Amino Acids : Towards Precise Nutrition in Monogastric Animals

❶ Amino Acids in Pigs and Broilers ❷ Updated Ideal Amino Acid Profiles ❸ Balance is Essential ❹ Reduction of Dietary Crude Protein AFMA 2016 CORRENT AMINO ACIDS 2016 All rights reserved AJINOMOTO EUROLYSINE S.A.S. 3

Overview of pathways in amino acid metabolism in monogastric Amino Acids ANABOLISM CATABOLISM Tissue proteins Hormones Bioactive molecules Amino group Carbon skeleton Gluconeogenesis Uric acid Urea AFMA 2016 CORRENT AMINO ACIDS 2016 All rights reserved AJINOMOTO EUROLYSINE S.A.S. 6

Overview of pathways in amino acid metabolism in monogastric Arg is an indispensable AA in poultry Difference in N metabolism (urea vs uric acid) In the uric acid pathway, more DAA are involved (more sensitive to interactions?) AFMA 2016 CORRENT AMINO ACIDS 2016 All rights reserved AJINOMOTO EUROLYSINE S.A.S. 7

Usual Amino Acids Classification in Swine and Broiler Indispensable LYS THR MET TRP VAL ILE LEU HIS PHE ARG Semi-Indisp. CYS TYR Dispensable GLY SER PRO ASP ASN ALA GLU GLN AFMA 2016 CORRENT AMINO ACIDS 2016 All rights reserved AJINOMOTO EUROLYSINE S.A.S. 8

Usual Amino Acids Classification in Swine and Broiler Indispensable LYS THR MET TRP VAL ILE LEU HIS Semi-Indisp. CYS Dispensable GLY SER PRO ASP ASN ALA GLU GLN Not in growing pigs PHE ARG TYR AFMA 2016 CORRENT AMINO ACIDS 2016 All rights reserved AJINOMOTO EUROLYSINE S.A.S. 9

Usual Amino Acids Classification in Swine and Broiler Indispensable Semi-Indisp. Dispensable LYS GLY THR SER MET CYS PRO L N A A TRP VAL ILE LEU HIS B C A A ASP ASN ALA GLU GLN PHE TYR ARG AFMA 2016 CORRENT AMINO ACIDS 2016 All rights reserved AJINOMOTO EUROLYSINE S.A.S. 10

CONCLUSIONS (1) ❶ Protein is not a predictor of performance ❷ Amino Acids metabolism should be more taken into account ❸ All amino acids are essential ❹ Interactions between amino acids might appear more often AFMA 2016 CORRENT AMINO ACIDS 2016 All rights reserved AJINOMOTO EUROLYSINE S.A.S. 11

L-Lys L-Thr DL-Met L-Trp L-Val 5 Feeduse amino acids = 7 co-limiting AA in the diet Example in piglet feeds : Lys, Thr, Met, (Cys), Trp, Val & the next one: Ile or Leu or His? Supply and source of dietary AA in % of the AA requirement 100 0 ❶ ❷ ❸ ❺ ❻ ❼ + ❹ Lys Thr Met + Cys Trp Val Ile Leu His Phe+Tyr AFMA 2016 CORRENT AMINO ACIDS 2016 All rights reserved AJINOMOTO EUROLYSINE S.A.S. 13

Amino Acids requirements in growing pigs Recent advances in AA Meta-analyses works: Simongiovanni et al., (Trp) van Milgen et al., (Val) van Milgen et al., (Ile) PhD program in FR Gloaguen et al.: Next limiting AA Very low CP diets Others works : Low CP diets and limiting AA (Jansman et al., NL). Ile, Val, Leu (Soumeh & Noorgard, DK) Leu, Trp, Low CP diets (Wessels et al., DE) AFMA 2016 CORRENT AMINO ACIDS 2016 All rights reserved AJINOMOTO EUROLYSINE S.A.S. 14

ADG in % of the best performance SID Valine requirement in piglets ADG response curve to dietary SID Val:Lys, meta-analysis based on 7 trials 100 95 90 85 Mavromichalis et al. (2001) - Exp. 5 - Barea et al. (2009)a - Exp. 4 - Dusel et al. (2008) Paulicks et al. (2008) Torrallardona et al. (2008) Vinyeta et al. (2010) 80 75 70 65 60 55 40% 43% 45% 48% 50% 53% 55% 58% 60% 63% 65% 68% 70% 73% 75% 78% 80% 83% 85% SID Val:Lys (%) AFMA 2016 CORRENT AMINO ACIDS 2016 All rights reserved AJINOMOTO EUROLYSINE S.A.S. 15

