L-VALINE: Release the potential of your feed!

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1 AJINOMOTO ANIMAL NUTRITION GO TO ESSENTIALS AJINOMOTO EUROLYSINE S.A.S. May 2009 INFORMATION N 33 LVALINE: Release the potential of your feed! An indispensable amino acid for piglet growth 70% SID Val:Lys EDITO Increased knowledge of amino acid nutrition goes a long way towards explaining the development of the use of amino acids by the feed industry. AJINOMOTO EUROLYSINE S.A.S. has pioneered the supply and use of amino acids since its creation in 1974, with specific advances with LThreonine in 1987 and LTryptophan in Feed use LValine is now available and this constitutes a precursor of major changes in animal nutrition and feed formulation practices for both pigs and poultry. Valine is a Branched Chain Amino Acid (BCAA) like isoleucine and leucine. Like lysine, threonine, methionine and tryptophan, valine is an essential and indispensable nutrient for pigs and poultry, and it has to be supplied through the feed since it cannot be synthesised by the animal. In European piglet diets, valine is the fifth limiting amino acid, and appears to be a deficient nutrient in regards to the minimum requirement for piglets. The decrease in crude protein levels in feed formulas is thus limited, yet the negative impact of an oversupply of dietary nitrogen on environment and piglet gut disorders is well documented. With availability of feed grade LValine, the nutritionist is presented with the opportunity to overcome the compromise between the pursuit of improving growth performance (technical and economic matters), health issues and environmental concerns. LValine provides three major advances in terms of nutrition. First, LValine supplementation allows the nutritional requirements of piglets to be met through better balanced diets in terms of amino acid profile, while at the same time allowing crude protein to remain restricted. Secondly, LValine gives the opportunity to increase dietary levels of lysine while maintaining the ideal amino acid balance, which is a strong lever to improve performance and feed efficiency of piglets. Finally, LValine opens the way to new formulations, allowing the use of locally produced raw materials and reducing dependence on imported protein feedstuffs. This technical bulletin compiles the information currently available on valine in piglets. The first part is dedicated to the valine requirement of the piglet. The comparison of the degree to which valine and the other amino acids are limiting is then covered. Practical implications of LValine supplementation are finally discussed with particular attention to the effect of the increase of SID Val:Lys ratio on performance.

2 Table of contents 1. A Minimum Dietary 70% SID Val:Lys Ratio is Required to Optimise Piglet Growth Literature Review Claims for a SID Val:Lys Ratio at 70% in Piglets Diets Recent Experiments Confirm 70% SID Val:Lys as a Minimum Requirement for Piglets 4 2. Valine is a Limiting Amino Acid in European Diets for Piglets Experimental Results Define Valine as a Limiting Amino Acids for Piglets In Practical European Formulas, Valine is the Fifth Limiting Amino Acid European Piglet Diets are Deficient in Valine Performance Improvement and Practical Interest of LValine Supplementation Response of Piglets to Dietary Valine Content and Practical Implications Significant improvements of performance with increased SID Val:Lys Confirmation of the effect of SID Val:Lys ratio increase on the Average Daily Gain Effect of increasing SID Val:Lys ratio under commercial conditions Further Decrease of Dietary Crude Protein with LValine Supplementation 18 Conclusion : LValine supplementation to release the potential of piglet feed 20 FOCUS 1 Nutritional Values of LValine 21 FOCUS 2 Amino Acid Requirement: Which Method and Which Value? 22 FOCUS 3 Interactions Between Amino Acids 24 FOCUS 4 Update of the Ideal Amino Acid Profile 27 FOCUS 5 Total Valine, Isoleucine and Leucine Contents and Digestible Coefficients in Various Feedstuffs 28 Reference List 30

3 AJINOMOTO EUROLYSINE S.A.S. Information N A Minimum Dietary 70% SID Val:Lys Ratio is Required to Optimise Piglet Growth For any nutrient, published requirements are often expressed in different units or within different nutritional systems, and this is also the case for valine. The experimental design determines to a large extent how an amino acid requirement must be expressed. Thus, the assessment of the valine requirement as a ratio to lysine from diverse trials is valid only on the basis of a compilation of results from trials that are consistent and have homogeneous experimental designs. The purpose of the following section is to determine the Standardized Ileal Digestible (SID) Val:Lys ratio requirement of piglets on the basis of both literature results and recent trials carried out by AJINOMOTO EUROLYSINE S.A.S. LValine: nutritional values. Focus 1 page 21 How to set a requirement in amino acids? Please read Focus 2 page Literature Review Claims for a SID Val:Lys Ratio at 70% in Piglets Diets Table 1. Comparison between recommended dietary SID Val:Lys in piglets. Various National Research Institutes give amino acid profile recommendations based on their own trials and/or literature reviews. Val:Lys recommendations from several countries are reported in Table 1. In average, 70% SID Val:Lys ratio is recommended. Country FR USA GB BR SP DK Reference INRA (Sève, 1994) NRC (1998) BSAS (Whittemore et al., 2003) UFV (Rostagno et al., 2005) FEDNA (de Blas et al., 2006) DSP (Jørgensen and Tybirk, 2008) SID Val:Lys (%) A review of studies on the valine requirement of piglets published after 1970 has been conducted (Table 2). 12 publications are available 7 of them were published in peerreviewed journals Results vary widely in the way of expressing the valine requirement Table 2. Published valine requirement of piglets, and comparison with NRC (1998) recommendations. Adapted from Barea et al., 2009a. Source Weight (kg) Tested valine levels (n) Published result % of NRC Chung and Baker, 1992 Mavromichalis et al., 2001 Exp. 4 Mavromichalis et al., 2001 Exp. 5 Warnants et al., 2001 Theil et al., 2004 Barea et al., 2009a Exp. 4 Wiltafsky et al., 2009 James et al., 2001 Kendall et al., 2004 Gaines et al., 2006 Gaines et al., 2006 Barea et al., 2009b Exp. 4 Journal Journal Journal Journal Journal Journal Journal Abstract Abstract Abstract Abstract Congress 10 5 to to 20 8 to 21 8 to to 25 8 to 22 9 to to 32 8 to to to % SID Val:Lys 0.60 g SID Val / MJ ME 0.53 g SID Val / MJ ME 68% SID Val:Lys 0.59 g AID / MJ ME 68 to 80% SID Val:Lys 65% SID Val:Lys 0.62 to 0.67 % SID Val 65% SID Val:Lys 0.92 % SID Val 0.78 % SID Val 69 to 74 % SID Val:Lys % 102% 106% % 128% to118% 96% 90 to 97 % 96% 114% 113% 101 to 109 % MJ: Mega Joule ME: Metabolizable Energy SID or AID : Standardized or Apparent Ileal Digestible To overcome the problem of mode of expression, we compare in Table 2 the different published results with the valine recommendations published by the National Research Council (NRC). Indeed the NRC itself published nutrient requirements for swine in different units in of the 12 publications reported a requirement equal or higher than the NRC recommendations (from to 128%). The average of all the quoted references sets the valine requirement of piglets at 105% of NRC recommendations.

