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Toxic pyrrolizidine alkaloids (PAs) are produced by flowering plant species throughout the world, including ragwort (Senecio jacobea). In animals, PAs are metabolised within the liver to form their corresponding dehydro-PAs or pyrroles. These compounds are highly reactive and are capable of alkylating and cross-linking DNA and proteins to lead to characteristic molecular and cellular insult within the liver. There are presently no metllOds for tlle definitive diagnosis of ragwort poisoning in animals. If biomarkers for ragwort poisoning could be identified in dIe early stages of ingestion dlen the onset of irreversible patllology might be preventable. The goal of this study was to explore methods for the detection and analysis of pyrrole-peptide and pyrrole-protein adducts· in vitro and to investigate techniques facilitating the identification of changes in equine blood as a result of PA poisoning in Vivo. Initial experiments aimed to elucida~e dIe range of responses of model proteins to alh.'ylating agents ill tll/ro. Intact mass analysis of dIe proteins by l-dimensional gel electrophoresis (lD-GE) and quadtupole-time-of-flight mass spectrometry (Q-ToF MS) demonstrated that different reagents are capable of distinctly modifying proteins, and that these reagents have varying degrees of specificity for dIe sulphydryl group of cysteine residues. Matri.x-assisted laser desorption/ionisation (MALDI)-ToF MS revealed the particular regions of proteins which were targeted by reagents and demonstrated that peptide adduct detection could be enhanced by guanidination of lysine-terminated peptides.

Colic (abdominal pain) is a relatively common disease of horses ranging from mild, transient episodes to a life-threatening syndrome that requires surgical intervention. Equine intestinal abnormalities that involve local stasis are often accompanied by marked proliferation of Gram negative enteric bacteria and breakdown of the mucosal barrier resulting in the transmural and transvascular migration of bacteria and their endotoxins into the circulation (endotoxaemia). Even if the original cause of colic is corrected or resolved, the presence of endotoxaemia can isult in significant morbidity and mortality in horses. This project aimed to characterise specific equine complement component, complement 1 esterase-inhibitor (Cl-inh) because it is considered a key inhibitor of the connplement, coagulation and contact activation pathways.

Domestic horses maintain many of their innate behavioural traits as a group-living, free-ranging herbivore that usually avoids predation by flight. Confinement, isolation, restraint, riding, training and exposure to management practices present a vastly different experience to their natural environment but the psychological need to respond to environmental factors may remain, even when the biological motivation has been removed. This disparity can lead to the development of physiological and behavioural abnormalities indicative of a negative affective state and poor welfare. The concept of animal welfare should include the animals’ physical and psychological health and harmony with their environment. There is an increasing call for subjective feelings and the assessment of emotion to be taken into account so that welfare can be enhanced by increasing the incidence of positive experiences and minimising negative ones. Reliably establishing the affective state of an animal is a challenging task but measuring physiology and behaviour in response to pleasant or unpleasant stimuli can provide evidence for the existence of affective state. Equine research has endeavoured to identify ‘reactivity’, temperament and emotionality but has not yet investigated underlying affective state in response to different stimuli, the existence of, or factors that might influence positive affective states. As such, there is no strong scientific knowledge of what equine emotional experiences are. Many physiological measures involve invasive procedures that contribute to the stress load of the individual and non-invasive methods often only determine the presence or absence of ‘stress’ or are dogged by interpretive problems. Heart rate variability (HRV) is a major growth area in the study of emotion and is an established parameter to quantify the state of the autonomic nervous system. Empirical evidence from human and animal research associates high levels of HRV with positive emotions and vice versa. This research aimed to evaluate the use of HRV alongside behavioural measurements as a non-invasive method of assessing affective state in horses. Equine HRV and behaviour were measured during anticipation of positive rewards (companionship and feeding), during a negatively valenced management procedure (sham-clipping), in four housing systems allowing increasing levels of social contact and potential factors affecting HRV were assessed. By exposing horses to housing and management conditions differing in their emotional valence it was possible to identify factors that significantly affected HRV and highlight incidences where a significant relationship existed between behaviour and HRV (significant if p≤0.05). Rewarding and contrasting circumstances were found to be significantly associated with HRV. Social interaction, ‘play’ behaviour and the provision of a haylage diet were all associated with high HRV whereas disruption to the horses’ leisure time was associated with low HRV. HRV provided an objective physiological measurement for interpreting behaviour and assessing underlying affective state; compliant behaviour in response to an aversive stimulus was not associated with low HRV and specific pre-feeding behaviours (behavioural transitions, ear movement and head nodding) could prove a useful indicator of negative affective state in future studies. Specific social and spatial factors significantly affecting HRV were also identified; tactile contact with neighbouring horses was related to high HRV and hay net position appeared to affect the horses’ capacity for environmental monitoring. A natural variation in HRV between equine sexes and between individuals was confirmed and changes in HRV were found to be situation-specific. It was possible to use these initial data to make suggestions for the establishment of a preferable habitat and management regime for horses. The importance of social interaction was highlighted, particularly ‘play’ behaviour. Horses housed in confinement and isolation may derive greater benefit from the ‘reward’ or contrast of social interaction than horses kept in more social conditions. Where negative experiences are encountered, these might be mitigated by rewarding with a preferred forage type and extending visual horizons towards neighbouring horses and the external environment. Ensuring that negative experiences are not prolonged or offsetting them with positive experiences could enhance quality of life. This study makes a unique contribution to equine welfare research as the use of HRV measurements to assess emotion in horses is a relatively new area of investigation. HRV was found to add vital physiological support to existing findings and although research of this nature is in its infancy, HRV appears to be a promising tool for assessing affective state and interpreting behavioural responses to stimuli in horses. There is extensive scope for further investigation into the use of HRV as a measure of emotional responses to management factors, environmental and ridden/training conditions in order to identify enjoyable or rewarding practices, so that the experience of positive emotion can be incorporated into management and training.

