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PHYSIOLOGIC MANIFESTATIONS OF STRESS FROM CAPTURE ANDRESTRAINT OF FREE-RANGING MALE AFRICAN GREEN MONKEYS(CERCOPITHECUS AETHIOPS)Author(s): Mbaruk A. SulemanB.V.M., Ph.D., Emmanuel WangoB.V.M., Ph.D., Robert M.SapolskyB.Sc., Ph.D., Hesbon OdongoDipl. H.N., and Jann HauM.D., Ph.D.Source: Journal of Zoo and Wildlife Medicine, 35(1):20-24. 2004.Published By: American Association of Zoo VeterinariansDOI: http://dx.doi.org/10.1638/01-025URL: http://www.bioone.org/doi/full/10.1638/01-025

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20

Journal of Zoo and Wildlife Medicine 35(1): 20–24, 2004Copyright 2004 by American Association of Zoo Veterinarians

PHYSIOLOGIC MANIFESTATIONS OF STRESS FROM CAPTUREAND RESTRAINT OF FREE-RANGING MALE AFRICAN GREENMONKEYS (CERCOPITHECUS AETHIOPS)

Mbaruk A. Suleman, B.V.M., Ph.D., Emmanuel Wango, B.V.M., Ph.D., Robert M. Sapolsky,B.Sc., Ph.D., Hesbon Odongo, Dipl. H.N., and Jann Hau, M.D., Ph.D.

Abstract: Adrenal gland weights, stomach mucosal lesions, and morning serum cortisol and prolactin levels weremeasured in 15 juvenile and adult male African green monkeys (Cercopithecus aethiops) that were shot by a hunter,euthanized after 24 hr of captivity, or euthanized after 45 days of captivity and intermittent blood sampling. Hormonelevels were measured in seven additional males that had been in captivity for 7 mo. Mean serum cortisol concentrationswere significantly lower in free-ranging wild monkeys at the time they were shot than in the monkeys after 1 day incaptivity. Cortisol concentrations were significantly higher in wild-caught monkeys on the day after capture than theywere in the same animals after 18 and 26 days of captivity. Cortisol concentrations were also significantly higher inthe wild-caught monkeys 18 days after capture than in the laboratory-habituated monkeys in captivity for 7 mo. Meanprolactin concentration was significantly lower in the wild-caught monkeys on day 2 after capture, and the levelsincreased gradually to 45 days in captivity and was highest in monkeys that had been captive for 7 mo.

Key words: Prolactin, hormone, cortisol, Cercopithecus aethiops, African green monkey, stress.

INTRODUCTION

Capture, relocation, and captive breeding meth-odologies can stress animals. De Brazza’s monkeys(Cercopithecus neglectus)10 are rare in East Africa,and optimal methodologies for capturing and relo-cating them are being sought. African green mon-keys (Cercopithecus aethiops), however, are rela-tively abundant and are excellent research models.They are highly susceptible to stress in laboratoryenvironments, experiencing high morbidity andmortality.4,6 Spontaneous gastric erosions and ulcersare common in captive C. aethiops,14 and adrenalcortical hypoplasia, hippocampal degeneration, andmultiple gastric lesions that may be stress relatedhave been observed.18 Ketamine anesthesia and re-peated blood collection from C. aethiops can in-crease circulating monocyte counts, probably fromglucocorticoid release after stress.19 Solitary captiveindividuals are more susceptible to stress and moreoften develop gastric lesions than do monkeyshoused in social groups.17

MATERIALS AND METHODS

The Institutional Scientific Research and EthicalCommittee (Institute of Primate Research [IPR],

From the Institute of Primate Research, P.O. Box 24481,Karen, Nairobi, Kenya (Suleman); the Department of An-imal Physiology, University of Nairobi, Box 30197, Nai-robi, Kenya (Wango, Odongo); the Department of Biolog-ical Sciences, Stanford University, Stanford, California94305-5020, USA (Sapolsky); and the Division of Com-parative Medicine, Uppsala University, BMC, Box 570,S-751 23 Uppsala, Sweden (Hau). Correspondence shouldbe directed to Dr. Suleman.

Nairobi, Kenya) approved this project. Shootingwild animals is an acceptable euthanasia methodaccording to the European Union5 and provides re-liable stress control corticosteroid data.11,20 All themonkeys in this study were obtained from the samelocation in Kibwezi, Kenya, and were members oftwo troops of feral monkeys that were destroyingfood crops and were scheduled to be killed in a pestcontrol program. Their ages were estimated fromdentition.

