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Behavioural and endocrine fear responses in Japanese quail upon presentation of a novel object in the home cage
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Behavioural Processes 77 (2008) 313–319
Behavioural and endocrine fear responses in Japanese quail upon presentation of a novel object in the home cage S. Richard a,∗ , N. Wacrenier-Cer´e b , D. Hazard b , H. Saint-Dizier a , C. Arnould a , J.M. Faure b a
Physiologie de la Reproduction et des Comportements, UMR85 INRA-CNRS-Universit´e de Tours-Haras Nationaux, 37 380 Nouzilly, France b Unit´ e de Recherches Avicoles, UR83 INRA, 37 380 Nouzilly, France Received 22 March 2007; received in revised form 27 June 2007; accepted 17 July 2007
Abstract Most tests used to study fear in birds involve transferring them to a novel environment, which constitutes a bias in studies aiming at identifying the neural correlates of a specific fear-inducing situation. In order to investigate fear in birds with minimum interference by humans, behavioural and endocrine responses to the presentation of a novel object in the home cage were investigated in two lines of Japanese quail divergently selected for long or short duration of tonic immobility, a behavioural index of fear. Presentation of the novel object induced typical fear responses (avoidance of the object, increased pacing and increased plasma corticosterone levels) that were similar in the two lines of quail. Presentation of a novel object in the home cage thus appears to be a suitable stimulus to induce fear reactions in quail, with minimum interference from other motivational systems. The fact that quail of both lines reacted similarly in this test, while they are known to differ greatly in their behavioural responses to other fear-inducing tests, illustrates the multidimensional nature of fear. © 2007 Elsevier B.V. All rights reserved. Keywords: Birds; Corticosterone; Coturnix japonica; Emotions; Escape reaction; Fear; Tonic immobility response
1. Introduction Growing concern for animal welfare and efforts to understand the evolution of emotions in vertebrates have motivated research into the neural mechanisms controlling fear reactions in birds. Most tests used to study fear in birds, such as the open-field test or the tonic immobility test, usually involve transferring them to a novel environment (Jones, 1996). In addition, while most tests are carried out in isolation, experimental birds are often housed in groups until the time of the test. Transfer to a novel environment and sudden social isolation are likely to influence not only the way the birds respond to the test itself, but also the associated patterns of brain activation (Murphy, 1978; Papa et al., 1993; Takeuchi et al., 1996; Bilc´ık et al., 1998). Therefore, to avoid these complications and control better the emotional drive that the test is intended to provoke, it would be better to use a standardised test carried out in the bird’s home cage. The home cage has been used before to test fear reactions in birds, ∗ Corresponding author at: Physiologie de la Reproduction et des Comportements, INRA, 37 380 Nouzilly, France. Tel.: +33 247 42 76 57; fax: +33 247 42 77 43. E-mail address: [email protected] (S. Richard).
0376-6357/$ – see front matter © 2007 Elsevier B.V. All rights reserved. doi:10.1016/j.beproc.2007.07.005
such as in laying hens and great tits (Verbeek et al., 1994; Jones, 1996; Bilc´ık et al., 1998). Most of these experiments involve the presentation of a novel object, the objects being usually presented manually by a human, who subsequently stays in front of the cage to carry out the behavioural observations. The reactions of the bird therefore reflect a combination of responses to the object and to the human. We set out initially in this study to evaluate the specific behavioural and endocrine responses of Japanese quail to the presentation of a novel object in the home cage from a distance, with minimum human interference. Such a test might prove useful to study the specific mechanisms of fear responses in birds, in particular at the level of the brain. Fear responses to novelty usually fade away rapidly and are likely to be associated with discrete and labile changes at the level of the brain, which may be difficult to detect (Duncan, 1985). These difficulties in studying the central mechanisms of fear responses would be overcome if we could induce fear responses that are sustained over time. To do this, we repeatedly presented the same object over 30 min, the object being presented suddenly to increase the intensity of the stimulus. Two lines of Japanese quail that have been divergently selected for either long (LTI) or short (STI) duration of tonic immobility (Mills and Faure, 1991) were used in this study. The
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selection process has been continued since the foundation of the lines and at the 36th generation, the mean duration of tonic immobility (±standard deviation) was 205 (±97) s in the LTI line and 10 (±14) s in the STI line. The duration of tonic immobility is positively correlated with other measures of fear in birds (Jones and Mills, 1983; Mills and Faure, 1986) and selection on this parameter has confirmed its relation to fear: LTI quail display higher levels of fear than STI quail when measured in a number of classical tests. LTI quail freeze more, vocalise and move less in an open field, emerge later from a ‘hole-in-thewall’ box (Jones et al., 1991) and struggle less during restraint in a ‘crush cage’ (Jones et al., 1994) than STI quail. In addition to their behavioural divergence, STI and LTI quail also differ in the activity of physiological systems that are involved in fear responses. As regards the activity of the autonomic nervous system, reflected by studies of heart rate variability, parasympathetic tone has been shown to be stronger in STI than in LTI quail (Valance et al., 2006). Moreover, differences have also been reported between STI and LTI quail in adrenocortical responses to stressful events. Notably, there is a greater increase in circulating corticosterone concentrations in STI than in LTI quail in response to restraint (Jones et al., 1994; R´emignon et al., 1996; Hazard et al., 2005). Studying the responses induced by the presentation of a novel object in the home cage in STI and LTI quail should help evaluate the ability of this test to induce fear in quail with different levels of underlying fearfulness. The two experiments presented here were conducted to assess behavioural and adrenocortical responses of STI and LTI quail to the presentation of a novel object in the home cage. The aim was to evaluate the ability of the test to induce fear in birds in the absence of human interference, as a prerequisite for using such a test to study the neural mechanisms of fear in birds. LTI quail were expected to exhibit stronger behavioural fear responses than STI quail upon presentation of a novel object in their home cage, and it was also thought likely that adrenocortical responses would differ between the two lines. 2. Materials and methods 2.1. Subjects Japanese quail (Coturnix japonica) of the 36th and 38th generations of the LTI and STI lines selected and maintained at the Station de Recherches Avicoles, Nouzilly, France (Mills and Faure, 1991), were used in the present experiment. On the day of hatching, chicks of both lines were wing-banded and transferred to communal floor pens, where they were reared in single line groups under continuous illumination for three weeks. On the 21st day after hatching, the chicks were sexed on the basis of plumage colour and transferred to communal floor pens, where they were reared in single line, single sex groups under a 16:8 h light:dark schedule. Unless otherwise specified, food and water were freely available at all times. The birds were treated according to the European Union’s Council Directive of November 24, 1986 (86/609/EEC) throughout. All procedures described here fully comply with French legislation on research involving animals.
Fig. 1. Diagram of an experimental cage viewed from the top with roof removed, showing the site at which the novel object was dropped as well as the boundaries of the three zones used for behaviour analysis.
