Non-Invasive Assessment of the Seasonal Stress Response to Veterinary Procedures and Transportation of Zoo-Housed Lesser Anteater (Tamandua tetradactyla)

Management procedures affect behavioural and physiological stress responses of wild mammals under human care. According to the Reactive Scope Model, normal values are presumed to exist within predictive and reactive ranges. First, stress parameters of zoo-housed adult Tamandua tetradactyla were evaluated in winter and summer (29 days each), determining the level of behaviour and/or physiological parameters needed to respond to predictable environmental changes. Secondly, the effects of veterinary procedures and transportation were studied in both seasons. Non-invasive methods were applied, assessing behaviour through videos and adrenocortical activity by faecal glucocorticoid metabolites (FGMs). Lesser anteaters exhibited seasonality (summer > winter) in some behavioural parameters, such as nocturnal activities, as well as in the activity cycle (e.g., acrophase) and FGMs. A veterinary check elicited an increase in total activity (TA), natural behaviours and repetitive locomotion and affected the activity cycle, particularly in summer. Transport produced changes in TA, nocturnal and natural activity and some variables of the activity cycle, mostly during summer. Although the effects of routine management procedures were different from each other and presumably stressful, they elicited changes only at the behavioural level, which was greater during summer. The differences observed according to non-invasive methodologies highlight the importance of a multidisciplinary approach in this context and suggest that it is unlikely that individual welfare was affected.

Behavioural diversity as a potential welfare indicator for professionally managed chimpanzees (Pan troglodytes): Exploring variations in calculating diversity using species-specific behaviours

Behavioural diversity may serve as a positive indicator of animal welfare that can be applied in long-term monitoring schemes in managed settings (eg zoos, laboratories, farms). Behavioural diversity is often higher when animals live in stimulating environments and experience positive events. Unfortunately, welfare researchers have not adopted consistent, standardised approaches to measuring behavioural diversity. The goal of this exploratory study was to utilise data from 41 adult chimpanzees (Pan troglodytes) housed across 16 zoological institutions to examine various models of Shannon's Diversity Index. Specifically, we investigated the impact of: combining versus splitting behaviours, including only positive behaviours, including human interaction, and considering recipient behaviours. We evaluate how the inclusion or exclusion of different behaviours impacts the relationship of behavioural diversity with: (i) concentrations of faecal glucocorticoid metabolites (GCM), a common indicator of adrenal activity; (ii) concentrations of immunoglobulin-A (IgA), an indicator of immune function and potential indicator of positive welfare; and (iii) stereotypic behaviour, a validated indicator of poor welfare. Most indices had significant negative relationships with faecal GCM. Animals that express a variety of behaviours from their species-typical repertoire have lower average faecal GCM concentrations and are likely experiencing better welfare. We did not find significant relationships between the behavioural diversity indices and IgA concentrations. Two indices were inversely associated with stereotypic behaviour. Our findings provide additional support for using Shannon's Diversity Index to calculate behavioural diversity as a robust, valid measure of positive welfare. However, future publications must justify the process for including or excluding behaviours from calculations.

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.

Influence of cereal harvest on adrenocortical activity in European hares (Lepus europaeus)

Anthropogenic disturbances, such as habitat modifications and machines, are associated with increased levels of faecal glucocorticoid metabolites (fGCMs) in mammals, an indicator of a stress response. One human-caused process provoking incisive habitat alterations is harvesting arable crops. We investigated the effect of cereal harvest on fGCM concentrations in European hares (Lepus europaeus) in arable landscapes in lower Austria during the year 2018 by collecting 591 faecal samples before, during and after cereal harvest. fGCMs were analysed using an enzyme immunoassay, and data were analysed using linear mixed-effects models. We found that neither cereal harvest nor farming practice (organic vs. conventional) caused an overall increase in the hares’ stress level. Lower vegetation density and higher proportions of bare ground were negatively correlated with fGCM concentrations, whereas the proportion of stubble fields was significantly positively correlated with fGCM concentrations in European hares. A change to more open landscapes might decrease time spent avoiding predation, and fallen grains may provide a beneficial additional food source for the hares. This indicates that European hares are well adapted to an opening up of the landscape and short-term disturbances such as cereal harvesting. In conclusion, cereal harvest had no large impact on European hares’ adrenocortical activity in an arable landscape with small average field size and enough available non-farmed areas.

