development of spatial memory in occlusion-experienced domestic chicks

0003

ANIMAL BEHAVIOUR, 2004, 67, 141e150doi:10.1016/j.anbehav.2003.03.015

Development of spatial memory in occlusion-experienced

domestic chicks

RAFAEL FREIRE*, HENG-WEI CHENG† & CHRISTINE J. NICOL‡

*Department of Animal Sciences, Purdue University

yUSDA, Animal Research Service

zUniversity of Bristol, Department of Clinical Veterinary Science

( Received 25 April 2002; initial acceptance 15 October 2002;

final acceptance 12 March 2003; MS. number: A9342R)

At around day 11 of life, domestic chicks show a tendency to move out of sight of their mother beforereturning and regaining social and visual contact. We conducted a series of experiments to investigate therole of this voluntary ‘out-of-sight’ behaviour on the development of spatial memory in young chicks. Wecompared the behaviour of chicks that were reared in environments that provided opportunities to moveout of sight of an imprinting stimulus (occlusion-experienced chicks) with the behaviour of chicks thatwere given minimal occlusion experience (controls). As in natural conditions, out-of-sight behaviourpeaked on day 11. When chicks were released into larger pens at 14 days of age, occlusion-experiencedchicks walked more than control chicks, but otherwise showed similar degrees of dispersal. Occlusion-experienced chicks tended to show better (although not significant, P ¼ 0:09) retrieval of a visuallydisplaced imprinting stimulus than control chicks. Time spent out of sight in the rearing pens wasnegatively related to the number of orientation errors in a detour test. Occlusion-experienced chicks alsotended to make fewer orientation errors in the first trial (P ¼ 0:07) and in subsequent trials (P ¼ 0:05). Incontrast, experimentally manipulating the amount of time that chicks were out of sight of an imprintingstimulus (by confining the chicks) had no effect on their performance in displacement or detour tests. Theresults presented here suggest that active experience of occlusion around day 11 improved egocentricorientation towards an out-of-sight goal, supporting the hypothesis that enrichment-induced behaviouralchanges are dependent on the interaction with objects.

� 2004 The Association for the Study of Animal Behaviour. Published by Elsevier Ltd. All rights reserved.

The notion that early environmental enrichment hasbeneficial effects on behaviour and brain structure hasreceived widespread interest. Scientific evidence for en-richment-induced behavioural and neural changes haswidespread applications in promoting child development(Carnegie Task Force on Meeting the Needs of YoungChildren 1994), improving recovery from brain damage(e.g. Stein & Glasier 1995), improving husbandry of ani-mals in zoos and farms (Mellen & MacPhee 2001) andensuring appropriate brain function and behaviour ofrodents used for research (Wurbel 2001). Spatial memoryand the corresponding neural systems, which maturerelatively late, appear particularly prone to enrichment-

Correspondence and present address: R. Freire, University of NewEngland, School of Biological Sciences, Centre for Neuroscience andAnimal Behaviour, Armidale NSW 2351, Australia (email: [email protected]). H.-W. Cheng is at the USDA-ARS, Poultry Building,Purdue University, West Lafayette, IN 47906, U.S.A. C. J. Nicol is atthe University of Bristol, Department of Clinical Veterinary Science,Langford House, Langford, North Somerset BS40 5DU, U.K.

141e3472/03/$30.00/0 � 2004 The Association

induced reconfiguration (Gould et al. 2000). Spatialmemory refers to the mental processes whereby spatialinformation about the surrounding environment isaccumulated, stored and retrieved to carry out spatialtasks. A large body of evidence has shown that exposureto an enriched environment (compared to a relativelybarren environment) improves spatial memory in humansand other mammals (e.g. Renner & Rosenzweig 1987;Sneddon et al. 2000). Interestingly, it may be that activelyinteracting with objects is necessary for the developmentof spatial memory (Renner & Pierre 1998).Identifying the critical environmental factors that

lead to enrichment-induced changes during developmentof spatial memory, however, has proved to be elusive(Rosenzweig et al. 1978). One exception is the recent workby Williams et al. (2001) in which mice that were reared ina ‘physically’ enriched environment (by adding nestingmaterial and toys) had improved performance in a watermaze compared with mice that were reared in socialgroups but without physical enrichment. AlthoughWilliams et al.’s (2001) study differentiated between the

for the Study of Animal Behaviour. Published by Elsevier Ltd. All rights reserved.

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ANIMAL BEHAVIOUR, 67, 1142

effects of social and physical factors on spatial memory, itdid not identify the critical factor in the physical environ-ment that gave rise to improved performance in the watermaze. Perhaps the change in locomotory patterns broughtabout by the presence of the toys or the disappearance andreappearance of toys as the mice moved around the cageand each other was the critical factor leading to improvedperformance in the water maze. It may be that enrich-ment has a nonspecific effect in reducing fear throughthe interaction with novel objects (as found in chicks;Candland et al. 1962), or increasing activity in a novelenvironment (as found in pigs; Wemelsfelder et al. 2000),both of which could lead to improvements in perfor-mance in the water maze. Clearly, identifying the criticalfactors in the development of spatial memory is method-ologically challenging and may have important applica-tions for ensuring complete spatial memory abilities incaptive animals.We propose that the domestic chicken, Gallus gallus

