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ANIMAL NUTRITION |
* Department of Animal Science and Interdepartmental Nutrition Program, North Carolina State University, Raleigh 27695-7621; and
Department of Animal and Poultry Sciences, Virginia Polytechnic Institute and State University, Blacksburg 24061-0306
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Abstract |
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 Top Abstract INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Key Words: rumen • omasum • parabiotic • proteinate • zinc
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INTRODUCTION |
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TopAbstractINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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). However, a limited amount of research has compared zinc proteinate (ZnProt), which is produced by chelation of ionized Zn from a soluble Zn salt with AA or partially hydrolyzed protein (Association of American Feed Control Officials, 2000), with inorganic Zn sources. Previously, ZnProt has improved performance and carcass characteristics in feedlot steers (Spears and Kegley, 2002) and hoof quality measurements in fattening bulls (Kessler et al., 2003), and has increased Zn concentrations in plasma, liver, and kidney of calves supplemented with high Zn concentrations (500 mg/kg of DM; Wright and Spears, 2004), relative to inorganic Zn sources (ZnSO4 or ZnO). Although the mechanisms responsible for these observed differences remain unclear, it has been hypothesized that Zn bound to organic compounds is more available for absorption than Zn from inorganic sources.
Mechanisms involved in the absorption of Zn by the small intestine have been extensively investigated. At least 4 transport proteins are involved in the transcellular movement of Zn through enterocytes (Liuzzi and Cousins, 2004). Whether these proteins are present in ruminal or omasal epithelia is not known. Zinc uptake and apparent absorption from ruminal tissue have been demonstrated in vivo by using 65Zn in lambs (Arora et al., 1969) and apparent absorption of Zn from the reticulo-rumen has been observed in wethers fitted with ruminal, abomasal, and ileal cannulas (Kennedy and Bunting, 1991; Kirk et al., 1994). Ashmead et al. (1985) suggested that metal-peptide complexes might be transported directly into the intestinal mucosa intact via peptide transport mechanisms. Evidence has been reported demonstrating that ruminal and omasal epithelia can translocate peptides (Matthews and Webb, 1995; McCollum and Webb 1998) and mRNA for a peptide transporter (PepT1) is present in these tissues (Pan et al., 1997, 2001; Chen et al., 2002). Absorption from the forestomach could reduce the potential for ionization or dissociation of Zn bound to organic compounds during the digestive process. A series of experiments was conducted to determine the effects of time, Zn concentration, and simulated ruminal digestion on uptake and transport of 65Zn from ZnSO4 and ZnProt by ruminal and omasal epithelia.
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MATERIALS AND METHODS |
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TopAbstractINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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The research reported herein was approved by the North Carolina State University Animal Care and Use Committee.
On any given day, tissues were collected from a wether lamb (Dorset or Katahdin) that was stunned by captive bolt and killed by exsanguination. The entire rumen and omasum were excised, digesta were removed, and tissues were rinsed with warm tap water and prewarmed (39°C) 0.85% saline. Tissues were transported to the laboratory in prewarmed (39°C), oxygenated serosal buffer (pH 7.4; Krebs-Ringer phosphate + 10 mM glucose). At the laboratory, tissues were prepared as described by Matthews and Webb (1995).
Uptake and Transport Measurement
Uptake and transport were measured by using parabiotic chambers, as described previously by Matthews and Webb (1995). Briefly, tissues were mounted in parabiotic units and the mucosal and serosal chambers were filled with 10 mL of Krebs-Ringer phosphate buffer (pH 7.4). Parabiotic units were then placed into a 39°C water bath before initiation of Zn uptake experiments. Uptake measurements were initiated (time 0) by replacing the initial buffer with the appropriate mucosal and serosal uptake buffers. All chambers were continuously oxygenated by bubbling an O2:CO2 (95:5, vol/vol) gas mixture through each at a similar rate. A preliminary experiment indicated that minimal Zn disappearance occurred beyond 240 min; thus, all experiments were conducted for 240 min. Samples were obtained at 30-min intervals from 0 to 240 min from the mucosal and serosal sides of each chamber throughout the incubation. Aliquots (0.2 mL) from each sample were transferred into 5-mL polyethylene tubes for gamma counting (Cobra II, Packard Instrument Company, Meriden, CT). The amount of Zn disappearing from the mucosal chambers was calculated as the product of the dpm quantified, the buffer volume, the Zn concentration in the time 0 mucosal buffer, and the specific activity of the time 0 mucosal buffer. After the final sample was taken, the remaining buffer was discarded and the tissue that was exposed to the buffer was excised, washed 3 times in ice-cold 5 mM EDTA solution, dried (24 h at 100°C), and weighed. Tissue samples were transferred to 5-mL polyethylene tubes for gamma counting. The amount of Zn in the tissue was calculated as the product of the dpm quantified, the dry tissue weight, the Zn concentration in the time 0 mucosal buffer, and the activity of the time 0 mucosal buffer.
