Monitoring Changes in Sperm Concentration and Quality Help Improve Captive Breeding Outcomes of the Endangered Amphibian Rawlinsonia littlejohni

Amphibians are among the most threatened taxonomic groups, with many species experiencing rapid declines that have resulted in small, isolated populations vulnerable to inbreeding depression and reduced fertility. One major outcome of inbreeding is lowered fecundity, often linked to morphological changes in gametes. Littlejohn's tree frog (Rawlinsonia littlejohni; previously Litoria) is a threatened species requiring targeted conservation management. To increase population numbers and restore genetic diversity, a captive breeding program has been established. This study aimed to optimize the timing of hormonally-induced sperm collection to improve outcomes for captive breeding, cryopreservation, and in vitro fertilization. Three experiments were conducted with ten captive males. Experiment one examined the effects of different human Chorionic Gonadotropin (hCG) and Gonadotropin-releasing hormone antagonist (GnRH-a) concentrations on spermiation response over 6 h. Both 5 and 10 IU hCG/g bwt yielded high sperm concentrations with similar motility and membrane viability, whereas GnRH-a (1 and 2 µg/g bwt) produced no response. Experiment two assessed seasonal effects on sperm traits over a year, showing high variability between individuals but no consistent seasonal pattern. Sperm motility and membrane viability remained high, with slight declines in January, April, and June. Experiment three compared captive and wild males during the breeding season. Sperm concentration was higher in June for wild frogs but variable across all months in both groups. Our findings demonstrate that sperm production and quality in R. littlejohni are shaped more by its breeding system than by seasonal cycles, informing strategies to enhance reproductive success and genetic recovery in captivity.