The objective of this work was to investigate whether or not olfactory clues play a role in host plant location by the polyphagous moth, Helicoverpa armigera. Volatiles collected from flowers of African marigold, Tagetes erecta, and sweet pea, Lathyrus odouratus, were found to elicit electroantennographic (EAG) responses from the antennae of female H. armigera. Compounds active in GC-EAG analyses of T. erecta floral headspace samples, identified by GC-MS and comparison of retention times on polar and non-polar GC columns with authentic standards, were (E)-myroxide, benzaldehyde, (f)-linalool, phenylacetaldehyde and (-)- piperitone. EAG-active compounds in L. odouratus floral headspace samples were identified as diacetone, (-)-linalool, phenylacetaldehdyde and benzyl alcohol. Increases in upwind flight to air entrained extracts of floral odours indicated that these cues caused attraction when presented to female H. armigera. A synthetic T. erecta blend comprising benzaldehyde, (f)- linalool, phenylacetaldehyde and (+)-limonene gave significant increases in upwind flight approaches. Limonene (either (+)- or (-)-) was found to be important for the behavioural response despite having low EAG-activity. There was no significant difference in upwind flight response to odours from the live flower and the synthetic floral blend. Significant increases in upwind flight were also obtained when insects were presented with a synthetic L. odouratus blend which contained the four EAG-active compounds identified from GCEAG studies. In field trapping experiments in Israel there was a significant difference in H. armigera catches in traps with a standard 4-component T. erecta lure compared with unbaited traps over the whole season. Mean H. armigera catch per trap per night (both sexes) over the whole season in unbaited traps, floral odour traps, pheromone traps and light traps were 0.004,0.11,8.8 and 1.35 respectively. The floral baited traps were non-selective catching large numbers of Ilymenoptera and Diptera as well as other moth species. Field trapping experiments in Pakistan indicated that the floral lure was significantly attractive to Earias spp. and other Lepidoptera although very few H. armigera were caught due to low population density. Olfactory cues are discussed in relation to host-plant finding behaviour of H. armigera. They are involved in early stages of host seeking behaviour prior to alighting on the plant and stimulate searching behaviour.

This study involves two village chicken populations sampled from Horro and Jarso regions of Western and Eastern Ethiopia respectively. This study maps the phenomic and genomic landscape of the two chicken populations using morphological markers and a high density (600K) SNP array. Although the two chicken populations tend to display nondescript morphological characteristics, they show a subtle variation except for rare morph variants that have been in most instances scored on Jarso chickens. Morphological analysis uncovers a vast array of intrapopulation variation. Genetic diversity and population structure analyses assign the two chicken populations to two distinct genepools representing their population of origin. A high intrapopulation genetic diversity is uncovered, which shows a broad genetic base (high genetic diversity) of the two chicken populations. We hypothesized that a clearly evident genetic divergence observed between the two chicken populations may be attributed to difference in demographic history, origin (routes of introduction to Africa), breeding history of the two chicken populations and demographic structure of subsistence farmers. Absence of gene flow owing to their distant geographic location and ecological variation may have also contributed to this divergence. A population structure analysis performed on a random subset of the two Ethiopian chicken populations along with village chickens sampled from other African countries, Asia and Latin America, commercial populations and the junglefowl species reveals a unique genetic structure of Ethiopian chickens, which implicates the need for further study on the genetic landscape of the latter. To infer the extent of inbreeding we performed a run of homozygosity analysis (ROH). Our analysis indicates that ROH is more intense in Jarso than Horro chickens and in macrochromosomes than microchromosomes. The extensive ROH mapped in some chickens implicates the need to restructure the existing traditional breeding practice of subsistence farmers. Our analysis confirms the commonness of ROH in genic regions. For the first time, we detect twenty three putative uniparental disomy in twenty two Ethiopian village chickens. Signature of selection analysis detects divergently selected genomic regions in the two chicken populations indicating a considerable divergent selection imposed on the two populations. Genes involving in melanogenesis pathway are among those subjected to a divergent selection. However, some overlapping regions were also mapped in the two chicken populations implicating the ubiquitous impact of natural selection on genes regulating vital biological processes. A genome-wide association study performed on pigmentation (earlobe, plumage and shank) traits and variants of crest, comb and a lightly feathered shank maps a number of putative loci that may underlie variations in these traits. Our GWAS analysis on pigmentation traits produced a long list of loci than that have been known to involve in the genetic control of pigmentation in the chicken, with most of these have been mapped in the mouse. We also refined further the causative variants underlying a lightly feathered shank mutation. Our GWAS analysis map a number of putative novel loci that may underlie the genetic control of the traits analysed and this has laid a foundation for subsequent work that would involve targeted sequencing and a candidate gene approach. This study is the first of its kind in Africa that uses a large number of samples and a high density SNP array to unlock phenomic and genomic landscape of the true type village chickens.

