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A Case Study: FecalCorticosteroid and Behavior asIndicators of Welfare DuringRelocation of an Asian ElephantNicole Laws a , Andre Ganswindt b , MichaelHeistermann b , Moira Harris c , Stephen Harris d &Chris Sherwin ca The Royal Veterinary College , London, UnitedKingdomb German Primate Centre , Göttingen, Germanyc Division of Clinical Veterinary Science , Universityof Bristol , United Kingdomd Department of Biological Sciences , University ofBristol , United KingdomPublished online: 05 Dec 2007.

To cite this article: Nicole Laws , Andre Ganswindt , Michael Heistermann , MoiraHarris , Stephen Harris & Chris Sherwin (2007) A Case Study: Fecal Corticosteroid andBehavior as Indicators of Welfare During Relocation of an Asian Elephant, Journal ofApplied Animal Welfare Science, 10:4, 349-358, DOI: 10.1080/10888700701555600

To link to this article: http://dx.doi.org/10.1080/10888700701555600

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BRIEF RESEARCH REPORT

A Case Study: Fecal Corticosteroid andBehavior as Indicators of Welfare During

Relocation of an Asian Elephant

Nicole LawsThe Royal Veterinary College

London, United Kingdom

Andre Ganswindt and Michael HeistermannGerman Primate Centre

Göttingen, Germany

Moira HarrisDivision of Clinical Veterinary ScienceUniversity of Bristol, United Kingdom

Stephen HarrisDepartment of Biological Sciences

University of Bristol, United Kingdom

Chris SherwinDepartment of Clinical Veterinary Science

University of Bristol, United Kingdom

This study was a preliminary investigation of an enzyme immunoassay for measuringfecal glucocorticoid metabolites in a male Asian elephant (Elephas maximus) by in-

JOURNAL OF APPLIED ANIMAL WELFARE SCIENCE, 10(4), 349–358Copyright © 2007, Lawrence Erlbaum Associates, Inc.

Correspondence should be sent to Chris Sherwin, Centre for Behavioral Biology, Division ClinicalVeterinary Science, University of Bristol, Bristol BS40 5DU, UK. Email: Chris.Sherwin@bristol.ac.uk

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vestigating changes in behavior and cortisol metabolite excretion associated with aputative stressful event. The study collected fecal samples for 10 days prior to, and 10days after, 24-hr transport and relocation of the elephant to a new herd. The studymeasured cortisol metabolites using 2 enzyme immunoassays indicating a 389% and340% increase in cortisol metabolite excretion following relocation. Maximalcortisol metabolite excretion occurred 2 days after relocation and remained elevatedduring establishment of the new herd. Stereotypic behavior increased approximately400% after relocation. The relocation disturbed sleep patterns, the elephant spent lesstime sleeping during the night, and the elephant slept standing up. These results pro-vide preliminary evidence that noninvasive monitoring of fecal cortisol metabolitescan be used to investigate adrenal activity in Asian elephants and may be a safe, prac-tical, and accurate welfare indicator.

Although there is no universally accepted definition of stress, it is generally ac-cepted to be “… reactions of the body to forces of a deleterious nature … thattend to disturb its normal physiological equilibrium” (Lane, 2006, p. 331). Thebody reacts to adverse events by activating the hypothalamic-pituitary-adrenalaxis and releasing catecholamine hormones, epinephrine and norepinephrine,and the glucocorticoid hormones, cortisol and cortisone, from the adrenal gland.Circulating glucocorticoids usually improve fitness by mobilizing energy or me-diating physiological and behavioral changes during emergency situations, al-though they can have negative consequences on health. Chronically high levelsmay decrease fitness by causing immunosuppression and atrophy of tissues.Therefore, the measurement of glucocorticoids can be valuable in studies ofnonhuman animal welfare. Increased cortisol concentrations have been recordedin a number of species after putatively stressful events:

1. Transport of cats (Felis catus [Farca, Pollicino, Massobrio, Badio, &Cavana, 2002]),

2. Transport of spotted hyenas (Crocuta crocuta [Goymann, Möstl, Van’tHof, East, & Hofer, 1999]),

3. Transport of tigers (Panthera tigris [Dembiec, Snider, & Zanella, 2004]),4. Transport of African elephants (Loxodonta African [Millspaugh et al.,

2007]),5. Restraint of cheetahs (Acinonyx jubatus [Jurke et al., 1997]), and6. Introduction of Asian elephants into a new herd (Elephas maximus [Dathe,

Kuckelhorn, & Minnemann, 1992; Schmid, Heistermann, Gansloßer, &Hodges, 2001]).

