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.

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.

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.

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.

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.

Use of urinary 13,14, dihydro-15-keto-prostaglandin F2α (PGFM) concentrations to diagnose pregnancy and predict parturition in the giant panda (Ailuropoda melanolecua).

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.

Ultrasound imaging improves hormone therapy strategies for induction of ovulation and in vitro fertilization in the endangered dusky gopher frog (Lithobates sevosa).

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.

Behavioral, semiochemical and androgen responses by male giant pandas to the olfactory sexual receptivity cues of females.

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.

Dietary changes during weaning shape the gut microbiota of red pandas (Ailurus fulgens).

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.

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'.

Phenological changes in bamboo carbohydrates explain the preference for culm over leaves by giant pandas (Ailuropoda melanoleuca) during spring.

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.

Urinary profiles of progestin and androgen metabolites in female polar bears during parturient and non-parturient cycles.

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.

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.