2.2. Testing apparatus and procedure 2.2.1. Experiment 1 32 LTI and 32 STI male quail were used in experiment 1. Within each line, half of the quail constituted a control group (CTRL LTI, n = 16 and CTRL STI, n = 16) and the other half were subjected to the presentation of a novel object (OBJ LTI, n = 16 and OBJ STI, n = 16). Experiment 1 was constituted by a series of six consecutive repeats comprising 10–12 quail each. Each repeat involved two or three quail from each of the four experimental groups. One week before testing, LTI and STI male quail were transferred to a testing room maintained at 20 ◦ C under a 16:8 h light:dark photoperiod. All quail were sexually mature at the time of testing (6–10 weeks). They were housed individually in PVC cages (length = 42 cm; width = 24 cm; height = 25 cm, Fig. 1) with wood-shavings on the floor and an opaque PVC roof. The front wall of each cage was made of clear perspex so that the quail could be observed with a video camera. The quail could see and hear other quail in the same room. In the corner of the cage adjacent to the food trough and to the back wall, a cylindrical PVC chimney built over the roof allowed the release of an object into the cage on the day of testing. The object used in the present experiment was a PVC cylinder (outer diameter = 4 cm; height = 21 cm) covered with 2-cm wide horizontal stripes of coloured tape (alternating blue, yellow, grey, red, black and white tape), similar to that previously used by Jones (1987). The object was suspended on a string, so that its base remained 5–6 cm above the floor once introduced into the cage. This apparatus allowed the object to be introduced into the cage and removed from a distance, with minimal disturbance to the quail, the experimenter remaining out of sight of the bird during the procedure. During the week preceding testing, the food trough of every cage was removed daily for 2 h, in order to habituate the quail to the procedure. This removal of the feeder was used to attract the quail momentarily to the part of the cage where the object would be dropped on the testing day. The quail were also habituated to
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was collected and stored at −20 ◦ C prior to the measurement of corticosterone levels following Etches’ (1976) specific radioimmunoassay. 2.3. Behaviour analysis
Fig. 2. Diagram illustrating the time-course of the test procedure for quail of the OBJ group, starting when a quail resumed pecking at the food after 40 min of feeder withdrawal. If a quail failed to peck at the food, the test procedure started 60 s after the return of the feeder.
the placement of a video camera and a cardboard screen in front of their cage, at a distance of approximately 1 m. On the day of testing, the cardboard screen prevented other quail in the room from seeing the novel object in the cage of the quail to be tested. On the day of testing, the food trough of each cage was removed for 40 min. When the food trough was replaced in the cage, as soon as a quail of the OBJ group pecked at the food, the multicoloured cylinder was dropped into the cage, as described above. If a quail failed to peck at the food, the multicoloured cylinder was dropped into the cage 60 s after the return of the food trough. The object was withdrawn 5 min after being dropped and the quail was left undisturbed for 1 min. This object presentation procedure, followed by the 1-min interval, was then repeated four times without taking into account the quail’s feeding behaviour (Fig. 2). A video recording was made of the behaviour of each OBJ quail throughout the object presentation procedure (total duration = 30 min). The behaviour of CTRL quail was recorded for 30 min, starting as soon as a quail pecked at the food after the return of the food trough. If a CTRL quail failed to peck at the food, the video recording started 60 s after the return of the food trough, without further disturbance. 2.2.2. Experiment 2 24 LTI and 24 STI male quail, other than those used in experiment 1, were used in experiment 2. Within each line, half of the quail constituted a control group (CTRL LTI, n = 12 and CTRL STI, n = 12) and the other half were subjected to the presentation of a novel object (OBJ LTI, n = 12 and OBJ STI, n = 12). Experiment 2 was constituted by a series of four consecutive repeats comprising 12 quail each. Each repeat involved three quail from each of the four experimental groups. The procedures were identical to those of experiment 1, except that all quail were decapitated either immediately after the first 5-min object presentation (OBJ group) or 5 min after the return of the feeder (CTRL group). Blood was collected from each quail directly after decapitation in a tube containing EDTA (2 mg/ml blood). Decapitation was used in this study because this sampling procedure has previously been shown to affect the least basal corticosterone levels, compared to venipuncture, cardiac or jugular puncture (Hazard et al., 2004). This procedure has proven particularly useful to reveal responses of very small amplitude, such as those described in the present study. The blood samples were temporarily stored on ice and subsequently centrifuged at 2000 × g for 15 min at 4 ◦ C. The plasma
The quail were coded so that all behaviour observations were performed ‘blind’ with respect to the line. The behaviour of OBJ quail was analysed during the first and fifth presentations of the object, i.e. during 5-min time windows starting when the object was dropped in the cage (P1 and P5, Fig. 2). The behaviour of CTRL quail was analysed at corresponding time points, i.e. 0–5 min and 24–29 min after the first peck at the food after the return of the food trough. If a CTRL or OBJ quail failed to peck at the food after the return of the food trough, the first time window for observations started 60 s after the return of the food trough. The cage was subdivided into three zones, zone 1 including the site of object presentation and zone 3 being furthest away from it (Fig. 1). The amounts of time spent in zone 1 and zone 3 and the amount of time spent eating were recorded. The occurrence of freezing behaviour and pecking at the object were also recorded. Freezing behaviour was defined as complete immobility, occurring suddenly with an abrupt interruption of ongoing activities and lasting for more than 3 s, sometimes including very small head movements such as those associated with breathing. Finally, the presence of pacing behaviour was recorded when a quail exhibited repetitive movements that were fixed in form and orientation: pacing only or pacing in conjunction with head movements or jumps in some cases. The occurrence of pacing was recorded for each 10-s interval of the 5-min period, using the one-zero sampling regime (Martin and Bateson, 1986). These records were then pooled into a pacing score (between 0 and 30) over 5 min. All observations were made by a single experimenter, using focal sampling. The Observer 3.0 software (Noldus Information Technology, The Netherlands, 1993) was used to record observations, except for pacing behaviour. 2.4. Statistical analysis As the data were not normally distributed, nonparametric statistical analyses were performed, using Statview 5.0 (SAS Institute Inc., USA, 1992–1998) and Excel 2000 (Microsoft Corporation). For both experiments, the effects of treatment (OBJ versus CTRL), line and interaction between line and treatment were assessed using 2 × 2 factorial analysis for unrelated datasets, with nonspecific hypotheses (Meddis, 1984). Whenever there was a significant interaction between line and treatment, the effects of treatment were evaluated independently within each line using a Mann–Whitney U-test. Comparison between the first and fifth object presentations (experiment 1) was carried out within each experimental group using a Wilcoxon test. The time spent in zone 1, time spent in zone 3 and time spent eating were necessarily related to some extent. However, considering that these three variables reflected separate aspects of the behaviour of the quail and were not highly
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correlated, we analysed them as independent variables. Freezing behaviour and pecking at the object occurred so rarely that these variables were not included in the statistical analysis. Box-plot diagrams were used to illustrate the results. Values illustrated by a box plot are the median, upper and lower quartiles, 10th and 90th percentiles and extreme observations. 3. Results 3.1. Experiment 1 (Fig. 3) 3.1.1. Behaviour during the first object presentation There was an effect of treatment on the time spent in zone 3 (Fig. 3B, H = 11.27, p < 0.001) and on the pacing score (Fig. 3C, H = 12.47, p < 0.001). OBJ quail spent more time in zone 3 and exhibited more pacing than CTRL quail. There was no significant effect of treatment on the time spent eating (Fig. 3D, H = 2.95, p > 0.05). There was no significant effect of line on the time spent in zone 3, on the pacing score, nor on the time spent eating (H < 1.7, p > 0.1 for each variable). However, there was an effect of interaction between line and treatment on the time spent in zone 1 (H = 4.22, p < 0.05). Indeed, in the STI line the time spent in zone 1 did not differ significantly between OBJ and CTRL quail (z = 0.98, p > 0.1) whereas in the LTI line OBJ
quail spent less time in zone 1 than did CTRL quail (z = 3.54, p < 0.001). There was no significant effect of interaction between line and treatment on any other variable (time spent in zone 3, pacing score, time spent eating: H < 2.2, p > 0.1 for each variable). 3.1.2. Behaviour during the fifth object presentation There was an effect of treatment on the time spent in zone 1 (Fig. 3A, H = 4.28, p < 0.05) and on the pacing score (Fig. 3D, H = 12.42, p < 0.001). OBJ quail spent less time in zone 1 and exhibited more pacing than CTRL quail. Neither the time spent in zone 3, nor the time spent eating differed significantly between CTRL and OBJ quail (Fig. 3B and C, H = 3.64, p > 0.05 and H = 0.43, p > 0.1, respectively). There was no significant effect of line on any of the variables of interest (H < 2.8, p > 0.05 for each variable). There was no significant effect of interaction between line and treatment on any of the variables studied (H < 1.1, p > 0.1for each variable). 3.1.3. Behaviour changes over time There was a decrease in the time spent in zone 1 between the first and fifth experimental periods in CTRL STI and LTI quail and OBJ STI quail, but not in OBJ LTI quail (Fig. 3A, CTRL STI, z = 2.79, p < 0.01; CTRL LTI, z = 2.28, p < 0.05; OBJ STI, z = 2.12, p < 0.05; OBJ LTI, z = 0.84, p > 0.1). The time
Fig. 3. Box-plot diagrams illustrating the behaviour of Japanese quail selected for long or short duration of tonic immobility (LTI and STI, respectively) in experiment 1. Zone 1 = area of the cage where the object was presented to quail of the OBJ group; zone 3 = area of the cage furthest away from the site of object presentation; P1 = first object presentation; P5 = fifth object presentation.