4 4 AJINOMOTO EUROLYSINE S.A.S. Information N Recent Experiments Confirm 70% SID Val:Lys as a Minimum Requirement for Piglets To achieve balanced amino acid profiles, the use of ratios of indispensable amino acids to lysine allows experimental findings to be transferred into practical formulations where lysine specifications might differ from the ones used in experiments. With the intention of establishing an optimal and practical SID Val:Lys ratio for piglets, consolidating trial results held in different facilities, environments, and using diets of different compositions is often the selected approach. Nevertheless, as all the experiments do not aim to express a requirement as a ratio, the consolidation needs to be selective regarding experimental designs. SELECTION OF THE EXPERIMENTS To accept a trial report, it must provide sufficient information about the amino acid and energy content of the experimental diets, and of course about performance. The dose response study must compare at least 4 levels of valine. All the available trials that aimed to refine the valine requirement for piglets (from 6 to 27 kg) and that satisfied the prior mentioned conditions, are listed in Table 3. The trials from the experimental program of AJINOMOTO EUROLYSINE S.A.S. are also included. In all these trials, a basal deficient diet was fortified with increasing levels of LValine. Table 3. Designs of the trials aimed at refining the valine requirement of piglets of various weights. Source Country Weight range (kg) Tested valine levels (n) SID Lys (%) Net Energy (MJ/kg) SID Lys (g/mj of NE) Recommended SID Lys (g/mj of NE) Mavromichalis et al., 2001 Exp. 4 Mavromichalis et al., 2001 Exp. 5 Barea et al., 2009a Exp. 4 Wiltafsky et al., 2009 Barea et al., 2009b Exp. 4 Dusel et al., 2008 Jansman et al., 2008 Paulicks et al., 2008 Torrallardona et al., 2008 Journal Journal Journal Journal Congress Trial report Trial report Trial report Trial report USA USA EU (FR) EU (DE) EU (FR) EU (DE) EU (NL) EU (DE) EU (SP) * 1.14* a 1.26b 1.26b 1.26b 1.26b 1.26b 1.26b 1.26b 1.26b *Assuming that Lys SID/ Lys Total = 88% a Garcia et al., 2008 b Kendall et al., 2008 Most of the abstracts cited in Table 2 page 3 did not give enough information regarding the experimental conditions and feed composition to be included in our analysis. As explained in Focus 2, for a determination of the requirement of valine as a ratio to lysine to be valid, lysine should be the second limiting factor. Lysine should be more limiting than dietary energy and all other indispensable amino acids should be supplied at levels above requirement (Boisen et al., 2003). To determine whether lysine was supplied at an adequate level, recent literature results were used. Garcia et al., 2008 studied in one trial the optimal dietary SID lysine level for piglets from 6 to 15 kg and Kendall et al., 2008 worked out (through 5 experiments) the SID lysine levels for piglets weighing from 11 to 27 kg. It was found that the dietary SID lysine need for piglets varies from 1.29 to 1.26 g Lys SID/MJ of NE, depending on body weight. Thus, comparing dietary lysine/energy ratios of the trials in Table 3 with the above recommendations, it appears that lysine in experiment 4 from Mavromichalis et al., 2001 (1.34 g SID Lys/MJ NE) was probably not the second limiting amino acid. Protocols, formulas and amino acid profiles of the remaining trials are reported in Tables 4 and 5 respectively. Average Daily Gain (ADG), Average Daily Feed Intake (ADFI) and Feed Conversion Ratio (FCR) of the selected trials are presented in Figures 1, 2 and 3 page 6. Table 4. Protocols. Weight range (kg) Tested valine levels (n) Replicates per treatment (n) SID Val:Lys ratio range (%) Breed Sex Number of animals per treatment (n) Number of animals in total (n) Mavromichalis et al., 2001 Exp. 5 Barea et al., 2009a Exp. 4 Wiltafsky et al., 2009 Barea et al., 2009b Exp. 4 Dusel et al., 2008 Jansman et al., 2008 Paulicks et al., 2008 Torrallardona et al., PIC castrated males + females Piétrain x (LWxLR) castrated males + females German Landrace x Piétrain castrated males + females Piétrain x (LWxLR) castrated males + females Cross Breed castrated males + females (GY x Pie) x "Dalland" castrated males German Landrace x Piétrain castrated males + females Duroc x Landrace entire males

