Stress in cynomolgus monkeys ( Macaca fascicularis ) subjected to long-distance transport and simulated transport housing conditions

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Stress in cynomolgus monkeys (Macaca fascicularis) subjected to long-distance transport and simulated transport housing conditionsA. L. Fernström ab; W. Sutian ac; F. Royo a; K. Westlund d; T. Nilsson a; H. -E. Carlsson a; Y. Paramastri c; J.Pamungkas c; D. Sajuthi c; S. J. Schapiro e; J. Hau ab

a Department of Neuroscience, Comparative Medicine, Uppsala University, Uppsala, Sweden b Department ofExperimental Medicine, University of Copenhagen and State Hospital, The Panum Institute, 2200 NCopenhagen, Denmark c Primate Research Centre, Bogor Agricultural University, Bogor, 16151, Indonesia d

The Swedish Institute for Infectious Disease Control, Stockholm, Sweden e The University of Texas M. D.Anderson Cancer Center, Bastrop, TX, USA

First Published:2008

To cite this Article Fernström, A. L., Sutian, W., Royo, F., Westlund, K., Nilsson, T., Carlsson, H. -E., Paramastri, Y., Pamungkas, J.,Sajuthi, D., Schapiro, S. J. and Hau, J.(2008)'Stress in cynomolgus monkeys (Macaca fascicularis) subjected to long-distancetransport and simulated transport housing conditions',Stress,11:6,467 — 476

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ORIGINAL RESEARCH REPORT

Stress in cynomolgus monkeys (Macaca fascicularis) subjectedto long-distance transport and simulated transport housing conditions

A. L. FERNSTROM1,5, W. SUTIAN1,3, F. ROYO1,†, K. WESTLUND2, T. NILSSON1,

H.-E. CARLSSON1, Y. PARAMASTRI3, J. PAMUNGKAS3, D. SAJUTHI3, S. J. SCHAPIRO4, &

J. HAU1,5

1Department of Neuroscience, Comparative Medicine, Uppsala University, BMC Box 572, 75123 Uppsala, Sweden,2The Swedish Institute for Infectious Disease Control, Stockholm, Sweden, 3Primate Research Centre, Bogor Agricultural

University, Jalan Lodaya II/5, Bogor, 16151, Indonesia, 4The University of Texas M. D. Anderson Cancer Center, Bastrop,

TX USA, and 5Department of Experimental Medicine, University of Copenhagen and State Hospital, The Panum Institute,

Blegdamsvej 3B, 2200 N Copenhagen, Denmark

(Received 24 July 2007; revised form 27 December 2007; accepted 8 January 2008)

AbstractThe stress associated with transportation of non-human primates used in scientific research is an important but almostunexplored part of laboratory animal husbandry. The procedures and routines concerning transport are not only importantfor the animals’ physical health but also for their mental health as well. The transport stress in cynomolgus monkeys (Macacafascicularis) was studied in two experiments. In Experiment 1, 25 adult female cynomolgus monkeys were divided into fivegroups of five animals each that received different diets during the transport phase of the experiment. All animals weretransported in conventional single animal transport cages with no visual or tactile contact with conspecifics. The animals weretransported by lorry for 24 h at ambient temperatures ranging between 208C and 358C. Urine produced before, during andafter transport was collected and analysed for cortisol by enzyme-linked immunosorbent assay (ELISA). All monkeysexhibited a significant increase in cortisol excretion per time unit during the transport and on the first day following transport.Although anecdotal reports concerning diet during transport, including the provision of fruits and/or a tranquiliser, wasthought likely to influence stress responses, these were not corrobated by the present study. In Experiment 2, behavioural datawere collected from 18 cynomolgus macaques before and after transfer from group cages to either single or pair housing, andalso before and after a simulated transport, in which the animals were housed in transport cages. The single housed monkeyswere confined to single transport cages and the pair housed monkeys were kept in their pairs in double size cages. Both pairhoused and singly housed monkeys showed clear behavioural signs of stress soon after their transfer out of their group cages.However, stress-associated behaviours were more prevalent in singly housed animals than in pair housed animals, and thesebehaviours persisted for a longer time after the simulated transport housing event than in the pair housed monkeys. Our dataconfirm that the transport of cynomolgus monkeys is stressful and suggest that it would be beneficial for the cynomolgusmonkeys to be housed and transported in compatible pairs from the time they leave their group cages at the source countrybreeding facility until they arrive at their final laboratory destination in the country of use.