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Table 1 Behaviour of Japanese quail selected for long or short duration of tonic immobility (LTI and STI, respectively) in experiment 2a Time spent in zone 1 (s)b
Time spent in zone 3 (s)c
Pacing score
Time spent eating (s)
LTI CTRL OBJ
185 (94) 106 (139)
49 (96) 98 (156)
0 (11) 11 (18)
30 (66) 12 (37)
STI CTRL OBJ OBJ vs. CTRLd
86 (118) 55 (92) p < 0.01
69 (82) 116 (148) p < 0.05
0 (5) 24 (11) p < 0.001
14 (21) 1 (19) p > 0.1
a
Values are medians with interquartiles in parentheses. Zone 1 = area of the cage where the object was presented to OBJ quail. c Zone 3 = area of the cage furthest away from the site of object presentation. d Results of factorial analysis for unrelated datasets as in Meddis (1984). There was no significant line effect and no significant effect of interaction between line and treatment. b
spent in zone 3 and the pacing score did not change significantly between the first and fifth experimental periods in any of the groups (Fig. 3B and D, z < 1.9, p > 0.05 in each group for each of these two variables). The most striking behaviour change between the first and fifth experimental periods was a decrease in the time spent eating in all groups (Fig. 3C, CTRL LTI, z = 3.30, p = 0.001; CTRL STI, z = 3.52, p < 0.001; OBJ LTI, z = 2.17, p < 0.05; OBJ STI, z = 2.73, p < 0.01).
3.2.2. Corticosterone (Fig. 4) Plasma corticosterone concentrations were higher in OBJ than in CTRL quail (H = 6.33, p < 0.05). There was no significant effect of line and no significant effect of interaction between line and treatment on plasma corticosterone concentrations (H < 0.01, p > 0.1 for each of these two effects).
3.2. Experiment 2
Presentation of a novel object in the home cage induced typical behavioural and endocrine fear responses in Japanese quail. Behaviourally, the first presentation of the object induced escape and avoidance reactions by the quail in both experiments. The quail in the OBJ group spent less time in the zone where the object was presented, than quail in the control group. They also spent more time than the control quail in the part of the cage furthest away from the object. This indicates avoidance of the object by the quail. Moreover, OBJ quail exhibited more pacing behaviour than control quail. Pacing behaviour has previously been observed in response to the presentation of fear stimuli in a restricted space, and in such a context it is usually thought to result from escape attempts (Murphy, 1977). Thus, the heightened pacing exhibited by OBJ quail in response to the presentation of the novel object may be interpreted in terms of escape from the object, whereas the low level of pacing observed in control quail probably reflected a more chronic response of the quail to their housing conditions, social isolation being a potential source of stress for some individuals (Dantzer, 1986; Mason and Latham, 2004). More generally, escape and avoidance are typically induced by presentation of a novel and intense stimulus and are considered as fear responses (Duncan, 1985). At the endocrine level, the novel object induced a slight, but significant, rise in plasma corticosterone levels, indicating that the hypothalamo–pituitary–adrenal axis was activated. As in mammals, the hypothalamo–pituitary–adrenal axis is a major route for the endocrine stress response in birds (Hill, 1983; Silverin, 1998). In particular, activation of this axis in response to fearful stimuli has previously been described in birds, including quail (Jones and Harvey, 1987; Hazard et al., 2005; Jones et al., 2005). Thus, the rise in plasma corticosterone levels induced by presentation of the novel object suggests that the test situation was
3.2.1. Behaviour (Table 1) As in experiment 1, there was an effect of treatment on the time spent in zone 1 (H = 4.96, p < 0.01), the time spent in zone 3 (H = 4.55, p < 0.05) and the pacing score (H = 17.18, p < 0.001). OBJ quail spent less time in zone 1, more time in zone 3 and exhibited more pacing than CTRL quail. The time spent eating did not differ significantly between OBJ and CTRL quail (H = 2.49, p > 0.1). There was no significant effect of line on any of the behaviour measurements made in experiment 2 (H < 3.6, p > 0.05 for each variable). Neither was there any significant effect of interaction between line and treatment (H < 3.5, p > 0.05 for each variable).
Fig. 4. Box-plot diagrams illustrating the plasma corticosterone concentrations of Japanese quail selected for long or short duration of tonic immobility (LTI and STI, respectively) in experiment 2.
