Cooling of porcine semen in an extender supplemented with isoespintanol

ABSTRACT: Spermatozoa experience oxidative, osmotic, chemical, and thermal stresses when cooled, which degrade the quality and fertilizing capacity of the cells. Adding antioxidants to the sperm extender mitigates these alterations. This study evaluated the effect of isoespintanol (ISO) on boar semen subjected to cooling. Fifteen ejaculates from five boars (Susscrofadomestica) were extended in Beltsville thawing solution (BTS) supplemented with 0 µM (control), 5 µM (ISO5), 10 µM (ISO10), 15 µM (ISO15), 20 µM (ISO20), 25 µM (ISO25), and 30 µM (ISO30) of ISO, which were then cooled for five days at 16 °C. Sperm kinetics, total motility (TM), and progressive motility (PM) were evaluated every 24 h using an IVOS computer-assisted sperm analysis (CASA) system. On day 1 and day 5 of cooling, a hypoosmotic test, spectrofluorometry, and flow cytometry were performed to evaluate the following: membrane functionality, measured as a function of hypoosmotic swelling (HOS); total antioxidant capacity (TAC); reactive oxygen species (ROS); and mitochondrial membrane potential (Δ¥M). Regression analysis and comparison of means using the Duncan test were performed. The ISO added had a slight impact on sperm motility, as evidenced by a reduction in TM at 24 h of cooling (but not prior) with the addition of 20 µM of ISO. Similarly, no effect of the ISO on the kinetics and functional integrity of the sperm membrane was observed at 96 h of cooling; however, the regression coefficients indicated that the ISO lowered the rate of decrease in sperm motility and the proportion of rapid spermatozoa relative to the concentration of ISO used. The ISO did not affect the TAC of the cooled semen; however, different concentrations of ISO lowered ROS production in the semen after 96 h of cooling. ISO also impacted the Δ¥M of the spermatozoa at 0 h of cooling, increasing the proportion of low Δ¥M cells and decreasing the proportion of high Δ¥M cells. In conclusion, ISO can reduce the loss of quality and oxidative stress occurring in boar semen during cooling and can modulate the mitochondrial activity of sperm.

摘要：精子在冷藏过程中会遭受氧化应激、渗透压应激、化学应激与热应激，导致细胞质量与受精能力下降。向精液稀释液中添加抗氧化剂可缓解此类损伤。本研究探讨了异松蒎酚(isoespintanol, ISO)对公猪冷藏精液的影响。选取5头家猪（Sus scrofa domestica）种公猪的15份精液样本，以添加0µM ISO（对照组）、5µM ISO(ISO5)、10µM ISO(ISO10)、15µM ISO(ISO15)、20µM ISO(ISO20)、25µM ISO(ISO25)及30µM ISO(ISO30)的贝尔茨维尔解冻液(Beltsville thawing solution, BTS)进行稀释，随后于16℃条件下冷藏5天。采用IVOS计算机辅助精子分析(computer-assisted sperm analysis, CASA)系统，每24小时检测精子动力学参数、总活力(total motility, TM)与前向运动活力(progressive motility, PM)。在冷藏第1天及第5天，通过低渗肿胀试验(hypoosmotic swelling test, HOS)、荧光分光光度法及流式细胞术，评估以下指标：以低渗肿胀率表征的精子膜功能、总抗氧化能力(total antioxidant capacity, TAC)、活性氧(reactive oxygen species, ROS)水平及线粒体膜电位(mitochondrial membrane potential, ΔΨM，原文作Δ¥M)。采用回归分析与邓肯多重比较检验进行统计分析。添加ISO对精子活力存在轻微影响：20µM ISO组在冷藏24小时时总活力出现下降（此前无此变化）。同样，冷藏96小时时未观察到ISO对精子动力学参数及精子膜功能完整性存在影响；但回归分析结果显示，随着ISO使用浓度升高，精子活力下降速率及快速运动精子占比的降低速率均有所减缓。ISO未对冷藏精液的总抗氧化能力产生影响，但不同浓度的ISO均可在冷藏96小时后降低精液内活性氧的生成量。冷藏0小时时，ISO同样会影响精子的线粒体膜电位：升高低线粒体膜电位细胞的占比，降低高线粒体膜电位细胞的占比。综上，异松蒎酚可降低公猪精液冷藏过程中的质量损失与氧化应激水平，并可调节精子的线粒体活性。