©2013 Poultry Science Association, Inc.
Different sources of dietary zinc for broilers submitted to immunological, nutritional, and environmental challenge M. M. Vieira,1 A. M. L. Ribeiro, A. M. Kessler, M. L. Moraes, M. A. Kunrath, and V. S. Ledur Department of Animal Science, Federal University of Rio Grande do Sul, Porto Alegre, RS 91540-000, Brazil Primary Audience: Nutritionists, Researchers, Processors, Producers SUMMARY A total of 1,248 one-day-old male Cobb chicks were used to evaluate the effect of different sources and levels of dietary zinc on the humoral immune response, zinc concentration in the tibia and carcass, footpad integrity, and performance. The birds were exposed to immunological (infectious bronchitis vaccine in the hatchery), nutritional (250 ppm of Cu in the diet) and environmental (reused litter sprayed daily with water; 1 to 21 d) challenges. The diets consisted of different levels of zinc from an organic source (10, 20, or 40 ppm of chelate Zn-HMTBa/ Mintrex Zn; Novus International Inc.), an inorganic source (10, 20, 40, or 100 ppm of zinc sulfate), and a negative control without zinc (0 ppm of Zn). The birds were immunized with 0.2 mg of BSA at 28 d and blood was collected at 41 d to measure antigen-specific antibody titer. Antibody titer, Zn concentration in the carcass, and performance (feed intake, BW gain, and feed conversion) were not affected by levels or sources of Zn. The absence of supplementary zinc in the diets significantly decreased its concentration in the tibia ash. Using 10 or 20 ppm of Zn from an organic source decreased the incidence of footpad lesions compared with the same levels of the inorganic source. It was concluded that both sources of Zn may be supplemented individually in broiler chicken diets, even in levels below 100 ppm, with no effect in carcass Zn concentration, performance, and humoral immunity, under the conditions tested. However, to keep footpad integrity, an organic source of Zn seems to be more efficient. Key words: footpad integrity, immune response, organic zinc, sulfate zinc 2013 J. Appl. Poult. Res. 22:855–861 http://dx.doi.org/10.3382/japr.2013-00753
DESCRIPTION OF PROBLEM Mineral supplementation of diets with organic or inorganic sources is required to meet the nutritional requirements of birds, because diets based only in natural ingredients produce clear trace mineral deficiencies [1]. Zinc requirements have been estimated to be 37 mg/kg from organic 1
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sources [2] and 84 mg/kg from inorganic sources [3]. The NRC [4] states that Zn requirements are 40 mg/kg, regardless of the source. The immune response of chickens may be modified by the level of zinc in the diet. Organic zinc supplementation had a positive effect on the immunological capacity of broilers by improving the levels of immunoglobulins IgA,
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856 IgM, and IgG [5], and may also improve the cellular response [6]. However, for this type of response, higher levels of supplementation may be required from organic [120 mg/kg (0 to 3 wk) and 90 mg/kg (4 to 6 wk)] [5] or inorganic (80 ppm to 5 wk) [7] sources. Zinc is absorbed with an efficiency from 15 to 60% [8] and the Zn plasma concentration varies according to its presence in tissues, being distributed among bone, muscle, liver, and pancreas [9]. Depending upon their chemical nature and complexation constants, organic chelators may affect the bioavailability of zinc [10]. The binding of zinc to low-molecular-weight ligands (amino acids) or chelators (e.g., EDTA) that can be absorbed also has a positive effect on zinc absorption because the solubility of zinc is increased [11]. The increase in the efficiency of Zn retention can be obtained with lower Zn supplementation in the diet, with consequent reduction of Zn concentration in the excreta [12]. Zinc excretion can also be reduced with the use of organic Zn in the diet [13]. The bone deposition of Zn increases linearly through the increment of Zn intake [1]. According to Ao et al. [14], dietary organic Zn led to higher accumulation of Zn in the tibia than Zn sulfate. Footpad integrity in broiler chickens is directly related to litter moisture [15], as an excess of moisture encourages the development of footpad lesions [16]. Zhao et al. [17] observed that 80 ppm of organic Zn in the diet improved footpad integrity and Rossi et al. [18] also observed higher tissue resistance with the organic source. This is due to the fact that Zn is involved in maintaining tissue integrity and is required for synthesis of structural proteins, such as collagen and keratin. Its deficiency can lead to severe dermatitis, especially on the bird’s footpad [9]. The aim of this study was to evaluate diets with different levels of organic or inorganic Zn for broiler chickens exposed to immunological, nutritional, and environmental challenges and its influence on humoral immunity, Zn concentration in the tibia and carcass, footpad integrity, and performance.
