Dietary carotenoid supplementation has long-term and community-wide effects on the amphibian skin microbiome.

The amphibian skin microbiome plays a crucial role in host immunity and pathogen defence, yet we know little about the environmental drivers of skin microbial variation across host individuals. Inter-individual variation in the availability of micro-nutrients such as dietary carotenoids, which are involved in amphibian immunity, may be one factor that influences skin microbial assembly across different life history stages. We compared the effect of four carotenoid supplementation regimes during different life stages on the adult skin microbiome using a captive population of the critically endangered southern corroboree frog, Pseudophryne corroboree. We applied 16S rRNA sequencing paired with joint-species distribution models to examine the effect of supplementation on taxon abundances. We found that carotenoid supplementation had subtle yet taxonomically widespread effects on the skin microbiome, even 4.5 years post supplementation. Supplementation during any life-history stage tended to have a positive effect on the number of bacterial taxa detected, although explanatory power was low. Some genera were sensitive to supplementation pre-metamorphosis, but most demonstrated either additive or dominant effects, whereby supplementation during one life history stage had intermediate or similar effects, respectively, to supplementation across life. Carotenoid supplementation increased abundances of taxa belonging to lactic acid bacteria, including Lactococcus and Enterococcus, a group of bacteria that have previously been linked to protection against the amphibian fungal pathogen Batrachochytrium dendrobatidis (Bd). While the fitness benefits of these microbial shifts require further study, these results suggest a fundamental relationship between nutrition and the amphibian skin microbiome which may be critical to amphibian health and the development of novel conservation strategies.

Cryopreservation Cooling Rate Impacts Post-Thaw Sperm Motility and Survival in Litoria booroolongensis.

The cryopreservation and storage of gametes (biobanking) can provide a long-term, low-cost option for the preservation of population genetic diversity and is particularly impactful when applied to manage selective breeding within conservation breeding programs (CBPs). This study aimed to develop a sperm cryopreservation protocol for the critically endangered Booroolong frog (Litoria booroolongensis) to capture founder genetics within the recently established (est. 2019) CBP for this species. Hormone-induced sperm release was achieved using established protocols, and spermic urine samples were collected over a 6-h period. Pooled spermic urine samples (n = 3 males) were divided equally between two cryoprotectant (CPA) treatments and diluted by 1:5 (sperm:CPA) with either 15% (v/v) dimethyl sulfoxide + 1% (w/v) sucrose in simplified amphibian Ringer's (SAR; CPAA) or 10% (v/v) dimethylformamide + 10% (w/v) trehalose dihydrate in SAR (CPAB). The samples were cryopreserved in 0.25 mL straws using either a programmable freezer (FrA) or an adapted dry shipper method (FrB). The thawed samples were activated via dilution in water and assessed for viability and motility using both manual assessment and computer-assisted sperm analysis (CASA; 0 h, 0.5 h post-thaw). Upon activation, the survival and recovery of motility (total motility, forward progression and velocity) of cryopreserved sperm suspensions were higher for sperm preserved using FrB than FrA, regardless of CPA composition. This work supports our long-term goal to pioneer the integration of biobanked cryopreserved sperm with population genetic management to maximize restoration program outcomes for Australian amphibian species.

The Increasing Role of Short-Term Sperm Storage and Cryopreservation in Conserving Threatened Amphibian Species.

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.

Application of Reproductive Technologies to the Critically Endangered Baw Baw Frog, Philoria frosti.

