Assessment of Myeloperoxidase activity in raw equine fresh semen

In previous studies [1], myeloperoxidase (MPO), a pro-oxidant enzyme, has been shown to be partially inactivated in commercial equine semen extenders. The aim of this study was to determine MPO concentration and activity in fresh equine semen and in supernatant of semen diluted with a cristalloïd solution after cushionned centrifugation.
Three stallions have been collected 5 times at 2 day-intervals. Semen concentration was assessed with Nucleo Counter sp100. A sample containing 100x106spz was then immediatly frozen (-80°C) before assays. A volume of semen containing 500x106spz was diluted 3 times with PBS (1v/3v). Samples were centrifuged (1000xg, 20 minutes). One milliliter of supernatant was sampled and stored at -80°C before assays. Total MPO concentration was assayed by commercial enzyme-linked immuno-sorbent assay and active MPO concentration was assayed by specific immunological extraction followed by enzymatic detection as previously described in our earlier paper [1]. As all values were not normally distributed, Kruskall-Wallis test was used to analyse differences between medians and Spearman test was used to determine correlations.
Median total and active MPO concentrations in raw semen were respectively 580500ng/mL and 1.098ng/ mL. Median total and active MPO concentration in semen supernatant extended with PBS (1v/3v) were respectively 107500ng/mL and 0.236ng/mL. In raw semen, total and active MPO concentrations were highly correlated (r=0.7096; p=0.0030). However, in semen supernatant extended with PBS (1v/3v), they were not (r=0.2121; p=0.4479).
Total MPO concentrations were similar to those observed in our previous papers [1]. When concentration observed in supernatant is compared to that observed in total raw semen (cells and supernatant), we observe that MPO in raw semen is higher than in supernatant, even after correction for dilution. This confirms that cellular part of semen contains MPO and releases it during cold-chock. Without any large proteins in the extender medium, MPO activity can be assessed in raw semen and in its supernatant. Active MPO concentrations are very low when compared to total MPO concentration, (nearly one million times). Concentration of active MPO in supernatant corrected for dilution was also lower than concentration observed in raw complete semen, also confirming active MPO release by cellular part of semen. Association of total and active MPO concentration is supporting this observation. This study confirms our previous assessments [1] that active and total MPO are released by non sperm-cells during cold shock.