2.1. Animals

Client-owned dogs were transferred to the Veterinary Clinic of the University Center of Veterinary Medicine JU-AU, University of Agriculture in Cracow, Poland in connection with reproductive problems, estrous monitoring, determination of mating date, or routine gynecological examination of breeding bitches. Eight healthy bitches (4 in proestrus/oestrus, 1 in diestrus and 3 in anestrus) and eight bitches with vaginitis (2 in proestrus and 6 in anestrus) were qualified for this study (mixed breeds, aged from 1 to 7 years). The qualification standards were set according to a protocol described by Golińska et al. [2]. Briefly, qualification was based on a clinical and gynecological examination, which included vaginal cytology. Subsequently, the phase of the estrous cycle was determined according to the guidelines described by Concannon [19]. These examinations were carried out by an experienced veterinarian. The bitches with mucusy, milky-white, yellowish, or greenish vaginal discharge and the presence of numerous neutrophils in a cytological smear were classified as animals with vaginitis. In bitches with vaginitis, owners often observed increased licking of the vulva by the animal and polydipsia/polyuria. Moreover, strands of mucus in the cytological smear were visible. The bitches without vaginitis had physiological vaginal discharge (from blood to straw colored) or no discharge at all. The presence of a few neutrophils in the cytological smear was acceptable in two cases: at the beginning of the proestrus phase (during bleeding) and during the diestrus phase, when the bleeding had ceased. To the best of our knowledge, no additional detailed classifications of canine vaginitis have been published to date. Only bitches in good condition, showing no systemic or organ diseases (excluding symptoms of vaginitis), were selected for this study, and bitches undergoing any treatment were excluded. The animals were not given antibiotics for at least 2 weeks prior to collection. The owners were informed about the purpose of the study and gave their written consent for their dogs to participate in the study.