TY - JOUR
AU - Luo, X. G.
AB - ABSTRACT This experiment was conducted to investigate the effect of iron source on Fe absorption and the gene expression of divalent metal transporter 1 (DMT1) and ferroportin 1 (FPN1) in the ligated duodenal loops of broilers. The in situ ligated duodenal loops from Fe-deficient broiler chicks (28-d-old) were perfused with Fe solutions containing 0 to 14.33 mmol Fe/L from 1 of the following: Fe sulfate (FeSO4∙7H2O), Fe methionine with weak chelation strength (Fe-Met W; chelation strength is expressed as quotient of formation [Qf] value, Qf = 1.37), Fe proteinate with moderate chelation strength (Fe-Prot M; Qf = 43.6), and Fe proteinate with extremely strong chelation strength (Fe-Prot ES; Qf = 8,590) for up to 30 min. The gene expression of DMT1 and FPN1 in the duodenal loops from the control group and the groups treated with 3.58 mmol Fe/L from 1 of 4 Fe sources was analyzed. The absorption kinetics of Fe from different Fe sources in the duodenum followed a saturated carrier-dependent transport process. The maximum transport rate (Jmax) values in the duodenum were greater (P < 0.03) for Fe-Prot ES and Fe-Prot M than for Fe-Met W and FeSO4∙7H2O. The Fe perfusion inhibited (P < 0.05) the mRNA expression of DMT1 but enhanced (P < 0.0008) the mRNA expression of FPN1 in the duodenum and had no effect (P > 0.14) on the protein expression levels of the 2 transporters. These results indicated that organic Fe sources with greater Qf values showed higher Fe absorption; however, all Fe sources followed the same saturated carrier-dependent transport process in the duodenum, and DMT1 and FPN1 might participate in Fe absorption in the duodenum of broilers regardless of Fe source. INTRODUCTION Iron is essential for DNA synthesis, respiration, and key metabolic reactions intrinsic to life (Dunn et al., 2007; Hentze et al., 2010; Ganz and Nemeth, 2011). Rapidly growing chicks have a high demand for Fe (Ma et al., 2016); therefore, some organic Fe sources, such as Fe AA complexes, chelates, or Fe proteinates, have been developed as supplements in livestock feeds. It has been reported that the bioavailabilities of organic Fe sources for broilers are closely correlated with their chelation strengths [quotient of formation (Qf) values] between Fe and their ligands (Ma et al., 2014; Zhang et al., 2016b). However, limited research in broilers has been done on the absorption and mechanisms of organic Fe with different Qf values. The divalent metal transporter 1 (DMT1), which is located on an apical transmembrane and actively transports reduced dietary Fe into intestinal enterocytes, is a protein recently shown to play a pivotal role in Fe uptake (Fleming et al., 1997; Gunshin et al., 1997; Hentze et al., 2010). Ferroportin 1 (FPN1) is the sole identified Fe transporter that is expressed on the basolateral membrane and is responsible for Fe transport from inside the cells to the bloodstream (Donovan et al., 2005; Mayr et al., 2010). Zoller et al. (2002) reported that the DMT1 and FPN1 mRNA levels decreased on treatment of CaCo-2 cells with Fe. Similar results reported that Fe deficiency induced DMT1 expression in the duodenum of mice (Abboud and Haile, 2000) and chickens (Tako et al., 2010). However, the effect of different Fe sources on the expression of DMT1 and FPN1 in the small intestine of chicks has not been elucidated yet. Therefore, the objective of this study was to investigate the Fe absorption kinetics of Fe from different Fe sources using in situ ligated duodenal loops of broilers, as well as the effect of different Fe sources on the gene expression of DMT1 and FPN1 in ligated duodenal loops. MATERIALS AND METHODS Materials The 2-(N-morpholino)ethanesulfonic was of biochemical grade (Beijing Jingke Chemical Reagent Co., Beijing, China). Phenol red was chemically pure (Sigma-Aldrich Co., Milwaukee, WI). The 4 Fe sources were inorganic Fe sulfate (FeSO4·7H2O, reagent grade, 19.5% Fe by analysis; Beijing Chemical Co., Beijing, China) and 3 organic Fe sources, including Fe methionine with weak chelation strength (Fe-Met W, feed grade, 14.7% Fe and Qf = 1.37 by analysis; DeBon Agri-TECH Group, Shanghai, China), Fe proteinate with moderate chelation strength (Fe-Prot M, feed grade, 14.2% Fe and Qf = 43.6 by analysis; Alltech Inc., Nicholasville, KY), and Fe proteinate with extremely strong chelation strength (Fe-Prot ES, feed grade, 10.2% Fe and Qf = 8,590 by analysis; Hebei Amino Acid Co., Baoding, China). All 3 organic Fe sources used in the present study and their Qf values and Fe concentrations are from a study on the chemical characteristics of organic Fe sources and their bioavailabilities for broilers (Zhang et al., 2016b). Chelation Properties of Fe Sources The Qf value is used to reflect the chelation strengths of the bonds between Fe and its organic ligands and was measured as described by Zhang et al. (2016b) using polarography with a hanging mercury drop electrode (Ag/AgCl reference electrode, model JM-01, Jiangsu Jiangfen Precision Instrument Factory, Taizhou, China; electrochemical analyzer, model CHI610D Shanghai Chenhua Instrument Co., Ltd., Shanghai, China). The chelation strengths of the organic Fe sources were classified into 4 classes: 1) weak chelation strength with Qf values between 0 and 10, 2) moderate chelation strength with the Qf values between 10 and 100, 3) strong chelation strength with the Qf values between 100 and 1,000, and 4) extremely strong chelation strength with Qf values over 1,000 (Holwerda et al., 1995). Biologically, the Qf value of 1.37 for Fe-Met W is not different from FeSO4, and it could be expected to dissociate very rapidly under typical low-pH conditions in the stomach. Birds, Diets, and Experimental Design The experimental procedures were approved by the Office of Beijing Veterinarians. One-day-old Arbor Acres commercial male chicks (Huadu Broiler Breeding Corp., Beijing, China) were housed in electrically heated and thermostatically controlled cages and were allowed ad libitum access to a corn–soybean meal diet supplemented with 40 mg Fe/kg as FeSO4·7H2O (Table 1; 146 mg Fe/kg by analysis, which is adequate for broilers; Ma et al., 2016) and tap water with no detectable Fe for the first 21 d. For the following 