The aim of this study was to research the effects of four antioxidants that had been added to semen diluents, on spermatological parameters, anti-oxidant enzymes activities and DNA integrity after the freeze-thaw procedures in Brown-Swiss bull semen. A total of 24 ejaculates were collected from three bulls. Each ejaculate was divided into five equal parts and they diluted to the Tris + 25 mM trehalose base
extender containing fetuin 1 mg/ml, dithioerithritol 1 mM, cysteamine 4 mM or linoleic acid 0.5 ml/50 ml, and no additives (control). Group dithioerithritol and linoleic acid showed the higher rates of CASA progressive motility, CASA sperm motility (P<0.05) compared with the other groups. Group linoleic acid provided the better protective effect for acrosome and total abnormalities (P<0.01, P<0.05; respectively). Also,
group dithioerithritol showed the highest values for the HOS test (P<0.01). In the comet test, group dithioerithritol enhanced tail with lesser chromatin damage than the other groups (P<0.05). Supplementation of dithioerithritol significantly affected the GSH activity (P<0.01). Also groups dithioerithritol and linoleic acid gave higher CAT values than the other groups (P<0.05). Additionally, supplementation of fetuin and
cysteamine showed the lowest total antioxidant activity value (P<0.05). In conclusion, we may said that, the addition of antioxidants, specially dithioerithritol and linoleic acid, to added with 25 mM trehalose Tris extender improve post-thaw sperm parameters.