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.

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.

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.

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.

Dietary Carotenoid Supplementation Enhances the Cutaneous Bacterial Communities of the Critically Endangered Southern Corroboree Frog (Pseudophryne corroboree).

The rapid spread of infectious disease has resulted in the decline of animal populations globally. Amphibians support a diversity of microbial symbionts on their skin surface that help to inhibit pathogen colonisation and reduce disease susceptibility and virulence. These cutaneous microbial communities represent an important component of amphibian immune defence, however, very little is known about the environmental factors that influence the cutaneous microbiome. Here, we characterise the cutaneous bacterial communities of a captive colony of the critically endangered Australian southern corroboree frog, Pseudophyrne corroboree, and examine the effect of dietary carotenoid supplementation on bacterial abundance, species richness and community composition. Individuals receiving a carotenoid-supplemented diet exhibited significantly higher bacterial abundance and species richness as well as an altered bacterial community composition compared to individuals that did not receive dietary carotenoids. Our findings suggest that dietary carotenoid supplementation enhances the cutaneous bacteria community of the southern corroboree frog and regulates the presence of bacteria species within the cutaneous microbiome. Our study is the second to demonstrate that carotenoid supplementation can improve amphibian cutaneous bacterial community dynamics, drawing attention to the possibility that dietary manipulation may assist with the ex situ management of endangered species and improve resilience to lethal pathogens such as Batrachochytrium dendrobatidis (Bd).

Sperm motility activation in the critically endangered booroolong frog: the effect of medium osmolality and phosphodiesterase inhibitors.

Effective activation of sperm motility is fundamental to successful artificial fertilisation; however, studies investigating optimal procedures in amphibians are lacking. This study found the optimal osmolality of activation media for sperm motility activation and evaluated the effect of phosphodiesterase (PDE) inhibitors on sperm activation and longevity in the critically endangered booroolong frog, Litoria booroolongensis. To assess the effect of medium osmolality (10, 25, 50, 75, 100 and 200mOsmolkg-1) and PDE inhibitors (control, 2.5mM caffeine, 5mM caffeine, 2.5mM pentoxifylline, 5mM pentoxifylline, 2.5mM theophylline and 5mM theophylline) on initial activation, percentage sperm motility and sperm velocity were quantified using computer-assisted sperm analysis. To assess the effect of PDE inhibitors (control, 2.5mM caffeine and 2.5mM theophylline) on sperm longevity, percentage motility and velocity were assessed hourly until 10h after activation. High (>60%) percentage motility was achieved in a broad range of activation-medium osmolalities (10-75mOsmolkg-1). PDE inhibitors did not have an effect on initial sperm motility or velocity, but caffeine and theophylline improved sperm longevity, significantly increasing motility and velocity at 8, 9 and 10h after activation. Data also show that sperm longevity in L. booroolongensis is extreme, with spermatozoa remaining motile more than twice as long as those of any other anuran amphibian.

The effect of gentamicin on sperm motility and bacterial abundance during chilled sperm storage in the Booroolong frog.

Antibiotics can inhibit bacterial contamination and extend sperm longevity during storage; a primary goal of captive facilities conducting biobanking and artificial fertilisation (AF). This study evaluated the effects of gentamicin on the short-term storage of Booroolong frog sperm. Sperm suspensions were obtained via either testis maceration, or as spermic urine, following hormonal induction of sperm-release. The effect of 0, 1, 2, 3 or 4mgmL-1 gentamicin on bacterial abundance (CFUmL-1) was determined and sperm motility assessed. In both testis macerate samples and spermic urine samples, gentamicin administered at intermediate-to-high doses (2, 3 & 4mgmL-1) eliminated, or significantly reduced, bacterial abundance. Sperm samples obtained via testis maceration exhibited significantly lower sperm motility at the highest doses (3 & 4mgmL-1). All remaining treatments (0, 1 & 2mgmL-1) were statistically similar and maintained sperm motility >55%. Sperm samples obtained as spermic urine exhibited no difference in sperm motility or velocity when treated with gentamicin at any dose. While antibiotic treatment did not improve sperm longevity as predicted, this is the first study to demonstrate that antibiotic treatment can reduce bacterial abundance without compromising sperm motility in an anuran amphibian. Antibiotic supplementation may be an important tool for reducing pathogen transmission where sperm samples are transferred between captive institutions for biobanking and AF.

Testing the effect of dietary carotenoids on larval survival, growth and development in the critically endangered southern corroboree frog.

