Refrigerated storage and cryopreservation of hormonally induced sperm in the threatened frog, Litoria aurea.

As sperm cryopreservation and other assisted reproductive technologies (ARTs) advance in common amphibian species, focus on applying non-lethal sperm collection methods to the conservation and genetic management of threatened species is imperative. The goal of this study was to examine the application of logistically practical ART protocols in a threatened frog (Litoria aurea). First, we tested the efficacy of various concentrations of human chorionic gonadotropin (hCG) (20, 40 IU/g bodyweight) and Gonadotropin releasing hormone antagonist (0.25 µg/g and 0.5 µg/g body weight GnRH-a) on the induction of spermatozoa. Using the samples obtained from the previous trials, we tested the effect of cold storage and cryopreservation protocols on long-term refrigerated storage and post-thaw sperm recovery. Our major findings include: (1) high quality sperm were induced with 20 and 40 IU/g bodyweight of (hCG); (2) proportions of live, motile sperm post-thaw, were recovered at higher levels than previously reported for L. aurea (>50%) when preserved with 15% v/v DMSO and 1% w/v sucrose; and (3) spermic urine stored at 5 °C retained motility for up to 14 days. Our findings demonstrate that the protocols developed in this study allowed for successful induction and recovery of high-quality spermatozoa from a threatened Australian anuran, L. aurea, providing a prime example of how ARTs can contribute to the conservation of rare and threatened species.

Paradigm shift in frog sperm cryopreservation: reduced role for non-penetrating cryoprotectants.

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. Abstract: 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.

The effect of salt dosing for chytrid mitigation on tadpoles of a threatened frog, Litoria aurea.

The novel fungal pathogen Batrachochytrium dendrobatidis (chytrid) is one of the greatest threats to amphibians worldwide. Small increases in water salinity (up to ca. 4 ppt) have been shown to limit chytrid transmission between frogs, potentially providing a way to create environmental refugia to reduce its impact at a landscape scale. However, the effect of increasing water salinity on tadpoles, a life stage confined to water, is highly variable. Increased water salinity can lead to reduced size and altered growth patterns in some species, with flow-on effects to vital rates such as survival and reproduction. It is thus important to assess potential trade-offs caused by increasing salinity as a tool to mitigate chytrid in susceptible frogs. We conducted laboratory experiments to examine the effects of salinity on the survival and development of tadpoles of a threatened frog (Litoria aurea), previously demonstrated as a suitable candidate for trialling landscape manipulations to mitigate chytrid. We exposed tadpoles to salinity ranging from 1 to 6 ppt and measured survival, time to metamorphosis, body mass and locomotor performance of post-metamorphic frogs as a measure of fitness. Survival and time to metamorphosis did not differ between salinity treatments or controls reared in rainwater. Body mass was positively associated with increasing salinity in the first 14 days. Juvenile frogs from three salinity treatments also showed the same or better locomotor performance compared to rainwater controls, confirming that environmental salinity may influence life history traits in the larval stage, potentially as a hormetic response. Our research suggests that salt concentrations in the range previously shown to improve survival of frogs in the presence of chytrid are unlikely to impact larval development of our candidate threatened species. Our study lends support to the idea of manipulating salinity to create environmental refugia from chytrid for at least some salt-tolerant species.

Modeling genetic benefits and financial costs of integrating biobanking into the conservation breeding of managed marsupials.

