Skip to main content
Download PDF

Antimicrobial peptides and proteins as alternative antibiotics for porcine semen preservation

BMC Veterinary Research volume 20, Article number: 257 (2024) Cite this article

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.

Peer Review reports

Introduction

Antimicrobial resistance (AMR) is nowadays one of the main global health threats that increases the risk of disease spread, severe illness, and death. In bacteria, where AMR naturally occurs, the misuse or overuse of antibiotics is accelerating the process jeopardizing the success of modern medicine in treating infections [1].

Sperm preservation also promotes the emergence of superbugs resistant to the most common antibiotics used for this purpose [2]. Pig breeding is mainly carried out by artificial insemination (AI) using refrigerated diluted semen and entails the collection, processing, and preservation of male gametes. Even with the highest hygienic standards, bacterial contamination frequently occurs during the semen collection and handling process [3]. In addition, the liquid-storage of boar semen at 15–17 ºC also favours microbes’ proliferation. Until very recently, all these steps make the addition of antibiotics to the semen samples a must. However, AMR to the most common antibiotics added to the semen extenders promotes the contamination of the seminal doses ranging from 14.73 to 32% [4, 5] where Gram-negative bacteria are predominant. On the other hand, most antibiotics exert negative effects per se (cytotoxicity) on sperm physiology [6, 7] and promote the release of bacterial endotoxins, such as lipopolysaccharide (LPS), which seriously compromises sperm function [8, 9]. The increasing evidence and derived problems of AMR worldwide have conducted the European Union (EU) to amend the regulations about the collection and processing of semen (Annex III (EU) 2020/686) towards the voluntary, more flexible, and prudent use of antibiotics (Regulation (EU) 2023/647 [10, 11]).

The use of alternative antimicrobial agents that delay or avoid AMR is therefore urgently needed. This kind of compounds includes antimicrobial peptides and proteins (AMPPs), which are a diverse group of molecules produced by a wide variety of organisms (prokaryotes and eukaryotes) as their first line of defence against pathogenic microorganisms [12]. While the great part of these AMPPs can directly kill a wide variety of microbial pathogens (e.g., bacteria, yeasts, fungi, viruses, etc.), others modulate the host immunity [13, 14]. However, many AMPPs have a limited spectrum of activity and are effective only at very high concentrations [15] and thus, increasing their cytotoxicity if any. In spite of this, through the application of different stimuli (e.g., heat treatment) and combination with other compounds (e.g., chelators, dextran), many AMPPs can broaden their effectiveness of killing/inhibiting both Gram-positive (G+) and Gram-negative (G-) bacteria and reduce the dosage needed for that purpose [15, 16].

Nisin, a polypeptide bacteriocin produced by Lactococcus lactis subsp. lactis, is widely used (over 50 countries) as a food preservative (E-234) of meat and dairy products and a promising compound for biomedical applications such as alternative antimicrobial and cancer therapeutic [17, 18]. On the other hand, lysozyme or muramidase was the first discovered antimicrobial protein (enzyme) by Alexander Fleming [19]. It is typically found in body fluids (e.g., saliva, tears, milk, semen, cervical mucus), organs, tissues, and cells (e.g., polymorphonuclear leukocytes) from many organisms mainly acting as an inborn immunological defence against a wide variety of pathogens [20,21,22]. The main antibacterial spectrum of both nisin and lysozyme is G+ bacteria, but, in the presence of chelating agents, like ethylenediaminetetraacetic acid (EDTA), they are also effective against G- bacteria [15]. The addition of EDTA to the semen extenders is a common praxis to block the action of calcium as a mediator of sperm capacitation and acrosome reaction [23], while it could be also harnessed to increase the antimicrobial spectrum of AMPPs. For this reason, semen extenders containing chelating agents such as EDTA could be suitable in the fight against AMR using alternative antibiotics for semen preservation.

The main goal of this study was to evaluate the potential use of nisin and lysozyme as alternative antibiotics for the preservation of boar semen at 17 ºC. For this purpose, we used Beltsville Thawing Solution (BTS) as semen extender that contains EDTA and sodium bicarbonate which have shown their own antimicrobial activity [24, 25] and broaden the spectrum of these AMPPs also to G- bacteria. This short-term extender allows the preservation of sperm cells for 1–3 days, although after 48 h of storage, there is a decline in the fertility rates and number of piglets born [23]. For this reason and because 85% of the AIs are carried out within the first two days after semen collection [26], the analyses of the present study were performed till 48 h of semen storage. The presence of lysozyme in several mammalian body fluids and the approval of nisin as a safe food preservative make these AMPPs suitable alternative antibiotics for boar semen preservation.

Material and methods

All reagents were purchased from Merck (Darmstadt, Germany), unless otherwise indicated.

Semen collection and processing

Semen was collected by the gloved hand method from fertile boars (Duroc breed) at a pig breeding company (Lipra Pork, a.s., Czech Republic). The semen from 12 boars was used in this study. Twenty mL of each ejaculate was transported to the laboratory in sterilised tubes. An aliquot of each sample was placed in 0.3% formaldehyde in phosphate-buffered saline (PBS) for assessing sperm abnormalities (200 sperm evaluated per sample). The ejaculates with more than 25% of sperm abnormalities were discarded for the experiments. Then, all ejaculates were supplemented with 5 mL of BTS (D-glucose 37 g/L, sodium citrate 6 g/L, EDTA 1.25 g/L, sodium bicarbonate 1.25 g/L, potassium chloride 0.75 g/L) without antibiotic. The BTS’ pH (adjusted with NaOH 10 M) and osmolality were ~ 7.2 (Five Easy F20, Mettler-Toledo, Greifensee, Switzerland) and ~ 330 mOsm/kg H2O (Osmomat 3000, Gonotec, Berlin, Germany), respectively. The BTS was prepared, under sterile conditions, in water (Carl Roth, Karlsruhe, Germany) and filtered (0.2 μm filter pore, Whatman plc, Buckinghamshire, United Kingdom) after preparation. For each experimental replicate, the semen from three boars was pooled and centrifuged at 167 × g for 3 min at 17 °C to remove debris and abnormal cells [27]. A sub-sample of the supernatant (fixed in 0.3% formaldehyde in PBS) was collected for the assessment of sperm concentration in a Bürker chamber. The pooled semen was then diluted to 40 × 106 spermatozoa/mL in BTS extender w/o antibiotic. Four replicates were used in the present study. The initial sperm motility in all replicates was > 75%.

