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Effects of a gentling programme on the behaviour of laboratory rats towards humans
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Applied Animal Behaviour Science 114 (2008) 554–571 www.elsevier.com/locate/applanim
Effects of a gentling programme on the behaviour of laboratory rats towards humans Barbara M. Maurer a, Dorothea Do¨ring a,*, Fabian Scheipl b, Helmut Ku¨chenhoff b, Michael H. Erhard a a
Department of Veterinary Sciences, Veterinary Faculty of Ludwig-Maximilians University, Schwere-Reiter-Str. 9, Munich 80637, Germany b Statistical Consulting Unit (STABLAB), Department of Statistics, Ludwig-Maximilians University, Akademiestr. 1, Munich 80799, Germany Accepted 21 April 2008 Available online 2 June 2008
Abstract The present study investigated the effects of a gentling programme on the later behaviour of laboratory rats towards humans. For that purpose, 24 female Wistar rats were purchased from a laboratory animal breeding facility at the age of 21 days and allocated, genetically balanced, to an experimental and a control group. The animals were kept under standard laboratory conditions in groups of three. The experimental group was subjected to a gentling programme twice daily for 10 min per cage in the fourth and fifth week of life. This involved gentling and hand-feeding the animals. In addition, the animals were talked to during gentling. At the beginning of the 6th, 8th, 10th and 14th week of life as well as at the age of 6, 6.5 and 9 months the animals of both the experimental and the control group were subjected to tests in order to assess the rats’ behaviour towards humans. In the 14th week of life as well as at the age of 6 months the same test was additionally carried out by a person not familiar to the rats for the purpose of studying the animals’ behaviour towards unfamiliar persons. At each testing point a precisely defined, standardised test procedure was carried out, including among other things, repeated catching of the animals, neck grip, a hand test and a modified open field test involving a human stressor. Evaluation of the results was based on five primary endpoints which defined and summarised the most important parameters of the test procedure for the assessment of ‘‘tameness’’ towards humans. Significantly higher values in the primary endpoints were noted in the experimental group when compared to the control group in the test performed during the sixth week of life. The statistically estimated difference between the experimental and control groups was present until the age of 6 months. This suggested a higher level of ‘‘tameness’’ in the experimental group and a long-term effect of the gentling
* Corresponding author. Tel.: +49 89 15 92 78 32; fax: +49 89 15 78 277. E-mail address: [email protected] (D. Do¨ring). 0168-1591/$ – see front matter # 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.applanim.2008.04.013
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programme. Contrary to the experimental group, the control group showed a significant increase in tameness over time, which can be attributed to the animals getting used to the repeated tests or an age-related effect. The analysis of the difference between familiar and unfamiliar experimenters showed a borderline significant effect. The experimental and control groups were both slightly tamer with the unfamiliar person. The gentling programme had a beneficial long-term effect on the behaviour of the rats and proved suitable to reduce the laboratory rats’ fear of humans. # 2008 Elsevier B.V. All rights reserved. Keywords: Gentling; Laboratory rats; Fear; Critical period; Behavioural test; Long-term effects of gentling
1. Introduction If laboratory animals are not used to humans even simple manipulations can elicit stress reactions in the animals (Kvetnansky et al., 1978; Brand, 1998; Mende, 1999). Avoiding the fear of humans in the animals is desirable not only from an animal welfare point of view, but also in terms of the reliability of animal experiments. Stress may result in changes of physiological parameters (Kvetnansky et al., 1978; Brand, 1998; Mende, 1999). In male Wistar rats kept under normal laboratory conditions, for instance, a marked increase in serum corticosterone levels from a mean resting value of 130.5 ng/ml to a mean value of 388.2 ng/ml was noted after fixation by neck grip (Mende, 1999). Kvetnansky et al. (1978) found that lifting the animal and touching it gently for half a minute increases the plasma epinephrine levels of rats sevenfold and causes a significant increase of their plasma costicosterone concentration. Stress can compromise the reliability of the test results (Shyu et al., 1987; Lawlor, 2002). Podberscek et al. (1991) concluded from their study on laboratory rabbits that programmes which involved being handled by and establishing contact with the scientists can reduce the fear reactions of animals. They suggest performing such programmes between experiments, stressing that these may be important for the well-being of laboratory animals. The European Council (1986), in Appendix A of the European Convention for the Protection of Vertebrate Animals used for Experimental and Other Scientific Purposes, points out that the behaviour of an animal during a procedure depends very much on its confidence in humans, and that this confidence has to be developed and once established has to be preserved. In addition, the Resolution on the Accommodation and Care of Laboratory Animals of the Multilateral Consultation of the Parties to the European Convention for the Protection of Vertebrate Animals used for Experimental and Other Scientific Purposes (Council of Europe, 1997) demands for laboratory animals that: ‘‘Animals should be handled or be in social contact with humans on a regular basis, with particular attention to the socialization period in species such as cats and dogs.’’ Although rats are known to be very social animals (Lore and Flannelly, 1977), no studies on the ‘‘critical period’’ of socialisation to humans have been conducted on them—as opposed to other animal species such as dogs and cats (Scott and Fuller, 1965; Karsh and Turner, 1988). In laboratory mice and rabbits (e.g. Dyer and Southwick, 1974; Wyly et al., 1975) as well as in farm animals (e.g. Hemsworth and Barnett, 1992; Jones and Waddington, 1993; Jones, 1994; Boivin and Braastadt, 1996; Krohn and Jago, 2001) efforts have been made to find the ‘‘critical period’’ for socialisation. In rats, instead of focusing on socialisation, the development of social behaviour was studied. It is a well-established fact that social isolation in the juvenile development phase in rats
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influences the later behaviour of the animals towards both peers and objects. A study by Hol et al. (1999), for instance, showed that rats kept isolated develop deficits in their social behaviour, depending on when isolation took place. If the animals were kept isolated during the fourth and the fifth week of life the resulting social deficits were irreversible. Any subsequent group housing was without effect on social behaviour. Isolation during the fifth week of life alone, however, had no recognisable effects when compared to animals which were always kept in a social group, whereas isolation during the fourth week alone also resulted in reduced social activity. The authors assume that the lack of social playing, which the animals learn during week 4 and which intensifies in the fifth week of life, is a decisive factor in the development of normal social behaviour of rats. According to Hol et al. (1999) playing can no longer be learned after the fifth week of life. Einon et al. (1978) discovered that rats which had been kept isolated during the fourth and fifth week of life showed differences in their behaviour during object contact in the open field compared to rats which had been housed in groups. The results of animals which during that time of life had only 1 h of social contact a day with another rat were between those of animals kept isolated and those housed in groups. This shows that 1 h of social contact helped reduce the isolation-related deficits. Even though a socialisation of rats to humans has so far not been studied, many studies investigated the effects of human–rat interactions such as handling and gentling. However, the authors of these studies focused on the effect of handling and gentling on the general later behaviour of rats and did not examine the later effect on the rats’ behaviour towards humans (e.g. Weininger, 1954, 1956; Weininger et al., 1954; Gertz, 1957; Levine, 1957; Levine et al., 1957; Candland et al., 1960; Eells, 1961; Candland et al., 1962; Abel, 1971; Francis et al., 1996; Nu´nez et al., 1996). Not in all studies a difference between experimental group and control group was found (Gertz, 1957), and some even discussed that gentling has a negative effect (Candland et al., 1960, 1962). It should be noted that the terms handling and gentling are interpreted differently and the time-frame during which the young animals were treated, varied from study to study. When the gentling procedure was performed under extremely standardised conditions and the rats were stroked while the animal was restrained, the action was a negative enforcement (Candland et al., 1962). In the majority of early handling and gentling studies however, the results showed decreased fear reactions during behavioural tests later in life and improved vital parameters. These results were attributed to changes in the physiological stress response caused by handling or gentling (Weininger, 1956; Levine, 1957; Levine et al., 1957; Eells, 1961; Nu´nez et al., 1996), changes in maternal care after handling (e.g. Francis et al., 1996; Liu et al., 1997) or habituation to being handled (Abel, 1971). Rats can become habituated to humans and it has been shown that rats are able to distinguish between different people and that they prefer familiar humans (McCall et al., 1969; Davis et al., 1997). In a study presented by Davis et al. (1997), this preference was retained in the absence of additional contact for at least 5 months. A study found that social interactions with humans can be very rewarding for rats, and that they can be even used as a reinforcer for a learning task (Davis and Perusse, 1988). Hirsja¨rvi and Junnila (1988) gentled male rats at the age of 10 weeks. Contrary to the gentled rats, the non-gentled animals presented with a different type of fear in the open field test (freezing, loose stools), suggesting fear towards the person performing the test. The authors believe that non-gentled rats see humans as a predator. Do¨ring (1999) gentled female Wistar rats for 5 min a day per cage (groups of 3) between the fourth and the sixth week of life for the specific purpose of making them more trusting. As early as during the fourth week of life, the animals became clearly tame enough to be handled, pushing themselves towards the hands and incorporating them into their play.
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Due to the indications in the literature of a ‘‘critical period’’ during the fourth and the fifth week of life this period was chosen for the study on hand. The objective of this study was to find out whether a gentling programme carried out in laboratory rats during the fourth and the fifth week of life would be able to achieve an effect resulting in a long-term reduction of fear reactions towards humans. According to Morton (1968) the term ‘‘gentling’’ is to be understood as gentle stroking, ‘‘taming’’. 2. Materials and methods 2.1. Animals and husbandry The study was conducted on 24 female Wistar rats from six different litters. The animals were purchased at the age of 21 days from Charles River Company (Sulzfeld, Germany), and housed under standard laboratory conditions (Council of Europe, 2006) in groups of 3 in Makrolon1 type IV cages with raised lid. Wood shavings (softwood granulates) were provided as bedding material. The rats were housed with a 12 h/12 h light/dark circle and given food (ssniff R/M-H 10 mm) and water ad libitum. The temperature was kept at an average of 22 2 8C. The light intensity was not more than 80 lx at all times. Division into experimental and control group was done under the aspect of genetic balancing (Rapp and Deerberg, 1987), i.e. four siblings each were evenly distributed to two cages of the experimental and two of the control group, respectively. This was done in a way that no siblings ended up in the same cage. Whether animals were put into the control or treatment group was determined by lot. All rats were marked on the tail using a standard human eyeliner pencil and the marking was refreshed every week at the time of the cage cleaning and after the tests. During each cage cleaning all rats were also lifted, weighed and subsequently transferred to the new cage. All routine husbandry procedures for both groups were carried out identically and by the female experimenter. The outfit worn during routine husbandry procedures (white lab coat, green surgical cap, mask and shoe covers) was the same that was used during the behavioural tests by the experimenter and the unfamiliar person. 2.2. Gentling method The gentling programme of the 12 rats in the experimental group was started on the day after the arrival of the animals. For gentling, as well as for animal care, no gloves were worn. Prior to gentling the animals of one cage, the experimenter washed and disinfected her hands. In order to make the smell of the disinfectant on her fingers less marked for the rats the hands were then briefly rubbed with fresh cage bedding. During gentling, a systematic ‘‘rotation system’’ was applied so that the same cage was not always treated first. Gentling was performed twice daily (1 h after the beginning of the light phase and 2 h before the end of the light phase) for 10 min per cage during a total period of 14 days. Gentling involved soft and gentle touching of the animals on their entire bodies and their tails. Care was taken to ensure that every animal was gentled for about the same amount of time. During the 10 min of gentling, each animal was briefly lifted by gripping the whole body from underneath after 3 and after 8 min. The animals were not allowed to balance on the rim of their open home cage, except on the side directly before which the experimenter was positioned. If they were found balancing on the rims of the other sides anyway, they were gently placed back inside the cage. In addition, the animals were hand-fed oat flakes (Ko¨llns1 ‘‘Echte Kernige’’ whole grain oat flakes): immediately at the beginning of gentling as well as after 4, 7 and 9 min each animal was offered one flake. If none of the animals accepted an oat flake at any point, three oat flakes were placed inside the cage, below the food rack, at the end of gentling. Throughout the entire gentling process – except during lifting and holding – the animals were talked to in a friendly and soothing manner. The animals in the control group remained without any human contact except for routine husbandry procedures such as control of food and water and the weekly transfer into fresh cages. The same applied to the animals in the experimental group after termination of gentling, i.e. from the sixth week of life.
