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Effects of zinc source and phytate on zinc absorption by in situ ligated intestinal loops of broilers
Effects of zinc source and phytate on zinc absorption by in situ ligated intestinal loops of broilers Y. Yu,*†‡ L. Lu,*† R. L. Wang,*†1 L. Xi,§ X. G. Luo,*†2 and B. Liu*† *Mineral Nutrition Research Division, Institute of Animal Science, Chinese Academy of Agricultural Sciences, Beijing 100193, P. R. China; †State Key Laboratory of Animal Nutrition, Beijing 100193, P. R. China; ‡Institute for Nutritional Sciences, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200031, P. R. China; and §Department of Animal Science, North Carolina State University, Raleigh 27695-7621 The changes of Zn absorption as different Zn sources in the ileum were also observed and were similar to those in the duodenum and jejunum. There was a significant interaction (P < 0.05) between phytate levels and Zn sources on Zn absorption in 3 intestinal segments. The absorption percentage of Zn as ZnSO4 in the duodenum with a phytate-added group (10:1) was 40% (P < 0.05) lower than that of Zn as ZnSO4 without a phytateadded group. With the increasing phytate levels, the changes of Zn absorption as organic Zn sources in 3 intestinal segments were similar to those of ZnSO4 in the duodenum. However, the reduction of Zn absorption as organic Zn sources was lessened with the increasing complex strengths, and the highest absorption of Zn as Zn Pro A was observed. These results indicated that the absorption of Zn as organic Zn was more effective than that of Zn as inorganic Zn. The organic Zn absorption increased with the increasing complex strengths as well. The simple mixture of ZnSO4 with amino acid did not increase Zn absorption. In addition, the phytate could reduce Zn absorption as different Zn sources in 3 intestinal segments. Organic Zn sources could lessen the negative effect of phytate on Zn absorption, and then the absorption of organic Zn, especially Zn Pro A with strong complex strength, was more effective than inorganic Zn under the high levels of phytate.
Key words: absorption, broiler, organic zinc, phytate, small intestine 2010 Poultry Science 89:2157–2165 doi:10.3382/ps.2009-00486
INTRODUCTION Zinc is an essential trace element involved in many physiological functions because of the role it plays in ©2010 Poultry Science Association Inc. Received October 1, 2009. Accepted June 21, 2010. 1 Current address: Department of Animal Science, Guangdong Ocean University, Zhanjiang 524088, P. R. China. 2 Corresponding author: [email protected]
numerous metalloenzyme systems (Vallee and Falchuk, 1993; Gaither and Eide, 2001). The utilization of Zn has become an increasing concern as the requirement of Zn in diets for chickens has increased as a result of the extremely rapid growth rate of commercial broiler strains, Zn deficiency in feed, and many factors limiting Zn absorption in feed (Lönnerdal, 2000; Huang et al., 2007, 2009). Some studies have indicated increased bioavailability in organic Zn sources compared with inorganic sources in rats (Dong, 2001), pigs (Matsui et
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ABSTRACT Two experiments were conducted to investigate the effects of Zn source and phytate on Zn absorption in broilers. In experiment 1, eight different Zn sources, including ZnSO4, Zn Gly chelate, Zn Met chelate, and Zn amino acid C complex with the weak complex strength (Zn AA C), Zn protein B complex with the moderate complex strength (Zn Pro B), and zinc protein A complex with the strong complex strength (Zn Pro A), and the mixtures of ZnSO4 with either Gly or Met (Zn + Gly or Zn + Met, respectively) were used to study the effects of Zn sources on Zn absorption by in situ ligated intestinal loops of broilers. In experiment 2, 3 × 4 factorial arrangements of treatments involving 3 phytate levels and 4 Zn sources were used to investigate the effect of phytate on Zn absorption. Three molar ratios of phytate to Zn were 0, 2:1, and 10:1, respectively, and 4 Zn sources included ZnSO4, Zn AA C, Zn Pro B, and Zn Pro A, respectively. No differences (P > 0.05) were found among the absorption percentages of Zn as Zn + Gly, Zn + Met, and ZnSO4 in 3 intestinal segments. The absorption percentages of Zn as Zn Gly chelate, Zn Met chelate, Zn AA C, Zn Pro B, and Zn Pro A in the duodenum and jejunum were 29 to 129% higher (P < 0.05) than those of Zn as ZnSO4, Zn + Gly, and Zn + Met in the following order: Zn Pro A > Zn Pro B > Zn AA C > Zn Gly chelate or Zn Met chelate > ZnSO4, Zn + Met, or Zn + Gly.
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MATERIALS AND METHODS Birds and Diets Birds were handled in accordance with guidelines (Yang and Diao, 1999) approved by the Office of the Beijing Veterinarian. Arbor Acres commercial male broilers (Huadu Broiler Breeding Corp., Beijing, P. R. China) were used in 2 experiments and managed according to Arbor Acres guidelines. All birds were randomly housed in stainless steel suspended cages with fiberglass feeders and plastic waterers. The chicks used in experiment 1 were fed a corn-soybean meal basal diet (90.49 mg of Zn/kg of diet, Table 1) from d 1 to
21 but were fed a semipurified diet (12.58 mg of Zn/ kg of diet, Table 1) after d 21 to deplete the body Zn stores. The chicks used in experiment 2 were fed a corn-soybean meal basal diet (94.28 mg of Zn/kg of diet, Table 2) from d 1 to 14 but were fed a semipurified diet (11.84 mg of Zn/kg of diet, Table 2) after d 14 to increase body sensitivity to Zn under high phytate level. All other nutrients in these diets met or exceeded the NRC (1994) requirements for broilers. The birds were allowed ad libitum access to feed and tap water containing 102 µg of Ca/mL, 48 µg of Mg/mL, 0 µg of Cu/mL, 0 µg of Fe/mL, 0.06 µg of Mn/mL, and 2.9 µg of Zn/mL before d 21. After d 21, water was deionized and contained undetectable levels of Zn. At 28 d of age, after an overnight fast, 90 birds were weighed (1,172 g, means) and allotted randomly to 1 of 9 groups with 10 replicates of 1 bird each in experiment 1. Alternatively, 96 birds were weighed (1,288 g, means) and allotted randomly to 1 of 12 groups with 8 replicates of 1 bird each in experiment 2.
Experimental Design Experiment 1 was carried out to evaluate the effect of Zn sources on the intestinal absorption of Zn. The 8 different Zn sources evaluated included ZnSO4·7H2O, Zn Gly chelate [Zn Gly (Fenyahua Bioengineering Co., Changzhi, P. R. China)], Zn Met chelate (Fenyahua Bioengineering Co., Changzhi, P. R. China), Zn AA C [formation quotient value (Qf) = 6.48, 11.93% Zn (Zinpro Corp., Eden Prairie, MN)], Zn Pro B [Qf = 30.73, 13.27% Zn (Fenyahua Bioengineering Co.)], Zn Pro A [Qf = 944.02, 18.61% Zn (Alltech Inc., Nicholasville, KY)], and the mixture of ZnSO4 with either Gly or Met mixed at a 1:2 molar ratio of Zn to Gly or Met (Zn + Gly or Zn + Met, respectively). The ratio was the same as that of Zn Gly and Zn Met. Complex strength of the ligand and metal bond was evaluated via Qf index as described by Holwerda et al. (1995), and Qf was determined by polarography with a hanging Hg drop electrode (Ag/AgCl reference electrode, Potentiostat/ Galvanostat model 283; EG&G Inc., Gaithersburg, MD). The Qf values and solubility data in buffers at pH 2 or 5 were all from a previous study conducted in our laboratory (Liang, 2006). In addition, the control group without Zn added to the media was designed to exclude the effect of endogenous Zn. In the treatment groups of different Zn sources, 0.616 mmol of Zn/L (40 mg of Zn/L) was added to the media. This amount was chosen based on the broiler’s dietary Zn requirement (NRC, 1994) and was intended to approximate the amount found in the intestine. Glycine is the lowest in molecular weight among all amino acids. Thus, it might be hypothesized that Zn as Zn Gly chelate would be the most easily absorbed of all Zn amino acid chelates if the chelates could be absorbed into the mucosal cell and transferred across the gut wall in intact form. Methionine is the first-limiting amino acid for broilers and the
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al., 1996), chicks (Wedekind et al., 1992), sheep (Rojas et al., 1995), and dogs (Lowe et al., 1994a,b). In an earlier study conducted in our laboratory (Huang et al., 2009), the bioavailabilities of Zn in 3 commercial organic Zn products [Zn amino acid C complex with the weak complex strength (Zn AA C), Zn protein B complex with the moderate complex strength (Zn Pro B), and zinc protein A complex with the strong complex strength (Zn Pro A)] were compared. The Zn Pro B with a moderate complex strength was found to be the most available, followed by the Zn AA C with the weak complex strength, and then the Zn Pro A with the strong complex strength. The Zn AA C also exhibited similar availability as ZnSO4 based on pancreas metallothionein mRNA levels. Differences might be due to the different intestinal absorption rates or tissue metabolism of different Zn sources. It has been reported that the absorption of organic Zn differs from inorganic Zn in rats, pigs, dogs, and humans, but results are inconsistent (Hill et al., 1987a,b; Lowe et al., 1994a,b; Beutler et al., 1998). Research on the effect of organic Zn sources on Zn absorption in distinct intestinal segments of broilers has not been conducted. It has been widely accepted that the phosphate groups in inositol hexaphosphate can form strong and insoluble complexes with cations such as Zn, Cu, Mn, and Ca (Lönnerdal, 2000). Previous studies have found that the phytate has an inhibitory effect on Zn metabolism in chicks (Bafundo et al., 1984; Edwards and Baker, 2000). In addition, a study from our laboratory has indicated that organic Zn is more available than inorganic Zn under high dietary phytate level in broilers (Huang et al., 2007, unpublished data). However, to date, the interaction of dietary phytate and various Zn sources on Zn absorption in broiler chicks has not been elucidated. The objective of the present study was to evaluate the effect of Zn sources and phytate on the absorption of Zn in different intestinal segments using the technique of in situ ligated loops. The technique of in situ ligated loops has been shown to be a rapid and useful method to predict absorptive responses in rats and chicks (Hempe and Cousins, 1989; Yu et al., 2008).
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EFFECTS OF ZINC SOURCE AND PHYTATE ON ZINC ABSORPTION
Preparation of Perfusion Solutions According to the previous study from our laboratory (Ji et al., 2006a; Yu et al., 2008), the solutions injected into the duodenal and jejunal loops were buffered with 15.5 mmol/L of morpholinoethanesulfonic acid at pH 6, and the solutions injected into the ileal loops were buffered with 15.5 mmol/L of Tris at pH 7. Eight Zn sources of experiment 1 and 4 Zn sources of experiment 2 were added to the medium as treatments. Phenol red, acting as a nonabsorbable marker in the luminal medium, was used to correct the changes of Zn concentration resulting from water absorption or intestinal secretion (see below). The content of phenol red in perfusion solutions was 20 mg/L (Schedl et al., 1966; Yu et al., 2008). All chemicals used were biochemical-grade.
