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Applied Animal Behaviour Science 135 (2011) 128– 137

Contents lists available at SciVerse ScienceDirect

Applied Animal Behaviour Science

journa l h o me pag e: www.elsev ier .com/ locate /applan im

hen is a Wistar a Wistar? Behavioral profiling of outbred Wistar ratsrom five different suppliers using the MCSF test

ara Palma,∗, Åsa Hävermarka, Bengt J. Meyersonb, Ingrid Nylandera,1, Erika Romana,1

Department of Pharmaceutical Biosciences, Neuropharmacology, Addiction and Behavior, Uppsala University, P.O. Box 591, SE-751 24 Uppsala, SwedenDepartment of Neuroscience, Pharmacology, Uppsala University, P.O. Box 593, SE-751 24 Uppsala, Sweden

r t i c l e i n f o

rticle history:ccepted 17 August 2011vailable online 15 September 2011

eywords:&K Universalody weightharles Riverarlanultivariate concentric square fieldTM

aconic

a b s t r a c t

The same strain of rats purchased from different suppliers may exhibit behavioral differ-ences that could give rise to variability and deficient repeatability. We have previouslydemonstrated that outbred Wistar rats from different suppliers display striking differencesin voluntary alcohol intake. Using outbred male Wistar rats from five suppliers, four sup-pliers in Europe (Charles River, Taconic, Harlan and B&K Universal) and one in the UnitedStates (Harlan), the present study investigated the variability in behavioral profiles of theWistar substrains. To this end, we used the multivariate concentric square fieldTM (MCSF)test. This test has an ethological foundation and is designed to provoke exploration andbehaviors associated with risk assessment, risk taking and shelter seeking. The term “mul-tivariate” refers to the test situation as well as the statistical analysis of the data. Our resultsdemonstrate significant differences in body weight and behavioral profiles between theage-matched groups of Wistar rats. A principal component analysis clearly separated Har-lan (US) and Charles River rats from the B&K Universal, Harlan (EU) and Taconic rats. Theparameters important for this separation included activity and risk-taking performance. A

trend analysis further confirmed this finding. The results emphasize the need for carefulspecification of the animals used in a given study. The utility of differences in explorativestrategies and behavioral profiles among various outbred strains derived from a single strainis discussed. In conclusion, the data support differences between substrains of Wistar ratsand illustrate the utility of using a multivariate strategy for behavioral profiling.

© 2011 Elsevier B.V. All rights reserved.

. Introduction

Behavior is the final measurable output of internal andxternal factors or stimuli. Many factors can give rise toxperimental variability, and it is important that the source

f variability is understood so that it can be prevented,inimized, controlled or used. For instance, even though

nimals from a specific strain are used, variability may

∗ Corresponding author. Tel.: +46 18 4714165, fax: +46 18 4714253.E-mail address: Sara.Palm@farmbio.uu.se (S. Palm).

1 Shared senior authorship.

168-1591/$ – see front matter © 2011 Elsevier B.V. All rights reserved.oi:10.1016/j.applanim.2011.08.010

arise within batches of animals, between batches fromthe same supplier and between animals from differentsuppliers (Hayakawa et al., 1980; Festing, 1993; Matsuoet al., 2010). Variability between batches may be a resultof variability over time due to genetic factors or variabilitybetween laboratory conditions (Crabbe et al., 1999). As aconsequence, the appropriate repeatability of results maybe difficult to achieve. One way of reducing genetic variabil-ity is to use inbred animals. However, this is sometimes not

optimal, and in some cases, the use of outbred animals canbe advantageous.

Outbred animals are maintained by non-siblingmatings, and most suppliers apply breeding schemes

S. Palm et al. / Applied Animal Behaviour Science 135 (2011) 128– 137 129

Table 1Overview of the five different suppliers and the respective nomenclature of the outbred Wistar rats as well as the abbreviations used in this paper.

