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Ultrasound images of Mountain yellow-legged frog show follicular development during three months of brumation. Females were ultrasounded once a month during the 3 months brumation period. In each month, different stages of development were detected. In the first month females could have, a) virtually no echogenic dots (yellow arrows), b) sparsely distributed echogenic dots at low densities and c) high-density echogenic dots and rounded, yoked medium to large follicles. In the second (def) and third (hej) months the density and distribution of echogenic dots and medium to large follicles also varied depending on the female. Ultrasound images were taken with a GE logiq Book XP and 8C-RS probe 4e10 MHz GE Medical Systems, USA). Sonigel (water soluble, salt-free) was used to enhance images during ultrasound (Mettler Electronics Corp CA, USA). (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)
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Amphibian evolution spans over 350 million years ago, consequently this taxonomic group displays a wide, complex array of physiological adaptations and their diverse modes of reproduction are a prime example. Reproduction can be affected by taxonomy, geographic and altitudinal distribution, and environmental factors. With some exceptions, amphibian...
Contexts in source publication
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... visualization of ovaries and/or eggs has not been extensively reported in amphibian literature [82,87,88]. However, our lab has used it to successfully confirm the presence of mature eggs and monitor possible egg retention in the Mountain yellow-legged frog (N. Calatayud, unpublished) and in the Alabama waterdog, Necturus alabamensis, Gulf Coast waterdog, Necturus beyeri and common mudpuppy, Necturus maculosus (M. Stoops personal observation). Ultrasound can provide a better under- standing of egg development and assist in determining the stage of the ovarian cycle (Figs. 2e5). In cases when eggs have been detected but only a few or no eggs are deposited, ultrasound also provides a useful tool for monitoring egg retention (Fig. ...
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... emergence from brumation, captive female Mountain yellow- legged frogs with ovaries classified as grade 3 (Fig. 2BeC) are ready for mating and will oviposit in 6e8 days (Fig. 3E). Grade 2 females (Fig. 2DeF), may require a longer time in amplexus than grade 3 fe- males before ovipositing (1e4 weeks; Calatayud personal observa- tion). Grade 2 females can also skip one or two breeding seasons and will not oviposit unless amplexed. The degree of receptivity of Grade 2 females at the time of emergence from brumation is individually variable. Grade 4 females are those that have produced mature eggs but fail to oviposit a complete egg mass. Egg retention is most often observed in females that are inconsistently amplexed or not amplexed at all. To avoid egg retention, females should always be examined to make ensure that all eggs have been oviposited by the end of amplexus (Fig. 5AeF). Some Necturus females can retain eggs for an entire year, depositing old eggs the following breeding season along with new eggs. Other females show what appears to be egg calcifi- cation (green arrows; Fig. 5CeF) and breakdown/reabsorption of amorphic structures along with fluid retention (white arrows; Fig. 5CeF) (M. Stoops personal ...
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... emergence from brumation, captive female Mountain yellow- legged frogs with ovaries classified as grade 3 (Fig. 2BeC) are ready for mating and will oviposit in 6e8 days (Fig. 3E). Grade 2 females (Fig. 2DeF), may require a longer time in amplexus than grade 3 fe- males before ovipositing (1e4 weeks; Calatayud personal observa- tion). Grade 2 females can also skip one or two breeding seasons and will not oviposit unless amplexed. The degree of receptivity of Grade 2 females at the time of emergence from brumation is individually variable. Grade 4 females are those that have produced mature eggs but fail to oviposit a complete egg mass. Egg retention is most often observed in females that are inconsistently amplexed or not amplexed at all. To avoid egg retention, females should always be examined to make ensure that all eggs have been oviposited by the end of amplexus (Fig. 5AeF). Some Necturus females can retain eggs for an entire year, depositing old eggs the following breeding season along with new eggs. Other females show what appears to be egg calcifi- cation (green arrows; Fig. 5CeF) and breakdown/reabsorption of amorphic structures along with fluid retention (white arrows; Fig. 5CeF) (M. Stoops personal ...
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... some females, a densely-packed ovary can persist in this stage even if oviposition does not occur. Females that skip one or more consecutive breeding seasons have ovaries resembling those shown in Fig. 3AeB. In females either emerging from or preparing to enter brumation, the ovary can have a variety of appearances including those shown in Fig. 2 AeJ. The stage of ovarian development at which females are observed entering brumation appears to be associated with age and individual (N. Calatayud personal obser- vation). Older, larger females often show more advanced staged of follicle growth than younger females although this has not been experimentally verified. In the first month of brumation, females may have different degrees of development including, early-gravid (Fig. 2AeB) or mid-gravid (Fig. ...
