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The effect of dietary supplementation with the natural carotenoids curcumin and lutein on broiler pigmentation and immunity
The effect of dietary supplementation with the natural carotenoids curcumin and lutein on broiler pigmentation and immunity N. Rajput,* M. Naeem,* S. Ali,† J. F. Zhang,* L. Zhang,* and T. Wang*1 *College of Animal Science and Technology, and †Key Laboratory of Meat Processing and Quality Control, College of Food Science and Technology, Nanjing Agricultural University, Nanjing 210095, P. R. China ABSTRACT The objective of this study was to compare the effects of supplementation with 2 carotenoids, curcumin and lutein, on pigmentation and immunity in lipopolysaccharide (LPS)-stimulated broiler chicks. Two hundred forty 1-d-old Arbor Acres broilers were randomly distributed into 3 dietary treatment groups: a basal diet without carotenoid supplementation (control), a basal diet supplemented with 200 mg/kg of curcumin (CRM), or a basal diet supplemented with 200 mg/kg of lutein (LTN) for 42 d. The birds were vaccinated against Newcastle disease (ND) and avian influenza on d 10. At 16, 18, and 20 d of age, half of the chicks in each group were injected in the abdominal region with either LPS (250 mg/kg of BW) or an equal volume of 0.9% NaCl. The intensity of the shank skin color (Roche color fan score) and the b* (yellow) values of the breasts and thighs were highest in lutein-supplemented broilers, followed by curcumin-supplemented and control broilers, whereas the a* (red) value of the thigh muscle was highest in curcumin-supplemented LPS-induced birds. At 42 d, the relative weight of the abdominal fat was lowest in the CRM-supplemented
group, followed by the LTN-supplemented and control groups; the spleen weight was lower in the non-LPS-induced LTN-supplemented group than the LPS-induced control group. The ND and avian influenza titers were significantly higher in the CRM-supplemented group than in the other groups at 20 d; at 30 d, the ND titer was significantly higher in the LPS-induced LTN group. Supplementation with curcumin significantly promoted B and T lymphocyte proliferation in both LPS- and non-LPS-induced birds at 21 d. Curcumin also promoted B lymphocyte proliferation in non-LPS-induced birds at 42 d. Curcumin significantly reduced alanine aminotransferase and aspartate aminotransferase activities at 42 d in non-LPS-treated birds, whereas lutein significantly increased the activities of these enzymes in LPS-induced birds. Both carotenoids significantly lowered lipid oxidation in the liver of supplemented birds. Thus, in broiler chickens, lutein-supplemented birds exhibited better pigmentation efficiency, whereas curcumin-supplemented birds exhibited improved immune responses.
Key words: curcumin, lutein, pigmentation, immunity, broiler 2013 Poultry Science 92:1177–1185 http://dx.doi.org/10.3382/ps.2012-02853
INTRODUCTION Broiler skin pigmentation is an important quality of poultry meat for consumers in China, the United States, Mexico, and many other countries (Liu et al., 2008). Carotenoids (or pigments) are additives that can enhance the color of animal products. Because hens cannot synthesize these pigments, they must be provided in the feed (Lokaewmanee et al., 2011). Recently, natural pigments have replaced synthetic pigments in broiler diets in China due to issues of safety and bioavailability and because these pigments have strong antioxidant and antiinflammatory properties (Liu et al., 2008). The ©2013 Poultry Science Association Inc. Received October 23, 2012. Accepted January 19, 2013. 1 Corresponding author: [email protected]
importance of carotenoids in promoting health is well documented in both humans and animals (Chew and Park, 2004), including birds (Shanmugasundaram and Selvaraj, 2011). Substantial evidence also shows that carotenoids can modulate the immune response (Chew and Park, 2004) by boosting immune activity and inducing lymphocyte proliferation (Ribaya-Mercado and Blumberg, 2004). Dietary supplementation with xanthophyll was shown to decrease oxidative damage and produce antiinflammatory effects in LPS-challenged turkeys (Shanmugasundaram and Selvaraj, 2011), and a review by Gautam et al. (2007) concluded that the available literature consistently demonstrates a positive effect of curcumin on lymphocyte proliferation in vitro. However, most of the previous studies on dietary carotenoids have focused on improving the pigmentation of animals and animal products; the few studies that have examined the immunomodulatory action of dietary ca-
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rotenoids in broiler chickens have produced conflicting results. The major natural sources of carotenoids that have been used in feed over the past several decades are yellow corn, corn gluten meal, algae meal, and dehydrated alfalfa meal. With the frequent introduction of new ingredients, poultry rations are constantly changing; in particular, special oxycarotenoid concentrates are continuously being developed, including curcumin- and lutein-based concentrates (Awang et al., 1992; Castañeda et al., 2005). Of the naturally produced carotenoids, curcumin and lutein are the most commonly used and accepted natural dietary carotenoids in commercial poultry feed; indeed, these carotenoids are considered safe due to their natural presence in edible plants. Curcumin, a xanthophyll carotenoid, is the principal active component of turmeric, which is derived from the plant Curcuma longa. In Southeast Asia, curcumin is used as a spice and a coloring agent in curries (Awang et al., 1992); it is not only a food coloring agent but is also a significant “cleanser of the body” used in Ayurvedic medicine. It has a number of salubrious properties, including antiinflammatory, antioxidant, anticarcinogenic, antibacterial, antiprotozoal, antiviral, and hypocholesterolemic properties (Niamsa and Sittiwet, 2009; Nair et al., 2010; Gandhi et al., 2011). Lutein is a xanthophyll carotenoid naturally present in marigolds (Lokaewmanee et al., 2011). Numerous studies have shown that lutein can enhance humoral and cellular immunity, increase the phagocytic activities of macrophages, and inhibit tumor cell propagation within animals (reviewed by Chew and Park, 2004). Some studies have shown that lutein enhances skin pigmentation, whereas others have suggested that lutein directly improves the immune response and antioxidant activity in several species (Bédécarrats and Leeson, 2006; Liu et al., 2008). To challenge the immune system, lipopolysaccharide (LPS), a key structural component of the outer membrane of gram-negative bacteria, is infused into birds as a stress-stimulating agent. The purpose of this study was to evaluate the possible immunomodulatory effect of dietary supplementation with natural carotenoids on the immune response to LPS injection in broiler chickens.
MATERIALS AND METHODS Birds and Management Two hundred forty 1-d-old commercial Arbor Acres broilers purchased from a local hatchery were initially weighed and randomly assigned to 1 of 6 treatment groups. Five replicate cages containing 8 chicks each were assigned to each treatment group. Throughout the entire experimental period, the chicks were kept in triple-stacked battery cages in an environmentally controlled house. The temperature and RH were ad-
justed to be within the optimal range for chickens of this age. The light regimen was a 12L:12D cycle (with light from 0600 to 1800 h). The Institutional Animal Care and Use Committee of Nanjing Agricultural University (Nanjing, People’s Republic of China) approved all of the procedures. A 2 × 3 factorial design was used as described by Zhang et al. (2010), with injection with LPS or NaCl solution as one factor and the use or nonuse of pigment supplementation as the other factor. The LPS (Escherichia coli serotype O55.B5; Sigma Chemical, St. Louis, MO) was dissolved in a sterile 9 g/L (wt/vol) NaCl solution at a concentration of 0.5 mg/mL to achieve the desired dosage with an injection of 0.5 ml/kg of BW. Half of the birds from each treatment group were injected in the lower abdominal region with LPS, whereas the remaining birds were injected with the 0.9% NaCl solution (250 µg/kg of BW) at 16, 18, and 20 d of age. The basal diet was a rice-soybean based diet (Table 1), formulated to fulfill the nutrient requirements of broiler chickens according to the recommendations of the NRC (1994). The birds were fed starter feed (1 to 21 d) and finisher feed (22 to 42 d). For the respective treatment groups, the basal diet was supplemented with either 200 mg/kg of curcumin or 200 mg/kg of lutein; the carotenoids had a purity level of 98% and were obtained from a commercial company (Guangzhou Leader Biotechnology Co. Ltd., Guangzhou, China). The birds were provided with feed and water ad libitum.
Sample Collection At the end of the 42 days, blood samples were taken from one randomly selected bird per replicate; plasma was separated by centrifugation at 300 × g for 10 min at 4°C and frozen at −20°C for further analysis. After collecting the blood samples, all birds were killed by exsanguination, and after decapitation, the immune organs (bursa, thymus, and spleen) and abdominal fat contents were collected and weighed; the relative weight was calculated according to the following formula: (weight of organ or fat/total live BW × 100).
Shank, Muscle Color, and pH At the end of 42 d, one randomly selected bird per replicate was used for the determination of breast and thigh muscle pH and color, and 3 birds per replicate were used for the evaluation of shank skin color. The intensity of the shank skin coloration was evaluated using a Roche color fan. The muscle pH was measured by directly inserting the probe of a pH meter (Thermo Scientific Orion 3 Star, Singapore) into the breast/thigh muscles. The color of the breast and thigh muscles was assessed for lightness (L*), redness (a*), and yellowness (b*) with a Konica Minolta chroma meter CR-400 (Japan) 45 min after air exposure to allow for blooming.
