Technical report: In vitro development of porcine parthenogenetic and cloned embryos: comparison of oocyte-activating techniques, various culture systems and nuclear transfer methods

Abstract

The present study compares the development of porcine embryos following several oocyte activation techniques and culture systems using in-vitro-matured porcine oocytes. Two different nuclear transfer techniques (electrofusion and nuclear injection) were also tested using adult somatic cell (nucleus) and enucleated cytoplasm. In Experiment 1, oocytes activated by electric pulse, electric pulse + 6-dimethylaminopurine (6-DMAP), or ionomycin + 6-DMAP showed higher pronuclear formation rates (P<0.01) than those in the other groups (negative control and ionomycin treatment). Of these three groups, oocytes activated by electric pulse + 6-DMAP showed greater developmental rate to the blastocyst stage and higher cell numbers in blastocysts than those activated by electric pulse or ionomycin + 6-DMAP (P<0.05). In Experiment 2, activated oocytes were grouped and cultured as follows: (i) NCSU-PVA for 6 days (PVA-PVA); (ii) NCSU-BSA for 6 days (BSA-BSA); (iii) NCSU-FBS for 6 days (FBS-FBS); (iv) NCSU-PVA for 4 days followed by NCSU-BSA for 2 days (PVA-BSA); (v) NCSU-PVA for 4 days followed by NCSU-FBS for 2 days (PVA-FBS); and (vi) NCSU-BSA for 4 days followed by NCSU-FBS for 2 days (BSA-FBS). Cleavage rates in all experimental groups were not significantly different, but the embryos cultured in PVA-BSA or BSA-BSA showed higher blastocyst formation rates than those in other groups (P<0.05). In Experiment 3, the pseudo-pronuclear formation rate tended to be higher in the electrofusion group than in piezo-driven nuclear injection group, but the difference was not statistically significant (P=0.12). In addition, there was no significant difference between groups in cleavage, blastocyst formation, or the number of cells in blastocysts. The results indicate that porcine adult somatic cell nuclear transfer can be performed by the nuclear injection technique with a piezo-driven micromanipulator. In addition, activation by electrical pulse followed by 6-DMAP and in vitro culture in BSA-supplemented medium throughout the culture period was found to be the most efficient system for the production of porcine parthenogenetic embryos in vitro.