Abstract：
Since the stallion acrosome is very small compared to other species and cannot be properly assessed without additional staining, several labelling techniques were developed to facilitate its assessment. The aim of this study was to compare the Spermac stain (Minitüb GmbH) and a PNA/PSA/PI triple-staining detected by flow cytometry with regard to method agreement for detecting non-intact acrosomes within two different extenders. For this purpose, eighteen stallion ejaculates were split in half and diluted with the semen extenders EquiPlus or Gent (Minitüb GmbH) to a final concentration of 50 × 106 sperm/mL, respectively. Subsequently, 126 semen samples were stained with both methods between 4 and 240 h (mean: 63.8 ± 48.9 h) after semen collection. Calculated Intraclass correlation coefficients revealed excellent correlations between both methods for EquiPlus (r = .77, p < .001) and fair correlations for Gent (r = .49, p < .001). Interestingly, flow cytometry detected more non-intact acrosomes in EquiPlus than in Gent (p < .001), whereas the Spermac stain showed no differences (p = .902) between extenders. The poorer method agreement in Gent could be caused by egg yolk artefacts, which made interpretation difficult, so flow cytometry might be preferred. The differences in detected non-intact acrosomes between extenders highlighted the importance of establishing adapted laboratory protocols for different extender types in order to generate comparable results.
摘要：
由于种马顶体与其他物种相比非常小，并且在没有额外染色的情况下无法正确评估，开发了几种标签技术来促进其评估。这项研究的目的是比较Spermac染色（MinitübGmbH）和通过流式细胞术检测到的PNA/PSA/PI三重染色，以确定在两种不同的延伸剂中检测不完整的顶体的方法协议。为此,将18种马射精分成两半，并用精液补充剂EquiPlus或Gent（MinitübGmbH）稀释至终浓度为50×106精子/mL，分别。随后，在收集精液后4至240小时（平均：63.8±48.9小时）之间，用两种方法对126份精液样品进行了染色。计算的类内相关系数揭示了两种方法之间的良好相关性（r=.77，p<.001）和Gent的公平相关性（r=.49，p<.001）。有趣的是,流式细胞术在EquiPlus中检测到比在Gent中更多的非完整顶体（p<.001），而Spermac染色显示延伸剂之间没有差异(p=.902)。Gent中较差的方法一致性可能是由蛋黄人工制品引起的，这使得解释变得困难，所以流式细胞术可能是首选。延伸剂之间检测到的非完整顶体的差异强调了为不同延伸剂类型建立适应的实验室方案以产生可比结果的重要性。