Experimental groups

Group I: Two 4-yr-old and two 18-mo-old maleAfrican green monkeys (C. aethiops) (group I)were shot in the head with a rifle (caliber 0.22 Mag-num, Sturm, Ruger & Co. Inc., Southport, Con-necticut 06490, USA) between 0700 and 0900hours in the morning in Kibwezi, Kenya. Theseserved as nonstressed control animals. The mon-keys were exsanguinated by jugular venipunctureimmediately following death and necropsied. Ad-renal glands were weighed and preserved in 10%buffered formalin, as were stomach tissue speci-mens.

Groups II and III: Before live capture, 28 Afri-can green monkeys (in a separate troop from thoseshot but in the same locality in Kibwezi) were ha-bituated to bait in large portable metal traps simul-taneously to the shooting exercise.9 Twenty-one ofthem were captured in the traps at sunrise as theydescended from their sleeping sites to feed andwere transported by truck to a holding facility lo-cated 8 km away. Within that facility they werehoused singly in metal cages (90 3 60 3 60 cm)and in full view of each other. A diet comprising

21SULEMAN ET AL.—STRESS PARAMETERS IN WILD-CAUGHT MONKEYS

locally available grains and potable water was pro-vided ad libitum and supplemented with fruits andvegetables from local markets. Twenty-four hoursafter capture, 10 of the monkeys, including all thefemales and their babies, were released at the orig-inal site of capture. Four (group II) of the remaining11 males, selected at random, were manually re-moved from their cages between 0700 and 0900hours and given injections of ketamine (KetaminoltVet, Veterinaria AG, Zurich, Switzerland; 15 mg/kg, i.m.). Blood samples were collected in vacutai-ner tubes (Vacuettet, Greiner Labortechnik GmbH,A-4550 Kremsmunster, Austria) for measurementof serum cortisol and prolactin levels. The monkeyswere then killed by exsanguination through the jug-ular vein, which was approached through a skinincision in the neck, while still under anesthesia.Necropsies were done immediately after death toidentify any pathologic changes in the stomachsand adrenal glands. The remaining seven males(group III) were maintained in their same cageswithin the holding facility for 45 days, and bloodsamples were collected from their femoral veins un-der ketamine anesthesia (as already described) ondays 2, 18, 26, and 45 of captivity.

Group IV: Seven other male C. aethiops hadbeen captured about 7 mo earlier in the same Kib-wezi location and transported to the IPR, Nairobi,200 km away. These animals had been quarantinedfor 3 mo during which they were tuberculin andblood tested and screened for infectious diseasesevery 2 wk following the Institute Standard Oper-ating Procedures. After the quarantine period, theywere moved to a research facility in the IPR, Nai-robi, and housed singly for a further 4 mo. Theywere then restrained and anesthetized with keta-mine injected at the same dosage as described pre-viously, and blood samples were drawn on day 210postcapture. Thereafter, they were allocated to otherexperiments.

Hormonal assays

Serum cortisol: Cortisol radioimmunoassays usedmethodology13 that has been used routinely in our lab-oratory for nonhuman primate samples and validatedfor C. aethiops.3 The test serum and cortisol stan-dards were assayed in triplicate using 50 ml pertube. Tubes for total count (TC), nonspecific bind-ing (NSB), and zero antigens (BO) were in dupli-cate, each containing 100 ml of the specific reagent.Glass distilled water (1 ml per tube) was added tothe 50 ml test serum. The mixture was incubated ina water bath at 608C for 30 min to denature cortisol-binding globulin and release protein-bound cortisoland then cooled to room temperature. Thereafter,

100 ml of sample and standard were pipetted intotriplicate assay tubes. To each tube, 100 ml tracer,100ml antiserum, and 100 ml steroid buffer wasadded and incubated at 48C overnight. Duplicateassay tubes of TC, NSB, and BO were prepared asdescribed in the World Health Organization (Hu-man Reproduction Program) manual.13 To eachtube, 200 ml of charcoal reagent was added, vortexmixed, and incubated at 48C for 30 min. The tubeswere centrifuged at 2,200 rpm for 5 min, and thesupernatant was decanted into scintillation vials im-mediately. Counting was done using a scintillationcounter at 1 min per tube. Inter- and intra-assaycoefficients of variation were 18 and 10%, respec-tively.

Serum prolactin: Enzyme immunoassay wasperformed using a commercial kit (ImmunometricsLtd. London, U.K.), which has been validated inour laboratory and is applicable for nonhuman pri-mates. Stored serum samples were analyzed in du-plicate or triplicate batches to minimize inter- andintra-assay variations. In addition, there were trip-licate sample controls of known values and sevenstandard samples as well as assay internal qualitycontrols provided by the manufacturers. Thirty-sixsamples were run at a time. To each assay tube,100 ml of serum was pipetted and 100 ml of washedmagnetic bead–coupled antibody added. Reconsti-tuted standards and internal quality controls wereadded into separate tubes. The tubes were shakento mix the contents and placed in a water bath at378C for 30 min. Mean interassay CV 5 16.3% andintra-assay CV 5 8%.

