Variation in the membrane transport properties and predicted optimal rates of freezing for spermatozoa of diploid and tetraploid pacific oyster, Crassostrea gigas

In the present study, a shape-independent differential scan ning calorimeter (DSC) technique was used to measure the de hydration response during freezing of sperm cells from diploid and tetraploid Pacific oysters, Crassostrea gigas. This represents the first application of the DSC technique to sperm cells from nonmammalian species. Volumetric shrinkage during freezing of oyster sperm cell suspensions was obtained at cooling rates of 5 and 20degreesC/min in the presence of extracellular ice and 8% (v/v) concentration of dimethyl sulfoxide (DMSO), a commonly used cryoprotective agent (CPA). Using previously published data, sperm cells from diploid oysters were modeled as a two-compartment "ball-on-stick" model with a "ball" 1.66 mum in diameter and a "stick" 41 mum in length and 0.14 mum wide. Similarly, sperm cells of tetraploid oysters were modeled with a "ball" 2.14 mum in diameter and a "stick" 53 mum in length and 0.17 mum wide. Sperm cells of both ploidy levels were assumed to have an osmotically inactive cell volume, V-b, of 0.6 V-o, where V. is the isotonic (or initial) cell volume. By fitting a model of water transport to the experimentally obtained volumetric shrinkage data, the best-fit membrane permeability parameters (L-pg and E,P) were determined. The combined-best-fit membrane permeability parameters at 5 and 20degreesC/min for haploid sperm cells (or cells from diploid Pacific oysters) in the absence of CPAs were: L-pg = 0.30 x 10(-15) m(3) /Ns (0.0017 mum/min-atm) and E-Lp = 41.0 kJ/mole (9.8 kcal/mole). The corresponding parameters in the presence of 8% DMSO were: L-pg[cpa] = 0.27 X 10(-15) m(3)/Ns (0.0015 mum/min-atm) and E-Lp[cpa] = 38.0 kJ/mole (9.1 kcal/mole). Similarly, the combined-best-fit membrane permeability parameters at 5 and 20degreesC/min for diploid sperm cells (or cells from tetraploid Pacific oysters) in the absence of CPAs were: L-pg = 0.34 X 10(-15) m(3)/Ns (0.0019 mum/min-atm) and E-Lp = 29.7 kJ/mole (7.1 kcal/mole). The corresponding parameters in the presence of 8% DMSO were: L-pg[cpa] = 0.34 X 10(-1)5 m(3)/Ns (0.0019 mum/min-atm) and E-Lp[cpa] = 37.6 kJ/mole (9.0 kcal/mole). The parameters obtained in this study suggest that optimal rates of cooling for Pacific oyster sperm cells range from 40 to 70degreesC/min. These theoretical cooling rates are in close con-formity with empirically determined optimal rates of cooling sperm cells from Pacific oysters, C gigas.