RESUMO
In the present study, we report the effects of cooling ejaculated and epididymal rhesus monkey (Macacamulatta) sperm with and without the presence of a cryoprotective agent, glycerol. Water transport data during freezing of ejaculated and epididymal sperm cell suspensions were obtained at a cooling rate of 20 degrees C/min in the absence of any cryoprotective agents and in the presence of 0.7 M of glycerol, as well. Using previously published values, the macaque sperm cell was modeled as a cylinder of length 73.83 microm with a radius of 0.40 microm and an osmotically inactive cell volume, V(b), of 0.772 V(o), where V(o) is the isotonic cell volume. This translated to a surface area, SA to initial water volume, WV ratio of approximately 22 microm(-1). By fitting a model of water transport to the experimentally determined volumetric shrinkage data, the best-fit membrane permeability parameters (reference membrane permeability to water at 0 degrees C, L(pg) or L(pg)[cpa] and the activation energy, E(Lp) or E(Lp)[cpa]) were found to range from: L(pg) or L(pg)[cpa]=0.0020-0.0029 microm/min-atm; E(Lp) or E(Lp)[cpa])=10.6-18.3 kcal/mole. By incorporating these membrane permeability parameters in a recently developed equation (optimal cooling rate, B(opt)=1009.5 x exp(-0.0546 x E(LP) x L(pg) x (SA/WV); where the units of B(opt) are degrees C/min, E(Lp) or E(Lp)[cpa] are kcal/mole, L(pg) or L(pg)[cpa] are mum/min-atm and SA/WV are mum(-1)), we determined the optimal rates of freezing macaque sperm to be approximately 23 degrees C/min (ejaculated sperm in the absence of CPAs), approximately 29 degrees C/min (ejaculated sperm in the presence of glycerol), approximately 24 degrees C/min (epididymal sperm in the absence of CPAs) and approximately 24 degrees C/min (epididymal sperm in the presence of glycerol). In conclusion, the subzero water transport response and consequently the subzero water transport parameters are not significantly different between the ejaculated and epididymal macaque spermatozoa under corresponding cooling conditions.