Managed breeding programs are an important tool in marsupial conservation efforts but may be costly and have adverse genetic effects in unavoidably small captive colonies. Biobanking and assisted reproductive technologies (ARTs) could help overcome these challenges, but further demonstration of their potential is required to improve uptake. We used genetic and economic models to examine whether supplementing hypothetical captive populations of dibblers (Parantechinus apicalis) and numbats (Myrmecobius fasciatus) with biobanked founder sperm through ARTs could reduce inbreeding, lower required colony sizes, and reduce program costs. We also asked practitioners of the black-footed ferret (Mustela nigripes) captive recovery program to complete a questionnaire to examine the resources and model species research pathways required to develop an optimized biobanking protocol in the black-footed ferret. We used data from this questionnaire to devise similar costed research pathways for Australian marsupials. With biobanking and assisted reproduction, inbreeding was reduced on average by between 80% and 98%, colony sizes were on average 99% smaller, and program costs were 69- to 83-fold lower. Integrating biobanking made long-standing captive genetic retention targets possible in marsupials (90% source population heterozygosity for a minimum of 100 years) within realistic cost frameworks. Lessons from the use of biobanking technology that contributed to the recovery of the black-footed ferret include the importance of adequate research funding (US$4.2 million), extensive partnerships that provide access to facilities and equipment, colony animals, appropriate research model species, and professional and technical staff required to address knowledge gaps to deliver an optimized biobanking protocol. Applied research investment of A$133 million across marsupial research pathways could deliver biobanking protocols for 15 of Australia's most at-risk marsupial species and 7 model species. The technical expertise and ex situ facilities exist to emulate the success of the black-footed ferret recovery program in threatened marsupials using these research pathways. All that is needed now for significant and cost-effective conservation gains is greater investment by policy makers in marsupial ARTs.

Los programas de reproducción controlada son una herramienta importante para los esfuerzos de conservación de marsupiales, aunque pueden resultar costosos y tener efectos genéticos adversos en las colonias cautivas incapaces de aumentar en tamaño. Los biobancos y las tecnologías de reproducción asistida (TRA) podrían ayudar a superar estos problemas, pero es necesario seguir demostrando su potencial para mejorar su adopción. Utilizamos modelos genéticos y económicos para analizar si la introducción de esperma fundador proveniente de biobancos mediante tecnologías de reproducción asistida a poblaciones cautivas hipotéticas de los marsupiales Parantechinus apicalis y Myrmecobius fasciatus podría reducir la endogamia, disminuir el tamaño efectivo de las colonias y reducir el costo de los programas. También pedimos a los profesionales del programa de recuperación en cautiverio del hurón de patas negras (Mustella nigripes) que respondieran un cuestionario para analizar los recursos y los métodos de investigación de las especies modelo necesarias para desarrollar un protocolo de biobanco optimizado para el hurón de patas negras. Utilizamos los datos de este cuestionario para diseñar métodos de investigación con costos similares para los marsupiales australianos. Con el biobanco y la reproducción asistida, la endogamia se redujo en promedio entre un 80 y un 98%, el tamaño de las colonias fue en promedio un 99% más pequeño y los costos del programa entre 69 y 83 veces menores. La integración del biobanco posibilitó los objetivos de retención genética en cautiverio a largo plazo en marsupiales (90% de heterocigosidad de la población de origen durante un mínimo de 100 años) dentro de un marco realista de costos. Entre el aprendizaje extraído del uso de la tecnología de biobancos que contribuyó a la recuperación del hurón de patas negras figuran la importancia de una financiación adecuada de la investigación (4.2 millones de dólares), colaboraciones profundas que faciliten el acceso a instalaciones y equipos, colonias de animales, especies modelo adecuadas para la investigación y el personal profesional y técnico necesario para abordar las lagunas de conocimiento y ofrecer un protocolo optimizado para los biobancos. Una inversión en investigación aplicada de 133 millones de dólares australianos para la investigación de los marsupiales podría proporcionar protocolos de biobancos para 15 de las especies de marsupiales australianos en mayor riesgo y 7 especies modelo. Existen los conocimientos técnicos y las instalaciones ex situ para emular el éxito del programa de recuperación del hurón de patas negras en marsupiales amenazados utilizando estas vías de investigación. Ahora sólo se necesita una mayor inversión por parte de los responsables políticos de las TRA para marsupiales para obtener beneficios de conservación significativos y rentables.

Modelling Genetic Benefits and Financial Costs of Integrating Biobanking into the Captive Management of Koalas.

