Proposal of criteria for the establishment of the profile of sperm subpopulations as qualitative standard for cryopreserved ram semen aiming assisted reproduction

• Grant number: 11/12548-5
• Support Opportunities: Scholarships in Brazil - Post-Doctoral
• Start date: September 01, 2011
• End date: August 31, 2013
• Field of knowledge: Agronomical Sciences - Veterinary Medicine - Animal Reproduction
• Principal Investigator: Sony Dimas Bicudo
• Grantee: Leandro Rodello
• Host Institution: Faculdade de Medicina Veterinária e Zootecnia (FMVZ). Universidade Estadual Paulista (UNESP). Campus de Botucatu. Botucatu , SP, Brazil

Abstract

The aims of this project were: to determine sperm subpopulations (ES) by four methodological criteria (MC) through computer-assisted semen analysis (CASA) of sperm kinetics (MC1 - five ES), to access plasma membrane integrity (MC2) and acrosome reaction (four ES); to quantify the percentage of apoptotic sperm (MC3 - four ES) and sperm undergoing capacitation (MC4) (membrane stability and fluidity - three ES) in cryopreserved ram semen supplemented with Trolox and catalase antioxidants. The results from the methodological criteria from evaluation of subpopulations will help determine the best method for separating and recovering or classification cryopreserved sperm, in order to use it in assisted reproductive biotechnologies. In Experiment 1, three ejaculates from Dorper breed rams (n = 10) will be collected by means of an artificial vagina and cryopreserved using Trolox and catalase antioxidants. Semen will be evaluated after collection (A1), post-cooling (A2) and post-thawing (A3) through CASA (MC1) and flow cytometry using different fluorochromes: propidium iodide (PI) and pisum sativum agglutinin conjugated to fluorescein isothiocyanate (FITC-PSA) to evaluate total integrity of sperm membranes, annexin-V for apoptosis and the combination of merocyanine-540 (M540) and Yo-Pro 1 to determine sperm capacitation (MC2, MC3 and MC4, respectively). During A3, lipid peroxidation will be accessed by TBARS technique. Experiment 2 will be carried out by the sperm separation and recovery methods dextran/ swim-up and Percoll gradient (70% and 35% densities) supplemented with Trolox or catalase antioxidants. Cryopreserved semen used in Experiment 2 will be obtained from Experiment 1 and genetic material storage centers, being ranked according to Experiment 1 ES. Semen will be evaluated by the same methods previously used in Experiment 1. (AU)