Effects of cryopreservation on sperm quality, nuclear DNA integrity, in vitro fertilization, and in vitro embryo development in the mouse

Abstract

Efficient freezing, archiving, and thawing of sperm are essential techniques to support large scale research programs using mouse models of human disease. The purpose of this study was to investigate the effects of variable combinations and concentrations of cryoprotectants on sperm-assessment parameters of frozen-thawed mouse sperm in order to optimize cryopreservation protocols. Sperm was frozen using combinations of 3% skim milkC0.2 or 0.3 M nonpermeating raffinose with either permeating glucose, fructose, propylene glycol, ethylene glycol, glycerol, or sodium pyruvate in CD-1, C3FeB6F1/J, B6129SF1, C57BL/6NCrIBR, 129S/SvPaslco, and DBA/2NCrIBR mice. Sperm-assessment parameters included progressive motility, plasma membrane integrity (SYBR-14CPI), in vitro fertilization rate, and in vitro embryo development rate to blastocyst. DNA content analysis of sperm was measured by the sperm chromatin structure assay (SCSA). 0.3 M raffinose with 0.1 M fructose significantly improved post-thaw sperm-assessment parameters for CD-1, C3B6F1, B6129SF1 mice (P!0.05-0.01), whereas 0.2 M raffinose with 0.1 M glycerol or 0.1 M fructose enhanced sperm assessment values for C57BL/6 and 129S mice (P!0.01), compared to 0.3 M raffinose alone. DNA fragmentation during cryopreservation was significantly increased in all strains evaluated when compared with fresh control sperm in a strain-dependent manner (P!0.01). Supplementation with permeating glycerol or fructose to the cryoprotectant (CPA) solution showed a significant protective effect to DNA integrity when cryopreserving sperm from C57BL/6 and 129S mice. Damage to sperm DNA significantly decreased the rate of in vitro embryo development to blastocyst in C57BL/6 mice. The type of monosaccharide sugar or polyols, CPA molarity, and combination of permeating and nonpermeating cryoprotectant are significant factors for improving progressive motility, plasma membrane integrity, DNA integrity, in vitro fertilization rate, and in vitro embryo development rate to blastocyst in cryopreserved mouse sperm. Reproduction (2007) 133 585-595

Vitrification Technique - New Possibilities for Male Gamete Low-Temperature Storage

“…The danger is that such spermatozoa are still able to fertilize the oocyte, however, the mutations and defects did not possible to discover until the embryo has divided and the fetus has developed (Twigg et al, 1998). At present exist the opinion that DNA decondensation or fragmentation may occur in different magnitudes, which will depend on the process or the kind of cryoprotectant used (Schuffner et al, 2001;Chohan et al, 2004;Ngamwuttiwong & Kunathikom, 2007;Yildiz et al, 2007). Unfortunately, until now this question is still open, because it is not entirely clear what the effect of cryopreservation on DNA integrity is, and what would be the ideal conditions of slow freezing to reduce this effect.…”

A Study on Freeze-Drying as a Method of Preserving Mouse Sperm

“…Sperm chromatin assessment is an independent measure of sperm quality that provides better diagnostic and prognostic capabilities for potential fertility than standard sperm parameters [15]. Janny and Menezo reported that development to the blastocyst stage is linked to sperm quality [16] and it has been reported that increased levels of DNA fragmentation significantly decrease embryonic development to the blastocyst stage in mice [17]. The flow cytometric SCSA, can detect DNA fragmentation of several thousand sperm [18], unlike conventional microscopy methods, and is currently the most accurate for predicting the outcome of various assisted reproduction technologies such as fertilization and implantation [19].…”

Sperm chromosomal aneuploidy and DNA integrity of infertile men with anejaculation

“…Damaged DNA and sperm chromosomal aberrations can have a significant negative impact on oocyte fertilization, the rate of embryo development and the live-birth rate. A significant correlation between the presence of nuclear DNA alterations in mature spermatozoa and poor sperm parameters or impaired reproductive efficiency, and a positive relationship between spermatozoa with total sperm head birefringence in their head and increased DNA fragmentation are reported in both humans and animals [48][49][50][51][52]. In the human ejaculate, there is a very heterogeneous sperm population, with various degrees of nuclear maturity.…”