Abstract: Mirror Self-Recognition (MSR) is an extremely rare capacity in the animal kingdom that reveals the emergence of complex cognitive capacities (de Waal 2008). So far, MSR has been reported only in humans, chimpanzees (Gallup, 1970), bottlenose dolphins (Reiss and Marino, 2001) and Asian elephants (Plotnik et al, 2006), all species characterized by a highly developed cognition. There is growing evidence that domestic horses posses high cognitive abilities, such as cross-modal individual recognition (Proops et al, 2009), triadic post-conflict reunion to maintain social homeostasis (Cozzi et al, 2010), complex communicative systems (Whatan and McComb, 2014), flexibility in problem-solving (Lovrovich et al, 2015), and long-term memory (Hanggi and Ingersoll, 2009). All these capacities make horses a good candidate to test the ability of MSR in a domestic species. Through a classical MSR experimental paradigm (de Waal 2008) we tested eight horses living in social groups under semi-natural conditions (from the Italian Horse Protection rescue centre). Animals showing MSR typically go through four stages (Plotnik et al, 2006): (i) social response, (ii) physical mirror inspection (e.g., looking behind the mirror), (iii) repetitive mirror-testing behaviour (i.e., the beginning of mirror understanding), and (iv) self-directed behaviour (i.e., recognition of the mirror image as self). The final stage, known as the “mark-test”, is verified when a subject spontaneously uses the mirror to check for a coloured artificial mark on its own body which it cannot perceive otherwise. The horses underwent a three-phase “mark-test”: 1) with sham mark (transparent ultrasound water gel) positioned on both side at jaw level, 2) mark (yellow eye shadow mixed with ultrasound water gel) positioned on left side of jaw (with sham mark on the right), 3) mark (yellow eye shadow mixed with ultrasound water gel) positioned on right side of jaw (with sham mark on the left)
The mirror was one 0.5-cm-thick piece of 140x220-cm plexiglass glue on wood. Each test lasted one hour, horses were tested once a day, in consecutive days and at the same time. Our preliminary result on 1 horse shows some changes in self-directed behaviours which can be attributed to presence of the coloured mark. Firstly, the presence of the coloured mark significantly increased the frequency of scratching on both sides of the muzzle (p < 0.0001). The most intriguing result (p < 0.0001) comes from the comparison of the scratching rates directed towards the coloured mark side (N = 41) and the sham mark side (N = 23). Under the control condition (i.e. sham mark on both sides) no statistical difference was found for the scratching rates directed to the muzzle sides (dx N = 8; sx N = 5). Although further analyses are needed to confirm these preliminary results, our finding opens new scenarios about the evolution of Mirror Self-Recognition. The capacity of horses to recognize themselves in a mirror may be the outcome of an evolutionary convergence process driven by the cognitive pressures imposed by a complex social system and maintained despite thousands years of domestication.
Keywords:
Domestic horse · Mark test · Socio-cognitive skills · Self-awareness
References
De Waal FBM (2008) The thief in the mirror. PloS Biol 6(8):e201
Gallup GG Jr (1970) Chimpanzees: Self-recognition. Science 167: 86-87.
Reiss D, Marino L (2001). Mirror self-recognition in the bottlenose dolphin: A case of cognitive convergence. Proc Natl Acad Sci U S A 98:5937-5942.
Plotnik J, de Waal FBM, Reiss D (2006) Self-recognition in an Asian elephant. Proc Natl Acad Sci U S A 103: 17053-17057.
Proops L, McComb K, Reby D. (2009) Cross-modal individual recognition in domestic horses (Equus caballus). Proc Nat Acad Sci USA;106:947-951.
Cozzi A, Sighieri C, Gazzano A, Nicol CJ, Baragli P. Post-conflict friendly reunion in a permanent group of horses (Equus caballus). Behav Process 2010;85:185-190.
Wathan J, McComb K. The eyes and ears are visual indicators of attention in domestic horses. Curr Biol 2014;24(15): R677-R679.
Lovrovich P, Sighieri C, Baragli P (2015) Following human-given cues or not? Horses (Equus caballus) get smarter and change strategy in a delayed three choice task. Appl Anim Behav Sci, in press.
