Applied Animal Behaviour Science 153 (2014) 26–35

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Applied Animal Behaviour Science

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Does stocking density modify affective state in pigs asassessed by cognitive bias, behavioural and physiologicalparameters?

Annalisa Scolloa,b,∗, Flaviana Gottardob, Barbara Contierob,Sandra A. Edwardsa

a School of Agriculture, Food and Rural Development, Newcastle University, Newcastle upon Tyne NE1 7RU, UKb Department of Animal Medicine, Production and Health, Padova University, Legnaro, 35020 Padova, Italy

a r t i c l e i n f o

Article history:Accepted 21 January 2014Available online 28 January 2014

Keywords:PigStocking densityWelfareCognitive biasBehaviourLearning

a b s t r a c t

Recent studies suggest that emotional state can affect cognitive abilities of humans andnon-human animals, determining biases in information processing. Negative mental states,such as anxiety or depression, induce pessimistic judgments of ambiguous stimuli. Theseassumptions may be used to derive indicators of emotional state in captive animals, pro-viding a novel approach to the assessment of animal welfare. This study used a spatialjudgement task, in which farmed pigs were trained to expect food inside a bowl in onelocation and not in another, to determine whether pigs housed in ways that might beexpected to result in relatively positive or negative emotional states respond differentlyto ambiguous stimuli of intermediate spatial locations. Forty growing pigs were housed ingroups of 10 at different density for 8 weeks prior to the start of the test. After training,the pigs successfully discriminated between the rewarded and the unrewarded locations asassessed by increased latency to arrive at the unrewarded location, with no rearing treat-ment difference. Then, pigs were tested on 3 days in which three ambiguous locations,intermediate between the known rewarded and the unrewarded sites, were introducedand latency recorded. In order to compare the novel cognitive bias task with other welfareindicators, during the 8 weeks of the study four behavioural observations, two measure-ments of skin lesions, two salivary samples for cortisol and �-amylase, and six individualweights to assess growth were collected.

Considering the mean of the three test days, there was no difference between the treat-ments in the pigs’ judgement of the three ambiguous locations. However, the latency trendduring the testing days led to difference between treatments on the third day (P = 0.026).Pigs housed a higher density seemed to learn faster that the ambiguous stimulus near theunrewarded location was also not reinforced by a reward and showed a higher latencyto approach on day 3 (44 vs 15.6 s). These animals were also observed to have a higherfrequency of sitting posture (P = 0.01), and more total skin lesions (P = 0.035) due to aggres-

siveness, in particular at the ear location (P = 0.009), but did not differ in other physiological parameters.

Although the results shdifferences in latencies tolearning process between

∗ Corresponding author at: University of Padova, Animal Medicine, ProductionTel.: +39 49 827 2620/+39 349 242 1613; fax: +39 49 827 2669.

E-mail addresses: annalisa.scollo@studenti.unipd.it, anna.polly@libero.it (A. Sc

0168-1591/$ – see front matter © 2014 Elsevier B.V. All rights reserved.http://dx.doi.org/10.1016/j.applanim.2014.01.006

owed no immediate effect of stocking density on cognitive bias, reach the bowl over the three testing days suggest a different

treatments.© 2014 Elsevier B.V. All rights reserved.

and Health, viale dell’Università 16, 35020 Legnaro, Italy.

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A. Scollo et al. / Applied Anima

. Introduction

The study of the emotional state of animals is increas-ngly attracting the attention of researchers and in recentears has also been reflected in public concern aboutnimal welfare (Dawkins, 2006; Mendl and Paul, 2004).urthermore, the recently adopted EU Strategy for therotection and Welfare of Animals 2012–2015 (Europeanommission, 2012) highlights the desirability of usingcientifically validated animal-based indicators to comple-ent the prescriptive requirements in EU legislation (EFSAHAW panel, 2012).

Currently it is not possible to obtain direct andbjectively measurable observations of the emotionalxperience of animals, and the investigation of emotionsocuses mainly on the assessment of physiological andehavioural indicators of stress and welfare (Abou-Ismailt al., 2007; Burman et al., 2007). However, interpretationf these indicators is complicated by the fact that a par-icular measure could reflect both a positive or negativemotional state. For example, an increase in locomotoryctivity or in heart rate can be associated both withscape from predation (aversive activity, negative valence)nd with sexual behaviour (pleasurable activity, positivealence). Furthermore, changes in physiological indicatorsf stress such as cortisol, could indicate an alteration of thesychological state but also pathological states or inflam-ation (Dawkins, 2001). Often the method of collection for

iological samples (e.g. blood) or the testing of spontaneousehaviour in an open field can cause emotional reactions innimals that may confuse the results (Broom and Johnson,993).

