B Forkman

A Boissy

M.-C Meunier-Salaün

E Canali

R B Jones

A critical review of fear tests used on cattle, pigs, sheep, poultry and horses

Keywords: Fear, Cattle, Sheep, Pig, Poultry, Horse, Open field, Tonic immobility, Novel object

Fear is arguably the most commonly investigated emotion in domestic animals. In the current review we attempt to establish the level of repeatability and validity found for fear tests used on cattle, pigs, sheep and goats, poultry and horses. We focus the review on the three most common types of fear tests: the arena test (open field), the novel object test, and the restraint test. For some tests, e.g. tonic immobility in poultry, there is a good and broad literature on factors that affect the outcome of the test, the validity of the test and its age dependency. However, there are comparatively few of these well defined and validated tests and what is especially missing for most tests is information on the robustness, i.e., what aspects can be changed without affecting the validity of the tests. The relative absence of standardized tests hampers the development of applied ethology as a science.

Part 1 -Justification and background

Fear and anxiety are two closely related emotions. Fear is generally defined as a reaction to the perception of actual danger, whereas anxiety is defined as the reaction to a potential danger that threatens the integrity of the individual [START_REF] Boissy | Fear and fearfulness in determining behavior[END_REF]. Fearrelated reactions are characterized by physiological and behavioral reactions preparing the animal to deal with the danger. From an evolutionary standpoint, defensive reactions promote fitness in wild animals: the life expectancy of an animal is obviously increased if it can react to avoid sources of danger such as predators. Although natural predators are largely absent for animals kept in captivity the mechanisms and the emotion persists, together with the behavioral responses (e.g. [START_REF] Dwyer | How has the risk of predation shaped the behavioural responses of sheep to fear and distress?[END_REF]). In addition populations of domestic animals reared in range environments may still experience severe predation by wild animals or dogs (e.g. [START_REF] Asheim | External effects of mitigating measures to reduce large carnivore predation on sheep[END_REF]). Routine management procedures can elicit fear-related responses. For example, shearing, castration, tail docking, dehorning, vaccination, herding and transportation have been reported to be stressful to cattle and sheep [START_REF] Wohlt | Cortisol increases in plasma of Holstein heifer calves from handling and method of electrical dehorning[END_REF][START_REF] Hargreaves | The effect of gentling on heart rate, flight distance and aversion of sheep to a handling procedure[END_REF]. Moreover, predator-avoidance behaviors against humans are still observed in farm species, though reduced fear of human beings is generally considered to be a major component of domestication [START_REF] Price | Behavioural aspects of animal domestication[END_REF]. In addition, besides its influence on the responses towards potentially dangerous situations, excessive fear may also lead to develop chronic stress known to alter fundamental behaviors (social, sexual and parental relationships) and reduce productivity in domestic herbivores. For instance, fear-related reactions affect sexual and maternal behaviors and social dominance ability in cattle and sheep [START_REF] Bouissou | The social behaviour of cattle[END_REF][START_REF] Fisher | The social behaviour of sheep[END_REF]. Reducing the frequency of potential aversive events or providing additional positive experiences to the animals, such as handling or training (reviewed by Hemsworth and Coleman [START_REF] Hemsworth | Human-livestock interactions[END_REF]) may help to make the environment more suitable for livestock. It may also allow adjusting animal fearfulness, defined as the propensity to experience fear or anxiety [START_REF] Wohlt | Cortisol increases in plasma of Holstein heifer calves from handling and method of electrical dehorning[END_REF]. In addition, heritability of fearfulness has been reported in laboratory species (reviewed by Ramos and Mormède, [START_REF] Ramos | Stress and emotionality: a multidimensional and genetic approach[END_REF]) and in farm species (reviewed by Boissy et al. [START_REF] Boissy | Genetics of fear in ruminant livestock[END_REF]). Therefore, environmental strategies and genetic selection both aimed at reducing fearfulness might be of economic and ethical significance for livestock production.

1.1. Diversity in the nature of fearful events According to Gray [START_REF] Gray | The psychology of fear and stress[END_REF], the fear-eliciting nature of an event is due to the general characteristics of the event, e.g. novelty and physical characteristics of its presentation, such as movement, intensity, duration, suddenness or proximity. Fear may be also be elicited by specific stimuli, such as height and darkness, in relation to the evolutionary history of the species (ancestral fears/innate fears). In addition, an event can elicit fear by being associated by previous experience with another fearful event (conditioned fear). Since the major characteristic of farm animals is their gregariousness [START_REF] Keeling | Social. behaviour. in. farm. animals[END_REF], fear may be induced through a variety of social stimuli. Triggering social signals can represent particular cases of the previous types of fear-eliciting stimuli. Some social signals are characterized by their novelty, as the novelty of the neonate that affects maternal behaviour in primiparous females [START_REF] Poindron | Comparaison du comportement maternel en bergerie à la parturition chez des brebis primipares ou multipares de race Romanov, Préalpes du Sud et Ile de. France[END_REF]. Other social signals, such as odors or alarm calls, can spontaneously elicit fear [START_REF] Boissy | Presence of cues from stressed conspecifics increases reactivity to aversive events in cattle: evidence for the existence of alarm substances in urine[END_REF]. Triggering social signals may also be acquired, as threats in the case of dominance hierarchies [START_REF] Bouissou | The social behaviour of cattle[END_REF]. In addition, social isolation is probably one of the most important stressful components for all social species that are generally tested individually for measuring fear. Various studies suggest that most of the domestic species have a high social motivation and consequently may suffer more from separation anxiety than from the fear-eliciting event per se.

Diversity in fear-related responses

Behavioral patterns related to fear vary greatly depending on the characteristics of the threat. They can be contradictory, since both active and passive strategies are observed in challenging situations: active defense (attack, threat) or active avoidance (flight, hiding, escape) and passive avoidance (immobility) can be viewed as expressing fear [START_REF] Erhard | Tonic immobility and emergence time in pigsmore evidence for behavioural strategies[END_REF]. Other behavioral patterns can also be considered as fear indicators, including some expressive movements, such as postures of the head and facial expressions, and some specific alarm calls, as well as odors or pheromones. These behavioral patterns play an important part in the social communication of herbivores by serving as signals to alert conspecifics. Fear-eliciting stimuli may also affect the activity in which the animal is engaged. When the level of fear is low, the activity may be enhanced: the administration of weak electric shocks leads to an increase in food intake. In contrast, when fear is intense, the activity can be disturbed or totally inhibited: regular noxious stimuli inhibit social interactions between animals. Finally, conflict between a negative emotional state and a positive motivation may result in a compulsive behaviour such as nibbling of a chain. The activation of the sympathetic nervous system-adrenal medulla and the hypothalamic-pituitary-adrenal system are the main neuroendocrine responses associated with negative emotions (reviewed respec-tively by von Borrel et al. [START_REF] Borrel | This volume[END_REF] and Mormède et al. [START_REF] Mormède | [END_REF] in this volume). A range of complex central nervous mechanisms, such as neural pathways and neurotransmitters, that occurred during fearful situations have also been widely studied (reviewed by Gray, [START_REF] Gray | The psychology of fear and stress[END_REF]).

Variety of experimental designs

The experimental situations that have been designed to study fear in farm animals are various. For the most of them, they were originally developed for laboratory species. Since the classic work of Hall in 1936 [START_REF] Hall | Emotional behaviour in the rat: III The relationship between emotionality and ambulatory activity[END_REF], the open-field or novel arena test has been extensively used in rodents [START_REF] Archer | Tests for emotionality in rat and mice: a review[END_REF]. Generally, a single animal is placed in a large novel area and the amount of defecation and activity is interpreted as reflecting the response to novelty. Subsequent work has shown that this test provides a number of other threatening events, such as absence of shelter and landmarks, social isolation and bright lighting. Many other tests were devised to assess fear in rodents, such as exposure to a predator or a novel object, confinement, handling by humans, administration of inescapable noxious stimuli, and passive or active avoidance conditioning [START_REF] Ramos | A multiple test study of anxiety related behaviours in six inbred rat strains[END_REF]. This is only more recently that most of these experimental paradigms were used in domestic animals. The open-field design, commonly called "Novel Arena test" (NAT), is applied to a wide range of farm animals, such as ruminants, pigs, horses and poultry. Fear of novelty is also evaluated through the exposure to a novel object ("Novel Object test", NOT). The exposure to a human has been specifically developed for farm animals to study their fearrelated responses, with the animal that is either approached by a human ("Forced Approach test", FAT) or is free to approach a human ("Voluntary Approach test", VAT). Confinement is also widely used in farm animals ("Tonic Immobility test", TI and "Restraint test", RT). Fear tests based on exposure to human tend to use forced vs. voluntary approach tests uncritically. According to Waiblinger et al. [START_REF] Waiblinger | Assessing the human animal relationship in farmed species: a critical review[END_REF], the forced approach test would probably increase the likelihood that an animal will respond more actively to the human whereas in the voluntary approach test, the chances of getting no response or a passive response would probably be higher. Finally, fear is also evaluated from the responses of the animals exposed either to a natural predator of the given domestic species ("Predator test", PT), or to a sudden sound or a visual stimulation ("Startle test", ST), or to a signal that had been previously associated with a nociceptive event such as electric shock ("Conditioned Fear test", CER).

The complexity of fear responses

Behavioral and endocrine reactions to fearful events are only indicators of fear and cannot be considered as direct measures of a subjective state of fear. Because of the complexity of the mechanisms underlying fear-related responses, it is not possible to attribute a given behavior to any single emotion [START_REF] Boissy | Fear and fearfulness in determining behavior[END_REF]. For instance, in cattle, the response to a novel object depends on the experimental designs. When the cows voluntarily approach the novel object, the most reactive animals to humans have the strongest tendency to approach it; by contrast, when the cows are forced to move toward the same novel object, just the opposite is true [START_REF] Murphey | Responses of cattle to humans in open spaces: breed comparisons and approach avoidance relationships[END_REF]. Therefore, a measurement used as an emotional indicator in one situation cannot be extrapolated directly to others, and it is impossible to assess simply the magnitude of concepts like fear on the basis of a single "objective and perfect" measurement.

The absence of validation in experimental designs

Apart for the tests implicating an exposure to humans, most of the tests used in farm animals for measuring fear have been previously designed for laboratory animals. However, such tests were generally used in applied ethology without taking into account their biological significance for farm animals. Clearly, there are limitations in extrapolating experimental paradigms between lab animals and domestic species, since differences of ecological characteristics and motivations across species have been identified. Laboratory animals are nocturnal whereas most domestic animals are diurnal. Laboratory animals show thigmotaxis whereas domestic animals often come from open areas (is open field in cattle an example of closed field?). In farm animals, young animals are generally precocious and the social dimension is extremely developed in most of farm animals that are highly gregarious and present exclusive mother-young relationship. Therefore, testing animals in inappropriate environmentsthat is likely to result in abnormal and maladaptive behaviourcan lead to an inaccurate estimation of fear and may explain the lack of links between studies. A reconsideration of the ecological context of the domestic species has to be conducted in order to elaborate and to validate more realistic conditions of fear stimulation.

In addition, studies in lab animals could shed light on how to better interpret the range of emotional reactions experienced by farm animals in aversive situations. A few studies in lab animals emphasize the relationship between emotions and cognition by showing how mental operations of evaluation are required to produce emotional reactions (reviewed by Boissy et al. in this volume [START_REF] Boissy | This volume[END_REF]). In addition to suddenness, unfamiliarity and unpredictability, the ability to cope with the challenging event can change the perception of the animal. Various forms of evaluative processing, ranging from rapid to subtle, occur to differentiate the emotional experiences. Suddenness, unfamiliarity and unpredictability have been recognized to produce, or at least affect, fear reactions of animals. For instance, sudden or unfamiliar events are often used in various species to assess the animals' fearfulness [START_REF] Boissy | Fear and fearfulness in determining behavior[END_REF]. From an ecological point of view, suddenness, unfamiliarity and unpredictability are the key features of a predatory attack, and domestic ungulates in range environments may still experience predation by wild animals or dogs [START_REF] Shelton | Predatory losses: a serious livestock problem[END_REF]. Tests for suddenness use the presentation of a stimulus such as a ball falling suddenly from the ceiling in front of the animal [START_REF] Romeyer | Assessment of fear reactions in domestic sheep, and influence of breed and rearing conditions[END_REF] or a blast of compressed air applied on the muzzle [START_REF] Boissy | Assessment of individual differences in behavioural reactions of heifers exposed to various fear-eliciting situations[END_REF]. As reported earlier, unfamiliarity is always included in the so-called open-field test. Finally, the unpredictability of an aversive stimulus is known to enhance stress responses [START_REF] Weiss | Psychological factors in stress and disease[END_REF]. For instance, in rats, the corticosterone response to repetitive exposure to a novel cage is more pronounced when the exposures are irregular than regular [START_REF] Muir | Time course of the corticosterone and prolactin response following predictable and unpredictable novelty stress in Rattus norvegicus[END_REF]. Not only aversive stimuli but also the unpredictability of pleasant stimuli can affect fear in animals. For instance, rats present stress-induced analgesia when food delivery is unpredictable [START_REF] Tazi | Prediction and control of food rewards modulate endogenous pain inhibitory systems[END_REF]. More generally, measures of cognitive processes, such as anticipation, can provide information about animal emotion: anticipatory behavior measured in a Pavlovian conditioning paradigm has been identified in rodents as a potential indicator of emotion [START_REF] Spruijt | A concept of welfare based on reward evaluating mechanisms in the brain: anticipatory behavior as an indicator for the state of reward systems[END_REF]. In addition to the intrinsic characteristics of the challenging event (i.e., its suddenness, unfamiliarity and unpredictability), the ability to cope with the event can change the emotional experience of the animal. For instance in cattle, the response to a novel object depends on the environmental context. When cows voluntarily approach a novel object, the most reactive ones to humans also have the strongest tendency to approach the object; but when the cows are forced to move toward the same novel object, the opposite is observed [START_REF] Murphey | Responses of cattle to humans in open spaces: breed comparisons and approach avoidance relationships[END_REF]. Therefore, the definition of experimental situations according to the basis of the evaluation abilities of the animals should help to interpret the variety of behavioral and physiological responses, and thus to better access the emotional world of these latter.

