There are extremely limited studies that have investigated DNA fragmentation in amphibians and even less information exists pertaining to DNA fragmentation in the spermatozoa. A DNA fragmentation assay applied and validated for amphibian spermatozoa would not only provide the first evidence of this phenomenon in a new major taxon, but also prove a valuable tool for developing improvements in ART used in the captive propagation of threatened and endangered species. Consequently, the fundamental aim of this project was the development and validation of the sperm chromatin dispersion (SCD) test for amphibian spermatozoa with the purpose of investigating the dynamic behaviour of amphibian sperm DNA in response to cryopreservation.
The SCD test was first applied to African clawed frog (Xenopus laevis) as an amphibian sperm model using a species-specific modified lysing solution for protein depletion. Sperm DNA fragmentation (SDF) was assessed immediately following activation of sperm motility (T0) and again after one hour and 24 hours of incubation in order to produce a range of spermatozoa with differing levels of DNA damage. The SCD procedure resulted in the production of three nuclear morphotypes; amphibian sperm morphotype 1 (ASM-1) and ASM-2 showed no evidence of DNA damage, whereas ASM-3 spermatozoa were highly fragmented with large halos of dispersed DNA fragments and a reduced nuclear core. Levels of SDF revealed by the SCD test were highly correlated with results produced using in situ nick hybridisation with DNA-specific molecular probes (ISNT) and the double comet assay (r = 0.9613). The alkaline step of the double-comet assay revealed the extensive presence of structural single-stranded DNA breaks (SSB) and provided grounds for the suggestion that alkali labile sites (ALS) are a constitutive feature of African clawed frog sperm chromatin.
SDF is a dynamic process and has been shown to increase with incubation at room temperature as well as following cryopreservation. However, the mechanisms of DNA cryoinjury and the resulting effect on the potential reproductive output of ART remain open to interpretation. Consequently, dynamic changes to the basal level of DNA fragmentation were examined in fresh African clawed frog spermatozoa in comparison with the post-thaw integrity of cryopreserved (-80 °C) and frozen (-20 °C) sperm DNA. SDF was examined initially at T0 and over incubation of up to 60 minutes. The difference in SDF was only marginal for fresh, frozen (-20 °C) and cryopreserved (-80 °C) spermatozoa examined at the onset of the experiment immediately upon thawing, however, a significant increase (p = 0.004) in SDF, albeit of a low magnitude, was observed in activated cryopreserved (-80 °C) spermatozoa following incubation. Although African clawed frog spermatozoa appeared to be highly resistant to cryopreservation-induced DNA damage, cryopreserved-thawed spermatozoa yielded a significantly lower (p = 0.0065) fertilisation rate than fresh spermatozoa.
In order to provide further insight into cryoinjury of amphibian sperm chromatin, the dynamic loss of DNA integrity in fresh and cryopreserved cane toad (Bufo marinus) spermatozoa was correlated with changes in sperm viability and motility. Analysis of SDF was conducted at T0 immediately following activation and/or thawing and again following T3 and T6 hours of incubation at room temperature. Sperm motility was activated by decreasing the osmolality of the sperm extender to < 50 mOsmol kg-1 with dH20. Sperm viability was assessed using a dual emission technique that incorporated the nucleic stains SYBR-14 and propidium iodide. Spermatozoa were classified as either live (green fluorescence) or dead (red fluorescence). Dead spermatozoa were further classified into two categories; dead-compact, where spermatozoa retained a normal elongated, rod-like morphology and dead-swollen, where spermatozoa appeared enlarged with less defined morphology. Assessment of SDF at the population level in fresh spermatozoa, as well as the dynamics of sperm chromatin longevity, revealed that cane toad sperm DNA was highly stable throughout the duration of the incubation period. Results demonstrated a surprisingly poor recovery of both motility and viability in cane toad spermatozoa following freeze-thawing and a significant increase in the proportion of spermatozoa showing nuclear swelling (p < 0.0001). The incidence of dead-swollen spermatozoa observed in the cane toad was interpreted to represent the transition from ASM-1 to ASM-2 described in African clawed frog spermatozoa and lent support to the hypothesis that ALS may exist in the sperm chromatin of both amphibians.