Semen proteome and transcriptome of the endangered black-footed ferret (Mustela nigripes) show association with the environment and fertility outcome.

The ex situ population of the endangered black-footed ferret (Mustela nigripes) has been experiencing declines in reproductive success over the past 30 years of human-managed care. A potential cause may be environmental-dependent inbreeding depression with diet being one of the contributing factors since ferrets are not fed their natural diet of prairie dogs. Here, we generated and analyzed semen proteome and transcriptome data from both wild and ex situ ferrets maintained on various diets. We identified 1757 proteins across all samples, with 149 proteins unique to the semen of wild ferrets and forming a ribosomal predicted protein-protein interaction cluster. Wild ferrets also differed from ex situ ferrets in their transcriptomic profile, showing enrichment in ribosomal RNA processing and potassium ion transport. Successful fertility outcomes documented for ex situ ferrets showed the strongest association with the semen transcriptome, with enrichment in genes involved in translation initiation and focal adhesion. Fertility also synergized with the effect of diet on differentially expressed transcriptomes, mainly affecting genes enriched in mitochondrial function. Our data and functional networks are important for understanding the causes and mechanisms of declining fertility in the ex situ ferret population and can be used as a resource for future conservation efforts.

Markers of fertility in reproductive microbiomes of male and female endangered black-footed ferrets (Mustela nigripes).

Reproductive microbiomes contribute to reproductive health and success in humans. Yet data on reproductive microbiomes, and links to fertility, are absent for most animal species. Characterizing these links is pertinent to endangered species, such as black-footed ferrets (Mustela nigripes), whose populations show reproductive dysfunction and rely on ex-situ conservation husbandry. To understand microbial contributions to animal reproductive success, we used 16S rRNA amplicon sequencing to characterize male (prepuce) and female (vaginal) microbiomes of 59 black-footed ferrets at two ex-situ facilities and in the wild. We analyzed variation in microbiome structure according to markers of fertility such as numbers of viable and non-viable offspring (females) and sperm concentration (males). Ferret vaginal microbiomes showed lower inter-individual variation compared to prepuce microbiomes. In both sexes, wild ferrets harbored potential soil bacteria, perhaps reflecting their fossorial behavior and exposure to natural soil microbiomes. Vaginal microbiomes of ex-situ females that produced non-viable litters had greater phylogenetic diversity and distinct composition compared to other females. In males, sperm concentration correlated with varying abundances of bacterial taxa (e.g., Lactobacillus), mirroring results in humans and highlighting intriguing dynamics. Characterizing reproductive microbiomes across host species is foundational for understanding microbial biomarkers of reproductive success and for augmenting conservation husbandry.

Epigenetic changes to gene pathways linked to male fertility in ex situ black-footed ferrets.

Environmental variation can influence the reproductive success of species managed under human care and in the wild, yet the mechanisms underlying this phenomenon remain largely mysterious. Molecular mechanisms such as epigenetic modifiers are important in mediating the timing and progression of reproduction in humans and model organisms, but few studies have linked epigenetic variation to reproductive fitness in wildlife. Here, we investigated epigenetic variation in black-footed ferrets (Mustela nigripes), an endangered North American mammal reliant on ex situ management for survival and persistence in the wild. Despite similar levels of genetic diversity in human-managed and wild-born populations, individuals in ex situ facilities exhibit reproductive problems, such as poor sperm quality. Differences across these settings suggest that an environmentally driven decline in reproductive capacity may be occurring in this species. We examined the role of DNA methylation, one well-studied epigenetic modifier, in this emergent condition. We leveraged blood, testes, and semen samples from male black-footed ferrets bred in ex situ facilities and found tissue-type specificity in DNA methylation across the genome, although 1360 Gene Ontology terms associated with male average litter size shared functions across tissues. We then constructed gene networks of differentially methylated genomic sites associated with three different reproductive phenotypes to explore the putative biological impact of variation in DNA methylation. Sperm gene networks associated with average litter size and sperm count were functionally enriched for candidate genes involved in reproduction, development, and its regulation through transcriptional repression. We propose that DNA methylation plays an important role in regulating these reproductive phenotypes, thereby impacting the fertility of male ex situ individuals. Our results provide information into how DNA methylation may function in the alteration of reproductive pathways and phenotypes in artificial environments. These findings provide early insights to conservation hurdles faced in the protection of this rare species.