Keywords: Behaviour, macaque, pair-housing, primate, urinary cortisol, welfare

Introduction

There is a strong scientific case for maintaining work on

non-human primates (NHPs) as models in carefully

selected research problems (Weatherall 2006), but

there is a critical shortage of captive-bred monkeys, in

particular Old World species, for use in biomedical

research (Cohen 2000; National Research Council

2003; Hau and Schapiro 2006). The present

inadequate supply of captive-bred NHPs in the USA

ISSN 1025-3890 print/ISSN 1607-8888 online q 2008 Informa USA, Inc.

DOI: 10.1080/10253890801903359

†Present address: Unit of Cytogenomics, CICBIOGUNE, Bizkaia Science Park, Derio, Spain

Correspondence: J. Hau, Department of Experimental Medicine, University of Copenhagen and State Hospital, 3 Blegdamsvej,2200 N Copenhagen, Denmark. Tel. þ45 35 32 73 63. Fax. þ45 35 32 73 99. Mobile: þ45 28 75 73 63. E-mail: [email protected]

Stress, November 2008; 11(6): 467–476

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and Europe necessitates the import of NHPs from

source countries in Asia and Africa. The use of primates

in scientific research is not expected to decrease

considerably in the coming years and the growing

demand in the USA and Europe will become less and

less attainable by production from domestic sources

(Carlsson et al. 2004; Weatherall 2006). As mentioned

in Weatherall (2006) report and recommended by Hau

and Schapiro (2006) establishment of accredited and

expert staffed NHP research laboratories in source

countries would benefit animal welfare and global

research. This is, however, a slow process and long

distance transportation of primates will thus continue,

and is associated with a number of animal welfare issues

(Honess et al. 2004; Guidelines for the Humane

Transportation of Research Animals 2006).

The typical transport event for a research NHP

approximates the following pattern. Shortly before

shipment, animals are removed from their social

groups (usually breeding groups) and placed in more

typical laboratory housing (single caging or perhaps

pair caging) for a period of pre-shipment monitoring

and conditioning. Following this period of pre-

shipping conditioning, the primates are normally

transported singly in small cages. Transport is

logistically complex, and the animals may travel

and/or wait for many hours under varying climatic

conditions. When the NHPs finally arrive at the

destination facility in a new country, they are again

quarantined – often in single cages. Long distance

transportation across multiple climate and time zones

is likely to induce substantial stress in the transported

animals (Wolfensohn 1997; Hau and Schapiro 2004;

Honess et al. 2004; Prescott and Jennings 2004).

While the effects of transport on farm animals have

been reasonably well investigated (Guide for Livestock

Exporters 1997), there have been few systematic

assessments of the impact of transportation on NHPs

(Wolfensohn 1997), although empirically derived

guidelines for the acquisition, care and breeding of

NHPs have been published by International Primato-

logical Society (IPS 2007). Monkeys caught from the

wild and confined to caging exhibit pronounced stress

(Moinde et al. 2004; Suleman et al. 2004), and

haematological parameters do not settle at new stable

values until after 6 months (Kagira et al. 2007).

However, little empirical data are available that identify

specific stress effects of transport in the literature,

although two recent publications have quantified

acclimation effects in NHPs brought into the

laboratory. Kim et al. (2005) have shown that

cynomolgus monkeys exhibited an increase in the

neutrophil-to-lymphocyte (N/L) ratio upon arrival

at their new laboratory. This response normalised a

week after arrival, suggesting a stress response to the

transportation. Similarly, Capitanio et al. (2006)

recently reviewed the effects of housing changes on a

variety of physiological parameters, and suggested that

captive NHPs may take up to 90 days to adapt to new

housing conditions, depending on the level of change

they experienced.

Provision of natural food items allows the animals to

focus on processing these and is generally rec-

ommended as an enhancement of environmental

complexity (Reinhardt 2002). Boinski et al. (1999)

demonstrated that access to a variety of foraging

enrichment positively affects behavioural and physio-

logical responses to stress and enhances psychological

well-being in singly housed brown capuchins. Conse-

quently, it may be possible to lower the stress

perception of transport in single housed monkeys by

providing them with different fruits and vegetables

during their journey.

