Label-Free Microfluidic Impedance Cytometry for Acrosome Integrity Assessment of Boar Spermatozoa.

Microfluidics and lab-on-chip technologies have been used in a wide range of biomedical applications. They are known as versatile, rapid, and low-cost alternatives for expensive equipment and time-intensive processing. The veterinary industry and human fertility clinics could greatly benefit from label-free and standardized methods for semen analysis. We developed a tool to determine the acrosome integrity of spermatozoa using microfluidic impedance cytometry. Spermatozoa from boars were treated with the calcium ionophore A23187 to induce acrosome reaction. The magnitude, phase and opacity of individual treated and non-treated (control) spermatozoa were analyzed and compared to conventional staining for acrosome integrity. The results show that the opacity at 19 MHz over 0.5 MHz is associated with acrosome integrity with a cut-off threshold at 0.86 (sensitivity 98%, specificity 97%). In short, we have demonstrated that acrosome integrity can be determined using opacity, illustrating that microfluidic impedance cytometers have the potential to become a versatile and efficient alternative in semen analysis and for fertility treatments in the veterinary industry and human fertility clinics.

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

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.

Droplet encapsulation of electrokinetically-focused analytes without loss of resolution.

Lab-on-chip electrokinetic focusing and separation techniques are widely used in several scientific fields. In a number of cases, these techniques have been combined with a selective analyte extraction for off-chip analysis. Nevertheless, the usability of the extracts is limited by diffusion which reduces the separation resolution. In this paper we propose the integration of a droplet generator capable of continuous or on-demand generation and extraction of electrokinetically separated and focused analytes. We demonstrate the selective droplet extraction of model analytes separated and concentrated via ion concentration polarization focusing (ICPF). We report extracted droplets with 1000-fold increased concentration. Importantly, the droplet generator does not interrupt the ICPF process making it suitable for integration with the majority of electrokinetic separation techniques.

Flow-Free Microfluidic Device for Quantifying Chemotaxis in Spermatozoa.

Current male fertility diagnosis tests focus on assessing the quality of semen samples by studying the concentration, total volume, and motility of spermatozoa. However, other characteristics such as the chemotactic ability of a spermatozoon might influence the chance of fertilization. Here we describe a simple, easy to fabricate and handle, flow-free microfluidic chip to test the chemotactic response of spermatozoa made out of a hybrid hydrogel (8% gelatin/1% agarose). A chemotaxis experiment with 1 µM progesterone was performed that significantly demonstrated that boar spermatozoa are attracted by a progesterone gradient.