Search

Ex situ amphibian populations can experience reproductive dysfunction due to the absence of environmental cues that trigger reproductive events. Assisted reproductive technologies (ART) for amphibians, specifically exogenous hormone regimens, can circumvent these external signals to induce gametogenesis and gamete release. Currently, the use of the mammalian reproductive hormones gonadotropin-releasing hormone (GnRH) and human chorionic gonadotropin (hCG) are used in a species-specific manner to stimulate amphibian breeding. Hormones or hormone mixtures that are effective in all breeding scenarios would provide the best option for conservation practitioners and some commercial products are already in use for breeding other ectotherms. Ovaprim®, which contains salmon GnRH analogue (sGnRHa) and the dopamine antagonist domperidone (DOM), is effective in fish aquaculture and may be effective for amphibians. To test this hypothesis, we treated Fowler's toads ( Anaxyrus fowleri ) with either sGnRHa alone, a high or low dose of Ovaprim® or hCG. We then compared spermiation response, sperm quantity and quality parameters, and changes in animal mass over time within each treatment. We found administration of Ovaprim® resulted in more males producing sperm with better motility compared to administration of sGnRHa alone. In addition, the Ovaprim® and sGnRHa treatments resulted in lower response rates, lower sperm motilities, more abnormal sperm, and higher aggregations of sperm compared to the hCG treatment. Furthermore, Ovaprim®-treated males gained significant mass, suggesting an anti-diuretic effect of DOM. Together, these results show that neither Ovaprim® nor sGnRHa, at the concentrations tested, are likely suitable replacements for hCG in ex situ bufonid breeding programmes and that hormone mixtures developed for fish may have limited transferability to new world toad species., Competing Interests: The authors have no conflicts of interest to declare., (© The Author(s) 2024. Published by Oxford University Press and the Society for Experimental Biology.)

More than 50% of caudates are threatened with extinction and are in need of ex-situ breeding programs to support conservation efforts and species recovery. Unfortunately, many salamander populations under human care can experience reproductive failure, primarily due to missing environmental cues necessary for breeding. Assisted reproductive technologies (ARTs) are a useful suite of techniques for overcoming or bypassing these missing environmental cues to promote breeding. Exogenous hormones are used to stimulate natural breeding behaviors or gamete expression for in-vitro fertilization or biobanking and are typically administered intramuscularly in caudates. While effective, intramuscular injection is risky to perform in smaller-bodied animals, resulting in health and welfare risks. This research investigated the spermiation response to hormone administration through a non-invasive oral bioencapsulation route using the tiger salamander (Ambystoma tigrinum) as a model species. Male salamanders were randomly rotated six weeks apart through four treatments (n = 11 males/treatment) in which animals received a resolving dose of gonadotropin-releasing hormone (GnRH) as follows: (1) Prime-Only (0.0 μg/g); (2) Low (0.25 μg/g); (3) Medium (1.0 μg/g); and (4) High (2.0 μg/g). All males were given a GnRH priming dose (0.25 μg/g) 24 hours prior to the resolving dose. Exogenous hormone was delivered inside of a cricket (Gryllodes sigillatus) that was presented as a food item by tweezers. Sperm samples were collected at 1, 3, 6, 9, 12, and 24 hours after the resolving dose and analyzed for quantity and quality. For all treatments, sperm concentration was produced in an episodic pattern over time. The Prime-Only treatment had a lower (p < 0.05) percent of sperm exhibiting normal morphology compared to treatments utilizing a resolving dose of GnRH. Overall, oral administration of GnRH is a feasible route of inducing spermiation in salamanders, yielding sperm of sufficient quantity and quality for in-vitro fertilization and biobanking efforts., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Chen et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Near infrared spectroscopy coupled with predictive modeling is a growing field of study for addressing questions in wildlife science aimed at improving management strategies and conservation outcomes for managed and threatened fauna. To date, the majority of spectroscopic studies in wildlife and fisheries applied chemometrics and predictive modeling with a single-algorithm approach. By contrast, multi-model approaches are used routinely for analyzing spectroscopic datasets across many major industries (e.g., medicine, agriculture) to maximize predictive outcomes for real-world applications. In this study, we conducted a benchmark modeling exercise to compare the performance of several machine learning algorithms in a multi-class problem utilizing a multivariate spectroscopic dataset obtained from live animals. Spectra obtained from live individuals representing eleven amphibian species were classified according to taxonomic designation. Seven modeling techniques were applied to generate prediction models, which varied significantly (p < 0.05) with regard to mean classification accuracy (e.g., support vector machine: 95.8 ± 0.8% vs. K-nearest neighbors: 89.3 ± 1.0%). Through the use of a multi-algorithm approach, candidate algorithms can be identified and applied to more effectively model complex spectroscopic data collected for wildlife sciences. Other key considerations in the predictive modeling workflow that serve to optimize spectroscopic model performance (e.g., variable selection and cross-validation procedures) are also discussed., Competing Interests: The authors declare no competing interests., (Copyright: © 2024 Chen et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Sperm cryopreservation and biobanking are emerging as tools for supporting genetic management of small and threatened populations in amphibian conservation programs. However, there is little to no evidence demonstrating reproductive maturity and viability of offspring generated with cryopreserved sperm, potentially limiting widespread integration of these technologies. The purpose of this report is to demonstrate that amphibian sperm can be cryopreserved and thawed to successfully produce individuals of an F1 generation that can reach adulthood and reproductive maturity, to generating viable gametes and an F2 generation. Species-specific exogenous hormones were administered to both F0 and F1 adults to stimulate spermiation and oviposition in the eastern tiger salamander ( Ambystoma tigrinum ), dusky gopher frog ( Lithobates sevosa ), and Puerto Rican crested toad ( Peltophryne lemur ). Sperm cells collected non-lethally from F0 adults were cryopreserved, thawed, and used for in vitro fertilization (IVF) to produce F1 offspring. Individuals of the F1 generation are shown to reach adulthood, express viable gametes, and produce offspring through facilitated breeding, or IVF. The production of amphibian F2 generations shown here demonstrates that amphibian sperm collected non-lethally can be banked and used to generate reproductively viable animals of subsequent generations, thus maintaining valuable genetic linages and diversity in threatened amphibian species. The incredible value that cryopreservation of sperm has for long-term genetic management aids in the sustainability of both in situ and ex situ conservation efforts for this taxon.

For amphibian species that display external fertilization in an aquatic environment, hypoosmotic shock to sperm cells can quickly result in damage to cellular structure and function. This study sought to determine how fertilization media osmolality, temperature, and time impact the stability of the mitochondrial vesicle's association with the sperm head and thus motility and quality of forward progression. The presence of the mitochondrial vesicle and its relationship with sperm motility and quality of forward progression were analyzed in sperm samples from the Fowler's toad (Anaxyrus fowleri) (n = 10) when held for six hours under two temperatures and four osmolalities. Results indicated that the presence of the mitochondrial vesicle is needed for sperm motility over time (p < 0.001), where higher osmolalities (p < 0.001) and lower temperatures (p < 0.001) correlated with maintaining the vesicle attachment to the spermatozoa. The higher osmolality of spermic urine was the most important factor for maintaining higher quality of forward progressive motility (p < 0.01) of spermatozoa. Sperm samples held at 4 °C and 40 mOsm/kg had the longest half-life for motility (540 min) and quality of forward progression (276 min), whereas sperm held at 22 °C and 2.5 mOsm/kg had the shortest half-life for motility (7 min) and quality of forward progression (18 min). Special attention should be given to the osmolality and temperature of fertilization solutions, or breeding tank water, when developing cold storage protocols for toad sperm or reproducing animals to ensure the retention of the mitochondrial vesicle for maximum fertilization capability., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

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., (© The Author(s) 2022. Published by Oxford University Press and the Society for Experimental Biology.)

