J. Dairy Sci. 2009. 92:2896-2905. doi:10.3168/jds.2008-1933
© 2009 American Dairy Science Association ®
Bioaccessibility of iron from soil is increased by silage fermentation
S. L. Hansen and
J. W. Spears1
Department of Animal Science, North Carolina State University, Raleigh 27695
1 Corresponding author: jerry_spears{at}ncsu.edu
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ABSTRACT
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High dietary Fe can negatively affect absorption of other minerals and cause tissue damage through the production of free radicals. Cattle are often exposed to high dietary Fe, and soil ingestion may represent a major dietary source of Fe. Iron in soil is often found in the ferric form bound in insoluble complexes; however, exposure to an acidic environment similar to that occurring during silage fermentation may cause this Fe to be reduced to the more soluble ferrous form. To test this theory, a 2 x 2 x 3 factorial arrangement examining time, level, and type of soil addition to greenchop was used. Factors included 2 times of soil addition (before or after ensiling), 2 levels of soil inclusion (1 and 5% contamination, wet basis) and 3 types of soil (Cecil clay loam, 3.4% Fe; Georgeville silt loam, 4.3% Fe; and Dyke clay loam, 6.9% Fe). In addition, greenchop with no soil added was ensiled to serve as a control. Fresh corn greenchop was mixed with the appropriate type and level of soil and tightly packed in experimental silos. Fermentation was allowed to proceed for 90 d before silos were opened and silage was freeze-dried and ground. To simulate contamination after ensiling, each soil type was added to control silage at the 2 levels of inclusion. Addition of soil to greenchop before ensiling resulted in greater amounts of water soluble Fe compared with soil addition after ensiling, suggesting that Fe-soil binding properties were altered by ensiling. To test the potential bioaccessibility of Fe during ruminant digestion, an enzymatic in vitro system was modified to simulate ruminal, abomasal, and intestinal digestion. The presence of soil, regardless of time of addition, type, or inclusion level, resulted in greater soluble or bioaccessible Fe concentrations after all 3 phases when compared with control silage. Ensiling further increased soluble Fe concentrations after each phase when compared with silage contaminated with soil after ensiling. In addition, dialyzable Fe concentration (15,000 Da molecular weight cut off) following intestinal phase simulation was greater due to ensiling. Iron that becomes soluble during the intestinal phase may be available to the animal for absorption, and ensiling resulted in increased concentrations of potentially bioavailable Fe. These results suggest that soil contamination of harvested feeds before ensiling may represent a major source of bioavailable Fe in the diets of cattle.
Key Words: bioavailability • cattle • iron • silage
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INTRODUCTION
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Iron is often found in high levels in ruminant diets due to naturally high levels of Fe in feedstuffs and (or) soil contamination of feedstuffs (Standish et al., 1971). Some common feedstuffs that are often high in Fe include alfalfa, soyhulls, and corn silage (NRC, 1996; DePeters et al., 2000). Humphries et al. (1983) noted that silages in the British Isles often contained more than 2,000 mg of Fe/kg of DM, which is clearly in excess of the recommendations for both beef cattle (50 mg of Fe/kg of DM; NRC, 1996) and dairy cattle (15–30 mg of Fe/kg of DM for a mature dairy cow across multiple stages of production; NRC, 2001). Soil contamination of silage is the most likely explanation for these high levels of Fe.
Soil contamination of feedstuffs or consumption by animals can be estimated using x-ray fluorescence analysis of titanium, a metal which is abundant in soils, but is present in only very low concentrations in plants (<1 mg of titanium/kg). Using this method, Rafferty et al. (1994) examined rates of soil contamination of both freshly harvested and stored feedstuffs in Ireland. Soil contamination of hay and grass silage was estimated at 4 time points; directly from the field, and 4, 8, and 12 mo after initial harvest and storage. Soil contamination was found to average less than 2% (wt/wt) for both hay and grass silage when samples were taken fresh from the field before harvest, while contamination rates as high as 8% were observed in some samples taken after 4 mo in storage. This sharp increase in soil contamination compared with samples taken directly from the field suggests that contamination probably occurred during harvest of the feedstuffs.
