Applying sperm collection and cryopreservation protocols developed in a model amphibian to three threatened anuran species targeted for biobanking management

The Increasing Role of Short-Term Sperm Storage and Cryopreservation in Conserving Threatened Amphibian Species

“…Additionally, where an unexpected death occurs within a colony of threatened species, or a planned euthanasia occurs, testes macerates are a valuable resource that should be collected and cryopreserved for future application. In recent years, the successful cryopreservation of sperm obtained non-invasively as spermic urine or milt has been achieved in a growing number of threatened species, including the Chinese giant salamander ( Andrias davidianus ) [ 146 ], the Wyoming toad ( A. baxteri ) [ 147 ], the Houston toad ( Anaxyrus houstonensis ) [ 148 ], a Harlequin frog ( Atelopus sp.) [ 149 ], the Panamanian golden frog ( Atelopus zeteki ) [ 150 ], the Hellbender ( C. alleganiensis ) [ 37 ], the Chiricahua leopard frog ( Lithobates chiricahensis ) [ 148 ], the dusky gopher frog ( L. sevosa ) [ 35 , 147 ], the green and golden bell frog ( L. aurea )(R. Upton, unpublished data), the Booroolong frog ( L. booroolongensis ) (R. J. Hobbs et al, unpublished data), the yellow-spotted bell frog ( Litoria castanea ) (R. J. Hobbs and J. K. O’Brien, unpublished data), Littlejohn’s tree frog ( Litoria littlejohni ) (R. Upton, unpublished data), the black-spotted newt ( Notophthalmus meridionalis ) [ 151 ], the Puerto Rican crested toad ( P. lemur ) [ 148 , 152 ], Baw Baw frog ( Philoria frosti ) (A. J. Silla and R. J. Hobbs et al, unpublished data), the Southern corroboree frog ( P. corroboree ), the Northern corroboree frog ( Pseudophryne pengilleyi ) (R. J. Hobbs and J. K. O’Brien, unpublished data), and the Kweichow newt ( Tylototriton kweichowensis ) [ 151 ].…”

“…In amphibians, a final concentration of 5–15% DMSO or DMF may provide this balance; however, susceptibility to CPA toxicity is species specific [ 35 ], which may provide an explanation for the differences in optimal concentrations among and within species. Furthermore, the addition of other components, including BSA and FBS (e.g., [ 38 , 55 , 56 , 60 , 148 , 151 , 168 , 170 ]), may further reduce adverse effects on sperm cells by acting as antioxidants, increasing membrane fluidity and further dehydrating the sperm cells [ 38 ], and so may explain the higher sperm parameters reported in some cases. Future studies should seek to test protocol transferability between model and endangered species, continue to test differing types and concentrations of CPAs in a wider range of amphibian species, and test whether the inclusion of additional components into the cryopreservation medium can improve post-thaw sperm outcomes.…”

“…Conversely, other studies demonstrated improved sperm motility following freezing at faster rates (>−30 °C/min), with Burger et al [ 38 ] freezing sperm 5 cm above liquid nitrogen (−32 to −45 °C/min) resulting in significantly higher sperm motility compared to freezing at 10 cm above liquid nitrogen (−20 to −29 °C/min) in Fowler’s toad ( A. fowleri ). A follow-up study [ 148 ] also found that freezing 5 cm above liquid nitrogen resulted in significantly higher post-thaw sperm motility in two additional species: the Puerto Rican crested toad ( P. lemur ) and the Chiricahua leopard frog ( L. chiricahuensis ). In addition, this method also resulted in higher sperm motility in the Houston toad ( A. houstonensis ), though this result was not statistically significant [ 148 ].…”

“…A follow-up study [ 148 ] also found that freezing 5 cm above liquid nitrogen resulted in significantly higher post-thaw sperm motility in two additional species: the Puerto Rican crested toad ( P. lemur ) and the Chiricahua leopard frog ( L. chiricahuensis ). In addition, this method also resulted in higher sperm motility in the Houston toad ( A. houstonensis ), though this result was not statistically significant [ 148 ]. The variation in post-thaw sperm parameters between freezing rates is difficult to explain due to large differences in freezing methods, including the composition of the cryopreservation medium, the packaging used, and the source of sperm (spermic urine or testes macerates).…”

Cryopreservation Cooling Rate Impacts Post-Thaw Sperm Motility and Survival in Litoria booroolongensis

“…The use of a faster cooling rate with DMSO/SUC as CPA in the present study produced similar proportions of motile sperm (~60% total motility) post-thaw as those published previously for testis macerates in a range of Litoria species (reviewed in: [6]). The higher survival and motility of spermic urine samples using a faster cooling rate has been demonstrated recently in three species [53], where cooling rates in the range of −30 to −45 • C min −1 proved beneficial. Furthermore, higher hatching rates were obtained following assisted fertilization (AF) using sperm cryopreserved at −30 to −45 • C min −1 than at −20 to −29 • C min −1 [27], though this may be due to higher numbers of motile sperm in aliquots for AF, rather than an improvement in sperm quality or velocity, as it is unclear whether the motile concentrations of samples were matched between groups prior to AF even though a split ejaculate design was employed.…”