44cc47baafcc5a31169a79643968027b ijms-27-00773.pdf 610bc09cb37d8ec8eda1a4b9e27cee9a9a0823df ijms-27-00773.pdf 6e3c49f8163c4d06f6631ee99aaf199911cca40e8fe2af531e61d8e88e9fbff9 ijms-27-00773.pdf Title: Addition of Copper Chloride and Zinc Chloride to Liquid-Stored Pig Semen Reduces Bacterial Growth Without Impairing Sperm Quality Subject: Bacterial contamination remains a challenge for multiple facets of modern life. While antibiotics are a primary tool for bacterial control, their overuse has accelerated the appearance of multidrug-resistant bacteria and raises global health concerns. In swine, semen is stored at 17 C in extenders that contain antibiotics to prevent bacterial growth. Apart from the potential consequences for the female, the proliferation of bacteria in liquid-stored semen is associated with a decline in sperm quality, ultimately reducing farrowing rates and litter sizes. With the aim of reducing the use of antibiotics while keeping bacterial growth under control, we herein investigated whether metal ions could exert an antimicrobial effect without impairing sperm quality. Separate metal ions (Ag, silver sulfadiazine; Al, aluminum chloride; Zn, zinc chloride; and Cu, and cooper chloride) were added at different concentrations (100 M, 300 M, 500 M, 1 mM, and 10 mM) to seminal doses, which were stored at 17 C for 48 h. Motility, viability, and the intracellular levels of reactive oxygen species (ROS) were tested to determine their effects on sperm quality maintenance. In addition, ions were added to bacterial strains and to extended seminal samples to assess the minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC). Results showed that, although silver sulfadiazine exerted an antimicrobial effect at all the concentrations tested, it also affected sperm quality negatively (p < 0.05). In contrast, aluminum chloride did not impair sperm quality but failed to inhibit bacterial growth at any of the tested concentrations (p > 0.05). Finally, 1 mM concentrations of copper and zinc chloride reduced microbial growth (p < 0.05) without affecting sperm quality. In spite of this, the inhibition of bacterial growth was not complete, thus suggesting that these two ions could contribute to reducing bacterial growth but should be combined with other strategies, such as a lower storage temperature and a decreased concentration of antibiotics. Further research is warranted to address whether copper and zinc chloride could have a synergistic effect when added together. Keywords: sperm; boar; preservation; contamination; antimicrobial Author: Judit Drago, Elia Bosch-Rué, Nasira Akrim, Marc Yeste and Jordi Ribas-Maynou Creator: LaTeX with hyperref Producer: pdfTeX-1.40.25; modified using OpenPDF 1.4.2 CreationDate: Tue Jan 13 08:39:43 2026 CET ModDate: Tue Jan 13 08:42:45 2026 CET Custom Metadata: yes Metadata Stream: no Tagged: no UserProperties: no Suspects: no Form: none JavaScript: no Pages: 17 Encrypted: no Page size: 595.276 x 841.89 pts (A4) Page rot: 0 File size: 5742648 bytes Optimized: no PDF version: 1.7 name type encoding emb sub uni object ID ------------------------------------ ----------------- ---------------- --- --- --- --------- LLMNHJ+CMSY10 Type 1 Builtin yes yes yes 121 0 TYTCIA+URWPalladioL-Roma Type 1 Custom yes yes yes 122 0 LZNGNM+URWPalladioL-Ital Type 1 Custom yes yes yes 123 0 RHFKGB+EURM10 Type 1 Builtin yes yes yes 124 0 HLFGXD+URWPalladioL-Bold Type 1 Custom yes yes yes 125 0 GNNHDF+PalatinoLinotype TrueType WinAnsi yes yes no 206 0 GNNHDH+PalatinoLinotype,Italic TrueType WinAnsi yes yes no 237 0 GNNHDF+PalatinoLinotype TrueType WinAnsi yes yes no 238 0 GNNHDF+PalatinoLinotype TrueType WinAnsi yes yes no 272 0 MVAPLY+URWPalladioL-BoldItal Type 1 Custom yes yes yes 312 0 GNNHDF+PalatinoLinotype TrueType WinAnsi yes yes no 304 0 GNNHDH+PalatinoLinotype,Italic TrueType WinAnsi yes yes no 327 0 GNNHDF+PalatinoLinotype TrueType WinAnsi yes yes no 328 0 PLOLAK+VnURWPalladioL Type 1 Custom yes yes yes 440 0 