The Effect of Oxidized Fish Oils on Growth Performance, Oxidative Status, and Intestinal Barrier Function in Broiler Chickens

The Effect of Oxidized Fish Oils on Growth Performance, Oxidative Status, and Intestinal Barrier... Abstract The objective of this study was to determine the effect of oxidized fish oils on growth performance, metabolic oxidative status, and intestinal barrier function in broiler chickens. A total of 240 1-d-old female broiler chickens were assigned to 4 dietary treatments. Dietary treatments comprised of a basal diet supplemented with 4% of non-oxidized (fresh) fish oil, low-oxidized fish oil (FLX), moderately oxidized fish oil (FMX), and highly oxidized fish oil (FHX). Serum corticosterone levels at day 14 and liver concentrations of malondialdehyde (MDA) at day 14 and 21 were higher in birds fed oxidized fish oil compared with those fed then non-oxidized fish oil diet (P < 0.01 in both cases). Furthermore, mRNA expression levels of claudin-1 and occludin were reduced, while those of IL-22 and catalase were increased, in the livers of birds fed the highly oxidized oils compared with those fed fresh fish oils (P ≤ 0.001 in all cases). These results indicate that supplementation of broiler diets with 4% oxidized fish oils can cause lipid peroxidation in the liver, involving increased concentrations of MDA, impaired gut barrier function as a result of increased intestinal permeability due to decreased expression of the tight junction proteins claudin-1/occludin, and intestinal inflammation mediated via upregulation of IL-22 expression in the mucosal tissues. DESCRIPTION OF PROBLEM Lipids derived from a range of animal and vegetable sources are routinely used in poultry feed formulation to increase the energy content of the diet [1]. This is particularly the case in broiler diets that require a high fat content in order to meet the bird's requirements for rapid growth [2]. In addition to increasing the energy content of the feed, lipid supplements reduce feed dustiness [3], are a source of fat soluble vitamins and essential fatty acids [4], and improve diet palatability [5]. Maintaining the high quality of lipid supplements intended for use in broiler diets is critical to maximizing the benefits in bird growth performance. A better understanding of their energy value and of how this can vary with quality will help nutritionists in the formulation of poultry diets [6]. Several previous studies have investigated the effects of fish oil on growth performance and gut health in a range of animal species, and have shown a range of beneficial effects [7]. However, it has also been shown that fish oils are easily oxidized because of their high polyunsaturated fatty acid content [8], where oxygen attacks the double bond in the fatty acids to form lipid [9]. Depending on their level of unsaturation, they may be prone to lipid peroxidation during heat-processing, because they are known to be thermally sensitive and unstable at high temperatures [5]. Several studies have demonstrated that this oxidation can have adverse effects on the growth performance of animals, including broilers and pigs: Yang et al. showed that fish oils impaired growth performance and induce oxidative stress in the whiteleg shrimp, Litopenaeus vannamei [10]; Dibner et al. [11] reported impaired growth performance in broilers and swine due to increased gastrointestinal epithelial cell turnover, hepatic cell proliferation, and decreased the concentration of immunoglobulin in intestinal tissues; and Liu et al. [12] demonstrated that feeding thermally oxidized lipids impaired metabolic oxidative status in young pigs by depleting serum levels of α-tocopherol and increasing serum levels of thiobarbituric acid reactive substance (TBARS). Furthermore, it has been shown that feeding peroxidized corn oils can decrease AMEn in broiler chicks [13]. However, there is limited published information on the effects of dietary supplementation with oxidized fish oil on oxidative stress and intestinal barrier function in broiler chickens. This study aimed to investigate the effect of supplementation of broiler diets with heated oxidized fish oils on growth performance, oxidative status, and intestinal barrier function. MATERIALS AND METHODS Fish Oil Preparation Fresh fish oil was purchased from the Shandong Fish Oil Production Company (Shangdong, China) and stored in a refrigerator at –30°C until use. To oxidize the fish oil, vessels containing the required amount of fresh fish oil were heated to 200°C for either 1, 5, or 9 h to produce low-oxidized fish oil (FLX), moderately oxidized fish oil (FMX), or highly oxidized fish oil (FHX), respectively. The processed oils were stored at –30°C prior to their addition to feed. (No antioxidant was added before or during diet preparation.) The oils were analyzed for their peroxide value (PV) and p-anisidine value (p-AV) according to ISO methods ISO 3960:2001 IDT [14] and ISO 6885:2006 IDT [15], respectively. Malondialdehyde (MDA) concentration was analyzed according to the method of Karatas et al. [16], and Vitamin E (VE) was analyzed according to the method of Yue [17]. Butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), and tertiary butylhydroquinone (TBHQ) content were detected by HPLC [18]. Analytical standards of BHA, BHT, TBHQ, and of α-, β-, γ-, and δ-VE were purchased from Sigma-Aldrich (USA). Animals, Housing, and Diets All experimental procedures were reviewed and approved by the China Agricultural University Animal Care and Use Committee, Beijing, China. A total of 240 1-d female avian broiler chicks were obtained from a commercial hatchery [19] assigned to 4 treatments in a completely randomized design, with 6 replicate cages per treatment and 10 birds per cage. The cage size was 120 × 100 × 60 cm. Treatments were applied to cages in completely randomized design. The treatments comprised of a basal corn–soybean meal-based diet which was formulated to meet or exceed the recommended nutritional requirements of the birds [20] (Table 1) that did not contain antibiotic growth promoters or added antioxidants. This basal diet was supplemented with 4 different fish oil products, at a 4% level of incorporation. The fish oil products included fresh fish oil (control treatment, FNX), low-oxidized fish oil (FLX), moderately oxidized fish oil (FMX), and highly oxidized fish oil (FHX). The trial lasted 21 d. Diets were offered ad libitum and water was freely available. Ambient temperature was initially 31°C and thereafter reduced by 0.5°C per day until 21°C was reached. Light was on continuously throughout the trial. Table 1. Ingredient and Nutrient Composition of the Basal Diet. Ingredient (g/kg) Day 1–21 Corn 531.80 Soybean meal 385.00 Choline chloride, 50% 2.60 Vitamin premix1 0.30 Trace mineral2 2.00 Fish oil 40.00 L-Lysine, 99% 0.80 DL-methionine, 98% 1.90 Dicalcium phosphate 20.00 Limestone 12.50 Sodium chloride 3.10 Calculated nutrient levels Metabolizable energy, kcal/kg 2,961 Crude protein3 211.0 Calcium 10.1 Available phosphorus 4.6 Lysine 12.1 Methionine 5.0 Ingredient (g/kg) Day 1–21 Corn 531.80 Soybean meal 385.00 Choline chloride, 50% 2.60 Vitamin premix1 0.30 Trace mineral2 2.00 Fish oil 40.00 L-Lysine, 99% 0.80 DL-methionine, 98% 1.90 Dicalcium phosphate 20.00 Limestone 12.50 Sodium chloride 3.10 Calculated nutrient levels Metabolizable energy, kcal/kg 2,961 Crude protein3 211.0 Calcium 10.1 Available phosphorus 4.6 Lysine 12.1 Methionine 5.0 1The vitamin premix provided the following per kilogram of complete diet: vitamin A, 9,500 IU; vitamin D3, 62.5 μg; vitamin E, 30 IU; vitamin K3, 2.65 mg; vitamin B1, 2 mg; vitamin B6, 6 mg; vitamin B12, 0.025 mg; biotin, 0.0325 mg; folic acid, 1.25 mg; pantothenic acid, 12 mg; nicotinic acid 50 mg. 2The trace mineral provided the following per kilogram of complete: copper, 8 mg (CuSO4·5H2O); iron, 80 mg (FeSO4); manganese, 100 mg (MnSO4·H2O), selenium, 0.15 mg (Na2SeO3); iodine, 0.35 mg (KI). 3Analyzed. View Large Table 1. Ingredient and Nutrient Composition of the Basal Diet. Ingredient (g/kg) Day 1–21 Corn 531.80 Soybean meal 385.00 Choline chloride, 50% 2.60 Vitamin premix1 0.30 Trace mineral2 2.00 Fish oil 40.00 L-Lysine, 99% 0.80 DL-methionine, 98% 1.90 Dicalcium phosphate 20.00 Limestone 12.50 Sodium chloride 3.10 Calculated nutrient levels Metabolizable energy, kcal/kg 2,961 Crude protein3 211.0 Calcium 10.1 Available phosphorus 4.6 Lysine 12.1 Methionine 5.0 Ingredient (g/kg) Day 1–21 Corn 531.80 Soybean meal 385.00 Choline chloride, 50% 2.60 Vitamin premix1 0.30 Trace mineral2 2.00 Fish oil 40.00 L-Lysine, 99% 0.80 DL-methionine, 98% 1.90 Dicalcium phosphate 20.00 Limestone 12.50 Sodium chloride 3.10 Calculated nutrient levels Metabolizable energy, kcal/kg 2,961 Crude protein3 211.0 Calcium 10.1 Available phosphorus 4.6 Lysine 12.1 Methionine 5.0 1The vitamin premix provided the following per kilogram of complete diet: vitamin A, 9,500 IU; vitamin D3, 62.5 μg; vitamin E, 30 IU; vitamin K3, 2.65 mg; vitamin B1, 2 mg; vitamin B6, 6 mg; vitamin B12, 0.025 mg; biotin, 0.0325 mg; folic acid, 1.25 mg; pantothenic acid, 12 mg; nicotinic acid 50 mg. 2The trace mineral provided the following per kilogram of complete: copper, 8 mg (CuSO4·5H2O); iron, 80 mg (FeSO4); manganese, 100 mg (MnSO4·H2O), selenium, 0.15 mg (Na2SeO3); iodine, 0.35 mg (KI). 3Analyzed. View Large Growth Performance Measurements Chicks and feed were weighed on a per pen basis at day of hatch, day 14 and 21. Feed intakes (FI), body weight gain (BWG), and feed conversion ratio (FCR) were calculated for each period. Sample Collection On day 14 and 21, 1 bird per cage was randomly selected and killed by venous administration of sodium pentobarbital (30 mg/kg of body weight) in order to obtain samples of blood, jejunum mucosa, ileum mucosa, and liver. The intestines were removed, the digesta flushed with 4% saline, and the mucous membranes gently scraped to obtain the samples. Samples were immediately frozen in liquid nitrogen and stored at –35°C until analysis. On day 14 only, a portion of the ileal mucosa samples was immediately frozen and stored at –80°C for mRNA determination. Additionally, on day 14 and 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 day 14, blood samples were collected by jugular exsanguination. Serum was isolated 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 with a radioimmunoassy kit [21], and the indication was measured 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 of ice-cold saline and centrifuged at 20,000× g for 10 min at 4°C. After appropriate dilution, the supernatant fractions were assayed for the activities of total superoxide dismutase (T-SOD), T-AOC, and glutathione peroxidase (GSH-PX), using enzymatic kits [22]. In order to prevent possible enzyme degradation, the samples were kept on ice throughout detection. Malondialdehyde concentration was analyzed as described above. Intestinal Morphology Analysis The fixed intestinal samples were dehydrated and embedded in paraffin wax, sectioned