Treatments

A stock solution (2 mg/mL) of both nisin (N5764; ≥900 IU/mg) and lysozyme (1052810001; from egg white; ≥30,000 FIP-U/mg) was freshly prepared in BTS. Then, the solutions were filtered by a syringe filter (0.2 μm pore; Whatman plc, Buckinghamshire, United Kingdom) and placed in sterile tubes. The diluted semen was split into 6 sterile tubes (diluted down 20 × 106 spermatozoa/mL in BTS w/ or w/o gentamicin or AMPPs) to reach the final concentrations as follows: Ctr (BTS w/o antibiotic), Gent (gentamicin, 250 µg/mL), Lys500 (lysozyme, 500 µg/mL), Lys50 (lysozyme, 50 µg/mL), Nis500 (nisin, 500 µg/mL), and Nis50 (nisin, 50 µg/mL). The choice of the used AMPPs concentrations was based on their antimicrobial activity in combination with EDTA as previously reported [15].

Sperm motility and kinetic parameters

A sperm aliquot (2 µL) was loaded into a pre-warmed Leja chamber (Leja Products BV, Nieuw-Vennep, The Netherlands, chamber depth: 20 μm). The sperm motility and kinetic parameters were evaluated, as previously reported [28], using a Computer Assisted Sperm Analysis (CASA) (NIS-Elements, Nikon, Tokyo, Japan and Laboratory Imaging, Prague, Czech Republic), which consists of an Eclipse E600 tri-ocular phase contrast microscope (Nikon, Tokyo, Japan), equipped with a warming stage set at 38 ºC (Tokai Hit, Shizuoka, Japan), and a DMK 23UM021 digital camera (The Imaging Source, Bremen, Germany). The analysis was carried out using a 10× negative phase-contrast objective (Nikon, Tokyo, Japan). A total of nine descriptors of sperm motility parameters were determined: total motility (TM, %), progressive motility (PM, %), average path velocity (VAP, µm/s), curvilinear velocity (VCL, µm/s), straight-line velocity (VSL, µm/s), amplitude of lateral head displacement (ALH, µm), beat-cross frequency (BCF, Hz), linearity (LIN, %), and straightness (STR, %). The standard CASA settings were as follows: frames per second, 60; minimum of frames acquired, 31; number of fields analysed, 6; VAP ≥ 10 μm/s to classify a spermatozoon as motile; STR ≥ 80% to classify a spermatozoon as progressive. A minimum of 200 motile sperm cells were analysed per sample. All videos were visually checked by the same researcher to remove debris or erroneously crossed sperm tracks. Sperm motile subpopulations were determined on the whole sperm population by cluster analysis.

Sperm plasma membrane integrity, acrosomal status, and mitochondrial activity

Sperm analyses were carried out as previously described [29]. Briefly, for the assessment of membrane integrity, the sperm samples were incubated with propidium iodide (stock solution: 0.5 mg/mL in phosphate-buffered saline, PBS), carboxyfluorescein diacetate (stock solution: 0.46 mg/mL in dimethyl sulfoxide, DMSO), and formaldehyde solution (0.3%) for 10 min at 38 ºC in the dark. Then, the spermatozoa were assessed under epi-fluorescence microscopy (Nikon Eclipse E600, Nikon, Japan; 40× objective), and those with green fluorescence over the entire head were considered to have an intact plasma membrane. For the acrosomal status, the percentage of sperm with a normal apical ridge (NAR) was determined. The sperm samples were fixed in a glutaraldehyde solution (2%) and evaluated under phase contrast microscopy (40× objective). To determine mitochondrial activity, the aliquots of the sperm samples were incubated with rhodamine 123 (5 mg/mL in DMSO) and propidium iodide (0.5 mg/mL in PBS) for 15 min at 38 ºC in the dark. After that, the samples were centrifuged at 500 × g for 5 min, the supernatant was removed, and the sperm pellet was resuspended in PBS. Then, the spermatozoa were evaluated using epi-fluorescence microscopy (40× objective), and the spermatozoa showing a bright green fluorescence over the midpiece were considered to have a high mitochondrial activity. Two-hundred sperm cells were assessed per analysis by the same observer.

Seminal oxidation-reduction potential (ORP)

The seminal ORP of the samples were determined as previously reported [28] with minor modifications. At the end of sperm incubation, a sample from each treatment was centrifuged at 16,300 × g for 5 min at room temperature. Then, 800 µL of the supernatant were transferred into a microcentrifuge tube and incubated at 38 °C. The ORP was measured using a micro ORP electrode with Argenthal™ reference system and platinum ring (InLab® Redox Micro, Mettler-Toledo, Greifensee, Switzerland) connected to a pH/mV meter (Five Easy F20, Mettler-Toledo, Greifensee, Switzerland). The ORP of each sample was recorded after embedding the microelectrode into the solution for 3 min. After each sample analysis, the probe was calibrated into a redox buffer solution (220 mV, pH 7, Mettler-Toledo, Greifensee, Switzerland) for 30 s. The assay was run in duplicate per each sample and expressed in millivolts (mV). The ORP levels were not normalized [30], because the experiments were performed at the same sperm concentration (i.e., 20 × 106/mL).

Isolation of contaminating bacteria and MALDI-TOF MS identification

Pseudomonas (PA) agar (Merck, Darmstadt, Germany), Blood (BA), MacConkey (MCA), Mannitol Salt (MSA), Plate count (PCA) agars (Oxoid, Basigstoke, United Kingdom) were used for the isolation of contaminating bacteria. Sample aliquots of 50 µl were plated in duplicate on 90 mm agar plates using spiral plate inoculator EasySpiral (Interscience, Saint Nom, France) and incubated aerobically at 37 °C for 24–48 h. Selected colonies with different morphology were further repassaged to ensure a pure culture.

Freshly grown colonies were harvested and subjected to the standard procedure recommended by Bruker Daltonics for the MALDI-TOF MS identification (ethanol-formic acid extraction procedure and then mixed with HCCA matrix). Protein spectra were measured and processed by Autoflex Speed MALDI-TOF MS using FlexControl 3.4; MALDI Biotyper Compass version 4.1; and flexAnalysis version 3.4 software (Bruker Daltonics, Bremen, Germany).

