Isoleucine Needs of Thirty

Isoleucine Needs of Thirty- to Forty-Day-Old Female Chickens: Immunity1

Hale, L L


Isoleucine is categorized as a branched-chain amino acid (BCAA). The BCAA consist of Val, Ile, and Leu, which are structurally related; Ile is the most hydrophobic of the BCAA. In addition, VaI and He share many of the same enzymes used for degradation. Degradation of Leu, He, and VaI in animals starts with transamination followed by oxidative decarboxylation of the respective keto acids. That reaction is carried out by the multienzyme complex referred to as the branched-chain α-ketoacid dehydrogenase complex (Mathews and van Holde, 1990). Although Ile metabolism is understood, little is known concerning its role in immunity.

Understanding the functions of the immune system and how these functions can be affected by environment, nutrition, or stress factors is imperative in maintaining successful innate or adaptive immunity. Feeding diets with amino acid imbalances or deficiencies may impair the bird’s ability to mount an effective immune response. The former could occur in birds fed diets limiting in He or excessive in Leu. Research in rats has shown that immunosuppressive effects of high Leu could be reversed by increasing levels of He and VaI (Aschkenasy, 1979). Because of structural similarities, Leu can antagonize VaI and He absorption or retention, or both, which may be the mechanism involved in Leu-induced immune suppression (Benton et al., 1956). Isoleucine and Val deficiencies appear to suppress immunity in mice (Petro and Bhattacharjee, 1981). It has been shown that a BCAA deficiency can significantly depress relative organ weights in chickens (Konashi et al., 2000). Deficiency in BCAA caused the most severe decrease in thymus and bursa weights (Konashi et al., 2000) compared with all other essential amino acid groups compared. Click et al. (1981; 1983) fed chicks a diet deficient in all required amino acids and measured immune response of the chicks. Depressed delayed-type hypersensitivity responses and secondary IgG responses to SRBC were noted between adequately fed controls and deficient chicks. Although select amino acid deficiencies in mammals have been shown to suppress the humoral immune response over the total cell mediated responses (Cook, 1991), the specific role of He on immunity in broilers is not fully understood. If dietary CP is reduced, He could become marginal because it is the fourth most limiting amino acid for broilers and its limitation could decrease immune system responses that require He (Konashi et al., 2000).

These decreasing immune responses may be observed when evaluating absolute counts of T and B cells. A CD4^sup +^ molecule is primarily expressed on a helper T cell. It binds to a class II major histocompatability molecule and serves as an activator for macrophages and assists B cells during humoral immune responses. A CD8^sup +^ molecule is primarily expressed on a cytotoxic T cell, a TCRl cell, or a natural killer cell during cell mediated responses. Cytotoxic cells express the CD8αβ heterodimer, and TCRl and natural killer cells express the CD8αα homodimer (Tregaskes et al., 1995; Luhtala, 1998). This cell subset is recognized by a class I major histocompatibility molecule and its primary role is to attack virus-infected cells. BU-1^sup +^ cells are considered B lymphocytes, which respond during humoral immune responses. Our research was conducted to assess He needs for immunity in female broiler chickens because this amino acid is fourth limiting and little is known concerning its need for immunity.


Typical poultry diets based on corn and soybean meal are not limiting in He if adequate CP levels are fed. Granulated blood cells3 (5.25% of diet) of mixed porcine and bovine origin, which are lie deficient, were added to corn and soybean meal to create lie deficient diets to test the birds’ immunological needs for He.

Experiment 1

Experimental Facilities. Experiment 1 was conducted in a closed-sided facility containing floor pens on a concrete pad. All floor pens measured 1.5 × 2.6 m. Each pen contained one tube feeder and one bell-type waterer, and used litter top-dressed with about 5 cm of new shavings. Supplemental pan feeders were used in each pen from d 1 to 7 to ensure ad libitum and feed consumption at placement. The facility was heated with electric lamps in each pen and supplemental gas brooders in the center aisle. The lighting program, consisted of 22 h of light throughout the trial.

Experimental Design, Diets, and Birds. Three strains (Arbor Acres+, Ross 508, and Ross 708) of commercial female broilers were obtained from a common hatchery. The strains consisted of a multipurpose (Arbor Acres+) feather-sexable strain and 2 high yield (Ross 508 and Ross 708) feather-sexable strains. The vaccination program consisted of Marek’s vaccination in ovo, and Newcastle and infectious bronchitis vaccines at d 1. Nine hundred broiler chicks (300/strain) were distributed into 30 pens, with 30 birds per pen. All birds were fed a common diet through d 29. On d 30, 2 diets were fed to broilers up to d 42 (6 replications/treatment). Initial BW was 0.954 kg/ bird, with a CV of 5.15%. All treatments (3 strains × 2 dietary He) were replicated 5 times. The test diet (Table 1) contained 0.42% lie. The second diet (Table 1) was an He-adequate diet containing 0.72% He, derived by the inclusion of 0.30% L-IIe to the test diet (0.42%) at the expense of the filler. The lie-deficient test diet was created with the inclusion of dried blood cells in a diet based on corn and soybean meal. Birds had continuous access to feed and water throughout the experiment.

