The Impact of Transportation on Physiological and Behavioral Parameters in Wistar Rats:Implications for Acclimatization PeriodsJohanna W. M. Arts, Klaas Kramer, Saskia S. Arndt, and Frauke Ohl

Johanna W. M. Arts, BSc, MSc, is a PhD student in the Department of Animals in Science & Society, Division of AnimalWelfare & Laboratory Animal Science, Veterinary Faculty, Utrecht University, Utrecht, NL, as well as a research scientistat Harlan Laboratories BV, NL. Klaas Kramer, PhD, is an associate professor in the Department of Animals in Science& Society, Division of Animal Welfare & Laboratory Animal Science, Veterinary Faculty, Utrecht University, Utrecht, NL,as well as the Animal Welfare Officer in the Department of Safety and Environmental Affairs, Free University,Amsterdam, NL. Saskia S. Arndt, PhD, is an assistant professor in the Department of Animals in Science & Society,Division of Animal Welfare & Laboratory Animal Science, Veterinary Faculty, Utrecht University, Utrecht, NL. FraukeOhl, PhD, is a professor in the Department of Animals in Science & Society, Division of Animal Welfare & LaboratoryAnimal Science, Veterinary Faculty, Utrecht University, Utrecht, NL

Address correspondence and reprint requests to Johanna W.M. Arts, BSc, MSc, Laboratory Animal Science, UtrechtUniversity, P.O. Box 80.166, 3508 TD, Utrecht, NL, or email jarts@harlan.com / j.arts@uu.nl.

Abbreviations that appear >3x throughout this article: ACT, activity, as measured by telemetry device; ANOVA, analysisof variance; BS, blood sample; BW, body weight; ΔBW, body weight gain; CK, creatine kinase; CORT, corticosterone;GRO, self-grooming; HR, heart rate; ip, intraperitoneal; LOC, locomotor activity, as scored by behavioral observations;MAP, mean arterial pressure; SD, standard deviation; SI, social interaction.

Abstract

Transportation of laboratory rodents unavoidably causes stress. Nevertheless, very little is known about the effects of transportationand how long it takes for the animal to recuperate. In the present study, we investigated physiological and behavioral parametersbefore and after transportation in both transported and nontransported animals. We took blood samples to analyze plasmacorticosterone and creatine kinase, and performed physiological measurements by means of telemetry, measuring heart rate, bloodpressure, and activity. Behavior was measured by means of home cage observations. This study revealed that plasma corticosteronelevels increased at least up to 16 days after transportation, blood pressure and heart rate showed a lasting decrease aftertransportation, grooming increased, and social interactions and locomotor activity decreased after transportation. With these data wedemonstrate that there is a long-lasting effect of transportation on physiological and behavioral parameters. Our results show that thestressful impact of transportation embraces all parts of the procedure, including for example the packing of the animals. Researchersmust be aware of this impact and provide a sufficient acclimatization period to allow for the (re-)stabilization of parameters.Insufficient acclimatization periods endanger not only the reliability of research results but also the welfare of the animal used.

Key Words: acclimatization; behavior; corticosterone; rats; rodents; telemetry; transportation

Introduction

Every day thousands of laboratory rodents are being transported all over the world. Especially common are transport betweeninstitutes (e.g., from a breeding to a research facility, which may last several days) and long distance transportation, which can beexpected to induce significant stress in animals (Swallow et al. 2005). However, evidence rarely exists about the effects oftransportation on the animals’ physiology and behavior or how long it takes for the animals to recuperate (Ruiven et al. 1998;Stemkens-Sevens et al. 2009). To obtain reliable scientific results from experiments using animals, it is necessary to normalize andstabilize their physiological status to a condition that can be defined as baseline. Using stressed animals is likely to result inconsiderable and unintended effects on research results (Obernier and Baldwin 2006).

Clear-cut legal recommendations concerning acclimatization periods for experimental animals do not exist. Article 5 of Appendix A ofthe European Convention for the Protection of Vertebrate Animals Used for Experimental and Other Scientific Purposes (ETS no.123) (Council of Europe 2006) states the following: “…even if the animals are seen to be in sound health it is good husbandry forthem to undergo a period of acclimatization before being used in a procedure. The time required depends on several factors, such asthe stress to which the animals have been subjected which in turn depends on several factors such as the duration of thetransportation and the age of the animal. This time shall be decided by a competent person.” Most experiments make use of anacclimatization period after transport to decrease the influence of transportation-related stress on results. However, the duration ofthis acclimatization period is scarcely based on scientific research, and it varies tremendously between facilities (from 1 day to asmuch as 3 weeks).

During a transportation procedure, many different stressors are present such as confinement, novelty, changes in temperature andlight condition, and exposure to new animal caretakers and procedures. In general, animals subjected to such stressors react withchanges in their physiology including body weight (BW ), hormone levels, heart rate (HR ), body temperature, and blood pressure(Capdevila et al. 2007; Obernier and Baldwin 2006; Ruiven et al. 1998; Stemkens-Sevens et al. 2009). Furthermore, severalinvestigators have found deteriorating effects of transportation on the immune system (Aguila et al. 1988; Drozdowicz et al. 1990)and on nutritional parameters (Ruiven et al. 1998). In general, stress is an adaptive response to stressful stimuli (stressors) thatdisrupts animals’ physiological balance (Stokes 2000). Activation of the hypothalamic-pituitary-adrenal axis results in the release ofstress hormones such as corticosterone (CORT ) from the adrenal glands (Herman et al. 2005; McEwen 2000). Elevated levels ofCORT are known to affect a variety of physiological parameters such as blood pressure, HR, immune system function, andbehavioral patterns (Drozdowicz et al. 1990; Harper et al.1996; Wood et al. 2003). Although long-lasting (chronic) stress may result inpathological conditions (Kloet et al. 2005; Marin et al. 2007; Sapolsky 2003), acute stress responses mostly are characterized by anadaptive value (i.e., preparing an individual to adequately respond to a challenging situation) (Marin et al. 2007; Ohl et al. 2008). Aftertermination of the stressful situation, the physiological and behavioral pattern returns to homeostasis within several hours (Kloet et al.2005).

