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

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

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.

Recovery and Characterization of Spermatozoa in a Neotropical, Terrestrial, Direct-Developing Riparian Frog (Craugastor evanesco) through Hormonal Stimulation.

The Vanishing Rainfrog (Craugastor evanesco) is an endemic and critically endangered frog species of Panama. It is suspected that 90% of the population has disappeared from the wild. Frogs were collected from the wild and brought to a Captive Breeding Program; however, accomplishing regular reproductive events for this species has been difficult. The objective of this study was to determine the effect of hormonal stimulation on the production and quality of C. evanesco spermatozoa, aiming to develop an efficient and safe sperm collection protocol as a tool to help reproduce this endangered species. Mature males received intra-peritoneal injections with one of six hormone treatments, including des-Gly10, D-Ala6, Pro-NHEt9-GnRH-A, Amphiplex or hCG. Urine samples were collected at 10 different time points post-injection. Quality assessments included sperm concentration, percentage motility, percentage forward progressive motility (FPM), osmolality, pH and morphology analysis. Our results indicate that the optimal treatment for the collection of highly concentrated sperm samples of C. evanesco is 4 µg/gbw GnRH, followed by Amphiplex and 2 µg/gbw GnRH as sub-optimal treatments and finally, 6 µg/gbw GnRH and 5 and 10 IU/gbw hCG as non-optimal treatments. GnRH-A at 4 µg/gbw and Amphiplex stimulated the production of samples with the highest sperm concentrations and quality, despite Amphiplex producing lower percentages of intact acrosome and tail. In contrast, hCG concentrations were not reliable inducers of sperm production, consistently showing lower concentrations, higher percentages of sperm abnormalities and more acidic spermic urine than that induced by Amphiplex and GnRH-A. Morphological assessments revealed that C. evanesco spermatozoa have a filiform shape with a large acrosome on the anterior part of an elongated head, a small midpiece and a long tail with two filaments joined together by an undulating membrane.

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

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

Using reproductive technologies to assess the development of secondary sexual characteristics, ovarian senescence and hermaphroditism in the endangered mountain yellow-legged frog Rana muscosa.

Anurans can display a host of intriguing sexual syndromes, including hermaphroditism and sex reversal. Using a multifaceted approach for diagnosing and characterising hermaphroditism in the endangered anuran species Rana mucosa, we tracked changes in female reproductive status using hormone monitoring, ultrasound examinations, individual life history, fertilisation records and post-mortem findings. Seven individuals originally sexed as females developed secondary male sexual characteristics, behaviour and hormone profiles and, in some cases, had testicular tissue despite having previously laid eggs. Our results suggest that reproductive technologies can shed light on life history patterns and reproductive anomalies that may affect endangered anuran survival.

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

Amphibians are becoming increasingly reliant on captive breeding programs for continued survival. Assisted reproductive technologies including gamete cryopreservation and IVF can help reduce costs of breeding programs, provide insurance against extinction and assist genetic rescue in wild populations. However, the use of these technologies to produce reproductively mature offspring has only been demonstrated in a few non-model species. We aimed to optimise sperm cryopreservation in the threatened frog Litoria aurea and generate mature offspring from frozen-thawed spermatozoa by IVF. We tested three concentrations (1.4, 2.1 and 2.8M) of the cryoprotectants dimethylsulfoxide (DMSO) and glycerol with 0.3M sucrose. Using DMSO was more likely to result in recovery of sperm motility, vitality and acrosome integrity than glycerol, regardless of concentration, with forward progressive motility being most sensitive to damage. The lowest concentrations of 1.4 and 2.1M provided the best protection regardless of cryoprotectant type. Spermatozoa cryopreserved in 2.1M DMSO outperformed spermatozoa cryopreserved in equivalent concentrations of glycerol in terms of their ability to fertilise ova, resulting in higher rates of embryos hatching and several individuals reaching sexual maturity. We have demonstrated that sperm cryopreservation and subsequent offspring generation via IVF is a feasible conservation tool for L. aurea and other threatened amphibians.

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.

Efficacy of hormone stimulation on sperm production in an alpine amphibian (Anaxyrus boreas boreas) and the impact of short-term storage on sperm quality.