In the United States, concentrations of Fe in soils range from less than 0.5% in the southern Gulf states to greater than 5.5% in mountainous areas and many parts of the western United States (United States Geological Survey, 2007). In general, Fe in soils is thought to be tightly bound to chelating agents and is therefore mostly unavailable for absorption by animals. However, in vitro work by Healy (1972) indicated that a considerable fraction of Fe from soil could become soluble in the ruminant digestive tract, suggesting that this Fe may be potentially available for absorption. It is also possible that exposure to an acidic environment, such as that found during fermentation of silage, may result in increased bioavailability of soil Fe through reduction of ferric Fe to ferrous Fe (Whitehead, 2000). Many studies demonstrated that excessive levels of dietary Fe decreased the Cu status of cattle (Standish et al., 1969; Humphries et al., 1983), and high dietary Fe might negatively affect Mn status of cattle. It is well accepted that high Fe decreases Mn absorption in rats (Davis et al., 1992); however, the relationship between Fe and Mn metabolism in ruminants remains unclear.
In the present study, we endeavored to determine the effect of silage fermentation on the bioaccessibility of Fe from various levels of contamination and types of soils using a simulated ruminant digestion system. Bioaccessibility has been defined as the fraction of a mineral that is soluble in the gastrointestinal environment and available for absorption by the animal (Ruby et al., 1996). Estimates of bioaccessibility of soil-bound Fe are probably greater than measures of bioavailability of Fe from soil; although bioaccessibility measures Fe that is soluble in specific gastrointestinal fluids, this soluble Fe is not necessarily absorbed by the animal. However, bioaccessibility may provide a good estimate of the potential bioavailability of soil-bound Fe.
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MATERIALS AND METHODS
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Experimental Design and Silage Preparation
To determine the effects of silage fermentation on the potential bioaccessibility of Fe from soil contamination a 2 x 2 x 3 factorial design with a control was used. Factors included time of soil addition (before or after ensiling of corn greenchop), level of soil addition [0 (control), 1, or 5% contamination on a wet basis], and type of soil (Cecil clay loam, Georgeville silt loam, or Dyke clay loam). Three soil types with differing soil properties and Fe content were examined: 1) Cecil clay loam (fine, kaolinitic, thermic type Kanhapludults; 3.4% Fe); 2) Georgeville silt loam (clayey, kaolinitic, thermic type Hapludults; 4.3% Fe); and 3) Dyke clay loam (clayey, mixed, mesic type Rhodudults; 6.9% Fe). Soils were air-dried and crushed by hand to pass through a 1-mm screen before being mixed with greenchop.
To simulate contamination of corn greenchop before ensiling, wide-mouth plastic jars (~2 L) with screw-top lids were used as experimental silos (2 silos per treatment). Each silo was tightly packed with 1,400 g (wet basis) of freshly harvested corn greenchop. Soil added before ensiling was thoroughly mixed with greenchop at the appropriate inclusion level (0, 1, or 5%) before packing. Greenchop was determined to contain 37% DM. Greenchop was allowed to ferment for 90 d, with occasional venting during the first few days to prevent gas buildup in the silos. On d 90 silos were opened and the top 5 cm of oxygen-exposed, spoiled silage was removed. The pH of silage from each silo was determined immediately after opening. Fifty grams of silage from each silo was added to 200 mL of deionized water and stirred on a stir plate for 30 min. The slurry was then strained through a double layer of cheesecloth and pH of the supernatant was measured (Corning pH meter 340, Corning Life Sciences Inc., Lowell, MA). The remaining silage was freeze-dried and ground to pass through a 1-mm sieve on a Wiley Mill (Model 4, Arthur H. Thomas Co., Philadelphia, PA). Silage samples from each silo were wet ashed for Fe analysis using microwave digestion (Mars 5, CEM Corp., Matthews, NC) as described by Gengelbach et al. (1994). Iron content of silage samples and digestion solutions were determined by flame atomic absorption spectroscopy (AA-6701F, Shimadzu Scientific Instruments, Kyoto, Japan). Control silage was analyzed for chemical content by a commercial laboratory (Dairy One, Ithaca, NY).
