www.elsevier.comrlocaterapplanim
Effects of diazepam on the behaviour of weaned
pigs in three putative models of anxiety
Inger Lise Andersen
a,), Gry Færevik
a, Knut Egil Bøe
a,
Andrew M. Janczak
b, Morten Bakken
ba
Agricultural UniÕersity of Norway, Department of Agricultural Engineering, P.O. Box 5065,
14 32 Aas, Norway b
Agricultural UniÕersity of Norway, Department of Animal Science, P.O. Box 5025, 1432 Aas, Norway
Accepted 21 December 1999
Abstract
Ž .
The present study examined the effects of diazepam a widely used anxiolytic benzodiazepine on the behavioural response of pigs to three novel experimental situations used to measure
Ž .
anxiety-related behaviour in rodents. Twelve weaned pigs two pairs from each of the three litters were tested in an elevated plus-maze at the age of 6 weeks, a lightrdark test at the age of 7 weeks and an open-field test at the age of 8 weeks. Six of the pigs were pre-treated with diazepam Žvalium and the other six with saline control . In the elevated plus-maze, diazepam-treated pigs. Ž .
Ž .
had a higher number of entries into open arms Ps0.04 , spent more time on open arms ŽPs0.07 , and had a higher number of total arm entries P. Ž s0.05 than pigs from the control.
Ž group. However, diazepam had no significant effects on behaviour in the lightrdark test i.e., latency to enter lit compartment, number of entries into lit compartment and the time spent in lit
. Ž .
compartment or the open-field test i.e., number of lines crossed, number of entries into centre . In summary, the anxiolytic effects of diazepam on the pigs’ behaviour were only demonstrated in the elevated plus-maze, where the time spent on open arms and the number of entries into open arms could be interpreted as measures of anxiety in pigs.q2000 Elsevier Science B.V. All rights reserved.
Keywords: Anxiety; Diazepam; Elevated plus-maze; Open-field test; Lightrdark test; Pigs
)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 .
1. Introduction
Fear and anxiety are important aspects of welfare in farm animals. The range of potentially fear or anxiety-eliciting events in pig husbandry include exposure to a new
Ž .
environment Mormede et al., 1984; Beattie et al., 1995; Erhard and Mendl, 1999 ,
`
Ž .
exposure to and handling by humans e.g., Hemsworth et al., 1986 , isolation from litter
Ž . Ž
mates Forkman et al., 1995 , and mixing with unfamiliar pigs Jensen, 1994; Jensen and
.
Yngvesson, 1998 . A common element in these situations is novelty, which is often
Ž .
associated with a negative emotional response in animals Boissy, 1995 . Different
Ž
methods have been developed to study fear responses of pigs during handling e.g.,
.
Hemsworth and Barnett, 1991, 1992; Hemsworth et al., 1986; Tanida et al., 1995 , but there is a considerable lack of validated methods for measuring fear-related behaviour in other novel situations. One approach to studying fear and anxiety in swine would be to apply the large body of knowledge provided by research on laboratory animals.
Two commonly used and well-validated models of anxiety in rodents are the elevated
Ž
plus-maze Pellow et al., 1985; Lister, 1987; Rodgers and Cole, 1993; Rodgers and
. Ž
Johnson, 1995; Cruz et al., 1994; File et al., 1994 and the lightrdark test Rodgers and Shepherd, 1993; Cheng et al., 1994; Costall and Naylor, 1994, 1995; Costall et al., 1987;
.
Smythe et al., 1996; Hendrie et al., 1997 . The elevated plus-maze is composed of four elevated arms of equal size radiating from a central platform. Two opposite arms have no walls while the other two have walls along their sides and ends. The model is based on the observation that although animals tend to explore a novel environment, the height
Ž
and the open space of open arms are perceived as aversive and avoided Montgomery,
.
1955; Pellow et al., 1985; File, 1992 . In the elevated plus-maze, increases in the number of entries into open arms and time spent on open arms are associated with a
Ž .
lower level of anxiety Pellow et al., 1985 . The lightrdark test, composed of a bright and dark room connected by a small opening, creates a conflict between the tendency to
Ž
explore a novel environment and to avoid the open, brightly-lit compartment Crawley
.
and Davis, 1982 . An anxiolytic effect in the lightrdark test is indicated by an increase
Ž
in the percent of activity and time spent in the aversive brightly-lit compartment Costall
.
et al., 1987; Onaivi and Martin, 1989 or the number of transitions between the two
Ž .
compartments Smythe et al., 1996 . Modifications of the open-field test developed by
Ž .
