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The effects of cage enrichment on agonistic behaviour and dominance in male laboratory rats (Rattus norvegicus)
Research in Veterinary Science 90 (2011) 346–351
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The effects of cage enrichment on agonistic behaviour and dominance in male laboratory rats (Rattus norvegicus) Usama A. Abou-Ismail * Department of Animal Husbandry, Faculty of Veterinary Medicine, Mansoura University, Gomhoria St., Mansoura, P.O. Box 35516, Egypt
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Article history: Received 18 January 2010 Accepted 8 June 2010
Keywords: Laboratory rats Enrichment Agonistic interaction Dominance score
a b s t r a c t This experiment was carried out to investigate the effects of enriching laboratory cages on agonistic interaction and dominance of rats. In a series of three replicates, 48 rats were housed in groups of four in either ‘standard’ or ‘enriched’ cages for 6 weeks. Successful aggressive and defensive behaviour that ended up in a clear winner and loser were sampled in the first hour of the dark phase of the light/dark cycle every other week. Rats in the ‘complex’ cages showed lower levels of both successful aggressive and successful defensive bouts compared to rats in the ‘standard’ cages. Enriching cages of laboratory rat did not change the social order of the animals in the cage. Thus, enhancing the complexity of cages of laboratory rats by the particular cage modification regimen implemented in this experiment could be considered enrichment and could therefore result in an improvement of welfare in these animals. Ó 2010 Elsevier Ltd. All rights reserved.
1. Introduction Experiments on laboratory rodents have demonstrated wide effects of environmental modifications, including physiological (Belz et al., 2003; Chamove, 1989; Roy et al., 2001), psychological (Chamove, 1989; Patterson-Kane et al., 1999), developmental (Davenport et al., 1976) and therapeutic effects (Hockly et al., 2002; Passineau et al., 2001). In addition to the physiological, psychological, developmental and therapeutic advantages that housing in enriched conditions may provide, research has elucidated behavioural benefits from being housed in enriched environments (Armstrong et al., 1998; Chamove, 1989; Orok-Edem and Key, 1994; Van Loo et al., 2002). An important behaviour in group-housed laboratory animals is agonistic behaviour. Damaging social behaviour between conspecifics, such as excessive agonistic behaviour, is a common problem related to housing male laboratory rodents in captivity (e.g. Hurst et al., 1999; Van Loo et al., 2002), leading to physical damage and associated social stress and poor welfare (e.g. Hurst et al., 1996, 1999). It is interesting to note that there is a conflict between the results of experiments regarding the effects of environmental enrichment on agonistic interaction in laboratory rodents. Some experiments reported that the addition of environmental enrichment can, sometimes, reduce excessive aggression between rodents kept under standard unenriched housing conditions (Chamove, 1989; Armstrong et al., 1998; Orok-Edem and Key, * Tel.: +2 050 0237 2593, mobile: +2 0162557996; fax: +2 050 0237 9952. E-mail address: [email protected] 0034-5288/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.rvsc.2010.06.010
1994; Van Loo et al., 2002; Kaliste et al., 2006). Similar results of reduced aggressive encounters between members of group-housed animals in enriched housing conditions have been reported in other animals; pigs (O’Connell and Beattie, 1999), laying hens (Gvaryahu et al., 1994) and captive primates (Kitchen and Martin, 1996). Others have reported no differences in levels of agonistic interaction between animals housed in standard versus enriched laboratory cages (Marashi et al., 2004) due mainly to the use of low animal number per cage and the kinship of animals. However, in contrast to these findings, there are data that also reported an increase in agonistic behaviours between rodents housed in groups in ‘enriched’ housing conditions (Haemisch and Gartner, 1997; Haemisch et al., 1994; McGregor and Ayling, 1999; Nevison et al., 1999; Van Loo et al., 2002; Kaliste et al., 2006). This increase in the agonistic interaction between animals housed in enriched cages has been reported in laboratory mice particularly and is either due to the encouragement of territorial behaviours (Haemisch and Gartner, 1997; Haemisch et al., 1994), rigidity and lack of manipulability of the objects that decreases the ability of the animal to control its environment (e.g. Van Loo et al., 2002) or due to the exposure of vulnerable body parts such as tails to biting (Nevison et al., 1999; Van Loo et al., 2002). The increase in the level of agonistic interaction might counteract the general goal of enrichment to improve animal welfare since high levels of aggression may cause physical and/or psychical injury. It is therefore also interesting to emphasize that the effects of different environmental modification regimens depend mainly on the species and strains of the animals experiencing it (Chapillon et al., 1999; Nevison et al., 1999; Kaliste et al., 2006). Scott (1966) reported that agonistic interaction in rats and mice are
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different and that both species are alike in their inability to form complex dominance hierarchies in which fighting is reduced to threat and avoidance. Van de Weerd et al. (1994) reported that environmental ‘‘enrichment” may actually increase the level of anxiety for some strains of laboratory mice. Similarly, Nicol et al. (2008) showed that changing enrichments regularly can adversely affect some strains but not others. To the authors’ knowledge, a vast majority of the experimental work carried out on the effects of environmental modification on the welfare of laboratory rodents was done using experimental designs that supplied the laboratory cages with a single enrichment item such as gnawing materials (Orok-Edem and Key, 1994), shelters (Townsend, 1997) nesting materials (Haemisch and Gartner, 1997; Haemisch et al., 1994; Van Loo et al., 2002) or foraging substrates (Johnson et al., 2004). There is a preliminary evidence from research that increasing the degree of complexity of laboratory environment (extent of enrichment) may increase the effects of enrichment and therefore improve the welfare of animals experiencing it (Marashi et al., 2004). It is therefore not known how increasing the complexity of laboratory cages, by providing multiple physical structures, can affect some key behavioural patterns indicative of welfare in laboratory rodents such as inter-male aggression. Another major factor that determines how a particular cage modification regimen produces a change in behaviour of the animals experiencing it is whether the regimen relies on supplying items that remain unchanged throughout the study (reintroduced to the cages uncleaned after cage cleaning every week) or items that are replaced regularly during the study (replaced with exactly the same new and clean items after cage cleaning every week). There is an evidence that with some territorial species of laboratory rodents such as mice, the use of nesting material as enrichment could improve the welfare of mice (Van Loo et al., 2004a) providing that they are transferred during cage cleaning (Van Loo et al., 2004b). When social animals are housed together, a form of dominance order emerges as a result of the interaction between them. That social interaction between animals discharges a dominant and subordinate animal(s). It has been shown that the dominant animal is the one that has the priority in gaining access to the valued resources or who supplants its opponent and remove it away when they meet (e.g. Berdoy et al., 1995; Hurst et al., 1996). Despite that clear way of assigning dominance within grouphoused animals, other methods have also been used in experimental work. One extensively used method is the outcome of the agonistic interaction between two animals. Takahashi (1986) described dominant rat as the one shows more offensive (aggressive) behaviours such as on- top, lateral display and biting, while the rat shows few or no offensive behaviours as a subordinate. It was also defined that a dominant rat in a colony is the male that never loses whilst the subordinate is the rat that last shows defensive behaviour at the end of social confrontation (Fokkema et al., 1995). In dyadic interactions between rats the difference in the number of aggressive acts initiated and received has been used to classify rats into different social status (Popova and Naumenko, 1972; Militzer, 1982; Hurst et al., 1996). The same method was used, with more or less modification, to determine the dominant and subordinate animal within a colony of animals of other species such as mice (Poole and Morgan, 1976; Van Loo et al., 2000), hamsters (Huhman et al., 1990) and pigs (McGlone, 1993; Tuchscherer et al., 1998). Despite the fact that a very large number of studies has been carried out to investigate the effects of environmental enrichment in laboratory rodents, nearly none of these research has considered whether environmental enrichment changes dominance order of
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animals within the cage or not. Changing the dominance order of a stable group of rats has been shown to be stressful (e.g. Burman et al., 2008) and social disorganization produced by forced contact (grouping) between unrelated individuals has been shown to induce agonistic interaction and serious fighting in laboratory rodents (Scott, 1966). This experiment was therefore carried out to investigate the effects of a particular cage modification regimen (recruiting laboratory cages with multiple items that incorporated both renewal and cleaning every week and re-introduction uncleaned to the cage every week) on agonistic interaction of laboratory rats. Another aim of the experiment was to investigate whether increasing the complexity of cages of laboratory rats using multiple items affects the form of the dominance order within the cage. A further aim of the experiment was to study the form of dominance order in stable groups of newly weaned male laboratory rats.