It is well recognised that membership in a

compatible group of conspecifics provides a sense of

security for NHPs (European Council 2002). Social

living also provides opportunities for a wide range of

positive species-specific social activities (Jolly 1985;

Cheney et al. 1987). For instance, it has been

demonstrated that social grooming has a relaxing

effect on the animal receiving grooming, lowering its

heart rate (Boccia et al. 1989). Similarly, cage mates

seem to serve as a buffer against stress in NHPs

subjected to experimental procedures (Mason 1960;

Coe and Franklin 1982; Coelho et al. 1991). It is thus

likely that pair housing during shipment of primates

may reduce some of the stress-related responses

commonly observed during and after transportation.

Cynomolgus monkeys constitute one of the most

important NHP species in biomedical research – 54%

of all NHPs used in the UK in the mid-1990s (Owen

et al. 1997) – and the use of this species is increasing

(Carlsson et al. 2004). Cynomolgus monkeys seem,

however, to exhibit more pronounced stress responses

to confinement in transport cages than do rhesus

monkeys (Macaca mulatta) or bonnet macaques

(Macaca radiata) (Clarke et al. 1988). The present

experiments were designed to quantify physiological

stress responses to transport and to assess the effects of

one manipulation (simulated transport housing in

single vs. pair enclosures) on behavioural measures

potentially indicative of stress. In both experiments,

the duration of the transport episode was chosen to

represent a typical transport scenario from South East

Asia to Europe or the USA.

The aims were addressed through two experiments

to (i) examine the urinary cortisol excretion as a

measure of stress in cynomolgus monkeys subjected

to standard transport conditions in standard single

cages and (ii) to analyse the behavioural response

A. L. Fernstrom et al.468

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of cynomolgus monkeys to transfer to single and pair

caging from group caging, and whether animals who

were subjected to a quarantine period and simulated

transport housing conditions in pairs behaved differ-

ently from singly housed animals subjected to the same

procedures.

Materials and methods

Both of the experiments described in this paper were

conducted at the quarantine facility of the Primate

Research Center (PRC) of Bogor Agricultural Univer-

sity, Java, Indonesia.

Experiment 1

Twenty-five captive bred (captive bred parents) adult

(6–7 years old) female cynomolgus monkeys (Macaca

fascicularis) obtained from New Inquatex (Rumpin,

Indonesia) were randomly divided into five groups of

five individuals each (R0–R4). Except during trans-

port, all animals were kept indoors in single cages

under quarantine conditions under ambient con-

ditions with approximately 12 h of daylight and 12 h of

darkness for 90 days to allow them to habituate to

single housing. The cages (w: 0.39 m, l: 0.39 m and h:

0.75 m), were made of stainless steel, and were located

on an upper or a lower tier of a two-tiered caging

system. All subjects had visual, auditory, and olfactory

contact with the other monkeys in the same room. The

cages were cleaned by hosing the tray under the cage

twice daily at 8 a.m. and 1 p.m. Fresh water bottles

were supplied at both cleaning times.

During transport, the temperature ranged from

258C to 358C and humidity ranged from 75% to 85%.

Although UK and USA recommendations advocate a

temperature range between 158C and 258C, the

transport in source countries is often performed in

lorries with no means of climate control. All groups of

animals were fed a commercial diet (Monkey chow,

Charogen, Phokphan, Thailand) supplemented with

fruit (banana and guava) and water was available ad

libitum. For the purposes of this study, the maintenance

diet is defined as the combination of the commercial

monkey chow, banana and guava. The space under the

wire mesh bottoms of the cages was modified to

separate faeces and urine from the individual animals.

During that time, they were fed the maintenance diet,

and water was available ad libitum. Two days prior to

transport, each group of monkeys was adapted to the

diet they were to be fed during transport:

Group R0 received only monkey chow.

Group R1 received the maintenance diet: monkey

chow, banana and guava.

Group R2 received the maintenance diet (monkey

chow, banana and guava) plus multi-vitamins.

Group R3 received sugar cane, banana, guava, apple

and orange.

