The aim of this study was to evaluate the effect of adding ascorbic acid (AA) to the vitrification medium of bovine preantral follicles (PFs) subjected previously to cooling at 4 °C for 4 h or 24 h. Ovaries were collected from Nelore heifers at 14 months of age. In the laboratory, ovarian fragments were removed from the cortical region and distributed to fragments as fresh control (C0h), and fragments to cooling at 4 ºC for 4 and 24 hours in TCM-199 plus HEPES and antibiotics. Of the cooled fragments, two were fixed as controls for each cooling time (C4h, C24h), and the remaining fragments were distributed in four vitrification treatments, using the TCM-199 medium associated with ethylene glycol and dimethyl sulfoxide (V), sucrose (VSUC) or ascorbic acid (VAA), and the treatment with ethylene glycol, dimethyl sulfoxide, sucrose and AA (VSUC+AA). After 72 h, fragments were war...