domesticus, provides a useful model for investigating thecritical factors in the development of spatial memory. Asa precocial animal, chicks hatch with well-developedbrains and rapidly learn a great deal about their environ-ment, including maternal (e.g. Bateson 1981) and foodrecognition (Nicol & Pope 1996). As in mammals, earlyvisual experience of the disappearance and reappearanceof objects (hereafter termed occlusion experience) iscrucial in the development of the ability to rememberthe location of hidden objects in birds (e.g. parrots,Pepperberg et al. 1997; magpies, Pollok et al. 2000). In thedomestic chick, improvement in search behaviour follow-ing visual displacement of an imprinting object was foundin occlusion-experienced chicks at 12 days of age but notat 4 days of age (Freire & Nicol 1999). The age effect in thedomestic chick may be related to a sensitive phase for thedevelopment of spatial memory at around 11 days of age.In brief, chicks reared in seminatural and indoor con-ditions show a sudden peak in moving out of sight of themother on day 11 (Workman & Andrew 1989; Vallortigaraet al. 1997) and a similarly timed viewing bias towards theleft eye (Dharmaretnam & Andrew 1994) and hence analmost exclusive involvement of the right hemisphere.The right hemisphere shows particular involvement intopographical orientation and spatial reasoning in chicks(Rashid & Andrew 1989; Andrew 1991) and the peak inout-of-sight behaviour, the left-eye viewing bias and righthemispheric dominance on day 11 suggest a period ofeffective spatial learning. Thus, the domestic chicks’precocial nature including the ability to imprint on aninanimate object and the apparent sensitive phase fordevelopment of spatial memory at around 11 days makesthe chick an excellent model for investigating the de-velopment of spatial memory.Additionally, considerable scientific effort is currently

directed towards improving farm animal welfare incommercial production systems. Such effort aims toimprove the match between the animal’s expectation ofthe environment and the actual environment, either bymodifying the animal through genetic selection (e.g. Muir& Craig 1998) or by modifying the environment (e.g.cages for hens, Appleby et al. 2002). Important beneficial

changes in behaviour have been achieved through en-richment during rearing of captive cats (Shepherdsonet al. 1993) and rats (reduced physiological stress inadulthood even after a short enrichment period; Anismanet al. 1998). However, the possibility of shaping behaviourduring development, so that the animal has a bettermatch with its environment has attracted relatively littleattention (but see Sneddon et al. 2000 for an exception).One possible benefit of enrichment-induced improvementin spatial memory is in reducing the problems associatedwith poor navigation and uneven distribution of chickensin large groups (see Introduction of experiment 1).

In the following series of experiments, we examined theeffect of occlusion experience on development of spatialmemory in domestic chicks. Chicks were not sexedbecause both sexes move out of sight on day 11(Workman & Andrew 1989), and have previously beenshown to succeed in visual displacement (Vallortigaraet al. 1998) and detour tests (Regolin et al. 1995). Oneadvantage of not sexing chicks is that it approximatesmore closely the method used in commercial rearing ofbroilers, which rarely involves separation of the sexes. Inexperiment 1, we sought to determine whether providingchicks with the opportunity for occlusion experienceduring development (1e14 days of age) influenced theirsubsequent behaviour and distribution when they werereleased into larger pens. We provided the chicks oppor-tunities for occlusion experience by allowing them tomove voluntarily out of sight behind opaque screenswithin the rearing pen. In experiment 2, we determinedwhether the development of spatial memory, assessedusing displacement and detour tests, differed betweenchicks that were given occlusion experience and chicksthat were given minimal occlusion experience. Additionalcontrols for the effects of the screens were included in thisexperiment. Lastly, we experimentally manipulated oc-clusion experience to investigate its effect on spatialmemory (experiment 3). To our knowledge, this procedureallows a hitherto unattained degree of reduction of thepossible critical experiential factors associated withenrichment-induced changes in spatial memory.

EXPERIMENT 1

The aim of experiment 1 was to examine the hypothesisthat dispersal in a large pen is dependent on the adequatedevelopment of spatial memory. In large group-produc-tion systems, laying hens sometimes crowd near the wallsand corners, leading to localized overcrowding that canincrease mortality due to smothering (Channing et al.2001). Additionally, some laying hens in large groupsystems may have difficulty relocating specific areas(Newberry & Hall 1990). It may be that the clumpeddistribution patterns and poor relocation observed arosebecause birds did not develop the appropriate cognitiveskills to navigate adequately in the centre of a pen. Theaim of experiment 1 was to determine whether occlusion-experienced birds (1) show less clumping, (2) move moreand (3) use the centre of the pen more than birds rearedwith minimal occlusion experience.

