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.

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.

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.

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.

Fertilization potential of cold-stored Fowler’s toad (Anaxyrus fowleri) spermatozoa: temporal changes in sperm motility based on temperature and osmolality.

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.

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.

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.

Short-term storage of tiger salamander (Ambystoma tigrinum) spermatozoa: The effect of collection type, temperature and time.

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.

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.

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.

Evaluation of different temporal periods between hormone-induced ovulation attempts in the female Fowler's toad Anaxyrus fowleri.

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.