G O TO E S S E N T I A L S

Information N 36 June 2010

oday broiler feed formulations are not only focused on minimising costs; they also have to take into consideration environmental issues and their impact on broiler health. European Union regulations limit and reduce nitrogen pollution in the environment (directive 96/61/EC) and have banned the use of antimicrobial growth promoters. Reducing excess dietary protein is an important way of addressing these issues. By reducing the nitrogen input to farms, reduced protein diets lower the nitrogen output to the environment. For instance in broilers, a reduction of 2 points of dietary pro tein content results in a 13% to 14% reduction in nitrogen excretion (Bregendahl et al. , 2002; Jialin et al. , 2004). In addition, health conditions can be improved by redu cing the dietary protein supply. The crude protein level is reported as a predisposing factor for necrotic enteritis (Dahiya et al., 2005; Mc Devitt et al., 2006; van Immerseel et al., 2004 and Drew et al., 2004). Moreover, an excess of protein would lead to a physiological need for an increase in water consumption to achieve efficient nitrogen excre tion. As a consequence, high crude protein diets lead to

higher levels of nitrogen and water excretion compared with low crude protein diets, leading to deterioration in litter quality and the birds environment (Lensing and van der Klis, 2006). Instead of formulating on dietary crude protein levels, the broiler feed formulator can focus on each individual amino acid since crude protein is an attempt to represent amino acid supply but not the quality of this supply. When redu cing crude protein, broiler performance will be limited by the indispensable amino acids that do not meet birds require ments. Amino acids which are said to be indispensable can not be synthesized by the organism. They must therefore be provided in the feed so that they can be used in the synthesis of body proteins and thus support the process of growth. When the supply of one of the indispensable amino acids does not meet the animals requirement, it is said to be limiting. The present bulletin aims to determine which amino acids are next limiting in broiler diets after sulphur amino acids, lysine and threonine, in order to help the feed formulator reduce dietary crude protein levels and formulate broiler feeds more precisely.

Update on the next limiting amino acids in broiler feeds Valine and Isoleucine Reducing dietary crude protein level
I. V  aline and isoleucine are the next limiting amino acids in broilers diets ................................................................................................................................................................................................. 3
A. Published valine and isoleucine ratios to lysine . ............................................................................................................... 5 B.  Explanation of the variability in published Val:Lys and Ile:Lys ratios ................................................... 5

II. Valine and isoleucine requirement in the literature .................................................................... 5

III. Valine requirements of broilers ......................................................................................................................................... 8

A. TD valine requirement as ratio to lysine ..................................................................................................................................... 9 B. TD valine requirement as amount of the feed .................................................................................................................. 11

IV. TD Ile:Lys requirement of broilers ............................................................................................................................ 13 V. Practical implications for broiler diets .............................................................................................................. 20 Conclusions ........................................................................................................................................................................................................... 22 References ............................................................................................................................................................................................................... 23

Focus 1: Focus 2: Focus 3: Focus 4:

Classification of amino acids for broilers ...................................................................................................................... 4 Interactions between branched chain amino acids ........................................................................................ 17 Broilers leucine requirements ................................................................................................................................................. 18 Branched chain amino acid profiles in feedstuffs are not consistent ..................................... 19

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I. V  aline and isoleucine are the next limiting amino acids in broilers diets
To reduce dietary crude protein (CP) levels in broiler feed, it is necessary to know which indispensable amino acids become limiting in diets and what the requirement of broilers is (Focus 1 p 4). Depending on the requirement assumed for each amino acid, valine, isoleucine, tryptophan and arginine are generally considered as the next limiting amino acids in broiler after sulphur amino acids, lysine and threonine.

> Feedstuffs used in broiler diets influence the 4th limiting amino acid
The amino acid composition of protein differs between feedstuffs and can impact the order in which amino acids become limiting in diets. Recent work of Corzo et al. (2007) tried to predict the 4th limiting amino acid in broiler diets. The authors made simulations for the whole broiler growing period with their respective minimum nutritional requirements. The summary of the simula tions is presented in Table 1.

Broiler age (days)

AME (kcal) TD* Lysine (%) TD Sulphur amino acids (%)

1-12
3050 1.27 0.92 0.80 1.00 0.86 1.30 0.20 Val Val Val Val Val Val

13-22
3075 1.09 0.81 0.70 0.87 0.75 1.13 0.18 Val Val Val Val Val Val

23-35
3100 1.00 0.76 0.65 0.80 0.71 1.05 0.16 Val Val Val Val-Ile Ile Val-Ile

36-48
3125 0.95 0.74 0.63 0.77 0.68 1.01 0.16 Val Val Val Val-Ile Ile Val-Ile

>49
3150 0.89 0.70 0.60 0.73 0.65 0.96 0.15 Val Arg Arg Ile Ile Ile

Nutritional constraints

TD Threonine (%) TD Valine (%) TD Isoleucine (%) TD Arginine (%) TD Tryptophan (%) Corn - SBM

4 limiting AA depending on the main feedstuffs

th

Wheat - SBM Sorghum - SBM Corn - SBM - MBM Wheat - SBM - MBM Sorghum - SBM - MBM

Table 1: Prediction of the 4th liming amino acid in broiler diets (Corzo et al., 2007) (SBM = soybean meal; MBM = meat and bone meal; TD True Digestible).

 In vegetable diets based on corn, wheat and sorghum, valine appears to be the 4th limiting amino acid.  When animal by products are included in the diets, isoleucine becomes the 4th co-limiting amino acid with valine. An equivalent formulation study (Kidd and Hackenhaar, 2005) done on broiler grower diets gave the same results. Valine appears to be the 4th limiting amino acid in vegetable diets, and it is isoleucine when animal by-products were used.

> The ranking of the limiting amino acids depends on the broiler requirement levels
A study carried out by Fernandez et al. (1994) on 8-21 day chicks determined which amino acids were limiting in corn soybean meal based diets. Amino acid levels of the control diet were according to Baker and Chung (1992) specifica tions. The authors used the deletion method (amino acid supply was reduced one after another) and observed average daily gain (ADG) and gain:feed ratio (G:F) of the birds (Figure 1 shows the results for G:F; results for weight gain gave the same ranking of limiting amino acids).

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Gain:Feed (g/g) 0.50

 Methionine, lysine and threonine were the 1st limiting amino acids for broiler performance  This trial confirmed that valine was the 4th limit ing amino acid for broilers ahead of arginine and tryptophan in a corn soybean meal diet. In another study carried out by Corzo et al. (2007) on 21-42 day broilers, not done by amino acids dele tions but by amino acids additions, confirmed this result. They fed a negative control diet (17.5% CP) based on corn and soybean meal with 1.0 kg/tonne of L-Valine or L-Isoleucine or L-Arginine. Best broiler performance was achieved with the diet supplemented with L-Valine. This study confirmed that valine was limiting before isoleucine and arginine. The present bulletin will focus on the valine and isoleu cine requirements of broilers because they appear to be limiting before tryptophan and arginine. By know ing precisely the requirements, feed formulators will be able to reduce dietary crude protein levels and to formulate broiler diets more accurately.

