Effect of Oxidized Soybean Oils on Oxidative Status and Intestinal Barrier Function in Broiler Chickens

The objective of this study was to evaluate the effect of oxidized soybean oils on the growth performance, metabolic oxidative status and intestinal barrier function of broiler chickens. A total of 240 one-day-old female broiler chickens were assigned to four dietary treatments with six replicates (cages) of 10 birds each. The dietary treatments comprised of a basal diet supplemented with 4% of: nonoxidized (fresh) soybean oil (control treatment, SNX); lowly-oxidized soybean oil (SLX) (oil heated for 10h at 200°C); moderately-oxidized soybean oil (SMX) (oil heated for 18h at 200°C); or highly-oxidized soybean oil (SHX) (oil heated for 30h at 200°C). Diets and water were offered ad libitum. The experiment was lasted 21d.The growth performance of broilers, determined from 1 to 14 d and from 1 to 21 d of age, was not affected by the dietary treatments (p>0.05). Broilers fed oxidized soybean oils presented higher corticosterone serum levels compared with those fed non-oxidized oil (p<0.05). Higher malondialdehyde (MDA) levels onday14 and 21 (p<0.05), and lower total antioxidant capacity (T-AOC) and totalsuperoxide dismutase (T-SOD) values on day 21were obtained in the liver of broiler fed oxidized oils relative to those fed the non-oxidized oil (p<0.05). Broilers fed the highly-oxidized soybean oil had higher (p<0.05) MDA levels in the jejunum on day 21 compared with those fed non-oxidized soybean oil. Chickens fed moderatelyand highlyoxidized soybean oil presented lower (p<0.05) T-SOD activity inileal mucosa compared with those fed non-oxidized soybean oil. Ileal mRNA expression of claudin-1 tended to be down regulated by the dietary addition of oxidized oils (p=0.056). The mRNA expression of interleukin-22 (IL-22) of broilers fed moderately-oxidized and highlyoxidized soybean oil was higher (p<0.05), and the mRNA expression of occludin and catalase was lower (p<0.05) than those fed nonoxidized soybean oil. However, the morphology of the jejunal and ileal mucosa was not influenced (p>0.05) by the dietary oxidized oil treatments. It was concluded that oxidized oils may cause oxidative stress by reducing intestinal and liver antioxidant capacity; increase intestinal permeability by reducing mRNA expression levels of tightjunction proteins claudin-1 and occludin; and cause inflammation by increasing mRNA expression level of the inflammation-related factor IL-22.


ABStRACt
The objective of this study was to evaluate the effect of oxidized soybean oils on the growth performance, metabolic oxidative status and intestinal barrier function of broiler chickens.A total of 240 one-day-old female broiler chickens were assigned to four dietary treatments with six replicates (cages) of 10 birds each.The dietary treatments comprised of a basal diet supplemented with 4% of: nonoxidized (fresh) soybean oil (control treatment, SNX); lowly-oxidized soybean oil (SLX) (oil heated for 10h at 200°C); moderately-oxidized soybean oil (SMX) (oil heated for 18h at 200°C); or highly-oxidized soybean oil (SHX) (oil heated for 30h at 200°C).Diets and water were offered ad libitum.The experiment was lasted 21d.The growth performance of broilers, determined from 1 to 14 d and from 1 to 21 d of age, was not affected by the dietary treatments (p>0.05).Broilers fed oxidized soybean oils presented higher corticosterone serum levels compared with those fed non-oxidized oil (p<0.05).Higher malondialdehyde (MDA) levels onday14 and 21 (p<0.05),and lower total antioxidant capacity (T-AOC) and totalsuperoxide dismutase (T-SOD) values on day 21were obtained in the liver of broiler fed oxidized oils relative to those fed the non-oxidized oil (p<0.05).Broilers fed the highly-oxidized soybean oil had higher (p<0.05)MDA levels in the jejunum on day 21 compared with those fed non-oxidized soybean oil.Chickens fed moderately-and highlyoxidized soybean oil presented lower (p<0.05)T-SOD activity inileal mucosa compared with those fed non-oxidized soybean oil.Ileal mRNA expression of claudin-1 tended to be down regulated by the dietary addition of oxidized oils (p=0.056).The mRNA expression of interleukin-22 (IL-22) of broilers fed moderately-oxidized and highlyoxidized soybean oil was higher (p<0.05), and the mRNA expression of occludin and catalase was lower (p<0.05)than those fed nonoxidized soybean oil.However, the morphology of the jejunal and ileal mucosa was not influenced (p>0.05) by the dietary oxidized oil treatments.It was concluded that oxidized oils may cause oxidative stress by reducing intestinal and liver antioxidant capacity; increase intestinal permeability by reducing mRNA expression levels of tightjunction proteins claudin-1 and occludin; and cause inflammation by increasing mRNA expression level of the inflammation-related factor IL-22.

