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Effects of Higher Levels of Zinc Supplementation on Performance, Mineral Availability, and Immune Competence in Broiler Chickens
©2008 Poultry Science Association, Inc.
Effects of Higher Levels of Zinc Supplementation on Performance, Mineral Availability, and Immune Competence in Broiler Chickens G. Shyam Sunder,1 A. K. Panda, N. C. S. Gopinath, S. V. Rama Rao, M. V. L. N. Raju, M. R. Reddy, and Ch. Vijay Kumar
Primary Audience: Nutritionists, Researchers SUMMARY Two feeding trials were simultaneously conducted utilizing 210 and 140 1-d-old commercial broiler chicks for evaluating the effect of Zn supplementation on performance, mineral availability (experiment I), and immune response (experiment II), respectively, up to 4 wk of age. A basal diet was formulated using corn-soybean meal without supplemental Zn. Six test diets were prepared in which the basal diet was supplemented with Zn at 10, 20, 40, 80, 160, or 320 ppm using ZnSO4ⴢ7H2O. Diets were fed ad libitum to broilers in cages from 8 to 28 d of age. Supplemental Zn did not influence body weight gain, feed intake, feed efficiency, ready-to-cook yields, leg scores, or tibia weight at 4 wk of age. Tibia ash was significantly lower only at 320 ppm level. The percent Ca and P in tibia increased steadily with supplemental Zn up to 80 and 40 ppm, respectively, and decreased thereafter. A linear increase in Zn deposition was observed in bone, liver, and kidney with Zn supplementation. The humoral and cell mediated immune responses were significantly higher at the 80-ppm level compared with lower levels. The heterophil and lymphocyte ratios were narrow, indicating less stress with Zn at 40 ppm and above. The weights of bursa and spleen were higher at 40 ppm compared with lower levels. This study showed that the basal diet Zn (29 ppm) was adequate to support optimum performance of broilers. For better mineral retention, immune response, and alleviation of stress, 80 ppm of Zn was required up to 4 wk of age. Key words: zinc, performance, mineral availability, immune competence, broiler chicken 2008 J. Appl. Poult. Res. 17:79–86 doi:10.3382/japr.2007-00029
DESCRIPTION OF PROBLEM Zinc plays a vital role in numerous biological processes as a component of many enzymes [1]. It is essential for growth, skeletal development [2], and immune competence [3]. In poultry, Zn deficiency causes reduction in weight gain, 1
Corresponding author: [email protected]
skeletal malformations, poor bone mineralization, and immunological dysfunctions [3, 4]. Therefore, Zn is often supplemented in practical poultry diets to elicit a positive response in broiler chickens, particularly during early age. Tissue uptake of Zn in chicks is linearly related to Zn levels in the diet [5, 6]. The Zn was found
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Project Directorate on Poultry, Rajendranagar, Hyderabad-30, India
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MATERIALS AND METHODS Two experiments were simultaneously conducted to evaluate the influence of Zn at levels that were higher than those recommended by NRC [8] on growth performance, mineral availability, and immune response in commercial broilers up to 4 wk of age. Experiment 1 Stock, Husbandry, and Experimental Diets. Two hundred ten 1-d-old commercial broiler chicks of both sexes [13] were equally distributed into 7 treatment groups with 6 replicates of 5 chicks each. The chicks were wing-banded individually and reared under uniform management care in stainless steel battery brooders (60 cm × 75 cm × 45 cm for 5 chicks) with raised wire floor in an open-sided house. They were provided with a rearing temperature of 34 ± 1°C up to 7 d of age and gradually reduced to 26 ± 1°C by 21 d of age after which no supportive heat was provided. The birds were vaccinated against Marek’s disease, Newcastle disease, and infectious bursal disease following the prescribed schedule. A basal diet was formulated with corn-soybean meal without Zn supplementation (Table 1). In addition, 6 experimental diets were prepared by adding measured quantities of
Table 1. Ingredient and nutrient composition of the basal diet (%) Ingredient Yellow maize Soybean meal Di-calcium phosphate Shell grit Common salt Choline chloride DL-Methionine Vitamins1 Trace minerals2 Coccidiostat Antibiotics Starch3 Nutrient composition (%) Metabolizable energy4 (kcal/kg) Crude protein5 Lys Met Calcium5 Nonphytate phosphorus5
Quantity 59.55 35.00 1.70 1.80 0.50 0.26 0.26 0.04 0.10 0.05 0.05 0.69 2,878 22.05 1.19 0.50 1.09 0.45
1
Supplies per kilogram of diet: vitamin A, 16,500 IU; vitamin D3, 3,150 ICU; vitamin E, 12 mg; vitamin K, 2 mg; vitamin B1, 1.2 mg; vitamin B2, 10 mg; vitamin B6, 2.4 mg; vitamin B12, 12 g; niacin, 18 mg; pantothenic acid, 12 mg. 2 Mn, 60 mg; Fe, 60 mg; Cu, 10 mg; I, 1.2 mg. 3 ZnSO4ⴢ7H2O salt of AR grade was used at appropriate levels quantitatively replacing starch by weight to maintain Zn supplemental levels between 0 and 320 ppm in test diets. 4 Calculated ME value. 5 Analyzed values.
ZnSO4ⴢ7H2O (analytical grade) to the basal diet for providing Zn supplementation at 10, 20, 40, 80, 160, or 320 ppm. Zinc was included in test diets at the expense of starch, whereas all the other feed ingredients remained unaltered. The diets were offered ad libitum to the respective groups from 8 to 28 d of age. The basal diet was fed to all chicks from 1 to 7 d of age for depleting the available Zn reserves. Body Weight, Feed Efficiency, and Leg Abnormality Scores. Individual body weights and feed intake by replicate were recorded at weekly intervals. Feed conversion efficiency was calculated as the ratio between feed consumed and weight gained. At the end of each week, leg abnormality scores were recorded on every bird by examining the hock joint for deformity [14]. Hock joints were assigned scores in the range of 1 to 5, depending upon the severity of deformity. The normal hock joint was scored as 1; slight swelling of tibiometatarsal joint as 2; marked swelling as 3; swelling of the joint with
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to be essential for normal functioning of the immune system by increasing the counts of thymocytes and peripheral T cells, an activity of natural killer cells [7]. It also enhances the production of neutrophils and antibodies, in addition to improving the functions of macrophages [3]. Because Zn supplementation was found to be essential, the NRC [8] recommended 40 ppm for broiler chickens, which appeared to be based on the results that considered growth performance as the only criterion [9, 10]. However, higher Zn levels (60 to 180 ppm) produced better immune status in broiler chickens [6, 11, 12], but clear information on Zn requirements for growth, mineral bioavailability, and immune response is scanty. Therefore, the present experiment was undertaken to study the effect of higher Zn levels on these factors in commercial broiler chickens during the juvenile period.
