Environmental, social, management and health factors associated with within- and between-individual variability in fecal glucocorticoid metabolite concentrations in zoo-housed Asian and African elephants

Identifying links between environmental, social, management, and health factors as they relate to physiological stress in captive elephants is crucial for the improvement of welfare and husbandry practices in North American zoos. Studies have examined the effects of short-term and chronic elevations in glucocorticoids in small groups of elephants, but few have examined adrenal activity on a large scale. This study evaluated 106 Asian (Elephas maximus) and 131 African (Loxodonta africana) elephants housed at 64 accredited facilities across North America. Fecal samples were collected every other week for 12 months and analyzed for glucocorticoid metabolite (FGM) concentrations. Risk factors for mean and individual variability (CV) in FGM were subjected to univariate and multivariable analyses using epidemiological methods. Independent variables that included individual traits, social environment, housing and management factors were chosen based on their identification as risk factors in previously published models for the same North American population of elephants. Results indicate that African elephants are more responsive to social stressors than Asians, and that poor joint health is a stress-related welfare problem for Asian, but not African elephants. For both species, higher FGM concentrations were associated with zoos located at more northern latitudes and having free access to indoor/outdoor spaces, whereas spending more time in managed interactions with staff were associated with lower FGM concentrations. Also important for captive management, elephants having diverse enrichment options and belonging to compatible social groups exhibited lower mean and reduced intra-individual variability in FGM. Our findings show that aspects of the zoo environment can be potential sources of stress for captive elephants, and that there are management activities that can facilitate coping and adapting to zoo conditions. Given species differences in factors that affected FGM, targeted, species-specific management approaches likely are needed to ensure good welfare for all elephants.


Introduction
Modern zoos strive to ensure animals under human care experience a high standard of welfare that 50 meets emotional and physical health needs [1]. Asian (Elephas maximus) and African (Loxodonta africana) elephants in zoos have received considerable scrutiny in the last decade due to concern over welfare and management practices [2]. To be successful, it is important that captive elephant programs evaluate the basic husbandry needs of individual animals, as well as the more complex factors that may affect welfare in a captive environment. An earlier study found no differences in serum cortisol 55 concentrations or cortisol variability in elephants managed in either free contact (elephants and people share the same space) or protected contact (elephants and people are separated by a barrier); however, there was a significant facility effect [3], suggesting that facility-specific differences in management exist that may affect adrenal activity and cortisol levels in captive elephants.
A recent 'Elephant Welfare Project (EWP)' took an epidemiological approach to investigating the 60 factors that impact zoo elephant welfare in North America [4]. That study, conducted by a multiinstitutional team of researchers and including 255 elephants at 68 Association of Zoos and Aquariums (AZA) accredited zoos, found that herd social structure, caretaker interactions, and enrichment, and feeding diversity correlated with a variety of welfare outcomes [5][6][7][8][9][10][11][12][13][14][15][16]. In particular, enrichment and social factors were important for reproductive activity and reducing stereotypic behaviors, diversity of 65 feeding practices and exercise reduced the likelihood that an elephant would be overweight, softer exhibit substrates were good for physical and behavioral health, and positive keeper-elephant relationships were mutually beneficial. Overall, environments that provided diversity and choice were of greater importance to elephant welfare than exhibit size alone. A remaining question is how these factors affect physiological stress responses in individual elephants, and their ability to cope with a zoo 70 environment. Models exhibiting multi-collinearity, as defined by a variance inflation factor of greater than 10 and a 175 Condition Index of greater than 30, were not considered for further analysis.
Unless otherwise indicated, differences were considered significant at P < 0.05. All analyses were conducted using IBM SPSS Statistics Version 25, IBM Corp., Armonk, NY, USA.

Results
The elephant study population ranged in age from 0 to 64 years (mean age: Asian, 34.3 ±1.5 180 years; African, 27.7 ±1.1). Table 2 presents seasonal mean FGM concentrations for each species.
Overall FGM concentrations were higher in Asian (124.41 ± 4.89 ng/g) than African (97.73 ± 3.01 ng/g) elephants. There was a significant main effect of species (F = 27.86, P = 0.000), but not season (F = 1.30, P = 0.000). In all seasons, Asian elephants had higher mean concentrations than Africans.

Multivariable analyses required the exclusion of Mean Daily Walking Distance and
Recumbent Rate because these variables were measured in only a sub-set of the elephants. Also, Social Experience was highly correlated (r = 0.899) with Social Group Contact and so also was not 220 included in the multivariable model building process due to collinearity problems. The final models are given in Table 6 for Asian and Table 7 for African elephants.  in space experience produces a 0.60 ng/g in FGMs.  Species confounds Enrichment Diversity and Social Group Contact due to Asian elephants receiving, on average, slightly more enrichment than Africans (see Table 4), and Africans having contact with more social groups than Asians (Table 4), primarily because Africans are kept more often in larger 270 groups.

