Impact of the withdrawal of antimicrobial growth promoters and health status on the amino acid requirement of the pig

GO TO ESSENTIALS INFORMATION N 29 Impact of the withdrawal of antimicrobial growth promoters and health status on the amino acid requirement of the pig In modern animal production, nutrition and sanitary conditions are crucial for optimum performance. More and more, consumer concern about the quality of food products, which starts with the feed given to livestock animals, is taken into account by the feed industry and the authorities. In this respect, the European authorities have, over the last few years, integrated many directives in order to take into account consumer demand. The latest being the complete ban of antimicrobial growth promoters (AGP) for pig and poultry feeds implemented in January 2006. The direct impact of these regulations on animal performance is difficult to quantify but it will likely increase variability and penalize technical and economic results. In fact, once AGP are removed, health status becomes an even more critical factor. In this context, it is important to evaluate if nutrient requirements are affected when health status is impaired. This issue has been adressed; the present bulletin outlines a series of trials recently performed either in growing pigs or in piglets to test these factors of variation on amino acids requirements. Impact of the withdrawal of AGP and health status : - On pig performances... 2 - On lysine requirement... 4 in young pigs in growing finishing pigs - On threonine requirement... 6 in growing finishing pigs The opinion of the researchers... 7 - On tryptophan requirement... 8 in young pigs Discussion and conclusions... 10

2 Impact of the withdrawal of AGP and health status on pig performance The negative impact of the deterioration of the health status on performance has clearly been demonstrated by Le Floc h et al., (2006) and Williams & Stahly (1995) Figures 1 & 2. When health status is deteriorated, the decrease in growth is not only due to a decrease in feed intake but also to the diversion of nutrients from growth towards tissues and cells involved in body defence. Thus, amino acid demand for functions other than growth could indeed be increased. In such a situation, amino acids could be used as an energy source for gluconeogenesis, immune cell proliferation and serve as building blocks for inflammatory protein and immunoglobulin synthesis. In addition, amino acids can enter specific metabolic pathways related to body defence. Figure 1: Daily weight gain (ADG) and feed intake (ADFI) of piglets with high sanitary status, reared in clean disinfected rooms and fed with diets containing antibiotic supplementation and piglets in low sanitary status reared in non sanitized rooms not disinfected after previous occupation by piglets from the same herd and fed the same basal diets without antibiotic supplementation (Le Floc'h et al., 2006). Antimicrobial growth promoters act mainly on the activity of intestinal bacteria by limiting the development of unfavourable bacteria. As a consequence, they increase the availability of nutrients and energy for growth. In addition, the production of biologically active compounds which are not necessarily valuable or even detrimental such as ammonia is reduced and gut integrity is better preserved when AGP are fed to the animals (Mellor et al., 2004). Figure 2: Daily weight gain (ADG) and feed intake (ADFI) of piglets reared via a medicated-early-weaning scheme and derived from sows that had been vaccinated for prevalent antigens in the herd of origin (high sanitary status) or reared in conventional scheme with maximum exposure to foreign antigens (low sanitary status) (Williams and Stahly, 1995). The 3 main functions of AGP can be summarized below (Blanchard, 2005) Figure 3. therapeutic role against various pathogens prophylactic effect, preventing occurrence of diseases improved performance such as growth and feed conversion Figure 3: The 3 main actions of antimicrobial growth promoters (AGP) and their interactions (Blanchard, 2005).

