L anserine nitrate

Hanks balanced salt solution (HBSS) containing 136 mM NaCl, 5.4 mM KCl, 0.8 mM MgSO₄, 1.26 mM CaCl₂, 4.2 mM NaHCO₃ and 5.6 mM glucose was used as sperm diluent. HBSS was incubated at 37°C for 10 min before semen collection. Semen was obtained during mating from mature male quails prior to ejaculation, in accordance with the procedure by Kuroki and Mori (1997) and suspended in 500 µl HBSS. Sperm cells were counted microscopically using a hemocytometer (BX 51, Olympus Optics, Tokyo, Japan) and prepared to reach a final concentration of 1×10⁸ sperm/ml. 100 µl of HBSS containing varying concentrations of anserine (L-Anserine Nitrate, Fujifilm Wako Pure Chemical Co., Osaka, Japan) or carnosine (L-Carnosine, Wako Pure Chemical Corporation) were added to the sperm suspension to achieve a final concentration of 2×10⁷ sperm/ml. Suspensions were incubated at 15°C upto 12 h. The optimum concentration of anserine and carnosine was determined from our preliminary experiments in which the ejaculated spermatozoa were incubated with either 1 µM, 3 µM, 10 µM, 30 µM or 100 µM of the dipepetides. We found that more than 3 µM of anserine and carnosine had no further potentiation effect on sperm motility (data not shown).