Conservation programs for threatened high- elevation amphibian species rely on hibernation to trigger appropriate male reproductive behaviours and gametogenesis. Although common practice and anecdotal observations have supported the practice of hibernation, there is limited empirical evidence documenting the effects on reproduction in these species. In this study, the effect of hibernation on sperm quantity and quality was evaluated for the alpine species Anaxyrus boreas boreas . Hibernated (n =19) and non-hibernated (n =21) male toads were administered 10IUg-1 body weight (BW) human chorionic gonadotropin (hCG) and spermic urine was collected over 24h. Hibernation had no effect on the number of males undergoing spermatogenesis, but hibernated males produced sperm in higher concentrations. Sperm quality was measured in terms of total motility, forward progressive motility and quality of forward progression. Although there was no difference in the total sperm motility of samples from hibernated and non-hibernated toads, the percentage of sperm exhibiting forward progressive motility and the quality of forward progression was significantly greater from hibernated toads. These results support our hypothesis that hibernation impacts both sperm quantity and quality in male boreal toads. This study will better inform captive breeding management decisions for threatened alpine species, in imminent danger of extinction.

Asynchrony of gamete release is problematic in amphibian captive breeding programs but can be overcome by short-term storage of spermatozoa. Hormonally induced sperm from the model species Anaxyrus fowleri were used to determine storage conditions for optimal fertilisation capacity. Sperm motility was measured over time, as a function of storage temperature (4°C or 22°C) and solution osmolality (7-40mOsm/kg). Sperm at 40mOsm/kg (spermic urine) stored at 4°C exhibited higher motility compared to 22°C. Also, sperm stored at 40mOsm/kg retained higher motility compared to sperm stored below 15mOsm/kg at both temperatures. Under optimal storage conditions (40mOsm and 4°C) a 30% decrease in sperm motility occurred within 24h, however, subsequent loss of sperm motility was lower (<10%/day) for days2-8 thereafter. Sperm samples stored for 1-8days under optimal conditions were tested for fertilising capacity by conducting in vitro fertilisation trials. Sperm stored for 8days yielded 48% neurula development, similar to sperm stored for 1day, which produced 60% neurula development. Overall, sperm stored for up to 8days at 4°C as spermic urine retained fertilising capacity and thus can be used to circumvent asynchronous gamete release in assisted breeding efforts for amphibians.

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.

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., (Copyright © 2021 The Authors. Published by Elsevier GmbH.. All rights reserved.)

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., (© 2021. The Author(s).)

The aims of this project were to characterize tiger salamander (Ambystoma tigrinum) spermatozoa motility over time, when excreted as either milt or spermic urine prior to packaging into a spermatophore, and to determine the effect of temperature on sperm motility. A split-plot design was utilized to assess the motility of the two pre-spermatophore sample types at two temperatures, 0°C and 20°C (n = 10 for each treatment). Spermiation was induced through exogenous hormone treatment of luteinizing hormone releasing hormone analog in order to collect both milt and spermic urine, which were evaluated for motility, divided into two separate aliquots, and subsequently stored in either an ice-bath (0°C) or on the benchtop (20°C). The decay rate of sperm motility was assessed by reevaluating subsamples at 0.5, 1, 2, 3, 5, 7, and 24 hours following the initial assessment. Results showed that sperm stored at 0°C had significantly higher progressive, non-progressive, and total motility for both sperm collection types over time. An interaction was found between collection type and time, with milt exhibiting lower initial motility that was more sustainable over time, compared to spermic urine. For both milt and spermic urine, motility decreased rapidly with storage duration, indicating samples should be used as soon as possible to maximize motility for in-vitro fertilization and cryopreservation. This is the first study to describe the differences in sperm motility between milt and spermic urine from an internally fertilizing caudate and demonstrates the benefits of near freezing temperatures on sperm longevity., Competing Interests: The authors have declared that no competing interests exist.

The aims of this project were to transfer hormone-induced spermiation and sperm cryopreservation protocols developed in the model salamander species, Ambystoma tigrinum, to three threatened newt species. Additionally, we tested if supplementation with trehalose or thawing at different temperatures impacts post-thaw sperm parameters. Hormone stimulation protocols were applied to male Notophthalmus meridionalis (N = 10), Neurergus kaiseri (N = 5) and Tylototriton kweichowensis (N = 6) with sperm collected periodically up to 24-28 h post-spermiation dose. Samples of adequate sperm concentration (>70%) were cryopreserved in solutions of 10% Me 2 SO + 1% BSA with or without a 10% trehalose cryodiluent. Frozen sperm samples were thawed at either 20 °C or 40 °C and examined for post-thaw motility parameters and abnormalities in head and tail structure. The spermiation response to exogenous hormone treatment was significantly different between newt species, with a success rate of 0% for N. kaiseri, 67% for T. kweichowensis, and 100% for N. meridionalis. Sperm concentration varied with time of collection after hormone administration in both T. kweichowensis and N. meridionalis. For N. meridionalis, structural abnormalities decreased in samples collected over the 24 h period (p < 0.0001) and a thaw temperature of 40 °C resulted in higher relative total sperm motility (p < 0.0001). This is the first study to describe the cryopreservation of sperm from two newt species and demonstrates the transferability of ART developed in a salamander to two newt species., Competing Interests: Declaration of competing interest None., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Many amphibian species reinitiate the processes of preparing for reproduction (e.g. oogenesis) soon after breeding indicating hormone-induced ovulation could potentially be achieved out-of-season, which would lead to higher annual fecundity compared to mono-seasonal breeding. Such strategies would be beneficial to captive breeding programs for threatened species that are short-lived, have aging populations or need large numbers of offspring to meet reintroduction goals for species recovery. Unfortunately, little is known regarding how female anurans respond to multiple ovulation events within a year, which could lead to higher annual fecundity compared to mono-seasonal breeding. Thus, we evaluated the effect of temporal period between exogenous hormone stimulation events on egg production using the Fowler's toad Anaxyrus fowleri as a model species. Female toads ( n  = 21) were administered hormone therapy twice in 1 year with toads randomly assigned to a treatment of either a 4-, 8- or 12-month recovery period between hormone stimulations. Ovulation was induced using two priming doses of human chorionic gonadotropin (100 IU; hCG) 72 h apart, followed by a resolving dose of hCG (500 IU) plus gonadotropin releasing hormone analogue (GnRHa; 15 μg) given 24 h after the second priming injection. Measured response variables include the number of females ovulating after treatment, total number of eggs produced and percent fertilization, neurula and tadpole development. No significant treatment effects were observed for any response variable ( P  > 0.05). Findings from this study suggest that hormone therapy can be administered in a bufonid species every 4 or 8 months without significantly affecting the number of ovulating females, egg production, fertilization, neurulation or tadpole development. By collecting gametes out-of-season or multiple times throughout the year, captive breeding programs could potentially increase tadpole production for reintroductions as well as extend the breeding window in captivity., (© The Author(s) 2020. Published by Oxford University Press and the Society for Experimental Biology.)

Background: Amphibian diversity is declining at an alarming rate due to habitat loss, invasive species, climate change and diseases. Captive assurance colonies have been established for some species at risk; however, many species do not breed well in captivity and the development of assisted reproductive technologies (ART) is critical to help sustain genetic diversity. To date, the majority of the work has been accomplished in species from the American continent and Australia, and there is a need to address similar breeding challenges in Eurasian and African species of amphibians., Methods: The aim of this study was to develop a hormone protocol for stimulation of spermiation in Epidalea calamita as a model for Eurasian bufonids. Hence, the effect on sperm production and quality of three doses of chorionic gonadotropin hormone (5, 10 and 15 IU hCG/g) over time (1 to 24 h) was evaluated. In addition, cold storage (at 5 °C) of sperm for 24 and 48 h and three frequencies for hormonal treatment (weekly, biweekly and monthly) were examined., Results: Hormone concentrations of 10 or 15 IU of hCG induced spermiation in 100% of males and produced sperm of comparable quality, while 5 IU hCG stimulated spermiation in only 40% of males. Total motility peaked between 1 to 4 h post-treatment with 10 IU hCG, whereas treatment with 15 IU hCG peaked between 2 to 6 h. After 24 h of cold storage total motility dropped by 20% and forward motility dropped by 10% for both the 10 and 15 IU treatments. Weekly hormone administration resulted in higher variation between trials in all motility parameters and a lower overall Total Motility and Forward Movement. Furthermore, the effect of exogenous hormone treatment overlapped between the last two trials in the weekly frequency. Sperm concentration was higher in the first trial for all frequencies but showed no differences among other trials., Conclusions: Overall, these results show that hormone concentration, time after treatment, frequency of hormone treatment and cold storage should be borne in mind when developing a hormone stimulation protocol for Eurasian amphibian species.

