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J. Anim. Sci. 2005. 83:2137-2145
© 2005 American Society of Animal Science

ANIMAL NUTRITION

Effects of iron supplementation on binding activity of iron regulatory proteins and the subsequent effect on growth performance and indices of hematological and mineral status of young pigs1,2

M. J. Rincker*, S. L. Clarke{dagger}, R. S. Eisenstein{dagger}, J. E. Link* and G. M. Hill*,3

* Department of Animal Science, Michigan State University, East Lansing 48824; and and {dagger} Department of Nutritional Sciences, University of Wisconsin, Madison 53706


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results and Discussion
 Implications
 Literature Cited
 
Two experiments were conducted to evaluate the effects of supplemental Fe on the binding activity of iron regulatory proteins (IRP) and the subsequent effect on growth performance and indices of hematological and mineral status of young pigs. In Exp. 1, male pigs (n = 10; 1.8 kg; age = 14 ± 1 h) were allotted by BW to two treatments (five pigs per treatment). Treatments administered by i.m. injection were as follows: 1) 1 mL of sterile saline solution (Sal); and 2) 1 mL of 200 mg Fe as Fe-dextran (Fe). Pigs were bled (d 0 and 13) to determine hemoglobin (Hb), hematocrit (Hct), transferrin (Tf), and plasma Fe (PFe), and then killed (d 13) to determine spontaneous and 2-mercaptoethanol (2-ME)-inducible IRP RNA binding activity in liver and liver and whole-body mineral concentrations. Contemporary pigs (n = 5; 2.2 kg; age = 14 ± 2 h) were killed at d 0 to establish baseline (BL1) measurements. In Exp. 2, pigs (six pigs per treatment; 6.5 kg; age = 19 ± 3 d) were fed a basal diet (Phase 1 = d 0 to 7; Phase 2 = d 7 to 21; Phase 3 = d 21 to 35) supplemented with 0 or 150 mg/kg of Fe as ferrous sulfate and killed at d 35 (18.3 kg; age = 54 ± 3 d). In addition, pigs (n = 5; 5.9 kg; age = 19 ± 3 d) were killed at the start of Exp. 2 to establish baseline (BL2) measurements, and liver samples were collected and analyzed for IRP RNA binding activity. In Exp. 1, no difference (P = 0.482) was observed in ADG. On d 13, Fe-treated pigs had greater (P = 0.001) Hb, Hct, and PFe and less (P = 0.002) Tf than Sal-treated pigs. Whole-body Fe concentration was greater (P = 0.002) in Fe- vs. Sal-treated pigs. Treated pigs (Fe or Sal) had greater (P = 0.006) whole-body Cu and less (P = 0.002) whole-body Ca, Mg, Mn, P, and Zn concentrations than BL1. Liver Fe concentration was greater (P = 0.001) in Fe- vs. Sal-treated pigs, but liver Fe concentration of Sal-treated pigs was less (P = 0.001) than that of BL1 pigs. Sal-treated pigs had greater (P = 0.004) spontaneous IRP binding activity than Fe-treated pigs. In Exp. 2, spontaneous and 2-ME inducible IRP binding activities were greater (P = 0.013 and 0.005, respectively) in pigs fed diets containing 0 vs. 150 mg of added Fe/kg of diet. Moreover, pigs fed either treatment for 35 d had greater (P = 0.001) 2-ME inducible IRP binding activity than BL2 pigs. Results indicate that IRP binding activity is influenced by Fe supplementation. Subsequently, other indicators of Fe status are affected via the role of IRP in posttranscriptional expression of Fe storage and transport proteins.

Key Words: Growth • Iron Regulatory Protein • Pig • Transferrin • Whole Body


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results and Discussion
 Implications
 Literature Cited
 
The use of an exogenous Fe source to prevent Fe deficiency in young pigs has been well documented (Ullrey et al., 1959Go; Pollmann et al., 1983Go), and it is standard practice in the swine industry. Iron regulatory proteins (IRP) are RNA binding proteins that control the posttranscriptional expression of proteins required for the uptake, storage, and use of Fe (Eisenstein, 2000Go).