The absence of current validation in experimental designs and the lack of standard protocols are hampering research into fear today. This review is an attempt to organize the methodology used to assess fear in farm animals. The species included in the review are: cattle, pig, sheep and goats, chicken and quail, and horse. The methods are briefly described, and the repeatability and validity for each test in each species given. Because of the recent publication of a review on human animal interaction [START_REF] Waiblinger | Assessing the human animal relationship in farmed species: a critical review[END_REF] the tests involving human contact have not been included in the present review. Additional information concerning the stability of the test, i.e. the effect of minor changes in the procedure, is discussed.

Discussing the efficiency of the most frequently used experimental designs for measuring fear in domestic animals is essentially based on two criteria, which are validity and reliability. Validity refers to the relation between a measured variable and what it is supposed to predict. Martin and Bateson [START_REF] Martin | Measuring behaviour, an introductory guide[END_REF] defined validity by accuracy (i.e., the degree of freedom from systematic errors that might over-or underestimate the measured variable), specificity (i.e., the extent to which a measured variable reflects what it is supposed to and nothing else) and scientific validity (i.e., the extent to which the method gives relevant information and answers to the hypothesis). Reliability is defined by Martin and Bateson [START_REF] Martin | Measuring behaviour, an introductory guide[END_REF] as being the degree to which measures are free from random errors. It is partly determined by the repeatability or the consistency of the measures (i.e., repeated measures of the same construct should produce the same result). Assessing repeatability for validating fear tests is a real unresolved problem. Because animals can react differently, some may habituate to the test-situation and some others may lower their threshold for expressing fear after being subjected to the same test situation several times. This is truer when taking into account tests based on novelty since test situations are not novel from the second exposure: Should we expect a habituation process that takes to reduce fear responses [START_REF] Brush | Genetic differences in avoidance learning by Rattus norvegicus: escape/ avoidance responding, sensitivity to electric shock, discrimination learning and open-field behavior[END_REF] reported that rats exhibiting low performance during active avoidance conditioning have a higher defecation rate in open-field. The defecation rates recorded on rats subjected to a conditioned fear then to an open-field are positively correlated [START_REF] Ley | Open-field behavior, emotionality during fear conditioning and fearmotivated instrumental performance[END_REF]. Good correlations among responses to a few kinds of aversive stimuli are also obtained in dogs [START_REF] Goddard | A factor analysis of fearfulness in potential guide dogs[END_REF]. Likewise, numerous correlations are found in birds among reactions to open-field, emergence and tonic immobility tests in chicks [START_REF] Jones | Estimation of fear in two lines of the domestic chick: correlations between various methods[END_REF], hens [START_REF] Jones | Repeatability of fear ranks among adult laying hens[END_REF] and Japanese quails [START_REF] Mills | The estimation of fear in domestic quail: correlations between various methods and measures[END_REF]. Concerning domestic ungulates, several correlations are found between behavioral patterns reflecting fear in different aversive situations: variables which express a high level of fear, are positively correlated through the various tests and negatively correlated with those expressing a low level of fear (heifers: [START_REF] Boissy | Effects of androgen treatment on behavioural and physiological responses of heifers to fear-eliciting situations[END_REF]; sheep: [START_REF] Romeyer | Assessment of fear reactions in domestic sheep, and influence of breed and rearing conditions[END_REF][START_REF] Vandenheede | Sex differences in fear reactions in sheep[END_REF]. Taken together, there is a strong tendency for individual characteristics to manifest themselves across a variety of aversive situations. Validation throughout inter-test consistency should thus be given more attention in futures studies.

Fear tests in cattle

The most commonly used fear test in cattle is the novel arena test, novel object and the voluntary approach, forced approach and different type of restraint tests are less used (Table 1). These tests are usually done one by one, but in some studies combined tests are carried out for example by including a novel object or a human, another calf or food in the novel environment right from the start [START_REF] Boissy | Assessment of individual differences in behavioural reactions of heifers exposed to various fear-eliciting situations[END_REF][START_REF] De Passillé | Interpreting the behaviour of calves in an open field test: a factor analysis[END_REF][START_REF] Lauber | The effects of age and experience on behavioural development in dairy calves[END_REF].

Novel arena test

Background

This test is often used in calves, heifers and cows and the variables most commonly recorded are: latency to enter and to leave the arena, locomotor activity (number of square entered, standing, walking), exploration (sniffing) eliminative behavior (defecation, miction), vocalizations [START_REF] Kilgour | The open field test as an assessment of the temperament of dairy cows[END_REF], and escape attempts [START_REF] Kilgour | Individual differences in the reaction of beef cattle to situations involving social isolation, close proximity of humans, restraint and novelty[END_REF]. The arena varies from 10-12 m 2 for calves and heifers up to 100 m 2 for cows. The arena has usually solid wall up to 2.6 m. Not only the size and the location (outdoor vs. indoor) but also the shapes differ among the studies. The time spent in the arena varies between 3 and 30 min. As already reviewed by [START_REF] Munksgaard | The use of "open field" tests in the assessment of welfare of cattle[END_REF] the duration of the test may influence the results obtained in the arena. Handling procedures and latency to enter the arena differ between authors. Cattle are tested at different ages: calves from 2 weeks of age since 6 months, heifers were tested at 15 and 18 months of age and in some studies cows were tested. The open-field test is sometime combined with others tests performed thereafter within the same arena or restraint test, or with behavior expressed in their pen. In the majority of the experiments the animals are tested alone, and only rarely in the presence of another calf.

Repeatability

The question of the relevance to expose repeatedly individuals to the same experimental designs when they are based on novelty has already been raised in the general background. Nevertheless, some open field studies reported intra-test consistency over time when the repetitions between tests are close and in cows [START_REF] Kilgour | The open field test as an assessment of the temperament of dairy cows[END_REF][START_REF] Kovalčikováč | Relationships between parameters of open field test of cows and their milk production in loose house[END_REF] while in calves there were no stability [START_REF] De Passillé | Interpreting the behaviour of calves in an open field test: a factor analysis[END_REF]. The most repeatable variables are movement, vocalizations, time spent immobile and exploration but at various levels according to studies. Locomotion seems more related to activity than fear of novelty in heifers [START_REF] Boissy | Assessment of individual differences in behavioural reactions of heifers exposed to various fear-eliciting situations[END_REF] and to social isolation in calves [START_REF] De Passillé | Interpreting the behaviour of calves in an open field test: a factor analysis[END_REF]. The repeatability studies have used very different time intervals, from no interval [START_REF] Kilgour | The open field test as an assessment of the temperament of dairy cows[END_REF] to 1 year [START_REF] Neindre | Influence of rearing conditions and breed on social behaviour and activity of cattle in novel environments[END_REF] and in all cases the animals were tested alone.

Validity

The overall picture is that the behavior of cattle in an open field situation does correlate with their behavior in other fear tests, at a low level [START_REF] Boissy | Assessment of individual differences in behavioural reactions of heifers exposed to various fear-eliciting situations[END_REF][START_REF] Jensen | Effect of single versus group housing and space allowance on responses of calves during open field tests[END_REF][START_REF] Kilgour | Individual differences in the reaction of beef cattle to situations involving social isolation, close proximity of humans, restraint and novelty[END_REF] or not at all, there are no correlations with reactions to humans. [START_REF] Kilgour | The open field test as an assessment of the temperament of dairy cows[END_REF][START_REF] Boissy | Effects of early handling on heifers' subsequent reactivity to humans and to unfamiliar situations[END_REF][START_REF] Boivin | Influence of breed and early management on ease of handling and open field behaviour of cattle[END_REF].

Conclusion

Because of the lack of strong correlation with other tests of fear the open field test can not be recommended as a general fear test for cattle. The repeatability for some of variables is good and the test seems to capture some part of the behavioral tendencies of an individual animal. However care should be taken in the interpretation of the test results. As pointed out [START_REF] Neindre | Influence of rearing conditions and breed on social behaviour and activity of cattle in novel environments[END_REF] results are very difficult to understand and many factors can lead to the same activity in the open field. This is the case of locomotion which can reflect fear but also investigation. For this reason correlations among behaviors or multivariate analysis could help in the interpretation of the different motivations. Social motivation can be the primary factor affecting the open arena behavior, especially when group-reared animals are tested individually [START_REF] Munksgaard | The use of "open field" tests in the assessment of welfare of cattle[END_REF]. Moreover the lack of consistency of responses over time may result an altered motivation over time (related or not to the rearing systems) or an habituation effect. The perception of the novel environment seem to change by aging (older calves are more active in open-field [START_REF] Boivin | Influence of breed and early management on ease of handling and open field behaviour of cattle[END_REF]). These changes may also occur when tests are repeated close in time.

Comment citer ce document : Forkman, B., Boissy On the other hand methodological problems can arise with repeated tests at long time span with the size of arena unadjusted to the changes in the body size of an animal. This test could be useful only as complementary test or to study other motivation such as the need for locomotion in confinement situation [START_REF] Jensen | A note on the effect of isolation during testing and length of previous confinement on locomotor behaviour during open-field test in dairy calves[END_REF].

Novel object test

The novel object test is seldom used independently in cattle, more commonly it is performed in the same pen used as a previous open-field test where different tests are combined. The novel stimulus tested are usually visual, an object introduced by human on the floor before the habituation period within the arena [START_REF] Dellmeier | Effect of changing housing on open field behaviour of calves[END_REF][START_REF] Boissy | Assessment of individual differences in behavioural reactions of heifers exposed to various fear-eliciting situations[END_REF], or dropped from the ceiling and left on the floor or after [START_REF] Miller | The effect of rearing systems on the response of calves to novelty[END_REF]. Cattle are tested alone. Habituation period to the test arena before the confrontation to a novel object varies between 1 and 15 min. Animals are tested at very different age calves and heifers. The variables most commonly recorded indicated the interest towards the novel stimulus measured by the latency to the contact, distance from the novel object, frequency or duration of contact, exploration (licking, smelling), body posture, vocalizations (Table 2).

Repeatability

One study have 8 repetitions at 1-week interval [START_REF] Dellmeier | Effect of changing housing on open field behaviour of calves[END_REF] but novel object test was done at the same time of the open field test, the authors found a decrease in the exploratory behavior in general. In another study the test was repeated three times at 3-4 weeks of interval and there was a decline in the interactions with novel object and the coefficient of repeatability was near to zero [START_REF] Kilgour | Individual differences in the reaction of beef cattle to situations involving social isolation, close proximity of humans, restraint and novelty[END_REF].

Validity

One study found a positive correlation between latency to exit the room and latency to approach novel object (Rho = 0.50) and a negative correlation between latency to exit the room and

Conclusion

There is very little done on novel substrate, and more work needs to be done before it can be said that it is a valid test.

Other tests

Only three papers used handling test and other fear test such as the novel area test [START_REF] Boivin | Influence of breed and early management on ease of handling and open field behaviour of cattle[END_REF][START_REF] Boissy | Effects of early handling on heifers' subsequent reactivity to humans and to unfamiliar situations[END_REF][START_REF] Kilgour | Individual differences in the reaction of beef cattle to situations involving social isolation, close proximity of humans, restraint and novelty[END_REF]. Handling tests involve human presence and consequently combine the potential stressful trait of the procedure of handling and the fear towards human. The animals were tested with the presence of conspecifics or individually. Various procedures were used in the handling tests including husbandry practice such to be separated by peers or the restraining it in a specific area or haltering, leading and tethering. In handling test the variables most commonly recorded concerns the ease, the attempts and time necessary to be separated from the other animals and the responsiveness to restraint both expressed as scores including motor reactivity.

Repeatabilityvalidity

There is a consistency between tests and physiological parameters [START_REF] Boissy | Effects of early handling on heifers' subsequent reactivity to humans and to unfamiliar situations[END_REF] but no relationships between tests [START_REF] Boivin | Influence of breed and early management on ease of handling and open field behaviour of cattle[END_REF] possibly due to different tests used and different breeds.

Conclusion

Type of handling and the human involvement in the handling procedures need to pay attention on the interaction between the confidence level of tested animals towards the handler and the responsiveness to handling itself and the importance of conspecifics during the tests.

Fear tests in pigs

The most commonly used fear tests in pigs are the open field, novel object and the voluntary approach tests. These tests are usually done one by one, but in many studies combined tests are carried out for example by letting a novel object fall into the open field arena after an habituation period within the test arena, or by including a novel object or a human in the novel environment right from the start. More recently new tests have been developed such as the back test, elevated-plus-maze test (EPM) or Dark/Light Test (D/L).

Novel arena test

Short background

This test is often used in pig [START_REF] Fraser | The vocalizations and other behaviour of growing pigs in an 'open field' test[END_REF] and the variables most commonly recorded are: locomotor activity (lying, standing, and exploration), eliminative behavior (defecation, miction) and vocalizations (squeals, grunts). The arena is usually between 5 to 10 m 2 for piglets (up to 8 weeks of age), adjusted to the body length of tested animals especially for older animals. The time spent in the arena varies between 5 and 20 min. In some cases a walkway or an alley just outside the box is used as an open field arena with "unusual" dimensions, very long and narrow, e.g. 1 × 7 m. Pigs are typically tested 1 or 2 weeks after weaning, or less frequently at older stage [START_REF] Von Borell | Relationship between behaviour and adrenocortical response pattern in domestic pigs[END_REF][START_REF] Thodberg | A general reaction pattern across situations in prepubertal gilts[END_REF][START_REF] Ruis | Personalities in female domesticated pigs: behaviouiral and physiological indications[END_REF]. The open-field test was usually combined with others tests performed thereafter within the same arena [START_REF] Jensen | Individual variation and consistency in piglet behaviour[END_REF][START_REF] Thodberg | A general reaction pattern across situations in prepubertal gilts[END_REF][START_REF] Van Kooij | Can we predict behaviour in pigs? Searching for consistency in behaviour over time and across situations[END_REF].