Hanggi EB, Ingersoll JF. (2009) Long-term memory for categories and concepts in horses (Equus caballus). Anim Cogn; 12:451-462.

Abstract: Stress responses play an important role in allowing animals to cope with challenges. Glucocorticoids, key elements in the neuroendocrine stress axis, are traditionally measured as a parameter for welfare assessment. As blood sample collection itself disturbs an animal, non-invasive or minimal invasive methods have gained importance for assessing stress. In horses saliva and faeces are most frequently used. Faecal samples offer the advantage that they can be collected easily and stress-free. In faecal samples circulating hormone levels are integrated over a certain period of time. As a consequence faecal glucocorticoid metabolites represent the cumulative secretion and they are less affected by short episodic fluctuations of hormone secretion.
However, in order to gain reliable information about an animal’s adrenocortical activity, certain criteria have to be met: Depending whether the impact of acute or chronic stressors is assessed, frequent sampling might be necessary whereas in other cases, single samples will suffice. Background knowledge regarding the metabolism and excretion of glucocorticoids is essential and a careful validation is obligatory. In addition, this presentation will address analytical issues regarding sample storage, extraction procedures, and immunoassays and various examples of a successful application in equids will be given. Applied properly, non-invasive techniques to monitor stress hormones are a useful tool for animal welfare assessment.

Abstract: An important question in the study of animal communication is whether non-human animals are able to produce communicative gestures, i.e. nonvocal bodily actions directed to a recipient, physically ineffective but with a meaning shared in the social group [1]. Passive gestures are instrumental, tuned to the mere presence/absence of others, whereas active informers recognize receivers as communicative agents and activate shared-attention mechanisms for identifying their attentional state (SAM [2]; e.g. Schwab and Huber [3]). Six operational criteria must be evaluated to classify a signal as referential and intentional [4]: (1) alternative gazes between the partner and the target; (2) apparent attention-getting behaviours are deployed; (3) an audience is required to exhibit the behaviour; (4) the attentional status of an observer influences the propensity to exhibit behaviours; (5) communication is persistent and (6) there is elaboration of communicative behaviour when apparent attempts to manipulate the partner fail. Dogs [5] and non-human primates (reviewed in Liebal and Call [6]) can tune a human receiver’s attention to the object of interest by combining directional and attention-getting signals, such as turning the head or body, gazing to the receiver, and/or establishing eye contact. Research on other species is scarce.
Horses rely on humans to survive in domestic settings and may have evolved skills for communicating flexibly with them [7]. Horses understand human attentional cues (such as body and head orientation, eyes opened/closed) [8], permanent pointing [9] and, to some extent, gazing [10]. Here we tested the ability of 14 outdoor, herd-living domestic horses to communicate referentially with a human partner about the location of a desired target, a bucket of food out of reach. After the baiting of two buckets placed in opposite, unreachable locations were shown by the experimenter, the subject would walk to one of the two buckets. Because approaching a bucket would reveal that the food is out of reach, we expected the horse to look back to the experimenter, then to the bucket, and alternate this gazing several times to indicate its intention. To test whether our prediction is correct and alternate gazing is indeed the result of the horse's referential communication, we video-recorded the behaviour of the subjects in the test (FORWARD) and three control conditions: (1) FORWARD: experimenter oriented to the center of the arena, (2) BACK: experimenter backward oriented in respect to the arena, (3) ALONE: experimenter absent, (4) MANY: as FORWARD plus a familiar human oriented to the subject behind the bucket (Figure 1). We used a conservative criterion of back gazing by considering only turning the head back more than 90 degrees. The results confirmed our prediction. The horses alternated gazes between the partner and the buck significantly more often in the FORWARD than in all the other conditions (Table 1), thus satisfying operational criteria #1, #3 and #4. They also alternated head nods with gazes to the partner significantly more often during the FORWARD condition. We thus considered head nods not an instrumental signal of arousal, but an attention-getting behaviour with communicative function. Subjects used both head nods and neck stretched toward the buck more often in the FORWARD than in the BACK and the ALONE conditions, thus satisfying criteria #2, #3 and #4. In condition MANY, the frequency of head nods did not differ from condition FORWARD, probably because nods were directed to the additional partner behind the buck. This also satisfies criteria #4. The horses gazed to the partner most often in the FORWARD than in the BACK and the MANY conditions, but not in the ALONE. In this condition, subjects could observe the partner walking further from the test arena. To test for the different functions of gazes in presence and in absence of the partner, we compared their average duration between the two conditions: the significantly longer duration of gazes when the subject was alone suggests the instrumental monitoring function of gazes in this experimental condition.
