1 Introduction

The intestine is an important organ in the defense against harmful endogenous and exogenous agents (Blikslager et al., 2007). However, many factors such as pathogens, polyunsaturated fatty acids, and mildewed or oxidized feed can induce the oxidative stress (Slater, 1984; Halliwell and Chirico, 1993; Moreto and Perez-Bosque, 2009), especially in neonatal piglets (Chamulitrat et al., 1996; Aw, 1999; Lackeyram et al., 2012). These “stressors” induce intestinal injury and dysfunction by producing excessive reactive oxygen species (ROS) and destroy the antioxidant capacity in neonatal piglets (Moreto and Perez-Bosque, 2009; Maeda et al., 2010). Also, a series of pro-inflammatory cytokines such as tumor necrosis factor-α (TNF-α) and interleukins-1α, -1β, -6 (IL-1α, IL-1β, IL-6) also are produced (Kelley, 2001). Oxidative stress involving ROS production is an important mediator of inflammation and immune activation (Aw, 1999; Ypsilantis et al., 2008), and ROS can cause injury to the intestinal mucosa (Mercer et al., 1996), but the mechanisms are not understood completely. The present study was designed to explore intestinal oxidative stress, provoked by including oxidized fish oil in the diet of young piglets, in an attempt to explain its influence on the development and immune function of the intestinal mucosa. Oxidative stress is lessened by the antioxidants (Otani et al., 2000; Kelley, 2001). Accumulating evidence suggests that supplementation of additives with antioxidant function (such as vitamin E, selenium, glutamine, and arginine) to the feed of piglets (Ban et al., 2004; Huang et al., 2011; Xi et al., 2011; Zhan et al., 2011) can reduce the oxidative stress and protect the intestinal function. Soybean isoflavones (ISFs) are stronger antioxidants, and have been studied extensively (Yousef et al., 2004; Foti et al., 2005; Lee et al., 2005; Jiang et al., 2007; Dixit et al., 2012). ISFs may inhibit the production and enhance the clearance of free radicals by inducing endogenous antioxidant enzymes such as superoxide dismutase (SOD), glutathione peroxidase (GSH-Px), catalase (CAT), etc. These results led us to speculate that dietary ISFs may reduce the intestinal oxidative stress in young piglets. The potential protective effects of ISFs on the intestinal oxidative stress in very young piglets have not been reported. The objective of the present study was to elucidate whether and how dietary ISFs affect the growth performance, intestinal antioxidant capacity, and intestinal function in young piglets fed oxidized fish oil.

2 Materials and methods

2.1 Animals and diets

Seventy-two 4-d-old male piglets Duroc×(Landrace×Yorkshire) were weighed ((1.80±0.04) kg) and randomly allotted to three treatments, each with six replicates of four animals. Each replicate of piglets was individually housed in 1.5 m×1.0 m pens with slatted stainless steel floors in a room held at (32±1) °C with constant humidity. Piglets were fed a diet formulated to meet the requirements of NRC (2012) recommendation for 3–5 kg piglets (Table 1). The lipid source for the control group was 50 g/kg fresh fish oil (Beihan Evergreen Aquatic Product Science & Technology Co. Ltd., Guangdong, China), and the other two groups received the same diet except for the substitution with 50 g/kg oxidized fish oil or 50 g/kg oxidized fish oil plus 20 mg/kg ISF (prepared at the Institute of Animal Science, Guangdong Academy of Agricultural Sciences, containing 98.4% glycitein). The fish oil was oxidized, as described by Andrew et al. (1960), and then stored at −20 °C. The peroxide values were 186.89 mmol/kg for the oxidized fish oil and 4.20 mmol/kg for fresh fish oil. Details of ingredient composition and the calculated nutrient content of the diet are provided in Table 1. The diets were mixed with four weight equivalents of water just before each feeding and were provided without restriction every 3 h from 06:00 to 24:00. Piglets had free access to water. The animals quickly became accustomed to the feeding method and consumed each meal without spillage. Feed intake (on an as-fed basis) per pen was calculated based on 25% of the weight difference of the feed before and after each feeding (dry weight/liquid weight=1:4). The body weights (BWs) of all piglets were measured on the 1st day and the 21st day of the experiment. Average daily gain (ADG), average daily feed intake (ADFI, dry weight), and gain/feed (G:F) ratios were calculated.

