A Modified-Herringbone Micromixer for Assessing Zebrafish Sperm (MAGS)

Abstract

Sperm motility analysis of aquatic model species is important yet challenging due to the small sample volume, the necessity to activate with water, and the short duration of motility. To achieve standardization of sperm activation, microfluidic mixers have shown improved reproducibility over activation by hand, but challenges remain in optimizing and simplifying the use of these microdevices for greater adoption. The device described herein incorporates a novel micromixer geometry that aligns two sperm inlet streams with modified herringbone structures that split and recombine the sample at a 1:6 dilution with water to achieve rapid and consistent initiation of motility. The polydimethylsiloxane (PDMS) chip can be operated in a positive or negative pressure configuration, allowing a simple micropipettor to draw samples into the chip and rapidly stop the flow. The device was optimized to not only activate zebrafish sperm but also enables practical use with standard computer-assisted sperm analysis (CASA) systems. The micromixer geometry could be modified for other aquatic species with differing cell sizes and adopted for an open hardware approach using 3D resin printing where users could revise, fabricate, and share designs to improve standardization and reproducibility across laboratories and repositories.

Keywords: PDMS; high-dilution; microfabricated; micromixer; zebrafish.

Figure 1

Detailed design of the Micromixer for Assessing zebrafish Sperm quality (MAGS) inlets, main channel, and symmetric herringbone geometries. The main channel width is 1.0 mm across, channel height and herringbone height are each 25 μm. The bottom right corner image depicts the MAGS setup and operation using a micropipette to pull sample into the device, where sperm motility was evaluated using video microscopy.

Figure 2

Design schematics of the “Symmetric mixer” geometry and inlet array used to create the tested microfluidic devices. The MAGS device included (a) the novel split inlet design and “Symmetric mixer” geometries and was compared to (b) a “Standard mixer” geometry for mixing efficiency. Single inlet versions for (c) the “Symmetric” and (d) “Standard mixer” geometries were compared to the split inlet design. All dimensions shown in millimeters.

Figure 3

COMSOL generated fluorescein concentration profiles for the single inlet designs of No Mixer, Standard Mixer, and Symmetric Mixer simulations, at 0.5, 1.0, and 5.0 μL/min. Average Re and Pe values were calculated after the last set of mixers in the main channel (x = 10,000 μm) and reported under the corresponding flow rates.

Figure 4

Concentration profiles produced from COMSOL simulations for the single inlet designs (top). Location of cross-sectional cut-lines used to as evaluation sites, with color of the line matching the concentration profiles above (bottom).

Figure 5

COMSOL generated fluorescein concentration profiles for the split inlet designs of No Mixer, Standard Mixer, and Symmetric Mixer simulations, at 0.5, 1.0, and 5.0 μL/min. Average Re and Pe values were calculated after the last set of mixers in the main channel (x = 10,000 μm) and reported at the corresponding flow rates.

Figure 6

Concentration profiles produced from COMSOL simulations for the split inlet designs. The location of cross-sectional cut-lines used to as evaluation sites, with color of the line matching the concentration profiles above are shown in Figure 4, bottom.

Figure 7

Velocity line plots produced from COMSOL simulations and concentration profile results for all evaluated designs. The location of cross-sectional cut lines used to as evaluation sites, with color of the line matching the concentration profiles above are shown in Figure 4, bottom.

Figure 8

Mixing efficiencies from COMSOL concentration plots at each of the cut-lines down the microchannel, where cut-line #1 was taken as the basis for the mixing efficiency calculations. Single inlets are shown with solid lines and split inlets with dashed lines.

Figure 9

Experimental intensity plots for “No Mixer” and MAGS devices at 1.0 and 5.0 μL/min. The images used to obtain line plots are above each plot and data were extracted using ImageJ. Images were captured at the beginning (Cut-line #1), middle (Cut-line #3), and end (Cut-line #5) of the main channel.

Figure 10

CASA motility tracking results of zebrafish sperm loaded into a Makler^® static counting chamber (left) and the MAGS device (right). Images contain sperm tracks from activity.