Abstract
Spontaneous eye blinks are brief closures of both eyelids. The spontaneous eye blink rate (SEBR) exceeds physiological corneal needs and is modulated by emotions and cognitive states, including vigilance and attention, in humans. In several animal species, the SEBR is modulated by stress and antipredator vigilance, which may limit the loss of visual information due to spontaneous eye closing. Here, we investigated whether the SEBR is modulated by attention in the domestic horse (Equus caballus). Our data supported previous studies indicating a tonic SEBR specific to each individual. We also found that, superimposed on a tonic SEBR, phasic changes were induced by cognitive processing. Attention downmodulated the SEBR, with the magnitude of blink inhibition proportional to the degree of attentional selectivity. On the other hand, reward anticipation upregulated the SEBR. Our data also suggested that horses possess the cognitive property of object permanence: they understand that an object that is no longer in their visual field has not ceased to exist. In conclusion, our results suggested that spontaneous eye blinks in horses are modulated by attentional cognitive processing.
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Introduction
Spontaneous eye blinking is a temporary closure of both eyelids and is an automatic process. It serves a physiological function in keeping the cornea moist and protecting the eye1,2,3. However, blinking frequency exceeds functional requirements4, suggesting that other factors affect the occurrence of spontaneous eye blinks (SEBs)5. Investigating SEBs in horses could provide information on their physiology and cognitive processes.
In humans, SEBs are modulated not only by environmental parameters such as temperature, humidity, air movement, and time of day6 but also by endogenous factors such as a person’s emotional or cognitive state7,8. Vigilance, fatigue and sleepiness9,10,11, stress and anxiety12, fear, sadness and pleasant stimuli13 all influence the spontaneous eye blink rate (SEBR), as do cognitive processes such as conversation and reading7, cognitive load14, task difficulty15 and attention16. Attention leads to a decrease in the blinking rate17 with the magnitude of blink inhibition proportional to the attentional demands13,18,19. Moreover, SEB characteristics are related to the allocation of attentional resources16,19. For example, while reading, SEBs tend to occur at the end of a phrase20, improving visual information capture, and can be strategically time-locked to saccadic eye movements21.
Animals spend a substantial amount of time monitoring their environment to detect potential threats. While blinking, the influx of visual information from the external world is lost for approximately 100–150 ms22. To circumvent this loss, the rate, duration and timing of SEBs can be adjusted. The SEBR is an indicator of antipredator vigilance in many species; it is related to the group size of primates23, including wild Anubis baboons (Papio anubis)24 and of red deer (Cervus elaphus)25. Chickens (Gallus gallus domesticus) modulate their blinking rate between feeding bouts to monitor their surroundings more effectively26. Peacocks (Pavo cristatus) tend to blink during gaze shifts when vision is degraded27. SEBRs and patterns can also be modulated by attention, such as in American crows (Corvus brachyrhynchos), which blink less when seeing a threatening person than when seeing a caring person28. Vigilance and attention may both be involved, similar to songbirds, which modify their blinking patterns relative to the perceived level of risk29.
Vigilance is a prerequisite for all upstream cognitive processing30 and is functionally distinct from selective aspects of attention31. Vigilance allows sensitivity to potential environmental changes32,33 and promotes an adequate reaction to ensure the survival of the individual34,35. Vigilance has been described as a state of high physiological efficiency36 and is related to the level of central nervous system activation37 through the ascending reticular activating system (ARAS)38,39. The ARAS is a network arising from the brainstem with widespread excitatory cortical projections40,41. In addition to the basal forebrain, the ARAS activates the thalamo-cortical system, which is phylogenetically shared among all vertebrates, suggesting similar functional states (such as vigilance)42. Neuronal activity in this network determines how receptive the brain is to external stimuli and how these stimuli influence the quality of perception43. In several species, the SEBR is a meaningful measure of vigilance23,24,25,26,27,44 that can be assessed through striato-thalamo-cortical projections44,45. Neural systems other than the ARAS, such as stress, motivation and novelty, may also modulate vigilance46.
Attention adds to vigilance by directing an individual towards spatially important stimuli33,47. When awake, the brain receives an overwhelming amount of information at all times. This excess requires the animal to distinguish the relevant information from irrelevant noise. Because the energy available to the brain48 and the capacity of the brain to process sensory information are limited49,50, the processing of relevant information is facilitated while distracting information is rejected51,52. To support this information selection, attention optimizes brain resource allocation53 and enables a more focused activation of the cerebral cortex that enhances target information processing53,54,55,56. Attention increases neuronal firing rates in neurons representing attended locations while inhibiting neuronal activity at unattended locations57. By filtering relevant information from the environment, spatial attention enhances the quality and speed of perceptual processing58 and enhances spatial resolution at the attended location (for a review, see Carrasco, 2011). SEBs rates and patterns are modulated by attention in several species13,17,18,19,28,29.
The attention filtering process is supported by at least two mechanisms. First, visual and auditory systems exhibit great congruence in orienting attention towards a specific target; for example, in sound localization59, the auditory system will acoustically guide visual orientation towards potentially important events. Second, as visual information is missed during spontaneous eye closing, modulation of the SEBR may limit its loss.
In this study, we investigated whether these two mechanisms are involved in the attentional processing of horses. We chose the horse (Equus caballus) as a biological model because, as prey animals, their survival strategy when facing a threat is to take flight. The success of this strategy depends on optimal threat detection, wherein vigilance and attention are key mechanisms. Horses have a wide visual field of approximately 340 degrees, with only a small area of nonvisibility behind them, allowing for extensive monitoring of their surroundings60. In addition to this perceptual anatomical conformation, the modulation of SEBs would improve event detection by limiting the loss of visual perception, and cognitive properties such as vigilance and attention would improve the selection of meaningful surrounding information. This study used data from a previous experiment that aimed to investigate horses’ intentionality in cross-species communication with humans. Here, we hypothesize that the SEBR of horses is modulated by vigilance and attentional processing and that these modulations occur in the human–animal social context.
