Effect of cooling and warming rates during cryopreservation on survival of in vitro-produced bovine embryos

The effect of cooling and warming rates during cryopreservation on subsequent embryo survival was studied in 607 bovine morulae and 595 blastocysts produced by in vitro maturation, fertilization and culture (IVM/IVF/IVC). Morulae and blastocysts were prepared by co-culturing presumptive zygotes with bovine oviductal epithelial cells (BOEC) in serum-free TCM199 medium for 6 and 7 d, respectively. The embryos in 1.5 M ethylene glycol in plastic straws were seeded at -7 degrees C, cooled to -35 degrees C at each of 5 rates (0.3 degrees, 0.6 degrees, 0.9 degrees, 1.2 degrees, or 1.5 degrees C/min) and then immediately plunged into liquid nitrogen. The frozen embryos were warmed either rapidly in a 35 degrees C water bath (warming rate >1,000 degrees C/min) or slowly in 25 degrees to 28 degrees C air (<250 degrees C/min). With rapid warming, 42.1% of the morulae that had been cooled at 0.3 degrees C/min developed into hatching blastocysts. The proportions of rapidly warmed morulae that hatched decreased with increasing cooling rates (30.4, 19.0, 15.8 and 8.9% at 0.6 degrees, 0.9 degrees, 1.2 degrees and 1.5 degrees C/min, respectively). With slow warming 25.9% of the morulae that had been cooled at 0.3 degrees C/min developed into hatching blastocysts, while <10% of the morulae that had been cooled faster developed. The hatching rate of blastocysts cooled at 0.3 degrees C/min and warmed rapidly (96.3%) was higher than those cooled at 0.6 degrees and 0.9 degrees C/min (82.7 and 84.6%, respectively), and was also significantly higher than those warmed slowly after cooling at 0.3 degrees, 0.6 degrees or 0.9 degrees C/min (69.1, 56.6 and 51.8%, respectively). Cooling blastocysts at 1.2 degrees or 1.5 degrees C/min resulted in lowered hatching rates either with rapid (71.2 or 66.0%) or slow warming (38.2 or 38.9%). These results indicate that the survival of in vitro-produced bovine morulae and blastocysts is improved by very slow cooling during 2-step freezing; nevertheless, slow warming appears to cause injuries to morulae and blastocysts even after very slow cooling.