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Applied Animal Behaviour Science 136 (2012) 57– 62
Contents lists available at SciVerse ScienceDirect
Applied Animal Behaviour Science
jou rna l h om epa ge: www.elsev ier .com/ locate /applan im
he effect of zoo visitors on the behaviour and faecal cortisol of theexican wolf (Canis lupus baileyi)
aría Pifarréa, Ricardo Valdezb, Carlos González-Rebelesa, Carlos Vázqueza,arta Romanob, Francisco Galindoa,∗
Facultad de Medicina Veterinaria y Zootecnia, Universidad Nacional Autónoma de México, Ciudad Universitaria, 04510 México D.F., MexicoDepartamento de Fisiología, Biofísica y Neurociencias, Centro de Investigación y de Estudios Avanzados, Apdo. Postal 14-740, 07360 México D.F., Mexico
r t i c l e i n f o
rticle history:ccepted 10 November 2011vailable online 1 December 2011
eywords:exican wolf
ehaviouraecal cortisolx situ conservation
a b s t r a c t
The effect of zoo visitors on the behaviour and physiological responses of the Mexicanwolf has not been documented but is worthy of investigation since it is a critically endan-gered native species and most of the population live in zoos. The effect of the number ofvisitors was assessed in 12 wolves (five males and seven females) in three zoos of cen-tral Mexico. Each wolf was observed for a total of 72 h on Saturdays, Sundays, Mondaysand Tuesdays (6 h/day) during a three-week period. Scan sampling was used to obtainindividual time budgets. A faecal sample was collected on the following morning of eachobservation day from 10 wolves to determine cortisol level (RIA). A repeated measuresanalysis of variance revealed that there was an effect of day on the proportion of timelying (P < 0.01), eating (P < 0.01) and in locomotion (P < 0.01). Wolves spent less time lyingon Saturdays and Sundays (0.2980 ± 0.012 and 0.3266 ± 0.012, respectively) than on Mon-days and Tuesdays (0.3821 ± 0.012 and 0.4075 ± 0.012, respectively), as well as less timeeating on weekend days (Saturdays 0.1214 ± 0.015, Sundays 0.0556 ± 0.015) than in theother 2 days (Mondays 0.816 ± 0.015 and Tuesdays 0.0915 ± 0.015). Mean faecal cortisolwas 203.7 ng/g of DM and ranged from 113.10 to 314.48 ng/g. There was an effect of dayon the faecal cortisol response (P < 0.01) with wolves having higher cortisol response on
Sundays (242.50 ng/g ± 8.48) compared to the other 3 days (Saturdays 192.71 ± 8.48, Mon-days 170.73 ng/g ± 7.80, Tuesdays 183.82 ng/g ± 8.48). This is the first study that measuresfaecal cortisol of Mexican wolves. These results indicate that the amount of visitors in zoosinfluence the behaviour and adrenal activity of these animals which could be undesirabletion ef
for ex situ conserva. Introduction
The Mexican wolf (Canis lupus baileyi) is the most dis-inctive of the five subspecies of North American wolves.ts natural distribution is the most southerly and it is
∗ Corresponding author at: Departamento de Etología y Fauna Silvestre,acultad de Medicina Veterinaria y Zootecnia, Universidad Nacionalutónoma de México, Ciudad Universitaria, Circuito Exterior, C.P. 04510oyoacán, México D.F., Mexico. Tel.: +52 55 5622 5941;ax: +52 55 5622 5859.
E-mail addresses: [email protected], [email protected]. Galindo).
168-1591/$ – see front matter © 2011 Elsevier B.V. All rights reserved.oi:10.1016/j.applanim.2011.11.015
forts of this endangered species.© 2011 Elsevier B.V. All rights reserved.
genetically different to the other four (García-Morenoet al., 1996; Parsons, 1996), but now considered extinctin the wild. Most remaining individuals live in zoos andare part of a Binational Conservation Programme betweenMexico and the US (Bernal and Packard, 1997; List, 2005).