Buffer Preparation
Serosal (pH 7.4; Krebs-Ringer phosphate + 10 mM glucose) and mucosal (pH 6.0; Krebs-Ringer phosphate + 10 mM mannitol and 500 µM phenol red) buffers and 0.85% saline were prepared the day before each experiment and warmed overnight in a 39°C water bath. On the day of each experiment, serosal and mucosal buffers were oxygenated by bubbling an O2:CO2 (95:5, vol/vol) gas mixture through each solution for 1 h. Labeled ZnProt was prepared by adding 65ZnCl2 (New England Nuclear, Boston, MA; specific activity 185 GBq/g) to a nonlabeled ZnProt (SoluKey, Chelated Minerals Corporation, Salt Lake City, UT) solution. The ligand mixture used to prepare unlabeled ZnProt was then added to ensure chelation of the 65Zn. Radiolabeled ZnSO4 was prepared by adding 65ZnCl2 to a ZnSO4 solution. The final stock solutions of 65ZnSO4 and 65ZnProt contained 1,000 mg of Zn/L, and 65Zn represented approximately 1% of the total Zn. Zinc (10, 20, or 200 µM) was added to the mucosal buffer by adding the appropriate amount of ZnSO4 or ZnProt (SoluKey, Chelated Minerals Corporation) and 7.4 kBq of 65Zn/mL from 65ZnSO4 or 65ZnProt.
Tissue Viability
In addition to the parabiotic chambers containing Zn substrates, 2 chambers were included with each incubation to evaluate tissue viability, as measured by butyrate metabolism to β-hydroxybutyrate. Mucosal buffer in these chambers was prepared containing 15.5 mM butyrate, and β-hydroxybutyrate was quantified (Williamson and Mellanby, 1974) in serosal buffer throughout the incubation.
Experiment 1
The influence of Zn source and incubation time on Zn uptake by ruminal and omasal epithelia was assessed. The experiment was run on 2 separate days using tissues from 1 lamb on each day. Mucosal buffer containing 20 µM Zn from either ZnSO4 or ZnProt and 7.4 kBq of 65Zn/mL from 65ZnSO4 or 65ZnProt, respectively, was added to the mucosal side of 4 replicate chambers for ruminal and omasal epithelium on each day (n = 8/treatment). Samples were taken and uptake was measured at 30-min intervals from 0 to 120 min and 60-min intervals from 120 to 240 min, as described above. The results of this experiment indicated that uptake of Zn by omasal tissue was affected by time (P = 0.04); however, the response was small and did not have a discernable pattern. Zinc uptake by omasal tissue was not affected by Zn source. Subsequent experiments were conducted solely with ruminal epithelium.
Experiment 2
To assess the effects of Zn source and concentration, ruminal epithelium was mounted in chambers and buffer containing Zn (10 or 200 mM) from either ZnSO4 or ZnProt, and 7.4 kBq of 65Zn/mL from 65ZnSO4 or 65ZnProt, respectively, was added to the mucosal side of chambers. Samples were taken and uptake was measured at 30-min intervals from 0 to 4 h, as described above. Each Zn source and Zn concentration combination consisted of 8 chambers (4/d).
Experiment 3
To assess the potential impact of the ruminal environment on Zn uptake from the 2 sources, ZnSO4 and ZnProt were subjected to incubation in a buffered ruminal inoculum before uptake was evaluated. Buffered medium (McDougall, 1948) was prepared fresh and warmed (39°C) before each incubation. Ruminal fluid was collected from fistulated steers and was strained through 8 layers of cheesecloth into a prewarmed (39°C), insulated container for transport to the laboratory. Upon arrival, ruminal inoculum and buffered medium were combined in a 2:1 (vol/vol) ratio and anaerobic conditions were maintained by bubbling CO2 through the mixture. Urea was added to provide a final concentration of 0.05%, and the solution was mixed well under CO2. Aliquots (10 mL) were transferred into 50-mL centrifuge tubes containing 0.118 g of substrate [74% corn, 11% soybean meal, 10% oat straw, and 5% CaCl2·2H2O ground in a Wiley mill (Arthur Thomas Co., Philadelphia, PA) to pass a 1 mm screen]. Before the experiment, ZnSO4 or ZnProt were combined with 65ZnSO4 and 65ZnProt, respectively, as described above, and were added to provide either 10 or 200 µM Zn and 18.5 kBq of 65Zn/mL of buffered ruminal inoculum.
