Interplay between environmental and genetic factors. in temperament / personality traits in horses (Equus caballus)

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1 Interplay between environmental Interplay between environmental and genetic factors in temperament / personality traits in horses (Equus caballus) Martine Hausberger, Cécile Bruderer, Nathalie Le Scolan and Jean-Sébastien Pierre UMR CNRS 6552, Ethologie - Evolution - Ecologie, Université de Rennes I Running head: Temperament in horses Keywords: temperament, personality, horses, genetics of behaviour We are grateful to Huguette Schuelke for her great help in preparing this manuscript and to Dr Ann Cloarec and Prof. Adrian Craig for checking the English. The final version benefitted greatly from comments made by Dr M. West, Dr E. Hanggi and an anonymous reviewer. This study was supported by a grant from the Haras Nationaux. Correspondence concerning this article should be addressed to Martine Hausberger, Université de Rennes 1, UMR CNRS 6552 Ethologie-Evolution-Ecologie, Campus de Beaulieu, F Rennes Cedex, France. Electronic mail may be sent to Martine.Hausberger@univ-rennes1.fr

2 Interplay between environmental Interplay between environmental and genetic factors in temperament / personality traits in horses (Equus caballus) Abstract The aim of the present study was to broach the question of the relative influence of different genetic and environmental factors on different temperament/personality traits of horses. A large sample of horses (702) was submitted to standardized experimental tests and multivariate and univariate statistical analyses enabled us to compare the influence of nine factors, either genetic (sex, breed, sire) or environmental (housing, food, type of work ). The results confirmed the interplay of genetic and environmental factors in determining behaviour and confirmed the multifaceted dimension of temperament. Thus, genetic factors, as sire or breed, seemed to influence more neophobic reactions, whereas environmental factors, such as the type of work, seemed to play a more dominant role in reactions to social separation or learning abilities. Additive effects were evident showing how environmental factors may modulate behavioural traits. Thus large differences were found between facilities in the reactions of horses to the tests, showing a context effect that can induce shift in genetically induced individual differences (e.g. breeds). This study constitutes a first step towards understanding the relative weights of genetic factors and how the environment may intervene in determining individual behavioural characteristics. The development of personality may be oriented according to the "openness" of some traits to environmental factors and the "conservativeness" of others, more dependent on genetic factors.

3 Interplay between environmental Interplay between environmental and genetic factors in temperament / personality traits in horses (Equus caballus) INTRODUCTION After a predominantly normative approach to behaviour during the past decades, the renewal of interest in individual distinctiveness is marked by new questions about concepts, definitions and methods. As Gosling (1998) mentioned, the terms temperament and personality appear in many cases to be used interchangeably. For Hall (1941), "temperament is the raw stuff of individuals" and is independent of environment and culture. For Lyons et al. (1988), it represents the basic response of an individual facing permanent changes and challenges in its environment. Personality is more often referred to as a "behavioural style" more "open" to modification by experience (Feaver et al., 1986; Lyons, 1989). This agrees with Hall's definition (1940): "when temperament is refined by environment and culture, it becomes personality". Some authors, however, consider that stability across time and situations is equally important for personality and for temperament (in Gosling, 1998). Various definitions of temperament have been proposed, especially in human studies. Most of the debates and differences refer to the degree to which genetic transmission is implied. The biological foundation of temperament is the main point in Bates definition (1989): The most general definition of temperament across these varied constituencies is that it consists of biologically rooted individual differences in behavior tendencies that are present early in life and are relatively stable across various kinds of situations and over the course of time.

4 Interplay between environmental According to Gosling (2001), animal studies should help us understand how an individual's personality is influenced by genetic, social and non-social environmental factors and their interactions. A better knowledge of the interplay between genetic and environmental factors in determining temperament/personality traits is crucial for an understanding of individual behavioural characteristics and refining the definitions of temperament and personality. Moreover, the multifaceted dimension of temperament has been emphasized (Mills, 1998) and different studies show that the degree of heritability may very according to the trait being considered (Ostaszewski et al., 1994; Weiss et al., 2000; Grignard et al., 2001). This suggests that the relative weight of genetic and environmental factors may vary according to the trait, but to our knowledge, no extensive study has been made up to now, that could take into account the large number of factors potentially influential in the daily life of animals. The aim of the present study was to investigate the relative weight of genetic and environmental factors on different temperament/personality traits using a very large sample of horses, in order to test the potential effects of a number of factors including genetic (breed, sex, sire) and environmental (housing, food, ) factors. We included amongst the environmental factors the type of work (dressage, jumping, ) which is particular to horses. On the theoretical point of view, it may be quite interesting to investigate how such an interspecies relation may affect personality traits. Moreover, the different types of training may stimulate or inhibit some traits, like learning and it seems important to see how this reflects on the horses' behaviour in other non working situations. Thus this particular human/horse interaction has appeared to have an influence on both the occurrence of stereotypies and emotional reactions in behavioural tests (Mc Greevy et al., 1995; Hausberger et al., 1996). Different breeds can be used for the same type of work and individual horses can change work during their life. Horses are an interesting species as we have some evidence for sire and

