ORIGINAL PAPER

G.H. Engelhard Æ S.M.J.M. Brasseur Æ A.J. HallH.R. Burton Æ P.J.H. Reijnders

Adrenocortical responsiveness in southern elephant seal mothersand pups during lactation and the effect of scientific handling

Accepted: 29 January 2002 / Published online: 11 April 2002� Springer-Verlag 2002

Abstract We examined the cortisol responses to chemi-cal and physical restraint stress in southern elephant sealMirounga leonina females and their pups at three stagesduring lactation. In anaesthetised females the serumcortisol levels changed moderately during the 45-minsampling period following restraint, with average peaksat 23 min after anaesthetic administration. Overall, cor-tisol was relatively low 2 days postpartum and increasedthroughout lactation. In physically restrained pups se-rum cortisol increased rapidly after capture; the responsewas milder at age 2 days than at 11 days and 21 days.Levels were higher in female pups than in males. In orderto test whether cortisol levels and/or responses becamechronically (i.e. days to weeks) altered due to restraint,we compared the cortisol response at a late stage oflactation between three groups of mother-pup pairspreviously given different levels of chemical (mothers) orphysical (pups) restraint stress: control (not handled

previously), moderate treatment (previously handledtwice), and high treatment (previously handled 3–4times). Pups of the three treatment groups showed sim-ilar adrenocortical responses suggesting no chronic effectof repeated physical restraint, despite the clear acute ef-fects. Mothers of the control and moderate treatmentgroups showed similar cortisol responses; however,mothers of the high treatment group showed significantlyattenuated responses. This indicated that elephant sealstolerated moderate degrees of handling disturbance;however, repeated (3–4) chemical immobilisations inlactating females may reduce their adrenocorticalresponsiveness for a period of days or weeks.

Keywords Cortisol Æ Hypothalamic-pituitary-adrenalaxis Æ Anaesthesia Æ Lactation Æ Mirounga leonina

Abbreviation HPA hypothalamic-pituitary-adrenal

Introduction

In pinniped research a certain degree of animal handlingcan in many cases not be avoided. For the collection ofphysiological data, or for the attachment of telemetrydevices needed for studies on diving behaviour, eitherphysical restraint by means of nets or chemical restraintby means of anaesthetics is usually required (reviewed byGales 1989; Lynch et al. 1999). It is important to un-derstand the stressful effects of research handling activ-ities, both direct or acute (during or immediately afterhandling) and indirect or chronic (days to months afterthe event). It will then be easier to assess to what extentthe base-line population values being studied (e.g. bloodparameters, behaviour) may be biased through impact ofstress. Moreover, the success of management and con-servation activities could be improved if any negativefitness consequences due to anthropogenic stress wereminimised (Hofer and East 1998). Such knowledge willbe particularly valuable in species with declining orvulnerable populations (Reijnders et al. 1993).

J Comp Physiol B (2002) 172: 315–328DOI 10.1007/s00360-002-0257-0

Communicated by G. Heldmaier

G.H. Engelhard (&)Centre for Ecological and Evolutionary Studies,Zoological Laboratory, Groningen University,P.O. Box 14, 9750 AA Haren, The NetherlandsE-mail: Georg.Engelhard@imr.noTel.: +47-55-238643Fax: +47-55-238687

G.H. Engelhard Æ S.M.J.M. Brasseur Æ P.J.H. ReijndersAlterra Institute, Marine and Coastal Zone Research,P.O. Box 167, 1790 AD Den Burg, The Netherlands

A.J. HallSea Mammal Research Unit,Gatty Marine Laboratory,University of St Andrews,Fife KY16 8LB Scotland, UK

H.R. BurtonAustralian Antarctic Division, Channel Highway,Kingston 7050, Tasmania, Australia

Present address: G.H. EngelhardInstitute of Marine Research,P.O. Box 1870 Nordnes,5817 Bergen, Norway

The southern elephant seal Mirounga leonina, a toppredator in the Southern Ocean ecosystem, is among themost extensively studied pinnipeds (e.g., Le Boeuf andLaws 1994). Despite this, it remains poorly understoodwhy populations of the species in the southern Indian andPacific Oceans have been declining over the past decadesor still are in decline (Hindell and Burton 1987; Guinetet al. 1999; McMahon et al. 1999; Pistorius et al. 1999),while South Atlantic populations have been stable (Laws1994). Declines appear to be primarily due to unfavour-able food conditions in the pelagic foraging ranges of theseals, but other factors that regulate populations mayalso be involved (Hindell 1991; Hindell et al. 1994).Southern elephant seals breed and moult on (sub)Ant-arctic islands. During the reproductive season (Septem-ber–November), females aggregate on beaches and formharems that are competed for by males (Carrick et al.1962). Females give birth to a pup some days after arrival,and nurse it intensively for approximately 24 days(McMahon et al. 1997) while not feeding themselves.Mothers then return to sea, weaning the pup abruptlywhen it becomes independent. Lactation is a period ofmaximum energetic demand for female mammals; hence,it might be a time when animals are more vulnerable tohuman disturbance. In particular, reproductive success islikely to be more directly affected during this period.Until recently, only a single survey studying disturbancein elephant seals had been carried out, where no effect wasfound based on population census data (Wilkinson andBester 1988). Since that study southern elephant sealshave continued to be the subject of considerable scientificinvestigation. This often includes physical or chemicalrestraint of the animals. We consider it important toexamine, at an individual level, how handling activitiesmay affect the physiology of these animals.

Endocrinology is a possible way to study levels ofstress in wild animals, and in many cases the responseof the hypothalamic-pituitary-adrenal (HPA) axis is areliable indicator of a stressed state (Sapolsky 1990;Wingfield et al. 1997; von Holst 1998). Stressful eventsare followed by increased levels of circulating gluco-corticoids, such as cortisol in pinnipeds (Liggins et al.1979). Elevated glucocorticoid levels cause manychanges in the body such as increased heart rate, bloodpressure and peripheral blood supply, analgesia, sup-pression of digestion, and mobilisation of glucose,nutrients and oxygen (Hadley 1996). Normal short-termadrenocortical responses usually are highly adaptive inunpredictable circumstances. However, prolonged highglucocorticoid levels can trigger harmful effects includ-ing immune suppression and neural degeneration(Axelrod and Reisine 1984). Single stressful events, ifsevere, may in some cases have long-lasting effects onphysiology and behaviour (Meerlo et al. 1996; Koolhaaset al. 1997). Prolonged stress responses are often mal-adaptive, ultimately resulting in a decrease in fitness.

