BackgroundSuckling and weaning arachidonic acid (ARA)+docosahexaenoic acid (DHA) supplementation promoted oral tolerance (OT) development in pups, however, the effect of it on the intestine to promote OT development remains unknown. ObjectiveWe aimed to explore the impact of this supplementation on intestinal fatty acid composition, structure, and indicators that are supportive of OT development. MethodsAllergy-prone Brown Norway dams were randomized to a control (0%ARA, 0%DHA) or ARA+DHA diet (0.45%ARA, 0.8%DHA) during suckling (0-3wks). At weaning (3-8wks), offspring were randomized to a control (0%ARA, 0%DHA) or ARA+DHA diet (0.5% ARA, 0.5%DHA). At 3wks, offspring in each group received an oral gavage of sucrose or ovalbumin (OVA) solution for 5 consecutive days. At 7wks, all offspring received an intraperitoneal OVA injection. At 8wks, offspring were terminated to evaluate jejunum morphology and measure mucosal food allergy-related sIgA and cytokines, ileum phospholipid and triglyceride fatty acid compositions and fecal calprotectin. ResultsWeaning ARA+DHA resulted in a higher percentage of DHA in ileum phospholipids and triglycerides (both P <0.001), without affecting the percentage of ARA. Despite no lasting effect of suckling ARA+DHA on the DHA content in ileum phospholipids, a programming effect was found on the allergy-related intestinal immune profile (higher levels of mucosal IL-2 (P =0.049) and sIgA (P =0.033)). OVA treatment resulted in a lower level of mucosal IL-6 (P =0.026) regardless of dietary interventions. Offspring fed ARA+DHA during suckling and/or weaning had a higher level of mucosal TGF-β after OVA treatment but this was not observed in offspring fed control diets during suckling and weaning (P =0.04). ConclusionsEarly-life dietary ARA+DHA supplementation to allergy-prone rats enhanced the DHA level in intestinal phospholipids (weaning period) and increased the mucosal sIgA, IL-2 and TGF-β levels (suckling and weaning period), indicating its ability to create a tolerogenic intestinal environment to support OT development.