Lipid peroxidation is considered to be an important cause of sperm membrane damage, resulting in an apparent reduction of reproductive fecundity. Recently, a new lipophilic fluorescent dye probe (BODIPY(581/591) C11; Invitrogen Singapore Pte Ltd, Singapore) has been demonstrated to be a highly sensitive indicator for the physiological oxidation of cell membrane fatty acids. The objectives of this study were: (i) to detect lipid peroxidation in frozen-thawed epididymal cat spermatozoa using the BODIPY(581/591) C11 and (ii) to study the effect of semen extender in protecting sperm membrane from lipid peroxidation [100-mm ferrous ion, ferrous sulphate (FeSO(4))]. Epididymal cat spermatozoa were collected from eight male cats. Two straws of sperm sample from each cat were cryopreserved. After thawing, the semen extender from the first straw was removed and the sperm pellet was resuspended with Tris buffer (control). The semen sample from the other straw was equally divided: one sample contained semen extender (treatment A) and one contained no extender (treatment B); both were incubated with FeSO(4). Semen samples were labelled with the BODIPY(581/591) C11 probe and evaluated by flow cytometry at 0 and 6 h after thawing (control), 6 h after the addition of FeSO(4) (treatment A), and 30 min and 6 h after the addition of FeSO(4) (treatment B), respectively. The percentage of lipid peroxidation was higher after treatment B (51.3 +/- 23.9) and 6-h incubation compared with the control and treatment A (p < 0.05). Furthermore, the percentage of lipid peroxidation after treatment B increased during the incubation time (p < 0.05). In conclusion, the high percentage of lipid peroxidation after treatment B indicated that FeSO(4) induced membrane damage in cat spermatozoa, which could be detected by BODIPY(581/591) C11. This marker is suggested to be a beneficial tool for the evaluation of lipid peroxidation. Furthermore, the use of semen extender seemed to protect cat spermatozoa membranes from lipid peroxidation.