Repeatability

The results from the open field studies are consistent over time, with the most repeatable variables being activity and vocalizations but at various levels according to studies [START_REF] Fraser | The vocalizations and other behaviour of growing pigs in an 'open field' test[END_REF][START_REF] Jensen | Individual variation and consistency in piglet behaviour[END_REF][START_REF] Von Borell | Relationship between behaviour and adrenocortical response pattern in domestic pigs[END_REF]. By contrast locomotion is more related to activity than fear of novelty and defecation does not represent a general response to frightening situation. The repeatability studies have used very different time intervals, from 2 days [START_REF] Jensen | Individual variation and consistency in piglet behaviour[END_REF] to 18 weeks [START_REF] Von Borell | Relationship between behaviour and adrenocortical response pattern in domestic pigs[END_REF][START_REF] Ruis | Personalities in female domesticated pigs: behaviouiral and physiological indications[END_REF] and in all cases the animals were tested alone. In a study in which the pigs were tested in group, the result deviates from those of other studies in that they did not find any consistency of behavior between tests [START_REF] Ruis | Personalities in female domesticated pigs: behaviouiral and physiological indications[END_REF].

Validity

The overall picture is that the behavior of pigs in an open field situation does correlate with their behavior in other fear tests, at low level (rs b 0.36) or not at all [START_REF] Thodberg | A general reaction pattern across situations in prepubertal gilts[END_REF][START_REF] Andersen | Effects of diazepam on the behaviour of weaned pigs in three putative models of anxiety[END_REF][START_REF] Giroux | Relationships between individual behavioural traits and post-weaning growth in segregated early-weaned piglets[END_REF][START_REF] Ruis | Personalities in female domesticated pigs: behaviouiral and physiological indications[END_REF]. Nor does the behavior in the open field appear to be sensitive to the administration of diazepama common anxiolytic that does affect the open field behavior in rodents [START_REF] Andersen | Behavioural evaluation of methods for assessing fear responses in weaned pigs[END_REF]. In return positive correlations were reported between the adrenocorticol reactivity measured in an ACTH challenge and ambulation or vocalization scores [START_REF] Von Borell | Relationship between behaviour and adrenocortical response pattern in domestic pigs[END_REF]. Relationships were also reported between the openfield responses and the maternal behavior [START_REF] Thodberg | A general reaction pattern across situations in prepubertal gilts[END_REF].

Conclusion

Because of the lack of strong correlation with other tests of fear the open field test can not be recommended as a general fear test for pigs. The repeatability for a number of variables is good and the test seems to capture some part of the behavioral tendencies of an individual animal. However care should be taken in the interpretation of the test results. Social motivation can be the primary factor affecting open-field behavior, at least when group-reared animals are tested individually. Moreover the lack of consistency of responses over time may result an altered motivation over time or a habituation effect. Although results suggested reduced fear related with a reduced threatening impact of leaving the home pen, it could be indicated an increased willingness to move from their home pen [START_REF] Geverink | Effects of regular moving and handling on the behavioural and physiological consequences to preslaughter treatment and consequences for subsequent meat quality[END_REF]. The perception of the novel environment may also be changed by aging, the animals becoming more experienced with various events. On the other hand methodological problems can be arise with repeated tests at long time span with the size of arena unadjusted to the changes in the body size of an animal.

In contrast to the often-described "emotionality" in laboratory animals characterized by freezing and defecation, the vocalization criteria give a relevant indication for the level of excitement in response to a novel situation. In further investigations the size of apparatus arena should be standardized or at least the size of a square, e.g. have it as the body length of the animal. In addition it should be important to pay attention before the test itself to the previous context especially when animals are tested outside their home pen, e.g. by training animals to move in free or limited stressful conditions to the test arena and to stay in the waiting box. Latency time to leave the home pen in the first test may be a useful measure of fear or timidity to leave the familiar environment [START_REF] Erhard | Tonic immobility and emergence time in pigsmore evidence for behavioural strategies[END_REF].

Novel object test

Short background

The novel object test is often performed after a habituation period to a barren open arena used in some case as a previous open-field test when different tests are combined [START_REF] Spoolder | Individual behavioural differences in pigs: intra and inter test consistency[END_REF]. The test can be also performed in home pen [START_REF] Forkman | Personality, coping patterns, and aggression in piglets[END_REF][START_REF] Van Kooij | Can we predict behaviour in pigs? Searching for consistency in behaviour over time and across situations[END_REF]. The novel stimulus tested are usually visual, an object introduced by human on the floor after the habituation period within the arena or dropped from the ceiling and left on the floor. The visual stimulus is typically a bucket, colored (red) or bright. The novel substrate can also concern olfactory cues, as unfamiliar odor or ammoniac [START_REF] Jones | Do pigs find a familiar odourant attractive in novel surroundings?[END_REF].

Animals are typically usually tested alone between 1 and 12 weeks after weaning, or more rarely older. Habituation period to the test arena before the confrontation to a novel object varies between 3 and 5 min or less. The arena is usually between 8 and 26 m 2 for piglets or gilts (3 weeks-16 weeks). The novel test can also be performed in the home pen and combined with other tests [START_REF] Van Kooij | Can we predict behaviour in pigs? Searching for consistency in behaviour over time and across situations[END_REF]. The variables most commonly recorded indicated the interest towards the novel stimulus measured by the latency, frequency or duration of contact, exploration and attention without physical contact.

Repeatability

Studies have used very different time intervals, from few days [START_REF] Spoolder | Individual behavioural differences in pigs: intra and inter test consistency[END_REF] to 12 weeks [START_REF] Van Kooij | Can we predict behaviour in pigs? Searching for consistency in behaviour over time and across situations[END_REF]. It should explain various results on the consistency over time. Vocalizations and object investigation appeared repeatable when measured in gilts over few days during a 2-week period [START_REF] Spoolder | Individual behavioural differences in pigs: intra and inter test consistency[END_REF], whereas Jensen et al. [START_REF] Jensen | Individual variation and consistency in piglet behaviour[END_REF] found no correlation in object investigation between repeated measures in piglets tested with 2 days between trials. When tests were carried out over a longer period (8 weeks-24 weeks) some consistency have been reported in duration and frequency of the object investigation (rs = 0.44 and rs = 0.53 respectively; [START_REF] Janczak | Aggression, fearfulness and coping styles in female pigs[END_REF]). In studies where animals were tested in group no consistency of behavior between tests has been found in pigs tested twice at 5 week interval (8 weeks and 13 weeks; [START_REF] Van Kooij | Can we predict behaviour in pigs? Searching for consistency in behaviour over time and across situations[END_REF]).

Validity

Very few correlations have been reported between the response to novel object and others tests, including tonic immobility (back test), voluntary approach a human or social test with congeners. In the case of significant correlation between human and novel object responses it was observed at 8 weeks of age but not 24 weeks later, the lack of correlation being related with a potential estrus effect [START_REF] Janczak | Aggression, fearfulness and coping styles in female pigs[END_REF].

Conclusion

The discrepancy of results in consistency over time may reflect an altered motivation over time or an habituation effect. Factors such as time (development) and situation (test) may also an important role in determining an individual's behavioral reaction. Behavioral increased human and object investigation observed in older pig may indicate that fear and anxiety decrease with age. Results should also indicate that the willingness to explore new and 'dangerous' object is parted from exploring behavior directed to the environment and that reactivity and curiosity are connected. This point is important in the case of various objects introduced in successive tests [START_REF] Hayne | Effects of regrouping on the individual behavioural characteristics of pigs[END_REF] where the novelty repetition should provide an enriched environment and enhance or fulfill the investigative motivation. Furthermore the nature of the novelty-related stimulus may modulate the responsiveness to novelty. Indeed [START_REF] Hutson | Development of a behavioural test of sensory responsiveness in the growing pig[END_REF] reported strongest and most consistent aversive responses to auditory stimuli rather than olfactory ones in individual growing pigs exposed to stimuli in a test pen.

On the other hand, the novel tests were in some case combined with suddenness when the object are dropped from the ceiling and left on the floor. Similarly the novel tests are classically associated with human intervention when the object has to be introduced or remove at the beginning and the end of the test respectively. This can partly explained the existing correlation between repeated measures of human and object investigation in young animals.

It is important to evaluate in a novel test the specific underlying effects of novelty and suddenness during the test. In addition the habituation period to the arena test is generally neglected and arbitrary fixed to a short period without investigation on the habituation process and its potential effects on the further responses.

Restraint test (back test)

Short background

The back test was used exclusively on piglets and could be named also tonic immobility test (as similar with the tonic immobility test done in poultry). In its original form the back test was done to characterize the fear responses or strategies of the animals rather than the level of fear per se [START_REF] Hessing | Individual behavioral and physiological strategies in pigs[END_REF], however it has since been compared with other traditional fear tests [START_REF] Forkman | Personality, coping patterns, and aggression in piglets[END_REF][START_REF] Andersen | Behavioural evaluation of methods for assessing fear responses in weaned pigs[END_REF]. The back test was carried out in two different ways, either by placing the animal in a cradle and then immobilizing it by placing a light weight on its chest [START_REF] Erhard | Individual differences in tonic immobility may reflect behavioural strategies[END_REF][START_REF] Janczak | Aggression, fearfulness and coping styles in female pigs[END_REF], or by simply placing the animal on its back on a flat surface, a table or the floor, and then pressing gently but firmly on its chest and hind legs [START_REF] Hessing | Individual behavioural characteristics in pigs[END_REF][START_REF] Forkman | Personality, coping patterns, and aggression in piglets[END_REF][START_REF] Ruis | Personalities in female domesticated pigs: behaviouiral and physiological indications[END_REF].

Repeatability

When repeated tests were carried out the time span varied between few days and one to 4 weeks. Repeatability for the test was either not found [START_REF] Forkman | Personality, coping patterns, and aggression in piglets[END_REF][START_REF] Ruis | Personalities in female domesticated pigs: behaviouiral and physiological indications[END_REF], was low (b 0.40; [START_REF] Van Kooij | Can we predict behaviour in pigs? Searching for consistency in behaviour over time and across situations[END_REF][START_REF] Van Kooij | Individual behavioural characteristics in pigs and their impact on production[END_REF]) or high [START_REF] Hessing | Individual behavioural characteristics in pigs[END_REF]. The discrepancy in results may be attributed to the time span between subsequent tests, shorter when high correlations were found.

Comment citer ce document : Forkman, B., Boissy

Validity

The back test for pigs has a varied record with some scientists reporting correlations between the back test and other fear tests related to human approach test [START_REF] Van Kooij | Can we predict behaviour in pigs? Searching for consistency in behaviour over time and across situations[END_REF], tests of aggression [START_REF] Hessing | Individual behavioural characteristics in pigs[END_REF]. Relationships were also reported between the subpopulations defined on the back test responses and physiological parameters [START_REF] Hessing | Individual behavioral and physiological strategies in pigs[END_REF], production parameters (lean meat percentagecarcass grading; [START_REF] Van Kooij | Individual behavioural characteristics in pigs and their impact on production[END_REF]).

For some authors, this test appears very arbitrary, superficial and has no theoretical justification in terms of motivational and functional basis [START_REF] Forkman | Personality, coping patterns, and aggression in piglets[END_REF][START_REF] Jensen | Behavioural strategies or just individual variation in behaviour?a lack of evidence for active and passive piglets[END_REF]. For instance authors are not convinced that such test can be considered as a non social test, one of the few normal situation in which a young pig would be found on its back being during a staged encounter. In addition the extrapolation of coping theory from the back test appears critical when intra and inter-situation consistency were low and the bimodal distribution was failed.

Conclusion

The results from studies suggest that the fear response should be evaluated through the TI duration. Nevertheless when associated with other tests, the order to apply the test can change the interpretation of the results. For instance the response to tonic immobility test performed after an emergence test can be regarded rather than reflecting a learned aversiveness or fear more reflecting an element of activity [START_REF] Erhard | Tonic immobility and emergence time in pigsmore evidence for behavioural strategies[END_REF]. Data on the validity of the test are conflicting and inconclusive. Some studies provide support for subpopulations interpreted as proactive and reactive coping pattern [START_REF] Hessing | Individual behavioural characteristics in pigs[END_REF][START_REF] Hessing | Individual behavioral and physiological strategies in pigs[END_REF][START_REF] Erhard | Tonic immobility and emergence time in pigsmore evidence for behavioural strategies[END_REF][START_REF] Erhard | Individual differences in tonic immobility may reflect behavioural strategies[END_REF]. The use of backtest as a valuable tool to be implemented by farmers for the formation of groups was suggested [START_REF] Ruis | Personalities in female domesticated pigs: behaviouiral and physiological indications[END_REF], nevertheless results remains inconclusive and their interpretations of results ambiguous depending in what the tests actually measure in the form they have applied.

Handling tests

Short background

In all cases handling tests involve human presence and consequently combine the potential stressful trait of the procedure of handling and the fear towards human. The animals were handled either as a group or tested individually. Various procedures were used in the handling tests including especially husbandry practice such as the transit of the animals, the weighting or the restraining nose used for veterinary inspection [START_REF] Lawrence | Individual differences in behavioural responses of pigs exposed to non-social and social challenges[END_REF]. In handling test the variables most commonly recorded concerns the ease of transit and the responsiveness to restraint both expressed as scores including motor reactivity and vocalizations.

Repeatabilityvalidity

Repeatability over time was not directly evaluated in the studies testing husbandry practice. Nevertheless consistent responses in different handling tests in group or individual situation were observed in female pig [START_REF] Lawrence | Individual differences in behavioural responses of pigs exposed to non-social and social challenges[END_REF]. On the other hand, animals with high responsiveness to handling (resistance to handling) tend to show greater attention to a novel object but without longer contact with it. This suggests that the novel object may also be perceived as aversive by high responders. In return no correlation was found between handling tests and social challenge test (Group feeding competition).