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Throughout the routine husbandry and experimental periods all animals were invariably lifted and carried by means of a grip around their body rather than by their tail. 2.3. Behaviour tests In order to investigate the effect of the gentling programme on behaviour towards humans tests were carried out at the beginning of the 6th, 8th, 10th and 14th week of life as well as at the age of 6, 6.5 and 9 months. In addition, in the 14th week of life and at the age of 6 months the same test was repeated 2 days later by a person unfamiliar to the rats who was familiar with the testing procedure. The original experimenter was present in the same room during these tests, but at a distance of at least one metre from the animals. Both humans were females and wore the same outfit during the tests. Each rat was tested individually and in a separate test room. Throughout the entire testing-session, the sequence of events was always the same and the test circumstances (handling, room-features, equipment used) were as standardised and controlled as possible. The entire test procedure lasted approx. 20 min per animal, contained the following, described sections and was recorded entirely on videotape to allow analysis at a later time. During the test procedure the animals were not talked to. Four cages, i.e. 12 animals, were tested on 1 of 2 consecutive days to ensure that the test was always performed at the same time, i.e. in the morning between 8:00 a.m. and 12:00 p.m. Six animals each from the experimental group and the control group were tested on the same day so that the influencing factors of each day were the same for both groups. During the tests a systematic ‘‘rotation system’’ was applied so that tests were not always started with the same cage, and not with the same animal within one cage, either. At the beginning of the test procedure, the cage to be studied was removed from the housing shelf and placed on a table in the animal room. Then, the first animal as per the systematic ‘‘rotation system’’ was caught from the cage and placed inside a Makrolon1 type III cage littered with bedding on a layer of gauze. Then, the animal was carried in this cage into the test room. In the test room, the animal was then transferred into a lidless Makrolon1 type IV cage which was littered with bedding on a layer of gauze and positioned inside a box with noise absorbing foam padding. The self-developed test ‘‘touch and grip’’ was performed: The animal was left alone for 5 s; then it was repeatedly touched (dabbed) softly on the neck for 10 s (see Fig. 1a). After a pause of 10 s, the animal was lifted with both hands, gently but firmly, and held for 10 s so that its front legs came to rest between index and middle fingers (see Fig. 1b). After a final rest of 10 s, a neck grip was performed for a total of 60 s (supporting the pelvis with the second hand, see Fig. 1c). After the neck grip, the rats were always placed in about the middle of the test cage floor. After a pause of 30 s, a ‘‘hand test’’ developed by Do¨ring (unpublished) was performed for 30 s. For that purpose, the experimenter’s right hand was placed in the middle of the cage floor, the fingers slightly splayed, and held motionless for 30 s (see Fig. 2). During that time the experimenter did not bend over the cage but turned her body and gaze away from the cage. If at the beginning of the hand test the animal was in the middle of the cage the hand was placed as close to the centre as possible without touching the animal. Then the animal was caught again from the test cage and placed inside the type III cage.
Fig. 1. Test ‘‘touch and grip’’: (a) neck touching, (b) lifting, and (c) neck grip.
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Fig. 2. ‘‘Hand test’’, animal making contact.
Afterwards, a modified ‘‘open field test’’ was performed. The open field used had a diameter of 80 cm; the walls were 35 cm high. The floor area was divided into 13 fields of equal size by milled lines on the underside. The height of the open field was increased by 14 cm to a total of 49 cm after the testing point at the age of 6 months using a sturdy drawing cardboard as the animals were able to reach the upper rim by jumping. The open field was illuminated using a neon lamp. The light had an intensity of 580 lx in the central field. The animals were invariably placed in the same outer field as start field, their head always pointing in the same direction. After 2 min the experimenter bent over the open field at the level of the start field, with her arms spread and her fingers splayed, for 5 s (open field stressor, Fig. 3). Other than that the experimenter was not visible to the rats during the entire test and was as noiseless as possible. Two minutes after the appearance of the stressor, the open field test was ended by catching the animal from the field. In this variation of the classic open field test, the results of Hirsja¨rvi and Junnila (1988), who described fearful
Fig. 3. Modified ‘‘Open field test’’ with open field stressor.
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behaviour of gentled versus non-gentled rats in an open field test with an unfamiliar experimenter, were taken into account. The animal was transferred onto a type II Makrolon1 cage tray directly from the open field. There, the individual tail marking was re-applied. Then the animal was placed inside a new, freshly littered type IV cage. This cage remained in the test room until all three animals of one cage had been subjected to the test and placed inside this new home cage. Then, the cage was placed back onto the shelf in the animal room. 2.4. Test analysis To minimise bias, the animals’ behaviour was analysed using the video recordings. The focus of analysis was placed on the observation of the rats’ behaviour towards humans. This involved among other things assessment of the animals’ behaviour in four different catching situations during the test procedure (catching from the home cage, catching after the ‘‘hand test’’, catching from the open field, and catching after tail marking), and dividing them into nine categories (see Table 1). The number of squeaks audible to humans which the animals emitted during the test ‘‘touch and grip’’ (touching at the neck, during lifting, and during the neck grip) were also evaluated. In addition, it was evaluated whether during the neck grip the animal bit the experimenter, or whether the neck grip had to be released due to the animal’s strong resistance. Analysis of the hand test focused on whether the respective animal had moved, had left the place where it had been at the beginning of the hand test, and whether it had made physical contact with the experimenter. Any motion apart from head swaying or briefly jerking the head was counted as movement. The animal was considered to have left its place if it had moved away from the place of origin with all four paws. The definition of contact was any touching of the experimenter with muzzle or paw. The animals’ behaviour during the open field stressor was divided into several categories including the following: ‘‘running away from the stressor’’, ‘‘freezing’’, ‘‘rearing’’, ‘‘moving towards the stressor’’. 2.5. Statistics From the parameters presented, five primary endpoints (PEs) were calculated as indicators for the level of ‘‘tameness’’ of the animals. For classification and the scale of points see Table 2. Values between 0 and 3 were possible for each primary endpoint. The higher the values in the primary endpoints, the higher was the implied level of ‘‘tameness’’ of the animals. Means of the five PEs per subject per test were analysed using a linear mixed-effects model with subject and litter-specific random intercepts. Models including random intercepts for the effects of cage or subject and litter/cage-specific random slopes led to identical conclusions and the results were practically identical. We used the function lme from the nlme-package (Pinheiro et al., 2007) for R (R Development Core Team, 2007). Table 1 Classification of behaviour during catching, plus the respective definition Category
Definition
Waits to be caught
Rears at the front rim of the cage and allows itself to be caught without trying to escape Does not try to escape and sits relaxedly in the hand Does not try to escape, does not freeze (see below) Only jerks its head or moves away 1–2 steps Moves away more than 2 steps or makes a quick evasive move More than one attempt is required to catch the animal Exhibits the species specific fear reaction: freezing behaviour involving rigid body and flattened ears Squeaks once (audible to the human ear) Squeaks several times (audible to the human ear)
Is relaxed during catching Does not try to escape Tries to escape to some extent Tries to escape Resists capture Freezes Emits one audible squeak Emits several audible squeaks
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Table 2 Overview of the primary endpoints (PEs) and the respective scale of points Primary endpoint
Scale of points
PE 1 (catching): squeaking and freezing during catching (sum of 4 captures)
3–4 squeaks = 0; 3 freezing acts or 1–2 squeaks = 1; 2 freezing acts and no squeaks = 2; neither squeaks nor freezing = 3 Biting or neck grip released = 0; squeaks upon touching the neck and/or holding = 1; 10–20 squeaks during neck grip = 2; <10 squeaks during neck grip = 3 Does not move, does not leave place, does not establish contact = 0; moves = 1; leaves place = 2; establishes contact = 3 Running away or freezing = 0; none of the four categories = 1; rearing or walking towards stressor = 2, rearing and walking towards stressor = 3 Resists capture, tries to escape or freezes = 0; tries to escape to some extent = 1; does not try to escape = 2; is relaxed during catching or waits to be caught = 3
PE 2 (neck grip): resistance and vocalisation during neck grip
PE 3 (hand test): hand test categories
PE 4 (open field stressor): reaction to human over open field
PE 5 (catching from open field): behaviour during catching
We used an identical model with an additional indicator for the familiarity of the experimenter and an interaction between familiarity and treatment group to analyse the differences between familiar and unfamiliar experimenters. In addition, the difference between experimental and control groups was estimated every 2 weeks after the beginning of testing and the p-value for multiple comparisons was adjusted using the Westfall method. These calculations were done using the R-package multcomp (Hothorn et al., 2008). To assess the reliability of the mean of the five PEs as a measure of tameness, we calculated Cronbach’s alpha. Correlations between the five PEs were not very strong, but the five items nevertheless achieved an acceptable Cronbach’s alpha of 0.598. In order to assess inter-rater reliability the test videos of 12 randomly selected animals were additionally analysed, and the primary endpoints 3 (hand test), 4 (open field stressor), and 5 (catching from open field) determined by another person. This other person did not know whether the rat in question was from the experimental or from the control group, but was familiar with the testing procedure. To assess concordance between the two raters Kruskal’s gamma (=number of concordant pairs minus number of discordant pairs, divided by the sum of concordant and discordant pairs; Sheskin, 2004) was determined for the three primary endpoints analysed. As the parameters were of an ordinal nature gamma was more suitable than Cohen’s kappa.
3. Results The experimental group had higher values during all test phases and in almost all PEs when compared to the control group (Table 3 and Figs. 4–8), which indicates a higher level of ‘‘tameness’’. In the tests which were performed in the 14th week of life and at the age of 6 months and which involved the unfamiliar person the animals in the experimental group exhibited higher values than the animals in the control group (see Table 4). In the first test involving the unfamiliar person the values of the majority of PEs in the experimental and control groups were slightly higher than those from the tests performed in the same age group (14th week of life) immediately beforehand by the familiar person.