Group R4 received the maintenance diet plus multi-

vitamins and a tranquilizer (Acepromazine Maleat

Injectione, 0.4 mg/kg body weight, intra-muscular

injection (Boehringer Ingelheim Vetmedica, Inc.,

St Joseph, MO, USA).

The monkeys were subjected to transport by lorry for

24 h. They were transported in standard transport

cages (IATA, 2007; w: 0.24 m, l: 0.4 m and h: 0.5 m)

made of plywood (Figure 1). After transportation, the

monkeys were returned to their home indoor single

cages under quarantine conditions, fed the mainten-

ance diet and provided with water ad libitum. Urine

was collected at 8 h intervals, from 16 h before

transport started, during the 24-h transport, and for

32 h after transport ended. The samples were stored

frozen at 2208C until analysed.

Quantification of cortisol. Urine samples were thawed,

centrifuged (1000g for 2 min) and the supernatants

were analysed without extraction. Cortisol was

measured with a pan-specific cortisol ELISA kit

(DRG Diagnostics, Marburg, Germany). The inter-

and intra-assay coefficients of variation were 9.3% and

1.9%, respectively.

Experiment 2

Subjects included 18 female cynomolgus monkeys from

the semi-natural breeding facility on Tinjil Island,

Indonesia (Kyes 1998). Prior to the project, the subjects

were housed at the PRC for 2 months in 32 m2

Figure 1. A typical transport box consisting of two single cage

units. The centre wall of the unit was removed to accommodate pair

housed monkeys.

Transport stress in cynomolgus monkeys 469

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group cages together with other (10–20) cynomolgus

monkeys. The subjects’ ages ranged from 2.5 to 5.5

years and body weights ranged from 1.6 to 2.1 kg.

The experimentbeganwhenthemonkeyswere removed

from their group cages and placed in either single or pair

cages in the indoor quarantine room. Room tempera-

ture and relative humidity varied between 258C and

318C and between 75% and 85%, respectively.

The quarantine room had no windows and the lights

were on daily between 8 a.m. and 5 p.m. The project

consisted of three phases. Ethological observations were

performed only during phases 1 and 3.

Phase 1: The first 21 days after the monkeys were

transferred from group cages into the PRC’s indoor

quarantine facility.

Phase 2: The transport housing simulation (48 h).

Phase 3: The first 21 days after the monkeys were

returned to their pair or single cages after the

transport housing simulation.

Phase 1 (days 1–21). On day 1, subjects were

transferred from a group cage to a room in the

quarantine facility. Ten monkeys were selected at

random to live in single cages and 10 monkeys were

selected to live in pairs (due to a computer breakdown,

the data from two of the pair-housed monkeys were

lost). Both single and pair cages were of the same size

(w: 0.39 m, l: 0.39 m and h: 0.75 m), were made of

stainless steel, and were located on an upper or a lower

tier of a two-tiered caging system. All subjects had

visual, auditory and olfactory contact with the other

monkeys in the same room. The cages were cleaned by

hosing the tray under the cage twice daily at 8 a.m. and

1 p.m. The monkeys were fed after every cleaning

episode. Monkey chow (Charoen Phokphan, Bankok,

Thailand) was provided in the morning and fresh

fruits or vegetables were given in the afternoon. Fresh

water bottles were supplied at both cleaning times.

Phase 2 (days 22 and 23). A partial transport

simulation was performed by transferring the

subjects to transport cages within the facility,

avoiding potentially confounding effects associated

with real transportation (e.g. noise, vibrations,

temperature changes, odours and air pressure

changes). The transport boxes used for single caged

monkeys were standard transport boxes (Figure 1).

Single transport boxes were modified into pair

transport boxes by simply removing the central wall

between adjoining compartments to accommodate

two monkeys. The single housed monkeys were placed

in single transport boxes and the pair housed animals

were placed in pair transport boxes. The boxes were

put in a separate section of the facility for 48 h without

any contact with other animals. The single and pair

housed monkeys were separated into different, but

identical rooms during this phase of the study.