IntRoduCtIon
Oils are added into poultry diets to supply energy and essential fatty acids, as a vitamin vehicle, and to alleviated acute heat stress (Mujahid et al., 2009).Vegetable oils contain large amounts of polyunsaturated fatty acid, such as soybean oil, which is susceptible to peroxidation (Liu  et al., 2014).The fatty-acid composition of lipids used in animal feeds is variable.Lipids used in animal feeds may contain various concentrations of primary and secondary lipid peroxidation products, depending on their fatty-acid composition, storage length, storage conditions, and processing (Totani et al., 2007;Totani et al., 2008).Heated oils contain various amounts of peroxidation products (Zhang et al., 2012), such as 4-hydroxynonenal, hydroperoxide, malondialdehyde, and 2,4-heptadienal (Choe & Min, 2007), which influence oil odor, palatability, and quality (Paul & Mittal, 1997;Poulli et al., 2009;Smyk, 2015).
The consumption of oxidized phosphatidylcholine can cause damage to organs and increase thiobarbituric acid reactive substances levels in the visceral organs of rats (Al-Orf, 2011).The end-product of n-3 PUFA oxidation, 4-hydroxy-2-hexenal (4-HHE), induced oxidative stress and inflammation in mice and human intestinal Caco-2/TC7 cells (Awada et al., 2012).In multiple animal species (Hayam et al., 1997;Kumagai et al., 2004;Yue et al., 2011;Ehr et al., 2015), oxidized oils decrease feed intake, depress growth, and even cause disease.Feeding oxidized fish oil impaired the growth performance and induced oxidative stress in Litopenaeus vannamei (Yang et al., 2015).Feeding auto-oxidized capelin oil impaired growth rates, antioxidant activities, and increased the occurrence of deformed fish in Siberian sturgeon (Acipenserbaeri) larvae (Fontagné et al., 2006).Feeding oxidized fats impaired growth performance by increasing gastrointestinal epithelium cell turnover and hepatic cell proliferation, and increasing the concentration of immunoglobulins in intestinal tissue of broilers and pigs (Dibner et al., 1996).Feeding heat-oxidized lipids impaired the metabolic oxidative status of young pigs by depleting serum α-T and increasing serum TBARS (Liuet al., 2014).
The biological mechanisms to explain these observations are largely unknown and little information has been reported regarding the effect of feeding oxidized soybean oil on the intestinal barrier function of broiler chickens.The following study was conducted to evaluate the effect of oxidized soybean oil on the performance, metabolic oxidative status, and intestinal barrier function of broiler chickens.

Fish Oil Preparation
Fresh soybean oil was purchased from the supermarket and stored in a freezer at -30°C until use.
In order to oxidize soybean oil, vesicles containing the required amount of fresh soybean oil were heated to 200°C for either 10h, 18h, or 30h to produce lowoxidized soybean oil (SLX), moderately-oxidized soybean oil (SMX) or highly-oxidized soybean oil (SHX), respectively.The processed oils were stored at -30°C prior to their addition to feed.No antioxidant was added before or during the manufacturing of the experimental diets.

Birds, Housing and Diets
All experimental procedures were reviewed and approved by the Animal Care and Use Committee of China Agricultural University, Beijing, China.
A total of 240 one-day female broiler chicks were obtained from a commercial hatchery, and assigned to four treatments with six replicate cages of 10 birds each, according to a completely randomized design.The treatments consisted of a basal diet based on corn-soybean meal which formulated to meet or exceed the recommended nutritional requirements of broilers (NRC, 1994; Table 1).No antibiotic growth promoters or antioxidants were added to the basal diet.The experimental diets were produced by adding 4% of four different soybean oil products.The soybean oil products included: fresh soybean oil (control treatment, SNX), lowly-oxidized soybean oil (SLX), moderately-oxidized soybean oil (SMX), and highly-oxidized soybean oil (SHX).The trial lasted 21 days during which birds had ad libitum access to feed and water.Initial environmental temperature (day 1) was 31°C, and daily reduced by 0.5°C until 21°C was reached.Continuous lighting was provided during the entire experimental period.

Growth Performance
Birds and feed offered were weighed per pen on the day of hatch, and day14 and day 21.Feed intake (FI), body weight gain (BWG) and feed conversion ratio (FCR) were calculated for each period. 3Analyzed.

