Effects of Liquid DL-2-Hydroxy-4-Methylthio Butanoic Acid on GrowthPerformance 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 deter-mine the effects of different doses of liquid DL-2-hydroxy-4-methylthio butanoic acid (LMA) on growth perfor-mance and immune response in broiler chickens. In anarrangement with 4 graded levels of LMA to meet 80,100, 120, and 140% of methionine requirements of broilersrecommended by Chinese feeding standards for chickens,256 one-day-old Arbor Acres male broiler chickens wererandomly divided into 4 treatments with 8 replicates of8 birds each. Growth performance, cellular immunity,and humoral immunity were determined. Results fromincreasing LMA levels were as follows. There were nosignificant differences (P > 0.05) in body weight gain andfeed intake among the treatments, but the ratio of feedto gain was linearly decreased and significantly greatest(P < 0.05) in the group fed at 80% of methionine require-ment. Serum globulin levels on d 21 and 42 were linearly

Key words: DL-2-hydroxy-4-methylthio butanoic acid, growth performance, immune response, broiler chicken

2008 Poultry Science 87:1370–1376doi:10.3382/ps.2007-00366

INTRODUCTION

Methionine and lysine are generally considered to bethe most limiting amino acid in commercial corn-soy-bean-based broiler chicken diets. There are two supple-mentary methionine sources commonly-used, DL-methio-nine powder (DLM, 99% pure) and liquid DL-2-hydroxy-4-methylthio butanoic acid (LMA, containing 88% activesubstance). Research on these two methionine sourceshas mainly focused on their relative bioavailability anddifferent metabolic pathways.

Dietary methionine levels affect the immune responsesof various animals. Dietary methionine deficiency led tomaldevelopment of lymphoid organs (Williams et al.,1979; Carew et al., 2003), reduced mitogen-induced lym-phocyte proliferation (van Heugten et al., 1994; Takahashiet al., 1997), and showed lower antibody production

©2008 Poultry Science Association Inc.Received August 31, 2007.Accepted March 31, 2008.1Corresponding author: guoyum@cau.edu.cn

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increased significantly (P < 0.05); phagocytosis of neutralred of peripheral blood lymphocyte was quadratic andwas lowest in the deficient group (P < 0.05). The prolifera-tion of peripheral blood lymphocytes in response to lipo-polysaccharide was quadratically influenced, and that ofthe 120% group on d 21 and the 100% group on d 42 wassignificantly greater than in the other groups (P < 0.05).Antibody titers to Newcastle disease virus on d 4 afterthe first inoculation of the vaccine were quadraticallyincreased, anti-bovine serum albumin antibody produc-tion on d 13 after the second immunization was quadratic,and antibody titers were greatest in the groups fed at100 or 120% of methionine requirement. In conclusion,methionine deficiency resulted in decreased feed utiliza-tion and decreased humoral and nonspecific immuno-competence of broiler chickens. The use of LMA to correcta methionine deficiency corrected these problems.

against SRBC and delayed hypersensitivity against phy-tohemagglutinin (PHA)-P in broiler chickens (Tsiagbe etal., 1987a). There are differing results about the effects ofhigh doses of methionine on humoral immunity. Bhar-gava et al. (1970) reported that antibody titers to Newcas-tle disease virus (NDV) were lower in chicks fed dietswith adequate methionine than in those with deficientlevels of methionine, and similar results were obtainedin rats immunized with SRBC (Kenney et al., 1970). How-ever, Swain and Johri (2000) showed that a methionineexcess did not alter the antibody response of broiler chick-ens immunized with SRBC. Panda et al. (2007) reportedthat LMA was comparable to DLM in White Leghornlayers as a source of methionine for production perfor-mance and immunity when the bioavailability of it wasconsidered to be 88% of DLM. Plasma ceruloplasmin,α-1 acid glycoprotein concentration, and heterophil tolymphocyte ratio in blood after lipopolysaccharide (LPS)injection were lower in chicks fed an LMA diet than inchicks fed a DLM diet, which suggested that dietary LMAhad a potential to alleviate certain stress responses (Mat-sushita et al., 2007). Martin-Venegas et al. (2006) showed