Altogether, the findings suggest that domestic horses possess the ability to use referential communication in an interspecific context, but additional analyses are needed to test for operational criteria #5 and #6. Flexible and voluntary use of communicative signals reveal sophisticated cognitive processes involved in the strategic emission of these signals, and the finding of referential communication skills in an ungulate species forces us to reconsider the evolutionary path of intelligence. Furthermore, ungulates are used intensively by humans (transportation, meat, agriculture, leisure activities), and their welfare is often compromised. Determining whether ungulates can communicate their needs and preferences is paramount to a proper ethical management.

Abstract: The complex housing environment and the close contact between humans and horses in equine sports place high demands on the learning capacity of horses. To date only limited information is available on the learning ability of the horse including higher order cognition. A type of higher order cognition is to perceive and discriminate quantities. Several mammals and birds have shown to be capable of discriminating objects due to their quantity (Brannon and Roitman, 2003). With regard to horses, there are only few studies available concerning their numerosity judgment (Uller and Lewis, 2009) and this ability is discussed controversially (Henselek et al, 2012). Possibly, the legacy of ‘‘Clever Hans’’ overshadowed further research on numerical capacity in horses, a horse to whom several psychologists incorrectly attributed the capacity of symbolic calculation (Pfungst, 1907; Rosenthal, 1965). In the present study we wanted to show whether Shetland ponies are able to transfer a previously learned concept of sameness to a numerosity judgment. The base of the test design was a “matching to sample” task, where the ponies had learned to relate abstract symbols to another which were presented on a LCD screen. Three Shetland ponies, which previously solved the matching to sample task, were tested in two test phases. In the first test phase different quantities of dots were presented (1 vs. 2; 2 vs. 3; 3 vs. 4; 4 vs. 5). To exclude discrimination due to the shape of the stimuli, the dots were varied in size and arrangement. The stimuli were presented in a triangular arrangement on the LCD screen; the sample stimulus was presented in the middle above and the discrimination stimuli in the two lower corners (S+ and S-). The pony received a food reward, by choosing the positive stimulus (S+). When the negative stimulus (S-) was chosen, the pony entered the next trial. Each learning session consisted of 20 decision trials. To investigate whether the numerosity judgment was transferable to mixed geometrical symbols (tri-, rectangle, rhomb, dot and cross) a second test phase was designed. All of the three Shetland ponies met the learning criterion of the first test phase (80% correct responses in two consecutive sessions) within the first eight sessions. One pony could transfer all judgments to the mixed symbols (up to 4 vs. 5), another pony up to 3 vs. 5 and the third on the level 2 vs. 3. These are the first reported findings that ponies are able to discriminate up to five objects. The numerosity judgment seemed to be easier for the ponies when homogenous objects were presented, than in the case of heterogeneous symbols. The reaction of the ponies occurred within few seconds, suggesting that the animals used subitizing for their numerosity judgment.
Keywords:
Cognition, numerical capacity, numerosity judgment, Shetland ponys
References:
Brannon E, Roitman J (2003) Nonverbal representations of time and number in animals and human infants. In: Meck WH (ed) Functional and neural mechanisms of interval timing. CRC Press, Boca Raton, pp 143–182
Henselek Y, Fischer J, Schloegl C (2012) Does the stimulus type influence horses’ performance in a quantity discrimination task? Front Psychol 3:504
Pfungst O (1907) Der Kluge Hans. Ein Beitrag zur nicht-verbalen Kommunikation. 3rd edn. (reprint of the original 1983) Frankfurter Fachbuchhandlung für Psychologie, Frankfurt
Rosenthal R (1965) Clever Hans: the horse of Mr. Von Osten. Holt, Rinehart and Winston, New York
Uller C, Lewis J (2009) Horses (Equus caballus) select the greater of two quantities in small numerical sets. Anim Cogn 12:733–738