Bacterial counts determination

The bacterial contamination rates in pooled boar ejaculates in the BTS extender were determined the first day following the insemination doses preparation (Ctr), and then after 24 and 48 h of semen storage at 17 °C for all treatments. The PCA and MCA agars were used for the enumeration of total aerobic mesophilic bacteria and Enterobacteriaceae, respectively. Sample aliquots of 100 µl were plated in duplicate using a spread plate technique. Samples diluted 10-fold in sterile peptone saline were plated in duplicate using a spiral plate inoculator with a 10− 5 dilution rate. The inoculated plates were incubated for 24–48 h at 37 °C. The microbial counts obtained by the spiral plate technique were interpreted according to the NF V08-100 Standard [31]. The final counts were expressed as log10 CFU/mL (CFU: colony forming unit) of an insemination dose. Because of the volume used for the initial microbial culture (100 µl; undiluted samples), a value of 0 in the bacterial counts is equivalent to < 10 CFU/mL.

Bacterial growth in Mueller-Hinton (MH) broth, BTS extender, and BTS extender supplemented with AMPPs

The bacterial genera most frequently isolated from the semen samples (see in the Results section) were selected to assess their sensitivity to AMPPs (lysozyme and nisin) in BTS. A modified broth microdilution method [32] was used for the sensitivity testing. Both AMPPs were serially two-fold diluted in BTS (concentration range 7.8 to 1,000 µg/mL) and inoculated with standardised bacterial suspension prepared from fresh overnight culture to achieve the concentration of 5 × 104 CFU/mL in the inoculated 96-well microtiter plates (the inoculum concentration was established based on the maximum contamination rates commonly detected in boar semen samples [33]). The plates were then incubated at 37 °C for 48 h. The minimum inhibitory concentrations were evaluated after 24 and 48 h as the lowest concentrations preventing bacterial growth. Cultures in Mueller-Hinton broth (Oxoid, Basingstoke, United Kingdom) as well as in BTS alone were used as positive growth controls. All concentrations were tested in triplicate.

Since all the tested AMPPs concentrations including BTS alone were shown to be inhibitory (no observable growth), bacterial counts in the wells containing 500 µg/mL of lysozyme or nisin were evaluated after 48 h and compared to controls. Aliquots of 40 µL were transferred from each replicate into one microtube, diluted 1:10 and plated on PCA plates in duplicate using a spiral plate inoculator. The plates were incubated at 37 °C for 24 h and the total counts were evaluated using an automatic colony counter Premium 90 h (VWR, Radnor, United States). The final counts were expressed as log10 CFU/mL and the detection limit was 2.3 log10 CFU/mL (200 CFU/mL).

Statistical analyses

The statistical analyses were carried out by the SPSS 24 statistical software package (IBM Inc, Chicago, IL, USA). To determine sperm motile subpopulations, a two-step cluster analysis was applied to the whole sperm population using VAP and STR as variables. The number of clusters was automatically determined using the Euclidean distance measure and the Schwarz’s Bayesian criterion. After that, the number of clusters previously obtained was used to set up the K-means cluster analysis by using the iteration and classification method. The Kruskal-Wallis test was used to check for differences between sperm motile subpopulations in kinetic variables. A generalized linear model (GZLM) was used to analyse the effects of time and treatment on the sperm variables and bacterial counts. The repeated measures analysis (Wilcoxon signed-rank test and Friedman test) was also conducted to check for differences in all parameters within each treatment during the semen storage period. The data concerning the bacterial load were log10-transformed to perform the analyses. The data are expressed as the mean ± standard error. The statistical significance was set at p < 0.05.

Results

Sperm motility and kinetic parameters

The average values of motility and kinetics of boar spermatozoa during semen storage are shown in Table 1. At 24 h, there were no significant differences between Gent and Ctr groups in the total motility (p > 0.05). Interestingly, Lys50 and Nis50 showed greater total motility than the Gent group (p < 0.05). On the other hand, a significant (p < 0.05) decrease in progressive motility was observed in Nis treatments in comparison with Gent group. Overall, most kinetic parameters (VAP, VCL, VSL, and ALH) of the Gent group were higher (p < 0.05) than those of other treatments (Ctr included). On the other hand, Lys and Nis treatments (500 µg/mL) showed higher values of VSL (Lys only), BCF (both treatments), and LIN (Lys only) than the Ctr group (p < 0.05).

Table 1 Effect of lysozyme and nisin on sperm motility and kinetic parameters during porcine semen storage at 17 ºC
Full size table

At 48 h, Lys treatments did not differ (p > 0.05) in any of the motility and kinetic parameters when compared with the Gent group. By contrast, Ctr and Nis treatments showed lower values than the Gent group in several kinetic parameters (p < 0.05). Similarly to the results observed at 24 h, Lys500 showed higher values of VSL, BCF, and LIN than the Ctr group (p < 0.05).

The motility subpopulation analyses rendered three groups (Table 2) as follows: SP1 (rapid and progressive spermatozoa), SP2 (rapid and no progressive spermatozoa), and SP3 (slow and no progressive spermatozoa). The percentage of each subpopulation for every treatment and during semen storage is shown in Table 3. Interestingly, there were no significant differences (p > 0.05) between Gent and Lys treatments in any of the sperm subpopulations during semen storage. In addition, Lys treatments showed more rapid and progressive sperm (SP1) than the Ctr group (p < 0.05). On the other hand, Ctr and Nis treatments showed a higher percentage of non-progressive spermatozoa (SP2 and SP3) when compared to the Gent and Lys treatments (p < 0.05).

Table 2 Sperm subpopulations based on kinetic parameters during porcine semen storage at 17 ºC
Full size table
Table 3 Effect of lysozyme and nisin on sperm subpopulations based on kinetic parameters during porcine semen storage at 17 ºC
Full size table

Sperm plasma membrane integrity, acrosomal status, and mitochondrial activity

The results are shown in Fig. 1A-C. At 24 h, the percentage of sperm with intact plasma membrane did not differ between treatments (p > 0.05). At the same incubation time, there were no differences between Gent and Ctr groups in the acrosomal status (p > 0.05). Interestingly, Lys500 better preserved the acrosome integrity (p < 0.05) when compared to Gent, while the remaining Lys and Nis treatments did not show any significant differences (p > 0.05) with the Gent group. Both Lys500 and Nis treatments showed a higher percentage of sperm with intact acrosome than the Ctr group (p < 0.01). The mitochondrial activity did not show any significant difference between treatments (p > 0.05).

Fig. 1
figure 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

Full size image

At 48 h, all Lys and Nis treatments showed a higher percentage of sperm with an intact plasma membrane when compared to the Gent group (p ≤ 0.01), whereas there were no differences between the Gent and Ctr groups (p > 0.05). Regarding the acrosomal status, both Gent and Ctr groups showed similar results (p > 0.05), while all Lys and Nis treatments better preserved the integrity of this organelle in comparison with the Ctr group (p ≤ 0.02). Moreover, Nis500 better preserved the acrosome integrity than Gent (p < 0.05). No significant differences were detected between groups in the mitochondrial activity (p > 0.05).

