Effects of excess DL- and L-methionine on growth performance of starter Pekin ducks

Effects of excess DL- and L-methionine on growth performance of starter Pekin ducks Abstract A dose-response experiment was conducted to investigate the effects of excess DL-methionine (DLM) and L-methionine (LM) on growth response of starter Pekin ducks from 7 to 21 days of age. A total of 462 seven-day-old male Pekin ducklings were allotted to 77 wire-floor pens with 6 birds per pen. There were 11 treatments including a methionine-adequate control diet (containing 0.49% methionine) and control diets supplemented with 5 levels of crystal DLM or LM (0.25, 0.50, 0.75, 1.0, and 1.25%) based on equal product weight. At 21 d of age, weight gain, feed intake, and feed/gain of ducks from each pen were measured. In our study, the weight gain and feed intake did not change markedly and kept a plateau when supplemental DLM or LM was below 0.50%, but the weight gain and feed intake decreased further as supplemental DLM or LM increased from 0.50 to 1.25%. When the maximum safe level (MSL) of supplemental methionine sources and methionine content of the control diets were combined, the total tolerable upper limits of methionine for weight gain and feed intake were 0.91 and 0.85% when DLM was used, respectively, and the corresponding values were 0.89 and 0.84% when LM was used, respectively. On the other hand, both excess DLM and LM reduced weight gain and feed intake (P < 0.05) but there were no significant differences (P > 0.05) in weight gain and feed intake between the ducks fed DLM- and LM-supplemented diets. Furthermore, linear slope-ratio assay was also used to evaluate the relative toxicity of excess DLM and LM. According to this assay, the efficiencies of DLM relative to LM for depression of weight gain and feed intake were 97 and 95%, respectively. In summary, excess DL- and L-methionine were toxic for starter Pekin ducks and both methionine sources were equally growth depressing. INTRODUCTION Methionine is the first-limiting amino acid in practical duck diets and optimal supplementation of crystalline methionine in diets could improve growth performance, carcass traits, and feather growth of White Pekin ducks (Xie et al., 2004; Xie et al., 2006; Zeng et al., 2015). However, excess methionine is toxic and growth depression was observed in growing Pekin ducks when 1 or 2% methionine was supplemented to control diets with the optimal methionine level (Xie et al., 2007). The tolerable upper limit of dietary methionine for growing Pekin ducks may be less than 1.38% (Xie et al., 2007), but the tolerable upper limit of dietary methionine for starter Pekin ducks is still unknown. In the past decades, DL-methionine (DLM) was usually supplemented to poultry diets to meet the total sulfur amino acid requirement of poultry and the toxicity of DLM was also specified in chicks and ducks. At present, as a new methionine source, L-methionine (LM) has been commercially available for feed formulation and LM is as efficacious as DLM for improvement of growth performance in broilers (Dilger and Baker, 2007). Like DLM, LM is also toxic when given in excess and growth depression takes place in chicks in such instances (Harter and Baker, 1978; Baker, 1979). In broilers, excess DLM and LM were equally growth depressing (Katz and Baker, 1975; Dilger, et al., 2007a). However, the information on the relative toxicity between DLM and LM was lacking in ducks. Therefore, in order to ensure the safe use of methionine sources and to establish the tolerable upper limit of methionine uptake for starter ducks, the objective of our study was to investigate the effects of excess DL- and L-methionine on the growth performance of starter White Pekin ducks and to assess and compare the toxicity of DLM and LM in ducks. MATERIAL AND METHODS All procedures of our experiment were approved by the animal care and use committee of Institute of Animal Sciences of Chinese Academy of Agricultural Sciences. Five hundred 1-day-old male Pekin ducklings from a commercial hatchery were randomly allocated to 50 wire-floor pens with 10 ducklings per pen. These ducklings were fed a control diet (Table 1) from hatch to 7 d of age. At 7 d of age, after all ducklings were weighed individually and some birds with lowest or highest body weight were removed, the 462 ducks with average body weight of 145 ± 20 g were selected and allocated to 77 wire-floor pens with 6 ducks per pen according to similar pen weight. There were 11 treatments (Table 2) including a methionine-adequate control diet and control diets supplemented with crystal DLM (Evonik Industries, Krefeld, Germany) or LM (CheilJedang Co., Seoul, Korea) according to 5 supplemental levels (0.25, 0.50, 0.75, 1.00, and 1.25%) based on equal product weight. Both crystal DLM and LM had 99% purity and they were supplemented to control diets at the expense of corn. Each treatment had 7 replicate pens of 6 birds each. During the period from hatch to 21 days of age, all ducks had free access to feed and water and feed was provided in the form of pellets. Lighting was continuous. The temperature was kept at 33°C for the first 3 d and then was reduced gradually to approximately 25°C until 21 d of age. Table 1. Composition of control diet from hatch to 21 d of age (% as fed). Item    Ingredient    Corn  62.07  Soybean meal  32.80  L-Met  0.18  Dicalcium