www.elsevier.comrlocaterapplanim
Behavioural evaluation of methods for assessing
fear responses in weaned pigs
Inger Lise Andersen
a,), Knut Egil Bøe
a, Gry Fœrevik
a,
Andrew M. Janczak
b, Morten Bakken
ba
Department of Agricultural Engineering, Agricultural UniÕersity of Norway, P.O. Box 5065,
˚
1432 As, Norway
b ˚
Department of Animal Science, Agricultural UniÕersity of Norway, P.O. Box 5025, 1432 As, Norway
Accepted 5 April 2000
Abstract
Models of anxiety and fear of novelty were evaluated using correlations and principal
Ž .
component analysis. A total of 84 pigs Landrace=Yorkshire from nine different litters were
Ž . Ž .
subjected to a tonic immobility TI test at the age of 2.5 weeks, an elevated plus-maze EPM at
Ž .
the age of 6 weeks, a lightrdark LrD exploration test at the age of 7 weeks and an open-field ŽOF test at the age of 8 weeks..
The first component from the principal component analysis had the highest correlation with number of entries into open arms in the EPM but was also highly correlated to variables from the other three tests confirming a common aversion-related element in the four experimental tests. The second component was negatively correlated with percent entries into and time spent on open arms in the EPM, but positively correlated with the number of entries into closed arms in the same test, number of lines crossed in the OF and time spent in the lit compartment of the LrD test. The last point illustrates a negative relationship between ‘‘anxiety’’ and ‘‘activity’’ in the EPM and OF. To achieve purer measures of fear of novelty and activity in the tests, the components were rotated using the Varimax criterion. The rotated factor pattern demonstrated a simple structure
Ž
where variables related to ‘‘anxiety’’ or ‘‘fear of novelty’’ i.e., percent entries into open arms and .
time spent on open arms of the EPM had the highest loading on factor 1, whereas variables Ž
related to activity i.e., number of entries into the closed arms in the EPM, number of lines crossed .
in the OF and time spent in the lit compartment of the LrD test had the highest loading on factor
Ž . Ž .
2. TI duration loaded more strongly on factor 1 ‘‘fear of novelty’’ than on factor 2 ‘‘activity’’ , but did not represent any pure measure of either fear of novelty or activity.
)Corresponding author. Tel.:q47-6494-8700; fax:q47-6494-8810.
Ž .
E-mail address: inger-lise.andersen@itf.nlh.no I.L. Andersen .
0168-1591r00r$ - see front matterq2000 Elsevier Science B.V. All rights reserved. Ž .
In conclusion, all of the test variables were related to one another. Open-arm avoidance represented the purest measure of fear of novelty, whereas entries into closed arms and number of lines crossed in the OF were the purest measures of activity. The EPM appeared to provide the best way to separate the fear of novelty and activity-related elements, indicating that the EPM may be a useful behavioural model of fear of novelty or avoidance in pigs.q2000 Elsevier Science B.V. All rights reserved.
Keywords: Fear of novelty; Pig, Fearful behaviour; Tonic immobility; Elevated plus-maze; Lightrdark test; Open-field
1. Introduction
Fear is an undesirable emotional state that reduces welfare, growth and reproductive Ž
performance in farm animals Hemsworth and Barnett, 1991; Hemsworth et al., 1989;
. Ž .
Jones, 1989; Mills and Faure, 1990; Seabrook, 1990; . According to Boissy 1995 , fear and anxiety can be defined as ‘‘emotional states that are induced by the perception of
Ž . Ž .
any actual danger fear state or potential danger anxiety that threatens the well-being of the individual’’. In commercial farms, pigs are frequently exposed to aversive conditions including different stock, people and changes in their physical and social
Ž
environments. Specific examples are mixing with unfamiliar conspecifics e.g., Jensen,
. Ž
1994; Jensen and Yngvesson, 1998 , inconsistent handling procedures Hemsworth
. Ž
et al., 1987 , and exposure to a new environment Mormede et al., 1984; Beattie et al.,
`
.1995; Erhard and Mendl, 1999 . In most situations, animals have conflicting motivations to avoid and investigate novel stimuli, and the balance is strongly dependent on the
Ž .
magnitude of novelty Gray, 1987 .
