Sperm longevity following pre-freeze sperm selection

Abstract

centrifugation improves cryosurvival after delayed cryopreservation, as compared to the standard centrifugation processing. Moreover, centrifugation processing directly after semen collection and storage of selected sperm was compared with centrifugation of stored sperm just prior to cryopreservation. For each processing method, twelve ejaculates were tested from stallions of the Hannoverian warmbloodbreed (twoejaculates fromeach of six stallions). Sperm motility was evaluated using computer assisted sperm analysis. Sperm stained with both propidium iodide and Sybr-14was analyzedbyflowcytometry todiscriminate between membrane damaged and intact cells, respectively. Fractions of normal double stranded and denatured singlestranded DNA were determined after acid treatment and acridine orange staining, as a measure for sperm chromatin stability and integrity. Two-layer iodixanol density centrifugation resulted in a 5% increase in the percentage of plasma membrane intact sperm, as compared to diluted sperm or sperm processed using ordinary centrifugation. In addition, chromatin stability was found to be increased for sperm obtained after iodixanol density centrifugation. Centrifugation processing at the day of collection or after storage resulted in similar percentagesof plasmamembrane intact sperm after one day storage at 4oC, whereas progressive motility of sperm processed after storage was slightlydecreased. Iodixanol densitycentrifugation resulted in increased cryosurvival rates as compared to ordinary centrifugation. Highest survival rates after freezing and thawingwere obtainedwhen centrifugationprocessing and cryopreservationwere performed at the day of collection. In case of delayed cryopreservation, survival rates could not be increased by centrifugation processing. Post-freeze plasma membrane integrity tended to be higher when sperm was selected via centrifugation processing directly after semen collection as compared to sperm processed just prior to cryopreservation, both for ordinary as well as for iodixanol density centrifugation. Taken together, survival after delayed cryopreservation can be increased when two-layer iodixanol density centrifugation is applied directly after semen collection.