7 d, the birds were fed a Fe-deficient casein-dextrose basal diet (Table 1; 4.94 mg Fe/kg by analysis) to deplete Fe stores in the body. In a previous study from our laboratory (Ma et al., 2016), when broilers were fed a Fe-deficient diet containing 67 mg Fe/kg for 21 d, they showed a moderate Fe deficiency, as reflected by the depressed growth performance, and meanwhile, the liver Fe and blood hemoglobin concentrations were 43.1 μg/g fresh tissue and 96.8 g/L, respectively. In the present study, the analyzed liver Fe and blood hemoglobin concentrations were decreased from 79.8 μg/g fresh tissue and 112.5 g/L on d 22 to 41.3 μg/g fresh tissue and 83.25 g/L on d 28 after a 7-d depletion, respectively, suggesting that the chickens should have reduced Fe status. The diet was formulated according to the NRC (1994) recommendations for starter or grower broilers except for Fe. The broilers were managed according to the guidelines for broiler management (Yang and Diao, 1999). At 28 d of age, after an overnight fast, 210 birds were weighed and selected by the average BW and then were randomly assigned to 1 of 21 treatments (10 birds each) in a completely randomized design involving 1 control (Fe-free solution) and a 4 × 5 (Fe sources × perfused Fe concentrations) factorial arrangement of treatments. These birds were used to prepare the in situ ligated duodenal loops following the in situ ligation procedure described below. The analyzed Fe concentration in the duodenal chyme was 0.45 mM (25 μg/mL) when the birds were fed a corn–soybean meal diet containing adequate Fe (146 mg/kg), and the Fe content in the duodenal chyme of broilers fed the casein-dextrose basal diet containing 5 mg/kg Fe was undetectable (below 0.05 μg/mL). However, there were no differences in Fe absorption among these Fe sources when the perfusate Fe concentration was 0.45 mM (25 μg/L), whereas significant differences in Fe absorption were detected among these Fe sources at a perfusate Fe concentration of 3.58 mM (200 mg/L), which is the common Fe concentration (200 mg/kg) in the conventional corn–soybean meal diet of broilers (Li et al., 2016). Therefore, we select 3.58 mM as the middle Fe concentration and then designed the following 5 perfusate Fe concentrations around the 3.58 mM Fe in the present experiment. The ligated duodenal loops were infused with Fe-free solution (control) or a solution containing 0.90, 1.79, 3.58, 7.16, or 14.33 mmol Fe/L of FeSO4·7H2O, Fe-Met W, Fe-Prot M, or Fe-Prot ES. Each treatment was repeated 10 times using 10 birds (1 bird each time), and each ligated duodenal loop was considered a replicate. Table 1. Composition and nutrition levels of the basal diets for broilers (as-fed basis) Item  Starter (d 1 to 21): Corn–soybean meal diet  Grower (d 22 to 28): Casein-dextrose diet  Ingredient, %          Corn  54.5  —      Soybean meal  37.5  —      Soybean oil  4.00  4.00      Dextrose1  —  63.7      Casein1  —  18.4      Cellulose1  —  3.00      CaHPO4·H2O  2.12  —      Ground limestone  1.06  —      NaCl  0.30  0.881      CaCO31  —  2.43      KH2PO41  —  1.61      NaHCO31  —  1.01      MgSO4·7H2O1  —  0.35      KHCO31  —  1.03      Met1  0.20  0.351      Arg1  —  0.99      Gly1  —  2.01      Micronutrients2,3  0.32  0.30  Calculated composition      ME, MJ/kg  12.5  12.8      CP,4 %  20.7  20.1      Lys, %  1.15  1.28      Met, %  0.50  0.82      Met + Cys, %  0.81  0.89      Arg, %  —  1.55      Gly, %  —  2.00      Ca,4 %  1.05  1.02      Nonphytate P, %  0.47  0.50      Fe,4 mg/kg  146  4.94      Cu,4 mg/kg  17.4  11.9      Zn,4 mg/kg  101  40.4      Mn,4 mg/kg  121  63.1  Item  Starter (d 1 to 21): Corn–soybean meal diet  Grower (d 22 to 28): Casein-dextrose diet  Ingredient, %          Corn  54.5  —      Soybean meal  37.5  —      Soybean oil  4.00  4.00      Dextrose1  —  63.7      Casein1  —  18.4      Cellulose1  —  3.00      CaHPO4·H2O  2.12  —      Ground limestone  1.06  —      NaCl  0.30  0.881      CaCO31  —  2.43      KH2PO41  —  1.61      NaHCO31  —  1.01      MgSO4·7H2O1  —  0.35      KHCO31  —  1.03      Met1  0.20  0.351      Arg1  —  0.99      Gly1  —  2.01      Micronutrients2,3  0.32  0.30  Calculated composition      ME, MJ/kg  12.5  12.8      CP,4 %  20.7  20.1      Lys, %  1.15  1.28      Met, %  0.50  0.82      Met + Cys, %  0.81  0.89      Arg, %  —  1.55      Gly, %  —  2.00      Ca,4 %  1.05  1.02      Nonphytate P, %  0.47  0.50      Fe,4 mg/kg  146  4.94      Cu,4 mg/kg  17.4  11.9      Zn,4 mg/kg  101  40.4      Mn,4 mg/kg  121  63.1  1Reagent grade. 2Provided per kilogram of diet (d 1 to 21): vitamin A (all-trans-retinyl acetate), 5.16 mg; cholecalciferol, 0.098 mg; vitamin E (all-rac-α-tocopherol acetate), 20 mg; vitamin K (menadione sodium bisulfate), 3.0 mg; thiamin (thiamin mononitrate), 2.4 mg; riboflavin, 9.0 mg; vitamin B6, 4.5 mg; vitamin B12, 0.021 mg; calcium pantothenate, 30 mg; niacin, 45 mg; folic acid, 1.2 mg; biotin, 0.18 mg; choline (choline chloride), 700 mg; Cu (CuSO4·5H2O), 8 mg; Mn (MnSO4·H2O), 100 mg; Zn(ZnSO4·7H2O), 60 mg; I (KI), 0.35 mg; and Se (Na2SeO3), 0.15 mg. 3Provided per kilogram of diet (d 22 to 28): vitamin A (all-trans-retinyl acetate), 1.79 mg; vitamin D3, 0.015 mg; vitamin E (DL-α-tocopheryl acetate), 13.36 mg; vitamin K3, 2 mg; vitamin B2, 10 mg; vitamin B1, 20 mg; vitamin B12, 0.04 mg; vitamin B6, 6 mg; calcium pantothenate, 30 mg; niacin, 50 mg; biotin, 0.60 mg; folic acid, 4 mg; ascorbic acid, 250 mg; choline chloride, 2,000 mg; Cu (CuSO4·5H2O), 8 mg; Mn (MnSO4·H2O), 60 mg; Zn (ZnSO4·7H2O), 40 mg; Se (Na2SeO3), 0.15 mg; I (KI), 0.35 mg; H3BO3, 9 mg; NaMoO4·2H2O, 9 mg. 4Analyzed values. Each value is based on triplicate determinations. View Large Table 1. Composition and nutrition levels of the basal diets for broilers (as-fed basis) Item  Starter (d 1 to 21): Corn–soybean meal diet  Grower (d 22 to 28): Casein-dextrose diet  Ingredient, %          Corn  54.5  —      Soybean meal  37.5  —      Soybean oil  4.00  4.00      Dextrose1  —  63.7      Casein1  —  18.4      Cellulose1  —  3.00      CaHPO4·H2O  2.12  —      Ground limestone  1.06  —      NaCl  0.30  0.881      CaCO31  —  2.43      KH2PO41  —  1.61      NaHCO31  —  1.01      MgSO4·7H2O1  —  0.35      KHCO31  —  1.03      Met1  0.20  0.351      Arg1  —  0.99      Gly1  —  2.01      Micronutrients2,3  0.32  0.30  Calculated composition      ME, MJ/kg  12.5  12.8      CP,4 %  20.7  20.1      Lys, %  1.15  1.28      Met, %  0.50  0.82      Met + Cys, %  0.81  0.89      Arg, %  —  1.55      Gly, %  —  2.00      Ca,4 %  1.05  1.02      Nonphytate P, %  0.47  0.50      Fe,4 mg/kg  146  4.94      Cu,4 mg/kg  17.4  11.9      Zn,4 mg/kg  101  40.4      Mn,4 mg/kg  121  63.1  Item  Starter (d 1 to 21): Corn–soybean meal diet  Grower (d 22 to 28): Casein-dextrose diet  Ingredient, %          Corn  54.5  —      Soybean meal  37.5  —      Soybean oil  4.00  4.00      Dextrose1  —  63.7      Casein1  —  18.4      Cellulose1  —  3.00      CaHPO4·H2O  2.12  —      Ground limestone  1.06  —      NaCl  0.30  0.881      CaCO31  —  2.43      KH2PO41  —  1.61      NaHCO31  —  1.01      MgSO4·7H2O1  —  0.35      KHCO31  —  1.03      Met1  0.20  0.351      Arg1  —  0.99      Gly1  —  2.01      Micronutrients2,3  0.32  0.30  Calculated composition      ME, MJ/kg  12.5  12.8      CP,4 %  20.7  20.1      Lys, %  1.15  1.28      Met, %  0.50  0.82      Met + Cys, %  0.81  0.89      Arg, %  —  1.55      Gly, %  —  2.00      Ca,4 %  1.05  1.02      Nonphytate P, %  0.47  0.50      Fe,4 mg/kg  146  4.94      Cu,4 mg/kg  17.4  11.9      Zn,4 mg/kg  101  40.4      Mn,4 mg/kg  121  63.1  1Reagent grade. 2Provided per kilogram of diet (d 1 to 21): vitamin A (all-trans-retinyl acetate), 5.16 mg; cholecalciferol, 0.098 mg; vitamin E (all-rac-α-tocopherol acetate), 20 mg; vitamin K (menadione sodium bisulfate), 3.0 mg; thiamin (thiamin mononitrate), 2.4 mg; riboflavin, 9.0 mg; vitamin B6, 4.5 mg; vitamin B12, 0.021 mg; calcium pantothenate, 30 mg; niacin, 45 mg; folic acid, 1.2 mg; biotin, 0.18 mg; choline (choline chloride), 700 mg; Cu (CuSO4·5H2O), 8 mg; Mn (MnSO4·H2O), 100 mg; Zn(ZnSO4·7H2O), 60 mg; I (KI), 0.35 mg; and Se (Na2SeO3), 0.15 mg. 3Provided per kilogram of diet (d 22 to 28): vitamin A (all-trans-retinyl acetate), 1.79 mg; vitamin D3, 0.015 mg; vitamin E (DL-α-tocopheryl acetate), 13.36 mg; vitamin K3, 2 mg; vitamin B2, 10 mg; vitamin B1, 20 mg; vitamin B12, 0.04 mg; vitamin B6, 6 mg; calcium pantothenate, 30 mg; niacin, 50 mg; biotin, 0.60 mg; folic acid, 4 mg; ascorbic acid, 250 mg; choline chloride, 2,000 mg; Cu (CuSO4·5H2O), 8 mg; Mn (MnSO4·H2O), 60 mg; Zn (ZnSO4·7H2O), 40 mg; Se (Na2SeO3), 0.15 mg; I (KI), 0.35 mg; H3BO3, 9 mg; NaMoO4·2H2O, 9 mg. 4Analyzed values. Each value is based on triplicate determinations. View Large Preparations of Perfusion Solutions and Ligated Loop Procedure The perfusion solution injected into the duodenal ligated loops consisted of 135 mmol/L of NaCl, 20 mg/L of phenol red (Bai et al., 2008, 2012), 15.5 mmol/L of 4-morpholineoethanesulfonic acid buffer (Ji et al., 2006a), and ascorbic acid (the mole ratio of ascorbic acid to Fe was 20:1; Tako et al., 2010; Li et al., 2016). Each Fe source was added to the medium to obtain the desired Fe concentrations, and the pH values of the perfusion solutions were moderated to 5.0 to keep the Fe ion in a soluble state and the pH of the solutions as close as possible to the physiological pH in the small intestine. Phenol red acted as a nonabsorbable marker in the luminal medium (Yu et al., 2008, 2010), which was used to correct the changes in Fe concentrations resulting from water absorption or intestinal secretion. The in situ ligation procedure of duodenal loops was performed as described previously (Zhang et al., 2016a). Sample Collections The feed ingredient and diet samples were taken and submitted for CP, Ca, Fe, Cu, Zn, and Mn analyses before the initiation of the trial to confirm CP, Ca, Fe, Cu, Zn, and Mn contents in diets. The tap water was collected for analysis of Fe content. The rate of Fe absorption was highest 30 min after perfusion (Thomson et al., 1971; Li et al., 2016; Zhang et al., 2016a); therefore, this time point was adopted to investigate the Fe absorption kinetics of Fe sources. Two milliliters of perfusates in the ligated duodenal loops were harvested with syringes 30 min after perfusion. Subsequently, the ligated duodenal loops of the control group and groups treated with 3.58 mM Fe from each Fe source were excised, flushed with ice-cold saline solution, and slit lengthwise, and then the mucosa was scraped with an ice-cold microscope slide, immediately frozen in liquid nitrogen, and stored at −80°C until further analysis. As mentioned above, 200 mg Fe/kg is the common Fe concentration in the conventional corn–soybean meal diet of broilers; therefore, we selected the ligated duodenal loops only from the control and 3.58 mM Fe treatment for analyses of gene expression to simply look at the effect of Fe source treatment on the gene expression of duodenal Fe transporters. The harvested solutions were divided into 2 parts, in which 1 part was stored at −4°C to analyze phenol red and the other part was frozen at −20°C until assays of Fe concentrations in solutions were performed. Sample Analyses Determinations of Fe, Cu, Mn, Zn, CP, Ca, and Phenol Red Contents The Fe, Cu, Mn and Zn contents in the feed ingredients, diet samples, and water were determined by inductively coupled plasma emission spectroscopy (IRIS Intrepid II, Thermal Jarrell Ash, Waltham, MA; Ji et al., 2006a,b; Bai et al., 2008, 2012; Yu et al., 2008, 2010; Li et al., 2015). Validation of the mineral analysis was conducted using bovine liver powder [GBW (E) 080193, National Institute of Standards and Technology, Beijing, China] as a standard reference. The CP and Ca concentrations in feed ingredients and diet samples were measured as described by AOAC (1990). The concentrations of phenol red in perfusion solutions were assayed by measuring absorbency at 520, 560, and 600 nm with an ultraviolet-visible spectrophotometer (Cary 100, Varian Inc., Palo Alto, CA; Li et al., 2016; Zhang et al., 2016a). Final volumes of solutions and Fe absorption velocities were calculated according to the equations listed in Table 2. Table 2. Formulas used for calculating final volume of perfusion solution and Fe absorption by the in situ ligated duodenal loops of broilers1 Item  Symbol  Formula  Final volume of perfusion solution, mL  V F     Fe absorption, nmol·min−1·cm−1  UV    Item  Symbol  Formula  