The monkeys were given water, fruits and vegetables

in the transport boxes at 9 a.m. at the start of the

simulation. Fresh water was provided daily at the usual

times (8 a.m. and 1 p.m.). Some fruit and vegetable

material was still present in the animals’ compartments

at the end of the transport simulation. Since the

monkeys were located in the smaller closed transport

boxes during this period and since we were attempting

to simulate transport conditions, no ethological

observations were conducted during Phase 2.

Phase 3 (days 24–44). After the 48-h transport

housing simulation, the monkeys were returned to

their original positions and social conditions in their

original home cages in the quarantine room and

observed for 21 days.

Behavioural observations

The ethogramme used (Table I) was derived from an

ethogramme designed to detect and monitor stress-

related behaviours in NHPs (Schapiro and Bloom-

smith 1994). An instantaneous time-sampling method

(Altmann 1974) was used to record appetitive,

affiliative and other active and inactive behaviours.

In an attempt to economically, yet representatively,

the sample activity from all of the subjects, behaviours

were recorded at 10 s intervals for 8 min daily

for each monkey. Additionally, since this technique

Table I. The ethogramme used in the study (modified from

Schapiro and Bloomsmith 1994).

Behavioural

categories Definition

Appetitive behaviours Eat, forage and drink

Affiliative behaviours Social groom, given and received

Autogroom Grooming own body

Locomotion Moving the whole body from one spot

to another

Rest/inactivity Sitting still with eyes not focusing

on object or other animal

Watch /exploratory

behaviours

Investigating object or other monkey

Abnormal behaviours Head toss, self plucking, self aggression,

faeces eating, and pacing

Aggressive behaviours Facial threat, approach and physical

aggression

Other behaviours Behaviours other than those described

above

A. L. Fernstrom et al.470

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over-represents states and under-represents events,

important events such as aggressive and abnormal

behaviours, were recorded using 1/0 sampling

(Altmann 1974). All observations were made by one

observer (ALF), and conducted between 8 a.m. and 3

p.m. and subjects for observation were randomised on

the first day (using Randomizer available at www.ran-

domizer.org), after which the order of observation

remained unchanged throughout the study. However,

observations for all subjects were evenly distributed

across time intervals during the daily observation

period. The monkeys were observed for a total of

101 h in the two phases (1 and 3).

Statistics

In Experiment 1, statistical analyses were performed

using SPSS v 12.0.1. Effect of transportation and diet

were tested by a mixed-model ANOVA with diet as a

between-subjects factor and time point (9 over the

3 days) as a within-subjects factor. The raw data were

not normally distributed and were subjected to a

square root transformation, normalising the data, for

analysis.

In Experiment 2, the data presented in results part

A (means of behavioural responses to caging and

transport simulation) were analysed with Fisher’s

exact test. The data presented in results part B

(adaptation to single and pair caging and effects of

transport housing simulation) were not normally

distributed and were analysed using Kruskal–Wallis

tests. P-values , 0.05 were considered significant for

all analyses.

Ethical approval

The protocols of the present studies were approved by

the Institutional Animal Care and Use Committee of

Bogor Agricultural University, Java, Indonesia.

Results

Experiment 1

There was a main effect of time (before vs. during vs.

after) on urinary cortisol concentration that increased

in all monkeys during and after transportation

(P ¼ 0.002) (Figure 2). Using polynomial contrast

analyses to compare groups R0, R1, R2, and R3 with

group R4, the last group had a significantly lower

concentration of cortisol in urine than the monkeys of

groups 1, 2, and 3 (P , 0.001). However, when

corrected for variation in urine volume voided, no

significant differences were found.

Experiment 2

Behavioural responses to caging and transport simulation

Appetitive behaviours. The singly housed monkeys

consistently spent more time engaged in appetitive

behaviours than did the pair housed animals during

both Phases 1 and 3 (Fishers test, two-tailed: P-values

,0.001). Little time was allocated to appetitive

behaviours on the first day of Phase 1. The monkeys

appeared to spend an increasing proportion of time in

appetitive activities throughout the entire observation

period.

Allogrooming. The pair housed monkeys spent on

average 1% of their time allogrooming during the

observation period. There was no adaptation effect

over-time during Phases 1 and 3 was no effect of the

transport housing simulation on allogrooming activity.

Autogrooming. The proportion of time spent in

autogrooming was significantly greater (Fishers test,

two-tailed: P , 0.001) among the pair housed

monkeys compared with that of the single housed

animals (Figure 3).

