Desenvolvimento de embriões in vitro na presença de IGF-1, GH e insulina nos meios de maturação de ovócitos e cultivo embrionário
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Data
2006-09-01
Autores
Ponchirolli, Carla Bianchini [UNESP]
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Universidade Estadual Paulista (Unesp)
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.
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.
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.
Descrição
Palavras-chave
Bovino - Reprodução, Bovines - Embryo culture
Como citar
PONCHIROLLI, Carla Bianchini. Desenvolvimento de embriões in vitro na presença de IGF-1, GH e insulina nos meios de maturação de ovócitos e cultivo embrionário. 2006. 81 f. Dissertação (mestrado) - Universidade Estadual Paulista, Faculdade de Medicina Veterinária e Zootecnia, 2006.