phosphate  1.55  Limestone  1.10  Sodium chloride  0.30  Premix1  1.00  Soybean oil  1.00  Calculated composition    Metabolizable energy,2 kcal/kg  2,879  Crude protein  19.94  Methionine  0.493  Cysteine  0.353  Lysine  1.03  Calcium  0.82  Nonphytate phosphorus  0.35  Item    Ingredient    Corn  62.07  Soybean meal  32.80  L-Met  0.18  Dicalcium phosphate  1.55  Limestone  1.10  Sodium chloride  0.30  Premix1  1.00  Soybean oil  1.00  Calculated composition    Metabolizable energy,2 kcal/kg  2,879  Crude protein  19.94  Methionine  0.493  Cysteine  0.353  Lysine  1.03  Calcium  0.82  Nonphytate phosphorus  0.35  1Supplied per kilogram of total diet: Cu (CuSO4•5H2O), 8 mg; Fe (FeSO4•7H2O), 60 mg; Zn (ZnO), 60 mg; Mn (MnSO4•H2O), 100 mg; Se (NaSeO3), 0.3 mg; I (KI), 0.4 mg; choline chloride, 1,000 mg; vitamin A (retinyl acetate), 1,376 μg; vitamin D3 (Cholcalciferol), 50 μg; vitamin E (DL-α-tocopheryl acetate), 20 mg; vitamin K3 (menadione sodium bisulfate), 2 mg; thiamin (thiamin mononitrate), 2 mg; riboflavin, 10 mg; pyridoxine hydrochloride, 4 mg; cobalamin, 0.02 mg; calcium-D-pantothenate, 20 mg; folic acid, 1 mg; and biotin, 0.15 mg. 2The value was calculated according to the apparent metabolizable energy of chickens (Ministry of Agriculture of China, 2004). 3The numbers were analyzed values. View Large Table 2. Effects of excess DL-methionine (DLM) and L-methionine (LM) on growth performance of starter Peking ducks from 7 to 21 days of age.1 Treatment  Weight gain (g/bird per d)  Feed intake (g/bird per d)  Feed/Gain (g/g)  Control(C)  69.9  117.4  1.68  C+0.25% DLM  70.0  118.1  1.69  C+0.25% LM  69.7  115.4  1.65  C+0.50% DLM  67.5  110.8  1.64  C+0.50% LM  65.7  107.2  1.63  C+0.75% DLM  58.6  96.9  1.65  C+0.75% LM  58.9  97.0  1.65  C+1.00% DLM  49.7  83.7  1.69  C+1.00% LM  49.7  83.0  1.67  C+1.25% DLM  42.0  71.5  1.70  C+1.25% LM  40.6  68.4  1.69  SEM  0.78  1.44  0.015  Probability        DLM vs. C  0.008  0.003  0.767  LM vs. C  0.004  0.001  0.261  DLM vs. LM  0.793  0.624  0.152  DLM broken-line  <0.001  <0.001  0.461  LM broken-line  0.002  0.002  0.368  Treatment  Weight gain (g/bird per d)  Feed intake (g/bird per d)  Feed/Gain (g/g)  Control(C)  69.9  117.4  1.68  C+0.25% DLM  70.0  118.1  1.69  C+0.25% LM  69.7  115.4  1.65  C+0.50% DLM  67.5  110.8  1.64  C+0.50% LM  65.7  107.2  1.63  C+0.75% DLM  58.6  96.9  1.65  C+0.75% LM  58.9  97.0  1.65  C+1.00% DLM  49.7  83.7  1.69  C+1.00% LM  49.7  83.0  1.67  C+1.25% DLM  42.0  71.5  1.70  C+1.25% LM  40.6  68.4  1.69  SEM  0.78  1.44  0.015  Probability        DLM vs. C  0.008  0.003  0.767  LM vs. C  0.004  0.001  0.261  DLM vs. LM  0.793  0.624  0.152  DLM broken-line  <0.001  <0.001  0.461  LM broken-line  0.002  0.002  0.368  1Results are means with n = 7 per treatment. View Large The methionine content of the control diet was 0.49% and it was adequate for duck growth (Xie et al, 2004). The contents of nutrients except crude protein in the control diet met the NRC (1994) recommendation of White Pekin ducks from 0 to 2 wk of age and the content of crude protein in the control diet met the requirements of starter Pekin ducks provided by Xie et al. (2017). All experimental diets were cold pelleted at room temperature. The methionine and cysteine of all experimental diets were analyzed according to the method recommended by Standardization Administration of China (2000). Briefly, methionine and cysteine were oxidized at 0°C by performic acid (formic acid: hydrogen peroxide = 9:1) for 16 hours and then hydrolyzed at 110°C by 6 M HCL for 24 hours. After that, the pH of these hydrolysates was adjusted to 2.2 and then analyzed by using ion-exchange chromatography with an amino acid analyzer (L-800, Hitachi, Tokyo, Japan). The supplemental DLM or LM levels of all diets were also confirmed by the difference in analyzed methionine contents between the control diet and the corresponding DLM or LM-supplemented control diets. The 5 confirmed supplemental DLM levels were 0.24, 0.50, 0.78, 0.98, and 1.26%, respectively, and the 5 confirmed supplemental LM levels were 0.26, 0.49, 0.73, 1.00, and 1.25%, respectively, which is very close to the formulated levels (0.25, 0.50, 0.75, 1.00, and 1.25%). At 21 d of age, weight gain, feed intake, and feed/gain of ducks from each pen were measured. Feed intake and feed/gain were all corrected for mortality. Data were analyzed as a completely randomized design by ANOVA procedure of SAS software (SAS Institute, 2003), with pen used as the experimental unit for analysis. The orthogonal contrasts were used to compare the difference between DLM and control, LM and control, and DLM and LM, and a probability level of P < 0.05 was considered to be statistically significant. In our study, the broken-line regression (Alhotan et al., 2017) was used to estimate the maximum safe level of supplemental DLM or LM for starter ducks using the NLIN procedure (SAS Institute, 2003). The broken-line linear model was provided as follows:   \begin{equation*}y = l + u\left( {x - r} \right)\end{equation*} where y = growth response (weight gain or feed intake), x = supplemental DLM or LM (%), r = breakpoint between two lines which was defined as the maximum safe level (MSL) of supplemental DLM or LM (%), u = the slope of the curve, l = maximum response if x < r and y = l + u (x−r) if x ≥ r. Furthermore, according to the statistical method suggested