Document Detail

Production of bovine cloned embryos with donor cells frozen at a slow cooling rate in a conventional freezer (-20 degrees C).
MedLine Citation:
PMID:  19500443     Owner:  NLM     Status:  MEDLINE    
SummaryUsually, fibroblasts are frozen in dimethyl sulphoxide (DMSO, 10% v/v) at a cooling rate of 1 degrees C/min in a low-temperature (-80 degrees 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 degrees 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.
Liliana Chacón; Martha C Gómez; Jill A Jenkins; Stanley P Leibo; Gemechu Wirtu; Betsy L Dresser; C Earle Pope
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Publication Detail:
Type:  Journal Article; Research Support, Non-U.S. Gov't; Research Support, U.S. Gov't, Non-P.H.S.     Date:  2009-06-08
Journal Detail:
Title:  Zygote (Cambridge, England)     Volume:  17     ISSN:  1469-8730     ISO Abbreviation:  Zygote     Publication Date:  2009 Nov 
Date Detail:
Created Date:  2009-10-16     Completed Date:  2009-12-01     Revised Date:  -    
Medline Journal Info:
Nlm Unique ID:  9309124     Medline TA:  Zygote     Country:  England    
Other Details:
Languages:  eng     Pagination:  341-51     Citation Subset:  IM    
School of Veterinary Medicine, Colombian National University, Bogotá, Colombia.
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MeSH Terms
Blastocyst / cytology
Cattle / embryology*
Cell Survival
Cloning, Organism / veterinary*
Cryopreservation / instrumentation*
Cryoprotective Agents
Dimethyl Sulfoxide
Fibroblasts / cytology*
Oocytes / metabolism
Reg. No./Substance:
0/Cryoprotective Agents; 67-68-5/Dimethyl Sulfoxide

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