Efeito dos meios básicos de cultivo, IGF-1 e GH, no desenvolvimento in vitro de folículos pré-antrais bovinos inclusos em tecido ovariano

Detalhes bibliográficos
Autor(a) principal: Jimenez, Carolina Rodriguez
Data de Publicação: 2014
Tipo de documento: Tese
Idioma: por
Título da fonte: LOCUS Repositório Institucional da UFV
Texto Completo: http://locus.ufv.br/handle/123456789/1877
Resumo: The objectives of this study were (I) to determine the best basic culture medium for bovine preantral follicles culture enclosed in ovarian cortical tissue (in situ), using the minimum essential medium (α-MEM), tissue medium culture 199 (TCM-199) and medium McCoy s (McCoy s); (II) to evaluate different concentrations of Insulin like growth factor 1 (IGF-1) and growth hormone (GH) in the development of bovine preantral follicles in situ cultured and (III) to evaluate a sequential medium containing GH and, or, IGF-1 on long-term in situ culture of bovine preantral follicles. Ovaries of cows without defined breed standard were collected from local slaughterhouse and transported to the laboratory in TCM-199 supplemented with HEPES, sodium bicarbonate, pyruvic acid, penicillin and streptomycin. In the laboratory the ovarian cortex was fragmented and was taken for short-term cultures (7 days) on phases I, II and long-term (14 days) on phase III. The culture was performed in an incubator at 37 °C and 5 % CO2 in a 24 well plate with complete medium exchange every 2 days. Uncultured (control) and cultured ovarian fragments were processed for histological evaluation (morphological and morphometric) and evaluated the percentage of normal morphologically follicles, primordial and developing (primary and secondary) follicles, beyond the follicular and oocyte diameter. In addition, viability was assessed by trypan blue staining, the follicles classified as being stained and unstained, dead and living, respectively. The morphological and morphometric variables were analyzed by ANOVA followed by comparisons of means by Tukey and Dunnett s test and viability variables were subjected to chi-square (χ2) test. The results of Phase I showed that α- MEM supplemented (α-MEM+) preserved follicular morphology and it highlighted in morphometric and viability analysis. Consolidating the results of phase I, on phase II we used the α-MEM+ with different concentrations of recombinant human IGF-1 (0, 30, 70, 100 ng/mL) or recombinant human GH (0, 10, 25, 50 ng/mL), composing the experiment 1 and 2, respectively. The results showed that IGF-1 (30 ng/ml) and GH (50 ng/mL) treatments were the most effective concentration to maintain morphological and morphometric functionality, as well as the viability of in situ cultured of bovine preantral follicles. On phase III ovarian fragments were cultured in α-MEM+ in the absence or presence of GH (50 ng/mL) and, or, IGF-1 (30 ng/mL) added as follows: (α- MEM+) for 14 days, (GH→IGF-1) GH from 1 st day to 7th day and IGF-1 from 8th day to 14th day, (GH→IGF-1) IGF-1 from 1st day to 7th day, and GH from8 th day to 14th day and (GH+IGF-1) for 14 days. The treatment (GH→IGF-1) maintained the percentage of normal morphology follicles during the whole culture period and the percentage of developing follicles increased compared to fresh control after 7 days of culture, while follicular diameter increased in both 7th and 14th days of culture. Therefore, it is concluded that in situ cultured bovine preantral follicles in sequential medium treated with GH→IGF-1 improved the development and follicular diameter until the first half of the culture and kept these parameters with normality and viability until the end of in vitro culture.
