Cryopreservation is widely used presently as a method of storing different cell types and tissues including male and female gametes and embryos. Since the late 1930s–1940s (Bernschtein and Petropavlovski, 1937
; Polge et al., 1949; Smirnov, 1949), it has been possible to cryopreserve the spermatozoa of several mammalian species effectively, particularly bovine and human sperm. This type of technique has important applications including the preservation of male fertility before radiotherapy and/or chemotherapy (Sanger et al., 1992), which may lead to testicular failure or ejaculatory dysfunction. However, due to the damage induced by freezing, the motility of cryopreserved spermatozoa after thawing is statistically reduced and shows wide interindividual variability (Critser et al., 1988; Yoshida et al., 1990). To date, the problems of cryoprotectant toxicity due to osmotic stress during the addition and removal of cryoprotectants and possible negative effects on the sperm’s genetic apparatus are unresolved (Critser et al., 1988; Perez-Sanchez et al., 1994; Gilmore et al., 1997). Further cryo-damage may also be attributed to the slow thawing process (Mazur et al., 1981).
Compared with the conventional ‘slow’ freezing method, the newly developed techniques of vitrification and ultrarapid freezing, in which cryopreservation is achieved by directly plunging spermatozoa into liquid nitrogen [vitrification (cooling rate 
720 000°K/min) Nawroth et al., 2002; Isachenko et al., 2003; ultrarapid freezing (cooling rate 300–600°C/min) Schuster et al., 2003)], seem to have certain benefits. This method of cryopreservation does not require the use of classic permeable cryoprotectants, and thus avoids the lethal effects of osmotic shock on the spermatozoa. Moreover, the entire freezing or thawing process only takes a few seconds. Before freezing, the simple ‘swim-up’ or density gradient centrifugation procedure allows the selection of spermatozoa with progressive motility, normal morphology or even those with non-damaged DNA. This pre-selection has been shown to improve sperm quality after thawing in terms of all the classic markers of quality including DNA integrity (Perez-Sanchez et al., 1994; Esteves et al., 2000; Sakkas et al., 2000; Donnelly et al., 2001b; Tomlinson et al., 2001; O’Connell et al., 2003). Indeed, we were able to report a significant improvement (11.6%; P in post-thaw sperm motility when vitrifying swim-up-prepared spermatozoa with no cryoprotectant (Nawroth et al., 2002; Isachenko et al., 2003) over best post-thaw results achieved after conventional freezing in the obligatory presence of a permeable cryoprotectant (glycerol). However, the factors ‘morphology’, ‘motile sperm recovery’, ‘viability after freezing’ and ‘acrosome-reacted cells’ were not statistically different for the two cryopreservation methods (P > 0.05). According to these data, the swim-up method of preparing the sperm resulted in a significant improvement in the quality of spermatozoa which was sufficient to match the final results obtained using the conventional freezing procedure.
The present study was designed to compare the effects of slow freezing and cryoprotectant-free vitrification on the motility and DNA integrity of spermatozoa from fertile men. The effects of cryoprotectants on fresh sperm and when used during the slow freezing and vitrification process were also evaluated.
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