Immune Measurements. Three chicks per pen were randomly chosen to evaluate a cutaneous basophil hypersensitivity test to phytohaemagglutinin-P (PHA-P) on d 37 and 38. At 37 d of age, the right toe web thickness was measured (in millimeters) with a constant tension caliper before injection of 100 µg of PHA-P suspended in 0.10 mL of sterile PBS. Twenty-four hours after the injection, the toe web was measured again. Relative swelling and toe web thickness were indicators of the cellular immune response (Corrier and DeLoach, 1990). A saline control was not used due to minor differences observed between the PHA-P and the saline-injected toe web (Murray et al., 1987).

Lymphoid organs (thymus, bursa, and spleen) were harvested and weighed from 2 randomly selected birds per pen at 42 d. Thymus weight represented the proximal 2 thymus lobes on the left side of the bird. Organ weights were expressed relative to BW to adjust for potential BW effects on organ weight.

Three broilers per pen were randomly selected to evaluate cell quantification of specific leukocytes. Half the birds were bled via the jugular vein on d 41 and the remaining birds on d 42 due to time constraints with the assay. Absolute counts of CD4^sup +^, CD8^sup +^, and BU-I^sup +^ cells were determined by flow cytometry as described (Burgess and Davison, 1999). Briefly, CD4^sup +^ and CD8^sup +^ T cells were identified with monoclonal antibodies CT4 and CTS (Chan et al., 1988) conjugated with fluorescein isothiocyanate and phycoerythrin, respectively.4 The T cells expressing the jo T cell receptor were detected with the antiT cell receptor (TCR)-I monoclonal antibody (Sowder et al., 1988) conjugated with fluorescein isothiocyanate . The BU-I^sup +^ B cells were identified with monoclonal antibody AV20 (Rothwell et al., 1996) conjugated with phycoerythrin. Whole blood was diluted 1:10 in Hank’s balanced salt solution containing 0.1% sodium azide and 1% BSA. A standard volume (20 µL) of diluted blood was added to 20 µL of blue-green fluorescent (430/465) 15-µm polystyrene beads (106/rnL).5 This mixture was then added to 50 µL of the appropriate antibody and incubated at 4°C for 10 min. After incubation, the mixtures were resuspended in 300 µL of Hank’s balanced salt solution containing 0.1% sodium azide and analyzed with a FACSCalibur flow cytometer.6 The electronic gates were set to count a standard number of polystyrene beads (500) and the concomitant fluorescently-labeled cells. Absolute numbers of CD4^sup +^, CDS^sup +^, and BU-I^sup +^ cells were calculated with the following method. Twenty microliters of a 1:10 dilution of whole blood was added to an equal volume of polystyrene beads resulting in a further 1:2 dilution. This resulted in a 1:20 final dilution of whole blood. Therefore, the number of fluorescent cells detected by the flow cytometer was multiplied by the final dilution factor (20) to give the absolute number of fluorescent cells per microliter of whole blood.

Experiment 2

Experimental Facilities. Broilers were raised in one floor pen and fed a nutritionally adequate diet. Supplemental gas brooders were used for heat. The birds were transferred to the test facility on d 17. The experimental period of experiment 2 was from d 30 to 42. The test facility for experiment 2 contained floor pens on a concrete pad and side wall curtains. All floor pens measured 0.9 × 1.2 m. Each pen contained one tube feeder, one nipple water line, and used litter. The lighting period was the same as in experiment 1.

Experimental Design, Diets, and Birds. One strain (Ross 508 feather-sexable) of high yield female broilers was obtained from a local hatchery. The same vaccination program was used as previously described in experiment 1. Five hundred seventy-six birds were distributed across 48 pens (12 birds per pen). All birds were fed a common diet through d 29. Initial BW and CV were 1.090 kg/bird and 5.31%, respectively. Experimental diets (7 pens per diet) consisted of 6 gradations of He (0.42, 0.50, 0.58, 0.66, 0.74, and 0.82% total of diet) in the blood cell based diet and one control diet (6 pens) based on corn and soybean meal (Table 1). Aliquots of He were achieved by adding L-IIe at the expense of sand.