Notably, it has been reported that an individual’s baseline condition (homeostasis) after stress may be different from that beforestress, a phenomenon referred to as allostasis (McEwen 2002). Broom (1986) defines animal welfare as “its state as regards itsattempts to cope with its environment.” Does this coping mean return to an internal set-point (homeostasis)? McEwen (2002)proposed that allostasis is a better term than homeostasis for physiological coping mechanisms because allostasis, which literallymeans “stability through change,” has the potential to replace homeostasis as the core model of physiological regulation. Thecapacity to change is crucial to physical and mental health and to good animal welfare (Korte et al. 2007).

To our knowledge, the length of time required for laboratory rodents to return to a (potentially new) baseline level after between-institute transportation has not yet been investigated systematically. To gain first insights into this question, we define a transportprocedure in this study as transporting animals from the breeding facility to a second animal facility by van, including all relatedprocedures and changes such as packing procedure, the change of animal chamber, cages, and caretakers. We then comparephysiological and behavioral measurements in male Wistar rats before and after transportation as well as in transported andnontransported animals.

Conventional measurements of physiological parameters often include potentially stressful procedures such as handling,immobilizing, and anesthesia, which may themselves have effects on the animals (Buñag 1983; Irvine et al. 1997; Kurtz et al. 2005;Vliet et al. 2000). Radiotelemetry provides a method to obtain accurate and reliable physiological measurements from conscious,freely moving animals (Kramer and Kinter 2003; Sgoifo et al. 1999) over a longer period of time (Buuse et al. 2001; Harper etal.1996; Kramer 2000; Krohn et al. 2003) and can thus be used to obtain nonconfounded data on physiological changes in laboratoryrats due to transportation (Capdevila et al. 2007). In the current study, we acquired mean arterial (blood) pressure (MAP ), HR, andlocomotor activity (ACT ) by means of radiotelemetry transmitters. In addition to these parameters, we measured home cagebehavior, BW and bodyweight gain (ΔBW ), plasma levels of CORT, and plasma levels of creatine kinase (CK ). We used plasma CKas an indicator for muscle damage (Goicoechea et al. 2008; Sanchez et al. 2002).

The main aim of our study was to investigate the stress effect in laboratory rats due to transportation. Furthermore, we aimed toevaluate the acclimatization period necessary for the animals to (re)establish a (potentially new) baseline status. This knowledge canbe expected not only to decrease the variation in research results in transported animals, resulting in a decrease in the number of

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animals required to obtain valid experimental results, but also to improve the welfare of laboratory rodents by defining the time thatthe animals need to recover from transportation stress.

Materials and Methods

Ethical Note

The scientific committee of the Department of Animals in Science & Society, Utrecht University, The Netherlands, peer reviewed theprotocol of our experiment (DEC-DGK 2008.I.01.004), which the Animal Experiments Committee of the Academic Biomedical Centre,Utrecht, The Netherlands, approved. The Animal Experiments Committee based its decision on “De Wet op de Dierproeven” (TheDutch “Experiments on Animals Act,” 1996) and on the “Dierproevenbesluit” (the Dutch “Experiments on Animals Decision,” 1996).Both documents are available online at: www.vet.uu.nl/nca_nl/legislation or http://wetten.overheid.nl. Furthermore, all animalexperiments followed the “Principles of Laboratory Animal Care” and refer to the Guidelines for the Care and Use of Mammals inNeuroscience and Behavioral Research (NRC 2003.)

Animals and Housing Conditions

Subjects were 108 male rats (HsdCpb:WU; Wistar Unilever, Harlan Laboratories BV, Horst, NL), 6 weeks old (150-160 g) on day -35(Table 1), randomly divided over 36 cages of three animals each. During the first part of the experiment, all animals were grouphoused inside the specific pathogen-free barrier of the breeder in a regular animal chamber at a constant temperature of 20˚C(19.5-22˚C) and humidity of >40% (42-55%). Animals had ad libitum access to food (Harlan Teklad irradiated 18% protein rodent diet2918) and water that was processed (acidified (pH 5.8-6.4), chlorinated (6-8 ppm), softened, and filtered (0.02 µm). A light-darkregime was maintained at 12:12 hours, “lights on” at 6:00 AM, with dimmed lights from 6:00 to 8:00 and 16:00 to 18:00. During thesecond part of the experiment after transportation, the animals of the transported group (group 1) were housed under standardlaboratory conditions of the animal facilities of Utrecht University in a temperature- (22 ± 2˚C) and humidity- (45-50%) controlledanimal chamber. The light-dark regime was maintained at 12:12 hours with lights on at 6:00 AM and no dimmed light period.

Table 1: Schedule of performed procedures

Expday

Group Procedure

-35 All Selection 108 animals and distribution over 36cages of 3 animals

-26,-25 1, 2 Surgery on 1 animal per cage in 18 of the 36cages

-25 –-12

1, 2 Recovery of surgery (transmitter animals)

-20 1, 2 Start telemetry measurements (transmitteranimals)

-9 – -2 1, 2 Baseline recordings of telemetry data intransmitter animals

-8 All Blood sample 1 (baseline):BS1

-7 1, 2, 4 Home cage observation 1 (baseline): OBS1

-1 1, 3 Packing and transportation to holding area

0 1 Transportation to second facility, continuationof telemetry measurements

3 Unpacking, weighing, blood sample 2: BS2,euthanization

1, 2, 4 Blood sample 2 after arrival of group 1: BS2

1, 2, 4 Home cage observation 2: OBS2

6 1, 2, 4 Blood sample 3: BS3

7 1, 2, 4 Home cage observation 3: OBS3

14 1, 2, 4 Home cage observation 4: OBS4

16 1, 2, 4 Blood sample 4: BS4

21 All End of telemetry measurements, euthanization

BS, blood sample; OBS, behavioral observation.