Watch/exploratory behaviour. The pair housed mon-

keys spent on average 58%, and the single housed

monkeys 68%, of their time engaged in exploratory/-

watch activity during the observation period. This

difference was not significant.

Figure 2. Urinary concentrations of cortisol in cynomolgus

monkeys fed different diets before, during and after transport by

lorry for 24 h. The transport period is represented as a solid line on

the x-axis. The diets were started 48 h before transport and

maintained until the end of the 24-h transport: group R0 received

only monkey chow; group R1 received the maintenance diet:

monkey chow, banana and guava; group R2 received the

maintenance diet (monkey chow, banana and guava) plus multi-

vitamins; group R3 received sugar cane, banana, guava, apple and

orange and group R4 received the maintenance diet plus multi-

vitamins and a tranquiliser. n ¼ 5 per group. Values are group means

^ SEM. Urinary cortisol concentration increased in all groups

during and after transportation (P ¼ 0.002).

Transport stress in cynomolgus monkeys 471

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Locomotion. The proportion of time spent in

locomotor activity was significantly higher (Fishers

test, two-tailed: P , 0.001) among the single housed

monkeys than among the pair housed animals

throughout the observation period. This was particu-

larly evident during the first several days after removal

from the group cages and placement in the indoor

quarantine room.

Aggressive behaviours. Aggressive behaviours were

almost exclusively threats directed against the obser-

ver.1 The frequency among the single housed monkeys

was significantly higher (Fishers test, two-tailed:

P , 0.001) than that of the pair housed monkeys

(Figure 4). The frequency of recorded aggressive

behaviours during the first two days after the transfer

to quarantine caging was significantly higher than the

rest of the period.

Abnormal behaviours. The average mean frequency

of abnormal behaviours recorded among the single

housed monkeys was 24.7 and 17.7 events/observation

hour among the pair housed monkeys throughout the

observation period. This difference was not statisti-

cally significant.

Adaptation to single and pair caging and effects of

transport housing simulation

Single housed monkeys. Time budgets were com-

pared for single housed monkeys during different time

periods: the early transition from group caging to

quarantine caging (days 1–2; Figure 5A); after

adaptation to the single cage (days 20–21;

Figure 5B); immediately after the transport simulation

(days 24–25; Figure 5C); and at the end of the study

(days 43–44; Figure 5D).

There were significant differences in the amount of

time spent in appetitive behaviours across the four time

periods (Friedman’s ANOVA: P , 0.05, U: 14.217).

Appetitive behaviour increased significantly (Wil-

coxon:P , 0.05) in the timeperiod after thequarantine

caging. At the end of the observation period, appetitive

Figure 3. Proportion of time spent in autogrooming by cynomolgus monkeys before and after simulated transport housing. Each monkey

was observed for 8 min daily between 8 a.m. and 3 p.m. Values are group mean percentage ^ SEM; n ¼ 10 single, 8 pair-housed.

The proportion of time spent in autogrooming was significantly greater (Fisher’s test, two-tailed: P , 0.001) among the pair housed than

among the single housed monkeys.

Figure 4. The frequency of aggressive behaviours in cynomolgus monkeys before and after transport housing simulation. Monkeys were

caged singly or in pairs. Each monkey was observed for 8 min daily between 8 a.m. and 3 p.m. Values are group mean number of events per day

þ SEM; n ¼ 10 single, 8 pair-housed. The frequency of aggressive behaviour was significantly greater (Fisher’s test, two-tailed: P , 0.001)

among the single housed than among the pair housed monkeys.

A. L. Fernstrom et al.472

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behaviours had increased significantly compared with

the time just after arrival (Wilcoxon: P , 0.05) in

quarantine and the time just after the transport housing

simulation (Wilcoxon: P , 0.05). There were signifi-

cant differences in the amount of time spent in

locomotor behaviours across the four time periods

(Friedman’s ANOVA: P , 0.05, U: 16.408). Loco-

motor behaviours decreased significantly after the

transfer to the quarantine room (Wilcoxon P , 0.05)

and increased again after the transport simulation

(Wilcoxon P , 0.05). Rest/inactive behaviours

occurred infrequently during the study.

