ANTIBIOTIC-FREE BOAR SEMEN EXTENDER FOR ARTIFICIAL INSEMINATION: SPERM QUALITY ASSESSMENT AND SURFACE ROUGHNESS CHARACTERISATION BY ATOMIC FORCE MICROSCOPY

Introduction: Antimicrobial resistance is a critical concern in pig production, particularly due to the widespread use of antibiotics routinely added to semen extenders formulations for artificial insemination [1]. Antibiotic-free formulations represent a sustainable alternative, requiring evaluation of bacterial control, sperm motility, and surface roughness properties, a nanoscale morphological indicator in which deteriorated spermatozoa exhibit irregular, elevated values associated with impaired fertilization outcomes [2] [3]. Aim: This study aims to compare a conventional antibiotic semen extender with an antibiotic-free formulation supplemented with natural antimicrobial biomolecules, assessing standard sperm quality parameters and sperm surface roughness properties. Methods: Raw ejaculates from three fertile Duroc boars were diluted to 30 × 10⁶ spermatozoa/mL in: (I) antibiotic-based extender; (II) antibiotic-free extender and stored at 17°C. Sperm quality parameters and surface roughness properties were analyzed at day 1 and 5 of storage: progressive motility was evaluated using CASA system (Microptic, Spain) and bacterial contamination was assessed on agar plates. Sperm surface roughness (nm) was detected using intermittent contact Atomic Force Microscopy (IC-AFM) [4]. Scans were processed with Gwyddion Software (v2.67) and statistics were performed with RStudio. Results: At day 1, progressive motility did not differ significantly between the antibiotic-based extender and the antibiotic-free extender. After five days of storage, progressive motility was significantly higher in antibiotic-free extender (55.9±4.6%) compared to the antibiotic one (45.7±7.9%; p<0.05). Bacterial growth was comparable across both formulations at both time points (day 1: 1.0 vs 1.15 log₁₀CFU; day 5: 1.15 vs 1.38 log₁₀CFU). AFM analysis showed no significant differences in surface roughness at day 1. At day 5, sperm surface roughness significantly increased in the antibiotic formulation (11.40±1.62 nm), while remaining stable in the antibiotic-free extender (8.03±1.50 nm; p<0.001). Conclusion: The antibiotic-free semen extender supplemented with natural antimicrobial biomolecules effectively preserves sperm quality parameters and nanoscale surface properties, highlighting its potential as a sustainable alternative for artificial insemination in the swine industry.