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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 degrees C/min and 20 degrees 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 microm and a radius of 0.66 microm with an osmotically inactive cell volume (V(b)) of 0.6V(o), where V(o) is the isotonic cell volume. Sperm samples were collected using water-insoluble Vaseline in the artificial vagina and slow cooled at &lt; or = 0.3 degrees C/min in an Equitainer-I from 37 degrees C to 4 degrees C. By fitting a model of water transport to the experimentally obtained DSC volumetric shrinkage data, the best-fit membrane permeability parameters (L(pg) and E(Lp)) were determined. The combined best-fit parameters of water transport (at both 5 degrees C/min and 20 degrees C/min) in Kenney extender (absence of CPAs) are L(pg) = 0.02 microm min(-1) atm(-1) and E(Lp) = 32.7 kcal/mol with a goodness-of-fit parameter R(2) = 0.96, and the best-fit parameters in the lactose-EDTA extender (the CPA medium) are L(pg)[cpa] = 0.008 microm min(-1) atm(-1) and E(Lp)[cpa] = 12.1 kcal/mol with R(2) = 0.97. These parameters suggest that the optimal cooling rate for equine sperm is approximately 29 degrees C/min and is approximately 60 degrees 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 approximately 5% of initial osmotically active water volume trapped inside the cells at -30 degrees C will form innocuous ice on further cooling. Numerical simulations also showed that in the lactose-EDTA extender, equine sperm trap approximately 3.4% and approximately 7.1% of the intracellular water when cooled at 20 degrees C/min and 100 degrees 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 degrees C/min, 20 degrees C/min, 50 degrees C/min, 70 degrees C/min, 130 degrees C/min, and 200 degrees C/min) to -80 degrees C in the CPA medium. Sperm viability was essentially constant between 20 degrees C/min and 130 degrees C/min.

biology of reproduction

Cryopreservation of Equine Sperm: Optimal Cooling Rates in the Presence and Absence of Cryoprotective Agents Determined Using Differential Scanning Calorimetry1