Comparison of chemical, electrical, and combined activation methods for in vitro matured porcine oocytes

  • Shuai Liu
  • Kuiqing Cui
  • Hong Li Li
  • Jun Ming Sun
  • Xing Rong Lu
  • Kai Yuan Shen
  • Qing You Liu
  • De Shun Shi
Article
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Abstract

Factors influencing porcine oocyte activation were systematically studied. This study included (1) the effect of ionomycin plus various chemical agents on activation, (2) comparison of different electrical activation parameters, (3) optimization of combined activation, and (4) evaluation of the optimized protocols. The results showed that (1) blastocyst rates of ionomycin (Ion) + 6-dimethylaminopurine (6-DMAP) (29.7 ± 1.1%), Ion + cytochalasin B (CB) + cycloheximide (CHX) (29.8 ± 1.2%), Ion + CB + 6-DMAP (30.4 ± 1.6%), and Ion + CB + CHX + 6-DMAP (30.2 ± 2.7%) were significantly higher than Ion + CHX (15.8 ± 1.5%, p < 0.05); (2) the parthenogenetic blastocyst formation of electrical activation was optimal when oocytes were activated by three direct current (DC) pulses of 1.00 kV cm−1 for 80 μs (39.5 ± 1.1%); (3) blastocyst rates of DC + CB + CHX (55.4 ± 1.2%) and DC + CB + 6-DMAP (50.4 ± 2.9%) were significantly higher than DC + 6-DMAP, DC + CB + CHX + 6-DMAP, electrical activation, and chemical activation alone (p < 0.05); and (4) approximately 84% of parthenogenetic blastocysts yielded by the optimized protocol were diploid, which was significantly higher than that of electrical activation blastocysts (40%). Using the optimized electrical and combined activation protocol, high blastocyst rates were generated by intracytoplasmic sperm injection (ICSI) (34.6 ± 1.1%), cytoplasmic microinjection (CI) (52.3 ± 2.2%), and handmade cloning (HMC) (31.2 ± 1.0%), respectively. This study concludes that the optimal activation protocol of in vitro matured porcine oocytes was combined activation with parameter as three DC pulses of 1.00 kV cm−1 for 80 μs plus CB and CHX treatment.

Keywords

Porcine oocytes Parthenogenetic activation Blastocyst development 
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Notes

Acknowledgments

This work was funded by the China 863 High-Tech Project (2011AA100607) and the National Natural Science Fund (NSFC 31260552).

Supplementary material

11626_2014_9819_MOESM1_ESM.tif (3.9 mb)
Additional Fig. 1 Blastocyst development after activated by ionomycin plus ethanol at 168 h (40×). (TIFF 4030 kb)
11626_2014_9819_Fig5_ESM.gif (140 kb)

High resolution image (GIF 139 kb)

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Additional Fig. 2 Blastocyst development after activated by different Ca2+ concentrations at 168 h (40×). (TIFF 6,037 kb)
11626_2014_9819_Fig6_ESM.gif (183 kb)

High resolution image (GIF 183 kb)

11626_2014_9819_MOESM3_ESM.tif (2.4 mb)
Additional Fig. 3 Conventional karyotyping of parthenogenetic porcine blastocysts. (×1,000). (TIFF 2,471 kb)
11626_2014_9819_Fig7_ESM.gif (10 kb)

High resolution image (GIF 10 kb)

11626_2014_9819_MOESM4_ESM.docx (16 kb)
ESM 4 (DOCX 16 kb)

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Copyright information

© The Society for In Vitro Biology 2014

Authors and Affiliations

  • Shuai Liu
    • 1
  • Kuiqing Cui
    • 1
  • Hong Li Li
    • 1
  • Jun Ming Sun
    • 1
  • Xing Rong Lu
    • 1
  • Kai Yuan Shen
    • 1
  • Qing You Liu
    • 1
  • De Shun Shi
    • 1
  1. 1.State Key Laboratory for Conservation and Utilization of Subtropical Agro-bioresourcesGuangxi UniversityNanningChina

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