There is considerable interest in the beneficial role of dietary polyunsaturated fatty acids (PUFA) on reproduction in ruminants. Detailed information regarding the mechanisms behind this beneficial effect is limited. The main objective of this thesis was to test the effects of dietary supplementation with omega-3 (n-3) or -6 (n-6) PUFA on gene expression, fatty acid (FA) composition and steroidogenesis in granulosa and theca cells and pre-implantation embryo development. A previous study in our laboratory reported increased follicular-fluid progesterone concentrations in ewes fed an n-3 compared to an n-6 PUFA-enriched diet, but detected no differential effect of n-3 and n-6 PUFA enriched high-density lipoproteins (HDL) on granulosa cell steroidogenesis in vitro. Also, n-6 PUFA enriched HDL reduced early embryo development, but in the absence of a net uptake of FA. In view of these observations it was hypothesised that (i) effects of n-3 PUFA on ovarian steroidogenesis are mediated by theca rather than granulosa cells and (ii) during embryo culture lipids are acquired solely from the albumin fraction of serum, so that albumin delivered n- 3 and n-6 PUFA would exert a greater differential effect on embryo development than either LDL or HDL delivered PUFA. Initial investigations into granulosa cell gene expression profiles using an ovine gonad-targeted cDNA macroarray were unsuccessful, highlighted by subsequent qRTPCR analysis. A thorough investigation confirmed that inconsistencies were due to poor array hybridisation. In vitro data confirmed that n-3 PUFA, via delivery by HDL, increase progesterone production solely in theca cells and that this is associated with an increase in STAR transcript expression. We also demonstrate that albumin is the only serum fraction that leads to a net uptake of FA during embryo culture. PUFA enriched serum and albumin accelerated the development of embryos and increased the yield of morphologically poorer quality blastocysts with increased transcript expression for the antioxidant enzyme SOD1. Important differential effects of n-3 and n-6 PUFA on ovarian steroidogenesis acting solely on theca cells are identified, but differentially effects of PUFA on embryo development are less apparent.

The aim was to assess the impact of milk somatic cell count (SCC) during the first lactation on the lifetime milk production of cows, and therefore estimate potential savings through heifer mastitis control. Cow level SCC over the first lactation was summarised as SCC between 5 and 30 days in milk (SCC1), and the geometric mean and variance of first lactation SCC. The impact of SCC1 on cumulative milk yield over different time periods was assessed for cows in Irish, English, and Welsh dairy herds. The impact of SCC1 and the geometric mean and variance of first lactation SCC on lifetime milk yield, and the association between SCC1 and disposal risk were assessed for cows in Irish dairy herds. Increase in SCC throughout the first lactation was associated with large reductions in the milk yield of cows, and increased disposal risk. Bayesian micro-simulation was used to demonstrate the impact in different herd scenarios. This was extended to synthesise evidence on potential savings using previous research, to estimate the economic impact of specific interventions to reduce the prevalence of cows with high SCC1. There was considerable variation between herds in the apparent impact of SCC1 on SCC throughout the first lactation, indicating the importance of a herd specific approach to control. ‘Cost effectiveness’ of interventions to reduce the prevalence of cows with high SCC1, was found to be highly dependent on the willingness of decision makers to pay for control measures. Increase in herd size was associated with increase in cow SCC, highlighting a need for improved management of mastitis when expansion is planned. An important component of this should be through monitoring and control of mastitis in heifers, especially those in spring-calving Irish dairy herds.

