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In on-farm conditions, frozen bovine embryos are frequently thawed at various environmental temperatures. Thawing temperature is an important factor affecting the viability of frozen-thawed bovine embryos. The present study investigated the effects of rapid temperature change on the viability of frozen IVM/IVF bovine embryos after thawing. Day 7- and 8- (Day 0 = day of insemination) expanded blastocysts were used in this study. Embryos were produced as previously described by Hamano & Kuwayama (1993 Theriogenology 39, 703–712,). Embryos were frozen in TCM-199 supplemented with 1.4 M glycerol, 20% calf serum (CS), and 0.25-M sucrose. The embryos were loaded into 0.25 mL straws. After equilibration, the straws were placed directly into a precooled alcohol chamber of a freezer at −6°C, seeded 1 min later, held at −6°C for 10 min, cooled to −25°C at a rate of 0.33°C/min, and then plunged into liquid nitrogen. Embryos were thawed by holding the straws in room temperature air for 10 s, and then immersing them in a 35°C water bath for 10 s. The thawed straws were randomly assigned to one of two groups. Some thawed straws were held for 5 min at −15, −5, 0, 5, or 15°C, and were then transferred directly into a water bath at 35°C for 5 min (Group 1). The remaining straws were subjected to the same post-thaw cooling step procedures as Group 1 two times (Group 2). The embryos were then directly rehydrated in PBS supplemented with 5% CS at 35°C, and cultured in TCM-199 supplemented with 5% CS and 0.1 mM β-mercaptoethanol. The morphology and hatching of embryos was assessed 72 h later. Data were analyzed using the chi-square method and Fisher’s exact test. The results are presented in the Table. There were no significant differences in the hatching rate among 5 temperatures in Group 1. Although there were no differences in the hatching rate of embryos held at −5, 0, 5, or 15°C after thawing, the rate for embryos held at −15°C was significantly lower than those of the other treatments in Group 2 (P < 0.05). The straws held at −15°C twice (Group 2), showed refreezing. These results suggest that exposing thawed straws to a broad range of environmental temperatures (−5 to 15°C) had no effect on the viability of frozen-thawed IVM/IVF bovine embryos. However, embryos might be irreversibly damaged when held at −15°C. Table 1 Effect of rapid temperature change on the viability of frozen-thawed IVM/IVF bovine embryos

97EFFECT OF RAPID TEMPERATURE CHANGE ON VIABILITY OF FROZEN-THAWED IVM/IVF BOVINE EMBRYOS