Final volume of perfusion solution, mL  V F     Fe absorption, nmol·min−1·cm−1  UV    1 C P(1) and CP(2) = initial and final concentrations (mg/L) of phenol red, respectively; VI = initial volume (mL) of injected dose; CFe(1) and CFe(2) = Fe concentrations (mM) of initial and final perfusion solutions, respectively; VF = final volume (mL) of injected dose; T = sampling time (min) after initiation of dosing; L = length (cm) of the ligated duodenal loops. View Large Table 2. Formulas used for calculating final volume of perfusion solution and Fe absorption by the in situ ligated duodenal loops of broilers1 Item  Symbol  Formula  Final volume of perfusion solution, mL  V F     Fe absorption, nmol·min−1·cm−1  UV    Item  Symbol  Formula  Final volume of perfusion solution, mL  V F     Fe absorption, nmol·min−1·cm−1  UV    1 C P(1) and CP(2) = initial and final concentrations (mg/L) of phenol red, respectively; VI = initial volume (mL) of injected dose; CFe(1) and CFe(2) = Fe concentrations (mM) of initial and final perfusion solutions, respectively; VF = final volume (mL) of injected dose; T = sampling time (min) after initiation of dosing; L = length (cm) of the ligated duodenal loops. View Large Quantification of Gene Expression by Real-Time PCR Total RNA was extracted from intestinal mucosa using TRIzol reagent (catalog number 15596018, Life Technologies, Carlsbad, CA) according to the manufacturer's proposals. The reverse transcriptions of mRNA were conducted using the PrimeScript RT reagent kit with gDNA Eraser (catalog number RR047A, Takara Bio Inc., Otsu, Japan) according to the manufacturer's instructions. Quantitative real-time PCR was performed in triplicate on an ABI 7900HT system (Life Technologies) using the SYBR Green PCR Master Mix (catalog number 4367659, Life Technologies) according to the manufacturer's instructions. The procedure used in the PCR program was an initial denaturation (2 min at 95°C) and then a 3-step amplification program (60 s at 95°C, 30 s at 60°C, and 30 s at 72°C) that was repeated 40 times. The genes for DMT1, FPN1, β-actin (housekeeping gene), and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) gene (housekeeping gene) were taken from GenBank, and their primers were chosen with Primer Express Software (Applied Biosystems Inc., Foster, CA, USA); their information is summarized in Table 3. The geometric mean of β-actin and GAPDH was used to normalize the expression of target genes (Livak and Schmittgen, 2001; Zhu et al., 2015; Zhang et al., 2016a,b; Qin et al., 2016). Table 3. Primer sequences for real-time PCR amplification1 Gene  GenBank ID2  Primer sequences  Position, bp  Length, bp  β-actin  NM_205518.1  F: 5′-CGGTACCAATTACTGGTGTTAGATG-3′  1,518 to 1,680  163      R: 5′-GCCTTCATTCACATCTATCACTGG-3′      GAPDH  NM_204305.1  F: 5′-CTTTGGCATTGTGGAGGGTC-3′  552 to 679  128      R: 5′-ACGCTGGGATGATGTTCTGG-3′      DMT1  NM_001128102.1  F: 5′-AGCCGTTCACCACTTATTTCG-3′  223 to 352  130      R: 5′-GGTCCAAATAGGCGATGCTC-3′      FPN1  XM_015289163.1  F: 5′-GGAGACTGGGTGGACAAGAACTC-3′  558 to 625  68      R:5′-GATGCATTCTGAACAACCAAGGA-3′      Gene  GenBank ID2  Primer sequences  Position, bp  Length, bp  β-actin  NM_205518.1  F: 5′-CGGTACCAATTACTGGTGTTAGATG-3′  1,518 to 1,680  163      R: 5′-GCCTTCATTCACATCTATCACTGG-3′      GAPDH  NM_204305.1  F: 5′-CTTTGGCATTGTGGAGGGTC-3′  552 to 679  128      R: 5′-ACGCTGGGATGATGTTCTGG-3′      DMT1  NM_001128102.1  F: 5′-AGCCGTTCACCACTTATTTCG-3′  223 to 352  130      R: 5′-GGTCCAAATAGGCGATGCTC-3′      FPN1  XM_015289163.1  F: 5′-GGAGACTGGGTGGACAAGAACTC-3′  558 to 625  68      R:5′-GATGCATTCTGAACAACCAAGGA-3′      1GAPDH = glyceraldehyde-3-phosphate dehydrogenase; DMT1 = divalent metal transporter 1; FPN1 = ferroportin 1; F = forward; R = reverse. 2ID = identity. View Large Table 3. Primer sequences for real-time PCR amplification1 Gene  GenBank ID2  Primer sequences  Position, bp  Length, bp  β-actin  NM_205518.1  F: 5′-CGGTACCAATTACTGGTGTTAGATG-3′  1,518 to 1,680  163      R: 5′-GCCTTCATTCACATCTATCACTGG-3′      GAPDH  NM_204305.1  F: 5′-CTTTGGCATTGTGGAGGGTC-3′  552 to 679  128      R: 5′-ACGCTGGGATGATGTTCTGG-3′      DMT1  NM_001128102.1  F: 5′-AGCCGTTCACCACTTATTTCG-3′  223 to 352  130      R: 5′-GGTCCAAATAGGCGATGCTC-3′      FPN1  XM_015289163.1  F: 5′-GGAGACTGGGTGGACAAGAACTC-3′  558 to 625  68      R:5′-GATGCATTCTGAACAACCAAGGA-3′      Gene  GenBank ID2  Primer sequences  Position, bp  Length, bp  β-actin  NM_205518.1  F: 5′-CGGTACCAATTACTGGTGTTAGATG-3′  1,518 to 1,680  163      R: 5′-GCCTTCATTCACATCTATCACTGG-3′      GAPDH  NM_204305.1  F: 5′-CTTTGGCATTGTGGAGGGTC-3′  552 to 679  128      R: 5′-ACGCTGGGATGATGTTCTGG-3′      DMT1  NM_001128102.1  F: 5′-AGCCGTTCACCACTTATTTCG-3′  223 to 352  130      R: 5′-GGTCCAAATAGGCGATGCTC-3′      FPN1  XM_015289163.1  F: 5′-GGAGACTGGGTGGACAAGAACTC-3′  558 to 625  68      R:5′-GATGCATTCTGAACAACCAAGGA-3′      1GAPDH = glyceraldehyde-3-phosphate dehydrogenase; DMT1 = divalent metal transporter 1; FPN1 = ferroportin 1; F = forward; R = reverse. 2ID = identity. View Large Tissue Preparations and Western Blotting Frozen intestinal mucosa samples (40 mg) were crushed and homogenized in 0.4 mL of ice-cold Radio Immunoprecipitation Assay (RIPA) lysis buffer (catalog number P0013B, Beyotime, Haimen, China), to which protease inhibitors (catalog number 4693159001, Roche, Penzberg, Germany) were added. The homogenates were centrifuged at 12,000 × g for 4 min at 4°C, and the supernatants were harvested to determine the total protein using a BCA Protein Assay kit (catalog number 23225, Pierce, Appleton,WI). Then, the supernatants were added to SDS-PAGE loading buffer (catalog number CW0027A, CWBIO, Beijing, China) and put into boiling water (100°C) for protein degeneration. The degenerated proteins (20 μg) were separated by electrophoresis on a 9% SDS-PAGE gel and transferred onto nitrocellulose membranes (catalog number IPVH00010, Merck-Millipore, Billerica, MA). After that, the membranes were blocked in blocking buffer with 5% skim milk at room temperature for 1 h and then incubated overnight at 4°C with the following primary antibodies and dilution rates: DMT1, bs-3577R, 1:500 (Bioss, Beijing, China); FPN1, bs-4906R, 1:500 (Bioss); and GAPDH, ab22555, 1:5,000 (Abcam, Cambridge, MA). After 