Pair housed monkeys. Time budgets were compared

for pair housed monkeys during the different time

periods as above (Figure 6). There were significant

differences in the amount of time spent in appetitive

behaviours across the four time periods (Friedman’s

ANOVA: P , 0.001, U: 17.830). At the end of the

observation period, appetitive behaviours had

decreased significantly compared with the first two

days after arrival in the indoor pair cages (Wilcoxon

P , 0.05).

There were significant differences in the amount of

time spent in locomotor behaviours across the four

time periods (Friedman’s ANOVA: P , 0.05, U:

9.160). At the end of the observation period, the

proportion of time spent in locomotor behaviours had

decreased significantly (Wilcoxon P , 0.05).

Discussion

Experiment 1 confirmed that standard single cage

transport is associatedwithan increasedurinary cortisol

excretion in the cynomolgus monkey, indicating HPA

axis activation. These data are taken to be indicative of a

stress response in these monkeys subjected to lorry

transport for 24 h. In the present study, the addition of

fruit treats, multi-vitamins, and/or tranquilisers had no

significant effect on urinary cortisol concentration

during transport, indicating that the provision of

various fruits and vegetables probably does not affect

the monkeys’ perception of, or response to the transport

stressor.

The second part of this study (Experiment 2) was

designed to mimic the sequence of cage changes

commonly experienced by monkeys produced in

South East Asia when they are designated for use in

research, and are subsequently transported from the

breeder to a laboratory in Europe or the US. In order

to minimise confounding variables and disturbances,

urine or faeces were not collected for cortisol

measurement in this behavioural study.

An absence of appetitive activities (anorexia) is a

sign of severe distress among captive NHPs (Morton

and Griffiths 1985). The trend toward spending an

increasing proportion of time in appetitive activities

observed for all monkeys in the present study may

indicate that they were adapting successfully to the new

Figure 5. Time-budgets for behaviours before and after transport housing simulation for the single housed macaques. (A) The early

transition from group caging to quarantine caging (days 1–2). (B) After adaptation to the single cage (days 20–21). (C) Immediately after the

transport simulation (days 24–25). (D) At the end of the study (days 43–44); n ¼ 10 monkeys.

Transport stress in cynomolgus monkeys 473

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conditions (habituation). The levels of allogrooming

activities were low in the present study. Judge and

De Waal (1997) described how levels of all behaviours

increased – aggressive as well as affiliative – between

female rhesus monkeys when subjected to crowding,

and there was no indication in the allogrooming results

from the present study to suggest stress from pair

housing caused by space constraints. Autogrooming is

essential for the wellbeing of all non-human primates

and an animal displaying an absence of autogrooming

is a general cause of worry for animal care staff.

However, “over-grooming” may also be indicative of

distress. The low autogrooming activity of the singly

housed animals compared with the pair housed

monkeys contrasts with our previous studies (personal

observation) and may suggest that even the pair

housed animals in the present study experienced

substantial stress.

In the wild, cynomolgus monkeys have a home

range size of about 1.25 km2 and daily path length

varies greatly between 150 and 1900 m (Wheatley

1980; Rowe 1996), and locomotor activity in semi-

wild conditions and large group cages is much greater

than in small cages (personal observation). In the

present study, opportunities for expressing natural

locomotion were inhibited during the entire study due

to the small cages. However, the singly housed

monkeys spent a higher proportion of their time

engaged in locomotor behaviours than did the pair

housed monkeys.

The presence of aggressive behaviours among

captive NHPs may be a sign of stress (Heinz 1999).

The present results on aggression suggest that the pair

housed animals were less stressed than the single

housed monkeys throughout the observation period.

It is possible that the results indicate that the initial

transfer from group caging to quarantine caging was a

more stressful event than was the transfer to and

period in the transport cages. However, information

about the baseline behaviour before transfer to the

quarantine caging is not available, and a more likely

reason for the frequent aggressive bouts during the

first several days of the study was due to the presence

of the observer, to which the monkeys were not yet

accustomed. The transport housing simulation did

result in a modest increase in aggressive behaviour,

although the monkeys at that point in time had

habituated somewhat to the new cages. Among the

single housed monkeys the aggressive behaviours

remained elevated for a longer period of time

compared with those of the pair housed animals in

which aggressive behaviours decreased rapidly and

reached a baseline faster than in the single housed

animals. This seems to support the interpretation that

a companion animal functions as a buffer against

stress (Mason 1960; Coe and Franklin 1982; Coelho

et al. 1991).

