Generation of reproductively mature offspring from the endangered green and golden bell frog Litoria aurea using cryopreserved spermatozoa

Rose Upton; Simon Clulow; Natalie E Calatayud; Kim Colyvas; Rebecca G Y Seeto; Lesley A M Wong; Michael J Mahony; John Clulow

doi:10.1071/RD20296

Generation of reproductively mature offspring from the endangered green and golden bell frog Litoria aurea using cryopreserved spermatozoa

Reprod Fertil Dev. 2021 Apr:33:562-572. doi: 10.1071/RD20296.

Authors

Rose Upton¹, Simon Clulow², Natalie E Calatayud³, Kim Colyvas⁴, Rebecca G Y Seeto⁵, Lesley A M Wong⁵, Michael J Mahony⁶, John Clulow⁶

Affiliations

¹ The Conservation Biology Research Group, School of Environmental and Life Sciences, The University of Newcastle, Callaghan, NSW 2308, Australia; and FAUNA Research Alliance, PO Box 5092, Kahibah, NSW 2290, Australia; and Corresponding author.
² FAUNA Research Alliance, PO Box 5092, Kahibah, NSW 2290, Australia; and Centre for Conservation Ecology and Genomics, Institute for Applied Ecology, University of Canberra, Bruce, ACT 2617, Australia.
³ FAUNA Research Alliance, PO Box 5092, Kahibah, NSW 2290, Australia; and Taronga Institute of Science and Learning, Taronga Conservation Society Australia, Taronga Western Plains Zoo, Dubbo, NSW 2830, Australia; and San Diego Zoo Global-Beckman Center for Conservation Research, 15600 San Pasqual Valley Road, Escondido, CA 92027, USA.
⁴ School of Mathematical and Physical Sciences, University of Newcastle, Callaghan, NSW 2308, Australia.
⁵ The Conservation Biology Research Group, School of Environmental and Life Sciences, The University of Newcastle, Callaghan, NSW 2308, Australia.
⁶ The Conservation Biology Research Group, School of Environmental and Life Sciences, The University of Newcastle, Callaghan, NSW 2308, Australia; and FAUNA Research Alliance, PO Box 5092, Kahibah, NSW 2290, Australia.

PMID: 33820600
DOI: 10.1071/RD20296

Abstract

Amphibians are becoming increasingly reliant on captive breeding programs for continued survival. Assisted reproductive technologies including gamete cryopreservation and IVF can help reduce costs of breeding programs, provide insurance against extinction and assist genetic rescue in wild populations. However, the use of these technologies to produce reproductively mature offspring has only been demonstrated in a few non-model species. We aimed to optimise sperm cryopreservation in the threatened frog Litoria aurea and generate mature offspring from frozen-thawed spermatozoa by IVF. We tested three concentrations (1.4, 2.1 and 2.8M) of the cryoprotectants dimethylsulfoxide (DMSO) and glycerol with 0.3M sucrose. Using DMSO was more likely to result in recovery of sperm motility, vitality and acrosome integrity than glycerol, regardless of concentration, with forward progressive motility being most sensitive to damage. The lowest concentrations of 1.4 and 2.1M provided the best protection regardless of cryoprotectant type. Spermatozoa cryopreserved in 2.1M DMSO outperformed spermatozoa cryopreserved in equivalent concentrations of glycerol in terms of their ability to fertilise ova, resulting in higher rates of embryos hatching and several individuals reaching sexual maturity. We have demonstrated that sperm cryopreservation and subsequent offspring generation via IVF is a feasible conservation tool for L. aurea and other threatened amphibians.