ANTIBIOTIC-FREE BOAR SEMEN EXTENDER FOR ARTIFICIAL INSEMINATION

Antibiotic-free boar semen extenders for artificial insemination represent an alternative strategy to mitigate bacterial contamination of semen doses while reducing the use of conventional antibiotics in the swine industry [1]. This approach has been investigated in response to the growing global concern over antimicrobial resistance, which represents a serious threat to both animal and human health [2]. The aim of this study was to compare the performance of a conventional antibiotic-based semen extender with an innovative antibiotic-free formulation supplemented with natural antimicrobial biomolecules. Raw ejaculates from three boars were diluted in (I) semen extender with conventional antibiotics and in (II) antibiotic-free semen extender. Semen doses were stored at 16 – 17 °C and semen quality was evaluated on days 1 and 5 of storage. Bacterial contamination was assessed on agar plates. Sperm motility was evaluated using CASA system (Microptic, Spain). Cell viability (Propidium Iodide) and functional parameters including acrosome integrity (FITC-PNA), mitochondrial activity (Mitotracker), and early apoptosis (YoPro-1) were acquired by flow cytometry. On day 1, total and progressive motility did not differ significantly between the extenders. After 5 days of storage, motility was significantly higher (p < 0.05) in the antibiotic-free extender (total: 96.3% ± 2.5, progressive: 55.9% ± 4.6) compared to the antibiotic extender (total: 85.2% ± 8.0, progressive: 45.7% ± 7.9). Flow cytometry analysis showed no significant differences in sperm viability, mitochondrial activity and early apoptosis between the formulations on both days of storage. The percentage of spermatozoa with acrosomal reaction was comparable on day 1 between the antibiotic-free (19.5% ± 8.2) and antibiotic-based extenders (22.2% ± 4.3), with an increase on day 5 in the antibiotic- free extender (30.1% ± 8.2 vs 22.0% ± 9.0). Bacterial growth was comparably controlled by the antibiotic-free and antibiotic extenders on both days (day 1: 1.15 log10 CFU ± 0.2 vs 1.0 ± 0.0 log10CFU; day 5: 1.38 log10CFU ± 0.54 vs 1.15 log10CFU ± 0.2 respectively). On day 1, only in ejaculate with a high initial bacterial load the antibiotic-free extender showed lower growth control (3.1 log10CFU vs. 1.0 log10CFU). Overall, these results suggest that the antibiotic-free semen extender supplemented with natural antimicrobial biomolecules effectively preserves sperm quality parameters and controls bacterial growth, highlighting its potential as a sustainable and applicable alternative for artificial insemination in the swine industry.