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Meat is still the most consumed protein source, especially in developed countries and ruminant meat has an important market share. However, it is marine food sources and plant-based food that are excellent suppliers for poly-unsaturated fatty acid (PUFA), particularly omega-3, which are recommended for human consumption due to their health attributes, nonetheless consumed to a lesser extent than meat. Hence, omega-3 enriched meat and animal products are coming to the market to compensate for this intake deficiency. The challenges of increasing omega-3 in red meat through lipids supplementation to ruminants lies in the lipolysis and biohydrogenation seen in the rumen, as the double-bonded molecules (i.e., in PUFA) create a toxic environment for the bacteria. This results in the formation of monounsaturated fatty acids (MUFA) and then saturated fatty acids (SFA). Dietary supplementation of some strains of microalgae have given good results in terms of enhancing omega-3 content in ruminant meat, however no comparison was made between the supplementation of docosahexaenoic acid-rich (DHA, C22:6), a long chain omega-3 PUFA, in different forms, but from the same genus. Consequently, this study investigated the effect of both DHA-rich microalgae cells and separately their extracted oil on the rumen microbiome via in vitro incubation. The project also provides more in-depth knowledge on the impact of Schizochytrium sp. supplementation on fatty acid profiles of different raw and cooked lamb meat cutlets, whilst also assessing the underlying changes in the gastrointestinal tract (GIT) microbiome. Finally, sensory parameters of enriched meat products are often assessed by a trained panel, and reported with "fishy", "oily", "seaweed" off-flavours, especially in the case of fish oil supplementations. Therefore, this study investigated the palatability and acceptability of lamb burgers, enriched with different levels of Schizochytrium sp. using a consumer panel. The in vitro incubations to study the effects of Schizochytrium sp. on rumen fermentation were conducted at equal DHA concentration, using Schizochytrium sp. cell form and extracted oil form, monitoring using ANKOM gas production system jars. The structure of Schizochytrium sp. cells were observed after in vitro incubation in a DaisyII incubator. Samples taken over time from the jars were analysed for volatile fatty acids (VFA), ammonia, protein and the rumen microbiome. Two in vivo studies were also conducted, one with 30 Easy-Care ram lambs allocated into 3 treatments (No supplementation (Control), 10.7 g biomass/head/day (Low) and 21.4 g biomass/head/day (High)) fed fresh grass and concentrates (50:50 energy based). The second one with 60 Texel crossed neutered lambs allocated into 4 treatments (No supplementation (Control), 10.7 g biomass/kg concentrates (Low), 21.4 g biomass/kg concentrates (Medium) and 32.1 g biomass/kg concentrates (High) fed grass silage and concentrates (50:50 energy based). From the first in vivo analysis, samples from different time points and different parts of the GIT (i.e., rumen, small intestine, large intestine) were kept to analyse VFAs, ammonia and the GIT microbiome. From both of the in vivo studies, the meat of the animals was collected for fatty acids analysis and from the second in vivo study, the meat was offered to a consumer panel for sensory assessment. The data obtained throughout this project showed no detrimental effect of the supplementation with whole cells or extracted Schizochytrium sp. oil on the rumen microbiome or its functioning. The data also suggested that the supplementation of Schizochytrium sp. as whole cells would limit the access to the unsaturated lipids due to the cell wall. A beneficial impact of the dietary supplementation with microalgae on the health properties of lamb meat was observed in both in vivo studies. In the first study, beneficial consumption was particularly emphasised in the loin and leg muscles. This enhanced quality meat caused no detriment to the lambs' growth or ruminal health. Finally, the project suggested the commercial potential of meat from lamb supplemented with Schizochytrium sp., as no difference in the palatability or appreciation was found in the enriched burgers as compared with the non-enriched, whilst improving human health at a medium inclusion concentration. To the best of our knowledge, this project provides novel data by comparing the supplementation of Schizochytrium sp. under two different forms in vitro, directly comparing the effect of the biomass supplementation in raw and cooked meat, from different cutlets and using a consumer panel for the assessment of the omega-3 enriched lamb meat.

The behaviour of sheep has large applications to the UK sheep industry to improve productivity to boost profits and meet global demand. This can be achieved by ensuring maximum reproductive output each year by maximising pregnancy rates, increasing lamb crop sizes and ensuring efficient lamb growth. The advancement in bio-logging technology has facilitated significant advancements in the measurement of animal behaviour and has made the continuous monitoring of the behaviour of farm animals in a commercial setting feasible. A growing body of work has begun to validate this technology in extensive grazing systems and there have been an increasing number of studies linking behaviour measurement to a range of production outcomes. However, evidence is scarce in 3 key areas, including (1) aging, (2) the detection of oestrous and (3) lamb growth and maternal ability which this thesis aimed to explore. Firstly, this thesis aimed to validate the use of bio-loggers in an extensive grazing system, using accelerometers and proximity sensors attached to sheep for 14-day periods at various points in the farm production cycle to collect behavioural data on a commercial sheep flock. Behavioural data was then combined with production records on a pedigree, performance-recorded sheep population and environmental data collected on-farm, to determine links with production traits to investigate how the sheep industry can improve production through maximising reproductive success. This thesis was able to bridge the gap between research and the UK sheep industry, by demonstrating the feasibility of continuously monitoring the behaviour of an entire commercial sheep flock at multiple stages of the production cycle using bio-loggers. Links between behavioural data and production revealed, (i) the presence of age effects on behaviour but no effects on long-term production, (ii) the feasibility of using bio-loggers for the automatic detection of oestrous either indirectly, from ewe-ram social behaviour or directly from ewe behaviour and (iii) lamb and maternal behavioural predictors of lamb growth. As a secondary aim, environmental effects on behaviour were also described, which may prove useful for quantifying the resilience of animals. Advancing our understanding of the measurement of behaviour in a commercial setting and uncovering links with production traits is key to facilitating commercial uptake by the UK sheep industry for real-time monitoring of behaviour and health of sheep flocks to improve performance.

Micronutrients play an important role in metabolic reactions in animals. To correct for potential micronutrient deficiencies in forages, the dominant feed source for ruminants, farmers routinely administer supplements to animals prophylactically. Micronutrients that are not absorbed from supplements are excreted, providing a potential flux of micronutrients to soil for uptake by forages. However, it is unclear if the form of supplemental minerals given to animals significantly affects the flux of micronutrients in pasture systems. This study investigated the impacts of supplement form (organic or inorganic), dose, excreta type (urine and/or faeces), and soil organic matter (OM) on the flux of micronutrients in a grazing pasture system. Over two weeks, 24 sheep were supplemented with organic or inorganic forms of Zn, Cu, Mn, and Se at two industrial doses. The excreta were applied to potted soils and seeded with perennial ryegrass (Lolium perenne L.). The redistribution of micronutrients was followed from feed to excreta to soil to grass. Over 90% of the Cu, Zn and Mn, and over 80% of the Se was excreted through faeces, regardless of supplement form or dose. The concentrations of bioavailable Zn, Cu, Mn and Se in faeces were not significantly affected by the supplement form, but recalcitrant fractions of Zn, Cu and Se were, implying a potential long-term effect of supplement form on micronutrient bioavailability in soils. Uptake of Zn, Cu and Mn by ryegrass was most affected by excreta type. Although faeces contributed 20-1200 times more Zn, Cu and Mn than urine, micronutrient uptake by ryegrass was significantly higher in soils applied with urine because N, K and S in urine increased grass growth. Uptake of Se by ryegrass was limited in high OM soils regardless of excreta type, revealing antagonism between the benefits of OM to plant growth and the bioavailability of Se.

Studies across different species have demonstrated the presence of an interplay between the brain, the gut and the microbiome, most commonly referred to as the gut-brain axis. Furthermore, it has been well documented that stress can affect neuroendocrinological and immunological systems, resulting in altered behaviours, as well as physiological dysregulations. In the past few decades, the effects of stress on the microbiome and the implications of the microbial community structure for the host have been the focus of many studies, aiming to shed light on this intricate and multi-pathway relationship. Despite this, most studies have been conducted on humans and rodents, with very few on farm animals, particularly ruminants. In ruminants, as in monogastrics, the gut accommodates high microbial concentrations and facilitates host-microbial interactions. What differentiates ruminants is the presence of the rumen, which hosts an equally important microbial community. This organ acts as a primary location for fermentation of feed and plays a pivotal role in animal metabolism, immunity and overall homeostasis. The effects of stress susceptibility, and more specifically psychological or behavioural stress, have been poorly explored in ruminants, despite their well-recognised and important effects on other aspects of animal health and welfare. This project therefore aimed to explore: 1) the effect of genetic predisposition to stress; 2) the long-term effect of prenatal and early life stressful events; and 3) effects of repeated and unpredictable management stress on the ovine gastrointestinal microbiome in conjunction with various physiology and behavioural aspects. The first experiment investigated genetic differences in gut and rumen microbial community structure and blood cortisol concentrations in 58 adult Romane ewes, previously selected on the basis of divergent reactivity to stress (30 ewes with high reactivity; 28 ewes with low reactivity). The two groups differed in their behavioural reactivity towards a temporary separation from congeners based on bouts of high bleats. Despite extensive analyses of the microbiota at the phylum, order and genus level, there were only small significant differences in the rumen and faecal microbiota, even when including cortisol levels in the analyses. For example, higher levels of cortisol were positively correlated with Ruminococcus abundance in faecal samples and Lactobacillus in the rumen, while Rikenellaceae abundance was positively correlated with reactive EBV scores in faecal samples. The second experiment investigated long-term effects of three prenatal stress treatments (Control, Negative and Alternative) and the effect of two early-life treatments (Isolation or Ewe Recognition tests) on the rumen microbial community structure of 35 8-month-old Scottish Mule lambs, at a stage when the rumen microbiome had assumed a relative stable and mature form. Sex and diet effects were confounded, while Prenatal Treatment did not appear to have an effect. Neonatal Treatment had an effect on relative abundances at the phylum level. The abundance of several bacterial species was correlated with higher or lower cortisol levels, such as Lactobacillus in Isolated females and Proteobacteria in Isolated males; these significant negative correlations suggested long-lasting effects of early life events. Finally, in the third experiment, we explored the effect of a 6-week mild unpredictable Chronic Stress paradigm on various behavioural (i.e., time budgets, reactions to a suddenness test) and physiology aspects (i.e. hormonal levels, heart rate and VFAs), as well as the rumen and gut microbiota structure. Forty-eight female Romane lambs were separated into two treatments: Non-Treated (NT, n = 24) and Mild Chronically Stressed Animals (MCS, n = 24). Amongst the most interesting results, indicating a treatment effect, were differences in synchronisation of animals resting and sleeping, duration of resting time, and reactions to novelty, as expressed by latency to approach the ball and contact time with the ball in the suddenness test. Microbiota diversity indices, particularly for the non-treated group, indicated a different development of the microbial community. In MCS animals, cortisol and serotonin levels indicated that several bacteria proliferate in the presence/absence of these hormones, but correlation scores were generally non-significant. In conclusion, it appears that the microbiota community structure in the rumen is not significantly affected by management stress or stress susceptibility, although the communication pathways between rumen bacteria and host behaviour warrant further exploration.