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Table 3 Results of the tests: arithmetic mean values for the respective primary endpoints (PE) in experimental (E) and control (C) group, n = 12 animals per group (OF, open field) increasing ‘‘tameness’’ from 0 to 3 Age at test
Week 6 Week 8 Week 10 Week 14 6 months 6.5 months 9 months
PE 1 (catching)
PE 2 (neck grip)
PE 3 (hand test)
PE 4 (OF stressor)
PE 5 (catching from OF)
E
C
E
C
E
C
E
C
E
C
2.3 1.9 1.5 2.1 2.3 2.5 2.0
0.4 0.5 1.1 0.9 1.3 2.2 1.2
1.8 1.8 1.4 1.5 1.2 1.7 1.3
0.9 1.3 1.0 1.0 1.0 1.1 0.9
2.9 2.6 2.1 2.0 2.1 1.7 2.1
1.7 1.3 1.3 2.0 1.8 1.8 2.1
1.1 1.4 1.2 1.2 1.2 0.7 0.8
0.5 0.2 0.2 0.8 0.6 0.4 0.8
0.6 0.5 0.8 0.8 1.1 1.3 1.3
0.2 0.0 0.6 0.5 1.0 1.4 1.0
An overview of the results of the various testing points can be found in Figs. 9 and 10. Differences in tameness between control and experimental groups at the beginning of the testing regimen (week 6) were large and highly significant (0.847; p < 0.001). While the control group showed a significant increase in tameness over time (0.020 per week; p < 0.001), no
Fig. 4. Results of the tests during the time-frame for PE 1 (catching): frequency of the individual scores in experimental and control groups, n = 12 animals per group, increasing ‘‘tameness’’ from 0 to 3.
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Fig. 5. Results of the tests during the time-frame for PE 2 (neck grip): frequency of the individual scores in experimental and control groups, n = 12 animals per group, increasing ‘‘tameness’’ from 0 to 3.
significant change in tameness over time was noted for the experimental group ( 0.004 per week; p = 0.345) in our data. An overview of the statistically estimated differences between the experimental and control groups during the studied time-frame is shown in Table 5. Up to an age of 26 weeks (6 months of age), there were significant differences between the groups ( p values for multiple comparisons were adjusted using the Westfall method, Hothorn et al., 2008). Additionally, also including the data gathered during the two occasions in which the testing was performed by an unfamiliar experimenter, we found a fairly strong and borderline significant Table 4 Results of the tests by unfamiliar person: arithmetic mean values for the respective primary endpoints (PE) in experimental (E) and control (C) group, n = 12 animals per group (OF, open field) increasing ‘‘tameness’’ from 0 to 3 Age at test
Week 14 6 months
PE 1 (catching)
PE 2 (neck grip)
PE 3 (hand test)
PE 4 (OF stressor)
PE 5 (catching from OF)
E
C
E
C
E
C
E
C
E
C
2.5 2.1
1.7 1.5
2.1 1.8
1.2 1.0
2.0 2.3
1.5 2.0
1.6 1.1
0.8 0.7
1.5 1.5
0.8 1.1
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Fig. 6. Results of the tests during the time-frame for PE 3 (hand test): frequency of the individual scores in experimental and control groups, n = 12 animals per group, increasing ‘‘tameness’’ from 0 to 3. Table 5 Statistically estimated differences between the experimental and control groups every 2 weeks from the beginning of the test (estimate, standard errors and p values) Week of age
Estimate
Standard error
p value
6 8 10 12 14 16 18 20 22 24 26 28 30 32 34 36
0.8468 0.7981 0.7495 0.7009 0.6522 0.6036 0.5550 0.5063 0.4577 0.4090 0.3604 0.3118 0.2631 0.2145 0.1659 0.1172
0.1764 0.1719 0.1682 0.1653 0.1632 0.1620 0.1616 0.1622 0.1637 0.1660 0.1692 0.1731 0.1778 0.1832 0.1891 0.1957
<0.0001*** <0.0001*** <0.0001*** <0.0001*** <0.0001*** 0.000194*** 0.000597*** 0.001802** 0.005173** 0.013746* 0.033145* 0.071725 0.138886 0.241536 0.380478 0.549071
The p values have been adjusted for multiple comparisons using the Westfall method (Hothorn et al., 2008). Significance codes: ‘***’ 0.001, ‘**’ 0.01, ‘*’ 0.05 (adjusted p values reported—Westfall method).
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Fig. 7. Results of the tests during the time-frame for PE 4 (open field stressor): frequency of the individual scores in experimental and control groups, n = 12 animals per group, increasing ‘‘tameness’’ from 0 to 3.
experimenter effect (0.188 for unfamiliar experimenter; p = 0.052). This means, that on average, the rats showed an increase of 0.188 in the average tameness score when the unfamiliar experimenter performed the tests. This effect did not differ significantly between the treatment groups: the interaction between experimental group and familiarity of the experimenter was not significant (0.103 for unfamiliar experimenter in experimental group; p = 0.4500). The concordance between the two raters for PE 3 (hand test), PE 4 (open field stressor) and PE 5 (catching from open field) was calculated and the pooled gamma for the three primary endpoints was 0.82. 4. Discussion Concordance between the two raters was sufficiently high to confirm the results obtained. In all tests performed up to the age of 6 months, including the two tests carried out by an unfamiliar person, the animals in the experimental group invariably achieved higher values in the primary endpoints than the animals in the control group. The higher values in the experimental group suggested a higher level of ‘‘tameness’’. Even in the statistical estimates made with the help of a linear mixed-effect model, a significant difference was noted between the groups up to an age of 6 months. The gentling programme had a long-term beneficial influence on the rats’ behaviour towards humans. This period of half a year seems remarkable, given that the gentling programme was limited to a mere two weeks during the rats’ juvenile development. Apart from the human
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Fig. 8. Results of the tests during the time-frame for PE 5 (catching from open field): frequency of the individual scores in experimental and control groups, n = 12 animals per group, increasing ‘‘tameness’’ from 0 to 3.
contact in the fourth and fifth week of life within the framework of the gentling programme, the animals in the experimental group, just like those in the control group, had not received any particular further human contact. Hence, a limited duration of gentling was able to achieve a long-lasting effect on the rats’ behaviour towards humans. These results coincide with the reports published by other authors such as Do¨ring (1999), Lawlor (2002) and Jones et al. (2003). Gentling experiences can have a long-term effect on rats as was demonstrated by Davis et al. (1997): the rats recognised a specific person who had gentled them 5 months earlier and preferred this person over an unfamiliar person. The increasing values noted in the control group are either the result of an ageing effect or the result of the animals getting used to the experimental routine. It is known, that repeated tests can lead to familiarisation, for example in the open field test (Doyle and Pratt Yule, 1959; Williams and Russell, 1972; Rebouc¸as and Schmidek, 1997 among others). Hirsja¨rvi et al. (1990) as well as Hirsja¨rvi and Va¨liaho (1995) found, that the behaviour of non-gentled rats became more similar to the behaviour of gentled animals with repeated tests in the open field. Gentling was performed at 10 weeks of age in male rats. Shyu et al. (1987) found that rats, which were accustomed to specific procedures (fixation), showed less stress-related reactions. The improvement of the results in the control group could be the result of an age-related effect. It is plausible, that rats become calmer as they become older, allowing easier handling. This explanation is supported by the fact, that during the initial tests performed frequently during the first three months of life (at 6, 8, 10 and 14 weeks of age), the control group did not show the
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Fig. 9. Overview of the results at the various testing points. Arithmetic mean values of all five primary endpoints (PEs) of experimental and control group, n = 12 animals per group, increasing ‘‘tameness’’ from 0 to 3.
results that the gentling group did. Only after half a year did the results begin to mirror those of the experimental group and this was after a testing break of two months. In order to differentiate the ageing and familiarity effects, additional tests performed at the same time with a separate control group that had no previous testing experience would be necessary. In summary, animals in a gentling program are as ‘‘tame’’ at 6 weeks of age as rats with repeated test experiences are at 6 months of age. Additionally, as gentling does not involve a specific testing situation, but has rather an unspecific taming effect, one can assume the gentled animals will react more tamely during other manipulations and procedures and would be easier to handle. The analysis of the difference between the familiar and unfamiliar experimenter showed a borderline significant experimenter effect in favour of the unfamiliar person. Accordingly, contrary to the results of Briese and De Quijada (1970), there was no evidence of any higher level of fear towards unfamiliar persons. The fact that the experimental group achieved higher values in the primary endpoints suggests a lingering effect of gentling, i.e. a higher level of ‘‘tameness’’ of the animals which had been subjected to the gentling programme. The altogether higher values of the experimental and the control group may be explained by a habituation effect in the animals. It is conceivable that the animals had grown accustomed to the test procedure by the repetition of the test after an interval of a mere 2 days. Another possible explanation is that the unfamiliar person may have caused the animals to behave more ‘‘tamely’’ due to a possibly slightly different way of handling the animals, such as touching them or catching them in a softer way. However, it was tried to keep the interactions of the experimenter and the unfamiliar person with the subjects as standardised as possible. Another reason might be personal factors of the experimenter such as smell, etc.
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Fig. 10. Overview of the results of the two tests with an unfamiliar experimenter. Arithmetic mean values of all five primary endpoints (PEs) of experimental and control group, n = 12 animals per group, increasing ‘‘tameness’’ from 0 to 3. For comparison, the results of the test 2 days earlier with the familiar experimenter are shown (gray).
In summary, the animals subjected to the gentling programme have shown a reduced level of fear reactions and consequently a higher degree of ‘‘tameness’’ towards humans. ‘‘Tameness’’ was associated not only with the person who had performed gentling and had become familiar to the animals through care, but also with a stranger. This means that the gentling effect was not restricted to one single person. The fact that the statistically estimated difference between the groups remained until the age of 6 months permits the conclusion that the gentling effect had persisted up to that point and can therefore be considered ‘‘long-lasting’’. In order to allow these findings to be put into practice the gentling programme used in this study should be further optimised. One particular objective should be to try and reduce the workload and staff requirement involved in conducting the gentling programme. For that reason, the period suitable for gentling should be further limited during any continuation studies. In addition, the effects of the individual factors (gentling–food–talking) of the gentling programme on the animals’ behaviour should be evaluated. Based on the results of this study, early gentling of laboratory rats can be recommended to achieve an immediate and long-lasting level of ‘‘tameness’’. As repeated tests lead to a familiarisation effect, these should be used to familiarise the animal with a specific test manipulation even at an advanced age of the rat. Both methods can be used to reduce fear and stress related to contact with humans. The results of this study should be taken into account when planning and conducting animal experiments as avoidable fear and stress reactions of laboratory rats are an issue of animal welfare on the one hand, while on the other hand – as has been shown in the literature again and again – they can influence test results.
