The importance of pre-freeze equilibration of glycerol in cryopreservation of human spermatozoa and the biochemical conversion of glycerol.

OBJECTIVE

To find optimal conditions for cryopreservation of human spermatozoa with glycerol as cryoprotectant.

MATERIALS AND METHODS

Pooled semen from groups of three normal donors was allowed to liquefy for 30 minutes at 37 degrees C. Samples were subjected to a prefreeze equilibration interval of 0, 30, or 60 minutes, with 5% or 10% glycerol, at 0 degrees C, 22 degrees C, or 37 degrees C. Samples were frozen in liquid nitrogen for 24 hours, thawed, and assessed for post-thaw motility. Uptake and metabolism of glycerol were studied using [3H] glycerol. Thin-layer chromatography of ethanol extracts of washed spermatozoa that had been incubated with the labeled glycerol was used to determine metabolic products.

RESULTS

Overall best cryopreservation was obtained at 37 degrees C with 5% glycerol and 30 minutes' prefreeze incubation. Labeled glycerol was incorporated into both polar and nonpolar metabolites. Two chromatographic peaks were found associated with the spermatozoa, and one peak was found in the incubation medium.