Zoo and wildlife hospital networks are set to become a vital component of Australia's contemporary efforts to conserve the iconic and imperiled koala (Phascolarctos cinereus). Managed breeding programs held across zoo-based networks typically face high economic costs and can be at risk of adverse genetic effects typical of unavoidably small captive colonies. Emerging evidence suggests that biobanking and associated assisted reproductive technologies could address these economic and genetic challenges. We present a modelled scenario, supported by detailed costings, where these technologies are optimized and could be integrated into conservation breeding programs of koalas across the established zoo and wildlife hospital network. Genetic and economic modelling comparing closed captive koala populations suggest that supplementing them with cryopreserved founder sperm using artificial insemination or intracytoplasmic sperm injection could substantially reduce inbreeding, lower the required colony sizes of conservation breeding programs, and greatly reduce program costs. Ambitious genetic retention targets (maintaining 90%, 95% and 99% of source population heterozygosity for 100 years) could be possible within realistic cost frameworks, with output koalas suited for wild release. Integrating biobanking into the zoo and wildlife hospital network presents a cost-effective and financially feasible model for the uptake of these tools due to the technical and research expertise, captive koala colonies, and ex situ facilities that already exist across these networks.

Common goals, different stages: the state of the ARTs for reptile and amphibian conservation.

Amphibians and reptiles are highly threatened vertebrate taxa with large numbers of species threatened with extinction. With so many species at risk, conservation requires the efficient and cost-effective application of all the tools available so that as many species as possible are assisted. Biobanking of genetic material in genetic resource banks (GRBs) in combination with assisted reproductive technologies (ARTs) to retrieve live animals from stored materials are two powerful, complementary tools in the conservation toolbox for arresting and reversing biodiversity decline for both amphibians and reptiles. However, the degree of development of the ARTs and cryopreservation technologies differ markedly between these two groups. These differences are explained in part by different perceptions of the taxa, but also to differing reproductive anatomy and biology between the amphibians and reptiles. Artificial fertilisation with cryopreserved sperm is becoming a more widely developed and utilised technology for amphibians. However, in contrast, artificial insemination with production of live progeny has been reported in few reptiles, and while sperm have been successfully cryopreserved, there are still no reports of the production of live offspring generated from cryopreserved sperm. In both amphibians and reptiles, a focus on sperm cryopreservation and artificial fertilisation or artificial insemination has been at the expense of the development and application of more advanced technologies such as cryopreservation of the female germline and embryonic genome, or the use of sophisticated stem cell/primordial germ cell cryopreservation and transplantation approaches. This review accompanies the publication of ten papers on amphibians and twelve papers on reptiles reporting advances in ARTs and biobanking for the herpetological taxa.

Factors influencing persistence of a threatened amphibian in restored wetlands despite severe population decline during climate change driven weather extremes.

Biodiversity is in global decline during the Anthropocene. Declines have been caused by multiple factors, such as habitat removal, invasive species, and disease, which are often targets for conservation management. However, conservation interventions are under threat from climate change induced weather extremes. Weather extremes are becoming more frequent and devastating and an example of this was the 2019/2020 Australian drought and mega-fires. We provide a case study the impacts of these extreme weather events had on a population of the threatened frog Litoria aurea that occurs in a constructed habitat which was designed to reduce the impact of introduced fish and chytrid-induced disease. We aimed to determine what factors influenced persistence so that the design of wetlands can be further optimised to future-proof threatened amphibians. We achieved this with 4 years (2016-2020) of intensive capture-recapture surveys during austral spring and summer across nine wetlands (n = 94 repeat surveys). As hypothesized, drought caused a sharp reduction in population size, but persistence was achieved. The most parsimonious predictor of survival was an interaction between maximum air temperature and rainfall, indicating that weather extremes likely caused the decline. Survival was positively correlated with wetland vegetation coverage, positing this is an important feature to target to enhance resilience in wetland restoration programs. Additionally, the benefits obtained from measures to reduce chytrid prevalence were not compromised during drought, as there was a positive correlation between salinity and survival. We emphasize that many species may not be able to persist under worse extreme weather scenarios. Despite the potential for habitat augmentation to buffer effects of extreme weather, global action on climate change is needed to reduce extinction risk. Supplementary Information: The online version contains supplementary material available at 10.1007/s10531-022-02387-9.

High clutch failure rate due to unpredictable rainfall for an ephemeral pool-breeding frog.