at 3 μm and stained with hematoxylin and eosin. Sections were observed for histomorphology [23]. Villus heights and crypt depths of 10 randomly selected complete villi per sample were measured at 40× 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 villous 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 samples (∼100 mg) using trizol reagent, in accordance with the manufacturer's instructions [24] The purity of the isolated total RNA was determined by measuring its optical density at 260 and 280 nm. Samples of the extracted total RNA (2 μg) were reverse transcribed using a reverse transcription kit [24], and the expression levels of targeted genes were detected according to quantitative real-time PCR assay with a 7,300 real-time PCR system [25] using Fast Start Universal SYBR Green Master [26] after generation of standard curves for each of 5 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 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 [27], and data were expressed as values relative to the control group. Table 2. Oligonucleotide Primers Used for Quantitative Real-Time PCR of Ileal Mucosa Tissue Samples. Gene Primer fragment Accession number β-actin 5‘-GGATTGGAGGCTCTATCCTGG-3’ NM_205518.1 5‘-GTTTAGAAGCATTTGCGGTGG-3’ Claudin-1 5‘-GATGCGGATGGCTGTCTTTG-3’ NM_001013611.2 5‘-GCTGGGTGGGTAGGATGTTTC-3’ Occludin 5‘-GCCGTAACCCCGAGTTGGAT-3’ NM_205128.1 5‘-TGATTGAGGCGGTCGTTGATG-3’ IL22 5 ‘-ACCCGTATGCTGAGGATGTGG-3 ’ NM_001199614.1 5‘-CTTGTTCCCTCCCTTCTTTGG-3’ CAT 5‘-AGCAGGTGCCTTTGGCTATT-3’ NM_001031215.1 5‘-CGAGGGTCACGAACTGTATCA-3’ Gene Primer fragment Accession number β-actin 5‘-GGATTGGAGGCTCTATCCTGG-3’ NM_205518.1 5‘-GTTTAGAAGCATTTGCGGTGG-3’ Claudin-1 5‘-GATGCGGATGGCTGTCTTTG-3’ NM_001013611.2 5‘-GCTGGGTGGGTAGGATGTTTC-3’ Occludin 5‘-GCCGTAACCCCGAGTTGGAT-3’ NM_205128.1 5‘-TGATTGAGGCGGTCGTTGATG-3’ IL22 5 ‘-ACCCGTATGCTGAGGATGTGG-3 ’ NM_001199614.1 5‘-CTTGTTCCCTCCCTTCTTTGG-3’ CAT 5‘-AGCAGGTGCCTTTGGCTATT-3’ NM_001031215.1 5‘-CGAGGGTCACGAACTGTATCA-3’ View Large Table 2. Oligonucleotide Primers Used for Quantitative Real-Time PCR of Ileal Mucosa Tissue Samples. Gene Primer fragment Accession number β-actin 5‘-GGATTGGAGGCTCTATCCTGG-3’ NM_205518.1 5‘-GTTTAGAAGCATTTGCGGTGG-3’ Claudin-1 5‘-GATGCGGATGGCTGTCTTTG-3’ NM_001013611.2 5‘-GCTGGGTGGGTAGGATGTTTC-3’ Occludin 5‘-GCCGTAACCCCGAGTTGGAT-3’ NM_205128.1 5‘-TGATTGAGGCGGTCGTTGATG-3’ IL22 5 ‘-ACCCGTATGCTGAGGATGTGG-3 ’ NM_001199614.1 5‘-CTTGTTCCCTCCCTTCTTTGG-3’ CAT 5‘-AGCAGGTGCCTTTGGCTATT-3’ NM_001031215.1 5‘-CGAGGGTCACGAACTGTATCA-3’ Gene Primer fragment Accession number β-actin 5‘-GGATTGGAGGCTCTATCCTGG-3’ NM_205518.1 5‘-GTTTAGAAGCATTTGCGGTGG-3’ Claudin-1 5‘-GATGCGGATGGCTGTCTTTG-3’ NM_001013611.2 5‘-GCTGGGTGGGTAGGATGTTTC-3’ Occludin 5‘-GCCGTAACCCCGAGTTGGAT-3’ NM_205128.1 5‘-TGATTGAGGCGGTCGTTGATG-3’ IL22 5 ‘-ACCCGTATGCTGAGGATGTGG-3 ’ NM_001199614.1 5‘-CTTGTTCCCTCCCTTCTTTGG-3’ CAT 5‘-AGCAGGTGCCTTTGGCTATT-3’ NM_001031215.1 5‘-CGAGGGTCACGAACTGTATCA-3’ View Large Statistical Analysis Data on growth performance were based on a per pen basis. All other data were based on individual birds. 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 ANOVA using the procedures of SPSS Version 18.0 statistical software [28]. Differences between means were identified using Duncan's multiple-range test. Differences were considered significant at P < 0.05. RESULTS AND DISCUSSION Chemical Characteristics of the Experimental Fish Oils The concentrations of PV, MDA, p-AV, VE, BHA, BHT, and TBHQ in the 4 experimental fish oils are shown in Table 3. Vitamin E, BHA, BHT, and TBHQ were not detected in any of the oils. As expected, with increased duration of heating, the PV of the oil gradually increased. Also as expected, compared to the FNX oil, the concentration of MDA and p-AV in the 3 oxidized oils was increased. Table 3. Biochemical Analysis of the Experimental Oils.1 Fish oil supplement Item FNX FLX FMX FHX Peroxide value (meq/kg) 20.77 140.41 183.54 277.35 Malondialdehyde (μg/kg) 65.67 145.40 150.39 154.73 p-anisidine value 22 66 149 192 Butylated hydroxyanisole (mg/kg) ND2 ND ND ND Butylated hydroxytoluene (mg/kg) ND ND ND ND Tertiary butylhydroquinone (mg/kg) ND ND ND ND Vitamin E (mg/kg) ND ND ND ND Fish oil supplement Item FNX FLX FMX FHX Peroxide value (meq/kg) 20.77 140.41 183.54 277.35 Malondialdehyde (μg/kg) 65.67 145.40 150.39 154.73 p-anisidine value 22 66 149 192 Butylated hydroxyanisole (mg/kg) ND2 ND ND ND Butylated hydroxytoluene (mg/kg) ND ND ND ND Tertiary butylhydroquinone (mg/kg) ND ND ND ND Vitamin E (mg/kg) ND ND ND ND 1FNX = non-oxidized fish oil (fresh fish oil); FLX = low-oxidized fish oil (heated for 1 h); FMX = moderately oxidized fish oil (heated for 5 h); FHX = highly oxidized fish oil (heated for 9 h). 2ND = not detected. View Large Table 3. Biochemical Analysis of the Experimental Oils.1 Fish oil supplement Item FNX FLX FMX FHX Peroxide value (meq/kg) 20.77 140.41 183.54 277.35 Malondialdehyde (μg/kg) 65.67 145.40 150.39 154.73 p-anisidine value 22 66 149 192 Butylated hydroxyanisole (mg/kg) ND2 ND ND ND Butylated hydroxytoluene (mg/kg) ND ND ND ND Tertiary butylhydroquinone (mg/kg) ND ND ND ND Vitamin E (mg/kg) ND ND ND ND Fish oil supplement Item FNX FLX FMX FHX Peroxide value (meq/kg) 20.77 140.41 183.54 277.35 Malondialdehyde (μg/kg) 65.67 145.40 150.39 154.73 p-anisidine value 22 66 149 192 Butylated hydroxyanisole (mg/kg) ND2 ND ND ND Butylated hydroxytoluene (mg/kg) ND ND ND ND Tertiary butylhydroquinone (mg/kg) ND ND ND ND Vitamin E (mg/kg) ND ND ND ND 1FNX = non-oxidized fish oil (fresh fish oil); FLX = low-oxidized fish oil (heated for 1 h); FMX = moderately oxidized fish oil (heated for 5 h); FHX = highly oxidized fish oil (heated for 9 h). 2ND = not detected. View Large Growth Performance Feed conversion ratio was significantly increased in birds fed the oxidized oils compared with the fresh fish oil (FNX), between day 0 and 14 (P < 0.05), but the differences were not significant for the overall period (day 0–21 (P = 0.09)) (Table 4). Dietary treatment had no effect on FI or BWG. This absence of effect on FI and BWG is consistent with the findings of other studies in which diets supplemented with oxidized oils did not affect animal performance [17, 29, 30]. No significant difference on body weight gain, FI in our study may be partly due to the lower oxidation levels of fish and less oxidized fish oil content of feed than in other studies [31, 32]. A reduced growth rate in animals fed thermally oxidized lipids may be caused by several factors. Firstly, the thermally oxidized lipids may cause rancidity that can reduce diet palatability and thereby decrease FI leading to a poor growth rate [12]. Secondly, secondary lipid peroxidation products, such as α,β-unsaturated hydroxyaldahydes, are of particular interest because some of them are highly toxic and readily absorbed [33], and are capable of modifying proteins in vivo by damaging the intestinal brush membrane [11]. Table 4. Growth Performance of Broilers Whose Diets were Supplemented With Fresh Fish Oil (FNX), Low-, Moderate-, and Highly Oxidized Fish Oils (FLX, FMX, and FHX).1,2 Day 1–14 Day 1–21 Treatment BWG (g) FI (g) FCR BWG (g) FI (g) FCR FNX 283 336 1.19b 675 905 1.34 FLX 280 367 1.31a 657 949 1.45 FMX 274 348 1.28a 663 930 1.41 FHX 269 358 1.33a 626 924 1.48 SEM 4 5 0.02 8 13 0.02 P-value 0.620 0.113 0.022 0.183 0.703 0.090 Day 1–14 Day 1–21 Treatment BWG (g) FI (g) FCR BWG (g) FI (g) FCR FNX 283 336 1.19b 675 905 1.34 FLX 280 367 1.31a 657 949 1.45 FMX 274 348 1.28a 663 930 1.41 FHX 269 358 1.33a 626 924 1.48 SEM 4 5 0.02 8 13 0.02 P-value 0.620 0.113 0.022 0.183 0.703 0.090 1Data are means of values from 10 birds per replicate cage (n = 6 replicate cages). FNX = fish oil non-oxidized group (fresh fish oil); FLX = fish oil low-oxidized group; FMX = fish oil moderately oxidized group; FHX = fish oil highly oxidized group. FCR = feed conversion ratio. SEM = standard error of the mean. 2Fish oils were incorporated into the diet at a level of 4%. a,bMeans in the same column with different superscript letters are significantly different (P < 0.05). View Large Table 4. Growth Performance of Broilers Whose Diets were Supplemented With Fresh Fish Oil (FNX), Low-, Moderate-, and Highly Oxidized Fish Oils (FLX, FMX, and FHX).1,2 Day 1–14 Day 1–21 Treatment BWG (g) FI (g) FCR BWG (g) FI (g) FCR FNX 283 336 1.19b 675 905 1.34 FLX 280 367 1.31a 657 949 1.45 FMX 274 348 1.28a 663 930 1.41 FHX 269 358 1.33a 626 924 1.48 SEM 4 5 0.02 8 13 0.02 P-value 0.620 0.113 0.022 0.183 0.703 0.090 Day 1–14 Day 1–21 Treatment BWG (g) FI (g) FCR BWG (g) FI (g) FCR FNX 283 336 1.19b 675 905 1.34 FLX 280 367 1.31a 657 949 1.45 FMX 274 348 1.28a 663 930 1.41 FHX 269 358 1.33a 626 924 1.48 SEM 4 5 0.02 8 13 0.02 P-value 0.620 0.113 0.022 0.183 0.703 0.090 1Data are means of values from 10 birds per replicate cage (n = 6 replicate cages). FNX = fish oil non-oxidized group (fresh fish oil); FLX = fish oil low-oxidized group; FMX = fish oil moderately oxidized group; FHX = fish oil highly oxidized group. FCR = feed conversion ratio. SEM = standard error of the mean. 2Fish oils were incorporated into the diet at a level of 4%. a,bMeans in the same column with different superscript letters are significantly different (P < 0.05). View Large Metabolic Oxidative Status The concentrations of serum CORT at day 14 were significantly affected (P < 0.01) by treatment (Table 5). Regardless of the level of oxidation, birds fed the oxidized fish oil had higher serum CORT levels than birds fed fresh fish oil (P < 0.01). The concentrations of MDA, T-AOC, and the activities of GSH-PX and T-SOD in the liver are showed in Table 5. The concentration of MDA in the liver was also affected by treatment at both day 14 and 21 (P < 0.01) (Table 5); at day 14, birds fed FLX oxidized fish oil had higher MDA concentrations in the liver than birds fed fresh fish oil (P < 0.001), but by day 21 MDA concentrations were significantly elevated in birds fed FLX, FMX, or FHX, compared with control-fed birds (P < 0.01). The oxidation levels of the fish oil did not affect the concentrations of T-AOC or the activities of GSH-PX and T-SOD in the liver on day 14 or 21 (Table 5). Table 5. Serum Corticosterone and Liver Concentrations of Antioxidant-Related Enzymes Broilers Fed Diets Supplemented With Fresh or Oxidized Fish Oils.1,2 Day 14 Day 21 Treatment Serum CORT (ng/mL) MDA (nmol/ mgprot) T-AOC (U/ mgprot) GSH-Px (U/ mgprot) T-SOD (U/ mgprot) MDA (nmol/ mgprot) T-AOC (U/ mgprot) GSH-Px (U/ mgprot) T-SOD (U/ mgprot) FNX 10.00b 1.407b 1.608 13.5 189 0.920b 0.988 18.4 152 FLX 11.50a 1.902a 1.464 14.2 205 1.384a 1.061 17.7 138 FMX 12.42a 1.857a,b 1.584 13.4 213 1.334a 1.045 18.6 151 FHX 11.99a 1.803a,b 1.720 13.0 207 1.312a 1.180 20.0 142 SEM 0.29 0.111 0.044 0.3 4 0.061 0.036 0.4 3 P-value 0.008 0.000 0.229 0.677 0.110 0.007 0.300 0.300 0.186 Day 14 Day 21 Treatment Serum CORT (ng/mL) MDA (nmol/ mgprot) T-AOC (U/ mgprot) GSH-Px (U/ mgprot) T-SOD (U/ mgprot) MDA (nmol/ mgprot) T-AOC (U/ mgprot) GSH-Px (U/ mgprot) T-SOD (U/ mgprot) FNX 10.00b 1.407b 1.608 13.5 189 0.920b 0.988 18.4 152 FLX 11.50a 1.902a 1.464 14.2 205 1.384a 1.061 17.7 138 FMX 12.42a 1.857a,b 1.584 13.4 213 1.334a 1.045 18.6 151 FHX 11.99a 1.803a,b 1.720 13.0 207 1.312a 1.180 20.0 142 SEM 0.29 0.111 0.044 0.3 4 0.061 0.036 0.4 3 P-value 0.008 0.000 0.229 0.677 0.110 0.007 0.300 0.300 0.186 1Data are means of values from 6 individual birds (1 per replicate cage). FNX = fish oil non-oxidized group; FLX = fish oil low-oxidized group; FMX = fish oil moderately oxidized group; FHX = fish oil highly oxidized group. serum CORT, serum corticosterone; MDA, malonaldehyde; T-AOC, total-antioxidative capacity; GSH-PX, glutathione peroxidase; T-SOD, total superoxide dismutase. mgprot, milligram protein. SEM, standard error of the mean. 2Fish oils were incorporated into the diet at a level of 4%. a,bMeans in the same column with different superscript letters are significantly different (P < 0.05). View Large Table 5. Serum Corticosterone and Liver Concentrations of Antioxidant-Related Enzymes Broilers Fed Diets Supplemented With Fresh or Oxidized Fish Oils.1,2 Day 14 Day 21 Treatment Serum CORT (ng/mL) MDA (nmol/ mgprot) T-AOC (U/ mgprot) GSH-Px (U/ mgprot) T-SOD (U/ mgprot) MDA (nmol/ mgprot) T-AOC (U/ mgprot) GSH-Px (U/ mgprot) T-SOD (U/ mgprot) FNX 10.00b 1.407b 1.608 13.5 189 0.920b 0.988 18.4 152 FLX 11.50a 1.902a 1.464 14.2 205 1.384a 1.061 17.7 138 FMX 12.42a 1.857a,b 1.584 13.4 213 1.334a 1.045 18.6 151 FHX 11.99a 1.803a,b 1.720 13.0 207 1.312a 1.180 20.0 142 SEM 0.29 0.111 0.044 0.3 4 0.061 0.036 0.4 3 P-value 0.008 0.000 0.229 0.677 0.110 0.007 0.300 0.300 0.186 Day 14 Day 21 Treatment Serum CORT (ng/mL) MDA (nmol/ mgprot) T-AOC (U/ mgprot) GSH-Px (U/ mgprot) T-SOD (U/ mgprot) MDA (nmol/ mgprot) T-AOC (U/ mgprot) GSH-Px (U/ mgprot) T-SOD (U/ mgprot) FNX 10.00b 1.407b 1.608 13.5 189 0.920b 0.988 18.4 152 FLX 11.50a 1.902a 1.464 14.2 205 1.384a 1.061 17.7 138 FMX 12.42a 1.857a,b 1.584 13.4 213 1.334a 1.045 18.6 151 FHX 11.99a 1.803a,b 1.720 13.0 207 1.312a 1.180 20.0 142 SEM 0.29 0.111 0.044 0.3 4 0.061 0.036 0.4 3 P-value 0.008 0.000 0.229 0.677 0.110 0.007 0.300 0.300 0.186 1Data are means of values from 6 individual birds (1 per replicate cage). FNX = fish oil non-oxidized group; FLX = fish oil low-oxidized group; FMX = fish oil moderately oxidized group; FHX = fish oil highly oxidized group. serum CORT, serum corticosterone; MDA, malonaldehyde; T-AOC, total-antioxidative capacity; GSH-PX, glutathione peroxidase; T-SOD, total superoxide dismutase. mgprot, milligram protein. SEM, standard error of the mean. 2Fish oils were incorporated into the diet at a level of 4%. a,bMeans in the same column with different superscript letters are significantly different (P < 0.05). View Large Table 6. Antioxidant-Related Activity Levels in the Jejunal Mucosa of Broilers Fed Diets With Fresh and Oxidized Fish Oils.1,2 Day 14 Day 21 Treatment MDA (nmol/mgprot) T-AOC (U/mgprot) T-SOD (U/mgprot) MDA (nmol/mgprot) T-AOC (U/mgprot) T-SOD (U/mgprot) FNX 0.655 1.149 103 1.221 3.029 138b FLX 0.679 1.120 104 1.304 3.165 159a,b FMX 0.469 1.177 91 1.076 3.400 163a FHX 0.628 2.129 94 1.163 4.393 172a SEM 0.053 0.133 2.776 0.050 0.202 4.004 P-value 0.521 0.193 0.259 0.451 0.060 0.007 Day 14 Day 21 Treatment MDA (nmol/mgprot) T-AOC (U/mgprot) T-SOD (U/mgprot) MDA (nmol/mgprot) T-AOC (U/mgprot) T-SOD (U/mgprot) FNX 0.655 1.149 103 1.221 3.029 138b FLX 0.679 1.120 104 1.304 3.165 159a,b FMX 0.469 1.177 91 1.076 3.400 163a FHX 0.628 2.129 94 1.163 4.393 172a SEM 0.053 0.133 2.776 0.050 0.202 4.004 P-value 0.521 0.193 0.259 0.451 0.060 0.007 1Data are means of values from 6 individual birds (1 per replicate cage). FNX = fish oil non-oxidized group; FLX = fish oil low-oxidized group; FMX = fish oil moderately oxidized group; FHX = fish oil highly oxidized group. MDA, malonaldehyde; T-AOC, total-antioxidative capacity; T-SOD, total superoxide dismutase. mgprot, milligram protein. SEM, standard error of the mean. 2Fish oils were incorporated into the diet at a level of 4%. a,bMeans in the same column with different superscript letters are significantly different (P < 0.05). View Large Table 6. Antioxidant-Related Activity Levels in the Jejunal Mucosa of Broilers Fed Diets With Fresh and Oxidized Fish Oils.1,2 Day 14 Day 21 Treatment MDA (nmol/mgprot) T-AOC (U/mgprot) T-SOD (U/mgprot) MDA (nmol/mgprot) T-AOC (U/mgprot) T-SOD (U/mgprot) FNX 0.655 1.149 103 1.221 3.029 138b FLX 0.679 1.120 104 1.304 3.165 159a,b FMX 0.469 1.177 91 1.076 3.400 163a FHX 0.628 2.129 94 1.163 4.393 172a SEM 0.053 0.133 2.776 0.050 0.202 4.004 P-value 0.521 0.193 0.259 0.451 0.060 0.007 Day 14 Day 21 Treatment MDA (nmol/mgprot) T-AOC (U/mgprot) T-SOD (U/mgprot) MDA (nmol/mgprot) T-AOC (U/mgprot) T-SOD (U/mgprot) FNX 0.655 1.149 103 1.221 3.029 138b FLX 0.679 1.120 104 1.304 3.165 159a,b FMX 0.469 1.177 91 1.076 3.400 163a FHX 0.628 2.129 94 1.163 4.393 172a SEM 0.053 0.133 2.776 0.050 0.202 4.004 P-value 0.521 0.193 0.259 0.451 0.060 0.007 1Data are means of values from 6 individual birds (1 per replicate cage). FNX = fish oil non-oxidized group; FLX = fish oil low-oxidized group; FMX = fish oil moderately oxidized group; FHX = fish oil highly oxidized group. MDA, malonaldehyde; T-AOC, total-antioxidative capacity; T-SOD, total superoxide dismutase. mgprot, milligram protein. SEM, standard error of the mean. 2Fish oils were incorporated into the diet at a level of 4%. a,bMeans in the same column with different superscript letters are significantly different (P < 0.05). View Large On the other hand, FMX and FHX group increased (P < 0.05) the level of T-SOD in jejunal mucosa on day 21 compared with chickens fed non-oxidized fish oil (Table 6). Dietary oxidized fish oil treatment did not affect the metabolic oxidative status in jejunal mucosa on day 14 (P > 0.05) (Table 6). The concentrations of MDA and activities of T-AOC and T-SOD in the ileal mucosa are showed in Table 7. There were no effects of dietary treatment on the metabolic oxidative status of the ileal mucosa on day 14 or 21 (P > 0.05). Table 7. Antioxidant-Related Activity Levels in the Ileal Mucosa of Broilers Fed Diets-With Fresh and Oxidized Fish Oils.1,2 Day 14 Day 21 Treatment MDA (nmol/mgprot) T-AOC (U/mgprot) T-SOD (U/mgprot) MDA (nmol/mgprot) T-AOC (U/mgprot) T-SOD (U/mgprot) FNX 0.534 1.274 60 0.813 1.979 110 FLX 0.540 1.564 65 0.728 1.789 106 FMX 0.453 1.547 63 0.725 1.986 100 FHX 0.427 1.609 59 0.660 2.160 106 SEM 0.021 0.046 1 0.035 0.057 1 P-value 0.135 0.182 0.077 0.481 0.126 0.108 Day 14 Day 21 Treatment MDA (nmol/mgprot) T-AOC (U/mgprot) T-SOD (U/mgprot) MDA (nmol/mgprot) T-AOC (U/mgprot) T-SOD (U/mgprot) FNX 0.534 1.274 60 0.813 1.979 110 FLX 0.540 1.564 65 0.728 1.789 106 FMX 0.453 1.547 63 0.725 1.986 100 FHX 0.427 1.609 59 0.660 2.160 106 SEM 0.021 0.046 1 0.035 0.057 1 P-value 0.135 0.182 0.077 0.481 0.126 0.108 1Data are means of values from 6 individual birds (1 per replicate cage). FNX = fish oil non-oxidized group; FLX = fish oil low-oxidized group; FMX = fish oil moderately oxidized group; FHX = fish oil highly oxidized group. MDA, malonaldehyde; T-AOC, total-antioxidative capacity; T-SOD, total superoxide dismutase. mgprot, milligram protein. SEM, standard error of the mean. 2Fish oils were incorporated into the diet at a level of 4%. View Large Table 7. Antioxidant-Related Activity Levels in the Ileal Mucosa of Broilers Fed Diets-With Fresh and Oxidized Fish Oils.1,2 Day 14 Day 21 Treatment MDA (nmol/mgprot) T-AOC (U/mgprot) T-SOD (U/mgprot) MDA (nmol/mgprot) T-AOC (U/mgprot) T-SOD (U/mgprot) FNX 0.534 1.274 60 0.813 1.979 110 FLX 0.540 1.564 65 0.728 1.789 106 FMX 0.453 1.547 63 0.725 1.986 100 FHX 0.427 1.609 59 0.660 2.160 106 SEM 0.021 0.046 1 0.035 0.057 1 P-value 0.135 0.182 0.077 0.481 0.126 0.108 Day 14 Day 21 Treatment MDA (nmol/mgprot) T-AOC (U/mgprot) T-SOD (U/mgprot) MDA (nmol/mgprot) T-AOC (U/mgprot) T-SOD (U/mgprot) FNX 0.534 1.274 60 0.813 1.979 110 FLX 0.540 1.564 65 0.728 1.789 106 FMX 0.453 1.547 63 0.725 1.986 100 FHX 0.427 1.609 59 0.660 2.160 106 SEM 0.021 0.046 1 0.035 0.057 1 P-value 0.135 0.182 0.077 0.481 0.126 0.108 1Data are means of values from 6 individual birds (1 per replicate cage). FNX = fish oil non-oxidized group; FLX = fish oil low-oxidized group; FMX = fish oil moderately oxidized group; FHX = fish oil highly oxidized group. MDA, malonaldehyde; T-AOC, total-antioxidative capacity; T-SOD, total superoxide dismutase. mgprot, milligram protein. SEM, standard error of the mean. 2Fish oils were incorporated into the diet at a level of 4%. View Large The primary physiological response of poultry when the body suffers oxidative stress is to activate the hypothalamic-pituitary-adrenal axis, which is characterized by adrenal cortical hypertrophy and increased synthesis and release of adrenal glucocorticoids, known as CORT [34]. Corticosterone levels are thus used as an indicator of stress in mammals [35]. The higher concentrations of serum CORT among birds fed the oxidized oils in this study are consistent with previous studies on mice [36], and raised CORT levels in broilers have previously been linked with conditions of stress [35]. Malondialdehyde is an endogenous genotoxic product of enzymatic and free radical-induced lipid peroxidation [37]. Therefore, raised MDA levels are an indication of lipid peroxidation in the tissues and of the presence of reactive oxygen species residing in the tissues [38]. In this study, while feeding oxidized fish oil to birds had no effect on concentrations in the jejunal or ileal mucosa, levels of MDA in the liver were significantly raised. These findings are broadly consistent with comparable evidence from other animal species. Feeding repeatedly heated soy oil has been reported to increase serum TBARS in ovariectomized rats [32], finishing barrows [39], young pigs [40], and the whiteleg shrimp [10]. However, feeding oxidized fish oil did not affect the TBARS values of blood serum and liver in the Chinese longsnout catfish [41]. In pigs, feeding oxidized oil was observed to increase hepatic nuclear concentration of the transcription factor erythroid-derived 2-like 2 (Nrf2) [3], which is an important transcription factor mediating cellular stress response. The action of heating oil leads to a loss in polyunsaturated fatty acids and degrades native nutritive compounds such as tocopherol and essential fatty acids [42]. Against this background, there are 2 possible reasons by which to explain the increased levels of MDA found in the liver of birds fed diets supplemented with oxidized oils. The first reason may be the activity of Nrf2 [43], leading to the increase of the concentrations of MDA in liver. The second reason of increasing MDA concentrations in liver may be the potential mutagenic effects of that peroxide which is produced as a result of the extended heating time of the oxidized fish oil [37]. Collectively, our results indicate that the dietary intake of oxidized fish oil caused oxidative liver damage that may have a direct