To simulate the effect of soil contamination of corn silage after ensiling, the following procedure was used. Freeze-dried and ground control silage was used in all analysis, to which was added the appropriate type (Cecil clay loam, Georgeville silt loam, or Dyke clay loam) and level (1 or 5% wet basis) of soil. Because control silage used was already dried, the level of soil added to each sample was adjusted to DM basis (approximately 3 and 15%, for 1 and 5% wet basis, respectively). This procedure would most closely simulate consumption of soil by a grazing animal or consumption of recently contaminated feedstuffs. Each sample was freshly prepared, in duplicate, before each in vitro procedure.
Water Solubility Procedures
Water solubility of Fe from soil added before or after ensiling was determined by adding 1 g of silage to 40 mL of deionized water in a 15-mL polypropylene tube and placing it in a 22°C shaking water bath for 5 h at 80 oscillations per min. Samples were then filtered through ashless Whatman 541 paper and the filtrate was analyzed for soluble Fe concentrations.
In Vitro Digestion System
The simulated digestion system used was a modification of the procedure described by Ward and Spears (1993). A mixture of enzymes was used to simulate ruminal digestion, because rumen fluid is variable and may contain high concentrations of trace minerals. In contrast, Ward and Spears (1993) used rumen fluid for the ruminal digestion stage. Preliminary experiments were conducted to determine the necessary pH and enzyme inclusion levels for optimal DM digestion in the ruminal stage (data not shown). These experiments were based on a target ruminal DM disappearance rate of ~70% for corn silage, as determined using the traditional Tilley and Terry (1963) method (Hunt et al., 1993). Digestion of silage in the rumen was simulated by adding 0.5 g of silage to a 50-mL Erlenmeyer flask. Thirty milliliters of an acetate buffer was then added to each flask. To make this buffer, 2.95 mL of glacial acetic acid was brought up to 500 mL with deionized water (part A) and 13.6 g of sodium acetate was brought up to 1 L with deionized water (part B; De Boever et al., 1986). Parts A and B were added together to reach a pH of ~5.1 for optimum enzyme activity and ruminal digestion of silage. All enzymes were purchased from Sigma-Aldrich (St. Louis, MO). Cellulase (EC 3.2.1.4) from Aspergillus niger was included at a level of 10 units/mL of buffer, where 1 unit liberates 1 µmol from cellulose in 1 h at pH 5.0 and 37°C (De Boever et al., 1986), hemicellulase (EC 232-799-9) from A. niger at a level of 1.67 units/mL of buffer (Nocek and Hall, 1984), and 50 µL of heat stable amylase (EC 3.2.1.1) was added to each flask. Flask openings were covered with parafilm, and agitated at 30 oscillations per min in a 39°C water bath for 24 h. After 24 h of digestion, flasks were swirled and a 3-mL aliquot of the fluid was removed and centrifuged at 580 x g for 5 min to remove any solid contents before analysis for Fe concentrations. Dry matter disappearance for control silage was determined following the 24 h ruminal stage. In addition, in vitro true DM disappearance was determined for control silage using fermentation vessels (Ankom Technology Corp., Fairport, NY) with rumen fluid inoculum as reported by Huntington and Burns (2007).
Abomasal digestion followed the ruminal stage, and included the addition of 5% pepsin (388 units/mg; EC 3.4.23.1) solution in 1 N HCl. Approximately 3 mL of this solution was required to lower the pH to ~2.5. Flasks were then agitated at 30 oscillations per min in a 37°C water bath for 1 h. Following the abomasal stage, a 3-mL aliquot was taken as described previously following the ruminal stage. To simulate intestinal digestion, the pH of the remaining solution was lowered through the drop-wise addition of 1 M NaOH to a pH of approximately 6.8 (approximately 2 mL of NaOH). Once the pH was stabilized, 0.4 mL of 10% pancreatin (P1500, Sigma, EC 232–468–9) solution in deionized water was added to each flask and flasks were again agitated at 30 oscillations per min in a 37°C water bath for 2 h. Dialyzable Fe concentrations were determined using dialysis tubing with a molecular weight cutoff of 15,000 Da (Spectra/Por 7 Dialysis Membrane, Spectrum Laboratories, Rancho Dominguez, CA). Following the intestinal stage of digestion, flasks were swirled, poured off into 50-mL polypropylene tubes, and centrifuged at 580 x g for 10 min. Eight milliliters of clarified supernatant was pipetted into a segment of dialysis tubing approximately 8 cm in length and the ends clamped off with clips. The tubing was suspended in 500 mL of deionized water in a 600-mL beaker, covered with parafilm and placed in a gently oscillating 37°C water bath for 2 h to simulate approximate retention time of digesta in the intestine. Soluble Fe concentrations of the remaining intestinal supernatant, dialysis tube contents, and the dialysate were analyzed. Dialyzable Fe concentrations were determined as the amount of soluble Fe that disappeared from the dialysis tubing during the 2-h time period (initial supernatant Fe content – dialysis tubing Fe content).