2. Materials and methods 2.1. General animal housing and husbandry Outbred newly weaned male Wistar rats (Hannover strain) purchased from Harlan Link Ltd. (Bicester, UK) were used in this study. Upon arrival to the laboratory the animals were housed in large metal cages (70 cm length 52 cm width 36 cm height) for 5 days allowing them to adapt to laboratory conditions. These cages were supplied with sawdust (LIGNOCEL, RS, grade 1–2) as a bedding material. All cages were kept in the same room to avoid exposing the rats to environments with potentials to differ and hence confounding room with treatment. A pellet food (Eurodent diet 22%, 5LF5, PMI Nutrition International LLC, Brentwood, MO) and tap water were provided ad libitum and were refreshed daily. The rats were 6 weeks of age, on arrival, and weighed 45–60 g. They were maintained under an artificial 12:12 h light:dark cycle, with white light on between 1200 and 2400 and a continuous dim red lighting (two 60 W bulbs) to facilitate dark phase observation, at a temperature (20 ± 2 °C) and humidity (46% relative humidity). Once a week, all rats were removed from their cages and rehoused in clean cages with new bedding material. The fur of each rat was marked with hair dye (Clairol Nice n’easy Natural Black) in one of four different patterns on the day of arrival to allow individual identification. These marks were refreshed after 3 weeks, allowing sufficient time after dyeing before behavioural observation to reduce any possible effects on behaviour of the dyeing process (e.g. Hurst et al., 1999). Tails were also marked, in one of eight distinguishable manners, with a permanent marker pen to provide an additional means of identification. Tail marks were renewed every week. 2.2. Housing systems Three separate identical experiments with a duration of 5 weeks were carried out. In each experiment 16 rats were randomly allocated into groups of four and housed in polypropylene cages (48.5 cm length 33 cm width 21 cm height). Two groups were housed in cages with only sawdust bedding and two groups were housed in cages supplied with various enrichment objects. 1. ‘‘Standard”: polypropylene cages without any additional cages structures. 2. ‘‘Complex”: standard cages that were supplied with a number of additional cage structures such as gnawing objects (aspen wood blocks, wood balls and nylabones), shelter (rodent retreat), devices for climbing (ladders and ropes) and other
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Table 1 Description of the additional cage structures used to enhance the cage complexity. Structure
Number supplied
Provision and renewal
Description
Shredded paper Nestlets Aspen wood block Rodent retreat (shelter) Percher Crawl ball Rope Nylabone Ladder Wood ball
Handful 4 1 1 1 1 1 1 1 1
Renewed every week Renewed every week Renewed every week Reintroduced uncleaned to the cage Reintroduced uncleaned to the cage For one week only For one week only For one week only For one week only For one week only
5 cm 5 cm sterilized cotton fibre pads, Lillico, UK 34 mm 70 mm long aspen wood block, Lillico, UK 20.5 cm L 15.7 cm W 11.5 cm H Guinea pig huts, red-tinted, Lillico, UK Pure cotton rope and natural wood, Percher Spider, 4260 115 mm, with 3 58 mm holes, red-tinted polycarbonate, Lillico, UK 60 cm length of 1.5 cm diameter hemp rope, UK Regular size, original flavour, (36 g), Nylabone, Lillico, UK 9 step wooden ladder 35.5 cm Roll ‘N’ chew (small), natural wood 9 cm diameter
objects to stimulate general activity and gnawing such as crawl balls and wood balls. The description of these additional cage structures and the frequency of their provision are presented in Table 1. In addition to enhancing cage complexity, foraging opportunities were also increased. This was done by mixing four intact food pellets, and four pellets that had been broken into numerous small pieces with the bedding material after the cages were cleaned every week (e.g. Johnson et al., 2004).
cessful aggressive and successful defensive bouts were tested using an independent t-test. Data are presented as estimated marginal means (EMM) ± SE. To study changes of both successful aggressive and successful defensive bouts over the observation weeks a GLM-repeated measures was used. A full factorial repeated measure model was used with observation week (1, 3, and 5) as a within-subjects factor, and housing condition (1, 2) and batch (1–3) as between- subjects factors.
