Techniques for improving the accuracy and efficiency of the sperm penetration assay

Six different techniques were evaluated for improving the accuracy and efficiency of the sperm penetration assay (SPA). Flushing and puncture of the oviduct were found to result in similar recovery of eggs from the oviduct, percentage of penetrated eggs, and penetrations per egg. However, the time required to flush the oviduct tended to be longer than for the puncture technique. Exposure of eggs to a 5% formalin solution prior to mounting resulted in fewer ruptured eggs, greater recovery of eggs, and a reduced time to mount and read the eggs compared to eggs not exposed to formalin. The percentage of penetrated eggs and penetrations per egg were similar for formalin exposed and nonexposed eggs. Significantly fewer penetrated eggs and penetrations per egg were noted when eggs were read as fresh mounts compared to eggs that were fixed and stained prior to evaluation. A significantly longer time was also required to read the freshly mounted eggs compared with the stained eggs. The results of these studies indicate that to maximize the accuracy and efficiency of the SPA, cumulus masses should be removed from the oviducts by puncture, eggs should be placed in a 5% formalin solution before mounting on slides, and eggs should be fixed and stained prior to evaluating for penetrations.