Comparison of cryoprotective effects of iodixanol, trehalose and cysteamine on ram semen

This study was conducted to improve cryosurvival of electroejaculated (EE) ram semen in the presence of iodixanol (OptiPrep™), trehalose or cysteamine. A tris-based extender was used to prepare 12 extenders containing OptiPrep™ (Op), trehalose (Tr) or cysteamine (Cy) alone, or different combinations of these compounds. Extenders were designated as follows: Tris (control), Op1.25 (1.25% Op, v/v), Op2.5 (2.5% Op, v/v), Op5 (5% Op, v/v), Tr50 (50 mM Tr), Tr100 (100 mM Tr), Cy (5 mM Cy), OpTr (2.5% Op and 100 mM Tr), OpCy (2.5% Op and 5 mM Cy), TrCy (100 mM Tr and 5 mM Cy), OpTrCy1 (2.5% Op, 100 mM Tr and 5 mM Cy) and OpTrCy2 (1.25% Op, 50 mM Tr and 2.5 mM Cy).A two-step dilution was used and glycerol was added at 5 °C in the second step. Diluted samples were equilibrated for 1 h, loaded in 0.25 mL straws and frozen in a programmable freezing machine. Supplementation of 5% OptiPrep™ significantly protected post-thaw progressive motility, membrane integrity, acrosomal integrity and morphological damages. Trehalose supplementation protected membrane integrity of ram sperm; however, it did not help post-thaw motility and morphology. Supplementation of 5 mM cysteamine had detrimental effect on cryosurvival of EE ram semen. These results demonstrate that the supplementation of iodixanol increases the cryosurvival of EE ram semen in a dose-dependent manner.