Cortisol output is often measured using blood or saliva, but this can be problem-atic for some studies. First, although this method of sampling is practical in ani-mals who are domesticated, it can be considerably more hazardous for some

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animals in the zoo. Second, the stress associated with handling during collectioncan elevate recorded cortisol concentrations, thereby invalidating the data. Third,due to the pulsatile release of cortisol, a single sample might not reflect the prevail-ing cortisol concentrations; accurate assessment can only be made with multiplesamples, but this can be particularly stressful for the animal—again invalidatingthe data. It is therefore advantageous to measure cortisol noninvasively and to usea technique that provides samples more representative of prevailing concentra-tions (Lane, 2006). To overcome these problems, methods for analyzing cortisolmetabolites in urine or feces have been developed in several species (Dembiec etal., 2004; Goymann et al., 1999; Lane, 2006; Millspaugh et al., 2002, 2007). Dueto the time lag of cortisol appearing in the samples, neither fecal nor urinarycortisol is useful as real-time measures of stress reactions, but they can be used formore significant or long-term changes in an animal’s life. Of these methods, fecalanalysis is often preferred because samples can be more easily obtained whileavoiding direct contact with a potentially dangerous animal.

Although the method does have drawbacks in that samples might be scarce, itcan be difficult to identify an individual when animals live in large groups andanalysis of fecal samples is often not as straightforward as that of urine or plasma.In elephants, cortisol has been measured in saliva (African and Asian elephants[Dathe et al., 1992]); urine (Asian elephants [Brown, Wemmer, & Lehnhardt,1995; Schmid et al., 2001]); and feces (African elephants [Ganswindt, Palme,Heistermann, Borragan, & Hodges, 2003; Millspaugh et al., 2007; Stead, Meltzer,& Palme, 2000; Wasser et al., 2000]). Although fecal glucocorticoid analysis hasbeen validated for the African elephant, we know of no published reports for theAsian species.

While working on a study examining the welfare of elephants in UK zoos, welearned one of the UK Asian elephants was to be transported overseas. Severalstudies have shown that transport and relocation of animals increase fecal cortisol(Dembiec et al., 2004; Farca et al., 2002, 2006; Goymann et al., 1999; Millspaughet al., 2007; Morrow, Kolver, Verkerk, & Matthews, 2002; Möstl, Maggs,Schrotter, Besenfelder, & Palme, 2002). The relocation of the elephant thereforeprovided the opportunity to examine whether glucocorticoid responses to a puta-tive stressful event could be measured noninvasively from fecal samples from anAsian elephant. The aim of this study was, therefore, to assess the suitability of twoenzyme immunoassays shown to be reliable for assessing glucocorticoid output inseveral other species (including the African elephant) to monitor adrenocorticalactivity in the Asian elephant using fecal measurements.

Studies on animal welfare are likely to provide the most robust findings if theyadopt a multidisciplinary approach. Abnormal behaviors, particularly stereo-typies, are often used as indicators of welfare. Stereotypies are abnormal, appar-ently nonfunctional, repetitive behaviors that often develop in animals housed inimpoverished environments or other adverse conditions (Mason, 1991). Most (ap-

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proximately 68%) situations that cause or increase stereotypies also decrease wel-fare (Mason & Latham, 2004). Stereotypy-eliciting situations are thus likely to bepoor for welfare, although exceptions exist. Mason and Latham proposed four pro-cesses that could link stereotypies to welfare but concluded that stereotypiesshould never be used as a sole indicator of welfare. In this study, we aimed to as-sess whether there was a relationship between glucocorticoid response and behav-ioral response to the putative stressful procedure of transport and relocation.

METHODS

Animal, Transport, and Husbandry

The study animal was a 23-year-old male Asian elephant, Chang, originallyhoused in Chester Zoo, United Kingdom. On May 8, 2005, he was transported toa zoo in France for breeding purposes. He was studied for 10 days prior to trans-portation and for 10 days after.

Prior to transportation, Chang was sedated using an etorphine and acepro-mazine maleate combination at a total dose of 2.8mg etorphine and 9.18mgacepromazine (Large Animal Immobilon, 2.25mg/ml etorphine base and 7.38mg/ml acepromazine base, C-Vet VP). Once Chang was loaded, the sedation was re-versed using 15 mg diprenorphine (Revivon, 3mg/ml, C-vet VP). He was thentransported by road and ship, which took a total of 24 hr.

The herd to which Chang was relocated consisted of four adult cows. For thefirst 2 days after arrival, he was housed in an outdoor bull pen during the day andindoors at night. Visual, auditory, and olfactory contact with the cows was possi-ble. After 3 days, he was released into the main outdoor enclosure by himself dur-ing the day and brought indoors at night. Four days after arrival, the gates betweenhis pen and the females were opened, allowing full physical contact indoors—hewas removed to his pen at night. Six days after arrival, he was allowed full contactwith the females in the outdoor enclosure but was housed in his pen indoors atnight, a routine that continued for the remainder of the study.

