Na pyruvate

All the nutrient media used for mouse sperm were prepared the day before the experiments and the balance of gases in the droplet of sperm was controlled overnight under suitable conditions (5% CO₂ and 37 °C). As a culture medium, human tubal fluid (HTF) was prepared, which included NaCl (102 mM; Wako), KCl (4.7 mM; Wako., Japan), MgSO₄·7H₂O (0.2 mM; Wako), KH₂PO₄ (0.4 mM; Wako), CaCl₂·2H₂O (2.0 mM; Wako), NaHCO₃ (25 mM; Wako), glucose (2.8 mM; Wako), Na lactate (23.2 mM; Wako), Na pyruvate (0.3 mM; Wako), 0.3% BSA (Sigma., Kanagawa, Japan), penicillin G (100 U/ml; Meiji., Tokyo, Japan) and streptomycin (100 µg/ml; Meiji). Male ICR mice (9−10 weeks old; Charles River Japan Inc., Yokohama-shi, Japan) were sacrificed by cervical dislocation. Spermatozoa collected from the cauda epididymides of ICR male mice were incubated for 1.5 h at 5% CO₂ and 37 °C. In this case, 1 × 10⁵ or 1 × 10⁶ cells/mL of spermatozoa were seeded in an HTF droplet of 200 μL before being cultured again in fresh HTF or Ca²⁺-free HTF (CaCl₂·2H₂O was removed from the prepared HTF) at 5% CO₂ and 37 °C. In general, the number of spermatozoa that can be collected from a male mouse is known to be about 1 × 10⁶ /mL or 5 × 10⁶ /mL at most. We obtained Institutional Review Board approval for this study. This study was overseen by an Animal Care and Use Committee at The University of Tokyo.