Current knowledge of the SEBR of horses suggests that it could be an indicator of stress61, and it has been correlated with salivary cortisol levels and heart rate variability62,63. The SEBR has also been associated with a horse’s temperament64. One study65 investigated the potential modulation of the SEBR by attention and unexpectedly revealed an increase. The authors hypothesized that the need to compensate for tear evaporation (induced by air movement associated with displacement) prevailed over an attention-induced decrease in SEBR.
In this study, we investigated whether the SEBR in horses is modulated by vigilance and/or attention in a human–horse social context. Horse attention was measured by ear position, which is a well-known behavioural marker of attention orientation while horses are selecting relevant information from their surroundings66,67,68,69,70. To date, most studies have investigated the direction of attention. Modulation of the SEBR would provide additional information about the attentional load and insight into some horses’ cognitive properties that are linked to SEBs.
We hypothesized that the SEBR would decrease in the presence of food as well as in the presence of a human experimenter attentively facing the horses. Our prediction was that SEBR could be a neurophysiological index of attention in horses. As attention is involved in a variety of cognitive processes, such as perception, decision-making, problem solving, learning, and memory, the SEBR could serve as a noninvasive marker for future studies, particularly those on horse communication, social cooperation and social learning, in both ecological conditions and human–horse interactions.
Results
Blinking
In all study conditions, the horses blinked an average of 12.3 + /− 6.3 times per minute. The SEBRs for each condition and descriptive statistics are displayed in (Table 1). There was no difference in the SEBR based on sex in all study conditions combined (V = 429, p = 0.812) or in each individual condition. This finding is similar to that in humans71 (Supplementary Table S2a online). No SEBR correlations were found with age (Supplementary Table S2b online). No differences in SEBRs were found between stables in the control condition (rank correlation Kruskal–Wallis test, H(2) = 1.7658, p = 0.4136), the food condition (H(2) = 1.9886, p = 0.370), or the experimenter condition (H(2) = 0.4341, p = 0.8049).
The SEBRs were significantly differentially modulated among the 3 conditions (F(2,42) = 5.81, p = 0.05) (Fig. 1).
(a) Boxplot showing the spontaneous eye blink rate during the three conditions. (b,c) Correlations of the spontaneous eye blink rate between the control and food conditions (b) and between the control condition and the experimenter condition (c). (d) Kernel graphs of the spontaneous eye blink rate density in the three conditions. CO control condition, FC food condition, EC experimenter condition. *p < 0.05.
Behavioural index of attention orientation
The orientations of the pinnae were first classified relative to the horse’s rostrocaudal axis (Table 2a) (Fig. 2a) (Supplementary Figure S1 online). The duration for which both ears were oriented forwards did not differ among the three conditions (general linear model: F(2,42) = 1.91, p = 0.385). The duration for which the ears were in the divided position with one ear forwards was significantly different among the three conditions (F(2,42) = 23.37, p < 0.001), with a greater duration in the EC than in the CO (Student test; t(21) = -5.81, p < 0.001) and in the FC (t(21) = −6.7, p < 0.001). The duration for which the ears were divided in any direction other than forwards was significantly different among the three conditions (F(2,42) = 36.3, p < 0.001), with a greater duration in the FC compared to the CO (Wilcoxon test: V = 6, p < 0.001) and to the EC (V = 253, p < 0.001) and a shorter duration in the EC compared to the CO (V = 204, p = 0.007). The ears were in the lateral position significantly more often in the CO (F(2,42) = 23.3, p < 0.001). The distribution of the ear positions was analysed for each condition (Supplementary Fig. S1). In CO, no significant differences were found among the positions, but significant differences were found in the FC and EC (Supplementary Table S3 online).
(a) Boxplot showing the ear position durations relative to the horse’s head rostrocaudal axis in the three conditions. (b) Boxplot showing the ear position durations relative to the experimenter location. CO control condition, FC food condition. *p < 0.05; **p < 0.01; ***p < 0.001.
Next, the orientations of the pinnae were classified relative to the position of the experimenter which is a fixed location in front of the horse, either occupied by the experimenter in the experimental condition, or unoccupied in the control and the food conditions (Table 2b) (Fig. 2b). The horses’ ears were oriented towards the experimenter position significantly more often in the EC (V = 5, p < 0.001) and less often in the FC (V = 232, p < 0.001), than in the control condition. The ears were oriented towards the outside of the experimenter significantly more often in the FC (F(2,42) = 36.1, p < 0.001) and in any other direction than towards the experimenter significantly less often in the EC (F(2,42) = 22.2, p < 0.001). The ears were oriented towards the experimenter in the EC significantly more often than towards the outside of the experimenter in the FC (V = 3, p < 0.001).
Effect of food out of reach on the SEBR
In the presence of food placed out of the horses’ reach (FC), the SEBRs were significantly lower than those in CO without food (V = 171, p = 0.042). The SEBR in CO was moderately correlated with the SEBR in FC (Pearson correlation test: r = 0.49, p = 0.02) (Fig. 1b). Moreover, the decrease in the SEBR between the FC and CO was moderately negatively correlated with the SEBR in CO (r = −0.48, p = 0.025), suggesting that the extent of the decrease in the SEBR depends on the initial blink rate.