Table 1 Ingredients and composition of the basal diet for young piglets (dry matter content)
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2.2 Sample collection

Two randomly chosen piglets from each pen were fasted for 12 h and then sacrificed after weighing on the 21st day of experiment by muscular injection of sodium pentobarbital (40 mg/kg of BW). The small intestine, immediately ligated from the pyloric sphincter to the ileocolonic junction, was rapidly removed along the mesenteric border on an ice-cold metal plate. The segment of small intestine proximal to the ligament of Treitz was designated as the duodenum; the remainder was divided into two equal lengths, jejunum proximal and ileum distal, as described by Burrin et al. (2000). A 5-cm piece from the middle of the jejunum was opened longitudinally and gently but thoroughly cleaned with ice-cold phosphate buffered saline (PBS) containing 137 mmol/L NaCl, 2.7 mmol/L KCl, 10 mmol/L Na2HPO4, and 2 mmol/L KH2PO4, pH 7.4. Jejunal mucosa was collected by scraping with a glass slide, and a portion was snap-frozen in liquid nitrogen.

2.3 Small intestinal morphology

Additional samples were collected from consistent locations (the duodenal sampling site was located 2 cm from the opening of the pancreatic duct, and the ileum sampling site was located 2 cm from the cecum) of each segment of the intestine, rinsed as above, and then fixed in 10% neutral buffered formalin overnight or longer. Paraffin-embedded sections were prepared from each sample in duplicate, stained with hematoxylin and eosin, and used for histometric measurements, as described by Jensen et al. (2001). The mean villous height, crypt depth, and intestinal circumference were measured using an Axiophot microscope (Philips, Holland) and National Institutes of Health (NIH) image software version 1.60 (Bethesda, MD, USA) in at least 20 vertically well-oriented villus-crypt columns. The villous area was calculated as described by Frankel et al. (1993).

2.4 Analyses of antioxidant enzymes and cytokines

The frozen jejunal mucosa was homogenized in PBS (9 ml added per gram tissue) at 4 °C using an Ultra-Turrax T8 homogenizer (IKA Labortechnik, Staufen, Germany) for 1–2 min at 3000–5000 r/min. The homogenates were centrifuged (4000g for 5 min at 4 °C) and the supernatants were used for determining the indices of antioxidant capacity and concentrations of cytokines. The antioxidant indicators, namely GSH-Px, SOD, CAT, GSH, oxidized glutathione (GSSG), and malondialdehyde (MDA), were quantified using assay kits (Nanjing Jiancheng Bioengineering Institute, China), previously validated in house for porcine tissue. Secretory immunoglobulin A (sIgA), IL-1β, IL-2, IL-4, IL-10, interferon-γ (IFN-γ), TNF-α, inducible nitric oxide synthase (iNOS), and nuclear factor κ B (NF-κB) were measured using porcine enzyme-linked immunosorbent assay (ELISA) kits (Groundwork Biotechnology Diagnosticate, San Diego, CA, USA) through an automated ELISA reader (Model 550, Bio-Rad, Hercules, CA, USA).

2.5 Statistical analysis

All data were analyzed using replicates as the experimental unit (n=6). The effect of treatment on each variable was analyzed by one-way analysis of variance (ANOVA) using the GLM procedure of SAS (Version 8.2, SAS Institute, Cary, NC, USA). P≤0.05 was considered to be statistically different.

3 Results

3.1 Growth performance

The effects of feeding ISF and oxidized fish oil for 21 d on growth performance of young piglets are shown in Table 2. The final BW, ADG, or G:F ratio of piglets fed oxidized fish oil was significantly lower than that of piglets in control group (P<0.05), while the final BW, ADG, or G:F ratio of piglets fed oxidized fish oil plus ISF has a trend to increase compared with those piglets fed oxidized fish oil. There was no difference in ADFI among treatments (P>0.05).