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spelling Jimenez, Carolina Rodriguezhttp://lattes.cnpq.br/3000028594640502Araújo, Valdevane Rochahttp://lattes.cnpq.br/8652126082521493Torres, Ciro Alexandre Alveshttp://buscatextual.cnpq.br/buscatextual/visualizacv.do?id=K4787213D4Guimarães, José Domingoshttp://buscatextual.cnpq.br/buscatextual/visualizacv.do?id=K4782270U6Neves, Mariana Machadohttp://lattes.cnpq.br/78801164996922312015-03-26T12:54:58Z2015-01-232015-03-26T12:54:58Z2014-02-26JIMENEZ, Carolina Rodriguez. Effects of basic culture medium, IGF-1 and GH in development in vitro of bovine preantral follicles enclosed in ovarian tissue. 2014. 95 f. Tese (Doutorado em Genética e Melhoramento de Animais Domésticos; Nutrição e Alimentação Animal; Pastagens e Forragicul) - Universidade Federal de Viçosa, Viçosa, 2014.http://locus.ufv.br/handle/123456789/1877The objectives of this study were (I) to determine the best basic culture medium for bovine preantral follicles culture enclosed in ovarian cortical tissue (in situ), using the minimum essential medium (α-MEM), tissue medium culture 199 (TCM-199) and medium McCoy s (McCoy s); (II) to evaluate different concentrations of Insulin like growth factor 1 (IGF-1) and growth hormone (GH) in the development of bovine preantral follicles in situ cultured and (III) to evaluate a sequential medium containing GH and, or, IGF-1 on long-term in situ culture of bovine preantral follicles. Ovaries of cows without defined breed standard were collected from local slaughterhouse and transported to the laboratory in TCM-199 supplemented with HEPES, sodium bicarbonate, pyruvic acid, penicillin and streptomycin. In the laboratory the ovarian cortex was fragmented and was taken for short-term cultures (7 days) on phases I, II and long-term (14 days) on phase III. The culture was performed in an incubator at 37 °C and 5 % CO2 in a 24 well plate with complete medium exchange every 2 days. Uncultured (control) and cultured ovarian fragments were processed for histological evaluation (morphological and morphometric) and evaluated the percentage of normal morphologically follicles, primordial and developing (primary and secondary) follicles, beyond the follicular and oocyte diameter. In addition, viability was assessed by trypan blue staining, the follicles classified as being stained and unstained, dead and living, respectively. The morphological and morphometric variables were analyzed by ANOVA followed by comparisons of means by Tukey and Dunnett s test and viability variables were subjected to chi-square (χ2) test. The results of Phase I showed that α- MEM supplemented (α-MEM+) preserved follicular morphology and it highlighted in morphometric and viability analysis. Consolidating the results of phase I, on phase II we used the α-MEM+ with different concentrations of recombinant human IGF-1 (0, 30, 70, 100 ng/mL) or recombinant human GH (0, 10, 25, 50 ng/mL), composing the experiment 1 and 2, respectively. The results showed that IGF-1 (30 ng/ml) and GH (50 ng/mL) treatments were the most effective concentration to maintain morphological and morphometric functionality, as well as the viability of in situ cultured of bovine preantral follicles. On phase III ovarian fragments were cultured in α-MEM+ in the absence or presence of GH (50 ng/mL) and, or, IGF-1 (30 ng/mL) added as follows: (α- MEM+) for 14 days, (GH→IGF-1) GH from 1 st day to 7th day and IGF-1 from 8th day to 14th day, (GH→IGF-1) IGF-1 from 1st day to 7th day, and GH from8 th day to 14th day and (GH+IGF-1) for 14 days. The treatment (GH→IGF-1) maintained the percentage of normal morphology follicles during the whole culture period and the percentage of developing follicles increased compared to fresh control after 7 days of culture, while follicular diameter increased in both 7th and 14th days of culture. Therefore, it is concluded that in situ cultured bovine preantral follicles in sequential medium treated with GH→IGF-1 improved the development and follicular diameter until the first half of the culture and kept these parameters with normality and viability until the end of in vitro culture.Os objetivos deste estudo foram (1) determinar o melhor meio-base para o cultivo de folículos pré-antrais bovinos inclusos no tecido cortical ovariano (in situ), utilizando o meio essencial mínimo (α-MEM), meio de cultivo de tecido 199 (TCM-199) e meio McCoy s (McCoy s); (2) avaliar diferentes concentrações do fator de crescimento semelhante à insulina 1 (IGF-1) e do hormônio do crescimento (GH) no desenvolvimento dos folículos pré-antrais bovinos cultivados in situ; e (3) avaliar um meio sequencial contendo GH e, ou, IGF-1 sobre o cultivo in situ de longa duração de folículos pré-antrais bovinos. Ovários de vacas sem padrão racial definido foram coletados em abatedouros locais e transportados ao laboratório em TCM-199 suplementado com HEPES, bicarbonato de sódio, ácido pirúvico, penicilina e estreptomicina. No laboratório