4. Discussion
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perceived by the quail as stressful. Overall, these behavioural and endocrine responses are characteristic of fear and similar to those described by Jones (1985, 1987) in laying hens upon presentation by a human of a novel object in the food trough. Thus, presentation of a novel object in the home cage with minimum human interference appears to be an appropriate design for studying the neural mechanisms of fear reactions in birds. Such a design should prove particularly useful for studying more specifically the relationships between the characteristics of a given stimulus and the reactions it induces, to understand better the way birds perceive their environment. Unlike most novel object tests, that involve a single presentation of a novel object (e.g. Miller et al., 2005), the present test consisted in repeated, sudden presentations of a novel object in order to induce sustained fear reactions over 30 min. In such a design, the quail exhibited very few signs of approach towards the object, but showed sustained fear responses over the successive presentations. Indeed, the fifth presentation of the object still induced significant escape and avoidance. The major behavioural change that was recorded between the first and the fifth experimental periods was a reduction in the time spent eating in all experimental groups, including controls. This was associated with a reduction in the time spent in zone 1, where the food trough was located. The reduction in the time spent eating over the experimental periods was linked to the test procedure: the 40-min feeder withdrawal preceding the first experimental period in all groups temporarily increased the quails’ motivation to eat. This motivation appeared to be similar in STI and LTI quail (no significant effect of line and no significant effect of interaction between line and treatment on the time spent eating), which is in agreement with previous work showing similar motivation to feed in the two lines (Turro-Vincent et al., 1995; Minvielle et al., 2002). At the beginning of the test, the motivation to eat attracted quail to zone 1, where the object was to be presented, but the attraction remained slight enough to allow the expression of fear behaviour in OBJ quail in both experiments. The only statistically significant variations observed between the first and fifth experimental periods may thus be attributed to the reduction in the motivation to eat over time and appear to be unrelated to the presentation of the object. However, there was an indication that avoidance of the object was weaker during the fifth than during the first presentation. Indeed, the time spent in the part of the cage furthest away from the object did not differ significantly between experimental groups during the fifth presentation, whereas it was higher in OBJ than in control quail during the first presentation. This slight reduction in avoidance behaviour may reflect the beginning of a habituation process. It is noteworthy that, in spite of this sign of habituation, typical fear responses were still evoked by the object during the fifth presentation since the quail continued to exhibit significant escape and avoidance behaviours. Such persistence of fear reactions over time is likely to be associated with sustained activation of specific brain regions (Schulkin et al., 2005). Thus, the design used in the present study may facilitate identification of the neural mechanisms underlying fear reactions in birds. Quail from the LTI and STI lines, which have been divergently selected on the duration of tonic immobility, exhibited
similar behavioural and endocrine fear responses to the novel object. The only difference between the two lines appeared in the first experiment to be an effect of interaction between line and treatment on the time spent in zone 1, where the object was presented: Avoidance of the object during the first experimental period was more pronounced in LTI than in STI quail. This result, indicative of stronger fear reactions in LTI than in STI quail, is consistent with several other studies demonstrating that LTI quail exhibited exaggerated fear behaviour, whereas STI quail exhibited reduced fear behaviour (Jones et al., 1991; Jones et al., 1994; Mills and Faure, 2000). However, in our case the difference in fear responses between the two lines was slight and was observed only in one of the two experiments, contrasting with preceding studies where the behaviour of STI and LTI quail in various fear-inducing situations differed greatly, both in chicks and in adults (Jones et al., 1991; Launay et al., 1993; Jones et al., 1994; Mills and Faure, 2000; Richard-Yris et al., 2005). This suggests that the test used in the present study reflects a dimension of fear that differs from that evoked in other fear tests previously used to compare STI and LTI quail. The latter tests differed from ours in at least one of two main aspects: placement of birds in a novel environment and obvious human intervention. STI and LTI quail have also been compared in situations where the quail were not transferred to a novel environment, but where human intervention was present. In such situations, which included capture by a human, manual presentation of a novel object or mere presence of a human in front of the cage, LTI quail exhibited stronger fear responses than STI quail (Mills and Faure, 2000; Richard-Yris et al., 2005). Thus, a new environment does not appear to be necessary to reveal the differences in fear behaviour between the STI and LTI lines, even if, in some cases, testing birds outside their home pen has been shown to exacerbate differences between experimental groups (Bilc´ık et al., 1998). By contrast, obvious human intervention is likely to have influenced the responses of the quail in the tests and may have played a major role in revealing the differences in fear behaviour between the two lines. Indeed, manipulation by an experimenter is the main component of the tonic immobility test that has been used to select the STI and LTI lines (Mills and Faure, 1991). Selection for tonic immobility duration has been accompanied by major differences in fear behaviour in a number of tests involving manipulation by humans or obvious human intervention, but the present study has revealed that the differences between the two lines appear to be weaker in a test designed to evaluate fear responses with minimum interference by humans. Similarly, Valance et al. (2007) have recently reported that STI and LTI quail did not differ in their behavioural response to an acoustic challenge in the absence of human intervention. These results are consistent with previous theoretical and experimental studies emphasizing the complex and multidimensional nature of emotions in general, and fear in particular (Thomson, 1979; Boissy, 1995; Ramos and Morm`ede, 1998; D´esir´e et al., 2002): even though fear of humans is correlated with some of the other dimensions of fear, it may be independent from others (Jones and Faure, 1981; Duncan, 1990; Lansade, 2005). The recognition of such complexity emphasizes the need for critical and detailed examination of experimental designs in
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studies aimed at understanding the determinants and the nature of emotions in animals. Acknowledgements The authors are grateful to M. Couty and S. Deniau for technical assistance and to J.M. Hervou¨et for care of the quail. References Bilc´ık, B., Keeling, L.J., Newberry, R.C., 1998. Effect of group size on tonic immobility in laying hens. Behav. Process. 43, 53–59. Boissy, A., 1995. Fear and fearfulness in animals. Q. Rev. Biol. 70, 165–191. Dantzer, R., 1986. Behavioral, physiological and functional aspects of stereotyped behavior: a review and a re-interpretation. J. Anim. Sci. 62, 1776–1786. D´esir´e, L., Boissy, A., Veissier, I., 2002. Emotions in farm animals: a new approach to animal welfare in applied ethology. Behav. Process. 60, 165–180. Duncan, J.H., 1985. How do fearful birds respond? In: 2nd European Symposium of Poultry Welfare 1985. World Poultry Science Association, pp. 96–106. Duncan, I.J.H., 1990. Reactions of poultry to human beings. In: Zayan, R., Dantzer, R. (Eds.), Social stress in domestic animals. Kluwer Academic Publishers, Dordrecht, The Netherlands, pp. 121–131. Etches, R.J., 1976. A radioimmunoassay for corticosterone and its application to the measurement of stress in poultry. Steroids 38, 763–773. Hazard, D., Faure, J.M., Gu´emen´e, D., 2004. Effects of sampling sites, photoperiod and age on adrenal responsiveness in Japanese quail. In: 8th International Symposium on Avian Endocrinology, June 6–11, 2004, Scottsdale, Arizona, p. 80. Hazard, D., Couty, M., Faure, J.M., Gu´emen´e, D., 2005. Relationship between hypothalamic-pituitary-adrenal axis responsiveness and age, sexual maturity status, and sex in Japanese quail selected for long or short duration of tonic immobility. Poult. Sci. 84, 1913–1919. Hill, J.A., 1983. Indicators of stress in poultry. World Poult. Sci. J. 39, 24–31. Jones, R.B., 1985. Fearfulness of hens caged individually or in groups in different tiers of a battery and the effects of translocation between tiers. Brit. Poult. Sci. 26, 399–408. Jones, R.B., 1987. Assessment of fear in adult laying hens: correlational analysis of methods and measures. Brit. Poult. Sci. 28, 319–326. Jones, R.B., 1996. Fear and adaptability in poultry: insights, implications and imperatives. World Poult. Sci. J. 52, 131–174. Jones, R.B., Faure, J.M., 1981. The effects of regular handling on fear responses in the domestic chick. Behav. Process. 6, 135–143. Jones, R.B., Mills, A.D., 1983. Estimation of fear in two lines of the domestic chick: correlation between various methods. Behav. Process. 8, 243–253. Jones, R.B., Harvey, S., 1987. Behavioural and adrenocortical responses of domestic chicks to systematic reductions in group size and to sequential disturbance of companions by the experimenter. Behav. Process. 14, 291–303. Jones, R.B., Mills, A.D., Faure, J.M., 1991. Genetic and experiential manipulation of fear-related behavior in Japanese quail chicks (Coturnix coturnix japonica). J. Comp. Psychol. 105, 15–24. Jones, R.B., Mills, A.D., Faure, J.M., Williams, J.B., 1994. Restraint, fear, and distress in Japanese quail genetically selected for long or short tonic immobility reactions. Physiol. Behav. 56, 529–534. Jones, R.B., Marin, R.H., Satterlee, D.G., 2005. Adrenocortical responses of Japanese quail to a routine weighing procedure and tonic immobility induction. Poult. Sci. 84, 1675–1677. Lansade, L., 2005. Le temp´erament du cheval: e´ tude th´eorique, application a` la s´election des chevaux destin´es a` l’´equitation. Universit´e Franc¸ois Rabelais, Tours, France, p. 298.
