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Nuclear transfer and electrofusion in bovine in vitro‐matured/in vitro‐fertilized embryos: Effect of media and electrical fusion parameters
Author(s) -
Van StekelenburgHamers Annelies E. P.,
Van Inzen Wouter G.,
Van Achterberg Tanja A. E.,
Kruip Theo A. M.,
De Laat Siegfried W.,
Weima Sjerp M.
Publication year - 1993
Publication title -
molecular reproduction and development
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.745
H-Index - 105
eISSN - 1098-2795
pISSN - 1040-452X
DOI - 10.1002/mrd.1080360304
Subject(s) - electrofusion , blastomere , biology , embryo , cytoplast , andrology , cytochalasin b , oocyte , in vitro , embryo transfer , embryo culture , microbiology and biotechnology , embryogenesis , genetics , medicine , materials science , metallurgy
In this study, micromanipulation and electrofusion conditions for the cloning of in vitro‐produced bovine embryos (here after termed IVM/IVF embryos) derived from in vitro‐matured (IVM) and in vitro‐fertilized (IVF) oocytes were established. The effect of DC field strength on the fusion rate was tested in a model system using pronuclear stage embryos in which a cytoplasmic vesicle was removed and reinserted. Efficient fusion (80%) was obtained by applying a pulse of 1.75 kV/cm for 40 μsec. In vitro development of manipulated pronuclear stage embryos was as efficient as that of unmanipulated control embryos. Different fusion media were compared in the cloning procedure, using IVM oocytes as recipients and blastomeres from day 6 IVM/IVF donor embryos. Zimmermann cell fusion medium reduced the lysis of nuclear transfer embryos compared to F300 (5% vs. 25%). The effects of drugs disrupting the microfilaments and microtubuli were determined. Neither the addition of cytochalasin B (CCB) for 1 hr in the postfusion medium nor incubation of donor blastomeres with nocodazole had a significant effect on the fusion or cleavage rate of the nuclear transfer embryos. Additional experiments demonstrated that there was no difference in developmental potential between nuclear transfer embryos allowed to develop in vitro or in vivo and that the embryos gave a 15% pregnancy rate in recipient cattle. © 1993 Wiley‐Liss, Inc.