SD Valine requirement in broilers AEL 2016, unpublished Level : 80% SD Val:Lys (Mack et al 1999) Response : - Varies with other BCAA? - Vaires due to endogenous losses? - Varies due to Thr / Gly interactions? AFMA 2016 CORRENT AMINO ACIDS 2016 All rights reserved AJINOMOTO EUROLYSINE S.A.S. 16

Updated Amino Acids Profile (2016) SID* Pigs Starter Grower Finisher Broilers 8-25 kg 25-60 kg 60-115 kg 0-44 days Thr:Lys % 65 67 68 Thr:Lys % 67 Trp:Lys % 22 20 19 Trp:Lys % 17 Met+Cys:Lys % 60 60 60 Met+Cys:Lys % 75 Val:Lys % 70 65 65 Val:Lys % 80 Ile:Lys % 53 53 53 Ile:Lys % 67 Leu:lys % 100 100 100 Leu:lys % 105 Phe+Tyr:Lys % 95 95 95 Phe+Tyr:Lys % 105 His:Lys % 32 32 32 His:Lys % 36 Ideal amino acids profiles in growing pigs and broilers. *SID : Standardized Ileal Digestibility, SD: Standardized Digestible. Based on Gloaguen et al. (2014), van Milgen et al. (2012, 2013), Mack et al. (1999), Lambert et al. (2015) and Ajinomoto Eurolysine works (2011, 2013, 2015). AFMA 2016 CORRENT AMINO ACIDS 2016 All rights reserved AJINOMOTO EUROLYSINE S.A.S. 17 SD* Arg:Lys % 105

Next limiting AA in SD a Lys=1.05%, broiler AMEn=3,000kcal/kg diet? What is the ranking of limiting AA in feed formulation? SD Lys=1.05% AMEn=3,000kcal/kg Valine is the 4 th limiting AA after Met, Lys and Thr Arginine and Ile (or Trp) are colimiting after Val AFMA 2016 CORRENT AMINO ACIDS 2016 All rights reserved AJINOMOTO EUROLYSINE S.A.S. 18

Next limiting AA in SD a Lys=1.05%, broiler AMEn=3,000kcal/kg diet? What is the ranking of limiting AA in feed formulation? Valine is the 4 th limiting AA after Met, Lys and Thr Arginine and Ile (or Trp) are colimiting after Val L-Valine allows a dietary CP reduction in broiler feeds of 0.6 to 0.9 points of CP (depending on the cereal used) -0.6% CP SD Lys=1.05% AMEn=3,000kcal/kg AFMA 2016 CORRENT AMINO ACIDS 2016 All rights reserved AJINOMOTO EUROLYSINE S.A.S. 19

ADG G:F Response to Thr, Val and Arg in broiler Loss of performance when the AA is dietary deficient by 20% (80% of the requirement) AEL Meta Analyses studies Arg -4% Arg:Lys : 105 85 % Val Thr -9% -12% Val:Lys : 80 65 % Thr:Lys : 67 55 % Arg -5% Val -13% Thr -15% Thr and Val response is stronger than Arg response (functions of Arginine?) AFMA 2016 CORRENT AMINO ACIDS 2016 All rights reserved AJINOMOTO EUROLYSINE S.A.S. 20

CONCLUSIONS (2) ❶ Valine is a limiting AA in broilers and piglets ❷ Meta-analyses studies allow to give an overview of the response and requirement to amino acids ❸ Ideal amino acids profile can be used in feed formulation ❹ Decision is based on level of deficiency and response AFMA 2016 CORRENT AMINO ACIDS 2016 All rights reserved AJINOMOTO EUROLYSINE S.A.S. 21

Branched-Chain Amino Acids have a common catabolism. 2016 All rights reserved Ajinomoto Eurolysine S.A.S. Leucine drives BCAA metabolism. AFMA 2016 CORRENT AMINO ACIDS 2016 All rights reserved AJINOMOTO EUROLYSINE S.A.S. 23