3 washes in Tris-buffered saline with Tween, the membranes were incubated with goat anti-rabbit horseradish peroxidase-conjugated secondary antibodies in a dilution of 1:5,000 (catalog number CW0103A; ComWin Biotech, Beijing, China) for 1 h at room temperature, and then, the membranes were washed 3 times using Tris-buffered saline with Tween. The signals were detected using a Super Signal West Pico Trial Kit (catalog number 34077, Pierce) and with an Image Quant LAS 4000 scanner (GE Healthcare Life Sciences, Pittsburgh, PA). The band density was analyzed using TotalLab Quant software (TotalLab, Newcastle on Tyne, UK), and the GAPDH protein was used to normalize the expression of the targeted proteins (Qin et al., 2016). Statistical Analyses The data for Fe absorption velocities were analyzed by 2-way ANOVA using the general linear model (GLM) procedure (SAS Inst. Inc., Cary, NC). The model included the effects of Fe source, perfused Fe concentration, and their interaction. The data for mRNA and protein expression levels of DMT1 and FPN1 were analyzed by single-factor ANOVA using GLM. If the variances were significant, differences among means were tested with the LSD method, and P ≤ 0.05 was considered to be statistically significant. Each ligated duodenal loop was used as the experimental unit. The kinetic analyses of Fe absorption in the ligated duodenal loops were performed as described by Zhang et al. (2016a). The kinetic parameters were analyzed by single-factor ANOVA using GLM. If the variances were significant, differences among means were tested using the LSD method. P ≤ 0.05 was considered to be statistically significant. RESULTS Effect of Fe Source on Fe Absorption in the Ligated Duodenal Loops of Broilers Data for Fe absorption are listed in Table 4. The Fe source and Fe concentration affected (P < 0.0001) Fe absorption in the ligated duodenum, but no interaction (P > 0.32) was observed. Iron absorption increased (P < 0.0001) as the perfused Fe concentrations increased. Iron absorption was higher (P < 0.05) for Fe-Pro ES and Fe-Pro M than for Fe-Met W and FeSO4·7H2O. However, no differences (P > 0.05) were detected in Fe absorption between Fe-Prot ES and Fe-Prot M or Fe-Met W and FeSO4·7H2O. Table 4. Effects of Fe source and Fe concentration on Fe absorption in the ligated duodenal loops of broilers1 Fe concentration, mM  Fe absorption, nmol·min−1·cm−1  Fe source2        FeSO4·7H2O  13.5B      Fe-Met W  14.8B      Fe-Prot M  17.6A      Fe-Prot ES  17.0A      SEM  0.7  Fe concentration3        0.90  5.3e      1.79  8.2d      3.58  16.7c      7.16  21.9b      14.33  28.3a      SEM  0.70  P-value4        Fe source  <0.0001      Fe concentration  <0.0001      Interaction  0.33  Fe concentration, mM  Fe absorption, nmol·min−1·cm−1  Fe source2        FeSO4·7H2O  13.5B      Fe-Met W  14.8B      Fe-Prot M  17.6A      Fe-Prot ES  17.0A      SEM  0.7  Fe concentration3        0.90  5.3e      1.79  8.2d      3.58  16.7c      7.16  21.9b      14.33  28.3a      SEM  0.70  P-value4        Fe source  <0.0001      Fe concentration  <0.0001      Interaction  0.33  A,BMean values lacking a common superscript within a column differ (P < 0.05). a–eMean values lacking a common superscript within a column differ (P < 0.05). 1Fe-Met W, Fe methionine with weak complex strength, quotient of formation (Qf) = 1.37 (DeBon Agri-TECH Group, Shanghai, China); Fe-Prot M, Fe proteinate with moderate chelation strength, Qf = 43.6 (Alltech Inc., Nicholasville, KY); Fe-Prot ES, Fe proteinate with extremely strong chelation strength, Qf = 8,590 (Hebei Amino Acid Co., Baoding, China). 2Data represent the means of 50 replicates (n = 50). 3Data represent the means of 40 replicates (n = 40). 4Probabilities of main effects. View Large Table 4. Effects of Fe source and Fe concentration on Fe absorption in the ligated duodenal loops of broilers1 Fe concentration, mM  Fe absorption, nmol·min−1·cm−1  Fe source2        FeSO4·7H2O  13.5B      Fe-Met W  14.8B      Fe-Prot M  17.6A      Fe-Prot ES  17.0A      SEM  0.7  Fe concentration3        0.90  5.3e      1.79  8.2d      3.58  16.7c      7.16  21.9b      14.33  28.3a      SEM  0.70  P-value4        Fe source  <0.0001      Fe concentration  <0.0001      Interaction  0.33  Fe concentration, mM  Fe absorption, nmol·min−1·cm−1  Fe source2        FeSO4·7H2O  13.5B      Fe-Met W  14.8B      Fe-Prot M  17.6A      Fe-Prot ES  17.0A      SEM  0.7  Fe concentration3        0.90  5.3e      1.79  8.2d      3.58  16.7c      7.16  21.9b      14.33  28.3a      SEM  0.70  P-value4        Fe source  <0.0001      Fe concentration  <0.0001      Interaction  0.33  A,BMean values lacking a common superscript within a column differ (P < 0.05). a–eMean values lacking a common superscript within a column differ (P < 0.05). 1Fe-Met W, Fe methionine with weak complex strength, quotient of formation (Qf) = 1.37 (DeBon Agri-TECH Group, Shanghai, China); Fe-Prot M, Fe proteinate with moderate chelation strength, Qf = 43.6 (Alltech Inc., Nicholasville, KY); Fe-Prot ES, Fe proteinate with extremely strong chelation strength, Qf = 8,590 (Hebei Amino Acid Co., Baoding, China). 2Data represent the means of 50 replicates (n = 50). 3Data represent the means of 40 replicates (n = 40). 4Probabilities of main effects. View Large Iron Absorption Kinetics of Different Fe Sources in the Ligated Duodenal Loops of Broilers The results of the regression analyses showed that the best models for Fe absorption kinetics of FeSO4·7H2O, Fe-Met W, Fe-Prot M, and Fe-Prot ES in the ligated duodenal loops of broilers were the saturated carrier-dependent transport process equations (Table 5; Akaike information criterion is 3.39, 3.17, 3.27, and 3.10, respectively; Akaike, 1976). The kinetic analyses of Fe absorption from different Fe sources were performed by graphical means (Fig. 1). The value of the maximum velocity of Fe (Jmax) of Fe-Prot M was greater (P < 0.05) than those of the other 3 Fe sources, and the Jmax value of Fe-Prot ES was greater (P < 0.05) than those of Fe-Met W and FeSO4·7H2O; however, no differences (P > 0.05) were found in the Jmax values between Fe-Met W and FeSO4·7H2O. In addition, no differences (P > 0.05) were detected among the Michaelis-Menten constants (Km) of these 4 Fe sources. Table 5. Kinetic and statistical parameters obtained after fitting Michaelis-Menten equations to the experimental data of Fe absorption in the ligated duodenal loops of broilers1 Fe source  Kinetic parameters2  Statistical parameters3  Jmax, nmol·min−1·cm−1  Km, mM  R2  AIC  FeSO4·7H2O  35.7 ± 5.3c  6.50 ± 2.11  0.62  3.39  Fe-Met W  36.3 ± 3.6c  5.51 ± 1.25  0.75  3.17  Fe-Prot M  46.9 ± 4.8a  6.31 ± 1.41  0.80  3.27  Fe-Prot ES  43.1 ± 3.4b  5.40 ± 0.96  0.84  3.10  Fe source  Kinetic parameters2  Statistical parameters3  Jmax, nmol·min−1·cm−1  Km, mM  R2  AIC  FeSO4·7H2O  35.7 ± 5.3c  6.50 ± 2.11  0.62  3.39  Fe-Met W  36.3 ± 3.6c  5.51 ± 1.25  0.75  3.17  Fe-Prot M  46.9 ± 4.8a  6.31 ± 1.41  0.80  3.27  Fe-Prot ES  43.1 ± 3.4b  5.40 ± 0.96  0.84  3.10  a–cMean values lacking a common superscript within a column differ (P < 0.05). 