*2.4. Periodontal Evaluation*

The periodontal examination was blinded and performed by a trained veterinarian. The clinician determined the dental plaque and gingivitis scoring and performed six measures of the distance between the gingival margin and the bottom of the periodontal pocket (periodontal pocket depth—PPD) of each tooth (three measures were performed in the vestibular face and the other three in the palatine face) [16,17]. Every tooth in each animal was evaluated. The indexes used are presented in Tables 2 and 3.


**Table 2.** Dental plaque index (DPI) based on Holmstrom et al. [16].

**Table 3.** Gingivitis index (GI) according to the modified Talbott method [17].


#### *2.5. Dental Plaque Sample Processing*

A dental plaque sample was collected from all dogs on day 0 and day 90 using a swab (AMIES, VWR, Amadora, Portugal), which was applied to the entire dental surface. Swabs were transported to the Laboratory of Microbiology and Immunology, Faculty of Veterinary Medicine, University of Lisbon, and processed for total bacterial quantification, according

to Belo et al. (2018) [18]. Briefly, the collected swabs were placed in test tubes with 1 mL of sterile saline and vortexed, and the resulting suspension was diluted (10−<sup>1</sup> to 10<sup>−</sup>8). From each dilution, 100 μL were collected and inoculated on Brain Heart Infusion agar (VWR, Amadora, Portugal) and incubated at 37 ◦C for 48 h. Afterward, bacterial quantification was performed by determining the colony-forming units [18].

#### *2.6. Statistical Analysis*

Statistical analysis of the data was carried out using RStudio® software version 1.1.383 (Boston, MA, USA) and Microsoft Excel 2016® (Redmond, WA, USA). Variables were evaluated by plotting the data into a histogram to confirm that they followed a normal distribution. Linear mixed models were used to evaluate differences in the dependent variables: mean PPD, GI, and DPI. In the mixed model, the variables group, timepoint, and tooth type (superior or inferior, incisive, canine, pre-molar, and molar) were defined as fixed effects, and the variables animal, weight, and tooth number were considered random effects. The interaction between fixed effects was investigated, and the Akaike Information Criterion was used to select the model.

Quantitative variables were expressed as mean values ± standard deviation. A confidence interval of 95% was considered in this study, with a *p*-value ≤ 0.05 indicating statistical significance.
