Quantitative and Qualitative Characteristics of Frozen-Thawed Bovine Spermatozoa Recovered Via a Conventional and a Standardized Swim-Up Technique

Abstract

The objective of this study was to use the bovine as a model to evaluate the recovery of frozen-thawed spermatozoa via a conventional and a standardized swim-up technique. Frozen-thawed semen specimens (3 bulls) were washed and reconstituted with 2.9% (w/v) sodium citrate extender containing 20% (v/v) chicken egg yolk (SC-EY). Reconstituted sperm specimens were used for selection via conventional swim-up and the standardized ZSC™ method. The swim-up method consisted of ovarlayering the sperm specimen with 0.7 to 1.0 ml of isolation media (Ham's F-b), followed by 1 hr of incubation. The ZSC™ consisted of a conical cavity on the bottom of a glass column. The sperm specimen was placed into the conical cavity until the surface of the specimen was at the same level as the upper boundaries of the conical cavity. The surrounding periconical and epiconical areas were filled with 0.7 to 1.0 ml of isolation media, followed by 1 hr of incubation. The isolation media was removed (harvesting) from swim-up (80% volume) and ZSC™ specimens (100% volume) at the end of incubation. Recovered specimens were assessed for volume (ml), sperm concentration (×10⁶ spermatozoa/ml), the percentage and grade of motility (0 to 4), the occurrence of osmotic shock and the percentage of spermatozoa reactive to the hypoosmotic swelling (HOS) test. Swim-up and ZSC™ selected specimens were qualitatively similar to each other. However, higher numbers of spermatozoa were recovered when sperm specimens were processed via the ZSC™ method (1.6 fold increase) than with the conventional swim-up technique. Because the ZSC™ method enabled the recovery of up to 100% of the overlayered media, it also enabled the recovery of most of the spermatozoa that migrated from the sperm specimen into the isolation media with no possibility of mixing the two, which was the case with the swim-up method, and which could also contaminate the recovered specimen with dead and immotile spermatozoa. Thus, the ZSC™ technique enabled the harvesting of the medium closest to the underlayered sperm specimen, which contributed to maximize the number of sperm recovered. When all assessed parameters were noted and all clinical improvements and efficiency of the method were compared to the swim-up technique, the sperm manipulation procedure of choice was clearly the ZSC™ method.