Hall 1936 have been used to a great extent to assess fear-related behaviour in many
Ž
domestic species pigs: Von Borell and Ladewig, 1992; Beattie et al., 1995, foxes: Bakken, 1994; poultry: Jones et al., 1995; Heiblum et al., 1998 and cattle: de Passille et
´
. Ž
al., 1995; Jensen et al., 1997 , but only a few have evaluated the method Boissy and
. Ž .
Bouissou, 1995; de Passille et al., 1995; Marin et al., 1997 . File 1992 states that
´
proper validation of behavioural models of anxiety should combine behavioural, phar-maceutical, and physiological methods. These procedures are ubiquitous in validating models of aversion for laboratory animals but are blatantly lacking in farm animal studies. Anxiolytic drugs should thus be helpful tools in revealing which behavioural elements are related to anxiety in different experimental tests for domestic species.Diazepam is considered to be one of the most powerful anxiolytic drugs. When used at low or moderate doses, the sedative and muscle relaxant effects are few and of low
Ž .
Ž .
without altering activity in the elevated plus-maze Cole and Rodgers, 1995 , and
Ž
reduces avoidance of the lit compartment in the lightrdark test Costall et al., 1987;
.
Hendrie et al., 1997 . On the contrary, diazepam is reported to have sedative effects on
Ž
motor activity in the open-field Hughes, 1993; Fisher and Hughes, 1996; Marin et al.,
.
1997 , which suggests that the effects of this drug can differ between tests. In pigs, diazepam reduces the aversive effect of non-reward and releases operant behaviour
Ž .
which has been suppressed by punishment Dantzer, 1976, 1978 . As anxiolytic effects
Ž .
of diazepam are documented in pigs Dantzer, 1976, 1977; Arnone and Dantzer, 1980 , this drug was chosen to assist in detecting anxiety-related behavioural elements in the present experiment.
The aim of the present experiment was thus to examine the effect of diazepam on the response of weaned pigs to three novel, aversive experimental tests to facilitate identification of anxiety-related behavioural elements.
2. Materials and methods
Twelve pigs were subjected to three experimental tests: an elevated plus-maze, a lightrdark test and an open-field test at the age of 6, 7 and 8 weeks of age, respectively. Half of the animals were pre-treated with diazepam and the other half with saline
Žcontrol ..
2.1. Animals
Ž .
Two pairs of weaned pigs Landrace=Yorkshire, castrated males and females from each of the three litters were chosen at the age of 6 weeks, so that pigs in each pair were
Ž .
of equal weight maximum weight difference was 0.5 kg . One pig from each pair was treated with diazepam, and the other with saline. The mean weight of pigs used in the experiment was 15.0"1.8 kg. After weaning at the age of 5 weeks, the three litters were moved to the research building. All pigs were kept in litter groups throughout the
Ž .
whole experimental period, and housed in pens 2.2=2.6 m containing a lying area with solid floor in 2r3 of the pen and slatted floor in the rest. An infrared lamp used as
Ž .
a heater was suspended in one corner of the pen, and a small amount of litter sawdust was supplied every morning. Solid pen partitions prevented any visual or physical contact between litters. The pigs were fed ad libitum with standard concentrate from a food dispenser, and had free access to water from a nipple drinker. Air temperature was between 188C and 208C, and artificial light was kept on between 0800 and 1500 h. During the rest of the day and night, the windows and the infrared heaters were the only sources of light.
2.2. Drug administration
The pigs were weighed 30 min before each test, and injected intramuscularly with
Ž w . Ž
diazepam Valium , Roche; 0.8 mgrkg from commercially available vials 10 mgr2
. Ž .
Ž
and 50 kg, 1 mgrkg diazepam is a common dosage level e.g., Dantzer, 1978; Arnone
.
and Dantzer, 1980 . The reason for reducing the dosage to 0.8 mgrkg in the present study, was that a preliminary study using 1 mgrkg resulted in severe side effects. These side effects included inactivity, problems with walking and shivering. At a dosage of 0.8 mgrkg, however, no side effects were observed.
