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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.
| Identifer | oai:union.ndltd.org:IBICT/oai:repositorio.ufrn.br:123456789/19810 |
| Date | 27 February 2015 |
| Creators | Lima, Gabriela Liberalino |
| Contributors | 70298254387, http://lattes.cnpq.br/1959482950237684, Pereira, Alexsandra Fernandes, 91307198368, http://lattes.cnpq.br/8114638410593492, Silva, L?cia Daniel Machado da, 65696972691, http://lattes.cnpq.br/3610262531647532, Lopes, Maria Denise, 98322290810, http://lattes.cnpq.br/6666129914663018, Oliveira, Moacir Franco de, 32594950459, http://lattes.cnpq.br/8843113233262619, Silva, Alexandre Rodrigues |
| Publisher | Universidade Federal do Rio Grande do Norte, PROGRAMA DE P?S-GRADUA??O EM BIOTECNOLOGIA, UFRN, Brasil |
| Source Sets | IBICT Brazilian ETDs |
| Language | Portuguese |
| Detected Language | English |
| Type | info:eu-repo/semantics/publishedVersion, info:eu-repo/semantics/doctoralThesis |
| Source | reponame:Repositório Institucional da UFRN, instname:Universidade Federal do Rio Grande do Norte, instacron:UFRN |
| Rights | info:eu-repo/semantics/openAccess |
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