The availability of feed-use amino acids such as. Information N 35 June 2010

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1 G O T O E S S E N T I A L S Information N 35 June 2010 The availability of feed-use amino acids such as L-Lysine, L-Threonine, L-Tryptophan and L-Valine, produced by AJINOMOTO EUROLYSINE S.A.S., allows the nutritional needs of piglets to be met with a high degree of precision and the dietary crude protein (CP) content of the feeds can be reduced with confidence. Besides its effects on health, and particularly diarrhoea (Lordelo et al., 2008), the further reduction of dietary CP through the use of feed-grade amino acids, is the best available technique to provide a balanced supply of essential amino acids (EAA) to animals while limiting the excretion of nitrogen (N) without compromising growth. From a practical point of view, it has been demonstrated that Val is the next limiting amino acid (AA) after Lys, Thr, Met and Trp for growth in piglets given feeds based on the feedstuffs normally used in European piglet feeds (Bulletin #33, 2009). A dietary deficiency of Val limits the expression of the growth potential of an animal. The recent usage of L-Valine in commercial feeds has broken through this barrier. Animal performance can now be considerably improved as the Val requirement is met at the same time as the CP level is reduced. Five feed-grade amino acids are used today in European animal feeds and this means that a nutritionist is able to formulate on six co-limiting AA in a diet. The sixth AA is called the next limiting AA, and determines the extent to which the dietary CP level can be further reduced. The identity of the next limiting AA depends both on the minimum dietary requirement to optimize piglets growth and the AA contents of the feedstuffs used in the feed formulation. In most commercial diets in Europe, Isoleucine (Ile) appears to be the sixth limiting AA. Among the twenty AAs that are necessary for protein synthesis, the Branched-Chain Amino Acids (BCAAs), Valine, Isoleucine and Leucine, are three of the nine AAs that cannot be synthesized by the pig. They are classified as indispensable nutrients and their continuous dietary supply is essential for maintenance and growth. Due to their common catabolism, BCAAs have to be studied together since there is an interaction between them. In this bulletin, the focus is on BCAAs and a review of the information that is available on them. The objective is to give clear recommendations about BCAAs in the formulations of piglet feeds based on the concept of ideal amino acid profiles and to evaluate the impact on formulations in terms of Lys levels and dietary CP reduction.

2 Branched-chain amino acids nutrition in piglets Valine Isoleucine Leucine Requirements and practical implications 1. Branched-Chain Amino Acid Metabolism Enzymes in BCAA catabolism processes and their regulation An excess of Leu decreases the performance of piglets receiving a Val or Ile deficient diet Branched-Chain Amino Acid Requirements of Piglets Valine requirement is confirmed to be at least 70% SID Val:Lys Isoleucine requirement is 53% SID Ile:Lys Leucine requirement: 100% SID Leu:Lys to avoid excess Branched-Chain Amino Acid Nutrition of Piglets in Practice Adjusting Lys levels within the ideal amino acid profile concept Valine is the most limiting AA in current European piglet feeds % SID Val:Lys improved ADG and FCR in commercial trials Reducing dietary Crude Protein by 2 points in piglet feed References Conclusions Focus 1: The anabolic pathway and its impact on nutrition... 8 Focus 2: Feed intake regulation in the case of BCAA imbalance... 8 Focus 3: Leucine interferes with the response to Val supply, but probably not with the Val requirement Focus 4: Estimation of the minimum dietary CP level in diets for piglets Focus 5: Feed-use amino-acids and risk management Information n 35 AJINOMOTO EUROLYSINE s.a.s.

3 1. Branched-Chain Amino Acid Metabolism Dietary nutrients are mainly absorbed (Figure 1, first flap) in the small intestine. The digestive processes lead to the presence of amino acids in the lumen, in a free form or as small peptides. Both forms are absorbed by the intestinal cells through different transporters. The majority of peptides are then hydrolysed by intracellular peptidases resulting in the release of single AAs in the hepatic portal blood (Krehbiel and Matthews, 2003). Having been taken up directly into the intracellular pool of a tissue, BCAAs, like any other AAs, cannot be stored. They must follow either the anabolism route (Focus1, protein synthesis), or go into the catabolic pathway. This catabolism is mainly a separation of nitrogen from the carbon skeleton, the latest being afterwards available for the glucogenic and/or ketogenic pathways, depending of the original AA. 1.1 Enzymes in BCAA catabolism processes and their regulation Branched-chain AA catabolism involves two initial enzymatic steps that are common to all three BCAAs. These enzymes are distributed widely and differently among body tissues (Hutson, 2005). The BCAAs are unusual in that they escape the first pass of metabolism in the liver and go directly to the muscle. The initial step in the degradation of the BCAAs involves a reversible transamination reaction by the BCAA Transaminase [BCAT]. In piglets, the BCAT is mainly located in the skeletal muscle (Wiltafsky, 2010) and accepts all three BCAA as substrates, yielding the following catabolites: Glutamate (Glu), precursor of Glutamine (Gln) and Alanine (Ala). These AAs subsequently join the AA pool for protein synthesis. NH 3, that will enter the urea cycle, and the Branched-Chain α-keto-acids [BCKA] (carbon skeleton): α-keto-isovalerate (KIV), from Val α-keto-β-methylvalerate (KMV), from Ile α-keto-isocaproate (KIC), from Leu The BCKA products formed then undergo oxidative decarboxylation catalyzed by BCKA Dehydrogenase [BCKDH] complex, mainly located in the liver in pigs (Wiltafsky, 2010). This reaction, which is irreversible, is the degradation of BCKA into Acyl-CoA derivatives. Afterwards, intermediate steps lead to the production of glucogenic (from Ile and Val) or ketogenic (from Ile and Leu) bodies. > Regulation of BCAA Transaminase (BCAT), the first step in the catabolism of BCAAs BCAT is the enzyme which catalyses the reversible reaction from BCAAs to their keto-acids. In Figure 2, the relation between plasma BCAA and their corresponding BCKA (adapted from Wiltafsky, 2010) shows that any increase in a BCAA is followed by an increase in its keto-acid. It confirms that the regulation of the BCAT depends primarily on the concentration of the enzyme, and its substrates (Harper, 1984). > Regulation of Branched-Chain Keto Acid Dehydrogenase (BCKDH), the second and limiting step in the catabolism of BCAAs Wiltafsky (2010) studied the effect of Leu excess on piglets, when either Ile or Val was the first limiting AA in the diet. This design allowed meaningful ratios to be determined for Leu, and to explore animals sensitivity to interactions between BCAA. The authors measured the effect of Leu on the activity of the BCKDH complex in the liver of piglets. The enzymatic activity was quantified through two criteria and it was observed that there was: No variation in the total activity (total amount of the enzyme), But a significant linear increase of the basal activity (enzyme activity state), Figure 3. AJINOMOTO EUROLYSINE s.a.s. Information n 35 5

4 gut flow VAL ILE LEU small intestine liver KIV KMV KIC BCKDH A-CoA 1 A-CoA 2 A-CoA 3 Glucogenic Ketogenic muscle VAL ILE LEU PROTEIN SYNTHESIS LYS VAL TRP BCAA CATABOLISM BCKA LEU THR ILE VAL KIV BCAT ILE KMV LEU KIC ALA GLN GLU NH3 Figure 1: Global pattern of the branched-chain amino acids metabolism. > Abbreviations A AA Amino acid AcoA Acyl-Coenzyme A ADFI Average daily feed intake ADG Average daily gain AID Apparent ileal digestible Ala Alanine Arg Arginine B BCAA Branched-chain amino acid BCKA Branched-chain α-keto-acid BCAT Branched-chain amino acid transaminase BCKDH BCKA dehydrogenase C CP Crude protein Cys Cysteine D d day E EAA Essential amino acid F FCR Feed conversion ratio FI Feed intake G Gln Glutamine Glu Glutamic acid H His Histidine I Ilvo Instituut voor Landbouw en Visserijonderzoek (Belgium) Ile Isoleucine INRA Institut National de la Recherche Agronomique (France) 3 Information n 35 AJINOMOTO EUROLYSINE s.a.s.