Group composition was maintained throughout the entire experimental period. Cage cleaning and weighing of the animals occurredweekly. All procedures were performed by either the researcher herself or by the animal caretakers at the facilities.

Blood Sampling

A total of four blood samples were taken, always on weekdays 3 to 4 hours after lights on. All 108 animals were sampled. The firstsample was taken 7 days before transportation (baseline: BS1). The second sample was taken on the day of transportation, directlyafter unpacking (BS2). The third sample was taken 7 days after transportation (BS3), and the fourth and last sample was taken 16days after transportation (BS4) (Table 1). Blood was sampled from the tail vein using a sterile razorblade (Gem-star, Solingen,Germany) and lithium heparin-coated microvette tube (Sarstedt, Nümbrecht, Germany) as described by Fluttert and colleagues(2000). The samples were taken in a separate, dedicated room within 2 minutes of retrieving the cage from the rack, never exceedingthe maximal allowed sampling quantity of 8 mg/kg BW/14 days. Samples were stored on ice until centrifugation in a tabletopcentrifuge (Eppendorf 5402, Boom B.V., Meppel) at 4ºC for 10 minutes at 14,000 rpm. Plasma was stored in a -18˚C freezer untilanalysis.

Plasma CORT levels were determined using a commercial radioimmunoassay kit (ImmuChem , MP biomedicals, LLC, Solon, OH)according to the protocol of the supplier. Besides plasma CORT, the samples were analyzed on plasma CK, using a commercial kitfor colorimetry (EnzyChrom , BioAssay Systems, Hayward, CA) according to the protocol of the supplier. Each sample for bothplasma CORT and plasma CK was analyzed in duplicate.

Telemetry

Surgery took place behind the barrier of the breeder (Harlan Laboratories B.V., Horst, NL) thus ensuring that no animals weretransported prior to the study. When the animals were 8 weeks old (average BW 204 g [186-218]), surgery was performed by anexperienced surgical technician on 18 randomly selected animals in a dedicated room in the breeding facility. In 18 cages, oneanimal was implanted with a 7.7-g radiotelemetry transmitter (model TA11PA-C40, Data Sciences International, St Paul, MN).Preoperatively the animals were given analgesia with carprofen (5 mg/kg subcutaneously), which was repeated every 12 hours until2 days after surgery. The animals were anesthetized with an intraperitoneal (ip ) injection of ketamine (75 mg/kg) followed by an ipinjection of médétomedine (Dexdomitor ® 10 mg/kg). During surgery, local analgesia was administered (Lidocaine®, Eurovet AnimalHealth, Cuijk, NL) and the eyes were protected with Duodrops® (Produlab Pharma, Raamsdonkveer, NL). The implantationprocedure was carried out under strictly aseptic conditions as described by Kramer and Kinter (2003) for mice and by Huetteman andBogie (2009) for laboratory rodents, in which the transmitter canulla was directly fixed into the abdominal aorta, and the body of thetransmitter was permanently fixed to the inside of the abdominal muscles. The still unconscious animals were brought back to theanimal chamber to recover. As soon as the animals regained consciousness after the surgical procedure, they were group housedwith their two nonimplanted cage mates.

The 18 cages containing transmitter animals were placed above a receiver station (RPC-1, DSi, St. Paul, MN), and the remaining 18cages were placed in a conventional rack. To prevent detection from adjacent transmitters, all receiver-cage couples were placed in aU-shaped stainless steel profile. After surgery, five of the transmitters did not measure correct MAP and HR values, but ACT wasmeasured correctly. The failure of the transmitters was probably due to improper surgical placement in the aorta. Variation wasexpected to be lowest in the control group, therefore these five animals were assigned to the control group 2 (Table 2). Of the other

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13 cages containing an animal with a functional transmitter, nine were randomly selected and assigned to group 1, while theremaining four cages were added to group 2. The other 18 cages containing only nonimplanted animals were randomly assigned totwo extra control groups (groups 3 and 4) to control for transmitter or surgery effects (Table 2). Autopsy of the animals after theexperiment showed no negative effect of the misplaced canulla in the aorta.

Table 2: Schedule of experimental groups and parameters

Group Transmitter n Transportation BW MAP/HR/ACT OBS. CORT/CK

1 =transported

Yes, oneanimal percage

9 Packed andtransported

Yes Yes Yes Yes

2 = control Yes, oneanimal percage

9 (4functionaltransmitters)

No Yes Yes Yes Yes

3 No 9 Packed Yes No No Yes

4 No 9 No Yes No Yes Yes

MAP, mean arterial pressure; HR, heart rate; ACT, activity, as measured by telemetry device; BW, body weight; OBS, behavioralobservation; CORT, corticosterone; CK, creatine kinase.

Behavioral Observations

All animals were observed in their home cage at a fixed location in the animal chamber (the same location in the rack for all animals)using a JVC digital video camera on a tripod. Observations were performed by continuous focal sampling for 5 minutes directly aftercage cleaning, 3 to 5 hours after lights on. All three animals per cage were scored. Because cleaning disturbs the animals andincreases the level of activity in rats (Burn et al. 2006; Saibaba et al. 1996), this offers an opportunity to observe active behaviorduring the daylight period. We scored 18 behavioral parameters using the software The Observer (Noldus, Wageningen, NL). Foranalysis we assigned the behavioral parameters to the following three different behavioral categories:

1. Activity/Locomotion (LOC ), consisting of the scored behaviors explore, rear, walk, shake, scratch, scan, and hop/jump;

2. Social interactions (SI ), consisting of social explore, social grooming, follow/chase, push, and pinn;

3. Self-grooming (GRO ), consisting of six behaviors that together compose the following cephalocaudal sequence that animalsperform in an undisturbed situation (Kalueff and Tuohimaa 2005): forepaw licking, face/nose wash, head wash, body wash/fur licking,hindleg licking, and tail/genitals licking.