5 Interplay between environmental breed effects on behaviour (Houpt & Kusunose, 2001; Hausberger & Richard-Yris, 2004). Thus, differences according to breed (Mader & Price, 1980; Budzynski et al., 1992; Hausberger et al., 1996) or sire (Wolff & Hausberger, 1994, 1996; Wolff et al., 1997; Lankin & Bouissou, 1998) have been mentioned both for emotional and learning tests. Moreover, individual horses tend to react differently to different emotional (Wolff et al., 1997; Anderson, 1999; Visser et al., 2001; Seaman et al., 2002) or learning (Wolff & Hausberger, 1996; Nicol, 2002) tests, which suggests that different traits may indeed be differently determined. In order to test our hypothesis, we used a battery of standardized behavioural tests. Methods assessing temperament or personality in animals are varied and include observations in the home environment or experimental behavioural tests associated with ethological measurements, observers' scorings or questionnaires borrowed from psychometrical tests for humans (Manteca & Deag, 1993, 2001; Gosling & Jonn, 1999). These approaches are complementary (Mendl & Harcourt, 2000) but experimental tests may increase the chances of identifying the biological bases as, for example, differences between genotypes of macropodes are best revealed in unusual environments (Gerlai & Csañyi, 1990). In the present case, we used experimental tests first developed by Wolff & Hausberger (1992) that proved useful in a number of further studies (Wolff & Hausberger, 1996; Wolff et al., 1997; Seaman et al., 2002; Visser et al., 2002). Given the aim of our study, where we had to compare a large number of animals, we needed simple quickly performed tests. This prevented us from including in our study learning tests of complex cognitive abilities. Although this would be highly interesting, given the recent findings of Hanggi (1999, 2003) and Sappington & Goldman (1994) showing that horses are able of categorization and concept learning, such studies require thorough complex tests that can only be performed on a limited number of animals (Hanggi, 2003).

6 Interplay between environmental We concentrated therefore on tests that had been shown to reveal individual behavioural learning abilities that correlated to those observed in the daily working situation (see also Fiske & Potter, 1979; Visser et al., 2003a). Thus, Le Scolan et al. (1997) found a correlation between the performances of individual horses in our learning test and ratings by riding teachers of their ability to learn new tasks. Finally, using multivariate statistical analysis, we were able to disentangle what relative importance the different factors may have on the different traits being considered and confirm the multifaceted dimension of temperament. This study opens new lines of thought about how personality construct may be constrained by the relative "openness" to environmental factors of some traits and "conservativeness" of others, more under genetic influence. MATERIAL AND METHODS Terminology The behavioural traits tested correspond to traits that have been reported regularly by riders and that have been mentioned in various studies on inter-individual variability in behaviour. Emotionality Archer (1973) defined emotionality as an inherited predisposition of the autonomic nervous system allowing an animal to react in a particularly strong and lasting way to some classes of stimuli. It can include different aspects as: - Fearfulness: a tendency to react with fear, which can be considered as a short term reaction to stimulation, inducing aversion (Jones, 1989). - Nervousness: a term used by most riders to describe the tendency of a horse to become excited easily. An increase in excitement level is correlated with an increase in

7 Interplay between environmental postural tonicity and in locomotor activity, but presents no situation-specificity (Kiley- Worthington, 1976). - Reaction to social separation: refers to the need to share activities and contacts with members of the same species (Buss, 1989) and to search for contact when isolated. It was included in the emotional traits as it describes the tendency of horses to show emotional reactions (whinnying, locomotion, vigilance) when separated from their group (Waring, 1983). It differs from the "prosocial" tendencies in humans described in psychometrics. Learning and memory abilities These abilities correspond to the capacity to acquire (learning) and memorize information or a task (Rosenzweig, 1976). Horses are able to learn complex tasks (e.g. Hanggi, 1999), but we chose to use only one simple test as our main aim was to compare the results of a learning test with those obtained in different emotional tests (see introduction). We used here, as in all other learning tests performed in other studies a food reward (Nicol, 2002). Since the animals had not been deprived and the results of this test are correlated with other evaluations of the horses' learning abilities, we were certain that instrumental learning was indeed involved and not merely food motivation, which would not imply that the horses succeed anyway. Subjects 702 horses and ponies were tested at 103 sites in different regions in France: 37 riding centres, 23 training centres, 9 national studs, 34 breeding farms. Only registered horses were chosen so that the precise genetic origin of each horse was known. Sexes (185 mares, 305 geldings, 212 stallions) and ages (2 to 26 years: X = 7.97 ± 4.67) of the subjects were relatively well distributed.

