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Poult Sci 2007. 86:877-887
© 2007 Poultry Science Association
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METABOLISM AND NUTRITION

Determining the Methionine Activity of Mintrex Organic Trace Minerals in Broiler Chicks by Using Radiolabel Tracing or Growth Assay1

G. F. Yi2, C. A. Atwell, J. A. Hume, J. J. Dibner, C. D. Knight and J. D. Richards3

Novus International Inc., St. Charles, MO 63304

3 Corresponding author: jdrich{at}novusint.com


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Mintrex Zn, Mintrex Cu, and Mintrex Mn organic trace minerals contain 16% Zn, 15% Cu, and 13% Mn with 80, 78, and 76% 2-hydroxy-4-(methylthio)butanoic acid (HMTBA) by weight as the organic ligand, respectively. Our objective was to determine if HMTBA from Mintrex was fully available as a Met source. In experiment 1, thirty-six broilers (7 to 10 d old) were orally gavaged with methyl-14C-labeled HMTBA, either as free HMTBA (Alimet feed supplement) or Zn bis(-2-hydroxy-4-methylthiobutyrate) (Mintrex Zn). Radiolabel incorporation from either source into protein was measured as a marker of bioavailable Met activity. Results demonstrated that the HMTBA from Mintrex Zn was equally available as free HMTBA to support protein synthesis. In experiment 2, five hundred seventy-six 1-d-old broilers were allotted to 12 dietary treatments (TRT) for a 21-d growth assay. A TSAA-deficient diet containing 0.70% total TSAA (TRT 1) was supplemented with 0.05, 0.10, 0.15, and 0.20% free HMTBA (TRT 2 to 5) to establish the standard Met response curve. Treatment 6 was analogous to TRT 2 but had an additional 160 ppm Zn, 80 ppm Cu, and 160 ppm Mn as sulfates. Treatments 7 to 12 were identical to TRT 2 but supplemented with 40 or 160 ppm Zn from Mintrex Zn, 20 or 80 ppm Cu from Mintrex Cu, or 40 or 160 ppm Mn from Mintrex Mn, respectively. For TRT 1 through 6, growth performance increased due to increasing Met addition (P < 0.01) but not to increasing inorganic trace minerals. For Mintrex Zn, Cu, and Mn (TRT 7 to 12), there was a linear increase in cumulative gain:feed ratio (P < 0.04), and for Mintrex Zn and Mn, there was a linear increase in cumulative gain (P < 0.03) to increasing Mintrex addition. A 1-slope broken-line model was used to calculate bioavailable Met activity from Mintrex for comparison with actual intake values. Results indicated that HMTBA from Mintrex was fully available as a Met source.

Key Words: Mintrex organic trace mineral • broiler • methionine • 2-hydroxy-4-(methylthio)butanoic acid


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Trace minerals such as Zn, Cu, and Mn are crucial for a wide variety of physiological processes in all animals (Underwood and Suttle, 2001). Organic trace minerals (OTM) are forms in which the mineral is complexed or chelated to an organic ligand, such as an amino acid, polysaccharide, or organic acid. Organic trace minerals are used in animal feeds to provide increased mineral bioavailability compared with inorganic trace mineral (ITM) salts, because mineral absorption from ITM might be limited by their tendency to form complexes with dietary constituents (e.g., phytic acid) or interfere with each other when multiple ITM salts are included in the diet (Leeson and Summers, 2001; Underwood and Suttle, 2001; Dibner et al., 2004). Indeed, organic forms of many minerals, including Zn, Cu, and Mn, are widely used in animal agriculture, and the increased availability of organic forms compared with inorganic forms has been demonstrated (Paik et al., 1999; Cao et al., 2000; Guo et al., 2001; Leeson, 2005; Predieri et al., 2005; Yan and Waldroup, 2006). A commonly used source of Met activity is 2-hydroxy-4-(methylthio)butanoic acid (HMTBA; e.g., 88% aqueous solution of HMTBA as Alimet feed supplement or 84% Ca salt of HMTBA as MHA feed supplement), because it can be converted to L-Met within the body of the animal through broadly distributed enzymatic systems (Dibner, 2003; Yi et al., 2006). However, HMTBA is not an amino acid (AA) but rather an organic acid in that it bears a hydroxyl group on the {alpha} carbon instead of the amino group found in Met. Its mineral binding is also similar to that of Met except that the hydroxyl group replaces the amino group in the formation of the mineral complex (Dibner et al., 2004). Recently, a family of OTM (Mintrex OTM) were developed in which 2 mol of HMTBA are reacted per mole of Zn, Cu, or Mn to create chelates containing 16% Zn, 15% Cu, and 13% Mn, with 80, 78, and 76% (by weight) HMTBA as the organic ligand, respectively. Fourier transform infrared spectroscopy and X-ray crystallography experiments indicate that the Zn, Mn, and Cu atoms in Mintrex are chelated by 2 molecules of HMTBA (Dibner et al., 2004; W.R. Harris and N. Rath, University of Missouri, St. Louis, and T. Blackburn and J. J. Dibner, Novus International, unpublished data). Recent data indicate that inclusion of Mintrex OTM into broiler diets can increase mineral bioavailability, enhance immune response to coccidiosis vaccination and lower lesion scores, increase intestinal breaking strength, and improve performance (Dibner et al., 2004, 2005; Richards et al., 2006; Yan and Waldroup, 2006).