Measurement of Fecal Cortisol Metabolites

Fecal samples were taken at various times through the day—from two to fourtimes each day—for 10 days before transport to obtain pre-transport controlglucocorticoid levels. Immediately after transport, the four freshest fecal boli inthe shipping container were sampled. After this, fecal samples were taken fourtimes each day for the next 3 days and twice each day for the remainder of thestudy. A mean concentration was calculated from these multiple samples to rep-resent that 24-hr period. Feces (approximately 30 g) were removed from the

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core of the fecal ball, usually within 30 min of being voided and placed intoplastic tubes with screw tops. Samples were stored at –20 °C within 15 min ofcollection and kept frozen until analysis.

Fecal extracts were analyzed for immunoreactive cortisol metabolites (CM) us-ing an 11-oxo-etiocholanolone (3�-11oxo-CM) (produced during a collaborationbetween Dr. M. Dehnhard from the Institute of Zoo and Wildlife Research, Berlin,and Dr. E. Möstl from the Institute of Biochemistry of the University of VeterinaryMedicine in Vienna, Austria, for the German Primate Centre, Göttingen) and11ß-hydroxy-etiocholanolone (3�-11�-dihydroxy-CM) (produced by Dr. E.Möstl from the Institute of Biochemistry of the University of Veterinary Medicinein Vienna, Austria for the German Primate Centre, Göttingen) enzyme immu-noassay (EIA), both of which previously have been shown to provide reliable in-formation on adrenocortical activity in several other species (e. g., Heistermann,Palme, & Ganswindt, 2006; Möstl et al., 2002), including the African elephant(Ganswindt et al., 2003). Details of the sample preparation and assays are de-scribed by Ganswindt et al.

Assay sensitivity at 90% binding was 3 pg for the 3-11oxo-CM EIA and 2 pgfor the 3-11ß-dihydroxy-CM EIA. Dilution curves of samples from the pre- andpost-transport periods ran parallel to the respective standard curve in both assays.Intra-assay coefficients of variation for quality controls with high (QC high) andlow (QC low) concentrations (n = 16 each) were all < 5% for both assays.Inter-assay coefficients of variation were 6.5% (QC high, n = 6) and 9.3% (QClow, n = 6) for the 3-11oxo-CM EIA and 8.4% (QC high, n = 6) and 7.9% (QC low,n = 6) for the 3-11ß-dihydroxy-CM EIA.

Behavioral Observations

So that the behavioral data represented activities throughout the 24-hr period,10-min behavioral observations were conducted at 07.30, 09.30, 11.30, 13.30,and 15.30hr and from nighttime video recordings at 21.00, 23.00, 01.00, 03.00and 05.00hr. Behavior was categorized according to a detailed ethogram (avail-able upon request) that included aggression, eating, drinking, dust-bathing, andvocalizations; however, for clarity and brevity, only stereotypies and sleepingare reported here.

RESULTS

Fecal Cortisol Metabolites

Both fecal CMs followed a similar pattern (Figures 1a and 1b) and were signifi-cantly correlated (r2 = 0.877, p < .001). Pre-transport concentrations were highly

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consistent, with mean concentrations of 290 and 210 ng/g DW for the3-11oxo-CM EIA and 3-11ß-dihydroxy-CM EIA, respectively. After transport,concentrations were more variable but markedly elevated, with maximum levelsof 1,129 ng/g DW (3-11oxo-CM) and 715 ng/g DW (3-11ß-dihydroxy-CM) be-ing recorded in the samples collected 2 days after transport. The mean concen-trations were significantly greater during the post-transport phase compared tothe pre-transport phase in both assays (Mann-Whitney test: U = 102, 105; Z =–4.539, –4.488; p < .0001, p < .0001, respectively), indicating an average 389%and 340% increase, respectively.

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FIGURE 1 Changes in immunoreactive fecal glucorticoid metabolites of a male Asianelephant in response to transport and relocating. Each data point represents the mean ± SEMvalue of two to four samples collected within that 24-hr period.

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Behavior

Chang displayed stereotypic pacing before and after relocation (Figure 2). Priorto relocation, this occurred during the day when he was waiting to be brought in-doors, whereas after relocation this occurred only at night. Peak stereotypic be-havior occurred on the day after he was allowed outside with the cows for thefirst time. During the immediate days after relocation, Chang’s usual keeperfrom the United Kingdom reported that Chang was unusually unresponsive tonormal training and bathing routines. Prior to relocation, Chang spent approxi-mately 30% of the time sleeping, all of this at night. After relocation, however,the maximum time spent asleep was 20%, recorded 7 days after the move, withChang—most nights—having little or no sleep. The amount of nighttime sleep-ing decreased, which was partly compensated for by daytime sleeping increasingby 60%. In addition, Chang’s usual sleeping position changed from 92% sleep-ing in lateral or sternal recumbency to 100% sleeping while standing.