2-HYDROXY-4-METHYLTHIO BUTANOIC ACID AND GROWTH PERFORMANCE 1371

Table 1. Composition and nutrient levels of basal diets

Item Week 0 to 3 Week 4 to 6

Ingredient (%)Corn 52.02 58.59Soybean meal 38.71 32.90Soybean oil 4.83 4.54Dicalcium phosphate 1.89 1.61Limestone 1.15 1.10Salt 0.35 0.35Mineral premix1 0.20 0.20Vitamin premix2 0.02 0.02Antioxidant 0.03 0.03Aureomycin 0.10 0.10Choline chloride 0.20 0.16Zeolite 0.50 0.40

Calculated composition3

ME, mcal/kg 3.00 3.05CP, % 21.12 19.05Ca, % 1.06 0.89Available P, % 0.45 0.41Lys, % 1.13 1.0Met, % 0.32 0.29Met + Cys, % 0.72 0.67

1Provided per kilogram of diet: Cu, 8 mg; Zn, 75 mg; Fe, 80 mg; Mn,100 mg; Se, 0.15 mg; I, 0.35 mg.

2Provided per kilogram of diet: vitamin A, 12,500 IU; vitamin D3,2,500 IU; vitamin K3, 2.65 mg; thiamin, 2 mg; riboflavin, 6 mg; vitaminB12, 0.025 mg; vitamin E, 30 IU; biotin, 0.0325 mg; folic acid, 1.25 mg;pantothenic acid 12 mg; niacin, 50 mg.

3Based on actual analysis of the individual feed ingredient.

that Cys and taurine synthesis after incubation with LMAis higher when compared with L-methionine incubation.Therefore, the data indicate that Cys and taurine forma-tion by chicken enterocytes could be favored when LMAis used as a methionine source, thereby suggesting thatthe LMA might be preferentially diverted to the transsul-furation pathway.

So far, little research has been published about theeffects of LMA on immunity in meat-type poultry. Thepresent study was conducted to examine the effects ofdifferent dietary doses of LMA on immune response ofbroiler chickens.

MATERIALS AND METHODS

Experimental Design and Diets

An arrangement with 4 graded levels of LMA (con-taining 88% active substance; Sumitomo Chemical,Tokyo, Japan) was designed in the present 42-d experi-ment. Two hundred fifty-six 1-d-old Arbor Acres malebroiler chickens were randomly divided into 4 treatmentswith 8 replicates of 8 birds each. The composition andnutrient levels of basal diets for starter period (wk 1 to3), grower period (wk 4 to 6) were showed in Table 1.The basal diets were supplemented with LMA to meet80, 100, 120, and 140% of methionine requirements forthe 2 phases of broiler chickens recommended by FeedingStandards of Chickens (Ministry of Agriculture of P. R.China, 2004). The methionine levels in the 4 treatmentswere, respectively, 0.4, 0.5, 0.6, and 0.7% for the starterperiod and 0.32, 0.40, 0.48, and 0.56% for the grower

period. The bioavailability of LMA was set at 80% [equiv-alent to 1.25-fold (wt/wt) the amount of methionine] ofDLM by weight (Bunchasak and Keawarun, 2006). TheLMA was added to aliquots of the basal diet at the expenseof zeolite. Chickens were raised in a temperature-con-trolled room with constant (24 h/d) light. The tempera-ture of the room was 35 to 33°C in the first 3 d anddeclined 3°C/wk until it reached 22 to 24°C. The birdshad free access to water and feed.

Feed ingredient samples were collected. All sampleswere analyzed for protein (AOAC, 1990; method 988.05),calcium (AOAC, 1990; method 927.02) and total phospho-rus (AOAC, 1990; method 965.05) according to the meth-ods presented by the Association of Official AnalyticalChemists (1990). Levels of methionine and cysteine weredetermined using HPLC (Cohen and Michaud, 1993).

