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Cattle are a very important part of the human food chain. Administration of veterinary drugs and other xenobiotic compounds to cattle can often result in the accumulation of metabolic residues in edible tissues that can potentially affect humans through the food chain. An understanding of drug metabolism in this species is vital to ensure safe use of drugs in cattle and eventually the provision of safer animal derived food products to man. Sulfation catalysed by sulfotransferases (SULTs) is an important phase 2 drug metabolising reaction. It is not only involved in the detoxification of drugs and xenobiotics but also in the bioactivation of procarcinogens. It is also important in the metabolism of several drugs used routinely in cattle. However, very little work has been carried out on SULTs in cattle. A variety of in vitro tools are available to study drug metabolising enzymes (DMEs) like SULTs. These include tissue microsomes, cytosol, recombinant enzymes and isolated cells such as the hepatocytes.Recombinant SULTs are important tools that can be used for the study of isoform specific drug biotransformation, drug-drug interactions and the effect of genetic polymorphisms on the activity of specific isoforms. As the liver is the major drug metabolising organ in the body, it is essential to study expression and activity of cytosolic liver sulfotransferases. Hepatocytes contain DMEs and drug transporters along with all the necessary cofactors that represent in vivo conditions. This makes hepatocytes a better representative of in vivo conditions as compared to microsomes, cytosol or recombinant enzymes. In this study we have characterised sulfotransferases in cytosol, recombinant enzymes and hepatocytes.Antibodies previously raised against human sulfotransferase isoforms were used in the detection of cytosolic bovine sulfotransferases. Probe substrates established for activity with human SULTs were used for assessing the activity of recombinant and cytosolic bovine sulfotransferases. Cytosol was prepared from 8 male livers and 12 female livers (8 untreated and 4 treated with an exogenous progestin). SULT1B1, SULT1E1 and SULT2A1 were detected in bovine liver cytosol. Expression of SULT2A1 in the bovine liver was sex specific with males expressing almost twice as much SULT2A1 compared to the females. However, no activity was detected with dehydroepiandrosterone (DHEA) which is used as a probe substrate for SULT2A1 in humans. Pregnenolone is metabolised by SULT2A1 and SULT2B1 in humans. Activity towards this substrate was detected in the bovine liver, however no sex related differences in activity were observed. 4-nitrophenol liver, however no sex related differences in activity were observed. 4-nitrophenol is metabolised by several members of the SULT1 family in humans such as SULT1A1, SULT1B1 and SULT1C. 17ß-estradiol is a probe substrate for human SULT1E1. Activity was detected with 4-nitrophenol in male and female bovine livers. Male liver cytosol followed Michaelis-Menten kinetics whereas the female liver cytosol displayed partial substrate inhibition. This suggests that different enzymes have been involved in the biotransformation of 4-nitrophenol in the male and female liver. Activity towards 17ß-estradiol in the female liver was almost 4 times higher than in the male liver.Recombinant bovine sulfotransferases (SULT1A1, SULT1B1, SULT1E1 and SULT2A1) were expressed in E. coli. All bovine SULTs except SULT2A1 were expressed in the soluble fraction. Like human SULT1A1, bovine SULT1A1 also displayed partial substrate inhibition, however the extent of inhibition (as seen with the Ki values) was lower compared to human SULT1A1. Bovine SULT1B1 followed Michaelis-Menten kinetics with 4-nitrophenol. Substrate specificity profiling carried out with equal amounts of bovine SULT1A1 and SULT1B1 revealed that SULT1B1 was better at sulfating phenolic compounds as compared to bovine SULT1A1. SULT1A1 is highly expressed in the human liver and is the major enzyme involved in drug metabolism in the human liver. This might not be the case in cattle given that SULT1B1 was found to be better at sulfation than SULT1A1 and expression of SULT1A1 was not detected in the bovine liver using antibodies. Human SULT1E1 is known to metabolise 17ß-estradiol with a very high affinity and with a Km in the low nanomolar range. Comparatively, bovine SULT1E1 metabolised 17ß-estradiol with a lower affinity, in the micromolar range.Expression and activity of bovine sulfotransferases differed from human sulfotransferases and some of the differences could be attributed to key amino acid residue substitutions in the active site of the bovine SULTs. For example, substitution of Phe141 in human SULT1E1 to Leu141 in bovine SULT1E1 restricts the ability of bovine SULT1E1 to form strong van der Waals interactions with the substrate due to loss of an aromatic hydrocarbon ring. This could explain the reduced affinity of bovine SULT1E1 for 17ß-estradiol. Substitutions of small uncharged residues with large charged ones in the active site of bovine SULT2A1 could have unforeseeable effects that could result in the formation of an insoluble protein. Substitutions in the active site of bovine SULT1A1 that bind the second molecule of 4-nitrophenol could be responsible for the reduced partial substrate inhibition effects observed in comparison to human SULT1A1. In order to further validate some of these findings it would be necessary to perform additional experiments that involve mutating the substituted residue to the original one as found in the human/mouse counterpart and looking for restoration of original properties. The work was extended to investigate conjugative metabolism of the steroid hormone 17ß-estradiol and its stereoisomer 17a-estradiol in microsomes, cytosol and cryopreserved hepatocytes all prepared from bovine liver. It was found that glucuronidation was the main route for estradiol metabolism in cattle since large amount of glucuronide metabolites were detected in microsomes and cryopreserved hepatocytes. In comparison no sulfate metabolites were detected in cytosol and hepatocytes.We now have a better understanding of some of the important phase 2 drug metabolism pathways in cattle.