Located within the cytoplasm, IRP bind to a highly conserved, 28-bp nucleotide sequence known as an iron responsive element (IRE) sequence (Aziz and Munro, 1987Go). Examples of proteins whose transcribed mRNA contain an IRE include erythroid-aminolevulinate synthase (eALAS), mitochondrial aconitase (m-acon), transferrin receptor (TfR), ferroportin, and ferritin (Eisenstein, 2000Go). Depending on the location of the IRE in either the 5'- or 3'-untranslated region (UTR) of mRNA, binding of an IRP to IRE inhibits or stabilizes translation, respectively. Two isoforms of IRP have been identified, IRP1 and IRP2, with both forms exhibiting similar affinity for an IRE sequence (Beard and Dawson, 1997Go). The binding activity of IRP1 is influenced by Fe status due to the bifunctional nature of the protein existing as either an RNA-binding protein or as the cytosolic isoform of the tricarboxylic acid enzyme aconitase (c-acon). Iron regulatory protein two does not exhibit aconitase activity, and its abundance is decreased during Fe sufficiency due to increased protein degradation (Guo et al., 1995Go). Iron deficiency increases binding activity, whereas Fe sufficiency decreases binding activity (Leibold and Munro, 1988Go; Rouault et al., 1988Go).

The objectives of our research were to evaluate the effects of supplemental Fe on IRP RNA binding activity and the subsequent effect on growth and indices of hematological and mineral status. Experiment 1 compared neonatal pigs administered Fe-dextran vs. saline, whereas Exp. 2 evaluated adding 0 and 150 mg of supplemental Fe as ferrous sulfate/kg of diet in nursery pigs.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results and Discussion
 Implications
 Literature Cited
 
Animal Use and Care
These experiments were conducted at the Michigan State University Swine Teaching and Research Facility. Use of animals in these experiments was approved by the All-University Committee on Animal Use and Care (Animal Use Form No. 12/02-164-00).

Experiment 1
Animals, Treatments, and Housing.
Ten male pigs (Duroc x [Landrace x Yorkshire]) were used during a 13-d experiment. Pigs were a product of pooled Duroc semen, representing six different boars, that was inseminated into a single sow. By having one sow as the source of milk (nutrition), the influence of environment was decreased. After parturition, pigs were allowed to nurse the sow for 13 to 15 h and then randomly allotted on the basis of initial BW (mean = 1.8 kg) to two treatments in a completely randomized design. Treatments were administered via i.m. injection and were as follows: 1) 1 mL of 0.9% (wt/vol) sterile saline solution (physiological saline solution, Phoenix Pharmaceutical, Inc., St. Joseph, MO; Sal); and 2) 1 mL of 200 mg Fe as Fe-dextran (Duravet Iron Dextran-200, Phoenix Pharmaceutical, Inc.; Fe). There were five pigs per treatment. After administration of the experimental treatment, pigs were returned to the sow’s pen and remained there until completion of the experiment. To prevent blood loss during the experiment, pigs were not castrated, tail docked, or ear notched. For identification purposes, a number was written on the back of each pig with a permanent marker. During the 13-d experiment, the sow was managed according to facility standard operating procedures. A colostrum sample was collected approximately 5 h after parturition, and milk samples were collected periodically during the 13-d experiment to determine Fe concentration. The colostrum and milk samples were collected from the same four teats each time, one anterior and one posterior on each side, and samples were collected at the initiation of a suckling period by pigs.

Pigs were housed in a temperature-controlled room and grouped in a farrowing pen that provided 3.25 m2 of total space. Within the farrowing pen, the sow was located in a hardened-steel-rod farrowing stall (Delphi Products Co., Delphi, IN) that occupied 1.24 m2 of space. Pens were located on hardened-steel-rod flooring (Nooyen Mfg., Inc., Mt. Sterling, KY). Room temperature was maintained at 19 to 21°C, and zone heating (0.45 m2 of space) was provided to neonatal pigs by an 85-W heat pad (Osborne Industries Inc., Osborne, KS) located on the floor on one side of the farrowing pen. Exogenous Fe contamination from environmental surroundings was avoided insofar as possible.

Performance, Blood, Tissue, and Whole-Body Collection.
Pigs were weighed 13 to 15 h after farrowing (d 0) and then at the conclusion of the 13-d experiment for the determination of ADG. Pigs were bled on d 0 and 13 by jugular venipuncture. Initial blood samples were collected using a 5-mL glass syringe (181 USP units of sodium heparin per syringe) with a 22-gauge, 2.54-cm needle. Before blood collection, glass syringes were soaked in 30% (vol/vol) nitric acid for 6 h, rinsed five times with double-deionized water, and allowed to air-dry. Blood samples on d 13 were drawn by jugular venipuncture into a 10-mL heparinized (143 USP units of sodium heparin per tube) Vacutainer (Becton Dickinson, Franklin Lakes, NJ) tube with a 21-gauge, 3.81-cm needle. From both collections, an aliquot of whole blood was transferred to a polypropylene tube and stored on ice until hemoglobin (Hb) and hematocrit (Hct) analysis could be performed later that day. The remaining blood was centrifuged at 2,000 x g, 4°C, for 10 min (Beckman GS-6KR, Palo Alto, CA). Plasma was stored in polypropylene tubes at –80°C until plasma Fe (PFe) and transferrin (Tf) analyses.