The Southern Rocky Mountain boreal toad (Anaxyrus boreas boreas) has disappeared from much of its range in the alpine regions of Central and Western North America, and restoration efforts are compromised by limited knowledge of this species' reproductive biology. This study aimed to establish whether assisted reproductive techniques could be used to improve breeding output in captive boreal toads by determining the most effective concentration of human chorionic gonadotropin (hCG) for induction of spermiation and viability of sperm during cold storage. Male toads (n = 21) were treated with a Low (3 IU g-1), Medium (10 IU g-1), or High (15 IU g-1) concentration of hCG and spermic urine samples were collected over 24 hrs. Treatment effectiveness was evaluated by measuring the response rate, Total Motility (TM), Forward Progressive Motility (FPM), Quality of FPM (QFPM), and concentration. For short-term cold storage, spermic urine samples (n = 13) were stored at 4 °C for 14 days and sperm TM and FPM monitored daily. All treatments induced spermiation; however, a greater number of toads produced sperm in the Medium and High treatments compared to the Low. Overall, TM, FPM, QFPM and sperm concentration were similar across all three treatments, but variation existed in the timing and duration of peak sperm production. Sperm motility was maintained for up to 14 days in cold storage, although the quality slowly decreased over time. An effective reproduction strategy for the boreal toad will provide a means to improve captive breeding efforts and increase our understanding of the reproductive physiology of alpine Bufonids.

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

Captive breeding is an important tool for amphibian conservation despite high economic costs and deleterious genetic effects of sustained captivity and unavoidably small colony sizes. Integration of biobanking and assisted reproductive technologies (ARTs) could provide solutions to these challenges, but is rarely used due to lack of recognition of the potential benefits and clear policy direction. Here we present compelling genetic and economic arguments to integrate biobanking and ARTs into captive breeding programs using modelled captive populations of two Australian threatened frogs, namely the orange-bellied frog Geocrinia vitellina and the white bellied frog Geocrinia alba . Back-crossing with frozen founder spermatozoa using ARTs every generation minimises rates of inbreeding and provides considerable reductions in colony size and program costs compared with conventional captive management. Biobanking could allow captive institutions to meet or exceed longstanding genetic retention targets (90% of source population heterozygosity over 100 years). We provide a broad policy direction that could make biobanking technology a practical reality across Australia's ex situ management of amphibians in current and future holdings. Incorporating biobanking technology widely across this network could deliver outcomes by maintaining high levels of source population genetic diversity and freeing economic resources to develop ex situ programs for a greater number of threatened amphibian species.

Evaluating amphibian biobanking and reproduction for captive breeding programs according to the Amphibian Conservation Action Plan objectives.

The Amphibian Conservation Action Plan (ACAP), published in 2007, is a formal document of international significance that proposed eleven relevant actions for global amphibian conservation. Action seven of the ACAP document addresses the use of amphibian captive programs as a conservation tool. Appendix material under this action explores the potential use of Genome Resource Banking (biobanking) as an urgently needed tool for these captive programs. ACAP proposed twelve objectives for Genome Resource Banking which exhibit little emphasis on reproduction as a vital underlying science for amphibian Captive Breeding Programs (CBP's). Here we have reassessed the original twelve ACAP objectives for amphibian reproduction and biobanking for CBP's as a contribution to future ACAP review processes. We have reviewed recent advances since the original objectives, as well as highlighted weaknesses and strengths for each of these objectives. We make various scientific, policy and economic recommendations based on the current reality and recent advances in relevant science in order to inform future ACAP towards new global objectives. The number of amphibian CBP'S has escalated in recent years and reproductive success is not always easily accomplished. Increases in applied and fundamental research on the natural history and reproductive biology of these species, followed by the appropriate development and application of artificial reproductive technologies (ART's) and the incorporation of genome resource banks (GRB's), may turn CBP's into a more powerful tool for amphibian conservation.

Reproductive Techniques for Ovarian Monitoring and Control in Amphibians.