implication for the ability of the bird to resist oxidative stress, as has been suggested previously for the Chinese mitten crab [44]. Glutathione peroxidase is important in metabolism and enzyme regulation as well as detoxification of cytotoxic materials [45], including the elimination of organic hydroperoxides and hydrogen peroxides [31]. No effects of treatment on liver concentrations of GSH-PX were observed in the present study. However, effects were seen on the levels of T-SOD in jejunal mucosa (at day 21). The findings are consistent with those of a recent study by Wang et al. [44], in which feeding oxidized oil to the Chinese mitten crab was observed to improve serum T-SOD activity. Raised levels of this antioxidant enzyme may reflect a compensatory effect or represent a precursor to the manifestation of oxidative stress in tissue cells [36, 44, 46]. Catalase is a major enzyme involved in the detoxification of H2O2 in cells [47]. Therefore, the decrease in CAT mRNA expression in the present study indicates a reduced capacity of the bird to eliminate free radicals. These findings are consistent with those of other studies conducted on broilers [38] and pigs [48] that have involved dietary supplementation with oxidized lipid. Overall, these findings suggest that oxidation stress occurred in the intestinal mucosa of broilers fed oxidized fish oil. Claudin-1, Occludin, IL-22, and CAT mRNA Expression in the Ileum The mRNA expression levels of claudin-1, occludin, IL-22, and CAT at day 14 in the ilea of birds fed the 4 experimental treatments are presented in Table 8. The mRNA expressions of claudin-1, occludin, IL-22, and CAT in ileum were affected by dietary oxidized oil treatment (P < 0.05). Birds fed oxidized fish oil, regardless of the level of oxidation, exhibited reduced mRNA expression levels of Claudin-1 compared with control-diet birds (P < 0.001). Birds in the low- and high-oxidized fish oil groups also exhibited reduced occludin expression (P < 0.01). Conversely, birds fed the moderate- and especially the highly oxidized fish oil exhibited increased expression levels of IL-22 compared with birds fed the fresh fish oil. Similarly, expression levels of CAT were reduced in birds fed the highly oxidized fish oil compared with control-diet birds (P < 0.01). Table 8. Relative mRNA Expression Levels of Claudin-1, Occludin, IL-22, and CAT.1,2 Treatment Claudin-1 Occludin IL-22 CAT FNX 0.912a 1.002a 1.102c 1.082a FLX 0.736b 0.335b 1.361c 1.102a FMX 0.651b 1.178a 2.443b 1.152a FHX 0.409c 0.473b 4.521a 0.737b SEM 0.045 0.115 0.381 0.054 P-value 0.000 0.001 0.000 0.001 Treatment Claudin-1 Occludin IL-22 CAT FNX 0.912a 1.002a 1.102c 1.082a FLX 0.736b 0.335b 1.361c 1.102a FMX 0.651b 1.178a 2.443b 1.152a FHX 0.409c 0.473b 4.521a 0.737b SEM 0.045 0.115 0.381 0.054 P-value 0.000 0.001 0.000 0.001 1Data are means of values from 6 individual birds (1 per replicate cage). FNX = fish oil non-oxidized group; FLX = fish oil low-oxidized group; FMX = fish oil moderately oxidized group; FHX = fish oil highly oxidized group. β-Actin was used as an endogenous reference gene, and mRNA expression was expressed as the relative value to the SNX group. IL-22, interleukin; CAT, catalase. SEM, standard error of the mean. 2Fish oils were incorporated into the diet at a level of 4%. a,bMeans in the same column with different superscript letters are significantly different (P < 0.05). View Large Table 8. Relative mRNA Expression Levels of Claudin-1, Occludin, IL-22, and CAT.1,2 Treatment Claudin-1 Occludin IL-22 CAT FNX 0.912a 1.002a 1.102c 1.082a FLX 0.736b 0.335b 1.361c 1.102a FMX 0.651b 1.178a 2.443b 1.152a FHX 0.409c 0.473b 4.521a 0.737b SEM 0.045 0.115 0.381 0.054 P-value 0.000 0.001 0.000 0.001 Treatment Claudin-1 Occludin IL-22 CAT FNX 0.912a 1.002a 1.102c 1.082a FLX 0.736b 0.335b 1.361c 1.102a FMX 0.651b 1.178a 2.443b 1.152a FHX 0.409c 0.473b 4.521a 0.737b SEM 0.045 0.115 0.381 0.054 P-value 0.000 0.001 0.000 0.001 1Data are means of values from 6 individual birds (1 per replicate cage). FNX = fish oil non-oxidized group; FLX = fish oil low-oxidized group; FMX = fish oil moderately oxidized group; FHX = fish oil highly oxidized group. β-Actin was used as an endogenous reference gene, and mRNA expression was expressed as the relative value to the SNX group. IL-22, interleukin; CAT, catalase. SEM, standard error of the mean. 2Fish oils were incorporated into the diet at a level of 4%. a,bMeans in the same column with different superscript letters are significantly different (P < 0.05). View Large The increased expression of claudin in birds fed the oxidized fish oils may be indicative of adverse effects on intestinal barrier function, which has not previously been reported in broilers. The most important structures of the intestinal barrier are the epithelial tight junctions (TJs) that connect adjacent enterocytes together to determine paracellular permeability through the lateral intercellular space [49]. Claudins are a family of nearly 2 dozen transmenbrane proteins that are a key part of the TJ barrier that regulates solute movement across the polarized epithelia [50]. Therefore, reduced claudin expression may indicate impaired functioning of TJ proteins and a consequent reduction in epithelial barrier integrity. Effects on occludin expression may have a similar result. Occludin regulates macromolecule flux across the intestinal epithelial TJ barrier [51]. The expression of occludin is known to be markedly decreased in intestinal permeability disorders, including in Crohn's disease, ulcerative colitis [52], and celiac disease [53], suggesting that reduced occludin expression may play a role in the increase in intestinal permeability. Against this background, it seems plausible that the reduced expression of claudin-1 and occludin in the ileal mucosa of birds fed oxidized fish oil in this study could be indicative of an increase in intestinal permeability, thus leading to intestinal barrier dysfunction. Similar findings have been observed in terms of reduced expression of TJ proteins in the human proximal small intestinal mucosa before and after Roux-en-Y gastric bypass surgery [54] and when hyperthermia-induced oxidative stress led to intestinal mucosa barrier dysfunction in mice [55]. However, other studies suggest an absence of effect; feeding thermally oxidized vegetable oils and animal fats had little influence on the ratio of urinary lactulose to mannitol in pigs, this being one of the most popular methods used to measure intestinal permeability [40]. There is little existing published information regarding the effects of dietary supplementation with oxidized oils on gut inflammation in broilers. Interleukin-22 is a pro-inflammatory cytokine that is predominantly produced by activated Th1 cells and signals through a receptor complex consisting of IL-22R1 and IL-10R2 [56]. Existing studies have revealed that IL-22 targets cells of the digestive, skin, and respiratory systems and plays an important role in the mucosal immunity [57]. In chronic Hepatitis C Viral (HCV), T cells producing IL-22 may migrate to the liver to reduce inflammation [58]. Furthermore, intestinal epithelial STAT3 activation can regulate immune homeostasis in the gut by promoting IL-22-dependent mucosal wound healing [59]. Many studies mouse model systems have identified a critical role for signaling by IL-22 through its receptor (IL-22R) in the promotion of antimicrobial immunity, inflammation, and tissue repair at barrier surfaces [60]. Therefore, the raised IL-22 expression levels in the ileal mucosa of birds fed highly oxidized fish oils in this study may be indicative of the induction of an inflammatory response. Intestinal Morphology in the Ileum and Jejunum The structure of the intestinal mucosa can reveal some information on gut health. The intestinal morphology in the jejunum and ileum were not significant affected (P > 0.05) by dietary oxidized oil treatment (data not shown). There is little existing published information regarding the effects of dietary supplementation with oxidized oils on gut morphology in broilers. The crypt is considered as the villus cell producer. In this respect, a deeper crypt shows rapid tissue turnover and a high demand for new tissue [61]. Stressors that are present in the digesta can lead relatively quickly to changes in the intestinal mucosa, due to the close proximity of the mucosal surface and the intestinal content. Changes in intestinal morphology, such as shorter villi and deeper crypts, have been associated with the presence of toxins or higher tissue turnover [62]. We did not see significant effects on intestinal morphology by different oxidation level of fish oils; this may be due to the low content of oil in feed and low oxidized degree of oil. CONCLUSION AND APPLICATION 1. Supplementation of broiler diets with oxidized fish oil vs. fresh fish oil reduced FCR between day 0 and 14 but had no other adverse effects on growth performance. 2. Serum corticosteroid and liver MDA concentrations were raised, metabolic oxidative status was impaired, and ileal mucosal expressions of claudin, occuldin-1, IL-22, and CAT were adversely affected in birds fed oxidized fish oils. 3. Based on this study, it is suggested that oxidized fish oil can reduce FCR, induce oxidative stress in the liver, and impair intestinal barrier function, through upregulation of IL-22. Acknowledgments The authors would like to thank Yuxin Shao, Yanyan Yang, Yuanyang Dong, Xuan Liu, and He Gao for their help with the experiments. This work was supported by the Beijing Higher Education Young Elite Teacher Project, the Yangtz River Scholar and Innovation Research Team Development Program (No. IRT0945), and the Chinese Universities Scientific Fund (No. 2015DK005). Footnotes Primary Audience: Poultry researchers REFERENCES AND NOTES 1. Li P. , Piao X. , Ru Y. , Han X. , Xue L. , Zhang H. . 2012 . Effects of adding essential oil to the diet of weaned pigs on performance, nutrient utilization, immune response and intestinal health . Asian Australas. J. Anim. Sci. 25 : 1617 – 1626 . Google Scholar CrossRef Search ADS PubMed 2. Wang L. , Zhang J. , Gao J. , Qian Y. , Ling Y. . 2016 . The effect of fish oil-based lipid emulsion and soybean oil-based lipid emulsion on cholestasis associated with long-term parenteral nutrition in premature infants . Gastroent. Res. 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Google Scholar CrossRef Search ADS PubMed © 2018 Poultry Science Association Inc. This article is published and distributed under the terms of the Oxford University Press, Standard Journals Publication Model (https://academic.oup.com/journals/pages/about_us/legal/notices) http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Journal of Applied Poultry Research Oxford University Press

The Effect of Oxidized Fish Oils on Growth Performance, Oxidative Status, and Intestinal Barrier Function in Broiler Chickens

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Applied Poultry Science, Inc.
