AMINONews. Information for the Feed Industry Special Edition ValAMINO August Editorial

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1 AMINONews Information for the Feed Industry Special Edition ValAMINO August 2016 Editorial Requirements and optimum dietary branched-chain amino acids to lysine ratios for pigs Page 2 14 Effect of dietary tryptophan and valine to lysine ratios on growth performance of 8 to 25 kg pigs Page Requirement of valine and optimal valine to lysine ratio in diets for 8 to 18 kg pigs Page Dear Reader, ValAMINO is now commercially available. This new milestone shows the commitment of Evonik to the feed and meat industries where we aim to provide solutions that increase efficiency and ensure sustainability. Bringing a product to the market is one piece of the puzzle. For Evonik as a science based company, we also want to progress the knowledgebase surrounding our products. We firmly believe that it is our responsibility to understand the metabolism and physiology of the animal in order to translate this knowledge into practical recommendations. Publishing information that you can apply in your daily operation is the recipe for success. In this AMINONews special issue you will find two review articles on the valine requirements of broilers and swine. This issue also contains some Facts & Figures articles reporting the results of experiments we conducted with ValAMINO. Enjoy reading Isoleucine requirement in a diet for broilers from 15 to 29 days of age Page Branched-chain amino acids in broiler nutrition Page Requirement of valine in a diet for broilers from 15 to 29 days of age Page Vincent Hess

2 2 PIG Requirements and optimum dietary branchedchain amino acids to lysine ratios for pigs Key information Based on the published literature, the average optimal standardized ileal digestible (SID) isoleucine to lysine ratio (Ile:Lys) in swine diets is 55 % when leucine (Leu) is not in excess. This estimate was consistent across different pig body weights, genetics and experimental conditions. An excess of Leu could lead to branched-chain amino acids (BCAA) antagonism resulting in increased catabolism of the other BCAA by stimulation of BCAA degrading enzyme, and consequently reduces their availabilities. The dietary SID Ile:Lys should be at least 60 % to maintain pig performance when diets contain excess Leu (i.e. SID Leu:Lys > 140 %). The published optimal SID valine (Val) to Lys ratio estimates were rather consistent among studies and an average optimal SID Val:Lys of 68 % is recommended in pig diets. Research showed that excess dietary Leu seems to have little effect on the utilization of Val. Because ingredients that are high in Leu are also high in Val, the impact of excess Leu on Val requirement is of less importance compared with the Ile requirement. Based on a few published data, the optimal SID Leu:Lys ratio of % seems to be adequate for pig diets. Although practical swine diets are unlikely to be deficient in Leu, more research is needed to re-evaluate Leu requirements, particularly the upper limit for Leu at which it becomes excess level and reduces performance. Least-cost formulations showed that Val becomes the next limiting amino acid before Ile (after Lys, threonine, methionine and tryptophan) in typical low protein US- and European-type diets for pigs. Depending on the ingredients used and the optimal ratios of Val and Ile used in diet formulations, Val and Ile may also be co-limiting in low CP diets. Introduction Pig producers have been facing major challenges in recent years including drastic increases in feed costs and changes in available ingredients which are largely impacted by the increased use of grains for the growing human population and for the biofuel production. At the same time, there is a growing environmental concern to reduce the excretions of nitrogen (N) and carbon footprint from animal production. Lowering the dietary crude protein (CP) content, while balancing with supplemental amino acids (AA) for an ideal protein (IP) ratio, is an established method to mitigate N excretion by swine. Lowering dietary CP can also improve gut health of weaned pigs by redu-cing the production of harmful microbial metabolites in the hindgut. Until recently, only four supplemental AA [Lys, threonine (Thr), methionine (Met) and tryptophan (Trp)] in a feed grade form were available for the feed industry. Now, Val is also available in the market and Ile has been recently permitted for use in the European Union (EU). The availability of these six supplemental AA gives nutritionists the opportunity to further reduce the dietary CP level and to meet animal requirements more precisely. This also gives more flexibility to take advantage of using cheaper raw materials to reduce the feed costs. The rising costs of conventional protein sources such as soybean meal (SBM) and fish meal have increased interest in using alternative protein sources including spray-dried blood cells (SDBC), a co-product of porcine plasma production, mainly in starter pig diets. Although SDBC contain high contents of Leu, Lys, and Val, a major disadvantage is their very low level of Ile (AMINODat 4.0, 2010). Pig performance was reduced when SDBC was included above 2 % in the diets (i. e. Kerr et al. 2004a), and this reduction was due to the BCAA antagonism (Harper et al., 1984). Research showed that Val or/and Ile become limiting after Lys, Thr, Met and Trp in low CP diets for pigs (i. e. Liu et al., 2000; Lordelo et al., 2008). Whether Val or Ile becomes the next limiting AA depends on the ingredients used and the IP ratio of Val and Ile used in the least cost feed formulations, and in many cases, they are co-limiting in low CP pig diets. With Val and Ile being available in the markets coupled with the use of SDBC, there is a need to re-evaluate the requirements and optimal ratios of Ile and Val. Because of the potential BCAA antagonism, it would be meaningful to discuss this issue for all three BCAA together. Therefore, the objective of this article was to review the requirements of BCAA and their dietary optimal ratios relative to Lys for pigs of various body weight (BW) categories based on published literature.

3 3 PIG Requirements of branched-chain amino acids in pigs Research on the Leu requirement is almost nonexistent, mainly because Leu has been thought unlikely to be deficient in typical pig diets. Research evaluating the requirements of Ile and Val in pigs is limited with most of the works having been conducted in the last decade. To come up with average requirement estimates of BCAA for pigs with varying BW ranges, we have gathered the relevant information from all available published papers and combined that with Evonik s in-house research. For a better comparison among studies and for providing results more consistently, the dietary levels as well as the requirement/ ratio estimates of Ile, Val and Leu are expressed on the SID basis. To do this, SID values were calculated as necessary from total values based on the established ingredient SID coefficients (AMINODat 4.0, 2010) or from apparent ileal digestible (AID) values corrected for endogenous loss (Rademacher et al., 2009). Optimal SID leucine to lysine ratios Eggert et al. (1954) first reported that the total Leu requirement for 2 5 wk old pigs fed a casein AA based diet was between 1.00 and 1.25 %. More recent research utilizing purified diets estimated the dietary optimal Leu:Lys of 100 % for kg starter pigs (Chung and Baker, 1992) and 110 % for kg growing pigs (Wang and Fuller, 1989). Augspurger and Baker (2004) using corn-peanut meal-whey-sbm based diets determined the SID Leu requirement for kg pigs to be approximately 1.05 % which corresponds to a SID Leu:Lys of 100 %. There seem to be optimal ratios between the three BCAA. The NRC (1998), using a factorial approach, estimated the dietary optimal SID ratios of Ile, Leu, and Val to Lys at 55, 100 and 68 %, respectively, with the corresponding optimal ratio for Ile:Leu:Val of 100:185:126. Using the indicator AA oxidation technique, Elango et al. (2004) reported that the optimal ratios among Ile:Leu:Val of 100:180:120 in diets for neonatal pigs (~ 1.5 kg initial BW) was adequate which agrees well with the NRC (1998) recommendations. Based on these data, the optimal SID Leu:Lys of 100 to 110 % seems to be adequate for pig diets. Effects of excess Leu on the requirements of Ile and Val In a typical corn-sbm based swine diet, BCAA comprise about 40 % of the total essential amino acids (EAA), and thus, it has been a common assumption that excess rather than deficiency of BCAA is more likely to occur (Harper et al., 1984). It is well known that excess Leu reduced feed intake and performance of pigs (i. e. Gatnau et al., 1995). Spray-dried blood cell contains a high level of Leu (12.2 %) and Val (8.1 %) but an extremely low content of Ile (0.35 %; AMINODat 4.0). Research showed that a low level of SDBC inclusion (up to 2 3 %) did not affect performance but performance was reduced when SDBC was included higher than 3 4 % in weaned pig diets and 4 5 % in finisher diets (Kerr et al. 2004a; Fu et al. 2006a; Hinson et al., 2007; Fruge et al., 2009). Supplementing L-Ile to the diets to increase the dietary SID Ile:Lys up to % fully restored pig performance (Kerr et al., 2004a; Hinson et al., 2007). Dietary inclusion of SDBC increased not only Leu and Val but also phenylalanine (Phe) and histidine (His) levels. Thus, the growth-depression effect could also be due to simultaneous excess levels of all AA. However, adding Leu only or all excess AA (Leu, Val, His, and Phe) to a corn-sbm diet resulted in reduced performance similar to that of pigs fed corn-sdbc diets (Fu et al., 2006a), which suggests that impaired performance was mainly due to excess Leu level. The underlying mechanism for impaired performance is because the addition of SDBC leads to excess level of Leu which then increases the catabolism of the other BCAA by stimulation of the BCAA degrading enzyme (BCAA transaminase) mainly in skeleton muscles and consequently reduces the amount of available Ile and Val.

4 4 PIG Regarding the interaction between Leu and Val, Fu et al. (2006a) found that adding Val alone or with Leu to corn-sbm control diet did not affect performance. Similarly, Langer and Fuller (2000) reported that increasing the dietary Val level alleviated the negative effect of excess Leu in Ile limiting diet on N-utilization of growing pigs, and excess Leu in a Val-deficient diet did not reduced the N-utilization. More recently, Gloaguen et al. (2010) confirmed that excess dietary Leu (SID Leu:Lys of 165 %) did not affect the performance of kg pigs when the dietary SID Val:Lys was maintained at 70 %. Overall, the excess dietary level of Leu has a greater negative impact on the utilization of Ile than that of Val. This suggests that the dietary requirement of Ile or optimal Ile:Lys is increased when diets contain excess level of Leu. Isoleucine requirements Isoleucine requirement estimates for pigs are summarized in Table 1. In these requirement studies, other EAA, with the exception of Ile, met or exceeded the requirement of the pigs of the corresponding BW. Some researchers used SDBC in their diets to obtain Ile-deficient diets, and this may have resulted in excess Leu, and influenced the Ile requirement estimates (Table 1). Table 1 Review of SID Ile requirement estimates for pigs of various body weight categories. BW Breed 1 ADG FI Level of AA in test diets, % 2 SID Ile requirement 3 Model 4 Reference kg g/day g/day SID Lys SID Val SID Leu % of diet mg/g ADG 5 12 H BLQ Becker et al., PIC BL James et al., PIC BLQ Kerr et al., 2004b 5 10 Average: Y BLQ Oestemer et al., PIC BL Fu et al., 2006b Average: n/a , n/a 5 n/a n/a 5 Lenis & van Diepen PIC 727 1, BLQ Parr et al., Average: H 758 2, BLQ Becker et al., D 606 1, BLQ Becker et al., Average: PIC 725 1, BL Parr et al., PIC 1,226 3, BL Kendall et al., LW/LR 842 3, BLQ Dean et al., Average: D = Duroc, H = Hampshire, LR = Landrace, LW = Large white, and Y = Yorkshire, PIC = pig improvement company 2 When SID value was not given, values from AMINODat 4.0 (2010) were used to derive AA content on SID basis 3 Requirements of Ile were estimated by taking the average of ADG, FCR and/or ADFI 4 BL = Broken-line, BLQ = Broken-line quadratic 5 n/a = Not available

