Motility and fertilizing ability of rat epididymal spermatozoa washed by a continuous gradient of Percoll

Removal of epididymal fluids from epididymal sperm suspension is an important step for the study of sperm motility, capacitation, and the acrosome reaction. The technique of washing should minimize damage to viable spermatozoa but at the same time efficiently remove debris, non-sperm cells, and biological fluids. We examined sperm motility and fertilizability in vitro of rat epididymal spermatozoa after washing with Percoll continuous gradient. Nine milliliters (ml) of 50% N-2-hydroxyethylpiperazine-N1-2-ethanesulfonic acid (HEPES) buffered Percoll solution was centrifuged at 20,000 g for 45 minutes to form a continuous gradient. One hundred to 300 microliters of sperm suspension was loaded onto the surface of the gradient and centrifuged at 150 g and 1,500 g for 10 minutes. Two main layers of spermatozoa were formed, one of high (lower layer) and one of low (upper layer) motility. At centrifugation 1,500 g, the sperm density and motility in the lower layer were greater than at 150 g. Spermatozoa from both layers at 150 g and at 1,500 g were diluted with modified Krebs-Ringer's bicarbonate solution (mKRB) and preincubated for 5 hours. Superovulated eggs collected from 21-25-day-old Wistar strain immature rats were introduced into the preincubated sperm suspension for insemination and fixed 5-5.5 hours later for observation of fertilization. Spermatozoa from both layers, 150 g and 1,500 g, showed the same fertilizability in vitro as control spermatozoa. From these results we conclude that Percoll gradients can be used for washing rat epididymal sperm for the study of sperm physiology including fertilization.