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Table 1. Feed ingredients and nutrient composition of basal diets for broiler chickens Item Ingredient (g/kg) Corn Soybean meal Corn gluten meal Soybean oil Limestone Dicalcium phosphate Mineral premix1 Sodium chloride l-Lysine dl-Methionine Analyzed chemical composition of diet (g/kg) ME (kcal/kg) CP Ether extract DM Lysine2 Methionine2 Methionine+cystine2 Ca2 Available P2
Starter (1 to 21 d)
Finisher (22 to 42 d)
607 300 25 28 11.9 17 5 3 1.6 1.5
660 240 30 30.5 12 17 5 3 1.5 1
3,055 219.2 54.8 890.7 10.6 5 8.2 10 4.3
3,120 196.5 61.3 880.1 9.5 4.3 7.3 9.1 4.0
1Mineral premix provided the following per kilogram of diet: trans-retinyl acetate, 25 mg; cholecalciferol, 6 mg; menadione, 1.2 mg; thiamine, 2.3 mg; riboflavin, 8 mg; nicotinamide, 42 mg; choline chloride, 400 mg; calcium pantothenate, 10 mg; pyridoxine HCl, 4 mg; biotin, 0.04 mg; folic acid, 1 mg; cobalamin, 0.012 mg; Fe (from ferrous sulfate), 82 mg; Cu (from copper sulfate), 7.5 mg; Mn (from manganese sulfate), 110 mg; Zn (from zinc oxide), 64 mg; I (from calcium iodate), 1.1 mg; Se (from sodium selenite), 0.28 mg. 2Calculated.
Immunization Against Newcastle Disease Virus and Avian Influenza Virus All of the birds were subcutaneously vaccinated against Newcastle disease (ND) virus (NDV IV strain vaccine) and avian influenza (AI) virus (AIV H5N2 strain vaccine) at 10 d of age. To evaluate the antibody response to ND and AI, blood samples were collected in nonheparinized tubes from the wing vein of one randomly selected bird per replicate at 16, 22, and 28 d of age. Blood samples were kept at 37°C for 2 h and centrifuged at 300 × g for 10 min at 4°C to separate serum. Serum samples were frozen at −20°C for assays. Antibody titers against ND and AI viruses were measured using the hemagglutination inhibition test.
Splenic Lymphocyte Isolation and Culture In Vitro The cellular immune response was measured in vitro using a lymphocyte isolation and blastogenesis assay. Splenic lymphocytes were tested for blastogenic response to concanavalin A (Sigma Chemical Co., St. Louis, MO, as the T-cell mitogen) and LPS (from E. coli, strain O55:B5, Sigma Chemical Co., as the B-cell mitogen) as described by Zhang et al. (2009b). Briefly, the spleens were aseptically removed, stored in PBS penicillin-streptomycin, minced into small pieces, and compressed with a 100-μm nylon mesh into a single-cell suspension. The mesh was flushed with 5 mL of RPMI1640 (Sangon Biological Engineering Technology and Services Co., Shanghai, China). The suspension was
immediately transferred into sterilized tubes with sodium citrate, diluted with an equal volume of Hanks’ solution, and coated onto the surface of a lymphocyte separation medium (Rongsheng Biostix Shanghai Inc., Shanghai, China; Ficoll-Hypaque, ρ: 1.077 ± 0.002, no. 030728). The suspensions were then centrifuged at 800 × g for 20 min until a white cloud-like lymphocyte band appeared; this band was collected and washed twice with RPMI-1640 media (supplemented with benzylpenicillin at 100 IU/mL, streptomycin at 100 IU/ mL) without fetal bovine serum. The resulting pellet was resuspended and diluted to 5 × 106/mL with RPMI-1640 media with 10% fetal bovine serum. Trypan blue exclusion was used to assess cell viability in the following manner: to each well of a 96-well culture plate, we added 180 μL of Trypan blue, 20 μL of concanavalin A at 10 μg/mL or LPS at 2.5 μg/mL, and relevant cell samples in triplicate. After 44 h of culture at 39.5°C in a humid atmosphere of 5% CO2 (Revco Technologies Inc., Asheville, NC), cell proliferation was determined by 3-(4, 5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (Sigma Chemical Co.) assay. Briefly, 4 h before the cell proliferation assay, 20 μL of 3-(4,5-dimethylthiazol-2-yl)-2, 5-diphenyl-tetrazolium bromide (5 μg/mL) was added to each well. The supernatant was removed, and 100 μL of dimethyl sulfoxide (Zhengxing Institute of Chemical Engineering, Suzhou, Jiangsu, Japan) was added to each well; the plates were then agitated for 5 min. A microplate reader (model 550, Bio-Rad, Tokyo, Japan) was used to measure the optical absorbance of cells in each well at a wavelength of 570 nm.
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Plasma Alanine Aminotransferase, Aspartate Aminotransferase, and Hepatic TBARS Determination Levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) enzymes, which are markers of oxidative damage sustained in hepatic tissue, were measured in the plasma using the corresponding diagnostic kits (Nanjing Jiancheng Bioengineering Institute, Nanjing, China) according to the manufacturer’s protocols. Oxidative stress in the liver was determined by measuring malondialdehyde (MDA) concentrations, which reflect TBA reactive substance (TBARS), according to the procedure described by Zhang et al. (2009a) using reagent kits (Nanjing Jiancheng Bioengineering Institute, Nanjing, China).
Statistical Analysis The data were analyzed by ANOVA using the GLM feature in SPSS 16.0 (SPSS Inc., Chicago, IL) followed by Duncan’s post hoc test. The differences among treatments were tested for significance by the least significant difference post hoc multiple comparisons test. Significant differences were based on a probability level <0.05.
RESULTS Muscle Color and pH The shank, muscle color, and pH results are presented in Table 2. Dietary supplementation with lutein produced an attractive dark yellow color in the broiler shank skin. The intensity of the shank skin color was strongest in the lutein-supplemented broilers, followed by the curcumin-supplemented and control broilers. The mean percent yellowness (b*) value of the breast meat in lutein-supplemented birds was 39.62 and 31.70% higher than that in the control and cur-
cumin-supplemented birds, respectively, in both LPSand non-LPS-challenged birds. There was no significant difference in lightness (L*) or redness (a*) among the groups at 42 d. For thigh meat, the a* value was 44.04% higher in the carotenoid-treated groups (both lutein and curcumin) than in the control group. In the LPS-challenged birds, the a* value in curcuminsupplemented birds was 14.38 and 11.47% higher than in control and lutein-supplemented birds, respectively. Similar to the values observed in breast meat, the b* values in the thigh meat were also significantly higher in lutein-supplemented birds (57.01 and 41.34% higher than in the control and curcumin-supplemented birds, respectively). There were no significant differences in the L* values of LPS- and non-LPS-challenged broiler chickens at 42 d in any group. Muscle pH was slightly but not significantly higher in both carotenoid-supplemented groups than in the control group.
Relative Weights of Abdominal Fat Contents and the Immune Organs The relative weights of the abdominal fat and immune organs of the broilers are presented in Table 3. In non-LPS-treated birds, the weight of the abdominal fat of curcumin-supplemented birds was 31.31 and 18.29% lower than that of control and lutein-supplemented birds, respectively. In LPS-challenged birds, however, the abdominal fat of curcumin-supplemented birds weighed 38.37 and 3.73% less than that of the control and lutein-supplemented birds, respectively. The relative weights of the bursa, thymus, and spleen of the broilers are shown in Table 3. The relative weight of the thymus was slightly higher in the curcumin-supplemented group than in the control and lutein-supplemented groups. In contrast, the relative weight of the bursa was higher in the lutein-supplemented group than in the control and curcumin-supplemented groups, with or without LPS challenge. The relative weight of the spleen was higher in the control group challenged
Table 2. The effects of dietary supplementation with the natural carotenoids curcumin and lutein on muscle color, pH, and shank color of broilers1 LPS (−) Item Breast muscle L* a* b* pH Thigh muscle L* a* b* pH Shank color a,bMean
LPS (+)
CONT
LTN
CRM
42.84 3.356 9.546b 6.268
40.144 4.794 16.898a 6.294
41.428 4.472 11.776b 6.424
51.602 4.382b 9.242b 6.512 6.940b
51.508 7.83a 15.296ab 6.574 8.523a
51.434 7.828a 13.12ab 6.64 9.023a
CONT
LTN
CRM
SEM
P-value
40.0920 4.766 10.294b 6.088
39.902 3.952 16.02a 6.250
41.064 4.116 10.72b 6.196
0.80266 0.48261 0.86851 0.08039
53.008 6.942ab 8.084b 6.372 6.836b
48.292 7.178ab 18.806a 6.558 8.363a
53.366 8.108a 11.032ab 6.406 8.970a
1.7718 0.78629 1.9097 0.07379 0.2005
0.1226 0.2959 0.0001 0.1305 0.4264 0.0280 0.0055 0.1227 0.0001
values in the same row that do not share a common letter differ significantly (P < 0.05). = basal diet without any carotenoid supplementation; LTN = basal diet with lutein supplementation at 200 mg/kg; CRM = basal diet with curcumin supplementation at 200 mg/kg; LPS (−) = without lipopolysaccharide (LPS) injection; LPS (+) = with LPS injection; L* = lightness; a* = redness; b* = yellowness; n = 5. 1CONT
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Table 3. The effects of dietary supplementation with the natural carotenoids curcumin and lutein on the relative immune organ weights of broilers at 42 d1 LPS (−)
LPS (+)
Item
CONT
LTN
CRM
42 d Spleen Bursa Thymus Abdominal fat
0.034ab 0.091 0.047 2.118ab
0.027b 0.068 0.052 1.908bc
0.0311ab 0.0554 0.074 1.613c
CONT
LTN
CRM
SEM
P-value
0.047a 0.087 0.069 2.286a
0.036ab 0.0608 0.048 1.714c
0.036ab 0.0808 0.055 1.652c
0.00402 0.01521 0.01069 0.07843
0.0364 0.4896 0.3593 0.0002
a–cMean
values in the same row that do not share a common letter differ significantly (P < 0.05). = basal diet without any carotenoid supplementation; LTN = basal diet with lutein supplementation at 200 mg/kg; CRM = basal diet with curcumin supplementation at 200 mg/kg; LPS (−) = without lipopolysaccharide (LPS) injection; LPS (+) = with LPS injection; n = 5. 1CONT
with LPS, but this difference was not significant at 21 d. The relative weight of the spleen in the control group was 15.26 and 23.84% higher than that of the curcumin- and lutein-supplemented groups, respectively. The weights of the bursa and thymus were higher in the LPS-induced control birds at 42 d, but the difference was not significant.
and ND titers that were 12.68 and 11.97% higher than those of control and lutein-supplemented birds, respectively. These results suggest that lutein may help maintain antibody levels in LPS-induced chickens for longer periods of time. However, both carotenoids failed to produce any significant effect on antibody production against AI at 30 d.