Male giant pandas identify female sexual receptivity through the detection of olfactory cues in estrous urine. However, it is yet unknown which specific days of the female estrous cycle may provoke male sexual-social responses and a physiological readiness to mate. We hypothesized that female urine from specific days of the estrous cycle will be positively associated with specific changes in male behaviors, urinary semiochemical production, and steroidogenic activity. Experimental simultaneous choice trials were conducted in captivity with four male giant pandas during the spring breeding season and during fall. Male interest was determined by a behavioral preference toward peri-estrual urine collected from a specific day of the estrous cycle encompassing proestrus (Day -13, Day -6, Day -3, Day -2), estrus (Day -1 and Day 0), and metestrus (Day four and Day nine) over that of anestrous urine. Provocation of male sexual motivation was examined by changes in urinary semiochemical composition and urinary androgen concentrations. During the spring, male investigative behaviors indicated a preference for Day -13, Day -3 and Day 0 urine over anestrous urine, while no significant preferences for estrous urine could be detected during fall. The relative abundance of only three compounds in male urine were significantly higher above baseline values after males were exposed to peri-estrual urine during spring; whereas 34 compounds significantly increased in the fall. Similarly, androgen concentrations increased above baseline in only two out of four males during spring, while all males had elevated androgen concentrations after exposure to Day -3 urine during the fall. Our results suggest that peri-estrual urine from Day -13, Day -3, and Day 0 elicited the greatest duration of male investigation, changes in the semiochemical profile, and elevations in androgen levels. These data suggest that managers should incorporate a combination of behavioral, semiochemical, and endocrinological assessment of males in the reproductive management of giant pandas to determine impending ovulation and pinpoint the best time for male-female introductions and artificial inseminations., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Pregnancy determination is difficult in the giant panda (Ailuropoda melanolecua), representing a challenge for ex situ conservation efforts. Research in other species experiencing pseudopregnancy indicates that urinary/fecal concentrations of 13,14, dihydro-15-keto-prostaglandin F2α (PGFM) can accurately determine pregnancy status. Our objective was to determine if urinary PGFM concentrations are associated with pregnancy status in the giant panda. Urinary PGFM concentrations were measured in female giant pandas (n = 4) throughout gestation (n = 6) and pseudopregnancy (n = 4) using a commercial enzyme immunoassay. Regardless of pregnancy status, PGFM excretion followed a predictable pattern: 1) baseline concentrations for 11-19 weeks following ovulation; 2) a modest, initial peak 14-36 days after the start of the secondary urinary progestagen rise; 3) a subsequent period of relatively low concentrations; and 4) a large, terminal peak at the end of the luteal phase. Pregnant profiles were distinguished by an earlier initial peak (P = 0.024), higher inter-peak concentrations (P < 0.001), and a larger terminal peak (P = 0.003) compared to pseudopregnancy profiles. Parturition occurred 23 to 25 days from the initial PGFM surge and within 24 hours of the start of the terminal increase. These pattern differences indicate that urinary PGFM monitoring can be used to predict pregnancy status and time parturition in the giant panda. Furthermore, this is the only species known to exhibit a significant PGFM increase during pseudopregnancy, suggesting a unique physiological mechanism for regulating the end of the luteal phase in the giant panda.

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. Methods for inducing ovulation in female DGFs using hormone therapies have not been evaluated. To address this need, we tested four exogenous hormone treatments to induce ovulation in DGFs ( n = 11/treatment), including: treatment (A) gonadotropin-releasing hormone agonist (GnRHa); (B) GnRHa with dopamine antagonist metoclopramide hydrochloride; (C) GnRHa and human chorionic gonadotropin (hCG) and (D) GnRHa with hCG following two low hCG priming doses. Treatments B, C and D resulted in a significantly greater ( P < 0.0125) number of ovulating females compared to the control (no hormone); Treatment A was not different from control. For ovulating females, the number of eggs, relative fecundity and cleavage rates of eggs were compared between the four hormone treatments and initial ultrasound grade. Between treatments, there was no difference in number of eggs or relative fecundity; however, Treatments A and D resulted in higher ( P < 0.05) cleavage rates than Treatment C, but were not different from Treatment B. Ultrasound imaging was used to assess the ovarian state of DGF females prior to and following hormone therapy. A grading scale (Grades 1-5) was developed to characterize ovarian states. Ultrasound grade was found to be a significant ( P = 0.002) predictor for ovulation following hormone treatment, with only high-grade females (Grades 3-4) ovulating in response to hormones. Ultrasound grade did not influence egg numbers or cleavage rate ( P > 0.05). Results demonstrate multiple hormone therapies are available for stimulating ovulation in female DGFs and ultrasonography is a valuable tool to inform hormone therapy. Ultimately, these reproductive technologies are critical to enhance breeding and reintroduction efforts for the DGF.

Mammalian herbivores have developed numerous adaptations to utilize their plant-based diets including a modified gastrointestinal tract (GIT) and symbiosis with a GIT microbiota that plays a major role in digestion and the maintenance of host health. The red panda ( Ailurus fulgens ) is a herbivorous carnivore that lacks the specialized GIT common to other herbivores but still relies on microorganisms for survival on its almost entirely bamboo diet. The GIT microbiota is of further importance in young red pandas, as high cub mortality is problematic and has been attributed to failure to meet nutritional requirements. To gain insight into the establishment of the GIT microbiota of red pandas, we examined microbial communities in two individuals following dietary changes associated with weaning using next-generation 16S rRNA Illumina MiSeq paired-end sequencing of faecal samples. Across all four stages (pre-weaning, during weaning, post-weaning and adult), the GIT microbial community displayed low diversity and was dominated by bacteria in the phylum Firmicutes with lesser contributions from the Proteobacteria. A core community was found consistently across all weaning stages and included species within the taxa Escherichia-Shigella, Streptococcus , Clostridium and an unclassified Clostridiaceae. Analysis of the overall community composition and structure showed that although the GIT microbiota is established early in red pandas, dietary changes during weaning further shape the community and are correlated with the presence of new bacterial species. This work is the first analysis of the GIT microbiota for red panda cubs during weaning and provides a framework for understanding how diet and host microbiota impact the development of these threatened animals.

Giant pandas have been described as mono-oestrus spring breeders, yet males exposed to aseasonal oestrous females in the autumn or winter exhibit breeding behaviours and interest in mating. In the present study, urinary androgens and sperm parameters were quantified for males exposed to females expressing oestrus during spring, autumn or winter to examine plasticity of reproductive seasonality in giant pandas. Monthly average androgen concentrations for two males exposed to females in either seasonal or aseasonal oestrus were greater (P<0.001) than baseline concentrations. Evaluation of daily androgen concentrations revealed a peak that was three- to fivefold greater than baseline, occurring an average of 5 days before ovulation for both seasonal and aseasonal cycles. There were no significant differences in testes volume, sperm motility, forward progression or sperm concentration in males between female seasonal and aseasonal cycle years. Male gonadal activity was more variable without a clear pattern in years when the female was anovulatory than when she was ovulatory (seasonal or aseasonal). These data show the flexible reproductive capacity of male giant pandas as demonstrated by a rapid physiological readiness to mate in response to female oestrous cues within or outside the normal breeding season and may suggest a facultative seasonal reproduction with a 'female-induced rut'.

Seasonal changes in the foodscape force herbivores to select different plant species or plant parts to meet nutritional requirements. We examined whether the search for calorie-rich carbohydrates explained giant panda's selection for bamboo culm over leaves during spring. Leaves and culms were collected from four Phyllostachys bamboos (P. aurea, P. aureosulcata, P. glauca, and P. nuda) once per month over 18-27 months. Monthly changes in annual plant part nutrients were examined, and compared to seasonal foraging behaviors of captive giant pandas. Although total fiber was greater (p<0.0001) in culm (85.6 ± 0.5%) than leaves (55.3 ± 0.4%) throughout the year, culm fiber was at its lowest in spring (79-85%) when culm selection by giant pandas exceeded 70% of their overall diet. Culm starch also was greatest (p = 0.044) during spring (5.5 ± 1.1%) and 2.5-fold the percentage of starch in leaves (2.2 ± 0.6%). The free sugars in spring culm consisted of a high proportion of glucose (35%) and fructose (47%), whereas sucrose made up 42% of the total free sugar content of spring leaves. Bound sugars in culm consisted of 60% glucose and 38% xylose likely representative of hemicellulose. The concentrations of bound sugars (hemicelluloses) in spring culms (543.7 ± 13.0 mg/g) was greater (p<0.001) than in leaves (373.0 ± 14.8 mg/g). These data help explain a long-standing question in giant panda foraging ecology: why consume the plant part with the lowest protein and fat during the energetically intensive spring breeding season? Giant pandas likely prefer spring culm that contains abundant mono- and polysaccharides made more bioavailable as a result of reduced fiber content. These data suggest that phenological changes in bamboo plant part nutrition drive foraging decisions by giant pandas.

