Development of a Single-Piece Sperm Counting Chamber (SSCC) for Aquatic Species

Abstract

Accurate determination of sperm concentration in aquatic species is important for assisted reproduction and cryopreservation, yet is challenging as current counting methods are costly or not suitable for many species. The goal of this work was to develop a simple (single-piece and single-layer photolithography) sperm counting chamber (SSCC) for aquatic species. Goldfish (Carassius auratus) and zebrafish (Danio rerio) sperm were used for evaluation in the device, which was created with soft lithography. Four designs with different geometries were evaluated for counting accuracy. Open-corner and open-midpoint designs were the most accurate with no significant differences (P > 0.05) for most of the target sperm concentrations (0.5-1.0 × 10⁸ cells/mL). The open-corner design was not significantly different from the Makler^® counting chamber intended for human sperm cells (P = 0.6) but was significantly different from a hemocytometer (P < 0.001) intended for other cell sizes. Material cost of device production was USD 16 per unit, including photolithography supplies, glass slide and coverslip, and polydimethylsiloxane. The cost can be reduced to USD 2 per unit with repeated wafer casts. This device could be further refined for resin 3-D printing and sharing via open-hardware approaches and modified to best suit species specific applications.

Keywords: PDMS; cell counting; goldfish; microfabrication; open hardware; sperm; zebrafish.

Figure 1.

Renderings of the 2-dimensional design of SSCC device. Overall design used to create the photomask for fabrication of the SSCC chips (a). Each device contained 25 arrays in a 5 × 5 pattern (a,b), with the enclosed design incorporating a border surrounding the grids (b). Each grid contained 100 squares arranged in a 10 × 10 pattern (c–e) with each square measuring 100 μm (L) × 100 μm (W) × 10 μm (H). Three different grid designs were produced: open-corners (c), open- midpoint (d), and closed-grids (e). Only the closed-grid design surrounding the array (b).

Figure 2.

Operation of the SSCC device. The PDMS chip was placed on a microscope stage (left), sperm suspension was pipetted onto the surface, and a coverslip was applied to form a monolayer (middle). The 3 × 3 arrangement of chambers is shown at 100-× magnification (right). Created with BioRender.com.

Figure 3.

Comparison of four different designs for cell counts of serial dilutions of goldfish sperm at 1 × 10⁸, 0.75 × 10⁸, and 0.5 × 10⁸ cells/mL (n = 5; mean ± SEM) with one-sample t-test using the serial dilution concentrations as the hypothetical mean (top). Comparison of open-midpoint and open-corner designs for counting of zebrafish sperm (1 × 10⁸ cells/mL) (bottom) (n = 3) with two-way ANOVA for multiple comparisons (Dunnet), compared to Average * indicates P < 0.05 and ** indicate P < 0.01. The dashed line indicates solution concentrations.

Figure 4.

Repeatability of SSCC devices with open-corner and open-midpoint grid configurations for counting of zebrafish sperm (1 × 10⁸ cells/mL) (n = 5; mean ± SEM) with two-way ANOVA multiple comparisons (Dunnet), compared to first use. The dashed line indicates solution concentrations.

Figure 5.

Comparison of the SSCC device with and without plasma bonding to a glass microscope slide for counting of zebrafish sperm (1.5 × 10⁸ cells/mL) (n = 5; mean ± SEM) with a t-test ** indicates P = 0.006. The dashed line indicates the solution concentration.

Figure 6.

Performance comparison of a Makler^® counting chamber, SSCC device, and hemocytometer with zebrafish sperm (n = 5, Mean ± SEM) with two-way ANOVA multiple comparisons (Dunnet). *** indicates P < 0.001 when compared to the Makler^® and SSCC.