5 Serum KIV, KMV or KIC (micromol/l) 60 Valine x KIV Isoleucine x KMV Leucine x KIC Plasmatic Val, Ile or Leu (micromol/l) Figure 2: Regulation of the first step of degradation of the BCAAs by the BCAA Transaminase (BCAT, reversible reaction). Plasmatic BCAAs (Val, Ile or Leu) and corresponding BCKAs (KIV, KMV or KIC). Adapted from Wiltafsky (2010). Level of the BCKDH activity (%) 40 Leu:Ile effect Leu:Val effect P < SID Leu:Ile or SID Leu:Val ratio Figure 3: Regulation of the second step of degradation of the BCAAs by the BCKDH (irreversible reaction). Effect of increasing Leu in diets either first limiting in Val or in Ile, on the BCKDH activity (%). Adapted from Wiltafsky (2010). K KIC α-keto-isocaproate KIV α-keto-isovalerate KMV α-keto-β-methylvalerate L Leu Leucine LNAA Large neutral amino acids LW Live weight Lys Lysine M Met Methionine MJ Megajoule N N Nitrogen NC Negative control NE Net energy NEAA NRC Non essential amino acid National Research Council (USA) P PC Positive control Phe Phenylalanine S SDBC Spray dried blood cells SID Standardised ileal digestible / digestibility T Thr Threonine Trp Tryptophan Tyr Tyrosine V Val Valine

6 The regulatory mechanism might be either an increase in the total amount of the BCKDH complex, (and) or an increase in its state of activity. This particular effect of Leu on the activity state of the BCKDH complex has been described in other species (Frick, 1981; Harper, 1984), and finds its origin in the KIC (substrate of BCKDH complex, issued from Leu). Indeed, the KIC specifically regulates, in a dose-dependent manner, the activity of the BCKDH complex (Murakami et al., 2005). In comparison to Leu, Val and Ile have (less) no effect on the regulation of the BCKDH complex activity (Cynober and Harris, 2006; Wiltafsky, 2010). This recent trial demonstrates the possible interactions between BCAAs: since the enzymes are common to the three BCAAs, the dietary intake of individual BCAAs may have an impact on the catabolism of all three. Emphasis is made on the second step in the catabolism, which is irreversible, and therefore determines the BCAA homeostasis. This means that in the case of an excess dietary Leu, the corresponding BCKAs of Val (KIV) and lle (KMV) will also be catabolised, even if they are not present in excess, resulting in a lower availability of Val and Ile for protein synthesis. AT A GLANCE! Branched-chain AA catabolism involves two initial enzymatic steps that are common to all three BCAAs. One of these enzymes (BCKDH) is highly regulated by the catabolite of Leu (KIC). Any increase in the activity of this enzyme increases the catabolism of all three BCAAs and lower their availability for protein synthesis. 1.2 An excess of Leu decreases the performance of piglets receiving a Val or Ile deficient diet In the experiments of Wiltafsky (2010), the effect on the growth of piglets of a dietary Leu excess, in a situation of either Val or Ile deficiency, was investigated as described in Table 1. In both situations, increasing levels of Leu (100 to 200% Leu of the basal diet, treatments 1.2 to 1.5) were achieved by L-Leucine supplementation. Since the BCAAs share a part of their catabolism, it can be hypothesised that the deficiency of one (i.e. Val) could be offset or increased by another (i.e. Ile). To address this issue, additional diets were formulated: on the one hand, the Ile content was doubled in the Val deficient diet (treatment1.6), and on the other hand, the Val content was doubled in the Ile deficient diet (treatment 2.6). Positive controls (PC) where formulated with a balanced BCAA supply (treatments 1.1 and 2.1). Piglets from 8 to 25 kg received the experimental diets. Growth performance (Figure 4), plasma BCAAs and serum BCKAs of Val, Ile and Leu were measured. Results are commented in Table 2. Experiment 1: Effect of Leu excess in piglets fed diets limiting in Val Diets Positive Control (PC) Negative Control (NC) NC + Leu (x1.5) NC + Leu (x1.75) NC + Leu (x2) NC + Leu (x2) + Ile (x2) SID Ile (%) SID Val (%) SID Leu (%) Experiment 2: Effect of Leu excess in piglets fed diets limiting in Ile Diets Positive Control (PC) Negative Control (NC) NC + Leu (x1.5) NC + Leu (x1.75) NC + Leu (x2) NC + Leu (x2) +Val (x2) SID Ile (%) SID Val (%) SID Leu (%) Table 1: Effect of dietary Leu in piglets fed diets either limiting in Val or Ile (Wiltafsky, 2010); experimental design. 6 Information n 35 AJINOMOTO EUROLYSINE s.a.s.

7 Val limiting levels (0.62% SID Val) Ile limiting levels (0.50% SID Ile) Performance of PC: ADFI 786 g/d, and ADG 570 g/d PC NC NC+ Leu (x1.5) NC+ Leu (x1.75) Performance of PC: ADFI 660 g/d, and ADG 478g/d 105 Linear effect (P < 0.05) Linear effect (P < 0.10) NC+ Leu (x2) NC+ Leu (x2) + Ile (x2) PC NC NC+ Leu (x1.5) ADG ( ) and ADFI ( ) in % of the Positive Control (PC) NC+ Leu (x1.75) Figure 4: Effect of SID Leu excess on piglets performance, in diets either Val or Ile first limiting. (Wiltafsky, 2010) NC+ Leu (x2) NC+ Leu (x2) + Val (x2) 1) Effect of Leu excess when Val supply is deficient 2) Effect of Leu excess when Ile supply is deficient Growth performance The Val deficiency decreased drastically ADG and ADFI (-28%, from 0.73% to 0.62% SID Val) Growth performance was then decreased further by Leu excess (-35%) The deficiency of Val was not affected by more Ile (diet 1.6) The Ile deficiency did not have a significant impact on growth performance Growth performances were decreased by Leu excess (-15%) The deficiency of Ile was not significantly affected (P > 0.05) by Val (diet 2.6) Plasma Leu and KIC Increased with dietary Leu excess Plasma Val and KIV No change (low) Decreased with Leu excess (= catabolism of Val) Plasma Ile and KMV Decreased with Leu excess (= catabolism of Ile) No change (low) Table 2: Effect of dietary Leu excess on piglets, comments on growth performance results, and plasma and serum measurements (Wilatfsky, 2010). Due to the greatest decrease in growth performance having been shown in the case of Val deficiency when compared with Ile deficiency, Val appears to be more limiting than Ile for piglets. This confirms the findings of Mavromichalis (1998), Figueroa (2003), and Barea (2009). In addition, the effect of an excess of Leu, and/or a BCAA deficiency, appears to be strongly driven by feed intake regulation, but this has to be interpreted with caution (Focus 2). As previously explained, these findings confirm that any surplus of Leu leads to its catabolism to the corresponding BCKA (KIC), which activates the catabolism of all three BCAAs. Indeed, while Val or Ile were not supplied above their requirements, their catabolism was increased by a Leu excess. However, the addition of Val did not restore performance, which is contradictory to the findings of Fu (2006), where Val addition to a diet with excess Leu gave a recovery in performance which made it equal to the positive control. A Leu excess lowers the availability of Val and Ile for protein synthesis, even if these AAs are already limiting in the diet. This suggests that Leu catabolism, in a situation of Leu excess, might have the priority over protein synthesis. AT A GLANCE! Branched-chain AA metabolism is unique and must be studied carefully since BCAAs (Val, Ile and Leu) interfere with each other. Leucine appears to be a strong regulator of BCAAs catabolism. As a consequence, minimum supply of Val and Ile must be ensured in the diet. The Leu contents have also to be monitored in the determination of Val and Ile requirements. AJINOMOTO EUROLYSINE s.a.s. Information n 35 7

