Subzero water transport characteristics of boar spermatozoa confirm observed optimal cooling rates

Abstract

Incomplete understanding of the water transport parameters (reference membrane permeability, L(pg), and activation energy, E(Lp)) during freezing in the presence of extracellular ice and cryoprotective agents (CPAs) is one of the main limiting factors in reconciling the difference between the numerically predicted value and the experimentally determined optimal rates of freezing in boar (and in general mammalian) gametes. In the present study, a shape-independent differential scanning calorimeter (DSC) technique was used to measure the water transport during freezing of boar spermatozoa. Water transport data during freezing of boar sperm cell suspensions were obtained at cooling rates of 5 and 20 degrees C/min in the presence of extracellular ice and 6% (v/v) glycerol. Using previously published values, the boar sperm cell was modeled as a cylinder of length 80.1 microm and a radius of 0.31 microm with an osmotically inactive cell volume, V(b), of 0.6 V(o), where V(o) is the isotonic cell volume. By fitting a model of water transport to the experimentally obtained data, the best-fit water transport parameters (L(pg) and E(Lp)) were determined. The "combined-best-fit" parameters at 5 and 20 degrees C/min for boar spermatozoa in the presence of extracellular ice are: L(pg) = 3.6 x 10(-15) m(3)/N. s (0.02 microm/min-atm) and E(Lp) = 122.5 kJ/mole (29.3 kcal/mole) (R(2) = 0.99); and the corresponding parameters in the presence of extracellular ice and glycerol are: L(pg)[cpa] = 0.90 x 10(-15) m(3)/N. s (0.005 microm/min-atm) and E(Lp)[cpa] = 75.7 kJ/mole (18.1 kcal/mole) (R(2) = 0.99). The water transport parameters obtained in the present study are significantly different from previously published parameters for boar and other mammalian spermatozoa obtained at suprazero temperatures and at subzero temperatures in the absence of extracellular ice. The theoretically predicted optimal rates of freezing using the new parameters ( approximately 30 degrees C/min) are in close agreement with previously published but experimentally determined optimal cooling rates. This analysis reconciles a long-standing difference between theoretically predicted and experimentally determined optimal cooling rates for boar spermatozoa.