Transportation Procedure

During the first week after surgery, the animals were weighed and checked daily for clinical abnormalities. After 5 days, MAP and HRmeasurements started at a collection rate of every 3 minutes for 10 seconds, 24 hours per day. ACT was expressed as counts perminute, with every count representing a movement of the rat of 1.5 to 2 cm, detected by the system as change in signal strength.ACT was recorded continuously and stored every 3 minutes (total activity of 3 minutes). Twenty-four days after surgery, the rats ofgroups 1 and 3 were prepared for transport in the room in which they were housed. The rats were removed from their cage, weighed,and allocated with their cage mates in solid floor plastic transport boxes with filters (64 × 42 × 16 cm, Williton Box Co., Taunton, UK).The boxes were prepared with wood bedding (Tierwohl, Classicâ bedding, Rettenmaier & Söhne, Rosenberg, Germany), diet pellets(Harlan Teklad Rodent Diet), and Hydrogel™ (Harlan, Indianapolis, IN) as a water source. The lids of the boxes were closed andtaped. The animals of groups 2 and 4 were left undisturbed.

The animals in groups 1 and 3 were packed in the transport boxes between 10:00 and 11:00 AM and placed back on the telemetryreceivers. One hour later, the boxes were taken to the holding area from which the loading of the vans occurred. The temperature inthe holding area was set to 17°C. The light-dark regime in the holding area was identical to that of the animal chambers. The next

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day at 6:00 AM, the animals of group 1 were loaded into an unlit, climate-controlled (15°C) van and transported to a second animalfacility. The journey lasted approximately 3 hours. As soon as the animals arrived at the receiving facility, they were placed on thetelemetry receivers in the destination animal chamber for 2 hours before they were unpacked by the researcher. At the receivingfacility, data recording resumed every 3 minutes for 10 seconds, 24 hours per day. Environmental conditions were similar to those inthe breeding facility. At the time group 1 was being transported, group 3 was unpacked and weighed, and blood samples were takenin the holding area by the researcher. Subsequently, the animals of group 3 were euthanized by an ip injection of barbiturate.Twenty-one days after transportation, measurements were ended and all animals of groups 1, 2, and 4 were euthanized, similarly togroup 3. In Table 1, an overview of all performed procedures is shown.

Statistical Analyses

The experimental (and statistical) unit is the entity that can be assigned at random to one of the treatments independently of all otherexperimental units. Any two experimental units must be capable of being assigned to different treatments (Festing and Altman 2002).In the present study the cage was the experimental unit. Therefore, we averaged body weight (gain) per cage using only thenontransmitter animals. For analysis of frequency and duration of behavioral parameters as well as blood plasma parameters, wealso used cage averages; however, these averages were based on three animals per cage. Data of the telemetry output wereaveraged to 24 hours per animal (=cage). Some plasma samples had corticosterone values below the detection limit. To be able touse these data, we gave the samples a value of one-half the detection limit (Antweiler and Taylor 2008).

We carried out all statistical analyses according to Petrie and Watson (2006), Quinn and Keough (2002), and/or Field (2009) using aSPSS® for Windows (version 16.0) software program (SPSS Inc., New York, NY), We estimated two-sided exact probabilitiesthroughout (i.e., for the nonparametric tests; [Mundry and Fischer 1998]), and we summarized continuous data (body weight, bloodplasma parameters, telemetry parameters, and duration of behavioral parameters) (see Tables 1 and 2, Figures 1-3) as means andstandard deviations (SDs ).

We used the Kolmogorov-Smirnov one-sample test to check Gaussianity of the continuous data. This check was done for eachexperimental group and led to the conclusion that several parameters were not normally distributed. We transformed all experimentalgroups of these non-normal distributed parameters to a Gaussian distribution by using a mathematical function (e.g., logarithmic,logistic, or exponential transformation). If the data did not fulfill the criterion via transformation, we rank-transformed the continuousparameter in question (Conover and Iman 1981).

Continuous data are tested for significant differences by multivariate repeated measures analysis of variance (ANOVA ). Tests ofsignificance are derived using the Wilks’ lambda criterion. The choice of a multivariate instead of an univariate statistic in therepeated measures ANOVA is based on the criteria given by Algina and Keselman (1997). For telemetry data, within the groups (tocompare the animal with itself), we compared a period of 7 days before transportation with an equal period after transportation, againusing a repeated measures ANOVA. It is known that sound and activities in animal facilities have an influence on the physiologicalvalues of laboratory animals (Schreuder et al. 2007). Therefore we performed before-after analyses by comparing only equal days(e.g., Monday before vs. Monday after) thus ensuring that no effect of environmental factors was biasing the results.

For all repeated measures ANOVAs, we tested homoscedasticity by the Levene’s test, which is a powerful and robust test based onthe F statistic (Lim and Loh 1996). When necessary, we equalized the variances by logarithmic or logistic transformation of thecontinuous data. After transformation, the variances should be similar and the transformed within-group data should be normallydistributed. If the data did not fulfill the criterion via logarithmic or logistic transformation, we rank-transformed the continuousparameter in question (Conover and Iman 1981).

If the repeated measures ANOVA detected significant effects, we further compared the group means of the continuous parameters.We performed between-subject post hoc comparisons (group differences) with unpaired Student’s t tests for normally distributeddata, or we performed the same comparisons for non-normally distributed data using a Mann-Whitney U-Wilcoxon rank sum W test.We performed the unpaired Student’s t tests using pooled (for equal variances) or separate (for unequal variances) varianceestimates, and we tested the equality of variances with the Levene’s test. For the unpaired Student’s t test with separate varianceestimates, SPSS® uses the Welch-Satterthwaite correction (Ruxton 2006). We made within-subject post hoc comparisons (timeeffects, i.e., before and after packing/transportation) using a paired Student’s t test when the difference between the two comparedgroups was normally distributed or, if not, we used the Wilcoxon matched-pairs signed ranks test.