Zoo enclosures are very different from a natural habitat(Seidensticker and Forthman, 1998) and can be stressfulenvironments, due to the lack of space and natural sub-strate, changes in the way food is obtained, and changes
in the social organisation of the species. This may hamperex situ conservation programmes because chronic adversestimuli can have negative behavioural and physiologicaleffects (Wingfield and Ramenofsky, 1999). For example,
58 M. Pifarré et al. / Applied Animal Behav
Table 1Number of wolves, males (M) and females (F), housed in the three zoos.
Zoo/exhibit Number of wolves Sex Age (Y)
M F
1 4 2 2 6–1–9–42 4 1 3 1–1–1–1
2 2 1 1 5–63 2 1 1 7–8chronic stress can inhibit the release of sexual steroids,interfering with reproduction (Creel, 2001; Dobson andSmith, 2000; Knol, 1991; Pottinger, 1999). Chronic stressimplies poor welfare (Broom, 1997) and some reproduc-tive problems in the Mexican wolf seem to have beenattributed to this condition by the zoos species survivalplan.
Although several studies on genetics, behaviour, andreproductive physiology have been carried out with theMexican wolf in captivity as part of the Binational Recov-ery Plan none have considered the effect of the public onbehaviour and adrenal activity. It has been hypothesizedthat the public could have an effect that ranges from stress-ful to enriching or have no effect at all (Chamove et al.,1988; Glatston et al., 1984; Hosey, 2000; Kuhar, 2008;Wells, 2005; Wormell et al., 1996). In carnivores, the onlyavailable studies are with felids and suggest no influenceof the public on their behaviour (O’Donovan et al., 1993). Itis important to mention that these studies which examinethe effect of the public on an animal’s welfare only mea-sure behavioural changes as a welfare indicator and notphysiological responses such as cortisol release.
Previous studies with domestic and non-domesticspecies proved that faecal cortisol is a useful non-invasiveindicator of animal welfare (Graham and Brown, 1996;Cavigelli, 1999; Monfort et al., 1998; Palme et al., 2000;Schatz and Palme, 2001). The aim of this study was toassess if the public presence in three zoos in Mexico hasan influence on the welfare of captive Mexican wolves bymeasuring behaviour and faecal cortisol concentrations.This information is useful to make recommendations toimprove the rescue strategy of this species on the vergeof extinction.
2. Methods
2.1. Subjects and housing
This study was carried out at three zoos: (1) AfricamSafari Park in Puebla, Mexico, (2) Chapultepec and (3) SanJuan de Aragon Zoo, both in Mexico City. Wolves are partof the Binational Conservation Programme Mexico – US,so zookeepers are specially trained and management issimilar across the zoos. Wolves are housed in four differ-ent exhibits and ground surface was 680, 776, 604, and790 m2, respectively (Table 1). All enclosures had natural
substrate to allow for digging and enough surface area forresting. Natural vegetation and shade during light hours inat least half of the enclosure was also provided. In all zoosfacilities were cleaned at 9:00 h and at 17:00 h. Wolvesiour Science 136 (2012) 57– 62
were fed 1.3 kg of chicken and beef combined. All wolveswere born in captivity and lived for more than 1 year intheir respective enclosures where observations were con-ducted.
2.2. Behavioural measurements
An ethogram was adapted from Scott and Fuller (1965),Servín (1991), and Gandelman (1992) using 40 h of adlibitum sampling. Scan sampling (Martin and Bateson,1993) was carried out to collect information on individualbehaviour. Seven observers sitting on the roofs of the nightenclosures observed the wolves simultaneously. Observerswere first trained in the use of the ethogram and samplingmethods. After this training period a 30 h pilot test wascarried out during a 2-week period. An inter-observer reli-ability correlation test with data from that pilot study wascarried out (rs = 0.95, P < 0.05) before behavioural data wereobtained. During the 3 weeks of the study each wolf wasobserved for 4 days per week (Saturday, Sunday, Mondayand Tuesday) for 6 h per day from 10:00 to 12:00, 12:30 to14:30 and 15:00 to 17:00 h. A total of 72 h of observationwere collected per wolf.