5 AJINOMOTO EUROLYSINE S.A.S. Information N 33 5 In most trials, piglets reached their best performance at ratios between 70 and 75 % of SID Val:Lys. In two trials (Jansman et al., 2008 & Wiltafsky et al., 2008), piglets had their growth limited at lower Val:Lys levels as lysine was probably not the second limiting amino acid: Looking at the dietary amino acid profile (Table 5), Jansman et al., 2008 had the lowest values for SID Ile:Lys (49%) and SID His:Lys (29%). The diets used in the trial of Wiltafsky et al., 2009 were recalculated through EvaPig. If the total and SID recalculated lysine levels are the same as in the article, the amino acid profile was slightly different than expected. Thus, SID Thr:Lys at 59% and SID Trp:Lys at 16% were possibly the factors that limited the piglets response to valine. These two trials have therefore been removed from our analysis to determine the valine requirement as a ratio to lysine. Table 5. Composition and nutritional values of the experimental diets. Mavromichalis et al Exp. 5 Barea et al a Exp. 4 Wiltafsky et al Barea et al b Exp. 4 Dusel et al Jansman et al Paulicks et al Torrallardona et al., 2008 LW range (kg) Feed Composition % Wheat Maize Barley Oats Maize Gluten Meal Wheat Middlings Maize Starch Soybean Meal Soybeans Extruded Peanut Meal Peas Whey Powder Sweet Milk Powder Lactose Gelatin Molasse Sucrose Vegetable/Animal Fat Others LLysine DLMethionine LThreonine LTryptophan LIsoleucine Other Crystalline AA Total Nutritional Values NE, MJ/kg CP, % Total Lys, % SID Lys, % SID Ratio to Lys, % Met+Cys Thr Trp Val Ile Leu His 10.4* 18.0* 1.30* 1.12* 65* 70* 20* 49* 55* 99* 38* * 17.7* 1.08* 1.00* 58* 59* 16* 46* 62* 96* 33* * 65* 22* 63* 65* 166* 34* * * Estimated with EvaPig 2008

6 6 AJINOMOTO EUROLYSINE S.A.S. Information N 33 Figure 1 : SID Val:Lys dose response Effect on Average Daily Gain (g/d). Figure 2 : SID Val:Lys dose response Effect on Average Daily Feed Intake (g/d) ADG g/day ADFI g/day % 45% 50% 55% 60% 65% 70% 75% 80% 85% 40% 45% 50% 55% 60% 65% 70% 75% 80% 85% SID Val:Lys ratio (%) SID Val:Lys ratio (%) Figure 3 : SID Val:Lys dose response Effect on Feed Conversion Ratio (g/g). FCR g/g Mavromichalis et al., 2001 Exp.5 Barea et al., 2009a Exp. 4 Wiltafsky et al., 2009 Barea et al., 2009b Exp. 4 Dusel et al., 2008 Jansman et al., 2008 Paulicks et al., 2008 Torrallardona et al., % 45% 50% 55% 60% 65% 70% 75% 80% 85% SID Val:Lys ratio (%) Table 6. SID Val:Lys requirement for piglets determined for each experiment. Models used ADG FI FCR Mavromichalis et al., 2001 Exp. 5 Barea et al., 2009a Exp. 4 Barea et al., 2009b Exp. 4 Dusel et al., 2008 Paulicks et al., 2008 Torrallardona et al., 2008 Linear Broken line and Curvilinear* Broken line and Curvilinear* Quadratic Curvilinear Curvilinear and Quadratic* Average Standard deviation 71% 73% 72% 70% 72% 69% 71% % 78% 72% 71% 73% % 72% 72% 68% 71% 72% 71% 1.4 * Average

7 AJINOMOTO EUROLYSINE S.A.S. Information N 33 7 CONSOLIDATION OF THE EXPERIMENTS In the 6 remaining trials, the means of ADG, ADFI and FCR at 70% SID Val:Lys were respectively 490 g/d (+/ 49), 795 g/d (+/ 78) and 1.63 g/g (+/ 0.15) for piglets from 8.0 to 26.0 kg LW. Mavromichalis et al., 2001 tested six dietary levels of valine for piglets. ADG, ADFI and feed efficiency were all improved linearly (p<0.05) indicating that the requirement was above the highest value of valine tested (71% SID Val:Lys). An increase of 50% in ADG was observed between the two extreme levels. Barea et al., 2009 (a and b), after establishing a sublimiting lysine level in a specific trial, studied the valine requirement through two trials: either with the actual recommended leucine level or with a leucine excess. The valine requirement was estimated to be the same in both cases, but the negative effect of a valine deficiency was exacerbated by an excess of leucine. This interaction is discussed later in the document (see Focus 3 page 24). Paulicks et al., in 2008 tested a valine dose response in piglets weighing from 12.0 to 25.0 kg. The experimental diets were very close to those of Barea et al., 2009a as one of the objectives was to repeat the trial in a different facility. Very consistent results were found in both facilities (INRA Saint Gilles, France and Technical University of Munich, Germany). Dusel et al., in 2008 carried out a trial with a low protein diet supplemented with four levels of LValine. During the 14 days after weaning (9.0 to 14.0 kg LW), the piglets received the same commercial prestarter feed (10 MJ/kg NE; 1.00 % Lys SID). Experimental diets were fed from 15 to 29 days after weaning (14.0 to 22.0 kg LW). The main benefit was in ADG when the SID Val:Lys ratio was increased to 69%. Four SID Val:Lys levels were also tested by Torrallardona et al., in 2008 in piglets weighing from 8.7 to 23.0 kg (28 days trial). Prestarter and starter feeds were fed to the piglets with 1.39 % and 1.12 % SID Lysine respectively. ADG was improved with an increase in the SID Val:Lys ratio and the best performance (FCR) was achieved at 75 %. The valine requirement was determined in each trial through the use of statistical models. The model chosen (broken line, quadratic, curvilinear ) influences the estimated requirement (see Focus 2). Table 6 reports the requirement determined by the authors. The average requirements for best ADG, ADFI and FCR were very similar with respective values of 71, 73 and 71% SID Val:Lys. This confirms that 70% SID Val:Lys is a minimum target in piglet diets. On the basis of Literature results, national recommendations, and recent trials, to optimise piglet growth between 8 and 25 kg, a minimum ratio of 70% Val:Lys SID is recommended. More about interactions between amino acids? Please read Focus 3 page 24 Update of the ideal amino acid profile. Please read Focus 4 page 27