RESULTS

Table 1 shows the mean and standard errors ofmeans of serum cortisol and prolactin concentra-tions in the four groups of C. aethiops. The meanserum cortisol concentration in the free-rangingwild monkeys (group I) was 391 6 115 nmol/L andin the wild-caught monkeys on day 1 postcapture(group II) was significantly higher (P , 0.05) at1,076 6 538 nmol/L. On day 2 postcapture, theserum cortisol level was lower (530 6 52 nmol/L)in the wild-caught monkeys (group III) and re-mained at similar levels throughout the study pe-riod. The differences in circulating cortisol valueswere highly significant between the wild-caughtgroups sampled on day 1 (group II, n 5 4) versusdays 18 (group III, n 5 7) and 26 postcapture (P# 0.01). The concentrations for day 45 were notsignificantly different (1,076 6 538 versus 838 6342 nmol/L).

The difference in prolactin serum concentrationsbetween the wild-caught monkeys on day 2 post-

22 JOURNAL OF ZOO AND WILDLIFE MEDICINE

Tab

le1.

Pla

sma

cort

isol

and

prol

acti

nco

ncen

trat

ions

inth

efo

urgr

oups

ofA

fric

angr

een

mon

keys

.

Gro

up(n

o.in

grou

p)

Ia(4

)II

b(4

)II

Ic(7

)IV

4(7

)

Sam

ple

day

Mea

nco

rtis

ol6

SD

e(n

mol

/L)

Mea

npr

olac

tin

6S

D(p

mol

/L)

039

16

230

399

636

4

11,

076

61,

076

361

623

9

253

06

138

234

610

9

1840

16

172

457

644

6

2644

06

125

549

626

6

4583

86

906

793

664

9

210

636

627

889

76

584

aF

ree

rang

ing,

kill

edby

rifl

esh

otto

the

head

.b

Wil

dca

ught

,1

day

post

capt

ure,

hous

edsi

ngly

.cW

ild

caug

ht,

fiel

dca

ptiv

e.d

Wil

dca

ught

,la

bora

tory

capt

ive.

eS

Dre

fers

tost

anda

rdde

viat

ion.

capture (group III, 234 6 101 pmol/L) and labo-ratory-habituated monkeys on day 210 (group IV,897 6 584 pmol/L) was highly significant (P #0.01).

Table 2 shows the mean and standard error of themean of body and adrenal gland weights. In thefree-ranging animals that were shot with a rifle(group I), the mean weights of the left and rightadrenal glands were 220 6 7.1 mg and 150 6 4.7mg, respectively, and in wild-caught monkeys eu-thanatized at 24 hr (group II) were 240 6 5.9 mgand 190 6 4.1 mg, respectively. The mean weightsof the left and right adrenals in the wild-caught,field-captive animals (group III) on day 45 were410 6 6.7 mg and 310 6 3.8 mg, respectively.

Table 3 shows the gross necropsy findings in thestomach and the mean individual cortisol and pro-lactin values of the C. aethiops on days 1 and 45postcapture. There were no gross pathologic lesionsin the free-ranging wild animals. One of four wild-caught animals (group II), 1 day postcapture, hadmultiple, bright red, circumscribed lesions distrib-uted in the mucosa of the body and fundus of thestomach.15 Three of seven (43%) wild-caught ani-mals (group III) exsanguinated on day 45 postcap-ture had dark red to brown, circular, depressed 2-to 5-mm diameter lesions.15 Mean cortisol levels ina juvenile male C. aethiops with stomach mucosallesions (number V 21) were low compared with thegroup members (V 1, 2, 5, and 20) that did notdevelop the lesions (640 nmol/L versus 2,000 nmol/L, respectively). Monkeys with stomach lesions hadsignificantly lower mean cortisol levels than didmonkeys without lesions (183 nmol/L versus 1,330nmol/L, respectively) (P # 0.01, Table 3).

DISCUSSION

Euthanasia by exsanguination avoided the use ofadditional chemical substances that might haveconfounded the results of the study, and ensuredthe availability of drained tissue samples for his-tologic analyses.

Previous studies of stress in a variety of nonhu-man primate species have been based on captive-conditioned or captive-bred animals, primarily.

Long-term studies involving behavioral obser-vations and hormonal analyses have focused onfree-ranging baboons.12 Such studies may ignorethe physiologic manifestations of stress.16

Experiments involving stress should includemultiple stressors over a fairly long period of time.