Ovine pulmonary adenocarcinoma (OPA) is a lung cancer that affects sheep, caused by jaagsiekte sheep retrovirus (JSRV). OPA is present in most sheeprearing countries of the world, but, at present, there are no reliable early-stage tests to diagnose the disease, and OPA continues to pose an animal welfare threat and cause substantial economic losses. In addition, OPA is a valuable animal model to study early oncogenic events in human lung cancer. Specifically, OPA and some types of human lung cancer present similarities in activated signalling pathways (Ras-MEK-ERK1/2 and PI3K-AKT-mTOR) and their association with type II pneumocytes. Nevertheless, study of the molecular pathogenesis of OPA has been hindered due to the lack of a permissive cell line for JSRV replication. JSRV encodes an unusual envelope protein (Env) which is actively oncogenic and sufficient to drive transformation in vivo and in vitro. Despite the lack of a permissive cell line, early oncogenic events induced by JSRV can be studied by transfection of cell lines with plasmids encoding JSRV Env. MicroRNAs (miRNAs) are short non-coding RNAs that regulate gene expression and essential cell processes such as cell proliferation and apoptosis. miRNAs are being extensively studied as biomarkers of several diseases, including cancer. The aims of this project were to investigate the role of miRNAs in the early oncogenic events induced by JSRV and to investigate their potential as OPA biomarkers. miRNA expression levels were investigated using small RNA sequencing in lung tissue from cases of experimentally induced OPA. No evidence of JSRV-encoded miRNAs was found, but levels of 40 miRNAs were found differentially expressed between affected and control sheep. Of those, upregulation of nine microRNAs (miR-135b, miR-182, miR-183, miR-21, miR-200b, miR-205, miR-31, miR-503 and miR-96) was confirmed by RT-qPCR in experimental and natural cases of OPA, suggesting that increased levels of these miRNAs were characteristic of OPA affected lung tissue. To investigate miRNAs as potential biomarkers, miRNA expression was measured in serum and bronchoalveolar lavage fluid (BALF) macrophages of OPA affected sheep. small RNA sequencing revealed 74 microRNAs and 85 miRNAs differentially expressed in serum and BALF macrophages, respectively. Interestingly, BALF macrophage microRNA expression was found to resemble more closely that of OPA affected sheep lungs. In addition, miRNA expression levels varied at different stages of the disease and no miRNAs were found to be consistently dysregulated in serum of OPA affected animals. Discordances in miRNA signatures in lung tissue and serum are not entirely unexpected. Lung tissue miRNAs might represent the tumour microenvironment and localised response to it, whereas miRNAs in serum may represent the global state of the animal, and tumour miRNAs might be released into circulation at low levels, making them difficult to detect. Expression of the nine upregulated miRNAs was then investigated in in vitro models to study their involvement in transformation. Lentiviral vectors encoding green fluorescent protein (GFP) or a GFP-2A-Env fusion protein were produced and used to transduce cell lines. Transformation was verified by immunocytochemical detection of the transformation markers P-Akt and PERK1/2. Nevertheless, miRNA expression levels in culture did not resemble those observed in lung tissue of OPA-affected sheep. These differences might be due to species variation, upregulation of miRNAs late in the transformation process, or involvement of other cell types in tissue besides the transformed cells. To study these questions further, JSRV and the GFP-2A-Env encoding lentiviral vectors were used to infect lung slices in culture. Expression levels of miRNAs did not, in any of the cases, resemble lung tissue findings. Fewer than 5% of cells in lung slices were found to be infected, suggesting that changes in miRNA expression could be masked by the background of normal cells. Nevertheless, increasing JSRV21 concentration did not yield higher infection levels, indicating that those might be more dependent on the availability of JSRV's target cells, dividing type II pneumocytes, than viral concentration. Taken together, this study has revealed new information on miRNA expression in OPA-affected sheep, including expression patterns in lung and serum. Future work should focus on developing a permissive replication system to allow the study of miRNAs in early JSRV-induced transformation events.

The survival of newborn lambs is highly dependent on the successful partnership between ewes and their offspring. High lamb mortality before one week of age is largely due to hypothermia, mismothering and starvation, which are indirectly consequences of poor establishment of a maternal bond between a ewe and her lambs. Maternal behaviour displayed by ewes towards their lambs is therefore crucial in ensuring the survival of the newborn lamb. However, maternal behaviour can be affected by many factors including the social environment, nutrition and husbandry routines. It is common in the UK for sheep to be kept indoors during winter from mid gestation until lambing to protect ewes and lambs from adverse conditions. Keeping the ewes indoor nonetheless, has its own challenges as the animals may have to be mixed with unfamiliar conspecifics, have limited floor and feeding space, changes to the diet and increased handling by humans. Therefore, the main objective of this study was to investigate the effect of housing systems experienced by ewes during gestation on mother-offspring interactions after parturition. The impact of different housing conditions were assessed by recording body weight, body condition score (BCS), behaviour, faecal glucocorticoid metabolite (FGM) concentrations and haematology parameters. After parturition, maternal behaviour was then assessed. Parity and the temperament of the ewes were also taken into account in assessing all parameters during gestation and after parturition. In the first experiment the effect of an indoor housing system on the behaviour and physiology of 41 primiparous and 36 multiparous ewes from 11 until 18 weeks of gestation was assessed. The ewes were divided into two groups: Control and RS-Mix (Restricted-Space and Mixed) where the RS-Mix ewes were allocated half the amount of space and feedface allowance given to Control group and were also subjected to three social mixing events. No significant treatment effects on physiology or behaviour during gestation were found except for higher aggression in RS-Mix ewes during the first week of observation which gradually declined to the same level as Control ewes at the end of experiment. RS-Mix ewes also displayed significantly higher ruminating behaviour at week 18 of gestation compared to Control ewes. However, primiparous ewes were more affected than multiparous ewes regardless of treatment group, with lower weight gain and higher FGM concentrations compared to multiparous ewes. In this study, the difference in housing system did not seem to have a lasting impact on the physiology and behaviour of pregnant ewes. The maternal behaviour of these ewes during parturition and lactation was investigated. RS-Mix ewes were significantly more likely to give birth to Lamb 2 (L2) while standing, compared to Control ewes and also more likely to give birth during the day whereas Control ewes were equally likely to give birth during the day or night. Grooming behaviour of these ewes was not affected by pregnancy treatment but RS-Mix ewes displayed significantly higher avoidance when the lambs reached the udder to suck compared to Controls. During tests of lamb recognition, primiparous ewes from RS-Mix group took longer to approach their own lambs and during lactation on the field, they kept the greatest distance from their neighbouring ewes, compared to all other groups. This suggests that maternal behaviour in RS-Mix ewes may have been affected by the stress experienced from the space restriction, regrouping and remixing during pregnancy. In the first experiment Control ewes as well as RS-Mix ewes displayed aggressive behaviour at the feedface during concentrate feeding. This implies that Control ewes may also experience competition at feeding which might explain the few behavioural effects of treatment. Therefore, in the next study two new housing treatments were set up: Alternative and Negative. Alternative ewes were provided with a larger space allowance of 4.57m2/ewe and 66cm/ewe of feedface allowance and had ad libitum access to food (grass silage) throughout the experiment. Negative ewes were provided with the same space and feedface allowance (1.28m2/ewe and 33cm/ewe respectively) as Control ewes from the first experiment but they were subjected to additional stressors, such as exposure to dogs and delayed feeding. The Control group in this experiment was as the RS-Mix group from the previous study but without the occurrence of social mixing. Surprisingly, Alternative ewes were found to be the most affected by the treatment. The BCS of Alternative ewes decreased significantly from week 15 to week 17 of gestation and this group had the least weight gain throughout gestation compared to Control and Negative ewes. FGM concentration in Alternative ewes was also the highest compared to other treatment groups from week 13 until week 17 of gestation, and at 12 hours postpartum. Interestingly however, the concentrations of beta-hydroxybutyrate and neutrophil-lymphocyte ratio in Alternative ewes were significantly lower than other groups. Alternative ewes also took a longer time to groom their lambs, spent less time grooming and were less cooperative with the lambs sucking attempts than other groups. This study found only minor evidence that housing systems, which mimicked commercial conditions, caused significant stress in pregnant ewes, although short-term aggression at feeding was observed in both Control and Negative groups. An alternative system, which was designed to reduce competition and aggression at feeding, by allowing continual access to feed, did reduce aggression but had other effects on the ewes, such as loss of body condition, which suggested this feeding system was not providing the ewes with their full requirements, perhaps because the ewes did not find the feed palatable. It was apparent, however, that primiparous ewes may have more difficulties in adjusting to the new environment than older and more experienced ewes, and this may contribute to greater stress in this group. Even though the behaviour and physiological parameters recorded during gestation were insufficient to verify the existence of stress, the alteration of certain aspects of maternal behaviour recorded after parturition suggested that management systems in indoor housing could cause stress to the ewes. Exposure to undernutrition during pregnancy was also shown to affect maternal behaviour in ewes. Therefore, special attention should be taken to provide the ewes with adequate housing condition and fulfil the nutrition requirements of gestating ewes in order to ensure the establishment of stronger ewe-lamb interaction which will eventually improve the survivability of newborn lambs.

The cardiovascular system is an essential component of mammalian biology. It is a complex network of various tissues and structures with unique functions. The function of the cardiovascular system is to supply nutrients including oxygen to the various cells, tissues and organs within the body, and remove waste products from them. Given the importance of this role, it is not surprising that there are countless regulatory mechanisms at the molecular, cellular and tissue levels that are required to support this functional system. Perturbations in parts of this system are likely to lead to abnormalities, and thus give rise to cardiovascular-related diseases. Despite the currently expanding list of genes reported to be involved in a variety of cardiovascular-related diseases, including calcific aortic valve disease (CAVD), the functions and associated pathways of these factors in both normal and pathological physiology have yet to be fully understood, such as at the transcriptomic level. In this thesis, a genome-wide transcriptomic atlas of the healthy mammalian cardiovascular system was generated using the sheep as a large animal model. This atlas was generated using RNA-seq, with the aim of further understanding normal gene expression patterns in the context of the known physiology of healthy mammalian tissues. Through this work, I identified novel gene networks and detailed functional clustering of co-expressed genes with region-specific expression and specialised cardiovascular roles. One interesting cluster was highly expressed in the cardiac valves, and shared genes found in physiological bone development, such as bone morphogenetic protein 4 (BMP4), collagen type I alpha 2 (COL1A2), Sry homeobox 8 (SOX8) and bone gamma-carboxyglutamate protein (BGLAP), some of which have been implicated in vascular calcification. Further to this work, I studied the expression profiles of these key cardiovascular genes during development in the sheep from foetal to adult stages. In addition, I investigated the gene expression patterns of various key vascular calcification genes. These studies showed differential expression of genes in the different cardiovascular tissues, demonstrating transcriptional differences between these different tissues known to have different functions. CAVD involves progressive valve leaflet thickening and severe calcification, resulting in impaired leaflet motion. The in vitro calcification of primary rat, human, porcine and bovine aortic valve interstitial cells (VICs) is commonly employed to examine the mechanisms of CAVD. However, to date, no published studies have utilised cell lines to investigate this process Thus, in this project, I generated and evaluated the calcification potential of an immortalised cell line derived from sheep aortic VICs (SAVICs). This novel large animal in vitro model of CAVD was demonstrated to calcify under high calcium and phosphate conditions. Changes in the expression of key calcification genes during VIC calcification was also observed, including increased mRNA expression of bone markers Runt-related transcription factor 2 (RUNX2) and sodium-dependent phosphate transporter 1 (PiT1), and a concomitant decrease in matrix Gla protein (MGP) mRNA expression. In addition, the role of extracellular nucleotides and their receptors (P2 receptors), which have been previously shown to be important in bone and vascular calcification, were investigated using SAVICs in vitro. This study has shown that extracellular nucleotides, particularly adenosine 5’-triphosphate (ATP) and uridine 5’-triphosphate (UTP) and other agonists of P2 receptors, reduced VIC calcification in vitro. Moreover, the cutting-edge gene-editing technology, clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein-9 nuclease (Cas9), was successfully applied to generate large animal models of cardiovascular-related diseases. In this project, I applied the CRISPR/Cas9 technology to edit ectonucleotide pyrophosphatase/phosphodiesterase 1 (ENPP1) and fibrillin 1 (FBN1) to generate two models of vascular calcification and Marfan Syndrome (MFS), respectively. In the ENPP1-edited animals, soft tissue calcification has been observed in the biallelic mutant and homozygous pigs. In this project, I have developed a range of novel in vitro and in vivo tools to advance the study of cardiovascular disease. These studies demonstrate that large animal models are highly valuable in the field of cardiovascular biology. The in vivo and in vitro experimental models described should facilitate detailed analysis of cardiovascular molecular biology and ultimately lead to therapies which will minimise the morbidity and mortality currently arising from cardiovascular pathology.