FREIRE ET AL.: SPATIAL MEMORY IN CHICKENS 143

Methods

The subjects were 32 unsexed broiler chicks obtainedfrom a commercial hatchery (Pine Manor Hatchery,Indiana, U.S.A.) at 1 day of age. Chicks were randomlypaired and placed in a cardboard box (55 ! 40 ! 60 cmhigh) with wood shavings and ad libitum access to foodand water. A water dispenser (10 cm diameter, 20 cm high)was placed in one corner so that birds could not walkaround it, and starter crumbs were placed in a shallow dish(10 cm diameter, 4 cm high). Temperature was maintainedat 30 (C and lighting was provided by 60-W incandescentbulbs on a 12:12 h light:dark cycle. When chicks were 8days old, we placed two wooden screens (20 ! 20 cm) ineight randomly chosen boxes, parallel to the shorter sidesof each box and 20 cm apart, to provide maximumexperience of occlusion (Treatment MO). Screens werenot added to the remaining eight ‘empty’ boxes (Treat-ment E). When chicks were 14 days old, we moved eachpair into a larger enclosure (370 ! 240 cm), which hadone wooden side and three wire sides. One side of theenclosure provided a view of a central corridor, and theother two sides provided views of identical enclosureswith two chicks (although for two enclosures, the view toone side was of an empty enclosure). A feeder was placedin the centre of the pen (diameter 40 cm, 60 cm high) anda water dispenser (diameter 35 cm, 20 cm high) wassuspended centrally and 100 cm from the feeder. A camerawas placed above the middle of each pen and was used torecord chick behaviour during the light periods on days14, 21, 28, 35 and 42. We conducted 1-min scan samplingsfor 5 min beginning on each hour to record the distanceof each chick from the feeder and its previous location (asmarked on the monitor), and the distance between eachchick. We used a blind procedure to record chick be-haviour by allocating each pen that was displayed onthe video recording a random number from 1 to 16.Corresponding rearing treatments were not disclosed untilcompletion of the behavioural observations. One chickdied at 40 days of age, so we did not collect data from thispen at 42 days of age. The data conformed to therequirements for parametric analysis and were analysedby a repeated measures analysis of variance (ANOVA)using a split-plot design, with rearing treatment asa between-subjects comparison and age as a within-sub-ject comparison (SPSS Base 10.0, SPSS 1999). Because thetwo chicks within a pair were not independent, we usedpairs as the unit of replication.

Results and Discussion

Chicks that had been given the opportunity toexperience maximum occlusion between 8 and 14 daysof age (Treatment MO) moved more when they werereleased into a large pen than chicks that had received noocclusion experience at 8e14 days of age (Treatment E)(ANOVA: F1;14 ¼ 11:9, P!0:01; Fig. 1). However, differ-ences in movement were not observed at 28 days of age orthereafter (ANOVA: treatment)age interaction: F4;55 ¼ 5:6,P!0:01). Indeed, movement in all birds declined rapidlywith age as is typical for broiler chickens due to fast

growth rate (ANOVA: age effect: F4;55 ¼ 14:0, P!0:0001;Preston & Murphy 1989). Mean distance from the centreof the pen when not feeding did not differ betweentreatments (XGSE: Treatment MO Z 88:3G11:8 cm;Treatment E Z 95:9G11:4 cm; ANOVA: F1;14Z 1:2, NS).Similarly, interbird distances did not differ between treat-ments (XGSE: Treatment MO Z 32:9G4:1 cm; TreatmentE = 29:2G4:5 cm; ANOVA: F1;14Z2:5, NS).In the following experiments, we investigated further

whether the increased activity of occlusion-experiencedchicks in the larger pens arose from improved knowledgeand skills for navigating in a large pen. First, we testedother chicks with differential experience of occlusionusing two spatial tests. Second, the addition of opaquescreens during rearing provided many changes in stimu-lation, making it difficult to determine whether experi-ence of occlusion was the critical factor. One possibility isthat the change in movement patterns induced by addingthe screens improved coordination, leading to increasedlocomotion once the chicks were released into the largepens. In the following experiments, we used additionalrearing treatments to reduce the possible causal factorsthat may be important in the development of spatialmemory. Furthermore, because the commercial practice ofobtaining day-old chicks from a hatchery made it difficultto determine what stimulation the chicks had receivedbefore their arrival, we obtained fertile eggs and hatchedchicks in our laboratory for the following experiments.

EXPERIMENT 2

The aims of experiment 2 were to (1) verify that day 11 isthe peak age at which chicks move out of sight of animprinting stimulus and (2) test the hypothesis thatocclusion experience at around 11 days of age improvesspatial memory. The tests of spatial memory were chosento be within the known capability of domestic chicks. Avisual displacement test involved confining the chick ina small cage and displacing the imprinting stimulusbehind one of two opaque screens. Once released, thechick’s retrieval of the displaced imprinting stimulus

0

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50

60

70

14 21 28 35 42

Age (days)

Dis

tan

ce m

oved

/min

(cm

) Treatment MOTreatment E

Figure 1. Mean G SE distance moved per minute by birds provided

with maximum experience of occlusion (Treatment MO) and limited

experience of occlusion (Treatment E).


indicates some memory for the stimulus and its location.A detour test involved placing the chick behind a clearscreen in view of the imprinting stimulus such thatcontact with the imprinting stimulus was possible only byleaving sight of it and walking around an occluding screen(Regolin et al. 1995).

Methods

The subjects were 48 broiler chicks (unsexed chicks,Pine Manor Hatchery) obtained as fertile eggs at 18 days ofincubation. Chicks were reared in isolation from about 2 hafter hatching to 7 days of age in a cardboard box(30 ! 25 ! 30 cm high). A yellow tennis ball was sus-pended by a string 10 cm above the floor in the centre ofthe box to provide an imprinting stimulus (broiler chickshave previously been found to imprint on tennis balls;Freire & Nicol 1999). Temperature was maintained at35 (C and lighting was provided on an LD 12:12 h cycle.To encourage pecking and eating, the floor of the box waslined with white paper and sprinkled with chick startercrumbs that we periodically tapped with a round dowel.Additional starter crumbs and water were available adlibitum from clear Perspex petri dishes.When chicks were 8 days old, we paired them, marking