For these reasons, the development of alternative meth-ds for the assessment of emotional states for whichehavioural and physiological measures may be incon-lusive or imprecise has been considered, not only bynimal welfare researchers but also in neuroscience andsychopharmacology (Lawrence, 2008; Rolls, 2005). One ofhese alternatives is the study of cognitive bias, a term usedor the first time by Shettleworth (1998) to indicate ‘the

echanisms by which animals acquire, process, store andct on information from the environment’. The literaturen human psychology provides the main source of infor-ation about the effects of background emotional states

n cognitive processes, influencing attention, memory andudgement (Paul et al., 2005). However, the ‘gold standard’ndicator of the subjective emotional state in humans iserbal report, which animals are unable to provide, thusaking evaluation more uncertain. Furthermore, interpre-

ation of tests, and translating them from one specie tonother, could be difficult and provide some uncertaintiesn research findings (Cryan and Mombereau, 2004; Janczakt al., 2002).

The first attempt to explore cognitive biases in animalsas conducted by Harding et al. (2004) in rats. Animals

xperiencing an unpredictable housing treatment showed reduced anticipation of a positive event, similar to pes-imistic cognitive bias seen in depressed humans. These

nitial results were followed by a number of studies car-ied out in different species, confirming the assumptionslso in starlings (Bateson and Matheson, 2007) and dogsMendl et al., 2010).

our Science 153 (2014) 26–35 27

In 1999, Laughlin et al. stated that cognitive processescould be involved in welfare problems also in farmed ani-mals, where housing and husbandry procedures may resultin stress or increased arousal of the animals. An initialexperiment demonstrated that the degree of environmen-tal enrichment in the housing of pigs could influencecognitive bias in a go/no-go discrimination task (Douglaset al., 2012). However, this task involved lengthy trainingand staff effort. In the present study we therefore decidedto investigate the promising approach of cognitive bias inpigs using a location judgement bias task based on theabsence/presence of a food reward.

A spatial location task was used because of its relevanceto foraging behaviour, a common cognitive task for animals(Thorpe et al., 2002; Wood et al., 1999). Furthermore, Heldet al. (2002) showed that pigs have well-developed spa-tial memory abilities. To induce a mild stress, we housedpigs at two different stocking densities. Reduced spaceallowance is reported to have negative effects in pigs andis detectable in both behavioural and physiological indica-tors of stress (Averós et al., 2010) and space is one of themost compromised needs in commercial housing systemsin the interests of efficiency of building utilization (Turneret al., 2000).

In order to compare results of the cognitive bias taskwith other commonly used indicators of affective state,concurrent behavioural and physiological measures weretaken. Among the behavioural parameters, we decided torecord vocalizations as a spontaneous behaviour indicat-ing the expectation of the food in the rewarded location,as reported by Waitt and Buchanan-Smith (2001) in pigsbefore the meal delivery. Vocal expression of apparentlyvalenced affective states has been identified in severalspecies and it is reported by Manteuffel et al. (2011) asa potential indicator of reduced welfare in pigs. More-over, we measured salivary cortisol and �-amylase. Thelatter has become an emerging biomarker for sympatheticnervous system activity related to depression in humans.Ishitobi et al. (2010) suggested that �-amylase may be astate-dependent marker of major depressive disorder inaddition to salivary cortisol, but its role in non-human ani-mals has not yet been investigated.

It was hypothesized that pigs with a lower spaceallowance would show a statistically significant differencein latency to approach an ambiguous stimulus as com-pared to pigs with a larger space allowance. Such differencewould be indicative of a negative or pessimistic emotionalstate. Moreover, it was also predicted that this difference inassumed emotional state would be associated with signifi-cantly different behavioural and physiological parametersmeasured during housing in the home pen.

2. Materials and methods

2.1. Animals, facilities and management

All experimental procedures and animal care were car-

ried out in accordance with the minimum standards forthe protection of pigs laid down by law (Council of TheEuropean Union, 2008), and were approved by the EthicalReview Committee of Newcastle University.

