Tvrdá Eva, Lukáč Norbert, Lukáčová Jana, Jambor Tomáš, Massányi Peter
Department of Animal Physiology, Faculty of Biotechnology and Food Sciences, Slovak University of Agriculture, Tr. A. Hlinku 2, 949 76, Nitra, Slovakia,
Biol Trace Elem Res. 2015 Sep;167(1):36-47. doi: 10.1007/s12011-015-0288-5. Epub 2015 Mar 12.
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.
本体外研究旨在评估二价铁（Fe(2+)）或三价铁（Fe(3+)）在体外培养特定时间间隔（0、2、8、16和24小时）对牛精子活力和氧化平衡的影响。从成年种公牛采集45份精液样本，并用补充了不同浓度（0、1、5、10、50、100、200、500、1000 μmol/L）FeCl2或FeCl3的生理盐水溶液进行稀释。使用SpermVision™计算机辅助精子分析（CASA）系统评估精子运动参数。通过代谢活性3-(4,5-二甲基噻唑-2-基)-2,5-二苯基四氮唑溴盐（MTT）试验检测细胞活力，并应用硝基蓝四氮唑（NBT）试验定量细胞内超氧化物的形成。二价铁和三价铁均对精子生理和氧化平衡表现出剂量和时间依赖性影响。FeCl2浓度≥50 μmol/L和FeCl3浓度≥100 μmol/L导致精子活力显著下降（P < 0.05）和线粒体活性显著下降（相对于200 - 1000 μmol/L FeCl2/FeCl3，P < 0.001；对于100 μmol/L FeCl2/FeCl3，P < 0.01），同时伴有超氧化物大量产生（就200 - 1000 μmol/L FeCl2和500 - 1000 μmol/L FeCl3而言，P < 0.001；相对于100 μmol/L FeCl2和100 - 200 μmol/L FeCl3，P < 0.01）。另一方面，FeCl2浓度低于10 μmol/L和FeCl3浓度低于50 μmol/L被证明可刺激精子活性，表现为活力和生存力特征显著保留（活力参数方面，P < 0.001；相对于精子生存力，P < 0.01），同时超氧化物生成显著下降（P < 0.05）。直接比较表明，二价铁比三价铁毒性更大。本体外研究结果表明两种形式的高浓度铁具有毒性，而低浓度可能具有促进精子活性的特性。当铁不再是必需的微量营养素而成为有毒风险因素时，体外可视为临界的二价或三价铁浓度分别为50 μmol/L FeCl2和100 μmol/L FeCl3。
