Diluent for cool storage of the Japanese eel (Anguilla japonica) spermatozoa

The appropriate ionic composition for the Japanese eel (Anguilla japonica) sperm diluent for cool storage was examined. Eel spermatozoa were immotile in isotonic NaCI, KCl, and mannitol solutions, and the motility was initiated when osmolality was increased. Spermatozoa were diluted 50 times with various isotonic solutions and stored for 3 h at 5 degrees C. Subsequently, this mixture was diluted 21 times with 450 mM NaCl to examine the percentage motility. Spermatozoa diluted with artificial seminal plasma, consisting of 149.3 mM NaCl+15.2 mM KCI+1.3 mM CaCl2+1.6 mM MgCl2 buffered with 5 mM NaHCO3-NaOH at pH 8.2, or with isotonic NaCl+KCl solution, showed high percentage motility after storage. However, spermatozoa diluted with isotonic NaCI, mannitol, NaCl+CaCl2, or NaCl+MgCl2 solutions showed significantly lower motility compared to the spermatozoa stored in the former two solutions. In subsequent experiments, spermatozoa were diluted with isotonic solutions of various potassium concentrations (0, 5, 15, 25, 35, 45, 75, and 164.5 mM) at 50 times and stored at 3 degrees C for up to 28 days to examine the optimal composition of diluent. Spermatozoa that were diluted with the 5-35 mM KCl solutions showed a decrease of percentage motility at 24 h, but they showed a marked recovery of motility by the seventh day. Increasing the potassium concentration in the isotonic diluent reduced the rate of decline in motility observed at 24 h of storage. Spermatozoa diluted with the 45, 75 or 164.5 mM KCI isotonic solutions did not show the temporal decrease of motility at 24 h. However, the percentage motility indices (percentage motility/initial percentage motility x 100) of spermatozoa in 75 or 164.5 mM KCI solutions at day 21 (26.2% and 0.6%, respectively) were significantly lower than those of 15-45 mM KCl solutions (47.9-64.8%). These results indicate that the potassium ion is an essential constituent of the isotonic diluent for cool storage of the Japanese eel spermatozoa. The optimal KCl concentration for short term storage (up to 7 days) is 45-75 mM,and that for longer storage is 15-45 mM.