Background: For reproductive biotechnology and genetic preservation, rooster sperm cryopreservation is essential. However, sperm quality is impacted by oxidative stress during freezing and thawing, which calls for the use of efficient cryoprotectants. Sugars that stabilize sperm membranes and reduce oxidative damage include sucrose, trehalose, and raffinose. Comparative analyses of these sugars are still lacking, though, especially when it comes to sperm characteristics and antioxidant activity. Objective: This study focused on computer-assisted sperm analysis characteristics and antioxidant enzyme activities (glutathione peroxidase, total antioxidant capacity, and superoxide dismutase) in order to examine the effectiveness of 2 disaccharides, sucrose and trehalose, as cryoprotectants in rooster sperm cryopreservation. Materials and Methods: This is an experimental study which was carried out in Tabriz University, Tabriz, Iran. 10 mature Ross roosters (30 wk) were used to obtain semen samples, which were then diluted in an extender enriched with either trehalose (0.2 M) or sucrose (0.2 M). Following a conventional freezing procedure, the samples were assessed after thawing. Analysis was done on sperm motility, velocity, and other computer assisted sperm analysis characteristics. Biochemical tests for glutathione peroxidas, total antioxidant capacity, and superoxide dismutase were used to quantify antioxidant activity. Results: When compared to sucrose, samples treated with trehalose exhibited noticeably greater velocity parameters, progressive motility (28.5), and total motility (61.7) (p < 0.05). Trehalose also offered better membrane integrity. In terms of antioxidant activity, the trehalose group exhibited much higher GPX and SOD activities, whereas TAC significantly outperformed sucrose. The findings show that during cryopreservation, trehalose performs better than sucrose at preserving functional sperm characteristics and reducing oxidative stress. Conclusion: For rooster sperm cryopreservation, trehalose works better than sucrose as a cryoprotectant, improving antioxidant defenses and CASA parameters. These results demonstrate its potential for improving poultry species cryopreservation procedures. Keywords: Sperm cryopreservation, Trehalose, Sucrose, Antioxidant enzymes.
