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he effect of social buffering on fear responsesn sheep (Ovis aries)
atilú Gonzáleza,b, Xavier Averósa, Ina Beltrán de Herediaa,oberto Ruiza, Josune Arranza, Inma Esteveza,c,∗
Neiker-Tecnalia, Arkaute Agrifood Campus, Department of Animal Production, 01080 Vitoria-Gasteiz, SpainUniversity of Córdoba, Zoology, Rabanales Campus, 14014 Córdoba, SpainIKERBASQUE, Basque Foundation for Science, 48011 Bilbao, Spain
a r t i c l e i n f o
rticle history:ccepted 17 September 2013vailable online 5 October 2013
eywords:heepovement
ocial interactionseactivityuffering effect
a b s t r a c t
Fear in farm animals has been extensively studied because of its close relation to animalwelfare. Numerous studies have categorized the behavioral responses of animals to stimulithat can elicit a fear reaction under social isolation conditions. However, farm animals arehighly social and therefore these responses could be conditioned by isolation. The objectiveof this study is to evaluate the potential buffering effect of the social environment on thefear responses of sheep, comparing the reactivity of individuals within different social envi-ronments. We studied the reactivity of 15 ewes (focal individuals) in isolation, and withingroups composed of 5 and 10 individuals, randomly alternating the presence of a suddenstimulus with a normal, non-stimulus situation (control). The XY coordinates and the behav-ior of each focal individual (inactivity, exploration, fast movements, attempt to escape, filialinteractions, agonistic interactions, and other activities) were recorded, at 1 min intervalsduring tests lasting 15 min, using the Chickitizer software. Euclidean distances were subse-quently obtained, and total traveled distance, net distance, minimum distance, maximumdistance, and path sinuosity (ND/TD) calculated. Results show a significant effect of both thepresence/absence of stimulus and the group size on the distance measures, exploration andfilial interactions (P ≤ 0.01). Moreover, the buffering effect of group size was demonstratedby the lower incidence of fast movements (P < 0.001) and escape attempts (P < 0.001). Theinteraction between the presence/absence of stimulus and the group size was significant
for inactivity (P = 0.001). Results show a marked effect of the stimulus presence, regardlessof group size. However, stress reactions due to social isolation were substantially greaterthan those elicited by the mere presence of the stimulus. These results highlight the impor-tance of the management procedures, particularly when animals need to be isolated fromthe group.
. Introduction
Public concern about how captive animals are treatedas increased tremendously over recent years. In this sense,
animal welfare has contributed to a better understandingof how animals perceive their social and physical environ-ment, their motivations, and physiological and behavioralneeds, allowing the design of environments to better satisfyall these needs (Webster, 2005).
Many studies have addressed the definition of animalwelfare, and the most suitable indicators to measure it(Mason and Mendl, 1993; Blokhuis et al., 2003; Mason andLatham, 2004). From all welfare aspects, the evaluation
of the animal’s mental state, such as freedom from fearand distress (FAWC, 2009) is perhaps the most difficult toassess. This is particularly the case in production underintensive conditions, where animals frequently face sit-uations implying behavioral and/or physiological stress.Some common stressors in farm animals include restric-tion of movements or social challenges that, dependingon the rearing system and management procedures, mayinclude social isolation, over-crowding, or regrouping. Theinability for captive animals to control their physical andsocial environment may be perceived as a threat affect-ing their welfare, and eventually their performance. Forexample, increased stocking densities in pigs increased thefrequency of aggression, competition, disease outbreak andresulted in lower daily feed consumption, daily weight gainand feed efficiency (Cho and Kim, 2011; Lebret et al., 2006;Hernández et al., 2006). In two different studies where thebehavior and the use of space of gestating ewes, main-tained at either 1, 2, or 3 m2/ewe resulted in movementsmore difficult and less effective movements, that translatedinto both a reduction in ewes’ activity, and into an increasein both positive and negative social interactions (Averóset al., 2013a,b). Regrouping and relocation of lactating ewesresulted in lower milk quality and quantity, probably as aconsequence of increased aggression and altered immuneresponses (Sevi et al., 2001).
