PERMEABILITY OF MACAQUE SPERM AT SUBZERO TEMP IN PRESENCE OF EXTACELLULAR ICE

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This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Background: In the present study, we report the effects of cooling ejaculated and epididymal rhesus monkey (Macaca mulatta) sperm with and without the presence of a cryoprotective agent, glycerol. Methods: Water transport data during freezing of ejaculated and epididymal sperm cell suspensions were obtained at a cooling rate of 20 ¿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 ¿m with a radius of 0.40 ¿m and an osmotically inactive cell volume, Vb, of 0.772Vo, where Vo is the isotonic cell volume. This translated to a surface area, SA to initial water volume, WV ratio of ~ 22 ¿m-1. Results/Discussion: 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 ¿C, Lpg or Lpg[cpa] and the activation energy, ELp or ELp[cpa]) were found to range from: Lpg or Lpg[cpa] = 0.00200.0029 ¿m/min-atm; ELp or ELp[cpa]) = 10.618.3 kcal/mole. By incorporating these membrane permeability parameters in a recently developed equation (optimal cooling rate, Bopt ¿ 1009:5 x exp(-0.0546 x ELp) x (Lpg) x (SA/WV); where the units of Bopt are ¿C/min, ELp or ELp[cpa] are kcal/mole, Lpg or Lpg[cpa] are ¿m/min-atm and SA/WV are ¿m-1), we determined the optimal rates of freezing macaque sperm to be ~ 23¿C/min (ejaculated sperm in the absence of CPAs), ~29 ¿C/min (ejaculated sperm in the presence of glycerol), ~24 ¿C/min (epididymal sperm in the absence of CPAs) and ~ 24 ¿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 spermatozoa under corresponding cooling conditions.