There was no significant difference in the abnormal

behaviours between single housed and pair housed

monkeys, and they were present throughout the

Figure 6. Time-budgets for behaviours before and after transport housing simulation for the pair housed macaques. (A) The early transition

from group caging to quarantine caging (days 1–2). (B) After adaptation to the single cage (days 20–21). (C) Immediately after the transport

simulation (days 2425). (D) at the end of the study (days 43–44); n ¼ 8 monkeys.

A. L. Fernstrom et al.474

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observation period. However, the presence of abnor-

mal behaviour such as stereotypies is interpreted to

reflect a reduced level of well-being and an inadequate

environment (Maple 1979; Bayne et al. 1992; Broom

and Johnson 1993; Olfert et al. 1993; IPS 2007).

In conclusion, Experiment 1 confirmed that trans-

port procedures result in activation of the HPA axis as

measured by urinary cortisol, and that the provision of

food treats and/or a tranquiliser associated with the

transport event had no significant influence on the

monkeys’ stress responses. Experiment 2 demonstrated

that both pair housed and singly housed monkeys

showed behavioural signs that may indicate elevated

stress levels just after transfer from group cages to

single- and pair-housing in indoor rooms. However,

stress-associated behaviours were in general more

prominent in singly housed than in pair housed

monkeys, and these behaviours remained consistently

more prominent throughout the observation period.

Although transport simulation, which in this case was

limited to 48 h in quiet, unmoved transport boxes,

seems to be a stressful event, it may not be as stressful as

the initial transfer from a group cage to a smaller single

or pair cage as judged by the behaviour of the animals in

the present study. At the time of the transport

simulation, the monkeys had had three weeks to adjust

to their new caging. This suggests that it may be

valuable for formerly group housed monkeys destined

for long distance transportation to spend some time in

cages, simply for the purpose of habituation, prior to

shipment. However, it has been described that

maintaining NHPs in extended social groups until

immediately prior to transportation may lower the

overall stress (magnitude and duration) associated with

transferringagroupofmonkeys fromonesocialgroupin

the source country to the same social grouping in the

receiving laboratory (Wolfensohn and Honess 2005).

Based on most of the behavioural data in the present

study, transport simulation seemed more stressful for

monkeys kept singly in transport cages compared with

monkeys kept housed in 3-week-old pairs. Also, after

transportation, the stress-related behaviours, with the

exception of the autogrooming results, were more

prominent in the single housed than in the pair housed

monkeys. However, the transport simulation of the

present study only involved the subjects being placed in

transport cages in quiet surroundings and not subjected

to noise, movements of the cage, changes in tempera-

ture and humidity, which they would experience during

real transport. The present approach was chosen in

order to avoid an over-abundance of confounding

variables in a single study. A real transportation episode

is thus likely to be more stressful.

Primates showphysiological and behavioural distress

reactions when exposed to life-threatening situations

over which they have no control (Reinhardt 2002).

Therefore, being removed from a familiar environment

and placed in a strange one may cause distress for the

animals (Hau and Schapiro 2004). Being housed with

another or other member(s) of the same species is

recognised as the single most important enrichment

practice for NHPs (Hau and Schapiro 2004).

The present study supports the contention that single

housed monkeys exhibit more stress-related behaviour

than pair housed monkeys. Our data suggest that it

would be beneficial for cynomolgus monkeys to be

housed and transported in compatible pairs from the

time they leave the group cages at the breeder until they

arrive at their final destination in the new laboratory.

Acknowledgements

This study was generously financed by the Swedish

National Board for Laboratory Animals (CFN).

We are grateful for the help provided by Diah Ariyanti,

and the staff at the quarantine facility of Primate

Research Center of Bogor Agricultural University.

Ingegerd and Viking Olof Bjork are acknowledged for

funding parts of Anna-Linnea Fernstrom’s travel

costs.

Note

1. Only on very rare occasions were threats observed to be aimedat other animals.

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