Parthenogenetic Activation of Porcine Oocytes and Isolation of Embryonic Stem Cells-like Derived from Parthenogenetic Blastocysts

Abstract

These experiments were carried out to optimize the parameters of electrical activation, methods of parthenogenetic activation and embryo culture in vitro and meanwhile to isolate embryonic stem cells-like (ESCs) derived from porcine parthenogenetic blastocysts (pPBs). These results showed that, as the electric field strength increased from 1.0 to 2.7 kV/cm, the cleavage rate of parthenogenetic embryos increased gradually but the rate of oocyte lysis was significantly increased when using 2.7 kV/cm field strength. The rate of cleavage in 2.2 and 2.7 kV/cm groups was significantly increased in comparison with that of the 1.0 kV/cm group. A voltage field strength of 2.2 kV/cm DC was used to investigate blastocyst development following activation with a single pulse of 30 or 60-μsec pulse duration. The optimum pulse duration was 30-μsec, with a blastocyst rate of 20.7%. Multiple pulses were inferior to a single pulse for blastocyst yield (8.0% vs. 29.9) (p<0.05). For porcine oocyte parthenogenetic activation methods, the rates of cleavage (79.0% vs. 59.8%) and blastocysts (19.4% vs. 3.4%) were significantly increased in electrical activation in contrast to chemical activation with ionomycin/6-DMAP (p<0.05). Rates of cleavage and blastocyst formation in NCSU-23 and PZM-3 embryo media were higher than those of G1.3/G2.3 serial culture media, but there was no significant difference among the three groups. The total cell number of blastocysts in PZM-3 embryo culture media containing 5 μg/ml insulin was significantly higher than that of the control (no insulin) (44.3±9.1 vs. 33.9±11.7). For isolation ofPESCs-like, the rates of porcine blastocysts attached to feeder layers and ICM colony formation in Method B (nude embryo culture) were better than those in Method A (intact embryo culture).