8 Interplay between environmental Information for each horse included its age, sex, breed, genetic origin, type of work, usual number of riders, food, housing, site (location with its management characteristics), i.e. nine factors (Table 1). Sixteen different breeds were involved, with more French saddlebreds, due to the over-representation of this breed in France and to their use for a further study on sire effect. Most variables in the horses' environments were thus taken into account (see appendix 1). Experimental tests We used a series of experimental tests that had previously proved to be good indicators of emotionality and learning abilities (Wolff & Hausberger, 1996; Wolff et al., 1997). Moreover, these tests could be easily and rapidly performed on different sites. Previously, the results of tests for a given horse and questionnaires filled by horse "users" of that horse proved to be strongly correlated, and confirmed that these tests were good indicators of behavioural traits (Le Scolan et al., 1997). Tests used to measure emotional reactions Three tests, described in Wolff et al. (1997) were used: - "Arena test": horses were released alone in a familiar arena and their behaviour was recorded every 10 s for 10 min (see also Anderson et al., 1999; Seaman et al., 2002). This differs from classical open-field tests, as the site is familiar and this test has been suggested to estimate the effects of social separation (Le Scolan et al., 1997). - "Novel object" test: an object was placed in the arena and a horse was released for 5 min: its behaviour, locomotion, gazes and approaches were recorded. Correlations had been found between reactions to this test and estimations of nervousness by users (Le Scolan et al., 1997).

9 Interplay between environmental "Bridge" test: a horse was led using a halter over an unknown obstacle built with a foam mattress (Le Scolan et al., 1997) (planks: Wolff et al., 1997; concrete blocks: Visser et al., 2001). Reactions to this test have been found to correlate with evaluations of fear by horse users (Le Scolan et al., 1997). Standing in front of the bridge was correlated with "spooky" in Visser et al. s (2002) study. The horses had worked or were in a paddock the day before a test. The tests were carried out before the horses worked that day. Each horse was used only once for each test, to avoid habituation (Jones, 1989; Budaev, 1997). General procedure for the «arena» and «novel object» tests The tests took place in arenas with sand or wood shavings. The horses were familiar with these arenas. The experimental horse was released as soon as it had gone through the gate and its behaviour was recorded by the observer who remained immobile in the arena, in a corner, far from the door while recording the observations on a voice cassette recorder. The horse s behaviour was recorded using the "instantaneous scan sampling" and "all occurrences" methods described by Altmann (1974). The procedure was the same as that used by Wolff et al. (1996): we observed the horses for 10 min (arena test) or 5 min (novel object test) after they had been released. Samples were taken every 10 s, yielding respectively 60 and 30 samples per horse. An object, unknown to the animals, was designed for the object test. Six metallic rails (square section: 9 cm) were assembled to form a cage (100 x 80 x 80 cm) onto which one long red fluorescent ribbon was attached, crossing the rails on each face. This object was placed to the side of the door so that the horse could see it only after it had entered the arena. A 10 m diameter circle was drawn on the sand around the object to help evaluate the distance between horse and object ( ± 5 m from the object).

10 Interplay between environmental Observation procedure. Rare or brief behavioural patterns were recorded every time they occurred: snorts, pawing, defecation, rolling, whinnying ("all occurrences method", Altmann, 1974). The behavioural patterns sampled using scan sampling were: (1) standing; (2) exploration: the horse walks slowly with its neck held horizontally or lowered, ready to stop and to sniff the ground or the wall. This is the characteristic slow walk of a quiet horse in a calm situation; (3) sustained walk: the horse walks energetically and looks ahead or around; (4) trot: a two-beat gait; (5) prancing: an animated form of trot when the legs are raised higher; (6) canter: a three-beat gait; (7) vigilance: the horse stands still holding its neck high, with intently oriented head and ears; (8) tail posture: it can hang down or be raised, the fleshy portion of the tail then held almost or completely upright with the long hairs of the tail stream making a showy display (Waring, 1983). Data analysis (see Wolff et al., 1996). Two different types of analyses were used. Frequencies of occurrence of behavioural patterns were calculated and compared between individuals. To «rank» reactivity of horses in each situation, we used an index based on both behavioural patterns and their frequencies of occurrence, this index had proved useful in previous studies. Values were attributed to the behavioural patterns according to their degree of specificity and corresponding level of arousal (see also Kiley-Worthington, 1970). These values were: exploration (slow walk) = 1, sustained walk = 2, trot or gallop = 3, vigilance = 4, whinnying = 5, prancing, snorting or tail raised = 6. These values were multiplied by the number of times the corresponding pattern was observed. It must be stressed that these values only give a rank indication and do not represent real data. Thus an animal with an index twice as high as that of another horse is not necessarily twice as reactive. General procedure for the bridge test