0.45

b c d

0.40

0.35

0.30

0.25

0.20

Control

Control -L-Val

Control -L-Arg

Control -L-Trp

Figure 1: Order of limiting amino acids in the protein of a corn soybean meal diet for chick growth (Fernandez et al., 1994). Columns with different letters are significantly different (P<0.05).

 Valine appears to be the 4th limiting amino acid in vegetable broiler diets after
sulphur amino acids, lysine and threonine.

 When animal by-products are used, isoleucine becomes co-limiting with valine.

Focus 1

Classification of amino acids for broilers

Animals, like plants, synthesize proteins which consist of 20 amino acids. However, unlike plants, animals cannot synthesize all the amino acids. The amino acids which cannot be syn thesized must be supplied in the diet to cover animal requirements. The 20 amino acids are classified as indispensable (10), semi-indispensable (2) and dispensable (8) (Table 2).
Indispensable Semi-indispensable Dispensable

(not synthesized in chickens) Methionine Lysine Threonine Valine Tryptophan Isoleucine Arginine Histidine Leucine Phenylalanine

(synthesized from limited substrates) Cystine

(Readily synthesized in chickens from simple substrates) Alanine Aspartic acid Asparagine Glutamic acid Glutamine Glycine Serine Proline

Tyrosine

Table 2: Nutritional classification of amino acids in chickens (Leeson and Summers, 2001).

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II. V  aline and isoleucine requirement in the literature

The first part of this section describes the available data on Val:Lys and Ile:Lys ratios in the literature and explains the existing variability between published data. The last part will describe the materials and methods used in this bulletin to determine valine and isoleucine requirements of broilers.

A.

Published valine and isoleucine ratios to lysine 

A literature survey was conducted to review valine and isoleucine published requirements. The ratio to lysine is a common way of expressing amino acid requirements and sixty Val:Lys and fifty-eight Ile:Lys (total and digestible) recommended ratios were found. Figure 2 presents the variability in the published recommendations.

Valine

Isoleucine 50 60 70 80 90 100 110 120 Ratios to lysine (%)

Figure 2: Compilation of available published requirements for valine and isoleucine expressed as Val:Lys and Ile:Lys ratios. Each point represents one published requirement.

 Published recommended Val:Lys ratios vary between 61% and 122%. The average and median ratios are identical at 81%. Published Ile:Lys ratios vary between 50% and 91%. The average and median ratios are identical at 70%.

B.

Explanation of the variability in published Val:Lys and Ile:Lys ratios 

The huge variability in the recommended broiler requirements could be attributed to different methods and modes of expression used to determine the requirement of a given amino acid. The following section describes how the design used in a study may impact the requirement that is reported by the study.

> Method used to determine the requirement of valine and isoleucine

Published valine and isoleucine ratios could have been estimated by the following methods:   Literature survey: Authors compile available data at the time of their publications.  Factorial approach: Generally several studies are done to determine maintenance and growth requirement of birds. Authors then calculate the requirement based on animal weight (maintenance) and a hypothetical growth (production).  Deletion method: The study consists of comparing birds performance between a positive control diet (balanced in all amino acids) and other treatments where the tested amino acids are supplied one after the other below the birds requirement. A statistical method is then used to estimate the requirement for the tested amino acids.  Dose response study: This method consists of testing different concentrations of an amino acid (at least 3 levels) and then determining through statistical methods which concentration gives the best performance.

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> Method of expressing amino acid requirements

Amino acid requirements can be expressed in several ways: as an amount in the feed, relative to energy, relative to weight gain or relative to lysine. They may also be expressed on a total, apparent digestible or standardised digestible basis. In addition to the different response criteria used (i.e. daily gain, feed conversion ratio, nitrogen retention, plasma urea nitrogen), different statistical models (Pesti et al., 2009) (linear plateau, curvilinear plateau, asymptotic model) are used to determine the requirement. Although all of these methods have their strengths and weaknesses, the desired mode of expression will determine the experimental design that has to be used in requirement studies. In growing animals, the ideal protein concept is often used to express animals amino acid requirements. This concept defines the amino acid profile (as g of the tested amino acid / 100 g lysine) which exactly meets the animals requirement for protein deposition and maintenance (Fuller et al., 1989). In this profile, all amino acids are presumed to be equally limi ting for all physiological functions. Lysine has traditionally been used as the reference because it is the 1st and 2nd limiting amino acid for growth respectively in pigs and broilers and is mainly used for muscle protein deposition. It is frequently assumed that the ideal protein profile does not change between different growth phases. In practical nutrition, this offers the advantage that the lysine requirement may be varied (per kg of feed or per unit of energy), but not the ideal protein profile expressed relative to lysine. Finally, one of the most important criteria which must be taken into account when a requirement to lysine is being determined is that lysine must be the 2nd limiting amino acid in the experimental diets, after the studied amino acid. In a dose-response study, different levels of the studied amino acid should be provided, while the lysine content in the diet should remain constant (and sub-limiting) (Figure 3). A two-level study will show if there is a response between the two levels tested but it will not be possible to determine the requirement for the tested amino acid from such a trial because no breakpoint would be demonstrated.

Response

Lys < requirement Lys << requirement Lys > requirement

Tested AA:Lys

Figure 3: Theoretical response to increasing levels of the tested AA:Lys when Lys is (or is not) the 2nd limiting amino acid after the tested one. The two arrows represent the estimated requirements. When a factor other than Lys is 2nd limiting for the response criterion, the calculated AA:Lys requirement ratio (a) will be underestimated in comparison to (b) where Lys is the 2nd limiting factor.

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  When lysine is 2nd limiting for performance (weight gain, feed efficiency), an increase in the tested amino acid supply will result in a increase in the response criterion up to the point that the tested amino acid supply is no longer limiting, which is indicated by the breakpoint. A further increase in the tested amino acid supply would not result in a change in the response criterion. If lysine is actually 2nd limiting, the plateau value will be determined by the lysine supply. At the breakpoint, the tested amino acid and lysine are co-limiting and the ratio between the two will be the requirement of the tested amino acid (b).  When lysine is not the 2nd limiting nutrient in the tested amino acid dose-response study, an increase in the tested amino acid supply will initially result in an increase in the response criterion. When the tested amino acid supply is further increased, another unknown factor (e.g., an amino acid other than the tested amino acid and lysine) will become limiting (indicated by the ooo-symbols in Figure 3). The breakpoint of that line corresponds to the tested amino acid requirement relative to the unknown factor (a). Interpreting this breakpoint as the tested amino acid requirement relative to lysine would underestimate the actual tested amino acid requirement. As shown in Figure 3, it is assumed that the optimal tested amino acid:lysine ratio is not affected by the lysine content in the diet when lysine is actually the second-limiting factor (Boisen, 2003). The constraint that lysine should be the 2nd limiting amino acid only applies to experimental studies in order to express the requirement relative to lysine whereas in practical nutrition, the lysine levels are higher to ensure that the animals can fully express their growth potential. Some studies have been carried out on amino acid requirements where lysine is not (or does not appear to be) the 2nd limiting amino acid. By itself, this does not invalidate these studies but the reported amino acid requirement should then be expressed relative to the 2nd limiting factor and not relative to lysine.