Conclusion

The responsiveness to handling in group situation might be ambiguous. Indeed handling effects can be confounded with the effects of social facilitation which may lead to overestimation of the handling effect itself. Pigs in group situation might also have competed to leave the home pen or to move in a corridor, according to the hierarchy pressure and the social cohesiveness within group. The human involvement in the handling procedures need to pay attention on the interaction between the confidence levels of tested animals towards the handler and the responsiveness to handling itself. According to the experience with human, the interaction with the handler might become either rewarding by a pleasurable experience which would exclude fear or additional stressor by aversive experience which would strengthen the handling effect [START_REF] Tanida | Behavioral responses of piglets to darkness and shadows[END_REF]. [START_REF] Cheng | Action of 5. hydroxytryptophan to inhibit and disinhibit mouse behaviour in the light/dark test[END_REF] and consists of two box, illuminated and dark, connected by an opened door. Time spent in the brightly lit compartment was firstly considered as a valid measure of aversion of light as shown in rodents [START_REF] Ramos | Stress and emotionality: a multidimensional and genetic approach[END_REF]. Nevertheless the anxiolytic effect of diazepam in the L/D has not been reported as did in rodents [START_REF] Andersen | Behavioural evaluation of methods for assessing fear responses in weaned pigs[END_REF] and time spent in the lit compartment appears more related to activity than fear of novelty [START_REF] Andersen | Effects of diazepam on the behaviour of weaned pigs in three putative models of anxiety[END_REF].

In the emergency test, the design consist of a start box connected by a door to a test arena [START_REF] Keeling | Social. behaviour. in. farm. animals[END_REF] and measure the reluctance to enter an arena from a start box, usually the home pen or a cage. This test is usually associated with the Novel Arena Test and belongs to the group of "timidity" test used in rats or mice [START_REF] Archer | Tests for emotionality in rat and mice: a review[END_REF]. It is expected that the more hesitant an individual is, the more reluctance it will be to enter the arena. This challenge situation should be different if the animal was physically placed into an unfamiliar environment (forced exploration) compared to situation given the opportunity to move around freely between a familiar and a novel environment (voluntary exploration, [START_REF] Misslin | Does neophobia necessarily imply fear or anxiety?[END_REF]).

3.5.1.2. Repeatabilityvalidity. Investigations on the L/D test are limited and were carried out once time at 8 weeks of age and so without evaluation of its repeatability [START_REF] Andersen | Effects of diazepam on the behaviour of weaned pigs in three putative models of anxiety[END_REF][START_REF] Andersen | Behavioural evaluation of methods for assessing fear responses in weaned pigs[END_REF]. Nevertheless significant correlations have been found with others tests carried out before (EPM at 6 weeks, NAT at 7 weeks) but appeared low in both cases (rs b 0.35). No correlation was shown with the tonic immobility tested at 2.5 weeks. In the emergency test, high consistency appeared between the four replicates, done on four consecutive days, except for the first 2 days (rs N 0.50; [START_REF] Erhard | Individual differences in tonic immobility may reflect behavioural strategies[END_REF]). A significant but mall correlation (rs = 0.37) was found between the first emergency test and the tonic immobility performed thereafter.

Comment citer ce document : Forkman, B., Boissy [START_REF] Andersen | Behavioural evaluation of methods for assessing fear responses in weaned pigs[END_REF]. Although wild pig are nocturnal with peak of activity at dusk and dawn [START_REF] Caley | Movements, activity patterns and habitat use of feral pigs (Sus scrofa) in a tropical habitat[END_REF][START_REF] Russo | Daily home range and activity of wild boar in a mediterranean area free from hunting[END_REF], there is very little documentation of preference for a dark area [START_REF] Braude | The effect of light on fattening pigs[END_REF]. Domestic pig should be adapted to the strong light in their production environment and consequently may not perceive bright light as aversive [START_REF] Andersen | Effects of diazepam on the behaviour of weaned pigs in three putative models of anxiety[END_REF]. Indeed [START_REF] Tanida | Behavioral responses of piglets to darkness and shadows[END_REF] reported that darkness frightens the piglets whereas the animals have a tendency to move towards a more brightly illuminated area. On the other hand pigs raised in totally enclosed and dimly illuminated windowless pen were observed to balk and refuse to walk into bright sunlight [START_REF] Fraser | Farm animal behaviour and welfare[END_REF]. Finally The Dark/Light test appears to be of less biological relevance for pigs, potentially controlled by the previous lighting environment before the test and thus cannot be relevant as a general fear test.

The interpretation of the emergency test is also conflicting. Indeed it depends to a large extent on the nature of the start box and of the arena which should reflect different fear-evoking situation: relatively safety if start box is the home pen but potential dangerous (unfamiliar and social isolation) if start box is novel for the animal. In the last case the arena is the only way out the box and therefore out of the dangerous. In addition, the consistency over time for tests carried on a short period can be explained by the effect of the experience on the animal's behavior, including the aversiveness of being handled immediately after having entered the arena the previous days and the learning to anticipate this aversiveness.

3.5.2. Elevated-plus maze test 3.5.2.1. Short background. The design is a modification of the apparatus commonly used as behavioral models of fear in rodents [START_REF] Pellow | Validation of open-closed arm entries in an elevated plus-maze as a measure of anxiety in the rat[END_REF][START_REF] Lister | The use of a plus maze to measure anxiety in the mouse[END_REF] adjusted to the size of young pigs (6-7 weeks, [START_REF] Andersen | Effects of diazepam on the behaviour of weaned pigs in three putative models of anxiety[END_REF][START_REF] Andersen | Behavioural evaluation of methods for assessing fear responses in weaned pigs[END_REF] This response was related to anxiety whereas the number of entries into closed arms unaffected by the diazepam treatment was related to activity [START_REF] Andersen | Effects of diazepam on the behaviour of weaned pigs in three putative models of anxiety[END_REF]. On the other hand the response level in the EPM test indicated that pigs did not show unconditioned avoidance of open arms relative to closed arms as shown in mice, suggesting that the subtype of anxiety measured in mice and pigs may not be directly synonymous [START_REF] Janczak | Factor analysis of behaviour in the porcine and murine elevated plus-maze models of anxiety[END_REF].

3.5.2.2. Repeatability, validity. Investigations on the elevatedplus maze test responses of pigs are limited and did not evaluated the test repeatability over time [START_REF] Andersen | Effects of diazepam on the behaviour of weaned pigs in three putative models of anxiety[END_REF], Low significant correlations were found between the number of entries into open arms and the responses to the Dark/light test (rs b 0.30), the tonic immobility (rs = -0.27), and in the Open-Field Test (rs = 0.33). A study showed no relationships between the activity in the open arms in the EMP test and factors prior to tests, including the age of pigs varied between 33 and 54 days, or the duration of transport between the home pen and the area test [START_REF] Janczak | Effects of variation in pre-test transport duration and animal age on behaviour in the porcine elevated plus-mazea brief report[END_REF]. Furthermore a blood sampling followed by transport compared to a "mere" transport before the EPM test did not affect the plus-maze behavior of the pigs tested [START_REF] Janczak | Effects of variation in pre-test transport duration and animal age on behaviour in the porcine elevated plus-mazea brief report[END_REF], nor did handling affect the behavior of the pigs [START_REF] Janczak | No effect of variation in handling on behaviour in a porcine elevated plusmazea brief report[END_REF].

3.5.2.3. Conclusion. The number of entries and the time spent on open arm was interpreted as the purest measures of fear novelty or avoidance, while the time spent in the lit compartment was more related to activity [START_REF] Andersen | Behavioural evaluation of methods for assessing fear responses in weaned pigs[END_REF]. The EPM test provides a stimuli gradient (aversive, open area, elevated, secure) and appears a way to separate fear and activity-related element [START_REF] Andersen | Effects of diazepam on the behaviour of weaned pigs in three putative models of anxiety[END_REF]. The validity of the test remains unclear though the measures appeared robust to the effects of procedure prior the test or the age of animals. Before any conclusive statement on the hypothesis that anxiety is the major emotional state measured in the EPM test, further investigation are needed with more detailed ethological observations, and repeated and over long period testing, as pointed out by [START_REF] Janczak | Factor analysis of behaviour in the porcine and murine elevated plus-maze models of anxiety[END_REF].

Fear tests in sheep and goats

The novel arena test is without doubt the most commonly used fear test for sheep and goats, but both novel object and handling tests are also frequently used. As for the others species the novel object tests are both carried out with the object being present from the start of the trial (e.g. [START_REF] Moberg | Effect of differential rearing on the behavioral and adrenocortical response of lambs to a novel environment[END_REF]), but also with the object suddenly appearing to the animal (e.g. [START_REF] Desire | On the way to assess emotions in animals: do lambs (Ovis aries) evaluate an event through its suddenness, novelty, or unpredictability?[END_REF][START_REF] Erhard | Effects of prenatal undernutrition on emotional reactivity and cognitive flexibility in adult sheep[END_REF]. There is an extensive literature on fear tests in sheep, especially for the novel arena test.

Novel arena test

Background

The variables most commonly recorded are similar to those for other species: locomotor activity (number of squares entered, standing, walking), rearing, exploration (sniffing) eliminative behavior and vocalizations. One measure which is being used in sheep and goats, but not for other species is latency to feed. There is however a very large difference in the number of variables used with e.g. [START_REF] Vandenheede | Fear reactions of ewes to photographic images[END_REF] recording 26 variables whereas [START_REF] Price | Behavioral responses to short term social isolation in sheep and goats[END_REF] only recorded three variables.

The arenas used vary in size, but many studies use one that is approximately 4 × 4 m. As with the size the time spent in the arena varies, but is often approximately 5 min. The overall level of standardization in most tests is greater than that found for e.g. pigs.

Repeatability

Many of the results from the novel arena studies are consistent over time, especially locomotion, vocalizations and defecation/urination [START_REF] Hernandez | An attempt to assess traits of emotionality in crossbred ewes[END_REF][START_REF] Lachaux | Behavior of Ile. de. France rams reared in different conditions in an open-field tests[END_REF]. The delays used in these studies are in the order of a few weeks.

Validity

There are good physiological as well as behavioral indications that the novel arena test induces fear (e.g. [START_REF] Parrott | Endocrine responses to acute stress in castrated rams: no increase in oxytocin but evidence for an inverse relationship between cortisol and vasopressin[END_REF]). For sheep, as for all the other species the reaction of the animals to the novel arena test is a mixture of the reaction to the physical situation itself, and social isolation. The strong reaction of sheep to isolation in their home pens makes it probable that a large part of the reactions observed in the novel arena tests are caused by the social isolation rather than the new environment [START_REF] Price | Behavioral responses to short term social isolation in sheep and goats[END_REF][START_REF] Neindre | Influence of breed on reactivity of sheep to humans[END_REF].

As for other species it seems that it is the number of vocalizations that is most affected by the presence/absence of conspecifics (e.g. [START_REF] Porter | Discriminative responsiveness by lambs to visual images of conspecifics[END_REF]).

Conclusion

Because of the large number of studies and the relatively good correlations found for other tests the novel arena test can be considered as a good fear test for sheep and goats, which include a social isolation component.

Novel object test

The novel object test is used much less frequently than is e.g. the novel arena test. Of special interest is a number of articles in which it is the manner of appearance of the object that is investigated (e.g. [START_REF] Romeyer | Assessment of fear reactions in domestic sheep, and influence of breed and rearing conditions[END_REF][START_REF] Desire | On the way to assess emotions in animals: do lambs (Ovis aries) evaluate an event through its suddenness, novelty, or unpredictability?[END_REF][START_REF] Vierin | Influence of maternal experience on fear reactions in ewes[END_REF][START_REF] Vandenheede | Sex differences in fear reactions in sheep[END_REF]). While it is not uncommon for novel objects to drop down in the novel arena tests for both cattle and pigs, no comparable systematic investigations have been done for these species to study the effect of the appearance, as opposed to the presence, of a novel object.

Repeatability

We have not found any studies on the repeatability of the test.

Validity

One study [START_REF] Romeyer | Assessment of fear reactions in domestic sheep, and influence of breed and rearing conditions[END_REF] found a good correlation to a surprise test and to the exposure to a stationary human. A higher reaction to a novel than a familiar object (heart rate and orienting response), and for the rapidly presented novel object a startle response with a transient increase in heart rate has also been found [START_REF] Desire | On the way to assess emotions in animals: do lambs (Ovis aries) evaluate an event through its suddenness, novelty, or unpredictability?[END_REF].

Conclusion

There has been little research on the reactions to novel object but these responses tend to correlate with the reactions of the animals to other putatively fear-inducing situations.

Restraint and human fear tests

As can be expected there is a huge variation in the performance of these tests. What they all have in common is the presence of a human, either as a stationary or moving object [START_REF] Lyons | Individual differences in temperament of domestic dairy goats: constancy and change[END_REF] or as someone who actively restraints the animal [START_REF] Parrott | Endocrine responses to acute stress in castrated rams: no increase in oxytocin but evidence for an inverse relationship between cortisol and vasopressin[END_REF]. When the animal is tested with a human the animals have often been habituated to the arena beforehand for anything from 3 days (e.g. [START_REF] Lyons | Individual differences in temperament of domestic dairy goats: constancy and change[END_REF]) to up to 10 days [START_REF] Romeyer | Assessment of fear reactions in domestic sheep, and influence of breed and rearing conditions[END_REF].

In a number of tests it is not the fear reaction of the animal to the human that is noted but rather the reduction of fear caused by another individual, albeit of another species (e.g. [START_REF] Price | Behavioral responses to short term social isolation in sheep and goats[END_REF][START_REF] Lyons | Relationships between heart rates and behavior of goats in encounters with people[END_REF]).

Repeatability

There is a good repeatability of the measures in these tests (e.g. [START_REF] Lyons | Individual differences in temperament of domestic dairy goats: constancy and change[END_REF][START_REF] Neindre | Influence of breed on reactivity of sheep to humans[END_REF][START_REF] Kilgour | Arena behaviour of ewes selected for superior mothering ability differs from that of unselected ewes[END_REF]). The variables that show the highest degree of repeatability are similar to those in the novel arena tests i.e. locomotion and vocalizations.

Validity

There are correlations between tests with a human present and surprise tests, isolation tests and [START_REF] Romeyer | Assessment of fear reactions in domestic sheep, and influence of breed and rearing conditions[END_REF][START_REF] Vandenheede | Sex differences in fear reactions in sheep[END_REF][START_REF] Vierin | Influence of maternal experience on fear reactions in ewes[END_REF]. As expected handling decreases the heart rate increase, and decreases the flight distance [START_REF] Hargreaves | The effect of gentling on heart rate, flight distance and aversion of sheep to a handling procedure[END_REF].

Conclusion

There are studies showing both good repeatability and validation of the restraint and handling tests. However since there is a large difference in the way they have been done, it is hard to say that restraint and handling tests for sheep and goats have been validated overall.