o córtex ovariano foi fragmentado e destinado a cultivos de curta duração (sete dias) nas fases I, II e de longa duração (14 dias) na fase III. O cultivo foi realizado em incubadora a 37 oC e 5% de CO2 em placa de 24 poços com troca total de meio a cada dois dias. Fragmentos ovarianos não cultivados (controle) e cultivados foram processados para avaliação histológica (morfológica e morfométrica), sendo avaliados o percentual de folículos morfologicamente normais, primordiais e em desenvolvimento (primários e secundários) além do diâmetro folicular e oocitário. Além disto, a viabilidade foi avaliada pela coloração de azul de tripan, sendo os folículos classificados como corados e não corados, mortos e vivos, respectivamente. As variáveis morfológicas e morfométricas foram submetidas à ANOVA, seguidas de comparações entre médias pelo teste de Tukey e Dunnett e as variáveis de viabilidade foram submetidas ao teste de Qui-quadrado (χ2). Os resultados da Fase I mostraram que o α-MEM suplementado (α-MEM+) preservou a morfologia folicular e se destacou na análise morfométrica e de viabilidade. Consolidando os resultados obtidos na fase I, na fase II utilizou-se o α-MEM+ com diferentes concentrações de IGF-1 recombinante humano (0, 30, 70, 100 ng/mL) ou GH recombinante humano (0, 10, 25, 50 ng/mL), compondo os experimentos 1 e 2, respectivamente. Os resultados mostraram que os tratamentos IGF-1(30 ng/mL) e GH (50 ng/mL) foram as concentrações mais eficientes para manter a funcionalidade morfológica e morfométrica, assim como, a viabilidade de folículos pré-antrais bovinos cultivados in situ. Na fase III, os fragmentos ovarianos foram cultivados em α-MEM+ na ausência ou presença de GH (50 ng/mL) e, ou, IGF-1 (30 ng/mL), adicionados da seguinte forma: (α-MEM+) durante 14 dias; (GH→IGF-1) GH do dia 1o ao 7o e IGF-1 do dia 8o ao 14o; (IGF-1→GH) IGF-1 do dia 1o ao 7o, e GH do dia 8o ao 14o; e (GH+IGF-1) durante 14 dias. O tratamento (GH→IGF-1) manteve o percentual de folículos morfologicamente normais durante todo o período de cultivo, e o percentual de folículos em desenvolvimento aumentou quando comparado com o controle a fresco após sete dias de cultivo, enquanto o diâmetro folicular aumentou em ambos, dia 7 e 14 de cultivo. Portanto, conclui-se que folículos pré-antrais bovinos cultivados in situ em meio sequencial, tratados com GH→IGF-1, melhoram o desenvolvimento e o diâmetro folicular até a primeira metade do cultivo e mantêm estes parâmetros com normalidade e viabilidade até o fim do cultivo in vitro.Conselho Nacional de Desenvolvimento Científico e Tecnológicoapplication/pdfporUniversidade Federal de ViçosaDoutorado em ZootecniaUFVBRGenética e Melhoramento de Animais Domésticos; Nutrição e Alimentação Animal; Pastagens e ForragiculBiotecnologiaBovinosFolículo pré-antralFoliculogêneseOvário in situ.BiotechnologyCattlePre-antral follicleFolliculogenesisOvary situCNPQ::CIENCIAS AGRARIAS::MEDICINA VETERINARIA::REPRODUCAO ANIMAL::FISIOPATOLOGIA DA REPRODUCAO ANIMALEfeito dos meios básicos de cultivo, IGF-1 e GH, no desenvolvimento in vitro de folículos pré-antrais bovinos inclusos em tecido ovarianoEffects of basic culture medium, IGF-1 and GH in development in vitro of bovine preantral follicles enclosed in ovarian tissueinfo:eu-repo/semantics/publishedVersioninfo:eu-repo/semantics/doctoralThesisinfo:eu-repo/semantics/openAccessreponame:LOCUS Repositório Institucional da UFVinstname:Universidade Federal de Viçosa (UFV)instacron:UFVORIGINALtexto completo.pdfapplication/pdf2813582https://locus.ufv.br//bitstream/123456789/1877/1/texto%20completo.pdf26d11929f6e3f39f72a4357dd61799d2MD51TEXTtexto completo.pdf.txttexto completo.pdf.txtExtracted texttext/plain199716https://locus.ufv.br//bitstream/123456789/1877/2/texto%20completo.pdf.txtc0c8bcc40817976b6e50ab427daa5bc8MD52THUMBNAILtexto completo.pdf.jpgtexto completo.pdf.jpgIM Thumbnailimage/jpeg3589https://locus.ufv.br//bitstream/123456789/1877/3/texto%20completo.pdf.jpg62ec126616f9bdffa81cc10d09af22f5MD53123456789/18772016-04-07 23:16:16.858oai:locus.ufv.br:123456789/1877Repositório InstitucionalPUBhttps://www.locus.ufv.br/oai/requestfabiojreis@ufv.bropendoar:21452016-04-08T02:16:16LOCUS Repositório Institucional da UFV - Universidade Federal de Viçosa (UFV)false
dc.title.por.fl_str_mv Efeito dos meios básicos de cultivo, IGF-1 e GH, no desenvolvimento in vitro de folículos pré-antrais bovinos inclusos em tecido ovariano
dc.title.alternative.eng.fl_str_mv Effects of basic culture medium, IGF-1 and GH in development in vitro of bovine preantral follicles enclosed in ovarian tissue
title Efeito dos meios básicos de cultivo, IGF-1 e GH, no desenvolvimento in vitro de folículos pré-antrais bovinos inclusos em tecido ovariano
spellingShingle Efeito dos meios básicos de cultivo, IGF-1 e GH, no desenvolvimento in vitro de folículos pré-antrais bovinos inclusos em tecido ovariano
Jimenez, Carolina Rodriguez
Biotecnologia
Bovinos
Folículo pré-antral
Foliculogênese
Ovário in situ.