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Launay, F., Mills, A.D., Faure, J.M., 1993. Effects of test age, line and sex on tonic immobility responses and social reinstatement behaviour in Japanese quail Coturnix japonica. Behav. Process. 29, 1–16. Martin, P., Bateson, P., 1986. Measuring Behaviour: An Introductory Guide. Cambridge University Press, Cambridge, p. 200. Mason, G.J., Latham, N.R., 2004. Can’t stop, won’t stop: is stereotypy a reliable animal welfare indicator? Anim Welfare 13, S57–S69. Meddis, R., 1984. Statistics Using Ranks: A Unified Approach. Basil Blackwell, Oxford, p. 449. Miller, K.A., Garner, J.P., Mench, J.A., 2005. The test-retest reliability of four behavioural tests of fearfulness for quail: a critical evaluation. Appl. Anim. Behav. Sci. 92, 113–127. Mills, A.D., Faure, J.M., 1986. The estimation of fear in domestic quail: correlations between various methods and measures. Biol. Behav. 11, 235–243. Mills, A.D., Faure, J.M., 1991. Divergent selection for duration of tonic immobility and social reinstatement behavior in Japanese quail (Coturnix coturnix japonica) chicks. J. Comp. Psychol. 105, 25–38. Mills, A.D., Faure, J.M., 2000. Ease of capture in lines of Japanese quail (Coturnix japonica) subjected to contrasting selection for fear or sociability. Appl. Anim. Behav. Sci. 69, 125–134. Minvielle, F., Mills, A.D., Faure, J.M., Monvoisin, J.L., Gourichon, D., 2002. Fearfulness and performance related traits in selected lines of Japanese quail (Coturnix japonica). Poult. Sci. 81, 321–326. Murphy, L.B., 1977. Responses of domestic fowl to novel food and objects. Appl. Anim. Ethol. 3, 335–349. Murphy, L.B., 1978. The practical problems of recognizing and measuring fear and exploration behaviour in the domestic fowl. Anim. Behav. 26, 422–431. Papa, M., Pellicano, M.P., Welzl, H., Sadile, A.G., 1993. Distributed changes in c-Fos and c-Jun immunoreactivity in the rat brain associated with arousal and habituation to novelty. Brain Res. Bull. 32, 509–515. Ramos, A., Morm`ede, P., 1998. Stress and emotionality: a multidimensional approach. Neurosci. Biobehav. Rev. 22, 33–57. R´emignon, H., Desrosiers, V., Bruneau, A., Gu´emen´e, D., Mills, A.D., 1996. Influence of an acute stress on muscle parameters in quail divergently selected for emotivity. In: XXth World’s Poultry Science Congress, 2–5 September, 1996, New Dehli, India, pp. 716–721. Richard-Yris, M.A., Michel, N., Bertin, A., 2005. Nongenomic inheritance of emotional reactivity in Japanese quail. Dev. Psychobiol. 46, 1–12. Schulkin, J., Morgan, M.A., Rosen, J.B., 2005. A neuroendocrine mechanism for sustaining fear. Trends Neurosci. 28, 629–635. Silverin, B., 1998. Stress responses in birds. Poult. Avian Biol. Rev. 9, 153–168. Takeuchi, H., Yazaki, Y., Matsushima, T., Aoki, K., 1996. Expression of fos-like immunoreactivity in the brain of quail chick emitting the isolation-induced distress calls. Neurosci. Lett. 220, 191–194. Thomson, R., 1979. The concept of fear. In: Sluckin, W. (Ed.), Fear in Animals and Man. Van Nostrand Reinhold Company, London, pp. 1–23. Turro-Vincent, I., Launay, F., Mills, A., Picard, M., Faure, J.M., 1995. Experiential and genetic influences on learnt food aversions in Japanese quail selected for high or low levels of fearfulness. Behav. Process. 34, 23–42. Valance, D., Despr`es, G., Boissy, A., Mignon-Grasteau, S., Constantin, P., Leterrier, C., 2006. Genetic selection on a behavioural fear trait is associated with changes in heart rate variability in quail. Genes Brain Behav. 6, 339–346. Valance, D., Boissy, A., Despr´es, G., Constantin, P., Leterrier, C., 2007. Emotional reactivity modulates autonomic responses to an acoustic challenge in quail. Physiol. Behav. 90, 165–171. Verbeek, M.E.M., Drent, P.J., Wiepkema, P.R., 1994. Consistent individual differences in early exploratory behaviour of male great tits. Anim. Behav. 48, 1113–1121.
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Behavioural and endocrine fear responses in Japanese quail upon presentation of a novel object in the home cage S. Richard a,∗ , N. Wacrenier-Cer´e b , D. Hazard b , H. Saint-Dizier a , C. Arnould a , J.M. Faure b a
Physiologie de la Reproduction et des Comportements, UMR85 INRA-CNRS-Universit´e de Tours-Haras Nationaux, 37 380 Nouzilly, France b Unit´ e de Recherches Avicoles, UR83 INRA, 37 380 Nouzilly, France Received 22 March 2007; received in revised form 27 June 2007; accepted 17 July 2007
Abstract Most tests used to study fear in birds involve transferring them to a novel environment, which constitutes a bias in studies aiming at identifying the neural correlates of a specific fear-inducing situation. In order to investigate fear in birds with minimum interference by humans, behavioural and endocrine responses to the presentation of a novel object in the home cage were investigated in two lines of Japanese quail divergently selected for long or short duration of tonic immobility, a behavioural index of fear. Presentation of the novel object induced typical fear responses (avoidance of the object, increased pacing and increased plasma corticosterone levels) that were similar in the two lines of quail. Presentation of a novel object in the home cage thus appears to be a suitable stimulus to induce fear reactions in quail, with minimum interference from other motivational systems. The fact that quail of both lines reacted similarly in this test, while they are known to differ greatly in their behavioural responses to other fear-inducing tests, illustrates the multidimensional nature of fear. © 2007 Elsevier B.V. All rights reserved. Keywords: Birds; Corticosterone; Coturnix japonica; Emotions; Escape reaction; Fear; Tonic immobility response
1. Introduction Growing concern for animal welfare and efforts to understand the evolution of emotions in vertebrates have motivated research into the neural mechanisms controlling fear reactions in birds. Most tests used to study fear in birds, such as the open-field test or the tonic immobility test, usually involve transferring them to a novel environment (Jones, 1996). In addition, while most tests are carried out in isolation, experimental birds are often housed in groups until the time of the test. Transfer to a novel environment and sudden social isolation are likely to influence not only the way the birds respond to the test itself, but also the associated patterns of brain activation (Murphy, 1978; Papa et al., 1993; Takeuchi et al., 1996; Bilc´ık et al., 1998). Therefore, to avoid these complications and control better the emotional drive that the test is intended to provoke, it would be better to use a standardised test carried out in the bird’s home cage. The home cage has been used before to test fear reactions in birds, ∗ Corresponding author at: Physiologie de la Reproduction et des Comportements, INRA, 37 380 Nouzilly, France. Tel.: +33 247 42 76 57; fax: +33 247 42 77 43. E-mail address: [email protected] (S. Richard).