Four replicate tubes were maintained for each source and concentration combination. The tubes were mixed gently, blanketed in CO2, and stoppers with one-way valves were installed to maintain an anaerobic environment. The tubes were then incubated in a water bath for 18 h at 39°C and were periodically mixed gently during the incubation. After incubation, 1-mL aliquots of the complete digestion mixture in each digestion tube were transferred into 5-mL polyethylene tubes for gamma counting. Digestion tubes were then centrifuged at 25,000 x g for 30 min. After centrifugation, a 0.2-mL aliquot from the aqueous fraction of each digestion solution was transferred into a 5-mL polyethylene tube for gamma counting. The proportion of Zn remaining soluble after ruminal fermentation was calculated from the activity of the digestion mixture before centrifugation and the aqueous fraction after centrifugation. The remaining supernatant fractions from the 4 replicates of each Zn source and concentration combination were pooled. Fifteen milliliters from each of the Zn source and concentration combination composites was added to the mucosal side of 4 replicate chambers prepared with ruminal tissue. Samples were taken and Zn uptake was measured at 30-min intervals for 4 h, as described above.
Statistical Analysis
Analysis of repeated measures data was conducted by ANOVA with the MIXED procedure (SAS Inst. Inc., Cary, NC), as described by Littell et al. (1998). Chamber was considered the experimental unit, and chamber within source was included as a random error term. Eight replicate chambers (4 replicates/lamb) were included in the analysis of time (Exp. 1) and concentration (Exp. 2). Four replicate chambers (from 1 lamb) were included in the analysis of data from the experiments after simulated digestion (Exp. 3). The model for Exp. 1 included source, time, and the source x time interaction. The models for Exp. 2 and Exp. 3 included concentration, source, time, and all appropriate interactions. Time was the repeated variable. Seven covariance structures were tested and, based on fit statistics, the covariance structures selected for analysis of data from Exp. 1, 2, and 3 were ar(1), ante, and toep, respectively. Analysis of final tissue Zn concentration was conducted by ANOVA with the GLM procedure of SAS. The model for Exp. 1 included Zn source and the models for Exp. 2 and 3 included source, concentration, and the source x concentration interaction.
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RESULTS |
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TopAbstractINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Experiment 1
When incubated with 20 µM Zn, Zn disappearance from mucosal buffer increased (P < 0.01) as incubation time increased (Figure 1). A preliminary experiment indicated that minimal Zn disappearance occurred beyond 240 min; thus, all experiments were conducted for 240 min. Zinc disappearance was also greater (P = 0.02) from mucosal buffer containing ZnProt compared with ZnSO4 (Figure 1). The ability of experimental buffers to maintain tissue metabolism was confirmed by the fact that β-hydroxybutyrate concentrations in serosal chamber buffer increased throughout the 240 min of incubation when butyrate was added to the mucosal chamber buffer.
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Zinc disappearance was affected by a source x concentration interaction (P = 0.05; Figure 2). At a Zn concentration of 200 µM, Zn disappearance was greater for ZnProt, whereas disappearance did not differ between the sources when the concentration was 10 µM. Zinc disappearance from mucosal fluid was also influenced by a concentration x time interaction (P < 0.01). Increasing incubation time resulted in increased Zn uptake when mucosal buffer contained 200 µM Zn; however, when mucosal buffer contained 10 µM Zn, uptake did not change significantly after 30 min.
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Following simulated ruminal digestion, the proportion of Zn remaining in a soluble form was influenced by a source x concentration interaction (P = 0.03). After 18 h of incubation, the percentage of Zn present in a soluble form in buffered ruminal inoculum containing 10 µM added Zn was not different between ZnSO4 and ZnProt (39.7 ± 11.5 and 14.7 ± 2.7%, respectively). However, when 200 µM Zn was added to buffered ruminal inoculum and incubated for 18 h, a greater proportion of Zn from ZnProt was soluble compared with ZnSO4 (41.5 ± 24.2 and 8.8 ± 2.8%, respectively). Zinc disappearance from aqueous fractions of simulated ruminal digestions containing 200 µM added Zn was greater (P < 0.01) than from those containing 10 µM added Zn (Figure 3). Uptake of Zn from ZnProt was numerically (P = 0.14) greater than from ZnSO4 for ruminal digestions containing 200 µM added Zn, but not those containing 10 µM of Zn. However, the Zn source x concentration interaction was not significant (P = 0.25).