3 As a consequence, removal of AGP may reduce the availability of nutrients and amino acids for growth. Thus, AGP removal from the feed may lead to a decrease in growth and an increase in feed conversion ratio and health damage, especially in farms which already have a low health status and in fragile periods like postweaning. This has been confirmed by Blanchard, 2005 predicting the response of AGP removal in UK pig herds. This simulation shows that the greatest effect of antibiotic withdrawal in the diet on growth and feed conversion ratio would be observed during the weaner period and in poor sanitary conditions (Blanchard, 2005) Figure 4. Figure 4 : Predicted response of antimicrobial growth promoters (AGP) removal on the increase in feed conversion ratio (FCR) in pigs from 6 to 40 kg and 40 to 100 kg depending on performance band parameter (optimal, excellent, good, average or poor) (Blanchard, 2005). Deterioration of health and withdrawal of AGP are likely to impact on growth Figure 5. Lysine and other amino acid requirements are closely related to growth performance and feed intake. On one hand, this lower performance could lead to a lower amino acid requirement for growth, but on the other hand, requirements for functions other than growth such as the immune system and maintenance are increased. For instance, in these situations, amino acids could be used in greater amounts to satisfy potentially higher uptake by intestinal flora due to bad environment and/or to cover higher usage for proteins involved in immune function. Finally, the amino acid requirement will depend not only on the level of growth performance but also on the way they are used by the body and the level of feed intake. The objective of this bulletin is to investigate through recent trials how far AGP withdrawal and sanitary conditions are impacting on amino acid requirements. Figure 5 : The effect of AGP withdrawal from the diet and poor sanitary status on growth. AJINOMOTO EUROLYSINE S.A.S. INFORMATION N 29

4 Impact of health status on lysine requirement in young pigs The impact of health status on lysine requirement in piglets was investigated by Piñeiro et al., 2005. In this trial, 1200 piglets reared in two Spanish farms with different sanitary status were used. EXPERIMENTAL DESIGN One commercial farm with low health status and one with high health status were selected. The criteria to discriminate low and high health status were productive performances, mortality and different pathologies serology. Four levels of standardised ileal digestible SID lysine in the diet were tested from 1.22 to 1.52% in prestarter piglets (21 to 40 days of age), and from 1.01 to 1.32% in starter piglets (41 to 63 days of age). All the other limiting amino acids, threonine, tryptophan and methionine + cysteine were kept in constant ratios to lysine with SID ratio at 65%, 22% and 60% respectively. The experimental diets were isoenergetic and isoproteic. They are described in appendix. No growth promoters were added to any of the experimental diets. RESULTS Feed intake and growth were lower in the low health status than in the high health status farm Table 1. Whatever health status and age, the lysine level had no significant effect on feed intake Figure 6a. There was a significant linear effect of lysine on average daily gain and body weight at 40 days of age in both farms. In both farms, maximum performance was achieved with the third step of lysine (1.42% in prestarter and 1.21% in starter). The magnitude of the effect of the dietary lysine content on performance was higher in the low health status farm than in the high one: during the prestarter phase, increasing SID lysine in the feed from 1.22 to 1.42% improved average daily gain by 22% in the low health status farm instead of 14% in the high health status farm Figure 6b. Similar response was obtained for FCR Figure 6c. In conclusion, during the whole experimental period (from 28 to 63 days of age), increasing SID lysine by 0.2 percentage point improved feed efficiency by 10% in the low health status farm and by 4% in the high health status farm. HHS LHS ADG (g/d) 441 341 AFDI (g/d) 616 492 FCR 1,39 1,44 Mortality < 4% > 6% Table 1 : Average performance parameters in the farm with high health status (HHS) and low health status (LHS) (Piñeiro et al., 2005). a) b) c) Figure 6 : Effect of lysine on average feed intake (ADFI, a), daily gain (ADG, b) and feed conversion ratio (FCR, c) relative to best or worst performance within each sanitary status group of prestarter piglets (Piñeiro et al., 2005). CONCLUSION In piglets, the dietary lysine content that optimizes growth and feed efficiency is not affected by health status. A lysine deficiency would penalize more severely the performance of animals with lower health status compared with high health status.

