The Impact of Transportation on Physiological and Behavioral Parameters in Wistar Rats: Implications for Acclimatization Periods

Johanna 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 Animal Welfare & Laboratory Animal Science, Veterinary Faculty, Utrecht University, Utrecht, NL, as well as a research scientist at 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.  Frauke Ohl, PhD, is a professor in the Department of Animals in Science & Society, Division of Animal Welfare & Laboratory Animal Science, Veterinary Faculty, Utrecht University, Utrecht, NL

Address correspondence and reprint requests to Johanna W.M. Arts, BSc, MSc, Laboratory Animal Science, Utrecht University, 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, analysis of 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 transportation and how long it takes for the animal to recuperate. In the present study, we investigated physiological and behavioral parameters before and after transportation in both transported and nontransported animals. We took blood samples to analyze plasma corticosterone and creatine kinase, and performed physiological measurements by means of telemetry, measuring heart rate, blood pressure, and activity. Behavior was measured by means of home cage observations. This study revealed that plasma corticosterone levels increased at least up to 16 days after transportation, blood pressure and heart rate showed a lasting decrease after transportation, grooming increased, and social interactions and locomotor activity decreased after transportation. With these data we demonstrate that there is a long-lasting effect of transportation on physiological and behavioral parameters. Our results show that the stressful impact of transportation embraces all parts of the procedure, including for example the packing of the animals. Researchers must 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 between institutes (e.g., from a breeding to a research facility, which may last several days) and long distance transportation, which can be expected to induce significant stress in animals (Swallow et al. 2005). However, evidence rarely exists about the effects of transportation 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 and stabilize their physiological status to a condition that can be defined as baseline. Using stressed animals is likely to result in considerable 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 of the 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 for them to undergo a period of acclimatization before being used in a procedure. The time required depends on several factors, such as the stress to which the animals have been subjected which in turn depends on several factors such as the duration of the transportation and the age of the animal. This time shall be decided by a competent person.” Most experiments make use of an acclimatization period after transport to decrease the influence of transportation-related stress on results. However, the duration of this acclimatization period is scarcely based on scientific research, and it varies tremendously between facilities (from 1 day to as much as 3 weeks).

During a transportation procedure, many different stressors are present such as confinement, novelty, changes in temperature and light condition, and exposure to new animal caretakers and procedures. In general, animals subjected to such stressors react with changes in their physiology including body weight (BW¹), hormone levels, heart rate (HR¹), body temperature, and blood pressure (Capdevila et al. 2007; Obernier and Baldwin 2006; Ruiven et al. 1998; Stemkens-Sevens et al. 2009). Furthermore, several investigators 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) that disrupts animals’ physiological balance (Stokes 2000). Activation of the hypothalamic-pituitary-adrenal axis results in the release of stress hormones such as corticosterone (CORT¹) from the adrenal glands (Herman et al. 2005; McEwen 2000). Elevated levels of CORT are known to affect a variety of physiological parameters such as blood pressure, HR, immune system function, and behavioral patterns (Drozdowicz et al. 1990; Harper et al.1996; Wood et al. 2003). Although long-lasting (chronic) stress may result in pathological conditions (Kloet et al. 2005; Marin et al. 2007; Sapolsky 2003), acute stress responses mostly are characterized by an adaptive value (i.e., preparing an individual to adequately respond to a challenging situation) (Marin et al. 2007; Ohl et al. 2008). After termination 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 before stress, a phenomenon referred to as allostasis (McEwen 2002). Broom (1986) defines animal welfare as “its state as regards its attempts 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 literally means “stability through change,” has the potential to replace homeostasis as the core model of physiological regulation. The capacity 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 transport procedure in this study as transporting animals from the breeding facility to a second animal facility by van, including all related procedures and changes such as packing procedure, the change of animal chamber, cages, and caretakers. We then compare physiological and behavioral measurements in male Wistar rats before and after transportation as well as in transported and nontransported 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 et al.1996; Kramer 2000; Krohn et al. 2003) and can thus be used to obtain nonconfounded data on physiological changes in laboratory rats due to transportation (Capdevila et al. 2007). In the current study, we acquired mean arterial (blood) pressure (MAP¹), HR, and locomotor activity (ACT¹) by means of radiotelemetry transmitters. In addition to these parameters, we measured home cage behavior, BW and bodyweight gain (ΔBW¹), plasma levels of CORT, and plasma levels of creatine kinase (CK¹). We used plasma CK as 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 to evaluate the acclimatization period necessary for the animals to (re)establish a (potentially new) baseline status. This knowledge can be expected not only to decrease the variation in research results in transported animals, resulting in a decrease in the number of animals required to obtain valid experimental results, but also to improve the welfare of laboratory rodents by defining the time that the 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 the protocol 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” (The Dutch “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 animal experiments followed the “Principles of Laboratory Animal Care” and refer to the Guidelines for the Care and Use of Mammals in Neuroscience 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 group housed 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 diet 2918) and water that was processed (acidified (pH 5.8-6.4), chlorinated (6-8 ppm), softened, and filtered (0.02 µm). A light-dark regime 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 the second part of the experiment after transportation, the animals of the transported group (group 1) were housed under standard laboratory conditions of the animal facilities of Utrecht University in a temperature- (22 ± 2˚C) and humidity- (45-50%) controlled animal 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
Exp day	Group	Procedure
-35	All	Selection 108 animals and distribution over 36 cages of 3 animals
-26,-25	1, 2	Surgery on 1 animal per cage in 18 of the 36 cages
-25 – -12	1, 2	Recovery of surgery (transmitter animals)
-20	1, 2	Start telemetry measurements (transmitter animals)
-9 – -2	1, 2	Baseline recordings of telemetry data in transmitter 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, continuation of 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 occurred weekly. 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 first sample was taken 7 days before transportation (baseline: BS1). The second sample was taken on the day of transportation, directly after unpacking (BS2). The third sample was taken 7 days after transportation (BS3), and the fourth and last sample was taken 16 days 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 exceeding the maximal allowed sampling quantity of 8 mg/kg BW/14 days. Samples were stored on ice until centrifugation in a tabletop centrifuge (Eppendorf 5402, Boom B.V., Meppel) at 4ºC for 10 minutes at 14,000 rpm. Plasma was stored in a -18˚C freezer until analysis.