At the conclusion of the experiment, pigs (mean BW = 4.7 kg; age = 13 d) were killed via cardiac injection of sodium pentobarbital (87 mg/kg of BW). Liver samples were excised and stored (–80°C) in Whirl-Pak bags (Nasco, Fort Atkinson, WI) until analyzed for minerals. A second liver aliquot approximately equal in amount to the first was flash-frozen in liquid N2 for the determination of IRP-RNA binding activity. The remaining whole-body of each pig was frozen and ground (model 4732 stainless steel meat chopper, Hobart Corp., Troy, OH) three times through a 12.7-mm aperture plate, mixed, and subsampled. Subsamples were freeze-dried (Tri-Philizer MP, FTS Systems Inc., Stone Ridge, NY) and further processed to decrease particle size by submersion in liquid N2 and blending in a 1.5-L stainless steel blender (Waring Products Co., New Hartford, CT). Dried, ground subsamples were then stored in Whirl-Pak bags until analyzed for minerals and chemical composition. Additionally, five 14-h-old male littermate pigs of the same farrowing group and similar genetic background (Duroc x [Landrace x Yorkshire]; mean BW = 2.2 kg; age = 14 ± 1 h) were killed to establish baseline (BL1) measurements for mineral concentrations, IRP binding activity, and chemical composition. Liver and whole-body samples were collected as previously described.

Mineral, Blood, and Whole-Body Chemical Composition Analyses.
Liver, whole-body, and milk samples were analyzed for minerals (Ca, Cu, Fe, Mg, Mn, P, and Zn) as previously described by Rincker et al. (2004)Go. Plasma samples were analyzed for Fe via graphite furnace atomic absorption spectrophotometry (GF90 plus; Thermo Elemental Corp., Franklin, MA). Analysis of whole-body (protein, lipid, and ash) and blood (Hb and Hct) samples also was performed by methods described earlier (Rincker et al., 2004Go). All analyses were performed in duplicate. Liver mineral concentrations were reported on a wet basis, whereas whole-body mineral concentrations were reported on a DM basis.

Preparation and Analysis of IRP Binding Activity.
Flash-frozen liver samples were minced with a sterile scalpel and homogenized in four volumes of HDGC buffer (20 mmol/L of HEPES pH 7.4, 1 mmol/L of dithiothreitol, 10% [vol/vol] glycerol, and 2 mmol/L of trisodium citrate) including protease inhibitors (40 mg/L of leupeptin, 0.4 mg/L of pepstatin, 34.8 mg/L of phenylmethylsulfonyl fluoride, 4.8 mg/L of MG132, and 100 mg/L of soybean trypsin inhibitor) using a 15-mL Potter-Elvehjem homogenizer (Tight pestle A, Wheaton Science Products, Millville, NJ). Liver cytosol was obtained by differential centrifugation. First, the homogenate was centrifuged at 10,000 x g, 4°C, for 15 min (Sorvall Superspeed RC2-B, Kendro Laboratory Products, Asheville, NC). The supernatant fraction was then transferred to a new tube and spun at 100,000 x g, 4°C, for 1 h (Beckman L-80 ultracentrifuge) to obtain the liver cytosol. Protein concentration of the liver cytosol was determined by the Bradford Reagent (Sigma, B-6916) assay using BSA (Sigma, A-2153) as a standard (Sigma-Aldrich Co., St. Louis, MO).

Protein-RNA binding activity was performed by gel shift analysis using [32P]RNA of the first 73 nucleotides of the rat L-ferritin 5'UTR (Schalinske and Eisenstein, 1996Go). Briefly, cell extracts to be analyzed for IRE binding activity were incubated with saturating levels of [32P]RNA (1 nmol/L) in the presence of a final concentration of 5% (vol/vol) glycerol, 1 mmol/L of magnesium acetate, 20 mmol/L of HEPES pH 7.5, 75 mmol/L of potassium chloride, and 20 mg/L of nuclease free BSA in a final volume of 30 µL. Binding reactions were started by the addition of [32P]RNA and were performed at room temperature for 10 min. Then, 3 µL of heparin (5 g/L in deionized diethylpyrocarbonate-treated water) were added, and the sample incubated for another 5 min, after which 25 µL of the sample was loaded onto a native 4% polyacrylamide (60:1 acrylamide/bisacrylamide) gel in 0.5x Tris-borate-EDTA buffer (Barton et al., 1990Go). The samples were electrophoresed at 160 V for 50 min. The optimal amount of protein used for determination of spontaneous RNA binding activity was 10 µg of cytosolic protein. The amount of IRP present in active and inactive (i.e., both RNA binding and c-acon) forms was assessed using 4% 2-mercaptoethanol (2-ME; Klausner et al., 1993Go). In the presence of 2-ME, 2.5 µg of cytosolic protein was the optimal amount used for the gel shift assay. Iron regulatory protein-RNA binding activity was quantified by liquid scintillation counting (Beckman LS 5000TD) as described by Schalinske and Eisenstein (1996)Go. Results are expressed as picomoles of [32P]RNA bound per milligrams of cytosolic protein. Additional details of the gel shift assay have been described previously (Barton et al., 1990Go).