Reproductive technologies (RTs) can assist integrated conservation breeding programs to attain propagation targets and manage genetic diversity more effectively. While the application of RTs to enhance the conservation management of threatened amphibians has lagged behind that of other taxonomic groups, a recent surge in research is narrowing the divide. The present study reports on the first application of RTs (hormone-induced spawning, hormone-induced sperm-release, and sperm cryopreservation) to the critically endangered Baw Baw frog, Philoria frosti. To determine the effect of hormone therapy on spawning success, male-female pairs were administered either 0 µg/g gonadotropin-releasing hormone agonist (GnRHa), 0.5 µg/g GnRHa, or 0.5 µg/g GnRHa + 10 µg/g metoclopramide (MET) (n = 6-7 pairs/treatment), and the number of pairs ovipositing, total eggs, and percent fertilisation success were quantified. To determine the effect of hormone therapy on sperm-release and to establish the peak time to collect sperm post-hormone administration, males were administered 0 IU/g (n = 4), or 20 IU/g hCG (n = 16). Total sperm, sperm concentration, and percent viability were quantified at 0, 2, 4, 6, 8, 10, and 12 h post-hormone administration. Overall, the percentage of pairs ovipositing was highest in the GnRHa + MET treatment, with 71% of pairs ovipositing, compared to 57% and 33% of pairs in the GnRHa and control treatments, respectively. The quantity of sperm released from males in response to hCG peaked at 4 h post-hormone administration, though it remained high up to 12 h. The percent sperm viability also peaked at 4 h post-administration (94.5%), exhibiting a steady decline thereafter, though viability remained above 77% throughout the 12 h collection period. The remaining sperm samples (n = 22) were cryopreserved using established protocols and biobanked for long-term storage and future conservation applications. The mean post-thaw sperm viability was 59%, and the percent total motility was 17%. The results from this preliminary study will direct further applications of RTs to the critically endangered Baw Baw frog to assist with species recovery.

A test for plasticity in sperm motility activation in response to osmotic environment in an anuran amphibian.

Evolutionary theory predicts that selection will favor phenotypic plasticity in sperm traits that maximize fertilization success in dynamic fertilization environments. In species with external fertilization, osmolality of the fertilization medium is known to play a critical role in activating sperm motility, but evidence for osmotic-induced sperm plasticity is limited to euryhaline fish and marine invertebrates. Whether this capacity extends to freshwater taxa remains unknown. Here, we provide the first test for plasticity in sperm-motility activation in response to osmotic environment in an anuran amphibian. Male common eastern froglets (Crinia signifera) were acclimated to either low (0 mOsmol kg-1) or high (50 mOsmol kg-1) environmental osmolality, and using a split-sample experimental design, sperm were activated across a range of osmolality treatments (0, 25, 50, 75, 100, and 200 ± 2 mOsmol kg-1). Unexpectedly, there was no detectable shift in the optimal osmolality for sperm-motility activation after approximately 13 weeks of acclimation (a period reflecting the duration of the winter breeding season). However, in both the low and high acclimation treatments, the optimal osmolality for sperm-motility activation mirrored the osmolality at the natural breeding site, indicating a phenotypic match to the local environment. Previously it has been shown that C. signifera display among-population covariation between environmental osmolality and sperm performance. Coupled with this finding, the results of the present study suggest that inter-population differences reflect genetic divergence and local adaptation. We discuss the need for experimental tests of osmotic-induced sperm plasticity in more freshwater taxa to better understand the environmental and evolutionary contexts favoring adaptive plasticity in sperm-motility activation.

Does dietary ß-carotene influence ontogenetic colour change in the southern corroboree frog?

Ontogenetic colour change occurs in a diversity of vertebrate taxa and may be closely linked to dietary changes throughout development. In various species, red, orange and yellow colouration can be enhanced by the consumption of carotenoids. However, a paucity of long-term dietary manipulation studies means that little is known of the role of individual carotenoid compounds in ontogenetic colour change. We know even less about the influence of individual compounds at different doses (dose effects). The present study aimed to use a large dietary manipulation experiment to investigate the effect of dietary ß-carotene supplementation on colouration in southern corroboree frogs (Pseudophryne corroboree) during early post-metamorphic development. Frogs were reared on four dietary treatments with four ß-carotene concentrations (0, 1, 2 and 3 mg g-1), with frog colour measured every 8 weeks for 32 weeks. ß-Carotene was not found to influence colouration at any dose. However, colouration was found to become more conspicuous over time, including in the control treatment. Moreover, all frogs expressed colour maximally at a similar point in development. These results imply that, for our study species, (1) ß-carotene may contribute little or nothing to colouration, (2) frogs can manufacture their own colour, (3) colour development is a continual process and (4) there may have been selection for synchronised development of colour expression. We discuss the potential adaptive benefit of ontogenetic colour change in P. corroboree. More broadly, we draw attention to the potential for adaptive developmental synchrony in the expression of colouration in aposematic species.

