There are numerous vaccines available for the control of infectious bronchitis virus (IBV) in poultry, however protection is short-lived and poorly cross-protective between strains. The vaccines must currently be grown in embryonated eggs, a cumbersome and expensive process. The ability to grow vaccines on a cell-line such as Vero cells would be highly advantageous. The spike (S) glycoprotein of IBV is comprised of two subunits, S1 and S2, has a vital role in virulence in vivo and is responsible for cellular tropism in vitro. This project aims to identify the amino acids present in the S glycoprotein involved in determination of cellular tropism and cell-to-cell fusion. The IBV Beaudette strain is able to replicate in both primary chick kidney (CK) cells and Vero cells, whereas the IBV M41 strain replicates in primary cells only. Recombinant IBVs with chimaeric S genes were generated using a reverse genetics system with the genomic background of Beaudette and part of the S gene from M41. Their growth characteristics and cellular tropism were investigated. The S2 subunit of Beaudette was found to be sufficient to confer the ability to grow on Vero cells and swapping just three amino acids with corresponding ones from M41 was sufficient to remove the ability of the Beaudette S glycoprotein for growth on Vero cells. Beaudette was further adapted to syncytia formation on Vero cells by serial passage and isolates were sequenced to identify amino acid changes between parent and Vero-adapted viruses that are potentially involved in cell-to-cell fusion. Understanding the way in which IBV infects host cells is vital in order to rationally design better vaccination and treatment strategies and help to reduce the prevalence of IBV infection in poultry worldwide. Using the IBV reverse genetics system, we now have the potential to grow IBV vaccines on Vero cells.

The avian coronavirus infectious bronchitis virus (IBV) expresses four nonstructural, non-gene 1 proteins (3a, 3b, 5a and 5b) which have been shown to be dispensable for virus replication in cell culture. These IBV accessory proteins have no sequence homology to any of the accessory proteins of the group I and II coronaviruses but are highly conserved among the group III coronaviruses. Characterisation of naturally occurring strains of IBV which do not express two or more of the accessory proteins and of genetically modified recombinant IBVs has demonstrated that these accessory proteins contribute at most a minor role to the pathogenicity of the virus. To understand the relevance of these proteins for IBV the subcellular location of the 3a, 5a and 5b proteins have been characterised along with the identification of potential protein-protein interactions for the 3a protein. The subcellular location and protein-protein interactions of the 3b protein were attempted but specific problems were encountered. Indirect immunofluorescence with confocal microscopy of IBV-infected chick kidney cells was used to study the subcellular location of the accessory proteins. The 3a protein displayed a punctate, cytoplasmic distribution pattern which colocalised with virally-induced double-stranded RNA. A diffuse, cytoplasmic distribution was observed for the 5b protein which produced limited colocalisation with an IBV structural protein. Expression of a FLAG-tagged 5a protein in transfected Vero cells resulted in a punctate, cytoplasmic pattern. The protein interactions of the 3a protein were identified using FLAG-tag pull-down experiments with tandem mass spectrometry. Six cellular proteins were identified as interacting with the FLAG/3a protein within transfected Vero cells, three of which, GCN1, PP2A and Exportin-1, may interact with native 3a protein in IBV-infected cells. The 3a protein could sequester the viral dsRNA to hide it from the innate immune system and the potential interactions with three cellular proteins indicate that the IBV 3a protein may contribute to attenuation of host cell translation, induce cell cycle arrest and/or attenuate the nuclear export of a specific subset of mRNAs.