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5. Conclusion The gentling programme performed proved suitable to achieve a long-lasting reduction of the animals’ fear of humans. The animals from the experimental group exhibited a higher level of ‘‘tameness’’ than those from the control group, even towards an unfamiliar person. Acknowledgement We wish to thank Ms. Pia Zausinger for performing the secondary evaluation of the data for calculating inter-rater reliability. References Abel, E.L., 1971. Habituation as a factor in early handling. J. Comp. Physiol. Psychol. 74, 219–221. Boivin, X., Braastadt, B.O., 1996. Effects of handling during temporary isolation after early weaning on goat kids’ later response to humans. Appl. Anim. Behav. Sci. 48, 61–71. Brand, U., 1998. Untersuchungen zu Streßparametern bei ma¨nnlichen Wistar-Ratten unter dem Einfluß einfacher Manipulationen. Thesis. Freie Universita¨t Berlin, D. Briese, E., De Quijada, M., 1970. Colonic temperature of rats during handling. Acta Physiol. Lat. Am. 20, 9–14. Candland, D.K., Faulds, B., Thoman, D.B., Candland, M.H., 1960. The reinforcing value of gentling. J. Comp. Physiol. Psychol. 53, 55–58. Candland, D.K., Horowitz, S.H., Culbertson, J.L., 1962. Acquisition and retention of acquired avoidance with gentling as reinforcement. J. Comp. Physiol. Psychol. 55, 1062–1064. Council of Europe, 1986. European Convention for the Protection of Vertebrate Animals used for Experimental and Other Scientific Purposes. ETS No. 123. Strasbourg, F. http://conventions.coe.int/Treaty/en/Treaties/Html/123.htm. Council of Europe, 1997. Multilateral Consultation of Parties to the European Convention for the Protection of Vertebrate Animals used for Experimental and Other Scientific Purposes (ETS 123)—Resolution on the Accommodation and Care of Laboratory Animals. http://www.coe.int/T/E/Legal_affairs/Legal_co-operation/Biological_safety%2C_use _of_animals/Laboratory_animals/Res%20accommodation.asp#TopOfPage. Council of Europe, 2006. Appendix A of the European Convention for the Protection of Vertebrate Animals used for Experimental and Other Scientific Purposes (ETS No. 123)—Guidelines for Accommodation and Care of Animals. Council of Europe, Strasbourg, France, June 15, 2006. Cons 123 (2006) 3. http://www.coe.int/t/e/legal_affairs/legal_ co-operation/biological_safety%2C_use_of_animals/laboratory_animals/2006/Cons123(2006)3AppendixA_en.pdf. Davis, H., Perusse, R., 1988. Human-based social interaction can reward a rat’s behavior. Anim. Learn. Behav. 16, 89–92. Davis, H., Taylor, A.A., Norris, C., 1997. Preference for familiar humans by rats. Psychon. Bull. Rev. 4, 118–120. Doyle, G., Pratt Yule, E., 1959. Grooming activities and freezing behaviour in relation to emotionality in albino rats. Anim. Behav. 7, 18–22. Do¨ring, D., 1999. Ka¨figraumausnutzung bei Laborratten. Thesis. Freie Universita¨t Berlin, D. http://www.diss.fuberlin.de/1999/82/. Dyer Jr., D.P., Southwick, C.H., 1974. A possible sensitive period for juvenile socialization in mice. Behav. Biol. 12, 551– 558. Eells, J.F., 1961. Inconsistency of early handling and its effects upon emotionality in the rat. J. Comp. Physiol. Psychol. 54, 690–693. Einon, D.F., Morgan, M.J., Kibbler, C.C., 1978. Brief periods of socialization and later behavior in the rat. Dev. Psychobiol. 11, 213–225. Francis, D., Diorio, J., Laplante, P., Weaver, S., Seckl, J., Meaney, M., 1996. The role of early environmental events in regulating neuroendocrine development: moms, pups, stress and glucocorticoid receptors. Ann. NY Acad. Sci. 794, 136–152. Gertz, B., 1957. The effect of handling at various age levels on emotional behavior of adult rats. J. Comp. Physiol. Psychol. 50, 613–616. Hemsworth, P.H., Barnett, J.L., 1992. The effects of early contact with humans on the subsequent level of fear of humans in pigs. Appl. Anim. Behav. Sci. 35, 83–90.
570
B.M. Maurer et al. / Applied Animal Behaviour Science 114 (2008) 554–571
Hirsja¨rvi, P.A., Junnila, M.A., 1988. The effects of gentling on open-field behaviour of rats. In: Beynen, A.C., Solleveld, H.A. (Eds.), New Developments in Biosciences: Their Implications for Laboratory Animal Science. Martinus Nijhoff Publishers, Dordrecht, pp. 399–403. Hirsja¨rvi, P.A., Va¨liaho, T., 1995. Gentled and nonhandled Wistar rats in a mildly novel open-field situation. Scand. J. Lab. Anim. Sci. 22, 265–269. Hirsja¨rvi, P.A., Junnila, M.A., Va¨liaho, T.U., 1990. Gentled and non-handled rats in a stressful open-field situation; differences in performance. Scand. J. Psychol. 31, 259–265. Hol, T., van den Berg, C.L., van Ree, J.M., Spruijt, B.M., 1999. Isolation during the play period in infancy decreases adult social interactions in rats. Behav. Brain Res. 100, 91–97. Hothorn, T., Bretz, F., Westfall, P., with contributions by Heiberger, R.M., 2008. Multcomp: Simultaneous Inference for General Linear Hypotheses. R Package Version 0.993-1. Jones, B.R., Waddington, D., 1993. Attenuation of the domestic chick’s fear of human beings via regular handling: in search of a sensitive period. Appl. Anim. Behav. Sci. 36, 185–195. Jones, T.A., Bury, S.D., Adkins-Muir, D.L., Luke, L.M., Allred, R.P., Sakata, J.T., 2003. Importance of behavioral manipulations and measures in rat models of brain damage and brain repair. ILAR J. 44, 144–152. Jones, B.R., 1994. Ontogeny of response to humans in handled and non-handled female domestic chicks. Appl. Anim. Behav. Sci. 42, 261–269. Karsh, E.B., Turner, D.C., 1988. Die Mensch/Katze Beziehung. In: Turner, D.C., Bateson, P. (Eds.), Die domestizierte Katze-eine wissenschaftliche Betrachtung ihres Verhaltens. Albert Mu¨ller Verlag, Ru¨schlikon-Zu¨rich, pp. 192–215. Krohn, C.C., Jago, J.G., 2001. The effect of early handling on the socialisation of young calves to humans. Appl. Anim. Behav. Sci. 74, 121–133. Kvetnansky, R., Sun, C.L., Lake, C.R., Thoa, N., Torda, T., Kopin, I.J., 1978. Effect of handling and forced immobilization on rat plasma levels of epinephrine, norepinephrine and dopamine-hydroxylase. Endocrinology 103, 1868–1874. Lawlor, M., 2002. Comfortable quarters for rats in research institutions. In: Reinhardt, V., Reinhardt, A. (Eds.), Comfortable Quarters for Laboratory Animals. 9th ed. Animal Welfare Institute, Washington, DC, pp. 26–32. Levine, S., 1957. Infantile experience and resistance to physiological stress. Science 126, 405. Levine, S., Alpert, M., Lewis, G.W., 1957. Infantile experience and the maturation of the pituitary adrenal gland. Science 126, 1347. Liu, D., Diorio, J., Tannenbaum, B., Caldji, C., Francis, D., Freedman, A., Sharma, S., Pearson, D., Plotsky, P.M., Meaney, M.J., 1997. Maternal care, hippocampal glucocorticoid receptors, and hypothalamic-pituitary-adrenal responses to stress. Science 277, 1659–1662. Lore, R., Flannelly, K., 1977. Rat society. Sci. Am. 236, 106–116. McCall, R.B., Lester, M.L., Corter, C.M., 1969. Caretaker effect in rats. Dev. Psychol. 1, 771. Mende, G., 1999. Untersuchung zur Beurteilung der Belastung von Laborratten durch einfache Manipulationen, an den Parametern Kortikosteron und Prolaktin. Thesis. Freie Universita¨t Berlin, D. http://www.diss.fu-berlin.de/1999/101/. Morton, J.R.C., 1968. Effects of early experience ‘‘handling and gentling’’ in laboratory animals. In: Fox, M.W. (Ed.), Abnormal Behaviour in Animals. W.B. Saunders Company, Philadelphia, pp. 261–292. Nu´nez, J.F., Ferre´, P., Escorihuela, R.M., Tobena, A., Fernande´z-Teruel, A., 1996. Effects of postnatal handling of rats on emotional, HPA-axis, and prolactin reactivity to novelty and conflict. Physiol. Behav. 60, 1355–1359. Pinheiro, J., Bates, D., DebRoy, S., Sarkar, D., 2007. Nlme: Linear and Nonlinear Mixed Effects Models. R Package Version 3. , pp. 1–83. Podberscek, A.L., Blackshaw, J.K., Beattie, A.W., 1991. The effects of repeated handling by familiar and unfamiliar people on rabbits in individual cages and group pens. Appl. Anim. Behav. Sci. 28, 365–373. R Development Core Team, 2007. R: A Language and Environment for Statistical Computing. R Foundation for Statistical Computing, Vienna, Austria. http://www.R-project.org. Rapp, K.G., Deerberg, F., 1987. Zur Problematik der Stichprobenentnahme und Versuchsgruppenzusammenstellung bei Auszuchtpopulationen. Z. Versuchstierkd. 29, 193–203. Rebouc¸as, R.C.R., Schmidek, W.R., 1997. Handling and isolation in three strains of rats affect open field, exploration, hoarding and predation. Physiol. Behav. 62, 1159–1164. Scott, J.P., Fuller, J.L., 1965. Genetics and the Social Behavior of the Dog. The University of Chicago Press, Chicago. Sheskin, D.J., 2004. Handbook of Parametric and Nonparametric Statistical Procedures. Chapman and Hall, New York. Shyu, W.C., Mordenti, J.L., Nightingale, C.H., Tsuji, A., Quintiliani, R., 1987. Effect of stress on the pharmacokinetics of Amikacin and Ticarcillin. J. Pharm. Sci. 76, 265–266. Weininger, O., 1954. Physiological damage under emotional stress as a function of early experience. Science 119, 285–286.
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Weininger, O., McClelland, W.C., Arime, R.K., 1954. Gentling and weight gain in the albino rat. Canad. J. Psychol. 8, 147–151. Weininger, O., 1956. The effects of early experience on behavior and growth characteristics. J. Comp. Physiol. Psychol. 1, 1–9. Williams, D.I., Russell, P.A., 1972. Open-field behaviour in rats: effects of handling, sex and repeated testing. Br. J. Psychol. 63, 593–596. Wyly, M.V., Denenberg, V.H., De Santis, D., Burns, J.K., Zarrow, M.X., 1975. Handling rabbits in infancy: in search of a critical period. J. Comp. Physiol. Psychol. 8, 179–186.
