Department of Biochemistry, Louisiana State University, Baton Rouge, Louisiana, USA.
Zebrafish. 2010 Jun;7(2):233-40. doi: 10.1089/zeb.2010.0655.
The goal of this study was to establish an efficient method for determination of sperm concentration requiring only 1-2 microL of sample by use of microspectrophotometry. The objectives were (1) determination of wavelengths with absorbance profiles appropriate for analysis of sperm suspensions from zebrafish Danio rerio, green swordtail Xiphophorus helleri, and medaka Oryzias latipes collected by crushing of dissected testis or by stripping of live males; (2) generation of standard curves and equations between sperm sample absorbance and sperm concentration estimated by hemocytometer counts; (3) accuracy verification of equations for estimating concentration by microspectrophotometry; and (4) analysis of the precision in generating equations and estimation of sperm concentration. Within the visible wavelengths (380-750 nm) there was no single maximal absorbance peak. For zebrafish, a linear correlation was established with an effective absorbance range of 0.034-0.936 for crushed samples, and 0.028-0.961 for stripped samples at 400 nm. For Xiphophorus, the effective absorbance range was 0.014-1.154 for crushed samples, and the effective range was 0.038-1.082 for stripped samples. For medaka, the effective range was 0.041-0.896 for crushed samples. The accuracy of these equations was verified by comparison of sample concentrations counted with hemocytometer and calculated with equations, and no significant differences (p = 0.447) were observed. Measurement of serially diluted aliquots from pooled samples verified the precision of techniques used. Overall, this confirmed that microspectrophotometric estimation of sperm concentration is accurate, efficient, and sample-saving for use with small-bodied fishes.
本研究旨在建立一种仅需 1-2 微升样本即可通过微分光光度法测定精子浓度的有效方法。目的是：(1)确定适合于分析斑马鱼 Danio rerio、绿剑尾鱼 Xiphophorus helleri 和青鳉 Oryzias latipes 精子悬液的吸收光谱曲线波长，这些精子悬液是通过解剖睾丸的研磨或活雄性个体的剥离收集的；(2)生成精子样品吸光度与血球计数板计数估计的精子浓度之间的标准曲线和方程；(3)验证通过微分光光度法估计浓度的方程的准确性；以及(4)分析生成方程和估计精子浓度的精密度。在可见波长范围内（380-750nm），没有单一的最大吸收峰。对于斑马鱼，建立了线性相关性，在 400nm 时，研磨样本的有效吸收范围为 0.034-0.936，而剥离样本的有效吸收范围为 0.028-0.961。对于剑尾鱼，研磨样本的有效吸收范围为 0.014-1.154，而剥离样本的有效吸收范围为 0.038-1.082。对于青鳉鱼，研磨样本的有效吸收范围为 0.041-0.896。通过比较血球计数板计数的样品浓度和用方程计算的样品浓度来验证这些方程的准确性，未观察到显著差异（p=0.447）。对混合样本的连续稀释等分试样的测量验证了所使用技术的精密度。总的来说，这证实了微分光光度法估计精子浓度是准确、高效且节省样本的，适用于小型鱼类。