Table 2 Effects of dietary soybean isoflavone addition on growth performance of young piglets fed a diet with oxidized fish oil
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3.2 Small intestinal morphology

The piglets fed oxidized fish oil had significantly lower villous height in the duodenum, jejunum, and ileum than the piglets fed fresh fish oil and those fed oxidized fish oil plus ISF (P<0.05; Table 3). Crypt depth, villous height/crypt depth, and villous area in the jejunum were not significantly different among the three treatments (P>0.05).

Table 3 Effect of dietary soybean isoflavone addition on morphology of intestine in young piglets fed a diet with oxidized fish oil
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3.3 Antioxidant capacity in jejunal mucosa

The effects of ISF and oxidized fish oil for 21 d of feeding on the antioxidant capacity in the jejunal mucosa of neonatal piglets are shown in Table 4. The levels of MDA and GSSG in piglets fed fresh fish oil diet and oxidized fish oil plus ISF diets were all significantly lower than those in piglets fed oxidized fish oil diet alone (P<0.05). The ratio of GSH to GSSG was higher in piglets fed fresh fish oil diets than in those fed oxidized fish oil alone or oxidized fish oil plus ISF (P<0.05). The activity of GSH-Px in piglets fed oxidized fish oil plus ISF was significantly higher than that in piglets fed oxidized fish oil along or fresh fish oil along. There were no differences in SOD, CAT, or GSH among three treatments (P>0.05).

Table 4 Effect of dietary soybean isoflavone addition on antioxidant indices of jejunal mucosa in young piglets fed a diet with oxidized fish oil
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3.4 Cell-mediated immunologic factors

Piglets fed oxidized fish oil diet had lower concentrations of sIgA and higher IL-1 β and IL-2 in jejunal mucosa than piglets fed fresh fish oil and those fed oxidized fish oil plus ISF (P≤0.05; Table 5). Piglets fed oxidized fish oil diet had higher concentrations of TNF-α in jejunal mucosa than piglets fed fresh fish oil (P<0.05), whereas its concentrations were numerically lower in piglets fed oxidized fish oil plus ISF diet, but did not differ from those fed fresh fish oil. All piglets fed oxidized fish oil diets had significantly lower IL-4 concentrations than those fed fresh fish oil, but no significant differences from the groups supplemented with oxidized fish oil and oxidized fish oil plus ISF. There were no significant differences in jejunal concentrations of IL-6, IL-8, IL-10, or IFN-γ among the three treatments.

Table 5 Effect of dietary soybean isoflavone addition on the cell-mediated immunologic factors of jejunal mucosa 1 in young piglets fed a diet with oxidized fish oil
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3.5 Jejunal mucosal NF-κB, iNOS, NO, and Caspase-3

Piglets fed oxidized fish oil diet had higher jejunal concentrations of NF-κB, iNOS and Caspase-3, and lower NO than piglets fed fresh fish oil (P≤0.05; Table 6), and supplemental ISF decreased the concentrations of NF-κB in jejunal mucosa and partially offset these differences on the concentrations of iNOS, Caspase-3 and NO between the two groups fed fresh fish oil and oxidized fish oil.

Table 6 Effect of dietary soybean isoflavone addition on the co-oxidation and immune-related factors of jejunal mucosa 1 in young piglets fed a diet with oxidized fish oil
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4 Discussion

This is the first study to demonstrate that dietary ISF could partly alleviate the oxidative stress induced by oxidized fish oil by improving the intestinal morphology, antioxidant capacity, and immune function in young piglets. Our findings support the potential of ISF as an intestinal protective agent in young piglets with its attenuation of intestinal damage induced by oxidative stress. Although several studies have evaluated the effectiveness of ISF on piglets (de Wilde et al., 2004; Mau et al., 2008; Goerke et al., 2012), none of these studies examined the effectiveness of ISF treatment on the oxidative stress and immune function of the piglets.