Serum Anti-ND Virus and Anti-AIV Antibody Titers in Response to LPS Challenge
Splenic B and T Lymphocyte Proliferation
The serum anti-ND virus and anti-AIV antibody results are presented in Table 4. At 20 d, curcuminsupplemented birds had antibody titers against ND that were 8.00 and 4.18% higher than those of control and lutein-supplemented non-LPS-challenged birds, respectively; similarly, curcumin-supplemented birds had AI titers that were 24.69 and 3.03% higher than those of control and lutein-supplemented birds, respectively. By 30 d, curcumin-supplemented birds had ND titers that were 26.49 and 4.01% higher and AI titers that were 18.14 and 16.06% higher than those of control and lutein-supplemented non-LPS challenged birds, respectively. Similarly, at 20 d, in LPS-induced birds, curcumin-supplemented birds had ND titers that were 12.39 and 1.76% higher and AI titers that were 9.63 and 1.59% than those of control and lutein-supplemented birds, respectively. At 30 d, curcumin-supplemented birds had AI titers that were 6.29 and 3.33% higher
The data on B and T lymphocyte proliferation in broiler chicks are presented in Table 5. Curcuminsupplemented birds experienced B lymphocyte proliferation levels that were 9.45 and 25.03% higher and T lymphocyte proliferation levels that were 36.55 and 38.85% higher than those of the lutein-supplemented group and the control group, respectively. Similarly, in LPS-induced broilers at 21 d, curcumin-supplemented birds had B lymphocyte proliferation levels that were 5.66 and 8.49% higher and T lymphocyte proliferation levels that were 30.45 and 46.42% higher than those of birds from the lutein-supplemented and control groups, respectively. In contrast, at 42 d, curcuminsupplemented birds had B lymphocyte proliferation levels that were 30.11 and 20.03% higher than those of birds from the lutein-supplemented and control groups, respectively. There were no significant differences in B or T lymphocyte proliferation levels among the LPSinduced treatment groups.
Table 4. The effects of dietary supplementation with the natural carotenoids curcumin and lutein on serum antibody titers of broilers1 LPS (−) Antibody titer log2 20 d ND AI 30 d ND AI a–cMean
LPS (+)
CONT
LTN
CRM
5.213ab 5.053ab
5.430ab 6.507ab
5.667a 6.710a
4.033b 5.370
5.267ab 5.507
5.487a 6.560
CONT
LTN
CRM
SEM
Stress
3.817c 4.910b
4.280bc 5.347ab
4.357abc 5.433ab
0.285 0.357
4.133b 4.967
4.733ab 5.123
4.167b 5.300
0.271 0.401
0.001 0.018 0.040 0.083
values in the same row that do not share a common letter differ significantly (P < 0.05). = basal diet without any carotenoid supplementation; LTN = basal diet with lutein supplementation at 200 mg/kg; CRM = basal diet with curcumin supplementation at 200 mg/kg; LPS (−) = without lipopolysaccharide (LPS) injection; LPS (+) = with LPS injection; ND = Newcastle disease; AI = avian influenza; n = 5. 1CONT
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Table 5. The effects of dietary supplementation with the natural carotenoids curcumin and lutein on splenic B- and T-lymphocyte proliferation of broilers1 LPS (−)
LPS (+)
Item
CONT
LTN
CRM
21 d LPS2 ConA3 Negative control 42 d LPS2 ConA3 Negative control
0.674ab 0.532ab 0.697 1.405ab 0.649 0.292
0.814ab 0.552ab 0.672
0.899a 0.870a 0.760
1.228b 0.249 0.330
1.757a 0.238 0.391
CONT
LTN
CRM
SEM
Stress
0.582b 0.359b 0.771
0.600ab 0.466b 0.602
0.636ab 0.670ab 0.921
0.066 0.076 0.106
0.006 0.126 0.558
1.157b 0.296 0.220
1.382b 0.174 0.233
1.175b 0.198 0.248
0.078 0.120 0.058
0.040 0.136 0.043
a,bMean
values in the same row that do not share a common letter differ significantly (P < 0.05). = basal diet without any carotenoid supplementation; LTN = basal diet with lutein supplementation at 200 mg/kg; CRM = basal diet with curcumin supplementation at 200 mg/kg; LPS (−) = without lipopolysaccharide (LPS) injection; LPS (+) = with LPS injection. n = 5. 2B lymphocytes. 3T lymphocytes. ConA = concanavalin A. 1CONT
Plasma ALT, AST, and Hepatic TBARS Activities in Response to LPS Challenge The results of the ALT and AST assays are shown in Figures 1 and 2. Curcumin supplementation significantly reduced ALT activity by 24.90 and 54.96% and AST activity by 21.41 and 8.52% compared with no supplementation (control group) and lutein supplementation, respectively. However, in LPS-induced broilers at 42 d, ALT activity was 51.07 and 46.80% higher and AST activity was 31.28 and 25.55% higher in lutein-supplemented broilers than in control and curcumin-supplemented broilers, respectively. There were no significant differences in ALT or AST activity among these groups at 21 d. The results of the hepatic TBARS assays, as evaluated using MDA concentrations, are shown in Figure 3. At 21 d, MDA levels were 11.26 and 6.38% lower in curcumin-supplemented birds than in the control and lutein-supplemented groups, respectively. Similarly, in LPS-induced birds, MDA levels were 5.99 and 2.25% lower in the curcumin-supplemented group than in the control and lutein-supplemented groups, respectively. In contrast, at 42 d, MDA levels were 13.71 and 3.38% higher in the curcumin-supplemented group than in the control and lutein-supplemented groups, respectively.
In the LPS-induced birds at 42 d, the MDA levels were 39.47 and 3.20% higher in the curcumin-supplemented group than in the control and lutein-supplemented groups, respectively.
DISCUSSION Carotenoids have positive antioxidant and immunomodulatory effects on the physical condition and performance of broilers (Surai, 2002). The dietary curcumin absorbed in the shank skin produces a dark yellow color, whereas lutein produces a yellowish-orange color. Castañeda et al. (2005) reported that a bright yellowish skin color was consistently produced by natural pigments. Similarly, Sirri et al. (2010) showed that carotenoid-supplemented chickens had greater skin yellowness in all body parts than nonsupplemented chickens. Similar findings were observed in our study. Our data on abdominal fat confirm that curcumin promotes fat metabolism, as reported in our previous work (Rajput et al., 2013); our study also showed that abdominal fat mass was lower in lutein-supplemented birds than in control birds. Furthermore, the LPS-challenged broilers without carotenoid supplementation had higher spleen weights than birds from the other treat-
Figure 1. Effect of dietary supplementation with the natural carotenoids curcumin and lutein on plasma ALT (alanine aminotransferase) levels of broilers at 42 d of age. Control = basal diet without any carotenoid supplementation; curcumin = basal diet with curcumin supplementation at 200 mg/kg; lutein = basal diet with lutein supplementation at 200 mg/kg; LPS (−) = without lipopolysaccharide (LPS) injection; LPS (+) = with LPS injection. n = 5. Mean values that do not share a common letter (a,b) are significantly different (P < 0.05).
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Figure 2. Effect of dietary supplementation with the natural carotenoids curcumin and lutein on plasma AST (aspartate aminotransferase) levels of broilers at 21 and 42 d of age. Control = basal diet without any carotenoid supplementation; curcumin = basal diet with curcumin supplementation at 200 mg/kg; lutein = basal diet with lutein supplementation at 200 mg/kg; LPS (−) = without lipopolysaccharide (LPS) injection; LPS (+) = with LPS injection. n = 5. Mean values that do not share a common letter (a,b) are significantly different (P < 0.05).
