Biological and Agricultural Engineering Department, Louisiana State University and LSU Agricultural Center, Baton Rouge, Louisiana, USA.
Theriogenology. 2012 Jul 15;78(2):334-44. doi: 10.1016/j.theriogenology.2012.02.008. Epub 2012 Apr 10.
Sperm viability in aquatic species is increasingly being evaluated by motility analysis via computer-assisted sperm analysis (CASA) following activation of sperm with manual dilution and mixing by hand. User variation can limit the speed and control over the activation process, preventing consistent motility analysis. This is further complicated by the short interval (i.e., less than 15 s) of burst motility in these species. The objectives of this study were to develop a staggered herringbone microfluidic mixer to: 1) activate small volumes of Danio pearl zebrafish (Danio albolineatus) sperm by rapid mixing with diluent, and 2) position sperm in a viewing chamber for motility evaluation using a standard CASA system. A herringbone micromixer was fabricated in polydimethylsiloxane (PDMS) to yield high quality smooth surfaces. Based on fluorescence microscopy, mixing efficiency exceeding 90% was achieved within 5 s for a range of flow rates (from 50 to 250 μL/h), with a correlation of mixing distances and mixing efficiency. For example, at the nominal flow rate of 100 μL/h, there was a significant difference in mixing efficiency between 3.5 mm (75±4%; mean±SD) and 7 mm (92±2%; P=0.002). The PDMS micromixer, integrated with standard volumetric slides, demonstrated activation of fresh zebrafish sperm with reduced user variation, greater control, and without morphologic damage to sperm. Analysis of zebrafish sperm viability by CASA revealed a statistically higher motility rate for activation by micromixing (56±4%) than manual activation (45±7%; n=5, P=0.011). This micromixer represented a first step in streamlining methods for consistent, rapid assessment of sperm quality for zebrafish and other aquatic species. The capability to rapidly activate sperm and consistently measure motility with CASA using the PDMS micromixer described herein will improve studies of germplasm physiology and cryopreservation.
精子活力在水生物种中越来越多地通过运动分析来评估，方法是通过手动稀释和混合激活精子，然后使用计算机辅助精子分析（CASA）进行分析。用户的变化会限制激活过程的速度和控制，从而无法进行一致的运动分析。这进一步复杂化了这些物种中爆发性运动的短暂间隔（即少于 15 秒）。本研究的目的是开发一种交错式人字形微混合器，以：1）通过与稀释剂快速混合来激活小体积的珍珠鱼斑马鱼（Danio albolineatus）精子，2）将精子定位在观察室中，以便使用标准 CASA 系统进行运动评估。人字形微混合器是在聚二甲基硅氧烷（PDMS）中制造的，以产生高质量的光滑表面。基于荧光显微镜，在 5 秒内实现了超过 90%的混合效率，混合距离和混合效率之间存在相关性。例如，在名义流量为 100 μL/h 的情况下，混合效率在 3.5 毫米（75±4%；均值±标准差）和 7 毫米（92±2%；P=0.002）之间存在显著差异。PDMS 微混合器与标准体积载玻片集成在一起，展示了通过减少用户变化、更大的控制以及对精子形态无损伤来激活新鲜斑马鱼精子的能力。通过 CASA 对斑马鱼精子活力进行分析，发现微混合激活的精子活力显著高于手动激活（56±4%比 45±7%；n=5，P=0.011）。这种微混合器代表了一种简化方法的第一步，可用于对斑马鱼和其他水生物种的精子质量进行一致、快速的评估。本文所述的 PDMS 微混合器能够快速激活精子并使用 CASA 进行一致的运动测量，这将改善种质生理学和冷冻保存的研究。