Milk recording consists in the regular, usually monthly, collection of a milk sample from all the lactating cows of a dairy herd. A large sample of milk recording data collected in England and Wales between 2004 and 2006 was used in this thesis. A sample of 8,211,988 recordings in 2,128 herds, representing 16 % of the dairy herds in activity in December 2006, were described and analysed. Calvings followed a seasonal pattern with 80 % more calving in September than in May. Milk production was highest in May (26.5 kg) and lowest in October (24.1 kg). Butterfat was stable, close to 4 % from October to March and reached a minimum at 3.7 % in June and July. Protein stayed between 3.2 and 3.3 % all the year. Geometric mean somatic cell count was between 177 and 180 between October and March and reached 205,000 cells/mL in July and August. At the individual cow level, the mean milk yield, percentage of butterfat, percentage of protein, fat to protein ratio and somatic cell count (geometric mean) were 26.4 kg, 3.96 %, 3.29 %, 1.21 and 90,000 cells/mL, between 5 and 305 days in milk. Changes in individual cow somatic cell counts around a threshold of 200,000 cells/mL between consecutive recording dates were used to predict bulk milk somatic cell count at both the herd-year and test-day levels. The main contributors to bulk milk somatic cell counts were cows staying above the threshold for 2 consecutive test-days. Milk yields and composition at the start of lactation were used to predict the calving to conception interval. Higher milk yield, lower percentage of protein, lower percentage of lactose, higher somatic cell count and higher percentage of butterfat were associated with lower probabilities of conception before 145 days in milk.

Oocyte cryopreservation represents one of the most recent developments in the field of reproductive technologies. However, despite of significant progress, the efficiency of oocyte cryopreservation is still very low. Cryopreservation of mature metaphase II (MIl) oocytes has been reported to induce disorganization of the meiotic spindle and chromosome damage. However, cryopreservation of immature oocyte at germinal vesicle (GV) stage may provide an alternative which avoids these problems. Slow freezing protocols have more recently been replaced by vitrification approaches. In this thesis, recovery, viability and subsequent developmental potential following in vitro fertilisation (IVF), parthenogenetic activation or somatic cell nuclear transfer (SCNT) of ovine oocytes vitrified at GV stage and matured in vitro were studied. Solid surface vitrification (SSV) and cryoloop technologies share the advantages of using a containerless system and small volumes of solution (less than t J.ll) which favours rapid cooling. Maturation, fertilisation, cleavage and blastocyst development were significantly decreased in SSV vitrified oocytes as compared to controls. Following cryoloop vitrification, frequencies of in vitro maturation (43.4 vs 63.2%), oocytes with normal spindle and chromosome configuration (50.0 vs 70.4%) and fertilisation (54.0 vs 74. t %) did not differ significantly between vitrified and control oocytes. Numbers of cleaved embryos that developed to the blastocyst stage following IVM/IVF/IVC did not differ significantly between vitrified and control groups (29.4 vs 45.1 %). In vitro matured ovine oocytes vitrified at GV stage using cryoloop were activated by two different protocols (I) a combination of calcium ionophore (A 23187), cycloheximide and cytochalasin 13 (CA+CHX/CI3), (2) strontium and CB (Sr/Cll). No blastocysts developed in vitrified oocytes activated by CA+CHX/CB; however, 3.8% were obtained following Sr/CI3 activation. Developmental competence of ovinc oocytes vitrified at GV stage and used as cytoplast recipients for SCNT was evaluated. Although the frequencies of cleaved embryos were significantly decreased in vitrified oocytes as compared to control, development to morula and blastocyst stage embryos was not significantly different. No significant differences were observed in total cell numbers, number of apoptotic nuclei as detected by Hoechst and TUNEL assay and proportions of diploid embryos in day 7 blastocysts produced following IVF or seNT of vitrified oocytes as compared to control. Pre-treatment of ovine GV-oocytes with cytochalasin 13 (7.5 J.lglml for 60 min) or demecolcine (0.1 flg/ml for 20 min) prior to vitrification improved frequencies of maturation, fertilisation and subsequent development following IVF or parthenogenetic activation. Caffeine treatment during IVM (10 mM for 6 h) increased the frequencies of blastocyst development in vitrified/thawed GV ovine oocytes. Taken together, these studies suggest that, ovine oocytes vitrified at GV stage can be matured, fertilised and develop in vitro to blastocyst stage embryos. Cryoloop vitrification resulted in higher maturation, fertilisation and subsequent development as compared to SSV. Strontium can be used effectively for parthenogenetic activation of vitrified/thawed ovine GV oocytes. Ovine oocytes vitrified at GV stage can be used effectively as cytoplast recipients for SCNT.