Models of the approach–avoidance conflict have been useful for developing
be-Ž .
havioural tests of fear in rodents see Gray, 1987 , but there are few standard and Ž well-validated experimental methods for studying aversion to novelty in pigs Erhard
. Ž . Ž .
and Mendl, 1999 . The elevated plus-maze EPM and the lightrdark LrD test are the Ž
most commonly used behavioural models of fear in rodents Cole and Rodgers, 1995; . Hendrie et al., 1997; Janczak et al., 1999; Lister, 1987; Pellow et al., 1985 . In the
Ž .
former, an anxiolytic anxiety reducing effect is indicated by an increase in the percent Ž
of entries into open arms and the percent of time spent on open arms Lister, 1987; .
Pellow et al., 1985 , whereas in the latter, it is the increase in the percent of activity and Ž
time spent in the aversive, brightly lit compartment Costall et al., 1987, 1989; Onaivi .
and Martin, 1989 . Performance in EPM is found to be predictive of behaviour in other
Ž .
models of anxiety for rodents Trullas and Skolnick, 1993 , and new experimental work
Ž .
suggests that this model may be used to measure anxiety in pigs Andersen et al., 1999 . Anxiolytic agents, such as diazepam, are found to reduce the avoidance of open arms in
Ž .
the EPM both in rodents and pigs Andersen et al., 1999; Cole and Rodgers, 1995 , but an anxiolytic effect of diazepam in the LrD test has only been reported in rodents ŽCostall et al., 1989 . The open-field OF test was originally developed for laboratory. Ž .
Ž .
animals Gray, 1987; Hall, 1936; Whimbey and Denenberg, 1967 , but has later been Ž
used to assess fear responses in domestic species pigs: Beattie et al., 1995; von Borell .
test, it is the conflict between exploration of and aversion to an open area, which
Ž .
determines the behaviour Asano, 1986; Crusio et al., 1989 . The usual interpretation of the OF data in rodents has been that a high defecation rate and low activity indicate a
Ž .
high level of fear or emotionality Broadhurst, 1957; Denenberg, 1964 . Other results on rodents, however, have shown that measures of defecation and locomotion in the OF loads on two different and independent components, which represent emotionality and
Ž .
activity, respectively Ramos et al., 1997; Whimbey and Denenberg, 1967 .
Ž . Ž .
The tonic immobility TI , motor inhibition response has been used to a great extent Ž
as an index of fearfulness in poultry Heiblum et al., 1998; Jones et al., 1997; Scott and .
Moran, 1993 . This is an involuntary, reflexive response to fear-inducing stimuli present
Ž . Ž . Ž .
in birds Heiblum et al., 1998 , toads Lupo et al., 1991 , rabbits Aloisi et al., 1995 ,
Ž . Ž .
rodents Bigal et al., 1994; Tikal, 1991a,b and humans Galliano et al., 1993 , and is
Ž .
sometimes referred to as a state of ‘‘pretended death’’ Tikal, 1991b . A longer duration of TI in rats appears to be typical for individuals tending to react with anxiety and
Ž .
submission in other situations Tikal, 1991b , whereas in pigs, the duration of this Ž
response is thought to reflect an active or passive coping strategy Erhard and Mendl, .
1999 .
Before applying these different experimental models to pigs, there is a need to evaluate the methods. This can be provided by using recognised anxiolytic drugs to
Ž .
detect fear-related behavioural elements Andersen et al., 1999 , and by examining the relationship between behaviours in different tests. The aim of the present study was to investigate the relationship between fear responses in different experimental tests of novelty in weaned pigs.
2. Materials and methods
Ž .
A total of 84 pigs Landrace=Yorkshire from nine different litters were subjected to four experimental tests, a TI test at the age of 2.5 weeks, an EPM at the age of 6 weeks, an LrD test at the age of 7 weeks and an OF test at the age of 8 weeks.
