Cloning for the Twenty-First Century and Its Place in Endangered Species Conservation.

Cloning as it relates to the animal kingdom generally refers to the production of genetically identical individuals. Because cloning is increasingly the subject of renewed attention as a tool for rescuing endangered or extinct species, it seems timely to dissect the role of the numerous reproductive techniques encompassed by this term in animal species conservation. Although cloning is typically associated with somatic cell nuclear transfer, the recent advent of additional techniques that allow genome replication without genetic recombination demands that the use of induced pluripotent stem cells to generate gametes or embryos, as well as older methods such as embryo splitting, all be included in this discussion. Additionally, the phenomenon of natural cloning (e.g., a subset of fish, birds, invertebrates, and reptilian species that reproduce via parthenogenesis) must also be pointed out. Beyond the biology of these techniques are practical considerations and the ethics of using cloning and associated procedures in endangered or extinct species. All of these must be examined in concert to determine whether cloning has a place in species conservation. Therefore, we synthesize progress in cloning and associated techniques and dissect the practical and ethical aspects of these methods as they pertain to endangered species conservation.

Resurrecting biodiversity: advanced assisted reproductive technologies and biobanking.

Biodiversity is defined as the presence of a variety of living organisms on the Earth that is essential for human survival. However, anthropogenic activities are causing the sixth mass extinction, threatening even our own species. For many animals, dwindling numbers are becoming fragmented populations with low genetic diversity, threatening long-term species viability. With extinction rates 1000-10,000 times greater than natural, ex situ and in situ conservation programmes need additional support to save species. The indefinite storage of cryopreserved (-196°C) viable cells and tissues (cryobanking), followed by assisted or advanced assisted reproductive technology (ART: utilisation of oocytes and spermatozoa to generate offspring; aART: utilisation of somatic cell genetic material to generate offspring), may be the only hope for species' long-term survival. As such, cryobanking should be considered a necessity for all future conservation strategies. Following cryopreservation, ART/aART can be used to reinstate lost genetics back into a population, resurrecting biodiversity. However, for this to be successful, species-specific protocol optimisation and increased knowledge of basic biology for many taxa are required. Current ART/aART is primarily focused on mammalian taxa; however, this needs to be extended to all, including to some of the most endangered species: amphibians. Gamete, reproductive tissue and somatic cell cryobanking can fill the gap between losing genetic diversity today and future technological developments. This review explores species prioritisation for cryobanking and the successes and challenges of cryopreservation and multiple ARTs/aARTs. We here discuss the value of cryobanking before more species are lost and the potential of advanced reproductive technologies not only to halt but also to reverse biodiversity loss. Lay summary: The world is undergoing its sixth mass extinction; however, unlike previous events, the latest is caused by human activities and is resulting in the largest loss of biodiversity (all living things on Earth) for 65 million years. With an extinction rate 1000-10,000-fold greater than natural, this catastrophic decline in biodiversity is threatening our own survival. As the number of individuals within a species declines, genetic diversity reduces, threatening their long-term existence. In this review, the authors summarise approaches to indefinitely preserve living cells and tissues at low temperatures (cryobanking) and the technologies required to resurrect biodiversity. In the future when appropriate techniques become available, these living samples can be thawed and used to reinstate genetic diversity and produce live young ones of endangered species, enabling their long-term survival. The successes and challenges of genome resource cryopreservation are discussed to enable a move towards a future of stable biodiversity.

Fungal microbiomes are determined by host phylogeny and exhibit widespread associations with the bacterial microbiome.