by Sterling et al. (2003), a Student t test was used to determine if a difference existed in maximum safe levels between supplemental DLM and LM. On the other hand, according to the statistical method of Littell et al. (1997), the linear ratio-slope regression was used to estimate the toxicity of DLM relative to LM by NLIN procedure of SAS software (SAS Institute, 2003). The linear ratio-slope model was provided as follows:   \begin{equation*} y = a + {b_1}{x_1} + {b_2}{x_2} \end{equation*} where y = growth response (weight gain or feed intake), a = intercept (duck performance with control diet), b1 = steepness coefficient for supplemental DLM, b2 = steepness coefficient for LM, and x1, x2 = supplemental level of DLM and LM, respectively. The relative toxicity of DLM compared with LM was given by b1/b2. RESULTS AND DISCUSSION In our study, the analyzed methionine content of the control diet was 0.49% (Table 1) and this value was similar to the methionine requirement of starter Pekin ducklings estimated by Xie et al. (2004), which ensured the maximum growth of ducks fed the control diets in our study. Furthermore, L-methionine could be incorporated directly into body proteins, but the D-methionine must first be converted into keto-methionine and then into the L-methionine before being incorporated into protein. In order to avoid the debate on the conversion of D-methionine to L-methionine, crystal LM (not DLM) was used in the control diet to meet the total methionine requirement of ducklings and the 0.18% crystal LM in the control diets was supposed to support duck growth through its incorporation into protein. Total mortality was less than 1% and dead birds came from different treatments, not only one treatment, which did not affect the accuracy of our results. In the present study, supplementation of excess DLM or LM in control diets had negative effects on weight gain (P < 0.05), feed intake (P < 0.05), but not feed/gain (P > 0.05), which indicated that growth depression resulted from reduction of feed intake. Our results confirmed the toxicity of excess DLM and LM in starter ducks and the toxicity of DLM was also observed in growing Pekin ducks (Xie et al., 2007). In order to avoid any detrimental effects of excess crystal methionine on the biological performance of the ducks, it was necessary to estimate the MSL of these methionine products when crystal methionine was supplemented into the diets. It is preferable to estimate MSL by linear broken-line regression rather than multiple range tests (Alhotan et al., 2017). In our study, the weight gain and feed intake did not change markedly and kept a plateau when supplemental DLM or LM was below 0.50%, but the weight gain and feed intake decreased further as supplemental DLM or LM increased from 0.50 to 1.25% (Table 2), which showed the broken-line response. Therefore, according to linear broken-line regression, the MSL of supplemental DLM and LM for weight gain were 0.42 and 0.40%, respectively, and the MSL of supplemental DLM and LM for feed intake were 0.36 and 0.35%, respectively (Table 3). Furthermore, when maximum safe level of supplemental methionine sources and methionine content (0.49%) of the control diets were combined, the total tolerable upper limits of methionine for weight gain and feed intake were 0.91 and 0.85% when DLM was used, respectively, and the corresponding values were 0.89 and 0.84% when LM was used, respectively. In broilers, the weight gain of broilers before 22 days of age post-hatch was not reduced significantly when 1% DLM was added to the control diets containing 0.57, 0.46, or 0.51% Met, respectively (Han and Baker, 1993; Scherer and Baker, 2000; Dilger et al., 2007b). Therefore, the ducks may be less able to tolerate excess methionine compared with broilers during the starter period. Table 3. Maximum safe levels of supplemental DL- and L-Methionine for satisfactory growth performance of White Pekin ducklings as estimated by broken-line linear model. Criteria  Methionine source  Broken-line linear model  R2  Estimated maximum response1  Maximum Safe levels1  Student t test2  Weight gain  DL-methionine  y = 69.9–34.2 × (x−0.42)  0.999  69.9 ± 0.3  0.42 ± 0.01  0.63  L-methionine  y = 69.8–33.9 × (x−0.40)  0.998  69.8 ± 0.5  0.40 ± 0.03  Feed intake  DL-methionine  y = 117.7–52.4 × (x−0.36)  0.999  117.7 ± 0.4  0.36 ± 0.01  0.24  L-methionine  y = 116.4–52.2 × (x−0.35)  0.996  116.4 ± 1.1  0.35 ± 0.04  Criteria  Methionine source  Broken-line linear model  R2  Estimated maximum response1  Maximum Safe levels1  Student t test2  Weight gain  DL-methionine  y = 69.9–34.2 × (x−0.42)  0.999  69.9 ± 0.3  0.42 ± 0.01  0.63  L-methionine  y = 69.8–33.9 × (x−0.40)  0.998  69.8 ± 0.5  0.40 ± 0.03  Feed intake  DL-methionine  y = 117.7–52.4 × (x−0.36)  0.999  117.7 ± 0.4  0.36 ± 0.01  0.24  L-methionine  y = 116.4–52.2 × (x−0.35)  0.996  116.4 ± 1.1  0.35 ± 0.04  1Expressed as means ± SE. 2Maximum safe levels considered significantly different if |t|> t (α = 0.05, df = 35) = 2.03. View Large On the other hand, the toxicities of DLM and LM in starter ducks were compared and excess DLM and LM were equally growth depressing based on the equal product weight. For orthogonal contrasts in our study, both excess DLM and LM reduced weight gain and feed intake (P < 0.05) but there were no significant differences (P > 0.05) in weight gain and feed intake