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.
Highlights
Considerable effort has been directed towards developing techniques for artificial insemination (AI) using frozen ram semen
The changes observed in motility were reflected in the recovery rate which indicated that method 1 preserved sperm motility better, showing significant difference with respect to method 2
It may be noted that the total motility was preserved better than the progressive motility with recovery rates of 0.89 and 0.74, respectively for method 1
Summary
Considerable effort has been directed towards developing techniques for artificial insemination (AI) using frozen ram semen. The widespread application of AI depends largely on the use of frozen semen and on the availability of techniques that result in acceptable fertility in a selective breeding control programme. AI with frozen semen dispensed through the cervix gives quite low fertility rates in ram and use of laparoscopy with thawed semen is more costly and time-consuming but it improves the fertility rate significantly. The need to prepare a large number of doses of ram’s semen each year requires the development of a rapid and effective method for freezing semen (Anel et al, 2003). Ram spermatozoa are susceptible to various stresses during cryopreservation (Anel et al, 2006). The physiological and functional changes that occur in spermatozoa such as an irreversible reduction in motility, viability and acrosome integrity (Watson, 1995; Salamon and Maxwell, 2000; Medeiros et al , 2002) cause
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