Seminal oxidation-reduction potential (ORP)

The seminal ORP was significantly lower in Nis500 treatment than in other groups (Fig. 1D; p ≤ 0.001) at both incubation times. The remaining groups did not show any significant differences between them at any incubation period (p > 0.05).

Bacteriological profile

The bacteriological profile of boar semen samples is shown in Table 4. A total of 17 species belonging to 11 bacterial genera were identified. The G- bacteria were the most prevalent contaminants in terms of frequency and the number of isolated genera (7/11). Thus, Pseudomonas aeruginosa (100%), Stenotrophomonas maltophilia (75%), and Klebsiella aerogenes (50%) were the most frequent isolated species. On the other hand, Staphylococcus spp. were present in all the replicates as the most recurrent G+ bacteria with a total of six species isolated.

Table 4 Bacteriological profile of diluted porcine semen samples
Full size table

Total bacterial and Enterobacteriaceae counts in the semen samples (TBC and TEC)

The data relative to bacterial load of the samples are shown in Fig. 2. We did not observe any bacterial growth (TBC and TEC) in the Gent group during semen storage.

Fig. 2
figure 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

Full size image

At 24 h of semen storage, the TBC were higher in Ctr and Nis treatments (p < 0.05) than in the Gent treatment, while the latter did not differ from both Lys concentrations (p > 0.05). At the same time, there were no significant differences in TEC between treatments (p > 0.05).

At 48 h, only Lys500 did not differ from the Gent group in the TBC (p > 0.05). Interestingly, there were no significant differences in TEC between the Gent group and Lys and Nis treatments at the highest concentration (500 µg/mL; p > 0.05).

Bacterial growth in Mueller-Hinton (MH) broth, BTS extender, and BTS extender supplemented with AMPPs

Bacterial counts of selected strains of K. aerogenes, P. aeruginosa, S. epidermidis and S. maltophilia in MH broth, BTS alone, and AMPPs-supplemented BTS are shown in Fig. 3. Although there was no observable microbial growth in BTS alone, the enumeration of bacterial counts revealed an increase in the counts of K. aerogenes, compared to initial inoculum (approximately 16 times higher), whereas a strong decrease was observed in the case of all other bacteria tested. On the contrary, BTS supplemented with lysozyme (500 µg/mL) caused over 99% decrease in the counts of all bacteria tested. The nisin-supplemented (500 µg/mL) BTS strongly reduced the numbers of all bacteria except K. aerogenes where the effect was comparable to BTS alone.

Fig. 3
figure 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

Full size image

Discussion

Our findings provide empirical evidence that both lysozyme and nisin enhance sperm parameters and reduce bacterial load during semen storage. Both AMPPs showed a higher percentage of motile sperm (at 24 h) and better-preserved sperm plasmalemma and acrosome integrity (24 h and 48 h) when compared to samples exposed to gentamicin. Moreover, lysozyme at 500 µg/mL did not show significant differences in the bacterial load (24 h and 48 h) and the percentage of sperm with rapid and progressive motility (SP1) compared to gentamicin treatment. Furthermore, this treatment (Lys500) reduced over 99% the bacterial counts (K. aerogenes, P. aeruginosa, S. epidermidis, and S. maltophilia) from the initial bacterial inoculum (5 × 104 CFU/mL). On the other hand, nisin at 500 µg/mL reduced the total number of Enterobacteriaceae but also decreased the percentage of sperm belonging to SP1 in comparison with the gentamicin group. The absence of toxicity of lysozyme to the sperm cells and its presence in the reproductive fluids of numerous animal species make this enzyme a suitable alternative to the common antibiotics used for boar semen preservation.

The use of AMPPs as alternative antibiotics is a promising approach for semen preservation as they are less likely to promote bacterial resistance because of their mechanism of action [34]. However, there are still challenges to cover such as their limited spectrum of antibacterial activity, noxious effects on sperm function, and their expensive and laborious production [34, 35]. Some AMPPs have been previously tested in boar semen showing a significant decrease in the bacterial load but also some toxicity to the sperm cells [36,37,38]. In the present study, lysozyme (500 µg/mL) kept the bacterial load at comparable levels to the samples treated with gentamicin without compromising the sperm function and even better preserving sperm acrosome and membrane integrity. In relation to sperm quality, the standard indicators established by breeding organizations worldwide for using preserved boar semen for AI are: 50–70% motile sperm and a bacterial load of < 1,000 CFU/mL [39]. Lysozyme at the highest concentration tested kept these parameters within the optimal range at 48 h of semen storage, with an averaged sperm motility of > 65% and a bacterial load of < 60 CFU/mL and 5 CFU/mL for total bacteria and Enterobacteriaceae counts, respectively. Nevertheless, the threshold on bacterial load in semen doses for AI is still widely debated. It is also important to bear in mind that the different microbes usually detected in boar semen have different toxicity to the sperm cells. For instance, bacteria such as Alcaligenes spp., Actinomyces spp., Streptococcus spp., and Staphylococcus spp. have almost no effects on sperm survival even in the presence of 1010–1012 CFU/mL; on the other hand, members of Enterobacteriaceae (i.e., Escherichia coli, Citrobacter spp., Klebsiella spp., and Serratia spp.) together with Proteus spp. and Pseudomonas spp. have been classified as the most harmful bacteria to spermatozoa [5, 33]. For instance, enteric bacteria like E. coli and Klebsiella spp. can drop the seminal pH to 5.2–5.7 that results in a drastic decrease of sperm motility and acrosome integrity [33]. Even though using antibiotics, up to 32% of the semen doses are contaminated with several bacterial genera mainly because of AMR [4]. In this regard, Úbeda et al. [5] in a quality control of boar seminal doses (supplemented with antibiotics) established an above cut-off of 3 × 102 CFU/mL for considering a semen sample as positive in bacterial contamination. On the other hand, some studies focusing on boar bacteriospermia [40] reported negative effects (litter size) when using semen for AI with more than 3.5 × 103 CFU/mL (E. coli). According to these cut-offs for bacteriospermia, our findings show that Lys500 (in all replicates) is below the range that considers a sample as positive for bacterial contamination or the one that compromises sperm function and fertility outcomes. Although nisin treatments and Lys50 enhanced some sperm parameters when compared with Gent and Ctr groups, they had a TBC higher than the recommended range worldwide (< 1,000 CFU/ml) and they would be considered as positive for bacterial contamination (> 300 CFU/mL; [5]).