SHYAM SUNDER: ZINC IN BROILER CHICKEN DIET
the digested samples were filtered, diluted to required volume, and then analyzed for Zn content [16]. Experiment 2 Simultaneously, a second experiment was conducted with one hundred forty 1-d-old commercial broiler chicks [13] employing the same levels of Zn as in experiment I. Chicks were equally distributed into 7 treatment groups with 4 replicates of 5 chicks each. Feeding practices, duration of experiment, and management conditions were similar to experiment 1. Humoral and cell-mediated immune response, weights of bursa and spleen, and the ratios between heterophils and lymphocytes were determined at 4 wk of age. Humoral Immune Response. Sheep red blood cells, a nonpathogenic antigen, was used for evaluating the humoral immune response in broiler chickens. Two birds (1 male and 1 female) from each replicate (8 birds/treatment) were injected with 0.1 mL of 0.5% SRBC suspension into brachial vein on the d 22, and blood samples were collected on d 5 of postinoculation. Subsequently, microhemagglutination activity of serum was estimated, and the antibody titers (log2) were measured following the standard procedure [17]. Cellular Immune Response. The cell-mediated immunity was assessed (8 birds/treatment) by measuring the hypersensitivity response of cutaneous basophils (CBH) to phytohemagglutinin-P. On d 28, phytohemagglutinin-P (100 g suspended in 0.1 mL of normal saline solution) was injected intradermally into chick wattles. The wattle thickness (mm) was measured with constant tension micrometer prior to (T0) and 24 h after injection (T24). The CBH response was calculated as posttreatment as the percentage of the pretreatment thickness of wattles using the following formula: CBH response = [(T24 ÷ T0) × 100]. Heterophil-Lymphocyte Ratio and Weight of Lymphoid Organs Blood smears were prepared from the birds of 1 replicate of each test group on d 22 prior to inoculation of SRBC and stained with MayGreenwald-Giemsa stain [18]. Heterophils and
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symptoms of slipped achilles tendon as 4, and swelling with marked degree of slipped tendon as 5. Ready-to-Cook Yield. On d 29, six birds (3 males and 3 females) from each treatment were killed by cervical dislocation, and data on readyto-cook yields were collected and expressed as grams per kilogram of live weight. The tibia, liver, and kidney were collected from the killed birds for estimation of Zn retention in tissues. Zn in Feed Sample. The Zn content in feed was determined by collecting a 2-g sample from the basal diet and finely ground to digest at 120°C using 5 mL of concentrated HNO3 for 1 h using the Tecator digestion system-2000 [15]. The digested sample was cooled and 3 mL of 70% HClO4 was added for further digestion at 200°C. The process continued until the contents appeared clear and colorless. A blank was also simultaneously run along with the test sample. The digested sample was carefully filtered into a volumetric flask. The contents of digestion tube were repeatedly washed with deionized water to obtain complete extract of the minerals. The samples were aspirated into an atomic absorption spectrophotometer [16] to determine the Zn content. Zn in Tibia. The right and left tibiae from each bird were pooled group-wise and pressurecooked for 1 h. They were cleaned manually, clearing off the attached muscle and cartilage, washed with distilled water, and oven dried. The tibiae were then weighed and ashed in a muffle furnace at 600 ± 5°C for 4 h. The total ash was estimated on percent weight basis. Approximately 0.2 g of ash sample from each replicate was solubilized in 5 mL of 50% HCl and the mineral extract was filtered into a volumetric flask. The extract was then diluted using deionized water to the required volume, and Zn content was determined [16]. Zn in Liver and Kidney Tissues. The liver and kidney samples from 6 birds per group were collected and stored at −20°C. They were thawed, oven dried at 100°C for 24 h, and finely ground for mineral analysis. Approximately 0.5 g of ground sample was predigested with 5 mL of concentrated HNO3 and continued digestion for 1 h at 120°C using the Tecator system-2000 [15]. The contents were further digested using 30% H2O2 at 200°C for 45 min. A blank and
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Table 2. Effect of supplemental Zn on body weight, feed efficiency, edible meat yield, and leg scores of broiler chicks at 28 d of age Supplemental Zn (ppm)
Feed intake (g)
Feed/weight gain
Ready-to-cook yield (g/kg live weight)
Leg scores
1,208 1,167 1,216 1,219 1,192 1,202 1,202 6.87 0.47
2,134 2,097 2,097 2,168 2,109 2,154 2,194 9.67 0.06
1.84 1.88 1.80 1.85 1.84 1.87 1.90 0.01 0.281
665 675 658 671 668 668 669 0.203 0.494
2.30 2.23 2.40 2.27 2.30 2.33 2.23 0.07 0.99
lymphocytes were counted to a total of 60 cells and expressed as heterophil-to-lymphocyte ratio, which indicated the relative stress in birds due to dietary Zn levels. Again, on d 29, 6 birds from each treatment were killed by cervical dislocation, and the weights of bursa and spleen were recorded and expressed as grams per kilograms of live weight. Statistical Analysis. The main effects included those of Zn levels used in the experiment. Statistical analysis of body weight and leg scores was done on individual bird data, whereas replicate data were used for feed intake and feed conversion efficiency. Similarly, the statistical analysis for other traits was based on the number of samples indicated under materials and methods. One-way ANOVA under completely randomized design was used [19]. The mean differences were compared using Duncan’s multiple range test [20]. Significance was considered at P ≤ 0.05 level.
