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Regular Article |
a Department of Animal Sciences, University of Missouri-Columbia, Columbia, Missouri 65211
The in vitro developmental potential of porcine nuclear transfer (NT) embryos was evaluated. Oocytes were matured for 42–44 h, and metaphase II-oocytes were enucleated. Fetal fibroblasts infected with the enhanced green fluorescent protein (EGFP) gene were serum-starved for 3–5 days. A single cell was injected into the perivitelline space of the enucleated oocytes. The reconstructed oocytes were allocated to different fusion and activation conditions. In experiment 1, two different fusion/activation conditions were compared: two pulses of 1.2 kV/cm for 30 µsec (group A), or one pulse of 1.6 kV/cm for 30 µsec followed in 30 min by one pulse of 1.2 kV/cm for 30 µsec (group B). Parthenogenetic controls were created by using the group A parameter. The fusion rate in group A (mean ± SEM, 68.4% ± 3.9%) was higher (P < 0.05) than in group B (59.4% ± 2.3%). The rates of cleavage (50.1% ± 4.6% to 62.8% ± 5.5%) were not different among control and treatment groups. However, the rate of parthenogenetic control embryos developing to the blastocyst stage (18.1% ± 3.1%) was higher (P < 0.05) than the rate of NT embryos (5.9% ± 1.7% and 4.9% ± 2.5%). In experiment 2, we compared two pulses of 1.2 kV/cm (group C) versus two pulses of 1.3 kV/cm (group D). For two control groups, the same pulses as those given to group C or D, respectively, were supplied. The fusion rate in group D (70.6% ± 4.2%) was higher (P < 0.05) than in group C (58.9% ± 2.7%). The cleavage rates were not different among control and treatment groups (58.1% ± 8.1% to 73.6% ± 6.0%). However, the rate of embryos developing to the blastocyst stage in group D (3.5% ± 1.7%) was lower (P < 0.05) than in controls and group C (11.4% ± 2.0% to 16.4% ± 1.1%). In experiment 3, we examined whether the presence of cytochalasin B (CB) during donor cell injection affects the development of NT embryos. The fusion rate of oocytes in the group with CB (78.4% ± 1.4%) was higher (P < 0.05) than in the group without CB (70.9% ± 0.2%). The cleavage rate of the control group (85.5% ± 4.9%) was higher (P < 0.05) than those of the treatment groups (61.6% ± 2.7% and 63.9% ± 4.3%). However, the rates of embryos developing to the blastocyst stage (8.1% ± 2.5% to 19.1% ± 6.0%) and the mean cell number of blastocysts (29.4 ± 5.2 to 45.7 ± 6.4) were not different among control and treatment groups. Green fluorescence was observed at all stages in NT embryos. These results indicate that two pulses of 1.2 kV/cm are enough for fusion/activation of NT embryos to develop to the blastocyst stage, and that the presence of CB during donor cell injection is not necessary for early development of NT embryos.
1 Supported by Food for the 21st Century and the NCRR13438.
2 Correspondence: Randall Prather, University of Missouri, 162 ASRC, 920 East Campus Drive, Columbia, MO 65211. FAX: 573 884 7827;pratherr{at}missouri.edu
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