Microfluidic mixing for sperm activation and motility analysis of pearl Danio zebrafish

Abstract

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.

Microfluidics for cryopreservation

“…Fortunately, both the designs for the staggered herringbone microfluidic mixer (Park et al, 2012) and the passive, planar micromixer based on logarithmic spirals (Scherr et al, 2015; Scherr et al, 2012) (Figure 7E), which are initially developed for sperm activation, can be potentially adopted for CPA processing, since where controllable extracellular mixing is supplied.…”

Microfluidics and numerical simulation as methods for standardization of zebrafish sperm cell activation

“…In the instance of the freshwater zebrafish, a widely used model for biomedical research (Lieschke and Currie 2007), a hypoosmotic environment is necessary for activation (Wilson-Leedy et al 2009; Park et al 2012). A postulated cascade of events has been published for turbot, a marine fish, spermatozoa activation upon encountering hyperosmotic conditions: the extracellular osmolality increases, water leaves the cell, internal osmolality increases along with internal ion concentrations, dynein motor proteins initiate flagellar movement, and eventually ATP stores become too low to maintain motility (Cosson 2004).…”

“…Several studies have been performed on isolating competent sperm from immotile sperm for fertilization (Cho et al 2003; Seo et al 2007; Tasoglu et al 2013). A micromixer was used to rapidly dilute the osmotic environment of zebrafish sperm, and analyze their motility using CASA (Park et al 2012). This represents a significant step in experimental protocols for studies of sperm cell activation; analysis times are significantly reduced, sample volumes are small, and cells can be exposed to highly reproducible and controlled conditions.…”

A numerical study on distributions during cryoprotectant loading caused by laminar flow in a microchannel

“…A variety of micromixers have been developed to overcome this problem and its associated challenges. [21][22][23][24][25] However, the laminar flow in microdevices has also been used advantageously. Spherical particles in laminar shear flow experience a lift force, which causes them to migrate to an equilibrium position.…”