Use of a synthetic antimicrobial peptide (PA-13) alone or in combination with reduced gentamicin levels to control Escherichia coli contamination in stored boar semen

Abstract

Background and Aim: Bacterial contamination during liquid storage of boar semen negatively affects sperm quality and fertility outcomes, necessitating the routine use of antibiotics in semen extenders. However, increasing concerns regarding antimicrobial resistance have encouraged the development of alternative antimicrobial strategies. Synthetic antimicrobial peptides (AMPs) have demonstrated broad-spectrum antibacterial activity and may serve as potential substitutes or adjuncts to conventional antibiotics. This study aimed to evaluate the antimicrobial efficacy of the synthetic peptide PA-13 alone and in combination with reduced gentamicin concentrations against Escherichia coli isolated from boar semen, as well as its effects on semen quality during storage at 18°C. Materials and Methods: Two experiments were conducted. In Experiment I, fresh semen samples collected from seven healthy adult boars were diluted in Beltsville Thawing Solution supplemented with gentamicin (200 µg/mL, positive control), without antibiotics (negative control), or PA-13 at concentrations of 62.5, 31.25, and 15.625 µg/mL. Total bacterial counts were measured at 0, 24, 48, and 72 h of storage, while semen quality parameters, including sperm motility, viability, acrosomal integrity, and mitochondrial membrane potential, were evaluated on days 1, 3, and 5. In Experiment II, isolated E. coli was incubated with PA-13 alone or in combination with varying gentamicin concentrations, and bacterial growth was monitored over 24 h using optical density measurements. Results: PA-13 effectively inhibited bacterial proliferation in extended semen during the first 24 h of storage, with lower concentrations (15.625 and 31.25 µg/mL) showing greater antibacterial activity than the higher concentration (62.5 µg/mL). Semen quality parameters were comparable among groups on day 1; however, prolonged storage demonstrated that the highest PA-13 concentration negatively affected sperm motility and viability. Lower PA-13 concentrations preserved semen quality similar to that of the gentamicin-treated control. In Experiment II, combination treatments exhibited synergistic effects, with PA-13 at 3.906 µg/mL combined with gentamicin at 100 µg/mL inhibiting E. coli growth equivalently to gentamicin at 200 µg/mL alone. Conclusion: PA-13 effectively controls bacterial contamination in stored boar semen while maintaining semen quality at appropriate concentrations. Its combination with gentamicin enables a substantial reduction in antibiotic dosage without compromising antibacterial efficacy. These findings support the use of AMPs as alternative or complementary antimicrobial agents in semen extenders to reduce antibiotic use in the swine industry.