■精液冷冻保存是用于牛人工授精和体外受精的精液生产中最流行的做法。精浆含有细胞外囊泡(spEV),可在卵母细胞繁殖过程中调节精子活力和功能。冻融精液剂量中的spEV研究可能会产生预测公牛生育能力的新指标,但是精液补充剂的存在可能会阻碍spEV的分子谱分析。这项研究的目的是提供在冷冻保存过程之前和之后从精浆中分离出的EV的广泛表征,并添加了商业动物无蛋白精液补充剂,以了解源自补充剂的EV在阻碍使用中的潜在影响SpEV衍生的生物标志物用于评估公牛生育力。
■从精浆中分离出EV(有或没有延伸剂),从没有精子的冷冻保存的稻草中,并使用两种不同的方法从扩展器,超速离心(UC)和尺寸排阻色谱(SEC),并以其结构和组成为特征。
■电动汽车的物理表征表明,大小和颗粒数与分离方法有关。spEV较大但较少(UC:168.9nm,n=2.68×109;SEC:197.0nm,n=6.42×109)与扩展器电动汽车(UC:129.0nm,n=2.68×1011;SEC:161.8nm,n=6.47×1011)。Western印迹分析(WB)证实spEVS中存在典型的EV标志物:膜结合CD9(25kDa)和腔内标志物Alix(96kDa)和TSG101(48KDa)。尽管透射电子显微镜证实了所有制剂中都存在脂质双层结构,当使用单分子阵列(SiMoa)时,在从延伸剂分离的囊泡中没有检测到特异性EV标记。在至少一种制剂中鉴定了总共724个Bos金牛miRNA。在来自延伸体的EV中鉴定的miRNA的百分比(总读段的0.05%-0.49%)低于含有spEV的制备物(总读段的10.56%-63.69%)。Edge-R通过两种方法鉴定了从延伸剂分离的EV之间的总共111个DE-miRNA。其中,11DE-miRNAs(bta-miR-11980,bta-miR-11987,bta-miR-12057,bta-miR-1246,bta-miR-125b,bta-miR-181b,bta-miR-2340,bta-miR-2358,bta-miR-2478,bta-miR-2898和bta-miR-345-3p)在从具有延伸剂的精浆制剂中分离的EV中也很丰富。
■这项研究清楚地表明,延伸剂的存在并不能阻止冷冻保存的精液中spEV的表征。然而,spEV的分子谱分析可能受到所用的分离方法和来自延伸剂的一些miRNA的存在的影响。因此,在这样的研究中,建议同时表征spEV和从延伸剂分离的囊泡。
UNASSIGNED: Semen cryopreservation is the most popular practice for semen production for artificial insemination and in vitro fertilization in cattle. The Seminal plasma contains extracellular vesicles (spEVs) which modulate sperm viability and function during oocyte fecundation. The study of spEVs in frozen-thawed semen doses may yield novel indicators for predicting bull fertility, but the presence of the semen
extender may hinder molecular profiling of spEVs. The aim of this study was to provide extensive characterization of EVs isolated from seminal plasma before and after the cryopreservation process and the addition of a commercial animal protein-free semen extender to understand the potential influence of EVs originating from the
extender in hindering the use of spEVs derived biomarkers for assessment of bull fertility.
UNASSIGNED: EVs were isolated from the seminal plasma (with or without the
extender), from the cryopreserved straw devoid of spermatozoa, and from the extender using two different methods, ultracentrifugation (UC) and size exclusion chromatography (SEC), and characterized for their structure and composition.
UNASSIGNED: Physical characterization of EVs showed that size and particle numbers were related to the method of isolation. spEVs were larger but less abundant (UC: 168.9 nm, n = 2.68 × 109; SEC: 197.0 nm, n = 6.42 × 109) compared to extender EVs (UC: 129.0 nm, n = 2.68 × 1011; SEC: 161.8 nm, n = 6.47 × 1011). Western blotting analysis (WB) confirmed the presence of typical EV markers in spEVS: the membrane bound CD9 (25 kDa) and the luminal markers Alix (96 kDa) and TSG101 (48 KDa). Although Transmission Electron Microscopy confirmed the presence of a lipid bilayer structure in all preparations, no specific EV markers were detected in the vesicles isolated from extender when the Single Molecule Array (SiMoa) was used. A total of 724 Bos taurus miRNAs were identified in at least one preparation. The percentage of miRNAs identified in EVs from the
extender (0.05%-0.49% of the total reads) was lower than in the preparation containing spEVs (10.56%-63.69% of the total reads). Edge-R identified a total of 111 DE-miRNAs between EVs isolated from the
extender by two methods. Among them, 11 DE-miRNAs (bta-miR-11980, bta-miR-11987, bta-miR-12057, bta-miR-1246, bta-miR-125b, bta-miR-181b, bta-miR-2340, bta-miR-2358, bta-miR-2478, bta-miR-2898, and bta-miR-345-3p) were also abundant in EVs isolated from seminal plasma preparations with extender.
UNASSIGNED: This study clearly demonstrates that the presence of the extender does not prevent the characterization of spEVs in cryopreserved semen. However, the molecular profiling of spEVs can be influenced by the isolation method used and by the presence of some miRNAs from the extender. Therefore, in such studies, it is advisable to characterize both spEVs and the vesicles isolated from the extender.