Amphibian reproductive technologies: approaches and welfare considerations.

Captive breeding and reintroduction programs have been established for several threatened amphibian species globally, but with varied success. This reflects our relatively poor understanding of the hormonal control of amphibian reproduction and the stimuli required to initiate and complete reproductive events. While the amphibian hypothalamo-pituitary-gonadal (HPG) axis shares fundamental similarities with both teleosts and tetrapods, there are more species differences than previously assumed. As a result, many amphibian captive breeding programs fail to reliably initiate breeding behaviour, achieve high rates of fertilization or generate large numbers of healthy, genetically diverse offspring. Reproductive technologies have the potential to overcome these challenges but should be used in concert with traditional methods that manipulate environmental conditions (including temperature, nutrition and social environment). Species-dependent methods for handling, restraint and hormone administration (including route and frequency) are discussed to ensure optimal welfare of captive breeding stock. We summarize advances in hormone therapies and discuss two case studies that illustrate some of the challenges and successes with amphibian reproductive technologies: the mountain yellow-legged frog (Rana muscosa; USA) and the northern corroboree frog (Pseudophryne pengilleyi; Australia). Further research is required to develop hormone therapies for a greater number of species to boost global conservation efforts.

Hormone-induced ovulation and artificial fertilisation in four terrestrial-breeding anurans.

The development and application of reproductive technologies has great potential to enhance the conservation management of threatened amphibians globally. The present study quantified the efficacy of protocols previously developed for Pseudophryne guentheri for hormonally inducing egg release and artificial fertilisation in three additional terrestrial-breeding species of Australian ground frog; namely Pseudophryne bibronii, Pseudophryne coriacea and Heleioporus eyrei. Females of each species were administered a priming dose of 0.4µg g-1 of gonadotropin-releasing hormone analogue (GnRHa), followed by an ovulatory dose of 2µg g-1 GnRHa. Egg release was achieved in all species (response 94.7-100%), but early embryo survival differed significantly, ranging from 23.8% to 89.3%. Overall, the protocols were considered effective in P. guentheri, P. bibronii and P. coriacea, but further protocol refinement is required for H. eyrei.

Evidence that genetic compatibility underpins female mate choice in a monandrous amphibian.

Mate choice for genetic benefits remains controversial, largely because few studies have estimated the relative contributions of additive and non-additive sources of genetic variation to offspring fitness. Moreover, there remains a deficit of these estimates for species where female-mate preferences have been quantified in the wild, especially species characterized by monandry or monogamy. Here, we use artificial fertilization techniques combined with a cross-classified breeding design to simultaneously test for "good genes" and "compatible genes" benefits of mate choice in the monandrous red backed toadlet (Pseudophryne coriacea). In addition, we used a genomic approach to estimate effects of parental-genetic relatedness (assessed using 27, 768 single nucleotide polymorphisms) on offspring fitness. Our results revealed no significant additive genetic effects (sire effects), but highly significant non-additive genetic effects (sire × dam interaction effects), on fertilization success, survival during embryonic development, and hatching success. We also found significant associations between parental genetic similarity and offspring survival (whereby survival was higher when parents were more related), and significant positive relationships between fertilization success and embryo survival through to hatching. These results indicate that offspring viability is significantly influenced by the genetic compatibility of parental genotypes, that more related parents are more genetically compatible, and that gametes with greater compatibility at fertilization produce more viable offspring. More broadly, our findings provide new quantitative genetic evidence that genetic incompatibility underpins female mate preferences. Continued quantitative genetic assessment of the relative importance of good genes versus compatible genes is needed to ascertain the general importance of genetic benefits as a driver of female mate choice.

