Kingdom b German Primate Centre, Göttingen, Germany. To link to this article:

Transcription

1 This article was downloaded by: [Dr Kenneth Shapiro] On: 09 June 2015, At: 07:23 Publisher: Routledge Informa Ltd Registered in England and Wales Registered Number: Registered office: Mortimer House, Mortimer Street, London W1T 3JH, UK Journal of Applied Animal Welfare Science Publication details, including instructions for authors and subscription information: A Case Study: Fecal Corticosteroid and Behavior as Indicators of Welfare During Relocation of an Asian Elephant Nicole Laws a, Andre Ganswindt b, Michael Heistermann b, Moira Harris c, Stephen Harris d & Chris Sherwin c a The Royal Veterinary College, London, United Kingdom b German Primate Centre, Göttingen, Germany c Division of Clinical Veterinary Science, University of Bristol, United Kingdom d Department of Biological Sciences, University of Bristol, United Kingdom Published online: 05 Dec To cite this article: Nicole Laws, Andre Ganswindt, Michael Heistermann, Moira Harris, Stephen Harris & Chris Sherwin (2007) A Case Study: Fecal Corticosteroid and Behavior as Indicators of Welfare During Relocation of an Asian Elephant, Journal of Applied Animal Welfare Science, 10:4, , DOI: / To link to this article: PLEASE SCROLL DOWN FOR ARTICLE Taylor & Francis makes every effort to ensure the accuracy of all the information (the Content ) contained in the publications on our platform. However, Taylor & Francis, our agents, and our licensors make no representations or warranties whatsoever as to the accuracy, completeness,

2 or suitability for any purpose of the Content. Any opinions and views expressed in this publication are the opinions and views of the authors, and are not the views of or endorsed by Taylor & Francis. The accuracy of the Content should not be relied upon and should be independently verified with primary sources of information. Taylor and Francis shall not be liable for any losses, actions, claims, proceedings, demands, costs, expenses, damages, and other liabilities whatsoever or howsoever caused arising directly or indirectly in connection with, in relation to or arising out of the use of the Content. This article may be used for research, teaching, and private study purposes. Any substantial or systematic reproduction, redistribution, reselling, loan, sub-licensing, systematic supply, or distribution in any form to anyone is expressly forbidden. Terms & Conditions of access and use can be found at

3 JOURNAL OF APPLIED ANIMAL WELFARE SCIENCE, 10(4), Copyright 2007, Lawrence Erlbaum Associates, Inc. BRIEF RESEARCH REPORT A Case Study: Fecal Corticosteroid and Behavior as Indicators of Welfare During Relocation of an Asian Elephant Nicole Laws The Royal Veterinary College London, United Kingdom Andre Ganswindt and Michael Heistermann German Primate Centre Göttingen, Germany Moira Harris Division of Clinical Veterinary Science University of Bristol, United Kingdom Stephen Harris Department of Biological Sciences University of Bristol, United Kingdom Chris Sherwin Department of Clinical Veterinary Science University of Bristol, United Kingdom This study was a preliminary investigation of an enzyme immunoassay for measuring fecal glucocorticoid metabolites in a male Asian elephant (Elephas maximus)by in- Correspondence should be sent to Chris Sherwin, Centre for Behavioral Biology, Division Clinical Veterinary Science, University of Bristol, Bristol BS40 5DU, UK. Chris.Sherwin@bristol.ac.uk

4 350 LAWS ET AL. vestigating changes in behavior and cortisol metabolite excretion associated with a putative stressful event. The study collected fecal samples for 10 days prior to, and 10 days after, 24-hr transport and relocation of the elephant to a new herd. The study measured cortisol metabolites using 2 enzyme immunoassays indicating a 389% and 340% increase in cortisol metabolite excretion following relocation. Maximal cortisol metabolite excretion occurred 2 days after relocation and remained elevated during establishment of the new herd. Stereotypic behavior increased approximately 400% after relocation. The relocation disturbed sleep patterns, the elephant spent less time sleeping during the night, and the elephant slept standing up. These results provide preliminary evidence that noninvasive monitoring of fecal cortisol metabolites can be used to investigate adrenal activity in Asian elephants and may be a safe, practical, and accurate welfare indicator. Although there is no universally accepted definition of stress, it is generally accepted to be reactions of the body to forces of a deleterious nature that tend to disturb its normal physiological equilibrium (Lane, 2006, p. 331). The body reacts to adverse events by activating the hypothalamic-pituitary-adrenal axis 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 mediating physiological and behavioral changes during emergency situations, although they can have negative consequences on health. Chronically high levels may decrease fitness by causing immunosuppression and atrophy of tissues. Therefore, the measurement of glucocorticoids can be valuable in studies of nonhuman animal welfare. Increased cortisol concentrations have been recorded in 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 t Hof, 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]), and 6. 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 problematic for some studies. First, although this method of sampling is practical in animals who are domesticated, it can be considerably more hazardous for some