Jhove (Rel. 1.28.0, 2023-05-18) Date: 2026-02-11 03:07:11 CET RepresentationInformation: ijms-27-00773.pdf ReportingModule: PDF-hul, Rel. 1.12.4 (2023-03-16) LastModified: 2026-02-10 12:48:22 CET Size: 5742648 Format: PDF Version: 1.7 Status: Well-Formed and valid SignatureMatches: PDF-hul MIMEtype: application/pdf PDFMetadata: Objects: 581 FreeObjects: 1 IncrementalUpdates: 0 DocumentCatalog: ViewerPreferences: HideToolbar: false HideMenubar: false HideWindowUI: false FitWindow: true CenterWindow: false DisplayDocTitle: false NonFullScreenPageMode: UseNone Direction: L2R ViewArea: CropBox ViewClip: CropBox PrintArea: CropBox PageClip: CropBox PageLayout: SinglePage PageMode: UseNone Outlines: Item: Title: Introduction Item: Title: Results Children: Item: Title: Effects of Metal Ion Treatments on Sperm Quality Item: Title: Effect of Metal Ion Treatments on Bacterial Growth Item: Title: Bacterial Growth on Extended Samples Item: Title: Discussion Item: Title: Materials and Methods Children: Item: Title: Experimental Design and Semen Samples Item: Title: Evaluation of the Effect of Ions on Sperm Quality Children: Item: Title: Motility Evaluation Item: Title: Flow Cytometry Item: Title: Evaluation of Antimicrobial Properties Children: Item: Title: Determination of the Minimum Inhibitory Concentration (MIC) Item: Title: Determination of the Minimum Bactericidal Concentration (MBC) Item: Title: Determination of Bacterial Growth in Rich Medium Item: Title: Statistical Analysis Item: Title: Conclusions Item: Title: References Info: Title: Addition of Copper Chloride and Zinc Chloride to Liquid-Stored Pig Semen Reduces Bacterial Growth Without Impairing Sperm Quality Author: Judit Drago, Elia Bosch-Rué, Nasira Akrim, Marc Yeste and Jordi Ribas-Maynou Subject: Bacterial contamination remains a challenge for multiple facets of modern life. While antibiotics are a primary tool for bacterial control, their overuse has accelerated the appearance of multidrug-resistant bacteria and raises global health concerns. In swine, semen is stored at 17 C in extenders that contain antibiotics to prevent bacterial growth. Apart from the potential consequences for the female, the proliferation of bacteria in liquid-stored semen is associated with a decline in sperm quality, ultimately reducing farrowing rates and litter sizes. With the aim of reducing the use of antibiotics while keeping bacterial growth under control, we herein investigated whether metal ions could exert an antimicrobial effect without impairing sperm quality. Separate metal ions (Ag, silver sulfadiazine; Al, aluminum chloride; Zn, zinc chloride; and Cu, and cooper chloride) were added at different concentrations (100 M, 300 M, 500 M, 1 mM, and 10 mM) to seminal doses, which were stored at 17 C for 48 h. Motility, viability, and the intracellular levels of reactive oxygen species (ROS) were tested to determine their effects on sperm quality maintenance. In addition, ions were added to bacterial strains and to extended seminal samples to assess the minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC). Results showed that, although silver sulfadiazine exerted an antimicrobial effect at all the concentrations tested, it also affected sperm quality negatively (p < 0.05). In contrast, aluminum chloride did not impair sperm quality but failed to inhibit bacterial growth at any of the tested concentrations (p > 0.05). Finally, 1 mM concentrations of copper and zinc chloride reduced microbial growth (p < 0.05) without affecting sperm quality. In spite of this, the inhibition of bacterial growth was not complete, thus suggesting that these two ions could contribute to reducing bacterial growth but should be combined with other strategies, such as a lower storage temperature and a decreased concentration of antibiotics. Further research is warranted to address whether copper and zinc chloride could have a synergistic effect when added together. 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