Gel Electrophoresis and Western Blot Analysis.
Tissue IRP1 and IRP2 presence was determined by SDS-PAGE followed by Western blot analysis using a rabbit polyclonal antibody raised against rat liver IRP1 and IRP2. Iron regulatory proteins were analyzed using 30 µg of liver homogenate protein. After proteins were electrophoretically separated in 8% polyacrylamide gels, they were electrophoretically transferred to polyvinylidene difluoride membranes. Next, membranes were blocked for 2 h at room temperature in PBS solution, pH 7.4 (5.0% [wt/vol] nonfat dry milk, 0.1% [vol/vol] Tween-20, 8.0 g/L of sodium chloride, 0.2 g/L of potassium chloride, 1.4 g/L of dibasic sodium phosphate, and 0.2 g/L of monobasic potassium phosphate). Membranes were then incubated with the anti-IRP1 and IRP2 polyclonal antibody (1:5,000 dilution) in PBS solution, followed by washing in PBS solution. Subsequently, membranes were incubated with goat anti-rabbit IgG conjugated with horseradish peroxidase (1:1,000 dilution) in PBS solution (Sigma-Aldrich Co). Immunoreactive proteins were visualized using the TMB membrane peroxidase substrate system (Kirkegaard & Perry Lab., Gaithersburg, MD).

Experiment 2
Pigs used in Exp. 2 were a subset of a larger data set previously reported and used to determine the effects of dietary Fe supplementation on growth performance, hematological status, and whole-body mineral concentrations of nursery pigs (Rincker et al., 2004Go). The subset of pigs consisted of pigs killed at the initiation of the study to establish baseline (BL2; n = 5; mean BW = 5.9 kg; age = 19 ± 3 d), and pigs fed the basal diet (Phase 1 = d 0 to 7; Phase 2 = d 7 to 21; Phase 3 = d 21 to 35) supplemented with 0 or 150 mg/kg of Fe as ferrous sulfate (six pigs per treatment; mean BW = 18.3 kg; age = 54 ± 3 d) killed at the conclusion of the 35-d feeding experiment. Liver samples were collected from these pigs and analyzed for IRP binding activity via the laboratory methods previously described in Exp. 1.

Statistical Analyses
In Exp. 1, data were analyzed as a completely randomized design using the MIXED procedures of SAS (SAS Inst., Inc., Cary, NC). Pig was the experimental unit. Two preplanned orthogonal comparisons were made: 1) BL1 pigs vs. pigs receiving a Sal or Fe i.m. injection; and 2) Sal vs. Fe i.m. injection. For blood response criteria, d 0 Hb, Hct, PFe, and Tf values were used as a covariate for their respective individual analyses. In Exp. 2, data were analyzed using the MIXED model procedures of SAS. Two preplanned orthogonal comparisons were made: 1) BL2 pigs vs. both Fe treatments (0 and 150 mg of added Fe/kg of diet); and 2) 0 vs. 150 mg of added Fe/kg diet. Differences were considered significant at the level of P < 0.05 and highly significant at the level of P < 0.01.


   Results and Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results and Discussion
Implications
 Literature Cited
 
The hepatic Fe stores of the neonatal pig combined with the low Fe concentration in sow’s milk are not sufficient to meet the rapid growth and increase in blood volume during this time. Braude et al. (1962)Go estimated that a suckling pig must retain 21 mg of Fe/kg of BW gain to maintain satisfactory Hb and storage Fe concentrations. In Exp. 1, the mean Fe concentration in sow’s milk was 1.02 mg/L (sow’s milk samples were collected on d 0, 4, 8, and 12). Thus, the amount of milk that neonatal pigs consumed did not meet their Fe requirement.

Whole blood Hb concentration and Hct percentage are commonly used as indicators of a pig’s Fe status because of their ease of measurement. A whole blood Hb concentration of 100 g/L is considered adequate, whereas 80 g/L suggests borderline anemia, and 70 g/L or less indicates anemia (Zimmerman, 1980Go). There were no differences (P > 0.10) in Hb, Hct, and PFe at the start of experiment Exp. 1 (Table 1Go). On d 13, Fe-treated pigs had greater (P = 0.001) Hct percentage and Hb and PFe concentration than Sal-treated pigs. The mean Hb concentration of Sal-treated pigs (59 g/L) indicated that they were anemic by d 13. Pollmann et al. (1983)Go reported a similar decrease in Hb concentration and Hct percentage at 10 d of age in pigs receiving no supplemental Fe compared with pigs receiving 200 mg of Fe via an i.m. injection of Fe-dextran at 1 d of age.


