Abstract: The use of molecular techniques has increased our understanding of biological systems; in particular how genes and proteins operate within a cell or organ in response to various stimuli. These techniques can be used to study modes of action or mechanisms in disease states, or in response to applied management or nutritional changes. While applied and basic research are often conducted independent of one another, the real advantages to the equine industry will come from the use of molecular techniques to address applied challenges and/or opportunities within the industry. Therefore, the overall goal of our research has been to use basic and molecular tools to investigate modes of action resulting from changes in nutrition or management. We investigated active nutrient transport capacities of the small and large intestine, the effects of feed withdrawal on intestinal transporter gene expression and function, as well as other measurements pertaining to gut health (gut barrier function and gastrointestinal morphology). This novel and thorough approach to characterize sectional differences (fore- and hind- gut) of nutrient absorption has resulted in quantitative measures of active transport capacity for glucose, phosphorus, glutamine, lysine, and di-peptides in the horse. This research demonstrates that by-products of fermentation in the cecum such as microbial proteins and hydrolyzed phytate phosphorus, can be absorbed in the colon. Previously unidentified in the hindgut of the horse, we reported the expression of the di-and tri-peptide transporter, PepT1, as well as the neutral amino acid transporter, ASCT2, and transport of their substrates. We observed that nutrient transporters for glucose (SGLT1), glutamine (ASCT2) and for di-peptides (PepT1) can be regulated by diet, implying biological relevance to feeding management strategies. Feed withdrawal in horses pre- or post-surgery, prior to or during horse shows, during transport, or for other management reason is common. It is therefore important to understand how the removals of nutrients impacts gut health and/or disease susceptibility. The use of molecular techniques has provided a basis for the roles of certain core nutrients during times of feed withdrawal. For example, nutrients such as glutamine play a major role in proliferation and repair in the gut, serve as a major energy substrate, and may provide a means of assessing nutritional stress in the gut. Recent data concludes that following a feed withdrawal, the need for glutamine increases up to 116% in the proximal jejunum, and the gene expression of its transporter (ASCT2) increases by 78%. Additional studies utilizing molecular techniques to address nutritional challenges in the horse include how hormone loss due to ovariectomization impacts the homeostatic regulation of calcium and phosphorus in the horse. To improve nutrition and feeding management practices in horses, knowledge regarding how and where nutrients are absorbed, utilized and regulated under varying physiologic stressors (disease, exercise, reproductive status, etc.) is needed. The integration of applied and basic research facilitates a better and more complete understanding of mechanisms underlying common health and nutritional challenges facing the equine industry.

Abstract: Pain evaluation is a key issue for equine welfare and it is often cause of concern because it is difficult to determine its intensity and duration. This is essential when different people are looking after the animals and they need to decide when or not giving analgesics to guarantee the welfare of the subject. The most widely used technique to determine pain in horses is identifying pain related behaviors. The aim of this study was to determine inter-observer reliability of two different assessors evaluating pain related behaviors in horses undergoing castration. 8 stallions of different breed, aged between 2 and 4 years, were included in the study. All the subjects underwent routine castration (closed technique in general anesthesia). The subjects were placed in an observation box for 5 days and their behavior was recorded for 15 minutes before the surgery and 4, 8, 16, 24 and 40 hours after intervention. Two blind observers, using a given ethogram of horse pain related behaviors modified from literature (for a review Ashley, 2005), analyzed horses behavior at each interval. Descriptive statistics and K Kendall test were performed. Observers agreed significantly assessing agitation, reluctance to move, kicking the abdomen, lethargy, rolling, attention and curiosity (P<0.05), however agreement was low for head movements, stretching, flank watching, lowered head carriage, weight shifting, abnormal movement, fixed stare. Our results show that assessing pain in horses should be a cause of concern, because different pain related behaviors are difficult to identify and to have agreement between two observers. Training of care takers of horses on identification of specific behaviors is needed to standardize pain assessment. Acknowledgements: The authors wish to thank the EU VII Framework programme (FP7-KBBE-2010-4) for financing the Animal Welfare Indicators (AWIN) project and for providing funds for Emanuela Dalla Costa and Michela Minero to present this paper.

