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.

Sperm morphology and forward motility are indicators of reproductive success and are not age- or condition-dependent in a captive breeding population of endangered snake.

The relationship between male ejaculate traits and reproductive success is an important consideration for captive breeding programs. A recovery plan for the endangered Louisiana pinesnake includes captive breeding for the release of young to the wild. Semen was collected from twenty captive breeding male snakes and ejaculate traits of motility, morphology, and membrane viability were measured for each male. Semen traits were analyzed in relation to the fertilization rate of eggs produced from pairings of each male with a single female (% fertility) to determine the ejaculate factors contributing to reproductive success. In addition, we investigated the age- and condition-dependence of each ejaculate trait. We found significant variation in the ejaculate traits of males and normal sperm morphology ([Formula: see text] = 44.4 ± 13.6%, n = 19) and forward motility ([Formula: see text] = 61.0 ± 13.4%, n = 18) were found to be the best predictors of fertility. No ejaculate traits were found to be condition-dependent (P > 0.05). Forward progressive movement (FPM) ([Formula: see text] = 4 ± 0.5, n = 18) was determined to be age-dependent (r2 = 0.27, P = 0.028), but FPM was not included in the best model for rate of fertilization. Male Louisiana pinesnakes do not appear to experience a significant decline in reproductive potential with age (P > 0.05). The observed average rate of fertilization in the captive breeding colony was below 50% and only those pairings with a male having >51% normal sperm morphology avoided a 0% rate of fertilization. Identification of the factors contributing to the reproductive success of captive breeding Louisiana pinesnakes is of considerable conservation value in the recovery of the species, and captive breeding programs should use assessments of ejaculate traits to plan breeding pairs for maximum reproductive output.

Ultrasound Description of Follicular Development in the Louisiana Pinesnake (Pituophis ruthveni, Stull 1929).

Accurate monitoring of reproductive activity is necessary for success of captive breeding and recovery of endangered species. Using ultrasonography, we aimed to describe the stages of follicle development of the endangered Louisiana pinesnake (Pituophis ruthveni). Ultrasound procedures were performed weekly for 11 females during the 2020 reproductive season by submerging the last half of an unanesthetized female in water and using a 3.0-10.0 MHz linear array transducer placed and moved along the gastrosteges to explore the whole reproductive tract. The presence of follicles, their size, echogenicity, and stage of development was assessed. We observed small, round, anechoic, linearly aligned previtellogenic follicles in the coelom at the beginning of the reproductive season and found that structures dramatically increased in size and shifted in echogenicity as follicles matured and developed before and after ovulation. We classified follicles according to ultrasonographic appearance into 7 different follicle categories: previtellogenic, early vitellogenic, vitellogenic, preovulatory, peri-ovulatory, post ovulatory, and shelled. Using ultrasound, we developed markers of progressive follicular maturation for the Louisiana pinesnake and identified signs of abnormal development and post ovulatory follicle reabsorption. Detailed description of follicular maturation will be useful to improve captive breeding successes, identify mechanisms of reproductive failure, and develop artificial insemination.

Intraspecific investigation of dehydration-enhanced innate immune performance and endocrine stress response to sublethal dehydration in a semi-aquatic species of pit viper.

Sublethal dehydration can cause negative physiological effects, but recent studies investigating the sub-lethal effects of dehydration on innate immune performance in reptiles have found a positive correlation between innate immune response and plasma osmolality. To investigate whether this is an adaptive trait that evolved in response to dehydration in populations inhabiting water-scarce environments, we sampled free-ranging cottonmouth snakes (n=26 adult cottonmouths) from two populations inhabiting contrasting environments in terms of water availability: Snake Key (n=12), an island with no permanent sources of fresh water, and Paynes Prairie (n=14), a flooded freshwater prairie. In addition to field surveys, we manipulated the hydration state of 17 cottonmouths (Paynes Prairie n=9, Snake Key n=8) in a laboratory setting and measured the response of corticosterone and innate immune performance to dehydration with the aim of identifying any correlation or trade-offs between them. We measured corticosterone of cottonmouths at a baseline level and then again following a 60 min stress test when at three hydration states: hydrated, dehydrated and rehydrated. We found that innate immune performance improved with dehydration and then returned to baseline levels within 48 h of rehydration, which agrees with previous research in reptiles. Despite the frequent exposure of cottonmouths on Snake Key to dehydrating conditions, we did not find cottonmouths inhabiting the island to show a greater magnitude or more prolonged immune response compared with cottonmouths from Paynes Prairie. We also found a positive association between dehydration and corticosterone values.

