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Interactions of Various Supplies of Isoleucine, Valine, Leucine and Tryptophan on the Performance of Laying Hens S. Peganova and K. Eder1 Institut fu¨ r Erna¨hrungswissenschaften, Martin-Luther-Universita¨t Halle-Wittenberg, Emil-Abderhalden-Strasse 26, D-06108 Halle/Saale, Germany ABSTRACT The present study was undertaken to investigate the interactions among the supplies of isoleucine, leucine, valine, and tryptophan in laying hens. A three-factor trial was conducted with laying hens in which the dietary concentrations of isoleucine (5.7, 8.0, and 11.5 g/kg), valine and leucine (6.3 and 7.2 g/kg and 10.1 and 11.5 g/kg, respectively), and tryptophan (1.5 and 2.4 g/ kg) were varied. At the lowest concentration of valine + leucine, an increase in dietary isoleucine concentration led to a dose-dependent reduction in feed consumption, daily egg mass, and body weight gain and an increase of the isoleucine concentration in plasma. At a high dietary concentration of valine + leucine, excess dietary isoleucine (Key words: laying hen, isoleucine, valine, leucine, interactions) 2003 Poultry Science 82:100–105 INTRODUCTION A recent study has shown that the margin between requirement and excess of the amino acid isoleucine is very narrow in laying hens (Peganova and Eder, 2002). For maximum daily egg mass from laying hens, a dietary isoleucine concentration of 4.0 g/kg is necessary. On the other hand, increasing the concentration slightly to 8.0 g/kg diet leads to a reduction in body weight; at 10.0 g/ kg, feed consumption and daily egg mass are reduced as well. The performance-depressant effect of excess dietary isoleucine might be due to antagonisms between this and the two other branched-chain amino acids, valine and leucine. The three structurally similar branched-chain amino acids (valine, leucine, and isoleucine) share common systems for transport through cellular membranes and use the same enzymes for degradation (Harper, 1984). Antagonisms among these three amino acids have already been demonstrated in several species such as rats, pigs, turkeys, and broilers (Sauberlich, 1961; Allen and Baker, 1972; Oestemer et al., 1973; Tuttle and Balloun, 2003 Poultry Science Association, Inc. Received for publication February 5, 2002. Accepted for publication August 23, 2002. 1To whom correspondence should be addressed: eder@ landw.uni-halle.de. 100 concentration caused only a weak depression of performance parameters; the isoleucine concentration in plasma was independent of the dietary isoleucine concentration. Increasing the dietary tryptophan concentration did not influence the effect of an excessive dietary isoleucine concentration on performance parameters. Increasing the tryptophan concentration from 1.5 to 2.4 g/kg diet did, however, lead to a significant increase in feed consumption, irrespective of the supply of isoleucine, valine, and leucine. In conclusion, our study demonstrates that the supply of valine + leucine influenced the effects of excess dietary isoleucine in laying hens, whereas the supply with tryptophan did not. 1976; Smith and Austic, 1978; Taylor et al., 1984). It has been shown in broiler chicks that increasing the dietary concentration of leucine alleviates the performancedepressant effect of an excessive isoleucine supply (Burnham et al., 1991). Similar interactions have not been studied in laying hens. Isoleucine and other large neutral amino acids also compete with tryptophan for transfer into the brain across the blood-brain barrier (Wurtman, 1980; Tackman et al., 1990). Tryptophan plays an important role in the brain as a precursor of the neurotransmitter serotonin, which has a major effect on the feeding behavior of animals among its many functions (Blundell and Latham, 1978; Tackman et al., 1990; Mullen