2.3. Behavioural sampling All behavioural observations were carried out by the same experienced researcher throughout the study. The observer entered the experimental room at least 5 min before the scheduled start time of the observation to allow the rats to habituate to his presence (e.g. Hurst et al., 1999). Behaviour sampling, or conspicuous behaviour recording, was used to record each occurrence of a complete aggressive and defensive bout between dyads of rats in each experimental group. All aggressive bouts that ended up with a clear winner (dominant ‘over’ position) and loser (submissive ‘on-back-posture’ position) were recorded. A rat was identified as a winner when it ended the confrontation adopting the dominant over position (an animal stands with its forepaws or all paws over its opponent who adopts the submissive on-back-posture, lies on its back and exposes the ventral surface of the body), (Baenninger, 1967; Meaney and Stewart, 1981; Panksepp, 1981; Takahashi, 1986). This form of behaviour was chosen because it is known to be a clear, easily scored, and quantified behaviour (Panksepp, 1981), but also because it represents the clear end point of aggressive encounters between rats in the laboratory situation. Behaviour sampling was carried out, using a check sheet, in one session for the first hour of the dark phase (1200) every other week. Each experimental group was observed for one day (1 h) per week (thus the four experimental groups were observed in 4 days every week; on Monday, Tuesday, Wednesday and Thursday). The day of observation was counterbalanced between different replicates to control for possible effect of observation day on behaviour. 2.4. Statistical analysis SPSS (version 12.0 for windows) was used for all statistical analyses. Data were checked for normality and homogeneity of variances to test for the suitability of using parametric tests. All data met assumptions of parametric statistics (normality, homogeneity of variance and linearity). 2.4.1. Differences between housing systems The group averages of total frequency of both successful aggressive and successful defensive bouts that ended in a clear winner and loser over the entire experimental period were calculated. Differences between the two housing systems in the frequency of suc-
2.4.2. Effects of housing systems on dominance and stability of dominance over time Successful aggressive (S.Ag) and successful defensive (S.Df) bouts were totalled for each rat in each observation week (1, 3, 5), and over the entire experimental period (T). For each housing system correlations between the total frequencies of successful aggressive bouts in each observation week and over the entire experimental period were measured using Pearson correlation test. Similarly, correlations between the total frequencies of successful defensive bouts were measured using Pearson correlation test. To determine the form of social dominance of laboratory rat, a further correlation between the total frequencies of both successful aggressive and successful defensive bouts in each observation week and over the entire experimental period were determined using a Partial correlation technique, partialling out the effect of housing system and replicate. 3. Results 3.1. Differences between housing systems There was a significant effect to the housing system on the frequency of successful aggressive bouts. Rats housed in the unenriched systems were found to be more frequently involved in successful aggressive bouts (t10 = 4.46, P < 0.01) as compared to those housed in the enriched systems (see Fig. 1). Similarly, rats of unenriched systems were found to be more frequently involved in successful defensive bouts (t10 = 3.82, P < 0.01) as compared to those housed in the enriched systems (see Fig. 2). There was however no effect to the observation week on the frequency of successful aggressive bouts (F2,24 = 2.01, NS) or the frequency of successful defensive bouts (F2,24 = 1.92, NS). Total frequency of successful aggressive and successful defensive bouts over the three observation weeks is shown in Table 2. 3.2. Effects of housing systems on dominance and stability of dominance over time For the enriched housing system, both total frequency of successful aggressive bouts and successful defensive bouts in the first observation week correlated positively with those of the fifth
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Average frequency of successful aggressive bouts
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Table 4 Pearson correlation coefficient of successful defensive bouts of both standard and complex housing system over the observation weeks and the entire experimental period.
***
18.00 16.00 14.00 12.00 10.00
S.Df1 S.Df3 S.Df5 S.DfT
8.00 6.00 4.00
S.Df1
S.Df3
S.Df5
S.DfT
1 NS 0.47* 0.49*
NS 1 0.44** 0.65**
0.47* 0.44** 1 0.62**
0.49* 0.65** 0.62** 1
NS = non-significant. P < 0.05. P < 0.01.
2.00
*
0.00
**
Enriched
Unenriched Treatment
Average frequency of successful defensive bouts
Fig. 1. EMM ± SE ‘Average frequency of successful aggressive bouts’ by the rats in the two housing systems. ***P < 0.001.
20.00
Table 5 Partial correlation coefficient of successful aggressive bouts over the observation weeks and the entire experimental period.
S.Ag1 S.Ag3 S.Ag5 S.AgT
***
18.00
S.Ag1 partial r
S.Ag3 partial r
S.Ag5 partial r
S.AgT partial r
1 NS 0.35* 0.67***
NS 1 0.39** 0.50***
0.35* 0.39** 1 0.51***
0.67*** 0.50*** 0.51*** 1
16.00 NS = non significant. P < 0.05. ** P < 0.01. *** P < 0.001.
14.00
*
12.00 10.00 8.00 6.00
Table 6 Partial correlation coefficient of successful defensive bouts over the observation weeks and the entire experimental period.
4.00 2.00 0.00 Enriched
Unenriched S.Df1 S.Df3 S.Df5 S.DfT
Treatment Fig. 2. EMM ± SE ‘Average frequency of successful defensive bouts’ by the rats in the two housing systems. ***P < 0.001.
S.Df1 partial r
S.Df3 partial r
S.Df5 partial r
S.DfT partial r
1 NS 0.41* 0.66***
NS 1 0.44** 0.49***
0.41* 0.44** 1 0.71***
0.66*** 0.49*** 0.71*** 1
NS = non-significant. P < 0.05. ** P < 0.01. *** P < 0.001. *
Table 2 Total frequency of successful aggressive bouts and successful defensive bouts over the three observation weeks.
Complex cages Standard cages
Total frequency of successful aggressive bouts
Total frequency of successful defensive bouts
Week 1
Week 3
Week 5
Week 1
Week 3
Week 5
74 129
57 113
62 113
74 129
57 114
62 114
Partial correlation technique also showed similar results. Regardless the treatment and replicate, there were positive correlations between the total frequencies of both successful aggressive and successful defensive bouts of the first observation week and those of the fifth observation week and the entire experimental period (Tables 5 and 6). 4. Discussion
Table 3 Pearson correlation coefficient of successful aggressive bouts of both standard and complex housing system over the observation weeks and the entire experimental period. S.Ag1 S.Ag1 S.Ag3 S.Ag5 S.AgT
1 NS 0.32* 0.44*
S.Ag3 NS 1 0.48* 0.65**
S.Ag5 *
0.32 0.48* 1 0.63**
S.AgT 0.44* 0.65** 0.63** 1
NS = non-significant. P < 0.05. P < 0.01.
*
**
observation week and with the total frequency of successful aggressive and defensive bouts of the entire experimental period (Tables 3 and 4). All of these findings were similarly evident when data of the unenriched housing system was analysed on its own.