Domestic sheep are highly gregarious animals, and thisdetermines their grazing activity, spatial distribution, andspeed of movement or proximity to other group members(Michelena et al., 2004). Like other ungulates, benefits ofgroup living in sheep may include assistance in findingmates or food resources, help with the care and protec-tion of younger animals, and protection from predators(Dwyer, 2009). In addition, from an animal welfare standpoint, group living may provide additional benefits, such asbetter recovery from distressing experiences, phenomenonknown as ‘social buffering’ (Kikusui et al., 2006; Rault,2012).
In rats, the benefits of social buffering, during, orafter the occurrence of a stressful situation, have alreadybeen described (Kiyokawa et al., 2007; Stanton, 1985).This is a particularly important aspect in domestic herbi-vores, where routine management procedures may elicitfear-related responses. Frequently or excessive fear dur-ing handling may lead to chronic stress, an alteration insocial, sexual and parental relationships, and a reductionin their productive performance (Forkman et al., 2007).In sheep, their strong need for companionship has beenused to determine their fear, anxiety and/or stress reactionstoward novel objects or situations (Beausoleil et al., 2005)through the use of behavioral indicators such as movement,walking frequency and speed, feeding, escape attempts,defecation, urination, bleats, and sniffing (Vandenheedeet al., 1998). In this sense, increased heart rates have beenobserved in sheep exposed to a novel object tests andappear to be a valid tool to measure the fear responseof sheep (Désiré et al., 2004). However, Forkman et al.
(2007) indicated that many of the observed reactionsmay be the consequence of the combined effects of theimposed situation and that of social isolation. In this sense,the effect of the presence of a group of conspecifics on
viour Science 149 (2013) 13– 20
the behavioral response to a stressful situation remainsunanswered.
The objective of this experiment was to assess the rele-vance of ‘social support’ in sheep (Ovis aries) when exposedto stressful, fear eliciting situations that may take place dur-ing management. It was hypothesized that the presenceof conspecifics (social buffering) during a stressful eventwould have calming effects on the individuals. Because ofthe gregarious nature of the domestic sheep the beneficialeffects were predicted to depend upon the size of the group.
2. Materials and methods
The experiment was carried out according to the Euro-pean Directive 86/609/ECC regarding the protection ofanimals used for experimental and other scientific pur-poses.
2.1. Animals and experimental set up
Fifteen 18 months old ewes (O. aries), were randomlyselected from the original experimental sheep flock (Latxabreed) at Neiker-Tecnalia (Vitoria-Gasteiz, Spain) that wascomposed of 135 ewes. The ewes used for the study wereseparated from the flock and housed together in an indoorpen (5 m × 5.8 m) for three days before the start of theexperiment. Purple numbers were sprayed on their backfor individual recognition. Ewes were maintained understandard feeding and handling practices during the wholeexperiment.
During the testing days, all individuals were initiallytransferred from the home pen to a pre-test holding penlocated close to the test arena. All tests were conductedin this test arena (4.6 × 4.4 m) after the morning feedingperiod, between 10:30 am and 12:30 pm. Feed was notprovided during the testing period but ewes had continu-ous access to water. Each experimental ewe was randomlytested under six scenarios: under social isolation, within asocial group of five ewes, and within a group of ten ewes,combined with or without exposure to a stressful fear elic-iting stimulus. Group sizes of five and ten correspondedto an attempt to simulate the effects of small versus largegroup sizes. Although more animals were available to workwith larger groups a maximum group size of ten was chosendue to time constrains for the experimental procedures.
During the days of the study, all experimental eweswere tested once per day during 5 min until completing allthe treatment combinations. For this, each day ewes weretested in isolation, five in groups of five, and five in groupsof ten, with or without the stimulus, in random order. Thedays and order in which each ewe completed each of thesix tests was totally randomized to prevent any potentialhabituation or learning effects. Four and nine additional,non-experimental ewes were randomly selected from theflock and introduced in the test arena to create the distinc-tive social group size situations. These ewes were familiarto the experimental ewes, as they all belonged to the initial
flock. In the stimulus treatment, either in isolation or in thecontext of a social group, an unfamiliar, orange umbrellawas suddenly opened right after the animal entered thetest arena. Once the umbrella was opened, it was placed
M. González et al. / Applied Animal Behaviour Science 149 (2013) 13– 20 15
Table 1Definition of the terms used in the study.
