Semen samples collected from 28 male partners of infertile couples were divided into three equal aliquots and prepared with three selected media, such as PureSperm (Nidacon, Gothenburg, Sweden), Sil-Select Plus (Fertipro, Beernem, Belgium) and SpermGrad (Vitrolife, Gothenburg, Sweden). The differences in mean percentages of semen parameters were assessed by repeated measures analysis. Correlations of sperm DNA damage, as measured by terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay, and of protamine deficiency, as measured by chromomycin A3 (CMA3) staining with sperm parameters, were determined by Pearson's correlation. After preparation with all three media, sperm concentrations decreased (P < 0.05) while percentages of sperm with normal morphology increased (P < 0.05). Percentages of sperm motility, rapid motility and progressive motile concentration (PMC) increased (P < 0.05) for each of these parameters, PureSperm preparation gave the best results (P < 0.05). The percentage of DNA damage decreased in the PureSperm and Sil-Select Plus preparations (17.9% and 31.3%, respectively, P < 0.05) and increased in the SpermGrad preparation (56.3%, P < 0.05). Protamine deficiency also decreased in all three kinds of media, 59.3%, 47.7% and 40.3% for PureSperm, Sil-Select Plus and SpermGrad preparations, respectively (P < 0.05). The percentage of DNA-damaged sperm was negatively correlated with the percentages of sperm motility, rapid motility and PMC, but was positively correlated with static motility (P < 0.05). This comparative study and correlation analysis revealed that PureSperm preparation yielded sperm with the best motility and the lowest percentage of protamine deficiency. The Sil-Select Plus preparation yielded sperm with the lowest amount of DNA damage. The SpermGrad preparation had a high percentage of sperm with normal morphology, but also had the highest percentage of sperm with DNA damage. Sperm DNA damage was correlated with percentages of sperm motility, rapid motility, static motility and PMC.