The corpus luteum (CL) is a tissue formed from the remnants of an ovulated follicle in the ovary, and it produces the progesterone needed for a healthy pregnancy. CL growth is highly dependent on a growing nutrient supply, and can be compared with the most aggressive vascular tumours. Angiogenesis, the growth of new blood vessels from existing ones, plays a key role in the growth and function of the CL. Inadequate angiogenesis has been linked to infertility in cows. The CL is composed of several vascular(e.g. endothelial cells (ECs), pericytes (PCs)), and avascular (e.g. luteal cells (LCs), immune cells) cell types, and several pro-angiogenic factors (e.g. Fibroblast Growth Factor 2, FGF2) found to be important in the angiogenic process. The objective of this thesis is to shed light on the cellular and extracellular level determinants of angiogenesis in the bovine CL. We begin with the relevant biological and mathematical literature in Chapter 1. In Chapter 2, an ordinary differential equation model of CL growth is introduced. We assume that the CL volume is a continuum of three cell types, ECs, LCs, and stromal cells (such as PCs). The fourth variable in the model, FGF2, enhances the EC proliferation rate. The model is able, by varying parameters such as the maximal proliferation rate of the ECs, to distinguish cases where the CL shifts from a ‘normal’ to a ‘pathological’ growth. In Chapter 3, we present in vitro CL published and novel studies from Robinson’s Lab. Preliminary results demonstrate interesting endothelial and pericyte behaviours regarding cell aggregation and sprout formation, which are the motivation for the next two Chapters. In these experimental studies, all the CL cell types were incorporated in the same in vitro culture, hence providing a closer approximation to the in vivo environment compared to other in vitro cultures which use only a single cell type (mainly ECs). However, this complicates matters in terms of distinguishing cell behaviours and factors which contribute on the overall cell dynamics. Therefore, in the Chapters 4 and 5 we use data from literature. In Chapter 4, by using the Cellular Potts Model (CPM) framework, we focus on EC-PC interactions, and particularly on the mechanism which is responsible for the EC growth inhibition. Our model incorporates two possible mechanisms for inhibition. That is, the mechanical cell-cell contact inhibition, and the inhibition mediated from diffusive TGF-b secreted once the two cell types come in contact. Interestingly, our model results suggest that the effective range of TGF-b is a crucial determinant of the degree of EC growth inhibition. Chapter 5, by using a CPM, is devoted to sprouting angiogenesis (the formation of new blood vessel). The dynamic interchange between stalk and tip EC phenotype is incorporated through the Notch signalling pathway, with the leading tip cell moving up macrophage-mediated VEGFA gradients in a non-uniform matrix environment. The model reproduces phenomena in sprouting angiogenesis, including sprout morphology, tip competition, and explains knockout experiments on the Notch signalling pathway. Finally, we close with Chapter 6 where we summarise the ain results from each chapter and propose model extensions for future directions.

Recent published work indicates a role for pre-slaughter dietary vitamin D supplementation to promote post mortem meat tenderization in cattle (Montgomery et al., 2000; Foote et al., 2004; Montogmery et al., 2004), pigs (Wilborn et al., 2004) and sheep (Wiegand et al., 2001; Boleman et al., 2004). The hypothesis being that vitamin D supplementation at supra-nutritional levels is able to cause increases in the calcium status of the animals, increasing the activity of the calcium-dependant proteolytic enzymes, the calpains, which are responsible for meat tenderization (Koohmaraie & Geesink, 2006). Muscle fibre type is a variable factor in muscle and is related to meat quality (Klont et al., 1998). Vitamin D has been suggested to play a role in regulating skeletal muscle function through the creation of vitamin D receptor knockout mice models (Endo et al., 2003) and observations that muscle weakness and falling risk in vitamin D deficient patients is linked to a loss of fast muscle fibres (Aniansson et al., 1986; Larsson et al., 1979; Sorenson et al., 1979; Sato et al., 2005). This thesis investigated two vitamin D pre-slaughter diet regimes on their effects on meat quality of the most economically important cut of the carcass, the longissimus dorsi (LD) muscle (Molina et al., 2005) and the expression of the calpain system. Trial 1 fed sheep vitamin D at 2.0 X 10[superscript]6 IU/day for four days prior to slaughter and found that this had no effect on shear force of LD chops. Calcium levels were unchanged but mRNA levels of calpain I and II were increased 3.7 and 10% respectively (P=0.099 and P=0.014) but there was no effect on calpastatin mRNA nor changes in the calpain system at the protein level. Trial 2 fed sheep the same dose of vitamin D for 7 days with an additional calcium bolus, resulting in a 10% increase in calcium concentrations of both serum and LD. Toughness of LD chops was increased (P<0.01), there was no effect on mRNA of the calpain system but there was an increase in the protein levels of calpain II and calpastatin by 16 and 17% respectively (P=0.05 and P=0.087). A microarray study of rat primary myoblasts treated with 1,25(OH)[subscript]2D[subscript]3 for 24 hours highlighted a number of responsive genes significantly up and down regulated 1.5 fold or more (P<0.05). Pathway analysis identified novel targets of 1,25(OH)[subscript]2D[subscript]3 with a possible relationship to muscle growth and function; these included C/EBPβ metallothionein 2A and the MAPK, ERK. Three muscle cell strains, the rat primary muscle cells, L6 Aston and C2C12, were assessed for myosin heavy chain (MHC) gene expression using semi-quantitative PCR and western blotting analysis. The muscle cell line demonstrating the broadest range of MHC genes relevant to mature muscle tissue was used for the final experiments; this was the C2C12 cell line demonstrating expression of the slow MHC 1/β, an isoform which was absent or showed much lower expression in the other cells. C2C12 cells treated with 1α(OH)D[subscript]3 for 48 hours at varying stages of development responded in changes in myogenic regulatory factors (MRFs), MHCs and novel target gene expression. Real time PCR analysis of C2C12 cells treated with the active vitamin D metabolite 1α(OH)D[subscript]3 affirmed C/EBPβ mRNA expression to be upregulated (P<0.001) and MAPK ERK 1/2 phosphorylation to be down regulated (P<0.001) by 1α(OH)D[subscript]3 in muscle cells. The effect of 1α(OH)D[subscript]3 in myoblasts was reduce proliferation and promote differentiation, as myotubes formed the effect of 1α(OH)D[subscript]3 was to promote MHC gene expression of an intermediate oxidative fibre type, increasing expression of MHC 1/β and 2A, decreasing MHC 2B. In conclusion, there is no apparent benefit of a pre-slaughter dietary vitamin D feeding regime on meat quality, but the active metabolites of vitamin D, 1α(OH)D[subscript]3 and 1,25(OH)[subscript]2D[subscript]3, exert changes in gene expression and MAPK signalling which are likely to affect muscle growth and fibre type, and is of relevance in terms of both meat quality and muscle function in the elderly.