Distances (cm) Definition
Total distance (TD) Sum of all Euclidean distances* in an observation periodNet distance (ND) The Euclidean distance* between first and last observed locationMinimum distance (MinD) The smallest Euclidian distance* between subsequent locations in an observation periodMaximus distance (MaxD) The largest Euclidian distance* between subsequent locations in an observation periodNet to total distance ratio (ND/TD) Ratio obtained by dividing net distance and total distanceBehaviors (frequency during the 5 min observation, %)Inactivity Standing without other activityExploration Normal or slow walking, low or medium position of the head, frequently sniffingFast movement Fast walking or trot, high position of the headAttempt to escape Attempts to escape by kicking the wall or jumpFilial interactions** Sniffing and groomingAgonistic interactions** Push, back, and hit with the body or headOther activities Lying on the hey, drinking, defecation, etc
t location, both
iv
2
1altt(itw
* Euclidean distance: distance between location i (xi, yi) and subsequen** Interaction measures were registered only in group size of five and te
mmobile in the wall of the arena during the 5 min of obser-ation.
.2. Observations
The test arena floor was divided into a grid of about.2 × 1.1 m rectangles, by placing placards along the wallsnd creating a visual grid that allowed precise animalocation. Once the experimental ewe was introduced intohe arena, it was observed for 5 min. During this timehe ewe’s location (using X Y coordinates) and behavior
Fig. 1. Effect of the surprise treatment on the total, net, max
n i + 1 (xi+1, Yi +1), defined as l (i,i+1) =√
((xi+1 − xi)2 + (yi+1 − yi)2).with and without stimulus.
were used to compute Euclidean distances (cm) betweensequential locations, which were used to calculate totaldistance (TD), net distance (ND), minimum (MinD) andmaximum (MaxD) distances (Leone and Estevez, 2008;Estevez et al., 2010) as different measures characterizingthe distance travelled, as well as the net to total distanceratio (ND/TD) as a measure of the trajectory sinuosity (seedefinitions in Table 1). All observations were collected bythe same observer, who was located immobile outside thetesting arena.
2.3. Statistical analysis
Parameters that met analysis of variance assumptionsof normality and variance homoscedasticity were analyzed
imum and minimum distance traveled (LSM ± SE).
16 M. González et al. / Applied Animal Behaviour Science 149 (2013) 13– 20
Table 2Results of the Mixed model ANOVA. Effects of surprise treatment, group size, and their interaction for parameters that meet normality and homoscedasticityof the variance.
Distances Surprise treatment Group size Surprise treatment × group size
F 1,84 P F 2,84 P F 2,84 P
Total distance (TD, cm) 6.78 0.0109 56.16 <0.0001 0.85 0.4327Net distance (ND, cm) 11.66 0.001 10.35 <0.0001 0.98 0.381Minimum distance (cm) 7.50 0.0075 7.64 0.0009 2.21 0.1164Maximum distance (cm) 9.22 0.0032 40.08 <0.0001 1.41 0.2507ND/TD 5.82 0.018 5.66 0.0049 1.41 0.2507Behaviors F 1,56 P F 2,84 P F 2,84 PInactivity (%) 27.04 <0.0001 13.96 <0.0001 7.12 0.0014
F
Exploration (%) 18.28 <0.0001
F 1,56 P
Filial interactions (%) 30.73 <0.0001
using a mixed model ANOVA with group size (isolation,group size 5, group size 10), surprise treatment (presence orabsence of surprise stimulus), and their interaction as fixedfactors and individual included as a within-subject, randomvariable. The studied parameters included TD, ND, MaxD,ND/TD, Inactivity and Exploration. Least square meanswere computed in case of statistically significant effects(P < 0.05), with P-values adjusted for multiple comparisonsby Tukey tests. The distribution of MinD was normal-ized using the logarithmic transformation, and analyzed aspreviously described. Attempts to escape, fast movement,aggressive interactions and other behaviors did not satisfythe assumptions of variance homogeneity and normality
distribution of the residuals, and were therefore analyzedusing the Kruskal–Wallis, non-parametric test. All analy-ses were performed using SAS 9.3 (SAS Institute, Cary, NC,USA).
