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Establishing captive breeding populations of amphibians is an important conservation strategy to safeguard against ongoing declines of wild populations and provide broodstock for reintroduction programs. The endangered dusky gopher frog (DGF) has never naturally reproduced in captivity and requires breeding intervention to sustain the population. M...
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... consistently describe the ovarian state across imaging, an ultrasound grading scale was developed, which categor- ized the imaged follicles and eggs on a scale from 1 to 5 based on the hyperechoic and hypoechoic (light and dark) patterns of the ultrasound image. A female of Ultrasound Grade 1 was considered to have low follicular development, while a female of Grade 5 had well-developed eggs ready for expres- sion. Images and descriptions for each grade are shown in Fig. 1. The patterns seen in Grades 1-4 ovaries were repre- sentative of follicle/oocyte complexes and are referred to as 'oocytes'. The differences in the Grade 5 images were shown to be the result of ovulation of mature eggs; thus, the gametes in these images are referred to as 'eggs'. The term 'egg' is also used for gametes that were oviposited by or manually expressed from a ...
Context 2
... consistently describe the ovarian state across imaging, an ultrasound grading scale was developed, which categor- ized the imaged follicles and eggs on a scale from 1 to 5 based on the hyperechoic and hypoechoic (light and dark) patterns of the ultrasound image. A female of Ultrasound Grade 1 was considered to have low follicular development, while a female of Grade 5 had well-developed eggs ready for expres- sion. Images and descriptions for each grade are shown in Fig. 1. The patterns seen in Grades 1-4 ovaries were repre- sentative of follicle/oocyte complexes and are referred to as 'oocytes'. The differences in the Grade 5 images were shown to be the result of ovulation of mature eggs; thus, the gametes in these images are referred to as 'eggs'. The term 'egg' is also used for gametes that were oviposited by or manually expressed from a ...
Similar publications
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 th...
Citations
... Refrigerated storage of anuran oocytes in the oviducts of live females [218] Reproductively mature anurans from cryopreserved testicular sperm [124,125] Ultrasound optimisation of anuran hormonal stimulation [219] 2019 ...
Intergenerational justice entitles the maximum retention of Earth’s biodiversity. The 2022 United Nations COP 15, “Ecological Civilisation: Building a Shared Future for All Life on Earth”, is committed to protecting 30% of Earth’s terrestrial environments and, through COP 28, to mitigate the effects of the climate catastrophe on the biosphere. We focused this review on three core themes: the need and potential of reproduction biotechnologies, biobanks, and conservation breeding programs (RBCs) to satisfy sustainability goals; the technical state and current application of RBCs; and how to achieve the future potentials of RBCs in a rapidly evolving environmental and cultural landscape. RBCs include the hormonal stimulation of reproduction, the collection and storage of sperm and oocytes, and artificial fertilisation. Emerging technologies promise the perpetuation of species solely from biobanked biomaterials stored for perpetuity. Despite significant global declines and extinctions of amphibians, and predictions of a disastrous future for most biodiversity, practical support for amphibian RBCs remains limited mainly to a few limited projects in wealthy Western countries. We discuss the potential of amphibian RBCs to perpetuate amphibian diversity and prevent extinctions within multipolar geopolitical, cultural, and economic frameworks. We argue that a democratic, globally inclusive organisation is needed to focus RBCs on regions with the highest amphibian diversity. Prioritisation should include regional and international collaborations, community engagement, and support for RBC facilities ranging from zoos and other institutions to those of private carers. We tabulate a standard terminology for field programs associated with RBCs for publication and media consistency.
... Refrigerated storage of anuran oocytes in the oviducts of live females [218] Reproductively mature anurans from cryopreserved testicular sperm [124,125] Ultrasound optimisation of anuran hormonal stimulation [219] 2019 ...