Applied Animal Behaviour Science 114 (2008) 554–571 www.elsevier.com/locate/applanim
Effects of a gentling programme on the behaviour of laboratory rats towards humans Barbara M. Maurer a, Dorothea Do¨ring a,*, Fabian Scheipl b, Helmut Ku¨chenhoff b, Michael H. Erhard a a
Department of Veterinary Sciences, Veterinary Faculty of Ludwig-Maximilians University, Schwere-Reiter-Str. 9, Munich 80637, Germany b Statistical Consulting Unit (STABLAB), Department of Statistics, Ludwig-Maximilians University, Akademiestr. 1, Munich 80799, Germany Accepted 21 April 2008 Available online 2 June 2008
Abstract The present study investigated the effects of a gentling programme on the later behaviour of laboratory rats towards humans. For that purpose, 24 female Wistar rats were purchased from a laboratory animal breeding facility at the age of 21 days and allocated, genetically balanced, to an experimental and a control group. The animals were kept under standard laboratory conditions in groups of three. The experimental group was subjected to a gentling programme twice daily for 10 min per cage in the fourth and fifth week of life. This involved gentling and hand-feeding the animals. In addition, the animals were talked to during gentling. At the beginning of the 6th, 8th, 10th and 14th week of life as well as at the age of 6, 6.5 and 9 months the animals of both the experimental and the control group were subjected to tests in order to assess the rats’ behaviour towards humans. In the 14th week of life as well as at the age of 6 months the same test was additionally carried out by a person not familiar to the rats for the purpose of studying the animals’ behaviour towards unfamiliar persons. At each testing point a precisely defined, standardised test procedure was carried out, including among other things, repeated catching of the animals, neck grip, a hand test and a modified open field test involving a human stressor. Evaluation of the results was based on five primary endpoints which defined and summarised the most important parameters of the test procedure for the assessment of ‘‘tameness’’ towards humans. Significantly higher values in the primary endpoints were noted in the experimental group when compared to the control group in the test performed during the sixth week of life. The statistically estimated difference between the experimental and control groups was present until the age of 6 months. This suggested a higher level of ‘‘tameness’’ in the experimental group and a long-term effect of the gentling
* Corresponding author. Tel.: +49 89 15 92 78 32; fax: +49 89 15 78 277. E-mail address: [email protected] (D. Do¨ring). 0168-1591/$ – see front matter # 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.applanim.2008.04.013
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programme. Contrary to the experimental group, the control group showed a significant increase in tameness over time, which can be attributed to the animals getting used to the repeated tests or an age-related effect. The analysis of the difference between familiar and unfamiliar experimenters showed a borderline significant effect. The experimental and control groups were both slightly tamer with the unfamiliar person. The gentling programme had a beneficial long-term effect on the behaviour of the rats and proved suitable to reduce the laboratory rats’ fear of humans. # 2008 Elsevier B.V. All rights reserved. Keywords: Gentling; Laboratory rats; Fear; Critical period; Behavioural test; Long-term effects of gentling
1. Introduction If laboratory animals are not used to humans even simple manipulations can elicit stress reactions in the animals (Kvetnansky et al., 1978; Brand, 1998; Mende, 1999). Avoiding the fear of humans in the animals is desirable not only from an animal welfare point of view, but also in terms of the reliability of animal experiments. Stress may result in changes of physiological parameters (Kvetnansky et al., 1978; Brand, 1998; Mende, 1999). In male Wistar rats kept under normal laboratory conditions, for instance, a marked increase in serum corticosterone levels from a mean resting value of 130.5 ng/ml to a mean value of 388.2 ng/ml was noted after fixation by neck grip (Mende, 1999). Kvetnansky et al. (1978) found that lifting the animal and touching it gently for half a minute increases the plasma epinephrine levels of rats sevenfold and causes a significant increase of their plasma costicosterone concentration. Stress can compromise the reliability of the test results (Shyu et al., 1987; Lawlor, 2002). Podberscek et al. (1991) concluded from their study on laboratory rabbits that programmes which involved being handled by and establishing contact with the scientists can reduce the fear reactions of animals. They suggest performing such programmes between experiments, stressing that these may be important for the well-being of laboratory animals. The European Council (1986), in Appendix A of the European Convention for the Protection of Vertebrate Animals used for Experimental and Other Scientific Purposes, points out that the behaviour of an animal during a procedure depends very much on its confidence in humans, and that this confidence has to be developed and once established has to be preserved. In addition, the Resolution on the Accommodation and Care of Laboratory Animals of the Multilateral Consultation of the Parties to the European Convention for the Protection of Vertebrate Animals used for Experimental and Other Scientific Purposes (Council of Europe, 1997) demands for laboratory animals that: ‘‘Animals should be handled or be in social contact with humans on a regular basis, with particular attention to the socialization period in species such as cats and dogs.’’ Although rats are known to be very social animals (Lore and Flannelly, 1977), no studies on the ‘‘critical period’’ of socialisation to humans have been conducted on them—as opposed to other animal species such as dogs and cats (Scott and Fuller, 1965; Karsh and Turner, 1988). In laboratory mice and rabbits (e.g. Dyer and Southwick, 1974; Wyly et al., 1975) as well as in farm animals (e.g. Hemsworth and Barnett, 1992; Jones and Waddington, 1993; Jones, 1994; Boivin and Braastadt, 1996; Krohn and Jago, 2001) efforts have been made to find the ‘‘critical period’’ for socialisation. In rats, instead of focusing on socialisation, the development of social behaviour was studied. It is a well-established fact that social isolation in the juvenile development phase in rats
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influences the later behaviour of the animals towards both peers and objects. A study by Hol et al. (1999), for instance, showed that rats kept isolated develop deficits in their social behaviour, depending on when isolation took place. If the animals were kept isolated during the fourth and the fifth week of life the resulting social deficits were irreversible. Any subsequent group housing was without effect on social behaviour. Isolation during the fifth week of life alone, however, had no recognisable effects when compared to animals which were always kept in a social group, whereas isolation during the fourth week alone also resulted in reduced social activity. The authors assume that the lack of social playing, which the animals learn during week 4 and which intensifies in the fifth week of life, is a decisive factor in the development of normal social behaviour of rats. According to Hol et al. (1999) playing can no longer be learned after the fifth week of life. Einon et al. (1978) discovered that rats which had been kept isolated during the fourth and fifth week of life showed differences in their behaviour during object contact in the open field compared to rats which had been housed in groups. The results of animals which during that time of life had only 1 h of social contact a day with another rat were between those of animals kept isolated and those housed in groups. This shows that 1 h of social contact helped reduce the isolation-related deficits. Even though a socialisation of rats to humans has so far not been studied, many studies investigated the effects of human–rat interactions such as handling and gentling. However, the authors of these studies focused on the effect of handling and gentling on the general later behaviour of rats and did not examine the later effect on the rats’ behaviour towards humans (e.g. Weininger, 1954, 1956; Weininger et al., 1954; Gertz, 1957; Levine, 1957; Levine et al., 1957; Candland et al., 1960; Eells, 1961; Candland et al., 1962; Abel, 1971; Francis et al., 1996; Nu´nez et al., 1996). Not in all studies a difference between experimental group and control group was found (Gertz, 1957), and some even discussed that gentling has a negative effect (Candland et al., 1960, 1962). It should be noted that the terms handling and gentling are interpreted differently and the time-frame during which the young animals were treated, varied from study to study. When the gentling procedure was performed under extremely standardised conditions and the rats were stroked while the animal was restrained, the action was a negative enforcement (Candland et al., 1962). In the majority of early handling and gentling studies however, the results showed decreased fear reactions during behavioural tests later in life and improved vital parameters. These results were attributed to changes in the physiological stress response caused by handling or gentling (Weininger, 1956; Levine, 1957; Levine et al., 1957; Eells, 1961; Nu´nez et al., 1996), changes in maternal care after handling (e.g. Francis et al., 1996; Liu et al., 1997) or habituation to being handled (Abel, 1971). Rats can become habituated to humans and it has been shown that rats are able to distinguish between different people and that they prefer familiar humans (McCall et al., 1969; Davis et al., 1997). In a study presented by Davis et al. (1997), this preference was retained in the absence of additional contact for at least 5 months. A study found that social interactions with humans can be very rewarding for rats, and that they can be even used as a reinforcer for a learning task (Davis and Perusse, 1988). Hirsja¨rvi and Junnila (1988) gentled male rats at the age of 10 weeks. Contrary to the gentled rats, the non-gentled animals presented with a different type of fear in the open field test (freezing, loose stools), suggesting fear towards the person performing the test. The authors believe that non-gentled rats see humans as a predator. Do¨ring (1999) gentled female Wistar rats for 5 min a day per cage (groups of 3) between the fourth and the sixth week of life for the specific purpose of making them more trusting. As early as during the fourth week of life, the animals became clearly tame enough to be handled, pushing themselves towards the hands and incorporating them into their play.
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Due to the indications in the literature of a ‘‘critical period’’ during the fourth and the fifth week of life this period was chosen for the study on hand. The objective of this study was to find out whether a gentling programme carried out in laboratory rats during the fourth and the fifth week of life would be able to achieve an effect resulting in a long-term reduction of fear reactions towards humans. According to Morton (1968) the term ‘‘gentling’’ is to be understood as gentle stroking, ‘‘taming’’. 2. Materials and methods 2.1. Animals and husbandry The study was conducted on 24 female Wistar rats from six different litters. The animals were purchased at the age of 21 days from Charles River Company (Sulzfeld, Germany), and housed under standard laboratory conditions (Council of Europe, 2006) in groups of 3 in Makrolon1 type IV cages with raised lid. Wood shavings (softwood granulates) were provided as bedding material. The rats were housed with a 12 h/12 h light/dark circle and given food (ssniff R/M-H 10 mm) and water ad libitum. The temperature was kept at an average of 22 2 8C. The light intensity was not more than 80 lx at all times. Division into experimental and control group was done under the aspect of genetic balancing (Rapp and Deerberg, 1987), i.e. four siblings each were evenly distributed to two cages of the experimental and two of the control group, respectively. This was done in a way that no siblings ended up in the same cage. Whether animals were put into the control or treatment group was determined by lot. All rats were marked on the tail using a standard human eyeliner pencil and the marking was refreshed every week at the time of the cage cleaning and after the tests. During each cage cleaning all rats were also lifted, weighed and subsequently transferred to the new cage. All routine husbandry procedures for both groups were carried out identically and by the female experimenter. The outfit worn during routine husbandry procedures (white lab coat, green surgical cap, mask and shoe covers) was the same that was used during the behavioural tests by the experimenter and the unfamiliar person. 2.2. Gentling method The gentling programme of the 12 rats in the experimental group was started on the day after the arrival of the animals. For gentling, as well as for animal care, no gloves were worn. Prior to gentling the animals of one cage, the experimenter washed and disinfected her hands. In order to make the smell of the disinfectant on her fingers less marked for the rats the hands were then briefly rubbed with fresh cage bedding. During gentling, a systematic ‘‘rotation system’’ was applied so that the same cage was not always treated first. Gentling was performed twice daily (1 h after the beginning of the light phase and 2 h before the end of the light phase) for 10 min per cage during a total period of 14 days. Gentling involved soft and gentle touching of the animals on their entire bodies and their tails. Care was taken to ensure that every animal was gentled for about the same amount of time. During the 10 min of gentling, each animal was briefly lifted by gripping the whole body from underneath after 3 and after 8 min. The animals were not allowed to balance on the rim of their open home cage, except on the side directly before which the experimenter was positioned. If they were found balancing on the rims of the other sides anyway, they were gently placed back inside the cage. In addition, the animals were hand-fed oat flakes (Ko¨llns1 ‘‘Echte Kernige’’ whole grain oat flakes): immediately at the beginning of gentling as well as after 4, 7 and 9 min each animal was offered one flake. If none of the animals accepted an oat flake at any point, three oat flakes were placed inside the cage, below the food rack, at the end of gentling. Throughout the entire gentling process – except during lifting and holding – the animals were talked to in a friendly and soothing manner. The animals in the control group remained without any human contact except for routine husbandry procedures such as control of food and water and the weekly transfer into fresh cages. The same applied to the animals in the experimental group after termination of gentling, i.e. from the sixth week of life.