8 8 AJINOMOTO EUROLYSINE S.A.S. Information N Valine is a Limiting Amino Acid in European Diets for Piglets Amino acid limitation to animal performance arises from the difference between the amino acid composition of feeds and the animals amino acid requirements. For each species, the ranking of limiting amino acids and the possibilities of crude protein reduction depend on the ideal amino acid profile set in the formulation and the feed materials available. The valine content of cereals is lower than that of soybean meal. Therefore, substituting soybean meal with cereals decreases the dietary valine supply. Consequently, at a given point, valine limits the reduction in dietary crude protein that is possible. Knowing now the requirement of valine by piglets, it is possible to evaluate to what extent valine is limiting for piglet growth compared to the other indispensable amino acids. What is Val, Ile & Leu contents in feedstuffs? Please read Focus 5 page Experimental Results Define Valine as a Limiting Amino Acids for Piglets Specific trials reported in the literature were designed to determine which amino acid of a tested series was limiting for piglets. These trials compared a positive control to a negative control where feed use amino acids were added alone or in a mix to reach the levels of the positive control. When the added amino acid has an effect on measured parameters, it is considered as limiting in the formula. The combination of diets allows the order in which the amino acids are limiting to be determined. In Table 7, various trials aimed at determining the limiting amino acids for pigs are reported. The 4 trials are designed around maize or sorghum based diet. Table 7. Protocols and results of 4 trials studying limiting amino acids in piglets. Weight (kg) Diet ingredients CP level (%) Tested amino acids Result: Limiting amino acid* Russel et al., to 40 Maize Soybean Meal LLys LThr LTrp 11.0 Met Val Ile Val Brudevold et al., to 20 Sorghum Soybean Meal L Lys LThr 12.0 Met Trp Val Ile His TrpValIleHis Mavromichalis et al., to 20 Maize Soybean Meal Dried Whey 13.5 Lys Thr Met Trp Val Ile His Glu 1 st Lys 2 nd (Met Thr Trp Val) Figueroa et al., to 45 Maize Soybean Meal LLys LThr DLMet LTrp 11.0 Val Ile His 1 st Val 2 nd (Ile & His) * In the absence of any indication, the listed amino acids are equally limiting. As a result, Lys is confirmed to be the first limiting amino acid (Mavromichalis et al., 1998). Val is reported to be a limiting amino acid before Ile and His (Russel et al., 1987; Figueroa et al., 2003) or equally limiting with Thr, Trp and Met (Brudevold et al., 1994; Mavromichalis et al., 1998).

9 AJINOMOTO EUROLYSINE S.A.S. Information N 33 9 Additional trials were run to determine which of the two amino acids, valine or isoleucine (both BCAAs), was first limiting. For instance, Barea et al., 2009a in their second experiment, tested the effect of SID valine and/or isoleucine ratios to lysine in 4 diets fed to piglets from 12 to 25 kg (Table 8). The basal diet contained 1.0 % SID Lys, 110% SID Leu:Lys and 10.2 MJ/Kg NE. SID valine to lysine ratio was increased from 57 to 70% and SID isoleucine to lysine ratio from 50 to 60%. Table 8. Effect of Val or lle on piglets performance (Barea et al., 2009a Exp. 2 ) Experimental design. Dietary concentration SID Lys (%) SID Val:Lys (%) SID Ile:Lys (%) Replicates (n) 1 Negative control Increase of SID Ile:Lys Increase of SID Val:Lys Increase of both SID Ile:Lys and Val:Lys Results are presented in Figure 4 ADG, feed intake and FCR were significantly improved by the valine increase No effect of increasing isoleucine (alone or after valine addition) was detected. A comparaison of this trial with other studies testing the same design is reported in Table 9 page 10. Figure 4 : Effect of Val and/or Ile on piglet ADG and FCR (Barea et al., 2009a Exp. 2 ) b 484b 450 ADG in g/day a 393a Negative control 1 Increase of SID lle:lys 2 Increase of SID Val:Lys 3 Increase of both SID lle:lys and Val:Lys a ab 1.70 FCR g/g c 1.67bc Negative control 1 Increase of SID lle:lys 2 Increase of SID Val:Lys 3 Increase of both SID lle:lys and Val:Lys 4 a,b,c : figures with different letters are significantly different (p<0.05).

10 10 AJINOMOTO EUROLYSINE S.A.S. Information N 33 Table 9. Effect of Val or lle on piglets performance complementary trials. Weight (kg) Diet ingredients CP (%) SID Lys (%) SID Ile:Lys ratios SID Val:Lys ratios Jansman et al., kg WBSBMPGMWP from 50 to 63 % from 55 to 70 % Lordelo et al., kg MWSBM from 50 to 61 % from 56 to 70 % Barea et al., 2009a Exp kg WMBSBM from 50 to 60 % from 57 to 70 % Fernandez et al., kg MWBSP from 53 to 61 % from 60 to 72 % W: Wheat; M: Maize; B: Barley; SBM: Soy Bean Meal; P: Pea; WP: Whey Powder; GM: Maize Gluten Meal; SP: Soja Protein For each test, the increase of ADG as a percentage of the performance obtained on the negative basal diet are presented in Figure 5. A systematic and significant effect was reported on ADG when Val:Lys ratio was increased There was no effect of an Ile:Lys increase or at the best, the effect was much more limited than the Val:Lys increase Figure 5 : Effect of Val or Ile on ADG, in % of the basal performance. 35 Response to lle:lys increase Response to Val:Lys increase SID lle:lys 50 to 63% SID lle:lys 50 to 61% SID lle:lys 50 to 60% SID lle:lys 53 to 61% SID Val:Lys 55 to 70% SID Val:Lys 56 to 70% SID Val:Lys 57 to 70% SID Val:Lys 60 to 72% Jansman et al., 2008 Lordelo et al., 2008 Barea et al., 2009a Exp.2 Fernandez et al., 2008 These results clearly demonstrate that valine is limiting before isoleucine in piglets Isoleucine requirement may be lower than actual recommendations SID Ile:Lys requirement in piglets? Please read Focus 3 page 24

