Differential gene expression, ultrastructural analysis and evaluation of the lipid profile of in vitro produced bovine blastocysts from oocytes matured conventionally or via SPOM system

Abstract

In vitro maturation (IVM) do not use superestimulatory hormones, which avoid excessive spending and manipulations of the animal and also the harmful iatrogenic effects of ovarian overstimulation. However, the efficiency of IVM does not meet expectations due to its inability to simulate the physiological events that occur in vivo (as the main example, the synchronization between the nuclear and cytoplasmic maturation of the oocyte). For this reason, the increment of IVM media is a target sought increasingly in reproductive studies. The evaluation of the lipid profile of embryos is a new and particularly important approach for gauging the embryo ability for cryopreservation. In addition, cell characterization through assessment of morphology and organelles distribution, provides consistent results on embryo characterization when examined ultrastructurally. Furthermore, the differential expression of genes - known as indicators of specific function and hence embryonic competence - can be evaluated in combination with such techniques in order to identify in vitro the blastocysts that have better quality (morphological and physiological) and competence for later implantation and development. Therefore, this study aims to compare the conventional IVM system with the new system simulated physiology oocyte maturation - SPOM), which uses drugs and control the specific timing of their administration (briefly: forskolin and IBMX on pre-maturation for 2 h, and cilostamide and FSH for the next 28 h of maturation). Oocytes will be matured, fertilized and the produced blastocysts will be evaluated for the lipid profile (mass spectrometry MALDI-TOF-MS, Thomson Laboratory, Campinas), the ultrastructure analysis (electron microscopy, Institute of Biosciences, Botucatu) and differential expression of 45 genes indicators of quality and competence of embryonic development (microfluidic system, Fluidigm). The methodology described will be held in Brazil (Institute of Biosciences, Botucatu), with Bos indicus cattle, and in Denmark (Department of Basic Animal and Veterinary Sciences, University of Copenhagen) with Bos taurus cattle in order to verify the efficiency and repeatability of the SPOM system with different scenarios and genotypes. The communion of the data to be obtained with this project aims to provide a general and reliable marker of embryo quality, covering the implantation, development and cryopreservation of embryos in commercial routines of IVP.