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. 2026 Sep 30;61(Suppl 3):e70282. doi: 10.1111/rda.70282

Silver Nanoparticles in Donkey Semen Stored at 17°C: Effect on Sperm Parameters and Bacterial Growth

Verónica Pérez Aguilera 1, Pilar Vallejo‐Soto 2, Carlos Montero‐Palma 2,3, Blenda Vilela 2, Antonio Romero‐Salmoral 3, Ángela Galán‐Relaño 3, Francisco Crespo‐Castejón 1, Antonio González‐Ariza 4, Manuel Hidalgo 2, Jesús Dorado 2, Isabel Ortiz 2,✉, Lidia Gómez‐Gascón 3, Inmaculada Luque 3
PMCID: PMC13627739  PMID: 42816940

ABSTRACT

The aim of this study was to evaluate the effect of silver nanoparticles (AgNPs) on donkey sperm parameters and their antimicrobial efficacy on bacteria isolated in the presence of antibiotics. Nine donkey ejaculates were collected and stored in a skim‐milk extender at different AgNPs concentrations (0, 0.01, 0.8, 1.6 and 3.12 mg/mL) at 5°C or 17°C. Sperm parameters, including total and progressive motility (CASA), plasma membrane integrity (PMI) and reactive oxygen species (rROS) production, were evaluated at 0, 24 and 48 h of storage. Additionally, bacteria isolated from donkey semen extended with antibiotics were exposed to AgNPs to determine the minimum inhibitory/bactericidal concentrations (MIC/MBC). Sperm motility parameters were higher at 17°C than 5°C after 48 h. AgNPs showed a dose‐ and time‐dependent cytotoxic effect, with concentrations ≥ 0.8 mg/mL significantly reducing motility, membrane integrity and increasing rROS after 24 h. AgNPs exhibited bactericidal activity at concentrations between 1.56 and ≥ 25 mg/mL. In conclusion, while liquid storage at 17°C is an effective strategy for preserving donkey semen, the use of AgNPs as a standalone antimicrobial is currently limited by their cytotoxicity at effective bactericidal doses.

Keywords: antibiotic resistance, bacteria, cytotoxicity, donkey sperm, silver nanoparticles

1. Introduction

Donkey semen does not appear to tolerate cooling protocols designed for stallion semen (Gobato et al. 2022). Some studies have suggested that liquid storage at 15°C–20°C may better preserve motility and viability (Serres et al. 2002; Vallejo‐Soto et al. 2025). However, storage at these temperatures facilitates microbial proliferation, even in the presence of antibiotics (Clulow and Gibb 2022). In this context, the integration of new antimicrobial alternatives to semen extenders is of the utmost importance.

Nanotechnology studies nanoparticles or nanomaterials (< 100 nm) applied to various fields of science (Feugang et al. 2019). In reproduction, nanoparticle formulations have been widely used for several purposes, including their antimicrobial properties (Odhiambo et al. 2014; Yousef et al. 2021).

Silver nanoparticles (AgNPs) have been shown to exert antibacterial, antifungal and antiviral action (Kanwar et al. 2023). However, no studies have evaluated the AgNPs concentration safe for liquid storage of donkey sperm or the effect of AgNPs on bacteria isolated from donkey semen.

Thus, the objectives of this study were to evaluate the effect of AgNPs on donkey sperm stored for up to 48 h and the in vitro efficacy of AgNPs against bacteria isolated from donkey sperm in an antibiotic‐containing extender.

2. Material and Methods

2.1. Silver Nanoparticles Preparation

Silver nanopowder (ref. 576,832, < 100 nm containing polyvinylpyrrolidone (PVP) as a dispersant, Sigma‐Aldrich; Merck SL, Madrid, Spain) was added to a skim‐milk extender (SM, INRA96; IMV Technologies, L'Aigle, France) to reach an AgNPs concentration of 25 mg/mL (stock solution). From this stock, four working solutions of 0.01, 0.8, 1.6 and 3.12 mg/mL were prepared. All AgNPs solutions were sonicated for 20 min and vortexed immediately before sperm suspension.

2.2. Semen Processing

Nine ejaculates from five jackasses were collected for this study. Following collection, the semen was centrifuged and the sperm pellets were then resuspended to a final concentration of 50 × 106 spermatozoa/mL in different concentrations of AgNPs: 0, 0.01, 0.8, 1.6 and 3.12 mg/mL. Samples were then cooled to 5°C or 17°C, according to treatment.

