Efeito do bloqueio meiótico sobre a maturação e fertilização de oócitos bovinos

Detalhes bibliográficos
Ano de defesa: 2016
Autor(a) principal: SANTANA, Breno Barros de lattes
Orientador(a): CARNEIRO, Gustavo Ferrer
Banca de defesa: CARNEIRO, Gustavo Ferrer, BATISTA, André Mariano, MAIA, Victor Netto
Tipo de documento: Dissertação
Tipo de acesso: Acesso aberto
Idioma: por
Instituição de defesa: Universidade Federal Rural de Pernambuco
Programa de Pós-Graduação: Programa de Pós-Graduação em Sanidade e Reprodução de Ruminantes
Departamento: Unidade Acadêmica de Garanhuns
País: Brasil
Palavras-chave em Português:
Área do conhecimento CNPq:
Link de acesso: http://www.tede2.ufrpe.br:8080/tede2/handle/tede2/8037
Resumo: Biotechnology of reproduction has been increasingly used in order to improve genetic material as well as germplasm preservation. Provision of viable oocytes in stage suitable for its use is essential to the success of this technique. An alternative would be to prevent resumption of meiosis after removal of oocytes from follicular environment inducing a meiotic blockage with the aim of providing the oocyte more time available so that it could be possible to suffer modifications required to support fertilization and normal embryonic development. Inhibitors, as Rolipram has been used with this objective. These blockers can improve oocyte competence through a better cytoplasmatic maturation. The goal of this project was to evaluate the effect of rolipram during maturation of bovine oocytes on quantity and quality of embryos produced in vitro. Ovaries was collected at slaughter and transported to the laboratory of Animal Reproduction at UAG/UFRPE - LABRAPE. The COC`s were selected and divided into 5 groups: Control 1 (time 0): oocytes fixed and stained immediately after selection to observe after collection status; control: in vitro maturation for 24 hours in the absence of Rolipram; Rolipram in 3 different treatments: blockage for 24 hours in maturation medium containing (100, 150 and 200 μM). After 24 hours, treatment and control groups were placed in maturation medium for over 24 hours to observe the reversal of the blockage. These oocytes were evaluated using an epifluorescent microscope to check maturation stage. After maturation, oocytes from control (within 24 hours of in vitro maturation) and treatments groups with Rolipram were in vitro fertilized 48 hours post fertilization to assess cleavage and blastocyst formation at D7 post fertilization. In addition, part of the oocytes were stored for expression of the following genes: Mater, BMP15 and BAX, to observe the effect of the blocker on expression of these genes in the embryos. As far as gene expression, no difference was seen between control group and treatments. The oocytes evaluated immediately after follicular aspiration 79.00% were in VG, QVGBD, or MI, characterized as immature and 13.40%, was in MII and 7.60%, were considered degenerate or not identified. In different concentrations of T100 and T200 T150 μ M, we observed significant differences in oocytes that have reached the MII phase comparing with control treatments (P = 0.003). As for the embryonic development, differences in the rate of cleavage (P < 0.05) were observed between T150 and T200 when compared to Control 24 hour Group. A difference was seen on blastocyst rate (P < 0.001) among the treatments compared to control group, being T100 μM the concentration which provided less negative effects during pre-maturing of bovine oocytes.