11 Interplay between environmental A foam mattress (200 x 100 x 10 cm) was covered with a brown and white checked oilcloth (squares: 2 x 2 cm), this was called the bridge. A starting line was drawn on the sand 2 m in front of the bridge. The experimenter led the horse using a halter with the lead attached to the ring and tried to make it cross the bridge. She was not allowed to touch or to talk to the animal. Her activity was limited to pulling slightly on the rope if necessary. All tests were made with the same person who was not familiar to the experimental horses. Many animals avoided walking on the bridge and passed by on one side. In this case, they were led back to the starting line and a new trial began (the stopwatch was stopped until the new start). The test was stopped when the horse crossed the bridge with at least three feet, 10 min was the maximum allowed for this trial. Data recorded were the total times required to cross the bridge. No data were available on the past history of the horses. Handling of foals and training procedures are very variable in France (in prep.) and therefore it would be quite impossible to have the individual life history for each of the 700 horses. Given the large sample though, and the results obtained (see further), it seems unlikely that this lack of knowledge has greatly affected the interpretation of the results. Learning and memory As mentioned above, we concentrated on one type of learning task, chosen because it led to easily measurable performances and showed correlations with learning abilities assessed by riders (Le Scolan et al., 1997). Our aim was only to evaluate comparatively the weight of genetic/environmental factors and not to give an exhaustive view of horses' learning abilities. Both learning and memorization were tested. In the latter case, it was a repetition of the same test 12 hours later.

12 Interplay between environmental The task consisted in opening a wooden chest to find food. The chest (60 x 50 x 40 cm) was closed with a lid fixed with two hinges. Test procedure was as follows: the chest, containing food (0.5 l), was placed in the horse s box near the trough. Positive reinforcement for both these tests was the horse s usual food (concentrated commercial pellets). The tests were made h before usual feeding time, to ensure a sufficient level of motivation. Their last pellet meal had been 8 to 10 hours before. The animals were given three successive trials and were considered to have failed when they did not succeed during these trials. Before the first trial, the observer demonstrated the task, as described above, in front of the horse to be tested while it was held and shown the food. The horse was then released and its behaviour was recorded for 3 min. If it was unsuccessful (this meant that the horse did not reach the food), the observer demonstrated the task again and, in addition, presented some food close to the animal who could then sniff it. The horse was then freed and its behaviour was again recorded for 3 min. If the animal was unsuccessful again, the procedure was repeated but this time the horse could take a mouthful of pellets. This third trial again lasted 3 min. This procedure was repeated three more times when a horse failed, in order to be able to test its memory in the memorization test. The ability to learn and memorize was estimated by numbers of trials and time required to perform the task. We measured the latency between the horse s first physical contact with the chest after release and eating out of the chest after having successfully lifted the lid (nose in the chest). Times for successive trials were summed (e.g.: 3 min for trial min for trial 2 = total time equals 6 min). The same procedure was followed for the memorization as for the learning tests, but on average 12 hours later.

13 Interplay between environmental In both cases (learning and memorization), horses that completed the task during the first three trials were considered «successful». Statistical analysis Two statistical approaches were used: univariate and multivariate analyses of variance and principal component analysis, a factorial method. Unbalanced ANOVA and MANOVA (General Linear Model) After the tests, each animal was characterized by a set of quantitative variables. The horses were also defined by several random or fixed classification factors (table 1). The number of subjects for each level of each factor was highly unbalanced due to the availability of the different categories in the horse farms or centres. Therefore, we used GLM (General Linear Model) SAS Procedure (SAS institute Inc, 1988). The statistical inference considered the SAS type III adjusted effects. The sequential sum of squares (SAS type I) and the corresponding adjusted squares for different sequences of factor declaration were compared to evaluate the importance of the bias induced by the non-orthogonality of factors. For random effects (for example, paternal origin), variance components were estimated by REML (Restricted Maximum Likelihood). The significance of each effect was assessed by considering the F ratio with the highest significant random effect as denominator for the nested parts of the model. Effects not significant at the α = 0.05 threshold were eliminated. F values for fixed effects and variance components for random effects were considered to compare the influence of each factor on the results. Univariate GLM procedures were applied for each behavioural test and multivariate (MANOVA) procedures for the whole set of tests. This approach evaluates the effects of factors on the horses general behaviour. Several statistical methods assessed the significance