On each observation day scan sampling was conductedevery 5 min to calculate individual time budgets. Thebehaviours recorded were: (1) lying – the subject assumeda recumbent posture with eyes opened or closed in anylocation of the enclosure; (2) standing – the subject sup-ported itself on all four legs without movement exceptfor eating or drinking; (3) eating – when the subject wasingesting food, either standing or lying down; (4) walking– when the subject was in locomotion at a slow gait; and(5) running – when the subject was in locomotion at a fastgait. The proportion of time in locomotion was consideredas the sum of walking + running.
For each wolf the time spent performing individualbehaviours was expressed as a proportion of time calcu-lated as follows: number of observations of a behaviourdivided by total number of scan samplings. The term timebudget was defined as the summed proportions (total100%) of these behaviours.
The three zoos were open to the public from 9:00 h to17:00 h and the distance from the visitor corridor to thelimits of the enclosures was in all cases between 6 and 8 m.Scan sampling was used every 5 min to record the numberof people that stood by the wolves exhibits during obser-vation sessions. The mean proportion of people in front ofthe exhibit was calculated for each day and related withthe total number of visitors recorded by the turnstiles ateach zoo entrance on the days of observation. A correlationof R = 0.90 (P < 0.05) was seen between these two and forfurther analysis the former was used.
The average number of visitors in zoo 1 on Saturdays,Sundays, Mondays, and Tuesdays was 2998.33, 3244.33,354.67 and 550.00, respectively. In zoo 2 the average num-ber of visitors was of 26567.00, 35450.83, 221.60 and
11009.83, respectively. For zoo 3 the average number wasof 21447.25, 36910.75, 283.00 and 6939.33, respectively.Both zoos 2 and 3 have a restricted number of visitors onMondays.
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M. Pifarré et al. / Applied Anima
.3. Faecal cortisol measurements
Prior to the start of the trial a test was carried outo compare faecal cortisol levels before and after a han-ling routine that involved physical restraint of the wolves.hree adult animals were chosen, one 6-year old male andwo females, 4 and 9 years old, respectively. These threeolves were moved to an enclosure for 3 days providedith a holding area for physical restraint. Faecal samplesere collected at 11:00 h 1 and 3 days after being moved
o that enclosure. Blood samples were obtained duringhysical restraint as part of an annual routine for clinicalxamination on day 2 after being moved to the enclosure.lood samples were obtained 5–7 min after restraint at2:00 h from the cephalic vein using a 20-gauge needlend EDTA tubes. Samples were immediately centrifuged at000 rpm and the plasma aliquots were frozen at −20 ◦C. Toetermine circulating levels of cortisol a commercial 125
-Radioimmunoassay (RIA) Kit (cort. CT2, CisBio Interna-ional, France) was used. Intra and inter-assay coefficientariation were 6.06% and 12.9% respectively. The secondaecal sample contained a significantly greater concentra-ion of cortisol than the first (t = −4.41, d.f. = 2, P < 0.05,able 2). This difference can be explained as a result of theolves’ physical restraint and clinical examination, vali-ating the method for faecal cortisol determination for theurpose of this study.
Faecal samples were collected from 10 of the observedolves. A total of 120 samples were collected, 12 per wolf
zoo 1 = 4, zoo 2 = 4, zoo 3 = 2), while the facilities wereleaned. On the day prior to collection the wolves wereiven natural colouring in their food for individual iden-ification except for the four wolves in zoo 2 where it wasot possible to separate them while eating. As these wolvesefecated always in specific areas of the enclosure, it wasossible to collect samples individually although the indi-idual wolf to which it belonged was unknown. The otherwo wolves in that zoo were not sampled, as it was logisti-ally impossible to collect their faeces. All wolves observedn this study defecated once daily so the measurements
ade represent the total amount of circulating cortisolxcreted in the interval between defecations (24 h). Fae-al cortisol determinations could therefore be related tohe behaviours recorded on the previous day of the sampleollection (Sands and Creel, 2004; Schatz and Palme, 2001).
