CRYOPRESERVATION OF RAINBOW-TROUT (ONCORHYNCHUS-MYKISS) SPERMATOZOA USING PROGRAMMABLE FREEZING

In an attempt to establish a protocol for the cryopreservation of the spermatozoa of the rainbow trout (Oncorhynchus mykiss), we studied the effect of various cryoprotective agents (CA) in spermatozoa motility and viability, before, during, and after freezing. Freezing was perfor med by using a controlled rate freezing system, which allows the accur ate setting of different cooling rates, as well as a proper recording of intra-sample temperatures throughout the procedure. Results obtaine d indicate that before the initiation of freezing, spermatozoa motilit y is affected more by the length of time of exposure to CA than by the chemical nature of the agents. Exposure periods longer than 10 min af fected motility irreversibly, which seems to be related to the high os molarity of the extender solutions, To study the changes in spermatozo a motility and viability during and after cryopreservation, cells in t he cryoprotective solution (glycerol, DMSO or DMSO-sucrose) were proce ssed in a programmable biological freezer at slow (1 degrees C and 10 degrees C min(-1)) or rapid (30 degrees C min(-1)) cooling rates. Resu lts obtained indicated that both during (-60 degrees C) and after comp letion of the freezing program (-80 degrees C, followed by storage und er liquid nitrogen), motility and viability was well preserved only in the cells in DMSO-sucrose when subjected to a rapid cooling rate. Und er these conditions, approximately 63% of spermatozoa were alive and s howed progressive motility, Together, the average fertilization potent ial of cryopreserved semen was 58% (47% to 85%) compared with that of fresh semen. The procedure described here provides consistency and pre cision, and permits processing, freezing and storage of trout spermato zoa in less than 15 min.