2.3. EleÕated plus-maze
The elevated plus-maze was a modification of the apparatus described by Lister
Ž1987 for mice, and adjusted to the size of 6-week-old pigs. The apparatus consisted of.
Ž .
two open, elevated 1.0 m above the floor arms with a 2-cm high threshold, 0.60=3.0 m, and two enclosed arms, 0.60=0.30=0.60 m, arranged in such a way that the two
Ž .
open arms were opposite to each other Fig. 1 . The walls of the enclosed arms were made of transparent plexiglas.
The pigs were placed individually into the centre of the maze facing one of the closed arms. From the time that the animal was placed onto the maze the following parameters were observed for a period of 5 min: number of entries into open arms, number of entries into closed arms, and time spent on open arms.
2.4. Lightrdark test
The lightrdark box used in this experiment was a modification of the apparatus used
Ž .
for mice by Cheng et al. 1994 , adjusted to the size of 7-week-old pigs. The box
Ž . Ž .
consisted of a brightly illuminated 60 W Halogen light compartment 1.2=1.2 m
Ž .
where the walls were painted white, a dark compartment 1.2=1.2 m with dark brown
Ž . Ž .
walls, and an opening 0.4=0.4 m between the two compartments Fig. 2 . An infrared light was positioned above the test apparatus. The light intensity in the lit and dark area was 90 and 2.5 lux, respectively.
The subjects were individually tested for 5 min at the age of 7 weeks. The pigs were placed into the dark compartment of the box, and the latency to enter the lit
Fig. 2. Lightrdark box.
ment, number of entries into the lit compartment and the time spent in the lit compartment were recorded.
2.5. Open-field test
Ž .
The open-field test was a modification of the test for rats developed by Hall 1936 , adjusted to the size of pigs at the age of 8 weeks. A circular open-field arena with a
Ž .
diameter of 3.0 m and an 0.6 m high wall of plywood was used Fig. 3 . The pigs were individually placed in area number 1 and the number of lines crossed and the number of entries into the centre of the arena were recorded during a 5-min period. After the pig was removed from the apparatus, the number of fecal boli dropped was counted
Ždefecation frequency ..
The following points were the same for all three behavioural tests. The test apparatus was situated in a separate test room without windows and having a solid door, which the handler immediately exited after placing the animal onto the test apparatus. All behaviour was observed from a monitor in an observation room, connected to a video camera vertically mounted above the centre of the test apparatus, and the apparatus was cleaned before each animal was tested.
2.6. Statistics
To analyse the differences in behaviour between the diazepam and control group, a pair-wise t-test was conducted.
3. Results and discussion
Diazepam treatment increased the number of entries onto open arms, time spent on
Ž .
open arms and the total number of arm entries Table 1 . This corresponds to a large
Ž
extent with the results from work on rodents Pellow et al., 1985; Lister, 1987; Rex et
.
al., 1993; Cole and Rodgers, 1995; Hendrie et al., 1997 , and suggests that open arm
Table 1
Ž . Ž .
Behavioural differences between diazepam-treated pigs ns6 and the control group ns6 in three experimental models of anxiety
Mean"S.E. and range are given.
Test variable Saline Diazepam P value
EleÕated plus-maze
Ž . Ž .
No. of entries into open arms 5"1 2–8 8"2 3–16 0.04
Ž . Ž . Ž .
Entries into the open arms % 56.0"5.2 40–80 55.8"7.2 36–71 0.49
Ž . Ž . Ž .
Time spent on open arms % 31.6"5.2 15.7–50.3 47.5"7.7 31.0–80.0 0.07
Ž . Ž .
Latency to enter light area s 148.2"49.3 10–293 238.5"40.8 68–300 0.11
Ž . Ž .
No. of lines crossed 90.7"10.2 540–117 98.8"14.1 54–117 0.31
Ž . Ž .
