Ultrasonic vibration (USV) of semen extender can be a novel method to modify the physico-chemical properties of semen extenders and improve their cryoprotective quality. This property may be further improved by combining with antioxidants. Therefore, this study examined the impact of a USV-treated extender enriched with Myo-inositol (MYO) on frozen ram sperm quality in a completely randomised block design with a 2 × 2 factorial arrangement. The main factors were USV (without and with) and MYO (0 and 7.5 mM) arranged in four treatments. Ultrasonic groups' diluents vibrated at 50% power for 15 min. In the breeding season, semen was collected from four Mehraban rams once a week, over five consecutive weeks. Ejaculates were mixed and divided equally into four parts and diluted up to the concentration of 200 × 106 spermatozoa/ml. The samples were frozen in liquid nitrogen. At least 72 h after freezing, samples were thawed and subjected to evaluation. Eosin stain, hypo-osmotic swelling test, computer-assisted sperm analysis, ferric reducing antioxidant power (FRAP), and thiobarbituric acid assay were methods for evaluation of viability, functional integrity of plasma membrane, sperm motility, total antioxidant capacity and lipid peroxidation respectively. Intracellular ROS level and mitochondrial membrane integrity were detected using 2′-7′-dichlorodihydrofluoresce in diacetate (DCFH-DA) and Rhodamine 123 coupled with propidium iodide (PI) stain by flow cytometry. The interaction effects of USV × MYO for none of the studied parameters were significant. Ultrasonic vibration increased viability, total and progressive motility and several kinematic factors; however, sperm antioxidant capacity was reduced and intracellular H2O2 level increased slightly (p < 0.05) without impaired lipid peroxidation or mitochondrial membrane integrity. Total and progressive motility was improved with MYO (p < 0.05). The values of VCL and VAP tended (p < 0.1) to increase with MYO treatment. The highest values of total motile and progressively motile spermatozoa and VCL were observed in the USV group enriched with MYO (p < 0.05). In conclusion, optimal ram sperm function can be preserved in the USV-treated or MYO-supplemented extenders. However, MYO enrichment of the USV-treated extender has no additional effect