Rural change and the ability of farming communities to respond and withstand change is a topic of ongoing concern in the current research agenda. ‘Rural community resilience’ is a concept that has become a core theme of academic, policy and lay discourses discussing dynamics of rural change, widely associated with community studies and allied to notions of social capital. This work reviews approaches to community relations developed within community studies and social capital scholarship, and suggests that the relational agency of the network ties might also be explored through the application of an assemblage approach. However, and unlike many previous approaches to community resilience that use the concept in a normative way and which understandably highlight agency of social relations, this research has been constructed in such a way that network ties established through day-to-day community practices are characterized both vital and far from passive. Developing this current line of thinking in rural studies, this project argues that more-than-social agency evoked by relations between human and non-human components of the rural assemblage is an important factor affecting community resilience. The empirical research feeds from two case studies and gathers evidence from two distinctive agricultural communities of Hungary and Wales, whilst also recognizing similarities in the context of globalization. It argues that rural community resilience lies in relations between the humans, the land and the agricultural commodities.

Animals are sentient beings, capable of experiencing emotions. Being able to assess emotional states in farm animals is crucial to improving their welfare. Although the function of emotion is not primarily for communication, the outward expression of an emotional state involves changes in posture, vocalisations, odours and facial expressions. These changes can be perceived and used as indicators of emotional state by other animals. Since emotions can be perceived between conspecifics, understanding how emotions are identified and how they can spread within a social group could have a major impact on improving the welfare of farmed species, which are mostly reared in groups. A recently developed method for the evaluation of emotions in animals is based on cognitive biases such as judgment biases, i.e. an individual in a negative emotional state will show pessimistic judgments while and individual in a positive emotional state will show optimistic judgments. The aims of this project were to (A) establish whether sheep and goats can discriminate between images of faces of familiar conspecifics taken in different positive and negative situations, (B) establish whether sheep and goats perceive the valence (positive of negative) of the emotion expressed by the animal on the image, (C) validate the use of images of faces in cognitive bias studies. The use of images of faces of conspecifics as emotional stimuli was first validated, using a discrimination task in a two-armed maze. A new methodology was then developed across a series of experiments to assess spontaneous reactions of animals exposed to video clips or to images of faces of familiar conspecifics. Detailed observations of ear postures were used as the main behavioural indicator. Individual characteristics (dominance status within the herd, dominance pairwise relationships and humananimal relationship) were also recorded during preliminary tests and included in the analyses. The impact of a low-mood state on the perception of emotions was assessed in sheep after subjecting half of the animals to unpredictable negative housing conditions and keeping the other half in good standard housing conditions. Sheep were then presented with videos of conspecifics filmed in situations of varying valence. Reactions to ambiguous stimuli were evaluated by presenting goats with images of morphed faces. Goats were also presented with images of faces of familiar conspecifics taken situations of varying emotional intensity. Sheep could discriminate images of faces of conspecifics taken either in a negative or in a neutral situation and their learning process of the discrimination task was affected by the type of emotion displayed. Sheep reacted differently depending on the valence of the video clips (P < 0.05); however, there was no difference between the control and the low-mood groups (P > 0.05). Goats also showed different behavioural reactions to images of faces photographed in different situations (P < 0.05), indicating that they perceived the images as different. Responses to morphed images were not necessarily intermediate to responses to negative and positive images and not gradual either, which poses a major problem to the potential use of facial images in cognitive bias experiments. Overall, animals were more attentive towards images or videos of conspecifics in negative situations, i.e., presumably, in a negative emotional state. This suggests that sheep and goats are able to perceive the valence of the emotional state. The identity of the individual on the photo also affected the animals’ spontaneous reaction to the images. Social relationships such as dominance, but also affinity between the tested and photographed individual seem to influence emotion perception.

Neurodegenerative diseases are associated with extensive physical and mental debilitation, significant costs to the healthcare system, as well as great emotional and financial burden to the patients, their families and care providers. Despite progress in our understanding of the mechanisms behind neurodegenerative diseases, the vast majority are still currently untreatable. Synapses are important pathological targets in a range of disorders, including Alzheimer’s disease, Parkinson’s disease, Huntington’s disease and lysosomal storage disorders, such as Batten disease. Loss of synaptic connections and impairments in synaptic function are present in the initial stages of neurodegenerative conditions and throughout the course of disease progression. Therefore, synaptoprotective strategies are regarded as a potentially key factor in the development of effective therapies aimed at preventing or halting neurodegeneration. Despite the continuously growing body of research elucidating the molecular mechanisms that modulate synaptic function and vulnerability, the contribution of these pathways to neurodegenerative diseases is far from fully characterized. In addition, there are frequent issues regarding the applicability of the research performed using in vitro and small animal models of disease to develop therapeutic strategies for use in human patients. In the work described in this thesis, we initially validated the involvement of a selection of key synaptic targets, previously identified as regulators of synaptic degeneration in lower animal models, including mice and Drosophila, in a large animal model of neurodegenerative disease: CLN5 Batten sheep. Subsequently, we explored two of these individual synaptic protein targets in more detail (calretinin and α-synuclein), to further investigate their contribution to synaptic function and stability. Calretinin is a poorly characterized protein, primarily known for its calcium buffering capacities and high levels of expression in a subpopulation of interneurons. In this work, we show calretinin is expressed in previously unreported cell populations, including motor axons and synapses from the peripheral nervous system, and that it is enriched in synapses in vitro. Furthermore, we show calretinin responds dynamically to synaptic activity and is directly involved in neurodegenerative pathways, as demonstrated by its ability to influence the course of Wallerian degeneration and apoptotic cell death. α-synuclein plays a central role in the pathophysiology of Parkinson’s disease and contributes to the maintenance of synaptic transmission and mitochondrial function. However, questions still remain about how to effectively manipulate α- synuclein to obtain therapeutic benefits. Therefore, we sought to explore downstream targets of α-synuclein in order to uncover new pathways through which this protein may influence synaptic stability. Using proteomics on mice lacking α-synuclein and in vitro cell systems we identified sideroflexin 3 (sfxn3). We show sfxn3 is localized at the inner mitochondrial membrane and that it functions outside the main canonical pathways of mitochondria energy production. In addition, overexpression of sfxn3 in Drosophila led to a significant loss of synaptic boutons at the level of the neuromuscular junction, suggesting regulated levels of sfxn3 are important for the maintenance of synaptic connections. Altogether, the work developed in this thesis provides novel insights into pathways regulating synaptic stability and function. We not only provide evidence that the molecular targets studied are affected in a large animal model of neurodegenerative disease, and are therefore likely to be relevant to studies in human conditions, but we also uncover two new molecular targets capable of independently regulating synaptic form and function.

Within the livestock sector, farmers and veterinarians are two groups of people who play a pivotal role in maintaining the health and welfare of animals. How the different welfare needs of farm animals are perceived and prioritised by these two caretaker groups will have direct implications for the animals in their care. People’s perceptions and attitudes directly influence their behaviour, and research has demonstrated that positive attitudes towards animals are paramount to ensuring good animal welfare. The prevention and mitigation of pain is an important component to ensuring good animal welfare, as pain has the potential to negatively affect both physical and mental health. How pain in animals is perceived by farmers and vets will influence how it is managed. Therefore, understanding how farmers and vets: view the capacity of animals to experience pain, perceive the pain severity associated with different conditions and procedures, view the importance of pain mitigation in relation to other welfare needs, and deem the necessity of analgesic use in livestock, is vital. Four separate questionnaire based studies were conducted to assess these attitudes in farmers and veterinarians as well as in agriculture and veterinary students, as these students will be the next generation of farmers and veterinarians. Overall, farmers and vets were found to have positive attitudes towards pain in livestock. Although the capacity of cattle and sheep to feel pain was perceived to be lower than that of humans it was still rated highly. In addition, positive beliefs about the benefits of pain alleviation, the negative impacts of pain on production and welfare, and the importance of prompt treatment and pain management for good welfare were held. Cattle farmers had more positive attitudes towards pain and analgesic use than sheep farmers. This difference was most evident around areas of resource availability, such as time and labour, and the practicalities associated with pain identification and drug provision. Farmers, vets and students perceived lameness to be a painful condition, with the perceived severity of pain being closely related to the perceived severity of the disease. In addition, participants reported a greater emotional reaction in instances where they rated lameness and pain more highly. Furthermore, a positive relationship was found between lameness, pain and emotional reaction scores and the decision to catch a lame sheep for inspection. The majority of students had positive views towards pain in farm animals, believing that: farm animals were capable of experiencing pain, prompt treatment and the provision of pain relief were the two most important elements of welfare, and that farm animals benefit from pain alleviation. However, there was a perceived difference between a number of animal species in their capacity to feel pain, with livestock species being viewed as having a lesser capacity than companion animals and humans. In addition effects of gender were found, with females reporting higher levels of empathy and compassion towards lame sheep, and rating pain higher. Furthermore, female students had a stronger belief that animals were sentient beings than did males. These four studies found that views on pain and analgesic use in livestock were generally positive. However, differences between individuals and between groups were found in a number of areas including how observers perceived the severity of painful conditions and procedures and in the capacity of different animal species to experience pain. These differences in attitudes may affect the decisions farmers and vets make regarding the treatment of pain, which is likely to have implications for farm animal welfare.