Gut bacterial composition shows sex-specific shifts during breeding season in ex situ managed black-footed ferrets.

The gut microbiome of mammals engages in a dynamic relationship with the body and contributes to numerous physiological processes integral to overall health. Understanding the factors shaping animal-associated bacterial communities is therefore paramount to the maintenance and management in ex situ wildlife populations. Here, we characterized the gut microbiome of 48 endangered black-footed ferrets (Mustela nigripes) housed at Smithsonian's National Zoo and Conservation Biology Institute (Front Royal, Virginia, USA). We collected longitudinal fecal samples from males and females across two distinct reproductive seasons to consider the role of host sex and reproductive physiology in shaping bacterial communities, as measured using 16S rRNA amplicon sequencing. Within each sex, gut microbial composition differed between breeding and non-breeding seasons, with five bacterial taxa emerging as differentially abundant. Between sexes, female and male microbiomes were similar during non-breeding season but significantly different during breeding season, which may result from sex-specific physiological changes associated with breeding. Finally, we found low overall diversity consistent with other mammalian carnivores alongside high relative abundances of potentially pathogenic microbes such as Clostridium, Escherichia, Paeniclostridium, and (to a lesser degree) Enterococcus - all of which have been associated with gastrointestinal or reproductive distress in mammalian hosts, including black-footed ferrets. We recommend further study of these microbes and possible therapeutic interventions to promote more balanced microbial communities. These results have important implications for ex situ management practices that can improve the gut microbial health and long-term viability of black-footed ferrets.

Exploring the Effect of Age on the Reproductive and Stress Physiology of Octopus bimaculoides Using Dermal Hormones.

Our goal was to validate the use of dermal swabs to evaluate both reproductive and stress physiology in the California two-spot octopus, Octopus bimaculoides. Our objectives were to (1) use dermal swabs to evaluate glucocorticoids and reproductive hormones of O. bimaculoides; (2) determine the influence of life stage on hormone production (glucocorticoids in all individuals; testosterone, estrogen, and progesterone in females; and testosterone in males) of reproductive (n = 4) and senescent (n = 8) individuals to determine the effect of age on hormonal patterns; and (3) determine whether these hormones change significantly in response to an acute stressor. For the stress test, individuals were first swabbed for a baseline and then chased around the aquarium with a net for 5 min. Afterward, individuals were swabbed for 2 h at 15 min intervals to compare to the pre-stress test swab. Reproductive individuals responded to the stressor with a 2-fold increase in dermal cortisol concentrations at 15 and 90 min. Six of the eight senescent individuals did not produce a 2-fold increase in dermal cortisol concentrations. Reproductive individuals had significantly higher sex hormone concentrations compared to senescent individuals (progesterone and estradiol measured in females, and testosterone for both sexes). After the stressor, only reproductive males produced a 2-fold increase in dermal testosterone concentrations, while sex hormones in females showed no change. The stress hormone cortisol was significantly higher in senescent than in reproductive individuals, independent of sex. Dermal corticosterone concentrations were highest in senescent females followed by senescent males, and lowest in reproductive individuals regardless of sex. Dermal swabs provide an effective and noninvasive means for evaluating octopus hormones. Application of these indicators may be imperative as cephalopods are more commonly cultured in captivity for experimentation, display, and consumption.

An empirical comparison of several commercial enzyme immunoassays for the non-invasive assessment of adrenocortical and gonadal function in mountain gorillas.