Sample Collection
On days14 and 21, one bird per cage was randomly selected and sacrificed by venous administration of sodium pentobarbital (30mg/kg of body weight) in order to collect blood, jejunal mucosa, ileal mucosa, and liver samples.After the intestines were removed, the digestawas flushed with 4% saline solution, and the mucous membrane was gently scraped to obtain the samples, which were immediately frozen in liquid nitrogen and stored at -35°C until analysis.On d 14 only, a portion of the ileal mucosa sample was immediately frozen and stored at -80°C for mRNA determination.Additionally, on d 14 and d 21, the right lobe of the liver was extracted and stored at -35°C for evaluation of oxidative stress enzyme activity and MDA concentration.On d 14, blood samples were collected by jugular exsanguination.Serum was separated by centrifugation at 3000×g for 10 min at 4°C, and stored at -35°C until analysis.

Serum analysis
Serum corticosterone (CORT) levels were measured using a radioimmunoassay (RIA) kit (Beijing Sino-uk Institute of Biological Technology, Beijing, China) by an automatic biochemical analyzer (Hitachi High-Technologies, Japan).

Oxidative Stress Enzyme Analysis
Intestinal (jejunal/ileal) mucosa and liver samples (~1 g) were homogenized in 10 mL ice-cold saline solution and centrifuged at 20,000×g for 10 min at 4°C.After appropriate dilution, the supernatant fractions were assayed for total superoxide dismutase (T-SOD) and glutathione peroxidase (GSH-PX activities), and total antioxidant capacity (T-AOC) and malondialdehyde (MDA) levels using enzymatic kits (Nanjing Jiancheng Bioengineering Institute, Nanjing, China).In order to prevent possible enzyme degradation, the samples were kept on ice throughout detection.

Intestinal morphology analysis
The fixed intestinal samples were dehydrated and embedded in paraffin wax, cut into 3-μm sections, and stained with hematoxylin and eosin.Villus height and crypt depth of 10 randomly selected complete villi per sample were measured at x40 magnification.Villus height was estimated by measuring the vertical distance from the villus tip to villus crypt junction.Crypt depth was measured as the vertical distance from the villus crypt junction to the lower limit of the crypt.The villus height/crypt depth ratio was then calculated from these measurements.

Gene Expression Analysis
Total RNA was extracted from ileal mucosa (~100mg) using trizol reagent, in accordance with the manufacturer's instructions (Invitrogen Life Technologies, Carlsbad, California, USA).The purity of the isolated total RNA was determined by measuring its optical density at 260 and 280nm.Samples of the extracted total RNA (2 mg) were reverse transcribed using a reverse transcription kit (Invitrogen Life Technologies, Carlsbad, California, USA).The expression levels of targeted genes were measured according to quantitative real-time PCR assay with a 7300 real-time PCR system (Applied Biosystems, Foster City, CA) using Fast Start Universal SYBR Green Master (Roche) after generation of standard curves for each of five selected gene products: claudin-1, occludin, interleukin-22 (IL-22), catalase (CAT), and β-actin.The primer pairs for the amplification of the appropriate cDNA fragments are listed in Table 2.The PCR program consisted of an initial denaturation step for 10 min at 95°C, an amplification step (40 cycles of 1 min at 95°C), an annealing and extension step for 5 min at 60°C, and a final extension step for 10 min 72°C.All measurements were carried out in triplicate, and values were averaged.The PCR products from each

Effect of Oxidized Soybean Oils on Oxidative Status and Intestinal Barrier Function in Broiler Chickens
primer pair were subjected to a melting curve analysis in order to confirm amplification specificity.The expression levels of the target genes were calculated using the comparative threshold cycle method (Livak & Schmittgen, 2001) and data were expressed as values relative to the control group.

Statistical Analysis
Data on growth performance were analyzed per pen basis.All other data were analyzed per individual bird.Data were subjected to Levene's test for homogeneity of variances before further statistical analysis, and expressed as mean values and associated standard errors.Data were analyzed by one-way analysis of variance using the procedures of SPSS Version 18.0 statistical software (SPSS Inc, Chicago, Illinois, USA).Differences among means were identified using Duncan's multiple-range test.Differences were considered significant at p<0.05.

Chemical Characteristics of the Experimental Soybean Oils
The concentrations of PV, MDA, p-AV, BHA, BHT, TBHQ, VE in the four experimental soybean oils are shown in Table 3. BHA, BHT, and TBHQ were not detected in any of the oils.As expected, with increased duration of heating, the peroxide value of the oil gradually increased.Compared with the SNX oil, the concentration of MDA and p-AV in the three oxidized oils increased.