A poor maternal diet leads to offspring with a greater risk of developing chronic diseases later in life. This thesis considered whether a low protein diet during pregnancy in sheep affected the development of the fetal kidney, and how this impacted upon adult renal function when challenged by obesity. Pregnant ewes were fed either a control diet or a diet that was isocaloric but contained only 50% of the protein, in either early or late gestation. The effects of the diet were assessed on the ewe, day 65 fetuses (0.44 gestation), and two year old offspring which had been subjected to an obesogenic environment (ab libitum feed and reduced exercise). Few effects were observed on the ewe, confirming that the nutritional insult was relatively mild. Fetal renal vasculature (assessed by vascular corrosion casts) was not different between groups, although the microvasculature was significantly reduced in the early protein group, as evidenced by CD34+ staining of endothelial cells. This was accompanied by a reduction in angiogenic factors compared to control animals. Protein-energy malnutrition in the ewe led to reduced urea in maternal and fetal plasma, along with a concomitant reduction in ornithine in the fetal plasma and amniotic fluid. Other amino acids were relatively unaffected. In the adult sheep there were no effects on long-term renal function in the group fed low protein late in pregnancy, despite the lambs having lower birth weights and a period of postnatal catch up growth compared to the other groups. However, the group fed low protein during early pregnancy had reduced nephron number, microalbumuria and reduced renal function as assessed by gamma scintigraphy. There was also evidence of microvascular rarefaction which may have been exacerbated by obesity. This study did not reveal any consistent sex-specific effects of the maternal low protein diet. This study emphasises the importance of diet quality rather than quantity, and the importance of consuming a well-balanced diet during pregnancy to protect against future chronic diseases.

The growth of ovarian follicles is well documented in terms of hormonal control, however the fluid dynamics of antral follicle growth is less well understood. Aquaporins (AQP) are transmembrane water channels which facilitate the passive movement of water. In mammals 13 AQPs have been identified in a vast range of tissue types. In terms of ovarian AQPs there is currently a paucity of information. Recent studies in rat, pig and human have revealed the presence of ovarian AQPs, but in doing so have also highlighted a lack of consensus on AQP-type and location. The main aim of this study was to investigate the potential role of AQP in antral follicle growth. The first objective was to identify tissue expression and localisation of AQP proteins in the bovine ovary. This required the characterisation of a panel of polyclonal serum antibodies. Immunohistochemistry (IHC) was then used to identify AQPs and to detect changes in protein expression during follicular growth. Aquaporin 1 was found in most vascular endothelium; it was plentiful in capillaries surrounding antral follicles and increased in abundance as vasculature increased with follicle development. Aquaporin 2 was not found in bovine ovarian tissue and the remaining antibodies were deemed too nonspecific to permit reliable conclusions. The second objective was to investigate, via RT-qPCR, mRNA levels of AQPs in granulosa and theca cells isolated from preantral, through to large preovulatory follicles. Transcripts of AQP 1, -3, -4, -5, -7 and -9 were detected in both the granulosa and theca of antral follicles with expression levels generally higher in theca. The expression of AQP 1, -5, -7 and -9 was initiated in the theca cells of early antral follicles. Finally, swelling assays using bovine and porcine granulosa cells demonstrated the ability of granulosa to swell. This was inhibited by HgCb which is characteristic of AQP function. Porcine granulosa cells incubated with androgen swelled by 27%, this effect was inhibited by hydroxyflutamide. Protein analysis of AQP5 via IHC and Western blotting showed possible up-regulation in porcine follicles. RTqPCR did not reveal AQP5 transcript, the reasons for this currently remain unclear. In conclusion, this study has revealed for the first time the involvement of AQPs in bovine ovarian follicle development, with AQPI, -5, -7 and -9 potentially playing a pivotal role in antrum formation. The AQP system in porcine granulosa cells is androgen sensitive however identification of the AQP/s responsible needs further investigation. The evidence from this investigation suggests a role for AQPs in facilitating follicle growth. The stage-dependent expression of certain AQPs and the androgen sensitive porcine granulosa cells reveals the possibility that AQPs may be modulated by follicle-regulating hormones.