2.1. Animals
The number of pigs per litter was 9.3"0.6 with a range of 7–12. After weaning at the age of 5 weeks, the pigs were moved from their home pens in the lactation unit to
Ž .
2.2. Tonic immobility
This test was conducted when the pigs were 2.5 weeks old in a similar manner as that Ž .
described by Erhard and Mendl 1999 . One pig at a time was carefully taken out of the lactation pen, put into a transport box, and moved to a separate test room. The pig was then lifted out of the box by its hind legs and placed on its back onto a 50-cm long, V-shaped, wooden cradle with an 808angle. The handler then put a sand-filled cloth bag Ž15=20 cm, 500 g on the pigs chest and gently stretched out its hind legs. The pigs. were restrained for 5–10 s prior to release. The time from when the experimenter
Ž .
released the pig to the first escape attempt when it struggled was recorded as duration
Ž . Ž .
of TI motor inhibition . Pigs showing active escape attempts struggling immediately after being put onto the cradle were given a TI duration of 0 s. If the pig did not respond within 5 min, the test was terminated, and a latency of 300 s was allocated. The procedure was repeated for all pigs in each litter. The observer was located in the same room, but out of sight of the pig during the test. After the test procedure, the pigs were returned to their lactation pen in the same manner as they were removed.
2.3. EleÕated plus-maze
Ž .
The EPM was a modification of the apparatus described by Lister 1987 for mice, and adjusted to the size of pigs at the age of 6 weeks. The apparatus consisted of two open arms, 0.60=3.0 m, and two enclosed arms 0.60=0.30=0.60 m, with an open roof, arranged in such a way that the two open arms were opposite each other. The open arms were equipped with 2-cm high wooden barriers around the edges to prevent the pigs from falling down to the floor. The walls of the enclosed arms were made of transparent Plexiglass. The maze was elevated 1.0 m above the floor, and it was placed in a separate testing room without windows.
The pigs behaviour was recorded with a video camera mounted vertically above the centre of the maze. The pigs were placed individually into the centre of the maze facing one of the closed arms, and the following variables were observed during the 5-min
Ž
period: the number of entries into open arms with both front legs placed onto one of the .
arms , time spent on open arms and the number of entries into closed arms. Derived measures were the percent of entries into open arms in respect to total arm entries and the percent of time on open arms. The observer was in a neighboring room during the test. The maze was cleaned between each test occasion.
2.4. LrD test
The LrD box used in this experiment was a modification of the apparatus used for Ž .
mice by Cheng et al. 1994 , adjusted to the size of pigs at the age of 7 weeks. The box
Ž .
infrared light was positioned near the camera. The light intensity in the lit and dark area was 90 and 2.5 lx, respectively.
The subjects were individually tested for 5 min at the age of 7 weeks. The pigs were
Ž .
placed into the dark compartment of the box, and the latency in seconds to enter the lit
Ž .
compartment both front legs in the lit compartment , number of entries into the lit
Ž .
compartment and the time spent in the lit compartment in seconds were recorded. The observer was in a neighboring room during the test. The box was cleaned between each test occasion.
3. OF test
Ž .
The OF test was a modification of the test developed for rats by Hall 1936 , adjusted to the size of pigs at the age of 8 weeks. A circular open field arena with a diameter of 3.0 and 0.6-m high wooden, solid, walls was used. A video camera was suspended above the centre of the arena. The pigs were individually placed in spot number 1 and
Ž .
during a 5-min test period, the number of lines crossed both front legs crossing a line
Ž .
and the number of entries into the centre both front legs within the centre of the arena were recorded. The observer was in a neighboring room during the test. After the pig
Ž
was removed from the apparatus, the number of fecal boli was counted defecation .
frequency . The arena was cleaned between each test.