Ligated Loop Procedure Chickens were fasted overnight and anesthetized by wing venous injection of sumianxin (a complex anesthetic, 0.1 mL/kg of BW). The abdomen was opened by midline incision. The duodenum was incised 1 cm distal to the pyloric sphincter, the jejunum was incised just anterior to the remnant of the yolk stem, and the
ileum was incised just anterior to the ileocecal junction (Melvin, 1984). Plastic cannulas (interior diameter, 2.5 mm; exterior diameter, 4 mm; length, 2.5 cm) were inserted into 3 incisions and secured by sutures. Loose ligatures were then placed 12 cm distal to the above tight ligatures to separate different intestinal segments. The isolated segments were flushed out with 40 mL of warm saline followed by 20 mL of air to eliminate food residues and debris, and the loose ligature of each intestinal segment was tightened. Then, a 5-cm3 syringe without a needle was inserted into the cannula of each intestinal segment and the 3.5 mL of Zn dose was injected. After administration of the dose, the syringe was removed and the cannula was clamped by hemostatic forceps. The intestine was put back into the abdomen cavity. The detailed operational process was described by Yu et al. (2008). Table 1. Composition of 2 diets for 1- to 28-d-old broilers in experiment 1 Amount Item Ingredient, % Ground yellow corn Soybean meal Fish meal dl-Methionine Corn starch Casein Cellulose Soybean oil Calcium carbonate1 Calcium hydrogen phosphate1 Sodium chloride1 Micronutrients Nutrient composition ME, MJ/kg CP,4 % Lysine, % Methionine, % Methionine + cystine, % Calcium,4 % Nonphytate phosphorus, % Zinc,4 mg/kg 1Feed
Basal diet (d 1 to 21)
Semipurified diet (d 22 to 28)
54.65 34.82 3.50 0.18 3.60 1.26 1.30 0.30 0.392 12.58 21.66 1.30 0.57 0.95 1.04 0.45 90.49
66.00 23.00 5.01 1.50 1.50 1.12 0.30 1.573 13.22 19.33 1.69 0.62 0.72 0.90 0.40 12.58
grade before d 21 and reagent grade after d 21. the following per kilogram of diet: vitamin A (all-transretinol acetate), 13,500 IU; vitamin D3 (cholecalciferol), 3,600 IU; vitamin E (all-rac-α-tocopherol acetate), 33 IU; vitamin K (menadione sodium bisulfate), 6 mg; thiamin (thiamin mononitrate), 4.5 mg; riboflavin, 10.5 mg; vitamin B6, 6 mg; vitamin B12, 0.03 mg; calcium pantothenate, 18 mg; niacin, 60 mg; folic acid, 1.8 mg; biotin, 0.165 mg; choline (choline chloride), 700 mg; Cu (CuSO4·5H2O), 8 mg; Zn (ZnSO4·7H2O), 60 mg; Mn (MnSO4·H2O), 100 mg; Fe (FeSO4·7H2O), 80 mg; I (KI), 0.35 mg; and Se (Na2SeO3), 0.15 mg. 3Provided the following per kilogram of diet: vitamin A (all-transretinol acetate), 13,500 IU; vitamin D3 (cholecalciferol), 3,600 IU; vitamin E (all-rac-α-tocopherol acetate), 33 IU; vitamin K (menadione sodium bisulfate), 6 mg; thiamin (thiamin mononitrate), 4.5 mg; riboflavin, 10.5 mg; vitamin B6, 6 mg; vitamin B12, 0.03 mg; calcium pantothenate, 18 mg; niacin, 60 mg; folic acid, 1.8 mg; biotin, 0.165 mg; choline (choline chloride), 700 mg; K (KCl), 3,000 mg; Mg (MgSO4·7H2O), 600 mg; Cu (CuSO4·5H2O), 8 mg; Fe (FeSO4·7H2O), 80 mg; Mn (MnSO4·H2O), 100 mg; I (KI), 0.35 mg; and Se (Na2SeO3), 0.15 mg. 4Determined values. 2Provided
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commonly used amino acid complex in broiler production practice is Zn Met. Thus, 4 organic Zn sources of the chelate and complex of Zn with Gly and the chelate and complex of Zn with Met were used in this study. Based on the assumption that Qf values were correlated with bioavailability and absorptivity, another 3 organic Zn sources with different Qf values were studied in this experiment. All sources were obtained from independent distributors rather than directly from the product manufacturers (see above). Each treatment was repeated 10 times using 10 birds (1 time in 1 bird). The duodenum, jejunum, and ileum of each bird were used as 1 replication of corresponding intestinal segments. A procedure involving an optimal sampling time of 30 min after the injection of solutions, as observed in our previous study (Yu et al. 2008), was adopted. In experiment 2, 3×4 factorial arrangement of treatments in a completely randomized design was conducted to study the effect of phytate on the absorption of Zn from organic and inorganic sources. Three molar ratios of phytate to Zn added to the media were 0, 2:1, and 10:1, respectively. Four Zn sources, ZnSO4·7H2O, and 3 organic Zn sources of Zn AA C, Zn Pro B, and Zn Pro A with the high Zn absorptivity based on the results in experiment 1 were added to the media to provide 0.616 mmol of Zn/L. Zinc concentration in fluids in ligated loops injected with Zn-free solution after dosing were too low to be tested in experiment 1; therefore, the control without additional Zn added to the media was not used in this experiment. The intestinal segments and the sampling time were the same as those in experiment 1.
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Determination of Zn and Phenol Red Concentrations in Perfusion Solutions
Table 2. Composition of 2 diets for 1- to 28-d-old broilers in experiment 2 Amount Item Ingredient, % Ground yellow corn Soybean meal Fish meal dl-Methionine Corn starch Casein Cellulose Soybean oil Calcium carbonate1 Calcium hydrogen phosphate1 Sodium chloride1 Micronutrients Nutrient composition ME, MJ/kg CP,4 % Lysine, % Methionine, % Methionine + cystine, % Calcium,4 % Nonphytate phosphorus, % Zinc,4 mg/kg 1Feed
Basal diet (d 1 to 14)
Semipurified diet (d 14 to 28)
51.21 37.00 5.20 0.15 3.60 1.00 1.30 0.30 0.342 12.46 22.32 1.35 0.54 0.91 1.06 0.46 94.28
66.00 23.00 5.01 1.51 1.50 1.12 0.30 1.533 13.22 20.21 1.69 0.62 0.72 0.93 0.40 11.84
grade before d 21 and reagent grade after d 21. 2Provided the following per kilogram of diet: vitamin A (all-transretinol acetate), 13,500 IU; vitamin D3 (cholecalciferol), 3,600 IU; vitamin E (all-rac-α-tocopherol acetate), 33 IU; vitamin K (menadione sodium bisulfate), 6 mg; thiamin (thiamin mononitrate), 4.5 mg; riboflavin, 10.5 mg; vitamin B6, 6 mg; vitamin B12, 0.03 mg; calcium pantothenate, 18 mg; niacin, 60 mg; folic acid, 1.8 mg; biotin, 0.165 mg; choline (choline chloride), 1,000 mg; Cu (CuSO4·5H2O), 8 mg; Zn (ZnSO4·7H2O), 60 mg; Mn (MnSO4·H2O), 100 mg; Fe (FeSO4·7H2O), 80 mg; I (KI), 0.35 mg; and Se (Na2SeO3), 0.15 mg. 3Provided the following per kilogram of diet: vitamin A (all-transretinol acetate), 13,500 IU; vitamin D3 (cholecalciferol), 3,600 IU; vitamin E (all-rac-α-tocopherol acetate), 33 IU; vitamin K (menadione sodium bisulfate), 6 mg; thiamin (thiamin mononitrate), 4.5 mg; riboflavin, 10.5 mg; vitamin B6, 6 mg; vitamin B12, 0.03 mg; calcium pantothenate, 18 mg; niacin, 60 mg; folic acid, 1.8 mg; biotin, 0.165 mg; choline (choline chloride), 700 mg; K (KCl), 3,000 mg; Mg (MgSO4·7H2O), 600 mg; Cu (CuSO4·5H2O), 8 mg; Fe (FeSO4·7H2O), 80 mg; Mn (MnSO4·H2O), 100 mg; I (KI), 0.35 mg; and Se (Na2SeO3), 0.15 mg. 4Determined values.
VF =
AP =
CP (1)×VI CP (2)
CZn (1) ´ VI - CZn (2) ´ VF CZn (1)´ VI
´ 100,
where VF = final volume of perfusion solution (mL); CP (1) and CP (2) = initial and final concentration (mg/L) of phenol red, respectively; VI = initial volume (mL) of injected dose; AP = absorption percentage of Zn (%); CZn (1) and CZn (2) = Zn concentration (mmol/L) of initial and final perfusion solution, respectively.
Statistical Analysis The GLM procedure of SAS (SAS Institute, 2003) was used to compare Zn absorption data of treatment groups. Data from experiment 1 were analyzed by 1-way ANOVA, whereas the data from experiment 2 was subjected to 2-way ANOVA with a model that included Zn source and phytate level as main effects and the interaction of Zn source × phytate level. If the variances were significant, differences between mean values were ascertained using Duncan’s multiple range test method. Significance was determined to be P < 0.05.
RESULTS Effect of Different Zn Sources on Zn Absorption in Ligated Duodenum, Jejunum, and Ileum The absorption percentages of Zn as both inorganic and organic Zn sources in the ileum were about 1.3 to 2.8 times (P < 0.05) those in the duodenum and jejunum (Table 3). No differences (P > 0.05) were found between the absorption percentages of Zn in the duodenum and jejunum. These results indicated that the ileum was the main absorption site of Zn in the small intestines of broilers. It was consistent with the findings of our earlier study (Yu et al., 2008). As for Zn absorption of different Zn sources in the same intestinal segment, no differences (P > 0.05) were
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The 2-mL perfusion solutions were collected using the syringes at 30 min after the administration of the Zn dose and then were frozen at −20°C for analyzing the concentrations of Zn and phenol red. Zinc concentrations in diets, water, and perfusion solutions were determined by inductively coupled Ar plasma spectroscopy (Model IRIS Intrepid II, Thermal Jarrell Ash, Waltham, MA) as described by Lu et al. (2007). Approximately 0.5 g of feed sample or 0.5 mL of perfusion solution sample was weighed in duplicate and digested with 10 mL of HNO3 in a 50-mL microwave digestion vessel using a microwave (Mars 5, CEM Corporation, Matthews, NC) under the manufacturer’s instructions, then it was evaporated to near dryness and diluted to
3 mL with 2% HNO3 before analysis. Validation of the mineral analysis was conducted using bovine liver powder [GBW (E) 080193, National Institute of Standards and Technology, Beijing, P. R. China] as a standard reference material. The concentrations of phenol red in perfusion solutions were assayed by measuring absorbance at 520, 560, and 600 nm with a UV-visible spectrophotometer (Model Cary 100, Varian Inc., Palo Alto, CA; Steel and Cousins, 1985). Final volumes of solutions and absorption percentages of Zn were calculated according to equations outlined as follows:
EFFECTS OF ZINC SOURCE AND PHYTATE ON ZINC ABSORPTION
Effect of Zn Source and Phytate on Zn Absorption in Broiler Intestines The absorption percentages of Zn as both inorganic and organic Zn sources at different phytate levels in the jejunum and ileum were significantly higher (P < 0.05) than those in the duodenum except for those in the jejunum with Zn AA C treatment at a 2:1 phytate level (Table 4). No differences (P > 0.05) were observed in Zn absorption in both the ileum and jejunum except Zn AA C and Zn Pro B treatments without phytate added (P < 0.05). This result agreed with the result of experiment 1 in that the ileum was the main absorption site of Zn in broilers. There were significant interactions (P < 0.05) between phytate levels and Zn sources on Zn absorption in 3 intestinal segments. When the phytate was added at a 2:1 molar ratio of phytate to Zn, the absorption percentages of Zn as ZnSO4, Zn AA C, Zn Pro B, and Zn Pro A in the duodenum were not affected (P > 0.05); however, the values of 4 forms of Zn in the jejunum and ileum were significantly decreased (P < 0.05) except for the group of Zn Pro A in the jejunum compared with those of groups without the phytate. When the phytate was added at a 10:1 molar ratio of phytate to Zn, the absorption percentages of Zn as ZnSO4 in the duodenum, jejunum, and ileum of broilers were 40, 26, and 21% (P < 0.05) lower than those of Zn as ZnSO4 without the phytate group, respectively. The absorption percentages of Zn as Zn AA C in the duodenum, jejunum, and ileum of broilers were 50, 21, and 19% (P < 0.05) lower than those of Zn as Zn AA C without the phytate group, respectively. The absorption percentages of Zn as Zn Pro B in the duodenum, jejunum, and ileum of broilers were 35, 24, and 23% (P < 0.05) lower than those of Zn as Zn Pro B without the phytate
Table 3. Absorption percentages of zinc from different sources in ligated duodenum, jejunum, and ileum of 28-d-old broilers1 Zinc absorption, % Added zinc source2 ZnSO4·7H2O ZnSO4·7H2O + Gly ZnSO4·7H2O + Met Zn Gly chelate Zn Met chelate Zn AA C (weak) Zn Pro B (moderate) Zn Pro A (strong) Pooled SE P-value a–dMeans A,BMeans 1n
Duodenum
Jejunum
Ileum
Pooled SE
P-value
26.63d,B 25.79d,B 26.43d,B 42.02bc,B 38.76b,B 43.21ab,B 50.00ac,B 52.26a,B
27.61d,B 21.12d,B 24.29d,B 44.17ac,B 35.63b,B 41.00bc,B 44.11ac,B 47.85a,B
62.47ac,A 58.08c,A 60.24bc,A 68.13ab,A 68.23ab,A 65.37ac,A 67.32ab,A 69.89a,A
2.69 3.38 3.25 3.11 1.98 2.78 2.53 3.39
<0.0001 <0.0001 <0.0001 <0.0001 <0.0001 <0.0001 <0.0001 0.0002
3.35 <0.0001
2.42 <0.0001
2.88 0.0491
with different superscripts within the same column differ significantly (P < 0.05). with different superscripts within the same row differ significantly (P < 0.05).
