Getting rid of blinkers: the case of mirror self-recognition in horses (Equus caballus).

The commentary by Gallup and Anderson (Anim Cogn https://doi.org/10.1007/s10071-021-01538-9 , 2021) on the original article by Baragli, Scopa, Maglieri, and Palagi (Anim Cogn https://doi.org/10.1007/s10071-021-01502-7 , 2021) raised some concerns about the methodological approach used by the authors to demonstrate Mirror Self-Recognition (MSR) in horses. The commentary does not take into account horse physiology and psychology, leading Gallup and Anderson to inappropriately discredit the findings obtained by Baragli et al. Anim Cogn 2021. In this reply, we underlined the importance of a blinker-free approach to understand the evolutionary processes at the basis of animal cognition.

If horses had toes: demonstrating mirror self recognition at group level in Equus caballus.

Mirror self-recognition (MSR), investigated in primates and recently in non-primate species, is considered a measure of self-awareness. Nowadays, the only reliable test for investigating MSR potential skills consists in the untrained response to a visual body mark detected using a reflective surface. Here, we report the first evidence of MSR at group level in horses, by facing the weaknesses of methodology present in a previous pilot study. Fourteen horses were used in a 4-phases mirror test (covered mirror, open mirror, invisible mark, visible colored mark). After engaging in a series of contingency behaviors (looking behind the mirror, peek-a-boo, head and tongue movements), our horses used the mirror surface to guide their movements towards their colored cheeks, thus showing that they can recognize themselves in a mirror. The analysis at the group level, which 'marks' a turning point in the analytical technique of MSR exploration in non-primate species, showed that horses spent a longer time in scratching their faces when marked with the visible mark compared to the non-visible mark. This finding indicates that horses did not see the non-visible mark and that they did not touch their own face guided by the tactile sensation, suggesting the presence of MSR in horses. Although a heated debate on the binary versus gradualist model in the MSR interpretation exists, recent empirical pieces of evidence, including ours, indicate that MSR is not an all-or-nothing phenomenon that appeared once in phylogeny and that a convergent evolution mechanism can be at the basis of its presence in phylogenetically distant taxa.

Validation of a Questionnaire for Equine Personality Assessement.

We propose a practical method of assessing the personality of horses using five personality axes. Four evaluators empirically judged 19 horse individuals on specific adjectives for each axis. To validate the questionnaire, four behavioral tests were conducted with these same animals (social interactions, novel object test, bridge test,and arena test). In this tests, the frequency of specific behaviors were evaluated to create a scale related to the same personality adjectives and judge the animals based on their reactions.The questionnaire was reliable in determining the personality of horses, since the results were consistent with those obtained through behavioral tests. Additionally, in this group of horses attention reactions were more frequent than fear reactions, but significant differences occurred among tests. This study proposes a practical questionnaire for owners and trainers to assess the personality of their horses. The application of this tool can improve the relationship between humans and horses, directing a more empathic approach in the everyday routine with these animals.

Heart and brain: Change in cardiac entropy is related to lateralised visual inspection in horses.

Cerebral lateralisation is the tendency for an individual to preferentially use one side of their brain and is apparent in the biased use of paired sensory organs. Horses vary in eye use when viewing a novel stimulus which may be due to different physiological reactions. To understand the interplay between physiology and lateralisation, we presented a novel object (an inflated balloon) to 20 horses while electrocardiogram traces were collected. We measured the amount of time each horse looked at the balloon with each eye. We calculated 'sample entropy' as a measure of non-linear heart rate variability both prior to and during the stimulus presentation. A smaller drop in sample entropy values between the habituation phase and the sample presentation indicates the maintenance of a more complex signal associated with a relaxed physiological state. Horses that spent longer viewing the balloon with their left eye had a greater reduction in sample entropy, while time spend looking with the right eye was unrelated to the change in sample entropy. Therefore, the horses that exhibited a greater reduction in sample entropy tended to use their right hemisphere more, which may take precedence in emotional reactions. These results may help to explain the variation in lateralisation observed among horses.