The results of this experiment demonstrated that rats housed in enriched cages showed lower levels of agonistic activities as compared to rats housed in standard cages. One possibility for the reduced agonistic interaction between rats in the complex cages is that in unenriched cages, due to the lack of any physical structures, rats themselves may be a more prominent focus of their cagemates’ attention, and therefore attract relatively more non-aggressive and aggressive interaction. Non-aggressive social investigation has previously been reported to promote aggressive responses (e.g. Robitaille and Bovet, 1976). The lack of cage structures may also simply increase the chance that two rats come into direct contact with each other. Close proximity of animals was reported to increase aggression in mice (Van Loo et al., 2001), and the haphazard collision of two running rats might also induce an aggressive response (e.g. Robitaille and Bovet, 1976). The provision of additional structures such as a shelter, bedding and nesting materials and crawling balls into enriched cages may also have provided rats
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with an increased opportunity to escape aggressive attacks by cage mates, whereas in unenriched cages, retreat may have been less easy to achieve, due to the lack of suitable shelters and refuges, resulting in a frustrated escape response (see Hurst et al., 1999). The ability to escape an attacker in enriched cages may have resulted in fewer decisive aggressive encounters whereas in unenriched cages, where escape may have been more difficult, loser rats may have ended up adopting the submissive posture more frequently, resulting in the dominant likely embarking in a bout of aggressive grooming (see Hurst et al., 1999). The reduction of agonistic behaviours in enriched conditions may also have been due to the nature of the husbandry procedures used in this experiment, cage cleaning in particular. Cages of all rats were cleaned out completely once a week. Whilst this process was carried out in exactly the same way for both housing types, it involved returning some of the enrichment items such as the shelter, uncleaned. The re-introduction of these items to the enriched cages may have imposed some level of familiarity to the environment (by providing familiar olfactory and visual cues), which may in turn have reduced agonistic interaction between rats in these cages. It has been shown in laboratory mice that incomplete cage cleaning regimes that allow retention of some uncleaned objects (e.g. nesting but not bedding materials) decreased inter-male aggression (Van Loo et al., 2000). Similarly, long-term enrichment with nesting material combined with the repeated transfer of nesting material when cleaning the cages reduced several parameters indicative of stress (Van Loo et al., 2004b). Orok-Edem and Key (1994) demonstrated that provision of some chewable items, such as gnawing blocks and wooden tongue depressors to the cages of laboratory rats, reduced the frequency of fighting, although the study only lasted 5 days and any novelty effects may not have worn off in this time. It is possible that allowing the animals to exert simple control over the environment (e.g. by providing objects that can promote the expression of species-specific behaviours such as gnawing), motivation for agonistic interactions may decrease. The finding of reduced agonistic interactions between groups of male laboratory rats housed in enriched cages reported in the current experiment is in accordance with the previous results reported by Chamove (1989), Gvaryahu et al. (1994), Orok-Edem and Key (1994), Kitchen and Martin (1996), Armstrong et al. (1998), O’Connell and Beattie (1999), Van Loo et al. (2002) and Kaliste et al. (2006) who found that animals reared in enriched environments displayed lower levels of agonistic interactions as compared to their counterparts reared in unenriched environments. In contrast, the result of the current experiment disagrees with those reported by Haemisch and Gartner (1997), Haemisch et al. (1994), Nevison et al. (1999), Van Loo et al. (2002) and Kaliste et al. (2006) who reported that housing laboratory mice in enriched conditions increased their agonistic interaction as compared to their counterparts housed in unenriched conditions. The increase of agonistic behaviours in enriched cages was interpreted as to be due to either close proximity and exposure of parts of the body that are vulnerable for biting, such as tails (Nevison et al., 1999; Van Loo et al., 2002), removal of scent marks necessary for territory formation (Kaliste et al., 2006) or stimulation of territorial behaviour (Haemisch and Gartner, 1997; Haemisch et al., 1994). This increased aggression in enriched environments appears to be a species difference in response to the housing environment, with male laboratory mice being less socially tolerant and more territorial than other species (e.g. Gray and Hurst, 1995; Rich and Hurst, 1997). The results of the correlation test showed that, in enriched cages, there were significant positive correlations between the frequency of both successful aggressive and successful defensive bouts of rats over the observation weeks and between them and
those of the total experimental period. The correlation between total frequencies of aggressive and defensive bouts of first and third week was not significant. These results were also evident for the rats in unenriched cages. This means that there was always a clear dominant and a clear subordinate animal in each cage of the enriched cages. It has previously been demonstrated that when laboratory rats are housed in groups in standard laboratory cages, they may behave aggressively towards each other in order to develop a stable social hierarchy (Grant and Chance, 1958; Scott, 1966; Stefanski, 2001; Stefanski et al., 2001). The results of this experiment demonstrate therefore that environmental enrichment, despite decreasing the agonistic interaction in laboratory cages, does not change dominance order. This could be due to the fact that the addition of physical items such as rodent retreat, wooden balls and perchers may compartmentalize the cages into various partitions which may in turn allow animals to escape their conspecific’s attacks and to mark their territories. The lack of positive correlations between aggressive and defensive bouts of the first and third week may reflect that the dominance order was not yet formed and that it needs more than 2 weeks to develop. The results of the partial correlation technique showed that there were also positive correlations between successful aggressive and successful defensive bouts over the observation weeks, except for the first and third week, and between them and those of the entire experimental period. This means that regardless, the housing system and replicate there was always a clear dominant and a clear subordinate animal within each cage. This finding is in accordance with those of Grant and Chance (1958) and Baenninger (1966) who reported that groups of rats (up to five animals per cage), if kept in harmony and undisturbed, can develop a stable form of nearly linear social hierarchy post weaning and over 9 weeks time. The way that was used in this experiment to assign dominance score in laboratory rats appears therefore reliable under different housing environment of standard and enriched laboratory cages. The findings of the present study thus strongly support the need of the current conventional housing systems of laboratory rats for re-evaluation to help provide better environment for the animals that can in turn result in an improvement in their welfare. The type of cage modification implemented in this study was of affordable cost, practical to use, clean and easy to replace, did not compromise the physical health of the rats, nor did it prevent ease of checking the animals. Importantly, this particular type of cage modification provided the rats with ample opportunities to cope with and to exert control over their environment; characteristics that resulted in improved welfare in the animals experiencing it. 5. Conclusion and implications for welfare Results of this experiment showed that environmental enrichment may affect some key behaviours of the laboratory rat such as agonistic interaction. Prolonged housing of rats at highly complex cages reduced their agonistic interaction and, most importantly, produced no change in the dominance order. Taking these findings together, it appears that enhancing the complexity of cages of laboratory rats by the particular cage modification regimen implemented in this experiment could be considered enrichment and could therefore result in an improvement of welfare in these animals. Acknowledgements This study was carried out as a part of a bigger project and was funded by the UK Home Office, as recommended by the Animal Procedures Committee (HOAPC). The author would like to thank two anonymous referees for their useful comments.
