Efficient in vitro production of porcine blastocysts by handmade cloning with a combined electrical and chemical activation.
The purpose of our work was to establish an efficient protocol for activation of porcine cytoplast-fibroblast constructs produced by the handmade cloning technique. Firstly, we investigated a combined electrical and chemical activation protocol for parthenogenetic development of in vitro matured zona-free oocytes. Oocytes were activated by one 80 micros pulse and subsequently cultured in cytochalasin B and cycloheximide. Developmental rates of blastocysts from activated oocytes were 49+/-1 and 40+/-2%, when using one 80 micros pulse of 0.85 or 1.25 kV/cm, respectively. The activation procedure was further confirmed by a simultaneous re-fusion and activation of bisected oocytes, resulting in a blastocyst rate of 41+/-8%. Secondly, the activation protocol was applied in the handmade cloning technique. In vitro matured zona-free porcine oocytes were bisected and halves containing no chromatin, i.e. the cytoplasts, were selected. Reconstructed embryos were produced by a two-step fusion procedure. At the first step, one cytoplast was fused to one fibroblast by one 80 micros pulse of 1.25 kV/cm. After 1h, the cytoplast-fibroblast pair and another cytoplast were fused and activated simultaneously by one 80 micros pulse of 0.85 kV/cm, and subsequently cultured in cytochalasin B and cycloheximide. The development of reconstructed embryos to the blastocyst stage was in average 21+/-4%, and total blastocyst cell counts were in average 48+/-3. Thus, the combined electrical and chemical activation procedure resulted in efficient blastocyst development in the handmade cloning technique.
Kragh
P M
PM
Population Genetics and Embryology, Department of Genetics and Biotechnology, Danish Institute of Agricultural Sciences, DK-8830 Tjele, Denmark. peterm.kragh@agrsci.dk
Du
Y
Y
Corydon
T J
TJ
Purup
S
S
Bolund
L
L
Vajta
G
G
D000818:Animals; D001755:Blastocyst; D019976:Cloning, Organism / Q000379:methods / Q000662:veterinary*; D003513:Cycloheximide / Q000008:administration & dosage; D003571:Cytochalasin B / Q000008:administration & dosage; D004558:Electric Stimulation; D005260:Female; D009865:Oocytes / Q000502:physiology*; D013552:Swine / Q000196:embryology*
D003571:Cytochalasin B; D003513:Cycloheximide