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© 2018 Poultry Science Association Inc.
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1056-6171
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1537-0437
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10.3382/japr/pfy013
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Abstract

Abstract The objective of this study was to determine the effect of oxidized fish oils on growth performance, metabolic oxidative status, and intestinal barrier function in broiler chickens. A total of 240 1-d-old female broiler chickens were assigned to 4 dietary treatments. Dietary treatments comprised of a basal diet supplemented with 4% of non-oxidized (fresh) fish oil, low-oxidized fish oil (FLX), moderately oxidized fish oil (FMX), and highly oxidized fish oil (FHX). Serum corticosterone levels at day 14 and liver concentrations of malondialdehyde (MDA) at day 14 and 21 were higher in birds fed oxidized fish oil compared with those fed then non-oxidized fish oil diet (P < 0.01 in both cases). Furthermore, mRNA expression levels of claudin-1 and occludin were reduced, while those of IL-22 and catalase were increased, in the livers of birds fed the highly oxidized oils compared with those fed fresh fish oils (P ≤ 0.001 in all cases). These results indicate that supplementation of broiler diets with 4% oxidized fish oils can cause lipid peroxidation in the liver, involving increased concentrations of MDA, impaired gut barrier function as a result of increased intestinal permeability due to decreased expression of the tight junction proteins claudin-1/occludin, and intestinal inflammation mediated via upregulation of IL-22 expression in the mucosal tissues. DESCRIPTION OF PROBLEM Lipids derived from a range of animal and vegetable sources are routinely used in poultry feed formulation to increase the energy content of the diet [1]. This is particularly the case in broiler diets that require a high fat content in order to meet the bird's requirements for rapid growth [2]. In addition to increasing the energy content of the feed, lipid supplements reduce feed dustiness [3], are a source of fat soluble vitamins and essential fatty acids [4], and improve diet palatability [5]. Maintaining the high quality of lipid supplements intended for use in broiler diets is critical to maximizing the benefits in bird growth performance. A better understanding of their energy value and of how this can vary with quality will help nutritionists in the formulation of poultry diets [6]. Several previous studies have investigated the effects of fish oil on growth performance and gut health in a range of animal species, and have shown a range of beneficial effects [7]. However, it has also been shown that fish oils are easily oxidized because of their high polyunsaturated fatty acid content [8], where oxygen attacks the double bond in the fatty acids to form lipid [9]. Depending on their level of unsaturation, they may be prone to lipid peroxidation during heat-processing, because they are known to be thermally sensitive and unstable at high temperatures [5]. Several studies have demonstrated that this oxidation can have adverse effects on the growth performance of animals, including broilers and pigs: Yang et al. showed that fish oils impaired growth performance and induce oxidative stress in the whiteleg shrimp, Litopenaeus vannamei [10]; Dibner et al. [11] reported impaired growth performance in broilers and swine due to increased gastrointestinal epithelial cell turnover, hepatic cell proliferation, and decreased the concentration of immunoglobulin in intestinal tissues; and Liu et al. [12] demonstrated that feeding thermally oxidized lipids impaired metabolic oxidative status in young pigs by depleting serum levels of α-tocopherol and increasing serum levels of thiobarbituric acid reactive substance (TBARS). Furthermore, it has been shown that feeding peroxidized corn oils can decrease AMEn in broiler chicks [13]. However, there is limited published information on the effects of dietary supplementation with oxidized fish oil on oxidative stress and intestinal barrier function in broiler chickens. This study aimed to investigate the effect of supplementation of broiler diets with heated oxidized fish oils on growth performance, oxidative status, and intestinal barrier function. MATERIALS AND METHODS Fish Oil Preparation Fresh fish oil was purchased from the Shandong Fish Oil Production Company (Shangdong, China) and stored in a refrigerator at –30°C until use. To oxidize the fish oil, vessels containing the required amount of fresh fish oil were heated to 200°C for either 1, 5, or 9 h to produce low-oxidized fish oil (FLX), moderately oxidized fish oil (FMX), or highly oxidized fish oil (FHX), respectively. The processed oils were stored at –30°C prior to their addition to feed. (No antioxidant was added before or during diet preparation.) The oils were analyzed for their peroxide value (PV) and p-anisidine value (p-AV) according to ISO methods ISO 3960:2001 IDT [14] and ISO 6885:2006 IDT [15], respectively. Malondialdehyde (MDA) concentration was analyzed according to the method of Karatas et al. [16], and Vitamin E (VE) was analyzed according to the method of Yue [17]. Butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), and tertiary butylhydroquinone (TBHQ) content were detected by HPLC [18]. Analytical standards of BHA, BHT, TBHQ, and of α-, β-, γ-, and δ-VE were purchased from Sigma-Aldrich (USA). Animals, Housing, and Diets All experimental procedures were reviewed and approved by the China Agricultural University Animal Care and Use Committee, Beijing, China. A total of 240 1-d female avian broiler chicks were obtained from a commercial hatchery [19] assigned to 4 treatments in a completely randomized design, with 6 replicate cages per treatment and 10 birds per cage. The cage size was 120 × 100 × 60 cm. Treatments were applied to cages in completely randomized design. The treatments comprised of a basal corn–soybean meal-based diet which was formulated to meet or exceed the recommended nutritional requirements of the birds [20] (Table 1) that did not contain antibiotic growth promoters or added antioxidants. This basal diet was supplemented with 4 different fish oil products, at a 4% level of incorporation. The fish oil products included fresh fish oil (control treatment, FNX), low-oxidized fish oil (FLX), moderately oxidized fish oil (FMX), and highly oxidized fish oil (FHX). The trial lasted 21 d. Diets were offered ad libitum and water was freely available. Ambient temperature was initially 31°C and thereafter reduced by 0.5°C per day until 21°C was reached. Light was on continuously throughout the trial. Table 1. Ingredient and Nutrient Composition of the Basal Diet. Ingredient (g/kg) Day 1–21 Corn 531.80 Soybean meal 385.00 Choline chloride, 50% 2.60 Vitamin premix1 0.30 Trace mineral2 2.00 Fish oil 40.00 L-Lysine, 99% 0.80 DL-methionine, 98% 1.90 Dicalcium phosphate 20.00 Limestone 12.50 Sodium chloride 3.10 Calculated nutrient levels Metabolizable energy, kcal/kg 2,961 Crude protein3 211.0 Calcium 10.1 Available phosphorus 4.6 Lysine 12.1 Methionine 5.0 Ingredient (g/kg) Day 1–21 Corn 531.80 Soybean meal 385.00 Choline chloride, 50% 2.60 Vitamin premix1 0.30 Trace mineral2 2.00 Fish oil 40.00 L-Lysine, 99% 0.80 DL-methionine, 98% 1.90 Dicalcium phosphate 20.00 Limestone 12.50 Sodium chloride 3.10 Calculated nutrient levels Metabolizable energy, kcal/kg 2,961 Crude protein3 211.0 Calcium 10.1 Available phosphorus 4.6 Lysine 12.1 Methionine 5.0 1The vitamin premix provided the following per kilogram of complete diet: vitamin A, 9,500 IU; vitamin D3, 62.5 μg; vitamin E, 30 IU; vitamin K3, 2.65 mg; vitamin B1, 2 mg; vitamin B6, 6 mg; vitamin B12, 0.025 mg; biotin, 0.0325 mg; folic acid, 1.25 mg; pantothenic acid, 12 mg; nicotinic acid 50 mg. 2The trace mineral provided the following per kilogram of complete: copper, 8 mg (CuSO4·5H2O); iron, 80 mg (FeSO4); manganese, 100 mg (MnSO4·H2O), selenium, 0.15 mg (Na2SeO3); iodine, 0.35 mg (KI). 3Analyzed. View Large Table 1. Ingredient and Nutrient Composition of the Basal Diet. Ingredient (g/kg) Day 1–21 Corn 531.80 Soybean meal 385.00 Choline chloride, 50% 2.60 Vitamin premix1 0.30 Trace mineral2 2.00 Fish oil 40.00 L-Lysine, 99% 0.80 DL-methionine, 98% 1.90 Dicalcium phosphate 20.00 Limestone 12.50 Sodium chloride 3.10 Calculated nutrient levels Metabolizable energy, kcal/kg 2,961 Crude protein3 211.0 Calcium 10.1 Available phosphorus 4.6 Lysine 12.1 Methionine 5.0 Ingredient (g/kg) Day 1–21 Corn 531.80 Soybean meal 385.00 Choline chloride, 50% 2.60 Vitamin premix1 0.30 Trace mineral2 2.00 Fish oil 40.00 L-Lysine, 99% 0.80 DL-methionine, 98% 1.90 Dicalcium phosphate 20.00 Limestone 12.50 Sodium chloride 3.10 Calculated nutrient levels Metabolizable energy, kcal/kg 2,961 Crude protein3 211.0 Calcium 10.1 Available phosphorus 4.6 Lysine 12.1 Methionine 5.0 1The vitamin premix provided the following per kilogram of complete diet: vitamin A, 9,500 IU; vitamin D3, 62.5 μg; vitamin E, 30 IU; vitamin K3, 2.65 mg; vitamin B1, 2 mg; vitamin B6, 6 mg; vitamin B12, 0.025 mg; biotin, 0.0325 mg; folic acid, 1.25 mg; pantothenic acid, 12 mg; nicotinic acid 50 mg. 2The trace mineral provided the following per kilogram of complete: copper, 8 mg (CuSO4·5H2O); iron, 80 mg (FeSO4); manganese, 100 mg (MnSO4·H2O), selenium, 0.15 mg (Na2SeO3); iodine, 0.35 mg (KI). 