Supplier Location Origin Nomenclature Abbreviation inthis paper

B&K Universal Ltd. Hull, the United Kingdom Zentralinstitut fürVersuchstierzucht,Hannover, Germany

Bkl:WI BK

Charles River (Europe) GmbH Germany The Wistar Institute,Philadelphia, PA, USA

Crl:WI Crl

Harlan Laboratories Inc. Indianapolis, IN, the UnitedStates

The Wistar Institute,Philadelphia, PA, USA

Hsd:WI Hsd

Harlan Laboratories B.V. Horst, the Netherlands Zentralinstitut fürVersuchstierzucht,Hannover, Germany

RccHanTM:WI Rcc

Taconic Farms (Europe) A/S Ejby, Denmark Zentralinstitut fürVersuchstierzucht,Hannover, Germany

HanTac:WH Tac

Modified from Palm et al. (2011).

that avoid crosses between closely related individualsto maintain heterozygosity. However, outbred stocks areessentially closed colonies, and many were originallyderived from a limited gene pool (Hedrich, 2000; Linderand Davisson, 2004), including Wistar rats from the WistarInstitute (Clause, 1993). The differences between suppliersmay arise from sampling variation when the colonieswere founded or from variations in breeding methods andenvironmental factors. These differences are sometimesforgotten when generic names such as “Wistar” and“Sprague-Dawley” rats and “Swiss” mice are used, butin fact these names mean very little without detailedinformation about breeder and animal. Thus, each breedercould be considered to supply a substrain of these animals.

It has been noted that phenotypic variability withinrat substrains has not been systematically investi-gated (Festing, 1993) even though it is a potentialexplanation for differences in results between labora-tories studying behaviors such as alcohol intake (Wise,1974). Previous studies in rats have revealed differencesbetween substrains in anxiety-like and fear-motivatedbehaviors using tests such as the open field and theelevated plus maze (Honndorf et al., 2011; Rex et al.,1996).

We have previously demonstrated that outbred Wistarrats from different suppliers display striking differencesin voluntary alcohol intake and preference (Palm et al.,2011). Using male Wistar rats from the different suppli-ers previously used by Palm et al. (2011), four suppliers inEurope (Charles River [Crl], Taconic [Tac], Harlan [Rcc] andB&K Universal [BK]) and one in the United States (Harlan[Hsd]), the present study investigated behavioral profilesusing the multivariate concentric square fieldTM (MCSF)(Meyerson et al., 2006; Roman and Colombo, 2009). This isan ethologically founded test designed to provoke explo-ration and behaviors associated with risk assessment, risktaking and shelter seeking by rodents in a novel environ-ment. The purpose of this design is to gather information

that, taken together, enables a behavioral profiling of theanimal in a single test situation (Meyerson et al., 2006).The test has been useful for behavioral profiling of dif-ferent outbred rats (Meyerson et al., 2006; Roman et al.,

2006), inbred mouse strains (Augustsson and Meyerson,2004; Augustsson et al., 2005), genetically modified mice(Wallen-Mackenzie et al., 2009) and rat lines selectivelybred for high and low voluntary oral alcohol intake andpreference (Roman et al., 2007, 2011; Roman and Colombo,2009).

2. Materials and methods

2.1. Animals

Fifty outbred male Wistar rats (n = 10/group) from fivedifferent suppliers (Table 1) arrived in the animal facil-ity at 7 weeks of age. All animals arrived at the sameweek, within 3 days of each other. Charles River Europeand Harlan Laboratories in the United States both receivedtheir stocks directly from the Wistar Institute (HarlanLaboratories, 2008; Charles River, 2010). B&K Univer-sal in the United Kingdom, Harlan Laboratories in theNetherlands and Taconic in Denmark received their stocksfrom the Zentralinstitut für Versuchstierzucht in Hannover(B&K Universal Ltd, personal communication, April 6, 2010;Harlan Laboratories, 2009; Taconic Farms, 2010). The ratswere housed two per cage in standard polysulfone cages(59 cm × 38 cm × 20 cm) containing wood-chip beddingmaterial and two paper sheets (Cellstoff, 40 cm × 60 cm,Papyrus Sverige AB, Mölndal, Sweden) in temperature-controlled (21 ± 1 ◦C) and humidity-controlled (50 ± 10%)cabinets in an animal room on a reversed 12 h light/darkcycle with lights off at 07:00 h. The paper sheet “enrich-ment” was selected to allow animals to use it in theirindividual way without affecting social hierarches in thecages by introducing limited resources, for example awooden house (Olsson and Dahlborn, 2002). They weremaintained on pellet food (R36, Lantmännen, Kimstad,Sweden) and water ad libitum. All animal experimentswere performed under a protocol approved by the Upp-

sala Animal Ethical Committee and followed the guidelinesof the Swedish Legislation on Animal Experimentation(Animal Welfare Act SFS1998:56) and the European Com-munities Council Directive (86/609/EEC).

