Fats and oils are usually provided for chicken as dietary energy source to enhance the productivity and fulfill the high energy demand of fast growing broilers. Among the different fats and oils, the high n-6 PUFA sources are cheaper and easier available. Incorporation of these sources resulted in high concentration of the n-6 PUFA in the meat or egg product, and less EPA, (20:5 n-3), docosapentaenioc (DPA 22:5 n-3) and DHA (22:6 n-3) . On the other hand, dietary n-3 PUFA enrichment and lowering n-6: n-3 PUFA ratio alters the fatty acid profile of meat toward long chain PUFA [22, 23]. In agreement, current study results showed that the total n-3 PUFA, EPA and DHA of plasma and breast muscle significantly increased along with ascending n-3 PUFA inclusion from T1 to T4. There was a relatively high level of arachidonic acid (AA, 20:4 n-6) to linoleic acid (LA, 18:2 n-6) in plasma of T1 on day 28 and 42. This phenomenon can be explained by the absence of linolenic acid (LNA, 18:3 n-3) in the diet of this group, while the other groups have LNA of 0.5, 0.6 and 0.6% (Table 2). Simopoulos  indicated that there is a competition between LA and LNA for conversion to the long-chain PUFA because they share the same enzymes and LNA is the preferred substrate. Moreover, there was no detectable DHA in plasma of T1 as their representative diet was also free of EPA and DHA and their precursor LNA.
Dietary n-3 PUFA enrichment also increased body weight and improved FCR as measured in T2 and T3 at 42 days of age. This may be due to the superior digestibility of unsaturated fatty acid compared to saturated type [24–27]. Newman  showed the modulatory effects of dietary n-3 and n-6 PUFA FCR in avian metabolism through the modulation of lipid deposition and oxidation. However, the enhancement was not observed in highest n-3 PUFA (T4) group, leading to the postulation that effect of n-3 PUFA enrichment is a dose-dependent response. Hulan  reported that isoenergetic and isonitrogenous diets enriched with redfish meal and redfish oil led to lower body weights than the control diet. They attributed this result to lower palatability and higher calcium levels, although no palatability problem as a result of n-3 PUFA supplementation in the diet was found in the present study. Evidently, significant differences due to dietary treatment are absent from the feed intake throughout the experimental period. In any case, in accordance Korver  demonstrated that low levels of dietary fish oil were more efficient than high levels in improving broiler growth rate and FCR, even when the birds were undergoing an inflammatory response.
Immune tissue development can in some cases reflect immune system response and functionality. In the current study, there was no affect of dietary treatment on the bursa of Fabricius weight and lesion score, but spleen weight increased as the level of n-3 PUFA in diet increased 7 days post-challenge. Some other studies reported that feeding PUFA to chickens  and mice  results in increased spleen weights. In the study of Wang , the author used single-comb White Leghorn layers fed sunflower oil, animal oil, linseed oil, or fish oil at 5% (wt/wt). The results demonstrated that chicks fed the 3 PUFA-rich diets had significantly higher weights of the thymus, spleen, and bursa compared with those of chicks fed the diet with animal oil. In our study, the spleen enlargement due to dietary n-3 PUFA enrichment accompanied with the same trends of IBD and ND antibody responses, boosting immune system and enhance spleen task. Increasing of the IBD and ND titer can be explained by the fact that IL-2 is able to facilitate production of immunoglobulins made by β cells. Anti-sheep red blood cell antibody responses of rats fed 17 g fish oil were significantly higher than corn oil fed rats . The β cell population is not affected by dietary fat in non-infected mice, but the fish oil enrichment results in the highest percentage of β cells in infected mice . Furthermore, fortifying the diet with n-3 PUFA from fish oil (5%) significantly increased serum IgG concentration and IgM+ B cells in chicken . Immunoglobulin M as the major antibody in the primary immune response reduces in circulating system during the acute lytic phase of the IBD virus [8, 10, 11]. The present study provides the evidences for immuno-stimulating properties of dietary n-3 PUFA in IBD infected chicken through antibody body response improvement and probably IgM-bearing B lymphocyte proliferation.
One of the interesting results of the current study is the ascending elevation of IL-2 and IFN-γ concentrations along with the n-3 PUFA enrichment from T2 to T4. Cytokine production plays an important role in mounting a complete and full immune response of both the innate and specific systems. These cytokines are produced by T helper lymphocytes (Th1) and activate macrophages, NK cells, and cytotoxic T lymphocytes and are the principal effectors of cell-mediated immunity. Interactions with bacteria, viruses, and fungi tend to induce Th1 activity. Because Th1 cytokines activate monocytes and macrophages, these cytokines may be regarded as pro-inflammatory. Interlukin-2 can enhance interferon production [34, 35]. Interferon-γ is a cytokine that is critical for innate and adaptive immunity against viral and intracellular bacterial infections. A close relationship exists between cytokine synthesis and n-3 fatty acids. It is possible that eicosanoid production derived from n-3 PUFA might stimulate the production of IL-2 and IFN-γ [36, 37]. At present, the mechanisms by which dietary n-3 PUFA modulate cytokine production is not clarified. However, the possible mechanism might be the decreased production of metabolites of n-6 PUFA, such as PGE2 and changes in phospholipids composition of immune cell membranes. Some studies suggested the PGE2 which is derived from arachidonic acid and n-6 PUFA as the possible candidate for inhibition of T cell proliferation, Th1 cell, IL-2 and IFN-γ production [38–40]. The PGE2 primes human naive T cells in a dose-dependent fashion for production of high levels of IL-4, IL-10, and IL-13 and very low levels of IL-2 and IFN-γ . Furthermore, some researchers noted that high levels of dietary fish oil apparently have different immunomodulatory effects than lower levels . It is reported that n-3 PUFA decreases production of PGE2 due to a competition between n-3 PUFA and n-6 PUFA for incorporation into the cell membrane phospholipids. This finding is in contrast with the result of dietary fish oil supplementation in human [4, 36] and rat . However, these findings are consistent with the effects of dietary supplementation in non-human primates  in human [43, 46] and mice . A possible explanation for the contrast in results between the present study and other reported studies may referred to the intake levels EPA and DHA, source and type of oil and the concentration of dietary fat. There is another highlight in the current study results on the IL-2 and IFN-γ concentration where high level of these cytokines observed in T1 chickens that received the lowest amount of n-3 PUFA. There is no clear explanation for the phenomenon, but it may speculated that this observation is contributed to the dose-response characteristic of the immune functions to n-3 PUFA levels [41, 42] or low level of linoleic acid in T1 compare to the other groups. Linoleic acid reported to inhibit the proliferation of rodent and human lymphocytes and decrease the production of IL-2 by mitogenstimulated rat and human lymphocytes [5, 48, 49], suggesting that it is potentially immunosuppressive.