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... some females, a densely-packed ovary can persist in this stage even if oviposition does not occur. Females that skip one or more consecutive breeding seasons have ovaries resembling those shown in Fig. 3AeB. In females either emerging from or preparing to enter brumation, the ovary can have a variety of appearances including those shown in Fig. 2 AeJ. The stage of ovarian development at which females are observed entering brumation appears to be associated with age and individual (N. Calatayud personal obser- vation). Older, larger females often show more advanced staged of follicle growth than younger females although this has not been experimentally verified. In the first month of brumation, females may have different degrees of development including, early-gravid (Fig. 2AeB) or mid-gravid (Fig. ...
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... some females, a densely-packed ovary can persist in this stage even if oviposition does not occur. Females that skip one or more consecutive breeding seasons have ovaries resembling those shown in Fig. 3AeB. In females either emerging from or preparing to enter brumation, the ovary can have a variety of appearances including those shown in Fig. 2 AeJ. The stage of ovarian development at which females are observed entering brumation appears to be associated with age and individual (N. Calatayud personal obser- vation). Older, larger females often show more advanced staged of follicle growth than younger females although this has not been experimentally verified. In the first month of brumation, females may have different degrees of development including, early-gravid (Fig. 2AeB) or mid-gravid (Fig. ...
Citations
... Acquisition of females outside the breeding season or immediately following oviposition will facilitate collection of longitudinal data on the effects of each experimental treatment on female reproductive state through the event of next oviposition. While the occurrence of, and time to, oviposition are indices of female reproductive behavior, biomarkers of female reproductive physiology can be measured regularly through ovarian monitoring (Calatayud et al., 2018) and hormone assays. ...
The annual onset of breeding activity by animals in seasonal environments is often accompanied by auditory signals such as stridulations in insects and vocalizations in birds, mammals, and anurans. In ectotherms, the seasonal timing of such activity by males signals to females the general favorability of environmental conditions for reproduction. Complementary to this, the acoustic characteristics of male auditory signals are presumed to indicate, primarily, the status or quality of males as mates or territorial competitors. Here, using male anurans as a case study, we present the novel hypothesis that characteristics of auditory signals that are modulated by temperature may also serve as bioclimatic indicators of the suitability of proximal abiotic conditions for reproduction by females. According to this hypothesis, thermal constraints on characteristics such as call rate and duration that are insuperable by males may imbue their calls with features that facilitate tracking of environmental conditions by females independently of information intended for communication by males. We integrate findings from empirical studies spanning multiple fields of ecology and evolution to demonstrate how temporal variation in call properties may directly influence female reproductive behavior and physiology. Finally, we outline how this proposed mechanism may enable environmental tracking, provide guidelines for future research to experimentally test this hypothesis, and discuss how findings from this research can translate into actionable conservation management for species of concern.
... While follicle maturation is precisely described in anurans (Necturus spp.), we were unable to discern the exact stage of follicular maturation in our females using our ultrasound frequencies. 29 Due to the substantial size of the ovaries, it was not always feasible to laterally differentiate the follicles, although this distinction is possible in some anuran species. 55 . ...
... 60 In comparison to other amphibians, the oviducts in axolotls were notably developed. 25,26,29 They exhibited a hyperechoic wall with inner hypoechoic tissue. The absence of distinct layer structure and peristaltic activity aided in distinguishing the genital tract from the digestive tract. ...
Axolotls (Ambystoma mexicanum) are extensively studied for their relevance in human medical research. Despite being critically endangered in the wild, they have gained popularity as household pets. Although they have been kept in captivity for over a century, detailed descriptions of their coelomic organ anatomy remain limited. Also, this species exhibits significant variations compared to other amphibians. Ultrasound is a non-invasive and painless medical imaging technique, ideally suited for investigating internal organs or structures. This study focused on describing the ultrasound appearance of the axolotl coelomic cavity. It details the identification, localization and parenchymal description of major organs in 28 neotenic axolotls using ultrasound frequencies ranging from 7 to 15 MHz. The accuracy of the results was validated by comparing ultrasound findings with necropsy results from one male and one female axolotl. The heart, lung surface, liver and reproductive tracts were visualized. Measurements, along with confidence intervals, were calculated for the spleen, kidneys, testicles, gastric wall, gallbladder, and pylorus. Occasional detection of hyperechoic millimetric particles in the gallbladder or ascites was noted. However, visualization of the pancreas and bladder was not possible. This research outcomes involve the development of a comprehensive atlas comprising images obtained throughout the study. Additionally, the experiment established a reproducible and readily accessible protocol for conducting anatomy-morphological assessments in axolotl medicine. This protocol stands as a crucial preliminary stage before advancing to lesion identification.