5 RELOCATION OF AN ASIAN ELEPHANT 351 animals in the zoo. Second, the stress associated with handling during collection can elevate recorded cortisol concentrations, thereby invalidating the data. Third, due to the pulsatile release of cortisol, a single sample might not reflect the prevailing cortisol concentrations; accurate assessment can only be made with multiple samples, but this can be particularly stressful for the animal again invalidating the data. It is therefore advantageous to measure cortisol noninvasively and to use a technique that provides samples more representative of prevailing concentrations (Lane, 2006). To overcome these problems, methods for analyzing cortisol metabolites in urine or feces have been developed in several species (Dembiec et al., 2004; Goymann et al., 1999; Lane, 2006; Millspaugh et al., 2002, 2007). Due to the time lag of cortisol appearing in the samples, neither fecal nor urinary cortisol is useful as real-time measures of stress reactions, but they can be used for more significant or long-term changes in an animal s life. Of these methods, fecal analysis is often preferred because samples can be more easily obtained while avoiding direct contact with a potentially dangerous animal. Although the method does have drawbacks in that samples might be scarce, it can be difficult to identify an individual when animals live in large groups and analysis 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 has been validated for the African elephant, we know of no published reports for the Asian species. While working on a study examining the welfare of elephants in UK zoos, we learned one of the UK Asian elephants was to be transported overseas. Several studies have shown that transport and relocation of animals increase fecal cortisol (Dembiec et al., 2004; Farca et al., 2002, 2006; Goymann et al., 1999; Millspaugh et al., 2007; Morrow, Kolver, Verkerk, & Matthews, 2002; Möstl, Maggs, Schrotter, Besenfelder, & Palme, 2002). The relocation of the elephant therefore provided the opportunity to examine whether glucocorticoid responses to a putative stressful event could be measured noninvasively from fecal samples from an Asian elephant. The aim of this study was, therefore, to assess the suitability of two enzyme immunoassays shown to be reliable for assessing glucocorticoid output in several other species (including the African elephant) to monitor adrenocortical activity in the Asian elephant using fecal measurements. Studies on animal welfare are likely to provide the most robust findings if they adopt a multidisciplinary approach. Abnormal behaviors, particularly stereotypies, are often used as indicators of welfare. Stereotypies are abnormal, apparently nonfunctional, repetitive behaviors that often develop in animals housed in impoverished environments or other adverse conditions (Mason, 1991). Most (ap-

6 352 LAWS ET AL. proximately 68%) situations that cause or increase stereotypies also decrease welfare (Mason & Latham, 2004). Stereotypy-eliciting situations are thus likely to be poor for welfare, although exceptions exist. Mason and Latham proposed four processes that could link stereotypies to welfare but concluded that stereotypies should never be used as a sole indicator of welfare. In this study, we aimed to assess whether there was a relationship between glucocorticoid response and behavioral 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, originally housed in Chester Zoo, United Kingdom. On May 8, 2005, he was transported to a zoo in France for breeding purposes. He was studied for 10 days prior to transportation and for 10 days after. Prior to transportation, Chang was sedated using an etorphine and acepromazine maleate combination at a total dose of 2.8mg etorphine and 9.18mg acepromazine (Large Animal Immobilon, 2.25mg/ml etorphine base and 7.38mg/ ml acepromazine base, C-Vet VP). Once Chang was loaded, the sedation was reversed using 15 mg diprenorphine (Revivon, 3mg/ml, C-vet VP). He was then transported by road and ship, which took a total of 24 hr. The herd to which Chang was relocated consisted of four adult cows. For the first 2 days after arrival, he was housed in an outdoor bull pen during the day and indoors at night. Visual, auditory, and olfactory contact with the cows was possible. After 3 days, he was released into the main outdoor enclosure by himself during the day and brought indoors at night. Four days after arrival, the gates between his pen and the females were opened, allowing full physical contact indoors he was removed to his pen at night. Six days after arrival, he was allowed full contact with the females in the outdoor enclosure but was housed in his pen indoors at night, 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 four times each day for 10 days before transport to obtain pre-transport control glucocorticoid levels. Immediately after transport, the four freshest fecal boli in the shipping container were sampled. After this, fecal samples were taken four times each day for the next 3 days and twice each day for the remainder of the study. A mean concentration was calculated from these multiple samples to represent that 24-hr period. Feces (approximately 30 g) were removed from the

