[Effect of incubation time in deep freezing human sperm]

For the protection of spermatozoa against deleterious influences during freezing and thawing, so-called "cryoprotective agents" are mixed with the ejaculate before freezing. Clinical experience indicated a decrease in the number of post-thaw motile sperms after increasing the incubation period of sperms with the added cryoprotective agents and prompted us to investigate this finding. In experiment 1 we used 21 ejaculates which had a mean motility rate of 63.3%. The ejaculates were divided into 3 portions (1 a, 1 b, 1 c), which were frozen after 10 (1 a), 30 (1 b), and 60 (1 c) minutes of equilibration respectively. After thawing, 18.4% (1 a), 12.8% (1 b), and 7.1% (1 c) of the sperms were motile. This decrease in sperm motility was highly significant (p = 0.0012). Cryosurvival was investigated more fully within the first 10 minutes of incubation in a second experiment with another 18 ejaculates. The ejaculates were divided into 2 portions; portion 2 a was mixed with the cryoprotectant and frozen immediately and portion 2 b was frozen after a 10 minute incubation time. There was no significant difference in post-thaw motility and in recovery rate between the groups 2 a and 2 b. Our results indicate that the cryoprotective agent has a toxic time-dependent negative influence on sperm quality during equilibration, which significantly increases after an incubation time of more than 10 minutes. We consider that the results of this study are applicable to other glycerol-containing cryoprotectants and we recommend that the incubation period is kept short.