Although the primary function of an OTM is to provide a highly bioavailable source of mineral, the organic ligand can also supply a nutritional benefit. In the case of Mintrex OTM, the HMTBA ligand would be expected to provide a significant source of Met activity. The objectives of this study were 2-fold: 1) to evaluate biochemically the Met activity provided by Mintrex when birds were gavaged with an equimolar amount of radiolabeled HMTBA either in the form of free HMTBA (supplied by Alimet feed supplement) or Mintrex Zn and 2) to determine if it is appropriate to recognize the Met content of Mintrex (as HMTBA) as a portion of the entire dietary Met activity when it is fed in conjunction with other sources of Met activity (e.g., Alimet) in broiler diets.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Experiment 1
A total of 50 one-day-old Cobb-500 male broilers were raised in pens of electrically heated battery brooders and fed a typical corn-soybean meal broiler starter diet from d 0 to 10 posthatch as described by Richards et al. (2005). Each pen was provided with water and an individual feeder. All birds were allowed to consume mash feed and water ad libitum. Chicks were exposed to 23L:1D from d 1 to 4 and 16L:8D from d 5 to 10. Room temperature was maintained at 32°C from d 0 to 5 posthatch and then gradually reduced according to standard brooding practice. From d 7 to 10, a total of 36 birds with similar BW were selected, and feed was removed 2 to 3 h before the gavage. Birds were orally gavaged with 80 mg of methyl-14C-labeled HMTBA (1 µCi ) in the form of Alimet (free HMTBA; American Radiolabeled Chemicals, St. Louis, MO) or Mintrex Zn [Zn bis(-2-hydroxy-4-methylthiobutyrate); Novus International Inc.]. Thus, Alimet and Mintrex doses were prepared to supply an equimolar amount of HMTBA. Birds were rested for 1, 1.5, or 4 h; killed by CO2 inhalation; and weighed. Experiments were performed over 6 d with 1 timepoint per day and 3 birds for each HMTBA source per day, such that there were 6 birds per time point for each HMTBA source. The animal protocol for this research was in accordance with the standard operating procedure of Novus International Inc. and approved by an internal safety committee.

For each bird, approximately 1 to 3 g of duodenum, jejunum, liver, leg muscle, and pancreas samples were collected. Digesta from duodenum and jejunum was gently rinsed out with PBS. Tissue masses were weighed and recorded. Tissues were placed in PBS + Complete Protease Inhibitor (Roche, Indianapolis, IN) at 1:2 (wt/vol), frozen at –20°C, and thawed. Tissues were homogenized with a polytron homogenizer and pipetted to homogeneity. The protein was precipitated by trichloroacetic acid (TCA) addition. Briefly, 900-µL aliquots of homogenate were precipitated by addition of 100 µL of 100% TCA, vortexed and then centrifuged in a Fisher Micro-Centrifuge model 235B (Fisher Scientific, Suwanee, GA) at full speed for 2 min. The pellet contained radiolabel incorporated into protein (Ausubel et al., 2002) and therefore represents HMTBA (from Alimet or Mintrex Zn) that was taken up by the tissue, converted to L-Met, and incorporated into protein (Dibner, 2003). The supernatant contained unincorporated radiolabel, in the form of HMTBA, plus HMTBA that had been converted to L-Met but not incorporated into protein. Supernatant was pipetted to scintillation vials, and 15 mL of EcoLite(+) scintillation fluid (ICN Biomedicals Inc., Costa Mesa, CA) was added. The TCA pellets were digested for 2 to 3 d at 37°C with 1 mL of 2.5 N NaOH, vortexed, and pipetted into scintillation vials. Additionally, 1.5 mL of 2 N HCl and then 15 mL of scintillation fluid were added. For Mintrex standards, 100 mg of radiolabeled Mintrex Zn (80 mg of HMTBA, approximately 1 µCi) was dissolved in 2 mL of 20% citric acid. Equal aliquots were pipetted into each of 4 scintillation vials, and 15 mL of scintillation fluid was added to each vial. For Alimet standards, 4 mg of free HMTBA (0.05 µCi) aliquots were mixed with 15 mL of scintillation fluid. The next day, all scintillation vials were counted with a Beckman LS6500 scintillation counter (Beckman Coulter Inc., Fullerton, CA). This scintillation counter automatically corrects for background, quench, and counter efficiency (Beckman Coulter, 1999). Radioactivity (dpm) per microgram of HMTBA (from Alimet or Mintrex Zn) was calculated by using the standards. Using these values, the micrograms of free and incorporated radiolabel per gram of tissue was calculated for all tissues, for both Alimet and Mintrex. Total radioactivity in each tissue was calculated by adding free plus incorporated counts.

Experiment 2
A total of 576 one-day-old Cobb 500 male chicks were allotted to 12 dietary treatments (TRT) in a completely randomized design with 6 replicate pens per TRT and 8 birds per pen for a 21-d growth assay. Chicks were hatched at the Novus International Research Center and put on test immediately after hatch. All birds were raised in electrically heated battery brooders. Each pen was provided with water and an individual feeder. All birds were allowed to consume mash feed and water ad libitum. Room temperature was maintained at 32°C from d 0 to 5 posthatch and then gradually reduced according to standard brooding practice. The study lasted 21 d. Chicks were exposed 23L:1D from d 1 to 4 and 16L:8D from d 5 to 21. The animal protocol for this research was in accordance with the standard operating procedure of Novus International Inc. and approved by an internal safety committee.