ment groups. Similar results were reported by Saino et al. (1999), who found that the addition of dietary lutein to broiler diets decreased spleen weight without affecting thymus or bursa weight and had a significant effect on the immune organs. In contrast, Brenes et al. (2008) found that the addition of dietary curcumin (a phenolic compound) did not affect the weight of the spleen or other organs in chickens. Koutsos et al. (2006) reported that a diet without lutein supplementation led to greater relative thymus, bursa, and spleen weights than a diet that included lutein supplementation and that nonsupplemented chicks suffered a severe systemic inflammatory response after LPS injection. Similar to our findings, Saino et al. (1999) showed that lutein supplementation augmented ND antibody persistence. In addition, a review by Gautam et al. (2007) indicated that natural food coloring agents improve antibody production. In contrast, Cheng et al. (2001) reported that antibody titers against fowl cholera vaccination were not affected by carotenoids. In addition, Haddad and Mashaly (1990) and Mashaly et al. (1993) reported that the T3 and T4 hormones enhanced lymphocyte production and increased the blastogenic response of lymphocytes to either concanavalin A or LPS mitogenic stimulation. Furthermore, they concluded that the endocrine function of lymphocytes plays a signifi-
cant role in the stimulation of antibody production in chickens, which provides a possible explanation for our results. Similarly, our previous study concluded that curcumin significantly increased thyroid hormone levels (Rajput et al., 2013), which might be responsible for the increased antibody production at 30 d as found in the present study. There are many factors (e.g., stress, maternal immunity, and environment) that can alter antibody titers. The lymphocyte proliferative response is commonly used to assess the immunomodulatory effects of a potential therapeutic agent. Curcumin has been investigated for its effects on the mitogen-induced proliferation of T cells in vitro and in vivo (Gautam et al., 2007). The results of these studies confirmed that curcumin reasonably boosts the number of T and B cells without changing the number of phagocytic macrophages (reviewed in Gautum et al., 2007). Various contradictory results on the influence of carotenoids on mitogen-induced B and T lymphocyte proliferation have been reported in species other than chickens; the goal of our study was, therefore, to assess the immunomodulatory properties of these compounds in broiler chickens. Curcumin is an effective immunomodulatory agent that can alter the activation of T and B cells (Jagetia and Aggarwal, 2007). In addition, higher B cell counts observed in the mucosa of the small intes-
Figure 3. Effect of dietary supplementation with the natural carotenoids curcumin and lutein on hepatic MDA (malondialdehyde) concentrations of broilers at 21 and 42 d of age. Control = basal diet without any carotenoid supplementation; curcumin = basal diet with curcumin supplementation at 200 mg/kg; lutein = basal diet with lutein supplementation at 200 mg/kg; LPS (−) = without lipopolysaccharide (LPS) injection; LPS (+) = with LPS injection. n = 5. Mean values that do not share a common letter (a–d) are significantly different (P < 0.05).
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tine of birds supplemented with curcumin suggest that this compound has an immunomodulatory effect on the mucosa (Gautum et al., 2007). Similarly, dietary lutein improves phytohemagglutinin-induced lymphocyte proliferation (Chew et al., 1996) and enhances the lymphocyte proliferative response to mitogens (Kim et al., 2000). Surprisingly, however, we observed that on d 21 and 42, the proliferation values of the control birds were greater than those of the birds stimulated with concanavalin A and LPS. Similar findings were reported by Abeer (2005), who observed that lymphocyte proliferation was 2- to 3-fold higher in unstimulated control spleen cell cultures of vaccinated chickens than in those of stimulated cell cultures collected 1 wk after vaccination. These results suggest that after vaccination, the immune cells were likely preactivated in live birds and that cell proliferation subsequently continued in vitro. Similarly, we found that the proliferation values of control birds were higher at 21 d and slightly higher at 42 d than those of treated birds. Because the birds were vaccinated at 10 d of age, vaccination may have led to an initial increase in cell proliferation followed by a decrease in activity. Interestingly, we found that the proliferation values of the control birds were higher than those of supplemented birds, which suggests that supplementation with carotenoids enhanced lymphocyte proliferation in vivo. Lymphocyte proliferation and antibody titers are indicators of the status and activity of cellular and humoral immunity (Zhang et al., 2009b). Our findings thus suggest that carotenoids act as comprehensive immunomodulatory agents (Jagetia and Aggarwal, 2007). Plasma ALT and AST concentrations have been found to be correlated with the decline in plasma antioxidant levels, particularly in the case of supplementation with carotenoids. Zamora et al. (1991) reported that β-carotene was effective in preventing liver damage and produced a concomitant reduction in plasma AST activity. Cheng et al. (2001) also reported that carotenoids boost the increase in ALT and AST activity that is stimulated by aflatoxin. In addition, free cholesterol levels are positively correlated with plasma ALT and AST levels, and our previous results (Rajput et al., 2013) showed that curcumin decreased cholesterol in a dose-dependent manner, whereas Kim et al. (2012) reported that lutein decreases total cholesterol. Therefore, birds in the control group experiencing high cholesterol levels were at risk of liver injury and an increase in plasma ALT and AST levels. Another experiment of ours corroborated the results of this study by showing that a combination of curcumin and lutein at 150 mg/kg reduced plasma ALT and AST levels in coccidiosis-induced broiler chickens (unpublished data). Kaur et al. (2006) measured the ALT, AST, and alkaline phosphatase levels in rats after LPS challenge and reported that curcumin alleviated LPS-induced hepatotoxicity and reduced serum ALT, AST, and alkaline phosphatase levels. Gandhi et al. (2011) also reported that curcumin improved liver function.
Free radicals are highly unstable molecules that can damage cellular structures through abnormal lipid oxidation reactions (Rao and Rao, 1996). Oxidative stress is considered to be a significant cause of various hepatic disorders. The extreme oxidation of lipids caused by high levels of free radicals is the main cause of oxidative stress; this oxidation, in turn, enhances the level of MDA, which is the end product of lipid peroxidation (Kim et al., 2012). In the present study, MDA concentrations were found to be lower in the curcumin- and lutein-supplemented birds than in the control birds; this observation suggests that these carotenoids may prevent or decrease lipid peroxidation in the liver by scavenging free radicals. In support of this hypothesis, Gowda et al. (2008) reported that curcumin significantly decreased lipid peroxidation in broilers. The results of our study showed that birds supplemented with curcumin and lutein carotenoids had lower hepatic MDA levels than the birds in the control group; we also observed that the effect of curcumin was slightly stronger than that of lutein. Thus, in broiler chickens, lutein results in better pigmentation, whereas curcumin produces a stronger immunomodulatory effect. We concluded that carotenoids (particularly curcumin) have positive effects on gut morphology, digestion, immunomodulation, and pigmentation of broiler chickens. Subsequently, we designed another experiment in which broilers were challenged with Eimeria parasites because it is well established that coccidiosis decreases cellular immunity, humoral immunity, digestion, and pigmentation of commercial broiler chickens and damages the intestinal lumen. In this trial, we used 300 mg/kg of curcumin and lutein and a combination of 150 + 150 mg/kg curcumin + lutein in chickens challenged with sporulated oocysts of Eimeria acervulina; we found that higher doses had no significant effect on growth performance, the 300 mg/kg dose of curcumin reduced fecal oocyst shedding, and the combination of both carotenoids enhanced cellular and humoral immunity.
ACKNOWLEDGMENTS The project was financially supported by China Scholarship Council, Beijing, People’s Republic of China, and the research was conducted at Nanjing Agricultural University, Nanjing, China.
REFERENCES Abeer, S. 2005. Investigation of the humoral and cellular immune responses of chickens to Salmonella typhimurium live vaccine. PhD Diss. Faculty of Veterinary Medicine Ludwig-MaximiliansUniversity, Munich, Germany. Awang, I. P. R., U. Chulan, and F. B. H. Ahmad. 1992. Curcumin for upgrading skin colour of broilers. Pertanika 15:37–38. Bédécarrats, G. Y., and S. Leeson. 2006. Dietary lutein influences immune response in laying hens. J. Appl. Poult. Res. 15:183– 189. Brenes, A., A. Viveros, I. Goñi, C. Centeno, S. G. Sáyago-Ayerdy, I. Arija, and F. Saura-Calixto. 2008. Effect of grape pomace con-