Cercopithecus aethiops is more prone to stressthan any other East African primate species.17

Our study methodology regarding capture, con-finement, handling, and blood sampling conformed

23SULEMAN ET AL.—STRESS PARAMETERS IN WILD-CAUGHT MONKEYS

Table 2. Mean and standard deviations of body and adrenal gland weights of three groups of African greenmonkeys.a

Group (no. in group)

Ib (4) IIc (4) IIId (7)

Sample dayMean body weight (kg 6 SD)

03 6 2.4

13.3 6 2.4

451.9 6 1.1

Mean adrenal weight (mg 6 SD)

LeftRight

220 6 14.2150 6 9.4

240 6 11.8190 6 8.2

410 6 18310 6 10.1

a Group IV described in the text was not euthanized; SD refers to standard deviation.b Free ranging wild, killed by rifle shot to the head.c Wild-caught, 1 day postcapture, housed singly.d Wild-caught (up to 45 days postcapture).

Table 3. Gross necropsy findings in the stomach and mean individual cortisol and prolactin values in wild maleCercopithecus aethiops on days 1 and 45 postcapture.a

Group

IIb IIIc

Identification no.Age groupStomach lesionMean cortisol (nmol/L)Mean prolactin (pmol/L)

75AN1,060

370

76JuvN2,000

522

77SAN620245

78JuvY640307

1AN400679

2AN2,000

413

3AY124747

5AN2,0002,128

11JuvY300476

20JuvN920601

21JuvY125516

a A, adult; M, male; Juv, juvenile; SA, sub-adult; N, no erosion or ulcer in the stomach mucosa; Y, lesion present in stomach mucosa.b 1 day postcapture.c 45 days postcapture.

to the routine procedures and methodologies fol-lowed in the IPR, Nairobi, Kenya.

Monkeys are always stressed during capture ordarting. The four free-ranging wild C. aethiops‘‘nonstressed’’ controls for this study were killedinstantly by a marksman. Two of them were dom-inant males, a status that implies comparativelyhigh plasma cortisol levels.7,8 The circulating cor-tisol (mean 391 6 115 against 530 6 52, respec-tively) and prolactin (mean 147 6 67 against 86 615, respectively) levels in these four free-rangingcontrol males were similar to those in wild-caughtmonkeys on day 2 postcapture (Table 1). This sug-gests that the high mean cortisol concentration inthe nonstressed controls was influenced by stressfactors unrelated to capture, which was the focusof this study.

Cortisol plays a role in the protein balance of thebody.1 During a stressful period, sustained hyper-cortisolemia activates protein catabolism and coun-teracts the action of insulin on glucose.2 This so-matic response may explain the significant loss ofbody weight during the early period in captivity of

group III monkeys (range of weight loss 38–50%),despite the provision of food ad lib.

Serum prolactin concentrations were low in thefour control monkeys and during the first 2 dayspostcapture in the wild-caught monkeys. Mean pro-lactin concentration increased progressively duringconfinement, confirming earlier reports of elevatedprolactin after capture in mangabey monkeys andpatas monkeys as well as in rhesus monkeys at-tached to a tethering device.7

Some stressed animals develop gastric mucosalhemorrhage and edema shortly after the onset ofstress. In the reindeer, abomasal ulcers developwithin 4 hr of capture.20 In the C. aethiops in ourstudy, acute superficial gastric mucosal lesions oc-curred within 24 hr of capture.

Increased adrenal gland weight has frequentlybeen associated with chronic stress and is primarilydue to hypertrophy of the adrenal cortex.9,20 In thepresent study, the enlarged adrenals in the wild-caught animals housed singly for 45 days confirmthat the capture and restraint of newly capturedmonkeys is stressful.

24 JOURNAL OF ZOO AND WILDLIFE MEDICINE

CONCLUSIONS

Capture and confinement are associated with sig-nificant stress in free-ranging wild primates. Mu-cosal hemorrhage and edema may develop in thestomach of African green monkeys within 24 hr ofcapture. Levels of circulating cortisol are inverselycorrelated with stomach lesions, suggesting thathigh hormone concentrations are not necessarily as-sociated with pathology.

Circulating prolactin concentration comparedwith cortisol rises gradually over time, whereas thelatter increases sharply.

Although free-ranging wild African green mon-keys are probably under continuous environmentalpressure and threat from predators, they are notfound to have elevated circulating cortisol and pro-lactin levels or stress-induced lesions in the stom-ach or enlarged adrenal glands.

Acknowledgments: We thank Prof. Hans-ErikCarlsson and Dr. Faisal Guhad for support. Mr. Ti-tus Ochieng Apindi and Mr. Tharcisse Ukizintam-bara provided technical assistance. The project waspartly supported by the Reproductive Biology Unit,University of Nairobi and the Human ReproductionProgramme, World Health Organization.

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Received for publication 2 April 2001