Effects of Liquid DL-2-Hydroxy-4-Methylthio Butanoic Acid on Growth Performance and Immune Responses in Broiler Chickens

L. B. Zhang and Y. M. Guo1

The State Key Laboratory of Animal Nutrition, College of Animal Science & Technology, China Agricultural University, Beijing 100094, P. R. China ABSTRACT An experiment was conducted to determine the effects of different doses of liquid DL-2-hydroxy4-methylthio butanoic acid (LMA) on growth performance and immune response in broiler chickens. In an arrangement with 4 graded levels of LMA to meet 80, 100, 120, and 140% of methionine requirements of broilers recommended by Chinese feeding standards for chickens, 256 one-day-old Arbor Acres male broiler chickens were randomly divided into 4 treatments with 8 replicates of 8 birds each. Growth performance, cellular immunity, and humoral immunity were determined. Results from increasing LMA levels were as follows. There were no signicant differences (P > 0.05) in body weight gain and feed intake among the treatments, but the ratio of feed to gain was linearly decreased and signicantly greatest (P < 0.05) in the group fed at 80% of methionine requirement. Serum globulin levels on d 21 and 42 were linearly Key words:
DL-2-hydroxy-4-methylthio

increased signicantly (P < 0.05); phagocytosis of neutral red of peripheral blood lymphocyte was quadratic and was lowest in the decient group (P < 0.05). The proliferation of peripheral blood lymphocytes in response to lipopolysaccharide was quadratically inuenced, and that of the 120% group on d 21 and the 100% group on d 42 was signicantly greater than in the other groups (P < 0.05). Antibody titers to Newcastle disease virus on d 4 after the rst inoculation of the vaccine were quadratically increased, anti-bovine serum albumin antibody production on d 13 after the second immunization was quadratic, and antibody titers were greatest in the groups fed at 100 or 120% of methionine requirement. In conclusion, methionine deciency resulted in decreased feed utilization and decreased humoral and nonspecic immunocompetence of broiler chickens. The use of LMA to correct a methionine deciency corrected these problems.

butanoic acid, growth performance, immune response, broiler chicken 2008 Poultry Science 87:13701376 doi:10.3382/ps.2007-00366

INTRODUCTION
Methionine and lysine are generally considered to be the most limiting amino acid in commercial corn-soybean-based broiler chicken diets. There are two supplementary methionine sources commonly-used, DL-methionine powder (DLM, 99% pure) and liquid DL-2-hydroxy4-methylthio butanoic acid (LMA, containing 88% active substance). Research on these two methionine sources has mainly focused on their relative bioavailability and different metabolic pathways. Dietary methionine levels affect the immune responses of various animals. Dietary methionine deciency 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

2008 Poultry Science Association Inc. Received August 31, 2007. Accepted March 31, 2008. 1 Corresponding author: guoyum@cau.edu.cn

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 decient 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

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2-HYDROXY-4-METHYLTHIO BUTANOIC ACID AND GROWTH PERFORMANCE

Table 1. Composition and nutrient levels of basal diets Item Ingredient (%) Corn Soybean meal Soybean oil Dicalcium phosphate Limestone Salt Mineral premix1 Vitamin premix2 Antioxidant Aureomycin Choline chloride Zeolite Calculated composition3 ME, mcal/kg CP, % Ca, % Available P, % Lys, % Met, % Met + Cys, % Week 0 to 3 52.02 38.71 4.83 1.89 1.15 0.35 0.20 0.02 0.03 0.10 0.20 0.50 3.00 21.12 1.06 0.45 1.13 0.32 0.72 Week 4 to 6 58.59 32.90 4.54 1.61 1.10 0.35 0.20 0.02 0.03 0.10 0.16 0.40 3.05 19.05 0.89 0.41 1.0 0.29 0.67

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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 33C in the rst 3 d and declined 3C/wk until it reached 22 to 24C. The birds had free access to water and feed. Feed ingredient samples were collected. All samples were analyzed for protein (AOAC, 1990; method 988.05), calcium (AOAC, 1990; method 927.02) and total phosphorus (AOAC, 1990; method 965.05) according to the methods presented by the Association of Ofcial Analytical Chemists (1990). Levels of methionine and cysteine were determined using HPLC (Cohen and Michaud, 1993).

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.

