DEVELOPMENT OF INNOVATIVE BUFFERS FOR BOAR SPERM WASHING ALTERNATIVES TO STANDARD PHOSPHATE-BUFFERED SALINE

Background and Objectives Removing seminal plasma from boar sperm through washing is a key step in many biotechnological applications for in vitro studies. To this end, phosphate-buffered saline (PBS) is commonly used since it prevents osmotic stress and does not interfere with DNA analysis; however, it may exert a negative effect on sperm cells. This study proposes alternative washing buffers to better preserve sperm cell viability upon washing, that was investigated by measuring progressive motility and cell surface roughness. Material and Methods Three aliquots of semen were centrifuged at 400g for 5 minutes, the supernatant was removed, and the pellet was resuspended in PBS, buffer A (polysaccharide base) or buffer B (monosaccharide base). Progressive motility (%) and surface roughness (nm) were evaluated after cells washing using SCA® (Microptic, Spain) and atomic force microscopy (IC-AFM mode), respectively. Statistical analysis was performed with RStudio. Results Sperm progressive motility, after pellet resuspension, showed statistically significant differences between PBS (51.5%) and buffers A (78.7%) and B (84.3%) (p <0.001). Cells surface roughness in PBS (6.67±0.27 nm) was significantly different compared to roughness observed in buffer A (7.03±0.29 nm) and B (7.53±0.31 nm) (p <0.05). Five days after washing, progressive motility in PBS (8.9%) was significantly lower than in buffers A (77.5%) and B (84.2%) (p < 0.01). Surface roughness in PBS (10.20±0.17 nm) showed a significant increase compared to the stable values observed in buffer A (7.76±0.22 nm) and buffer B (8.15±0.22 nm) (p < 0.001). Discussion and Conclusion This study demonstrates that PBS increases cell surface roughness and reduces progressive motility, whereas buffer A and B can maintain cell viability for up to five days after washing. This provides a significant advantage for biotechnological applications and in vitro studies; therefore, proposed buffers can be considered better alternatives to PBS for sperm cell washing procedures.