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Regular Article |
a Department of Mechanical Engineering,
b College of Veterinary Medicine, and
c Department of Urologic Surgery, University of Minnesota, Minneapolis, Minnesota 55455
Optimization of equine sperm cryopreservation protocols requires an understanding of the water permeability characteristics and volumetric shrinkage response during freezing. A cell-shape-independent differential scanning calorimeter (DSC) technique was used to measure the volumetric shrinkage during freezing of equine sperm suspensions at cooling rates of 5°C/min and 20°C/min in the presence and absence of cryoprotective agents (CPAs), i.e., in the Kenney extender and in the lactose-EDTA extender, respectively. The equine sperm was modeled as a cylinder of length 36.5 µm and a radius of 0.66 µm with an osmotically inactive cell volume (Vb) of 0.6Vo, where Vo is the isotonic cell volume. Sperm samples were collected using water-insoluble Vaseline in the artificial vagina and slow cooled at 
0.3°C/min in an Equitainer-I from 37°C to 4°C. By fitting a model of water transport to the experimentally obtained DSC volumetric shrinkage data, the best-fit membrane permeability parameters (Lpg and ELp) were determined. The combined best-fit parameters of water transport (at both 5°C/min and 20°C/min) in Kenney extender (absence of CPAs) are Lpg = 0.02 µm min-1 atm-1 and ELp = 32.7 kcal/mol with a goodness-of-fit parameter R2 = 0.96, and the best-fit parameters in the lactose-EDTA extender (the CPA medium) are Lpg[cpa] = 0.008 µm min-1 atm-1 and ELp[cpa] = 12.1 kcal/mol with R2 = 0.97. These parameters suggest that the optimal cooling rate for equine sperm is 
29°C/min and is 60°C/min in the Kenney extender and in the lactose-EDTA extender. These rates are predicted assuming no intracellular ice formation occurs and that the 5% of initial osmotically active water volume trapped inside the cells at -30°C will form innocuous ice on further cooling. Numerical simulations also showed that in the lactose-EDTA extender, equine sperm trap 3.4% and 7.1% of the intracellular water when cooled at 20°C/min and 100°C/min, respectively. As an independent test of this prediction, the percentage of viable equine sperm was obtained after freezing at 6 different cooling rates (2°C/min, 20°C/min, 50°C/min, 70°C/min, 130°C/min, and 200°C/min) to -80°C in the CPA medium. Sperm viability was essentially constant between 20°C/min and 130°C/min.
1 This work was supported by a grant from the Academic Health Center, University of Minnesota, Minneapolis, MN.
2 Correspondence: K.P. Roberts, Department of Urologic Surgery, University of Minnesota, 420 Delaware Street S.E., Minneapolis, MN 55455. FAX: 612 626 0428; roberts{at}med.umn.edu
3 Current address: Department of Mechanical Engineering, Louisiana State University, Baton Rouge, LA 70803.
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