Animal welfare researchers are committed to identifying novel measures for enhancing the quality of life of individual animals. Recently, welfare scientists have emphasized the need for tracking multiple indicators of an animal’s behavioral, emotional and mental health. Researchers are currently focused on integrating non-invasive physiological biomarkers to gain insight into an individual’s welfare status. Most commonly, the animal welfare community has analyzed glucocorticoid hormones and their metabolites as a measure of stress. While glucocorticoids provide valuable information about hypothalamic–pituitary–adrenal (HPA) axis activity, there are limitations to utilizing these hormones as the sole measure of long-term stress and welfare. Other biomarkers, such as dehydroepiandrosterone and its sulfate ester—collectively referred to as DHEA(S)—help provide a more complete picture of HPA activity. DHEA(S) counteracts the effects glucocorticoids by having anti-aging, immune-enhancing and neuroprotective properties. Recent studies have examined the ratio of glucocorticoids to DHEA(S) as a way to better understand how the HPA axis is functioning. There is evidence that this ratio serves as an indicator of immune function, mental health, cognitive performance and overall welfare. We review studies that employed the glucocorticoid:DHEA(S) ratio, outline methodological considerations and discuss how researchers can integrate glucocorticoids, DHEA(S) and the glucocorticoid:DHEA(S) ratio into welfare assessments.

Citation -Razal, C. B., Bryant, J., & Miller, L. J. (2017). Monitoring the behavioral and adrenal activity of giraffe (Giraffa camelopardalis) to assess welfare during seasonal housing changes. Animal Behavior and Cognition, 4(2), 154-164. https://doi.org/10.12966/abc.03.05.2017Abstract -In inclement weather, northern zoos are required to provide animals from warmer climates with indoor exhibits. These indoor exhibits are typically smaller, lack natural substrate, and have lower levels of stimulation for the animals. The purpose of this study was to examine the welfare of four reticulated giraffe (Giraffa camelopardalis reticulata) exhibited at the Chicago Zoological Society -Brookfield Zoo -during the summer in an outdoor enclosure compared to the winter in an indoor enclosure. A combination of direct behavioral observations, percentage of time spent recumbent, and adrenal hormone monitoring through fecal samples were utilized for a more comprehensive look at welfare. Individual variation was observed between the giraffe. An adult female giraffe engaged in Active Forage behavior significantly more in the winter compared to summer (mean difference = 0.23, p = 0.049). An adult female and a juvenile male displayed significantly more Active Non-Forage behavior in the summer than in the winter (mean difference = 0.32, p = 0.048; mean difference = 0.31, p = 0.048). The predominant behavior in the summer for the group as a whole was Active Non-Forage (59%), whereas Active Forage was most prevalent in the winter (60%). There was also a significant positive correlation between time spent recumbent per hour for each 24-hour day measured in the summer and winter (r = 0.51, p = 0.010). Although no significant differences were found for individual FGM concentrations between the seasons, average FGM concentrations for the group were 2306.67 ng/g in the summer and 4261.64 ng/g in the winter. Information gained from this study can help aid animal managers to make informed decisions to ensure each individual giraffe is thriving year-round. In addition, we hope this study can serve as a model to examine the seasonal welfare of other animals in zoological institutions with similar conditions.

Accredited zoological facilities are committed to fully understanding the behavioral, mental, and physical needs of each species to continuously improve the welfare of the animals under their professional care and detect when welfare has diminished. In order to accomplish this goal, internally consistent and externally valid indicators of animal welfare are necessary to advance our understanding of the current welfare status of individual animals. Historically, efforts have focused on monitoring visible or observable signs of poor health or problem behavior, but lack of signs or problems does not necessarily demonstrate that an individual animal is thriving. The current study examined fecal hormone metabolite levels and behavior for two species of bottlenose dolphins (Tursiops truncatus and Tursiops aduncus) from 25 different accredited zoological facilities. At the time of the study, all facilities were accredited by the Alliance of Marine Mammal Parks and Aquariums and/or the Association of Zoos and Aquariums. This was part of the multi-institutional study ‘Towards understanding of the welfare of cetaceans in zoos and aquariums” commonly referred to as the Cetacean Welfare Study. Behavioral diversity was calculated using the Shannon Diversity Index on species-appropriate behavioral events. Behavioral diversity was compared to the fecal metabolites of cortisol, aldosterone, and the ratio of cortisol to dehydroepiandrosterone (DHEA) as well as the stereotypic behavior of route tracing. Similar to previous studies on other species, there was a significant inverse relationship between behavioral diversity and both fecal cortisol metabolites and route tracing. Additionally, a significant inverse relationship also exists between behavioral diversity and the ratio of fecal cortisol to DHEA metabolites. Behavioral diversity and fecal aldosterone metabolites were not associated. Additional research is still needed to validate behavioral diversity as an indicator of positive animal welfare for bottlenose dolphins and across species. However, based on current results, facilities could utilize behavioral diversity combined with other measures of welfare to more comprehensively evaluate the welfare of bottlenose dolphins.