7 RELOCATION OF AN ASIAN ELEPHANT 353 core of the fecal ball, usually within 30 min of being voided and placed into plastic tubes with screw tops. Samples were stored at 20 C within 15 min of collection and kept frozen until analysis. Fecal extracts were analyzed for immunoreactive cortisol metabolites (CM) using an 11-oxo-etiocholanolone (3a-11oxo-CM) (produced during a collaboration between 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 Veterinary Medicine in Vienna, Austria, for the German Primate Centre, Göttingen) and 11ß-hydroxy-etiocholanolone (3a-11b-dihydroxy-CM) (produced by Dr. E. Möstl from the Institute of Biochemistry of the University of Veterinary Medicine in Vienna, Austria for the German Primate Centre, Göttingen) enzyme immunoassay (EIA), both of which previously have been shown to provide reliable information 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 described by Ganswindt et al. Assay sensitivity at 90% binding was 3 pg for the 3-11oxo-CM EIA and 2 pg for the 3-11ß-dihydroxy-CM EIA. Dilution curves of samples from the pre- and post-transport periods ran parallel to the respective standard curve in both assays. Intra-assay coefficients of variation for quality controls with high (QC high) and low (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% (QC low, 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, and 05.00hr. Behavior was categorized according to a detailed ethogram (available upon request) that included aggression, eating, drinking, dust-bathing, and vocalizations; however, for clarity and brevity, only stereotypies and sleeping are reported here. RESULTS Fecal Cortisol Metabolites Both fecal CMs followed a similar pattern (Figures 1a and 1b) and were significantly correlated (r 2 = 0.877, p <.001). Pre-transport concentrations were highly

8 354 LAWS ET AL. FIGURE 1 Changes in immunoreactive fecal glucorticoid metabolites of a male Asian elephant in response to transport and relocating. Each data point represents the mean ± SEM value of two to four samples collected within that 24-hr period. consistent, with mean concentrations of 290 and 210 ng/g DW for the 3-11oxo-CM EIA and 3-11ß-dihydroxy-CM EIA, respectively. After transport, concentrations were more variable but markedly elevated, with maximum levels of 1,129 ng/g DW (3-11oxo-CM) and 715 ng/g DW (3-11ß-dihydroxy-CM) being recorded in the samples collected 2 days after transport. The mean concentrations were significantly greater during the post-transport phase compared to the 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.

9 RELOCATION OF AN ASIAN ELEPHANT 355 FIGURE 2 elephant. Behavior Changes in behavior in response to transport and relocation of a male Asian Chang displayed stereotypic pacing before and after relocation (Figure 2). Prior to relocation, this occurred during the day when he was waiting to be brought indoors, whereas after relocation this occurred only at night. Peak stereotypic behavior occurred on the day after he was allowed outside with the cows for the first time. During the immediate days after relocation, Chang s usual keeper from the United Kingdom reported that Chang was unusually unresponsive to normal training and bathing routines. Prior to relocation, Chang spent approximately 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, with Chang most nights having little or no sleep. The amount of nighttime sleeping decreased, which was partly compensated for by daytime sleeping increasing by 60%. In addition, Chang s usual sleeping position changed from 92% sleeping in lateral or sternal recumbency to 100% sleeping while standing. DISCUSSION This study demonstrates that transport and relocation of a male Asian elephant to a new herd increased glucocorticoid output measured from fecal samples. Measures of the repeatability, sensitivity, and specificity of the assays as well as the parallelism to the standard curve for both assays indicated that the results were valid. This report is clearly a case study, but the data strongly indicate

