The aim of this study was to determine the effect of butylated hydroxytoluene (BHT) supplemented cryopreservation medium on sperm parameters during the freeze-thaw process. In addition, sperm lipid peroxidation, DNA damage, and the amount of reactive oxygen species (ROS) were determined. Semen samples were obtained from 75 donors. Fifteen semen samples were used for optimizing BHT concentration and incubation time and 60 samples were used for the final experiments. After the determination of basic parameters, groups of three sample with similar parameters were pooled and processed by Pure Sperm gradient centrifugation. The semen samples were then diluted with normal freezing medium (control) or a medium containing 0.5 mM BHT (test) for 5 minute and stored in liquid nitrogen. Frozen cryovials were thawed individually for 20 seconds in a water bath (37°C) for evaluation. Freezing extenders supplemented with 0.5 mM BHT led to higher sperm motility and viability compared with control samples (p < 0.001). Furthermore, the addition of BHT decreased malondialdehyde (MDA) formation, DNA fragmentation, and ROS content compared with controls (p < 0.001). Our results showed that the addition of BHT to the freezing medium could be of advantage in reducing ROS and preventing the detrimental effect of ROS on the human sperm function.
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