Arroyo-Salvo Camila, Sanhueza Francisco, Fuentes Fernanda, Treulén Favián, Arias María Elena, Cabrera Paulina, Silva Mauricio, Felmer Ricardo
Laboratory of Reproduction, Centre of Reproductive Biotechnology (CEBIOR-BIOREN), Universidad de La Frontera, Temuco, Chile.
School of Medical Technology, Faculty of Sciences, Universidad Mayor, Temuco, Chile.
Reprod Domest Anim. 2019 Feb;54(2):184-194. doi: 10.1111/rda.13328. Epub 2018 Sep 29.
Conventional in vitro fertilization has not yet been implemented in the equine species. One of the main reasons has been the inability to develop a culture medium and incubation conditions supporting high levels of stallion sperm capacitation and hyperactivation in vitro. Although different culture media have been used for this purpose, human tubal fluid (HTF) medium, widely used in the manipulation of human and mice gametes, has not been reported so far in stallion sperm culture. The first part of this study aimed to compare HTF and Whitten's media on different stallion sperm quality and capacitation variables. Additionally, the effect of procaine, aminopyridine and caffeine in both media was evaluated on sperm motility parameters at different incubation times. Integrity and destabilization of the plasma membrane were evaluated by merocyanine 540/SYTOX Green (MC540), mitochondrial membrane potential (∆Ψm) using tetramethylrhodamine methyl ester perchlorate (TMRM), acrosome membrane integrity by PNA/FITC and tyrosine phosphorylation by P-tyrosine mouse mAb conjugated to Alexa Fluor® by flow cytometry. Motility parameters were evaluated using the integrated semen analysis system (ISAS®). We found no differences between Whitten's and HTF media and incubation time in terms of sperm viability, uninduced acrosome membrane damage or mitochondrial membrane potential at 30- and 120-min incubation. Membrane fluidity (MC540) increased in both media at 30- and 120-min incubation compared to noncapacitating conditions. Similarly, tyrosine phosphorylation increased in both media in capacitating conditions at 2- and 4-hr incubation compared to noncapacitating conditions. Although procaine showed the best result in terms of sperm hyperactivated motility in both media, aminopyridine also showed parameters consistent with the hyperactivation including an increase in curvilinear velocity and decrease in straightness. In conclusion, HTF medium and aminopyridine equally support capacitation-related parameters in stallion sperm.
常规体外受精尚未在马属动物中实施。主要原因之一是无法开发出一种培养基和培养条件，以支持体外高水平的种马精子获能和超活化。尽管已为此目的使用了不同的培养基，但广泛用于人类和小鼠配子操作的人类输卵管液（HTF）培养基，迄今尚未见在种马精子培养中的报道。本研究的第一部分旨在比较HTF和惠顿培养基对不同种马精子质量和获能变量的影响。此外，还评估了两种培养基中普鲁卡因、氨基吡啶和咖啡因在不同孵育时间对精子活力参数的影响。通过部花青540/ SYTOX Green（MC540）评估质膜的完整性和去稳定化，使用四甲基罗丹明甲酯高氯酸盐（TMRM）评估线粒体膜电位（∆Ψm），通过PNA/FITC评估顶体膜完整性，并通过流式细胞术使用与Alexa Fluor®偶联的抗酪氨酸小鼠单克隆抗体评估酪氨酸磷酸化。使用综合精液分析系统（ISAS®）评估活力参数。我们发现在孵育30分钟和120分钟时，惠顿培养基和HTF培养基以及孵育时间在精子活力、未诱导的顶体膜损伤或线粒体膜电位方面没有差异。与非获能条件相比，在孵育30分钟和120分钟时，两种培养基中的膜流动性（MC540）均增加。同样，与非获能条件相比，在获能条件下孵育2小时和4小时时，两种培养基中的酪氨酸磷酸化均增加。尽管在两种培养基中，就精子超活化活力而言，普鲁卡因显示出最佳结果，但氨基吡啶也显示出与超活化一致的参数，包括曲线速度增加和直线性降低。总之，HTF培养基和氨基吡啶同样支持种马精子中与获能相关的参数。
