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Title: Morphologic, viability and ultrastructural analysis of vitrified sheep preantral follicles enclosed in ovarian tissue
Authors: Lunardi, Franciele Osmarini
Araújo, Valdevane Rocha
Faustino, Luciana Rocha
Carvalho, Adeline de Andrade
Gonçalves, Raphael Fernando Braga
Bassa, Casie Shantel
Báo, Sônia Nair
Name, Khesller Patrícia Olázia
Campello, Claudio Cabral
Figueiredo, José Ricardo de
Rodrigues, Ana Paula Ribeiro
Assunto:: Ovelha
Ovários
Criopreservação de órgãos, tecidos, etc.
Folículos pré-antrais
Issue Date: 2012
Publisher: Elsevier
Citation: LUNARDI, Franciele Osmarini, et al. Morphologic, viability and ultrastructural analysis of vitrified sheep preantral follicles enclosed in ovarian tissue. Small Ruminant Research, v. 107, p. 121-130, 2012. Disponível em:<http://www.sciencedirect.com/science/article/pii/S0921448812001599>. Acesso em: 03 fev. 2015.
Abstract: The main objective was to compare the efficiency of vitrification techniques and solutions on the preservation of morphology, ultrastructure and viability of sheep preantral follicles enclosed in ovarian tissue fragments. The fragments were cryopreserved by using macrotube vitrification (MTV), solid-surface vitrification (SSV) or conventional vitrification (CV). These techniques were combined with one of the six solutions containing 6 M ethylene glycol (EG) and with or without sucrose (SUC) (0.25 or 0.50 M) and with or without fetal calf serum (FCS) (10%). After one week, samples were warmed and histological analysis was performed, showing that the percentage of normal follicles after CV (66.20 ± 8.87%) using a solution containing 6 M EG, 0.25 M SUC and 10% FCS (vitrification solution 4 – VS4) was similar to fresh control (79.40 ± 7.83%), MTV (53.40 ± 10.60%) and SSV (56.75 ± 15.33%), all of them with the same vitrification solution (P < 0.05). For follicular viability evaluation, ovarian fragments were vitrified as described above. After warming, follicles were assessed by trypan blue dye. Controversially, the highest percentage of viable follicles was observed in MTV (97.06%) and was similar to fresh control (92.62%) (P < 0.05), but was significantly different from SSV (81.08%) and CV (83.81%) (P < 0.05). These results were validated by transmission electron microscopy that showed normal follicles observed in MTV and in fresh control. In addition, to verify the MTV with VS4 (a combination of the best technique plus the best solution), follicle viability was evaluated after 48 h in vitro culture. The viability assay was performed by fluorescence microscopy (calcein-AM and ethidium homodimer-1) analysis as follows: follicles isolated from fresh tissue were forthwith analyzed or underwent 48 h in vitro culture before analysis, whereas others fragments were vitrified/warmed and immediately analyzed or underwent 48 h in vitro culture before analysis. These results showed that, although follicular viability after MTV/VS4 (65%) was reduced when compared to the non-vitrified follicles at day 0 (100%), follicular viability after MTV/VS4 at day 2 (36.5%) was similar to follicles vitrified at day 0 (65%) and similar to non-vitrified follicles at day 2 (62.5%) (P > 0.05). As the decrease of viability in non-vitrified follicles at day 2 was similar to the decrease of MTV/VS4 in the same time, follicle viability at day 2 is not affected by MTV/VS4. In conclusion, using the experimental conditions of the present study, an efficient solution (VS4: 6 M EG, 0.25 M SUC and 10% FCS) and technique (MTV) were successfully used to vitrify ovine ovarian tissue.
Licença:: Este Manuscrito do Autor Aceito para Publicação (AAM) é protegido por direitos autorais e publicado pela Elsevier. Ele esta disponível neste Repositório, por acordo entre a Elsevier e a Universidade de Brasília. As alterações decorrentes do processo de publicação - como a edição, correção, formatação estrutural, e outros mecanismos de controle de qualidade - não estão refletidas nesta versão do texto. A versão definitiva do texto foi posteriormente publicado em [Small Ruminant Research, Volume 107, Número 2-3, Outubro 2012, Pages 121–130 , doi:10.1016/j.smallrumres.2012.04.009]. Você pode baixar, copiar e utilizar de outra forma o AAM para fins não comerciais , desde que sua licença seja limitada pelas seguintes restrições: (1) Você pode usar este AAM para fins não comerciais apenas sob os termos da licença CC- BY- NC-ND. (2) A integridade do trabalho e identificação do autor, detentor dos direitos autorais e editor deve ser preservado em qualquer cópia. (3) Tem de atribuir este AAM no seguinte formato: [acordo na linguagem atribuída, incluindo o link para CC BY-NC-ND licença Digital + DOI do artigo publicado na revista Elsevier ScienceDirect ® da plataforma].
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