RESULTS AND DISCUSSION Performance and Leg Scores Supplementation of Zn to the basal diet at graded levels had no significant influence on body weight, feed intake, feed conversion efficiency, ready-to-cook yields, or leg scores at 4 wk of age (Table 2). This was in agreement with the findings of Burell et al. [10] who found that a practical diet of maize-soybean meal containing 30 ppm of Zn was adequate to support optimum performance during the initial 3 wk of age. Similarly, others [9] reported that a basal diet containing 37 ppm of Zn was optimum for
realizing good growth in chicks and additional supplementation had no added advantage. In another study [21] with male broiler chicks, little effect was found on body weight, feed efficiency, or livability with supplementation of Zn up to 6 wk of age because the basal diet contained 44 ppm of Zn. The present study also indicated that the Zn content available in cornsoybean diet (29 ppm) was adequate to sustain growth and other related parameters at par with those supplemented with Zn up to 4 wk of age. Obviously, this level was lower than that recommended by NRC [8] (40 ppm) for broiler chicks. The absence of any difference in performance between groups fed diets with or without supplemental Zn could be due to slower rate of Zn utilization, necessitating no further replenishment in diets [22]. Tibia Weight and Percent Ash, Ca, and P Supplemental Zn did not influence tibia weights at 4 wk of age (Table 3). Tibia ash content also did not vary with the level of Zn in feed up to 160 ppm, but declined significantly (P ≤ 0.05) at 320 ppm, indicating the possibility of negative effect of higher Zn levels on bone mineralization. The Ca retention in tibia was significantly (P ≤ 0.05) lower in birds fed no supplemental Zn and improved as its level increased in the diets. Maximum Ca retention was observed at the 80-ppm level, and receded thereafter, suggesting the limitations of Zn supplementation at lower and higher levels. Similarly, the P retention in the tibia was significantly (P ≤ 0.05) higher at 40 ppm compared with all other levels, except 80 ppm, indicating that Zn
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0 10 20 40 80 160 320 SEM P-value
Body weight (g)
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Table 3. Influence of dietary Zn level on bone and tissue mineralization in commercial broilers at 28 d of age Supplemental Zn (ppm)
Tibia ash (%)
Tibia Ca (%)
Tibia P (%)
3.15 3.27 3.20 3.05 3.04 3.24 3.18 0.04 0.64
54.22a 54.63a 54.20a 54.38a 54.75a 54.17a 52.70b 0.13 0.0002
35.77c 36.51b 36.57b 36.56b 37.41a 37.08ab 36.60b 0.09 0.0002
16.86d 17.11cd 17.19bc 17.59a 17.46ab 17.22bc 17.07cd 0.04 0.0001
0 10 20 40 80 160 320 SEM P-value
Means in a column with different superscripts are significant (P ≤ 0.05).
a–d
supplementation at specific levels was essential to optimize P concentration in bone. Higher levels of Zn in the diet appeared to interfere with the absorption and utilization of Ca and P particularly above 80 ppm [23], which was also observed in our study. Zn Uptake by Bone, Liver, and Kidney Tissues The Zn uptake by bone, liver, and kidney tissues is presented in Figure 1. A linear increase
(P ≤ 0.05) in Zn deposition was observed in bone (251 to 482 ppm), liver (117 to 143 ppm), and kidney tissues (88 to 113 ppm). Although, the rate of Zn retention in bone was higher (P ≤ 0.05) with each incremental raise, there was no difference between the 80- and 160-ppm and 160- and 320-ppm levels. However, the proportional increase in Zn deposition was high at lower levels of Zn supplementation (up to 40 ppm) compared with that between 80 to 160 ppm and 160 to 320 ppm. The Zn retention in
Figure 1. Variations in the deposition of Zn in tibia, liver, and kidney tissues of broiler chickens at 4 wk of age due to incremental increase in Zn supplementation to diets. a–fMeans on a line having no common letter are significantly different (P < 0.05).
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Tibia weight (g)
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Table 4. Influence of supplemental Zn on immune response, heterophil-to-lymphocyte (H/L) ratios, and weight of lymphoid organs Supplemental Zn (ppm)
H/L ratio
Bursa (g/kg)
Spleen (g/kg)
3.40b 3.40b 3.70b 4.40ab 5.30a 5.60a 5.30a 0.205 0.002
156 c 178 b 215 ab 224 ab 247 a 265 a 259 a 9.24 0.003
0.68a 0.64a 0.59a 0.46b 0.41b 0.47b 0.45b 0.016 0.0001
1.28b 1.26b 1.39b 2.31a 2.12a 2.19a 2.00a 0.077 0.0001
1.17c 1.13c 1.46bc 2.23a 2.39a 1.94ab 2.11a 0.087 0.0001
0 10 20 40 80 160 320 SEM P-value
Means in a column with different superscripts are significant (P ≤ 0.05). CBH = cutaneous basophils.
a–c 1
liver and kidney tissues also showed a consistent increase with Zn level in the diets. Henry et al. [24] and Sandoval et al [25] observed a linear accumulation of Zn in bone, liver, and kidney in chicks fed 0 to 1,500 ppm, which was similar to the results observed in our trial. Chicks fed no supplemental Zn had poor retention in bones. Therefore, for appreciating the bioavailability of Zn in broiler chicks, supplementation of higher levels of the mineral than that required for optimum performance is essential [22]. In this study, we also observed a consistent increase in Zn uptake by bone, liver, and kidney in relation to its concentration in diets. Bone accumulated more Zn than liver or kidney. This finding is further supported by the earlier report of Sandoval et al. [25]. It has been reported that Zn from bone can be utilized when Zn deficiency occurs; thus, bone appears to be a functional reserve of Zn, which could be readily mobilized for any immediate need [26]. However, it must be remembered that mineral metabolism is highly dynamic and that constant redistribution keeps occurring among the relevant tissues within the body, regardless of the dietary intake of an element [25]. Immune Competence The antibody titers against SRBC inoculation, a measure of humoral immune response, varied significantly (P ≤ 0.05) with the level of supplemental Zn at 4 wk of age (Table 4). Highest antibody titers were observed at 80 ppm, above which no further improvement in antibody
titers was noticed, implying that 80 ppm was adequate to provide optimum response. Similarly, others [11] reported a higher response of T-dependent humoral immunity in chickens fed 160 ppm of Zn. It was also observed [6] that higher antibody titers to SRBC with 181 ppm of Zn compared with lower levels (34 to 68 ppm) of supplementation. In our study, maximum immune response was observed at the 80 ppm level, which was comparatively lower than those reported earlier. Higher levels of Zn beyond the physiological limits of an individual may have no beneficial effect [27]. Thus, it could be hypothesized that the genetic stocks used in our study elicited better response at a lower physiological limits. Nevertheless, Zn plays an important role in immunomodulation by increasing the counts of thymocytes and peripheral T cells and also by enhancing the production of interferon, which might have been elevated by the dietary increases of Zn in our study [3]. The CBH response as measured by the thickness of wattles following the injection of phytohaemagglutinin also showed a steady increase (P ≤ 0.05) with Zn supplementation in diets (Table 4). Birds that received no supplemental Zn from the diet elicited the lowest CBH response. Although peak response was observed at the 160-ppm level, it was statistically not different from 20 to 80 ppm levels, suggesting the relevance of even lower levels of Zn supplementation in triggering immune response at par with higher levels. However, a numerical increase in CBH response was observed as the Zn levels
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Antibody titers (log2)
CBH1 response, % of pretreatment
SHYAM SUNDER: ZINC IN BROILER CHICKEN DIET
CONCLUSIONS AND APPLICATIONS 1. Zinc available from the corn-soybean diet (29 ppm) was adequate to maintain optimum growth, feed conversion efficiency, ready-to-cook yields, and normal hock joints to 4 wk of age. 2. Supplementation of Zn to broiler diets was essential for enhancing immune response, mineral retention, and alleviation of stress in broiler chickens up to 4 wk of age.
3. Maximum response in broiler chicken was observed at the 80-ppm level of Zn supplementation, which was twice the level recommended by NRC (40 ppm).
