- Title
- Development of sperm cryopreservation and assisted reproductive technologies for the conservation of threatened Australian tree frogs
- Creator
- Upton, Rose
- Relation
- University of Newcastle Research Higher Degree Thesis
- Resource Type
- thesis
- Date
- 2020
- Description
- Research Doctorate - Doctor of Philosophy (PhD)
- Description
- As global amphibian declines surpass those of other taxa in the event recognised widely as the 6th mass extinction, amphibians are becoming increasingly reliant on captive breeding programs for the survival of many species. Assisted reproductive technologies (ARTs), such as sperm cryopreservation, in vitro fertilisation (IVF), short-term cold storage of sperm and induction of gamete release using exogenous hormones, can provide additional tools to increase efficiency and decrease costs of captive breeding and provide genetic rescue to small wild breeding populations. There are relatively few examples where ARTs have been applied to conservation in practice, and no examples of the restoration of genetic diversity of threatened amphibian species in captive or wild populations. This gap in the application of technology to conservation programs needs to be addressed if the potential of ARTs and biobanking for amphibian conservation are to be realised. To begin to bridge this gap, development of reliable protocols and proofs-of-concept, including the production of sexually mature progeny by ARTs, will help to demonstrate the legitimacy of the technologies to the conservation of amphibians. This thesis, therefore, aimed to: (1) Compare the use of two penetrating cryoprotectants, dimethyl sulfoxide (DMSO) and glycerol at three different concentrations (10, 15 and 20% v/v, equivalent to 1.4, 2.1 and 2.8 M) for the recovery of motile and live sperm of a threatened pelodryadid, Litoria aurea. Further, 15% v/v DMSO and glycerol cryopreserved sperm were tested in an IVF system to compare fertilisation and development through the various stages of embryogenesis; (2) Compare the use of varying concentrations of the penetrative cryoprotectant DMSO (at 10 and 15% v/v) and the non-penetrative cryoprotectant sucrose (at 1 and 10% w/v) for the recovery of motile and live sperm from a range of pelodryadid species; and (3) Compare hormonal induction protocols for non-lethal collection of L. aurea sperm for collection of concentrated and motile sperm. Further, the cryopreservation protocols tested in aims 1 and 2 were tested to determine whether cryopreservation of urinic sperm post-thaw recovery is comparable to that of testicular macerates. Finally, the effect of storage of L. aurea urinic sperm at 5℃ for 14 days on motility was investigated. It was found that cryopreservation of testicular macerates of L. aurea was successful using 10-15% DMSO (with a base of 10% w/v sucrose), with recovery of up to ~40% forward progressive motility and vitality (assessed by membrane integrity) and 53% intact acrosomes using these cryoprotectants. Cryopreserved sperm achieved fertilisation rates of ~12% using the treatments 15% DMSO and 15% glycerol, however only embryos fertilised with sperm from DMSO treatments were able to continue development to hatched tadpoles (~2%). This study also demonstrated that individuals fertilised with cryopreserved sperm (15% DMSO) were able to reach sexual maturity. A major modification of the amphibian sperm cryopreservation paradigm was introduced by reducing the sucrose concentration in cryodiluents to 1% w/v. This resulted in an improved post-thaw preservation of motility and vitality in an additional six pelodryadid species (L. dentata, L. fallax, L. citropa, L. nudidigitus, L. lesuerii and L tyleri). While the recovery varied between these species (with post-thaw forward-progressive motility as low as ~10% in L. lesuerii and as high as 50-65% in L. dentata, L. fallax and L. tyleri), the best recovery was always present with the lowered sucrose concentration. This held true when testing the post-thaw recovery of L. aurea urinic sperm in the final study, with post-thaw recovery of ~55% motility and vitality using this treatment. Induction of sperm release using 20 IU/g human chorionic gonadotropin (hCG) yielded the highest concentration of sperm (~1.44x108 cells/ml at peak timepoint, 3 hours post injection) and total numbers (~3.61x106 cells/sample at peak timepoint, 2 hours post-injection) in this study and was also associated with significantly higher motility than sperm induced by other treatments. Finally, urinic sperm samples from L. aurea were successfully stored at 5℃ for up to 14 days with an average retention of ~50% motility at day 7 and ~20% motility by day 14. These results represent a significant contribution to the development of ARTs in Australian pelodryadids. With increasing interest in biobanking, development of effective protocols and proof-of concepts, such as production of sexually mature individuals through IVF, is an imperative if ARTs are to be recognised as a legitimate tool in the management of threatened species. The results of this study therefore demonstrate the successful development of sperm cryopreservation protocols for a range of different pelodryadids as well as IVF, hormone induction and short-term cold storage protocols for sperm of L. aurea. This may provide an avenue for inclusion of ARTs in conservation of threatened pelodryadids, including L. aurea and other species.
- Subject
- amphibian; frog; Anura; assisted reproductive technolgies; sperm cryopreservation; IVF; conservation
- Identifier
- http://hdl.handle.net/1959.13/1493460
- Identifier
- uon:53557
- Rights
- Copyright 2020 Rose Upton
- Language
- eng
- Full Text
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