14 Interplay between environmental of the effects revealed by MANOVA. The four statistics (Wilk s λ criterion, Pillai s trace, Hotelling-Lawley trace and Roy s greater root) given by the software (SAS GLM) were always consistent, therefore, only the most common, Wilks λ, is given with the appropriate F and associated degrees of freedom. F and the degrees of freedom are the result of an iterative calculus and have generally nothing to do with the univariate degrees of freedom. General ANOVAs were complemented by multiple means comparisons for each significant factor. Two conservative tests were used: the Scheffe and the Bonferroni-Dunn tests. Redundancy Analysis (RDA) A more descriptive but very informative approach consists of using a factorial analysis. We chose a recent method called Redundancy Analysis (RDA) first devised by Ter Braak (1987). When contingency tables are used, this method is known as Canonical Correspondence Analysis (CCA). The same method, but calculated with a different algorithm, was described as PCA-IV (Principal Components Analysis on Instrumental Variables) by Chessel & Lebreton (Chessel et al., 1987; Lebreton et al., 1988), and as reduced-rank regression by Davies & Tso (1982). The method, comparable to PCA in its principle and interpretation is well suited for analysing the effect of data in one table on data in another table. The first table (called X) is a set of p explanatory variables, that can be either quantitative (predictors, instrumental variables) or qualitative (factors) and defined for n individuals. The second table (called Y) is a set of q variables of either quantitative (PCA-IV) or qualitative (CCA, CA-IV) interest measured on the same number of individuals (n). This technique consists of a Principle Components Analysis (PCA) of the table estimated by the multiple linear regression (or ANOVA in the case of factors) of each column in Y on every column in X. It yields a simultaneous plot of both groups of variables and a visualization of their relationships. Each axis on the plots can be interpreted as a factor in Thurstone s sense

15 Interplay between environmental (i.e. a linear relation between correlated descriptors). These axes can be interpreted by considering the factor loadings of initial variables, that means squared correlation coefficients between each variable and each axis, just as in an ordinary PCA. Furthermore, factor loadings (or relative contributions) can also be affected to the qualitative predictors ( factors in the analysis of variance sense = set of separate levels) as follows: the factor loading of a qualitative predictor is defined as the sum of the factor loadings of all its levels. These factor loadings are used for interpreting the relationships between each axis ( factor in Thurstone s sense) and the qualitative predictors. A factorial plot of individuals can also be drawn and can be used to detect clusters of individuals. A home-made software GTABM (Quris, 1989) was used. The data used were the indices of emotionality for the arena and new object tests and the time required for completion of the task for the bridge and learning tests (plus number of trials). Of course these data, given the low number of variables could give us a partial knowledge of the structure of horse personality and a higher number of test scores would improve a factorial analysis conceived as the research of latent tendencies. However the use of a very large sample of horses clustered in several locations implies such a cost, that a balance between the number of subjects and the number of test applied to each subject was necessary. The tests we used, although limited in number, were carefully designed to enlight fundamental tendencies of personalities. The large number of horses used in the study gives some insurance on the generality of the underlying factors found if any. RESULTS General approach: multivariate analysis

16 Interplay between environmental GLM and ANOVA revealed that among all the factors taken into account in this study, two, sex and age, had no significant effects. Two other factors (sire and site) had to be analysed separately, as their N was greater than 100 (N = 398 and 103 respectively) and therefore too high for the general factorial analyses. A simple model with no interaction was computed on the five remaining factors. Behavioural factors The Principle Component Analysis (PCA) table crossed the responses measured for each individual test, in columns, with each individual horse, in rows. Three factors accounted for 90.5% of the total inertia (variance). The first two axes were linked to factors accounting for 81.7% of the inertia. These first two factors can be clearly interpreted in terms of linear combinations of test scores (Fig. 1). Tables 2 a and b summarize, respectively, the factor loadings of test scores and of qualitative predictors. The first factor (55 % of the inertia) was entirely defined by the learning and memory tests, the second by the arena and novel object test and the third factor was entirely defined by the bridge test (Table 2b). Therefore factor 1 represents learning and memory abilities whereas factors 2 and 3 are more representative of emotional reactions. The fact that the factors were defined by the experimental test used is in accordance with the finding that each of these tests measures a different facet of personality: reaction to social separation for the arena test, nervousness for the novel object, fear while handled for the bridge, learning for the chest opening test (Le Scolan et al., 1997; Visser et al., 2003). Breed and type of work had a strong influence on axes 1 and 2, but axis 2 was also influenced by housing. Consideration of the separate levels yields more details.

