Captive breeding is an important tool for amphibian conservation despite high economic costs and deleterious genetic effects of sustained captivity and unavoidably small colony sizes. Integration of biobanking and assisted reproductive technologies (ARTs) could provide solutions to these challenges, but is rarely used due to lack of recognition of the potential benefits and clear policy direction. Here we present compelling genetic and economic arguments to integrate biobanking and ARTs into captive breeding programs using modelled captive populations of two Australian threatened frogs, namely the orange-bellied frog Geocrinia vitellina and the white bellied frog Geocrinia alba. Back-crossing with frozen founder spermatozoa using ARTs every generation minimises rates of inbreeding and provides considerable reductions in colony size and program costs compared with conventional captive management. Biobanking could allow captive institutions to meet or exceed longstanding genetic retention targets (90% of source population heterozygosity over 100 years). We provide a broad policy direction that could make biobanking technology a practical reality across Australia’s ex situ management of amphibians in current and future holdings. Incorporating biobanking technology widely across this network could deliver outcomes by maintaining high levels of source population genetic diversity and freeing economic resources to develop ex situ programs for a greater number of threatened amphibian species.

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.

Amphibians are the most threatened vertebrate class globally based on recent rates of decline and extinction. Sperm cryopreservation and other assisted reproductive technologies have the potential to help manage small and threatened populations and prevent extinctions. There are a growing number of reports of recovery of amphibian sperm after cryopreservation, but relatively few published reports of amphibian embryos generated from frozen sperm developing beyond metamorphosis to the adult stage and achieving sexual maturation. In this study on the Eastern dwarf tree frog (Litoria fallax), a temperate amphibian species from eastern Australia, a small number of viable metamorphs and one sexually mature male frog (itself producing sperm) were produced from cryopreserved sperm, demonstrating the capacity of embryos generated from cryopreserved sperm to complete the life cycle to sexual maturity. Low progression rates between developmental stages were not deemed to be due to effects of cryopreservation, since control embryos from unfrozen sperm had a similarly low progression rate through development.

The problem of global amphibian declines has prompted extensive research over the last three decades. Initially, the focus was on identifying and characterizing the extent of the problem, but more recently efforts have shifted to evidence‐based research designed to identify best solutions and to improve conservation outcomes. Despite extensive accumulation of knowledge on amphibian declines, there remain knowledge gaps and disconnects between science and action that hamper our ability to advance conservation efforts. Using input from participants at the ninth World Congress of Herpetology, a U.S. Geological Survey Powell Center symposium, amphibian on‐line forums for discussion, the International Union for Conservation of Nature Assisted Reproductive Technologies and Gamete Biobanking group, and respondents to a survey, we developed a list of 25 priority research questions for amphibian conservation at this stage of the Anthropocene. We identified amphibian conservation research priorities while accounting for expected tradeoffs in geographic scope, costs, and the taxonomic breadth of research needs. We aimed to solicit views from individuals rather than organizations while acknowledging inequities in participation. Emerging research priorities (i.e., those under‐represented in recently published amphibian conservation literature) were identified, and included the effects of climate change, community‐level (rather than single species‐level) drivers of declines, methodological improvements for research and monitoring, genomics, and effects of land‐use change. Improved inclusion of under‐represented members of the amphibian conservation community was also identified as a priority. These research needs represent critical knowledge gaps for amphibian conservation although filling these gaps may not be necessary for many conservation actions.

Multidisciplinary approaches to conserve threatened species are required to curb biodiversity loss. Globally, amphibians are facing the most severe declines of any vertebrate class. In response, conservation breeding programs have been established in a growing number of amphibian species as a safeguard against further extinction. One of the main challenges to the long-term success of conservation breeding programs is the maintenance of genetic diversity, which, if lost, poses threats to the viability and adaptive potential of at-risk populations. Integrating reproductive technologies into conservation breeding programs can greatly assist genetic management and facilitate genetic exchange between captive and wild populations, as well as reinvigorate genetic diversity from expired genotypes. The generation of offspring produced via assisted fertilisation using frozen–thawed sperm has been achieved in a small but growing number of amphibian species and is poised to be a valuable tool for the genetic management of many more threatened species globally. This review discusses the role of sperm storage in amphibian conservation, presents the state of current technologies for the short-term cold storage and cryopreservation of amphibian sperm, and discusses the generation of cryo-derived offspring.

: In brief: Sperm cryopreservation has been recognised as a tool for preventing loss of genetic diversity in amphibians; however, the combined effect of penetrative and non-penetrative cryoprotectants in cryodiluents is poorly understood. We demonstrate a clear benefit of using low concentrations of non-penetrative cryoprotectants when cryopreserving sperm of Australian tree frogs. Sperm cryopreservation protocols have been developed for an increasing number of amphibian species since the recognition of a global amphibian decline. Yet, the development of these protocols has neglected to elucidate the combined effect of the penetrative(PCP) and non-penetrative cryoprotectant (NPCP) on the recovery of live, motile sperm. The two-factor hypothesis of cryoinjury recognises a trade-off between cooling cells slowly enough to allow osmotic dehydration and therefore avoid intracellular ice formation, but fast enough to minimise effects from increasing extracellular osmolality as the frozen fraction of the media increases during freezing. We tested the effect of two concentrations of a PCP (10 and 15% v/v dimethyl sulfoxide (Me2SO)) and two concentrations of an NPCP (1 and 10% w/v sucrose) in various combinations on the sperm of six pelodryadid frogs. In all species, 15% v/v Me2SO with 1% w/v sucrose provided superior post-thaw recovery with high proportions of forward progressive motility, live cells and intact acrosomes (typically >50% for each). Theoretically, it has been suggested that increased NPCP concentration should improve cell survival by increasing the rate and extent of cell dehydration. We suggest, however, that the elevated osmolality in the unfrozen water fraction when 10% sucrose is used may be causing damage to cells via excessive cell shrinkage and solute effects as proposed in the two-factor hypothesis of cryoinjury. We showed this response in sperm across a range of frog species, providing compelling evidence for this hypothesis. We suggest protocol development using the PCP/NPCP ratios demonstrated in our study will be broadly applicable to many amphibian species.