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spelling CARNEIRO, Gustavo FerrerCARNEIRO, Gustavo FerrerBATISTA, André MarianoMAIA, Victor Nettohttp://lattes.cnpq.br/6478689237895888SANTANA, Breno Barros de2019-05-21T15:45:26Z2016-07-29SANTANA, Breno Barros de. Efeito do bloqueio meiótico sobre a maturação e fertilização de oócitos bovinos. 2016. 59 f. Dissertação (Programa de Pós-Graduação em Sanidade e Reprodução de Ruminantes) - Universidade Federal Rural de Pernambuco, Garanhuns.http://www.tede2.ufrpe.br:8080/tede2/handle/tede2/8037Biotechnology of reproduction has been increasingly used in order to improve genetic material as well as germplasm preservation. Provision of viable oocytes in stage suitable for its use is essential to the success of this technique. An alternative would be to prevent resumption of meiosis after removal of oocytes from follicular environment inducing a meiotic blockage with the aim of providing the oocyte more time available so that it could be possible to suffer modifications required to support fertilization and normal embryonic development. Inhibitors, as Rolipram has been used with this objective. These blockers can improve oocyte competence through a better cytoplasmatic maturation. The goal of this project was to evaluate the effect of rolipram during maturation of bovine oocytes on quantity and quality of embryos produced in vitro. Ovaries was collected at slaughter and transported to the laboratory of Animal Reproduction at UAG/UFRPE - LABRAPE. The COC`s were selected and divided into 5 groups: Control 1 (time 0): oocytes fixed and stained immediately after selection to observe after collection status; control: in vitro maturation for 24 hours in the absence of Rolipram; Rolipram in 3 different treatments: blockage for 24 hours in maturation medium containing (100, 150 and 200 μM). After 24 hours, treatment and control groups were placed in maturation medium for over 24 hours to observe the reversal of the blockage. These oocytes were evaluated using an epifluorescent microscope to check maturation stage. After maturation, oocytes from control (within 24 hours of in vitro maturation) and treatments groups with Rolipram were in vitro fertilized 48 hours post fertilization to assess cleavage and blastocyst formation at D7 post fertilization. In addition, part of the oocytes were stored for expression of the following genes: Mater, BMP15 and BAX, to observe the effect of the blocker on expression of these genes in the embryos. As far as gene expression, no difference was seen between control group and treatments. The oocytes evaluated immediately after follicular aspiration 79.00% were in VG, QVGBD, or MI, characterized as immature and 13.40%, was in MII and 7.60%, were considered degenerate or not identified. In different concentrations of T100 and T200 T150 μ M, we observed significant differences in oocytes that have reached the MII phase comparing with control treatments (P = 0.003). As for the embryonic development, differences in the rate of cleavage (P < 0.05) were observed between T150 and T200 when compared to Control 24 hour Group. A difference was seen on blastocyst rate (P < 0.001) among the treatments compared to control group, being T100 μM the concentration which provided less negative effects during pre-maturing of bovine oocytes.As biotecnologias da reprodução são cada vez mais utilizadas com o objetivo de aumentar a oferta de material genético assim como preservação de germoplasma. A oferta de oócitos viáveis no estágio adequado para sua utilização é fundamental para o sucesso na produção in vitro de embriões. Uma alternativa seria evitar a retomada prematura da meiose após a remoção dos oócitos do ambiente folicular induzindo um bloqueio meiótico com o intuito de fornecer ao oócito mais tempo para que o mesmo possa sofrer modificações necessárias para suportar a fertilização e ter desenvolvimento embrionário normal. Inibidores, como o Rolipram, tem sido utilizado com esse intuito. Estes bloqueadores podem possivelmente melhorar a competência oocitária através de uma melhor maturação citoplasmática. O objetivo desse projeto foi avaliar o efeito do rolipram durante a maturação de oócitos bovinos na quantidade e qualidade