1 Provided per kilogram of diet: Cu, 8 mg; Zn, 75 mg; Fe, 80 mg; Mn, 100 mg; Se, 0.15 mg; I, 0.35 mg. 2 Provided per kilogram of diet: vitamin A, 12,500 IU; vitamin D3, 2,500 IU; vitamin K3, 2.65 mg; thiamin, 2 mg; riboavin, 6 mg; vitamin B12, 0.025 mg; vitamin E, 30 IU; biotin, 0.0325 mg; folic acid, 1.25 mg; pantothenic acid 12 mg; niacin, 50 mg. 3 Based on actual analysis of the individual feed ingredient.

Relative Lymphoid Organ Weights

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. 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 g for 10 min at 4C. The serum was stored at 30C until assay. Serum albumin was quantied 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 L 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, nally diluted to 20 mL by bacterium

MATERIALS AND METHODS Experimental Design and Diets

An arrangement with 4 graded levels of LMA (containing 88% active substance; Sumitomo Chemical, Tokyo, Japan) was designed in the present 42-d experiment. Two hundred fty-six 1-d-old Arbor Acres male broiler chickens were randomly divided into 4 treatments with 8 replicates of 8 birds each. The composition and nutrient levels of basal diets for starter period (wk 1 to 3), grower period (wk 4 to 6) were showed in Table 1. 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

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Table 2. Effect of dietary liquid DL-2-hydroxy-4-methylthio butanoic acid on growth performance1 of chickens in starter and grower periods (n = 8) Day 0 to 21 Met level, % 80 100 120 140 SEM Linear Quadratic
a,b 1

Day 21 to 42 F:G 1.52 1.44b 1.45b 1.43b 0.00 <0.001 0.006

a

Day 0 to42 F:G 1.96 1.87b 1.85b 1.82b 0.00 <0.001 0.952
a

BWG (g) 602 607 610 621 5.8 0.308 0.761

FI (g) 917 872 884 892 8.9 0.287 0.145

BWG (g) 1,336 1,379 1,391 1,425 19.7 0.082 0.124

FI (g) 2,621 2,584 2,578 2,598 24.3 P-value 0.548 0.345

BWG (g) 1,938 1,986 2,001 2,046 22.6 0.099 0.972

FI (g) 3,538 3,456 3,462 3,490 30.5 0.676 0.360

F:G 1.83a 1.74b 1.73b 1.71b 0.00 0.001 0.886

Means in the same column without common superscripts differ signicantly (P < 0.05). BWG = BW gain; FI = feed intake; F:G = feed: gain.

solvent. After 15 min in 37C water followed by 3 min in 0C water, the solutions were read via a spectrophotometer at 530 nm.

reader (model 550 Microplate Reader, Bio-Rad Pacic Ltd., Hong Kong, China) at 570 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 g at 4C. 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 (nal concentration, 100 U/mL), 0.5% streptomycin (nal concentration, 100 g/mL), and 1% N-(2-hydroxyethyl)-piperazine-N-2-ethane-sulfonic acid (HEPES, nal 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 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 nal concentration of 45 g/ mL (Con A) or 25 g/mL (LPS). Cells then were stored at 37C with 5% CO2 in an incubator (MCO-18AIC CO2 incubator, Sanyo Electric Biomedical Co. Ltd., Tokyo, Japan). After 68 h, 15 L of 5 mg/mL MTT was added to each well and the plates were incubated for another 4 h. Subsequently, 100 L 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

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 37C 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 L of cell dissolving uid [1:1 (vol/vol) ethanol: acetic acid] was added. After refrigeration at 4C 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 NDVIV 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 30C for assays. Briey, 2fold serial dilutions of serum were made in a 96-well, Vshaped bottom microtiter plate containing 25 L of PBS without Ca2+ and Mg2+ in all wells; then, 50 L 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 L of 1% rooster erythrocyte suspension was added to each well for 60 min. The greatest dilution of

2-HYDROXY-4-METHYLTHIO BUTANOIC ACID AND GROWTH PERFORMANCE

Table 3. Effect of dietary liquid (n = 8)
DL-2-hydroxy-4-methylthio

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butanoic acid on relative lymphoid organ weight Day 21

Day 10 Met level, % 80 100 120 140 SEM Linear Quadratic

ac

Thymus 1.83 2.14 2.30 2.13 0.087 0.179 0.186

Spleen 0.70 0.75 0.86 0.77 0.036 0.294 0.338

BF 1.96 1.66c 2.20a 1.77bc 0.059 0.976 0.488

ab

Thymus 2.70 2.61 2.94 2.49 0.097 P-value 0.743 0.376

Spleen 0.80 0.85ab 0.98b 0.89ab 0.026 0.073 0.155

a

BF 2.95 2.44 2.68 2.73 0.088 0.603 0.124

Means in the same column without common superscripts differ signicantly (P < 0.05).

