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Ex situ conservation breeding for species recovery often must address small founder number. Here we investigated the long‐term effects of small founder size in the endangered black‐footed ferret Mustela nigripes using 20 years of reproductive data. Specifically, we studied seminal traits, pregnancy success and litter size in the context of the population's low genetic diversity. Detailed evaluation of 539 electroejaculates from 262 pedigreed donors with varied inbreeding coefficients (F = 0.000–0.250) revealed a wide range in sperm motility (0.0–100.0%), forward progression (type of forward movement on a scale 0–5; 5 = best; 0.0–4.5), cell concentration (7.2–6348.0 × 106 mL−1 of ejaculate), structural normality (0.0–89.0%) and normal apical ridges (critical for fertilization; 4.8–100.0%). Over this two decade interval, there was an increase (P < 0.05) in F and a decrease (P < 0.05) in sperm forward progression and structurally normal cell morphology. From analysis of artificial insemination (AI) data, higher values for sperm motility, forward progression and structural normality or spermatozoa that retained a normal apical ridge had a positive influence (P < 0.05) on litter size. Males producing higher values for these four traits also sired more kits. Results demonstrate that sperm motility and morphology are decreasing in black‐footed ferrets managed intensively ex situ. These two metrics appear associated with an increasing F and likely are at least partially responsible for lowered reproductive success in this closed, endangered population. These results illustrate the potential physiological and offspring production impact of having few founders available in an ex situ conservation breeding program. [ABSTRACT FROM AUTHOR]

One of the most significant challenges in the recovery of threatened species is the ability to maintain genetic diversity, avoid inbreeding and sustain population health and reproduction. Assisted reproductive techniques, including artificial insemination ( AI), have been touted for decades as approaches that could contribute to the demographic and genetic management of rare species. Here, we report the first successful integration of AI with frozen semen into a formal recovery program and the positive impact on genetic diversity for the critically endangered black-footed ferret M ustela nigripes. Techniques developed in the taxonomically related domestic ferret Mustela putorius furo and Siberian polecat M ustela eversmannii were applied over time to selected black-footed ferrets, including semen banking from six of the last 18 survivors. After evaluation, processing and storage in liquid nitrogen (−196° C/−321° F), for as long as 20 years, sperm samples were thawed and transabdominally inseminated into the uterine horns of female conspecifics. Eight black-footed ferret offspring were produced using thawed sperm samples (including after two decades of cryopreservation) with inseminates containing as few as 3.4 × 106 motile spermatozoa. The incorporation of these offspring and/or their descendants into the ex situ breeding program prevented heterozygosity loss in the population and actually enhanced gene diversity ( GD) significantly by 0.2% and lowered measures of inbreeding by 5.8%. This study demonstrates the utility and genetic diversity benefits of applying AI with cryopreserved spermatozoa 20 generations removed from the contemporary population for a wild animal revival program. [ABSTRACT FROM AUTHOR]

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., (© The Author(s) 2023. Published by Oxford University Press on behalf of The American Genetic Association. All rights reserved. For commercial re-use, please contact reprints@oup.com for reprints and translation rights for reprints. All other permissions can be obtained through our RightsLink service via the Permissions link on the article page on our site—for further information please contact journals.permissions@oup.com.)

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., (© 2024. The Author(s).)

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., Competing Interests: The authors have no known conflicts of interest., (© 2024 The Authors. Evolutionary Applications published by John Wiley & Sons Ltd.)