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  		Table 1. Effects of Fe administration on neonatal pig growth performance and hematological status in Exp. 1a
 
Examples of proteins whose expression is regulated by IRP include ferritin (Aziz and Munro, 1987Go), TfR (Casey et al., 1988Go), eALAS (May et al., 1990Go), and ferroportin (Donovan et al., 2000Go). These proteins are critical in maintaining Fe homeostasis. Because of the role of IRP in the posttranscriptional expression of these proteins, the determination of IRP binding activity, along with the traditional measurements (i.e., Hb concentration and Hct percentage) used to establish Fe status, a more sensitive assessment of a young pig’s Fe state can be made during this rapid growth period.

A representative immunoblot of IRP1 is shown in Figure 1Go. The reported molecular weight of rat IRP1 is 98 kDa (Yu et al., 1992Go). The molecular mass of porcine IRP1 is similar to that of rats (Figure 1Go). The presence of IRP2 in liver was not detected via SDS-PAGE followed by Western blot analysis. Iron regulatory protein-2 is temperature sensitive, and degradation occurs rapidly (R. S. Eisenstein, Univ. Wisconsin, personal communication); however, extreme caution was taken to minimize these risks during laboratory analysis. A representative autoradiograph of spontaneous and 2-ME-inducible RNA binding activity of cytosolic IRP is shown in Figure 2Go. Previous reports indicate that human IRP1 and IRP2 migrate in a similar band during gel shift analysis, whereas rat IRP1 and IRP2 migrate differently (Rothenberger et al., 1990Go). In Exp. 1, pigs receiving a Sal injection had greater (P = 0.004) spontaneous IRP binding activity than Fe-treated pigs (Table 2Go). This finding agrees with the results of Chen et al. (1997)Go, who reported greater IRP RNA binding activity in weanling rats fed diets containing 2 mg of Fe/kg of diet compared with rats fed adequate-Fe diets (37 mg of Fe/kg of diet). Yu et al. (1992)Go also reported that IRP RNA binding activity of Fe-deficient rats increased by 50% after 16 h compared with rats administered Fe via an intraperitoneal injection. These authors also reported that IRP mRNA remained constant during Fe treatment, suggesting that the decrease in IRP RNA binding activity by Fe in rat liver was due to posttranslational changes in the RNA binding affinity of IRP and not due to a decrease in the transcription of the IRP gene or to the destabilization of the IRP mRNA. Both treatment groups had greater (P = 0.018) spontaneous IRP binding activity compared with BL1 pigs. Based on the role of IRP, the greater spontaneous IRP binding activity noted in Sal-treated pigs suggests that the synthesis of proteins involved in Fe storage was inhibited, whereas the synthesis of proteins involved in Fe transport and uptake was increased to meet erythropoietic demands of tissues compared with Fe-treated pigs.



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  		Figure 1. A representative immunoblot of iron regulatory protein 1 (IRP1) is shown. In Exp. 1, treatments were: BL1 (n = 5; mean BW = 2.2 kg; age = 14 ± 1 h); Sal (n = 5; mean BW = 4.7 kg; age = 13 d; 1 mL of 0.9% sterile saline solution via an i.m. injection; physiological saline solution, Phoenix Pharmaceutical, Inc., St. Joseph, MO); and Fe (n = 5; mean BW = 4.7 kg; age = 13 d; 1 mL of 200 mg Fe as Fe-dextran via an i.m. injection; Duravet Iron Dextran-200, Phoenix Pharmaceutical, Inc.). In Exp. 2, treatments were: BL2 (n = 5; mean BW = 5.9 kg; age = 19 ± 3 d); for 0 and 150 mg of added Fe/kg of diet (n = 6 per treatment; mean BW = 18.3 kg; age = 54 ± 3 d). Lane A represents the pre-stained molecular weight markers ß-galactosidase and bovine serum albumin that have a molecular weight of 115 and 97 kDa, respectively. Lanes B through D represent IRP1 in Sal, Fe, and BL1 pigs in Exp. 1, respectively, whereas Lanes E through G represent IRP1 in 0, 150, and BL2 pigs in Exp. 2, respectively. Lane H represents IRP1 in rat (control).