The success of captive breeding programs (CBPs) for threatened species is often limited due to a lack of knowledge of the nutritional conditions required for optimal growth and survival. Carotenoids are powerful antioxidants known to accelerate vertebrate growth and reduce mortality. However, the effect of carotenoids on amphibian life-history traits remains poorly understood. The aim of our study was to use a manipulative laboratory experiment to test the effect of dietary-carotenoid supplementation during the larval life stage on the survival, growth and development of the critically endangered southern corroboree frog (Pseudophryne corroboree). Larvae were fed either a carotenoid supplemented diet or an unsupplemented diet and the survival, growth and development of individuals was monitored and compared. There was no significant effect of dietary treatment on larval survival, growth rate, time taken to reach metamorphosis, or body size at metamorphosis. Our findings provide no evidence that carotenoid supplementation during the larval life stage improves the growth and development of southern corroboree frogs. However, because the carotenoid dose used in our study did not have any detrimental effects on P. corroboree larvae, but has previously been shown to improve adult coloration, immunity, and exercise performance, carotenoid supplementation should be considered when evaluating the nutritional requirements of P. corroboree in captivity. Carotenoid supplementation studies are now required for a diversity of anuran species to determine the effects of carotenoids on amphibian survival, growth and development. Understanding the effects of dietary carotenoids on different life-history traits may assist with amphibian captive breeding and conservation.

Planning for ex situ conservation in the face of uncertainty.

Ex situ conservation strategies for threatened species often require long-term commitment and financial investment to achieve management objectives. We present a framework that considers the decision to adopt ex situ management for a target species as the end point of several linked decisions. We used a decision tree to intuitively represent the logical sequence of decision making. The first decision is to identify the specific management actions most likely to achieve the fundamental objectives of the recovery plan, with or without the use of ex-situ populations. Once this decision has been made, one decides whether to establish an ex situ population, accounting for the probability of success in the initial phase of the recovery plan, for example, the probability of successful breeding in captivity. Approaching these decisions in the reverse order (attempting to establish an ex situ population before its purpose is clearly defined) can lead to a poor allocation of resources, because it may restrict the range of available decisions in the second stage. We applied our decision framework to the recovery program for the threatened spotted tree frog (Litoria spenceri) of southeastern Australia. Across a range of possible management actions, only those including ex situ management were expected to provide >50% probability of the species' persistence, but these actions cost more than use of in situ alternatives only. The expected benefits of ex situ actions were predicted to be offset by additional uncertainty and stochasticity associated with establishing and maintaining ex situ populations. Naïvely implementing ex situ conservation strategies can lead to inefficient management. Our framework may help managers explicitly evaluate objectives, management options, and the probability of success prior to establishing a captive colony of any given species.

Antibiotics and oxygen availability affect the short-term storage of spermatozoa from the critically endangered booroolong frog, Litoria booroolongensis.

Sperm-storage technologies aim to extend sperm longevity and increase the time available to achieve artificial fertilisation. The aim of the present study was to quantify the effects of antibiotic supplementation (4mgmL(-1) gentamicin) and altered gaseous storage environment (100%, 20% and 0% O2) on sperm longevity in the critically endangered booroolong frog, Litoria booroolongensis. A split-sample experimental design was adopted, whereby each sperm suspension (n=10) was evenly divided among six experimental treatments (100% O2 with antibiotic, 20% O2 with antibiotic, 0% O2 with antibiotic, 100% O2 without antibiotic, 20% O2 without antibiotic, 0% O2 without antibiotic). Sperm suspensions were refrigerated at 5°C for the duration of the 21-day storage period. Percentage sperm motility and sperm velocity were quantified every 3 days using a computer-assisted sperm analysis system. Treatments aerated with either 100% or 20% oxygen, without the addition of the antibiotic gentamicin, consistently exhibited the highest percentage sperm motility. On Day 21 of storage, sperm suspensions in these two treatments (100% O2 without antibiotic, 20% O2 without antibiotic) maintained 61.3% and 52.0% sperm motility, respectively, whereas all remaining experimental treatments exhibited <30% sperm motility. Sperm velocity did not differ significantly among storage treatments, at any of the sampling periods, with the exception of day 21. Overall, the results from this study indicate that increased oxygen availability is beneficial to sperm longevity, but that gentamicin inhibits sperm motility in L. booroolongensis.

Artificial fertilisation in a terrestrial toadlet (Pseudophryne guentheri): effect of medium osmolality, sperm concentration and gamete storage.

