Oxidative stress contributes to inadequate in vitro maturation of porcine oocytes which leads to a failure of successful fertilization and embryo development. Therefore, the overall objective of this research was to characterize the mechanisms of oxidative stress in maturing oocytes and determine how oocytes alleviate oxidative stress with the assistance of supplemental antioxidants. A preliminary study was conducted to evaluate the effects of glutathione (GSH), N-acetyl-cysteine (NAC), and N-acetyl-cysteine-amide (NACA) supplemented to the maturation medium on intracellular GSH concentrations, nuclear maturation, fertilization success and embryo development. Antioxidants GSH, NAC and NACA (1.0 mM) were supplemented to the media during oocyte maturation. Intracellular GSH concentrations were recorded at 48 h of maturation and nuclear maturation and fertilization were analyzed 12 h after IVF. Embryo development was analyzed at 48 h and 144 h after IVF or intracytoplasmic sperm injection (ICSI). Supplementation of antioxidants had no effect on intracellular levels of GSH, nuclear maturation or fertilization traits. Blastocyst formation for NAC (35.0 ± 7.4%) and NACA (40.0 ± 7.4%) supplementation were higher (P < 0.05) than the control (20.0 ± 7.4%) and GSH supplemented (20 ± 7.4%) oocytes. The same pattern was seen for ICSI-derived embryos: blastocyst formation for NAC (22.0 ± 5.9%) and NACA (25.0 ± 4.6%) supplementation were higher (P < 0.05) than the un-supplemented (10.0 ± 6.0%) oocytes. There were no differences between NAC and NACA supplementation and there were no differences between the cleavage rates for any of the treatment groups. These results indicate that supplementing 1.0 mM of NAC or NACA to the oocyte maturation medium and the ICSI medium increased the percentage of viable embryos reaching the blastocyst stage of development, and could warrant further investigation. The next study was conducted to evaluate the effects of different concentrations of NAC supplemented to the maturation medium on embryo development. Comparisons of significant concentrations of NAC and NACA on embryo development were evaluated for nuclear maturation, fertilization success and embryo development. Concentrations of NAC (0, 0.5, 1.0, 1.5, 2.0, 2.5, 5.0 mM) were supplemented to maturing oocytes and embryo development was analyzed at 48 h and 144 h post-fertilization. There were no differences between cleavage rates for any of the treatment groups. Blastocyst formation for 1.5 mM NAC (56.5 ± 9.2%) was significantly higher (P < 0.05) than all other supplementations. There were no differences in nuclear maturation or fertilization when comparing 1.5 mM NAC and 1.5 mM NACA supplementation to the maturation media. There was no difference between cleavage rates of 1.5 NAC and 1.5 mM NACA supplementation to the maturation media. Blastocyst formation for 1.5 mM NAC (44.4 ± 4.7%) and 1.5 mM NACA (46.2 ± 3.4%) supplementation were significantly higher (P < 0.05) than the control (32.1 ± 6.2%) oocytes. These results indicate that supplementing 1.5 mM of NAC or NACA to the oocyte maturation medium increased the percentage of viable embryos reaching the blastocyst stage of development and could be used during the oxidative stress experiments. In the final study, the mechanisms of oxidative stress in maturing oocytes were studied in addition to evaluating the effects of antioxidant supplementation to the media. This study focused on superoxide dismutase (SOD), GSH peroxidase, catalase and intracellular GSH concentrations with respect to DNA fragmentation evaluated using the single cell Comet assay. Results indicate that when SOD was inhibited, the GSH peroxide levels and length of DNA migration significantly increased (P < 0.05). Catalase levels significantly decreased (P < 0.05) and intracellular GSH remained unchanged. When GSH peroxidase was inhibited, the SOD levels and catalase levels significantly decreased (P < 0.05) but the intracellular GSH and DNA migration length significantly increased (P < 0.05). The supplementation of 1.5 mM NAC and 1.5 mM NACA had multiple effects on the enzyme levels. Specifically, supplementation of 1.5 mM NAC or 1.5 mM NACA significantly decreased (P < 0.05) the length of DNA migration when other enzymes were inhibited compared to no antioxidant supplementation. These results indicate that antioxidant supplementation may alleviate the free radicals associated with oxidative stress in the maturing porcine oocyte. In conclusion, supplementing the antioxidants NAC or NACA to the oocyte maturation media does not have negative effects on IVF or embryo culture. Supplementation of NACA increases the number of oocytes reaching the blastocyst stage of development. Glutathione, SOD, catalase, and GSH peroxidase are all required to be functional during oocyte development to alleviate oxidative stress on the oocyte. Antioxidants enhance the enzyme activity during oocyte maturation and may even contribute to protecting the oocyte when enzyme activity is impaired. / Ph. D.

published_or_final_version / Zoology / Doctoral / Doctor of Philosophy

published_or_final_version / Anatomy / Doctoral / Doctor of Philosophy

published_or_final_version / Biological Sciences / Doctoral / Doctor of Philosophy

The Best PhD Thesis in the Faculties of Dentistry, Engineering, Medicine and Science (University of Hong Kong), Li Ka Shing Prize,2008-2009 / published_or_final_version / Biological Sciences / Doctoral / Doctor of Philosophy