Intergenerational justice entitles the maximum retention of Earth’s biodiversity. The
2022 United Nations COP 15, “Ecological Civilisation: Building a Shared Future for All Life on Earth”, is committed to protecting 30% of Earth’s terrestrial environments and, through COP 28, to mitigate the effects of the climate catastrophe on the biosphere. We focused this review on three core themes: the need and potential of reproduction biotechnologies, biobanks, and conservation breeding programs (RBCs) to satisfy sustainability goals; the technical state and current application of RBCs; and how to achieve the future potentials of RBCs in a rapidly evolving environmental and cultural landscape. RBCs include the hormonal stimulation of reproduction, the collection and storage of sperm and oocytes, and artificial fertilisation. Emerging technologies promise the perpetuation of species solely from biobanked biomaterials stored for perpetuity. Despite significant global declines
and extinctions of amphibians, and predictions of a disastrous future for most biodiversity, practical support for amphibian RBCs remains limited mainly to a few limited projects in wealthy Western countries. We discuss the potential of amphibian RBCs to perpetuate amphibian diversity and prevent extinctions within multipolar geopolitical, cultural, and economic frameworks. We argue
that a democratic, globally inclusive organisation is needed to focus RBCs on regions with the highest amphibian diversity. Prioritisation should include regional and international collaborations, community engagement, and support for RBC facilities ranging from zoos and other institutions to those of private carers. We tabulate a standard terminology for field programs associated with RBCs
for publication and media consistency.
... Also, they have studied how environmental factors such as photoperiod, temperature, and altitude influence seasonality, affecting reproductive or hibernation processes [17,18] (Figure 2a). In recent years, hormonal therapies for inducing ovulation and in vitro fertilization have been developed in indoor breeding facilities beyond their natural habitats for conservation purposes [19,20]. The cryopreservation of amphibian sperm and eggs has also been actively pursued in conservation efforts [21,22]. ...
... Researchers have investigated endocrine compounds related to life history and reproduction, such as neuropeptide gonadotropin-releasing hormone (GnRH), luteinizing hormone (LH), and sex steroids, to understand the factors and processes influencing reproductive success and contribute to amphibian conservation [23,24]. In recent years, hormonal therapies for inducing ovulation and in vitro fertilization have been developed in indoor breeding facilities beyond their natural habitats for conservation purposes [19,20]. The cryopreservation of amphibian sperm and eggs has also been actively pursued in conservation efforts [21,22]. ...
Simple Summary
Over the years, conservation physiologists have attempted to evaluate the threat factors affecting populations and manage ecosystems effectively by understanding their physiological responses. Amphibians are currently considered to be one of the most endangered vertebrate groups, and many researchers are contributing to their conservation efforts through physiological analysis. Our review aimed to examine current research trends in the conservation physiology of amphibians and identify areas for potential future studies. We categorized the 796 studies into 10 research topics. In each topic, strategies to achieve the goals of conservation physiology were identified. Additionally, we propose four comprehensive directions for future research to advance amphibian conservation physiology.
Abstract
Analysis of physiological responses can be used to assess population health, identify threat factors, and understand mechanisms of stress. In addition to this, conservation physiologists have sought to establish potential management strategies for environmental change and evaluate the effectiveness of conservation efforts. From past to present, the field of conservation physiology is developing in an increasingly broader context. In this review, we aim to categorize the topics covered in conservation physiology research on amphibians and present the measured physiological parameters to provide directions for future research on conservation physiology. Physiological responses of amphibians to environmental stressors are the most studied topic, but conservation physiological studies on metamorphosis, habitat loss and fragmentation, climate change, and conservation methods are relatively lacking. A number of physiological indices have been extracted to study amphibian conservation physiology, and the indices have varying strengths of correlation with each subject. Future research directions are suggested to develop a comprehensive monitoring method for amphibians, identify interactions among various stressors, establish physiological mechanisms for environmental factors, and quantify the effects of conservation activities on amphibian physiology.
... In addition, a hormone therapy strategy is temporally explicit when the timing between the spatial arrangements is important in order to allow appropriate maturation of the ova prior to treating the salamander with the ovulation dose. For example, Graham et al. [33] demonstrated that ultrasound analysis could be used as a decision tool to determine the optimal exogenous hormone regimen in the Mississippi gopher frog (Lithobates sevosa) or whether animals should even be treated with hormones. Such non-invasive decision tools have not been developed for caudates and would shed light on ovarian cyclicity in Ambystomid salamanders, relative to oogenesis. ...
