Cu nps

The Cu-NPs were purchased from Shanghai Aladdin Co., Ltd. China, with an average particle diameter of 20±10nm and 99.9% purity (manufacturer’s specification). Cu-NPs suspension containing 1.0 g Cu L^-1 was prepared by dispersing nanoparticles in ultrapure water(Millipore, ion free and unbuffered), sonicated for 30 min and stirred for 1 h at room temperature to increase dispersion before use[10 (link),17 (link)]. Copper ion stock solution (1.0 g Cu L^-1) was prepared by dissolving 3.929 g CuSO₄·5H₂O in 1 L of ultrapure water.
As in our previous study [18 (link)], the particle size was characterized using transmission electron microscopy (TEM, JEOL JEM-2100, Japan) and nanoparticle tracking analysis (NTA, NanoSight LM₁₀). For TEM analysis, Cu-NPs were diluted in ultrapure water (Millipore, ion free and unbuffered) and sonicated 30 min to keep the particles in solution and avoid aggregation [19 (link),20 (link)]. The primary range of particle sizes was determined from micrographs through analysis of 50 NPs selected randomly. Additionally, particle size distribution in the cell culture medium[DMEM/F12 medium in the presence of 15% (v/v) serum, 100 IU mL^-1penicillin and 100 IU mL^-1 streptomycin] were measured by NTA in 20 mg Cu L^-1 to give sufficient particle tracks (>100 tracks per sample)[21 (link)].

The copper nanoproducts (NPs) of Cu NPs and CuO NPs of similar primary particle size of around 50 nm and ionic form CuSO₄ were purchased from Sigma Aldrich. The basic suspension of compounds 1 g Cu·L⁻¹ was prepared in artificial seminal plasma (ASP: 125 mM NaCl, 40 mM KCl, 1 mM CaCl₂, 1 mM MgCl₂, 50 mM Tris, pH 8.5, 363 mOsm·kg⁻¹, composition by Morisawa and Morisawa [55 (link)] modified by Dziewulska) and sonicated in an ultrasonic bath for 30 min. Following stock suspension: 0, 1, 5, 10, 25, 50 mg Cu·L⁻¹ of testing compounds were prepared by mixing in artificial seminal plasma. Four milt samples were mixed individually with artificial seminal plasma containing an adequate nanoproduct concentration in the ratio 1:10 and stored in thin layers (5 mm), at 6 °C, for 96 h. The samples were mixed every 12 h. At defined incubation times: 0, 2, 12, 24, 48, 72, and 96 h activation of spermatozoa motility was determined in CASA following the method used in Kowalska-Góralska et al. [23 (link)].

In this study, three bacterial strains were tested for various responses to the exposure of selected metallic nanoparticles. They included gram-negative Escherichia coli (ATCC^® 25922™) and gram-positive Bacillus cereus (ATCC^® 11778™), and Staphylococcus epidermidis (ATCC^® 12228™) strains purchased from the American Type Culture Collection (ATCC). E. coli was maintained using Bacto™ Tryptic Soy Broth (cat. 211825; pancreatic digest of casein 17.0 g L⁻¹, papaic digest of soybean 3.0 g L⁻¹, dextrose 2.5 g L⁻¹, sodium chloride 5.0 g L⁻¹, dipotassium phosphate g L⁻¹); however, B. cereus and S. epidermidis were passaged in Difco™ Nutrient Broth (cat. 234000; beef extract 3.0 g L⁻¹, peptone 5.0 g L⁻¹).
All these strains were exposed to four types of nanoparticles (NPs): Ag-NPs (cat. 576832), Cu-NPs (cat. 774081), ZnO-NPs (cat. 677450) obtained from Sigma-Aldrich company and TiO₂-NPs (cat. US1019F) acquired from US Research. The size of Ag-NPs, Cu-NPs and ZnO-NPs ranged in <100 nm, 25 nm and <50 nm, respectively, while TiO₂-NPs were 20 nm in size. All NPs were characterised by 97–99.5% purity. Before starting the actual experiment, the stock solutions of NPs in sterile Millipore Water were sonicated (Vibra-Cell™, 20 kHz) for 10–20 min to avoid their aggregation/agglomeration.