The bacterial profile in boar semen closely depends on the hygienic conditions during the sample collection, the season, and the environmental characteristics where the animals are raised [41, 42]. Thus, raw boar semen is usually contaminated with one or more bacterial species and can result in the presence of aerobic bacteria in 99% of the ejaculates [43,44,45,46]. In our work and like other studies [4], the most frequently isolated bacteria were P. aeruginosa (G-), S. maltophilia (G-), Klebsiella spp. (G-), and Staphylococcus spp. (G+). According to the abundance of Staphylococcus spp. observed in our study, it was reported that the presence of lysozyme in boar semen (2.4 µg/mL) might have been related to a bactericidal effect especially against S. aureus [47]. This finding, together with the role of lysozyme in the innate immunity and the great sperm-tolerance to this compound at high concentrations, indicates the suitability of this enzyme as an antimicrobial agent for boar semen preservation. The antimicrobial spectrum of lysozyme and nisin mainly includes G+ bacteria but in combination with chelators (e.g., EDTA) they can broaden their activity against G-, as shown in the present study. Semen extenders containing EDTA, such as the BTS, have been recently defined as “antimicrobially active extenders” as they allow to reduce the amount of antibiotic needed and act themselves as bacteriostatic in the absence of other antimicrobial agents [48], as also confirmed by our study. Our results support these previous findings as both lysozyme and nisin (500 µg/mL) reduced the bacterial counts (Enterobacteriaceae) from 5.6 × 104 CFU/mL (Ctr) to < 14 CFU/mL (lysozyme: 5 CFU/mL; nisin: 13.75 CFU/mL). Both lysozyme and nisin have also shown the ability to reduce the endotoxic activity of LPS [49, 50], which is released by G- bacteria under antibiotic treatments (bacteriolysis) and negatively affects sperm quality. Gentamicin, on the other hand, cannot reduce the toxicity of LPS to the sperm cells at least at the concentration commonly used (250 µg/mL) for boar semen storage [35]. However, the combination of AMPPs with the common antibiotics used for sperm preservation neutralizes this bacteria-released endotoxin and increases sperm quality during semen storage [51]. The improved preservation of sperm membrane and acrosome integrity during semen storage, both for Lys and Nis treatments, could be therefore related to the capacity of these AMPPs to neutralise the detrimental effects of LPS on sperm function.

The presence of lysozyme in the semen of a wide range of invertebrate and vertebrate species is well known [52, 53]. In the seminal plasma, the abundance of this enzyme has been associated with good sperm quality [54, 55]. By contrast, human patients with chronic prostatitis have lower concentration of lysozyme than healthy men [56]. In spermatozoa, a lysozyme c-like protein (SLLP1) is located in the acrosome and involved in the fertilization process [57]. Similarly, the presence of a seminal vesicle-secreted lysozyme c-like protein (SVLLP) has been reported in mice. This protein binds to spermatozoa and suppresses bovine serum albumin-induced sperm capacitation and inhibits acrosome reaction [58]. The enhanced sperm quality associated with an increased amount of lysozyme could be related not only to its antimicrobial properties but also to its ameliorative effect against oxidative stress. For instance, the oxidative damage caused to the sperm cells by the advanced glycation end-products (AGE), which is promoted by extenders containing high glucose concentration [59, 60], is cushioned by lysozyme activity [61]. It seems also plausible that this enzyme has no cytotoxic effects on sperm cells as gentamicin has [6, 7] because of its physiological presence in several body fluids including semen. On the other hand, nisin has shown spermicidal action (fast inhibition of sperm motility) in several mammalian species, including humans, in a range of concentrations from 50 to 400 µg/mL [62]. In our study, we did not observe such phenomena as we even found a significant enhancement in some sperm parameters (i.e., sperm motility − 24 h- and acrosome/membrane integrity) compared to the gentamicin group. These differences could be attributed to nisin preparation (purification vs. direct dilution in BTS), a high tolerance to this peptide in the porcine species, and reduced drug potency because of the presence of seminal plasma [62]. The enhancement of some sperm parameters by nisin treatments found in our study may be related to the recently reported antioxidant properties of this AMPP [63, 64]. This explanation is supported by the lower ORP values found at the highest concentration of nisin in the present study. However, in comparison to the gentamicin treatment, we also observed a decrease in some sperm velocity parameters and in the percentage of rapid and progressive spermatozoa (SP1), which might be explained by the impaired redox status [65] found in the samples treated with this AMPP. The bacterial load in nisin treatments (~ 1,700-3,000 CFU/mL) on the second day could have also influenced the drop observed in kinetic parameters as the decline in sperm parameters due to bacterial contamination is more evident at 48 h of semen storage [66, 67].

Even though we found a significant reduction in bacterial load and growth inhibition of the most frequently isolated bacterial genera, we cannot ensure the same effectiveness of lysozyme in case of different bacterial species and/or higher rates of contamination than the ones detected in the present study. To prevent or reduce bacterial contamination, in addition to EU regulation, the World Organisation for Animal Health (WOAH) legislation urges to follow strict hygiene measures during semen collection, processing, and storage (Annex 7; Chap. 4.6 [68]). The achievement of high hygienic standards together with the use of alternative compounds (like AMPPs) or methods (e.g., colloid centrifugation [69]) to reduce the bacterial load can avoid the use of antibiotics in AI doses.

Conclusions

Lysozyme (500 µg/mL) significantly reduces the bacterial load at comparable levels of samples treated with gentamicin (250 µg/mL). In addition, the sperm parameters (motility subpopulations, mitochondrial activity and, redox status) were unaltered or even better preserved (acrosome − 24 h- and membrane integrity − 48 h-) than in the gentamicin group. The presence of this enzyme in several body fluids (including semen and cervical mucus) and its sperm tolerance even at high concentrations, makes lysozyme an interesting alternative antimicrobial agent for boar semen preservation. Even though the bacterial load was low (< 60 CFU/mL), our next steps are directed towards finding a natural compound that offers synergy against a broader spectrum of bacteria and the assessment of sperm fertilizing ability, both in vitro and in vivo, treated with this enzyme.

Data availability

Data are provided within the manuscript or supplementary information files.

References

  1. World Health Organization (WHO). Antimicrobial resistance. https://www.who.int/news-room/fact-sheets/detail/antimicrobial-resistance. Accessed March 2023.

  2. Bresciani C, Cabassi CS, Morini G, Taddei S, Bettini R, Bigliardi E, et al. Boar semen bacterial contamination in Italy and antibiotic efficacy in a modified extender. Ital J Anim Sci. 2014;13:83–7.