Numerous studies have shown that feeding oxidized fish oil to experimental animals can result in excessive production of ROS and MDA, the reduction of SOD, and the induction of oxidative stress (John et al., 1959; Liu and Lee, 1998; Aw, 1999; VázquezAñón and Jenkins, 2007). In the present study, the content of MDA in the jejunal mucosa of piglets fed oxidized fish oil was significantly higher, and the activity of GSH-Px and ratio of GSH/GSSG were lower than those of piglets fed diets containing fresh fish oil. The lower intestinal morphometry, especially the reduction in duodenal villous height, is consistent with oxidative damage to the intestine, thus providing a potentially valuable model of the influence of oxidative stress on intestinal development and the associated function.

Previous studies have indicated that ISF improved the antioxidant activity in the breast muscle of male broilers fed oxidized fish oil (Jiang et al., 2007). Liu et al. (2005) also used soy ISF extracts to elevate the activities of many antioxidant enzymes in rats fed diets containing partially oxidized oil and proved that ISF alleviated the negative effect of oxidized fish oil. Our present results show that the balance of antioxidant/oxidant status in the intestine of piglets is disturbed by feeding oxidized fish oil and partially reverted by feeding ISF. The concentrations of MDA and GSSG in piglets fed oxidized fish oil increased, while they were decreased by the inclusion of ISF in the diet. The activity of GSH-Px changed in the opposite direction. These findings indicate that intestinal oxidative stress provoked by feeding oxidized fish oil is reduced by ISF.

Our present results show that the final BW and ADG are significantly lower in piglets fed oxidized fish oil diet, but the addition of ISF restores BW and ADG to the levels of the control group fed fresh fish oil. Note that the high values of the G:F ratio result from the feed being of very high dry matter (DM) content compared to the much lower expected DM content of these young pigs. As reported earlier with broilers (Jiang et al., 2007) and piglets (Yuan, 2007), the addition of ISF offsets the deleterious effect of oxidized fish oil on growth performance.

Growth retardation of weaned piglets was related to the reduction of villous height (Cera et al., 1988). Yuan (2007) found that oxidative stress caused by feeding oxidized fish oil decreased the villous dimensions in piglets and their growth. The present study demonstrates that the addition of oxidized fish oil to piglets decreases the duodenal villous height, and 20 mg/kg ISF protects the duodenal villi from the deleterious effects of oxidized fish oil. Presumably, ISF, by stimulating antioxidant function, confers some degree of protection on the intestinal mucosa, helping to improve the growth performance.

It has been reported that atrophic changes in the intestinal mucosa, like those caused here by oxidized fish oil, provoke immediate or delayed-type hypersensitivity and mucosal inflammation (Li et al., 1990; 1991). Our results show that sIgA is decreased by feeding oxidized fish oil, whereas supplemental ISF markedly increases the intestinal IgA levels, probably by augmenting B- and T-cell counts (Fan et al., 2010). The IgA-mediated intestinal immune response is controlled by the balance between opposing actions of the Th2-type IgA-stimulating and the Th1-type IgA-inhibiting cytokines from their respective lymphocyte subsets (Kudsk, 2001; Pruett, 2003; Shang et al., 2004). Th1-mediated cytokines such as IL-1β, IL-2, and TNF-α enhance cell-mediated immunity, and are produced by Th1 lymphocytes. A predominant Th1 effect results in the activation of T lymphocytes. Th2 cytokines such as IL-4 and IL-10 enhance humoral immunity, and are produced by Th2 lymphocytes (Kalinski and Moser, 2005). A predominant effect of Th2 lymphocytes results in the activation of B lymphocytes and upregulation of antibody production. There was a shift from Th1 to Th2 profiles of cytokines in the jejunal extracts from the ISF-fed piglets, as the levels of IL-4 declined significantly and those of IL-1β, IL-2, and TNF-α increased, compared to piglets fed oxidized fish oil alone. The pattern of changes in IL-4 levels closely approximated those of IgA (Fan et al., 2010). Deng et al. (2010) reported that diets supplemented with antioxidants decreased the serum levels of inflammatory cytokines in post-weaning piglets. Morimoto et al. (2009) found less inflammation and colonic tissue damage in mice supplemented with ISF than in control mice with experimental colitis, and Kim et al. (2004) found that ISF inhibited IL-1-induced upregulation of NF-κB activation.