= 10. AA C (weak) = zinc amino acid C complex with the weak complex strength [Qf = 6.48, 11.93% Zn (Zinpro Corp., Eden Prairie, MN)]; Zn Pro B (moderate) = zinc protein B complex with the moderate complex strength [formation quotient value (Qf ) = 30.73, 13.27% Zn (Fenyahua Bioengineering Co., Changzhi, P. R. China)]; Zn Pro A (strong) = zinc protein A complex with the strong complex strength [Qf = 944.02, 18.61% Zn (Alltech Inc., Nicholasville, KY)]. 2Zn
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detected among ZnSO4, the Zn + Gly mixture, and the Zn + Met mixture in 3 intestinal segments. In addition, the differences of Zn absorption between organic and inorganic sources decreased as the gut position became more distal to the pylorus. Zinc absorption percentages in the duodenum, Zn Gly chelate, and Zn Met chelate were 45 to 63% higher (P < 0.05) than ZnSO4, the Zn + Gly mixture, and the Zn + Met mixture, but there was no difference (P > 0.05) between Zn Met chelate, Zn Gly chelate, and Zn AA C. The absorption percentages of Zn as Zn Pro B and Zn Pro A were 88 to 102% higher (P < 0.05) than those of Zn as ZnSO4, the Zn + Gly mixture, and the Zn + Met mixture and 19 to 35% higher than those of Zn as Zn Met chelate (P < 0.05) and Zn Gly chelate (P ≤ 0.0961). No difference (P > 0.05) was observed among the absorption percentage of Zn as Zn AA C, Zn Pro A, and Zn Pro B. In the jejunum, the absorption percentages of Zn as Zn Gly chelate and Zn Met chelate were 29 to 110% higher (P < 0.05) and Zn AA C, Zn Pro B, and Zn Pro A were 49 to 129% higher (P < 0.05) than those of Zn as ZnSO4, the Zn + Gly mixture, and the Zn + Met mixture. The Zn absorption as Zn Pro A was significantly higher (P < 0.05) than that of Zn as Zn AA C and Zn Met chelate. The Zn absorption percentages of Zn as Zn Pro B and Zn Gly chelate were significantly higher (P < 0.05) than that of Zn as Zn Met chelate. No differences (P > 0.05) were found among other Zn sources. Finally, in the ileum, the absorption percentages of Zn as Zn Pro B, Zn Gly chelate, and Zn Met chelate were significantly higher (P < 0.05) than that of Zn as the Zn + Gly mixture. The absorption percentage of Zn as Zn Pro A increased significantly (P < 0.05) in comparison with those of Zn as the Zn + Gly mixture and the Zn + Met mixture. No differences (P > 0.05) were detected among other groups.
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Yu et al. Table 4. Absorption percentages (%) of zinc as different forms in the ligated duodenum, jejunum, and ileum of 28-d-old broilers perfused with different phytate levels1 Zinc absorption, % Item2
a–fMeans
Jejunum
Ileum
Pooled SE
P-value
82.81ab,B 82.42ab,C 84.71a,C 84.77a,B
92.41ab,A 89.07cd,B 93.30ab,B 94.08a,A
94.25ab,A 93.85ab,A 95.38a,A 95.61a,A
0.89 1.35 0.68 1.14
<0.0001 <0.0001 <0.0001 <0.0001
75.39b,B 78.62ab,B 78.66ab,B 80.48ab,B
86.61de,A 83.49e,AB 88.61cd,A 90.74bc,A
90.80cd,A 88.51de,A 89.97de,A 92.58bc,A
1.87 2.17 1.41 1.93
<0.0001 0.0148 <0.0001 0.0004
49.50cd,B 41.21d,B 55.32c,B 78.06ab,B 3.17
68.70f,A 70.87f,A 70.73f,A 86.54de,A 1.15
74.00f,A 75.69f,A 73.10f,A 87.48e,A 0.90
3.46 3.60 2.21 0.85
<0.0001 <0.0001 <0.0001 <0.0001
83.68 78.29 56.02 1.59
92.22 87.36 74.21 0.58
94.77 90.46 77.57 0.45
69.24 67.42 72.90 81.10 1.83
82.58 81.14 84.21 90.45 0.66
86.35 86.02 86.15 91.89 0.52
<0.0001 <0.0001 <0.0001
<0.0001 <0.0001 <0.0001
<0.0001 <0.0001 <0.0001
with different superscripts within the same column differ significantly (P < 0.05). with different superscripts within the same row differ significantly (P < 0.05).
A–CMeans 1n
= 8. AA C (weak) = zinc amino acid C complex with the weak complex strength [formation quotient value (Qf ) = 6.48, 11.93% Zn (Zinpro Corp., Eden Prairie, MN)]; Zn Pro B (moderate) = zinc protein B complex with the moderate complex strength [Qf = 30.73, 13.27% Zn (Fenyahua Bioengineering Co., Changzhi, P. R. China)]; Zn Pro A (strong) = zinc protein A complex with the strong complex strength [Qf = 944.02, 18.61% Zn (Alltech Inc., Nicholasville, KY)]. 2Zn
group, respectively. The absorption percentages of Zn as Zn Pro A in the jejunum and ileum of broilers were 8% (P < 0.05) and 9% (P < 0.05) lower than those of Zn as Zn Pro A without the phytate group, respectively. Moreover, Zn absorption of 4 sources in all 3 segments was impaired (P < 0.05) except for the group of Zn Pro A in the duodenum compared with those of groups at a 2:1 phytate level.
DISCUSSION It has been reported that organic Zn absorption is different from inorganic Zn absorption in rats, humans, and dogs, but the results were inconsistent (Hill et al., 1987a,b; Lowe et al., 1994a,b; Beutler et al., 1998). The discrepancies among the reports might be explained by differences in the various species of animals, the techniques used, and the quality of organic Zn used in these studies. It has been documented that the complex strength is the most important factor for evaluating the
quality of organic mineral sources, and the quality is related to the stabilization and absorption of organic mineral sources in the gut (Ashmead and Graff, 1985). Ji et al. (2006a) found that organic Mn was more efficiently absorbed than inorganic Mn in the broiler small intestine using the technique of in situ ligated loops. Moreover, the absorption of organic Mn with strong and moderate complex strengths was greater than that of the organic Mn, which was weak, and the absorption of organic Mn with strong complex strength was greater than that of the organic Mn with moderate complex strength. Results of the present study showed that the simple mixture of ZnSO4 with either Met or Gly did not increase the absorption of Zn in broiler small intestines unless they were chelated. The absorption percentages of organic Zn as Zn Gly chelate, Zn Met chelate, Zn AA C, Zn Pro B, and Zn Pro A in the duodenum, jejunum, and ileum were higher than those of Zn as ZnSO4. Moreover, the absorption of organic Zn with strong and moderate complex strengths in 3 intestinal
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Molar ratio of phytate to zinc 0 ZnSO4·7H2O Zn AA C (weak) Zn Pro B (moderate) Zn Pro A (strong) 2:1 ZnSO4·7H2O Zn AA C (weak) Zn Pro B (moderate) Zn Pro A (strong) 10:1 ZnSO4·7H2O Zn AA C (weak) Zn Pro B (moderate) Zn Pro A (strong) Pooled SE Molar ratio of phytate to zinc 0 2:1 10:1 Pooled SE Added zinc source ZnSO4·7H2O Zn AA C (weak) Zn Pro B (moderate) Zn Pro A (strong) Pooled SE P-value Phytate level Zinc source Zinc source × phytate level
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of organic Zn absorption was the same as that of inorganic Zn. Results of our previous study suggested that inorganic Zn absorption in the ileum occurred mainly by a nonsaturable diffusive pathway, which is different from that in the other 2 intestinal segments, depending on the regulation of Zn transporter expression (Yu et al., 2008). However, the detailed mechanism of organic Zn absorption in broiler intestines was now not known and needed to be further elucidated. The complex formation capability of cations and phytate is Cu > Zn > Co > Mn > Fe > Ca in turn (from strong to weak), and the stability of complexes is Zn > Cu > Ni > Co > Mn > Ca in turn (from strong to weak). Thus, Zn is affected the most significantly by the phytate (Lönnerdal, 2000). Bafundo et al. (1984) found that the addition of phytate to diets at levels from 0.6 to1.1% increased Zn concentration in broiler feces by 20 to 50% and the requirement for Zn. Turnlund et al. (1984) also demonstrated that the addition of 2.34 g of phytate to food with 15 mg of Zn reduced Zn absorption from 34 to 17.5% by using the stable isotope technology. Similarly, the Zn absorption increased from 27 to 45% when the phytate content in food decreased from 0.621 to 0.067 mmol/L. In agreement with these results, the present study indicated that the addition of phytate in perfusates reduced the absorption of Zn with different Zn sources in 3 intestinal segments of broilers. Greater reduction of Zn absorption was observed with the increasing molar ratio of phytate to Zn, with the most obvious changes in the duodenum. It was reported that the phytate and Zn molar ratio of 10:1 or above has been considered as the minimum ratio to reduce Zn absorption in rats (Morris and Ellis, 1980). In addition, Lönnerdal et al. (1988) found that a 6:1 molar ratio of phytate to Zn had a negative effect on Zn absorption in humans. However, Zn absorption in the current study reduced significantly when the molar ratio of phytate and Zn was 2:1, and greater reduction was observed when the molar ratio of phytate and Zn was 10:1. These observations might be due to the difference in the media from which the phytate was supplied. The phytate was supplied to the diet or food in early reports, whereas it was added to the perfusates in this study, therefore resulting in a more direct and sensitive inhibiting effect of phytate on Zn. In addition, the Zn absorption was significantly higher in experiment 2 than in experiment 1. This could be explained by the degree of Zn deficiency in chicks, which was higher in experiment 2 than in experiment 1 because the chicks in experiment 1 were fed a Zn-deficient diet for 1 wk, whereas the chicks of experiment 2 were fed a Zn-deficient diet for 2 wk. Thus, chicks in experiment 2 became more sensitive to Zn, resulting in a higher Zn absorption. Similarly, Smith and Cousins (1980) found that the absorption velocity of Zn was 627 nmol/h in rats fed with a diet lacking of Zn but reduced to 229 nmol/h in rats fed with a diet containing Zn. This result demonstrated that Zn deficiency in the diet increased
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segments was greater than that of the organic Zn with weak complex strength. These results were similar to the conclusions of Ji et al. (2006a, see above). Some evidence suggests positively charged Zn ions are not able to easily penetrate the negatively charged cell wall and therefore are transported across the mucosal surface via a low molecular weight ligand mediated process. Amino acids, proteins, and peptide fragments are the primary low molecular weight ligands during the absorptive process and are produced in sufficient amounts by the endogenous or dietary protein digestion. Zinc has a high affinity to these ligands. It is thought that the chelation of Zn by low molecular weight ligands leads to the formation of a soluble complex that can move across mucosal pores or the mucosal membrane by passive or active transport mechanisms or prevents Zn ions from precipitating at physiological pH and chelating by the phytate or other compounds. Finally, these Zn chelates can facilitate the intestinal absorption of Zn in vivo (Wapnir et al., 1983; Wapnir and Steel, 1986; Ashmead, 1993). Strength of Zn complex is important for facilitating absorption. Organic Zn with weak complex strength dissociated easily, but organic Zn sources with moderate and strong complex strengths were difficult to dissociate so they were absorbed easily (Huang et al., 2009). The above findings provided a good explanation for our data that the absorption of organic Zn chelated with amino acid or protein was greater than that of inorganic Zn and the absorption of organic Zn with strong and moderate complex strengths in the duodenum, jejunum, and ileum was greater than that of the organic Zn with weak complex strength. Ji et al. (2006b) reported that the absorption of Mn increased when the mixture of MnSO4 with Met or Gly mixed at a 1:2 molar ratio of Mn to Met or Gly, and Met as a ligand, was more effective in facilitating Mn absorption than Gly. However, the results of this study indicated that the simple mixture of ZnSO4 with Met or Gly at a 1:2 ratio did not increase Zn absorption and were not in line with those of Ji et al. (2006b). How the ligands affect the mineral absorption depend on many factors including the ratio of ligand to the minerals, the degree of chelation, the kind of ligand, and the techniques used (Seal and Heaton, 1983; Wapnir et al., 1983). The results of the main absorption site of Zn obtained in these experiments were consistent with the previous work from our laboratory (Yu et al., 2008). Once again, the experiments have clearly demonstrated the importance of the ileum as the main site at which Zn is absorbed. From the data of experiment 1, we found that there was a consistent change in Zn absorption of organic and inorganic Zn sources in 3 segments of broilers. The largest Zn absorption of different sources was observed in the ileum, and no differences were found between the Zn sources in it, but the Zn absorption percentages with organic Zn in the duodenum and jejunum were obviously greater than those of inorganic Zn. Based on these findings, it seemed that the mechanism
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ACKNOWLEDGMENTS Supported by the Earmarked Fund for Modern AgroIndustry Technology Research System (project no. nycytx-42-G2-04; Beijing, P. R. China), the Program of the National Natural Science Foundation of China (project no. 30871798; Beijing, P. R. China), and the Research Program of the Key Laboratory of Animal Nutrition (project no. 2004DA125184G0812; Beijing, P. R. China).