REFERENCES AND NOTES 1. Vallee, B. L., and D. S. Auld. 1980. The metallobiochemistry of zinc enzymes. A. Meister, ed. Adv. Enzymol. 283–429. 2. Brandeo-Neto, J., V. Stefan, B. Mendonca, W. Bloise, and A. Castro. 1995. The essential role of zinc in growth. Nutr. Res. 15:335–358. 3. Kidd, M. T., P. R. Ferket, and M. A. Qureshi. 1996. Zinc metabolism with special reference to its role in immunity. World’s Poult. Sci. J. 52:309–323. 4. Blamberg, D. L., U. B. Blackwood, W. C. Supplee, and G. F. Combs. 1960. Effect of zinc deficiency in hens on hatchability and embryonic development. Proc. Soc. Exp. Biol. Med. 104:217–220. 5. Sandoval, M., P. R. Henry, C. B. Ammerman, R. D. Miles, and R. C. Littell. 1997. Relative bioavailability of supplemental inorganic zinc sources for chicks. J. Anim. Sci. 75:3195–3205. 6. Bartlett, J. R., and M. O. Smith. 2003. Effects of different levels of zinc on the performance and immune competence of broilers under heat stress. Poult. Sci. 82:1580–1588. 7. Dardenne, M., and J. M. Bach. 1993. Rationale for the mechanism of zinc interaction in the immune system. Pages 501–509 in Nutrient Modulation of the Immune Response. S. Cunningham-Rundles, ed. Marcel Dekker Inc., New York, NY. 8. National Research Council. 1994. Nutrient Requirements of Poultry, 9th ed. Natl. Acad. Press, Washington, DC. 9. Stahl, J. L., M. E. Cook, and M. L. Sunde. 1986. Zinc supplementation: Its effect on egg production, feed conversion, fertility and hatchability. Poult. Sci. 65:2104– 2109. 10. Burrell, A. L., W. A. Dozier, A. J. Davis, M. M. Compton, M. E. Freeman, P. F. Vendrell, and T. L. Ward. 2004. Responses of broilers to dietary zinc concentrations and sources in relation to environmental implications. Br. Poult. Sci. 45:255–263. 11. Bertuzzi, S., G. Manfreda, and A. Franchini. 1998. Influence of dietary inorganic zinc and vitamin E on broiler immune response. Nuovi aspetti della profilassi vaccinale in avicoltura. XXXVI Convegno della Societa Italiana di Patalogia Aviares, Forli, 25–26 September 1997. SelezioneVeterinaria. No. 8–9:627–636. 12. Kidd, M. T., M. A. Qureshi, P. R. Ferket, and L. N. Thomas. 2000. Turkey hen zinc source affects progeny immunity and disease resistance. J. Appl. Poult. Res. 9:414–423. 13. Hubbards, National Regional Office for Asia and Pacific, Bangkok, Thailand. 14. Watson, L. T., C. B. Ammerman, S. M. Miller, and R. H. Harms. 1970. Biological assay of inorganic manganese for chicks. Poult. Sci. 49:1548–1554. 15. Digestion system, Tecator 2000, Perstorp Analytical Tecator, Hoganas, Sweden. 16. Atomic absorption spectrophotometer, Perkin-Elmer model AA 100, Perkin-Elmer Life and Analytical Sciences, Shelton, CT.
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increased from 20 to 160 ppm. The cell-mediated immunity was possibly related to the production of interleukin-2, which was supported by higher Zn level in the diet [3]. It was possible that the cell-mediated response in our study was more intense at 80 and 160 ppm than with lower levels, probably due to higher production of interleukin-2. The weights of spleen and bursa were influenced by the dietary Zn levels (Table 4). They were significantly (P ≤ 0.05) heavier in birds fed supplemental Zn at 40 ppm compared with lower levels. Further increase of Zn to 80 ppm and above did not have any additional advantage in improving the size of either organ, which supported better humoral and cell-mediated responses. These findings were similar to those observed by Bartlett and Smith [6]. On the other hand, low levels of supplemental Zn showed a relative reduction in the size of lymphoid organs with the possible decrease in T-cell function [3]. The ratio between heterophils and lymphocytes, an index of stress in birds, was significantly (P ≤ 0.05) wider when the diets were not supplemented with Zn. However, a perceptible decline in the ratios was observed with the inclusion of Zn at the 40-ppm level, which was significantly (P ≤ 0.05) better than lower levels. Higher Zn levels had similar effect as that of 40 ppm, suggesting that supplementation of Zn was useful in reducing stress in young broilers. Zinc is required for the normal development of lymphocytes, and a deficiency of Zn leads to thymocyte depletion in the thymus and reduction in peripheral T-cell numbers and T-cell helper functions [3]. Probably Zn supplemented at 40 ppm was adequate to support optimum development of lymphocytes, which alleviated stress, as observed from the present study.
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23. Underwood, E. J. 1981. The mineral nutrition of livestock. 2nd ed. Commonwealth Agriculture Bureau, Slough, UK. 24. Henry, P. R., C. B. Ammerman, and R. D. Miles. 1987. Effect of dietary zinc on tissue mineral concentration as a measure of zinc bioavailability in chicks. Nutr. Rep. Int. 35:15–23. 25. Sandoval, M., P. R. Henry, X. G. Luo, R. C. Littell, R. D. Miles, and C. B. Ammerman. 1998. Performance and tissue zinc and metallothionein accumulation in chicks fed a high dietary level of zinc. Poult. Sci. 77:1354–1363. 26. Harland, B. F., M. R. S. Fox, and B. E. Fry Jr. 1975. Protection against zinc deficiency by prior excess dietary zinc in young Japanese quail. J. Nutr. 105:1509–1518. 27. Wellinghausen, N., M. Martin, and L. Rink. 1997. Zinc inhibits IL-I dependent T cell stimulation. Eur. J. Immunol. 27:2529–2535.
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17. Wegmann, T. G., and O. Smithies. 1966. A simple hemagglutination system requiring small amounts of red cells and antibodies. Transfusion 6:67–73. 18. Gross, W. B., and P. B. Siegel. 1983. Evaluation of the heterophil to lymphocyte ratio as a measure of stress in chickens. Avian Dis. 27:972–979. 19. Snedecor, G. W., and W. G. Cochran. 1989. Statistical Methods. Oxford and IBH Publishing Company, New Delhi, India. 20. Duncan, D. B. 1955. Mutiple range and F test. Biometrics 11:1–42. 21. Collins, N. E., and E. T. Moran, Jr. 1999. Influence of supplemental manganese and zinc on live performance and carcass quality of broilers. J. Appl. Poult. Res. 8:222–227. 22. Emmert, J. L., and D. H. Baker. 1995. Zinc stores in chickens delay the onset of zinc deficiency symptoms. Poult. Sci. 74:1011–1021.