Fear tests in hens and quail

The two most common fear tests for poultry are the tonic immobility test (a specialized restraint test) and the novel arena test. Much less frequent are the novel object test and the human approach tests, as well as the emergence test. For poultry a number of related tests, primarily designed to measure sociality have been used, these have not been included in the present review however. Generally speaking the fear tests in poultry are well validated [START_REF] Jones | Fear and adaptability in poultry: insights, implications and imperatives[END_REF], only the most relevant papers published on the fear tests in poultry are therefore presented in the tables.

Novel arena test

Background

There is a general consensus that what is tested in the open field is both the general fear of the bird but that there is also a strong effect of social isolation/dependence. The discussion focuses on the degree to which the different variables measure social dependency and general fear/antipredator responses (e.g. [START_REF] Gallup | An ethological analysis of open-field behaviour in chickens[END_REF]).

These different findings make it do indicate that there are two different motivational states that influence the behavior of the birds. This means that e.g. a low number of escape attempts might either be because the birds are very afraid or because they have a low social reinstatement motivation. There are some behaviors that seem to be more heavily influenced by fear than by the social motivation, e.g. the duration of freezing, and ambulation [START_REF] Suarez | Social reinstatement and open-field testing in chickens[END_REF].

As for the tonic immobility test the effect of an observer is disputed. While one study [START_REF] Gallup | An ethological analysis of open-field behaviour in chickens[END_REF] found an effect of observer present on latency to distress call, ambulation, as well as fewer lines crossed and fewer escape attempts, another failed to do so [START_REF] Jones | Open field behaviour in domestic chicks (Gallus domesticus): the influence of the experimenter[END_REF]. The presence of a new coat however did affect the chickens; the conclusion of the study is that it is the overall novelty of the situation that determines the response of the birds [START_REF] Jones | Open field behaviour in domestic chicks (Gallus domesticus): the influence of the experimenter[END_REF].

Steps and areas entered are both good measures of ambulation and have been shown to be closely correlated [START_REF] Jones | Open field behavior in domestic chicks tested individually or in pairs: differential effects of painted lines delineating subdivisions of the floor[END_REF].

Comment citer ce document : Forkman, B., Boissy Chicks tested singly were not affected by the presence of lines, tested in pairs were [START_REF] Jones | Open field behavior in domestic chicks tested individually or in pairs: differential effects of painted lines delineating subdivisions of the floor[END_REF].

Repeatability

There is a strong genetic component to the open field behavior, ranging from 0.2 for flying to 0.5 for ambulation [START_REF] Rodenburg | Heritability of feather pecking and open-field response of laying hens at two different ages[END_REF]. As for the tonic immobility a QTL has been identified that is closely related to the responses in the open field, although the exact QTL seems to be different for adult animals compared to young ones [START_REF] Buitenhuis | Identification of QTLs involved in openfield behavior in young and adult laying hens[END_REF].

Validity

High novelty in the test arena gives longer latency to movement and lower activity and fewer vocalizations [START_REF] Jones | Open field behaviour of male and female domestic chicks as a function of housing conditions, test situations and novelty[END_REF]. Electrical shock prior to the testing suppresses distress calling, walking and jumping [START_REF] Gallup | An ethological analysis of open-field behaviour in chickens[END_REF], see also [START_REF] Suarez | Social reinstatement and open-field testing in chickens[END_REF]. Birds from enriched environments showed shorter latencies for vocalizations and were more active [START_REF] Jones | Modification of fear in domestic chicks, Gallus gallus domesticus, via regular handling and early environmental enrichment[END_REF]. Birds that are tested individually showed higher levels of activity, vocalization and defecation (and corticosterone), but longer latency to ambulate, preened and pecked less [START_REF] Jones | Individual or paired exposure of domestic chicks to an open field: some behavioural and adrenocortical consequences[END_REF].

Conclusion

The open field test is a well validated test. However, what is tested is a combination of two motivations, fear and social reinstatement. This means that the test is less easy to interpret than other fear tests for poultry, especially since isolation per se might cause fear. Tentatively it can be said that it is the latency to move and number of steps that are most indicative of fear whereas the number of peeps and jumps seem to be more related to the social reinstatement.

Novel object test

Background

The novel object test is often used to test the level of fearfulness of hens in cages, in that the novel object can be placed in front of the cage and the reaction of the bird or birds noted. This makes this a very practical test for testing large number of birds when these are kept in cages [START_REF] Jones | Fear responses of individually caged laying hens as a function of cage level and aisle[END_REF][START_REF] Jones | Fearfulness of hens caged individually or in groups in different tiers of a battery and the effects of translocation between tiers[END_REF][START_REF] Barnett | Fear of humans by laying hens in different tiers of a battery: behavioural and physiological responses[END_REF].

Repeatability

The degree of aversion shown in a novel object test is repeatable between days [START_REF] Jones | Repeatability of fear ranks among adult laying hens[END_REF] and weeks [START_REF] Hocking | Age-related changes in fear, sociality and pecking behaviours in two strains of laying hen[END_REF].

Validity

Birds from an enriched environment approach the novel object more readily [START_REF] Jones | Modification of fear in domestic chicks, Gallus gallus domesticus, via regular handling and early environmental enrichment[END_REF], but there is no effect of handling on the response of the birds.

Birds from cages on top tiers are generally found to have greater avoidance of a novel object [START_REF] Jones | Fearfulness of hens caged individually or in groups in different tiers of a battery and the effects of translocation between tiers[END_REF][START_REF] Barnett | Fear of humans by laying hens in different tiers of a battery: behavioural and physiological responses[END_REF]. The same birds also showed longer TI ( [START_REF] Jones | Fear responses of individually caged laying hens as a function of cage level and aisle[END_REF][START_REF] Jones | Fearfulness of hens caged individually or in groups in different tiers of a battery and the effects of translocation between tiers[END_REF], but see [START_REF] Barnett | Fear of humans by laying hens in different tiers of a battery: behavioural and physiological responses[END_REF]), and slower approach to a novel object or a human when tested in a pen.

Conclusion

The novel object test is a fast and practical fear test. It seems not to be sensitive to handling of the birds and so is possibly a better measure for general fearfulness than is the TI test.

Restraint testtonic immobility

Background

The rationale for the tonic immobility test is that the experimenter simulates a predator thereby eliciting an antipredator response -"death feigning". The idea is that the prey animal plays dead to be able to escape when/if the predator relaxes its concentration. Indeed [START_REF] Sargent | Death feigning by ducks in response to predation by red foxes (Vulpes fulva)[END_REF] showed that "death feigning" birds often took advantage of escape opportunities. Later Thompson et al. [START_REF] Thompson | Tonic immobility in Japanese quail can reduce the probability of sustained attack by cats[END_REF] showed that TI in quail reduced the probability of the birds being predated by cats.

In a systematic study [START_REF] Jones | Sex and strain comparisons of tonic immobility ("righting time") in the domestic fowl and the effects of various methods of induction[END_REF] six different positions/materials were tested for inducing TI: table, table with head hanging, cloth and cloth with head hanging, cradle and cradle with cloth. The results show that the number of inductions necessary to induce TI is lowest for the cradle. There was no effect of the cloth on the number of inductions, or on the duration of the TI or latency of first movement. Today most studies on adult hens use a cradle, whereas studies on chicks typically use cloth on e.g. a table top. The bird is restrained for 15 s with one hand on the sternum and one on the head. They are then released, the variables observed are typically the number of inductions necessary to obtain at least 10 s of TI, latency to first head movement and time until righting (commonly called the duration of the TI) [START_REF] Jones | The tonic immobility reaction of the domestic fowl: a review[END_REF].

There are contradictory findings as regards the presence of an observer and the direction of the gaze of the observer. One study [START_REF] Gallup | The experimenter as an independent variable in studies of animal hypnosis in chickens (Gallus gallus)[END_REF] found an effect of the presence of the experimenter, and also found a strong effect of the direction of the gaze (direct vs. averted), another study [START_REF] Jones | Experimenter visibility, spectacles and tonic immobility in the domestic fowl[END_REF] on the other hand, found that In most cases the tonic immobility test is performed on an animal that is isolated from its conspecifics. Under some circumstances it might however be desirable to test the animals in group. One study [START_REF] Bilĉik | Effect of group size on tonic immobility in laying hens[END_REF] tested the effect of the presence on conspecifics, in groups of 15, 30, 60 and 120 individuals. The TI duration was almost four times shorter when tested with the group present than when the animals were tested in isolation. The same response pattern emerged however. When only one other animal is present the respective relationship of the animals seems to be relevant [START_REF] Jones | Tonic immobility in the domestic fowl as a function of social rank[END_REF]. Regular handling has a strong effect on the duration of TI (e.g. [START_REF] Jones | The effects of regular handling on fear responses in the domestic chick[END_REF][START_REF] Jones | Attenuation of the domestic chick's fear of human beings via regular handling: in search of a sensitive period[END_REF]), as does enriched home cages [START_REF] Jones | Modification of fear in domestic chicks, Gallus gallus domesticus, via regular handling and early environmental enrichment[END_REF].

Repeatability

The repeatability of the tonic immobility test is good [START_REF] Jones | Repeatability of fear ranks among adult laying hens[END_REF][START_REF] Hocking | Age-related changes in fear, sociality and pecking behaviours in two strains of laying hen[END_REF]. There is also a study that indicates a QTL specific for the tonic immobility response [START_REF] Schütz | Major growth QTLs in fowl are related to fearful behavior: possible genetic links between fear responses and production traits in a red junglefowl x white leghorn intercross[END_REF], something that also indicates stability in the behavioral response.

Validity

There is ample evidence that the duration of the tonic immobility response is a measure of the level of fear. Aversive treatment prior to the induction is known to increase the duration of TI [START_REF] Jones | The nature of handling immediately prior to test affects tonic immobility fear reactions in laying hens and broilers[END_REF][START_REF] Marin | Effects of an acute stressor on fear and on the social reinstatement responses of domestic chicks to cagemates and strangers[END_REF], whereas regular handling results in a decrease of TI [START_REF] Jones | The effects of regular handling on fear responses in the domestic chick[END_REF][START_REF] Jones | Attenuation of the domestic chick's fear of human beings via regular handling: in search of a sensitive period[END_REF]. Birds from enriched environments also tend to show shorter TI durations [START_REF] Jones | Modification of fear in domestic chicks, Gallus gallus domesticus, via regular handling and early environmental enrichment[END_REF]. Overall the duration of TI seems to be a more sensitive measure than the number of inductions needed (but see [START_REF] Schütz | Domestication effects on foraging strategy, social behaviour and different fear responses: a comparison between the red junglefowl (Gallus gallus) and a modern layer strain[END_REF]).

There are various reports on the effect of age on tonic immobility. There is some agreement that it is hard, some say impossible, to induce a TI response very early in the life of the chick (e.g. [START_REF] Ratner | Immobility reactions (fear) of domestic fowl as a function of age and prior experience[END_REF]). Later studies have however shown that it is a quantitative rather than qualitative difference, and that while it is easier to induce it later in life (after day 3) it is also possible to do so at a very young age [START_REF] Heiblum | Tonic immobility and open field responses in domestic fowl chicks during the first week of life[END_REF].

Conclusion

The tonic immobility test is a well validated fear test that can be used for both quail and chickens. It works well under a range of conditions, but these need to be standardized if the result is to be valid. Of particular practical importance is that the birds are caught and handled in the same way prior to the test, and that the presence/absence of the experimenter and the direction of the experimenters gaze is standardized.

Other tests

Emergence test

There are a number of alternative fear tests that have been used in chickens and in quail. One of these, the emergence test, is based on the logic of the Light/Dark test in rodents [START_REF] Cheng | Action of 5. hydroxytryptophan to inhibit and disinhibit mouse behaviour in the light/dark test[END_REF]. There is little evidence to suggest that light is aversive to poultry (unless it is very bright) and the test can perhaps best be seen as a variety of the novel arena test.

Validity

Overall the emergence test is well validated with good correlations to e.g. the novel arena test and TI (e.g. [START_REF] Jones | Estimation of fear in two lines of the domestic chick: correlations between various methods[END_REF][START_REF] Jones | Genetic and experiential manipulation of fear-related behavior in Japanese quail chicks (Coturnix coturnix japonica)[END_REF]).

Repeatability

Little is known about the repeatability of the test.

Conclusion

The emergence test is well validated, but it is uncertain how much novel information is gained from this test, compared to a novel arena test. The novel arena is better validated and more commonly used and is thus to be preferred.

Fear test in horses

Novel object, restraint, novel arena, voluntary and forced approach to human are all fear tests used in horses. These tests are usually done one by one, but in some studies combined tests are carried out for example by introducing a novel object into the open field arena after an habituation period within the test arena or by including a novel object or a human in the novel environment right from the start.

Repeatability

There is only a repeated study which used 9 days interval and the results were consistent over the times [START_REF] Seaman | How reliable is temperament assessment in the domestic horse (Equus caballus)?[END_REF]. The animals are always tested alone.

Validity

The behaviour of horses in a novel arena situation does correlate with their behavior in other fear tests at low level (rs = 0.305; [START_REF] Scolan | Stability over situations in temperament traits of horses as revealed by experimental and scoring approach[END_REF]) only in males (r = 0.71; [START_REF] Wolff | Experimental tests to assess emotionality in horses[END_REF]) or not at all [START_REF] Seaman | How reliable is temperament assessment in the domestic horse (Equus caballus)?[END_REF].

Conclusion

Open field is not widely used to test fear reaction in horses and for this reason it is difficult to draw clear conclusions due to the discrepancy in the obtained results. However due to the lack of strong correlation with other tests of fear the open field test can not be recommended as a general fear test for horses.

Novel object test

Background

The novel object test is sometimes performed after a habituation period to a barren open arena used in some case as a previous open-field test when different tests are combined [START_REF] Seaman | How reliable is temperament assessment in the domestic horse (Equus caballus)?[END_REF][START_REF] Wolff | Experimental tests to assess emotionality in horses[END_REF][START_REF] Scolan | Stability over situations in temperament traits of horses as revealed by experimental and scoring approach[END_REF].