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 30C until assays. Indirect ELISA was performed on serum samples using 96-well plates coated with 8 g 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 37C 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 37C in an incubator for 15 min. Plates were rinsed and a substrate solution containing 100 L of dimethyl sulfoxide with 1 mg of tetramethylbenzidine in 10 mL of sodium acetate buffer (pH 5.5) was added. After 15 min at 37C in an incubator, the reaction was stopped by adding 50 L 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 signicant difference test, and differences were signicant at P < 0.05.

RESULTS Growth Performance

As shown in Table 2, there were no signicant 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 inuenced by dietary LMA level.

Lymphoid Organ Development

There were no signicant differences in thymus relative weight, but the greatest relative weights of bursa of Fabricius on d 10 and spleen on d 21 were in the group fed at 120% of methionine requirement (Table 3).

Immunological Measures
As shown in Table 4, serum albumin was not inuenced by LMA supplementation. However, serum globulin and

Table 4. Effect of dietary liquid DL-2-hydroxy-4-methylthio butanoic acid on serum albumin and globulin (n = 8) Day 21 Met level, % 80 100 120 140 SEM Linear Quadratic
ac

Day 42 Globulin (g/L) 5.74a 7.71ab 11.06c 10.60bc 0.65 P-value 0.001 0.256 Total protein (g/L) 35.14ab 33.37a 36.14bc 38.39c 0.54 0.004 0.032 Albumin (g/L) 27.34 26.03 26.82 27.22 0.35 0.903 0.245 Globulin (g/L) 7.91a 8.55ab 8.87ab 10.77b 0.42 0.016 0.425

Total protein (g/L) 30.80a 32.97ab 35.92b 33.70ab 0.58 0.015 0.034

Albumin (g/L) 24.47 23.94 25.66 23.55 0.61 0.852 0.538

Means in the same column without common superscripts differ signicantly (P < 0.05).

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Table 5. Effect of dietary liquid DL-2-hydroxy-4-methylthio butanoic acid on proliferation of peripheral blood lymphocytes1 [stimulating index (SI); n = 6] Day 21 Met level, % 80 100 120 140 SEM Linear Quadratic
a,b 1

Day 42 SI (LPS) 1.08 1.10a 1.21b 1.10a 0.018 P-value 0.263 0.079
a

SI (Con A) 1.08 1.12 1.06 1.11 0.014 0.868 0.932

SI (Con A) 1.12 1.16 1.14 1.11 0.011 0.532 0.158

SI (LPS) 1.09a 1.19b 1.14ab 1.09a 0.018 0.733 0.050

Means in the same column without common superscripts differ signicantly (P < 0.05). Con A = concanavalin A; LPS = lipopolysaccharide.

total protein on d 21 and 42 were linearly increased signicantly as the dietary LMA supplementation level was elevated. As shown in Table 5, dietary LMA dosage did not signicantly inuence the proliferation of peripheral blood lymphocyte in response to Con A; however, proliferation was quadratically inuenced 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 signicantly 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-decient group (Table 6). Serum lysozyme activity was not inuenced by dietary LMA dosage, but lysozyme activity was greatest in the group fed at 100% of methionine requirement (Table 6). Antibody titers to NDV were not inuenced by dietary LMA level; however, titers were quadratically inuenced on d 13. The antibody titers to BSA (Table 7) 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 inuenced signicantly.

DISCUSSION Growth Performance

Even though feed intake and BW gain were not signicantly inuenced as supplemental LMA increased, feed

utilization was signicantly improved. The dietary methionine levels of the group receiving 80% of methionine requirement were 0.40 and 0.32%, respectively, for the 2 phases, and levels were marginally decient for broiler chickens. Chickens in this group showed no differences from all other treatments in feed intake and BW gain but had poorer feed utilization. Earlier researchers reported that methionine addition reduced feed intake compared with a diet decient in sulfur-containing amino acids (Esteve-Garcia and Llaurado, 1997). However, improvements in feed utilization as a result of methionine supplementation have been widely observed in broiler chickens, and increases in methionine levels promoted an increase of approximately 12 to 14% in BW gain compared with broilers receiving a methionine-decient diet (Solberg et al., 1971; Garlich, 1985; Lin et al., 1996). In agreement with the results of those researchers, the present study found that better feed utilization was achieved when LMA was supplied, and dietary methionine at 0.4 and 0.32% was adequate for minimum growth requirement during the starter and grower phases, respectively, but the broilers receiving a marginally decient diet needed to obtain similar growth by overeating. Lin and Shih (2000), Carew et al. (2003), and Attia et al. (2005) showed that a marginal methionine deciency is often compensated for by increased feed intake with little change in the rate of gain. Broiler chickens fed diets marginally decient in methionine could overeat slightly to meet the adequate amounts

