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

Published online by Cambridge University Press:  08 June 2009

Liliana Chacón ,
Martha C. Gómez ,
Jill A. Jenkins ,
Stanley P. Leibo ,
Gemechu Wirtu ,
Betsy L. Dresser  and
C. Earle Pope
Liliana Chacón
Affiliation:
School of Veterinary Medicine, Colombian National University, Bogotá, Colombia. Audubon Center for Research of Endangered Species, New Orleans, Louisiana, USA. Department of Biological Sciences, University of New Orleans, New Orleans, Louisiana, USA.
Martha C. Gómez*
Affiliation:
Audubon Center for Research of Endangered Species, 14001 River Road, New Orleans, Louisiana 70131, USA. Audubon Center for Research of Endangered Species, New Orleans, Louisiana, USA. Institute of Genetics, Colombian National University, Bogotá, Colombia.
Jill A. Jenkins
Affiliation:
National Wetlands Research Center, US Geological Survey, Lafayette, Louisiana, USA.
Stanley P. Leibo
Affiliation:
Audubon Center for Research of Endangered Species, New Orleans, Louisiana, USA. Department of Biological Sciences, University of New Orleans, New Orleans, Louisiana, USA.
Gemechu Wirtu
Affiliation:
Audubon Center for Research of Endangered Species, New Orleans, Louisiana, USA.
Betsy L. Dresser
Affiliation:
Audubon Center for Research of Endangered Species, New Orleans, Louisiana, USA. Department of Biological Sciences, University of New Orleans, New Orleans, Louisiana, USA.
C. Earle Pope
Affiliation:
Audubon Center for Research of Endangered Species, New Orleans, Louisiana, USA.
*
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: mgomez@auduboninstitute.org
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Summary

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.

Keywords

BovineCloningConventional FreezerFibroblasts−20 °C
Type
Research Article
Information
Zygote , Volume 17 , Issue 4 , November 2009 , pp. 341 - 351
Copyright
Copyright © Cambridge University Press 2009

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