Effects of Cooling Rate on Membrane Integrity and Motility Parameters of Cryopreserved Ram Spermatozoa

In this study we aimed to determinate the effects of three different cooling rates from +26 degrees C to +5 degrees C at (0.3 degrees C/min 0.6 degrees C/min and 0.9 degrees C/min) on spermatologic and ultrastructure properties of ram semen. For this purpose semen from 6 rams was collected by electroejaculator and was pooled in a +26 degrees C waterbath. Pooled semen was diluated with tris based extender and divided into three equal parts according cooling rates (0.3 degrees C/min., 0.6 degrees C/min. and 0.9 degrees C/min). Cooled semen was reextended with extender B +5 degrees C in the second step. Diluated samples were equilibrated for 1 h and then were loaded in 0.25 mL straws and freezed in liquid nitrogen vapor. After each freezing stage semen was evaluated motility with computer-assisted semen analysis (CASA). Electron microscobic evaluation was done for pooled and chilled samples. It has been observed that 0.3 degrees C/min. cooled group had meaningfully higher values of motility and progressive motility at +5 degrees C after equilibration and post-thaw stages when compared with the 0.9 degrees C/min. group (P<0.05). When compared to the 0.6 degrees C/min., the 0.3 degrees C/min. cooled group had higher total motility values at after cooling to +5 degrees C (P<0.05), equilibration (P<0.05) and post thaw stages (P>0.05) and had higher progressive motility at after cooling to + 5 degrees C (P<0.05), equilibration (P>0.05) and post-thaw stage (P<0.05). The TEM evaluation showed that at cooling to the +5 degrees C increases the total damaged spermatozoa in all groups (P<0.05). In conclusion, cooling the ram semen to +5 degrees C with a rate above 0.3 degrees C/min. affected negatively the spermatological characteristics. Reaching the cooling rates of 0.6 and 0.9 degrees C/min. increasingly deteriorated the post-thaw motility and progressive motility values. Also, low temperature related to ultrastructural damage was observed at the first dilution step and localized at different regions of the sperm head depends upon the processes and cooling rates.