Due to the environmental and anthropogenic impacts that continue to threaten the reproductive success of polar bears, a more detailed understanding of their reproductive cycle is needed. Captive populations of polar bears provide an excellent opportunity to learn more about the reproductive physiology of the species. Progestin (P4) and androgen (T) metabolites in urine, and their ratio (P4:T), were examined during 11 reproductive cycles of captive female polar bears ( n = 4) to characterize the steroid hormone profile during pregnancy and determine possible variations related to reproductive failure. The concentration of hormone metabolites in urine were determined through enzyme immunoassay. Reproductive cycles were classified as pregnant ( n = 3), anovulatory ( n = 4) and ovulatory-non-parturient ( n = 4) based on the changes in urinary hormone metabolite values and cub production. In the absence of a lactational suppression of estrus, elevated androgen concentrations suggested resumption of follicular development within 3 weeks of parturition. Breeding behaviours were most often observed when androgen values were at their highest or in decline. Ovulation was identified by a return to basal androgen concentration and elevation of progestins within 1-4 weeks after breeding. As a result, urinary concentrations of progestins were greater than androgens (P4:T ratio ≥ 1.0) during ovulatory cycles whereas the P4:T ratio was <1.0 when females were anovulatory. Progestins and the P4:T ratio of parturient cycles were greatest beginning in June/July (17-20 weeks after breeding) and reached a peak at 24-37 weeks (mid-October/mid-November, 4-9 weeks before birth of cubs). Non-invasive monitoring of hormone metabolites in urine provided a rapid determination of endocrine function for improved husbandry and reproductive management of polar bears in captivity. Further research is warranted to understand the reproductive endocrinology of polar bears and its impact on conservation and management of this species in captivity and the wild.

There is growing evidence that pathogens play a role in population declines and species extinctions. For small populations, disease-induced extinction may be especially probable. We estimated the susceptibility of two amphibian species of conservation concern (the dusky gopher frog [Lithobates sevosus] and boreal toad [Anaxyrus boreas boreas]) to an emerging pathogen (ranavirus) using laboratory challenge experiments, and combined these data with published demographic parameter estimates to simulate the potential effects of ranavirus exposure on extinction risk. We included effects of life stage during pathogen exposure, pathogen exposure interval, hydroperiod of breeding habitat, population carrying capacity, and immigration in simulations. We found that both species were highly susceptible to ranavirus when exposed to the pathogen in water at environmentally relevant concentrations. Dusky gopher frogs experienced 100 % mortality in four of six life stages tested. Boreal toads experienced 100 % mortality when exposed as tadpoles or metamorphs, which were the only life stages tested. Simulations showed population declines, greater extinction probability, and faster times to extinction with ranavirus exposure. These effects were more evident with more frequent pathogen exposure intervals and lower carrying capacity. Immigration at natural rates did little to mitigate effects of ranavirus exposure unless immigration occurred every 2 years. Our results demonstrate that disease-induced extinction by emerging pathogens, such as ranavirus, is possible, and that threat may be especially high for species with small population sizes. For the species in this study, conservation organizations should incorporate ranavirus surveillance into monitoring programs and devise intervention strategies in the event that disease outbreaks occur.

Background: Accurate sex identification techniques are important for wildlife demographic studies and for genetic management of captive breeding colonies. Various non-invasive methods for identification of biological sex in the weakly dimorphic endangered dusky gopher frog (DGF; Lithobates sevosa) were explored to support planned recovery efforts for this species including breeding and augmentation of wild populations., Methods: Body size (snout-vent length and body weight) measurements, observation of nuptial pads, ultrasound imaging, and urinary hormone analysis for testosterone and estrone were performed on 27 male and 19 female DGFs. For each method, the mean and range of measurement values were determined for male and female DGFs housed in a captive breeding population. The ability of these methods to accurately predict the true biological sex of the individuals was assessed retrospectively., Results: Body size measurements were of limited use for sex identification purposes, as males and females demonstrated overlapping body lengths and weights. Observation of the presence/absence of nuptial pads in males and females, respectively, proved to be accurate and easy to perform in most cases. Ultrasound imaging was useful for predicting the sex of female frogs, particularly when females were gravid. Commercial enzyme immunoassay kits were validated to measure urinary hormones in the DGF. Mean urinary testosterone (males: 2.22 ± 0.38 ng/ml; females: 0.92 ± 0.11 ng/ml) and estrone (males: 0.08 ± 0.01 ng/ml; females: 1.50 ± 0.39 ng/ml) concentrations were significantly (p < 0.05) different between the sexes. However, there was some overlap in hormone concentrations between the sexes. When a ratio of testosterone (T) to estrone (E) concentrations was calculated for each individual, males demonstrated significantly greater T/E ratios compared to females (p < 0.05). Use of this ratio showed greater accuracy in predicting the sex of the animal compared to using testosterone or estrone concentrations alone., Conclusions: Monitoring for presence/absence of nuptial pads and using urinary testosterone to estrone hormone ratios were the most accurate methods for identifying the biological sex of adult DGFs. Urinary hormone measurements for sex identification may be useful in other weakly dimorphic and monomorphic amphibian species in both ex situ and in situ settings.

Captive rearing and reintroduction / translocation are increasingly used as tools to supplement wild populations of threatened species. Reintroducing captive-reared Chinese giant salamanders may help to augment the declining wild populations and conserve this critically endangered amphibian. We released 31 captive-reared juvenile giant salamanders implanted with VHF radio transmitters at the Heihe River (n = 15) and the Donghe River (n = 16) in the Qinling Mountains of central China. Salamanders were monitored every day for survival from April 28th 2013 to September 3rd 2014. We attempted to recapture all living individuals by the end of the study, measured their body mass and total body length, and checked for abnormalities and presence of external parasites. Two salamanders at the Heihe River and 10 animals at the Donghe River survived through the project timeline. Nine salamanders were confirmed dead, while the status of the other 10 animals was undetermined. The annual survival rate of giant salamanders at the Donghe River (0.702) was 1.7-fold higher than that at the Heihe River (0.405). Survival increased as individuals were held longer following surgery, whereas body mass did not have a significant impact on survival rate. All salamanders recaptured from the Donghe River (n = 8) increased in mass (0.50 ± 0.13 kg) and length (5.5 ± 1.5 cm) after approximately 11 months in the wild, and they were only 7% lighter than wild animals of the same length (mean residual = -0.033 ± 0.025). Our results indicate that captive-reared Chinese giant salamanders can survive in the wild one year after release and adequate surgical recovery time is extremely important to post-release survival. Future projects may reintroduce older juveniles to achieve better survival and longer monitoring duration.

Amphibians are experiencing a global extinction crisis and captive assurance colonies, along with reintroduction programs, are necessary to prevent further losses. Assisted reproductive technologies (ART), such as hormone-stimulated gamete collection and in vitro fertlisation (IVF), are conservation methods that can be used to increase reproductive output for breeding and reintroduction programs when animals fail to breed naturally. In order to maximise the production of offspring using ART, it is important to establish the physiological limitations on the frequency that hormone therapy can be used to collect gametes for IVF or assisted breeding. The present study examined the effects of the frequency of hormone-induced spermiation on sperm quantity and quality in Fowler's toad (Bufo fowleri) by comparing four levels of hormone injection frequencies: twice a week, once a week, every other week, and every 3 weeks. Sperm release was induced with an intraperitoneal injection of 300IU human chorionic gonadotropin (hCG). Spermatozoa were collected at three time points after injection (5, 7 and 9h) and sperm concentration, motility and quality of forward progressive movement were measured. A significant decrease in sperm concentration (P<0.01) was observed with the most frequent treatment (twice a week hormone injections). However, there was no negative effect of the treatments on sperm motility (P=0.06) or forward movement (P=0.06). We also observed a significant decrease in the concentration (P<0.01), motility (P=0.02) and quality of forward progressive movement (P=0.01) of spermatozoa at the 9h collection compared with earlier collection times. These results have clear implications for amphibian captive breeding programs, where more frequent hormone-induced spermiation could have a negative effect on male performance. We recommend that hormone injections be spaced a minimum of 2 weeks apart to optimise the health of the animals, assisted breeding, IVF or collection of gametes for genome resource banking.