5 5 PIG Becker et al. (1963) first reported the Ile requirement for 5 12 kg pigs fed dextrose-blood meal based diets to be 0.73 % based on average daily gain (ADG) and gain:feed (G:F) responses. James et al. (2000) estimated the SID Ile requirement to optimize performance in 6 9 kg pigs fed a corn- SBM-whey-based diet containing 4 % SDBC was 0.69 %. More recently Kerr et al. (2004b) determined the SID Ile requirement of 7 11 kg pigs fed a corn-sbm-whey-based diet containing 7.5 % SDBC was 0.73 % based on an average of performance and plasma urea nitrogen (PUN) data. In 6 14 kg pigs, Oestemer et al. (1973) determined the optimal SID Ile level to be 0.63 % using a cornstarch-glucose-blood flour-based diet. Based on G:F responses, Fu et al. (2006b) came up with the SID Ile requirement of 0.66 % for kg pigs fed corn-sbm-based diets. In kg growing pigs, Lenis and van Diepen (1997) estimated the SID Ile requirement to be 0.57 % based on ADG and feed conversion ratio (FCR) responses. Using corn-sbm diets containing 7.5 % SDBC, Parr et al. (2003) reported that the SID Ile requirement was 0.50 % for kg growing pigs based on ADG and average daily feed intake (ADFI) responses. Published Ile requirements for growing pigs from kg BW are outdated. Based on the ADG and G:F responses, Becker et al. (1963) estimated the SID Ile requirement to be 0.48 % for kg pigs fed cornstarch-glucose-blood flour-based diets. In another trial, the authors estimated a slightly lower SID Ile requirement of 0.37 % for kg pigs fed corn-starch-dextrose-blood flour based diets. Parr et al. (2004) estimated the SID Ile requirement to be 0.31 % in kg finishing pigs that were fed corn- SBM diet that contained 5.0 % SDBC. Kendall et al. (2004) estimated the SID Ile requirement of 0.36 % in kg pigs fed diets that contained corn and 5.0 % SDBC. Dean et al. (2005) also reported the SID Ile requirement of 0.36 % for kg pigs fed corn-sdbc (5.0 %) diets. Conclusion: Overall, the average SID Ile requirement estimate was 0.71, 0.65, 0.54, 0.43 and 0.34 % for growing-finishing pigs with a BW range of 5 10 kg, kg, kg, kg and kg, respectively. These estimates were slightly higher than the NRC (1998) requirement estimates of 0.65, 0.55, 0.45, 0.37 and 0.29 %, respectively for the corresponding BW categories. Optimal SID isoleucine to lysine ratios A brief overview of the optimal dietary Ile:Lys ratio estimates are given in Table 2. In the diets used in these studies, Ile was first limiting and Lys was second limiting to make sure that the estimated Ile ratio was not underestimated. It should be mentioned that about half of the studies used no or little inclusion of SDBC in the diets resulting in a normal level of Leu while the remaining utilized a higher level of SDBC resulting in excess Leu.

6 6 PIG Table 2 Review of optimal SID Ile:Lys ratio estimates for pigs of various body weight categories. BW Breed 1 Lys Leu: Lys kg Leu+Val: Lys SDBC % Response criteria Optimal Ile:Lys SID, 2 % SID basis %, SID Model 3 Reference 6 9 PIC ADG, ADFI, G:F 54 BLQ James et al., PIC ADG, ADFI, G:F 61 BLQ Kerr et al., 2004b 8 25 LR x P ADG, ADFI 54 BLQ Wiltafsky, et. al., 2009a Y ADG 51 n/g 4 Htoo et al., PIC ADG 56 BL Bergstorm et al., PIC ADG 55 BL Bergstorm et al., LR x P ADG, ADFI 59 BL Wiltafsky et al., 2009a PIC G:F 62 BL Fu et al., 2006b PIC ADG, ADFI, G:F 55 E Lindemann et al., PIC ADG, G:F 56 BL, BLQ Fu et al., 2005a PIC ADG, G:F 60 BL, BLQ Fu et al., 2005a PIC ADG, FI 53 BL Fu et al., 2005b PIC ADG, G:F 60 BL Fu et al., 2005b PIC ADG, G:F 62 BL, BLQ Fu, LR = Landrace, P = Pietrain, and Y = Yorkshire, PIC = pig improvement company 2 When SID value was not given, values from AMINODat 4.0 (2010) were used to derive AA content on SID basis 3 BL = Broken-line, BLQ = Broken-line quadratic, E = Exponential model (95 % of maximum response) 4 n/s = Not suitable for regression analysis James et al. (2000) determined the optimal SID Ile:Lys to be 54 % in 6 9 kg pigs fed corn-srmwhey and SDBC (4 %) based diets (SID Leu:Lys of 1.38). Kerr et al. (2004b) reported the SID Ile:Lys of 61 % to maximize performance of 7 10 kg pigs fed corn-sbm-whey-sdbc (7.5 %) diets (SID Leu:Lys of 1.68). Recently, Wiltafsky et al. (2009a) utilized a corn-barley-corn gluten feed diet (SID Leu:Lys of 1.11) and determined that the optimal SID Ile:Lys was 54 % based on ADG and FI responses of 8 25 kg pigs. Similarly, Htoo et al. (2009) reported that the performance of kg pigs was maximized at SID Ile:Lys ratio of 51 % in a wheatbarley-sbm-sdbc (3.7 %) diet ( SID Leu:Lys of 1.29). Bergstrom et al. (1997) evaluated the optimal Ile ratio in starter pigs at two dietary Lys levels. Based on ADG responses, the optimal SID Ile:Lys in kg pigs was estimated to be 56 % when the diet contained 0.81 % SID Lys (SID Leu:Lys of 1.31) and 55 % when the dietary SID Lys was 1.18 % (SID Leu:Lys of 110 %). In another trial, Wiltafsky et al. (2009a) determined the effect of a high Leu on the Ile:Lys ratio in 8 25 kg pigs fed a wheat-barley-corn-sdbc (7.5 %) diet ( SID Leu:Lys of 159 %). Based on the responses of ADG and FI, an optimal SID Ile:Lys was estimated to be 59 %. Similarly, Fu et al. (2006b) reported an optimal SID Ile:Lys of 62 % in kg pigs fed diets added with 10.7 % SDBC ( SID Leu:Lys of 181 %). In kg pigs, Lindemann et al. (2010) determined the optimal Ile ratio using wheatbarley-sbm-sdbc (4.5 %) diets (SID Leu:Lys of 129 %). Based on the average of ADG, FI and G:F responses, the optimal SID Ile:Lys was 55 % for these pigs.

7 7 PIG In kg pigs, the ADG and G:F were optimized at the SID Ile:Lys of 56 % when corn-sbm diets contained SID Leu:Lys of 123 % (Fu et al., 2005a). However, the optimal SID Ile:Lys was estimated to be 60 % when 7.15 % SDBC was added to the corn-sbm diets to create an excess level of Leu (200 % SID Leu:Lys). In kg pigs, Fu et al. (2005b) determined the SID Ile:Lys was estimated to be 53 % using corn-based diets (SID Leu:Lys of 148 %). However, they found in another trial that the optimal SID Ile:Lys was increased to 60 % when 3.9 % SDBC was added to the corn-based diets with SID Leu:Lys of 238 % in kg pigs. Furthermore, Fu (2005) estimated the optimal SID Ile:Lys to be 62 % in kg pigs when 3.9 % SDBC was added to the corn-based diets that contained an excess level of Leu (SID Leu:Lys of 238 %). Conclusion: It should be mentioned that increased dietary needs of SID Ile:Lys caused by excess Leu were consistent across different BW groups, genetics and experimental conditions. Based upon the tested levels of AA (12 dose response data sets) mentioned in Table 2, a regression equation: y = x x (R 2 = 0.86; where x = SID Leu:Lys, and y = SID Ile:Lys in %), can be developed for estimating an optimal Ile:Lys ratio (Figure 1). This suggests that the optimal Ile ratio is not constant and affected by the dietary Leu level. To come up with a precise estimate of the upper threshold level of Leu at which it becomes an excess level is difficult as it may depend on the levels of Ile and Val in the diets. For practical applications, the optimal SID Ile:Lys ratio is recommended at 55 % when diets contain a normal Leu level (i. e. SID Leu:Lys of %). However, a higher SID Ile:Lys ratio of at least 60 % is recommended when diets contain an excess Leu level (i.e. SID Leu:Lys of > 140 %). SID Ile:Lys ratio [%] Figure 1 Effect of SID Leu:Lys level on optimal SID Ile:Lys ratio (Source: Table 2) y = x x R = SID Leu:Lys ratio [%] Valine requirements A summary of Val requirement estimates are given in Table 3. In a series of dose response trials utilizing corn-sbm-whey diets, Mavromichalis et al. (2001) reported that the SID Val requirement of 6 10 kg pigs to optimize ADG was 0.86 %. Based on the ADG responses, the authors estimated the SID Val requirement for kg pigs to be 0.78 %. More recently, Gaines et al. (2006) determined the Val requirement of 8 12 kg starter pigs that were fed a corn-sbm based diet to be 0.92 %. Gaines et al. (2006) also reported that the SID Val requirement was 0.78 % to optimize performance of kg pigs. In 8 21 kg pigs, Warnants et al. (2001) determined the requirement of Val by feeding corn, SBM, tapioca, barley, whey and fish meal-based diets. Using graded levels of IP wherein Val appeared to be the first limiting AA, the SID Val requirement to optimize ADG and FCR was 0.70 %. Liu et al. (2000) reported that the SID Val requirement was 0.37 % in kg pigs. Based on ADG, G:F and PUN responses, Lewis and Nishimura (1995) estimated the SID Val requirement to be 0.42 % for kg pigs fed corn-corn starch-gelatin diets.