Anurans exhibit a greater reproductive diversity than any other vertebrate order. However, studies investigating the effects of the external fertilisation environment on fertilisation success are limited to aquatic-breeding species. This study investigated the effects of fertilisation medium osmolality, sperm concentration and short-term oocyte storage on fertilisation success in a terrestrial-breeding anuran, Pseudophryne guentheri. Split-clutch experimental designs were used to determine optimal fertilisation conditions. To determine the effect of short-term sperm storage, sperm viability was assessed using fluorescence microscopy and percentage sperm motility and velocity quantified with a computer-assisted sperm analysis system. Fertilisation success was highest in media ranging in osmolality from 25 mOsm kg⁻¹ to 100 mOsm kg⁻¹, representing a broader range and higher optimal osmolality than previously reported for aquatic breeders. High rates of fertilisation (>75%) were achieved in relatively low sperm concentrations (2.5×104 mL⁻¹). Oocytes stored in isotonic solutions (200 mOsm kg⁻¹) retained fertilisation capacity (32%) after 8h of storage, while sperm suspensions maintained motility (≥26%) for 13 days. Additional studies on terrestrial-breeding anurans will be required to ascertain whether the optimal fertilisation conditions reported reflect adaptations to achieve fertilisation in a terrestrial environment.

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.

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.

Investigating patterns in the spermiation response of eight Australian frogs administered human chorionic gonadotropin (hCG) and luteinizing hormone-releasing hormone (LHRHa).

Exogenous LHRHa and hCG are routinely employed to induce spermiation in vivo in anurans. To date, however, few studies have directly compared the efficacy of these two hormones. The aim of this study was threefold. First to quantify the spermiation response of eight Australian anuran species (Crinia glauerti, Crinia georgiana, Crinia pseudinsignifera, Geocrinia rosea, Heleioporus albopunctatus, Heleioporuseyrei, Neobatrachus pelobatoides and Pseudophryne guentheri) administered LHRHa and hCG. Second, to determine whether variance in spermiation responses is related to a species' reproductive mode (aquatic vs. terrestrial) or family (Limnodynastidae vs. Myobatrachidae). Third, to compare the quantity and quality of spermatozoa obtained via hormone administration (LHRHa and hCG) to spermatozoa obtained via testis removal and maceration. There was no significant difference in the viability of spermatozoa obtained from hCG or LHRHa administration in any of the eight study species. The sperm viability of samples ranged from 28-84% in C. georgiana and G. rosea, respectively. The hormone that induced the release of the highest number of spermatozoa differed among species, with all five species belonging to the family Myobatrachidae responding better to LHRHa, and the three species from the family Lymnodynastidae releasing a greater number of spermatozoa in response to hCG. Importantly, these results provide the first preliminary evidence that hCG and LHRHa efficacy in anurans may be predicted by phylogeny. Understanding such broad-scale patterns in the response of anurans to exogenous hormones will expedite the application of assisted reproductive technologies to novel species.

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.

Effect of priming injections of luteinizing hormone-releasing hormone on spermiation and ovulation in Gϋnther's toadlet, Pseudophryne guentheri.

BACKGROUND: In the majority of vertebrates, gametogenesis and gamete-release depend on the pulsatile secretion of luteinizing hormone-releasing hormone (LHRH) from the hypothalamus. Studies attempting to artificially stimulate ovulation and spermiation may benefit from mimicking the naturally episodic secretion of LHRH by administering priming injections of a synthetic analogue (LHRHa). This study investigated the impact of low-dose priming injections of LHRHa on gamete-release in the Australian toadlet Pseudophryne guentheri. METHODS: Toadlets were administered a single dose of two micrograms per. gram LHRHa without a priming injection (no priming), or preceded by one (one priming) or two (two priming) injections of 0.4 micrograms per. gram LHRHa. Spermiation responses were evaluated at 3, 7 and 12 hrs post hormone administration (PA), and sperm number and viability were quantified using fluorescent microscopy. Oocyte yields were evaluated by stripping females at 10-11 hrs PA. A sub-sample of twenty eggs per female was then fertilised (with sperm obtained from testis macerates) and fertilisation success determined. RESULTS: No priming induced the release of the highest number of spermatozoa, with a step-wise decrease in the number of spermatozoa released in the one and two priming treatments respectively. Peak sperm-release occurred at 12 hrs PA for all priming treatments and there was no significant difference in sperm viability. Females in the control treatment failed to release oocytes, while those administered an ovulatory dose without priming exhibited a poor ovulatory response. The remaining two priming treatments (one and two priming) successfully induced 100% of females to expel an entire clutch. Oocytes obtained from the no, or two priming treatments all failed to fertilise, however oocytes obtained from the one priming treatment displayed an average fertilisation success of 97%. CONCLUSION: Spermiation was most effectively induced in male P. guentheri by administering a single injection of LHRHa without priming. In contrast, female P. guentheri failed to ovulate without priming. A single priming injection induced the release of oocytes of high viability compared to oocytes obtained from females in the two priming treatment which underwent a process of over-ripening.

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.

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.