Growth Performance

At 21 and 42 d of age, the following performance vari-ables were determined: BW gain, feed intake, and ratioof feed to gain. Chickens of each replicate cage wereweighed after overnight feed deprivation, and the re-maining feed was weighed. All pens were checked dailyfor deaths.

Relative Lymphoid Organ Weights

At 10 and 21 d of age, 8 healthy chickens (1 per replicate)were randomly chosen from each treatment. Chickenswere humanely killed after weighing. Thymus, spleen,and bursa of Fabricius were collected and weighed. Rela-tive lymphoid organ weights were calculated aslymphoid 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 sam-ples were collected from the wing vein and centrifugedat 3,000 × g for 10 min at 4°C. The serum was storedat −30°C until assay. Serum albumin was quantified bybromocresol green colorimetry using an albumin kit (Jian-cheng Bioengineering Institute, Nanjing, China). Twentymicroliters of serum sample was added to 5 mL of bromo-cresol green colorimetry solution. After 10 min, the solu-tions were read via a spectrophotometer at 628 nm. Serumtotal protein was determined with a Coomassie BrilliantBlue kit (Jiancheng Bioengineering Institute). Serum wasdiluted 1:50 with saline; then, 50 �L was added to 3 mLof Coomassie Brilliant Blue solution. After placement for10 min, the solutions were read via a spectrophotometerat 595 nm. Total serum globulin was calculated as serumtotal protein minus serum albumin. Serum lysozymeactivity was determined with a lysozyme kit (JianchengBioengineering Institute). Five milligrams of bacteriumpowder was dissolved by 1 mL of bacterium solvent, andthen ground slowly, finally diluted to 20 mL by bacterium

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Table 2. Effect of dietary liquid DL-2-hydroxy-4-methylthio butanoic acid on growth performance1 of chickensin starter and grower periods (n = 8)

Day 0 to 21 Day 21 to 42 Day 0 to42

Met level, % BWG (g) FI (g) F:G BWG (g) FI (g) F:G BWG (g) FI (g) F:G

80 602 917 1.52a 1,336 2,621 1.96a 1,938 3,538 1.83a

100 607 872 1.44b 1,379 2,584 1.87b 1,986 3,456 1.74b

120 610 884 1.45b 1,391 2,578 1.85b 2,001 3,462 1.73b

140 621 892 1.43b 1,425 2,598 1.82b 2,046 3,490 1.71b

SEM 5.8 8.9 0.00 19.7 24.3 0.00 22.6 30.5 0.00P-value

Linear 0.308 0.287 <0.001 0.082 0.548 <0.001 0.099 0.676 0.001Quadratic 0.761 0.145 0.006 0.124 0.345 0.952 0.972 0.360 0.886

a,bMeans in the same column without common superscripts differ significantly (P < 0.05).1BWG = BW gain; FI = feed intake; F:G = feed: gain.

solvent. After 15 min in 37°C water followed by 3 minin 0°C water, the solutions were read via a spectropho-tometer at 530 nm.

Peripheral Blood Lymphocyte Proliferation

A 3-[4,5-dimethylthiazol]-2,5-diphenyltetrazolium bro-mide (MTT, Sigma Chemical Co., St. Louis, MO) assaywas used to determine the peripheral blood lymphocyteproliferation response at 21 and 42 d of age. Eight healthychickens (1 per replicate) were randomly chosen fromeach treatment. Heparinized blood samples were col-lected from the wing vein. Then, each blood sample wasadded to isometric lymphocyte separation medium (den-sity = 1.077; HaoYang Biological Manufacture Co. Ltd.,Tianjin, China). Lymphocytes were isolated after 30 min,and centrifugation was at 1,006 × g at 4°C. The lymphocytefraction was collected from the interface and washed 3times with RPMI 1640 (Invitrogen Corp., Grand Island,NY) incomplete culture medium. Lymphocytes were thenresuspended in 2 mL of RPMI 1640 complete culture me-dium supplemented with 5% (vol/vol) of fetal calf serum,0.5% penicillin (final concentration, 100 U/mL), 0.5%streptomycin (final concentration, 100 �g/mL), and 1%N-(2-hydroxyethyl)-piperazine-N-2-ethane-sulfonic acid(HEPES, final concentration, 24 mM; Amresco 0511, Amr-esco Inc., Cleveland, OH). Cells were detected by trypanblue dye exclusion and counted to adjust the density ofthe cells to 1 × 107cells per milliliter of culture medium.