 An exhaustive literature review show that there is huge variability in published

requirements Val:Lys = 81% 19.7 Ile:Lys = 70% 17.0

 This variability is dependent on a multitude of factors and at this stage does not
allow conclusions to be reached on practical recommendations.

> Database construction for determination of valine and isoleucine requirement

Due to the wide variation in published requirements, it is very difficult to interpret and reach conclusions about the valine and isoleucine requirements of broilers. In order to investigate this further, a database was built in which the available data on the valine and isoleucine responses and requirements of broilers were compiled. An exhaustive search was made of world literature and 28 valine and 66 isoleucine trials were collected into the database (articles or abstracts from scientific journals). To enter a trial in the database, the following minimum information was required:  Ages of the tested chickens  Composition of the basal experimental diet. Nutritional values (apparent metabolisable energy and amino acid expressed in true digestible) were re-calculated with INRA tables (Sauvant et al., 2004) (where possible) so that the analysis could be based on complete amino acid profiles drawn from a common source, and to remove the variability arising from published values (expected analyzed).  Measures of growth performance: Average daily gain, average daily feed intake, gain:feed ratio. Carcass weight and yield, breast meat weight and yield were also entered when was available.

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III. Valine requirements of broilers

Valine is one of the branched-chain amino acids (BCAA) along with isoleucine and leucine. The way in which the requirement is expressed depends on the experimental design used in studies.
The lysine levels used in the trials (expressed on a true digestible (TD) basis) is of particular importance because:  If TD lysine level is sub-limiting, the valine requirement can be expressed as a ratio to lysine (Val:Lys)  If TD lysine level is not limiting, the valine requirement should be expressed as a percentage of the feed (%) The TD lysine of the 28 basal diets entered in the database were compared graphically to the TD lysine requirement at different ages of modern broiler genotypes (adapted from Ross 308 (2007) and Cobb 700 (2008) management guides) (Figure 4).

TD Lysine (%) 1.60 Lysine requirement of modern genetics

1.40

1.20

1.00

0.80

0.60

0.40 0 10 20 30 Age (days) 40 50 60

Figure 4: Comparison of TD lysine levels in valine trials and TD lysine requirements of modern broilers.

Dietary TD lysine levels for the growth period studied were higher than the birds requirement in 4 trials (Corzo et al., 2008 exp.1, 2 and 3 and Mack et al., 1999). In these trials, lysine was probably not the 2nd limiting amino acid after valine and therefore the requirement could not be expressed as a ratio to lysine. Nevertheless, these trials used an experimental design that allowed an estimate of the valine requirement as a percentage of the feed according to the age of bird.

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A.

TD valine requirement as ratio to lysine

> Selection of the trials

The selection of trials based on lysine level in the feed (24 trials) must be further refined. No other amino acids must be at a level that would limit the broilers response to TD Val:Lys. TD amino acids profiles of basal diets were compared to Baker and Han (1994) to determine if any amino acids other than valine were limiting in the diet. If one or more amino acids were deficient, the study was eliminated from the review. The example of TD Arg:Lys levels is given in Figure 5. Trials where TD Arg:Lys was lower than 105% (in red in Figure 5) were eliminated from the compilation.

TD Arg:Lys (%) 145 135 125 115 105 95 85 Trials

TD Arg:Lys recommended by Baker and Han (1994)

Figure 5: TD Arg:Lys levels in trials entered the valine database. Red dots highlight trials where TD Arg:Lys was lower than recommended by Baker and Han (1994).

In addition, because a 2-level study does not allow the determination of a requirement, two trials were eliminated from the compilation (Thornston et al., 2006 exp1 and Corzo et al. 2007 exp1). The selection process is summarized in Figure 6. In the end, 10 dose-responses were used to determine the TD Val:Lys requirement of broilers.

28 trials Non sub-limiting lysine levels Sub-limiting lysine levels

4 trials

24 trials

6 Lack of information 6 De ciency in one or more amino acids

2 2-level TD Val:Lys studies

4 trials for TD Val (%)

10 trials for TD Val:Lys

Figure 6: Summary of reasons for selection or elimination of trials.

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The experimental designs, the nutritional values of the basal feed and the growth performance of the best treatment in each of the 10 trials kept for estimating the TD Val:Lys requirement of broilers are presented in Table 3. The 10 trials differ in the broilers ages, sex, genetics and in the feeds nutritional values. This explains the differences in ADG, ADFI and G:F ratio between trials.

Allen et al. 1972 Exp 1 Age range (days) Genetics Sex 8 - 14 Allen et al. 1972 Exp 2 8 - 14 Mendonca et al. 1989 21 - 42 Leclercq 1997 20 - 40 ISA 220 M Bae et al. 1999 8 - 22 Arbor Acres M Nutritional Values AME (kcal) CP (%) TD Lys (%) TD Thr:Lys (%) TD SAA:Lys (%) TD Ile:Lys (%) TD Leu:Lys (%) TD Arg:Lys (%) TD Val:Lys (%) 4350 14.3 0.93 69 74 65 443 128 38 - 59 4450 16.6 0.93 69 74 65 443 128 54 - 75 3120 15.2 0.90 74 84 75 140 124 69 - 91 3140 15.6 0.57 80 77 91 207 135 107 - 154 4050 17.3 1.07 73 83 73 110 114 20 - 104 3040 23.8 1.18 66 79 76 193 108 89 - 228 3170 17.3 0.75 98 103 100 221 126 79 - 107 3115 14.6 0.93 73 87 74 131 110 63 - 136 3115 14.6 0.93 73 87 74 131 110 63 - 136 3125 17.3 0.95 67 82 74 124 139 62 - 88 Ekermans et al. 2001 7 - 21 MF Corzo et al. 2004 Exp 2 42 - 56 Ross 308 M Thornston Thornston et al. et al. 2006 Exp 2 21 - 42 Ross 508 M 2006 Exp 3 21 - 42 Ross 508 F Corzo et al. 2007 21 - 42 M

New Hampshire X Columbian Plymouth Rock M M

Best performance ADG (g/day) ADFI (g/day) G:F (g/g) 5.2 11 0.471 5.0 11 0.463 54.1 113 0.482 78.1 134 0.585 28.6 43 0.670 36.4 53 0.693 103.6 246 0.422 69.6 178 0.411 60.0 174 0.349 66.2 141 0.485

Table 3: Experimental designs, recalculated nutritional values of the basal feed and broiler performance of the best treatment in each of the 10 trials kept for the determination of the TDVal:Lys requirement of broilers. (M=Male; F=Female).