Immune Measurements. Two chicks per pen were randomly chosen to evaluate toe web swelling after injection with PHA-P on d 35 and 36 as described in experiment 1. To test antibody responses, SRBC were obtained, washed twice, and a 10% solution was prepared for intravenous injection into 2 randomly selected chicks per pen at d 35. On d 42, each bird was bled via the jugular vein and serum was obtained for evaluation of the primary antibody response. The scrum was inactivated at 56°C for 30 min. Twenty-five microliters of saline was added to every well and 25 µL of serum was added to the first well of a microtiter plate. Serial dilutions (from 2- to 1024-fold) were performed, followed by an addition of 25 µL of 2.5% SRBC to each well. The plate was sealed and incubated for 60 min at 37°C. Primary antibody titers were read and measured 24 h later by a microhemagglutination assay in 96-well plates by observing the well where total agglutination occurred. The titers were expressed as Iog2 of the highest serum dilution that agglutinated 0.05 mL of 2% suspension of SRBC in sterile saline.

On d 42, lymphoid organs (thymus, bursa, and spleen) were harvested and weighed from one randomly selected bird per pen. Thymus weight represented the proximal 3 thymus lobes on the left side of the bird. Organ weights were expressed relative to BW to adjust for potential BW effects on organ weight. Quantification of T cell surface proteins CDSα, CO8β, and TCRl were done in 3 birds per pen (except the control pens) in 2 replications per day over 2 d using the flow cytometry method previously described in experiment 1.

Statistical Analysis

A 2 × 3 factorial arrangement of treatments was used in experiment 1. Experiment 2 was a completely randomized design. Data in all experiments were analyzed by the GLM procedure of SAS (SAS Institute, 1996). Because blood used for determination of absolute counts of CD4^sup +^, CDS^sup +^, and Bu-I^sup +^ cells were collected over 2 d, day was included in the statistical model. Means were compared for significant (P ≤ 0.05) differences using the LSMEANS option of SAS (SAS Institute, 1996). Statements of significance are based on P ≤ 0.05 unless otherwise noted. Linear and quadratic (P ≤ 0.05) models for the parameter tested in the dietary treatments containing blood cells and varying in He in experiment 2 were fitted using the GLM procedure of SAS (SAS Institute, 1996).


Experiment 1

No He × strain interactions occurred for any parameters measured in experiment 1. Impact of He and female broiler strain on relative lymphoid organ weights at d 42 is presented in Table 2. Broiler strain had no effect on lymphoid organ weights. Broilers fed 0.42% He had reduced relative thymus weights (38% smaller than birds fed 0.72% Ile). However, differences in relative bursa and spleen weights did not occur between He treatments.

The impact of He and female broiler strain on the cutaneous basophil hypersensitivity test to PHA-P at d 38 is shown in Table 3. The toe web increase in birds fed 0.42% He was 24% smaller than in birds fed 0.72% He. However, relative increases in toe web swelling mediated by PHA-P (P = 0.052) were not found to differ between He treatments. Differences among broiler strains to the cutaneous basophil hypersensitivity test were not observed.

Results of flow cytometric analyses of whole blood for T and B cell markers are shown in Table 4. The number of CDS^sup +^ T cells was lower in blood from birds fed 0.42% He than from those fed 0.72% lie. The number of CD4^sup +^ and BU-I^sup +^ lymphocytes did not differ between birds fed the He diets.

Experiment 2

Initial (d 30) and ending BW (d 42), respectively, were 0.954 and 1.893kg/bird in experiment 1, and 1.090 and 1.720kg/bird in experiment 2. Immunity measurements in birds fed surfeit He (0.66 and 0.74% of diet) in the titration diets were equal to birds fed the control diet (Tables 5 and 6). The impact of He gradations on female broiler relative lymphoid organ weights is shown in Table 5. Linear responses to increasing crystalline He in diets containing blood cells were obtained for the bursa. Thymus and spleen weights were not affected by increasing He. The impact of lie gradations on cutaneous basophil hypersensitivity to PHA-P and the primary antibody response to SRBC is shown in Table 6. No linear or quadratic responses were noted for these parameters. Table 7 shows the impact of He gradations on female broiler lymphocyte populations expressing CDSα, CDSβ, and TCR1. No Tie linear or quadratic responses were noted.


The 2 experiments presented herein tested the effect of He-deficient and adequate diets (NRC, 1994) on immunity in female broilers. Because He is the fourth limiting amino acid for broilers, research delineating He requirements is important so that broiler diets can be formulated to more closely meet the bird’s needs. Although some research has addressed He requirements for growth of male broilers from 30 to 42 d (Kidd et al., 2004), very little has addressed lie requirements for immunity of male or female broilers. Therefore, the major objectives of this study were to measure the impact of He deficiencies on immunity and estimate levels of lie needed to improve immunity in broilers through dose response methodology.

