SummaryRam spermatozoa are very sensitive to any cold shock or oxidative damage, therefore making them unsuitable for prolonged storage or distant transport to specialized laboratories for flow-cytometric analysis. The aim of this study was to stain ram semen samples with several fluorescent markers and analyse their stability during formaldehyde fixation. Briefly, freshly collected semen samples were stained for apoptosis (annexin V-FITC, YO-PRO™-1 and FLICA), acrosomal damage (PNA-AF488 and FITC-conjugated antibody against GAPDHS), mitochondrial activity (Mitotracker probes), oxidative damage [dihydroethidium (DHE) and CellROX™ Green] and cell viability (live/dead fixable viability dyes). Next, samples were fixed in buffer containing formaldehyde and then washed. Stained sample were analyzed using flow cytometer before fixation, immediately after fixation, and at 5 h and 20 h post-fixation. Fluorescent signals and the proportion of positively stained spermatozoa were compared statistically in fresh and post-fixed samples. All examined markers, except YO-PRO-1 (decreased significantly, P < 0.05), retained their fluorescence intensities after fixation. In conclusion, several tested markers were able to withstand formaldehyde fixation of ram semen samples as follows: annexin V and FLICA for apoptosis; PNA for acrosomal status; MitoTracker Red CMXRos for mitochondrial activity; and CellROX Green for oxidative status in combination with a suitable live/dead fixable viability dye. This optimized methodology could help to comprehensively analyse the quality of ram semen from local farms countrywide.

中文翻译：

---

固定后数小时可通过流式细胞术评估公羊精液质量

摘要 Ram 精子对任何冷休克或氧化损伤都非常敏感，因此不适合长时间储存​​或远距离运输到专业实验室进行流式细胞术分析。本研究的目的是用几种荧光标记对公羊精液样本进行染色，并分析它们在甲醛固定过程中的稳定性。简而言之，对新鲜收集的精液样本进行细胞凋亡（膜联蛋白 V-FITC、YO-PRO™-1 和 FLICA）、顶体损伤（PNA-AF488 和 FITC 结合的 GAPDHS 抗体）、线粒体活性（Mitotracker 探针）、氧化损伤染色[二氢乙锭 (DHE) 和 CellROX™ Green] 和细胞活力（活/死可固定活力染料）。接下来，将样品固定在含有甲醛的缓冲液中，然后洗涤。在固定前使用流式细胞仪分析染色样品，固定后立即，固定后 5 小时和 20 小时。在新鲜样品和固定后样品中对荧光信号和阳性染色精子的比例进行了统计比较。所有检查的标记，除了 YO-PRO-1（显着减少，磷< 0.05)，固定后保留其荧光强度。总之，几种测试标记物能够承受公羊精液样品的甲醛固定，如下所示：膜联蛋白 V 和 FLICA 用于细胞凋亡；顶体状态的 PNA；MitoTracker Red CMXRos 用于线粒体活性；和 CellROX Green 用于氧化状态，与合适的活/死可固定活力染料结合使用。这种优化的方法有助于全面分析全国当地农场的公羊精液质量。