28 Cryopreserved sperm obtained from live donor males in four amphibian species produces F₁ generation sexually mature adults with subsequent F₂ generation offspring

Assisted reproductive technologies (ART) are used for various amphibian species to manage genetic diversity and sustainability efforts to stabilise captive and wild populations threatened with extinction. Hormone therapy, sperm cryopreservation, and IVF are key elements in the ART toolbox to assist captive populations with low reproductive output. To date, there are few examples where threatened amphibian species have been produced using cryopreserved sperm obtained from live donor males. Even more uncommon are examples where F₂ generations of threatened amphibians have been produced from those offspring that were originally generated using frozen-thawed sperm. Thus, our objective was to evaluate the potential for cryopreserved sperm to produce reproductively viable F₁ offspring. To show that F₁ generations could be produced using cryopreserved sperm, we selected four species, including the threatened Puerto Rican crested toad (Peltophryne lemur); Dusky gopher frog (Lithobates sevosa); Houston toad (Anaxyrus houstonensis); and common tiger salamander (Ambystoma tigrinum). Spermatozoa from these four species were collected via exogenous hormone therapy, mixed with cryoprotectants for a final dilution of 10% trehalose with either 10% dimethylformamide or 10% dimethylsulfoxide, and subsequently loaded into 0.25-mL straws for freezing. Sperm were frozen in a two-step process by exposing straws to liquid nitrogen (LN₂) vapour for 10 min, followed by plunging into LN₂, which provided a freezing rate of −29 to −32°C min⁻¹. Cryopreserved sperm were thawed for 5 s at 40°C, reactivated in distilled water, and applied to eggs for 5 min before flooding the Petri dish with water. Here, we show the production of F₁ generation of offspring for all four species using cryopreserved sperm from live donor males, followed by IVF; see Table 1 below for offpsring production for each species. A total of 91 adults were generated using cryopreserved sperm from live donor males, 90 of them from threatened species. Only three of the species had reached sexual maturity by the time of this report and could be used to show proof of concept for production of the F₂ generation. Sexually mature F₁ males from L. sevosa and P. lemur were used, whereas one female A. tigrinum was used to show viable reproduction. Upon sexual maturity, the males and females were treated with exogenous hormones as described above to obtain gametes, and IVF was conducted as previously described. Following IVF, F₂ generation offspring were produced for L. sevosa (n = 48 metamorphs), P. lemur (n = 5095 tadpoles), and A. tigrinum (n = 5 metamorphs). Overall, we demonstrate that four amphibian species can be produced using cryopreserved sperm, and that upon sexual maturity, three of the species are reproductively viable producing their own offspring, highlighting the conservation value of these technologies for threatened amphibians.

Table 1.  Offspring production for each species