between the ducks fed DLM- and LM-supplemented diets (Table 2). Furthermore, the Student t test also showed that the MSL of DLM was not significantly different from the MSL of LM (P > 0.05, Table 3), which also indicated DLM and LM had the same toxic effects on duck performance. Our results were also supported by Dilger et al. (2007a) who found that DLM was no more growth depressing than LM when 1.5 or 3.0% methionine was supplemented to chick diets. DL-methionine is a racemic (50:50) mixture of D- and L-methionine. L-methionine could be incorporated directly into body proteins, and it is assumed to be 100% efficacious, but D-methionine must be converted to L-methionine before it is incorporated into protein. It is well established that the chick can use D-Met with greater than 90% efficacy relative to L-Met (Wretlind and Rose, 1950; Sugahara et al., 1967; Sunde, 1972; Baker, 1986) and it has been traditionally accepted that DL-Met is 95% efficacious relative to L-Met due to equal dietary contributions of the D- and L-isomers (Baker, 2006). Our results in linear slope-ratio assay for relative toxicity of excess methionine sources supported the aforementioned hypothesis. In our study, linear slope-ratio assay was also used to compare relative toxicity of excess DLM and LM because the responses to the factors in the dose-response studies should be continuous, not discrete. According to this assay, on the equal supplemental levels, the efficiencies of DLM relative to LM for depression of weight gain and feed intake were 97 and 95%, respectively (Figures 1 and 2), which also indicated that DLM was as toxic as LM. Furthermore, our results were also supported by the results in chicks at nontoxic methionine levels in which the efficacy of DLM relative to LM was 98.5 and 97.2% for improvement of weight gain and gain/feed, respectively (Dilger et al., 2007a). Figure 1. View largeDownload slide Slope-ratio comparison of toxicity between supplemental DL-methionine (DLM) and L-methionine (LM) based on the weight gain of starter Pekin ducklings from 7 to 21 days of age. Values in brackets indicate 95% confidence intervals. Figure 1. View largeDownload slide Slope-ratio comparison of toxicity between supplemental DL-methionine (DLM) and L-methionine (LM) based on the weight gain of starter Pekin ducklings from 7 to 21 days of age. Values in brackets indicate 95% confidence intervals. Figure 2. View largeDownload slide Slope-ratio comparison of toxicity between supplemental DL-methionine (DLM) and L-methionine (LM) based on the feed intake of starter Pekin ducklings from 7 to 21 days of age. Values in brackets indicate 95% confidence intervals. Figure 2. View largeDownload slide Slope-ratio comparison of toxicity between supplemental DL-methionine (DLM) and L-methionine (LM) based on the feed intake of starter Pekin ducklings from 7 to 21 days of age. Values in brackets indicate 95% confidence intervals. In conclusion, excess DL-methionine and L-methionine reduced the growth response of starter Pekin ducks and DL-methionine was as growth depressing as L-methionine at equal product weight. The total tolerable upper limits of methionine for starter Pekin ducks was about 0.90%. ACKNOWLEDGEMENTS This work was sponsored by the earmarked fund for China Agriculture Research System (CARS-42) and the science and technology innovation project of Chinese Academy of Agricultural Sciences (CXGC-IAS-09). REFERENCES Alhotan R. A., Vedenov D. V., Pesti G. M.. 2017. Estimation of the maximum safe level of feed ingredients by spline or broken-line nonlinear regression models. Poult. Sci.  96: 904– 913. Google Scholar CrossRef Search ADS PubMed  Baker D. H. 1979. Efficacy of the D- and L-isomers of N-acetylmethionine for chicks fed diets containing either crystalline amino acids or intact protein. J. Nutr.  109: 970– 974. Google Scholar CrossRef Search ADS PubMed  Baker D. H. 1986. Problems and pitfalls in animal experiments designed to establish dietary requirements for essential nutrients. J. Nutr.  116: 2339– 2349. Google Scholar CrossRef Search ADS PubMed  Baker D. H. 2006. Comparative species utilization and toxicity of sulfur amino acids. J. Nutr.  136: 1670S– 1675S. Google Scholar CrossRef Search ADS PubMed  Dilger R. N., Baker D. H.. 2007. DL-Methionine is as efficacious as L-methionine, but modest L-cystine excesses are anorexigenic in sulfur amino acid-deficient purified and practical-type diets fed to chicks. Poult. Sci.  86: 2367– 2374. Google Scholar CrossRef Search ADS PubMed  Dilger R. N., Kobler C., Weckbecker C., Hoehler D., Baker D. H.. 2007a. 2-Keto-4-(Methylthio)Butyric Acid (Keto Analog of Methionine) Is a Safe and Efficacious Precursor of l-Methionine in Chicks. J. Nutr.  137: 1868– 1873. Google Scholar CrossRef Search ADS   Dilger R. N., Toue S., Kimura T., Sakai R., Baker D. H.. 2007b. Excess dietary L-cysteine, but not L-cystine, is lethal for chicks but not for rats or pigs. J. Nutr.  137: 331– 338. Google Scholar CrossRef Search ADS   Han Y., Baker D. H.. 1993. Effects of excess methionine or lysine for broilers fed a corn-soybean meal diet. Poult. Sci.  72: 1070– 1074. Google Scholar CrossRef Search ADS PubMed  Harter J. 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Nutr.  130: 3055– 3058. Google Scholar CrossRef Search ADS PubMed  Standardization Administration of China. 