Beneficial effect of oocyte activation prior to and during nuclear transfer in cattle using in vitro matured oocytes 24 h of age.
This research was designed to study in vitro development of bovine nuclear transferred embryos using enucleated young in vitro matured oocytes 24 h of age as recipient cytoplasts activated prior to nuclear transfer. The oocytes were enucleated and then activated with electric pulse at 24 h of age followed by incubation with cycloheximide for 6-7 h before nuclear transfer and membrane fusion with a blastomere of the 16-32 cell stage. This new protocol effectively improved cleavage (68 vs 17%, p < 0.05) and morula/blastocyst development of reconstructed embryos (29 vs 6%, p < 0.05), compared to similar nuclear transfer procedure without prior activation of recipient oocytes. Corresponding rates of cleavage and morula/blastocyst development for oocytes activated similarly without nuclear transfer were 49 and 19%. Factors affecting nuclear transfer were also compared. Two electric pulses for fusion increased rates of fusion (76 vs 60%, p < 0.05) and subsequent development of cloned embryos (32 vs 11%, p < 0.05). Cytochalasin B treatment following nuclear transfer manipulation seemed not to be beneficial in improving development of cloned embryos (p > 0.05). Both co-culture systems with buffalo rat liver (BRL) cells and cumulus cells promoted development of cloned embryos compared to the controls (28, 21 vs 0%, p < 0.05). The BRL cell system seemed to be better for manipulated embryos by reducing embryolysis.
Department of Animal Science, Cornell University, Ithaca, NY 14853-4801, USA.
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Du
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Jiang
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Yang
D000818:Animals; D001755:Blastocyst / Q000502:physiology; D001757:Blastomeres / Q000648:ultrastructure; D002417:Cattle; D002970:Cleavage Stage, Ovum; D046508:Culture Techniques; D003513:Cycloheximide / Q000494:pharmacology; D003571:Cytochalasin B / Q000494:pharmacology; D004558:Electric Stimulation; D005260:Female; D008099:Liver; D008561:Membrane Fusion; D009028:Morula / Q000502:physiology; D053652:Nuclear Transfer Techniques; D009865:Oocytes / Q000502:physiology* / Q000648:ultrastructure*; D051381:Rats; D011915:Rats, Inbred BUF
D003571:Cytochalasin B; D003513:Cycloheximide