Dimethylacetamide can be used as an alternative to glycerol for the successful cryopreservation of koala (Phascolarctos cinereus) spermatozoa

Abstract

Swelling of koala sperm chromatin following cryopreservation has largely been attributed to the absence of intermolecular disulfide cross-linkages in the marsupial sperm nucleus. Fish spermatozoa also lack disulfide bonds within their chromatin, but have been successfully cryopreserved. The present study examined the hypothesis that the cryoprotectants used for fish sperm cryopreservation would confer a similar degree of protection on koala spermatozoa. Three concentrations each of five cryoprotectants (dimethyl sulfoxide, methanol, propylene glycol, ethylene glycol and dimethylacetamide (DMA)) were evaluated. Each treatment was compared against an established koala sperm cryopreservation protocol that uses 14% glycerol. Post-thaw assessment of progressive motility, plasma membrane integrity and mitochondrial membrane potential (MMP) revealed that protocols using 15% DMA achieved 62.2 +/- 3.6% (P < 0.05) sperm survival, of which 79% (P < 0.05) had high MMP, an improvement of 32% and 40%, respectively, over sperm frozen in 14% glycerol. The percentage of spermatozoa with swollen nuclei was also lowest when frozen in 15% DMA, both immediately after thawing (18.0 +/- 3.5%; P < 0.05) and after 2 h incubation at 35 degrees C (35.8 +/- 4.4%; P < 0.05). A second study was conducted to determine the optimal concentration of DMA for use in the cryopreservation of koala spermatozoa. High DMA concentrations (17.5% and 20%) resulted in significantly lower proportions of live spermatozoa showing high MMP immediately after thawing compared with spermatozoa frozen in the lower concentrations. The percentage of koala spermatozoa with swollen chromatin following cryopreservation was not affected by DMA concentration.

Osmotic stress and cryoinjury of koala sperm: an integrative study of the plasma membrane, chromatin stability and mitochondrial function

“…These results are consistent with other studies which show that the osmotic stress associated with thawing is likely to be most detrimental to the spermatozoa during the cryopreservation procedure (Holt et al 1992). The post-thaw membrane integrity reported in this study was higher than that reported in other studies on koala sperm examined immediately after cryopreservation (Johnston et al 2006, Zee et al 2008, 2009a and reflects the fact that spermatozoa in the current study were examined 30 min after thawing; however, it should be noted that these studies have also reported a rapid decline in PMI of these same spermatozoa after 120 min of incubation at 35 8C. These observations draw attention to the importance of induced damage associated with ex vivo handling (so-called 'iatrogenic damage') and the variable survival time of spermatozoa when incubated within in vitro environments that attempt to mimic the physiology of the female reproductive tract.…”

“…Koala semen cryopreservation has been described previously (Johnston et al 2006, Zee et al 2008. Briefly, each ejaculate was initially diluted (1:1) with warm (35 8C -normal koala body temperature) TCG buffer in a pre-warmed 1.5 ml microcentrifuge tube (Eppendorf AG, Hamburg, Germany) and allowed to cool to room temperature (25 8C) over a period of 10 min.…”

“…The koala (Phascolarctos cinereus) is one species in which the effects of cryopreservation have been investigated (Johnston et al 1994, 2006, 2008, 2009a. For example, koala spermatozoa have been shown to have a post-thaw viability of up to 60-70% if frozen in high levels of glycerol (14% v/v) combined with a relatively slow freezing rate of 6 8C/min.…”

“…For example, koala spermatozoa have been shown to have a post-thaw viability of up to 60-70% if frozen in high levels of glycerol (14% v/v) combined with a relatively slow freezing rate of 6 8C/min. However, extended incubation of thawed koala spermatozoa for a further 2 h rapidly reduces the percentage of live spermatozoa and results in a corresponding increase in the number of cells with swollen or relaxed chromatin (Zee et al 2008(Zee et al , 2009b. It is uncertain whether this modified form of chromatin is either a consequence of physical swelling or distortion of the DNA/protamine complex in response to osmotically induced injury or is associated with an increase in the fragmentation of the DNA molecule , Zee et al 2009b.…”

“…In fact, the aim of this investigation was to examine whether osmotic flux is one of the primary causes of koala sperm cryoinjury. The first objective was to investigate the phenomenon of koala sperm chromatin relaxation postcryopreservation associated with a loss of membrane integrity (Johnston et al 2006, Zee et al 2008, 2009a and to determine whether it was linked to a corresponding increase in sperm DNA fragmentation (SDF). Our second objective was to examine the effect of varying anisosmotic media on the MMP of koala spermatozoa and directly compare the observations of spermatozoa from the same ejaculate following cryopreservation.…”