3.1. Statistics
To determine the relationship between different behavioural elements, a Pearson Ž .
correlation analysis PCA including all test variables was conducted. The pattern of correlation between test variables was studied by performing a principal component analysis resulting in an orthogonal solution, and the extracted components were rotated using the Varimax criterion. A detailed description of the PCA is provided in Goddard
Ž . Ž .
and Beilharz 1984 and Hatcher and Stepanski 1994 . Loadings greater than 0.40 were considered for interpretation.
4. Results
4.1. Tonic immobility
Ž . Ž
Twenty pigs 23.8% did not show the TI response struggled immediately when
. Ž .
released from restrainment . The frequency distribution of TI was unimodal Fig. 1 , with a mean duration of 28.0"4.4 s.
4.2. EleÕated plus-maze
Mean number of entries into open arms was 5.3"0.3, which amounts to 45.7"1.8%
Ž .
Ž .
Fig. 1. Frequency distribution of TI duration s .
entries into open arms, closed arms and the time spent on open arms were all normally
Ž .
distributed Fig. 2a,b,c . The number of entries into open arms was strongly correlated
Ž .
with the total number of arm entries rs0.89, P-0.001 , but only moderately
Ž .
correlated with the number of entries into closed arms rs0.61, P-0.001 and time
Ž .
spent on open arms rs0.48, P-0.001 . There was a moderate, positive relationship
Ž .
between the percent and the number of entries into open arms rs0.48, P-0.001 .
4.3. LrD test
Ž .
The pigs spent on average 29.5"2.8% 88.4"8.4 s of their time in the lit
Ž .
compartment, and 23 pigs 27.4% did not enter this compartment at all. The frequency distribution of this variable was difficult to interpret, with 32% of the pigs spending less than 20 s in the lit compartment, whereas for the rest, it was approximately normally
Ž .
distributed Fig. 3 . Mean number of entries into the lit compartment was 1.8"0.2. The correlation between the number of entries into the lit compartment and the time spent
Ž .
there was 0.51 P-0.001 .
4.4. OF test
Ž . Ž .
The number of lines crossed 71.3"2.3 was normally distributed Fig. 4 . The mean number of entries into the centre of the OF and the defecation frequency were 2.6"0.2 and 1.5"0.2, respectively. There was a moderate, positive correlation between the
Ž .
number of lines crossed and the number of entries into the centre rs0.45, P-0.001 . There was no relationship between the number of lines crossed and the frequency of
Ž .
defecation rs0.13 , nor was there any relationship between the number of entries into
Ž .
the centre and the defecation frequency rs0.09 .
4.5. Interrelationship of the four experimental tests
4.5.1. Correlation between testÕariables
Ž . Ž .
Fig. 2. a Frequency distribution of the number of entries into open arms. b Frequency distribution of the
Ž . Ž .
Ž .
Fig. 3. Frequency distribution of the time spent in the illuminated compartment s .
Ži.e., number of entries into open and closed arms , entered the lit compartment of the. Ž
LrD box more frequently, spent more time there, and were more active i.e., number of
. Ž .
lines crossed in the OF Table 1 . The number of lines crossed in the OF was positively correlated with time spent in the lit compartment of the LrD test. A long duration of TI was associated with a lower number of entries into open and closed arms and less time on open arms, but there was no significant relationship between TI duration and
Ž .
variables from the OF and the LrD tests Table 1 . These correlations indicate several common fear-related elements in the different novelty tests and a general negative
Ž .
relationship between activity and fear of novelty avoidance between tests.
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Table 1
Pearson correlation coefficients between variables from the four experimental tests
EPM OF LrD
Entries into No. of entries Time on open No. of entries No. of lines No. of entries Time in No. of entries
Ž . Ž . Ž .
open arms % into open arms arms % into closed arms crossed into centre light % into light
) ) )
Ž .
Tonic immobility s y0.16 y0.27 y0.34 y0.29 y0.10 0.07 y0.07 y0.11
) ) )
No. of entries into light 0.05 0.23 0.25 0.15 0.35 0.19
) ) )
Ž .
