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Abstract The effects of extracellular ions on the acquisition and maintenance of the potential for motility in the spermatozoa of the ayu, Plecoglossus altivelis Temminck et Schlegel (Osmeridae), were examined. Testicular spermatozoa and milt spermatozoa were obtained from fully matured males and diluted with buffered solution (BS, 20 mM HEPES–NaOH, pH 7.5). Testicular spermatozoa showed a significantly low rate of motility (0.8 ± 0.4%), whereas milt spermatozoa showed a high rate (89.4 ± 2.1%). The spermatozoa were incubated with various isotonic media for 2 h, diluted with BS, and changes in the rates of motility were then compared. When incubated for 2 h with artificial seminal plasma (ASP), corresponding in terms of ionic constituents to seminal plasma buffered at pH 8.0, both spermatozoa showed a high rate of motility. Testicular spermatozoa acquired and milt spermatozoa maintained the potential for motility in response to the HCO3– ion concentrations (between 0 and 20 mM) in the ASP. The differences in the pH of the ASP had a significant effect on the acquisition and maintenance of the potential for motility, and spermatozoa showed the highest rate of motility with the ASP at pH 8.0 and 8.5. These results suggest that the quality of milt in the ayu can be regulated by controlling the concentration of bicarbonate and the pH of the incubating media. [ABSTRACT FROM AUTHOR]