Sperm parameters were evaluated in fresh semen and after 24 and 48 h of storage. Sperm motility was assessed using computer‐assisted sperm analysis (CASA, SCA v.6.5; Microptic) (Ortiz et al. 2015). The percentage of plasma membrane integrity (PMI) and the reactive oxygen species (rROS) production in viable sperm relative to control were determined by flow cytometry (BDAccuri C6 Plus; Becton, Dickinson and Company, Franklin Lakes, NJ, USA). A minimum of 10,000 gated events was evaluated per sample and sperm parameter. PMI was determined by staining sperm diluted in Dulbecco's phosphate‐buffered saline (DPBS, Sigma‐Aldrich; Merck SL, Madrid, Spain) with propidium iodide (PI, 1.3 μM) and Arachis hypogaea lectin (PNA, 0.05 mg/mL) for 10 min at room temperature (RT) in the dark. After this, sperm were diluted with DPBS to a final concentration of approximately 1–5 × 106 sperm/mL before flow cytometric analysis (Ortiz et al. 2021). rROS was evaluated using the double staining PI (1.3 μM) and 2′,7′‐dichlorodihydrofluorescein diacetate (DCFH‐DA, 140 mM). After 30 min of incubation at RT in the dark, sperm were diluted to 1 × 106 sperm/mL (Catalán et al. 2022). The relative to control median fluorescence intensity in viable sperm (PI−) was calculated for analysis (Garriga et al. 2026).

2.3. Microbiological Analysis

Microorganisms were isolated from cryopreserved semen straws from nine donkeys frozen in different farms using an extender with antibiotics (Gent, Minitube, Tiefenbach, Germany). This methodology allowed to obtain antibiotic‐resistant microorganisms from diverse environments and improve representativeness. Semen suspensions were plated on MacConkey agar, mannitol agar, blood agar, Columbia and Sabouraud agar for 72 h (Corona and Cherchi 2009; Jesus et al. 2025). Presumptive identification of isolates was performed by Gram staining, oxidase and catalase tests and glucose fermentation, and they were further accurately identified by MALDI‐TOFF analysis by Bruker MALDI Biotyper with score values ≥ 2.0 and classified as G+/− and animal/environmental (Pérez‐Sancho et al. 2017). The antimicrobial susceptibility was determined by the broth microdilution method (Clinical and Laboratory Standards Institute 2016). From a stock solution of 50 mg/mL AgNPs, serial twofold dilutions (range of 25–0.02 mg/mL) were dispensed in U‐bottom 96‐well microtitre plates (Lab‐Center, Spain). An equal volume of adjusted bacterial inoculum (5 × 105 CFU/mL) in Mueller Hinton Broth (MHB) was added to each well. Microdilution plates were read after 24 h incubation at 37°C in aerobic atmosphere. The minimum inhibitory concentration (MIC, the lowest antimicrobial concentration with no evident bacterial growth) and MIC50 and MIC90 (MIC that inhibits the visible growth of 50% and 90% of tested bacterial isolates, respectively) values were determined. After this, a sample from wells where no visible growth was observed were obtained to test for bactericidal activity (MBC).

2.4. Statistical Analysis

Sperm parameters assessed were compared among temperatures and AgNPs concentrations using general linear model (PROC MIXED, SAS Institute Inc., Cary, NC, USA) in which AgNPs concentration and storage time were considered fixed factors, whereas donkey and ejaculate within donkey were considered random effects. Tukey's post hoc test was performed to assess the differences among AgNPs concentrations and time. Results were expressed as mean ± SEM. Significant differences were considered when p < 0.05.

3. Results

Sperm motility was significantly decreased in samples stored at 5°C in comparison to 17°C (Figure 1). AgNPs cytotoxicity was observed at concentrations ≥ 0.8 mg/mL after 24 h (Table 1, Table S1). The dose‐ and time‐dependent effect of AgNPs on bacteria isolated from cryopreserved donkey semen using an extender with antibiotics is shown in Table 2.

FIGURE 1.

FIGURE 1

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Effect of storage temperature (5°C vs. 17°C) on sperm motility (TMOT, PMOT and VCL). Different subscripts (a–c) indicate significant differences (p < 0.05) among time (0, 24 and 48 h). PMOT, progressive motility; TMOT, total motility; VCL, curvilinear velocity.

TABLE 1.

Effect of silver nanoparticles concentrations ([AgNPs]) on donkey sperm parameters after 48 h of storage at 17°C.