Statistical Analysis
Statistical analysis of all data was performed by ANOVA using the MIXED procedure of SAS (SAS Institute Inc., Cary, NC). The model included the fixed effects of time of soil addition (before or after ensiling), level of soil inclusion (1 or 5%) and type of soil added (Cecil clay loam, Georgeville silt loam, or Dyke clay loam) nested within control treatment (factor) and all appropriate interactions. When the F test was significant, the LSMEANS procedure of SAS with a Tukey’s adjustment was used to separate treatment means. P-values 
0.05 were considered statistically significant, and interactions that were not significant (P > 0.20) for the measurement of interest were removed from the model. Least squares means are presented and represent the mean of duplicate analysis of each replicate.
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RESULTS
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Silage Characteristics
The wide-mouth plastic jars used as experimental silos in the present study appeared to work quite well. A small amount of silage at the mouth of each jar was spoiled due to oxygen exposure, but beneath this layer the silage looked and smelled like normal silage. Silage pH did not differ because of level or type of soil inclusion and averaged 4.0, suggesting that silage fermentation was normal. The control silage was analyzed and found to contain 8.3% CP, 24.4% ADF, 43.9% NDF, and 69% total digestible nutrients. Major mineral concentrations were: 0.23% Ca, 0.23% P, 0.23% Mg, 1.0% K, and 0.004% Na. Trace mineral concentrations in the control silage were (mg of mineral/kg of DM): 52 Fe, 25 Zn, 5 Cu, 38 Mn, 0.5 Mo and 0.1% S.
Water-Soluble Iron Concentrations
Water-soluble concentrations of Fe were affected by a time by level of soil addition interaction (P = 0.005; Table 1). Soluble Fe concentrations were greater (P = 0.001), regardless of level of soil contamination (1 or 5%), when soil was added to greenchop before ensiling. However, level of soil contamination did not affect (P > 0.05) water-soluble concentrations of Fe if soil was added after ensiling (Table 1). The percentage of total Fe that was water soluble decreased (P = 0.008) as total Fe concentrations increased, and was greater (P = 0.001) if soil was added before ensiling compared with soil added after ensiling.
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Table 1. The effect of time and level of soil addition to corn silage on total and water-soluble concentrations of Fe and percentage of water soluble Fe1
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There was a time of soil addition by soil type interaction (P = 0.005) observed with water-soluble Fe concentrations (Table 2). No differences between soil types were observed if soil was added after ensiling, however, concentrations of water soluble Fe were greater (P < 0.05) with the Dyke clay loam compared with the Georgeville silt loam and Cecil clay loam soils when soil was added before ensiling. The percentage of total Fe soluble in water was increased (P = 0.001) across soil types when soil was exposed to the acidic environment of silage fermentation for 90 d.
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Table 2. The effect of time and type of soil addition to corn silage on total and water-soluble Fe concentrations and percentage of water-soluble Fe1
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In Vitro Bioaccessible Iron Concentrations
Dry matter disappearance of the control silage following the 24-h simulated ruminal digestion averaged 70% (n = 4). To confirm that enzymatic ruminal digestion was similar to that of digestion estimates using rumen fluid (Tilley and Terry, 1963), in vitro true DM disappearance was determined. In vitro true DM disappearance averaged 76% (n = 4) for control silage, suggesting that our enzymatic technique provided a good approximation of ruminal DM disappearance. For all stages of simulated digestion, 2- or 3-way interactions between time, level, and type of soil addition were observed. These interactions are described in Figures 1, 2, 3, and 4, and main effects as well as control silage Fe concentrations at all stages are summarized in Table 3.
