Effects of cationic antimicrobial peptides on liquid-preserved boar spermatozoa.

Martin Schulze,Stephanie Speck,Karin Mueller,Christof Junkes,Peter Mueller,Karin Ruediger,Margitta Dathe,Axel Cloeckaert

doi:10.1371/journal.pone.0100490

Abstract

Antibiotics are mandatory additives in semen extenders to control bacterial contamination. The worldwide increase in resistance to conventional antibiotics requires the search for alternatives not only for animal artificial insemination industries, but also for veterinary and human medicine. Cationic antimicrobial peptides are of interest as a novel class of antimicrobial additives for boar semen preservation. The present study investigated effects of two synthetic cyclic hexapeptides (c-WFW, c-WWW) and a synthetic helical magainin II amide derivative (MK5E) on boar sperm during semen storage at 16°C for 4 days. The standard extender, Beltsville Thawing Solution (BTS) containing 250 µg/mL gentamicin (standard), was compared to combinations of BTS with each of the peptides in a split-sample procedure. Examination revealed peptide- and concentration-dependent effects on sperm integrity and motility. Negative effects were more pronounced for MK5E than in hexapeptide-supplemented samples. The cyclic hexapeptides were partly able to stimulate a linear progressive sperm movement. When using low concentrations of cyclic hexapeptides (4 µM c-WFW, 2 µM c-WWW) sperm quality was comparable to the standard extender over the course of preservation. C-WFW-supplemented boar semen resulted in normal fertility rates after AI. In order to investigate the interaction of peptides with the membrane, electron spin resonance spectroscopic measurements were performed using spin-labeled lipids. C-WWW and c-WFW reversibly immobilized an analog of phosphatidylcholine (PC), whereas MK5E caused an irreversible increase of PC mobility. These results suggest testing the antimicrobial efficiency of non-toxic concentrations of selected cyclic hexapeptides as potential candidates to supplement/replace common antibiotics in semen preservation.

Highlights

Antimicrobial substances are necessary and mandatory for the liquid preservation of boar semen
The last decades have seen an intensification of efforts at producing antimicrobial peptides (AMPs) for application in clinical and veterinary settings [2]
Hydrophobic interactions allow the peptides to intrude into the hydrophobic region of the lipid bilayer and, in contrast to conventional antibiotics, to destabilize the bacterial cell membrane directly [5]

Summary

Introduction

Antimicrobial substances are necessary and mandatory for the liquid preservation of boar semen. In order to combat the increase in bacterial resistance, new agents for new targets have to be developed [1]. For this reason, the last decades have seen an intensification of efforts at producing antimicrobial peptides (AMPs) for application in clinical and veterinary settings [2]. The spatial separation of the cationic and hydrophobic constituents is an essential prerequisite for their effective interaction with bacterial membranes. This structural feature enables AMPs to interact with the lipids of asymmetrical bacterial membranes in detergence-similar way [4]. Hydrophobic interactions allow the peptides to intrude into the hydrophobic region of the lipid bilayer and, in contrast to conventional antibiotics, to destabilize the bacterial cell membrane directly [5]

Methods

Results

Discussion

Conclusion