8 8 PIG Table 3 Review of SID Val requirement estimates for pigs of various body weight categories. BW Breed 1 ADG FI, SID Lys 2 Optimal SID Val Model 3 Reference kg g/day % % mg/g gain 6 10 PIC BL Mavromichalis et al., PIC BL Gaines et al., Average: P LW/LR BL Warnants et al., PIC BL Mavromichalis et al., PIC BL Gaines et al., Average: n/a 4 n/a n/a 4 n/a 4 Lui et al.., LW/LR D Q Lewis & Nishimura, Average: D = Duroc, LR = Landrace, LW = Large white, and P = Pietrain, PIC = pig improvement company 2 When SID value was not given, values from AMINODat 4.0 (2010) were used to derive AA content on SID basis 3 BL = Broken-line, and Q = Quadratic 4 n/a = Not available Conclusion: Based on these results, the average SID Val requirement estimates were 0.86, 0.75, and 0.40 % for pigs with BW range of 5 10 kg, kg, and kg, respectively. The estimates for starter pigs were slightly higher than the NRC (1998) estimates of 0.81 and 0.69 % respectively for 5 10 kg and kg pigs. Optimal SID valine to lysine ratios The overview on the dietary Val:Lys ratio estimates are given in Table 4. In these studies, Val was first limiting and Lys was second limiting in the diets used. The estimation of optimal Val ratios has been focused only in 8 33 kg BW and trials were conducted in the last decade. Empirical data for finishing pigs are lacking. Table 4 Review of optimal SID Val:Lys ratio estimates for 8 33 kg pigs. BW Breed 1 Lys % kg Leu:Lys SID basis Leu+Ile: Lys Response criteria Optimal Val:Lys SID, % Model 2 Reference 8 21 P LW/LR ADG, FCR 68 BL Warnants et al., n/a n/a 3 n/a 3 ADG, FCR 73 4 n/s 5 Theil et al., LR x P ADG, ADFI 67 BL Wiltafsky et al., 2009b 9 22 n/a n/a 3 ADG, FCR 69 Q Trautwein et al., LR P ADG, ADFI, FCR 71 BLQ Trautwein et al., P LW/LR ADG, ADFI, FCR 71 BL Barea et al., PIC 1.10 n/a 3 n/a 3 ADG, G:F 65 BL Kendall et al., LR P N-retention 65 BL Wiltafsky et al., 2009b PIC ADG, G:F 65 BL Gaines et al., PIC 1.10 n/a 3 n/a 3 ADG, G:F 66 BL Kendall et al., 2004 Average: 68 1 LR = Landrace, LW = Large white, and P = Pietrain, PIC = pig improvement company 2 BL = Broken-line, BLQ = Broken-line quadratic, and Q = Quadratic 3 n/a = Not available 4 SID values were converted from AID values by correcting endogenous loss (Rademacher et al., 2009) 5 n/s = Not suitable for regression analysis

9 9 PIG Warnants et al. (2001) estimated the optimal supply of Val in 8 21 kg pigs fed corn, SBM, tapioca, barley, whey, fish meal-based diets with graded levels of IP wherein Val appeared to be the first limiting AA. Based on the average of ADG and FCR responses, the SID Val:Lys ratio was estimated at 68 %. Theil et al. (2004) utilized barley-wheat-soy protein concentrate diets and determined that the performance in 8 17 kg pigs was maximized at a SID Val:Lys of 73 %. Recently, Wiltafsky et al. (2009b) utilized both growth assay and N-balance technique to evaluate the optimal Val ratio in starter pigs fed wheat-corn-barleypea-whey based diets. The ADG and FCR were optimized at a SID Val:Lys of 67 % in 8 22 kg pigs, and N-retention was maximized at an optimal SID Val:Lys of 65 % in kg pigs. Unlike the Ile ratio studies, SDBC was not used in the Val ratio studies and the tested Leu levels were not at excess (SID Leu:Lys ranges from %). As mentioned previously, excess dietary Leu seems to have little effect on the utilization of Val (Gloaguen et al., 2010). Because raw materials that are high in Leu are also high in Val (AMINODat 4.0, 2010), the impact of excess Leu on Val requirement is of less importance compared with Ile requirement. Nevertheless, more research is needed to draw a reliable conclusion on the effect of excess Leu on Val-requirement in pigs. Conclusion: Overall, the estimated optimal SID Val:Lys is rather consistent among studies and averaged at 68 % (62 73 %) for starter and growing pigs. Based on these results, a SID Val:Lys of 68 % is recommended in swine diets. Results of Trautwein et al. (2009) showed that maximal ADFI and ADG of 9 22 kg pigs were achieved at a SID Val:Lys of 68 and 70 %, respectively. In another study, Trautwein et al. (2009) found that the average SID Val:Lys ratio to optimize the ADG, ADFI and FCR of kg pigs was 71 %. Using corn-wheatbarley-sbm diets, Barrea et al. (2009) estimated an optimal SID Val:Lys of approximately 71 % based on the average optimal of ADG, ADFI and G:F of 8 25 kg starter pigs. Recently, Gaines et al. (2011) determined the optimal SID Val:Lys to be 65 % in kg pigs fed corn-sbm based diets. Kendall et al. (2004) conducted two dose response growth trials using starter pigs fed corn- SBM based diets. Based on ADG and G:F responses, the optimal SID Val:Lys ratio was 66 % in kg pigs, and 65 % in kg pigs, respectively.

10 10 PIG Figure 2 Considerations for practical application of low protein diets The level to which CP can be lowered in a pig diet is dictated by the next AA that becomes limiting for performance. In order to know how low CP can be reduced in diets fortified with supplemental AA, least cost diets were formulated (corn-sbm based US-type or wheat-barley-sbm-based European type diets) for different pig BW ranges [pre-starter (PS): 5 10 kg, starter (S): kg; grower (G): kg; finisher (F): kg] as shown in Table 5. Diets were formulated to meet the SID Lys and IP recommendation of Evonik (2009). All diets were formulated to contain adequate levels of Leu and ratios of SID Ile and Val to Lys were kept constant at 55 and 68 %, respectively. For the current formulations, ingredient prices of August 2010 (Feedinfo) and L-Val and L-Ile prices of $/kg and US$/kg, respectively were used. Content of branched-chain amino acids in selected ingredients (AMINODat 4.0, 2010). Based upon the nutrient specifications given in Table 5, Val becomes the 5th limiting AA (after Lys, Thr, Met, Trp) followed by Ile as the 6th limiting AA in all US-type diets. In all European-type diets, Val, Ile and Leu, respectively, become the 5th, 6th and 7th limiting AA (after Lys, Thr, Met, Trp). Least-cost formulations showed that the CP levels can be kept as low as 18.5, 16.9, 13.7 and 12.0 %, respectively (without resulting in AAdeficiency), by using six supplemental AA (Lys, Met, Thr, Trp, Val, Ile) in the US-type pre-starter, starter, grower and finisher diets. Similarly, the CP levels can be lowered to 19.5, 18.6, 15.0 and 12.4 % in the corresponding European-type diets (Table 5). Because of growth-depressing effect of excess Leu, nutritionists should watch for the Leu level in their diets. Some ingredients used in piglet diets particularly SDBC, blood meal, and corn gluten meal contain a very high level of Leu while Ile content is extremely low (Figure 2), which may result in excess Leu with their uses in the diets. Conclusion: Based on the specifications for prestarter diets mentioned in Table 5, inclusions of SDBC at 4 %, blood meal at 5 % or corn gluten meal at 5 % can lead to unexcess Leu level (i.e. SID Leu:Lys of > 140 %) in the US-type pre-starter diet (Table 6). The inclusion level of SDBC, blood meal and corn gluten meal at which the Leu level becomes excessive in the European pre-starter diet are slightly higher at 6, 8 and 8 %, respectively (Table 6). This is because the content of Leu in wheat and barley are lower relative to their Ile and Val content compared with corn (Figure 2). In fact, Leu becomes limiting (after Lys, Thr, Met, Trp, Val and Ile) in low CP wheat-barley based diets. As noted above, excess Leu in the diet increases the nutritional need of Ile. Thus, the SID Ile:Lys should be increased to at least 60 % when the diets contain excess Leu in order to maintain pig performance.

11 11 PIG Table 5 Composition of low protein, AA-supplemented swine diets. US-type diets European-type diets PS 1 S 2 G 3 F 4 PS 1 S 2 G 3 F 4 Corn Wheat Whey powder Barley Skim milk powder Wheat middlings Soybean meal, 48 % Spray-dried blood plasma 8.35 Tallow/Soy oil DCP, limestone, salt Premix Biolys DL-Met L-Thr L-Trp L-Val L-Ile Calculated nutrient composition NE (MJ/kg) CP, % (20.8) 16.9 (19.5) 13.7 (15.0) 12.0 (12.6) 19.5 (21.0) 18.6 (19.7) 15.0 (15.9) 12.4 (12.9) SID Lys, % SID Val:Lys, % SID Ile:Lys, % PS:Pre-starter (5 10 kg) 2 S:Starter (10 20 kg) 3 G:Grower (20 50 kg) 4 F:Finisher ( kg) 5 Values given in the parenthesis are the minimum CP levels (without AA-deficiency) by using Lys, Met, Thr and Trp Table 6 Inclusion level (%) of selected ingredients at which Leu level becomes excessive in pre-starter diets. US-type pre-starter diets European-type pre-starter diets Raw materials inclusion % SID Leu:Lys, % CP, % % SID Leu:Lys, % CP, % Spray-dried blood cell (> 1.4) (> 1.4) 23.0 Blood meal (> 1.4) (> 1.4) 23.0 Corn gluten meal (> 1.4) (> 1.4) 23.0

12 12 PIG References AMINODat 4.0. Platinum version (2010): Evonik Degussa GmbH, Hanau-Wolfgang, Germany. Augspurger, N. R. and D. H. Baker (2004): An estimate of the leucine requirement for young pigs. Animal Science 79: Barea, R., L. Brossard, N. Le Floc h, Y. Primont, D. Melchior, and J. van Milgen (2009): The standardized ileal digestible valine to lysine requirement ratio is at least 70 % in post-weaning piglets. Journal of Animal Science 87: Becker, D. E., I. D. Smith, S. W. Terrill, A. H. Jensen, and H. W. Norton (1963): Isoleucine need of swine at two stages of development. Journal of Animal Science 22: Bergstrom, J. R., J. L. Nelssen, M. D. Tokach, and R. D. Goodband (1997): Determining the optimal isoleucine:lysine ratio for the SEW-reared, 10 to 20 kg pig. Journal of Animal Science 75: 60. Chung, T. K. and D. H. Baker (1992): Ideal amino acid pattern for 10-kg pigs. Journal of Animal Science 70: Dean, D. W., L. L. Southern, B. J. Kerr, and T. D. Bidner (2005): Isoleucine requirement of 80- to 120-kilogram barrows fed corn-soybean meal or corn-blood cell diets. Journal of Animal Science 83: Eggert, R. G., H. H. Williams, B. E. Sheffy, E. G. Sprague, J. K. Loosli, and L. A. Maynard (1954): The quantitative leucine requirement of the suckling pig. Journal of Nutrition 53 (2): Elango, R., L. A. Goonewardene, P. B. Pencharz, and R. O. Ball (2004): Parenteral and enteral routes of feeding in neonatal piglets require different ratios of branched-chain amino acids. Journal of Nutrition 134 (1): Evonik (2009): Recommendations for Swine ( Fruge, E. D., T. D. Bidner, and L. L. Southern (2009): Effect of incremental levels of red blood cells on growth performance and carcass traits of finishing pigs. Journal Animal Science 87: Fu, S. X. (2005): Isoleucine requirement and imbalance in swine. PhD thesis, University of Missouri, Columbia. Fu, S. X., R. W. Fent, P. Srichana, B. W. Ratliff, G. L. Gary, and J. L. Usry (2005a): Effects of protein source on true ileal digestible (TID) isoleucine:lysine ratio in pigs from 58 to 76 kg. Journal of Animal Science 83: 213. Fu, S. X., D. C. Kendall, R. W. Fent, G. L. Allee, and J. L. Usry (2005b): True ileal digestible (TID) isoleucine:lysine ratio of late-finishing barrows fed corn-blood cell or corn-amino acid diets. Journal of Animal Science 83: 67. Fu, S. X., R. W. Fent, G. L. Allee, and J. L. Usry (2006a): Branched chain amino acid interactions increase isoleucine requirement in late-finishing pigs. Journal of Animal Science 84: Fu, S. X., A. M. Gaines, R. W. Fent, G. L. Allee, and J. L. Usry (2006b): True ileal digestible isoleucine requirement and ratio in 12 to 22 kg pigs. Journal of Animal Science 84: 283. Gaines, A. M., P. Srichana, B. W. Ratliff, G. L. Allee, and J. L. Usry (2006): Evaluation of the true ileal digestible (TID) valine requirement of 8 to 20 kg pigs. Journal of Animal Science 84: 284. Gaines, A. M., D. C. Kendall, G. L. Allee, J. L. Usry, and B. J. Kerr (2011): Estimation of the standardized ileal digestible valine-to-lysine ratio in 13- to 32-kilogram pigs. Journal of Animal Science 89:

13 13 PIG Gatnau, R., D. R. Zimmerman, S. L. Nissen, M. Wannemuehlert and R. C. Ewan (1995): Effects of excess dietary leucine and leucine catabolites on growth and immune responses in weanling pigs. Journal of Animal Science 73: Gloaguen, M., N. Le Floc h, L. Brossard, Y. Primot, E. Corrent, and J. Van Milgen (2010): An excessive supply of leucine aggravates the effect of a valine deficiency in post-weaned piglets. Pages in Energy and protein metabolism and nutrition, edited by G. M. Crovetto, Wageningen Academic Publishers, The Netherlands. Harper, A. E., R. H. Miller, and K. P. Block (1984): Branched-chain amino acid metabolism. Annual Review of Nutrition 4: Hinson, R. B., G. L. Allee, and J. D. Crenshaw (2007): Use of spray-dried blood cells and isoleucine supplementation in pig starter diets. Journal Animal Science 85: 93. Htoo, J. K., C. Zhu, and C. de Lange (2009): Optimum isoleucine to lysine ratio in a barley and wheat based diet fed to starter pigs. Journal of Animal Science 87 (E-Suppl. 2): James, B. W., R. D. Goodband, M. D. Tokach, J. L. Nelssen, and J. M. Derouchey (2000): The optimum isoleucine: lysine ratio in starter diets to maximize growth performance of the earlyweaned pig. Swine day, Kansas State University. Kendall, D. C., B. J. Kerr, R. W. Fent, S. X. Fu, J. L. Usry, and G. L. Allee (2004): Determination of the true ileal digestible isoleucine requirement for 90 kg barrows. Journal of Animal Science 82: 156. Kerr, B. J., M. T. Kidd, J. A. Cuaron, K. L. Bryant, T. M. Parr, C. V. Maxwell, and E. Weaver (2004a): Utilization of spray-dried blood cells and crystalline isoleucine in nursery pig diets. Journal of Animal Science 82: Kerr, B. J., M. T. Kidd, J. A. Cuaron, K. L. Bryant, T. M. Parr, C. V. Maxwell, and J. M. Campbell (2004b): Isoleucine requirements and ratios in starting (7 to 11 kg) pigs. Journal of Animal Science 82: Langer, S. and M. F. Fuller (2000): Interactions among the branched-chain amino acids and their effects on methionine utilization in growing pigs: effects on nitrogen retention and amino acid utilization. British Journal of Nutrition 83: Lenis, N. P. and J. T. M. van Diepen (1997): Requirement for apparent ileal digestible isoleucine of young pigs. Journal of Animal Science 75: 185. Lewis, A. J. and N. Nishimura (1995): Valine requirement of the finishing pig. Journal of Animal Science 73: Lindemann, M. D., A. D. Quant, J. H. Cho, B. J. Kerr, G. L. Cromwell, and J. K. Htoo (2010): Determining the optimum ratio of standardized ileal digestible isoleucine to lysine for growing pigs fed wheat-barley based diets. Journal of Animal Science 88 (E-Suppl. 3): Liu, H., G. L. Allee, K. J. Touchette, J. W. Frank, and J. D. Spencer (2000): Effects of reducing protein and adding amino acids on performance, carcass characteristics, and nitrogen excretion, and the valine requirement of early-finishing barrows. Journal of Animal Science 78: 184. Lordelo, M. M., A. M. Gaspar, L. Le Bellego, and J. P. B. Freire (2008): Isoleucine and valine supplementation of a low-protein corn-wheatsoybean meal-based diet for piglets: Growth performance and nitrogen balance. Journal of Animal Science 86: Mavromichalis, I., B. J. Kerr, T. M. Parr, D. M. Albin, V. M. Gabert, and D. H. Baker (2001): Valine requirement of nursery pigs. Journal of Animal Science 79:

14 14 PIG NRC (1998): Nutrient Requirements of Swine, 10th revised edn. National Academy Press, Washington, DC. Oestemer, G. A., L. E. Hanson, and R. J. Meade (1973): Re-evaluation of the isoleucine requirement of the young pig. Journal Animal Science 36: Parr, T. M., B. J. Kerr, and D. H. Baker (2003): Isoleucine requirement of growing (25 to 45 kg) pigs. Journal Animal Science 81: Parr, T. M., B. J. Kerr, and D. H. Baker (2004): Isoleucine requirement for late-finishing (87 to 100 kg) pigs. Journal of Animal Science 82: Rademacher, M., W. S. Sauer, and A. J. M. Jansman (2009): Standardized ileal digestibility of amino acids in pigs. Evonik Degussa GmbH. Wang, T. C. and M. F. Fuller (1989): The optimum dietary amino acid pattern for growing pigs 1. Experiments by amino acid deletion. British Journal of Nutrition 62: Wiltafsky, M. K., J. Bartelt, C. Relandeau, and F. X. Roth (2009a): Estimation of the optimum ratio of standardized ileal digestible isoleucine to lysine for eight- to twenty-five-kilogram pigs in diets containing spray-dried blood cells or corn gluten feed as a protein source. Journal of Animal Science 87: Wiltafsky, M. K., B. Schmidtlein, and F. X. Roth (2009b): Estimates of the optimum dietary ratio of standardized ileal digestible valine to lysine for eight to twenty-five kilograms of body weight pigs. Journal of Animal Science 87: Theil, P. K., J. A. Fernandez, and V. Danielsen (2004): Valine requirement for maximal growth rate in weaned pigs. Livestock Production Science 88: Trautwein, J., G. Dusel, and J. Bartelt (2009): Releasing potentials in piglet feed. Feed Magazine. Issue 11-12: Dr. John Htoo john.htoo@evonik.com Warnants, N., M. J. van Oeckel, and M. de Paepe (2001): Study of the optimum ideal protein level for weaned piglets. Journal of Animal Physiology and Animal Nutrition 85:

15 15 PIG Effect of dietary tryptophan and valine to lysine ratios on growth performance of 8 to 25 kg pigs Conclusions Performance of 8 to 25 kg pigs was improved by increasing the standardized ileal digestible (SID) tryptophan (Trp):lysine (Lys) ratio from 17 to 22 %. Increasing the dietary SID Trp:Lys ratio from 17 to 22 % increased average daily gain (ADG) by 5 % and improved feed conversion ratio (FCR) by 6 %. Performance of pigs fed 68 % SID valine (Val):Lys was greater than that of pigs fed 63 % SID Val:Lys diets, but not different from pigs fed 73 % SID Val:Lys diets. Increasing the dietary SID Val:Lys ratio from 63 to 68 % increased ADG by 6 % and improved FCR by 5 %. Introduction Tryptophan is considered as the 3 rd or 4 th limiting amino acid (AA) in typical pig diets after Lys, threonine (Thr) and methionine (Met). The published estimates for the optimal SID Trp:Lys ratio for 9 to 25 kg weaned pigs range from 16 (NRC, 2012) to 22 % (Jansman et al., 2010). Because Trp is involved in the regulation of immune response, the nutritional need of Trp may be increased for pigs fed antibiotic-free diets and raised under commercial conditions (Zhang et al., 2012). Valine is usually the 5 th limiting AA in typical low crude protein (CP) pig diets. However, the recommended optimal SID Val:Lys ratios for weaned pigs are inconsistent and range from 63 (GfE, 2008; NRC, 2012) to 70 % (Barea et al., 2009). Reliable estimates for optimal dietary ratios of Val and Trp to Lys are needed to assure maximum pig performance. Objective This experiment was conducted at the Brzezinski commercial Pig farm, Bersteland, Germany to evaluate the effects of dietary ratios of SID Trp:Lys and SID Val:Lys on the performance of 8 to 25 kg weaned pigs fed antibiotic-free diets and raised under commercial conditions. Materials and Methods A 28-day (d) growth study was conducted with 210 mixed-sex pigs [DANZucht Piétrain; initial body weight (BW) = 8.2 ± 0.56 kg] which were selected from a pool of 290 piglets (weaned at 25 ± 3 d). Pigs were given an adaptation period of 7 d and fed a commercial pre-starter diet. Pigs were blocked by the initial BW, litter, and gender, and assigned to 6 dietary treatments with 7 pens (5 pigs/pen) per treatment using a 2 3 factorial design with 2 levels of SID Trp:Lys ratio (17 and 22 %) and 3 levels of SID Val:Lys ratio (63, 68 and 73 %). The average room temperature was maintained at 27 C during week 1 and was gradually reduced by 1 C per week. The experimental diets were formulated based on corn, wheat and soybean meal using analyzed ingredient AA contents and published SID coefficients (AMINODat 4.0) to meet or exceed AA requirements except for Trp, Val and Lys (AMINODat 4.0; NRC, 2012). All diets were formulated to contain 1.18 % SID Lys (90 % of requirement) and adequate in net energy (NE; MJ/kg). Diets 1 to 3 contained 0.20 % SID Trp (63, 68 and 73 % SID Val:Lys) and diets 4 to 6 contained 0.26 % SID Trp (63, 68 and 73 % SID Val:Lys) by supplementing L-Trp and L-Val at the expense of corn starch (Table 1). The diets did not contain antibiotics or enzymes and were pelleted at a die temperature of 58 C. Individual BW and feed disappearance were recorded weekly to calculate ADG, feed intake, and FCR. Data were analyzed by ANOVA using GLM procedure of SAS and the model included initial BW as a covariant. Differences were considered significant if p < 0.05 and orthogonal-polynomial contrasts were used to determine linear effects of ratios of SID Trp:Lys (17 vs. 22 %) and SID Val:Lys (63 vs. 68 % and 68 vs. 73 %) on pig responses.