Cryopreservation of Sperm from an Endangered Snake with Tests of Post-Thaw Incubation in Caffeine.

Cryopreservation of sperm to preserve the genetic diversity of declining populations is a promising technique to aid in the recovery of endangered species such as the Louisiana pinesnake (Pituophis ruthveni). However, this technique has been performed on only a handful of snake species and with limited success. Here, we tested a cryoprotective agent (CPA) mixture containing Lake's buffer with 10% N,N-dimethyl formamide (DMF), 2% methanol, 5% clarified egg yolk, (v/v% final concentration) against 16 other CPA-treatment mixtures. These contained either Lake's buffer or TEST egg yolk buffer as the base diluent with a penetrating or non-penetrating CPA on the post-thaw recovery of sperm motility and viability. We also investigated the effect of post-thaw incubation treatment in TL HEPES supplemented with 10% fetal bovine serum (H10) alone or with caffeine on post-thaw motility parameters. Sperm from 16 Louisiana pinesnakes was cryopreserved, and the effectiveness of the CPA treatment mixtures and post-thaw treatments was determined based on measurements of sperm motility and viability. Sperm cryopreservation significantly reduced initial post-thaw sperm quality for all of the extender treatments. Viability of sperm was best maintained when cryopreserved in an CPA treatment mixture containing Lake's buffer with 10% DMF, 2% methanol, and 5% clarified egg yolk with the addition of 5 mg/mL bovine serum albumin (BSA). For several extender mixtures a similar percent of post-thaw motility was observed, but no forward motility returned in any post-thaw samples prior to incubation in dilution treatments. Following incubation in both post-thaw treatments, the percent of forward motility and the index of forward progressive movement improved significantly. Post-thaw dilution with H10 containing caffeine improved motility parameters over H10 alone, suggesting further investigation of post-thaw treatment in caffeine could be beneficial. Although, cryopreservation of sperm from the Louisiana pinesnake continues to present a challenge, post-thaw dilution and the addition of BSA to CPA mixtures provides areas for improving cryopreservation methods for this endangered species.

Toxicity of cryoprotective agents to semen from two closely related snake species: The endangered Louisiana pinesnake (Pituophis ruthveni) and bullsnake (Pituophis cantenifer).

