¹Istanbul University-Cerrahpasa, Cerrahpasa Faculty of Medicine, Department of Histology and Embryology, TR-34098 Istanbul - TURKEY DOI : 10.9775/kvfd.2021.25952 Cryopreservation techniques achieved by optimization of the composition and concentration of cryoprotectants not only reduce cryoinjury to ovarian tissue but also enhance survival. Anti-Freeze Proteins (AFPs) have been acknowledged to have positive effects on the survival of oocytes, embryos and ovaries during cryopreservation. AFPs drop the freezing point beneath the balance point of melting by binding ice crystals; restricting their expansion and recrystallization. This study aimed to investigate the protective effects of AFP I and III separately or in combination on the vitrification and warming procedures of ovarian tissues which were vitrified for one week and one month. Ovaries were obtained from rats and randomly assigned to four groups according to the AFP supplements in the media used during freeze/warm procedures: 1) control group (medium without AFP); 2) AFP I; 3) AFP III; 4) AFP I + AFP III groups. The groups were further organized according to the duration of vitrification, 1 week or 1 month. Ovaries were evaluated morphologically by a grading system, TUNEL assay and by transmission electron microscopy. AFP supplementation decreased apoptosis and follicular damage. Supplementation of AFP type I and type III has cryoprotective roles in ovaries which were vitrified for 1 week and 1 month. Additionally, AFP I+AFP III supplementation were shown to have more protection in long term. Keywords : Anti-freeze protein, Cryopreservation, Ovary, Vitrification, Rat