Millions of people globally depend on camelids, which demands an increased knowledge of their reproduction. We used zoo-housed Bactrian camels (Camelus bactrianus) to better understand camelid reproductive physiology. Our specific objectives were to: 1) validate the use of fecal hormone metabolite analysis to characterize camel reproductive physiology during sexual maturity and pregnancy; and 2) determine the influence of season on male and female reproduction. We collected fecal samples from 1 male and 3 females housed at Lincoln Park Zoo (Chicago, IL, USA) 1 to 2 times per week for 3.5 years. Extracted hormones were analyzed using enzyme immunoassays for progestogen (FPM), estrogen (FEM), and androgen (FAM) metabolite concentrations. One female sexually matured during our study as evidenced by increased FEM baseline. Results demonstrated seasonal effects on male androgen production with FAMs higher (P < 0.05) January to June (mean ± SEM: 664.6 ± 22.6 ng/g wet feces), compared to July to December (401.6 ± 17.5 ng/g wet feces). One female experienced a persistent corpus luteum, a reproductive abnormality, which was identified by prolonged elevated FPM. FPMs increased during pregnancy for two females (452.9 ± 24.9 and 294.4 ± 19.8 ng/g wet feces) with a gestation of 404 d and 442 d, respectively. The third female never conceived. The FEMs varied (P < 0.05) during the year with no clear seasonal patterns (monthly mean range: 213.1-371.0 ng/g wet feces). Fecal hormone metabolite analysis is a validated method for assessing male seasonality and female pregnancy in the Bactrian camel and can for their management and conservation in zoos and the wild., (Copyright © 2022 Elsevier Inc. All rights reserved.)

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., (© 2021 Wiley Periodicals LLC.)

Living in a rapidly changing environment can alter stress physiology at the population level, with negative impacts on health, reproductive rates, and mortality that may ultimately result in species decline. Small, isolated animal populations where genetic diversity is low are at particular risks, such as endangered Virunga mountain gorillas ( Gorilla beringei beringei ). Along with climate change-associated environmental shifts that are affecting the entire population, subpopulations of the Virunga gorillas have recently experienced extreme changes in their social environment. As the growing population moves closer to the forest's carrying capacity, the gorillas are coping with rising population density, increased frequencies of interactions between social units, and changing habitat use (e.g., more overlapping home ranges and routine ranging at higher elevations). Using noninvasive monitoring of fecal glucocorticoid metabolites (FGM) on 115 habituated Virunga gorillas, we investigated how social and ecological variation are related to baseline FGM levels, to better understand the adaptive capacity of mountain gorillas and monitor potential physiological indicators of population decline risks. Generalized linear mixed models revealed elevated mean monthly baseline FGM levels in months with higher rainfall and higher mean maximum and minimum temperature, suggesting that Virunga gorillas might be sensitive to predicted warming and rainfall trends involving longer, warmer dry seasons and more concentrated and extreme rainfall occurrences. Exclusive use of smaller home range areas was linked to elevated baseline FGM levels, which may reflect reduced feeding efficiency and increased travel efforts to actively avoid neighboring groups. The potential for additive effects of stress-inducing factors could have short- and long-term impacts on the reproduction, health, and ultimately survival of the Virunga gorilla population. The ongoing effects of environmental changes and population dynamics must be closely monitored and used to develop effective long-term conservation strategies that can help address these risk factors.

In the current global health climate, many conservation biologists are managing crisis situations, including increased species extinction rates. One strategy for securing wildlife populations into the future is to preserve biomaterials in genome resource banks (GRB; or 'biobanks'). However, for GRBs to be successful we must understand the fundamental reproductive biology of species, along with developing assisted reproductive techniques (ARTs), including AI and semen cryopreservation. ART has been successfully used for several taxa, from amphibians to mammals, including ungulates, carnivores and primates. Not all these success stories implemented the use of a biobank, but one example that discussed herein is the black-footed ferret (Mustela nigripes) GRB. From a founder population of seven individuals, this species has been breeding in a managed setting for nearly 30 years. The goal of the breeding program is to maintain genetic integrity by ensuring each individual has the opportunity to pass his/her genes onto the next generation, while simultaneously providing animals for release into the wild. Scientists have used ART (e.g. AI) in the recovery program. Recently, semen from an individual of the founder population that was cryopreserved for up to 20 years was used successfully for AI, which improved the genetic diversity of the population. The black-footed ferret recovery program can serve as a model for other endangered species and demonstrates the usefulness of ART and GRBs to maintain highly endangered species into the future.