Salmonella enterica subspecies enterica (S. enterica) infection remains a global problem in a wide range of animals and in man. Poultry-derived food is a common source of human infection with the non-host-adapted Salmonella strains while fowl typhoid and pullorum disease are serious diseases in poultry. Development of novel immune-based control strategies against Salmonella infection necessitates a better understanding of the host-pathogen interactions at the cellular level. This study characterizes, in vitro and in vivo, the immune responses that develop following infection of avian species with typhoid and non-typhoid Salmonella serotypes. Salmonella serovars Typhimurium, Enteritidis, Hadar and Infantis showed a greater level of invasion and/or uptake characters to both chicken macrophages (HD11) and chicken kidney epithelial cells (CKC), when compared with S. Pullorum or S. Gallinarum. Nitrate and reactive oxygen species were greater in Salmonella-infected HD11 cells compared with the non-infected controls. HD11 cells revealed higher mRNA gene expression for CXCLi2 (IL-8), IL-6 and iNOS genes in response to S. Enteritidis infection when compared to S. Pullorum-infected cells. S. Typhimurium- and S. Hadar-infected HD11 showed higher gene expression for CXCLi2 versus S. Pullorum-infected cells. Higher mRNA gene expression levels of pro-inflammatory cytokine IL-6, chemokines CXCLi1 (K60) and CXCLi2 and iNOS genes were detected in S. Typhimurium- and S. Enteritidis-infected CKC followed by S. Hadar and S. Infantis while no significant changes were observed in S. Pullorum or S. Gallinarum-infected CKC. Epithelial cell response and production of pro-inflammatory cytokines and chemokines were greatly influenced by Salmonella virulence markers, including Salmonella pathogenicity island type-1 (SPI-1), SPI-2 and bacterial flagella. In chicken infections, S. Enteritidis and S. Infantis colonized the caeca more efficiently than S. Gallinarum and S. Pullorum. High numbers of B-lymphocytes and macrophages were observed in the caecal tonsils of infected birds. S. Enteritidis infection in newly hatched birds elicited the expression of CXCLi1 and CXCLi2 chemokines in the caecal tonsils, while S. Gallinarum up-regulated the expression of LITAF. In older chickens, S. Enteritidis infection resulted in a significantly higher expression of CXCLi2, iNOS, LITAF and IL-10 while S. Pullorum appeared to down-regulate CXCLi1 expression in the caecal tonsils. Data from spleens showed either no expression or down-regulation of the tested genes. In conclusion, data from the present study provide further insights on the interaction of Salmonella with poultry, and while both S. Typhimurium and S. Enteritidis are strong inflammatory serotypes, S. Pullorum and S. Gallinarum are not.

