Andrology: Effect of freezing method, thawing temperature and post-thaw dilution/washing on motility (CASA) and morphology characteristics of high-quality human sperm

Abstract

Sixteen semen samples, 12 donor and four patient samples of high initial quality, were processed to compare the effect of two freezing methods, two thawing temperatures and the effect of dilution and washing on sperm motility and morphology characteristics. Sperm 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 treatments. No effect of the freezing method and thawing temperature was observed on motility characteristics evaluated by computer-assisted semen analysis, nor on light-microscopical morphology parameters. Post-thaw dilution and washing, however, exerted a deleterious effect on sperm motility, by reducing percentage motility by 50% compared to unwashed thawed specimens. Linearity and percentage of morphologically normal spermatozoa were obviously impaired, while percentage of abnormal tails and beat cross frequency increased significantly. In general, freeze-thawing was most successful when rapid vapour freezing was followed by 37 degrees C thawing, and when slower computer-controlled freezing was combined with 22 degrees C thawing, causing significant interactions between the freezing method and the thawing temperature. For semen samples of high initial quality, vapour and computer-controlled freezing were equally effective in terms of recovery of morphologically normal, motile spermatozoa.

Effects of cryopreservation on semen quality and the expression of sperm membrane hexose transporters in the spermatozoa of Iberian pigs

“…Several other authors have also observed an increase of 50-60% altered acrosomes after thawing (Bwanga et al 1990, Fiser & Fairfull 1990, Hofmo & Almlid 1991, Verheyen et al 1993.…”

Effects of cryopreservation on sperm parameters and ultrastructural morphology of human spermatozoa

“…Thus, osmotic and chemical toxicity of CPAs would be avoided if viability after thawing can be maintained [43][44][45][46][47]. It has been reported that, while freezing sperm on liquid nitrogen vapour is sufficient for healthy donors, it is more desirable to use slow, controlled freezing devices for those who are sub-or infertile [48,49]. In our opinion, different and novel cryopreservation methods (slow freezing, vitrification, vapour freezing, etc.)…”

Fertilization and early embryology: Ongoing pregnancies and birth after intracytoplasmic sperm injection with frozen—thawed epididymal spermatozoa

“…[Straws were placed horizontally -10 cm above the liquid nitrogen surface for 10 min (room temperature to -80°C; rate -10°C/min), and then plunged into the liquid nitrogen (-196°C).] For thawing, the straws were removed from the liquid nitrogen and placed in a 37°C chamber for 10 min (Verheyen et al, 1993). To remove the cryoprotectant, thawed specimens were put on a two-layer Percoll gradient (95.0-47.5%) and centrifuged for 20 min at 300 g. The 95% fraction was washed by centrifugation with Earle's medium at 1800 g for 5 min.…”