Effect of pyruvate on lactate-induced spontaneous acrosome reaction in stallion sperm

Stallion sperm metabolism is thought to be largely dependent on oxidative phosphorylation.

Our recent study suggested that under “capacitating-like conditions” (presence of calcium, bovine serum albumin (BSA), and bicarbonate) only samples with pyruvate (as an energy substrate) had higher motility of stallion sperm. Presence of lactate alone was associated with substantial increases in spontaneous acrosome reaction rate in viable sperm, to almost half of all viable sperm at 4 hour incubation. However, motility was reduced in lactate treatment at 4 hours. We determined whether addition of pyruvate to a lactate-only-containing medium may increase stallion sperm motility while maintaining acrosome reaction rate in viable sperm. Fresh stallion ejaculates (n = 9) were washed and diluted to 30 x 106  sperm/ml using a Modified Whitten’s medium (MW) containing 7 mg/ml BSA and 10 mM lactate (Control), or 10 mM lactate with 0.5, 1, 5, or 10 mM added pyruvate. To control the effect of added substrate treatments, additional lactate was added at similar concentrations. Diluted sperm were incubated under a 5% CO 2 atmosphere for 4 hours. After incubation, samples were analyzed for total and progressive motility, %TMOT, and PMOT by CASA. Samples were stained with a fixable live/dead stain, followed by FITC-PSA and assessed for viability and acrosome status by flow cytometry. Data were rank-transformed prior to analysis by General Linear Model. The % TMOT was higher (p < 0.05) in 5 and 10 mM pyruvate (47 - 50%) than in lactate-only media (30 - 37%). Similarly, % PMOT was higher (p < 0.05) in 5 and 10 mM pyruvate (16%) than in lactate-only media (10 - 11 %,). The % viable (55 - 57%) was not affected (p > 0.05) by any treatment tested. Addition of pyruvate was associated with a dose-dependent decrease in the proportion of viable sperm that were acrosome-reacted (AR; 42, 36, 31, 18, and 11% for 0, 0.5, 1, 5, or 10 mM added pyruvate), as compared to lactate (42, 40, 42, 39, and 40%, respectively). Results suggested that presence of pyruvate increased motility and decreased proportion of viable sperm undergoing spontaneous AR. Increases in motility and decreases in AR occurred at the same pyruvate concentration (5 mM added pyruvate). Increasing lactate concentration in the medium was not associated with an increase in AR. Findings may help to formulate media for stallion sperm capacitation, and highlighted the delicate energy balance required to support key functions of stallion sperm.

Keywords: Stallion sperm, acrosome reaction, energy substrate, pyruvate, lactate

This manuscript was originally published in the journal Clinical Theriogenology Vol 12(3) Sept 2020.  Clinical Theriogenology is the official journal of the Society for Theriogenology (SFT) and the American College of Theriogenologists (ACT).  This content has been reproduced on the IVIS website with the explicit permission of the SFT/ACT.