11 AJINOMOTO EUROLYSINE S.A.S. Information N In Practical European Formulas, Valine is the Fifth Limiting Amino Acid In order to assess the ranking of limiting amino acids under European conditions (wheat based diets), a piglet diet was formulated based on average dietary nutrient levels for piglets between 12 and 25 kg live weight (9.9 MJ/kg NE ; 1.10 % SID Lys according to AEL survey 2006). The amino acid profile was according to Table 19 page 27 (Focus 4). Diets contained fixed amounts of barley (8%), maize (10%) and rape seed meal (5%) (Nutritional values according to Sauvant et al., 2004). A progressive reduction in crude protein (CP) was achieved by addition of wheat at the expense of soybean meal. Figure 6 illustrates the ranking of limiting amino acids and the protein levels at which each amino acid becomes limiting. Figure 6 : Dietary crude protein (CP) in a standard starter diet for piglet (soybean meal is gradually replaced by wheat): Ranking of limiting amino acids (from left to right) and lowest crude protein level achievable without supplementation with the corresponding amino acid LLysine + LThreonine + DLMethionine + LTryptophan CP level (%) LValine ) Lys 2) Thr 3) Met+Cys 4) Trp 5) Val 6) lle 7) Leu 8) His Ranking of limiting amino acids These calculations confirm that lysine, threonine, sulphur amino acids and tryptophan are the first limiting amino acids in a wheat based diet. Valine at 70% SID Val:Lys is the next limiting amino acid, followed by Ile, Leu and His. In this example, the LValine supplementation allowed a further reduction in crude protein by 1.6 points of CP per kg of feed (from 19.0 to 17.4 %).

12 12 AJINOMOTO EUROLYSINE S.A.S. Information N European Piglet Diets are Deficient in Valine In practice, piglet diets are usually designed with low crude protein levels (18% or below) for better health status and reduced nitrogen output to the environment. In such diets, valine content might be below the recommendation of 70% SID Val:Lys. A survey was conducted in 2006 by AJINOMOTO EUROLYSINE S.A.S. on piglet prestarter and starter commercial feeds. The 317 samples were collected in 18 countries all over Europe. Crude protein was analysed using nitrogen determination by Dumas and total amino acids were determined by ion exchange chromatography. Results presented in Figure 7 (total valine and CP, %) confirmed that in the absence of LValine supplementation, dietary valine content decreases as crude protein level is reduced. Figure 7 : Total valine (%) according to Crude Protein (%). Source : AEL Survey Prestarter diets Starter diets Total valine (%) Crude Protein (%) The average Val:Lys and Ile:Lys ratios of the deficient samples for both prestarter and starter feeds are presented in Table 10. Table 10. AEL Survey 2006: Average Val and lle content relatively to Lys in the valine deficient diets (Prestater and Starter). Occurrence Mean of the deficient population Total Val:Lys Equivalent SID Val:Lys* Total Ile:Lys Equivalent SID Ile:Lys* Valdeficient diets Prestarter Feeds 88% 65% 61% 56% 53% Starter Feeds 73% 67% 64% 57% 55% In prestarter feeds, 88% of the samples were deficient in valine with an average content of 61% SID Val:Lys ratio In starter feeds, 73% of the samples were deficient in valine with an average content of 64% SID Val:Lys ratio In both prestarter and starter diets deficient in Valine, SID Ile:Lys was 54% on average *Estimation SID/Total = 95%

13 AJINOMOTO EUROLYSINE S.A.S. Information N Figures 8 and 9 plot total Val:Lys with total lysine, either for prestarter or starter feeds. The 70% SID Val:Lys requirement of piglets is equivalent to 73% in total and is represented by the line in blue. The deficiency in valine is demonstrated whatever the lysine level Figure 8 : Total Val:Lys dietary content in Piglet Prestarter diets. Source : AEL Survey % Valine deficient diets 90% Total Val:Lys content (%) 85% 80% 75% 70% 65% 60% 55% Total Val:Lys = 73% 50% 45% Total Lys content (%) Figure 9 : Total Val:Lys dietary content in Piglet Starter diets. Source : AEL Survey 2006 Valine deficient diets 95% 90% 85% Total Val:Lys content (%) 80% 75% 70% 65% 60% 55% Total Val:Lys = 73% 50% 45% Total Lys content (%) It is concluded that: Valine was a limiting and a deficient amino acid in most of the piglet diets collected Isoleucine was not a deficient amino acid in these feeds LValine supplementation is an opportunity to rebalance the piglets diets according to the ideal amino acid profile Valine is the fifth limiting amino acid in European diets, before isoleucine. Valine is normally deficient in prestarter and starter piglet diets formulated in Europe.

14 14 AJINOMOTO EUROLYSINE S.A.S. Information N Performance Improvement and Practical Interest of LValine Supplementation The availability of LValine offers a new opportunity to formulate more efficient diets through the optimisation of the ideal amino acid profile. Consequently, all nutrients will be used in a more efficient way. The growth potential of a piglet is extremely high and should be fully expressed during the postweaning period to optimise performance in the later stages of growth. Higher SID Val:Lys ratios will make possible the improvement of the performance of piglets, therefore it is necessary to quantify this response and particularly its effect on ADG (Part 3.1). Moreover, LValine supplementation enables the formulation of diets with lower crude protein levels and higher lysine to energy ratios (Part 3.2) Response of Piglets to Dietary Valine Content and Practical Implications Significant improvements of performance with increased SID Val:Lys On the basis of the trials used to determine the valine requirement (section 1), the performance improvements in response to increases in SID Val:Lys levels were calculated. An example of the observed improvement is given in Figure 10 (The valine response in Barea et al., 2009b with an excess of leucine was not taken into account in this study). Figure 10 : Observed ADG, ADFI and FCR improvement when SID Val:Lys is increased in a range from 65 to 70% 12.0% ADG ADFI FCR 10. 0% 8.0% 6.0% 4.0% Torrallardona et al., 2008 SID Val:Lys 63 to 69 % Mavromichalis et al., 2001 Exp.5 SID Val:Lys 67 to 71 % 2.0% 0.0% 2.0% SID Val:Lys 63 to 69 % Dusel et al., % 6.0% SID Val:Lys 65 to 70 % Paulicks et al., 2008 SID Val:Lys 62 to 69 % Barea et al., 2009a Exp.4 With this set of trials, ADG and FCR were always improved while the effect on the feed intake was not consistent. Polynomial equations were calculated for ADG and FCR and are presented in Figures 11 and 12 together with the trials results. These are presented as percentages of the best result within each trial (The best result being expressed as %), in order to reduce the variation due to experimental conditions.