REFERENCES Ashmead, D. H. 1993. Page 32 in The Role of Metal Amino Acid Chelate. American Academic Press, Cleveland, OH. Ashmead, D. H., and D. J. Graff. 1985. Page 121 in Intestinal Absorption of Metal Ions and Chelates. Charles C. Thomas, Springfield, IL.
Bafundo, K. W., D. H. Baker, and P. R. Fitzgerald. 1984. Zinc utilization in the chick as influenced by dietary concentrations of calcium and phytate and by Eimeria acervulina infection. Poult. Sci. 63:2430–2437. Beutler, K. T., O. Pankewycz, and D. L. Brautigan. 1998. Equivalent uptake of organic and inorganic zinc by monkey kidney fibroblasts, human intestinal epithelial cells, or perfused mouse intestine. Biol. Trace Elem. Res. 61:19–31. Dong, X. H. 2001. Study on zinc absorption and metabolism of zinc amino acid chelate and effecting factors. Pages 73–82 in PhD Diss. Northeast Agricultural University, Harbin, P. R. China. Edwards, H. M., and D. H. Baker. 2000. Zinc bioavailability in soybean meal. J. Anim. Sci. 78:1017–1021. Gaither, L. A., and D. J. Eide. 2001. Eukaryotic zinc transporters and their regulation. Biometals 14:251–270. Hempe, J. M., and R. J. Cousins. 1989. Effect of EDTA and zincmethionine complex on zinc absorption by rat intestine. J. Nutr. 119:1179–1187. Hill, D. A., E. R. Peo Jr., and A. J. Lewis. 1987a. Effect of zinc source and picolinic acid on 65zinc uptake in an in vitro continuous flow perfusion system for pig and poultry intestinal segments. J. Nutr. 117:1704–1707. Hill, D. A., E. R. Peo Jr., and A. J. Lewis. 1987b. Influnce of picolinic acid on the uptake of 65zinc-amino acid complexes by the everted rat gut. J. Anim. Sci. 65:173–178. Holwerda, R. A., R. C. Albin, and F. C. Madsen. 1995. Chelation effectiveness of zinc proteinates demonstrated. Feedstuffs 67:12– 13. Huang, Y. L., L. Lu, S. F. Li, B. Liu, and X. G. Luo. 2009. Relative bioavailabilities of organic zinc sources with different chelation strengths for broilers fed a conventional corn-soybean meal diet. J. Anim. Sci. 87:2038–2046. Huang, Y. L., L. Lu, X. G. Luo, and B. Liu. 2007. An optimal dietary zinc level of broiler chicks fed a corn-soybean meal diet. Poult. Sci. 86:2582–2589. Ji, F., X. G. Luo, L. Lu, B. Liu, and S. X. Yu. 2006a. Effect of manganese source on manganese absorption by the intestine broilers. Poult. Sci. 85:1947–1952. Ji, F., X. G. Luo, L. Lu, B. Liu, and S. X. Yu. 2006b. Effects of manganese source and calcium on manganese uptake by in vitro everted gut sacs of broiler’s intestinal segments. Poult. Sci. 85:1217–1225. Liang, J. G. 2006. The study of characterization of organic zinc sources and their bioactivities and modes of action in cows. PhD Diss. Chinese Academy of Agricultural Science, Beijing, P. R. China. Lönnerdal, B. 2000. Dietary factors influencing zinc absorption. J. Nutr. 130:1378S–1383S. Lönnerdal, B., J. G. Bell, R. A. Burns, and C. L. Keen. 1988. Effect of phytate removal on zinc absorption from soy formula. Am. J. Clin. Nutr. 48:1301–1306. Lowe, J. A., J. Wiseman, and D. J. Cole. 1994a. Absorption and retention of zinc when administered as an amino-acid chelate in the dog. J. Nutr. 124(Suppl.):2572S–2574S. Lowe, J. A., J. Wiseman, and D. J. Cole. 1994b. Zinc source influences zinc retention in hair and hair growth in the dog. J. Nutr. 124:2575S–2576S. Lu, L., X. G. Luo, C. Ji, B. Liu, and S. X. Yu. 2007. Effect of manganese supplementation and source on carcass traits, meat quality, and lipid oxidation in broilers. J. Anim. Sci. 85:812–822. Matsui, T., T. Ishiguro, S. Suzaki, H. Yano, and S. Fujita. 1996. Supplementation of zinc as amino acid-chelated zinc for piglets. Page 754 in Proceedings of the 8th AAAP Animal Science Congress, Tokyo, Japan. Vol 2. Melvin, J. S. 1984. Page 359 in Duke’s Physiology of Domestic Animals. 10th ed. Cornell University Press, Ithaca, NY. Morris, E. R., and R. Ellis. 1980. Effect of dietary/zinc molar ratio on growth and bone zinc response of rats fed semipurified diets. J. Nutr. 110:1037–1045. NRC. 1994. Nutrient Requirements of Poultry. 9th rev. ed. Natl. Acad. Press, Washington, DC. Rojas, L. X., L. R. McDowell, R. J. Cousins, F. G. Martin, N. S. Wilkinson, A. B. Johnson, and J. B. Velasquez. 1995. Relative
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Zn absorption in the intestines of rats. When compared with inorganic Zn, the organic Zn sources with different complex strengths inhibited the chelate function by the phytate and therefore reduced the negative effect of the phytate on Zn absorption. Of the organic Zn sources, the reduced degree of Zn absorption of Zn Pro A under the high phytate level was the minimum because it was the most difficult to dissociate and chelate by the phytate in the intestines of broilers. However, Zn Pro B and Zn AA C were not as large as Zn Pro A in the effects of resisting the chelate by phytate of Zn; therefore, the Zn absorption reduced more than that of Zn Pro A. These results were in agreement with the conclusions of Dong (2001), who suggested that the disappearance of 65ZnCl2, 65Zn Lys, and 65Zn Met in the rat ligated duodenum was reduced by 64.44, 15.17, and 14.95%, respectively, when 1% phytate was added to the semipurified diet. These results indicated that the absorption of organic Zn was more effective than inorganic Zn under the high phytate level. In conclusion, the ileum was the main site of Zn absorption in the intestines of broilers. The absorption of organic Zn was more effective than that of inorganic Zn. Zinc absorption increased with the increasing complex strength with the order of Zn Pro A > Zn Pro B > Zn AA C > Zn Gly chelate or Zn Met chelate > ZnSO4, Zn + Met, or Zn + Gly in the duodenum and jejunum. The simple mixture of ZnSO4 with amino acid could not increase Zn absorption unless they were chelated. These results further indicated that Zn absorption was affected by the degree of chelation between ligands and minerals regardless of the ligands action in improving or restraining Zn absorption. Phytate could reduce the absorption of Zn as different Zn sources in 3 intestinal segments, and Zn absorption decreased with the increasing phytate levels, with the most obvious effects in the duodenum. The absorption of organic Zn, especially Zn Pro A, was higher than inorganic Zn with the high levels of phytate, further confirming the superior availability of organic Zn compared with inorganic Zn.
EFFECTS OF ZINC SOURCE AND PHYTATE ON ZINC ABSORPTION
Vallee, B. L., and K. H. Falchuk. 1993. The biochemical basis of zinc physiology. Physiol. Rev. 73:79–118. Wapnir, R. A., D. E. Khani, M. A. Bayne, and F. Lifshitz. 1983. Absorption of zinc by the rat ileum: Effects of histidine and other low-molecular-weight ligands. J. Nutr. 113:1346–1354. Wapnir, R. A., and L. Steel. 1986. Zinc intestinal absorption in rats: Specificity of amino acids as ligands. J. Nutr. 116:2171–2179. Wedekind, K. J., A. E. Hortin, and D. H. Baker. 1992. Methodology for assessing zinc bioavailability: Efficacy estimates for zinc-methionine, zinc sulfate, and zinc oxide. J. Anim. Sci. 70:178–187. Yang, Q. M., and Y. X. Diao. 1999. The Handbook for Raising of Broilers. China Agricultural University Press, Beijing, P. R. China. Yu, Y., L. Lu, X. G. Luo, and B. Liu. 2008. Kinetics of zinc absorption by in situ ligated intestinal loops of broilers involved in zinc transporters. Poult. Sci. 87:1146–1155.
