The anti-inflammatory properties of CBD make hemp oil a promising adjunct therapy in veterinary medicine. A formulation of hemp oil containing CBD and CBDA may have superior antiinflammatory effects than CBD alone. Notably, CBDA inhibits cyclooxygenase-2 (COX-2), attenuating inflammation. However, specific anti-inflammatory effects of a mixed CBD/CBDA hemp oil remain unknown in the dog. This study includes three arms of testing to broadly assess the impact of CBD/CBDA on ex vivo and in vitro inflammatory responses using blood from 6 research colony dogs. First, we will test the effects of CBD, CBDA, and mixed CBD/CBDA (mCBD) on canine neutrophil function ex vivo. PMA-induced reactive oxygen species (ROS) generation, phagocytosis of fluorescent Ig-coated latex beads, and eicosanoid concentrations (PGE2 and LTB4) will be measured with commercially available kits my lab routinely uses. The Boyden chamber method will quantify neutrophil chemotaxis to recombinant canine IL-8. Second, we will evaluate the effect of CBD/CBDA on T cell function. We will measure the proliferative response of mitogen-stimulated T cells producing pro inflammatory (IFNgamma, IL-2, IL-4, IL-10, IL-17) and anti-inflammatory (IL-10) cytokine production ex vivo with optimized canine-specific EliSpot assays. Third, we will measure the effects of CBD, CBDA, and mCBD on canine fibroblast response as resident cells of most tissue types can elaborate eicosanoids and cytokines in response to pathogen components such as LPS from gram-negative bacteria. We will measure the effect of CBD/CBDA on LPS stimulated fibroblast eicosanoid production, cytokine production with a commercially available inflammatory array, and proliferation with the MTT assay in vitro. We hypothesize that the combination of CBD and CBDA will have superior anti-inflammatory effects compared to CBD alone. The proposed study lays the groundwork for future studies evaluating the anti-inflammatory effects of CBD/CBDA in vivo.