3Analyzed. View Large Growth Performance Measurements Chicks and feed were weighed on a per pen basis at day of hatch, day 14 and 21. Feed intakes (FI), body weight gain (BWG), and feed conversion ratio (FCR) were calculated for each period. Sample Collection On day 14 and 21, 1 bird per cage was randomly selected and killed by venous administration of sodium pentobarbital (30 mg/kg of body weight) in order to obtain samples of blood, jejunum mucosa, ileum mucosa, and liver. The intestines were removed, the digesta flushed with 4% saline, and the mucous membranes gently scraped to obtain the samples. Samples were immediately frozen in liquid nitrogen and stored at –35°C until analysis. On day 14 only, a portion of the ileal mucosa samples was immediately frozen and stored at –80°C for mRNA determination. Additionally, on day 14 and 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 day 14, blood samples were collected by jugular exsanguination. Serum was isolated 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 with a radioimmunoassy kit [21], and the indication was measured 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 of ice-cold saline and centrifuged at 20,000× g for 10 min at 4°C. After appropriate dilution, the supernatant fractions were assayed for the activities of total superoxide dismutase (T-SOD), T-AOC, and glutathione peroxidase (GSH-PX), using enzymatic kits [22]. In order to prevent possible enzyme degradation, the samples were kept on ice throughout detection. Malondialdehyde concentration was analyzed as described above. Intestinal Morphology Analysis The fixed intestinal samples were dehydrated and embedded in paraffin wax, sectioned at 3 μm and stained with hematoxylin and eosin. Sections were observed for histomorphology [23]. Villus heights and crypt depths of 10 randomly selected complete villi per sample were measured at 40× 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 villous 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 samples (∼100 mg) using trizol reagent, in accordance with the manufacturer's instructions [24] The purity of the isolated total RNA was determined by measuring its optical density at 260 and 280 nm. Samples of the extracted total RNA (2 μg) were reverse transcribed using a reverse transcription kit [24], and the expression levels of targeted genes were detected according to quantitative real-time PCR assay with a 7,300 real-time PCR system [25] using Fast Start Universal SYBR Green Master [26] after generation of standard curves for each of 5 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 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 [27], and data were expressed as values relative to the control group. Table 2. Oligonucleotide Primers Used for Quantitative Real-Time PCR of Ileal Mucosa Tissue Samples. Gene Primer fragment Accession number β-actin 5‘-GGATTGGAGGCTCTATCCTGG-3’ NM_205518.1 5‘-GTTTAGAAGCATTTGCGGTGG-3’ Claudin-1 5‘-GATGCGGATGGCTGTCTTTG-3’ NM_001013611.2 5‘-GCTGGGTGGGTAGGATGTTTC-3’ Occludin 5‘-GCCGTAACCCCGAGTTGGAT-3’ NM_205128.1 5‘-TGATTGAGGCGGTCGTTGATG-3’ IL22 5 ‘-ACCCGTATGCTGAGGATGTGG-3 ’ NM_001199614.1 5‘-CTTGTTCCCTCCCTTCTTTGG-3’ CAT 5‘-AGCAGGTGCCTTTGGCTATT-3’ NM_001031215.1 5‘-CGAGGGTCACGAACTGTATCA-3’ Gene Primer fragment Accession number β-actin 5‘-GGATTGGAGGCTCTATCCTGG-3’ NM_205518.1 5‘-GTTTAGAAGCATTTGCGGTGG-3’ Claudin-1 5‘-GATGCGGATGGCTGTCTTTG-3’ NM_001013611.2 5‘-GCTGGGTGGGTAGGATGTTTC-3’ Occludin 5‘-GCCGTAACCCCGAGTTGGAT-3’ NM_205128.1 5‘-TGATTGAGGCGGTCGTTGATG-3’ IL22 5 ‘-ACCCGTATGCTGAGGATGTGG-3 ’ NM_001199614.1 5‘-CTTGTTCCCTCCCTTCTTTGG-3’ CAT 5‘-AGCAGGTGCCTTTGGCTATT-3’ NM_001031215.1 5‘-CGAGGGTCACGAACTGTATCA-3’ View Large Table 2. Oligonucleotide Primers Used for Quantitative Real-Time PCR of Ileal Mucosa Tissue Samples. Gene Primer fragment Accession number β-actin 5‘-GGATTGGAGGCTCTATCCTGG-3’ NM_205518.1 5‘-GTTTAGAAGCATTTGCGGTGG-3’ Claudin-1 5‘-GATGCGGATGGCTGTCTTTG-3’ NM_001013611.2 5‘-GCTGGGTGGGTAGGATGTTTC-3’ Occludin 5‘-GCCGTAACCCCGAGTTGGAT-3’ NM_205128.1 5‘-TGATTGAGGCGGTCGTTGATG-3’ IL22 5 ‘-ACCCGTATGCTGAGGATGTGG-3 ’ NM_001199614.1 5‘-CTTGTTCCCTCCCTTCTTTGG-3’ CAT 5‘-AGCAGGTGCCTTTGGCTATT-3’ NM_001031215.1 5‘-CGAGGGTCACGAACTGTATCA-3’ Gene Primer fragment Accession number β-actin 5‘-GGATTGGAGGCTCTATCCTGG-3’ NM_205518.1 5‘-GTTTAGAAGCATTTGCGGTGG-3’ Claudin-1 5‘-GATGCGGATGGCTGTCTTTG-3’ NM_001013611.2 5‘-GCTGGGTGGGTAGGATGTTTC-3’ Occludin 5‘-GCCGTAACCCCGAGTTGGAT-3’ NM_205128.1 5‘-TGATTGAGGCGGTCGTTGATG-3’ IL22 5 ‘-ACCCGTATGCTGAGGATGTGG-3 ’ NM_001199614.1 5‘-CTTGTTCCCTCCCTTCTTTGG-3’ CAT 5‘-AGCAGGTGCCTTTGGCTATT-3’ NM_001031215.1 5‘-CGAGGGTCACGAACTGTATCA-3’ View Large Statistical Analysis Data on growth performance were based on a per pen basis. All other data were based on individual birds. 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 ANOVA using the procedures of SPSS Version 18.0 statistical software [28]. Differences between means were identified using Duncan's multiple-range test. Differences were considered significant at P < 0.05. RESULTS AND DISCUSSION Chemical Characteristics of the Experimental Fish Oils The concentrations of PV, MDA, p-AV, VE, BHA, BHT, and TBHQ in the 4 experimental fish oils are shown in Table 3. Vitamin E, BHA, BHT, and TBHQ were not detected in any of the oils. As expected, with increased duration of heating, the PV of the oil gradually increased. Also as expected, compared to the FNX oil, the concentration of MDA and p-AV in the 3 oxidized oils was increased. Table 3. Biochemical Analysis of the Experimental Oils.1 Fish oil supplement Item FNX FLX FMX FHX Peroxide value (meq/kg) 20.77 140.41 183.54 277.35 Malondialdehyde (μg/kg) 65.67 145.40 150.39 154.73 p-anisidine value 22 66 149 192 Butylated hydroxyanisole (mg/kg) ND2 ND ND ND Butylated hydroxytoluene (mg/kg) ND ND ND ND Tertiary butylhydroquinone (mg/kg) ND ND ND ND Vitamin E (mg/kg) ND ND ND ND Fish oil supplement Item FNX FLX FMX FHX Peroxide value (meq/kg) 20.77 140.41 183.54 277.35 Malondialdehyde (μg/kg) 65.67 145.40 150.39 154.73 p-anisidine value 22 66 149 192 Butylated hydroxyanisole (mg/kg) ND2 ND ND ND Butylated hydroxytoluene (mg/kg) ND ND ND ND Tertiary butylhydroquinone (mg/kg) ND ND ND ND Vitamin E (mg/kg) ND ND ND ND 1FNX = non-oxidized fish oil (fresh fish oil); FLX = low-oxidized fish oil (heated for 1 h); FMX = moderately oxidized fish oil (heated for 5 h); FHX = highly oxidized fish oil (heated for 9 h). 2ND = not detected. View Large Table 3. Biochemical Analysis of the Experimental Oils.1 Fish oil supplement Item FNX FLX FMX FHX Peroxide value (meq/kg) 20.77 140.41 183.54 277.35 Malondialdehyde (μg/kg) 65.67 145.40 150.39 154.73 p-anisidine value 22 66 149 192 Butylated hydroxyanisole (mg/kg) ND2 ND ND ND Butylated hydroxytoluene (mg/kg) ND ND ND ND Tertiary butylhydroquinone (mg/kg) ND ND ND ND Vitamin E (mg/kg) ND ND ND ND Fish oil supplement Item FNX FLX FMX FHX Peroxide value (meq/kg) 20.77 140.41 183.54 277.35 Malondialdehyde (μg/kg) 65.67 145.40 150.39 154.73 p-anisidine value 22 66 149 192 Butylated hydroxyanisole (mg/kg) ND2 ND ND ND Butylated hydroxytoluene (mg/kg) ND ND ND ND Tertiary butylhydroquinone (mg/kg) ND ND ND ND Vitamin E (mg/kg) ND ND ND ND 1FNX = non-oxidized fish oil (fresh fish oil); FLX = low-oxidized fish oil (heated for 1 h); FMX = moderately oxidized fish oil (heated for 5 h); FHX = highly oxidized fish oil (heated for 9 h). 2ND = not detected. View Large Growth Performance Feed conversion ratio was significantly increased in birds fed the oxidized oils compared with the fresh fish oil (FNX), between day 0 and 14 (P < 0.05), but the differences were not significant for the overall period (day 0–21 (P = 0.09)) (Table 4). Dietary treatment had no effect on FI or BWG. This absence of effect on FI and BWG is consistent with the findings of other studies in which diets supplemented with oxidized oils did not affect animal performance [17, 29, 30]. No significant difference on body weight gain, FI in our study may be partly due to the lower oxidation levels of fish and less oxidized fish oil content of feed than in other studies [31, 32]. A reduced growth rate in animals fed thermally oxidized lipids may be caused by several factors. Firstly, the thermally oxidized lipids may cause rancidity that can reduce diet palatability and thereby decrease FI leading to a poor growth rate [12]. Secondly, secondary lipid peroxidation products, such as α,β-unsaturated hydroxyaldahydes, are of particular interest because some of them are highly toxic and readily absorbed [33], and are capable of modifying proteins in vivo by damaging the intestinal brush membrane [11]. Table 4. Growth Performance of Broilers Whose Diets were Supplemented With Fresh Fish Oil (FNX), Low-, Moderate-, and Highly Oxidized Fish Oils (FLX, FMX, and FHX).1,2 Day 1–14 Day 1–21 Treatment BWG (g) FI (g) FCR BWG (g) FI (g) FCR FNX 283 336 1.19b 675 905 1.34 FLX 280 367 1.31a 657 949 1.45 FMX 274 348 1.28a 663 930 1.41 FHX 269 358 1.33a 626 924 1.48 SEM 4 5 0.02 8 13 0.02 P-value 0.620 0.113 0.022 0.183 0.703 0.090 Day 1–14 Day 1–21 Treatment BWG (g) FI (g) FCR BWG (g) FI (g) FCR FNX 283 336 1.19b 675 905 1.34 FLX 280 367 1.31a 657 949 1.45 FMX 274 348 1.28a 663 930 1.41 FHX 269 358 1.33a 626 924 1.48 SEM 4 5 0.02 8 13 0.02 P-value 0.620 0.113 0.022 0.183 0.703 0.090 1Data are means of values from 10 birds per replicate cage (n = 6 replicate cages). FNX = fish oil non-oxidized group (fresh fish oil); FLX = fish oil low-oxidized group; FMX = fish oil moderately oxidized group; FHX = fish oil highly oxidized group. FCR = feed conversion ratio. SEM = standard error of the mean. 2Fish oils were incorporated into the diet at a level of 4%. a,bMeans in the same column with different superscript letters are significantly different (P < 0.05). View Large Table 4. Growth Performance of Broilers Whose Diets were Supplemented With Fresh Fish Oil (FNX), Low-, Moderate-, and Highly Oxidized Fish Oils (FLX, FMX, and FHX).1,2 Day 1–14 Day 1–21 Treatment BWG (g) FI (g) FCR BWG (g) FI (g) FCR FNX 283 336 1.19b 675 905 1.34 FLX 280 367 1.31a 657 949 1.45 FMX 274 348 1.28a 663 930 1.41 FHX 269 358 1.33a 626 924 1.48 SEM 4 5 0.02 8 13 0.02 P-value 0.620 0.113 0.022 0.183 0.703 0.090 Day 1–14 Day 1–21 Treatment BWG (g) FI (g) FCR BWG (g) FI (g) FCR FNX 283 336 1.19b 675 905 1.34 FLX 280 367 1.31a 657 949 1.45 FMX 274 348 1.28a 663 930 1.41 FHX 269 358 1.33a 626 924 1.48 SEM 4 5 0.02 8 13 0.02 P-value 0.620 0.113 0.022 0.183 0.703 0.090 1Data are means of values from 10 birds per replicate cage (n = 6 replicate cages). FNX = fish oil non-oxidized group (fresh fish oil); FLX = fish oil low-oxidized group; FMX = fish oil moderately oxidized group; FHX = fish oil highly oxidized group. FCR = feed conversion ratio. SEM = standard error of the mean. 2Fish oils were incorporated into the diet at a level of 4%. a,bMeans in the same column with different superscript letters are significantly different (P < 0.05). View Large Metabolic Oxidative Status The concentrations of serum CORT at day 14 were significantly affected (P < 0.01) by treatment (Table 5). Regardless of the level of oxidation, birds fed the oxidized fish oil had higher serum CORT levels than birds fed fresh fish oil (P < 0.01). The concentrations of MDA, T-AOC, and the activities of GSH-PX and T-SOD in the liver are showed in Table 5. The concentration of MDA in the liver was also affected by treatment at both day 14 and 21 (P < 0.01) (Table 5); at day 14, birds fed FLX oxidized fish oil had higher MDA concentrations in the liver than birds fed fresh fish oil (P < 0.001), but by day 21 MDA concentrations were significantly elevated in birds fed FLX, FMX, or FHX, compared with control-fed birds (P < 0.01). The oxidation levels of the fish oil did not affect the concentrations of T-AOC or the activities of GSH-PX and T-SOD in the liver on day 14 or 21 (Table 5). Table 5. Serum Corticosterone and Liver Concentrations of Antioxidant-Related Enzymes Broilers Fed Diets Supplemented With Fresh or Oxidized Fish Oils.1,2 Day 14 Day 21 Treatment Serum CORT (ng/mL) MDA (nmol/ mgprot) T-AOC (U/ mgprot) GSH-Px (U/ mgprot) T-SOD (U/ mgprot) MDA (nmol/ mgprot) T-AOC (U/ mgprot) GSH-Px (U/ mgprot) T-SOD (U/ mgprot) FNX 10.00b 1.407b 1.608 13.5 189 0.920b 0.988 18.4 152 FLX 11.50a 1.902a 1.464 14.2 205 1.384a 1.061 17.7 138 FMX 12.42a 1.857a,b 1.584 13.4 213 1.334a 1.045 18.6 151 FHX 11.99a 1.803a,b 1.720 13.0 207 1.312a 