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Poult Sci 2007. 86:1995-1999
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METABOLISM AND NUTRITION: Research Note

Effect of Excess Methionine and Methionine Hydroxy Analogue on Growth Performance and Plasma Homocysteine of Growing Pekin Ducks

M. Xie*,{dagger}, S. S. Hou*,{dagger},1, W. Huang*,{dagger} and H. P. Fan*,{dagger}

* Institute of Animal Science, Chinese Academy of Agricultural Sciences, Beijing, China 100094; and {dagger} State Key Laboratory of Animal Nutrition, Beijing, China 100094

1 Corresponding author: houss{at}263.net


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS AND DISCUSSION
 REFERENCES
 
One experiment was conducted to study the effect of excess DL-methionine (DLM) and DL-2-hydroxy-4-methylthiobutanoic acid free acid (DL-HMB-FA) on duck growth. One-day-old male white Pekin ducklings were fed common starter diets from hatch to 21 d of age and then fed the experimental diets from 21 to 42 d of age. Three hundred twenty 21-d-old birds were allotted to 40 raised wire-floor pens with 8 birds per pen according to similar pen weight. There were 5 dietary treatments that included a methionine-adequate control diet and control diets supplemented with 2 levels of dry DLM (1 or 2%) or 2 equimolar levels of liquid DL-HMB-FA (1.13 or 2.26%). Each dietary treatment was replicated 8 times. At 42 d of age, weight gain, feed intake, and gain/feed were measured and plasma was collected to analyze homocysteine. Compared with ducks fed control diets, excess DLM or DL-HMB-FA supplementation reduced weight gain and feed intake of birds significantly. However, on the equimolar basis, at 1 or 2% supplemental methionine activity, DL-HMB-FA was less growth-depressing than DLM. According to the growth response to excess methionine, the tolerable upper limit of dietary methionine for growing ducks may be less than 1.38% when the methionine level of the control diet (0.38%) was considered. On the other hand, plasma homocysteine was elevated markedly when 2% DLM or 2.26% DL-HMB-FA was added to control diets, but plasma homocysteine of ducks fed 2.26% DL-HMB-FA supplemented diets was lower significantly than birds fed equimolar DLM-supplemented diets, which indicated the toxicity of excess methionine sources and less toxicity of DL-HMB-FA relative to DLM.

Key Words: methionine • methionine hydroxy analogue • duck • toxicity


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS AND DISCUSSION
 REFERENCES
 
Methionine is the first-limiting amino acid in poultry diets and optimal supplementation of methionine can improve growth performance and carcass quality of poultry. Therefore, synthetic methionine sources, mainly DL-methionine (DLM) or DL-2-hydroxy-4-methylthiobutanoic acid free acid (DL-HMB-FA), are commonly supplemented to poultry diets. However, excess methionine was toxic for poultry, and growth depression was observed in broilers and turkeys when this amino acid was above 1% (Katz and Baker, 1975a; Hafez et al., 1978; Harter and Baker, 1978; Edmonds and Baker, 1987; Han and Baker, 1993; Carew et al., 1998; Scherer and Baker, 2000; Acar et al., 2001). However, many previous studies on methionine toxicity were conducted by using DLM but few with methionine hydroxy analogues. Excess methionine hydroxy analogue was also toxic. Some research reported the growth depression in broilers when excess DL-HMB-FA or its calcium salt was consumed, and it was also found that methionine hydroxy analogue was less growth-depressing than methionine in these birds (Katz and Baker, 1975b; Baker and Boebel, 1980; Boebel and Baker, 1982; Dibner et al., 2004). Presently, synthetic methionine has already been used in duck diets, and methionine requirement was estimated by some researchers (He et al., 2003; Xie et al., 2004, 2006). However, these studies were conducted at nontoxic levels up to 0.685%, and no information about the toxicity of methionine or its hydroxy analogue in ducks was reported until now. Therefore, to ensure the safe use of methionine sources and establish the tolerable upper limit of methionine uptake for ducks, it is essential to study the effect of excess methionine and methionine hydroxy analogue on duck growth.

Homocysteine may be a toxic methionine metabolite and the mechanism of homocysteine toxicity was reviewed by Perna et al. (2003). Plasma homocysteine increased markedly when above 1% methionine was consumed by rats (Finkelstein and Martin, 1986; Toue et al., 2006). Recently, to screen toxicity biomarker for methionine excess in rats, the relationship between plasma measurements (amino acids, enzymes, and methionine metabolites) and toxic change (growth suppression, hemolytic anemia, and splenic hemosideroses) was examined by cluster analysis of multivariate correlations when rats were fed different supplemental methionine levels from 0 to 2.4%, and plasma homocysteine was found to be the most plausible biomarker to assess methionine excess (Toue et al., 2006). Presently, the effect of excess methionine hydroxy analogue on plasma homocysteine was unknown. However, considering the conversion of methionine hydroxy analogue to methionine in vivo (Dibner, 2003), plasma homocysteine may be a useful biomarker to evaluate methionine hydroxy analogue excess.