Fig. 2. Effect of group size on the total, net, maximum and minimum distancedifferent letters.
10.77 <0.0001 1.49 0.2304 1,56 P F 1,56 P
7.19 0.0096 0.53 0.4696
3. Results
Results of this study indicate that all distance traveledparameters, were affected by both group size and the sur-prise treatment but not by their interaction (Table 2). TD,ND, MinD and MaxD were longer when the surprise stim-ulus was not present (Fig. 1), and when ewes were testedunder isolation as compared to groups of five and ten indi-viduals (Fig. 2). Likewise, ND/TD ratio was smaller with nostimulus (mean ± standard error; 0.16 ± 0.02), as comparedwith its presence (0.25 ± 0.03; P < 0.05), indicating highersinuosity in the movement path when the surprise stim-ulus was absent. ND/TD ratio was also affected by group
size, with significant lower values for ewes when tested inisolation (0.11 ± 0.03) as compared with groups of five orten animals (0.26 ± 0.04 and 0.23 ± 0.04 for groups of fiveand ten, respectively, P < 0.01).
traveled (LSM ± SE). Significant differences (P < 0.05) are designated by
M. González et al. / Applied Animal Behaviour Science 149 (2013) 13– 20 17
Fs(
eqeortiaaiaana
sbt
Table 3Effect of surprise treatment and group size on the non-parametric studiedparameters.
Behaviors Surprise treatment Group size
Chi-square P Chi-square P
Fast movements (%) 0.007 0.9309 49.927 <0.0001Attempts to escape (%) 0.917 0.3381 50.456 <0.0001Agonistic interactions (%) 0.191 0.6614 0.008 0.9276
TM
W
ig. 3. Effects of the interaction between surprise treatment and groupize on the frequency of inactivity (LSM ± SE). Significant differencesP < 0.05) are designated by different letters.
Regarding the behavioral effects, we found a significantffect of the surprise treatment and group size on the fre-uency of inactivity, with significant differences betweenwes with and without stimulus only observed in groupsf ten (Table 2 and Fig. 3). On the other hand, the explo-ation, filial interactions were significantly affected by bothhe surprise treatment and group size but not by theirnteraction (Tables 2 and 4), whereas non parametric vari-bles such as fast movements and attempts to escape wereffected by group size only (Table 3). Individuals tested insolation showed much higher values of fast movementsnd attempts to escape as compared when in groups of fivend ten (Table 4). Neither the frequency of fast movementsor attempts to escape were affected by the presence orbsence of the surprise treatment (Tables 3 and 4).
Social interactions analyzed for groups of five and tenhowed that filial interactions were significantly affectedoth by group size and surprise treatment, but not by theirwo-way interaction (Table 2). Ewes showed fewer filial
able 4ean values (±SE) of the behaviors, according to surprise treatment and group si
Variable Surprise treatment
Without stimulus
Mean (±SE)
Inactivity (%) 36.99 ± 3.20
Exploration (%) 26.84 ± 1.98
Fast movements (%) 4.78 ± 1.34
Attempts to escape (%) 5.26 ± 1.64
Filial interactions (%) 24.64 ± 2.43
Agonistic interactions (%) 3.85 ± 1.11
Other activities (%) 7.14 ± 1.79
Variable Group size
1
Mean (±SE)
Inactivity (%) 33.82 ± 4.37a
Exploration (%) 30.03 ± 3.12a
Fast movements* (%) 12.71 ± 1.95
Attempts to escape* (%) 17.98 ± 2.98
Filial interactions (%)
Agonistic interactions (%)
Other activities (%) 5.46 ± 1.12
ithin each row, significant differences (P < 0.05) are designated by different lett* Variables with significant differences (P < 0.05) obtained with Kruskal–Wallis
Other activities (%) 2.412 0.1204 4.041 0.1325
Statistical effect obtained using Kruskal–Wallis tests.
interactions when the sudden stimulus was present andwhen housed in groups of five as compared to ten (Table 4).No effects of the surprise treatment or group size weredetected for the frequency of agonistic interactions or otheractivities (Tables 3 and 4).