Time in light % 0.01 0.29 0.20 0.24 0.34 0.13
) )
No. of lines crossed 0.01 0.33 0.18 0.31
No. of entries into centre y0.07 0.02 0.01 0.13
)
4.5.2. Principal component analysis
A principal component analysis was conducted to summarise the correlation matrix ŽTable 1 . From the initial analysis which resulted in seven independent components, the. two components with an eigenvalue of more than one were retained for interpretation ŽTable 2 . The first component PC 1 accounted for 37% of the total variation, and was. Ž . highly correlated to all test variables thus supporting the correlations in showing a common motivational element in the four aversive tests. Except for the TI response, the
Ž .
loadings on PC 1 were all positive Table 2 , indicating that pigs with a short TI Ž
duration are more active i.e., higher number of entries into closed arms and number of .
lines crossed and show less avoidance of aversive elements in the EPM and the LrD
Ž .
test i.e., open arms and lit compartment, respectively . PC 2 explained 21.8% of the total variation, and was positively correlated with closed entries in the EPM and lines
Ž .
crossed in the OF Table 2 . On the other hand, this component was negatively correlated with percent entries into open arms and the time spent on open arms. In other
Ž words, pigs showing less aversion to open arms had higher locomotor activity i.e.,
. number of lines crossed and number of entries into closed arms .
In order to achieve purer measures of aversion and activity in these tests, the components were rotated using the Varimax criterion. This rotation does not alter the total variance explained by the two components, but makes the dimensions of the factor
Ž .
solution easier to interpret Hatcher and Stepanski, 1994 . The rotated factor pattern demonstrated a simple structure, where most of the variables had a relatively high
Ž .
loading on only one factor Table 2 . Both percent open entries and time spent on open arms in the plus-maze loaded strongly on factor 1, but weakly on factor 2. When
Ž
discounting the two variables which loaded on both factors i.e., number of entries into .
open arms and TI duration , the first factor now consisted of percent entries into and
Ž .
time spent on open arms Table 2 . These variables may thus be considered as the purest
Table 2
Factors retained from the initial PCA before and after a Varimax rotation
Initial Rotated
PC 1 PC 2 Factor 1 Factor 2
EPM
) ) )
No. of entries into open arms 0.87 y0.01 0.63 0.59
) ) )
Ž .
Entries into open arms % 0.48 y0.80 0.90 y0.25
) ) )
Time spent on open arms 0.71 y0.45 0.83 0.17
) ) )
No. of entries into closed arms 0.59 0.62 0.00 0.86
OF
) ) )
No. of lines crossed 0.52 0.41 0.09 0.65
LrD
) )
Time spent in lit compartment 0.47 0.36 0.10 0.58
TI
) )
Tonic immobility duration y0.51 0.07 y0.42 y0.30
Eigenvalues 2.6 1.5 2.08 2.04
( )
Variance explained by each factor % 37.0 21.8 29.7 29.1
Ž . )
measures of aversion to novelty and elevation in pigs. Number of entries into closed arms, number of lines crossed, and time spent in the lit compartment had high loadings
Ž .
on factor 2, but very low loadings on factor 1 Table 2 . These variables may thus represent the purest measures of activity. Number of entries into open arms loaded almost equally on both factors, and does not provide any pure measure of either fear of novelty or activity. TI duration loaded more strongly on factor 1 than factor 2, but the strength of the loadings were rather low.
The number of entries into the centre of the open field was not correlated with any of
Ž .
the factors from the rotated PCA factor 1: rs0.14, factor 2: rs y0.09 , and there
Ž .
was no correlation between defecation score and factor 1 rs0.08 or factor 2 Žrs0.03 ..
