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

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review

3 Citations (Scopus)
3 Downloads (Pure)


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 2.8 M) of the cryoprotectants dimethylsulfoxide (DMSO) and glycerol with 0.3 M 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 2.1 M provided the best protection regardless of cryoprotectant type. Spermatozoa cryopreserved in 2.1 M 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.

Original languageEnglish
Pages (from-to)562-572
Number of pages11
JournalReproduction, Fertility and Development
Issue number9
Early online date6 Apr 2021
Publication statusPublished - 2021
Externally publishedYes

Bibliographical note

Copyright the Publisher 2021. Version archived for private and non-commercial use with the permission of the author/s and according to publisher conditions. For further rights please contact the publisher.


  • anuran
  • artificial fertilisation
  • captive survival-assurance colonies
  • cryoconservation
  • genome resource bank
  • green and golden bell frog
  • Pelodryadidae
  • spermatozoa


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