Background: Sperm cryopreservation, a key technique in as- sisted reproductive technology, often results in significant damage to sperm quality. Adding antioxidants to the cryo- preservation medium can help mitigate these adverse effects. Theobromine, a plant alkaloid derived from methylxanthine, is widely used and known for its antioxidant properties, which positively affect sperm parameters. This study investigated the effect of theobromine supplementation on sperm parameters during the cryopreservation of semen samples from astheno- zoospermic men. Materials and Methods: Semen samples were obtained from 30 asthenozoospermic men. Each sample was then divided into three groups: control (fresh), freeze (treated with cryoprotect- ant alone), and freeze + theobromine (treated with cryoprotect- ant and 10 mmol/L theobromine). The samples in the freez- ing groups were cryopreserved using a human sperm freezing medium and a rapid freezing method. For each sample, sperm motility was assessed according to World Health Organization (WHO) criteria using light microscopy. Viability was evaluated using eosin-nigrosin staining, and sperm morphology was ex- amined using the Diff-Quick kit. Data were statistically ana- lyzed using the Repeated Measures Analysis method. Results: Mean sperm motility, viability, and normal morphol- ogy significantly decreased in the Freeze group compared to the Control group (P<0.05). In the Freeze + Theobromine group, a significant increase in sperm motility and viability was ob- served compared to the Freeze group (P <0.05). However, there was no significant difference in normal morphology between the Freeze + Theobromine group and the Freeze group (P>0.05). Conclusion: Our data indicate that theobromine reduces the ad- verse effects of cryopreservation on sperm motility and viabil- ity in asthenozoospermic men, but it has no impact on normal morphology.