Paratuberculosis is a chronic enteropathy of ruminants that presents as two distinct disease forms in sheep; paucibacillary (or tuberculoid) and multibacillary (or lepromatous) disease. The immunopathology of paucibacillary and multibacillary sheep paratuberculosis has been linked to inflammatory Th1/Th17 cell and Th2/macrophage responses respectively. IL23 and IL25 are key to the development of these responses by interaction with their complex receptors, IL23R/IL12RB1 and IL17RA/IL17RB. Furthermore, the polarization of T cells and the development of appropriate immune responses is controlled by the master regulator transcription factor; T-bet, GATA3, RORγt and RORα. In humans, variations in the structure, sequence and/or expression of the genes encoding these proteins have been implicated in the different pathological forms of tuberculosis and leprosy, and gastrointestinal inflammatory disorders such as Crohn’s disease. In the current study, sequencing has identified multiple transcript variants of sheep IL23R, IL12RB1 and IL17RB and a single IL17RA transcript. RT-qPCR assays were developed for the cytokine receptor variants identified in this study and known transcript variants of the transcription factor genes. Expression levels were compared in the ileo cecal lymph node of paucibacillary or multibacillary paratuberculosis diseased sheep. Of the cytokine receptors; the IL12RB1v3 variant, which lacks the receptor activation motif, was differentially expressed and was significantly increased in multibacillary disease; this may contribute to high Th2 responses. Full length IL17RB was differentially expressed and was significantly increased in multibacillary pathology, which may also contribute to Th2 polarization. IL17RA was significantly increased in paucibacillary disease. The contrast between the IL17RA and IL17RB results may indicate that, in addition to Th1 cells, Th17 T cells are also involved in paucibacillary pathology. Of the transcription factor transcripts; full length TBX21 (T-bet) was differentially expressed and was significantly increased in paucibacillary disease; this may explain increased Th1 responses in these sheep. Full length GATA3 was significantly increased in paucibacillary compared to multibacillary sheep, suggesting a loss of Th2 responses in late-stage multibacillary pathology. RORAv1 variant was differentially expressed and was significantly increased in paucibacillary pathology, indicating a role of Th17 T cells in paucibacillary pathology.

The increased public interest in the welfare of animals used in food production has led to the emergence of welfare assessment schemes for a range of livestock species. There are currently over 100 million sheep in the EU which are primarily bred for milk, meat and wool production and the majority of these animals are managed extensively. The general perception of sheep in extensive systems living “natural lives” with few welfare compromises, along with the practical challenges of adequately assessing their welfare, has caused them to be largely ignored in comparison to other species. It was only relatively recently that the lack of animalbased welfare assessments for extensively kept small ruminants was recognised. Thus, the aim of this project was to evaluate potential animal-based welfare indicators for use during on-farm welfare assessments of extensively managed sheep. The current project used the Welfare Quality® 4 principles and 12 criteria as a foundation for selecting indicators for the assessment of extensively managed sheep. Following a comprehensive review of the scientific literature and a meeting attended by experts from across the EU, 16 indicators were selected for evaluation. Each principle and criteria were covered by at least one of these 16 indicators ensuring the main aspects of sheep welfare were addressed. The indicators selected for investigation could either be applied without handling or gathering the animals during an 'Assessment in the Field', or during a more thorough hands-on 'Assessment at Gather'. The reliability, feasibility and validity of some indicators (e.g. body condition scoring) are already established. However for others (e.g. Qualitative Behavioural Assessment approach), at least one of these criteria required further investigation before the indicator could be accepted. The reliability of selected measures was evaluated by assessing their repeatability and inter-observer agreement. Face validity was assumed for the indicators selected during the expert meeting, and further cross validation was performed when appropriate using additional information collected on the animal’s physical health status. During the Assessments at Gather blood samples were collected for the assessment of metabolic profiles, and faecal samples provided gastro-intestinal parasite counts. The Assessments at Gather were performed on the same 100-135 Scottish Blackface ewes across a two year period (2011-2013) on a Scottish hill farm. The Assessments at Gather occurred five times a year coinciding with key points in the ewes’ reproductive cycle: pre-mating, mid-pregnancy, late pregnancy, mid lactation and weaning. During the assessments data were collected on the ewe’s body condition, coat cleanliness, faecal soiling score, respiratory conditions, anaemia, lameness and udder condition (udders assessed during lactation only). Current stage in the reproductive cycle and seasonality were found to have significant impacts upon the long-term reliability of the following measures: body condition score, tooth loss, nasal discharge and anaemia scores (P < 0.001) with variation both within, and between years. On commercial farms older and less productive ewes tend to be removed from the flock once a year prior to mating. Of the indicators applied to the ewes during the Assessments at Gathers, tooth loss and body condition score were the best predictors for the ewe’s exclusion from the flock, predicting the future removal of a ewe from the flock 12 months in advance of the shepherd’s decision. For the Assessments in the Field, indicators which did not necessitate close contact were required. A whole-animal method (Qualitative Behavioural Assessment (QBA) was therefore particularly useful as it can be performed with minimal disturbance. Rather than quantitatively scoring the behaviour patterns of an animal the assessor focusses on how the animal interacts with their environment. This information is translated in to qualitative descriptors such as “calm” or “agitated”. Good interobserver reliability was found when three observers assessed 49 individual ewes on two occasions (W=0.77, P < 0.001). When QBA was applied 13 times to 50 individual ewes over a six-month time period (spanning late pregnancy to post-weaning) four meaningful Principal Components were identified; the first two accounted for more than half of the explained variation between sheep. The two main components were ‘General Mood’ (PC1), describing the overall affective state of the ewe, and ‘Arousal’ (PC2) reflecting energy levels. General Mood scores significantly increased in the post-lambing period compared to pre-lambing observations, and significantly increased again post-weaning (P < 0.001). Ewes were significantly experiencing significantly higher Arousal in post-lambing and post-weaning conditions compared to pre-lambing (P < 0.001), but there was no difference between post-lambing and post-weaning. During the Assessments in the Field data were also collected on: the ewe’s response to human approach, a surprise test, the ewe’s social group size, group demographics and behavioural synchrony. Ewes with lower mood scores tended to have larger distances between them and other ewes (P=0.023). The distance to which a human could approach before the ewe fled was significantly related to Arousal (P=0.05), as ewes in a higher energy state fled from the approaching human sooner than those who were in lower Arousal states. Ewes in social groups with higher numbers of ewe and lamb vocalisations tended to have lower General Mood scores (P=0.014), and lower Arousal scores (P < 0.001) than those in smaller groups. Indicators which met the conditions of feasibility, reliability and validity (for example, those reported above) proved to be suitable for use when assessing the welfare of extensively managed sheep. The effect of time on the reliability of the indicators applied during the assessments have important implications for understanding temporary fluctuations in the animal’s welfare caused by either internal (reproductive state) or external (environmental) factors. These fluctuations may not be representative of a farm’s overall welfare levels in the long term and therefore further careful consideration of the most appropriate time to apply the selected indicators is required.

Teladorsagia circumcincta is a common parasitic nematode of the sheep abomasum, causing reduced growth in young lambs. The widespread development of anthelmintic resistant parasites has driven the need for alternative control strategies. Resistant immunity is acquired through repeated exposure to the parasite. The immune response and clinical outcome vary greatly between animals, but resistance is heritable. The aims of this project were: 1) understand how sheep respond to nematode infection; 2) understand and identify genes associated with the response; 3) identify variation within those genes which may contribute to resistance. Using an artificially infected animal model, transcriptomic analysis in resistant and susceptible lambs identified genes involved in T helper cell polarization as integral to disease outcome. T helper cell (Th)1 and Th17 activation was associated with susceptibility (low antibody, high worm numbers) while a Th2 response was associated with resistance (high antibody levels and clearance of infection). The Th cell transcription factors (GATA3, TBX21, RORC2 and RORA) were sequenced with splice variants and SNPs identified. Analysis of gene expression in the abomasal lymph node identified RORAv2 as associated with susceptibility and RORAv5 as associated with resistance. In the abomasal mucosa, GATA3 expression was linked to resistance. Expression analysis of cytokine receptors expressed by Th cells identified IL17RB and IL17RBv2 as associated with resistance in the abomasal mucosa. Analysis of the SNPs within these genes in 3 naturally infected populations identified a significant association between SNPs in IL23R with weight and FEC. This project has provided an in-depth analysis of the ovine transcriptome and identified several genes associated with the development of resistance to nematodes.

Fasciola hepatica infections cause morbidity and mortality in sheep and have a significant economic impact on farmers. The commonly used diagnostic tests; faecal egg count (FEC), anti-Fasciola antibody ELISA (AbELISA) and the biochemical assays (measuring GLDH and GGT) all have limitations, particularly in detection of pre-patent infections in sheep. A coproantigen ELISA (cELISA) is reported to detect low burdens of infection from 4 weeks post-challenge (wpc) and to only detect current infection. A faecal PCR has been used for early detection of infection, but is limited by inhibitory factors in faecal samples. Loop-mediated isothermal amplification (LAMP) is more resistant to inhibitory factors and has the potential to be a pen-side assay. Triclabendazole (TCBZ) is the drug of choice to treat immature F. hepatica but there have been increasing reports of TCBZ treatment failure in the UK. Treatment outcome is determined using a FEC reduction test (FECRT). A cELISA reduction test (CRT) has recently been proposed. Within this thesis the cELISA, along with FEC, and where feasible the AbELISA and the use of GLDH and GGT concentrations, are evaluated in (1) an experimental challenge model in sheep, (2) individual naturally exposed sheep, in early infection, pre- and post-treatment situations, (3) groups of naturally exposed sheep, including composite samples, in pre- and post-treatment situations and evaluating the FECRT and CRT, lastly a LAMP assay is developed for the detection of F. hepatica, and evaluated against cELISA, FEC and PCR based detection. Two groups of 6 sheep were challenged with F. hepatica metacercarial cysts. In both studies, AbELISA was first to detect infection (3-4 weeks post-challenge (wpc)), followed by cELISA (3-10 wpc) and then FEC (9-10 wpc). Minor fluctuations were seen in both FEC and cELISA levels over both studies and a transient increase in cELISA levels was seen in the first study at 3-8 wpc. All animals were dosed with TCBZ 2 weeks prior to slaughter. The highest FECR was 37% and all sheep had live fluke present in their livers post-mortem. 27 lambs were sampled monthly between June and November with AbELISA, GLDH, GGT, FEC and cELISA tests performed. GLDH and GGT concentrations were above reference ranges from June. AbELISA detected infection in most animals by September and in all but one animal by November. FEC and cELISA both detected some very early positive results, most likely false-positive results, but the majority of animals became positive in November. Twelve lambs were followed to slaughter and all had low burdens of fluke (≤10). A cross-sectional study was conducted including 36 British farms, comprising 812 and 528 sheep pre- and post-treatment, respectively. Low FEC and cELISA results were seen, with better agreement between the two tests pre- than post-treatment. Disagreements between the two tests were more frequently seen where the FEC detected infection but the cELISA did not. This was true both before and after treatment. 80 animals from 2 Scottish farms were confirmed to be infected with liver fluke and given either a TCBZ or closantel treatment and followed for 56 days. A closantel treatment was given to animals that were still infected at 21 days post-treatment (dpt). The highest FECR and CR of the TCBZ-treated groups was 60.3% and 56.4%, respectively, and the lowest FECR and CR of the closantel-treated groups was 83.7% and 94.9%, respectively. A small proportion of closantel-treated animals maintained a low FEC following treatment. Both the FECRT and CRT indicated treatment outcome from 7 dpt. In a postal survey, 41 sample packs were sent to British farmers, of which 25 farmers participated. Samples from 44 and 36 groups were submitted pre- and post-treatment, respectively. Individual and composite faecal samples from each group were tested by FEC and cELISA. Group mean FECs were low and prevalence of infection on farms did not follow a normal distribution. The composite cELISA was more sensitive than the average cELISA, whilst the opposite was true for FEC. The composite cELISA was less sensitive than the composite FEC in low burden situations. A modified version of the composite CRT showed good agreement with the composite FECRT and appears promising in situations where burden was sufficiently high. A faecal LAMP assay, specific to F. hepatica, was developed and evaluated using samples from one of the groups of 6 experimentally challenged animals described above. FEC, cELISA and PCR testing were also performed and compared to the LAMP results. LAMP first detected infection at 3 wpc, followed by cELISA (7 wpc), FEC (10 wpc) and PCR (13 and 14 wpc). The studies within this thesis (1) confirm that cELISA can detect experimental infection of sheep with F. hepatica later than AbELISA but earlier than FEC, and confirm the TCBZ resistant status of a British isolate (Moredun isolate), (2) demonstrate that in animals naturally exposed to F. hepatica, the cELISA does not have an advantage of earlier detection over FEC and is not as sensitive as FEC in established infections (3) show that the modified CRT and composite CRT appear to give a good indication of treatment outcome from 7 dpt, but is of limited use in flocks with a low burden of infection, and (4) demonstrate that a faecal LAMP can detect F. hepatica infection at 3 wpc.