and Martin, 1992). As excess dietary isoleucine was associated with a marked reduction in feed consumption, the obvious assumption was that effects of excess dietary isoleucine might also be due to a secondary tryptophan deficiency in the brain. The present study, designed as a three-factor experiment, was undertaken to investigate the interactions among the supplies of isoleucine, valine + leucine, and tryptophan. In particular, we wanted to find out whether the performance depression caused by an excessive supply of isoleucine could be alleviated by increasing the supply of the other two branched-chain amino acids or increasing the supply of tryptophan. We Abbreviation Key: BCKA = branched-chain α-keto acid. INTERACTIONS OF AMINO ACIDS IN LAYING HENS 101 TABLE 1. Composition of the basal experimental diet Ingredient Amount (g/kg) Wheat 300 Barley 336 Peas 137 Cellulose 41 Soybean oil 54 Calcium carbonate 85 Calcium phosphate 15 Vitamin and mineral premix1 10 Salt 1.5 L-Lysine-HCl 2.5 DL-Methionine 1.9 L-Threonine 1.0 L-Tryptophan 0.3 L-Isoleucine 1.9 L-Valine 1.5 L-Aspartic acid 6.5 L-Glutamic acid 6.6 Analysis (g/kg) Crude protein 131 Methionine 3.7 Methionine + cysteine 6.1 Lysine 6.1 Tryptophan 1.5 Threonine 4.2 Isoleucine 5.7 Valine 6.3 Leucine 7.2 Calcium 37.0 Total phosphorus 6.9 Energy (MJ ME/kg), calculated2 11.44 1Supplied per kilogram of diet: calcium, 1.70 g; sodium, 0.80 g; vitamin A, 12,000 IU; cholecalciferol, 2,500 IU; DL-α-tocoopherol acetate, 20 mg; thiamine, 5 mg; riboflavine, 3 mg; pyridoxine, 3 mg; vitamin B12, 20 μg; vitamin K3, 1.2 mg; pantothenic acid, 8 mg; niacin, 30 mg; folic acid, 0.5 mg; choline chloride 150 mg; iron, 25 mg; zinc, 60 mg, manganese, 100 mg; copper, 5 mg; cobalt, 0.1 mg; iodine, 1 mg; selenium, 0.2 mg. 2Calculated according to data provided by Jahrbuch fu¨ r die Geflu¨ - gelwirtschaft (2000). proposed to determine feed consumption, other performance parameters, and the concentrations of free amino acids in the blood plasma. MATERIALS AND METHODS An experiment was conducted with 144 Lohmann Brown layers from 25 to 28 wk of age. The laying hens were assigned to 12 treatment groups. We intended to use a diet in which the concentrations of branched-chain amino acids and tryptophan were in accordance with the recommendations of the German Nutrition Society (Gesellschaft fu¨ r Erna¨hrungsphysiologie, 1999). To avoid an excess concentration of dietary leucine, a basal diet with low protein was used. The basal diet was composed primarily of cereal and peas and contained 11.4 MJ ME/ kg (Table 1). The basal dietary concentrations of isoleu- 2Lohmann Animal Health, Cuxhaven, Germany. 3Biotronik LC 3000, Eppendorf, Hamburg, Germany. 4Laborservice Onken, Gruendau, Germany. 5Hewlett Packard HPLC system, Palo Alto, CA, equipped with an RP-18-e column (5-μm particle size, 250 4 mm). cine, valine, leucine, and tryptophan were 5.7, 6.3, 7.2, and 1.5 g/kg, respectively. In accordance with a threefactor trial design, the dietary concentrations of (I) isoleucine, (II) valine + leucine, and (III) tryptophan were varied. The dietary isoleucine concentrations were 5.7, 8.0, or 11.5 g/kg diet; the dietary concentrations of valine and leucine were 6.3 and 7.2 g/kg or 10.1 and 11.5 g/kg, respectively. The dietary concentration of tryptophan was 1.5 or 2.4 g/kg. The concentrations of those amino acids in the diets were varied by supplementing the basal diets individually with L-isoleucine, L-valine, L-leucine, or Ltryptophan. 