Dietary shifts can result in changes to the gastrointestinal tract (GIT) microbiota, leading to negative outcomes for the host, including inflammation. Giant pandas (Ailuropoda melanoleuca) are physiologically classified as carnivores; however, they consume an herbivorous diet with dramatic seasonal dietary shifts and episodes of chronic GIT distress with symptoms including abdominal pain, loss of appetite and the excretion of mucous stools (mucoids). These episodes adversely affect the overall nutritional and health status of giant pandas. Here, we examined the fecal microbiota of two giant pandas' non-mucoid and mucoid stools and compared these to samples from a previous winter season that had historically few mucoid episodes. To identify the microbiota present, we isolated and sequenced the 16S rRNA using next-generation sequencing. Mucoids occurred following a seasonal feeding switch from predominately bamboo culm (stalk) to leaves. All fecal samples displayed low diversity and were dominated by bacteria in the phyla Firmicutes and to a lesser extent, Proteobacteria. Fecal samples immediately prior to mucoid episodes had lower microbial diversity as compared to mucoids. Mucoids were mostly comprised of common mucosal-associated taxa including Streptococcus and Leuconostoc species, and exhibited increased abundance for bacteria in the family Pasteurellaceae. Taken together, these findings indicate that mucoids may represent an expulsion of the mucosal lining that is driven by changes in diet. We suggest that these occurrences serve to reset their GIT microbiota following changes in bamboo part preference, as giant pandas have retained a carnivorous GIT anatomy while shifting to an herbivorous diet.

During the annual period of bamboo shoot growth in spring, free-ranging giant pandas feed almost exclusively on the shoots while ignoring the leaves and full- height culm. Little is known about the nutritional changes that occur during bamboo shoot growth, if nutritional changes differ among species, or how these changes might influence forage selection. Our objective was to examine the nutrient and mineral composition during three phases of shoot growth (<60, 90-150 and >180 cm) for seven species of bamboo (Phyllostachys (P.) aurea, P. aureosulcata, P. bissetii, P. glauca, P. nuda, P. rubromarginata, Pseudosasa japonica) fed to captive giant pandas at the Memphis Zoo. Total dietary fiber content of bamboo shoots increased (p < 0.0001) from an overall species average of 61% dry matter (DM) at < 60 cm to 75% DM at shoot heights > 180 cm, while crude protein, fat and ash exhibited significant declines (p < 0.05). Phyllostachys nuda had the overall greatest (p = 0.007) crude protein (21% DM) and fat (4% DM) content, and lowest overall total fibre (61% DM) content compared to the other species examined. In contrast, Pseudosasa japonica had the overall lowest crude protein and fat, and relatively higher fibre content (9%, 3% and 74% respectively). Concentrations of Zn and Fe were highest in shoots <60 cm (10-50 μg/g DM) and decreased (p < 0.05) during growth in all species examined. Concentrations of Ca, Cu, Mn, Na and K varied among species and were largely unaffected by growth stage. Due to their higher concentrations of nutrients and lower fibre content in comparison to culm and leaf, bamboo shoots should be a major component of captive giant panda diets when available., (Journal of Animal Physiology and Animal Nutrition © 2015 Blackwell Verlag GmbH.)

Captive African (Loxodonta africana) and Asian (Elephas maximus) elephants can experience foot pathologies and arthritis. As a preventative measure against these pathologies and to alleviate the potential discomfort due to concrete substrates, some zoological institutions have renovated elephant housing to increase the amount of natural or shock-absorbent substrates. The objective of this study was to compare behavioral (diurnal and nocturnal) and glucorticoid (e.g., serum cortisol) responses of three female African elephants before, during, and after renovation to their indoor housing floor to assess whether renovations had short-term effects on the elephants' behavior and stress physiology. Behavioral data were collected using scan-sampling methods, and activity budgets were constructed for each of the three elephants. In addition, the duration of all lying rest activities were recorded. Weekly serum cortisol concentrations were determined with enzyme immunoassay (EIA). Overall, eating was the most prevalent behavior exhibited outdoors during the day, while resting (either in a lying or standing position) were most common during the indoor, nocturnal periods. Although variation existed among the three elephants, all three females spent significantly more time walking and less time eating during the day after the completion of the renovations. The extent to which the three elephants exhibited nocturnal lying rest behavior varied among the elephants, with the oldest elephant exhibiting the least amount (an average of 13.2 ± 2.8% of the nightly behavioral scans) compared to the two younger elephants (an average of 34.5 ± 2.1% and 56.6 ± 2.8% of the nightly behavioral scans). There was a significant increase in lying rest behavior for one elephant and standing rest for a second elephant following renovations. Baseline cortisol concentrations prior to renovations were 3.0 ± 0.4 ng/ml, 4.5 ± 0.5 ng/ml, and 4.9 ± 0.5 ng/ml for the three elephants. Cortisol concentrations remained baseline for two of the elephants throughout and after the renovation period, while one elephant that was pregnant had elevated cortisol during construction. Cortisol concentrations for the pregnant elephant remained higher than baseline once she was introduced to the new flooring and allowed back into the building, but these values were closer to the cortisol concentrations before renovations than during construction. Our findings demonstrate that individual elephants can vary in their behavioral and physiological responses to exhibit modifications. Given that the elephants walked more during the day, two of the three elephants had an increase in rest behavior during the night, and there were minimal changes in cortisol response after the flooring renovations, we conclude that the flooring renovations overall had a positive impact on animal welfare.

Declines of the southern Rocky Mountain population of boreal toad (Anaxyrus boreas boreas) have led to the establishment of a captive assurance population and reintroduction program, in an attempt to preserve and propagate this geographically isolated population. One of the unique adaptations of this species is its ability to survive in cold environments by undergoing long periods of hibernation. In captivity, hibernation can be avoided altogether, decreasing morbidity caused by compromised immune systems. However, it is not entirely clear how essential hibernation is to reproductive success. In this study, the effects of hibernation versus nonhibernation, and exogenous hormones on oviposition, were examined in boreal toad females in the absence of males. In the summers of 2011 and 2012, 20 females housed at Mississippi State University were treated with a double priming dose of hCG and various ovulatory doses of hCG and LH-releasing hormone analog but denied hibernation. Exogenous hormones, in the absence of hibernation, could not induce oviposition over two breeding seasons (2011-2012). In contrast, during the summer of 2012 and 2013, 17 of 22 females (77%) housed at the Native Aquatic Species Restoration Facility (Alamosa, CO, USA) oviposited after they were treated with two priming doses of hCG (3.7 IU/g each) and a single ovulation dose of hCG (13.5 IU/g) and LH-releasing hormone analog (0.4 μg/g) after hibernation. There was a significant difference in oviposition between females that were hibernated and received hormones (2012, P < 0.05 and 2013, P < 0.01) compared to hibernated control females. In 2013, 12 of 16 remaining Mississippi State University females from the same group used in 2011 and 2012 were hibernated for 1, 3, and 6 months, respectively and then treated with the same hormone regimen administered to females at the Native Aquatic Species Restoration Facility. Together, hibernation and hormone treatments significantly increased oviposition (P < 0.05), with 33% of females ovipositing. These results suggest that (1) hibernation is a key factor influencing oviposition that cannot be exclusively circumvented by exogenous hormones; (2) females do not require the presence of a male to oviposit after hormone treatments; and (3) longer hibernation periods are not beneficial for oviposition. The hormonal induction of oviposition in the absence of males and shorter hibernation periods could have important captive management implications for the boreal toad. Furthermore, the production of viable offspring by IVF where natural mating is limited could become an important tool for genetic management of this boreal toad captive population., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Each amphibian species is evolutionarily distinct, having developed highly specialized and diverse reproductive strategies in both terrestrial and aquatic environments. These unique reproductive patterns and mechanisms, key to species propagation, have only been explored in a limited number of laboratory models. Although the development of applied reproductive technologies for amphibians has proven useful for a few threatened species, the real benefit of this technology has been new insights into the reproductive adaptations, behavior, endocrinology, and physiological mechanisms that have evolved over millions of years. As the basic fundamental database on amphibian reproductive physiology has grown, so has the applied benefit for species conservation. In particular, technologies such as non-invasive fecal and urinary hormone assays, hormone treatments for induced breeding or gamete collection, in vitro fertilization, and the ability to establish genome resource banks have all played important roles in monitoring or managing small populations of captive species. Amphibians have the ability to produce a large excess of germplasm (up to 10,000 ovulated eggs in a single reproductive event) that if not collected and preserved, would represent a wasted valuable resource. We discuss the current state of knowledge in assisted reproductive technologies for amphibians and why their extinction crisis means these available tools can no longer be implemented as small-scale, last-ditch efforts. The reproductive technologies must be established early as a key component of large-scale species recovery.