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bioavailability of two organic and two inorganic zinc sources fed to sheep. J. Anim. Sci. 73:1202–1207. SAS Institute. 2003. SAS User’s Guide: Statistics. Version 9.0. SAS Institute Inc., Cary, NC. Schedl, H. P., D. Miller, and D. White. 1966. Use of polyethylene glycol and phenol red as unabsorbed indicators for intestinal absorption studies in man. Gut 7:159–163. Seal, C. J., and F. W. Heaton. 1983. Chemical factors affecting the intestinal absorption of zinc in vitro and in vivo. Br. J. Nutr. 50:317–324. Smith, K. T., and R. J. Cousins. 1980. Quantitative aspects of zinc absorption by isolated vascularly perfused rat intestine. J. Nutr. 110:316–323. Steel, L., and R. J. Cousins. 1985. Kinetics of zinc absorption by luminally and vascularly perfused rat intestine. Am. J. Physiol. 248:G46–G53. Turnlund, J. R., J. C. King, W. R. Keyes, and M. C. Michel. 1984. A stable isotope study of zinc absorption in young men: Effects of phytate and α-cellulose. Am. J. Clin. Nutr. 40:1071–1077.
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Key words: absorption, broiler, organic zinc, phytate, small intestine 2010 Poultry Science 89:2157–2165 doi:10.3382/ps.2009-00486
INTRODUCTION Zinc is an essential trace element involved in many physiological functions because of the role it plays in ©2010 Poultry Science Association Inc. Received October 1, 2009. Accepted June 21, 2010. 1 Current address: Department of Animal Science, Guangdong Ocean University, Zhanjiang 524088, P. R. China. 2 Corresponding author: [email protected]
numerous metalloenzyme systems (Vallee and Falchuk, 1993; Gaither and Eide, 2001). The utilization of Zn has become an increasing concern as the requirement of Zn in diets for chickens has increased as a result of the extremely rapid growth rate of commercial broiler strains, Zn deficiency in feed, and many factors limiting Zn absorption in feed (Lönnerdal, 2000; Huang et al., 2007, 2009). Some studies have indicated increased bioavailability in organic Zn sources compared with inorganic sources in rats (Dong, 2001), pigs (Matsui et
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ABSTRACT Two experiments were conducted to investigate the effects of Zn source and phytate on Zn absorption in broilers. In experiment 1, eight different Zn sources, including ZnSO4, Zn Gly chelate, Zn Met chelate, and Zn amino acid C complex with the weak complex strength (Zn AA C), Zn protein B complex with the moderate complex strength (Zn Pro B), and zinc protein A complex with the strong complex strength (Zn Pro A), and the mixtures of ZnSO4 with either Gly or Met (Zn + Gly or Zn + Met, respectively) were used to study the effects of Zn sources on Zn absorption by in situ ligated intestinal loops of broilers. In experiment 2, 3 × 4 factorial arrangements of treatments involving 3 phytate levels and 4 Zn sources were used to investigate the effect of phytate on Zn absorption. Three molar ratios of phytate to Zn were 0, 2:1, and 10:1, respectively, and 4 Zn sources included ZnSO4, Zn AA C, Zn Pro B, and Zn Pro A, respectively. No differences (P > 0.05) were found among the absorption percentages of Zn as Zn + Gly, Zn + Met, and ZnSO4 in 3 intestinal segments. The absorption percentages of Zn as Zn Gly chelate, Zn Met chelate, Zn AA C, Zn Pro B, and Zn Pro A in the duodenum and jejunum were 29 to 129% higher (P < 0.05) than those of Zn as ZnSO4, Zn + Gly, and Zn + Met in the following order: Zn Pro A > Zn Pro B > Zn AA C > Zn Gly chelate or Zn Met chelate > ZnSO4, Zn + Met, or Zn + Gly.
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MATERIALS AND METHODS Birds and Diets Birds were handled in accordance with guidelines (Yang and Diao, 1999) approved by the Office of the Beijing Veterinarian. Arbor Acres commercial male broilers (Huadu Broiler Breeding Corp., Beijing, P. R. China) were used in 2 experiments and managed according to Arbor Acres guidelines. All birds were randomly housed in stainless steel suspended cages with fiberglass feeders and plastic waterers. The chicks used in experiment 1 were fed a corn-soybean meal basal diet (90.49 mg of Zn/kg of diet, Table 1) from d 1 to
21 but were fed a semipurified diet (12.58 mg of Zn/ kg of diet, Table 1) after d 21 to deplete the body Zn stores. The chicks used in experiment 2 were fed a corn-soybean meal basal diet (94.28 mg of Zn/kg of diet, Table 2) from d 1 to 14 but were fed a semipurified diet (11.84 mg of Zn/kg of diet, Table 2) after d 14 to increase body sensitivity to Zn under high phytate level. All other nutrients in these diets met or exceeded the NRC (1994) requirements for broilers. The birds were allowed ad libitum access to feed and tap water containing 102 µg of Ca/mL, 48 µg of Mg/mL, 0 µg of Cu/mL, 0 µg of Fe/mL, 0.06 µg of Mn/mL, and 2.9 µg of Zn/mL before d 21. After d 21, water was deionized and contained undetectable levels of Zn. At 28 d of age, after an overnight fast, 90 birds were weighed (1,172 g, means) and allotted randomly to 1 of 9 groups with 10 replicates of 1 bird each in experiment 1. Alternatively, 96 birds were weighed (1,288 g, means) and allotted randomly to 1 of 12 groups with 8 replicates of 1 bird each in experiment 2.
Experimental Design Experiment 1 was carried out to evaluate the effect of Zn sources on the intestinal absorption of Zn. The 8 different Zn sources evaluated included ZnSO4·7H2O, Zn Gly chelate [Zn Gly (Fenyahua Bioengineering Co., Changzhi, P. R. China)], Zn Met chelate (Fenyahua Bioengineering Co., Changzhi, P. R. China), Zn AA C [formation quotient value (Qf) = 6.48, 11.93% Zn (Zinpro Corp., Eden Prairie, MN)], Zn Pro B [Qf = 30.73, 13.27% Zn (Fenyahua Bioengineering Co.)], Zn Pro A [Qf = 944.02, 18.61% Zn (Alltech Inc., Nicholasville, KY)], and the mixture of ZnSO4 with either Gly or Met mixed at a 1:2 molar ratio of Zn to Gly or Met (Zn + Gly or Zn + Met, respectively). The ratio was the same as that of Zn Gly and Zn Met. Complex strength of the ligand and metal bond was evaluated via Qf index as described by Holwerda et al. (1995), and Qf was determined by polarography with a hanging Hg drop electrode (Ag/AgCl reference electrode, Potentiostat/ Galvanostat model 283; EG&G Inc., Gaithersburg, MD). The Qf values and solubility data in buffers at pH 2 or 5 were all from a previous study conducted in our laboratory (Liang, 2006). In addition, the control group without Zn added to the media was designed to exclude the effect of endogenous Zn. In the treatment groups of different Zn sources, 0.616 mmol of Zn/L (40 mg of Zn/L) was added to the media. This amount was chosen based on the broiler’s dietary Zn requirement (NRC, 1994) and was intended to approximate the amount found in the intestine. Glycine is the lowest in molecular weight among all amino acids. Thus, it might be hypothesized that Zn as Zn Gly chelate would be the most easily absorbed of all Zn amino acid chelates if the chelates could be absorbed into the mucosal cell and transferred across the gut wall in intact form. Methionine is the first-limiting amino acid for broilers and the
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al., 1996), chicks (Wedekind et al., 1992), sheep (Rojas et al., 1995), and dogs (Lowe et al., 1994a,b). In an earlier study conducted in our laboratory (Huang et al., 2009), the bioavailabilities of Zn in 3 commercial organic Zn products [Zn amino acid C complex with the weak complex strength (Zn AA C), Zn protein B complex with the moderate complex strength (Zn Pro B), and zinc protein A complex with the strong complex strength (Zn Pro A)] were compared. The Zn Pro B with a moderate complex strength was found to be the most available, followed by the Zn AA C with the weak complex strength, and then the Zn Pro A with the strong complex strength. The Zn AA C also exhibited similar availability as ZnSO4 based on pancreas metallothionein mRNA levels. Differences might be due to the different intestinal absorption rates or tissue metabolism of different Zn sources. It has been reported that the absorption of organic Zn differs from inorganic Zn in rats, pigs, dogs, and humans, but results are inconsistent (Hill et al., 1987a,b; Lowe et al., 1994a,b; Beutler et al., 1998). Research on the effect of organic Zn sources on Zn absorption in distinct intestinal segments of broilers has not been conducted. It has been widely accepted that the phosphate groups in inositol hexaphosphate can form strong and insoluble complexes with cations such as Zn, Cu, Mn, and Ca (Lönnerdal, 2000). Previous studies have found that the phytate has an inhibitory effect on Zn metabolism in chicks (Bafundo et al., 1984; Edwards and Baker, 2000). In addition, a study from our laboratory has indicated that organic Zn is more available than inorganic Zn under high dietary phytate level in broilers (Huang et al., 2007, unpublished data). However, to date, the interaction of dietary phytate and various Zn sources on Zn absorption in broiler chicks has not been elucidated. The objective of the present study was to evaluate the effect of Zn sources and phytate on the absorption of Zn in different intestinal segments using the technique of in situ ligated loops. The technique of in situ ligated loops has been shown to be a rapid and useful method to predict absorptive responses in rats and chicks (Hempe and Cousins, 1989; Yu et al., 2008).
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Preparation of Perfusion Solutions According to the previous study from our laboratory (Ji et al., 2006a; Yu et al., 2008), the solutions injected into the duodenal and jejunal loops were buffered with 15.5 mmol/L of morpholinoethanesulfonic acid at pH 6, and the solutions injected into the ileal loops were buffered with 15.5 mmol/L of Tris at pH 7. Eight Zn sources of experiment 1 and 4 Zn sources of experiment 2 were added to the medium as treatments. Phenol red, acting as a nonabsorbable marker in the luminal medium, was used to correct the changes of Zn concentration resulting from water absorption or intestinal secretion (see below). The content of phenol red in perfusion solutions was 20 mg/L (Schedl et al., 1966; Yu et al., 2008). All chemicals used were biochemical-grade.