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Throughout the routine husbandry and experimental periods all animals were invariably lifted and carried by means of a grip around their body rather than by their tail. 2.3. Behaviour tests In order to investigate the effect of the gentling programme on behaviour towards humans tests were carried out at the beginning of the 6th, 8th, 10th and 14th week of life as well as at the age of 6, 6.5 and 9 months. In addition, in the 14th week of life and at the age of 6 months the same test was repeated 2 days later by a person unfamiliar to the rats who was familiar with the testing procedure. The original experimenter was present in the same room during these tests, but at a distance of at least one metre from the animals. Both humans were females and wore the same outfit during the tests. Each rat was tested individually and in a separate test room. Throughout the entire testing-session, the sequence of events was always the same and the test circumstances (handling, room-features, equipment used) were as standardised and controlled as possible. The entire test procedure lasted approx. 20 min per animal, contained the following, described sections and was recorded entirely on videotape to allow analysis at a later time. During the test procedure the animals were not talked to. Four cages, i.e. 12 animals, were tested on 1 of 2 consecutive days to ensure that the test was always performed at the same time, i.e. in the morning between 8:00 a.m. and 12:00 p.m. Six animals each from the experimental group and the control group were tested on the same day so that the influencing factors of each day were the same for both groups. During the tests a systematic ‘‘rotation system’’ was applied so that tests were not always started with the same cage, and not with the same animal within one cage, either. At the beginning of the test procedure, the cage to be studied was removed from the housing shelf and placed on a table in the animal room. Then, the first animal as per the systematic ‘‘rotation system’’ was caught from the cage and placed inside a Makrolon1 type III cage littered with bedding on a layer of gauze. Then, the animal was carried in this cage into the test room. In the test room, the animal was then transferred into a lidless Makrolon1 type IV cage which was littered with bedding on a layer of gauze and positioned inside a box with noise absorbing foam padding. The self-developed test ‘‘touch and grip’’ was performed: The animal was left alone for 5 s; then it was repeatedly touched (dabbed) softly on the neck for 10 s (see Fig. 1a). After a pause of 10 s, the animal was lifted with both hands, gently but firmly, and held for 10 s so that its front legs came to rest between index and middle fingers (see Fig. 1b). After a final rest of 10 s, a neck grip was performed for a total of 60 s (supporting the pelvis with the second hand, see Fig. 1c). After the neck grip, the rats were always placed in about the middle of the test cage floor. After a pause of 30 s, a ‘‘hand test’’ developed by Do¨ring (unpublished) was performed for 30 s. For that purpose, the experimenter’s right hand was placed in the middle of the cage floor, the fingers slightly splayed, and held motionless for 30 s (see Fig. 2). During that time the experimenter did not bend over the cage but turned her body and gaze away from the cage. If at the beginning of the hand test the animal was in the middle of the cage the hand was placed as close to the centre as possible without touching the animal. Then the animal was caught again from the test cage and placed inside the type III cage.
Fig. 1. Test ‘‘touch and grip’’: (a) neck touching, (b) lifting, and (c) neck grip.
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Fig. 2. ‘‘Hand test’’, animal making contact.
Afterwards, a modified ‘‘open field test’’ was performed. The open field used had a diameter of 80 cm; the walls were 35 cm high. The floor area was divided into 13 fields of equal size by milled lines on the underside. The height of the open field was increased by 14 cm to a total of 49 cm after the testing point at the age of 6 months using a sturdy drawing cardboard as the animals were able to reach the upper rim by jumping. The open field was illuminated using a neon lamp. The light had an intensity of 580 lx in the central field. The animals were invariably placed in the same outer field as start field, their head always pointing in the same direction. After 2 min the experimenter bent over the open field at the level of the start field, with her arms spread and her fingers splayed, for 5 s (open field stressor, Fig. 3). Other than that the experimenter was not visible to the rats during the entire test and was as noiseless as possible. Two minutes after the appearance of the stressor, the open field test was ended by catching the animal from the field. In this variation of the classic open field test, the results of Hirsja¨rvi and Junnila (1988), who described fearful
Fig. 3. Modified ‘‘Open field test’’ with open field stressor.
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behaviour of gentled versus non-gentled rats in an open field test with an unfamiliar experimenter, were taken into account. The animal was transferred onto a type II Makrolon1 cage tray directly from the open field. There, the individual tail marking was re-applied. Then the animal was placed inside a new, freshly littered type IV cage. This cage remained in the test room until all three animals of one cage had been subjected to the test and placed inside this new home cage. Then, the cage was placed back onto the shelf in the animal room. 2.4. Test analysis To minimise bias, the animals’ behaviour was analysed using the video recordings. The focus of analysis was placed on the observation of the rats’ behaviour towards humans. This involved among other things assessment of the animals’ behaviour in four different catching situations during the test procedure (catching from the home cage, catching after the ‘‘hand test’’, catching from the open field, and catching after tail marking), and dividing them into nine categories (see Table 1). The number of squeaks audible to humans which the animals emitted during the test ‘‘touch and grip’’ (touching at the neck, during lifting, and during the neck grip) were also evaluated. In addition, it was evaluated whether during the neck grip the animal bit the experimenter, or whether the neck grip had to be released due to the animal’s strong resistance. Analysis of the hand test focused on whether the respective animal had moved, had left the place where it had been at the beginning of the hand test, and whether it had made physical contact with the experimenter. Any motion apart from head swaying or briefly jerking the head was counted as movement. The animal was considered to have left its place if it had moved away from the place of origin with all four paws. The definition of contact was any touching of the experimenter with muzzle or paw. The animals’ behaviour during the open field stressor was divided into several categories including the following: ‘‘running away from the stressor’’, ‘‘freezing’’, ‘‘rearing’’, ‘‘moving towards the stressor’’. 2.5. Statistics From the parameters presented, five primary endpoints (PEs) were calculated as indicators for the level of ‘‘tameness’’ of the animals. For classification and the scale of points see Table 2. Values between 0 and 3 were possible for each primary endpoint. The higher the values in the primary endpoints, the higher was the implied level of ‘‘tameness’’ of the animals. Means of the five PEs per subject per test were analysed using a linear mixed-effects model with subject and litter-specific random intercepts. Models including random intercepts for the effects of cage or subject and litter/cage-specific random slopes led to identical conclusions and the results were practically identical. We used the function lme from the nlme-package (Pinheiro et al., 2007) for R (R Development Core Team, 2007). Table 1 Classification of behaviour during catching, plus the respective definition Category
Definition
Waits to be caught
Rears at the front rim of the cage and allows itself to be caught without trying to escape Does not try to escape and sits relaxedly in the hand Does not try to escape, does not freeze (see below) Only jerks its head or moves away 1–2 steps Moves away more than 2 steps or makes a quick evasive move More than one attempt is required to catch the animal Exhibits the species specific fear reaction: freezing behaviour involving rigid body and flattened ears Squeaks once (audible to the human ear) Squeaks several times (audible to the human ear)
Is relaxed during catching Does not try to escape Tries to escape to some extent Tries to escape Resists capture Freezes Emits one audible squeak Emits several audible squeaks
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Table 2 Overview of the primary endpoints (PEs) and the respective scale of points Primary endpoint
Scale of points
PE 1 (catching): squeaking and freezing during catching (sum of 4 captures)
3–4 squeaks = 0; 3 freezing acts or 1–2 squeaks = 1; 2 freezing acts and no squeaks = 2; neither squeaks nor freezing = 3 Biting or neck grip released = 0; squeaks upon touching the neck and/or holding = 1; 10–20 squeaks during neck grip = 2; <10 squeaks during neck grip = 3 Does not move, does not leave place, does not establish contact = 0; moves = 1; leaves place = 2; establishes contact = 3 Running away or freezing = 0; none of the four categories = 1; rearing or walking towards stressor = 2, rearing and walking towards stressor = 3 Resists capture, tries to escape or freezes = 0; tries to escape to some extent = 1; does not try to escape = 2; is relaxed during catching or waits to be caught = 3
PE 2 (neck grip): resistance and vocalisation during neck grip
PE 3 (hand test): hand test categories
PE 4 (open field stressor): reaction to human over open field
PE 5 (catching from open field): behaviour during catching
We used an identical model with an additional indicator for the familiarity of the experimenter and an interaction between familiarity and treatment group to analyse the differences between familiar and unfamiliar experimenters. In addition, the difference between experimental and control groups was estimated every 2 weeks after the beginning of testing and the p-value for multiple comparisons was adjusted using the Westfall method. These calculations were done using the R-package multcomp (Hothorn et al., 2008). To assess the reliability of the mean of the five PEs as a measure of tameness, we calculated Cronbach’s alpha. Correlations between the five PEs were not very strong, but the five items nevertheless achieved an acceptable Cronbach’s alpha of 0.598. In order to assess inter-rater reliability the test videos of 12 randomly selected animals were additionally analysed, and the primary endpoints 3 (hand test), 4 (open field stressor), and 5 (catching from open field) determined by another person. This other person did not know whether the rat in question was from the experimental or from the control group, but was familiar with the testing procedure. To assess concordance between the two raters Kruskal’s gamma (=number of concordant pairs minus number of discordant pairs, divided by the sum of concordant and discordant pairs; Sheskin, 2004) was determined for the three primary endpoints analysed. As the parameters were of an ordinal nature gamma was more suitable than Cohen’s kappa.
3. Results The experimental group had higher values during all test phases and in almost all PEs when compared to the control group (Table 3 and Figs. 4–8), which indicates a higher level of ‘‘tameness’’. In the tests which were performed in the 14th week of life and at the age of 6 months and which involved the unfamiliar person the animals in the experimental group exhibited higher values than the animals in the control group (see Table 4). In the first test involving the unfamiliar person the values of the majority of PEs in the experimental and control groups were slightly higher than those from the tests performed in the same age group (14th week of life) immediately beforehand by the familiar person.