Conservation breeding programs (CBPs) for threatened salamander species depend on reliable reproduction. We followed ovarian maturation and oviposition in eastern tiger salamander (Ambystoma tigrinum) females treated with exogenous hormone regimens developed within a framework of grading their initial follicular stages based on oocyte size and echogenicity (G0 = little/no development; G1 = minor development; G2 = moderate development; G3 = extensive development). The exogenous hormones used in this study included human chorionic gonadotropin (hCG) and gonadotropin releasing hormone (GnRH), where lower concentrations of hCG were used as a priming hormone compared to the ovulatory dose, which included both hCG and GnRH. The three hormone treatment regimens consisted of: (1) an extended prime plus ovulatory dose; (2) a short prime plus an ovulatory dose; and (3) an ovulatory dose only. The three exogenous hormone regimens were trialed for optimal spawning success based on ovarian ultrasound grades 1–3, in which higher doses of priming hormones were administered to salamanders with less developed follicles to stimulate oogenesis, prior to a final ovulatory dose. The three exogenous hormone regimens and three ovarian grades were used in a 3 × 3 factorial design, with n = 6 females for each factorial combination, and efficacy assessed by ovarian grade and/or exogenous hormone regimen on proportion of animals laying eggs, egg numbers, and latency to egg deposition. The salamanders in the most advanced initial stage of follicular development (G3) were most likely to ovulate and lay eggs and those with low follicular development (G1) the least likely. Initial follicular stages also affected the egg numbers, with G3 females laying the most eggs and G1 laying the fewest. Moreover, we found a significant interaction effect of exogenous hormone treatment and ovarian grade, in which G1 females required the greatest numbers and levels of exogenous hormone doses to induce oviposition and G3 the least. These findings show that ultrasound can be used with Ambystomid species to develop optimal spatial and temporal hormone regimens for ovulation and oviposition, depending on follicular maturation, thereby enhancing the efficiency of CBPs, improving animal welfare, and advancing genetic diversity management.
... Ultrasonography was used to select females for IVF according to follicle maturation and development as previously described for Lithobates sevosa (Graham et al., 2018). All ultrasound exams were graded on a 0-3 scale, with 0 being no egg development and 3 being an even level of development across the entire ovary. ...
Sperm cryopreservation is a vital tool in amphibian assisted reproductive technologies that aids in genetic and population management, specifically for at-risk species. Significant advancements have been made in the cryopreservation of amphibian sperm, yet there is little information on how the cryopreservation process influences fertilization and embryonic development. In this study, we tested several cryoprotective agents (CPAs) and freezing rates on sperm recovery, fertilization potential and embryo development using Fowler's toads (Anaxyrus fowleri) as a model amphibian species for application to at-risk anurans. Three cryoprotectant treatments were tested, which included 10% trehalose + 0.25% bovine serum albumin with (1) 5% N,N-dimethylformamide (DMFA); (2) 10% DMFA; or (3) 10% dimethyl sulfoxide (DMSO). Additionally, sperm in each cryoprotectant was frozen at two different rates, -32 to -45°C/min and -20 to -29°C/min. Post-thaw sperm analysis included motility, morphology, viability, fertilization success and embryo development. Results show that 10% DMFA produced significantly higher (P = 0.005) post-thaw sperm motility than 5% DMFA and was similar to 10% DMSO. Furthermore, sperm frozen at -32 to -45°C/min had significantly higher post-thaw motility (P < 0.001) compared to sperm frozen at -20 to -29°C/min. We also found that embryos fertilized with sperm frozen with 5% DMFA resulted in significantly higher (P = 0.02) cleavage than 10% DMSO, yet there was no other effect of CPA on fertilization or embryo development. Furthermore, embryos fertilized with sperm frozen at -32 to -45°C/min resulted in significantly higher cleavage (P = 0.001), neurulation (P = 0.001) and hatching (P = 0.002) numbers than sperm frozen at a rate of -20 to -29°C/min. Overall, eggs fertilized with frozen-thawed sperm produced 1327 tadpoles. These results provide insight towards a biobanking strategy that can be applied to imperilled species to preserve genetic lineages and bolster offspring genetic diversity for reintroduction.