Animals that reproduce in temporary aquatic systems expose their offspring to a heightened risk of desiccation, as they must race to complete development and escape before water levels recede. Adults must therefore synchronise reproduction with the changing availability of water, yet the conditions they experience to trigger such an event may not relate to those offspring face throughout development, potentially leading to clutch failure. The sandpaper frog (Lechriodus fletcheri) breeds in ephemeral pools that dry within days to weeks after rainfall has ceased. We examined whether spawning frequency and offspring survival differed across two consecutive breeding seasons based on (1) rainfall at the moment of oviposition and throughout offspring development, and (2) pool volume, given their combined effect on hydroperiod. Reproduction was triggered by rainfall, with more spawn laid during periods of greater rainfall and in larger pools. While pool size was a predictor of offspring survival, rainfall during oviposition was not. Rather, follow-up rain events were required to prevent pools drying prior to metamorphosis, with rainfall evenness during development the strongest predictor of reproductive success. High clutch failure rates recorded in both seasons suggest that adults do not have the capability to predict rainfall frequency post-oviposition. We thus conclude that unpredictable rainfall leading to premature desiccation of spawning sites is the primary source of pre-metamorphic mortality for this species. Understanding the influence of rainfall predictability on offspring survival could be critical in predicting the effects of altered hydroperiod regimes due to climate change for species that exploit temporary waters.

Seasonal variation in the prevalence of a fungal pathogen and unexpected clearance from infection in a susceptible frog species.

The amphibian chytrid fungus Batrachochytrium dendrobatidis (Bd) causes the disease chytridiomycosis, which is a primary driver for amphibian population declines and extinctions worldwide. For highly susceptible species, such as the green and golden bell frog Litoria aurea, large numbers of Bd-related mortalities are thought to occur during the colder season (winter), when low temperatures favour the growth of the pathogen. However, extant L. aurea populations are persisting with Bd. We measured Bd prevalence and infection levels of wild L. aurea using capture-mark-recapture and radio-tracking methods. Using this information, we sought to determine host and environmental correlates of Bd prevalence and infection load. Mean ± SE infection load was higher in frogs sampled in autumn (431.5 ± 310.4 genomic equivalents; GE) and winter (1147.5 ± 735.8 GE), compared to spring (21.8 ± 19.3 GE) and summer (0.9 ± 0.8 GE). Furthermore, prevalence of Bd infection in L. aurea was highest in winter (43.6%; 95% CI 33.1-54.7%) and lowest in summer (11.2%; 95% CI 6.8-17.9%). Both prevalence and infection load decreased with increasing temperature. Seven frogs cleared their fungal infection during the coolest months when Bd prevalence was highest; however, these clearances were not permanent, as 5 frogs became infected again. Understanding the factors that allow amphibians to clear their Bd infections when temperatures are optimal for Bd growth presents the potential for manipulating such factors and provides an important step in future research.

Resetting the paradigm of reproductive science and conservation.

In the application of reproductive science to conservation breeding, it has long been assumed that artificial insemination using frozen thawed sperm would be the default technology. This has always been problematic considering the wide range of tolerance to freeze thawing among vertebrate sperm. Furthermore, those providing leadership for genome banking should be proactive to preserve maximum genetic diversity, however, for many species there is little or no sperm motility after thawing of cryopreserved sperm. In this review article, there is the contention that a much wider range of tissues should be banked, and the range of evolving advanced reproductive and developmental technologies should be considered for conservation breeding programs, to realize the maximum opportunities of genome banking to contribute to conservation of animal species.

Genetic evidence for polyandry in the threatened green and golden bell frog.

Identifying which species exhibit polyandry may lead to further insights into evolutionary biology and social behaviour. However, confirming polyandry can be difficult. High-resolution genetics provides a useful means to gain evidence. Although the threatened Pelodryadid frog, the green and golden bell frog Litoria aurea, has been subject to numerous ecological studies, there is uncertainty surrounding its reproductive ecology. Polyandry has not been formally identified in L. aurea or any species within the Pelodryadidae family. We aimed to identify if there was genetic evidence of polyandry in a population occurring in a wetland complex on Kooragang Island, New South Wales. To accomplish this, we collected genetic samples of tadpoles within the same size cohort about 20-30 days after explosive breeding events. Genotypes of 14 females, nine males and 70 tadpoles were analysed with COLONY (1988 single nucleotide polymorphisms after filtering) to identify parentage, full-siblings and half-siblings. We found support for the hypothesis that L. aurea is polyandrous. Based on previous observations of multi-male matings and the narrow time periods that breeding occurred in, it is likely this species exhibits simultaneous polyandry. We discuss these results in regards to behavioural adaptive processes and avenues for further research.

