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Production of bovine cloned embryos with donor cells frozen at a slow cooling rate in a conventional freezer (−20 °C)

  • Liliana Chacón (a1) (a2) (a3), Martha C. Gómez (a4) (a2) (a5), Jill A. Jenkins (a6), Stanley P. Leibo (a2) (a3), Gemechu Wirtu (a2), Betsy L. Dresser (a2) (a3) and C. Earle Pope (a2)...


Usually, fibroblasts are frozen in dimethyl sulphoxide (DMSO, 10% v/v) at a cooling rate of 1 °C/min in a low-temperature (−80 °C) freezer (LTF) before storage in liquid nitrogen (LN2); however, a LTF is not always available. The purpose of the present study was to evaluate apoptosis and viability of bovine fibroblasts frozen in a LTF or conventional freezer (CF; −20 °C) and their subsequent ability for development to blastocyst stage after fusion with enucleated bovine oocytes. Percentages of live cells frozen in LTF (49.5%) and CF (50.6%) were similar, but significantly less than non-frozen control (88%). In both CF and LTF, percentages of live apoptotic cells exposed to LN2 after freezing were lower (4% and 5%, respectively) as compared with unexposed cells (10% and 18%, respectively). Cells frozen in a CF had fewer cell doublings/24 h (0.45) and required more days (9.1) to reach 100% confluence at the first passage (P) after thawing and plating as compared with cells frozen in a LTF (0.96 and 4.0 days, respectively). Hypoploidy at P12 was higher than at P4 in cells frozen in either a CF (37.5% vs. 19.2%) or in a LTF (30.0% vs. 15.4%). A second-generation cryo-solution reduced the incidence of necrosis (29.4%) at 0 h after thawing as compared with that of a first generation cryo-solution (DMEM + DMSO, 60.2%). The percentage of apoptosis in live cells was affected by cooling rate (CF = 1.9% vs. LFT = 0.7%). Development of bovine cloned embryos to the blastocyst stage was not affected by cooling rate or freezer type.


Corresponding author

All correspondence to: Martha C. Gómez. Audubon Center for Research of Endangered Species, 14001 River Road, New Orleans, Louisiana 70131, USA. Tel: +1 504 398 3159. Fax: +1 504 391 7707. e-mail:


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Production of bovine cloned embryos with donor cells frozen at a slow cooling rate in a conventional freezer (−20 °C)

  • Liliana Chacón (a1) (a2) (a3), Martha C. Gómez (a4) (a2) (a5), Jill A. Jenkins (a6), Stanley P. Leibo (a2) (a3), Gemechu Wirtu (a2), Betsy L. Dresser (a2) (a3) and C. Earle Pope (a2)...


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