4. Discussion
The purpose of our study was to determine whetherthe presence of conspecifics might help reducing fearresponses of Latxa ewes that can arise as a consequence ofa sudden stimulus, and to determine the extent to whichgroup size may play a role in promoting social buffering.Our results clearly show that the movement patterns andthe behavior of isolated ewes differ from those observedwhen they are located within a social group. Similarly, theirresponse to a surprise stimulus when in isolation differsfrom that observed when they are within a group.
Movement pattern-related parameters (TD, ND, MinD,MaxD and ND/TD ratio) differed according to the pres-
ence/absence of a surprise stimulus. In this sense, themeasured distances were longer when no stimulus waspresented. Different mechanisms could explain theseresults. It might be possible that ewes not presented with
the surprise stimulus had a bigger ‘effective’ available areacompared to those presented with the stimulus. This couldbe due to the physical space reduction caused by the stimu-lus itself and consequently to the flight distance establishedaround it, as shown in sheep (Hutson, 1982), and otherprey animals such as birds (Møller, 2008) and deer (Boeret al., 2004). Nevertheless, it is more likely that the observeddecrease in the walked distances was due to increasedimmobility and vigilance, likely due to the sudden pres-ence of the stimulus (Torres-Hernandez and Hohenboken,1979).
Distance traveled by ewes tested in isolation werehigher than those tested within groups of five or ten indi-viduals, and this would be explained by fear reactionstriggered by social isolation, as it is known to occur ingregarious species such as sheep (Deiss et al., 2009). Den-sity has been shown to have important effects in terms oftraveled distances in farm animals, with distances traveledbeing generally larger at low density (Leone and Estevez,2008; Mallapur et al., 2009). Although it must be acknowl-edged that group size and density are confounded in thisstudy (Estevez et al., 2007), the enclosure in which the testwere conducted was sufficiently large for the individuals tomove around (Department for Environment Food and RuralAffairs (DEFRA), 2003). Therefore, the findings of this studysuggest that the effects found in this study were due mostlikely to changes in group size, results that would agreewith previous findings in studies conducted with broilerchickens (Newberry and Hall, 1990; Estevez et al., 1997;Leone and Estevez, 2008; Mallapur et al., 2009) and merinosheep (Michelena et al., 2008a,b). The detrimental effectof social isolation has been described in prey and socialanimals such as horses, with isolated animals showinghigher motivation for movement, traveling further, spend-ing more time trotting, and performing a greater numberof different activities than those in a group of conspecifics(Mal et al., 1991). Social support has been suggested to bea source of positive emotions for farmed animals, and tocontribute to increase their animal welfare (Rault, 2012).Social isolation has also been found to increase locomotionin sheep (Beausoleil et al., 2012; Romeyer and Bouissou,1992). In addition, the detrimental effect of social isolationmay have been particularly powerful due to the fact that,although ewes were unable to establish visual contact withother conspecifics during testing, they were able to hearother animals located in adjacent enclosures.
The preceding explanation would be further supportedby the occurrence of behaviors such as fast movementsand attempts to escape, that occurred at higher frequen-cies under isolation conditions than in groups. This wouldconcur with results from other studies regarding the effectsof short-term isolation in sheep, and would been linked toincreased fear and anxiety levels (Romeyer and Bouissou,1992; Vandenheede et al., 1998). Nevertheless, it would beerroneous to assume that an increase in the traveled dis-tance and high activity are always synonymous of higherfear and anxiety levels (Carter et al., 2012), as this is likely to
occur for example under conditions of higher space avail-ability or higher level of environmental complexity. Asmentioned before, the longest distances were traveled byewes without a surprise stimulus situation. Certainly, it is
viour Science 149 (2013) 13– 20
possible that our results could be affected by ewes’ indi-vidual temperament, with the highest activity scores andthe longest distances corresponding to active ewes. Activeewes appear to be less fearful than less active ones, andhad lower blood cortisol levels as well (Beausoleil et al.,2008, 2012). To distinguish high activity trigger by fearfrom environmental conditions that trigger higher activ-ity levels, perhaps characterization of the sinuosity of thetrajectory patterns or speed might be necessary.