de embriões produzidos in vitro. Para tanto, ovários foram coletados em abatedouro e transportados para o Laboratório de Reprodução Animal- LABRAPE da UFRPE/UAG. Os complexos cúmulos oócitos (CCOs) foram selecionados e divididos em 5 grupos: 1 Controle 0 hora: oócitos fixados e corados logo após a seleção para observarmos o status pós-coleta; Controle A: maturação por 24horas na ausência de Rolipram; Rolipram 3 tratamentos: bloqueio da maturação por 24horas em meio de maturação contendo (100, 150 e 200 μM). Após a maturação, uma parte dos oócitos dos grupos tratamentos e o grupo controle foram colocados em meio de maturação base por mais 24 horas para observar a reversão do bloqueio. Estes oócitos foram avaliados em microscópio equipado com epifluorescência. Após a maturação, o restante dos oócitos do grupo Controle e os tratamentos com Rolipram foram fecundados in vitro para avaliação das taxas de clivagem 48 horas pós fecundação e de blastocisto no D7 pós fecundação. Além disso, parte dos oócitos foram desnudados e armazenados para avaliação de expressão dos seguintes genes: Mater, BMP15 e BAX, para observação do efeito do bloqueador na expressão desses genes nos embriões. Não houve diferença estatística na expressão gênica entre grupo controle e tratamentos. Dos oócitos avaliados logo após a aspiração folicular, 79,00% encontravam-se em VG, QVGBD, ou MI, caracterizando-se como imaturos enquanto 13,40%, encontrava-se em MII e 7,60%, considerados degenerados ou não identificados. Nas diferentes concentrações de T100, T150 e T200 μM foram observadas diferenças em oócitos que atingiram a fase de MII comparando os tratamentos com o controle (P= 0,003). Quanto ao desenvolvimento embrionário houve diferenças na taxa de clivagem (P < 0,05) entre T150 e T200 quando comparados ao grupo C/24 e a taxa de blastocisto obteve diferença (P < 0,001) entre os tratamentos comparados ao grupo controle, sendo T100 μM a concentração que proporcionou menos efeitos negativos como diminuição da taxa de clivagem e blastocisto durante a pré-maturação de oócitos bovinos.Submitted by Mario BC (mario@bc.ufrpe.br) on 2019-05-21T15:45:26Z No. of bitstreams: 1 Breno Barros de Santana.pdf: 1122974 bytes, checksum: b19bc70484957e1d686d3c51dffd0298 (MD5)Made available in DSpace on 2019-05-21T15:45:26Z (GMT). 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dc.title.por.fl_str_mv Efeito do bloqueio meiótico sobre a maturação e fertilização de oócitos bovinos
title Efeito do bloqueio meiótico sobre a maturação e fertilização de oócitos bovinos
spellingShingle Efeito do bloqueio meiótico sobre a maturação e fertilização de oócitos bovinos
SANTANA, Breno Barros de
Rolipram
Maturação oocitária
Bovino
Fertilização in vitro
MEDICINA VETERINARIA::REPRODUCAO ANIMAL
title_short Efeito do bloqueio meiótico sobre a maturação e fertilização de oócitos bovinos
title_full Efeito do bloqueio meiótico sobre a maturação e fertilização de oócitos bovinos
title_fullStr Efeito do bloqueio meiótico sobre a maturação e fertilização de oócitos bovinos
title_full_unstemmed Efeito do bloqueio meiótico sobre a maturação e fertilização de oócitos bovinos
title_sort Efeito do bloqueio meiótico sobre a maturação e fertilização de oócitos bovinos
author SANTANA, Breno Barros de
author_facet SANTANA, Breno Barros de
author_role author
dc.contributor.advisor1.fl_str_mv CARNEIRO, Gustavo Ferrer
dc.contributor.referee1.fl_str_mv CARNEIRO, Gustavo Ferrer
dc.contributor.referee2.fl_str_mv BATISTA, André Mariano
dc.contributor.referee3.fl_str_mv MAIA, Victor Netto
dc.contributor.authorLattes.fl_str_mv http://lattes.cnpq.br/6478689237895888
dc.contributor.author.fl_str_mv SANTANA, Breno Barros de
contributor_str_mv CARNEIRO, Gustavo Ferrer
CARNEIRO, Gustavo Ferrer
BATISTA, André Mariano
MAIA, Victor Netto
dc.subject.por.fl_str_mv Rolipram
Maturação oocitária
Bovino
Fertilização in vitro
topic Rolipram
Maturação oocitária
Bovino
Fertilização in vitro
MEDICINA VETERINARIA::REPRODUCAO ANIMAL
dc.subject.cnpq.fl_str_mv MEDICINA VETERINARIA::REPRODUCAO ANIMAL
