Abstract: Relatively few large-scale faecal DNA studieshave been initiated due to difficulties inamplifying low quality and quantity DNAtemplate. To improve brown bear faecal DNA PCRamplification success rates and to determinepost collection sample longevity, fivepreservation methods were evaluated: 90%ethanol, DETs buffer, silica-dried, oven-driedstored at room temperature, and oven-driedstored at -20 °C. Preservationeffectiveness was evaluated for 50 faecalsamples by PCR amplification of a mitochondrialDNA (mtDNA) locus (~146 bp) and a nuclear DNA(nDNA) locus (~200 bp) at time points of oneweek, one month, three months and six months. Preservation method and storage timesignificantly impacted mtDNA and nDNAamplification success rates. For mtDNA, allpreservation methods had >= 75% success atone week, but storage time had a significantimpact on the effectiveness of the silicapreservation method. Ethanol preserved sampleshad the highest success rates for both mtDNA(86.5%) and nDNA (84%). Nuclear DNAamplification success rates ranged from 26-88%, and storage time had a significant impacton all methods but ethanol. Preservationmethod and storage time should be importantconsiderations for researchers planningprojects utilizing faecal DNA. We recommendpreservation of faecal samples in 90% ethanolwhen feasible, although when collecting inremote field conditions or for both DNA andhormone assays a dry collection method may beadvantageous.

Abstract: The Y chromosome directly reflects male genealogies, but the extremely low Y chromosome sequence diversity in horses has prevented the reconstruction of stallion genealogies [1, 2]. Here, we resolve the first Y chromosome genealogy of modern horses by screening 1.46 Mb of the male-specific region of the Y chromosome (MSY) in 52 horses from 21 breeds. Based on highly accurate pedigree data, we estimated the de novo mutation rate of the horse MSY and showed that various modern horse Y chromosome lineages split much later than the domestication of the species. Apart from few private northern European haplotypes, all modern horse breeds clustered together in a roughly 700-year-old haplogroup that was transmitted to Europe by the import of Oriental stallions. The Oriental horse group consisted of two major subclades: the Original Arabian lineage and the Turkoman horse lineage. We show that the English Thoroughbred MSY was derived from the Turkoman lineage and that English Thoroughbred sires are largely responsible for the predominance of this haplotype in modern horses.

Abstract: 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.

Abstract: A previous study demonstrated that horses can learn socially from observing humans, but could not draw any conclusions about the social learning mechanisms. Here we develop this by showing horses four different human action sequences as demonstrations of how to press a button to open a feed box. We tested 68 horses aged between 3 and 12 years. 63 horses passed the habituation phase and were assigned either to the group Hand Demo (N = 13) for which a kneeling person used a hand to press the button, Head Demo (N = 13) for which a kneeling person used the head, Mixed Demo (N = 12) for which a squatting person used both head and hand, Foot Demo (N = 12) in which a standing person used a foot, or No Demo (N = 13) in which horses did not receive a demonstration. 44 horses reached the learning criterion of opening the feeder twenty times consecutively, 40 of these were 75% of the Demo group horses and four horses were 31% of the No Demo group horses. Horses not reaching the learning criterion approached the human experimenters more often than those who did. Significantly more horses used their head to press the button no matter which demonstration they received. However, in the Foot Demo group four horses consistently preferred to use a hoof and two switched between hoof and head use. After the Mixed Demo the horses' actions were more diverse. The results indicate that only a few horses copy behaviours when learning socially from humans. A few may learn through observational conditioning, as some appeared to adapt to demonstrated actions in the course of reaching the learning criterion. Most horses learn socially through enhancement, using humans to learn where, and which aspect of a mechanism has to be manipulated, and by applying individual trial and error learning to reach their goal.

Abstract: This field study tested the hypothesis that domestic horses living under putatively challenging-to-welfare conditions (for example involving social, spatial, feeding constraints) would present signs of poor welfare and co-occurring pessimistic judgement biases. Our subjects were 34 horses who had been housed for over 3 years in either restricted riding school situations (e.g. kept in single boxes, with limited roughage, ridden by inexperienced riders; N = 25) or under more naturalistic conditions (e.g. access to free-range, kept in stable social groups, leisure riding; N = 9). The horses' welfare was assessed by recording health-related, behavioural and postural indicators. Additionally, after learning a location task to discriminate a bucket containing either edible food ('positive' location) or unpalatable food ('negative' location), the horses were presented with a bucket located near the positive position, near the negative position and halfway between the positive and negative positions to assess their judgement biases. The riding school horses displayed the highest levels of behavioural and health-related problems and a pessimistic judgment bias, whereas the horses living under more naturalistic conditions displayed indications of good welfare and an optimistic bias. Moreover, pessimistic bias data strongly correlated with poor welfare data. This suggests that a lowered mood impacts a non-human species' perception of its environment and highlights cognitive biases as an appropriate tool to assess the impact of chronic living conditions on horse welfare.