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

  • Xu, X.M. (Northwest A & F University, Shaanxi Branch of National Stem cell Engineering and Technology) ;
  • Hua, J.L. (Northwest A & F University, Shaanxi Branch of National Stem cell Engineering and Technology) ;
  • Jia, W.W. (Northwest A & F University, Shaanxi Branch of National Stem cell Engineering and Technology) ;
  • Huang, W. (Northwest A & F University, Shaanxi Branch of National Stem cell Engineering and Technology) ;
  • Yang, C.R. (Northwest A & F University, Shaanxi Branch of National Stem cell Engineering and Technology) ;
  • Dou, Z.Y. (Northwest A & F University, Shaanxi Branch of National Stem cell Engineering and Technology)
  • Received : 2006.12.16
  • Accepted : 2007.05.21
  • Published : 2007.10.01

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 $60-{\mu}sec$ pulse duration. The optimum pulse duration was 30-${\mu}sec$, 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 $5{\mu}g/ml$ insulin was significantly higher than that of the control (no insulin) ($44.3{\pm}9.1$ vs. $33.9{\pm}11.7$). For isolation of PESCs-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).

Keywords

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