A TSAA-deficient basal diet containing 0.70% TSAA (TRT 1) was supplemented with 0.05, 0.10, 0.15, or 0.20% HMTBA as Alimet (TRT 2 to 5) to establish the standard Met response curve, in which all the other nutrients of the basal diet were formulated to meet or exceed NRC (1994) estimated nutrient recommendations (dietary formulation and nutrient analysis shown in Tables 1Go and 2Go). Treatment 6 was analogous to TRT 2 but had an additional 160 ppm Zn from ZnSO4·H2O, 80 ppm Cu from Cu-SO4·5H2O, and 160 ppm Mn from MnSO4·H2O added. Treatments 7 to 12 were the same as TRT 2 but supplemented with 40 or 160 ppm Zn from Mintrex Zn, 20 or 80 ppm Cu from Mintrex Cu, and 40 or 160 ppm Mn from Mintrex Mn, respectively (detailed experimental design presented in Table 2Go). All birds were observed at least twice daily, and mortality was recorded. Initial BW at day of hatch and BW and cumulative feed intake at d 21 posthatch were recorded. Cumulative gain, cumulative gain:feed ratio, and actual supplemental Met intake (the analyzed HMTBA content from Alimet in TRT 1 to 6 or from Alimet plus Mintrex in TRT 7 to 12 multiplied by the cumulative feed intake) were calculated for the 21-d posthatching period.


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  		Table 1. Dietary composition and nutrient profile of broiler chick diets (as-fed basis)1
 

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  		Table 2. Experimental treatments (TRT) and dietary supplemental Met activity and mineral concentrations1
 
Diet Analyses
All dietary amino acids except Met; cystine, Cys, or both; and Trp were determined after acid hydrolysis [method 982.30E (a); AOAC International, 2000], whereas TSAA content was determined by performic acid oxidation followed by acid hydrolysis [method 982.30E (b); AOAC International, 2000], and Trp content was determined after alkaline hydrolysis [method 982.30E (c); AOAC International, 2000]. Dietary HMTBA (at least 2 analyses per sample) was determined using HPLC methods adapted from Ontiveros et al. (1987). Results of HMTBA analysis confirmed that supplemental HMTBA from Alimet (TRT 1 to 6) or from Alimet plus Mintrex (TRT 7 to 12) was added correctly with a correlation of analyzed to theoretical inclusion levels of greater than 95% (Tables 1Go and 2Go). Samples of all diets were sent to an external laboratory (Eurofins Scientific Inc., Des Moines, IA) for mineral (Zn, Cu, and Mn) analysis by inductively coupled plasma optical emission spectrometry (method 985.01; AOAC International, 2000; 1 analysis per sample). The following analytical methods were also used (1 analysis per sample): CP (method 990.03; AOAC International, 2000), moisture (method 934.01; AOAC International, 2000), crude fat (method 954.02; AOAC International, 2000), crude fiber (method 978.10; AOAC International, 2000), and ash (method 942.05; AOAC International, 2000).

Statistical Analyses
All the data were subjected to ANOVA appropriate for completely randomized designs by using the GLM procedure of SAS (SAS Institute, 2003). Statistical difference of TRT means comparisons were made with Fisher’s protected least significant difference test. In experiment 1, the amount of HMTBA-derived radiolabel incorporation into protein in individual tissues or pooled across tissues was used to evaluate the HMTBA source effect within a given timepoint, and the amount of HMTBA-derived radiolabel incorporation into protein pooled across time was used to evaluate the HMTBA source effect within a given tissue. The individual bird was used as the experimental unit. A t-test was used to determine whether rates of incorporation of HMTBA-derived radiolabel into protein were different (P ≤ 0.05) between HMTBA sources. In experiment 2, TRT was the main effect for the growth performance criterion, with pen used as experimental unit. Orthogonal polynomial contrast coefficients were used to determine the linear effect of increasing dietary Mintrex Zn (TRT 2, 7, and 8), Mintrex Cu (TRT 2, 9, and 10), and Mintrex Mn (TRT 2, 11, and 12), respectively (Snedecor and Cochran, 1989). For the 21-d growth period, the cumulative gain or cumulative gain:feed of TRT 1 to 5 as the dependent variable was regressed on the supplemental Met intake and fit to a 1-slope broken-line model (Robbins et al., 1979). With measured cumulative gain or gain:feed of TRT 7 to 12, the 1-slope broken-line prediction equation (below the breakpoint) was used to calculate the bioavailable Met activity (on an intake basis) released from supplemental Alimet plus Mintrex. The bioavailable Met activity was then divided by the actual intake of Met activity (the analyzed HMTBA content from supplemental Alimet plus Mintrex multiplied by the cumulative feed intake) and then multiplied by 100% to generate the relative bioefficacy. An {alpha} level of P ≤ 0.05 was used as the criterion for statistical significance. In addition, the PROC NLIN method of SAS (SAS Institute, 2003) was used to construct 95% confidence intervals around the linear-plateau-predicted values for the Alimet standard growth curve (TRT 1 to 5) for both gain and gain:feed. Mintrex + Alimet means (TRT 7 to 12) were compared with the confidence intervals to determine whether they differed significantly (P ≤ 0.05) from the Alimet standard curve.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Experiment 1
Incorporation of methyl-14C-labeled HMTBA from free HMTBA (supplied by Alimet) or Zn bis(-2-hydroxy-4-methylthiobutyrate) (Mintrex Zn) into cellular protein in duodenum, jejunum, liver, leg muscle, and pancreas was measured at 1, 1.5, and 4 h after gavage. Data are expressed as micrograms of HMTBA from either source that was absorbed, converted to L-Met, and incorporated into protein per gram of tissue sampled. The results indicated that there were no significant differences between the 2 HMTBA sources across tissues at any timepoint (Figure 1Go), indicating that HMTBA from Alimet and Mintrex Zn provided the same Met activity on an equimolar (HMTBA) basis. However, when we measured radiolabel incorporation into protein in individual tissues across time, we did observe that more precipitable Met activity was provided to the duodenum by Mintrex Zn than by Alimet (P ≤ 0.05; Figure 2Go). This difference was also seen at 1 and 4 h (P ≤ 0.05), but the difference was not significant at the 1.5-h timepoint (data not shown). In contrast, there was no difference in the Met activity provided by the 2 HMTBA sources in any of the other tissues tested across time. In fact, the kinetics of radiolabel incorporation into protein was generally similar between the 2 sources, as shown in jejunum (Figure 3Go) and leg muscle (Figure 4Go).