Table 6. Effect of dietary liquid DL-2-hydroxy-4-methylthio butanoic acid on phagocytosis of neutral red of peripheral blood lymphocyte (optical density at 540 nm; n = 6) and serum lysozyme activity (n = 8) Phagocytosis Met level, % 80 100 120 140 SEM Linear Quadratic
a,b

Lysozyme (mg/L) Day 42 0.212 0.306b 0.249ab 0.259ab 0.013 P-value 0.463 0.098
a

Day 21 0.051 0.093b 0.080b 0.081b 0.005 0.007 0.003

a

Day 21 3.16 3.31 3.07 3.12 0.11 0.879 0.202

Day 42 3.47ab 4.34a 3.03b 3.04b 0.21 0.151 0.288

Means in the same column without common superscripts differ signicantly (P < 0.05).

2-HYDROXY-4-METHYLTHIO BUTANOIC ACID AND GROWTH PERFORMANCE

Table 7. Effect of dietary liquid DL-2-hydroxy-4-methylthio butanoic acid on serum antibody titers to BSA (optical density at 490 nm; n = 8) Post rst immunization Met level, % 80 100 120 140 SEM Linear Quadratic
a,b

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Post secondary immunization 14 d 0.635 0.584 0.638 0.640 0.013 0.552 0.305 6d 0.328 0.350a 0.309b 0.357a 0.007 P-value 0.436 0.302
ab

7d 0.291 0.299a 0.338b 0.307ab 0.006 0.092 0.084

a

10 d 0.584 0.579 0.574 0.538 0.010 0.101 0.424

9d 0.479 0.484a 0.453b 0.475ab 0.005 0.322 0.383

ab

13 d 0.605a 0.615ab 0.643b 0.600a 0.006 0.785 0.037

Means in the same column without common superscripts differ signicantly (P < 0.05).

of methionine needed, and the increased feed intake did not cause an increase in BW gain because the added caloric intake might be converted to body fat to replace body water (Carew and Hill, 1961; Carew et al., 2003), nally resulting in lower feed utilization.

Immunological Index
Dietary methionine deciency 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 inuenced by diets marginally decient in methionine. Nonspecic 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 nonspecic immunocompetence, and marginal methionine deciency 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 benecial to immunocompetence, even though the antibody titers to NDV were not inuenced. Takahashi et al. (1993) reported that there were no signicant 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 benet 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 signicant 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 specically 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 abovementioned 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 nonspecic immunocompetence of broiler chickens.

ACKNOWLEDGMENT
The authors thank Sumitomo Chemical Co. Ltd. (Tokyo, Japan) for supplying the LMA product and for partial nancial support. This work was supported in part by the Project nyhyzx07039 from the Ministry of Agriculture, P. R. China.

REFERENCES
AOAC (Association of Ofcial Analytical Chemists). 1990. Ofcial Methods of Analysis. 15th ed. AOAC, Washington, DC. Attia, Y. A., R. A. Hassan, M. H. Shehatta, and S. B. A. El-Hady. 2005. Growth, carcass quality and serum constituents of slow growing chicks as affected by betaine addition to diets containing 2. Different levels of methionine. Int. J. Poult. Sci. 4:856865.

