A Mobile Sperm Analyzer with User-Friendly Microfluidic Chips for Rapid On-Farm Semen Evaluation

Abstract

This study presents a mobile-based sperm analysis system featuring a user-friendly, droplet-loaded microfluidic chip that enables non-specialist users to perform the rapid and accurate quantitative evaluation of boar semen directly on the farm. The iSperm system integrates a tablet, optical module, heater, and real-time image analysis app to deliver automated measurements of sperm concentration, motility, and progressive motility in under one minute. Precision and user variability tests demonstrated high concordance with CASA and the hemocytometer, with minimal differences between trained and untrained users. A method comparison using 77 farm-collected samples confirmed agreement through Passing-Bablok regression and Bland-Altman analysis. ROC curve analyses further validated diagnostic accuracy for all parameters, with AUC values exceeding 0.95. The iSperm platform offers a reliable, user-friendly, and field-deployable solution for on-site semen quality assessment, improving decision-making in swine artificial insemination.

Keywords: ROC and regression analysis; boar semen analysis; image processing; microfluidic chip; on-farm point-of-care testing; portable sperm analysis system; user variability assessment.

Figure 1

The process flow for semen analysis using iSperm prior to sow artificial insemination. (a) iSperm system for evaluating sperm concentration, motility, and progressive motility in boar semen, ensuring semen quality at sow farms before AI. (b) Schematic diagram of the iSperm analyzer, including an exploded assembly drawing showing how the components of iSperm fit together. (c) Installation and operation of the droplet-loaded microchips within the iSperm system. (i) Semen is gently mixed to ensure uniformity before sampling. (ii) A dropper transfers ~50 µL of sample into the cover chip. (iii) The base chip is mounted onto the illumination module. (iv) The base and cover chips are assembled to encapsulate the sample. (v) The completed microchip module is inserted into the optical unit for analysis. (d) Software interface displaying analysis results and sperm tracks visualized in four distinct colors.

Figure 2

Optical module of the iSperm system. (a) The assembled optical module (3 cm × 2 cm), designed for compact and portable use with a tablet camera. (b) Components of the optical module shown in an exploded view. (c) Images captured using the iSperm optical module: (i) 5 µm latex beads and (ii) boar spermatozoa.

Figure 3

Droplet-Loaded Microchips used in the iSperm. (a) The base and cover chips, injection-molded from polycarbonate (PC), form the droplet-loaded microchip. (b) Assembled microchips ready for insertion into the iSperm optical module. (c) Schematic of the sample chamber with a 20 µm thickness and 2.6 mm diameter, designed for precise sperm analysis.

Figure 4

Heater module for the iSperm. (a) Exploded view showing the construction of the heater module, secured with key components and housed in a compact 5.5 cm × 3 cm enclosure. (b) Layered assembly of the heater system, showing the thermally conductive adhesive tape, flexible heater, thermistor, polyimide tape, and sponge used for insulation. (ci) Exploded schematic showing the placement of the heater module and temperature sensors in relation to the test device. (cii) Photograph of the experimental setup, demonstrating the heater system configuration during testing. (cii) Temperature profiles demonstrating the system’s ability to maintain the target temperature of 37.5 °C within approximately 5 min.

Figure 5

Software application interface and analysis process of the iSperm. (a) The main screen of the iSperm software on an iPad mini 6, providing easy access to key functions such as “Analyze Now.” (b) User interface guiding the alignment of the semen sample. (c) Visual feedback during analysis, guiding users to adjust the sample position across multiple fields of view. (d) Representative view of multiple fields displayed in a nine-grid layout to assist visual inspection. (e) Analysis results displayed on the iSperm interface, including sperm concentration, motility, progressive motility. (f(i)) Example of sperm tracking output generated by the iSperm system, highlighting different trajectories; (red: static sperm; green: motile sperm; blue: progressive sperm; yellow: late-track sperm). (f(ii)) Schematic diagram of velocity parameters.

Figure 6

Image processing pipeline used in the iSperm system.

Figure 7

Precision and user variability testing. (a) Sperm concentration (M/mL) results obtained from the hemocytometer, iSperm, and CASA systems, for concentrations ranging from 10 to 75 M/mL. (bi) Box plot comparing median sperm concentration measurements by trained and untrained users using the iSperm analyzer, and by CASA with slides loaded by untrained users and analyzed by trained personnel. Trained iSperm users reported a median value of 43.91 M/mL (IQR: 2.59), untrained iSperm users had 43.28 M/mL (IQR: 3.74), and CASA analysis based on untrained user-loaded slides yielded 42.41 M/mL (IQR: 36.69). (bii) Coefficient of variation (CV) in sperm concentration measurements by trained users using iSperm (4.58%), untrained users using iSperm (6.19%), and untrained users loading CASA slides (23.25%, analysis performed by trained personnel), illustrating the variability observed across trained iSperm users, untrained iSperm users, and untrained CASA loading.

Figure 8

Comparative Analysis Between iSperm and CASA. Passing–Bablok analysis comparing (a) sperm concentration, (c) total motility, and (e) progressive motility as measured by iSperm and CASA across all boar semen samples. The solid blue line represents the regression line, the solid red line indicates the identity line, and the dashed red lines represent the confidence band. Bland–Altman analysis comparing (b) sperm concentration, (d) total motility, and (f) progressive motility between iSperm and CASA. In the Bland–Altman plots, the blue dotted lines represent the mean difference between the methods, while the red dashed line represents the 95% limits of agreement (LOAs).

Figure 9

Effect evaluation. Device evaluation using boar semen samples based on farm-defined thresholds: 30 M/mL for concentration, 70% for total motility, and 30% for progressive motility. (a,c,e) Scatter plots of sperm concentration, total motility, and progressive motility, respectively, showing “Positive” and “Negative” groups separated by the blue threshold line. (b,d,f) Corresponding ROC curves illustrating the diagnostic performance of iSperm for each parameter. Area under the curve (AUC) values confirm the system’s high classification accuracy.