Plasma CORT levels were determined using a commercial radioimmunoassay kit (ImmuChem^TM, 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 kit for colorimetry (EnzyChrom^TM, BioAssay Systems, Hayward, CA) according to the protocol of the supplier. Each sample for both plasma 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 were transported prior to the study. When the animals were 8 weeks old (average BW 204 g [186-218]), surgery was performed by an experienced surgical technician on 18 randomly selected animals in a dedicated room in the breeding facility. In 18 cages, one animal 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 until 2 days after surgery. The animals were anesthetized with an intraperitoneal (ip¹) injection of ketamine (75 mg/kg) followed by an ip injection of médétomedine (Dexdomitor ® 10 mg/kg). During surgery, local analgesia was administered (Lidocaine®, Eurovet Animal Health, Cuijk, NL) and the eyes were protected with Duodrops® (Produlab Pharma, Raamsdonkveer, NL). The implantation procedure was carried out under strictly aseptic conditions as described by Kramer and Kinter (2003) for mice and by Huetteman and Bogie (2009) for laboratory rodents, in which the transmitter canulla was directly fixed into the abdominal aorta, and the body of the transmitter was permanently fixed to the inside of the abdominal muscles. The still unconscious animals were brought back to the animal chamber to recover. As soon as the animals regained consciousness after the surgical procedure, they were group housed with 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 18 cages were placed in a conventional rack. To prevent detection from adjacent transmitters, all receiver-cage couples were placed in a U-shaped stainless steel profile. After surgery, five of the transmitters did not measure correct MAP and HR values, but ACT was measured correctly. The failure of the transmitters was probably due to improper surgical placement in the aorta. Variation was expected to be lowest in the control group, therefore these five animals were assigned to the control group 2 (Table 2). Of the other 13 cages containing an animal with a functional transmitter, nine were randomly selected and assigned to group 1, while the remaining four cages were added to group 2. The other 18 cages containing only nonimplanted animals were randomly assigned to two extra control groups (groups 3 and 4) to control for transmitter or surgery effects (Table 2). Autopsy of the animals after the experiment 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, one animal per cage	9	Packed and transported	Yes	Yes	Yes	Yes
2 = control	Yes, one animal per cage	9 (4 functional transmitters)	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, behavioral observation; 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 after cage cleaning, 3 to 5 hours after lights on. All three animals per cage were scored. Because cleaning disturbs the animals and increases the level of activity in rats (Burn et al. 2006; Saibaba et al. 1996), this offers an opportunity to observe active behavior during the daylight period. We scored 18 behavioral parameters using the software The Observer (Noldus, Wageningen, NL). For analysis we assigned the behavioral parameters to the following three different behavioral categories:

