In this study, we observed and evaluated the sperm of the Neurergus derjugini species for the first time. Our primary aim was to explore methods for preserving sperm for future fertilization studies under laboratory conditions and to address spawning asynchrony in captivity. This research also established a connection between sperm dilution media, motility duration, and storage temperatures. Furthermore, we investigated long-term sperm storage using different cryoprotectants to develop optimal protocols for amphibian genetic diversity programs in captivity. Our study highlights the critical role of selecting an appropriate dilution solution, as it significantly impacts both sperm motility and morphology. Notably, this research is pioneering in its use of a DNA fragmentation kit to assess sperm health following the freezing process. This approach underscores the importance of integrating advanced technologies into sperm storage strategies for breeding programs and the conservation of endangered species.
Few studies have explored extenders for diluting sperm in caudate amphibians. For instance, Mansour et al., 201138 examined sperm motility in Ambystoma mexicanum and reported a motility duration of 4 hours at 0°C and 1 hour at room temperature when diluted in water or fertilization solution. These findings are consistent with our results on the effectiveness of various dilutions at low temperatures. Marcec 201633 used 10% Holt’s solution and 1% BSA for sperm dilution in Ambystoma tigrinum, while Guy et al., 2020 9 employed 2% trehalose and 0.2% BSA for three newt species. McGinnity et al., 2021 39 used SAR solution for sperm dilution, which aligns with our findings showing SAR as the second most effective diluent at investigated temperatures. Additionally, Gillis et al., 202127 demonstrated that 2% trehalose + 0.2% BSA maintained sperm motility better at 0°C compared to 20°C, a result that corroborates our findings favoring lower temperatures for sperm storage. Similarly, Arregui et al., 2020 26 showed that sperm from Anaxyrus fowleri stored at 4°C maintained motility and fertilizing capacity for up to 8 days, and Browne et al.,2002 10 observed over 50% motility for Bufo marinus sperm stored at 0°C for up to 7 days. Our study confirms that 10% Holt’s solution is effective for sperm dilution, particularly for extended storage at 4°C, and that it is superior to other extenders like SAR, PBS, and HBSS at this temperature. At 9°C, 10% Holt’s solution showed the highest daily motility, followed by SAR, HBSS, PBS, and 1% BSA, respectively. At 21°C, 10% Holt’s solution and HBSS maintained motility for two days, whereas other extenders like sucrose and distilled water showed less than 24 hours of motility. Our study builds upon recent research, such as Coxe et al., 2024 14 which compared deionized water and HBSS for sperm dilution and found that HBSS provided better motility. Chen et al., 2024 31 demonstrated that 10% Holt’s solution was effective for maintaining progressive motility. We expanded on these findings by evaluating multiple diluents at three different temperatures, confirming that 10% Holt’s solution supports long-term sperm storage at 4°C.
Research has shown that the choice and concentration of cryoprotectants significantly impact the post-thaw motility and survival of sperm. The use of specific cryoprotectants, such as DMSO and DMFA, at certain concentrations can lead to high rates of recovery of motility and fertilizing capacity in amphibian sperm 40. Our study found that Cryoprotectants C14, C15, and C16 were the most effective in preserving sperm motility and morphology. Cryoprotectant C16 showed the best results in terms of motility, morphology, and DNA integrity, highlighting the importance of selecting optimal cryoprotectants for long-term sperm storage. The studies that have been used in recent years for freezing the sperm of caudate amphibians have used a combination of both penetrative and non-penetrative cryoprotectants, which have brought different results on motility and morphology sperm 9,33,39. The combination of penetrative and non-penetrative cryoprotectants is crucial in achieving superior post-thaw recovery with high proportions of forward progressive motility, live cells, and intact acrosomes in various amphibian species 41. Our results showed that no significant motility was observed for mountain newt sperm using the compounds that were previously used to investigate the motility after thawing of tailed amphibian sperm (C1-C3). The mechanism of cold protection for amphibian sperm involves the use of cryoprotectants to minimize the detrimental effects of cooling, freezing, and thawing on sperm function. The use of dimethyl sulfoxide (Me2SO) as a cryoprotectant is effective in reducing sperm lysis and promoting post-thaw recovery of sperm motility and vitality 16. Additionally, the combination of Me2SO and sucrose is particularly effective in preserving sperm viability and motility in various amphibian species. However, the concentration of non-penetrative cryoprotectants such as sucrose should be carefully considered, as excessive osmolality may cause damage to cells 42. Overall, the use of cryoprotectants, particularly Me2SO in combination with sucrose and different chemical compounds, demonstrated feasibility in preserving amphibian sperm and is a valuable tool for the conservation and genetic management of endangered species.
Sperm DNA fragmentation can have significant implications for ART outcomes. Studies have shown that high levels of sperm DNA fragmentation are associated with decreased pregnancy rates in ART 43. The use of DNA fragmentation analysis kits is potentially useful 44. However, it's important to note that the relationship between sperm DNA fragmentation and ART outcomes remains controversial. Therefore, while DNA fragmentation analysis kits can be valuable tools in evaluating male infertility and predicting IVF success, further research is warranted to explore the mechanisms underlying DNA fragmentation and to develop standardized assessment methods 45. DNA fragmentation in thawed sperm can have significant effects on amphibian reproduction. Studies have shown that cryopreserved sperm can undergo structural changes, leading to compromised chromatin integrity and susceptibility to DNA fragmentation. This can impact the reproductive viability of offspring generated using assisted reproductive technologies, potentially limiting the widespread integration of these technologies 4. However, the specific impact of DNA fragmentation on the reproductive outcome of amphibian species is not yet fully understood, and further research is needed to determine the extent of its influence on the success of assisted fertilization and the development of cryo-derived offspring 5. These findings demonstrate the importance of cryoprotectants and freezing protocols in preserving the motility and viability of amphibian sperm for assisted reproductive technologies and conservation efforts.
Our findings, which demonstrate that the most effective cryoprotectants also resulted in lower DNA fragmentation, emphasize the potential for using these methods in conservation efforts for endangered species. By improving sperm preservation techniques, we can advance breeding programs and contribute to the recovery of endangered amphibian populations 5,46. Our study not only provides new insights into the preservation of Neurergus derjugini sperm but also establishes a foundation for future research on amphibian reproductive biology. By integrating advanced technologies and refining cryopreservation protocols, we aim to support the conservation of this species and enhance our understanding of amphibian reproductive processes. The potential evolutionary consequences of DNA fragmentation in amphibian sperm are not explicitly investigated in empirical studies, but there are theoretical explanations and potential implications to consider. The establishment of genetic resource banks and the use of ART for amphibian conservation highlight the importance of understanding the potential significance of sperm DNA damage in the assessment of amphibian spermatogenesis and sperm preservation procedures. These considerations suggest that DNA fragmentation in amphibian sperm may have implications for sexual selection, reproductive success, and genetic diversity within amphibian populations.