Interactions between hosts and their resident microbial communities are a fundamental component of fitness for both agents. Though recent research has highlighted the importance of interactions between animals and their bacterial communities, comparative evidence for fungi is lacking, especially in natural populations. Using data from 49 species, we present novel evidence of strong covariation between fungal and bacterial communities across the host phylogeny, indicative of recruitment by hosts for specific suites of microbes. Using co-occurrence networks, we demonstrate marked variation across host taxonomy in patterns of covariation between bacterial and fungal abundances. Host phylogeny drives differences in the overall richness of bacterial and fungal communities, but the effect of diet on richness was only evident in the mammalian gut microbiome. Sample type, tissue storage and DNA extraction method also affected bacterial and fungal community composition, and future studies would benefit from standardized approaches to sample processing. Collectively these data indicate fungal microbiomes may play a key role in host fitness and suggest an urgent need to study multiple agents of the animal microbiome to accurately determine the strength and ecological significance of host-microbe interactions.

Reproductive males are effective at managing conflict in captive Sulawesi crested macaques (Macaca nigra).

Group size, density, and composition significantly influence the expression of agonism and, as demography is frequently manipulated in captivity, natural mechanisms of conflict management may be disrupted. Here, we examine how changes to the social and physical environment of a captive group of Sulawesi crested macaques, Macaca nigra, influenced mechanisms of conflict management. The initial group was comprised of females, juveniles, and castrated males; under a series of management interventions, the group was moved to a new enclosure, 75% of the castrated adult males were removed, 67% of the females were contracepted, a novel, intact male was introduced, and the group was allowed access to an outdoor enclosure. Despite a decreasing trend in aggression following the changes to the social group, we found no significant differences in aggression or intervention behavior in the 5-week periods immediately following changes to the social group, apart from a significant decrease in aggression following the introduction of the novel male. This decrease in the frequency of aggression was still evident 10 weeks after the interventions occurred, and was coupled with a decrease in the intensity of aggression. Moreover, the intensity of aggression as well as the frequency of aggression and intervention were significantly lower under low-density conditions. Our results highlight how management changes to the social and physical environment can influence aggressive behavior, albeit following a period of acclimation. We discuss the relative impacts of social group composition, social density, and individual reproductive status on the management of conflict behavior in a captive setting.

Irregular ovarian cyclicity is associated with adrenal activity in female eastern black rhinoceros (Diceros bicornis michaeli).

To achieve self-sustaining and genetically diverse populations ex situ, captive breeding programmes must ensure good overall rates of reproduction, and equal contribution across individuals. Previous research in the critically endangered eastern black rhinoceros (Diceros bicornis michaeli) revealed a high incidence of irregular oestrous cyclicity; in particular extended cycle duration among nulliparous females and acyclic periods in parous females that have not bred for several years. Irregular ovarian activity could play a role in reduced reproductive output; however, the mechanisms underlying these anomalies are poorly understood. The aim of this study was to measure faecal glucocorticoid metabolite (fGCM) concentrations and variability prior to and during periods of regular and irregular ovarian activity, and determine if adrenal activity influences the occurrence of different cycle types in this species. Faecal samples were collected every other day from parous (N = 6) and nulliparous (N = 12) females at eight European institutions for periods of 9-15 months. Concentration and variability in fGCM were compared between periods of regular and irregular cyclicity and between different cycle types (<20 days, 20-40 days, >40 days, acyclic) using generalized linear mixed models. Concentrations of fGCM were influenced by season and higher during the luteal than the follicular phase of the oestrous cycle. Taking this into account, fGCMs were increased during periods of irregular cyclicity (all types combined and during cycles >40 days in length) compared to 20-40 day cycles. This was predominantly driven by nulliparous females. The variation in fGCM concentration also differed between periods of regular and irregular cyclicity; higher standard deviation in fGCM preceded irregular cycles and >40 day cycles compared to 20-40 day cycles. These results suggest that although fGCM concentrations fluctuate across the oestrous cycle in this species, changes in adrenal activity at specific times could be one factor associated with irregular ovarian activity in the black rhinoceros.

Rare gut microbiota associated with breeding success, hormone metabolites and ovarian cycle phase in the critically endangered eastern black rhino.