Biotechnology
Cattle
Pre-antral follicle
Folliculogenesis
Ovary situ
CNPQ::CIENCIAS AGRARIAS::MEDICINA VETERINARIA::REPRODUCAO ANIMAL::FISIOPATOLOGIA DA REPRODUCAO ANIMAL
title_short Efeito dos meios básicos de cultivo, IGF-1 e GH, no desenvolvimento in vitro de folículos pré-antrais bovinos inclusos em tecido ovariano
title_full Efeito dos meios básicos de cultivo, IGF-1 e GH, no desenvolvimento in vitro de folículos pré-antrais bovinos inclusos em tecido ovariano
title_fullStr Efeito dos meios básicos de cultivo, IGF-1 e GH, no desenvolvimento in vitro de folículos pré-antrais bovinos inclusos em tecido ovariano
title_full_unstemmed Efeito dos meios básicos de cultivo, IGF-1 e GH, no desenvolvimento in vitro de folículos pré-antrais bovinos inclusos em tecido ovariano
title_sort Efeito dos meios básicos de cultivo, IGF-1 e GH, no desenvolvimento in vitro de folículos pré-antrais bovinos inclusos em tecido ovariano
author Jimenez, Carolina Rodriguez
author_facet Jimenez, Carolina Rodriguez
author_role author
dc.contributor.authorLattes.por.fl_str_mv http://lattes.cnpq.br/3000028594640502
dc.contributor.author.fl_str_mv Jimenez, Carolina Rodriguez
dc.contributor.advisor-co1.fl_str_mv Araújo, Valdevane Rocha
dc.contributor.advisor-co1Lattes.fl_str_mv http://lattes.cnpq.br/8652126082521493
dc.contributor.advisor1.fl_str_mv Torres, Ciro Alexandre Alves
dc.contributor.advisor1Lattes.fl_str_mv http://buscatextual.cnpq.br/buscatextual/visualizacv.do?id=K4787213D4
dc.contributor.referee1.fl_str_mv Guimarães, José Domingos
dc.contributor.referee1Lattes.fl_str_mv http://buscatextual.cnpq.br/buscatextual/visualizacv.do?id=K4782270U6
dc.contributor.referee2.fl_str_mv Neves, Mariana Machado
dc.contributor.referee2Lattes.fl_str_mv http://lattes.cnpq.br/7880116499692231
contributor_str_mv Araújo, Valdevane Rocha
Torres, Ciro Alexandre Alves
Guimarães, José Domingos
Neves, Mariana Machado
dc.subject.por.fl_str_mv Biotecnologia
Bovinos
Folículo pré-antral
Foliculogênese
Ovário in situ.
topic Biotecnologia
Bovinos
Folículo pré-antral
Foliculogênese
Ovário in situ.