Chicken meat is an important source of high quality protein in the diet of most people in the world. Consequently, the increasing demand for this meat has made chicken meat production the most important growth sector among other meat species. This has been achieved by a half century of intensive genetic selection for growth traits; however, this was coupled with poor reproductive performance of broiler breeders. Ross 708 represents a broiler breeder strain that has been developed for breast-meat yield, and has been reported to exhibit poor reproductive performance in comparison to Ross 308, a typical broiler breeder strain. Accordingly, the current study investigated key points involved in the reproductive process that might influence variation in reproductive performance. Ovarian follicles number was the first point to investigate, as they are the main material of the egg. Liver fatty acid profiles were also investigated in order to identify lipid metabolism and the efficiency of dietary fat utilisation, as the liver is the main site that supplies different body tissues with fatty acids. Carcass fat content was also examined as its negative relationship with reproduction is well documented. Finally, the content of calcium in the tibia bone was examined to identify whether variation in egg production was associated with differences in the metabolism of this element. Accordingly, broiler breeder females from Ross 308 and 708 strains, reared under the standard production system on two different commercial farms of PD Hook, were collected throughout the reproductive cycle; starting at 25-week-old and in five weeks interval until 55-weeks of age. Findings showed no difference in the number of both the large yellow follicles (P=0.332), and the small yellow follicles (P=0.134); whereas the number of large white follicles was higher in the 708 ovaries (P=0.005). Differences in lipid metabolism were identified with a strong tendency for the 708s towards having lower content of linoleic acid (P=0.056) in addition to significantly lower a-linolenic acid (P=0.005). Of particular importance is the latter fatty acid as it is the precursor to (n-3) fatty acids, some of which were found to be less (P<0.001) in the 708s including docosapentaenoic (DPA) and docosahexaenoic (DHA). The importance of these fatty acids in follicular maturation is well documented in addition to the importance of the linoleic acid; these findings indicate that the 708s were not receiving adequate levels of the essential fatty acids which might have contributed to their poor reproductive performance. The 708s also laid down significantly more fat (P<0.001) in comparison to the 308s which could be another factor that has impaired their reproduction performance. This could indicate different levels of metabolic hormones which, in turn, have been found to act in concert with the reproductive hormones. 708s also exhibited a trend towards lower content of calcium in their tibiae, with an age by strain interaction and thus suggesting a difference in the metabolism of this element at some ages. The current study has addressed changes of the investigated parameters with age, but the effect of genetic selection on reproductive performance was difficult to address. Rather, some physiological differences have been identified; 708s were found to be receiving inadequate amounts of essential fatty acids, calcium content was found to be less at some ages and they exhibited a higher content of carcass fat. All these factors have the potential to contribute to poor reproductive performance, and once they are taken into consideration better assessment for the effect of the continued genetic selection for more growth traits can investigated.

Human epidemiological studies have indentified obesity as an independent risk factor for renal disease. In addition, maternal nutrient restriction (MNR) during gestation results in a series of adaptations that may predispose those offspring to obesity and hypertension. Recent reports demonstrated that obese sheep exposed to MNR during early to mid gestation have a predisposition to ectopic lipid deposition in the heart and a rise in necrotic adipocytes, which are markers of severe metabolic dysfunction. Surprisingly, in this model of MNR, the renal tissue of those offspring showed an apparent reduction in cell apoptosis. However, renal diseases associated with obesity have a slow progression and their mechanisms are not completely understood. In the light of these results, the main hypothesis of my thesis is that the renal amelioration observed in those nutrient restricted (NR) obese offspring is a product of post-injury responses, inducing scarring and others adaptations to obesity. Therefore, some of the main regulatory factors in renal and perirenal adipose tissue (PAT) development were analysed in seven-day-old and one-year-old obese sheep offspring exposed to MNR (3.5 KJ/days) from 30 to 80 gestational days (term ≈ 145 days). At one week of age, the renal composition and gene expression showed small changes between NR offspring and those born to control mothers. However, in PAT of NR offspring, an increased in expression of the methyltransferase DNMT-1 and a decrease in mRNA abundance of IGF-2 were observed. At six months of age, obesity onset was accompanied by raised plasma cortisol and leptin concentration in NR offspring compared to control. By one year of age, whilst plasma leptin concentration was similar between the obese groups, in the PAT of the NR offspring there was an increase in gene expression of pro-inflammatory factors and DNMT-1, suggesting advanced adipose tissue remodelling. In kidney, regardless of in utero diet, obesity induced similar amounts of oxidative stress, activation of cellular proliferative factors and collagen deposition. Although, both obese groups had equal activation of pro-apoptotic factors (e.g p-53 and Bax), renal iron and mRNA abundance of the death receptor, Fas only increased in obese offspring born to control fed mothers. A major finding in the NR kidney was increased ectopic triglyceride deposition, indicating early onset post-injury in response to sympathetic activation and lipotoxity. The main conclusion of my thesis is that functional changes observed in the adipose tissue lead the kidney to an initial cycle of cell proliferation, apoptosis and arrest, followed by tissue remodelling, characterised by the presence of collagen. However, this adaptation to obesity is accompanied by an increase in lipid deposition in the kidney of the NR group that may be a sign of an advanced state of metabolic dysfunction.

