Effect of freezing rate on acrosome and chromatin integrity in ram semen

The objective of the present study was to investigate the effect of different freezing rates on post-thaw sperm motility, acrosome defect, and sperm chromatin structure and apoptotic activity in ram semen. Collected semen was diluted at 1:5 (semen/extender) with Bioxel (R) (IMV technologies France) at 30 degrees C and then cooled to 5 degrees C within 1h. Cooled semen was subjected to the equilibration for 2 hours. Equilibrated semen was frozen in 0.25 ml straw at two different cooling rates (slow: 0.5 degrees C/min from 5 to -20 degrees C and fast: 5 degrees C/min from 5 to -20 degrees C). Both groups were frozen from -20 to -120 degrees C at 25 degrees C/min and stored in liquid nitrogen until use. Post-thaw (37 degrees C/30 min) sperm motility, defected acrosome (Pisum sativum agglutinin fluorescein conjugate, FITC PSA), sperm chromatin structure determined by Acridin Orange (AO) and apoptotic activity using terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) were evaluated. Post-thaw sperm motility, acrosome defect, AO and TUNEL for slow frozen semen were 42.8 +/- 8.8%, 31.6 +/- 12.9%, 2.9 +/- 2.4% and 2.8 +/- 1.6%, and for fast frozen semen were 36.5 +/- 9.9%, 24.7 +/- 11.1%, 3.3 +/- 2.2% and 6.3 +/- 3.4%, respectively. Post-thaw semen analyses showed that there was no significant difference between two freezing curves in terms of acrosome defect, sperm chromatin damage (AO). However, a significant difference was found for post-thaw semen motility between two groups (P<0.05). In conclusion, while the slow freezing procedure improved post-thaw sperm motility, acrosome and chromatin integrities and apoptotic index in ram spermatozoa did not show any significant difference between freezing rates.