Egyptien Sophie, Dewals Benjamin, Ectors Fabien, Brutinel Flore, Ponthier Jérôme, Deleuze Stéfan
Fundamental and Applied Research for Animals and Health Research Unit (FARAH), Comparative Veterinary Medicine, Faculty of Veterinary Medicine, University of Liège, Avenue de Cureghem 1, 4000 Liège, Belgium.
FARAH, Veterinary Public Health, Faculty of Veterinary Medicine, University of Liège, Avenue de Cureghem 1, 4000 Liège, Belgium.
Animals (Basel). 2023 Jun 4;13(11):1874. doi: 10.3390/ani13111874.
Many fluorochromes routinely used in semen quality analysis emit in the green and red channels, limiting their possible combination for multiple parameter analysis. The use of fluorophores emitting in different light channels broadens the possibilities of combination to expand the range of simultaneously evaluated criteria. This is of great interest in cases of small ejaculated volumes, such as those naturally occurring in roosters, small dog breeds and drones (). The purpose of this experiment is to establish Calcein Violet (CaV), a blue fluorochrome, as a marker of viability and acrosomal integrity in domestic animals in order to free the red and green channels. SYBR14/Propidium Iodide (PI) was used as reference dye, heat-treated samples as negative controls, serial staining combination for validation and epifluorescence microscopy for observation. Dead spermatozoa marked in red with PI showed no blue fluorescence either from the head or the tail. Live spermatozoa showed a decreasing blue emission from head to tail when single stained with CaV. Unreacted acrosomes showed intense blue fluorescence irrespective of plasma membrane integrity. This needs to be further confirmed for species with small and difficult to observe heads. Establishment of CaV as a marker of membrane integrity by fluorescence microscopy is a decisive first step towards further technical development and use with flow cytometry.
许多精液质量分析中常用的荧光染料在绿色和红色通道中发出荧光，这限制了它们用于多参数分析的可能组合。使用在不同光通道中发出荧光的荧光团拓宽了组合的可能性，从而扩大了同时评估标准的范围。这对于射精量小的情况非常有意义，比如公鸡、小型犬种和雄蜂自然出现的情况。本实验的目的是确立蓝色荧光染料钙黄绿素（CaV）作为家畜活力和顶体完整性的标志物，以便释放红色和绿色通道。使用SYBR14/碘化丙啶（PI）作为参考染料，热处理样本作为阴性对照，采用系列染色组合进行验证，并使用落射荧光显微镜进行观察。用PI染成红色的死精子头部和尾部均未显示蓝色荧光。用CaV单染时，活精子从头部到尾部的蓝色荧光逐渐减弱。无论质膜完整性如何，未反应的顶体均显示强烈的蓝色荧光。对于头部小且难以观察的物种，这一点需要进一步证实。通过荧光显微镜将CaV确立为膜完整性的标志物，是朝着进一步技术发展以及与流式细胞术联用迈出的决定性的第一步。