8 Focus 1 The anabolic pathway and its impact on nutrition Body proteins (muscular proteins, functional proteins ) are the main outcome of the anabolism pathway of AA. All proteins are made from the ubiquitous set of 20 AAs. The AA sequence of a protein is dictated by a linear sequence of a continuous triplet of nucleotides (CODON). The absence or poor availability of one AA is enough to slow down or stop the synthesis of protein (Nelson and Cox, 2008). Lysine must be supplied before any other amino acid to support muscle protein deposition in piglets (Mavromichalis, 1998) and is therefore described as the first limiting AA. As Lys is one of the least available AAs in vegetable protein, its correct supplementation is strategic for the growth of piglets. An increase in dietary Lys supply, until its requirement is reached, with a balanced AA profile, will result in an improvement in performance. Once the Lys supply is optimised, the next limiting AA is the one which most limits protein synthesis. This depends mainly on the concentration of AA in feeds. For instance, in a corn based diet, Trp is normally limiting due to the low level of this AA in corn. If Trp is not supplied at an adequate level, protein synthesis slows down. In addition, due to the specific usage of Trp for immune functions (Le Floch, 2008; Trevisi, 2008), the effect of a Trp deficiency might be exacerbated in pigs suffering from poor health. These observations are the fundamentals of the ideal amino acid profile concept where the optimum essential AA supply is described in terms of ratios to Lys. In this concept, any deficiency of one of the EAAs will compromise growth and/or health. Focus 2 Feed intake regulation in the case of BCAA imbalance A drastic decrease in the ADFI of piglets fed a dietary excess of Leu, with either a Val or Ile deficiency, was shown by Wiltafsky (2010). This has also been observed in Val doseresponse trials (Barea, 2009), or under situations of imbalances between BCAAs (Fu et al., 2005). Interpreting the ADFI response is difficult because it cannot be easily determined if the response is due to a nutritional deficiency (i.e. Val), a nutritional excess (i.e Leu), or an imbalance between AAs (i.e. Val and Leu). Results from the literature show that animals are able to recognise when diets have an imbalanced AA pattern, or have a single AA deficiency (Ettle and Roth, 2004). It has been described (Gietzen,1993) that the decline in the concentration of an EAA is detected by brain tissues (prepyriform cortex). The accumulation of the trna for the deficient AA acts as a signal which regulates the nutrient intake (Hao, 2005). Therefore, concerning BCAAs, an excess of Leu may create an additional requirement for Val or Ile, leading to their relative deficiency. Besides, Leu seems to be a nutrient signal that informs the organism about the protein intake. High plasma Leu concentration would signal that sufficient protein had been ingested, resulting in feed intake regulation (Wiltafsky, 2009). Branched-chain AAs are also in the group of the Large Neutral AAs (LNAAs) which compete for the same transporters across the cell membrane, intestinal apical membrane and blood brain barrier (Fu, 2006). It can be assumed that a large excess of one of the BCAAs will result in a reduced uptake of the other BCAAs, as well as other LNAAs, from blood into the brain, and thus influence the feed intake. These hypotheses need further investigation; however, from a commercial point of view, the feed intake response linked to BCAAs may vary for each specific context, and must not be regarded as a performance parameter that can be systematically improved. 8 Information n 35 AJINOMOTO EUROLYSINE s.a.s.

9 2. Branched-Chain Amino Acid Requirements of Piglets Branched-Chain AAs are EAA that have not been as extensively studied as Lys, Thr or Trp. In Table 3, the BCAAs pattern (ratios to Lys) of sow s milk and pigs whole body composition are shown together with published ideal AA profiles. Based on sow s milk composition, relatively high estimates of the BCAAs requirements would be given. Concerning the EAA composition of pigs, Mahan and Shields (1998) reported that there was a wide variation in AA values between studies. Val Ile Leu Composition of sow s milk and pigs whole body BCAAs ratios to Lys (%) Sow s Milk (Boisen, 2003) Pigs whole body (Mahan and Shields, 1998) min/max values in their literature review Proposed ideal AA pattern SID BCAAs ratios to Lys (%) Wang and Fuller (1989) Chung and Baker (1992) Cole and van Lunen (1994) Sève (1994) Boisen (2000) Average Coefficient of variation 4% 7% 6% Table 3: Comparison of published ideal amino acid profiles to sow s milk and pigs whole body composition. The different published ideal AA profiles (in SID values) highlight that, while there is a consensus concerning the SID Val:Lys requirement at 70%, the SID Ile:Lys findings are more variable. Recent findings concerning Ile claim either that the requirement is lower (Barea, 2009; Wiltafsky, 2009), or considerably higher (Kerr, 2004; Dean, 2005; Fu, 2006), than the average. In animal nutrition, the term requirement has no precise definition, and for growing animals it is usually associated with the maximization of growth. Dose-response trials are frequently used to determine AAs requirements. The requirements of the three BCAAs have therefore been reviewed through the compilation of dose-response trials that aimed to maximize the growth performance of piglets. 2.1 Valine requirement is confirmed to be at least 70% SID Val:Lys Until the release of feed grade L-Valine in 2009, Val was the next limiting AA in piglets feed. Before the availability of L-Valine, constraints on dietary CP led to formulations with either an imbalanced AA profile - where Val was deficient - or with low Lys dietary concentrations to keep a balanced AA profile. In both cases, growth performance was lower than piglets potential. Current feeds using L-Valine increase their value by meeting Val requirements, and thereby allowing complete utilisation of the other nutrients. Consequently Lys levels can be increased to levels that allow piglets to express their full potential for growth. The Val requirements of piglets have been the subject of an increasing number of studies and have been the topic of recent reviews (Barea, 2009; Corrent, 2009). Since new data about Val:Lys requirements are available, the objective of this section is to discuss these new trials and to synthesize them through a meta-analytical method (Sauvant, 2008). AJINOMOTO EUROLYSINE s.a.s. Information n 35 9

10 > New dose-response trials In addition to those presented in Bulletin #33, three new dose-responses to SID Val:Lys in piglets (8.5 to 25.0 kg LW) have been carried out in different research institutes, in collaboration with AJINOMOTO EUROLYSINE S.A.S. (protocols and results are presented in Table 4). Figure 5 presents the ADG, ADFI and FCR reported in these trials, together with the dose-response trials already selected in Bulletin #33. Gloaguen (2010) performed an experiment in the INRA facilities in France to study the Val requirements of piglets with diets containing an excessive supply of SID Leu:Lys (165%) through the use of corn gluten meal. The Lys level was sublimiting (1.00 % SID Lys), and five SID Val:Lys levels varying from 60 to 80% were tested. The diet was based on corn and soybean meal (Table 12, final flap: detailed formulas). It must be noticed that SID Ile:Lys was 63% avoiding any interactions between Leu excess and Ile deficiency. A significant effect of Val was observed on ADFI, ADG and FCR. On average, for the three response criteria, the curvilinear-plateau model estimated that 72% SID Val:Lys was required to reach the plateau. No effect of Leu was demonstrated on the estimate of the Val:Lys requirement (Focus 3). Gloaguen (2010) Vinyeta (2010) Millet (2010) INRA Schothorst Feed Research ILVO Country France Netherlands Belgium Weight range (kg) SID Lys (%) (90% of CVB 2008) 1.06 AID Lys (%) (90% of CVB 2008) 1.02 Net Energy (MJ/kg) SID Lys (g/mj of NE) Tested valine levels (n) SID Val:Lys ratio range (%) Breed Pietrain x (LW x LR) Topigs Tempo x (GYorkshire x FLR) Pietrain x rattlerow seghers Sex Barrows + gilts Barrows + gilts Barrows + gilts Replicates per treatments (n) Number of animals per treatments (n) Number of animals in total (n) Age at weaning (days) Age at start of the exp. (days) Age at end of the exp. (days) Duration of the experiment (days) Weight at weaning (kg) Weight at the start of exp. (kg) Final weight (kg) ADFI at 70% SID Val:Lys (g/d) ADG at 70% SID Val:Lys (g/d) FCR at 70% SID Val:Lys (g/d) SID Val:Lys requirements ADG (average = 72%) 72% 74% 69% ADFI (average = 72%) 73% 74% 70% FCR (average = 71%) 71% - - Table 4: Main Characteristics and results of the Valine new dose-response trials. 10 Information n 35 AJINOMOTO EUROLYSINE s.a.s.