Very little is known about cattle vocalizations. The few studies available in the literature have been conducted using animals under stress or very intensive husbandry conditions. Similarly, the individual consistency of behaviour in cattle has rarely been considered except in applied studies of constrained and isolated animals, and no previous research has attempted to address a possible association between vocal communication and temperament in cattle. The studies reported here address these gaps in our knowledge. I found that cattle contact calls have acoustic characteristics that give them individualized distinctiveness, in both adult cows and calves. These results were confirmed using playback experiments, where I found that there is bidirectional mother-offspring recognition, as has been recorded in other “weak hider” ungulates. Additionally, using visual and acoustic stimuli, I assessed individual cattle temperament. The results showed that there was no individual behavioural consistency in responses to a novel object presentations. However, calves behaved consistently more boldly than cows. Furthermore, there was significant individual consistency in responses to vocalisations of heterospecifics, when they were played back through a speaker in the field. Surprisingly, no correlations were found between the ability of cattle to identify their own mother/offspring and the acoustic features of their vocalisations, or behavioural responses in any other context. There were, however, significant correlations between one characteristic of vocalisations in adult cows (formant spacing) and the boldness of behavioural responses to both novel objects and auditory stimuli. Additionally, higher F0 in calf contact vocalizations correlated with boldness in the auditory stimuli experiment. These relationships imply that vocalisations may encode information about individual temperament, something which has rarely been documented. Surprisingly, no strong correlations were found between the behavioural responses to visual and acoustic stimuli, suggesting that individual consistency in behaviour across contexts was limited, and that behavioural plasticity could play an important role in determining responses in different environmental contexts. Overall, my results contribute to our knowledge of animal communication in mammals from a bioacoustic point of view, and they are also potentially relevant to studies of vocalizations as indicators of cattle welfare.

An investigation of 5186 hock maps, from 3691 cows with hair loss on the hocks from 76 UK dairy farms, identified six risk factors associated with area of hair loss: higher locomotion score; poor cleanliness; higher mean milk yield; low body condition score, prolonged winter housing and certain combinations of stall base and bedding materials. Another finding was significantly poor correlation between a categorical scale and the continuous measure of hair loss. Hair loss on the hocks of 70 heifers (three herds) was observed monthly from Sep 2008-Mar 2010. The results imply that lameness precedes hair loss on the hock and not vice versa, i.e. lame animals develop hair loss rather than hock hair loss leads to future lameness. Poor cleanliness score and higher milk yield were associated positively with the risk of having hair loss on the hock. Based on the total 6889 observations from 731 cows in four herds, cows with a greater decrease in BCS (compared to BCS at calving) had higher probability of becoming lame and lower probability of recovering. Also, increase in BCS from calving was associated with lower probability of cows moving from non-lame to lame state and higher probability from lame to non-lame. Days of lactation, months of calving and parity impacted upon both non-lame to lame and lame to non-lame transitions. Analysis of 678 cows from four herds found that cows with chronic lameness had a lower probability of conception and a longer calving to conception period compared with healthy cows. Further, cows with lower average BCS had a lower probability of conception and were more likely to be lame. However, these factors didn’t influence the likelihood of an individual AI leading to pregnancy.