Ligated Loop Procedure Chickens were fasted overnight and anesthetized by wing venous injection of sumianxin (a complex anesthetic, 0.1 mL/kg of BW). The abdomen was opened by midline incision. The duodenum was incised 1 cm distal to the pyloric sphincter, the jejunum was incised just anterior to the remnant of the yolk stem, and the
ileum was incised just anterior to the ileocecal junction (Melvin, 1984). Plastic cannulas (interior diameter, 2.5 mm; exterior diameter, 4 mm; length, 2.5 cm) were inserted into 3 incisions and secured by sutures. Loose ligatures were then placed 12 cm distal to the above tight ligatures to separate different intestinal segments. The isolated segments were flushed out with 40 mL of warm saline followed by 20 mL of air to eliminate food residues and debris, and the loose ligature of each intestinal segment was tightened. Then, a 5-cm3 syringe without a needle was inserted into the cannula of each intestinal segment and the 3.5 mL of Zn dose was injected. After administration of the dose, the syringe was removed and the cannula was clamped by hemostatic forceps. The intestine was put back into the abdomen cavity. The detailed operational process was described by Yu et al. (2008). Table 1. Composition of 2 diets for 1- to 28-d-old broilers in experiment 1 Amount Item Ingredient, % Ground yellow corn Soybean meal Fish meal dl-Methionine Corn starch Casein Cellulose Soybean oil Calcium carbonate1 Calcium hydrogen phosphate1 Sodium chloride1 Micronutrients Nutrient composition ME, MJ/kg CP,4 % Lysine, % Methionine, % Methionine + cystine, % Calcium,4 % Nonphytate phosphorus, % Zinc,4 mg/kg 1Feed
Basal diet (d 1 to 21)
Semipurified diet (d 22 to 28)
54.65 34.82 3.50 0.18 3.60 1.26 1.30 0.30 0.392 12.58 21.66 1.30 0.57 0.95 1.04 0.45 90.49
66.00 23.00 5.01 1.50 1.50 1.12 0.30 1.573 13.22 19.33 1.69 0.62 0.72 0.90 0.40 12.58
grade before d 21 and reagent grade after d 21. the following per kilogram of diet: vitamin A (all-transretinol acetate), 13,500 IU; vitamin D3 (cholecalciferol), 3,600 IU; vitamin E (all-rac-α-tocopherol acetate), 33 IU; vitamin K (menadione sodium bisulfate), 6 mg; thiamin (thiamin mononitrate), 4.5 mg; riboflavin, 10.5 mg; vitamin B6, 6 mg; vitamin B12, 0.03 mg; calcium pantothenate, 18 mg; niacin, 60 mg; folic acid, 1.8 mg; biotin, 0.165 mg; choline (choline chloride), 700 mg; Cu (CuSO4·5H2O), 8 mg; Zn (ZnSO4·7H2O), 60 mg; Mn (MnSO4·H2O), 100 mg; Fe (FeSO4·7H2O), 80 mg; I (KI), 0.35 mg; and Se (Na2SeO3), 0.15 mg. 3Provided the following per kilogram of diet: vitamin A (all-transretinol acetate), 13,500 IU; vitamin D3 (cholecalciferol), 3,600 IU; vitamin E (all-rac-α-tocopherol acetate), 33 IU; vitamin K (menadione sodium bisulfate), 6 mg; thiamin (thiamin mononitrate), 4.5 mg; riboflavin, 10.5 mg; vitamin B6, 6 mg; vitamin B12, 0.03 mg; calcium pantothenate, 18 mg; niacin, 60 mg; folic acid, 1.8 mg; biotin, 0.165 mg; choline (choline chloride), 700 mg; K (KCl), 3,000 mg; Mg (MgSO4·7H2O), 600 mg; Cu (CuSO4·5H2O), 8 mg; Fe (FeSO4·7H2O), 80 mg; Mn (MnSO4·H2O), 100 mg; I (KI), 0.35 mg; and Se (Na2SeO3), 0.15 mg. 4Determined values. 2Provided
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commonly used amino acid complex in broiler production practice is Zn Met. Thus, 4 organic Zn sources of the chelate and complex of Zn with Gly and the chelate and complex of Zn with Met were used in this study. Based on the assumption that Qf values were correlated with bioavailability and absorptivity, another 3 organic Zn sources with different Qf values were studied in this experiment. All sources were obtained from independent distributors rather than directly from the product manufacturers (see above). Each treatment was repeated 10 times using 10 birds (1 time in 1 bird). The duodenum, jejunum, and ileum of each bird were used as 1 replication of corresponding intestinal segments. A procedure involving an optimal sampling time of 30 min after the injection of solutions, as observed in our previous study (Yu et al. 2008), was adopted. In experiment 2, 3×4 factorial arrangement of treatments in a completely randomized design was conducted to study the effect of phytate on the absorption of Zn from organic and inorganic sources. Three molar ratios of phytate to Zn added to the media were 0, 2:1, and 10:1, respectively. Four Zn sources, ZnSO4·7H2O, and 3 organic Zn sources of Zn AA C, Zn Pro B, and Zn Pro A with the high Zn absorptivity based on the results in experiment 1 were added to the media to provide 0.616 mmol of Zn/L. Zinc concentration in fluids in ligated loops injected with Zn-free solution after dosing were too low to be tested in experiment 1; therefore, the control without additional Zn added to the media was not used in this experiment. The intestinal segments and the sampling time were the same as those in experiment 1.
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Determination of Zn and Phenol Red Concentrations in Perfusion Solutions
Table 2. Composition of 2 diets for 1- to 28-d-old broilers in experiment 2 Amount Item Ingredient, % Ground yellow corn Soybean meal Fish meal dl-Methionine Corn starch Casein Cellulose Soybean oil Calcium carbonate1 Calcium hydrogen phosphate1 Sodium chloride1 Micronutrients Nutrient composition ME, MJ/kg CP,4 % Lysine, % Methionine, % Methionine + cystine, % Calcium,4 % Nonphytate phosphorus, % Zinc,4 mg/kg 1Feed
Basal diet (d 1 to 14)
Semipurified diet (d 14 to 28)
51.21 37.00 5.20 0.15 3.60 1.00 1.30 0.30 0.342 12.46 22.32 1.35 0.54 0.91 1.06 0.46 94.28
66.00 23.00 5.01 1.51 1.50 1.12 0.30 1.533 13.22 20.21 1.69 0.62 0.72 0.93 0.40 11.84
grade before d 21 and reagent grade after d 21. 2Provided the following per kilogram of diet: vitamin A (all-transretinol acetate), 13,500 IU; vitamin D3 (cholecalciferol), 3,600 IU; vitamin E (all-rac-α-tocopherol acetate), 33 IU; vitamin K (menadione sodium bisulfate), 6 mg; thiamin (thiamin mononitrate), 4.5 mg; riboflavin, 10.5 mg; vitamin B6, 6 mg; vitamin B12, 0.03 mg; calcium pantothenate, 18 mg; niacin, 60 mg; folic acid, 1.8 mg; biotin, 0.165 mg; choline (choline chloride), 1,000 mg; Cu (CuSO4·5H2O), 8 mg; Zn (ZnSO4·7H2O), 60 mg; Mn (MnSO4·H2O), 100 mg; Fe (FeSO4·7H2O), 80 mg; I (KI), 0.35 mg; and Se (Na2SeO3), 0.15 mg. 3Provided the following per kilogram of diet: vitamin A (all-transretinol acetate), 13,500 IU; vitamin D3 (cholecalciferol), 3,600 IU; vitamin E (all-rac-α-tocopherol acetate), 33 IU; vitamin K (menadione sodium bisulfate), 6 mg; thiamin (thiamin mononitrate), 4.5 mg; riboflavin, 10.5 mg; vitamin B6, 6 mg; vitamin B12, 0.03 mg; calcium pantothenate, 18 mg; niacin, 60 mg; folic acid, 1.8 mg; biotin, 0.165 mg; choline (choline chloride), 700 mg; K (KCl), 3,000 mg; Mg (MgSO4·7H2O), 600 mg; Cu (CuSO4·5H2O), 8 mg; Fe (FeSO4·7H2O), 80 mg; Mn (MnSO4·H2O), 100 mg; I (KI), 0.35 mg; and Se (Na2SeO3), 0.15 mg. 4Determined values.
VF =
AP =
CP (1)×VI CP (2)
CZn (1) ´ VI - CZn (2) ´ VF CZn (1)´ VI
´ 100,
where VF = final volume of perfusion solution (mL); CP (1) and CP (2) = initial and final concentration (mg/L) of phenol red, respectively; VI = initial volume (mL) of injected dose; AP = absorption percentage of Zn (%); CZn (1) and CZn (2) = Zn concentration (mmol/L) of initial and final perfusion solution, respectively.
Statistical Analysis The GLM procedure of SAS (SAS Institute, 2003) was used to compare Zn absorption data of treatment groups. Data from experiment 1 were analyzed by 1-way ANOVA, whereas the data from experiment 2 was subjected to 2-way ANOVA with a model that included Zn source and phytate level as main effects and the interaction of Zn source × phytate level. If the variances were significant, differences between mean values were ascertained using Duncan’s multiple range test method. Significance was determined to be P < 0.05.
RESULTS Effect of Different Zn Sources on Zn Absorption in Ligated Duodenum, Jejunum, and Ileum The absorption percentages of Zn as both inorganic and organic Zn sources in the ileum were about 1.3 to 2.8 times (P < 0.05) those in the duodenum and jejunum (Table 3). No differences (P > 0.05) were found between the absorption percentages of Zn in the duodenum and jejunum. These results indicated that the ileum was the main absorption site of Zn in the small intestines of broilers. It was consistent with the findings of our earlier study (Yu et al., 2008). As for Zn absorption of different Zn sources in the same intestinal segment, no differences (P > 0.05) were
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The 2-mL perfusion solutions were collected using the syringes at 30 min after the administration of the Zn dose and then were frozen at −20°C for analyzing the concentrations of Zn and phenol red. Zinc concentrations in diets, water, and perfusion solutions were determined by inductively coupled Ar plasma spectroscopy (Model IRIS Intrepid II, Thermal Jarrell Ash, Waltham, MA) as described by Lu et al. (2007). Approximately 0.5 g of feed sample or 0.5 mL of perfusion solution sample was weighed in duplicate and digested with 10 mL of HNO3 in a 50-mL microwave digestion vessel using a microwave (Mars 5, CEM Corporation, Matthews, NC) under the manufacturer’s instructions, then it was evaporated to near dryness and diluted to
3 mL with 2% HNO3 before analysis. Validation of the mineral analysis was conducted using bovine liver powder [GBW (E) 080193, National Institute of Standards and Technology, Beijing, P. R. China] as a standard reference material. The concentrations of phenol red in perfusion solutions were assayed by measuring absorbance at 520, 560, and 600 nm with a UV-visible spectrophotometer (Model Cary 100, Varian Inc., Palo Alto, CA; Steel and Cousins, 1985). Final volumes of solutions and absorption percentages of Zn were calculated according to equations outlined as follows:
EFFECTS OF ZINC SOURCE AND PHYTATE ON ZINC ABSORPTION
Effect of Zn Source and Phytate on Zn Absorption in Broiler Intestines The absorption percentages of Zn as both inorganic and organic Zn sources at different phytate levels in the jejunum and ileum were significantly higher (P < 0.05) than those in the duodenum except for those in the jejunum with Zn AA C treatment at a 2:1 phytate level (Table 4). No differences (P > 0.05) were observed in Zn absorption in both the ileum and jejunum except Zn AA C and Zn Pro B treatments without phytate added (P < 0.05). This result agreed with the result of experiment 1 in that the ileum was the main absorption site of Zn in broilers. There were significant interactions (P < 0.05) between phytate levels and Zn sources on Zn absorption in 3 intestinal segments. When the phytate was added at a 2:1 molar ratio of phytate to Zn, the absorption percentages of Zn as ZnSO4, Zn AA C, Zn Pro B, and Zn Pro A in the duodenum were not affected (P > 0.05); however, the values of 4 forms of Zn in the jejunum and ileum were significantly decreased (P < 0.05) except for the group of Zn Pro A in the jejunum compared with those of groups without the phytate. When the phytate was added at a 10:1 molar ratio of phytate to Zn, the absorption percentages of Zn as ZnSO4 in the duodenum, jejunum, and ileum of broilers were 40, 26, and 21% (P < 0.05) lower than those of Zn as ZnSO4 without the phytate group, respectively. The absorption percentages of Zn as Zn AA C in the duodenum, jejunum, and ileum of broilers were 50, 21, and 19% (P < 0.05) lower than those of Zn as Zn AA C without the phytate group, respectively. The absorption percentages of Zn as Zn Pro B in the duodenum, jejunum, and ileum of broilers were 35, 24, and 23% (P < 0.05) lower than those of Zn as Zn Pro B without the phytate
Table 3. Absorption percentages of zinc from different sources in ligated duodenum, jejunum, and ileum of 28-d-old broilers1 Zinc absorption, % Added zinc source2 ZnSO4·7H2O ZnSO4·7H2O + Gly ZnSO4·7H2O + Met Zn Gly chelate Zn Met chelate Zn AA C (weak) Zn Pro B (moderate) Zn Pro A (strong) Pooled SE P-value a–dMeans A,BMeans 1n
Duodenum
Jejunum
Ileum
Pooled SE
P-value
26.63d,B 25.79d,B 26.43d,B 42.02bc,B 38.76b,B 43.21ab,B 50.00ac,B 52.26a,B
27.61d,B 21.12d,B 24.29d,B 44.17ac,B 35.63b,B 41.00bc,B 44.11ac,B 47.85a,B
62.47ac,A 58.08c,A 60.24bc,A 68.13ab,A 68.23ab,A 65.37ac,A 67.32ab,A 69.89a,A
2.69 3.38 3.25 3.11 1.98 2.78 2.53 3.39
<0.0001 <0.0001 <0.0001 <0.0001 <0.0001 <0.0001 <0.0001 0.0002
3.35 <0.0001
2.42 <0.0001
2.88 0.0491
with different superscripts within the same column differ significantly (P < 0.05). with different superscripts within the same row differ significantly (P < 0.05).