11 A preliminary experiment was run in the Schothorst Feed Research facilities in Netherlands, by Vinyeta (2010) to establish a Val:Lys deficient diet. On the basis of this diet, five SID Val:Lys ratios were tested from 61 to 80 % in Val dose-response study. The SID Lys level was set at 90% of the CVB (2008) recommendations in order to express the requirement as a ratio to Lys, as defined by Boisen (2003). In this trial, the growth performance was very high (562 g/day ADG when 70% Val:Lys SID). The requirement was determined in both units, AID and SID, using the linear-plateau and the curvilinear-plateau models, with an overall minimum of 70% SID Val:Lys (68% AID Val:Lys). The FCR was linearly (p<0.001) improved during the first phase of the trial (from 8.3 to 9.7 kg LW). A dose response study was held at the ILVO Institute in Belgium by Millet (2010) to determine the piglet (8 to 25 kg LW) response to increases in the Val:Lys ratio. A basal diet (cereals and soybean meal) was formulated with 1.06 % SID Lys (considered as slightly limiting). As described in Table 4, five Val:Lys ratios were tested. The requirements were found to range from 68 to 71% SID Val:Lys, depending on the analytical model used. Higher requirements were determined in the first two weeks after weaning (from 74 to 83%). The Val requirement determined with the curvilinear-plateau model in each trials confirms the data already published with an average value of 72 % SID Val:Lys. g/day 600 g/day SID Val:Lys ratio (%) Figure 5.1: SID Val:Lys dose-responses Effect on Average Daily Gain (g/d) SID Val:Lys ratio (%) Figure 5.2: SID Val:Lys dose-responses Effect on Average Daily Feed Intake (g/d) g/g Mavromichalis (2001) - Exp. 5 Barea (2009)a - Exp. 4 Barea (2009)b - Exp. 4 Dusel (2008) - Trautwein (2010) Paulicks (2008) Torrallardona (2008) Vinyeta (2010) Millet (2010) Gloaguen (2010) SID Val:Lys ratio (%) Figure 5.3: SID Val:Lys dose-responses Effect on Feed Conversion Ratio (g/g) AJINOMOTO EUROLYSINE s.a.s. Information n 35 11

12 > The meta-analysis confirms that SID Val:Lys requirement for piglets is at least 70% Trials must be repeated and compared to make a decision on a recommendation of a minimum ratio that optimizes performance. The meta-analysis method described by Sauvant (2008) takes into account the between-trial variability. A meta-analysis study was therefore performed using a curvilinear-plateau model to determine the SID Val:Lys ratio which maximizes ADG and minimizes FCR. It is shown that Leu excess exacerbates the effect of a Val deficiency (Focus 3), thus trials where Leu was in excess were not used to avoid overestimating the response and the optimum Val:Lys ratio (Barea, 2009 and Gloaguen, 2010). The meta-analysis compiles 7 of the 9 trials presented in Figure 5. In the selected trials, Val was the first limiting amino acid in the diet, followed by Lys as the second limiting factor and this allowed the Val requirement to be expressed as a ratio to Lys. Results of the meta-analysis (Figure 6), indicate that 77% SID Val:Lys is necessary to achieve the plateau value for ADG, and 73% SID Val:Lys for FCR. It was calculated from the response curve that a balanced diet using 70% SID Val:Lys in comparison to a deficient one (i.e. 60% SID Val:Lys), leads to an average increase of 15% in ADG in piglets from 12 to 25 kg (no other AAs being limiting), and improves FCR by 7%. This confirms the findings of Bulletin #33. This approach clearly confirms that 70% SID Val:Lys must be considered as a minimum specification for piglets diets. ADG in % of the best performance SID Val:Lys (%) FCR in % of the best performance SID Val:Lys (%) Mavromichalis (2001) - Exp. 5 Dusel (2008) Torrallardona (2008) Millet (2010) Barea (2009)a - Exp. 4 Paulicks (2008) Vinyeta (2010) Average Response (meta-analysis) Figure 6.1: ADG response curve to dietary SID Val:Lys, meta-analysis based on 7 trials. Figure 6.2: FCR response curve to dietary SID Val:Lys, meta-analysis based on 7 trials. The magnitude of the effect of Val on feed intake (Focus 2) was not consistent in all trials and it was not possible to perform the meta-analysis on this parameter. The FCR response also varied between trials. For instance, in Vinyeta (2010) and Millet (2010), the FCR was improved during the first weeks of the experiment, but not during the whole period. However, the FCR of piglets fed basal diets in these trials was already very good (below 1.60 g/g), and may have been difficult to improve. In Section II, commercial trials are presented and they confirm the general improvement of feed efficiency with L-Valine supplementation. AT A GLANCE! The growth response of piglets to Val supply is confirmed by three new dose-response trials, and a minimum requirement of 70% SID Val:Lys is necessary to optimize piglet feeds. The meta-analysis of the dataset depicts a strong response to Val dietary supply through L-Valine supplementation. The minimum of 70% SID Val:Lys should be respected in commercial piglet diets since Val is likely to be the most limiting AA in these diets. 12 Information n 35 AJINOMOTO EUROLYSINE s.a.s.

13 Focus 3 Leucine interferes with the response to Val supply, but probably not with the Val requirement Due to the recognized interactions between BCAAs, it is important to consider if Leu interferes with Val in terms of the estimate of requirement and responses to Val. The comparison of Val dose-response trials of Barea (2009a) and Gloaguen (2010) is relevant since these trials were performed in the same facilities and conditions. However, Leu and Ile supply were higher (165% SID Leu:Val, 63% SID Ile:Lys) in Gloaguen (2010). Average daily gain and FCR are given in Figures 5.1 and 5.3. The SID Val:Lys requirements were the same (>70%). However, the comparison of performance at a deficient level of 60% SID Val:Lys reveals that, on average, performance was 13% lower when Leu was supplied in excess compared with when a balanced Leu supply was provided. To validate these findings, Gloaguen (2010) ran an additional trial where four diets (Table 5) were formulated on the basis of a factorial design (Val x Leu), and fed to (n= 4 x 16) piglets individually caged (12 to 25 kg LW). Val and Leu effects were observed, confirming the strong benefits of, at least, 70% SID Val:Lys, and showing that dietary Leu excesses are detrimental to growth performance. Although the interaction was not significant, maybe due to the low number of animals involved, it is clear that the effect of a surplus of Leu was greater when Val was deficient (-26% vs. -10% for ADG, -11% vs. -3% for ADFI, and +19% vs. +7% for FCR). Diet 1 Diet 2 Diet 3 Diet 4 SID Val:Lys % P value SID Leu:Lys % ValxLeu ADFI (g/d) Leu effect, for the same Val level -11% -3% ADG (g/d) Leu effect, for the same Val level -26% -10% FCR (g/g) Leu effect, for the same Val level +19% +7% ns ns ns Data split to Val or Leu contents SID Val:Lys % SID Leu:Lys % P value Val Leu ADFI (g/d) Val or Leu effect +21% -7% ADG (g/d) Val or Leu effect +56% -16% FCR (g/g) Val or Leu effect -23% +14% Table 5: Effect of Val and Leu on piglet performance (Gloaguen, 2010). < <0.01 <0.01 <0.01 <0.01 The antagonism between BCAAs, explained in Section I, have consequences on growth performance. This must be taken into account in feed formulation. In commercial diets, among the three BCAAs, Val is first limiting. Therefore, minimum requirement must be set (70% SID Val:Lys), to avoid any detrimental effects of the antagonism between BCAAs. AJINOMOTO EUROLYSINE s.a.s. Information n 35 13