We evaluated the suitability of a corticosterone enzyme immunoassay (EIA) to monitor excretion of fecal glucocorticoid metabolites (FGM) in response to Adrenocorticotropic hormone (ACTH) and saline injections in three desert rodent species (Gerbillus andersoni allenbyi (GA), Gerbillus nanus (GN), and Gerbilis piridium (GP). We exposed 24 gerbils (N = 9 for GA, N = 7 for GN, N = 8 for GP) to an ACTH and a saline injection at different times. Fecal samples were collected hourly for 24 hours after injection. The average starting concentration (baseline) FGM concentration was 797 ng/g for GA, 183 ng/g for GN, and 749 ng/g for GP. The average peak concentration was 2377 ng/g for GA, 589 ng/g for GN, and 1987 ng/g for GP. We were able to provide a physiological validation for the chosen assay in GAs and GPs, however, our results for GNs were less clear. We found an increase in FGM concentrations on average after 5.5 hours in GA, 3.1 hours in GN, and 3.8 hours in GP. We found a peak in FGM concentration on average after 8.8 hours in GA, 5.6 hours in GN, and 10.3 hours in GP. We determined that FGM concentration returned to starting value on average after 14.4 hours in GA, 9.1 hours in GN, and 15.1 hours in GP. The outcomes of this study can help establish trapping protocols and time frames for FGM monitoring of these wild small mammal populations. The time course for excretion of FGM is similar between the species in this study, and comparable to some non-desert rodents. We found high variation in the time course of excretion within species. This variation needs to be taken into account when monitoring stress responses in the field. By assessing adrenocortical activity using FGM monitoring, stress responses to varying ecological and environmental factors can be reliably examined in the field.

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.

Visitors and the accompanying increased sound levels are two of the factors of daily life for many zoo-housed animals. Here the impact different seasons have on the physiological and behavioral responses of two African elephants, Loxodonta africanus, and three Black rhinoceros, Diceros bicornis, were evaluated at the Brookfield Zoo. All study animals showed the highest fecal glucocorticoid metabolite (FGM) concentrations during the summer when exhibit attendance is the highest, and lowest FGM concentrations in the winter when exhibit attendance is the lowest. Interestingly, black rhinoceros were less active when FGM levels and exhibit attendance were higher and elephants more active. Unexpectedly, ambient sound levels followed no clear pattern between seasons. Friedman's tests showed FGM concentrations and exhibit attendance was significantly higher in the summer than in winter. The results highlight that exhibit attendance may be associated with some changes in adrenal and behavioral responses of zoo animals, but there is also substantial individual variation within and across species in their responses to the same environmental factors.

The goal of the current study was to create reference intervals and values for several common and one potential novel physiological indicators of animal welfare for four species of cetaceans. The subjects included 189 bottlenose dolphins (Tursiops truncatus), 27 Indo-Pacific bottlenose dolphins (Tursiops aduncus), eight Pacific white-sided dolphins (Lagenorhynchus obliquidens), and 13 beluga whales (Delphinapterus leucas) at Alliance of Marine Mammal Parks and Aquariums and/or Association of Zoos and Aquariums accredited facilities. During two sampling time periods between July and November of 2018 and between January and April of 2019, fecal samples were collected weekly for five weeks from all animals. Samples were processed and analyzed using enzyme immunoassay for fecal cortisol, aldosterone, and dehydroepiandrosterone (DHEA) metabolites. Linear mixed models were used to examine demographic and time factors impacting hormone metabolite concentrations. Age, sex, and time of year were all significant predictors for some of the models (p < 0.01). An iOS mobile application ZooPhysioTrak was created for easy access to species-specific reference intervals and values accounting for significant predictors. For facilities without access to this application, additional reference intervals and values were constructed without accounting for significant predictors. Information gained from this study and the use of the application can provide reference intervals and values to make informed management decisions for cetaceans in zoological facilities.

Knowledge regarding the relationship between endocrine parameters and reproductive activity can offer important insights into how social and environmental factors influence the reproductive success of mammals. Although components of both the physical and social environment affect endocrine regulation of reproduction, less is understood about the potential role of interactions between different endocrine axes on reproductive activity. We evaluated temporal patterns of reproductive and adrenocortical steroids in two male and three female aardwolves (Proteles cristata) housed in captivity at Brookfield Zoo, Chicago, USA. We found seasonal variation in faecal androgens, estrogens, and progestagens, which provide support for previous observations of the aardwolf as a seasonal breeder. However, the timing of peak endocrine activity did not correspond to observations from wild populations. Our interpretation is that this discrepancy is caused by photoperiodic regulation of reproductive activity. We found a positive relationship between faecal androgens and faecal glucocorticoid metabolites in males and a positive relationship between faecal estrogens and faecal glucocorticoid metabolites in females when housed with conspecifics but not when housed alone. We also found a positive but asymptotic relationship between faecal progestagens and faecal glucocorticoid metabolites. We argue that these observations indicate a potential effect of reproductive endocrine activity on the hypothalamic-pituitary adrenal axis, which could result in interesting physiological trade-offs in male reproductive tactics and female prepartum maternal investment because of the negative effects of long-term glucocorticoid elevation on reproductive performance. Finally, our results suggest that social and environmental factors interact in regulating many aspects of endocrine fluctuations in this mostly solitary species. Key Words aardwolf, hypothalamic-pituitary adrenal axis, seasonality, stress, reproductive timingActa Theriologica, In Press 2 IntroductionKnowledge of how social and environmental factors influence animal reproductive physiology is important for our ability to understand the regulation of reproductive success. Reproductive activity in mammals is often either seasonal or non-seasonal, with subsequent different longitudinal patterns of reproductive hormones. In non-seasonal breeders, females exhibit ovulatory cycles and males are typically reproductively active throughout the year (Brown 2006). Contrarily, in seasonally breeding species reproductive hormones are elevated but highly variable during a defined period of time in both males and females (Monfort et al. 1989;Wingfield 1990;Monfort et al. 1997;Kraaijeveld-Smit et al. 2002;Kretzschmar et al. 2004;Hesterman et al. 2005; Dloniak et al. 2006b; Fanson et al. 2010a, b). The ultimate causes for seasonal breeding strategies are linked to temporal variation in resource abundance, whereas the most important proximate regulator of reproductive activity is photoperiod (Scott 1986; Gold...