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28 A. Scollo et al. / Applied Anima

A total of 40 entire male and female crossbred pigs(Large White × Landrace), which had previously experi-enced the same housing and management conditions, wereselected at approximately 14 weeks of age and 45 kg liveweight. All the pigs were ear tagged for individual iden-tification. Animals were housed in 4 matched pens of 10pigs each (5 females and 5 males). Pen floors were 83%solid and, to allow drainage, 17% slatted. Pens were locatedin the same controlled environment, fan ventilated roomwith the temperature set for 20 ◦C. Artificial lighting pro-vided at least 40 lx for a period of 11 h per day. Pigs weremanually fed once daily with a dry pelleted concentrate(A-One Feeds Ltd, Thirsk, UK) distributed on the solid penfloor and spread evenly so that all pigs could feed simul-taneously. In order to increase the animals’ motivationtoward the food reward during experimental testing, mealswere delivered after the completion of the experimentalprocedures at approximately 16:00 h. Water was availablead libitum through one automatic nipple drinker per pen.Pens were provided with suspended metal chains as basicenvironmental enrichment.

2.2. Treatments

Two pens of pigs were housed at each of two dif-ferent stocking densities. For the determination of spaceallowance, an allometric approach (Gonyou et al., 2006;Petherick, 1983) was used, calculating the area required(A) in relation to the animals’ body weight (BW) accord-ing to the equation A = k × BW0.667, where the constant (k)represents a space allowance coefficient. For the low spaceallowance treatment k = 0.030, approximating the currentEuropean Union minimum standard required (Council ofThe European Union, 2008). For the high space allowancetreatment k = 0.048, according to the recommendation ofthe Scientific Panel on Animal Health and Welfare of theEuropean Food Safety Authority (EFSA AHAW panel, 2005)for permitting the animals to lie in full recumbency. Thespace allowance was maintained by a weekly assessmentof mean live-weight and the movement of a partition toextend the pen width accordingly whilst maintaining thesame ratio of solid to slatted floor.

2.3. Cognitive bias test

The testing procedure was modified from that used byBurman et al. (2008) in the rat, in order to apply it in swine.Training and testing were performed in a concrete flooredarena (9 × 6.5 m) located in a separate building. The wallsand gate were solid and approximately 1.5 m high in orderto avoid external visual distractions. The entrance througha manually opened solid access door was positioned alongone short side of the arena, permitting individual access toa crate which allowed the pig to observe into the arena.The pig was waiting in the crate until the opening of asecond gate to commence the test run into the arena.The solid access door was connected with a waiting pen

adjacent to the arena where animals remained until theirturn. The goal point was a clearly identifiable yellow bowl,placed in the arena in one of five possible locations assuggested by Burman et al. (2008). The two ‘reference’

our Science 153 (2014) 26–35

locations (rewarded or unrewarded) were equidistant fromthe crate (8 m) and from the side wall (20 cm), whereas thethree ambiguous ‘probe’ locations were located at equidis-tant intermediate locations (1.5 m apart) between the tworeference locations (Fig. 1). In order to return the bowl tothe correct place after each cleaning between trials, the fivelocations were marked on the floor. For the rewarded loca-tion, 60 g of a pelleted weaner diet (A-One Feeds Ltd, Thirsk,UK) was used as this contained ingredients known to be ofelevated hedonic value (Clouard et al., 2012) and provedattractive to pigs in pilot trials.

2.3.1. Habituation to the apparatusPigs were pre-exposed to the arena for 5 days to let the

animals became familiar with the arena, the waiting pen,the start crate and the test conditions, in order to avoid anyinfluencing stressors caused by the methodology (Mendlet al., 2009). On the first pre-exposure day, the 10 pigs fromeach pen were introduced into the arena at the same time,allowing free exploration of the environment as a group.Over the two subsequent days, each pig was individuallyexposed to the apparatus, entering through the start crate.Initially, the bowl with food was placed in front of the cageat a distance of two meters, to encourage approach andincrease the chance to discover the reward. This proce-dure was repeated on the fourth and the fifth pre-exposuredays but, once the pig began feeding, a researcher movedthe bowl and replaced it at a random location within thearena, leaving the pig to approach the bowl again. Pre-exposure days were completed after all pigs were followingthe bowl to four different locations. At the end of the finalpre-exposure day, all pigs were successful in following thebowl.

2.3.2. TrainingDuring the 5 weeks following habituation, pigs learned

to discriminate between the two reference locations. Ineach individual training trial, the bowl was present eitherin the rewarded location (containing food pellets) or in theunrewarded location (without food pellets). For half of theanimals in each treatment, the rewarded location was tothe right of the start cage and the unrewarded was to theleft, vice versa for the remaining pigs. To consolidate theassociation learnt in the pre-exposure phase between thebowl and reward, during the first week of training the bowlwas placed only in the rewarded location. In the remainingfour training weeks, pigs underwent four trials per day,half in the rewarded location and half in the unrewardedone. To avoid any sequence memorization, the order inwhich the four trials occurred was modified daily accordingto a pseudo-randomization procedure. The first constraintadopted for the pseudo-random procedure was to makethe rewarded-unrewarded ratio equal for the two groupsand for each day of training. A second constraint ensuredthat the rewarded-unrewarded ratio was equal also for thefirst and the last trial of the sequences within group.

