Rose Upton

Amphibians are among the most threatened taxonomic groups, with many species experiencing rapid declines that have resulted in small, isolated populations vulnerable to inbreeding depression and reduced fertility. One major outcome of inbreeding is lowered fecundity, often linked to morphological changes in gametes. Littlejohn's tree frog ( Rawlinsonia littlejohni ; previously Litoria ) is a threatened species requiring targeted conservation management. To increase population numbers and restore genetic diversity, a captive breeding program has been established. This study aimed to optimize the timing of hormonally‐induced sperm collection to improve outcomes for captive breeding, cryopreservation, and in vitro fertilization. Three experiments were conducted with ten captive males. Experiment one examined the effects of different human Chorionic Gonadotropin (hCG) and Gonadotropin‐releasing hormone antagonist (GnRH‐a) concentrations on spermiation response over 6 h. Both 5 and 10 IU hCG/g bwt yielded high sperm concentrations with similar motility and membrane viability, whereas GnRH‐a (1 and 2 µg/g bwt) produced no response. Experiment two assessed seasonal effects on sperm traits over a year, showing high variability between individuals but no consistent seasonal pattern. Sperm motility and membrane viability remained high, with slight declines in January, April, and June. Experiment three compared captive and wild males during the breeding season. Sperm concentration was higher in June for wild frogs but variable across all months in both groups. Our findings demonstrate that sperm production and quality in R. littlejohni are shaped more by its breeding system than by seasonal cycles, informing strategies to enhance reproductive success and genetic recovery in captivity.

Amphibians are among the most threatened vertebrates, yet assisted reproductive technologies (ARTs) remain underutilised in their conservation. We developed and evaluated a biologically relevant, non-lethal sperm collection protocol for Rana muscosa, a critically endangered frog in a long-term conservation breeding program. Thirteen hormone treatments were tested across six post-injection time points, and sperm quality was assessed via concentration, motility, osmolality, and pH. Generalised linear mixed models revealed that gonadotropin-releasing hormone (GnRHa) alone significantly outperformed hCG-based regimens. The 3 µg/g GnRHa dose yielded the highest sperm concentration and sustained motility from 3 to 24 hours post-injection. Motility was highest under moderately acidic (pH 6.5–7.0) and hypoosmotic (75–100 mOsm/kg) conditions. To support decision-making, we applied a refined Wildlife Sperm Index (WSI) that weights motility and concentration as primary functional-yield traits, with motility given greater relative weight given its direct role in fertilisation, and treats pH and osmolality as secondary environmental-compatibility traits. Under this revised composite score, 3 µg/g GnRHa achieved the highest overall WSI score, outperforming 2 µg/g and 4 µg/g GnRHa, which ranked second and third, respectively, despite their more favourable pH and osmolality profiles. These findings provide the first evidence-based ART protocol for R. muscosa and offer a transferable framework for optimising gamete collection, IVF, and cryopreservation in line with other amphibian species, advancing both genetic management and species recovery goals.

Environmental change is reshaping wildlife reproduction through increasing temperatures and often leading to the spread of emerging infectious diseases, yet the physiological consequences of managing these stressors remain poorly understood. Amphibians are particularly vulnerable due to their ectothermy and high susceptibility to chytridiomycosis, caused by Batrachochytrium dendrobatidis (Bd). Here, we examine how Bd infection and thermal treatment interact to influence sperm quality and reproductive investment in male green and golden bell frogs (Ranoidea aurea), a species that has suffered severe population declines. Moderate Bd infection was associated with elevated sperm concentration relative to uninfected and heavily infected males, consistent with increased short-term reproductive investment under elevated mortality risk. However, severe infection led to pronounced reductions in sperm concentration and motility. Thermal treatment successfully eliminated Bd infection but imposed reproductive costs: sperm concentration declined following treatment and remained significantly reduced six months later, despite partial recovery of sperm motility and membrane integrity. Our findings reveal that disease and thermal stress jointly shape amphibian reproductive outcomes through context-dependent trade-offs between immune defence and gamete production. While mild infection may trigger short-lived increases in reproductive output, both severe infection and pathogen clearance via thermal exposure impose lasting constraints on fertility. These results highlight an underappreciated cost of disease mitigation and suggest that increasing thermal extremes associated with climate change may further limit amphibian reproductive resilience, with important implications for conservation management and population persistence.