17 Interplay between environmental Breed influenced both axes 1 and 2 (axis 1: Arab, Haflinger and French pony, axis 2: French Saddlebred, Mérens, and Camargue). Breed represents respectively 33% and 30% of the total loadings of the qualitative variables on axes 1 and 2. - Type of work influenced separately the two first axes. High-school had the major loading on axis 1 (10%), followed by jumping, riding center and school training. Axis 2 was explained more by dressage (7.88%), leisure time riding, riding centre, and stallion. The equal loadings of riding centre on axes 1 and 2 suggests that horses assigned to this type of work would be characterized both by reasonable learning aptitudes and reasonably low levels of emotionality. - Housing had a weak influence on axis 1 (0.84%) but a stronger influence on axis 2 (27.8%). According to the usual restriction on general linear models (sum of level effects equal to zero), the two levels, box and paddock had the same loadings, but on opposite sides of the axis. Food had a negligible influence on axes 1 and 2, but it had a greater influence on axis 3 (not interpreted here). - Number of riders showed a noticeable influence on axes 1 and 2 (respectively 19.33% and 13.06%). Axis 1 was loaded mainly by the opposition between horses experiencing no rider and those experiencing many. The positions of these variables on the plot defined by axes 1 and 2 supported these comments (Fig. 1). Their precise effects on behaviour are analysed more precisely by ANOVA and MANOVA below. In summary, horses were distributed along the two factors on the plots. Axis 1 separated dressage, school or unridden horses as well as Arab horses, from Haflinger, voltige or western horses that tended to perform very well in the learning tests. Axis 2 opposed Fjord, Camargue or Merens and leisure horses in general to dressage, breeding stallions and French saddlebreds that tended to show higher indices of emotionality.

18 Interplay between environmental General tendencies emerged: show horses tended to present more emotional reactions than voltige, western, driving or unridden horses. Breeds like Quarter horses, French Trotters, Merens, Fjord, Camargue or New Forest in particular appeared to have less emotional reactions in tests as compared to French Saddlebreds, Anglo-Arabs, Arabs or Connemaras. Horses housed in boxes and fed with pellets were placed on the lower part of the plot. Obviously, some of these categories are linked (e.g. Quarter horses, appaloosas and western riding, young horses, breeding mares and paddock, etc.) and only detailed statistical analyses can disentangle the relative weights of these effects on the temperamental traits studied, which was indeed the next step in our study looking at the relative influence of the different factors. Relative influence of different factors Five factors were highly significant, amongst which three had predominant effects: One environmental factor: site (Wilks λ = 0.056, F = , df1 = 469, df2 = 1240, p = ) and two genetic factors (or at least factors involving genetic differences): breed (Wilks λ = 0.476, F = 1.82, df1 = 77, df2 = 1062, p = ) and sire (Wilks λ = , F = 1.225, df1 = 2527, df2 = 1247, p = ) appeared as highly important. Two other environmental factors were significant: type of work (Wilks λ = 0.517, F = , df1 = 84, df2 = 1086, p = ) and number of riders (Wilks λ = 0.759, F = 1.805, df1 = 28, df2 = 636, p = ) Only one interaction emerged, although it was not significant: between type of work and breed (Wilks λ = 0.228, F = 1.146, df1 = 224, df2 = 1068, p = 0.088). This interaction indicated that type of work could be a significant factor and that breeds react differently according to type of work.

19 Interplay between environmental Three factors were not significant at the chosen level (α = 0.05): sex (Wilks λ = 0.914, df1 = 14, df2 = 352, p = 0.311), way of life (Wilks λ = 0.944, F = , df1 = 7, df2 = 176, p = 0.166), and age (Wilks λ = 0.432, df1 = 133, df2 = 1174, p = 0.075). This result is rather surprising concerning the effects of sex and age. The strong sire effect is remarkable because there were many sires (N = 398). However, all the French ponies shared the same sire (a pure-bred Arab stallion), but their mothers came from different breeds (French saddlebred, Connemaras, Anglo-Arabs, etc.): the coefficients of variation for the comparisons among these siblings in the different tests were amongst the lowest, indicating important homogeneity of responses (N = 11 arena test: CV = 60%, all horses: , novel object test: CV = 87%, all horses: ). The most interesting result is probably that different factors influenced the responses of horses in relation to test, with predominance of type of work (F = 1.46; p = 0.025) in the arena test, predominance of sire effect (F = 1.55; p = 0.005) in the new object test, and predominance of breed effect (F = 2.15; p = 0.02) in the bridge test. Type of work (F = 2.47; p = ) and, to a lesser degree, number of riders (F = 2.35; p = 0.055) played a role in the memorization test. Genetic factors seemed to influence more neophobic responses, whereas environmental factors seemed to predominate more in gregariousness or learning (Fig. 2). Examples of differences in performance according to factor are given in figures 3 and 4. Although our data were not really appropriate for testing heritability (unequal samples: e.g. 1 to 20 offspring of each stallion), we calculated tentative coefficients and found values between 0.2 and 0.5. These values seem indicative of a good heritability of these behavioural traits but must be considered with caution, given the large variance due to the number of sires and breeds (Hausberger & Ricard, 2002). An example of additive gene-environment effects