... 25,26 By taking ethical considerations into account, ultrasounds allows organs exploration and diseases monitoring without pain in amphibians. 25,[27][28][29] This ability is vital for both wild populations and pet axolotls, as early detection of illnesses can facilitate timely treatment. Moreover, ultrasonography can assist in studying reproductive organs and processes, aiding both conservation efforts for wild populations and breeding programs for pet amphibians. ...
... Although follicles maturation is precisely described in anurans (Necturus spp.), we were unable to determine the precise stage of follicular maturation in our females with our ultrasound frequencies. 29 Due to the large size of the ovaries, it was not always possible to lateralize the follicles, whereas it can be differentiated in some anuran species. 50 Close to ovaries, convoluted and coarser oviducts appeared slightly hyperechoic compared to the liver. ...
... 55 In comparison to other amphibians, they were strongly developed in axolotls. 25,26,29 They presented hyperechoic wall with inner hypoechoic tissue. The absence of both layer structure and peristaltic activity was helpful in distinguishing the genital tract from the digestive tract. ...
Axolotls (Ambystoma mexicanum) are widely studied for human medical research. While critically endangered in wildlife, they have also become popular as household pets. Although they have been kept in captivity for over a century, coelomic organs description of their anatomy remains limited, and this species presents significant variations compared to other amphibians. Ultrasound is a non-invasive, painless medical imaging technique, well-suited for investigating internal organs or other structures. This study describes ultrasound appearance of the axolotl coelomic cavity. Identification, localization and parenchyma description of major organs are reported on 28 neotenic axolotls by using frequencies between 7 to 15 MHz. Results were confirmed by comparing ultrasound and necropsy on one male and one female. The heart, lung surface, liver and reproductive tracts were observed. Measurements with confidence intervals were calculated for spleen, kidneys, testicles, gastric wall, gallbladder, and pylorus. Hyperechoic millimetric particles in gallbladder or ascites were occasionally detected. Pancreas and bladder could not be observed. The outcomes of this research include the production of images combined in an atlas. The experiment showed a repeatable and easily accessible protocol for anatomy-morphological assessment in axolotl medicine and is the necessary step before identification of lesions.
... Anura (frogs and toads), Caudata (salamanders), and Gymnophiona (caecilians) are three orders of Amphibia, and their reproduction can be categorized into seasonal and continuous breeders [36]. Amphibians have very different reproductive strategies based on the temperature and environment. ...
Ovarian aging leads to menopause, loss of fertility and other disorders in multiple organs, which brings great distress to women. For ethical reasons, it is impossible to use humans as direct study subjects for aging research. Therefore, biomedical researchers have employed different non-human organisms to study ovarian aging, including worms, fruit flies, fishes, amphibians, birds, mice, rats, cavies, rabbits, pigs, sheep, cows, horses, monkeys, and apes. Because each of these model organisms has its own features, multiple factors, such as size, anatomical structure, cost, ease of operation, fertility, generation time, lifespan, and gene heredity, should be carefully considered when selecting a model system to study ovarian aging. An appropriate model organism would help researchers explore the risk factors and elucidate molecular mechanisms underlying declined ovarian functions, which might be conducive to preventing or delaying the ovarian aging process. This article will offer an overview on several currently available and commonly used model organisms for ovarian aging research by comparing their pros and cons. In doing so, we hope to provide useful information for ovarian aging researchers.
... Determining the reproductive readiness of female anurans has most commonly been assessed visually, by palpation and transillumination [43]. More recently, preovulatory status (gravidity), in several species has also been identified by ultrasound [40e42] and hormone monitoring [33,43e47]. ...
... More recently, preovulatory status (gravidity), in several species has also been identified by ultrasound [40e42] and hormone monitoring [33,43e47]. Staging ovarian development has been previously described in R. muscosa and broadly categorized into three stages [43]. Females that are categorized as Grade 1 show the presence of echogenic dots that are assumed to correspond to a developing ovary containing pre-vitellogenic follicles, while females categorized as Grades 2 and 3 have more prominent looking oocytes suggestive of vitellogenic development (Fig. 3). ...
