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

Authors: LAMPERT, SHAINA - Mississippi State University; BURGER, ISABELLA - Mississippi State University; CHEN, LI-DUNN - Mississippi State University; SMITH, DUSTIN - North Carolina Zoo; KOUBA, ANDREW - Mississippi State University; BARBER, DIANE - Fort Worth Zoo; VANCE, CARRIE - Mississippi State University; COBOS, CASSIDI - Turner Enterprises, Inc

Submitted to: Biological Conservation
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 12/5/2022
Publication Date: 12/26/2022
Citation: Lampert, S., Burger, I., Chen, L., Smith, D., Kouba, A., Barber, D., Vance, C.K., Cobos, C. 2022. Applying sperm collection and cryopreservation protocols developed in a model amphibian to three threatened anuran species targeted for biobanking management. Biological Conservation. 227:109850-56. https://doi.org/10.1016/j.biocon.2022.109850.
DOI: https://doi.org/10.1016/j.biocon.2022.109850

Interpretive Summary: This study highlights the value of ART and biobanking for amphibian conservation and genetic management by showing: 1) the transferability of protocols developed in our model species, A. fowleri, to other anuran species; and 2) the use of a faster freezing rate to produce higher levels of post-thaw sperm recovery. We also introduce the idea that freezing rate protocols can be applied not only within a family, but also between families, with a potential explanation of how morphological distinctions between Bufonidae and Ranidae (e.g., presence of a fragile mitochondrial vesicle on Bufonid sperm, but not Ranid sperm) may be the cause of the differences in recovered motility rates. Continuing to apply the cryopreservation protocols developed here to other species, while also continuing to refine current ART strategies in model organisms, will not only further validate the possibility of protocol transmission, but also limit the number of species-specific protocols that need to be developed to assist in population and genetic management for threatened amphibians.

Technical Abstract: The challenge of replicating environmental cues in amphibian captive assurance colonies has been shown to be one of the contributing factors leading to limited breeding success. To circumvent this challenge, assisted reproductive technologies (ART) have been developed in model organisms and can now be applied to at-risk species. This study tested the transferability of cryopreservation protocols developed in the Fowler's toad (Anaxyrus fowleri) to three threatened anurans, the Chiricahua leopard frog (Lithobates chiricahuensis), Puerto Rican crested toad (Peltophryne lemur), and Houston toad (Anaxyrus houstonensis). Males were administered human chorionic gonadotropin and/or gonadotropin releasing hormone-analog to elicit spermiation, and spermic urine was collected and analyzed for motility. Samples were cryopreserved with 10 % N,N-dimethylformamide + 10 % trehalose + 0.25 % bovine serum albumin and frozen at two rates, -32 to -45 °C/min and - 20 to -29 °C/min. Results show that sperm frozen at -32 to -45 °C/min recovered significantly higher post-thaw sperm motility than -20 to -29 °C/min for P. lemur (p = 0.04) and L. chiricahuensis (p = 0.03). When compared across species, L. chiricahuensis had significantly higher post-thaw motility at both freezing rates (p < 0.05) than the other species, yet there was no difference between sperm motility between the three bufonid species for either freezing rate (p > 0.06). These results indicate that transferability of a cryopreservation protocol between species is possible, and differences in sensitivity and response to cryopreservation may exist between genera.