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 degreesC/min and 20 degreesC/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 mum and a radius of 0.66 mum with an osmotically inactive cell volume My 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 less than or equal to0.3 degreesC/min in an Equitainer-I from 37 degreesC to 4 degreesC. 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 degreesC/min and 20 degreesC/min) in Kenney extender (absence of CPAs) are L-pg = 0.02 lm 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 mum 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 similar to 29 degreesC/min and is similar to 60 degreesC/min in the Kenney extender and in the lactose-EDTA extender. These rates are predicted assuming no intracellular ice formation occurs and that the similar to5% of initial osmotically active water volume trapped inside the cells at -30 degreesC will form innocuous ice on further cooling. Numerical simulations also showed that in the lactose-EDTA extender, equine sperm trap similar to3.4% and similar to7.1% of the intracellular water when cooled at 20 degreesC/min and 100 degreesC/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 degreesC/min, 20 degreesC/min, 50 degreesC/min, 70 degreesC/min, 130 degreesC/min, and 200 degreesC/min) to -80 degreesC in the CPA medium. Sperm viability was essentially constant between 20 degreesC/min and 130 degreesC/min.