Antimicrobial peptides and proteins as alternative antibiotics for porcine semen preservation

Abstract

Background: Antimicrobial resistance (AMR) is nowadays a major emerging challenge for public health worldwide. The over- and misuse of antibiotics, including those for cell culture, are promoting AMR while also encouraging the research and employment of alternative drugs. The addition of antibiotics to the cell media is strongly recommended in sperm preservation, being gentamicin the most used for boar semen. Because of its continued use, several bacterial strains present in boar semen have developed resistance to this antibiotic. Antimicrobial peptides and proteins (AMPPs) are promising candidates as alternative antibiotics because their mechanism of action is less likely to promote AMR. In the present study, we tested two AMPPs (lysozyme and nisin; 50 and 500 µg/mL) as possible substitutes of gentamicin for boar semen preservation up to 48 h of storage.

Results: We found that both AMPPs improved sperm plasma membrane and acrosome integrity during semen storage. The highest concentration tested for lysozyme also kept the remaining sperm parameters unaltered, at 48 h of semen storage, and reduced the bacterial load at comparable levels of the samples supplemented with gentamicin (p > 0.05). On the other hand, while nisin (500 µg/mL) reduced the total Enterobacteriaceae counts, it also decreased the rapid and progressive sperm population and the seminal oxidation-reduction potential (p < 0.05).

Conclusions: The protective effect of lysozyme on sperm function together with its antimicrobial activity and inborn presence in body fluids, including semen and cervical mucus, makes this enzyme a promising antimicrobial agent for boar semen preservation.

Keywords: Antimicrobial resistance; Boar semen; Gentamicin; Lysozyme; Nisin; Sperm function.

Fig. 1

Effect of lysozyme and nisin on sperm parameters during porcine semen storage at 17 ºC. (A) Sperm plasma membrane integrity; (B) Sperm acrosomal status; (C) Sperm mitochondrial activity; (D) Seminal oxidation-reduction potential. Different superscripts (lower case letters) indicate significant differences (p < 0.05) between treatments within each given time. Different superscripts (capital letters) indicate significant differences (p < 0.05) between the incubation times within each given treatment. Gent: Gentamicin; Ctr: Control; Lys: Lysozyme; Nis: Nisin; Treatments: Lys500 (500 µg/mL); Lys50 (50 µg/mL); Nis500 (500 µg/mL); Nis50 (50 µg/mL). The data are shown as the mean ± standard error of four replicates

Fig. 2

Effect of lysozyme and nisin on microbiological analysis of porcine semen during storage at 17 ºC. (A) Total bacterial count; (B) Total Enterobacteriaceae count. Different superscript letters indicate significant differences (p < 0.05) between treatments within each given time. There were not significant differences between the incubation times within each given treatment (p > 0.05). Gent: Gentamicin; Ctr: Control; Lys: Lysozyme; Nis: Nisin; Treatments: Lys500 (500 µg/mL); Lys50 (50 µg/mL); Nis500 (500 µg/mL); Nis50 (50 µg/mL). The data are shown as the mean ± standard error of four replicates

Fig. 3

Bacterial growth in different culture media. BTS: Beltsville Thawing Solution; CFU: Colony Forming Unit; Lys500: Lysozyme 500 µg/mL; MH: Mueller-Hinton Broth; Nis500: Nisin 500 µg/mL; tntc: Too Numerous to Count. *: <200 CFU/mL