Made available in DSpace on 2014-06-11T19:29:15Z (GMT). No. of bitstreams: 0 Previous issue date: 2012-02-27Bitstream added on 2014-06-13T20:19:15Z : No. of bitstreams: 1 rocha_nas_me_jabo.pdf: 503011 bytes, checksum: f1714a5fae847fcec5ec5165da511227 (MD5) / Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) / O estudo foi conduzido com o objetivo de avaliar os efeitos da suplementação com antioxidantes intracelulares e extracelulares em diferentes etapas da PIV (MIV e/ou CIV), e da tensão de oxigênio durante o CIV sobre o desenvolvimento e criotolerância de embriões bovinos. No Exp.1 foi realizada a suplementação com antioxidantes {0,6 mM cisteína (CIST), 0,6 mM cisteína associado á 100 μM de cisteamina (C+C) e 100 UI catalase (CAT)} durante todo o período de CIV, em diferentes atmosferas gasosas {5% CO2 em ar (20% O2) ou atmosfera controlada (7% O2, 5% CO2 e 88% N2)}. Já, no Exp. 2 foi realizada a suplementação com antioxidantes {0,6 mM cisteína (CIST), 100 UI catalase (CAT) e 100 μM de β-mercaptoetanol (β-ME)} durante 72 horas de CIV, nas diferentes atmosferas gasosas. Posteriormente, após definir a tensão de oxigênio, bem como, o período de suplementação adequado para o CIV, foi realizada a adição de antioxidantes durante a maturação in vitro (MIV) e/ou 72 horas de CIV (Exp.3). No Exp.1, a taxa de desenvolvimento embrionário foi adversamente afetada (P<0,05) pelos tratamentos CIST (11,2%) e C+C (1,4%), em relação ao Controle (26,6%), e pela tensão de oxigênio (17,2% e 11,1%; 20 e 7% O2, respectivamente). Em relação à taxa de re-expansão, após reaquecimento e cultivo in vitro por 24 horas, não houve diferença significativa (P>0,05) entre os tratamentos avaliados (66,7% a 100%). No Exp.2, as taxas de blastocistos não foram afetadas (P>0,05) pelos tratamentos CIST, β-ME e CAT (43,7% a 48,5%), porém a baixa tensão de oxigênio afetou adversamente (P<0,05) o desenvolvimento embrionário (52,1% e 38,4%; 20 e 7% O2 respectivamente). A mensuração dos níveis intracelulares de ROS não foi afetada (P>0,05) pelas variáveis tratamentos (0,95 a 0,78) e tensão de oxigênio... / This study was conducted to evaluate the effects of intracellular and extracellular antioxidants supplementation, in different stages of IVP (IVM and/or IVC), and oxygen tension during IVC on development, quality and cryotolerance of bovine embryos. Exp.1 was performed with the supplementation with antioxidants {0.6 mM cysteine (CIST); 0.6 mM cysteine associated to 100 μM cysteamine (C+C); 100 UI catalase (CAT)} during entire period of IVC in different gaseous atmospheres {5% CO2 in air (20% O2) or controlled atmosphere (7% O2, 5% CO2 and 88% N2)}. Already, in Exp.2 was performed the antioxidant supplementation {0.6 mM cysteine (CIST); 100 μM β-mercaptoethanol (β-ME); 100 UI catalase (CAT)} for 72 hours of IVC in different gaseous atmospheres. Later, after setting the oxygen tension as well as the supplementation period suitable for IVC, was carried out the addition of antioxidants during in vitro maturation (IVM) and/or 72 hours of IVC (Exp.3). In Exp.1, the rate of embryo development was adversely affected (P<0.05) by the treatments CIST (11.2%) and C+C (1.4%), compared to Control (26.6%), and oxygen tension (17.2% and 11.1%, 20 and 7%O2, respectively). Regarding the re-expansion rate after warming and in vitro culture for 24 hours, no difference (P>0.05) between the treatments were found (66.7% to 100%). In Exp.2, blastocysts rates were not affected (P>0.05) by treatments CIST, β-ME and CAT (43.7% to 48.5%), but the low oxygen tension adversely affected (P<0.05) embryo development (52.1% to 38.4%, 20 and 7%O2, respectively). The quantification of intracellular levels of ROS was not affected (P>0.05) by the variables treatments (0.95 to 0.78) and oxygen tension... (Complete abstract click electronic access below)

