The aim of the present study is to assess sperm cells quality before and after chilling and freezing processes in modified TRIS extender supplemented with two different antioxidants (vitamin E and/ or N-acetyl-cysteine). Twenty experimental units (EU) will be used. Each EU will correspond to a spermatic "pool" recovered from the epididimus of three healthy male dogs aged between 1 to 5 years, previously submitted to elective orchiectomy. Nevertheless, three groups composed by 7 EU each will be formed. Group I (GI), Group II (GII), and Group III (GIII), will be supplemented with 0,5mM, 2,5mM e 5mM of antioxidants, respectively. In all groups, fresh semen will be evaluated on motility, progressive motility, and vital staining. Sperm morphology will be assessed by Pope's staining and membrane integrity will be evaluated by hypo-osmotic swelling test, and fluorescent probes. In addition thermo-resistance test will be performed, and reactive oxygen species (ROS) will be measured as well as mitochondrial activity. Subsequently each EU will be divided in 4 aliquots of equal volume and centrifuged. The supernatant will be discarded and sperm cells resuspended in TRIS extender described by Peña and Linde-Forsberg (2000) and modified by Martins (2005), supplemented by vitamin E, N-acetyl-cysteine, or both. Each aliquot will be divided in two samples one for chilling (for 48 hours), and another for freezing. All samples will be evaluated after chilling and thawing. One way ANOVA with Tukey test as a post hoc test will be performed to compare means (P<0.05).
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