The effect of injection and topical application of hCG and GnRH agonist to induce sperm-release in the roseate frog, Geocrinia rosea.

Reproductive technologies may assist amphibian conservation breeding programs (CBPs) to achieve propagation targets and genetic management goals. However, a trial-and-error approach to protocol refinement has led to few amphibian CBPs routinely employing reproductive technologies with predictable outcomes. Additionally, while injections can be safely administered to amphibians, perceived animal welfare risks, such as injury and disease transmission, warrant the development of alternative hormone administration protocols. The present study investigated the spermiation response of roseate frogs, Geocrinia rosea, administered various doses of human chorionic gonadotropin (hCG) and gonadotropin-releasing hormone agonist (GnRH-a) via subcutaneous injection. This study also quantified the spermiation response of frogs administered both hormones via topical application. Total sperm, sperm concentration and sperm viability were assessed over a 12-h period post hormone administration. Males released sperm in response to the injection of hCG (88-100% response; 5, 10 or 20 IU), but all samples collected from males administered hCG topically (100, 100 + DMSO or 200 IU hCG) were aspermic. In contrast, males consistently released sperm in response to both the injection (100% response; 1, 5 or 10 µg), or topical application (80-100% response; 50, 50 + DMSO or 100 µg) of GnRH-a. Overall, the administration of GnRH-a was more effective at inducing spermiation than hCG. Mean total sperm and sperm concentration were highest in response to the optimal topically applied dose of 100 µg GnRH-a (mean total sperm = 2.44 × 103, sperm concentration = 1.48 × 105 sperm/ml). We provide novel evidence that topical application provides a viable alternative to injection for the administration of GnRH-a to induce spermiation in amphibians.

Genetic benefits of extreme sequential polyandry in a terrestrial-breeding frog.

Sequential polyandry may evolve as an insurance mechanism to reduce the risk of females choosing mates who are genetically inferior (intrinsic male quality hypothesis) or genetically incompatible (genetic incompatibility hypothesis). The prevalence of such indirect benefits remains controversial, however, because studies estimating the contributions of additive and nonadditive sources of genetic variation to offspring fitness have been limited to a small number of taxonomic groups. Here, we used artificial fertilization techniques combined with a crossclassified breeding design (North Carolina Type II) to simultaneously test the "good genes hypothesis" and the "genetic incompatibility hypothesis" in the brown toadlet (Pseudophryne bibronii); a terrestrial-breeding species with extreme sequential polyandry. Our results revealed no significant additive or nonadditive genetic effects on fertilization success. Moreover, they revealed no significant additive genetic effects, but highly significant nonadditive genetic effects (sire by dam interaction effects), on hatching success and larval survival to initial and complete metamorphosis. Taken together, these results indicate that offspring viability is significantly influenced by the combination of parental genotypes, and that negative interactions between parental genetic elements manifest during embryonic and larval development. More broadly, our findings provide quantitative genetic evidence that insurance against genetic incompatibility favors the evolution and maintenance of sequential polyandry.

Sperm collection and storage for the sustainable management of amphibian biodiversity.

Current rates of biodiversity loss pose an unprecedented challenge to the conservation community, particularly with amphibians and freshwater fish as the most threatened vertebrates. An increasing number of environmental challenges, including habitat loss, pathogens, and global warming, demand a global response toward the sustainable management of ecosystems and their biodiversity. Conservation Breeding Programs (CBPs) are needed for the sustainable management of amphibian species threatened with extinction. CBPs support species survival while increasing public awareness and political influence. Current CBPs only cater for 10% of the almost 500 amphibian species in need. However, the use of sperm storage to increase efficiency and reliability, along with an increased number of CBPs, offer the potential to significantly reduce species loss. The establishment and refinement of techniques over the last two decades, for the collection and storage of amphibian spermatozoa, gives confidence for their use in CBPs and other biotechnical applications. Cryopreserved spermatozoa has produced breeding pairs of frogs and salamanders and the stage is set for Lifecycle Proof of Concept Programs that use cryopreserved sperm in CBPs along with repopulation, supplementation, and translocation programs. The application of cryopreserved sperm in CBPs, is complimentary to but separate from archival gene banking and general cell and tissue storage. However, where appropriate amphibian sperm banking should be integrated into other global biobanking projects, especially those for fish, and those that include the use of cryopreserved material for genomics and other research. Research over a broader range of amphibian species, and more uniformity in experimental methodology, is needed to inform both theory and application. Genomics is revolutionising our understanding of biological processes and increasingly guiding species conservation through the identification of evolutionary significant units as the conservation focus, and through revealing the intimate relationship between evolutionary history and sperm physiology that ultimately affects the amenability of sperm to refrigerated or frozen storage. In the present review we provide a nascent phylogenetic framework for integration with other research lines to further the potential of amphibian sperm banking.