No. of entries into centre 4.5"1.2 0–7 3.2"0.7 1–6 0.17
Ž . Ž .
entries and time spent on open arms are indicators of anxiety in pigs. The number of and percent entries into open arms and time and percent of time spent on open arms are considered the most important indicators of anxiety for rodents in the plus-maze, as they
Ž .
to a lower extent are sensitive to changes in locomotor activity Hogg, 1996 . In factor analysis of murine elevated plus-maze behaviour these four variables all load strongly
Ž .
on the factor which is anxiety-related see Rodgers and Johnson, 1995 . In the present experiment, however, diazepam did not increase the percent of entries into open arms as one would expect if this element was anxiety-related. The reason for this is unclear. Since the interpretation of behavioural elements for rodents in the elevated plus-maze is to a large degree based on the results from factor analysis, this discrepancy also
Ž
illustrates a need for factor analysis of porcine elevated plus-maze behaviour see
. Ž .
Andersen et al., 1999 . Similar to the findings of Cole and Rodgers 1995 for mice, the number of entries into closed arms was not significantly affected by diazepam treatment. As closed entries are considered a purer measure of the locomotor activity than total
Ž
number of arm entries Lister, 1987; File, 1992; Rodgers and Johnson, 1995; Hogg,
.
1996 , it can be inferred that diazepam did not affect the activity level per se. This could also explain why there were no significant effects of diazepam on open-field activity
Ži.e., number of lines crossed, the number of entries into the centre; Table 1 . It is also.
possible that rodents and pigs show different behavioural responses in the open-field test. This can only be addressed by including more behavioural parameters when studying open-field behaviour in pigs. Furthermore, we need to analyse the behavioural alteration in pigs after administration of anxiolytic drugs and how these changes are related to the pigs fear responses in other experimental tests. Some authors have reported
Ž
sedative effects of diazepam on motor activity in the open-field Hughes, 1993; Fisher
.
and Hughes, 1996; Marin et al., 1997 , but others suggest that this effect is dose
Ž . Ž .
dependent Schmitt and Hiemke, 1998 . As found by Pohorecky and Roberts 1991 , diazepam treatment resulted in a higher frequency of defecation in the open-field, which may be interpreted as a side effect of the drug. There were no differences between the
Ž .
diazepam and the control group in any of the variables in the lightrdark test Table 1 . In fact, the diazepam-treated pigs tended to show a greater latency to enter the lit compartment. The wild boar and feral pig show a peak of activity at dusk and dawn
ŽCaley, 1997; Russo et al., 1997 , but unlike rodents, there is very little documentation.
Ž .
of preference for a dark area in the domestic pig Braude et al., 1958 . The lack of documentation of aversion to light in swine, combined with the fact that diazepam did not influence the response to light in the present experiment, indicates that pigs may not perceive light as aversive. The lightrdark test may thus have less biological relevance and practical value for pigs than for rodents.
Acknowledgements
We gratefully acknowledge our research technician,Byvind Vartdal, for building the
pens and experimental apparatus. We also want to thank Ronald Knustad for the nice illustrations.
References
Andersen, I.L., Boue, K.E., Færevik, G., Janczak, A.M., Bakken, M., 1999. Behavioural evaluation of methods for assessing fear responses in weaned pigs. Appl. Anim. Behav. Sci., Submitted.
Arnone, M., Dantzer, R., 1980. Effects of diazepam on extinction induced aggression in pigs. Pharmacol. Biochem. Behav. 13, 27–30.
Bakken, M., 1994. Infanticidal behaviour and reproductive performance in relation to competition capacity among farmed silver fox vixens, VulpesÕulpes. Doctoral thesis.
Beattie, V.E., Walker, N., Sneddon, I.A., 1995. Effect of rearing environment and change of environment on the behaviour of gilts. Appl. Anim. Behav. Sci. 46, 57–65.
Boissy, A., 1995. Fear and fearfulness in animals. Q. Rev. Biol. 70, 165–191.
Boissy, A., Bouissou, M.-F., 1995. Assessment of individual differences in behavioural reactions of heifers exposed to various fear-eliciting situations. Appl. Anim. Behav. Sci. 46, 17–31.
Braude, R. et al., 1958. The effect of light on fattening pigs. Proc. Nutr. Soc. 17, 38.
Ž .
Caley, P., 1997. Movements, activity patterns and habitat use of feral pigs Sus scrofa in a tropical habitat. Wildl. Res. 24, 77–87.
Cheng, C., Costall, B., Kelly, M.E., Naylor, J.R., 1994. Actions of 5-hydroxytryptophan to inhibit and disinhibit mouse behaviour in the lightrdark test. Eur. J. Pharmacol. 225, 39–49.