130 S. Palm et al. / Applied Animal Behaviou

Fig. 1. The multivariate concentric square fieldTM test arena(100 cm × 100 cm) and the defined zones, numbered as follows: 1.CENTER, 70 cm × 70 cm, open area; 2. CENTRAL CIRCLE, 25 cm diameter,risk area; 3–5. CORRIDORs, transit areas; 6. dark corner room (DCR),area for shelter seeking; 7. HURDLE, high passage to hole board withpta

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hotocell to count head dips, exploratory incentive; 8. SLOPE, leading upo BRIDGE, risk assessment area; 9. BRIDGE ENTRANCE, risk assessmentrea; 10. BRIDGE, elevated and illuminated, risk area.

.2. Procedure

The rats were handled during the week before test-ng began. The handling procedure was similar to that ofrevious experiments (Roman et al., 2006; Roman andolombo, 2009) and consisted of individual handling,eighing, and adaptation to the transportation bucket thatas used to take the animals from the home cage to the

esting room. The behavioral testing was conducted simul-aneously for all groups and the animals were 11–12 weeksld when the testing started. The rats were tested using aounterbalanced running schedule to avoid time of day andrder biases. All testing was performed in a room separaterom the housing room, with a masking background noise.he conditions in the testing room were similar with regardo temperature and humidity to those in the animal room.ll behavioral observations were carried out during theark period of the light/dark cycle. The experimental pro-edure followed that of previous experiments as much asossible (Roman et al., 2007; Roman and Colombo, 2009).

.3. The MCSF test

The MCSF test has been described in detail else-here (Meyerson et al., 2006; Roman and Colombo,

009). The MCSF arena (Fig. 1) consists of a squareeld (100 cm × 100 cm) with a smaller square field70 cm × 70 cm) located in the center of the larger one. Thealls are 25 cm high except for the walls surrounding the

RIDGE (see below), which are 40 cm high. The arena isivided into zones, which forms the basis of the descrip-ion and the variables of the animals’ performance in thisest. The defined zones (Fig. 1) were as follows: CENTER,

r Science 135 (2011) 128– 137

the center field of the arena; CORRIDORs, the corridors sur-rounding the center field; DCR, the dark corner room wherethe animal can seek shelter; HURDLE, an elevated passageto a hole board with a photocell device located under thehole board floor, allowing recording of head dips into theholes; BRIDGE, the elevated and illuminated bridge con-struction, associated with risk-taking behavior; SLOPE, theslope leading up to the BRIDGE, associated with risk assess-ment; BRIDGE ENTRANCE, the initial part of the BRIDGEused as an additional risk-assessment area; and CENTRALCIRCLE, the circular zone (diameter 25 cm) in the middleof the CENTER, used for assessment of risk-taking behav-ior in the open central part of CENTER versus thigmotaxis.The animal to be tested was transferred in a bucket fromthe home cage to the MCSF and was placed in the CENTERfacing the wall between the CENTER and BRIDGE. The testsession lasted 20 min. There was no prior habituation tothe arena before the session. After each session, the arenawas wiped with 10% alcohol solution and allowed to drybefore the next animal was tested. The approximate lightconditions (l×) in the MCSF arena were as follows: DCR: <1;CENTER: <20; CORRIDORs and HURDLE: <10; SLOPE: <30;and BRIDGE: 600–650.

2.4. Behavioral recordings

The animals were monitored by video cameras mountedon the ceiling above the apparatus. The numbers ofstretched attend postures (SAPs; from the CORRIDORsinto the CENTER) and rearing and grooming actions wererecorded by direct observation. The number of fecal boliand urinations were counted after each trial, and the num-ber of head dips into the hole board of the HURDLE wasnoted. Manual scoring of the videotaped behavior in theMCSF was performed using Score 3.3 (Pär Nyström, Copy-right Soldis, Uppsala, Sweden). The latency (LAT, s) of firstvisiting, frequency (FRQ) of visits, duration (DUR, s) oftime spent in each defined zone, and the number of ani-mals visiting each zone (OCCURRENCE) were registered.The Ethovision system (version 2.3; Noldus InformationTechnology, Wageningen, the Netherlands) was used fortracking total distance (cm) moved in the CENTER, CEN-TRAL CIRCLE and the total arena and the mean velocity(cm/s) in the total arena. Visits to the defined zones wereonly scored as such if both hind legs had crossed over intothat section. The sum of frequencies to the CORRIDORs (FRQTOTCORR) and to all zones (TOTACT), the total time spentin the CORRIDORs (DUR TOTCORR), the mean duration pervisit to each zone (DUR/FRQ, s), and the percentage dura-tion and the percentage number of visits in the zones werecalculated.

