The use of nuclear transfer (NT) techniques to create transgenic offspring capable of producing valuable proteins may have a major impact on the pharmaceutical market. Our objective was to compare the in vivo developmental potential of NT embryos produced from the fusion of transgenic donor cells with cytoplasts prepared from either FSH-stimulated ovaries or nonstimulated abattoir-derived ovaries. Donor cells were prepared from a transgenic fetus carrying the gene for human antithrombin III as a marker and used within four to eight subpassages. Cells were serum deprived for 4 days prior to cytoplast transfer. Oocytes were enucleated by removing the metaphase plate using a DNA stain and epifluorescent illumination. Donor cells were fused to enucleated oocytes by electric pulse and then chemically activated. There was no difference in the number of transferable embryos produced from cytoplasts of FSH-stimulated ovaries or from the fusion of cytoplasts from abattoir ovaries, nor was there a difference in the number of pregnancies established per recipient with either treatment. All pregnancies from both groups culminated in the births of healthy female kids (five total). To our knowledge, this is the first report of cloned goats produced from NT using cytoplasts derived from abattoir ovaries.
PNMSs of 1, 2 or 3 do not correlate with live birth rates when assessing unique PNMS embryo transfers. In particular, previously considered poor (type 3) embryos can result in pregnancy with normal live birth rates. Whether type 4 embryos are compatible with normal development remains to be shown.
To evaluate the effects of a three gas mixture of 5% O2, 5% CO2 and 90% N2 (OCN) on preimplantation embryo development, bovine in-vitro fertilization (IVF) oocytes were cultured in a defined medium (mBECM) with various supplements either under 5% CO2 in air or under OCN. When cultured in mBECM alone, embryo development was significantly stimulated in OCN compared to 5% CO2 in air (experiment 1). In the OCN atmosphere, blastocyst formation was further increased after addition of fetal bovine serum (FBS; 10%) or FBS + cumulus granulosa cells (CGC) to mBECM. The ratio of blastocysts to 8-cell embryos, number of hatched blastocysts and embryo diameter were markedly increased, and zona thickness was decreased after FBS addition. However, development up to the morula stage was fully supported by mBECM alone. There was no significant effect of beta-mercaptoethanol (ME; 10 microM) in OCN. In the 5% CO2 atmosphere, embryo development was significantly (P < 0.05) enhanced after addition of FBS + CGC + ME. In experiment 2, in OCN, FBS added at 60 h post-insemination was effective in stimulating blastocyst formation, but changes in medium volume per oocyte from 13.6 to 1.36 microliters had only a marginal effect. In conclusion, OCN gas mixture provides a suitable atmosphere for early embryo growth in vitro and mBECM + FBS in the optimal culture medium under this atmosphere.
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