Methodological Factors Affecting Canine Vaginal Cytology: The Influence of Sampling Site, Swab Condition, and...

Vaginal cytology is widely used for cycle staging in bitches because of its practicality and low cost; however, its diagnostic reliability is strongly influenced by methodological variability. The present study aimed to evaluate the impact of sampling site, swab type, and fixation method on the interpretation of exfoliative vaginal cytology.

Exfoliative vaginal cytology samples were obtained from 332 clinically healthy bitches representing all stages of the estrous cycle. Samples were collected from different locations (vestibulum vs. vagina) using either dry or saline-moistened swabs and prepared as air-dried or spray-fixed smears, followed by eosin-thiazine staining. Clinical examination and serum progesterone concentrations served as the reference method for cycle staging.

The proportions of different cell types were significantly influenced by the sampling location. Vestibular smears contained higher proportions of keratinized and intermediate cells (p < 0.0001), whereas basal cells (p = 0.0078) and anuclear superficial cells (p < 0.0001) were more frequently observed in vaginal smears. Overall, sampling location significantly affected the distinction between proestrus and estrus. Swab condition significantly influenced smear quality and cellular presentation but had minimal impact on cycle stage classification. Smears obtained using saline-moistened swabs showed significantly higher cellular yield per microscopic field (p < 0.0001). Bacteria, cellular debris, and secretory material were less frequently observed in moistened smears (p < 0.0001), whereas free nuclei were detected more frequently (p < 0.0001). Cotton fibers were more common in dry swab preparations (p < 0.0001). Cellular arrangement also differed between sampling methods (p = 0.003). The fixation method influenced certain morphological features but did not affect cytological stage classification. No significant differences in total cellular yield were observed between air-dried and spray-fixed smears.

Among the factors evaluated, only the sampling location exerted a clinically relevant effect on cycle stage determination, particularly for distinguishing proestrus from estrus.