Effect of carotenoid class and dose on the larval growth and development of the critically endangered southern corroboree frog.

Dietary carotenoids are expected to improve vertebrate growth and development, though evidence for beneficial effects remains limited. One reason for this might be that few studies have directly compared the effects of carotenoids from different classes (carotenes versus xanthophylls) at more than one dose. Here, we tested the effect of two doses of dietary ß-carotene and lutein (representing two different carotenoid classes) on the growth and development of larval southern corroboree frogs (Pseudophryne corroboree). Individuals were supplemented with either ß-carotene or lutein at one of two doses (0.1 mg g-1, 1 mg g-1), or given a diet without carotenoids (control). Each dietary treatment included 36 replicate individuals, and individuals remained on the same diet until metamorphosis (25-39 weeks). We measured larval survival, larval growth (body length), time to metamorphosis, metamorphic body size (mass and SVL), and body condition. Lutein had no detectable effect on larval growth and development. However, larvae receiving a high dose (1 mg g-1) of ß-carotene metamorphosed significantly faster than all other dietary treatments, despite no significant differences in growth rate. This result indicates that ß-carotene supplementation in P. corroboree has positive effects on development independent of growth effects. Our study provides new evidence for differential effects of carotenoid class and dose on vertebrate development. From a conservation perspective, our findings are expected to assist with the recovery of P. corroboree by expediting the generation of frogs required for the maintenance of captive insurance colonies, or the provision of frogs for release. More broadly, our study highlights the potential for dietary manipulation to assist with the ex situ management of threatened amphibian species worldwide.

Hormone-induced sperm-release in the critically endangered Booroolong frog (Litoria booroolongensis): effects of gonadotropin-releasing hormone and human chorionic gonadotropin.

Research into the development of reproductive technologies for amphibians has increased in recent years due to the rapid decline of amphibian species globally. Reproductive technologies have great potential to overcome captive breeding failure and improve the propagation and genetic management of threatened species. However, the incorporation of these technologies into conservation breeding programs has been protracted, primarily as a result of trial-and-error approaches to the refinement of hormone therapies. The present study investigated the effects of: (1) GnRH-a dose (0, 0.5, 1, 2, 4, 8 or 16 µg g-1), and (2) hCG dose (0, 2.5, 5, 10, 20 or 40 IU g-1), on the sperm-release response of the critically endangered Booroolong frog. Administration of GnRH-a at a dose of 0.5 µg g-1 resulted in the greatest number of sperm released (mean total sperm = 3.5 ×106, n = 11). Overall, hCG was more effective at eliciting spermiation in Booroolong frogs, with peak sperm release (mean total sperm = 25.1 ×106, n = 10) occurring in response to a dose of 40 IU g-1. Sperm output in response to 40 IU g-1 hCG was greatest between 1 and 6 h and steadily declined between 8 and 24 h post-hormone administration. Percent sperm motility peaked between 4 and 10 h (58.1-62.7%), and sperm velocity between 4 and 12 h (24.3-27.2 µm s-1). Booroolong frogs join a small, but growing number of amphibian species that exhibit improved spermiation in response to hCG. Further research is required to identify optimal hormone-induction protocols for threatened amphibians and expedite the incorporation of reproductive technologies into CBPs.

The Role of Reproductive Technologies in Amphibian Conservation Breeding Programs.