Improving efficiency within the agricultural industry is vital to maintain the food demands of the increasing population, as well the current preference for a more protein rich diet. One avenue for addressing these issues is to study animal-based growth to determine if the efficiency of the production system can be improved by increasing lean muscle mass. The aim of this thesis is to provide an alternative exploration to experimental work to provide an insight into how muscle metabolism in pigs is altered by the administration of a beta-agonist which induces muscle hypertrophy. This will be incorporated into a wider body of work to determine specific pathways to target for improving feed conversion efficiency, contributing to the necessary research into global food security. We begin by compiling a selection of statistical methods to analyse muscle microarray data, which enables the identification of a selection of genes whose expressions are altered by the exposure to a beta-agonist. These differentially expressed transcripts are then grouped via a k-means algorithm, with log likelihood and the Bayesian Inference Criterion calculations providing an optimal selection of clusters. This results in selecting a group of 51 transcripts and partitioning them into 9 clusters, and identifying several pathways which appear key to the regulation of muscle metabolism in the presence of beta-agonist. We have proceeded to incorporate this information into a mathematical model for glycolysis and the TCA cycle, in an effort to analyse biological hypotheses about how the promoters work. The equations describe the concentrations of metabolites within the cytosolic and mitochondrial compartments of a cell using mass balance ODEs. An initial model is presented, which is then increased in complexity, to keep up with developments in the experimental side of the overarching project. We make use of a selection of methods to analyse the model in an attempt to determine the effects that the different parameters cause. Through steady state analysis, we determine parameter ranges which permit positive steady states. In finding these regions, we also determine the existence of time dependent solutions, which occur when critical values of certain parameters are exceeded, and result in the build up of specific metabolites. We use asymptotic analysis to generate approximate solutions when steady states do not exist. The model parameters of most interest are those which were identified through the microarray work, namely the upregulated transcripts of PCK2 and those within the serine synthesis pathway, the control mechanism for the first half of the TCA cycle, the proportion of GTP producing enzyme from the second half of the TCA cycle, and the flux into the glycolytic pathway. We find that critical values for the glycolytic flux, and the GTP production parameter exist, determining whether the model lies within the steady state regime. In a large number of cases, the parameters we choose to represent the beta-agonist case push the system into the time dependent state. The model does not exhibit any interesting behaviour when the parameter controlling the PCK2 pathway is studied, indicating that initial intuition of the key controlling reaction mechanisms were incomplete. Whilst there are shortfalls in the model, which highlight areas for investigation, the system is set up for validation and parameter fitting when appropriate experimental data become available. We have been able to determine specific metabolic pathways within the cell which may be of significance to improving feed efficiency.