20 Interplay between environmental Particular attention was paid to interactions between breed and site. We compared data for the 98 breeding stallions tested in different national facilities. These national facilities are government establishments where stallions are kept and made available for breeding. Either mares are brought there or sperm is taken and frozen. Each of these facilities has stallions of two to five different breeds, all kept under the same conditions. The different studs tend to present the same housing conditions (food, box, little or no training for riding). Housing and feeding were similar and the main activity was breeding. Stallions were tested outside the breeding period. The most probable differences between facilities were handling routines and behaviour of caretakers. A redundancy analysis (RDA) on these data showed (Fig. 5) that the first two axes contributed 66.5% to the variance and corresponded to the memorization test for axis 1 (41.2% of the variance) and the bridge test (handling test) for axis 2 (25.33% of the variance). All the horses from a given stud were clustered together. However, emotional reactions in the bridge test revealed the same gradient between breeds within each stud cluster: French Saddlebreds/Merens < Anglo-Arabs < Arabs. Thus, breed effect was similar everywhere, but an environmental factor (as facility effect) added to this effect and could induce a general shift. For example, facility 9 Arabs did not appear more reactive than facility 3 French Saddlebreds. Environmental conditions appeared to add to breed differences in the expression of emotional reactions. DISCUSSION Experimental tests performed on a large set of horses both confirmed the interplay of genetic and environmental factors in determining behaviour and revealed that this interplay varied according to test, confirming the multifaceted dimension of temperament (Mills 1998). Thus, genetic factors, as sire or breed, seemed to influence more neophobic reactions, whereas environmental factors, as type of work, seemed to play more determinant role in the

21 Interplay between environmental reactions to social separation or in learning abilities. Additive effects were evidenced, showing how environmental factors may modulate behavioural traits. Personality construct may be constrained by the relative "openness" to environmental factors of some traits and the "conservativeness" of others, more dependent on genetic factors. Genetic and environmental factors as determinants of horses behaviour Although, until recently, studies of horse temperament were scarce and genetics of behaviour in horses was still in its infancy (Houpt and Kusunose, 2001), evidence of some genetic bases of behaviour in this species are increasing (Hausberger & Richard 2004). No selection on the sole basis of behaviour, as those performed in quail (Mills et al., 1994), mink (Hansen, 1996), foxes (Belyaev & Trut, 1975) or great tits (Drent et al., 2002; van Oers et al., 2003) has been performed in horses, but some evidence exists through comparisons of breeds or bloodlines. Thus, individual differences in behaviour can be observed at an early age, and some seem to be related to sire (Wolff & Hausberger, 1994). A sire influence has been found on learning abilities (Wolff & Hausberger, 1996), emotional reactions (Wolff et al., 1997; Lankin & Bouissou, 1998; Houpt & Kusunose, 2001) or the tendency to develop stereotypic behaviour (Luescher et al., 1998, Vecchioti & Galanti, 1986). Breed differences have been observed in stereotypic behaviour (Redbo et al., 1998; Luescher et al., 1998; Hausberger et al., 1996), emotional reactions or learning abilities (Budzynski et al., 1992). The relative weights of these genetic factors and the way the environment may intervene are however largely unexplored and the present study is a first step towards understanding their interactions. In an earlier study, Gardner (1937), using an instrumental learning task, observed that age, breed and type of work had an influence. Moreover, the tendency to express stereotypic behaviour seems to be transmitted as a susceptibility that may or may not be