Cryopreservation of sperm is an important tool for the conservation of threatened species. Many species of reptile are under considerable threat of extinction and there has been limited investigation of sperm cryopreservation in this taxonomic group. We performed a comparative test of toxicity to sperm of six commonly used cryoprotective agents (CPAs) at three concentrations (5%, 10%, 20%) from the Louisiana pinesnake, Pituophis ruthveni (n = 11), and the closely related bullsnake, Pituophis cantenifer (n = 8). Our objective was to determine the general toxicity of CPAs for cryopreservation in snakes and the cryoprotective ability of CPAs for sperm from the endangered Louisiana pinesnake. We conducted three experiments to: 1) evaluate the short-term in vitro toxicity of common CPAs in two closely related snake species, 2) determine the effectiveness of cryoprotectants for freezing and thawing semen in the Louisiana pinesnake, and 3) test the possible reduction in toxic effects of individual CPAs on semen of the Louisiana pinesnake by combining two of them. We used measures of motility including total motility, forward motility, and forward progressive motility index to characterize toxic effects and cryoprotective ability of each CPA. The results of our three experiments provide several important findings: 1) sperm of the bullsnake and Louisiana pinesnake responded differently to CPAs, 2) few CPAs provided any cryoprotection, as measured by percent recovered motility, in Louisiana pinesnakes, and 3) using mixtures of CPAs did not reduce toxicity relative to the best performing CPA on its own. Motility was best maintained at a concentration of 5% for CPAs tested; however, cryoprotection was best achieved with glycerol at 20% followed by DMA and DMF at 10%. These results provide further insight into the challenges faced by researchers attempting to cryopreserve sperm from snakes. Further comparative studies are required to determine the generality of cryopreservation methods in reptiles and suggest caution should be taken when developing cryopreservation protocols across species, particularly in snakes. All CPAs tested in this study were permeating CPAs and showed a significant acute toxic effect on motility at concentrations that provided cryoprotection. Future work in snakes might consider additional avenues of cryoprotection and combinations of multiple approaches.

A tale of two islands: evidence for impaired stress response and altered immune functions in an insular pit viper following ecological disturbance.

The frequency and intensity of ecological perturbations affecting wild animal populations is expected to increase in the future with animals facing numerous global threats. Seahorse Key is a continental island off mainland Florida that has historically been a major rookery for several species of waterbirds. As a result of an unknown disturbance, the entire rookery abandoned Seahorse Key in April 2015 and shifted nesting activities to nearby Snake Key, resulting in an influx of food resources in the form of fish carrion to resident Florida cottonmouth snakes (Agkistrodon conanti), while snakes on Seahorse Key experienced a drastic reduction in food resources. Our objective was to assess plasma corticosterone concentrations, corticosterone negative feedback using dexamethasone, blood glucose, body condition, packed cell volume, natural antibody agglutination, white blood cell counts and ratios and erythrocyte sedimentation rate to characterize the long-term effects of differential resource availability in these two snake populations 3 years after this major ecological disturbance. We collected blood samples at three time points from cottonmouths on Seahorse Key (n = 6 individuals) and Snake Key (n = 13 individuals) in fall 2018. In due consideration of the small sample size, our study shows evidence that 3 years after the shift in waterbird nesting Seahorse Key cottonmouths exhibit a dampened acute stress response and presumptive impaired innate immune functions relative to cottonmouths on Snake Key. These results highlight the context-dependent nature of biomarkers and implicate the significant decrease in food resources on Seahorse Key in altering hormonal stress responses and innate immune functions, possibly leading to unknown long-term downstream effects. This study assessed the response of a wild population of pit viper to ecological disturbance in situ with the aim to improve our understanding of how animals cope with such perturbations and improve our capacity to make informed decisions for conservation.

Water relations of an insular pit viper.

Colonization of novel habitats often requires plasticity or adaptation to local conditions. There is a critical need to maintain hydration in terrestrial environments having limited water. Atypical populations of Florida cottonmouth snakes, Agkistrodon conanti, inhabit continental islands with no permanent sources of fresh water. Here, we report investigations related to how these insular snakes maintain water balance considering the mainland conspecifics are semi-aquatic and typically associate with freshwater mesic habitats. We tested three hypotheses related to water relations of insular populations of cottonmouth snakes compared with those on the mainland. (1) Voluntary drinking of fresh water in free-ranging insular snakes should reflect a relationship to recency of rainfall more strongly than in mainland snakes. (2) Insular snakes will tolerate greater dehydration before drinking than will mainland snakes. (3) Insular snakes will avoid drinking seawater more strongly than will those from the mainland. Between 2001 and 2018, we quantitatively estimated the hydration status of 337 individual cottonmouth snakes from insular populations and 30 cottonmouth snakes from mainland Florida, as judged by the tendency of wild-caught snakes to drink fresh water immediately following capture. We found that insular cottonmouth snakes had a higher incidence of dehydration than did mainland cottonmouth snakes (64% versus 23%), and the hydration status of the insular snakes correlated with patterns of precipitation. We also determined experimentally the dehydration threshold for drinking fresh water in insular (mean±s.d. -5.64±4.3%, n=34) and mainland cottonmouth snakes (-5.74±4.5%, n=21), and these were not significantly different. Discrimination tests for drinking serially from a graded series of brackish water showed that mainland snakes did not discriminate against the highest brackish value (10.5 ppt or 30% seawater), whereas insular snakes showed a preference for <15% seawater. Naive neonates from insular and mainland cohorts behaved similarly. The preference of insular snakes for fresh water represents an important aspect of the maintenance of water balance that differs from the mainland conspecifics and is likely a habituated or adaptive response to dependence on rainfall.