Here I investigate, using autosomal whole-genome sequence data, the signature of positive selection and/or introgression in the indigenous domestic village chickens from three countries (Ethiopia, Saudi Arabia, Sri Lanka), three fancy birds, three junglefowl species (red junglefowl Gallus gallus, grey junglefowl G. sonneratii, Ceylon Junglefowl G. lafayettii) and the Javan red junglefowl G. g. bankiva. All the new sequencing data were obtained from Illumina HiSeq 2000/2500 DNA sequencers with an individual bird depth of genome coverage ranging from 10 X to 30 X. The analyses in this thesis have been completed using the reference genome Galgal 4.0. For the detection of signatures of positive selection, this analysis excluded the three fancy birds and the grey junglefowl due to small sample size. Using the pool heterozygosity and SweeD composite likelihood selection signature methods, I identified two candidate selected regions shared between all the three indigenous domestic village chicken populations and the red junglefowl (chapter 2). These regions contain genes that are associated with the development of the central nervous system and adaptation to hypoxic environments. Five candidate regions were shared among the three indigenous village chicken populations, and they represent candidate domestication regions. Unique regions in each domestic chicken population were also identified. Functional genes have not been assigned to most of these regions but in those where the genes have been annotated, the gene function may be related to production and reproductive traits as well as adaptation to cold/hot temperatures and hypoxia. In chapter 3, I analysed only the Ceylon and green junglefowl whole genome sequences for the detection of candidate signatures of positive selection using both the pool heterozygosity and Tajima’s D. In both species, I identified candidate selected regions that contained genes which may be linked to adaptation to different environmental challenges e.g disease resistance, stress, thermoregulation and hypoxia. In the genome of green junglefowl, candidate selected regions associated with skeletal formation and ovarian follicle development were significantly detected. In chapter 4, I identified introgressed candidate regions from the grey and Ceylon junglefowls in domestic chicken (including the three indigenous chicken populations and fancy birds) using the ABBA – BABA four taxon method. Our result shows that, domestic chickens shared 75.8% of their genome with the red junglefowl, 4% with the grey junglefowl and 1.1% with the Ceylon junglefowl. I observed introgression in both directions, namely from the grey/Ceylon junglefowls into domestic chickens and vice versa. While from the grey junglefowl, introgression was present in all the domestic chicken populations as well as interestingly in the red junglefowl, for the Ceylon junglefowl, introgression was more restricted to the domestic chicken from Sri Lanka. From the ABBA – BABA analysis between the grey junglefowl and the domestic chicken, I also identified a single candidate introgressed region from the green junglefowl G. varius in two domestic birds from Sri Lanka. Future study should therefore consider investigating the genome-wide analysis of introgression from the green junglefowl into the domestic chicken. In chapter 5, I ended our introgression study by investigating if a distantly related subspecies of red junglefowl, the Javan red junglefowl Gallus gallus bankiva, has contributed to the gene pool of the domestic chicken. Alongside the three indigenous domestic chicken populations, I included the genome sequences of three domestic chickens sampled from the Java island of Indonesia. Our result shows a significant 10.6% genome admixture between the domestic chicken and the Javan red junglefowl. Overall, our results indicate that the genetic make-up of the domestic chicken is rather complex with multiple species and subspecies influences. These introgression events have contributed to the genetic diversity of these domesticates. Our results also support the geographic difference of introgression and indicate that these introgression events may have contributed to the adaptive traits of the domestic chicken. However, this requires further investigation.

Animal models of musculoskeletal ageing are required for the investigation of a number of conditions which may be difficult or unethical to investigate in humans. For this purpose, rodent models have been widely utilised, however, there are a number of differences between rodents and humans which may limit their usefulness as a model of human disease. Osteoarthritis (OA) is a musculoskeletal joint disorder characterised by degenerative loss of the articular cartilage and bone remodelling. In attempting to better understand the processes that mediate cartilage degeneration animal models have been utilised, however, OA in these models is typically induced either chemically or surgically and poorly mimics the pathological features of human OA. Spontaneous models also exist, one such model is the Dunkin Hartley guinea pig, but questions have been raised about the utility of these models due to issues with translatability of research into meaningful treatments in humans. Therefore, in order to better understand the key drivers of musculoskeletal decline and to identify and develop new therapeutics there is a high unmet need to develop more translatable animal models of musculoskeletal ageing. Importantly, the pig shares a high degree of physiological and anatomical similarity with that of humans and as a result, the pig is considered more closely related to humans than any other non-primate mammalian species. The high prevalence of degenerative joint conditions including osteochondrosis and osteoarthritis in commercial pig breeds suggests that these animals may represent an improved model. Therefore, in this study we assessed whether the commercial pig might present an alternative model of musculoskeletal ageing and early OA. For this purpose, this study investigated commercial pigs at a number of ages up to 4yrs paying particular attention to body composition changes, muscle changes and knee OA development within the age range. The utility of porcine cartilage and bone for ex-vivo studies was also examined. The investigation of body composition changes in these pigs did not reveal significant age-related deterioration, likewise, in muscle, no indication of early sarcopenic changes were identified. Together these findings suggest that the pigs used in this study were too young to be undergoing age-related muscle decline. Examination of OA in porcine knee joints revealed development of OA-like lesions and proteoglycan loss suggesting that these commercial pigs spontaneously develop OA at a relatively young age. Similarities between porcine and human cartilage and bone were also revealed with loss of chondrocyte phenotype in 2D culture and the discovery of an altered osteoblast phenotype in cells obtained from older, damaged knee joints; this osteoblast phenotype was similar to that found in sclerotic human bone. In summary, this work has shown that commercial pigs within this age range are too young to exhibit early indications of age-linked muscle decline, such as sarcopenia. However, pigs within this age range exhibit early OA changes in the knee joint and both porcine cartilage and bone may be of use in ex-vivo studies investigating OA. Together this suggests that pig could represent an appropriate animal model for the investigation of early OA.