2.5. Data handling

The overall analysis began with presentations of thedescriptive parameters followed by the multivariate dataanalysis. In order to make the presentation of descrip-

tive parameters clearer to the reader the parameters wereoperationally categorized into functional categories (i.e.,general activity, exploration, risk assessment, risk takingand shelter seeking) (Augustsson, 2004; Meyerson et al.,

ehaviour Science 135 (2011) 128– 137 131

S. Palm et al. / Applied Animal B

2006; Roman and Colombo, 2009). Finally, an interpreta-tion of mental states that were expressed by the behaviorswas made (Roman et al., 2011).

2.6. Statistical analysis

Body weight data were normally distributed and wereanalyzed using the parametric repeated measures ANOVA,followed by Tukey’s HSD post hoc test. The majority ofthe behavioral data did not show a normal distribution,and analysis was done using non-parametric statistics. TheKruskal–Wallis test followed by the Mann–Whitney U-test was used for between-group comparisons of MCSFparameters. When an animal did not enter a zone, this wasconsidered to be a missing value. Instead, the Pearson Chi-squared test was used for analysis of OCCURRENCE, i.e., thenumber of animals entering a zone or performing a behav-ior. For the comparisons of percentage number of visits,percentage duration of time spent in each zone and thetrend analysis the zero value was used. Differences in totalactivity and distance over time were analyzed using theFriedman test followed by the Wilcoxon signed-rank test.

The relationship between time spent in the shelteredarea (DCR) and the risk area (BRIDGE) in relation to the timespent in both zones (the risk/shelter index) is used as a mea-sure of anxiety-like behavior. After entering the SLOPE, theanimal has to assess the risk of entering the BRIDGE. Basedon this, the latency to first enter the BRIDGE in relation tofirst enter the SLOPE (the SLOPE/BRIDGE interval) is usedas a measure of impulsive-like behavior.

A trend analysis was used for analysis of MCSF perfor-mance. The trend analysis, explained in detail elsewhere(Daoura et al., 2010; Ekmark-Lewen et al., 2010; Romanet al., 2011; Akerberg, 2009), uses parameters that are cor-related and summarizes the individual rank values withinthe functional categories general activity (TOTAL ACTIV-ITY, FRQ TOTCORR and CENTER, and DUR/FRQ TOTCORRDIST), exploratory activity (DUR TOTCORR, CENTER andHURDLE, REARING, and number of PHOTOCELL COUNTSin the hole board), risk assessment (DUR/FRQ SLOPE andBRIDGE ENTRANCE, and SAP to CENTER), risk-taking behav-ior (FRQ BRIDGE and CENTRAL CIRCLE, DUR BRIDGE andCENTRAL CIRCLE, and DUR/FRQ BRIDGE and CENTRAL CIR-CLE) and shelter-seeking behavior (FRQ, DUR, and DUR/FRQDCR). The individuals were ranked against each other, andthe rank values were then summed into a sum rank for eachfunctional category.

Differences were considered statistically significant atp < 0.05. Statistica 8.0 (StatSoft Inc., Tulsa, OK) was used forthe statistical analyses.

In addition to the conventional statistical tests,multivariate data analysis was used to examine MCSF per-formance. A principal component analysis (PCA) (Erikssonet al., 2006) was performed to illustrate the relationshipbetween groups of rats in the MCSF test. The PCA is a multi-variate projection-based approach designed to extract anddisplay the systematic variation in a data set. The most

important use of PCA is to obtain an overview of the data,for example groups of observations, trends and outliers,and also to uncover the relationships between observationsand variables and among the variables themselves. The PCA

Fig. 2. The mean body weights ± SEM over time for the different groups ofWistar rats. BK, B&K Universal; Hsd, Harlan, the United States; Rcc, Harlan,the Netherlands; Crl, Charles River; Tac, Taconic.

creates a score plot showing a summary of the relation-ship between the individuals and a loading plot identifyingvariables important for creating these relationships, i.e., theparameters recorded in the MCSF. The direction of the scoreplot corresponds to the direction in the loading plot. Whenan animal did not enter a zone, this was considered to be amissing value in the PCA. The SIMCA-P+ 12.0 (Umetrics AB,Umeå, Sweden) was used.

3. Results

3.1. Body weights

A repeated measures ANOVA revealed significant dif-ferences in mean body weight between the groups[F(4,45) = 55.82; p < 0.001] and a significant interactionbetween time and group [F(16,180) = 18.43; p < 0.001], indi-cating differences in body weight gain (Fig. 2). The posthoc test revealed differences (p < 0.05) between most ofthe groups throughout the experimental period. At age 7weeks, the BK group weighed less than all other groups.Furthermore, the Rcc rats weighed less than the Hsd rats.At 18 weeks of age, the BK group weighed less than allother groups, and the Crl group weighed more than all othergroups. The Hsd group also weighed more than the Rcc andTac groups. At 18 weeks of age the body weight increasecompared to 7 weeks of age was as follows: BK 231%, Hsd203%, Rcc 212%, Crl 254% and Tac 201%.

