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Dilated cardiomyopathy (DCM) or round heart disease is a muscle disease of the heart characterized by left ventricular dilatation and abnormal systolic and diastolic ventricular function. In animals, including turkeys and humans, DCM is a major cause of morbidity and mortality that results in heart failure. In the turkey, DCM can be idiopathic or induced. Since idiopathic or spontaneous DCM occurs in about 2-4 % of normal turkeys, it is of significant concern to the poultry industry. This dissertation was designed to increase our understanding of the pathophysiology of DCM in commercial turkeys. Specific objectives included: evaluating the influence of dietary selenium (Se) and vitamin E on poults fed toxic levels of furazolidone (Fz). Evaluating differences among reciprocal crosses of turkey varieties in susceptibility to a toxic level of Fz that induces DCM were used to assess the role of genetics in DCM. Using glutathione (GSH), glutathione peroxidase (GPx), malondialdehyde (MDA), and plasma uric acid (PUA) as biomarkers, oxidative stress (OS) levels were evaluated. Oxidative stress was also evaluated in poults fed diets containing varying concentration and combinations of vitamin E (0, 50 and 100 IU/kg) and Se (0.0, 0.3 and 0.5 mg/kg). Results from echocardiography measurements at four weeks of age, for poults fed toxic levels of Fz, showed the Narragansett x Bourbon Red reciprocal cross had the lowest internal-diastolic (LVIDd) and systolic dimensions (LVISd), while the Bourbon Red x Narragansett reciprocal cross had the largest LVIDd and LVISd. Left ventricular internal-diastolic and systolic dimension were lower for cross bred than parental poults. In treatment poults, heterosis for ventricular dilation was most significant for Bourbon Red x Narragansett cross. The data suggest that reciprocal crosses respond differently to toxin that induces DCM and genetics may influence a turkey's response to toxic levels of Fz that causes DCM. Results from OS measurements in poults fed normal and those fed normal diets with Fz at two weeks of age, showed no significant differences in MDA and GPx levels. PUA and GSH levels were however significantly increased for poults fed Fz-containing diets. At four weeks of age, no differences were observed for MDA and GPx measurements between poults fed normal and Fz-containing diets. PUA levels increased for poults fed normal diets with Fz, while GSH levels increased only for those fed normal diets. Differences between poults fed normal and Fz-containing diets were significant for GPx measurements. Results of this study showed that, feeding diets with Fz does not increase OS. Measure of the influence of feeding diets supplemented with different concentrations and combinations of Se and vitamin E to poults fed either normal or normal diets with Fz at two and four wks of age, showed higher MDA levels for poults fed Fz-containing diets supplemented with 0.3 mg/kg Se and 100 IU/kg vitamin E. For antioxidant biomarkers, GPx activity were increased for poults fed normal diets with Fz supplemented with 0.5 mg/kg Se and those fed 100 IU/kg vitamin E. Poults fed normal diets supplemented with 100 IU/kg vitamin E had the highest GPx. PUA levels were higher for poults fed normal diets with Fz supplemented with 0.5 mg/kg Se at two wks of age. At four wks of age, PUA concentrations were higher for poults fed Fz-containing diets supplemented with 100 IU/kg vitamin E. Increased PUA were also observed for poults fed diets supplemented with 0.5 mg/kg Se and 50 IU/kg vitamin E and 0.5 mg/kg and 100 IU/kg vitamin E. Poults fed diets supplemented with 50 and 100 IU/kg vitamin E had the highest GSH at two wks of age. Measurements taken at 2 wks of age, for poults fed normal diets supplemented with different concentrations and combinations of Se and vitamin E had increased GSH levels when compared with those fed diets with Fz at four wks of age. In this study, we showed that supplementation of poults fed normal diets with Fz with different concentrations and combinations of Se and vitamin E did not reduce DCM at 2 wks of age. However, at 4 wks of age, though DCM was not decreased by feeding diets supplemented with different concentrations and combinations of Se and vitamin E, reduced oxidant and antioxidant biomarkers were observed. / Ph. D.