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We first rank-transformed discrete data on the ordinal scale (total numbers of the behavioral parameters) (Conover and Iman 1981)and subsequently subjected the data to multivariate repeated measures ANOVA. We made post hoc comparisons with eitherWilcoxon matched-pairs signed ranks test for paired data (time effect) or Mann-Whitney U-Wilcoxon rank sum W tests for unpaireddata (group differences).

We compared the variability of data (i.e., the continuous data, except duration of behavioral parameters) form the different groups byusing the Levene’s test. To achieve independence from average values, we used the coefficient of variation (SD/mean value) insteadof the variance (SD ) to compare the variation between the groups.

To take into account the greater probability of a Type I error due to multiple hypotheses, a more stringent criterion should be used forstatistical significance (i.e., for the paired and unpaired Student’s t tests, Mann-Whitney U-Wilcoxon rank sum W tests, and Wilcoxonmatched-pairs signed ranks test). We approached this problem by calculating so-called Dunn-Šidák corrections ([Ludbrook 1998]; α= 1 – [1 – 0.05] ; λ = number of comparisons). In all other cases, we took the probability of a Type I error < 0.05 as the criterion ofsignificance.

Results

Bodyweight (BW) and Bodyweight Gain (ΔBW)

There was no overall difference in BW or ΔBW between the groups (group effect: p = 0.176, ΔBW: p = 0.081). There was a Timeeffect (group BW: p < 0.001, ΔBW: p < 0.001) and a Time × Transport interaction effect (group BW: p < 0.001, ΔBW: p < 0.001). Onthe day of transportation, there was a significant difference in ΔBW compared with the moment of packing between the transportedgroup 1 and the nontransported group 2 (mean group 1 = -5.44 g, mean group 2 = + 4.53 g, p < 0.001). Group 1 decreased 1.51% inBW, and group 2 increased 1.28% in BW during the same period between the packing and unpacking of group 1, approximately 27hours. Results of group 3 were similar to those in group 1, and results of group 4 were similar to those in group 2, indicating thatthere was no effect of transmitter on BW or ΔBW.

Plasma CORT

For groups 1 and 2, which contained animals with transmitters, a significant elevated level of plasma CORT was evident even beforetransportation ( λ = 3 : α = 0.017) (BS1: groups 1 (mean: 98.5 ng/mL) and 2 (mean: 93.1 ng/mL), in contrast to animals from group 3(mean: 24.8 ng/mL) and group 4 (mean: 17.2 ng/mL) (p < 0.017), which had no transmitter (Figure 1). This increased level of plasmaCORT was found not only in the animals that had received a transmitter but also in their cage mates (data not shown). The averageplasma CORT level of the control group (group 2) was on baseline level at BS2 after transportation of group 1.

Figure 1. Plasma corticosterone (CORT) levels (mean in ng/mL + SD) of cages at four

sampling moments. At BS1 (baseline) group 1 (black square) and 2 (black circle) (with

transmitters) are significantly higher than groups 3 (white square) and 4 (white circle) (without

transmitters). At BS2 (directly after transport) group 1 (packed and transported) and 3

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(packed) are significantly higher than the nontransported control groups 2 and 4. Gr1

continuously stays significantly higher until BS4 (16 days after transportation). SD, standard

deviation.

There was an overall Transport- , Time-, and Time × Transport effect (all effects: p < 0.017). Directly after transportation (BS2),plasma CORT levels of group 1 were significantly higher than the levels in control group 2 (mean group 1: 115.9 ng/mL, mean group2: 35.7 ng/mL, p > 0.017). Although still declining at BS4 (Figure 1), plasma CORT levels of group 1 stayed continuously significantlyhigher during the rest of the experiment (p < 0.017). Results of group 3 at BS2 were similar to those of group 1. There was nosignificant difference between groups 1 and 3 at BS2 (mean group 3: 96.26 ng/mL, p = 0.333). Like group 1, group 3 was significantlydifferent from group 2 (p = 0.001) and 4 (mean group 4: 31.8 ng/mL, p > 0.001).

Creatine Kinase

No significant differences were found between plasma CK levels of transported versus control animals on either of the samplingmoments ( λ = 3 : α = 0.017, mean group 1= 83.12, mean group 2 = 92.46, p = 0.331)). Plasma CK levels within the groups did notdiffer significantly between consecutives samples (all p > 0.017, data not shown).

Telemetry

Mean Arterial Pressure (MAP)

In Figure 2, the time course of the average 24-hour values of the MAP is shown, starting 1 week before transportation and continuinguntil 3.5 weeks after transportation. Data of MAP show an overall gradual increase, mirrored by a Time-effect in the repeatedmeasures ANOVA (7 days before vs. 7 days after transportation, p = 0.031). During transportation there was a significant decrease inMAP in the transported group 1 (Rep measures ANOVA, before-after × Transport interaction, p < 0.001). MAP in group 1 did notrecover (to the before-transportation level) during the time span of this experiment.

Figure 2. Mean arterial pressure (MAP) in mmHg and heart rate (HR) in bpm (mean 24 hours + SD). No overall differences appear

between group 1 (transported=TP) and group 2 (control=CO). Group 1 shows a significant decrease during transportation (day 0) and

continues on this level until the end of the experiment (day 21). Group 2 shows an increase in MAP and a decrease in HR over time.

SD, standard deviation.

Heart Rate (HR)

The 24-hour averaged HR values (Figure 2) indicate an overall but slight gradual decrease (repeated measures time effect, p =0.014). During transportation there was a significant decline in HR (before-after × Transport interaction, p < 0.001) that did not returnto the before-transportation level during the remaining time of the experiment.

Activity (ACT)

ACT levels of the nontransported group 2 were stable during the time course of the experiment. Group 1 tended to increase in ACTduring transportation (repeated measures ANOVA, before-after × Transport interaction, p = 0.102), and the elevated level persistedduring the remaining period of the experiment.