Autonomic nervous system balance in parturient mares: Spontaneous vs induced delivery.

Delivery is not easily predictable in horses and the consequences of dystocia can be serious for both the mare and foal. An induction protocol with low doses of oxytocin has been reported as a safe procedure. This study investigates the effect of induced delivery on at-term mares' sympathetic-vagal balance. Fourteen mares were included and divided into two groups, one subjected to spontaneous delivery (SD), and one to induced delivery (ID). In both groups, an ECG was recorded using an elastic belt with integrated smart textile electrodes. The recording started before the delivery (Basal), continued close to delivery (Pre-delivery) and during delivery (Delivery), and ended after parturition (Placental expulsion). From the ECGs, Heart Rate Variability (HRV) parameters relating to time and frequency domains and non-linear analysis were extrapolated. The HRV analysis was performed both within the same group (IntraGA) and between the two groups (InterGA). In the present study, spontaneous and induced delivery did not appear to differ in autonomic nervous system functioning. In IntraGA analysis, both for SD and ID mares, delivery and placental expulsion periods were parasympathetic dominated since vagal-related HRV parameters increased. Moreover, no differences were found in InterGA comparison between SD and ID mares, except for the pre-delivery period of ID mares, during which both branches of the autonomic nervous system were activated. These results are in line with the literature on parasympathetic dominance during parturition and no change in Heart Rate Variability following exogenous oxytocin administration in parturient mares.

Detour test performance of cloned minipigs from three different clone populations.

Genetics, the uterine environment, maternal behavior, and rearing conditions can all influence animal behavioral phenotypes. Some studies on cloned pigs have found no differences between the behavioral patterns of cloned and non-cloned animals. Other studies conducted on dogs have reported similarities in the behavior of cloned subjects. This study evaluated the performance of 12 cloned minipigs from three different clone populations (A, B, C) in a detour test around symmetric and asymmetric barriers. We measured the detour time and patterns, in order to investigate the pigs' cognitive abilities.The detour time and the detour entry/exit pattern were recorded. All the animals tended to keep a fixed entry/exit pattern instead of modifying it to accommodate changes in the working set. Significant differences in detour time were found among the populations, with animals belonging to population B being faster than the others, and also within each population.Our study is one of the few to assess the cognitive abilities of cloned minipigs. The results indicate that even animals belonging to the same cloned population may develop different cognitive, hence behavioral characteristics. Whether cloning can be utilized to obtain similar behavioral phenotypes therefore remains a matter of debate.

Rider Variables Affecting the Stirrup Directional Force Asymmetry during Simulated Riding Trot.

Riders' asymmetry may cause back pain in both human and equine athletes. This pilot study aimed at documenting in a simple and quick way asymmetry in riders during a simulation of three different riding positions on wooden horseback using load cells applied on the stirrup leathers and identifying possible associations between riders' asymmetry and their gender, age, level of riding ability, years of riding experience, riding style, motivation of riding, primary discipline and handedness. After completing an interview to obtain the previously mentioned information, 147 riders performed a standardized test on a saddle fixed on a wooden horseback-shaped model. The riding simulation was split into three phases of 1 min each: (1) sit in the saddle, (2) standing in the stirrups and (3) rising trot. The directional force on the left and the right stirrup leathers was recorded every 0.2 s. A paired t-test was performed on the recorded data to test the difference (i.e., asymmetry) in each phase. In phases 1, 2 and 3, 99.3% (53.4% heavier on the right (R)), 98% (52.8% heavier on the left (L)) and 46.3% (51.5% heavier on the left (L)) of the riders were asymmetrical, respectively. Chi-square tests showed a significant association between riding ability and riding experience, but no significant association between reported handedness and calculated leg-sidedness (p > 0.05). Univariate logistic (1: asymmetry, 0: symmetry) regression analysis was performed only on the phase 3 data. One-hand riders were found twice more likely to be asymmetrical than two-hand riders (Odds Ratio (OR): 2.18, Confidence Interval (CI): 1.1−4.29; p = 0.024). This preliminary study confirmed that the majority of the riders are asymmetrical in load distribution on stirrups and suggested the riding style as a possible risk factor for asymmetry.

