Enhancing predictive performance for spectroscopic studies in wildlife science through a multi-model approach: A case study for species classification of live amphibians.

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.

Spermiation response to exogenous hormone therapy in hibernated and non-hibernated boreal toads (Anaxyrus boreas boreas).

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.

The impact of time and environmental factors on the mitochondrial vesicle and subsequent motility of amphibian sperm.

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.

Sperm collection and cryopreservation for threatened newt species.

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% Me2SO + 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.

Hormonal induction of spermiation in a Eurasian bufonid (Epidalea calamita).

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.

Changes in urinary androgen concentration indicate that male giant pandas (Ailuropoda melanoleuca) respond to impending female oestrus during and outside the typical spring breeding season.

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'.

Biological sex identification in the endangered dusky gopher frog (Lithobates sevosa): a comparison of body size measurements, secondary sex characteristics, ultrasound imaging, and urinary hormone analysis methods.

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.

Ranavirus could facilitate local extinction of rare amphibian species.

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.

Frequency of exogenous hormone therapy impacts spermiation in male Fowler's toad (Bufo fowleri).

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.

Amphibian declines in the twenty-first century: why we need assisted reproductive technologies.

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.

High susceptibility of the endangered dusky gopher frog to ranavirus.

Amphibians are one of the most imperiled vertebrate groups, with pathogens playing a role in the decline of some species. Rare species are particularly vulnerable to extinction because populations are often isolated and exist at low abundance. The potential impact of pathogens on rare amphibian species has seldom been investigated. The dusky gopher frog Lithobates sevosus is one of the most endangered amphibian species in North America, with 100-200 individuals remaining in the wild. Our goal was to determine whether adult L. sevosus were susceptible to ranavirus, a pathogen responsible for amphibian die-offs worldwide. We tested the relative susceptibility of adult L. sevosus to ranavirus (103 plaque-forming units) isolated from a morbid bullfrog via 3 routes of exposure: intra-coelomic (IC) injection, oral (OR) inoculation, and water bath (WB) exposure. We observed 100% mortality of adult L. sevosus in the IC and WB treatments after 10 and 19 d, respectively. Ninety-five percent mortality occurred in the OR treatment over the 28 d evaluation period. No mortality was observed in the control treatment after 28 d. Our results indicate that L. sevosus is susceptible to ranavirus, and if adults in the wild are exposed to this pathogen, significant mortality could occur. Additionally, our study demonstrates that some adult amphibian species can be very susceptible to ranavirus, which has been often overlooked in North American studies. We recommend that conservation planners consider testing the susceptibility of rare amphibian species to ranavirus and that the adult age class is included in future challenge experiments.

Oral administration of GnRH via a cricket vehicle stimulates spermiation in tiger salamanders (Ambystoma tigrinum).

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.

Fecal estrogen, progestagen and glucocorticoid metabolites during the estrous cycle and pregnancy in the giant anteater (Myrmecophaga tridactyla): evidence for delayed implantation.

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-17ß, 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.

A comparison of human chorionic gonadotropin and luteinizing hormone releasing hormone on the induction of spermiation and amplexus in the American toad (Anaxyrus americanus).

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.

International Society for Wildlife Endocrinology: the future of endocrine measures for reproductive science, animal welfare and conservation biology.

Hormone analysis is a precise and widely accepted tool for monitoring reproductive function and responses to stressors. Although hormones are present and can be measured in various biological matrices, non-invasive methods have gained popularity over the past 30 years as a more practical approach for assessing ovarian, testicular and, more recently, adrenocortical activity in intractable wildlife species. Non-invasive hormone monitoring also has been key to understanding biological mechanisms related to observed behaviours of captive and free-ranging animals. Despite the increasing popularity of this research field, wildlife endocrinologists have not had a specific forum for sharing and discussing their latest findings, technical developments and common challenges. To provide such a communication platform, the International Society for Wildlife Endocrinology (ISWE) was established in 2010, followed by an international meeting held on 3-4 November 2011 at the Toronto Zoo, Canada. Over several sessions, keynote speakers and participants discussed recent developments of new and innovative methods for hormone monitoring, as well as the latest advances in basic endocrinology as applied to adrenal function, reproductive physiology, animal health, ecology and evolution. Here, we introduce ISWE to the scientific community and discuss how this new society will serve as a resource for wildlife endocrinologists worldwide.

Development of an amphibian sperm biobanking protocol for genetic management and population sustainability.

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.

Sperm Cryopreservation as a Tool for Amphibian Conservation: Production of F2 Generation Offspring from Cryo-Produced F1 Progeny.

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.

Influence of oviposition-inducing hormone on spawning and mortality in the endangered Panamanian golden frog (Atelopus zeteki).

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.

Near-Infrared Spectroscopy (NIRS) as a Method for Biological Sex Discrimination in the Endangered Houston Toad (Anaxyrus houstonensis).

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.

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

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