Figure 1
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  		Figure 1. Incorporation of radiolabel from 2-hydroxy-4-(methylthio)-butanoic acid (HMTBA) into protein across tissues over time. Broilers were gavaged with radiolabeled HMTBA as free HMTBA (supplied by Alimet feed supplement) or as Zn bis(-2-hydroxy-4-methylthiobutyrate) (Mintrex Zn). Alimet and Mintrex doses were prepared to supply an equimolar amount of HMTBA. The incorporation of radiolabel into cellular protein in duodenum, jejunum, liver, leg muscle, and pancreas was measured at 1, 1.5, and 4 h and was pooled for analysis. There were 27 observations per mean at the 1- and 1.5-h timepoints and 30 observations per mean at the 4-h timepoint. Data are expressed as micrograms of HMTBA from either source that were absorbed, converted to L-Met, and incorporated into protein per gram of tissue sampled. There were no significant differences between the 2 HMTBA sources at any timepoint.

 

Figure 2
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  		Figure 2. Incorporation of radiolabel from 2-hydroxy-4-(methylthio)butanoic acid (HMTBA) into protein in individual tissues across time. Broilers were gavaged with radiolabeled HMTBA as free HMTBA (supplied by Alimet feed supplement) or as Zn bis(-2-hydroxy-4-methylthiobutyrate) (Mintrex Zn). Alimet and Mintrex doses were prepared to supply an equimolar amount of HMTBA. The incorporation of radiolabel into cellular protein in duodenum, jejunum, liver, leg muscle, and pancreas was measured at 1, 1.5, and 4 h and was pooled across time. There were 18 observations per mean for all tissues except pancreas, where there were 12 observations per mean. Data are expressed as micrograms of HMTBA from either source that were absorbed, converted to L-Met, and incorporated into protein per gram of tissue sampled. Bars containing different letters were significantly different (P ≤ 0.05). The only significant difference between HMTBA sources was observed in duodenum (P ≤ 0.05), which was observed over time, and at 1 and 4 h (data not shown).

 

Figure 3
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  		Figure 3. Kinetics of radiolabel incorporation from 2-hydroxy-4-(methylthio)butanoic acid (HMTBA) into protein in jejunum. The rate of incorporation of radiolabel from HMTBA as free HMTBA (supplied by Alimet feed supplement) or as Zn bis(-2-hydroxy-4-methylthiobutyrate) (Mintrex Zn) into protein in the jejunum is shown. There were 6 observations per mean. There was no significant difference in incorporation rates between the 2 HMTBA sources.

 

Figure 4
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  		Figure 4. Kinetics of radiolabel incorporation from 2-hydroxy-4-(methylthio)butanoic acid (HMTBA) into protein in leg muscle. The rate of incorporation of radiolabel from HMTBA as free HMTBA (supplied by Alimet feed supplement) or as Zn bis(-2-hydroxy-4-methylthiobutyrate) (Mintrex Zn) into protein in leg muscle is shown. There were 6 observations per mean. There was no significant difference in incorporation rates between the 2 HMTBA sources.

 
In addition to HMTBA incorporation into protein, we also measured total radioactivity (including free HMTBA, free HMTBA converted to L-Met but not incorporated into protein, and HMTBA converted to L-Met and incorporated into protein) in individual tissues across time (Figure 5Go). This represents total uptake of methyl-14C-labeled HMTBA from either source, regardless of conversion to L-Met or incorporation into protein. Similar to the radiolabel incorporation into protein in individual tissues across time (shown in Figure 2Go), the total radioactivity in the duodenum was greater in birds gavaged with Mintrex Zn than those gavaged with Alimet (P < 0.001), although this difference was not seen in the other tissues across time.