The samples were labelled and frozen at −20 ◦C untilnalysis. Dry matter (DM) was determined by drying sam-les in the oven for 4 h at 65 ◦C. A total of 0.18–0.2 g of thery sample was used for cortisol extraction with ethanolt 90% as reported by Brown et al. (1994). To obtain theecovery range of this process a known amount of cor-isol (23,000 cpm) was added to six samples, recovering8.9 ± 7.1% (N = 6). The samples were re-suspended withiluted buffer RIA and ethanol 2:1 and were kept at −4 ◦Cntil processed by radioimmunoassay (RIA).
The RIA was done following the method described byarrott et al. (1989). Rabbit anticortisol (3/CMO Chemicon)
as used as an antibody. Tritiated hydrocortisone was useds a marker [1,2,6,7-3H(N)] 70–100 Ci/mmol (1 mCi mLf ethanol of NEN Life Science Products, Inc., Frederick,O, U.S.A.). The standard curve had seven points: 200,
iour Science 136 (2012) 57– 62 59
100, 50, 25, 12.5, 6.25 and 3.125 pg/mL of hydrocortisone.Inter-assay coefficient of variation was of 12.20 ± 1.38%(N = 9) while intra-assay coefficient of variation was of5.96 ± 0.86% (N = 9) (Bedolla et al., 1984).
2.4. Data analysis
All data were analysed using Statistical Analysis Soft-ware SAS® 9.0. A paired sample t test was used tocompare faecal samples before and after physical restraintas part of the assay validation. These samples werenot used for subsequent analysis. Data of the observedwolves was transformed to comply with the assumptionsof normality and independence. Cortisol was trans-formed as
√(cortisol + 1) and the proportion of time lying,
standing, eating, and locomotion was transformed asARCSINE
√(proportion). A repeated measures model was
used represented by:
Yijkl = � + Zi + Aj(i) + ı(ij) + Dk + ZDik + ε(ijk)l
where Yijkl is the transformed dependant variable, corti-sol, lying, standing, eating, and locomotion; � is the overallmean; Zi the effect of zoo; Aj(i) is the ith animal nested inthe jth zoo, ı(ij) restriction error due to the process of ran-domization of wolves in each zoo; Dk, the effect of the day(Saturday, Sunday, Monday, Tuesday), ZDik is the interac-tion between zoo and day; ε(ijk)l is the random error NID(0, �2).
3. Results
The repeated measures model showed that there was aneffect of day on the proportion of time lying (F = 5.64, d.f. = 3,P < 0.01; Tables 3 and 4), eating (F = 2.92, d.f. = 3, P < 0.01;Tables 3 and 4) and locomotion (F = 16.98, d.f. = 3, P < 0.01;Tables 3 and 4). The effects of the different sources of varia-tion are shown in Table 3. The coefficient of determination(R2) for each variable shows that the model explains a highproportion of variation as those values were: 0.86, 0.67,0.7, and 0.9 for lying, standing, eating, and locomotion. Theleast square means values for those behaviours are shownin Table 4. There was also an effect of zoo on the pro-portion of time lying (F = 5.15, d.f. = 2, P < 0.05) and eating(F = 4.61, d.f. = 2, P < 0.05). Wolves in zoo 3 spent less timelying (0.2576 ± 0.013) than in zoos 1 and 2 (0.4012 ± 0.009and 0.4018 ± 0.007, respectively) and also less time eating(0.0380 ± 0.017) than in zoos 1 and 2 (0.11.05 ± 0.012 and0.1141 ± 0.010, respectively).
