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METABOLISM AND NUTRITION |
The State Key Laboratory of Animal Nutrition, College of Animal Science & Technology, China Agricultural University, Beijing 100094, P. R. China
1 Corresponding author: guoyum{at}cau.edu.cn
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key Words: DL-2-hydroxy-4-methylthio butanoic acid • growth performance • immune response • broiler chicken
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Dietary methionine levels affect the immune responses of various animals. Dietary methionine deficiency led to maldevelopment of lymphoid organs (Williams et al., 1979; Carew et al., 2003), reduced mitogen-induced lymphocyte proliferation (van Heugten et al., 1994; Takahashi et al., 1997), and showed lower antibody production against SRBC and delayed hypersensitivity against phytohemagglutinin (PHA)-P in broiler chickens (Tsiagbe et al., 1987a). There are differing results about the effects of high doses of methionine on humoral immunity. Bhargava et al. (1970) reported that antibody titers to Newcastle disease virus (NDV) were lower in chicks fed diets with adequate methionine than in those with deficient levels of methionine, and similar results were obtained in rats immunized with SRBC (Kenney et al., 1970). However, Swain and Johri (2000) showed that a methionine excess did not alter the antibody response of broiler chickens immunized with SRBC. Panda et al. (2007) reported that LMA was comparable to DLM in White Leghorn layers as a source of methionine for production performance and immunity when the bioavailability of it was considered to be 88% of DLM. Plasma ceruloplasmin, 
-1 acid glycoprotein concentration, and heterophil to lymphocyte ratio in blood after lipopolysaccharide (LPS) injection were lower in chicks fed an LMA diet than in chicks fed a DLM diet, which suggested that dietary LMA had a potential to alleviate certain stress responses (Matsushita et al., 2007). Martin-Venegas et al. (2006) showed that Cys and taurine synthesis after incubation with LMA is higher when compared with L-methionine incubation. Therefore, the data indicate that Cys and taurine formation by chicken enterocytes could be favored when LMA is used as a methionine source, thereby suggesting that the LMA might be preferentially diverted to the transsulfuration pathway.
So far, little research has been published about the effects of LMA on immunity in meat-type poultry. The present study was conducted to examine the effects of different dietary doses of LMA on immune response of broiler chickens.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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. The basal diets were supplemented with LMA to meet 80, 100, 120, and 140% of methionine requirements for the 2 phases of broiler chickens recommended by Feeding Standards of Chickens (Ministry of Agriculture of P. R. China, 2004). The methionine levels in the 4 treatments were, respectively, 0.4, 0.5, 0.6, and 0.7% for the starter period and 0.32, 0.40, 0.48, and 0.56% for the grower period. The bioavailability of LMA was set at 80% [equivalent to 1.25-fold (wt/wt) the amount of methionine] of DLM by weight (Bunchasak and Keawarun, 2006). The LMA was added to aliquots of the basal diet at the expense of zeolite. Chickens were raised in a temperature-controlled room with constant (24 h/d) light. The temperature of the room was 35 to 33°C in the first 3 d and declined 3°C/wk until it reached 22 to 24°C. The birds had free access to water and feed.
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Growth Performance
At 21 and 42 d of age, the following performance variables were determined: BW gain, feed intake, and ratio of feed to gain. Chickens of each replicate cage were weighed after overnight feed deprivation, and the remaining feed was weighed. All pens were checked daily for deaths.
Relative Lymphoid Organ Weights
At 10 and 21 d of age, 8 healthy chickens (1 per replicate) were randomly chosen from each treatment. Chickens were humanely killed after weighing. Thymus, spleen, and bursa of Fabricius were collected and weighed. Relative lymphoid organ weights were calculated as lymphoid organ weight divided by body weight.
Serum Albumin, Globulin, and Lysozyme Activity
At 21 and 42 d of age, 8 healthy chickens (1 per replicate) were randomly chosen from each treatment. Blood samples were collected from the wing vein and centrifuged at 3,000 x g for 10 min at 4°C. The serum was stored at –30°C until assay. Serum albumin was quantified by bromocresol green colorimetry using an albumin kit (Jiancheng Bioengineering Institute, Nanjing, China). Twenty microliters of serum sample was added to 5 mL of bromocresol green colorimetry solution. After 10 min, the solutions were read via a spectrophotometer at 628 nm. Serum total protein was determined with a Coomassie Brilliant Blue kit (Jiancheng Bioengineering Institute). Serum was diluted 1:50 with saline; then, 50 µL was added to 3 mL of Coomassie Brilliant Blue solution. After placement for 10 min, the solutions were read via a spectrophotometer at 595 nm. Total serum globulin was calculated as serum total protein minus serum albumin. Serum lysozyme activity was determined with a lysozyme kit (Jiancheng Bioengineering Institute). Five milligrams of bacterium powder was dissolved by 1 mL of bacterium solvent, and then ground slowly, finally diluted to 20 mL by bacterium solvent. After 15 min in 37°C water followed by 3 min in 0°C water, the solutions were read via a spectrophotometer at 530 nm.