2 The purity of those amino acids was at least 98%. The concentrations of the other essential amino acids were adjusted to an adequate level as recommended by Gesellschaft fu¨ r Erna¨hrungsphysiologie (1999) by supplementation with synthetic amino acids. The hens were maintained one bird per cage in an environmentally controlled room at 18 C. Lighting was 14 h daily at 20 to 30 lx. Feed (in ground form) and water (via nipple drinkers) were available ad libitum. Because our prestudies showed that feed consumption and daily egg mass of laying hens respond quickly to amino acid antagonism in the diet, this experiment was restricted to 3 wk. All procedures followed established guidelines for the care and handling of animals and were approved by the veterinary council of Saxony-Anhalt. The following data were recorded: BW at the start and end of the trial, feed consumption weekly, and number of eggs daily. Egg weight was determined on two eggs from each hen at the end of each week. At the end of the experiment, 4 h after feed withdrawal, blood samples were drawn from the vena jugularis from each bird to determine the concentrations of free amino acids in the blood (Kirchgessner et al., 1995). The crude nutrient concentrations of the diets were analyzed according to official VDLUFA methods (Naumann and Bassler, 1993). Concentrations of amino acids were determined by hydrolyzing the diets with 6 N hydrochloric acid; the pH of the hydrolysate was adjusted to 2.2. Amino acids were separated and quantified by ion exchange chromatography in an amino acid analyzer3 using a special separating column with cation exchanger resin.4 The tryptophan concentration of the diets was determined by reverse-phase-HPLC5 (Fontaine et al., 1998). The determination of free amino acids in blood plasma was performed with the same amino acid analyzer as used for amino acid analysis of the diets. After precipitation of the plasma-protein compounds with 10% sulfosalicylic acid, samples were centrifuged (15,600 g), pH of the solution adjusted to 2.2 by addition of a dilution buffer, and samples were applied to a special polyether ether ketone separating column with cation exchanger resin.4 The statistical analysis of the data was performed with the software Statistica for Windows (StatSoft, Inc., 2000). The data were tested for normal distribution and homogeneity of the variances. Data were evaluated by three-way analysis of variance with the factors isoleucine, valine + leucine, and tryptophan and the interactions of these 102 PEGANOVA AND EDER TABLE 2. Performance of laying hens receiving diets with various concentrations of isoleucine, valine + leucine, and tryptophan from 25 to 28 wk of age1 Dietary treatment Feed Egg Egg Daily egg Feed/egg Body weight Isoleucine Valine/leucine Tryptophan consumption production weight mass mass change (g/kg) (g/kg) (g/kg) (g/hen/d) (%) (g) (g/hen) (g/g) (g) 5.7 6.3/7.2 1.5 115abc 94.4a 55.5bcd 52.3ab 2.18ab +8abc 8.0 6.3/7.2 1.5 93d 86.1abc 55.4bcd 47.8b 1.95b −154bcd 11.5 6.3/7.2 1.5 74e 73.8bc 53.0d 40.4c 1.95b −228d 5.7 10.1/11.5 1.5 119ab 90.1ab 58.5ab 52.8ab 2.25ab +33ab 8.0 10.1/11.5 1.5 118ab 94.0a 56.8abc 53.5ab 2.19ab −28abcd 11.5 10.1/11.5 1.5 101bcd 84.9abc 58.0ab 49.6ab 2.12ab −97abcd 5.7 6.3/7.2 2.4 130a 97.6a 57.7ab 56.3a 2.28ab +71a 8.0 6.3/7.2 2.4 97cd 80.2abc 55.8abcd 48.0b 2.05ab −90abcd 11.5 6.3/7.2 2.4 77e 71.0c 54.3cd 39.7c 2.11ab −199cd 5.7 10.1/11.5 2.4 130a 97.a 58.9a 57.3a 2.24ab +73ab 8.0 10.1/11.5 2.4 120ab 90.5ab 57.3abc 52.0ab 2.36a +23ab 11.5 10.1/11.5 2.4 107bcd 90.9ab 58.0ab 52.6ab 1.99b −89abcd Pooled SEM 5 4.5 0.8 1.8 0.08 51 Main effects Isoleucine (g/kg) 5.7 123a 94.8a 57.6a 54.7a 2.24a 46a 8.0 107b 87.7b 56.3b 50.4b 2.14ab −62b 11.5 90c 80.2c 55.9b 45.7c 2.04b −153c Pooled SEM 3 2.4 0.4 1.0 0.04 26 Valine/leucine (g/kg) 6.3/7.2 98b 83.9b 55.3b 47.5b 2.09b −99b 10.1/11.5 116a 91.3a 57.9a 53.0a 2.19a −14a Pooled SEM 3 2.0 0.3 0.8 0.03 23 Tryptophan (g/kg) 1.5 103b 87.2 56.2 49.5 2.11 −77 2.4 110a 87.9 57.0 51.1 2.18 −35 Pooled SEM 3 2.0 0.3 0.8 0.03 23 Interactions (P ) Isoleucine valine/leucine 0.001 0.05 0.05 0.001 0.05 NS a–eMeans with the same superscript within a column do not differ significantly (P 0.05). 