First successful artificial insemination of the endangered Louisiana pinesnake, Pituophis ruthveni.

The Louisiana pinesnake (Pituophis ruthveni) is considered one of the rarest snakes in North America and was federally listed under the Endangered Species Act in 2018. Captive breeding and reintroduction of zoo-bred hatchlings has been successful, however, limited founders in the captive population and the inability to bring new, wild genes into the captive colony presents a major concern for the conservation of this species. The use of artificial insemination (AI) was first applied to snakes in the 1980s but further development of the technique has since received little attention. Our goal was to develop a method of AI for use in breeding Louisiana pinesnakes to facilitate gene flow from wild to captive populations. We inseminated two captive Louisiana pinesnakes with semen collected from one donor male, novel to both females. Timing of AI occurred following the emergence of females from brumation, and when large, distinct follicles were detected using digital palpation. Females were inseminated four and five times over a period of 14 and 19 days, respectively, using fresh and 2-day refrigerator stored semen. One female laid seven eggs, which resulted in four fertile eggs and two viable hatchlings, while the second female produced three fertile of seven eggs laid but no viable hatchlings. Genetic analyses confirmed the donor male was the sire of hatchlings. This is the first successful AI of an endangered snake species and provides a framework for the use and optimization of assisted reproductive technologies for use in conservation breeding programs.

Effects of age on sperm quality metrics in endangered Mississippi gopher frogs (Lithobates sevosus) from captive populations used for controlled propagation and reintroduction efforts.

A decline in sperm quality with age is a common prediction of senescence-based hypotheses and empirical studies. While widely studied across taxa, there is little known on the effect of ageing on sperm quality in amphibians, especially in captive populations used for controlled propagation and reintroduction efforts. Here, we investigated variation in sperm quality metrics (i.e., motility, concentration, and morphology) in the endangered Mississippi gopher frog (Lithobates sevosus) among males of three age categories using individuals from captive breeding populations housed at three different zoological institutions. Different aged males across the species expectant lifespan (1-9, 1-2, 3-4, and 8-9-year-old subcategories) were chosen in an attempt to identify an optimal breeding age relevant for captive breeding programs. Moreover, we explored and statistically controlled for potential differences in sperm quality which may be attributed to the type of induction hormones and source populations that differed among institutions. Results indicated that males of different ages did not differ in sperm motility or concentration. However, we did find that older males (8-9 years old) had significantly longer sperm than other age categories and younger males (1-2 years old) had significantly more atypical sperm than other age categories. Furthermore, we found no significant differences in any sperm quality metrics between the different induction hormones or source populations used at the different institutions. Within a captive breeding program, this information is especially valuable as our results indicate that males that have only recently sexually matured may not be ready to breed, while older males maintain sperm quality metrics presumably related to fertilization success.

Inbreeding depression in sperm quality in a critically endangered amphibian.

Small, isolated populations often experience increased inbreeding and decreased heterozygosity, which increases the potential risk of inbreeding depression. The relationship between inbreeding and sperm health is well-documented in a variety of taxa, but has yet to be explored in amphibians. The dusky gopher frog, Lithobates sevosus, is a critically endangered species with years of documented inbreeding and low genetic variability as a consequence of isolation and population size reduction. This study investigated the effects of inbreeding on sperm quality in captive L. sevosus using an outbred, sister species (Lithobates pipiens) as a standard for comparison. We found L. sevosus to have severely reduced sperm quality in terms of total motility, forward progressive motility, concentration, and viability. Additionally, we observed a significant, negative relationship between total sperm motility and mean kinship within captive-bred individuals. These data serve to enhance our understanding of the role inbreeding plays in amphibians, and to provide valuable insight into new risk factors declining amphibian populations may face.

Corrigendum to: Sperm output and body condition are maintained independently of hibernation in an endangered temperate amphibian.

Hibernation is an integral part of the life history of species living in seasonal environments. However, our knowledge about the link between hibernation and reproductive success in amphibians remains limited, which poses an obstacle for critical conservation efforts. To fill this gap, we quantified the effects of captive hibernation on sperm quality, sperm quantity and body condition in an endangered anuran, the Wyoming toad (Anaxyrus baxteri), and used naturally hibernated wild toads as a standard for comparison. We hypothesised that hibernation is essential for optimal sperm output but is detrimental to body condition. Sperm collection was performed using assisted reproductive technologies for both captive and wild toads. Contrary to our hypotheses, no differences were observed in sperm metrics (total number of cells, concentration, motility and viability) or in body condition across captive treatment groups (0, 30 or 60 days of hibernation). Moreover, no difference was found between sperm metrics of captive toads and wild toads. These unexpected findings suggest that hibernation may not be an essential process for spermiation in A. baxteri while using exogenous hormones, and illustrate the potential of temperate amphibians to adapt to varying environmental conditions during winter months.

Sperm output and body condition are maintained independently of hibernation in an endangered temperate amphibian.