The novel stimulus tested are usually visual, an object introduced by human on the floor before or after the habituation period within the arena, or dropped from the ceiling and left on the floor [START_REF] Scolan | Stability over situations in temperament traits of horses as revealed by experimental and scoring approach[END_REF] or rotating from the ceiling [START_REF] Momozawaa | Assessment of equine temperament by a questionnaire survey to caretakers and evaluation of its reliability by simultaneous behavior test[END_REF]. The visual stimulus varies from a cage which contains a colorful inflated ball [START_REF] Scolan | Stability over situations in temperament traits of horses as revealed by experimental and scoring approach[END_REF] to a red plastic child's sledge [START_REF] Seaman | How reliable is temperament assessment in the domestic horse (Equus caballus)?[END_REF] to a rotation equipment with two balloons [START_REF] Momozawaa | Assessment of equine temperament by a questionnaire survey to caretakers and evaluation of its reliability by simultaneous behavior test[END_REF] or a colored open umbrella [START_REF] Visser | Responses of horses in behavioural tests correlate with temperament assessed by riders[END_REF]. Horses are usually tested alone. Habituation period to the test arena before the confrontation to a novel object varies The variables most commonly recorded indicated the interest towards the novel stimulus measured by the latency to the contact, distance from the novel object, frequency or duration of contact, exploration, body posture.

Repeatability

Studies have used no interval [START_REF] Visser | Responses of horses in behavioural tests correlate with temperament assessed by riders[END_REF] or at very different time intervals, from 9 days [START_REF] Seaman | How reliable is temperament assessment in the domestic horse (Equus caballus)?[END_REF] to 1 month up to 1 year [START_REF] Visser | Heart rate and heart rate variability during a novel object test and a handling test in young horses[END_REF][START_REF] Visser | Quantifying aspects of young horses' temperament: consistency of behavioural variables[END_REF]. This difference in time interval may explain the differing results on the consistency over time. Whereas one study [START_REF] Seaman | How reliable is temperament assessment in the domestic horse (Equus caballus)?[END_REF], found no consistency between the behaviors another study in which the tests were carried out over a longer period did find such a consistency [START_REF] Visser | Quantifying aspects of young horses' temperament: consistency of behavioural variables[END_REF].

Validity

Few correlations have been reported between the response to novel object and others tests, such as handling test. A positive correlation was found between reactivity to novel object, questionnaire about horse temperament and hr frequency during the test [START_REF] Momozawaa | Assessment of equine temperament by a questionnaire survey to caretakers and evaluation of its reliability by simultaneous behavior test[END_REF].

Conclusion

Factors such as time (development) and situation (test) may also an important role in the individual's behavioral reaction. In all cases handling tests involve human presence and consequently combine the potential stressful trait of the procedure of handling and the fear towards human. The animals were tested individually. Various procedures were used in the handling tests including husbandry practice such as the transit of the horse on a bridge, or the restraining nose used for veterinary inspection In handling test the variables most commonly recorded concerns the ease, the attempts and time necessary to cross the bridge and the responsiveness to restraint both expressed as scores including motor reactivity and vocalizations and expressions.

Repeatabilityvalidity

Repeatability over time was evaluated only in two studies which are contradictory: [START_REF] Visser | Quantifying aspects of young horses' temperament: consistency of behavioural variables[END_REF] found a consistency after a year in the responses to cross the bridge while [START_REF] Jezierski | Effects of handling on behaviour and heart rate in Konik horses: comparison of stable and forest reared youngstock[END_REF] found no consistency. This discrepancy could be due to different kind of handling as in both studies the horses were young.

Conclusion

Type of handling and the human involvement in the handling procedures need to pay attention on the interaction between the confidence level of tested animals towards the handler and the responsiveness to handling itself.

Concluding discussion

Fear is a negative emotion and as such is often included in assessments and recommendations of animal welfare, in e.g. the [START_REF] Waiblinger | Assessing the human animal relationship in farmed species: a critical review[END_REF] for tests of fear towards humans). For sheep and poultry some of the traditional tests are on the other hand well validated (specifically the arena and the novel object test, and for poultry also the tonic immobility test, although this probably is affected by fear of humans). For pigs finally there exists a wide variety of different tests, these have however relatively low inter-test correlations and cannot be said to be well validated.

The novel arena test, also called open-field test, is a common fear test which has been used for all species in this review. As stated in the introduction it is very probable that different aspects of the arena are the main cause of fear in the different species. So might it e.g. represent a risk of predation for a species evolved to hide in the undergrowth (the jungle fowl/ domestic chicken) whereas it for a species evolved for more open ground (e.g. the horse) might represent something else, e.g. confinement. Using the novel arena test as a fear test for species whose ancestors have evolved for open areas is therefore not recommended.

The high validity for the novel arena test for sheep is somewhat surprising for the reason given above. It might how-ever be that the isolation component is so important for this species that it provokes the fear response [START_REF] Price | Behavioral responses to short term social isolation in sheep and goats[END_REF]. For poultry the novel arena test has a high validity, whereas for pigs the results are conflicting. In the arena tests it is generally the latency to move which is the best validated response for general fear (ref). Vocalizations are more correlated to the isolation component, at least in sheep and poultry [START_REF] Porter | Discriminative responsiveness by lambs to visual images of conspecifics[END_REF][START_REF] Jones | Open field behavior in domestic chicks tested individually or in pairs: differential effects of painted lines delineating subdivisions of the floor[END_REF]. Locomotion is probably related to curiosity and/or social reinstatement.

The novel object test is an intuitively appealing test. It uses the animal's reaction to a novel stimulus, the novelty of which according to most theories should elicit a fear reaction (e.g. [START_REF] Gray | The psychology of fear and stress[END_REF]). One of the problems with the test however is the same as for the voluntary approach test in the testing of humanrelationships [START_REF] Waiblinger | Assessing the human animal relationship in farmed species: a critical review[END_REF], a non-curious/indifferent animal and a fearful animal will both show a long latency to approach the novel object. In the approach test this can be solved by using a forced approach in which the experimenter moves towards the animal and it is the avoidance reaction of the animal which is measured, however we have not found any corresponding test with a moving novel object.

Restraining an animal can at least in some species be thought to represent a predator attack and as such is an innate fear evoking stimulus. In poultry there are good indications that the freezing seen in the birds is related to an anti-predator response with birds showing a shorter freezing response being more prone to being killed by predators [START_REF] Thompson | Tonic immobility in Japanese quail can reduce the probability of sustained attack by cats[END_REF]. The situation in the pig is more complex, in the most common restraint test in the pig, the backtest, the piglet is put on its back and either restrained manually [START_REF] Hessing | Individual behavioural characteristics in pigs[END_REF] or by means of a small weight [START_REF] Erhard | Tonic immobility and emergence time in pigsmore evidence for behavioural strategies[END_REF], and it is hard to see how this situation directly relates to a predation situation. This does not mean that the test is invalid for pigs, it might be that the novelty or strangeness of the situation which might cause a fear response. There is less ecological validity for the response than for poultry however, and the results found are much more varied, ranging from good correlations with other tests [START_REF] Erhard | Tonic immobility and emergence time in pigsmore evidence for behavioural strategies[END_REF] to no significant correlations [START_REF] Forkman | Personality, coping patterns, and aggression in piglets[END_REF].

In most of the studies reported throughout the present review the emphasis is on the sensitivity of a given test. What is tested is if a putative difference in fear levels exists, e.g. if different levels of handling give different results. There has been much less emphasis on the specificity of the test, i.e. whether the correlation between two different tests is caused by a difference in a second factor, e.g. overall activity, rather than a difference in fear (but see e.g. [START_REF] Andersen | Behavioural evaluation of methods for assessing fear responses in weaned pigs[END_REF]).

In some cases these second factors are welfare irrelevant, as in the example above, but in other cases it is less clear, one of the factors might be the effect of social isolation. As stated in the introduction in many tests the animal is tested alone, and it is probable that some of the fear response shown is an effect of this social isolation. If the fear test is done to evaluate an aspect of animal welfare then it is only relevant to use the test with isolated animals if the animal is often kept alone, i.e. in a situation in which that specific fear inducing stimulus is present. One example might be e.g. the domestic dog in which separation anxiety is a recurring problem [START_REF] Mikkelsen | Euthanasia of dogs because of behavioural problems. An epidemiological study on euthanasia of dogs in Denmarkwith particular reference to aggression problems[END_REF]. On the other hand a production animal strain with a high level of social dependency might show a strong fear reaction when tested in isolation. This is however probably not relevant from an animal welfare perspective since the animals in almost all production systems will encounter the fear provoking stimulus when together with other individuals.

In most cases it is recommended that the situation/fear provoking stimulus is as simple as possible. There is an unfortunate tendency to mix tests, or alternatively to use several tests in a rapid succession, this is especially true for tests of pigs and to some extent cattle (it is less common in poultry). In [START_REF] Thodberg | A general reaction pattern across situations in prepubertal gilts[END_REF] an "open field test" consists of the gilt being put into a pen, left for 5 min, a bucket dropped from the ceiling, after 5 min three persons enter the pen, the gilt is fixated with a nose sling and blood sample is taken, the gilt is covered with a blanket with one person present in the pen, blanket is removed after 5 min, left alone for 5 min and a spool is dropped from the ceiling (for further examples see e.g. [START_REF] Jensen | Individual variation and consistency in piglet behaviour[END_REF][START_REF] Lawrence | Individual differences in behavioural responses of pigs exposed to non-social and social challenges[END_REF]). Such procedures can lead to difficulties for the interpretation of result according to the order of the successive tests. Indeed the response measured in the following test may reflect an underlying tendency to bring about more or less quickly when animals faced with a new challenging situation. If the response for each type of test was evaluated it would not only help in the interpretation of the results of study, but also be more valuable in a comparative context.

The basis for the studies in which validation is measured as the correlation between behavior patterns in different tests is that an animal responds to fear evoking stimuli in a consistent manner and that fear is a unitary concept. This might however very well be an oversimplification, for e.g. domestic dogs there are a number of examples of both differing behavior and physiological patterns depending on the fear eliciting response [START_REF] Hydbring-Sandberg | Physiological reactions to fear provocation in dogs[END_REF], as well as different fears which do not correlate with each other but which are internally consistent [START_REF] Svartberg | Personality traits in the domestic dog (Canis familiaris)[END_REF][START_REF] Svartberg | Consistency of personality traits in dogs[END_REF]. There is no strong a priori reason for believing that fear is unitary concept, and if different tests yield different fear-rankings the alternative that they might be measuring different aspects of fear or "feartypes" has to be considered. Because of this we would like to emphasise a broad approach to the validation of fear tests, ideally using pharmacological, physiological and behavioral methods.

To conclude, we believe that if more effort is put into the development, and validation of standardized behavioral tests to assess fear in farm animals, then this would strengthen the area of applied ethology. While it is important that different tests are developed we would also like to urge researchers to gather information on factors influencing the outcome of relatively simple and standardized tests, which can then later be used "out of the box". (Tables 3,4

  Physiology & Behavior 92 (2007) 340 -374 ⁎ Corresponding author. Tel.: +45 3528 3581; fax: +45 3528 3022. E-mail address: bjf@kvl.dk (B. Forkman). 0031-9384/$ -see front matter © 2007 Elsevier Inc. All rights reserved. doi:10.1016/j.physbeh.2007.03.016 Comment citer ce document : Forkman, B., Boissy, A., Meunier-Salaün, M.-C., Canali, E., Jones, R.B. (2007). A critical review of fear tests used on cattle, pigs, sheep, poultry and horses. Physiology and Behavior, 92, 340-374. , DOI : 10.1016/j.physbeh.2007.03.016

  Comment citer ce document : Forkman, B., Boissy, A., Meunier-Salaün, M.-C., Canali, E., Jones, R.B. (2007). A critical review of fear tests used on cattle, pigs, sheep, poultry and horses. Physiology and Behavior, 92, 340-374. , DOI : 10.1016/j.physbeh.2007.03.016

  Abbreviations: Lat. = latency, Dur. = duration, No. = number, Corr. = correlation, PCA = principal component analysis. Abbreviations for the tests: NAT = novel arena test, NOT = novel object test, RT = Restraint test, VAT = voluntary approach test, FAT = forced approach test.

  Comment citer ce document : Forkman, B., Boissy, A., Meunier-Salaün, M.-C., Canali, E., Jones, R.B. (2007). A critical review of fear tests used on cattle, pigs, sheep, poultry and horses. Physiology and Behavior, 92, 340-374. , DOI : 10.1016/j.physbeh.2007.03.016

  3.5. Others tests 3.5.1. 1 Light/Darkemergency test 3.5.1.1. Short background. The design Light/Dark test is a modification of the apparatus use for mice

  ). It consists of two open arms and two enclosed arms opposite each other. The walls of the enclosed arms were made of transparent plexiglas. The maze is elevated 1 m above the floor and placed in a room without other stimuli. The pigs are placed individually into the centre of the maze facing one of the closed arms and the measured parameters for a period of 5 min are the number of entries into open arms, the number of entries into closed arms and the time spent on open arms. An anxiolytic effect of diazepam are found to reduce the avoidance of open arms indicated by an increase in the percent of entries into open arms and the percent of time spent on open arms.

  Comment citer ce document : Forkman, B., Boissy, A., Meunier-Salaün, M.-C., Canali, E., Jones, R.B. (2007). A critical review of fear tests used on cattle, pigs, sheep, poultry and horses. Physiology and Behavior, 92, 340-374. , DOI : 10.1016/j.physbeh.2007.03.016

  Abbreviations: Lat. = latency, Dur. = duration, No. = number, Corr. = correlation, PCA = principal component analysis. Abbreviations for the tests: NAT = novel arena test, NOT = novel object test, RT = Restraint test, VAT = voluntary approach test, FAT = forced approach test.

  Abbreviations: Lat. = latency, Dur. = duration, No. = number, Corr. = correlation, PCA = principal component analysis. Abbreviations for the tests: NAT = novel arena test, NOT = novel object test, RT = restraint test, TI = tonic immobility, VAT = voluntary approach test, FAT = forced approach test.

6. 1 .