Therefore, the objective of our study was to examine the effects of excess methionine and methionine hydroxy analogue on growth performance and plasma homocysteine of growing Pekin ducks and to assess the toxicity of these 2 compounds.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS AND DISCUSSION
 REFERENCES
 
All procedures of our experiment were approved by the animal care and welfare committee of our institute. Four hundred twenty 1-d-old male white Pekin ducklings from one commercial hatchery were assigned to 21 raised wire-floor pens with 20 birds per pen. These ducklings were raised with a common starter diet (Table 1Go) until 21 d of age. During this period, feed and water were provided ad libitum and lighting was continuous; the temperature was kept at 33°C for the first 3 d and then was reduced gradually to room temperature until 21 d of age.


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  		Table 1. Composition of common starter diet from hatch to 21 d of age and Met-adequate control diet from 21 to 42 d of age (% as fed)
 
At 21 d of age, when all birds were weighed individually and some birds with lowest or highest BW were removed, 320 birds were selected on the basis of average BW of remaining birds. After that, three hundred twenty 21-d-old birds were divided to 40 raised wire-floor pens with 8 birds per pen according to similar pen weight. Our study comprised 5 dietary treatments that included a methionine-adequate control diet (Table 1Go) and control diets supplemented with 2 levels of dry DLM (1 or 2%) or 2 equimolar levels of liquid DL-HMB-FA (1.13 or 2.26%). Each dietary treatment was replicated 8 times using 8 pens. The DLM was calculated as 99% purity and DL-HMB-FA was calculated as 88% purity. Control diet was methionine-adequate corn-soybean-based diet, and the sulfur amino acid in the diet was analyzed according to the method used by Xie et al. (2004). Briefly, methionine and cystine in the control diet were oxidized by mixing 88% formic acid and 30% hydrogen peroxide at a ratio of 9:1 and then hydrolyzed at 110°C by 6 M HCl for 24 h. Next, the pH of the hydrolyzate was adjusted to 2.2 and was then analyzed using an amino acid analyzer (L-800, Hitachi, Tokyo). Supplemental DLM in diets was verified by the method used by Xie et al. (2004), and supplemental DL-HMB-FA in diets was verified by the method used by Ontiveros et al. (1987). The ducks were raised with feed and water provided ad libitum from 21 to 42 d of age.

At 42 d of age, weight gain, feed intake, and gain/ feed of ducks from each pen were measured, and 2 birds selected randomly from each pen were bled by heart puncture. Feed intake and gain/feed were all corrected for mortality. To obtain plasma, blood samples were collected into Na2EDTA-containing tubes and were centrifuged at 3,000 rpm for 10 min. Plasma samples were kept at –20°C until analyzed for homocysteine.

Plasma homocysteine was determined using reversed phase HPLC with precolumn derivation and fluorescence detection according to the method of Ubbink et al. (1991) as modified by Gilfix et al. (1997) and Pfeiffer et al. (1999). The modification used tris(2-carboxylethyl)- phosphine as a reductant.

Data were analyzed as a completely randomized design by 1-way ANOVA procedure of SAS software (SAS Institute, 2003). To investigate the effects of methionine source and supplemental level, data were also analyzed as 2 (methionine source: DLM or DL-HMB-FA) x 2 (supplemental methionine activity: 1 or 2%) factorial design excluding control treatment by 2-way ANOVA procedure of SAS software (SAS Institute, 2003). When dietary treatment was significant (P < 0.05), means were compared by Duncan’s multiple-range test.


   RESULTS AND DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS AND DISCUSSION
REFERENCES
 
The methionine level of control diet was 0.38%, and this value was equal to the requirement of growing ducks estimated by Xie et al. (2006), which ensured the maximum growth of ducks fed control diets in our study. Although excess DLM and DL-HMB-FA were used in our experiment, the mortality (4%) was low and dead birds came from all treatments, which did not affect the accuracy of our results.

Excess methionine or methionine hydroxy analogue was growth-depressing for growing ducks. In our study, compared with ducks fed control diets, excess DLM or DL-HMB-FA supplementation caused a significant decrease in weight gain, feed intake, and gain/feed (Table 2Go), which was in agreement with many previous studies on the toxicity of methionine and its hydroxy analogue in broilers (Katz and Baker, 1975a,b; Hafez et al., 1978; Harter and Baker, 1978; Baker and Boebel, 1980; Boebel and Baker, 1982; Edmonds and Baker, 1987; Han and Baker, 1993; Carew et al., 1998; Scherer and Baker, 2000; Acar et al., 2001; Dibner et al., 2004). At present, no information about methionine toxicity in ducks was reported. When both methionine sources of each supplemental methionine activity were taken together, the 2 x 2 factorial analysis showed that 2% supplemental methionine activity caused less weight gain, feed intake, and gain/feed of ducks than 1% supplemental methionine activity (Table 2Go). Therefore, on the basis of the growth response to excess methionine in our study, the tolerable upper limit of dietary methionine for growing ducks may be less than 1.38% when methionine level of control diet (0.38%) was considered, and this value suggested that poultry was less tolerable to excess methionine after 21 d of age post-hatching than before this period because the weight gain of broilers before 22 d of age posthatching was not depressed significantly when 1% DLM was added to control diets containing 0.57, 0.46, or 0.51% Met, respectively (Han and Baker, 1993; Scherer and Baker, 2000; Dilger et al., 2007).