16 16 PIG Table 1 Ingredient and nutrient composition of experimental diets (%, as-fed). Diet 1 Diet 2 Diet 3 Diet 4 Diet 5 Diet 6 SID Trp:Lys, % SID Val:Lys, % Corn Wheat Soybean meal Corn starch Min-vitamin premix Others L-Lysine HCl L-Threonine DL-Methionine L-Tryptophan L-Valine (ValAMINO ) Calculated contents NE, MJ/kg SID Lys, % SID Met + Cys, % SID Thr, % SID Trp, % SID Val, % SID Ile, % Analyzed values CP /% Total Lys, % Total Trp, % Total Val, % including mono-calcium phosphate (1.12 %), limestone (1.05 %), soybean oil (3.33 %) and salt (0.05 %)

17 17 PIG Table 2 Effects of dietary valine and tryptophan levels on performance and fecal scores (d 0 28). Diet 1 Diet 2 Diet 3 Diet 4 Diet 5 Diet 6 Contrast p-values SID Trp:Lys, % Val:Lys SID Val:Lys, % SEM Trp 63 vs vs 73 Trp Val:Lys Body weight, kg d d ADG, kg/d Feed intake, kg/d FCR, g/g Fecal consistency score* d P-value (ANOVA) * Fecal scores: 1 = liquid diarrhea; 2 = mild diarrhea; 3 = soft feces; 4 = well-formed feces; 5 = hard and dry Results The analyzed contents of CP and total AA in the experimental diets were consistent and close to calculated values (Table 1). Therefore, the calculated SID Trp:Lys and SID Val:Lys ratios were used for presenting the results. The overall performance results (d 0-28) are presented in Table 2. The overall feed intake was not affected by the treatments. The average feed intake during the 28-d period was kg/d and similar among the treatments. Increasing dietary SID Trp:Lys from 17 to 22 % increased (p = 0.008) the average final BW (d 28; Table 2). Similarly, the ADG of pigs fed diets that contained 22 % SID Trp:Lys were greater (p = 0.007) than their counterparts fed a lower (17 %) SID Trp:Lys ratio. The ADG maximized at the SID Val:Lys ratio of 68 % when the diets contained 17 % SID Trp:Lys ratio but at a higher SID Trp:Lys ratio of 22 % the greatest ADG was achieved with 73 % SID Val:Lys. Overall, the ADG of pigs fed diets containing 68 % SID Val:Lys were greater (p = 0.004) than that of pigs fed 63 % SID Val:Lys diets, but was not different (p = 0.790) from pigs fed the 73 % SID Val:Lys diets (Table 2; Figure 1). The FCR was optimized (1.330) at SID Val:Lys ratio of 68 % when the diets contained a lower (17 %) SID Trp:Lys ratio. Among the 22 % SID Trp:Lys diets the best FCR (1.255) was achieved with a SID Val:Lys ratio of 73 %. Based on the orthogonal-contrast comparison, FCR was improved by increasing the SID Val:Lys ratios from 63 to 68 % (p = 0.009) but FCR of pigs fed 68 % SID Val:Lys diets were not different (p = 0.783) from those fed 73 % SID Val:Lys diets (Table 2; Figure 2). The dietary Trp level Val:Lys interaction effect was not observed for all measured parameters (p > 0.05) which indicates that the optimal Val:Lys ratio observed was independent of dietary Trp level, and the effect of Trp:Lys ratio was not affected by the Val:Lys ratios. Figure 1 Effects of dietary Val and Trp levels on ADG of 8 to 25 kg pigs (d 0 28). Figure 2 Effects of dietary Val and Trp levels on FCR of 8 to 25 kg pigs (d 0 28) ADG /g/d FCR /g/g % SID Trp:Lys 22 % SID Trp:Lys Dietary SID Val:Lys /% % SID Trp:Lys 22 % SID Trp:Lys Dietary SID Val:Lys /% Trp:Lys (17 vs 22): P = Val:Lys (63 vs 68): P = Val:Lys (68 vs 73): P = 0.79 Trp:Lys (17 vs 22): P = Val:Lys (63 vs 68): P = Val:Lys (68 vs 73): P = 0.78

18 18 PIG Only few piglets exhibited diarrhea and the overall fecal scores ranged from 3.3 to 3.8 (soft feces to well-formed feces; Table 2) which indicates that pigs were in good gut health status. Probably for this reason the feed intake was not affected by the dietary Trp level in the current study which disagrees with Zhang et al. (2012) who reported a linear increase in feed intake by increasing dietary SID Trp:Lys from 13 to 22 %. However, the final BW, ADG and FCR of pigs fed diets containing 22% SID Trp:Lys were significantly better than those fed diets containing 17 % SID Trp:Lys ratio. Overall, growth performance of 8 to 25 kg weaned pigs fed antibiotic-free diets and raised under commercial conditions maximized at 22 % SID Trp:Lys and 68 % SID Val:Lys in the diet. Source Effect of dietary valine and tryptophan on productive performance of 8 to 25 kg post-weaning piglets. Evonik Trial report # References AMINODat 4.0. Platinum version Evonik Degussa GmbH, Hanau-Wolfgang, Germany. Barea, R., L. Brossard, N. Le Floc h, Y. Primot, D. Melchior and J. van Milgen The standardized ileal digestible -to-lysine requirement ratio is at least seventy percent in postweaned piglets. J. Anim. Sci. 87: GfE Empfehlungen zur Energie und Nährstoffversorgung von Schweinen. DLG-Verlag. Jansman, A. J. M., J. Th. M. van Diepen and D. Melchior The effect of diet composition on tryptophan requirement of young piglets. J. Anim. Sci. 88, NRC, Nutrient Requirements of Swine. 11 th ed., Washington D.C. USA. Zhang, G. J., Q. L. Song, C. Y. Xie, L. C. Chu, P. A. Thacker, J. K. Htoo, and S. Y. Qiao Estimation of the ideal ratio of standardized ileal digestible tryptophan to lysine for growing pigs fed low crude protein diets supplemented with crystalline amino acids. Livestock Sci. 149,

19 19 PIG Requirement of valine and optimal valine to lysine ratio in diets for 8 to 18 kg pigs Conclusions The final body weight (BW) and average daily gain (ADG) maximized at a dietary standardized ileal digestible (SID) valine (Val):lysine (Lys) of 66 % while the feed conversion ratio (FCR) was optimized at 72 % SID Val:Lys. Based on the two-slope broken-line regression the optimal dietary SID Val:Lys ratio to improve ADG and FCR was estimated at 67 %. When the diets contained adequate SID Lys level (1.34%), a dietary SID Val content of 0.94 % (SID Val:Lys ratio of 71 %) maximized the growth performance of 8 18 kg pigs. Overall, the results of ratio and requirement approaches provide an optimal SID Val:Lys ratio recommendation of at least 67 % for 8 18 kg pigs. Introduction Valine and/or isoleucine (Ile) are the next (5th and 6th) limiting amino acids (AA) in typical low crude protein (CP) pig diets. The use of L-Valine facilitates lowering the CP level and to meet the optimal Val level more closely in the diets. However, the recommendations for SID Val:Lys for starter pigs are inconsistent, ranging from 64 % (NRC, 2012) to 70 % (Barea et al., 2009). Thus, reliable estimates on the Val requirement or optimal ratio of Val to Lys in diets that contain no antibiotic growth promoters (AGP) are needed to assure optimal pig performance. Objective To evaluate the dietary SID Val:Lys ratio and SID Val level that optimizes the growth performance of 8 to 18 kg pigs fed corn-wheat-soybean meal diets that contained no AGP. Materials and Methods A 28-day (d) growth study (co-operation with Prof. Georg Dusel, University of Bingen) was conducted with 192 Topigs weaned pigs with initial body weight (BW) of 8.5 kg which were blocked by gender and BW, and allotted to 8 dietary treatments with 6 pen replicates (2 barrows and 2 gilts/pen) per treatment. A cornwheat-soybean meal-lupine-based basal diet was formulated using analyzed ingredient AA contents and published SID coefficients (AMINODat 4.0), to meet requirements of AA other than Val (0.69 % SID Val). Lysine was second limiting (1.15 % SID Lys). Graded levels of supplemental L-Val (ValAMINO ) were added to the basal diet to obtain 5 SID Val:Lys ratios (60, 64, 68, 72 and 76 %). In addition, diets 6, 7 and 8 were produced to be adequate in Lys (1.34 % SID Lys) but varied in Val content (0.86, 0.91 and 0.97 %). All diets contained the same net energy content (10.35 MJ/kg). The analyzed Val contents in the diets were slightly higher than calculated values. Based on the analyzed AA, the corrected SID Val:Lys ratios in diets 1 to 5 were 63, 66, 72, 76 and 78 %, and corrected SID Val contents in diets 6 to 8 were 0.86, 0.94 and 1.01 % (SID Val:Lys ratios of 66, 71 and 76 %), respectively. Pigs had free access to pelleted feed and water. Results The final BW (d 28) and ADG of pigs fed diet 7 (71 % SID Val:Lys; 1.34 % SID Lys) were greater than that of diet 3 (72 % SID Val:Lys; 1.15 % SID Lys) indicating that Lys was second limiting in diets 1 to 5 (Table 1). Table 1 Effect of SID Val:Lys on performance of 8 18 kg pigs fed Lys-deficient diets vs. positive control. Experimental diets Diet 1 Diet 2 Diet 3 Diet 4 Diet 5 Diet 7 SID Val:Lys, % SEM Linear Quad. Lys effect SID Lys, % diet 1 5 diet vs. 7 BW (d 0), kg BW (d 28), kg a abc ab ab ab c ADG, g/d 258 a 284 abc 278 ab 270 ab 260 ab 322 c ADFI, g/d FCR, g/d b ab ab ab ab a a,b,c Values in the same row with different superscripts differ significantly (P < 0.05).

20 20 PIG Increasing dietary SID Val:Lys from 63 to 78 % (Diet 1 to 5) increased (p = 0.046) the final BW quadratically, and tended to increase the ADG (d 0-28) quadratically (p = 0.075). The greatest final BW (16.47 vs kg) and ADG (284 vs. 258 g/d) in comparison to the basal diet were achieved at a SID Val:Lys ratio of 66 %. The overall (d 0 28) FCR tended to improve (p = 0.078; quadratic) and the best FCR (1.377 vs ) in comparison to the basal diet was achieved at a SID Val:Lys ratio of 72 %. Feed intake was similar and not affected by dietary treatments. Using a two-slope broken-line regression model the optimal dietary SID Val:Lys ratio of 67 % was estimated to optimize ADG and FCR of 8 18 kg pigs (Figures 1 and 2). Figure 1 Optimal dietary SID Val:Lys ratio to improve ADG by two-slope broken-line model. Figure 2 Optimal dietary SID Val:Lys ratio to improve FCR by two-slope broken-line model. ADG, g/d Two-slope brocken-line model: SID Val:Lys = 67 % y = (67.0 x) 2.86 (x 67.0) r 2 = Dietary SID Val:Lys ratio, % FCR, g/g Two-slope brocken-line model: SID Val:Lys = 67 % y = (66.8 x) (x 66.8) r 2 = Dietary SID Val:Lys ratio, % When the diets contained adequate level of Lys (diets 6 to 8), increasing SID Val from 0.86 to 0.94 % tended to increase (p = 0.059) the final BW on d 28 (Table 2). The ADG was not affected by the Val content but maximized at a SID Val level of 0.94 %. Increasing the SID Val level from 0.86 to 0.94 % (increasing SID Val:Lys from 66 to 71 %) significantly improved (p = 0.011) the FCR. These results tested under adequate Lys condition support the optimal SID Val:Lys ratio of 67 % which was estimated in pigs fed diets in which Lys was second limiting. Table 2 Requirement of SID Val to optimize performance of 8 to 20 kg weaned pigs fed Lys-adequate diets. Experimental diets 1 Diet 6 Diet 7 Diet 8 P-values SID Val, % SIDVal: Lys, % SEM ANOVA BW (d 0), kg BW (d 28), kg ADG, g/d ADFI, g/d FCR, g/d b a ab All diets contained adequate Lys (1.34 % SID Lys). Source Evonik Trial report # Optimal Val to Lys ratio in diets for 8 to 18 kg starter pigs. References AMINODat 4.0. Platinum version, Evonik Degussa GmbH, Hanau-Wolfgang, Germany. Barea, R., L. Brossard, N. Le Floc h, Y. Primot and J. van Milgen (2009): The standardized ileal digestible isoleucine-to-lysine requirement ratio may be less than fifty percent in eleven- to twenty-three-kilogram piglets. J. Anim. Sci. 87: NRC (2012): Nutrient Requirements of Swine. 11 th ed. Washington (D.C): The National Academic Press.

