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Made available in DSpace on 2014-06-11T19:29:16Z (GMT). No. of bitstreams: 0 Previous issue date: 2006-09-01Bitstream added on 2014-06-13T20:59:35Z : No. of bitstreams: 1 ponchirolli_cb_me_botfmvz.pdf: 354035 bytes, checksum: 36e40571a8b77281cc6690316cdc226b (MD5) / Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) / As técnicas de produção in vitro de embriões bovinos ainda precisam ser melhoradas em relação às taxas de embriões produzidos e sua qualidade. A adição de fatores de crescimento aos meios de cultivo de embriões bovinos tem sido estudada, com o intuito de melhorar a produção e a qualidade embrionária. O objetivo deste experimento foi testar a adição de GH, IGF-1 e Insulina em duas etapas do processo de produção in vitro: na maturação dos ovócitos e no cultivo dos embriões, visando aumentar as taxas de produção de blastocistos e a qualidade dos embriões produzidos, além de verificar a viabilidade de uma coloração com acridina orange e iodeto de propídeo para avaliação da qualidade dos embriões. No experimento 1, avaliou-se as taxas de clivagem e formação de blastocistos após maturação ovocitária em meio 199 (sais de Earle e L-glutamina e 2,2 mg/ml de bicarbonato de sódio, médium 199, Sigma, Saint Louis, EUA, # M-4530 ), na presença de 10% de SFB, de sódio, 1 mg/ml de 17 estradiol (E2 - Sigma, Saint Louis, EUA, # E-2758), 50 g/ml de hCG (Profasi® 5.000UI), 5 g/ml de FSH (Folltropin-V®, Vetrepharm, Ontário, Canadá), e 75æg/ml de sulfato de gentamicina, suplementado com IGF-1 , GH, Insulina, adicionados separadamente ou em conjunto. No experimento 2 foram avaliadas as taxas de clivagem e formação de blastocistos em meio composto por HTF (Human Tubal Fluid, HTF , Irvane) e BME (Basal Medium Eagle, BME , Sigma, Saint Louis, EUA), na proporção de 1:1 (HTF:BME) acrescido de 0,6% de BSA, 0,01% de Mioinositol e 75 g/ml de gentamicina, incubados em estufa a 38, 5oC, a 5% de CO2 e umidade absoluta, com suplementação de 10% de SFB no dia 3 de cultivo. O meio foi suplementado com IGF-1, GH, Insulina,adicionados separadamente ou em conjunto. No experimento 3, a coloração com acridina orange e iodeto de propídeo foi testada para avaliar a qualidade das estruturas produzidas. / In vitro embryo production techniques in bovines still need to be improved due to the embryo production rate and the quality. The addition of growth factors to the bovine embryo culture medium has been studied due to their effects on the production and embryo quality. The objective of this experiment was to study the addition of GH, IGF-1 and Insulin at two steps of the in vitro embryo production such as oocyte maturation and embryo culture, aiming at to increase the blastocysts rates as well their quality, as evidenced by the viability after staining with acridine orange and propideium iodete. In experiment 1, the cleavage rate and blastocyst production were evaluated after oocyte maturation in medium 199 with Earle's salt, L-glutamine and 2.2 mg/ml sodium bicarbonate, medium 199, Sigma, Saint Louis, USA, # M-4530) plus 10% BFS, 50 æg /ml sodium piruvate, 1 mg/ml estradiol 17 ß (E2 - Sigma, Saint Louis, USA., # E-2758), 50æg/ml hCG (Profasi® 5.000UI), 5 æg/ml FSH (Foltropin-V®, Vetrepharm, Ontário, Canada), and 75æg/ml gentamicin sulfate, supplemented either with IGF-1, GH, Insulin alone or in combination. In experiment 2, the cleavage rate and blastocyst formation were evaluated after in vitro embryo culture in HTF (Human Tubal Fluid, HTF ®, Irvane) and BME (Basal Medium Eagle, BME ®, Sigma), in 1:1 proportion (HTF: BME) plus 0.6% of BSA, 0.01% of Mioynositol and 75 æg/ml of gentamicin sulfate, at 38, 5ºC, 5% of CO2 and absolute humidity atmosphere, supplemented with 10% FCS at day 3 of culture. The medium was supplemented with IGF-1, GH, Insulin alone or in combination. In experiment 3, the acridine orange and propideium iodete stain was used to evaluate the quality of produced morulae produced as in experiment 1 or 2. The number and percentage of cells with DNA fragmentation was used to determine marulae quality.