one chick in each pair with blue spray paint on the back,and randomly assigned each pair to one of four rearingtreatments. Six pairs were reared in ‘empty’ boxes with animprinting stimulus suspended in the middle of the box(Treatment E). In the other three treatments, the rearingboxes contained two screens (20 ! 20 cm), which pro-vided varying degrees of occlusion depending on the typeof screen used, placed centrally and parallel to the shorterside of the box, 10 cm from the imprinting stimulus in themiddle of the box. For chicks that received ‘maximumocclusion’ (Treatment MO, N ¼ 6 pairs), the screens wereopaque and made of wood (0.5 cm thick). For chicks thatreceived ‘some occlusion’ (Treatment SO, N ¼ 6 pairs),one screen was wooden and the other was transparent(0.3-cm-thick acrylic sheet). For chicks that received‘minimal occlusion’ (control, N ¼ 6 pairs), both screenswere transparent acrylic. All boxes measured 55 !40 ! 60 cm high and contained wood shavings. Foodand water were provided ad libitum as in experiment 1.Temperature was maintained at 30 (C and lighting wasprovided by 60-W incandescent light bulbs on an LD12:12 h cycle. Chicks were videotaped from above duringthe entire 12-h light phase on days 10, 11 and 12. Weconducted scan samplings every 15 min during the lightphase to record whether a chick was out of sight of eitherthe imprinting stimulus or the other chick.When chicks were 13 days old, they received two tests

in a balanced order. Chicks were placed in their rearingpen for at least 3 h between each test. Chicks werevideotaped from above during the tests to record behav-iour. We used a blind procedure to record chick behaviourby allocating each chick that was displayed on the videorecording a random number from 1 to 48. Correspondingrearing treatments were not disclosed until completion ofthe behavioural observations.

Visual displacement testThe test apparatus consisted of a circular plastic arena

(90 cm in diameter, 70 cm high) with wood shavings onthe floor (Fig. 2). A circular wire start cage (20 cmdiameter, 30 cm high) was placed at one end of the arenaand two opaque screens (wooden, 20 ! 20 cm) wereplaced 20 cm to the right and left of the centre (point Yin Fig. 2) and 40 cm from the start cage. Before each test,we placed a tennis ball behind one of the screens (with thedirection, left or right, balanced between treatments). Atthe start of each test, we confined a chick in the start cagein view of a suspended tennis ball (at point X on Fig. 2).When the chick was oriented towards the ball, we movedthe ball away from the chick (to point Y on Fig. 2), thenplaced it behind the other screen. We released the chickfrom the start cage and recorded its response as ‘correct’ ifit approached to within 5 cm of the displaced ball or‘incorrect’ if it approached the other ball. A test wasdeemed invalid if a chick did not approach either ballwithin 5 min. The outcome (correct, incorrect or invalid)and the time taken from release to approach of a ball werenoted for each chick (identified from the assigned numberfrom the video recordings).

Detour testThe test apparatus consisted of an 80 ! 80-cm square

pen 70 cm high, with wood shavings on the floor (Fig. 3).A tennis ball was suspended centrally on one side of theapparatus and four wooden posts (10 ! 10 cm) wereplaced 15 cm apart in the centre of the apparatus. Thestart position of the chick was between two wooden postswith an acrylic screen (0.3 cm thick) between the chickand the ball. To reach the ball, the chick had to movearound one of the posts. When the chick approached towithin 5 cm of the imprinting stimulus, it was allowed toremain there for 15 s before being returned to the startposition. Each chick was displaced four times (and thusreceived five detour trials). If a chick failed to reach theball within 25 min, the test was terminated and the chickwas returned to its rearing box. From the overhead videorecordings, we noted the time taken to approach the ballfor each trial. An orientation error was counted if a chickthat was out of view of the ball moved behind the acrylic

Y

Start cage X

Ball start position

Figure 2. Plan of the visual displacement apparatus. The chick was

confined in the start cage while the imprinting object was moved

from its start position X to point Y and then made to disappearbehind one of the screens.


screen to view the ball, or if a chick moved away from theball (i.e. approached or crossed the dashed line in Fig. 3).

Analysis

Latencies to reach the correct imprinting stimulus in thedisplacement test were normally distributed following logtransformation. Success rate in the displacement test wasanalysed in a 4 ! 2 table, with four rearing treatments andeither a correct category or an incorrect pooled category(incorrect and invalid outcomes combined). The purposeof the above pooling of categories for chi-square analysiswas to reduce the number of small (!5) observedfrequencies to below 20% as required for this test (SPSSBase 10.0, SPSS 1999). Detour latencies conformed to therequirements for parametric analysis following log trans-formation and were analysed by a repeated measuresANOVA using a split-plot design, with rearing treatmentas a between-subjects comparison and trial as a within-subject comparison (SPSS Base 10.0). The number of errorsmade during detour trials did not conform to parametricrequirements, so we analysed the data for the first trial(representing spontaneous detour behaviour) and latertrials (i.e. mean number of errors from trials 2, 3, 4 and 5for each chick) using the KruskaleWallis test.Test order (displacement test versus detour test) had no

effect on the outcome in the displacement test (compar-ing the first and second tests: 12 and 9 chicks chosecorrectly, 4 and 1 chicks chose incorrectly and 8 and 14chicks had invalid tests, respectively; chi-square test:c22 ¼ 3:9, NS). We also found no evidence that test order

affected the chicks’ latency to approach the correctdisplaced imprinting object (XGSE: first testZ 87:3G22:8 s; second testZ 43:7G23:6 s; ANOVA: F1;20Z2:2,NS). Similarly, test order had no effect on detour latency(XGSE: first test Z 110:3G16:2 s; second testZ 88:6G16:2 s; ANOVA: F1;39Z1:1, NS) or on the number of errorsmade (median and interquartile range: first testZ 1.0(0e2.5), second testZ 1.0 (0e1.8); ManneWhitney Utest: UZ191:5, N1Z20, N2Z21, NS). Because we found noevidence that order of presentation of the tests had

Chick start position

Ball

X Transparent screen

Figure 3. Plan of the detour test apparatus. The chick was placed atX in view of an imprinting object behind a transparent acrylic screen.