0376-6357/$ – see front matter © 2007 Elsevier B.V. All rights reserved. doi:10.1016/j.beproc.2007.07.005
such as in laying hens and great tits (Verbeek et al., 1994; Jones, 1996; Bilc´ık et al., 1998). Most of these experiments involve the presentation of a novel object, the objects being usually presented manually by a human, who subsequently stays in front of the cage to carry out the behavioural observations. The reactions of the bird therefore reflect a combination of responses to the object and to the human. We set out initially in this study to evaluate the specific behavioural and endocrine responses of Japanese quail to the presentation of a novel object in the home cage from a distance, with minimum human interference. Such a test might prove useful to study the specific mechanisms of fear responses in birds, in particular at the level of the brain. Fear responses to novelty usually fade away rapidly and are likely to be associated with discrete and labile changes at the level of the brain, which may be difficult to detect (Duncan, 1985). These difficulties in studying the central mechanisms of fear responses would be overcome if we could induce fear responses that are sustained over time. To do this, we repeatedly presented the same object over 30 min, the object being presented suddenly to increase the intensity of the stimulus. Two lines of Japanese quail that have been divergently selected for either long (LTI) or short (STI) duration of tonic immobility (Mills and Faure, 1991) were used in this study. The
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selection process has been continued since the foundation of the lines and at the 36th generation, the mean duration of tonic immobility (±standard deviation) was 205 (±97) s in the LTI line and 10 (±14) s in the STI line. The duration of tonic immobility is positively correlated with other measures of fear in birds (Jones and Mills, 1983; Mills and Faure, 1986) and selection on this parameter has confirmed its relation to fear: LTI quail display higher levels of fear than STI quail when measured in a number of classical tests. LTI quail freeze more, vocalise and move less in an open field, emerge later from a ‘hole-in-thewall’ box (Jones et al., 1991) and struggle less during restraint in a ‘crush cage’ (Jones et al., 1994) than STI quail. In addition to their behavioural divergence, STI and LTI quail also differ in the activity of physiological systems that are involved in fear responses. As regards the activity of the autonomic nervous system, reflected by studies of heart rate variability, parasympathetic tone has been shown to be stronger in STI than in LTI quail (Valance et al., 2006). Moreover, differences have also been reported between STI and LTI quail in adrenocortical responses to stressful events. Notably, there is a greater increase in circulating corticosterone concentrations in STI than in LTI quail in response to restraint (Jones et al., 1994; R´emignon et al., 1996; Hazard et al., 2005). Studying the responses induced by the presentation of a novel object in the home cage in STI and LTI quail should help evaluate the ability of this test to induce fear in quail with different levels of underlying fearfulness. The two experiments presented here were conducted to assess behavioural and adrenocortical responses of STI and LTI quail to the presentation of a novel object in the home cage. The aim was to evaluate the ability of the test to induce fear in birds in the absence of human interference, as a prerequisite for using such a test to study the neural mechanisms of fear in birds. LTI quail were expected to exhibit stronger behavioural fear responses than STI quail upon presentation of a novel object in their home cage, and it was also thought likely that adrenocortical responses would differ between the two lines. 2. Materials and methods 2.1. Subjects Japanese quail (Coturnix japonica) of the 36th and 38th generations of the LTI and STI lines selected and maintained at the Station de Recherches Avicoles, Nouzilly, France (Mills and Faure, 1991), were used in the present experiment. On the day of hatching, chicks of both lines were wing-banded and transferred to communal floor pens, where they were reared in single line groups under continuous illumination for three weeks. On the 21st day after hatching, the chicks were sexed on the basis of plumage colour and transferred to communal floor pens, where they were reared in single line, single sex groups under a 16:8 h light:dark schedule. Unless otherwise specified, food and water were freely available at all times. The birds were treated according to the European Union’s Council Directive of November 24, 1986 (86/609/EEC) throughout. All procedures described here fully comply with French legislation on research involving animals.
Fig. 1. Diagram of an experimental cage viewed from the top with roof removed, showing the site at which the novel object was dropped as well as the boundaries of the three zones used for behaviour analysis.
2.2. Testing apparatus and procedure 2.2.1. Experiment 1 32 LTI and 32 STI male quail were used in experiment 1. Within each line, half of the quail constituted a control group (CTRL LTI, n = 16 and CTRL STI, n = 16) and the other half were subjected to the presentation of a novel object (OBJ LTI, n = 16 and OBJ STI, n = 16). Experiment 1 was constituted by a series of six consecutive repeats comprising 10–12 quail each. Each repeat involved two or three quail from each of the four experimental groups. One week before testing, LTI and STI male quail were transferred to a testing room maintained at 20 ◦ C under a 16:8 h light:dark photoperiod. All quail were sexually mature at the time of testing (6–10 weeks). They were housed individually in PVC cages (length = 42 cm; width = 24 cm; height = 25 cm, Fig. 1) with wood-shavings on the floor and an opaque PVC roof. The front wall of each cage was made of clear perspex so that the quail could be observed with a video camera. The quail could see and hear other quail in the same room. In the corner of the cage adjacent to the food trough and to the back wall, a cylindrical PVC chimney built over the roof allowed the release of an object into the cage on the day of testing. The object used in the present experiment was a PVC cylinder (outer diameter = 4 cm; height = 21 cm) covered with 2-cm wide horizontal stripes of coloured tape (alternating blue, yellow, grey, red, black and white tape), similar to that previously used by Jones (1987). The object was suspended on a string, so that its base remained 5–6 cm above the floor once introduced into the cage. This apparatus allowed the object to be introduced into the cage and removed from a distance, with minimal disturbance to the quail, the experimenter remaining out of sight of the bird during the procedure. During the week preceding testing, the food trough of every cage was removed daily for 2 h, in order to habituate the quail to the procedure. This removal of the feeder was used to attract the quail momentarily to the part of the cage where the object would be dropped on the testing day. The quail were also habituated to
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was collected and stored at −20 ◦ C prior to the measurement of corticosterone levels following Etches’ (1976) specific radioimmunoassay. 2.3. Behaviour analysis
Fig. 2. Diagram illustrating the time-course of the test procedure for quail of the OBJ group, starting when a quail resumed pecking at the food after 40 min of feeder withdrawal. If a quail failed to peck at the food, the test procedure started 60 s after the return of the feeder.
