Sperm storage is a basic tool for reproductive biotechnology that causes cell damage at different levels. Our objective was to detect DNA strand breaks or nucleotide oxidization promoted by short and long-term storage in spermatozoa from sex-reversed rainbow trout, using the Comet assay and previous digestion with specific endonucleases. Short-term storage promotes strand breaks and oxidization, especially in guanines. Cryopreservation increases DNA fragmentation, but apparently does not cause oxidization, suggesting possible apoptotic events.