Two times are critical for the horse ¾ the first few days of its life and the last few moments of a race. Vitamin E is critical in regard to immune competence in the first and antioxidant status in the latter. Studies conducted at the Middleburg Agricultural Research and Extension (MARE) Center include the development of horse feeds that replace sugar and starch with fat and fiber. The previous fat source of the pasture supplement under development was corn oil, which contains much vitamin E, was replaced with a cereal by product, which contains relatively little. Vitamin E has been studied in horses to a limited degree but not in grazing Thoroughbreds, thus the MARE Center gave me the opportunity to study vitamin E in Thoroughbred mares and foals. Middleburg is located in the Blue Ridge Mountains on Northern Virginia close to the site of one of the toughest endurance races in the world. This allowed me the opportunity to study vitamin E and antioxidant status in the horse during endurance racing. Initial studies of vitamin E supplementation to mares during the last trimester of gestation were disconcerting with no changes in serum concentrations of a-tocopherol (vitamin E). Studies conducted during the post-partum period revealed evidence of responses to vitamin E supplementation, as increased a-tocopherol concentrations were observed in mares' milk and in foal serum. Foals are born with virtually no circulatory antibodies and the supplementation of a synthetic form of vitamin E to mares demonstrated an increased passive transfer of immunoglobulins to foals. Natural vitamin E has shown a greater bioavailability than synthetic forms, in other species and was tested here at the MARE Center on mares. A greater passive transfer of immunoglobulins was observed with natural vitamin E supplementation compared with the synthetic forms, with immunoglobulin M concentrations in foal serum remaining higher for a longer period after birth compared to foals of non-supplemented mares. The transfer of a-tocopherol via the milk was also increased in concentration and duration in mares supplemented with natural vitamin E. Bioavailability of five oral forms of vitamin E (3 natural and 2 synthetic) were tested and one natural form was also administered intravenously so that clearance of vitamin E could be used to calculate the efficiency of absorption of the oral forms. Efficiency of absorption for oral treatments was not determined because of the slow turnover time of the intravenously administered vitamin E, which confounded all subsequent baseline serum a-tocopherol concentrations. Of the salvageable data, serum a-tocopherol concentrations were higher in grouped treatments at 9 and 12 h post dosing. Lipid fractions revealed possible insufficient absorption of the oral doses of vitamin E and possibly tissue saturation following intravenous doses of vitamin E. Serum concentrations of a-tocopherol were generally higher following natural forms of oral vitamin E administration. As vitamin E is the most important antioxidant in cells, it is often supplemented to endurance horses competing in 80 and 160 km races. Vitamin E protects lipid cell membranes from peroxidation by free radicals, which are increased during strenuous exercise resulting in oxidative stress. The antioxidant status of horses is severely tested during endurance racing and so a study was conducted to monitor changes in circulating antioxidants during three endurance races. Interesting novel findings in the horse were the maintenance of serum a-tocopherol and the depletion of erythrocyte glutathione and plasma ascorbate during two 80 km and one 160 km races. Associations were found between increased muscle cell enzyme leakage and decreased antioxidant status during endurance exercise and although associations do not prove a causation of oxidative stress, they do provide motivation to search for a cause and it is tempting to propose that oxidative stress damaged muscle cell membranes in endurance horses. Further, these findings propose a connection between muscle cell damage and a new form of exertional rhabdomyolysis (ER) that has been observed in endurance horses, where oxidative fibers are damaged compared to the typical glycolytic fiber damage associated with known forms or ER. An increased understanding of vitamin E utilization in the horse will improve the health and welfare of all horses, but especially newborn foals and the athletic endurance horses. / Ph. D.

A series of studies were conducted to examine the antioxidant status, drought and disease tolerance, and growth response to foliar application of soluble seaweed (Ascophyllum nodosum) extracts (SE) and humic acid (HA; 25% active HA or 2.9% active HA) in tall fescue (Festuca arundinacea Schreb), Kentucky bluegrass (Poa pratensis L.) and creeping bentgrass (Agrostis palusttis Huds.) grown under low (-0.5 MPa) and high (-0.03 MPa) soil moisture environments. Foliar application of humic acid (2.9 % active HA) at 23.7 and 47.4 l/ha improved leaf water status, shoot and root development in tall fescue, Kentucky bluegrass and creeping bentgrass grown under drought. Humic acid (2.9% active HA) at 15.5 l/ha or SE at 326 g/ha significantly reduced dollarspot incidence and improved turf quality in creeping bentgrass. Drought stress induced an increase of antioxidants alpha-tocopherol and ascorbic acid concentrations in the three turfgrass species. In the experiment with Kentucky bluegrass, drought stress increased beta-carotene concentration, but did not significantly influence superoxide dismutase (SOD) activity. Foliar application of humic acid (25% active HA) at 5 l/ha and/or SE at 326 g/ha consistently enhanced alpha-tocopherol and ascorbic acid concentrations, leaf water status, and growth in the three cool-season turfgrass species grown under low and high soil moisture environments. In the experiment with Kentucky bluegrass, application of HA at 5 l/ha plus SE at 326 g/ha also increased beta-carotene content and SOD activity under low and high soil moisture environments. There were close positive correlations between the antioxidant status and shoot or root growth in the three turfgrass species regardless of soil moisture levels. The antioxidant SOD activity, photosynthetic capacity in terms of Fvm690, and chlorophyll content in terms of Fm730/Fm690 exhibited a seasonal fluctuation in endophyte [Neotiphodium coenophialum (Morgan Jones and Gams) Glenn, Bacon, Price and Hanlin] -free and endophyte-infected tall fescue. Application of SE enhanced SOD activity, photosynthetic capacity, and chlorophyll content in tall fescue, especially at 4 weeks after SE treatment. The SOD activity, photosynthetic capacity and chlorophyll content were not significantly influenced by the endophyte infection. A close positive correlation between SOD and photosynthetic capacity during the summer was found in endophyte-free and endophyte-infected tall fescue. / Ph. D.

