Refrigerated storage and cryopreservation of hormone induced sperm in a threatened frog

There are strong potential benefits of incorporating assisted reproductive technologies (ARTs) into conservation programs for the management of threatened amphibians as the global amphibian decline continues. As sperm cryopreservation and other ARTs advance in common species, focus on non-lethal sperm collection methods for threatened amphibians is imperative. We aimed to realise this goal by testing various doses of exogenous hormones for non-lethal induction of spermiation in a threatened frog (Litoria aurea) and develop cold storage and cryopreservation protocols following the recovery of urinic sperm. Our major findings include: (1) that sperm release could be induced in high concentrations with 20 IU/g bodyweight of human chorionic gonadotrophin (hCG); (2) high levels (>50%) of live, motile sperm could be recovered post-cryopreservation by treating the sperm with 15% v/v DMSO and 1% w/v sucrose pre-freeze; and (3) urinic sperm stored at 5{degrees}C retained motility over a 14-day period. Our findings demonstrate that it is possible to obtain and store large quantities of quality sperm from a threatened amphibian via non-lethal means, representing an important step forward for the use of ARTs in conservation programs for rare and threatened species. Lay SummaryAmphibians are undergoing an extinction crisis unparalleled in any other taxa. The development of assisted reproductive technologies, such as sperm cryopreservation and IVF have an important role to play in the conservation management of amphibians globally. Here we report non-lethal methods of sperm collection and storage in a highly threatened amphibian. Major findings include successfully induced sperm release in high concentrations, retention of [~]50% live, motile sperm after 7 days storing it at 5{degrees}C, and successful recovery of of more than 50% live, motile sperm following cryopreservation. Our results demonstrate the viability of obtaining and storing sperm from rare and threatened species via non-lethal means, providing an important step forward for the use of ARTs in conservation programs around the globe.