In a field experiment, we studied the cortisol stressresponse to typical research handling activities insouthern elephant seals during the reproductive season.

The animals were distributed over two geographicallysimilar, almost adjacent study areas that were differentin the general levels of human presence (Engelhard et al.2001). The research handling activities consisted ofchemical immobilisation of adult lactating females andphysical restraint of pups. Such handling procedures arealso representative of studies on several other pinnipedspecies.

First, we investigated the acute effects of restraint onthe levels of circulating cortisol during the 45-min timespans following the start of handling. Second, weexamined ‘natural’ factors that may affect levels ofcortisol, including the stage of lactation, the mass of theanimals, and the sex of the pup; we also studied whetherthere was evidence of circadian and/or seasonal varia-tion in glucocorticoids, which is known to exist in arange of species including the harbour seal Phoca vitu-lina (Gardiner and Hall 1997). Third, we studiedwhether different levels of restraint stress may lead tochronically altered cortisol levels or responses (i.e. over aperiod lasting days to weeks): to test this we compared,at a final stage of the lactation period, the stress responsebetween mother-pup pairs that at earlier stages of lac-tation had either experienced relatively high, moderate,or no handling. Fourth, we compared cortisol levels andresponses between the seals located in the areas of lowand high human presence. Based on results in the fore-going investigations, we assessed whether there is reasonto assume an impact of stress on blood parametersobtained in earlier seal studies.

Materials and methods

The study was carried out on sub-Antarctic Macquarie Island(54�300S, 158�570E), which has the third largest breeding popula-tion of southern elephant seals in the world (Laws 1994). Since1948, the Australian Antarctic Division has run a permanentresearch station on the island situated on the Isthmus. For most ofthis time the elephant seal population has been monitored, and wasfound to be in long-term decline (Hindell and Burton 1987;McMahon et al. 1999).

Areas of high and low human presence

During September–November 1996 we monitored eight elephantseal harems, which at the peak of the breeding season (around16 October) on average numbered 154 females (range 28–360females). Four harems were on the east coast of the Isthmus(Isthmus East) adjacent to the research station; and four were onMiddle Beach, an east coast site 2 km south of the station. The twoareas were similar in natural features such as beach topography,wind and surf exposure, and characteristics of elephant sealharems, but mainly different in the levels of human presence(Engelhard et al. 2001). Most human activities on MacquarieIsland (scientific, maintenance, sightseeing, and other) have been ator near the research station on the Isthmus (area of high humanpresence); there is far less frequent human visitation at any othersite of the island, including Middle Beach (area of low humanpresence). Moreover, during the study season, elephant seal haremson the Isthmus were visited daily by a second, five-person teamcarrying out long-term population monitoring studies. Teammembers weighed and tagged pups within a day of birth, and laterre-weighed these at weaning (methods as in McMahon et al. 1997);

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this sample included approximately 500 pups on Isthmus East.These activities were not carried out on Middle Beach. The amountof disturbance in harems caused by our own, separate team(comprising four persons) was about equal for all eight studyharems.

Levels of experimental disturbance: treatment groups

Twenty-six mother-pup pairs distributed over these harems weremarked with a paint dot on one of the first days of lactation toallow individual recognition. Of these, 10 pairs were early breeders(pup born 17–27 September) and 16 pairs were late breeders (pupborn 9–23 October).

Within each harem, some study animals were given low andothers high levels of experimental disturbance. As a control group,13 pairs were captured once around 21 days postpartum, i.e.,towards the end of the lactation period. As an experimental group,13 pairs were captured 3–5 times at early, middle, and late stages oflactation, i.e. around 2 days, 11 days, (14 days, 18 days), and21 days postpartum. Within the experimental group, five pairscaptured three times during lactation are here considered a mod-erate treatment group; and eight pairs captured four or five timesthroughout lactation are considered a high treatment group. Thevariability of the number of captures within the experimental groupwas due to practical circumstances resulting in not all plannedcaptures being carried out. In several cases, the study animal wassituated in the centre of a large harem for a number of consecutivedays; this made it impossible to approach the individual withoutcausing unacceptable levels of disturbance in the harem. All han-dling procedures were in the light phase of the day between8.00 a.m. and 3.00 p.m.

Handling procedure of adult females

Adult females were immobilised with a mixture of tiletamine andzolazepam in a 1:1 ratio (Zoletil 100, Virbac). At present this is oneof the more commonly used anaesthetic combinations in wildpinnipeds (Gales 1989; Baker et al. 1990; Lynch et al. 1999;McMahon et al. 2000b); it provides a safe, predictable and reliablemeans of immobilising elephant seals (McMahon et al. 2000b). Theapproximate combined dose was 1 mg Zoletil (dry mass) per kgseal body mass; this was based on visual estimation of the mass of astudy animal. As a result, the average dosage was 386±79 mg(mean±SD, range 250–550 mg, n=52); based on body massmeasured afterwards, this was equivalent to 0.97±0.13 mg kg–1 ofseal body mass (range 0.69–1.29 mg kg–1).

Preparation of anaesthetics was at a site distant from the harem;the animal was then quickly approached and within 1 min the drugwas administered intramuscularly into the pelvic region by means ofan anaesthetic dart delivered by blowpipe (Fedak et al. 1996). Depthof anaesthesia was recorded on a scale of 1–5 (light sedation tosurgical anaesthesia; Baker et al. 1990). Once the degree of sedationwas an estimated 3–5, a timed series of four blood samples (1–5 mleach) was collected from the extradural vein by means of a 3.50018GYale spinal needle in 6 ml untreated Plain Plus Plastic vacutainers(Beckton Dickinson UK). The timing of the first blood sample inrelation to the start of experimental disturbance (i.e. anaestheticadministration) ranged from 6 min to 59 min (median 18 min); thethree consecutive samples were taken 7, 14 and 21 min after theinitial sample. Simultaneously, additional blood samples were col-lected for further studies on blood parameters. Once blood samplingwas complete, nose-to-tail length wasmeasured with a tapemeasure.The female was weighed in a net attached to a load cell (Salter DC2,West Bromwich, UK), suspended under a tripod. Finally, the sealwas released and monitored until fully recovered from anaesthesia.

Handling procedure of pups

Pups were immobilised by physical restraint in either a pup bag or,if heavier than approximately 75 kg, in a pole net (Laws 1953).