Cole, J.C., Rodgers, R.J., 1995. Ethological comparison of the effects of diazepam and acuterchronic imipramine on the behaviour of mice in the elevated plus-maze. Phamacol. Biochem. Behav. 52, 473–478. Costall, B., Hendrie, C.A., Kelly, M.E., Neylor, R.J., 1987. Actions of sulpiride and tiapride in a simple model
of anxiety in mice. Neuropharmacology 26, 195–200.
Costall, B., Naylor, R.J., 1994. Atypical neuroleptic agents attenuate the inhibitory profile of 5-hydroxytrypto-phan in the mouse lightrdark test. Br. J. Pharmacol. 112, U114.
Costall, B., Naylor, R.J., 1995. Behavioral interactions between 5-hydroxytryptophan, neuroleptic agents and 5-HT receptor antagonists in modifying rodents responding to aversive situations. Br. J. Pharmacol. 116, 2989–2999.
Crawley, J.N., Davis, L.G., 1982. Baseline exploratory activity predicts anxiolytic responsiveness to diazepam in five mouse strains. Brain Res. Bull. 8, 609–612.
Cruz, A.P.M., Frei, F., Graeff, F.G., 1994. Ethopharmacological analysis of rat behavior on the elevated plus-maze. Pharmacol. Biochem. Behav. 49, 171–176.
Dantzer, R., 1976. Effect of diazepam on performance of pigs in a progressive ratio schedule. Brief communication. Physiol. Behav. 17, 161–163.
Dantzer, R., 1977. Effects of diazepam on behaviour suppressed by extinction in pigs. Pharmacol. Biochem. Behav. 6, 157–161.
Dantzer, R., 1978. Antipunishment effects of diazepam: interaction with shock and food deprivation levels in pigs. Psychopharmacology 58, 99–104.
de Passille, A.M., Rushen, J., Martin, F., 1995. Interpreting the behaviour of calves in an open field test: a´ factor analysis. Appl. Anim. Behav. Sci. 45, 201–213.
Erhard, H., Mendl, M., 1999. Tonic immobility and emergence time in pigs — more evidence for behavioural strategies. Appl. Anim. Behav. Sci. 61, 227–239.
Ž .
File, S.E., 1992. Behavioural detection of anxiolytic action. In: Elliot, J.M., Heal, D.J., Marsden, C.A. Eds. , Experimental Approaches to Anxiety and depression. Wiley, Chichester, UK, pp. 25–44.
Fisher, C.E., Hughes, R.N., 1996. Effects of diazepam and cyclohexyladenosine on open-field behaviour in rats perinatally exposed to caffeine. Life Sci. 58, 701–709.
Flaten, M.A., Grawe, R.W., 1986. Evaluation of the effects of diazepam on anxiety after conditioning with˚
Ž .
internal and external stimuli. Res. Rep. — Dept. Psychiatry Behav. Med., Univ. Trondheim 9 52 , pp. 75. Forkman, B., Furuhaug, I.L., Jensen, P., 1995. Personality, coping patterns, and aggression in pigs. Appl.
Anim. Behav. Sci. 45, 31–42.
Hall, C.S., 1936. Emotional behaviour in the rat: III. The relationship between emotionality and ambulatory activity. J. Comp. Psychol. 22, 345–352.
Heiblum, R., Aizenstein, O., Gvaryahu, G., Voet, H., Robinzon, B., Snapir, N., 1998. Tonic immobility and open field responses in domestic fowl chicks during the first week of life. Appl. Anim. Behav. Sci. 60, 347–357.
Hemsworth, P.H., Barnett, J.L., 1991. The effects of aversively handling pigs, either individually or in groups, on their behaviour, growth and corticosteroids. Appl. Anim. Behav. Sci. 30, 61–72.
Hemsworth, P.H., Barnett, J.L., 1992. The effects of early contact with humans on the subsequent level of fear of humans. Appl. Anim. Behav. Sci. 35, 83–90.
Hemsworth, P.H., Barnett, J.L., Hansen, C., Gonyou, H.W., 1986. The influence of early contact with humans on subsequent behavioural response of pigs to humans. Appl. Anim. Behav. Sci. 15, 55–63.
Hendrie, C.A., Eilam, D., Weiss, S.M., 1997. Effects of diazepam and buspirone on the behaviour of wild voles. Pharmacol. Biochem. Behav. 58, 573–576.
