Effect of MnTBAP on sperm ultra-rapid freezing and its proteomics study

MnTBAP is a new synthetic antioxidant that has been used for the cryopreservation of sperm. However, the exact mechanism of its cryoprotection at the molecular level is largely unknown. Therefore, in this study, normal human semen samples were selected and MnTBAP (0, 5, 10, 20, 40 μM) was added to sperm freezing medium to assess changes in kinetics parameters, apoptosis, reactive oxygen species (ROS), and DNA fragmentation index (DFI) after sperm ultra-rapid freezing. The tandem masstagging (TMT) proteomics technique was used to further investigate the changes in proteins after sperm ultra-rapid freezing. The kinetic parameters of sperm after ultra-rapid freezing and thawing were significantly reduced and apoptosis, ROS production and DFI were significantly increased. The addition of 40 μM MnTBAP improved the kinetic parameters, while it reduced apoptosis, ROS production, and DFI of sperm after ultra-rapid freezing and thawing (P < 0.05). Compared with the fresh semen, 1978 differential proteins were identified in the frozen-thawed sperm without MnTBAP and 1888 differential proteins were identified in the frozen-thawed sperm with MnTBAP (40 μM) added. The proteins affected during ultra-rapid freezing were mainly related to sperm metabolism, flagellar structure motility, apoptosis, intracellular signaling, capacitation and fertilization, while the addition of MnTBAP reduced the alterations of these proteins.