Blood samples were collected as in adult females, however,restricted to a series of three samples and by means of 1.50019Gneedles. The interval between the start of experimental disturbance(i.e. pup capture) and the first blood sample was in the range1–44 min (median 7 min); the two consecutive samples were taken10 min and 20 min after the initial sample. Next, nose-to-tail lengthand mass were measured. The pup was released near its mother andmonitored until re-establishment of mother-pup contact.

Laboratory analysis

Blood samples were allowed to clot, and the serum was thenremoved and stored in 2 ml cryotubes at –20 �C. Samples werelater analysed in duplicate for cortisol concentrations using a radioImmunoassay kit (Coat-A-Count Cortisol Kit, Diagnostic Prod-ucts Corporation), at an incubation temperature of 37 �C. Theinter- and intra-assay coefficients of variation were 5% and6%, respectively. Laboratory analysis was carried out by BCOAnalytical Services, Breda, The Netherlands.

Statistics

Our dataset on cortisol measurements was characterised by ahierarchically nested or clustered structure. First, during eachhandling event repeated blood samples were collected from oneanimal, so that such serial data points were not fully independent ofeach other. Second, at three stages of lactation series of bloodsamples were taken from animals of the experimental group, so thatin addition, different sets of samples collected from one individualwere not entirely independent of each other. Third, individual studyanimals could be situated, and hence clustered, within the samebreeding harem. The structure of the data was further complicatedsince it was unbalanced due to unequal sample sizes per handlingevent, per individual or per harem; therefore conventional repeatedmeasures analysis could not be applied. Because of these compli-cations, we used hierarchical linear modelling (Goldstein et al.1998; Loonen et al. 1999) for analysing factors affecting cortisol,while taking into account the dependency or clustering among themeasurements. A hierarchical linear model is a special type ofregression model designed for data with a hierarchical structure. Byincorporating a random error term for each level of clustering, itallows analysis of variances and covariances of factors acting atdifferent hierarchical levels. We applied the MLwiN 1.10 software(Multilevel Models Project, Institute of Education: Goldstein et al.1998) based on iterative generalised least-squares regression anal-ysis. The models used here included three or four hierarchicallevels:

1. Restraint time level; single blood samples collected during45 min restraint or one handling event (mothers n=155, pupsn=139 samples).

2. Lactation stage level; sets of blood samples collected either atearly, middle, or late stages of the lactation period (2 days,11 days, 21 days postpartum; mothers n=50, pups n=52 sets ofsamples).

3. Individual level; individual mothers (n=26) and pups (n=26).4. Harem level; harems (n=8) over which individuals were dis-

tributed.

Where comparisons between the different treatment groupswere made, only data for the late stage of lactation were included(no data on earlier stages being available for the control group);accordingly, the respective models did not contain the hierarchicallevel of lactation stage.

The dependent variable was cortisol (nmol l–1). In model-building, a forward stepwise selection procedure was used (seeappendix in Loonen et al. 1999). We first tested for a significantlinear change in cortisol due to the direct stress response, byincorporating the covariate restraint time (min) since initiation oftreatment. Next, we tested for a quadratic change in cortisol levels(such as an increase followed by a decrease), by including the

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covariate squared restraint time (min2). We then examined theeffects of other factors and covariates on cortisol levels. Categoricalfactors included the stage of lactation (early, middle, or late),treatment group (control, moderate or high treatment), pup sex,time of day, season (pup born either in September or October), andarea (Middle Beach or Isthmus East). Covariates included bodymass (kg), nose-to-tail length (m), body condition index (mass:length ratio, kg m–1), mass loss in mothers (kg), and mass gain inpups (kg).

Results

Mothers of experimental group at three stagesof lactation

For the 13 adult female elephant seals comprising theexperimental group, changes in serum cortisol duringimmobilisation with Zoletil at three stages of lactationare shown in Fig. 1.

Direct response to immobilisation stress

Due to the interval between anaesthetic administrationand the first possible collection of a blood sample, basallevels of cortisol were not directly measured. Over thesampled time-spans (in most cases, 10–40 min followinginitial disturbance) there were moderate, but significant,changes in the levels of serum cortisol. Hierarchicallinear model analysis (Table 1) indicated a quadraticeffect (P=0.004) of immobilisation time (min) on thelevels of serum cortisol (nmol l–1), following the rela-tionship (±SE): cortisol=initial value+8.16

(±3.05)·restraint time–0.174 (±0.059)·restraint time2.Cortisol levels peaked on average 23 min after anaes-thetic administration (see regression lines in Fig. 1).

Effect of the stage of lactation

There was, regardless of the direct effect of chemicalrestraint, a strong increase in the levels of serum cortisolthroughout the lactation period. Around 2 days,11 days and 21 days, postpartum cortisol values (±SD)averaged 126±64 nmol l–1 (n=35), 280±99 nmol l–1

(n=35), and 358±100 nmol l–1 (n=41), respectively.Both the difference in cortisol between early and middlelactation (P=0.0005) and that between middle and latelactation (P=0.009) were significant (Table 1). Whileoverall levels of cortisol increased throughout lactation,the shape of the quadratic response-curve was not sig-nificantly different between the three stages of lactation(Table 1, see rejected interactions of restraint time andsquared restraint time with early and late lactationstage).

Diurnal and seasonal effects

The longitudinal data set for the experimental groupshowed no evidence for an effect of time of day(P=0.669) or season (P=0.783) on the levels of serumcortisol (Table 1).

Mass change

Around 2 days postpartum, the 13 experimental femalesweighed 498±79 kg (mean±SD). Body mass averaged433±64 kg around 11 days postpartum and 346±53 kgaround 21 days postpartum. Throughout lactation,females lost, on average, 190±30 kg (37.0±2.2% ofinitial body mass). There was no association between thetotal loss of mass during lactation and the levels ofserum cortisol (P=0.849; Table 1).

Fig. 1. Cortisol response during 45 min of chemical immobilisa-tion in adult female elephant seals of the experimental group,sampled repeatedly at early, middle and late stages of lactation(2 days, 11 days, 21 days postpartum). Marked and open symbolsrepresent samples taken from early or late breeding females (pupborn either in September or October), respectively. Thin linesconnect sequential samples taken from the same individual. Heavylines represent regressions, based on hierarchical linear modelling(Table 1). Letters a–c refer to significant differences in cortisollevels

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Mothers of different treatment groups at late lactation

Figure 2 shows the cortisol response to immobilisationstress for mothers at late lactation (around 21 dayspostpartum), that either belonged to the control, mod-erate, or high treatment group. There was no differencein the dosages of Zoletil anaesthetics given to thesefemales during the handling event (mean±SD: control321±45 mg, moderate treatment 340±55 mg, hightreatment 344±75 mg, 1-way ANOVA, F2,23=0.466,P=0.633) or in the dosage of Zoletil given per kilogramseal body mass (mean±SD: control 0.98±0.18 mg kg–1,moderate treatment 1.02±0.07 mg kg–1, high treatment0.97±0.08 mg kg–1, F2,23=0.286, P=0.754). Moreover,there was no difference in the degree of sedation betweenthe treatment groups (scaled 1–5, mean±SD: control4.1±0.7, moderate treatment 3.7±0.8, high treatment3.8±0.9, F2,23=0.659, P=0.527).

