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To investigate the differences in semen quality at different times of reanalysis and the correlation of sperm DNA fragmentation index (DFI) with sperm motility alteration using semen samples completely liquefied and normal in initial examination.We analyzed 127 semen samples up to the inclusion criteria with the computer-assisted semen analysis (CASA) system at 15, 30 and 60 min after semen collection, and obtained sperm morphology parameters and DFI by Shorr staining and acridine orange test (AOT) , respectively.Sperm concentration, and the percentages of grades a and b sperm showed no statistically significant differences at the three time points (P0.05). The percentages of grades a + b and a + b + c sperm were significantly higher at 15 min than at 30 and 60 min after semen collection (P0.05), but with no significant difference between the latter two time points (P0.05). The incidence of alternation from normal to abnormal in at least one index of sperm motility at different times was 25.2%, but there were no significant differences in sperm DFI and morphology between the normal and abnormal groups (P0.05). Among the altered parameters of sperm motility from 15 to 60 min, the percentages of grades a, a + b and a + b + c sperm were all positively correlated with sperm DFI (P0.05).Semen samples completely liquefied within 15 min after collection and normal in initial examination, when reanalyzed at 30 and 60 min, showed significant decreases in the percentages of grades a + b and a + b + c sperm, but not in the percentages of grades a and b sperm, and the parameters of sperm motility might be abnormal. Thus, at least 2 sperm analyses are required for a comprehensive evaluation of fertility. Significant difference between the results of the two analyses, and particularly a markedly reduced percentage of rapidly progressive sperm, might indicate sperm DNA damage, and thus the necessity of sperm DNA damage detection.