BACKGROUND Chronic respiratory inflammation is a major cause of recurrent airway obstruction (RAO) in mature horses. RAO has aetiological and clinical similarities to human asthma. Remodelling of airway tissue after bronchial inflammation is evident in RAO and human asthma. Severe asthma in humans is associated with defective lipoxin A4 (LXA4) synthesis and abnormal expression of specific lipoxin receptor (ALX). Arachidonic acid metabolite LXA4 modulates acute inflammation in a number of species and models of acute inflammation. Dysfunctional LXA4 synthesis and/or expression of the ALX receptor may contribute to the chronic inflammatory response seen in asthma. Abnormal LXA4 and/or ALX expression may also be present in horses with RAO thereby promoting airway remodelling. HYPOTHESIS Equine inflammation and its resolution can be modelled in vitro using respiratory epithelial and smooth muscle models. AIMS To develop an in vitro equine respiratory model of respiratory epithelium and airway smooth muscle. Characterise the response of this model to exogenous lipolysaccharide (LPS) and lipoxin A4 on selected molecules associated with inflammation and inflammatory resolution. METHODS Primary equine tracheal epithelial (ETE) cells were obtained from trypsindissociation of tracheal epithelial tissue derived from healthy horses with no sign of inflammation at post-mortem. Primary airway smooth muscle (ASM) cells were cultured from explants of equine trachealis muscle. Near confluent (70-75%) ETE and ASM cells were stimulated with 0.1, 10 and 100µg/ml LPS at 4 and 24 hrs (ETE cells) or 12, 24 and 72hrs (ASM cells). Expression of COX-2 mANA in these cells was used to determine a suitable time point and LPS concentration to induce inflammation. Inflammatory resolution was then examined by comparing the selected time points and LPS concentrations with the response of ETE and ASM cells to 15 minutes incubation with 100µM LXA4 and LXA4/LPS treatment. Finally, the inflammatory relationship between the epithelium and smooth muscle layer was examined by a co-culture model of ETE and ASM cells. Conditioned media from ETE cells treated with 0.1µg/ml LPS, 100µM LXA4 and LXA4/LPS treatment for 24hrs was used to culture ASM cells for a further 24hrs. To examine inflammation and its resolution, selected genes, namely ALX, toll-like receptor 4 (TLA-4), tumour-necrosis factor alpha (TNF-α), interleukin-1 beta (IL-1β), inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) were amplified and quantified by real time PCR. ALX and COX-2 proteins were monitored by Western blot and nitric oxide (NO) levels measured by fluorometric analysis. Values for statistical analysis of ETE and ASM cells were obtained with a two and one-way ANOVA and Tukey's multiple comparisons test. RESULTS Primary ETE and ASM cells were identified by positive immunocytochemical staining with pan cytokeratin-26 and alpha-smooth muscle actin respectively. Treatment of ETE cells with 0.1µg/ml LPS for 24hrs increased iNOS and COX-2 mRNA levels, and significantly increased mRNA expression for ALX, TLR-4 TNF-α and IL-1 j β mRNA (p values <0.05). ASM cells incubated over 72hrs incubation with 0.1119/ml LPS showed increased expression of selected genes but only significant increases in COX-2 mRNA were observed (p values <0.05). Incubation of ETE and ASM cells with LXA4 did not significantly increase or inhibit inflammation as measured by real time PCR, Western blot and fluorometric anlaysis. Western blotting showed some inhibition of COX-2 protein in ASM cells but not ETE cells at 72hrs after LXA4 treatment. Fluorometric analysis of NO levels in ETE and ASM cells showed no significant difference after treatment in either cell type. No noticeable evidence of inflammation or inhibition of inflammation was observed in the co-culture model of ETE and ASM cells. CONCLUSION It was concluded that an in vitro equine model of respiratory epithelium and smooth muscle was successfully established. It was possible to induce partial inflammation in ETE and ASM cells but inflammatory resolution could not be definitively shown in either cell type.