Once the chick had moved out of view, each crossing or moving

towards the dashed lines was recorded as an orientation error.

a significant effect on any of the variables, we omittedorder effect from further analysis.


Out-of-sight behaviour in rearing pensChicks frequently moved around the wooden and

transparent screens, and in this respect, the woodenscreens proved to be effective in providing experience ofocclusion. We expected occlusion both of the imprintingstimulus and of the other chick to be most important inlearning about occlusion. Therefore, we compared thepercentage of time that each chick spent out of sight ofeither the imprinting stimulus or the other chick in MOand SO treatments using the mean percentage of timespent out of sight for each box (since pairs of chicks werenot independent). The time out of sight of either theimprinting stimulus or the other chick was greater inTreatment MO than in Treatment SO (ANOVA: F1;10 ¼25:8, P!0:001; Fig. 4). Percentage of time spent out ofsight was also greater on day 11 than on days 10 or 12(F2;20 ¼ 20:2, P!0:001; Fig. 4). The peak in out-of-sightbehaviour on day 11 was also greater in Treatment MOthan in Treatment SO (treatment)day interaction:F2;20 ¼ 5:0, P!0:05; Fig. 4).

Displacement testAll chicks that moved past the screens approached an

imprinting stimulus within 5 min. In contrast, all chicksthat failed to move past the screens within 5 min (invalidresponses) never moved within sight of either ball. Allchicks that made invalid responses also produced locali-zation calls, suggesting that their failure to approach a ballwas not the result of a lower motivation to reach theimprinting stimuli.There was a nonsignificant tendency for Treatment MO

chicks to perform better in the displacement test thanchicks from the other three treatments (chi-square

0

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40

50

60

70

80

10 11 12Days after hatching

% T

ime

out

of s

igh

t

Treatment MOTreatment SO

Figure 4. Mean G SE percentage of time that chicks provided withone (Treatment SO) or two (Treatment MO) occluding screens spent

out of sight of either the imprinting stimulus or the other chick at 10,

11 and 12 days of age.


test: treatment versus success rate: c23 ¼ 6:3, P ¼ 0:09;

Fig. 5). Because only two of the chicks that were reared inTreatment E correctly reached the displaced imprintingstimulus, we combined Treatment E and Treatment C(since both provided minimal occlusion experience) forthe purpose of analysing the latency to approach thecorrect imprinting stimulus. The latency to reach thecorrect imprinting object did not differ significantlybetween treatments (XGSE: Treatment MO Z 49:0G25:6 s; Treatment SOZ 68:7G34:7 s; Treatments C and Ecombined Z 91:0G30:6 s; ANOVA: F2;18Z1:2, NS). Thus,we found no evidence that latency to reach the correctimprinting stimulus was influenced by rearing treatment.

Detour testMost chicks successfully completed five detour trials,

with only two chicks from each of Treatments MO, SOand E and one chick from Treatment C failing to approachthe imprinting stimulus. All chicks that gained anunobstructed (i.e. not through the acrylic screen) view ofthe imprinting object approached it in all five trials. Incontrast, the seven chicks that did not complete the trialsfailed to obtain an unobstructed view of the imprintingstimulus. The chicks that did not complete the trials gavelocalization calls and showed some pacing while facingthe acrylic screen, suggesting that they were motivated toreach the imprinting stimulus.No difference in latency to approach the imprinting

stimulus was found between treatments (ANOVA: treat-ment: F3;37 ¼ 1:8, NS). Latency to approach the imprint-ing stimulus was generally lower in subsequent trials, andparticularly between the first two trials and the remainder(ANOVA: trial: F4;148 ¼ 28:5, P!0:0001; Fig. 6). No evi-dence was found that the general decline in latency insubsequent trials was differentially influenced by treat-ment (ANOVA: treatment)trial interaction: F12;148 ¼ 0:7,NS). Thus, we found no evidence that rearing treatmentinfluenced the latency to detour around a post to reach animprinting stimulus.The difference in the number of orientation errors made

by chicks in each treatment during their first andsubsequent trials approached significance (P ¼ 0:070 and0.050, respectively), with Treatment MO chicks makingthe least number of errors (Table 1). Furthermore, forTreatments MO and SO, chicks that spent more time outof sight on day 11 in the rearing boxes made fewer

Figure 5. Outcome of displacement test for chicks reared inmaximum occlusion (MO), some occlusion (SO), control (C) and

empty (E) treatments. -: Correct ball approached; ,: incorrect ball

approached; G: neither ball approached (invalid).

orientation errors in their first trial (Spearman rankcorrelation: rS ¼ �0:58, N ¼ 20 (four chicks failed toapproach the imprinting object), P!0:01; Fig. 7). How-ever, there was no significant correlation with the meannumber of errors made in subsequent trials (Spearmanrank correlation: rS ¼ 0:03, N ¼ 20, NS). The formercorrelation analysis was probably strongly influenced bythe two chicks that showed two and four orientationerrors in the first trial (Spearman rank correlation omittingthese two points: rS ¼ �0:41, N ¼ 18, P!0:05; Fig. 7). Afurther comparison was provided by comparing the timeout of sight for chicks that showed no errors (N ¼ 12) withthat for chicks that made at least one error (N ¼ 8). Chicksthat showed no errors on their first detour trial spent moretime out of sight on day 11 than chicks that made at leastone error (XGSE percentage of time out of sight: 59:3G6:2% and 32:7G8:7%, respectively; ANOVA: F1;18 ¼ 6:5,P!0:05).