> Results from the compilation for growth performance

In order to estimate the TD Val:Lys ratio which optimises ADG and G:F ratio of broilers, the performance in the ten selected trials was represented in a curvilinear plateau model. The model took into account a trial effect by estimating a plateau for each trial separately. The results are presented graphically in Figures 7 and 8.
ADG (% of the plateau) 110 105 100 95 90 85 80 75 55 60 65 70 75 80 TD Val:Lys (%) 85 90 95 100

G:F (% of the plateau) 110 105 100 95 90 85 80 75 55 60 65 70 75 80 TD Val:Lys (%) 85 90 95 100

Figure 7: Effect of TD Val:Lys ratio on ADG.

Figure 8: Effect of TD Val:Lys ratio on G:F.

 There is a clear response in ADG and G:F ratio to increased TD Val:Lys ratios, which validates the selection procedure.  The TD Val:Lys ratio which optimises broiler growth and feed efficiency is 80%.

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> Results from the compilation for carcass parameter

There is a strong effect of TD Val:Lys ratio on weight of carcass and breast meat (Corzo et al., 2004; 2007). However, carcass yield (Thornston et al., 2006; Corzo et al., 2004; 2007) and breast meat yield were not affected (Leclerq, 1998; Thornston et al., 2006; Corzo et al., 2004; 2007). As carcass weight and breast meat weight increased together with increased Val:Lys ratios, the ratio between breast meat and carcass weight did not change. However, meeting the birds requirement for valine is of key importance in ensuring the optimal usage of lysine which is well known to increase breast meat yield (Berri et al., 2008).

> Validation of the average response curve

Thornston et al. (2006, exp1) and Corzo et al. (2007, exp1) were eliminated from the compilation because they tested only two TD Val:Lys ratios. However, these trials can be used to estimate birds responses to valine, and to validate the average response curves in Figures 9 and 10. The improvement of performance observed in the 2-step trials was very much in line with the average response curves and validates their representation of birds responses to increasing TD Val:Lys ratios.
ADG (% of the plateau) 110 105 100 95 90 85 80 75 55 60 65 70 75 80 TD Val:Lys (%)
Corzo et al. (2007) +9% Thornston et al. (2006) +8%

G:F (% of the plateau) 110 105 100 95 90 85 80 75 55 60 65 70 75 80 TD Val:Lys (%)

Corzo et al. (2007) +5% Thornston et al. (2006) +4%

85

90

95

100

85

90

95

100

Figure 9: ADG response to TD Val:Lys ratio in 2-step trials.

Figure 10: G:F response to TD Val:Lys ratio in 2-step trials.

 Increasing the TD Val:Lys ratio in broiler diets improves performance until the
valine requirement is met.

 A TD Val:Lys of at least 80% is needed to optimize broiler performance.

B.

TD valine requirement as amount of the feed

Four trials allow the optimum valine requirement of broilers to be assessed as a percentage of the feed according to the age of birds. The procedure to determine the requirement as a percentage of the feed was different in each publication. In order to homogenise the results, recalculated nutritional values and a common statistical model (curvilinear-plateau model) were used to determine the broiler requirement as a percentage of the feed for ADG and G:F in each trial (Table 4).

Authors Mack et al., 1999 exp1 Corzo et al., 2008 exp1 Corzo et al., 2008 exp2 Corzo et al., 2008 exp3

Age (days) 20-40 1-14 15-28 29-42

Genetics ISA 220 Ross 308 Ross 308 Ross 308

Best ADG (g/day) 77.9 28.6 78.3 99.7

Best ADFI (g/day) 136 34 105 180

Best G:F (g/g) 0.585 0.841 0.714 0.557

TD Valine requirement (% of the feed) ADG 0.79 1.00 0.92 0.80 G:F 0.76 1.05 0.83 0.75

Table 4: ADG and G:F requirement as % of the feed in each trial.

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  The re-estimated requirements are similar for ADG and feed efficiency within and between studies for the same broiler ages (Mack et al., 1999 and Corzo et al., 2008 exp3), whatever the genetic strain used.  The decrease in requirement with age is mainly explained by the increase in feed intake in older birds. Four requirements were determined for ADG and feed efficiencies for 4 different periods. With the objective to model the TD valine requirement in relation to broiler age, it is essential to take into account the feed intake effect. This effect can be modelled through a logarithmic function. The re-estimated requirements from each trial for ADG at different ages were plotted in a single graph. A logarithmic curve was then used to take into account the increase in feed intake with broiler age (Figure 11). The same method was used to determine the valine requirement of broilers for feed efficiency (Figure 12).
TD Val (%) 1.60 1.50 1.40 1.30 1.20 1.10 1.00 0.90 0.80 0.70 0.60 0.50 0.40 0
Corzo et al. 2008a y = -0.1351ln(x) + 1.2843 R2 = 0.87 Corzo et al. 2008b Corzo et al. 2008c Mack et al. 1999

10

15

20 25 Age (days)

30

35

40

TD Val (%) 1.60 1.50 1.40 1.30 1.20 1.10 1.00 0.90 0.80 0.70 0.60 0.50 0.40 0

Corzo et al. 2008a y = -0.2028ln(x) + 1.4561 R2 = 0.99 Corzo et al. 2008b Corzo et al. 2008c Mack et al. 1999

10

15

20 25 Age (days)

30

35

40

Figure 11: TD valine requirement for ADG (% of the feed).

Figure 12: TD valine requirement for G:F (% of the feed).

By using the equations modelling the valine requirement as % of the feed according to age of broiler (given in Figures 11 and 12), it is possible to calculate the TD valine requirement for a targeted age of bird (Table 5). However, these results should be used with care as the compilation regroups only 4 trials.

Age (days) 10 20 30

TD Valine requirement (% of the feed) ADG 0.97 0.87 0.82 G:F 0.99 0.83 0.76

Table 5: Example of TD valine requirements % of feed depending on the age of broiler.