Prior to each trial, pigs were moved in their group

through an external walkway from the housing pen to thewaiting pen adjacent to the arena, where they remaineduntil it was their turn for testing. Once the first solidgate was opened, the pig entered into the start crate

A. Scollo et al. / Applied Animal Behaviour Science 153 (2014) 26–35 29

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Fig. 1. The experimental arena, showing the five pos

hich allowed it to observe the placing of the bowl by aesearcher. Subsequent to the bowl placement, the crateoor was manually opened to permit the entrance of theig into the arena. The time taken by the animal to touchhe bowl with the nose was recorded. In case of a rewardedrial, the pig was allowed a standard time of 5 s to eat aart of the food pellets. During the first week of training,rials were open-ended and continued until the pig reachedhe bowl, whereas trials in the remaining weeks had a cut-ff of 2 min, after which time the pig was removed fromhe arena. Once all the pen-mates had completed the fouraily trials, the group was returned to the housing pen andnother group taken for training. Pigs were deemed to haveearnt the discrimination between the two reference loca-ions when a significant difference (P < 0.05) was shown inhe average latencies to reach the bowl from the last threeewarded trials and from the last three unrewarded trials.ll pigs achieved the learning criteria during the 5 weeks.

.3.3. TestingPigs were tested in two batches on alternate days,

ithin which the two treatments were counterbalanced.n this way, on each day only one batch was tested but thether one was submitted to a ‘refresher-day’ with a pseu-orandom sequence of the reference locations, similar tohose of the training, in order to reinforce the discrimina-ion for the subsequent day. Testing lasted 6 days in total:

ach batch was submitted to 3 testing days alternating with

‘refresher-days’ (Table 1). During the testing days, the ani-als of the tested batch were individually exposed once

er day to each of the three ambiguous probe locations

able 1chedule of the testing procedure.

Batch 1a Batch 2a

Day 1 1st Testing day Refresher-dayDay 2 Refresher-day 1st Testing dayDay 3 2nd Testing day Refresher-dayDay 4 Refresher-day 2nd Testing dayDay 5 3rd Testing day Refresher-dayDay 6 Refresher-day 3rd Testing day

a Within each batch, the two treatments were counterbalanced.

d bowl locations and the distances between them.

(near rewarded: NR; middle: M; near unrewarded: NU),spread within a pseudorandom sequence of the two refer-ence locations. The sequence consisted of a total of six trialsstarting with the rewarded location. Daily sequences wereplanned to ensure that, for both treatments, the numberof times each ambiguous location followed immediatelyafter a rewarded location and immediately after an unre-warded one was the same. As suggested by Burman et al.(2008), ambiguous locations were unrewarded to minimizeany association between them and the reward outcomes,which could occur faster if ambiguous locations had beenrewarded.

During testing, the latency to reach the bowl and thenumber of vocalizations emitted in 10 s before opening thestart crate were recorded. Regarding latency, trials had acut-off of 2 min after which time the pig was removed fromthe arena.

2.4. Behavioural and physiological measurements

All the behavioural and physiological measurementswere carried out during rest days in order to avoid anyinfluence of the training/testing procedures.

2.4.1. Behavioural observationsBehaviour was evaluated four times (once per week

from week 5 to week 8 of the study), by direct observa-tion of animals in their home pens using a scan-samplingtechnique (Martin and Bateson, 2007) with a 2-min inter-val between scans. At each scan the number of animals perpen inactive (sternal lying, lateral lying, sitting, standingup, sleeping, awake) and performing an activity (exploringenvironment or chains or nipple drinker, belly nosing) wasrecorded using the ethogram shown in Table 2. Each obser-vation day was carried out by two trained observers, eachof whom recorded the behaviour performed by the pigs intwo pens scanned alternately. The pigs were observed for

two uninterrupted hours in the morning (from 10:00 h to12:00 h). Furthermore, the number of events of conflicts,ear biting and tail biting were recorded during observationusing the behaviour sampling technique (Altmann, 1974).

30 A. Scollo et al. / Applied Animal Behaviour Science 153 (2014) 26–35

Table 2Ethogram of behaviours observed and methods adopted for their recording.

