Effects of cationic antimicrobial peptides on liquid-preserved boar spermatozoa

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.

Figure 1. Motion parameters of boar spermatozoa after 5 min of incubation with increasing concentrations (5 µM, 10 µM, 20 µM, and 40 µM) of c-WFW, c-WWW, and MK5E (n = 12).

Before the application of peptides, semen was preserved for 24°C in BTS containing 250 µg/mL gentamicin (standard). * Significant difference compared to standard (see text for the exact P-values).

Figure 2. Motion parameters and percentage of damaged boar spermatozoa during BTS preservation at 16°C in the presence of 250 µg/mL gentamicin (standard), 4 µM and 8 µM c-WFW, 2 µM and 4 µM c-WWW, and 1 µM and 2 µM MK5E (n = 9).

The percentage, the velocity (VAP), and the linearity (VSL/VCL) of progressive motile spermatozoa after 12 h and 96 h storage are shown (A, C, E). The percentage and the velocity (VAP) of progressive motile spermatozoa after 48 h storage are shown in a thermoresistance test (TRT, incubation at 38°C) for 30 and 300 min (B, D). The percentage of damaged spermatozoa was determined after 12 h, 48 h, and 96 h storage by staining with propidium iodide and/or markers for the acrosomal membrane and contents (F). Significant difference (see text for the exact P-values) compared to standard after 12 h (▪), 48 h (#), 96 h (▴) storage, and after TRT 30 min (○) and TRT 300 min (•) incubation at 38°C.