The poultry industry’s continuous improvements in broiler genetics have changed the bird’s ability to respond to infection. Modern broilers have increased cell-mediated and inflammatory responses, but responses have decreased in the adaptive arm of the immune system (Cheema et al., 2003). Such differences warrant testing of immune responses in the modern commercial broiler. Our first experiment demonstrated that the immune responses measured did not differ among commercial broiler strains (known to differ in terms of growth rate) when fed diets that were adequate or marginal in He. Commercial broilers fed diets limiting in CP may have marginally reduced He, and although He was shown to impact immunity, its impact was not shown to differ across broiler strains.

Konashi et al. (2000) demonstrated that a BCAA-deficiency in broiler chickens significantly depressed relative lymphoid organ weights. Results suggested that the type of essential amino acid or the degree of its deficiency modifies weights of lymphoid organs. Furthermore, the weights of thymus and bursa are more susceptible to dietary amino acid deficiencies than is spleen weight. Of the amino acids tested, the BCAA deficiency caused the most severe decrease in both thymus and bursa weights in chickens (Konashi et al., 2000). Aschkenasy (1975) showed that deficiencies of He and VaI in rats inhibited leukopoiesis, with involution of lymphoid organs (especially the thymus), and caused a dramatic drop in the blood lymphocyte numbers. Marginal reduction in SRBC titers were noted when BCAA were fed at deficient levels (Konashi et al., 2000).

In agreement with Konashi et al. (2000), experiment 1 showed that a deficiency in the BCAA He caused a significant decrease in the weight of the thymus. In addition, a decrease in CDS+ T cells isolated from whole blood in birds fed a diet deficient in He was observed. This decrease could be directly related to the decrease in thymus weight, the organ responsible for the production of T cells. Cell mediated response differences to PHA-P were noted between birds fed He-adequate and deficient diets in experiment 1. Broilers fed a diet deficient in He had a smaller increase in toe web thickness after PHA-P injection than broilers fed a diet adequate in He. Millimeter decreases in toe web thickness due to deficient dietary He as mediated by intradermal injection of PHA-P during a cutaneous basophil hypersensitivity test were shown. Cutaneous basophil hypersensitivity is a good indicator of T cell activity in broilers (Corrier and DeLoach, 1990).

In chickens, cytotoxic T cells express the CD8αβ heterodimer, whereas TCRl T cells and natural killer cells express the CD8αα homodimer (Tregaskes et al., 1995; Luhtala, 1998). In experiment 1, birds fed low He had a reduction in the number of peripheral blood lymphocytes expressing the antigen recognized by the monoclonal antibody CT8, which recognizes the CD8α surface protein. Although no hypothesis was proposed for why a deficiency in He decreased expression of a surface protein, experiment 2 was designed to evaluate the numbers of the different lymphocyte populations expressing CO8αβ, CDSαα, or TCRl in birds fed graduated levels of He, in addition to organ weights, and humoral and cell-mediated immune responses. The difference (P = 0.08) in CDSαα homodimer caused by feeding a diet with 0.42 or 0.74% He level in experiment 2 was similar to the effects of 0.42 or 0.72% He on CDS+ T cells in experiment 1. The bursa of Fabricius relative weight increased as He deficiency increased, resulting in a significant linear response to He gradations. Relative bursa size decreases as a bird ages and it may be that the reduced growth due to lie deficiency slowed its maturation, resulting in an increased relative bursa size.

Dietary deficiency of He has been shown to decrease circulating numbers of white blood cells (Aschkenasy, 1975). Furthermore, deficient He has been shown to decrease immunity to a greater extent than total dietary protein deprivation (Aschkenasy, 1975). Hence, He may play an important role in protein synthesis critical for T cell development, as well as T-dependent and independent reactions (Aschkenasy, 1975). The negative impact on immunity as mediated by 0.42 vs. 0.72% dietary He reported herein may be a result of decreased thymic cellularity. Moreover, use of the most limiting amino acids (i.e., TSAA, Lys, and Thr) was reduced to the degree of lie use because He is fourth limiting, pointing to the possibility that observed depressions in immunity may be a result of suppressed protein synthesis.

No quadratic responses to He levels occurred; therefore, no lie requirements could be recommended for immunity. Because analyses of immune responses as affected by the titrated lie levels in experiment 2 were done by regression rather than by mean separation, the impact of deficient lie in experiment 2 was not observed. However, marginal He limitations, as observed when broilers consume diets reduced in CP, should not result in immunosuppression. Furthermore, these studies showed that deficient levels of He could have a depressive effect on immunity in female broilers from 30 to 42 d of age.


The authors thank Ajinomoto Heartland and Degussa Corporation for providing amino acid analyses of ingredients and test diets. Provision of supplemental amino acids used in dietary formulation by Kyowa Hakko, Ajinomoto Heartland, and Degussa Corporation is greatly appreciated. Appreciation is extended to American Protein Corporation for the donation of blood cells used in dietary formulation.

Copyright Poultry Science Association Dec 2004

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