The experiments reported in this thesis investigated the use of temperature measurement using infrared thermography (IRT) as an objective, non-invasive method to identify the physiological stress response in the horse. The primary area of investigation was the eye area within the medial posterior palpebral border of the lower eyelid and the lacrimal caruncle as in existing work in other species. The application of these findings to horse management and welfare was discussed. Horses were exposed to potentially stressful situations that were acute (<20 seconds), short term (ten minutes) and long term or repetitive (one week) in duration. Temperature was measured using IRT in addition to measurement of salivary cortisol, faecal corticosterone and behavioural assessment, all of which are currently accepted measures of the stress response. IRT was shown to be an unsuitable method to assess acute stress in the horse due to the species specific behavioural response of flight. Rapid evasive movement of the horse meant that recording temperature using a thermal camera was difficult. A modified experimental design may have made it possible to capture the thermal response to acute stress however restraint of the horse would have been stressful in itself and confounded results. A significant (p=0.005), positive correlation was found between eye temperature and the stress hormone salivary cortisol when horses were exposed to the short term potentially stressful husbandry procedure of clipping. Behavioural assessment of the horses during clipping did not support the physiological findings. Finally IRT was shown to be an unsuitable method to identify long term or repetitive stress associated with restrictive housing. Faecal corticosterone and behavioural assessment were found to be more appropriate methods to monitor stress chronic duration. These experiments provide evidence that IRT is able to identify temperature change associated with short term stress and offers an objective, non-invasive and instant physiological measure of the equine stress response. Use of IRT as a research tool will allow a better understanding of how horses perceive short term husbandry procedures and management techniques and allow alterations to be made if necessary in order to improve equine welfare and maintain well being.

No description available.

This thesis assessed the factors influencing body condition and subsequent obesity risk in UK horses and ponies, using an interdisciplinary approach. It was novel in considering social and behavioural risk factors for obesity, using modelling to generate testable predictions, epidemiological risk factor studies to quantify the relative role of behavioural versus other factors, and behavioural fieldwork studies to support model predictions. In a partially outdoor-living population the prevalence of obesity was 27% at the end of winter, rising to 35% during summer. Regional nuchal crest adiposity had the opposite seasonal pattern. Breed was the most important factor in determining obesity risk. As horses and ponies became obese, usual seasonal variation in body condition was reduced, something not previously reported. The role of social and behavioural risk factors in determining body condition and subsequent obesity was highlighted. Dominance was strongly associated with body condition in two studies, where more dominant individuals tended to have a higher body condition score and greater obesity risk. Theoretical modelling predicted that differences in foraging efficiency could lead to stable and predictable body condition differences within pairs and trios of individuals. When tested empirically, foraging efficiency was associated with both dominance and body condition in domestic herds. Animals with a greater foraging efficiency generally had a higher body condition and were more dominant. A potential 'fattening cycle' is described whereby a greater body condition leads to a higher dominance status, allowing for a greater foraging efficiency, and further body condition increase. Herd dynamics and social relationships could therefore determine obesity risk. The direction of association between dominance, body condition and foraging efficiency requires future longitudinal investigation. Social and behavioural risk factors should be considered in future clinical investigations in group-living animals. A series of practical recommendations for horse owners are detailed.

