Iodixanol supplementation during sperm cryopreservation improves protamine level and reduces reactive oxygen species of canine sperm

The objective of this study was to analyze the protective effects of iodixanol on dog spermatozoa during cryopreservation. The optimal concentration of iodixanol, 1.5%, was determined using fresh spermatozoa and was applied in the following experiments. The 1.5% iodixanol group showed significantly increased sperm motility from that in the control (p < 0.05). Lower mitochondrial reactive oxygen species (ROS) modulator (ROMO1) and pro-apoptotic gene (BAX) expressions, together with higher expressions of protamine-2 (PRM2), protamine-3 (PRM3), anti-apoptotic B-cell lymphoma-2 (BCL2), and sperm acrosome associated-3 (SPACA3) genes were detected in the iodixanol-treated group. In addition, decreased protamine deficiency and cryocapacitation were observed in the treatment group. Our results show that supplementation with 1.5% iodixanol is ideal for reducing production of ROS and preventing detrimental effects during the canine sperm cryopreservation process, effects manifested as increased motility and reduced cryocapacitation in frozen-thawed spermatozoa.