Occlusion-experienced chicks tended to perform betterin displacement and detour tests, suggesting improvedspatial memory relative to control chicks. The use of clearscreens as a control allowed us to eliminate the possibilitythat the experience of moving around screens led to thechanges in performance in the spatial memory tests.

0

100

200

300

400

500

600

Trial 1 Trial 2 Trial 3 Trial 4 Trial 5

Mea

n la

ten

cy (

s)

MOSOCE

Figure 6. MeanG SE latency to approach the imprinting stimulus in

the detour test for chicks reared in maximum occlusion (MO), someocclusion (SO), control (C) and empty (E) treatments.

Table 1. Median (interquartile range) number of orientation errorsmade during the first and subsequent detour trials for chicks fromdifferent rearing treatments, and results of the KruskaleWallis test

Treatment

Errors

NTrial 1 Trials 2e5

MO 0 (0e0.3) 0 (0e0.75) 10SO 1.0 (0e1.3) 0.1 (0e0.6) 10C 1.0 (0e2.0) 0.3 (0e0.5) 11E 1.0 (0e1.0) 0.8 (0.5e0.9) 10

KruskaleWallis, c23 7.0 7.6

P 0.070 0.050

MO: Maximum occlusion; SO: some occlusion; C: control; E: empty.


Therefore, we next sought to determine which aspects ofocclusion experience, as determined by the chicks’ ownbehaviour (e.g. exploration of unseen areas of the pen,their time spent out of sight of the imprinting stimulusand their control over occlusion events), were critical inthe development of spatial memory.

EXPERIMENT 3

The aim of experiment 3 was to investigate whether oneaspect of occlusion, the time that the imprinting stimulusis out of view (i.e. occlusion time), is a critical factor in thedevelopment of spatial memory. Chicks were restrained ina circular wire cage and occlusion time was manipulatedby placing either a wooden screen or a clear screenbetween the chicks and the imprinting stimulus. Dis-placement and detour tests were used to assess spatialmemory as in experiment 2.

Methods

The subjects were 36 unsexed broiler chicks, incubatedand reared in isolation with a tennis ball until 7 days ofage as in experiment 2. At 8 days of age, chicks were pairedand placed in a box measuring 55!40!60 cm high. Theposition of the imprinting stimulus, feeder and waterdispenser, and temperature and lighting were as inexperiment 2. On days 9, 10, 11 and 12 posthatching,chicks were restrained in a circular wire cage for four 1-hperiods, starting at 0900, 1200, 1500 and 1800 hours(lights on at 0800 hours). The circular cage with the chickswas placed in the box adjacent to one of the short sides.Twelve chicks were randomly assigned to one of threetreatments as follows. (1) A wooden screen (20!20 cm)

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60

70

80

90

100

Number of errors

Tim

e ou

t of

sig

ht

(%)

0 1 2 3 4

Figure 7. The relationship between the percentage of time spent

out of sight on day 11 and the number of orientation errors made bychicks in the first detour trial.

was placed between the restrained chicks and theimprinting stimulus during the four restraining periodsso that the imprinting stimulus was completely occludedfrom view of the chicks for 4 h (Treatment F). (2) Awooden screen was placed between the chicks and theimprinting stimulus during two restraining periods, anda transparent acrylic screen (20!20 cm) was placedbetween the chicks and the imprinting stimulus duringthe other two restraining periods so that the imprintingstimulus was completely occluded for 2 h (Treatment T).The choice of screen at each period was determined bya semirandom sequence, with the same type of screenapplied to all Treatment T chicks at each restrainingperiod. (3) A transparent acrylic screen was placed be-tween the chicks and the ball during the four restrainingperiods (Treatment C, control).At 13 days of age, chicks received a visual displacement

and a detour test in a balanced order as in experiment 2.We again used a blind procedure to record behaviour byallocating each chick that was displayed on the videorecording a random number from 1 to 36. Correspondingrearing treatments were not disclosed until completion ofthe behavioural observations.

Analysis

The latencies to reach the imprinting object in thedisplacement and detour tasks were again log transformedto meet the requirements for parametric analysis, andwere analysed as in experiment 2. The order of pre-sentation of the displacement and detour tests had noeffect on the outcome in the displacement test (compar-ing the first and second tests: 10 and 11 chicks chosecorrectly, with 7 and 6 invalid tests, respectively; chi-square test: c2

2 ¼ 0:13, NS). We found no evidence thattest order affected the chicks’ latency to approach thecorrect displaced imprinting object (XGSE: firsttest = 88:4G22:6 s; second test Z 72:8G33:4 s; ANOVA:F1;20Z3:4, NS). Similarly, test order had no effect ondetour latency (XGSE: first test Z 144:8G25:8 s; secondtestZ 109:2G17:7 s; ANOVA: F1;29Z0:5, NS) or on thenumber of errors made (median and interquartile range:first test Z 0.5 (0.2e1.5); second test Z 0.5 (0.2e0.8);ManneWhitney U test: UZ100:5, N1Z14, N2Z16, NS).Because we found no evidence that order of presentationof the tests had a significant effect on any of the variables,we omitted order effect from further analysis.