Background: Declining numbers of wild giant anteaters highlight the importance of sustainable captive populations. Unfortunately, captive reproductive management is limited by the lack of external physical indicators of female reproductive status and the aggressive behavior of males. We examined the endocrinology of the estrous cycle and pregnancy, and whether delayed implantation is a gestational strategy for giant anteaters as described for other xenarthrans., Methods: Feces were collected from seven captive females 3-5 times weekly and mating was recorded. Concentrations of estrogen (estrone-glucuronide, E1, and estradiol-17β, E2), progestagen (20-oxo-progestagens, P4), and glucocorticoid (GC) metabolites were examined in fecal extracts by enzyme immunoassay., Results: Estrous cycles for nulliparous females (6 cycles, n = 2) compared to the multiparous female (6 cycles, n = 1) were shorter (47.3 +/- 4.3 days versus 62.5 +/- 2.6 days) with relatively lower luteal phase concentrations of P4 (49.4 +/- 2.9 ng/g versus 136.8 +/- 1.8 ng/g). The four remaining females had unclear ovarian activity: two females exhibited apparent luteal activity but unclear fluctuations in estrogens, while the other two females had parallel fecal P4 and estrogens concentrations. Pregnancy ranged 171-183 days with females returning to estrus post-partum as early as 60 days (n = 3, 1.8-4 years of age at mating). Delayed implantation was indicated by a biphasic elevation in fecal P4 metabolites: the initial 4-fold increase occurred for 81-105 days and was followed by a 26-fold secondary rise in P4 metabolites lasting 66-94 days prior to parturition. Fecal GC was correlated with fecal estrogens and greatest during estrus, late pregnancy, and six days prior to parturition (estrous cycle GC, 14.4-62.8 ng/g; pregnancy GC, 13.6-232.7 ng/g)., Conclusions: Estrous cycles of giant anteaters occurred year-round, but were shorter and more intermittent in younger nulliparous animals compared to a multiparous female. A pronounced elevation in fecal P4, estrogen, and GC occurred during late gestation after an initial post-mating delay providing evidence for delayed implantation. Adrenocorticoid activity indicated impending parturition. Differences in estrous cycle characteristics with age and the protracted but variable gestation length must be considered to improve reproductive success and neonatal survival in giant anteaters.

Background: Captive breeding programs for endangered amphibian species often utilize exogenous hormones for species that are difficult to breed. The purpose of our study was to compare the efficacy of two different hormones at various concentrations on sperm production, quantity and quality over time in order to optimize assisted breeding., Methods: Male American toads (Anaxyrus americanus) were divided into three separate treatment groups, with animals in each group rotated through different concentrations of luteinizing hormone releasing hormone analog (LHRH; 0.1, 1.0, 4.0 and 32 micrograms/toad), human chorionic gonadotropin (hCG; 50, 100, 200, and 300 IU), or the control over 24 hours. We evaluated the number of males that respond by producing spermic urine, the sperm concentration, percent motility, and quality of forward progression. We also evaluated the effects of hCG and LHRH on reproductive behavior as assessed by amplexus. Data were analyzed using the Generalized Estimating Equations incorporating repeated measures over time and including the main effects of treatment and time, and the treatment by time interaction., Results: The hormone hCG was significantly more effective at stimulating spermiation in male Anaxyrus americanus than LHRH and showed a dose-dependent response in the number of animals producing sperm. At the most effective hCG dose (300 IU), 100% of the male toads produced sperm, compared to only 35% for the best LHRH dose tested (4.0 micrograms). In addition to having a greater number of responders (P < 0.05), the 300 IU hCG treatment group had a much higher average sperm concentration (P < 0.05) than the treatment group receiving 4.0 micrograms LHRH. In contrast, these two treatments did not result in significant differences in sperm motility or quality of forward progressive motility. However, more males went into amplexus when treated with LHRH vs. hCG (90% vs. 75%) by nine hours post-administration., Conclusion: There is a clear dichotomy between the two hormones' physiological responses on gamete production and stimulation of amplexus. Understanding how these two hormones influence physiology and reproductive behaviors in amphibians will have direct bearing on establishing similar breeding protocols for endangered species.

Giant panda (Ailuropoda melanoleuca) monitoring and research often require accurate estimates of population size and density. However, obtaining these estimates has been challenging. Innovative technologies, such as fecal near infrared reflectance spectroscopy (FNIRS), may be used to differentiate between sex, age class, and reproductive status as has been shown for several other species. The objective of this study was to determine if FNIRS could be similarly used for giant panda physiological discriminations. Based on samples from captive animals in four U.S. zoos, FNIRS calibrations correctly identified 78% of samples from adult males, 81% from adult females, 85% from adults, 89% from juveniles, 75% from pregnant and 70% from non-pregnant females. However, diet had an impact on the success of the calibrations. When diet was controlled for plant part such that "leaf only" feces were evaluated, FNIRS calibrations correctly identified 93% of samples from adult males and 95% from adult females. These data show that FNIRS has the potential to differentiate between the sex, age class, and reproductive status in the giant panda and may be applicable for surveying wild populations.

The giant panda (Ailuropoda melanoleuca David, 1869) is an iconic species for global conservation, yet field research has only recently advanced to the point where adaptive management is possible. Here, we review recent developments in giant panda conservation science and propose a strategic plan for moving panda conservation forward. Because of scientific, funding, political, and logistical hurdles, few endangered species management programs have embraced adaptive management, wherein management decisions are shaped iteratively by targeted scientific research. Specific threats, such as habitat destruction, anthropogenic disturbance and fragmented nonviable populations, need to be addressed simultaneously by researchers, managers and policy-makers working in concert to understand and overcome these obstacles to species recovery. With the backing of the Chinese Government and the conservation community, the giant panda can become a high-profile test species for this much touted, but rarely implemented, approach to conservation management., (© 2011 ISZS, Blackwell Publishing and IOZ/CAS.)

After ovulation, non-pregnant female giant pandas experience pseudopregnancy. During pseudopregnancy, non-pregnant females exhibit physiological and behavioral changes similar to pregnancy. Monitoring hormonal patterns that are usually different in pregnant mammals are not effective at determining pregnancy status in many animals that undergo pseudopregnancy, including the giant panda. Therefore, a physiological test to distinguish between pregnancy and pseudopregnancy in pandas has eluded scientists for decades. We examined other potential markers of pregnancy and found that activity of the acute phase protein ceruloplasmin increases in urine of giant pandas in response to pregnancy. Results indicate that in term pregnancies, levels of active urinary ceruloplasmin were elevated the first week of pregnancy and remain elevated until 20-24 days prior to parturition, while no increase was observed during the luteal phase in known pseudopregnancies. Active ceruloplasmin also increased during ultrasound-confirmed lost pregnancies; however, the pattern was different compared to term pregnancies, particularly during the late luteal phase. In four out of the five additional reproductive cycles included in the current study where females were bred but no birth occurred, active ceruloplasmin in urine increased during the luteal phase. Similar to the known lost pregnancies, the temporal pattern of change in urinary ceruloplasmin during the luteal phase deviated from the term pregnancies suggesting that these cycles may have also been lost pregnancies. Among giant pandas in captivity, it has been presumed that there is a high rate of pregnancy loss and our results are the first to provide evidence supporting this notion.

