Nisin and lysozyme as antibiotic alternatives in an extender for chilled canine semen

Chilling is an effective method for the short-term preservation of fertilization potential in male gametes. Because bacteriospermia is a common phenomenon, antibiotics are routinely added to extenders to ensure microbiological safety, particularly for insemination doses used in international trade. However, increasing bacterial resistance in veterinary medicine is driving the search for alternative additives for semen preservation. This study evaluated the bacteriostatic activity of nisin with ethylenediaminetetraacetic acid (EDTA) and lysozyme and their effects on canine sperm characteristics during storage at 4 °C for 48 and 96 h. One ejaculate from each of fifteen animals was diluted in Tris-citrate-fructose-egg yolk (TCF-EY) extender, and each ejaculate was divided into three groups: control-Antb (0.06 g/100 mL benzylpenicillin + 0.1 g/100 mL streptomycin), Nis (50 µg/mL nisin + 1.25 µg/mL EDTA), and Lys (500 µg/mL lysozyme). Fresh and chilled samples were assessed for sperm motility, concentration, membrane integrity, mitochondrial activity, ROS levels, and DNA fragmentation. Then, microbiological analyses were conducted, included plating, identification, and antibiotic susceptibility testing. No differences in sperm parameters were observed between the groups at either time point. In ejaculates with bacteriospermia ≤ 3 × 10⁶ CFU/mL, nisin and lysozyme demonstrated bactericidal effects comparable to those of antibiotics. Forty percent of the fresh samples had no bacterial growth, whereas some isolates were antibiotic-resistant. These results indicate that both nisin with EDTA and lysozyme exhibit promising bacteriostatic activity in chilled canine semen without compromising sperm characteristics.