BACKGROUND: Host microbiomes play a role in hormone production and subsequent fertility in humans, but this is less well understood in non-model organisms. This is of particular relevance to species in zoo-based conservation breeding programmes, as relationships between host microbiome composition and reproductive output may allow for the development of microbial augmentation strategies to improve success. Here, we characterise faecal bacterial communities of breeding and non-breeding eastern black rhino (Diceros bicornis michaeli) using 16S rRNA gene amplicon sequencing and quantify progestagen and glucocorticoid metabolite concentrations through enzyme immunoassays to identify such relationships. RESULTS: We identified significant differences in black rhino gut microbiome composition according to ID, institution, breeding success and ovarian cycle phase. In particular, the gut microbiome during pregnancy and post-parturition was significantly altered. Around a third of bacterial genera showed more than ± 10% correlation with either progestagen and/or glucocorticoid concentration, and in general, microbial genera correlated with both hormones in the same direction. Through a combination of analyses, we identified four genera (Aerococcaceae, Atopostipes, Carnobacteriaceae and Solobacterium) that were significantly associated with breeding success, pregnancy and/or post-parturition, and higher faecal progestagen metabolite concentrations. These genera had a lower-than-average relative abundance in the gut microbiome. CONCLUSION: Our results indicate that many members of the gut microbiome of black rhino are associated with hormone production and breeding success, and some members of the rare microbiota appear to be particularly important. Although the directionality of the relationship is unclear, the variation in gut microbiome communities represents a potential biomarker of reproductive health. We identified four genera that were associated with multiple indicators of reproductive output; these could be candidate probiotics to improve the breeding success of black rhino in zoo-based conservation breeding programmes. Further work is required to understand the efficacy and feasibility of this, either directly through microbial augmentation (e.g. probiotics) or indirectly via dietary manipulation or prebiotics.

ASSESSING THE EFFICACY OF DESLORELIN ACETATE IMPLANTS (SUPRELORIN) IN ALTERNATIVE PLACEMENT SITES.

Hormonal contraception is being increasingly used to manage captive animals in zoological collections. Many of the animals placed on contraception are of genetic importance within captive breeding programs; therefore, it is imperative that the application of contraceptive products minimize potential side effects and facilitate a return to fertility if required. Deslorelin acetate implants (Suprelorin®) are one example of a hormonal contraceptive that is frequently used in captivity as they are easy to use and effective in most species. It is hypothesized that removing implants may hasten reversal of contraception treatment; therefore, placement in a location where they can easily be recovered is advocated. In this report, the efficacy and safety of Suprelorin implants placed in their recommended site between the scapulae is compared with alternative placement sites where implants can more easily be located for removal. Using the European Association of Zoos and Aquariums (EAZA) Group on Zoo Animal Contraception (EGZAC) Contraception Database, rates of success, failure, and reversal in 561 records of Suprelorin use in European collections are compared. Of these, 357 have information relating to the location of implant placement. When correctly applied, rates of efficacy were high (>99%) irrespective of placement site. Rates of reversal were 33.3% higher in alternative placement sites, although in most cases it is unknown whether implants were removed or not. In conclusion, the placement of Suprelorin implants in alternative sites does not negatively affect the efficacy, facilitating at the same time implant removal, minimizing potential side effects, and reducing reversal time, allowing for effective use in captive conservation breeding programs.

Characterizing the reproductive biology of the female pygmy hippopotamus (Choeropsis liberiensis) through non-invasive endocrine monitoring.