22 Interplay between environmental expressed in favourable environments (Luescher et al., 1998; Houpt & Kusunose, 2001). Our study on stallions shows that both influences are additive. Interestingly, type of work appeared as a major environmental factor. Although some breeds were selected for some types of work, in many cases different breeds were represented here for the same type of work (see appendix). Moreover, it appeared often that horses may have changed for example from racing to leisure, breeding or jumping. Our study takes only into account the present situation. Show horses, and particularly dressage horses, presented higher emotional reactions than most of the other horses. Dressage horses have also been shown to present higher frequencies of stereotypies in other studies (McGreevy et al., 1995; Hausberger et al., 1996). The fact that one-hour daily training may have such an important influence on general behaviour in the box or in behavioural tests, totally unrelated to the working situation, must be stressed. Apart from the fact that show horses live under more restricted conditions than leisure horses (box/paddock in particular), the importance of human-animal relationships on the general internal state of an animal is an interesting question to raise here, especially as totally untrained animals presented less emotional reactions in our tests. This could suggest either that being with a human (at least as far as going to the test arena) is more stressful for trained animals or that untrained animals have no expectations, contrary to the other horses that may be excited by the prospect of working. Caretakers seem to have an influence on the way horses react to being approached by humans (Hausberger & Muller, 2002) and human factors may well be responsible for the differences in behaviour we observed in stallions in relation to stud (this study) showing as well that the human influence can reinforce or on the contrary diminish the emotional state of a horse. Handling may also reduce fear in young horses (e.g. Jeziersky, 1999). On the other hand, unridden horses, as high-school horses, showed lower scores in the learning test. This may be because, during training, horses are stimulated to learn or learn to learn. However, high-

23 Interplay between environmental school horses, which have to perform highly sophisticated exercises as elevated paces with a high level of temporal precision, showed the lowest level of learning performance. One hypothesis may be that, given the precision required, riders give little freedom to the horse that may then not learn to learn. These findings would certainly deserve further consideration. Temperament/personality as a multifactorial system The most important finding in this study is certainly the fact that different factors do not have the same weights in determining behaviour in relation to the situation. As mentioned by Gosling (2001), animal studies provide a useful framework in which to examine how an individual s personality is influenced by its biology, genes, social and non-social environment and the interaction among these factors. Whereas it is usual in psychometrical approaches to rate different facets of temperamental traits, variability in reactions in relation to behavioural test are easily interpreted as either a lack of individual consistency across situations or the tests are invalidated. Several studies in horses have shown that results to different tests were not correlated either when emotional reactions (Wolff et al., 1997; Seaman et al., 2002; Visser et al., 2001) or learning abilities (Wolff & Hausberger, 1996; Nicol, 2002; Visser et al., 2003b) were involved. Anderson et al. (1999) found no correlations between cortisol levels and measurements of emotionality. These findings agree with those obtained in other species like beef cattle (Boivin et al., 1992) or pigs (Lawrence, 1991). Even guppies do not show the same patterns of emotionality in response to an open-field, a predator or the visual presence of conspecifics (Budaev, 1997), whereas fear reactions to humans and to novel objects are not correlated in wolves (McDonald, 1983). Interestingly, in horses, correlations were found between reactions to behavioural tests and riders or observers ratings (Le Scolan et al., 1997; Visser et al., 2003a). Thus, Le Scolan et al. (1997) found that horses reacting strongly

24 Interplay between environmental to the arena test were difficult to separate from others in the home working situation or that horses that had difficulties crossing the unknown bridge were also rated as being more fearful, which does indeed suggest some consistency across situations. In fact, as suggested by Budaev s (1997) study in guppies, predators, novel objects, novel spaces or social separation may not elicit the same internal states, nor should humans necessarily elicit the same emotions as novel inanimate objects. Studies like the present one analysing the weights of factors in determining the reactions in given behavioural tests, may prove useful in clarifying such findings. Thus Whitney (1970) found significant effects due to genotype, sex and environmental stimulation on open-field defecation scores in mice. Those with the highest scores (more fearful ) however emerged faster from their home cage (less timid ). Ostaszewski et al. (1994) found that the need in rats for kinaesthetic-tactile stimulation was more strongly genetically determined than their need for light stimulation. Grignard et al. (2001) found a stronger sire effect in a docility test than in a crush test in beef cattle, while the degree of heritability differed according to test (separation/restraint) and breed (German Angus/Simmental cattle) in Gauly et al. s (2001) study. In a study on chimpanzees, based on personality ratings, Weiss et al. (2000) found that only dominance was significantly heritable whereas estimates for agreeableness and emotional stability were small. All these studies converge on the fact that determination of behavioural traits varies differentially with genetic influence. Our study on horses confirms these findings but, in addition, attempted to examine which environmental factors may interact with each other or with the genetic bases. We confirm the multifaceted aspect of temperament and recommend caution when attempting to generalize concepts like fear or emotionality. Whether psychometrical approaches (e.g. Gosling, 1998; Morris et al., 2002) or behavioural tests are used is probably not crucial in that both approaches may lead to complementary sets of data.

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