This study evaluated the effect of daily oral administration of CBD oil and CBD gel, each at a daily dose of 4 mg/kg body weight, over a 14-day period on hematological and biochemical parameters, including cortisol, in shelter dogs exposed to acute confinement stress. The selected dose was based on previous studies that demonstrated the safety and therapeutic potential of oral CBD in dogs within the 2–4 mg/kg/day range [23,26]. Based on this evidence, 4 mg/kg was selected as a representative dose for evaluating both safety and biological effects, particularly under chronic stress conditions relevant to the shelter population. Overall, most hematological and biochemical parameters remained within reference ranges for dogs [23] and did not differ significantly between treatment groups or across sampling days (Table 1, Table 2). These findings support the safety profile of CBD at the tested dose, with no adverse effects observed in liver function markers (ALT, AST, ALP) or hematological indices, aligning with previous reports in healthy dogs [6,25,27,28]. Notably, Vaughn et al. (2020) [27] employed a comparable small group size (n = 4 per treatment) and reported no clinically relevant adverse effects even at higher doses up to 16.8 mg/kg, providing additional support for the safety data observed in the present study despite its limited sample size. To compensate for this limitation, repeated measures ANOVA was applied to increase the analytical sensitivity when evaluating time-dependent effects. This design is especially suitable for small animal studies, where using each subject as its own control helps minimize between-subject variability.

It is worth noting that baseline hemoglobin (Hb), hematocrit (HCT), and platelet counts were lower than the standard reference ranges for healthy dogs, a finding commonly reported in shelter populations, where suboptimal health, chronic stress, and nutritional deficiencies are prevalent [24,29,30]. However, these values remained stable throughout the study and did not differ significantly between treatment groups, indicating that CBD supplementation did not negatively impact or further deteriorate the hematological status of these dogs. Furthermore, no adverse hematological effects, such as anemia or leukopenia, were detected during the study period, reinforcing the safety of CBD supplementation in dogs with pre-existing physiological challenges.

Another interesting observation relates to lymphocyte counts, which showed a numerical increase in the CBD-treated groups by Day 14, despite the absence of statistically significant differences between treatment groups (Table 1). This upward pattern in lymphocyte counts, combined with the observation that IgG levels remained comparable across all groups (Table 2), raises the possibility that CBD supplementation may have influenced T-cell populations more than B-cell-mediated antibody production. This finding aligns with previous research showing that cannabinoids modulate immune function primarily through T-cell regulation and cytokine signaling, which may help maintain immune resilience under chronic stress conditions [31,32]. Additionally, these effects may be indirectly mediated by the stress-attenuating properties of CBD, which could help preserve lymphocyte homeostasis by mitigating cortisol-induced immunosuppression [33]. Beyond its role in stress-related immune modulation, CBD has been shown to regulate lymphocyte activity by interacting with immune signaling cascades that influence cell proliferation and apoptosis pathways, supporting its potential immunomodulatory effects [34,35].

This indirect immunomodulatory pathway is further supported by the observed reduction in cortisol levels in CBD-treated dogs in this study, reinforcing the role of CBD in attenuating stress responses. Both CBD oil and CBD gel significantly reduced post-stress cortisol levels compared to controls, demonstrating that CBD may help modulate acute stress responses induced by solitary confinement [25]. On Day 14, for instance, the mean post-stress cortisol level was 5.78 ± 1.33 µg/dL in the control group, compared to 4.98 ± 0.88 µg/dL in the CBDO group and 3.58 ± 0.21 µg/dL in the CBDG group, highlighting a greater reduction in the gel-treated dogs (Table 2). These findings are consistent with previous studies reporting that CBD can attenuate HPA axis activation, reducing stress-induced elevations in cortisol in both animals and humans [6,36,37], with emerging evidence suggesting possible benefits in reducing aggressive behavior in shelter dogs [38].

