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The Influence of the Locationof a Nest Box in an IndividuallyVentilated Cage on thePreference of Mice to Use ItNikolaos G. Kostomitsopoulos a , Euthimios Paronis a

, Paul Alexakos a , Evangelos Balafas a , Pascalle vanLoo b & Vera Baumans ba Center for Experimental Surgery, Foundation forBiomedical Research, Academy of Athens , Greeceb Department of Animals, Science, and Society,Division of Laboratory Animal Science , UtrechtUniversity , The NetherlandsPublished online: 05 Dec 2007.

To cite this article: Nikolaos G. Kostomitsopoulos , Euthimios Paronis , Paul Alexakos ,Evangelos Balafas , Pascalle van Loo & Vera Baumans (2007) The Influence of theLocation of a Nest Box in an Individually Ventilated Cage on the Preference ofMice to Use It, Journal of Applied Animal Welfare Science, 10:2, 111-121, DOI:10.1080/10888700701313256

To link to this article: http://dx.doi.org/10.1080/10888700701313256

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The Influence of the Locationof a Nest Box in an Individually

Ventilated Cage on the Preferenceof Mice to Use It

Nikolaos G. Kostomitsopoulos, Euthimios Paronis,Paul Alexakos, and Evangelos Balafas

Center for Experimental SurgeryFoundation for Biomedical Research

Academy of Athens, Greece

Pascalle van Loo and Vera BaumansDepartment of Animals, Science, and Society

Division of Laboratory Animal ScienceUtrecht University, The Netherlands

The improvement of housing conditions for mice by using environmental enrichmentmaterials is of high concern for the scientific community. Plastic, autoclavable nestboxes are commercially available and ready to use for specific cases such as in indi-vidually ventilated cages, metabolic cages, or during toxicological studies. The aim ofthis study was to see if the location of the nest box within the cage could influence themice to prefer and use it. Located on the cage floor or hung from the cage lid, a nestbox (MPLEX, Otto Environmental, Milwaukee, Wisconsin), enriched the cages. Thestudy concluded that the location of the nest boxes in the individually ventilated cageplays a significant role in the mice preferring to use it or to avoid it. It is also importantto use environmental enrichment items that provide animals with the possibility of ex-pressing their preferences and manipulating them in a way to cope better with their en-vironmental conditions.

JOURNAL OF APPLIED ANIMAL WELFARE SCIENCE, 10(2), 111–121Copyright © 2007, Lawrence Erlbaum Associates, Inc.

Correspondence should be sent to Nikolaos G. Kostomitsopoulos, Center for Experimental Surgery,Foundation for Biomedical Research, Academy of Athens, 4, Soranou Ephesius Street, Athens, 115 27Greece. Email: nkostom@bioacademy.gr

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During the last decade, there has been an increased scientific interest in the im-provement of housing conditions for nonhuman animals in the laboratory byproviding them with opportunities to perform more species-specific behavioralrepertoires through enriching their environment.

Environmental enrichment is, by definition, any modification in the environ-ment of the captive animals that seeks to enhance their physical and physiologicalwell being by providing stimuli that meet the animals’ species-specific needs(Baumans, 2000). Providing the animals with different kinds of materials or de-vices such as nest boxes, tubes, nesting materials, and shelters can focus an enrich-ment scheme on the social environment through the presence of socialpartners—including human beings—as well as on the physical environment (Vande Weerd & Baumans, 1995).

The use of environmental enrichment schemes is supported by recommenda-tions and guidelines from animals in the laboratory associations and by laws andlegal regulations (Baumans et al., 2006; Canadian Council on Animal Care, 1993;Jennings et al., 1998). However, there is still concern among experts in the fieldwho believe that environmental enrichment could influence the experimental out-comes (Eskola & Kaliste-Korhonen, 1999; Frank, 2004; Tsai, Stelzer, Hedrich, &Hackbarth, 2003). On the other hand, there is scientific evidence that standardiza-tion of the environmental enrichment could contribute positively to the quality ofanimal experimentation and, of course, to the quality of the obtained results (Tsai,Pachowsky, Stelzer, & Hackbarth, 2002). In addition, environmental enrichmentdoes not influence individual variability in behavioral tests and does not increasethe risk of obtaining conflicting data in replicate studies (Meshi et al., 2006;Wolfer et al., 2004). However, it should be kept in mind that the impact of environ-mental enrichment on experimental data depends on the type of enrichment, strainand sex of the animals, and the parameters measured (Baumans et al., 2006).

