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In vitro embryonic development and gene expression of bovine embryos produced from oocytes cultured in maturation medium containing plasma enriched with platelet factors and macromolecules

Grant number: 13/07260-8
Support type:Scholarships in Brazil - Scientific Initiation
Effective date (Start): June 01, 2013
Effective date (End): May 31, 2014
Field of knowledge:Agronomical Sciences - Veterinary Medicine
Principal Investigator:Alicio Martins Júnior
Grantee:Tairini Erica da Cruz
Home Institution: Faculdade de Medicina Veterinária (FMVA). Universidade Estadual Paulista (UNESP). Campus de Araçatuba. Araçatuba , SP, Brazil

Abstract

This study will be carry out to investigate the effects of blood plasma enriched with platelet factors (PPF) alone or in combination with FCS or BSA to the bovine in vitro oocyte maturation (IVM) medium on the subsequent embryonic development, total cell number (TCN), percentages of live/dead cells, and gene expression. The following experiments will be performed: exp. I: groups PPF at concentrations of 5 and 10%, and two control groups referred here as A (IVM medium + 10% FCS) and B (IVM medium + 8 mg/ml de BSA); exp. II: PPF at concentrations of 5 and 10% supplemented with 10% FCS, and other group with the best result obtained in exp. I; exp. III: PPF at concentrations of 5 and 10% supplemented with 8 mg/ml BSA, and other group with the best result obtained in exp. II. Oocytes, obtained from slaughtered cows' ovaries, will be selected in base maturation medium (b-MM; TCM 199 plus bicarbonate, pyruvate, glutamine, and penicillin, supplemented with FCS or BSA). Groups of 20 to 25 oocytes per drop will be cultured in b-MM with additions of FSH and LH (IVM medium). Sperm selection will be performed in discontinuous Percoll gradient; IVF carried out in TALP medium plus PHE and heparin. Embryo culture will be performed in modified SOF medium; humidified atmosphere with 5% CO2, at 38.7o C in air will be used for IVM, IVF, and IVC steps. The number of cleaved oocytes reaching morula/blastocyst and blastocyst/expanded blastocyst stages will be recorded at 72, 144, and 168h post-insemination, respectively. ANOVA and Bonferroni t test will be used to determine differences among groups for embryo development, TCN and percentage of live/dead cells. The results of gene expression will be analyzed by x2 test. Differences of P<0.05 will be taken as significant. (AU)