3.2. The MCSF test

Profound differences between the groups were detectedfor parameters operationally categorized into the follow-ing functional categories: general activity, exploration,risk assessment, risk taking and shelter seeking. Theresults of the 20 min session in the MCSF test are shownin Supplementary Table 1. Measures of general activity

(TOTACT, FRQ TOTCORR, FRQ, DUR and DUR/FRQ CEN-TER, DISTANCE and VELOCITY in the arena) revealedsignificant differences, in particular, a generally higheractivity in Hsd and Crl rats compared to the other groups.

132 S. Palm et al. / Applied Animal Behaviour Science 135 (2011) 128– 137

A

B

Fig. 3. Principal component analysis (PCA) for the different groups of Wistar rats. The score plot (upper panel) illustrates the individual rats, and the loadingplot (lower panel) illustrates the MCSF variables that were included in the analysis. Variables located further away from the origin are most important.BE, BRIDGE ENTRANCE; CTRCl, CENTRAL CIRCLE; D, duration; DCR, dark corner room; D/F, duration/frequency; F, frequency; Groom, number of groomingactions; L, latency; PC counts, photocell counts; Rear, number of rearings; SAP, stretched attend posture; TOTACT, sum of visits to all zones in the arena;TOTCORR, sum of performances in the CORRIDORs. BK, B&K Universal; Hsd, Harlan, the United States; Rcc, Harlan, the Netherlands; Crl, Charles River; Tac,Taconic.

ehaviour Science 135 (2011) 128– 137 133

Fig. 4. The trend analysis in which individual rank values for the param-eters included in the functional categories are summed for each group of

S. Palm et al. / Applied Animal B

Measures of exploration indicated significant differencesfor the parameters LAT LEAVE, DUR and DUR/FRQ, TOT-CORR, LAT HURDLE and REARING. Hsd rats left the CENTERsignificantly faster than all other groups and differed incertain parameters of explorative activity compared toBK, Rcc and Tac rats. Tac rats were less explorative com-pared to BK and Crl rats. Parameters of relevance forrisk-assessment behavior (LAT, FRQ, DUR, DUR/FRQ SLOPEand BRIDGE ENTRANCE, and SAPs) indicated differencesbetween the groups. The Hsd and Crl rats displayed thelowest risk-assessment behavior, which was significantlydifferent from the BK and Tac rats and, to some extent, theRcc rats. Measures of risk-taking behavior (LAT, FRQ, DUR,DUR/FRQ BRIDGE and CENTRAL CIRCLE, VELOCITY CEN-TRAL CIRCLE and CENTER) revealed differences betweenthe groups. In terms of duration per visit on the BRIDGE, Hsdrats showed significantly lower risk-taking behavior com-pared to all other groups. A similar pattern in the CENTRALCIRCLE was shared with Crl rats. Hsd and Crl rats had signif-icantly higher velocity in the CENTER and CENTRAL CIRCLEcompared to the other groups. Parameters of relevance toshelter-seeking behavior (LAT, FRQ, DUR, DUR/FRQ DCR)gave a general indication of lower shelter-seeking behav-ior in Hsd and Crl rats compared to the other groups. Finally,significantly more Tac rats defecated, and Tac rats urinatedsignificantly more than Hsd and BK rats.

All experimental groups visited all zones of the arena,but the proportion of time spent in the different parts of theMCSF arena was different, as illustrated in SupplementaryFig. 1. BK rats spent less time in the CORRIDORs than Hsd,Crl and Tac rats, and Tac rats spent more time in the COR-RIDORs compared to Rcc and Crl rats. BK and Crl rats spentmore time on the BRIDGE than Hsd and Tac rats.

Hsd and Crl rats made a larger percentage of visits tothe CENTER than the other groups and fewer visits to theHURDLE compared to Tac rats. BK rats made more visitsto the risk area BRIDGE than Hsd, Crl and Tac rats. Finally,Tac rats made fewer visits to the risk area CENTRAL CIRCLEthan BK and Hsd rats. Supplementary Fig. 2 illustrates thepercentage of visits to the different zones in relation to theactivity (TOTACT) of each individual.

