Measuring Sperm Glutathione Levels

GSH plays a key role in maintaining cellular redox homeostasis. According to Zhu et al [20 (link)], a commercial GSH assay kit (A061-1, Nanjing Jiancheng Bioengineering Institute, China) was used to measure sperm GSH content. For the determination of total glutathione (T-GSH), the sperm samples were homogenized and centrifugated at 3,500 rpm/min and the supernatant was collected. Then, reagents were added and mixed with the supernatant based on the manufacturer’s instructions. The absorbance (A1) was measured at 532 nm using a microplate reader after waiting for 30 s, while absorbance (A2) was measured after standing at room temperature for 10 min. To detect oxidized glutathione (GSSG), reagents were added, mixed with the supernatant, and incubated at 37°C for 30 min according to the manufacturer’s instructions. Thereafter, reagents were added and mixed again, and absorbance (A1) and absorbance (A2) were read at 450 nm based on manufacturer instructions. GSH content was determined using the formula: GSH = T-GSH-2×GSSG. The analyses were performed in triplicate.