Effect of microfluidic processing on the viability of boar and bull spermatozoa

Abstract

The use of microfluidics in artificial reproductive technologies for manipulation or assessment of spermatozoa is unique in the sense that it is not always an end point measurement and the sample may be used afterward. During microfluidic processing, spermatozoa are exposed to shear stress, which may harm viability and functioning of spermatozoa. The shear stresses during general microfluidic processing steps were calculated and compared to estimated shear stresses during ejaculation. The viability of boar and bull spermatozoa after microfluidic processing was studied and compared to the typical handling method (centrifugation) and to a control (the sample in a tube at the same temperature). The boar spermatozoa showed a small but significant decrease in viability of 6% after microfluidic handling. Bull spermatozoa proved to be less susceptible to shear stress and were not significantly affected by microfluidic processing. These data indicate that the impact of microfluidic processing on the viability of boar and bull spermatozoa is less than the literature values reported for flow cytometry and comparable to the impact of centrifugation.

FIG. 1.

Schematic representation of the microfluidic chips used to process spermatozoa. (a) Straight channel with 300 μm width and 50 μm height (length 2 cm) and (b) PFF chip, with 100 μm wide inlets, 50 μm wide pinched section, and 2500 μm wide broadened section (total length of 8 mm).

FIG. 2.

Schematic overview of the three different tested conditions for microfluidic processing of (1) tubing, (2) straight channel, (3) PFF channel, and (4) centrifuge for both bull and boar spermatozoa. P_in [or P₁ (sample) and P₂ (buffer)] was varied, while P_out was kept at atmospheric pressure. Each sample group was compared to the control with ANOVA to determine the significance of the change in viability. Features are not to scale. Pressure (P).

FIG. 3.

Representative images of boar (a) and bull (b) spermatozoa treated with live/dead staining after being processed with the microfluidic chip (straight channel, 1000 mbar). Live and dead cells are represented in green and red, respectively.

FIG. 4.

The percentage of normalized viability after microfluidic processing of boar (left) and bull (right) spermatozoa with connection to tubing (a) and (e), the microfluidic chip with a straight channel (b) and (f), a pinch flow fractionation (PFF) chip (c) and (g), and centrifugation (d) and (h). Error bars = 1 SD, N = 3 (° N = 2). In all experimental conditions, no trend in viability decrease with increasing applied pressure/centrifugal force was observed (p > 0.05).

FIG. 5.

Average normalized viability (±1 SD) of the boar and bull spermatozoa after (microfluidic) processing. The viability of spermatozoa after microfluidic processing was almost 100% when taking the standard deviation into account. Therefore, the effect of microfluidic processing on the viability of spermatozoa is negligible. * indicates p < 0.05.