Orientador: João Batista Alcântara Oliveira / Banca: Anice Maria Vieira de Camargo Martins / Banca: Claudia Guilhermino Petersen / Resumo: Apesar do conhecimento de que os processos de desenvolvimento e implantação embrionários de mamíferos não ocorrem em concentração atmosférica in vivo, é prática comum em laboratórios de fertilização in vitro (FIV) a cultura de embriões em concentração próxima a 20% de oxigênio. A discrepância entre as concentrações de oxigênio leva a importantes discussões sobre seus efeitos no desenvolviemento embrionário in vitro. Acredita-se que o cultivo de embriões em ambientes com concentração atmosférica de oxigênio pode ser prejudicial à qualidade dos mesmos por predispô-los à interferência negativa de radicais livres de oxigênio (RLO), resultantes do estresse oxidativo, levando a ação deletéria sobre o metabolismo embrionário e a expressão gênica de blastocitos cultivados naquelas condições. Frente aos argumentos favoráveis e contrários a cultura de embriões in vitro em concentrações de oxigênio semelhantes às encontradas in vivo, este artigo de revisão pretende condensar e debater os dados da literatura acerca da influência da concentração de oxigênio sobre a qualidade de embriões cultivados in vitro / Abstract: Despite the fact that the processes of embryonic development and deployment of mammals do not occur in atmospheric concentration in vivo, it is common practice in laboratories for in vitro fertilization (IVF) the culture of embryos in a concentration close to 20% of oxygen. The discrepancy between the concentrations of oxygen leads to important discussions about their effects on in vitro embryonic development. It is believed that the cultivation of embryos in atmospheric concentration of oxygen can be detrimental to the quality of the data for predisposing them to the negative interference of reactive oxygen species (ROS), resulting in oxidative stress, leading to deleterious effects on metabolism embryonic gene expression and blastocysts grown in those conditions. Faced with arguments for and against the culture of embryos in vitro oxygen concentrations similar to those found in vivo, this review article aims to condense and discuss the literature concerning the influence of oxygen concentration on the quality of embryos cultured in vitro / Mestre

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Orientador: Fernanda da Cruz Landim-Alvarenga / Banca: Alício Martins Junior / Banca: Yeda Fumie Watanabe / Resumo: As técnicas de produção in vitro de embriões bovinos ainda precisam ser melhoradas em relação às taxas de embriões produzidos e sua qualidade. A adição de fatores de crescimento aos meios de cultivo de embriões bovinos tem sido estudada, com o intuito de melhorar a produção e a qualidade embrionária. O objetivo deste experimento foi testar a adição de GH, IGF-1 e Insulina em duas etapas do processo de produção in vitro: na maturação dos ovócitos e no cultivo dos embriões, visando aumentar as taxas de produção de blastocistos e a qualidade dos embriões produzidos, além de verificar a viabilidade de uma coloração com acridina orange e iodeto de propídeo para avaliação da qualidade dos embriões. No experimento 1, avaliou-se as taxas de clivagem e formação de blastocistos após maturação ovocitária em meio 199 (sais de Earle e L-glutamina e 2,2 mg/ml de bicarbonato de sódio, médium 199, Sigma, Saint Louis, EUA, # M-4530 ), na presença de 10% de SFB, de sódio, 1 mg/ml de 17 estradiol (E2 - Sigma, Saint Louis, EUA, # E-2758), 50 g/ml de hCG (Profasi® 5.000UI), 5 g/ml de FSH (Folltropin-V®, Vetrepharm, Ontário, Canadá), e 75æg/ml de sulfato de gentamicina, suplementado com IGF-1 , GH, Insulina, adicionados separadamente ou em conjunto. No experimento 2 foram avaliadas as taxas de clivagem e formação de blastocistos em meio composto por HTF (Human Tubal Fluid, HTF , Irvane) e BME (Basal Medium Eagle, BME , Sigma, Saint Louis, EUA), na proporção de 1:1 (HTF:BME) acrescido de 0,6% de BSA, 0,01% de Mioinositol e 75 g/ml de gentamicina, incubados em estufa a 38, 5oC, a 5% de CO2 e umidade absoluta, com suplementação de 10% de SFB no dia 3 de cultivo. O meio foi suplementado com IGF-1, GH, Insulina,adicionados separadamente ou em conjunto. No experimento 3, a coloração com acridina orange e iodeto de propídeo foi testada para avaliar a qualidade das estruturas produzidas. / Abstract: In vitro embryo production techniques in bovines still need to be improved due to the embryo production rate and the quality. The addition of growth factors to the bovine embryo culture medium has been studied due to their effects on the production and embryo quality. The objective of this experiment was to study the addition of GH, IGF-1 and Insulin at two steps of the in vitro embryo production such as oocyte maturation and embryo culture, aiming at to increase the blastocysts rates as well their quality, as evidenced by the viability after staining with acridine orange and propideium iodete. In experiment 1, the cleavage rate and