1Values were expressed as means ± SD, n = 10. 2Jmax represents the maximum absorption rate of Fe; Km is the Michaelis-Menten constant. 3R2 is the coefficient of determination; AIC is Akaike's information criterion. View Large Table 5. Kinetic and statistical parameters obtained after fitting Michaelis-Menten equations to the experimental data of Fe absorption in the ligated duodenal loops of broilers1 Fe source  Kinetic parameters2  Statistical parameters3  Jmax, nmol·min−1·cm−1  Km, mM  R2  AIC  FeSO4·7H2O  35.7 ± 5.3c  6.50 ± 2.11  0.62  3.39  Fe-Met W  36.3 ± 3.6c  5.51 ± 1.25  0.75  3.17  Fe-Prot M  46.9 ± 4.8a  6.31 ± 1.41  0.80  3.27  Fe-Prot ES  43.1 ± 3.4b  5.40 ± 0.96  0.84  3.10  Fe source  Kinetic parameters2  Statistical parameters3  Jmax, nmol·min−1·cm−1  Km, mM  R2  AIC  FeSO4·7H2O  35.7 ± 5.3c  6.50 ± 2.11  0.62  3.39  Fe-Met W  36.3 ± 3.6c  5.51 ± 1.25  0.75  3.17  Fe-Prot M  46.9 ± 4.8a  6.31 ± 1.41  0.80  3.27  Fe-Prot ES  43.1 ± 3.4b  5.40 ± 0.96  0.84  3.10  a–cMean values lacking a common superscript within a column differ (P < 0.05). 1Values were expressed as means ± SD, n = 10. 2Jmax represents the maximum absorption rate of Fe; Km is the Michaelis-Menten constant. 3R2 is the coefficient of determination; AIC is Akaike's information criterion. View Large Figure 1. View largeDownload slide Effect of Fe source on the kinetic curves of Fe absorption in the ligated duodenal loops of Fe-deficient broilers. The ligated duodenal loops (n = 10) were perfused with the Fe-free solution (control) or 1 of the solutions containing 0.90 to 14.33 mmol Fe/L from Fe sulfate (FeSO4∙7H2O), Fe methionine with weak chelation strength (Fe-Met W, quotient of formation [Qf] = 1.37; DeBon Agri-TECH Group, Shanghai, China), Fe proteinate with moderate chelation strength (Fe-Prot M, Qf = 43.6; Alltech Inc., Nicholasville, KY), or Fe proteinate with extremely strong chelation strength (Fe-Prot ES, Qf = 8,590; Hebei Amino Acid Co., Baoding, China). At 30 min after perfusion, Fe absorption (disappearance of Fe from the ligated duodenal loop) was determined, and the initial rate of Fe absorption was calculated. Values of Fe absorption rates are means, with their SD represented by vertical bars. All kinetic curves of Fe absorption from different Fe sources in the duodenum are described by the Michaelis-Menten equation (a saturated process). Figure 1. View largeDownload slide Effect of Fe source on the kinetic curves of Fe absorption in the ligated duodenal loops of Fe-deficient broilers. The ligated duodenal loops (n = 10) were perfused with the Fe-free solution (control) or 1 of the solutions containing 0.90 to 14.33 mmol Fe/L from Fe sulfate (FeSO4∙7H2O), Fe methionine with weak chelation strength (Fe-Met W, quotient of formation [Qf] = 1.37; DeBon Agri-TECH Group, Shanghai, China), Fe proteinate with moderate chelation strength (Fe-Prot M, Qf = 43.6; Alltech Inc., Nicholasville, KY), or Fe proteinate with extremely strong chelation strength (Fe-Prot ES, Qf = 8,590; Hebei Amino Acid Co., Baoding, China). At 30 min after perfusion, Fe absorption (disappearance of Fe from the ligated duodenal loop) was determined, and the initial rate of Fe absorption was calculated. Values of Fe absorption rates are means, with their SD represented by vertical bars. All kinetic curves of Fe absorption from different Fe sources in the duodenum are described by the Michaelis-Menten equation (a saturated process). Effect of Fe Source on the Gene Expression of DMT1 and FPN1 in the Ligated Duodenal Loops of Broilers The DMT1 mRNA levels were 30% to 50% lower (P < 0.05) in the duodenal loops perfused with solutions containing different Fe sources than in those perfused with the Fe-free control solution (Fig. 2). Although the duodenal DMT1 mRNA levels in the Fe-Prot M and Fe-Prot ES groups were numerically higher than those in the FeSO4·7H2O and Fe-Met W groups, no differences (P > 0.15) were detected among the Fe treatment groups. The FPN1 mRNA levels of duodenal loops in the Fe treatment groups were 2 to 3 times (P < 0.0008) those in the control group (Fig. 2), but no differences (P > 0.88) were detected among the Fe treatment groups. In addition, there were no significant differences (P > 0.14; Fig. 3) in the duodenal DMT1 and FPN1 protein expression levels among all of these groups. Figure 2. View largeDownload slide Effect of Fe source on the divalent metal transporter 1 (DMT1) and ferroportin 1 (FPN1) mRNA levels in the ligated duodenal loops of Fe-deficient broilers at 30 min after perfusion as determined by real-time quantitative PCR. The treatments included a Fe-free basal solution (control) and the basal solutions supplemented with 3.58 mmol Fe/L from Fe sulfate (FeSO4∙7H2O), Fe methionine with weak chelation strength (Fe-Met W, quotient of formation [Qf] = 1.37; DeBon Agri-TECH Group, Shanghai, China), Fe proteinate with moderate chelation strength (Fe-Prot M, Qf = 43.6; Alltech Inc., Nicholasville, KY), and Fe proteinate with extremely strong chelation strength (Fe-Prot ES, Qf = 8,590; Hebei Amino Acid Co., Baoding, China). Data are presented in arbitrary units as relative mRNA abundances normalized to the geometric means of β-actin (housekeeping gene) and glyceraldehyde 3-phosphate dehydrogenase (GAPDH; housekeeping gene) mRNA abundances. Values are means, with their SE represented by vertical bars (n = 10). a,bMean values with different letters were