5 Impact of the withdrawal of AGP on lysine requirement in growing finishing pigs The effect of the withdrawal of AGP on lysine requirement in growing finishing pigs was tested by Bikker et al., (2006). Trial 1 EXPERIMENTAL DESIGN The first trial was a 2 x 2 factorial design with diets differing in balanced lysine levels (0.67% or 0.74% SID) and AGP content (0 or 30 ppm of salinomycin). For this trial a total of 288 pigs from 40 to 110 kg were used. In all treatments, other amino acids were kept in constant ratio to lysine (SID Thr:Lys 64%, Met + Cys:Lys 69% and Trp:Lys 20%). RESULTS Increasing dietary balanced SID lysine level from 0.67% to 0.74% significantly improved weight gain and feed efficiency Table 2. Moreover, the effect of dietary balanced lysine on weight gain was significantly higher in the group of pigs fed the diet without AGP than in pigs fed a diet with 30 ppm of salinomycin. Feed intake was not affected by either AGP or lysine Table 2. salinomycin, ppm 0 30 effects SID lysine, g/kg 6.7 7.4 6.7 7.4 AA AGP AA x AGP ADFI, kg/d 2.59 2.60 2.60 2.60 ns ns ns ADG, g/d 887 924 931 940 ** ** * FCR 2.96 2.85 2.83 2.77 ** ** ns Fæcal consistency score 5.4 5.4 5.8 5.8 ns ** ns Table 2 : Effect of AGP and dietary lysine content on feed conversion ratio (FCR), average daily gain (ADG), average daily feed intake (ADFI) and faecal consistency in pigs from 40-110 kg (Bikker et al., 2006). Trial 2 EXPERIMENTAL DESIGN In the second trial, the objective was to quantify, with four dietary lysine levels (0.49-0.59-0.69-0.79% SID), the effect of the withdrawal of AGP on growth response to lysine. Other amino acids were kept in constant ratio to lysine (Thr:Lys 65%, Met + Cys 62% and Trp:Lys 19% SID). The experimental diets are described in appendix. RESULTS In the second experiment daily gain was optimized at the third step of lysine (0.69%) in the group with salinomycin. In the group without salinomycin, daily gain was optimized at the fourth step of lysine (0.79% SID) Figure 7a. Similar results were obtained for feed conversion ratio Figure 7b. Quadratic and linear regression models reached higher optimum levels of lysine for growth and FCR without AGP compared to diet with AGP. Feed intake was not affected by either AGP or lysine. a) b) Figures 7a & 7b : Effect of AGP and dietary lysine content on average daily gain (ADG, a) and feed conversion ratio (FCR, b), in pigs from 40-110 kg (Bikker et al., 2006). CONCLUSION In growing finishing pigs amino acid requirement for growth rate and feed efficiency is increased by 6% in diets without AGP. AJINOMOTO EUROLYSINE S.A.S. INFORMATION N 29

6 Impact of the withdrawal of AGP on threonine requirement in growing finishing pigs The threonine fraction that is absorbed by the ileum is not entirely delivered in the portal blood which collects the nutrients from the digestion process. Indeed, a significant part of the digestible threonine may be utilized by the digestive tract itself. This important uptake of threonine by the gut is consistent with the high threonine content of digestive secretions, amongst which is mucus (Le Bellego et al., 2002). Withdrawal of AGP from animal diets is known to increase microbial growth in the digestive tract and the activity of lymphatic tissue. In turn, endogenous secretions (mucus, enzymes, bacteria...), amino acid utilisation by bacteria and protein turnover would be increased. Consequently ileal digestibility of amino acids may be reduced (Mellor et al., 2004). Furthermore, in 17kg BW pigs, it was shown that higher levels of dietary threonine were needed to maximize humoral antibody production and IgG levels than to optimize growth (Li Defa et al., 1999). Therefore, increased endogenous losses and higher activation of the immune system following withdrawal of AGP may increase the demand for threonine for functions other than growth. EXPERIMENTAL DESIGN Four dietary threonine levels were tested in growing and finishing pigs fed a diet with (30 ppm) or without salinomycin (Bikker et al., 2006). The grower diet was distributed to pigs from 20 to 45 kg BW and the finisher diet was distributed from 45 to 110 kg BW. Basal SID Thr:Lys ratios were 54 and 55% respectively for grower and finisher phases. The diets, both for grower and finisher, were formulated to contain 9.5 MJ NE/kg. The three other steps of threonine were achieved by addition of free threonine to the basal diet (Appendix). RESULTS Antimicrobial growth promoters or threonine levels did not significantly affect feed intake whatever the period of experiment. Inclusion of the AGP significantly improved the growth of the pigs (P<0,01). During the growing period linear effect of threonine was significant in both groups with or without AGP (P<0.001). Best performances were obtained for a ratio Thr:Lys at minimum 65%. There were no significant interaction between threonine response and AGP nevertheless FCR was minimum for a ratio Thr:Lys higher in the group without AGP in the diet than in the group with AGP Figure 8. In the finishing phase and the whole experimental period, the response of pigs to threonine was affected by the removal of AGP in the diet. Indeed, during the whole experimental period (45-110 kg), threonine had no significant effect on daily gain and FCR when the diet contained the AGP, whereas dietary threonine linearly increased daily gain (P=0.03) and quadratically decreased FCR (P=0.03) when the diet did not contain AGP Figure 9. In the group without dietary AGP, increasing Thr:Lys ratio up to 70% allows to reach the same level of performances of the animals fed with dietary AGP. Figure 8 : Effect of the Thr:Lys ratio on weight gain (ADG) and feed conversion ratio (FCR) in pigs fed a diet with 0 (AGP -) or 30 ppm of salinomycin (AGP +) from 25 to 45 kg (Bikker et al., 2006). Figure 9 : Effect of the Thr:Lys ratio on weight gain (ADG) and feed conversion ratio (FCR) in pigs fed a diet with 0 (AGP -) or 30 ppm of salinomycin (AGP +) from 25 to 110 kg (Bikker et al., 2006). CONCLUSION Threonine supply has more effect on pig performance in diets without AGP. Withdrawal of AGP increases the threonine requirement of growing finishing pigs. In the group without dietary AGP, increasing SID Thr:Lys ratio up to 70% allows to reach the level of performance of animals with dietary AGP.

7 Their opinion... Dr Nathalie Le Floc'h D.V.M., PhD., INRA UMR SENAH F 35590 Saint Gilles. France Nathalie.Lefloch@rennes.inra.fr Alteration of health and sanitary status is associated with the retardation of growth rate. This is partly explained by modifications of nutrient utilisation. In such conditions, some amino acids could become less available for growth and any other biological functions in which they are involved. When these amino acids are not limiting for optimising performance of pigs kept in excellent sanitary conditions, their supplies may be not adapted for pigs submitted to immune stress even if moderate. Lastly, it is also important to keep in mind that essential amino acid as well, such as tryptophan, can be involved in body defence processes. Consequently, by preserving an adequate ratio of amino acids, such as tryptophan for example, to lysine, one expects to limit the consequences of health deterioration on both growth and health. Dr Paul Bikker PhD, Schothorst Feed Research PO Box 533, 8200 AL Lelystad The Netherlands pbikker@schothorst.nl Antimicrobial growth promoters (AGP) have been banned from pig diets in the EU; other countries may follow in the near future. Schothorst Feed Research conducted a series of experiments to study interactions between AGP and amino acid requirements for growing-finishing pigs. The results showed that without AGP the pigs required a higher amino acids content for maximum growth performance. Further studies indicated that especially threonine requirements may be increased. Possible reasons for these effects include increased microbial degradation of dietary protein in the digestive tract and increased utilisation of amino acids, especially threonine for endogenous protein secretion. In addition, stimulation of the immune system without AGP may increase protein turnover and amino acid requirements for immune functions. This emphasises the importance of a better understanding of amino acid requirements in relation to diet composition and farm circumstances. Feedstuffs and dietary ingredients that reduce microbial growth in the digestive tract may improve amino acid digestion and utilisation, whereas feedstuffs that increase endogenous losses may increase requirements of specific amino acids. Poor hygienic farm conditions may increase the need for specific amino acids involved in the immune response of pigs. On the other hand, growing pigs with a high health status (e.g. SPF, specific pathogen free) are able to realise a higher protein deposition per kg of feed and therefore most likely need a higher amino acid to energy ratio for optimal use of their growth potential. Improvement of health status on pig farms is required for future social acceptance and to increase the profit of pig production. Adjusting dietary composition and amino acid content to farm circumstances will be a prerequisite for optimal technical and economical results. This is a