Effect of Freezing Method, Thawing Temperature and Post-thaw Dilution/Washing on Motility and Morphology Characteristics of High-quality Human Sperm

Paick, Jae Seung; Lee, Jin Haeng; Kim, Chung Mi

Abstract

Ten semen samples of high initial quality donated by fertile men were processed to compare the effect of two freezing methods, two thawing temperatures and the effect of dilution and washing on sperm motility (CASA) and morphologic characteristics (stricter criteria). Semen samples were divided in two equal parts and frozen either by fast vapour freezing or by slow computer-controlled freezing. For each freezing method, half of the straws were thawed at room temperature (22 degrees C), the other half were thawed at 37 degrees C. From each freeze-thawing treatment, one straw was evaluated immediately post-thawing; another straw was washed to remove the cryoprotectant solution. In this way, each semen sample was subjected to eight freeze-thawing treatment. The freezing and thawing of each semen sample was uniformly associated with a decrease in a sperm quality. The most commonly observed adverse effect was severe impairment of sperm motility. No effect of the freezing method and thawing temperature was observed on motility characteristics evaluated by computer-assisted semen analysis, nor on morphology parameters evaluated by stricter criteria. Only in regard to normal morphology was computer-controlled freezing slightly superior to fast vapour freezing. Post-thaw dilution and washing exerted a detrimental effect on sperm motility by reducing percentage motility by 30% - 40% compared to unwashed thawed specimens. Post-thaw dilution and washing obviously impaired normal morphology of spermatozoa by increasing percentage of small heads while linearity was increased significantly. Freezing-thawing was most effective when fast vapour freezing was followed by 37 degrees C thawing , and when slower computer-controlled freezing was combined with 22 degrees C thawing, causing linearity increased significantly Otherwise significant interactions between the freezing method and the thawing temperature were not observed. From these data, we think that vapour freezing is similar to computer-controlled freezing for high-quality semen in terms of recovery of morphologically normal with adequate progressive motility. For cryopreservation flow quality spermatozoa from patients, the effect of each freeze-thawing treatment is eligible for testing in another study.