MATERIALS AND METHODS All procedures used in this experiment were approved by the Ethics Committee on Animal Use from the Federal University of Rio Grande
do Sul (Porto Alegre, Brazil). The experiment was conducted in a poultry house where 1,248 Cobb male chicks (42.7 ± 0.26 g) were housed in 48 pens from 1 to 41d. The inside temperature was controlled by gas hoods, fans, and curtain managements, to keep the thermoneutral zone during the entire experiment. The birds received water and food ad libitum and a lighting program of 16 h. The experimental diets were divided into 4 phases: prestarter: 1 to 7 d, starter: 7 to 21 d, grower: 21 to 35 d, and final: 35 to 41 d. Different levels of supplemental zinc in the diet (10, 20, or 40 ppm) from organic (chelated Zn-HMTBa [19]) and inorganic sources (zinc sulfate), a negative control without supplemental zinc (0 ppm of Zn) and a positive control with zinc sulfate at 100 ppm were tested. The experimental design was completely randomized, composed of 8 treatments and 6 replications. Each pen was an experimental unit, with 26 birds/pen and 10 birds/m2. The diets were produced using the same basal diet for all treatments to avoid mixing problems and changes in the nutritional composition, besides levels and sources of Zn, formulated by WUFFDA [20], according to the recommendations of Rostagno et al. [21] (Table 1). The mineral premix in the basal diet did not have Zn. Methionine presented in chelated ZnHMTBa was considered in the formulation, as shown in Table 2. The Zn level analyzed in the basal diet in each phase was, respectively, 58, 46, 41, and 38 mg/kg. Broilers were challenged nutritionally, environmentally, and immunologically. For nutritional challenge, all diets were formulated with the inclusion of high levels of Cu (250 ppm of Cu from copper sulfate) to antagonize Zn supplemented [20]. The environmental challenge consisted of utilizing reused litter (used 3 times), which was sprayed daily with 1 L of water/m2 from 1 to 21 d to provide an environment conducive to challenge footpad integrity and to result in the development of footpad lesions [16]. Also, birds were immunologically challenged by vaccination in the hatchery with a full dose of 3,150 particles [22] against infectious bronchitis, with the aim of promoting a decline in postvaccination performance, as Rubin et al. [23] demonstrated. Analyzed responses were antibody titer against BSA, measured by optical density; Zn
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Table 1. Ingredient composition and nutritional levels of the basal diets by phases Basal diet Item Ingredient (%) Corn Soybean meal, 46 CP Corn gluten meal, 60 CP Vegetable oil Monobicalcium phosphate Limestone Salt MHA 84%1 l-Lysine HCl Premix minerals, no Zn2 Premix treatments3 Premix vitamins4 l-Threonine Cl− choline Monensin 40% NaHCO3 Nutrient ME (kcal/kg) CP (%) Calcium (%) Available phosphorus (%) Sodium (%) Available lysine (%) Available Met + Cys (%) Available arginine (%) Available tryptophan (%) Available threonine (%) Choline (mg/kg) Na+K-Cl (mEq/kg)
Prestarter, d 1–7 56.34 32.07 5.0 1.55 1.80 1.44 0.45 0.315 0.48 0.15 0.10 0.04 0.07 0.05 0.028 0.12 3,000 23.00 1.0 0.46 0.23 1.30 1.02 1.19 0.27 0.86 1,500 200
Starter, d 7–21 57.27 34.91 — 3.32 1.71 1.35 0.51 0.27 0.26 0.15 0.10 0.04 0.02 0.06 0.028 — 3,050 21.00 0.95 0.44 0.23 1.16 0.82 1.19 0.22 0.80 1,500 211
Grower, d 21–35 59.81 32.39 — 3.63 1.48 1.37 0.46 0.25 0.23 0.15 0.10 0.04 — 0.06 0.028 — 3,100 20.00 0.90 0.40 0.20 1.08 0.78 1.12 0.20 0.72 1,450 200
Final, d 35–41 64.63 27.90 — 3.46 1.45 1.20 0.46 0.26 0.29 0.15 0.10 0.03 — 0.06 — — 3,150 18.40 0.82 0.39 0.20 1.03 0.75 1.0 0.18 0.66 1,350 178