Effect of the presence of the experimenter on the SEBR
In the presence of the experimenter (in addition to the food), the SEBR was not significantly different from that in the CO (V = 138, p = 0.721). The SEBR in the EC was moderately marginally correlated with the SEBR in CO (Spearman correlation test: ρ = 0.41, p = 0.056), with individuals exhibiting a high initial SEBR maintaining a higher rate in these conditions and individuals exhibiting a lower initial SEBR maintaining a lower rate in these conditions (Fig. 1c). Although the SEBR was not significantly different between the EC and the CO, the high standard deviation of the SEBR in the EC suggested different modulation patterns (Table 1, Fig. 1a). A strong positive correlation was found between the change in SEBR between the EC and CO and the SEBR in the EC (ρ = 0.715, p < 0.001), suggesting that the horses exhibited different modulation patterns in the EC (Kendall test: W = 3.495, t = 0.554, p < 0.001). No correlation was found with the SEBR in CO (r = −0.268, p = 0.227), suggesting that there is no relationship between modulation of the SEBR and the SEBR in CO. The kernel density graphs show different distributions among conditions, suggesting that none of the horses modulated their SEBR in the same way in the EC relative to CO (Fig. 1d). Cluster analysis revealed two groups of horses based on the SEBRs in the EC (Cluster 1: n = 14; Cluster 2: n = 8). The two groups differed significantly in the EC (V = 112, p < 0.001).
In Cluster 1, the SEBRs were significantly different among the three conditions (F (2, 26) = 3.59, p = 0.042) (Fig. 3a). Post hoc tests indicated that there was no difference between CO and FC (t (13) = 1.13, p = 0.209) or between the FC and EC (t (13) = 1.17, p = 0.63), but there was a significant decrease in the EC compared to CO (t (13) = 3.20, p = 0.021). The SEBRs were not correlated between the two conditions (ρ = 0.11, p = 0.702). This result suggests that the decrease in the SEBR was not linearly induced by the presence of the experimenter but rather was related to greater attention directed towards the experimenter, as indicated by the significant increase in ear orientation towards the experimenter position (t(13) = −6,46, p < 0.0001).
Boxplot showing spontaneous eye blink rate during the three conditions for Cluster 1 (a) and Cluster 2 (b). CO control condition, FC food condition, EC experimenter condition. *p < 0.05.
The decrease in the SEBR between CO and EC was not correlated with the increase in the amount of time spent with both ears forwards towards the experimenter (ρ = 0.11, p = 0.71). However, the blink rate was moderately negatively correlated with the amount of time spent in the ears forwards position (ρ: −0.55, p = 0.043) in CO but not in the EC (ρ = −0.19, p = 0.505). The SEBR in CO was moderately positively correlated with the time spent with the ears backwards (r = 0.53, p = 0.049). The ears were oriented in the forwards N position significantly less often in the EC than in the CO (V = 105, p < 0.001), and the decrease in the blink rate was not correlated with the decrease in duration of the ears in the forwards N position (ρ = -0.06, p = 0.84).
The decrease in the blink rate between CO and EC was strongly negatively correlated with the blink rate in the control condition (r = −0.846, p < 0.001). This result suggests that a lower (physiological) limit of the blink rate could be responsible for the lack of correlation between the decrease in SEBR and the increase in duration of ears oriented towards the experimenter (r = 0.001, p = 0.99).
In Cluster 2, the SEBRs were significantly different among the three conditions (F(2,14) = 12.2, p = 0.002) (Fig. 3b) (Table 3). According to the post hoc tests, the SEBRs were significantly lower in the presence of food than in CO (V = 28.0, p = 0.022) and EC (V = 0, p = 0.014). The SEBR was significantly greater in EC than in CO (V = 3, p = 0.04). The SEBR in CO was strongly positively correlated with the SEBR in FC (ρ = 0.871, p = 0.004) but not with the SEBR in EC (ρ = 0.44 p = 0.27). The SEBR in the EC was strongly negatively correlated with the time spent with the ears in the divided position with one ear oriented towards the experimenter (ρ = −0.97, p < 0.001). The increase in the SEBR between the EC and CO was strongly negatively correlated with the increase in the time spent in the position with both ears forwards towards the experimenter (ρ = −0.92, p = 0.001) and was positively correlated with time spent in the both ears backwards orientation (ρ = 0.77, p = 0.025). This finding suggested that the horses with the greatest increase in the time spent in the ears forwards position in the EC had the lowest increase in the SEBR. Interestingly, the increase in the SEBR was not correlated with the change in the ears oriented towards a target other than the experimenter (ρ = −0.38, p = 0.34).
No differences were found in the ear positions between the clusters in each condition (Table 4).
Behavioural measures
Behavioural indicators were measured to evaluate potential frustration. No differences in the following occurrences were found between the conditions: body displacement (F(2,42) = 0.949, p = 0.622), forwards body displacement (F(2,21) = 0.189, p = 0.911), and trampling movement (F(2,21) = 1.279, p = 0.528). No differences were found between the clusters (see Supplementary data, Table S4 online).
The height of the neckline was measured as an index of vigilance72. A decrease in the time spent with the ears in the lateral position was moderately negatively correlated with an increase in the incidence of a high neckline between CO and EC (r = −0.42, p = 0.05). No correlation was found between CO and FC (r = −0.262, p = 0.236).
Correlation of SEBR modulation with behavioural indices of attention orientation
The SEBR was moderately negatively correlated with all ear positions, with the exception of lateral and backwards orientations (r = −0.44, p = 0.043) in CO. This correlation was not found in FC (r = −0.08, p = 0.72) or EC (ρ = 0.005, p = 0.98) (Fig. 4). In CO, the SEBR was also moderately negatively correlated with the time spent with the ears in the divided position with one ear forwards (r = −0.383, p = 0.046) but not with both ears forwards (r = 0.336, p = 0.126) or with ears divided (one ear lateral and one ear backwards) (r = 0.17, p = 0.43). No correlations were found in FC with the exception of ears in the lateral position (r = 0.49, p = 0.019). No correlations were found in EC.