Abstract: Like many other herbivores equids feed on rather evenly distributed resources. Especially in ruminants several studies have proved the influence of social organisations, rank, sex and the depletion of feeding sites on the feeding behaviour of individuals. However, it is not yet understood whether social aspects affect horses´ feeding decisions. Horses roam on vast habitats with constantly changing vegetation. In non-competitive situations domestic horses tend to return to the same feeding site until it is overgrazed. Whereas, for competition over limited food the social status of the individuals appears to be important. Curiosity about the influence of social rank and different social feeding conditions on the horses´ feeding decisions between two buckets, equally filled with high-quality surplus food, led us to create the test situation described here. The observer horses were alternately tested with a dominant and a subordinate demonstrator placed in one of three different positions. We conclude that domestic horses use cognitive strategic decision making in order to decide where to feed in a social feeding situation. When possible they tend to return to the same, continuously supplied feeding site and switch to an “avoidance tendency” when another horse is already feeding from it or in the presence of a dominant horse. Thus the position and the social rank of conspecifics affect the feeding strategy of horses.

Abstract: We tested untrained horses (foals and breeding mares) in 3 different breeding centres. Horses were kept in a pasture during daytime in bigger groups (median 12, min: 6, max 35 / pasture) according to age, gender and breed (hungarian halfbred, hucul, arabian and thoroughbred). To measure their reactions to an unfamiliar person we conducted an active and a passive human test. In the active human test the test person (TP) approached a focal animal in the group from a predetermined direction: from their front, from their side (left or right) or from the rear. As TP reached the horse (of app. 0.5 m distance), she tried to pet the animal’s head. The TP always chose and approached a standing or a grazing horse (ie. those that were not walking, galloping, playing etc.). The horse’s reaction to the approaching human was scored 1-5. Score 1: the horse moved away and the TP could not even approach it within 0.5 m; Score 2: the horse made max. 2 steps away, but could be reached and petted; Score 3 and Score 4: the horse stood in its place but showed different signs of discomfort (head turn – Score 3; backing ears, tail slash – Score 4); Score 5: the horse stood and did not show any sign of discomfort or actively approached the TP. There was no significant difference in the horses’ reaction between approaching from their left or right side. Approaching from their front or from their side (left or right) did not differ significantly either. However, the odds of walking away from the TP (score 1 or 2) was 2.7 (Fisher-test, p=0.039) and 3.3 (p=0.012) times higher when TP approached from the rear compared to approaching from their front or from their side (left or right), respectively. In the passive human test the TP stood immobile for 4 minutes in 5 or 10 m far from the horses during their active (grazing) or inactive (standing idle during noon) period. Horses approached TP significantly sooner (general linear model, p=0.017) when she stood 5m distance (38 ±63 s) compared to 10m (97 ±52 s). The arrival of a second horse after the first horse approached the TP showed significant high correlation with the latency of the first horse arrival (Pearson correlation, r=0.96, p<0.001). The horses were less keen (p=0.008) to approach the human when they were tested in an inactive period (177 ±110 s) compare to active period (38 ±63 s).

Abstract: The living equids display a variety of different forms of social organisation; plains (Equus quagga) and mountain zebra (Equus zebra) associate in small, closed harems, whereas the asses and Grevy’s zebra (Equus grevyi) are found in looser, more ephemeral associations. Feral domestic horses appear to be somewhat intermediate: although stallions generally maintain harems, there is some movement of individuals between groups. This talk will use several statistical approached to explore evolutionary explanations for these differences. Firstly, we will explore the relationship between brain architecture and social organisation both among the equids and across all Perissodactyls. Equids have larger relative neocortices than other Perissodactyls; within the equids, species typically found in more stable groups have larger neocortices than equids in more unstable ephemeral groups. The strong relationship between group stability and brain size and architecture mirrors that found in other mammals, including primates. Thus, living in stable social groups consistently appears to impose cognitive demands across species. In order to understand the historical trajectory resulting in the relatively large, social brains found in some species, we then reconstruct the evolutionary pathway of social living in the equids. Finally, we discuss how we can use network theory to measure social structure and how these differences may impose different cognitive demands on individuals.