No description available.

No description available.

Colic is an important cause of death in managed horse populations and is a health issue of great concern to horse owners. Epidemiological studies that have investigated risk factors for colic have identified a number of factors that increase the likelihood of colic, most of which are related to change in management including diet. The precise relationship between diet and colic is currently unknown but it has been hypothesised that changes in gut microbiota composition are responsible for the development of colic associated with change in management. The work presented in this thesis has further investigated risk factors for colic and postoperative intestinal dysfunction (postoperative colic and postoperative reflux) and has explored the gut microbiota in specific groups of horses at increased risk of colic. Risk factors for colic were investigated in a previously unstudied working horse population in Egypt using a cross-sectional epidemiological study. The study recruited 350 working horses from two provinces in Egypt and data about their management and history of colic in the preceding 12 months were collected. Risk factors identified included stereotypic behaviour, severe dental disease, feeding ground corn during the ‘dry season’, anthelmintic treatment in previous six months and feeding rice bran during the ‘green season’. A prospective cohort study of 264 surgical colic patients that were admitted to the Philip Leverhulme Equine Hospital, University of Liverpool in 2012–2014 was conducted. The study investigated risk factors for morbidity and mortality following laparotomy. Factors identified to be significantly associated with altered risk of recurrent postoperative colic episodes included strangulating large colon volvulus, epiploic foramen entrapment, postoperative reflux (POR), repeat laparotomy and heart rate on admission. A subset of these horses were used to investigate risk factors for POR in horses that underwent laparotomy for treatment of small intestinal lesions. The effect of systemic perioperative lidocaine treatment on the prevalence, duration and net volume of POR was compared with a previous cohort of horses (2004–2006). Risk factors associated with POR included packed cell volume on admission, pedunculated lipoma obstruction, pelvic flexure enterotomy, side-to-side jejunocaecal anastomosis, duration of surgery and the period of hospital admission. Lidocaine treatment had no effect on the prevalence, duration and net volume of POR nor was it a risk factor for postoperative survival. The relationship between gut microbiota composition and previously reported risk factors for colic/postoperative colic were investigated in a series of faecal microbiota studies. Temporal variation in faecal microbiota composition in a group of horses managed at pasture was investigated. Faecal samples were collected every 14 days from 7 horses for a total of 52 weeks. Samples were processed to extract bacterial DNA before creation of amplicon libraries and sequencing using the Ion Torrent PGM next-generation sequencing technology. Analysis of these sequence data revealed that the horse faecal microbiota is dominated by members of the phylum Firmicutes and Bacteroidetes. Significant effects of season, diet and ambient weather conditions on the composition of the horse faecal microbiota were identified. The study concluded that the horse faecal microbiota is in a continuous state of adaptive process to external stimuli and that fluctuations in the composition of horse faecal microbiota over time is normal. The temporal stability of the faecal microbiota of periparturient mares was also investigated. Weekly faecal samples were collected from 7 broodmares for a total of 17 weeks (5 weeks pre-foaling – 12 weeks post-foaling). Foaling had minimal effect on the composition of the horse faecal microbiota and variation in data over time was associated with changes in management. This suggested that minimising changes in management during the periparturient period may be important in stabilising the gut microbiota of periparturient mares. The faecal microbiota of surgical colic patients that underwent laparotomy for treatment of primary large colon diseases was compared to a group of emergency orthopaedic patients that underwent surgery under general anaesthesia. Samples were collected from both groups on admission, during hospitalisation and following hospital discharge for a period of 3 months. The results suggested that a 3-month period was not enough to fully investigate changes in faecal microbiota composition of surgical colic patients compared with samples collected on admission. This thesis provides new information about risk factors for colic in a working horse population, risk factors for complications following intestinal surgery and has questioned the efficacy of lidocaine in management of POR. The equine faecal microbiota in 3 different horse populations has been studied and has generated new hypotheses about the relationship between the equine gut microbiota and the risk of colic.