IN-VITRO DEVELOPMENT OF MOUSE EMBRYOS AFTER CRYOPRESERVATION IN PHOSPHATE OR HEPES BUFFERED MEDIA IN 2 DIFFERENT SIZE STRAWS

We compared in vitro survival rate of mouse embryos after cryopreservation in phosphate buffered saline (PBS) with that in Hepes buffered Tyrode's (HeTy) medium. We also examined the effect of size of straw (0.25 ml versus 0.50 ml) in which embryos were frozen on the in vitro survival post thawing. Early to mid-morula (n=823) from superovulated females were randomly assigned to 1 of 4 treatment groups: Group 1 = PBS in 0.25-ml straws, Group 2 = PBS in 0.50-ml straws, Group 3 = HeTy in 0.25-ml straws, Group 4 = HeTy in 0.50-ml straws. Embryos exposed to glycerol in 3 steps (0.44 M, 0.88 M and 1.4 M) at 22 degrees C were loaded into straws and placed in a programmable freezer at -6 degrees C, manually seeded during a 6 min holding period and cooled to -34 degrees C at 0.5 degrees C/min. After a 15-min holding period at -34 degrees C, the embryos were plunged into liquid nitrogen. Thawing was done by exposing each straw to air (22 degrees C) for 10 sec before plunging into a 37 degrees C water bath for 15 to 20 sec. Embryos were rehydrated for 10 min at 37 degrees C in 0.8 M sucrose, washed (3 times) and cultured for 72 h in modified Ham's F-10 + 10% fetal calf serum (FCS) at 38 degrees C in 5% CO2/air. The percentages of expanding blastocysts at 72 h were 68% (140/206), 68% (135/197), 77% (164/212) and 69% (143/208) for Groups 1, 2, 3 and 4, respectively. The survival rate of embryos frozen in HeTy (Groups 3 and 4) was slightly higher (73%; P = 0.07) than those frozen in PBS (68%, Groups 1 and 2). Straw size did not have an affect (P = 0.19) on post-thaw development, nor was there an interaction between media and straw size (P = 0.13). We conclude that mouse embryos can be successfully frozen in either 0.25- or 0.50-ml straws, and that HeTy is a suitable medium for mouse embryo cryopreservation; however, further studies with embryos of other species are warranted.