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a Bioheat and Mass Transfer Laboratory, Department of Mechanical Engineering,
b Department of Cell Biology and Neuroanatomy,
c Department of Urologic Surgery, University of Minnesota, Minneapolis, Minnesota 55455
Optimization of techniques for cryopreservation of mammalian sperm is limited by a lack of knowledge regarding water permeability characteristics during freezing in the presence of extracellular ice and cryoprotective agents (CPAs). Cryomicroscopy cannot be used to measure dehydration during freezing in mammalian sperm because they are highly nonspherical and their small dimensions are at the limits of light microscopic resolution. Using a new shape-independent differential scanning calorimeter (DSC) technique, volumetric shrinkage during freezing of ICR mouse epididymal sperm cell suspensions was obtained at cooling rates of 5 and 20°C/min in the presence of extracellular ice and CPAs. Using previously published data, the mouse sperm cell was modeled as a cylinder (122-µm long, radius 0.46 µm) with an osmotically inactive cell volume (Vb) of 0.61Vo, where Vo is the isotonic cell volume. By fitting a model of water transport to the experimentally obtained volumetric shrinkage data, the best-fit membrane permeability parameters (Lpg and ELp) were determined. The "combined best-fit" membrane permeability parameters at 5 and 20°C/min for mouse sperm cells in solution are as follows: in D-PBS: Lpg = 1.7 x 10-15 m3/Ns (0.01 µm/min-atm) and ELp = 94.1 kJ/mole (22.5 kcal/mole) (R2 = 0.94); in "low" CPA media (consisting of 1% glycerol, 6% raffinose, and 15% egg yolk in D-PBS): Lpg[cpa] = 1.7 x 10-15 m3/Ns (0.01 µm/min-atm) and ELp[cpa] = 122.2 kJ/mole (29.2 kcal/mole) (R2 = 0.98); and in "high" CPA media (consisting of 4% glycerol, 16% raffinose, and 15% egg yolk in D-PBS): Lpg[cpa] = 0.68 x 10-15 m3/Ns (0.004 µm/min-atm) and ELp[cpa] = 63.6 kJ/mole (15.2 kcal/mole) (R2 = 0.99). These parameters are significantly different than previously published parameters for mammalian sperm obtained at suprazero temperatures and at subzero temperatures in the absence of extracellular ice. The parameters obtained in this study also suggest that damaging intracellular ice formation (IIF) could occur in mouse sperm cells at cooling rates as low as 25–45°C/min, depending on the concentrations of the CPAs. This may help to explain the discrepancy between the empirically determined optimal cryopreservation cooling rates, 10–40°C/min, and the numerically predicted optimal cooling rates, greater than 5000°C/min, obtained using suprazero mouse sperm permeability parameters that do not account for the presence of extracellular ice. As an independent test of this prediction, the percentages of viable and motile sperm cells were obtained after freezing at two different cooling rates ("slow" or 5°C/min; "fast," or 20°C/min) in both the low and high CPA media. The greatest sperm motility and viability was found with the low CPA media under fast (20°C/min) cooling conditions.
2 Correspondence: John C. Bischof, Bioheat and Mass Transfer Laboratory, Department of Mechanical Engineering, University of Minnesota, 111 Church Street S.E., Minneapolis, MN 55455. FAX: 612 624 1398; bischof{at}maroon.tc.umn.edu
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