Ovarian control and monitoring in amphibians require a multi-faceted approach. There are several applications that can successfully induce reproductive behaviors and the acquisition of gametes and embryos for physiological or molecular research. Amphibians contribute to one-quarter to one-third of vertebrate research, and of interest in this context is their contribution to the scientific community's knowledge of reproductive processes and embryological development. However, most of this knowledge is derived from a small number of species. In recent times, the decimation of amphibians across the globe has required increasing intervention by conservationists. The captive recovery and assurance colonies that continue to emerge in response to the extinction risk make existing research and clinical applications invaluable to the survival and reproduction of amphibians held under human care. The success of any captive population is founded on its health and reproduction and the ability to develop viable offspring that carry forward the most diverse genetic representation of their species. For researchers and veterinarians, the ability to monitor and control ovarian development and health is, therefore, imperative. The focus of this article is to highlight the different assisted reproductive techniques that can be used to monitor and, where appropriate or necessary, control ovarian function in amphibians. Ideally, any reproductive and health issues should be reduced through proper captive husbandry, but, as with any animal, issues of health and reproductive pathologies are inevitable. Non-invasive techniques include behavioral assessments, visual inspection and palpation and morphometric measurements for the calculation of body condition indices and ultrasound. Invasive techniques include hormonal injections, blood sampling, and surgery. Ovarian control can be exercised in a number of ways depending on the application required and species of interest.

Oestrogen blocks the nuclear entry of SOX9 in the developing gonad of a marsupial mammal.

BACKGROUND: Hormones are critical for early gonadal development in nonmammalian vertebrates, and oestrogen is required for normal ovarian development. In contrast, mammals determine sex by the presence or absence of the SRY gene, and hormones are not thought to play a role in early gonadal development. Despite an XY sex-determining system in marsupial mammals, exposure to oestrogen can override SRY and induce ovarian development of XY gonads if administered early enough. Here we assess the effect of exogenous oestrogen on the molecular pathways of mammalian gonadal development. RESULTS: We examined the expression of key testicular (SRY, SOX9, AMH and FGF9) and ovarian (WNT4, RSPO1, FOXL2 and FST) markers during gonadal development in the marsupial tammar wallaby (Macropus eugenii) and used these data to determine the effect of oestrogen exposure on gonadal fate. During normal development, we observed male specific upregulation of AMH and SOX9 as in the mouse and human testis, but this upregulation was initiated before the peak in SRY expression and 4 days before testicular cord formation. Similarly, key genes for ovarian development in mouse and human were also upregulated during ovarian differentiation in the tammar. In particular, there was early sexually dimorphic expression of FOXL2 and WNT4, suggesting that these genes are key regulators of ovarian development in all therian mammals. We next examined the effect of exogenous oestrogen on the development of the mammalian XY gonad. Despite the presence of SRY, exogenous oestrogen blocked the key male transcription factor SOX9 from entering the nuclei of male somatic cells, preventing activation of the testicular pathway and permitting upregulation of key female genes, resulting in ovarian development of the XY gonad. CONCLUSIONS: We have uncovered a mechanism by which oestrogen can regulate gonadal development through the nucleocytoplasmic shuttling of SOX9. This may represent an underlying ancestral mechanism by which oestrogen promotes ovarian development in the gonads of nonmammalian vertebrates. Furthermore, oestrogen may retain this function in adult female mammals to maintain granulosa cell fate in the differentiated ovary by suppressing nuclear translocation of the SOX9 protein. See commentary: http://www.biomedcentral.com/1741-7007/8/110.

Ontogeny of the oestrogen receptors ESR1 and ESR2 during gonadal development in the tammar wallaby, Macropus eugenii.

Oestrogen has wide ranging effects in development mediated mainly via the two oestrogen receptors, alpha (ESR1, also known as ERalpha) and beta (ESR2, also known as ERbeta). Oestrogen is the key factor that directs the indifferent gonad to become an ovary in many non-mammalian vertebrates. Oestrogen is not required for early ovarian differentiation in mammals but can disrupt normal testicular development in eutherians. Surprisingly, exogenous oestrogen can cause sex reversal of an XY gonad in two marsupials, the North American opossum and the tammar wallaby. To understand the mechanism by which oestrogen induces sex reversal, we characterised the genes for ESR1 and ESR2 and examined their expression during gonadal differentiation in the tammar wallaby, Macropus eugenii. Both receptors were expressed in the somatic cells and germ cells of the indifferent gonad in both XX and XY foetuses throughout all stages of development, and persisted in these cells into adulthood. ERs were also present in many other tissues including kidney, pituitary and mammary gland. ER mRNA was not significantly altered by exogenous oestrogen in cultured XY gonads but the receptors translocated to the nucleus in its presence. These findings confirm that there is conserved expression of the ERs in the indifferent gonad despite the lack of available ligand during early gonadal development. The receptors can respond to exogenous estrogen at this early stage and are capable of transducing signals in the early mammalian gonad. However, the selective forces that maintained conserved ER expression in this tissue remain unknown.