Figure 5
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  		Figure 5. Total radioactivity in individual tissues across time in birds gavaged with radiolabeled 2-hydroxy-4-(methylthio)butanoic acid (HMTBA) as free HMTBA (supplied by Alimet feed supplement) or as Zn bis(-2-hydroxy-4-methylthiobutyrate) (Mintrex Zn). Broilers were gavaged with radiolabeled HMTBA as free HMTBA (supplied by Alimet feed supplement) or as Zn bis(-2-hydroxy-4-methylthiobutyrate) (Mintrex Zn). Alimet and Mintrex doses were prepared to supply an equimolar amount of HMTBA. Total radioactivity in duodenum, jejunum, liver, leg muscle, and pancreas was measured at 1, 1.5, and 4 h and was pooled across time. There were 18 observations per mean for all tissues except pancreas, where there were 12 observations per mean. Data are expressed as micrograms of HMTBA absorbed per gram of tissue. Bars containing different letters were significantly different (P ≤ 0.05). The only significant difference between HMTBA sources was observed in duodenum (P < 0.001). This difference was seen also at 1, 1.5, and 4 h (data not shown).

 
Experiment 2
Diets for TRT 1 to 5 were formulated with increasing Met activity from Alimet to establish a Met response curve (Table 2Go). Treatment 6 was the same as TRT 2 but supplemented with an additional amount of ITM to test if there was a growth response to high levels of ITM addition per se. Treatments 7 to 12 were supplemented with different amounts of Mintrex Zn, Cu, and Mn on the top of 0.05% Met activity from ALIMET to evaluate the bioavailability of Met activity released from Mintrex. The growth performance of birds fed different dietary TRT are shown in Table 3Go (d 0 to 21 posthatching).


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  		Table 3. Growth performance of broilers in different treatments (TRT; d 0 to 21 posthatch)1
 
For BW and cumulative gain, there were significant differences among dietary TRT (P < 0.01). Birds fed the TSAA-deficient diet (TRT 1) exhibited the lowest performance, and birds fed diets supplemented with 0.10 to 0.20% Met activity from Alimet (TRT 3, 4, and 5), 160 ppm Zn from Mintrex Zn (TRT 8), and 160 ppm Mn from Mintrex Mn (TRT 12) had the highest performance. However, there was no growth response to increasing ITM addition (TRT 6) per se. There was a linear increase of BW and cumulative gain to increasing Met activity addition in TRT 1 to 5 (P < 0.01) and to increasing OTM addition by Mintrex Zn (TRT 2, 7, and 8; P < 0.01) and Mintrex Mn (TRT 2, 11, and 12; P < 0.03). There was no difference in cumulative feed intake among different dietary TRT, indicating that Met activity addition from Alimet or Alimet plus Mintrex had no effect on feed intake.

For cumulative gain:feed ratio, there were significant differences among dietary TRT (P < 0.01). Birds fed the TSAA-deficient diet (TRT 1) had the poorest feed efficiency, whereas birds fed diets supplemented with 0.10 to 0.20% Met activity from Alimet (TRT 3, 4, and 5) and the highest level of Mintrex Zn (160 ppm; TRT 8), Mintrex Cu (80 ppm; TRT 10), and Mintrex Mn (160 ppm; TRT 12) demonstrated the best feed efficiency. However, there was no feed efficiency response to increasing ITM addition (TRT 6) per se. There was a linear increase of feed efficiency to increasing Met activity addition in TRT 1 to 5 (P < 0.01) and to increasing OTM addition by Mintrex Zn (TRT 2, 7, and 8; P < 0.01), Mintrex Cu (TRT 2, 9, and 10; P < 0.02), and Mintrex Mn (TRT 2, 11, and 12; P < 0.04).

As expected, there were significant TRT differences (P < 0.01) for actual supplemental Met intake (calculated by the analyzed HMTBA content from Alimet or Alimet plus Mintrex multiplied by the feed intake). There was a linear increase of actual supplemental Met intake to increasing Alimet addition in TRT 1 to 5 (P < 0.01), as well as to increasing addition of Mintrex Zn (TRT 2, 7, and 8; P < 0.01), Mintrex Cu (TRT 2, 9, and 10; P < 0.01), and Mintrex Mn (TRT 2, 11, and 12; P < 0.01).

A fitted 1-slope broken-line analysis of cumulative gain for TRT 1 to 5 as a function of supplemental Met intake (supplied by Alimet) is shown in Figure 6Go. Based on the 1-slope broken-line prediction equation (below the breakpoint) with measured cumulative gain, the predicted bioavailable Met activity (on an intake basis) from supplemental Alimet plus Mintrex Zn (40 ppm, TRT 7), Mintrex Cu (20 and 80 ppm, TRT 9 and 10), and Mintrex Mn (40 ppm, TRT 11) was calculated to be 0.90, 0.56, 0.79, and 0.78 g. As divided by the actual analyzed supplemental Met activity (see Table 2Go), the relative bioefficacy of Met activity released from Alimet plus Mintrex of TRT 7, 9, 10, and 11 was 113, 95, 86, and 98%, respectively, with an average of 98%, indicating that all the potential Met activity present in Mintrex was fully bioavailable to the chicks. The cumulative gain of TRT 8 (Mintrex Zn, 160 ppm) and TRT 12 (Mintrex Mn, 160 ppm) was beyond the breakpoint and therefore not used for calculation. In addition, 95% confidence intervals around the linear-plateau-predicted values for the Alimet standard growth curve (TRT 1 to 5) were constructed, and the Mintrex + Alimet means (TRT 7 to 12) were compared with these confidence intervals to determine whether they differed significantly (P ≤ 0.05) from the Alimet standard curve. This analysis indicated that at every Met intake level measured (including HMTBA from both Alimet and Mintrex), none of the Mintrex + Alimet means were significantly different than the Alimet-only standard curve (Figure 7Go).