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Figure 1. The effect of time, level, and type of soil addition to corn silage on bioaccessible Fe concentrations following simulated ruminal digestion. Time x level x type interaction (P = 0.01). Bars with differing letters differ (P < 0.05).
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Figure 2. The effect of time and level of soil addition (A; P = 0.001) and time and type of soil addition to corn silage (B; P = 0.001) on bioaccessible Fe concentrations following simulated ruminal and abomasal digestion. Bars with different letters differ (P < 0.05).
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Figure 3. The effect of time, level, and type of soil addition to corn silage on bioaccessible Fe concentrations following simulated ruminal, abomasal, and intestinal digestion (P = 0.001). Bars with different letters differ (P < 0.05).
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Figure 4. The effect of time, level, and type of soil addition to corn silage on dialyzable Fe concentrations (<15,000 Daltons in molecular weight) following simulated ruminal, abomasal, and intestinal digestion (P = 0.02). Bars with different letters differ (P < 0.05).
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Table 3. Summarized least squares means for the main effects of time, level, and type of soil addition on bioaccessible Fe fractions following simulated ruminant digestion
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Bioaccessible Fe fractions following simulated ruminal digestion were affected by a time by level by type of soil addition interaction (P = 0.01; Figure 1). Across soil inclusion levels and soil types, Fe concentrations were increased (P = 0.001) when soil was added before ensiling compared with when soil was added after ensiling. Concentrations of soluble Fe did not differ between 1 or 5% soil addition when soil was added after ensiling, suggesting that level of soil contamination was not as important when soil was not exposed to the acidic environment of silage fermentation. However, when soil was added before ensiling, soluble Fe concentrations increased (P = 0.001) because of the addition of 5% soil compared with 1%. Soluble Fe concentrations did not differ among soil types at the 1% inclusion level if soil was added before ensiling, but at the 5% inclusion level Dyke clay loam had higher (P < 0.05) soluble Fe concentrations compared with the other 2 soils.
Regardless of soil level or type, bioaccessible Fe concentrations were much higher (P = 0.001) following ruminal and abomasal digestion when soil was added before ensiling compared with soil addition after ensiling (Table 3). Following simulated ruminal and abomasal digestion, bioaccessible Fe fractions were affected by time of soil by type of soil addition (P = 0.001; Figure 2A) and time of soil by level of soil addition (P = 0.001; Figure 2B) interactions. No differences in bioaccessible Fe were observed between soil types when soil was added to silage after ensiling (Figure 2A). However, when soil was added before ensiling, greater amounts of soluble Fe from the Dyke clay loam were observed at this stage compared with the other 2 soils. This may be partially explained by the greater Fe content (6.9%) of the Dyke clay loam soil. Conversely, when soil was added before ensiling, more soluble Fe (P = 0.01) was observed when the contaminating soil was Cecil compared with the Georgeville soil, suggesting that chemical properties of the soils may be affecting availability of Fe, as the Georgeville soil analyzed 4.3% Fe compared with 3.4% Fe for the Cecil soil. Increasing the level of soil contamination from 1 to 5% increased (P = 0.001) bioaccessible Fe concentrations when soil was added before ensiling but not when soil was added after ensiling (P = 0.71; Figure 2B).
Bioaccessible Fe concentrations following simulated ruminal, abomasal, and intestinal digestion are shown in Figure 3. Soluble Fe concentrations were affected by a time by level by type of soil addition interaction (P = 0.001). For both 1 and 5% soil contamination levels, the addition of soil before ensiling resulted in increased (P < 0.05) concentrations of soluble Fe compared with the addition of soil after ensiling. Soluble Fe concentrations at the 1% inclusion level did not differ between soil types if soil was added after ensiling; however, when soil was added before ensiling, both Cecil and Georgeville soils resulted in lower (P < 0.05) soluble Fe concentrations compared with the Dyke soil. At the 5% inclusion level, an increase (P = 0.01) in soluble Fe concentrations was observed as soil Fe concentrations increased (Cecil < Georgeville < Dyke) when soils were added before ensiling. When soils were added after ensiling at the 5% inclusion level, Fe concentrations were greatest from Cecil clay loam (P = 0.01) compared with Georgeville), though minimal differences were observed between the 3 soil types at this inclusion level.
