Dose- and Time-Dependent In Vitro Effects of Divalent and Trivalent Iron on the Activity of Bovine Spermatozoa.

This in vitro study was designed to assess the impact of divalent (Fe(2+)) or trivalent (Fe(3+)) iron on the activity and oxidative balance of bovine spermatozoa at specific time intervals (0, 2, 8, 16, and 24 h) during an in vitro culture. Forty-five semen samples were collected from adult breeding bulls and diluted in physiological saline solution supplemented with different concentrations (0, 1, 5, 10, 50, 100, 200, 500, 1000 μmol/L) of FeCl2 or FeCl3. Spermatozoa motion parameters were assessed using the SpermVision™ computer-aided sperm analysis (CASA) system. Cell viability was examined with the metabolic activity 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, and the nitroblue-tetrazolium (NBT) test was applied to quantify the intracellular superoxide formation. Both divalent and trivalent iron exhibited a dose- and time-dependent impact on the spermatozoa physiology and oxidative balance. Concentrations ≥50 μmol/L FeCl2 and ≥100 μmol/L FeCl3 led to a significant decrease of spermatozoa motility (P < 0.05) and mitochondrial activity (P < 0.001 with respect to 200-1000 μmol/L FeCl2/FeCl3; P < 0.01 in case of 100 μmol/L FeCl2/FeCl3), accompanied by a significant superoxide overproduction (P < 0.001 in terms of 200-1000 μmol/L FeCl2 and 500-1000 μmol/L FeCl3; P < 0.01 with respect to 100 μmol/L FeCl2 and 100-200 μmol/L FeCl3). On the other hand, concentrations below 10 μmol/L FeCl2 and 50 μmol/L FeCl3 proved to stimulate the spermatozoa activity, as shown by a significant preservation of the motility and viability characteristics (P < 0.001 in case of the motility parameters; P < 0.01 with respect to the spermatozoa viability), alongside a significant decline of the superoxide generation (P < 0.05). In a direct comparison, divalent iron has been shown to be more toxic than trivalent iron. Results from this in vitro study show that high concentrations of both forms of iron are toxic, while their low concentrations may have spermatozoa activity-promoting properties. In vitro concentrations of divalent or trivalent iron that could be regarded as critical are 50 μmol/L FeCl2 and 100 μmol/L FeCl3 when iron ceases to be an essential micronutrient in order to become a toxic risk factor.