This work concerns infection of avian erythrocytes by influenza and two other viruses. The first section investigates the production of viral polypeptides and infectious progeny virions from avian erythrocytes infected with fowl plague virus, Newcastle disease virus and Semliki Forest virus. In the second section a closer examination is made of viral polypeptide synthesis in avian erythrocytes following infection with avian and/or human influenza A strains. The third section investigates the distribution of newly synthesized influenza viral polypeptides between the erythrocyte nucleus and cytoplasm. The fourth and fifth sections are concerned with viral replication in erythrocytes at different stages of differentiation.

The herd-level incidence of Mycobacterium bovis has been increasing in the United Kingdom (UK) and Republic or Ireland (RoI) for the past thirty years, resulting in substantial economic and animal welfare issues. Failure to control this pathogen in cattle is in part due to European badgers (Meles meles), a wildlife reservoir that are responsible for a proportion of transmission of M. bovis to cattle. Monitoring infection in badger populations is currently limited due to the need to trap badgers, which requires highly trained field staff and is expensive. In addition, although contact with infected badger faeces is a potential transmission route to cattle, very little is known about the extent and variability of the environmental pool of M. bovis shed by badgers. In this project we evaluated the suitability of using environmental badger faeces and a quantitative PCR (qPCR) assay to diagnose and monitor M. bovis in badger populations and described the extent of this environmental pool of potential infection. The first study identified that intensive environmental faecal sampling and analysis with qPCR is at least, if not more, sensitive at diagnosing M. bovis in badger populations than the currently used immunoassays. This study also identified that even within a high prevalence population, the levels of shedding of M. bovis in faeces are highly variable between groups and between seasons, suggesting that there may be heterogeneity in transmission risk throughout the year. Using this non-invasive qPCR method to monitor the first field trial of oral BCG vaccination identified a trend of decreasing levels of M. bovis in faeces with increasing vaccination levels however, these results failed to reach statistical significance, highlighting the importance of adequate sample sizes when implementing this method. Finally, characterisation of the gut and faecal microbiota from animals shedding M. bovis in faeces confirmed that the source of faecal M. bovis is most likely sputum that has been expelled from the lungs, and not from colonisation of the gut. The work presented here suggests that this non-invasive monitoring method can be applied to examine the variable pool of M. bovis over periods of time and large areas, providing an epidemiological tool which has the potential to be implemented to monitor infection in badger populations and disease intervention strategies.

Doctor of Philosophy / Department of Animal Sciences and Industry / Evan C. Titgemeyer / Ractopamine is a repartitioning agent that can increase muscle growth and has led to our interest in determining the mechanisms involved in enhancing muscle growth. Therefore, three studies were conducted to determine the impact of ractopamine on growth in cattle. The first experiment evaluated the impact of increasing metabolizable protein supply to finishing heifers fed ractopamine. Three different diets were fed to increase the amount of metabolizable protein reaching the small intestine, and the diets contained urea, solvent soybean meal, or expeller soybean meal as the primary supplemental protein source. From this study it was determined that increasing metabolizable protein supply above that present in typical feedlot diets containing urea and steam-flaked corn is not necessary to improve responsiveness to ractopamine. The second experiment evaluated the effect of feeding ractopamine to growing Holstein steers implanted with trenbolone acetate/estradiol. Half of the steers were implanted 28 days prior to all steers receiving ractopamine for the final 28 days; this model represents an intense implant program. The mode of action of the ractopamine and of steroidal implants was different based on their different effects on serum insulin-like growth factor (IGF)-I and longissimus expression of IGF-I mRNA. Additionally, administering a combination of the two growth promotants, based on nitrogen retention, yields a less than additive response using our model of growing Holstein steers. The third study was conducted to evaluate the differential response to ractopamine of implanted, finishing steers and heifers. Treatments were steer vs. heifer and 0 vs. 200 mg/d ractopamine fed for the final 28 days. This study attempted to address some questions generated from the previous study, which were how serum and local tissue production of IGF-I are affected by ractopamine. Ractopamine had different effects on serum IGF-I between steers and heifers and numerically increased IGF-I mRNA abundance in longissimus and biceps femoris muscles. Additionally, ractopamine impacted protein turnover differently in different muscles and changed myosin heavy chain IIA expression. The effect of ractopamine on IGF-I warrants more research. These experiments aid in our understanding of the mode of action of ractopamine in cattle.