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Zinc accumulation in tissues after incubation was measured. Zinc source did not affect Zn accumulation in ruminal epithelium in any experiment (Table 1). In both experiments in which Zn concentrations were tested, the higher concentration of Zn resulted in greater (P < 0.01) accumulations of Zn in tissue.
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DISCUSSION |
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TopAbstractINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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investigated the chemical characteristics and bioavailability of several organic Zn sources relative to ZnSO4. In gel filtration experiments, Zn from all of the organic Zn products eluted in the same range of fractions as Zn from ZnSO4 when examined at pH 2 and pH 5. However, in deionized H2O, small peaks appeared for each organic Zn source before that of ZnSO4, indicating that a small percentage (ranging from 2.2 to 11.7%) of the organic products remained chelated or complexed when not buffered. Values for the 3 different ZnProt compounds examined ranged from 10.2 to 11.7% and numerically were 3 of the 4 greatest values, suggesting that ZnProt compounds remain partially intact in a nonbuffered solution. Given the observations of Cao et al. (2000), it is reasonable to assume that uptake of Zn from the dissociated fractions would be similar between inorganic and organic Zn sources. Ashmead et al. (1985) suggested that it may be possible for metal ions to be transported into the intestinal mucosa as part of metal-peptide complexes via mechanisms distinct from ionic Zn. Furthermore, researchers have demonstrated the ability of ruminal and omasal tissue to effectively absorb and translocate methionine and the dipeptides carnosine and methionylglycine (Matthews and Webb, 1995). After simulated ruminal digestion, Zn uptake was not significantly influenced by Zn source; however, when 200 µM Zn was added to digestion solutions, Zn uptake was numerically greater from ZnProt than from ZnSO4. Ionic Zn from ZnSO4 or ZnProt was likely bound by one of many potential ligands, many of which may negatively affect solubility and uptake. However, ionization of Zn was not measured in this study. In contrast, intact ZnProt may remain soluble and available for uptake by reducing or eliminating interactions with dietary antagonists.
Tissue Zn accumulation in ruminal epithelium increased as Zn concentration increased. At low Zn concentrations (10 and 20 µM) in Exp. 1 and 2, the amount of Zn recovered in ruminal epithelium agreed closely with values obtained by using mucosal buffer Zn disappearance. However, at the high Zn concentration (200 µM) evaluated in Exp. 2, recovery of Zn in ruminal tissue was approximately 60% of the Zn that disappeared from mucosal buffer. The discrepancy between Zn disappearance from buffer and Zn recovery in ruminal tissues at the high Zn concentration may relate to removal of Zn when tissues were washed with 5 mM EDTA solution. Tissues were washed 3 times with EDTA (a potent Zn chelator), and this may have removed loosely bound Zn from tissues. At the lower Zn concentrations evaluated, Zn may have been tightly bound to tissue and thus not removed by washing. In Exp. 3, in which Zn uptake was measured after simulated ruminal fermentation, the quantity of Zn recovered in tissues was very low at low (20 µM) and high (200 µM) Zn concentrations relative to Zn uptake measured by mucosal buffer Zn disappearance. It is possible that various ligands present in the simulated digestion system resulted in more Zn being loosely bound to tissue, and thus susceptible to removal by EDTA washing.
Regardless of Zn source or concentration, Zn was not detectable in the serosal chambers of the parabiotic chambers during these incubations. This indicates that despite Zn uptake by ruminal tissue, Zn transport did not occur. Such preparations have been used extensively to examine the absorption of many nutrients, including AA and peptides from the rumen and omasum (Matthews and Webb, 1995; McCollum and Webb, 1998) and minerals from the intestine (Song et al., 1988; Mineo et al., 2001). The tissues mounted in the chambers in the present study appear to have remained metabolically functional based on the fact that they produced β-hydroxybutryate throughout the incubation period. Thus, it is reasonable to assume that other physiological functions, including nutrient transport, were functional as well.