 


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  		Figure 2. A representative autoradiograph of spontaneous and 2-mercaptoethanol (2-ME) inducible RNA binding activity of cytosolic iron regulatory protein (IRP) is shown. Treatments were: BL1 (n = 5; mean BW = 2.2 kg; age = 14 ± 1 h); Sal (n = 5; mean BW = 4.7 kg; age = 13 d; 1 mL of 0.9% sterile saline solution via an i.m. injection; physiological saline solution, Phoenix Pharmaceutical, Inc., St. Joseph, MO); and Fe (n = 5; mean BW = 4.7 kg; age = 13 d; 1 mL of 200 mg Fe as Fe-dextran via an i.m. injection; Duravet Iron Dextran-200, Phoenix Pharmaceutical, Inc.). Lanes A through C represent spontaneous IRP binding activity in BL1, Sal, and Fe pigs, respectively, whereas Lanes D through F represent 2-mercaptoethanol inducible IRP binding activity in BL1, Sal, and Fe pigs, respectively. The optimal amount of protein used for the gel shift assay when determining spontaneous IRP binding activity was 10 µg of cytosolic protein. In the presence of 4% 2-ME, 2.5 µg of cytosolic protein was the optimal amount for the gel shift assay.

 

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  		Table 2. Effects of Fe administration on young pig liver iron regulatory protein (IRP) binding activity in Exp. 1 and 2
 
In the presence of 2-ME, inactive IRP (i.e., c-acon) are converted to an active, high-affinity RNA binding form. Thus, the amount of 2-ME inducible RNA binding activity is a measure of the total amount of IRP protein present (Hentze et al., 1989Go). No difference (P = 0.237) in 2-ME inducible IRP binding activity was observed between injection treatments; however, both treatment groups had less (P = 0.002) 2-ME inducible IRP binding activity than BL1 pigs. This suggests that neonatal pigs are born with a portion of the IRP protein in an inactive form. If pigs are not supplemented with Fe soon after birth to meet the body’s demands, inactive IRP protein is converted to an active form and an increase in IRP binding activity occurs, as was observed in Sal-treated pigs. Chen et al. (1997)Go reported that the amount of 2-ME-inducible IRP binding activity in rats ingesting diets containing 2 or 11 mg of Fe/kg of diet was greater than that of controls (37 mg of Fe/kg of diet).

During the initial stage of Fe depletion, liver and spleen Fe stores (i.e., ferritin and hemosiderin) are decreased (Underwood and Suttle, 1999Go). Liver Fe concentration (Table 3Go) of BL1 pigs was considered adequate for neonatal pigs as reported by Underwood and Suttle (1999)Go. Liver Fe concentrations of Sal-treated pigs were less (P = 0.001) than those of BL1 pigs, whereas Fe-treated pigs had a greater (P = 0.001) liver Fe concentration than BL1 pigs. Iron-treated pigs had a 25-fold greater (P = 0.001) liver Fe concentration than Sal-treated pigs. Similar results were reported by Pollmann et al. (1983)Go, who noted a 15-fold increase in nonheme liver Fe at 3 wk of age in pigs receiving 200 mg of Fe via an i.m. Fe-dextran injection at 1 d of age compared with pigs receiving no supplemental Fe. As previously noted, Sal-treated pigs had increased IRP binding activity. The increase in binding activity of IRP results in IRP binding to IRE in the mRNA and inhibition of ferritin synthesis. Chen et al. (1997)Go reported that a biphasic relationship existed between IRP binding activity and ferritin concentration in rat liver, and these authors suggested that at a critical concentration of Fe, ferritin synthesis is either rapidly and fully activated or repressed depending on the direction of change in Fe concentration.


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  		Table 3. Effects of Fe administration on neonatal pig liver mineral concentrations (wet basis), and whole-body mineral concentrations (DM basis) and chemical composition in Exp. 1a
 
Transferrin is the primary means of interorgan transport of nonheme Fe. The abundance of plasma Tf is inversely related to Fe status in rats (Morton and Tavill, 1977Go; Zakin, 1992Go) and chicks (McKnight et al., 1980Go). On d 13, Sal-treated pigs had greater (P = 0.002) plasma Tf concentration than Fe-treated pigs (Table 1Go). We previously reported (Rincker et al., 2004Go) a linear decrease in plasma Tf concentration in pigs fed increasing dietary Fe concentrations (0, 25, 50, 100, and 150 mg of added Fe/kg). Results from the present experiment suggest that Sal-treated pigs had a greater need than Fe-treated pigs to transport nonheme Fe to and between tissues. The uptake of nonheme Fe by tissue is a coordinated process between Tf and TfR. The mRNA encoding TfR contains IRE that IRP bind to and stabilize for translation of the protein. Thus, the increased IRP binding activity observed in Sal-treated pigs suggests that TfR synthesis was increased, along with the noted increase in plasma Tf concentration, to meet the demands of Fe-dependent tissue. Cox and Adrian (1993)Go reported that human Tf synthesis is regulated by Fe posttranscriptionally, and that similarities may exist between the mechanisms (i.e., IRP binding activity) regulating Tf and ferritin synthesis; however, this has not been studied in the pig. Transcription also is likely important in the synthesis of Tf (McKnight et al., 1980Go).

