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. 2015 Dec 1;5:17643. doi: 10.1038/srep17643

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Copyright © 2015, Macmillan Publishers Limited

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(a) Effects of lactic acid on sperm motility in vitro. Ejaculated sperm were suspended in media containing various concentrations of either L-lactic acid or D-lactic acid, and sperm motility was evaluated on a scale of 0 to 5. Values shown are means ± SEM of three independent experiments. *P < 0.05 vs. 0 mM. **P < 0.01 vs. 0 mM. (b) Changes in the pH_e or pH_i of sperm in response to the addition of L-lactic acid. pH_e of the medium was directly measured using a pH meter. Sperm pH_i was determined using pHrodo^TM. The results shown are representative of results from five independent experiments. (c) ATP levels in sperm incubated with L-lactic acid. ATP concentrations of sperm are expressed as mean values ± SEM of three independent experiments. (d,e) Photographs of pHrodo^TM-loaded sperm swimming in Hank’s balanced salt solution (d) and resident sperm in SSTs (e). Dashed line indicates the outline of SSTs. Scale bar = 50 μm. (f) Representative standard curve of sperm pH_i equilibrated with the indicated pH buffer containing valinomycin and nigericin. (g) pH_i values of sperm swimming in Hank’s balanced salt solution (Extender) and resident sperm in SSTs (SSTs). Values shown are means ± SEM of three independent experiments. Asterisk denotes a significant difference (P < 0.00001). (h) Effects of pH on ATPase activity of sperm. De-membraned sperm were incubated with 1 mM ATP, and the free phosphoric acid in the incubation mixture was measured using a microplate reader. (i) Effects of pH on axonemal sliding of sperm. Demembraned sperm were mounted on chamber slides, treated with trypsin, and an assay buffer that adjusted the pH to either 7.4, 6.4, 5.7, or 5.4 was perfused for equilibration. The ATP solution was perfused and axonemal sliding was recorded. Percentages of axonemal sliding were calculated and expressed as mean values ± SEM of three independent experiments. **denotes a significant difference from pH 7.4 (P < 0.0000001).