71 research outputs found
Effect of priming injections of luteinizing hormone-releasing hormone on spermiation and ovulation in GĎnther's Toadlet, Pseudophryne guentheri
<p>Abstract</p> <p>Background</p> <p>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 <it>Pseudophryne guentheri</it>.</p> <p>Methods</p> <p>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.</p> <p>Results</p> <p>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%.</p> <p>Conclusion</p> <p>Spermiation was most effectively induced in male <it>P. guentheri </it>by administering a single injection of LHRHa without priming. In contrast, female <it>P. guentheri </it>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.</p
Effects of luteinizing hormone-releasing hormone and arginine-vasotocin on the sperm-release response of GĂźnther's Toadlet, Pseudophryne guentheri
<p>Abstract</p> <p>Background</p> <p>Luteinizing hormone-releasing hormone (LHRH) is an exogenous hormone commonly used to induce spermiation in anuran amphibians. Over the past few decades, the LHRH dose administered to individuals and the frequency of injection has been highly variable. The sperm-release responses reported have been correspondingly diverse, highlighting a need to quantify dose-response relationships on a species-specific basis. This study on the Australian anuran <it>Pseudophryne guentheri </it>first evaluated the spermiation response of males administered one of five LHRHa doses, and second, determined whether AVT administered in combination with the optimal LHRHa dose improved sperm-release.</p> <p>Methods</p> <p>Male toadlets were administered a single dose of 0, 1, 2, 4 or 8 micrograms/g body weight of LHRHa. A 4 micrograms/g dose of AVT was administered alone or in combination with 2 micrograms/g LHRHa. Spermiation responses were evaluated at 3, 7 and 12 h post hormone administration (PA), and sperm number and viability were quantified using fluorescent microscopy.</p> <p>Results</p> <p>LHRHa administration was highly effective at inducing spermiation in <it>P. guentheri</it>, with 100% of hormone-treated males producing sperm during the experimental period. The number of sperm released in response to 2 micrograms/g LHRHa was greater than all other doses administered and sperm viability was highest in the 1 microgram/g treatment. The administration of AVT alone or in combination with LHRHa resulted in the release of significantly lower sperm numbers.</p> <p>Conclusion</p> <p>Overall, results from this study suggest that in <it>P. guentheri</it>, LHRHa is effective at inducing spermiation, but that AVT inhibits sperm-release.</p
Hormonal induction of gamete release, and in-vitro fertilisation, in the critically endangered Southern Corroboree Frog, Pseudophryne corroboree
<p>Abstract</p> <p>Background</p> <p>Conservation Breeding Programs (CBP's) are playing an important role in the protection of critically endangered anuran amphibians, but for many species recruitment is not successful enough to maintain captive populations, or provide individuals for release. In response, there has been an increasing focus on the use of Assisted Reproductive Technologies (ART), including the administration of reproductive hormones to induce gamete release followed by <it>in vitro </it>fertilisation. The objective of this study was to test the efficacy of two exogenous hormones to induce gamete release, for the purpose of conducting <it>in vitro </it>fertilisation (IVF), in one of Australia's most critically endangered frog species, <it>Pseudophryne corroboree</it>.</p> <p>Methods</p> <p>Male frogs were administered a single dose of either human chorionic gonadotropin (hCG) or luteinizing hormone-releasing hormone (LHRHa), while female frogs received both a priming and ovulatory dose of LHRHa. Spermiation responses were evaluated at 3, 7, 12, 24, 36, 48, 60 and 72 h post hormone administration (PA), and sperm number and viability were quantified using fluorescent microscopy. Ovulation responses were evaluated by stripping females every 12 h PA for 5 days. Once gametes were obtained, IVF was attempted by combining spermic urine with oocytes in a dilute solution of simplified amphibian ringer (SAR).</p> <p>Results</p> <p>Administration of both hCG and LHRHa induced approximately 80% of males to release sperm over 72 h. Peak sperm release occurred at 12 h PA for hCG treated males and 36 h PA for LHRHa treated males. On average, LHRHa treated males released a significantly higher total number of live sperm, and a higher concentration of sperm, over a longer period. In female frogs, administration of LHRHa induced approximately 30% of individuals to release eggs. On average, eggs were released between 24 and 48 h PA, with a peak in egg release at 36 h PA. IVF resulted in a moderate percentage (54.72%) of eggs being fertilised, however all resultant embryos failed prior to gastrulation.</p> <p>Conclusions</p> <p>Hormone treatment successfully induced spermiation and ovulation in <it>P. corroboree</it>, but refinement of gamete induction and IVF techniques will be required before ART protocols can be used to routinely propagate this species.</p
The importance of quantifying fitness-determining traits throughout life to assess the application of reproductive technologies for amphibian species recovery
The application of reproductive technologies to amphibian conservation breeding programs is gaining momentum and the field is poised to contribute significantly toward amphibian species recovery. We briefly discuss the opportunities for reproductive technologies to enhance conservation breeding outcomes, including their potential to enhance the genetic management, and in turn, the fitness of threatened species. Despite this potential, an important consideration that is not yet well understood is the degree to which specific reproductive technologies might influence (either enhance, or in some instances potentially decrease) individual fitness and lead to shifts in population viability. The development of a standardised approach to monitoring offspring throughout life-stages to detect changes to morphology, behaviour, physiology, survivorship, and developmental trajectories is essential. The primary focus of this review is to provide a âbest-practiseâ framework for quantifying key fitness determining traits expected to contribute to the fitness of individuals and long-term viability of populations, which will ultimately allow us to progress the field of amphibian reproductive technologies and assess the impact of protocol refinement
