Submitted by Automa??o e Estat?stica (sst@bczm.ufrn.br) on 2016-02-17T22:37:29Z No. of bitstreams: 1 GabrielaLiberalinoLima_TESE.pdf: 6308180 bytes, checksum: b658a30fc2afc3a608b4b5b454c6d36a (MD5) / Approved for entry into archive by Arlan Eloi Leite Silva (eloihistoriador@yahoo.com.br) on 2016-02-19T22:18:54Z (GMT) No. of bitstreams: 1 GabrielaLiberalinoLima_TESE.pdf: 6308180 bytes, checksum: b658a30fc2afc3a608b4b5b454c6d36a (MD5) / Made available in DSpace on 2016-02-19T22:18:54Z (GMT). No. of bitstreams: 1 GabrielaLiberalinoLima_TESE.pdf: 6308180 bytes, checksum: b658a30fc2afc3a608b4b5b454c6d36a (MD5) Previous issue date: 2015-02-27 / O objetivo da presente tese foi utilizar a manipula??o de o?citos inclusos em fol?culos ovarianos pr? antrais (MOIFOPA) como ferramenta para o resgate e otimiza??o do uso de gametas femininos oriundos de esp?cies silvestres do bioma Caatinga. A tese foi dividida em quatro experimentos. No primeiro, foi realizada a estimativa e descri??o das caracter?sticas histol?gicas e ultraestruturas dos fol?culos pr? antrais (FOPA) de cutias (Dasyprocta leporina), nos quais foram estimados 4419.8 ? 532.26 e 5397.52 ? 574.91 fol?culos para os ov?rios direito e esquerdo, respectivamente, sendo a maioria (86,63%) pertencente a categoria de fol?culos primordiais (P<0,05). A maior parte da popula??o consiste de fol?culos morfologicamente normais (70,78%), apresentando n?cleo oocit?rio grande e central, com citoplasma uniforme. Na avalia??o ultraestrutural verificou-se a presen?a de um grande n?mero de mitoc?ndrias arredondadas e gotas de lip?dios. No segundo experimento, foi realizada a estimativa e a descri??o das caracter?sticas dos FOPA de pre?s (Galea spixii), bem como avalia??o do efeito da vitrifica??o em superf?cie s?lida (VSS) sobre a morfologia de FOPA in situ. O total de 416,0 ? 342,8 FOPA foi estimado por par de ov?rios e a presen?a de uma grande quantidade de fol?culos prim?rios foi evidenciada (P<0,05). A maior parte dos FOPA apresentou-se morfologicamente normal (94,6%), possuindo n?cleo oocit?rio contendo gr?nulos condensados de heterocromatina. Mitoc?ndrias com formato arredondado ou alongado representaram as organelas mais abundantes. Quanto a VSS, o protocolo utilizando o dimetilsulf?xido (DMSO) 3M possibilitou a preserva??o de 69,5% de FOPA morfologicamente normais, sendo evidenciado atrav?s da an?lise por microscopia de luz e eletr?nica de transmiss?o. No terceiro experimento, foi realizada a avalia??o do efeito da VSS sobre a morfologia e viabilidade de FOPA in situ de catetos (Pecari tajacu). N?o foram observadas diferen?as entre os tratamentos, onde o uso dos crioprotetores DMSO, etilenoglicol (EG) e dimetilformamida (DMF), independente da concentra??o utilizada (3 ou 6 M) promoveu a preserva??o da morfologia de mais de 70% dos FOPA. Quanto a viabilidade, os crioprotetores DMSO e EG, demonstraram melhor manuten??o da mesma. O quarto experimento teve por objetivo avaliar o efeito do ? MEM+ ou TCM199 associados ou n?o a 50 ng de FSHr sobre a morfologia, ativa??o e crescimento de FOPA de catetos, cultivados in vitro (CIV) durante 1 ou 7 dias e o efeito sobre a matriz extracelular (MEC). Ap?s 7 dias de CIV apenas o TCM199/FSH manteve a propor??o de FOPA intactos similar ao dia 1 (63,2%), contudo nenhuma diferen?a foi observada entre os tratamentos (P>0,05). Adicionalmente, um aumento na propor??o de FOPA em desenvolvimento foi verificada (P>0.05). Atrav?s da an?lise com Ag-NOR, observou-se que apenas o tratamento com TCM199/FSH manteve a propor??o de prolifera??o celular similar ao dia 1 (P>0.05). A colora??o com picrosirius red revelou que a MEC permaneceu intacta em todos os tratamentos (P>0.05). Assim, como conclus?o geral, o uso da MOIFOPA nas referidas esp?cies permitiu o conhecimento de aspectos relacionados a sua morfofisiologia reprodutiva, possibilitando tanto a conserva??o do material gen?tico destas esp?cies, com a possibilidade de forma??o de bancos de germoplasma, como a elucida??o de mecanismos relacionados a sobreviv?ncia e desenvolvimento dos FOPA in vitro. / 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 ? 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.