Conservação de material genético de espécies silvestres do bioma caatinga utilizando a manipulação oócitos inclusos em folículos ovarianos pré antrais (MOIFOPA)
The objective of the present thesis was to use the manipulation of oocytes enclosed in preantral follicles (MOEPF) as a tool for the female gametes rescue and optimization, from wild species of Caatinga biome. The thesis was divided into 4 experiments. At first experiment, it was performed the es...
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Folículos pré antrais Vitrificação em superfície sólida Cultivo in vitro FSHr α MEM+ TCM199 CNPQ::CIENCIAS BIOLOGICAS |
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Folículos pré antrais Vitrificação em superfície sólida Cultivo in vitro FSHr α MEM+ TCM199 CNPQ::CIENCIAS BIOLOGICAS Lima, Gabriela Liberalino Conservação de material genético de espécies silvestres do bioma caatinga utilizando a manipulação oócitos inclusos em folículos ovarianos pré antrais (MOIFOPA) |
description |
The objective of the present thesis was to use the manipulation of oocytes
enclosed in preantral follicles (MOEPF) as a tool for the female gametes rescue and
optimization, from wild species of Caatinga biome. The thesis was divided into 4
experiments. At first experiment, it was performed the estimative and description of the
agouti (Dasyprocta leporina) preantral follicles (PF) histologic and ultrastructural
features, in which it was estimated 4419.8 ± 532.26 and 5397.52 ± 574.91 follicles for
the right and left ovary, respectively, and the majority (86,63%) belonged to the
primordial follicles category (P<0.05). Most of the population consists of
morphologically normal follicles (70.78%), presenting a large and central nuclei and
uniform cytoplasm. At ultrastructural evaluation it was verified the presence of a great
number of round mitochondrias associated to lipid droplets. In the second experiment, it
was performed the estimative and description of yellow-toothed cavies (Galea spixii) PF
characteristics, also, the evaluation of the effect of solid surface vitrification (SSV) on the
in situ PF morphology. The total of 416.0 ± 342.8 PF was estimated for the ovary pair
and the presence of a large quantity of primary follicles (P<0.05) was evidenced. Most of
the PF was morphologically normal (94.6%), in which the oocyte nuclei presented
condensed granules of heterochromatin. Round or elongated shaped mitochondria
constituted the most abundant organelles. In regard of the SSV, the protocol using the
dimethylsulfoxide (DMSO) 3M possibility the preservation of 69.5% of morphologically
normal PF, which was evidenced by the light and transmission electronic microscopy. At
third experiment, the evaluation of the SSV procedure on the morphology and viability
in situ PF form collared peccaries (Pecari tajacu) was performed. No differences were
observed among treatments, in which the use of DMSO, ethylene glycol (EG) and
dimethylformamide (DMF) as cryoprotectants, regardless its concentration, promoted the
morphology preservation of much than 70% of PF. Concerning the PF viability, the
DMSO and EG promoted the best preservation. The fourth experiment aimed to evaluate
the effect of α MEM+ or TCM199 associated or not to 50 ng of FSHr on the morphology,
activation and growth of collared peccaries PF, in vitro cultured (IVC) during 1 or 7 days
and the effect on the extracellular matrix (ECM). After 7 days of IVC only the use of
TCM199/FSH maintained the proportion of intact PF, similar to day 1(63.2%), however,
no differences were observed among treatments (P>0.05). Also, an improvement of the
proportion of intact growing PF was verified (P>0.05). By the Ag-NOR analysis it was
observed that only the treatment using TCM199/FSH promoted the maintenance of cell
proliferation similar to day 1 (P>0.05). The picrosirius red stain revealed that ECM
remained intact in all treatments (P>0.05). Thus, as the general conclusion, the use of
MOEPF in the refereed species allowed the knowledge of aspects related to its
reproductive morphology and physiology, enabling the germplasm conservation, with the
possibility of germplasm bank formation, as the elucidation of mechanisms related to the
PF survive and in vitro development. |
author2 |
Silva, Alexandre Rodrigues |
author_facet |
Silva, Alexandre Rodrigues Lima, Gabriela Liberalino |
format |
doctoralThesis |
author |
Lima, Gabriela Liberalino |
author_sort |
Lima, Gabriela Liberalino |
title |
Conservação de material genético de espécies silvestres do bioma caatinga utilizando a manipulação oócitos inclusos em folículos ovarianos pré antrais (MOIFOPA) |
title_short |
Conservação de material genético de espécies silvestres do bioma caatinga utilizando a manipulação oócitos inclusos em folículos ovarianos pré antrais (MOIFOPA) |