... Females were assessed by ultrasound immediately after the NIR spectral data were collected, and ultrasound grades were also incorporated post hoc in the PCA scores plot to evaluate females of varying reproductive states with consideration of male scores. Ultrasound data were collected for each female using a 0-5 grading scale adapted from Graham et al. [34], where 0 indicated a lack of follicular development and 5 indicated advanced folliculogenesis with large, fully developed eggs. A simplified schematic of the workflow involved in spectral data collection and analysis is outlined in Figure 2. to test for mathematical pre-processing and avoid modeling bias. ...
... Females were assessed by ultrasound immediately after the NIR spectral data were collected, and ultrasound grades were also incorporated post hoc in the PCA scores plot to evaluate females of varying reproductive states with consideration of male scores. Ultrasound data were collected for each female using a 0-5 grading scale adapted from Graham et al. [34], where 0 indicated a lack of follicular development and 5 indicated advanced folliculogenesis with large, fully developed eggs. A simplified schematic of the workflow involved in spectral data collection and analysis is outlined in Figure 2. ...
Biological sex is one of the more critically important physiological parameters needed for managing threatened animal species because it is crucial for informing several of the management decisions surrounding conservation breeding programs. Near-infrared spectroscopy (NIRS) is a non-invasive technology that has been recently applied in the field of wildlife science to evaluate various aspects of animal physiology and may have potential as an in vivo technique for determining biological sex in live amphibian species. This study investigated whether NIRS could be used as a rapid and non-invasive method for discriminating biological sex in the endangered Houston toad (Anaxyrus houstonensis). NIR spectra (N = 396) were collected from live A. houstonensis individuals (N = 132), and distinct spectral patterns between males and females were identified using chemometrics. Linear discriminant analysis (PCA-LDA) classified the spectra from each biological sex with accuracy ≥ 98% in the calibration and internal validation datasets and 94% in the external validation process. Through the use of NIRS, we have determined that unique spectral signatures can be holistically captured in the skin of male and female anurans, bringing to light the possibility of further application of this technique for juveniles and sexually monomorphic species, whose sex designation is important for breeding-related decisions.
... Ovarian development stages for PRCTs are shown in Figure 2. Only females with mature oocytes at an ultrasound grade 3 were administered the 0.4 μg/g BW GnRHa in order to induce ovulation and egg laying (Bronson & Vance, 2019;Graham, Langhorne, Vance, Willard, & Kouba, 2018). ...
Genetic variation of the endangered Puerto Rican crested toad (PRCT; Peltophryne lemur) has dwindled over time in both wild and captive populations, leading to long‐term sustainability issues for the recovery program. To address this challenge, we propose that PRCTs can be used as a model species to show how in‐situ and ex‐situ populations can be linked through sperm biobanking and gamete transfer, expanding genetic variability of both populations. Male toads (n = 10) in Guayanilla, Puerto Rico were administered human chorionic‐gonadotropin (hCG) and luteinizing‐hormone‐releasing‐hormone analog (GnRHa) to stimulate spermiation. Sperm was collected noninvasively from 9/10 males, cryopreserved in 10% trehalose with either 10% N,N‐dimethylformamide (DMFA) or 10% dimethyl sulfoxide (DMSO), and transported in liquid nitrogen vapor to the National Amphibian Genome Resource Bank at Mississippi State University. Ultrasonography was used to identify females (n = 3) with mature oocytes for GnRHa‐induced oviposition of eggs for in‐vitro fertilization (IVF). Post‐thaw sperm motility was 28% and 25% for sperm cryopreserved with DMFA or DMSO, respectively. Of the 9,672 eggs used for IVF, 4% (n = 306/6,981) were fertilized with frozen–thawed sperm, compared with 20% (n = 525/2691) fertilized with fresh sperm controls. Overall, 46 toadlets were produced from frozen–thawed sperm. After 4.5 months of headstarting, 14 juvenile toads produced from various genetic crosses using frozen–thawed sperm were released to new sites in the wild, introducing unique genetic representation and new founder lines. After 1.5 years, 24 adult toads produced using frozen–thawed sperm continue to thrive in the captive collection, and one of these males has now produced an F2 generation of offspring with 5,085 tadpoles released to the wild. This transformational study is the first to produce reproductively viable adult PRCTs using cryopreserved sperm from hormonally‐induced wild males and captive females, introducing innovative methods that link in‐situ and ex‐situ populations of endangered amphibians to revolutionize genetic management. Genetic variation of the endangered Puerto Rican crested toad (PRCT; Peltophryne lemur) has dwindled over time in both wild and captive populations, leading to long‐term sustainability issues for the recovery program. To address this challenge, we show how in‐situ and ex‐situ populations can be linked through sperm biobanking and gamete transfer, expanding genetic variability of both populations. This transformational study led to the production of 46 PRCTs produced from frozen–thawed sperm that were released to the wild or held in the captive colony, introducing new genetic representation and founder lines to both populations. Furthermore, these animals have reached adulthood and are shown to be reproductively viable, producing a second generation of >5,000 offspring for reintroduction.