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  		Table 2. Effect of excess DL-methionine (DLM) and DL-2-hydroxy-4-methylthiobutanoic acid free acid (DL-HMB-FA) on growth performance and plasma homocysteine of male growing Pekin ducks1
 
However, excess methionine hydroxy analogue was less growth depressing than methionine at equimolar level (Katz and Baker, 1975b; Baker and Boebel, 1980; Boebel and Baker, 1982; Dibner et al., 2004), which was confirmed further by our study. In our study, weight gain and feed intake were depressed by 14 and 15%, respectively, when 1% DLM was supplemented to control diets, but equimolar DL-HMB-FA (1.13%) supplementation reduced weight gain and feed intake by 6 and 8%, respectively, and the similar phenomena were observed when 2% DLM or equimolar DL-HMB-FA (2.26%) was added to control diets (Table 2Go). When both supplemental levels of each methionine source were taken together, the factorial analysis also confirmed the less growth depression caused by excess DL-HMB-FA than DLM (Table 2Go).

Homocysteine may be a toxic methionine metabolite because its toxicity was reported by Perna et al. (2003), and plasma homocysteine was elevated markedly in rats when excess methionine was consumed (Finkelstein and Martin, 1986; Toue et al., 2006). Recently, plasma homocysteine was found to be the most plausible biomarker to assess methionine excess when amino acids, methionine metabolites, enzymes, and other measurements in plasma were selected to screen toxicity biomarkers for methionine excess in rats (Toue et al., 2006). Therefore, considering the conversion of DL-HMB-FA and D-methionine to L-methionine (Dibner, 2003), plasma homocysteine was used to evaluate the toxicity of methionine sources in our study. Compared with the ducks fed control diets, a 1.5-fold and 1.2-fold increase in plasma homocysteine was observed for ducks fed 2% DLM and equimolar DL-HMB-FA (2.26%), respectively (P < 0.05, Table 2Go). In our study, the significantly elevated plasma homocysteine caused by highest supplemental level of both methionine sources may be because the accumulation of homocysteine went beyond the removal of homocysteine, which was supported by Toue et al. (2006) who observed a marked decrease in the ratio of cystathionine (a product of homocysteine degradation) to homocysteine in rat plasma when excess methionine was added to control diets. Therefore, the markedly elevated plasma homocysteine in our study indicated the toxicity of DLM and DL-HMB-FA in ducks.

Moreover, due to the role of homocysteine as biomarker of methionine toxicity, less growth depression was accompanied by less plasma homocysteine. In our study, although excess methionine or its hydroxy analogue elevated plasma homocysteine significantly, plasma homocysteine of ducks fed 2.26% DL-HMB-FA was lower significantly than birds fed equimolar DLM (2%), and the 2 x 2 factorial analysis also indicated that excess DLM caused more plasma homocysteine than DL-HMB-FA (Table 2Go), which further supported less toxicity of DL-HMB-FA compared with DLM.

When some physiological reasons for incomplete use of methionine hydroxy analogue were considered, less toxicity of DL-HMB-FA may be due to its less efficient absorption compared with DLM. Maenz and Engele-Schaan (1996a) and Drew et al. (2003) found that intestinal bacteria affected methionine hydroxy analogue absorption more severely than methionine. Brachet and Puigserver (1989) and Maenz and Engele-Schaan (1996b) studied the uptake of methionine and methionine hydroxy analogue by chick small intestinal brush border membrane vesicles, and they found that methionine hydroxy analogue was absorbed less rapidly than methionine. In addition, both methionine hydroxy analogue and D-methionine must be converted to L-methionine with {alpha}-keto-methionine as intermediate before they were utilized as methionine source, and Dupuis et al. (1989) and Dibner and Ivey (1992) suggested that D-methionine was oxidized to {alpha}-keto-methionine more rapidly than methionine hydroxy analogue, which may be another reason for less toxicity of DL-HMB-FA relative to DLM.

In conclusion, excess DLM or DL-HMB-FA reduced duck growth and DL-HMB-FA was less growth-depressing than equimolar DLM, which was indicated by the change of plasma homocysteine. On the basis of the growth response to excess methionine, the tolerable upper limit of dietary methionine for growing ducks may be less than 1.38%.


   ACKNOWLEDGMENTS
 
Our research was sponsored by National Scientific and Technical Supporting program (2006BAD14B06) and Basic Science Research Program in National Institute (ywf-td-04) of Ministry of Science and Technology of China.

Received for publication February 10, 2007. Accepted for publication May 24, 2007.


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 RESULTS AND DISCUSSION
 REFERENCES
 
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