challenge for nutritionists and consultants in the near future. Carlos Piñeiro R+D Manager PigCHAMP Pro Europa Carretera San Rafael 42 40006 SEGOVIA, Spain carlos.pineiro@pigchamp-pro.com The banning in the use of antibiotic growth promoters has been one of the changes with a big impact in pig performance and, consequently, in the economic results of the farms. The best option to attenuate the repercussions under commercial conditions seems to be to improve all of the aspects related to pig management and nutrition. Under this new approach, nutritional requirements have been modified, in particular those related with the immune system. Very recent papers are showing us the importance of a different amino acid profile depending on pig health status, mainly affecting lysine, tryptophan and threonine. This will very likely affect diet formulation in the near future. AJINOMOTO EUROLYSINE S.A.S. INFORMATION N 29

8 The impact of health deterioration on tryptophan requirement in young pigs In piglets, situations such as chronic lung inflammation (Melchior et al., 2004), intraperitoneal infection by E. coli (Yoo et al., 1997) or intravenous administration of E. coli LPS modify tryptophan metabolism. All of these situations decrease plasma tryptophan concentration suggesting increased utilisation of this amino acid. Two key areas of utilisation of tryptophan have been shown to be through IDO (indoleamine 2,3 dioxygenase) pathway and incorporation into acute phase proteins (Melchior et al., 2004). Recently, it was shown that poor sanitary conditions (dirty environment and AGP whithdrawal from the diet) also negatively affect plasma tryptophan concentration in piglets (Le Floc h et al., 2004). Two levels of dietary tryptophan were tested: low (total Trp:Lys 19% and 17% for prestarter and starter respectively) or L-tryptophan supplemented (total Trp:Lys 22% and 21% in prestarter and starter respectively). In both treatments, tryptophan supplementation significantly increased weight gain and feed intake and decreased feed conversion ratio. The response to tryptophan was not significantly different for both treatments. However, in the dirty environment, the significant positive effect of tryptophan on growth and plasma concentration suggests that growth depression caused by unsanitised conditions could be limited by increasing dietary tryptophan level. Therefore, Le Floc h et al., (2006) conducted a trial to test whether the growth response to increased dietary tryptophan levels varied with health status. EXPERIMENTAL DESIGN In a total of 160 piglets, two levels of sanitary status were tested: low and high. Low sanitary status was achieved by transferring piglets in rooms that were not cleaned after previous occupation by pigs from the same herd. Moreover, these pigs were fed a diet without antibiotic supplementation. By comparison, control high health status pigs were kept in clean and disinfected rooms and were fed a diet containing a preventive antibiotic medication (avilamycin 20 at 2 g/kg of diet until 5th week then oxytetracyclin was added at 4 g/kg of diet for the two last weeks). In each environment, 4 levels of dietary tryptophan were tested starting with a basal diet without addition of free tryptophan. The three other steps of tryptophan were achieved by addition of free tryptophan to the basal diet (Table 3 & Appendix). weeks postweaning Phase 1 Phase 2 Phase 3 0 to 3 3 to 5 5 to 8 weight, kg 8 to 13 13 to 23 23 to 31 diet type Prestarter Starter Table 3 : Design: repartition of animals according to weight, phase and type of diet. RESULTS Sanitary status significantly impacted on feed intake and growth in all phases. During the two first phases (phases 1+2), in the clean and in the dirty environments, average daily gain was maximized for the third step of dietary tryptophan meaning a ratio SID Trp:Lys at 21% in phase 1 and 2. Feed intake was significantly increased with tryptophan addition (P < 0.02) during phase 2 and phase 3. The interaction between dietary tryptophan and health status was not significant. However, it is worth noting that during the two first phases (phases 1+2), increasing dietary tryptophan from the first step to the third induced an increase in weight gain by 9% in the clean environment and by 12% in the dirty environment. Looking at the whole experimental period (phases 1+2+3), increasing dietary tryptophan from the first step to the third induced an increase in weight gain of 6% and 16% respectively in the clean and in the dirty environment Figure 10. The effect of tryptophan on growth was partly mediated through an increase in feed intake.

