Background: Human milk contains both arachidonic acid (ARA) and docosahexaenoic acid (DHA). Supplementation of infant formula with ARA and DHA results in fatty acid (FA) profiles, neurodevelopmental outcomes, and immune responses in formula-fed infants that are more like those observed in breastfed infants. Consequently, ARA and DHA have been historically added together to infant formula. This study investigated the impact of ARA or DHA supplementation alone or in combination on tissue FA incorporation, immune responses, and neurodevelopment in the young pig. Methods: Male pigs (N = 48 total) received one of four dietary treatments from postnatal day (PND) 2-30. Treatments targeted the following ARA/DHA levels (% of total FA): CON (0.00/0.00), ARA (0.80/0.00), DHA (0.00/0.80), and ARA+DHA (0.80/0.80). Plasma, red blood cells (RBC), and prefrontal cortex (PFC) were collected for FA analysis. Blood was collected for T cell immunophenotyping and to quantify a panel of immune outcomes. Myelin thickness in the corpus callosum was measured by transmission electron microscopy and pig movement was measured by actigraphy. Results: There were no differences in formula intake or growth between dietary groups. DHA supplementation increased brain DHA, but decreased ARA, compared with all other groups. ARA supplementation increased brain ARA compared with all other groups but did not affect brain DHA. Combined supplementation increased brain DHA levels but did not affect brain ARA levels compared with the control. Pigs fed ARA or ARA+DHA exhibited more activity than those fed CON or DHA. Diet-dependent differences in activity suggested pigs fed ARA had the lowest percent time asleep, while those fed DHA had the highest. No differences were observed for immune or myelination outcomes. Conclusion: Supplementation with ARA and DHA did not differentially affect immune responses, but ARA levels in RBC and PFC were reduced when DHA was provided without ARA. Supplementation of either ARA or DHA alone induced differences in time spent asleep, and ARA inclusion increased general activity. Therefore, the current data support the combined supplementation with both ARA and DHA in infant formula and raise questions regarding the safety and nutritional suitability of ARA or DHA supplementation individually.

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