Behaviour Definition of the behaviour Method of observation

Sternal lying Pigs in sternal lying, awake or sleeping

Scan sampling

Lateral lying Pigs in lateral lying, awake or sleepingSitting Pigs in sitting position (as a dog)Standing up Pigs standing up inactive, doing nothingSleeping Pigs lying (sternal or lateral) and sleepingAwake inactive Pigs lying (sternal or lateral) but awakeExploring environment Pigs sniffing, licking, rooting the environmentExploring chains Pigs sniffing, rooting and chewing chainsExploring nipple drinker Pigs sniffing, licking and sucking nipple drinkerBelly nosing Pigs thrusting their noses towards the belly of

another pig, nuzzling the teat and flank areasConflicts Aggressive interaction among two or more pigs

Behaviour samplingEar biting A pig has in the mouth an ear of a pen mateand bites it

other p

Tail biting A pig bites the tail of an

2.4.2. Skin lesionsSkin lesions were visually assessed on two occasions (at

week 6 and week 8) using the method of Weng et al. (1998).The body was divided into eight areas: face, ears, neck,shoulder, main body, rump, genitals, tail, and the numberof lesions on each area recorded.

2.4.3. Saliva samplesSaliva samples were collected from individual pigs on

two occasions (at week 6 and week 8) for the evalu-ation of cortisol and �-amylase levels. Both collectionswere performed between 10:00 h and 11:00 h, in order toavoid circadian variations in the considered parameters.A researcher entered the home pen and held out a largecotton bud to each pig in turn, allowing spontaneous chew-ing in order to minimize any stress due to the collectionprocedure. Samples were immediately transported to thelaboratory, centrifuged at 2000 × g for 15 min, and stored at−20 ◦C before analysis. For the analysis of cortisol level, anenzyme linked immunosorbent assay (Salivary Cortisol Kit,Salimetrics Europe Ltd., Suffolk, UK) was used. The protocoldetailed in the kit specifications and features (SalimetricsEurope Ltd, 2012a) was applied. The minimum detectableconcentration of cortisol that could be distinguished from0 was 0.007 �g/dL; the intra- and inter-assay coefficientsof variations were 4.7% and 4.1%, respectively.

For the analysis of �-amylase level, a kinetic enzymeassay (Salivary �-amylase Kit, Salimetrics Europe Ltd.,Suffolk, UK) was used. The protocol detailed in the kit spec-ifications and features (Salimetrics Europe Ltd, 2012b) wasapplied.

2.4.4. GrowthEach pig was weighed on six occasions (once per week

from week 3 to week 8 of the study) to compare the growthrates between treatments. This also permitted a check thatpigs had no statistically significant differences in their bodyweight during testing, as suggested by Mendl et al. (2010),

to avoid the possibility that differences in latencies to reachthe bowl could be due to intrinsic differences between thepigs in their running speeds, linked to different animalsizes.

ig

2.5. Statistical analyses

All tests used SAS 9.2 (SAS Institute Inc., Cary, NC). Thetraining data were analyzed before the beginning of thetest phase, to determine when the pigs were ready for test-ing. The average latency to reach the bowl from the lastthree rewarded trials and from the last three unrewardedtrials was calculated using a PROC GLM. After training, sta-tistical analysis was focused on the test phase. For eachpig we calculated its mean latency to touch the food bowlduring each of the three types of probe test trial (NR, M,NU) and the two reference locations (rewarded and unre-warded). Furthermore, we considered the daily latency foreach pig to achieve the bowl in these five locations. Ifthe cut-off of 2 min was reached during the test phase, alatency of 120 s was considered for that pig in the analy-sis. Data were analyzed for their Gaussian distribution, andcoefficients of skewness and kurtosis were computed usingPROC UNIVARIATE to measure distribution asymmetry andpeakedness. Variables with Shapiro–Wilk values (W) ≥0.95were considered normal, whereas all other variables weretransformed before analysis to adjust for their lack of nor-mality (using natural logarithm transformation: latenciesfor reference, NU and M locations, amylase; using box–coxtransformation with � = −1.46: latencies for NR location).Gaussian and transformed data were statistically processedusing PROC MIXED. Analysis of repeated measurement datawas based on the mixed model with a compound sym-metric parametric structure on the covariance matrix; penwithin treatment was the repeated random effect. Themodel considered the fixed effects of treatment (low spaceallowance vs high space allowance) and testing day (fourfor behaviour, two for physiological parameters and skinlesions), and their interactions. Least square (LS) meanswere compared using the Bonferroni test.

Behavioural data from scan sampling were transformedinto frequencies by dividing, for each scan, the number ofanimals that were engaged in a given behaviour by the

total number of animals housed in that pen. The meanbehavioural, salivary and growth data were then processed,adopting the same statistical model used for the otherparameters.

