Effects of media and promoters on different lipid peroxidation assays in stallion sperm

Abstract

Effects of different media and promoters on lipid peroxidation (LPO) in viable stallion sperm have not been reported. Aims of this study were to determine effects of three media (INRA-96™, Equipro CoolGuard™, and Biggers, Whitten and Whittingham [BWW]), and promoters (iron sulfate-Fe; ultraviolet light-UV; or control-no exposure to promoters) on viable sperm LPO using four different flow cytometric assays (i.e., BODIPY, Liperfluo, 4-hydroxylnonenal [4HNE], malonaldehyde [MDA]). Significant media x promoter interactions were detected using the Liperfluo, 4HNE, and MDA assays (P < 0.05); therefore, data were sorted by media and by promoters. With inclusion of milk-based media, there were similar concentrations of LPO in control samples with use of all LPO assays. The effect of iron, as a promoter of LPO production, was media dependent, and milk-based media protected sperm from iron-induced LPO production when there were assessments with all assays. In contrast, iron promoted LPO in sperm diluted in BWW when there was use of in all assays, except BODIPY, probably because of the different target molecule with use of this assay. Ultraviolet light was the most potent LPO promoter with all media and assays evaluated. Data indicate milk-based extenders are generally more LPO-protective than BWW early in the LPO production pathway (based on BODIPY and Liperfluo assays), but are less protective during the later stages of LPO production (based on 4HNE and MDA assays). The use of different media and promoters of LPO allowed for determination of early and late stages of LPO in viable stallion sperm.

Keywords: Lipid peroxidation; Media; Promoters; Reactive oxygen species; Stallion sperm.