Cryopreservation of rainbow trout (Oncorhynchus mykiss) spermatozoa using programmable freezing

Abstract

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 performed by using a controlled rate freezing system, which allows the accurate setting of different cooling rates, as well as a proper recording of intra-sample temperatures throughout the procedure. Results obtained indicate that before the initiation of freezing, spermatozoa motility 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 affected motility irreversibly, which seems to be related to the high osmolarity of the extender solutions. To study the changes in spermatozoa motility and viability during and after cryopreservation, cells in the cryoprotective solution (glycerol, DMSO or DMSO-sucrose) were processed in a programmable biological freezer at slow (1°C and 10°C min-1) or rapid (30°C min-1) cooling rates. Results obtained indicated that both during (-60°C) and after completion of the freezing program (-80°C, followed by storage under liquid nitrogen), motility and viability was well preserved only in the cells in DMSO-sucrose when subjected to a rapid cooling rate. Under these conditions, approximately 63% of spermatozoa were alive and showed progressive motility. Together, the average fertilization potential of cryopreserved semen was 58% (47% to 85%) compared with that of fresh semen. The procedure described here provides consistency and precision, and permits processing, freezing and storage of trout spermatozoa in less than 15 min