Potential methods for cryopreservation of mouse spermatozoa are freeze-drying, desiccation, and suspension in EGTA Tris–HCl buffered solution (ETBS: 50 mM NaCl, 50 mM EGTA, and 10 mM Tris–HCl). To determine the duration that mouse spermatozoa suspended in ETBS-based solutions could retain their normal characteristics without freezing, spermatozoa collected from the cauda epididymis were suspended in ETBS or in ETBS supplemented with the antioxidants, dimethyl sulfoxide (DMSO), or _DL-α-tocopherol acetate (Vitamin E acetate; VEA) diluted in DMSO, then held at ambient temperature (22–24 °C) for up to 9 days. When oocytes were injected with spermatozoa preserved in ETBS alone, activation rates of oocytes and chromosome integrity at the first cleavage metaphase decreased at 1 day (P<0.001) and 2–4 days (P<0.01) following treatment. When oocytes were injected with spermatozoa preserved in ETBS supplemented with DMSO or VEA/DMSO, chromosome integrity did not decrease significantly (through 9 days of preservation). Although DMSO maintained sperm chromosome integrity more effectively than VEA/DMSO up to 2–4 days (91 and 67%, normal karyotypes in DMSO and VEA/DMSO, respectively), VEA/DMSO helped to maintain the ability of spermatozoa to activate oocytes, but did not enhance the maintenance of sperm chromosome integrity. These results suggested that deterioration of spermatozoa preserved in ETBS alone was delayed by supplementation with antioxidants.
