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.

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.

Widespread Behavioral Responses by Mammals and Fish to Zoo Visitors Highlight Differences between Individual Animals.

The impact that humans have on zoo animals can vary based on the species of animal, exhibit design, and individual differences in behavioral responses. We independently analyzed data from 10 never-published studies that examined the impact of zoo visitors on zoo animal behavior. Of the 16 species studied, 90.9% of the mammal species and 60.0% of the fish species demonstrated a change in at least one behavior based on zoo visitor abundance or visitor behavior (e.g., noise, solicitation of interactions from zoo animals). In addition, behavioral changes associated with zoo visitors were present in animals housed in exhibits where there was direct contact with zoo visitors, as well as in exhibits where there was indirect contact and no direct contact. Individuals often varied in their behavioral responses, and some individuals appeared to seek out interactions with visitors. Our findings demonstrate that short-term research projects can provide valuable insight into individual animal-level and species-level responses to visitor abundance and visitor behavior in the zoo setting. We recommend that behavioral assessments focus on the analysis of behaviors of individual animals whenever possible, and we recommend that exhibits provide areas that allow for animals to retreat from the public view.