1Results are means with n = 12 per treatment. factors. When F-values were statistically significant (P 0.05), means were compared by the Newman-Keuls test. To test the correlations among feed consumption, daily egg mass, and BW change, linear correlation analysis was performed. RESULTS All performance parameters of the laying hens were affected by the dietary isoleucine concentration and by the concentrations of valine + leucine (Table 2). Significant interactions were observed between the concentration of isoleucine and the concentrations of valine + leucine for all tested parameters except body weight change. At the low concentration of valine + leucine, an increase in the dietary isoleucine concentration led to a significant reduction in performance parameters. Increasing the isoleucine concentration from 5.7 to 11.5 g/kg diet reduced feed consumption by 38%, egg production by 25%, egg weight by 5%, and daily egg mass by 26%. The feed conversion ratio for egg mass on the other hand improved, probably as a result of the marked reduction in BW. At the high concentration of valine + leucine, an increase in the dietary isoleucine concentration had less effect on all performance parameters than at the low concentration of valine + leucine. Increasing the isoleucine concentration from 5.7 to 11.5 g/kg diet reduced feed consumption by 16%, egg production by 6%, egg weight by 1%, and daily egg mass by 7%. The dietary tryptophan concentration also had a significant effect on feed consumption. At the high concentration of dietary tryptophan, feed consumption was 6% higher than at the low concentration of dietary tryptophan. The parameters egg production, egg weight, daily egg mass, feed/egg mass, andBWchange were not different between the dietary tryptophan concentrations. Interactions between the isoleucine concentration and the tryptophan concentration did not occur. There were significant correlations between feed consumption and BW change (Figure 1) and between feed consumption and daily egg mass (Figure 2). Table 3 shows the concentrations of different free amino acids in the plasma in relation to the dietary concentrations of isoleucine, leucine, valine, and tryptophan. As expected, the strongest treatment response was recorded for the concentrations of the amino acids isoleucine, valine, and leucine. In contrast, the concentration of tryptophan in plasma was not influenced by any of the dietary treatment factors. The effect of the dietary isoleucine concentration on the concentration of isoleucine in plasma was dependent on the dietary level of valine + leucine but not on the dietary tryptophan concentration. At low INTERACTIONS OF AMINO ACIDS IN LAYING HENS 103 FIGURE 1. Relationship between feed consumption and bodyweight change of laying hens (y = body weight change, g; x = feed consumption, g/d). levels of valine + leucine, increase of the dietary isoleucine concentration led to a marked dose-dependent rise in the isoleucine concentration of the plasma. At high dietary levels of valine + leucine, increasing the dietary isoleucine supply did not alter the plasma isoleucine concentration. The dietary isoleucine concentration had no effect on the concentrations of valine, leucine, phenylalanine, methionine, or tryptophan in plasma. In contrast, the concentration of lysine in plasma was significantly reduced by high dietary isoleucine concentration. Increasing the dietary concentrations of valine + leucine in the diet significantly increased the concentrations of those amino acids and of phenylalanine and lysine. In contrast, increasing the dietary tryptophan concentration did not alter the concentration of any of the amino acids measured in plasma. DISCUSSION This study shows that an excessive concentration of