The Cat-Owner Relationship: Validation of the Italian C/DORS for Cat Owners and Correlation with the LAPS.

Globally, most people now own a pet. Scales have been developed to understand the impact of pet ownership on people's lives and to measure specific aspects of the owner-pet relationship. The Cat-/Dog-Owner Relationship Scale (C/DORS) is a tool developed to investigate this relationship in both dog and cat owners. The aim of the study was to refine and validate the C/DORS for cat owners in Italian. Exploratory Factor Analysis and Confirmatory Factor Analysis (CFA) were used to determine the most appropriate factor model. Construct validity was confirmed by correlating the C/DORS subscales with the Lexington Attachment to Pets (LAPS) subscales. Results confirmed the original structure of the English version (i.e., three factors: Pet Owner Interaction = POI, Perceived Emotional Closeness = PEC, Perceived Costs = PC) and CFA confirmed the structure of LAPS and C/DORS scales. Cronbach's alpha demonstrated the Italian version of the two scales to have good internal reliability for all domains. Owners of cats living exclusively indoors reported higher scores on POI and PEC compared to indoor/outdoor cats. Owning both cats and dogs was correlated with lower scores on POI, and fewer perceived costs (i.e., PC) of cat ownership. Finally, behaviour problems, not being neutered, and lack of previous experience with cat ownership were associated with lower scores on PC.

Horses show individual level lateralisation when inspecting an unfamiliar and unexpected stimulus.

Animals must attend to a diverse array of stimuli in their environments. The emotional valence and salience of a stimulus can affect how this information is processed in the brain. Many species preferentially attend to negatively valent stimuli using the sensory organs on the left side of their body and hence the right hemisphere of their brain. Here, we investigated the lateralisation of visual attention to the rapid appearance of a stimulus (an inflated balloon) designed to induce an avoidance reaction and a negatively valent emotional state in 77 Italian saddle horses. Horses' eyes are laterally positioned on the head, and each eye projects primarily to the contralateral hemisphere, allowing eye use to be a proxy for preferential processing in one hemisphere of the brain. We predicted that horses would inspect the novel and unexpected stimulus with their left eye and hence right hemisphere. We found that horses primarily inspected the balloon with one eye, and most horses had a preferred eye to do so, however, we did not find a population level tendency for this to be the left or the right eye. The strength of this preference tended to decrease over time, with the horses using their non-preferred eye to inspect the balloon increasingly as the trial progressed. Our results confirm a lateralised eye use tendency when viewing negatively emotionally valent stimuli in horses, in agreement with previous findings. However, there was not any alignment of lateralisation at the group level in our sample, suggesting that the expression of lateralisation in horses depends on the sample population and testing context.

The Influence of Oxytocin on Maternal Care in Lactating Dogs.

In recent years, many studies on the role of oxytocin (OXT) in the onset of parental care, regulation of social bonding, and modulation of the emotional state have been published. However, its possible regulation of maternal behavior in lactating dogs has not been investigated yet. For this reason, the present study aimed at assessing potential correlations between salivary oxytocin and maternal behavior in 25 lactating Labrador Retriever dogs. Salivary concentrations of OXT (sOXT) were unrelated to the amount of maternal care except for a weak negative correlation with sniffing/poking behavior. Moreover, sOXT was positively correlated with the percentage of male puppies. Sniffing/poking behavior, in turn, was positively correlated with the duration of time the mothers spent out of the whelping box, while the number of male puppies showed a positive correlation with lateral nursing, a position known to provide puppies the easiest access to the milk. Taken together, these results suggest that sOXT may not be strongly associated with maternal care dynamics but could be correlated with sex-biased parental investment in dogs.

Smart textiles biotechnology for electrocardiogram monitoring in horses during exercise on treadmill: Validation tests.