Drinking by sea snakes from oceanic freshwater lenses at first rainfall ending seasonal drought.

Acquisition of fresh water (FW) is problematic for FW-dependent animals living in marine environments that are distant from sources of FW associated with land. Knowledge of how marine vertebrates respond to oceanic rainfall, and indeed the drinking responses of vertebrates generally following drought, is extremely scant. The Yellow-bellied Sea Snake (Hydrophis platurus) is the only pelagic species of squamate reptile and ranges across the Indo-Pacific oceans, having one of the largest geographic distributions of any vertebrate species. It requires FW and dehydrates at sea during periods of drought. Here we report drinking behaviors of sea snakes precisely at the transition from dry to wet season when rainfall first impacted the ocean following 6 months of seasonal drought. We show that the percentage of sea snakes that voluntarily drank FW in the laboratory when captured over eight successive days decreased from 80% to 13% before and after rainfall commenced, respectively. The percentage of snakes that drank immediately following capture exhibited a significant linear decline as the earliest rains of the wet season continued. Drinking by snakes indicates thirst related to dehydration, and thus thirsty snakes must have dehydrated during the previous six months of drought. Hence, the progressive decline in percentage of thirsty snakes indicates they were drinking from FW lenses associated with the first rainfall events of the wet season. These data reinforce the importance of accessing oceanic FW from precipitation, with implications for survival and distribution of pelagic populations that might be subjected to intensifying drought related to climate change.

A serosurvey for Brucella suis, classical swine fever virus, porcine circovirus type 2, and pseudorabies virus in feral swine (Sus scrofa) of eastern North Carolina.

As feral swine (Sus scrofa) populations expand their range and the opportunity for feral swine hunting increases, there is increased potential for disease transmission that may impact humans, domestic swine, and wildlife. From September 2007 to March 2010, in 13 North Carolina, USA, counties and at Howell Woods Environmental Learning Center, we conducted a serosurvey of feral swine for Brucella suis, pseudorabies virus (PRV), and classical swine fever virus (CSFV); the samples obtained at Howell Woods also were tested for porcine circovirus type 2 (PCV-2). Feral swine serum was collected from trapped and hunter-harvested swine. For the first time since 2004 when screening began, we detected B. suis antibodies in 9% (9/98) of feral swine at Howell Woods and <1% (1/415) in the North Carolina counties. Also, at Howell Woods, we detected PCV-2 antibodies in 59% (53/90) of feral swine. We did not detect antibodies to PRV (n=512) or CSFV (n=307) at Howell Woods or the 13 North Carolina counties, respectively. The detection of feral swine with antibodies to B. suis for the first time in North Carolina warrants increased surveillance of the feral swine population to evaluate speed of disease spread and to establish the potential risk to commercial swine and humans.

Bartonella spp. in feral pigs, southeastern United States.

In conjunction with efforts to assess pathogen exposure in feral pigs from the southeastern United States, we amplified Bartonella henselae, B. koehlerae, and B. vinsonii subsp. berkhoffii from blood samples. Feral pigs may represent a zoonotic risk for hunters or butchers and pose a potential threat to domesticated livestock.