18 All aboard the polar express: transferability of a cryopreservation protocol between anuran species

Sperm cryopreservation is a crucial tool in preserving genetic lineages to increase diversity within populations. While cryopreservation has been readily explored in livestock and aquaculture, its use in amphibian conservation is relatively recent. Due to the vast number of amphibian species, the likelihood of developing a single protocol for each group is nearly impossible. Determining whether cryopreservation practices are transferable between species will streamline efforts and maximise the amount of genetic material stored across individuals. The purpose of this study was to determine transferability of a sperm cryopreservation procedure developed using Fowler’s toads (Anaxyrus fowleri) as a model species on two threatened and endangered species, the Houston toad (Anaxyrus houstonensis) and Puerto Rican crested toad (Peltophryne lemur). A. fowleri (N = 32) were housed at Mississippi State University for the model study, and based on the results, we selected 10% N,N-dimethylformamide as the cryoprotectant and tested two freezing rates: 20 to 29°C min⁻¹ and 32 to 45°C min⁻¹. We hypothesised that the faster freezing rate would lead to a higher post-thaw motility, as observed in A. fowleri. A. houstonensis (N = 11) were housed at the Fort Worth Zoo, and P. lemur were housed at the Fort Worth Zoo (N = 9) and the North Carolina Zoo (N = 10). Toads were administered species-specific hormone dosages using human chorionic gonadotrophin and gonadotrophin-releasing hormone analogue, followed by sperm collection and motility analysis. Sperm samples were divided into 100-µL aliquots, mixed in a 1:1 ratio with a stock cryoprotectant for a final concentration of 10% trehalose + 0.25% bovine serum albumin + 10% N,N-dimethylformamide, and cryopreserved at the two freezing rates. A subset of cryopreserved sperm (A. houstonensis: N = 8; P. lemur: N = 12) was selected for thawing to determine how the two rates affected post-thaw sperm motility. We used generalised linear mixed models to determine variation in post-thaw motility between species separated by freezing rate, as well as generalised additive model for location scale and shape tests to determine variation in post-thaw sperm motility between freezing rates separated by species. We did not find any significant differences in post-thaw motilities between species for the faster freezing rate (t > 0.05; P > 0.6; A. fowleri: 30 ± 3%; A. houstonensis: 32 ± 5%; P. lemur: 32 ± 4%) or slower rate (t > 0.04; P > 0.5; A. fowleri: 23 ± 2%; A. houstonensis: 27 ± 4%; P. lemur: 27 ± 4%), indicating that the effect of freezing rates on motility could be consistent across species. Furthermore, we did not find any significant differences between post-thaw sperm motility for either freezing rate in A. houstonensis (t = 1.8; P = 0.09) or P. lemur (t = 2.1; P = 0.05). However, on average, the faster rate produced higher levels of post-thaw motility than the slower rate for all groups. Although we did not find any significant variation between treatments, these results indicate that there may be an emerging trend supporting the possibility for protocol transmission within the Bufonid family.