Open Access Open Badges Research article

Effect of controlled and uncontrolled cooling rate on motility parameters of cryopreserved ram spermatozoa

Iraj Ashrafi1*, Hamid Kohram23, Hamid Naijian2, Majid Bahreini4 and Hamid Mirzakhani4

Author Affiliations

1 Young Researchers Club, Science and Research Branch, Islamic Azad University, Tehran, Iran

2 Department of Animal Science, Faculty College of Agriculture and Natural Resources, University of Tehran, Karaj, Iran

3 Department of Clinical Sciences, Faculty of Veterinary Medicine, Shahid Chamran University, Ahvaz, Iran

4 Animal Breeding Center of Iran

For all author emails, please log on.

BMC Research Notes 2011, 4:547  doi:10.1186/1756-0500-4-547

Published: 20 December 2011



Ram spermatozoa are sensitive to extreme changes in temperature during the freeze-thaw process. The degree of damage depends on a combined effect of various factors including freezing temperature. 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 three replicates of the ejaculates were diluted with a Tris-based extender and packed in 0.25 ml straws. Then, sample processed according to the two methods. Method 1: straws 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, then the straws were frozen at rate of -0.3°C/min from 5°C to -10°C and -25°C/min from -10°C to -150°C and plunged into liquid nitrogen for storage. Method 2: straws were transferred to 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. Computer-assisted sperm motility analysis was used to analyze sperm motion characteristics.


Controlled rate of freezing (Method 1) significantly improve the pre-freezing and post-thaw total and progressive motility compared to uncontrolled rate (Method 2). In specific kinetic parameters, Method 1 gives significantly higher value for VSL and VCL in comparison with Method 2. There are no significant differences between the two methods for VAP and LIN. In conclusion, controlled rate of cooling conferred better cryopreserving ability to ram spermatozoa compared to uncontrolled rate of cooling prior to programmable freezing.