Behavior

Overall there was a significant difference between groups 1 and 2 in total number of behaviors performed (sum of LOC, SI, and GROparameters) and the total number of different behaviors performed during the observations period (p = 0.024 and p < 0.001). Group 1performed significantly more different behaviors before transportation (OBS1: mean rank group 1= 39.11, mean rank group 2 =23.89, p = 0.041) but significantly less after transportation at OBS2 and OBS4 (OBS2: mean rank group 1 = 12.00, mean rank group2 = 42.00, p < 0.000; OBS4: mean rank group 1 = 15.11, mean rank group 2 = 57.22, p < 0.001).

The average duration per performed behavior also differed significantly between the groups (p < 0.001). In group 1 the relativeduration of behaviors was significantly shorter before transportation (OBS1: mean group 1 = 6.2%, mean group 2 = 7.3%, p =0.050), in comparison with the relative duration after transportation at OBS2, OBS3, and OBS4 (OBS2: mean group 1 = 8.12%, meangroup 2 = 6.2%, p < 0.001; OBS3: mean group 1 = 7.3%, mean group 2 = 5.9%, p = 0.038; OBS4: mean group 1= 8.3%, mean group2 = 5.5%, p = 0.001). Results in group 4 were similar to those in group 2.

Locomotor Activity (LOC)

When looking at the frequency of observed locomotion-related behaviors, we found significant differences between groups 1 and 2.Baseline values were equal (OBS1: mean group 1= 74.70, mean group 2 = 66.56, p = 0.073). LOC was significantly lower in thetransported animals than in the control group at the second observation moment directly after transportation (OBS2 mean group 1 =57.74, mean group 2 = 82.19, p < 0.001) and also at the last observation moment 2 weeks after transportation (OBS4: mean group1= 68.78, mean group 2 = 79.48, p = 0.013). No effect was found at OBS3. Comparing the observation moments within the groupsshowed that there was a significant decrease between OBS1 and OBS2 within group 1 (p = 0.004), whereas there was a significantincrease within group 2 (p = 0.027). This increased LOC within group 2 was also present at OBS4 when compared with OBS1 (p =0.020). When we compared OBS2 and OBS3 within group 1, we observed a significant increase back to baseline level (OBS2 vs.OBS3, p = 0.004; OBS1 vs. OBS3, p = 0.652).

We found no differences in the duration of LOC (in percentage of total observation time) either between the two experimental groupsor within the groups. The duration of LOC was nearly significantly higher in the control animals of group 2 at OBS2 (OBS2: meangroup 1 = 75.73%, mean group 2 = 87.00%, p = 0.064).

Social Interaction (SI)

When comparing the frequency of SI between the groups, we found significantly less SI with the transported animals of group 1 thanwith the control animals of group 2 at OBS2 (mean rank group 1 = 11.19, mean rank group 2 = 19.07, p = 0.004) and OBS4 (meanrank group 1 = 16.07, mean rank group 2 = 20.63, p = 0.038). The number of observed SIs within group 1 showed a significantdecrease between OBS1 and OBS2 (mean rank OBS1= 22.15, mean rank OBS2= 11.16, p = 0.008) followed by a significantincrease between OBS2 and OBS3 (mean rank OBS3 = 16.74, p = 0.008) back to baseline level (OBS1 vs. OBS3, p = 0.098).

Relative durations (in percentage of total observation time) of SI in group 1 were significantly reduced in comparison with group 2(Figure 3) at all observation times, except at baseline level (OBS1: mean group 1 = 12.02%, mean group 2 = 8.78%, p = 0.297;OBS2: mean group 1 = 4.10%, mean group 2 = 10.13%, p = 0.026; OBS3: mean group 1 = 4.42%, mean group 2 = 10.85%, p =0.003; OBS4: mean group 1 = 3.51% , mean group 2 = 11.24%, p < 0.000). The relative durations of SI within group 1 decreasedbetween OBS1 and OBS2 (p = 0.006) and stayed continuously decreased for all observations after transportation (OBS1 vs. OBS3,p = 0.010, OBS1 vs. OBS4, p = 0.007). Within group 2 we found no significant differences between the four observation moments. Finally when we looked at the individual behavioral parameters in more detail, we concluded that all of the individual parameters ofwhich SI consisted contributed to the overall decrease of SI in group 1 (data not shown).

Figure 3. Duration of social interaction (SI) at four observation moments in groups 1 and 2

(percentage of observed time). At OBS2, OBS3, and OBS4 (day 0, 7, 14) SI of group 1

(transported, blocked) is significantly lower than group 2 (control, black) and significantly

lower than OBS1 (baseline: day -7). No significant differences appeared within control group

2. Within group 1 OBS2, OBS, and OBS4 are significantly lower than OBS1 (baseline). OBS,

behavioral observation.

Self-grooming (GRO)

GRO frequencies showed a significant difference at OBS2, wherein the transported group 1 had a higher level of GRO than group 2(OBS2: mean group 1 = 14.37, mean group 2 = 8.74 ). GRO increased significantly between OBS1 and OBS2 within group 1 (OBS1:mean group 1 = 7.52, p = 0.020) followed by a significant decrease between OBS2 and OBS3 (OBS3: mean group 1 = 7.16, p =0.008). Within control group 2, there were no significant differences between the observation moments.

The duration of grooming showed a significant difference between groups 1 and 2 at the baseline observation moment. Animals ofgroup 1 spent more time on grooming (OBS1: mean group 1 = 10.73%, mean group 2 = 6.46%). Within group 1, there was asignificant decrease in grooming duration between OBS 1 and OBS2 (OBS2: mean group 1 = 6.21%, p = 0.009), OBS1 and OBS3(OBS3: mean group 1 = 5.97%, p = 0.003) and OBS1 and OBS4 (OBS4: mean group 1 = 2.94%, p < 0.000). In both groups 1 and 2,there was a significant decrease in grooming duration between OBS2 and OBS4 (group 1: p = 0.026, mean group 2: OBS2 = 7.80%,OBS4 = 4.90%, p = 0.041).