Orientador: Gisele Zoccal Mingoti / Banca: Joaquim Mansano Garcia / Banca: Juliana Corrêa Borges Silva / Resumo: O estudo foi conduzido com o objetivo de avaliar os efeitos da suplementação com antioxidantes intracelulares e extracelulares em diferentes etapas da PIV (MIV e/ou CIV), e da tensão de oxigênio durante o CIV sobre o desenvolvimento e criotolerância de embriões bovinos. No Exp.1 foi realizada a suplementação com antioxidantes {0,6 mM cisteína (CIST), 0,6 mM cisteína associado á 100 μM de cisteamina (C+C) e 100 UI catalase (CAT)} durante todo o período de CIV, em diferentes atmosferas gasosas {5% CO2 em ar (20% O2) ou atmosfera controlada (7% O2, 5% CO2 e 88% N2)}. Já, no Exp. 2 foi realizada a suplementação com antioxidantes {0,6 mM cisteína (CIST), 100 UI catalase (CAT) e 100 μM de β-mercaptoetanol (β-ME)} durante 72 horas de CIV, nas diferentes atmosferas gasosas. Posteriormente, após definir a tensão de oxigênio, bem como, o período de suplementação adequado para o CIV, foi realizada a adição de antioxidantes durante a maturação in vitro (MIV) e/ou 72 horas de CIV (Exp.3). No Exp.1, a taxa de desenvolvimento embrionário foi adversamente afetada (P<0,05) pelos tratamentos CIST (11,2%) e C+C (1,4%), em relação ao Controle (26,6%), e pela tensão de oxigênio (17,2% e 11,1%; 20 e 7% O2, respectivamente). Em relação à taxa de re-expansão, após reaquecimento e cultivo in vitro por 24 horas, não houve diferença significativa (P>0,05) entre os tratamentos avaliados (66,7% a 100%). No Exp.2, as taxas de blastocistos não foram afetadas (P>0,05) pelos tratamentos CIST, β-ME e CAT (43,7% a 48,5%), porém a baixa tensão de oxigênio afetou adversamente (P<0,05) o desenvolvimento embrionário (52,1% e 38,4%; 20 e 7% O2 respectivamente). A mensuração dos níveis intracelulares de ROS não foi afetada (P>0,05) pelas variáveis tratamentos (0,95 a 0,78) e tensão de oxigênio... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: This study was conducted to evaluate the effects of intracellular and extracellular antioxidants supplementation, in different stages of IVP (IVM and/or IVC), and oxygen tension during IVC on development, quality and cryotolerance of bovine embryos. Exp.1 was performed with the supplementation with antioxidants {0.6 mM cysteine (CIST); 0.6 mM cysteine associated to 100 μM cysteamine (C+C); 100 UI catalase (CAT)} during entire period of IVC in different gaseous atmospheres {5% CO2 in air (20% O2) or controlled atmosphere (7% O2, 5% CO2 and 88% N2)}. Already, in Exp.2 was performed the antioxidant supplementation {0.6 mM cysteine (CIST); 100 μM β-mercaptoethanol (β-ME); 100 UI catalase (CAT)} for 72 hours of IVC in different gaseous atmospheres. Later, after setting the oxygen tension as well as the supplementation period suitable for IVC, was carried out the addition of antioxidants during in vitro maturation (IVM) and/or 72 hours of IVC (Exp.3). In Exp.1, the rate of embryo development was adversely affected (P<0.05) by the treatments CIST (11.2%) and C+C (1.4%), compared to Control (26.6%), and oxygen tension (17.2% and 11.1%, 20 and 7%O2, respectively). Regarding the re-expansion rate after warming and in vitro culture for 24 hours, no difference (P>0.05) between the treatments were found (66.7% to 100%). In Exp.2, blastocysts rates were not affected (P>0.05) by treatments CIST, β-ME and CAT (43.7% to 48.5%), but the low oxygen tension adversely affected (P<0.05) embryo development (52.1% to 38.4%, 20 and 7%O2, respectively). The quantification of intracellular levels of ROS was not affected (P>0.05) by the variables treatments (0.95 to 0.78) and oxygen tension... (Complete abstract click electronic access below) / Mestre

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Graduation date: 1996

In order to complete their life cycles, vertebrates require oxygen and water. However, environments are not always forgiving when it comes to constantly providing these basic needs for vertebrate life. The annual killifish Austrofundulus limnaeus is possibly the most well described extremophile vertebrate and its embryos have been shown to tolerate extremes in oxygen, salinity, and water availability. This phenotype is likely a result of the annual killifish life history, which includes periods of temporary habitat desiccation and oxygen deprivation, and requires the production of stress-tolerant embryos that depress metabolism in a state of suspended animation, known as diapause. Over the past several decades, the basic morphology and physiology of annual killifish development has become better characterized. However, there are still basic cellular processes that remain to be described in annual killifish such as A. limnaeus. Specifically, changes in DNA structure, expression, and copy number are known to have profound impacts on the phenotype and survival of an organism. Little is known as to how A. limnaeus maintains genome integrity during cell stress, nor how the A. limnaeus nuclear and mitochondrial genomes may have evolved under the unpredictable conditions in which A. limnaeus thrive. Early annual killifish embryonic development is also characterized by a complete dispersion and subsequent reaggregation of embryonic blastomeres prior to formation of the embryonic axis. This unusual period of early development may provide a functional adaptation that allows annual killifish embryos to survive these extreme conditions. The overall goals of this project were to (1) characterize the ability of A. limnaeus to tolerate and repair DNA damage through enzymatic and developmental mechanisms, (2) to determine possible consequences of mitochondrial DNA sequence and copy number on the metabolism of A. limnaeus, and (3) to establish a draft genome of A. limnaeus for future comparative genome studies. The results of this project show that embryos of A. limnaeus have an impressive ability to survive and reverse high doses of DNA damage induced by ultraviolet-C (UV-C) radiation, especially when allowed to recover under photoreactivating light. Surprisingly, embryos that survived irradiation during blastomere dispersion phases were able to develop normally. Characterization of gene expression during embryonic development for genes important for axis formation and cellular differentiation suggests that A. limnaeus embryos may delay axis formation until several days after epiboly is complete, thus allowing time for cells that become damaged to be replaced by surrounding pluripotent cells. This outcome would represent first case of a developmental buffering stage in a vertebrate. A. limnaeus embryos are also unique in their mitochondrial response to anoxia. Whereas in other species the amount of mitochondrial DNA (mtDNA) copy number fluctuates following extremes in oxygen availability, A. limnaeus embryonic mtDNA remains stable. Additionally, characterization of the fully sequenced A. limnaeus mitochondrial genome reveals possible evolutionary adaptations that may have facilitated dormancy and anoxia tolerance when compared to other species within the Order Cyprinodontiformes. The final chapter of this project characterizes the draft genome of A. limnaeus and I provide evidence suggesting that epigenetic DNA methylation that may be involved in regulating diapause.

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Call number: LD2668 .T4 1984 S88 / Master of Science