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.

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.

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.

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'.

Nutritional effects on reproductive performance of captive adult female coyotes (Canis latrans).

Interactions between animals and their environment are fundamental to ecological research. Field studies of coyote (Canis latrans) reproductive performance suggest mean litter size changes in response to prey abundance. However, this relationship has been assessed primarily by using carcasses collected from trappers. The objective of this study was to assess whether nutritional manipulation prior to mating affected reproduction in adult female coyotes. We examined the effects of caloric restriction during the 7 months prior to estrus on the reproductive rates of 11 captive female coyotes and the subsequent initial survival of pups through two reproductive cycles. This was a 2-year study with a cross-over design so each female was monitored for reproductive performance on each of the two diet treatments. We assessed the number of implantation scars, number of pups born, sex ratios of pups, average pup weight at birth and 2- and 6-weeks of age, and the survival rates between implantation and 2-weeks of age for two diet treatments. We found the mean number of implantation sites and pups whelped during a reproductive cycle was influenced by food-intake prior to conception. Additionally, we found evidence suggesting the effects of nutritional stress may persist for additional breeding cycles. We also provided evidence suggesting well-fed females tended to have more male pups. Understanding how environmental factors influence reproductive output may improve model predictions of coyote population dynamics.

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

BACKGROUND: Declining numbers of wild giant anteaters highlight the importance of sustainable captive populations. Unfortunately, captive reproductive management is limited by the lack of external physical indicators of female reproductive status and the aggressive behavior of males. We examined the endocrinology of the estrous cycle and pregnancy, and whether delayed implantation is a gestational strategy for giant anteaters as described for other xenarthrans. METHODS: Feces were collected from seven captive females 3-5 times weekly and mating was recorded. Concentrations of estrogen (estrone-glucuronide, E1, and estradiol-17ß, E2), progestagen (20-oxo-progestagens, P4), and glucocorticoid (GC) metabolites were examined in fecal extracts by enzyme immunoassay. RESULTS: Estrous cycles for nulliparous females (6 cycles, n = 2) compared to the multiparous female (6 cycles, n = 1) were shorter (47.3 +/- 4.3 days versus 62.5 +/- 2.6 days) with relatively lower luteal phase concentrations of P4 (49.4 +/- 2.9 ng/g versus 136.8 +/- 1.8 ng/g). The four remaining females had unclear ovarian activity: two females exhibited apparent luteal activity but unclear fluctuations in estrogens, while the other two females had parallel fecal P4 and estrogens concentrations. Pregnancy ranged 171-183 days with females returning to estrus post-partum as early as 60 days (n = 3, 1.8-4 years of age at mating). Delayed implantation was indicated by a biphasic elevation in fecal P4 metabolites: the initial 4-fold increase occurred for 81-105 days and was followed by a 26-fold secondary rise in P4 metabolites lasting 66-94 days prior to parturition. Fecal GC was correlated with fecal estrogens and greatest during estrus, late pregnancy, and six days prior to parturition (estrous cycle GC, 14.4-62.8 ng/g; pregnancy GC, 13.6-232.7 ng/g). CONCLUSIONS: Estrous cycles of giant anteaters occurred year-round, but were shorter and more intermittent in younger nulliparous animals compared to a multiparous female. A pronounced elevation in fecal P4, estrogen, and GC occurred during late gestation after an initial post-mating delay providing evidence for delayed implantation. Adrenocorticoid activity indicated impending parturition. Differences in estrous cycle characteristics with age and the protracted but variable gestation length must be considered to improve reproductive success and neonatal survival in giant anteaters.