... It would be prudent to investigate the effect of disease and population of origin on females of varying age to ensure that the effects seen here and in Brannelly et al. (2016c) are consistent with age-dependent responses to pathogen exposure. Such an approach could be adopted in field studies using skeletochronology to determine age (Scheele et al. 2017c;Brannelly et al. 2018), and ultrasound to estimate if females are gravid (Graham et al. 2018;Calatayud et al. 2019). ...
The devastating infectious disease chytridiomycosis has caused declines of amphibians across the globe, yet some populations are persisting and even recovering. One understudied effect of wildlife disease is changes in reproductive effort. Here we aimed to understand if disease has plastic effects on reproduction and if reproductive effort could evolve with disease endemism. We compared the effects of experimental pathogen exposure (trait plasticity) and population‐level disease history (evolution in trait baseline) on reproductive effort using gametogenesis as a proxy in the declining and endangered frog Litoria verreauxii alpina. We found that unexposed males from disease‐endemic populations had higher reproductive effort, which is consistent with an evolutionary response to chytridiomycosis. We also found evidence of trait plasticity, where males and females were affected differently by infection: pathogen exposed males had higher reproductive effort (larger testes), whereas females had reduced reproductive effort (smaller and fewer developed eggs) regardless of the population of origin. Infectious disease can cause plastic changes in reproductive effort at an individual level, and population‐level disease exposure can result in changes to baseline reproductive effort; therefore, individual‐ and population‐level effects of disease should be considered when designing management and conservation programs for threatened and declining species. This article is protected by copyright. All rights reserved
... For other ectothermic species such as reptiles, health concerns related to dystocia can often be circumvented through hormone therapy, such as the use of oxytocin to stimulate oviduct contractions in turtles [18]. Similarly, exogenous hormones have been used to promote oocyte development and spawning in amphibians [19][20][21][22][23][24][25][26][27]. The development of an effective exogenous hormone therapy to stimulate spawning for the PGF population would have several potential benefits including increased reproductive output, less time in amplexus, reduced mortality, and the acquisition of more knowledge about this species' unique reproductive biology in order to optimize care. ...
... For example, GnRHa induces ovulation when administered at 0.4 μg/g body weight (BW) for many amphibian species, but responses to a wide range of GnRHa concentrations (0.1-2 μg/g BW) have also been reported [20,23,27,34,36,37]. Amphiplex, which is a combined mixture of GnRHa plus the dopamine antagonist metoclopramide (MET), has been used to induce spawning in Northern leopard frogs (Lithobates pipiens) [21,38] and dusky gopher frogs (Lithobates sevosa) [24], as well as spermatogenesis in male PGFs [39]. The addition of MET is proposed to limit dopamine inhibition of GnRH, thus enhancing LH/ FSH release and gametogenesis [21,38]. ...
... However, there are amphibian species in which priming or pulses of hormones are required to facilitate the spawning process. The beneficial impacts of primed or pulsed hormones on increased spawning rates have been described for Wyoming toad (Anaxyrus baxteri) [19], Fowler's toad (Anaxyrus fowleri) [25], Günther's toadlet (Pseudophryne guentheri) [22], corroboree frog (Pseudophryne corroboree) [20], dusky gopher frog [24,40], Puerto Rican crested toad (Peltophryne lemur) [37] and Chiricahua leopard frog (Lithobates chiricahuensis) (Kouba per. communication). ...