5. Discussion
The results showed that there was a relationship between the variables from the different experimental tests, and that most behavioural responses contained both a fear and an activity element. There was a large variation in maze-activity among the pigs. According to the rotated factor pattern, time spent on open arms and percent entries into
Ž .
open arms were the purest measures of fear of novelty or avoidance factor 1 in pigs, whereas number of entries into closed arms, number of lines crossed and time spent in
Ž .
the lit compartment were related to activity factor 2 . From this, it can be suggested that factor 2 reflects locomotion in novel environments. These results correspond to a large extent with the findings of work in rodents. The EPM provides a stimulus gradient represented by aversive, open, elevated arms and more secure closed arms. Because of this gradient, the test provides a better way to separate fear and activity-related elements than the other three behavioural models of fear. Although total arm entries is sometimes
Ž
used as the activity variable in the EPM Pellow et al., 1985; Rex et al., 1993; Trullas .
and Skolnick, 1993 , the number of entries into closed arms is considered the purest Ž
measure of locomotive activity in rodents File, 1992; Ramos et al., 1997; Rodgers and .
Johnson, 1995 . The results of the present experiment are strongly supported by Ž .
Andersen et al.’s 1999 finding that moderate doses of diazepam increased the time spent on open arms in weaned pigs without significantly affecting OF or LrD behaviour.
The present finding that OF locomotion is more related to activity than fear of Ž
novelty is also supported by findings in rodents Ramos et al., 1997; Whimbey and
. Ž
Denenberg, 1967 . In contrast to rodent experiments, Ramos et al., 1997; Weijers and .
Weyers, 1998; Whimbey and Denenberg, 1967 , however, there was no relationship Ž
between defecation score and locomotive activity i.e., number of lines crossed, number .
of entries into the centre in the present study, nor was there any correlation between
Ž .
defecation score and the rotated factors from the PCA ‘‘fear of novelty’’ or ‘‘activity’’ . This may indicate that defecation in pigs does not represent a general response to frightening situations in the same manner as for rodents.
While time spent in the lit compartment of the LrD test is sometimes considered a Ž
.
et al., 1997; Onaivi and Martin, 1989; Ramos et al., 1997 , this variable was more related to activity in the present study. The reasons for this are unclear. The frequency distribution of this variable showed that 32% of the pigs had an extremely low duration Ž0–20 s in the lit compartment, whereas for the rest it was approximately normally. distributed. Besides, diazepam treated pigs spent more time in the dark compartment
Ž .
than the control group Andersen et al., 1999 , which is contradictory to what is
Ž .
expected and reported from rodent experiments Costall et al., 1989 . These results question the validity of this test model for pigs. Although wild pigs are nocturnal, and
Ž
thus similar to rodents, with peaks of activity at dusk and dawn Caley, 1997; Russo et .
al., 1997 , it is possible that domestic pigs are adapted to the strong light in their production environment. If so, pigs may not perceive bright light as aversive.
Although the TI duration consisted of a larger fear than activity component, this response cannot be interpreted as a pure measure of either fear of novelty or activity.
Ž .
This partly supports Erhard and Mendl’s 1999 conclusions that the TI test may not be a good behavioural model of fear of novelty in pigs. However, the rotated factor pattern in the present study suggests that TI is more related to fear than the locomotor activity in the OF, but less related to fear than open arm avoidance in the EPM. The duration of TI
Ž
in the present study was equally correlated with activity i.e., number of entries into .
closed arms and open arm avoidance in the EPM. Pigs with a short duration of TI
Ž .
showed less avoidance of open arms i.e., spent more time on the open arms and were
Ž .
more active in the plus-maze i.e., number of open and closed arm entries than pigs with a long duration. The lack of relationship between TI duration and OF activity in the present study coincides with the results reported from domestic fowl chicks by Heiblum
Ž . et al. 1998 .
In conclusion, most test variables can be interpreted as more or less fear-related activities. Open arm avoidance represented the purest measure of fear of novelty or avoidance, whereas the number of entries into closed arms and the number of lines crossed in the OF were the purest measures of activity. Time spent in the lit compart-ment was more related to activity than fear of novelty, but the test appeared to be of less biological relevance for pigs. The EPM provided a better way of separating the fear and activity elements than the OF and the TI tests. Altogether, the EPM appeared to be the best behavioural model to measure fear of novelty or avoidance in pigs.
Acknowledgements
We gratefully acknowledge our research technician, Øyvind Vartdal, for building the pens and experimental apparatus. We also want to thank Ronald Knustad for the nice illustrations.
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