description Biotechnology of reproduction has been increasingly used in order to improve genetic material as well as germplasm preservation. Provision of viable oocytes in stage suitable for its use is essential to the success of this technique. An alternative would be to prevent resumption of meiosis after removal of oocytes from follicular environment inducing a meiotic blockage with the aim of providing the oocyte more time available so that it could be possible to suffer modifications required to support fertilization and normal embryonic development. Inhibitors, as Rolipram has been used with this objective. These blockers can improve oocyte competence through a better cytoplasmatic maturation. The goal of this project was to evaluate the effect of rolipram during maturation of bovine oocytes on quantity and quality of embryos produced in vitro. Ovaries was collected at slaughter and transported to the laboratory of Animal Reproduction at UAG/UFRPE - LABRAPE. The COC`s were selected and divided into 5 groups: Control 1 (time 0): oocytes fixed and stained immediately after selection to observe after collection status; control: in vitro maturation for 24 hours in the absence of Rolipram; Rolipram in 3 different treatments: blockage for 24 hours in maturation medium containing (100, 150 and 200 μM). After 24 hours, treatment and control groups were placed in maturation medium for over 24 hours to observe the reversal of the blockage. These oocytes were evaluated using an epifluorescent microscope to check maturation stage. After maturation, oocytes from control (within 24 hours of in vitro maturation) and treatments groups with Rolipram were in vitro fertilized 48 hours post fertilization to assess cleavage and blastocyst formation at D7 post fertilization. In addition, part of the oocytes were stored for expression of the following genes: Mater, BMP15 and BAX, to observe the effect of the blocker on expression of these genes in the embryos. As far as gene expression, no difference was seen between control group and treatments. The oocytes evaluated immediately after follicular aspiration 79.00% were in VG, QVGBD, or MI, characterized as immature and 13.40%, was in MII and 7.60%, were considered degenerate or not identified. In different concentrations of T100 and T200 T150 μ M, we observed significant differences in oocytes that have reached the MII phase comparing with control treatments (P = 0.003). As for the embryonic development, differences in the rate of cleavage (P < 0.05) were observed between T150 and T200 when compared to Control 24 hour Group. A difference was seen on blastocyst rate (P < 0.001) among the treatments compared to control group, being T100 μM the concentration which provided less negative effects during pre-maturing of bovine oocytes.
publishDate 2016
dc.date.issued.fl_str_mv 2016-07-29
dc.date.accessioned.fl_str_mv 2019-05-21T15:45:26Z
dc.type.status.fl_str_mv info:eu-repo/semantics/publishedVersion
dc.type.driver.fl_str_mv info:eu-repo/semantics/masterThesis
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dc.identifier.citation.fl_str_mv SANTANA, Breno Barros de. Efeito do bloqueio meiótico sobre a maturação e fertilização de oócitos bovinos. 2016. 59 f. Dissertação (Programa de Pós-Graduação em Sanidade e Reprodução de Ruminantes) - Universidade Federal Rural de Pernambuco, Garanhuns.
dc.identifier.uri.fl_str_mv http://www.tede2.ufrpe.br:8080/tede2/handle/tede2/8037
identifier_str_mv SANTANA, Breno Barros de. Efeito do bloqueio meiótico sobre a maturação e fertilização de oócitos bovinos. 2016. 59 f. Dissertação (Programa de Pós-Graduação em Sanidade e Reprodução de Ruminantes) - Universidade Federal Rural de Pernambuco, Garanhuns.
url http://www.tede2.ufrpe.br:8080/tede2/handle/tede2/8037
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language por
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dc.rights.driver.fl_str_mv info:eu-repo/semantics/openAccess
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dc.publisher.none.fl_str_mv Universidade Federal Rural de Pernambuco
dc.publisher.program.fl_str_mv Programa de Pós-Graduação em Sanidade e Reprodução de Ruminantes
dc.publisher.initials.fl_str_mv UFRPE
dc.publisher.country.fl_str_mv Brasil
dc.publisher.department.fl_str_mv Unidade Acadêmica de Garanhuns
publisher.none.fl_str_mv Universidade Federal Rural de Pernambuco
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