= 10. AA C (weak) = zinc amino acid C complex with the weak complex strength [Qf = 6.48, 11.93% Zn (Zinpro Corp., Eden Prairie, MN)]; Zn Pro B (moderate) = zinc protein B complex with the moderate complex strength [formation quotient value (Qf ) = 30.73, 13.27% Zn (Fenyahua Bioengineering Co., Changzhi, P. R. China)]; Zn Pro A (strong) = zinc protein A complex with the strong complex strength [Qf = 944.02, 18.61% Zn (Alltech Inc., Nicholasville, KY)]. 2Zn
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detected among ZnSO4, the Zn + Gly mixture, and the Zn + Met mixture in 3 intestinal segments. In addition, the differences of Zn absorption between organic and inorganic sources decreased as the gut position became more distal to the pylorus. Zinc absorption percentages in the duodenum, Zn Gly chelate, and Zn Met chelate were 45 to 63% higher (P < 0.05) than ZnSO4, the Zn + Gly mixture, and the Zn + Met mixture, but there was no difference (P > 0.05) between Zn Met chelate, Zn Gly chelate, and Zn AA C. The absorption percentages of Zn as Zn Pro B and Zn Pro A were 88 to 102% higher (P < 0.05) than those of Zn as ZnSO4, the Zn + Gly mixture, and the Zn + Met mixture and 19 to 35% higher than those of Zn as Zn Met chelate (P < 0.05) and Zn Gly chelate (P ≤ 0.0961). No difference (P > 0.05) was observed among the absorption percentage of Zn as Zn AA C, Zn Pro A, and Zn Pro B. In the jejunum, the absorption percentages of Zn as Zn Gly chelate and Zn Met chelate were 29 to 110% higher (P < 0.05) and Zn AA C, Zn Pro B, and Zn Pro A were 49 to 129% higher (P < 0.05) than those of Zn as ZnSO4, the Zn + Gly mixture, and the Zn + Met mixture. The Zn absorption as Zn Pro A was significantly higher (P < 0.05) than that of Zn as Zn AA C and Zn Met chelate. The Zn absorption percentages of Zn as Zn Pro B and Zn Gly chelate were significantly higher (P < 0.05) than that of Zn as Zn Met chelate. No differences (P > 0.05) were found among other Zn sources. Finally, in the ileum, the absorption percentages of Zn as Zn Pro B, Zn Gly chelate, and Zn Met chelate were significantly higher (P < 0.05) than that of Zn as the Zn + Gly mixture. The absorption percentage of Zn as Zn Pro A increased significantly (P < 0.05) in comparison with those of Zn as the Zn + Gly mixture and the Zn + Met mixture. No differences (P > 0.05) were detected among other groups.
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a–fMeans
Jejunum
Ileum
Pooled SE
P-value
82.81ab,B 82.42ab,C 84.71a,C 84.77a,B
92.41ab,A 89.07cd,B 93.30ab,B 94.08a,A
94.25ab,A 93.85ab,A 95.38a,A 95.61a,A
0.89 1.35 0.68 1.14
<0.0001 <0.0001 <0.0001 <0.0001
75.39b,B 78.62ab,B 78.66ab,B 80.48ab,B
86.61de,A 83.49e,AB 88.61cd,A 90.74bc,A
90.80cd,A 88.51de,A 89.97de,A 92.58bc,A
1.87 2.17 1.41 1.93
<0.0001 0.0148 <0.0001 0.0004
49.50cd,B 41.21d,B 55.32c,B 78.06ab,B 3.17
68.70f,A 70.87f,A 70.73f,A 86.54de,A 1.15
74.00f,A 75.69f,A 73.10f,A 87.48e,A 0.90
3.46 3.60 2.21 0.85
<0.0001 <0.0001 <0.0001 <0.0001
83.68 78.29 56.02 1.59
92.22 87.36 74.21 0.58
94.77 90.46 77.57 0.45
69.24 67.42 72.90 81.10 1.83
82.58 81.14 84.21 90.45 0.66
86.35 86.02 86.15 91.89 0.52
<0.0001 <0.0001 <0.0001
<0.0001 <0.0001 <0.0001
<0.0001 <0.0001 <0.0001
with different superscripts within the same column differ significantly (P < 0.05). with different superscripts within the same row differ significantly (P < 0.05).
A–CMeans 1n
= 8. AA C (weak) = zinc amino acid C complex with the weak complex strength [formation quotient value (Qf ) = 6.48, 11.93% Zn (Zinpro Corp., Eden Prairie, MN)]; Zn Pro B (moderate) = zinc protein B complex with the moderate complex strength [Qf = 30.73, 13.27% Zn (Fenyahua Bioengineering Co., Changzhi, P. R. China)]; Zn Pro A (strong) = zinc protein A complex with the strong complex strength [Qf = 944.02, 18.61% Zn (Alltech Inc., Nicholasville, KY)]. 2Zn
group, respectively. The absorption percentages of Zn as Zn Pro A in the jejunum and ileum of broilers were 8% (P < 0.05) and 9% (P < 0.05) lower than those of Zn as Zn Pro A without the phytate group, respectively. Moreover, Zn absorption of 4 sources in all 3 segments was impaired (P < 0.05) except for the group of Zn Pro A in the duodenum compared with those of groups at a 2:1 phytate level.
DISCUSSION It has been reported that organic Zn absorption is different from inorganic Zn absorption in rats, humans, and dogs, but the results were inconsistent (Hill et al., 1987a,b; Lowe et al., 1994a,b; Beutler et al., 1998). The discrepancies among the reports might be explained by differences in the various species of animals, the techniques used, and the quality of organic Zn used in these studies. It has been documented that the complex strength is the most important factor for evaluating the
quality of organic mineral sources, and the quality is related to the stabilization and absorption of organic mineral sources in the gut (Ashmead and Graff, 1985). Ji et al. (2006a) found that organic Mn was more efficiently absorbed than inorganic Mn in the broiler small intestine using the technique of in situ ligated loops. Moreover, the absorption of organic Mn with strong and moderate complex strengths was greater than that of the organic Mn, which was weak, and the absorption of organic Mn with strong complex strength was greater than that of the organic Mn with moderate complex strength. Results of the present study showed that the simple mixture of ZnSO4 with either Met or Gly did not increase the absorption of Zn in broiler small intestines unless they were chelated. The absorption percentages of organic Zn as Zn Gly chelate, Zn Met chelate, Zn AA C, Zn Pro B, and Zn Pro A in the duodenum, jejunum, and ileum were higher than those of Zn as ZnSO4. Moreover, the absorption of organic Zn with strong and moderate complex strengths in 3 intestinal
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Molar ratio of phytate to zinc 0 ZnSO4·7H2O Zn AA C (weak) Zn Pro B (moderate) Zn Pro A (strong) 2:1 ZnSO4·7H2O Zn AA C (weak) Zn Pro B (moderate) Zn Pro A (strong) 10:1 ZnSO4·7H2O Zn AA C (weak) Zn Pro B (moderate) Zn Pro A (strong) Pooled SE Molar ratio of phytate to zinc 0 2:1 10:1 Pooled SE Added zinc source ZnSO4·7H2O Zn AA C (weak) Zn Pro B (moderate) Zn Pro A (strong) Pooled SE P-value Phytate level Zinc source Zinc source × phytate level
Duodenum
EFFECTS OF ZINC SOURCE AND PHYTATE ON ZINC ABSORPTION
of organic Zn absorption was the same as that of inorganic Zn. Results of our previous study suggested that inorganic Zn absorption in the ileum occurred mainly by a nonsaturable diffusive pathway, which is different from that in the other 2 intestinal segments, depending on the regulation of Zn transporter expression (Yu et al., 2008). However, the detailed mechanism of organic Zn absorption in broiler intestines was now not known and needed to be further elucidated. The complex formation capability of cations and phytate is Cu > Zn > Co > Mn > Fe > Ca in turn (from strong to weak), and the stability of complexes is Zn > Cu > Ni > Co > Mn > Ca in turn (from strong to weak). Thus, Zn is affected the most significantly by the phytate (Lönnerdal, 2000). Bafundo et al. (1984) found that the addition of phytate to diets at levels from 0.6 to1.1% increased Zn concentration in broiler feces by 20 to 50% and the requirement for Zn. Turnlund et al. (1984) also demonstrated that the addition of 2.34 g of phytate to food with 15 mg of Zn reduced Zn absorption from 34 to 17.5% by using the stable isotope technology. Similarly, the Zn absorption increased from 27 to 45% when the phytate content in food decreased from 0.621 to 0.067 mmol/L. In agreement with these results, the present study indicated that the addition of phytate in perfusates reduced the absorption of Zn with different Zn sources in 3 intestinal segments of broilers. Greater reduction of Zn absorption was observed with the increasing molar ratio of phytate to Zn, with the most obvious changes in the duodenum. It was reported that the phytate and Zn molar ratio of 10:1 or above has been considered as the minimum ratio to reduce Zn absorption in rats (Morris and Ellis, 1980). In addition, Lönnerdal et al. (1988) found that a 6:1 molar ratio of phytate to Zn had a negative effect on Zn absorption in humans. However, Zn absorption in the current study reduced significantly when the molar ratio of phytate and Zn was 2:1, and greater reduction was observed when the molar ratio of phytate and Zn was 10:1. These observations might be due to the difference in the media from which the phytate was supplied. The phytate was supplied to the diet or food in early reports, whereas it was added to the perfusates in this study, therefore resulting in a more direct and sensitive inhibiting effect of phytate on Zn. In addition, the Zn absorption was significantly higher in experiment 2 than in experiment 1. This could be explained by the degree of Zn deficiency in chicks, which was higher in experiment 2 than in experiment 1 because the chicks in experiment 1 were fed a Zn-deficient diet for 1 wk, whereas the chicks of experiment 2 were fed a Zn-deficient diet for 2 wk. Thus, chicks in experiment 2 became more sensitive to Zn, resulting in a higher Zn absorption. Similarly, Smith and Cousins (1980) found that the absorption velocity of Zn was 627 nmol/h in rats fed with a diet lacking of Zn but reduced to 229 nmol/h in rats fed with a diet containing Zn. This result demonstrated that Zn deficiency in the diet increased
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segments was greater than that of the organic Zn with weak complex strength. These results were similar to the conclusions of Ji et al. (2006a, see above). Some evidence suggests positively charged Zn ions are not able to easily penetrate the negatively charged cell wall and therefore are transported across the mucosal surface via a low molecular weight ligand mediated process. Amino acids, proteins, and peptide fragments are the primary low molecular weight ligands during the absorptive process and are produced in sufficient amounts by the endogenous or dietary protein digestion. Zinc has a high affinity to these ligands. It is thought that the chelation of Zn by low molecular weight ligands leads to the formation of a soluble complex that can move across mucosal pores or the mucosal membrane by passive or active transport mechanisms or prevents Zn ions from precipitating at physiological pH and chelating by the phytate or other compounds. Finally, these Zn chelates can facilitate the intestinal absorption of Zn in vivo (Wapnir et al., 1983; Wapnir and Steel, 1986; Ashmead, 1993). Strength of Zn complex is important for facilitating absorption. Organic Zn with weak complex strength dissociated easily, but organic Zn sources with moderate and strong complex strengths were difficult to dissociate so they were absorbed easily (Huang et al., 2009). The above findings provided a good explanation for our data that the absorption of organic Zn chelated with amino acid or protein was greater than that of inorganic Zn and the absorption of organic Zn with strong and moderate complex strengths in the duodenum, jejunum, and ileum was greater than that of the organic Zn with weak complex strength. Ji et al. (2006b) reported that the absorption of Mn increased when the mixture of MnSO4 with Met or Gly mixed at a 1:2 molar ratio of Mn to Met or Gly, and Met as a ligand, was more effective in facilitating Mn absorption than Gly. However, the results of this study indicated that the simple mixture of ZnSO4 with Met or Gly at a 1:2 ratio did not increase Zn absorption and were not in line with those of Ji et al. (2006b). How the ligands affect the mineral absorption depend on many factors including the ratio of ligand to the minerals, the degree of chelation, the kind of ligand, and the techniques used (Seal and Heaton, 1983; Wapnir et al., 1983). The results of the main absorption site of Zn obtained in these experiments were consistent with the previous work from our laboratory (Yu et al., 2008). Once again, the experiments have clearly demonstrated the importance of the ileum as the main site at which Zn is absorbed. From the data of experiment 1, we found that there was a consistent change in Zn absorption of organic and inorganic Zn sources in 3 segments of broilers. The largest Zn absorption of different sources was observed in the ileum, and no differences were found between the Zn sources in it, but the Zn absorption percentages with organic Zn in the duodenum and jejunum were obviously greater than those of inorganic Zn. Based on these findings, it seemed that the mechanism
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ACKNOWLEDGMENTS Supported by the Earmarked Fund for Modern AgroIndustry Technology Research System (project no. nycytx-42-G2-04; Beijing, P. R. China), the Program of the National Natural Science Foundation of China (project no. 30871798; Beijing, P. R. China), and the Research Program of the Key Laboratory of Animal Nutrition (project no. 2004DA125184G0812; Beijing, P. R. China).