The PCA is shown in Fig. 3. The PCA confirms the tradi-tional statistical analysis and separated the experimentalgroups (score plot). The Hsd and Crl groups were groupedin the left quadrant. The BK, Rcc and Tac rats were foundin the right quadrants. The BK and Rcc rats grouped clos-est together, mainly in the upper right quadrant. The MCSFparameters important for this separation were located far-ther away from the origin in the loading plot and includedactivity parameters (generally loading corresponding tothe Hsd and Crl groups) and measures of risk-taking behav-ior (generally more evident in the BK and Rcc groups).

A trend analysis of the functional categories generalactivity, exploration, risk assessment, risk taking and shel-ter seeking is shown in Fig. 4. This analysis revealedsignificant differences in the functional categories generalactivity [H = 19.42; p < 0.001] and risk taking [H = 13.35;

p = 0.01]. No differences were found in the categoriesexploration [H = 7.21; p = 0.13], risk assessment [H = 8.04;p = 0.09] and shelter seeking [H = 1.39; p = 0.85]. The Hsdgroup had higher activity than the BK, Rcc and Tac groups,

rats. Values represent the mean ± SEM. *p < 0.05, **p < 0.01. BK, B&K Uni-versal; Hsd, Harlan, the United States; Rcc, Harlan, the Netherlands; Crl,Charles River; Tac, Taconic.

while the Crl group had higher activity than the Rcc and Tacgroups. The BK group displayed higher risk-taking behaviorthan the Hsd and Tac groups. The Rcc group showed higherrisk-taking behavior than the Tac group.

Interpretation as to the constructs of anxiety-like andimpulsive-like behavior (Supplementary Table 1) revealedno significant differences between the groups.

3.3. Total activity and distance over time

The 20 min MCSF trials were divided into four 5 minbrackets to explore differences in total activity (TOTACT)and distance over time. The total activity over time foreach group is shown in Fig. 5A, and the distance movedover time is shown in Fig. 5B. Differences in activityover time were found in the BK [�2 = 11.00; p = 0.01], Hsd[�2 = 16.56; p < 0.001] and Rcc [�2 = 9.87; p = 0.02] groups.There were no differences in the Crl [�2 = 4.76; p = 0.19]and Tac [�2 = 7.08; p = 0.07] groups. The BK rats increasedactivity over time while the Hsd rats decreased their activ-ity during the 20 min. The Rcc rats had a lower activity at0–5 min than at 5–10 min. No differences were found indistance over time (BK [�2 = 4.33; p = 0.23], Hsd [�2 = 7.56;p = 0.06], Rcc [�2 = 6.84; p = 0.08], Crl [�2 = 3.84; p = 0.28],Tac [�2 = 5.88; p = 0.12]).

3.4. Associations between activity in the MCSF and bodyweight

The correlation between body weight and total activ-ity is shown in Fig. 6A, and the correlation between bodyweight and total distance moved in the arena is shown inFig. 6B. No significant correlations were found for any groupbetween either body weight and activity (BK [r = 0.05;p = 0.90], Hsd [r = 0.05; p = 0.89], Rcc [r = 0.17; p = 0.65], Crl

[r = −0.46; p = 0.18], Tac [r = 0.20; p = 0.58]) or body weightand distance moved (BK [r = 0.62; p = 0.08], Hsd [r = 0.09;p = 0.80], Rcc [r = 0.03; p = 0.92], Crl [r = −0.36; p = 0.31], Tac[r = 0.11; p = 0.77]).

134 S. Palm et al. / Applied Animal Behaviour Science 135 (2011) 128– 137

F < 0.01. (t arlan, t

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ig. 5. (A) The total activity over time for each group of rats. *p < 0.05, **phe mean ± SEM. BK, B&K Universal; Hsd, Harlan, the United States; Rcc, H

. Discussion

The strength of this study is the direct comparisonetween outbred Wistar rats from different suppliers with-ut confounding factors in study design, experimentalonditions or handling. The results provide evidence for aarked variability in substrains of outbred male Wistar ratsith regard to explorative strategies and behavioral pro-les at least within the context of the particular test used

n this study. The main finding is that the groups that origi-ate from the Wistar Institute (Hsd and Crl) display a cleareparation in the PCA from the BK, Rcc and Tac rats, whichre of Hannover origin. The separation between the groupsan be traced to differences in general activity and risk-aking behavior, functional categories that also revealed

ignificant differences in the trend analysis.