blastocyst production were evaluated after oocyte maturation in medium 199 with Earle's salt, L-glutamine and 2.2 mg/ml sodium bicarbonate, medium 199, Sigma, Saint Louis, USA, # M-4530) plus 10% BFS, 50 æg /ml sodium piruvate, 1 mg/ml estradiol 17 ß (E2 - Sigma, Saint Louis, USA., # E-2758), 50æg/ml hCG (Profasi® 5.000UI), 5 æg/ml FSH (Foltropin-V®, Vetrepharm, Ontário, Canada), and 75æg/ml gentamicin sulfate, supplemented either with IGF-1, GH, Insulin alone or in combination. In experiment 2, the cleavage rate and blastocyst formation were evaluated after in vitro embryo culture in HTF (Human Tubal Fluid, HTF ®, Irvane) and BME (Basal Medium Eagle, BME ®, Sigma), in 1:1 proportion (HTF: BME) plus 0.6% of BSA, 0.01% of Mioynositol and 75 æg/ml of gentamicin sulfate, at 38, 5ºC, 5% of CO2 and absolute humidity atmosphere, supplemented with 10% FCS at day 3 of culture. The medium was supplemented with IGF-1, GH, Insulin alone or in combination. In experiment 3, the acridine orange and propideium iodete stain was used to evaluate the quality of produced morulae produced as in experiment 1 or 2. The number and percentage of cells with DNA fragmentation was used to determine marulae quality. / Mestre

Made available in DSpace on 2014-06-11T19:29:51Z (GMT). No. of bitstreams: 0 Previous issue date: 2011-08-22Bitstream added on 2014-06-13T18:59:45Z : No. of bitstreams: 1 gomessobrinho_db_me_botfm.pdf: 442419 bytes, checksum: 1fc08e81e15251258b8a19119e2337c2 (MD5) / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / Apesar do conhecimento de que os processos de desenvolvimento e implantação embrionários de mamíferos não ocorrem em concentração atmosférica in vivo, é prática comum em laboratórios de fertilização in vitro (FIV) a cultura de embriões em concentração próxima a 20% de oxigênio. A discrepância entre as concentrações de oxigênio leva a importantes discussões sobre seus efeitos no desenvolviemento embrionário in vitro. Acredita-se que o cultivo de embriões em ambientes com concentração atmosférica de oxigênio pode ser prejudicial à qualidade dos mesmos por predispô-los à interferência negativa de radicais livres de oxigênio (RLO), resultantes do estresse oxidativo, levando a ação deletéria sobre o metabolismo embrionário e a expressão gênica de blastocitos cultivados naquelas condições. Frente aos argumentos favoráveis e contrários a cultura de embriões in vitro em concentrações de oxigênio semelhantes às encontradas in vivo, este artigo de revisão pretende condensar e debater os dados da literatura acerca da influência da concentração de oxigênio sobre a qualidade de embriões cultivados in vitro / Despite the fact that the processes of embryonic development and deployment of mammals do not occur in atmospheric concentration in vivo, it is common practice in laboratories for in vitro fertilization (IVF) the culture of embryos in a concentration close to 20% of oxygen. The discrepancy between the concentrations of oxygen leads to important discussions about their effects on in vitro embryonic development. It is believed that the cultivation of embryos in atmospheric concentration of oxygen can be detrimental to the quality of the data for predisposing them to the negative interference of reactive oxygen species (ROS), resulting in oxidative stress, leading to deleterious effects on metabolism embryonic gene expression and blastocysts grown in those conditions. Faced with arguments for and against the culture of embryos in vitro oxygen concentrations similar to those found in vivo, this review article aims to condense and discuss the literature concerning the influence of oxygen concentration on the quality of embryos cultured in vitro

This study investigated the development potential of equine embryos in vitro after biopsy and vitrification. Twenty embryos were obtained from Quarter Horse, Thoroughbred, and mix-breed light mares between three and ten years old. The twenty embryos were divided into a biopsy (n=10) and control group (n=10). The biopsy group underwent microaspiration biopsy using a micromanipulator to obtain a small tissue sample from the embryo. Both groups were then vitrified using a commercially available technique originally described by Carnevale (2006) at Colorado State \ University. All 20 embryos were cultured in DMEM/Hams F-12 medium under oil at 37°C in 5% CO2 in air (Hinrichs et al., 1990). Embryos were monitored for expansion and hatching. Embryo development was statistically different between the two groups (p<0.05). The biopsy procedure did result in a much lower development potential in the biopsy group as compared to the control group (20% vs. 80%). However, embryos in the biopsy group did show expansion and hatching therefore the combined procedure did not preclude development potential in vitro. Based on these findings, more research needs to be done to increase the success of the combined procedure and the ultimate viability of the embryos needs to be confirmed with the establishment of successful pregnancies.