title_full |
Conservação de material genético de espécies silvestres do bioma caatinga utilizando a manipulação oócitos inclusos em folículos ovarianos pré antrais (MOIFOPA) |
title_fullStr |
Conservação de material genético de espécies silvestres do bioma caatinga utilizando a manipulação oócitos inclusos em folículos ovarianos pré antrais (MOIFOPA) |
title_full_unstemmed |
Conservação de material genético de espécies silvestres do bioma caatinga utilizando a manipulação oócitos inclusos em folículos ovarianos pré antrais (MOIFOPA) |
title_sort |
conservação de material genético de espécies silvestres do bioma caatinga utilizando a manipulação oócitos inclusos em folículos ovarianos pré antrais (moifopa) |
publisher |
Universidade Federal do Rio Grande do Norte |
publishDate |
2016 |
url |
https://repositorio.ufrn.br/jspui/handle/123456789/19810 |
work_keys_str_mv |
AT limagabrielaliberalino conservacaodematerialgeneticodeespeciessilvestresdobiomacaatingautilizandoamanipulacaooocitosinclusosemfoliculosovarianospreantraismoifopa AT limagabrielaliberalino germplasmconservationfromwildspeciesofcaatingabiomeusingthemanipulationofoocytesenclosedinpreantralfolliclesmoepf |
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1773964539254013952 |
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ri-123456789-198102019-01-30T01:44:14Z Conservação de material genético de espécies silvestres do bioma caatinga utilizando a manipulação oócitos inclusos em folículos ovarianos pré antrais (MOIFOPA) Germplasm conservation from wild species of caatinga biome using the manipulation of oocytes enclosed in preantral follicles (MOEPF) Lima, Gabriela Liberalino Silva, Alexandre Rodrigues http://lattes.cnpq.br/0329054086208548 http://lattes.cnpq.br/1959482950237684 Pereira, Alexsandra Fernandes http://lattes.cnpq.br/8114638410593492 Silva, Lúcia Daniel Machado da http://lattes.cnpq.br/3610262531647532 Lopes, Maria Denise http://lattes.cnpq.br/6666129914663018 Oliveira, Moacir Franco de http://lattes.cnpq.br/8843113233262619 Folículos pré antrais Vitrificação em superfície sólida Cultivo in vitro FSHr α MEM+ TCM199 CNPQ::CIENCIAS BIOLOGICAS The objective of the present thesis was to use the manipulation of oocytes enclosed in preantral follicles (MOEPF) as a tool for the female gametes rescue and optimization, from wild species of Caatinga biome. The thesis was divided into 4 experiments. At first experiment, it was performed the estimative and description of the agouti (Dasyprocta leporina) preantral follicles (PF) histologic and ultrastructural features, in which it was estimated 4419.8 ± 532.26 and 5397.52 ± 574.91 follicles for the right and left ovary, respectively, and the majority (86,63%) belonged to the primordial follicles category (P<0.05). Most of the population consists of morphologically normal follicles (70.78%), presenting a large and central nuclei and uniform cytoplasm. At ultrastructural evaluation it was verified the presence of a great number of round mitochondrias associated to lipid droplets. In the second experiment, it was performed the estimative and description of yellow-toothed cavies (Galea spixii) PF characteristics, also, the evaluation of the effect of solid surface vitrification (SSV) on the in situ PF morphology. The total of 416.0 ± 342.8 PF was estimated for the ovary pair and the presence of a large quantity of primary follicles (P<0.05) was evidenced. Most of the PF was morphologically normal (94.6%), in which the oocyte nuclei presented condensed granules of heterochromatin. Round or elongated shaped mitochondria constituted the most abundant organelles. In regard of the SSV, the protocol using the dimethylsulfoxide (DMSO) 3M possibility the preservation of 69.5% of morphologically normal PF, which was evidenced by the light and transmission electronic microscopy. At third experiment, the evaluation of the SSV procedure on the morphology and viability in situ PF form collared peccaries (Pecari tajacu) was performed. No differences were observed among treatments, in which the use of DMSO, ethylene glycol (EG) and dimethylformamide (DMF) as cryoprotectants, regardless its concentration, promoted the morphology preservation of much than 70% of PF. Concerning the PF viability, the DMSO and EG promoted the best preservation. The fourth experiment aimed to evaluate the effect of α MEM+ or TCM199 associated or not to 50 ng of FSHr on the morphology, activation and growth of collared peccaries PF, in vitro cultured (IVC) during 1 or 7 days and the effect on the extracellular matrix (ECM). After 7 days of IVC only the use of TCM199/FSH maintained the proportion of intact PF, similar