275
Because Enrichment Diversity is calculated on a zoo-level, Figure 1 shows the correlation between a zoo's enrichment diversity score and the average CV FGM of the elephants at a zoo. because it increases an animal's perceived control over its environment [42] and being given a choice of moving between indoor and outdoor areas at will has been associated with reduced stereotypic behaviors in polar bears [43], Asian elephants [44], and giant pandas [45]. However, in a separate Joint health was associated with FGM concentrations among Asian elephants. Elephants with joint problems had higher FGMs than those that did not, presumably due to pain. This was the case for zoo-housed Asian elephants, which spent more time on hard surfaces and were older on average than 320 zoo-housed Africans [12], both risk factors for joint health problems, although there was no difference in the muscoskeletal scores assigned to individuals of these two populations [12]. This species difference may mean that joint health has been unintentionally diagnosed differently for each species, or is differentially experienced as more painful by Asian elephants.
Latitude of Zoo was a risk factor for higher FGMs in African elephants, increasing as a zoo 325 location was more northwards. For Asians, this effect was only identified in the spring months. There are a variety of elephant management modifications that take place as seasons change, such as elephants spending more time confined inside or outside, with potential changes in social density or social contact that could account for increased social stress. Carlstead et al. [15] also found that Latitude of Zoo was a predictor of higher serum cortisol for the North American population of Asian 330 elephants. Latitude as a risk factor of FGMs may be indicative of sensitivity to climatological changes that we would expect to be more pronounced the further north an elephant resides. Higher glucocorticoids have been reported during colder seasons among small numbers of zoo-housed Asian [46] and African [47] elephants. It remains unclear if latitude effects in the U.S. are due to climatological factors such as temperature and day length, or husbandry differences that cause more 335 stressful conditions for elephants. In Thailand, mean FGM concentrations were ~28% higher in winter compared to the summer and rainy seasons, and were negatively associated with environmental factors: temperature and rainfall, but not humidity [48]. The need for more energy to maintain optimum body temperature and ensure survival in cooler temperatures could be related to this finding.
This likely is an adaptive mechanism to ensure maintenance of anormal body temperature. In other 340 ungulates, higher GC levels during winter have been found in white-tailed deer (Odocoileus virginianus) [49] and mule deer (Odocoileus hemionus) [50]. Elevated circulating GC levels as a response to cold stress also were documented in reindeer (Rangifer tarandus) [51] and in farm animals [52]. Seasonal trends in reproductive activity also have been documented. For example, a group of African elephants housed indoors because of cold temparatures at a zoo in Rhode Island showed 345 prolonged non-luteal phases before re-initiating normal ovarian cycles in the spring [53] that could have been due to increased social stress or proximity effects, although GCs were not evaluated in that study. Therefore, while the overall population effect of choice appears to be stress-reducing, it leads to 380 slightly increased variability in individuals. We speculate that this may be due to movements of other elephants in the herd going in and out in an unpredictable manner. A given individual might benefit from having increased choice and control over its own situation, but it has no control over the whereabouts of other elephants, potentially resulting in more variable stress responses. Cochrem [57] points out that CV needs to be included in studies of GCs because the factors that account for within-385 individual variation and their adaptive significance for a species, such as personality, coping styles, genetic or maternal influences, are little known for most species. For example, increased variability in FGMs was correlated with abnormal reproductive function, rates of fighting, and institutional mortality rates in rhinoceros [39], leading to the conclusion that the variability of FGMs is a valuable measure of stress responsiveness that has biological costs to the animal. The subject of individual variation in GC 390 responses to stressors has included studies investigating different coping styles and disease susceptibility [58], and a better understanding of inter-and intra-individual variation in HPA reactivity would be beneficial to our use of GCs as a welfare measure [16].
Enrichment Diversity was strongly associated with a reduction in CV of FGMs, but not with the provision of manipulatable objects such as balls, tires and hanging objects, to feeding items such as browse and treat boxes/bags, and scents, music and problem-solving tasks [6]. We found the zoo average FGM CVs to be negatively correlated with the frequency of only three of the 30 enrichment types: problem-solving (r = -0.348, n = 57, p = 0.007), hanging objects (r = -0.261, p = 0.048) and 415 scratching posts (r = -0.340. p = 0.009); three enrichments that intensely engage elephants. All evidence together strongly suggests that enrichment has a "calming" effect on stress responses of elephants, most likely by providing additional behavioral options and/or cognitive opportunities to cope with their daily lives.
Last, being a member of more social groups (Social Group Contact) also was associated with 420 lower variability in FGMs. Therefore, being a familiar and accepted member of multiple social groups may also stabilize HPA-axis activity in a manner similar to Enrichment Diversity, effectively increasing social enrichment diversity, a clear benefit for elephant welfare.

Conclusions
425 Results elucidate species differences in adrenal responses of elephants in zoo environments. African elephants appear to be more responsive to social stressors than Asians. It is well known that Asian elephants are not as bonded to large social groups as their African cousins and, therefore, have more limited hierarchical stratification, whereas African elephants live and interact in multi-tiered groups with presumably more social constraints [62]. Another species difference is that Asians might be more 430 sensitive to stress caused by joint pain than Africans, but rates of joint problems and age differences between the two populations complicate this conclusion. In any case, the evidence points to poor joint health being a stress-related welfare problem for the U.S. population of Asian elephants. For both species, zoos located at more northern latitudes were characterized by elephants with slightly to significantly higher FGMs. It is unclear if these responses are due to climatological or management 435 factors, or both. One factor that reduced FGMs for both species was more time being managed, suggesting time spent with keepers has a positive effect. More time being managed also was associated with reduced stereotypy [7], perhaps related to less boredom. Finally having diverse enrichment options and contact with multiple social groups also appears to be calming for elephants, reducing intra-individual variability in FGMs. Together, all evidence points to the beneficial effects of diverse 440 enrichment opportunities, including cognitive enrichment for zoo-housed elephants. We conclude that there are many avenues for further research on stress in zoo-housed elephants, and monitoring FGMs longitudinally is a proven non-invasive method for determining factors contributing to adrenal function, stress and coping responses in elephants. The species differences observed in adrenal responses to zoo factors suggests that a one-size-fits-all management strategy may not be the best for 445 both Asian and African elephans, and that more species-specific approach to husbandry may be needed.