the placement of a video camera and a cardboard screen in front of their cage, at a distance of approximately 1 m. On the day of testing, the cardboard screen prevented other quail in the room from seeing the novel object in the cage of the quail to be tested. On the day of testing, the food trough of each cage was removed for 40 min. When the food trough was replaced in the cage, as soon as a quail of the OBJ group pecked at the food, the multicoloured cylinder was dropped into the cage, as described above. If a quail failed to peck at the food, the multicoloured cylinder was dropped into the cage 60 s after the return of the food trough. The object was withdrawn 5 min after being dropped and the quail was left undisturbed for 1 min. This object presentation procedure, followed by the 1-min interval, was then repeated four times without taking into account the quail’s feeding behaviour (Fig. 2). A video recording was made of the behaviour of each OBJ quail throughout the object presentation procedure (total duration = 30 min). The behaviour of CTRL quail was recorded for 30 min, starting as soon as a quail pecked at the food after the return of the food trough. If a CTRL quail failed to peck at the food, the video recording started 60 s after the return of the food trough, without further disturbance. 2.2.2. Experiment 2 24 LTI and 24 STI male quail, other than those used in experiment 1, were used in experiment 2. Within each line, half of the quail constituted a control group (CTRL LTI, n = 12 and CTRL STI, n = 12) and the other half were subjected to the presentation of a novel object (OBJ LTI, n = 12 and OBJ STI, n = 12). Experiment 2 was constituted by a series of four consecutive repeats comprising 12 quail each. Each repeat involved three quail from each of the four experimental groups. The procedures were identical to those of experiment 1, except that all quail were decapitated either immediately after the first 5-min object presentation (OBJ group) or 5 min after the return of the feeder (CTRL group). Blood was collected from each quail directly after decapitation in a tube containing EDTA (2 mg/ml blood). Decapitation was used in this study because this sampling procedure has previously been shown to affect the least basal corticosterone levels, compared to venipuncture, cardiac or jugular puncture (Hazard et al., 2004). This procedure has proven particularly useful to reveal responses of very small amplitude, such as those described in the present study. The blood samples were temporarily stored on ice and subsequently centrifuged at 2000 × g for 15 min at 4 ◦ C. The plasma
The quail were coded so that all behaviour observations were performed ‘blind’ with respect to the line. The behaviour of OBJ quail was analysed during the first and fifth presentations of the object, i.e. during 5-min time windows starting when the object was dropped in the cage (P1 and P5, Fig. 2). The behaviour of CTRL quail was analysed at corresponding time points, i.e. 0–5 min and 24–29 min after the first peck at the food after the return of the food trough. If a CTRL or OBJ quail failed to peck at the food after the return of the food trough, the first time window for observations started 60 s after the return of the food trough. The cage was subdivided into three zones, zone 1 including the site of object presentation and zone 3 being furthest away from it (Fig. 1). The amounts of time spent in zone 1 and zone 3 and the amount of time spent eating were recorded. The occurrence of freezing behaviour and pecking at the object were also recorded. Freezing behaviour was defined as complete immobility, occurring suddenly with an abrupt interruption of ongoing activities and lasting for more than 3 s, sometimes including very small head movements such as those associated with breathing. Finally, the presence of pacing behaviour was recorded when a quail exhibited repetitive movements that were fixed in form and orientation: pacing only or pacing in conjunction with head movements or jumps in some cases. The occurrence of pacing was recorded for each 10-s interval of the 5-min period, using the one-zero sampling regime (Martin and Bateson, 1986). These records were then pooled into a pacing score (between 0 and 30) over 5 min. All observations were made by a single experimenter, using focal sampling. The Observer 3.0 software (Noldus Information Technology, The Netherlands, 1993) was used to record observations, except for pacing behaviour. 2.4. Statistical analysis As the data were not normally distributed, nonparametric statistical analyses were performed, using Statview 5.0 (SAS Institute Inc., USA, 1992–1998) and Excel 2000 (Microsoft Corporation). For both experiments, the effects of treatment (OBJ versus CTRL), line and interaction between line and treatment were assessed using 2 × 2 factorial analysis for unrelated datasets, with nonspecific hypotheses (Meddis, 1984). Whenever there was a significant interaction between line and treatment, the effects of treatment were evaluated independently within each line using a Mann–Whitney U-test. Comparison between the first and fifth object presentations (experiment 1) was carried out within each experimental group using a Wilcoxon test. The time spent in zone 1, time spent in zone 3 and time spent eating were necessarily related to some extent. However, considering that these three variables reflected separate aspects of the behaviour of the quail and were not highly
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correlated, we analysed them as independent variables. Freezing behaviour and pecking at the object occurred so rarely that these variables were not included in the statistical analysis. Box-plot diagrams were used to illustrate the results. Values illustrated by a box plot are the median, upper and lower quartiles, 10th and 90th percentiles and extreme observations. 3. Results 3.1. Experiment 1 (Fig. 3) 3.1.1. Behaviour during the first object presentation There was an effect of treatment on the time spent in zone 3 (Fig. 3B, H = 11.27, p < 0.001) and on the pacing score (Fig. 3C, H = 12.47, p < 0.001). OBJ quail spent more time in zone 3 and exhibited more pacing than CTRL quail. There was no significant effect of treatment on the time spent eating (Fig. 3D, H = 2.95, p > 0.05). There was no significant effect of line on the time spent in zone 3, on the pacing score, nor on the time spent eating (H < 1.7, p > 0.1 for each variable). However, there was an effect of interaction between line and treatment on the time spent in zone 1 (H = 4.22, p < 0.05). Indeed, in the STI line the time spent in zone 1 did not differ significantly between OBJ and CTRL quail (z = 0.98, p > 0.1) whereas in the LTI line OBJ
quail spent less time in zone 1 than did CTRL quail (z = 3.54, p < 0.001). There was no significant effect of interaction between line and treatment on any other variable (time spent in zone 3, pacing score, time spent eating: H < 2.2, p > 0.1 for each variable). 3.1.2. Behaviour during the fifth object presentation There was an effect of treatment on the time spent in zone 1 (Fig. 3A, H = 4.28, p < 0.05) and on the pacing score (Fig. 3D, H = 12.42, p < 0.001). OBJ quail spent less time in zone 1 and exhibited more pacing than CTRL quail. Neither the time spent in zone 3, nor the time spent eating differed significantly between CTRL and OBJ quail (Fig. 3B and C, H = 3.64, p > 0.05 and H = 0.43, p > 0.1, respectively). There was no significant effect of line on any of the variables of interest (H < 2.8, p > 0.05 for each variable). There was no significant effect of interaction between line and treatment on any of the variables studied (H < 1.1, p > 0.1for each variable). 3.1.3. Behaviour changes over time There was a decrease in the time spent in zone 1 between the first and fifth experimental periods in CTRL STI and LTI quail and OBJ STI quail, but not in OBJ LTI quail (Fig. 3A, CTRL STI, z = 2.79, p < 0.01; CTRL LTI, z = 2.28, p < 0.05; OBJ STI, z = 2.12, p < 0.05; OBJ LTI, z = 0.84, p > 0.1). The time
Fig. 3. Box-plot diagrams illustrating the behaviour of Japanese quail selected for long or short duration of tonic immobility (LTI and STI, respectively) in experiment 1. Zone 1 = area of the cage where the object was presented to quail of the OBJ group; zone 3 = area of the cage furthest away from the site of object presentation; P1 = first object presentation; P5 = fifth object presentation.
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Table 1 Behaviour of Japanese quail selected for long or short duration of tonic immobility (LTI and STI, respectively) in experiment 2a Time spent in zone 1 (s)b
Time spent in zone 3 (s)c
Pacing score
Time spent eating (s)
LTI CTRL OBJ
185 (94) 106 (139)
49 (96) 98 (156)
0 (11) 11 (18)
30 (66) 12 (37)
STI CTRL OBJ OBJ vs. CTRLd
86 (118) 55 (92) p < 0.01
69 (82) 116 (148) p < 0.05
0 (5) 24 (11) p < 0.001
14 (21) 1 (19) p > 0.1
a
Values are medians with interquartiles in parentheses. Zone 1 = area of the cage where the object was presented to OBJ quail. c Zone 3 = area of the cage furthest away from the site of object presentation. d Results of factorial analysis for unrelated datasets as in Meddis (1984). There was no significant line effect and no significant effect of interaction between line and treatment. b
spent in zone 3 and the pacing score did not change significantly between the first and fifth experimental periods in any of the groups (Fig. 3B and D, z < 1.9, p > 0.05 in each group for each of these two variables). The most striking behaviour change between the first and fifth experimental periods was a decrease in the time spent eating in all groups (Fig. 3C, CTRL LTI, z = 3.30, p = 0.001; CTRL STI, z = 3.52, p < 0.001; OBJ LTI, z = 2.17, p < 0.05; OBJ STI, z = 2.73, p < 0.01).
3.2.2. Corticosterone (Fig. 4) Plasma corticosterone concentrations were higher in OBJ than in CTRL quail (H = 6.33, p < 0.05). There was no significant effect of line and no significant effect of interaction between line and treatment on plasma corticosterone concentrations (H < 0.01, p > 0.1 for each of these two effects).