 1 Novel arena test 6.1.1. Background This test is sometimes used in horses and the variables most commonly recorded are: locomotor activity (standing, walk, trot, and exploration), eliminatory behavior (defecation, urination) tail position and vocalizations. The arena is usually 30 × 15 m. the time spent in the arena varies between 5 and 20 min. Horses tested are adult. The open field was usually combined with other tests performed thereafter within the same arena[START_REF] Seaman | How reliable is temperament assessment in the domestic horse (Equus caballus)?[END_REF][START_REF] Wolff | Experimental tests to assess emotionality in horses[END_REF][START_REF] Scolan | Stability over situations in temperament traits of horses as revealed by experimental and scoring approach[END_REF].

  Comment citer ce document : Forkman, B., Boissy, A., Meunier-Salaün, M.-C., Canali, E., Jones, R.B. (2007). A critical review of fear tests used on cattle, pigs, sheep, poultry and horses. Physiology and Behavior, 92, 340-374. , DOI : 10.1016/j.physbeh.2007.03.016 between 0 and 4 min. Animals are tested at very different ages young (till 3-4 years old) and adult from 5 to 17 years old.

  Abbreviations: Lat. = latency, Dur. = duration, No. = number, Corr. = correlation, PCA = principal component analysis. Abbreviations for the tests: NAT = novel arena test, NOT = novel object test, RT = restraint test, TI = tonic immobility, VAT = voluntary approach test, FAT = forced approach test.

  Abbreviations: Lat. = latency, Dur. = duration, No. = number, Corr. = correlation, PCA = principal component analysis. Abbreviations for the tests: NAT = novel arena test, NOT = novel object test, RT = restraint test, TI = tonic immobility, VAT = voluntary approach test, FAT = forced approach test.

  , 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 and 15). Comment citer ce document : Forkman, B., Boissy, A., Meunier-Salaün, M.-C., Canali, E., Jones, R.B. (2007). A critical review of fear tests used on cattle, pigs, sheep, poultry and horses. Physiology and Behavior, 92, 340-374. , DOI : 10.1016/j.physbeh.2007.03.016

  , A., Meunier-Salaün, M.-C., Canali, E., Jones, R.B. (2007). A critical review of fear tests used on cattle, pigs, sheep, poultry and horses. Physiology and Behavior, 92, 340-374. , DOI : 10.1016/j.physbeh.2007.03.016

Table 2

 2 Novel object test in cattle

	Reference Nature Arena	Time	Age (week) Replicate, n	Variables	Validity		Procedures and
		size (m)	(Min)		Interval/test				other factors
				Sex	-Within		Internal validity	External validity
				Breed	-Between
	[50]	Suspended	10	Heifers	3 rep	Lat: approach,		Lack of inter-correlations	PCA analysis
		tambourine				contact		between tests. Time in
		and ball						contact corr between rep.
				F	13 weeks	Dur: in contact,			Also tested with NAT,
						in locomotion			VAT, RT
				Friesian		No: voc
	[51]	Suspended	10	Heifers	3 rep	Lat: approach,		Positive corr latency to	PCA analysis
		tambourine				contact		contact and cortisol,
		and container						negative corr time spent
								in contact and cortisol
				F	3 -13 weeks Dur: in contact,			Also tested with NAT
						in locomotion
				Friesian		No: voc, defecations,
						urination
						Other: Cortisol
						response to ACTH,
						ACTH and cortisol
						response to
						CRH, heart rate
	[43]	4.4 × 3	3	2-6 weeks 1 rep	Lat: approach,	Decrease latency to	Lower HR at 2 weeks	HR, NAT, Startle,
							contact with age,		learning test
							Increase number of
							interactions
		ball 80 cm		M	1 month	Dur: in contact
		diameter
				Friesian		No: interactions
						with the ball
	[44]	6 × 9	5	6-8 months 3 rep	No: investigations,	Decreased		NAT, RT, VAT,
							interactions		FAT startle, crush test,
									lateralisation,, flight time,
									ease of sorting, following;
									PCA analysis
		metal cube
		0.

  By contrast to rodents domestic pigs seems to not perceive the bright light as aversive

, A., Meunier-Salaün, M.-C., Canali, E., Jones, R.B. (2007). A critical review of fear tests used on cattle, pigs, sheep, poultry and horses. Physiology and Behavior, 92, 340-374. , DOI : 10.1016/j.physbeh.2007.03.016 3.5.1.3. Conclusion.

Table 3

 3 Restraint and handling test in cattle

	Reference Types of	Time	Age	Replicate, n	Variables	Validity		Procedures and other
		restraint	(Min)	(week)	Interval/test				factors
				Sex	-Within		Internal	External
				Breed	-Between		validity	validity
	[47]	Capture, leading,	No time	15 months		Lat: Time to capture		No relationships	3 levels of handling
		tethering	given					between RT and
								other tests
				F		Dur: moving: relaxed,			PCA
						struggling, immobile:
						rope stretched/not
						stretched/ pulling
				Friesian		Other: Ease of leading (1-5),			Other tests: NAT,
						Cortisol levels, heart rate			VAT, FAT
	[49]	Separating,	3	3.5 months 4 rep	Time need to restrain		No relationship	Other tests: NAT
		following,				or separate the animal		between tests
		restraining
				?
				Aubrac
	[50]	Tethered	10	Heifers	3 rep	Duration moving		Lack of inter-	PCA analysis
						head or legs, rope		correlations
						stretched		between tests
				F	13 weeks
				Friesian
	[44]	Restrain	2	6-	3 rep	Movement (score 1-7)			NAT, RT, VAT, FAT
		in a crush		8 months					startle, lateralisation,,
									flight time, ease of sorting,
									following; PCA analysis
		Restrain	1.5	M, Steers 3, 4 weeks	Time need to restrain
		with rope				and separate the animals
				Angus

Table 4

 4 sunglasses nor the presence/absence of the experimenter affected the TI in adult Isa Brown hens.

	Novel arena test on pigs
	Reference Arena	Time	Age	Interval	Variables	Validity		Procedures and other
		size (m)	(min)	Sex Breed	between tests		Internal validity	External validity	factors
	[64]	3 × 7	10	10 weeks	18 weeks,	Lat: start box	Corr test1-test	Corr locomotion, vocalization	Individual transport,
					2 rep	emergence	2 (vocaliz.)	-adrenal reactivity
				Castrated males		No: sections entered			ACTH challenge
						vocalization, defecation			(24 weeks)
				German LW
	[67]	2.4 × 3.4 10	3 weeks		No: vocalization,		No corr with aggression test Combined tests:
						locomotion, exploration,			NAT, NOT1, NOT2.
						defecations
				F,M					Other test: aggression test
				H × (Y × Ld)
	[65]	4.4 × 5.6 5	4-4.5 months		Lat: posture		Low corr with VAT/ FAT	Combined tests NAT/
								and aggression test	NOT1/RT/ VAT/ NOT2
				F		Dur: posture			Other tests: combined
									VAT/FAT; aggression test
				Ld × Y		No: posture,
						crossed squares
	[69]	Ø: 3	5	8 weeks		No: lines crossed, entries		Low corr with variables of	Other tests: TI, Elevated
						within centre, defecations			plus maze, Light/Dark test
				F,Castrated				Elevated plus maze,
				males				Light/Dark
				Ld × Y				no corr IT
	[70]	Ø: 3	5	8 weeks		No: crossed lines, entries		No effect of diazepam on	Diazepam treatment
						within centre, defecations		entries to center and
								lines crossed
				F,Castrated					Other test: Elevated-plus
				males					maze, Light/Dark test
				Ld × Y
	[66]	7 × 1	10	10 weeks	14 weeks,	No: locomotion		No corr with TI	Test arena: corridor
					2 rep
				F					Combined test: NAT/VAT
				GY × (GY × DLd)					Others test: TI
									Animals tested in group

Abbreviations: Lat. = latency, Dur. = duration, No. = number, Corr. = correlation, PCA = principal component analysis. Abbreviations for the tests: NAT = novel arena test, NOT = novel object test, RT = restraint test, TI = tonic immobility, VAT = voluntary approach test, FAT = forced approach test. Comment citer ce document : Forkman, B., Boissy, A., Meunier-Salaün, M.-C., Canali, E., Jones, R.B. (2007). A critical review of fear tests used on cattle, pigs, sheep, poultry and horses. Physiology and Behavior, 92, 340-374. , DOI : 10.1016/j.physbeh.2007.03.016 Abbreviations: Lat. = latency, Dur. = duration, No. = number, Corr. = correlation, PCA = principal component analysis. Abbreviations for the tests: NAT = novel arena test, NOT = novel object test, RT = restraint test, TI = tonic immobility, VAT = voluntary approach test, FAT = forced approach test. Comment citer ce document : Forkman, B., Boissy, A., Meunier-Salaün, M.-C., Canali, E., Jones, R.B. (2007). A critical review of fear tests used on cattle, pigs, sheep, poultry and horses. Physiology and Behavior, 92, 340-374. , DOI : 10.1016/j.physbeh.2007.03.016 neither spectacles/

Table 6

 6

	Restraint test in pigs
	Reference Type of	Time	Age	Interval	Variables	Validity		Procedures and other
		test		Sex Breed	between tests		Internal validity	External validity	factors
	[82]	Backtest	60 s	1-3 weeks	5 rep	Lat: escape attempts	High	High with	Other test :
		(manual						aggression test	aggression test
		restraint)
				F, M	2-3 days	No: escape attempts,
						voc
				Y × DLd
	[77]	Backtest	60 s	1 week	5 rep	No: escape attempts		High with	Other test :
		(manual						aggression test,	aggression test, NOT
		restraint)
				F,M	2-3 days			High with NOT
				Y × DLd
	[74]	Backtest	60 s	1 week	4 rep	No: escape attempts,	Low	No corr with AS	Test in home pen
		(manual				voc.		and NOT
		restraint)
				F	1 week				Other test: NOT,
									aggression test
				Y × SLd × H × D
	[68]	Backtest	60 s	3 or 10 days	1 rep: 7 days No: escape attempts	Low	High corr with some	Measures on
		(manual						production parameters	production
		restraint)							parameters
				F, C
				DLd × GY
	[66]	Backtest	60 s	2-4 days	1 rep	Dur: escape attempts		No corr with NAT/VAT Other test: combined
		(manual							NAT/VAT
		restraint)
				F	4 weeks	No: escape attempts,
						voc
				GY × (GY × DLd)
	[68]	Backtest	60 s	3 days	2 rep	No: escape attempts	Low	Corr with VAT and	Combined tests: VAT,
		(manual						NOT, only at 7d	NOT, Emergence
		restraint)							in home pen
				F, C	7-20 days
				DLd × GY
	[81]	Backtest	5 min max 2.5 weeks	no rep	Dur: immobility		Corr with RT and NAT Other tests: RT, NAT
		(weight)
				F, B		No: inductions
				LW × Ld; LW
	[17]	Backtest	5 min max 2.5 weeks	3 rep	Dur: immobility	High for	Low and limited to	Other tests:
		(weight)					duration	the first Emergence test	Emergence start box
				F, B	1 day	No: inductions
				LW × Ld; LW
	[70]	Backtest	5-10 s	2.5 weeks	No rep	Dur: immobility		Low with	Other tests: NAT, Light/
		(weight)						Elevated-plus maze	Dark, Elevated-plus maze
				F, CM		No: inductions		No with NAT
								or Light/Dark
	[76]	Restraint 60 s	7 months	No rep	Response score		Corr with Social test	Restraining nose
						according to physical
						resistance
				F					Combined tests:
									FAT, Social test, RT
				Ld × LW					Other test: NOT

Abbreviations: Lat. = latency, Dur. = duration, No. = number, Corr. = correlation, PCA = principal component analysis. Abbreviations for the tests: NAT = novel arena test, NOT = novel object test, RT = restraint test, TI = tonic immobility, VAT = voluntary approach test, FAT = forced approach test. Comment citer ce document : Forkman, B., Boissy, A., Meunier-Salaün, M.-C., Canali, E., Jones, R.B. (2007). A critical review of fear tests used on cattle, pigs, sheep, poultry and horses. Physiology and Behavior, 92, 340-374. , DOI : 10.1016/j.physbeh.2007.03.016 (continued on next page) Comment citer ce document : Forkman, B., Boissy, A., Meunier-Salaün, M.-C., Canali, E., Jones, R.B. (2007). A critical review of fear tests used on cattle, pigs, sheep, poultry and horses. Physiology and Behavior, 92, 340-374. , DOI : 10.1016/j.physbeh.2007.03.016 Abbreviations: Lat. = latency, Dur. = duration, No. = number, Corr. = correlation, PCA = principal component analysis. Abbreviations for the tests: NAT = novel arena test, NOT = novel object test, RT = restraint test, TI = tonic immobility, VAT = voluntary approach test, FAT = forced approach test. Comment citer ce document : Forkman, B., Boissy, A., Meunier-Salaün, M.-C., Canali, E., Jones, R.B. (2007). A critical review of fear tests used on cattle, pigs, sheep, poultry and horses. Physiology and Behavior, 92, 340-374. , DOI : 10.1016/j.physbeh.2007.03.016

Table 8

 8 Handling and restraint tests in sheep and goats

	Reference Test	Time	Age (week)	Interval	Variables	Validity		Procedures and other factors
			(Min)	Sex	between tests		Internal validity	External validity
				Breed
	[101]	1.3 × 2	5	2.5 months	1 rep	No: rearings, voc		Comparison between presence	Comparison between sheep and goat
		(truck)			(delay			and absence of human:
					unknown)
		Passive		M		Dur: rearings, contact		Reduction of vocalizations and	Comparison between home pen and
		human				with human		rearings in presence of a human	truck (animals reared by pairs: one
									animal tested in NAT while the other
									maintained in home pen)
				Dorset (sheep) Alpine,				Species comparison:	Comparison between presence and
				Saanen, Toggenburg					absence of human (2 times alone and
				and LaMancha (goat)					2 times in presence of a human)
								Goats spend more time in contact
								of the human than sheep
	[112]	Sta. human 5	14 weeks		No: voc		Reduction of Vocalization in	Comparison between human-reared
								presence of a human in	and dam-reared
								human-reared goat
		1 × 1 m		M/F		Other: HR		No difference in HR between
								human-reared and dam-reared
				Alpine goat
	[111]	2 tests:	10 and	14 weeks	2 rep:	1. Stationary human	Good corr (ranged from		Comparison between human-reared
			3.5				0.36 to 0.92		and dam-reared
		1) stat		F	-22 weeks	Lat: to proximity,			3-day period of familiarization
		human				Dur: proximity, avoidance			to the holding pen
		1.8 × 9.8
		2) mov		Alpine goat	-30 weeks	2. Moving human
		human
		Ø 2 m				No: mean flight distance
						Other: Cortisol
	[6]	Cage		Adult		Flight distance test	Flight distance and HR of gentled sheep are less than	Effect of five previous
		(1.7 × 0.5)				No: flight distance	those of control sheep		handing (routine)
						Other: HR
		Human		Wethers		Aversion test
		appr
				Merino		Dur: transit time
	[27]	10 × 10	4	5-22 months		15 items		Corr with surprise test and	10-day period of habituation
								exposure to a stationary human	(i.e., free access with
									partners and food)
		Stat human		M/F		Lat: to enter, feeding			3 other tests:
				Ile-de-France		Dur: feeding			surprise, novel arena and object
				and Romanov
									(continued on next page)