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Table 3 Results of the tests: arithmetic mean values for the respective primary endpoints (PE) in experimental (E) and control (C) group, n = 12 animals per group (OF, open field) increasing ‘‘tameness’’ from 0 to 3 Age at test
Week 6 Week 8 Week 10 Week 14 6 months 6.5 months 9 months
PE 1 (catching)
PE 2 (neck grip)
PE 3 (hand test)
PE 4 (OF stressor)
PE 5 (catching from OF)
E
C
E
C
E
C
E
C
E
C
2.3 1.9 1.5 2.1 2.3 2.5 2.0
0.4 0.5 1.1 0.9 1.3 2.2 1.2
1.8 1.8 1.4 1.5 1.2 1.7 1.3
0.9 1.3 1.0 1.0 1.0 1.1 0.9
2.9 2.6 2.1 2.0 2.1 1.7 2.1
1.7 1.3 1.3 2.0 1.8 1.8 2.1
1.1 1.4 1.2 1.2 1.2 0.7 0.8
0.5 0.2 0.2 0.8 0.6 0.4 0.8
0.6 0.5 0.8 0.8 1.1 1.3 1.3
0.2 0.0 0.6 0.5 1.0 1.4 1.0
An overview of the results of the various testing points can be found in Figs. 9 and 10. Differences in tameness between control and experimental groups at the beginning of the testing regimen (week 6) were large and highly significant (0.847; p < 0.001). While the control group showed a significant increase in tameness over time (0.020 per week; p < 0.001), no
Fig. 4. Results of the tests during the time-frame for PE 1 (catching): frequency of the individual scores in experimental and control groups, n = 12 animals per group, increasing ‘‘tameness’’ from 0 to 3.
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Fig. 5. Results of the tests during the time-frame for PE 2 (neck grip): frequency of the individual scores in experimental and control groups, n = 12 animals per group, increasing ‘‘tameness’’ from 0 to 3.
significant change in tameness over time was noted for the experimental group ( 0.004 per week; p = 0.345) in our data. An overview of the statistically estimated differences between the experimental and control groups during the studied time-frame is shown in Table 5. Up to an age of 26 weeks (6 months of age), there were significant differences between the groups ( p values for multiple comparisons were adjusted using the Westfall method, Hothorn et al., 2008). Additionally, also including the data gathered during the two occasions in which the testing was performed by an unfamiliar experimenter, we found a fairly strong and borderline significant Table 4 Results of the tests by unfamiliar person: arithmetic mean values for the respective primary endpoints (PE) in experimental (E) and control (C) group, n = 12 animals per group (OF, open field) increasing ‘‘tameness’’ from 0 to 3 Age at test
Week 14 6 months
PE 1 (catching)
PE 2 (neck grip)
PE 3 (hand test)
PE 4 (OF stressor)
PE 5 (catching from OF)
E
C
E
C
E
C
E
C
E
C
2.5 2.1
1.7 1.5
2.1 1.8
1.2 1.0
2.0 2.3
1.5 2.0
1.6 1.1
0.8 0.7
1.5 1.5
0.8 1.1
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Fig. 6. Results of the tests during the time-frame for PE 3 (hand test): frequency of the individual scores in experimental and control groups, n = 12 animals per group, increasing ‘‘tameness’’ from 0 to 3. Table 5 Statistically estimated differences between the experimental and control groups every 2 weeks from the beginning of the test (estimate, standard errors and p values) Week of age
Estimate
Standard error
p value
6 8 10 12 14 16 18 20 22 24 26 28 30 32 34 36
0.8468 0.7981 0.7495 0.7009 0.6522 0.6036 0.5550 0.5063 0.4577 0.4090 0.3604 0.3118 0.2631 0.2145 0.1659 0.1172
0.1764 0.1719 0.1682 0.1653 0.1632 0.1620 0.1616 0.1622 0.1637 0.1660 0.1692 0.1731 0.1778 0.1832 0.1891 0.1957
<0.0001*** <0.0001*** <0.0001*** <0.0001*** <0.0001*** 0.000194*** 0.000597*** 0.001802** 0.005173** 0.013746* 0.033145* 0.071725 0.138886 0.241536 0.380478 0.549071
The p values have been adjusted for multiple comparisons using the Westfall method (Hothorn et al., 2008). Significance codes: ‘***’ 0.001, ‘**’ 0.01, ‘*’ 0.05 (adjusted p values reported—Westfall method).
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Fig. 7. Results of the tests during the time-frame for PE 4 (open field stressor): frequency of the individual scores in experimental and control groups, n = 12 animals per group, increasing ‘‘tameness’’ from 0 to 3.
experimenter effect (0.188 for unfamiliar experimenter; p = 0.052). This means, that on average, the rats showed an increase of 0.188 in the average tameness score when the unfamiliar experimenter performed the tests. This effect did not differ significantly between the treatment groups: the interaction between experimental group and familiarity of the experimenter was not significant (0.103 for unfamiliar experimenter in experimental group; p = 0.4500). The concordance between the two raters for PE 3 (hand test), PE 4 (open field stressor) and PE 5 (catching from open field) was calculated and the pooled gamma for the three primary endpoints was 0.82. 4. Discussion Concordance between the two raters was sufficiently high to confirm the results obtained. In all tests performed up to the age of 6 months, including the two tests carried out by an unfamiliar person, the animals in the experimental group invariably achieved higher values in the primary endpoints than the animals in the control group. The higher values in the experimental group suggested a higher level of ‘‘tameness’’. Even in the statistical estimates made with the help of a linear mixed-effect model, a significant difference was noted between the groups up to an age of 6 months. The gentling programme had a long-term beneficial influence on the rats’ behaviour towards humans. This period of half a year seems remarkable, given that the gentling programme was limited to a mere two weeks during the rats’ juvenile development. Apart from the human
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Fig. 8. Results of the tests during the time-frame for PE 5 (catching from open field): frequency of the individual scores in experimental and control groups, n = 12 animals per group, increasing ‘‘tameness’’ from 0 to 3.
contact in the fourth and fifth week of life within the framework of the gentling programme, the animals in the experimental group, just like those in the control group, had not received any particular further human contact. Hence, a limited duration of gentling was able to achieve a long-lasting effect on the rats’ behaviour towards humans. These results coincide with the reports published by other authors such as Do¨ring (1999), Lawlor (2002) and Jones et al. (2003). Gentling experiences can have a long-term effect on rats as was demonstrated by Davis et al. (1997): the rats recognised a specific person who had gentled them 5 months earlier and preferred this person over an unfamiliar person. The increasing values noted in the control group are either the result of an ageing effect or the result of the animals getting used to the experimental routine. It is known, that repeated tests can lead to familiarisation, for example in the open field test (Doyle and Pratt Yule, 1959; Williams and Russell, 1972; Rebouc¸as and Schmidek, 1997 among others). Hirsja¨rvi et al. (1990) as well as Hirsja¨rvi and Va¨liaho (1995) found, that the behaviour of non-gentled rats became more similar to the behaviour of gentled animals with repeated tests in the open field. Gentling was performed at 10 weeks of age in male rats. Shyu et al. (1987) found that rats, which were accustomed to specific procedures (fixation), showed less stress-related reactions. The improvement of the results in the control group could be the result of an age-related effect. It is plausible, that rats become calmer as they become older, allowing easier handling. This explanation is supported by the fact, that during the initial tests performed frequently during the first three months of life (at 6, 8, 10 and 14 weeks of age), the control group did not show the
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Fig. 9. Overview of the results at the various testing points. Arithmetic mean values of all five primary endpoints (PEs) of experimental and control group, n = 12 animals per group, increasing ‘‘tameness’’ from 0 to 3.
results that the gentling group did. Only after half a year did the results begin to mirror those of the experimental group and this was after a testing break of two months. In order to differentiate the ageing and familiarity effects, additional tests performed at the same time with a separate control group that had no previous testing experience would be necessary. In summary, animals in a gentling program are as ‘‘tame’’ at 6 weeks of age as rats with repeated test experiences are at 6 months of age. Additionally, as gentling does not involve a specific testing situation, but has rather an unspecific taming effect, one can assume the gentled animals will react more tamely during other manipulations and procedures and would be easier to handle. The analysis of the difference between the familiar and unfamiliar experimenter showed a borderline significant experimenter effect in favour of the unfamiliar person. Accordingly, contrary to the results of Briese and De Quijada (1970), there was no evidence of any higher level of fear towards unfamiliar persons. The fact that the experimental group achieved higher values in the primary endpoints suggests a lingering effect of gentling, i.e. a higher level of ‘‘tameness’’ of the animals which had been subjected to the gentling programme. The altogether higher values of the experimental and the control group may be explained by a habituation effect in the animals. It is conceivable that the animals had grown accustomed to the test procedure by the repetition of the test after an interval of a mere 2 days. Another possible explanation is that the unfamiliar person may have caused the animals to behave more ‘‘tamely’’ due to a possibly slightly different way of handling the animals, such as touching them or catching them in a softer way. However, it was tried to keep the interactions of the experimenter and the unfamiliar person with the subjects as standardised as possible. Another reason might be personal factors of the experimenter such as smell, etc.
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Fig. 10. Overview of the results of the two tests with an unfamiliar experimenter. Arithmetic mean values of all five primary endpoints (PEs) of experimental and control group, n = 12 animals per group, increasing ‘‘tameness’’ from 0 to 3. For comparison, the results of the test 2 days earlier with the familiar experimenter are shown (gray).
In summary, the animals subjected to the gentling programme have shown a reduced level of fear reactions and consequently a higher degree of ‘‘tameness’’ towards humans. ‘‘Tameness’’ was associated not only with the person who had performed gentling and had become familiar to the animals through care, but also with a stranger. This means that the gentling effect was not restricted to one single person. The fact that the statistically estimated difference between the groups remained until the age of 6 months permits the conclusion that the gentling effect had persisted up to that point and can therefore be considered ‘‘long-lasting’’. In order to allow these findings to be put into practice the gentling programme used in this study should be further optimised. One particular objective should be to try and reduce the workload and staff requirement involved in conducting the gentling programme. For that reason, the period suitable for gentling should be further limited during any continuation studies. In addition, the effects of the individual factors (gentling–food–talking) of the gentling programme on the animals’ behaviour should be evaluated. Based on the results of this study, early gentling of laboratory rats can be recommended to achieve an immediate and long-lasting level of ‘‘tameness’’. As repeated tests lead to a familiarisation effect, these should be used to familiarise the animal with a specific test manipulation even at an advanced age of the rat. Both methods can be used to reduce fear and stress related to contact with humans. The results of this study should be taken into account when planning and conducting animal experiments as avoidable fear and stress reactions of laboratory rats are an issue of animal welfare on the one hand, while on the other hand – as has been shown in the literature again and again – they can influence test results.
