Effect of staining methods on human sperm morphometrics using HT CASA II.

Objective: To utilize computer-assisted semen analysis (CASA) for comparison of sperm morphometrics between fresh sperm and three different staining methods METHODS: Semen samples from 140 volunteers were stained with Diff-Quik (DQ), SpermBlue (SB), and black glutinous rice extract (BR). Morphometry by automatic CASA evaluated head defects, width, length and presence of normal sperm and acrosomes.

Results: Comparison of width and length between fresh sperm heads and those stained by the three methods found that DQ produced averages that were higher (p<0.05), whereas results obtained from SB and BR dyes were consistent with those of fresh sperm. Parameter measurements between SB and BR did not statistically significantly differ. In contrast, sperm head defects were found to be significantly varied among all techniques. Also, percentages of normal sperm and normal acrosomes were significantly different (p<0.05), with the exception that percentage of normal acrosomes from DQ were consistent to those from SB and BR.

Conclusion: The implemented stains will impact sperm morphometrics. This dilemma can be mitigated if specific criteria of CASA parameters are defined for each stain; this would be particularly applicable for evaluation of novel dyes.