SORTYX Operational Group: Development of a new microfluidic system to control the sperm population of pigs

This study aims to characterize porcine sperm and identify differences between X and Y chromosome carriers. To achieve this, a comprehensive study was conducted, including the analysis of zeta potential, sperm size and quality, and the evaluation of sperm survival under different ionic conditions. Cytometry and PCR protocols were developed and optimized to separate and quantify X/Y populations, and the influence of calcium and magnesium on their differential viability was studied. Furthermore, potential differences in zeta potential between the two populations were validated. These results provide a solid scientific basis for the development of sex-selection technologies in swine.

This study aims to design a microfluidic system capable of separating sperm by sex in swine. A prototype is developed using 3D printing and biocompatible materials, integrating microfluidic channels and an electrode system to apply controlled electric fields. Different geometries and physical parameters are analyzed to optimize sperm targeting and separation. The system is validated through functional assays that evaluate motility, viability, acrosomal integrity, and genetic sexing. The results allow for adjustments to the design and improvements in the efficiency and safety of the process, moving towards an optimized device for sperm sex selection.

This result aims to optimize the microfluidic chip design through computational simulation. A model was developed in COMSOL Multiphysics that integrates experimental data to adjust fluid parameters and sperm behavior, achieving a high correlation between simulation and real-world results. Subsequently, electrical parameters were optimized to induce the separation of X and Y sperm based on their charge, evaluating voltage, electrode arrangement, and distance. Finally, advanced simulations of the final chip designs were performed to define the optimal operating conditions, maximizing the efficiency of the sperm sex selection system.

This study aims to verify the efficiency of the microfluidic system for safely obtaining sperm samples enriched in the desired sex. The final device is manufactured and validated through biological assays on 50 ejaculates, evaluating sperm quality before and after the process (motility, viability, acrosomal integrity, and kinetic parameters). The proportion of selected sperm and the sexing efficiency are also analyzed using zeta potential, cytometry, and PCR. Finally, the device is optimized, and a statistical analysis of the results is performed, generating a comprehensive report on the system's effectiveness.

This activity validates the efficiency of the microfluidic system for producing embryos and offspring of the desired sex without affecting fertility. In vitro fertilizations are performed with 25 samples, evaluating parameters such as penetration, decondensation, pronuclear formation, cell division, and development to the blastocyst stage, with embryonic sex determination. The system is implemented in artificial insemination centers (AICs), evaluating time, ease of use, technical problems, and sperm loss. Finally, an in vivo pilot study is conducted with 40 females using postcervical inseminations, with pregnancy diagnosis at days 21 and 30 and analysis at farrowing, recording the number of live births, stillbirths, mummified piglets, and their sex.

RD1 focuses on designing an effective dissemination and transfer strategy for the project, including the necessary tools for its implementation. Strategic and operational foundations are established for communicating and leveraging the results, developing channels, content, and materials that comply with the project's visual identity and current regulations (e.g., GDPR). This outcome ensures professional communication from the outset, with a consistent image, effective channels, and resource planning to guarantee dissemination and compliance with the call's publicity requirements.

RD2 aims to implement effective outreach to the scientific and technical community within the swine sector. It will generate and disseminate original knowledge on the development of a microfluidic system for sperm sex selection in swine, with the goal of obtaining offspring of the desired sex. Furthermore, information about the project, its objectives, expected results, and the funding received from the Ministry will be communicated to the general public, ensuring visibility and understanding of the project's impact.

RD3 aims to ensure the transfer and application of results to pig farmers and technicians. The adoption of developed innovations and solutions will be facilitated by demonstrating their applicability and economic, environmental, and animal welfare benefits under real-world conditions. Furthermore, the application of and access to the results will be communicated through an Implementation Plan, promoting the practical application of the project's advancements.