    Article  CAS  Google Scholar 

  3. Althouse GC, Pierdon MS, Lu KG. Thermotemporal dynamics of contaminant bacteria and antimicrobials in extended porcine semen. Theriogenology. 2008;70:1317–23.

    Article  CAS  PubMed  Google Scholar 

  4. Kuster CE, Althouse GC. The impact of bacteriospermia on boar sperm storage and reproductive performance. Theriogenology. 2016;85:21–6.

    Article  CAS  PubMed  Google Scholar 

  5. Úbeda JL, Ausejo R, Dahmani Y, Falceto MV, Usan A, Malo C, et al. Adverse effects of members of the Enterobacteriaceae family on boar sperm quality. Theriogenology. 2013;80:565–70.

    Article  PubMed  Google Scholar 

  6. Khaki A. Assessment on the adverse effects of aminoglycosides and flouroquinolone on sperm parameters and male reproductive tissue: a systematic review. Iran J Reprod Med. 2015;13:125–34.

    PubMed  PubMed Central  Google Scholar 

  7. Aurich C, Spergser J. Influence of bacteria and gentamicin on cooled-stored stallion spermatozoa. Theriogenology. 2007;67:912–8.

    Article  CAS  PubMed  Google Scholar 

  8. Li Z, Zhang D, He Y, Ding Z, Mao F, Zhang X. Lipopolysaccharide compromises human sperm function by reducing intracellular cAMP. Tohoku J Exp Med. 2016;238:105–12.

    Article  CAS  PubMed  Google Scholar 

  9. Eley A, Hosseinzadeh S, Hakimi H, Geary I, Pacey AA. Apoptosis of ejaculated human sperm is induced by co-incubation with Chlamydia trachomatis lipopolysaccharide. Hum Reprod. 2005;20:2601–7.

    Article  CAS  PubMed  Google Scholar 

  10. Commission Delegated Regulation (EU). 2020/686. https://eurlex.europa.eu/eli/reg_del/2020/686/oj. Accessed 3 May 2024.

  11. Commission Delegated Regulation (EU). 2023/647. https://eur-lex.europa.eu/legal-content/EN/TXT/?uri=CELEX:32023R0647. Accessed 3 May 2024.

  12. Zhang LJ, Gallo RL. Antimicrobial peptides. Curr Biol. 2016;26:R14–9.

    Article  CAS  PubMed  Google Scholar 

  13. Mahlapuu M, Håkansson J, Ringstad L, Björn C. Antimicrobial peptides: an emerging category of therapeutic agents. Front Cell Infect Microbiol. 2016;6:1–12.

    Article  Google Scholar 

  14. Pulido D, Nogús MV, Boix E, Torrent M. Lipopolysaccharide neutralization by antimicrobial peptides: a gambit in the innate host defense strategy. J Innate Immun. 2012;4:327–36.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  15. Branen JK, Davidson PM. Enhancement of nisin, lysozyme, and monolaurin antimicrobial activities by ethylenediaminetetraacetic acid and lactoferrin. Int J Food Microbiol. 2004;90:63–74.

    Article  CAS  PubMed  Google Scholar 

  16. Nakamura S, Kato A, Kobayashi K. Novel Bifunctional Lysozyme–Dextran Conjugate that acts on both Gram-negative and Gram-positive Bacteria. Agric Biol Chem. 1990;54:3057–9.

    CAS  Google Scholar 

  17. Younes M, Aggett P, Aguilar F, Crebelli R, Dusemund B, Filipič M et al. Safety of nisin (E 234) as a food additive in the light of new toxicological data and the proposed extension of use. EFSA J. 2017;15.

  18. Shin JM, Gwak JW, Kamarajan P, Fenno JC, Rickard AH, Kapila YL. Biomedical applications of nisin. J Appl Microbiol. 2016;120:1449–65.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  19. Fleming A. On a remarkable bacteriolytic element found in tissues and secretions. Proc R Soc B-Biol Sci. 1922;93:306–17.

    CAS  Google Scholar 

  20. Jiang L, Li Y, Wang L, Guo J, Liu W, Meng G, et al. Recent insights into the prognostic and therapeutic applications of Lysozymes. Front Pharmacol. 2021;12:1–16.

    Article  Google Scholar 

  21. Chimura T, Hirayama T, Takase M. Lysozyme in cervical mucus of patients with chorioamnionitis. Jpn J Antibiot. 1993;46:726–9.

    CAS  PubMed  Google Scholar 

  22. Ohno N, Morrison DC. Lipopolysaccharide interaction with lysozyme. J Biol Chem. 1989;264:4434–41.

    Article  CAS  PubMed  Google Scholar 

  23. Gadea J. Review: semen extenders used in the artificial insemination of swine. Span J Agric Res. 2003;1:17–27.

    Article  Google Scholar 

  24. Caballero Gómez N, Manetsberger J, Benomar N, Castillo Gutiérrez S, Abriouel H. Antibacterial and antibiofilm effects of essential oil components, EDTA and HLE disinfectant solution on Enterococcus, Pseudomonas and Staphylococcus sp. multiresistant strains isolated along the meat production chain. Front Microbiol. 2022;13:1–20.

    Article  Google Scholar 

  25. Corral LG, Post LS, Montville TJ. Antimicrobial activity of sodium bicarbonate. J Food Sci. 1988;53:981–2.

    Article  CAS  Google Scholar 

  26. Yeste M. State-of-the-art of boar sperm preservation in liquid and frozen state. Anim Reprod. 2017;14:69–81.

    Article  Google Scholar 

  27. Pintus E, Jovičić M, Kadlec M, Ros-Santaella JL. Divergent effect of fast- and slow-releasing H2S donors on boar spermatozoa under oxidative stress. Sci Rep. 2020;10:6508.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  28. Pintus E, Chinn AF, Kadlec M, García-Vázquez FA, Novy P, Matson JB, et al. N-thiocarboxyanhydrides, amino acid-derived enzyme-activated H2S donors, enhance sperm mitochondrial activity in presence and absence of oxidative stress. BMC Vet Res. 2023;19:52.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  29. Ros-Santaella JL, Kadlec M, Pintus E. Pharmacological activity of Honeybush (Cyclopia intermedia) in Boar Spermatozoa during Semen Storage and under oxidative stress. Animals. 2020;10:463.

    Article  PubMed  PubMed Central  Google Scholar 

  30. Agarwal A, Gupta S, Sharma R. Oxidation–reduction potential measurement in Ejaculated Semen samples. In: Agarwal A, Gupta S, Sharma R, editors. Andrological evaluation of male infertility. Switzerland: Springer, Cham;; 2016. pp. 165–70.