Large animal species, such as cattle, sheep and pigs, have great potential value to scientific research. This is due to their physiological similarity to humans, meaning they make excellent disease models in addition to their inherent agricultural value. However, the efficiency with which such animals can be created has been a critical barrier to their use in bioscience. Research into creating genetically modified large animals has not progressed as rapidly as research on smaller mammals, such as mice, for two main reasons. Firstly, technologies such as pluripotent stem cells, which are well established in rodents, are lacking for large animals. Secondly, large animals cannot produce as many offspring within a given time frame as mice or rats. This, combined with the low efficiencies and lack of precision of current transgenic methods, severely reduces the likelihood of obtaining an animal with a desired genotype within a viable amount of time. Recently, new tools known as ’genome editors’ have been developed to facilitate genetic modification of animals. The vastly enhanced efficiency of these editors in comparison to previous gene targeting methods, combined with the fact that genome editors do not require marker genes to be used, mean that creating genetically modified livestock is now far more feasible. This thesis investigates whether two types of genome editor, TALENs and CRISPR/Cas9, can be used to produce genetically modified large animals for a range of applications. Genome editors were combined with interspecific blastocyst complementation techniques to produce chimeric rodents where the haematopoietic system is partially or fully derived from the donor cells. This work was carried out with a long-term aim of producing chimeric animals which could produce human organs suitable for transplantation. Initial blastocyst complementation experiments were carried out by injecting murine ESCs into wildtype rat blastocysts. One animal resulting from these injections showed chimerism in several tissues. Further experiments were carried out using rat ESCs and mouse blastocysts which were either Runx1-/- or Rag1-/-, however no additional chimeras were identified. In addition to these experiments, TALENs and sgRNAs were designed against Runx1 and Rag1 in sheep and pigs in order to create a large animal model for future blastocyst complementation experiments. Increasing animal productivity is a key step in meeting the demands of an increasing global population and tackling future food insecurities. TALENs and sgRNAs for use in the CRISPR/ Cas9 system were created to target the myostatin gene in sheep. Myostatin is a negative regulator of muscle growth and animals which acquire natural inactivating mutations in both myostatin alleles exhibit a well-characterised double-muscled phenotype, where total muscle mass is about 20% greater than that of a wildtype animal. Embryo microinjections were carried out using both types of genome editor and two edited lambs were produced, one from each editor. The TALEN-edited lamb was mosaic for a deletion of arginine 283 which, upon further analysis of the muscle, did not appear to cause a significant phenotype. The CRISPR-edited lamb was heterozygous for a 20bp deletion, causing the formation of a premature stop codon and severe truncation of the mature myostatin protein. Based on data from other myostatin-knockout animals, including the Belgian Blue cattle breed, this truncated protein is not thought to be functional. To determine if this is indeed the case, the CRISPR-edited lamb is now part of a breeding programme to amplify the edited allele. To discover if genome editors could be applied to create disease-resistant animals, the project focused on foot and mouth disease. Through a literature search and bioinformatic analysis of the bovine and porcine proteomes, three host genes which are cleaved by the virus were identified; eIF4A1, eIF4G1 and IKBKG. TALENs were designed to bind and cut at the FMDV protease cleavage sites in all three genes in order to disrupt protease cleavage and reduce viral replication by slowing viral disruption of the host translation and innate immune response pathways. Although none of the TALENs showed any signs of activity, this thesis sets out some potential directions for future work. In conclusion, this thesis shows that, despite some technical issues, genome editors are a promising technology for the creation of genetically modified livestock.

Sheep and goat formed the foundation of pastoral activity across the Central Asian steppe through the Bronze Age. Theories of pastoral activity have assumed that flocks were uniform in association with ethnic groups that crossed the steppe with new ceramic forms and technologies. This study investigated differences between flocks of sheep and goat across the eastern Kazakh steppe in the Late and Final Bronze Age to elucidate the potential for animal exchange and mobility. Geometric morphometric techniques were applied to archaeological astragali from Ovis aries and Capra hircus. The methods for measurement and analysis were carefully developed to control only for inherited characteristics that relate to environmentally driven adaptations in the movement of the hind limb. Efficiency of movement in this limb is tied to survival and reproductive success of animals. Specimens were selected from three archaeological sites located in different ecozones across the steppe to maximize ecological variability. Geometric morphometric results revealed that flocks of sheep exhibited unique astragalus morphology, indicating that crossbreeding and exchange did not occur between sites. These sites were also subjected to full zooarchaeological analyses to investigate variability of economic subsistence patterns. The total number of species as well as investigations into survival and skeletal body part representation revealed that each site had unique subsistence patterns that were related to local ecological resource availability, despite material culture links. This variability in subsistence patterns and flock uniformity indicate that animal trade was not a feature of steppe networks. Local lifeways were specific to small patches of the steppe, despite overarching shared material cultures.

The U.S. Hair Sheep Index was designed to increase total weight of lamb weaned per ewe lambing (TW) using estimated breeding values (EBV) for direct (DWWT) and maternal (MWWT) weaning weight, and number of lambs born (NLB) and weaned (NLW), as selection criteria. Producers are interested in adding post-weaning fecal egg count EBV (PFEC), an indicator of parasite resistance, to the index to achieve genetic change in both TW and PFEC. First, the effects of adding PFEC alongside TW to the breeding objective of indexes that used either phenotypes or EBV as selection criteria were studied. Since MWWT is not phenotypically recorded, it was combined with DWWT as a selection criterion. The genetic (co)variance matrix among the criteria and goal traits was singular and, thereby, corrected with bending. Indexes were developed using TW as the breeding objective without PFEC as a selection criterion (benchmark), including PFEC as a selection criterion, or adding PFEC as both a selection criterion and goal trait. A scaled economic value (EV) of +3 for TW and -1 for PFEC achieved balanced genetic gains in both traits. Relative to the benchmark, TW retained more than 94% of its genetic response, PFEC favorably decreased by >5%/yr, while ram and ewe selection decisions were similar. To mimic the current United States Hair sheep index, indexes were then developed with DWWT and MWWT as separate selection criteria. Like before, due to its singularity, the genetic (co)variance matrix was modified (bent). The EV of +3 for TW and -1 for PFEC remained the ‘best’ choice: TW retained 97% of its benchmark response, PFEC was reduced by 8.00%/yr, and 73% and 83% of males and females, respectively, were chosen in common with the benchmark. However, index coefficients differed substantially from those in the United States Hair Sheep Index. The coefficient for NLB went from negative to positive while that for NLW was reduced. Those changes were likely caused by the decreased genetic correlation between NLB and NLW after bending. Validation of the effects of applying the revised index on selection decisions in commercial flocks is needed before its implementation by hair sheep breeders.

Breeding programmes that use elite sires with the best estimated breeding values for muscling traits have achieved significant improvement in lamb production in the UK. Further acceleration of the rate of genetic gain for the desirable production traits could be achieved using DNA marker-assisted selection (MAS) breeding strategies. The underlying causal genetic variants associated with improved muscling may be unknown and lying between a cluster of genes known as quantitative trait loci (QTL) or could be single nucleotide polymorphisms (SNP). LoinMAXTM, Texel muscling QTL (TM-QTL) and c.*1232G > A myostatin mutation were genetic variants that reported to be associated with improved muscling characteristics and hence subjected to further analysis in this project. It is essential before incorporating segregating genetic variants in any breeding scheme to comprehensively evaluate their effects on carcass traits. In-vivo scanning (ultrasound scanning (US) and computed tomography scanning (CT)), and carcass video image analyses (VIA) were used in the current studies. Objective VIAprediction weights of the carcass primal cuts could be the backbone of a value-based marketing system that is suggested to replace the current Meat and Livestock Commission (MLC) carcass grades for conformation scores (MLC-C) and fat class (MLC-F). The effect of a single copy of LoinMAXTM QTL (LM-QTL) compared to noncarriers was evaluated in UK crossbred lambs out of Scottish Mule ewes. M. longissimus lumborum (MLL) width, depth and area, as measured by CT scanning, were significantly greater in lambs heterozygous for LM-QTL compared to noncarriers. VIA detected a significant effect of the LM-QTL on the predicted weight of saleable meat yield in the loin primal cut (+2.2%; P < 0.05). The effects of the ovine c.*1232G > A myostatin mutation (MM), found on sheep chromosome 2, on carcass traits in heterozygous crossbred lambs sired by Texel and Poll Dorset rams were studied. Texel crossbred lambs carrying MM had increased loin depth and area. In both crossbred lambs, MM-carriers had significantly higher CT-estimated lean weight and proportion (2 to 4%) and muscle to bone ratios (by ~3%). Poll Dorset heterozygous crossbred animals had higher muscle to fat ratio (28%) and significantly lower fat-related measurements. The c.*1232G > A (MM) mutation as well as TM-QTL effects were evaluated in a different genetic background of Texel x Welsh Mountain crossbreed lambs. Carrying two copies of MM was associated with a significant positive effect on 8 week weight, a negative effect on ultrasound fat depth, a substantial decrease in MLC-fat score, positive impact on VIA-estimated weight of the hind leg, chump and loin primal cuts, as well as the muscularity of the hind leg and loin regions with greater loin muscle width, depth and area. Two copies of MM altered lambs‟ morphological traits with significantly wider carcasses across the shoulders, breast and hind legs and greater areas of the back view of the carcass when measured by VIA. TM-QTL significantly increased US-muscle depth and TM-QTL carriers had significantly greater loin muscle width and area measurements. Comparing TM-QTL genetic groups (homozygote allele carriers (TM/TM), heterozygote carriers of paternal and maternal origin of allele (TM/+ and +/TM, respectively) and homozygote non-carriers (+/+)) and TM-QTL mode of action were then studied. TM/TM carcasses were significantly heavier than non-carriers by 1.6 kg and scored higher conformation values when compared to heterozygote groups only. TM/+ lambs had significantly higher VIA-predicted weight and muscularity in the hind leg and loin, and higher loin dimensions relative to some other genotypic groups. The effect of TM-QTL on some carcass shape measurements was significant. TM-QTL mode of action results on the loin muscling traits supports the earlier reports of polar over dominance. In the light of growing calls to replace the current subjective carcass payment system with the objective VIA system that values the carcass according to the superiority of its cuts, I investigated the ability of US and CT measurements to predict the VIAestimated weights of the carcass primal cuts. Several prediction equations were examined but the best could be achieved when ultrasound measurement, CT linear measurements and live weight were fitted in the model. Since CT scanning information of elite sires is now being used for genetic selection for carcass merit, genetic parameters and genetic relationships between CT scanning measurements and post mortem traits (VIA and MLC-FC) were estimated. However, results were not sufficiently accurate to be of practical use due to lack of data.