1. Activity/Locomotion (LOC¹), consisting of the scored behaviors explore, rear, walk, shake, scratch, scan, and hop/jump;

2. Social interactions (SI¹), consisting of social explore, social grooming, follow/chase, push, and pinn;

3. Self-grooming (GRO¹), consisting of six behaviors that together compose the following cephalocaudal sequence that animals perform 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 HR measurements started at a collection rate of every 3 minutes for 10 seconds, 24 hours per day. ACT was expressed as counts per minute, 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 of groups 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 and taped. 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 telemetry receivers. One hour later, the boxes were taken to the holding area from which the loading of the vans occurred. The temperature in the 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 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 animal facility. The journey lasted approximately 3 hours. As soon as the animals arrived at the receiving facility, they were placed on the telemetry receivers in the destination animal chamber for 2 hours before they were unpacked by the researcher. At the receiving facility, data recording resumed every 3 minutes for 10 seconds, 24 hours per day. Environmental conditions were similar to those in the breeding facility. At the time group 1 was being transported, group 3 was unpacked and weighed, and blood samples were taken in 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 to group 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 other experimental 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 the nontransmitter animals. For analysis of frequency and duration of behavioral parameters as well as blood plasma parameters, we also used cage averages; however, these averages were based on three animals per cage. Data of the telemetry output were averaged to 24 hours per animal (=cage). Some plasma samples had corticosterone values below the detection limit. To be able to use 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 a SPSS® for Windows (version 16.0) software program (SPSS Inc., New York, NY), We estimated two-sided exact probabilities throughout (i.e., for the nonparametric tests; [Mundry and Fischer 1998]), and we summarized continuous data (body weight, blood plasma parameters, telemetry parameters, and duration of behavioral parameters) (see Tables 1 and 2, Figures 1-3) as means and standard deviations (SDs¹).

We used the Kolmogorov-Smirnov one-sample test to check Gaussianity of the continuous data. This check was done for each experimental group and led to the conclusion that several parameters were not normally distributed. We transformed all experimental groups 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 continuous parameter in question (Conover and Iman 1981).

Continuous data are tested for significant differences by multivariate repeated measures analysis of variance (ANOVA¹). Tests of significance are derived using the Wilks’ lambda criterion. The choice of a multivariate instead of an univariate statistic in the repeated measures ANOVA is based on the criteria given by Algina and Keselman (1997). For telemetry data, within the groups (to compare the animal with itself), we compared a period of 7 days before transportation with an equal period after transportation, again using a repeated measures ANOVA. It is known that sound and activities in animal facilities have an influence on the physiological values 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 on the F statistic (Lim and Loh 1996). When necessary, we equalized the variances by logarithmic or logistic transformation of the continuous data. After transformation, the variances should be similar and the transformed within-group data should be normally distributed. If the data did not fulfill the criterion via logarithmic or logistic transformation, we rank-transformed the continuous parameter 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 distributed data, 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) variance estimates, and we tested the equality of variances with the Levene’s test. For the unpaired Student’s t test with separate variance estimates, SPSS® uses the Welch-Satterthwaite correction (Ruxton 2006). We made within-subject post hoc comparisons (time effects, i.e., before and after packing/transportation) using a paired Student’s t test when the difference between the two compared groups was normally distributed or, if not, we used the Wilcoxon matched-pairs signed ranks test.

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 either Wilcoxon matched-pairs signed ranks test for paired data (time effect) or Mann-Whitney U-Wilcoxon rank sum W tests for unpaired data (group differences).

We compared the variability of data (i.e., the continuous data, except duration of behavioral parameters) form the different groups by using the Levene’s test. To achieve independence from average values, we used the coefficient of variation (SD/mean value) instead of the variance (SD²) 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 for statistical significance (i.e., for the paired and unpaired Student’s t tests, Mann-Whitney U-Wilcoxon rank sum W tests, and Wilcoxon matched-pairs signed ranks test). We approached this problem by calculating so-called Dunn-Šidák corrections ([Ludbrook 1998]; α = 1 – [1 – 0.05]^1/λ; λ = number of comparisons). In all other cases, we took the probability of a Type I error < 0.05 as the criterion of significance.