Figure 6
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  		Figure 6. Fitted 1-slope broken line (shown as a solid line) of cumulative gain (g) for treatments (TRT) 1 to 5 as a function of supplemental Met intake (g). The breakpoint of supplemental Met intake was 1.16 g [prediction equation below the breakpoint Y = 838.6 – 82.5 x (1.16 – X); R2 = 0.66]. The dashed line represents the baseline performance of birds fed diets supplemented with 0.05% Met activity (TRT 2) from Alimet feed supplement [an 88% aqueous solution of 2-hydroxy-4-(methylthio)-butanoic acid (HMTBA)]. Symbols for TRT 7 to 12 represent measured cumulative gain at actual supplemental Met intake levels. The predicted bioavailable Met activities (on intake basis, g) from supplemental Alimet plus Mintrex Zn (40 ppm, TRT 7), Mintrex Cu (20 and 80 ppm, TRT 9 and 10), and Mintrex Mn (40 ppm, TRT 11) were calculated to be 0.90, 0.56, 0.79, and 0.78 g, indicating the relative bioefficacy of Met activity released from Alimet plus Mintrex was 113, 95, 86, and 98%, respectively, with an average of 98%. The cumulative gain of TRT 8 (Mintrex Zn, 160 ppm) and TRT 12 (Mintrex Mn, 160 ppm) was beyond the breakpoint and therefore not used for calculation. Mintrex Zn, Cu, and Mn contain 16% Zn, 15% Cu, and 13% Mn and 80, 78, and 76% HMTBA, respectively. There were 6 observations (replicate pens) per mean. ITM = inorganic trace minerals; CI = confidence interval.

 

Figure 7
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  		Figure 7. The fitted 1-slope broken line (shown as a solid line) of cumulative gain (g) for the Alimet standard curve [treatments (TRT) 1 to 5] as a function of supplemental Met intake (g). Cumulative gain in the Mintrex + Alimet TRT is not significantly different than the gain predicted by the Alimet standard curve. Upper and lower 95% confidence interval (CI) curves for the Alimet standard curve are also shown, as dotted and dashed lines, respectively. Actual cumulative gain as a function of supplemental Met intake [including 2-hydroxy-4-(methylthi-o)butanoic acid (HMTBA) from both Alimet and Mintrex] of the 6 Mintrex + Alimet TRT (TRT 7 to 12) is also plotted as filled triangles. Cumulative gain from the Mintrex + Alimet TRT fall well within the upper and lower CI curves, demonstrating no difference in performance between the Mintrex + Alimet vs. Alimet only TRT and suggesting that the HMTBA portion of Mintrex is fully available as a source of Met activity.

 
A broken-line prediction equation (below the breakpoint) was also determined for cumulative gain:feed, as shown in Figure 8Go. In this analysis, the predicted bioavailable Met activity (on an intake basis) from supplemental Alimet plus Mintrex in TRT 7, 9, 10, and 11 was calculated to be 0.72, 0.54, 0.98, and 0.84 g; thus, the relative bioefficacy of Met activity released from Alimet plus Mintrex of these TRT was 91, 91, 106, and 106%, respectively, with an average of 99%. As was the case with cumulative gain, the cumulative gain:feed of TRT 8 (Mintrex Zn, 160 ppm) and TRT 12 (Mintrex Mn, 160 ppm) was beyond the breakpoint and therefore not used for calculation. Furthermore, as with gain, the confidence interval analysis indicated no significant differences between the Alimet-only standard curve and the Mintrex + Alimet TRT at every measured Met intake level (Figure 9Go).


Figure 8
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  		Figure 8. Fitted 1-slope broken line (shown as a solid line) of cumulative gain:feed (g:g) for treatments (TRT) 1 to 5 as a function of supplemental Met intake (g). The breakpoint of supplemental Met intake was 1.28 g [prediction equation below the breakpoint Y = 0.7443 – 0.043 x (1.28 – X); R2 = 0.68]. The dashed line represents the baseline performance of birds fed diets supplemented with 0.05% Met activity (TRT 2) from Alimet feed supplement [an 88% aqueous solution of 2-hydroxy-4-(methylthio)butanoic acid (HMTBA)]. Symbols for TRT 7 to 12 represent measured cumulative gain:feed at actual supplemental Met intake levels. The predicted bioavailable Met activities (on intake basis, g) from supplemental Alimet plus Mintrex Zn (40 ppm, TRT 7), Mintrex Cu (20 and 80 ppm, TRT 9 and 10), and Mintrex Mn (40 ppm, TRT 11) were calculated to be 0.72, 0.54, 0.98, and 0.84 g, indicating the relative bioefficacy of Met activity released from Alimet plus Mintrex was 91, 91, 106, and 106%, respectively, with an average of 99%. The cumulative gain:feed of TRT 8 (Mintrex Zn, 160 ppm) and TRT 12 (Mintrex Mn, 160 ppm) was beyond the breakpoint and therefore not used for calculation. Mintrex Zn, Cu, and Mn contain 16% Zn, 15% Cu, and 13% Mn and 80, 78, and 76% HMTBA, respectively. There were 6 observations (replicate pens) per mean. ITM = inorganic trace minerals; CI = confidence interval.