To test the amount of Fe from soil contamination of corn silage which might truly be of an available size for absorption by the animal, dialyzable Fe concentrations following ruminal, abomasal, and intestinal digestion were determined (Figure 4). Dialyzable Fe concentrations were affected by a time by level by type of soil addition interaction (P = 0.02). No differences due to soil inclusion level or soil type were observed if soil was added to corn silage after ensiling. Dialyzable Fe concentrations were the greatest (P < 0.05) when 5% Dyke clay loam was added to corn silage before ensiling, while Cecil and Georgeville soils at the 5% inclusion level did not differ from any of the soils at the 1% inclusion level.
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DISCUSSION
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Several potential sources of excessive dietary Fe may be found in ruminant diets, including drinking water, soil ingestion, and feedstuffs naturally high in Fe (NRC, 1996; DePeters et al., 2000). Little is known about the bioavailability of Fe from feedstuffs such as alfalfa and soyhulls that are often high in Fe. It has long been assumed that soil contamination would not represent a highly bioavailable form of dietary Fe (Whitehead, 2000). The results of the present study appear to support this assumption. In general, the bioaccessibility of Fe from soil contamination was very low if soil was not first exposed to the acidic environment of silage fermentation. These results suggest that consumption of soil by grazing ruminants may not have a dramatic effect on mineral status of the animal. However, bioaccessibility of Fe from soil contamination of harvested corn greenchop was increased following ensiling, suggesting that exposure of soil to the low pH associated with silage fermentation may alter the composition of Fe bound in the soil. It remains unclear as to how quickly this change may occur following exposure to an acidic environment—it may take days or weeks.
Approximately 14.7% of Fe in the control silage was soluble in water following the 5 h incubation time (Table 1). Ivan and Veira (1981) reported similar findings, with approximately 17.6 ± 1.46% of Fe in a corn silage-based diet becoming soluble during extraction with deionized water over a 24-h period. The slightly higher percentage Fe solubility in water reported by Ivan and Veira (1981) may be caused by their 24-h water extraction procedure compared with the 5 h used in our study.
In the present study, the soluble concentrations of Fe in water were dramatically increased when soil was added to corn greenchop before ensiling compared with after ensiling (16- and 29-fold greater for the 1% and 5% soil inclusion levels, respectively; Table 1). In addition, bioaccessibility of Fe from all soil types was increased following all 3 stages of digestion when soil was added before ensiling of corn greenchop compared with after ensiling (Figures 1–3). These results suggest that significant changes in the chemical makeup of Fe in the soil-silage mix occurred at some point in the process of silage fermentation, during which lactic and acetic acids produced by anaerobic bacteria causes the pH to decrease to approximately 4. Exposure of the soils to this low pH may have induced reduction of soil-bound Fe from the ferric to ferrous forms, as well as dissociation of some Fe from insoluble complexes such as hydrous oxides (Whitehead, 2000). It should also be noted that any ruminant feedstuff with a low pH that may come into contact with soil during harvesting, processing, or storage should also be considered as a potential source of bioaccessible Fe. Both Rooke et al. (1983) and Ibrahim et al. (1990) observed that the solubility of several minerals was elevated in grass silage and corn silage compared with unfermented feedstuffs, suggesting that the acidic environment of ensiling also promoted increased mineral release of elements naturally present in the silages. However, as previously stated, it is unclear how rapidly this change may occur and the extent to which an acidic environment may affect mineral bioavailability from feedstuffs.