14 2.2 Isoleucine requirement is 53% SID Ile:Lys A literature review of trials on the Ile requirement of pigs (piglets, grower and finisher pigs) indicates that about 40 datasets have been published and studied since 1957 (Barea, 2009); this is much more than Val (about 12 data sets). Most of these trials have been carried out in North America (85%) where a particular problem relating to Ile appeared. The use of blood products mainly Spray Dried Blood Cells (SDBC) - in pig s formulations was associated with deterioration in performance as soon as the inclusion levels of these raw materials increased (Frugé, 2009). His:CP Val:CP Lys:CP Ile:CP His:CP Spray dried blood cells Blood meal Soybean meal Leu:CP Val:CP Lys:CP Leu:CP Ile:CP Figure 7: Amino acid profile of blood products vs Soyabean Meal, in % of crude protein. Sauvant (2004); Kerr (1999); Bulletin#32 (2008). In comparison to soyabean meal s AA profile, blood meal and SDBC s are very deficient in Ile but extremely rich in Leu, Val and His (Figure 7). Unsurprisingly, Ile was limiting in these particular diets using SDBC and the question of the Ile requirement therefore arose. For dose-response studies, SDBC were used to provide diets deficient in Ile, but with the other AAs in excess, there was a danger of responses to Ile being confounded with the effects of imbalances of the other AA as discussed in Section I. In particular, the detrimental effect of an oversupply of Leu on performance has to be recognised (Wiltafsky, 2010). > Isoleucine requirement is influenced by specific feedstuffs Concerning Ile requirements, the use of blood products in the dose-response trials must be addressed. To illustrate this issue, the response to dietary Ile level of pigs (from 7 to 115 kg LW) fed a diet with SDBC or without is presented in Figure 8. Because trials were performed with various pig types (sex, age and body weight), the tested Ile levels could not be compared directly. Levels were therefore expressed as a percentage of the dietary Lys level in each experiment (ratios express on SID basis after calculation using the INRA database of literature J. van Milgen, Pers. Comm.). Since no selection of trials was done with regards to Lys (sub-limiting or not), all the ratios must be assumed as minimum requirements. In every trial, increasing the dietary Ile level resulted in increased performance (feed intake, growth rate and feed efficiency), up to a plateau or summit. Figure 8 shows that the optimum Ile level depends on whether or not the animals were fed a diet with SDBC: In the experiments using a blood-free diet (green dots), the best treatment was, in most cases, the first or second tested level. In these diets, the requirement was not higher than 55% SID Ile:Lys and probably between 45% and 55% (green area in Figure 8). In the experiments using a diet with blood products (red dots), the best treatment was never the basal diet. When SDBC were used, the requirement was not less than 58% SID Ile:Lys and probably between 58% and 68% (red area in Figure 8). This difference is probably due to interactions between BCAAs. The study of the Ile requirement of piglets must take into account this factor. If piglets (from 5 to 25 kg) are focused on, seven researchers recently studied the Ile requirement through dose-responses. In Table 6, the information relative to these studies and the published results are presented. As already discussed in Bulletin #33, the method of expressing the requirement depends on the protocol which is implemented. Therefore, variability is quite important with regards to the unit (AID vs. SID) and the expression of the results (% of the feed or ratio to Lys). A comparison of published results with the NRC (1998) recommendations indicates considerable variation: from 73 to 148 % of NRC Ile requirement. Lastly, the trials were mostly associated with the use of blood products and consequently only a few trials are based on blood-free diets. 14 Information n 35 AJINOMOTO EUROLYSINE s.a.s.

15 Trial number SID Ile:Lys ratio (%) Figure 8: Tested (white balls) and best (coloured balls) SID Ile:Lys ratios. The red balls indicate the presence of SDBC in the diets; the green balls indicate diets without blood products. The red and green areas show the optimal SID Ile:Lys ranges when SDBC are used or not, respectively. Live Weight Estimated Ile requirement (in the published unit) % NRC Use of blood by products Lys SID % 1 Doubts on the first limiting AA 2 Is it possible to express the results as Ile:Lys ratio? Bergstrom (1997)a 5 to 8 kg 0.69% AID 113 2% Blood meal 1.19 Thr no Bergstrom (1997)a 5 to 8 kg 0.90% AID 148 2% Blood meal 1.50 Thr no Bergstrom (1997)b 10 to 20 kg 0.38% AID 73 2% Blood meal 0.75 Thr no Bergstrom (1997)b 10 to 20 kg 0.55% AID 106 2% Blood meal 1.10 Thr no Lenis (1997) 18 to 40 kg 0.54% AID % SDBC 1.04 Thr no James (2000)a 6 to 25 kg 55% Ile:Lys AID % SDBC YES James (2000)b 6 to 25 kg 0.63% Ile AID % SDBC no Kerr (2004)a 7 to 11 kg 0.68% Ile AID % SDBC no Kerr (2004)b 7 to 11 kg 61% Ile:Lys AID % SDBC YES Fu (2006)a 11 to 22 kg 0.60% Ile SID Trp no Fu (2006)b 12 to 22 kg 0.51% Ile SID Trp no Fu (2006)c 12 to 22 kg 68% Ile:Lys SID % SDBC YES Wiltafksy (2009)a 8 to 25 kg 54% Ile:Lys SID YES Wiltafksy (2009)b 8 to 25 kg 59% Ile:Lys SID % SDBC YES Htoo (2009) 10 to 20 kg 51% Ile:Lys SID YES AID : Apparent Ileal digestibility; SID: Standardized Ileal Digestibility SDBC: Spray Dried Blood Cells 1: In bold, Lys is not sublimiting for the considered live weight category 2: The named AA is likely limiting before Ile in the basal diet. Table 6: Characteristics, results and selection of the dose-response studies on the Ile requirement in piglets. Adapted from Barea (2009). Based on a study of the dietary AA profile and dietary Lys content (Table 6 and Table 12), only two trials in which no blood products were used were selected and four trials where blood products were used. Due to the limited information available, additional trials which were not dose-responses were also studied in order to confirm the general trend (Paulicks et al.,2008; Wiltafsky,2010; Barea,2009; Jansman,2008; Lordelo, 2008; and Nørgaard and Fernandez, 2009). Growth performance in the selected trials are presented as a function of the use (or not) of SDBC in Figure 9. AJINOMOTO EUROLYSINE s.a.s. Information n 35 15

16 ADG (g/d) 600 Without SDBC ADG (g/d) 600 With SDBC SID Ile:Lys ratio (%) SID Ile:Lys ratio (%) Figure 9.1: Response to SID Ile:Lys increase Effect on Average Daily Gain (g/d). ADFI (g/d) 1200 Without SDBC ADFI (g/d) 1200 With SDBC SID Ile:Lys ratio (%) SID Ile:Lys ratio (%) Figure 9.2: Response to SID Ile:Lys increase Effect on Average Daily Feed Intake (g/d). FCR (g/g) 2.0 Without SDBC FCR (g/g) 4.0 With SDBC SID Ile:Lys ratio (%) Figure 9.3: Response to SID Ile:Lys increase Effect on Feed Conversion Ratio (g/g) SID Ile:Lys ratio (%) Wiltafsky (2009) Exp3. Jansman (2008) Barea (2009) Barea (2009) Exp1. Barea (2009) Exp2. Barea (2009) Exp3. Htoo (2009) Lordelo (2008) Noorgard and Fernandez (2009) Barea (2009) Exp1. Barea (2009) Exp2. Wiltafsky (2010) Exp1. James (2000) Paulicks (2008) Wiltafsky (2009) Exp1. Barea (2009) Exp3. Paulicks (2008) Kerr (2004) Fu (2006) 16 Information n 35 AJINOMOTO EUROLYSINE s.a.s.

17 > Isoleucine requirement in blood-free diets : 53% SID Ile:Lys As an overall comment, for the two selected dose-response trials (Htoo, 2009 and Wiltafsky, 2009), the maximum performance (ADFI, ADG and FCR) was reached at levels of SID Ile:Lys between 50 and 55%. In these trials, SID Ile:Lys requirements were found to be 51% (Htoo, 2009) and 54% (Wiltafsky, 2009), as an overall estimate. To compare these dose-responses to those found in the two-level trials, ADG and FCR were expressed as a percentage of the best performance (Figure 10). An average response curve was determined for ADG on the basis of the two dose-response trials, with a maximum ADG reached at 55% SID Ile:Lys. However, the two-level trials did not indicate an improvement of ADG above 50% SID Ile:Lys. It was not possible to determine an average curve for FCR since responses were flat. There was a response only in Wiltafsky (2010), and no further improvement in FCR was achieved above a value of 53% SID Ile:Lys. ADG (% of the best performance) SID Ile:Lys ratio (%) FCR (%) SID Ile:Lys ratio (%) Wiltafsky (2009) Exp3. Barea (2009) Barea (2009) Exp2. Htoo (2009) Noorgard and Fernandez (2009) Barea (2009) Exp2. Jansman (2008) Barea (2009) Exp1. Barea (2009) Exp3. Lordelo (2008) Barea (2009) Exp1. Wiltafsky (2010) Exp1. Figure 10: SID Ile:Lys dose-responses, effect on Average Daily Gain anf Feed Conversion Ratio (% of the best performance). These estimates are much lower than the levels recommended in most of the AA profiles presented in Table 3 page 9 (57% SID Ile:Lys on average). Mavromichalis (1998) suggested that the Ile requirement was probably lower than 60% SID Ile:Lys. More recently, Barea (2009) suggested that the SID Ile:Lys requirement may be even lower than 50%. The previous recommendation of AJINOMOTO EUROLYSINE S.A.S. was 55% SID Ile:Lys. In the light of this review, this recommendation has been revised to 53% SID Ile:Lys for piglets on blood-free diets. This information is an important consideration in designing efficient feeds and determines to what extent dietary CP can be reduced without a negative effect on performance. AJINOMOTO EUROLYSINE s.a.s. Information n 35 17

