Lectin Functionalised Iron Magnetic Nanoparticle-Based Sperm Selection: A Potential Technique to Improve Bull Sperm Quality In Vitro.

Abstract

Premature acrosomal exocytosis in cryopreserved semen is one of the reasons attributed to low fertility among livestock. In the present study, we attempted to enhance the cryopreserved semen quality by selective removal of acrosome-reacted spermatozoa using FITC-PNA conjugated iron magnetic nanoparticles (MNPs). Further, the effect of nano purification on other sperm functional attributes was also assessed. Iron MNPs were prepared using co-precipitation method and dextran-coated MNPs were conjugated with FITC-PNA (0.04 mg/mL). A preliminary experiment was conducted to standardise the dose of FITC-PNA conjugated iron MNPs (0.02, 0.05, 0.1, 0.15, 0.2, 0.4 and 0.6 mg). Among the different doses used, 0.6 mg FITC-PNA conjugated iron MNPs significantly (p < 0.05) removed higher acrosomal reacted spermatozoa from the semen, and therefore, this dose was used in further experiments. Cryopreserved semen from Holstein Friesian breeding bulls (n = 6) were thawed and washed using Sperm-TALP to remove residual extender. Washed spermatozoa (2 × 10⁶) were exposed to 0.6 mg of FITC-PNA conjugated iron MNPs for 10 min at 37°C. The nano purified semen was assessed for various vital sperm parameters viz., viability, intracellular calcium, apoptosis, mitochondrial ROS and mitochondrial membrane potential using flow cytometry. We found that nanopurification using FITC-PNA conjugated iron MNPs significantly (p < 0.05) improved the sperm quality. The proportion of viable non-apoptotic spermatozoa with low intracellular calcium levels was significantly (p < 0.05) enriched in nano purified semen. Nano purification did not affect sperm mitochondrial membrane potential and ROS production. In conclusion, these preliminary findings indicate that FITC-PNA coated iron MNPs effectively removed acrosome reacted spermatozoa and significantly improved sperm functional attributes in the purified fraction.