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Background and Aim: Chlamydophila spp. affect Leydig and Sertoli cells by dysregulating spermatogenesis, inducing apoptosis and sperm DNA fragmentation, as well as benign prostate hyperplasia. Canid herpes virus 1 (CHV-1) infection in male dogs is manifested by lesions on the base of the penis and foreskin. There is a lack of information on the influence of these microorganisms on the quality of canine semen. Seroprevalence of Chlamydophila spp. (55%-61%) and CHV-1 (22%-81%) in Europe is high. The prevalence of Chlamydophila spp. and CHV-1 has been evaluated using polymerase chain reaction (PCR) only in Sweden and Croatia, respectively. No positive samples were detected in either case. The aim of this study was to evaluate the epidemiological situation in Polish male dogs (PMDs) to provide a solution to limit the spread of these microorganisms using assisted reproduction techniques or elimination from the reproduction of CHV-1 carriers. In addition, we assessed the semen quality of Chlamydophila spp. carriers and CHV-1 carriers. Materials and Methods: Cotton swabs were collected from prepuce or semen from each dog (n = 130). Real-time PCR for Chlamydophila spp. and CHV-1, as well as semen analysis, was performed using the computer-assisted semen analysis system. Results: To the best of our knowledge, this is the first report of Chlamydophila spp. infection in PMD confirmed by realtime PCR. All parameters, except progressive movement in Chlamydophila semen carriers, were normal. Conclusion: The average velocity values for a dog with Chlamydia are detailed. No CHV-1 was detected. The results achieved should be verified on the basis of a larger number of studies. However, the high prevalence of these pathogens in the PMD population has not been established. [ABSTRACT FROM AUTHOR]