No visual symptoms of Fe deficiency (i.e., labored breathing, rough hair coat, wrinkly skin, or listlessness) were observed among the experimental pigs. Amine et al. (1972)Go suggested that BW gain is not a sensitive indicator of Fe adequacy because a decrease in growth is one of the last signs of an Fe deficiency after the development of hypochromic-microcytic anemia. For the 13-d experiment, no difference (P = 0.482) was observed in ADG between pigs receiving a Sal or Fe injection (Table 1Go). Talbot and Swenson (1970)Go reported no difference in BW at 14 d of age between pigs receiving an i.m. injection of 0.85% NaCl solution or 150 mg of Fe via an i.m. Fe-dextran injection at 1 d of age. Ullrey et al. (1959)Go did not observe a decrease in BW until 5 wk of age in pigs receiving no supplemental Fe compared with pigs receiving a 100-mg i.m. injection of ferrous Fe at 3 or 4 d of age. Similar results were reported by Kernkamp et al. (1962)Go, who compared the growth of pigs receiving various Fe sources, including ferric ammonium citrate, Fe-dextran, and Fe-dextrin at 3 d of age with pigs receiving no supplemental Fe. These authors did not observe a decrease in growth after 14 d; however, pigs receiving no supplemental Fe had lower BW gain after 28 d than pigs receiving any of the other Fe sources. In pigs, the life of the red blood cell is approximately 72 d (Withrow and Bell, 1969Go). Because the present study lasted 13 d, the animals did not have complete red blood cell turnover. A decrease in growth performance would most likely have occurred if the duration of Exp. 1 had been extended.

Pigs 13 d of age had greater liver Cu (P = 0.050), Zn (P = 0.001), Mg (P = 0.037), Ca (P = 0.001), and P (P = 0.001) concentrations compared with BL1 pigs (Table 3Go). Sow milk nutrient composition changes with stage of lactation. Hill et al. (1983)Go reported that micromineral concentrations (Cu, Fe, and Zn) are greater in the colostrum and then decrease as lactation progresses, whereas macromineral concentrations (Ca, Mg, and P) increase during the progression of lactation. Thus, the greater hepatic mineral stores in pigs 13 d of age are due to more appreciable quantities of milk consumption (mineral source) as pigs matured. Pigs receiving a Fe injection had a greater (P = 0.002) liver Mn concentration than Sal-treated pigs. Hill and Matrone (1970)Go reported that an interrelationship exists between Fe and Mn because of their similar chemical and physical properties (i.e., similar electron structure). In contrast to our results, a previous report indicated that Mn absorption by the proximal intestine is increased during Fe deficiency in rats (Thomson et al., 1971Go).

Whole-body mineral concentrations of neonatal pigs are shown in Table 3Go. Pigs administered a Fe injection had a 2.5-fold greater (P = 0.002) whole-body Fe concentration than Sal-treated pigs. No differences (P > 0.10) were observed in Cu, Zn, Mg, Mn, Ca, and P whole-body concentrations between pigs receiving a Sal or Fe injection at 1 d of age. Nonetheless, pigs 13 d of age had a greater whole-body Cu (P = 0.006) and less whole-body Zn (P = 0.002), Mg (P = 0.001), Mn (P = 0.001), Ca (P = 0.001), and P (P = 0.001) concentrations than BL1 pigs. In contrast, Mahan and Shields (1998)Go reported that Ca, Mg, Mn, P, and Zn whole-body concentrations (expressed on a fat-free, empty BW basis) increased during the nursing period because of the transfer of minerals from maternal tissue stores to the tissue of nursing pigs via milk consumption. If we had expressed our mineral concentrations on a fat-free basis, an increase from d 0 to 13 in whole-body mineral concentrations (Ca, Mg, Mn, P, and Zn) would have been noted.

Whole-body chemical composition of neonatal pigs in Exp. 1 also is presented in Table 3Go. Baseline pigs had a greater percentage of water and ash and a smaller percentage of protein and lipid (P = 0.001) than 13-d-old pigs. The increased percentage of lipid in pigs at 13 d of age is the result of the consumption of sow’s milk, which contains 30 to 40% fat on a DM basis (deMann and Bowland, 1963Go). Given the close associations among body protein, water, and ash, most of the variation in chemical body composition between different groups of pigs can be attributed to variation in body lipid content (Hendriks and Moughan, 1993Go; Bikker et al., 1996aGo,bGo). Thus, the 4.5-fold increase in the percentage of lipid in pigs 13 d of age compared with BL1 pigs is the reason the percentage of water and ash decreased from d 0 to 13. No differences (P > 0.10) were observed in whole-body chemical composition between Fe- vs. Sal-treated pigs.

