This study focused on the identification of naturally occurring bacteria in the reproductive fluid and impact on the quality of ejaculates obtained from the turkey breed British United Turkeys (BUT) Big 6 (n = 60). We determined possible relationships between the bacterial load and advanced sperm quality parameters that are important for effective artificial insemination and high fertility, as well as the concentration of selected antimicrobial proteins and pro-inflammatory markers of turkey semen. Sperm motility was assessed with computer-assisted sperm analysis (CASA), while the membrane and acrosome integrity were examined with smearing and staining methods. Reactive oxygen species (ROS) generation was quantified via luminometry, sperm DNA fragmentation was evaluated using the TUNEL assay, and the JC-1 assay was applied to evaluate the mitochondrial membrane potential. Cell lysates were prepared to investigate the extent of lipid and protein oxidation. Furthermore, levels of interleukins 1 and 6 (IL-1, IL-6), C-reactive protein, cathelicidin, and β-defensin were quantified in the seminal plasma using the ELISA method. The most dominant species identified by the matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry was Escherichia coli, Proteus mirabilis, Staphylococcus lentus, and Citrobacter braakii. The bacterial load had a negative effect on the sperm motility (p < 0.001), as well as membrane (p < 0.05) and acrosome integrity (p < 0.01). A strong positive relationship between the bacterial load and DNA fragmentation (p < 0.001) was detected as well. Positive associations were recorded between the increasing presence of bacteria, ROS overgeneration (p < 0.001), and a subsequent oxidative damage to the proteins (p < 0.001) and lipids (p < 0.01). It was revealed that the antimicrobial peptides β-defensin (p < 0.001) and cathelicidin (p < 0.001) had a positive relationship with the motility. In contrast, pro-inflammatory markers, such as IL-1 (p < 0.001) and IL-6 (p < 0.001), had a negative impact on the motion behavior of turkey spermatozoa. Our results suggest that the semen quality may be notably affected by the bacterial quantity as well as quality. It seems that bacteriospermia is associated with inflammatory processes, oxidative stress, sperm structural deterioration, and a subsequent risk for a failed artificial insemination in turkey breeding.

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