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5. Conclusion The gentling programme performed proved suitable to achieve a long-lasting reduction of the animals’ fear of humans. The animals from the experimental group exhibited a higher level of ‘‘tameness’’ than those from the control group, even towards an unfamiliar person. Acknowledgement We wish to thank Ms. Pia Zausinger for performing the secondary evaluation of the data for calculating inter-rater reliability. References Abel, E.L., 1971. Habituation as a factor in early handling. J. Comp. Physiol. Psychol. 74, 219–221. Boivin, X., Braastadt, B.O., 1996. Effects of handling during temporary isolation after early weaning on goat kids’ later response to humans. Appl. Anim. Behav. Sci. 48, 61–71. Brand, U., 1998. Untersuchungen zu Streßparametern bei ma¨nnlichen Wistar-Ratten unter dem Einfluß einfacher Manipulationen. Thesis. Freie Universita¨t Berlin, D. Briese, E., De Quijada, M., 1970. Colonic temperature of rats during handling. Acta Physiol. Lat. Am. 20, 9–14. Candland, D.K., Faulds, B., Thoman, D.B., Candland, M.H., 1960. The reinforcing value of gentling. J. Comp. Physiol. Psychol. 53, 55–58. Candland, D.K., Horowitz, S.H., Culbertson, J.L., 1962. Acquisition and retention of acquired avoidance with gentling as reinforcement. J. Comp. Physiol. Psychol. 55, 1062–1064. Council of Europe, 1986. European Convention for the Protection of Vertebrate Animals used for Experimental and Other Scientific Purposes. ETS No. 123. Strasbourg, F. http://conventions.coe.int/Treaty/en/Treaties/Html/123.htm. Council of Europe, 1997. Multilateral Consultation of Parties to the European Convention for the Protection of Vertebrate Animals used for Experimental and Other Scientific Purposes (ETS 123)—Resolution on the Accommodation and Care of Laboratory Animals. http://www.coe.int/T/E/Legal_affairs/Legal_co-operation/Biological_safety%2C_use _of_animals/Laboratory_animals/Res%20accommodation.asp#TopOfPage. Council of Europe, 2006. Appendix A of the European Convention for the Protection of Vertebrate Animals used for Experimental and Other Scientific Purposes (ETS No. 123)—Guidelines for Accommodation and Care of Animals. Council of Europe, Strasbourg, France, June 15, 2006. Cons 123 (2006) 3. http://www.coe.int/t/e/legal_affairs/legal_ co-operation/biological_safety%2C_use_of_animals/laboratory_animals/2006/Cons123(2006)3AppendixA_en.pdf. Davis, H., Perusse, R., 1988. Human-based social interaction can reward a rat’s behavior. Anim. Learn. Behav. 16, 89–92. Davis, H., Taylor, A.A., Norris, C., 1997. Preference for familiar humans by rats. Psychon. Bull. Rev. 4, 118–120. Doyle, G., Pratt Yule, E., 1959. Grooming activities and freezing behaviour in relation to emotionality in albino rats. Anim. Behav. 7, 18–22. Do¨ring, D., 1999. Ka¨figraumausnutzung bei Laborratten. Thesis. Freie Universita¨t Berlin, D. http://www.diss.fuberlin.de/1999/82/. Dyer Jr., D.P., Southwick, C.H., 1974. A possible sensitive period for juvenile socialization in mice. Behav. Biol. 12, 551– 558. Eells, J.F., 1961. Inconsistency of early handling and its effects upon emotionality in the rat. J. Comp. Physiol. Psychol. 54, 690–693. Einon, D.F., Morgan, M.J., Kibbler, C.C., 1978. Brief periods of socialization and later behavior in the rat. Dev. Psychobiol. 11, 213–225. Francis, D., Diorio, J., Laplante, P., Weaver, S., Seckl, J., Meaney, M., 1996. The role of early environmental events in regulating neuroendocrine development: moms, pups, stress and glucocorticoid receptors. Ann. NY Acad. Sci. 794, 136–152. Gertz, B., 1957. The effect of handling at various age levels on emotional behavior of adult rats. J. Comp. Physiol. Psychol. 50, 613–616. Hemsworth, P.H., Barnett, J.L., 1992. The effects of early contact with humans on the subsequent level of fear of humans in pigs. Appl. Anim. Behav. Sci. 35, 83–90.
570
B.M. Maurer et al. / Applied Animal Behaviour Science 114 (2008) 554–571
Hirsja¨rvi, P.A., Junnila, M.A., 1988. The effects of gentling on open-field behaviour of rats. In: Beynen, A.C., Solleveld, H.A. (Eds.), New Developments in Biosciences: Their Implications for Laboratory Animal Science. Martinus Nijhoff Publishers, Dordrecht, pp. 399–403. Hirsja¨rvi, P.A., Va¨liaho, T., 1995. Gentled and nonhandled Wistar rats in a mildly novel open-field situation. Scand. J. Lab. Anim. Sci. 22, 265–269. Hirsja¨rvi, P.A., Junnila, M.A., Va¨liaho, T.U., 1990. Gentled and non-handled rats in a stressful open-field situation; differences in performance. Scand. J. Psychol. 31, 259–265. Hol, T., van den Berg, C.L., van Ree, J.M., Spruijt, B.M., 1999. Isolation during the play period in infancy decreases adult social interactions in rats. Behav. Brain Res. 100, 91–97. Hothorn, T., Bretz, F., Westfall, P., with contributions by Heiberger, R.M., 2008. Multcomp: Simultaneous Inference for General Linear Hypotheses. R Package Version 0.993-1. Jones, B.R., Waddington, D., 1993. Attenuation of the domestic chick’s fear of human beings via regular handling: in search of a sensitive period. Appl. Anim. Behav. Sci. 36, 185–195. Jones, T.A., Bury, S.D., Adkins-Muir, D.L., Luke, L.M., Allred, R.P., Sakata, J.T., 2003. Importance of behavioral manipulations and measures in rat models of brain damage and brain repair. ILAR J. 44, 144–152. Jones, B.R., 1994. Ontogeny of response to humans in handled and non-handled female domestic chicks. Appl. Anim. Behav. Sci. 42, 261–269. Karsh, E.B., Turner, D.C., 1988. Die Mensch/Katze Beziehung. In: Turner, D.C., Bateson, P. (Eds.), Die domestizierte Katze-eine wissenschaftliche Betrachtung ihres Verhaltens. Albert Mu¨ller Verlag, Ru¨schlikon-Zu¨rich, pp. 192–215. Krohn, C.C., Jago, J.G., 2001. The effect of early handling on the socialisation of young calves to humans. Appl. Anim. Behav. Sci. 74, 121–133. Kvetnansky, R., Sun, C.L., Lake, C.R., Thoa, N., Torda, T., Kopin, I.J., 1978. Effect of handling and forced immobilization on rat plasma levels of epinephrine, norepinephrine and dopamine-hydroxylase. Endocrinology 103, 1868–1874. Lawlor, M., 2002. Comfortable quarters for rats in research institutions. In: Reinhardt, V., Reinhardt, A. (Eds.), Comfortable Quarters for Laboratory Animals. 9th ed. Animal Welfare Institute, Washington, DC, pp. 26–32. Levine, S., 1957. Infantile experience and resistance to physiological stress. Science 126, 405. Levine, S., Alpert, M., Lewis, G.W., 1957. Infantile experience and the maturation of the pituitary adrenal gland. Science 126, 1347. Liu, D., Diorio, J., Tannenbaum, B., Caldji, C., Francis, D., Freedman, A., Sharma, S., Pearson, D., Plotsky, P.M., Meaney, M.J., 1997. Maternal care, hippocampal glucocorticoid receptors, and hypothalamic-pituitary-adrenal responses to stress. Science 277, 1659–1662. Lore, R., Flannelly, K., 1977. Rat society. Sci. Am. 236, 106–116. McCall, R.B., Lester, M.L., Corter, C.M., 1969. Caretaker effect in rats. Dev. Psychol. 1, 771. Mende, G., 1999. Untersuchung zur Beurteilung der Belastung von Laborratten durch einfache Manipulationen, an den Parametern Kortikosteron und Prolaktin. Thesis. Freie Universita¨t Berlin, D. http://www.diss.fu-berlin.de/1999/101/. Morton, J.R.C., 1968. Effects of early experience ‘‘handling and gentling’’ in laboratory animals. In: Fox, M.W. (Ed.), Abnormal Behaviour in Animals. W.B. Saunders Company, Philadelphia, pp. 261–292. Nu´nez, J.F., Ferre´, P., Escorihuela, R.M., Tobena, A., Fernande´z-Teruel, A., 1996. Effects of postnatal handling of rats on emotional, HPA-axis, and prolactin reactivity to novelty and conflict. Physiol. Behav. 60, 1355–1359. Pinheiro, J., Bates, D., DebRoy, S., Sarkar, D., 2007. Nlme: Linear and Nonlinear Mixed Effects Models. R Package Version 3. , pp. 1–83. Podberscek, A.L., Blackshaw, J.K., Beattie, A.W., 1991. The effects of repeated handling by familiar and unfamiliar people on rabbits in individual cages and group pens. Appl. Anim. Behav. Sci. 28, 365–373. R Development Core Team, 2007. R: A Language and Environment for Statistical Computing. R Foundation for Statistical Computing, Vienna, Austria. http://www.R-project.org. Rapp, K.G., Deerberg, F., 1987. Zur Problematik der Stichprobenentnahme und Versuchsgruppenzusammenstellung bei Auszuchtpopulationen. Z. Versuchstierkd. 29, 193–203. Rebouc¸as, R.C.R., Schmidek, W.R., 1997. Handling and isolation in three strains of rats affect open field, exploration, hoarding and predation. Physiol. Behav. 62, 1159–1164. Scott, J.P., Fuller, J.L., 1965. Genetics and the Social Behavior of the Dog. The University of Chicago Press, Chicago. Sheskin, D.J., 2004. Handbook of Parametric and Nonparametric Statistical Procedures. Chapman and Hall, New York. Shyu, W.C., Mordenti, J.L., Nightingale, C.H., Tsuji, A., Quintiliani, R., 1987. Effect of stress on the pharmacokinetics of Amikacin and Ticarcillin. J. Pharm. Sci. 76, 265–266. Weininger, O., 1954. Physiological damage under emotional stress as a function of early experience. Science 119, 285–286.
B.M. Maurer et al. / Applied Animal Behaviour Science 114 (2008) 554–571
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Weininger, O., McClelland, W.C., Arime, R.K., 1954. Gentling and weight gain in the albino rat. Canad. J. Psychol. 8, 147–151. Weininger, O., 1956. The effects of early experience on behavior and growth characteristics. J. Comp. Physiol. Psychol. 1, 1–9. Williams, D.I., Russell, P.A., 1972. Open-field behaviour in rats: effects of handling, sex and repeated testing. Br. J. Psychol. 63, 593–596. Wyly, M.V., Denenberg, V.H., De Santis, D., Burns, J.K., Zarrow, M.X., 1975. Handling rabbits in infancy: in search of a critical period. J. Comp. Physiol. Psychol. 8, 179–186.