significantly different (P < 0.05). Figure 2. View largeDownload slide Effect of Fe source on the divalent metal transporter 1 (DMT1) and ferroportin 1 (FPN1) mRNA levels in the ligated duodenal loops of Fe-deficient broilers at 30 min after perfusion as determined by real-time quantitative PCR. The treatments included a Fe-free basal solution (control) and the basal solutions supplemented with 3.58 mmol Fe/L from Fe sulfate (FeSO4∙7H2O), Fe methionine with weak chelation strength (Fe-Met W, quotient of formation [Qf] = 1.37; DeBon Agri-TECH Group, Shanghai, China), Fe proteinate with moderate chelation strength (Fe-Prot M, Qf = 43.6; Alltech Inc., Nicholasville, KY), and Fe proteinate with extremely strong chelation strength (Fe-Prot ES, Qf = 8,590; Hebei Amino Acid Co., Baoding, China). Data are presented in arbitrary units as relative mRNA abundances normalized to the geometric means of β-actin (housekeeping gene) and glyceraldehyde 3-phosphate dehydrogenase (GAPDH; housekeeping gene) mRNA abundances. Values are means, with their SE represented by vertical bars (n = 10). a,bMean values with different letters were significantly different (P < 0.05). Figure 3. View largeDownload slide Effect of Fe source on divalent metal transporter 1 (DMT1) ferroportin 1 (FPN1) protein levels in the ligated duodenal loops of Fe-deficient broilers at 30 min after perfusion as determined by Western blotting. The treatments included a Fe-free basal solution (control) and the basal solution supplemented with 3.58 mmol Fe/L from Fe sulfate (FeSO4∙7H2O), Fe methionine with weak chelation strength (Fe-Met W, quotient of formation [Qf] = 1.37; DeBon Agri-TECH Group, Shanghai, China), Fe proteinate with moderate chelation strength (Fe-Prot M, Qf = 43.6; Alltech Inc., Nicholasville, KY), or Fe proteinate with extremely strong chelation strength (Fe-Prot ES, Qf = 8,590; Hebei Amino Acid Co., Baoding, China). Data are presented in arbitrary units as relative protein levels normalized to the glyceraldehyde 3-phosphate dehydrogenase (GAPDH; housekeeping gene) protein levels. Values are means, with their SE represented by vertical bars (n = 10). Figure 3. View largeDownload slide Effect of Fe source on divalent metal transporter 1 (DMT1) ferroportin 1 (FPN1) protein levels in the ligated duodenal loops of Fe-deficient broilers at 30 min after perfusion as determined by Western blotting. The treatments included a Fe-free basal solution (control) and the basal solution supplemented with 3.58 mmol Fe/L from Fe sulfate (FeSO4∙7H2O), Fe methionine with weak chelation strength (Fe-Met W, quotient of formation [Qf] = 1.37; DeBon Agri-TECH Group, Shanghai, China), Fe proteinate with moderate chelation strength (Fe-Prot M, Qf = 43.6; Alltech Inc., Nicholasville, KY), or Fe proteinate with extremely strong chelation strength (Fe-Prot ES, Qf = 8,590; Hebei Amino Acid Co., Baoding, China). Data are presented in arbitrary units as relative protein levels normalized to the glyceraldehyde 3-phosphate dehydrogenase (GAPDH; housekeeping gene) protein levels. Values are means, with their SE represented by vertical bars (n = 10). DISCUSSION A series of studies on organic Fe bioavailabilities have demonstrated that organic Fe sources were more bioavailable for chicks than inorganic sources (Shinde et al., 2011; Ma et al., 2014; Kwiecień et al., 2015; Zhang et al., 2016b). Chemical characteristics are considered to be important in predicting the bioavailabilities of complexed or chelated metals. Several studies have shown that the bioavailabilities of organic Mn, Zn, Cu, and Fe sources are closely related to their Qf values (Li et al., 2004, 2005; Huang et al., 2009, 2013; Liu et al., 2012, 2013; Ma et al., 2014; Zhang et al., 2016b). In the present study, Fe absorption of Fe-Pro ES and Fe-Prot M in the ligated duodenum of broilers was greater than that of FeSO4·7H2O and Fe-Met W, indicating that the organic Fe sources with greater Qf values had more Fe absorption. Our findings are consistent with the results from our earlier studies on Fe bioavailabilities for broilers (Ma et al., 2014; Zhang et al., 2016b) and thus provide further support for the higher bioavailabilities of organic Fe sources with greater Qf values. The results from the current study showed that Fe absorption in the ligated duodenum increased as supplemental Fe concentration increased, regardless of Fe source. Similar findings have been observed by Li et al. (2016), who reported that supplemental Fe could increase duodenal Fe absorption in broilers with deficient Fe stores. There are 2 hypotheses concerning the absorption and utilization mechanisms of mineral complexes (Ashmead, 1993). The first hypothesis is that an organic mineral complex or chelate with optimal Qf could resist interference from dietary and nutritional factors in the digestive tract and directly reach the intestinal brush border, where it is hydrolyzed and absorbed as ions, resulting in higher bioavailability of the complexed or chelated metal than the inorganic form of the metal (Cook et al., 1972). The second hypothesis is that an organic mineral complex or chelate with optimal Qf could maintain its structural integrity in the digestive tract and arrive at absorptive sites in the small intestine as the original intact molecules (Ashmead, 2001). If the second hypothesis is true, Fe-Prot M and Fe-Prot ES, which were more difficult to dissociate, might be chelated in the digestive tract and absorbed as the original intact molecules in the small intestine, especially the duodenum, the main site of Fe absorption (Zhang et al., 2016a), whereas Fe-Met W, being easy to dissociate, might be absorbed mainly as Fe ions. However, until now, there has been no direct evidence supporting either of these hypotheses, mainly because of the lack of effective methods to test how the organic mineral complexes or chelates are absorbed. Kinetic results of Fe absorption in the present study suggest that Fe absorption from different Fe sources is a saturated carrier-dependent transport process in the ligated duodenum of broilers regardless of Fe source. Nevertheless, in comparing the Jmax values, we found that the Jmax values for Fe-Prot ES and Fe-Prot M were greater than those for Fe-Met and FeSO4·7H2O, suggesting