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ANIMAL NUTRITION |
* University of Kentucky, Lexington 40546;
and
Degussa AG, Feed Additives Division, P.O. Box 1345, 63403 Hanau, Germany; and
and
Maple Leaf Foods Agresearch, 150 Research Lane—Suite 200, Guelph, Ontario N1G 4T2, Canada
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Abstract |
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 Top Abstract INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Key Words: bioefficacy • methionine • methionine hydroxy analog • pig
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INTRODUCTION |
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TopAbstractINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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), and it is commonly supplemented as dry DL-methionine (DLM, 99% pure) or as liquid DL-methionine hydroxy analog-free acid (MHA-FA, containing 88% of the compound 2-hydroxy-4-methylthiobutanoic acid). Potentially greater amounts of Met sources will be supplemented in the future as more producers adopt low-CP diets to decrease N in swine manure.
The relative effectiveness of MHA-FA and DLM in pigs is less clear than in broilers due to the lack of relevant studies (Jansman et al., 2003). Many studies have investigated the relative effectiveness of MHA-FA and DLM as Met sources for poultry (Esteve-Garcia and Llaurado, 1997; Lemme et al., 2002; Liu et al., 2004); however, there are relatively few refereed publications of the biological efficacy of MHA-FA relative to DLM in pigs. Studies reported by Roth and Kirchgessner (1986), Chung and Baker (1992), and Knight et al. (1998) compared biological efficacy with growth assays. Actual N balance studies comparing the biological efficacy of both sources are rare (Römer and Abel, 1999; Zimmerman et al., 2005), and wheat was used as the principal cereal grain in the studies. Thus, the objective of the present studies was to further determine the relative efficacy of MHA-FA compared with DLM to support growth performance and N retention by young pigs, using diets with corn as the principal cereal grain.
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MATERIALS AND METHODS |
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TopAbstractINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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. The trial was conducted according to all applicable Maple Leaf Agresearch standard operating procedures. The second experiment was conducted in stainless steel metabolism cages in environmentally controlled rooms at the University of Kentucky under experimental protocols approved by the Institutional Animal Care and Use Committee of the University of Kentucky.
Experiment 1—Animals and Dietary Treatments
In a 28-d experiment, a total of 245 crossbred [(Duroc x Yorkshire) x (Landrace x Yorkshire)] barrows and gilts with an initial BW of 6.4 kg (SD = 0.5) were allotted to 7 treatments (Table 1) in a randomized complete block design based on initial BW. Pigs were housed in groups of 5 pigs per pen (1.2 m x 1.2 m) with 75% slatted and 25% solid floors. Each pen was equipped with a nipple waterer, and pigs were allowed ad libitum access to feed and water. The BW of individual pigs and feed disappearance were recorded at weekly intervals.
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) was formulated to be deficient in Met (0.21% Met and 0.61% Met + Cys, which was below the Met requirement of 0.30% total Met for pigs weighing 10 to 20 kg; NRC, 1998) but adequate in all other essential nutrients. Ingredients were analyzed for AA content before diet formulation. The BD was then supplemented with 3 graded levels of either DLM (0.030, 0.060, and 0.090%, as-fed basis) or MHA-FA (0.0342, 0.0684, and 0.1026%) on an equimolar basis. The corresponding supplementation levels of MHA-FA were based on a DL-MHA-FA content of 88% in the commercial product. To minimize unintended variations from potential weighing or mixing errors, test diets were prepared by adding the products to a single common batch of the BD before pelleting the diets. Both test supplements, DLM and MHA-FA, were purchased locally by Maple Leaf Foods Agresearch, and analyses verified the concentrations of 99.7 and 88.6% active substance in DLM and MHA-FA, respectively. The dietary nutrient levels as well as concentrations of either DLM or MHA-FA were confirmed by analysis.
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) based on analyzed CP and AA of the ingredients. Dietary treatments included 1) BD, 2) BD + 0.030% DLM, 3) BD + 0.060% DLM, 4) BD + 0.046% MHA-FA, and 5) BD + 0.092% MHA-FA. The levels of MHA-FA used (Table 2) were based on an attempt to provide approximately equal bioequivalents of the 2 compounds and assumed that MHA-FA would have a relative bio-equivalence of about 65% compared with the DLM (Zimmerman et al., 2005). The term bioequivalence used herein describes the biological equivalence, or similarity of a response measure, of 2 or more compounds; it incorporates all aspects of absorption and metabolism that combine to provide the biological response in question. A single batch of BD was mixed that was of sufficient amount to feed all pigs on all treatments for the entire study. The necessary quantities of DLM or MHA-FA were then weighed and blended into the BD to create the additional treatments. The Met equivalents that were added for the dietary treatments were as DLM or a dry, free-flowing MHA-FA product. The normal MHA-FA product is a liquid product that can have some handling and mixing problems. Therefore, a dry product was developed for the study by adding the liquid MHA-FA, which is a minimum of 88% actual MHA-FA, to the carrier (silicon dioxide) at a ratio of 2:1. The subsequent analyzed DL-MHA-FA content was 59.4%, which equated to a liquid MHA-FA equivalent of 67.5% (59.4/ 0.88), confirming the mixing procedures.