Atherosclerosis, one of the major causative factors of cardiovascular diseases (CVD), is thought to be initiated by oxidative stress. Particular attention has been paid to the atherogenic effects of oxidative damage on low density lipoproteins (LDL). Current research shows that dietary antioxidant supplementation protects against oxidative stress, and therefore may present preventative measures and treatments for patients with diseases influenced by oxidative stress. Isoflavones found in soy, such as genistein and daidzein are reported to have potent antioxidant properties and have been shown to inhibit LDL oxidation in vitro. Although there is a strong base of data that supports the correlation between soy consumption, cholesterol reduction and cardiovascular protection, it remains to be elucidated whether it is the soy protein, the isoflavone, or a combination of both that confers benefits. This study investigated the effect of isoflavone supplementation on the following parameters: plasma genestein levels, oxidized LDL levels, plasma cholesterol, vitamin E, and C-reactive protein. Elevated serum cholesterol and C-reactive protein (CRP) have been identified as risk factors for cardiovascular disease. In a randomized, double-blind, placebo-controlled study, 150 mg/d isoflavone was supplemented for four weeks by 30 healthy, yet sedentary male subjects who underwent 30 minute exercise sessions at 80% VO2pk before and after a 28 day period of supplementation. The purpose of the exercise was to induce oxidative stress. The average plasma genistein and daidzein concentrations increased significantly after isoflavone supplementation from 0 ng/ml to 561.6 ± 39.3 and 466.3 ± 35.5 ng/ ml (SE) respectively (P < 0.0001), compared to 0 ng/ml in the placebo group throughout the study. There was no significant beneficial effect of isoflavone supplementation on oxidized LDL, plasma vitamin E concentrations, total cholesterol, LDL, HDL, or triglycerides. Isoflavone supplementation resulted in an average increase in CRP levels by 44% (P = 0.014), which was opposite from expectations. This study supports the theory that it may not be soy isoflavones alone that benefit lipid profiles, or offer protection from oxidative stress. / Master of Science

The decline of honey bee (Apis mellifera) colony numbers in recent years presents an economic and ecological threat to agriculture. One outstanding threat to honey bees is the unintended exposure to agricultural pesticides. Previous studies report that acute exposures to the common-use herbicide atrazine elicit oxidative stress in non-target insects; however, little information is currently available on the exposure risk of atrazine to honey bees. This project examined biochemical and molecular oxidative stress response markers of honey bees following laboratory and field treatments of atrazine. Laboratory experiments were conducted with honey bees exposed to increasing concentrations of atrazine for 24 h whereas hive experiments were conducted with bees exposed to one sub-lethal concentration of atrazine for 28 d. The overall antioxidant enzyme activities of atrazine-treated honey bees were decreased compared to the untreated honey bees in both the laboratory and hive experiments. After exposure to atrazine in the laboratory and field, semi-quantitative RT-PCR analysis of antioxidant-encoding genes reveals the differential expression of genes in atrazine-treated bees that are important for oxidative stress tolerance in the laboratory and field experiments. Here, we provide evidence that the laboratory and hive exposure of honey bees to the common-use herbicide atrazine results in oxidative stress responses that can compromise the health of bee colonies. The data will be discussed with regard to the protection of these pollinators against the untended exposure of agricultural pesticides. / Master of Science in Life Sciences

Gossypol , a yellow polyphenolic pigment found in cottonseeds, is known to promote the production of reactive species of oxygen in vitro, and has toxic actions on spermatogenic epithelium, hepatocytes and cardiac myocytes in vivo. Species vary in tissue sensitivity to the toxic effects of gossypol. The spermatogenic epithelium is the most sensitive tissue to gossypol in rats, followed by the liver. Toxic effects to the rat heart are found only after prolonged administration of gossypol. The antioxidant defense system that protects cells from injury by reactive species of oxygen was examined in the present study to determine a possible pathogenesis for gossypol associated tissue damage. The concentrations of several hepatic antioxidants including catalase, glutathione peroxidase, ascorbate and copper-zinc superoxide dismutase were decreased in gossypol treated rats. Catalase, glutathione peroxidase, ascorbate and glucose-6-phosphate dehydrogenase were decreased in the