Correlations between spontaneous eye blink rate and all ear positions, with the exception of the lateral and backwards orientations, in the three conditions. CO control condition, FC food condition, EC experimenter condition.
The change in SEBR between CO and FC was moderately negatively correlated with the change in the ears divided positions (ears divided and ears divided with one ear forwards) (r = −0.43, p = 0.045). This change in SEBR was not correlated with the change in ears forwards position, which is consistent with the lack of difference in the ears forwards position between the conditions (Table 1a) (r = −0.14, p = 0.52). The change in the SEBR between CO and EC did not correlate with the change in the ears in the divided position (ρ = −0.45, p = 0.27) or with the change in the ears forwards position (r = −0.15, p = 0.52). This result was consistent with the divergence of the SEBR modulation of all individuals between CO and EC.
Discussion
The present study revealed that horses exhibit a tonic spontaneous eye blink rate (SEBR), which can be modulated by cognitive processes, such as vigilance, attention and reward anticipation. Moreover, our data indicate strong complementarity between the visual and auditory systems in the attentional processing of horses.
High and low blinkers
High SEBR variability was observed among individuals. This variability is widely recognized in humans7 and has also been found in primates23, dogs73 and horses62. Additionally, the SEBRs were correlated between conditions, with the high/low blinkers remaining the same throughout the conditions. This finding is consistent with the relationship between a horse’s temperament and its modulation by dopamine (DA), to which the SEBR is correlated74. Momozawa et al.75 reported that single-nucleotide polymorphisms (SNPs) of the equine DA receptor DRD4 gene might be related to individual differences in equine temperament for vigilance and curiosity. Roberts et al.64 reported low and high blinkers. ‘Anxiety’ was positively correlated with the SEBR and high DA within the amygdala, a structure involved in anxiety, whereas ‘docility’ was negatively correlated with SEBR, ‘docile’ horses being also more even-tempered and self-controlled, indicating reduced DA activity.
In humans, tonic and phasic SEBRs have been described. Tonic SEBR refers to the baseline blink rates at rest in neutral conditions, and phasic SEBR refers to blink rates in response to stimulus conditions74,76. As in animals, human SEBRs are also related to various aspects of personality77. A higher baseline SEBR has been associated with better cognitive flexibility and distractibility78,79. However, these correlations were not always replicated74,80. Compared to humans, a stronger association among temperament, DA and SEBR in horses was supported by the fact that the equine temperament is considered to be less influenced by individuals’ mothers since horses are more mature at birth. An equine temperament emerges at a young age and appears to be stable over both time and situation81. According to our data, the consistency of the high/low blinkers across all conditions suggests that, in horses, a tonic SEBR could also be an intrinsic property of each individual (even if it may be modulated by phasic changes). Although we did not evaluate the temperament of the horses in our study, these data support the results of Roberts et al.64.
Complementarity between the visual and auditory systems
In the control condition, we identified a negative correlation between the horse’s SEBR and the combination of all ear positions oriented in directions where the auditory and visual fields overlapped. The more often the horses had their ears oriented towards directions covered by their visual field, the less they blinked. For many species of mammals, one key function of directional hearing systems is to acoustically guide vision towards potentially important events23,24,25,27,59, suggesting cooperation between the visual and auditory systems. Our data suggest that this complementarity between visual and auditory systems is also implemented in horses.
Among the ear orientations overlapping the visual field, the SEBR was correlated with the ears in a divided position with one ear forwards, a position indicative of surrounding monitoring, suggesting a relationship between the SEBR and the vigilance level. No correlation was found for both ears forwards, a position that could be highly adopted in the nocturnal resting state82, or during positive experiences such as grooming83, suggesting that the ears forwards position in our control condition indicated a quiet and comfortable condition for the horses without specific monitoring. No correlation was found with divided ear position (including one ear oriented backwards), a direction in which no complementarity between audition and vision is feasible.
The lack of correlation of the SEBR with the lateral ear position is consistent with the resting state associated with this position in which no surrounding monitoring is performed67.
The SEBR was marginally correlated with the ears backwards position. This result is consistent with a disconnection between the auditory and visual systems in this position, with the horses being blind to what happens at their back. This disconnection is an opportunity to perform the physiologically needed SEBR without any risk of missing visual information. This strategically timed pattern was also observed in other species showing preferential blink occurrence during low visual information periods, such as humans at the end of reading a sentence84, in the vicinity of line change saccades85, during scene changes86 or during intervisual task intervals87. Eye blinks also synchronize with other behaviours impairing visual perception, such as eye saccades21, combined eye-head movements in monkeys (Macaca mulatta)88, gaze shifts between targets in peacocks (Pavo cristatus)27 or strategies based on the perceived level of risk in chickens (Gallus gallus domesticus)26 and song birds (Quiscalus mexicanus)29.
Under our experimental conditions, these correlations were lost. Ear orientations were no longer homogenously distributed, as in the control condition, and the ears selectively pointed at a specific target (a food item or the experimenter). The time spent with ears in the lateral and backwards orientations was reduced by 47% in the food condition and 45% in the experimenter condition relative to the control condition. Our hypothesis is that the time spent with the ear orientation disconnected from the visual field was too short to allow the expression of SEBs relative to physiological needs and temperament64.
Horse SEBR modulated by vigilance
Although in some species, a vigilant state has been reported without outwards signs89, vigilance can generally be measured through observable behavioural markers such as visual scanning, e.g., gaze shifts, ear pointing and/or head raising34,35.
Horses have laterally placed eyes and a wide visual field due to the high density distribution of retinal ganglion cells along the horizontal visual streak90,91,92,93. This allows extended visual scanning without the need for gaze direction shifts92; thus, in horses, other markers would be better candidates for indicating the level of vigilance.