2000. Determination of Amino Acids in Feeds . Standards Press of China, Beijing, China. Sterling K. G., Pesti G. M., Bakalli R. I.. 2003. Performance of broiler chicks fed various levels of dietary lysine and crude protein. Poult. Sci.  82: 1939– 1947. Google Scholar CrossRef Search ADS PubMed  Sugahara M., Morimoto T., Kobayashi T., Ariyoshi S.. 1967. The nutritional value of D-amino acid in the chick nutrition. Agric. Biol. Chem.  31: 77– 84. Google Scholar CrossRef Search ADS   Sunde M. L. 1972. Utilization of D- and DL-amino acids and analogs. Poult. Sci.  51: 44– 55. Google Scholar PubMed  Wretlind K. A., Rose W. C.. 1950. Methionine requirement for growth and utilization of its optical isomers. J. Biol. Chem.  187: 697– 703. Google Scholar PubMed  Xie M., Hou S. S., Huang W.. 2006. Methionine requirements of male white Peking ducks from twenty-one to forty-nine days of age. Poult. Sci.  85: 743– 746. Google Scholar CrossRef Search ADS PubMed  Xie M., Hou S. S., Huang W., Fan H. P.. 2007. Effect of excess methionine and methionine hydroxy analogue on growth performance and plasma homocysteine of growing Pekin ducks. Poult. Sci.  86: 1995– 1999. Google Scholar CrossRef Search ADS PubMed  Xie M., Hou S. S., Huang W., Zhao L., Yu J. Y., Li W. Y., Wu Y. Y.. 2004. Interrelationship Between Methionine and Cystine of Early Peking Ducklings. Poult. Sci.  83: 1703– 1708. Google Scholar CrossRef Search ADS PubMed  Xie M., Jiang Y., Tang J., Zhang Q., Huang W., Hou S. S.. 2017. Starter and subsequent grower response of Pekin ducks to low-protein diets in starter phase. Livest. Sci.  203: 92– 96. Google Scholar CrossRef Search ADS   Zeng Q. F., Zhang Q., Chen X., Doster A., Murdoch R., Makagon M., Gardner A., Applegate T. J.. 2015. Effect of dietary methionine content on growth performance, carcass traits, and feather growth of Pekin duck from 15 to 35 days of age. Poult. Sci.  94: 1592– 1599. Google Scholar CrossRef Search ADS PubMed  © 2017 Poultry Science Association Inc. http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Poultry Science Oxford University Press

Effects of excess DL- and L-methionine on growth performance of starter Pekin ducks

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Abstract

Abstract A dose-response experiment was conducted to investigate the effects of excess DL-methionine (DLM) and L-methionine (LM) on growth response of starter Pekin ducks from 7 to 21 days of age. A total of 462 seven-day-old male Pekin ducklings were allotted to 77 wire-floor pens with 6 birds per pen. There were 11 treatments including a methionine-adequate control diet (containing 0.49% methionine) and control diets supplemented with 5 levels of crystal DLM or LM (0.25, 0.50, 0.75, 1.0, and 1.25%) based on equal product weight. At 21 d of age, weight gain, feed intake, and feed/gain of ducks from each pen were measured. In our study, the weight gain and feed intake did not change markedly and kept a plateau when supplemental DLM or LM was below 0.50%, but the weight gain and feed intake decreased further as supplemental DLM or LM increased from 0.50 to 1.25%. When the maximum safe level (MSL) of supplemental methionine sources and methionine content of the control diets were combined, the total tolerable upper limits of methionine for weight gain and feed intake were 0.91 and 0.85% when DLM was used, respectively, and the corresponding values were 0.89 and 0.84% when LM was used, respectively. On the other hand, both excess DLM and LM reduced weight gain and feed intake (P < 0.05) but there were no significant differences (P > 0.05) in weight gain and feed intake between the ducks fed DLM- and LM-supplemented diets. Furthermore, linear slope-ratio assay was also used to evaluate the relative toxicity of excess DLM and LM. According to this assay, the efficiencies of DLM relative to LM for depression of weight gain and feed intake were 97 and 95%, respectively. In summary, excess DL- and L-methionine were toxic for starter Pekin ducks and both methionine sources were equally growth depressing. INTRODUCTION Methionine is the first-limiting amino acid in practical duck diets and optimal supplementation of crystalline methionine in diets could improve growth performance, carcass traits, and feather growth of White Pekin ducks (Xie et al., 2004; Xie et al., 2006; Zeng et al., 2015). However, excess methionine is toxic and growth depression was observed in growing Pekin ducks when 1 or 2% methionine was supplemented to control diets with the optimal methionine level (Xie et al., 2007). The tolerable upper limit of dietary methionine for growing Pekin ducks may be less than 1.38% (Xie et al., 2007), but the tolerable upper limit of dietary methionine for starter Pekin ducks is still unknown. In the past decades, DL-methionine (DLM) was usually supplemented to poultry diets to meet the total sulfur amino acid requirement of poultry and the toxicity of DLM was also specified in chicks and ducks. At present, as a new methionine source, L-methionine (LM) has been commercially available for feed formulation and LM is as efficacious as DLM for improvement of growth performance in broilers (Dilger and Baker, 2007). Like DLM, LM is also toxic when given in excess and growth depression takes place in chicks in such instances (Harter and Baker, 1978; Baker, 1979). In broilers, excess DLM and LM were equally growth depressing (Katz and Baker, 1975; Dilger, et al., 2007a). However, the