Jäkel Helen, Henning Heiko, Luther Anne-Marie, Rohn Karl, Waberski Dagmar
Unit for Reproductive Medicine of the Clinics/Clinic for Pigs and Small Ruminants, University of Veterinary Medicine Hannover, Hannover, Germany.
Faculty of Veterinary Medicine, Department of Clinical Sciences, Utrecht University, Utrecht, The Netherlands.
Cytometry A. 2021 Oct;99(10):1033-1041. doi: 10.1002/cyto.a.24301. Epub 2021 Jan 26.
Hypothermic storage of boar semen may allow antibiotic-free semen preservation but is limited due to chilling sensitivity of boar spermatozoa. Progress in this area requires sensitive tools to detect chilling injury. Therefore, multiparameter flow cytometry panels were evaluated to ascertain whether they are useful tools for identifying sublethal damage of sperm function at a single cell level, thus considering the high intrinsic sperm heterogeneity in a sample. The first fluorochrome panel consisted of Hoechst 33342 to identify DNA-containing events, Yo-Pro 1 to detect viability, merocyanine 540 to describe membrane fluidity, and PNA-Alexa Fluor™ 647 to identify acrosomic integrity. The second fluorochrome panel consisted of SiR700-DNA to identify DNA-containing events, JC-1 to characterize the mitochondrial transmembrane potential (MMP), and Calbryte 630 to assess the intracellular calcium level. Extended boar semen was stored either at 17°C (control) or 5°C (chilled). It is shown that chilling increased membrane fluidity in the viable (Yo-Pro 1 negative) sperm population at 24 h (p < 0.05). At 144 h, the viable, acrosomic intact sperm population with low membrane fluidity was similar for both storage temperatures. Moreover, chilling reduced the main sperm population with high MMP, medium fluorescence for JC-1 monomer and low intracellular calcium level (p < 0.05). However, after in vitro sperm capacitation, this population did not differ between the two storage temperatures. Exemplary computational data visualization in t-distributed stochastic neighbor embedding (t-SNE) maps and moving radar plots revealed similar subpopulations as identified by three-dimensional stacked bar charts. In conclusion, sperm surviving an initial chilling injury withstand long-term storage and respond in a similar manner to capacitation conditions as sperm stored conventionally at 17°C. Multicolor flow cytometry is a valuable tool for detecting chilling-induced alterations of cell function in sperm subpopulations.
公猪精液的低温保存可能允许无抗生素的精液保存，但由于公猪精子对冷刺激敏感而受到限制。该领域的进展需要灵敏的工具来检测冷损伤。因此，对多参数流式细胞术面板进行了评估，以确定它们是否是在单细胞水平上识别精子功能亚致死损伤的有用工具，从而考虑到样本中高度固有的精子异质性。第一个荧光染料面板由用于识别含DNA事件的Hoechst 33342、用于检测活力的Yo-Pro 1、用于描述膜流动性的部花青540和用于识别顶体完整性的PNA-Alexa Fluor™ 647组成。第二个荧光染料面板由用于识别含DNA事件的SiR700-DNA、用于表征线粒体跨膜电位（MMP）的JC-1和用于评估细胞内钙水平的Calbryte 630组成。稀释后的公猪精液分别储存在17°C（对照）或5°C（冷藏）。结果表明，在24小时时，冷刺激增加了存活（Yo-Pro 1阴性）精子群体的膜流动性（p < 0.05）。在144小时时，两种储存温度下具有低膜流动性的存活、顶体完整精子群体相似。此外，冷刺激减少了具有高MMP、JC-1单体中等荧光和低细胞内钙水平的主要精子群体（p < 0.05）。然而，在体外精子获能后，这两个储存温度下的该群体没有差异。t分布随机邻域嵌入（t-SNE）图和移动雷达图中的示例性计算数据可视化显示了与三维堆叠柱状图所识别的相似亚群。总之，在初始冷损伤中存活的精子能够经受长期储存，并且对获能条件的反应与传统上在17°C储存的精子相似。多色流式细胞术是检测精子亚群中冷诱导的细胞功能改变的有价值工具。
