Intracellular pH of bovine sperm increases during capacitation

The effect of heparin‐induced capacitation on the intracellular pH (pH i ) of individual bovine sperm was determined with image analysis. Sperm were loaded with the acetoxymethyl ester of the pH sensitive fluorescent indicator, 2′,7′‐bis(carboxyethyl)‐5(6)‐carboxy‐fluorescein (BCECF). The pH i of 5303 sperm was evaluated from a total of five bulls at .5, 2, 3, 4, and 5 h of incubation. The pH i did not differ between the sperm head and mid‐piece ( P > 0.05). An increase in sperm head pH i was seen in heparin‐treated sperm at 3, 4, and 5 h of incubation relative to sperm incubated without heparin (control, P < 0.05). At 5 h of incubation, the pH i in heparin‐treated sperm was 6.92 ± 0.07, while control‐treated sperm pH i was 6.70 ± 0.03. Initially a normal frequency distribution was seen for sperm pH i in both heparin‐ and control‐treated sperm. As the incubation progressed, the frequency distribution began to skew towards higher pH i in both samples but was more dispersed for the heparin‐treated sperm. Following an NH 4 Cl‐induced alkaline load, the pH i of both control‐ and heparin‐treated sperm recovered toward the resting pH i with a half‐time of recovery of 1.5–1.7 min. The recovery of sperm pH i was not due to leakage of NH 4+ into sperm because recovery also occurred with trimethylamine. The instantaneous velocity of the pH i recovery (v i ) was dependent on pH i and decreased as pH i decreased. Capacitation by heparin was associated with an 81% decrease in v i at a pH i of 7.00, but there was no effect of capacitation on the proton buffering power of the sperm, which was 87 ± 8 mM/pH unit. Results demonstrate that both the regulation of pH i and resting pH i were altered during capacitation of bovine sperm by heparin. © 1995 Wiley‐Liss, Inc.