Anthropogenic environmental change has led to unprecedented rates of species extinction, presenting a major threat to global biodiversity. Among vertebrates, amphibians have been most severely impacted, with an estimated 41% of species now threatened with extinction. In response to this biodiversity crisis, a moral and ethical obligation exists to implement proactive interventionist conservation actions to assist species recovery and decelerate declines. Conservation breeding programs have been successfully established for several threatened amphibian species globally, aiming to prevent species' extinction by maintaining genetically representative assurance colonies ex situ while providing individuals for population augmentation, translocation, and reestablishment in situ. Reproductive technologies have enormous potential to enhance the propagation and genetic management of threatened species. In this review, we discuss the role of reproductive technologies in amphibian conservation breeding programs and summarize technological advancements in amphibian hormone therapies, gamete storage, and artificial fertilization.

Dose and life stage-dependent effects of dietary beta-carotene supplementation on the growth and development of the Booroolong frog.

Carotenoids are known for their antioxidant capacity and are considered to play an important role in vertebrate growth and development. However, evidence for their beneficial effects remains limited, possibly because very few studies have tested for dose effects across different life stages. The present study investigated the effect of various doses of dietary beta-carotene supplements on the growth and development of larval and post-metamorphic Booroolong frogs (Litoria booroolongensis). Larval and post-metamorphic basal diets (containing 0.015 and 0.005 mg g-1 total carotenoids, respectively) were supplemented with beta-carotene at one of four concentrations: 0 mg g-1, 0.1 mg g-1, 1 mg g-1 and 10 mg g-1. Each treatment included 72 replicate individuals, and individuals remained on the same diet treatment over both life stages (spanning 53 experimental weeks). Our results show that larvae receiving an intermediate (1 mg g-1) beta-carotene supplement dose grew faster than unsupplemented larvae (0 mg g-1), and metamorphosed earlier. After metamorphosis, there was no effect of the lowest supplement dose (0.1 mg g-1) on growth and development. However, juveniles fed the highest supplement dose (10 mg g-1) displayed significantly smaller body mass and lower body condition, compared to all other supplement doses, from 4-months through to sexual maturity (7-months). These findings indicate that beta-carotene supplementation has positive effects on growth and development, but only at intermediate doses, and only in the larval life stage. This knowledge may assist with amphibian conservation by expediting the rate that metamorphs can be generated in captive breeding programmes. More broadly, this is the first study to demonstrate both dose and life stage-dependent effects of dietary beta-carotene supplementation on vertebrate growth and development.

The unexpected genetic mating system of the red-backed toadlet (Pseudophryne coriacea): A species with prolonged terrestrial breeding and cryptic reproductive behaviour.

Molecular technologies have revolutionized our classification of animal mating systems, yet we still know very little about the genetic mating systems of many vertebrate groups. It is widely believed that anuran amphibians have the highest reproductive diversity of all vertebrates, yet genetic mating systems have been studied in <1% of all described species. Here, we use single nucleotide polymorphisms to quantify the genetic mating system of the terrestrial breeding red-backed toadlet Pseudophryne coriacea. In this species, breeding is prolonged (approximately 5 months), and males construct subterranean nests in which females deposit eggs. We predicted that females would display extreme sequential polyandry because this mating system has been reported in a closely related species (P. bibronii). Parentage analysis revealed that mating success was heavily skewed towards a subset of males (30.6% of potential sires) and that nearly all females (92.6%) mated with one male. In a high percentage of occupied nests (37.1%), the resident male was not the genetic sire, and very few nests (4.3%) contained clutches with multiple paternity. Unexpectedly, these results show that sequential polyandry is rare. They also show that there is a high frequency of nest takeover and extreme competition between males for nest sites, but that males rarely sneak matings. Genetic analysis also revealed introgressive hybridization between P. coriacea and the red-crowned toadlet (Pseudophryne australis). Our study demonstrates a high level of mating system complexity, and it shows that closely related anurans can vary dramatically in their genetic mating system.