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 gâ1 total carotenoids, respectively) were supplemented with beta-carotene at one of four concentrations: 0 mg gâ1 , 0.1 mg gâ1 , 1 mg gâ1 and 10 mg gâ1 . 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 gâ1 ) beta-carotene supplement dose grew faster than unsupplemented larvae (0 mg gâ1 ), and metamorphosed earlier. After metamorphosis, there was no effect of the lowest supplement dose (0.1 mg gâ1 ) on growth and development. However, juveniles fed the highest supplement dose (10 mg gâ1 ) 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 stagedependent effects of dietary beta-carotene supplementation on vertebrate growth and development
Characterisation of the urinary microbiome of a frog, and the effect of antibiotics on bacterial abundance and sperm viability during refrigerated storage
Reproductive technologies are increasingly being adopted to improve the conservation management of threatened species. The storage and transport of sperm is a vital aspect of the practical implementation of reproductive technologies, however, bacterial contamination during the storage and transport of sperm samples presents a biosecurity risk and can contribute to a reduction in sperm longevity during storage. The present study: 1) characterised the urinary microbiome (bacterial species composition and abundance) using culture-independent 16S rRNA sequencing; 2) quantified the effect of various doses of gentamicin and streptomycin-penicillin on bacterial abundance (colony-forming units; CFUs) and; 3) quantified the effect of antibiotic supplementation on the sperm viability (proportion live/dead) of spermic urine during a 12-day cold-storage period, using the common eastern froglet, Crinia signifera. Overall, urine samples were found to host a diverse array of bacteria, dominated by the phyla Proteobacteria, Firmicutes, and Bacteroidetes. Bacterial abundance was significantly reduced in all antibiotic treatment groups compared to the control group. Antibiotic supplementation had no effect on sperm viability between day 0 and day 6 of storage, however both antibiotic treatments significantly improved sperm viability from days 9 to 12 of storage compared to the control group. Overall, the results of this study provide novel insight into the urinary microbiome, being the first to use a metagenomics approach to characterise the bacterial community present in the urine of an amphibian. Importantly, this study provides evidence that antibiotic supplementation with either gentamicin or streptomycin-penicillin, minimises bacterial proliferation and improves sperm viability during cold storage. These findings will contribute to the development of biosecurity protocols aimed at reducing the risk of disease transmission and cross-infection from unwanted bacteria and infectious agents in amphibian captive breeding programs
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.The study was funded by an Australian Research Council grant awarded to PB, SK and AS (Linkage Grant LP140100808). Work was completed while DO was supported by an Australian Government Research Training Program (RTP) scholarship
Dietary carotenoid supplementation has longâterm and communityâwide 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. Interâindividual variation in the availability of microânutrients 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 jointâspecies 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 lifeâhistory 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 preâmetamorphosis, 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
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
Priority research needs to inform amphibian conservation in the Anthropocene
The problem of global amphibian declines has prompted extensive research over the last three decades. Initially, the focus was on identifying and characterizing the extent of the problem, but more recently efforts have shifted to evidenceâbased research designed to identify best solutions and to improve conservation outcomes. Despite extensive accumulation of knowledge on amphibian declines, there remain knowledge gaps and disconnects between science and action that hamper our ability to advance conservation efforts. Using input from participants at the ninth World Congress of Herpetology, a U.S. Geological Survey Powell Center symposium, amphibian onâline forums for discussion, the International Union for Conservation of Nature Assisted Reproductive Technologies and Gamete Biobanking group, and respondents to a survey, we developed a list of 25 priority research questions for amphibian conservation at this stage of the Anthropocene. We identified amphibian conservation research priorities while accounting for expected tradeoffs in geographic scope, costs, and the taxonomic breadth of research needs. We aimed to solicit views from individuals rather than organizations while acknowledging inequities in participation. Emerging research priorities (i.e., those underârepresented in recently published amphibian conservation literature) were identified, and included the effects of climate change, communityâlevel (rather than single speciesâlevel) drivers of declines, methodological improvements for research and monitoring, genomics, and effects of landâuse change. Improved inclusion of underârepresented members of the amphibian conservation community was also identified as a priority. These research needs represent critical knowledge gaps for amphibian conservation although filling these gaps may not be necessary for many conservation actions
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