Examining the different behavioral parameters of which GRO is composed made clear that the increased level of GRO (frequency)after transportation (OBS2) within group 1 compared with baseline (OBS1) was caused by an increase in forepaw licking (frequency:p = 0.004, duration: p < 0.001) and face/nose wash (frequency: p = 0.006, duration: p < 0.001), the first two grooming components ofthe cephalocaudal sequence. None of the other grooming components were significantly increased.

Discussion

Overall, our results show that transportation from the breeder to another institute significantly affects physiology and behavior inlaboratory rats. Some parameters appear to stabilize at a level different from initial baseline, which indicates allostasis rather thanhomeostasis. More specifically, MAP and HR decreased after transportation and did not return to pretransportation levels, insteadgaining a level at which they stabilized after approximately 4 days. Increased CORT levels did not return to baseline within theobservation period of this study. The effects of transportation on several behavioral parameters were still visible after 2 weeks.

Corticosterone

Plasma CORT levels in the transported animals were higher overall than those of control animals. Levels of the control groups withnonoperated animals were comparable with those of other studies measuring plasma corticosterone levels in rats during the lightphase (Korte et al. 1992; Retana-Marquez et al. 2003). At baseline there was an elevated level of CORT in both experimental groups

with transmitters but not in the extra control groups without transmitters. This increase may still represent a postsurgery effect,although Greene and colleagues (2007) found that a recovery period of 1 week was sufficient after transmitter implantation inSprague-Dawley rats. Based on plasma CORT values in the transmitter control group in the present study, recovery from surgeryshould have been continued for 1 more week before sampling for “baseline” plasma CORT levels. Group 3 showed that packed-but-not-transported animals also have a significantly increased plasma CORT level compared with nonpacked control animals.

Differences in management and animal care between facilities can cause differences in effects on experimental parameters. Forexample, caretakers were constantly present (during the light phase) at the breeding facility, whereas in the receiving facility,caretakers were present for only about 30 minutes per day. Next to this difference in daily routine, the transported animals also had toadapt to the new housing conditions. Both factors might induce increased levels of plasma CORT. Segar and colleagues (2009)found increased plasma CORT in male F344 and BN rats, Marin and colleagues (2007) in Wistar rats, and Croft and colleagues(2008) in male TO mice after exposure to a novel environment. The transported animals of group 1 may as well have been sensitizedby transportation and, therefore, may have developed an increased stress responsivity compared with the nontransported animals inthe control group, resulting in higher plasma CORT levels. Although we cannot exclude the possibility of different baseline CORTlevels in experimental groups, such a difference is unlikely. The fact that CORT levels did not return to pretransport baseline in ourstudy may be explained by a process of allostasis (i.e., establishment of a new baseline level after transport) (McEwen 2002).However, no definite conclusion can be drawn about the homeo- or allostasis of the plasma CORT levels after transportationbecause levels appear to decline toward the end of the study instead of stabilizing.

Blood Pressure and Heart Rate

In contrast to earlier studies reporting elevations in blood pressure due to both acute (Bechtold et al. 2009) and chronic (Fokkema etal. 1995; Scheuer 2010) stress, blood pressure (MAP values) in our study were shown to decrease after transportation. However,Adams and colleagues (1987) found a similar acute decrease in MAP after a repeated social stressor (defeat) in male S/JR rats.Furthermore, we found no positive correlation between plasma CORT levels and MAP as described by Scheuer and colleagues(2004).

MAP levels in transported animals stabilized within the first week after transportation at 107 mmHg. MAP values of the control groupincreased within 4 weeks from 106 mmHg in the week before transportation to 110 mmHg in the last week. These values are inagreement with other studies that found an age effect on MAP in rats in which MAP was increased in aged rats compared with youngrats (El-Mas and Abdel-Rahman 2005; Korte et al. 1992). Compared with baseline levels reported in the literature (370-375 bpm:[DiMicco et al. 2006]; 350 bpm [Korte et al. 1992]), HR levels in both groups 1 and 2 were rather high during the first weeks of theexperiment (mean group 1 and 2 = 413 bpm). Several studies found an age effect on HR in rats, in which HR was lower in agedanimals (23/26 months) than in young rats (3-4 months) (Buwalda et al. 1992; Korte et al. 1992).

The first week after transportation, HR in both groups stabilized. In the control group, HR levels stabilized at 375 bpm; in thetransported group, at 351 bpm. Bradycardia has been reported in several studies concerning transportation. Stemkens-Sevens andcolleagues (2009) found a decreased HR in guinea pigs after transportation, and Capdevila and colleagues (2007) found this effect inrats. Similar findings have been reported for rats after exposure to an emotional stressor (footshock and social defeat) (Korte et al.1990; Nyakas et al. 1990; Roozendaal et al. 1990) and after exposure to a mild stressor (Buwalda et al. 1992). In the present study,only resting levels of HR (during lights-on) were significantly decreased, making the amplitude of HR larger between light on andlights off. This decrease is comparable with the findings of Harper and colleagues (1996), who found an increase in HR amplitude inLE rats after surgical and social stress. Sgoifo and colleagues (2005) found a reduction in amplitude of daily HR in Wistar rats aftersocial stress that lasted up to 3 weeks. Finally, Meerlo and colleagues (2002) found a decrease in HR amplitude in wild type rats aftersocial conflict that lasted 2 weeks.

Both MAP and HR appear to stabilize on a lower level after transportation, again indicating an allostasis effect rather than a return topretransport baseline levels in these parameters.

Behavior

We performed behavioral observations during lights-on conditions (i.e., the resting period of rats). By cleaning the cages directlybefore observations, animals were well awake and triggered to perform behavior in this seminovel environment (Duke et al. 2001).