1
MHA = methionine hydroxy analog. Contained (per kilogram of diet) 250 mg of Cu, 55 mg of Fe, 70 mg of Mn, 1.0 mg of I, and 0.4 mg of Se. 3 Contained (per kilogram of diet) ZnSO4·H2O: 27.5, 54.9, 109.9, or 274.7 mg (10, 20, 40, or 100 ppm); and chelated ZnHMTBa (Mintrex Zn; Novus International Inc., St. Charles, MO): 62.5, 125.0, or 250.0 mg (10, 20, or 40 ppm); Zn values in the basal diet: 58, 46, 41, and 38 mg/kg in each phase, respectively. 4 Contained (per kilogram of diet) 8,000 IU of vitamin A, 2,000 IU of vitamin D3, 30 mg of vitamin E, 2 mg of vitamin K, 2 mg of vitamin B1, 6 mg of vitamin B2, 2.5 mg of vitamin B6, 0.012 mg of vitamin B12, 0.08 mg of biotin, 15 mg of pantothenic acid, 35 mg of niacin, and 1 mg of folic acid. 2
Table 2. Treatment compositions according to the addition of different levels and sources of Zn (g/1,000 kg of diet) Treatment 0 ppm of Zn 10 ppm of Zn (ZnSO4·H2O) 20 ppm of Zn (ZnSO4·H2O) 40 ppm of Zn (ZnSO4·H2O) 10 ppm of Zn (Zn-HMTBa) 20 ppm of Zn (Zn-HMTBa) 40 ppm of Zn (Zn-HMTBa) 100 ppm of Zn (ZnSO4·H2O) 1
MHA 84%1
Starch
ZnSO4·H2O
Chelated Zn-HMTBa2
238.1 238.1 238.1 238.1 178.6 119.0 0.0 238.1
761.9 734.4 707.0 652.0 758.9 756.0 750.0 487.2
0.0 27.5 54.9 109.9 0.0 0.0 0.0 274.7
0.0 0.0 0.0 0.0 62.5 125.0 250.0 0.0
MHA = methionine hydroxy analog. Mintrex Zn (Novus International Inc., St. Charles, MO).
2
858 concentration in tibia ash at 21 d and in the carcass at 41 d; score of footpad lesions at 21 and 41 d; and performance responses (feed intake, weight gain, and FCR). The humoral immunity of birds was measured using BSA [24]. At 28 d, birds were immunized with a solution containing 0.2 mg of BSA dissolved in 0.2 mL of PBS and 0.2 mL of Incomplete Freund’s Adjuvant [25]. Three birds per replication were inoculated with this solution (0.4 mL/bird) in the pectoralis major muscle. Blood samples were taken from the inoculated birds at 41 d and antibody production to BSA was determined by ELISA in 96 plates [26]. Each plate blood sample was coated with 100 μL of BSA (500 µg/mL) in a buffer solution (0.2 M sodium carbonate, pH 9.2) and incubated at 4°C overnight. Serum samples were tested in duplicate (100 µL/pool), diluted 1:2,000 and incubated for 2 h. Then, the plates were incubated with 100 µL of IgG serum conjugated with peroxidase [27] diluted 1:8,000, for 30 min. We added 100 µL of a revealing solution [o-phenylenediamine (OPD)] [28]. After 15 min of rest in a darkroom, the reaction was blocked with 50 µL/plate with sulfuric acid (2 M). After each step, plates were washed 3 times with 100 µL of PBS Tween 0.1% and incubated in a moist chamber at 37°C. Serum from a BSA hyperimmune fowl was used as a positive control and serum of specific pathogen-free bird was used as a negative control. The anti-BSA IgG production was quantified by optical density measurements performed with an ELISA plate reader (Anthos 2020, Biochrome, Cambridge, UK) at 495 nm. The results were calculated by the average of the experimental samples tested in duplicate, discounting the average of the negative control duplicate samples. Zinc concentration in tibia ash was analyzed from 1 bird/replicate and 1 bird/replication was killed for carcass analysis (whole bird with feathers and organs). These tibia and carcass samples were burned in an oven at 550°C for 4 h and analysis of the ashes was done by atomic absorption spectrophotometry [29]. To evaluate the footpad integrity, footpad lesion scores were determined in 10 birds per replication at 21 and 41 d based on Angelo et al. [30] scores. Performance (feed intake, BW gain, and FCR) were evaluated weekly.