    Chapter  Google Scholar 

  31. AFNOR, NF V08-100. Microbiology of food and animal feeding stuffs - Plating out and enumeration of microorganisms by spiral plate technique. 2001. https://m.boutique.afnor.org/Store/Preview/DisplayExtract?ProductID=17973&VersionID=6.

  32. CLSI. Methods for Dilution Antimicrobial Susceptibility Tests for Bacteria That Grow Aerobically. CLSI standard M07. 9th ed. Wayne, PA: Clinical and Laboratory Standards Institute; 2012.

    Google Scholar 

  33. Sone M. Investigations on the Control of Bacteria in Boar Semen. Japanese J Anim Reprod. 1990;36:P23–9.

    Article  Google Scholar 

  34. Espeche JC, Varas R, Maturana P, Cutro AC, Maffía PC, Hollmann A. Membrane permeability and antimicrobial peptides: much more than just making a hole. Pept Sci. 2023;1–14.

  35. Schulze M, Grobbel M, Müller K, Junkes C, Dathe M, Rüdiger K, et al. Challenges and limits using antimicrobial peptides in Boar Semen Preservation. Reprod Domest Anim. 2015;50:5–10.

    Article  CAS  PubMed  Google Scholar 

  36. Jakop U, Hensel B, Orquera S, Rößner A, Alter T, Schröter F, et al. Development of a new antimicrobial concept for boar semen preservation based on bacteriocins. Theriogenology. 2021;173:163–72.

    Article  CAS  PubMed  Google Scholar 

  37. Hensel B, Jakop U, Scheinpflug K, Mühldorfer K, Schröter F, Schäfer J, et al. Low temperature preservation of porcine semen: influence of short antimicrobial lipopeptides on sperm quality and bacterial load. Sci Rep. 2020;10:1–12.

    Article  Google Scholar 

  38. Puig-Timonet A, Castillo-Martín M, Pereira BA, Pinart E, Bonet S, Yeste M. Evaluation of porcine beta defensins-1 and – 2 as antimicrobial peptides for liquid-stored boar semen: effects on bacterial growth and sperm quality. Theriogenology. 2018;111:9–18.

    Article  CAS  PubMed  Google Scholar 

  39. Waberski D, Riesenbeck A, Schulze M, Weitze KF, Johnson L. Application of preserved boar semen for artificial insemination: past, present and future challenges. Theriogenology. 2019;137:2–7.

    Article  PubMed  Google Scholar 

  40. Maroto Martín LO, Muñoz EC, De Cupere F, Van Driessche E, Echemendia-Blanco D, Rodríguez JMM, et al. Bacterial contamination of boar semen affects the litter size. Anim Reprod Sci. 2010;120:95–104.

    Article  PubMed  Google Scholar 

  41. Contreras MJ, Núñez-Montero K, Bruna P, García M, Leal K, Barrientos L, et al. Bacteria and Boar Semen Storage: Progress and challenges. Antibiotics. 2022;11:1–14.

    Article  Google Scholar 

  42. Ciornei Ş, Drugociu D, Ciornei LM, Mareş M, Roşca P. Total aseptization of Boar Semen, to increase the biosecurity of reproduction in Swine. Molecules. 2021;26(20):6183.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  43. Gączarzewicz D, Udała J, Piasecka M, Błaszczyk B, Stankiewicz T. Bacterial contamination of boar semen and its relationship to sperm quality preserved in commercial extender containing gentamicin sulfate. Pol J Vet Sci. 2016;19:451–9.

    Article  PubMed  Google Scholar 

  44. Bennemann E, Machado PA, Girardini SK, Sonálio L, Tonin KA. Contaminantes bacterianos y perfil de susceptibilidad del semen porcino en centros de recogida em Brasil. Rev MVZ Córdoba. 2018;23:6637–48.

    Article  CAS  Google Scholar 

  45. Dalmutt AC, Moreno LZ, Gomes VTM, Cunha MPV, Barbosa MRF, Sato MIZ, et al. Characterization of bacterial contaminants of boar semen: identification by MALDI-TOF mass spectrometry and antimicrobial susceptibility profiling. J Appl Anim Res. 2020;48:559–65.

    Article  CAS  Google Scholar 

  46. Tvrdá E, Bučko O, Rojková K, Ďuračka M, Kunová S, Kováč J, et al. The efficiency of selected extenders against bacterial contamination of Boar Semen in a swine breeding facility in Western Slovakia. Animals. 2021;11:3320.

    Article  PubMed  PubMed Central  Google Scholar 

  47. Schulze M, Czirják GÁ, Müller K, Bortfeldt R, Jung M, Jakop U. Antibacterial defense and sperm quality in boar ejaculates. J Reprod Immunol. 2019;131:13–20.

    Article  CAS  PubMed  Google Scholar 

  48. Luther AM, Nguyen TQ, Verspohl J, Waberski D. Antimicrobially active semen extenders allow the reduction of antibiotic use in pig insemination. Antibiotics. 2021;10(11):1319.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  49. Mouritzen MV, Andrea A, Qvist K, Poulsen SS, Jenssen H. Immunomodulatory potential of Nisin A with application in wound healing. Wound Repair Regen. 2019;27:650–60.

    Article  PubMed  Google Scholar 

  50. Takada K, Ohno N, Yadomae T. Detoxification of lipopolysaccharide (LPS) by egg white lysozyme. FEMS Immunol Med Microbiol. 1994;9:255–63.

    Article  CAS  PubMed  Google Scholar 

  51. Okazaki T, Mihara T, Fujita Y, Yoshida S, Teshima H, Shimada M. Polymyxin B neutralizes bacteria-released endotoxin and improves the quality of boar sperm during liquid storage and cryopreservation. Theriogenology. 2010;74:1691–700.

    Article  CAS  PubMed  Google Scholar 

  52. Rowe M, Czirják GÁ, Lifjeld JT, Giraudeau M. Lysozyme-associated bactericidal activity in the ejaculate of a wild passerine. Biol J Linn Soc. 2013;109:92–100.

    Article  Google Scholar 

  53. Otti O, Naylor RA, Siva-Jothy MT, Reinhardt K. Bacteriolytic activity in the ejaculate of an insect. Am Nat. 2009;174:292–5.

    Article  PubMed  Google Scholar 

  54. Tvrdá E, Lovíšek D, Gálová E, Schwarzová M, Kováčiková E, Kunová S, et al. Possible implications of Bacteriospermia on the sperm quality, oxidative characteristics, and seminal Cytokine Network in Normozoospermic Men. Int J Mol Sci. 2022;23(15):8678.