Time [AgNPs] (mg/mL) Sperm parameters
TMOT PMOT VCL PMI rROS
24 h 0 68.85 ± 8.30a 58.11 ± 8.34a 108.38 ± 3.01a 57.36 ± 10.22a 1.00 ± 0a,b
0.01 67.73 ± 7.33a 53.83 ± 8.09a,b 101.16 ± 4.88a 53.83 ± 8.47a,b 1.10 ± 0.07a,b
0.8 30.48 ± 9.49c,d 18.73 ± 8.85c,d,e 64.33 ± 11.54b,c 36.59 ± 10.47a,b,c,d 1.16 ± 0.05a,b
1.6 13.26 ± 8.56d,e 7.06 ± 5.69d,e 31.92 ± 11.43d,e 24.81 ± 10.37c,d 0.77 ± 0.15b,c
3.12 1.29 ± 1.29e 0.10 ± 0.10e 2.93 ± 2.93e,f 25.46 ± 11.44c,d 0.41 ± 0.14c,d
48 h 0 64.88 ± 8.10a,b 54.70 ± 8.54a 99.82 ± 3.52a 59.37 ± 9.06a 1.13 ± 0.13a,b
0.01 65.63 ± 7.04a,b 54.21 ± 7.92a 103.96 ± 4.31a 57.83 ± 8.78a 1.08 ± 0.19a,b
0.8 14.98 ± 9.06d,e 10.04 ± 7.44c,d,e 50.55 ± 13.61c,d 28.52 ± 11.47b,c,d 1.08 ± 0.17a,b
1.6 1.67 ± 1.61e 0.47 ± 0.46e 11.08 ± 7.37e,f 15.23 ± 8.31c,d 0.80 ± 0.18b,c
3.12 0e 0e 0 ± 0f 36.00 ± 18.28d 0d
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Note: Values are expressed as mean ± SEM. Different superscripts (a–f) indicate significant differences (p < 0.05) among [AgNPs].

Abbreviations: PMI, intact plasma membrane (%); PMOT, progressive motility (%); rROS, reactive oxygen species in viable sperm relative to control; TMOT, total motility (%); VCL, curvilinear velocity (μm/s).

TABLE 2.

Effect of silver nanoparticles (AgNPs) on bacteria isolated from cryopreserved donkey semen using an extender with antibiotics.

N AgNPs (mg/mL) MIC50 MIC90
1.56 12.5 25 > 25
Gram+ Animal 5 2 1 1 1 12.5 > 25
Environment 4 0 0 1 3 > 25 > 25
Total G+ 9 2 1 2 4 25 > 25
Gram− Animal 2 1 1 0 0 1.56 12.5
Environment 4 0 2 2 0 12.5 25
Total G— 6 1 3 2 0 12.5 25
Total 15 3 4 4 4 12.5 > 25
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Abbreviations: N, number of bacterial isolates; MIC50, minimum inhibitory concentration 50%; MIC90, minimum inhibitory concentration 90%.

The MIC distribution ranged from ≥ 25 to 1.56 mg/mL (Table 2). In all cases, the MIC and MBC values coincided, indicating that AgNPs exerted a bactericidal effect.

4. Discussion

The results of this study showed that motility parameters of donkey semen did not decrease after 48 h of storage at 17°C. These results agreed with previous studies performed on donkey and stallion (Clulow and Gibb 2022; Serres et al. 2002). However, storage at these temperatures could increase bacterial proliferation, even if antibiotics are added into semen extenders (Clulow and Gibb 2022).

Silver nanoparticles emerge as a potential alternative to conventional antibiotics (Morrell and Wallgren 2014). The evaluation of the cytotoxicity of AgNPs revealed a dose‐ and time‐dependent effect (Pérez et al. 2024). AgNPs started showing deleterious effects on all sperm parameters at 0.8 mg/mL. These detrimental effects were found first in sperm motility (24 h). PMI and rROS were significantly affected at concentrations higher than 1.6 and 0.8 mg/mL at 24 and 48 h, respectively. These findings agree with previous studies showing that the adhesion of AgNPs to the acrosomal region of the sperm membrane compromises its integrity, leading to reduced motility (Pérez et al. 2024) and increased ROS production (Pérez‐Duran et al. 2020). Thus, concentrations higher than 0.8 mg/mL of this AgNPs formulation should not be used for storage times longer than 24 h.