One hundred microliters of cell suspension and thelymphocyte mitogen concanavalin A (Con A, SigmaChemical Co.) or LPS (Sigma Chemical Co.) were addedto a 96-well microtiter plate (Costar 3599, Corning Inc.,Corning, NY) to provide a final concentration of 45 �g/mL (Con A) or 25 �g/mL (LPS). Cells then were storedat 37°C with 5% CO2 in an incubator (MCO-18AIC CO2

incubator, Sanyo Electric Biomedical Co. Ltd., Tokyo, Ja-pan). After 68 h, 15 �L of 5 mg/mL MTT was added toeach well and the plates were incubated for another 4h. Subsequently, 100 �L of 10% sodium dodecyl sulfatedissolved in 0.04 mol/L HCl solution was added intoeach 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 PacificLtd., Hong Kong, China) at 570 nm.

Phagocytosis of Neutral Redof Peripheral Blood Lymphocyte

Lymphocyte suspensions were collected for the phago-cytosis of neutral red assay at 21 and 42 d of age. Onehundred microliters of cell suspension was added perwell in 96-well culture plates. Blank control wells wereincluded that contained culture medium alone. Cells wereincubated at 37°C with 5% CO2 in an incubator for 2 hand were then washed 3 times with culture medium. Onehundred microliters of neutral red [0.1%, 100 mg/100 mLof saline water (0.9%)] was added into each well and thenincubated. After 15 min, the supernatant was discarded,excessive neutral red was washed with saline water, and100 �L of cell dissolving fluid [1:1 (vol/vol) ethanol: aceticacid] was added. After refrigeration at 4°C overnight,the plates were read via an automated ELISA reader at540 nm.

Serum Antibody Titers to NDV and BSA

Antibody titers against NDV were detected by hemag-glutination-inhibition test using 4 hemagglutinin units ofthe ND antigen. All chickens were vaccinated with NDV-IV strain vaccine (Intervet Co., Boxmeer, Holland)through intranasal and intraocular administration on d9, and blood samples were collected from the wing veinat 13, 17, and 21 d of age. All chickens were vaccinatedagain through drinking water on d 23 and blood sampleswere collected at 27, 31, and 35 d of age. Serum sampleswere 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 �L of PBSwithout Ca2+ and Mg2+ in all wells; then, 50 �L of theNDV antigen (4 hemagglutination units; China Instituteof Veterinary Drug Control, Beijing, China) was addedinto all wells except for the last 2 rows, which served ascontrols. Serum dilutions ranged from 1:2 to 1:1,024. After20 min, 25 �L of 1% rooster erythrocyte suspension wasadded to each well for 60 min. The greatest dilution of

2-HYDROXY-4-METHYLTHIO BUTANOIC ACID AND GROWTH PERFORMANCE 1373

Table 3. Effect of dietary liquid DL-2-hydroxy-4-methylthio butanoic acid on relative lymphoid organ weight(n = 8)

Day 10 Day 21

Met level, % Thymus Spleen BF Thymus Spleen BF

80 1.83 0.70 1.96ab 2.70 0.80a 2.95100 2.14 0.75 1.66c 2.61 0.85ab 2.44120 2.30 0.86 2.20a 2.94 0.98b 2.68140 2.13 0.77 1.77bc 2.49 0.89ab 2.73SEM 0.087 0.036 0.059 0.097 0.026 0.088

P-value

Linear 0.179 0.294 0.976 0.743 0.073 0.603Quadratic 0.186 0.338 0.488 0.376 0.155 0.124

a–cMeans in the same column without common superscripts differ significantly (P < 0.05).

serum causing complete inhibition was considered to bethe end point. The antibody titers were expressed as recip-rocal log2 values for the greatest dilution that displayedhemagglutination inhibition.