In Exp. 2, liver spontaneous IRP binding activity was greater (P = 0.013) in pigs fed diets containing 0 mg/kg added Fe than those fed 150 mg/kg added Fe (Table 2Go). These results further support those we previously published, in which Hct percentage and Hb, PFe, and liver Fe concentrations were less, and plasma Tf concentration was greater, in pigs fed diets supplemented with 0 vs. 150 mg/kg diet added Fe (Rincker et al., 2004Go). This finding suggests that the dietary Fe was inadequate to meet the Fe requirement of pigs fed 0 mg of added Fe/kg diet. Thus, IRP binding activity was increased in pigs fed diets supplemented with 0 mg of added Fe/kg diet, and the synthesis of Fe storage proteins was inhibited, whereas the synthesis of proteins involved in Fe transport and uptake was increased to meet the erythropoietic tissue demands.

No difference (P = 0.230) in spontaneous IRP binding activity was observed between BL2 pigs and pigs fed the experimental diets for 35 d. We previously reported (Rincker et al., 2004Go) that the plasma Tf and liver Fe concentrations for BL2 pigs (52.20 g/L and 247 mg/kg, respectively) and pigs fed diets containing 0 or 150 mg of added Fe/kg of diet (44.66 or 39.08 g/L and 35 or 113 mg/kg, respectively). The high liver Fe concentration in BL2 pigs is indicative of the Fe stores that were accumulated after administration of an i.m injection of Fe-dextran at 1 to 2 d of age. Nonetheless, the high IRP binding activity and plasma Tf concentration suggest that BL2 pigs were drawing on their Fe stores to meet the rapid growth demands and increase in blood volume at this age.

Not only was 2-ME inducible IRP binding activity greater (P = 0.005) in pigs fed diets containing 0 vs. 150 mg of added Fe/kg of diet, but in contrast to BL2 pigs, it also was greater (P = 0.001) in both groups fed dietary treatments for 35 d. Chen et al. (1997)Go also reported an increase in the amount of 2-ME inducible RNA binding activity in Fe-deficient rats and suggested that more of the newly synthesized binding protein is diverted to the high-affinity RNA binding form in Fe-deficient animals.

Based on the traditional measurements of Fe status (i.e., Hb concentration and Hct percentage), Fe deficiency was induced in Sal-injected pigs. The increased IRP binding activity suggests that Sal-treated pigs were not storing Fe, but instead, they were transporting Fe to meet the erythropoietic demands of tissues; this is evident by the decreased liver Fe and increased plasma Tf concentrations observed in these pigs. Results of Exp. 2 suggest that the dietary Fe was inadequate to meet the Fe requirement of pigs fed 0 mg/kg diet added Fe. Thus, IRP binding activity was increased in these pigs, which would be expected to decrease the synthesis of Fe storage proteins and increase the synthesis of proteins involved in Fe transport and uptake; this is supported by the lower liver Fe and greater plasma Tf concentrations of pigs fed diets containing 0 vs. 150 mg of added Fe/kg of diet that we reported previously (Rincker et al., 2004Go).

In conclusion, Fe supplementation influences IRP binding activity in young pigs. By having post-transcriptional control over proteins involved in Fe transport and storage, IRP are key regulators of Fe homeostasis in the young pig.


   Implications
Top
 Abstract
 Introduction
 Materials and Methods
 Results and Discussion
 Implications
Literature Cited
 
Our research shows that the use of an exogenous iron source to prevent iron deficiency in neonatal pigs influences the binding activity of iron regulatory proteins. Based on their role in modulating the posttranscriptional expression of proteins required for the uptake, storage, and use of iron, an increase in iron regulatory protein binding activity can indicate the initial stage of iron depletion not found by measuring hemoglobin and hematocrit. In the neonatal phase, the dietary iron source influences binding activity of iron regulatory proteins and maintains iron homeostasis via the expression of iron transport and storage proteins.


   Footnotes
 
1 Partially funded by Initiative for Future Agriculture and Food Systems grant. Back

2 Our laboratory thanks P. M. Coussen and M. E. Doumit for their assistance with laboratory techniques. Back

3 Correspondence: 2209 Anthony Hall (phone: 517-355-9676; fax: 517-432-0190; e-mail: hillgre{at}msu.edu).

Received for publication February 16, 2005. Accepted for publication June 3, 2005.


   Literature Cited
Top
 Abstract
 Introduction
 Materials and Methods
 Results and Discussion
 Implications
 Literature Cited
 


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