Linking in-situ and ex-situ populations of threatened amphibians through genome banking

Genetic variation of the endangered Puerto Rican crested toad (PRCT; Peltophryne lemur) has dwindled over time in both wild and captive populations, leading to long-term sustainability issues for the recovery program. To address this challenge, we propose that PRCTs can be used as a model species to show how in-situ and ex-situ populations can be linked through sperm biobanking and gamete transfer, expanding genetic variability of both populations. Male toads (n = 10) in Guayanilla, Puerto Rico were administered human chorionic-gonadotropin (hCG) and luteinizing-hormone-releasing-hormone analog (GnRHa) to stimulate spermiation. Sperm was collected noninvasively from 9/10 males, cryopreserved in 10% trehalose with either 10% N,N-dimethylformamide (DMFA) or 10% dimethyl sulfoxide (DMSO), and transported in liquid nitrogen vapor to the National Amphibian Genome Resource Bank at Mississippi State University. Ultrasonography was used to identify females (n = 3) with mature oocytes for GnRHa-induced oviposition of eggs for in-vitro fertilization (IVF). Post-thaw sperm motility was 28% and 25% for sperm cryopreserved with DMFA or DMSO, respectively. Of the 9,672 eggs used for IVF, 4% (n = 306/6,981) were fertilized with frozen-thawed sperm, compared with 20% (n = 525/2691) fertilized with fresh sperm controls. Overall, 46 toadlets were produced from frozen-thawed sperm. After 4.5 months of headstarting, 14 juvenile toads produced from various genetic crosses using frozen-thawed sperm were released to new sites in the wild, introducing unique genetic representation and new founder lines. After 1.5 years, 24 adult toads produced using frozen-thawed sperm continue to thrive in the captive collection, and one of these males has now produced an F2 generation of offspring with 5,085 tadpoles released to the wild. This transformational study is the first to produce reproductively viable adult PRCTs using cryopreserved sperm from hormonally-induced wild males and captive females, introducing innovative methods that link in-situ and ex-situ populations of endangered amphibians to revolutionize genetic management.