One of the characteristic features of parasitic infections is the chronicity and persistence of the infection for long time. Their success is due to its ability to deviate the immune response of their host toward the immunoregulatory or immunosuppressive state. IL-10 is a multifunctional cytokine with both immunosuppressive and immunostimulatory effects during parasite infection, playing a crucial role in the regulation of the immunity by ameliorating the destructive immunopathology associated with excessive inflammation but often suppressing effective immune responses against pathogens and impairing parasite clearance. Several transcription factors have been characterised that act on the promoter region of human and mouse IL-10 and their role in the transcription of this cytokine has been experimentally verified. In our study, a 570 bp fragment of the 5' UTR region flanking the bovine il-10 gene was cloned and characterised by sequencing. Several SNPs relative to the reference sequence are described, the putative polymorphisms lie within the transcription factor binding sites Sp1, Cap, HSF, ADR1, MZF1, GATA1 and Hb. We have investigated the role of GATA1 in regulation of IL-10 expression from fluke naïve PBMCs in response to LFH and FhTLM and we demonstrate that both LFH and FhTLM treatment induced GATA1 expression from PBMCs. These molecules showed distinct effects on PBMC functions i.e. stimulation of IL-10 in response to FhTLM and inhibition when LFH is used. However, both factors inhibit PBMCs proliferation and IFN-γ expression. We have also found that FhTLM binds to bovine TGFβ-RIED and TGFβ-RIIED fusion proteins but with a higher avidity to TGFβ-RIIED. These results highlight the role of FhTLM as a potent immunomodulatory of the bovine immune system by binding to bovine cytokine receptors. Neospora caninum infection is the major cause of abortion in dairy cattle. Infection with this parasite leads to a polarized Th1 response with high IFN-γ and IL-12 which provide protection aginst N. caninum infection. We have determined the effects of IL-10 in the regulation of IFN-γ and its association with production parameters (milk yield, lactation numbers) by measuring the levels of these cytokines in serum of infected animals and relating these to the key production parameters. Our results have shown that 84 % of high seropositive dams gave birth to seropositive calves and IL-10 concentration was higher in seropositive animals compared to seronegative. However, no correlation was found between the IL-10 concentration of seropositive dams and their calves. Together these indicated that IL-10 is an important cytokine during neosporosis, which modulate the immune response and allow the transmission of the infection to the offspring. Finally, our results for association of the IL-10 with lifetime daily yield demonstrated that IL-10 has a negative association with milk production during infection with N. caninum. Results of our study demonstrate a key role for bovine IL-10 in the regulation of multiple facets of parasite infection and immunity.

Cytochrome P450s (CYPs) are a superfamily of enzymes involved in the phase I metabolism of endogenous and exogenous substances. They are present in almost all forms of life and have been studied extensively, particularly in relation to human medicine, where knowledge of their activities is essential for predicting drug-drug interactions. In the horse, little is currently known about CYP-specific drug metabolism, which holds importance for animal welfare and for doping control within the horseracing industry where drug-specific metabolites are tested for on race days. Recently the first recombinant equine CYPs have been produced, allowing specific data on equine P450 activity to be gathered for the first time. During the current study,46 full-length P450 sequences were identified from the equine genome. RT-PCR analysis was then carried out on equine liver in order to detect hepatic expression of P450s across various families. After this, cold-induction (pCold) E. coli were used for production of recombinant P450 proteins for subsquent functional testing. Four recombinant equine P450s were successfully expressed (CYP1A1, CYP2A13, CYP2C92 and CYP2D50). Due to being the isoforms most likely to be involved in drug metabolism, rCYP2D50 and rCYP2C92 were selected to be screened against ten of the most commonly used horse drugs to identify potential substrates. rCYP2C92 appeared to metabolise all four NSAIDs tested (flunixin, ketoprofen, phenylbutazone and diclofenac), however presence of the known hydroxylated metabolites of diclofenac and phenylbutazone (4-hydroxydiclofenac and oxyphenbutazone, respectively) could not be confirmed despite being present within equine liver microsome and human recombinant CYP2C9 samples. In spite of the apparant acivity displayed by rCYP2C92 towards all four NSAIDs, no conclussions can be made about this enzyme’s role in NSAID metabolism due to a lack of known hydroxylated metabolite production.