An automated modular open-technology device to measure and adjust concentration of aquatic sperm samples for cryopreservation

Abstract

Repositories for aquatic germplasm are essential for safeguarding valuable genetic diversity for species relevant to aquaculture, biomedical research, and conservation. Development of aquatic germplasm repositories is impeded by a lack of standardization within laboratories and across the research community. Protocols for cryopreservation are often developed ad hoc and without close attention to variables, such as cell concentration, that strongly affect the success and reproducibility of cryopreservation. The wide dissemination and use of specialized tools and devices as open hardware can improve processing reliability and save costs. The goal of the present work was to develop and prototype a modular and open-technology approach to help to standardize the cell concentration of germplasm samples prior to cryopreservation. The specific objectives were to: 1) design and fabricate prototypes of the automated concentration measurement and adjustment system (CMAS), incorporating custom peristaltic pumps and optical evaluation modules, and 2) evaluate the performance of the CMAS with biological samples. Linear regression models were obtained for estimation of aquatic sperm concentration >10⁸ cells/mL and for algae concentration > (3 × 10⁵) cells/mL. Algae were diluted with extender medium by an automated process, resulting in a dilution precision of ±12.6% and ±6.7% in two trials, attaining means of 89% and 71% of the target cell concentration. The development of the CMAS as open technology can provide opportunities for community-level standardization in cryopreservation of aquatic germplasm and can invite new users, makers, and developers into the open-technology community. This will increase the reach and capabilities of much-needed aquatic germplasm repositories.

Keywords: 3-D Printing; Aquatic; Concentration; Cryopreservation; Dilution; Open technology.

Fig. 1.

Design concept for the CMAS. A fluidic sample flows from the input to the output reservoir, with the measurement of sample concentration and the addition of diluents. The microcontroller mediates among the user, user-interface components, sensing components for concentration evaluation, and electromechanical components for fluid flow. The built-in concentration measurement after adjustment (at right) was not implemented in the present work.

Fig. 2.

Illustrations of the optical enclosure. (a) The optics housing was designed to measure the intensity of light passing through a sample in clear PVC tubing. The LED, photodiode, and tubing were enclosed in a 3-D printed housing to exclude ambient light. (b) A cutaway illustration to show internal features. (c) Light (red arrow) passed from a light-emitting diode (LED), through the walls of the tubing and through the sample, to a photodiode. Note that the tubing internal diameter and the slit through which light was admitted were both 1 mm.

Fig. 3.

Three prototype iterations of the custom peristaltic pump for the CMAS. All versions were designed to be driven by a NEMA 17 stepper motor. (a) The first version was designed on a 3-D printed mount meant for attachment to metal DIN rails, common in networking and power cabinets. Scale bar is approximately accurate at the rotor (⌀ ~30 mm in all versions). (b) The second version featured stronger springs and was designed to mount on a laser-cut acrylic panel. (c) The third version featured a strengthened frame and a redesigned rotor assembly.

Fig. 4.

The third CMAS prototype. The input sample is pumped through an optical module, which estimates the sample sperm cell concentration. The diluents are then pumped into tubing T-junctions that meet the sample flow in a ratio calculated to achieve a target dilution, and the mixed fluid stream is produced into the diluted sample container. Scale bar is approximately accurate at the center of the front face of the case.

Fig. 5.

Optical measurements on the CMAS of serial dilutions of channel catfish sperm allowed fitting of a regression line. (a) Serial dilutions of sperm are shown, ranging from most dilute (7.8 × 10⁶ cells/mL) at left to the starting concentration (4.0 × 10⁹ cells/mL) at right. A faint red discoloration is visible in the high-concentration aliquots due to blood present in the sample, a normal artifact of the collection method. (Blood content ≤5% v/v changed the concentration estimates by <2%.) A sheet of paper with black lines was placed in the background to help to demonstrate the increasing opacity of the samples. (b) Sensor voltage from the CMAS optical evaluation module was plotted against calculated cell concentration based on serial dilutions on a log scale. All data points (5 per concentration) were plotted. A regression line was fitted to all data points >10⁸ cells/mL. The left-to-right order of samples in (a) matches the left-to-right order of data points in (b). (c) Calculated sperm concentration in a blind trial correlated closely with known concentration. A point falling exactly on the y = x line would indicate an exact match between the ‘known’ dilution of the sample and the value produced by calculations with data from the CMAS. A best fit regression line was fitted to all concentrations ≥10⁸ cells/mL. The points corresponding to samples at 5 × 10⁷ cells/mL appeared on the plot directly below the samples at 1 × 10⁸ cells/mL, suggesting that the CMAS may have reached the lower bound of its sensitivity at 1 × 10⁸ cells/mL.

Fig. 6.

(a) Optical signals from algal samples evaluated on the CMAS were fitted by linear regression. The fitted equation allowed the CMAS to calculate the cell concentration of samples, with the aim of finding a suitable dilution ratio for the sample and adjusting to a target concentration. (b) The transmittance of the same algal samples as in panel a, evaluated on a Nanodrop 1000 spectrophotometer (λ = 625 nm). The linear regression served to indicate that the transmittance data obtained with the CMAS can be matched to a simple linear fit more readily than the Nanodrop data. Qualitatively, the sensitivity and technical replicability achieved with the CMAS sensor compare favorably to the Nanodrop in the tested range. (c) Results of automatic dilutions by the CMAS of algal samples. The target concentration from both starting concentrations was 1.0 × 10⁶ cells/mL. The symbol “ × ” indicates individual data points; whiskers indicate one standard deviation from the mean.