In each trial, 10 pigs were assigned based on BW and, if possible, sire of litter as 2 blocks of 5 dietary treatments. Pigs were placed in metabolism crates for a 7-d adaptation to their diet and crate. Feed allowance was equalized within block at approximately 3.5% of the average BW. One-third of the daily feed allowance was provided at 0600, 1300, and 2000, respectively, for the 7-d adaptation period and the 5-d collection period, mixed with a sufficient quantity of water to create a gruel. Feed allowance for the collection period was adjusted based on the final BW for the adaptation period. The beginning and end of the collection period were marked by the addition of 0.5% indigo carmine (Adeola, 2000) to the morning feed allotment. After consumption of each meal, water was added to the metabolism crate feeder to allow ad libitum access to water between meals.
During the collection periods, the total quantities of feces excreted were collected daily, stored in plastic bags, and frozen at –20°C until the end of the collection period. The total feces collected for 5 d were dried in a forced-air drying oven for 72 h at 55°C. The dried fecal samples were ground to pass a 1-mm screen in a Wiley Mill (Model 3; Arthur H. Thomas, Philadelphia, PA) for analysis of DM and N. The collection of urine was initiated 14 h after the feeding of the first marked meal and was completed 14 h after the feeding of the second marked meal at the end of the collection period. A total of 150 mL of 3 N HCl were added to the collection container at the beginning of each collection to prevent volatilization of urinary N. Urine was collected every 24 h and stored at –20°C. At the end of the collection, the total quantity of urine collected from each pig was allowed to thaw, then measured and pooled. Two aliquots (approximately 200 mL) of urine from each pig were subsampled for N analysis. The average of the analyzed dietary values (5 dietary treatments were analyzed in duplicate) was used for feed N content calculation because of the mixing of a common BD for all experimental treatments.
Laboratory Analyses
The DM in feed and feces was determined after oven-drying for 4 h at 103°C (AOAC, 2000). The N content of the diets, feces, and urine was determined using a gas combustion method (AOAC, 1998; FP-2000, Leco Corp., St. Joseph, MI). Ethylenediaminetetraacetic acid was used as a reference standard before and after all N analyses. Dietary AA concentrations were determined by ion-exchange chromatography with postcolumn derivatization with ninhydrin. Amino acids were oxidized with performic acid, which was neutralized with sodium metabisulfite (Llames and Fontaine, 1994; European Community, 1998). Amino acids were liberated from the protein by hydrolysis with 6 N HCl for 24 h at 110°C and quantified with the internal standard method by measuring the absorption of reaction products with ninhydrin at 570 nm. Tryptophan was determined by HPLC with fluorescence detection (extinction 280 nm, emission 356 nm) after alkaline hydrolysis with barium hydroxide octahydrate for 20 h at 110°C (European Community, 2000). Tyrosine was not determined. Supplemented AA were determined after extraction with 0.1 N HCL (European Community, 1998). Supplemented MHA-FA was analyzed using the method described by VDLUFA (1997).
Statistical Analyses
Experiment 1.
Growth performance data were analyzed using GLM procedures of SAS 8 (SAS Inst., Inc., Cary, NC). The pen was considered the experimental unit for statistical analyses. Preplanned contrasts were performed to test the effects of Met sources (BD vs. DLM, BD vs. MHA-FA, and DLM vs. MHA-FA). A nonlinear exponential model was used to determine the effectiveness of MHA-FA relative to pure DLM from weight gain and feed conversion data. Although supplemented levels of DLM and MHA-FA were adjusted on an equimolar basis, the regression analysis was performed on a weight-supplementation basis. The following nonlinear equation was applied:
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in which y = performance criterion (weight gain, feed conversion); a = intercept (animal performance with BD); b = asymptotic response, a + b = common asymptote (maximum performance level), c1 = steepness coefficient for DLM, c2 = steepness coefficient for MHA-FA, and x1, x2 = dietary level of DLM and MHA-FA, respectively
According to Littell et al. (1997), bioefficacy values for MHA-FA relative to DLM were given by the ratio of their c-values (100 x [c2/c1]). The 
level used for determination of statistical significance was 0.05.
