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KMID : 0900920000240040407
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Korean journal of Animal Reproduction
2000 Volume.24 No. 4 p.407 ~ p.417
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In Vitro Development of Bovine Nuclear Transfer Embryos Reconstructed with Fetal Fibroblasts
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Koo D.-B.
Park Y.-H.
Park J.-S.
Kim H.-N.
Kang Y.-K.
Lee C.-S.
Han Y.-M.
Park H.-D.
Lee K.-K.
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Abstract
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The present study was to examine effects of various electrical stimulus treatments used for electro-fusion on the preimplantation development of bovine nuclear transfer (NT) embryos with fetal fibroblast cells. Fetal fibroblast cells were isolated from one fetus at day 45 of gestation in Holstein cow, and passaged 3 to 4 times before being transferred into enucleated oocytes. Single fibroblast cells were individually placed into the perivitelline space of enucleated oocytes by using a micromanipulator. At first, the fusion and developmental rates of reconstructed oocytes were compared between different electric stimulation conditions. When fusion of the reconstructed oocyte was induced by different electric pulse periods (15, 30 and 45 musec) at a DC pulse of 1.8 kV/cm, 15 (45.5%, 120/264) or 30 mu sec group (43.9%, 106/241) showed a higher fusion rate than 45 musec group (23.2%, 58/250, P＜0.05). However, no difference was detected in the development rate of the fused oocytes to blastocysts between groups. Next experiment was to examine the effects of different electrical field strengths (1.5, 1.8 and 2.1 kV/cm) for 15 musec at electrofusion on in vitro development of the NT embryos. As results, there was no difference in the fusion and developmental rates of the NT embryos between electrical strength (P＞0.05). Finally, developmental competence of bovine NT embryos with somatic cells was compared with IVF-derived embryos. Of enucleated oocytes fused with fibroblast cells, 27.4% (75/274) developed to the blastocyst stage, which is similar to that (24.5%, 58/237) of IVF-derived embryos. However, mean nuclei number of NT blastocysts was smaller than that of IVF-derived blastocysts. Thus, we have established an optimal condition (1.8 kV/cm, 15 musec) for electric fusion of bovine NT oocytes with somatic cells. The present study indicates that bovine reconstructed embryos with somatic cells normally develop to blastocyst stage in vitro, although having smaller nuclei numbers of blastocysts as compared to IVF-derived embryos.
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KEYWORD
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Electrofusion, Nuclear transfer, Fetal fibroblasts, Bovine
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