Ex vivo characterization of NETs induced by sperm and evaluation of their impact on the quality of post-insemination uterine lavage with frozen/thawed semen

Abstract

The cryopreservation of swine semen holds great potential for growth in global artificial insemination. Some of the favorable aspects of this technique include semen preservation for extended periods and its transfer over long distances, which can contribute to the productive efficiency of intensive pig farming. However, a challenge faced in its utilization is the modulation of the intense inflammatory response occurring in the endometrium after intrauterine insemination with thawed semen. Therefore, this current project aims to evaluate the influence of optimized cryopreserved semen use (using an optimized diluent) on the innate immunity response in female pigs and its effects on spermatozoa. To this end, 50 gilts will be inseminated with optimized cryopreserved thawed semen in 5 different treatments (1. Optimized diluent; 2. Optimized diluent plus NETs inhibitor; 3. Optimized diluent plus antioxidant; 4. Conventional cryopreservation diluent; 5. Control medium). After 3 hours of AI, uterine lavage will be collected, centrifuged, and the samples processed for analysis via scanning electron microscopy (SEM) to determine NETs (neutrophil extracellular traps). Different NETs phenotypes will be identified through dual immunofluorescence staining. NETs quantification will be performed via a luminometer after extracellular DNA staining with PicoGreen® and through PCR. Macrophages and neutrophils will be identified through immunophenotypic analysis for NET cell subpopulations. Inflammatory markers will be analyzed using Luminex MAGPIX® equipment and the Bio-Plex ProTM kit.