BACKGROUND: There are several bioengineering solutions aimed at improving human health and welfare. Smart electrodes based on textile substrates have met the growing demand for comfort, reliability, and robustness when acquiring physiological signals. OBJECTIVES: Given the importance of good quality electrocardiograms (ECG) in equine sports medicine, this study focuses on the validation of smart textile electrodes to acquire ECG signals in horses during treadmill exercise. STUDY DESIGN: The performance of the smart textile electrodes is compared with standard silver/silver chloride (Ag/AgCl) electrodes in terms of signal quality. METHODS: Five healthy Standardbred mares were fitted with two identical electronic systems for the simultaneous recording of ECGs during a standardised exercise test (SET) on a treadmill. One system was equipped with smart textile electrodes, whereas the second was equipped with standard Ag/AgCl electrodes. The Ag/AgCl electrodes were positioned on shaved skin with self-adhesive pads, and without (SET1) or with glue (SET2). The textile electrodes were positioned without shaving the skin. The Kurtosis (k) value for each ECG trace recorded was calculated as an index of ECG signal quality. RESULTS: For the textile electrodes, k values were higher, and closer to ideal compared to Ag/AgCl electrodes. The median values of the Signal Quality Indexes (kSQI) were higher for textile compared to Ag/AgCl electrodes. These differences were significant in SET 2 (P < .001), but not in SET 1 (P = .08). MAIN LIMITATIONS: This study was limited to treadmill exercise that did not include a rider or harness. CONCLUSIONS: During treadmill exercise, textile electrodes are a practical solution for collecting good quality ECG traces.

Rein Tension Signals Elicit Different Behavioral Responses When Comparing Bitted Bridle and Halter.

When a rider maintains contact on the reins, rein tension will vary continuously in synchronicity with the horse's gait and stride. This continuous variation makes it difficult to isolate the rein tension variations that represent a rein tension signal, complicating interpretation of rein tension data from the perspective of horse-rider interaction. This study investigated (1) the characteristics of a rein tension signal and (2) horse response to a rein tension signal for backing, comparing pressure applied by a bit (bridle), or by a noseband (halter). Twenty Warmblood horses (10 young, 10 adult) wearing a rein tension meter were trained to step back in the aisle of a stable. The handler stood next to the horse's withers, applying tension on the reins until the horse stepped back. This was repeated eight times with the bridle and eight times with the halter. Data analysis was performed using mixed linear and logistic regression models. Horses displaying behaviors other than backing showed significantly increased response latency and rein tension. Inattentive behavior was significantly more common in the halter treatment and in young horses, compared with the bridle treatment and adult horses. Evasive behaviors with the head, neck, and mouth were significantly more common in the bridle treatment than in the halter treatment and the occurrence of head/neck/mouth behaviors increased with increasing rein tension and duration of the rein tension signal. When controlling for behavior, the horses responded significantly faster and to a lighter rein tension signal in the bridle treatment than in the halter treatment. By scrutinizing data on rein tension signals in relation to horse behavior and training exercise, more can be learnt about the horse's experience of the pressures applied and the timing of the release. This can assist in developing ways to evaluate rein tension in relation to correct use of negative reinforcement.

Interspecific two-dimensional visual discrimination of faces in horses (Equus caballus).

In social animals, recognizing conspecifics and distinguishing them from other animal species is certainly important. We hypothesize, as demonstrated in other species of ungulates, that horses are able to discriminate between the faces of conspecifics and the faces of other domestic species (cattle, sheep, donkeys and pigs). Our hypothesis was tested by studying inter-and intra-specific visual discrimination abilities in horses through a two-way instrumental conditioning task (discrimination and reversal learning), using two-dimensional images of faces as discriminative stimuli and food as a positive reward. Our results indicate that 8 out of 10 horses were able to distinguish between two-dimensional images of the faces of horses and images showing the faces of other species. A similar performance was obtained in the reversal task. The horses' ability to learn by discrimination is therefore comparable to other ungulates. Horses also showed the ability to learn a reversal task. However, these results were obtained regardless of the images the tested horses were exposed to. We therefore conclude that horses can discriminate between two dimensional images of conspecifics and two dimensional images of different species, however in our study, they were not able to make further subcategories within each of the two categories. Despite the fact that two dimensional images of animals could be treated differently from two dimensional images of non-social stimuli, our results beg the question as to whether a two-dimensional image can replace the real animal in cognitive tests.