to day 1(63.2%), however, no differences were observed among treatments (P>0.05). Also, an improvement of the proportion of intact growing PF was verified (P>0.05). By the Ag-NOR analysis it was observed that only the treatment using TCM199/FSH promoted the maintenance of cell proliferation similar to day 1 (P>0.05). The picrosirius red stain revealed that ECM remained intact in all treatments (P>0.05). Thus, as the general conclusion, the use of MOEPF in the refereed species allowed the knowledge of aspects related to its reproductive morphology and physiology, enabling the germplasm conservation, with the possibility of germplasm bank formation, as the elucidation of mechanisms related to the PF survive and in vitro development. O objetivo da presente tese foi utilizar a manipulação de oócitos inclusos em folículos ovarianos pré antrais (MOIFOPA) como ferramenta para o resgate e otimização do uso de gametas femininos oriundos de espécies silvestres do bioma Caatinga. A tese foi dividida em quatro experimentos. No primeiro, foi realizada a estimativa e descrição das características histológicas e ultraestruturas dos folículos pré antrais (FOPA) de cutias (Dasyprocta leporina), nos quais foram estimados 4419.8 ± 532.26 e 5397.52 ± 574.91 folículos para os ovários direito e esquerdo, respectivamente, sendo a maioria (86,63%) pertencente a categoria de folículos primordiais (P<0,05). A maior parte da população consiste de folículos morfologicamente normais (70,78%), apresentando núcleo oocitário grande e central, com citoplasma uniforme. Na avaliação ultraestrutural verificou-se a presença de um grande número de mitocôndrias arredondadas e gotas de lipídios. No segundo experimento, foi realizada a estimativa e a descrição das características dos FOPA de preás (Galea spixii), bem como avaliação do efeito da vitrificação em superfície sólida (VSS) sobre a morfologia de FOPA in situ. O total de 416,0 ± 342,8 FOPA foi estimado por par de ovários e a presença de uma grande quantidade de folículos primários foi evidenciada (P<0,05). A maior parte dos FOPA apresentou-se morfologicamente normal (94,6%), possuindo núcleo oocitário contendo grânulos condensados de heterocromatina. Mitocôndrias com formato arredondado ou alongado representaram as organelas mais abundantes. Quanto a VSS, o protocolo utilizando o dimetilsulfóxido (DMSO) 3M possibilitou a preservação de 69,5% de FOPA morfologicamente normais, sendo evidenciado através da análise por microscopia de luz e eletrônica de transmissão. No terceiro experimento, foi realizada a avaliação do efeito da VSS sobre a morfologia e viabilidade de FOPA in situ de catetos (Pecari tajacu). Não foram observadas diferenças entre os tratamentos, onde o uso dos crioprotetores DMSO, etilenoglicol (EG) e dimetilformamida (DMF), independente da concentração utilizada (3 ou 6 M) promoveu a preservação da morfologia de mais de 70% dos FOPA. Quanto a viabilidade, os crioprotetores DMSO e EG, demonstraram melhor manutenção da mesma. O quarto experimento teve por objetivo avaliar o efeito do α MEM+ ou TCM199 associados ou não a 50 ng de FSHr sobre a morfologia, ativação e crescimento de FOPA de catetos, cultivados in vitro (CIV) durante 1 ou 7 dias e o efeito sobre a matriz extracelular (MEC). Após 7 dias de CIV apenas o TCM199/FSH manteve a proporção de FOPA intactos similar ao dia 1 (63,2%), contudo nenhuma diferença foi observada entre os tratamentos (P>0,05). Adicionalmente, um aumento na proporção de FOPA em desenvolvimento foi verificada (P>0.05). Através da análise com Ag-NOR, observou-se que apenas o tratamento com TCM199/FSH manteve a proporção de proliferação celular similar ao dia 1 (P>0.05). A coloração com picrosirius red revelou que a MEC permaneceu intacta em todos os tratamentos (P>0.05). Assim, como conclusão geral, o uso da MOIFOPA nas referidas espécies permitiu o conhecimento de aspectos relacionados a sua morfofisiologia reprodutiva, possibilitando tanto a conservação do material genético destas espécies, com a possibilidade de formação de bancos de germoplasma, como a elucidação de mecanismos relacionados a sobrevivência e desenvolvimento dos FOPA in vitro. 2016-02-19T22:18:54Z 2016-02-19T22:18:54Z 2015-02-27 doctoralThesis LIMA, Gabriela Liberalino. Conservação de material genético de espécies silvestres do bioma caatinga utilizando a manipulação oócitos inclusos em folículos ovarianos pré antrais (MOIFOPA). 2015. 225f. Tese (Doutorado em Biotecnologia) - Centro de Tecnologia, Universidade Federal do Rio Grande do Norte, Natal, 2015. https://repositorio.ufrn.br/jspui/handle/123456789/19810 por Acesso Aberto application/pdf Universidade Federal do Rio Grande do Norte Brasil UFRN PROGRAMA DE PÓS-GRADUAÇÃO EM BIOTECNOLOGIA |