Semi-Quantitative Assessment of Superoxide in the Human Neat Ejaculate Using a Nitro Blue Tetrazoilum Based Assay: A Comparison of Visual, Spectrophotometric and Image Analysis Approaches

Abstract

Purpose: To evaluate the efficacy of three colorimetric methods for detecting the presence of SuperOxide (SOx) in the neat human ejaculate based on the transformation of Nitro Blue Tetrazolium (NBT) to formazan. Methods: The resultant color after reacting 230 neat semen samples for 30 min after ejaculation was assessed semi-quantitatively and the results compared using (1) a simple visual assessment, (2) spectrophotometry and (3) an image analysis methodology based on direct scanned digital images. Results: After comparing visual classification with the spectrophotometric values and image analysis, 8 % and 6 %, of cases were misclassified, respectively. However, Pearson correlation calculated between values obtained by visual versus spectrophotometric or image analysis assessments were extremely high (P=0.000). The strongest correlation was observed when spectrophotometric and image analysis data were compared. Conclusion: Color assessment produced after reacting NBT with neat sperm samples can be used as semiquantitative methodology for non-subjective assignment of SOx presence in the human neat ejaculate. The methodologies tested here, allow discrimination amongst ejaculates, with a margin of error among the different methodologies of less than 10%, irrespective of whether the assessment is performed visually, spectrophotometrically or via image analysis.