Pathogens that cause acute inflammation activate the NF-κB and iNOS pathways, thereby regulating genes encoding pro-inflammatory cytokines, chemokines, and adhesion molecules (Jelínková et al., 2004). Gookin et al. (2002) demonstrated that the villous epithelium is the predominant site of iNOS expression and that the lack of iNOS in porcine decreases the inflammatory responses (Pampusch et al., 1998). Epithelial iNOS expression has likewise been reported in mice experimentally infected with Cryptosporidium parvum (Leitch and He, 1999). Other reports also showed that the epithelium is capable of rapid synthesis of iNOS after acute gut injury (Banan et al., 2001; Gookin et al., 2002). The novelty of the present study is that the concentrations of iNOS and Caspase-3 were increased by oxidized fish oil but the increase was partially alleviated by supplementation of ISF. Caspase-3 also is activated by oxidative stress and helps to increase the inflammatory response (Ozacmak et al., 2014), which is consistent with our result. In addition, the concentration of NF-κB in this present study increased significantly in the young piglets fed oxidized fish oil diets, consistent with the hypothesis that oxidative stress in the intestinal mucosa increases ROS and then increases expression of proinflammatory cytokines via activating NF-κB (Aw, 1999). NF-κB was partially reduced in the piglets fed oxidized fish oil plus ISF compared to that in the piglets fed oxidized fish oil alone, a finding consistent with that from other models (Valachovicova et al., 2004; Li et al., 2005). The most striking benefit was the reduction in NF-κB seen in piglets fed oxidized fish oil plus ISF diets. It appears that NO modulates the phenotype of T-helper cells to promote a shift from Th1 to Th2, accompanied by a decline in pro-inflammatory mediators including IL-2 and by an increase in anti-inflammatory mediators such as IL-4 (Chang et al., 1997; Taylor-Robinson, 1997; Daniel et al., 2006). NO decreased in the jejunal mucosa of piglets fed oxidized fish oil alone, while the decrease was less in the piglets fed oxidized fish oil plus ISF. NO is also involved in activation of Caspase-3 (Parikh et al., 2003), and in this present research NO was decreased in piglets fed oxidized fish oil; probably, the activation of Caspase-3 needs the consumption of NO.

The antioxidant properties of ISF are both direct, by scavenging or quenching oxygen free radicals thereby inhibiting MDA production, and indirect, by preventing increased membrane permeability resulting from oxidative stress, in many tissues including liver and plasma (Jiang et al., 2007; 2011). In conclusion, our results demonstrated that addition of oxidized fish oil increased MDA and decreased GSH-Px activity in the jejunal mucosa, changed the cytokines in jejunal extracts from a pro- to an antiinflammatory profile, and increased sIgA and decreased NF-κB concentrations. Collectively, these data show that dietary inclusion of moderate amounts (20 mg/kg) of ISF is an effective way of reducing oxidative stress in the small intestine of young piglets fed diets containing oxidized fish oil.

Compliance with ethics guidelines

Lin HUANG, Xian-yong MA, Zong-yong JIANG, You-jun HU, Chun-tian ZHENG, Xue-fen YANG, Li WANG, and Kai-guo GAO declare that they have no conflict of interest.

This study was conducted according to the Chinese Guidelines for the Use of Experimental Animals and Animal Welfare (Science and Technology Ministry of China, 2006) and approved by Animal Experimental Committee of Institute of Animal Science, Guangdong Academy of Agricultural Sciences. All institutional and national guidelines for the care and use of laboratory animals were followed.