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 Time effect (group BW: p < 0.001, ΔBW: p < 0.001) and a Time × Transport interaction effect (group BW: p < 0.001, ΔBW: p < 0.001). On the day of transportation, there was a significant difference in ΔBW compared with the moment of packing between the transported group 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% in BW, and group 2 increased 1.28% in BW during the same period between the packing and unpacking of group 1, approximately 27 hours. Results of group 3 were similar to those in group 1, and results of group 4 were similar to those in group 2, indicating that there 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 before transportation ( 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 plasma CORT was found not only in the animals that had received a transmitter but also in their cage mates (data not shown). The average plasma CORT level of the control group (group 2) was on baseline level at BS2 after transportation of group 1.

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 group 2: 35.7 ng/mL, p > 0.017). Although still declining at BS4 (Figure 1), plasma CORT levels of group 1 stayed continuously significantly higher during the rest of the experiment (p < 0.017). Results of group 3 at BS2 were similar to those of group 1. There was no significant difference between groups 1 and 3 at BS2 (mean group 3: 96.26 ng/mL, p = 0.333). Like group 1, group 3 was significantly different 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 sampling moments ( 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 not differ 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 continuing until 3.5 weeks after transportation. Data of MAP show an overall gradual increase, mirrored by a Time-effect in the repeated measures ANOVA (7 days before vs. 7 days after transportation, p = 0.031). During transportation there was a significant decrease in MAP in the transported group 1 (Rep measures ANOVA, before-after × Transport interaction, p < 0.001). MAP in group 1 did not recover (to the before-transportation level) during the time span of this experiment.

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 return to 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 ACT during transportation (repeated measures ANOVA, before-after × Transport interaction, p = 0.102), and the elevated level persisted during 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 GRO parameters) and the total number of different behaviors performed during the observations period (p = 0.024 and p < 0.001). Group 1 performed 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 group 2 = 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 relative duration 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%, mean group 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 group 2 = 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 the transported 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 group 1= 68.78, mean group 2 = 79.48, p = 0.013). No effect was found at OBS3. Comparing the observation moments within the groups showed that there was a significant decrease between OBS1 and OBS2 within group 1 (p = 0.004), whereas there was a significant increase 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 groups or within the groups. The duration of LOC was nearly significantly higher in the control animals of group 2 at OBS2 (OBS2: mean group 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 than with 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 (mean rank group 1 = 16.07, mean rank group 2 = 20.63, p = 0.038). The number of observed SIs within group 1 showed a significant decrease between OBS1 and OBS2 (mean rank OBS1= 22.15, mean rank OBS2= 11.16, p = 0.008) followed by a significant increase 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 decreased between 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 of which SI consisted contributed to the overall decrease of SI in group 1 (data not shown).

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 of group 1 spent more time on grooming (OBS1: mean group 1 = 10.73%, mean group 2 = 6.46%). Within group 1, there was a significant 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 of the 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 in laboratory rats. Some parameters appear to stabilize at a level different from initial baseline, which indicates allostasis rather than homeostasis. More specifically, MAP and HR decreased after transportation and did not return to pretransportation levels, instead gaining a level at which they stabilized after approximately 4 days. Increased CORT levels did not return to baseline within the observation 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 with nonoperated animals were comparable with those of other studies measuring plasma corticosterone levels in rats during the light phase (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 in Sprague-Dawley rats. Based on plasma CORT values in the transmitter control group in the present study, recovery from surgery should 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. For example, 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 to adapt 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 sensitized by transportation and, therefore, may have developed an increased stress responsivity compared with the nontransported animals in the control group, resulting in higher plasma CORT levels. Although we cannot exclude the possibility of different baseline CORT levels in experimental groups, such a difference is unlikely. The fact that CORT levels did not return to pretransport baseline in our study 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 transportation because 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 et al. 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 group increased within 4 weeks from 106 mmHg in the week before transportation to 110 mmHg in the last week. These values are in agreement with other studies that found an age effect on MAP in rats in which MAP was increased in aged rats compared with young rats (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 the experiment (mean group 1 and 2 = 413 bpm). Several studies found an age effect on HR in rats, in which HR was lower in aged animals (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 the transported group, at 351 bpm. Bradycardia has been reported in several studies concerning transportation. Stemkens-Sevens and colleagues (2009) found a decreased HR in guinea pigs after transportation, and Capdevila and colleagues (2007) found this effect in rats. 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 and lights off. This decrease is comparable with the findings of Harper and colleagues (1996), who found an increase in HR amplitude in LE rats after surgical and social stress. Sgoifo and colleagues (2005) found a reduction in amplitude of daily HR in Wistar rats after social stress that lasted up to 3 weeks. Finally, Meerlo and colleagues (2002) found a decrease in HR amplitude in wild type rats after social 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 to pretransport 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 directly before 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-related behaviors (e.g., “play/fight,” “follow/chase,” and “pinn”) (Vanderschuren et al. 1995) were reduced to almost zero after transportation and stayed absent during the remaining period of this study. During the same period, levels of these parameters in nontransported control animals stayed on baseline level. Notably, social play is considered a positive indicator of welfare that is absent in stressful situations (Oliveira et al. 2010). In addition, another parameter of SI, social grooming, also decreased significantly after transportation. This effect on social behavior in transported rats could indicate that the welfare of these animals is compromised by transportation for a period of at least 2 weeks. More research is needed to evaluate these newly acquired levels of SI and whether or not 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 highly sensitive 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. Such grooming is supposed to play a role in the de-arousal of the animal. After 1 week, grooming frequency levels in this study were back to before-transportation baseline.