As with any in vitro experiment, our digestive simulation may not account for all potential interactions and environments that Fe may encounter in vivo. During the ruminal phase of our in vitro digestion system, a pH of ~5.1 was selected to optimize activities of the enzymes used in the procedure. It is likely that Fe solubility is increased at this pH when compared with a normal ruminal fluid pH of >6. However, because the rumen is not an important site of Fe absorption, more emphasis should be placed on estimates of bioaccessible Fe following the intestinal phase of digestion.
Dialyzable Fe, using a similar molecular weight cutoff to that used in the present study, has been used by several researchers to more accurately predict the in vivo bioavailability of Fe from various foods. After comparing several in vitro and in vivo methods of Fe bioavailability estimation, Forbes et al. (1989) determined that dialyzable Fe provided a good estimate of in vivo bioavailability. Additionally, Schricker et al. (1981) found that dialyzable Fe was well correlated (>0.90) with in vivo indicators of Fe availability. In our study, dialyzable Fe concentrations following the 3-stage simulated digestion procedure were greatest with 5% Dyke clay loam soil added before ensiling (Figure 4). It is unclear why such a large fraction of the soluble Fe from this soil was of such small size. Differing compositions of the soils likely affected both the potential solubility of Fe as well as the size of Fe released. A variety of factors can affect the mineral content and availability of soils, including organic matter content, binding of minerals as insoluble complexes, weathering and leaching of minerals, and pH of the soil (Whitehead, 2000). Our work suggests that if soil contamination of a feedstuff occurs before exposure to an acidic environment, not only is Fe bioaccessibility increased, but at least some fraction of that Fe is of a small enough size to potentially be absorbed by the animal.
Increased bioaccessible concentrations of Fe resulting from soil contamination may have deleterious effects on the metabolism of other essential trace metals, either through interactions before absorption or through post-absorptive mechanisms. Hidiroglou et al. (1990) noted lower serum Mn concentrations and an increased incidence of a condition known as congenital joint laxity and dwarfism in calves born to cows wintered on clover or grass silage as compared with those fed hay. It is interesting to note that the Mn content of both the grass silage and clover silage fed in this experiment was greater than 60 mg of Mn/kg of DM, suggesting that fetal demands for Mn should have been met. It is quite possible that Mn availability from silages may have been affected by the presence of an antagonist such as Fe in the diet, possibly due to soil contamination before ensiling.
Recent advances in molecular biology have increased our knowledge about pathways of absorption and metabolism of trace minerals such as Fe, Cu, and Mn (Sharp, 2004; Mackenzie and Garrick, 2005). These advances have shed light on the molecular mechanisms that result in antagonisms between these trace minerals. For example, the transport of ferrous Fe into the absorptive enterocyte of the small intestine has been shown to require the action of divalent metal protein 1 (DMT1) in rodents and in vitro models. Interestingly, Mn is also transported into the enterocyte by DMT1, and some evidence suggests that Cu may also use this route of absorption to some extent (Arredondo et al., 2003; Garrick et al., 2006). Concentrations of intestinal DMT1 are regulated by Fe status in the body as well as dietary Fe concentrations. As a result, high dietary Fe may lead to impaired absorption of Mn or Cu. We have recently demonstrated the presence of DMT1 in the duodenum of beef cattle (Hansen et al., 2008), suggesting that the molecular machinery potentially responsible for the antagonism between Fe, Mn, and possibly Cu, is present in ruminants.
In summary, in vitro bioaccessibility of Fe from soil contamination of corn silage was increased when soil was exposed to the acidic environment of silage fermentation. Iron is a well-known antagonist of Cu in ruminants, and appears to share a similar route of intestinal absorption with Mn. Therefore, situations of high dietary Fe may result in increased competition for intestinal absorption with Mn and Cu, negatively affecting the status of these essential trace minerals in ruminants. Although there are many known sources of elevated Fe in the diets of cattle, our findings suggest that soil contamination of fermented feedstuffs may represent an overlooked source of bioavailable Fe in ruminant diets.
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ACKNOWLEDGMENTS
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Appreciation is extended to Dean Askew and Jim Turner (North Carolina State University) for assistance in procurement of soil samples, as well as to Robert Fry, Karen Lloyd, and Leon Legleiter (North Carolina State University) for assistance in the laboratory.
Received for publication November 26, 2008.
Accepted for publication January 24, 2009.
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