JAPR: Research Report The ANOVA was performed by generalized least squares for all quantitative responses using the SAS program [31] and means with normal distribution were tested at a probability of 5% through the Student-Newman-Keuls test. The optical density response was considered with normal distribution and was not transformed. Footpad lesion scores were analyzed by chisquared test.
RESULTS AND DISCUSSION The response of antibody titer against BSA was not affected by Zn level and source (Table 3). Some researchers found improvement in chicken antibody titers against SRBC with 80 ppm of Zn sulfate [7, 32]. However, Mohanna and Nys [12] found no effect of Zn levels (20 to 190 mg/kg) or source (Zn sulfate or Zn-methionine) on antibody titers against the same antigen. In the research of Moghaddam and Jahanian [6], supplementation of 40 mg of Zn-methionine/kg led to higher antibody titer against SRBC than the same dose of Zn sulfate or Zn oxide. Bartlett and Smith [33] found a higher antibody titer against SRBC only at higher levels of Zn complexed with amino acid (181 mg/kg). Different responses may be related to the type of antigen used in the immune protocol. The BSA, despite having been used to assess the development of immunocompetence in broilers by Mast and Goddeeris [34] and having been considered suitable for assessment of broiler humoral immunity by Ribeiro et al. [35], can induce a different immune response from other antigens used. Opposite results for immune response are commonly reported [36]. Because of the great variability and specificity of cells and molecules that regulate the immune system and their interactions, a certain level or source of a specific nutrient may not affect equally all types of immune response (innate, humoral, and cell-mediated immune response) [37]. Zinc concentration in the tibia was more sensitive in detecting differences between treatments than the concentration in the carcasses (Table 3). This response increased with the increase of Zn in the diet, showing that the negative control resulted in bones with less Zn. On the other hand, it was observed that the level at 100 ppm of inorganic Zn did not bring additional
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Table 3. Antibody titer against BSA (41 d), Zn concentration in tibia ash (21 d), and carcass (41 d) of broiler chickens supplemented with Zn in the diet Antibody titer2
Item1 Treatment 0 ppm of Zn 10 ppm of Zn (ZnSO4·H2O) 20 ppm of Zn (ZnSO4·H2O) 40 ppm of Zn (ZnSO4·H2O) 10 ppm of Zn (Zn-HMTBa) 20 ppm of Zn (Zn-HMTBa) 40 ppm of Zn (Zn-HMTBa) 100 ppm of Zn (ZnSO4·H2O) P-value SEM
0.904 0.658 0.982 0.739 0.901 0.825 0.930 0.992 NS 0.49
Tibia Zn (mg/kg)
Carcass Zn (mg/kg)
295.8c 381.1ab 396.7ab 409.8ab 364.7b 407.1ab 422.9a 404.4ab 0.001 4.55
368.6 342.8 308.5 333.2 374.7 392.2 341.7 315.0 NS 107.67
a–c
Means followed by different superscript letters within a column are significantly different (SNK P < 0.05). Chelated Zn-HMTBa: Mintrex Zn (Novus International Inc., St. Charles, MO). 2 Optical density measured by ELISA. 1
observed that litter moisture favored footpad lesions, as expected [15, 16]. After this age (21 d), the wetting procedure was interrupted, reducing the challenge by moisture spraying. In 41-d-old birds, supplementation with 20 ppm of organic Zn also showed lower incidence of footpad lesions (score zero = 90%). Zhao et al. [17] noted that using 80 ppm of organic Zn improved the footpad integrity. In our study, 20 ppm of organic Zn already resulted in lower footpad lesion incidence compared with the same level of the inorganic source. This result may be associated with the increasing tissue resistance provided by organic source supplementation [18]. Performance responses were not significantly different between levels and sources of Zn used in any phase or in the total experimental period
benefit, with Zn values statistically similar to the treatment with 40 mg/kg of organic or inorganic source. There were no differences in carcass Zn between sources or levels of Zn tested. The concentration of Zn in the body is strongly regulated by endogenous fecal Zn, which increases when the need for excretion increases [38], as in the situation of high Zn intake. Zn in the body is also controlled by plasma Zn concentration that regulates Zn in the tissues [39]. Footpad lesions in 21-d-old broilers (Table 4) showed a higher incidence of moderate inflammation (score I ≥60%) in diets without or with 10 ppm of Zn from both sources. However, when the birds were supplemented with 20 ppm of organic Zn, a lower incidence of footpad lesions (score zero = 56.7%) was noted. It was also
Table 4. Incidence of footpad lesions in broiler chickens at 21 and 41 d (0 = absence of lesion; 1 = moderate inflammation; 2 = severe inflammation) Footpad lesions at 21 d Item1 Treatment 0 ppm of Zn 10 ppm of Zn (ZnSO4·H2O) 20 ppm of Zn (ZnSO4·H2O) 40 ppm of Zn (ZnSO4·H2O) 10 ppm of Zn (Zn-HMTBa) 20 ppm of Zn (Zn-HMTBa) 40 ppm of Zn (Zn-HMTBa) 100 ppm of Zn (ZnSO4·H2O) Chi-squared test 1
Footpad lesions at 41 d
0
1
2
0
1
2
30.0 25.0 41.7 51.7 38.3 56.7 48.3 45.0
70.0 61.7 55.0 43.3 60.0 41.7 48.3 53.3 P < 0.001
0.0 13.3 3.3 5.0 1.7 1.6 3.4 1.7
51.7 51.7 66.7 66.7 60.0 90.0 70.0 65.0
43.3 48.3 30.0 31.7 38.3 10.0 23.3 31.7 P < 0.001
5.0 0.0 3.3 1.6 1.7 0.0 6.7 3.3
Chelated Zn-HMTBa: Mintrex Zn (Novus International Inc., St. Charles, MO).
