111 Improvement of cat and bull sperm quality using nanotechnology as a model for wild species

Assisted reproductive techniques (ART) are widely used in domestic species, with increasing applications in wildlife conservation. However, the currently available techniques for semen preparations are not fully reliable or applicable in all species to ensure successful ART outcomes. Recent developments in nanotechnology offer new horizons for further optimization of sperm preparations. The use of conjugated magnetic nanoparticles allows for selective targeting and removal of moribund spermatozoa through the technique known as nanopurification, which has shown to be beneficial in domestic boars and bulls. Nanopurification is a rapid, straightforward, effortless, and noninvasive technique that, when applied in wildlife or endangered species, is expected to have a tremendous impact on ART outcomes. In this study, we evaluated the effectiveness of magnetic (iron oxide) nanoparticles designed to target moribund spermatozoa (e.g. acrosome reacted) on domestic felid and threatened bovid species. Fresh epididymal spermatozoa of domestic cats and ejaculated spermatozoa from genetically valuable threatened bulls (Ankole and Sahiwal) were collected and mixed with iron-oxide magnetic nanoparticles (MNP). After 30min of incubation at 38°C, semen mixtures were placed on an electromagnetic field (10-15min) to trap MNP-bound spermatozoa (moribund), followed by the elution of viable nontrapped spermatozoa (nanopurified). All samples were analysed for motility characteristics using a computer-assisted sperm analyzer. Aliquots of cat spermatozoa were subjected to (1) morphology and acrosome analyses (n=4-8 replicates), (2) cryotolerance to liquid nitrogen storage (n=4 replicates), and (3) IVF (n=1 replicate). Data were analysed with Student’s t-test and P<0.05 indicated significant differences. Regardless of the species, both control and nanopurified spermatozoa revealed comparable motility characteristics (P>0.05). Sperm MNP-bound samples exhibited higher proportions of moribund spermatozoa as compared to their nanopurified and control nonpurified counterparts (57.5v. 47.5 and 49.5%, respectively; P<0.05). Cryopreservation revealed a trend for higher post-thaw motility of nanopurified cat spermatozoa (18%) v. the controls (12%). The use of frozen-thawed control v. nanopurified cat spermatozoa to in vitro fertilize oocytes (n=13 per group) resulted in comparable embryo cleavage (15v. 31%, respectively) and blastocyst (15v. 8%, respectively) rates. This preliminary study indicates the successful interactions of MNP nanoparticles with feline and bovine spermatozoa. The nanopurification seems to improve the cryotolerance of cat spermatozoa without affecting their fertilization potential. Additional research is being conducted to confirm the current findings and optimize this novel technique for future implementation in conservation breeding programs. Work supported by USDA-ARS 58-6402-3-018 and Morris Animal Veterinary Student Scholarship - D18ZO-608.