18 > The Isoleucine requirement is above 60% SID Ile:Lys in diets using blood products The requirement determined in each selected Ile dose-response has been reported in Table 7. On average, the Ile requirement determined in dose-response trials where blood products were used are higher (61% SID Ile:Lys) than in the trials using blood-free diets (53% SID Ile:Lys), and confirms the outcome of Figure 8 presented previously. Nevertheless, a more restricted study of the dose-response with high SDBC contents (Fu, 2006; Wiltafsky, 2009) shows that there is a linear trend for ADFI and ADG and consequently the Ile requirement might be higher than the highest Ile level tested. The inconsistency in the results makes difficult to clearly conclude on a recommendation for Ile specifications in diets using blood products. Amount of blood product SID Ile:Lys requirement Average SID Ile:Lys requirement Htoo, % 51% Wiltafsky, % 54% James, % 55% Kerr, % 61% Wiltafsky, % 59% Fu, % > 68% 53% 61% Table 7: SID Ile:Lys (%) requirements estimated in the selected dose-response trials. The Wiltafsky (2009) team is one of those that has studied, in the same facility, the Ile dose-response according to the use of SDBC or not, but is the only one that get a response to Ile in both cases. A comparison of these trials is therefore the most informative. The team used piglets from 8 to 25 kg with the same Lys (1.00 % SID) and Net Energy (10.0 MJ/kg) levels. The performance results were already presented in Figure 9. But, in order to evaluate the difference in the efficiency with which Ile was used in these different contexts, the ingested Ile (%SID Ile x ADFI g/d) has been plotted against the corresponding ADG (g/d) in Figure 11. This allows the optimum amount of SID Ile ingested per gram of ADG to be calculated. ADG (g/d) mg SID Ile / g ADG 9.7 mg SID Ile / g ADG Ingested Ile (g/d) Without SDBC With SDBC Figure 11: ADG (g/d) and ingested Ile (g/d) with and without SDBC. Adapted from Wiltafsky (2009). 18 Information n 35 AJINOMOTO EUROLYSINE s.a.s.

19 8.1 mg SID Ile /g ADG were needed for piglets fed diets free of SDBC to maximize their ADG. The corresponding value was 9.7 mg/g ADG when the diet used 7.5% SDBC. Thus, piglets fed SDBC-containing diets needed to ingest 20% more SID Ile to reach the same ADG as those on diets free of SDBC. On the basis of the minimum requirement determined for blood-free diets (SID 53% Ile:Lys), and from our calculations based on the Wiltafsky (2009) dataset (+20%), it is suggested that a minimum specification of 63% Ile:Lys SID is required to optimize piglet feeds that contain blood products. This recommendation is very dependent on the type and quantity of feedstuffs used in the diet. AT A GLANCE! The response of piglets to Ile supply and the estimate of the Ile requirement are influenced by the presence of specific feedstuffs, such as blood products, particularly rich in Leu. The published requirements must be studied as a function of this factor. In blood-free diets, 53% SID Ile:Lys is recommended to optimize piglet feeds, while a minimum of 63% SID Ile:Lys is proposed in diets using blood products, especially blood meal and spray-dried blood cells. AJINOMOTO EUROLYSINE s.a.s. Information n 35 19

20 2.3 Leucine requirement: 100% SID Leu:Lys to avoid excess Recommendations given for the Leu requirement in piglets are very consistent largely because very few studies on the requirement for this AA have been done in piglets. In most of cases, the recommendations are based on the work of Chung and Baker (1992), and Wang and Fuller (1989) who determined minimum SID Leu:Lys of 100 and 110% respectively. Other teams (Oestemer,1973; Mitchell, 1968) studied Leu responses but no requirement could be concluded because the basal diets were not deficient in Leu. More recently, Augspurger and Baker (2004), studied in 3 trials the requirement of Leu in young pigs (10 to 20kg). The first experiment validated a basal deficient diet (Table 12) and this was followed by two dose-response trials. The study of the formula and AA profiles indicates that Trp and Val (slightly under their requirement, Table 12) were possible limiting factors in these dose-responses. Therefore, Lys (1.12% SID) was likely not the second limiting factor in these diets but even so, in this bulletin, the results (Figure 12) were expressed in ratio to Lys to compare to previous estimates of the Leu requirements. ADG (g/d) FCR (g/g) SID Leu:Lys (%) SID Leu:Lys (%) Figure 12: SID Leu:Lys (%) dose-responses Effect on ADG (g/d) and FCR (g/g) Augspurger and Baker (2004). The first dose-response trial resulted in a linear increase in performance and no requirement could therefore be determined. The second dose-response trial used larger increases between Leu levels to achieve a plateau in response. In the second experiment the use of a curvilinear model (AEL s calculations, in red) allowed a requirement of 96% SID Leu:Lys to be determined. This result expressed in ratio to Lys, is a minimum since Lys was likely not the second limiting factor in the diet. Even if additional dose-response trials might be performed, 100% SID Leu:Lys remains a good estimate of the Leu requirement of piglets. AT A GLANCE! Only a few dose-response trials on Leu have been published and 100% SID Leu:Lys appears to be a safe estimate of the requirement in pigs. However, the study of BCAA catabolism clearly indicates that Leu excesses are detrimental to growth performance at least in situations where Val or Ile are deficient. Formulators should therefore ensure minimum supply of dietary Val since it is likely the first limiting AA among the BCAA. 20 Information n 35 AJINOMOTO EUROLYSINE s.a.s.

21 3. Branched-Chain Amino Acid Nutrition of Piglets in Practice This review on BCAA nutrition allows minimum requirements for Val, Ile and Leu to be determined that ensure optimal growth of piglets. Particular interactions between BCAAs have been underlined and Leu appears to be the factor with the greatest influence. From this scientific background, the integration of these new data into the ideal AA profile concept will now be focused on. Then, an overview of the BCAA content of current feeds for piglets will allow the most limiting BCAAs to be identified. A synthesis of commercial trials that have tested L-Valine responses will then be presented and the potential to reduce dietary CP will finally be discussed. 3.1 Adjusting Lys levels within the ideal amino acid profile concept In growing animals, ideal protein is a concept in which the AA pattern (defined as g of AA / 100 g Lys) that maximizes nitrogen retention is defined. In this profile, all AAs are considered to be equally limiting on performance, just covering the requirements for all physiological functions. Lysine has traditionally been used as the reference because it is the first limiting AA for growth in piglets. Although the concept of ideal protein was developed in the late 1950s and early 1960s, one of the first reference studies in pigs was carried out by Wang and Fuller (1989). It is normally assumed that the ideal protein profile does not change for a given growth phase. In practical nutrition, this offers the advantage that while the Lys requirement will vary (per kg of feed or per MJ of energy), the ideal AA profile expressed relative to Lys will remain the same. If formulating a diet using the concept of ideal AA profile suggests that essential AAs must be supplied at least at their required ratio to make full use of the dietary Lys, it also implies that excesses must be avoided as well as imbalances within a category of AA (i.e. BCAAs). In addition, the lack of any one of the AAs in the profile will impact the efficiency with which the whole AA profile is used. > Relevance of the ideal AA profile concept In a recent trial, Jansman (2010) studied the interactions between Trp and Val. Tryptophan is a limiting AA in European diets and must be supplied through the feed to cover the requirement of piglets. This requirement has recently been validated by a meta-analysis study of 37 trials (Simongiovanni, 2010) showing that 22% SID Trp:Lys is needed to maximize growth performance. In this bulletin, the requirement of Val has been reviewed and a minimum of 70% SID Val:Lys has been recommended. The aim of the study was to evaluate the impact of the fulfilment of the Trp and Val requirements in a low CP diet. Two levels of dietary Trp (17% and 22% SID Trp:Lys) were combined with two levels of Val (64 and 70% SID Val:Lys) in a factorial design along with a positive control treatment. Diets, based on cereals and soybean meal, were formulated with 1.06% SID Lys, and 9.7 MJ/kg Net Energy. L-Tryptophan and L-Valine were used to increase the Trp and Val levels. The positive control had the same nutritional characteristics except that it had a higher CP content (19.5% vs. 17.7%). The experimental feeds were fed to 320 castrated piglets (8 24 kg LW, SPF status) split into 8 replicates per treatment. The weight gain and feed intake were measured after 2 and 4 weeks. Results of the first period (34 to 48 days of age), where the most significant effects appeared, are presented in Figure 13. AJINOMOTO EUROLYSINE s.a.s. Information n 35 21