    Article  PubMed  PubMed Central  Google Scholar 

  55. Lahnsteiner F, Radner M. Lysozyme activities and immunoglobulin concentrations in seminal plasma and spermatozoa of different teleost species and indications on its significance for sperm function. Theriogenology. 2010;74:246–54.

    Article  CAS  PubMed  Google Scholar 

  56. Mårdh PA, Colleen S. Lysozyme in seminal fluid of healthy males and patients with Prostatitis and in tissues of the male uro-genital tract. Scand J Urol Nephrol. 1974;8:179–83.

    Article  PubMed  Google Scholar 

  57. Mandal A, Klotz KL, Shetty J, Jayes FL, Wolkowicz MJ, Bolling LC, et al. SLLP1, a unique, intra-acrosomal, non-bacteriolytic, c lysozyme-like protein of human spermatozoa. Biol Reprod. 2003;68:1525–37.

    Article  CAS  PubMed  Google Scholar 

  58. Ou C, Lee RK, Lin M, Lu C, Yang T, Yeh L, et al. A mouse seminal vesicle-secreted lysozyme c‐like protein modulates sperm capacitation. J Cell Biochem. 2021;122:653–66.

    Article  CAS  PubMed  Google Scholar 

  59. Peña FJ, Ortiz-Rodríguez JM, Gaitskell-Phillips GL, Gil MC, Ortega-Ferrusola C, Martín-Cano FE. An integrated overview on the regulation of sperm metabolism (glycolysis-Krebs cycle-oxidative phosphorylation). Anim Reprod Sci. 2022;246:106805.

    Article  PubMed  Google Scholar 

  60. Ortiz-Rodríguez JM, Martín-Cano FE, Gaitskell-Phillips GL, Silva A, Ortega-Ferrusola C, Gil MC, et al. Low glucose and high pyruvate reduce the production of 2-oxoaldehydes, improving mitochondrial efficiency, redox regulation, and stallion sperm function. Biol Reprod. 2021;105:519–32.

    PubMed  Google Scholar 

  61. Liu H, Zheng F, Cao Q, Ren B, Zhu L, Striker G, et al. Amelioration of oxidant stress by the defensin lysozyme. Am J Physiol - Endocrinol Metab. 2006;290:824–32.

    Article  Google Scholar 

  62. Aranha C, Gupta S, Reddy KVR. Contraceptive efficacy of antimicrobial peptide Nisin: in vitro and in vivo studies. Contraception. 2004;69:333–8.

    Article  CAS  PubMed  Google Scholar 

  63. Khazaei Monfared Y, Mahmoudian M, Caldera F, Pedrazzo AR, Zakeri-Milani P, Matencio A, et al. Nisin delivery by nanosponges increases its anticancer activity against in-vivo melanoma model. J Drug Deliv Sci Technol. 2023;79:104065.

    Article  CAS  Google Scholar 

  64. Yuan N, Wang Y, Guan Y, Chen C, Hu W. Effect of Nisin on the quality and antioxidant activity of fresh-cut pumpkins (Cucurbita moschata Duch). Horticulturae. 2023;9(5):529.

    Article  Google Scholar 

  65. Panner Selvam MK, Agarwal A, Henkel R, Finelli R, Robert KA, Iovine C, et al. The effect of oxidative and reductive stress on semen parameters and functions of physiologically normal human spermatozoa. Free Radic Biol Med. 2020;152:375–85.

    Article  CAS  PubMed  Google Scholar 

  66. Ngo C, Suwimonteerabutr J, Prapasarakul N, Morrell JM, Tummaruk P. Bacteriospermia and its antimicrobial resistance in relation to boar sperm quality during short-term storage with or without antibiotics in a tropical environment. Porc Heal Manag. 2023;9:21.

    Article  Google Scholar 

  67. Althouse GC, Kuster CE, Clark SG, Weisiger RM. Field investigations of bacterial contaminants and their effects on extended porcine semen. Theriogenology. 2000;53:1167–76.

    Article  CAS  PubMed  Google Scholar 

  68. World Organisation for Animal Health (WOAH). Terrestrial Animal Health Code. https://www.woah.org/en/what-we-do/standards/codes-and-manuals/#ui-id-1. Accessed 3 May 2024.

  69. Morrell JM, Cojkic A, Malaluang P, Ntallaris T, Lindahl J, Hansson I. Antibiotics in semen extenders – a multiplicity of paradoxes. Reprod Fertil Dev. 2024;36.

Download references

Funding

This research was funded by the Czech National Agency for Agricultural Research (NAZV QK21010327).

Author information

Authors and Affiliations

  1. Department of Veterinary Sciences, Faculty of Agrobiology, Food and Natural Resources, Czech University of Life Sciences Prague, Prague, 165 00, Czech Republic

    Jose Luis Ros-Santaella, Maria Scaringi & Eliana Pintus

  2. Department of Food Science, Faculty of Agrobiology, Food and Natural Resources, Czech University of Life Sciences Prague, Prague, 165 00, Czech Republic

    Pavel Nový

Authors
  1. Jose Luis Ros-Santaella
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Pavel Nový
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Maria Scaringi
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Eliana Pintus
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

JLR-S, PN, and EP: conceptualization; JLR-S: funding acquisition; JLR-S, PN, MS, and EP: methodology; JLR-S, MS, and EP: sperm analysis; PN and MS: microbiological analysis; JLR-S: writing the first draft of the manuscript; PN, MS, and EP: review and editing. All authors contributed to manuscript revision, read, and approved the submitted version.

Corresponding authors

Correspondence to Jose Luis Ros-Santaella or Eliana Pintus.

Ethics declarations

Ethics approval and consent to participate

This study did not involve animal handling because the sperm samples were purchased as artificial insemination doses from a pig breeding company (Lipra Pork, a.s., Czech Republic).

Consent for publication

Not applicable.

Competing interests

The authors declare no competing interests.

Additional information

Publisher’s Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Electronic supplementary material

Below is the link to the electronic supplementary material.

Supplementary Material 1

Rights and permissions

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data.

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Ros-Santaella, J.L., Nový, P., Scaringi, M. et al. Antimicrobial peptides and proteins as alternative antibiotics for porcine semen preservation. BMC Vet Res 20, 257 (2024). https://doi.org/10.1186/s12917-024-04105-9

Download citation

  • Received:

  • Accepted:

  • Published:

  • DOI: https://doi.org/10.1186/s12917-024-04105-9

Keywords

BMC Veterinary Research

ISSN: 1746-6148

Contact us