The first aim of the research conducted as part of this thesis was to investigate the expression and activities of 110HSD enzymes in porcine mural granulosa cells and COCs from small, medium and large antral follicles, as well as from ovarian cysts. Both cloned lipHSD enzymes (lipHSDl and 110HSD2) were expressed in porcine granulosa cells however the results of the expression studies in COCs were inconclusive. In granulosa cells from antral follicles, the 11-dehydrogenase (11P-DH) activities of the lipHSD enzymes increased by approximately 3-fold with antral follicle growth (P<0.01). Similarly, COCs isolated from large follicles had approximately 10-fold higher lip-DH activities than COCs from small follicles (PO.001). These results suggested that antral follicle growth occurred alongside increasing levels of intracellular Cortisol metabolism in both granulosa cells and COCs. In granulosa cells from ovarian cysts, there were significantly decreased net lip-DH activities compared to cells from large antral follicles (P<0.01), suggesting that decreased Cortisol metabolism occurred in the cells of porcine ovarian cysts. The next aim of this thesis was to investigate the levels of ovarian enzyme modulators in FF from small, medium and large antral follicles and in the fluid from ovarian cysts. The levels of inhibitors appeared to decrease in FF with follicle growth but were significantly increased in ovarian cysts (PO.01). These intrafollicular 1 lpHSDl inhibitors in FF and cyst fluid were also able to modulate lipHSD activities in the granulosa cells and COCs. Furthermore, cyst fluid, and the hydrophobic components thereof (which were likely to have included the ovarian enzyme modulators), were shown to significantly increase the rates of porcine oocyte maturation (PO.001). In summary, the findings of the studies conducted in this thesis suggest that the intrafollicular modulators of lipHSDl could influence local cortisol-cortisone inter-conversion in granulosa cells and oocytes during antral follicle growth. In addition the ovarian enzyme inhibitors could possibly influence oocyte maturation with follicle growth. In ovarian cysts however, the high levels of intra-follicular lipHSDl inhibitors in the cyst fluid, and the decreased levels of intracellular Cortisol metabolism observed in the granulosa cells, could be factors contributing to cyst development.

The submitted collection of papers represents my work in the area of equine ovarian function and dysfunction. During spring transition, the endocrinological events responsible for recruitment of large anovulatory follicles appear to resemble recruitment of preovulatory follicles in the natural breeding season. These large follicles contain only low concentrations of progesterone and oestradiol. They have low expression of mRNA encoding steriodogenic enzymes, have poor development of the theca interna and are poorly vascluarised. These follicles are therefore showing signs of atresia while they are actively increasing in size. In preovulatory follicles, concentrations of inflammatory mediators increase as ovulation approaches, and fluid from preovulatory follicles is chemotactic for leucocytes. Intrafollicular treatment with indomethacin delayed ovulation which supports the central role for inflammation in the ovulatory process. It is also likely the matrix metalloproteinases are involved in the profound tissue remodelling that occurs around ovulation. Control of follicular growth has been studied and equine follicles are dependent on gonadotrophin stimulation when they reach 10 mm in diameter and on LH stimulation for final growth and maturation. The equine CL is dependent, at least in part, on trophic support by LH and luteal cells bind LH <i>in vitro</i>. Steroidogenesis in the CL varies before and after development of the endometrial cups in pregnancy. StAR protein increases after endometrial cup formation, allowing greater mobilisation of substrate for steroid synthesis. Although P450_arom is consistently present in the equine CL, P450_C17 increases after the endometrial cups form, allowing oestrogen synthesis by the CL in the pregnant mare. The cell types in the equine CL appear to co-operate in steriodogenesis, with P450_C17 located in small luteal cells, and P450_arom in large luteal cells. Maintenance of the CL in early pregnancy appears to be caused by the inhibition of endometrial PG synthesis by the conceptus, although the conceptus itself produces PGs. The demise of the CL involves apoptosis and is preceded by a decrease in angiogenesis.

