A microfluidic device for motility and osmolality analysis of zebrafish sperm

Abstract

A microfluidic chip is described that facilitates research and quality control analysis of zebrafish sperm which, due to its miniscule (i.e., 2-5 μl) sample volume and short duration of motility (i.e., <1 min), present a challenge for traditional manual assessment methods. A micromixer molded in polydimethylsiloxane (PDMS) bonded to a glass substrate was used to activate sperm samples by mixing with water, initiated by the user depressing a transfer pipette connected to the chip. Sample flow in the microfluidic viewing chamber was able to be halted within 1 s, allowing for rapid analysis of the sample using established computer-assisted sperm analysis (CASA) methods. Zebrafish sperm cell activation was consistent with manual hand mixing and yielded higher values of motility at earlier time points, as well as more subtle time-dependent trends in motility, than those processed by hand. Sperm activation curves, which indicate sample quality by evaluating percentage and duration of motility at various solution osmolalities, were generated with on-chip microfabricated gold floor electrodes interrogated by impedance spectroscopy. The magnitude of admittance was linearly proportional to osmolality and was not affected by the presence of sperm cells in the vicinity of the electrodes. This device represents a pivotal step in streamlining methods for consistent, rapid assessment of sperm quality for aquatic species. The capability to rapidly activate sperm and consistently measure motility with CASA using the microfluidic device described herein will help improve the reproducibility of studies on sperm and assist development of germplasm repositories.

Low-Cost Resin 3-D Printing for Rapid Prototyping of Microdevices: Opportunities for Supporting Aquatic Germplasm Repositories

“…A master mold of the testing channels was patterned with SU-8 photoresist (Mi-croChem Corp., Newton, MA, USA) on a silicon wafer (Universitywafer.com, South Boston, MA, USA) with an existing protocol [9]. A 10:1 mixture (elastomer:curing agent) of Sylgard-184 (Dow Corning, Midland, MI, USA) polydimethylsiloxane (PDMS) was cast onto the master mold, degassed in a vacuum chamber, and cured in an oven at 65 • C for 3 h.…”

“…In recent years, microfabrication ("Lab-on-a-chip") technologies have been applied for the creation of novel devices (microdevices) to enable quality assessment of samples to facilitate routine quality control programs. For example, microdevices have been developed for evaluation of sperm motility [8,9] and concentration [7] of aquatic species. These devices can be standardized with community-level efforts and customized to accommodate certain species.…”

Microfabrication of low-cost customisable counting chambers for standardised estimation of sperm concentration

“…To fabricate the coverslip for the counting chamber, a master mould was made by standard single-layer photolithography and a permanent epoxy-based negative photoresist (SU-8 2025; MicroChem; Beckham et al 2018).…”

“…Microfabrication techniques provide great potential to produce standardisable, low-cost and disposable devices for reproducible analysis of sperm cells (Park et al 2012;Beckham et al 2018). The need for this was identified by the recent Cryopreservation of Aquatic Biomedical Models Workshop (7-8 January 2017; Birmingham, AL, USA) hosted by the Office of Research Infrastructure Programs of the National Institutes of Health (NIH).…”

Rheotaxis quality index: a new parameter that reveals male mammalian in vivo fertility and low sperm DNA fragmentation

“…20,21 Further, CASA parameters do not necessarily evaluate the fertilization capacity of semen and it does not reflect the role that the female reproductive tract plays in sperm selection. 22 In the recent decade, burgeoning microfluidic devices have allowed for higher precision and point-of-care (POC) analysis [23][24][25][26] and made biological automation easier. 27 Microfluidics has been used for precise identification of normal sperm morphology, 28 beating amplitude, 29 and frequency 30 at the single-cell level by differential impedance measurement and single-cell trapping.…”