21 21 POULTRY Isoleucine requirement in a diet for broilers from 15 to 29 days of age Conclusion The first three limiting amino acids in broilers are well known to be methionine, lysine and threonine. However, identification of the 4 th -limiting amino acid is still being discussed and seems to depend upon diet composition. Feed formulation exercises showed a potential for isoleucine (Ile) to be 4 th -limiting in diets based on corn/wheat, soybean meal and certain animal protein sources (poultry/meat meal). To estimate the Ile requirement of boilers from day 15 to 29 in two studies 960 (6 replicates, 20 birds/replicate; Germany) or 1280 (5 replicates, 32 birds/replicate; Hungary) male Ross 308 broilers, respectively, were allocated randomly to wood-shavings littered floor pens. A wheatcorn-soybean meal based basal diet deficient in Ile but adequate in all other nutrients and energy was divided to produce diets of increasing content of standardized ileal digestible (SID) Ile (0.59, 0.62, 0.65, 0.68, 0.71, 0.74, 0.78, 0.82 % SID Ile) by supplementation with L-isoleucine. Feed and water were supplied ad libitum. Only small numerical improvements of animal performance were achieved with increasing dietary Ile in trial 1. The second study showed significant (p < 0.05) effects of dietary Ile supply especially on body weight, weight gain, and feed conversion ratio. Regression analysis revealed optimal SID Ile levels of 0.71 to 0.72 % in the diet which was slightly lower than currently recommended for growing broilers from 15 to 29 days of age. Introduction Methionine, lysine and threonine are investigated to be the first three limiting amino acids in broiler nutrition. With regard to the next limiting amino acid valine, isoleucine, and arginine are discussed. It seems that the identity of the 4th limiting amino acid depends on the diet composition. Corzo (2008) formulated various broiler diets based on different raw material combinations. He identified isoleucine to be the most probable 4th limiting amino acid in diets based on corn/wheat, soybean meal and certain animal protein sources like poultry and meat meal. Therefore, two dose-response-trials with identical experimental design were conducted in order to determine the isoleucine (Ile) requirement of boilers. Materials and Methods At the facility of feedtest, Germany, 960 (6 replicates, 20 birds/replicate) and at the facility of the University of Kaposvár, Hungary, 1280 (5 replicates, 32 birds/replicate) male Ross 308 broilers were randomly allocated to 8 treatments. In both facilities birds were housed in wood-shavings littered floor pens, light and temperature were managed according to breeder s recommendations (Aviagen 2007) and birds received a commercial starter diet adequate in nutrients and energy until start of experiment at 15 days of age. The basal diets were compounded for the German or the Hungarian trial of or % wheat, % corn, or % soybean meal (48 % crude protein), 3.98 or 4.31 % soybean oil, 2.10 or 2.03 % corn gluten meal, 3.89 or 3.83 % premix and minerals, 1.32 or 1.51 % glutamic acid, 1.10 or 1.01 % Biolys, 0.35 or 0.38 % DL- Methionine, 0.34 % L-Arginine, 0.34 or 0.74 % glycine, 0.26 % L-Threonine, 0.26 or 0.28 % L-Valine, 0.02 or 0.01 % L-Tryptophan, and 0.03 % L-Histidine, respectively. Standardized ileal digestible (SID) amino acid contents were specified to exceed the recommendations (Evonik, 2010) by 10 % except for Ile (1.17 % SID lysine, 0.87 % SID methionine+cystine, 0.75 SID threonine, 0.19 % SID tryptophan, 1.21 % SID arginine, 1.24 % SID leucine, 0.94 % SID valine, 0.40 % SID histidine, and 0.73 % SID phenylalanine). Starting with an SID Ile content of 0.59 % in basal diet, the Ile levels of the other treatments were adjusted by adding L-Isoleucine to contain 0.62, 0.65, 0.68, 0.71, 0.74, 0.78, 0.82 % SID Ile, respectively. Experimental diets were fed from day 15 to 29 ad libitum, water was freely accessible by bell drinkers. Expected amino acid levels were well confirmed by analysis. Body weights were recorded individually, feed consumption per pen. At trial termination, four birds with body weights closest to the pen average were selected from each pen for carcass evaluation. Experimental data were evaluated by standard ANOVA procedures and, where suitable, by exponential regression analysis using the following model: a + b * 1 exp ( c*x) ). Results In the German trial no significant response could be detected for body weight gain (BWG) and feed conversion ratio (FCR) whereas breast meat yield (BMY) was significantly improved (p < 0.05) at a SID Ile level of 0.65 % (Table 1). The Hungarian

22 22 POULTRY trial showed a significant impact of increasing SID Ile on BWG, FCR and BMY but no significant effects on carcass weight (Table 1). At higher Ile supplementation levels no further performance improvement could be observed. However, data set allowed for an exponential regression and detected an optimum SID Ile level of 0.72 % for BWG and 0.71 % for FCR (Figure 1). Discussion and conclusion The results of the German trial indicated that the lowest tested level of SID Ile was sufficient to meet the requirement. However, the significantly higher carcass weights and the corresponding numerical improvements of other performance parameters at 0.65 % SID Ile lead to the assumption that a level somewhere between the lowest tested level of 0.59 % SID Ile and the current recommended level of 0.74 % SID Ile might be optimal. A nonlinear exponential regression suggested an optimum isoleucine concentration of 0.65 %, at which 95% of the increase of performance was realised, which is in line with the numerically highest performance of this trial. The Hungarian trial suggested a higher optimal level of SID Ile of 0.71 to 0.72 %. There is no clear explanation for the differences in the magnitude of responses between trials. However, broilers of the German trial achieved final body weights which were about 400 g higher compared to the Hungarian trial while the expectations according to Aviagen (2007) was 1597 g. The optimal SID Ile values of both trials are below the currently recommended level of 0.74 % SID Ile for male broilers from 15 to 29 days of age (Evonik 2010). Table 1 Responses of growing broilers from 15 to 29 days of age to graded levels of dietary standardised ileal digestible isoleucine (SID Ile) on body weight, body weight gain, feed conversion ratio, carcass weight, and breast meat yield obtained from two trials performed in Germany and Hungary. SID Ile Trial at feedtest, Germany Body weight, g Body weight gain, g/d Feed conversion ratio, g/g Carcass weight, g 1220 a 1256 abc 1295 c 1252 abc 1264 abc 1279 bc 1234 ab 1228 ab Breast meat yield, % of CW 35.0 ab 34.0 a 35.8 b 35.2 ab 35.3 ab 35.3 ab 34.5 ab 34.6 ab Trial at University of Kaposvár, Hungary Body weight, g 1537 b 1568 ab 1575 ab 1592 a 1600 a 1588 a 1586 a 1589 a Body weight gain, g/d 82.8 b 85.0 ab 85.6 ab 86.6 a 87.3 a 86.4 a 76.3 a 86.6 a Feed conversion ratio, g/g 1.49 b 1.43 a 1.43 a 1.41 a 1.39 a 1.40 a 1.41 a 1.41 a Carcass weight, g Breast meat yield, % of CW 35.2 b 36.4 ab 37.2 a 37.1 a 37.4 a 36.3 ab 36.1 ab 36.5 ab Different superscripts ( a-c ) indicate significant differences within a row (P < 0.05). SID = standardized ileal digestible, Ile = isloleucine, CW = carcass weight Figure 1 Exponential regression to investigate the requirement of standardized ileal digestible isoleucine in male broilers from 15 to 29 days of age according to results for body weight gain and feed conversion ratio of a trial conducted in Hungary; trial results in white rhomb, estimated optimum in black rhomb. Body weight gain, g/d y = (1 e x ) R 2 = Standardized ileal digestible isoleucine, % Standardized ileal digestible isoleucine, % FCR, g/g y = (1 e x ) R 2 = 0.92 References Aviagen Inc Ross 308 Management Guide. Corzo A Maximizing production efficiency by a better understanding of the 4 th limiting amino acid in broiler formulation. Zimmermann N.G. 6 th Mid-Atlantic Nutrition Conference. Timonium. M.D AMINONews, Volume 14, Number 01, August Revised amino acid recommendations. Evonik Degussa GmbH, Germany.

23 23 POULTRY Branched-chain amino acids in broiler nutrition Key information The branched-chain amino acids valine and isoleucine are regarded as the next performance limiting amino acids after methionine+cysteine, lysine, and threonine in broiler nutrition. An evaluation both of recent research from literature and of own research data on branchedchain amino acid responses in broiler chickens was conducted. The survey confirmed current recommendations for valine and isoleucine in broilers as suggested by amino acid recommendation tool QuickChick (2006) from Evonik. Introduction Valine (Val), isoleucine (Ile), and leucine (Leu) are categorised as branched-chain amino acids (BCAA) and are essential for broilers. While they serve as building blocks for protein synthesis they also have functions in metabolism. Metabolism of and interactions between these three branchedchain amino acids are explained in detail in a separate article by Htoo and Wiltafsky (2011) in this AMINONews. In his survey from 2007 Corzo calculated various types of broiler diets on the basis of the recommendations given by Degussa (today Evonik) in order to figure out the next performance limiting amino acid. Accordingly, in most of the scenarios valine was identified to be the next limiting amino acid and isoleucine became co-limiting in many diets as well. Moreover, the commercial availability of supplemental amino acids like L-Valine and L-Isoleucine today offers a higher flexibility to nutritionists in terms of balancing the dietary amino acid profile. One consequence might be a reduced use of high priced raw materials, hence avoiding oversupply with amino acids of the third and forth limiting level. Since diets are ideally formulated according to the ideal protein concept (Mack et al., 1999; Baker et al., 2002) which eventually defines minimum specification for the essential amino acids in least-cost feed formulation, the improved flexibility with the above mentioned free amino acids call for updating or substantiating current amino acid recommendations. Therefore, data obtained from valine and isoleucine researchs in broilers reported in international scientific literature and from Evonik s own research were evaluated. In the following, the procedure and the outcome of this survey as well as our conclusions will be presented. Dose-response data were collected, assessed and evaluated in a survey Dose-response data published in international literature and results of research supported by Evonik was collected and standardised according to a certain procedure in order to make them comparable. First of all, experimental diets of published trials were recalculated with AMINODat 4.0 (2010) as this allowed to assess the responses on basis of standardised ileal digestible (SID) amino acids. Moreover, recalculated amino acid profiles and dietary contents compared with our current amino acid recommendation for broilers (QuickChick, 2006) allowed to categorise the respective trial either as requirement or ratio study (for details see below). Once single data sets were assessed suitable for the review, response data were analysed by exponential regression [ y = a + b (1 exp (- c X) )] defining 95 % of the asymptotic response as optimum. Finally, the resulting optima were compared to our current recommendations on a relative scale setting QuickChick (2006) recommendations of 100 % (Table 1). In order to properly examine amino acid doseresponse studies and their results, it is crucial to understand whether an optimal dietary level of Val or Ile (requirement study) or an optimal ratio between Val or Ile and lysine (Lys) according to ideal protein concept (ratio study) was determined. The difference shall briefly be explained. In case of a requirement study, the basal experimental diet is formulated to be as low as possible regarding the nutrient under investigation. In contrast all other essential (and non-essential) amino acids shall be clearly above levels which are assumed to be optimal for covering the requirement. This is to minimise the probability that any other amino acid except the amino acid under investigation limits the response to graded dietary Val or Ile levels. It is thus assumed that the finally determined optimal dietary supply is not biased by any other amino acid and represents therefore the dose for meeting the requirement. Compared to