Paratuberculosis (Johne’s disease) is a chronic intestinal disease of ruminants caused by <i>Mycobacterium avium </i>subspecies <i>paratuberculosis</i> (MAP). Three forms have been described in sheep – multibacillary, paucibacillary and asymptomatic. Real-time RT-PCR (qPCR) and microarray analyses were used to compare gene expression in ileal tissue from sheep with the three forms of the disease to try to understand the immune responses underpinning these three defined pathologies. All animals from the infected flocks were IS900 positive by qPCR and therefore infected with MAP. Asymptomatic sheep had no clinical signs of disease, showed no evidence of acid-fast bacteria (ZN-), exhibited normal histology of the terminal ileum and were seronegative. Paucibacillary sheep were ZN- and showed lymphocyte/eosinophil infiltrate into the lamina propria. 2/6 of the paucibacillary animals were seropositive. Multibacillary sheep had high numbers of ZN+ bacteria associated with infiltrating sheets of epithelioid macrophages and were seropositive. Control sheep were IS900 negative and thus uninfected with MAP. qPCR experiments confirmed that pauci- and multibacillary forms are linked to the differential expression of IFNγ and IL-10 respectively. Increased levels of the proinflammatory cytokines IL-1β, IL-8, IL-18, TNFα and TRAF-1, indicative of persistent inflammatory lesions, were observed in clinical tissues. IL-3 was detected at low levels in all infected animals but never in uninfected control samples. IGFBP-6 was upregulated and CXCR4 downregulated in paucibacillary samples compared to multibacillary samples. Microarray experiments discovered 64 differentially expressed genes. Ten genes were found to be differentially expressed in infected tissue compared to uninfected controls, and a further eight in clinical tissues compared to uninfected controls. Fifteen genes were differentially expressed in clinical tissue compared to asymptomatic tissue. Six genes were quantified by qPCR and validated in microarray data well. Pathway analysis of the microarray data identified several immune pathways that are involved in pathogenesis. Infected tissues displayed upregulation of the genes involved in TCR signalling and complement activation, and downregulation of MHC class II genes. In addition, clinical tissues displayed upregulation of genes involved in the JAK-STAT and TLR2 signalling pathways, NK cell cytotoxicity and antibody production. Multibacillary tissues also displayed upregulation of genes involved in leukocyte migration. Overall, these data confirm that multibacillary pathology is linked to type 2 and paucibacillary pathology is linked to type 1 immune responses and identify novel genes and gene pathways for future analyses.

The mycotoxin ochratoxin A (OA) can contaminate a wide variety of feedstuffs and be nephrotoxic to domestic animals and poultry. The objective of this work was to study the clinical, pathological and immunological effect induced by feeding OA to broilers and turkeys from hatch as well as its effects on production characteristics and teratogenicity in quail. Some basic immune mechanisms were studied. OA caused growth depression in broilers and turkeys but not in quail. There was a loss of carotenoid pigments in the shank of broilers. The principal pathological effects were in the kidney especially in the proximal convoluted tubules (PCT) where the mitochondria were the organelles most sensitive to OA damage although other intracellular elements were involved together with a marked increase in lysosomal activity. OA localised within PCT and glomeruli. Ring-form mitochondria in the PCT and the accumulation of glycogen in the liver were considered to be of diagnostic significance. In the lymphoid organs, widespread degeneration was a consistent feature of OA-toxicity, suggesting a direct effect on immunity. OA induced a marked depression of both humoral and c~e~ mediated immune (CMI) responses in broilers and turkeys. Total serum levels were reduced in broilers but only IgM levels were depressed in turkeys. Tissue immunoglobulins were also depressed. Delayed hypersensitivity responses were reduced in broilers and turkeys. Graft-versus-host reactions, evaluated by a chick embryo splenomegaly test, were depressed in OA-fed broilers. It was not possible to elicit a distinct Arthus reaction in turkeys in contrast to broilers. In general, granulocytes, particularly heterophils and eosinophils, and vascular endothelial cells appeared to play a significant role in CMI responses in poultry. OA-contaminated feed resulted in reduced egg production, fertility and hatchability in a dose-related fashion in Japanese quail and an increase in embryonic mortality and a number of developmental defects.

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