10 356 LAWS ET AL. that analysis of fecal cortisol metabolites can be used as a noninvasive monitor of adrenal activity in male Asian elephants. In this respect, both assays tested appear to be of similar value as patterns of excretion were highly significantly correlated and indicated a change in cortisol metabolite concentrations of similar magnitude as a result of translocation. The marked increase in fecal CM concentrations 2 days after transport was expected, given that the delay in glucocorticoid excretion is correlated with the transit time of digesta and peaks after 12 to 56 hr in the African elephant (Ganswindt et al., 2003; Stead et al., 2000). The increase supports previous studies indicating that transport and/or relocation of animals increases fecal corticosteroids (Dembiec et al., 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 increase is also comparable to other studies. In this study, cortisol metabolites increased by 389% and 340%, 2 days after transport. Dembiec et al. (2004) reported that for inexperienced tigers, fecal corticosteroid levels peaked at 482% above baseline levels, 3 to 6 days after transport. Goymann et al. (1999) reported a fivefold increase in fecal cortisol occurred 3 days after translocating a male, spotted hyena. Morrow et al. (2002) reported an increase in fecal corticosteroids of cattle within 6 hr of transport and a return to baseline levels within 24 hr. This could reflect the different digestive physiology of ruminants compared to nonruminants. The time of the maximal concentration in this study might also have reflected additional stimulation due to the novelty of Chang s new social and physical environment when placed into the French zoo. In captive elephants, the use of a validated assay to determine fecal cortisol metabolites could help in the assessment of welfare by evaluating responses to new enclosure designs, enrichment practices, training techniques, introduction of new animals, and other management practices. The suitability of fecal cortisol metabolites as a welfare indicator was supported by behavioral observations. After relocation, stereotypic activity increased approximately 400%. Stereotypies are widely regarded as indicating that an animal is frustrated or experiencing some other negative affective state (Mason, 1991). Stereotypies in elephants have previously been noted to occur in conjunction with specific events such as being given food or water (Friend, 1999), being moved between different sections of the enclosure (Wilson et al., 2004), or even associated with changes in ambient temperature (Rees, 2004). The increases in Chang s stereotypical behavior appeared more long-term than these types of stereotypies and were possibly due to Chang s frustration at being unable to contact the females for the first few days. Alternatively, stereotypies can become emancipated from their original cause and might simply have appeared in Chang s repertoire as a consequence of previous experience. In addition, translocation altered Chang s sleeping behavior. Prior to relocation, the majority of his sleeping was either sternal or lateral; post-relocation, sleeping duration was substantially reduced and was always

11 RELOCATION OF AN ASIAN ELEPHANT 357 standing. The temporal proximity of increases in cortisol metabolites and stereotypies (Figures 1a, 1b, and 2) indicate that transport and introduction of a male Asian elephant into a novel herd was a stressful event. CONCLUSION These results showed that when a male Asian elephant was exposed to a putatively stressful experience (24 hr of transportation and relocation to a novel herd), there were physiological and behavioral changes indicative of this having a temporary negative impact on his welfare. The timing and intensity of the physiological responses, supported by the behavioral changes, indicated that fecal glucocorticoid measurement is an accurate and appropriate method to noninvasively and safely assess the welfare of Asian elephants. ACKNOWLEDGMENTS This study was jointly funded by the Department for Environment, Food and Rural Affairs; British and Irish Association of Zoos and Aquariums; International Fund for Animal Welfare; Royal Society for the Prevention of Cruelty to Animals; and Universities Federation for Animal Welfare. We gratefully thank the staff at Chester Zoo (United Kingdom); Zoo Le Pal in Dompierre-sur-Besbre (France) for their valued support and assistance; and Professor Rupert Palme from the Department of Natural Sciences at the University of Veterinary Medicine in Vienna, Austria, for the antibody and label for the 3-11oxo-CM EIA. We also thank an anonymous referee for helpful comments. REFERENCES Brown, J. L., Wemmer, C. M., & Lehnhardt, J. (1995). Urinary cortisol analysis for monitoring adrenal activity in elephants. Zoo Biology, 14, Dathe, H., H., Kuckelhorn, B., & Minnemann, D. (1992). Salivary cortisol assessment for stress detection in the Asian elephant (Elephas maximus): A pilot study. Zoo Biology, 11, Dembiec, D. P., Snider, R. J., & Zanella, A. J. (2004). The effects of transport stress on tiger physiology and behavior. Zoo Biology, 23, Farca, A. M., Cavana, P., Badino, P., Barbero, R., Odore, R., & Pollicino, R. (2006). Measurement of fecal corticoid metabolites in domestic dogs. Schweizer Archiv Fur Tierheilkunde, 148, Farca, A. M., Pollicino, P., Massobrio, M., Badio, P., & Cavana, P. (2002, September). Fecal cortisol metabolite measurement in stressed domestic cats. Proceedings of the 12th European Society for Veterinary Internal Medicine Congress, Munich, Germany.

