ATOMIC FORCE MICROSCOPY APPLICATION TO ASSESS THE EFFECT ON SPERM CELLS OF ALTERNATIVE BIOMELECULES TO ANTIBIOTICS IN BOAR SEMEN STORAGE FORMULATIONS

Atomic force microscopy (AFM) is a powerful nanoscale imaging technique that enables high- resolution three-dimensional characterization of surfaces by direct mechanical contact between the sample surface and a flexible cantilever tip without the need for staining or coating. Since AFM also offers the advantage of directly observing biological samples, this technique has found several applications in many biology-related fields. In reproductive biology AFM has proven to be a very useful tool for directly observing spermatozoa, opening the possibility of analyzing structural aspects at the sub-molecular level, such as membrane integrity and surface roughness, which are crucial for the fertilization ability. [1-3]. This study employed AFM to evaluate surface roughness changes in boar spermatozoa stored for five days in a semen extender containing conventional antibiotics compared to an antibiotic-free extender supplemented with antimicrobial biomolecules. Semen was collected from a fertile Duroc boar with the double-glove technique [4]. The sperm-rich fraction was diluted in 3 different extenders corresponding to the treatment groups to a final concentration of 30 × 10⁶ spermatozoa/mL. Diluted semen doses were stored at 16 °C for 7 days under gentle mixing. The experimental groups included: (I). Boar semen extender with two conventional antibiotics (Positive control: Formula5®, Medinova, Italy); (II). Boar semen extender containing antimicrobial biomolecules (MediBio®, Medinova, Italy); (III). Boar semen extender without antimicrobials compounds (Negative control formulation). Analyses were performed using intermittent contact atomic force microscopy (IC-AFM) from day 1 and after 5 days of storage. For each sample, 50 µL were deposited on a coverslip, allowed to stand for two minutes, then gently washed and dried using a stream of nitrogen. The slides were fixed to a metal base with adhesive tape and placed on the AFM scanning platform. The topographic scans obtained were processed with Gwyddion software (v2.67), which allowed three-dimensional visualization and analysis of the surface roughness of the sperm cells. Statistical analysis was performed with RStudio (v2024.09.0+375). AFM scans revealed significant differences in sperm surface roughness across the extenders over time. On day 1, the mean roughness values were 7.69 ± 0.90 nm for (I) Formula5®, 6.90 ± 1.65 nm for (II) MediBio® , and 10.52 ± 1.39 nm for (III) Negative control formulation. At day 5 of storage, surface roughness increased significantly in (I) Formula5® (11.40 ± 1.62 nm) and in (III) Negative control formulation (14.14 ± 1.10 nm) groups, while it remained stable in (II) MediBio® group (8.03 ± 1.50 nm). Statistical analysis confirmed that sperm cells stored with MediBio® had significantly lower surface roughness than those in Formula5® and Negative control groups at all time points (p < 0.001) indicating a higher effectiveness in preserving cell membrane integrity during storage. In this study, AFM technique allowed the detection of changes in sperm surface roughness, highlighting the effectiveness of antimicrobial biomolecules in preserving the structural features of sperm cells during storage. This further supports the value of AFM in the investigation of nanoscale biological processes in biomedical and biotechnology research. References [1]. Awczyk-Wołoszyn A, Roczkowski D, Reich A, Żychowska M. Applying the Atomic Force Microscopy Technique in Medical Sciences - A Narrative Review. Biomedicines(2024). https://doi.org/10.3390/biomedicines12092012 [2]. Carvalho JO, Silva LP, Sartori R, Dode MAN. Nanoscale Differences in the Shape and Size of X and Y Chromosome- Bearing Bovine Sperm Heads Assessed by Atomic Force Microscopy. PLoS ONE (2013). 10.1371/journal.pone.0059387 [3]. Kumar, S., Chaudhury, K., Sen, P. Atomic force microscopy: a powerful tool for high-resolution imaging of spermatozoa. J Nanobiotechnol (2005). https://doi.org/10.1186/1477-3155-3-9 [4]. Lopez Rodriguez, A., Van Soom, A., Arsenakis, I. et al. Boar management and semen handling factors affect the quality of boar extended semen. Porc Health Manag (2017). https://doi.org/10.1186/s40813-017- 0062-5