Changes in the Antioxidant Capacity and Iron-Binding Properties of Bovine Spermatozoa Following In Vitro Incubation with Ferrous or Ferric Iron

The aim of this study was to assess the impact of ferrous (Fe2+) or ferric (Fe3+) iron on the antioxidant capacity and the ability to bind iron of bovine spermatozoa at specific time intervals (0h, 2h, 8h, 16h and 24h) during an in vitro culture. 35 semen samples were collected from 7 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 motility was assessed using the SpermVisionTM CASA (Computer aided sperm analysis) system. The ferric reducing ability of plasma (FRAP) assay was applied to study the antioxidant capacity of the samples, while the ability of the sample to bind excess iron was determined using the Total iron-binding capacity (TIBC) test. Both ferrous and ferric iron exhibited a dose- and time-dependent impact on the spermatozoa motility. Concentrations ≥50 µmol/L FeCl2 and ≥100 µmol/L FeCl3 led to a significant decrease of spermatozoa motion (P<0.001), while concentrations below 10 µmol/L FeCl2 and 50 µmol/L FeCl3 proved to preserve the parameter (P<0.001). The FRAP assay revealed that both ferrous as well as ferric iron had a similar effect on the FRAP marker of the samples: high concentrations led to a dramatic and significant (P<0.001) increase of the parameter, followed by a notable decrease of the reducing ability in the subsequent time periods, whose intensity was dependent upon the time, oxidation state of iron, as well as the time of analysis. Furthermore, supplementation of FeCl2 and FeCl3 had an impact on the capacity of the sperm culture to bind free iron, reflected in a significant decrease of the parameter (P<0.001) early on (Time 2h) in case of high doses of both oxidative states of this biometal. In a direct comparison, ferrous iron has been shown to be more toxic than ferric 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. 50 µmol/L FeCl2 and 100 µmol/L FeCl3 could be regarded as critical in vitro concentrations of ferrous or ferric iron when it critically accumulates with toxic outcomes.