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Table 5. Feed intake (FI), weight gain (WG), and FCR of broiler chickens in the starter phase (1 to 21 d) and in the total period (1 to 41d) Starter phase, 1 to 21 d Item1 Treatment 0 ppm of Zn 10 ppm of Zn (ZnSO4·H2O) 20 ppm of Zn (ZnSO4·H2O) 40 ppm of Zn (ZnSO4·H2O) 10 ppm of Zn (Zn-HMTBa) 20 ppm of Zn (Zn-HMTBa) 40 ppm of Zn (Zn-HMTBa) 100 ppm of Zn (ZnSO4·H2O) P-value SEM
FI (g)
WG (g)
1,339 1,323 1,325 1,387 1,345 1,328 1,347 1,361 NS 48.40
955 950 948 975 962 969 957 977 NS 25.29
Total period, 1 to 41 d
FCR (g/g) 1.403 1.393 1.398 1.422 1.398 1.370 1.410 1.393 NS 0.05
FI (g)
WG (g)
4,709 4,717 4,828 4,850 4,758 4,703 4,830 4,721 NS 179.62
2,891 2,899 2,954 2,925 2,924 2,909 2,937 2,894 NS 81.37
FCR (g/g) 1.629 1.627 1.634 1.658 1.627 1.617 1.643 1.631 NS 0.03
1
Chelated Zn-HMTBa: Mintrex Zn (Novus International Inc., St. Charles, MO).
(Table 5). There was no decrease in performance in the absence of supplemental Zn, which agrees with the report by Rossi et al. [18]. It is inferred that the negative control did not show worsened performance because (1) the birds could have eaten litter resulting in Zn recycling or (2) the Zn contained in the ingredients (basal diet) was enough for the bird’s performance. Note that Zn values in the basal diet (58, 46, 41, and 38 mg/kg in each phase, respectively) are close to NRC [4] requirements. In agreement with this idea, Shyam Sunder et al. [32] observed that a diet containing 29 ppm of Zn was sufficient for maximum bird performance. The diet with 100 ppm of Zn from ZnSO4·H2O did not differ from the other treatments, demonstrating that is possible to reduce Zn in the diet without losses in performance. The reduction in dietary Zn levels for broilers has been indicated [40] because excessive intake of Zn results in a higher concentration of this mineral in the manure [41]. In this study, neither the nutritional or immunological challenge increased Zn requirements for broilers. It is important to note that the challenges created were more closely related to practical situations than to academic ones, as vaccination and excessive use of mineral antagonists are common in poultry production. Responses to 100 ppm of Zn were no better than responses to levels of 20 or 40 ppm, regardless of the source used. The lack of differences observed between levels and sources of Zn in these responses evaluated showed that it is feasible to use either source in low concentration.
CONCLUSIONS AND APPLICATIONS
1. It is feasible to use less than 100 ppm of supplemental Zn (organic or inorganic) in broiler diets without affecting humoral immunity, zinc concentration in the carcass) and bird performance. 2. An organic source of zinc is more effective in maintaining the footpad integrity of birds. Inorganic Zn supplementation below 20 ppm decrease Zn concentration in the tibia and can damage footpad integrity.
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