Rodent grooming is an intricately patterned behavior that generally proceeds in a cephalocaudal direction. The pattern can be disrupted by both acute and chronic stress (Denmark et al. 2010) and is sensitive to different levels of stress (Kalueff and Tuohimaa 2005). High or low levels of stress, respectively, have a divergent effect on the grooming microstructure in which high stress levels show an increase in interruptions and incorrect transitions in the cephalocaudal sequence. The increased nose and head wash found in the present study thus indeed indicates elevated stress levels in the transported animals. Although both groups were confronted with a novel situation (clean cage), the regional distribution of grooming of the transported rats was markedly affected by transportation and manifested in increased rostral grooming (nose and head wash). However, the transportation effect on GRO in this study appears to last less than 1 week because rostral grooming increased only during OBS2, after which grooming levels in the transported 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, to increased defecation (Kalueff and Tuohimaa 2005; Kim et al. 2008; O’Malley et al. 2010), or to an increased need for energy resulting in the use of body fat reserves during transportation. However, the differences in BW gain disappeared within 1 week. Packed animals showed identical weight loss to the transported animals, therefore weight loss might be due more to packing than to transportation.

Conclusions

In pursuit of the concept of homeostasis, it can be concluded that animals that undergo transportation reveal partially persisting changes in physiological and behavioral parameters. Applying the concept of allostasis on these results would result in a different view on acclimatization periods, shifting anticipation from a return to baseline levels to stabilization (adaptation) of parameters at potentially different levels. If stabilization would be considered acclimatization, physiological and behavioral parameters would probably indicate a much shorter acclimatization period compared with acclimatization by achieving before-transportation levels. A similar study with a longer continuation of measurements after transportation should be performed to give insight into the possibility of 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 the results of this study. Due to the minimum BW of 200 g for implanting the transmitter, and the recovery period after surgery, the age at which the animals in this study were transported was a few weeks greater than the average age animals normally are being transported from the breeder (6-7 weeks). Furthermore, internal (in-house) transportation is also a disruption of environment that can have effects similar to external transportation (Swallow et al. 2005) and should thus be investigated in more detail. Additionally, it should be taken into account that international transport may necessitate an extended period of acclimatization due to disturbance of the diurnal rhythm of the animals (Council of Europe 2006). More research is needed to complete the picture of transportation-related stress 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 physiological parameters (Capdevila et al. 2007). Rowland and colleagues (2000) recommended an acclimatization period of 7 days based on myocardial antioxidant enzyme activity. Both Van Ruiven and colleagues (1998) and Rowland and colleagues (2000) measured only after transportation, so no inferences can be made about whether or not the advised acclimatization periods are based on homeostasis or allostasis. In the report  by Capdevilla and colleagues (2007), HR and ACT measures returned to baseline levels before transportation, but animals were transported back to the point of departure. No study has thus far  combined the effects of transportation 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 study occurs after approximately 1 week if exclusively based on MAP and HR values. However, because plasma CORT was not stable after 3 weeks, a second prolonged study is needed to be able to draw a conclusion as to whether or not plasma CORT and behavioral 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 physiological parameters in rats. Our results show that the stressful impact of transportation embraces all parts of the procedure, including but not limited to the packing of the animals. Researchers must be aware of this impact and provide a sufficient acclimatization period to allow for the (re-)stabilization of parameters. Insufficient acclimatization periods will endanger not only the reliability of animal research 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 study design, 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 the Surgical Team Harlan Laboratories B.V. at the Harlan Laboratories B.V. breeding facility is gratefully acknowledged.

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