Wildlife researchers seeking to non-invasively examine endocrine function in their study species are presented with a dense and technical 'garden of forking paths' to navigate between collecting a biological sample and obtaining a final measurement. In particular, the choice of which enzyme immunoassay (EIA) to use with collected fecal samples, out of the many options offered by different manufacturers and research laboratories, may be one of the most consequential for final results. However, guidance for making this decision is still emerging. With this gap in mind, we performed a head-to-head comparison of results obtained from four different EIAs for fecal glucocorticoid metabolites (FGCMs), and three different EIAs for fecal androgen metabolites (FAMs), applied to the same set of fecal samples collected from the mountain gorillas (Gorilla beringei beringei) monitored by the Dian Fossey Gorilla Fund in Volcanoes National Park, Rwanda. We provide a) an analytical validation of the different EIAs via tests of parallelism and linearity; b) an estimate of inter-assay correlation between EIA kits designed for the same metabolites; and c) a test of the kits' ecological validity, in which we examine how well each captures endocrine changes following events that theory predicts should result in elevated FGCM and/or FAM concentrations. Our results show that kits differ to some degree in their performance; at the same time, nearly all assays exhibited at least moderate evidence of validity and covariance with others for the same analyte. Our findings, which differ somewhat from similar comparisons performed in other species, demonstrate the need to directly assess assay performance in a species- and context-specific manner as part of efforts to develop the burgeoning discipline of wildlife endocrinology.

Integrating Measures of Fecal Glucocorticoid Metabolites and Giving-Up Densities to Assess Adrenocortical Activity and Well-Being in Zoo-Housed Three-Banded Armadillos (Tolypeutes matacus).

To monitor adrenocortical activity in zoo-housed species, we propose using physiological and behavioral indicators that are non-invasive and practical to implement. We explore this model in the southern three-banded armadillo (Tolypeutes matacus; armadillo), which is a near-threatened species commonly found in zoos. We aimed to (1) deploy food patches to quantify foraging behavior (via giving-up densities, GUDs); (2) determine the effects of food patch and environmental modifications on individuals' GUDs and adrenocortical activity (via fecal glucocorticoid metabolites, FGMs); and (3) examine the relationship between GUDs and FGMs. Three males and four females received food patches under varying experimental conditions at the Lincoln Park Zoo (Chicago, IL, USA). Fecal samples were collected before, during, and after foraging experiments to examine FGMs. Armadillos did not respond to patch modifications but did forage more when given increased cover. Individual mean FGMs and GUDs were highly variable, and individuals had consistent FGM and GUD ranks across experiments. FGMs and GUDs did not vary across the experiments nor did they relate to each other. Armadillos and species with a limited behavioral repertoire (i.e., constant movement) can benefit from this multi-trait model to determine the effect of environmental modifications on individuals and provide meaningful information about adrenocortical activity.

Designing an assay to evaluate behavioral responses to opposite-sex conspecifics in the endangered black-footed ferret (Mustela nigripes).

The endangered black-footed ferret (ferret; Mustela nigripes) is a North American carnivore that is actively managed to reestablish self-sustaining wild populations. Behavioral abnormalities have been reported in the breeding program and may be a limiting factor for the species' success. Our goal was to design and test an assay that examines the ferret's exploratory response to odor cues in the form of soiled bedding from opposite-sex conspecifics. Across two breeding seasons, males and females were tested using a T-maze that connected their home nest box to two novel nest boxes containing two different conspecific's soiled bedding. For a control, we provided two clean bedding samples. We ran linear mixed models to determine the effect of sex, type of odor cue (soiled, clean), and order of trial (first, second) on time exploring and proportion of that time spent in each behavior. Ferrets spent the majority of time in the novel nest boxes sniffing (44%), standing alert (27%) and scratching (14%). Males explored for longer than females; however, both displayed similar behaviors. Type of cue influenced behavior, with ferrets sniffing more among soiled cues than clean cues. Habituation to the assay was also observed, with less exploration and more standing alert during the second trial of the day. This study is the first step in characterizing the ferret's exploratory response and provides information regarding vital investigatory and vigilance behaviors. The continual development of this assay to further evaluate reproductive and mate choice behaviors will facilitate more successful breeding of the species.

A novel field method for preserving African lion (Panthera leo) fecal samples for noninvasive hormone metabolite analysis.