Direct response to immobilisation stress

Basal serum cortisol levels remained unknown due to thetime lag between anaesthetic administration and the firstpossible collection of a blood sample. Over the sampledtime-spans (in most cases, 10–40 min after anaestheticadministration), cortisol levels generally decreasedaccording to linear relationships with the time ofrestraint (P<0.0001, Table 2; see regression lines in

Fig. 2). In this data set restricted to late lactation, therewas no evidence of a quadratic relationship with time(squared restraint time, P=0.590).

Diurnal and seasonal effects

We did not detect an effect of time of day on levels ofserum cortisol at late lactation (P=0.361). However,there was a significant effect of season; females that gavebirth in September showed lower cortisol levels at latelactation than females that gave birth in October(average difference±SE: 109±32 nmol l–1; P=0.003).

No effect of moderate, but significant effectof high previous treatment

There was no evidence indicating an effect of moderateprevious treatment on the cortisol response to immo-bilisation stress (Table 2). At late lactation, females ofthe control group (not handled previously) and moder-ate treatment group (immobilised twice before) showedsimilar levels of cortisol (P=0.509) and similar lineardeclines of cortisol with immobilisation time (P=0.622).In contrast, both the levels of cortisol (P=0.024) and thecortisol change during restraint time (P=0.039) weresignificantly different in the high treatment group (after3–4 previous immobilisations) when compared to the

Deviance df Estimate SE P

Null model 1287.6 110Final model 1243.9 106

Random parametersVariance harem level 2 0 0Variance individual level 8 0 0Variance lactation stage level 32 5331 1451Variance restraint time level 106 2173 357

Fixed parametersConstant 106 187 45 <0.0001Restraint time (min) 106 8.16 3.05 0.009(Restraint time)2 106 –0.174 0.059 0.004Early lactation (2 days postpartum) 32 –126 33 0.0005Late lactation (21 days postpartum) 32 89 32 0.009

Rejected fixed parametersEarly lactation·restraint time 31 1.10 1.31 0.406Early lactation·(restraint time)2 30 0.139 0.116 0.240Late lactation·restraint time 31 –1.76 1.26 0.171Late lactation·(restraint time)2 30 –0.114 0.123 0.361Time of day (0–24 h) 31 3.22 7.45 0.669Season (birth in Sep=0, birth in Oct=1) 7 8.4 29.4 0.783Sex of pup (male=0, female=1) 7 –2.8 27.7 0.922Mass loss during lactation (kg) 7 –0.089 0.451 0.849Area (Middle Beach=0, Isthmus East=1) 1 19.0 25.9 0.598

Table 1. Hierarchical linear model describing the effect of chemi-cal restraint time (min since Zoletil administration) on serum cor-tisol (nmol l–1) in adult female elephant seals at early, middle andlate stages of lactation (around 2 days, 11 days and 21 days post-partum, respectively), based on the data set for the experimentalgroup (compare with Fig. 1). Note: the factors restraint time andsquared restraint time describe the change over time in cortisollevels. The decrease in deviance from the null model including the

constant only, to the final model including all parameters signifi-cantly affecting cortisol was significant (change in deviance –43.7,change in df=4, P<0.0001). A forward selection process was used.The significance of parameters was tested by adding these to themodel one at a time; when testing the significance of higher orderterms (interactions, squares), the lower order terms were includedinto the model, regardless of their significance. P values <0.05 areshown in bold type

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control and moderate treatment groups. The effect ofmilder responsiveness to immobilisation stress in thehigh treatment group appeared to be consistent for thesubsets of females that gave birth either in September

or October, although there was a trend (P=0.072)indicating an interaction between high previous treat-ment and season. Within the high treatment group, thefour early breeding females tended to have relativelylesser reduced levels of cortisol than the four latebreeding females (see also Fig. 2). There was no evidencethat the degree of sedation affected the levels of cortisol(P=0.946) or the cortisol response (P=0.920; Table 2).

Maternal size and condition

Body length was not significantly different betweenmothers of the three treatment groups (mean±SE:

Fig. 2. Comparison of the cortisol response during 45 min ofchemical immobilisation between adult female elephant seals at latelactation of the control, moderate and high treatment groups. Thinlines connect sequential samples taken from the same individual.Marked and open symbols represent early or late breeding females(pup born either in September or October), respectively. Regressionlines are based on hierarchical linear modelling (Table 2); contin-uous regression lines represent early breeders, dotted regression linesrepresent late breeders. Letters a–d refer to significant differences inthe cortisol response

Table 2. Hierarchical linearmodel comparing the effect ofchemical restraint time (minsince Zoletil administration) onserum cortisol (nmol l–1)between adult females of thecontrol, moderate and hightreatment groups at late lacta-tion (21 days postpartum;compare with Fig. 2). Themodel shows: (1) that the cor-tisol response in the high treat-ment group was significantlylower than in both the moderatetreatment and control groupsand (2) that cortisol levels werehigher for late-breeding whencompared to early breedingfemales. See note in Table 1; thechange in deviance from thenull model to the final modelwas –31.1 (change in df=4,P<0.0001)

Deviance df Estimate SE P

Null model 988.3 84Final model 957.2 80

Random parametersVariance harem level 3 160 1739Variance individual level 21 5132 2381Variance restraint time level 80 2451 451

Fixed parametersConstant 80 422 37 <0.0001Restraint time (min) 80 –4.14 0.86 <0.0001Season (birth in Sep=0, birth in Oct=1) 21 109 32 0.003High treatment (‡3 previous captures) 21 –128 52 0.024High treatment · restraint time 21 3.39 1.54 0.039