The lack of Zn appearance in the serosal buffer may also be due to a requirement for some factors found in the plasma that were not present in the serosal buffer used in the present study. A high proportion of Zn in blood plasma is bound to albumin (Bax and Bloxam, 1997). To test whether the presence of albumin in the serosal buffer influenced movement of Zn into that fluid, BSA was included in the serosal buffer of some chambers. In spite of the presence of the albumin in the chambers, there was no detectable appearance of Zn in the serosal fluid.
Arora et al. (1969) measured the accumulation of Zn in ruminal tissues after administration of 65ZnCl2 into the rumen and concluded that ruminal tissue has the ability to absorb an appreciable amount of Zn. In their study, however, very little, if any, Zn was actually translocated across the ruminal epithelium, as indicated by a lack of appearance of Zn in the blood. The change in concentration of Zn they observed in ruminal tissue most likely was a measure of Zn adhering to the tissue or accumulated in the cells rather than a measure of the ability of the ruminal epithelium to absorb Zn. In their study, samples of ruminal tissue were rinsed with tap water and, in the present study, tissues were rinsed with a 5 mM EDTA solution. Because EDTA is known to be a strong Zn chelator, loosely bound Zn should have been removed from the epithelial tissue and any remaining Zn was likely located inside cells. Apparent absorption of Zn from the reticulo-rumen was observed in wethers fitted with ruminal, abomasal, and ileal cannulas (Kennedy and Bunting, 1991; Kirk et al., 1994). Because the translocation of Zn across the epithelial cells of the rumen was not measured in these studies, the apparent absorption that was observed may also be due to adherence to or accumulation in ruminal tissue. Data obtained in the present study lend credence to the possibility that Zn is not transported across ruminal or omasal epithelia.
Translocation of Zn across enterocytes in the small intestine is known to be mediated by multiple transport proteins (Liuzzi and Cousins, 2004). Members of the Zip family of transport proteins, including Zip1, 2, and 4, mediate the transport of Zn across the brush-border membrane. A member of the ZnT family of transport proteins, ZnT1, mediates movement of Zn across the basolateral membrane out from the enterocyte. Whether these proteins are present in ruminal or omasal epithelia is not known. It may be possible that a transport protein is present in ruminal epithelium that mediates transport of Zn into the cell, but that no transporter is present to mediate movement out from the cell. This could explain the lack of appearance of Zn in serosal buffer in the present study. It is also possible that Zn uptake occurs via nonspecific binding to ruminal mucosa; thus, no transporter is present in the ruminal epithelium that specifically mediates Zn transport into the cell.
Binding of Zn by intracellular ligands such as metallothionein (MT) may also partially explain the accumulation of Zn in ruminal tissue. In vivo, excess Zn accumulated with MT in liver, kidney, pancreas, and small and large intestine, but not with MT in heart, testes, ruminal papilla, abomasal mucosa, and choroid plexus in cattle and sheep fed 2,000 mg of Zn/kg of dietary DM (Whanger et al., 1981). Hempe and Cousins (1992) observed that more Zn was associated with MT in the cytosol of intestinal tissues from rats fed 180 µg of Zn/ g of diet than those fed 1 µg of Zn/g of diet. Although MT was not quantified in the current study, in vivo ruminal MT concentration was not affected by dietary Zn source or concentration (Wright and Spears, 2004). Zinc may have entered the epithelium and become bound to MT that was already present. Although ZnProt may have been taken up intact, as suggested above, cytosolic MT may have bound Zn from both Zn sources. Metallothionein has a strong affinity for Zn (Cousins, 1985); thus, MT may have removed Zn from the ZnProt chelate.
Results of the current experiments suggest that Zn absorption into blood does not occur from the rumen and omasum, but that in the absence of dietary inhibitors, Zn uptake by ruminal tissue is greater from ZnProt than from ZnSO4 at Zn concentrations of 20 or 200 µM. Ruminal Zn concentrations of 200 µM are not likely to occur under practical conditions. However, a Zn concentration of 20 µM is consistent with ruminal Zn concentrations that have been observed in cattle fed high-concentrate diets (Starnes et al., 1984; Spears and Kegley, 2002).
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Footnotes |
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2 Appreciation is extended to Grahame Leach for technical assistance in preparing labeled zinc sources.
3 Current address: Department of Animal Science, South Dakota State University, Brookings, SD 57007.
4 Corresponding author: Jerry_Spears{at}ncsu.edu
Received for publication September 22, 2006. Accepted for publication February 14, 2008.
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LITERATURE CITED |
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TopAbstractINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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