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Background: Tracheal collapse (TC), a common disease in dogs, is characterized by cough; however, little is known about the serum biomarkers that can objectively evaluate the severity of cough in canine TC. Furthermore, studies elucidating the relationship of fluoroscopic characteristics with the severity of cough are lacking. Therefore, this study aimed to evaluate the relationship between cough severity and clinical characteristics, fluoroscopic images, and new serum biomarkers in canine TC. Results: Fifty-one client-owned dogs diagnosed with TC based on fluoroscopic and clinical signs were enrolled in this study and divided into three groups according to the severity of cough (grade of cough: 0, 1, and 2). Signalments, comorbidities, and fluoroscopic characteristics were compared among the groups retrospectively. The serum matrix metalloproteinase-9 (MMP-9), interleukin-6 (IL-6), surfactant protein-A (SP-A), and syndecan-1 (SDC-1) levels were measured in all groups. No significant differences in age, breed, sex, or clinical history were observed among the groups. Concomitant pharyngeal collapse increased significantly with the severity of cough (p =.031). Based on the fluoroscopic characteristics, the TC grade of the carinal region increased significantly and consistently with the grade of cough (p =.03). The serum MMP-9 level was significantly higher in the grade 2 group than that in the grade 0 group (p =.014). The serum IL-6 level was significantly lower in the grade 1 group than that in the grade 0 group (p =.020). The serum SP-A and SDC-1 levels did not differ significantly among the groups. Conclusions: The severity of cough with the progression of TC can be predicted with the fluoroscopic TC grade at the carinal region. MMP-9 may be used as an objective serum biomarker that represents cough severity to understand the pathogenesis. [ABSTRACT FROM AUTHOR]