Hormone-induced spawning of the critically endangered northern corroboree frog Pseudophryne pengilleyi.

Fundamental knowledge of the optimal hormone concentrations required to stimulate amplexus and spawning in breeding pairs of amphibians is currently lacking, hindering our understanding of the proximate mechanisms underpinning mating behaviour. The present study investigated the effects of: (1) the dose of a gonadotropin-releasing hormone analogue (GnRH-a) administered; (2) male-female hormone administration interval; and (3) topical application of GnRH-a, on spawning success in the northern corroboree frog. Administration of GnRH-a at doses of 0.5, 1.0 and 2.0µgg-1 were highly successful, with a significantly greater proportion of hormone-treated pairs ovipositing (89-100%) compared with the 0µgg-1 treatment (22%). Of the hormone-treated pairs, those receiving 0.5µgg-1 GnRH-a exhibited the highest fertilisation success (61%). Administration of GnRH-a to males and females simultaneously (0h) was more effective than injecting males either 48 or 24h before the injection of females. Overall, administration of GnRH-a was highly successful at inducing spawning in northern corroboree frogs. For the first time, we also effectively induced spawning following the topical application of GnRH-a to the ventral pelvic region. Topical application of GnRH-a eliminates the need for specialised training in amphibian injection, and will allow assisted reproductive technologies to be adopted by a greater number of captive facilities globally.

Effect of long-term dietary beta-carotene supplementation on sperm concentration and motility in an endangered amphibian.

Dietary carotenoids have a high antioxidant capacity, so it has been hypothesised that carotenoid supplimentation will improve sperm production and quality by protecting sperm from oxidative damage. The effects of carotenoids on sperm have only been assessed in three vertebrate species, and evidence for improved sperm concentration and motility remains equivocal. One reason for this might be that in most studies there has not been an assessment of the effects of single carotenoid compounds over a range of doses. Applied research focused on developing ways to improve sperm quality could benefit the captive breeding and conservation of threatened species. The aim of the present study was to assess a dose-dependent effect of beta-carotene supplementation on sperm concentration and motility in the endangered booroolong frog (Litoria booroolongensis). Individuals were supplemented with one of four beta-carotene doses (0, 0.1, 1 and 10 mg/g) from hatching until sexual maturity (53 weeks). Sperm concentration was determined prior to activation, and percent sperm motility and sperm velocity were measured at 0, 3 and 6 h post-activation using computer-assisted sperm analysis. Unexpectedly, beta-carotene had no significant effect on sperm concentration, percent motility or velocity at any time point, providing no evidence for beneficial effects. Findings of the present study indicate there are likely to be species-specific differences in sperm production and motility that influence the risk of oxidative damage to sperm and dependence on dietary antioxidants to inhibit these detrimental effects.

The effect of dietary antioxidants and exercise training on the escape performance of Southern Corroboree frogs.

Escape-response behaviour is essential to ensure an individual's survival during a predator attack, however, these behaviours are energetically costly and may cause oxidative stress. Oxidative stress can be reduced by supplementing an individual's diet with exogenous antioxidants or through regular moderate exercise training, which stimulates the upregulation of the endogenous antioxidant system. Two studies have tested the simultaneous effects of dietary antioxidant supplementation and exercise training on animal escape-response behaviour. The present study investigated the effects of dietary carotenoids and exercise training on the escape-response behaviour of Southern Corroboree frogs. Frogs were fed either a carotenoid-supplemented or unsupplemented diet and were exposed to repeated escape-response trials (training) for five consecutive weeks. Carotenoid-supplemented individuals outperformed unsupplemented individuals in initial hopping speed, length of the first hop and hopping distance, however, the performance of frogs in each treatment group became statistically similar after training. Within treatment groups, exercise training significantly improved the hopping speed of unsupplemented frogs, with speeds almost doubling between training weeks one and five. By contrast, exercise training did not significantly improve the hopping speed of carotenoid-supplemented frogs. Our results provide some of the first evidence that exercise training improves escape performance, and that dietary antioxidants may inhibit training-induced benefits.