IMMUNOMODULATORY EFFECTS OF NATURAL CAROTENOIDS centrate and vitamin E on digestibility of polyphenols and antioxidant activity in chickens. Poult. Sci. 87:307–316. Castañeda, M. P., E. M. Hirschler, and A. R. Sams. 2005. Skin pigmentation evaluation in broilers fed natural and synthetic pigments. Poult. Sci. 84:143–147. Cheng, Y. H., T. F. Shen, V. F. Pang, and B. J. Chen. 2001. Effects of aflatoxin and carotenoids on growth performance and immune response in mule ducklings. Comp. Biochem. Physiol. C Toxicol. Pharmacol. 128:19–26. Chew, B. P., and J. S. Park. 2004. Carotenoid action on the immune response. J. Nutr. 134:257S–261S. Chew, B. P., M. W. Wong, and T. S. Wong. 1996. Effects of lutein from marigold extract on immunity and growth of mammary tumors in mice. Anticancer Res. 16:3689–3694. Gandhi, P., Z. Khan, and N. Chakraverty. 2011. Soluble curcumin: A promising oral supplement for health management. J. Appl. Pharmacol. Sci. 01:01–07. Gautam, S. C., X. Gao, and S. Dulchavsky. 2007. Immunomodulation by curcumin. Adv. Exp. Med. Biol. 595:321–341. Gowda, N. K. S., D. R. Ledoux, G. E. Rottinghaus, A. J. Bermudez, and Y. C. Chen. 2008. Efficacy of turmeric (Curcuma longa), containing a known level of curcumin, and a hydrated sodium calcium aluminosilicate to ameliorate the adverse effects of aflatoxin in broiler chicks. Poult. Sci. 87:1125–1130. Haddad, E. E., and M. M. Mashaly. 1990. Effect of thyrotropin-releasing hormone, triiodothyronine, and chicken growth hormone on plasma concentrations of thyroxine, triiodothyronine, growth hormone, and growth of lymphoid organs and leukocyte populations in immature male chickens. Poult. Sci. 69:1094–1102. Jagetia, G. C., and B. B. Aggarwal. 2007. “Spicing up” of the immune system by curcumin. J. Clin. Immunol. 27:19–35. Kaur, G., N. Tirkey, S. Bharrhan, V. Chanana, P. Rishi, and K. Chopra. 2006. Inhibition of oxidative stress and cytokine activity by curcumin in amelioration of endotoxin-induced experimental hepatoxicity in rodents. Clin. Exp. Immunol. 145:313–321. Kim, H. W., B. P. Chew, T. S. Wong, J. S. Park, B. B. C. Weng, K. M. Byrne, M. G. Hayek, and G. A. Reinhart. 2000. Dietary lutein stimulates immune response in the canine. Vet. Immunol. Immunopathol. 74:315–327. Kim, J. E., R. M. Clark, Y. Park, J. Lee, and M. L. Fernandez. 2012. Lutein decreases oxidative stress and inflammation in liver and eyes of guinea pigs fed a hypercholesterolemic diet. Nutr. Res. Pract. 6:113–119. Koutsos, E. A., J. C. García Lopez, and K. C. Klasing. 2006. Carotenoids from in ovo or dietary sources blunt systemic indices of the inflammatory response in growing chicks (Gallus gallus domesticus). J. Nutr. 136:1027–1031. Liu, G.-D., G.-Y. Hou, D.-J. Wang, S.-J. Lv, X.-Y. Zhang, W.-P. Sun, and Y. Yang. 2008. Skin pigmentation evaluation in broilers fed different levels of natural okra and synthetic pigments. J. Appl. Poult. Res. 17:498–504. Lokaewmanee, K., K. Yamauchi, T. Komori, and K. Saito. 2011. Enhancement of yolk color in raw and boiled egg yolk with lutein
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from marigold flower meal and marigold flower extract. Jpn. Poult. Sci. 48:25–32. Mashaly, M. M., J. M. Trout, and G. L. Hendricks. 1993. The endocrine function of the immune cells in the initiation of humoral immunity. Poult. Sci. 72:1289–1293. Nair, H. B., B. Sung, V. R. Yadav, R. Kannappan, M. M. Chaturvedi, and B. B. Aggarwal. 2010. Delivery of anti-inflammatory nutraceuticals by nanoparticles for the prevention and treatment of cancer. Biochem. Pharmacol. 80:1833–1843. Niamsa, N., and C. Sittiwet. 2009. Antimicrobial activity of curcuma longa aqueous extract. J. Pharmacol. Toxicol. 4:173–177. NRC. 1994. Nutrient Requirements of Poultry. 9th rev. ed. Natl. Acad. Press, Washington, DC. 1994. Rajput, N., M. Naeem, R. Yan, X. Zhong, and T. Wang. 2013. Effect of dietary supplementation of curcumin on growth performance, intestinal morphology and nutrients utilization of broiler chicks. Jpn. Poult. Sci. 50:44–52. Rao, N. S., and M. N. A. Rao. 1996. Free radical scavenging activity of curcuminoids. Arzneim. Forsch. Drug Res. 46:169–171. Ribaya-Mercado, J. D., and J. B. Blumberg. 2004. Lutein and zeaxanthin and their potential roles in disease prevention. J. Am. Coll. Nutr. 23:567S–587S. Saino, N., R. Stradi, P. Ninni, E. Pini, and A. P. Møller. 1999. Carotenoid plasma concentration, immune profile, and plumage ornamentation of male barn swallows (Hirundo rustica). Am. Nat. 154:441–448. Shanmugasundaram, R., and R. K. Selvaraj. 2011. Lutein supplementation alters inflammatory cytokine production and antioxidant status in F-line turkeys. Poult. Sci. 90:971–976. Sirri, F. N., M. B. Petracci, and A. Meluzzi. 2010. Survey of skin pigmentation of yellow-skinned broiler chickens. Poult. Sci. 89:1556–1561. Surai, P. F. 2002. Natural Antioxidants in Avian Nutrition and Reproduction. Nottingham University Press, Nottingham, UK. Zamora, R., F. J. Hidalgo, and A. L. Tappel. 1991. Comparative antioxidant effectiveness of dietary β-carotene, vitamin E, selenium and coenzyme Q10 in rat erythrocytes and plasma. J. Nutr. 121:50–56. Zhang, G. F., Z. B. Yang, Y. Wang, W. R. Yang, S. Z. Jiang, and G. S. Gai. 2009a. Effects of ginger root (Zingiber officinale) processed to different particle sizes on growth performance, antioxidant status, and serum metabolites of broiler chickens. Poult. Sci. 88:2159–2166. Zhang, X. H., X. Zhong, Y. Zhou, H. Du, and T. Wang. 2009b. Effect of RRR-α-tocopherol succinate on the growth and immunity in broilers. Poult. Sci. 88:959–966. Zhang, X. H., X. Zhong, Y. Zhou, G. Wang, H. Du, and T. Wang. 2010. Dietary RRR-a-tocopherol succinate attenuates lipopolysaccharide-induced inflammatory cytokines secretion in broiler chicks. Br. J. Nutr. 104:1796–1805.
group, followed by the LTN-supplemented and control groups; the spleen weight was lower in the non-LPS-induced LTN-supplemented group than the LPS-induced control group. The ND and avian influenza titers were significantly higher in the CRM-supplemented group than in the other groups at 20 d; at 30 d, the ND titer was significantly higher in the LPS-induced LTN group. Supplementation with curcumin significantly promoted B and T lymphocyte proliferation in both LPS- and non-LPS-induced birds at 21 d. Curcumin also promoted B lymphocyte proliferation in non-LPS-induced birds at 42 d. Curcumin significantly reduced alanine aminotransferase and aspartate aminotransferase activities at 42 d in non-LPS-treated birds, whereas lutein significantly increased the activities of these enzymes in LPS-induced birds. Both carotenoids significantly lowered lipid oxidation in the liver of supplemented birds. Thus, in broiler chickens, lutein-supplemented birds exhibited better pigmentation efficiency, whereas curcumin-supplemented birds exhibited improved immune responses.
Key words: curcumin, lutein, pigmentation, immunity, broiler 2013 Poultry Science 92:1177–1185 http://dx.doi.org/10.3382/ps.2012-02853
INTRODUCTION Broiler skin pigmentation is an important quality of poultry meat for consumers in China, the United States, Mexico, and many other countries (Liu et al., 2008). Carotenoids (or pigments) are additives that can enhance the color of animal products. Because hens cannot synthesize these pigments, they must be provided in the feed (Lokaewmanee et al., 2011). Recently, natural pigments have replaced synthetic pigments in broiler diets in China due to issues of safety and bioavailability and because these pigments have strong antioxidant and antiinflammatory properties (Liu et al., 2008). The ©2013 Poultry Science Association Inc. Received October 23, 2012. Accepted January 19, 2013. 1 Corresponding author: [email protected]
importance of carotenoids in promoting health is well documented in both humans and animals (Chew and Park, 2004), including birds (Shanmugasundaram and Selvaraj, 2011). Substantial evidence also shows that carotenoids can modulate the immune response (Chew and Park, 2004) by boosting immune activity and inducing lymphocyte proliferation (Ribaya-Mercado and Blumberg, 2004). Dietary supplementation with xanthophyll was shown to decrease oxidative damage and produce antiinflammatory effects in LPS-challenged turkeys (Shanmugasundaram and Selvaraj, 2011), and a review by Gautam et al. (2007) concluded that the available literature consistently demonstrates a positive effect of curcumin on lymphocyte proliferation in vitro. However, most of the previous studies on dietary carotenoids have focused on improving the pigmentation of animals and animal products; the few studies that have examined the immunomodulatory action of dietary ca-
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rotenoids in broiler chickens have produced conflicting results. The major natural sources of carotenoids that have been used in feed over the past several decades are yellow corn, corn gluten meal, algae meal, and dehydrated alfalfa meal. With the frequent introduction of new ingredients, poultry rations are constantly changing; in particular, special oxycarotenoid concentrates are continuously being developed, including curcumin- and lutein-based concentrates (Awang et al., 1992; Castañeda et al., 2005). Of the naturally produced carotenoids, curcumin and lutein are the most commonly used and accepted natural dietary carotenoids in commercial poultry feed; indeed, these carotenoids are considered safe due to their natural presence in edible plants. Curcumin, a xanthophyll carotenoid, is the principal active component of turmeric, which is derived from the plant Curcuma longa. In Southeast Asia, curcumin is used as a spice and a coloring agent in curries (Awang et al., 1992); it is not only a food coloring agent but is also a significant “cleanser of the body” used in Ayurvedic medicine. It has a number of salubrious properties, including antiinflammatory, antioxidant, anticarcinogenic, antibacterial, antiprotozoal, antiviral, and hypocholesterolemic properties (Niamsa and Sittiwet, 2009; Nair et al., 2010; Gandhi et al., 2011). Lutein is a xanthophyll carotenoid naturally present in marigolds (Lokaewmanee et al., 2011). Numerous studies have shown that lutein can enhance humoral and cellular immunity, increase the phagocytic activities of macrophages, and inhibit tumor cell propagation within animals (reviewed by Chew and Park, 2004). Some studies have shown that lutein enhances skin pigmentation, whereas others have suggested that lutein directly improves the immune response and antioxidant activity in several species (Bédécarrats and Leeson, 2006; Liu et al., 2008). To challenge the immune system, lipopolysaccharide (LPS), a key structural component of the outer membrane of gram-negative bacteria, is infused into birds as a stress-stimulating agent. The purpose of this study was to evaluate the possible immunomodulatory effect of dietary supplementation with natural carotenoids on the immune response to LPS injection in broiler chickens.