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Research in Veterinary Science journal homepage: www.elsevier.com/locate/rvsc
The effects of cage enrichment on agonistic behaviour and dominance in male laboratory rats (Rattus norvegicus) Usama A. Abou-Ismail * Department of Animal Husbandry, Faculty of Veterinary Medicine, Mansoura University, Gomhoria St., Mansoura, P.O. Box 35516, Egypt
a r t i c l e
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Article history: Received 18 January 2010 Accepted 8 June 2010
Keywords: Laboratory rats Enrichment Agonistic interaction Dominance score
a b s t r a c t This experiment was carried out to investigate the effects of enriching laboratory cages on agonistic interaction and dominance of rats. In a series of three replicates, 48 rats were housed in groups of four in either ‘standard’ or ‘enriched’ cages for 6 weeks. Successful aggressive and defensive behaviour that ended up in a clear winner and loser were sampled in the first hour of the dark phase of the light/dark cycle every other week. Rats in the ‘complex’ cages showed lower levels of both successful aggressive and successful defensive bouts compared to rats in the ‘standard’ cages. Enriching cages of laboratory rat did not change the social order of the animals in the cage. Thus, enhancing the complexity of cages of laboratory rats by the particular cage modification regimen implemented in this experiment could be considered enrichment and could therefore result in an improvement of welfare in these animals. Ó 2010 Elsevier Ltd. All rights reserved.
1. Introduction Experiments on laboratory rodents have demonstrated wide effects of environmental modifications, including physiological (Belz et al., 2003; Chamove, 1989; Roy et al., 2001), psychological (Chamove, 1989; Patterson-Kane et al., 1999), developmental (Davenport et al., 1976) and therapeutic effects (Hockly et al., 2002; Passineau et al., 2001). In addition to the physiological, psychological, developmental and therapeutic advantages that housing in enriched conditions may provide, research has elucidated behavioural benefits from being housed in enriched environments (Armstrong et al., 1998; Chamove, 1989; Orok-Edem and Key, 1994; Van Loo et al., 2002). An important behaviour in group-housed laboratory animals is agonistic behaviour. Damaging social behaviour between conspecifics, such as excessive agonistic behaviour, is a common problem related to housing male laboratory rodents in captivity (e.g. Hurst et al., 1999; Van Loo et al., 2002), leading to physical damage and associated social stress and poor welfare (e.g. Hurst et al., 1996, 1999). It is interesting to note that there is a conflict between the results of experiments regarding the effects of environmental enrichment on agonistic interaction in laboratory rodents. Some experiments reported that the addition of environmental enrichment can, sometimes, reduce excessive aggression between rodents kept under standard unenriched housing conditions (Chamove, 1989; Armstrong et al., 1998; Orok-Edem and Key, * Tel.: +2 050 0237 2593, mobile: +2 0162557996; fax: +2 050 0237 9952. E-mail address: [email protected] 0034-5288/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.rvsc.2010.06.010
1994; Van Loo et al., 2002; Kaliste et al., 2006). Similar results of reduced aggressive encounters between members of group-housed animals in enriched housing conditions have been reported in other animals; pigs (O’Connell and Beattie, 1999), laying hens (Gvaryahu et al., 1994) and captive primates (Kitchen and Martin, 1996). Others have reported no differences in levels of agonistic interaction between animals housed in standard versus enriched laboratory cages (Marashi et al., 2004) due mainly to the use of low animal number per cage and the kinship of animals. However, in contrast to these findings, there are data that also reported an increase in agonistic behaviours between rodents housed in groups in ‘enriched’ housing conditions (Haemisch and Gartner, 1997; Haemisch et al., 1994; McGregor and Ayling, 1999; Nevison et al., 1999; Van Loo et al., 2002; Kaliste et al., 2006). This increase in the agonistic interaction between animals housed in enriched cages has been reported in laboratory mice particularly and is either due to the encouragement of territorial behaviours (Haemisch and Gartner, 1997; Haemisch et al., 1994), rigidity and lack of manipulability of the objects that decreases the ability of the animal to control its environment (e.g. Van Loo et al., 2002) or due to the exposure of vulnerable body parts such as tails to biting (Nevison et al., 1999; Van Loo et al., 2002). The increase in the level of agonistic interaction might counteract the general goal of enrichment to improve animal welfare since high levels of aggression may cause physical and/or psychical injury. It is therefore also interesting to emphasize that the effects of different environmental modification regimens depend mainly on the species and strains of the animals experiencing it (Chapillon et al., 1999; Nevison et al., 1999; Kaliste et al., 2006). Scott (1966) reported that agonistic interaction in rats and mice are
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different and that both species are alike in their inability to form complex dominance hierarchies in which fighting is reduced to threat and avoidance. Van de Weerd et al. (1994) reported that environmental ‘‘enrichment” may actually increase the level of anxiety for some strains of laboratory mice. Similarly, Nicol et al. (2008) showed that changing enrichments regularly can adversely affect some strains but not others. To the authors’ knowledge, a vast majority of the experimental work carried out on the effects of environmental modification on the welfare of laboratory rodents was done using experimental designs that supplied the laboratory cages with a single enrichment item such as gnawing materials (Orok-Edem and Key, 1994), shelters (Townsend, 1997) nesting materials (Haemisch and Gartner, 1997; Haemisch et al., 1994; Van Loo et al., 2002) or foraging substrates (Johnson et al., 2004). There is a preliminary evidence from research that increasing the degree of complexity of laboratory environment (extent of enrichment) may increase the effects of enrichment and therefore improve the welfare of animals experiencing it (Marashi et al., 2004). It is therefore not known how increasing the complexity of laboratory cages, by providing multiple physical structures, can affect some key behavioural patterns indicative of welfare in laboratory rodents such as inter-male aggression. Another major factor that determines how a particular cage modification regimen produces a change in behaviour of the animals experiencing it is whether the regimen relies on supplying items that remain unchanged throughout the study (reintroduced to the cages uncleaned after cage cleaning every week) or items that are replaced regularly during the study (replaced with exactly the same new and clean items after cage cleaning every week). There is an evidence that with some territorial species of laboratory rodents such as mice, the use of nesting material as enrichment could improve the welfare of mice (Van Loo et al., 2004a) providing that they are transferred during cage cleaning (Van Loo et al., 2004b). When social animals are housed together, a form of dominance order emerges as a result of the interaction between them. That social interaction between animals discharges a dominant and subordinate animal(s). It has been shown that the dominant animal is the one that has the priority in gaining access to the valued resources or who supplants its opponent and remove it away when they meet (e.g. Berdoy et al., 1995; Hurst et al., 1996). Despite that clear way of assigning dominance within grouphoused animals, other methods have also been used in experimental work. One extensively used method is the outcome of the agonistic interaction between two animals. Takahashi (1986) described dominant rat as the one shows more offensive (aggressive) behaviours such as on- top, lateral display and biting, while the rat shows few or no offensive behaviours as a subordinate. It was also defined that a dominant rat in a colony is the male that never loses whilst the subordinate is the rat that last shows defensive behaviour at the end of social confrontation (Fokkema et al., 1995). In dyadic interactions between rats the difference in the number of aggressive acts initiated and received has been used to classify rats into different social status (Popova and Naumenko, 1972; Militzer, 1982; Hurst et al., 1996). The same method was used, with more or less modification, to determine the dominant and subordinate animal within a colony of animals of other species such as mice (Poole and Morgan, 1976; Van Loo et al., 2000), hamsters (Huhman et al., 1990) and pigs (McGlone, 1993; Tuchscherer et al., 1998). Despite the fact that a very large number of studies has been carried out to investigate the effects of environmental enrichment in laboratory rodents, nearly none of these research has considered whether environmental enrichment changes dominance order of
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animals within the cage or not. Changing the dominance order of a stable group of rats has been shown to be stressful (e.g. Burman et al., 2008) and social disorganization produced by forced contact (grouping) between unrelated individuals has been shown to induce agonistic interaction and serious fighting in laboratory rodents (Scott, 1966). This experiment was therefore carried out to investigate the effects of a particular cage modification regimen (recruiting laboratory cages with multiple items that incorporated both renewal and cleaning every week and re-introduction uncleaned to the cage every week) on agonistic interaction of laboratory rats. Another aim of the experiment was to investigate whether increasing the complexity of cages of laboratory rats using multiple items affects the form of the dominance order within the cage. A further aim of the experiment was to study the form of dominance order in stable groups of newly weaned male laboratory rats.
