Abstract

Ram spermatozoa are sensitive to extreme changes in temperature during the freeze-thaw process. The aim of this study was to determine the effects of two cooling method (controlled-rate and uncontrolled-rate) on pre-freezing and post-thaw sperm motility parameters. Ejaculates were collected using the artificial vagina from four Chal rams and diluted with a Tris-based extender and packed in 0.25 ml straws. Then, the sample was processed according to the two methods; method 1: straws were cooled from 37 to 5°C, at a liner rate of -0.3°C/min in a controlled-rate cooling machine (custom-built) and equilibrated at 5°C for 80 min and then were frozen at the rate of -0.3°C/min from 5 to -10 and -25°C from -10 to -150°C and plunged into liquid nitrogen for storage; method 2: straws were transferred to a refrigerator and maintained at 5°C for 3 h, then, the straws were frozen in liquid nitrogen vapor 4 cm above the liquid nitrogen, for 15 min and plunged into liquid nitrogen. A computer-assisted sperm motility analysis was used to analyze sperm motion characteristics. The controlled rate of freezing (method 1) significantly improved the pre-freezing and post-thaw total and progressive motility compared with the uncontrolled rate (method 2). In specific kinetic parameters, method 1 gave significantly higher value for straight linear velocity (VSL) and curvilinear velocity (VCL) in comparison with method 2. There were no significant differences between the two methods for average path velocity (VAP) and linearity (LIN). In conclusion, the controlled rate of cooling conferred better cryopreserving ability to ram spermatozoa compared with the uncontrolled rate of cooling prior to programmable freezing.

Key words: Controlled cooling rate, uncontrolled cooling rate, motility parameters, ram spermatozoa.