Ghorbani Marzieh, Amiri Iraj, Khodadadi Iraj, Fattahi Amir, Atabakhsh Mojgan, Tavilani Heidar
Student Research Committee .
Syst Biol Reprod Med. 2015 Jan;61(1):57-61. doi: 10.3109/19396368.2014.968267. Epub 2014 Oct 6.
The aim of this study was to determine the effect of butylated hydroxytoluene (BHT) supplemented cryopreservation medium on sperm parameters during the freeze-thaw process. In addition, sperm lipid peroxidation, DNA damage, and the amount of reactive oxygen species (ROS) were determined. Semen samples were obtained from 75 donors. Fifteen semen samples were used for optimizing BHT concentration and incubation time and 60 samples were used for the final experiments. After the determination of basic parameters, groups of three sample with similar parameters were pooled and processed by Pure Sperm gradient centrifugation. The semen samples were then diluted with normal freezing medium (control) or a medium containing 0.5 mM BHT (test) for 5 minute and stored in liquid nitrogen. Frozen cryovials were thawed individually for 20 seconds in a water bath (37°C) for evaluation. Freezing extenders supplemented with 0.5 mM BHT led to higher sperm motility and viability compared with control samples (p < 0.001). Furthermore, the addition of BHT decreased malondialdehyde (MDA) formation, DNA fragmentation, and ROS content compared with controls (p < 0.001). Our results showed that the addition of BHT to the freezing medium could be of advantage in reducing ROS and preventing the detrimental effect of ROS on the human sperm function.
本研究的目的是确定在冻融过程中，添加丁基羟基甲苯（BHT）的冷冻保存培养基对精子参数的影响。此外，还测定了精子脂质过氧化、DNA损伤及活性氧（ROS）含量。从75名捐献者处获取精液样本。15份精液样本用于优化BHT浓度和孵育时间，60份样本用于最终实验。在测定基本参数后，将参数相似的三份样本分为一组，通过Pure Sperm梯度离心法进行处理。然后将精液样本用正常冷冻培养基（对照组）或含0.5 mM BHT的培养基（试验组）稀释5分钟，并储存在液氮中。冷冻的冻存管在37°C水浴中单独解冻20秒以进行评估。与对照样本相比，添加0.5 mM BHT的冷冻稀释液可使精子活力和存活率更高（p < 0.001）。此外，与对照组相比，添加BHT可降低丙二醛（MDA）生成、DNA片段化及ROS含量（p < 0.001）。我们的结果表明，在冷冻培养基中添加BHT可能有助于降低ROS，并防止ROS对人类精子功能产生有害影响。