A. Scollo et al. / Applied Animal Behaviour Science 153 (2014) 26–35 31

Fig. 2. Average latency to reach the bowl during testing for both the treat-ments, in the two reference locations (U = unrewarded; R = rewarded) andtN

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he three ambiguous locations (NU = nearest unrewarded; M = middle;R = nearest rewarded).

Skin lesions and vocalizations, reported as frequencyount data, were analyzed using a generalized estimatingquation using PROC GENMOD with a Poisson model.

. Results

.1. The cognitive bias task

All data are reported as back-transformed. At the endf the training all the pigs had learnt the discriminationetween the two reference locations, showing a signifi-ant effect of location (P < 0.01, F = 6.121, 109) in the latencyo approach the bowl. Animals took significantly longero reach the unrewarded location than the rewarded one42.5 s vs 4.2 s, respectively), without effect of housingreatment.

In the analysis of testing data, the difference in averageatency to approach the two reference locations (rewardednd unrewarded) was maintained, but there were no differ-nces between treatments in the average latency to reachhe bowl in the different ambiguous locations (P > 0.05,R: 5.3 s, F = 1.221, 111; M: 16.1 s, F = 0.091, 109; NU: 18.2 s,

= 1.881, 109) (Fig. 2). However, investigating the latenciesor each day of test, there was a significant effect of thereatment on the approach to the probe nearest the unre-arded location (NU) (P = 0.0027, F = 6.252, 109). During the

hird (and last) testing day, pigs with low space allowanceook longer to reach the bowl than pigs with high spacellowance (Adjusted Bonferroni P = 0.026, t = −3.211, 109;ow space allowance: 44.0 s; high space allowance: 15.6 s)Fig. 3).

No statistical difference was observed between treat-ents in the number of vocalizations emitted by the pigs

n 10 s before opening the weighing crate (P > 0.05, df = 1;R: �2 = 1.52, M: �2 = 0.32, NU: �2 = 1.50), with very high

ndividual variability.

.2. Behavioural and physiological measurements

For all the behavioural and physiological data, the inter-

ction between treatment and day of observation did nothowed any significant interaction. Animals housed withow space allowance were more frequently sitting than pigs

Fig. 3. Average latency to reach the bowl per testing day, for the threeambiguous locations (NU = nearest unrewarded; M = middle; NR = nearestrewarded). a,b P = 0.026.

with high space allowance (P = 0.01, F = 76.541, 2) and thiswas the only significant treatment effect (Table 3).

The average number of skin lesions recorded for eachtreatment is reported in Table 4. Pigs housed in pens withlow space allowance showed a greater total number oflesions (P = 0.035, df = 1, �2 = 4.44) and in particular at theear location (P = 0.009, df = 1, �2 = 6.66).

Pigs had no statistically significant differencesbetween treatments in their growth (high spaceallowance = 486.7 g/d; low space allowance = 448.4 g/d;P > 0.05, F = 1.471, 38) and in the salivary parameters(cortisol: high space allowance = 0.20 �g/dL; low spaceallowance = 0.19 �g/dL; P > 0.05, F = 0.661, 55. �-amylase,back-transformed data: high space allowance = 1.21 U/mL;low space allowance = 1.29 U/mL; P > 0.05, F = 0.021, 51).

4. Discussion

After training, pigs were able to discriminate betweenthe rewarded and the unrewarded locations, as indicatedby differences in the latencies to reach the bowl. This is in

32 A. Scollo et al. / Applied Animal Behaviour Science 153 (2014) 26–35

Table 3Average frequency of animals involved in each behaviour (%) for different space allowance treatments.

Behaviour High space allowance(%)

Low space allowance(%)

P-value SEM F-value1, 2

Sternal lying 44 45 0.91 4.7 0.01Lateral lying 23.9 20.4 0.48 2.8 0.74Sitting 5.3 3.2 0.01 0.2 76.54Standing up 13.4 17.9 0.49 3.8 0.72Sleeping 47 42 0.15 1.8 5.24Awake inactive 24.3 24.8 0.91 2.2 0.02Exploring environment 12.4 18.0 0.22 2.3 3.03Exploring chains 0.7 0.4 0.62 0.4 0.34Exploring nipple drinker 0.3 0.5 0.24 0.1 2.69Belly nosing 0.4 0.9 0.23 0.2 2.85

Conflicts 1.1 1.0Ears biting 0.7 0.8Tail biting 0.3 0.2

agreement with the response obtained using spatial loca-tion as a discriminatory stimulus in rats by Burman et al.(2008) and confirms the findings of Held et al. (2002) onthe well-developed spatial memory abilities of the pig. Assuggested by Burman et al. (2008), the lack of differences intraining performance between treatments indicates simi-lar levels of food motivation, spatial memory abilities andlocomotory behaviour despite the housing with differentspace allowances.

