Brazilian Agricultural Research Corporation, EMBRAPA/EMPARN, Natal-RN, Brazil.
Anim Reprod Sci. 2010 Oct;122(1-2):118-23. doi: 10.1016/j.anireprosci.2010.08.004. Epub 2010 Aug 11.
The objective of this study was to evaluate the effect of addition of the antioxidants Trolox and catalase to a ram semen cryopreservation extender on lipid peroxidation and hydrogen peroxide generation on the extender and in the thawed semen. Semen was collected from 23 Santa Inês rams (one ejaculate per ram) and diluted at 32°C to a concentration of 400×10⁶ cells/ml in one of the following solution: Tris-egg yolk extender (control), or the same extender supplemented with either 50μM Trolox/10⁸ sperm (Trolox), 50μgcatalase/ml (Catalase) or a combination of Trolox and catalase (Tro+cat, 50μM Trolox/10⁸ sperm and 50μg catalase/ml). The semen was loaded into 0.25ml straws, cooled and frozen in a programmable freezer and subsequently stored in liquid nitrogen. Prior to evaluation, frozen straws were thawed in a water bath (42°C for 20s). Lipid peroxidation (LPO), both spontaneous and catalyzed, on the semen and the extender were measured using the thiobarbituric acid (TBA) assay in accordance with the method described by Buege and Aust (1978). Hydrogen peroxide (H₂O₂) generation was measured using the horseradish peroxidase-dependent oxidation of phenol red to a derivative with absorbance at 610nm, according to the method described by Pick and Keisari (1980). Spontaneous LPO resulted in the least production of thiobarbituric acid-reactive substances (TBARS) in the Tro+cat (1.37±0.02nMol/10⁸ sperm), compared to amounts in the other treatments groups. In the catalyzed LPO experiments, the least (P<0.05) amounts of TBARS were observed in Trolox (2.52±0.02nMol/10⁸ sperm) and Tro+cat (2.54±0.02nMol/10⁸ sperm) groups, compared to the control (3.81±0.02nMol/10⁸ sperm) and catalase (3.83±0.02nMol/10⁸ sperm) groups. Hydrogen peroxide generation was less (P<0.05) in the Trolox (6.00±0.18nMol/40×10(6)sperm/±40min) and Tro+cat (6.08±0.18nMol/40×10⁶sperm/±40min) groups than in the control (6.97±0.18nMol/40×10⁶ sperm/±40min) and catalase (6.53±0.18nMol/40×10⁶ sperm/±40min) groups. Compared to the control group, Trolox and catalase treatment significantly reduced TBARS in catalyzed LPO and hydrogen peroxide concentrations in the samples (P<0.05). ROS (reactive oxygen species) generation occurred in all extenders, without sperm cells. The data presented provide evidence that ROS are produced in ram semen, both in the extender and during the freezing and thawing process. In addition, the data suggest that the antioxidants Trolox and catalase may be used to control the oxidative stress imposed on ram spermatozoa by the cryopreservation process.
本研究的目的是评估在绵羊精液冷冻保存稀释液中添加抗氧化剂 Trolox 和过氧化氢酶对脂质过氧化作用和过氧化氢生成的影响，即在稀释液中和解冻精液中。从 23 只 Santa Inês 公羊（每只公羊采集一次精液）中采集精液，并在 32°C 下以 400×10⁶ 个细胞/ml 的浓度稀释到以下溶液中的一种：Tris-卵黄稀释液（对照），或相同的稀释液中添加 50μM Trolox/10⁸ 精子（Trolox）、50μg 过氧化氢酶/ml（Catalase）或 Trolox 和过氧化氢酶的组合（Tro+cat，50μM Trolox/10⁸ 精子和 50μg 过氧化氢酶/ml）。将精液装入 0.25ml 细管中，冷却并在可编程冷冻机中冷冻，然后在液氮中储存。在评估之前，将冷冻的细管在水浴（42°C 20s）中解冻。根据 Buege 和 Aust（1978）的方法，使用硫代巴比妥酸（TBA）测定法测量精液和稀释液中的自发和催化的脂质过氧化作用（LPO）。根据 Pick 和 Keisari（1980）的方法，使用辣根过氧化物酶依赖性氧化酚红生成在 610nm 处具有吸光度的衍生物来测量过氧化氢（H₂O₂）的生成。自发的 LPO 导致 Tro+cat（1.37±0.02nMol/10⁸ 精子）中产生的硫代巴比妥酸反应性物质（TBARS）最少，与其他处理组的量相比。在催化的 LPO 实验中，与对照（3.81±0.02nMol/10⁸ 精子）和过氧化氢酶（3.83±0.02nMol/10⁸ 精子）组相比，Trolox（2.52±0.02nMol/10⁸ 精子）和 Tro+cat（2.54±0.02nMol/10⁸ 精子）组中观察到的 TBARS 最少（P<0.05）。与对照（6.97±0.18nMol/40×10⁶ 精子/±40min）和过氧化氢酶（6.53±0.18nMol/40×10⁶ 精子/±40min）组相比，Trolox（6.00±0.18nMol/40×10⁶ 精子/±40min）和 Tro+cat（6.08±0.18nMol/40×10⁶ 精子/±40min）组中的过氧化氢生成较少（P<0.05）。与对照组相比，Trolox 和过氧化氢酶处理显著降低了催化的 LPO 中的 TBARS 和样本中过氧化氢的浓度（P<0.05）。ROS（活性氧）在所有的稀释液中产生，而没有精子细胞。提供的证据表明，ROS 在绵羊精液中产生，无论是在稀释液中还是在冷冻和解冻过程中。此外，数据表明，抗氧化剂 Trolox 和过氧化氢酶可用于控制冷冻保存过程对绵羊精子产生的氧化应激。