 

Figure 9
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  		Figure 9. Cumulative gain:feed in the Mintrex + Alimet treatments (TRT) is not significantly different than the gain:feed predicted by the Alimet standard curve. The fitted 1-slope broken line (shown as a solid line) of cumulative gain:feed (g/g) for the Alimet standard curve [TRT 1 to 5] as a function of supplemental Met intake (g) is shown. Upper and lower 95% confidence interval (CI) curves for the Alimet standard curve are also shown, as dotted and dashed lines, respectively. Actual cumulative gain:feed as a function of supplemental Met intake [including 2-hydroxy-4-(methylthio)butanoic acid (HMTBA) from both Alimet and Mintrex] of the 6 Mintrex + Alimet TRT (TRT 7 to 12) is also plotted as filled triangles. Cumulative gain:feed from the Mintrex + Alimet TRT fall well within the upper and lower CI curves, demonstrating no difference in performance between the Mintrex + Alimet vs. Alimet only TRT and suggesting that the HMTBA portion of Mintrex is fully available as a source of Met activity.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Historically, trace minerals have been supplemented in poultry diets using ITM salts, which were used in the development of mineral requirements (NRC, 1994). However, it has been reported that use of ITM salts can result in poor bioavailability of the minerals, primarily because of numerous nutrient and ingredient antagonisms that impair absorption (Leeson and Summers, 2001; Underwood and Suttle, 2001; Dibner, 2005). The ITM salts can be easily dissociated in the relatively low pH of the upper gastrointestinal tract (GIT), allowing the mineral to be lost to dietary antagonists like phytic acid, thereby reducing absorption by the tissue of the animal. The advantage of using OTM is that binding of minerals by the organic ligand provides stability of the complex in the upper GIT, thereby resisting dissociation in the crop, proventriculus, and gizzard, thus allowing the intact OTM complex to be delivered to the absorptive epithelium of the small intestine (Leeson and Summers, 2001; Dibner, 2005). The most common OTM forms available are mineral-AA complexes using specific AA (e.g., Met) as a binding ligand or mineral proteinates using multiple AA (including Met) derived from hydrolyzed protein as binding ligands (Underwood and Suttle, 2001). However, the bioavailability of Met activity of these OTM is rarely evaluated or reported in the literature.

As a novel OTM, Mintrex chelates Zn, Cu, or Mn with HMTBA (a known L-Met precursor; Dibner and Knight, 1984; Dibner, 2003) at a 2:1 ligand-to-mineral ratio (Dibner et al., 2004; W. R. Harris and N. Rath, University of Missouri, and T. Blackburn and J. J. Dibner, Novus International, unpublished data). As with other OTM, the Mintrex chelates have been demonstrated to increase mineral bioavailability (Dibner et al., 2004, 2005; Yan and Waldroup, 2006). The focus of the current study was to evaluate the bioavailability of the Met activity provided by Mintrex, because Mintrex Zn, Cu, and Mn contain 80, 78, and 76% HMTBA by weight, respectively.

In experiment 1, to measure the Met activity of Mintrex, broilers were orally gavaged with methyl-14C-labeled HMTBA in the form of free HMTBA (supplied by Alimet) or as Zn bis(-2-hydroxy-4-methylthiobutyrate) (Mintrex Zn) on an iso-HMTBA basis. The amount of radiolabeled HMTBA incorporated per gram of tissue sampled represented HMTBA from either source that was absorbed, converted to L-Met, and incorporated into protein. The results showed that the incorporation of radiolabel into protein across various tissues over time was not different between Alimet and Mintrex, which demonstrated that both HMTBA sources provided the same Met activity on an equimolar HMTBA basis.

The underlying mechanism of Mintrex absorption at the intestinal epithelial level is not completely understood. Most likely, the organic ligand (HMTBA) and minerals become dissociated in the acidic environment of the unstirred layers along the intestinal mucosa of the proximal GIT (Dibner and Buttin, 2002). Dissociation of a given chelate molecule may be mediated by the acidic microclimate itself or more likely by the mineral transporters in the intestinal epithelium. It is important to note that the mineral transporters have an affinity for their respective metals, that is orders of magnitude greater than the affinity for metals exhibited by commercial ligands, including HMTBA, single amino acids, or short polypeptides (Gaither and Eide, 2001; Dufner-Beattie et al., 2003). Mineral uptake into the intestinal epithelium will most likely be mediated by their corresponding metal transporters (for example, the ZIP transporters for Zn; Gaither and Eide, 2001; Dufner-Beattie et al., 2003; Eide, 2004; Liuzzi and Cousins, 2004; Petris, 2004). The HMTBA released from Mintrex would then be absorbed via passive diffusion or via carrier-mediated uptake (Knight and Dibner, 1984; Brachet and Puigserver, 1987, 1989; Dibner, 2003; Richards et al., 2005).