During testing, the discrimination between the tworeference locations continued to not be influenced bytreatment but, contrary to our predictions, there were nodifferences also in the average latency to reach the bowlin the three ambiguous locations. However, analysing thelatency for each testing day, the performance in relationto the probe nearest to the unrewarded (NU) was different.On the first testing day, the two treatments showed similarlatency, but the time taken by pigs housed with low spaceallowance increased up to the third day, to achieve a signifi-cant difference compared with pigs housed with high spaceallowance. These findings might be related to the effect ofemotional state on learning output and fall within a pos-sible confounding explanation for predicted biases tackledby Mendl et al. (2009). They discussed the possibility thatanimals in a negative state learn faster that the ambiguouslocations provide no reinforcement and thus are quicker tostop responding, versus the reverse hypothesis that a rela-

tively positive state is generally thought to have a beneficialeffect on learning abilities (Van Praag et al., 2000; Young,2003). Mendl et al. (2009) suggested that animals are influ-enced by stimuli previously received and then they make

Table 4Average number of lesions per pig for each anatomical area.

Body area High spaceallowance

Low spaceallowance

Face 0.2 0.3

Ears 1.2 2.2

Neck 0.5 0.9

Shoulder 0.2 0.5

Main body 0.1 0.5

Rump 0.5 1.1

Genitals 0.1 0.1

Tail 0.1 0.1

Total 2.9 5.7

0.44 0.1 0.920.33 0.1 1.600.69 0.1 0.22

the appropriate response to an ambiguous probe biasedby a ‘latent learning effect’. For example, animals withputative negative state might learn that unpredictableevents are usually negative, while those with putativepositive state might learn the opposite. According to thisassertion, pigs that in the present experiment were housedwith low space allowance might have learnt faster thatbowl nearest to the unrewarded (NU) location was non-reinforced and stopped responding to this sooner than pigshoused with high space allowance.

Although there was treatment difference in latencies onthe third day, and the evidence of a learned discriminationbetween the rewarded and the unrewarded locations, wefailed to observe similar differences in the number of voca-lizations. The lack of difference was found both in referencelocations and in probe locations, indicating that vocaliza-tions were not associated to the pigs’ anticipation of a foodreward as expected. There seemed to be a clear individ-ual difference between pigs, either they were vocalizers ornon-vocalizers as observed by Burman et al. (2008) in rats,and it is probable that this impeded a meaningful compar-ison.

In the analysis of behavioural and physiological mea-surements, pigs housed with low space allowance showeda greater sitting behaviour frequency and higher num-ber of ear lesions and total lesions. Regarding sittingbehaviour, results are in agreement with findings of Pearce

et al. (1989), who observed that pigs which experiencedunpleasant handling by humans during rearing spent moretime sitting than pigs handled pleasantly. They also inves-tigated growth performance and adrenal hypertrophy as

P-Value SEM Chi-Sq (1df)

0.684 0.4 0.170.009 0.1 6.660.202 0.3 1.630.102 0.4 2.680.073 0.4 3.210.203 0.4 1.620.272 0.6 1.210.381 0.5 0.770.035 0.4 4.44

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A. Scollo et al. / Applied Anima

hysiological indicators of reduced welfare, but no differ-nces were found between treatments, similarly to theresent study. Pearce et al., in agreement with Beattie et al.1996) and Wood-Gush and Beilharz (1983), suggested thathis result could indicate a chronic stress and that thedoption of an inactive ‘cut-off’ strategy through increaseditting may have protected the pigs from the physiologi-al consequences of unpleasant experience. Furthermore,he increased sitting behaviour could be also related to theow space allowance that, below the threshold of k = 0.046,oes not permit the concurrent full recumbent lying of allhe pigs (Petherick, 1983), forcing animals to modify theirehavioural pattern.

The greater number of ear lesions in pigs housed withow space allowance can be interpreted as the result of ago-istic behaviours that provoke retaliation, expressed withhe typical aggressive head-to-head interaction (Jensen,980). Results are in agreement also with Anil et al. (2007)ho showed that reduced space allowance resulted inigher injury scores in growing-finishing pigs.