In seasonal mammals the duration of nocturnal melatonin secretion accurately reflects the environmental photoperiod. The endocrine rhythm is decoded by a specialised portion of the pituitary gland (the pars tuberalis, PT) which then relays this information to the pars distalis and hypothalamus, precipitating huge annual changes in physiology and behaviour. However how the PT decodes the melatonin signal is currently unknown. Melatonin influences gene transcription in the ovine PT at its onset and offset, and the phase relationship of these two groups is believed to form the underlying mechanism by which the PT integrates seasonal time. The transcripts induced at melatonin offset are understood to be under the control of a seasonally gated cAMP mechanism. Conversely processes involved in melatonin-mediated gene induction are currently not understood.The work in this thesis ultimately aims to reveal how the seasonal melatonin signal is decoded by the PT. To this end melatonin-mediated gene induction has been characterised through RNAseq, the highly displaced cohorts submitted to bioinformatic promoter analysis and the paradigm tested though in vitro modelling techniques.In this study a 1.5 h infusion with melatonin acutely regulated 219 transcripts in the ovine PT (115 induced, 104 repressed, >1.5 fold change), confirming previous association of several genes (including Cry1, MT1, Gadd45g, Nampt and Npas4) to rapid melatonin control. Gross promoter analysis of these groups indicated that the induced gene cohort was significantly enriched for GC content and CpG islands suggesting the involvement of epigenetic mechanisms of transcriptional control. Further bioinformatic analysis specifically implicated the importance of transcription factors ZFP161 and PAX5 in melatonin-mediated gene induction in the PT. Several immortalised cell lines were screened for the presence of a functional melatonin receptor. Two strains (MCF7 oMT1 and NES2Y) exhibited significant attenuation of forskolin-mediated cAMP accumulation when co-treated with melatonin, a hallmark of melatonin Gαi-coupled protein receptor signalling. These lines were subsequently evaluated as models of melatonin-mediated gene induction of the sheep PT through ovine promoter reporter assays of Cry1, Nampt, NeuroD1 and Npas4. However, treatment with melatonin failed to evoke a reporter response suggesting that the cell line models were inadequately equipped to reflect PT biology. Subsequently a protocol was established to culture ovine PT explants culture which faithfully recapitulated melatonin mediated transcriptional dynamics in vitro, providing a possible tool for the future investigation of the PT. Lastly, previous work has shown the transcriptional profile of Npas4 to peak highly and transiently, pre-empting the expression of other melatonin-induced genes. Using a COS7 cell line model, heterologously-expressed NPAS4 was shown to form functional heterodimeric partnerships with ARNT and ARNTL and transactivate both Cry1 and Nampt promoter reporters through novel binding sites. Collectively these data indicated NPAS4 to act as an immediate activator of melatonin regulated circuits

Control strategies against the parasitic nematode Teladorsagia circumcincta are problematic under current sheep management systems. Infection with the parasite, particularly in young lambs, results in significant production losses therefore sustainable worm control is being sought. It has been established that variation in resistance to T. circumcincta is under genetic control and the development of resistance is an acquired characteristic and has an immunological basis. This project investigated the immunological response to infection, of lambs with predicted resistance or susceptibility to T. circumcincta. Specifically, the study aimed to identify immune response-associated genes that were differentially-expressed in resistant and susceptible lambs and attempted to identify mutations in these genes. This study was part of a long term project that aims to identify genetic marker/s to aid in marker-assisted selection (MAS) for resistance to T. circumcincta. Real time reverse transcription-quantitative polymerase chain reaction (real time RTqPCR) was performed on abomasal mucosa and lymph nodes from 55 lambs used in a previous experiment. The lambs had been either trickle-infected with 2,300 infective larvae every two days over three months (infected resistant/susceptible, n=45) or sham-dosed (non-infected control, n=10). Lambs were ranked in relation to faecal egg count (FEC) and adult worm count (AWC) at post mortem; zero or low FEC (resistant) to high FEC (susceptible). Histopathology showed only mild pathological changes in the abomasal mucosa of resistant lambs but heavy lymphocytic inflammatory infiltration in the mucosa and submucosa of infected susceptible animals. Measurements of a range of cytokine transcripts and cell markers associated with the four major CD4+ T cell subsets identified IL6, IL21, and IL23A as significantly increased by at least two-fold in abomasal lymph nodes and abomasal mucosa of susceptible lambs in comparison to resistant animals. Highly significant (P<0.02) positive correlations were found between IL6 (ρ=0.35), IL21 (ρ=0.54) and IL23A (ρ=0.38) transcript levels and AWC. Similarly, there were highly significant (P<0.01) positive correlations between FEC and IL6 (ρ=0.41), IL21 (ρ=0.65) and IL23A (ρ=0.31). In contrast, significant negative correlation (P<0.04) between IL23A with IgA antibody levels (ρ=-0.31) was found. There was also a significant positive correlation (P<0.03) of TGFB1 levels with AWC (ρ=0.42) and FEC (ρ=0.32) in the abomasal mucosa. These data suggests that susceptibility to T. circumcincta is linked to the activation of the inflammatory TH17 T cell subset and that this chronic inflammatory response was inappropriate to clear worm infection. High resolution melt analysis failed to identify single nucleotide polymorphisms in the coding regions of IL21 and IL21R. This is the first report of the involvement of TH17 response in GI worm infection in sheep. Similar gene expression studies involving the known upstream and downstream players of the TH17 response could be done.

The abomasal helminth Teladorsagia circumcincta is one of the most economically important parasites to affect the farming of sheep and goats. T.circumcincta infection is particularly detrimental to lambs, in which it can cause pronounced morbidity and severe production losses. Due to the spreading resistance of this parasite to all currently available classes of anthelmintic drugs, it is having an increasingly severe impact on the sheep industry with significant implications for sheep welfare. Infection of sheep with T.circumcincta triggers local changes in the abomasum characteristic of a T helper type-2 (Th2) driven immune response, including local eosinophilia, mastocytosis and increased mucus production, which leads to expulsion of the parasite. However, this protective immunity develops slowly during repeated exposure, wanes rapidly, and does not appear to be evident in young lambs. Vaccination to provoke early onset of protective immunity has therefore been suggested as an alternative means of control in the face of spreading anthelmintic resistance. Greater understanding of the development of immunity to T.circumcincta, and why this is delayed in lambs, would be useful in vaccine development. This thesis focuses on cytokine transcription profiling of the ovine abomasal mucosa and local lymphatic tissues. Changes in cytokine transcription over the course of a challenge infection with T.circumcincta were defined in helminth naïve sheep, and in previously infected sheep which have developed a degree of immunity during an eight week trickle infection, to clarify the mechanisms by which this immunity is orchestrated. This work demonstrated a clear Th2 cytokine response in the abomasal mucosa over the course of infection, which developed earlier and was more pronounced in the previously infected sheep; possibly owing to a population of polarised Th2-type cells built up during the previous infection. Suppression of Th1 cytokine transcription was also a prominent finding in the draining lymph node, which likewise occurred earlier in the previously infected sheep. Repetition of this experiment using younger lambs provided a possible explanation for the reduced resistance to T.circumcincta in this age group. While Th2 and proinflammatory cytokine responses in the abomasal mucosa demonstrated similar trends to those found in the older sheep, little suppression of Th1 cytokine transcription was observed in the draining lymph node. It is therefore suggested that the increased susceptibility of young lambs to T.circumcincta is not due to an inability to generate adequate Th2 responses, but an inability to suppress transcription of antagonistic Th1 cytokines.

Teladorsagia circumcincta is a major financial burden on the UK sheep farming industry. Disease control is becoming increasingly difficult due to the rapid emergence of anthelmintic resistance. This has prompted the search for alternative, sustainable control measures, including vaccination. Vaccine design would be aided by a thorough knowledge of the mechanisms involved in immunity to T.circumcincta. Most research has focussed on humoral and cellular responses to infection with this nematode. This thesis focuses on the impact of infection with regards to the proteins found locally within the abomasum. Using a well established infection model, proteomic analysis of lymph draining the abomasum was carried out by means of 2-dimensional electrophoresis (2-DE). The identity of many of the proteins in gastric lymph was revealed by means of MALDI-TOF analysis. The relative quantities of the lymph proteins were monitored over time using gel analysis software in both primary infection and immune challenged infection models. This study revealed a number of proteins of interest, including the acute phase proteins serum amyloid A, alpha-1 acid glycoprotein and haptoglobin, as well as the actin depolymerising protein, gelsolin. The effect of infection and immunity to T.circumcincta on these proteins was investigated further by means of biochemical assays, western blotting and real-time PCR. The impact of infection on the permeability of the abomasal mucosa will affect the resultant gastric lymph proteome. This “leak lesion” phenomenon is well documented in T.circumcincta infection but the underlying cause is unknown. Tight junction proteins in the abomasum were studied, using immunofluorescence techniques, in an attempt to define the role of these proteins in this important immunological/pathological event. The aim of this thesis is to contribute to the knowledge of innate immune responses and local pathology occurring within the abomasum during T.circumcincta infection.