Interestingly, in experiment 1, when we measured radiolabel incorporation into protein in individual tissues across time, we observed that more radiolabel from Zn bis(-2-hydroxy-4-methylthiobutyrate) (Mintrex Zn) than from free HMTBA (Alimet) was incorporated into protein in the duodenum. Similarly, total HMTBA absorption into duodenum was greater from Mintrex Zn than from Alimet. The most likely explanation for this is simply that Mintrex and Alimet are absorbed at different sites along the GIT. Free HMTBA is an organic acid and as such, it is absorbed rapidly into cells mainly via passive diffusion, especially at the low pH levels that are found in the crop, proventriculus, and gizzard (Dibner, 2003). Indeed, free HMTBA is absorbed primarily in the upper GIT of the chick (Richards et al., 2005). In contrast, the HMTBA in Mintrex Zn is complexed to Zn and therefore is not a free, lipophilic organic acid that would readily diffuse into cells even in the low-pH environment of the upper GIT. The HMTBA in Mintrex would therefore be preferentially absorbed in the small intestine like most other nutrients, as described in the previous paragraph. In contrast to the duodenum, there were no differences in the incorporation of radiolabel into protein of other issues (e.g., jejunum, liver, leg muscle, and pancreas) across time. Furthermore, incorporation kinetics were generally similar between sources in the tissues, as shown for jejunum and leg muscle. In sum, these biochemical data confirm that both HMTBA sources supplied similar Met activity.

In experiment 2, TRT 1 through 5 were used to establish a standard Met response curve, and cumulative gain or cumulative gain:feed ratio was regressed on supplemental Met intake to fit a 1-slope broken line (Robbins et al., 1979). For TRT 1 to 5, there was a linear (P < 0.01) increase of growth performance and feed efficiency to increasing Met activity addition by Alimet, indicating the basal diet was deficient in TSAA (0.70%). The reason for adding 0.05% Met activity from Alimet for TRT 6 to 12 was to ensure a detectable growth response to the ITM and different levels of Mintrex addition over the basal diet (TRT 1), because, theoretically, the maximal Met activity that could be released from Mintrex varied from 0.007 to 0.087% (details shown in Table 2Go). In other words, our goal was to ensure that even the lowest levels of HMTBA addition from Mintrex supplementation would be in the middle portion of the linear part of the standard Met response curve. Therefore, the bioavailable Met activity released from Mintrex supplementation after mineral absorption should contribute to the growth performance beyond the baseline level of 0.05% Met activity addition from Alimet. Cumulative gain increased linearly with increasing Mintrex Zn and Mn addition, and cumulative gain:feed increased linearly with increasing Mintrex Zn, Cu, and Mn inclusion. These results indicated that the Met activity from supplemental Mintrex was bioavailable. Concerning the broken-line analysis, the average relative bioefficacy of Met activity released from supplemental Alimet plus Mintrex was 98% based on cumulative gain and 99% based on cumulative gain:feed, respectively. Furthermore, the confidence-interval analyses demonstrated that for both gain and gain:feed, there were no performance differences between the Alimet and Mintrex + Alimet TRT. These data indicate that the Met activity provided by supplemental Mintrex was fully bioavailable in addition to the HMTBA provided by the Alimet. These data are in agreement with a recent study by Yan and Waldroup (2006) to evaluate the bioavailability of Mintrex Mn, in which the Met activity of Mintrex Mn was also found to be fully available.

It is also formally possible that differences in the levels of the trace elements across the diets could partially explain the performance responses observed in TRT 7 to 12. However, this explanation is far less likely than a Met response for a variety of reasons. First, all diets exceeded NRC (1994) recommendations for Zn, Cu, and Mn. Whereas trace mineral requirements can far exceed NRC levels under commercial conditions, there is no evidence to support a higher requirement for trace minerals in the nonchallenging growth environment of our battery cages. Thus, we would not expect to see a performance response to additional inorganic or OTM supplementation per se in this experiment. Indeed, in the TRT included to examine the potential for such a trace mineral response we observed no significant performance effects (i.e., TRT 6 vs. TRT 2). Second, the radiolabel experiment provided direct evidence that, following absorption, the HMTBA from Mintrex is converted to L-Met and supports protein synthesis. Therefore, the most likely explanation for the observed performance effects in TRT 7 to 12 is that the HMTBA from Mintrex Zn, Cu, and Mn is serving as a source of Met activity.

In conclusion, radiolabel experiments demonstrated that the amount and kinetics of radiolabel incorporation from HMTBA as free HMTBA (supplied by Alimet feed supplement) or Zn bis(-2-hydroxy-4-methylthiobutyrate) (Mintrex Zn) into protein across tissues were similar, demonstrating that both HMTBA sources supplied an equivalent amount of Met activity when compared on an iso-HMTBA basis. The relatively lower amount of HMTBA absorption and radiolabel incorporation into protein in duodenum across time from free HMTBA vs. Mintrex Zn suggests different sites of absorption for free HMTBA vs. Mintrex Zn. For the growth assay, the HMTBA from supplemental Mintrex + Alimet was found to be fully bioavailable as a source of Met activity. Therefore, for practical formulation, the Met activity supplied by the HMTBA content of Mintrex Zn (80% by weight), Cu (78%), and Mn (76%) should be taken into account.


   ACKNOWLEDGMENTS
 
Appreciation is extended to B. Wuelling, M. Wehmeyer, S. Hampton, and D. Kratzer for assistance in this research project.


   FOOTNOTES
 
1 Data in this paper were partially presented at the 2005 International Poultry Scientific Symposia, Atlanta, Georgia (abstract 132) and the 2006 International Poultry Scientific Symposia, Atlanta, Georgia (abstract T147). Alimet, Mintrex, and MHA are trademarks of Novus International Inc. and are registered in the United States and other countries. Back

2 Current address: DaChan Northeast Asia Corp., Beijing, P.R. China. Back

Received for publication July 31, 2006. Accepted for publication January 4, 2007.


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