocysts. In Experiment 3, three different volumes (5, 10 and 20 pl) were microinjected into porcine embryos to determine the most appropriate volume. Out of 3 groups, significantly higher development rates of cleavage (68.3, 58.0, 29.3%), blastocysts (11.7, 12.7, 0.5%) and GFP expressed blastocysts (2.9, 7.8, 0.0%) were shown in the 10 pl group (P<0.05). In conclusion, these results imply that 20 ng/μl concentration, 10 pl of volume and injection at 4 hrs after post activation for parthenogenetic and 2∼4 hrs after IVF, 20 ng/μl concentration and 10 pl volume for IVF embryos were more effective microinjection conditions.

In order to generate transgenic goats expressing human growth hormone (hGH) in their mammary glands, goat β-casein/hGH hybrid gene was introduced into goat zygotes by pronuclear microinjection. DNA-injected embryos were transferred to the oviduct of recipients at 2-cell stage or to the uterus at morula/blastocyst stage after cultivation in glutathione-supplemented mSOF medium in vitro. Pregnancy and survival rate were not significantly different between 2-cell embryos and morula/blastocysts transferred to oviduct and uterus, respectively. One transgenic female goat was generated from 153 embryos survived from DNA injection. Southern blot analysis revealed that the transgenic goat harbored single-copy transgene with a partial deletion in its sequences. Despite of the partial sequence deletion, the transgene was successfully expressed hGH at the level of 72.1±15.1 μg/ml in milk throughout lactation period, suggesting that the sequence deletion had occurred in non-essential part of the transgene for the transgene expression. Unfortunately, however, the transgene was not transmitted to her offspring during three successive breeding seasons. These results demonstrated that goat β-casein/hGH gene was integrated into the transgenic goat genome in a mosaic fashion with a partial sequence deletion, which could result in a low level expression of hGH and a failure of transgene transmission.