Peripheral Blood Lymphocyte Proliferation
A 3-[4,5-dimethylthiazol]-2,5-diphenyltetrazolium bromide (MTT, Sigma Chemical Co., St. Louis, MO) assay was used to determine the peripheral blood lymphocyte proliferation response at 21 and 42 d of age. Eight healthy chickens (1 per replicate) were randomly chosen from each treatment. Heparinized blood samples were collected from the wing vein. Then, each blood sample was added to isometric lymphocyte separation medium (density = 1.077; HaoYang Biological Manufacture Co. Ltd., Tianjin, China). Lymphocytes were isolated after 30 min, and centrifugation was at 1,006 x g at 4°C. The lymphocyte fraction was collected from the interface and washed 3 times with RPMI 1640 (Invitrogen Corp., Grand Island, NY) incomplete culture medium. Lymphocytes were then resuspended in 2 mL of RPMI 1640 complete culture medium supplemented with 5% (vol/vol) of fetal calf serum, 0.5% penicillin (final concentration, 100 U/mL), 0.5% streptomycin (final concentration, 100 µg/mL), and 1% N-(2-hydroxyethyl)-piperazine-N-2-ethane-sulfonic acid (HEPES, final concentration, 24 mM; Amresco 0511, Amresco Inc., Cleveland, OH). Cells were detected by trypan blue dye exclusion and counted to adjust the density of the cells to 1 x 107cells per milliliter of culture medium.
One hundred microliters of cell suspension and the lymphocyte mitogen concanavalin A (Con A, Sigma Chemical Co.) or LPS (Sigma Chemical Co.) were added to a 96-well microtiter plate (Costar 3599, Corning Inc., Corning, NY) to provide a final concentration of 45 µg/mL (Con A) or 25 µg/mL (LPS). Cells then were stored at 37°C with 5% CO2 in an incubator (MCO-18AIC CO2 incubator, Sanyo Electric Biomedical Co. Ltd., Tokyo, Japan). After 68 h, 15 µL of 5 mg/mL MTT was added to each well and the plates were incubated for another 4 h. Subsequently, 100 µL of 10% sodium dodecyl sulfate dissolved in 0.04 mol/L HCl solution was added into each well to lyse the cells and solubilize the MTT crystals. Finally, plates were read using an automated ELISA reader (model 550 Microplate Reader, Bio-Rad Pacific Ltd., Hong Kong, China) at 570 nm.
Phagocytosis of Neutral Red of Peripheral Blood Lymphocyte
Lymphocyte suspensions were collected for the phagocytosis of neutral red assay at 21 and 42 d of age. One hundred microliters of cell suspension was added per well in 96-well culture plates. Blank control wells were included that contained culture medium alone. Cells were incubated at 37°C with 5% CO2 in an incubator for 2 h and were then washed 3 times with culture medium. One hundred microliters of neutral red [0.1%, 100 mg/100 mL of saline water (0.9%)] was added into each well and then incubated. After 15 min, the supernatant was discarded, excessive neutral red was washed with saline water, and 100 µL of cell dissolving fluid [1:1 (vol/vol) ethanol: acetic acid] was added. After refrigeration at 4°C overnight, the plates were read via an automated ELISA reader at 540 nm.
Serum Antibody Titers to NDV and BSA
Antibody titers against NDV were detected by hemagglutination-inhibition test using 4 hemagglutinin units of the ND antigen. All chickens were vaccinated with NDV-IV strain vaccine (Intervet Co., Boxmeer, Holland) through intranasal and intraocular administration on d 9, and blood samples were collected from the wing vein at 13, 17, and 21 d of age. All chickens were vaccinated again through drinking water on d 23 and blood samples were collected at 27, 31, and 35 d of age. Serum samples were prepared and frozen at –30°C for assays. Briefly, 2-fold serial dilutions of serum were made in a 96-well, V-shaped bottom microtiter plate containing 25 µL of PBS without Ca2+ and Mg2+ in all wells; then, 50 µL of the NDV antigen (4 hemagglutination units; China Institute of Veterinary Drug Control, Beijing, China) was added into all wells except for the last 2 rows, which served as controls. Serum dilutions ranged from 1:2 to 1:1,024. After 20 min, 25 µL of 1% rooster erythrocyte suspension was added to each well for 60 min. The greatest dilution of serum causing complete inhibition was considered to be the end point. The antibody titers were expressed as reciprocal log2 values for the greatest dilution that displayed hemagglutination inhibition.
Half of the chickens of each treatment were injected with 1 mL of 0.5% BSA (Roche 738328, Roche, Basel, Switzerland) in sterilized saline (0.9%) in the thigh muscle on d 15, and blood samples were collected at 22, 25, and 29 d of age. The same chickens were injected again on d 29, and blood samples were collected at 35, 38, and 42 d of age. Blood was collected from the wing vein, and serum samples were stored at –30°C until assays. Indirect ELISA was performed on serum samples using 96-well plates coated with 8 µg of BSA per well. Following overnight incubation, plates were rinsed with PBS-Tween (pH 7.4, 0.05% Tween 20). Serum was added and incubated at 37°C in an incubator for 2 h. Plates were rinsed, and polyvalent, peroxidase-labeled, rabbit anti-chicken IgG (Sigma Chemical Co.) was added to each well and the plates were incubated at 37°C in an incubator for 15 min. Plates were rinsed and a substrate solution containing 100 µL of dimethyl sulfoxide with 1 mg of tetramethylbenzidine in 10 mL of sodium acetate buffer (pH 5.5) was added. After 15 min at 37°C in an incubator, the reaction was stopped by adding 50 µL of 2 mol/L sulfuric acid. Absorbance was read via an automated ELISA reader at 490 nm.
