Steinholt H C, Chandler J E, Tirado V
Department of Dairy Science, Louisiana State University Agricultural Center, Baton Rouge 70803.
J Dairy Sci. 1991 Nov;74(11):3822-6. doi: 10.3168/jds.s0022-0302(91)78574-3.
Acrosomal integrity of bovine spermatozoa was evaluated using naphthol yellow S-erythrosine B stain with bright-field microscopy, .2% glutaraldehyde fixation with Nomarski optics, and live wet smears with Nomarski optics to compare the evaluation techniques. Four ejaculates per two Holstein bulls were enriched through a Percoll gradient, capacitated with heparin, and acrosome-reacted with lysophosphatidylcholine to prepare spermatozoa for in vitro fertilization. Spermatozoa samples were taken after each preparatory step, and percentage of intact acrosomes was evaluated with the stain, .2% glutaraldehyde, and live wet smear. Acrosomal integrity decreased with each preparatory step across all techniques. The decrease in integrity from heparin to lysophosphatidylcholine indicated that all methods detected the acrosome reaction. Technique means across preparation steps showed no differences. Correlations between microscopy techniques were high. The live wet smear used no fixation methods. Both the naphthol yellow S-erythrosine B stain and the .2% glutaraldehyde techniques employed fixation steps that may have introduced artifacts that could influence the data. The live wet smear evaluated by Nomarski optics allowed a comparable evaluation of the acrosome-reacted bovine spermatozoa. Comparing brightfield with differential interference contrast microscopy for detecting the acrosome reaction in bovine spermatozoa showed that both methods were equally accurate.
使用萘酚黄S-赤藓红B染色结合明场显微镜、0.2%戊二醛固定结合诺马斯基光学显微镜以及活的湿涂片结合诺马斯基光学显微镜来评估牛精子的顶体完整性，以比较评估技术。从两头荷斯坦公牛身上采集的每份四份射精样本通过Percoll梯度进行富集，用肝素诱导获能，并用溶血磷脂酰胆碱诱导顶体反应，以制备用于体外受精的精子。在每个制备步骤后采集精子样本，并用染色法、0.2%戊二醛法和活的湿涂片法评估完整顶体的百分比。在所有技术中，随着每个制备步骤的进行，顶体完整性均下降。从肝素处理到溶血磷脂酰胆碱处理，完整性的下降表明所有方法都能检测到顶体反应。各制备步骤间的技术平均值无差异。显微镜技术之间的相关性很高。活的湿涂片法未使用固定方法。萘酚黄S-赤藓红B染色法和0.2%戊二醛技术都采用了固定步骤，这可能会引入影响数据的假象。通过诺马斯基光学显微镜评估的活的湿涂片法能够对发生顶体反应的牛精子进行可比的评估。比较明场显微镜和微分干涉对比显微镜检测牛精子顶体反应的结果表明，两种方法同样准确。
