# Addition of Copper Chloride and Zinc Chloride to Liquid-Stored Pig Semen Reduces Bacterial Growth Without Impairing Sperm Quality

## TL;DR

Adding copper and zinc chloride to pig semen reduces bacteria without harming sperm quality, offering an alternative to antibiotics.

## Contribution

Copper and zinc chloride are shown to reduce bacterial growth in pig semen without impairing sperm quality.

## Key findings

- Copper and zinc chloride at 1 mM reduced bacterial growth without affecting sperm quality.
- Silver sulfadiazine and aluminum chloride either harmed sperm or failed to inhibit bacteria.
- Combining metal ions with other strategies may further reduce bacterial growth.

## Abstract

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 μM, 300 μM, 500 μM, 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.