Statistical Analysis
The results were reported as means ± SEM and all data were statistically analyzed by one-way ANOVA of SPSS 10.0 for Windows (SPSS, 1995). Differences among each treatment group were tested by least significant difference test, and differences were significant at P < 0.05.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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, there were no significant differences in BW gain and feed intake among the treatments. The ratio of feed to gain decreased linearly as LMA supplementation increased during any phase of growth. The average mortality was 1.6% for the whole experiment and was not influenced by dietary LMA level.
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, serum albumin was not influenced by LMA supplementation. However, serum globulin and total protein on d 21 and 42 were linearly increased significantly as the dietary LMA supplementation level was elevated. As shown in Table 5, dietary LMA dosage did not significantly influence the proliferation of peripheral blood lymphocyte in response to Con A; however, proliferation was quadratically influenced when the cells were exposed to LPS, and proliferation levels in the group fed at 120% on d 21 and in the group fed at 100% on d 42 were significantly greater than in other groups. For phagocytosis of neutral red of peripheral blood lymphocyte, the effect of dietary LMA was quadratic and the least effect in the Met-deficient group (Table 6). Serum lysozyme activity was not influenced by dietary LMA dosage, but lysozyme activity was greatest in the group fed at 100% of methionine requirement (Table 6).
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) were greater in the groups fed at 100 or 120% of methionine requirement, and on d 13 after the secondary immunization titers to BSA were quadratically influenced significantly.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Immunological Index
Dietary methionine deficiency could cause the maldevelopment of lymphoid organs and their normal function (Konashi, et al., 2000; Carew et al., 2003). In the present study, the greatest relative weights of lymphoid organs were in the group with a dietary methionine level of 0.60% for the starter period. Even though the difference in the spleen of birds aged 21 d was much more obvious (0.05 < P < 0.10), development of lymphoid organs was not influenced by diets marginally deficient in methionine.
Nonspecific immunity was assessed by serum lysozyme activity and phagocytosis of neutral red of peripheral blood lymphocytes. Our results showed that supplemental LMA to meet 100% of methionine requirement was required to achieve the greatest nonspecific immuno-competence, and marginal methionine deficiency would result in low phagocytic function of peripheral blood lymphocytes.
Humoral immunity was evaluated by antibody response to NDV and BSA, and cellular immunity was measured by lymphocyte proliferation. Serum globulin increased linearly as dietary LMA level elevated, which was in agreement with the results of Attia et al. (2005). The greatest level of antibody to BSA in the groups fed at 100 or 120% of methionine requirement in the present research suggested that additional LMA was beneficial to immunocompetence, even though the antibody titers to NDV were not influenced. Takahashi et al. (1993) reported that there were no significant differences in the responses to SRBC of methionine intake when the chickens were fed diets of equal energy and protein values. Many earlier researchers showed no benefit in improving the antibody response by additional methionine in pigs (van Heugten et al., 1994) and broiler chickens (Lin and Shih, 2000; Swain and Johri, 2000), but the antibody titers against SRBC and NDV were enhanced when dietary methionine supplementary levels increased from 4.5 to 6 g/kg (Rama Rao et al., 2003; Panda et al., 2007). Moreover, methionine supplementation resulted in significant dose-related increases in total antibody and IgG, which suggested that methionine is required for some components of the antibody response and might be required for thymus-derived (T)-cell helper function (Tsiagbe et al., 1987b).
Concanavalin A and LPS specifically stimulate lymphocyte proliferation. The quadratically enhanced proliferation in response to LPS by dietary LMA supplementation could also contribute to the improved antibody or globulin production. In earlier studies, additional methionine did not affect wing-web PHA response in adult quail (Dabbert et al., 1996) or the Con A-induced proliferative response of thymus mononuclear cells (Takahashi et al., 1997) but did enhance cutaneous wing-web or wattle response to PHA in young broiler chickens (Tsiagbe et al., 1987b; Rama Rao et al., 2003) and mitogen-induced proliferation of T cells in rats (Williams et al., 1979). The strain, age, and basal and supplementary methionine levels were partly responsible for the different outcomes of the above-mentioned studies.
The 0.4 and 0.32% dietary methionine levels were adequate for maximum growth requirement during the starter and grower phases, respectively. Dietary LMA supplementation improved feed utilization and humoral and nonspecific immunocompetence of broiler chickens.
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ACKNOWLEDGMENTS |
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Received for publication August 31, 2007. Accepted for publication March 31, 2008.
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REFERENCES |
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