Post-thaw ATP supplementation enhances cryoprotective effect of iodixanol in rat spermatozoa

Background: Successful cryopreservation of rat spermatozoa from various strains still remains a challenge. The objective of this study was to determine if combinations of OptiPrep (TM) (iodixanol) and adenosine 5'-triphosphate (ATP) can improve rat sperm function during the cryopreservation procedure. Methods: Epididymal rat spermatozoa were frozen under different OptiPrep (TM) concentrations (0, 1, 2, 3 or 4 %) and were diluted with media supplemented with or without 2 mM ATP after thawing. Post-thaw sperm motility, acrosomal membrane integrity (AMI) and mitochondrial membrane potential (MMP) were then evaluated. In addition, the effect of different OptiPrep (TM) concentrations on fresh and cooled rat spermatozoa was tested via motility. Results: There was no effect of OptiPrep (TM) on motility of fresh and cooled spermatozoa. The supplementation of 1 and 2 % OptiPrep (TM) increased motility of frozen spermatozoa at 10 min after thawing, while it did not improve motility of spermatozoa at 3 h after thawing in the absence of ATP. During incubation of thawed spermatozoa, the ATP addition protected time-dependent decrease in motility after thawing in OptiPrep (TM)-treated samples. OptiPrep (TM) had no effect on AMI and MMP in frozen-thawed spermatozoa but combinations of OptiPrep (TM) and ATP improved MMP in frozen-thawed spermatozoa. Conclusions: Iodixanol has cryoprotective effects during rat sperm freezing without any toxic effect. Moreover, the combinations of iodixanol and ATP have a beneficial role in maintaining function of frozen-thawed rat spermatozoa for long period of incubation post-thaw.