Highly efficient and reliable chemically assisted enucleation method for handmade cloning in cattle.
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 microg 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.
Population Genetics and Embryology, Department of Genetics and Biotechnology, Danish Institute of Agricultural Sciences, DK-8830 Tjele, Denmark.
Gábor
G
Vajta
Poul
P
Maddox-Hyttel
Christina T
CT
Skou
R Tayfur
RT
Tecirlioglu
Teija T
TT
Peura
Liangxue
L
Lai
Clifton N
CN
Murphy
Randall S
RS
Prather
Peter M
PM
Kragh
Henrik
H
Callesen
D000818:Animals; D001947:Breeding / Q000379:methods*; D002417:Cattle; D002467:Cell Nucleus / Q000187:drug effects*; D002843:Chromatin / Q000378:metabolism; D019976:Cloning, Organism / Q000379:methods* / Q000662:veterinary*; D003703:Demecolcine / Q000494:pharmacology*; D046529:Microscopy, Electron, Transmission / Q000662:veterinary; D008856:Microscopy, Fluorescence / Q000662:veterinary; D008870:Microtubules / Q000187:drug effects; D053652:Nuclear Transfer Techniques; D009865:Oocytes / Q000187:drug effects* / Q000648:ultrastructure
D002843:Chromatin; D003703:Demecolcine