In relation to hill sheep, the perception is that they live to a high welfare standard with minimal labour input per ewe, and that there is little reason for concern.  This perception has however not been confirmed by science to date, chiefly due to a lack of research interest into the hill sheep sector.  Meanwhile, extensive sheep farming faces increasing economic pressures, with low returns and falling government subsidy that may compromise animal welfare.  Labour has been shown to be a key input factor for both productivity and welfare in extensive production systems. Despite this, little research has been done to try and optimise the labour input for productivity and welfare. This study is a first attempt to link labour input, productivity and welfare in extensive sheep systems through empirical observation and computer modelling.  The fieldwork was undertaken from 2007 to 2009 on three research farms and three commercial farms.  Labour use was analysed within a number of defined tasks.  Linear programming (LP) modelling was then used to explore eight different scenarios with regard to sheep welfare, labour and productivity, and the potential impacts of specific management changes. Data analysis and model results showed hill sheep live to a high welfare standard according to the low incidence of welfare problems with low risks to productivity, confirming public expectations.  Farmers, are however, aware their attention to an individual sheep’s welfare brings poor returns in terms of flock productivity.  With this in mind, there is some scope for welfare-beneficial or –neutral reductions in labour, particularly if within-farm movement, e.g. between grazing areas, can be reduced.  However, larger reductions of labour, even within legal guidelines, could seriously endanger the welfare of individual sheep, though without great risks to productivity at flock or farm level.  Thus considerable savings in labour might, in some circumstances, reduce costs and hence improve profitability.

A large database of domestic mammal bone measurements from sites across Greenland, Iceland, the Faroe Islands, and the Northern and Western Isles of Scotland is presented. The reasons for variations in bone growth of domestic ungulates are examined in detail; nutrition is identified as a key factor in the determination of adult bone size and shape. Possible sources of variation in bone size in both time and space in the North Atlantic region are identified. Four hypotheses are proposed; firstly that bone dimensions, particularly breadth, will decrease with increasing latitude in the study region; secondly that higher status sites will raise larger livestock than lower status sites within the same time period and region; thirdly the size of domestic mammals in the Northern and Western Isles of Scotland will increase in the Later Iron Age, possibly in relation to increased fodder supply; finally at times of environmental degradation (climatic and/or landscape) domestic mammal size will decrease. The latitude hypothesis could only be partly upheld; there is no evidence for increased size with site status; a small increase in size is noted at some Scottish Iron Age sites and varying results are found for the environmental degradation hypothesis. The results are discussed with particular reference to how changes in the skeletal proportions of domestic mammals affect their human carers and beneficiaries. The potential of further expanding the dataset and integrating biometrical data with other forms of evidence to create a powerful tool for the examination of economic and environmental changes at archaeological sites is discussed.

Experiments were carried out to test the effects of maternal nutrition (0.5 versus 1.0 x maintenance) during early foetal life and peri-ovulatory nutrition on the reproductive performance and associated parameters of Scottish hillbreeds (North Country Cheviot and Scottish Blackface) crossed with Texel sires carrying or not carrying the X-linked Inverdale (FecXI) gene. Undernutrition of the dam resulted in reduced birthweights of single male lambs, reduced plasma progesterone concentrations of ewe lambs and a tendency for reduced ovulation rates in ewe lambs and shearlings (non-carriers) but litter size was not affected. Heterozygous carriers of the Inverdale gene showed a mean increase in ovulation rate of approximately 0.35 as ewe lambs, and approximately 0.65 the following year, the latter resulting in a mean litter size increase of approximately 0.6. Plasma progesterone concentrations were lower for FecXI gene-carriers as ewe lambs and also as adults in a flock that had been flushed prior to mating, suggesting that the increased feed intake could have had a suppressing effect on the concentrations of this hormone. It is postulated that this could have resulted in the large number of peri-natal losses associated with gene-carrier ewes in the same flock, since neonatal vigour of offspring of gene-carriers was not affected in the SAC experimental flock that had not been flushed pre-mating. Presence of the FecXI gene is associated with a reduction in birthweight of approximately 0.5 kg which could be due to the reduced placental efficiency of gene-carrier dams. At puberty and as shearlings gene-carriers remained lighter than their non-carrier counterparts. Throughout the shearling breeding season the increase in ovulation rate associated with this prolificacy gene remained constant.

The studies within this thesis have made a valuable contribution to our understanding of anthelmintic resistance in Scotland and in particular to the prevalence of benzimidazole (BZ) and ivermectin (IVM) resistance, the expression of multiple resistance and its management. Parasitic gastroenteritis (PGE) is a major welfare issue not only for Scottish, UK and European farmers but also for livestock producers throughout the world. Parasites such as Haemonchus, Trichostrongylus and Teladorsagia are estimated to cost the sheep industry hundreds of millions of dollars annually. To date control has largely been achieved using anthelmintics, but over reliance on anthelmintics has led to the development of multi class anthelmintic resistance (AR) and the realization that intensive chemoprophylaxis is not a sustainable approach for the control of nematodoses. The first two papers contributing to this thesis assessed the prevalence of benzimidazole (BZ) and ivermectin (IVM) resistance within ovine gastrointestinal nematode populations in Scotland. The prevalence of BZ resistance in selected Scottish lowland sheep farms was around 24% in 1991 but this had risen to over 80% by 2001. The first cases of ivermectin resistance in sheep were only detected in 2001 but a small scale survey in 2004 showed that 35% of the farms (6 from 17) surveyed had IVM resistance, with Teladorsagia and Trichostrongylus being identified as the resistant genera. The isolation of a triple class resistant T. circumcincta (MTci5) population has enabled research to focus on the important issue of the therapeutic and prophylactic management of this emerging problem. The third and fourth papers detail a series of controlled efficacy tests conducted on MTci5 that confirmed, in the short term at least, it should be possible to use a milbemycin (moxidectin; MOX) or combination treatments, with IVM and one other class of anthelmintic to control nematodoses (>90% efficacy) caused by adult and/or immature worms. However the study examining larval susceptibility highlighted the important role that immature stages can play in the selection and transmission of resistance. Currently there are no tests that can detect the presence of these resistant larval stages. The fifth paper outlines parasitological findings from the farm where MTci5 was isolated following the confirmation of multiple class resistance. Substantial efforts were made to find solutions to maintain sustainability and profitability of the enterprise though ultimately the use of MOX selected for a, predominately Teladorsagia, population against which the persistent activity of the compound was only negligible with the reappearance of eggs in faeces occurring between 21 and 28 days post treatment. Effective sustainable control of AR populations not only requires an understanding of the phenotypic and genotypic mechanisms that underpin resistance but also improved means of ensuring that our farmers are made aware of and utilize identified best practice approaches. The written and verbal responses of the farmers to questions relating to best practice advice (papers six and seven) would suggest that many of the recommendations for delaying the selection and transmission of AR (ACME, Moredun Foundation and sustainable control of parasites of sheep (SCOPS), DEFRA) are not being followed, recommendations such as the effective quarantine treatment of newly purchased animals and dosing animals at the manufacturers’ recommended dose rate were followed by only 20% and 56% of farmers respectively.

In this study, data collected in a survey of Scottish sheep farmers was used to corroborate and augment secondary data available in the literature on sheep ectoparasites and their control in 1999/2000.  The data was used to design and construct a decision tree model, which was used to determine probability weighted profit-maximising control strategies for six flock types/size groups that were representative of Scottish sheep farms.  Organophosphate (OP) based dips, applied in both the autumn and spring/summer, were found to be the profit-maximising control strategy for five flock types/size groups.  The exception was for small (100-ewe) lowground flocks, where two applications of cypermethrin in pour-on formulation maximised the avoidable disease losses. Each of these strategies can give rise to animal welfare, human health and /or environmental externalities.  OP dips can damage human health and the environment but minimise animal welfare losses, and cypermethrin pour-on, while non-damaging to human health and the environment, can result in some avoidable welfare losses, as ectoparasite control is less effective than for OP dips. Using multi-criteria analysis (MCA), the economics of sheep ectoparasite control from a social standpoint has also been examined.  Some profit-maximising control strategies do not necessary maximise social benefits.  A conflict can arise between the farmer and society.  The minimum cost that society would need to be willing to accept in order to finance possible incentives for sheep farmers to switch from the profit-maximising control strategy to strategies that provide different bundles of social benefits is estimated.

In female sheep, the release of gonadotropin-releasing hormone (GnRH) from the hypothalamus triggers luteinizing hormone (LH) to be released from the pituitary. Luteinizing hormone, in turn, acts on the ovaries to elicit estradiol (E2) release. E2 acts in a negative feedback manner to control the amount of LH released. The increase in LH release that drives puberty onset results from a decrease in sensitivity to E2 as the animal ages. However, the neural mechanisms mediating this change in sensitivity are not known, as is where in the hypothalamus E2-negative feedback occurs. In the arcuate nucleus (ARC) of the hypothalamus, a population of neurons coexpressing kisspeptin, neurokinin B (NKB), and dynorphin are critical for reproduction and are thought to be responsible for pulsatile GnRH release. Data from my initial work showed that there is no change in the number of ARC kisspeptin or NKB immunopositive cells, or percent colocalization of the two, with pubertal development. This, in conjunction with a previous finding that the receptor for estradiol, ERa increases instead of decreases within KNDy cells during development, raised the possibility that E2-negative feedback does not directly take place within these cells and that increased production of kisspeptin or NKB is not a limiting factor to puberty onset. However, in my second study, I found that ablating about 75% of KNDy cells with injections of NK3-saporin led to a significant delay in puberty onset. A subsequent experiment in that study also showed that KNDy cells are necessary for the full amplitude of the LH surge, but in a third experiment, ablation of NK3R-containing cells in the ARC was not needed for a positive response to intravenous injections of the NK3R agonist, senktide. Interestingly, in this last experiment, ewes showed clear evidence of normal pulsatile LH secretion despite the delay in puberty onset and previous work in adult ewes showing that ARC NK3-SAP injections in adult ewes blocked pulsatile LH release for at least 4 weeks. This novel work clearly indicated that KNDy cells are critical for timely puberty onset in female sheep but may not be a point of initiation for the decline in E2-negative feedback. Thus, there may be an upstream population or populations of cells that serve to transduce input regarding E2 and other important cues (such as nutritional information) to the reproductive system via KNDy cells. Indeed, in subsequent an increase in KNDy neuron activity was associated with puberty-related changes in LH secretion, further supporting this idea. In another set of experiments, two hypothalamic areas were assessed as possible sites for E2-negative feedback via placement of E2 microimplants into the hypothalamus of ovariectomized ewes or the ERa antagonist ICI 182,780 into the hypothalamus of intact ewes: the preoptic area (POA) and mediobasal hypothalamus (MBH). While the results of this study were not completely clear, they pointed to a role of both the POA and MBH in E2-negative feedback. The E2 microimplants within either area were not able to suppress LH to the level that subcutaneous E2 implants did on their own, indicating that the two areas act in concert, perhaps with other regions of the brain, to communicate E2 input. Further work will be needed to identity these regions. Nutritional state and puberty onset are intimately connected, as animals only undergo puberty onset after reaching a critical energetic state. One pathway through which these metabolic signals are communicated to the reproductive system likely occurs via neuronal populations in the ARC that contain neuropeptide Y (NPY), agouti-related peptide (AgRP), and proopiomelanocortin (POMC). The latter controls satiety and increases energy expenditure, and injection of the POMC analogue a-melanocyte stimulating hormone (aMSH) leads to an increase in LH. AgRP and NPY trigger food intake, decrease energy expenditure, and decrease LH. aMSH and AgRP both act via MC3R and MC4R receptors (commonly referred to as the melanocortin system), which have been observed in KNDy neurons. NPY acts on multiple receptor isoforms (Y1-6), but it is unknown if these receptors are expressed by KNDy neurons. In my final study, POMC and NPY immunopositive cell numbers did not change and AgRP cell numbers decreased significantly with pubertal development. The fiber density for NPY in the ARC through development was also determined, but there was no significant change. The activity of POMC neurons increased with puberty-associated changes in LH secretion, and AgRP neuronal activity decreased. AgRP or NPY contacts onto KNDy did not change with development, but there was an increase in POMC contacts onto KNDy cells. Finally, the percentage of POMC and AgRP that expressed ERa through development did not change. This work adds significant knowledge regarding potential neural mechanisms that regulate ovine puberty. The melanocortin system is involved in receiving and transducing important information on the nutritional state of the individual to KNDy neurons, allowing for a pubertal increase in GnRH and LH secretion that drives puberty onset. KNDy neurons are a critical penultimate input to GnRH neurons and ultimately mediate the decline in sensitivity to E2 that leads to puberty onset. However, where and how E2-negative feedback is harbored in the hypothalamus remains a critical but open question.