Rejected fixed parameters(Restraint time)2 79 –0.033 0.061 0.590Moderate treatment (‡2 previous captures) 20 –30 44 0.509Moderate treatment·restraint time 19 –1.07 2.13 0.622Moderate treatment·season n.a. n.a. n.a. n.a.High treatment·season 20 –77 40 0.072Season·restraint time 20 –2.12 1.42 0.150Degree of sedation (scaled 1–5) 20 1.45 21.01 0.946Sedation·restraint time 19 –0.10 1.00 0.920Time of day (0–24 h) 20 10.7 11.5 0.361Sex of pup (male=0, female=1) 20 35 32 0.287Length (m) 20 –18.5 101.5 0.857Mass (kg) 20 0.18 0.42 0.671Condition index (kg m–1) 20 1.16 1.44 0.430Pup weaning mass (kg) 20 –0.17 0.72 0.819Area (Middle Beach=0, Isthmus East=1) 2 –7.1 32.3 0.847Area·restraint time 1 2.14 1.43 0.376

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control 2.42±0.04 m, moderate treatment 2.54±0.06 m,high treatment 2.53±0.06 m; 1-way ANOVA, F2,23=1.501, P=0.244) and did not co-vary with levels of serumcortisol (Table 2, P=0.857). At 21 days postpartum,neither body mass nor condition index were significantlydifferent between females of the three treatment groups(mean±SE, mass: control 332±13 kg, moderate treat-ment 332±20 kg, high treatment 355±21 kg, F2,23=0.532, P=0.594; condition index: control 137±4 kg m–1,moderate treatment 130±5 kg m–1, high treatment140±5 kg m–1, F2,23=0.676, P=0.519). There was noevidence that body mass (P=0.671) or body conditionindex (P=0.430) affected levels of cortisol (Table 2).Mothers of the moderate and high treatment groupsshowed similar loss of mass over the entire lactation pe-riod (mean±SE: moderate treatment 183±11 kg, hightreatment 194±12 kg, independent samples t-test,t11=–0.637, P=0.537). The mass loss during lactationwas unknown for control females, since no data on massat early lactation had been collected for this group.

Pup sex and maternal cortisol

Mothers of male or female pups did not differ in thelevels of serum cortisol (Table 2, P=0.287).

Comparison between areas different in frequencyof human visits

At late lactation, adult females on Isthmus East (areaof high human presence; n=15) and Middle Beach

(area of low human presence; n=11) showed similarlevels of serum cortisol (P=0.847) and similar changesin cortisol during immobilisation time (P=0.376;Table 2).

Pups of experimental group at three stages of lactation

For nine female and six male pups comprising theexperimental group, changes in serum cortisol duringphysical restraint at three stages of lactation are illus-trated in Fig. 3.

Direct stress response at early, middle and late lactation

Capture and handling generally caused rapid increases inthe levels of serum cortisol. Over the sampled intervals(in most cases, 5–35 min post-capture) the cortisolresponses followed linear patterns (Table 3: effect ofrestraint time, P<0.0001), with no evidence of a qua-dratic effect (squared restraint time, P=0.665). Pupsshowed a weaker (P=0.003) cortisol response at earlylactation (age 2 days) than at the middle or late stages oflactation (age 11 days, 21 days, respectively); theresponse was not significantly different between the mid-dle and late stages (interaction late lactation·restrainttime, P=0.660). At early lactation, serum cortisolincreased on average (±SE) with 1.95±1.07 nmol l–1

min–1 over the sampled intervals following restraint; atmiddle and late lactation, the average increase rateswere 5.60±1.10 nmol l–1 min–1 and 6.33±1.13 nmol l–1

min–1, respectively.

Diurnal and seasonal effects

Within the experimental group of pups there was noevidence for an effect of time of day (P=0.371) orseason (P=0.417) on the levels of serum cortisol(Table 3).

Fig. 3. Cortisol response during 45 min of physical restraint inelephant seal pups of the experimental group at early, middle andlate lactation (2 days, 11 days, 21 days postpartum). Thin linesconnect sequential samples taken from the same individual. Closedand open symbols refer to male and female pups, respectively.Regression lines are based on hierarchical linear modelling(Table 3); continuous and dotted lines represent male and femalepups. Letters a–d refer to significant differences in the cortisolresponse

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Sex difference

Increase rates of cortisol during physical restraint werehighly similar for the pups of the two sexes (Table 3,P=0.925). However, absolute levels of serum cortisolwere significantly higher in females than males, regard-less of the time since capture (Table 3, P=0.023).The difference averaged throughout lactation was72±27 nmol l–1.

Mass change

The 13 pups of the experimental group weighed40±4 kg and 117±27 kg at birth and at weaning,respectively, on average gaining 77±25 kg of massthroughout the lactation period (means±SD). Wedetected no effect of serum cortisol on total mass gainduring lactation (Table 3, P=0.860).

Pups of different treatment groups at late lactation

We compared serum cortisol responses to physicalrestraint between pups aged 21 days of the control,moderate and high treatment groups (Fig. 4). Hierar-chical linear modelling comparing the treatment groups(Table 4) confirmed the sex difference in absolute cor-tisol levels already found within the experimental group(females greater than males; P=0.0002). The rate ofincrease of serum cortisol during handling was similarfor male and female pups (P=0.369). The average dif-ference (±SE) regardless of the effect of restraint timewas 123±28 nmol l–1. There was no evidence of diurnal(P=0.145) or seasonal (P=0.860) effects on serumcortisol in pups.

Effect of previous treatment

There was no evidence suggesting an effect of either amoderate or high level of handling of pups during earlyand middle lactation on their levels of serum cortisol atlate lactation (Table 4). When compared to the controlgroup, pups of the moderate treatment group showedsimilar absolute levels (P=0.814) and rates of increase(P=0.299) of serum cortisol during physical restraint atlate lactation. Neither did pups of the high treatmentgroup differ from the other groups in absolute levels(P=0.612) or increase rates (P=0.169) of serum cortisolduring restraint at late lactation.