In our study, the SEBR modulation between the conditions was associated with changes in ear and head markers of vigilance. The lateral orientation of the ears, a position associated with the resting state and lack of active monitoring of surroundings67, was significantly less pronounced in the experimental conditions than in the control conditions. Moreover, the decrease in the time spent with the ears in the lateral position was negatively correlated with the increase in the incidence of high necklines between the control and experimenter conditions, suggesting that vigilance increased between these conditions. No correlation was found between the control and food conditions, but vigilance may have been modulated without clear behavioural observable cues89, especially if the degree of vigilance modulation was low. This could have been the case in the food condition, as the chance of getting the food was lower than that in the presence of the experimenter. Our results suggest that the SEBR in horses is modulated by vigilance, which is consistent with previous studies in other species23,24,25,27.
Effect of food (carrot) out of reach on the spontaneous eye blink rate
Attention adds to vigilance a direction towards spatially important stimuli33,47. In the presence of a carrot, which was clearly visible but unreachable for the horses, the diffuse distribution of the ear positions found in the control condition was lost, and the horses expressed more selective orientation of their ears. This finding suggests the involvement of attention towards a specific target, adding a stimulus-driven orientation to vigilance. We hypothesized that an increase in ear orientation towards the carrot would occur, but surprisingly, the reverse was observed. The horses did not selectively orient their ears towards the carrot but rather oriented them more towards the experimenter’s outside from the experimental area. This result suggests that the horses were attentive to the potential return of the experimenter and were aware of the need for the experimenter’s intervention to obtain the carrot. This finding shows that horses possess the cognitive property of object permanence94,95 that humans gain at approximately one year of age96. They understand that an object that is no longer in their visual field has not ceased to exist.
The SEBR was significantly lower in the food condition than in the control condition. One explanation could be that the out-of-reach carrot was frustrating to the horses. A decrease in SEBR was observed when a reward was inaccessible in dogs97. Frustration may induce displacement and repetitive behaviours98. However, no behavioural signs of frustration were observed in our study. No changes in body displacement, specific forwards displacement towards the carrot or trampling were observed relative to those in the control condition. Although frustration could not be excluded68, a more likely explanation might be that, as discussed by Daniel Kahneman16, focused attention is closely linked to eye physiology. A reduced SEBR allows improvement in visual information capture, as the closing of the eyes during a blink prevents visual information from reaching the retina22. In our study, the association of attention focused on the experimenter’s outside and SEBR downregulation suggested that, as in humans17, horse attention leads to a decrease in SEBR.
Moreover, the negative correlation between the decrease in the SEBR between the food and control conditions and the SEBR in the control condition suggests that the importance of the decrease in the SEBR depends on the initial blink rate. Two explanations can be suggested. First, there may be a minimal limit of the SEBR, so there is more room for the SEBR to decrease if it is higher under control conditions. A second hypothesis could be that, as in humans, a high tonic SEBR predicts a reduced threshold for responding to the saliency of stimuli80, with individuals with a high tonic SEBR expressing greater modulation of the SEBR. Supporting this last hypothesis, horses that exhibited the most significant decrease in SEBR also exhibited the most significant increase in time spent with the ears in the divided positions. However, there was no correlation between the decrease in SEBR and the decrease in the ears in the resting position or with the increase in ear orientation towards the experimental outside. These results partially support the hypothesis of greater attentional modulation by individuals with high tonic SEBR. This possibility should be further investigated.
Spontaneous eye blink rate modulation in the presence of food and the experimenter
The horses oriented their ears significantly longer towards the experimenter than in control condition, suggesting that selective attention was oriented towards her as a visual stimulus66,99. However, the respective attention towards the carrot or the experimenter could not be dissociated because the carrot was in front of the horses and was aligned on the same axis as the experimenter. However, when a food reward is inaccessible, the horses solicit humans with touches from the nose, and they increasingly look at the experimenter100,101. The use of a food reward as positive reinforcement increases horses’ selective attention towards their trainer102. Additionally, in our study, when only carrots were present, the horses mainly monitored the return of the experimenter and not the carrot itself. This result suggests that, in the experimenter condition, the horses’ attention was probably directed more towards the experimenter than towards the carrot.
The SEBR was modulated relative to the control condition differently for all horses. Two subgroups of SEBRs were observed.
The SEBR decreased for the horses in Cluster 1. A study revealed that the SEBR of horses decreases with stress62. One explanation could be that the out-of-reach carrot and the experimenter remaining stationary induced frustration in the horses. However, there was no increase in stress- or frustration-related behaviours. A more likely hypothesis would be that the decrease in SEBR would improve visual information capture. Moreover, the ears were significantly more selectively oriented towards the visual targets in the experimenter condition (the experimenter and the food) than in the food condition (the experimenter’s outside). Relative to the control, the SEBRs decreased more sharply in the experimenter condition than in the food condition. This finding is consistent with what has been observed in humans103,104, in whom the magnitude of SEB inhibition is proportional to attentional demands13,18,19. Similar findings have been reported for macaques (Macaca mulatta), which inhibited their blinking more when their attention was better captured by more visually rich videos105. Our results suggest that, as in humans106 and macaques105, a horse’s blinking rate has an inverse relationship with attention selectivity, with the SEBR being more inhibited when attention is more focused.