Faecal cortisol levels ranged from 113.10 to 314.48 ng/gof DM with an average value of 203.7 ng/g. The analysis alsorevealed an effect of day on faecal cortisol values (F = 16.15,d.f. = 3, P < 0.01; Tables 3 and 4) as the mean faecal corti-sol values were higher on Sundays compared to the other
three days. The effects of the different sources of variationare shown in Table 3. The coefficient of determination (R2)for cortisol was 0.9, the least square mean values for thisvariable are shown in Table 4.
60 M. Pifarré et al. / Applied Animal Behaviour Science 136 (2012) 57– 62
Table 2Blood and faecal cortisol concentrations in three wolves sampled after physical restraint.
Animal First faecal sample (ng/g) Blood sample (nmol/L) Second faecal sample (ng/g)
1 76.59a Physical restraint 213.6 131.86b
2 91.13a 233.5 129.65b
3 93.16a 251.9 177.69b
Different letters between faecal samples represent statistical differences (t = −4.41, d.f. = 2, P < 0.05).
Table 3The effect of zoo, animal within zoo, day, and interaction between zoo and day on faecal cortisol levels and the proportion of time lying, standing, eating,and in locomotion of Mexican wolves in three zoos.
Source of variation d.f. Cortisol d.f. Lying Standing Eating Locomotion
Zoo 2 11.11 (0.07) 2 999.25 (0.032) 163.97 (0.10) 61.89 (0.04) 269.2 (0.16)Animal (zoo) 1 3.15 1 194.00 56.99 13.40 120.46Days 3 11.15 (0.001) 3 180.77 (0.004) 19.70 (0.39) 24.22 (0.05) 96.63 (0.001)Zoo × days 6 0.74 (0.417) 6 132.53 (0.004) 17.88 (0.48) 33.39 (0.005) 110.95 (0.001)Error 29 0.69 27 32.00 19.17 8.27 5.69
The first and second columns of degrees of freedom (d.f.) correspond to cortisol and the behaviour variables respectively. P values are presented inparenthesis.
Table 4Least square means for faecal cortisol levels (ng/g) and proportion of time lying, standing, eating, and in locomotion of Mexican wolves in three zoos.
Day Cortisol Lying Standing Eating Locomotion
Saturday 192.71 ± 8.48a 0.2980 ± 0.012a 0.0320 ± 0.009 0.1214 ± 0.015a 0.0982 ± 0.010aSunday 242.50 ± 8.48b 0.3266 ± 0.012ab 0.0531 ± 0.009 0.0556 ± 0.015ab 0.1209 ± 0.010aMonday 170.73 ± 7.8a 0.3821 ± 0.012b 0.0478 ± 0.009 0.0816 ± 0.015b 0.1568 ± 0.010bTuesday 183.82 ± 8.48a 0.4075 ± 0.012b 0.0499 ± 0.009 0.0915 ± 0.015b 0.1843 ± 0.010b
Different letters in each column are statistically significant.
4. Discussion
It has been hypothesized that the effect of the public inzoos could induce stress (Chamove et al., 1988; Fa, 1992;Glatston et al., 1984; Lambeth et al., 1997; Maki et al., 1987;Mitchell et al., 1991; Wells, 2005; Wormell et al., 1996).Conversely, it can have no effect at all on the welfare ofcaptive animals (Hosey, 2000; O’Donovan et al., 1993). Thisis the first such study on the Mexican wolf and is relevantespecially because this species is critically endangered andthe majority of the population live in zoos.