Half of the chickens of each treatment were injectedwith 1 mL of 0.5% BSA (Roche 738328, Roche, Basel,Switzerland) in sterilized saline (0.9%) in the thigh muscleon d 15, and blood samples were collected at 22, 25, and29 d of age. The same chickens were injected again on d29, and blood samples were collected at 35, 38, and 42 dof age. Blood was collected from the wing vein, and serumsamples were stored at −30°C until assays. Indirect ELISAwas performed on serum samples using 96-well platescoated with 8 �g of BSA per well. Following overnightincubation, plates were rinsed with PBS-Tween (pH 7.4,0.05% Tween 20). Serum was added and incubated at37°C in an incubator for 2 h. Plates were rinsed, andpolyvalent, peroxidase-labeled, rabbit anti-chicken IgG(Sigma Chemical Co.) was added to each well and theplates were incubated at 37°C in an incubator for 15 min.Plates were rinsed and a substrate solution containing100 �L of dimethyl sulfoxide with 1 mg of tetrameth-ylbenzidine in 10 mL of sodium acetate buffer (pH 5.5)was added. After 15 min at 37°C in an incubator, thereaction was stopped by adding 50 �L of 2 mol/L sulfuricacid. Absorbance was read via an automated ELISAreader at 490 nm.

Table 4. Effect of dietary liquid DL-2-hydroxy-4-methylthio butanoic acid on serum albumin and globulin (n = 8)

Day 21 Day 42

Total protein Albumin Globulin Total protein Albumin GlobulinMet level, % (g/L) (g/L) (g/L) (g/L) (g/L) (g/L)

80 30.80a 24.47 5.74a 35.14ab 27.34 7.91a

100 32.97ab 23.94 7.71ab 33.37a 26.03 8.55ab

120 35.92b 25.66 11.06c 36.14bc 26.82 8.87ab

140 33.70ab 23.55 10.60bc 38.39c 27.22 10.77b

SEM 0.58 0.61 0.65 0.54 0.35 0.42P-value

Linear 0.015 0.852 0.001 0.004 0.903 0.016Quadratic 0.034 0.538 0.256 0.032 0.245 0.425

a–cMeans in the same column without common superscripts differ significantly (P < 0.05).

Statistical Analysis

The results were reported as means ± SEM and all datawere statistically analyzed by one-way ANOVA of SPSS10.0 for Windows (SPSS, 1995). Differences among eachtreatment group were tested by least significant differencetest, and differences were significant at P < 0.05.

RESULTS

Growth Performance

As shown in Table 2, there were no significant differ-ences in BW gain and feed intake among the treatments.The ratio of feed to gain decreased linearly as LMA sup-plementation increased during any phase of growth. Theaverage mortality was 1.6% for the whole experiment andwas not influenced by dietary LMA level.

Lymphoid Organ Development

There were no significant differences in thymus relativeweight, but the greatest relative weights of bursa of Fab-ricius on d 10 and spleen on d 21 were in the group fedat 120% of methionine requirement (Table 3).

Immunological Measures

As shown in Table 4, serum albumin was not influencedby LMA supplementation. However, serum globulin and

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Table 5. Effect of dietary liquid DL-2-hydroxy-4-methylthio butanoic acid on proliferation of peripheral bloodlymphocytes1 [stimulating index (SI); n = 6]

Day 21 Day 42

Met level, % SI (Con A) SI (LPS) SI (Con A) SI (LPS)

80 1.08 1.08a 1.12 1.09a

100 1.12 1.10a 1.16 1.19b

120 1.06 1.21b 1.14 1.14ab

140 1.11 1.10a 1.11 1.09a

SEM 0.014 0.018 0.011 0.018P-value

Linear 0.868 0.263 0.532 0.733Quadratic 0.932 0.079 0.158 0.050

a,bMeans in the same column without common superscripts differ significantly (P < 0.05).1Con A = concanavalin A; LPS = lipopolysaccharide.

