The ability of sucrose to protect spermatozoa against mitochondrial damage, artificial acrosome reaction and DNA fragmentation during ultra-rapid cryopreservation in canine sperm was investigated. Swim-up selected spermatozoa of second-fraction semen were vitrified with different concentrations of sucrose (0.1, 0.25 and 0.4 M) in proportion 1 : 1 v/v with HTF-BSA 1%. From each group, 30-mu l suspensions of cells were dropped directly into liquid nitrogen and stored for at least 24 h. Cells were thawed by submerging the spheres in HTF with 1% BSA at 37 degrees C. The number of progressively motile spermatozoa was significantly higher in the sucrose 0.25 M + HTF-BSA 1% (42.5 +/- 2.3%, P < 0.01) than in HTF only (1.66 +/- 0.3%). The same combination of sucrose 0.25 m + HTF-BSA 1% (42.7 +/- 1.5%) had a stronger cryoprotective effect on the integrity of mitochondrial membrane potential (P < 0.05) and decreased the DNA fragmentation (2.8 +/- 0.5%) as compared with HTF only (1.93 +/- 0.6% and 5.6 +/- 0.6% respectively). With respect to acrosome-reacted spermatozoa, no significant difference was found between the groups investigated (P > 0.05). It is concluded that sucrose, a nonpermeable cryoprotectant, can effectively preserve important physiological parameters such as mitochondrial membrane potential and DNA integrity during ultra-rapid cryopreservation.