Nesting material is the most commonly used environmental enrichment for lab-oratory mice. Female and male mice of several strains and ages strongly prefer touse various kinds of nesting materials (Van de Weerd, Van Loo, Van Zutphen,Koolhaas, & Baumans, 1997; Van de Weerd, Van Loo, Van Zutphen, Koolhaas, &Baumans, 1998) to build their nests during breeding activities and to regulate lightand temperature levels. Evidence shows that reducing levels of stress and aggres-sion in males enhances the well being of group-housed mice (Armstrong, Clark, &Peterson, 1998; Van Loo et al., 2002; Van Loo et al., 2004). Especially wheretransparent cages are used, the provision of nesting material is crucial; it providesanimals with the possibility of building a nest where they can hide or retreat fromconspecifics or protect themselves from light (Brain & Rajendram, 1986; Van deWeerd et al., 1997). The most easily available and cheap nesting material is softpaper or compressed cotton fibers.

In the last few years, there has been an increase in commercially available nestboxes for mice. These are usually made from plastic, transparent, autoclavable

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material, are relatively easy to use, and are standardized as types of environmentalenrichment for mice (Van Loo, Blom, Meijer, & Baumans, 2005). Depending onthe sex, age, and number of animals housed in the cages, nest boxes have a varietyof potential benefits for the animals. The boxes give the mice control of their expo-sure to cold, light, or aggression—especially in the case of group housing of malemice (Nevison, Hurst, & Barnard, 1999; Van Loo, Van Zutphen, & Baumans,2003). The boxes also allow the animals to jump or climb. Despite the fact thatmice prefer nesting material to nest boxes (Van de Weerd & Baumans, 1995; Vande Weerd et al., 1997), the design and use of environmental enrichment devices foranimals in the laboratory are mainly based on human demands for standardizationof environmental enrichment—especially in metabolic cages and toxicologicalstudies—as well as for economic and ergonomic reasons.

Pressurized, individually ventilated caging systems were designed to protectanimals from cross-contamination and to improve their microenvironment. Withindividually ventilated caging systems, animals are thought to experience re-duced stress and discomfort because of the improved microenvironment, re-duced handling by personnel, fewer bedding changes, and reduced risk ofcontamination. However, high intracage ventilation rates could induce chronicstress and heat loss.

The use of nest boxes or nesting material seems to have a positive impact on thewell being of the animals housed in these cages by offering them protection in caseof high intracage ventilation rate as well as the opportunity to cope better with theirenvironment (Baumans, Schlingmann, Vonck, & Van Lith, 2002; Blom et al.,1992).

An additional benefit of nondisposal, intracage device selection is the reductionin clogging of exhaust air vents and filters, something that frequently occurs whenproviding manipulative enrichment such as paper or cotton fibers.

The aim of the current observation was to study the influence of the location ofa nest box placed into an individually ventilated cage on the preference of the ani-mals to use this box. In both groups, animals had to choose between the acceptanceof the nest box or its avoidance.

METHOD

The study was performed in the animal facility of the Center for ExperimentalSurgery of the Foundation for Biomedical Research of the Academy of Athens,Greece. The facility is registered as a “breeding” and “experimental” facility bythe Veterinary Service of the Prefecture of Athens, Greece, according to thePresidential Decree 160/91 in harmonization to the European Directive 86/609/EEC for the protection of animals used for experimental and other scientificpurposes. A health-monitoring program based on the Federation of European

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Laboratory Animal Science Association’s recommendations is being imple-mented (Nicklas et al., 2002).

Animals and Housing

One hundred ten female mice of the outbred strain Crl:CD1 (ICR), at the age of5 to 7 weeks, from the breeding colony of the animal facility of the Foundationwere randomly divided into groups of five and housed in 22, H-Temp™polysulfone type III, individually ventilated cages (800 cm2, Tecniplast, Milan,Italy), with 70 air changes per hour under positive pressure.

Animals were handled according to the guidelines of the Guide for the Care andUse of Laboratory Animals of the U.S. National Research Council.

Environment

All animals were housed in animal rooms under Specific Pathogen Free (SPF)conditions at a room temperature 22 ± oC, with 55 10% relative humidity, 12 hrlight starting at 07:00 followed by 12 hr dark, and with a light density of 300 lxmeasured 1 m above the floor in the middle of the room. Animal rooms were op-erated with a positive air of 0.6 Pa.