Pup size and condition

There was no evidence that body length (P=0.943), mass(P=0.882) or condition index (P=0.988) affected levelsof serum cortisol in pups aged 21 days (Table 4). Pups ofthe three treatment groups showed similar body lengths(means±SE: control 1.36±0.02 m, moderate treatment1.36±0.03 m, high treatment 1.30±0.06 m; 1-wayANOVA, F2,20=0.834, P=0.449), similar body masses(control 112±9 kg, moderate treatment 120±9 kg, hightreatment 108±12 kg, F2,23=0.262, P=0.771), and sim-ilar body condition indices (control 79±6 kg m–1, mod-erate treatment 88±5 kg m–1, high treatment 81±7 kgm–1, F2,20=0.379, P=0.689). The mass of pups at wean-ing was not significantly different between the treatmentgroups (control 119±8 kg, moderate treatment122±7 kg, high treatment 114±12 kg, F2,23=0.157,P=0.856). The gain of mass from birth to weaning wassimilar for pups of the moderate and high treatmentgroups (moderate treatment 81±7 kg, high treatment75±11 kg, independent samples t-test, t11=0.391,

Table 3. Hierarchical linearmodel describing the directeffect of physical restraint time(min since capture) on serumcortisol (nmol l–1) in male andfemale pups at early, middleand late stages of lactation (agearound 2 days, 11 days and21 days, respectively), based onthe data set for the experimentalgroup (compare with Fig. 3).Cortisol levels increased linearlyduring restraint, more rapidlyat middle and late stages oflactation than at early lactation.See note in Table 1; the changein deviance from the null modelto the final model was –70.3(change in df=4, P<0.0001)

Deviance df Estimate SE P

Null model 1324.2 108Final model 1253.9 104

Random parametersVariance harem level 2 0 0Variance individual level 8 0 0Variance lactation stage level 34 4879 1393Variance restraint time level 104 3227 544

Fixed parametersConstant 104 194 29 <0.0001Restraint time (min) 104 6.07 0.76 <0.0001Early lactation (2 days postpartum) 34 –39 37 0.307Early lactation·restraint time 34 –4.12 1.31 0.003Sex of pup (male=0, female=1) 8 75 27 0.023Rejected fixed parameters(Restraint time)2 103 0.020 0.046 0.665Late lactation (21 days postpartum) 33 –36 30 0.247Late lactation·restraint time 32 0.68 1.53 0.660Sex of pup·restraint time 7 0.13 1.34 0.925Time of day (0–24 h) 33 –6.56 7.24 0.371Season (birth in Sep=0, birth in Oct=1) 7 –26 30 0.417Mass gain during lactation (kg) 7 0.11 0.60 0.860Area (Middle Beach=0, Isthmus East=1) 1 –21.1 26.0 0.566

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P=0.703); the total mass gain was unknown for controlpups due to absence of data at early lactation. The effi-ciency of mass transfer from mother to pup, expressed asthemass gained by the pup as a percentage of themass lostby the mother throughout lactation, averaged 44.0±1.1% and 37.8±3.5% (±SE) for mother-pup pairs of themoderate and high treatment groups; the difference wasnot significant (Mann-Whitney, U=12.0, P=0.242).

Comparison between areas different in frequencyof human visits

There was no difference in cortisol levels (P=0.796) orin cortisol responses during restraint (P=0.227) betweenthe 11 pups in the remote area of Middle Beach and 15pups in the frequently visited area of Isthmus East(Table 4).

Discussion

All levels of serum cortisol in southern elephant sealmothers and pups on sub-Antarctic Macquarie Island(range 47–684 nmol l–1) found in this study, were lowerthan the plasma cortisol levels found previously in two

Fig. 4. Comparison of the cortisol response during 45 min ofphysical restraint between elephant seal pups aged around 21 daysof the control, moderate, and high treatment groups. Thin linesconnect sequential samples taken from the same individual. Closedand open symbols represent male and female pups, respectively.Regression lines are based on hierarchical linear modelling(Table 4); continuous and dotted lines represent male and femalepups, respectively. Letters a–b refer to significant differences in thecortisol response

Table 4. Hierarchical linearmodel comparing the effect ofphysical restraint (min sincecapture) on serum cortisol(nmol l–1) between pups of thecontrol, moderate, and hightreatment groups at age 21 days(compare with Fig. 4). Both thefactors moderate and hightreatment and the interactionsof moderate and high treatmentwith restraint time were reject-ed. See note in Table 1; thechange in deviance from thenull model to the final modelwas –47.0 (change in df=2,P<0.0001)

Deviance df Estimate SE P

Null model 825.7 66Final model 778.7 64

Random parametersVariance harem level 64 335 1274Variance individual level 23 2260 1685Variance restraint time level 5 4647 1021

Fixed parametersConstant 64 125 30 <0.0001Restraint time (min) 64 7.00 0.91 <0.0001Sex of pup (male=0, female=1) 23 123 28 0.0002

Rejected fixed parameters(Restraint time)2 63 –0.062 0.076 0.403Moderate treatment (‡2 previous captures) 22 6.5 27.3 0.814Moderate treatment·restraint time 21 –1.94 1.82 0.299High treatment (‡3 previous captures) 22 –14.9 28.9 0.612High treatment · restraint time 21 –2.69 1.89 0.169Season (birth in Sep=0, birth in Oct=1) 22 –5.1 28.7 0.860Season·restraint time 21 2.48 1.85 0.195Time of day (0–24 h) 22 –14.9 9.8 0.145Sex of pup·restraint time 22 1.74 1.90 0.369Length (m) 22 –15.1 140.0 0.943Mass (kg) 22 –0.069 0.461 0.882Condition index (kg m–1) 22 –0.011 0.798 0.988Pup weaning mass (kg) 22 0.229 0.500 0.651Area (Middle Beach=0, Isthmus East=1) 4 –11.6 27.6 0.796Area·restraint time 3 –1.94 1.82 0.227

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male subadult southern elephant seals sampled nearPalmer Base, Antarctica (Liggins et al. 1993: 1420 nmoll–1 and 820 nmol l–1). The levels were considerably lowerthan the plasma cortisol levels of typical Antarcticphocid seals (about 1500–4500 nmol l–1), such asWeddell seals Leptonychotes weddellii (Liggins et al.1979; Barrell and Montgomery 1989; Liggins et al.1993), crabeater seals Lobodon carcinophagus and leop-ard seals Hydrurga leptonyx (Liggins et al. 1993). Levelswere comparable to those in northern temperate andArctic phocids, such as grey seals Halichoerus grypus(Engelhardt and Ferguson 1980; Nordøy et al. 1990;Boily 1996), harbour seals Phoca vitulina (Riviere et al.1977; Gardiner and Hall 1997; P.J.H. Reijnders,unpublished results), larga or spotted seals Phoca largha(Ashwell-Ericksson et al. 1986), harp seals Phocagroenlandica (Engelhardt and Ferguson 1980) and rin-ged seals Phoca hispida (St. Aubin and Geraci 1986). Inthese species, base-line plasma or serum levels are gen-erally in the range of 50–800 nmol l–1. The very highcortisol levels in Antarctic Weddell, crabeater andleopard seals have been related to their high divingcapabilities, as potentially being protective against highpressure nervous syndrome (Liggins et al. 1993). Ourdata do not provide correlative evidence supporting thehypothesis. Diving performance of adult female south-ern elephant seals generally exceeds that of typicalAntarctic phocid seals (Hindell et al. 1991; Jonker andBester 1994), while the cortisol levels we found were notexceptionally high. It should be remarked that ourresults do not concern the pelagic phase of the annualcycle of the seals.