Growth Performance
Different oxidation level of soybean oil did not affect broiler growth performance from 1 to 14 d of age and from 1 to 21 d of age (p>0.05,Table 4).

Metabolic Oxidative Status
The serum concentration of corticosterone was affected (p<0.05) by dietary oil treatment (Table 5).Chickens fed oxidized soybean oil, regardless of the oxidation levels in soybean oil, presented significant higher corticosterone serum levels compared with those fed the non-oxidized soybean oil diet.
Liver MDA and T-AOC concentrations and GSH-PX and T-SOD activities were shown in the Table 5.The content of MDA in the liver on days 14 and   The concentrations of MDA in jejunum on day 21 were affected (p<0.05) by dietary oxidized oil treatment (Table 6).The jejunum of chickens fed highly-oxidized soybean oil had higher (p<0.05)MDA concentrations on day 21 compared with those fed non-oxidized soybean oil diet.Dietary oxidized oil treatment did not influence the metabolic oxidative status of the jejunal mucosa on day 14 (p>0.05).The activities of T-SOD in the ileal mucosa both on days 14 and 21 were affected (p<0.05) by dietary oxidized oil treatment (Table 7).Chickens fed moderately-and highly-oxidized soybean oil presented lower (p<0.05)T-SOD activity in the ileal mucosa compared with those fed non-oxidized soybean oil diet.The different oxidation levels of soybean oils did not affect MDA concentrations on days 14 and 21 or T-AOC activity in the ileal mucosa on days 14 and 21 (p>0.05).

Intestinal morphology
The intestinal morphology of the jejunum and ileum were not significantly affected (p>0.05,Table 8 and 9) by dietaryoxidized-oil treatments.
Claudin-1, Occludin, TNF-α, IL-22, and CAT mRNA Expression in the ileum The mRNA expression of claudin-1 in the ileum tended (p=0.056) to be down regulated by the dietary addition of oxidized oils (Figure1).The mRNA

Effect of Oxidized Soybean Oils on Oxidative Status and Intestinal Barrier Function in Broiler Chickens
expression of occludin, IL-22, and CAT in ileum were affected by dietary oxidized oil treatment (p<0.05).Chickens fed moderately-and highly-oxidized soybean oil had higher (p<0.05) mRNA expression of IL-22, but lower (p<0.05)mRNA expression of occludin and CAT compared with those fed the nonoxidized soybean oil diet.The mRNA expression of CAT was down regulated by oxidized soybean oil treatments (p<0.05).
2 Soybean oils were included in the diet at a level of 4%.

