Abstract
Nano-graphene oxide (NGO) is a nanomaterial that has been frequently used in the fields of health and bioengineering in recent years. However, its potential use in semen cryopreservation is still in the exploratory phase. In this study, Angora bucks, a breed with low resistance to cold shock, were used. Sperm was collected from five different Angora bucks, pooled, diluted with a Tris-based egg yolk diluent, and frozen with the addition of NGO at two different sizes (50 and 500 nm) and doses (10 and 50 µg/mL). Nanoparticle characterization was performed using field emission scanning electron microscopy (FE-SEM), dynamic light scattering (DLS), and Fourier-transform infrared spectroscopy (FTIR). Post-thaw sperm analyses were evaluated based on motility and kinematic parameters, mitochondrial membrane potential (MMP), plasma membrane and acrosome integrity (PMAI), and DNA fragmentation. Applying 50 nm NGO at a dose of 50 µg/mL led to statistically significant improvements in motility and PMAI (p < 0.05). The same dose of 500 nm NGO, however, only showed a statistically significant improvement in the PMAI parameter (p < 0.05). No significant differences were observed between the groups for MMP and kinematic parameters (p > 0.05). Conversely, it was found that all sizes and doses of NGO significantly protected post-thaw sperm regarding DNA integrity (p < 0.05). These findings indicate that the NGO, at a size of 50 nm and a dose of 50 µg/mL, improves the post-thaw quality of Angora buck sperm and provides a cryoprotective effect that depends on size and dose. This study provides preliminary data on the potential effects of NGO; however, comprehensive mechanistic and in vivo validation studies are required to establish the biological and clinical validity of these findings.