DETERMINATION OF OPTIMAL CRYOPROTECTANTS AND PROCEDURES FOR THEIR ADDITION AND REMOVAL FROM HUMAN SPERMATOZOA

The objective was to test the hypothesis that the optimal cryoprotecti ve agent for cryopreservation of human spermatozoa would be a solute f or which cells have the highest plasma membrane permeability, resultin g in the least amount of volume excursion during its addition and remo val. To test this hypothesis, theoretical simulations were performed u sing membrane permeability coefficients to predict optimal procedures for the addition and removal of a cryoprotectant. Simulations were per formed using data from four different cryoprotectants: (i) glycerol, ( ii) dimethyl sulphoxide, (iii) propylene glycol and (iv) ethylene glyc ol. Thermodynamic formulations were applied to determine approaches fo r the addition and removal of 1 M and 2 M final concentrations of cryo protectant, allowing the spermatozoa to maintain a cell volume within their osmotic tolerance limits. Based on these data, ethylene glycol w as predicted to be optimal for minimizing volume excursions among the solutes evaluated. These predictions were then experimentally tested u sing glycerol as the control cryoprotectant and ethylene glycol as the experimental cryoprotectant. The results indicate that there was a hi gher (P <0.05) recovery of motile spermatozoa after cryopreservation w hen using 1 M ethylene glycol than with 1 M glycerol, supporting the h ypothesis that use of the cryoprotectant for which the cell has the hi ghest permeability will result in higher cell survival.