information on the relative toxicity between DLM and LM was lacking in ducks. Therefore, in order to ensure the safe use of methionine sources and to establish the tolerable upper limit of methionine uptake for starter ducks, the objective of our study was to investigate the effects of excess DL- and L-methionine on the growth performance of starter White Pekin ducks and to assess and compare the toxicity of DLM and LM in ducks. MATERIAL AND METHODS All procedures of our experiment were approved by the animal care and use committee of Institute of Animal Sciences of Chinese Academy of Agricultural Sciences. Five hundred 1-day-old male Pekin ducklings from a commercial hatchery were randomly allocated to 50 wire-floor pens with 10 ducklings per pen. These ducklings were fed a control diet (Table 1) from hatch to 7 d of age. At 7 d of age, after all ducklings were weighed individually and some birds with lowest or highest body weight were removed, the 462 ducks with average body weight of 145 ± 20 g were selected and allocated to 77 wire-floor pens with 6 ducks per pen according to similar pen weight. There were 11 treatments (Table 2) including a methionine-adequate control diet and control diets supplemented with crystal DLM (Evonik Industries, Krefeld, Germany) or LM (CheilJedang Co., Seoul, Korea) according to 5 supplemental levels (0.25, 0.50, 0.75, 1.00, and 1.25%) based on equal product weight. Both crystal DLM and LM had 99% purity and they were supplemented to control diets at the expense of corn. Each treatment had 7 replicate pens of 6 birds each. During the period from hatch to 21 days of age, all ducks had free access to feed and water and feed was provided in the form of pellets. Lighting was continuous. The temperature was kept at 33°C for the first 3 d and then was reduced gradually to approximately 25°C until 21 d of age. Table 1. Composition of control diet from hatch to 21 d of age (% as fed). Item    Ingredient    Corn  62.07  Soybean meal  32.80  L-Met  0.18  Dicalcium phosphate  1.55  Limestone  1.10  Sodium chloride  0.30  Premix1  1.00  Soybean oil  1.00  Calculated composition    Metabolizable energy,2 kcal/kg  2,879  Crude protein  19.94  Methionine  0.493  Cysteine  0.353  Lysine  1.03  Calcium  0.82  Nonphytate phosphorus  0.35  Item    Ingredient    Corn  62.07  Soybean meal  32.80  L-Met  0.18  Dicalcium phosphate  1.55  Limestone  1.10  Sodium chloride  0.30  Premix1  1.00  Soybean oil  1.00  Calculated composition    Metabolizable energy,2 kcal/kg  2,879  Crude protein  19.94  Methionine  0.493  Cysteine  0.353  Lysine  1.03  Calcium  0.82  Nonphytate phosphorus  0.35  1Supplied per kilogram of total diet: Cu (CuSO4•5H2O), 8 mg; Fe (FeSO4•7H2O), 60 mg; Zn (ZnO), 60 mg; Mn (MnSO4•H2O), 100 mg; Se (NaSeO3), 0.3 mg; I (KI), 0.4 mg; choline chloride, 1,000 mg; vitamin A (retinyl acetate), 1,376 μg; vitamin D3 (Cholcalciferol), 50 μg; vitamin E (DL-α-tocopheryl acetate), 20 mg; vitamin K3 (menadione sodium bisulfate), 2 mg; thiamin (thiamin mononitrate), 2 mg; riboflavin, 10 mg; pyridoxine hydrochloride, 4 mg; cobalamin, 0.02 mg; calcium-D-pantothenate, 20 mg; folic acid, 1 mg; and biotin, 0.15 mg. 2The value was calculated according to the apparent metabolizable energy of chickens (Ministry of Agriculture of China, 2004). 3The numbers were analyzed values. View Large Table 2. Effects of excess DL-methionine (DLM) and L-methionine (LM) on growth performance of starter Peking ducks from 7 to 21 days of age.1 Treatment  Weight gain (g/bird per d)  Feed intake (g/bird per d)  Feed/Gain (g/g)  Control(C)  69.9  117.4  1.68  C+0.25% DLM  70.0  118.1  1.69  C+0.25% LM  69.7  115.4  1.65  C+0.50% DLM  67.5  110.8  1.64  C+0.50% LM  65.7  107.2  1.63  C+0.75% DLM  58.6  96.9  1.65  C+0.75% LM  58.9  97.0  1.65  C+1.00% DLM  49.7  83.7  1.69  C+1.00% LM  49.7  83.0  1.67  C+1.25% DLM  42.0  71.5  1.70  C+1.25% LM  40.6  68.4  1.69  SEM  0.78  1.44  0.015  Probability        DLM vs. C  0.008  0.003  0.767  LM vs. C  0.004  0.001  0.261  DLM vs. LM  0.793  0.624  0.152  DLM broken-line  <0.001  <0.001  0.461  LM broken-line  0.002  0.002  0.368  Treatment  Weight gain (g/bird per d)  Feed intake (g/bird per d)  Feed/Gain (g/g)  Control(C)  69.9  117.4  1.68  C+0.25% DLM  70.0  118.1  1.69  C+0.25% LM  69.7  115.4  1.65  C+0.50% DLM  67.5  110.8  1.64  C+0.50% LM  65.7  107.2  1.63  C+0.75% DLM  58.6  96.9  1.65  C+0.75% LM  58.9  97.0  1.65  C+1.00% DLM  49.7  83.7  1.69  C+1.00% LM  49.7  83.0  1.67  C+1.25% DLM  42.0  71.5  1.70  C+1.25% LM  40.6  68.4  1.69  SEM  0.78  1.44  0.015  Probability        DLM vs. C  0.008  0.003  0.767  LM vs. C  0.004  0.001  0.261  DLM vs. LM  0.793  0.624  0.152  DLM broken-line  <0.001  <0.001  0.461  LM broken-line  0.002  0.002  0.368  1Results are means with n = 7 per treatment. View Large The methionine content of the control diet was 0.49% and it was adequate for duck growth (Xie et al, 2004). The contents of nutrients except crude protein in the control diet met the NRC (1994) recommendation of White Pekin ducks from 0 to 2 wk of age and the content of crude protein in the control diet met the requirements of starter Pekin ducks provided by Xie et al. (2017). All experimental diets were cold pelleted at room temperature. The methionine and cysteine of all experimental diets were analyzed according to the method recommended by Standardization Administration