9 a) Moreover, increasing dietary tryptophan from step 1 to 3 decreased the difference of average daily gain between clean and dirty environment Figure 11. Plasma tryptophan concentrations 6 and 43 days after weaning were significantly lower (P < 0.05) in pigs kept in poor sanitary conditions than in pigs reared in clean environment Figure 12. Although the interaction between tryptophan level and environment is not significant, tryptophan accumulation with higher dietary tryptophan levels seemed to be lower in pigs kept in poor sanitary conditions. This last result confirms the hypothesis of a higher tryptophan utilisation in poor sanitary conditions. b) Figure 11 : Effect of the ratio Trp:Lys on weight gain in pigs from 8 to 33 kg with two sanitary status: dirty AGP (dirty environment and no antibiotics in the diet) or clean AGP + (clean environment and antibiotics in the diet). c) Figure 10 : Effect of the Trp:Lys ratio on average daily gain (ADG, a), feed intake (ADFI, b) and feed conversion ratio (FCR, c)(relative to best or worst performance within each sanitary status group) in pigs from 8 to 25 kg with two sanitary status: dirty AGP (dirty environment and no antibiotics in the diet) or clean AGP + (clean environment and antibiotics in the diet). Figure 12 : Effect of the ratio Trp:Lys on plasma tryptophan concentration in pigs (43 days after weaning) with two sanitary status: dirty AGP (dirty environment and no antibiotics in the diet) or clean AGP + (clean environment and antibiotics in the diet). CONCLUSION Sanitary status is a significant factor of variation of the tryptophan requirement in piglets. In poor sanitary conditions, a deficiency in tryptophan affects the growth of piglets more severely. Additional dietary tryptophan, through L-Tryptophan supplementation, partly prevents the consequences of poor sanitary conditions on growth rate. AJINOMOTO EUROLYSINE S.A.S. INFORMATION N 29

10 Synthesis of the results and discussion Despite lower growth performance when health status deteriorates or when AGP are removed from the feed, the requirement for amino acids rather increases. In growing finishing pigs, it is shown that not only balanced lysine but also threonine requirement as such is higher in pigs fed a diet without salinomycin compared to pigs fed the diet with 30 ppm of salinomycin. In addition, in piglets reared in poor sanitary conditions balanced lysine and tryptophan requirement for optimal growth is not changed, however for both amino acids, a deficiency will affect more severely the performance parameters of the piglets with low health status compared to high health status. Furthermore, supplying adequate levels of these amino acids results in a decrease in the difference between the groups differing in sanitary status. Consequently, adequate supply of amino acids in conditions of poor sanitary status, and also when AGP are removed from the feed, helps to reduce the impact of a decrease in feed intake, change in nutrient utilisation and to maintain a correct level of growth. In piglets, maintaining adequate amino acids level helps to preserve performances in poor health conditions In growing-finishing pigs, AGP withdrawal SID Thr:Lys 65% SID Trp:Lys 22% Amino acid requirement Lys +8% SID Thr:Lys 70% GENERAL CONCLUSION In growing pigs fed diets without AGP, despite lower growth performance, lysine and threonine requirements are higher. In piglets, the magnitude of the response to increased levels of dietary amino acids on performance parameters is higher in poor sanitary conditions or without AGP. Adequate AA supply is even more important to consider in low health status and animals fed diets without AGP.