2. Materials and methods 2.1. General animal housing and husbandry Outbred newly weaned male Wistar rats (Hannover strain) purchased from Harlan Link Ltd. (Bicester, UK) were used in this study. Upon arrival to the laboratory the animals were housed in large metal cages (70 cm length 52 cm width 36 cm height) for 5 days allowing them to adapt to laboratory conditions. These cages were supplied with sawdust (LIGNOCEL, RS, grade 1–2) as a bedding material. All cages were kept in the same room to avoid exposing the rats to environments with potentials to differ and hence confounding room with treatment. A pellet food (Eurodent diet 22%, 5LF5, PMI Nutrition International LLC, Brentwood, MO) and tap water were provided ad libitum and were refreshed daily. The rats were 6 weeks of age, on arrival, and weighed 45–60 g. They were maintained under an artificial 12:12 h light:dark cycle, with white light on between 1200 and 2400 and a continuous dim red lighting (two 60 W bulbs) to facilitate dark phase observation, at a temperature (20 ± 2 °C) and humidity (46% relative humidity). Once a week, all rats were removed from their cages and rehoused in clean cages with new bedding material. The fur of each rat was marked with hair dye (Clairol Nice n’easy Natural Black) in one of four different patterns on the day of arrival to allow individual identification. These marks were refreshed after 3 weeks, allowing sufficient time after dyeing before behavioural observation to reduce any possible effects on behaviour of the dyeing process (e.g. Hurst et al., 1999). Tails were also marked, in one of eight distinguishable manners, with a permanent marker pen to provide an additional means of identification. Tail marks were renewed every week. 2.2. Housing systems Three separate identical experiments with a duration of 5 weeks were carried out. In each experiment 16 rats were randomly allocated into groups of four and housed in polypropylene cages (48.5 cm length 33 cm width 21 cm height). Two groups were housed in cages with only sawdust bedding and two groups were housed in cages supplied with various enrichment objects. 1. ‘‘Standard”: polypropylene cages without any additional cages structures. 2. ‘‘Complex”: standard cages that were supplied with a number of additional cage structures such as gnawing objects (aspen wood blocks, wood balls and nylabones), shelter (rodent retreat), devices for climbing (ladders and ropes) and other
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Table 1 Description of the additional cage structures used to enhance the cage complexity. Structure
Number supplied
Provision and renewal
Description
Shredded paper Nestlets Aspen wood block Rodent retreat (shelter) Percher Crawl ball Rope Nylabone Ladder Wood ball
Handful 4 1 1 1 1 1 1 1 1
Renewed every week Renewed every week Renewed every week Reintroduced uncleaned to the cage Reintroduced uncleaned to the cage For one week only For one week only For one week only For one week only For one week only
5 cm 5 cm sterilized cotton fibre pads, Lillico, UK 34 mm 70 mm long aspen wood block, Lillico, UK 20.5 cm L 15.7 cm W 11.5 cm H Guinea pig huts, red-tinted, Lillico, UK Pure cotton rope and natural wood, Percher Spider, 4260 115 mm, with 3 58 mm holes, red-tinted polycarbonate, Lillico, UK 60 cm length of 1.5 cm diameter hemp rope, UK Regular size, original flavour, (36 g), Nylabone, Lillico, UK 9 step wooden ladder 35.5 cm Roll ‘N’ chew (small), natural wood 9 cm diameter
objects to stimulate general activity and gnawing such as crawl balls and wood balls. The description of these additional cage structures and the frequency of their provision are presented in Table 1. In addition to enhancing cage complexity, foraging opportunities were also increased. This was done by mixing four intact food pellets, and four pellets that had been broken into numerous small pieces with the bedding material after the cages were cleaned every week (e.g. Johnson et al., 2004).
cessful aggressive and successful defensive bouts were tested using an independent t-test. Data are presented as estimated marginal means (EMM) ± SE. To study changes of both successful aggressive and successful defensive bouts over the observation weeks a GLM-repeated measures was used. A full factorial repeated measure model was used with observation week (1, 3, and 5) as a within-subjects factor, and housing condition (1, 2) and batch (1–3) as between- subjects factors.