In a vast number of studies, behavioral phenotypesave been compared in inbred mouse strains (Crawleyt al., 1997; Jackson, 2003), inbred rat strains (Lad et al.,

240220200180160140120100806040Total activity (sum of all frequencies)

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440A B

ig. 6. (A) The correlation between body weight and total activity. (B) The correlaarlan, the United States; Rcc, Harlan, the Netherlands; Crl, Charles River; Tac, Ta

B) The distance moved over time for each group of rats. Values representhe Netherlands; Crl, Charles River; Tac, Taconic.

2010), and substrains of inbred C57BL/6 and 129 mice(Izidio et al., 2005; Matsuo et al., 2010). Fewer studieshave been designated to study outbred animals, and it hasbeen stressed that the phenotypic variability between out-bred rats should be systematically investigated (Festing,1993). As mentioned previously, examples of studies thathave been done revealed differences between substrains inanxiety-like and fear-motivated behavior using tests suchas the open field and the elevated plus maze (Honndorfet al., 2011; Rex et al., 1996). However, these studies utilizea combination of several different behavioral tests that arefocused on specific predetermined mental states. Limita-tions with this approach include carry over effects that mayinfluence the results. The inter-test intervals may impactthe outcome, and repeated laboratory staff handling of the

animals is a known source of variance (Crabbe et al., 1999;McIlwain et al., 2001). Attempts have been made to solvethis problem by for example integrating several tests (e.g.,the open field, elevated plus maze and light/dark box) into

110001000090008000700060005000400000Total distance (cm)

BK Hsd Rcc Crl Tac

tion between body weight and distance moved. BK, B&K Universal; Hsd,conic.

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one (Ramos et al., 2008) or to develop new tests for theassessment of anxiety-like behavior (Ennaceur et al., 2006).Another approach is to use test situations that are notlimited to a specific predetermined mental condition, butinstead investigates a more extended behavioral repertoire(Blanchard and Blanchard, 1989; Meyerson et al., 2006). Inthe present study, the MCSF test was used to address thephenotypic variability within substrains of outbred Wistarrats. The MCSF test with its multivariate design is based onthe circumstances and challenges met by an animal undernatural conditions and allows the animal to explore on avoluntary basis. Compared to many commonly used behav-ioral tests, the MCSF test offers a diversity of choices andallows the expression of a greater behavioral repertoire,which can be tested in one test situation. The multivari-ate strategy, using statistical approaches including PCA andtrend analysis, emphasizes the overall behavioral profile ofthe animals rather than a single construct, such as anxiety-like behavior (Meyerson et al., 2006; Paylor et al., 2006).Therefore, the MCSF test can be viewed as an alternative toseveral commonly used behavioral tests.

In previous studies, the MCSF test has revealed dif-ferences between inbred male and female C57Bl/6/Scaand BALB/c/Sca mice (Augustsson and Meyerson, 2004;Augustsson et al., 2005) and strain differences in mice com-monly used as background for genetically modified mice(Marques et al., 2008). Using an approach of repeated test-ing the MCSF has also been used to show differences incognitive behavior in mice subjected to traumatic braininjury (Ekmark-Lewen et al., 2010). In rats, differences hasbeen found between outbred male Sca:SD and Sca:Wistarrats (Meyerson et al., 2006). Furthermore, striking differ-ences between outbred Sca:Wistar and Hsd:WI were foundwhen comparing two separate studies (Roman et al., 2010).A drawback in that study was that the animals were testedin two different labs at different time points. In the presentstudy, all age-matched rats were tested concurrently in thesame lab, at the same time and by the same experimenter.Pronounced differences were observed depending on thesupplier. The BK, Rcc and Tac groups are of Hannover originand are grouped fairly close together in the PCA. How-ever, the Rcc and the Tac groups originate from the same156 breeding pairs sent to the Research and ConsultingCompany Ltd. (RCC) from the Zentralinstitut für Versuch-stierzucht in Hannover in 1989 (Harlan Laboratories, 2009;Taconic Farms, 2010). These groups should therefore sharemore genetic similarity compared to the other groups. Thetrend analysis does, however, reveal differences betweenthese groups with respect to risk taking, where the Rccgroup is ranked higher. In fact, the Rcc and BK groups arelocated closer together in the PCA than the Rcc and Tacgroups. The Hsd rats were found to have higher generalactivity than all other groups except the Crl group. Thispattern was also found for some zones when individualactivity was taken into account. The Hsd rats were mostactive in the first 5 min of the MCSF test when looking atthe total activity, followed by a steady decrease in activity

over the rest of the 20 min session. This pattern contraststo the other groups that were generally more active duringthe second 5 min period. In this respect, it is interestingto note that the Hsd rats also display different alcohol

r Science 135 (2011) 128– 137 135

consumption patterns than the other Wistar substrains(Palm et al., 2011). Interpretations about the constructs ofanxiety-like and impulsive-like behavior in the MCSF aremade based on performance in risk and sheltered areas,respectively (Roman et al., 2011). No differences in eitheranxiety-like or impulsive-like behavior between the sub-strains were found.

