Abstract:
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 _-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.