Biotechnology
Cattle
Pre-antral follicle
Folliculogenesis
Ovary situ
CNPQ::CIENCIAS AGRARIAS::MEDICINA VETERINARIA::REPRODUCAO ANIMAL::FISIOPATOLOGIA DA REPRODUCAO ANIMAL
dc.subject.eng.fl_str_mv Biotechnology
Cattle
Pre-antral follicle
Folliculogenesis
Ovary situ
dc.subject.cnpq.fl_str_mv CNPQ::CIENCIAS AGRARIAS::MEDICINA VETERINARIA::REPRODUCAO ANIMAL::FISIOPATOLOGIA DA REPRODUCAO ANIMAL
description The objectives of this study were (I) to determine the best basic culture medium for bovine preantral follicles culture enclosed in ovarian cortical tissue (in situ), using the minimum essential medium (α-MEM), tissue medium culture 199 (TCM-199) and medium McCoy s (McCoy s); (II) to evaluate different concentrations of Insulin like growth factor 1 (IGF-1) and growth hormone (GH) in the development of bovine preantral follicles in situ cultured and (III) to evaluate a sequential medium containing GH and, or, IGF-1 on long-term in situ culture of bovine preantral follicles. Ovaries of cows without defined breed standard were collected from local slaughterhouse and transported to the laboratory in TCM-199 supplemented with HEPES, sodium bicarbonate, pyruvic acid, penicillin and streptomycin. In the laboratory the ovarian cortex was fragmented and was taken for short-term cultures (7 days) on phases I, II and long-term (14 days) on phase III. The culture was performed in an incubator at 37 °C and 5 % CO2 in a 24 well plate with complete medium exchange every 2 days. Uncultured (control) and cultured ovarian fragments were processed for histological evaluation (morphological and morphometric) and evaluated the percentage of normal morphologically follicles, primordial and developing (primary and secondary) follicles, beyond the follicular and oocyte diameter. In addition, viability was assessed by trypan blue staining, the follicles classified as being stained and unstained, dead and living, respectively. The morphological and morphometric variables were analyzed by ANOVA followed by comparisons of means by Tukey and Dunnett s test and viability variables were subjected to chi-square (χ2) test. The results of Phase I showed that α- MEM supplemented (α-MEM+) preserved follicular morphology and it highlighted in morphometric and viability analysis. Consolidating the results of phase I, on phase II we used the α-MEM+ with different concentrations of recombinant human IGF-1 (0, 30, 70, 100 ng/mL) or recombinant human GH (0, 10, 25, 50 ng/mL), composing the experiment 1 and 2, respectively. The results showed that IGF-1 (30 ng/ml) and GH (50 ng/mL) treatments were the most effective concentration to maintain morphological and morphometric functionality, as well as the viability of in situ cultured of bovine preantral follicles. On phase III ovarian fragments were cultured in α-MEM+ in the absence or presence of GH (50 ng/mL) and, or, IGF-1 (30 ng/mL) added as follows: (α- MEM+) for 14 days, (GH→IGF-1) GH from 1 st day to 7th day and IGF-1 from 8th day to 14th day, (GH→IGF-1) IGF-1 from 1st day to 7th day, and GH from8 th day to 14th day and (GH+IGF-1) for 14 days. The treatment (GH→IGF-1) maintained the percentage of normal morphology follicles during the whole culture period and the percentage of developing follicles increased compared to fresh control after 7 days of culture, while follicular diameter increased in both 7th and 14th days of culture. Therefore, it is concluded that in situ cultured bovine preantral follicles in sequential medium treated with GH→IGF-1 improved the development and follicular diameter until the first half of the culture and kept these parameters with normality and viability until the end of in vitro culture.
publishDate 2014
dc.date.issued.fl_str_mv 2014-02-26
dc.date.accessioned.fl_str_mv 2015-03-26T12:54:58Z
dc.date.available.fl_str_mv 2015-01-23
2015-03-26T12:54:58Z
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dc.identifier.citation.fl_str_mv JIMENEZ, Carolina Rodriguez. Effects of basic culture medium, IGF-1 and GH in development in vitro of bovine preantral follicles enclosed in ovarian tissue. 2014. 95 f. Tese (Doutorado em Genética e Melhoramento de Animais Domésticos; Nutrição e Alimentação Animal; Pastagens e Forragicul) - Universidade Federal de Viçosa, Viçosa, 2014.
dc.identifier.uri.fl_str_mv http://locus.ufv.br/handle/123456789/1877
identifier_str_mv JIMENEZ, Carolina Rodriguez. Effects of basic culture medium, IGF-1 and GH in development in vitro of bovine preantral follicles enclosed in ovarian tissue. 2014. 95 f. Tese (Doutorado em Genética e Melhoramento de Animais Domésticos; Nutrição e Alimentação Animal; Pastagens e Forragicul) - Universidade Federal de Viçosa, Viçosa, 2014.
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dc.publisher.department.fl_str_mv Genética e Melhoramento de Animais Domésticos; Nutrição e Alimentação Animal; Pastagens e Forragicul
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