Hibernation is an integral part of the life history of species living in seasonal environments. However, our knowledge about the link between hibernation and reproductive success in amphibians remains limited, which poses an obstacle for critical conservation efforts. To fill this gap, we quantified the effects of captive hibernation on sperm quality, sperm quantity and body condition in an endangered anuran, the Wyoming toad (Anaxyrus baxteri), and used naturally hibernated wild toads as a standard for comparison. We hypothesised that hibernation is essential for optimal sperm output but is detrimental to body condition. Sperm collection was performed using assisted reproductive technologies for both captive and wild toads. Contrary to our hypotheses, no differences were observed in sperm metrics (total number of cells, concentration, motility and viability) or in body condition across captive treatment groups (0, 30 or 60 days of hibernation). Moreover, no difference was found between sperm metrics of captive toads and wild toads. These unexpected findings suggest that hibernation may not be an essential process for spermiation in A. baxteri while using exogenous hormones, and illustrate the potential of temperate amphibians to adapt to varying environmental conditions during winter months.

Cryopreservation and hormonal induction of spermic urine in a novel species: The smooth-sided toad (Rhaebo guttatus).

Global amphibian declines have fueled an increased interest in amphibian assisted reproductive technologies. Within the genus Rhaebo, half of the species are experiencing decreasing population trends; however, insufficient information is available on many of these species' reproductive biology. Using the smooth-sided toad, Rhaebo guttatus, we present effective methods for collecting and cryopreserving an example of Rhaebo sperm. Specifically, our findings show that administering 10 IU/g body weight of hCG (human chorionic gonadotropin) yields the most motile and concentrated sperm and that cryopreserving spermic urine in a solution of 5% DMFA (N,N-Dimethylformamide) and 10% trehalose returns sperm with a 33 ±â€¯3% average post-thaw motility. These findings may represent an important step forward in developing techniques that can be safely applied to other, more vulnerable species within the Rhaebo genus.

Initial founders of captive populations are genetically representative of natural populations in critically endangered dusky gopher frogs, Lithobates sevosus.

The rapid rate of decline in amphibian populations has urged many researchers and conservationists to establish captive, or ex situ, populations. Such populations are guarded against effects of habitat loss and degradation, and if actively managed, can serve as a reservoir for rare alleles that might be lost in the wild. Without proper management, ex situ population sizes can dwindle and will no longer perform this function. The dusky gopher frog, Lithobates sevosus, is a critically endangered species, imperiled by habitat loss and population isolation. To assist in recovery of the species and prevent further genetic erosion, a captive breeding program was initiated. We investigated how well natural genetic variation was captured within the ex situ population and determined relatedness within each ex situ population. We genotyped individuals from two natural populations and two founding, captive populations to compare metrics of genetic variation and relatedness. The data show the initial founder populations are genetically representative of the natural populations, although variation is low in each, and that relatedness values are similar. Therefore, founding captive populations were successful at capturing genetic variation in the wild. Future research should continue to compare genetic variation of captive and natural populations to monitor efficacy of their management programs. Zoo Biol. 35:378-384, 2016. © 2016 Wiley Periodicals, Inc.

Using dermal glucocorticoids to determine the effects of disease and environment on the critically endangered Wyoming toad.

Amphibian populations are declining worldwide, and increased exposure to environmental stressors, including global climate change and pathogens like Batrachochytrium dendrobatidis (Bd), may be contributing to this decline. Our goal was to use a novel dermal swabbing method to measure glucocorticoid (GC) hormones and investigate the relationship among disease and environmental conditions in the critically endangered Wyoming toad (Anaxyrus baxteri). Our objectives were to (i) validate the use of dermal swabs to measure GCs using an adrenocorticotropic hormone (ACTH) challenge on eight captive toads (4 ACTH: 2 M, 2F and 4 saline as a control: 2 M, 2F), (ii) investigate stress physiology and disease status of toads across six reintroduction sites and (iii) compare dermal cortisol between reintroduced and captive toads. Dermal cortisol peaked immediately after the ACTH and saline injections. Faecal GC metabolites (FGMs) were significantly higher one week after the ACTH injection compared with the week before. Saline-injected toads had no change in FGM over time. Toads were only found in three reintroduction sites and dermal cortisol was similar across sites; however, reintroduced toads had higher dermal cortisol in August compared with June and compared with captive individuals. Bd status did not influence dermal cortisol concentrations. Dermal and faecal hormonal metabolite analyses can be used to study amphibian stress physiology and learn how environmental conditions are impacting population success.

Temporal trends in genetic data and effective population size support efficacy of management practices in critically endangered dusky gopher frogs (Lithobates sevosus).

Monitoring temporal changes in population genetic diversity and effective population size can provide vital information on future viability. The dusky gopher frog, Lithobates sevosus, is a critically endangered species found only in coastal Mississippi, with low genetic variability as a consequence of isolation and population size reduction. Conservation management practices have been implemented, but their efficacy has not been addressed. We genotyped individuals collected 1997-2014 to determine temporal trends in population genetic variation, structure, and effective size. Observed and expected heterozygosity and allelic richness revealed temporally stable, but low, levels of genetic variation. Positive levels of inbreeding were found in each year. There was weak genetic structure among years, which can be attributed to increased effects of genetic drift and inbreeding in small populations. L. sevosus exhibited an increase in effective population size, and currently has an estimated effective size of 33.0-58.6 individuals, which is approximately half the census size. This large ratio could possibly be explained by genetic compensation. We found that management practices have been effective at maintaining and improving effective size and genetic diversity, but that additional strategies need to be implemented to enhance viability of the species.