Food and Water

Tap water in drinking bottles and an irradiated, vacuum-packed, pelleted foodcontaining 20.0% protein, 4.5% fat, 4.7% fiber, 6.0 %ash (5061 Pico-Vac LabRodent Diet, PMI Nutrition International, Brentwood, Missouri) was providedad libitum to the animals.

Bedding

Approximately 140 g of 8-in. corncob bedding (IPS, United Kingdom) was usedas bedding. Cages and bedding were changed once a week.

Nest Box

All cages were enriched with a nest box (MPLEX, Otto Environmental, Milwaukee,Wisconsin). MPLEX is made from blue, transparent, autoclavable polycarbonate

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(7–8.5 cm long × 5 cm wide × 5.5 cm deep), with a weight of approximately 31.6 g. Ithas two arch openings—one small (3.5 × 4 cm) and one large (7.5 × 4 cm)—and ven-tilation holes for enhancing airflow.

Procedure and Data Collection

Cages were divided into two groups. In Group A (n = 11), nest boxes wereplaced on the cage floor opposite the food hopper (Figure 1). In Group B (n =11), nest boxes were clipped onto the stainless steel wire bar lid of the cage alsoopposite the food hopper (Figure 2).

All animals used in the observation study were unfamiliar with any kind of en-vironmental enrichment material. To minimize any influence of the different posi-tion of the individually ventilated cages on the rack, the study was carried out intwo parts: (a) for floor placement, and (b) for lid placement. Cages of both groupswere placed on the upper two rows of the rack.

During the study, animals were not disturbed. The access to the animal roomwas permitted only for the person who carried out the observations. Dirty bed-ding was removed just before and after the study. Twenty-four hours after theplacement of nest boxes in the cages, the use of nest boxes by the animals wasmonitored.

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FIGURE 1 In the cages of Group A, nest boxes were placed on the cage floor, at the opposite tothe feeder site.

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Observation was performed twice during the day phase of the light cycle, whichis considered as the normal resting period for mice, at 10:00 and 15:00 for a con-secutive period of 8 days. The hypotheses (H0: p = 0, and HA: p ≠ 0) for each group(A, B, A10:00, A15:00, B10:00, B15:00) were tested by using one-proportion test.The hypotheses (H0: p1 – p2 = 0, and HA: p1 – p2 ≠ 0) between groups (A–B,A10:00–A15:00, B10:00–B15:00, A10:00–B10:00, A15:00–B15:00) were testedby using two-proportions test (significance level = 95%). For each group, 95%confidence intervals (CIs) were calculated.

RESULTS

All the observations carried out during the study are shown in Table 1. A statis-tically significant difference on the preference of the mice to use the nest boxwas noticed between Group A and Group B (p < .05). From the 1st day of theobservation, animals showed a preference for using nest boxes that were placedon the floor of the cage. The percentage of cages in which the animals used thenest boxes was 69.31% (95% CI = .62–.76) for Group A and 17.61% (95% CI =.12–.24) for Group B (Figure 3).

From the 1st day of observation, all nest boxes in the cages of Group A wereturned upside down. Nest boxes were also moved by the animals to differentplaces in the cages. Mainly after the 4th day of observation, the animals moved thenest box to the front of the cage, under the food hopper (Figures 4 and 5).

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FIGURE 2 In cages of Group B, nest boxes were clipped on the stainless steel lid of the cagebehind the feeder site.

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Limited preference for the hanging nest boxes was noticed in all cages of GroupB. This preference was slightly increased after the 6th day of observation (up to36.36%; see Figure 3).

No statistical significant difference was noticed between the observations per-formed at 10:00 compared with 15:00 for either Groups A or B (p > .05).

DISCUSSION

The main objective of this study was to investigate the influence of the locationof a nest box within an individually ventilated cage on the preference of labora-tory mice to use it or avoid it. MPLEX, a plastic nest box, was used. Accordingto the instructions of the manufacturer, the box could be placed either on thefloor of the cage or clipped on the wire bar lid. After a consecutive period of 8days, it was noticed that there was a statistically significant preference of the an-imals to use nest boxes that were placed on the floor of the cages (Group A),compared with those hanging from the cage lid (Group B).