 TD valine requirement as % of feed varies with broiler age  TD valine requirement as % of the feed may be estimated from the following
equations: For ADG: TDVal(%) = -0.1351ln(day) + 1.2843 For G:F: TDVal(%) = -0.2028ln(day) + 1.4561

 These results are an indication of a minimum TD Valine level required in feed

formulations since they are based on only 4 studies. The use of the valine ratio to lysine is preferred: TD Val:Lys = 80%

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IV. TD Ile:Lys requirement of broilers

As explained in previous sections, trials must be selected based on TD lysine levels to determine the method of expressing the isoleucine requirement (Figure 13).

TD Lysine (%) 1.60 Lysine requirement of modern genetics

1.40

1.20

1.00

0.80

0.60

0.40 0 10 20 30 Age (days) 40 50 60

Figure 13: Comparison of TD lysine levels in isoleucine trials and TD lysine requirement of modern broiler genotypes.

> Selection for % of the feed

Six trials used higher TD lysine levels than recommended for the studied growth period. Lysine was therefore probably not the 2nd limiting amino acid after isoleucine in these studies. The isoleucine requirement found in these trials should therefore not be expressed as a ratio to lysine but as a percentage of the feed. Five of the six eliminated trials were 2-dose isoleucine trials. The sixth trial (Mack et al.,1999) was the only trial left with an experimental design that allowed the TD isoleucine requirement to be expressed as a percentage of the feed. Because there was only one, it was not possible to make a compilation of multi-trial data. The isoleucine requirement, estimated by a curvilinear-plateau model as was done for valine above, was found to be 0.72% for ADG and 0.60% for G:F in 20-40 day old ISA 220 broilers.

> Selection for ratio to lysine

Sixty trials used sub-limiting lysine levels and therefore potentially allowed an expression of broilers isoleucine require ment as a ratio to lysine. TD amino acid profiles of basal diets were compared to Baker and Han (1994) to determine if any other amino acids were limiting in the diet and therefore restrict the response to increasing TD Ile:Lys ratios. If one or more amino acids were found to be deficient, the study was eliminated from the compilation. Also, all the 2-dose tri als (Kidd et al., 2004 exp 1, 2 and 3 ; Park and Austic, 2000 exp 1, 2, 3 and 4) were eliminated for reasons described in previous sections. The selection procedure is summarized in Figure 14. In the end, only six trials were kept to determine the isoleucine requirement of broilers expressed as a TD Ile:Lys ratio.

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66 trials Non sub-limiting lysine levels Sub-limiting lysine levels

6 trials

60 trials

12 Lack of information 5 2-level studies 35 De ciency in one or more amino acids

7 2-level TD Ile:Lys studies

1 trial for TD Ile (%)

6 trials for TD Ile:Lys

Figure 14: Summary of reasons for selection or elimination of trials.

The main characteristics of the trials selected are presented in Table 6. The 6 trials differ in the broilers ages, sex, genetics and nutritional values of the feed. This explains the differences in ADG, ADFI and G:F ratio between trials.

Corzo et al. 2004 Age range (days) Genetics Sex 42 - 56 Ross 308 M

Hale et al. 2004 30 - 42 Ross 508 F

Kidd et al. 2004 18 - 30 Ross 308 M Nutritional values

Kidd et al. 2004 30 - 42 Ross 308 M

Kidd et al. 2004 42 - 56 Ross 308 M

Corzo et al. 2008 35 - 54 Ross 708 -

AME (MJ) CP (%) TD Lys (%) TD Thr:Lys (%) TD SAA:Lys (%) TD Val:Lys (%) TD Arg:Lys (%) TD Leu:Lys (%) TD Ile:Lys (%)

3010 15.6 0.81 90 89 98 122 202 65 - 95

3000 15.7 0.87 79 96 92 114 177 48 - 96

3310 17.7 0.88 79 98 105 119 195 57 - 91 Best performance

3030 16.2 0.87 79 94 91 115 175 54 - 89

3040 15.1 0.73 88 99 107 126 209 57 - 98

3110 16.7 0.95 69 77 76 100 147 65 - 74

ADG (g/day) ADFI (g/day) G:F (g/g)

93.8 226 0.415

60.4 142 0.436

59.1 104 0.578

79.4 157 0.524

52.8 177 0.302

99.0 208 0.483

Table 6: Experimental designs, recalculated nutritional values of the basal feed and broiler performance of the best treatment in each of the 6 trials kept for the determination of the TDIle:Lys requirement of broilers.

Moreover, it is important to notice that in four trials (Hale et al., 2004 and Kidd et al., 2004 exp 1, 2 and 3) the authors used blood cells (animal product). Blood cells have a specific imbalance in their amino acid profiles; they are rich in valine and leucine and particularly poor in isoleucine (Figure 15 and Focus 4). This raw material is often used in isoleucine dose response trials because it easely creates a deficiency. Howewer, the imbalance between these three amino acids has been reported to be a factor that impacts animal response (Focus 2 p 17).

14 | Information n 36 | AJINOMOTO EUROLYSINE s.a.s.

% of CP Lys
12 % 10 % 8% 6%

% of CP Lys
12 % 10 % 8% 6%

Leu

4% 2%

Thr

Leu

4% 2%

Thr

Blood cell, Kidd et al., 2004 Soybean meal 48, INRA 2004

Val

Ile

Val

Ile

Figure 15: Lysine, threonine, isoleucine, valine and leucine content in percentage of CP of blood cell (Kidd et al., 2004) and soybean meal 48 (INRA tables, Sauvant et al., 2004).

> Results of the compilation

In order to estimate the TD Ile:Lys ratio that optimises ADG and G:F ratio in broilers, growth performance from the remain ing six trials were compiled using the same method as for valine. The graphical results of the compilation of these trials are presented in Figures 16 and 17.

ADG (% of the plateau) 110 105 100 95 90 85 80 75 70 65 45 50 55 60 65 70 TD IIe:Lys (%) 75 80 85 90 G:F (% of the plateau) 110 105 100 95 90 85 80 75 70 65 45 50 55 60 65 70 TD IIe:Lys (%) 75 80 85 90

Figure 16: Effect of TD Ile:Lys ratio on ADG.

Figure 17: Effect of TD Ile:Lys ratio on G:F.

There is a strong effect of TD Ile:Lys ratio on weight of carcass and breast meat (Kidd et al., 2004 trials 1 and 2). However, carcass yield (Kidd et al., 2004 trials 1 and 2; Corzo et al., 2004; 2008) and breast meat yield were not affected.