The SEBR of the horses in Cluster 2 increased in the presence of the experimenter. This increase in the SEBR has also been observed in chimpanzees when their keepers are approaching107. One study revealed that a horse’s SEBR increases with stress61,63, but again, no behavioural indices of stress were observed in the experimenter condition. A more likely hypothesis is related to dopaminergic activity. Changes in the SEBR have been associated with dopamine (DA) levels in humans108, primates108,109,110, and rats111 and have been suggested to occur in horses64. Therefore, the SEBR is a useful predictor of central DA function, with a higher SEBR predicting greater DA function74. DA is a pivotal neurotransmitter involved in the reward neuronal network not only at the time of receiving a reward but also in reward anticipation112 and reward prediction113. In humans, reward anticipation is associated with an increase in SEBR114. Our results suggest that this could also be the case in horses. If the DA neurons are first activated by the primary features of a reward (e.g., the carrot in our study), after learning, their activation could then be associated with environmental cues (such as the presence of the experimenter) in anticipation of the reward, which would activate DA release and elevate the SEBR115. The SEBR increase of the horses in Cluster 2 suggested that they had associated anticipation of receiving a reward with the presence of the experimenter.
In support of the DA hypothesis, the increase in attentional selectivity towards the experimenter was also consistent with the role of DA in enhancing the selectivity of attention towards a reward. DA contributes to target selection through visual attention in macaques (Macaca fuscata)116, and its release is correlated with the orientation of attention to previous reward-associated stimuli115. DA makes the visual representations of reward-associated stimuli more salient by improving the neuronal signal-to-noise ratio117,118, providing a mechanism by which reward “sharpens” attentional control119. Horses also exhibit gaze alternations between the experimenter and an out-of-reach reward120. This condition could not be measured in our data, as the carrot and the experiment were aligned in front of the horses.
If attention oriented towards the experimenter and the carrot increased, no direct correlation was found between the duration for which the ears were oriented towards them and the SEBR. The same result has been observed in humans, who differ both in the degree of influence of reward history on attention and in the amount of striatal dopamine released in response to reward115. Our data suggest that these variabilities are also present in horses. These findings could explain why the SEBR was not directly correlated with any of the ear positions in the experimenter condition.
Interestingly, the decrease in the SEBR under food condition in Cluster 2 suggested that the carrot alone did not induce a reward effect. This result is consistent with the fact that the horses’ attention was more strongly oriented towards the outside of the experimenter than towards the carrot. The horses seem to have prioritized the need for the experimenter’s help to obtain the carrot over the potential reward of the carrot itself. In Cluster 2, we also observed a negative correlation between the increase in the orientation of the ear towards the experimenter and the increase in the SEBR, suggesting that the horses that have the most increased attention towards the experimenter have the least increased SEBR. This finding suggests that the SEBR of these horses was modulated by both reward anticipation and attention in opposite directions. Additionally, the increase in SEBR was not correlated with a change in the ears oriented towards a target other than the experimenter. Two hypotheses could be suggested: first, for the correlation to achieve a significant level, more individuals need to be included, as the duration of ears oriented towards another target was very short in the presence of the experimenter. Second, the attractivity of the carrot and the experimenter induced a greater attentional load than the surroundings.
In humans, individual differences in basal dopamine levels in the striatum can predict the strength of reward-related motivation121. Individuals with a higher SEBR experience a stronger reward effect122. This suggests that horses with a high tonic SEBR could be more motivated towards the reward value of the food through their higher DA level in the neural reward network. In our study, we found no correlation between SEBR in the control condition and SEBR modulation in the experimenter condition. Although the horses in Cluster 2, which exhibited increased SEBR in the experimenter condition, had an SEBR at or above the mean SEBR with all individuals included, our results are not as clear, as a few of the horses with high SEBR in the control group did not maintain a high SEBR in the experimenter condition. This finding suggests that some horses did not consider the carrot as a potential reward but simply modulated their SEBR according to the attention level. Moreover, the respective contribution of reward anticipation versus the involvement of attention associated with the target could vary among individuals with several differentially involved factors, such as the perception of the carrot as a reward, varying thresholds in the estimated probability of receiving the reward, and the assessment of the perceived stimulus. All of these factors remain to be further investigated.
Den Daas et al.123 reported that people in an impulsive state focus their attention on salient information, whereas people in a reflective state distribute their attention. In horses, the SEBR is correlated with levels of impulsivity124 and anxiety64. The orientation of both ears forwards is indicative of focused attention, while the divided position is indicative of more distributed attention66,67. In our study, for the horses with a greater SEBR in Cluster 2, we detected a correlation between the SEBR and the duration at which the ears were in a divided position with one ear forwards in the control condition. There was also a strong correlation between the SEBR and the duration of ears in the divided position with one ear towards the experimenter in the experimenter condition. There was no correlation with both ears forwards. In contrast, in the horses of Cluster 1 with a low SEBR, we observed the reverse situation: a correlation between the SEBR and both ears forwards but not with divided ears. This result suggests that, contrary to humans, high-SEBR horses are more likely to distribute their attentional resources in monitoring both a salient stimulus (the experimenter) and their surroundings (in the control condition). This result is consistent with a greater sensitivity of anxious horses to potential environmental stressors and greater investment in monitoring their surroundings in addition to stimuli of interest.
Our results suggest that the SEBR of the horses in Cluster 1 decreased to avoid missing visual information, while the SEBR of the horses in Cluster 2 increased in anticipation of a potential reward (the carrot) from the experimenter.
This study has several limitations. The temperament of the horses was not tested, which would have reinforced (or not) the hypothesis of a potential relationship among tonic SEBR, temperament and DA-related functioning, such as in the reward context. Another limitation is the limited number of horses included in this study. As we did not expect to find two different phasic modulations of the SEBR in the experimenter condition and due to the high variability of the SEBR among individuals, we underestimated the number of individuals needed to consolidate the statistical results. Some correlation between the ear position and the SEBR may be lacking due to the different limits of occurrence/duration linked to the physiological systems of the individuals.