The pygmy hippopotamus (Choeropsis liberiensis) is endangered in the wild and very little is known about its reproductive biology. In zoological facilities, this species experiences a number of reproductive issues that complicate breeding management, including a high rate of stillbirths and failure of many pairs to reproduce. We conducted a comprehensive study to evaluate reproductive cycles and pregnancy in this species using enzyme immunoassays (EIAs) for fecal hormone metabolite analysis. Fresh fecal samples were collected twice weekly for a one to three year period from 36 female pygmy hippos housed at 24 zoological institutions. Samples were analyzed in three separate laboratories. Three progestogen metabolite EIAs (Pg-diol: 5ß-pregnane-3α,20α-diol 3HS:BSA; PdG: pregnanediol-3-glucuronide R13904; mono-P4: Quidel clone 425) and three estrogen metabolite EIAs (E2a: estradiol-17ß-OH 17-HS:BSA; E2b: estradiol 17ß R0008; E2c: estradiol 17ß R4972) accurately reflected reproductive events. Average estrous cycle length was 31.8 ± 7.4 days based on estrogen metabolite peaks and 30.9 ± 7.3 days based on nadir to nadir progestogen metabolite concentrations. Cyclical patterns in both estrogen and progestogen metabolites were detected throughout the year, indicating a lack of seasonality. Estrogen metabolite peaks were also observed during pregnancy and lactation, suggesting that follicular development occurs during both reproductive states. Pregnancy was most reliably demonstrated by elevation in progestogen metabolites (Pg-diol or PdG) in the second half of gestation. Average gestation length based on breeding to calving date was 203 ± 4 days for 15 pregnancies. This comprehensive overview of the reproductive biology of the female pygmy hippo provides valuable data for guiding long-term breeding management for this endangered species and serves as a baseline for future studies addressing the potential influence of social structure, diet, body condition, and other husbandry factors on estrous cycling and reproduction.

Fecal Glucocorticoid Analysis: Non-invasive Adrenal Monitoring in Equids.

Adrenal activity can be assessed in the equine species by analysis of feces for corticosterone metabolites. During a potentially aversive situation, corticotrophin releasing hormone (CRH) is released from the hypothalamus in the brain. This stimulates the release of adrenocorticotrophic hormone (ACTH) from the pituitary gland, which in turn stimulates release of glucocorticoids from the adrenal gland. In horses the glucocorticoid corticosterone is responsible for several adaptations needed to support equine flight behaviour and subsequent removal from the aversive situation. Corticosterone metabolites can be detected in the feces of horses and assessment offers a non-invasive option to evaluate long term patterns of adrenal activity. Fecal assessment offers advantages over other techniques that monitor adrenal activity including blood plasma and saliva analysis. The non-invasive nature of the method avoids sampling stress which can confound results. It also allows the opportunity for repeated sampling over time and is ideal for studies in free ranging horses. This protocol describes the enzyme linked immunoassay (EIA) used to assess feces for corticosterone, in addition to the associated biochemical validation.

Concentrations of faecal glucocorticoid metabolites in Asian elephant's dung are stable for up to 8 h in a tropical environment.

The use of faecal glucocorticoid metabolites (fGCMs) has facilitated the development of non-invasive methods to study physiological conditions of endangered wildlife populations. One limitation is that fGCM concentrations are known to change over time and to vary according to different environmental conditions. The aim of this study was to perform a controlled dung decay experiment to understand the impact of time (since defecation) and two common environmental variables (exposure to water and direct sunlight) on fGCM concentrations of Asian elephants (Elephas maximus). Eighty dung piles from 10 Malaysian elephants were randomly exposed to a 2 × 2 combination of treatments (wet-shade, dry-shade, wet-sun and dry-sun) and repeatedly subsampled from the time of defecation through to 2 days post-defecation (n = 685 faecal subsamples). Overall, the mean concentration of fGCMs was stable in samples of up to 8 h old from defecation time, regardless of environmental treatment (water or direct sunlight); thereafter, the overall mean fGCM concentrations increased, peaking 1 day after defecation (31.8% higher than at defecation time), and subsequently decreased (reaching values 9.2% below defecation time on the second day). Overall, the treatment of sun exposure resulted in higher fGCM concentration compared with shade, whereas water exposure (compared with no water exposure) had no impact on fGCM concentrations. Hence, in field studies we recommend collecting dung samples <8 h old and recording shade conditions (e.g. sun vs. shade) as a covariate for the subsequent interpretation of fGCM measurements. This study has helped to identify the optimal window for sampling in which we can have a higher confidence in interpreting the results as being a genuine reflection of glucocorticoid status in the elephant.

