Optimisation of porcine oocyte activation following nuclear transfer.
Experiments were conducted to examine the effects of (a) different activation methods, (b) incubation time in calcium-free medium and (c) bisbenzimide staining on the activation and subsequent development of pig oocytes. Oocytes were matured in vitro and activated by one of the following methods: combined thimerosal/dithiothreitol (DTT) treatment, calcium ionophore A23187 treatment followed by incubation in the presence of 6-dimethylaminopurine (6-DMAP), electroporation, and electroporation followed by incubation with cytochalasin B. There were no significant differences in the activation rate (ranging from 70.0% to 88.3%) and the percentage of cleaved embryos after activation (ranging between 48.8% and 58.8%) among the four treatment groups (p < 0.05). The rate of development of the blastocyst stage in oocytes activated by thimerosal/DTT (10.0%) or electroporation followed by cytochalasin B treatment (12.3%) was significantly higher (p < 0.05) than in the group activated with A23187/6-DMAP (2.5%). Both the activation rate and the rate of blastocyst formation in oocytes that were incubated in Ca(2+)-free medium for 8 h before thimerosal/DTT activation were significantly lower (p < 0.05) than in those incubated for 0, 1 or 4 h. Intracellular Ca2+ measurements revealed that the Ca2+ homeostasis in these oocytes were severely altered. Staining of oocytes with 5 micrograms/ml bisbenzimide for 2 h decreased the quality of blastocysts and increased the rate of degenerated embryos at day 6. Two activation protocols (thimerosal/DTT and electroproation) were used for activation after nuclear transfer; the rate of nuclear formation did not differ in the oocytes activated by the two different methods.
Tao
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Department of Animal Sciences, University of Missouri-Columbia 65211, USA.
Macháty
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Abeydeera
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Day
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BN
Prather
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RS
D000225:Adenine / Q000031:analogs & derivatives / Q000494:pharmacology; D000818:Animals; D006690:Bisbenzimidazole / Q000494:pharmacology; D000001:Calcimycin / Q000494:pharmacology; D002118:Calcium / Q000378:metabolism; D003470:Culture Media; D004229:Dithiothreitol / Q000494:pharmacology; D004558:Electric Stimulation; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005456:Fluorescent Dyes / Q000494:pharmacology; D066298:In Vitro Techniques; D007476:Ionophores / Q000494:pharmacology; D008540:Meiosis; D053652:Nuclear Transfer Techniques; D009865:Oocytes / Q000166:cytology / Q000187:drug effects / Q000502:physiology*; D013552:Swine; D013849:Thimerosal / Q000494:pharmacology
D003470:Culture Media; D004791:Enzyme Inhibitors; D005456:Fluorescent Dyes; D007476:Ionophores; D013849:Thimerosal; D000001:Calcimycin; C001020:N(6),N(6)-dimethyladenine; D000225:Adenine; D006690:Bisbenzimidazole; D002118:Calcium; D004229:Dithiothreitol