Worldwide, there are only a handful of reintroduction programs for threatened salamander species, and very few have conducted postrelease studies to examine survival, habitat selection, and dispersal. Limitations in postrelease monitoring are primarily due to size constraints of amphibians and to dimensions of the radiotransmitters available for implantation. However, due to the large size of the critically endangered Chinese giant salamander (Andrias davidianus), these animals make optimal candidates for surgical implantation of radiotransmitters prior to reintroduction or translocation. The objective of this study was to develop an anesthetic protocol using tricane methanesulfonate (MS-222) and test a surgical procedure for coelomic implantation of radiotransmitters for this species. A total of 32 Chinese giant salamanders from two age groups (Group A: 4.7 yr old, n = 16; Group B: 2.7 yr old, n = 16) were implanted with 4-g radiotransmitters designed for underwater monitoring of fish. Group A w...

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.

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 28^th 2013 to September 3^rd 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.

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.

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. 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. 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. 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.

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.