DISCUSSION

This study demonstrates that transport and relocation of a male Asian elephantto a new herd increased glucocorticoid output measured from fecal samples.Measures of the repeatability, sensitivity, and specificity of the assays—as wellas the parallelism to the standard curve for both assays—indicated that the re-sults were valid. This report is clearly a case study, but the data strongly indicate

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FIGURE 2 Changes in behavior in response to transport and relocation of a male Asianelephant.

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that analysis of fecal cortisol metabolites can be used as a noninvasive monitorof adrenal activity in male Asian elephants. In this respect, both assays testedappear to be of similar value as patterns of excretion were highly significantlycorrelated and indicated a change in cortisol metabolite concentrations of similarmagnitude as a result of translocation.

The marked increase in fecal CM concentrations 2 days after transport was ex-pected, given that the delay in glucocorticoid excretion is correlated with the tran-sit time of digesta and peaks after 12 to 56 hr in the African elephant (Ganswindt etal., 2003; Stead et al., 2000). The increase supports previous studies indicating thattransport and/or relocation of animals increases fecal corticosteroids (Dembiec etal., 2004; Farca et al., 2002, 2006; Goymann et al., 1999; Millspaugh et al., 2007;Morrow et al., 2002; Möstl et al., 2002). The magnitude and timing of the increaseis also comparable to other studies. In this study, cortisol metabolites increased by389% and 340%, 2 days after transport. Dembiec et al. (2004) reported that for in-experienced tigers, fecal corticosteroid levels peaked at 482% above baseline lev-els, 3 to 6 days after transport. Goymann et al. (1999) reported a fivefold increasein fecal cortisol occurred 3 days after translocating a male, spotted hyena. Morrowet al. (2002) reported an increase in fecal corticosteroids of cattle within 6 hr oftransport and a return to baseline levels within 24 hr. This could reflect the differ-ent digestive physiology of ruminants compared to nonruminants. The time of themaximal concentration in this study might also have reflected additional stimula-tion due to the novelty of Chang’s new social and physical environment whenplaced into the French zoo. In captive elephants, the use of a validated assay to de-termine fecal cortisol metabolites could help in the assessment of welfare by eval-uating responses to new enclosure designs, enrichment practices, trainingtechniques, introduction of new animals, and other management practices.

The suitability of fecal cortisol metabolites as a welfare indicator was supportedby behavioral observations. After relocation, stereotypic activity increased ap-proximately 400%. Stereotypies are widely regarded as indicating that an animal isfrustrated or experiencing some other negative affective state (Mason, 1991).Stereotypies in elephants have previously been noted to occur in conjunction withspecific events such as being given food or water (Friend, 1999), being moved be-tween different sections of the enclosure (Wilson et al., 2004), or even associatedwith changes in ambient temperature (Rees, 2004). The increases in Chang’s ste-reotypical behavior appeared more long-term than these types of stereotypies andwere possibly due to Chang’s frustration at being unable to contact the females forthe first few days. Alternatively, stereotypies can become emancipated from theiroriginal cause and might simply have appeared in Chang’s repertoire as a conse-quence of previous experience. In addition, translocation altered Chang’s sleepingbehavior. Prior to relocation, the majority of his sleeping was either sternal or lat-eral; post-relocation, sleeping duration was substantially reduced and was always

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standing. The temporal proximity of increases in cortisol metabolites andstereotypies (Figures 1a, 1b, and 2) indicate that transport and introduction of amale Asian elephant into a novel herd was a stressful event.

CONCLUSION

These results showed that when a male Asian elephant was exposed to a puta-tively stressful experience (24 hr of transportation and relocation to a novelherd), there were physiological and behavioral changes indicative of this havinga temporary negative impact on his welfare. The timing and intensity of thephysiological responses, supported by the behavioral changes, indicated that fe-cal glucocorticoid measurement is an accurate and appropriate method tononinvasively and safely assess the welfare of Asian elephants.

ACKNOWLEDGMENTS

This study was jointly funded by the Department for Environment, Food and RuralAffairs; British and Irish Association of Zoos and Aquariums; International Fundfor Animal Welfare; Royal Society for the Prevention of Cruelty to Animals; andUniversitiesFederationforAnimalWelfare .Wegratefully thank thestaffatChesterZoo (United Kingdom); Zoo Le Pal in Dompierre-sur-Besbre (France) for their val-ued support and assistance; and Professor Rupert Palme from the Department ofNaturalSciencesat theUniversityofVeterinaryMedicine inVienna,Austria, for theantibody and label for the 3-11oxo-CM EIA. We also thank an anonymous refereefor helpful comments.

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