12 358 LAWS ET AL. Friend, T. H. (1999). Behavior of picketed circus elephants. Applied Animal Behavior Science 62, Ganswindt, A., Palme, R., Heistermann, M., Borragan, S., & Hodges, J. K. (2003). Non-invasive assessment of adrenocortical function in the male African elephant (Loxodonta africana) and its relation to musth. General and Comparative Endocrinology, 134, Goymann, W., Möstl, E., Van t Hof, T., East, M. L., & Hofer, H. (1999). Noninvasive fecal monitoring of glucocorticoids in spotted hyenas (Crocuta crocuta). General and Comparative Endocrinology, 114, Heistermann, M., Palme, R., & Ganswindt, A. (2006). Comparison of different enzyme immunoassays for assessment of adrenocortical activity in primates based on fecal analysis. American Journal of Primatology, 68, Jurke, M. H., Czekala, N. M., Lindburg, D. G., & Millard, S. E. (1997). Faecal corticoid metabolic measurement in the Cheetah (Acinonys jubatus). Zoo Biology, 16, Lane, J. (2006). Can non-invasive glucocorticoid measures be used as reliable indicators of stress in animals? Animal Welfare, 15, Mason, G. J. (1991). Stereotypies: A critical review. Animal Behavior, 41, Mason, G. J., & Lathan, N. R. (2004). Can t stop, won t stop: Is stereotypy a reliable animal welfare indicator? Animal Welfare, 13, S57 S69. Millspaugh, J. J., Burke, T., Van Dyk, G., Slotow, R., Washburn, B. E., & Woods, R. J. (2007). Stress response of working African elephants to transportation and safari adventures. Journal of Wildlife Management, 71, Millspaugh, J. J., Washburn, B. E., Milanick, M. A., Beringer, J., Hansen, L. P., & Meyer, T. M. (2002). Non-invasive techniques for stress assessment in white-tailed deer. Wildlife Society Bulletin, 30, Morrow, C. J., Kolver, E. S., Verkerk, G. A., & Matthews, L. R. (2002). Fecal glucocorticoid metabolites as a measure of adrenal activity in dairy cattle. General and Comparative Endocrinology, 126, Möstl, E., Maggs, J. L., Schrötter, G., Besenfelder, U., & Palme, R. (2002). Measurement of cortisol metabolites in faeces of ruminants. Veterinary Research Communications, 26, Rees, P. A. (2004). Low environmental temperature causes an increase in stereotypic behavior in captive Asian elephants (Elephas maximus). Journal of Thermal Biology, 29, Schmid, J., Heistermann, M., Gansloßer, U., & Hodges, J. K. (2001). Introduction of foreign female Asian Elephants (Elephas maximus) into an existing group: behavioral reactions and changes in cortisol levels. Animal Welfare, 10, Stead, S. K., Meltzer, D. G. A., & Palme, R. (2000). The measurement of glucocorticoids concentrations in the serum and faeces of captive African elephants (Loxodonta africana) after ACTH stimulation. Journal of the South African Veterinary Association, 71, Wasser, S. K., Hunt, K. E., Brown, J. L., Cooper, K., Crockett, C. M., Bechert, U., et al. (2000). A generalized fecal glucocorticoid assay for use in a diverse array of nondomestic mammalian and avian species. General and Comparative Endocrinology, 120, Wilson, M. L, Bloomsmith, M. A., & Maple, T. L. (2004). Stereotypic swaying and serum cortisol concentrations in three captive African elephants (Loxodonta africana). Animal Welfare, 13,