JAPR: Research Report
Effects of Higher Levels of Zinc Supplementation on Performance, Mineral Availability, and Immune Competence in Broiler Chickens G. Shyam Sunder,1 A. K. Panda, N. C. S. Gopinath, S. V. Rama Rao, M. V. L. N. Raju, M. R. Reddy, and Ch. Vijay Kumar
Primary Audience: Nutritionists, Researchers SUMMARY Two feeding trials were simultaneously conducted utilizing 210 and 140 1-d-old commercial broiler chicks for evaluating the effect of Zn supplementation on performance, mineral availability (experiment I), and immune response (experiment II), respectively, up to 4 wk of age. A basal diet was formulated using corn-soybean meal without supplemental Zn. Six test diets were prepared in which the basal diet was supplemented with Zn at 10, 20, 40, 80, 160, or 320 ppm using ZnSO4ⴢ7H2O. Diets were fed ad libitum to broilers in cages from 8 to 28 d of age. Supplemental Zn did not influence body weight gain, feed intake, feed efficiency, ready-to-cook yields, leg scores, or tibia weight at 4 wk of age. Tibia ash was significantly lower only at 320 ppm level. The percent Ca and P in tibia increased steadily with supplemental Zn up to 80 and 40 ppm, respectively, and decreased thereafter. A linear increase in Zn deposition was observed in bone, liver, and kidney with Zn supplementation. The humoral and cell mediated immune responses were significantly higher at the 80-ppm level compared with lower levels. The heterophil and lymphocyte ratios were narrow, indicating less stress with Zn at 40 ppm and above. The weights of bursa and spleen were higher at 40 ppm compared with lower levels. This study showed that the basal diet Zn (29 ppm) was adequate to support optimum performance of broilers. For better mineral retention, immune response, and alleviation of stress, 80 ppm of Zn was required up to 4 wk of age. Key words: zinc, performance, mineral availability, immune competence, broiler chicken 2008 J. Appl. Poult. Res. 17:79–86 doi:10.3382/japr.2007-00029
DESCRIPTION OF PROBLEM Zinc plays a vital role in numerous biological processes as a component of many enzymes [1]. It is essential for growth, skeletal development [2], and immune competence [3]. In poultry, Zn deficiency causes reduction in weight gain, 1
Corresponding author: [email protected]
skeletal malformations, poor bone mineralization, and immunological dysfunctions [3, 4]. Therefore, Zn is often supplemented in practical poultry diets to elicit a positive response in broiler chickens, particularly during early age. Tissue uptake of Zn in chicks is linearly related to Zn levels in the diet [5, 6]. The Zn was found
Downloaded from http://japr.oxfordjournals.org/ at Serials Section, Dixson Library on October 29, 2014
Project Directorate on Poultry, Rajendranagar, Hyderabad-30, India
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MATERIALS AND METHODS Two experiments were simultaneously conducted to evaluate the influence of Zn at levels that were higher than those recommended by NRC [8] on growth performance, mineral availability, and immune response in commercial broilers up to 4 wk of age. Experiment 1 Stock, Husbandry, and Experimental Diets. Two hundred ten 1-d-old commercial broiler chicks of both sexes [13] were equally distributed into 7 treatment groups with 6 replicates of 5 chicks each. The chicks were wing-banded individually and reared under uniform management care in stainless steel battery brooders (60 cm × 75 cm × 45 cm for 5 chicks) with raised wire floor in an open-sided house. They were provided with a rearing temperature of 34 ± 1°C up to 7 d of age and gradually reduced to 26 ± 1°C by 21 d of age after which no supportive heat was provided. The birds were vaccinated against Marek’s disease, Newcastle disease, and infectious bursal disease following the prescribed schedule. A basal diet was formulated with corn-soybean meal without Zn supplementation (Table 1). In addition, 6 experimental diets were prepared by adding measured quantities of
Table 1. Ingredient and nutrient composition of the basal diet (%) Ingredient Yellow maize Soybean meal Di-calcium phosphate Shell grit Common salt Choline chloride DL-Methionine Vitamins1 Trace minerals2 Coccidiostat Antibiotics Starch3 Nutrient composition (%) Metabolizable energy4 (kcal/kg) Crude protein5 Lys Met Calcium5 Nonphytate phosphorus5
Quantity 59.55 35.00 1.70 1.80 0.50 0.26 0.26 0.04 0.10 0.05 0.05 0.69 2,878 22.05 1.19 0.50 1.09 0.45
1
Supplies per kilogram of diet: vitamin A, 16,500 IU; vitamin D3, 3,150 ICU; vitamin E, 12 mg; vitamin K, 2 mg; vitamin B1, 1.2 mg; vitamin B2, 10 mg; vitamin B6, 2.4 mg; vitamin B12, 12 g; niacin, 18 mg; pantothenic acid, 12 mg. 2 Mn, 60 mg; Fe, 60 mg; Cu, 10 mg; I, 1.2 mg. 3 ZnSO4ⴢ7H2O salt of AR grade was used at appropriate levels quantitatively replacing starch by weight to maintain Zn supplemental levels between 0 and 320 ppm in test diets. 4 Calculated ME value. 5 Analyzed values.
ZnSO4ⴢ7H2O (analytical grade) to the basal diet for providing Zn supplementation at 10, 20, 40, 80, 160, or 320 ppm. Zinc was included in test diets at the expense of starch, whereas all the other feed ingredients remained unaltered. The diets were offered ad libitum to the respective groups from 8 to 28 d of age. The basal diet was fed to all chicks from 1 to 7 d of age for depleting the available Zn reserves. Body Weight, Feed Efficiency, and Leg Abnormality Scores. Individual body weights and feed intake by replicate were recorded at weekly intervals. Feed conversion efficiency was calculated as the ratio between feed consumed and weight gained. At the end of each week, leg abnormality scores were recorded on every bird by examining the hock joint for deformity [14]. Hock joints were assigned scores in the range of 1 to 5, depending upon the severity of deformity. The normal hock joint was scored as 1; slight swelling of tibiometatarsal joint as 2; marked swelling as 3; swelling of the joint with
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to be essential for normal functioning of the immune system by increasing the counts of thymocytes and peripheral T cells, an activity of natural killer cells [7]. It also enhances the production of neutrophils and antibodies, in addition to improving the functions of macrophages [3]. Because Zn supplementation was found to be essential, the NRC [8] recommended 40 ppm for broiler chickens, which appeared to be based on the results that considered growth performance as the only criterion [9, 10]. However, higher Zn levels (60 to 180 ppm) produced better immune status in broiler chickens [6, 11, 12], but clear information on Zn requirements for growth, mineral bioavailability, and immune response is scanty. Therefore, the present experiment was undertaken to study the effect of higher Zn levels on these factors in commercial broiler chickens during the juvenile period.