Investigating temporary acyclicity in a captive group of Asian elephants (Elephas maximus): Relationship between management, adrenal activity and social factors.

Routine faecal steroid monitoring has been used to aid the management of five captive Asian elephant (Elephas maximus) females at Chester Zoo, UK, since 2007. Progestagen analysis initially revealed synchronised oestrous cycles among all females. However, a 14- to 20-week period of temporary acyclicity subsequently occurred in three females, following several management changes (increased training, foot-care and intermittent matriarch removal for health reasons) and the initiation of pregnancy in another female. The aim of this study was to retrospectively investigate whether these management changes were related to increased adrenal activity and disruption of ovarian activity, or whether social factors may have been involved in the temporary cessation of cyclicity. Faecal samples collected every other day were analysed to investigate whether glucocorticoid metabolites were related to reproductive status (pregnant, cycling, acyclic) or management (training, foot-care, matriarch presence). Routine training and foot-care were not associated with adrenal activity; however, intensive foot-care to treat an abscess in one female was associated with increased glucocorticoid concentration. Matriarch presence influenced adrenal activity in three females, being lower when the matriarch was separated from the group at night compared to being always present. However, in the females that exhibited temporary acyclicity, there was no consistent relationship between glucocorticoids and cyclicity state. Although the results of this study do not fully explain this occurrence, the highly synchronised nature of oestrous cycles within this group, and the concurrent acyclicity in three females, raises the question of whether social factors could have been involved in the temporary disruption of ovarian activity.

The use of altrenogest to avoid hyperestrogenism after eCG-hCG ovulation induction in southern tigrina (Leopardus guttulus).

The goal of this study was to optimize an ovulation induction protocol for use with artificial insemination in the southern tigrina (Leopardus guttulus). The specific aims were to report the efficacy of using altrenogest, an oral progestin (Regumate, MSD Animal Health, Merck Animal Health), to suppress ovarian activity and prevent follicular hyperstimulation and hyperestrogenism after the administration of exogenous eCG and hCG. To monitor ovarian responses, fecal estrogen and progestogen metabolites were quantified by enzyme immunoassay in females before and after intramuscular administration of 200-IU eCG and 150-IU hCG in two trials, 4 months apart. During the first trial, there was no use of altrenogest, only the eCG-hCG ovulation induction protocol. In the second trial, the ovulation induction protocol was preceded by the administration of oral altrenogest for 14 days (minimum of 0.192 mg per kg per day). Altrenogest administration resulted in a suppression of follicular activity in three out of six females before eCG-hCG administration on the basis of lower mean estrogen concentrations (P < 0.05). It also resulted in four out of six females presenting lower fecal estrogen metabolite concentrations (P < 0.05) after ovulation induction, and two out of six individuals showed a reduction (P < 0.05) in postovulatory fecal progestogen metabolite concentrations, all when compared to the same female's cycles without the progestin. Fecal estrogen metabolite concentrations were closer to baseline in 50% of these individuals after altrenogest and eCG-hCG treatments when compared to basal concentrations before gonadotropins without the use of altrenogest. This study reported that use of altrenogest in southern tigrina can suppress ovarian activity and avoid hyperestrogenism after administration of eCG and hCG treatment. However, not all females responded uniformly, so more studies are needed to increase the efficacy of ovulation induction for use with artificial insemination in this species.

Domesticated horses differ in their behavioural and physiological responses to isolated and group housing.