Mice housed in cages of Group A quickly became familiar with the nest box onthe floor. During the first observation, 24 hours after the placement of the nestboxes into the cages, animals in 4 of 11 cages started using the nest box. This rateincreased up to 10 cages during the next 5 days but went down to 8 cages after-ward. All animals housed in cages of Group B were more cautious about using the

PREFERENCE OF MICE FOR NEST BOX LOCATION 117

TABLE 1Number of Cages Where the Use of the Nest Box Was Observed

Per Total Number of Cages

Day of Observation Time of Observation Group A Group B

1st 10:00 4 of 11 1 of 1115:00 8 of 11 1 of 11

2nd 10:00 7 of 11 1 of 1115:00 7 of 11 1 of 11

3rd 10:00 7 of 11 0 of 1115:00 7 of 11 1 of 11

4th 10:00 6 of 11 2 of 1115:00 7 of 11 0 of 11

5th 10:00 10 of 11 2 of 1115:00 10 of 11 1 of 11

6th 10:00 8 of 11 3 of 1115:00 10 of 11 3 of 11

7th 10:00 8 of 11 4 of 1115:00 8 of 11 4 of 11

8th 10:00 7 of 11 4 of 1115:00 8 of 11 3 of 11

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nest box. The preference rate varied from 1 of 11 cages for the 1st day of observa-tion to 4 of 11 cages during the 7th day of observation (Table 1).

From the 1st day of observation, all nest boxes of Group A were turned upsidedown. This appeared to be accidental, occurring when the mouse entered the nestbox. However, this new position of the nest boxes gave the animals an opportunityto use boxes for jumping, climbing, or squeezing. Another advantage for animalsof Group A was their having the possibility to move nest boxes—probably in orderto cope better with their microenvironment. This behavior could indicate that nestboxes, as well as other environmental enrichment devices, should be designed toprovide animals the maximum possibility for manipulating them—depending onthe animals’ behavioral needs and not just on human demands for standardization,economy, or ergonomy.

Moving the position of the nest boxes to the front of the cage was clearly shownin all cages of Group A during the last 2 days of the study. Mice preferred to movethe nest box to the front of the cage, where possibly the ventilation rate is lower orbecause the food hopper provided extra shelter and less light. This animal behaviorwas also observed in other preference studies (Baumans et al., 2002). In cages ofGroup B, where the nest boxes were hanging from the cage lid without any possi-bility of moving them, animals needed more time to become familiar with them.The high intracage ventilation rate was regulated up to 70 air changes per hour, andthis airflow could be the main reason for the movement of the nest boxes to a placeless affected by airflow. In cages of Group B, the nest boxes were placed close tothe air inlet and outlet ports in the cover of the cage, resulting in a higher air

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FIGURE 3 Percentage of cages with nest boxes in use during the observations.

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FIGURE 4 A preference, mainly after the eighth observation (4th day of the experiment), wasexpressedbytheanimals toplace thenestboxin thefrontsideof thecage,under thefoodhopper.

FIGURE 5 In most of the cages of Group A, nest boxes were turned upside down and movedby the animals to the front of the cage, under the food hopper.

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velocity and airflow making the animals avoid using the hanging nest box. Itshould be noted that, over time, the hanging nest box was used more frequently,meaning that the benefits of a nest box might have overruled the anxiety aboutnovelty and possible increased airflow.

Housing conditions of laboratory mice, especially when they are housed in in-dividually ventilated cages, are of great importance for their physical and mentalhealth and for the quality of experimental results. The placement of different kindsof materials into the cage aims to improve the quality of microenvironment so thatthe mice have a greater choice of activity and some control over their social andphysical environment (Newberry, 1995; Stauffacher, 1995). Despite a clearly ex-pressed preference of laboratory animals for using nesting material, the use of nestboxes is sometimes contraindicated, namely, in metabolic cages, individually ven-tilated cages, and toxicological studies.

Based on the results of this study, it is concluded that the location of the nestboxes in the individually ventilated cages can give animals a choice to modify theirenvironment and to choose what to use—especially when the object is not fixed.

ACKNOWLEDGMENT

The authors acknowledge Dimitrios Zavras (National School of Public Health,Athens, Greece) for his help with the statistical analysis of data.

REFERENCES

Armstrong, K. R., Clark, T. R., & Peterson, M. R. (1998). Use of corn-husk nesting material to reduceaggression in caged mice. Contemporary Topics in Laboratory Animal Sciences, 37, 64–66.

Baumans, V. (2000). Environmental enrichment: A right of rodents. In M. Balls, A. M. Van Zeller, & M.E. Halder (Eds.), Progress in reduction, refinement, replacement of animal experimentation (pp.1251–1255). Amsterdam: Elsevier.

