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.

Integrating microbiome science and evolutionary medicine into animal health and conservation.

Microbiome science has provided groundbreaking insights into human and animal health. Similarly, evolutionary medicine - the incorporation of eco-evolutionary concepts into primarily human medical theory and practice - is increasingly recognised for its novel perspectives on modern diseases. Studies of host-microbe relationships have been expanded beyond humans to include a wide range of animal taxa, adding new facets to our understanding of animal ecology, evolution, behaviour, and health. In this review, we propose that a broader application of evolutionary medicine, combined with microbiome science, can provide valuable and innovative perspectives on animal care and conservation. First, we draw on classic ecological principles, such as alternative stable states, to propose an eco-evolutionary framework for understanding variation in animal microbiomes and their role in animal health and wellbeing. With a focus on mammalian gut microbiomes, we apply this framework to populations of animals under human care, with particular relevance to the many animal species that suffer diseases linked to gut microbial dysfunction (e.g. gut distress and infection, autoimmune disorders, obesity). We discuss diet and microbial landscapes (i.e. the microbes in the animal's external environment), as two factors that are (i) proposed to represent evolutionary mismatches for captive animals, (ii) linked to gut microbiome structure and function, and (iii) potentially best understood from an evolutionary medicine perspective. Keeping within our evolutionary framework, we highlight the potential benefits - and pitfalls - of modern microbial therapies, such as pre- and probiotics, faecal microbiota transplants, and microbial rewilding. We discuss the limited, yet growing, empirical evidence for the use of microbial therapies to modulate animal gut microbiomes beneficially. Interspersed throughout, we propose 12 actionable steps, grounded in evolutionary medicine, that can be applied to practical animal care and management. We encourage that these actionable steps be paired with integration of eco-evolutionary perspectives into our definitions of appropriate animal care standards. The evolutionary perspectives proposed herein may be best appreciated when applied to the broad diversity of species under human care, rather than when solely focused on humans. We urge animal care professionals, veterinarians, nutritionists, scientists, and others to collaborate on these efforts, allowing for simultaneous care of animal patients and the generation of valuable empirical data.

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.

Gut microbiomes of captive primates show phylosymbiosis, respond to dietary sugar reduction, and select for host-specific dietary microbes.

Host-associated microbiomes are influenced by evolutionary history and proximate factors such as diet and environment. Zoos house animals in relatively standardized and manipulatable environments, making zoo populations valuable for studying microbiomes. Using a small population of five, closely related primate species housed under nearly identical environments, we investigated gut microbiome variation regarding (a) congruence between host evolutionary history and gut bacterial composition (i.e. phylosymbiosis), (b) a longitudinal reduction in dietary sugar intake, and (c) ingestion of bacteria from dietary sources. We found that the primate gut microbiomes varied across individuals and showed phylosymbiosis. When animals were fed diets with reduced sugar and increased fibre, we found host-specific changes in taxonomically distinct microbes (Phascolarctobacterium, Megasphaera, and Sharpea). Yet, these bacterial genera share similar functional potential (fibre degradation), indicating that the distinct bacterial communities may fulfill similar functions. Although all individuals received the same diet, the diet-associated bacteria in primate gut microbiomes were distinct across individuals of different species, suggesting a mechanism that selects for unique dietary microbes to persist in animal guts. Our findings show that the microbiomes of a small, captive primate population housed under uniform environmental conditions still show patterns congruent with combined influences of evolutionary history and diet.

Fecal microbiota transplants modulate the gut microbiome of a two-toed sloth (Choloepus didactylus).

The microbes inhabiting an animal's gastrointestinal tracts, collectively known as the gut microbiome, are vital to animal health and wellbeing. For animals experiencing gut distress or infection, modulation of the gut microbiome, for example, via fecal microbiota transplant (FMT), provides a possible disease prevention and treatment method. The beneficial microbes present in the donor's transplanted feces can help combat pathogens, assist in digestion, and rebalance the recipient's microbiota. Investigating the efficacy of FMTs in animal health is a crucial step toward improving management strategies for species under human care. We present a case study of the use of FMTs in a two-toed sloth experiencing abnormally large, clumped, and frequent stools. We used 16 S rRNA amplicon sequencing of fecal samples to (a) compare the microbiomes of the FMT donor, a healthy, cohoused conspecific, and the FMT recipient and (b) assess the influence of multiple rounds of FMTs on the recipient's microbiome and stool consistency and frequency over time. In response to the FMTs, we found that the recipient's microbiome showed trends toward increased diversity, shifted community composition, and altered membership that more resembled the community of the donor. FMT treatment was also associated with marked, yet temporary, alleviation of the recipient's abnormal bowel movements, suggesting a broader impact on gut health. Our results provide valuable preliminary evidence that FMT treatments can augment the recipient's gut microbiome, with potential implications for animal health and management.

Challenges of devising a milk recipe in a hand-reared hippopotamus (Hippopotamus amphibius).