of China (2000). Briefly, methionine and cysteine were oxidized at 0°C by performic acid (formic acid: hydrogen peroxide = 9:1) for 16 hours and then hydrolyzed at 110°C by 6 M HCL for 24 hours. After that, the pH of these hydrolysates was adjusted to 2.2 and then analyzed by using ion-exchange chromatography with an amino acid analyzer (L-800, Hitachi, Tokyo, Japan). The supplemental DLM or LM levels of all diets were also confirmed by the difference in analyzed methionine contents between the control diet and the corresponding DLM or LM-supplemented control diets. The 5 confirmed supplemental DLM levels were 0.24, 0.50, 0.78, 0.98, and 1.26%, respectively, and the 5 confirmed supplemental LM levels were 0.26, 0.49, 0.73, 1.00, and 1.25%, respectively, which is very close to the formulated levels (0.25, 0.50, 0.75, 1.00, and 1.25%). At 21 d of age, weight gain, feed intake, and feed/gain of ducks from each pen were measured. Feed intake and feed/gain were all corrected for mortality. Data were analyzed as a completely randomized design by ANOVA procedure of SAS software (SAS Institute, 2003), with pen used as the experimental unit for analysis. The orthogonal contrasts were used to compare the difference between DLM and control, LM and control, and DLM and LM, and a probability level of P < 0.05 was considered to be statistically significant. In our study, the broken-line regression (Alhotan et al., 2017) was used to estimate the maximum safe level of supplemental DLM or LM for starter ducks using the NLIN procedure (SAS Institute, 2003). The broken-line linear model was provided as follows:   \begin{equation*}y = l + u\left( {x - r} \right)\end{equation*} where y = growth response (weight gain or feed intake), x = supplemental DLM or LM (%), r = breakpoint between two lines which was defined as the maximum safe level (MSL) of supplemental DLM or LM (%), u = the slope of the curve, l = maximum response if x < r and y = l + u (x−r) if x ≥ r. Furthermore, according to the statistical method suggested by Sterling et al. (2003), a Student t test was used to determine if a difference existed in maximum safe levels between supplemental DLM and LM. On the other hand, according to the statistical method of Littell et al. (1997), the linear ratio-slope regression was used to estimate the toxicity of DLM relative to LM by NLIN procedure of SAS software (SAS Institute, 2003). The linear ratio-slope model was provided as follows:   \begin{equation*} y = a + {b_1}{x_1} + {b_2}{x_2} \end{equation*} where y = growth response (weight gain or feed intake), a = intercept (duck performance with control diet), b1 = steepness coefficient for supplemental DLM, b2 = steepness coefficient for LM, and x1, x2 = supplemental level of DLM and LM, respectively. The relative toxicity of DLM compared with LM was given by b1/b2. RESULTS AND DISCUSSION In our study, the analyzed methionine content of the control diet was 0.49% (Table 1) and this value was similar to the methionine requirement of starter Pekin ducklings estimated by Xie et al. (2004), which ensured the maximum growth of ducks fed the control diets in our study. Furthermore, L-methionine could be incorporated directly into body proteins, but the D-methionine must first be converted into keto-methionine and then into the L-methionine before being incorporated into protein. In order to avoid the debate on the conversion of D-methionine to L-methionine, crystal LM (not DLM) was used in the control diet to meet the total methionine requirement of ducklings and the 0.18% crystal LM in the control diets was supposed to support duck growth through its incorporation into protein. Total mortality was less than 1% and dead birds came from different treatments, not only one treatment, which did not affect the accuracy of our results. In the present study, supplementation of excess DLM or LM in control diets had negative effects on weight gain (P < 0.05), feed intake (P < 0.05), but not feed/gain (P > 0.05), which indicated that growth depression resulted from reduction of feed intake. Our results confirmed the toxicity of excess DLM and LM in starter ducks and the toxicity of DLM was also observed in growing Pekin ducks (Xie et al., 2007). In order to avoid any detrimental effects of excess crystal methionine on the biological performance of the ducks, it was necessary to estimate the MSL of these methionine products when crystal methionine was supplemented into the diets. It is preferable to estimate MSL by linear broken-line regression rather than multiple range tests (Alhotan et al., 2017). In our study, the weight gain and feed intake did not change markedly and kept a plateau when supplemental DLM or LM was below 0.50%, but the weight gain and feed intake decreased further as supplemental DLM or LM increased from 0.50 to 1.25% (Table 2), which showed the broken-line response. Therefore, according to linear broken-line regression, the MSL of supplemental DLM and LM for weight gain were 0.42 and 0.40%, respectively, and the MSL of supplemental DLM and LM for feed intake were 0.36 and 0.35%, respectively (Table 3). Furthermore, when maximum safe level of supplemental methionine sources and methionine content (0.49%) of the control diets were combined, the total tolerable upper limits of methionine for weight gain and feed intake were 0.91 and 0.85% when DLM was used, respectively, and the