Baumans, V., Clausing, P., Hubrecht, R., Reber, A., Vitale, A., Wyffels, E., et al. (2006). Report of theFELASA’s working group on standardization of enrichment. In Web guides. Retrieved June 26,2006, from http://www.lal.org.uk/felasa.html

Frank, M. (2004). Public affairs: European welfare standards may affect U.S. research. The Physiolo-gist, 47, 1–3.

Jennings, M., Batchelor, G. R., Brain, P. F., Dick, A., Elliott, H., Francis, R. J., et al. (1998). Report ofthe joint working party on refinement: Refining rodent husbandry: The mouse. Laboratory Animals,32, 233–259.

Meshi, D., Drew, M. R., Saxe, M., Ansorge, M. S., David, D., Santarelli, L., et al. (2006). Hippocampalneurogenesis is not required for behavioural effects of environmental enrichment. Nature Neurosci-ence, 9, 729–731.

Nevison, C. M., Hurst, J. L., & Barnard, C. J. (1999). Strain-specific effects of cage enrichment in malelaboratory mice (Mus musculus). Animal Welfare, 8, 361–379.

120 KOSTOMITSOPOULOS ET AL.

Dow

nloa

ded

by [

Dr

Ken

neth

Sha

piro

] at

07:

06 0

9 Ju

ne 2

015

Newberry, R. C. (1995). Environmental enrichment: Increasing the biological relevance of captive en-vironment. Applied Animal Behaviour Science, 44, 229–243.

Nicklas, W., Baneux, P., Boot, R., Decelle, T., Deeny, A. A., Fumanelli, M., et al. (2002). Recommenda-tions for the health monitoring of rodent and rabbit colonies in breeding and experimental units. Lab-oratory Animals, 36, 20–42.

Stauffacher, M. (1995). Environmental enrichment, fact and fiction. Scandinavian Journal of Labora-tory Animal Science, 22, 39–42.

Tsai, P. P., Pachowsky, U., Stelzer, H. D., & Hackbarth, H. (2002). Impact of environmental enrichmentin mice: Effect of housing conditions on body weight, organ weights and haematology in differentstrains. Laboratory Animals, 36, 411–419.

Tsai, P. P., Stelzer, H. D., Hedrich, H. J., & Hackbarth, H. (2003). Are the effects of different designs onthe physiology and behaviour of DBA/2 mice consistent? Laboratory Animals, 31, 314–327.

Van de Weerd, H. A., & Baumans, V. (1995). Environmental enrichment in rodents: Environmental en-richment information resources for laboratory animals. AWIC Resources Series, 2, 145–149.

Van de Weerd, H. A., Van Loo, P. L. P., Van Zutphen, L. F. M., Koolhaas, J. M., & Baumans, V. (1997).Preferences for nesting material as environmental enrichment for laboratory mice. Laboratory Ani-mals, 31, 133–143.

Van de Weerd, H. A., Van Loo, P. L. P., Van Zutphen, L. F. M., Koolhaas, J. M., & Baumans, V. (1998).Strength of preference for nesting material as environmental enrichment for laboratory mice. Ap-plied Animal Behaviour Science, 55, 369–382.

Van Loo, P. L. P., Blom, H. J. M., Meijer, M. K., & Baumans, V. (2005). Assessment of the use of twocommercially available environmental enrichment by laboratory mice in preference testing. Labo-ratory Animals, 39, 58–67.

Van Loo, P. L. P., Kruitwagen, C. L. J. J., Koolhaas, J. M., Van de Weerd, H. A., Van Zutphen, L. F. M.,& Baumans, V. (2002). Influence of cage enrichment on aggressive behaviour and physiological pa-rameters in male mice. Applied Animal Behaviour Science, 76, 65–81.

Van Loo, P. L. P., Van der Meer, E., Kruitwagen, C. L. J. J., Koolhaas, J. M., Van Zutphen, L. F. M., &Baumans, V. (2004). Long term effects of husbandry procedures on stress-related parameters inmale mice of two strains. Laboratory Animals, 39, 169–177.

Van Loo, L. P. L., Van Zutphen, L. F. M., & Baumans, V. (2003). Male management: Coping with ag-gression problem in male laboratory mice. Laboratory Animals, 37, 300–313.

Wolfer, P. D., Litvin, O., Morf, S., Nitsch, R. M., Lipp, H. P., & Wurbel, H. (2004). Cage enrichment andmouse behaviour. Nature, 432, 821–822.

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