The purpose of this study was to observe the effect of different thawing methods on semen parameters such as motility and morphology and sperm chromatin integrity as assessed by toluidine blue (TB) staining. A total of 20 frozen sperm straws from the same Holstein bull were used. While the 30 sec thawing protocol at 37 degrees C, which is used for thawing frozen sperm straws, constitutes our slow thawing group (n=10), the 6 sec thawing protocol at 70 degrees C constitutes our fast-thawing group (n=10). The motility, viability, morphology, plasma membrane integrity, and sperm chromatin condensation parameters of all thawed sperm were investigated. There was a significant difference (P<0.05) in sperm plasma membrane integrity, head defect, and total abnormal sperm morphology. The chromatin decondensation rate detected by TB in bull semen thawed in the slow thaw system, and the decondensation rate in the fast thaw system, differed significantly from each other in line with the literature data (P<0.05). According to the evaluations made in terms of chromatin decondensation rate, the rate obtained in slow thawing (7 +/- 0.39) shows an increase up to two times compared to the fast-thawing rate (3.3 +/- 0.33) (P<0.05). The TB staining procedure can be used to evaluate infertility and chromatin integrity, especially in cases that are suspicious and require rapid evaluation.