Experiment 2.
One replicate was removed from the experiment because of problems with the pig fed the BD. This pig had an extremely high N retention, which resulted in a negative slope for the N-retention line, which violated the assumptions of a first-limiting nutrient study (i.e., a positive slope). Additionally, the slope of the regression line for this replicate was >4 SEM from the mean slope, whereas the next largest deviation was 2.51 SEM from the mean. Based on the statistical deviations from mean response and violation of the assumptions of a first-limiting nutrient study, the entire replicate was removed and the slopes recomputed. Following recomputation of treatment mean slopes, no individual replicate slope exceeded 2.6 SEM from the mean.
The experimental data were analyzed using GLM procedures of SAS. The model included treatment, collection group, and replicate. Single df contrasts were used to evaluate specific items of interest (i.e., linear and quadratic effects within each source). The results also were subjected to linear regression analysis within each Met source using the REG procedure of SAS. Calculation of relative bioequivalence was made according to Littell et al. (1997), wherein the slope of the response line of the test ingredient is divided by the slope of the response line of the standard ingredient. Data were evaluated to verify that the assumptions for validity of the slope ratio assay were met (i.e., linear response in the absence of curvilinearity and a common intercept for both sources) before the computation of relative bio-equivalence. The level used for determination of statistical significance was 0.05.
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RESULTS |
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TopAbstractINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Experiment 1
Supplementation of either DLM or MHA-FA enhanced ADG, ADFI, and feed:gain ratio (P < 0.001; Table 3) compared with pigs fed the BD. During d 0 to 14, pigs fed diets supplemented with DLM had 12.6% greater ADG and 7.8% lower feed:gain ratio than the pigs fed diets supplemented with MHA-FA (P = 0.011 and 0.009, respectively). During the overall period, DLM treatments had 9.7% greater ADG and 5.3% lower feed:gain ratio than MHA-FA treatments (P = 0.012 and 0.029, respectively). Pigs fed the BD achieved an ADG of 124 g and feed:gain ratio of 1.860 (Table 3), whereas the maximum potential response for ADG and feed:gain ratio was 465 g (375% of basal-fed pigs) and 1.290 (30% less than basal-fed pigs), respectively, based on the equations developed from the data for the overall period (Figures 1 and 2); realized ADG was less than predicted potential, whereas realized feed:gain was very close to the predicted potential. In the 28-d growth experiment, the bioefficacy of MHA-FA compared with DLM for ADG (Figure 1) was 73% on a product-to-product (wt/wt) basis (83% on an equimolar basis). The bioefficacy of MHA-FA to DLM for feed:gain ratio (Figure 2) was 54% on a product-to-product basis (61% on an equimolar basis; P < 0.05).
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The effects of dietary DLM and MHA-FA level on N digestibility and retention are shown in Table 4. The supplementation of Met sources did not affect fecal N output; however, urinary N (g) excretion linearly decreased (P = 0.034 for DLM, and P = 0.007 for MHA-FA) with increasing concentrations of both Met sources, which resulted in a linear increase in retained N (g) for both DLM (P = 0.012) and MHA-FA (P = 0.005). In addition, N retention rate (%) linearly increased with increasing level of DLM (P = 0.014) and MHA-FA (P = 0.007). There were no quadratic effects observed with dietary DLM or MHA-FA level on urinary N, retained N, or N retention rate.
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) and 66.3% based on the N retained per day (Figure 4). Assuming an 88% inclusion rate of DL-MHA-FA in the product and a mean response of 65.25%, this would equate to a relative bioequivalence of MHA-FA to DLM of 74.1% on an equimolar basis.
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DISCUSSION |
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TopAbstractINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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). This criterion was met. In Exp. 1, as the inclusion rate of both Met sources increased,
ADG, ADFI, and feed:gain ratio were improved (P < 0.001) during d 0 to 14, d 14 to 28, and the entire period (Table 3). An exponential regression fit the data better than linear regression, and thus, exponential regression was used to determine the effectiveness of the 2 Met sources for weight gain and feed conversion. In Exp. 2, the N balance trial, N retention also responded to each unit of addition of each product, and a linear regression model fit the data best.