total protein on d 21 and 42 were linearly increased sig-nificantly as the dietary LMA supplementation level waselevated. As shown in Table 5, dietary LMA dosage didnot significantly influence the proliferation of peripheralblood lymphocyte in response to Con A; however, prolif-eration was quadratically influenced when the cells wereexposed to LPS, and proliferation levels in the group fedat 120% on d 21 and in the group fed at 100% on d42 were significantly greater than in other groups. Forphagocytosis of neutral red of peripheral blood lympho-cyte, the effect of dietary LMA was quadratic and theleast effect in the Met-deficient group (Table 6). Serumlysozyme activity was not influenced by dietary LMAdosage, but lysozyme activity was greatest in the groupfed at 100% of methionine requirement (Table 6).

Antibody titers to NDV were not influenced by dietaryLMA level; however, titers were quadratically influencedon d 13. The antibody titers to BSA (Table 7) were greaterin the groups fed at 100 or 120% of methionine require-ment, and on d 13 after the secondary immunization titersto BSA were quadratically influenced significantly.

DISCUSSION

Growth Performance

Even though feed intake and BW gain were not signifi-cantly influenced as supplemental LMA increased, feed

Table 6. Effect of dietary liquid DL-2-hydroxy-4-methylthio butanoic acid on phagocytosis of neutral red ofperipheral blood lymphocyte (optical density at 540 nm; n = 6) and serum lysozyme activity (n = 8)

Phagocytosis Lysozyme (mg/L)

Met level, % Day 21 Day 42 Day 21 Day 42

80 0.051a 0.212a 3.16 3.47ab

100 0.093b 0.306b 3.31 4.34a

120 0.080b 0.249ab 3.07 3.03b

140 0.081b 0.259ab 3.12 3.04b

SEM 0.005 0.013 0.11 0.21P-value

Linear 0.007 0.463 0.879 0.151Quadratic 0.003 0.098 0.202 0.288

a,bMeans in the same column without common superscripts differ significantly (P < 0.05).

utilization was significantly improved. The dietary methi-onine levels of the group receiving 80% of methioninerequirement were 0.40 and 0.32%, respectively, for the 2phases, and levels were marginally deficient for broilerchickens. Chickens in this group showed no differencesfrom all other treatments in feed intake and BW gain buthad poorer feed utilization. Earlier researchers reportedthat methionine addition reduced feed intake comparedwith a diet deficient in sulfur-containing amino acids (Es-teve-Garcia and Llaurado, 1997). However, improve-ments in feed utilization as a result of methionine supple-mentation have been widely observed in broiler chickens,and increases in methionine levels promoted an increaseof approximately 12 to 14% in BW gain compared withbroilers receiving a methionine-deficient diet (Solberg etal., 1971; Garlich, 1985; Lin et al., 1996). In agreement withthe results of those researchers, the present study foundthat better feed utilization was achieved when LMA wassupplied, and dietary methionine at 0.4 and 0.32% wasadequate for minimum growth requirement during thestarter and grower phases, respectively, but the broilersreceiving a marginally deficient diet needed to obtainsimilar growth by overeating. Lin and Shih (2000), Carewet al. (2003), and Attia et al. (2005) showed that a marginalmethionine deficiency is often compensated for by in-creased feed intake with little change in the rate of gain.Broiler chickens fed diets marginally deficient in methio-nine could overeat slightly to meet the adequate amounts

2-HYDROXY-4-METHYLTHIO BUTANOIC ACID AND GROWTH PERFORMANCE 1375

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 first immunization Post secondary immunization

