Sperm cryopreservation, as a key technique in assisted reproductive technology, often results in significant damage to sperm quality. Theobromine has many positive effects on sperm quality due to its antioxidant properties. In this study, the effect of theobromine on sperm of asthenospermic men during sperm Freezing-Thawing was investigated. Semen samples were collected from 30 men with asthenozoospermia, and then each sample was divided into three groups: control, cryopreservation, and cryopreservation + theobromine (10 mM). Acrosomal reaction was assessed by staining with FITC- PSA labeled with fluorescein isothiocyanate, mitochondrial membrane potential was assessed by staining with rhodamine123, and DNA fragmentation analysis was performed using the SDFA kit. Statistical analysis was performed using the Repeated Measure Analysis method and Bonferroni post hoc test. In the freeze group, there was a significant increase in the mean level of sperm DNA fragmentation and a significant decrease in both the percentage of sperm with intact acrosomes and mitochondrial membrane potential, compared to the control group (P<0.001). In the freeze + theobromine group, DNA fragmentation increased significantly (P<0.05), and mitochondrial membrane potential improved significantly (P<0.001) compared to the freeze group. However, the percentage of sperm with intact acrosomes showed no significant change (P>0.05). The results of this study demonstrated that sperm freezing impairs quality by increasing DNA damage and reducing acrosome integrity and mitochondrial function. Theobromine improved mitochondrial potential but failed to protect acrosome integrity and increased DNA fragmentation.Keywords: Asthenozoospermia, Cryopreservation, Theobromine, Acrosome Reaction, mitochondrial membrane potential, DNA fragmentation.