SHYAM SUNDER: ZINC IN BROILER CHICKEN DIET
the digested samples were filtered, diluted to required volume, and then analyzed for Zn content [16]. Experiment 2 Simultaneously, a second experiment was conducted with one hundred forty 1-d-old commercial broiler chicks [13] employing the same levels of Zn as in experiment I. Chicks were equally distributed into 7 treatment groups with 4 replicates of 5 chicks each. Feeding practices, duration of experiment, and management conditions were similar to experiment 1. Humoral and cell-mediated immune response, weights of bursa and spleen, and the ratios between heterophils and lymphocytes were determined at 4 wk of age. Humoral Immune Response. Sheep red blood cells, a nonpathogenic antigen, was used for evaluating the humoral immune response in broiler chickens. Two birds (1 male and 1 female) from each replicate (8 birds/treatment) were injected with 0.1 mL of 0.5% SRBC suspension into brachial vein on the d 22, and blood samples were collected on d 5 of postinoculation. Subsequently, microhemagglutination activity of serum was estimated, and the antibody titers (log2) were measured following the standard procedure [17]. Cellular Immune Response. The cell-mediated immunity was assessed (8 birds/treatment) by measuring the hypersensitivity response of cutaneous basophils (CBH) to phytohemagglutinin-P. On d 28, phytohemagglutinin-P (100 g suspended in 0.1 mL of normal saline solution) was injected intradermally into chick wattles. The wattle thickness (mm) was measured with constant tension micrometer prior to (T0) and 24 h after injection (T24). The CBH response was calculated as posttreatment as the percentage of the pretreatment thickness of wattles using the following formula: CBH response = [(T24 ÷ T0) × 100]. Heterophil-Lymphocyte Ratio and Weight of Lymphoid Organs Blood smears were prepared from the birds of 1 replicate of each test group on d 22 prior to inoculation of SRBC and stained with MayGreenwald-Giemsa stain [18]. Heterophils and
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symptoms of slipped achilles tendon as 4, and swelling with marked degree of slipped tendon as 5. Ready-to-Cook Yield. On d 29, six birds (3 males and 3 females) from each treatment were killed by cervical dislocation, and data on readyto-cook yields were collected and expressed as grams per kilogram of live weight. The tibia, liver, and kidney were collected from the killed birds for estimation of Zn retention in tissues. Zn in Feed Sample. The Zn content in feed was determined by collecting a 2-g sample from the basal diet and finely ground to digest at 120°C using 5 mL of concentrated HNO3 for 1 h using the Tecator digestion system-2000 [15]. The digested sample was cooled and 3 mL of 70% HClO4 was added for further digestion at 200°C. The process continued until the contents appeared clear and colorless. A blank was also simultaneously run along with the test sample. The digested sample was carefully filtered into a volumetric flask. The contents of digestion tube were repeatedly washed with deionized water to obtain complete extract of the minerals. The samples were aspirated into an atomic absorption spectrophotometer [16] to determine the Zn content. Zn in Tibia. The right and left tibiae from each bird were pooled group-wise and pressurecooked for 1 h. They were cleaned manually, clearing off the attached muscle and cartilage, washed with distilled water, and oven dried. The tibiae were then weighed and ashed in a muffle furnace at 600 ± 5°C for 4 h. The total ash was estimated on percent weight basis. Approximately 0.2 g of ash sample from each replicate was solubilized in 5 mL of 50% HCl and the mineral extract was filtered into a volumetric flask. The extract was then diluted using deionized water to the required volume, and Zn content was determined [16]. Zn in Liver and Kidney Tissues. The liver and kidney samples from 6 birds per group were collected and stored at −20°C. They were thawed, oven dried at 100°C for 24 h, and finely ground for mineral analysis. Approximately 0.5 g of ground sample was predigested with 5 mL of concentrated HNO3 and continued digestion for 1 h at 120°C using the Tecator system-2000 [15]. The contents were further digested using 30% H2O2 at 200°C for 45 min. A blank and
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Table 2. Effect of supplemental Zn on body weight, feed efficiency, edible meat yield, and leg scores of broiler chicks at 28 d of age Supplemental Zn (ppm)
Feed intake (g)
Feed/weight gain
Ready-to-cook yield (g/kg live weight)
Leg scores
1,208 1,167 1,216 1,219 1,192 1,202 1,202 6.87 0.47
2,134 2,097 2,097 2,168 2,109 2,154 2,194 9.67 0.06
1.84 1.88 1.80 1.85 1.84 1.87 1.90 0.01 0.281
665 675 658 671 668 668 669 0.203 0.494
2.30 2.23 2.40 2.27 2.30 2.33 2.23 0.07 0.99
lymphocytes were counted to a total of 60 cells and expressed as heterophil-to-lymphocyte ratio, which indicated the relative stress in birds due to dietary Zn levels. Again, on d 29, 6 birds from each treatment were killed by cervical dislocation, and the weights of bursa and spleen were recorded and expressed as grams per kilograms of live weight. Statistical Analysis. The main effects included those of Zn levels used in the experiment. Statistical analysis of body weight and leg scores was done on individual bird data, whereas replicate data were used for feed intake and feed conversion efficiency. Similarly, the statistical analysis for other traits was based on the number of samples indicated under materials and methods. One-way ANOVA under completely randomized design was used [19]. The mean differences were compared using Duncan’s multiple range test [20]. Significance was considered at P ≤ 0.05 level.
