EVALUATION OF PERMEATING CRYOPROTECTANTS FOR THE CRYOPRESERVATION OF BOVINE TROPHOBLASTIC VESICLES

In the first experiment, bovine trophoblastic vesicles (bTV; 46/treatment) were exposed to 1.5 M glycerol, dimethyl sulfoxide, propylene glycol or ethylene glycol for 30 min at room temperature (27 degrees C). Cryoprotectants were removed from bTV by sequential exposure to 0.75 M of the appropriate cryoprotectant plus 0.75 M sucrose for 10 min, followed by 0.75 M sucrose for 10 min. The bTV were then cultured in RPMI 1640 medium and evaluated daily (for 3 d) for morphological quality. Post-exposure morphological quality of bTV indicated ethylene glycol was the least toxic cryoprotectant to bTV, followed by glycerol, propylene glycol and dimethyl sulfoxide. In the second experiment, bTV (60 bTV/treatment) were exposed to the same cryoprotectants for 30 min, loaded into 0.25-ml plastic straws (3 bTV/straw) and frozen, using a bovine embryo protocol (1 degrees C/min from 20 degrees C to -6 degrees C; seeded and held at -6 degrees C for 10 min; 0.3 degrees C/min to -28 degrees C; 0.1 degrees C/min to -35 degrees C; plunge into liquid nitrogen). After a few days storage, the straws were allowed to thaw in air (27 degrees C), the cryoprotectants were removed as described in Experiment 1 and the bTV were placed into culture. Daily evaluation for 3 d post-thawing indicated bTV cryopreserved in ethylene glycol were of higher morphological quality than those cryopreserved in either glycerol, propylene glycol or dimethyl sulfoxide. The post-thaw viability of bTV (20 bTV/treatment) cryopreserved in either 1.0 M, 1.5 M or 2.0 M glycerol or ethylene glycol was compared in the third experiment. The bTV were cryopreserved, thawed, cultured and evaluated as described for Experiment 2. Post-thaw morphological quality was highest for bTV cryopreserved in 1.5 M ethylene glycol, followed by 1.0 M ethylene glycol, 1.5 M glycerol, 2.0 M ethylene glycol or glycerol and 1.0 M glycerol, respectively. The results of this study indicate that ethylene glycol is less toxic to bTV than the other cryoprotectants evaluated. Based on post-thaw morphological quality, 1.5 M ethylene glycol is the cryoprotectant of choice for bTV.