24 24 POULTRY Table 1 Current recommendations for isoleucine (Ile), valine (Val), and leucine (Leu) concentrations in broiler diets (QuickChick, 2006). Days ME/kg Ile Val Leu Ile Val Leu Male MJ/kcal SID levels, % per kg Ratio in % to Lys /3, /3, /3, /3, > /3, Female /3, /3, /3, /3, > /3, Mixed /3, /3, /3, /3, > /3, ME = metabolizable energy SID = standardized ileal digestible Lys = lysine that the nutritional settings for a ratio study differ slightly as the investigator needs to ensure that lysine remains the second limiting amino acid in the experimental diet. Typically, the chosen dietary lysine level is marginally below the assumed optimum (recommendation) whereas all other amino acids except the amino acid under test (Val, Ile) are clearly above. Although this exercise appears easy on paper, it is not necessarily sure that lysine is indeed second limiting in the experiment. In order to prove that, an additional treatment is needed in which extra lysine is supplemented to the experimental diet with the highest dose of the test amino acid. If this extra lysine results in a further performance improvement of the broilers, evidence is provided that lysine was second limiting in the dose-response set-up. Otherwise, it cannot be excluded that lysine was not second limiting which then would suggest an underestimation of the calculated Val or Ile to lysine ratio. In turn, if lysine was proven to be the second limiting amino acid, the response cannot be used for determining a requirement because the response was limited by lysine and thus the requirement for the amino acid under investigation would be underestimated. It needs to be noted for our survey, that in most cases (especially in data obtained from literature) the control treatment with extra lysine was missing and therefore the categorisation of the trial (requirement or ratio study) was based on the re-calculated amino acid composition based on AMINODat 4.0 (2010).

25 25 POULTRY Broiler responses to valine Corzo et al. (2007) conducted a trial looking into the response of male Ross 708 broilers to dietary Val from days of age based on all-vegetable diets with reduced Lys content (0.95 % SID Lys compared to recommended 1.00 %). The trial was thus assessed a ratio study. For the parameters body weight gain (BWG) and feed conversion ratio (FCR) a ratio of 88 % SID Val/Lys and for breast meat yield (BMY) a SID Val/Lys ratio of 78 % yielded optimal performance (Table 2). In a follow-up study with male Ross 308 broilers the same authors (Corzo et al., 2008) found optimal SID Val/Lys ratios to be 79 % and 85 % for BWG and FCR, respectively, during 1 14 days of age. This is well in line with the recommendations of 79 % suggested by QuickChick (2006). In the experiments with growing (14 28 days of age) and finishing broilers (28 42 days of age), calculated SID Lys (1.10 and 1.04 %) as well as other essential amino acids exceeded levels considered adequate for optimal performance. Owing to lysine being non-limiting the experimental design fulfilled the condition of a requirement study but was for the same reason not suitable to derive an optimum Val/Lys ratio. For the period days of age the researchers found 0.90 % and 0.80 % SID Val to be optimal for BWG and FCR, respectively, which compares well to the 0.84 % SID Val recommended by QuickChick (2006). For the period days of age optimal performance was achieved at 0.76 % and 0.69 % SID Val for BWG and FCR, respectively, which is lower than the 0.78 % SID Val recommended by QuickChick (2006). However, the most recent study conducted by Berres et al. (2011) using male Cobb 500 broilers during days of age suggests an optimum of 0.89 % and 0.82 % SID Val for BWG and FCR, respectively. Those results are based on diets formulated to a non-limiting SID Lys content of 1.10 %. In cooperation with Evonik two experiments with 1400 male Cobb 500 broilers for the starter (8 21 days of age) and 1120 broilers for the finisher phase (30 43 days of age; Rostagno et al., 2008) were conducted at the University of Viçosa, Brazil. A basal diet adequate in all nutrients and energy, except valine, was added in increments with L-valine to test the range of 0.71 to 0.86 % SID Val in starter diets and 0.66 to 0.81 % SID Val in finisher diets, respectively. The exponential regression detected optima of 0.86 % and 0.82 % SID Val for the starter and 0.75 % and 0.76 % SID Val for the finisher phase for BWG and FCR, respectively. At the research facility of feedtest, Germany, an experiment to investigate the requirement of Val was designed with wheat-corn-soybean meal based diets including 0.68, 0.71, 0.74, 0.77, 0.80, 0.83, 0.87, and 0.91 % SID Val and being adequate in all other nutrients and energy (Helmbrecht et al., 2010a). Nine hundred and sixty male Ross 308 broilers in 6 replicates with 20 birds per replicate received those diets during the age period days. The exponential regression of the response data suggested a level of 0.86 % SID Val for optimum BWG, 0.80 % SID Val for optimum FCR and 0.88 % SID Val for optimum carcass weight (Figure 1; Table 2). These Val levels agree well with the 0.87 % recommended by Quick- Chick (2006) for broilers of the tested phase. Finally, a total of eight suitable valine doseresponse studies providing 17 data sets of relevant performance criteria were included in this survey and are summarised in Table 2. In the table data as extracted and determined from the individual studies including the re-calculated diet compositions according to AMINODat 4.0 (2010) are displayed as well as recommended figures for SID Lys, SID Val, and SID Val:SID Lys ratio according to QuickChick (2006). The determined optima from the requirement and ratio represent on average 98 and 105 % of Evonik s current recommendation, respectively. Although there was also a certain variation between studies and performance criteria, it was concluded that the data do not provide reason for changing Evonik s current recommendations for valine in broilers.

26 26 POULTRY Figure 1 Optimal dietary standardized ileal digestible valine levels derived from body weight gain and feed conversion ratio data of growing broilers from 15 to 29 days of age; trial results are shown with white circles, the suggested optimum with a black rhomb (Helmbrecht et al., 2010a). Table 2 Results of broiler trials investigating responses to valine either in requirement studies or in ratio studies attained from literature and own research; literature results were related to amino acid values recalculated with AMINODat 4.0 (2010). Source Period days Gender strain ME MJ/kcal per kg SID Lys Trial, % SID Lys Quick- Chick, % SID Val, % SID Val/Lys, % Result of regression analysis Performance Corresponding QuickChick recom. Relation of trial to QuickChick recom. Result of regression analysis Corresponding QuickChick recom. Relation of trial to QuickChick recom. Ratio studies Corzo et al., Male/ Ross / 3, BWG FCR BMY Corzo et al., Male/ Ross / 3, BWG FCR Requirement studies Corzo et al., Male/ Ross / 3, BWG FCR Corzo et al., Male/ Ross / 3, BWG FCR Berres et al., Male/ Cobb / 3, BWG FCR Rostagno et al., Male/ Cobb / 3, BWG FCR Male/ Cobb / 3, BWG FCR Helmbrecht et al., 2010a Male/ Ross / 3, BWG FCR Average of comparison between trial results and current recommendation ME = metabolizable energy, SID = standardized ileal digestible, Lys = lysine, Val = valine BWG = body weight gain, FCR = feed conversion ratio, BMY = breast meat yield, recom. = recommendation

27 27 POULTRY Twenty data sets were available from isoleucine studies with broilers Kidd et al. (2004) investigated the Ile requirement of male Ross 308 broilers during the periods 18 30, 30 42, and days of age, based on diets providing a limiting amount of SID Lys (0.91, 0.90 and 0.75 % as compared to Evonik s recommendations of 1.04, 0.98 and 0.91 %), hence suitable to derive optimum Ile/Lys ratios. Exponential regression analysis resulted in optimum SID Ile/Lys ratios of 69 and 76 % (18 30 days of age), 56 and 58 % (30 42 days of age) and 72 and 81 % (42 56 days of age) for BWG and FCR, respectively. These values are in line with recommended SID Ile/Lys ratios of 70, 71 and 73 % for the respective periods except for the period days of age. In this particular data a very flat response curve resulting in a low optimum level for the tested amino acid ratio was found. However, as no obvious confusions during the course of the trial were described and the design met the premises for a ratio study the results were considered in the present review. Despite, from the biological point of view there is no explanation why the optimal SID Ile/Lys ratio should be that much lower in days of age old broilers compared to younger or older birds. Hale et al. (2004) studied growth and carcass performance responses of female Ross 508 broilers from days of age. Dietary Lys was formulated close to Evonik recommendations (0.86 % actual vs % SID Lys recommended). The experiment resulted in optimum performance responses at 0.66, 0.58, and 0.62 % SID Ile for BWG, FCR, and BMY, respectively. Determined optima were thus somewhat lower than Evonik s currently recommended 0.70 % SID Ile. Two experiments were conducted at the University of Viçosa, Brazil (Rostagno et al., 2008), in cooperation with Evonik looking into this topic by using male Cobb 500 broilers during the starter (1400 birds, 7 21 days of age) and the finisher periods (1120 birds, days of age) (Helmbrecht et al., 2010b). Diets were formulated to be below Evonik s Lys recommendations (1.00 % and 0.90 % SID Lys vs % and 0.98 % in starter and finisher feed, respectively) in order to meet the requirements for a ratio study with Lys being second limiting. In this case a positive control with extra Lys was included to which broilers responded positively confirming the suitability of the trial as a ratio study. Tested ranges of SID Ile/Lys covered % in the starter phase and % in the finisher phase by adding L-isoleucine. Exponential regression of the response data suggested an optimum ratio of 63 % for BWG and FCR and 64 % SID Ile/Lys for BMY in starter diets and 68 % and 78 % for BWG and FCR, respectively, in finisher diets. In the second phase BMY was not affected by changing SID Ile/Lys. The determined values are in line with QuickChick (2006) recommendations (69 % SID Ile/Lys for the age range of 7 21 days and 71 % for days of age).