corresponding values were 0.89 and 0.84% when LM was used, respectively. In broilers, the weight gain of broilers before 22 days of age post-hatch was not reduced significantly when 1% DLM was added to the control diets containing 0.57, 0.46, or 0.51% Met, respectively (Han and Baker, 1993; Scherer and Baker, 2000; Dilger et al., 2007b). Therefore, the ducks may be less able to tolerate excess methionine compared with broilers during the starter period. Table 3. Maximum safe levels of supplemental DL- and L-Methionine for satisfactory growth performance of White Pekin ducklings as estimated by broken-line linear model. Criteria  Methionine source  Broken-line linear model  R2  Estimated maximum response1  Maximum Safe levels1  Student t test2  Weight gain  DL-methionine  y = 69.9–34.2 × (x−0.42)  0.999  69.9 ± 0.3  0.42 ± 0.01  0.63  L-methionine  y = 69.8–33.9 × (x−0.40)  0.998  69.8 ± 0.5  0.40 ± 0.03  Feed intake  DL-methionine  y = 117.7–52.4 × (x−0.36)  0.999  117.7 ± 0.4  0.36 ± 0.01  0.24  L-methionine  y = 116.4–52.2 × (x−0.35)  0.996  116.4 ± 1.1  0.35 ± 0.04  Criteria  Methionine source  Broken-line linear model  R2  Estimated maximum response1  Maximum Safe levels1  Student t test2  Weight gain  DL-methionine  y = 69.9–34.2 × (x−0.42)  0.999  69.9 ± 0.3  0.42 ± 0.01  0.63  L-methionine  y = 69.8–33.9 × (x−0.40)  0.998  69.8 ± 0.5  0.40 ± 0.03  Feed intake  DL-methionine  y = 117.7–52.4 × (x−0.36)  0.999  117.7 ± 0.4  0.36 ± 0.01  0.24  L-methionine  y = 116.4–52.2 × (x−0.35)  0.996  116.4 ± 1.1  0.35 ± 0.04  1Expressed as means ± SE. 2Maximum safe levels considered significantly different if |t|> t (α = 0.05, df = 35) = 2.03. View Large On the other hand, the toxicities of DLM and LM in starter ducks were compared and excess DLM and LM were equally growth depressing based on the equal product weight. For orthogonal contrasts in our study, both excess DLM and LM reduced weight gain and feed intake (P < 0.05) but there were no significant differences (P > 0.05) in weight gain and feed intake between the ducks fed DLM- and LM-supplemented diets (Table 2). Furthermore, the Student t test also showed that the MSL of DLM was not significantly different from the MSL of LM (P > 0.05, Table 3), which also indicated DLM and LM had the same toxic effects on duck performance. Our results were also supported by Dilger et al. (2007a) who found that DLM was no more growth depressing than LM when 1.5 or 3.0% methionine was supplemented to chick diets. DL-methionine is a racemic (50:50) mixture of D- and L-methionine. L-methionine could be incorporated directly into body proteins, and it is assumed to be 100% efficacious, but D-methionine must be converted to L-methionine before it is incorporated into protein. It is well established that the chick can use D-Met with greater than 90% efficacy relative to L-Met (Wretlind and Rose, 1950; Sugahara et al., 1967; Sunde, 1972; Baker, 1986) and it has been traditionally accepted that DL-Met is 95% efficacious relative to L-Met due to equal dietary contributions of the D- and L-isomers (Baker, 2006). Our results in linear slope-ratio assay for relative toxicity of excess methionine sources supported the aforementioned hypothesis. In our study, linear slope-ratio assay was also used to compare relative toxicity of excess DLM and LM because the responses to the factors in the dose-response studies should be continuous, not discrete. According to this assay, on the equal supplemental levels, the efficiencies of DLM relative to LM for depression of weight gain and feed intake were 97 and 95%, respectively (Figures 1 and 2), which also indicated that DLM was as toxic as LM. Furthermore, our results were also supported by the results in chicks at nontoxic methionine levels in which the efficacy of DLM relative to LM was 98.5 and 97.2% for improvement of weight gain and gain/feed, respectively (Dilger et al., 2007a). Figure 1. View largeDownload slide Slope-ratio comparison of toxicity between supplemental DL-methionine (DLM) and L-methionine (LM) based on the weight gain of starter Pekin ducklings from 7 to 21 days of age. Values in brackets indicate 95% confidence intervals. Figure 1. View largeDownload slide Slope-ratio comparison of toxicity between supplemental DL-methionine (DLM) and L-methionine (LM) based on the weight gain of starter Pekin ducklings from 7 to 21 days of age. Values in brackets indicate 95% confidence intervals. Figure 2. View largeDownload slide Slope-ratio comparison of toxicity between supplemental DL-methionine (DLM) and L-methionine (LM) based on the feed intake of starter Pekin ducklings from 7 to 21 days of age. Values in brackets indicate 95% confidence intervals. Figure 2. View largeDownload slide Slope-ratio comparison of toxicity between supplemental DL-methionine (DLM) and L-methionine (LM) based on the feed intake of starter Pekin ducklings from 7 to 21 days of age. Values in brackets indicate 95% confidence intervals. In conclusion, excess DL-methionine and L-methionine reduced the growth response of starter Pekin ducks and DL-methionine was as growth depressing as L-methionine at equal product weight. The total tolerable upper limits of methionine for starter Pekin ducks was about 0.90%. 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Poultry ScienceOxford University Press

Published: Mar 1, 2018

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