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Variation in the Membrane Transport Properties and Predicted Optimal Rates of Freezing for Spermatozoa of Diploid and Tetraploid Pacific Oyster, Crassostrea gigas¹

Bioengineering Laboratory,³ Department of Mechanical Engineering, Louisiana State University, Baton Rouge, Louisiana 70803 Aquaculture Research Station,⁴ Louisiana Agricultural Experiment Station, Louisiana State University Agricultural Center, Baton Rouge, Louisiana

In the present study, a shape-independent differential scanning calorimeter (DSC) technique was used to measure the dehydration 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 20°C/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 µm in diameter and a "stick" 41 µm in length and 0.14 µm wide. Similarly, sperm cells of tetraploid oysters were modeled with a "ball" 2.14 µm in diameter and a "stick" 53 µm in length and 0.17 µm 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_o 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_Lp) were determined. The combined-best-fit membrane permeability parameters at 5 and 20°C/min for haploid sperm cells (or cells from diploid Pacific oysters) in the absence of CPAs were: L_pg = 0.30 x 10^–15 m³/Ns (0.0017 µm/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 m³/Ns (0.0015 µm/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 20°C/min for diploid sperm cells (or cells from tetraploid Pacific oysters) in the absence of CPAs were: L_pg = 0.34 x 10^–15 m³/Ns (0.0019 µm/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^–15 m³/Ns (0.0019 µm/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 70°C/min. These theoretical cooling rates are in close conformity with empirically determined optimal rates of cooling sperm cells from Pacific oysters, C. gigas.

¹ Supported in part by funding from the Louisiana Board of Regents (LEQSF 2002-05-RD-A-03) and the USDA-SBIR program. This manuscript was approved for publication by the Director of the Louisiana Agricultural Experiment Station as manuscript 03-11-1573.

² Correspondence: Ram Devireddy, Department of Mechanical Engineering, Louisiana State University, Baton Rouge, LA 70803. FAX: 225 578 5924; devireddy{at}me.lsu.edu

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