Conclusion
In conclusion, our data support previous studies suggesting a tonic SEBR in horses. Superimposed on tonic SEBR, phasic changes can be induced by cognitive processing. Attention downregulates the SEBR with the magnitude of the blink inhibition proportional to the degree of attentional selectivity. On the other hand, reward anticipation increases SEBR.
Additionally, we also found complementarity between the visual and auditory systems. The horses’ SEBRs were lower when the ears were oriented in directions where the visual and auditory fields overlapped. This result suggests a substantial congruence between hearing and vision when horses are monitoring the surrounding environment, e.g., in sound localization, where the auditory system acoustically guide the orientation of the vision towards potentially important events.
Our data also suggest that horses possess the cognitive property of object persistence: they understand that an object that is no longer in their visual field has not ceased to exist.
Methods
Participants
Twenty-two horses (Equus caballus) (11 mares, 9 geldings and 2 stallions) aged 4 to 26 years (mean age, 14.8 ± 5.6 years) of various breeds participated in the study. Information about each individual can be found in Supplementary Table S1 online. The horses were housed in three different stables. They went outdoors in fields daily and were kept in the stables at night. They had daily contact with their human caretaker/rider/owner (being fed, groomed, led between their stall and the fields and, for most horses, ridden on a regular basis). They were not deprived of food.
Experimental design
The experiments took place in the grooming area, an isolated and quiet space in the stables with which the horses were familiar. Both sides of their halter were loosely and safely attached allowing some freedom of movement (Fig. 5). All experiments took place in the afternoon on sunny days in June. The doors and windows in the experimental area were closed to ensure that no air movements were present.
Experimental area.
The same two experimenters, who were unfamiliar with the participating horses, were involved in all recording sessions: one experimenter kept time and managed the recording devices, and the second experimenter conducted the experimental interactions with the horses.
Three cameras fixed on camera stands were used for recording: a computer camera recording the full stage was placed behind and slightly laterally from the experimenter, and two cameras were placed to focus on the left and right sides of the horse’s head at an angle of 45° relative to the horse’s body and a distance of 2 m from the horse’s head (Fig. 5).
Preliminary phase
In the familiarization phase (0–5 min), the horses were brought to the grooming area individually and were progressively accustomed to the area with desensitization actions when necessary (keeping the cameras at a distance until the horse became familiar with them while one of the experimenters gently stroked the horse before placing the cameras at the final experimental position). Once the horse remained quiet and calm and showed no specific interest in the cameras, he was attached and left alone. The horse was evaluated through the consensus of all the experimenters (all of whom were familiar with horses) who looked for the following behaviours: no frightened immobility69, no tension in the facial muscles61 or body muscles, a horizontal or slightly elevated position of the neckline125, and no signs of attention specifically oriented towards the devices, including the head, eyes and ears pointed towards them67. Once these conditions were met, the experiment was started.
Experimental series
This experiment included two control conditions and seven experimental conditions to investigate whether horses adapt their behaviours according to the attentional state of the human experimenter. The control conditions (hereafter referred to as “control condition” and “food condition”) and one of the experimental conditions (hereafter referred to as “experimenter condition”) were used for the SEBR analysis. In the control condition, the horse was left alone for one minute. Then, the experimenter approached the horse holding a carrot, let the horse smell the carrot, and put the carrot on a high stool placed in front of the horse, clearly visible but unreachable. Then, the experimenter left the horse’s sight, and the food condition started and lasted for one minute. Immediately after these control conditions, the sequence of experimental conditions started. In the experimental conditions, the experimenter entered the experimental area and stood in front of the horse at 0.25 m behind the stool (Fig. 5). The experimenter adopted a different position for each experimental condition: (1) attentive, (2) inattentive, (3) eyes closed, (4) back turned to the horse with the head straight or (5) 1/4 turned, or (6) body 3/4 turned relative to the horse with the head straight or (7) 1/4 turned. The experimental condition where the experimenter looked attentively at the horse was selected for SEBR analysis because it was the optimal and simplest condition for observing the horse’s attentional state oriented towards the experimenter without additional variables. In this condition, the experimenter stood upright in front of the horse with her arms at her sides; she looked at the chamfer of the horse and followed its movement with her eyes but not her head without making eye contact, and she did not move. In each experimental condition, the experimenter maintained the position without moving for 1 min, left the experimental area (out of sight of the horse) for one minute and returned to stand in the next position. This process was repeated successively for the 7 experimental conditions. The order of the experimental conditions was randomly selected for each horse (order provided by the Android application “Tirage au sort sur papier”).
A brief vocal signal (“GO”) was given by the timekeeper to mark the recordings and indicate to the experimenter the onset/offset of the test condition.
Data collection and processing
All tests were recorded by three cameras: a computer camera recording the full stage and two cameras (JVC GZ-V500BE and JVC GZ-EX515BE) focused on the left and right sides of the horse’s head at an angle of 45° relative to the horse’s body and a distance of 2 m from the head (Fig. 5). The recordings from the right lateral camera were used to analyse the SEBR.
The videos were analysed with BORIS software (for Windows Portable v.7.9.8)126 in frame-by-frame mode at a rate of 25 images/sec.
