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The role of exogenous melatonin on in vitro maturation medium for bovine and porcine oocytes: determination and quantification of the main metabolites by mass spectrometry.

Grant number: 12/20843-0
Support Opportunities:Scholarships in Brazil - Post-Doctoral
Effective date (Start): March 01, 2013
Effective date (End): February 28, 2014
Field of knowledge:Agronomical Sciences - Veterinary Medicine - Animal Reproduction
Principal Investigator:Cláudia Lima Verde Leal
Grantee:Mirela Batista Coelho
Host Institution: Faculdade de Zootecnia e Engenharia de Alimentos (FZEA). Universidade de São Paulo (USP). Pirassununga , SP, Brazil
Associated research grant:10/51677-2 - Development of new mass spectrometry techniques and their general applications in science: chemistry, biochemistry, material sciences, forensics, medicine, food science, pharmaceutical and veterinary medicine, AP.TEM


The development of new technologies in animal production as well as the knowledge of biological systems research are relevant to the advancement of biotechnology in the agricultural sector in Brazil. In this activity, the area of animal reproduction is of special interest because of its impact on animal production. In this project, we propose the analysis of the metabolomic profile of exogenous melatonin and its possible metabolites generated during in vitro maturation of bovine and swine oocytes, using for such development, the validation and application of an analytical method based on a high reliability liquid chromatography and mass spectrometry sequence (HPLC-MS/MS).Melatonin is a hormone of significant biological importance, but with not well defined roles in oocyte and embryo development of in cattle and pigs. The HPLC-MS/MS method developed will be applied also in the comparative analysis between the follicular fluid samples obtained from these same species at different stages of development in order to elucidate the role of melatonin in oocyte competence and confirm the physiological range of these metabolites. Studies in the literature show that in vitro maturation may be impaired by oxidative stress caused by reactive oxygen species, which melatonin may produce an inactivating effect.Previous results with bovine follicular fluid analysis showed that the proposed method (HPLC-MS/MS) showed linearity with a regression coefficient greater than 0.99 for both the metabolites analyzed. The limits of quantitation (LOQ) were 2.5 pg.mL-1 for AFMK and AMK metabolites, 5-pg.mL-1 for melatonin and 1 to 12.5 to 1 pg.mL-1 6HMEL and NAS, respectively. The limits of detection (LOD) were 0.75 pg.mL-1 for the metabolites AFMK and AMK, 1.5 pg.mL-1 for melatonin and 3.75 for 6HMEL pg.mL-1 and NAS. These data provide an initial understanding of the physiological patterns of melatonin and its metabolites in bovine follicular fluid and allow us to question the role of melatonin in in vitro experiments and in oocyte maturation. These data also allow us to delineate the possible mechanism and interactions of melatonin and its metabolites with the different stress conditions and to try to elucidate the possible pathways used by these compounds for the success of in vitro production of bovine and porcine embryos.

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Scientific publications
(References retrieved automatically from Web of Science and SciELO through information on FAPESP grants and their corresponding numbers as mentioned in the publications by the authors)
COELHO, MIRELA B.; RODRIGUES-CUNHA, MARIA CAROLINA; FERREIRA, CHRISTINA R.; CABRAL, ELAINE C.; NOGUEIRA, GUILHERME P.; EBERLIN, MARCOS N.; LEAL, CLAUDIA L. V.; SIMAS, ROSINEIDE C.. Assessing melatonin and its oxidative metabolites amounts in biological fluid and culture medium by liquid chromatography electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS). ANALYTICAL METHODS, v. 5, n. 24, p. 6911-6918, . (10/51677-2, 12/20843-0, 10/18023-9)

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