In January 2017, a Nile hippopotamus (Hippopotamus amphibious) was born approximately six weeks premature at the Cincinnati Zoo & Botanical Garden. Due to the calf's weakened condition and lack of interest from the dam, management at the zoo made the decision to hand-rear the calf. Limited published information was available on hand-rearing this species of hippopotamus (hippo). To devise a nutritionally appropriate recipe, milk samples were acquired from the dam on Days 1, 3, 8, and 9 postpartum, and assayed for sugar, protein, fat, mineral, and water content using standard methods validated for multiple species of mammals at the Smithsonian National Zoo's Nutrition Science Laboratory. The sugar content stayed relatively constant (mean = 4.5%; range: 4.3%-4.7%). The fat consistently increased from 0.48% to 4.24% (mean = 2%). Excluding Day 9, the protein content gradually decreased from 9.56% to 6.39% (mean = 8%). The dry matter (DM) ranged from 14.38% to 16.72% (i.e., water content of 85.62%-83.28%), with the sum of the solids (sugar, protein, fat, and ash) averaging 98.5% of measured DM. Fat content was lower than expected but within the range of other artiodactyls. Between Days 1 and 8, the trend of decreasing protein and increasing fat was consistent with a change from colostrum to mature milk. The sharp increase in fat and protein with a decrease in sugar on Day 9 may indicate the beginning of the involution of the mammary gland due to lack of nursing stimulus. Utilizing this information, the Cincinnati Zoo was able to formulate a successful milk replacement recipe that allowed the calf to be raised through weaning to maturity.

Bamboo Specialists from Two Mammalian Orders (Primates, Carnivora) Share a High Number of Low-Abundance Gut Microbes.

Bamboo specialization is one of the most extreme examples of convergent herbivory, yet it is unclear how this specific high-fiber diet might selectively shape the composition of the gut microbiome compared to host phylogeny. To address these questions, we used deep sequencing to investigate the nature and comparative impact of phylogenetic and dietary selection for specific gut microbial membership in three bamboo specialists-the bamboo lemur (Hapalemur griseus, Primates: Lemuridae), giant panda (Ailuropoda melanoleuca, Carnivora: Ursidae), and red panda (Ailurus fulgens, Carnivora: Musteloideadae), as well as two phylogenetic controls-the ringtail lemur (Lemur catta) and the Asian black bear (Ursus thibetanus). We detected significantly higher Shannon diversity in the bamboo lemur (10.029) compared to both the giant panda (8.256; p = 0.0001936) and the red panda (6.484; p = 0.0000029). We also detected significantly enriched bacterial taxa that distinguished each species. Our results complement previous work in finding that phylogeny predominantly governs high-level microbiome community structure. However, we also find that 48 low-abundance OTUs are shared among bamboo specialists, compared to only 8 OTUs shared by the bamboo lemur and its sister species, the ringtail lemur (Lemur catta, a generalist). Our results suggest that deep sequencing is necessary to detect low-abundance bacterial OTUs, which may be specifically adapted to a high-fiber diet. These findings provide a more comprehensive framework for understanding the evolution and ecology of the microbiome as well as the host.

Feeding anatomy, filter-feeding rate, and diet of whale sharks Rhincodon typus during surface ram filter feeding off the Yucatan Peninsula, Mexico.

The feeding anatomy, behavior and diet of the whale shark Rhincodon typus were studied off Cabo Catoche, Yucatan Peninsula, Mexico. The filtering apparatus is composed of 20 unique filtering pads that completely occlude the pharyngeal cavity. A reticulated mesh lies on the proximal surface of the pads, with openings averaging 1.2mm in diameter. Superficial to this, a series of primary and secondary cartilaginous vanes support the pads and direct the water across the primary gill filaments. During surface ram filter feeding, sharks swam at an average velocity of 1.1m/s with 85% of the open mouth below the water's surface. Sharks on average spent approximately 7.5h/day feeding at the surface on dense plankton dominated by sergestids, calanoid copepods, chaetognaths and fish larvae. Based on calculated flow speed and underwater mouth area, it was estimated that a whale shark of 443 cm total length (TL) filters 326 m(3)/h, and a 622 cm TL shark 614 m(3)/h. With an average plankton biomass of 4.5 g/m(3) at the feeding site, the two sizes of sharks on average would ingest 1467 and 2763 g of plankton per hour, and their daily ration would be approximately 14,931 and 28,121 kJ, respectively. These values are consistent with independently derived feeding rations of captive, growing whale sharks in an aquarium. A feeding mechanism utilizing cross-flow filtration of plankton is described, allowing the sharks to ingest plankton that is smaller than the mesh while reducing clogging of the filtering apparatus.

Behavior, nutrition, and veterinary care of patagonian cavies (Dolichotis patagonum).

Patagonian cavies (Dolichotis patagonum) are large South American rodents well adapted for cursorial life (well adapted for running). They are monogamous but can live in groups of up to 70 individuals who maintain communal burrows. They are primarily herbivorous and may be maintained on commercially produced rodent or primate diets. Their long, thin legs and skittish nature make them difficult to restrain. Common medical problems include malocclusion of cheek teeth, gastrointestinal parasites, hypertrophic cardiomyopathy, and traumatic leg fractures.