Impact of the BCAA catabolism on performance in piglet Gloaguen et al. (2012) and Barea et al. (2009) performed doseresponses to Val (@INRA) Comparison show same requirement but different responses Higher response was obtained when Leu was in excess Deficient Valine diets are detrimental for piglets performance [ADFI, ADG and FCR]. 70% SID Val:Lys ensures the piglet to sustain maximal growth ADG FCR 100 95 90 85 80 75 70 65 60 Leu 55 50 55% 60% 65% 70% 75% 80% 85% SID Val:Lys ratio (%) Barea et al. (2009) Low Leu 115.0 Leu 112.5 110.0 107.5 105.0 102.5 100.0 55% 60% 65% 70% 75% 80% 85% SID Val:Lys ratio (%) Glaoguen et al. (2012) High Leu AFMA 2016 CORRENT AMINO ACIDS 2016 All rights reserved AJINOMOTO EUROLYSINE S.A.S. 24

g/day Amino Acids Imbalance and Feed Intake in Piglets Effect on Average Daily Feed Intake (g/d) - WHEN THE FEED IS IMBALANCED IN BCAA 1000 900 800 700 600 PIGLET DOES NOT EAT 500 400 SID Val:Lys ratio (%) 300 40% 50% 60% 70% 80% AFMA 2016 CORRENT AMINO ACIDS 2016 All rights reserved AJINOMOTO EUROLYSINE S.A.S. 25

Impact of Amino Acids interactions on Ile requirement PIGLETS Amount of blood product SID Ile:Lys requirement Average Htoo et al., 2009 0% 51% Wiltafsky et al., 2009 0% 54% Gloaguen et al., 2013 0% 49% 51.5% Noorgard et al., 2013 0% 52% James et al., 2009 3.2% 55% Kerr et al., 2004 7.5% 61% Wiltafsky et al., 2009 7.5% 59% 61% Fu et al., 2006 10.8% >68% The presence of BC increases the Ile requirement in growing pigs. Meta-analysis work of van Milgen et al. (2012) concludes that 50% SID Ile:Lys is sufficient to cope with the Ile need AFMA 2016 CORRENT AMINO ACIDS 2016 All rights reserved AJINOMOTO EUROLYSINE S.A.S. 26

Impact of Amino Acids interactions on Ile requirement BROILERS Without blood cells With blood cells Without blood cells 2016 All rights reserved Ajinomoto Eurolysine S.A.S. With blood cells SD Ile:Lys (%) AFMA 2016 CORRENT AMINO ACIDS 2016 All rights reserved AJINOMOTO EUROLYSINE S.A.S. 27

The Glycine issue in broiler When (very) low protein diets are fed to broiler, only the addition of Gly is efficient Why? (Gly is a «dispensable» amino acid) Very high variability in the recommendations : 145 to 245 % Gly+Ser:Lys! AFMA 2016 CORRENT AMINO ACIDS 2016 All rights reserved AJINOMOTO EUROLYSINE S.A.S. 28

The Glycine issue in broiler 4 precursors Serine : reversible Choline Glyoxylate Threonine Based on Mélendez-Hevia et al. (2009); Akinde (2014a) and Nelson and Cox (2008) AFMA 2016 CORRENT AMINO ACIDS 2016 All rights reserved AJINOMOTO EUROLYSINE S.A.S. 29

Interaction between Thr and Gly in broiler Gly requirement is high when Thr is low When Thr is at the requirement, Gly is not limiting AFMA 2016 CORRENT AMINO ACIDS 2016 All rights reserved AJINOMOTO EUROLYSINE S.A.S. 30

CONCLUSIONS (3) ❶ When dietary crude protein is reduced, interactions can appear, and must be taken into account ❷ BCAA : Leu increases the catabolism of Valine which is a limiting AA in current piglet and broiler diets ❸ When a feed is imbalanced in amino acids, feed intake is reduced. ❹ In broiler, Gly requirement is lower when Threonine is correctly supplied AFMA 2016 CORRENT AMINO ACIDS 2016 All rights reserved AJINOMOTO EUROLYSINE S.A.S. 31

Dietary protein reduction allows to save feed costs Example in broiler diet Formulation exercise from January 2013 to January 2016 Moving from a CP constraint to a formulation on each AA Up to : - 1.6 points of CP - 186 ZAR/T (-11 /mt) - 8 points of SBM inclusion SD Lys=1.05%, mini CP=20%, AMEn=3,000kcal/kg AFMA 2016 CORRENT AMINO ACIDS 2016 All rights reserved AJINOMOTO EUROLYSINE S.A.S. 33