28 28 POULTRY Table 3 Composition and nutrient content of basal diets used for requirement studies in Germany and Hungary to investigate the requirement of standardized ileal digestible (SID) isoleucine (Ile) in male Ross 308 broilers from 15 to 29 days of age; through the addition of 0.03, 0.06, 0.09, 0.12, 0.15, 0.19, and 0.23 % L-Isoleucine eight treatments with different levels of Ile were achieved. Composition of basal diet Germany Hungary Nutrients of basal diet Germany Hungary Ile content is equivalent to 80 % of current recommendation Ile content is equivalent to 80 % of current recommendation Wheat Crude protein Corn ME, kcal 3,155 3,155 Soybean meal, 48 % CP ME, MJ Soybean oil SID Lysine Corn gluten meal SID Methionine Premix* + Minerals** SID Cystine Glutamic acid SID M+C Biolys SID Threonine MetAmino SID Tryptophan L-Arginine SID Arginine Glycine SID Isoleucine ThreAmino SID Leucine L-Valine SID Valine TrypAmino SID Histidine L-Histidine SID Phenylalanine L-Isoleucine * Premix composition: 12,000 IE retinol, 2,400 IE cholecalciferol, 50 mg DL-a-tocopherol, 1.5 mg menadione, 2.0 mg thiamine, 7.5 mg riboflavine, 3.5 mg pyridoxine, 20 mcg cobalamins, 35 mg niacin, 10 mg D-pantothenic acid, 460 mg choline chloride, 1.0 mg folic acid, 0.2 mg biotin, 80 mg iron, 12 mg copper, 85 mg manganese, 60 mg zinc, 0.40 mg cobalt, 0.8 mg iodine, 0.1 mg selenium, 125 mg anti-oxidant mixture ** Minerals: Germany: 2.78 % di-calcium phosphate 22, 0.65 % calcium carbonate, 0.25 % sodium carbonate, 0.21 % sodium chloride; Hungary: 2.77 % di-calcium phosphate 22, 0.65 % calcium carbonate, 0.10 % sodium carbonate, 0.31 % sodium chloride The SID Ile requirement was tested in two doseresponse trials conducted at the facilities of feedtest, Germany (Elwert, 2010), and the University of Kaposvár, Hungary (Tossenberger et al., 2010). By adding L-isoleucine to wheatcorn-soybean meal based diets eight levels of SID Ile (0.59, 0.62, 0.65, 0.68, 0.71, 0.74, 0.78, and 0.82 %) were achieved and fed to male Ross 308 broilers (Table 3). Compared to Evonik s recommendations the basal diets of each trial were clearly deficient in Ile but exceeding recommendations for all other amino acids. Other nutrients and energy were adequate and based on breeder recommendations (Aviagen, 2007). The trial conducted at feedtest showed no significant effects for BWG and FCR but the best numerical performance was observed at the second supplementation level. At this level a significant (P < 0.05) improvement for BMY compared with the basal diet could be detected. In contrast the trial performed at the University of Kaposvár showed significant (P < 0.05) impact of increasing dietary SID Ile which was of exponential nature. Exponential regression revealed an optimum SID Ile level of 0.72 % for BWG and 0.71 % for FCR and BMY. QuickChick (2006) recommends 0.74 % SID Ile for the period days of age. It is interesting to note that diet specifications were identical in both trials and diet compositions were very similar (after adjustments of the compositions according to an ingredient analysis) but final body weights at day 29 ranged between 1920 and 1995 g in the German trial whereas they ranged between 1537 and 1600 g in the Hungarian trial. Still, the performance level of the Hungarian trial was above the performance objectives suggested by the breeding company (Aviagen, 2007). This difference opens room for speculations whether general performance level and experimental conditions interact with the isoleucine requirement which, however, cannot be answered by these experiments.

29 29 POULTRY In Table 4 the results of the Ile survey is summarised. Similar to the valine data evaluation, results were related to our current recommendations and accordingly optimal dietary levels and ratios were on average 95 and 96 % of Evonik s current recommendations (QuickChick, 2006). However, the variation around these means was much higher compared to the valine responses with the above mentioned studies by Kidd et al. (2004; days of age) and Elwert (2010) having a strong impact on the outcome. Leaving these two studies out because of the uncertainties mentioned earlier would result in close to a 100 % agreement with current recommendations. We feel that the evidence is not strong enough for changing our current recommendations. In this context interaction with Leu may play a role as described for swine (see Htoo and Wiltafsky, 2011) but the effect of Leu on Ile and Val requirements is not well studied in broilers. Burnham et al. (1990) found a positive performance response of broilers to Leu at low dietary Ile which, however, disappeared as soon as Ile (and also Val) met the requirement. High Leu did not impact the Ile requirement in that study which appears to be in contrast to findings in swine. Table 4 Results of broiler trials investigating responses to isoleucine either in requirement studies or in ratio studies attained from literature and own research; literature results were related to amino acid values recalculated with AMINODat 4.0 (2010). Source Period days Gender strain ME MJ/kcal per kg SID Lys trial, % SID Lys Quick Chick, % SID Ile, % SID Ile/Lys, % Result of regression analysis Performance Corresponding QuickChick recom. Relation of trial to QuickChick recom. Result of regression analysis Corresponding QuickChick recom. Relation of trial to QuickChick recom. Ratio studies Kidd, et al., Male/ Ross / 3, BWG FCR Male/ Ross / 3, BWG FCR Male/ Ross / 3, BWG FCR Helmbrecht, et al., Male/ Cobb / 3, BWG FCR BMY Male/ Cobb / 3, BWG FCR Requirement studies Hale, et al., Female/ Ross / 3, BWG FCR BMY Elwert, Male/ Ross / 3, BWG FCR BMY Tossenberger, Male/ Ross / 3, BWG FCR BMY Average of comparison between trial results and current recommendation ME = metabolizable energy, SID = standardized ileal digestible, Lys = lysine, Val = valine, BWG = body weight gain FCR = feed conversion ratio, BMY = breast meat yield, recom. = recommendations

30 30 POULTRY In summary, Evonik s current recommendations for dietary SID Val and SID Ile contents as well as their ratios to SID Lys in broilers were confirmed by this survey and will thus not be changed (see Table 5). However, the survey also revealed a lack of data and variability between studies which suggests a need for further studies to better understand broiler nutrition with respect to branched-chain amino acids. Table 5 Amino acid recommendations for male and female broilers (AMINOChick, 2011). Male Days ME MJ /kcal/kg Lys Met M+C Thr Trp Arg Ile Val Leu His Phe + Tyr Standardized ileal digestible amino acid levels (% of diet) 0 12 * 12.7/3, /3, /3, /3, > /3, Amino acids to lysine ratios 0 12 * 12.7/3, /3, /3, /3, > /3, Female Standardized ileal digestible amino acid levels (% of diet) 0 12 * 12.7/3, /3, /3, /3, > /3, Amino acids to lysine ratios 0 12 * 12.7/3, /3, /3, /3, > /3, Mixed Standardized ileal digestible amino acid levels (% of diet) 0 12 * 12.7/3, /3, /3, /3, > /3, Amino acids to lysine ratios 0 12 * 12.7/3, /3, /3, /3, > /3, ME = metabolizable energy, Lys = lysine, Met = methionine, M+C = methionine+cystine, Thr = threonine, Trp = tryptophan, Arg = arginine, Ile = isoleucine, Val = valine, Leu = leucine, His = histidine, Phe +Tyr = phenylalanine + tyrosine * equals a cumulated feed intake of about g/broiler

31 31 POULTRY References AMINOChick (2011), Evonik Industries, Health & Nutrition, Hanau-Wolfgang, Germany. AMINODat 4.0 (2010): 50 years amino acid analysis, Platinum Version, Evonik Degussa GmbH, Health & Nutrition, Hanau-Wolfgang, Germany. Aviagen (2007): Ross 308 Broiler Nutrition Specification. Baker, D. H., A. B. Batal, T. M. Parr, N. R. Augspurger, and S. M. Parsons (2002): Ideal Ratio (Relative to Lysine) of Tryptophan, Threonine, Isoleucine, and Valine for Chicks during the Second and Third Weeks Posthatch. Poultry Science 81: Berres, J., S. L. Vieira, A. Favero, D. M. Freitas, J. E. M. Peña, and E. T. Nogueira (2011): Digestible valine requirements in high protein diets for broilers from twenty-one to forty-two days of age. Animal Feed Science and Technology 165: Burnham, D., G. C. Emmans, and R. M. Gous (1990): Isoleucine requirements of the chicken: the effect of excess leucine and valine on the response to isoleucine. British Poultry Science 33: Corzo, A., M. T. Kidd, W. A. Dozier III, and S. L. Vieira (2007): Marginality and Needs of Dietary Valine for broilers fed Certain All-Vegetable Diets. Journal of Applied Poultry Research 16: Corzo, A. (2007): Valine and Isoleucine: their importance in broiler feed formulation. AMINONews 9: 1-7. Corzo, A., W. A. Dozier III, and M. T. Kidd (2008): Valine Nutrient Recommendations for Ross x Ross 308 Broilers. Poultry Science 87: Elwert, C. (2010): Limitations of Isoleucine in a broiler diet dose-response study. Trial report. Helmbrecht, A., C. Elwert, and A. Lemme (2010a): Requirement of valine in a diet for broilers from 15 to 29 days of age. Proceedings 11. Tagung Schweine- und Geflügelernährung, November 2010 Lutherstadt Wittenberg, Germany, Institute for Agricultural and Food Science: Helmbrecht, A., F. de Castro Tavernari, H. S. Rostagno, L. F. Teixeira Albino, and A. Lemme (2010b): Optimal Digestible Isoleucine to Lysine Ratios in Starter and Finisher Broilers. Proceedings XIIIth European Poultry Conference, August 2010, Tours, France. Htoo, J. K. and M. Wiltafsky (2011): Roles, metabolisms and antagonisms of branched-chain amino acids in animal nutrition. AMINONews 16: 1-8. Kidd, M. T., D. J. Burnham and B. J. Kerr (2004): Dietary isoleucine responses in male broiler chickens. British Poultry Science 45: Mack, S., D. Bercovici, G. de Groote, B. Leclercq, M. Lippens, M. Pack, J. B. Schutte, and S. Van Cauwenberghe (1999): Ideal amino acid profile and dietary lysine specification for broiler chickens of 20 to 40 days of age. British Poultry Science 40: QuickChick V (2006), Evonik Degussa GmbH, Health & Nutrition, Hanau-Wolfgang, Germany. Rostagno, H. S., L. F. T. Albino, and F. de Castro Tavernari (2008): Dietary digestible valine/ lysine ratios for boiler chickens. Trial report. Tossenberger, J. (2010): Limitations of one of the 4 th limiting amino acids isoleucine. Trial report. Dr. Ariane Helmbrecht ariane.helmbrecht@evonik.com Hale, L. L., S. J. Barber, A. Corzo, and M. T. Kidd (2004): Isoleucine of Thirty- to Forty-Two-Day- Old Female Chickens: Growth and Carcass Responses. Poultry Science 83:

32 32 POULTRY Requirement of valine in a diet for broilers from 15 to 29 days of age Conclusion The three first-limiting amino acids in broilers are well known to be methionine, lysine and threonine. But the identity of the fourth-limiting amino acid is still discussed. It seems that this depends upon diet composition. Corzo (2008) summarized the results of trials investigating this object. Valine showed a potential to be of limiting character in diets based on soybean meal and corn or wheat. To estimate the valine requirement of boilers from 15 to 29 days of age, a basal corn/soybean based diet was supplemented with valine to produce six diets of increasing valine content (0.68, 0.71, 0.74, 0.77, 0.80, 0.83, 0.87, 0.910% standardized ileal digestible (SID) valine). Every other amino acid was supplied 10% above recommendation of Evonik (2009), thus valine was the only deficient amino acid and every parameter decrease should be a direct result of valine. After the birds showed the same body weight at start (592 g) an increase of final body weight, body weight gain, and daily feed consumption with increasing valine supplementation was observed. Exponential regression for body weight and body weight gain returned a level of 0.80 % and for feed conversion ratio of 0.88 % SID valine in order to achieve a performance equal to 95 % of the asymptote. This agrees well with the 0.87 % recommended by Evonik (2010) for broilers from 15 to 29 days of age. Introduction The three first limiting amino acids in broilers are well known as methionine, lysine and threonine. But the position as 4th limiting amino acid is still discussed. It seems that the identification of the 4th limiting amino acid depends on the diet composition. Corzo (2008) summarized the results of trials investigating this subject. Valine appeared to be 4 th limiting in diets based on corn-soybean meal, and wheat-soybean meal. To estimate the valine requirement of boilers in a dose-response trial six levels of valine were fed (Table 1). Table 1 Experimental design to investigate valine requirement in broilers from 15 to 29 days of age. Treatment L-Valine supplementation SID Valine SID = standardized ileal digestibility