Supplemental Materials: Reisinger et al., 2025

n-3 Fatty acids have anti-inflammatory effects across species, and within the n-3 fatty acid family, different fatty acids have unique effects on immune responses. The objective of this study was to assess circulating leukocyte phenotypes and their ex vivo immune response to challenge following abomasal infusions of the n-3 fatty acid docosahexaenoic acid (DHA; C22:6). Eight mid-lactation Holstein cows (97 ± 37 DIM) were randomly assigned to treatment sequence in a 4 × 4 Latin square design. Treatments were 0, 2, 4, and 6 g/d DHA (supplied via an enriched oil containing 65% DHA) infused into the abomasum for 11 d. Blood samples were collected on the final 2 d of each treatment period. Circulating leukocytes and phenotypes were first assessed in the absence of any challenge. Additionally, whole blood was stimulated with <i>Escherichia coli</i> or <i>Staphylococcus aureus</i> bioparticles in the presence of dihydrorhodamine 123 to assess bacterial uptake and oxidative burst of neutrophils by flow cytometry. A separate whole-blood sample was stimulated with heat-killed <i>S. aureus</i> and plasma was collected to quantify cytokine concentrations. Increasing DHA dose reduced circulating eosinophil concentrations in a linear manner and tended to affect lymphocyte concentrations in a quadratic manner. Cytotoxic T and helper T cell populations, along with the abundance of L-selectin on these populations, showed quadratic effects, with peak abundance in cows receiving 2 to 4 g/d DHA. The γδ T cells responded in a cubic manner, with peak concentrations in cows receiving the 4 g/d DHA dose, whereas L-selectin expression in γδ T cells linearly decreased with increasing DHA dose. Unstimulated blood samples were affected by DHA dose in a cubic manner for IL-1α and VEGF-A and a quadratic manner for TNF and IL-36RA, where highest concentrations were observed at 2 g/d DHA. During whole-blood stimulation with <i>S. aureus</i>, MCP-1 concentration increased linearly with increasing DHA dose and TNF responded in a cubic manner, with a nadir at the 6 g/d DHA dose. Following whole-blood stimulation, DHA dose linearly increased the proportion of neutrophils with uptake of <i>E. coli</i> bioparticles, whereas DHA dose did not affect the proportion of neutrophils with uptake of <i>S. aureus.</i> These results indicate that despite the anti-inflammatory effects of DHA, neutrophil response is not blunted following bacterial stimulation. Further studies are required to assess the effects of DHA on in vivo immune responses.