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ZHANG AND GUO methylthio) butanoic acid to sulfur-containing amino acids in the chicken small intestine. Poult. Sci. 85:19321938. Matsushita, K., K. Takahashi, and Y. Akiba. 2007. Effects of adequate or marginal excess of dietary methionine hydroxy analogue free acid on growth performance, edible meat yields and inammatory response in female broiler chickens. Jpn. Poult. Sci. 44:265272. Ministry of Agriculture of P. R. China. 2004. Feeding standard of chickens. Beijing, China. Panda, A. K., S. V. Rao, M. V. Raju, and S. Bhanja. 2007. Relative performance and immune response in White Leghorn layers fed liquid DL-methionine hydroxy analogue and DL-methionine. Asian-australas. J. Anim. Sci. 20:948953. Rama Rao, S. V., N. K. Praharaj, V. Ramasubba Reddy, and A. K. Panda. 2003. Interaction between genotype and dietary concentrations of methionine for immune function in commercial broilers. Br. Poult. Sci. 44:104112. Solberg, J., P. J. Buttery, and K. N. Boorman. 1971. Effect of moderate methionine deciency of food, protein and energy utilisation in the chick. Br. Poult. Sci. 12:297304. SPSS. 1995. SPSS version 10.0 for Windows. SPSS Inc., Chicago, IL. Swain, B. K., and T. S. Johri. 2000. Effect of supplemental methionine, choline and their combinations on the performance and immune response of broilers. Br. Poult. Sci. 41:8388. Takahashi, K., S. Konashi, Y. Akiba, and M. Horiguchi. 1993. Effects of marginal excess or deciency of dietary methionine on antibody production in growing broilers. Anim. Sci. Technol. 64:1319. Takahashi, K., N. Ohta, and Y. Akiba. 1997. Inuences of dietary methionine and cysteine on metabolic responses to immunological stress by Escherichia coli lipopolysaccharide injection, and mitogenic response in broiler chickens. Br. J. Nutr. 78:815821. Tsiagbe, V. K., M. E. Cook, and A. E. Harper. 1987a. Enhanced immune response in broiler chicks fed methionine-supplement diets. Poult. Sci. 66:11471154. Tsiagbe, V. K., M. E. Cook, and A. E. Harper. 1987b. Efcacy of cysteine in replacing methionine in the immune responses of broiler chicks. Poult. Sci. 66:11381146. van Heugten, E., J. W. Spears, M. T. Coffey, E. B. Kegley, and M. A. Qureshi. 1994. The effect of methionine and aatoxin on immune function in weanling pigs. J. Anim. Sci. 72:658664. Williams, E. A., B. M. Gebhardt, and B. Morton. 1979. Effects of early marginal methionine-choline deprivation on the development of the immune system in the rats. Am. J. Clin. Nutr. 32:12141223.

Bhargava, K. K., R. P. Hanson, and M. L. Sunde. 1970. Effect of methionine and valine on antibody production in chicks infected with Newcastle disease virus. J. Nutr. 100:241248. Bunchasak, C., and N. Keawarun. 2006. Effect of methionine hydroxy analogue-free acid on growth performance and chemical composition of liver of broiler chicks fed a cornsoybean diet from 0 to 6 weeks of age. Jpn. J. Anim. Sci. 77:95102. Carew, L. B., and F. W. Hill. 1961. The effect of methionine deciency on the utilization of energy by the chick. J. Nutr. 74:158190. Carew, L. B., J. P. Mcmurtry, and F. A. Alster. 2003. Effect of methionine deciencies on plasma levels of thyroid hormones insulin-like growth factors-I and -II, liver and body weights, and feed intake in growing chickens. Poult. Sci. 82:19321938. Cohen, S. A., and D. P. Michaud. 1993. Synthesis of a uorescent derivatizing reagent, 6-aminoquinolys-N-hydroxysuccin-imidyl carbamate, and its application for the analysis of hydrolysate amino acid via high-performance liquid chromatography. Anim. Biochem. 211:279287. Dabbert, C. B., R. L. Lochmiller, P. W. Waldroup, and R. G. Teeter. 1996. Examination of the dietary methionine requirements of breeding Northern Bobwhite, Colinus virginianus. Poult. Sci. 75:991997. Esteve-Garcia, E., and L. L. Llaurado. 1997. Performance, breast meat yield and abdominal fat of male broiler chickens fed diets supplemented with DL-methionine or DL-methionine hydroxy analogue free acid. Br. Poult. Sci. 34:397404. Garlich, J. D. 1985. Response of broiler to DL-methionine hydroxy analogue free acid, DL-methionine, and L-methionine. Poult. Sci. 64:15411548. Kenney, M. A., J. L. Magee, and F. Pedad-Pascual. 1970. Dietary amino acid and immune response in rats. J. Nutr. 100:10631072. Konashi, S., K. Takahashi, and Y. Akiba. 2000. Effects of dietary essential amino acid deciencies on immunological variables in broiler chickens. Br. J. Nutr. 83:449456. Lin, Y. F., B. J. Chen, and T. F. Shen. 1996. The effects of methionine-supplemented diets on the growth performance and immune response of Taiwanese native chicks and broiler chicks. J. Chin. Anim. Sci. 25:357372. Lin, Y. F., and B. L. Shih. 2000. Effects of methionine-supplementation on growth performance and immune response of Taiwan native chicken at 58 weeks of age. J. Chin. Anim. Sci. 29:110. n-Venegas, R., P. A. Geraert, and R. Ferrer. 2006. ConverMart sion of the methionine hydroxy analogue DL-2-hydroxy-(4-