Fenton S E, Ax R L, Cowan C M, Coyle T, Gilbert G R, Lenz R W
Department of Dairy Science, University of Wisconsin, Madison 53706.
J Dairy Sci. 1990 Nov;73(11):3118-25. doi: 10.3168/jds.S0022-0302(90)79000-5.
Spectrophotometers are used for estimating sperm concentrations from raw ejaculates in semen processing laboratories. Unfortunately, these instruments have a limited detection spectrum and do not permit accurate quantification of sperm numbers in highly diluted or concentrated samples. The objectives of this study were to validate a DNA assay for quantification of sperm numbers in extended or undiluted semen samples and to determine precision of the assay. The principle of the assay is based upon a fluorescent dye that binds to adenine-thymine base pairs in double-stranded DNA. Semen samples and calf thymus DNA standards were sonicated in 2 M NaCl buffer with 1 mM EDTA. The DNA content of samples was compared to standards of calf thymus DNA using fluorometry. Sensitivity of the assay was determined to be 1.4 x 10(5) sperm cells. Concentrations of sperm estimated from DNA assay values did not differ from flow cytometric cell counts. Assays were performed in three different laboratories, using different equipment, to assess the assay's repeatability. Estimates of sperm concentrations determined by the DNA assay were similar, regardless of location and source of equipment used to perform the assays. This assay fulfills statistical criteria for being sensitive, accurate, and repeatable, and it can be employed in laboratories processing semen for artificial insemination as a tool for spectrophotometer calibration, a check for straw filling accuracy, or to quantify sperm numbers in extended, packaged semen.
在精液处理实验室中，分光光度计用于估算原始射精精液中的精子浓度。不幸的是，这些仪器的检测光谱有限，无法准确量化高度稀释或浓缩样本中的精子数量。本研究的目的是验证一种DNA检测方法，用于量化稀释或未稀释精液样本中的精子数量，并确定该检测方法的精密度。该检测方法的原理基于一种荧光染料，它能与双链DNA中的腺嘌呤 - 胸腺嘧啶碱基对结合。精液样本和小牛胸腺DNA标准品在含有1 mM EDTA的2 M NaCl缓冲液中进行超声处理。使用荧光测定法将样本的DNA含量与小牛胸腺DNA标准品进行比较。该检测方法的灵敏度确定为1.4×10⁵个精子细胞。通过DNA检测值估算的精子浓度与流式细胞术细胞计数结果无差异。在三个不同实验室使用不同设备进行检测，以评估该检测方法的重复性。无论进行检测的设备的位置和来源如何，通过DNA检测确定的精子浓度估计值都相似。该检测方法符合灵敏、准确和可重复的统计标准，可用于人工授精精液处理实验室，作为分光光度计校准工具、检查细管填充准确性或量化稀释、包装精液中的精子数量。
