Determination of the membrane permeability characteristics of Pacific oyster, Crassostrea gigas, oocytes and development of optimized methods to add and remove ethylene glycol

In order for cryopreservation to become a practical tool for aquaculture, optimized protocols must be developed for each species and cell type. Knowledge of a cell's osmotic tolerance and membrane permeability characteristics can assist in optimized protocol development. In this study, these characteristics were determined for Pacific oyster oocytes and modified methods for loading and unloading ethylene glycol (EG) were tested. Oocytes were found to behave as ideal osmometers and their osmotically inactive fraction (V(b)) was calculated to be 0.48. Oocytes exposed to NaCl solutions of 0.6 to 2.3 Osm fertilized at rates equivalent to oocytes left in seawater. This corresponds to volume changes of +27.3 and -38.1+/-1.2%. The permeability of the oocytes to water (L(p)) was determined to be 3.8+/-0.4 x 10(-2), 5.7+/-0.8 x 10(-2), and 13.2+/-1.3 x 10(-2) microm min(-1)atm(-1), when measured at temperatures of 5, 10 and 20 degrees C. The respective EG permeability values (P(s)) were 9.5+/-0.1 x 10(-5), 14.6+/-1.2 x 10(-5), and 41.7+/-2.4 x 10(-5) cm min(-1). The activation energies for L(p) and P(s) were determined to be 14.5 and 17.5 kcal mol(-1), respectively. Different models for EG loading and unloading from oocytes were developed and tested. Post-thaw fertilization did not differ significantly between a published step addition method and single step addition at 20 degrees C. This represents a considerable reduction in handling. The results of this study demonstrate that the cryobiological characteristics of a given cell type should be taken into account when developing cryopreservation methods.