Acetic acid

A thiobarbituric acid-reactive substances (TBARS) assay was employed to assess the production of malondialdehyde (MDA), considered to be a primary byproduct of lipid peroxidation (LPO). The sperm lysates (100 μL) were pre-treated with 100 μL 5% SDS (sodium dodecyl sulfate; Sigma-Aldrich, St. Louis, MO, USA) and 4 mL 0.53% thiobarbituric acid (Sigma-Aldrich, St. Louis, MO, USA), previously dissolved in 20% acetic acid (pH 3.5; Centralchem, Slovakia). Subsequently, the samples were exposed to heat (100 °C) in a water bath for 60 min. To cease the ongoing reaction, the samples were cooled down for 10 min and subsequently centrifuged (1300× g, 10 min, 4 °C). The supernatants were pipetted into a 96-well plate, and the absorbances were measured with a Glomax plate spectrophotometer at 540 nm. MDA levels were calculated with the help of a standardization curve constructed from pre-prepared MDA standards. The extent of LPO is expressed in µmol/L MDA/g protein [22 (link)].

An aliquot of each sample was centrifuged (800× g, 10 min) and the sperm pellet was sonicated (28 kHz, 30 s) in the presence of the RIPA buffer (Sigma-Aldrich, St. Louis, MO, USA) and protease inhibitor (Sigma-Aldrich, St. Louis, MO, USA). Following the second round of centrifugation (11,828× g, 4 °C, 10 min) and purification, the lysates were subjected to the quantification of malondialdehyde (MDA), considered to be the principal marker of lipid peroxidation (LPO).
The sperm lysates were pre-treated with 5% sodium dodecyl sulphate (Sigma-Aldrich, St. Louis, MO, USA) and subsequently exposed to 0.53% thiobarbituric acid (TBA; Sigma-Aldrich, St. Louis, MO, USA) dissolved in 20% acetic acid (pH 3.5; Centralchem, Bratislava, Slovakia) under high-temperature conditions (90–100 °C) for 1 h. Afterwards, the samples were cooled down for 10 min and centrifuged (1750× g, 10 min). The supernatants (150 μL) were transferred into a transparent 96-well plate and the levels of MDA were assessed at 540 nm using the Glomax microplate spectrophotometer (Promega, Madison, WI, USA). MDA levels are expressed as μmol/L [71 (link)].

Acetic acid (concentrated 96%), Acetone p.a., Aluminum chloride, Ethyl acetate p.a., Folin–Ciocalteu reagent (phosphomolybdic-phosphotungstic acid), Hide powder CRS, Hydrochloric acid (concentrated 36%), Methanol p.a., Methenamine (hexamethylenetetraamine), Sodium carbonate and Sodium sulphate (anhydrous) were purchased from Centralchem, Slovakia. Distilled decarbonized water was used for all experiments.
Standards cyanidin 3-O-glucoside chloride, hyperoside and pyrogallol were purchased from Sigma – Aldrich, MO, USA.