MATERIALS AND METHODS Birds and Management Two hundred forty 1-d-old commercial Arbor Acres broilers purchased from a local hatchery were initially weighed and randomly assigned to 1 of 6 treatment groups. Five replicate cages containing 8 chicks each were assigned to each treatment group. Throughout the entire experimental period, the chicks were kept in triple-stacked battery cages in an environmentally controlled house. The temperature and RH were ad-
justed to be within the optimal range for chickens of this age. The light regimen was a 12L:12D cycle (with light from 0600 to 1800 h). The Institutional Animal Care and Use Committee of Nanjing Agricultural University (Nanjing, People’s Republic of China) approved all of the procedures. A 2 × 3 factorial design was used as described by Zhang et al. (2010), with injection with LPS or NaCl solution as one factor and the use or nonuse of pigment supplementation as the other factor. The LPS (Escherichia coli serotype O55.B5; Sigma Chemical, St. Louis, MO) was dissolved in a sterile 9 g/L (wt/vol) NaCl solution at a concentration of 0.5 mg/mL to achieve the desired dosage with an injection of 0.5 ml/kg of BW. Half of the birds from each treatment group were injected in the lower abdominal region with LPS, whereas the remaining birds were injected with the 0.9% NaCl solution (250 µg/kg of BW) at 16, 18, and 20 d of age. The basal diet was a rice-soybean based diet (Table 1), formulated to fulfill the nutrient requirements of broiler chickens according to the recommendations of the NRC (1994). The birds were fed starter feed (1 to 21 d) and finisher feed (22 to 42 d). For the respective treatment groups, the basal diet was supplemented with either 200 mg/kg of curcumin or 200 mg/kg of lutein; the carotenoids had a purity level of 98% and were obtained from a commercial company (Guangzhou Leader Biotechnology Co. Ltd., Guangzhou, China). The birds were provided with feed and water ad libitum.
Sample Collection At the end of the 42 days, blood samples were taken from one randomly selected bird per replicate; plasma was separated by centrifugation at 300 × g for 10 min at 4°C and frozen at −20°C for further analysis. After collecting the blood samples, all birds were killed by exsanguination, and after decapitation, the immune organs (bursa, thymus, and spleen) and abdominal fat contents were collected and weighed; the relative weight was calculated according to the following formula: (weight of organ or fat/total live BW × 100).
Shank, Muscle Color, and pH At the end of 42 d, one randomly selected bird per replicate was used for the determination of breast and thigh muscle pH and color, and 3 birds per replicate were used for the evaluation of shank skin color. The intensity of the shank skin coloration was evaluated using a Roche color fan. The muscle pH was measured by directly inserting the probe of a pH meter (Thermo Scientific Orion 3 Star, Singapore) into the breast/thigh muscles. The color of the breast and thigh muscles was assessed for lightness (L*), redness (a*), and yellowness (b*) with a Konica Minolta chroma meter CR-400 (Japan) 45 min after air exposure to allow for blooming.
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Table 1. Feed ingredients and nutrient composition of basal diets for broiler chickens Item Ingredient (g/kg) Corn Soybean meal Corn gluten meal Soybean oil Limestone Dicalcium phosphate Mineral premix1 Sodium chloride l-Lysine dl-Methionine Analyzed chemical composition of diet (g/kg) ME (kcal/kg) CP Ether extract DM Lysine2 Methionine2 Methionine+cystine2 Ca2 Available P2
Starter (1 to 21 d)
Finisher (22 to 42 d)
607 300 25 28 11.9 17 5 3 1.6 1.5
660 240 30 30.5 12 17 5 3 1.5 1
3,055 219.2 54.8 890.7 10.6 5 8.2 10 4.3
3,120 196.5 61.3 880.1 9.5 4.3 7.3 9.1 4.0
1Mineral premix provided the following per kilogram of diet: trans-retinyl acetate, 25 mg; cholecalciferol, 6 mg; menadione, 1.2 mg; thiamine, 2.3 mg; riboflavin, 8 mg; nicotinamide, 42 mg; choline chloride, 400 mg; calcium pantothenate, 10 mg; pyridoxine HCl, 4 mg; biotin, 0.04 mg; folic acid, 1 mg; cobalamin, 0.012 mg; Fe (from ferrous sulfate), 82 mg; Cu (from copper sulfate), 7.5 mg; Mn (from manganese sulfate), 110 mg; Zn (from zinc oxide), 64 mg; I (from calcium iodate), 1.1 mg; Se (from sodium selenite), 0.28 mg. 2Calculated.
Immunization Against Newcastle Disease Virus and Avian Influenza Virus All of the birds were subcutaneously vaccinated against Newcastle disease (ND) virus (NDV IV strain vaccine) and avian influenza (AI) virus (AIV H5N2 strain vaccine) at 10 d of age. To evaluate the antibody response to ND and AI, blood samples were collected in nonheparinized tubes from the wing vein of one randomly selected bird per replicate at 16, 22, and 28 d of age. Blood samples were kept at 37°C for 2 h and centrifuged at 300 × g for 10 min at 4°C to separate serum. Serum samples were frozen at −20°C for assays. Antibody titers against ND and AI viruses were measured using the hemagglutination inhibition test.
Splenic Lymphocyte Isolation and Culture In Vitro The cellular immune response was measured in vitro using a lymphocyte isolation and blastogenesis assay. Splenic lymphocytes were tested for blastogenic response to concanavalin A (Sigma Chemical Co., St. Louis, MO, as the T-cell mitogen) and LPS (from E. coli, strain O55:B5, Sigma Chemical Co., as the B-cell mitogen) as described by Zhang et al. (2009b). Briefly, the spleens were aseptically removed, stored in PBS penicillin-streptomycin, minced into small pieces, and compressed with a 100-μm nylon mesh into a single-cell suspension. The mesh was flushed with 5 mL of RPMI1640 (Sangon Biological Engineering Technology and Services Co., Shanghai, China). The suspension was
immediately transferred into sterilized tubes with sodium citrate, diluted with an equal volume of Hanks’ solution, and coated onto the surface of a lymphocyte separation medium (Rongsheng Biostix Shanghai Inc., Shanghai, China; Ficoll-Hypaque, ρ: 1.077 ± 0.002, no. 030728). The suspensions were then centrifuged at 800 × g for 20 min until a white cloud-like lymphocyte band appeared; this band was collected and washed twice with RPMI-1640 media (supplemented with benzylpenicillin at 100 IU/mL, streptomycin at 100 IU/ mL) without fetal bovine serum. The resulting pellet was resuspended and diluted to 5 × 106/mL with RPMI-1640 media with 10% fetal bovine serum. Trypan blue exclusion was used to assess cell viability in the following manner: to each well of a 96-well culture plate, we added 180 μL of Trypan blue, 20 μL of concanavalin A at 10 μg/mL or LPS at 2.5 μg/mL, and relevant cell samples in triplicate. After 44 h of culture at 39.5°C in a humid atmosphere of 5% CO2 (Revco Technologies Inc., Asheville, NC), cell proliferation was determined by 3-(4, 5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (Sigma Chemical Co.) assay. Briefly, 4 h before the cell proliferation assay, 20 μL of 3-(4,5-dimethylthiazol-2-yl)-2, 5-diphenyl-tetrazolium bromide (5 μg/mL) was added to each well. The supernatant was removed, and 100 μL of dimethyl sulfoxide (Zhengxing Institute of Chemical Engineering, Suzhou, Jiangsu, Japan) was added to each well; the plates were then agitated for 5 min. A microplate reader (model 550, Bio-Rad, Tokyo, Japan) was used to measure the optical absorbance of cells in each well at a wavelength of 570 nm.
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Plasma Alanine Aminotransferase, Aspartate Aminotransferase, and Hepatic TBARS Determination Levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) enzymes, which are markers of oxidative damage sustained in hepatic tissue, were measured in the plasma using the corresponding diagnostic kits (Nanjing Jiancheng Bioengineering Institute, Nanjing, China) according to the manufacturer’s protocols. Oxidative stress in the liver was determined by measuring malondialdehyde (MDA) concentrations, which reflect TBA reactive substance (TBARS), according to the procedure described by Zhang et al. (2009a) using reagent kits (Nanjing Jiancheng Bioengineering Institute, Nanjing, China).
Statistical Analysis The data were analyzed by ANOVA using the GLM feature in SPSS 16.0 (SPSS Inc., Chicago, IL) followed by Duncan’s post hoc test. The differences among treatments were tested for significance by the least significant difference post hoc multiple comparisons test. Significant differences were based on a probability level <0.05.
RESULTS Muscle Color and pH The shank, muscle color, and pH results are presented in Table 2. Dietary supplementation with lutein produced an attractive dark yellow color in the broiler shank skin. The intensity of the shank skin color was strongest in the lutein-supplemented broilers, followed by the curcumin-supplemented and control broilers. The mean percent yellowness (b*) value of the breast meat in lutein-supplemented birds was 39.62 and 31.70% higher than that in the control and cur-
cumin-supplemented birds, respectively, in both LPSand non-LPS-challenged birds. There was no significant difference in lightness (L*) or redness (a*) among the groups at 42 d. For thigh meat, the a* value was 44.04% higher in the carotenoid-treated groups (both lutein and curcumin) than in the control group. In the LPS-challenged birds, the a* value in curcuminsupplemented birds was 14.38 and 11.47% higher than in control and lutein-supplemented birds, respectively. Similar to the values observed in breast meat, the b* values in the thigh meat were also significantly higher in lutein-supplemented birds (57.01 and 41.34% higher than in the control and curcumin-supplemented birds, respectively). There were no significant differences in the L* values of LPS- and non-LPS-challenged broiler chickens at 42 d in any group. Muscle pH was slightly but not significantly higher in both carotenoid-supplemented groups than in the control group.