testis. In contrast, antioxidants including catalase and glutathione reductase were increased in the hearts of gossypol treated rats. The selective inhibition of testis and hepatic antioxidants may account for the greater sensitivity of these organs to reactive oxygen species generated by gossypol. The rat heart may adapt to oxidative insult by inducing the production of antioxidants. Glucose-6-phosphate dehydrogenase activity was decreased in the testis but not liver or heart of gossypol treated rats. This important enzyme is known to produce NADPH reducing equivalents for testosterone biosynthesis and the glutathione antioxidant system. In the present study, micromolar concentrations of gossypol inhibited glucose-6-phosphate dehydrogenase in a competitive manner with respect to glucose-6-phosphate. This may explain the degeneration of spermatogenic epithelium as well as decreases in serum testosterone concentrations in gossypol treated rats. Gossypol is known to cause infertility in women and female rats. The present study found irregularities in the estrous cycles and ultrastructural changes in endometrial macula adherentes of gossypol treated female rats. / Ph. D.

Background: Dietary and serum antioxidants and physical activity can effect inflammation, which is associated with breast cancer risk and recurrence. This study investigated the relationship between diet, serum antioxidant capacity, physical activity, and inflammation in breast cancer survivors and individuals without cancer. Methods: Existing demographic, dietary intake, and physical activity data of 78 breast cancer survivors and 30 individuals without cancer from the Day and Night Study conducted at Virginia Commonwealth University were used. Participants were recruited from southern Virginia. Metabolic equivalents were calculated through type, intensity, and duration of physical activity. Dietary antioxidant intake (FRAP) was calculated from Harvard Food Frequency Questionnaire data. Serum samples were analyzed for inflammation (hsCRP,IL-6,IL-1,and TNF alpha) and serum antioxidant capacity (ORAC) at Virginia Tech. Results: Anthropometrics and inflammation were higher, and FRAP and ORAC lower in breast cancer survivors compared to individuals without cancer, although not significant. There was a significant direct relationship between FRAP and ORAC and inverse relationship between FRAP and hsCRP. Breast cancer survivors 6+ years since diagnosis showed significant direct FRAP and IL-1 association, and inverse ORAC and TNF-alpha association. BMI was directly associated with IL-6 and CRP. Inflammation was not associated with METs or weekly activity, although there was an increasing inverse relation between METs, IL-1 and TNF- α with increasing ORAC. Conclusion: There is a significant relationship between dietary antioxidant intake and serum antioxidant capacity and inflammation. Increased body mass index increases inflammation. Diets high in antioxidants and maintaining a healthy weight may help reduce inflammation in breast cancer survivors. / Master of Science / Background: Dietary and serum antioxidants and physical activity can effect inflammation, which is associated with breast cancer risk and recurrence. This study investigates the relationship between diet, serum antioxidant capacity, physical activity, and inflammation in breast cancer survivors and individuals without cancer. Methods: Demographic, dietary intake, and physical activity data of 78 breast cancer survivors and 30 individuals without cancer from the Day and Night Study conducted at Virginia Commonwealth University were used. Participants were recruited in southern Virginia. Metabolic equivalents, a measure of physical activity, were calculated from type, intensity, and duration of physical activity. Dietary antioxidant intake (FRAP) was calculated from Harvard Food Frequency Questionnaire data. Serum samples were analyzed for inflammation (hsCRP, IL-6, IL-1, and TNF-alpha) and serum antioxidant capacity (ORAC) at Virginia Tech. Results: Anthropometrics and inflammation were higher in breast cancer survivors while FRAP and ORAC were lower. Significance existed between dietary antioxidant intake and serum antioxidant capacity as well as dietary antioxidant intake and hsCRP. Higher body mass index was associated with increased inflammation. Breast cancer survivors 6+ years since diagnosis with higher dietary antioxidant intake had lower IL-1, and with serum antioxidant capacity and TNF-alpha. Conclusion: In this population there is a significant relationship between dietary and serum antioxidant capacity, as well as dietary antioxidant capacity and hsCRP. In breast cancer survivors 6+ years since diagnosis there are significant associations in antioxidant capacity and inflammation. This is evidence that dietary antioxidants and maintaining a healthy weight can help reduce inflammation.