Dietary crude protein reduction in piglets Efficient and Safe Reduction of undigested protein supply: Lower proliferation of bacteria and reduction of diarrhea Formulation based on EAA instead of CP: Lower proteic feedstuffs Possible cost savings Balanced AA profile Implementation of risk management Reduction of nitrogen output Body weight (kg) Lordelo et al. (2008) 7-23 Jansman et al. (2008) 10-25 Norgaard and Fernandez (2009) 9-21 Vinyeta et al. (2010) 8-25 Gloaguen et al. (2013) 12-23 Jansman et al. (2013) 8-25 Dietary CP (%) SID Lys (%) ADFI (g/d) ADG (g/d) FCR P AEL Ideal AA profile 20.5 1.12 932 583 1.60 ns 17.0 1.12 941 571 1.65 19.0 1.03 869 567 1.54 ns 16.0 1.03 866 568 1.52 19.0 1.10 627 445 1.40 ns 17.0 1.10 635 449 1.41 17.5 0.94 857 564 1.52 ns 15.5 0.94 873 567 1.54 17.6 1.00 766 450 1.70 ns 15.6 1.00 775 454 1.71 16.8 1.00 835 548 1.49 ns 15.4 1.00 816 564 1.48 AFMA 2016 CORRENT AMINO ACIDS 2016 All rights reserved AJINOMOTO EUROLYSINE S.A.S. 34

Dietary crude protein reduction in fattening pigs Net energy system ADG (g/d) 1300 FCR 3.80 1200 1100 3.60 3.40 3.20 1000 900 3.00 2.80 2.60 800 700 2.40 2.20 2.00 600 20 19 18 17 16 15 14 13 12 Dietary Crude Protein level (%) 11 10 9 1.80 20 19 18 17 16 15 14 13 12 Dietary Crude Protein level (%) 11 10 9 AFMA 2016 CORRENT AMINO ACIDS 2016 All rights reserved AJINOMOTO EUROLYSINE S.A.S. 35

ADFI (g/d) 200 190 180 170 160 150 140 INRA 2015 (Exp 1) INRA 2015 (Exp 2) 130 14 15 16 17 18 19 20 FCR (g/g) 1.80 1.75 1.70 1.65 1.60 1.55 Dietary crude protein reduction in broilers (21-35 days) * * No effect (all AA at their requirement) 115 110 105 100 ADG (g/d) 95 INRA 2015 (Exp 1) INRA 2015 (Exp 2) b 90 14 15 16 17 18 19 20 ADFI/BW : no effect of CP reduction FCR : Reducing CP from 19% down to 16.5% - 17% did not result in higher FCR when Thr is on the safe side and other IAA controlled b a 1.50 14AFMA 2016 15 CORRENT 16 AMINO 17 ACIDS 18 19 20 2016 All rights reserved AJINOMOTO EUROLYSINE S.A.S. 36

CONCLUSIONS (4) ❶ Formulation on each indispensable AA allows 1) to save feed costs, 2) to reduce the impact of livestock production on the environment, 3) to improve health status of animals ❷ As soon as amino acids are controlled, performance are not affected by dietary crude protein reduction ❸ It is possible to reduce dietary CP in piglets (-2 points), fattening pigs (-1.5 point), and in broilers (-1 to -2 points), it varies with the Lys level AFMA 2016 CORRENT AMINO ACIDS 2016 All rights reserved AJINOMOTO EUROLYSINE S.A.S. 37

CONCLUSIONS ❶ Determining the next limiting AA in each context [Requirement Feedstuffs Animal status] ❷ Valine is the next limiting AA in piglet (70% SID Val:Lys), Trp is the next limiting AA in fattening pigs (20% SID Trp:Lys) ❸ Valine is the next limiting AA in broiler (80% SD Val:Lys), Arg requirement depends on context ❹ Interactions between AA must be considered when CP is reduced [BCAA in piglet and broilers] [Thr and Gly in broiler] ❺ Dietary protein reduction leads to a more precised amino acids nutrition AFMA 2016 CORRENT AMINO ACIDS 2016 All rights reserved 2016 All rights AJINOMOTO reserved Ajinomoto EUROLYSINE Eurolysine S.A.S. 38