Bovine tuberculosis (bTB) is one of the principal concerns currently facing the livestock industry in England. The disease has spread dramatically in recent years and is costing the country millions of pounds each year. Tens of thousands of cattle are being slaughtered annually; a huge financial and emotional burden to affected farmers. While various measures to control the disease have been taken, none have been successful in bringing it under control. Instead bTB continues to spread unabated. The essence of the bTB problem is that it necessitates industry buy-in in order to implement disease control measures. It is therefore not simply an issue of regulation. Current government bTB control policy emphasises communication and cooperative working across the government and the farming industry, coupled with cost and responsibility sharing. However, previous studies have shown that relationships between farmers and the government are already strained, engendered by a sense of distrust and a lack of confidence. Although some social science work has been conducted within the field of disease control and particularly bTB, the majority focuses on farmers‘ attitudes towards government policy and disease control. However, in order to implement successful disease control measures it is necessary to explore the ways in which farmers currently respond to bTB, and how their responses may be recognised by, and incorporated into, successful policy. While previous research has identified the important role of the wider social context in influencing farmers‘ attitudes and behaviour, no studies have yet provided an in-depth analysis of farmers‘ social networks in relation to bTB. In response, this study uses the lens of social capital to explore the various social ties which enhance or constrain farmers‘ capacity to respond to bTB. An iterative, mixed methods approach is adopted across two phases of research. The first incorporates twenty in-depth qualitative farmer interviews, exploring various aspects of bTB risk and response strategies as well as the core features of social capital. This informs a second, quantitative phase, in which data are iii gathered through a self-completion postal survey of 374 farmers in the South West of England. A farmer segmentation model is developed using factor and cluster analysis and two farmer groups are identified. The first group represents vulnerable farmers who are concerned about the negative impacts of bTB, and who are internally focused with respect to their networks. Characteristically, they exhibit strong relationships with others from within the farming community. In comparison, the second group are more resilient and less concerned about the impacts of bTB on their farm business. These farmers are externally focused, mainly seeking information from the government, the National Farmers‘ Union and their vet. The role of various forms of social capital is explored and an important distinction between the two farmer groups is found. Vulnerable farmers tend to be members of close networks of other farmers (bonding social capital), while resilient farmers are more likely to enjoy positive relationships with those from outside the farming community including vets (bridging social capital) and the government (linking social capital). However, while the research findings suggest that bridging and linking social capital can positively influence farmers‘ attitudes towards bTB, they do not necessarily lead to positive disease control behaviour. Statistical analysis of the data reveals no significant differences between the farmer groups in terms of their uptake of biosecurity measures, which represents an important disease avoidance strategy. A disjuncture between farmers‘ attitudes and their behaviour is therefore identified. The research concludes that investment in social capital between the government and farmers should form a core area of policy through providing opportunities for consistent and regular contact, allowing for the development of trusting and productive relationships. The current situation, characterised by low levels of trust and limited uptake of recommended disease control measures by farmers, indicates incoherence with contemporary policy discourses. A better understanding of the role of social capital in influencing farmer attitudes and behaviour will enable policy makers to increase the ability of farmers to respond to bTB risk, either through disease avoidance or through more effective management and coping mechanisms.

Clinical reasoning is the skill used when veterinary surgeons make a decision regarding the diagnosis, treatment plan or prognosis of a patient. Despite its necessity and ubiquity within clinical practice, very little is known about the development of clinical reasoning during undergraduate training. Even less is understood about how veterinary schools should be helping students improve this skill. The aim of the research presented within this thesis was to, firstly, examine the development of clinical reasoning ability within veterinary students and, secondly, to investigate possible methods to aid this process. The University of Nottingham School of Veterinary Medicine and Science (SVMS) was used as a case study for this research. In study one, focus groups and interviews were conducted with SVMS staff, students and graduates to investigate the development of clinical reasoning. A curriculum document content analysis was also performed. The findings suggested that clinical reasoning development is not optimal, with alumni facing a steep learning curve when entering practice. These results were used to design study two, in which a simulated consultation exercise utilizing standardised clients was created and implemented for final year students. The success of the simulation was measured using both quantitative and qualitative methods – all of which supported the use of the session for clinical reasoning development. The final study, also building on the findings of study one, aimed to improve the accessibility of veterinary surgeons’ decision-making processes during student clinical extramural studies placements (CEMS). A reflective Decision Diary was created and trialled with third and fourth year SVMS students. Diary content analysis showed the study aim was met, triangulated by survey and focus group findings. During the research, wider issues relating to clinical reasoning integration into veterinary curricula were unearthed. These included low student awareness of the subject and the misalignment between the skill learnt during training and the skill required when in practice. Several recommendations have been made to improve the design of the undergraduate curriculum in relation to clinical reasoning.