Effect of Oxidized Soybean Oils on Oxidative Status and Intestinal Barrier Function in Broiler Chickens
any negative effects (Dong et al., 2011).This may be related to differences in dietary oil content and degree of oil oxidation (Yue et al., 2010, Zhang et al., 2011).
In our study, no significant effects of feeding diets with different oxidation levels of soybean oils on broiler growth performance were detected, possibly due to the low oil content of the experimental diets and their low oxidation levels.The primary physiological response of poultry when the body suffers oxidative stress is to activate the hypothalamic-pituitary-adrenal (HPA) axis.The response is characterized by adrenal cortical hypertrophy and increased synthesis and release of adrenal glucocorticoids, primarily corticosterone (Zhang et al., 2009).The increase in corticosterone levels by feeding oxidized oils in current study indicated that the broilers were under oxidative stress.
MDA is a lipid peroxidation marker, and increasing levels are related to lipid peroxidation and oxidation stress (Aytekin et al., 2015, Esgalhado et al., 2015).Our data showed that the dietary inclusion of highlyoxidized soybean oil increased the concentration of MDA in the jejunum and the liver, indicating lipid peroxidation in these tissues.Similar results were obtained in pigs (Ringseis et al., 2007) and broilers (Zhang et al., 2011;Liang et al., 2015).MDA level changes observed in the birds fed oxidized oils maybe triggered by free radicals of lipid peroxidation.
Other biomarkers of metabolic oxidative status are total antioxidant capacity (T-AOC) levels, and total superoxide dismutase (T-SOD) and glutathione peroxidase (GSH-PX) activities.Liver T-AOC level on day 21 was reduced in SMX-and SHX-fed broilers compared with those fed SNX, but no changes in these parameters at other evaluated time points or tissues.In addition, liver T-AOC activity of SMX and SHX diet treatments was reduced between d 14 and d 21, which may suggest that their liver suffered oxidative stress (Gao et al., 2013, Zhang et al., 2015).Total superoxide dismutases (T-SOD) are important antioxidant enzymes that accept an electron from superoxide anion (O 2-) and H 2 O to generate hydrogen peroxide (H 2 O 2 ) (Tan et al., 2015).Glutathione peroxidase (GHS-Px) may prevent cell membrane oxidative damage caused by lipid peroxides (LOOH).In the present study, T-SOD levels both inileum (d 14 and 21) and liver (d 21) and the expression of CAT gene in ileum significantly decreased when oxidized oils were fed.These results were consistent with those reported in another oxidative stress study (Liang et al., 2015).The present study demonstrated that feeding oxidized oil decreased the activity of antioxidation enzymes (T-SOD), indicating the lower capacity of scavenging free radicals, and suggested that the oxidized oil might cause metabolic oxidative stress in intestine of broiler chickens.
Claudins and occludin play a critical roles in the intestinal barrier function and paracellular permeable selectivity in the intestinal tissue (Cani et al., 2008;Lee 2015).Claudins, including claudin-1, -3, -5, -11, and -19, have been associated with a more general barriertightening function (Overgaard et al., 2011).Occludin influences the tight junction structure and permeability of the intestinal epithelia (Al-Sadi et al., 2011).Disruption of the tight junctions increases intestinal permeability.The mRNA expression of occludin and claudin-1 in intestines of broilers were detected by realtime PCR.The results showed that feeding oxidized oils down regulated occludin and claudin-1 mRNA expression in the ileum and suggest that the regulation of occludin and claudin-1 expression by oxidized oils may be increase intestinal permeability, leading intestinal barrier dysfunction.These results were in agreement with other reports on animals suffering oxidative stress (Casselbrant et al., 2015, Nevado et al., 2015, Suzuki & Hara, 2010).According to John et al. (2011), excessive levels of reactive oxygen species in intestines may contribute to barrier dysfunction, as indicated by a disruption of composition of the tight junctions.
21, and T-AOC and T-SOD activities on day 21wereaffected by dietary oil treatment (p<0.05).Chickens fed highly-oxidized soybean oil presented higher (p<0.05)MDA levels compared with those fed the non-oxidized soybean oil diet.Both SMX-and SHX-fed birds presented lower (p<0.05)T-AOC and T-SOD levels in the liver on day 21 compared with the SNXfed birds.The oxidation levels of soybean oil did not affect T-AOC, T-SOD and GSH-PX levels in liver on day 14 (p>0.05).

a, b
Means in the same column with different superscript letters are significantly different (p<0.05).

Figure 1 -
Figure 1 -Relative mRNA expression levels of Claudin-1, Occludin, IL-22, and CAT.Data are mean values of 6 individual birds (1 per replicate cage).SNX = birds fed non--oxidized soybean oil; SLX = birds fed lowly-oxidized soybean oil; SMX = birds fed moderately-oxidized soybean oil; SHX = birds fed highly-oxidized soybean oil.β-actin was used as an endogenous reference gene, and mRNA expression is expressed relative value to the SNX group.IL-22 = interleukin; CAT = catalase.SEM = standard error of the mean.Soybean oils were included in the diet at a level of 4%.a, b, c and d: means in the same column with different superscript letters are significantly different (p<0.05).

Table 1 -
Ingredients and calculated nutritional composition of the basal diet.

Table 2 -
Oligonucleotide primers used for quantitative real-time PCR of ileal mucosa tissue samples.

Table 3 -
Biochemical analysis of the experimental oils 1 .

Table 5 -
Serum corticosterone levels and liver levels of antioxidant-related compounds and enzymes of broilers fed diets with fresh or oxidized soybean oils 1, 2 .

Table 6 -
Levels of antioxidant-related compounds in the jejunal mucosa of broilers fed diets with fresh or oxidized soybean oils1, 2. Data are presented as the mean value of 10 birds per replicate cage (n = 6 replicate cages).SNX = non-oxidized soybean oil (fresh soybean oil); SLX = lowly-oxidized soybean oil (heated for 10 h); SMX = moderately-oxidized soybean oil (heated for 18 h); SHX = highly-oxidized soybean oil (heated for 30 h).MDA = malonaldehyde; T-AOC = total antioxidant capacity; T-SOD = total superoxide dismutase.mg prot =milligram protein.SEM = standard error of the mean.
1 2 Soybean oils were included in the diet at a level of 4%.a,b Means in the same column with different superscript letters are significantly different (p<0.05).

Table 8 -
Intestinal morphology in the jejunum mucosa of broilers fed diets with fresh or oxidized soybean oils 1, 2 .

Table 9 -
Intestinal morphology in the ileal mucosa of broilers fed diets with fresh and oxidized soybean oils 1, 2 .