The present thesis aims to provide a critical analysis of the immaterial resurrection proposed by the Governing Body of the Watchtower Bible and Tract Society and, in doing so, demonstrates the superiority of the orthodox Christian position on the bodily resurrection because of its consideration of key aspects such as Christology, the resurrection, gospel, and eschatology. The first chapter explains how the Watchtower Bible and Tract Society became a global religious organization. In chapter two, Jehovah's Witness documentation on the immaterial resurrection of Christ is presented, with occasional commentary on issues. Chapter three delves into the historical perspective of the bodily resurrection, building on the minimal facts approach developed by Gary Habermas. The argument demonstrates that the earliest eyewitnesses believed they encountered the risen Jesus in bodily form.

The epidemiology of ectoparasitic mites within the genus Psoroptes infesting domestic British livestock was investigated. The ear mite P. cuniculi was found to be common in sheep, goats, rabbits and probably horses. The body mite P. ovis was endemic within the National sheep flock, but was a sporadic, imported problem for cattle, with the last case caused by P. natalensis and not P. ovis. Equine mange (P. equi) has been eradicated from Britain. With the exception of P. ovis, mites from all other host species were not infestive to sheep. Comparative measurements of the male L4 outer opisthosomal setae revealed no statistical differences between ovine and bovine P. ovis and rabbit P. cuniculi, but P. cuniculi from the ears of sheep and goats, P. equi and P. natalensis were all statistically different from populations of sheep P. ovis and rabbit P. cuniculi. Setal lengths for goat and sheep ear mites were predominantly below 74.0 μm but mites isolated from the ears of rabbits fell into short ("typical ear canker") and long (generally "extra auricular") setal forms. Some "extra auricular" populations of P. cuniculi infesting rabbits were shown to contain sub-populations of P. cuniculi (non infestive to sheep) and P. ovis (infestive to sheep) and these populations could be selected for by ivermectin. A narrow band of bovine and ovine P. ovis divided the two forms of rabbit P. cuniculi. Subclinical, rapid growth and decline phases of disease were recorded for P. ovis infesting sheep, the duration of these phases varying with the virulence of the infesting population. Highly significant differences were observed between setal lengths for low, medium or high virulence population (73.6 μm, 80.2 and 90.5 μm, respectively). The scab mite P. ovis was also recorded in the ear canals of 38.6% of infested sheep, and prevalence was greater the higher the virulence of the infesting population. The majority of infestations were recorded in early rapid growth phase when lesions only covered 11.0 to 43.9% of the body and as far away as the mid back (and as early as 28 days after challenge) and only 22.6 % recorded in the decline phase (lesions 98.9% to 100.0% body cover), when the pinnae themselves could be infested. P. ovis and P. cuniculi are therefore synoxenous, occuring sympatrically on the same host (sheep) and may be syntopic (sharing the same habitat, ie the ear canal). P. ovis and P. cuniculi infesting sheep are not reproductively or ecologically isolated but are phenotypic variants of the same species. Thus populations high in P. cuniculi act almost as commensals of the ear canal and populations high in P. ovis act as highly pathogenic agents of mange. It is also suggested that the type species Psoroptes communis should be re-instated, with two variants infesting sheep, P. communis var ovis and P. communis var cuniculi.

The quantitative relationship between the urinary excretion of TBA and the uptake of PPA and CHCA was examined by continuous intraruminal infusion of PPA (8, 16 or 24 mmol/d) either alone or with CHCA (8 or 16 mmol/d) in sheep nourished by intragastric infusions of all nutrients. The daily excretion of TBA was linearly correlated (r 0.99, P<0.001) with the amounts of PPA and CHCA infused. The urinary recovery of infused PPA and CHCA as TBA was 0.79 (s.e. 0.01). Recovery of infused SA (8 mmol/d) as urinary total salicylic acid (TSA) was 0.89 (SD 0.08). Both TBA and TSA excretions were found to be exclusively of exogenous origin, with negligible faecal excretion. It is concluded that urinary TBA is a potential estimator of PPA + CHCA absorption from the digestive tract. SA may have potential as a marker of urine volume. The relationship between urinary excretion of TBA and forage intake was assessed with steers fed different amounts of fresh herbage from pastures comprising oat, rye, vetch and lucerne. Herbage samples collected daily were incubated in vitro with buffered rumen fluid for 48 h. The PPA production per unit of herbage DM incubated was fitted to the exponential function of time Y = a + [b. (1 - e-c.l)]. Regression analysis showed that the TBA:creatine concentration ratio was a good explanatory variable (r2 0.92-095; P<0.001) of herbage intake. TBA:creatinine concentration ratio divided by the product b.c, both constants from the fitted curves of PPA production of herbage cultures, was another good index (r2 up to 0.98). It is concluded that a method for the prediction of herbage intake for grazing animals based on TBA excretion in urine seems promising.

Artificial insemination (AI) and embryo transfer (ET) in the ewe are severely restricted by the rigid, impassable cervix that resists catheterization. This thesis deals with the ripening effect of intravaginal prostaglandin E2 (PGE2) in the luteal phase ovine cervix. The potential of this drug to induce cervical ripening has been extensively studied in periparturient sheep and other mammalian species to facilitate labour, but not as a mean of softening the cervix during the cycle. Physiological cervical ripening is characterised by macroscopical, ultrasonographical, microscopical, mechanical and biochemical changes. Studies during pregnancy and at term suggest that PGE2 mimics these changes, but there are no studies during the cycle. The sensitivity of the cervix to PGE2 during the cycle may be lower than at term, but there have been no attempts to characterise the PGE2 receptors (EP1- EP4) in the cervix. The ultrasonographic appearance of the cervix has been described in other species, but not in sheep. In chapter V, ultrasound is evaluated in the monitoring of cervical changes during the cycle, the ovine cervix was imaged at oestrus and during the early luteal phase. This was achieved employing the transrectal technique and two probes of 5 MHz and 7.5 MHz (Toshiba) and a linear scanner (Capasee, Toshiba). In chapter III, several sheep in the luteal phase and early pregnancy (n=15), were treated with different regimes of PGE2 alone or together with oestradiol, to induce cervical ripening. Catheterization was incomplete, but deeper scores (4 cm) than in control sheep (0.1-3 cm) were achieved. Following PGE2 treatment on day 6 of the cycle, microscopical changes similar to an inflammatory response and therefore similar to those during labour were observed. There was activation of polymorphonuclear leukocytes (PML), eosinophils, mast cells and platelets. Vasodilatation and extravasation of red blood cells were also prominent. The findings in chapter III, suggested that there was a response to PGE2 during the luteal phase and that receptors for PGE2 may be present in the cervix. The observation of PML degranulation is in line with current theories of physiological cervical ripening. These cells, when activated, release collagenolytic enzymes, like elastase, MMP-8 and MMP-9 capable of softening the cervix by degrading the extracellular matrix. It was hypothesised here that the EP3R, one of the four PGE2 receptor subtypes, may be involved in the activation of PMLs. At the beginning of these studies, the only reagents available for the characterisation of the EP3R in the ovine cervix were a few cDNA sequences in other mammalian species. The first objective therefore was to isolate and sequence cDNA encoding for the ovine EP3R, using RT-PCR techniques and bovine primers. The successful isolation and sequencing of a cDNA from kidney, using RT-PCR, is described in the first part of chapter IV. The sequence of the fragment isolated was highly homologous to the bovine cDNA sequence, indicating very strongly that it corresponded to the ovine homologue. In the second part of chapter IV, mRNA expression studies are described. The same primers as the cDNA isolation were used to study mRNA expression, DNAse I incubations of the RNA were necessary because the fragment amplified was found to be intronless. Using this method, the presence of mRNA was demonstrated in kidney, liver, uterus, adrenal gland and skin. This distribution pattern is similar to other species and further confirms the identity of the cDNA fragment as the ovine EP3 receptor. Expression of this message was also found for the first time in the oestrous and luteal cervix, by RT-PCR and on the peri parturient cervix by Southern analysis. These preliminary data suggest that the EP3R may have a function in the ovine cervix. It is reasoned, based on these findings and other publications, that EP3R may act as a proinflammatory receptor, activating the cellular response observed in chapter III.

The ovine endometrial oxytocin receptor (OTR) plays a pivotal role in both luteolysis and maternal recognition of pregnancy. The purpose of this PhD thesis was to investigate structure, function, localisation and regulation of OTR in light of this role. An ovine endometrial OTR cDNA was sequenced. The encoded receptor was a 391 amino acid polypeptide, 94% homologous to a previously reported human myometrial OTR, and was assigned to the G-protein-coupled seven transmembrane domain receptor family. The ovine OTR contained two extra amino acids compared to the human OTR, located in the third intracytoplasmic loop; the region associated with G-protein coupling. Northern blot and primer extension analysis revealed multiple OTR transcript sizes in oestrous endometrium of 1.65, 2.5, 3.9 and 6.2 kb and a possible transcription start site 90 base pairs upstream of the ATG translation initiation codon. The OTR cDNA was transiently expressed in Cos-7 cells and shown to be functional. Binding parameters for the expressed receptor were consistent with native OTR. Accumulation of inositol phosphates in OTR cDNA transfected Cos-7 cells was measured in response to treatment with ligand, non-specific G-protein analogues (A1F4-, GTP?S), pertussis toxin (PTX) and a phospholipase C (PLC) inhibitor to characterise oxytocin-induced stimulation of PLC activity. OTR mRNA was localised, using in situ hybridisation, primarily to the epithelial cells. It first appeared in the luminal epithelium (LE) on day 14 of the cycle and reached a peak at oestrus. OTR mRNA was absent during early pregnancy, appearing only on day 21 which coincides with the time at which the inhibition of OTR by trophoblast interferon (IFN?) ceases. In situ hybridisation on uterine sections from ovariectomized steroid-treated ewes demonstrated that OTR mRNA was under transcriptional control such that progesterone loses its inhibitory influence in the LE on day 14, whereas development in the myometrium at oestrus is oestrogen-dependent. Oxytocin had no effect on its own receptor at the level of gene transcription, suggesting its reported inhibitory effect occurs at the level of translation or down-regulation of functional OTR.