Highly efficient and reliable chemically assisted enucleation method for handmade cloning in cattle
Gábor Vajta A G , Poul Maddox-Hyttel B , Christina T. Skou B , R. Tayfur Tecirlioglu C , Teija T. Peura D , Liangxue Lai E , Clifton N. Murphy E , Randall S. Prather E , Peter M. Kragh A F and Henrik Callesen AA Population Genetics and Embryology, Department of Genetics and Biotechnology, Danish Institute of Agricultural Sciences, DK-8830 Tjele, Denmark.
B Department of Animal and Veterinary Basic Sciences, The Royal Veterinary and Agricultural University, DK-1870 Frederiksberg C, Denmark.
C Centre for Early Human Development, Monash Institute of Medical Research, Monash University, Clayton, Vic. 3168, Australia.
D Sydney IVF, Sydney, NSW 2001, Australia.
E Department of Animal Sciences, University of Missouri-Columbia, Columbia, MO 65211-5300, USA.
F Department of Human Genetics, University of Aarhus, DK-8000 Aarhus, Denmark.
G Corresponding author. Email: gabor.vajta@agrsci.dk
Reproduction, Fertility and Development 17(8) 791-797 https://doi.org/10.1071/RD05066
Submitted: 20 June 2005 Accepted: 30 September 2005 Published: 16 December 2005
Abstract
The purpose of the present study was to find an efficient and reliable chemically assisted procedure for enucleation related to the handmade cloning (HMC) technique. After in vitro maturation oocytes were incubated in 0.5 μg mL−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 microtubules. 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.
Extra keywords: bovine, demecolcine, nuclear transfer.
Acknowledgments
The authors thank Anette M. Pedersen, Klaus Villemoes and Ruth Kristensen for excellent technical assistance in the embryology part of the work. Moreover, Mette Rahbek, Marie Louise Davidsen and Alexis Patry are acknowledged for their participation in the fluorescence and transmission electron microscopy analyses. This research was supported by grant no. 53-00-0294 from the Danish Agricultural and Veterinary Research Council, grant no. FREM98-8 from the Danish Directorate for Development and the National Institutes of Health (RR13438), and Food for the 21st Century.
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