The predominant housing system used for domestic horses is individual stabling; however, housing that limits social interaction and requires the horse to live in semi-isolation has been reported to be a concern for equine welfare. The aim of the current study was to compare behavioural and physiological responses of domestic horses in different types of housing design that provided varying levels of social contact. Horses (n = 16) were divided equally into four groups and exposed to each of four housing treatments for a period of five days per treatment in a randomized block design. The four housing treatments used were single housed no physical contact (SHNC), single housed semi-contact (SHSC), paired housed full contact (PHFC) and group housed full contact (GHFC). During each housing treatment, adrenal activity was recorded using non-invasive faecal corticosterone metabolite analysis (fGC). Thermal images of the eye were captured and eye temperature was assessed as a non-invasive measure of the stress response. Behavioural analysis of time budget was carried out and an ease of handling score was assigned to each horse in each treatment using video footage. SHNC horses had significantly higher (p = 0.01) concentrations of fGC and were significantly (p = 0.003) more difficult to handle compared to the other housing types. GHFC horses, although not significantly different, had numerically lower concentrations of fGC and were more compliant to handling when compared to all other housing treatments. Eye temperature was significantly (p = 0.0001) lower in the group housed treatment when compared to all other treatments. These results indicate that based on physiological and behavioural measures incorporating social contact into the housing design of domestic horses could improve the standard of domestic equine welfare.

Male reproductive success is correlated with testosterone in the eastern black rhinoceros (Diceros bicornis michaeli).

Among natural populations of polygynous species, males often vary in their lifetime reproductive success. However, in managed populations of endangered species, either in situ or as part of captive breeding programmes, it is important to understand why differences in reproductive success occur. The European captive population of the critically endangered eastern black rhinoceros is currently under-performing relative to their wild counterparts, with low reproductive output and high reproductive skew limiting growth and genetic diversity. To investigate why over 40% of captive males fail to breed, faecal samples were collected weekly from 23 males at 12 institutions across Europe for 4-32 months. Testosterone metabolite concentration was compared between proven and non-proven males and a number of intrinsic and extrinsic factors that could influence reproductive success were also investigated. Males that sired within the last 3½ years had significantly higher androgen concentrations than non-proven males, and average testosterone was positively correlated with the number of offspring sired per year spent in the reproductive age class. Proven and non-proven males did not differ in their body condition, or in average faecal glucocorticoid concentration. Differences in individual temperament were associated with adrenal activity, but did not correlate with reproductive category. Highest testosterone concentrations were observed in proven males that were housed with females during oestrus, and lowest concentrations in non-proven females not housed with females at all during the study period. Further work is necessary to determine whether proven males had higher testosterone due to underlying differences associated with quality, or whether external stimuli such as access to females could influence testosterone concentration and increase a male's chances of becoming a successful breeder.

Irregular ovarian activity, body condition and behavioural differences are associated with reproductive success in female eastern black rhinoceros (Diceros bicornis michaeli).

Ex situ populations of endangered species such as the black rhinoceros play an important role in global conservation strategies. However, the European captive population of eastern black rhinoceros is performing sub-optimally, with growth rates and genetic viability limited by low birth rates and high reproductive skew. We investigated several intrinsic differences between parous and nulliparous females that may underlie differences in reproductive success, including ovarian cyclicity, adrenal activity, behaviour and body condition. Faecal samples were collected from 39 females (17 parous, 15 nulliparous and 7 pre-reproductive) at 11 zoological institutions, every other day for between 4months and 6years. Progestagen metabolite concentration indicated that although all non-pregnant females exhibited ovarian activity, irregular cyclicity was common. Longer cycles (>40days) were more common in nulliparous females and periods of acyclicity observed more often in females that had not bred for at least 7years. Even when endocrine data indicated clear ovarian activity, overt behavioural signs of oestrus were not always apparent, particularly among nulliparous females. Faecal glucocorticoids did not differ between parous and nulliparous females, although did differ according to individual temperament. More unpredictable temperaments were associated with higher glucocorticoids, and nulliparous females tended to be rated as more unpredictable. Finally, nulliparous females had higher body condition scores than parous females. This is the first comprehensive survey of the reproductive physiology of this European captive population, and highlights a number of intrinsic differences related to parity, which may underlie differences in reproductive success among captive female black rhinoceros.

A practical field extraction method for non-invasive monitoring of hormone activity in the black rhinoceros.