22 ADG (g/d) ADG (g/d) FCR (g/g) FCR (g/g) c 427c bc 1.39bc 1.40bc ab 392ab 396abc a a % CP 22% SID Trp:Lys 70% SID Val:Lys 17.7% CP 17% SID Trp:Lys 64% SID Val:Lys 17.7% CP 17% SID Trp:Lys 70% SID Val:Lys 17.7% CP 22% SID Trp:Lys 64% SID Val:Lys 17.7% CP 22% SID Trp:Lys 70% SID Val:Lys 1.26 Figure 13: Effect of L-Tryptophan and/or L-Valine supplementation to a low crude protein (CP) diet (piglets, from 8 to 14 kg) Jansman, Figures associated with different letters are significantly different (p<0.05). If both Trp and Val are below their requirement levels in a low CP diet, performance decreases in comparison with a high CP diet where Trp and Val are not limiting (positive control), Both L-Tryptohan and L-Valine must be added to the Low CP diet to get the same, or even better, performance than with the positive control. This trial confirms that the requirements determined for Trp and Val, as well as the methods used to achieve these levels, are relevant to the ideal AA profile concept. AT A GLANCE! The relevance of the ideal AA profile concept has again been demonstrated. Low dietary CP diets must therefore ensure that the minimum requirements of both Trp and Val are met through supplementation of the feeds by L-Tryptophan and L-Valine to take maximum advantage of the nutritive value of the diets. This concept is increasingly important in the context of close control of the dietary CP level. 22 Information n 35 AJINOMOTO EUROLYSINE s.a.s.

23 > Adapting Lys levels according to the targeted performance Since the ideal AA profile is well established, if the AA levels in feed follow those described in the ideal protein, the only limiting factor with regards to AA nutrition of the animal is the ingestion of Lys. Lys intake is dependent on feed intake of (different) animals raised in (different) local contexts, with different energy levels. A global recommendation for Lys is therefore inappropriate and levels must be adapted to each local objective. However, to overcome the variability due to feed intake, it is possible to express the ADG response as a function of the ingested Lys measured in independent Lys dose-response trials. A review of recent Lys trials (after the year 2000) is presented in Figure 14. The best ADG and the corresponding amounts of ingested Lys were highlighted with the red circles on the graph. By using a linear model to depict this dataset, it has been estimated that 19 mg SID Lys/g of ADG is needed, which was recently confirmed by the experiments of Schneider (2010). ADG (g/day) 700 Kendall (2008) Exp kg y = 50.42x R 2 = 0.97 Kendall (2008) Exp kg Kendall (2008) Exp kg Kendall (2008) Exp kg Kendall (2008) Exp kg Garcia (2008) 6-15 kg Torrallardona (2006) 7-14 kg Barea (2009) kg Warnants (2001) 8-25 kg Beltranena and Patience (2000) 5-20 kg Gaudré (2007) kg Barea (2009)d kg Barea. (2009)d kg Schneider (2010) kg Schneider (2010) kg Best ADG = f(ingested Lys) lngested Lys (SID g/day) Figure 14: Effect of ingested Lys (SID g/day) on ADG of piglets; a compilation of trials from 2000 to It is possible to estimate a range of the minimum SID Lys levels needed by modern genotypes. For piglets growing between 12 and 25 kg LW, with an ADG of 510 g/d (plateau value of the meta-analysis performed on Val, Section II), the SID Lys requirement is: 19 mg SID Lys /g x 510 g/d = 9.7 g SID Lys /d Assuming an average ADFI of 810 g/d, a minimum dietary content of 1.20% SID Lys is required to maximize the growth of piglets between 12 and 25 kg LW. The value of 1.20% SID Lys is the same as suggested by Warnants (2001), and in line with the results of Gaudré (2007) who found an increase of performance above 1.2 g SID Lys/MJ NE. This level of Lys remains indicative and depends mainly on the level of feed intake. Nevertheless, it gives an idea of the dietary SID Lys level required by current genotypes. AT A GLANCE! The SID Lys requirement in the feed of modern genotypes is dependent on the level of feed intake but should provide at least 19 mg SID Lys / g of targeted ADG. The Lys supply is efficiently utilised only if it is accompanied by a balanced AA profile, avoiding deficiencies and excesses. AJINOMOTO EUROLYSINE s.a.s. Information n 35 23

24 3.2 Valine is the most limiting AA in current European piglet feeds Two surveys were conducted in 2006 and 2008 by AJINOMOTO EUROLYSINE S.A.S. on commercial piglet pre-starter and starter feeds. The 467 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 (methods are described in Bulletin #32). The survey performed in 2006 has been presented in Bulletin #33. The following analysis collates the two surveys. In Figure 15, total BCAA content (ratio to Lys) were plotted against total Lys content. The requirement for each AA, in blood-free based diets, is represented by the green line (73% Total Val:Lys; 54% Total Ile:Lys; and 101% Total Leu:Lys) : 81% of the diets were below the Val requirement, 78% of the diets were above the Ile requirement, 80% of the diets were above the Leu requirement. Val:Lys Total % % of the samples < 70 % SID Val:Lys Total Lys content (%) Figure 15.1: Total Val:Lys dietary content in piglet diets (Prestarter and Starter). Source: AEL surveys 2006 & Ile:Lys Total % % of the samples > 53 % SID ILe:Lys Total Lys content (%) Figure 15.2: Total Ile:Lys dietary content in piglet diets (Prestarter and Starter). Source: AEL surveys 2006 & Information n 35 AJINOMOTO EUROLYSINE s.a.s.

25 Leu:Lys Total % % of the samples > 100 % SID Leu:Lys Total Lys content (%) Figure 15.3: Total Leu:Lys dietary content in piglet diets (Prestarter and Starter). Source: AEL surveys 2006 & 2008 Prestarter Feeds Val-deficient diets Starter Feeds Occurrence 88% 73% Total Val:Lys 65% 67% Total Ile:Lys 56% 57% Total Leu:Lys 106% 107% Equivalent SID Val:Lys 1 61% (-13%) 2 64% (-9%) 2 Equivalent SID Ile:Lys 1 53% (0%) 2 54% (2%) 2 Equivalent SID Leu:Lys 1 101% (1%) 2 102% (2%) 2 1) Estimation SID/Total = 95% 2) Difference to the requirement in SID Table 8: AEL Surveys 2006 and 2008 : Average Val, Ile and Leu contents relative to Lys, in the Val-deficient diets (Prestarter and Starter diets). In Table 8, characteristics of the Val-deficient feeds: In Prestarter diets, 88% of the samples were deficient in Val, and 73% in starter diets. In deficient Prestarter diets, the Val content was, on average, 13% below requirement. In deficient Starter diets, the Val content was, on average, 9% below requirement. This deficiency was not related to the Lys level. In the Val-deficient feeds, Ile and Leu requirements were covered, on average. Considering the average analyzed levels of Val in the deficient prestarter and starter diets, it has been calculated that raising the level to 70% SID Val:Lys through L-Valine supplementation and no other EAA being limiting, ADG could be increased by 10% and FCR by 5%. AT A GLANCE! In more than 80% of the analysed piglet feeds, Val was the most limiting AA (61% SID Val:Lys in prestarter and 64% in starter diets). L-Valine supplementation to correct the Val deficiency to the requirement level (70% SID Val:Lys) would improve ADG by 10% and FCR by 5%. In most of the cases, a further reduction of dietary CP could have been possible since Ile and Leu were not deficient. AJINOMOTO EUROLYSINE s.a.s. Information n 35 25

