Laboratory of Reproductive Physiopathology, Center for Translational Medicine (CEMT-BIOREN), Chile; Escuela de Medicina Veterinaria, Facultad de Recursos Naturales y Medicina Veterinaria, Universidad Santo Tomas, Chile.
Laboratory of Reproductive Physiopathology, Center for Translational Medicine (CEMT-BIOREN), Chile; Department of Preclinical Sciences, Faculty of Medicine, Universidad de La Frontera, Temuco, Chile.
Res Vet Sci. 2021 May;136:143-150. doi: 10.1016/j.rvsc.2021.02.006. Epub 2021 Feb 16.
The addition of antioxidants to the cryopreservation medium has been shown to exert a positive effect on the quality of frozen-thawed sperm in different species. The objective of the present study was to evaluate the effects of supplementing the freezing medium with butylhydroxytoluene (BHT) and melatonin (MEL) in frozen-thawed pig spermatozoa. With this purpose, six ejaculates coming from six separate boars were cryopreserved in traditional freezing medium (i.e. lactose/egg-yolk/glycerol; Control) supplemented with 1.0 mM BHT (BHT-1), 2.0 mM BHT (BHT-2), 0.01 μM MEL (MEL-1) and 1.0 μM MEL (MEL-2). We evaluated sperm viability, membrane lipid disorder, acrosome integrity, mitochondrial membrane potential, lipid peroxidation, oxidation of thiol groups, and levels of total reactive oxygen species (ROS), peroxynitrite and superoxide anion (·O). We also analysed total (TM) and progressive sperm motilities (PM), and kinetic parameters at post-thaw (T0, T30 and T60). The BHT-2 and MEL-2 groups presented higher viability and acrosome integrity, and lower levels of peroxynitrite, ·O and lipid peroxidation than the control (P < 0.05), whereas MEL-2 diminished the levels of total ROS (P < 0.05). TM and PM were not affected by the treatment, while, LIN and STR shows differences between experimental groups. In conclusion, the addition of BHT and MEL to cryopreservation medium diminishes oxidative and nitrosative stress markers, which has repercussions for the integrity of plasma and acrosomal membranes of frozen-thawed spermatozoa.
抗氧化剂添加到冷冻保护剂中已被证明对不同物种的冷冻-解冻精子质量有积极影响。本研究的目的是评估在冷冻猪精子中添加丁羟甲苯（BHT）和褪黑素（MEL）对冷冻-解冻精子的影响。为此，从 6 头公猪的 6 个不同精液中，用传统冷冻液（即乳糖/蛋黄/甘油；对照）冷冻保存，其中添加 1.0mM BHT（BHT-1）、2.0mM BHT（BHT-2）、0.01μM MEL（MEL-1）和 1.0μM MEL（MEL-2）。我们评估了精子活力、膜脂紊乱、顶体完整性、线粒体膜电位、脂质过氧化、硫基氧化和总活性氧（ROS）、过氧亚硝酸盐和超氧阴离子（·O）的水平。我们还分析了总（TM）和渐进精子运动（PM），以及解冻后（T0、T30 和 T60）的动力学参数。与对照组相比，BHT-2 和 MEL-2 组的活力和顶体完整性更高，而过氧亚硝酸盐、·O 和脂质过氧化水平更低（P<0.05），而 MEL-2 降低了总 ROS 水平（P<0.05）。处理组对 TM 和 PM 没有影响，而 LIN 和 STR 显示出实验分组之间的差异。总之，在冷冻保护剂中添加 BHT 和 MEL 可降低氧化应激和硝化应激标志物的水平，这对冷冻-解冻精子的质膜和顶体膜的完整性有影响。
