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Melatonin on meiosis resumption and bovine embryo development in vitro

Abstract

Melatonin (MLT) is an indoleamine that can act on reproductive competence, has antioxidant and antiapoptotic activities and influences different cell signaling pathways. In vitro studies have shown beneficial effects of MLT use on in vitro maturation (IVM) and culture (IVC) of bovine oocytes and embryos, respectively. In addition, MLT is able to induce the resumption of meiosis, but the mechanism by which this stimulus occurs is still unknown. In this study, we propose to investigate the involvement of maturation promoting factor (MPF) in meiosis resumption stimulated by MLT, as well as its effect on the development, quality and cryotolerance of bovine embryos produced in vitro. In experiment 1, cumulus-oocyte complexes (COCs) will be submitted to IVM with MLT (10-9M) or FSH (0.5 µg/ml control) for 0, 6, 9 and 12 h IVM when samples will be collected and evaluated for the presence of the phosphorylated CDK1 protein (active MPF catalytic unit) and total CDK1 protein. In experiment 2, MLT will be supplemented to the IVM medium for 24 h in association with gonadotropins: 1) FSH; 2) FSH+LH; 3) MLT; 4) FSH+MLT and 5) FSH+LH+MLT. Oocytes will be evaluated for meiosis progression (% MII). The best combination of MLT and gonadotropins (highest % MII) will be used in experiment 3 in which matured COCs will be submitted to in vitro fertilization (IVF) followed by IVC with MLT (0, 10-11, 10-9 and 10-7 M). The embryos will be evaluated for cleavage and blastocysts rates, and quality analysis by TUNEL. In experiment 4, embryo cryotolerance will be assessed for the treatment with best development rates from experiment 3. Embryos produced will vitrified, thawed and submitted to IVC for up to 72 h to determine the re-expansion and embryonic hatching rates. Embryos in the control group will be produced without MLT. The study will contribute to the understanding the mechanism by which MLT may stimulate meiosis resumption and investigate its use during embryo IVP aiming to improve embryo quality in cattle. (AU)

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