15 AJINOMOTO EUROLYSINE S.A.S. Information N Figure 11 : Val:Lys SID dose response ADG in % of the best performance within each trial Figure 12 : Val:Lys SID dose response FCR in % of the best performance within each trial Mavromichalis et al., 2001 Exp. 5 Barea et al., 2009a Exp. 4 Dusel et al., 2008 Paulicks et al., 2008 Torrallardona et al., 2008 Average Response Curve ADG % 85 FCR % % 55% 60% 65% 75% 80% 50% 55% 60% 65% 75% 80% Val:Lys SID ratio (%) Val:Lys SID ratio (%) PRACTICAL IMPLICATION From these curves, an estimate of the improvement of ADG and FCR when SID Val:Lys ratio increases is reported in Table 11. Table 11. Average performance improvement with increasing SID Val:Lys. SID Val:Lys From 61 to 70% From 64 to 70% ADG improvement +14% +7% FCR improvement 7% 4% Source: Average response curve (Figures 11 and 12) Table 12. Pratical implications of increasing SID Val:Lys ratio (weight gain). 61% Sid Val:Lys 64% Sid Val:Lys 70% Sid Val:Lys ADG g/d Weight gain after 28 days duration (between 12 and 25 kg) 430* kg 460* kg kg * According to average performance in the trial data set used in this review. Based on the Val:Lys levels in today s practical diets, an increase to 70% SID Val:Lys may lead to 1 to 2 kg greater weight gain in piglets reared between 12 to 25 kg over 28 days.

16 16 AJINOMOTO EUROLYSINE S.A.S. Information N Confirmation of the effect of SID Val:Lys ratio increase on the Average Daily Gain To confirm the average daily gain response to valine, a compilation was made of trials comparing 2 or 3 levels of dietary Val:Lys. A brief description of these trials is given in table 13. Table 13. Summary table of the trial characteristics used in the compilation of Figure 13. Trial code Objective Dietary SID Lys Weight range 1 Lordelo et al., 2008 Reduction of CP with LValine 1.12%* 723 kg 2 Theil et al., 2004 Reduction of CP with LValine 1.34%* 821 kg 3 Fernandez et al., 2008 Next limiting amino acid 1.10% 921 kg 4 Jansman et al., 2008 Next limiting amino acid 1.03% 1025 kg 5 Barea et al., 2009a Exp. 2 (Diets A & C) Next limiting amino acid 1.00% 1225 kg 6 Barea et al., 2009a Exp. 2 (Diets A & C) Next limiting amino acid 1.00% 1225 kg 7 Barea et al., 2009a Exp. 3 (Diets A, B & D) Bioequivalency 1.00% 1225 kg 8 Barea et al., 2009a Exp. 3 (Diets A, C & E) Bioequivalency 1.00% 1225 kg * Estimated with EvaPig 2008 In Figure 13, the improvement of ADG is reported as a function of the ADG obtained by the piglets fed the basal deficient diets. Figure 13 : Effect of SID Val:Lys on ADG improvement, according to the performance of the basal level. Improvement: % of the ADG of the basal diet (g/d) In each trial, a positive ADG response to additional dietary valine is observed. An effect on the feed intake was also noticed.

17 AJINOMOTO EUROLYSINE S.A.S. Information N Effect of increasing SID Val:Lys ratio under commercial conditions PROTOCOL With the goal of a practical assessment of the effect of SID Val:Lys ratio, a field trial was conducted in a commercial facility in Spain. It involved 180 animals from 9 to 25 kg blocked in 3 experimental groups differing in their SID Val:Lys ratio: 63, 68 and 72%. The basal diet contained 1,18% SID lysine and 10.5 MJ/kg NE (i.e g SID Lys / MJ of NE) and was mainly composed of barley, corn, soybean meal and whey concentrate. Piglets were individually weighed at the start and the end of the trial. Feed consumption was reported by pen. ADG results between 0 and 28 days after weaning are reported in Figure 14. RESULTS The level of performance was high: ADG of piglets receiving the basal diet was 488 g/d. There was a linear increase in the ADG with LValine supplementation : From 63 to 72% SID Val:Lys: a significant effect (p<0,05) was reported for ADG (+7%), and a numerical effect of 3% for FCR. From 68 to 72% SID Val:Lys there was a numerical effect of +5 % for ADG and a significant effect for FCR with the saving of 0.14 kg of feed per kg of weight gain i.e. an improvement of 9.2 %. After 28 days, there was a difference of 1 kg of weight gain between the two extreme treatments (63 and 72% Val:Lys SID): 13.7 kg and 14.6 kg of gain respectively. Figure 14 : Effect of dietary SID Val:Lys on starter piglet weight gain (from 9 to 25 kg). + + ADG (g/d) Dietary SID Val:Lys (%) a,b: figures with different letters are significantly different (p<0.05) CONCLUSIONS The response to dietary valine was in line with the previous estimates and was even higher for FCR between 68 and 72% SID Val:Lys. Even with good basal performance, an improvement is possible between 68 and 72 % of SID Val:Lys. There is a strong effect of LValine supplementation on ADG in a high lysine diet.