In agreement with previous studies using rats from thepresent suppliers (Palm et al., 2011), profound differencesin body weight were found. In general, the highest bodyweight was found in the Crl group, while the lowest bodyweight was found among the BK rats. These differencesoccurred using the same type of ad libitum feeding regi-men. This finding was secondary to the aim of this study.Though not tested, it cannot be excluded that the differentgroups of Wistar rats also differ in consummatory behavior.The differences in body weight are interesting consideringthe regular practice to order and describe research ani-mals by weight instead of age. This study clearly showsthat rats of the same weight can differ by several weeksin age depending on origin, which may contribute to dis-crepancies between studies. As body weights differed anda substantial difference in general activity in the MCSF waspresent between the groups, it was of interest to investigatethe association between activity and body weight. Contraryto the expectation that heavier rats are less mobile, thepresent data show that the heaviest rats, the Hsd and Crlrats, scored higher in activity measures. However, whencalculated by group, no associations between body weightand activity were evident. This finding is in agreement withprevious studies in which no association between bodyweight and activity in the MCSF has been demonstrated(Roman and Colombo, 2009; Roman et al., 2011).

Some of the profound differences in body weight andbehavioral profiles revealed herein could be attributed todifferences in genetic make-up due to genetic drift, whichwill occur when breeding rats at different facilities formany years (Kloting et al., 2003). However, informationabout the genetic variation between different outbred ratsand mice is rare. In a comparison of randomly selectedmicrosatellite markers among outbred Wistar (Crl:WIST),Sprague-Dawley (Rj:SD) and unrelated wild rats, a loss ofalleles was found in both outbred stocks compared withwild rats. Thus, the designation “outbred” is not a guar-antee of genetic and phenotypic variability (Kloting et al.,2003). The observed differences between the Wistar groupsmay also be a result of different environmental conditions,either alone or in combination with genetic factors. Wehave previously shown that early life environment affectsthe behavioral profile of adult rats (Roman et al., 2006) aswell as their neurobiology (Ploj et al., 2003; Gustafssonet al., 2008; Oreland et al., 2009). To control for environ-mental factors, one direct approach would be to keep thefive stocks in the lab and breed them to produce rats thatwere conceived and reared under the same conditions.However, this study was designed to mimic the way inwhich the majority of behavioral studies are carried out.

Usually, researchers buy a limited number of animals fromone supplier and ship them to the lab where the behavioraltest is conducted. The results are then compared to previ-ous results obtained within the same lab (using separate

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36 S. Palm et al. / Applied Animal B

atches of animals) or to results presented in the litera-ure. The results of the present study highlight the problemith comparing results presented in the literature with-

ut taking into account the origin of the animals. However,ven though large differences within Wistar strains cre-te problems in comparative studies, they also provide theossibility of choosing specific substrains of outbred Wis-ar rats for different purposes. Selective breeding betweenxtremes and within high/low-performing strains in rela-ion to activity, explorative behavior and risk-associatederformance have been demonstrated to provide interest-

ng experimental tools (Burghardt et al., 2011; Dellu et al.,996; Lofgren et al., 2006, 2009).

In conclusion, profound differences were revealedetween Wistar rats from different suppliers using theCSF test. The specific behavioral profiles of Wistar sub-

trains may be an obstacle, but when known, they can alsorovide experimental opportunities. This study empha-izes the importance of better knowledge and informationbout phenotypic variation in laboratory animals and theeed for careful specification of the animals used, similaro standard practices for analytical methods and chemicaluppliers.

cknowledgements

The authors gratefully acknowledge technical assis-ance from Ms. Marita Berg. The Alcohol Research Council ofhe Swedish Alcohol Retailing Monopoly (SRA 07-21:3), the

agnus Bergvall Foundation, the Fredrik and Ingrid Thur-ng Foundation, Facias Foundation, Quality and Renewalppsala University 2007 (E.R.), and the Swedish Medicalesearch Council (K2008-62X-12588-11-3; IN) supportedhis study.

ppendix A. Supplementary data

Supplementary data associated with this arti-le can be found, in the online version, atoi:10.1016/j.applanim.2011.08.010.

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