Evaluation of AFP III-Conjugated Fe₃O₄ Nanoparticles on Bull Sperm Cryopreservation

Antifreeze proteins are naturally occurring proteins that are known for their ability to inhibit ice formation. Although effective in nature, they have failed to produce the expected results in cryobiological studies. However, the lack of a significant effect when used alone has raised the following question: Could conjugation with Fe₃O₄ nanoparticles (Fe₃O₄ NPs), which are frequently used in drug delivery studies, enable closer interaction with spermatozoa and provide cumulative protection? To address this question, bull spermatozoa were used. First, Fe₃O₄ NPs (~6 nm) and AFP III-conjugated Fe₃O₄ NPs (AFP III@Fe₃O₄ NPs) were synthesised and characterised by dynamic light scattering, zeta potential analysis, and FTIR spectroscopy to confirm the conjugation. Then, sperm samples collected from five different Holstein bulls for five replications were diluted with AndroMed extender and divided into five groups: control, Fe₃O₄ NPs only, AFP III only (0.1 and 0.5 μg/mL), and AFP III@Fe₃O₄ NPs (0.042 and 0.075 μg/mL). After cryopreservation, spermatological parameters and DNA condensation were analysed. The AFP III@Fe₃O₄ NPs (0.075 μg/mL) exhibited significantly higher motility and DNA integrity compared to the other treatment groups (p < 0.05). However, no statistically significant differences were observed in mitochondrial membrane potential, membrane, and acrosomal integrity. Analyses of motility and DNA damage supported the protective effect of the AFP III@Fe₃O₄ NPs 0.075 μg/mL formulation. Thus, we have demonstrated the applicability of AFP III@Fe₃O₄ NPs, for the first time, in bull sperm cryopreservation and have introduced a novel nano-biotechnological approach for improving the success of assisted reproductive technologies in livestock.