Mild cortisol response in immobilised lactating females

No direct data on base-line cortisol in lactating femaleelephant seals were available for this study, due to thetime lag between administration of Zoletil and sedation(�10 min, Baker et al. 1990). This is unfortunate sincethe initial rise in cortisol from base-line levels is animportant component of the adrenocortical response.Over the measured time-spans (about 10–40 min afteranaesthetic administration) we found significant butmoderate changes in the levels of serum cortisol. Mildcortisol responses in anaesthetised adult female elephantseals are consistent with a comparative study on Africanland mammals (Morton et al. 1995), where in six out ofseven artiodactyl species the plasma cortisol levels wererelatively low in chemically immobilised animals whencompared to physically restrained individuals. Ratherinconsistent and generally moderately variable cortisolprofiles were also found in spotted hyenas Crocuta cro-cuta immobilised with Zoletil (Van Jaarsveld and Skin-ner 1992).

However, the moderate cortisol responses to immo-bilisation stress in lactating female elephant seals foundin this study do not necessarily imply that the procedureitself was a mild stressor. It is likely that during lactation

a variety of physiological responses to stress are sup-pressed, including responsiveness of the adrenocorticalaxis; hyporesponsiveness during lactation has beendescribed for rats Rattus norvegicus (e.g. Windle et al.1997) and humans (Altemus et al. 1995). If the adreno-cortical axis is also toned down in the nursing elephantseal, which is likely since lactation is particularly inten-sive in this species and the adults fast throughout thisperiod (Fedak et al. 1996), then our results on thechanges in cortisol levels may underestimate the mag-nitude of the entire stress response. A more appropriatedescription of the full impact of disturbance might begiven by measures on the sympathetic-adrenal-medul-lary response to stress, or by measures of immuno-competence.

Cortisol increase during lactation in mothers

Cortisol levels in elephant seal mothers, regardless of theeffect of restraint, increased over the 24-day lactationperiod (Fig. 1). Glucocorticoids have profound effectson metabolism in fasting animals, and on lactogenesis(Hadley 1996). Engelhardt and Ferguson (1980) foundhigh plasma cortisol levels in nursing female harp sealswhen compared to non-nursing females, suggesting thisstimulates extrahepatic lipid mobilisation necessary forthe production of fat-rich milk. During the first 20 daysof lactation, southern elephant seal milk becomesincreasingly rich in fat content, from about 12% toapproximately 52% (Carlini et al. 1994). This may cor-respond with increasing cortisol levels. Glucocorticoidsare specifically involved in lipid and protein degradationto produce glucose, therefore increasing basal cortisolfrom initially low levels may be involved in maintainingadequate lactation in a fasting mother. However,glucocorticoid levels that continue to increase are alsolikely, in the end, to facilitate and induce foraging be-haviour and food intake (e.g. Honma et al. 1984;Leibowitz et al. 1984; Wingfield 1994). In emperorpenguins Aptenodytes forsteri which fast for up to4 months while breeding on the sea ice, body stores aregradually depleted while low plasma corticosterone lev-els are maintained and the birds show little activity; at athreshold body condition however, both plasma corti-costerone rises and activity increases, which then mayact as a ‘refeeding signal’ stimulating birds to departfrom the breeding group, return to sea and start foragingagain (Robin et al. 1998). In lactating and fasting ele-phant seal mothers, cortisol levels may similarly increaseup to a threshold level, and may at that stage be involvedin motivating the female to wean the pup and enter thepelagic foraging stage.

In addition to, or alternatively, the increasing levelsdocumented here may be related to oestrus, sexualactivities, and harem dynamics. Mating usually occursduring the last days of the lactation period (Carrick et al.1962), when we observed an increase in cortisol levels. Inharems, sexual activities are far more frequent late in the

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breeding season than at early stages (McCann 1981); asa result, harassment of females due to male sexualactivity is more frequent during late October and earlyNovember than earlier in the season (McCann 1981;Galimberti et al. 2000). This increase in ‘natural’ dis-turbance over the season may be a crucial factorexplaining why cortisol levels increased during lactation(Fig. 1); in particular, it may explain why late breedingfemales showed higher cortisol levels when compared toearly breeding females (Fig. 2). It is remarkable that ingrey seals, exhibiting harem systems, lactational oestrusand an approximate lactation period of 18 days, cortisolin females decreases over the lactation period (P.J.H.Reijnders, unpublished results).

In phocid seals, cortisol might be involved in theregulation of moult, although the evidence of its role isnot fully clear (Riviere et al. 1977; Ashwell-Ericksonet al. 1986; Boily 1996). Changes in cortisol over thelactation period (September–November) in southernelephant seals are unlikely to be associated with moult,since the females undergo a distinct moulting fast inJanuary–February (Hindell and Burton 1988).

Direct stress response in pups

While in chemically immobilised mothers, blood samplesusually could not be collected within 10 min of anaes-thetic administration and, hence, the initial rise incortisol from baseline levels was likely to be missed,physically restrained pups could mostly be blood sam-pled fairly rapidly after the start of handling. Thus thecortisol profiles reported here for pups (Figs. 3, 4) inmost cases are likely to include the initial rise from base-line levels. The profiles are similar to typical adreno-cortical responses to capture and handling stressdescribed in a number of studies (e.g. Thomson andGeraci 1986; Harlow et al. 1987; Gardiner and Hall1997). The adrenocortical responses of pups werestronger around ages 11 days and 21 days than around2 days after birth (Fig. 3). This may reflect the devel-opment of the adrenocortical system during the first fewweeks of life (Walker et al. 1991) and suggests a hypo-responsive period of the HPA axis shortly after birth; theexistence of postnatal hyporesponsiveness has been welldocumented in rodents (Sapolsky and Meaney 1986). Ifcortisol is involved in regulation of moult, the increasingresponsiveness may in addition be related with the onsetof the first moult, or the replacement of the black lanugoby the grey hairs typical of weaned pups (Ling andThomas 1967). In suckling harp seal pups, increasingcortisol levels during lactation have been related to thefirst moult (Engelhardt and Ferguson 1980).

