Seasonal Variation in Fecal Glucocorticoid Levels and Their Relationship to Reproductive Success in Captive Populations of an Endangered Parrot

Many species are threatened with extinction, and captive breeding programs are becoming more common to avoid this outcome. These programs serve to prevent extinction and produce individuals for eventual reintroduction to natural populations in historical habitat. Captive animals experience different energetic demands than those in the wild, however, and as a result may have different levels of glucocorticoid hormones. Glucocorticoids help with responses to energetically expensive and potentially stressful situations. Elevated glucocorticoid levels can also potentially alter reproduction and other key behaviors, thus complicating successful captive breeding. The Puerto Rican parrot (Amazona vittata) is a critically endangered parrot that currently exists in only two wild and two captive populations. Its recovery program provides a good platform to better understand how glucocorticoid levels may relate to reproductive success under captive conditions. We validated a corticosterone assay in this species and used non-invasive techniques of measuring fecal glucocorticoid metabolites of males and females from two captive populations (Rio Abajo and El Yunque) of Puerto Rican parrots over two consecutive breeding seasons, 2017 and 2018, and the pre-breeding season of 2018, which occurred just after Hurricane Maria struck Puerto Rico. Our results show that levels of fecal glucocorticoid metabolites of males measured during the breeding season of 2018 negatively correlated to the number of total eggs and fertile eggs laid by pairs. In contrast, there was a positive relationship of female fecal glucocorticoid metabolite levels during the pre-breeding season of 2018 with total eggs laid. In males from the Rio Abajo population, we found seasonal differences in fecal glucocorticoid metabolite levels, with higher levels during the pre-breeding season of 2018 compared to both 2017 and 2018 breeding seasons. There was no difference in the mean value of male fecal glucocorticoid metabolites between the 2017 breeding season and 2018 breeding season which started four months after Hurricane Maria struck Puerto Rico. We did find sex differences during the pre-breeding season of 2018 in birds from the Rio Abajo population. Adjustments in the care routine of both populations that could reduce circulating baseline glucocorticoids and avoid frequent, sudden elevations of glucocorticoids should be considered. These results provide a baseline for future comparison with reintroduced populations of this endangered species and other species with captive breeding programs.

How is Western Lowland Gorilla (Gorilla gorilla gorilla) Behavior and Physiology Impacted by 360 Degree Visitor Viewing Access?

The effect visitors may have on the welfare of professionally managed animals is vital to consider. The present study utilized an experimental approach to examine how 360° visitor viewing access (i.e., viewing from all sides of the habitat) impacts the behavior and physiology of Western lowland gorillas (Gorilla gorilla gorilla, N = 7). Data collection included behavioral observations and obtaining fecal samples to examine physiological indicators of welfare. The study included a baseline period observing the gorillas’ behavior and physiology during the typical 360° visitor viewing access and a treatment period examining the same indicators but reducing viewing access by approximately 70%. Behavioral states, behavioral events, and hormone data were compared across conditions using Wilcoxon signed-rank tests. Restricted viewing did not impact social or solitary grooming (as a state behavior), social or solitary play, foraging, stereotypies, locomotion, inactivity, or behavioral diversity. However, restricted viewing significantly reduced solitary grooming events, fecal glucocorticoid metabolites, and the ratio of glucocorticoid metabolites to dehydroepiandrosterone metabolites. These results suggest that 360° visitor viewing access may have an impact on the welfare of gorillas. However, further research is necessary to thoroughly understand the influence of allowing 360° visitor viewing on behavior and its possible interaction with variables of the zoo environment.

498 Late-Breaking: Identifying Meaningful Measures of Dam and Puppy Welfare in Commercial-breeding (CB) Kennels

Maternal factors such as fear and stress influence offspring welfare in many species, but this area of research has been underexplored in dogs. As a large proportion of society’s dogs originate from commercial-breeding (CB) kennels, further investigation in this environment is crucial to ensure canine welfare. The current study aimed to refine a set of welfare metrics used to explore associations between maternal factors and litter welfare in CB kennels. Dams (n = 28) from five USDA-licensed CB kennels were tested at 6 and 1-week prepartum, and 4 and 8-weeks postpartum using a stranger approach test, physical health assessment, exposure to novel stimuli, social isolation test, hair cortisol concentration, fecal glucocorticoid metabolites (FGM) and fecal immunoglobulin A (IgA) concentrations. Puppies (n = 138) were tested at 4 (4W) and 8-weeks (8W) old using equivalent measures. Preliminary analyses of the metrics collected during puppy social isolation revealed that locomotion was positively correlated with exploration (4W: rs=0.46, P < 0.001; 8W: rs=0.39, P < 0.001), escape attempt duration (4W: rs=0.26, P = 0.002; 8W: rs=0.31, P < 0.001), frequency of stress behaviors (4W: rs=0.22, P = 0.01; 8W: rs=0.40, P < 0.001), vocalization (4W: rs=0.18, p=0.04; 8W: rs=0.20, P = 0.02), and fecal IgA (4W: rs=0.24, P = 0.05). Stationary behavior was negatively correlated with escape attempt duration (4W: rs=-0.37, P < 0.001; 8W: rs=-0.43, P < 0.001), frequency of stress behaviors (4W: rs=-0.22, P = 0.01; 8W: rs=-0.32, P < 0.001), and physiologic metrics (4W: fecal IgA rs=-0.25, P = 0.04; 8W: FGM rs=-0.23, P = 0.01). Results suggest that, in response to social isolation, 4W and 8W old puppies exhibit one of two coping styles, proactive or passive, and the latter is associated with lower activation of the immune and stress response. The outcomes of this study will inform future research exploring maternal risk factors, including behavioral and physiologic stressors, affecting puppy developmental welfare in CB kennels.