The establishment and management of ex situ breeding and assurance populations around the globe are meant to provide short-term solutions to the formidable loss of amphibian diversity presently occurring. Large multi-scaled facilities, such as zoos and aquariums, can provide the infrastructure to safeguard species and populations. However, often even large, economically viable facilities lack the knowledge to efficiently cater to the plethora of environmentally controlled physiological strategies that amphibians possess. Anurans present a class of amphibians that have often been viewed as easy to maintain ex situ. However, while adult survival may be relatively successful it is rarely accompanied by good reproductive output, health, and fitness. Even more conspicuous is the low survivorship of offspring produced ex situ once they are translocated back into the wild. The mountain yellow-legged frog (R. muscosa) ex situ breeding program EBP is a prime example of the challenges that amphibians EBPs face. Although more research is needed, the R. muscosa program has increased reproductive output and health of its colony by incorporating reproductive technologies and strategic genetic management in conjunction with a greater understanding of the species' natural history, to produce and translocate viable animals each year. This paper highlights the EBPs past decade of research featuring the program's contribution to building empirical, multidisciplinary approaches that boost the robustness of an endangered species, by safeguarding existing genetic diversity and maximizing fitness and survival outcomes.
... In juvenile and 1-year old common toad (Bufo bufo), common frog, (Rana temporaria) (Tattersall and Ultsch 2008), and European green toads (Bufo viridis) (Jorgensen 1986) brumation is important for growth and fat deposition. Furthermore, some high-altitude species experience growth during brumation, indicating that growth is not exclusively driven by nutrition but that other internal processes such as circadian rhythms are at play (Calatayud et al. 2018). Despite growing evidence for the importance of brumation in amphibians, it is often omitted from husbandry practices due to lingering concerns that brumation may be associated with increased mortality (Carey et al. 2005). ...
At high altitudes, amphibians brumate (over winter) during the winter months, an adaptation that provides protection from harsh weather and minimizes metabolic demand when food resources are scarce. However, brumation in ex situ populations is often avoided due to concerns regarding slow growth rates, compromised immunity, and increased morbidity, and to accelerate growth and sexual maturation. Running counter to these ideas is the hypothesis that husbandry that mimics the environmental conditions under which a species evolved may benefit animal health and reproduction. This may be particularly critical for animals slated for release into the wild. Here, we evaluated the effects of brumation on juvenile southern mountain yellow-legged frogs (Rana muscosa) in a conservation breeding and release program. Growth measurements, (weight and snout-urostyle length (SUL)), were examined in three experimental groups: Non-brumated, 1 or 3-month brumation. Post-release survival was also analyzed and compared between non-brumated and 3-month brumated frogs. This study indicates that brumated R. muscosa juveniles grow to sizes and weights similar to controls within 3 to 4 months following brumation. Mark-recapture models suggested that short-term post-release survival was not lower and in fact, may be higher in brumated compared to non-brumated frogs. Results of this study indicate that although brumation entails short-term costs to growth, this species possesses compensatory growth mechanisms following brumation which allow them to attain similar body size to non-brumated conspecifics in time for the next winter and that for frogs destined for translocation to the wild, brumation could improve survival outcomes.
... 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). ...
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.
... 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). ...
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.
... 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). ...
Simple Summary: Amphibians are of critical importance among vertebrates. They play a critical role in their ecosystems, are commonly used as environmental health indicators and are also considered as exotic pets throughout the world. Among amphibians, many anuran species are included in active conservation programs as they are listed as endangered species. Thus, it is important for veterinarians and biologists to examine their sanitary status and to find a non-invasive tool to evaluate the health status of these individuals, particularly the state of their reproductive system and to be able to carry out a sex determination in case of no sexual dimorphism. For the first time, we demonstrate that benchtop magnetic resonance imaging and high-resolution ultrasound are suitable non-invasive imaging techniques for an accurate sex determination of two anuran species. Both techniques allowed the identification of ovaries and testes. Therefore, our data constitute an important contribution for clinical diagnostic and conservation purposes in amphibians, as it is possible to distinguish males and females in a quick, safe and relatively easy way. Abstract: 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.
... The development of hormonal treatments for the collection of eggs has been more complicated than those of sperm collection since they usually require the administration of priming doses, possible due to the more complex hormonal cycles present in females [49,76,82,83]. After the selection of the hormones and administration method, the assessment of response time with and without priming doses should be explored, considering combinations of different hormones and concentrations for priming and ovulation doses. ...
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.