The application of population genetic metrics to evaluate the health of a population after undergoing anthropogenic management and experiencing environmental stochasticity remains underexplored in amphibians. Several metrics can be used to measure loss of genetic diversity and decreased gene flow to determine population health, including heterozygosity, inbreeding, and structure. This study assessed the genetic health of the endangered frog, Rawlinsonia littlejohni using genetic metrics in a population exposed to human modifications (dam creation) and extreme climatic events (droughts followed by heavy rainfall). We detected strong population structuring, with two distinct sub‐populations located only 2.5 km apart but exhibiting limited gene flow. Genetic differentiation between these sub‐populations, determined by PCoA analysis, decreased after high rainfall in 2021, but returned during drought conditions in 2023, indicating that environmental conditions influence dispersal dynamics. The northern sub‐population, which includes both constructed ponds and natural creek pools, exhibited significantly higher heterozygosity and lower mean kinship. In contrast, the southern sub‐population, dominated by constructed ponds and lacking natural creek connectivity, showed reduced heterozygosity and a marked increase in inbreeding (0.003 in 2020 to 0.162 in 2023). These finding suggest that limited connectivity is constraining gene flow and contributing to reduced genetic health in the southern sub‐population. We propose that additional ponds spaced between sub‐populations may act as stepping‐stones to enhance connectivity and reduce inbreeding. However, our results reveal a key conservation trade‐off: constructed ponds support breeding and population persistence, while natural stream systems facilitate dispersal and genetic connectivity. These findings highlight that restoring long‐term population viability will require multiple, complementary management actions that balance breeding habitat provision with landscape connectivity.

Environmental change is reshaping wildlife reproduction through increasing temperatures and the spread of emerging infectious diseases, yet the physiological consequences of managing these stressors remain poorly understood. Amphibians are particularly vulnerable due to their ectothermy and high susceptibility to chytridiomycosis, caused by Batrachochytrium dendrobatidis ( Bd ). Here, we examine how Bd infection and thermal treatment interact to influence sperm quality and reproductive investment in male green and golden bell frogs ( Ranoidea aurea ), a species that has suffered severe population declines. Moderate Bd infection was associated with elevated sperm concentration relative to uninfected and heavily infected males, consistent with increased short-term reproductive investment under elevated mortality risk. However, severe infection led to pronounced reductions in sperm concentration and motility. Thermal treatment successfully eliminated Bd infection but imposed substantial reproductive costs: sperm concentration declined following treatment and remained significantly reduced six months later, despite partial recovery of sperm motility and membrane integrity. These results indicate persistent impairment of spermatogenic capacity rather than transient suppression. Our findings reveal that disease and thermal stress jointly shape amphibian reproductive outcomes through context-dependent trade-offs between immune defence and gamete production. While mild infection may trigger short-lived increases in reproductive output, both severe infection and pathogen clearance via thermal exposure impose lasting constraints on fertility. These results highlight an underappreciated cost of disease mitigation and suggest that increasing thermal extremes associated with climate change may further limit amphibian reproductive resilience, with important implications for conservation management and population persistence.

As amphibian populations continue to decline, there is a need to establish procedures to store and recover genetic diversity. Nuclear transplantation (NT) is a potentially useful reproductive technology with applications to conservation in specific circumstances. Although sperm have been successfully cryopreserved and used in in vitro fertilization to produce sexually mature offspring, there are many endangered and even extinct amphibian species that only exist as stored frozen adult or larval tissues. The only way to recover that genetic diversity is through NT. Nuclear transplantation can introduce new genes into captive breeding programs from individuals where no viable gametes from that individual have been cryopreserved. Amphibian NT research was common during the middle to late 20 ^th century; however, it was never conducted for amphibian conservation. Early amphibian NT experiments were conducted for the purpose of determining whether differentiation was a terminal process that involved the loss of reprogramming genes. However, following the success of mammalian NT, amphibian NT research declined, and the field has been at a standstill for decades. This study developed the first nuclear transplantation protocol for a native Australia frog using Limnodynastes peronii , as a model. Specifically, this paper aimed to test: 1) nuclear transplantation with fresh embryonic cells in Limnodynastes peronii and trial the implementation of different egg enucleation methods (pricked eggs only, UV only, and pricked + UV), 2) use of cryopreserved cells in nuclear transplantation and 3) use single nucleotide polymorphisms to determine if the nuclear genomes of nuclear transplant embryos were derived from the injected donor cell nuclei. With both fresh and cryopreserved embryonic cells as donor cell nuclei, development of the nuclear transplant embryos was low compared to the fertilized controls. Only one nuclear transplant embryo derived from a fresh embryonic cell (injected into a UV only enucleated egg) developed into a tadpole. This attained stage 36 of the Gosner Staging System for Anurans (hind limbs) before dying. As an egg activation technique, pricking the eggs resulted in higher cleavage rates and development to blastula than UV only and pricked + UV nuclear transplants (in trials with cryopreserved cells), but none of the nuclear transplants from the pricked only group developed beyond blastula. None of the nuclear transplants derived from cryopreserved embryonic cells developed to tadpoles. The most advanced stage attained from cryopreserved embryonic cells was stage 13 (neurula) from UV only eggs. Single nucleotide polymorphism (SNP) analysis testing for genotype mismatches was used to determine if the genomes of developing nuclear transplant embryos were derived from the injected donor cell nucleus. SNP analysis confirmed that two true clones were generated in this study. This is the first confirmed nuclear transplantation in any Australian native frog.

