The present study aimed to define functional changes as sperm motility, dead spermatozoa and sperm abnormalities and chromatin damage of sperm by toluidine blue stain, acridine orange stain and flowcytometry during different stages of freezing semen. Semen ejaculates from five Friesian bulls were pooled, diluted in tris-citric acid extender, cooled to 4°C over 2 hours, equilibrated at 4°C for 4 hours and frozen  immediately into a liquid nitrogen (-196°C). Samples were evaluated just after dilution (at 37°C), at 4 hours as equilibration period, and after thawing at 37°C for 30s in water bath. The obtained results showed that, the percentages of sperm motility were significantly (P<0.05) decreased, while the percentages of dead spermatozoa and sperm abnormalities revealed significantly (P<0.05) increased with the different stages of raw semen, diluted semen, cooled semen, equilibrated semen and frozen-thawed semen. In addition, the percentage of chromatin damage was significantly (P<0.05) higher during thawing after freezing using toludine blue, acridine orange and flowcytometry techniques as compared to other steps of freezing bull semen. In conclusion, the present study indicated marked difference in impairment of semen quality and chromatin damage during different steps of Friesian bull frozen spermatozoa, in particular, from equilibration to freezing/thawing.