REFERENCES Ashmead, D. H. 1993. Page 32 in The Role of Metal Amino Acid Chelate. American Academic Press, Cleveland, OH. Ashmead, D. H., and D. J. Graff. 1985. Page 121 in Intestinal Absorption of Metal Ions and Chelates. Charles C. Thomas, Springfield, IL.
Bafundo, K. W., D. H. Baker, and P. R. Fitzgerald. 1984. Zinc utilization in the chick as influenced by dietary concentrations of calcium and phytate and by Eimeria acervulina infection. Poult. Sci. 63:2430–2437. Beutler, K. T., O. Pankewycz, and D. L. Brautigan. 1998. Equivalent uptake of organic and inorganic zinc by monkey kidney fibroblasts, human intestinal epithelial cells, or perfused mouse intestine. Biol. Trace Elem. Res. 61:19–31. Dong, X. H. 2001. Study on zinc absorption and metabolism of zinc amino acid chelate and effecting factors. Pages 73–82 in PhD Diss. Northeast Agricultural University, Harbin, P. R. China. Edwards, H. M., and D. H. Baker. 2000. Zinc bioavailability in soybean meal. J. Anim. Sci. 78:1017–1021. Gaither, L. A., and D. J. Eide. 2001. Eukaryotic zinc transporters and their regulation. Biometals 14:251–270. Hempe, J. M., and R. J. Cousins. 1989. Effect of EDTA and zincmethionine complex on zinc absorption by rat intestine. J. Nutr. 119:1179–1187. Hill, D. A., E. R. Peo Jr., and A. J. Lewis. 1987a. Effect of zinc source and picolinic acid on 65zinc uptake in an in vitro continuous flow perfusion system for pig and poultry intestinal segments. J. Nutr. 117:1704–1707. Hill, D. A., E. R. Peo Jr., and A. J. Lewis. 1987b. Influnce of picolinic acid on the uptake of 65zinc-amino acid complexes by the everted rat gut. J. Anim. Sci. 65:173–178. Holwerda, R. A., R. C. Albin, and F. C. Madsen. 1995. Chelation effectiveness of zinc proteinates demonstrated. Feedstuffs 67:12– 13. Huang, Y. L., L. Lu, S. F. Li, B. Liu, and X. G. Luo. 2009. Relative bioavailabilities of organic zinc sources with different chelation strengths for broilers fed a conventional corn-soybean meal diet. J. Anim. Sci. 87:2038–2046. Huang, Y. L., L. Lu, X. G. Luo, and B. Liu. 2007. An optimal dietary zinc level of broiler chicks fed a corn-soybean meal diet. Poult. Sci. 86:2582–2589. Ji, F., X. G. Luo, L. Lu, B. Liu, and S. X. Yu. 2006a. Effect of manganese source on manganese absorption by the intestine broilers. Poult. Sci. 85:1947–1952. Ji, F., X. G. Luo, L. Lu, B. Liu, and S. X. Yu. 2006b. Effects of manganese source and calcium on manganese uptake by in vitro everted gut sacs of broiler’s intestinal segments. Poult. Sci. 85:1217–1225. Liang, J. G. 2006. The study of characterization of organic zinc sources and their bioactivities and modes of action in cows. PhD Diss. Chinese Academy of Agricultural Science, Beijing, P. R. China. Lönnerdal, B. 2000. Dietary factors influencing zinc absorption. J. Nutr. 130:1378S–1383S. Lönnerdal, B., J. G. Bell, R. A. Burns, and C. L. Keen. 1988. Effect of phytate removal on zinc absorption from soy formula. Am. J. Clin. Nutr. 48:1301–1306. Lowe, J. A., J. Wiseman, and D. J. Cole. 1994a. Absorption and retention of zinc when administered as an amino-acid chelate in the dog. J. Nutr. 124(Suppl.):2572S–2574S. Lowe, J. A., J. Wiseman, and D. J. Cole. 1994b. Zinc source influences zinc retention in hair and hair growth in the dog. J. Nutr. 124:2575S–2576S. Lu, L., X. G. Luo, C. Ji, B. Liu, and S. X. Yu. 2007. Effect of manganese supplementation and source on carcass traits, meat quality, and lipid oxidation in broilers. J. Anim. Sci. 85:812–822. Matsui, T., T. Ishiguro, S. Suzaki, H. Yano, and S. Fujita. 1996. Supplementation of zinc as amino acid-chelated zinc for piglets. Page 754 in Proceedings of the 8th AAAP Animal Science Congress, Tokyo, Japan. Vol 2. Melvin, J. S. 1984. Page 359 in Duke’s Physiology of Domestic Animals. 10th ed. Cornell University Press, Ithaca, NY. Morris, E. R., and R. Ellis. 1980. Effect of dietary/zinc molar ratio on growth and bone zinc response of rats fed semipurified diets. J. Nutr. 110:1037–1045. NRC. 1994. Nutrient Requirements of Poultry. 9th rev. ed. Natl. Acad. Press, Washington, DC. Rojas, L. X., L. R. McDowell, R. J. Cousins, F. G. Martin, N. S. Wilkinson, A. B. Johnson, and J. B. Velasquez. 1995. Relative
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Zn absorption in the intestines of rats. When compared with inorganic Zn, the organic Zn sources with different complex strengths inhibited the chelate function by the phytate and therefore reduced the negative effect of the phytate on Zn absorption. Of the organic Zn sources, the reduced degree of Zn absorption of Zn Pro A under the high phytate level was the minimum because it was the most difficult to dissociate and chelate by the phytate in the intestines of broilers. However, Zn Pro B and Zn AA C were not as large as Zn Pro A in the effects of resisting the chelate by phytate of Zn; therefore, the Zn absorption reduced more than that of Zn Pro A. These results were in agreement with the conclusions of Dong (2001), who suggested that the disappearance of 65ZnCl2, 65Zn Lys, and 65Zn Met in the rat ligated duodenum was reduced by 64.44, 15.17, and 14.95%, respectively, when 1% phytate was added to the semipurified diet. These results indicated that the absorption of organic Zn was more effective than inorganic Zn under the high phytate level. In conclusion, the ileum was the main site of Zn absorption in the intestines of broilers. The absorption of organic Zn was more effective than that of inorganic Zn. Zinc absorption increased with the increasing complex strength with the order of Zn Pro A > Zn Pro B > Zn AA C > Zn Gly chelate or Zn Met chelate > ZnSO4, Zn + Met, or Zn + Gly in the duodenum and jejunum. The simple mixture of ZnSO4 with amino acid could not increase Zn absorption unless they were chelated. These results further indicated that Zn absorption was affected by the degree of chelation between ligands and minerals regardless of the ligands action in improving or restraining Zn absorption. Phytate could reduce the absorption of Zn as different Zn sources in 3 intestinal segments, and Zn absorption decreased with the increasing phytate levels, with the most obvious effects in the duodenum. The absorption of organic Zn, especially Zn Pro A, was higher than inorganic Zn with the high levels of phytate, further confirming the superior availability of organic Zn compared with inorganic Zn.
EFFECTS OF ZINC SOURCE AND PHYTATE ON ZINC ABSORPTION
Vallee, B. L., and K. H. Falchuk. 1993. The biochemical basis of zinc physiology. Physiol. Rev. 73:79–118. Wapnir, R. A., D. E. Khani, M. A. Bayne, and F. Lifshitz. 1983. Absorption of zinc by the rat ileum: Effects of histidine and other low-molecular-weight ligands. J. Nutr. 113:1346–1354. Wapnir, R. A., and L. Steel. 1986. Zinc intestinal absorption in rats: Specificity of amino acids as ligands. J. Nutr. 116:2171–2179. Wedekind, K. J., A. E. Hortin, and D. H. Baker. 1992. Methodology for assessing zinc bioavailability: Efficacy estimates for zinc-methionine, zinc sulfate, and zinc oxide. J. Anim. Sci. 70:178–187. Yang, Q. M., and Y. X. Diao. 1999. The Handbook for Raising of Broilers. China Agricultural University Press, Beijing, P. R. China. Yu, Y., L. Lu, X. G. Luo, and B. Liu. 2008. Kinetics of zinc absorption by in situ ligated intestinal loops of broilers involved in zinc transporters. Poult. Sci. 87:1146–1155.
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bioavailability of two organic and two inorganic zinc sources fed to sheep. J. Anim. Sci. 73:1202–1207. SAS Institute. 2003. SAS User’s Guide: Statistics. Version 9.0. SAS Institute Inc., Cary, NC. Schedl, H. P., D. Miller, and D. White. 1966. Use of polyethylene glycol and phenol red as unabsorbed indicators for intestinal absorption studies in man. Gut 7:159–163. Seal, C. J., and F. W. Heaton. 1983. Chemical factors affecting the intestinal absorption of zinc in vitro and in vivo. Br. J. Nutr. 50:317–324. Smith, K. T., and R. J. Cousins. 1980. Quantitative aspects of zinc absorption by isolated vascularly perfused rat intestine. J. Nutr. 110:316–323. Steel, L., and R. J. Cousins. 1985. Kinetics of zinc absorption by luminally and vascularly perfused rat intestine. Am. J. Physiol. 248:G46–G53. Turnlund, J. R., J. C. King, W. R. Keyes, and M. C. Michel. 1984. A stable isotope study of zinc absorption in young men: Effects of phytate and α-cellulose. Am. J. Clin. Nutr. 40:1071–1077.
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