1.180 20.0 142 SEM 0.29 0.111 0.044 0.3 4 0.061 0.036 0.4 3 P-value 0.008 0.000 0.229 0.677 0.110 0.007 0.300 0.300 0.186 Day 14 Day 21 Treatment Serum CORT (ng/mL) MDA (nmol/ mgprot) T-AOC (U/ mgprot) GSH-Px (U/ mgprot) T-SOD (U/ mgprot) MDA (nmol/ mgprot) T-AOC (U/ mgprot) GSH-Px (U/ mgprot) T-SOD (U/ mgprot) FNX 10.00b 1.407b 1.608 13.5 189 0.920b 0.988 18.4 152 FLX 11.50a 1.902a 1.464 14.2 205 1.384a 1.061 17.7 138 FMX 12.42a 1.857a,b 1.584 13.4 213 1.334a 1.045 18.6 151 FHX 11.99a 1.803a,b 1.720 13.0 207 1.312a 1.180 20.0 142 SEM 0.29 0.111 0.044 0.3 4 0.061 0.036 0.4 3 P-value 0.008 0.000 0.229 0.677 0.110 0.007 0.300 0.300 0.186 1Data are means of values from 6 individual birds (1 per replicate cage). FNX = fish oil non-oxidized group; FLX = fish oil low-oxidized group; FMX = fish oil moderately oxidized group; FHX = fish oil highly oxidized group. serum CORT, serum corticosterone; MDA, malonaldehyde; T-AOC, total-antioxidative capacity; GSH-PX, glutathione peroxidase; T-SOD, total superoxide dismutase. mgprot, milligram protein. SEM, standard error of the mean. 2Fish oils were incorporated into the diet at a level of 4%. a,bMeans in the same column with different superscript letters are significantly different (P < 0.05). View Large Table 5. Serum Corticosterone and Liver Concentrations of Antioxidant-Related Enzymes Broilers Fed Diets Supplemented With Fresh or Oxidized Fish Oils.1,2 Day 14 Day 21 Treatment Serum CORT (ng/mL) MDA (nmol/ mgprot) T-AOC (U/ mgprot) GSH-Px (U/ mgprot) T-SOD (U/ mgprot) MDA (nmol/ mgprot) T-AOC (U/ mgprot) GSH-Px (U/ mgprot) T-SOD (U/ mgprot) FNX 10.00b 1.407b 1.608 13.5 189 0.920b 0.988 18.4 152 FLX 11.50a 1.902a 1.464 14.2 205 1.384a 1.061 17.7 138 FMX 12.42a 1.857a,b 1.584 13.4 213 1.334a 1.045 18.6 151 FHX 11.99a 1.803a,b 1.720 13.0 207 1.312a 1.180 20.0 142 SEM 0.29 0.111 0.044 0.3 4 0.061 0.036 0.4 3 P-value 0.008 0.000 0.229 0.677 0.110 0.007 0.300 0.300 0.186 Day 14 Day 21 Treatment Serum CORT (ng/mL) MDA (nmol/ mgprot) T-AOC (U/ mgprot) GSH-Px (U/ mgprot) T-SOD (U/ mgprot) MDA (nmol/ mgprot) T-AOC (U/ mgprot) GSH-Px (U/ mgprot) T-SOD (U/ mgprot) FNX 10.00b 1.407b 1.608 13.5 189 0.920b 0.988 18.4 152 FLX 11.50a 1.902a 1.464 14.2 205 1.384a 1.061 17.7 138 FMX 12.42a 1.857a,b 1.584 13.4 213 1.334a 1.045 18.6 151 FHX 11.99a 1.803a,b 1.720 13.0 207 1.312a 1.180 20.0 142 SEM 0.29 0.111 0.044 0.3 4 0.061 0.036 0.4 3 P-value 0.008 0.000 0.229 0.677 0.110 0.007 0.300 0.300 0.186 1Data are means of values from 6 individual birds (1 per replicate cage). FNX = fish oil non-oxidized group; FLX = fish oil low-oxidized group; FMX = fish oil moderately oxidized group; FHX = fish oil highly oxidized group. serum CORT, serum corticosterone; MDA, malonaldehyde; T-AOC, total-antioxidative capacity; GSH-PX, glutathione peroxidase; T-SOD, total superoxide dismutase. mgprot, milligram protein. SEM, standard error of the mean. 2Fish oils were incorporated into the diet at a level of 4%. a,bMeans in the same column with different superscript letters are significantly different (P < 0.05). View Large Table 6. Antioxidant-Related Activity Levels in the Jejunal Mucosa of Broilers Fed Diets With Fresh and Oxidized Fish Oils.1,2 Day 14 Day 21 Treatment MDA (nmol/mgprot) T-AOC (U/mgprot) T-SOD (U/mgprot) MDA (nmol/mgprot) T-AOC (U/mgprot) T-SOD (U/mgprot) FNX 0.655 1.149 103 1.221 3.029 138b FLX 0.679 1.120 104 1.304 3.165 159a,b FMX 0.469 1.177 91 1.076 3.400 163a FHX 0.628 2.129 94 1.163 4.393 172a SEM 0.053 0.133 2.776 0.050 0.202 4.004 P-value 0.521 0.193 0.259 0.451 0.060 0.007 Day 14 Day 21 Treatment MDA (nmol/mgprot) T-AOC (U/mgprot) T-SOD (U/mgprot) MDA (nmol/mgprot) T-AOC (U/mgprot) T-SOD (U/mgprot) FNX 0.655 1.149 103 1.221 3.029 138b FLX 0.679 1.120 104 1.304 3.165 159a,b FMX 0.469 1.177 91 1.076 3.400 163a FHX 0.628 2.129 94 1.163 4.393 172a SEM 0.053 0.133 2.776 0.050 0.202 4.004 P-value 0.521 0.193 0.259 0.451 0.060 0.007 1Data are means of values from 6 individual birds (1 per replicate cage). FNX = fish oil non-oxidized group; FLX = fish oil low-oxidized group; FMX = fish oil moderately oxidized group; FHX = fish oil highly oxidized group. MDA, malonaldehyde; T-AOC, total-antioxidative capacity; T-SOD, total superoxide dismutase. mgprot, milligram protein. SEM, standard error of the mean. 2Fish oils were incorporated into the diet at a level of 4%. a,bMeans in the same column with different superscript letters are significantly different (P < 0.05). View Large Table 6. Antioxidant-Related Activity Levels in the Jejunal Mucosa of Broilers Fed Diets With Fresh and Oxidized Fish Oils.1,2 Day 14 Day 21 Treatment MDA (nmol/mgprot) T-AOC (U/mgprot) T-SOD (U/mgprot) MDA (nmol/mgprot) T-AOC (U/mgprot) T-SOD (U/mgprot) FNX 0.655 1.149 103 1.221 3.029 138b FLX 0.679 1.120 104 1.304 3.165 159a,b FMX 0.469 1.177 91 1.076 3.400 163a FHX 0.628 2.129 94 1.163 4.393 172a SEM 0.053 0.133 2.776 0.050 0.202 4.004 P-value 0.521 0.193 0.259 0.451 0.060 0.007 Day 14 Day 21 Treatment MDA (nmol/mgprot) T-AOC (U/mgprot) T-SOD (U/mgprot) MDA (nmol/mgprot) T-AOC (U/mgprot) T-SOD (U/mgprot) FNX 0.655 1.149 103 1.221 3.029 138b FLX 0.679 1.120 104 1.304 3.165 159a,b FMX 0.469 1.177 91 1.076 3.400 163a FHX 0.628 2.129 94 1.163 4.393 172a SEM 0.053 0.133 2.776 0.050 0.202 4.004 P-value 0.521 0.193 0.259 0.451 0.060 0.007 1Data are means of values from 6 individual birds (1 per replicate cage). FNX = fish oil non-oxidized group; FLX = fish oil low-oxidized group; FMX = fish oil moderately oxidized group; FHX = fish oil highly oxidized group. MDA, malonaldehyde; T-AOC, total-antioxidative capacity; T-SOD, total superoxide dismutase. mgprot, milligram protein. SEM, standard error of the mean. 2Fish oils were incorporated into the diet at a level of 4%. a,bMeans in the same column with different superscript letters are significantly different (P < 0.05). View Large On the other hand, FMX and FHX group increased (P < 0.05) the level of T-SOD in jejunal mucosa on day 21 compared with chickens fed non-oxidized fish oil (Table 6). Dietary oxidized fish oil treatment did not affect the metabolic oxidative status in jejunal mucosa on day 14 (P > 0.05) (Table 6). The concentrations of MDA and activities of T-AOC and T-SOD in the ileal mucosa are showed in Table 7. There were no effects of dietary treatment on the metabolic oxidative status of the ileal mucosa on day 14 or 21 (P > 0.05). Table 7. Antioxidant-Related Activity Levels in the Ileal Mucosa of Broilers Fed Diets-With Fresh and Oxidized Fish Oils.1,2 Day 14 Day 21 Treatment MDA (nmol/mgprot) T-AOC (U/mgprot) T-SOD (U/mgprot) MDA (nmol/mgprot) T-AOC (U/mgprot) T-SOD (U/mgprot) FNX 0.534 1.274 60 0.813 1.979 110 FLX 0.540 1.564 65 0.728 1.789 106 FMX 0.453 1.547 63 0.725 1.986 100 FHX 0.427 1.609 59 0.660 2.160 106 SEM 0.021 0.046 1 0.035 0.057 1 P-value 0.135 0.182 0.077 0.481 0.126 0.108 Day 14 Day 21 Treatment MDA (nmol/mgprot) T-AOC (U/mgprot) T-SOD (U/mgprot) MDA (nmol/mgprot) T-AOC (U/mgprot) T-SOD (U/mgprot) FNX 0.534 1.274 60 0.813 1.979 110 FLX 0.540 1.564 65 0.728 1.789 106 FMX 0.453 1.547 63 0.725 1.986 100 FHX 0.427 1.609 59 0.660 2.160 106 SEM 0.021 0.046 1 0.035 0.057 1 P-value 0.135 0.182 0.077 0.481 0.126 0.108 1Data are means of values from 6 individual birds (1 per replicate cage). FNX = fish oil non-oxidized group; FLX = fish oil low-oxidized group; FMX = fish oil moderately oxidized group; FHX = fish oil highly oxidized group. MDA, malonaldehyde; T-AOC, total-antioxidative capacity; T-SOD, total superoxide dismutase. mgprot, milligram protein. SEM, standard error of the mean. 2Fish oils were incorporated into the diet at a level of 4%. View Large Table 7. Antioxidant-Related Activity Levels in the Ileal Mucosa of Broilers Fed Diets-With Fresh and Oxidized Fish Oils.1,2 Day 14 Day 21 Treatment MDA (nmol/mgprot) T-AOC (U/mgprot) T-SOD (U/mgprot) MDA (nmol/mgprot) T-AOC (U/mgprot) T-SOD (U/mgprot) FNX 0.534 1.274 60 0.813 1.979 110 FLX 0.540 1.564 65 0.728 1.789 106 FMX 0.453 1.547 63 0.725 1.986 100 FHX 0.427 1.609 59 0.660 2.160 106 SEM 0.021 0.046 1 0.035 0.057 1 P-value 0.135 0.182 0.077 0.481 0.126 0.108 Day 14 Day 21 Treatment MDA (nmol/mgprot) T-AOC (U/mgprot) T-SOD (U/mgprot) MDA (nmol/mgprot) T-AOC (U/mgprot) T-SOD (U/mgprot) FNX 0.534 1.274 60 0.813 1.979 110 FLX 0.540 1.564 65 0.728 1.789 106 FMX 0.453 1.547 63 0.725 1.986 100 FHX 0.427 1.609 59 0.660 2.160 106 SEM 0.021 0.046 1 0.035 0.057 1 P-value 0.135 0.182 0.077 0.481 0.126 0.108 1Data are means of values from 6 individual birds (1 per replicate cage). FNX = fish oil non-oxidized group; FLX = fish oil low-oxidized group; FMX = fish oil moderately oxidized group; FHX = fish oil highly oxidized group. MDA, malonaldehyde; T-AOC, total-antioxidative capacity; T-SOD, total superoxide dismutase. mgprot, milligram protein. SEM, standard error of the mean. 2Fish oils were incorporated into the diet at a level of 4%. View Large The primary physiological response of poultry when the body suffers oxidative stress is to activate the hypothalamic-pituitary-adrenal axis, which is characterized by adrenal cortical hypertrophy and increased synthesis and release of adrenal glucocorticoids, known as CORT [34]. Corticosterone levels are thus used as an indicator of stress in mammals [35]. The higher concentrations of serum CORT among birds fed the oxidized oils in this study are consistent with previous studies on mice [36], and raised CORT levels in broilers have previously been linked with conditions of stress [35]. Malondialdehyde is an endogenous genotoxic product of enzymatic and free radical-induced lipid peroxidation [37]. Therefore, raised MDA levels are an indication of lipid peroxidation in the tissues and of the presence of reactive oxygen species residing in the tissues [38]. In this study, while feeding oxidized fish oil to birds had no effect on concentrations in the jejunal or ileal mucosa, levels of MDA in the liver were significantly raised. These findings are broadly consistent with comparable evidence from other animal species. Feeding repeatedly heated soy oil has been reported to increase serum TBARS in ovariectomized rats [32], finishing barrows [39], young pigs [40], and the whiteleg shrimp [10]. However, feeding oxidized fish oil did not affect the TBARS values of blood serum and liver in the Chinese longsnout catfish [41]. In pigs, feeding oxidized oil was observed to increase hepatic nuclear concentration of the transcription factor erythroid-derived 2-like 2 (Nrf2) [3], which is an important transcription factor mediating cellular stress response. The action of heating oil leads to a loss in polyunsaturated fatty acids and degrades native nutritive compounds such as tocopherol and essential fatty acids [42]. Against this background, there are 2 possible reasons by which to explain the increased levels of MDA found in the liver of birds fed diets supplemented with oxidized oils. The first reason may be the activity of Nrf2 [43], leading to the