Non-invasive hormone analysis is a vital tool in assessing an animal's adrenal and reproductive status, which can be beneficial to in situ and ex situ conservation. However, it can be difficult to employ these techniques when monitoring in situ populations away from controlled laboratory conditions, when electricity is not readily available. A practical method for processing faecal samples in the field, which enables samples to be extracted soon after defaecation and stored in field conditions for prolonged periods prior to hormone analysis, is therefore warranted. This study describes the development of an optimal field extraction method, which includes hand-shaking faecal material in 90% methanol, before loading this extract in a 40% solvent onto HyperSep™ C8 solid-phase extraction cartridges, stored at ambient temperatures. This method was successfully validated for measurement of adrenal and reproductive hormone metabolites in faeces of male and female black rhinoceros (Diceros bicornis) and was rigorously tested in controlled laboratory and simulated field conditions. All the hormones tested demonstrated between 83 and 94% and between 42 and 89% recovery of synthetic and endogenous hormone metabolites, respectively, with high precision of replication. Furthermore, results obtained following the developed optimal field extraction method were highly correlated with the control laboratory method. Cartridges can be stored at ambient (cool, dry or warm, humid) conditions for periods of up to 6 months without degradation, before re-extraction of hormone metabolites for analysis by enzyme immunoassay. The described method has great potential to be applied to monitor faecal reproductive and adrenal hormone metabolites in a wide variety of species and allows samples to be stored in the field for up to 6 months prior to analysis. This provides the opportunity to investigate hormone relationships within in situ populations, where equipment and facilities may previously have been limiting.

Effects of visible implanted elastomer marking on physiological traits of frogs.

Amphibians possess innate immune defences, including antimicrobial peptides and symbiotic bacterial communities, that can protect them from infectious diseases, including chytridiomycosis. On-going research is attempting to use amphibian symbiotic bacteria to develop probiotic treatments that can protect hosts from the causative agent of chytridiomycosis, the fungal pathogen Batrachochytrium dendrobatidis. Events that cause disruption of symbiotic bacterial communities or deplete peptide stores could increase the susceptibility of individuals to disease and may have implications for amphibians involved in probiotic trials or time course studies that investigate symbiotic bacterial communities. It has previously been shown that passive integrated transponder tagging of frogs causes a rapid (within 24 h) and major proliferation of micro-organisms on the skin. Here, we show that marking of red-eyed tree frogs (Agalychnis callidryas) with visible elastomer has no effect on adrenal response (represented by faecal glucocorticoid metabolite concentrations) or peptide production, although there was evidence of a slightly greater microbial abundance associated with the skin of marked frogs 2 weeks after tagging. The results indicate that visible elastomer may be a preferable marking technique to passive integrated transponder tagging, particularly in the context of probiotic trials or time course studies that investigate symbiotic bacterial communities. More work is required to determine the effects of different marking techniques on physiological responses of amphibians, whether these physiological responses are consistent across host species and whether such 'non-invasive' marking methods affect the susceptibility of amphibians to infectious pathogens, such as B. dendrobatidis.

Development of a versatile enzyme immunoassay for non-invasive assessment of glucocorticoid metabolites in a diversity of taxonomic species.

