van Munster E B, Stap J, Hoebe R A, te Meerman G J, Aten J A
Holland Genetics, Arnhem, The Netherlands.
Theriogenology. 1999 Dec;52(8):1281-93. doi: 10.1016/s0093-691x(99)00217-4.
Volume-based sorting of X- and Y-chromosome-bearing sperm cells could be an interesting alternative to the existing technique based on DNA content. Advantages would be that DNA staining and ultraviolet excitation, used in the existing technique, could be avoided. To assess the possibilities and limitations of sperm-head volume as sorting criterion, achievable purity and yield are determined for bull sperm. Two important parameters in this respect are the magnitude of the volume difference and the biological variation within each (X or Y) population. Earlier, we established a difference in volume matching the difference in DNA content (3.8%) between X- and Y-bearing bull sperm heads by comparing thicknesses and areas of high numbers of pre-sorted X- and Y-bearing bull sperm heads by interference microscopy and subsequent image analysis. Unfortunately, despite the high number of measurements, a direct determination of biological variations was not possible due to an unknown contribution of instrumental variations. In this paper, we determine the contribution of instrumental errors by measuring a single sperm head, varying parameters such as location in the image, orientation angle, focusing etc., simulating the behavior of the measuring system. After correction, both for the instrumental variation, and for the fact that the original samples were not pure, biological variations in volume of 5.9 +/- 0.8% were found. Our results indicate that when 10% of the bull sperm are sorted on basis of their head volume, a theoretical enrichment of 80% could be achieved. Expected purity and yield are lower than what is standard for the existing technique. At the moment, a technique to physically separate X- and Y-bearing sperm cells based on volume is not available. However, for applications for which the potential hazards of DNA staining and UV excitation are problematic, the development of such technique should be considered.
基于体积对携带X和Y染色体的精子细胞进行分选，可能是现有基于DNA含量技术的一个有趣替代方案。其优点是可以避免现有技术中使用的DNA染色和紫外线激发。为了评估精子头部体积作为分选标准的可能性和局限性，我们测定了公牛精子可实现的纯度和产量。在这方面的两个重要参数是体积差异的大小以及每个（X或Y）群体内的生物学变异。此前，我们通过干涉显微镜和后续图像分析，比较了大量预分选的携带X和Y染色体的公牛精子头部的厚度和面积，确定了携带X和Y染色体的公牛精子头部之间的体积差异与DNA含量差异（3.8%）相匹配。不幸的是，尽管进行了大量测量，但由于仪器变异的未知贡献，无法直接测定生物学变异。在本文中，我们通过测量单个精子头部，改变图像中的位置、取向角、聚焦等参数，模拟测量系统的行为，来确定仪器误差的贡献。经过校正，既校正了仪器变异，也考虑到原始样品不纯的事实，发现体积的生物学变异为5.9±0.8%。我们的结果表明，当根据头部体积对10%的公牛精子进行分选时，理论上可以实现80%的富集。预期的纯度和产量低于现有技术的标准。目前，还没有一种基于体积物理分离携带X和Y染色体精子细胞的技术。然而，对于DNA染色和紫外线激发的潜在危害存在问题的应用，应考虑开发这种技术。
