*4.1. Lucigenin and Tetrazolium Salts*

The use of NAD(P)H in conjunction with either lucigenin or tetrazolium salt techniques has been previously discussed here in Section 3.1. In this case, the main concern is that several tissue reductases, including sperm cytochromes (CP450R and Cb5R) [101,102], can reduce both probes and, therefore, lead to artefactual NAD(P)H-dependent reduction and the generation of O2•<sup>−</sup> by autoxidation [117,118] (Figures 2 and 3). However, despite these consistent factors, many studies have used this approach to indirectly report the presence of O2•<sup>−</sup> in sperm and further correlate it with semen quality [119–121], capacitation [122], hyperactivation [123], DNA integrity [120,124,125], apoptosis [120], IVF outcomes [121], among others. Yet, caution and a deep understanding of the limitations of both detection methods must guide the interpretation of these data.