AJINOMOTO EUROLYSINE s.a.s. | Information n 36 | 15

> Validation of the average response curve

The trials of Kidd et al. (2004; exp 1, 2 and 3) and Park and Austic (2000; exp 1, 2, 3 and 4) were not used in the compila tion because they tested only two isoleucine levels. However, these trials can be used to estimate the birds responses to isoleucine and to validate the average response curves in Figures 18 and 19.
ADG (% of the plateau) 110 100 90 80 70 60 50 40 25 35 45 55 65 TD Ile:Lys (%) 75 85 95 G:F (% of the plateau) 110 100 90 80 70 60 50 40 25 35 45 55 65 TD Ile:Lys (%) 75 85 95

Kidd et al. (2004) exp1 Kidd et al. (2004) exp2 Kidd et al. (2004) exp3

Park and Austic (2000) exp1 Park and Austic (2000) exp2 Average curve in g 16 and 17

Park and Austic (2000) exp3 Park and Austic (2000) exp4

Figure 18: ADG response to TD Ile:Lys ratio in 2-step trials.

Figure 19: G:F response to TD Ile:Lys ratio in 2-step trials.

There is a response to isoleucine increase in all the trials but two trial populations appears through the differences in the magnitude of the response to increasing TD Ile:Lys ratio. The Kidd et al. (2004) trials showed a greater response (more than +30% for ADG and +25% for G:F) than those of Park and Austic (2000). The main differences in experimental design between these publications come from the diet composi tion (particularly the use of blood cells). Consequently, the TD Leu:Lys ratios are much higher in Kidd et al. (2004) (average TD Leu:Lys of 193%) than in Park and Austic (2000) (average TD Leu:Lys 147%) (Table 7). A consideration of interactions between BCAA is presented in Focus 2 p 17.

Publication

Exp 1

Blood cells Present Present Present Absent Absent Absent Absent

Age (days) 18-30 30-42 42-56 2-14 2-14 2-14 2-14

TD Lys level (%) 0.88 0.87 0.73 1.12 1.12 1.12 1.12

TD Val:Lys ratio (%) 105 91 107 84 123 84 123

TD Leu:Lys ratio (%) 195 175 209 129 164 129 164

Kidd et al., 2004

2 3 1

Park and Austic, 2000

2 3 4

Table 7: TD lysine, Val:Lys and Leu:Lys levels in feed with and without blood cells.

 Increasing the TD Ile:Lys ratio in broiler diets improves performance until the
isoleucine requirement is met.

 A TD Ile:Lys dietary level of at least 67% is needed to optimize broiler performance.  The response to isoleucine is modified by specific feedstuff particulary rich in
leucine.

16 | Information n 36 | AJINOMOTO EUROLYSINE s.a.s.

Focus 2

Interactions between branched-chain amino acids

It has been reported by several authors (DMello and Lewis, 1970ab; Burnham et al., 1992; Witalfsky et al., 2010) that there is an antagonism between the branched-chain amino acids (valine, isoleucine and leucine) in broilers. The first two steps in their catabolism are common to all three amino acids and involve the BCAA aminotransferase (BCAT) and branched-chain -keto acid dehydrogenase (BCKDH). Their further metabolism employs distinct pathways to different end products (glucose and/or ketone bodies) (Figure 20).

Branched Chain Amino Acids Leu - Ile - Val

-Ketoglutarate Glu

Reversible transamination

BCAT enzyme

Branched-chain -keto acids

Irreversible oxydative decarboxylation BCKDH enzyme

-ketoacyl-CoA Thiesters
-oxydation Acetyl-CoA from Leu Acetyl-CoA + Propionyl-CoA from Ile Propionyl-CoA from Val

Figure 20: Catabolism of branched chain amino acids (valine, isoleucine, leucine).

In practical terms, any supply of one of the three BCAA leads to the activation of the common enzymes and the catabolism of all three BCAA (Harper et al., 1984; Langer and Fuller, 2000; Witalfsky et al., 2010). Moreover, the -keto acid of leucine plays a particular role in the acti vation of the BCKDH complex (Witalfsky et al., 2010). DMello and Lewis (1970a) observed in growing broilers (7-21 days) that circulating levels of isoleucine and valine were lowered by an excess of leucine. They also described that valine and isoleucine requirements increased with leucine supply (DMello and Lewis, 1970b). Burnham et al. (1992) observed that dietary leucine set at 1.76 times the requirement level depressed chick growth. However an excess of valine does not seem to impact birds response to isoleucine (DMello and Lewis, 1970a). This is also confirmed by the comparison of the growth responses reported by Kidd et al. (2004, exp 1, 2, 3) and Park and Austic (2000, exp 1, 2, 3, 4) in Table 7. The dataset used to determine the valine requirement of broilers did not show any interaction between valine and leucine, even with an extremely high level of leucine. Nevertheless, Barea et al. (2009) showed that an excess of leucine impacted the growth response in piglets but not the valine requirement level itself. However, in contrast to valine, the data presented in the isoleucine trial compilation suggest that there is an interaction between leucine and isoleucine and raise doubts on the estimate of TD Ile:Lys requirement determined in this compilation. Most of the trials (4 out of 6) used blood cells which have a high leucine content. Moreover, the basal diet used by Corzo et al. (2004) contained corn gluten meal (Focus 3 p 18) which is a raw material extremely rich in leucine and this could have created an imbalance between isoleucine and leucine (Table 6).

AJINOMOTO EUROLYSINE s.a.s. | Information n 36 | 17

Focus 3

Broilers leucine requirements

As for valine and isoleucine, there is a large range in the published requirements for leucine as expressed as Leu:Lys ratios (Figure 21). However, taking data only from publications since 1994, the Leu:Lys requirement varies between 92% and 123% with average and median val ues respectively of 107% and 108%. These levels are very much in line with the TD Leu:Lys of 105% given in AEL bulletin 27 (2004).

Leucine 96 104 112 120 128 Ratios to lysine (%) 136 144

Figure 21: Compilation of available published requirements for leucine expressed as Leu:Lys ratios. Each point represents one published requirement.

With the objective to determine how leucine is limiting in a practical broiler diet, different formulas of a broiler grower feed (3200 kcal AME and 1.05% TD lysine) were formulated:  In a corn based diet (corn 55%, wheat 10%, soybean meal 30% and supplemental amino acids), the TD Leu:Lys ratio was 140%.  In a wheat based diet (wheat 70%, soybean meal 25% and supplemental amino acids), the TD Leu:Lys ratio was approximately 120%.

 Leucine levels in todays feed formulations are much higher

than the recommended requirement level (TD Leu:Lys=105%).

18 | Information n 36 | AJINOMOTO EUROLYSINE s.a.s.

