Highly efficient and reliable chemically assisted enucleation method for handmade cloning in cattle

Research output: Contribution to journalJournal articleResearchpeer-review

  • Gábor Vajta
  • Hyttel, Poul
  • Christina T. Skou
  • R. Tayfur Tecirlioglu
  • Teija T. Peura
  • Liangxue Lai
  • Clifton N. Murphy
  • Randall S. Prather
  • Peter M. Kragh
  • Henrik Callesen
The purpose of the present study was to find an efficient and reliable assisted procedure for enucleation related to the handmade cloning (HMC) technique. After in vitro maturation oocytes were incubated in 0.5 µgmL-¹ demecolcine for 2 h. Subsequently, zonae pellucidae were digested with pronase, and one-third of the cytoplasm connected to an extrusion cone was removed by hand using a microblade. The remaining two-thirds were used as recipients for HMC, and reconstructed and activated embryos were cultured for 7 days. The time-dependent manner of the development of extrusion cones, the efficiency (oriented bisection per oocyte; 94%), reliability (success per attempted enucleation; 98%), and the blastocyst per reconstructed embryo rates (48%) were measured. Ultrastructural analyses demonstrated that demecolcine treatment resulted in disoriented and haphazardly orientated icrotubules. The general ultrastructure of the oocyte organelles, however, appeared to be unaltered by the treatments. Considering that no oocyte selection based on polar body presence was performed, this system seems to be more efficient and reliable than any other enucleation method. Moreover, expensive equipment (inverted fluorescence microscope) and a potentially harmful step (staining and ultraviolet illumination) can be eliminated from the HMC procedure without compromising the high in vitro efficiency.
Original languageEnglish
JournalReproduction, Fertility and Development
Issue number8
Pages (from-to)791-797
Number of pages7
Publication statusPublished - 2005

ID: 7997464