Bacteria were isolated from cryopreserved donkey doses extended with antibiotic‐supplemented extenders, showing the importance of finding new antimicrobial alternatives. Interestingly, Gram‐positive isolates exhibited higher resistance to AgNPs. Their thicker peptidoglycan layer could be less sensitive to the three main AgNPs antibacterial mechanisms: (i) silver ion release disrupting the respiratory chain, (ii) non‐oxidative membrane destabilization and (iii) ROS‐induced oxidative stress (Wang et al. 2017). Nonetheless, future research should evaluate the antibacterial effect of silver nanoparticles co‐incubated with donkey sperm at 17°C on a larger scale.

Thus, the AgNPs concentrations needed to inhibit the growth of bacteria able to survive in the presence of antibiotics are cytotoxic to donkey sperm. One potential strategy to overcome this limitation might be to exploit the synergistic interaction between AgNPs and conventional antibiotics. Such synergy could enhance antibacterial efficacy, making it possible to reduce the minimum inhibitory concentrations of both AgNPs and antibiotics (Elmasry et al. 2026; Faheem et al. 2026). Further research is warranted to optimize these AgNPs‐conventional antibiotic combinations in order to enable the safe use of AgNPs in donkey semen samples.

In conclusion, liquid storage at 17°C preserves donkey semen quality for up to 48 h compared to 5°C. AgNPs are non‐toxic to donkey spermatozoa at concentrations lower than 0.8 mg/mL. AgNPs exhibit antibacterial activity against bacteria isolated from semen samples in the presence of antibiotics. However, at the concentrations effective in vitro, they negatively affected the viability of donkey sperm.

Author Contributions

V.P.A., P.V.‐S., F.C.‐C., J.D., I.O., L.G.‐G., I.L.: conceptualization. V.P.A., P.V.‐S., I.O., L.G.‐G., I.L.: methodology. V.P.A., P.V.‐S., I.O., L.G.‐G., I.L.: formal analysis. V.P.A., P.V.‐S., C.M.‐P., B.V., A.R.‐S., A.G.‐R., A.G.‐A., I.O., L.G.‐G., I.L.: investigation. P.V.‐S., C.M.‐P., B.V., A.R.‐S., I.O., L.G.‐G., I.L.: data curation. I.O., I.L.: formal analysis. F.C.‐C., M.H., J.D., I.L.: resources. M.H., J.D.: funding acquisition. V.P.‐A., I.O., I.L.: writing – original draft. V.P.‐A., F.C.‐C., J.D., I.O., I.L.: writing – review and editing.

Funding

This study was supported by the project grant PID2020‐116090RB‐I00 from MICIN (Ministerio de Ciencia e Innovación), which also supported C.M.‐P. P.V.‐S. was supported by the Spanish FPU fellowship (FPU23/03472) from Ministerio de Universidades. B.V. was funded by Coordenação de Aperfeiçoamento de Pessoal de Nível Superior—Brazil (CAPES) – Finance Code 001. Funding for open access charge: Universidad de Cordoba/CBUA.

Conflicts of Interest

The authors declare no conflicts of interest.

Supporting information

Table S1: Effect of silver nanoparticles concentrations ([AgNPs]) on donkey sperm kinetic parameters after 48 h of storage at 17°C. ALH, amplitude of lateral head displacement (%); BCF, beat cross frequency (Hz); LIN, linearity (%); STR, straightness (%); VAP, average path velocity (μm/s); VCL, curvilinear velocity (μm/s); VSL, straight‐line velocity (μm/s); WOB, wobble (%). Different superscripts (a–f) indicate significant differences among [AgNPs].

RDA-61-e70282-s001.docx (35.2KB, docx)

Acknowledgements

The authors would like to thank the Military Horse Breeding Centre of Ávila of the Spanish Army and the Provincial Agropecuary Centre of Córdoba for allowing access to their herds.

Data Availability Statement

The data that support the findings of this study are available from the corresponding author upon reasonable request.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Table S1: Effect of silver nanoparticles concentrations ([AgNPs]) on donkey sperm kinetic parameters after 48 h of storage at 17°C. ALH, amplitude of lateral head displacement (%); BCF, beat cross frequency (Hz); LIN, linearity (%); STR, straightness (%); VAP, average path velocity (μm/s); VCL, curvilinear velocity (μm/s); VSL, straight‐line velocity (μm/s); WOB, wobble (%). Different superscripts (a–f) indicate significant differences among [AgNPs].

RDA-61-e70282-s001.docx (35.2KB, docx)

Data Availability Statement

The data that support the findings of this study are available from the corresponding author upon reasonable request.

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