Hogg, S., 1996. A review of the validity and variability of the elevated plus-maze as an animal model on anxiety. Pharmacol. Biochem. Behav. 54, 21–30.
Hughes, R.N., 1993. Effects on open-field behaviour of diazepam and buspirone alone and in combination with chronic caffeine. Life Sci. 53, 1217–1225.
Jensen, P., 1994. Fighting between unacquainted pigs — effects of age and individual reaction pattern. Appl. Anim. Behav. Sci. 41, 37–52.
Jensen, M.B., Vestergaard, K.S., Krohn, C.C., Munksgaard, L., 1997. Effect of single versus group housing and space allowance on responses of calves during open-field tests. Appl. Anim. Behav. Sci. 54, 109–121. Jensen, P., Yngvesson, J., 1998. Aggression between unacquainted pigs — sequential assessment and effects
of familiarity and weight. Appl. Anim. Behav. Sci. 58, 49–63.
Jones, R.B., Blokhuis, H.J., Beuving, G., 1995. Open-field and tonic immobility responses in domestic chicks of two genetic lines differing in their propensity to feather peck. Br. Poult. Sci. 36, 525–530.
Lister, R.G., 1987. The use of a plus maze to measure anxiety in the mouse. Psychopharmacology 92, 180–185.
Marin, R.H., Martijena, I.D., Arce, A., 1997. Effect of diazepam and Ab-carboline on open-field and T-maze behaviours in 2-day-old chicks. Pharmacol. Biochem. Behav. 58, 915–921.
Montgomery, K.C., 1955. The relation between fear induced by novel stimulation and exploratory behaviour. J. Comp. Phys. Psychol. 48, 254–260.
Mormede, P., Dantzer, R., Bluthe, R.M., 1984. Differences in adaptive abilities of three breeds of chinese pigs.` ´ Behavioural and neuroendocrine studies. Genet. Sel. Evol. 16, 85–102.
Onaivi, E.S., Martin, B.R., 1989. Neuropharmacological and physiological validation of a computer-controlled two-compartment black and white box for the assessment of anxiety. Prog. Neuropsychopharmacol. Biol. Psychiatry 13, 963–976.
Pellow, S., Chopin, P., File, S.E., Briley, M., 1985. Validation of openrclosed arm entries in an elevated plus-maze as a measure of anxiety in the rat. J. Neur. Meth. 14, 149–167.
Pohorecky, L.A., Roberts, P., 1991. Activity in a modified open field apparatus — effect of diazepam and prenatal stress. Neurotoxicol. Teratol. 13, 129–133.
Rex, A., Marsden, C.A., Fink, H., 1993. Effect of diazepam on cortical 5-HT release and behaviour in the guinea-pig on exposure to the elevated plus maze. Psychopharmacology 110, 490–496.
Rodgers, R.J., Cole, J.C., 1993. Influence of social isolation, gender, strain, and prior novelty on plus-maze behavior in mice. Physiol. Behav. 54, 729–736.
Rodgers, R.J., Shepherd, J.K., 1993. Influence of prior maze experience on behavior and response to diazepam in the elevated plus-maze and lightrdark tests of anxiety in mice. Psychopharmacology 113, 237–242. Rodgers, R.J., Johnson, N.J.T., 1995. Factor analysis of spatiotemporal and ethological measures in the murine
Russo, L., Massei, G., Genov, P.V., 1997. Daily home range and activity of wild boar in a Mediterranean area free from hunting. Ethol. Ecol. Evol. 9, 287–294.
Schmitt, U., Hiemke, C., 1998. Combination of open field and elevated plus-maze: a suitable test battery to assess strain as well as treatment differences in rat behaviour. Prog. Neuropsychopharmacol. Biol. Psychiatry 22, 1197–1215.
Smythe, J.W., Murphy, D., Bhatnagar, S., Timothy, C., Costall, B., 1996. Muscarinic antagonists are anxiogenic in rats tested in the black–white box. Pharmacol. Biochem. Behav. 54, 57–63.
Tanida, H., Miura, A., Tanaka, T., Yoshimoto, T., 1995. Behavioural response to humans in individually handled weanling pigs. Appl. Anim. Behav. Sci. 42, 249–259.