Cryopreservation of intact fish and amphibian eggs and embryos is challenging due to the sheer size and yolk content, which prevents proper dehydration and causes lethal intracellular ice formation during cooling. Alternatively, cryopreservation of dissociated embryonic cells allows to biobank diploid genomes. However, amphibian and fish embryos have cells of varying sizes throughout the developing embryo and cell size distribution remains understudied in cryopreservation. This study examined cell size effects during cryopreservation of dissociated embryonic cells from two amphibian species. Most work used Limnodynatses peronii blastula, gastrula, and neurula cells cryopreserved with 10% dimethyl sulfoxide (DMSO) and sucrose at 0%, 1%, or 10%. Increasing sucrose concentration improved post-thaw recovery of membrane intact cells and cell concentrations, with gastrula and neurula cells having better recovery than blastula cells. An interaction amongst cryoprotectant concentration, cell size, and embryonic stage was observed. Higher sucrose improved recovery of larger cells, but reduced recovery of smaller cells. These results supported a “Goldilocks” model of cryoinjury, in which larger cells require more time to dehydrate adequately, while smaller cells are damaged by excessive dehydration and solute effects, implying that optimal cryoprotectant conditions differ by cell size. Based on post-thaw recovery, 10% DMSO + 10% sucrose was optimal and successfully applied to the cryopreservation of neurula cells from the threatened Rawlinsonia littlejohni , marking the first report of embryonic cell cryopreservation in a threatened amphibian. Our results demonstrated the importance of cell size effects on cryoinjury and their consideration in the application of cryopreservation for amphibian conservation. In brief: Since intact amphibian eggs and embryos cannot be cryopreserved, dissociated embryonic cell cryopreservation provides an alternative avenue for biobanking embryonic diploid genomes; however, cell size effects on cryoinjury is poorly understood and the developing amphibian embryo consists of different sized cells. We demonstrated that cell size influences cryopreservation outcomes by cryopreserving embryonic cells from three developmental stages under varying cryoprotectant concentrations.

The application of population genetic metrics to evaluate the health of a population after undergoing anthropogenic management and experiencing environmental stochasticity remains underexplored in amphibians. Several metrics can be used to measure loss of genetic diversity and decreased gene flow to determine population health, including heterozygosity, inbreeding, and structure. This study assessed the genetic health of the endangered frog, Litoria littlejohni using genetic metrics in a population exposed to human modifications (dam creation) and extreme climatic events (droughts followed by heavy rainfall). We found strong population structuring, with two distinct subpopulations, one in the northern and another in the southern area of occupancy. Despite being only 2.5 kilometres apart, there was limited gene flow between the sub-populations. Genetic differentiation between these sub-populations, determined by PCA analysis, decreased after high rainfall in 2021. However, strong genetic structuring returned in 2023 during drought conditions. In the northern sub-population, where breeding habitats included both constructed ponds and natural creek pools, heterozygosity was significantly higher and mean kinship significantly lower compared to the southern sub-population where there are fewer natural creek lines nearby. Furthermore, in the southern sub-population, inbreeding metrics increased significantly from 0.003 in 2020 to 0.162 in 2023. As the southern sub-population predominantly relies on constructed ponds for breeding, the absence of natural creek pools may be limiting dispersal and affecting the genetic health of this sub-population. To improve heterozygosity and connectivity, additional ponds could be constructed between the two sub-populations, acting as stepping-stones to enhance gene flow and reduce inbreeding.

Amphibians are among the most threatened vertebrates yet assisted reproductive technologies (ARTs) remain underutilized in their conservation. We developed and evaluated a biologically optimized, non-lethal sperm collection protocol for Rana muscosa , a critically endangered frog in a long-term conservation breeding program. Thirteen hormone treatments were tested across six post-injection time points, and sperm quality was assessed via concentration, motility, osmolality, and pH. Generalized linear mixed models revealed that gonadotropin-releasing hormone (GnRHa) alone significantly outperformed hCG-based regimens. The 3 µg/g GnRHa dose yielded the highest sperm concentration and sustained motility from 3 to 24 hours post-injection. Motility was highest under moderately acidic (pH 6.5–7.0) and hypoosmotic (75–100 mOsm/kg) conditions. To support decision-making, we used a Wildlife Sperm Index (WSI) developed by our team that integrates sperm traits and environmental parameters into a single composite score. While 3 µg/g GnRHa ranked highest by concentration alone, 4 µg/g GnRHa achieved the top WSI score due to greater environmental (pH and osmolality) compatibility. These findings provide the first evidence-based ART protocol for R. muscosa and offer a transferable framework for optimizing gamete collection, IVF, and cryopreservation in line with other amphibian species, advancing both genetic management and species recovery goals.