It has been suggested that cellular damage from oxygen radicals is one of the processes leading to cardio-vascular disease and cancer. Natural antioxidants prevent uncontrolled oxidative reactions by decreasing molecular oxygen levels, scavenging chain-initiating and chain-propagating free radicals, chelating metals, or decomposing peroxides. Beer is rich in antioxidants, derived both from malt and hops, consisting mostly of flavanoids and phenolic secondary plant metabolites. Much research has been conducted concerning antioxidant activity of beer in relationship to flavor stability. Yeast cells possess both enzymatic and non-enzymatic antioxidant systems to defend against oxygen radicals, in addition to scavenging and absorbing molecular oxygen for cell synthesis. It is well known that bottle-conditioned beer has a longer shelf life than conventional beer in terms of flavor stability and freshness. This is likely due to a complex relationship between the yeasts inherent ability to scavenge oxygen species, produce SO₂, chelate transition metals and employ other methods to defend against molecular oxygen. The objective of this research was to determine whether bottle-conditioned beer (which contains live yeast) has a higher antioxidant activity compared to that of conventional beer. Initial experiments were conducted to establish a baseline of antioxidant potentials. The first experiment consisted of determining the antioxidant potential of commercially available beers and how those values compared to common foods and drinks. Next, live yeast was added to commercially available artificially carbonated beer, to determine whether the presence of live yeast alone had an impact on antioxidant potential. Lastly, in the first set of beer trials wort was prepared, brewers yeast added, and then allowed to complete primary fermentation. The beer was bottle-conditioned (naturally carbonated) by the addition of a second yeast strain and either a 'high' or 'low' level of sugar, or artificially carbonated. Treatments consisted of using three different yeasts. A control was prepared by artificially carbonating beer without live yeast. Antioxidant potentials were determined using Ferric Reducing Antioxidant Potential (FRAP) analysis. Results indicated that there was an increase in the level of antioxidant activities between the bottle-conditioned beers compared to the control beer, whether or not the high or low level of yeast was present. In the second set of beer trials wort was prepared, brewers yeast added, and then allowed to complete primary fermentation, as in the first set of beer trials. The beer was bottle-conditioned by the addition of a second yeast strain and sugar, or artificially carbonated followed by the addition of a second yeast but no sugar. Treatments consisted of using three different yeasts. A control was prepared by artificially carbonating beer without live yeast. Dissolved oxygen, free and total sulfite, yeast viability, and antioxidant capacities were again determined. Results indicated that there was an increase in the level of sulfite, a decrease in dissolved oxygen, yeast remained viable for a longer period of time, and antioxidant activities were higher in the bottle-conditioned beers when compared to the control beer. Furthermore, while the differences were not as great, the same trends were observed for all parameters when comparing the artificially carbonated beers containing live yeast to the control beer. The elevation in antioxidant activities of beer with live yeast present (live beer) was significant. The third and final phase of experiments focused on the contribution of antioxidant potential specifically from the yeast. Yeast cells for each strain were cultivated in beer wort, harvested, washed, and cell extracts prepared. The crude yeast extracts were subjected to heat treatment, size fractionation followed by heat and protease treatments, glutathione determination, and lipid extraction, and then analyzed for antioxidant activity. Results indicated a complex interaction between many different yeast components that contributed to the total antioxidant activity provided specifically from yeast. Rather than one single compound, the yeast contributed heat stable components, consisting of proteins and enzymes, molecules with high, medium, and low molecular weights, and active lipid portions. The overall results suggest, that while the malt and hops components likely play the major role in antioxidant activity of beer, beer containing live yeast has a significant increase on that antioxidant activity. Consequently, the flavor stability and health benefits from beer containing live yeast would be increased. / Graduation date: 2003

No description available.

No description available.

The provitamin A carotenoid β-carotene is an attractive candidate for the prevention of cancer. Indeed, abundant evidence suggests that β-carotene inhibits carcinogenesis. β-Carotene is thought to inhibit carcinogenesis by scavenging free radicals involved in tumor formation. However, there is no direct evidence that β-carotene traps radicals under conditions where it inhibits carcinogenesis. The overall objective of this dissertation research was to identify β-carotene oxidation products from β-carotene antioxidant reactions in model systems. Identification of such products will enable the direct measurement of β-carotene antioxidant activity in systems where it inhibits neoplastic transformation. In hexane solution, β-carotene was oxidized by peroxyl radicals to 5,6-epoxy-β, β-carotene, 15,15'-epoxy-β, β-carotene, a previously unreported product, and several unidentified polar products. Studies on the kinetics of product formation suggested that polar products are formed by both epoxide-dependent and -independent pathways. Because β-carotene may be localized within lipid bilayers in vivo, peroxyl radical oxidation of β-carotene in model membranes was examined. In soy phosphatidylcholine liposomes, β-carotene was oxidized by peroxyl radicals to the 5,6-epoxide and to unidentified polar products. β-Carotene antioxidant activity in the liposome system was the same at 15 torr and 160 torr O₂ and decreased at 760 torr O₂. These results suggest that β-carotene provides equal antioxidant protection in all tissues in vivo. The relative rates of product formation and β-carotene oxidation at different pO₂ suggested that β-carotene antioxidant activity is governed by the relative proportions of β-carotene radical trapping and autoxidation reactions, which do not contribute to radical trapping. Therefore, the loss of β-carotene antioxidant action at 760 torr O₂ may result from an increase in β-carotene oxidation by autoxidation pathways. The 5,6-epoxide was formed during both antioxidant reactions and autoxidation reactions and may be marker for the peroxyl radical oxidation of β-carotene. Attempts to study β-carotene antioxidant reactions in biological membranes were only partially successful. In vitro incorporation of β-carotene into microsomes was attempted by several methods. However, these efforts resulted in only modest β-carotene antioxidant activity in microsomes. These studies provide a basic understanding of β-carotene antioxidant chemistry in model systems. Their results will enable further investigation of β-carotene antioxidant action in biological systems.

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