Effects of melatonin on development and cryopreservation of porcine parthenogenetic embryos produced in vitro

• Grant number: 12/06180-8
• Support Opportunities: Scholarships in Brazil - Doctorate
• Start date: October 01, 2012
• End date: July 31, 2015
• Field of knowledge: Agronomical Sciences - Veterinary Medicine - Animal Reproduction
• Principal Investigator: Cláudia Lima Verde Leal
• Grantee: Maria Helena Coelho Cruz
• Host Institution: Faculdade de Zootecnia e Engenharia de Alimentos (FZEA). Universidade de São Paulo (USP). Pirassununga , SP, Brazil

Abstract

In vitro production of embryos (IVPE) in porcine has some limitations, due to the high vulnerability to free radicals and oxidative stress provided by their significantly higher levels of lipids. Melatonin is a substance that has antioxidant and anti-apoptotic effects and which has been suggested as an alternative for improvements in the rates of IVPE of porcine species. This study aims to evaluate the effects of melatonin during IVM of porcine oocyets on the development and cryopreservation of parthenogenetic embryos produced in vitro. Four experiments will be conducted using TCM199 medium as basic maturation medium. In the first experiment, oocytes submitted to maturation medium containing three concentrations of melatonin: without addition of melatonin (T0), 10-6 M melatonin (T6) and 10-9 M melatonin (T9) and will be evaluated for meiotic progression and cortical granule distribution at three times of maturation (36, 40 and 44 h). In the second experiment, mature oocytes and their cumulus cells will be evaluated for the expression of genes of the antioxidant enzymes catalase, Cu,Zn-SOD, Mn-SOD and GPx. In the third experiment, parthenogenetic embryos obtained from oocytes matured under the conditions defined in experiments 1 and 2 will be evaluated for the effect of melatonin in IVM on parthenogenetic embryo production. The embryos will be evaluated for quality in terms of DNA fragmentation and total cell number, as well as for the expression of genes of pro-and anti-apoptotic proteins (Bax and Bcl2). In the fourth experiment, the parthenogenetic embryos will be cryopreserved/thawed and will be evaluated for the rate of re-expansion, hatching and apoptosis to evaluate embryo survival. Statistical evaluations for differences between matured oocytes, cleavage and blastocyst rates will be performed using Chi-square test and for the differences between gene expression Student's t test will be used. The total number of TUNEL positive cells will be compared by Tukey's test. The data will be subjected to analysis of variance using GLM procedure of SAS (2003). A value of P<0.05 will be considered statistically significant. (AU)

Academic Publications

(References retrieved automatically from State of São Paulo Research Institutions)

CRUZ, Maria Helena Coelho. Effects of melatonin on viability and gene expression in porcine oocytes and cumulus cells matured in vitro. 2016. Doctoral Thesis - Universidade de São Paulo (USP). Faculdade de Zootecnica e Engenharia de Alimentos (FZE/BT) Pirassununga.