Percoll gradient and swim-up are the most common methods used to obtain high quality intact sperm to be used for the in vitro fertilization procedures. In this study, we evaluated the effects of swim-up and percoll gradient methods on DNA integrity and other spermatological parameters. To determine DNA breakdown, toluidine blue staining was used. Motility, and progressive motility were analyzed via computer assisted semen analyzer (CASA) system and the others (morphology, viability, membrane and acrosome integrity) were evaluated via phase contrast and florescent microscopy. The membrane integrity was tested by hypo-osmotic swelling test. Also, the separated sperms were tested for in vitro embryos production. Our results showed no differences between the two methods of separation compared to the fresh samples in regard of sperm motility (P>0.05), however, in the separated sperms with both methods, progressive motility was higher than the fresh sperm and the highest results were obtained in Percoll method (P<0.05). In comparison to swim-up, Percoll gradient method maintained the highest normal sperm morphology rate and the lowest acrosome defects (P<0.05). In addition, the membrane integrity, intact acrosome and intact chromosome rates were also higher in the sperm samples separated by percoll gradient method (P<0.05). The blastocyst rate produced with sperm separated by Percoll gradient method was significantly higher than the swim-up group (P<0.001). Sperm samples obtained after Percoll gradient centrifugation could improve motility, morphological intactness, and DNA integrity. Accordingly, it was concluded that the Percoll gradient separation method is more useful then the swim-up for in vitro sheep embryo production. Also, Toluidine blue staining could be a suitable technique for determination of DNA integrity.