26 3.3 70% SID Val:Lys improved ADG and FCR in commercial trials L-Valine supplementation has been tested under commercial conditions. In each trial (Table 9), a commercial basal diet was supplemented with the corresponding amount of L-Valine necessary to achieve higher Val ratios to Lys. Performance of basal treatments are reported together with the improvement achieved thanks to the L-Valine supplementation in Table 9. In Figure 18, ADG results have been compared with the average response curve to Val reported in section II by meta-analysis. Weight of piglets (kg) duration (d) SID Val:Lys tested levels (%) Effect on ADG Effect on ADFI Effect on FCR Effect on final body weight Save of feed to reach the same body weight Basal ADG (g/d) Basal ADFI (g/d) Basal FCR (g/d) 9 to % + 4% -3.5% kg -0.8 kg Spain to % 0% -9.8% kg -2.6 kg to % + 2% -7.0% kg -1.8 kg Belgium to % + 5% -12.0% kg -3.2 kg to % + 7% -16.0% kg -4.4 kg Belgium to % + 9% -2.0% kg -1.0 kg ) 08SP03 Nuri i Espadaler S.I. 2) Kul 2009/44 DSM Nutritional Products, Belgium 3) 09BE07 VIMIX NV - VDS BVBA Table 9: Effect of L-Valine supplementation tested under commercial conditions. ADG (% of the best performance) SID Val:Lys % ADG response curve to SID Val:Lys 1. Spain 2. Belgium 3. Belgium 3. Belgium Figure 16: Effect of L-Valine supplementation: Comparison of the commercial trials and the average expected response (ADG). The increase of Val:Lys ratio resulted, in all the commercial trials, in a strong increase of the ADG and FCR of the piglets. In the third trial, animals were split according to their starting weight. It is shown that the effect of providing a balanced diet (higher SID Val:Lys) on the lighter piglets was exacerbated. The average ADG response curve to Val is validated by the commercial trials (Figure 16). 26 Information n 35 AJINOMOTO EUROLYSINE s.a.s.

27 Average SID Val:Lys 1 Source Average Effect on ADG Average Effect on FCR Average Effect on Weight Gain Measured performance 1 11% -8% +1.3 kg 62 to 70 % Predicted improvement 12% -5% +1.4 kg = + = 1) From the trials presented in Table 9 Table 10: Effect of L-Valine supplementation: Predicted and measured performance. In Table 10, predicted and measured effects were compared: The ADG response is clearly described by the model: increasing from 62 to 70% SID Val:Lys gives an increase in ADG of 12% and field trials showed a similar response. The FCR improvement measured in field tests was -8% on average when SID Val:Lys was increased from 62 to 70% while the model predicted -5%. In all the field trials, the feed intake response was lower than in the doseresponse trials held in controlled conditions (individual housing, controlled environment ), and the FCR was improved by more than that predicted by the model. This confirms the value of L-Valine supplementation under commercial conditions. On average, at the end of the experimental period, piglets fed optimal SID Val:Lys levels (70%), weighed 1.3 kg more than those fed deficient diets (62%), while the feed saving calculated was 2.3 kg of feed per piglet. AT A GLANCE! The piglet response to an increase in SID Val:Lys levels under practical conditions is consistent in terms of ADG and FCR improvement. On average, L-Valine supplementation resulted in +1.3 kg of live weight for piglets between 5 and 25 kg and a saving of 2.3 kg of feed. AJINOMOTO EUROLYSINE s.a.s. Information n 35 27

28 3.4 Reducing dietary Crude Protein by 2 points in piglet feed Reducing the dietary CP level (N x 6.25) and supplementing the diet with the limiting AAs reduces N excretion (Lenis et al., 1999) and limits the frequency and severity of gut disorders in piglets (Lordelo, 2008). A successful reduction of dietary CP is achievable under the following conditions: sufficient levels of all essential AAs, constant net energy levels. After this complete review of BCAA requirements, it is important to recall particularly that the Ile requirement for piglets is established at 53% SID Ile:Lys in blood-free diets. This piece of information is crucial for practical formulations since it determines the extent to which dietary CP levels can be decreased. The reduction of dietary CP levels with L-Valine supplementation has been tested in several recent trials. Three trials were presented in Bulletin #33 and showed that L-Valine allowed a further reduction of dietary CP by 2 points. Two additional trials can now be added to the first findings (Table 11). Experimental treatments Weight of piglets (kg) Experimental treatments CP (%) Net Energy (MJ/kg) SID Lys (%) SID Val:Lys (%) SID Ile:Lys (%) SID Leu:Lys (%) EAA N: Total N (%) Free Lys/ SID Lys (%) Lordelo (2008) Norgaard and Fernandez (2009) Jansman (2008) Vinyeta (2010) 7 to 23 9 to to 25 8 to 25 High CP Low CP (-3.5 points) Low CP kg/t L-Valine High CP Low CP (-2 points) Low CP kg/t L-Valine High CP Low CP (-3 points) Low CP kg/t L-Valine High CP Low CP (-2.5 points) Low CP kg/t L-Valine SP to 23 High CP Low CP kg/t L-Valine Results Experimental treatments CP (%) SID Val:lys (%) ADG (g/day) ADFI (g/day) FCR (g/g) Lordelo (2008) Norgaard and Fernandez (2009) Jansman (2008) Vinyeta (2010) High CP a 932a 1.60 Low CP (-3.5 points) b 820b 1.61 Low CP kg/t L-Valine a 941a 1.65 High CP a b Low CP (-2 points) b a Low CP kg/t L-Valine a b High CP a 869a 1.54a Low CP (-3 points) b 682b 1.57b Low CP kg/t L-Valine a 866a 1.52a High CP a 857a 1.52 Low CP (-2.5 points) b 697b 1.56 Low CP kg/t L-Valine a 873a SP08 1 Low CP kg/t L-Valine High CP ) 08SP08 Nuri i Espadaler S.I. In the same column, figures associated with different letters are significantly different (p<0.05). Table 11: Effect of lowering dietary crude protein (CP) with or without L-Valine supplementation on piglet performance. 28 Information n 35 AJINOMOTO EUROLYSINE s.a.s.

29 The experimental design of the trials was generally the same: A positive control with high CP A negative control where dietary CP was reduced and where Val was limiting (below 70% SID Val:Lys). Isoleucine and Leu were not limiting even after the 2 points CP reduction. The negative control was supplemented with the appropriate amount of L-Valine to cover the animals needs. Information about the Val content of the feeds is given in Table 11, as well as the Ile and Leu levels. In addition, ratios between Nitrogen from Essential AA (EAA N ) and Total Nitrogen (Total N ) were calculated (Focus 4). Finally, the contribution of free Lys (from L-Lysine HCL,) to the SID dietary Lys is reported. The reduction of dietary CP has a strong negative effect on piglet performance if L-Valine is not supplemented (Table 11). The supplementation of L-Valine allows a reduction in the dietary CP by 2 to 3 points without any detrimental effect on performance. Dietary CP levels have no influence on growth performance as long as the AA profile is balanced and feed is supplemented with AAs (Figure 17). The minimum ratio of EAA N /Total N is 46% on average so that N is not deficient for the synthesis of non-essential AAs (Focus 4). The amount of L-Lysine added to feed does not affect performance as long as the AA profile is balanced (Figure 18). Feed-use amino acids are powerful tools that reduce the risk of nutritional imbalances in feed formulations (Focus 5). ADG and ADFI (g/d) FCR (g/g) Dietary CP (%) ADG and ADFI (g/d) Free Lys/SID Lys (%) FCR (g/g) Lordelo, 2008 Norgaard and Fernandez, 2009 Jansman, 2008 Vinyeta, SP08 Figure 17: Effect of dietary crude protein on the growth Figure 18: Effect of the contribution of free Lysine of piglets while maintaining a constant ideal AA profile to SID dietary Lys on the growth of piglets (8 to 25 kg LW). (8 to 25 kg LW). AT A GLANCE! Dietary CP is not a nutrient and is just the product of N x It does not describe the quality of the AA supply yet it is crucial to monitor the EAA contents. The reduction of 2 points of dietary CP is feasible if EAA:Lys ratios are maintained at the Ideal Protein ratios, particularly Val at 70% SID Val:Lys. The reduction of SID Ile:Lys to 53% has no detrimental effect on growth performance. This is the lowest Ile requirement and relates to diets that do not contain blood products. An increase in the proportion of added free Lys (from feed-use L-Lysine) does not affect pig performance. AJINOMOTO EUROLYSINE s.a.s. Information n 35 29