RESULTS AND DISCUSSION Performance and Leg Scores Supplementation of Zn to the basal diet at graded levels had no significant influence on body weight, feed intake, feed conversion efficiency, ready-to-cook yields, or leg scores at 4 wk of age (Table 2). This was in agreement with the findings of Burell et al. [10] who found that a practical diet of maize-soybean meal containing 30 ppm of Zn was adequate to support optimum performance during the initial 3 wk of age. Similarly, others [9] reported that a basal diet containing 37 ppm of Zn was optimum for
realizing good growth in chicks and additional supplementation had no added advantage. In another study [21] with male broiler chicks, little effect was found on body weight, feed efficiency, or livability with supplementation of Zn up to 6 wk of age because the basal diet contained 44 ppm of Zn. The present study also indicated that the Zn content available in cornsoybean diet (29 ppm) was adequate to sustain growth and other related parameters at par with those supplemented with Zn up to 4 wk of age. Obviously, this level was lower than that recommended by NRC [8] (40 ppm) for broiler chicks. The absence of any difference in performance between groups fed diets with or without supplemental Zn could be due to slower rate of Zn utilization, necessitating no further replenishment in diets [22]. Tibia Weight and Percent Ash, Ca, and P Supplemental Zn did not influence tibia weights at 4 wk of age (Table 3). Tibia ash content also did not vary with the level of Zn in feed up to 160 ppm, but declined significantly (P ≤ 0.05) at 320 ppm, indicating the possibility of negative effect of higher Zn levels on bone mineralization. The Ca retention in tibia was significantly (P ≤ 0.05) lower in birds fed no supplemental Zn and improved as its level increased in the diets. Maximum Ca retention was observed at the 80-ppm level, and receded thereafter, suggesting the limitations of Zn supplementation at lower and higher levels. Similarly, the P retention in the tibia was significantly (P ≤ 0.05) higher at 40 ppm compared with all other levels, except 80 ppm, indicating that Zn
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0 10 20 40 80 160 320 SEM P-value
Body weight (g)
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Table 3. Influence of dietary Zn level on bone and tissue mineralization in commercial broilers at 28 d of age Supplemental Zn (ppm)
Tibia ash (%)
Tibia Ca (%)
Tibia P (%)
3.15 3.27 3.20 3.05 3.04 3.24 3.18 0.04 0.64
54.22a 54.63a 54.20a 54.38a 54.75a 54.17a 52.70b 0.13 0.0002
35.77c 36.51b 36.57b 36.56b 37.41a 37.08ab 36.60b 0.09 0.0002
16.86d 17.11cd 17.19bc 17.59a 17.46ab 17.22bc 17.07cd 0.04 0.0001
0 10 20 40 80 160 320 SEM P-value
Means in a column with different superscripts are significant (P ≤ 0.05).
a–d
supplementation at specific levels was essential to optimize P concentration in bone. Higher levels of Zn in the diet appeared to interfere with the absorption and utilization of Ca and P particularly above 80 ppm [23], which was also observed in our study. Zn Uptake by Bone, Liver, and Kidney Tissues The Zn uptake by bone, liver, and kidney tissues is presented in Figure 1. A linear increase
(P ≤ 0.05) in Zn deposition was observed in bone (251 to 482 ppm), liver (117 to 143 ppm), and kidney tissues (88 to 113 ppm). Although, the rate of Zn retention in bone was higher (P ≤ 0.05) with each incremental raise, there was no difference between the 80- and 160-ppm and 160- and 320-ppm levels. However, the proportional increase in Zn deposition was high at lower levels of Zn supplementation (up to 40 ppm) compared with that between 80 to 160 ppm and 160 to 320 ppm. The Zn retention in
Figure 1. Variations in the deposition of Zn in tibia, liver, and kidney tissues of broiler chickens at 4 wk of age due to incremental increase in Zn supplementation to diets. a–fMeans on a line having no common letter are significantly different (P < 0.05).
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Tibia weight (g)
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Table 4. Influence of supplemental Zn on immune response, heterophil-to-lymphocyte (H/L) ratios, and weight of lymphoid organs Supplemental Zn (ppm)
H/L ratio
Bursa (g/kg)
Spleen (g/kg)
3.40b 3.40b 3.70b 4.40ab 5.30a 5.60a 5.30a 0.205 0.002
156 c 178 b 215 ab 224 ab 247 a 265 a 259 a 9.24 0.003
0.68a 0.64a 0.59a 0.46b 0.41b 0.47b 0.45b 0.016 0.0001
1.28b 1.26b 1.39b 2.31a 2.12a 2.19a 2.00a 0.077 0.0001
1.17c 1.13c 1.46bc 2.23a 2.39a 1.94ab 2.11a 0.087 0.0001
0 10 20 40 80 160 320 SEM P-value
Means in a column with different superscripts are significant (P ≤ 0.05). CBH = cutaneous basophils.
a–c 1
liver and kidney tissues also showed a consistent increase with Zn level in the diets. Henry et al. [24] and Sandoval et al [25] observed a linear accumulation of Zn in bone, liver, and kidney in chicks fed 0 to 1,500 ppm, which was similar to the results observed in our trial. Chicks fed no supplemental Zn had poor retention in bones. Therefore, for appreciating the bioavailability of Zn in broiler chicks, supplementation of higher levels of the mineral than that required for optimum performance is essential [22]. In this study, we also observed a consistent increase in Zn uptake by bone, liver, and kidney in relation to its concentration in diets. Bone accumulated more Zn than liver or kidney. This finding is further supported by the earlier report of Sandoval et al. [25]. It has been reported that Zn from bone can be utilized when Zn deficiency occurs; thus, bone appears to be a functional reserve of Zn, which could be readily mobilized for any immediate need [26]. However, it must be remembered that mineral metabolism is highly dynamic and that constant redistribution keeps occurring among the relevant tissues within the body, regardless of the dietary intake of an element [25]. Immune Competence The antibody titers against SRBC inoculation, a measure of humoral immune response, varied significantly (P ≤ 0.05) with the level of supplemental Zn at 4 wk of age (Table 4). Highest antibody titers were observed at 80 ppm, above which no further improvement in antibody
titers was noticed, implying that 80 ppm was adequate to provide optimum response. Similarly, others [11] reported a higher response of T-dependent humoral immunity in chickens fed 160 ppm of Zn. It was also observed [6] that higher antibody titers to SRBC with 181 ppm of Zn compared with lower levels (34 to 68 ppm) of supplementation. In our study, maximum immune response was observed at the 80 ppm level, which was comparatively lower than those reported earlier. Higher levels of Zn beyond the physiological limits of an individual may have no beneficial effect [27]. Thus, it could be hypothesized that the genetic stocks used in our study elicited better response at a lower physiological limits. Nevertheless, Zn plays an important role in immunomodulation by increasing the counts of thymocytes and peripheral T cells and also by enhancing the production of interferon, which might have been elevated by the dietary increases of Zn in our study [3]. The CBH response as measured by the thickness of wattles following the injection of phytohaemagglutinin also showed a steady increase (P ≤ 0.05) with Zn supplementation in diets (Table 4). Birds that received no supplemental Zn from the diet elicited the lowest CBH response. Although peak response was observed at the 160-ppm level, it was statistically not different from 20 to 80 ppm levels, suggesting the relevance of even lower levels of Zn supplementation in triggering immune response at par with higher levels. However, a numerical increase in CBH response was observed as the Zn levels
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Antibody titers (log2)
CBH1 response, % of pretreatment
SHYAM SUNDER: ZINC IN BROILER CHICKEN DIET
CONCLUSIONS AND APPLICATIONS 1. Zinc available from the corn-soybean diet (29 ppm) was adequate to maintain optimum growth, feed conversion efficiency, ready-to-cook yields, and normal hock joints to 4 wk of age. 2. Supplementation of Zn to broiler diets was essential for enhancing immune response, mineral retention, and alleviation of stress in broiler chickens up to 4 wk of age.
3. Maximum response in broiler chicken was observed at the 80-ppm level of Zn supplementation, which was twice the level recommended by NRC (40 ppm).
