The beneficial effects of FO have been documented abundantly [13–15]. And evidences concerning n-3 PUFA alleviating inflammation were persuasive [16–18]. However, the effect of dietary FO on attenuating mammary inflammation has not been verified. Thus, the present study was focused on the mammary glands to test the anti-inflammatory effect of dietary FO.
Given that inflammatory responses are in strong association with FA types such as n-6 and n-3 PUFA, we firstly examined the plasma FA profile at different reproductive stages of rats receiving different diets. An amplified plasma concentration of n-3 PUFA was observed following FO consumption for 14 days in this study, which agreed well with the previous study in horses by Hall et al. Hall et al also found decreased plasma n-6 FA concentration in the FO-fed horses, but little variation of plasma n-6 PUFA concentration was observed in the FO-fed rats in this study. Noting that the horses used in the study of Hall et al consumed FO for 6 weeks, a shorter consumption period (24 days) in this study may explain the little variation of n-6 PUFA. In addition, it was verified in the present study that substituting SO with FO resulted in higher plasma n-3 PUFA concentration and lower concentrations of SFA and n-6 PUFA. Amira et al reported that decreased n-6/n-3 ratio led to higher plasma n-3 PUFA concentration and lower n-6 PUFA concentration, which was consistent with our results considering that the n-6/n-3 ratio in our experimental diets was approximately 0.5:1 in the FO diet and 10:1 in the SO diet.
Another result in the present study was that up-regulated plasma FFA emerged with the advance of gestation. As FFA have been reported to be associated with inflammation including mastitis [20, 21], we further studied whether the advance of gestation was related to the expression of inflammation mediators. In both diet groups, the mRNA abundances of XOR and IL-8, protein levels of IL-1β and TNF-α as well as PMN prevalence all increased from day 0 of gestation to day 14 of gestation. TNF-α, IL-8 and IL-1β are all known as pro-inflammatory cytokines. Stimulated by a range of agents, TNF-α induces other inflammatory mediators that participated in inflammatory responses . IL-8 can activate neutrophils to degranulate and induce tissue damage . Moreover, IL-1β and TNF-α were elucidated to be key mediators participating in the neutrophil recruitment into the udder . XOR is also an inflammatory indicator that highly expressed in mammary tissues during pregnancy and lactation [25, 26]. Hence, we proposed that the advance of pregnancy was accompanied by inflammatory responses of the udder. Notably, compared with the SO-fed rats, the FO-fed rats had lower mRNA levels of XOR and TNF-α but higher mRNA levels of IL-10 and PPAR-γ, both of which are acknowledged as anti-inflammatory mediators [27, 28]. The down-regulation of XOR and TNF-α may be induced by the lower SFA and n-6 PUFA concentrations and higher n-3 PUFA concentrations in plasma. Consistent with this notion, it has been shown that the decreased production of TNF was accompanied by a decreased ratio of C20:4n6 to C20:5n3 in the membrane phospholipids of mononuclear cells, which indicates the significance of systemic n-6/n-3 PUFA profile in inflammatory responses .
Excited by the potential effect of FO in decreasing pro-inflammatory cytokines in mammary glands, we further determined the anti-inflammatory effect of FO with a rat mastitis model. Rats at day 3 postpartum were infused with LPS or saline. LPS challenge resulted in elevated plasma FFA in both groups indicating the systemic inflammation induced by LPS. However, only in the SO-fed rats did LPS infusion stimulate the expression of IL-1β, XOR and TNF-α. During the process of mammary glands inflammation induced by advance of gestation as well as by LPS infusion, the relatively lower level of mammary pro-inflammatory mediators in the FO-fed rats may result from higher total n-3 PUFA concentration and lower SFA concentration in plasma. During lactation, the mammary blood flow increased sharply  and the mammary glands may become more susceptible to substances in the blood. Therefore, we assumed that the relatively higher SFA concentration in the SO-fed rats may lead to aggravated toxicity of LPS. On one hand, LPS could activate the TLR4 pathway, free NF-κB  and finally facilitate the expression of pro-inflammatory cytokines in the mammary glands of the SO-fed rats. On the other hand, the elevated XOR expression may enhance the generation of ROS which may participate in the TLR4 dimerization and recruitment of TLR4 into lipid rafts on condition that SFA were metabolized into ceramide [9, 10]. The higher levels of PPAR-γ and IL-10 in the FO-fed rats may block the TLR4 pathway, as elucidated previously [32, 33]. It has been demonstrated that DHA specifically enhanced anti-inflammatory IL-10 secretion . Moreover, it has been shown in HK-2 cells that DHA and EPA can activate the mRNA expression of PPAR-γ . Meanwhile, n-3 PUFA and their metabolites are natural ligands for PPAR-γ  and DHA for example can be metabolized by oxygenase to 17-OH and 7-OH-DHA thus facilitates PPAR-γ activation . Therefore, n-3 PUFA can upregulate PPAR-γ expression and promote PPAR-γ functioning. Noticeably, maternal PPAR-γ was demonstrated to be pivotal for protecting the nursing newborns by suppressing the production of inflammatory lipids in the lactating mammary glands . Additionally, it was assumed that IL-10 may inhibit the production of TNF-α and IL-6 in the mammary glands . Therefore, we postulated that FO might down-regulate the mRNA expression of IL-1β, XOR and TNF-α through enhancing the expression of IL-10 and PPAR-γ.