It is recognised that the environment to which the fetus is exposed in utero, after implantation, can program longer term health outcomes and alter the possibility of disease onset later in life. It is becoming evident that the environment, to which the pre-implantation embryo is exposed, can also affect the ability of the embryo to form a viable pregnancy as well as altering fetal growth. Despite this understanding, little is known about the mechanism by which the environment can ‘program’ the pre-implantation embryo. Using model stress systems, either ammonium or DMO in the culture medium, this thesis addressed the hypothesis that suboptimal environmental conditions may alter mitochondrial homeostasis and function and/or epigenetic parameters and these are the possible mechanisms responsible for the altered fetal outcomes seen. While common measures of embryo quality such as on time blastocyst development were not affected by either stress, more in-depth investigations found several striking differences. Exposure to DMO significantly decreased blastocyst cell number and allocation to the inner cell mass and trophectoderm, as well as increased blastocyst apoptosis. After exposure to DMO, blastocysts were transferred to pseudopregnant recipients, and both the ability of the embryos to implant and develop into a fetus was impaired as well as fetal weights and crown rump length were significantly reduced indicative of altered growth. Similar results have also been demonstrated after pre-implantation embryos are exposed to ammonium in vitro. Exposure to ammonium during pre-implantation embryo development also altered placental gene expression and function, indicating a possible mechanism of the observed reduced fetal growth parameters. Interestingly, the pre-implantation embryo appears to be the most vulnerable to an environmental stress during the pre-compaction stage, in particular the zygote to 2-cell transition, as exposure to either stress during this stage alone shows similar perturbations to if the stress was present for the entire pre-implantation developmental period. At this early stage of embryo development, mitochondria are the sole energy generators and are therefore critical for embryo function. This study determined that either ammonium or DMO stress exposure, during the first cleavage division, significantly perturbed mitochondrial distribution, membrane potential and ATP/ADP levels. Removal of the stress did not allow these effects to be completely reversed, implicating mitochondrial perturbations as a possible mechanism behind altered embryo programming. During pre-implantation embryo development there are also significant epigenetic changes which are vital for re-programming the embryonic genome. Both in vitro stresses significantly altered DNA de-methylation at the 2-cell stage and reduced blastocyst gene expression levels of DNA methyltransferases (Dnmt3a and Dnmt3b), which are responsible for de novo methylation. Together these data highlight the importance of pre-implantation embryo development as a critical period of growth in which the presence of environmental stress can have an impact on metabolic homeostasis and critical epigenetic events that may be responsible for the downstream effects seen on fetal growth. These results are not only important for assisted reproductive therapy, where the presence of an in vitro laboratory stress can potentially alter embryo programming, but are also important for in vivo embryo development where the health and wellbeing of the mother can also potentially influence the in utero environment and thus the long-term health outcomes of her child. / http://proxy.library.adelaide.edu.au/login?url= http://library.adelaide.edu.au/cgi-bin/Pwebrecon.cgi?BBID=1522143 / Thesis (Ph.D.) -- University of Adelaide, School of Paediatrics and Reproductive Health, 2010