that the saturated transport system for Fe-Prot ES and Fe-Prot M has a greater capacity than that for Fe-Met and FeSO4·7H2O in the duodenum. In addition, the results showing no differences in the Km values among Fe sources imply that transport systems for different Fe sources might have similar affinities for Fe ions and chelated Fe. The mechanism for the transport of inorganic and heme Fe across enterocytes has been well studied (Gunshin et al., 1997; Ferris et al., 1999; Donovan et al., 2000; McKie et al., 2000; Qiu et al., 2006). Inorganic Fe3+ must be reduced to Fe2+ first before it can be utilized (McKie, 2008), and then, Fe2+ is transported into the intestinal epithelium via DMT1 (Gunshin et al., 1997). After Fe reaches the basolateral membrane of enterocytes, cytosolic Fe will be exported into the bloodstream by the basolateral Fe exporter FPN1 (Donovan et al., 2000; McKie et al., 2000) in conjunction with the ferroxidase hephaestin (Ma et al., 2014). In the present study, duodenal absorption of Fe was greater for Fe-Prot ES and Fe-Prot M than for Fe-Met W and FeSO4·7H2O, which might explain why Fe-Prot ES and Fe-Prot M had higher bioavailabilities than Fe-Met W and FeSO4·7H2O in our previous study (Zhang et al., 2016b). However, how organic Fe sources such as Fe AA or proteinates are transferred to the bloodstream is unclear. Some studies have suggested that chelates or complexes of minerals and amino acids might be absorbed intact (Suso and Edwards, 1971, 1972; Kratzer and Vohra, 1986; Ashmead, 1993). Chelates of metals and AA or proteinates can cause the metal atoms to remain safely bound or protected within organic molecular structures or ligands (Kratzer and Vohra, 1986; Ashmead, 1993). Additionally, Zn-EDTA with extremely strong chelation strength is transported across enterocytes to the bloodstream as an intact complex (Suso and Edwards, 1971, 1972). In the current study, it is still unclear whether organic Fe with higher Qf was absorbed in the duodenum as ions or as intact chelates or both. However, our findings provide indirect evidence for the differences between inorganic and organic Fe absorption in the small intestine of chicks. Previous studies (Combs and Pesti, 1976; Li et al., 2016; Liu et al., 2016; Zhang et al., 2016a) have confirmed that intact intestinal morphology and functions were well maintained in the ligated small intestinal loops of chicks. To a larger extent, these results relieve the concern about the viability of enterocytes after ligation. The divalent metal transporter 1 is abundantly expressed in the intestine (Canonne-Hergaux et al., 2001a,b; Qian and Shen, 2001; Mete et al., 2005), and its mRNA expression is upregulated by the low Fe status in intestinal epithelial cells of rats (Gunshin et al., 1997). Subsequently, a similar result was observed by Fleming et al. (1999). Trinder et al. (2000) demonstrated that compared with Fe-abundant rats, Fe-deficient rats had higher DMT1 mRNA expression levels in the duodenum. Yeh et al. (2000) also reported that a bolus of dietary Fe resulted in a gradual decrease of duodenal DMT1 mRNA. Some studies in bull calves and pigs have also shown a depressive effect of high dietary Fe on duodenal DMT1 and FPN1 expression (Hansen et al., 2009; 2010a,b). The Fe2+ ion is highly reactive and can be easily oxidized to induce cell and tissue injury (Ganz and Nemeth, 2012); therefore, the decrease in DMT1 mRNA levels in the ligated duodenal loops might be partially due to high mucosal Fe accumulation, which signaled for the downregulation of duodenal DMT1 mRNA expression to prevent toxic accumulation of Fe. Furthermore, the elevations in FPN1 mRNA expression in duodenal loops treated with Fe in the present study suggest that the Fe flux from the epithelial cells to the bloodstream might increase, and then damage from Fe accumulation in the duodenal mucosa would be avoided. A previous study showed that compared with those perfused with FeSO4, the rats perfused with Fe-Gly chelate had lower DMT1 and FPN1 mRNA, or DMT1 protein expression but higher FPN1 protein expression in the duodenum 4 h after perfusion (Zhuo et al., 2014). However, in the present study, no differences were detected in the duodenal DMT1 and FPN1 mRNA or protein expression levels among the groups of different Fe sources, which might be due to the very short time (30 min) of Fe perfusion. Some researchers demonstrated that the maximal abundance of protein might occur a few hours after the increased amounts of corresponding mRNA (Blalock et al., 1988; Luo et al., 2007; Li et al., 2011). Similarly, Knutson et al. (2003) observed that the FPN1 mRNA level in the mouse macrophages increased 2 h after erythrophagocytosis, although the FPN1 protein expression did not change. These findings might explain the phenomenon in the present study that the DMT1 and FPN1 protein levels in the duodenum were less sensitive than their mRNA levels in reflecting the changes in Fe status in the small intestine of chicks. The data for the duodenal DMT1 and FPN1 gene expression in the present study failed to explain the differences among Fe sources in the duodenal Fe absorption of broilers but gave a strong hint that besides DMT1 and FPN1, other carriers such as AA or small peptide carriers might be involved in Fe transport of organic Fe sources in the duodenum of broilers. Therefore, further studies are needed to address these matters in the future. In conclusion, organic Fe sources with greater Qf values showed increased Fe absorption in the duodenum of broilers. Kinetic models indicated that the absorption of organic or inorganic Fe in the duodenum followed the same saturated carrier-dependent transport process. The Fe supplementation decreased DMT1 mRNA expression and enhanced FPN1 mRNA expression but did not affect DMT1 and FPN1 protein expression in the in situ ligated duodenum of broilers. 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TI - Effect of iron source on iron absorption and gene expression of iron transporters in the ligated duodenal loops of broilers
JF - Journal of Animal Science
DO - 10.2527/jas.2016.1147
DA - 2017-04-01
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VL - 95
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