3.2. Experiment 2
Presentation of a novel object in the home cage induced typical behavioural and endocrine fear responses in Japanese quail. Behaviourally, the first presentation of the object induced escape and avoidance reactions by the quail in both experiments. The quail in the OBJ group spent less time in the zone where the object was presented, than quail in the control group. They also spent more time than the control quail in the part of the cage furthest away from the object. This indicates avoidance of the object by the quail. Moreover, OBJ quail exhibited more pacing behaviour than control quail. Pacing behaviour has previously been observed in response to the presentation of fear stimuli in a restricted space, and in such a context it is usually thought to result from escape attempts (Murphy, 1977). Thus, the heightened pacing exhibited by OBJ quail in response to the presentation of the novel object may be interpreted in terms of escape from the object, whereas the low level of pacing observed in control quail probably reflected a more chronic response of the quail to their housing conditions, social isolation being a potential source of stress for some individuals (Dantzer, 1986; Mason and Latham, 2004). More generally, escape and avoidance are typically induced by presentation of a novel and intense stimulus and are considered as fear responses (Duncan, 1985). At the endocrine level, the novel object induced a slight, but significant, rise in plasma corticosterone levels, indicating that the hypothalamo–pituitary–adrenal axis was activated. As in mammals, the hypothalamo–pituitary–adrenal axis is a major route for the endocrine stress response in birds (Hill, 1983; Silverin, 1998). In particular, activation of this axis in response to fearful stimuli has previously been described in birds, including quail (Jones and Harvey, 1987; Hazard et al., 2005; Jones et al., 2005). Thus, the rise in plasma corticosterone levels induced by presentation of the novel object suggests that the test situation was
3.2.1. Behaviour (Table 1) As in experiment 1, there was an effect of treatment on the time spent in zone 1 (H = 4.96, p < 0.01), the time spent in zone 3 (H = 4.55, p < 0.05) and the pacing score (H = 17.18, p < 0.001). OBJ quail spent less time in zone 1, more time in zone 3 and exhibited more pacing than CTRL quail. The time spent eating did not differ significantly between OBJ and CTRL quail (H = 2.49, p > 0.1). There was no significant effect of line on any of the behaviour measurements made in experiment 2 (H < 3.6, p > 0.05 for each variable). Neither was there any significant effect of interaction between line and treatment (H < 3.5, p > 0.05 for each variable).
Fig. 4. Box-plot diagrams illustrating the plasma corticosterone concentrations of Japanese quail selected for long or short duration of tonic immobility (LTI and STI, respectively) in experiment 2.
4. Discussion
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perceived by the quail as stressful. Overall, these behavioural and endocrine responses are characteristic of fear and similar to those described by Jones (1985, 1987) in laying hens upon presentation by a human of a novel object in the food trough. Thus, presentation of a novel object in the home cage with minimum human interference appears to be an appropriate design for studying the neural mechanisms of fear reactions in birds. Such a design should prove particularly useful for studying more specifically the relationships between the characteristics of a given stimulus and the reactions it induces, to understand better the way birds perceive their environment. Unlike most novel object tests, that involve a single presentation of a novel object (e.g. Miller et al., 2005), the present test consisted in repeated, sudden presentations of a novel object in order to induce sustained fear reactions over 30 min. In such a design, the quail exhibited very few signs of approach towards the object, but showed sustained fear responses over the successive presentations. Indeed, the fifth presentation of the object still induced significant escape and avoidance. The major behavioural change that was recorded between the first and the fifth experimental periods was a reduction in the time spent eating in all experimental groups, including controls. This was associated with a reduction in the time spent in zone 1, where the food trough was located. The reduction in the time spent eating over the experimental periods was linked to the test procedure: the 40-min feeder withdrawal preceding the first experimental period in all groups temporarily increased the quails’ motivation to eat. This motivation appeared to be similar in STI and LTI quail (no significant effect of line and no significant effect of interaction between line and treatment on the time spent eating), which is in agreement with previous work showing similar motivation to feed in the two lines (Turro-Vincent et al., 1995; Minvielle et al., 2002). At the beginning of the test, the motivation to eat attracted quail to zone 1, where the object was to be presented, but the attraction remained slight enough to allow the expression of fear behaviour in OBJ quail in both experiments. The only statistically significant variations observed between the first and fifth experimental periods may thus be attributed to the reduction in the motivation to eat over time and appear to be unrelated to the presentation of the object. However, there was an indication that avoidance of the object was weaker during the fifth than during the first presentation. Indeed, the time spent in the part of the cage furthest away from the object did not differ significantly between experimental groups during the fifth presentation, whereas it was higher in OBJ than in control quail during the first presentation. This slight reduction in avoidance behaviour may reflect the beginning of a habituation process. It is noteworthy that, in spite of this sign of habituation, typical fear responses were still evoked by the object during the fifth presentation since the quail continued to exhibit significant escape and avoidance behaviours. Such persistence of fear reactions over time is likely to be associated with sustained activation of specific brain regions (Schulkin et al., 2005). Thus, the design used in the present study may facilitate identification of the neural mechanisms underlying fear reactions in birds. Quail from the LTI and STI lines, which have been divergently selected on the duration of tonic immobility, exhibited
similar behavioural and endocrine fear responses to the novel object. The only difference between the two lines appeared in the first experiment to be an effect of interaction between line and treatment on the time spent in zone 1, where the object was presented: Avoidance of the object during the first experimental period was more pronounced in LTI than in STI quail. This result, indicative of stronger fear reactions in LTI than in STI quail, is consistent with several other studies demonstrating that LTI quail exhibited exaggerated fear behaviour, whereas STI quail exhibited reduced fear behaviour (Jones et al., 1991; Jones et al., 1994; Mills and Faure, 2000). However, in our case the difference in fear responses between the two lines was slight and was observed only in one of the two experiments, contrasting with preceding studies where the behaviour of STI and LTI quail in various fear-inducing situations differed greatly, both in chicks and in adults (Jones et al., 1991; Launay et al., 1993; Jones et al., 1994; Mills and Faure, 2000; Richard-Yris et al., 2005). This suggests that the test used in the present study reflects a dimension of fear that differs from that evoked in other fear tests previously used to compare STI and LTI quail. The latter tests differed from ours in at least one of two main aspects: placement of birds in a novel environment and obvious human intervention. STI and LTI quail have also been compared in situations where the quail were not transferred to a novel environment, but where human intervention was present. In such situations, which included capture by a human, manual presentation of a novel object or mere presence of a human in front of the cage, LTI quail exhibited stronger fear responses than STI quail (Mills and Faure, 2000; Richard-Yris et al., 2005). Thus, a new environment does not appear to be necessary to reveal the differences in fear behaviour between the STI and LTI lines, even if, in some cases, testing birds outside their home pen has been shown to exacerbate differences between experimental groups (Bilc´ık et al., 1998). By contrast, obvious human intervention is likely to have influenced the responses of the quail in the tests and may have played a major role in revealing the differences in fear behaviour between the two lines. Indeed, manipulation by an experimenter is the main component of the tonic immobility test that has been used to select the STI and LTI lines (Mills and Faure, 1991). Selection for tonic immobility duration has been accompanied by major differences in fear behaviour in a number of tests involving manipulation by humans or obvious human intervention, but the present study has revealed that the differences between the two lines appear to be weaker in a test designed to evaluate fear responses with minimum interference by humans. Similarly, Valance et al. (2007) have recently reported that STI and LTI quail did not differ in their behavioural response to an acoustic challenge in the absence of human intervention. These results are consistent with previous theoretical and experimental studies emphasizing the complex and multidimensional nature of emotions in general, and fear in particular (Thomson, 1979; Boissy, 1995; Ramos and Morm`ede, 1998; D´esir´e et al., 2002): even though fear of humans is correlated with some of the other dimensions of fear, it may be independent from others (Jones and Faure, 1981; Duncan, 1990; Lansade, 2005). The recognition of such complexity emphasizes the need for critical and detailed examination of experimental designs in
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