Lipid peroxidation, mitochondrial membrane potential and DNA integrity of spermatozoa in relation to intracellular reactive oxygen species in liquid and frozen-thawed buffalo semen

Several factors affect sperm motility and functional integrity during preservation; the damage caused by reactive oxygen species (ROS) being an important factor. The present study investigated intracellular ROS generation and its relationship with sperm motility, lipid peroxidation (LPO), mitochondrial membrane potential (MMP) and DNA integrity during preservation (liquid preservation at 4 °C and cryopreservation) of buffalo semen. Fifty-six ejaculates, eight each from seven buffalo bulls (Bubalus bubalis) were utilized for the study. Intracellular ROS level was detected using 2′-7′-dichlorodihydrofluorescein diacetate (DCFH-DA) and propodium iodide (PI) by flow cytometry. 3,3′-Dihexyloxacarbocyanine iodide [DiOC6 (3)]/PI and acridine orange were used for detection of MMP and DNA integrity of spermatozoa, respectively. Results revealed that ROS and LPO level in sperm increased linearly between 0 h and 72 h of liquid preservation at 4 °C, with significantly (P < 0.01) higher levels at 48 h and 72 h of storage compared to fresh semen. The ROS level in viable sperm in frozen-thawed semen did not differ significantly from fresh semen, but the LPO was significantly (P < 0.05) higher in frozen-thawed sperm compared to fresh sperm. There was a linear reduction in the sperm with high MMP and DNA integrity in liquid semen, which was significantly (P < 0.01) higher at 48 h and 72 h of storage compared to fresh semen. The intracellular ROS was strongly associated to sperm motility, LPO, MMP and DNA integrity during liquid preservation, while this association did not exist in frozen-thawed sperm. The study concluded that ROS generation and its associated effects are likely to be an important contributor to the reduced sperm motility and functional integrity during liquid preservation of buffalo semen at 4 °C, but ROS generation and its damage had only minor effects during freezing and thawing process.