Pup sex

There is a sex difference in the activity of the HPA axisof many species (Kitay 1961; Montano et al. 1991;

Vierhapper et al. 1998; Canny et al. 1999). In line withthis, female elephant seal pups showed significantlyhigher cortisol values than males; it is interesting to notethat a substantial difference in levels was already presentwithin days to weeks of birth (Figs. 3, 4). Althoughadult southern elephant seals are among the most sex-ually dimorphic mammal species (Laws 1953), thisdimorphism is still extremely small during the sucklingperiod (Campagna et al. 1992; McMahon et al. 1997).However, at the stage of weaning there are small butsignificant differences in mean weight and tooth devel-opment between the sexes; higher cortisol levels infemale pups might be involved in their more precociousdevelopment of teeth when compared to male pups(McMahon et al. 1997).

Effects of repeated handling

In laboratory rat pups, repeated handling can causelong-term changes in the adrenocortical responsivenessto stress that persist into adulthood (Liu et al. 1997;Durand et al. 1998; Meerlo et al. 1999). In the presentstudy on wild southern elephant seals in their naturalenvironment, no chronic effect of repeated physicalrestraint of pups on their adrenocortical axis was foundfor the duration of the lactation period. At late lacta-tion, cortisol responses to physical restraint were similarfor elephant seal pups of the control, moderate and hightreatment groups (Fig. 4). Despite the absence of achronic effect of repeated handling of juveniles over aperiod of weeks, the possibility of a long-term effect onadrenocortical responsiveness during adulthood cannotbe fully excluded (Meerlo et al. 1999).

By contrast, the study shows that even though it islikely that the HPA axis is naturally toned down in thelactating elephant seal, repeated chemical immobilisa-tions in lactating females may nevertheless, over a periodof weeks, result in chronic changes in adrenocorticalresponsiveness. First, this was shown by the finding thatalthough at late lactation there was no significant dif-ference in the cortisol stress response between females ofthe control and moderate treatment groups, thisresponse was significantly attenuated in females of thehigh treatment group (Fig. 2; Table 2). Second, alteredresponsiveness may also be indicated by the discrepancybetween the comparison of lactational stages which waslimited to the experimental group (quadratic responsesindicative of mild cortisol peaks after around 20–30 min:Fig. 1; Table 1), and the comparison of treatmentgroups at late lactation (linearly decreasing trendsdespite partial overlap of data: Fig. 2; Table 2). Over themeasured time-spans (�10–40 min after anaestheticadministration), the negative trends were particularlysubstantial for the control and moderate treatmentgroups; this suggests that these females may have beencharacterised by a relatively rapid cortisol response suchthat blood sampling missed the initial increase and onlycaught the subsequent decline. However, the trend for

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the high treatment group was almost horizontal; thismay indicate that in this group not only was the plateauof the response reduced, but the speed of the responsewas also delayed. There was no evidence that the dosageof anaesthetics used during the handling event, or theresulting degree of sedation were different in the hightreatment group; therefore these factors are unlikely toexplain the attenuated cortisol responses. This indicatesthat the high degree of treatment previously given tothese females may have dampened their HPA respon-siveness to stress (Dhabhar et al. 1997), either due to ageneral desensitisation of the adrenals as a by-effect ofthe anaesthetic, or to a degree of habituation to therepeated handling procedure, or through other mecha-nisms.

A reduction in physiological condition is likely tohave adverse effects on chances of survival and repro-ductive success (e.g. Fedak et al. 1996; Guinet et al.1998; Pomeroy et al. 1999; McMahon et al. 2000a; Hallet al. 2001). We compared a set of mass- and condition-related measures between the treatment groups, thatmay be interpreted as indicating potential or likely fit-ness consequences of disturbance. There was no effect ofeither a moderate or a high degree of treatment on themass and condition of mothers and pups at late lacta-tion, the mass lost by mothers throughout lactation, thetotal mass gained by pups from birth to weaning, andthe mass gain of pups proportional to the mass loss ofmothers. Moreover, there was no effect on the mass ofthe pup at weaning; this is of particular significance,since a positive association of pup weaning mass with1st-year survival has been shown for the southern ele-phant seal (McMahon et al. 2000a) and for several otherpinniped species (Baker and Fowler 1992; Craig andRagen 1999; Hall et al. 2001). Hence, based on proxymeasures there was no indication of an adverse fitnesseffect of disturbance. However, negative consequencesfor true Darwinian fitness cannot be directly excluded bythis study, since no data on survival of study animals, oron reproductive success of study females during laterbreeding seasons were available.

Area comparison

Cortisol levels and responses were similar for elephantseals in the two areas different in the frequency ofhuman visits, i.e. Isthmus East and Middle Beach (cf.Engelhard et al. 2001), indicating that the human pres-ence and activities involved with the research carried outat the station on the Isthmus of Macquarie Island do notpose a long-term stress effect on the residing elephantseals, as measured by cortisol. Absence of a difference incortisol levels gave no reason to assume that earliercollected chemical and physical blood parameter base-line values are seriously biased by stress-related arte-facts.

Moderate to high levels of physical restraint in pups,and moderate levels of chemical immobilisations in

lactating mothers, caused direct cortisol stress responsesduring the period of handling, but no chronic changes inthe adrenocortical response over a period of days toweeks. Hence, elephant seals appear to tolerate moder-ate levels of handling disturbance. However, higherlevels of handling, especially immobilisation treatmentsin lactating females, may result in altered adrenocorticalresponsiveness, and should be minimised either for sci-entific or other purposes.

Acknowledgements This study is a collaboration of the Nether-lands Antarctica Program of NWO (Project 751 49 505), theAlterra Institute (Marine and Coastal Zone Research Team Project35 007 05), and the Australian Antarctic Division (Human ImpactsProject 1007 and Biological Sciences Project 2265). Fieldwork wasconducted as part of the 49th Australian National AntarcticResearch Expeditions. We thank the ANARE staff and partici-pating expeditioners on Macquarie Island, in particular PirieConboy, Paul Klemes and Jeremy Smith. Rupert Davies, PaulDavis, Frans Jonker, Margaret Morrice, David Slip, and John vanden Hoff supported us in the field. Maarten Loonen and PopkoWiersma contributed in discussions on statistics. The manuscriptwas greatly improved through the comments of Leo Bruinzeel,Jeroen Creuwels, Rudi Drent, Katalin M. Horvath, Clive McMa-hon, Lucia Privitera, Jeroen Reneerkens, Jan Verhees, Wim Wolff,and an anonymous referee. The Tasmanian National Parks andWildlife Service supplied permits to work on Macquarie Island.Our activities involving the handling of animals followed theGuidelines of the Antarctic Animal Ethics Committee.

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