Background
With Panamanian golden frogs (Atelopus zeteki; PGFs) likely extirpated from the wild, ensuring long-term sustainability of captive populations is crucial in order to conserve this critically endangered species. Unfortunately, PGFs display a unique reproductive behavior involving a prolonged period of amplexus leading to challenges in their successful captive propagation. The Maryland Zoo in Baltimore has observed high levels of mortality during the breeding season and suboptimal reproductive success leading to the use of hormone stimulation to aid in reproduction and health management.
Methods
This project aimed to develop induced ovulation and health management protocols by (1) evaluating different doses of gonadotropin releasing hormone analogue (GnRHa), (2) comparing the efficacy of GnRHa and GnRHa + metoclopramide, (3) determining latency periods and the effects of pulsed hormone sequences; and (4) establish if mortality is impacted by hormone therapy. Female PGFs (n = 174) were given GnRHa either in various concentrations (Experiment 1) or combined with metoclopramide (Experiment 2), and oviposition success, latency, and mortality were measured as binary response variables.
Results
Overall, the use of exogenous hormones significantly decreased mortality when compared to the control data of natural egg-laying females. GnRHa doses of 0.05 μg/g body weight produced similar ovulation rates compared to higher doses, and the addition of metoclopramide did not increase oviposition success compared to GnRHa alone. Lastly, results indicate the majority of female PGFs will release eggs within 48 h following the initial pulse of hormones with a small percentage ovipositing after a second pulse.
Conclusion
Findings from this study will benefit captive management of PGFs by documenting the increased survival of females when given hormone stimulation and defining appropriate GnRHa doses and expected latency to spawning.
... Several articles describe the use of ultrasonography in amphibians for medical purposes [11][12][13], assessment of reproductive status and sex identification in frogs with better specificity for female identification [14][15][16][17]. Magnetic resonance imaging (MRI) has been previously used in frogs focusing on different functional aspects of the Central Nervous System (CNS) [18,19]. ...
... Ultrasonographic visualization of reproductive organs in anurans has been considered difficult, especially in males [13][14][15][16][17]. However, in our study, ultrasound examination allowed an easy identification of testes and ovaries in both anuran species (Figures 5-7). ...
... Yet, the identification of testes required trained ultrasonographers to make a correct interpretation of the images. In our study, we used state-of-the-art HR-US equipment and two transducers with a frequency range up to 17 and 22 MHz, respectively, which provided excellent quality images from the skin to 3-5 cm deep, while the transducers previously used in similar studies had lower frequency ranges [14,16,17,37]. The high technical characteristics of the HR-US equipment and transducers used in this study can explain the discrepancies with previous studies on the feasibility of ultrasonography to visualize testes and ovaries in anurans, as pointed out in studies pertaining to reptiles [38]. ...
The objective of the present study was to evaluate whether gender determination in two
amphibian species (Kaloula pulchra and Xenopus laevis) can be reliably carried out by means of magnetic resonance imaging (benchtop magnetic resonance imaging; BT-MRI) or ultrasound (high-resolution ultrasound; HR-US) techniques. Two species of healthy, sexually mature anurans have been used in the present study. Eight Kaloula (blind study) and six Xenopus were used as controls. Magnetic resonance imaging experiments were carried out on a low-field (1 Tesla) benchtop-MRI (BT-MRI) system. HR-US examination was performed with high-resolution equipment. Low-field BT-MRI images provided a clear and quantifiable identification of all the sexual organs present in both genders and species. The HR-US also allowed the identification of testes and ovaries in both species.
Results indicate that BT-MRI allowed a very precise sex identification in both anuran species, although its use is limited by the cost of the equipment and the need for anesthesia. HR-US allowed an accurate identification of ovaries of both species whereas a precise identification of testes is limited by the ultrasonographer experience. The main advantages of this technique are the possibility of performing it without anesthesia and the higher availability of equipment in veterinary and zoo institutions.