Effects of Stroking on Salivary Oxytocin and Cortisol in Guide Dogs: Preliminary Results.

This pilot study aimed at investigating how salivary oxytocin levels are affected by human interaction and isolation in eight guide dogs (six Labrador retrievers and two golden retrievers; four males and four females, 21.87 ± 1.36 months old) just before assignment to the blind person. Each dog engaged, at one-week intervals, in a positive (5 min of affiliative interaction with their trainer) and a negative (5 min of isolation) condition. Saliva samples used for Enzyme Immunoassay (EIA) quantification of salivary oxytocin were collected before and immediately after both experimental conditions. In order to assess potential hypothalamic pituitary adrenal (HPA) axis activation that could have affected oxytocin levels, saliva samples were collected 15 min after both experimental conditions for EIA quantification of salivary cortisol and a behavioral assessment was performed during the negative condition. The results were compared using the Wilcoxon test (p < 0.05). Oxytocin concentrations showed a statistically significant increase after the positive interaction (p = 0.036) and no difference after the negative one (p = 0.779). Moreover, no difference (p = 0.263) was found between the cortisol concentrations after each experimental condition and no signs of distress were observed during the isolation phase. These preliminary findings support the hypothesis that stroking dogs has positive effects on their emotional state independently of hypothalamic pituitary adrenal axis activation.

Inside the Interaction: Contact With Familiar Humans Modulates Heart Rate Variability in Horses.

A human-animal relationship can be developed through subsequent interactions, affected by the positive or negative emotional valence of the proceeding one. Horses implement a process of categorization to classify humans with whom they interact as positive, negative, or neutral stimuli by evaluating the kind of approach and the nature of the contact. In these terms, human-animal interactions are emotionally charged events, eliciting specific emotional states in both subjects involved. Although the human-horse relationship has been mainly investigated through behavioral analysis, physiological indicators are needed for a more objective assessment of the emotional responses. Heart rate variability (HRV) is a commonly used autonomic nervous system (ANS) correlate estimating the sympathovagal balance as a psychophysiological marker of emotion regulation in horses. We have assumed that long-term positive relationships with humans may have a positive and immediate impact on the emotional arousal of the horse, detectable, via ANS activity, during the interaction. We analyzed horses' heartbeat dynamics during their interaction with either familiar or unfamiliar handlers, applying a standardized experimental protocol consisting of three different conditions shifting from the absence of interaction to physical contact. The ANS signals were monitored through an innovative non-invasive wearable system, not interfering with the unconscious emotional response of the animal. We demonstrated that horses appeared to feel more relaxed while physically interacting (e.g., grooming on the right side) with some familiar handlers compared to the same task performed by someone unfamiliar. The shift of the sympathovagal balance toward a vagal predominance suggests that the horses experienced a decrease in stress response as a function not only of the handler's familiarity but also of the type of interaction they are experiencing. These results constitute the objective evidence of horses' capacity to individually recognize a familiar person, adding the crucial role of familiarity with the handler as a paramount component of human-animal interaction. Our rigorous methodological approach may provide a significant contribution to various fields such as animal welfare while further investigating the emotional side of the human-animal relationships.

Emotional Transfer in Human-Horse Interaction: New Perspectives on Equine Assisted Interventions.

