Freezing of human spermatozoa is done to increase the success of assisted reproductive techniques (Agarwal & Majzoub, 2017). In the freezing method, intracellular ice crystal formation and the concentration of solute are problematic, and the survival of frozen cells depends on the type of cell and freezing rate (Tomás et al., 2019). One way to avoid damage by forming ice crystals is to use the vitrification method (Arav et al., 2018). In the vitrification method, freezing is performed by immersing the samples directly into the liquid nitrogen tank (Isachenko et al., 2018). This method reduces ice crystal formation and intracellular injuries (Adib et al., 2018). Vitrification is delivered in less time than conventional sperm freezing methods and is also safer and less costly (Horta et al., 2017; Spis et al., 2019). Evidence has shown that in semen, ROS are produced in the freezing and thawing processes. Although the average level of ROS can regulate sperm functions when it exceeds the detoxification level, it leads to oxidative stress (Lone et al., 2018). Free radicals from oxidative stress can disrupt motility after thawing, viability, cell membrane, mitochondrial membrane potential, DNA fragmentation, intracellular enzymatic activity, sperm function and fertility (Bustamante Filho et al., 2018; Zhang et al., 2019). Recent studies have indicated that ultra-rapid freezing reduces destructive effects on sperm

Objective: Reactive oxygen species (ROS) are produced during cryopreservation of human sperm and impair sperm function. Antioxidant compounds, such as fennel and purslane, reduce the damaging effects of ROS. This study aimed to evaluate motility parameters, plasma membrane integrity (PMI), mitochondrial membrane potential (MMP), intracellular ROS, and DNA damage to determine the optimum concentrations of hydroalcoholic extracts of fennel and purslane for human spermatozoa cryopreservation. Methods: Twenty human sperm samples were used and divided into seven equal groups consisting of fennel hydroalcoholic extract (5, 10, and 15 mg/L), purslane hydroalcoholic extract (25, 50, and 100 mg/L), and no additive. Results: Supplementation of 25 mg/L and 50 mg/L purslane extract and 10 mg/L fennel extract in cryopreservation extender significantly increased the motility and PMI of sperm with a significant reduction in intracellular ROS compared to control groups (p<0.05). A 50 mg/L concentration of purslane extract elevated progressive motility and MMP compared to the control group (p<0.05). No significant differences were seen for motion patterns and DNA damage of frozen–thawed human sperm in extender containing these extracts. Conclusion: The results showed that supplementation of 50 mg/L purslane extract and 10 mg/L fennel extract in semen cryopreservation extender has the potential to decrease intracellular ROS and subsequently elevate the motility and PMI of human sperm.