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 followingseveral oocyte activation techniques and culture systems usingin-vitro-matured porcine oocytes. Two different nucleartransfer techniques (electrofusion and nuclear injection) were also testedusing 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 higherpronuclear formation rates (P<0.01) than those in theother groups (negative control and ionomycin treatment). Of these threegroups, oocytes activated by electric pulse + 6-DMAP showed greaterdevelopmental rate to the blastocyst stage and higher cell numbers inblastocysts than those activated by electric pulse or ionomycin + 6-DMAP(P<0.05). In Experiment 2, activated oocytes weregrouped 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-FBSfor 2 days (PVA-FBS); and (vi) NCSU-BSA for 4 daysfollowed by NCSU-FBS for 2 days (BSA-FBS). Cleavage rates in all experimentalgroups were not significantly different, but the embryos cultured in PVA-BSAor BSA-BSA showed higher blastocyst formation rates than those in other groups(P<0.05). In Experiment 3, the pseudo-pronuclearformation rate tended to be higher in the electrofusion group than inpiezo-driven nuclear injection group, but the difference was not statisticallysignificant (P=0.12). In addition, there was nosignificant difference between groups in cleavage, blastocyst formation, orthe number of cells in blastocysts. The results indicate that porcine adultsomatic cell nuclear transfer can be performed by the nuclear injectiontechnique with a piezo-driven micromanipulator. In addition, activation byelectrical pulse followed by 6-DMAP and in vitro culturein BSA-supplemented medium throughout the culture period was found to be themost efficient system for the production of porcine parthenogenetic embryosin vitro.Keywords: nuclear injection, piezo-drivenmicromanipulator.