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

• Published in: Zygote (Cambridge) Vol. 17; no. 4; pp. 341 - 351
• Main Authors: Chacón, Liliana, Gómez, Martha C., Jenkins, Jill A., Leibo, Stanley P., Wirtu, Gemechu, Dresser, Betsy L., Pope, C. Earle
• Format: Journal Article
• Language: English
• Published: Cambridge, UK Cambridge University Press 01.11.2009
• Subjects: 20 °C; Animals; Blastocyst - cytology; Bovine; Cattle - embryology; Cell Survival; Cloning; Cloning, Organism - veterinary; Conventional Freezer; Cryopreservation - instrumentation; Cryoprotective Agents; Dimethyl Sulfoxide; Fibroblasts; Fibroblasts - cytology; Freezing; Oocytes - metabolism; Conventional Freezer; Fibroblasts; 20 °C; Bovine; Cloning
• ISSN: 0967-1994; 1469-8730
• DOI: 10.1017/S0967199409005474

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.