Equine assisted interventions (EAIs) include all therapeutic interventions aimed at improving human wellbeing through the involvement of horses. Due to the prominent emotional involvement traditionally characterizing their relation with humans, horses developed sophisticated communicative skills, which fostered their ability to respond to human emotional states. In this review, we hypothesize that the proximate causation of successful interventions could be human-animal mutual coordination, through which the subjects bodily and, most importantly, emotionally come into contact. We propose that detecting emotions of other individuals and developing the capacity to fine-tune one's own emotional states accordingly (emotional transfer mechanism), could represent the key engine triggering the positive effects of EAIs. We provide a comprehensive analysis of horses' socio-emotional competences according to recent literature and we propose a multidisciplinary approach to investigate this inter-specific match. By considering human and horse as a unique coupling system during the interaction, it would be possible to objectively measure the degree of coordination through the analysis of physiological variables of both human and animal. Merging the state of art on human-horse relationship with the application of novel methodologies, could help to improve standardized protocols for animal assisted interventions, with particular regard to the emotional states of subjects involved.

Inter- and Intra-Species Communication of Emotion: Chemosignals as the Neglected Medium.

Human body odors contain chemosignals that make species-specific communication possible. Such communication is without communicative intent and is generally below the threshold of consciousness. Human recipients of these chemosignals produced during emotional conditions display a simulacrum of the emotional state under which the chemosignal was produced. The investigation of an inter-species transfer of emotions via chemosignals was initiated by considerations of the historically anchored interdependence between humans and domesticated species, such as dogs and horses. Indeed, experiments with dogs have demonstrated that human body odors produced under emotional conditions of happiness and fear led dogs to manifest corresponding emotions to those experienced by humans. Preliminary data from horses also show that human body odors collected under fear and happiness conditions activate the autonomic nervous system of horses differentially. These studies indicate the possibility of a road to open our understanding of inter-species emotional communication via chemosignals.

Physiological outcomes of calming behaviors support the resilience hypothesis in horses.

To manage a stressful stimulus animals react both behaviorally and physiologically to restore the homeostasis. In stable horses, a stressful stimulus can be represented by social separation, riding discomfort or the presence of novel objects in their environment. Although Heart Rate Variability is a common indicator of stress levels in horses, the behavioral mechanisms concurrently occurring under stressful conditions are still unknown. The sudden inflation of a balloon was administered to 33 horses. Video-recording of self-directed behaviors (snore, vacuum chewing, snort, head/body shaking) and monitoring of heart activity (HR and SDRR) were conducted for five minutes before (Pre-test) and after the stimulus administration (Stress-test). During the Stress-test, only snore and vacuum chewing increased and a significant increase was also recorded in both HR and SDRR. Moreover, the snore variation between the two conditions showed a significant correlation with the variation of both HR and SDRR. With the snore acting as stress-releasing behavior to restore basal condition, the homeostasis recovered via the enactment of such behavior could be physiologically expressed by an increasing vagal activity. Hence, the capacity to maintain homeostasis (resilience) could correspond to a prevalence of parasympathetic control on heart activity, intervening when certain behaviors are performed.

Real-time Evaluation of ECG Acquisition Systems through Signal Quality Assessment in Horses during Submaximal Treadmill Test.

This paper reports on a novel real time index designed to assess the quality of electrocardiographic (ECG) traces recorded in a group of five horses during a submaximal treadmill test procedure. During the experimental protocol two ECG monitoring systems were simultaneously applied to the animals. The first system was equipped with textile electrodes while the second one with standard red-dot electrodes. The procedure comprised four phases with an increased treadmill velocity, specifically, Walk 1, Trot 1, Trot 2 and Gallop. Three signal quality levels have been fixed according to the amount of noise present in the ECG trace: good (G), acceptable (A), and unacceptable (U). Moreover, a statistical comparison between textile and red-dot electrodes has been performed in terms of percentage of signal belonging to each class. Even if preliminary, results showed that in each experimental phase textile electrodes are more robust to movement artifacts with respect to the reddot showing a significant evidence of their better performance. These results enable to design robust wearable monitoring systems suitable to improve the quality of collected ECG, reducing the great amount of motion artifacts due to red-dot electrode application and leading to a more accurate diagnosis of high speed arrhythmias.