A key element of our study to explain those apparentlycontradictory results is the existence of a significant inter-action between the stimulus treatment and the group sizeon the inactivity levels. The lowest inactivity scores cor-responded to ewes tested in isolation and within groupsof ten. No significant differences were found under isola-tion conditions either in the presence or absence of thestimulus. On the contrary, ewes tested in groups of tenshowed the highest inactivity scores when the stimuluswas presented, and they also showed the highest differ-ences in inactivity scores when stimulus was either presentor absent. Differences in the effect of the stimulus betweenisolated ewes and those tested in groups of ten suggestthat, in each case, inactivity obeys to different mecha-nisms. Behaviors displayed by socially isolated sheep, suchas walking frequency, time spent walking, trotting, or thenumber of escape attempts, have been positively related tofear responses in fear-eliciting situations, with social iso-lation considered as one of the main factors contributingto fear in this domestic species (Vandenheede et al., 1998).The combination of movement and behavioral results inthe present study suggests that the emotional fear responsedue to isolation was much stronger than that of the poten-tial fear elicited by the presence of the stimulus, the latterresulting in an escape response.
On the other hand, it is clear from our results that thebehavior of the tested animals changed dramatically whentested in either group sizes of five or ten. When tests wereconducted under a group situation, attempts to escape andfast movements were punctual events in comparison tothose observed under isolation conditions, while higherfrequencies of filial behaviors were observed. In addition,the reduction in movement, as indicated by TD, ND, MinDand MaxD, would suggest that both group sizes wouldequally satisfy the social needs of ewes, even under thepotentially fearful situation of being in a new enclosure.These results would agree with the ideal of the social grouphaving a positive buffering effect under situations of expo-sure to stress fear eliciting events by achieving a reductionin the fear reactions through the presence of conspecifics(Vandenheede and Bouissou, 1994).
However, we found clear differences in the responseof ewes when in groups of ten, mostly associated to ahigher frequency of filial interactions compared to groupsof five, as well as an increase in the activity levels of eweswhen no stimulus was used (Fig. 3). It is possible that,even when tested in groups of five without stimulus, theexperimental conditions elicited some fear reaction, with
the higher inactivity levels reflecting increased vigilancetoward the environment. It would therefore be possibleto speculate that vigilance in a mildly stressful situationwould have been lower, and this would be explained by the
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act that larger group sizes provide a higher level of pro-ection against potential predators. These results would,o a certain extent agree with the findings of Michelenat al. (2008b), who found that sheep in larger groups devoteore time to activities other than vigilance. However, con-
rary to their findings, no differences in distance traveledere detected across group sizes of five and ten in the cur-
ent study. Although the different conditions of in whichoth studies were conducted make the comparison diffi-ult.
The origin of the differences in the frequency of filialnteractions between groups of five or ten remains unclear,ut it might be explained by the fact that ewes in the largerroup may have had more opportunities to interact withther conspecifics. Alternatively, the average area/ewe inroups of ten was smaller, and so was the proximity tother ewes, potentially increasing chances for the occur-ence of social interactions as found in previous studiesAverós et al., 2013b).
In conclusion, results of this study evidenced thelear differences in movement patterns and behavioralesponses derived from isolation as compared to groupousing. The reactivity shown by ewes tested in isolations measured by the frequency in exploration, attemptso escape and fast movements as compared to housed inroups of five or ten would support the idea of an under-ying social buffering effect. These results would highlighthe importance of quiet management procedures, particu-arly when animals need to be isolated from the group. Theack of group size effects in this study would suggest thathe benefits of social buffering would unlikely depended onhe size of the social group, at least for the ranges of groupizes tested in the present study..
cknowledgments
The authors gratefully acknowledge the EU financialupport provided to conduct the study and salary for X.verós under the VII Framework Program for Research,echnological Development and Demonstration Activitiesf the project Animal Welfare Indicators (FP7-KBBE-2010-). The authors also thank Imanol Etxebarría and Juanarlos Ochoa de Zuazola for the excelent care of the ani-als during the experiment and to Alexander Weiss and
n anonymous reviewer for their helpful and constructiveomments on an earlier version of this manuscript.
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