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

• Published in: Biology of reproduction Vol. 66; no. 1; pp. 222 - 231
• Main Authors: DEVIREDDY, R. V, SWANLUND, D. J, OLIN, T, VINCENTE, W, TROEDSSON, M. H. T, BISCHOF, J. C, ROBERTS, K. P
• Format: Journal Article
• Language: English
• Published: Madison, WI Society for the Study of Reproduction 01.01.2002
• Subjects: Algorithms; Animals; Biological and medical sciences; Calorimetry, Differential Scanning; Cell Membrane - physiology; Cell Membrane - ultrastructure; Cell Survival - physiology; Computer Simulation; Cryopreservation; Cryoprotective Agents - pharmacology; Freezing; Horses - physiology; In Vitro Techniques; Male; Semen Preservation; Spermatozoa - drug effects; Spermatozoa - physiology; Temperature; Water - metabolism
• ISSN: 0006-3363; 1529-7268
• DOI: 10.1095/biolreprod66.1.222

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 R 2 = 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 R 2 = 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.