The giant panda (Ailuropoda melanoleuca) is classified as a carnivore, yet subsists on a diet comprised almost exclusively of bamboo. Wild and captive giant pandas use highly selective foraging behaviors for processing and consuming bamboo. These behaviors are for the first time quantified in captive giant pandas over a 5-year period of time showing highly specific seasonal trends. Giant panda feeding behavior was recorded using live video observations of two giant pandas housed at the Memphis Zoo from November 2003 to June 2008. Leaf was the primary plant part consumed from June to December, whereas culm was consumed primarily from February to May, with both bears displaying similar seasonal shifts in plant part consumption. From May to June, leaf consumption increased significantly (P-values<0.001); from June to August, leaf consumption remained high and stable. From December to March, leaf consumption decreased significantly (P-values<0.001). Specific behaviors for bamboo leaf and culm consumption were also observed. Both bears formed wads of leaves before ingestion while feeding on leaf, but the male employed this feeding behavior more often than the female (54 and 33%, respectively). Both bears used similar culm-stripping behavior (26 and 25%), used to remove the outer layer and isolate the pith for consumption. This study indicates that unique seasonal foraging behaviors observed in wild pandas are also apparent in captive animals in relation to plant part selectivity and feeding behaviors., ((c) 2009 Wiley-Liss, Inc.)

The giant panda is a conservation icon, but science has been slow to take up its cause in earnest. In the past decade, researchers have been making up for lost time, as reflected in the flurry of activity reported at the symposium Conservation Science for Giant Pandas and Their Habitat at the 2009 International Congress for Conservation Biology (ICCB) in Beijing. In reports addressing topics ranging from spatial ecology to molecular censusing, from habitat recovery in newly established reserves to earthquake-induced habitat loss, from new insights into factors limiting carrying capacity to the uncertain effects of climate change, this symposium displayed the vibrant and blossoming application of science to giant panda conservation. Collectively, we find that we have come a long way, but we also reach an all-too-familiar conclusion: the more we know, the more challenges are revealed. While many earlier findings are supported, many of our assumptions are debatable. Here we discuss recent advancements in conservation science for giant pandas and suggest that the way forward is more direct application of emerging science to management and policy.

As amphibian populations continue to decline, both government and non-government organisations are establishing captive assurance colonies to secure populations deemed at risk of extinction if left in the wild. For the most part, little is known about the nutritional ecology, reproductive biology or husbandry needs of the animals placed into captive breeding programs. Because of this lack of knowledge, conservation biologists are currently facing the difficult task of maintaining and reproducing these species. Academic and zoo scientists are beginning to examine different technologies for maintaining the genetic diversity of founder populations brought out of the wild before the animals become extinct from rapidly spreading epizootic diseases. One such technology is genetic resource banking and applied reproductive technologies for species that are difficult to reproduce reliably in captivity. Significant advances have been made in the last decade for amphibian assisted reproduction including the use of exogenous hormones for induction of spermiation and ovulation, in vitro fertilisation, short-term cold storage of gametes and long-term cryopreservation of spermatozoa. These scientific breakthroughs for a select few species will no doubt serve as models for future assisted breeding protocols and the increasing number of amphibians requiring conservation intervention. However, the development of specialised assisted breeding protocols that can be applied to many different families of amphibians will likely require species-specific modifications considering their wide range of reproductive modes. The purpose of this review is to summarise the current state of knowledge in the area of assisted reproduction technologies and gene banking for the conservation of amphibians.

Amphibian populations in the wild are experiencing massive die-offs that have led to the extinction of an estimated 168 species in the last several decades. To address these declines, zoological institutions are playing an important role in establishing captive assurance colonies to protect species in imminent danger of extinction. Many of the threatened species recently placed into captivity are failing to reproduce before they expire, and maintaining founder populations is becoming a formidable challenge. Assisted reproductive technologies, such as hormone synchronization, gamete storage and artificial fertilization, are valuable tools for addressing reproductive failure of amphibians in captive facilities. Artificial fertilization has been commonly employed for over 60 years in several keystone laboratory species for basic studies in developmental biology and embryology. However, there are few instances of applied studies for the conservation of threatened or endangered amphibian species. In this review, we summarize valuable technological achievements in amphibian artificial fertilization, identify specific processes that need to be considered when developing artificial fertilization techniques for species conservation, and address future concerns that should be priorities for the next decade.

Very little is known about the effects of manipulating toad sperm activity in vitro, and such information is important in the development of a genetic resource bank for bufonid species. The specific objectives of this study were to: 1). identify the optimal inactivation and reactivation solutions for toad spermatozoa collected in urine; 2). establish the length of time toad spermatozoa can be exposed to an inactivation buffer and still resume motility upon reactivation; 3). evaluate the consequence of inactivation on specific sperm characteristics; and 4). characterize the sperm mitochondria vesicle (MV) and its relationship to motility. Reactivated sperm motility was similar after inactivation in either Simplified Amphibian Ringers (SAR) solution or DeBoer's (DB) solution. Diluting the buffer by 80% with water provided the best method for reactivating sperm. Dilutions with NaCl solutions (10-50 mM) produced inferior results. SAR-inactivated spermatozoa could remain suspended up to 4 hr and still regain 25% of initial motility upon reactivation in water. Compared to the controls, sperm motility was greater (P<0.01) over time for samples treated with SAR, although forward progression was significantly lower. Furthermore, SAR treatment resulted in sperm samples with a greater number of viable, morphologically normal, and intact MVs over time. Electron microscopy and fluorescent staining confirmed that the toad sperm's MV contains a large number of active mitochondria with very few other cytoplasmic structures. Nearly all spermatozoa exhibiting motility had an intact MV, and dissociation of this structure was clearly related to motility loss. In conclusion, toad spermatozoa can be effectively inactivated and reactivated by varying the osmolality of the external solutions and, although sperm forward progression is reduced, all other characteristics are well maintained. Moreover, the increased number of spermatozoa with intact MV after inactivation suggests the process may help preserve this important structure., (Copyright 2003 Wiley-Liss, Inc.)

The purpose of this study was to evaluate cryopreserved fringe-eared (FE) oryx (Oryx gazella callotis) sperm function using a heterologous in vitro fertilization (IVF) system previously developed to study scimitar-horned (SH) oryx (Oryx dammah) spermatozoa. Semen was collected by electroejaculation from FE oryx (n = 2) and SH oryx (n = 2), evaluated immediately postcollection, and cryopreserved. Thawed spermatozoa were evaluated for motility, forward progression, and acrosomal status immediately post-thaw, after Percoll-separation, and 1, 2, 3, and 8 h after culture in IVF medium. In vitro-matured cow oocytes (n = 924) were inseminated with either domestic bull, FE, or SH oryx spermatozoa and after an 8-h coincubation period, half the oocytes were fixed and examined for sperm penetration, polyspermy, and male pronuclear formation. The remaining oocytes were placed into in vitro culture and evaluated for cleavage after 48 h. Overall, there were no between-species differences in sperm motility and acrosome integrity. However, an effect of time (P < 0.05) and a species-by-time interaction (P < 0.05) were detected for both parameters. Penetration, male pronuclear formation, and embryo cleavage were high (>90%, >85%, and >70%, respectively) for oocytes inseminated with domestic bull and SH oryx spermatozoa and did not differ (P > 0.05) between species. In contrast, very few oocytes (2.8%, 4 of 141) inseminated with FE oryx sperm were penetrated. Cleavage was rare (8.0%, 16 of 200) in oocytes inseminated with FE oryx spermatozoa and did not differ (P > 0.05) from that in parthenogenetic controls (4.2%, 3 of 72). Furthermore, FE oryx spermatozoa were incapable of penetrating zona-free cow oocytes. These results indicate that species-specific differences in gamete interaction may exist even between very closely related nondomestic bovids.