Concentrations of faecal glucocorticoid metabolites in South African Mutton Merino sheep fed various sources of roughage

The objective of this study was to determine whether various sources of roughage in nutritionally balanced feedlot diets would evoke a stress response in sheep. A nutritional stress response test was performed on 20 ewes, randomly divided into four treatment groups with 5 ewes per group. Ewes were kept individually in metabolic crates for thirty days. Rations were balanced nutritionally, and various sources of roughage were included; T1: alfalfa hay, T2: maize stover, T3: soya hulls; and T4: Eragrostis tef. Faeces was removed manually from the caudal rectum of each ewe at 05h00 and 19h00. Samples were frozen at -20°C until analyses. A total of 520 faecal samples were analysed to determine the concentrations of faecal glucocorticoid metabolites (fGCM) by enzyme immunoassay (EIA). These concentrations were expressed as mass/g dry weight (ng/g DW). Baseline values were determined, and those greater than the mean plus 2 standard deviations were removed. Baseline values were compared between the groups with one-way ANOVA analysis. The average concentrations of fGCM were 178.77 ± 21.7 in the morning and 183.2 ± 14.4 in the evening. Sheep fed T4 had significantly higher fGCM concentrations in both morning (302.0 ± 86.1 ng/g DW) and evening (237.0 ± 48.1 ng/g DW) compared with the other treatment groups. Thus, fGCM concentrations in sheep were related to the source of roughage, and Eragrostis tef caused a stress response. Keywords: animal welfare, cortisol, dietary fibre, selective grazer, stress response

Effect of Dietary Inclusion of Live Microbial Cultures on Faecal Glucocorticoid Metabolites in Safari Asian Elephants

Background: Wildlife tourism has been shown to increase stress in a variety of species and can negatively affect the survival, reproduction, welfare, and behaviour of individuals. The use of faecal glucocorticoid metabolites has favoured the development of non-invasive methods to study physiological conditions and could aid in successful and effective strategies for the management and conservation of endangered species. Methods: An experiment was conducted to determine the effect of dietary inclusion of live microbial cultures to find patterns of physiological stress hormone in faeces of 18 safari Asian elephants of 30-62 years age and body weight (3495 ± 133.34 Kg) for two months. They were randomly divided into three groups, with six elephants in each, i.e. control with no probiotic (T1) and the other two with probiotics, which contained 1 × 109 CFU/g concentrate of Lactobacillus acidophilus (T2) and Saccharomyces cerevisiae (T3) @ every 50 kg body weight per day. Faecal samples were taken on 0, 30th, 40th and 50th day of the experiment. Result: It was observed that the mean faecal glucocorticoid metabolites, demonstrated by the elephants of T2, and T3 groups were not significantly (P > 0.05) different from the control group (T1) and remained unaffected on the supplementation of live microbial cultures.

Comparison of the Glucocorticoid Concentrations between Three Species of Lemuridae Kept in a Temporary Housing Facility

We compared the glucocorticoid concentrations in response to various types of potential stressors present during standard operation of a temporary housing facility between three species, namely, ring-tailed lemurs, collared brown lemurs and white-headed lemurs. The levels of faecal glucocorticoid metabolites (FGMs) were measured non-invasively on a daily basis during a 30-day period. A total of 510 faecal samples were collected. Concentrations of immunoreactive glucocorticoid hormone metabolites were measured in the obtained extracts by using an enzyme immunoassay. The polyclonal antibodies used in this assay were directed against the metabolite 11-oxo-etiocholanolone I. We found all three monitored lemur species to respond to specific potentially stressful situations by increasing (p < 0.05) the FGM levels within one to two days after the event. Although housed in the same room, differences in response to potentially stressful situations were found in white-headed lemurs compared to ring-tailed lemurs. Increased mean levels of the FGMs were found more frequently in white-headed lemurs than in ring-tailed lemurs. The results suggest that this species may be more sensitive to changes in its surroundings. In general, the levels of the FGMs showed a similar pattern during 30 days of monitoring suggesting that all groups of lemurs responded in a similar manner to the same events. However, we recorded the differences in the absolute values of glucocorticoid concentrations between the monitored species likely due to the differences in sex ratios in the groups and presence of lactating females in the ring-tailed lemurs.

Non-invasive stress evaluation in domestic horses ( Equus caballus ): impact of housing conditions on sensory laterality and immunoglobulin A

The study aimed to evaluate sensory laterality and concentration of faecal immunoglobulin A (IgA) as non-invasive measures of stress in horses by comparing them with the already established measures of motor laterality and faecal glucocorticoid metabolites (FGMs). Eleven three-year-old horses were exposed to known stressful situations (change of housing, initial training) to assess the two new parameters. Sensory laterality initially shifted significantly to the left and faecal FGMs were significantly increased on the change from group to individual housing and remained high through initial training. Motor laterality shifted significantly to the left after one week of individual stabling. Faecal IgA remained unchanged throughout the experiment. We therefore suggest that sensory laterality may be helpful in assessing acute stress in horses, especially on an individual level, as it proved to be an objective behavioural parameter that is easy to observe. Comparably, motor laterality may be helpful in assessing long-lasting stress. The results indicate that stress changes sensory laterality in horses, but further research is needed on a larger sample to evaluate elevated chronic stress, as it was not clear whether the horses of the present study experienced compromised welfare, which it has been proposed may affect faecal IgA.