TY - JOUR

T1 - Highly efficient and reliable chemically assisted enucleation method for handmade cloning in cattle

AU - Vajta, Gábor

AU - Maddox-Hyttel, Poul

AU - Skou, Christina T.

AU - Tecirlioglu, R. Tayfur

AU - Peura, Teija T.

AU - Lai, Liangxue

AU - Murphy, Clifton N.

AU - Prather, Randall S.

AU - Kragh, Peter M.

AU - Callesen, Henrik

PY - 2005

Y1 - 2005

N2 - 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.

AB - 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.

KW - Former LIFE faculty

KW - bovine

KW - demecolcine

KW - nuclear transfer

U2 - 10.1071/RD05066

DO - 10.1071/RD05066

M3 - Journal article

VL - 17

SP - 791

EP - 797

JO - Australian journal of scientific research. Ser. B: Biological sciences

JF - Australian journal of scientific research. Ser. B: Biological sciences

SN - 1031-3613

IS - 8

ER -