18 18 AJINOMOTO EUROLYSINE S.A.S. Information N Further Decrease of Dietary Crude Protein with LValine Supplementation Reducing the dietary CP level and supplementing the diet with the corresponding limiting crystalline amino acids (e.g. LValine), reduces N excretion (Relandeau et al., 2000) and limits the frequency and the severity of gut disorders in piglets. Indeed, diarrhoea often occurs when a high level of CP is fed to piglets. A part of the dietary protein remains undigested and a significant amount reaches the distal intestine. This enhances bacterial proliferation, causing significant digestive disorders. Furthermore, LValine supplementation allows additional opportunity and flexibility to make greater use of locally produced feedstuffs (wheat DDGS, rape seed meal, cereals ) at the expense of imported soybean meal. The use of such low protein diets has no detrimental effect on performance when amino acids are correctly supplied (ideal amino acid ratios maintained) and using constant net energy levels. PROTOCOL Table 14 gathers the results of three trials with the same experimental designs, where 3 types of diets were tested: a positive control with a high level of crude protein a negative control with a low level of crude protein and low SID Val:Lys ratio a third diet, the negative control supplemented with LValine to achieve the SID Val:Lys ratio of the positive control. Within a trial, SID lysine and net energy were the same in all the experimental diets. The negative controls were supplemented with 1.0 to 1.5 kg/t of LValine, depending on the positive control. The diets were also supplemented with LLysine, LThreonine, DLMethionine, and LTryptophan. As crude protein decreased, the ratio Lys:CP increased. RESULTS Effect on performance Table 14. Effect of lowering dietary crude protein (CP) with or without LValine supplementation on piglets performance. Experimental treatments CP (%) Net Energy (MJ/kg) SID Lys (%) Lys/CP SID Val:lys (%) ADG (g/day) FI (g/day) FCR (g/g) Lordelo et al., kg High CP Low CP (3.5 points) Low CP kg/t LValine * 1.12* 1.12* a 509b 571a 932a 820b 941a Fernandez et al., kg High CP Low CP (2 points) Low CP kg/t LValine * 10.4* 10.4* a 391b 449a b 1.52a 1.41b Jansman et al., kg High CP Low CP (3 points) Low CP kg/t LValine a 434b 568a 869a 682b 866a 1.54a 1.57b 1.52a * Estimated with EvaPig 2008 a,b:in the same column, figures with different letters are significantly different (p<0.05). The use of reduced nitrogen diet with no supplementation of LValine leads to a significant depletion of ADG, feed intake and FCR in all the trials. When LValine was added, the piglets recovered the performance level of the positive control.

19 AJINOMOTO EUROLYSINE S.A.S. Information N Effect on diarrhoea incidence and nitrogen excretion Lordelo et al., 2008 scored the faeces on a weekly basis on a scale from 0 to 3 (0 = normal; 3 = severe diarrhoea) and determined the incidence of diarrhoea. They also measured the effect of the experimental diets on the nitrogen (N) balance. The results are presented in Figures 15 and 16. Figure 15 : Effect of lowering dietary crude protein (CP) on diarrhoea incidence (Lordelo et al., 2008). Figure 16 : Effect of lowering dietary crude protein (CP) on nitrogen excretion (Lordelo et al., 2008). Diarrhoea incidence: higher is worse a 0.8a 0.6b 0. 9a 0.4b 0.3b 0.7a 0.5ab 0.4b High CP Low CP (3.5 points) Low CP kg/t LVal 0.7a 0.4b 0.1c Nitrogen excretion (g/d) a High CP Low CP (3.5 points) Low CP kg/t LVal 3.9b 5.0a 9.0a 5.3b 4.6b Week 1 Week 2 Week 3 Week Feces Urine Both a,b,c: figures with different letters are significantly different (p<0.05) The incidence of diarrhoea was significantly decreased by the use of the reduced CP diet and reduced CP diet supplemented with LValine The N excretion was significantly reduced by the low CP diets; confirming that with LValine addition, the reduction of 1 point of CP leads to 10% of N excretion, without detrimental effects on performance. CONCLUSIONS LValine helps to formulate diets with a CP reduced by more than 2 points LValine allows the use of reduced CP diets without a detrimental effect on performance The use of reduced CP diets supplemented with feeduse amino acids is an efficient way to reduce nitrogen excretion and the risk of digestive disorders. By increasing the SID Val:Lys ratio from 64% up to 70%, weight gain is improved by 7% and feed efficiency by 4% LValine supplementation is cost effective in commercial conditions LValine offers an additional opportunity to formulate reduced crude protein diets: with an additional CP decrease of 2 points with improvement of the piglets health status with lower excretion of nitrogen into the environment

20 20 AJINOMOTO EUROLYSINE S.A.S. Information N 33 Conclusion LValine supplementation to release the potential of piglet feed LValine supplementation improves piglet growth performance by: Satisfying the nutritional requirement of piglets at 70% SID Val:Lys. Allowing further decrease of dietary crude protein levels for health and environmental reasons. Rebalancing the ideal amino acid profile, leading to better feed efficiency. Allowing dietary lysine levels to be increased while maintaining the ideal amino acid balance, so that the high growth potential of piglets can be expressed without increasing the health risk due to excess protein. 70% SID Val:Lys