In addition to its effects on cortisol, Principal Component Analysis (PCA) provided further insight into how CBD supplementation influenced overall hematological and biochemical profiles. The PC1 vs. PC3 biplot demonstrated a clear clustering of samples based on treatment, with dogs in both CBD-treated groups separating distinctly from controls, particularly along PC3. This separation suggests that CBD supplementation—regardless of delivery form—contributed to a shift in blood profiles, potentially reflecting broader physiological adaptations to chronic supplementation in combination with stress reduction. The accompanying PCA loadings plot (Figure 4) revealed that samples from the CON group were more closely associated with neutrophil counts, post-stress cortisol, and ΔCortisol, indicating a heightened acute stress response accompanied by a stress leukogram [39,40]. In contrast, samples from the CBD oil group aligned more closely with albumin, ALP, and WBC counts, while the CBD gel group was associated with BUN, IgG, and lymphocytes, suggesting potential differences in immune modulation and renal-related responses between formulations [6,31,32,41,42]. The successful classification by Support Vector Machine (SVM) further confirms that these multivariate shifts were treatment-dependent, highlighting the value of combining multivariate analysis with machine learning classifiers to capture the comprehensive physiological footprint of CBD supplementation in stressed dogs. An interesting pattern emerges when comparing our multivariate findings with those reported by Mills et al. (2024) [43], particularly regarding ALP responses in dogs receiving CBD oil formulations. Although ALP levels in our study did not differ significantly between treatment groups in univariate analysis, they showed a clear association with the CBD oil group in PCA loadings. Mills et al. (2024) [43] reported a statistically significant increase in ALP following oral administration of a 4 mg/kg CBD-rich hemp extract in MCT oil over a 36-day period in dogs. Their findings—together with our multivariate pattern—suggest that oil-based formulations may may exert a more noticeable effect on hepatic biomarkers. In contrast, the gel-based formulation in our study showed no such ALP alignment and was instead associated with immunological markers, which may indicate a more favorable systemic profile in chronically stressed dogs. It is important to note that PCA was performed on pooled data from all dogs across the three sampling timepoints, allowing for the analysis of integrated hematological and biochemical variation rather than isolated comparisons at each timepoint. While univariate statistical tests, as reported in Table 1 and Table 2, assess differences in individual parameters across groups, PCA, as a multivariate method, summarizes covariation patterns among all measured variables. Although this approach does not directly enhance statistical power, it enables the detection of multivariate trends and group-level clustering that may not be apparent from univariate comparisons alone.

Interestingly, although both formulations provided comparable cortisol-lowering effects, the literature suggests that gel-based formulation may offer additional advantages over the oil in terms of both bioavailability enhancement and practical administration. As a semi-solid oleogel, the gel matrix enhances gastrointestinal retention and promotes lymphatic absorption of lipophilic compounds like CBD, which may contribute to more consistent systemic exposure [12,16,18,44]. This pharmacokinetic advantage could partly explain the observed pattern of lower ΔCortisol in the gel group compared to the oil group, particularly on later sampling days (Figure 3). The data from Table 2 show that on Day 7 and Day 14, both post-stress cortisol levels and ΔCortisol (the change from pre- to post-stress) were numerically lower in the CBD gel group compared to the oil group, even though the differences were not statistically significant. This pattern may suggest that the CBD-infused gel formulation provided more stable or sustained cortisol modulation over time; however, due to the small sample size, further studies are needed to validate this hypothesis. Such patterns are consistent with the proposed stress-attenuating effects of CBD, whereby chronic supplementation helps mitigate acute stress responses, potentially through modulation of the hypothalamic-pituitary-adrenal (HPA) axis and regulation of neuroendocrine signaling [36]. Similar findings have been reported in both canine and human studies investigating CBD’s anxiolytic and anti-stress properties under acute stress challenges [6,45].

In addition to its pharmacokinetic advantages, the gel formulation may also offer practical benefits, including greater ease of handling and improved compatibility with palatable delivery methods. Compared to conventional oil or tablet formulations, oleogel-based delivery systems provide better adhesion within soft food carriers and reduce the likelihood of spillage [46,47], making them particularly attractive for companion animals and other populations with swallowing difficulties, where ensuring complete oral intake is essential for treatment adherence and therapeutic success [46].

Future studies incorporating larger sample sizes and longer supplementation periods are warranted to validate these preliminary findings and explore the long-term immunological and physiological effects of CBD gel in dogs under chronic stress conditions. Expanding the scope of investigation to include functional assays, such as T-cell subset profiling or cytokine analysis, could also provide deeper mechanistic insights into the immunomodulatory actions of CBD. Additionally, research on the impact of gel-based CBD supplementation on feeding behavior would be valuable in assessing its potential role in appetite modulation and nutritional intake in stress-prone dogs.