The traditional methods of preserving fecal samples to suspend hormone-degrading bacteria are not always options during remote fieldwork when studying wildlife endocrinology. Our goal was to develop a field method for preserving African lion (Panthera leo) feces for hormone metabolite analysis by determining the stability of fecal hormone metabolites: 1) when exposed to the natural environment, and 2) in silica beads at ambient temperatures. We collected fresh feces from zoo-housed lions and combined them into two (male and female) homogenous samples. Each was divided into eight samples to undergo a cross-designed treatment.•We immediately lyophilized one sample to serve as the control.•We then exposed seven samples outside to natural environmental conditions for 0, 12, 24, 36, 48, 60, or 72 h. After outdoor exposure, we desiccated a subsample in silica beads for an additional 5, 7, or 10 days.•We analyzed the fecal hormone metabolite concentrations in each sample using enzyme immunoassays for corticosterone, cortisol, testosterone, progesterone, and estradiol.We determined that male and female fecal hormone metabolites in fresh African lion fecal samples are stable and comparable to a standardized desiccation method if dried in silica beads for 5 to 10 days prior to storing them at -20℃.

Preliminary Investigation into Developing the Use of Swabs for Skin Cortisol Analysis for the Ocean Sunfish (Mola mola).

The ocean sunfish (mola; Mola mola) is the heaviest bony fish in the world. This slow-moving fish often is injured by fishing boats that use drift gillnets attributing to its listing as Vulnerable by the IUCN. The Monterey Bay Aquarium (Monterey, CA, USA) has a program that brings in smaller molas from the ocean and acclimates them for an exhibit. When they grow too large for the million-gallon Open Seas exhibit, they are returned to Monterey Bay through a "reverse" acclimatization. Our overall goal was to use skin swabs to evaluate mola stress physiology to better understand the effects of this program. Our objectives were to validate this non-invasive method by taking opportunistic swabs throughout acclimatization and during stressful events. We swabbed each individual (n = 12) in three different body locations. Swabs were analyzed using a cortisol enzyme immunoassay. We averaged the three swabs and examined the absolute change of cortisol from the first taken upon handling to during treatments and the different acclimation stages. We considered elevated cortisol concentrations to be ≥1.5-fold higher than the first sample. Overall, mean (±SEM) cortisol varied among individuals (564.2 ± 191.5 pg/mL swab (range, 18.3-7012.0 pg/mL swab). The majority (four of six) of molas swabbed within the first week or month had elevated skin cortisol compared to their first sample. All seven molas that were being treated for an injury or illness had elevated skin cortisol (range, 1.7- to 127.6-fold higher) compared to their post-acclimation sample. This is the first step in validating the use of non-invasive skin swabs for glucocorticoid analysis in the mola. Further biochemical analysis is needed to determine the specific steroids that are being measured.

RECOMBITEK CANINE DISTEMPER VACCINE AS AN ALTERNATIVE FOR PUREVAX DISTEMPER VACCINE IN ENDANGERED BLACK-FOOTED FERRETS (MUSTELA NIGRIPES).

Black-footed ferrets (Mustela nigripes) are an endangered species in North America that are highly sensitive to canine distemper virus (CDV) infections and any exposure could be devastating to species recovery. The U.S. Fish and Wildlife Service recovery program has safely used a recombinant DNA (rDNA) canarypox-vectored CDV vaccine, Purevax® Ferret Distemper (PFD), to vaccinate black-footed ferrets. Because of a PFD shortage in 2015, an rDNA vaccine labeled for use in dogs, Recombitek® CDV (rCDV), was chosen to vaccinate black-footed ferrets. Our goal was to compare the serum neutralizing (SN) titers after vaccination of 17 captive and 18 wild black-footed ferrets with rCDV or PFD, respectively, considering ≥1:128 as a protective titer. Both vaccines produced comparable 1 yr postvaccination protective titers in captive and wild black-footed ferrets. In wild black-footed ferrets, one PFD vaccination produced SN titers similar to two PFD vaccinations at 1 yr postvaccination. One year after vaccination with rCDV, SN titers in captive black-footed ferrets were higher than in wild ferrets. These results indicate rCDV may be an effective alternative CDV vaccine in captive black-footed ferrets and PFD should be prioritized for wild ferrets because one dose was effective for animals that can be difficult to recapture.

The Gombe Ecosystem Health Project: 16 years of program evolution and lessons learned.

