The present study permitted us to observe highly contrasting responses from horses in terms of their behaviors, facial expressions and locomotion between the P + and N- conditions. Overall, in the P + condition, horses interacted less with the experimenters and exhibited a typical facial expression with low neck position, upper lip advanced and ears forward; they moved faster and exhibited an increase in locomotor activity compared to that during the N- condition. In contrast, in the N- condition, horses interacted more with the experimenters by looking at or touching them, blinked more and exhibited more ear movements. The typical facial expression in the N- condition consisted of ears backward or to the side with a high neck position. They also moved slower and showed a lower level of locomotor activity in this condition.
The valence of the two conditions was confirmed by the general speed of the horses, which was higher in the P + condition than in the N- condition. In the positive condition (P+), horses may have accelerated because they wanted to reach the reward, which may reflect the activation of the reward acquisition system3; in contrast, in the negative condition (N-), horses may have slowed down because they were unable to reach the food despite being aware that it was available.
Regarding behaviors, we observed clear differences between the P + and N- conditions. Visual and tactile interactions with the experimenter were significantly greater in the N- condition than in the P + condition. This difference has been previously reported in horse-human communication paradigms. In Ringhofer’s protocol, when horses could not open the box that they knew contained food, they exhibited tactile interactions with the experimenter, which could be explained as a request for help45. In another protocol, the horses solicited more assistance (by looking and touching) from the experimenter that witnessed which sealed bucket contained the food46. Additionally, horses showed perseverance in their communication with the experimenter by changing between visual and tactile signals to obtain the desired but inaccessible food47. Attention toward the experimenter is also a behavior observed in dogs, with an increase in “side orientation to the location of the human” when they felt frustrated in a reinforcement-extinction task involving inaccessible food16. In the present study, these behaviors could therefore be a way for horses to communicate with the experimenter when they experience frustration in the N- condition. Additionally, horses showed more ear movements in the N- condition than in the P + condition. In some other mammals, such as pigs, it has been proposed that ear movements are an indicator of negative emotions48, which could also be the case in horses. The frequency of tail swishing did not differ between the two situations, perhaps because in the literature, even if this behavior is expressed in negative situations, it appears when the horse is worked or ridden and may reflect physical discomfort rather than frustration 35,49,50.
Regarding facial expressions, two facial expression patterns were found specific to the P + and N- conditions. The expression with ears forward, a low neck and advanced upper lip were the main characteristics of horses in the P + condition, i.e., the condition used to induce positive anticipation. These facial cues are generally described in the scientific literature as indicators of positive situations. For instance, in horses, forward ears are correlated with indicators of positive emotion, such as snorting51 or play behavior, such as when foals “prance” with the ears forward52. Additionally, other animals, such as foxes, showed more upright ears during positive anticipation38. An advanced upper lip is also reported in horses in response to positive tactile contact with a human31. Upper lip advancement is described in the EquiFACS as Facial Action Unit 18, "lip pucker"30. It appears in situations such as when the horse rubs against something to scratch itself; scratching is known to produce pleasure and activation of the reward system53 and to trigger short-lasting positive emotions. The presence of an advanced upper lip in these different situations with the same valence (positive) suggests that this indicator might be very useful for identifying positive emotions in horses. This is also the case for the low neck position. During gentle grooming, low neck position is exhibited more frequently by the horses31, as is the case in the present study with the anticipation of food reward.
In contrast, higher neck position and ears backward or to the side were the principal features of the horses’ expression during the N- condition. In the literature, high neck position in horses is associated with vigilance and hypervigilance44,54 or an alert posture55, emotions with negative valence. Ears backward are also commonly described in negative situations, such as pain after a surgical operation34 or when ridden35 with musculoskeletal pain28. Ears backward is also associated with negative behaviors such as aggressiveness56 or conflict behaviors when horses are trained57. This indicator appears in the literature in a variety of situations with the same valence (negative). It is therefore interesting to observe this same indicator in the present study in the N- condition, used to induce frustration. Other species, such as dogs, also express frustration by moving their ears caudally4. However, the position of the ears, whether forward or backward, can also indicate that the horse is hearing the sound of the feed bucket, which is one of the limitations of the present study. Finally, eye blinks were more frequent in the N- condition than in the P + condition, similar to findings in the induced frustration protocol for dogs; dogs in the negative condition, who could not access food4, exhibited more eye blinks than those in the positive anticipation condition. Blinking is also used as a negative indicator of pain in ridden horses35 and could be an indicator of negative emotions in other negative situations, such as stressful situations58. Frequencies of dilated nostrils, wide eyes and exposed sclera did not differ between the P + and N- conditions. These indicators have been reported in situations of fear or pain27,34,35 but appear not to be specific to situations of frustration or positive anticipation. Overall, two very different patterns of facial expression were clearly expressed in the conditions with opposite valence. Each individual facial expression unit corresponds to current understanding of horse behavior (for example, ears backward in negative situations), but the combination of them during positive anticipation and frustration is novel and can serve as an indicator of positive anticipation and frustration in horses.
Regarding locomotion, speed, stride frequency and total activity were all significantly higher in the P + condition than in the N- condition. Horses moved faster in the P + condition than in the N- condition, which could translate into a strong desire to reach the food bucket in the P + condition and therefore a positive anticipation state, whereas the slower speed in the N- condition could indicate a refusal to move away from the food bucket. Examining speed in greater depth, there was an increase in stride frequency but not in stride length, indicating that the horse did not increase its speed by lengthening its strides but by increasing their frequency. This increase in locomotor activity seems consistent with the increase in activity observed in many species during positive anticipation7,10,38. Additionally, in humans, locomotion and kinematics of happiness are expressed as more “energetic” behavior than those associated with other emotions, and speed and kinematic changes are used to recognize emotion59. These results are novel in the horse.
To conclude, in this study, we describe two very different patterns of behavior, including facial expressions and locomotion, in two conditions with opposing valence: one typical of a positive emotion (positive anticipation) and the other typical of a negative emotion (frustration). In the literature, positive anticipation has been suggested as a potential indicator of positive emotion60 and may enrich animals’ positive experiences 61. On the other hand, frustration has many physical and behavioral repercussions for animals as the appearance of stereotypies62. These results could serve as a basis for developing a horse repertoire to identify these two emotions in the field and could be incorporated into the larger framework of animal welfare.