REFERENCES AND NOTES 1. Vallee, B. L., and D. S. Auld. 1980. The metallobiochemistry of zinc enzymes. A. Meister, ed. Adv. Enzymol. 283–429. 2. Brandeo-Neto, J., V. Stefan, B. Mendonca, W. Bloise, and A. Castro. 1995. The essential role of zinc in growth. Nutr. Res. 15:335–358. 3. Kidd, M. T., P. R. Ferket, and M. A. Qureshi. 1996. Zinc metabolism with special reference to its role in immunity. World’s Poult. Sci. J. 52:309–323. 4. Blamberg, D. L., U. B. Blackwood, W. C. Supplee, and G. F. Combs. 1960. Effect of zinc deficiency in hens on hatchability and embryonic development. Proc. Soc. Exp. Biol. Med. 104:217–220. 5. Sandoval, M., P. R. Henry, C. B. Ammerman, R. D. Miles, and R. C. Littell. 1997. Relative bioavailability of supplemental inorganic zinc sources for chicks. J. Anim. Sci. 75:3195–3205. 6. Bartlett, J. R., and M. O. Smith. 2003. Effects of different levels of zinc on the performance and immune competence of broilers under heat stress. Poult. Sci. 82:1580–1588. 7. Dardenne, M., and J. M. Bach. 1993. Rationale for the mechanism of zinc interaction in the immune system. Pages 501–509 in Nutrient Modulation of the Immune Response. S. Cunningham-Rundles, ed. Marcel Dekker Inc., New York, NY. 8. National Research Council. 1994. Nutrient Requirements of Poultry, 9th ed. Natl. Acad. Press, Washington, DC. 9. Stahl, J. L., M. E. Cook, and M. L. Sunde. 1986. Zinc supplementation: Its effect on egg production, feed conversion, fertility and hatchability. Poult. Sci. 65:2104– 2109. 10. Burrell, A. L., W. A. Dozier, A. J. Davis, M. M. Compton, M. E. Freeman, P. F. Vendrell, and T. L. Ward. 2004. Responses of broilers to dietary zinc concentrations and sources in relation to environmental implications. Br. Poult. Sci. 45:255–263. 11. Bertuzzi, S., G. Manfreda, and A. Franchini. 1998. Influence of dietary inorganic zinc and vitamin E on broiler immune response. Nuovi aspetti della profilassi vaccinale in avicoltura. XXXVI Convegno della Societa Italiana di Patalogia Aviares, Forli, 25–26 September 1997. SelezioneVeterinaria. No. 8–9:627–636. 12. Kidd, M. T., M. A. Qureshi, P. R. Ferket, and L. N. Thomas. 2000. Turkey hen zinc source affects progeny immunity and disease resistance. J. Appl. Poult. Res. 9:414–423. 13. Hubbards, National Regional Office for Asia and Pacific, Bangkok, Thailand. 14. Watson, L. T., C. B. Ammerman, S. M. Miller, and R. H. Harms. 1970. Biological assay of inorganic manganese for chicks. Poult. Sci. 49:1548–1554. 15. Digestion system, Tecator 2000, Perstorp Analytical Tecator, Hoganas, Sweden. 16. Atomic absorption spectrophotometer, Perkin-Elmer model AA 100, Perkin-Elmer Life and Analytical Sciences, Shelton, CT.
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increased from 20 to 160 ppm. The cell-mediated immunity was possibly related to the production of interleukin-2, which was supported by higher Zn level in the diet [3]. It was possible that the cell-mediated response in our study was more intense at 80 and 160 ppm than with lower levels, probably due to higher production of interleukin-2. The weights of spleen and bursa were influenced by the dietary Zn levels (Table 4). They were significantly (P ≤ 0.05) heavier in birds fed supplemental Zn at 40 ppm compared with lower levels. Further increase of Zn to 80 ppm and above did not have any additional advantage in improving the size of either organ, which supported better humoral and cell-mediated responses. These findings were similar to those observed by Bartlett and Smith [6]. On the other hand, low levels of supplemental Zn showed a relative reduction in the size of lymphoid organs with the possible decrease in T-cell function [3]. The ratio between heterophils and lymphocytes, an index of stress in birds, was significantly (P ≤ 0.05) wider when the diets were not supplemented with Zn. However, a perceptible decline in the ratios was observed with the inclusion of Zn at the 40-ppm level, which was significantly (P ≤ 0.05) better than lower levels. Higher Zn levels had similar effect as that of 40 ppm, suggesting that supplementation of Zn was useful in reducing stress in young broilers. Zinc is required for the normal development of lymphocytes, and a deficiency of Zn leads to thymocyte depletion in the thymus and reduction in peripheral T-cell numbers and T-cell helper functions [3]. Probably Zn supplemented at 40 ppm was adequate to support optimum development of lymphocytes, which alleviated stress, as observed from the present study.
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23. Underwood, E. J. 1981. The mineral nutrition of livestock. 2nd ed. Commonwealth Agriculture Bureau, Slough, UK. 24. Henry, P. R., C. B. Ammerman, and R. D. Miles. 1987. Effect of dietary zinc on tissue mineral concentration as a measure of zinc bioavailability in chicks. Nutr. Rep. Int. 35:15–23. 25. Sandoval, M., P. R. Henry, X. G. Luo, R. C. Littell, R. D. Miles, and C. B. Ammerman. 1998. Performance and tissue zinc and metallothionein accumulation in chicks fed a high dietary level of zinc. Poult. Sci. 77:1354–1363. 26. Harland, B. F., M. R. S. Fox, and B. E. Fry Jr. 1975. Protection against zinc deficiency by prior excess dietary zinc in young Japanese quail. J. Nutr. 105:1509–1518. 27. Wellinghausen, N., M. Martin, and L. Rink. 1997. Zinc inhibits IL-I dependent T cell stimulation. Eur. J. Immunol. 27:2529–2535.
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17. Wegmann, T. G., and O. Smithies. 1966. A simple hemagglutination system requiring small amounts of red cells and antibodies. Transfusion 6:67–73. 18. Gross, W. B., and P. B. Siegel. 1983. Evaluation of the heterophil to lymphocyte ratio as a measure of stress in chickens. Avian Dis. 27:972–979. 19. Snedecor, G. W., and W. G. Cochran. 1989. Statistical Methods. Oxford and IBH Publishing Company, New Delhi, India. 20. Duncan, D. B. 1955. Mutiple range and F test. Biometrics 11:1–42. 21. Collins, N. E., and E. T. Moran, Jr. 1999. Influence of supplemental manganese and zinc on live performance and carcass quality of broilers. J. Appl. Poult. Res. 8:222–227. 22. Emmert, J. L., and D. H. Baker. 1995. Zinc stores in chickens delay the onset of zinc deficiency symptoms. Poult. Sci. 74:1011–1021.
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