In the current study, the relative bioefficacy of MHA-FA to DLM was lower than 88% on a product-to-product basis for all responses measured (ADG, feed:gain, N retention); thus, the relative effectiveness of MHA-FA was less than DLM on an equimolar basis. Our results are consistent with a recent review (Jansman et al., 2003), which analyzed 5 studies to calculate the biological efficacy for MHA-FA relative to DLM in pigs. Even though the data were variable, ranging from 54 to 104% among the studies, the mean value of the biological efficacy for MHA-FA relative to DLM was 82% on an equimolar basis (which equates to 72% on a product-to-product basis). According to the review by Jansman et al. (2003), more publications were available with broilers, and the calculated efficacy values were slightly less variable (53 to 97% in 20 studies), and the mean value of the biological efficacy for MHA-FA relative to DLM was 77% on an equimolar basis, which equates to 68% on a product-to-product basis.
Roth and Kirchgessner (1986) evaluated the biological efficacy for MHA-FA relative to DLM in growing pigs from 8 to 55 kg with a slope-ratio comparison of the growth and feed:gain response. The mean value of the biological efficacy for MHA-FA relative to DLM was 78.4% on an equimolar basis for ADG (P < 0.05; equates to 69.0% on a product-to-product basis) and 89.6% on an equimolar basis for feed:gain (equates to 78.8% on a product-to-product basis). However, Knight et al. (1998) reported that MHA-FA had the same effectiveness as DLM on an equimolar basis in liver cell culture and growth performance of early-weaned pigs from 4 to 14 kg of BW. Chung and Baker (1992) tested the molar efficacy of Met isomers including DLM and MHA-FA with a growth assay in swine and also failed to observe different growth to equimolar levels of MHA-FA and DLM. However, their study included only one MHA-FA supplementation level above the basal concentration.
In trials conducted by Schmidt (2000) and Zimmerman et al. (2005), the N balance technique was used as a precise method to establish the efficacy of MHA-FA compared with DLM in pigs. Schmidt (2000) used pigs in the BW range from 20 to 50 kg. A BD, clearly deficient in Met, was supplemented with 3 graded levels of DLM or MHA-FA on an equimolar basis. Based on equal feed intake, the effectiveness of MHA-FA was calculated to be 63% compared with DLM on a product-to-product basis. Zimmerman et al. (2005) used weanling pigs weighing 11 to 12 kg BW with a BD containing 0.22% Met and 0.51% Met + Cys based on wheat, peas, and barley as the principle cereal components. The Met-deficient BD was supplemented with three graded levels of either DLM or MHA-FA on an equimolar basis. They reported that the relative effectiveness of MHA-FA to DLM on a product-to-product basis was 62%. Contrary to these reports, Römer and Abel (1999) concluded that the 2 sources of Met did not differ with regard to their biological efficacy for improving N retention by heavier pigs (29 to 35 kg) fed wheat-based diets containing 0.19% Met.
Questions remain regarding the physiological reasons for the possible incomplete use of the hydroxy analog. The conversion of the D and L isomers of MHA-FA and D-Met enables these precursors to become biologically available as L-Met. The enzymes required for the transformations are in various tissues and organs including liver and kidney (Dibner, 2003). After conversion, MHA-FA acquires the same characteristics as L-Met; however, whether absorption of MHA-FA and DLM is similar is less clear. Because L-Met has a greater affinity for its transporter and greater maximal velocity of transport compared with L-MHA-FA (Maenz and Engele-Schaan, 1996), L-Met has less exposure to intestinal bacteria, resulting in increased Met uptake across the brush border membrane. Maenz and Engele-Schaan (1996) concluded that there is a substantial conversion of dietary MHA-FA during passage through the small intestine to compounds that cannot be utilized as a source of Met by poultry. Recently, Drew et al. (2003) demonstrated that intestinal bacteria decreased (P < 0.05) the apparent MHA-FA absorption from the intestinal tract in a poultry model. Alterations in the intestinal tract would explain why liver cell culture would demonstrate equal molar bioequivalence (Knight et al., 1998) that did not translate to in vivo model bioequivalence (Jansman et al., 2003).
In conclusion, our data demonstrated a product-to-product bioefficacy of MHA-FA relative to DLM of 73, 54, and 65% (83, 61, and 74% on a equimolar basis) for ADG, feed:gain ratio, and N retention, respectively. Additional studies would be useful to add clarity to this important area of swine nutrition.
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Footnotes |
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2 Appreciation is expressed to D. Higginbotham for help in diet preparation and to APC, Inc., Ames, IA, and Akey, Inc., Lewisburg, OH, for ingredients used in the experiments at the Univ. of Kentucky.
3 Corresponding author: mdlind1{at}uky.edu
Received for publication April 22, 2005. Accepted for publication August 27, 2005.
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LITERATURE CITED |
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TopAbstractINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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