The SEBR was defined as the onset and offset of the eye closing, allowing the calculation of frequency. A blinded rater without knowledge of the study coded 5 horses out of the 22 in the three conditions and exhibited an interrater agreement of 96%. The ear position classification was adapted from Reefmann et al.127 and included lateral (pinna opening oriented perpendicular to the body on the rostrocaudal axis), forwards (pinna opening oriented towards the front at an angle of more than 60° from perpendicular to the body axis) or backwards (pinna opening towards the back at more than 60° from the perpendicular); the asymmetric position was defined as the two ears being differentially oriented. The asymmetric positions where one of the ears was forwards-oriented according to the previous criterion were separately classified from all other asymmetric positions. Additionally, instead of being coded relative to the rostrocaudal body axis, the ear positions were coded relative to the specific directions of interest for this study as follows: 1) the position taken by the experimenter in the experimental condition, 2) the direction taken by the experimenter to leave the experimental area and 3) the ears oriented in any direction other than (1) and (2). The ear positions were classified as both ears in the direction of interest, asymmetrical with one ear oriented in the direction of interest and no ears oriented in the direction of interest. In the data analysis, the ear positions either relative to the horse’s rostrocaudal body axis or to the direction of interest (experimenter position/experimenter outside) were collected. These data allowed us to identify whether the attentional orientation of the horses increased towards the experimenter/the outside in specific conditions.
The ear position termed “towards experimenter position” included both the position with two ears forwards-oriented towards experimenter position and the position with at least one ear forwards-oriented towards experimenter position (including the asymmetric position with one ear forwards and situations when only one ear forwards was visible), with the two positions being mutually exclusive. The ear positions termed “towards the direction taken by the experimenter to leave the experimental area” included both the position with two ears forwards-oriented towards the outside direction of the experimenter and the position with at least one ear forwards-oriented towards the outside direction (including the asymmetric position with one ear forwards and situations when only one ear forwards was visible), with the two positions being mutually exclusive (see Supplementary Table S5 online).
The total amount of time in which only one ear was visible was 7.4% in the control condition and 7.5% in the experimental condition. The total duration of ear invisibility was 2.9% in the control condition and 0.7% in the experimental condition.
Additionally, body displacement occurrences128 (classified as any forwards, backwards or lateral displacement of at least one step), forwards movement occurrences of at least one step, and trampling movement occurrences were measured as potential behavioural indicators of stress. To assess the degree of attention, the height of the neckline72 was classified as low when the horse’s eye was below its withers, intermediate when the eye was at the same height as the withers and high when the eye was higher than the withers.
Statistical analysis
First, we retained only the image sequences where the complete SEB, that is, the lowering, closing and full opening of the eyelids, was identifiable; sequences in which the eye was transiently no longer visible, for example, if the horse turned its head to the side or lowered its neck out of the camera field, were not included. Next, we determined that we had a sufficient duration of visible data for all subjects to avoid over- or underestimation of occurrences when normalizing the data. We set the limit at 30% based on what is typically applied in similar studies. All the data for each condition and for each individual were normalized for one minute.
The Shapiro‒Wilk normality test was performed to determine the normality of the data. Differences in SEBRs were analysed using generalized linear models (GLMs) with condition as a fixed factor and subject as a random factor. The Friedman test and repeated-measures ANOVA were performed, followed by a post hoc test. Pairwise comparisons were performed using either the parametric Student’s t test or the nonparametric Wilcoxon signed-rank test with an error risk of Type I of 5%. Bonferroni corrections were applied except for the intracluster statistical analysis of Cluster 2 due to its low sample size because we had no a priori assumption that SEBRs would be differentially modulated by the presence of the experimenter. In the cluster 2 analysis, we reported exact p values with no adjustments, as we did not consider any correction of this type in the cluster a priori129,130,131. The significant results were compared via paired comparisons. Statistical computing was performed using RStudio version 4.0.3 (2020-10-10).
Because the high standard deviation of the SEBR in the EC could suggest different modulation patterns, univariate kernel density estimations with the Gaussian kernel function were calculated using Stata/SE 16.0 software. K-means clustering was performed. A Hartigan’s dip test was used to evaluate the multimodality of the SEBR distribution.
To evaluate any potential bias between the stables, rank correlation Kruskal‒Wallis tests were performed. The correlations between age and SEBR were tested with Spearman/Pearson correlation tests, and the effect of sex on SEBR was tested with Wilcoxon signed-rank tests or t tests.
Ethical considerations
All experimental procedures and protocols used in this investigation were reviewed and approved by the Institutional Animal Care and Use Ethics Committee of the University of Liège (Belgium), reference #22-2507. The “Guide for the Care and Use of Laboratory Animals,” prepared by the Institute of Laboratory Animal Resources, National Research Council, and published by the National Academy Press, was followed carefully as well as European and local legislation. The authors complied with the ARRIVE guidelines and regulations. Owners of the horses provided informed consent prior to participation.
Data availability
The raw data and analysis of this study are available from the corresponding author on request.
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Acknowledgements
The authors thank the Prince Laurent Foundation for their financial support, Prof. Tatiana Art for her valuable support, Ms Sophie Pellon and Ms Héloïse Ganier for their help in the acquisition of the data, Ms Chloé Redouin for being the naïve rater, Prof. Jean-Louis Deneubourg and Prof. Christian Melot for their advices in statistics, and Ms Clémentine Querton for her help improving English writing. We would like to thank the owners and their horses for their participation to the study with a special tribute to Spirit, Cosima, Rhapsodie, Mahony and Ouma who passed away since the data were recorded. The funding sources had no involvement in the writing of the article or in the decision.
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The study concept and design were set by C.T.. C.T. conducted the experiments. M.P. and L.d.S. coded the videos for the SEBR, CT coded the videos for the ears positions. C.T. and L.d.S., conducted statistical analysis and interpreted the data. C.T. wrote the manuscript. L.d.S. prepared all figures with original drawings and pictures. L.d.S. contributed to the structure and contents of the results and the discussion. All authors contributed and approved the final manuscript.
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Tomberg, C., Petagna, M. & de Selliers de Moranville, LA. Spontaneous eye blinks in horses (Equus caballus) are modulated by attention. Sci Rep 14, 19336 (2024). https://doi.org/10.1038/s41598-024-70141-y
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DOI: https://doi.org/10.1038/s41598-024-70141-y
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