title: Production of bovine cloned embryos with donor cells frozen at a slow cooling rate in a conventional freezer (-20 degrees C).
abstract: 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.
authors.affiliation: School of Veterinary Medicine, Colombian National University, Bogotá, Colombia.
authors.forename: Liliana
authors.initials: L
authors.lastname: Chacón
authors.forename: Martha C
authors.initials: MC
authors.lastname: Gómez
authors.forename: Jill A
authors.initials: JA
authors.lastname: Jenkins
authors.forename: Stanley P
authors.initials: SP
authors.lastname: Leibo
authors.forename: Gemechu
authors.initials: G
authors.lastname: Wirtu
authors.forename: Betsy L
authors.initials: BL
authors.lastname: Dresser
authors.forename: C Earle
authors.initials: CE
authors.lastname: Pope
mesh_terms: D000818:Animals; D001755:Blastocyst / Q000166:cytology; D002417:Cattle / Q000196:embryology*; D002470:Cell Survival; D019976:Cloning, Organism / Q000662:veterinary*; D015925:Cryopreservation / Q000295:instrumentation*; D003451:Cryoprotective Agents; D004121:Dimethyl Sulfoxide; D005347:Fibroblasts / Q000166:cytology*; D005615:Freezing; D009865:Oocytes / Q000378:metabolism
chemical_list: D003451:Cryoprotective Agents; D004121:Dimethyl Sulfoxide