Development of an open hardware 3-D printed conveyor device for continuous cryopreservation of non-batched samples.

A great challenge among communities participating in germplasm repository development is to obtain suitable cryopreservation equipment and devices. Commercial programmable freezers are costly and thus unaffordable to many users. Self-made devices have substantial variability among users, resulting in few opportunities for standardization across communities. The development of open hardware with the increasing accessibility of three-dimensional (3-D) printing offers rapid prototyping and easy fabrication of devices by users around the world at low cost. The present study explored the feasibility of developing operational prototypes of 3-D printed motorized cryopreservation devices for continuous freezing of non-batched samples. A controlled cooling conveyor device (CCCD) was designed and fabricated to cryopreserve sperm samples in straws that were loaded onto chain links suspended over liquid nitrogen held in a Styrofoam box. Cooling rates of 5 to 34 °C/min for 0.5-ml French straws were produced by adjusting the height of conveyor chains, slopes, and liquid nitrogen mass. The plunge temperature (-47 °C to -61 °C) was controlled by adjustment of conveyor speed. The cooling curves from the CCCD were comparable to a commercial programmable freezer. There were no significant differences in post-thaw motility of sperm from ornamental (Koi) common carp (Cyprinus carpio) among samples frozen with the CCCD and those frozen with a commercial programmable freezer. The post-thaw sperm motility was consistent among samples frozen in the CCCD across a 15-min time span. The CCCD prototypes in the present study proved to be feasible and functional as low-cost, customizable, portable, and yet standardizable options for freezing of individual (non-batched) samples. Additional design alternatives are proposed to facilitate further adaptation and development by diverse user communities.

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

Evaluation of sperm concentration is essential for research and procedures involving AI, cryopreservation and sperm quality assessment. Microfabrication technologies have shown tremendous potential for rapid prototyping and fabrication of devices to assist reproduction and fertility research, but such utility has not yet been made available for most reproduction laboratories. The aim of this study was to evaluate the feasibility of using microfabrication techniques to produce counting chambers for estimation of sperm concentration. Zebrafish (Danio rerio) spermatozoa were used as a model for evaluation of functionality of the chambers. These microfabricated enumeration grid chambers (MEGC) were composed of a polydimethylsiloxane (PDMS) coverslip with grid patterns (100 µm×100 µm) and a PDMS base platform to create a known volume with a 10-µm height to restrict the cells to a single layer. The results of cell counts estimated by two of three prototype MEGC devices tested were not significantly different from the control device, a commercially available Makler chamber. The material cost for a MEGC was less than US$0.10 compared with product costs of approximately US$100 for a standard haemocytometer and US$700 for a Makler counting chamber. This study demonstrates the feasibility of microfabrication in creating low-cost counting chambers to enhance standardisation and strengthen interdisciplinary collaborations.

A microfluidic device for motility and osmolality analysis of zebrafish sperm.

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.

Challenges in Development of Sperm Repositories for Biomedical Fishes: Quality Control in Small-Bodied Species.

Quality control (QC) is essential for reproducible and efficient functioning of germplasm repositories. However, many biomedical fish models present significant QC challenges due to small body sizes (<5 cm) and miniscule sperm volumes (<5 µL). Using minimal volumes of sperm, we used Zebrafish to evaluate common QC endpoints as surrogates for fertilization success along sequential steps of cryopreservation. First, concentrations of calibration bead suspensions were evaluated with a Makler® counting chamber by using different sample volumes and mixing methods. For sperm analysis, samples were initially diluted at a 1:30 ratio with Hanks' balanced salt solution (HBSS). Motility was evaluated by using different ratios of sperm and activation medium, and membrane integrity was analyzed with flow cytometry at different concentrations. Concentration and sperm motility could be confidently estimated by using volumes as small as 1 µL, whereas membrane integrity required a minimum of 2 µL (at 1 × 106 cells/mL). Thus, <5 µL of sperm suspension (after dilution to 30-150 µL with HBSS) was required to evaluate sperm quality by using three endpoints. Sperm quality assessment using a combination of complementary endpoints enhances QC efforts during cryopreservation, increasing reliability and reproducibility, and reducing waste of time and resources.

Microfluidics and numerical simulation as methods for standardization of zebrafish sperm cell activation.

Sperm cell activation plays a critical role in a range of biological and engineering processes, from fertilization to cryopreservation protocol evaluation. Across a range of species, ionic and osmotic effects have been discovered that lead to activation. Sperm cells of zebrafish (Danio rerio) initiate motility in a hypoosmotic environment. In this study, we employ a microfluidic mixer for the purpose of rapidly diluting the extracellular medium to initiate the onset of cell motility. The use of a microchannel offers a rapid and reproducible mixing profile throughout the device. This greatly reduces variability from trial to trial relative to the current methods of analysis. Coupling these experiments with numerical simulations, we were able to investigate the dynamics of intracellular osmolality as each cell moves along its path through the micromixer. Our results suggest that intracellular osmolality, and hence intracellular ion concentration, only slightly decreases, contrary to the common thought that larger changes in these parameters are required for activation. Utilizing this framework, microfluidics for controlled extracellular environments and associated numerical modeling, has practical applicability in standardizing high-throughput aquatic sperm activation, and more fundamentally, investigations of the intracellular environment leading to motility.

Sperm quality assessments for endangered razorback suckers Xyrauchen texanus.

Flow cytometry (FCM) and computer-assisted sperm motion analysis (CASA) methods were developed and validated for use with endangered razorback suckers Xyrauchen texanus collected (n=64) during the 2006 spawning season. Sperm motility could be activated within osmolality ranges noted during milt collections (here 167-343 mOsm/kg). We hypothesized that sperm quality of milt collected into isoosmotic (302 mOsm/kg) or hyperosmotic (500 mOsm/kg) Hanks' balanced salt solution would not differ. Pre-freeze viabilities were similar between osmolalities (79%±6 (s.e.m.) and 76%±7); however, post-thaw values were greater in hyperosmotic buffer (27%±3 and 12%±2; P=0.0065), as was mitochondrial membrane potential (33%±4 and 13%±2; P=0.0048). Visual estimates of pre-freeze motility correlated with total (r=0.7589; range 23-82%) and progressive motility (r=0.7449) by CASA and were associated with greater viability (r=0.5985; P<0.0001). Count (FCM) was negatively correlated with post-thaw viability (r=-0.83; P=0.0116) and mitochondrial function (r=-0.91; P=0.0016). By FCM-based assessments of DNA integrity, whereby increased fluorochrome binding indicated more fragmentation, higher levels were negatively correlated with count (r=-0.77; P<0.0001) and pre-freeze viabilities (r=-0.66; P=0.0004). Fragmentation was higher in isotonic buffer (P=0.0234). To increase reproductive capacity of natural populations, the strategy and protocols developed can serve as a template for use with other imperiled fish species, biomonitoring, and genome banking.