Focus 4

Branched-chain amino acid profiles in feedstuffs are not consistent

Because of the interactions between BCAA, it is interesting to compare the BCAA composition of various feedstuffs. The valine, isoleucine and leucine ratios to total BCAA (based on INRA table, Sauvant et al., 2004) of several feedstuffs have been calculated and are presented in Figure 22.
Leu:BCAA (%) 0.70 0.65 0.60 0.55 0.50 0.45 0.40 0.70 0.10 0.20 Ile:BCAA (%)
Barley Corn Sorghum Triticale Wheat Wheat gluten feed Corn gluten feed Corn gluten meal Blood meal Corn Sorghum Corn gluten meal

0.15 0.20 0.25 0.30 0.15 0.35 0.25 0.30 0.40 Val:BCAA (%)

Pea Rapeseed meal Soybean meal Sun ower meal Fish meal Blood meal Feather meal Meat and bone meal

Figure 22: Val:BCAA, Ile:BCAA and Leu:BCAA ratios of the main feedstuffs used in broiler diets (INRA Tables, Sauvant et al., 2004).

 A large group of feedstuffs have a similar profile of BCAA (average of all listed feedstuffs; Ile:BCAA = 22%, Val:BCAA = 30% and Leu:BCAA = 48%)  However, corn (59% Leu:BCAA), corn gluten meal (65% Leu:BCAA), sorghum (58% Leu:BCAA), and blood meal (6% Ile:BCAA and 56% Leu:BCAA) have very imbalanced profiles.

 Corn, corn gluten meal and sorghum have a very high

proportion of leucine and may therefore contribute to an excess of dietary leucine. isoleucine and may contribute to a branched-chain imbalanced even at moderate incorporation rates.

 Blood products have an extremely low proportion of

AJINOMOTO EUROLYSINE s.a.s. | Information n 36 | 19

V. Practical implications for broiler diets

A sound knowledge of amino acid requirements is a prerequisite for any investigation on the possibility of reducing dietary crude protein levels. This new review concerning the valine and isoleucine requirements of broilers allows the ideal protein proposed in AEL bulletin 27 (2004) to be updated (Table 8). Todays greater knowledge of individual indispensable amino acid requirements allows diets to be formulated not on crude protein but on each amino acid (precision protein). Feed formulators can now formulate more accurate diets and thereby help to reduce the number of health challenges that broilers have to face.
Lysine Sulphur amino acids Threonine Valine Isoleucine Arginine Tryptophan Histidine Leucine Phenylalanine + Tyrosine 100 75 65 80 67 105 17 40 105 105

Table 8: Proposed ideal protein for broilers (true digestible amino acids relative to lysine).

By using a minimum specification for each of these amino acids in formulations, it is possible to progressively determine the next limiting amino acid in broiler feed and to establish the extent to which it is possible to reduce dietary crude protein through supplementation with amino acids. As an example, three grower broiler diets (14-28 days) were formulated to contain 3200 kcal AME and 1.05% TD lysine, using the AA profile presented in Table 8. This exercise was done with different feed ingredients:  to reflect European formulation practice,  to understand the ranking of limiting amino acids,  and to estimate the extent of crude protein reduction through supplementation with feed-use amino acids. The first simulations are based on a wheat-soybean meal diet and the second ones on a corn-wheat-soybean meal diet (Figure 23). The last one is based on a corn-soybean meal-blood meal-sunflower meal diet (Figure 24).

> Cereal-based diet simulations:

Wheat 72%/Soybean meal 23% CP level (%) 35 30 25 20 15 10 29.2
+DL-Met +L-Lys

Corn 56%/Wheat 10%/Soybean meal 23% CP level (%) 35 30 25 28.7

+DL-Met

22.4

21.5

+L-Thr

20.3

+L-Val

21.3 19.0 18.9 18.0 17.3 20 15 10

+L-Lys

19.3

20.2

+L-Thr

19.0

+L-Val

18.0

17.7

17.5

16.1

16.0

M+C

Lys

Thr

Val

Arg

Ile

His

Leu

Trp

M+C

Lys

Thr

Val

Arg

Ile

His

Leu

Trp

Figure 23: Dietary CP level (%) in a standard grower broiler diet (soybean meal was gradually replaced by wheat or corn). Ranking of limiting amino acids (from left to right) and lowest crude protein level achievable without supplementation with the corresponding amino acid.

20 | Information n 36 | AJINOMOTO EUROLYSINE s.a.s.

  For clarification, with wheat and soya, the lowest CP level that could be achieved without DL-Methionine supple mentation was 29.2%. When methionine was added to the formulation, the next limiting amino acid was lysine and, without L-Lysine supplementation, the lowest CP level that could be reached was 22.4%. When L-Lysine was offered, the CP level dropped to 21.5% and threonine became limiting.  In both simulations, sulphur amino acids, lysine and threonine were the first limiting amino acids.  L-Threonine supplementation allowed a reduction of approximately 1 percentage point of CP in both diet types.  In vegetable diets, valine was the next limiting amino acid before arginine and isoleucine, as already described in section I.  Supplementation of L-Valine allowed a further reduction of 1 percentage point of dietary CP.

>  Diet containing blood product

(Corn 55%/Wheat 10%/Soybean meal 18%/Sunflower meal 34%/Blood meal 2%)

CP level (%) 35 30 25 20 15 10 27.8

+DL-Met

22.1

+L-Lys

20.4

+L-Thr

+L-Ile

19.2

18.4

18.0

16.3

16.7

15.8

M+C

Lys

Thr

Ile

Arg

Val

Trp

His

Leu

Figure 24: Dietary CP level (%) in a standard grower broiler diet with blood meal and sunflower meal (soybean meal was gradually replaced by corn). Ranking of limiting amino acids (from left to right) and lowest crude protein level achievable without supplementation with the corresponding amino acid.

 The order of the first limiting amino acids was the same as in the vegetable diets (1 st sulphur amino acids, 2nd lysine and 3rd threonine). L-Threonine supplementation allowed a reduction of 1.2 percentage points of CP.  Isoleucine was the 4th limiting amino acid in this diet, ahead of arginine and valine. As already described in previous sections, blood meal is very low in isoleucine and rich in leucine. Even a moderate usage results in a limitation on the reduction in dietary crude protein.  The supplementation of L-Isoleucine would allow a CP reduction of 0.8%, down to 18.4% CP in a grower broiler diet.

 This practical formulation exercise confirmed the position of threonine as a

limiting amino acid in corn and wheat based diets. L-Threonine supplementation helps achieve better formulations. vegetable diets, followed by arginine and isoleucine.

 Valine at 80% TD Val:Lys is the next limiting amino acid after threonine in  Isoleucine at 67% TD Ile:Lys is the next limiting amino acid after threonine in feed
containing blood meal, followed by arginine and valine.

 Broiler nutritionists are now able to formulate on at least the first five limiting
amino acids and to reduce dietary crude protein level through amino acid supplementation. L-Valine supplementation is a powerful tool in optimizing vegetable broiler feed formulations.

AJINOMOTO EUROLYSINE s.a.s. | Information n 36 | 21

Conclusions
Valine is the 4th limiting amino acid in vegetable broiler diets based
on wheat or corn.

