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Strategies for minimizing oxidative stress in in vitro production system of bovine embryos destined for vitrification

Grant number: 12/10083-8
Support type:Scholarships in Brazil - Doctorate
Effective date (Start): January 01, 2013
Effective date (End): February 29, 2016
Field of knowledge:Agronomical Sciences - Veterinary Medicine - Animal Reproduction
Principal researcher:Gisele Zoccal Mingoti
Grantee:Nathália Alves de Souza Rocha Frigoni
Home Institution: Faculdade de Medicina Veterinária (FMVA). Universidade Estadual Paulista (UNESP). Campus de Araçatuba. Araçatuba , SP, Brazil
Associated scholarship(s):14/06885-7 - Protective effects of melatonin on oocyte competence in vitro, BE.EP.DR

Abstract

Since bovine embryos in vitro produced are more susceptible to oxidative damage, antioxidant supplementation has been shown effective to improve embryo quality. In addition, other factors have been shown to play an important role in embryonic development in adverse conditions, such as growth factors. To improve the quality, development potential and the success of cryopreservation of bovine embryos, this study will be conducted with the main objective to promote changes in some steps of the in vitro production system to minimize the damage caused by oxidative stress. Therefore, in a first step (Experiment I), the oocytes are in vitro matured in TCM-199 bicarbonate (B-199) supplemented with 0.6 mM cysteine associated with 100 mM cysteamine (C + C), 100 IU catalase (CAT) or 0.6 mM cysteine associated with 100 mM cysteamine and 100 IU catalase (CAT + C + C) or without supplementation with antioxidants (Control) and will be evaluated for competence and oocyte quality. The experimental group that provides the best results (oocyte quality and subsequent embryo development) will be used in subsequent experiments. In Experiment II, will be assessed the effects of supplementation with growth factor during IVC in generating oxidative stress situation (in the presence of menadione) on development and oxidative potential of bovine embryos in vitro produced. Therefore, oocytes will in vitro matured in medium pre-defined in the Experiment I and the presumptive zygotes IVC in SOF supplemented with 100 ng/mL IGF-I (IGF-I) in the presence or absence of 1 mM of menadione. The embryos will be evaluated for their quality (levels of GSH, the presence of ROS or apoptosis rate) and groups that provide the most and least promising results will be used in Experiment III, when the effects of antioxidants during IVM associated with IGF-I supplementation during the IVC on embryonic development and cryotolerance and the pattern of gene expression will be assessed. Therefore, oocytes will be in vitro matured in medium pre-defined in Experiment I and zygotes will be IVC under conditions defined on Experiment II, in the presence or absence of menadione. The obtained blastocysts will be vitrified or evaluated for expression of genes Bcl-2, BAX, and GPX MnSOD.

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