Met level, % 7 d 10 d 14 d 6 d 9 d 13 d

80 0.291a 0.584 0.635 0.328ab 0.479ab 0.605a

100 0.299a 0.579 0.584 0.350a 0.484a 0.615ab

120 0.338b 0.574 0.638 0.309b 0.453b 0.643b

140 0.307ab 0.538 0.640 0.357a 0.475ab 0.600a

SEM 0.006 0.010 0.013 0.007 0.005 0.006P-value

Linear 0.092 0.101 0.552 0.436 0.322 0.785Quadratic 0.084 0.424 0.305 0.302 0.383 0.037

a,bMeans in the same column without common superscripts differ significantly (P < 0.05).

of methionine needed, and the increased feed intake didnot cause an increase in BW gain because the added calo-ric intake might be converted to body fat to replace bodywater (Carew and Hill, 1961; Carew et al., 2003), finallyresulting in lower feed utilization.

Immunological Index

Dietary methionine deficiency could cause the malde-velopment of lymphoid organs and their normal function(Konashi, et al., 2000; Carew et al., 2003). In the presentstudy, the greatest relative weights of lymphoid organswere in the group with a dietary methionine level of0.60% for the starter period. Even though the differencein the spleen of birds aged 21 d was much more obvious(0.05 < P < 0.10), development of lymphoid organs was notinfluenced by diets marginally deficient in methionine.

Nonspecific immunity was assessed by serum lyso-zyme activity and phagocytosis of neutral red of periph-eral blood lymphocytes. Our results showed that supple-mental LMA to meet 100% of methionine requirementwas required to achieve the greatest nonspecific immuno-competence, and marginal methionine deficiency wouldresult in low phagocytic function of peripheral blood lym-phocytes.

Humoral immunity was evaluated by antibody re-sponse to NDV and BSA, and cellular immunity wasmeasured by lymphocyte proliferation. Serum globulinincreased linearly as dietary LMA level elevated, whichwas in agreement with the results of Attia et al. (2005).The greatest level of antibody to BSA in the groups fedat 100 or 120% of methionine requirement in the presentresearch suggested that additional LMA was beneficialto immunocompetence, even though the antibody titers toNDV were not influenced. Takahashi et al. (1993) reportedthat there were no significant differences in the responsesto SRBC of methionine intake when the chickens werefed diets of equal energy and protein values. Many earlierresearchers showed no benefit in improving the antibodyresponse by additional methionine in pigs (van Heugtenet al., 1994) and broiler chickens (Lin and Shih, 2000;Swain and Johri, 2000), but the antibody titers againstSRBC and NDV were enhanced when dietary methioninesupplementary levels increased from 4.5 to 6 g/kg (Rama

Rao et al., 2003; Panda et al., 2007). Moreover, methioninesupplementation resulted in significant dose-related in-creases in total antibody and IgG, which suggested thatmethionine is required for some components of the anti-body response and might be required for thymus-derived(T)-cell helper function (Tsiagbe et al., 1987b).

Concanavalin A and LPS specifically stimulate lympho-cyte proliferation. The quadratically enhanced prolifera-tion in response to LPS by dietary LMA supplementationcould also contribute to the improved antibody or globu-lin production. In earlier studies, additional methioninedid not affect wing-web PHA response in adult quail(Dabbert et al., 1996) or the Con A-induced proliferativeresponse of thymus mononuclear cells (Takahashi et al.,1997) but did enhance cutaneous wing-web or wattle re-sponse to PHA in young broiler chickens (Tsiagbe et al.,1987b; Rama Rao et al., 2003) and mitogen-induced prolif-eration of T cells in rats (Williams et al., 1979). The strain,age, and basal and supplementary methionine levels werepartly responsible for the different outcomes of the above-mentioned studies.

The 0.4 and 0.32% dietary methionine levels were ade-quate for maximum growth requirement during thestarter and grower phases, respectively. Dietary LMAsupplementation improved feed utilization and humoraland nonspecific immunocompetence of broiler chickens.

ACKNOWLEDGMENT

The authors thank Sumitomo Chemical Co. Ltd.(Tokyo, Japan) for supplying the LMA product and forpartial financial support. This work was supported inpart by the Project nyhyzx07–039 from the Ministry ofAgriculture, P. R. China.

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