Infectious disease outbreaks pose a significant threat to the conservation of chimpanzees (Pan troglodytes) and all threatened nonhuman primates. Characterizing and mitigating these threats to support the sustainability and welfare of wild populations is of the highest priority. In an attempt to understand and mitigate the risk of disease for the chimpanzees of Gombe National Park, Tanzania, we initiated a long-term health-monitoring program in 2004. While the initial focus was to expand the ongoing behavioral research on chimpanzees to include standardized data on clinical signs of health, it soon became evident that the scope of the project would ideally include diagnostic surveillance of pathogens for all primates (including people) and domestic animals, both within and surrounding the National Park. Integration of these data, along with in-depth post-mortem examinations, have allowed us to establish baseline health indicators to inform outbreak response. Here, we describe the development and expansion of the Gombe Ecosystem Health project, review major findings from the research and summarize the challenges and lessons learned over the past 16 years. We also highlight future directions and present the opportunities and challenges that remain when implementing studies of ecosystem health in a complex, multispecies environment.

Determining the effectiveness of using dermal swabs to evaluate the stress physiology of laboratory cephalopods: A preliminary investigation.

To better comprehend the physiology of cephalopods, we used a minimal invasive technique of skin mucus swabs to measure immunoreactive corticosteroids in three cephalopod species commonly kept in captivity and promoted as new model organisms: Euprymna berryi, Sepia bandensis, and Octopus chierchiae. We compared results between sexes and age classes and then evaluated their stress responses during acclimation to a new habitat. To better understand glucocorticoid production, we conducted an adrenocorticotropic hormone, using Cosyntropin (an adrenocorticotropin (ACTH) analogue) challenge with a saline control and swabbed their mantles at 15-minute intervals for 2 h. Results showed cortisol was higher for younger individuals. Additionally, cortisol and corticosterone concentrations decreased by 2-fold after 2 to 4 days of acclimation to a new habitat. We were able to successfully measure 2-fold increase in immunoreactive corticosteroids which reacted with cortisol and corticosterone assays for all the species following ACTH injection, although not all individuals responded similarly. With further investigation, this technique can increase our understanding and management of cephalopods in captivity.

Group structure, but not dominance rank, predicts fecal androgen metabolite concentrations of wild male mountain gorillas (Gorilla beringei beringei).

Androgens are important mediators of male-male competition in many primate species. Male gorillas' morphology is consistent with a reproductive strategy that relies heavily on androgen-dependent traits (e.g., extreme size and muscle mass). Despite possessing characteristics typical of species with an exclusively single-male group structure, multimale groups with strong dominance hierarchies are common in mountain gorillas. Theory predicts that androgens should mediate their dominance hierarchies, and potentially vary with the type of group males live in. We validated the use of a testosterone enzyme immunoassay (T-EIA R156/7, CJ Munro, UC-Davis) for use with mountain gorilla fecal material by (1) examining individual-level androgen responses to competitive events, and (2) isolating assay-specific hormone metabolites via high-performance liquid chromatography. Males had large (2.6- and 6.5-fold), temporary increases in fecal androgen metabolite (FAM) after competitive events, and most captured metabolites were testosterone or 5α-dihydrotestosterone-like androgens. We then examined the relationship between males' dominance ranks, group type, and FAM concentrations. Males in single-male groups had higher FAM concentrations than males in multimale groups, and a small pool of samples from solitary males suggested they may have lower FAM than group-living peers. However, data from two different time periods (n = 1610 samples) indicated there was no clear relationship between rank and FAM concentrations, confirming results from the larger of two prior studies that measured urinary androgens. These findings highlight the need for additional research to clarify the surprising lack of a dominance hierarchy/androgen relationship in mountain gorillas.

Validating the use of fecal glucocorticoid metabolite analysis to assess the adrenocortical activity of the zoo-housed Sichuan takin (Budorcas taxicolor tibetana).

With limited information known about the zoo-housed Sichuan takin (Budorcas taxicolor tibetana), there is a need to gain more knowledge about their basic physiology to be able to better assess their well-being. Our goal was to develop noninvasive methods to evaluate stress physiology in the Sichuan takins housed under human care. The objectives were: (1) validate the use of fecal glucocorticoid metabolite (FGM) analysis to monitor adrenocortical activity and (2) determine the relationship between FGM concentrations and changes in various factors including environmental conditions, reproductive hormones, and social factors. Three (one male and two females) adult Sichuan takins were included in the study from Lincoln Park Zoo. A cortisol enzyme immunoassay was used to analyze FGM from samples that were collected 2-4 times per week. FGM was biochemically validated in the laboratory and biologically validated using pregnancy and parturition. Results showed that 1 day after birth one female had a more than a six-fold increase in FGM. A positive relationship (p < .05) was observed between FGM and temperature for two of the three takins. Finally, FGM values tended to increase during times when aggression (p = .09) and reproductive (p = .08) behaviors were observed. In conclusion, environmental, reproductive, and behavioral factors could affect adrenocortical activity in zoo-housed Sichuan takins; therefore, these methods can be used to assist in the management and conservation of this threatened species both in zoos and potentially in the wild.