This thesis presents research on the dynamics and control of porcine reproductive and respiratory syndrome (PRRS) caused by the PRRS virus (PRRSV) in the pig population of Great Britain (GB). The roles of the metapopulation of pig herds (metaherd) and individual herd characteristics are examined, and different control and intervention strategies assessed. A novel stochastic model of a metaherd was created, incorporating the births, deaths, slaughter, culls and movement of pigs within and between herds. The metaherd was structured to have characteristics representative of the GB metaherd: the distribution of herd sizes, ‘source’ herds per herd, and numbers of pigs moved per movement. The metaherd was arranged into a typical pyramidal structure. A stochastic infectious process of PRRSV was included. Herd size was found to be key to within herd persistence of PRRSV, with infection failing to persist in smaller (~250 sow) herds. Fadeout did not occur in larger herds once infection established in the rearing herd. PRRSV reduces productivity of herds and the metaherd. There was variability in productivity both between herds and within herds over time. The number of source herds did not influence the dynamics, persistence or prevalence of infection within a herd. Breeding herd production was further decreased by PRRSV when the herd also had a rearing herd (breeder finisher). The model was extended to test the effects of control and intervention strategies. Vaccination effect increased with herd size, and reduced variability in production. Vaccination in small herds was ineffective in increasing production due to PRRSV failing to persist regardless of vaccination. Vaccination of the breeding herd produced higher gains per vaccine dose than vaccination of the rearing herd only. Vaccination of the rearing herd only resulted in higher total herd and metaherd gains, with less variability. Partial de-population combined with vaccination increased the probability of increasing herd performance unless the herd was small (<100 sows) or very large (>1000 sows). Results highlighted the value of modelling to support the decisions of individual farmers to vaccinate and partially depopulate, showing that the optimal decision is influenced by the herd size. Results also demonstrated that the decision to introduce interventions is different for individual farmers.

Constructive alignment (CA) describes an approach to education where teaching, learning and assessment are aligned to allow the learner to achieve the intended learning outcomes (ILOs) of the course. Assessment has a strong influence on learning and therefore the potential to have either a positive or negative impact on CA. The aim of the research in this thesis is to explore the effects of assessment on CA. The context for the research is the final year of study within the School of Veterinary Medicine and Science (SVMS), at the University of Nottingham. Five mixed methods studies were conducted utilising questionnaires, semi-structured interviews and focus groups. In study one the ILOs of the course were defined and subsequently used in study two as a framework for an alumni survey to determine how well prepared graduates feel for clinical practice. Studies three and four investigated the impact of assessments on learning behaviour, namely multiple-choice questions (MCQ), directly observed procedural skills (DOPS) and the script concordance test (SCT). Finally in study five the influence of the transition to practice on learning behaviour during final year was explored. Graduates felt well prepared by the SVMS curriculum for a career in practice with respect to all ILOs. However, assessments were not rated so highly when considered alone. DOPS and to some extent the SCT are considered to be authentic assessment formats and encourage a deep approach to learning. The MCQ in this context results in surface learning strategies being adopted. The imminent transition to practice has a positive effect on learning behaviour, however this conflicts with the preparation required for final year examinations. Elements of the assessment strategy that have a positive and negative impact on CA are identified and discussed. Changes to the curriculum are proposed to enhance CA and ease the transition to practice.