Displacement testAs in experiment 2, all chicks that moved past the

screens approached an imprinting stimulus within 5 min.We found no evidence that treatment influenced successrate (chi-square test: c2

2 ¼ 0, NS; Fig. 8). Latency toapproach the correct imprinting object did not differsignificantly between treatments (XGSE: TreatmentFZ 72:1G29:5 s; Treatment TZ 115:6G41:7 s; TreatmentCZ 53:0G33:2 s; ANOVA: F2;18Z1:2, NS).


Detour testAll but two chicks approached the imprinting stimulus

within 25 min in the first trial, and four chicks thatapproached the imprinting stimulus in the first trial failedin subsequent trials. We found no evidence that treatmentaffected the number of failures to approach the imprintingstimulus, with two, three and one failures for treatmentsF, T and C, respectively (chi-square test: c2

2 ¼ 1:9, NS).Chicks were generally faster in approaching the imprint-ing object in subsequent trials (ANOVA: trial effect:F4;113 ¼ 34:5, P!0:0001). Rearing treatment had no effecton the latency to approach the imprinting stimulus(ANOVA: treatment effect: F2;34 ¼ 2:4, NS; ANOVA: treat-ment)trial interaction F8;113 ¼ 1:4, NS). The number oforientation errors made in the first trial did not varysignificantly between Treatments F, T and C (median andinterquartile range: 1.0 (0e2.0), 1.0 (0e2.0) and 2.0(1.0e3.0), respectively; KruskaleWallis test: H2 ¼ 2:5,NS). Similarly, treatment had no effect on the meannumber of orientation errors made in trials 2, 3, 4 and 5(Treatment FZ 0.3 (0e0.8), Treatment TZ 0.5 (0e1.1),Treatment CZ 0.3 (0.3e0.8); KruskaleWallis test: H2 ¼0:9, NS). Thus, we found no evidence that treatment hadany significant effect on response in the detour test.

GENERAL DISCUSSION

In summary, we found that the amount of time thatchicks spent out of sight of the imprinting object andother chicks peaked on day 11. In experiment 1, chicksreared in an environment providing the opportunityto experience occlusion moved more following releaseinto a larger pen than chicks reared in an environmentproviding minimal experience of occlusion. In experiment2, chicks that spent more time out of sight in the rearingpens made fewer orientation errors in the detour test thanchicks that spent relatively less time out of sight.Additionally, chicks given the opportunity to experienceocclusion tended to perform (nonsignificantly) better inthe displacement test (P ¼ 0:09), and made fewer orienta-tion errors in the first (P ¼ 0:07) and subsequent (P ¼0:05) detour trials than chicks reared with minimalexperience of occlusion. In our opinion, the above non-

Figure 8. Outcome of displacement test for chicks provided with 4,

2 or 0 h per day out of sight of the imprinting stimulus. -: Correct

imprinting object approached; ,: incorrect imprinting objectapproached; G: neither imprinting object approached (‘invalid’).

significant finding merits further discussion since all threetests suggested that occlusion-experienced chicks orientedbetter towards an out-of-sight goal than did chicks rearedwith minimal experience of occlusion. In contrast, noevidence was found that passive experience of occlusionimproved performance in visual displacement and detourtests (experiment 3). The question of whether chicks’success in visual displacement and detour tests involvesspatial memory is the subject of considerable research (e.g.Vallortigara et al. 1998; Freire & Nicol 1999). Vallortigaraet al. (1998) found that chicks could solve successivevisual displacement tests even after an occluding screenblocked the view of possible hiding locations for 60 s,suggesting that chicks form a representation independentof local cues. They concluded that the chick’s ability tocontinuously update the content of the representationfrom trial to trial fits the description of ‘working memory’in mammals. More recently, impairment in relocationbehaviour in chicks following specific lesions of thehippocampus (Tommasi et al. 2000) supports the assertionthat there are similar spatial memory processes in birdsand mammals (Colombo & Broadbent 2000). Addition-ally, spontaneous detour behaviour (i.e. the first trial) doesnot rely on local cues but instead is based on the chick’suse of egocentric coordinates, which again implies someform of memory for an out-of-sight goal (Regolin et al.1995). In the remainder of the discussion, therefore, weconsider the role that performing out-of-sight behaviourplays in the development of ‘spatial memory’.

Our finding of a peak in out-of-sight behaviour on day11 complements Vallortigara et al.’s (1997) finding ofa similar timed peak in moving out of sight of a motherhen in various environments. Because the present studyused an imprinting object, we can rule out the possibilitythat out-of-sight behaviour is dependent on the mother. Itis worth stressing that out-of-sight behaviour may notarise from a motivation to experience visual occlusion, butcould arise from a different motivation such as to exploreunseen areas of the pen so that visual occlusion is anincidental effect. It should also be stressed that althoughwe made an effort to minimize experience of occlusion inthe control and empty rearing treatments, chicks may stillhave obtained some limited experience of occlusionbehind the other chick or imprinting object.