increase of the concentrations of MDA in liver. The second reason of increasing MDA concentrations in liver may be the potential mutagenic effects of that peroxide which is produced as a result of the extended heating time of the oxidized fish oil [37]. Collectively, our results indicate that the dietary intake of oxidized fish oil caused oxidative liver damage that may have a direct implication for the ability of the bird to resist oxidative stress, as has been suggested previously for the Chinese mitten crab [44]. Glutathione peroxidase is important in metabolism and enzyme regulation as well as detoxification of cytotoxic materials [45], including the elimination of organic hydroperoxides and hydrogen peroxides [31]. No effects of treatment on liver concentrations of GSH-PX were observed in the present study. However, effects were seen on the levels of T-SOD in jejunal mucosa (at day 21). The findings are consistent with those of a recent study by Wang et al. [44], in which feeding oxidized oil to the Chinese mitten crab was observed to improve serum T-SOD activity. Raised levels of this antioxidant enzyme may reflect a compensatory effect or represent a precursor to the manifestation of oxidative stress in tissue cells [36, 44, 46]. Catalase is a major enzyme involved in the detoxification of H2O2 in cells [47]. Therefore, the decrease in CAT mRNA expression in the present study indicates a reduced capacity of the bird to eliminate free radicals. These findings are consistent with those of other studies conducted on broilers [38] and pigs [48] that have involved dietary supplementation with oxidized lipid. Overall, these findings suggest that oxidation stress occurred in the intestinal mucosa of broilers fed oxidized fish oil. Claudin-1, Occludin, IL-22, and CAT mRNA Expression in the Ileum The mRNA expression levels of claudin-1, occludin, IL-22, and CAT at day 14 in the ilea of birds fed the 4 experimental treatments are presented in Table 8. The mRNA expressions of claudin-1, occludin, IL-22, and CAT in ileum were affected by dietary oxidized oil treatment (P < 0.05). Birds fed oxidized fish oil, regardless of the level of oxidation, exhibited reduced mRNA expression levels of Claudin-1 compared with control-diet birds (P < 0.001). Birds in the low- and high-oxidized fish oil groups also exhibited reduced occludin expression (P < 0.01). Conversely, birds fed the moderate- and especially the highly oxidized fish oil exhibited increased expression levels of IL-22 compared with birds fed the fresh fish oil. Similarly, expression levels of CAT were reduced in birds fed the highly oxidized fish oil compared with control-diet birds (P < 0.01). Table 8. Relative mRNA Expression Levels of Claudin-1, Occludin, IL-22, and CAT.1,2 Treatment Claudin-1 Occludin IL-22 CAT FNX 0.912a 1.002a 1.102c 1.082a FLX 0.736b 0.335b 1.361c 1.102a FMX 0.651b 1.178a 2.443b 1.152a FHX 0.409c 0.473b 4.521a 0.737b SEM 0.045 0.115 0.381 0.054 P-value 0.000 0.001 0.000 0.001 Treatment Claudin-1 Occludin IL-22 CAT FNX 0.912a 1.002a 1.102c 1.082a FLX 0.736b 0.335b 1.361c 1.102a FMX 0.651b 1.178a 2.443b 1.152a FHX 0.409c 0.473b 4.521a 0.737b SEM 0.045 0.115 0.381 0.054 P-value 0.000 0.001 0.000 0.001 1Data are means of values from 6 individual birds (1 per replicate cage). FNX = fish oil non-oxidized group; FLX = fish oil low-oxidized group; FMX = fish oil moderately oxidized group; FHX = fish oil highly oxidized group. β-Actin was used as an endogenous reference gene, and mRNA expression was expressed as the relative value to the SNX group. IL-22, interleukin; CAT, catalase. SEM, standard error of the mean. 2Fish oils were incorporated into the diet at a level of 4%. a,bMeans in the same column with different superscript letters are significantly different (P < 0.05). View Large Table 8. Relative mRNA Expression Levels of Claudin-1, Occludin, IL-22, and CAT.1,2 Treatment Claudin-1 Occludin IL-22 CAT FNX 0.912a 1.002a 1.102c 1.082a FLX 0.736b 0.335b 1.361c 1.102a FMX 0.651b 1.178a 2.443b 1.152a FHX 0.409c 0.473b 4.521a 0.737b SEM 0.045 0.115 0.381 0.054 P-value 0.000 0.001 0.000 0.001 Treatment Claudin-1 Occludin IL-22 CAT FNX 0.912a 1.002a 1.102c 1.082a FLX 0.736b 0.335b 1.361c 1.102a FMX 0.651b 1.178a 2.443b 1.152a FHX 0.409c 0.473b 4.521a 0.737b SEM 0.045 0.115 0.381 0.054 P-value 0.000 0.001 0.000 0.001 1Data are means of values from 6 individual birds (1 per replicate cage). FNX = fish oil non-oxidized group; FLX = fish oil low-oxidized group; FMX = fish oil moderately oxidized group; FHX = fish oil highly oxidized group. β-Actin was used as an endogenous reference gene, and mRNA expression was expressed as the relative value to the SNX group. IL-22, interleukin; CAT, catalase. SEM, standard error of the mean. 2Fish oils were incorporated into the diet at a level of 4%. a,bMeans in the same column with different superscript letters are significantly different (P < 0.05). View Large The increased expression of claudin in birds fed the oxidized fish oils may be indicative of adverse effects on intestinal barrier function, which has not previously been reported in broilers. The most important structures of the intestinal barrier are the epithelial tight junctions (TJs) that connect adjacent enterocytes together to determine paracellular permeability through the lateral intercellular space [49]. Claudins are a family of nearly 2 dozen transmenbrane proteins that are a key part of the TJ barrier that regulates solute movement across the polarized epithelia [50]. Therefore, reduced claudin expression may indicate impaired functioning of TJ proteins and a consequent reduction in epithelial barrier integrity. Effects on occludin expression may have a similar result. Occludin regulates macromolecule flux across the intestinal epithelial TJ barrier [51]. The expression of occludin is known to be markedly decreased in intestinal permeability disorders, including in Crohn's disease, ulcerative colitis [52], and celiac disease [53], suggesting that reduced occludin expression may play a role in the increase in intestinal permeability. Against this background, it seems plausible that the reduced expression of claudin-1 and occludin in the ileal mucosa of birds fed oxidized fish oil in this study could be indicative of an increase in intestinal permeability, thus leading to intestinal barrier dysfunction. Similar findings have been observed in terms of reduced expression of TJ proteins in the human proximal small intestinal mucosa before and after Roux-en-Y gastric bypass surgery [54] and when hyperthermia-induced oxidative stress led to intestinal mucosa barrier dysfunction in mice [55]. However, other studies suggest an absence of effect; feeding thermally oxidized vegetable oils and animal fats had little influence on the ratio of urinary lactulose to mannitol in pigs, this being one of the most popular methods used to measure intestinal permeability [40]. There is little existing published information regarding the effects of dietary supplementation with oxidized oils on gut inflammation in broilers. Interleukin-22 is a pro-inflammatory cytokine that is predominantly produced by activated Th1 cells and signals through a receptor complex consisting of IL-22R1 and IL-10R2 [56]. Existing studies have revealed that IL-22 targets cells of the digestive, skin, and respiratory systems and plays an important role in the mucosal immunity [57]. In chronic Hepatitis C Viral (HCV), T cells producing IL-22 may migrate to the liver to reduce inflammation [58]. Furthermore, intestinal epithelial STAT3 activation can regulate immune homeostasis in the gut by promoting IL-22-dependent mucosal wound healing [59]. Many studies mouse model systems have identified a critical role for signaling by IL-22 through its receptor (IL-22R) in the promotion of antimicrobial immunity, inflammation, and tissue repair at barrier surfaces [60]. Therefore, the raised IL-22 expression levels in the ileal mucosa of birds fed highly oxidized fish oils in this study may be indicative of the induction of an inflammatory response. Intestinal Morphology in the Ileum and Jejunum The structure of the intestinal mucosa can reveal some information on gut health. The intestinal morphology in the jejunum and ileum were not significant affected (P > 0.05) by dietary oxidized oil treatment (data not shown). There is little existing published information regarding the effects of dietary supplementation with oxidized oils on gut morphology in broilers. The crypt is considered as the villus cell producer. In this respect, a deeper crypt shows rapid tissue turnover and a high demand for new tissue [61]. Stressors that are present in the digesta can lead relatively quickly to changes in the intestinal mucosa, due to the close proximity of the mucosal surface and the intestinal content. Changes in intestinal morphology, such as shorter villi and deeper crypts, have been associated with the presence of toxins or higher tissue turnover [62]. We did not see significant effects on intestinal morphology by different oxidation level of fish oils; this may be due to the low content of oil in feed and low oxidized degree of oil. CONCLUSION AND APPLICATION 1. Supplementation of broiler diets with oxidized fish oil vs. fresh fish oil reduced FCR between day 0 and 14 but had no other adverse effects on growth performance. 2. Serum corticosteroid and liver MDA concentrations were raised, metabolic oxidative status was impaired, and ileal mucosal expressions of claudin, occuldin-1, IL-22, and CAT were adversely affected in birds fed oxidized fish oils. 3. Based on this study, it is suggested that oxidized fish oil can reduce FCR, induce oxidative stress in the liver, and impair intestinal barrier function, through upregulation of IL-22. Acknowledgments The authors would like to thank Yuxin Shao, Yanyan Yang, Yuanyang Dong, Xuan Liu, and He Gao for their help with the experiments. This work was supported by the Beijing Higher Education Young Elite Teacher Project, the Yangtz River Scholar and Innovation Research Team Development Program (No. IRT0945), and the Chinese Universities Scientific Fund (No. 2015DK005). Footnotes Primary Audience: Poultry researchers REFERENCES AND NOTES 1. Li P. , Piao X. , Ru Y. , Han X. , Xue L. , Zhang H. . 2012 . Effects of adding essential oil to the diet of weaned pigs on performance, nutrient utilization, immune response and intestinal health . Asian Australas. J. Anim. Sci. 25 : 1617 – 1626 . Google Scholar CrossRef Search ADS PubMed 2. Wang L. , Zhang J. , Gao J. , Qian Y. , Ling Y. . 2016 . The effect of fish oil-based lipid emulsion and soybean oil-based lipid emulsion on cholestasis associated with long-term parenteral nutrition in premature infants . Gastroent. Res. 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Journal of Applied Poultry ResearchOxford University Press

Published: May 14, 2018

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