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.

Corticosterone Metabolite Concentration Is Not Related to Problem Solving in the Fawn-Footed Mosaic-Tailed Rat Melomys Cervinipes.

Animals can respond physiologically, such as by adjusting glucocorticoid hormone concentrations, to sudden environmental challenges. These physiological changes can then affect behavioural and cognitive responses. While the relationships between adrenocortical activity and behaviour and cognition are well documented, results are equivocal, suggesting species-specific responses. We investigated whether adrenocortical activity, measured using corticosterone metabolite concentration, was related to problem solving in an Australian rodent, the fawn-footed mosaic-tailed rat (Melomys cervinipes). Mosaic-tailed rats live in complex environments that are prone to disturbance, suggesting a potential need to solve novel problems, and have been found to show relationships between physiology and other behaviours. We measured problem solving using five food-baited puzzles (matchbox and cylinder in the home cage, and activity board with pillars to push, tiles to slide and levers to lift in an open field), and an escape-motivated obstruction task in a light/dark box. Faecal samples were collected from individuals during routine cage cleaning. Adrenocortical activity was evaluated non-invasively by measuring faecal corticosterone metabolites using an enzyme immunoassay, which was biochemically and biologically validated. Despite varying over time, adrenocortical activity was not significantly related to problem solving success or time spent interacting for any task. However, as adrenocortical activity is reflective of multiple physiological processes, including stress and metabolism, future studies should consider how other measures of physiology are also linked to problem solving.

Influence of vitamin E and carcass feeding supplementation on fecal glucocorticoid and androgen metabolites in male black-footed ferrets (Mustela nigripes).

In recent years, the ex situ population of the endangered black-footed ferret (Mustela nigripes; ferret) has experienced a decline in normal sperm morphology (from 50% to 20%), which may be linked to inbreeding depression and/or a dietary change. We examined the effects of adding carcass and vitamin E to the diet on stress and reproductive biomarkers in male ferrets (n = 42 males including 16 juveniles and 26 adults) housed at the U.S. Fish and Wildlife National Black-footed Ferret Conservation Center (Carr, CO, USA). Fecal samples (3x/week) were collected from November and December (pre-breeding season, no diet change), February through May (breeding season, diet change) and June (post-breeding season, diet change) and analyzed for fecal glucocorticoid metabolites (FGM) via a corticosterone enzyme immunoassay (EIA). A subset of samples from adult males (n = 15) were analyzed for fecal androgen metabolites (FAM) via a testosterone EIA. We first used a linear mixed effects model to identify the important fixed effects among meat treatment, vitamin E treatment, age class (juvenile or adult), and all possible interactions on each hormone. We then examined the important factor's effects across seasons using the non-parametric Friedman test. We found that age did not influence (p = 0.33) FGMs; however there was a significant effect of meat treatment on FGM (p = 0.04) and an effect of vitamin E on FAMs (p<0.10). When fed carcass, FGMs declined (p<0.001) from pre- to the during the breeding season time period, but was similar (p>0.05) between during and post-breeding season periods. Males that were not fed carcass had higher (p<0.05) FGMs during the breeding season compared to pre- and post-breeding season and FGMs were lower (p<0.05) in the post-breeding season compared to pre-breeding season. Males fed with carcass had lower (p<0.001) FGM than males that were not fed carcass during both the pre-breeding and the breeding season but not during the post-breeding season (p>0.05). Males supplemented with vitamin E had higher (p<0.001) FAM than non-supplemented males during the breeding season only. For both groups, FAM was highest (p<0.05) during the breeding season. In conclusion, adding carcass to the diet can reduce glucocorticoid production and adding vitamin E can increase testosterone during the breeding season, which may influence reproductive success.