2.3. Behavioural sampling All behavioural observations were carried out by the same experienced researcher throughout the study. The observer entered the experimental room at least 5 min before the scheduled start time of the observation to allow the rats to habituate to his presence (e.g. Hurst et al., 1999). Behaviour sampling, or conspicuous behaviour recording, was used to record each occurrence of a complete aggressive and defensive bout between dyads of rats in each experimental group. All aggressive bouts that ended up with a clear winner (dominant ‘over’ position) and loser (submissive ‘on-back-posture’ position) were recorded. A rat was identified as a winner when it ended the confrontation adopting the dominant over position (an animal stands with its forepaws or all paws over its opponent who adopts the submissive on-back-posture, lies on its back and exposes the ventral surface of the body), (Baenninger, 1967; Meaney and Stewart, 1981; Panksepp, 1981; Takahashi, 1986). This form of behaviour was chosen because it is known to be a clear, easily scored, and quantified behaviour (Panksepp, 1981), but also because it represents the clear end point of aggressive encounters between rats in the laboratory situation. Behaviour sampling was carried out, using a check sheet, in one session for the first hour of the dark phase (1200) every other week. Each experimental group was observed for one day (1 h) per week (thus the four experimental groups were observed in 4 days every week; on Monday, Tuesday, Wednesday and Thursday). The day of observation was counterbalanced between different replicates to control for possible effect of observation day on behaviour. 2.4. Statistical analysis SPSS (version 12.0 for windows) was used for all statistical analyses. Data were checked for normality and homogeneity of variances to test for the suitability of using parametric tests. All data met assumptions of parametric statistics (normality, homogeneity of variance and linearity). 2.4.1. Differences between housing systems The group averages of total frequency of both successful aggressive and successful defensive bouts that ended in a clear winner and loser over the entire experimental period were calculated. Differences between the two housing systems in the frequency of suc-
2.4.2. Effects of housing systems on dominance and stability of dominance over time Successful aggressive (S.Ag) and successful defensive (S.Df) bouts were totalled for each rat in each observation week (1, 3, 5), and over the entire experimental period (T). For each housing system correlations between the total frequencies of successful aggressive bouts in each observation week and over the entire experimental period were measured using Pearson correlation test. Similarly, correlations between the total frequencies of successful defensive bouts were measured using Pearson correlation test. To determine the form of social dominance of laboratory rat, a further correlation between the total frequencies of both successful aggressive and successful defensive bouts in each observation week and over the entire experimental period were determined using a Partial correlation technique, partialling out the effect of housing system and replicate. 3. Results 3.1. Differences between housing systems There was a significant effect to the housing system on the frequency of successful aggressive bouts. Rats housed in the unenriched systems were found to be more frequently involved in successful aggressive bouts (t10 = 4.46, P < 0.01) as compared to those housed in the enriched systems (see Fig. 1). Similarly, rats of unenriched systems were found to be more frequently involved in successful defensive bouts (t10 = 3.82, P < 0.01) as compared to those housed in the enriched systems (see Fig. 2). There was however no effect to the observation week on the frequency of successful aggressive bouts (F2,24 = 2.01, NS) or the frequency of successful defensive bouts (F2,24 = 1.92, NS). Total frequency of successful aggressive and successful defensive bouts over the three observation weeks is shown in Table 2. 3.2. Effects of housing systems on dominance and stability of dominance over time For the enriched housing system, both total frequency of successful aggressive bouts and successful defensive bouts in the first observation week correlated positively with those of the fifth
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Average frequency of successful aggressive bouts
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Table 4 Pearson correlation coefficient of successful defensive bouts of both standard and complex housing system over the observation weeks and the entire experimental period.
***
18.00 16.00 14.00 12.00 10.00
S.Df1 S.Df3 S.Df5 S.DfT
8.00 6.00 4.00
S.Df1
S.Df3
S.Df5
S.DfT
1 NS 0.47* 0.49*
NS 1 0.44** 0.65**
0.47* 0.44** 1 0.62**
0.49* 0.65** 0.62** 1
NS = non-significant. P < 0.05. P < 0.01.
2.00
*
0.00
**
Enriched
Unenriched Treatment
Average frequency of successful defensive bouts
Fig. 1. EMM ± SE ‘Average frequency of successful aggressive bouts’ by the rats in the two housing systems. ***P < 0.001.
20.00
Table 5 Partial correlation coefficient of successful aggressive bouts over the observation weeks and the entire experimental period.
S.Ag1 S.Ag3 S.Ag5 S.AgT
***
18.00
S.Ag1 partial r
S.Ag3 partial r
S.Ag5 partial r
S.AgT partial r
1 NS 0.35* 0.67***
NS 1 0.39** 0.50***
0.35* 0.39** 1 0.51***
0.67*** 0.50*** 0.51*** 1
16.00 NS = non significant. P < 0.05. ** P < 0.01. *** P < 0.001.
14.00
*
12.00 10.00 8.00 6.00
Table 6 Partial correlation coefficient of successful defensive bouts over the observation weeks and the entire experimental period.
4.00 2.00 0.00 Enriched
Unenriched S.Df1 S.Df3 S.Df5 S.DfT
Treatment Fig. 2. EMM ± SE ‘Average frequency of successful defensive bouts’ by the rats in the two housing systems. ***P < 0.001.
S.Df1 partial r
S.Df3 partial r
S.Df5 partial r
S.DfT partial r
1 NS 0.41* 0.66***
NS 1 0.44** 0.49***
0.41* 0.44** 1 0.71***
0.66*** 0.49*** 0.71*** 1
NS = non-significant. P < 0.05. ** P < 0.01. *** P < 0.001. *
Table 2 Total frequency of successful aggressive bouts and successful defensive bouts over the three observation weeks.
Complex cages Standard cages
Total frequency of successful aggressive bouts
Total frequency of successful defensive bouts
Week 1
Week 3
Week 5
Week 1
Week 3
Week 5
74 129
57 113
62 113
74 129
57 114
62 114
Partial correlation technique also showed similar results. Regardless the treatment and replicate, there were positive correlations between the total frequencies of both successful aggressive and successful defensive bouts of the first observation week and those of the fifth observation week and the entire experimental period (Tables 5 and 6). 4. Discussion
Table 3 Pearson correlation coefficient of successful aggressive bouts of both standard and complex housing system over the observation weeks and the entire experimental period. S.Ag1 S.Ag1 S.Ag3 S.Ag5 S.AgT
1 NS 0.32* 0.44*
S.Ag3 NS 1 0.48* 0.65**
S.Ag5 *
0.32 0.48* 1 0.63**
S.AgT 0.44* 0.65** 0.63** 1
NS = non-significant. P < 0.05. P < 0.01.
*
**
observation week and with the total frequency of successful aggressive and defensive bouts of the entire experimental period (Tables 3 and 4). All of these findings were similarly evident when data of the unenriched housing system was analysed on its own.