In experiment 1, chicks reared with the opportunity toexperience occlusion moved more following release intoa larger pen than control chicks. Insertion of two woodenscreens into the chicks’ pen increased the complexity ofthe pens (relative to an identical environment withoutwooden screens). Similar high levels of activity followingrelease into a novel environment have been found inother animals such as rats (Zimmermann et al. 2001) andpigs (Wemelsfelder et al. 2000) reared in relativelycomplex environments. The latter study concluded thatenrichment affected the pigs’ propensity to explore andinteract with their environment. In the present study,treatment differences in activity persisted for at least 7days, suggesting that the effect of rearing in a relativelycomplex environment was reasonably persistent, al-though activity levels ultimately decreased. Gunnarssonet al. (2000) also found that chickens given early access to


perches showed increased dispersal, which was attribut-able to the more complete development of spatialcognition arising from an increase in the use of highperches relative to birds without early access to perches. Inthe present study, there was no effect of treatment on thechicks’ use of the centre of the pen or on interbirddistances, suggesting that dispersal is not simply related tospatial memory, but is likely to incorporate other factorssuch as the degree of concealment (Newberry & Shackle-ton 1997). Thus, practical measures to encourage moreeven dispersal in birds in large groups should address allcontributing factors.In experiment 2, occlusion-experienced chicks tended

to perform better (although P ¼ 0:09) than chicks rearedwith minimal experience of occlusion. Improvement inthe displacement test may however arise due to somecharacteristic other than improved spatial memory. It maybe that chicks in Treatments C and E were more fearful ofthe wooden screens in the displacement test because thescreens were seen for the first time, and that fearfulnessinterfered with performance in the test (as has been foundin chicks in other tests; Candland et al. 1962). Severalfindings presented here contradict the hypothesis thatincreased fearfulness impaired performance in the tests.(1) Treatment SO chicks in experiment 2 had someexperience with the wooden screens but did not performas well as Treatment MO chicks in the displacement test.(2) Chicks that had no experience with the clear screens(Treatments MO and E) did not perform worse in thedetour test even though these chicks saw the clear screensfor the first time during the detour test. To complicatematters further, explanations for impaired performancebased on impaired spatial memory or increased fear arisingfrom seeing the wooden screen for the first time are notnecessarily mutually exclusive. It may be that fear inTreatment C and E chicks was elicited by the imprintingstimulus moving out of sight behind the wooden screensince this would have been the first experience of occlusionof the imprinting stimulus. To clarify, it could be thatocclusion of the imprinting object (whichwould have beena new event), rather than the occluding screen, increasedfear in chicks reared in pens without wooden screens andimpaired performance in the displacement test.Experiment 2 showed that time spent out of sight was

negatively related to the number of orientation errors inthe detour test. Additionally, rearing treatment (non-significantly) influenced the number of orientation errorsin the first (P ¼ 0:07) and subsequent (P ¼ 0:05) detourtrials. In rats, egocentric movement appears to be encodedby cells in the hippocampus that respond to the animal’sdirection of movement (direction cells; Taube 1998).Given the avian and mammalian similarities in theinvolvement of the hippocampus in spatial memory(Colombo & Broadbent 2000), similar responding di-rection cells may be present in the chick brain. Consid-ering that moving towards an out-of-sight imprintingstimulus in the rearing pens and detour test presumablyinvolves similar egocentric orientation processes, perfor-mance of the former may lead to improvements in thelatter. These findings suggest that the action of movingtowards an out-of-sight stimulus, rather than merely

viewing the stimulus being moved out of sight (as inexperiment 3), is instrumental in the development ofegocentric orientation. The above findings are in agree-ment with the hypothesis that interaction with objectsgives rise to enrichment-induced changes (Renner &Rosenzweig 1986; Renner 1987; Wemelsfelder et al.2000). The importance of occlusion experience in ego-centric orientation is further supported by our finding thatchicks with more (as opposed to less) occlusion experienceon day 11 made significantly fewer orientation errors inthe first detour trial. However, occlusion per se may not bethe relevant part of active occlusion experience. Activeexperience of moving out of sight involves many aspects,such as frequent reuniting with the imprinting stimulusand control over occlusion events compared with a lack ofcontrol over such events and time spent out of sight of theimprinting stimulus. Although the results of experiment 3showed that time spent out of sight of the imprintingstimulus was not the critical factor involved in thedevelopment of spatial memory, it may be relevant incombination with some other aspect of occlusion. Furtherexperiments are needed to determine which types ofexperience or combination of experiences associated withthe out-of-sight behaviour are critical in the developmentspatial memory.Surprisingly, no treatment differences were found in the

detour latencies to reach the imprinting stimulus inexperiment 2. It may be that the distance of the detourwas too short for orientation errors to delay significantlythe chick’s approach to the imprinting stimulus. Latenciesto reach the imprinting stimulus did, however, decrease insubsequent trials for all rearing treatments, a finding alsoreported in other studies and considered to indicate thatroute learning is required in order to reach the imprintingstimulus (Vallortigara et al. 1999). However, in the presentstudy, chicks continued to show a similar number oforientation errors in subsequent trials, suggesting thatchicks were not learning the route, but instead may haveshown reduced fear or learned the futility of pushingagainst the clear screen in subsequent trials (Regolin et al.1995).In conclusion, the series of experiments presented here

provide behavioural evidence that the domestic chick isa useful model for investigating developmental plasticity.In particular, the apparent sensitive phases for develop-ment and the possibility of imprinting chicks on anartificial object allow, to our knowledge, unprecedentedsubtlety in the types of manipulations that can be appliedfor identifying critical environmental factors in develop-ment. The results presented here suggest that activeexperience of occlusion around day 11 improves egocen-tric orientation, supporting the hypothesis that enrich-ment-induced behavioural changes are dependent on theinteraction with objects.

Acknowledgments

We thank Sue Healy for helpful comments on this studyand UFAW for financial support to R.F. (grant no. 28-99)for an earlier pilot study.


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development of spatial memory in occlusion-experienced domestic chicks

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