Evaluation of the spatial disposition of cholesterol by filipin in different states of sperm capacitation of bovines

• Grant number: 22/12878-0
• Support Opportunities: Scholarships in Brazil - Scientific Initiation
• Start date: December 01, 2022
• End date: November 30, 2023
• Field of knowledge: Agronomical Sciences - Veterinary Medicine - Animal Reproduction
• Principal Investigator: Eneiva Carla Carvalho Celeghini
• Grantee: Gabriel de Miranda Teodoro Soares
• Host Institution: Faculdade de Medicina Veterinária e Zootecnia (FMVZ). Universidade de São Paulo (USP). São Paulo , SP, Brazil

Abstract

Sperm must undergo a series of biochemical events during transport through the female's reproductive tract in order to be able to fertilize the oocyte. These events culminate in sperm modifications that characterize sperm capacitation; in view of the importance of this event for the fertilization of the oocyte, there is therefore a need for new forms of sperm evaluation that can detect these events and relate them to the fertility potential of the semen. One of the steps of capacitation is the depletion of cholesterol from the plasma membrane and the redistribution of the remaining cholesterol from the equatorial to the apical region, forming lipid rafts. The objectives of this work are to propose a protocol for bovine semen using Filipina to evaluate the spatial disposition of cholesterol at different stages of sperm capacitation, and to evaluate the changes in the disposition of these molecules after cryopreservation. Three ejaculates from four Nelore bulls (n=12) will be used. Each ejaculate will be subjected to conventional analyzes (motility, vigor, concentration and morphology) and then the semen will be distributed in four treatments: 1. in natura (IN), non-capacited sperm; 2. induction of sperm capacitation for two hours and 30 minutes (MT, half the time of total induction); 3. induction of sperm capacitation for five hours (PC, full induction time); 4. post-cryopreservation (CR). After the treatments, all samples will be evaluated for cholesterol disposition using the Philippine fluorescent probe and for the acrosomal reaction using FITC-PSA. Data will be submitted to ANOVA and linear regression analysis, considering a significance level of 5%. It is expected that, in the IN sample, the normal distribution of cholesterol molecules on the plasma membrane (control) is observed, and that after half of the capacitation induction time (2.5h), in the MT sample, a rearrangement of the cholesterol molecules, after the complete capacitation induction time (5h), in the CP sample, cholesterol depletion has occurred; in addition, in the post-cryopreservation (CR) sample, it is expected to find a disorganization of the cholesterol molecules due to the lesions caused by cryopreservation.