Isoleucine becomes the 4th limiting amino acid, when blood cells
or blood meal are used in formulations.

The TD Val:Lys and TD Ile:Lys requirements of broilers Knowing the requirements of individual amino acids,

are respectively 80% and 67% to optimize performance.

feed formulators are able to reduce dietary crude protein levels.

L-Valine supplementation in combination with L-Threonine

provides the opportunity to formulate technically, economically and environmentally better broiler feed.

Amino acid profile for broiler feed
Lysine Sulphur amino acids Threonine Valine Isoleucine Arginine Tryptophan Histidine Leucine Phenylalanine + Tyrosine 100 75 65 80 67 105 17 40 105 105

Disclaimer
The estimation of nutrient requirement is not an exact science. The company AJINOMOTO EUROLYSINE S.A.S. has taken all steps to check the authenticity and relevance of the information provided in this bulletin. However, AJINOMOTO EUROLYSINE S.A.S. declines all responsibility for any use made of this data and may not, under any circumstances, be held liable for any damage suffered by third parties.

22 | Information n 36 | AJINOMOTO EUROLYSINE s.a.s.

References
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 Fuller, M. F., R. McWilliam, T. C. Wang, and L. R. Giles. 1989. The optimum dietary amino acid pattern for growing pigs. Br. J. Nutr. 62:225-267.  Hale, L. L., S. J. Barber, A. Corzo, and M. T. Kidd. 2004. Isoleucine needs of thirty- to forty-two-day-old female chickens: growth and carcass responses. Poult. Sci. 83:1986-1991.  Harper, A. E., R. H. Miller, and K. P. Block. 1984. Branchedchain amino acid metabolism. Annu. Rev. Nutr. 4:409-454.  Jianlin, S., C. A. Fritts, D. Burnham, and A. L. Waldroup. 2004. Extent to which crude protein maybe reduced in corn soyabean meal broiler diets through amino acid supplementation. Int. J. Poult. Sci. 3:46-50.  Kidd, M. T., D. J. Burnham, and B. J. Kerr. 2004. Dietary isoleucine responses in male broiler chickens. Br. Poult. Sci. 45:67-75.  Langer, S. and M. F. Fuller. 2000. Interactions among the branched-chain amino acids and their effects on methionine utilization in growing pigs: effects on nitrogen retention and amino acid utilization. Br. J. Nutr. 83:43-48.  Leclercq, B. 1998. Specific effects of lysine on broiler pro duction: comparison with threonine and valine. Poult. Sci. 77:118-123.  Leeson, S. and J. D. Summers. 2001. Proteins and amino acids. Pages 102-175 in: Scotts nutrition of the chicken. Ed. University Books, Guelph, Ontario, Canada.  Lensing, M. and J. D. Van der Klis. 2006. The effect of synthetic threonine on broiler performance under suboptimal dietary con ditions. Schothorst Feed Research. Trial report.  Mack, S., D. Bercovici, G. de Groote, B. Leclercq, M. Lippens, M. Pack, B. Schutte, and S. van Cauwenberghe. 1999. Ideal amino acid profile and dietary lysine specification for broiler chickens of 20 to 40 days of age. Br. Poult. Sci. 40:257-265.  Mcdevitt, R. M., C. R. Brooker, Acamovic, T. and N.H.C. Sparks. 2006. Necrotic enteritis, a continuing challenge for the poultry industry. World. Poult. Sci. J. 62:221-248.  Mendona, C. X. Jr., and L. S. Jensen. 1989. influence of valine level on performance of older broilers fed a low protein diet supplemented with amino acids. Nutr. Rep. Int. 40:247-252.  Park, B. C. and R. E. Austic. 2000. Isoleucine imbalance using selected mixtures of imbalancing amino acids in diets of the broiler chick. Poult. Sci. 79:1782-1789.  Pesti, G. M., D. V. Vedenov, J. A. Cason, and L. Billard. 2009. A comparison of methods to estimate nutritional requirements from experimental data. Br. Poult. Sci. 50:16-32.  Relandeau, C. and L. Le Bellego. 2004. Amino acid nutrition of the broiler chicken, update on lysine, threonine and other amino acids. AJINOMOTO EUROLYSINE technical information 27  Sauvant, D., J. M. Perez, and G. Tran. 2004. Tables of com position and nutritional value of feed materials. Wageningen Academic Publishers, INRA Editions and AFZ, Paris.  Thornton, S. A., A. Corzo, G. T. Pharr, W. A. Dozier, D. M. Miller, and M. T. Kidd. 2006. Valine requirements for immune and growth responses in broilers from 3 to 6 weeks of age. Br. Poult. Sci. 47:190-199.  Van Immerseel, F., J. de Buck, F. Pansmans, G. Huyghebaert, F. Haesebrouck, and R. Ducatelle. 2004. Clostridium perfringens in poultry: an emerging threat for animal and public health. Avian Pathol. 33:537-549.  Wiltafsky, M. K., M. W. Pfaffl, and F. X. Roth. 2010. The effects of branched-chain amino acid interactions on growth perfor mance, blood metabolites, enzyme kinetics and transcriptomics in weaned pigs. Br. J. Nutr. 103:964-976.

AJINOMOTO EUROLYSINE s.a.s. | Information n 36 | 23

Already issued:
Information N36 June 2010 Information N29 June 2006

Update on the next limiting amino acids in broiler feed: Valine and Isoleucine Reducing dietary crude protein level
Information N35 June 2010

Impact of the withdrawal of antimicrobial growth promoters and health status on the amino acid requirement of the pig
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Branched chain amino acids nutrition in piglets: Valine, Isoleucine and Leucine Requirements and practical implications
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Tryptophan in young pigs: a key nutrient for growth and feed intake
Information N27 June 2004

SID Trp:Lys requirement for piglets determined by meta-analysis

Information N33 May 2009

Amino acid nutrition of the broiler chicken Update on lysine, threonine and other amino acids
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Information N32 November 2008

Threonine requirement in pigs Benefits of L-Threonine supplementation

Information N25 March 2002

AJINOMOTO EUROLYSINE S.A.S. Formulators Handbook Measuring and predicting amino acids contents in feedingstuffs
Information N31 June 2008

Low protein diets for piglets

Information N24 May 2001

Managing growth and carcass quality of growing pigs fed low protein diets
Information N23 September 2000

Threonine in pigs and broilers: a crucial amino acid for growth and gut function
Information N30 March 2008

L-Tryptophan supplementation to enhance piglet growth

Information N22 June 2000
Realisation Magamo Illustrations Nathalie Morvillier

Tryptophan in young pigs: an essential nutrient with numerous biological functions

Prevention of nitrogen pollution from pig husbandry through feeding measures

Yvan PRIMOT, Etienne CORRENT and Aude SIMONGIOVANNI, June 2010.

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