The results of this experiment demonstrated that rats housed in enriched cages showed lower levels of agonistic activities as compared to rats housed in standard cages. One possibility for the reduced agonistic interaction between rats in the complex cages is that in unenriched cages, due to the lack of any physical structures, rats themselves may be a more prominent focus of their cagemates’ attention, and therefore attract relatively more non-aggressive and aggressive interaction. Non-aggressive social investigation has previously been reported to promote aggressive responses (e.g. Robitaille and Bovet, 1976). The lack of cage structures may also simply increase the chance that two rats come into direct contact with each other. Close proximity of animals was reported to increase aggression in mice (Van Loo et al., 2001), and the haphazard collision of two running rats might also induce an aggressive response (e.g. Robitaille and Bovet, 1976). The provision of additional structures such as a shelter, bedding and nesting materials and crawling balls into enriched cages may also have provided rats
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with an increased opportunity to escape aggressive attacks by cage mates, whereas in unenriched cages, retreat may have been less easy to achieve, due to the lack of suitable shelters and refuges, resulting in a frustrated escape response (see Hurst et al., 1999). The ability to escape an attacker in enriched cages may have resulted in fewer decisive aggressive encounters whereas in unenriched cages, where escape may have been more difficult, loser rats may have ended up adopting the submissive posture more frequently, resulting in the dominant likely embarking in a bout of aggressive grooming (see Hurst et al., 1999). The reduction of agonistic behaviours in enriched conditions may also have been due to the nature of the husbandry procedures used in this experiment, cage cleaning in particular. Cages of all rats were cleaned out completely once a week. Whilst this process was carried out in exactly the same way for both housing types, it involved returning some of the enrichment items such as the shelter, uncleaned. The re-introduction of these items to the enriched cages may have imposed some level of familiarity to the environment (by providing familiar olfactory and visual cues), which may in turn have reduced agonistic interaction between rats in these cages. It has been shown in laboratory mice that incomplete cage cleaning regimes that allow retention of some uncleaned objects (e.g. nesting but not bedding materials) decreased inter-male aggression (Van Loo et al., 2000). Similarly, long-term enrichment with nesting material combined with the repeated transfer of nesting material when cleaning the cages reduced several parameters indicative of stress (Van Loo et al., 2004b). Orok-Edem and Key (1994) demonstrated that provision of some chewable items, such as gnawing blocks and wooden tongue depressors to the cages of laboratory rats, reduced the frequency of fighting, although the study only lasted 5 days and any novelty effects may not have worn off in this time. It is possible that allowing the animals to exert simple control over the environment (e.g. by providing objects that can promote the expression of species-specific behaviours such as gnawing), motivation for agonistic interactions may decrease. The finding of reduced agonistic interactions between groups of male laboratory rats housed in enriched cages reported in the current experiment is in accordance with the previous results reported by Chamove (1989), Gvaryahu et al. (1994), Orok-Edem and Key (1994), Kitchen and Martin (1996), Armstrong et al. (1998), O’Connell and Beattie (1999), Van Loo et al. (2002) and Kaliste et al. (2006) who found that animals reared in enriched environments displayed lower levels of agonistic interactions as compared to their counterparts reared in unenriched environments. In contrast, the result of the current experiment disagrees with those reported by Haemisch and Gartner (1997), Haemisch et al. (1994), Nevison et al. (1999), Van Loo et al. (2002) and Kaliste et al. (2006) who reported that housing laboratory mice in enriched conditions increased their agonistic interaction as compared to their counterparts housed in unenriched conditions. The increase of agonistic behaviours in enriched cages was interpreted as to be due to either close proximity and exposure of parts of the body that are vulnerable for biting, such as tails (Nevison et al., 1999; Van Loo et al., 2002), removal of scent marks necessary for territory formation (Kaliste et al., 2006) or stimulation of territorial behaviour (Haemisch and Gartner, 1997; Haemisch et al., 1994). This increased aggression in enriched environments appears to be a species difference in response to the housing environment, with male laboratory mice being less socially tolerant and more territorial than other species (e.g. Gray and Hurst, 1995; Rich and Hurst, 1997). The results of the correlation test showed that, in enriched cages, there were significant positive correlations between the frequency of both successful aggressive and successful defensive bouts of rats over the observation weeks and between them and
those of the total experimental period. The correlation between total frequencies of aggressive and defensive bouts of first and third week was not significant. These results were also evident for the rats in unenriched cages. This means that there was always a clear dominant and a clear subordinate animal in each cage of the enriched cages. It has previously been demonstrated that when laboratory rats are housed in groups in standard laboratory cages, they may behave aggressively towards each other in order to develop a stable social hierarchy (Grant and Chance, 1958; Scott, 1966; Stefanski, 2001; Stefanski et al., 2001). The results of this experiment demonstrate therefore that environmental enrichment, despite decreasing the agonistic interaction in laboratory cages, does not change dominance order. This could be due to the fact that the addition of physical items such as rodent retreat, wooden balls and perchers may compartmentalize the cages into various partitions which may in turn allow animals to escape their conspecific’s attacks and to mark their territories. The lack of positive correlations between aggressive and defensive bouts of the first and third week may reflect that the dominance order was not yet formed and that it needs more than 2 weeks to develop. The results of the partial correlation technique showed that there were also positive correlations between successful aggressive and successful defensive bouts over the observation weeks, except for the first and third week, and between them and those of the entire experimental period. This means that regardless, the housing system and replicate there was always a clear dominant and a clear subordinate animal within each cage. This finding is in accordance with those of Grant and Chance (1958) and Baenninger (1966) who reported that groups of rats (up to five animals per cage), if kept in harmony and undisturbed, can develop a stable form of nearly linear social hierarchy post weaning and over 9 weeks time. The way that was used in this experiment to assign dominance score in laboratory rats appears therefore reliable under different housing environment of standard and enriched laboratory cages. The findings of the present study thus strongly support the need of the current conventional housing systems of laboratory rats for re-evaluation to help provide better environment for the animals that can in turn result in an improvement in their welfare. The type of cage modification implemented in this study was of affordable cost, practical to use, clean and easy to replace, did not compromise the physical health of the rats, nor did it prevent ease of checking the animals. Importantly, this particular type of cage modification provided the rats with ample opportunities to cope with and to exert control over their environment; characteristics that resulted in improved welfare in the animals experiencing it. 5. Conclusion and implications for welfare Results of this experiment showed that environmental enrichment may affect some key behaviours of the laboratory rat such as agonistic interaction. Prolonged housing of rats at highly complex cages reduced their agonistic interaction and, most importantly, produced no change in the dominance order. Taking these findings together, it appears that enhancing the complexity of cages of laboratory rats by the particular cage modification regimen implemented in this experiment could be considered enrichment and could therefore result in an improvement of welfare in these animals. Acknowledgements This study was carried out as a part of a bigger project and was funded by the UK Home Office, as recommended by the Animal Procedures Committee (HOAPC). The author would like to thank two anonymous referees for their useful comments.
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