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Effects of bovine sperm selection through centrifugation in Percoll before freezing and Percoll or TALP after freezing on the sperm kinetic and viability and IVF rates

Grant number: 14/19261-1
Support type:Scholarships in Brazil - Scientific Initiation
Effective date (Start): April 01, 2015
Effective date (End): October 31, 2015
Field of knowledge:Agronomical Sciences - Veterinary Medicine
Principal Investigator:Alicio Martins Júnior
Grantee:Jaqueline Poleto Bragato
Home Institution: Faculdade de Medicina Veterinária (FMVA). Universidade Estadual Paulista (UNESP). Campus de Araçatuba. Araçatuba , SP, Brazil

Abstract

The present study was carried out to verify the effects of sperm selection through single layer centrifugation in Percoll® (SLCP; at 90%), before freezing, as well as in discontinuous gradient and SLCP, after thawing, evaluating the sperm parameters and rate of in vitro fertilization (IVF). The sperm of three Nelore bulls (12 samples in a total, four for each experimental group), maintained at the center of collection and processing of semen (CCPS), will be collected by artificial vagina. In exp. I, the semen of each bull will be mixed and equally divided (1,000 x 106 sperm/on the top of gradient column) between the control groups (without SLCP, P0) and those subjected to SLCP (volumes of 9 mL or 6 mL, groups P9 and P6, respectively), while P9 and P6 will be centrifuged at 700 x g/12 min before freezing. In exp. II, four samples of frozen semen from each group, obtained in exp. I, will be subject to sperm selection before IVF; thus, after thawing, the semen will be put on the top of discontinuous gradient of Percoll (45 and 90%) and centrifuged at 700 x g/15 min and then at 250 x g/5 min., the latter for sperm washing. In exp. III, the best result obtained in exp. II will be compared with semen samples of the groups P6 and P9, however, this time subjected to SLCP at 90%. Assessments of motility, vigour, concentration and spermatic morphology will be performed before, after centrifugation and post-thawing. The extender and all procedures will be those of routine at CCPS. The post-thawing evaluations will be made through computer analysis of sperm movement (CASA), membrane integrity (propidium iodide and carboxyfluorescein) as well as through the rate of IVF (at 72 h post-insemination) which the latter being performed in all experiments, from bovine oocytes matured, fertilized and cultured in vitro. ANOVA and Tukey`s test will be used to determine statistical differences among groups, with P<0.05 taken as significant.