Optimal cooling rates for sperm cryopreservation in a threatened lizard conform to two-factor hypothesis of cryo-injury.

Assisted reproductive technologies provide important tools for wildlife conservation but have rarely been developed for reptiles. A critical step in developing cryopreservation protocols is establishing optimal cooling rates for cell survival. The two-factor hypothesis explaining cryoinjury to cells originates from an inverted 'U' shape of recovery curves generated in many cell types thawed after cryopreservation, due to cell recovery declining at cooling rates either side of a single optimum. We generated such a curve for the yellow-spotted monitor lizard Varanus panoptes, the first for any reptile. We cryopreserved sperm using two cooling devices (LN2 dry shipper; LN2 bath vapour) and two sperm-holding vessels (Cassou sperm straws; Nunc CryoTubes) to generate four different cooling-rate curves during freezing. Sperm motility and viability (47.3% and 76.5% respectively) were highest when frozen in straws suspended in a LN2 bath at an intermediate cooling rate of 73 °C/min between 0 and -50 °C, whereas sperm frozen in straws suspended in a dry shipper at the fastest cooling rate (231 °C/min between 0 and -50 °C) produced the lowest recovery (10.4% and 36.4% motility and viability, respectively). Sperm frozen in cryotubes at the lowest cooling rates in either LN2 bath vapour or dry shipper produced intermediate recovery. The shape of the optimal cooling curve conformed to the two-factor hypothesis of cryoinjury, the first such evidence in reptile sperm. This in turn led to the identification of simple cryopreservation setups (LN2 vapour with straws and cryotubes; dry shipper with cryotubes but not straws) suitable for cryopreserving lizard sperm in the field.

Large area used by squirrel gliders in an urban area, uncovered using GPS telemetry.

The squirrel glider (Petaurus norfolcensis) is a threatened, gliding marsupial that persists in fragmented landscapes despite its restricted capacity to cross large gaps. As measures to maintain and/or restore suitable habitat depend on knowledge about the species' ecological requirements, we investigated the area used by squirrel gliders in an urban area near Newcastle, Australia. Using GPS telemetry data and the autocorrelated kernel density estimator, we estimated area used to average 10.8 ha and varied from 4.6 to 15 ha, which is equal to or greater than found in previous studies that spanned longer time periods. This has implications when identifying the minimum patch size necessary for ensuring the long-term conservation of a squirrel glider population.

Rapid population increase of the threatened Australian amphibian Litoria aurea in response to wetlands constructed as a refuge from chytrid-induced disease and introduced fish.

Amphibians have declined due to multiple impacts including invasive fish and the disease chytridiomycosis caused by the chytrid fungus Batrachochytrium dendrobatidis (Bd). Wetland restoration can be used to increase amphibian populations. However the design of created wetlands must account for threats such as Bd and introduced fish. There have been no attempts on a landscape level to manage these threats with habitat design. Here we monitored the green and golden bell frog (Litoria aurea) in 2.6 ha of constructed wetlands designed to enhance breeding and increase survival through passive mitigation of Bd and exotic fish. We compared the fecundity, adult population sizes, introduced fish occupancy, Bd prevalence and survival rates of frogs in created wetlands (CW) to three control sites to determine if and why the habitat design was successful. Monitoring involved weekly capture-recapture during the austral spring and summer for three L. aurea breeding seasons. We hypothesised that (1) if the CWs were successful in passively limiting fish colonisation, a larger number of breeding events would be detected compared to control sites which are known to be widely colonised by introduced fish. (2) If the wetlands were successful in passively mitigating Bd, then we would observe an equal or greater survival rate and equal to or lower Bd prevalence compared to control wetlands. We observed a 3.3-fold increase in adult population size in CW from season 1 to 2, and the population increased further in season 3.We found strong support for hypothesis (1) and weak support for (2). Based on these results, we conclude that this design was beneficial shortly after their formation primarily due to fish exclusion, but further study is required to determine if these benefits extend long-term. Future amphibian restoration studies are needed to improve the design of wetlands to enhance suppression of Bd.

Using citizen science in the photo-identification of adult individuals of an amphibian based on two facial skin features.