The absence of differences in both salivary cortisolnd �-amylase between treatments is in agreement witheunier-Salaün et al. (1987), Pearce and Paterson (1993),

nd Warnier and Zayan (1985), who showed no differ-nce in plasma cortisol levels at different space allowances.owever, data in growing/finishing pigs are very limitednd it cannot be excluded that changes in these phys-ological parameters could be appreciable only above aertain overcrowding threshold. Von Borrell (1984), inn unpublished dissertation cited by the EFSA AHAWanel (2005), reported that the cortisol concentration inounger pigs tended to be higher for animals housed atery low space allowance (0.23 m2 per pig) than at higherpace allowances (0.30 m2 or 0.45 m2 per pig). Results ofhe present study suggest that the low space allowanceelected in the current experiment might have inducedltered behaviour and greater ear lesions, in accordanceith the literature, but was not sufficient either for higher

ortisol and �-amylase levels or for decreased growth per-ormance. In their meta-analysis, Gonyou et al. (2006)nvestigated the critical value of the allometric constantk) for defining space allowance which affected pig per-ormance. They identified a range from 0.0317 to 0.0348elow which the negative effects of crowding result in aecreased average daily gain. In the present study, the kalue used (0.030) was therefore relatively close to thehreshold suggested to result in decreased growth per-ormance. However, the low space allowance used in theresent study seems to be responsible for a difference in

earning ability, permitting some considerations.The relationship between the reduction in space

llowance and the onset of chronic stress is well knownSutherland et al., 2006; Kornegay et al., 1993), with chronictress also shown to be responsible for impaired spa-ial learning and memory deficits in rats and in primatesWright and Conrad, 2008). De Jong et al. (2000) statedhat the stress-induced shortfall in pig learning could be

aused by the variation in corticosteroid level; however,ven in the absence of an evident physiological response,hronic stress might be responsible for alterations in neu-al structures which are essential in spatial learning and

our Science 153 (2014) 26–35 33

memory (Conrad, 2010). Collectively there is clear evidencethat chronic stress can significantly alter hippocampusstructure in humans (McKittrick et al., 2000; Donohueet al., 2006; McLaughlin et al., 2005), result in cell death(Uno et al., 1989) and consistently activate the anteriorcingulate cortex in neuroimaging investigations of stressreactivity (Dedovic et al., 2009). In particular, the anteriorcingulate cortex seems to be involved in the facilitationof negative event learning in stress-vulnerable individuals.In a recent study conducted in humans (Cavanagh et al.,2011), a higher in learning rate on punishment trials wasshown in these subjects, defining the term ‘punishment’ asphenomena which decrease the probability of behaviouralresponse. In the present study, the unrewarded probenearest to the known unrewarded position could have rep-resented such a ‘punishment’ phenomenon.

Although data collected in the present study do notallow confirmation of a neural alteration caused by chronicstress due to a low space allowance, nor a clear associationbetween the k value selected and the onset of a sufficientchronic stress, it is possible that this condition might havefacilitated negative event learning. However, even if theresults obtained over the three testing days seem to suggesta different learning process between experimental groupsregarding the near unrewarded probe, there seems to beno clear demonstration of a pessimistic mood affectingcognitive bias. The absence of such a difference betweentreatments in cognitive bias, expected to show in the aver-age latency, may be because the stress of the reduced spaceallowance tested here was not sufficiently strong to inducea negative mental state, as similarly suggested by the phys-iological parameters. Furthermore, the training experienceitself could have impacted the influence of chronic stress(Ghiglieri et al., 1997) with undetermined influences fromrepeated training sessions.

5. Conclusions

An increase in space allowance above that cur-rently specified in EU legislation resulted in onlyminor behavioural change and no measured physiologi-cal response. The lower space allowance selected for thepresent study was not sufficient to cause a clear cognitivebias suggesting negative mental state. However, resultsobtained from analysis over the three testing days seemto show a different learning process between experimen-tal groups regarding the negative event of an unrewardedprobe; the hypothesis of a mild chronic stress-inducedalteration in the neural structures essential in spatial learn-ing and memory needs further investigation.

Acknowledgements

We are grateful to Jessica Gough, Alicja Rogusz andMark Brett for assistance in carrying out the experimen-tal work. Thanks to the pig unit staff of Cockle Farm,Newcastle University. Thanks also to Christopher Bulman

and Lorna Dawson for supervising the laboratory anal-ysis, and to Yolande Seddon for the friendly assistance.The present work was performed within the integratedproject QPorkChains FOOD-CT-2007-036245, financially

l Behavi

34 A. Scollo et al. / Applied Anima

supported by the EU under the Sixth Framework Pro-gramme for Research, Technological Development andDemonstration Activities. The content of the paper reflectsonly the views of the authors and the EU Commission isnot liable for any use that may be made of the informationcontained therein.

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