Relative Weights of Abdominal Fat Contents and the Immune Organs The relative weights of the abdominal fat and immune organs of the broilers are presented in Table 3. In non-LPS-treated birds, the weight of the abdominal fat of curcumin-supplemented birds was 31.31 and 18.29% lower than that of control and lutein-supplemented birds, respectively. In LPS-challenged birds, however, the abdominal fat of curcumin-supplemented birds weighed 38.37 and 3.73% less than that of the control and lutein-supplemented birds, respectively. The relative weights of the bursa, thymus, and spleen of the broilers are shown in Table 3. The relative weight of the thymus was slightly higher in the curcumin-supplemented group than in the control and lutein-supplemented groups. In contrast, the relative weight of the bursa was higher in the lutein-supplemented group than in the control and curcumin-supplemented groups, with or without LPS challenge. The relative weight of the spleen was higher in the control group challenged
Table 2. The effects of dietary supplementation with the natural carotenoids curcumin and lutein on muscle color, pH, and shank color of broilers1 LPS (−) Item Breast muscle L* a* b* pH Thigh muscle L* a* b* pH Shank color a,bMean
LPS (+)
CONT
LTN
CRM
42.84 3.356 9.546b 6.268
40.144 4.794 16.898a 6.294
41.428 4.472 11.776b 6.424
51.602 4.382b 9.242b 6.512 6.940b
51.508 7.83a 15.296ab 6.574 8.523a
51.434 7.828a 13.12ab 6.64 9.023a
CONT
LTN
CRM
SEM
P-value
40.0920 4.766 10.294b 6.088
39.902 3.952 16.02a 6.250
41.064 4.116 10.72b 6.196
0.80266 0.48261 0.86851 0.08039
53.008 6.942ab 8.084b 6.372 6.836b
48.292 7.178ab 18.806a 6.558 8.363a
53.366 8.108a 11.032ab 6.406 8.970a
1.7718 0.78629 1.9097 0.07379 0.2005
0.1226 0.2959 0.0001 0.1305 0.4264 0.0280 0.0055 0.1227 0.0001
values in the same row that do not share a common letter differ significantly (P < 0.05). = basal diet without any carotenoid supplementation; LTN = basal diet with lutein supplementation at 200 mg/kg; CRM = basal diet with curcumin supplementation at 200 mg/kg; LPS (−) = without lipopolysaccharide (LPS) injection; LPS (+) = with LPS injection; L* = lightness; a* = redness; b* = yellowness; n = 5. 1CONT
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Table 3. The effects of dietary supplementation with the natural carotenoids curcumin and lutein on the relative immune organ weights of broilers at 42 d1 LPS (−)
LPS (+)
Item
CONT
LTN
CRM
42 d Spleen Bursa Thymus Abdominal fat
0.034ab 0.091 0.047 2.118ab
0.027b 0.068 0.052 1.908bc
0.0311ab 0.0554 0.074 1.613c
CONT
LTN
CRM
SEM
P-value
0.047a 0.087 0.069 2.286a
0.036ab 0.0608 0.048 1.714c
0.036ab 0.0808 0.055 1.652c
0.00402 0.01521 0.01069 0.07843
0.0364 0.4896 0.3593 0.0002
a–cMean
values in the same row that do not share a common letter differ significantly (P < 0.05). = basal diet without any carotenoid supplementation; LTN = basal diet with lutein supplementation at 200 mg/kg; CRM = basal diet with curcumin supplementation at 200 mg/kg; LPS (−) = without lipopolysaccharide (LPS) injection; LPS (+) = with LPS injection; n = 5. 1CONT
with LPS, but this difference was not significant at 21 d. The relative weight of the spleen in the control group was 15.26 and 23.84% higher than that of the curcumin- and lutein-supplemented groups, respectively. The weights of the bursa and thymus were higher in the LPS-induced control birds at 42 d, but the difference was not significant.
and ND titers that were 12.68 and 11.97% higher than those of control and lutein-supplemented birds, respectively. These results suggest that lutein may help maintain antibody levels in LPS-induced chickens for longer periods of time. However, both carotenoids failed to produce any significant effect on antibody production against AI at 30 d.
Serum Anti-ND Virus and Anti-AIV Antibody Titers in Response to LPS Challenge
Splenic B and T Lymphocyte Proliferation
The serum anti-ND virus and anti-AIV antibody results are presented in Table 4. At 20 d, curcuminsupplemented birds had antibody titers against ND that were 8.00 and 4.18% higher than those of control and lutein-supplemented non-LPS-challenged birds, respectively; similarly, curcumin-supplemented birds had AI titers that were 24.69 and 3.03% higher than those of control and lutein-supplemented birds, respectively. By 30 d, curcumin-supplemented birds had ND titers that were 26.49 and 4.01% higher and AI titers that were 18.14 and 16.06% higher than those of control and lutein-supplemented non-LPS challenged birds, respectively. Similarly, at 20 d, in LPS-induced birds, curcumin-supplemented birds had ND titers that were 12.39 and 1.76% higher and AI titers that were 9.63 and 1.59% than those of control and lutein-supplemented birds, respectively. At 30 d, curcumin-supplemented birds had AI titers that were 6.29 and 3.33% higher
The data on B and T lymphocyte proliferation in broiler chicks are presented in Table 5. Curcuminsupplemented birds experienced B lymphocyte proliferation levels that were 9.45 and 25.03% higher and T lymphocyte proliferation levels that were 36.55 and 38.85% higher than those of the lutein-supplemented group and the control group, respectively. Similarly, in LPS-induced broilers at 21 d, curcumin-supplemented birds had B lymphocyte proliferation levels that were 5.66 and 8.49% higher and T lymphocyte proliferation levels that were 30.45 and 46.42% higher than those of birds from the lutein-supplemented and control groups, respectively. In contrast, at 42 d, curcuminsupplemented birds had B lymphocyte proliferation levels that were 30.11 and 20.03% higher than those of birds from the lutein-supplemented and control groups, respectively. There were no significant differences in B or T lymphocyte proliferation levels among the LPSinduced treatment groups.
Table 4. The effects of dietary supplementation with the natural carotenoids curcumin and lutein on serum antibody titers of broilers1 LPS (−) Antibody titer log2 20 d ND AI 30 d ND AI a–cMean
LPS (+)
CONT
LTN
CRM
5.213ab 5.053ab
5.430ab 6.507ab
5.667a 6.710a
4.033b 5.370
5.267ab 5.507
5.487a 6.560
CONT
LTN
CRM
SEM
Stress
3.817c 4.910b
4.280bc 5.347ab
4.357abc 5.433ab
0.285 0.357
4.133b 4.967
4.733ab 5.123
4.167b 5.300
0.271 0.401
0.001 0.018 0.040 0.083
values in the same row that do not share a common letter differ significantly (P < 0.05). = basal diet without any carotenoid supplementation; LTN = basal diet with lutein supplementation at 200 mg/kg; CRM = basal diet with curcumin supplementation at 200 mg/kg; LPS (−) = without lipopolysaccharide (LPS) injection; LPS (+) = with LPS injection; ND = Newcastle disease; AI = avian influenza; n = 5. 1CONT
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Table 5. The effects of dietary supplementation with the natural carotenoids curcumin and lutein on splenic B- and T-lymphocyte proliferation of broilers1 LPS (−)
LPS (+)
Item
CONT
LTN
CRM
21 d LPS2 ConA3 Negative control 42 d LPS2 ConA3 Negative control
0.674ab 0.532ab 0.697 1.405ab 0.649 0.292
0.814ab 0.552ab 0.672
0.899a 0.870a 0.760
1.228b 0.249 0.330
1.757a 0.238 0.391
CONT
LTN
CRM
SEM
Stress
0.582b 0.359b 0.771
0.600ab 0.466b 0.602
0.636ab 0.670ab 0.921
0.066 0.076 0.106
0.006 0.126 0.558
1.157b 0.296 0.220
1.382b 0.174 0.233
1.175b 0.198 0.248
0.078 0.120 0.058
0.040 0.136 0.043
a,bMean
values in the same row that do not share a common letter differ significantly (P < 0.05). = basal diet without any carotenoid supplementation; LTN = basal diet with lutein supplementation at 200 mg/kg; CRM = basal diet with curcumin supplementation at 200 mg/kg; LPS (−) = without lipopolysaccharide (LPS) injection; LPS (+) = with LPS injection. n = 5. 2B lymphocytes. 3T lymphocytes. ConA = concanavalin A. 1CONT
Plasma ALT, AST, and Hepatic TBARS Activities in Response to LPS Challenge The results of the ALT and AST assays are shown in Figures 1 and 2. Curcumin supplementation significantly reduced ALT activity by 24.90 and 54.96% and AST activity by 21.41 and 8.52% compared with no supplementation (control group) and lutein supplementation, respectively. However, in LPS-induced broilers at 42 d, ALT activity was 51.07 and 46.80% higher and AST activity was 31.28 and 25.55% higher in lutein-supplemented broilers than in control and curcumin-supplemented broilers, respectively. There were no significant differences in ALT or AST activity among these groups at 21 d. The results of the hepatic TBARS assays, as evaluated using MDA concentrations, are shown in Figure 3. At 21 d, MDA levels were 11.26 and 6.38% lower in curcumin-supplemented birds than in the control and lutein-supplemented groups, respectively. Similarly, in LPS-induced birds, MDA levels were 5.99 and 2.25% lower in the curcumin-supplemented group than in the control and lutein-supplemented groups, respectively. In contrast, at 42 d, MDA levels were 13.71 and 3.38% higher in the curcumin-supplemented group than in the control and lutein-supplemented groups, respectively.
