title: Factors affecting survival of deep frozen bovine embryos in vitro: The effect of freezing container and method of removing cryoprotectant.
abstract: The effect of freezing container and method of glycerol removal on in vitro survival of frozen-thawed Day 7 bovine embryos was investigated. Two hundred and fifteen embryos were frozen in ampules or straws, in either vertical or horizontal position and at a cooling rate of 0.3 degrees C/minute from -7 degrees C to -35 degrees C, before being plunged into liquid nitrogen. Samples were thawed in a water bath at +35 degrees C and glycerol was removed by either step-wise dilution (increments 0.25 M) or by exposure to 1.0 M sucrose for 10 minutes. A total of 197 embryos was recovered post-thaw (91%) with an overall survival after 1, 3, 6 and 24 hours in culture of 87, 81, 71, and 23%, respectively. Embryonic quality and percent survival, as assessed morphologically, did not change significantly between 1 and 3 hours but decreased significantly between 6 and 24 hours in culture (p < 0.05). Survival at 24 hours was significantly higher after removal of the cryoprotectant with sucrose when compared to the step-wise glycerol dilution (p < 0.05). Overall, embryonic survival in straws equaled that in ampules; freezing orientation of straws did not affect results. Further, glycerol removal with sucrose tended to yield survival superior to that provided by a step-wise dilution technique.
authors.affiliation: Western College of Veterinary Medicine, University of Saskatchewan Saskatoon, Saskatchewan, Canada S7N 0W0.
authors.forename: A
authors.initials: A
authors.lastname: Bielanski
authors.forename: U
authors.initials: U
authors.lastname: Schneider
authors.forename: V P
authors.initials: VP
authors.lastname: Pawlyshyn
authors.forename: R J
authors.initials: RJ
authors.lastname: Mapletoft