Cryopreservation of equine sperm: Optimal cooling rates in the presence and absence of cryoprotective agents determined using differential scanning calorimetry

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 (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 ≤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 (L_pg and E_Lp) 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 L_pg = 0.02 μm min^-1 atm^-1 and E_Lp = 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 L_pg[cpa] = 0.008 μm min^-1 atm^-1 and E_Lp[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.