Study of the efficiency of chemically assisted enucleation method for handmade cloning in goat (Capra hircus).
The present investigation was carried out to find an efficient chemically assisted procedure for enucleation of goat oocytes related to handmade cloning (HMC) technique. After 22-h in vitro maturation, oocytes were incubated with 0.5 μg/ml demecolcine for 2 h. Cumulus cells were removed by pipetting and vortexing in 0.5 mg/ml hyaluronidase, and zona pellucida were digested with pronase. Oocytes with extrusion cones were subjected to oriented bisection. One-third of the cytoplasm with the extrusion cone was removed with a micro blade. The remaining cytoplasts were used as recipients in HMC. Goat foetal fibroblasts were used as nuclear donors. The overall efficiency measured as the number of cytoplasts obtained per total number of oocytes used was significantly (p < 0.05) higher in chemically assisted handmade enucleation (CAHE) than oriented handmade enucleation without demecolcine (OHE) (80.02 ± 1.292% vs. 72.9 ± 1.00%, respectively, mean ± SEM). The reconstructed and activated embryos were cultured in embryo development medium (EDM) for 7 days. Fusion, cleavage and blastocyst development rate were 71.63 ± 1.95%, 92.94 ± 0.91% and 23.78 ± 3.33% (mean ± SEM), respectively which did not differ significantly from those achieved with random handmade enucleation and OHE. In conclusion, chemically assisted enucleation is a highly efficient and reliable enucleation method for goat HMC which eliminates the need of expensive equipment (inverted fluorescence microscope) and potentially harmful chromatin staining and ultraviolet (UV) irradiation for cytoplast selection.
Animal Biotechnology Centre, National Dairy Research Institute, Karnal, India.
Y S
YS
Akshey
D
D
Malakar
A K
AK
De
M K
MK
Jena
S
S
Sahu
R
R
Dutta
D000818:Animals; D002467:Cell Nucleus / Q000187:drug effects; D019976:Cloning, Organism / Q000379:methods / Q000662:veterinary*; D003703:Demecolcine / Q000494:pharmacology*; D006041:Goats; D053652:Nuclear Transfer Techniques; D009865:Oocytes / Q000166:cytology* / Q000502:physiology*
D003703:Demecolcine