In the LPS-induced birds at 42 d, the MDA levels were 39.47 and 3.20% higher in the curcumin-supplemented group than in the control and lutein-supplemented groups, respectively.
DISCUSSION Carotenoids have positive antioxidant and immunomodulatory effects on the physical condition and performance of broilers (Surai, 2002). The dietary curcumin absorbed in the shank skin produces a dark yellow color, whereas lutein produces a yellowish-orange color. Castañeda et al. (2005) reported that a bright yellowish skin color was consistently produced by natural pigments. Similarly, Sirri et al. (2010) showed that carotenoid-supplemented chickens had greater skin yellowness in all body parts than nonsupplemented chickens. Similar findings were observed in our study. Our data on abdominal fat confirm that curcumin promotes fat metabolism, as reported in our previous work (Rajput et al., 2013); our study also showed that abdominal fat mass was lower in lutein-supplemented birds than in control birds. Furthermore, the LPS-challenged broilers without carotenoid supplementation had higher spleen weights than birds from the other treat-
Figure 1. Effect of dietary supplementation with the natural carotenoids curcumin and lutein on plasma ALT (alanine aminotransferase) levels of broilers at 42 d of age. Control = basal diet without any carotenoid supplementation; curcumin = basal diet with curcumin supplementation at 200 mg/kg; lutein = basal diet with lutein supplementation at 200 mg/kg; LPS (−) = without lipopolysaccharide (LPS) injection; LPS (+) = with LPS injection. n = 5. Mean values that do not share a common letter (a,b) are significantly different (P < 0.05).
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Figure 2. Effect of dietary supplementation with the natural carotenoids curcumin and lutein on plasma AST (aspartate aminotransferase) levels of broilers at 21 and 42 d of age. Control = basal diet without any carotenoid supplementation; curcumin = basal diet with curcumin supplementation at 200 mg/kg; lutein = basal diet with lutein supplementation at 200 mg/kg; LPS (−) = without lipopolysaccharide (LPS) injection; LPS (+) = with LPS injection. n = 5. Mean values that do not share a common letter (a,b) are significantly different (P < 0.05).
ment groups. Similar results were reported by Saino et al. (1999), who found that the addition of dietary lutein to broiler diets decreased spleen weight without affecting thymus or bursa weight and had a significant effect on the immune organs. In contrast, Brenes et al. (2008) found that the addition of dietary curcumin (a phenolic compound) did not affect the weight of the spleen or other organs in chickens. Koutsos et al. (2006) reported that a diet without lutein supplementation led to greater relative thymus, bursa, and spleen weights than a diet that included lutein supplementation and that nonsupplemented chicks suffered a severe systemic inflammatory response after LPS injection. Similar to our findings, Saino et al. (1999) showed that lutein supplementation augmented ND antibody persistence. In addition, a review by Gautam et al. (2007) indicated that natural food coloring agents improve antibody production. In contrast, Cheng et al. (2001) reported that antibody titers against fowl cholera vaccination were not affected by carotenoids. In addition, Haddad and Mashaly (1990) and Mashaly et al. (1993) reported that the T3 and T4 hormones enhanced lymphocyte production and increased the blastogenic response of lymphocytes to either concanavalin A or LPS mitogenic stimulation. Furthermore, they concluded that the endocrine function of lymphocytes plays a signifi-
cant role in the stimulation of antibody production in chickens, which provides a possible explanation for our results. Similarly, our previous study concluded that curcumin significantly increased thyroid hormone levels (Rajput et al., 2013), which might be responsible for the increased antibody production at 30 d as found in the present study. There are many factors (e.g., stress, maternal immunity, and environment) that can alter antibody titers. The lymphocyte proliferative response is commonly used to assess the immunomodulatory effects of a potential therapeutic agent. Curcumin has been investigated for its effects on the mitogen-induced proliferation of T cells in vitro and in vivo (Gautam et al., 2007). The results of these studies confirmed that curcumin reasonably boosts the number of T and B cells without changing the number of phagocytic macrophages (reviewed in Gautum et al., 2007). Various contradictory results on the influence of carotenoids on mitogen-induced B and T lymphocyte proliferation have been reported in species other than chickens; the goal of our study was, therefore, to assess the immunomodulatory properties of these compounds in broiler chickens. Curcumin is an effective immunomodulatory agent that can alter the activation of T and B cells (Jagetia and Aggarwal, 2007). In addition, higher B cell counts observed in the mucosa of the small intes-
Figure 3. Effect of dietary supplementation with the natural carotenoids curcumin and lutein on hepatic MDA (malondialdehyde) concentrations of broilers at 21 and 42 d of age. Control = basal diet without any carotenoid supplementation; curcumin = basal diet with curcumin supplementation at 200 mg/kg; lutein = basal diet with lutein supplementation at 200 mg/kg; LPS (−) = without lipopolysaccharide (LPS) injection; LPS (+) = with LPS injection. n = 5. Mean values that do not share a common letter (a–d) are significantly different (P < 0.05).
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tine of birds supplemented with curcumin suggest that this compound has an immunomodulatory effect on the mucosa (Gautum et al., 2007). Similarly, dietary lutein improves phytohemagglutinin-induced lymphocyte proliferation (Chew et al., 1996) and enhances the lymphocyte proliferative response to mitogens (Kim et al., 2000). Surprisingly, however, we observed that on d 21 and 42, the proliferation values of the control birds were greater than those of the birds stimulated with concanavalin A and LPS. Similar findings were reported by Abeer (2005), who observed that lymphocyte proliferation was 2- to 3-fold higher in unstimulated control spleen cell cultures of vaccinated chickens than in those of stimulated cell cultures collected 1 wk after vaccination. These results suggest that after vaccination, the immune cells were likely preactivated in live birds and that cell proliferation subsequently continued in vitro. Similarly, we found that the proliferation values of control birds were higher at 21 d and slightly higher at 42 d than those of treated birds. Because the birds were vaccinated at 10 d of age, vaccination may have led to an initial increase in cell proliferation followed by a decrease in activity. Interestingly, we found that the proliferation values of the control birds were higher than those of supplemented birds, which suggests that supplementation with carotenoids enhanced lymphocyte proliferation in vivo. Lymphocyte proliferation and antibody titers are indicators of the status and activity of cellular and humoral immunity (Zhang et al., 2009b). Our findings thus suggest that carotenoids act as comprehensive immunomodulatory agents (Jagetia and Aggarwal, 2007). Plasma ALT and AST concentrations have been found to be correlated with the decline in plasma antioxidant levels, particularly in the case of supplementation with carotenoids. Zamora et al. (1991) reported that β-carotene was effective in preventing liver damage and produced a concomitant reduction in plasma AST activity. Cheng et al. (2001) also reported that carotenoids boost the increase in ALT and AST activity that is stimulated by aflatoxin. In addition, free cholesterol levels are positively correlated with plasma ALT and AST levels, and our previous results (Rajput et al., 2013) showed that curcumin decreased cholesterol in a dose-dependent manner, whereas Kim et al. (2012) reported that lutein decreases total cholesterol. Therefore, birds in the control group experiencing high cholesterol levels were at risk of liver injury and an increase in plasma ALT and AST levels. Another experiment of ours corroborated the results of this study by showing that a combination of curcumin and lutein at 150 mg/kg reduced plasma ALT and AST levels in coccidiosis-induced broiler chickens (unpublished data). Kaur et al. (2006) measured the ALT, AST, and alkaline phosphatase levels in rats after LPS challenge and reported that curcumin alleviated LPS-induced hepatotoxicity and reduced serum ALT, AST, and alkaline phosphatase levels. Gandhi et al. (2011) also reported that curcumin improved liver function.
Free radicals are highly unstable molecules that can damage cellular structures through abnormal lipid oxidation reactions (Rao and Rao, 1996). Oxidative stress is considered to be a significant cause of various hepatic disorders. The extreme oxidation of lipids caused by high levels of free radicals is the main cause of oxidative stress; this oxidation, in turn, enhances the level of MDA, which is the end product of lipid peroxidation (Kim et al., 2012). In the present study, MDA concentrations were found to be lower in the curcumin- and lutein-supplemented birds than in the control birds; this observation suggests that these carotenoids may prevent or decrease lipid peroxidation in the liver by scavenging free radicals. In support of this hypothesis, Gowda et al. (2008) reported that curcumin significantly decreased lipid peroxidation in broilers. The results of our study showed that birds supplemented with curcumin and lutein carotenoids had lower hepatic MDA levels than the birds in the control group; we also observed that the effect of curcumin was slightly stronger than that of lutein. Thus, in broiler chickens, lutein results in better pigmentation, whereas curcumin produces a stronger immunomodulatory effect. We concluded that carotenoids (particularly curcumin) have positive effects on gut morphology, digestion, immunomodulation, and pigmentation of broiler chickens. Subsequently, we designed another experiment in which broilers were challenged with Eimeria parasites because it is well established that coccidiosis decreases cellular immunity, humoral immunity, digestion, and pigmentation of commercial broiler chickens and damages the intestinal lumen. In this trial, we used 300 mg/kg of curcumin and lutein and a combination of 150 + 150 mg/kg curcumin + lutein in chickens challenged with sporulated oocysts of Eimeria acervulina; we found that higher doses had no significant effect on growth performance, the 300 mg/kg dose of curcumin reduced fecal oocyst shedding, and the combination of both carotenoids enhanced cellular and humoral immunity.
ACKNOWLEDGMENTS The project was financially supported by China Scholarship Council, Beijing, People’s Republic of China, and the research was conducted at Nanjing Agricultural University, Nanjing, China.
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