Background: To evaluate the efficiency of bacterial clearance of different sperm preparation procedure. Methods: A total of 46 semen samples were collected in this study, each semen sample was divided into four equal parts (0.5 mL each). The first part was prepared by density-gradient centrifugation (D-group), the second part was prepared by swim-up (S-group), the third part was treated with density-gradient centrifugation combined with swim-up (D+S-group), and the fourth group was the original raw semen group (O-group) regarded as the control. After each semen preparation procedure, the O-group and suspensions were inoculated and incubated for bacterial identification and colony counting. Results: Initially only 8.7% (4/46) of samples were bacteria free. After processing, the bacterial clearance rates were 23.8% for D-group, 57.4% for S-group and 97.8% for D+S-group (p < 0.001). Multiple bacterial strains were observed in 37 samples with 117 different bacterial strains in all identified in the original semen. 44.4% of the staphylococci and 89.7% of streptococci were not eliminated in D-group. In S-group, the corresponding rates were 24.4% and 35.9%. In D+S-group there were no remaining strains of staphylococci and only 2.6% of streptococci remained. After the combined procedure, the number of bacterial colonies fell dramatically after processing. Conclusion: The combined D+S protocol appears to be substantially more efficient than either method alone in eliminating bacteria from semen samples.
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