Endocrinology is a useful tool for conservation biologists and animal managers, and measuring glucocorticoids can help understand biological mechanisms associated with species decline and animal welfare. The current study describes the development and optimization of a glucocorticoid enzyme immunoassay (EIA) to non-invasively assess adrenal activity in a variety of taxa. The antiserum (CJM006) was raised in rabbits to a corticosterone-3-CMO-BSA immunogen and used in a standard competitive EIA system. However, the EIA initially produced results with unacceptably high inter-assay variation, attributed to consistent patterns observed within the optical density of developing plates. To determine the cause of this variability, a number of factors were examined using synthetic corticosterone standard and endogenous faecal extract, including: plate type (Nunc MaxiSorp® II versus Immulon IB plates); the use of non-specific secondary antibody; type (artificial versus natural) and presence (light versus dark) of light during incubation; plate loading temperature (4°C versus room temperature); and substrate reagent temperature (4°C versus room temperature). Results indicated that variability was associated with plate location effects, which were not initially detected because control samples were always run in the same positions across plates. Light and temperature were the two major factors that affected EIA reliability. For this assay, the standard protocol required slight modification, with the optimal protocol using Nunc MaxiSorp® plates, room temperature substrate reagents and dark incubation conditions. Following optimization, this EIA was then validated biochemically for 38 species, through parallel displacement curves and interference assessment tests of faecal and urine samples. Additionally, biological validation was performed opportunistically in a subset of species, with use of this EIA demonstrating significant elevations in faecal glucocorticoid metabolites following potentially challenging events. In summary, this glucocorticoid EIA cross-reacts with excreted glucocorticoid metabolites across a wide range of taxa, including ungulates, primates, felids, birds, rodents and amphibians. We conclude that when used with optimal reagent and incubation conditions, this EIA will be useful for non-invasive monitoring of adrenal activity in a wide range of wildlife species.

Associations between social behaviour and adrenal activity in female Barbary macaques: consequences of study design.

Faecal glucocorticoid metabolite (fGCM) concentrations have been used to evaluate adrenal activity in a variety of species; including as an indicator of the physiological response to social stress. However, across studies, the relationships between dominance rank, social behaviours and adrenal responses can be inconsistent. Differences in the relationship between rank and glucocorticoids may be due to the relative costs of social status, and the relative frequencies of social stressors and potential coping mechanisms. However, the differences in observed relationships between specific social behaviours and glucocorticoids may be partly explained by sampling frequency, as studies often use average fGCM concentrations collected over a period of weeks or months, rather than fGCM concentrations that are temporally-matched with behavioural data. In this study, we directly compared long-term average and temporally-matched data to determine whether particular social behaviours were related to adrenal activity in female Barbary macaques (Macaca sylvanus) at Trentham Monkey Forest, UK; and whether observed relationships were consistent using these two approaches. Average rates of autogrooming were positively correlated with average fGCM; however, this relationship was not robust in temporally-matched samples. Instead, specific social behaviours associated with agonism were associated with fGCM in temporally-matched samples within individuals. These results indicate that analyses of relationships using long-term average fGCM and temporally-matched samples do not necessarily provide comparable results, highlighting that study design is critical in determining associations between an individual's social behaviour and the relative physiological costs involved.

Relationship between behavior, adrenal activity, and environment in zoo-housed western lowland gorillas (Gorilla gorilla gorilla).

Monitoring adrenal activity through noninvasive fecal hormone sampling is rapidly gaining popularity as a tool to assess zoo animal welfare. However, few studies have sought to investigate the interrelationships between behavior, adrenal activity, and environment, and ask whether both behavioral and adrenal monitoring strategies are required to assess welfare sufficiently. We present the findings of a 9-month study of a small group (one male, two females) of Western lowland gorillas, Gorilla gorilla gorilla. First, we examined the effect of environmental variables on gorilla behavior. Second, we examined the effect of environmental variables on the concentration of fecal glucocorticoid metabolites (FGC) and the relationship between behavior and FGC. Environmental variables had similar effects on all three gorillas. Negative vigilance of visitors (NVV; staring, posturing, and charging at visitors) significantly increased in all subjects as environmental noise levels increased, and food-related behavior significantly decreased in all subjects as crowd size increased. Exhibit modifications had a number of positive effects on behavior. Notably, when privacy screens were used, NVV significantly decreased in two subjects. We found no significant effects of environmental variables on FGC. However, we did find significant relationships between behavior and FGC in one female. Specifically, her NVV was significantly higher one day before, and on the same day as, raised FGC. Also, hair plucking significantly increased in the two days following raised FGC. Overall, this study demonstrates how concurrent noninvasive fecal and behavioral monitoring can be used for gorilla welfare assessment.