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

Abstract

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 < 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.