City sanitation and socioeconomics predict rat zoonotic infection across diverse neighbourhoods.

Rat-associated zoonoses transmitted through faeces or urine are of particular concern for public health because environmental exposure in homes and businesses may be frequent and undetected. To identify times and locations with greater public health risks from rats, we investigated whether rat characteristics, environmental features, socioeconomic factors, or season could predict rat infection risk across diverse urban neighbourhoods. In partnership with a pest management company, we sampled rats in 13 community areas along an income gradient in Chicago, a large city where concern about rats has increased in recent years. We collected kidneys for Leptospira spp. testing and colon contents for aerobic bacteria such as Salmonella spp. and Escherichia coli. Of 202 sampled rats, 5% carried Leptospira spp. and 22% carried E. coli. Rats were significantly more likely to carry Leptospira spp. on blocks with more standing water complaints in higher-income neighbourhoods (OR = 6.74, 95% CI: 1.54-29.39). Rats were significantly more likely to carry E. coli on blocks with more food vendors (OR = 9.94, 2.27-43.50) particularly in low-income neighbourhoods (OR = 0.26, 0.09-0.82) and in the spring (OR = 15.96, 2.90-88.62). We detected a high diversity of E. coli serovars but none contained major virulence factors. These associations between environmental features related to sanitation and infection risk in rats support transmission through water for Leptospira spp. and faecal-oral transmission for E. coli. We also found opposing relationships between zoonotic infection risk and income for these two pathogens. Thus, our results highlight the importance of sanitation for predicting zoonotic disease risks and including diverse urban areas in pathogen surveillance to mitigate public health risks from rats.

Factors affecting reproductive traits in male snow leopards (Unciauncia).

ABSTRACT: The population of snow leopards (Unciauncia) maintained in US zoos is no longer sustainable due to poor reproductive success. Our objective was to assess reproductive traits in male snow leopards and identify factors (markers of oxidative stress in seminal fluid, surveys of husbandry practices, gonadal and adrenocortical activity, dietary intake of various nutrients, and genetics) that may affect ejaculate traits and subsequent fertility. Ejaculates (2.9 ± 0.2 mL) from 32 male snow leopards (9.8 ± 0.7 years; 38.6 ± 0.8 kg) housed at 27 institutions contained 119.2 + 26.0 x 106 spermatozoa, of which 75.1 ± 2.3% were motile and 28.6 ± 2.6% exhibited normal morphology. Overall, 34% of males produced <5 million spermatozoa and 27% of males produced spermatozoa with <20% normal morphology. Activity of superoxide dismutase (SOD) in the seminal fluid was negatively correlated (P < 0.05, r2 = 0.90) with normal sperm morphology. Husbandry practices, mean concentrations of fecal androgen metabolites (fAM), and baseline concentrations of fecal glucocorticoid metabolites (fGM), inbreeding coefficients, and generations each male was removed from the founders in their lineages were not correlated (P > 0.05) with the total number of spermatozoa or the proportion of spermatozoa with normal morphology. Total sperm count was positively correlated (P < 0.05, R2 = 0.86) with the weekly intake of polyunsaturated fatty acids (PUFA) and the proportion of spermatozoa with normal morphology tended (P < 0.10, R2 = 0.31) to be positively correlated with copper intake. Altering the nutrient composition of snow leopard diets could provide managers with a possible method of improving reproductive traits in this endangered species. LAY SUMMARY: The population of snow leopards (Uncia uncia) maintained in US zoos has been declining since 1993 due to poor breeding success. Our objective was to assess the reproductive traits of male snow leopards and identify factors (e.g. hormones, diet, genetics) that may be affecting the quality of semen produced and therefore subsequent fertility. Within a cohort of 32 male snow leopards maintained at 27 US zoos, we found that 34% produced less than 5 million sperm and 27% of males produced sperm where less than 20% looked normal. The quantity and quality of the recovered sperm was not correlated with husbandry practices, concentrations of hormones (androgens and glucocorticoids) in feces, or genetics. However, the number of sperm was positively correlated with polyunsaturated fatty acids in the diet. Altering the nutrient composition of snow leopard diets could provide managers with a possible method of improving reproductive traits in this endangered species.