Results show that when wolves were exposed to ahigher number of visitors on Sundays there were higherlevels of faecal cortisol and changes in the proportionof time lying, eating and in locomotion. The relation-ships between time budgets and adrenal activity cannotbe conclusive, as the wolves were not observed during thewhole day. To know more about the relationships betweenindividual time budgets and faecal cortisol values it is sug-gested that further studies be conducted on behaviour thatreflect 24-h cycles. Also, in future studies the use of spacewould be an important aspect to consider as Kuhar (2008)found that gorillas exposed to large crowds were scoredas not visible more often. This could explain why wolvesin zoo 3 spent also more time lying, possibly as a result ofhow shade and vegetation were distributed in the enclo-sure. This could be a tool for the animal to have some type
of control over its environment and should be consideredwhen designing zoo enclosures.As an assessment of animal welfare should ideally inte-grate more than one indicator it was decided to measure
faecal cortisol concentrations, a non-invasive indicatorof the adrenal activity. These measurements representthe total amount of circulating cortisol excreted in theinterval between defecations. In this study, this intervalcorresponded to 24 h meaning that the cortisol measuredrepresented adrenal activity levels from the day prior tothe faeces collection (Brousset et al., 2005). Faecal cortisolhas been reported in the grey wolf of North America (Canislupus lupus) under free living conditions and the valuesfound ranged from 872 to 1468 ng/g DM (Creel et al., 2002;Sands and Creel, 2004). The range differences betweenstudies can be explained by different factors. Firstly, the factthat the Mexican wolf is a genetically different subspeciesfrom that of the grey wolf (García-Moreno et al., 1996)may influence the amount of cortisol released (Schatz andPalme, 2001) and secondly, the diet composition and tran-sit through the digestive tract, which can influence faecalconcentrations (von der Ohe and Servheen, 2002), weredifferent as previous studies were carried out with wildwolves while this study used captive wolves fed a con-trolled diet.
Faecal cortisol values were higher on Sundays. It isknown that cortisol is released when the organism needsmore energy to surpass environmental challenges of anyorigin (Nelson, 2000). The fact that Sunday’s cortisol val-ues increased, as well as the time spent lying, perhaps as aconsequence of attempting to be less visible to the public
(Kuhar, 2008), suggests that large crowds of visitors couldbe having an effect on the welfare of the wolves. Theseresults are in accordance with a study carried out withclouded leopards (Neofelis nebulosa) where faecal cortisol
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ncreased in animals exposed to the public in comparison tother animals not exposed to visitors (Wielobnowski et al.,002).
It is important to mention that most of the studieshat have attempted to measure the effect of visitors onhe welfare of captive animals in zoos have not relatedehaviour with physiological responses, particularly withortisol values. This is relevant as in some cases there istill controversy on the ‘visitors effect’ on behaviour (seeoss et al., 2007; Wells, 2005, 2007) and, hence, it is rec-mmended to consider other welfare indicators apart fromehaviour in order to better understand how animals copeith these stimuli.
Captive breeding has been an important tool forndangered species recovery plans since emergence andevelopment of conservation biology as a discipline.owever, limitations are being recognized and generat-
ng growing concern among conservationists (e.g., costs,eproductive success, feasibility for reintroduction, dis-ase transmission, genetic and behaviour modification)Entwistle and Dunstone, 2000; Jimenez, 1996; Snydert al., 1996). Balmford (2000) prompts for a critical reviewf this approach focused primarily on large charismaticammalian species. The Mexican Wolf is one of several
riority species recently considered for Mexico’s Nationaltrategy for the Conservation of Wildlife (INE, 2000;EMARNAP, 2000). For this reason the results presentedere are relevant to provide further data to review andetter direct this type of conservation approaches.
. Conclusions
This is the first study that measures faecal cortisol levelsf Mexican wolves. Days with high attendance of zoo vis-tors had an effect on the behaviour and faecal cortisol ofhese animals. Wolves spent less time lying and eating andad higher faecal cortisol levels in days of high attendancehan in days with a lower attendance. These results indi-ate that the condition of captive Mexican wolves could bendesirable for ex situ conservation efforts of this endan-ered species.
cknowledgements
We would like to thank the General Direction of Zoosf Mexico City as well as the Africam Safari Zoo for theirupport and collaboration. A special acknowledgement isade to the DGAPA of the National Autonomous University
f Mexico (UNAM) for the support provided to Dr. Franciscoalindo while finishing this paper.
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