A Longitudinal Study of Hematology and Stress Biomarker Profiles in Young Asian Elephants (Elephas maximus) in Relation to Elephant Endotheliotropic Herpesvirus (EEHV) in Thailand

Elephant endotheliotropic herpesvirus hemorrhagic disease (EEHV-HD) is a virulent disease that causes severe hemorrhage and sudden death in Asian elephant calves. A change in hematology profiles is one indicator of infection before clinical signs appear; however, to be effective, individual baselines and age-matched reference values are needed. Stress has been speculated to be a factor in clinical EEHV cases, but relationships have not been demonstrated empirically. This study evaluated blood hematology and several stress response markers—salivary cortisol, fecal glucocorticoid metabolites (FGM), salivary Immunoglobulin A (SIgA), and fecal IgA (FIgA) in samples collected for 1 year from three healthy calves with no EEHV history (non-EEHV), and six that had previously been infected, developed clinical signs and survived (prior-EEHV). Hematology values between non-EEHV and prior-EEHV elephants were not different and within published reference ranges. Concentrations of salivary cortisol, FGM, SIgA, and FIgA also were variable and showed seasonal differences, but no relationships to prior EEHV status. One of the prior EEHV calves became re-infected, developed hemorrhagic disease (HD), and died during the study period. That calf exhibited lymphocytopenia, monocytopenia, and thrombocytopenia. Additionally, all stress biomarker concentrations were lower in the 12 days before viremia was observed. Thus, as in other studies, changes in hematology occur with EEHV infection, while preliminary data in one calf suggests that stress-response measures might also be informative and should be studied further.

Fecal glucocorticoid metabolites as an indicator of adrenocortical activity in Arctic foxes (Vulpes lagopus) and recommendations for future studies

Measuring fecal glucocorticoid metabolites (fGCMs) is a widely used, non-invasive method for studies of stress in vertebrates. To study physiological responses in wild Arctic foxes (Vulpes lagopus) to perceived stressors such as fluctuating food availability, occurrence of competitors and predators and disturbance from human activities, a species-specific physiological validation of a method to evaluate adrenocortical activity is needed. Here we used 15 captive Arctic foxes (both males and females and juveniles and adults) to investigate fGCM concentrations following ACTH injection (physiological validation), or handling alone and compared them with their respective baseline concentrations prior to the treatments. A 5α-pregnane-3ß,11ß,21-triol-20-one enzyme immunoassay measured significant fGCM increases following both treatments. The time lags to reach peak fGCM values were 9.3 ± 1.3 h and 12.8 ± 1.7 h for ACTH and handling treatment, respectively. Concentrations of fGCMs varied a lot between individuals, but not attributed to sex nor age of the foxes. However, we found a negative relationship between boldness and fGCM concentrations. Faecal glucocorticoid metabolites concentrations did not change significantly over a period of 48 h in samples kept at temperatures reflecting winter and summer means. This would allow the collection of samples up to two days old in the wild regardless of the season. We conclude that our successfully validated method for measuring fGCMs can be used as a non-invasive tool for studies exploring various stressors both in wild and captive Arctic foxes.

Early-life social experience affects offspring DNA methylation and later life stress phenotype

Studies in rodents and captive primates suggest that the early-life social environment affects future phenotype, potentially through alterations to DNA methylation. Little is known of these associations in wild animals. In a wild population of spotted hyenas, we test the hypothesis that maternal care during the first year of life and social connectedness during two periods of early development leads to differences in DNA methylation and fecal glucocorticoid metabolites (fGCMs) later in life. Here we report that although maternal care and social connectedness during the den-dependent life stage are not associated with fGCMs, greater social connectedness during the subadult den-independent life stage is associated with lower adult fGCMs. Additionally, more maternal care and social connectedness after den independence correspond with higher global (%CCGG) DNA methylation. We also note differential DNA methylation near 5 genes involved in inflammation, immune response, and aging that may link maternal care with stress phenotype.

DNA Damage as a Potential Non-Invasive Indicator of Welfare: A Preliminary Study in Zoo-Housed Grizzly Bears (Ursus arctos horribilis)

Measures of oxidative stress have potential for integrating positive and negative life experiences into comprehensive cellular indicators of animal welfare. We explored this possibility when three adult grizzly bear brothers at the Detroit Zoo were temporarily moved to a smaller habitat while their primary home was expanded. We expected that the spatial compression and construction activity might be sources of stress. We observed increased social play and other affiliative behavior in the smaller habitat, and we used daily fecal samples (17 to 24 per bear) to examine whether concentrations of fecal glucocorticoid metabolites (FGM) and 8-hydroxy-2′-deoxyguanosine (8-OHdG, a by-product of DNA damage) were correlated with social behavior. Our overall aim was to explore 8-OHdG as a potential indicator of welfare based on the prediction that 8-OHdG would be lower when more positive social interactions occurred. Concentrations of fecal 8-OHdG increased significantly with higher FGM concentrations, supporting a potential relationship between adrenal activity and rates of DNA damage. However, we found that on days when they engaged in higher rates of affiliative interactions, there were trends for 8-OHdG concentrations to increase for one bear and decrease for another, and no relationship for the third bear. These preliminary results should be interpreted with caution, but suggest a potential relationship between social behavior and 8-OHdG that is modulated by health, personality, or other individual factors. Further validation research is needed, but 8-OHdG may have promise as a non-invasive, cumulative indicator of animal welfare.