Comment citer ce document : Forkman, B., Boissy, A., Meunier-Salaün, M.-C., Canali, E., Jones, R.B. (2007). A critical review of fear tests used on cattle, pigs, sheep, poultry and horses. Physiology and Behavior, 92, 340-374. , DOI : 10.1016/j.physbeh.2007.03.016

Table 8

 8

		(continued)
	Reference Test	Time	Age (week)	Interval	Variables	Validity		Procedures and other factors
			(Min)	Sex	between tests		Internal validity	External validity
				Breed
	[41]	Stat human		8 months		24 items		Corr. with other fear tests	4-day period of habituation (i.e., free
								(isolation, suddenness)	access with partners and food)
				M/F		Lat: to sniff the hum, to eat		Sex diff.
				Rom and Ile-de-France		No: glances at human,
						sniffing the human
	[110]	4 × 6	4 ×2	12 months	2 steps:	No: section entered, sniffings,	High corr for section entered,	Corr between section entered	2 other tests: animal alone,
			times			vocalizations, defecations,	vocalizations and	and vocalizations, and between	animal with pen-mates
						urinations, rearings	eliminations	section entered and eliminations
		Stat human		F	1. H stat				Comparison between genotypes
				Mérinos d'Arles,	2. H stat
				Romanov, M × R	with
				and RxM	pen-mates
	[113]	Stat human	10	Adult	2 rep:	No: distance to human,	Highly repeatable:	Lines comparison:	Lines comparison (Trangie fertility
		in front of				section entered,	Section entered: 0.48,		flock vs. random flock)
		pen-mates				voc, elimination	Vocalisation: 0.57
				F	12 m after			Fertility flock are closer to human
					and 3 m after			and move less than random flock
				Trangie fertility and
				random flocks
	[114]	Stat human 2 and 4 4-10 weeks	1 rep:	Lat: contact	For each type of test, no		Comparison between human-reared
							change between tests		and dam-reared lambs
							performed before
							and after weaning
		1) in home		F	5 weeks	No: voc, agitation
		pen			after the
					first test
					(after
					weaning)
		2) in test		INRA401		Dur: contact
		pen
	[109]	4 × 4	4	Adult		15 items		Corr with isolation test	A sat and immobile human
								and surprise test	behind the trough.
				48M/155F		Lat: to enter, feeding			2-day period of habituation (i.e., free
									access with partners and food)
				Ile-de-France		Dur: feeding
	[105]	Suspending	120	Adult		Other: Cortisol		First 30 min: cortisol response	Restraint test
		in a canvas						to isolation is higher than
		sling						after restraint
				Wethers		Vasopressin		After: no difference	Comparison with isolation
									test (Latin square)
				Clun forest		Prolactin

Abbreviations: Lat. = latency, Dur. = duration, No. = number, Corr. = correlation, PCA = principal component analysis. Abbreviations for the tests: NAT = novel arena test, NOT = novel object test, RT = restraint test, TI = tonic immobility, VAT = voluntary approach test, FAT = forced approach test. Comment citer ce document : Forkman, B., Boissy, A., Meunier-Salaün, M.-C., Canali, E., Jones, R.B. (2007). A critical review of fear tests used on cattle, pigs, sheep, poultry and horses. Physiology and Behavior, 92, 340-374. , DOI : 10.1016/j.physbeh.2007.03.016 (continued on next page) Comment citer ce document : Forkman, B., Boissy, A., Meunier-Salaün, M.-C., Canali, E., Jones, R.B. (2007). A critical review of fear tests used on cattle, pigs, sheep, poultry and horses. Physiology and Behavior, 92, 340-374. , DOI : 10.1016/j.physbeh.2007.03.016

Table 10

 10 Lat. = latency, Dur. = duration, No. = number, Corr. = correlation, PCA = principal component analysis. Abbreviations for the tests: NAT = novel arena test, NOT = novel object test, RT = restraint test, TI = tonic immobility, VAT = voluntary approach test, FAT = forced approach test. Comment citer ce document : Forkman, B., Boissy, A., Meunier-Salaün, M.-C., Canali, E., Jones, R.B. (2007). A critical review of fear tests used on cattle, pigs, sheep, poultry and horses. Physiology and Behavior, 92, 340-374. , DOI : 10.1016/j.physbeh.2007.03.016 Comment citer ce document : Forkman, B., Boissy, A., Meunier-Salaün, M.-C., Canali, E., Jones, R.B. (2007). A critical review of fear tests used on cattle, pigs, sheep, poultry and horses. Physiology and Behavior, 92, 340-374. , DOI : 10.1016/j.physbeh.2007.03.016

	Novel object test in chickens and quail
	Reference Stimulus	Time	Age (day)	Interval	Variables	Validity		Procedures and
			(min)	Sex Species:	between tests		Intrinsic validity	Extrinsic validity	other factors
				Breed
	[124]	Varying	3	1, 6, 13, 20,	5-10	Presence in 4		Good correlation between weeks	All birds in
		novel objects		and 30 weeks	weeks,	proximity zones			each pen tested
					5 rep				in group
				F
				Chicken:
				Isa Brown,
				Tetra SL
	[125]	Light bulb +	4	13 days		Lat: approach		Strong effect on all variables by	Handling and
		rosette over						enrichment, none by handling	Enrichment
		drinker						(except freezing)
				M/F		Dur: freezing			RIR × LS =
									Rhode Island
				Chicken:		No: pecks, position
				RIR × LS
	[138]	Novel food	2 min,	31 weeks		Lat: head out, feed		M showed more avoidance of novel	Intact males
			15 min,					food and ate less, but more active	and capons
			4 h					behaviour in approach test, no diff in TI
				M/Capons		Dur: feeding
				Chicken:		No: feeding, pecks,
						scrapes, shakes, preening
				Warren SSL		Amount food eaten
	[135]	Rod	2	36 weeks		Degree of avoidance		Top tiers reacted more fearfully,	Housed singly,
								as in TI, novel object in pen and	tested in home
								human in pen	cage 45 × 30 cm
				F
				Chicken:
				Golden
				Comet
	[139]	Colored	3	27	None	Approach/avoidance		Avoidance reduced by vitamin C	Scan sampling
		fishing float						(anti-stress) treatment	of position at
									10 s intervals.
				M/F
				Quail
	[140]	Colored	3	26	None	Approach/avoidance		Agreement with line effects	Low and high
		fishing float						in TI fear tests	body weight lines
				M/F					Scan sampling of
									position at
									10 sec intervals.
				Quail
	Abbreviations:

Abbreviations: Lat. = latency, Dur. = duration, No. = number, Corr. = correlation, PCA = principal component analysis. Abbreviations for the tests: NAT = novel arena test, NOT = novel object test, RT = restraint test, TI = tonic immobility, VAT = voluntary approach test, FAT = forced approach test. Comment citer ce document : Forkman, B., Boissy, A., Meunier-Salaün, M.-C., Canali, E., Jones, R.B. (2007). A critical review of fear tests used on cattle, pigs, sheep, poultry and horses. Physiology and Behavior, 92, 340-374. , DOI : 10.1016/j.physbeh.2007.03.016

Table 12

 12 Despite this, several of the tests commonly used to assess fear in farm animals have not been well validated.The lack of good fear tests is most acute in cattle and horses, and at present there are no well validate tests for general fear for these species (but seeWaiblinger et al.

	Emergence test in chickens and quail
	Reference Box (m)	Time	Age (day)	Interval	Variables	Validity	Procedures and other
			(min)	Sex Species: Breed	between tests		Intrinsic validity	Extrinsic validity	factors
	[37]	0.7 × 0.4	10	7 days		Lat: step, peep		Sign overall fear rank	RIR × LS = Rhode Island
								corr with NAT, TI	Red × Light Sussex
				F		Dur: freezing,
						sitting, eye closure
				Chicken: White		No: peeps, ambulation,
				Leghorn RIR × LS		pecks, preens, jumps
	[125]	30 × 20/comp	?	14 days		Lat: peep before and		Strong effect on all	H ndling and Enrichment
						after raising door, head		variables by enrichment,
						emergence, full emergence		less by handling
				M/F				RIR × LS = Rhode
								Island × Light Sussex
				Chicken:				2 min acclim before
				RIR × LS				raising door
	[130]	0.23 × 0.23 × 0.20 2 + 5	5-6	None	Vocalization		Agreement with line	Non-selected, high and
								effects in other fear tests	low activity lines
				M/F		Head emergence
				Quail		Full emergence		Acclimation for 2 min
								before trapdoor opened
	[39]	0.24 × 0.22 × 0.20 1.66 +	10	None	Vocalization		Corr with scores in	Acclimation for 100 sec
			3					startle, TI, NAT tests	before trapdoor opened to
								allow access to large arena
				M/F		Head emergence
				Quail		Full emergence
	[131]	0.25 × 0.22 × 0.17 1 + 5	8-9	None	Vocalization		Strong PCA corr with	Short and long tonic
								NAT and TI scores	immobility lines
				M/F		Head emergence		Acclimation for 2 min
								before trapdoor opened
				Quail		Full emergence		Emergence slower in
								high fear long TI birds
	[159]	0.21 × 0.21 × 0.21 1 + 10 23	None	Vocalization,		Emergence faster after	Low and high stress lines
								vitamin C (anti-stress)
								treatment
				M/F		Head emergence		Emergence faster in	Acclimation for 2 min
								low-fear low stress than	before trapdoor opened
								high-fear high stress quail
				Quail		Full emergence

Abbreviations: Lat. = latency, Dur. = duration, No. = number, Corr. = correlation, PCA = principal component analysis. Abbreviations for the tests: NAT = novel arena test, NOT = novel object test, RT = restraint test, TI = tonic immobility, VAT = voluntary approach test, FAT = forced approach test. Comment citer ce document : Forkman, B., Boissy, A., Meunier-Salaün, M.-C., Canali, E., Jones, R.B. (2007). A critical review of fear tests used on cattle, pigs, sheep, poultry and horses. Physiology and Behavior, 92, 340-374. , DOI : 10.1016/j.physbeh.2007.03.016 five freedoms.

Table 13

 13 Lat. = latency, Dur. = duration, No. = number, Corr. = correlation, PCA = principal component analysis. Abbreviations for the tests: NAT = novel arena test, NOT = novel object test, RT = restraint test, TI = tonic immobility, VAT = voluntary approach test, FAT = forced approach test.

	Novelty arena test in horses
	Reference Arena	Time	Age (w)	Replicate, n Interval/test Variables	Validity	Procedures and other
		size (m)	(Min)	Sex Breed	-Within -Between		Internal validity	External validity	factors
	[160]	30 × 21 20	b5-20 years 3	No: Voc, defecation,	High between	No correlation between	PCA, factor analysis
						urination	3 repetitions	novel object and OF
								or unknown person; no
								correlation between
								of and startle test
				F, Gelding	Within: 9 days	Dur: Stand, Explor,		Questionnaires, startle
						Sustained walk, Trot,		test water spray test
						Vigilance, tail position,
						snort, paw
				TB and
				saddle horses
	[161]	30 × 15 10	1-3 years		Dur: Standing, exploration,		Correlation between	Horses tested in a
						sustained walk, trot,		reactivity in O F and	familiar arena
						passage, gallop, vigilance,		novel object in males
						tail position, locomotion
				F,M
				French
				saddle-breeds
				F, M
	[162]	30 × 15f 5	3-17 years		Dur: Standing, exploration,		Correlation between	Horses tested in a
						sustained walk, trot,		indices in O F and	familiar arena
						passage, gallop, vigilance,		gregariuousness
						tail position, locomotion
				Different				Use of indices, ratings
				breeds				of temperament by
								riding teachers
				F Gelding
	Abbreviations:

Comment citer ce document : Forkman, B., Boissy, A., Meunier-Salaün, M.-C., Canali, E., Jones, R.B. (2007). A critical review of fear tests used on cattle, pigs, sheep, poultry and horses. Physiology and Behavior, 92, 340-374. , DOI : 10.1016/j.physbeh.2007.03.016

Table 15

 15

	Restraint and handling tests in horses
	Reference Nature	Time Age (week)	Replicate, n	Variables	Validity	Procedures and
		area size			Interval /test			other factors
		(m)		Sex	-Within		Internal validity External validity
				Breed	-Between
	[164]	Indoor	12	5-15 years	3 no	Lat: time to approach	Heart rate neg. corr. to standing still	NOT test
		arena			interval		in front of the bridge, number of trials
		18 × 21 m					to cross the bridge, and resistance
							behaviour in approaching the bridge
		Bridge		Swedish		No: vocalization	Handling test correlated	PCA
		2 m wide		warmblood			with riders' scores
		4 long
				F, geldings		Dur: Locomotion,		Riders scores
						position within the arena,
						posture of head and tail
						Other: heart rate
	[166]	Indoor	12	9-10 and
		arena		21-22 months
		18 × 21 m		FM (G)

B. Forkman et al. / Physiology & Behavior 92 (2007) 340-374

Acknowledgement

This review/paper/report is based on the conclusions of COST Action 846 Measuring and monitoring farm animal welfare supported by the EU Commission. The text represents the authors' views and does not necessarily represent a position of the Commission who will not be liable for the use made of such information.