026 Cryoconservation: Successful sperm cryopreservation and develop-mental outcomes using endangered North American amphibians

In response to the dramatic global decline of amphibian species, captive assurance colonies are being established to safeguard genetic diversity; however, many of these founder populations fail to reproduce. Therefore, assisted reproductive technologies such as gamete cryopreservation and artificial fertilisation are valuable conservation tools in the genetic management and breeding of these non-reproducing populations. The following study aimed to develop a successful sperm cryopreservation protocol for two endangered amphibian species maintained in captivity: the threatened boreal toad (Anaxyrus boreas boreas) and the critically endangered Mississippi gopher frog (Lithobates sevosa). Spermiation was induced by exogenous hormone administration of human chorionic gonadotropin at 10IU/g for A. b. boreas and by a cocktail of 500 IU hCG and 15 μg luteinizing hormone-releasing hormone for L. sevosa. Fresh sperm quality was assessed using the following parameters: (1) sperm concentration (2) percentage motile sperm, and (3) percentage sperm moving progressively forward. Sperm samples were subsequently extended 1:1 in chilled cryoprotectant containing 10% (v/v) N-N-dimethylformamide and 10% (w/v) trehalose. The chilled sperm suspensions were held at 4 °C for an acclimation period of 10 min before being loaded into 0.25 mL plastic freezing straws and frozen in a step-wise slow-rate freezing process. Straws were thawed in a 40 °C water bath before a 1:10 dilution with aged tap water to activate the sperm for re-evaluation. Exogenous hormone induction stimulated spermiation in A. b. boreas and L. sevosa, producing mean fresh sperm concentrations of 4.6 × 106/mL and 2.4 × 106/mL respectively, with high rates of motility for both A. b. boreas (86 ± 4%) and L. sevosa (92 ± 2%) (mean ± SE). Upon activation, motility recovery from cryopreserved sperm suspensions was high for A. b. boreas (62 ± 2%) and L. sevosa (70 ± 3%) and sperm forward progression was maintained for both species (57 ± 3% and 29 ± 6%, for A. b. boreas and L. sevosa, respectively). Furthermore, frozen-thawed spermatozoa were used for fertilisation trials and developmental stages analysis. Preliminary investigations using cryopreserved L. sevosa sperm indicated that 14% of fertilised oocytes developed to 2-cell stage embryos with 6% continuing to tadpole stage. The results of this study demonstrate that sperm from both A. b. boreas and L. sevosa can be successfully cryopreserved and maintain high rates of sperm motility and forward progressive movement recovery. Additionally, fertilisation can be achieved in the critically endangered Mississippi gopher frog with subsequent tadpole production. The ability to successfully cryopreserve sperm from endangered amphibian species has enormous potential in augmenting declining populations by facilitating gene flow, both between breeding facilities and between in situ and ex situ populations, without removing source animals. Moreover, this methodology may be directly applicable to a wide range of amphibian taxa, affording a viable option for securing the remaining genetic diversity of endangered species in a global genetic resource bank. Source of funding: Institute of Museum and Library Services. Conflict of interest: None declared. cjl224@msstate.edu