APPENDIX 11 Composition and main nutritional values of the experimental diets C. Piñeiro N. Le Floc'h P.Bikker (Lysine trial 2) P.Bikker (Threonine) Stage Prestarter Starter Prestarter Starter Grower / Finisher Grower Finisher Feed composition, % Wheat 8 13.8 19 36 34.1 Corn 5.9 23.1 28.9 54.6 1.50 Cooked corn 25.8 1.5 Barley 15.1 30.4 37.5 25 Pea 19 20 10 Tapioca 40-27 Rye 15 Corn gluten meal 5.7 1.5 0-1.80 Wheat middlings 10 Soybean meal 2.1 17 19 18.5 4.5-15.5 12 8.5 Rapeseed meal 2.70 Sunflower meal Palmoil byproducts 2 Extruded soybean 14 5 Soybean hulls 5 8.9 Molasses 0.49-0.79 4 4 4 Dried Whey 13.50 20 Delactosed whey 5 Fish meal 7.5 4.1 0.7 Soya oil 0.40-0 1 0.85 Oil 1 3 Animal fat 3.5 2.13-3.3 1.6 1.75 Calcium carbonate 0.82 0.82 1.47 1.93 0.19-0.25 1.07 0.63 Salt 0.36 0.36 0.19-0.18 0.26 0.26 Calcium phosphate 1.8 1.98 0.14-0.01 0.68 0.27 Dicalcium phosphate 0.97 Others (minerals, vitamins, enzymes ) 0.38 0.38 0.5 0.5 1.24-1.30 0.4 0.4 Amino acid, % L-Lys HCl 0.37-0.81 0.27-0.70 0.4 0.35 0.049-0.063 0.31 0.25 L-Thr 0.17-0.41 0.13-0.36 0.07 0.1 0.018-0 0-0.15 0-0.12 DL-Met 0.192-0.37 0.15-0.37 0.17 0.15 0.006-0.25 0.11 0.07 L-Trp 0.09-0.15 0.04-0.12 0-0.105 0-0.09 0.002-0 0.02 0.02 Crude protein, % 19.6 19 21.5 19 11.6-17.5 15.3 13.3 SID amino acids, % Lys 1.22-1.52 1.01-1.32 1.30 1.13 0.49-0.80 0.79 0.67 Thr 0.76-0.94 0.67-0.80 0.83 0.75 0.32-0.50 0.41-0.57 0.37-0.47 Trp 0.25-0.31 0.22-0.28 0.207-0.308 0.177-0.268 0.092-0.152 0.17 0.15 AJINOMOTO EUROLYSINE S.A.S. INFORMATION N 29

12 REFERENCE LIST Bikker, P., J. Fledderus, L. Le Bellego, and M. Rovers. 2006. AGB ban effects on amino acid requirements. Feed Mix 14:22-24. Blanchard, P. J. 2005. Assessing AGP removal. Pig Progress 21:30-31. Defa, L., X. Changting, Q. Shiyan, Z. Jinhui, E. W. Johnson, and P. A. Thacker. 1999. Effects of dietary threonine on performance, plasma parameters and immune function of growing pigs. Animal Feed Science & Technology 78:179-188. Le Bellego, L.; Relandeau, C.; Van Cauwenberg, S.; 2002. Threonine requirement in pigs. Benefits of L-threonine supplementation. Ajinomoto Eurolysine Information n 26. Le Floc'h, N., D. Melchior, and B. Sève. Effet de la détérioration du statut sanitaire et de la teneur en tryptophane de l'aliment sur les performances de croissance des porcelets après le sevrage. 37[37 èmes Journées de la Recherche Porcine], 231-238. 2005. ITP - INRA. Journées de la Recherche Porcine. 1-2-2005. Le Floc'h, N. Impact of alteration of sanitary status on growth performance, feed intake and tryptophan metabolism in weaned pigs fed with different levels of tryptophan. INRA SENAH. Trial report n 3 - Contract B 04462, 1-20. 2006. Melchior, D., B. Sève, and N. Le Floc'h. Chronic lung inflammation affects plasma amino acid concentrations in pigs. Journal of Animal Science 82[4], 1091-1099. 2004. Melchior, D., N. Le Floc'h, and C. Relandeau. 2005. Tryptophan requirements and health status of piglets. In: P. C. Garnsworthy and J. Wiseman (Eds.) Recent Advances in Animal Nutrition 2005. pp. 43-50. Notthingham University Press, Nottingham. Mellor, S. 2004. Overcome EU hurdles without controversy. Feed Mix 12:27-29. Piñeiro, C. N. Effect of lysine and threonine supply on performance in weaned piglets. Pig Champ Pro Europa. 1-28 + Annexes p. 1-69. 2005. Indukern, S.A. Williams, N. H. and T. S. Stahly. Impact of immune system activation on the lysine and sulfur amino acid needs of pigs. ISU. ASL-R1268, 31-34. 1995. ISU Swine Research Report. Yoo, S. S., C. J. Field, and M. I. McBurney. 1997. Glutamine supplementation maintains intramuscular glutamine concentrations and normalizes lymphocyte function in infected early weaned pigs. J. Nutr. 127:2253-2259. Melchior D., Le Bellego L. & Relandeau C., June 2006 Art mony Graphic - 01 44 08 02 20 www.ajinomoto-eurolysine.com 153, RUE DE COURCELLES 75817 PARIS CEDEX 1 TEL. (33) 01 44 40 12 12 FAX (33) 01 44 40 12 13