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Simple Summary  Livestock semen is often contaminated by opportunistic bacterial pathogens originating from an intrinsic environment of the urogenital tract. Particularly, species classified in the Staphylococcus genus are predominantly represented in bovine ejaculates. Until recently, it was believed that these are a negligible part of the bovine ejaculate; however, recent studies revealed their potentially adverse effects on the sperm quality. Hereby, we simulated staphylococcal infection of bovine semen under laboratory conditions and analyzed its consequences on the sperm quality.    Abstract  Bacterial contamination of bovine ejaculates intended for artificial insemination may be reflected in a significant economic loss due to unsuccessful fertilization as well as health issues of the recipients. The  Staphylococcus  genus represents a large part of bacteriocenosis of bovine ejaculates. Therefore, this study aims to get a closer look on the effects of  Staphylococcus -induced bacteriospermia under in vitro conditions on bovine sperm quality. Prior to inducing bacteriospermia, spermatozoa were separated from each ejaculate using Percoll ®  Plus gradient medium in order to limit the effects only to the selected bacterial species. Seven  Staphylococcus  species previously isolated from bovine semen were used for our experiments at a turbidity of 0.5 McFarland (equivalent to 1.5 × 10 8  colony-forming units per mL). The contaminated semen samples were incubated at 37 °C and at times of 0, 2, and 4 h, motility, mitochondrial membrane potential, reactive oxygen species (ROS) generation, sperm DNA fragmentation, and magnesium (Mg) and calcium (Ca) extracellular concentration were analyzed and compared with the control group (uncontaminated). The results showed no significant changes at the initial measurement. However, significant adverse effects were observed after 2 h and 4 h of incubation. Most notably, the presence of  S. aureus ,  S. warneri ,  S. kloosii , and  S. cohnii  caused a significantly increased ROS production, leading to sperm DNA fragmentation, changes in the mitochondrial membrane potential, and a decreased sperm motility. Furthermore, the presence of  Staphylococcus  species led to lower extracellular concentrations of Mg and Ca. In conclusion, the overgrowth of  Staphylococcus  bacteria in bovine semen may contribute to oxidative stress resulting in sperm DNA fragmentation, altered mitochondrial membrane potential, and diminished sperm motility.

Staphylococcus-Induced Bacteriospermia In Vitro: Consequences on the Bovine Spermatozoa Quality, Extracellular Calcium and Magnesium Content