isoleucine in laying hen diets leads to a marked performance depression, which can be significantly alleviated by increasing the concentrations of the two other branched-chain amino acids, leucine and valine. This observation confirms the existence of antagonisms among these three amino acids, which has also been described in other studies (Sauberlich, 1961; D’Mello and Lewis, 1970; Tuttle and Balloun, 1976; Shinnick and Harper, 1977). A major factor in this phenomenon is probably the competition between isoleucine and other, structurally similar, amino acids for transfer into the brain across the blood-brain barrier. It has been shown in rats (Peng et al., 1973) and broilers (Harrison and D’Mello, 1986) that an excess of one branched-chain amino acid leads to depletion of other structurally similar amino acids in the brain, causing secondary anorexia. It is likely that the increase in the isoleucine concentration in the blood due to a high dietary isoleucine supply and low levels of valine + leucine inhibits transfer of leucine, valine, and other structurally similar amino acids into the brain, resulting in a significant reduction of the feed consumption as a secondary effect. The finding of high correlations between feed consumption and performance parameters suggests that the lower feed consumption is probably also the main factor responsible for the depressant effect on performance caused by excess dietary isoleucine. A pair-feeding regime, in which animals of all the treatment groups receive identical amounts of food, would be helpful in distinguishing between the effects of an excess intake of isoleucine per se and the effects of reduced feed consumption. In the present study, basal diets were supplemented with free amino acids to study the interaction between isoleucine and valine + leucine. Free amino acids are more available than protein-bound amino acids (Izquierdo et al., 1988; Han et al., 1990). It is therefore assumed that in practical diets with higher protein concentrations, antagonism between isoleucine and valine + leucine will appear at greater concentrations of those amino acids than those found in the present study. Studies in broilers have shown that excess branchedchain amino acids are associated with reduced formation of serotonin in the brain (Harrison and D’Mello, 1986). FIGURE 2. Relationship between feed consumption and daily egg mass of laying hens (y = daily egg mass, g/d; x = feed consumption, g/d). 104 PEGANOVA AND EDER TABLE 3. Concentrations of free amino acids in plasma of laying hens receiving diets with various concentrations of isoleucine, valine + leucine, and tryptophan from 25 to 28 wk of age1 Dietary treatment Isoleucine Valine/leucine Tryptophan Isoleucine Valine Leucine Phenylalanine Lysine Methionine Tryptophan (g/kg) (g/kg) (g/kg) (μmol/L) (μmol/L) (μmol/L) (μmol/L) (μmol/L) (μmol/L) (μmol/L) 5.7 6.3/7.2 1.5 84bc 162bc 144ab 75 310 81ab 28 8.0 6.3/7.2 1.5 100b 144c 133b 72 306 78ab 24 11.5 6.3/7.2 1.5 141a 157bc 136b 76 317 84a 25 5.7 10.1/11.5 1.5 73c 213ab 158ab 87 384 80ab 28 8.0 10.1/11.5 1.5 80bc 211ab 155ab 86 355 78ab 26 11.5 10.1/11.5 1.5 79bc 171bc 139b 79 289 63b 22 5.7 6.3/7.2 2.4 73c 149c 132b 77 327 74ab 25 8.0 6.3/7.2 2.4 100bc 139c 131b 84 328 76ab 25 11.5 6.3/7.2 2.4 124a 155bc 133b 75 299 83a 27 5.7 10.1/11.5 2.4 72c 188abc 146ab 80 368 77ab 26 8.0 10.1/11.5 2.4 92bc 231a 174a 88 423 82ab 26 11.5 10.1/11.5 2.4 81bc 174bc 134b 76 314 68ab 25 Pooled SEM 30 54 29 15 103 14 1 Main effects Isoleucine (g/kg) 5.7 75c 178 145 80 347a 78 27 8.0 93b 181 149 82 353ab 79 25 11.5 107a 164 136 77 305b 74 25 Pooled SEM 4 8 4 2 15 2 1 Valine/leucine (g/kg) 6.3/7.2 104a 151b 135b 77b 315b 79 26 10.1/11.5 80b 198a 151a 83a 356a 75 25 Pooled SEM 3 6 3 2 12 21 Tryptophan (g/kg) 1.5 93 177 144 79 327 77 25 2.4 91 172 142 80 343 76 26 Pooled SEM 4 6 4 2 12 2 1 Interactions (P ) Isoleucine valine/leucine 0.001 0.01 0.05 NS NS 0.001 NS a–cMeans with the same superscript within a column do not differ significantly (P 0.05). 