Magnetic nanoparticle based Bovine sperm sorting

Abstract

Artificial insemination (AI) using cryopreserved semen is the most widely used method to accelerate the production of livestock population. Sperm selection is a crucial component of assisted reproductive methodologies which helps in sorting out dead and damaged sperms from viable fraction. The low conception rate associated with using cryopreserved semen can be attributed to cryodamage that lowers the sperm viability. The presence of dead and defective sperms in the semen population limits artificial insemination to be exploited efficiently. The competent removal of dead sperms before cryopreservation can significantly increase the conception percentage. This thesis discusses a novel approach of magnetically activated bovine sperm cell sorting to get an enriched eluent comprising majorly of healthy and live sperms, which can further be used for AI. Interaction between phosphatidyl serine moieties of dead sperms and Annexin-V microbeads in a magnetic environment have been incorporated to work out a protocol for the separation of dead spermatozoa. Co-staining with PI/Hoechst and fluorophore-conjugated Annexin V were then employed to visualize dead sperms via fluorescence microscopy. This is a simple, rapid and cost-effective method for live sperm enrichment post-thawing of cryopreserved semen. Optical Tweezer Raman spectroscopy (OTRS) for differentiating the X-Spermatozoa and the Y-Spermatozoa has not been reported. This novel approach helped us in obtaining Raman Spectrum from healthy live cells. This technique is staining free, non-invasive, and is significantly better than using FACS based sperm sorting. Keywords: Sperm enrichment assay, Dead and live Assay, Phosphatidylserine, Annexin V, Annexin V conjugated microbeads, Raman Spectrum, Optical Tweezer Raman spectroscopy.