Among amphibians, adults have traditionally been identified in capture-mark-recapture studies using invasive marking techniques with associated ethical, cost and logistical considerations. However, species in this group may be strong candidates for photo-identification based on natural skin features that removes many of these concerns, with this technique opening up opportunities for citizen scientists to be involved in animal monitoring programs. We investigated the feasibility of using citizen science to distinguish between individuals of an Australian anuran (the sandpaper frog, Lechriodus fletcheri) based on a visual analysis of their natural skin features. We collected photographs of marked individuals in the field over three breeding seasons using a smartphone device. This photo-database was used to create an online survey to determine how easily members of the general public could photo-match individuals by a comparison of two facial skin features; black banding that runs horizontally above the tympanum and a background array of tubercles present in this region. Survey participants were provided with 30 closed, multiple choice questions in which they were asked to match separate images of a query frog from small image pools of potential candidate matches. Participants were consistently able to match individuals with a low matching error rate (mean ± SD of 26 ± 5) despite the relatively low quality of photographs taken from a smartphone device in the field, with most query frogs being matched by a majority of participants (mean ± SD of 86.02 ± 9.52%). These features were found to be unique and stable among adult males and females. Thus, photo-identification is likely to be a valid, non-invasive method for capture-mark-recapture for L. fletcheri, and likely many anurans that display similar facial skin features. This may become an important alternative to artificial marking techniques, with the challenges of manual photo-matching reduced by spreading workloads among members of the public that can be recruited online.

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

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.

Efficacy of short-term cold storage prior to cryopreservation of spermatozoa in a threatened lizard.

Assisted reproductive technologies (ARTs) have a significant role to play in reptile conservation, yet are severely lacking. Previous attempts to cryopreserve spermatozoa in the threatened lizard Varanus panoptes achieved approximately 48% motile sperm post-thaw for samples frozen immediately after collection. However, the feasibility of extended cold storage before cryopreservation has not been tested. We held V. panoptes spermatozoa at either 25°C or 4°C for 8 days, assessing sperm motility at days 1, 2, 4 and 8. Subsamples were cryopreserved on days 1 and 4 following the previously reported protocol for this species. Percentage motility decreased rapidly at 25°C, but did not decrease significantly until 4 days after collection at 4°C, with >30% motility maintained after 8 days. There was no significant difference in post-thaw motility or viability of samples cryopreserved after 1 or 4 days storage at 4°C, yielding substantial results for both parameters (mean motility 23.8% and 28.1% and mean viability 50.1% and 57.5% after 1 and 4 days respectively). We demonstrate the capacity to extend sperm viability for up to 8 days in unfrozen samples and to produce acceptable post-thaw motility in samples frozen after 4 days of storage, contributing to the development of valuable ARTs for lizards and other reptiles.

Integrating biobanking could produce significant cost benefits and minimise inbreeding for Australian amphibian captive breeding programs.

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.

Corrigendum to: Efficacy of short-term cold storage prior to cryopreservation of spermatozoa in a threatened lizard.

Assisted reproductive technologies (ARTs) have a significant role to play in reptile conservation, yet are severely lacking. Previous attempts to cryopreserve spermatozoa in the threatened lizard Varanus panoptes achieved approximately 48% motile sperm post-thaw for samples frozen immediately after collection. However, the feasibility of extended cold storage before cryopreservation has not been tested. We held V. panoptes spermatozoa at either 25°C or 4°C for 8 days, assessing sperm motility at days 1, 2, 4 and 8. Subsamples were cryopreserved on days 1 and 4 following the previously reported protocol for this species. Percentage motility decreased rapidly at 25°C, but did not decrease significantly until 4 days after collection at 4°C, with >30% motility maintained after 8 days. There was no significant difference in post-thaw motility or viability of samples cryopreserved after 1 or 4 days storage at 4°C, yielding substantial results for both parameters (mean motility 23.8% and 28.1% and mean viability 50.1% and 57.5% after 1 and 4 days respectively). We demonstrate the capacity to extend sperm viability for up to 8 days in unfrozen samples and to produce acceptable post-thaw motility in samples frozen after 4 days of storage, contributing to the development of valuable ARTs for lizards and other reptiles.