1Results are means with n = 12 per treatment. This finding led us to suspect that a diminished absorption of tryptophan into the brain in the presence of excess isoleucine resulting in a reduced formation of serotonin probably plays a major role in lowering of feed consumption. This supposition was not confirmed, however. The observation that increasing the dietary tryptophan concentration did not alleviate the effects of excess isoleucine suggests that tryptophan and its metabolites do not play a primary role in the reduced feed consumption at high concentrations of dietary isoleucine. At the high dietary concentration of valine + leucine, an excessive supply of isoleucine did not lead to a significant increase in the blood plasma isoleucine level, probably due to increased oxidation of the three branched-chain amino acids as a result of the increased leucine intake. High intake of leucine stimulates the activity of branchedchain α-keto acid (BCKA) dehydrogenase, the key enzyme involved in degradation of all three branched-chain amino acids (Harris et al., 2001). The performance-lowering effect of excessive leucine is therefore partially due to increased oxidation of valine and isoleucine and the resulting deficiency of these amino acids (Smith and Austic, 1978; Calvert et al., 1982; Block and Harper, 1984). Excess isoleucine does not apparently stimulate BCKA dehydrogenase activity because at normal intake levels of leucine and valine the concentrations of these amino acids in plasma were independent of the dietary isoleucine concentration. In rats, too, a high isoleucine intake does not lead to enhanced oxidation of valine and leucine (Shinnick and Harper, 1977; Block and Harper, 1984). An excess of isoleucine does not presumably cause a secondary deficiency of valine + leucine, but the reduced concentrations of valine + leucine in plasma clearly indicate that at high dietary levels of valine + leucine a high dietary concentration of isoleucine seems additionally to stimulate the activity of BCKA dehydrogenase. Our study, moreover, shows that increasing the dietary tryptophan concentration from 1.5 to 2.4 g/kg diet did not increase the concentration of this amino acid in plasma or increase the daily egg mass of laying hens. This finding demonstrates that dietary tryptophan at 1.5 g/kg diet, corresponding to a tryptophan intake of 175 mg, is sufficient for maximum daily egg mass. This result agrees with other published studies. Harms and Russell (2000) calculated a daily tryptophan requirement of 149 mg for a daily egg mass of 50 g. The recommendations based on the study by Jensen et al. (1990) are approximately 124 to 168 mg tryptophan daily. The National Research Council (1994) recommends a daily tryptophan intake of 175 mg for brown-egg layers. Interestingly, an increase of dietary tryptophan concentration from 1.5 to 2.4 g/kg significantly increased the feed consumption of laying hens. INTERACTIONS OF AMINO ACIDS IN LAYING HENS 105 This effect could be due to the function of tryptophan as a precursor of serotonin. It is well known that serotonin, which is formed in the brain, influences feed consumption of animals (Tackman et al., 1990; Mullen and Martin, 1992). Our study, in conclusion, demonstrated the existence of pronounced antagonisms between the amino acids isoleucine and valine + leucine. Interactions between tryptophan and isoleucine, however, could not be detected. ACKNOWLEDGMENT Financial support by Lohmann Animal Health (Cuxhaven, Germany) is gratefully acknowledged. REFERENCES Allen, N. K., and D. H. Baker. 1972. 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