The most striking result of our study was that rats reduced their social behaviors significantly after transportation. Play-relatedbehaviors (e.g., “play/fight,” “follow/chase,” and “pinn”) (Vanderschuren et al. 1995) were reduced to almost zero after transportationand stayed absent during the remaining period of this study. During the same period, levels of these parameters in nontransportedcontrol animals stayed on baseline level. Notably, social play is considered a positive indicator of welfare that is absent in stressfulsituations (Oliveira et al. 2010). In addition, another parameter of SI, social grooming, also decreased significantly aftertransportation. This effect on social behavior in transported rats could indicate that the welfare of these animals is compromised bytransportation for a period of at least 2 weeks. More research is needed to evaluate these newly acquired levels of SI and whether ornot these levels are allostatic.

Self-grooming, a common behavior in rodents, is considered as a behavioral marker for stress and has been shown to be highlysensitive to experimental factors (Kalueff et al. 2007). In this study, transported rats showed an increased frequency, but not duration,of self-grooming behavior directly after transportation. Self-grooming behavior often occurs in response to (mild) stressors. Suchgrooming is supposed to play a role in the de-arousal of the animal. After 1 week, grooming frequency levels in this study were backto before-transportation baseline.

Rodent grooming is an intricately patterned behavior that generally proceeds in a cephalocaudal direction. The pattern can bedisrupted by both acute and chronic stress (Denmark et al. 2010) and is sensitive to different levels of stress (Kalueff and Tuohimaa2005). High or low levels of stress, respectively, have a divergent effect on the grooming microstructure in which high stress levelsshow an increase in interruptions and incorrect transitions in the cephalocaudal sequence. The increased nose and head wash foundin the present study thus indeed indicates elevated stress levels in the transported animals. Although both groups were confrontedwith a novel situation (clean cage), the regional distribution of grooming of the transported rats was markedly affected bytransportation and manifested in increased rostral grooming (nose and head wash). However, the transportation effect on GRO in thisstudy appears to last less than 1 week because rostral grooming increased only during OBS2, after which grooming levels in thetransported animals returned to baseline level.

Bodyweight

Decreased or even negative BW gain in the transported animals might have been due to a reduced intake of food and/or water, toincreased defecation (Kalueff and Tuohimaa 2005; Kim et al. 2008; O’Malley et al. 2010), or to an increased need for energy resultingin the use of body fat reserves during transportation. However, the differences in BW gain disappeared within 1 week. Packedanimals showed identical weight loss to the transported animals, therefore weight loss might be due more to packing than totransportation.

Conclusions

In pursuit of the concept of homeostasis, it can be concluded that animals that undergo transportation reveal partially persistingchanges in physiological and behavioral parameters. Applying the concept of allostasis on these results would result in a differentview on acclimatization periods, shifting anticipation from a return to baseline levels to stabilization (adaptation) of parameters atpotentially different levels. If stabilization would be considered acclimatization, physiological and behavioral parameters wouldprobably indicate a much shorter acclimatization period compared with acclimatization by achieving before-transportation levels. Asimilar study with a longer continuation of measurements after transportation should be performed to give insight into the possibilityof a return to baseline levels on a long-term basis.

No conclusions can be drawn about the effects of transportation on other strains, female individuals, or other species based on theresults of this study. Due to the minimum BW of 200 g for implanting the transmitter, and the recovery period after surgery, the age atwhich the animals in this study were transported was a few weeks greater than the average age animals normally are beingtransported from the breeder (6-7 weeks). Furthermore, internal (in-house) transportation is also a disruption of environment that canhave effects similar to external transportation (Swallow et al. 2005) and should thus be investigated in more detail. Additionally, itshould be taken into account that international transport may necessitate an extended period of acclimatization due to disturbance ofthe diurnal rhythm of the animals (Council of Europe 2006). More research is needed to complete the picture of transportation-relatedstress and acclimatization periods in laboratory rodents.

Recommendations for sufficient acclimatization periods strongly depend on the parameters investigated. Investigators of studies

using rats recommend an acclimatization period of 3 days based on nutritional parameters (Ruiven et al. 1998) and physiologicalparameters (Capdevila et al. 2007). Rowland and colleagues (2000) recommended an acclimatization period of 7 days based onmyocardial antioxidant enzyme activity. Both Van Ruiven and colleagues (1998) and Rowland and colleagues (2000) measured onlyafter transportation, so no inferences can be made about whether or not the advised acclimatization periods are based onhomeostasis or allostasis. In the report by Capdevilla and colleagues (2007), HR and ACT measures returned to baseline levelsbefore transportation, but animals were transported back to the point of departure. No study has thus far combined the effects oftransportation on physiology, behavior, and blood values.

If acclimatization is defined as a period of 3 successive days in which parameters are on a stable level, acclimatization in this studyoccurs after approximately 1 week if exclusively based on MAP and HR values. However, because plasma CORT was not stableafter 3 weeks, a second prolonged study is needed to be able to draw a conclusion as to whether or not plasma CORT andbehavioral parameters return to baseline level or stabilize at a new level.

We have demonstrated that there is a significant and often long-lasting effect of transportation on behavioral and physiologicalparameters in rats. Our results show that the stressful impact of transportation embraces all parts of the procedure, including but notlimited to the packing of the animals. Researchers must be aware of this impact and provide a sufficient acclimatization period toallow for the (re-)stabilization of parameters. Insufficient acclimatization periods will endanger not only the reliability of animalresearch results but also the welfare of the animal.

Acknowledgments

This work was supported by a research grant from Harlan Laboratories BV, The Netherlands. The funders had no role in studydesign, data collection and analysis, decision to publish, or preparation of the manuscript.

We thank Dr. Hein van Lith for his contribution to the statistical analysis. In addition, the assistance of ing. Kees van Berkel and theSurgical Team Harlan Laboratories B.V. at the Harlan Laboratories B.V. breeding facility is gratefully acknowledged.

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