21 AJINOMOTO EUROLYSINE S.A.S. Information N FOCUS 1 Nutritional Values of LValine To add LValine to your formulation matrix and fill its nutritional values FOCUS 2 Amino Acid Requirement: Which Method and Which Value? To understand what is behind a requirement value FOCUS 3 Interactions Between Amino Acids Branched Chain Amino Acids share the same metabolic pathway, what are the consequences? FOCUS 4 Update of the Ideal Amino Acid Profile Ideal Amino Acid profile to exactly meet the requirement of piglets FOCUS 5 Total Valine, Isoleucine and Leucine Contents and Digestible Coefficients in Various Feedstuffs BCAAs content of the raw materials FOCUS 1 Nutritional Values of LValine Molecule C5H11NO2 Minimum LValine content in product* 96.5% as is 98.0% on dry matter Crude Protein (N x 6.25) 72.1% H H H C H H C CH H 0 H C C 0 H H N H Digestible Energy Pig Metabolizable Energy Pig Net Energy Pig Metabolisable Energy Poultry 24.4 MJ.kg MJ.kg MJ.kg MJ.kg Kcal.kg Kcal.kg Kcal.kg Kcal.kg 1 Digestibility coefficient % * Commercial guarantee in product

22 22 AJINOMOTO EUROLYSINE S.A.S. Information N 33 FOCUS 2 Amino Acid Requirement: Which Method and Which Value? Experimental designs and statistical models Dose response trials are mainly used to determine nutrient requirements. Depending on the statistical model applied to determine the requirement, the experimental design must compare a minimum number of levels of the studied nutrient. A 3levels study does not allow a convincing determination of the requirement (linear or quadratic response?). Four levels will lead to more accuracy and 5 levels will avoid any doubt on the type of response (Figure 17). The choice of the number of levels to be tested must be done as a function of an a priori knowledge of the requirement: at least 2 levels below to describe the shape and at least 2 levels higher to reach a plateau value. Figure 17 : Assessment of Val:Lys requirement by a curvilinear model Requirements should be determined through the use of regression models. Pointbypoint comparisons using classical analysis of variance, do not take into account that response variables (body weight, feed efficiency ) are continuous rather than discrete (Pesti et al., 2009). The broken line model provides a minimum value for the requirement but this is more accurate for individuals than for a population. Nutrient supplies below this value have a very strong and negative effect on the predicted parameter. The curvilinear model determines the requirement of the whole population. Experimental designs and expression of the result Amino acid requirements in the literature are often variable and are reported in a wide range of units : amount of the feed (in %, or in g/mj of NE), relative to weight gain (mg/g/day), ratio relative to lysine (g/ g of lysine), in total or digestible, standardized or apparent values, using broken line or curvilinear models All of them have strengths and weaknesses. However, the basal diet must be designed according to the way in which the requirement will be expressed (Table 15). At the time of making decisions regarding nutritional recommendations, the nutritionist should use the most advanced nutritional concepts and express requirement on the same basis to formulate consistent feeds (for instance : SID system, Net Energy system, ideal amino acid profile). The scope of valine requirement determined as an amount of feed is generally limited to the particular conditions of the trial. In such experiments, none of the nutrients should be limiting to make sure that the observed response is only due to the increase in the valine concentration. The best performance indicates the optimal dietary valine concentration for the specific situation. Nevertheless, the increase in performance could be limited by another factor which is unknown (dietary factor, genetic, environment ). As a result, the published requirements vary over a wide range: an average of the published values is given in Table 15 but with a high coefficient of variation (17%). Moreover, as lysine is in excess of requirement, it is not possible to express the valine requirement from these trials as a ratio to lysine. All the valine is used but not all the lysine; the ratio is then based on a wrong lysine reference and the optimal valine to lysine ratio is likely to be underestimated (Boisen, 2003). Lysine is considered as the first limiting amino acid in formulas and is used mainly for growth. Its requirement should be first established in an optimal amount of feed. But, as protein accretion will be then limited by energy, the practical recommendation for lysine requirement should be expressed relative to the energy content of the feed.

23 AJINOMOTO EUROLYSINE S.A.S. Information N A common way to express amino acid requirements is to use their ratios to lysine. For establishing the optimal ratio between a specific amino acid, e.g. valine and lysine, lysine needs to be the second limiting amino acid to ensure that the optimal valine to lysine ratio corresponds to a maximum use of both lysine and valine. This ratio corresponds to the ratio at which valine and lysine are equally limiting. To achieve this condition, 1) lysine should be limiting to energy, 2) the other indispensable amino acids must be provided in a sufficient amount relative to lysine. Thus, lysine becomes sublimiting in the diet. These controlled trial conditions are also necessary to avoid any other nutrients or external parameters limiting performance. As a result, the requirements established in different trials leads to very homogeneous dataset with a very low coefficient of variation (2.1%). The resulting optimal ratio is not considered to be influenced by the concentration of lysine (Boisen, 2003). Indeed, in practice, lysine levels are adjusted upwards to allow the full expression of piglet potential for growth. Table 15. Comparison of trial characteristics aiming to determine the valine requirement of piglets, either in amount of the feed or in ratio to lysine. Mode of expression Objective Characteristics of the basal experimental diets (Example: study of valine requirement) Valine Lysine Others amino acids Energy Examples of results Strenghs Weaknesses Amount of the Feed % of the feed g/unit of energy To determine the valine concentration which leads to the best performance in the particular situation Limiting Not limiting Not limiting Not limiting % SID Val in average 0.75 % +/ 0.13 (cv 17%) 0.53 g SID Val/MJ ME 3 Allows to optimize a specific diet Good way to produce lysine reference Specific to the studied diet (lysine level, energy level...) Unknown limiting factors Results are very variable Ideal Amino Acid Profile g/ g of lysine To determine the ratio at which valine and lysine are equally limiting Limiting Sublimiting in the diet Slightly oversupplied (in ratio to Lys) Not limiting 71% +/ 1.5 (cv 2.1%) SID Val:Lys (This compilation) Easy to apply in practice Homogeneous results Not specific to the experiment and the diet Assumes that the requirement is the same whatever the stage of life of the animal 1 James et al., 2001 (9 to 15 kg) 2 Gaines et al., 2006 (8 to 12 kg) 3 Mavromichalis et al., 2001 (10 to 20 kg) In this bulletin, the valine requirement for piglets relative to lysine is expressed in standardized ileal digestibility. Trials with different lysine levels are presented to confirm the use of the recommended ratio in commercial feeds with higher lysine levels.

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