Recent identification of plasminogen activator inhibitor-1 (PAI-1) in the pig oviduct has prompted an evaluation of its mRNA, protein synthesis, and hormonal regulation during the estrous cycle and early pregnancy, defined as time prior to and after maternal recognition of pregnancy. To examine PAI-1 protein synthesis, oviductal tissue was collected from European Large White and Chinese Meishan gilts on days 0, 2, and 5 of early pregnancy, divided into three functional segments, and cultured. Culture media was collected and de novo synthesized PAI-1 analyzed by 2D-SDS-PAGE, fluorography, and densitometry. To determine hormonal regulation of PAI-1 synthesis and secretion, four groups of ovariectomized (OVX) cross-bred gilts were each treated with one of four steroid regimens (corn oil, estrogen, progesterone, or estrogen + progesterone) and tissue collected for RNA or cultured. Steady-state mRNA levels of PAI-1 were evaluated throughout the estrous cycle in cross-bred gilts. To compare steady-state PAI-1 mRNA levels between cyclic and pregnant cross-bred gilts, tissue was collected on days 0, 2, and 12. Quantitative analysis of steady-state levels of PAI-1 mRNA were analyzed by dot-blot hybridization and densitometry. A greater (P < 0.01) synthesis and secretion of PAI-1 protein was found in the isthmus portion of the oviduct relative to either the ampulla or infundibulum regardless of day of pregnancy or breed. No difference could be detected for PAI-1 protein between breeds. The Large White had a greater (P < 0.05) secretion of PAI-1 on day 2 of early pregnancy relative to other days examined. Whole oviductal tissue from cross-bred gilts was found to have a significantly greater amount of PAI-1 mRNA on days 1 and 2 compared to other days examined, while the isthmus had significantly greater levels of mRNA on days 2 and 12. A significant effect of day and segment was detected for levels of PAI-1 mRNA from cyclic and early pregnant cross-bred gilts. PAI-1 mRNA was found to be significantly greater in the isthmus than other segments, regardless of day of the estrous cycle or pregnancy. An interaction was detected for estrogen and progesterone on PAI-1 mRNA (P < 0.05) and protein (P = 0.09). Estrogen was found to inhibit PAI-1 protein synthesis and also inhibited progesterone-mediated stimulation of PAI-1 mRNA. Our results demonstrate expression of PAI-1 mRNA and protein are highest on day 2 of early pregnancy, which is consistent with its proposed function of protecting the oocyte/embryo from enzymatic degradation and/or extracellular matrix remodeling of both oviduct and early cleavage-stage embryo., (Copyright 2000 Wiley-Liss, Inc.)

This study evaluated the effects of porcine oviduct-specific glycoprotein (pOSP) on in vitro fertilization (IVF), polyspermy, and development to blastocyst. Experiment 1 evaluated the effects of various concentrations (0-100 microgram/ml) of purified pOSP on fertilization parameters, including penetration, polyspermy, male pronuclear formation, and mean number of sperm penetrated per oocyte. Experiment 2 examined the ability of an anti-pOSP immunoglobulin G to inhibit the observed effects of pOSP on fertilization parameters. Experiments 3 and 4 examined various concentrations of pOSP (0-100 microgram/ml) on zona pellucida solubility and sperm binding, respectively. Lastly, experiment 5 assessed the effects of various concentrations of pOSP (0-100 microgram/ml) on the in vitro embryo cleavage rate and development to blastocyst. Pig oocytes matured and fertilized in vitro were used for all experiments. An effect of treatment (P < 0.05) was detected for pOSP on penetration, polyspermy, and mean number of sperm per oocyte. Concentrations for pOSP of 0-50 microgram/ml had no effect on sperm penetration rates; however, compared with the control, 100 microgram/ml significantly decreased the penetration rate (74% vs. 41%). Addition of 10-100 microgram/ml significantly reduced the polyspermy rate compared with the control (61% vs. 24-29%). The decrease in polyspermy achieved by addition of pOSP during preincubation and IVF was blocked with a specific antibody to pOSP. No effect of treatment was observed on zona digestion time relative to the control; however, the number of sperm bound to the zona pellucida was significantly decreased by treatment (P < 0.05). Compared with the control, all concentrations of pOSP examined reduced the number of sperm bound per oocyte (45 vs. 19-34). A treatment effect (P < 0.05) was observed for pOSP on embryo development to blastocyst but not on cleavage rates. Addition of pOSP during preincubation and fertilization significantly increased postcleavage development to blastocyst, but a synergistic stimulation on development was not detected when pOSP was included during in vitro culture. These results indicate that exposure to pOSP before and during fertilization reduces the incidence of polyspermy in pig oocytes, reduces the number of bound sperm, and increases postcleavage development to blastocyst.

The porcine oviduct synthesizes de novo and secretes a number of proteins into culture medium, many of which are unidentified. The objectives of the present study were to 1) semipurify and identify a M(r) 45 000 secreted protein of the oviduct, 2) examine its synthesis within the three functional segments (infundibulum, ampulla, and isthmus), and 3) evaluate its distribution throughout the oviduct. Oviductal tissue was collected during early pregnancy, divided into functional segments, and subsequently cultured. Medium was collected, and the M(r) 45 000 protein was concentrated by gel-filtration chromatography. The semipurified protein was transferred onto a polyvinylidene fluoride membrane and subjected to N-terminal amino acid analysis. The 26-amino acid sequence was 96% identical to that of pig plasminogen activator inhibitor (PAI)-1. Analysis by 1-dimensional SDS-PAGE and fluorography of rabbit anti-human PAI-1-immunoprecipitated product confirmed PAI-1. Subsequent 2-dimensional SDS-PAGE and fluorographic analyses of media revealed greater PAI-1 synthesis by the isthmus than by the ampulla or infundibulum. PAI-1 was immunolocalized throughout the oviduct and was heavily concentrated in the apical region of epithelial cells. Immunogold electron microscopy localized PAI-1 within putative secretory granules in the epithelial apical region and also associated with cilia in the isthmus. Isthmic PAI expression suggests a crucial role in protecting the preimplantation embryo from proteolytic degradation as well as in regulation of extracellular matrix turnover and remodeling.

During late follicular development and estrus, the mammalian oviduct undergoes specific physiological and biochemical modifications which contribute to an optimization of the microenvironment for fertilization and early cleavage-stage embryonic development. These changes appear to be hormonally regulated by ovarian steroids, most importantly, estrogen. The hundreds of macromolecules found within the oviductal lumen are contributed by selective serum transudation and active biosynthesis and secretion from nonciliated epithelial cells. Recent studies have indicated temporal and regional (infundibulum, ampulla and isthmus) differences in steady-state levels of specific mRNAs and in de novo protein synthesis and secretion by the oviduct. One protein synthesized de novo, the estrogen-dependent oviductal secretory glycoprotein (OSP), has been shown to be unique to the oviduct and is conserved across a number of mammalian species. This protein associates with the zona pellucida, perivitelline space and vitelline or blastomere membrane of ovulated eggs and preimplantation embryos. OSPs have been shown to enhance sperm binding and penetration in oocytes and may regulate development in early preimplantation embryos. Other regulatory molecules, protease inhibitors, growth factors, cytokines, binding proteins, enzymes and immunoglobulins have been identified in the oviductal microenvironment. The identification and potential roles for oviduct-secreted proteins will be reviewed and discussed. Current research focuses on continued identification and characterization of specific oviductal proteins and a determination of the molecular basis of their interactions with the oocyte, sperm or embryo.

Insulin-like growth factor I (IGF-I) has been identified in human seminal plasma. This study was conducted to determine whether IGF-I is present in bovine seminal plasma, whether sperm cells express the IGF-I receptor (IGF-IR), and whether IGF-I affects sperm motility. Semen samples were collected from bulls by electroejaculation and maintained at 37 degrees C, and motility of sperm was assessed. After centrifugation to separate sperm cells from seminal plasma, the seminal plasma was submitted to a validated heterologous RIA for IGF-I. Significant concentrations of IGF-I (116.29 +/- 40.83 ng/ml expressed as mean +/- SD) were measured in bovine seminal plasma. Sperm cells were washed with buffer and subjected to either radioreceptor assay (RRA) or immunocytochemistry (IC). RRA revealed a single high affinity for the IGF-IR with a Kd of 0.83 nM as determined by the computer program LIGAND. IC, using three monoclonal antibodies, localized the IGF-IR to the acrosomal region of the sperm. Computer-assisted sperm-motion analysis was used to determine the effects of IGF-I and IGF-II on bovine sperm motility parameters. Both IGF-I and IGF-II increased sperm motility and straight-line velocity (p < 0.05) relative to the control. The presence of IGF-IR on sperm, the presence of IGF-I in semen, and the ability of IGF-I to stimulate sperm motility provide evidence that the IGF system may be involved in the fertilization process in the bovine species.