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Objective To compare the degree of efficiency between density gradient centrifugation (DGC) method and an extended horizontal swim-up (SU) method. Methods A total of 97 couples undergoing in vitro fertilization were enrolled in the study. Semen samples were divided into three aliquots and treated using DGC, extended horizontal SU, and combined methods. DNA fragmentation and chromatin decondensation were detected in native semen samples and their three corresponding aliquots. The corresponding mature oocytes of each semen sample were divided into two sibling cultures. The first sibling culture was microinjected with semen pellets from DGC, and the second sibling culture was microinjected with semen pellets from the combination of both methods. Fertilization rate and embryonic development were assessed at day 3. Results DNA fragmentation and chromatin decondensation was significantly low in DGC and extended horizontal SU samples; however, the rates of DNA fragmentation and chromatin decondensation were significantly lower in extended horizontal SU samples than in DGC samples. The lowest rates of DNA fragmentation and chromatin decondensation corresponded to the samples treated with both methods. The highest rates of DNA fragmentation and chromatin decondensation corresponded to the samples treated with DGC. No significant difference was found in the fertilization rate or day 3 embryos between sibling cultures. Conclusion The combination of DGC and the extended horizontal SU techniques is best for giving the lowest rates of sperm DNA fragmentation and chromatin decondensation.