Refrigerated storage and cryopreservation of hormonally induced sperm in the threatened frog, Litoria aurea

As sperm cryopreservation and other assisted reproductive technologies (ARTs) advance in common amphibian species, focus on applying non-lethal sperm collection methods to the conservation and genetic management of threatened species is imperative. The goal of this study was to examine the application of logistically practical ART protocols in a threatened frog (Litoria aurea). First, we tested the efficacy of various concentrations of human chorionic gonadotropin (hCG) (20, 40 IU/g bodyweight) and Gonadotropin releasing hormone antagonist (0.25 µg/g and 0.5 µg/g body weight GnRH-a) on the induction of spermatozoa. Using the samples obtained from the previous trials, we tested the effect of cold storage and cryopreservation protocols on long-term refrigerated storage and post-thaw sperm recovery. Our major findings include: (1) high quality sperm were induced with 20 and 40 IU/g bodyweight of (hCG); (2) proportions of live, motile sperm post-thaw, were recovered at higher levels than previously reported for L. aurea (>50%) when preserved with 15% v/v DMSO and 1% w/v sucrose; and (3) spermic urine stored at 5 °C retained motility for up to 14 days. Our findings demonstrate that the protocols developed in this study allowed for successful induction and recovery of high-quality spermatozoa from a threatened Australian anuran, L. aurea, providing a prime example of how ARTs can contribute to the conservation of rare and threatened species.