![]() subjects that showed a higher level of response were tested more than others). According to these authors, the studies exploring MSR in non-primate species suffer some methodological biases such as very reduced sample size, not always mirror naive animals, asymmetric arenas, presence of conspecifics during the test, irritating marks, non-blind video analyses and lack of standardization in the number of trials proposed to the different subjects for each step (e.g. Some weaknesses in the experimental protocols have been highlighted by de Waal ( 2019) and Gallup and Anderson ( 2019). ![]() One of the most criticized issues is the methodological procedure adopted for the demonstration of MSR. Overall, the tests on all these species seem to indicate the presence of MSR ability in at least a few subjects, although the issue is still under debate (for extensive review see de Waal 2019 Gallup and Anderson 2019). ( 2017) considered face scraping and rubbing against the substrate as indicators of mark-directed behaviors. In their pilot study on the domestic horse ( Equus caballus), Baragli et al. In cleaner wrasses ( Labroides dimidiatus), Kohda and coworkers ( 2019) considered rubbing marked-throat against the substrate as a reliable measure of self-directed behaviors. While in other studies touching/scraping the mark has been considered the self-directed behavior proving self-recognition (elephants, Elephas maximus, Plotnik et al. In bottlenose dolphins ( Tursiops truncatus) the authors considered as mark-directed behaviors those movements in which the animal positioned itself towards the reflective surface to visually explore the mark (Reiss and Marino 2001). Starting from 2000 the studies on MSR have expanded to many vertebrate taxa beside primates, although the application of the classic MSR experimental paradigm has introduced important methodological variants according to the different species tested (de Waal 2019). Within the primate order, the experiments conducted on monkeys revealed no presence of MSR (Gallup 1977a, b), although some recent studies challenge these first results (Rajala et al. The first successful experiments showing the presence of the phenomenon in non-human primates were obtained on Pan and Pongo genera (for an extensive review Anderson and Gallup 2015). The only reliable data informing us of the presence of MSR is the untrained response to a visual body mark detected with the assistance of a reflective surface (de Waal 2019). In these terms, this shift has been interpreted as a mirror-induced demonstration of self-recognition ability (Suárez and Gallup 1981). Other-directed behaviors are elicited by the perception of the presence of a conspecific, while self-directed behaviors involve the investigation of body parts normally not visible without the aid of a reflective surface. The methodology is based on a test in which subjects shift from other-directed behaviors towards self-directed behaviors after a certain amount of time of exposure to a mirror. Since the 1960′s mirror self-recognition (MSR) has been introduced as a measure of the “awareness of self” in great apes and humans (Gallup 1968, 1977a). 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. 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. 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. 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. Fourteen horses were used in a 4-phases mirror test (covered mirror, open mirror, invisible mark, visible colored mark). 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. 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. Mirror self-recognition (MSR), investigated in primates and recently in non-primate species, is considered a measure of self-awareness.
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